April 2010 One File

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In The Name of Allah, The Most Gracious, The Most Merciful
101. Verily, those for whom the good has preceded from Us, they will be
removed far therefrom (Hell). 102. They shall not hear the slightest sound of it
(Hell), while they abide in that which their ownselves desire. 103. The greatest
terror (on the Day of Resurrection) will not grieve them, and the angles will
meet them (with the greeting): " This is your Day which you were promised"
104. And (remember) the Day when We shall roll up the heaven like a scroll
rolled up for books. As We began the first creation, We shall repeat it. (It is)
a promise binding up Us Truly, We shall do it.
(Surah 21, Al-Anbiya, Part 17, The Noble Quran)

JOURNAL OF THE EGYPTIAN SOCIETY OF PARASITOLOGY
Volume (40), Number (1), April, 2010

Contents Page

1- POTENTIAL OF MEDICINAL PLANTS IN MOSQUITO CONTROL By
SAHAR A.B. FALLATAH AND EMAD I.M. KHATER.......

2-SURVEY ON DIPTEROUS FLIES AND THEIR DENSITIES IN ALEXA-
NDARIA AND HURGADA, Egypt By AZZA S. ABD EL-HALIM.

3- SUSCEPTIBILITY OF BROMADIOLONE ANTICOAGULANT RODE-
NTICIDE IN TWO RODENT SPECIES AND ITS HAEMATOLOGIC EFF-
ECT By MICHEAL.W. MIKHAIL AND YOUSRYA M. ABDEL- HAMID ......

4- SUBCLINICAL AUTOIMMUNE THYROID DISORDERS IN EGYPTI-
AN PATIENTS WITH UNTREATED CHRONIC HEPATITIS C VIRUS IN-
FECTION By ZAKARIA Y. MOHRAN, NADIA A. ABDEL KADER, AMAL
T. ABDEL MOEZ and AMAL A. ABBAS

5- A STUDY ON THE PREVALENCE OF HOUSE DUST MITES IN AL-
ARISH CITY, NORTH SINAI GOVERNORATE, EGYPT By GIHAD T. El-
SHERBINY, EMAN T. El-SHERBINI, NAGLA MOSTAFA KAMEL SAL-
ED, FOUAD M. HARIDY and AYMAN T.A. MORSY.

6- ANTIMICROBIAL RESISTANT BACTERIA AMONG HEALTH CARE
WORKERS IN INTENSIVE CARE UNITS AT AIN SHAMS UNIVERSITY
HOSPITALS By AMANY TH. ABDEL RAHMAN, SHEREEN F. HAFEZ,
SARA M. ABDELHAKAM, ZAINAB A. ALI-ELDIN, IBRAHIM M.E.
ABDEL-HAMID ESMAT, MARWA S. ELSAYED, AND AISHA ABOUL-
FOTOUH..
1

27

35

45

57

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II

7- EIMERIA KHOBAHENSIS SP. N. (APICOMPLEXA: EIMERIIDAE)
FROM GUINEA FOWL, NUMIDA MELEAGRIS IN SAUDI ARABIA By
MOHAMED S. ALYOUSIF AND YASER R. AL-SHAWA

8- HUMAN INFECTION WITH BERTIELLA STUDERI (CESTODE: AN-
OPLOCEPHALIDAE) IN AN EGYPTIAN WORKER RETURNING BACK
FROM SAUDI ARABIA By EBTESAM M. Al-MATHAL, NAGLA MOSTA-
FA KAMEL SALED AND TOSSON A. MORSY.

9- EFFECT OF TREATMENT WITH NEEM (AZADIRACHTA INDICA)
COMPARED WITH BAYCOX DRUG ON THE CAECUM OF CHICKEN
EXPERIMENTALLY INFECTED WITH EIMERIA TENELLA By FAWZIA
H. TOULAH, HAYAT A. ISMEEL AND SAMAR KHAN ......

10- RODENT BORNE DISEASES AND THEIR FLEAS IN MENOUFIA
GOV-ERNORATE, EGYPT By MOHAMED ISMAIL SOLIMAN, AZZA S.
ABD EL-HALIM AND MICHEAL W. MIKHAIL

11- MIRAZID IN TREATMENT OF 3 ZOONOTIC TREMATODES IN
BENI-SWEIF AND DAKHALIA GOVERNORATES By AHMAD M.A. MA-
SSOUD, EMAN T. El-SHERBINI, NAGLA MOSTAFA KAMEL SAEH,
MOHAMED FATHY ABOUEL-NOUR AND AYMAN T.A. MORSY

12- DETECTION OF SOME INTESTINAL PROTOZOA IN COMM-
ERCIAL FRESH JUICES By SHEREEN F. MOSSALLAM

13- EFFECTS OF RICINUS COMMUNIS, BRASSICA NIGRA AND
MINERAL OIL KEMESOL ON SOME BIOCHEMICAL ASPECTS OF
LARVAE STAGE OF SPODOPTERA LITTORALIS (BOISD) (LEPI-
DOPTERA: NOCTUIDAE) By NAJAT A. KHATTER AND FATEN F.
ABULDAHB

14- CHOLEOEIMERIA RIYADHAE SP.N. (APICOMPLEXA: EIMERII-
DAE) FROM THE LIZARD, SCINCUS SCINCUS (SAURIA: SCINCIDAE)
IN SAUDI ARABIA By MOHAMED S. ALYOUSIF AND YASER R. AL-
SHAWA

15- DISINFECTION EFFICACY OF SODIUM DICHLOROISOCYANU-
RATE (NADCC) AGAINST COMMON FOOD-BORNE INTESTINAL
PROTOZOA By LOBNA A. EL ZAWAWY,

DOAA EL-SAID, SAFIA M.
ALI AND FOUAD M. FATHY.

16- EFFECT OF PLANT MOLLUSCICIDES ON SELECTED ENZYMES
RE-LATED TO ENERGY METABOLISM IN BIOMPHALARIA ARABICA
SNAILS MOLLUSCAN HOSTS TO SCHISTOSOMA MANSONI IN
SAUDI ARABIA By SOOAD AL-DAIHAN

17- EFFECTS OF SCHISTOSOMA MANSONI EXPERIMENTAL INFE-
CTION ON SOME INORGANIC ELEMENTS IN THE SNAIL HOST BIO-

151

71

83

89

93

107

119

135

159

165

187
III

MPHALARIA ALEXANDRINA By OSAMA M. S. MOSTAFA AND SAAD
M. BIN DAJEM..

18- THE EFFECT OF A SUBLETHAL CONCENTRATION OF SOLAN-
UM NIGRUM ON SOME ANTIOXIDANTS IN BIOMPHALARIA ARABICA
By SOOAD K. AL-DAIHAN, J.S.KAGGWA AND AFAF K. EL-ANSARY..

19- DISTRIBUTION AND SEASONAL ACTIVITY OF MOSQUITOES IN
AL MADINAH AL MUNWWRAH, SAUDI ARABIA By S.M.KHEIR, A.M.
ALAHMED, M.A AL KURIJI AND SALEEM F. AL ZUBYANI..

20- EFFICACY OF PUNICA GRANATUM EXTRACT ON IN-VITRO AND
IN-VIVO CONTROL OF TRICHOMONAS VAGINALIS By GEHAD M.
EL-SHERBINI, KHAD-RA M. IBRAHIM, EMAN T. EL SHERBINY, NE-
VEIN M. ABDEL-HADY

AND TOSSON A. MORSY.

21- EVALUATION OF "MYRRH EXTRACT" AGAINST SCHISTOSOMA
MANSONI: A HISTOLOGICAL STUDY By AHMED M.A. MASSOUD,
FAIKA H. EL EBIARY, SUZI H. IBRAHIM, HANAN A.A. SALEH AND
HAZEM H.M. KHALIL .

22- THE IMPACT OF EXAMS ANXIETY ON THE LEVEL OF TRIGLYC-
ERIDES IN UNIVERSITY FEMALE STUDENTS By TAHIA A. MAIMAN-
NEE.

23-SEM STUDY OF TROPHOZOITE AND CYST STAGES OF NAEGLER-
IA FOWLERI By SANAA N. ANTONIOS

Society News.
WHO IS WHO

THE EGYPTIAN SOCIETY OF PARASITOLOGY
1 Ozoris Street, Garden City, Cairo, Egypt

IV

THE EXECUTIVE AND ADVISORY BOARD 2010
(The Society Annual Meeting 3/3/2010)

President:
Prof. Dr. Abdel Hameed Abdel Tawab Sabry,
Al-Fayoum University , Vice President for Community Service and Environmental
Development and the Representative of the Society (World Federation of
Parasitologists, Member at Large, Africa)
Vice President:
Prof. Dr. Atef Mohamed Aly El Shazly,
Head Department of Parasitology, Faculty of Medicine, Mansoura University.
Treasurer:
Assist-Prof. Dr. Nahla M. Shoukry,
Assistant Professor of Zoology, Faculty os Science, Suez Canal University, Suez.
Secretary General:
Prof. Dr. Tosson Aly Morsy,
Professor of Parasitology and formerly WHO/Consultant, Faculty of Medicine,
Ain-Shams University, Cairo 11566.
Members:
Prof. Dr. Ahmad Aly Aly Sabah,
Professor of Parasitology, Faculty of Medicine, Al-Azhar University, Nasr City.
Dr. Fouad Mohamed Sayed Haridy,
Enironmental Health Consultant and formerly General Organization for Veterinary
Services,
Prof. Dr. Magdy Abdel Latif Saleh Arafa,
Professor of Parasitology, Faculty of Medicine, Ain-Shams University, Cairo
11566.
Prof. Dr. Mohamed El-Husseiny Fayad,
Professor of Parasitology, Faculty of Medicine, Ain-Shams University, Cairo
11566.
Prof. Dr. Sanaa Nageeb Antonios Boctor,
Professor of Parasitology, Faculty of Medicine, Tanta University, Tanta.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
*For CVs please refer to http://www.parasitology.eg.net

1

Journal of the Egyptian Society of Parasitology, Vol. 40, No. 1, April 2010

J. Egypt. Soc. Parasitol., 40 (1), 2010: 1 - 26

POTENTIAL OF MEDICINAL PLANTS IN MOSQUITO CONTROL
By
SAHAR A.B. FALLATAH
1*
AND EMAD I.M. KHATER
2,3
Department of Zoology, Science College for Girls, Dammam University,
Kingdom of Saudi Arabia
1
, Dammam, 31113, P.O. Box 838, E-mail: sa-
haraf14@hotmail.com.
2
College of Food & Agricultural Sciences, King
Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia, ekha-
ter@ksu.edu.sa, and
3
Faculty of Science, Ain Shams University, Cai-
ro11655, Egypt, eikhater@yahoo.com,
*
corresponding author

Abstract

Medicinal plants have long history as important components in traditional
medicine, and food of humans since ancient Egyptians and Chinese. Naturally
occurring botanical compounds contain a broad range of chemical active ingre-
dients can intervene in all biological processes of the mosquito, thus interrupt
its life cycle and dispersal and reduce harms to humans and animals. Many me-
dicinal plants were tested for their pesticide and repellent potential, as crude
material, essential oils or individual active ingredients.
This article reviewed studies on the efficacy of many well known and com-
monly used safe medicinal plants or their products in controlling the mosqui-
toes; Aedes aegypti, Anopheles gambiae, An. stephensi and Culex quinquefascia-
tus and the ticks, Dermacentor variabilis, Amblyomma americanum, Ixodes
scapularis and I. ricinus. Promising and encouraging results were obtained
against these arthropod-vectors of zoonotic diseases.
Keywords: Medicinal plants, mosquitocides, pesticides, phytochemicals.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Mosquitoes are vectors of many
important parasitic and arboviral
diseases such as malaria, filariasis,
yellow fever, dengue fever and the
Rift Valley fever. These diseases
cause millions of deaths every year
and threaten >50% of the world
population with huge economic
losses (WHO, 2003, 2004). The in-
sect vector is a critical dynamic
element in the disease transmission
cycle, and therefore, any small ef-
fect on the vector population results
in significant impact on the overall
disease prevalence and severity
(Sinden, 2007). The control of vec-
tors is largely dependent on chemi-
cal insecticides, which have many
problems, as resistance, harmful
effects on non-target organisms and
their persistence in environment
(WHO, 1997). The risk of chemical
2

control is that they render the con-
trol programs ineffective, very cost-
ly and may fail completely with se-
rious consequences of disease re-
surgence and epidemics. Therefore,
alternative natural resources more
effective, safer and cheaper than
synthetic chemicals such as medi-
cinal plants are needed.
Many medicinal plants were used
for protection against pests and dis-
ease vectors (Karunamoorthi et al.,
2009). Bioactive compounds have
been extracted from plant parts or
from their essential oils. These
plants are renewable, safe and cheap
resources for therapeutics (anti-
cancer, anti-diabetes, anti-bacterial
and anti-parasitic drugs) and pesti-
cides. Phytochemicals are secondary
metabolites which might be specific
to plant species or genera or fami-
lies, which apparently play little role
in plant developmental processes.
They are important means of plant
interactions with other plants or or-
ganism and important in plant de-
fence mechanisms against insects or
pathogens or in plant tolerance to
stress conditions (reviewd in Balan-
drin et al., 1985). Plant secondary
metabolites are localized in specific
plant cells or tissues and in small
amounts and have complex chemi-
cal structures. These characteristics
make it difficult to extract them and
determine their functional domains
to laboratory synthesis or process
them for large-scale production and
commercialization at reasonable
prices. This is why many of these
compounds are called high value-
low volume compounds, such as
nicotine and opium-derived chemi-
cals and pyrethrins (Isman, 2008).
Due to their small molecular
weights and amounts present of
most plant secondary metabolites,
they require various methods of ex-
traction, which in turn results in
varying degrees in the number, type
and quality of extracted active in-
gredients. Extraction methods in-
clude water- and organic solvents-
based and steam-distillation (Bak-
kali et al., 2008). The availability of
large number of trees helped to
overcome the problems of large-
scale production and affordability of
high value products (Gertsch, 2009).
Commercial compounds have
been produced from medicinal
plants, such as pyrethrins from pyre-
thrum flowers, rotenoids and alka-
loids. Other pesticides as rotenoids
and nicotine-based were important
pesticides, but their use became lim-
ited or halted completely due to
their low efficacy and high toxicity
to non-target organisms as mam-
mals. Phytochemicals were used as
pesticides, repellents and essential
oils. Quinine is one of the first plant
products known in the 18
th
century
as antimalarial drug from Cinchona
trees, nicotine from Nicotiana ta-
bacum seeds and artemisinins from
the Chinese tree Artemisia annua
and azadirachtin from the neem tree
Azadirachta indica. Phytochemicals
extracted from medicinal plants
proved effective as insecticides, re-
pellents and oviposition-deterrents
against many insect-borne diseases.
3

Pyrethrins are of the most impor-
tant botanical products used in vec-
tor control based on which the syn-
thetic pyrethroids were produced.
Well-known examples are azadi-
rachtin, neem oil and artemisinins
were proved very effective against
mosquito-borne diseases (Nathan et
al., 2005, Okumu et al., 2007).
Few comprehensive reviews are
available on medicinal plants that
contain bioactive chemicals used as
pesticides or repellents against dis-
ease vectors and therapeutics and
addressing the role of biotechno-
logy, commercialization and public
acceptance (Mulla and Su, 1999;
Boeke et al., 2004; Shaalan, et al.,
2005; Katz et al., 2006; Isman,
2008; Bakkali et al., 2008; Pavela et
al., 2008; Krishna et al., 2008;
Gertsch, 2009).
Pyrethrum is the oleo-resin of
dried flowers of pyrethrum daisy
(Chrysanthemum) Tanacetum cin-
erariaefolium (Trev.) Schultz-Bip
(Glynne-Jones, 2001), is composed
of 3 esters of chrysanthemic acid
and pyrethric acid. Those esters in-
corporating the alcohol pyrethro-
lone, pyrethrin I & pyrethrin II, are
the most abundant in the extract and
account for most of the insecticidal
activities. Pyrethrum is the most
used botanical insecticide and con-
stitutes >80% of global botanical
insecticides market. Pyrethrins tar-
get the sodium-gated ion channels
in nerve axons causing knockdown
effect as DDT and organo-chlorines.
Technical grade pyrethrum has low
mammalian toxicity at 1500 mg/kg
rat. Due to their labiality to sunlight
and low short half-life, they are of
limited use outdoors. Synthetic py-
rethroids are the only choice used in
insecticide treated materials as mos-
quito nets.
Artemisia (wormwoods) is a large,
diverse genus of plants belonging to
the daisy family of hardy herbs and
shrubs, with wide geographical dis-
tribution in temperate climates and
in dry or semi-dry habitats. These
plants have volatile oils, strong
aromas and bitter tastes due to the
presence of terpenoids and the
sesquiterpene lactones. Artemisinins
include total of six products,
dihydroartemisinin, artemerther,
artesunate, artemisone, artemerther
and artelinic acid. Artemisinin is a
sesquiterpene lactone with an
endoperoxide bridge, which is
produced semi-synthetically as an
antimalarial drug. These constituents
are considered an adaptation of the
plant against herbivores, as insects.
Artemisinin (Qinghaosu) from A.
annua (L., annual wormwood or sweet
sagewort), is the active ingredient in
the anti-malarial combination
therapy 'CoArtem' commonly used
in the tropics by people who can
afford it, preferentially as a
combination cocktail with other
anti-malarial in order to prevent the
development of parasite resistance.
Laxaminarayan et al. (2006) recom-
mended the use of artemisinin-based
combination therapy (ACT) to treat
uncomplicated malaria.
Many species of family Meliaceae
are sources of botanical pesticides.
4

These include A. indica A. Juss, Me-
lia azadarcha L. and M. volkensii
Grke (reviewed in Mulla and Su,
1999). Azadirachtin is the predomi-
nant active principle with insecti-
cidal activity, found in seeds, leaves
and other parts of Meliaceae plants.
There are two derivatives; oil pro-
duced by cold-pressing of seeds and
azadirachtin obtained by polar ex-
traction of seeds after oil removal.
Many medicinal plants were tested
as mosquitocides under laboratory
and field conditions, of which pyre-
thrum and neem were the most
tested due to their wide geo-
graphical distribution and importa-
tion from indigenous areas into new
areas, where they are not naturally
growing
This paper aimed to evaluate the
potential of medicinal plants in con-
trolling mosquitoes and other arth-
ropods, with emphasis on the role of
biotechnology and ethical perspec-
tives in production and application
of botanical-based pesticides.

1. Mosquitoes tested, medicinal
plants and bioassays:

The major mosquito species tested
were; Aedes aegypti, Anopheles
gambiae, An. stephensi and Culex.
quinquefasciatus.Comparative stu-
dies included the flea, Ctenocepha-
lides felis and four ticks, Dermacen-
tor variabilis, Amblyomma ameri-
canum, Ixodes scapulari and I. rici-
nus Crude, essential oils and various
water- and organic-solvent extracts
of medicinal plants as well as prepa-
rations of purified chemicals were
used (Tabs. 1, 2). The chemical
constituents were iden-tified by high
performance liquid chromatography
(HPLC) and mass spectrometry.
The compounds were applied in
standard or modified bioassays
(WHO, 1981) against mosquito lar-
vae, pupae and adults or applied on
natural larval habitats in the field.
Mosquitoes, fleas and ticks were
given plant material in sugar or
blood meals using artificial mem-
brane feeding system (Lucantoni, et
al., 2006; Jayasuriya et al., 2004).
Plant material was applied in dif-
ferent concentrations to larvae or
pupae, exposed for 24 or 48 hr or
longer, and then mortality percen-
tages were calculated and tabulated.
A logprobit regression line was es-
tablished from which median lethal
concentrations or doses that kill 50
& 90% of the tested population
(LC
50
/LC
90
or LD
50
/LD
50
) were also
deduced. Other values could also be
deduced from the line and used to
interpret the data and determine the
susceptibility level and homogenei-
ty of the tested insect population.
Some repellent formulations pre-
pared in laboratory or from com-
mercial sources were tested using
human volunteers (arm-in-cage,
moving-object, head-to-head assays
or in the field). Materials were ap-
plied directly on human skin or on
clothes. Choice tests were used for
ticks exposed to filter papers im-
pregnated with the plant material
and the time spent on each material
was calculated (Fradin and Day,
5

2002, Witting-Bissinger et al.,
2008, 2009). In these assays, DEET
(7, 15, 30, 50 & 98.11 %) was used
as a reference repellent.

2. The factors determining mos-
quito susceptibility to botanical
pesticides:
All the mosquitoes tested were
highly susceptible to plant crude
preparations, water- or organic sol-
vent-extracts or purified active in-
gredients. However, there were con-
siderable variations due to: 1. mos-
quito species, stage, age, physio-
logical condition and geographic
origin; 2. type and concentration of
plant formulation, and 3. experi-
mental conditions. All these make it
difficult to compare the efficacy of
one compound against related mos-
quito species in different countries.
The only possible way is to compare
the efficacy of one plant material on
different species or stages of one
species under the same conditions
or the efficacy of many plants on
the same mosquito species or stage
under the same conditions. From the
results obtained the following points
can be made: 1. Young larval (I-II)
instars were more susceptible than
old (III-IV) instars; 2. Non-fed lar-
vae were more susceptible than fed
larvae; and 3. Exposure period in-
creased mosquito susceptibility.
These results were reported Commi-
phora molmol (myrrh) oil and oleo-
resin extract proved valuable against
Cx. pipiens and Ae. caspius (Mas-
soud and Labib, 2000). Myrrh con-
tains volatile oils (myrrhol) resin
(myrrhins), gum and other macro-
molecules, which are responsible
for its broad bioactivities; antibac-
terial, anti-helminthic, cytotoxic,
anti-carcinogenic and anti-mosquito
effects (Massoud et al., 1998;
Hamed and Hetta, 2005). Quercus
lusitania and AkseBio2 against Cx.
pipiens (Cetin et al., 2004; Redwane
et al., 2002), A. indica (neem) de-
rivative neemarin against An. ste-
phensi and Cx. quinquefasciatus
(Vatandoost and Vaziri, 2004);
neem limnoids against An. stephensi
larvae, pupae and adults; larval-
pupal periods were increased with
decreased adult longevity and fe-
cundity (Nathan et al., 2005); neem
oil containing 0.03% azadirachtin,
crude neem powder or neem water-
extract against An. gambiae larvae,
with reduction of adult emergence
(Okumu et al., 2007, Gianotti et al.,
2008, Howard, et al., 2009); neem
oil commercial emulsified concen-
trate against, Ae. aegypti, An. ste-
phensi and Cx. quinquefasciatus
(Dua et al., 2009) and Calotropis
procera (milkweed or ushaar) crude
latex or purified laticifer proteins
against An. labranchiae mosquito
larvae (Markouk et al., 2000) or
larvae of Ae. aegypti, An. stephensi,
Cx. quinquefasciatus (Singh et al.,
2005) or different heat-treated latex
or chemical dialysis fractions on Ae.
aegypti, with effect in preventing
egg hatching and arrest of larval
growth at the instar I-II and death
(Ramos et al., 2006).
The susceptibility of mosquitoes
to plant-products varied due to
6

chemical active ingredients and me-
thod of extractions. Azadirachta and
Melia species of Meliaceae contain
triterpenoids (Siddiqui, et al., 2002)
and limnoids as azadirachtin, salan-
nin, gedunins, with broad larvicidal,
pupicidal, adulticidal and anti-
ovipositional activities against An.
stephensi (Nathan et al., 2005;
Howard, et al., 2009). Freitas et al.
(2007) found that cysteine protei-
nase and bacteriolytic enzymes from
laticifer proteins C. procera had
lethal effect against insects includ-
ing mosquitoes.). Pomegranate, Pu-
nica granatum contained various
ingredients that vary with the plant
part, cultivar, processing and sto-
rage conditions (Lansky and New-
man, 2007; Syed et al., 2007). It is
rich in complex phenolic ingredients
such as flavenoids, hydrolyzable
tannins, which account for the most
of its anti-oxidant activities. The
flavenoids prevented lipid peroxida-
tion of the cell or liposome mem-
branes and chelate metal ions as
iron (Halvorsen et al., 2002; Afaq et
al., 2005). Potential anti-oxidants in
pomegranate interfered with cellular
and developmental functions of Cx.
quinquefasciatus larvae (Fallatah,
2009a).
These results encouraged the con-
duct of few field studies that re-
ported high anti-mosquito efficacy
of neem products. In Iran, neemarin
(2 L/ hectare) caused maximum ef-
ficacy at 7 days post-treatment, with
pupation and adult emergence are
the most affected processes (Vatan-
doost and Vaziri, 2004). In Niger,
Gianotti et al. (2008) found that lo-
cal crude neem seed powder re-
duced An. gambiae adults emerged
by 50% compared to a nearby un-
treated village and compared to pre-
vious studies. They sprinkled the
dried powder twice weekly on larval
habitats, which represent a simple
and cheap method for mosquito
control that can be conducted by
local people using available plant
sources.
3. Medicinal plant essential oils
active ingredients and efficacy
3.1. Active ingredients:
Essentials oils are produced by
steam distillation of various plant
parts. Chromatography and mass
spectrometry and chemical frac-
tionation analysis revealed the pres-
ence of various active ingredients,
which are usually mixtures of
monoterpenes, phenols and ses-
quiterpenes and other classes. Most
of these ingredients have strong in-
secticidal and repellent action
against mosquitoes and many vec-
tors. Important botanical essential
oils active ingredients (table 2) in-
cluded piperitenone oxide (PO)
(Methna spicata L. variety viridis)
(Tripathi et al., 2004); p-menthane
aldehyde eucamalol (Santolina insu-
laris) (Fattorusso et al., 2004); pre-
cocenes I a& II, (Cymbopogon win-
terianus), which possess anti-
juvenile hormone activity and could
seriously interfere with insect me-
tamorphosis via down-regulation of
juvenile hormones-controlled gene
expression (De Mendonca et al.,
2005); 1,8-cineole (Rosemarinus
7

officinale & Eucalyptus globus),
eugenol (Syzygium aromaticum),
thymol (Thymbra vulgaris) and
menthol (Mentha spp)(Bakkali et
al., 2008); 2-undecanone (wild to-
mato Lycopersicon hirsutum gla-
bratum, which provides plant resis-
tance to insect herbivores (Witting-
Bissinger et al., 2008, 2009); limo-
nene, sabinene and beta-myrcene
(Japanese pepper Zanthoxylum pi-
peritum)(Choochote et al., 2007);
thymol and sabinene (parsley Petro-
selinum crispum), carvacrol (Th.
vulgaris), anethole (Pimpinella ani-
sum) and linalool (Coriander sati-
vum)(Knio et al., 2008); carvone
(Carum carvi), limonene (Apium
graveoles and Za. limonella), ane-
thole (Foeniculum vulgare), 1,8-
cineole, p-cymene & -phellandrene
(Curcuma zedoaria)(Pitasawat et
al., 2007; Bakkali et al., 2008);
thymoquinone, thymol, carvacrol,
carvone, p-cymeme (Nigella sativa),
juvacemene (O. basilicum); ju-
vabiones (Abies balsamea), chro-
menes and farnesol (Ageratum hu-
ostonianum) (Baland-rin et al.,
1985; Bakkali et al., 2008).
Essential oils have rapid chemical
insecticide-like mode of action on
insect nervous system and sensory
receptors, and therefore they can be
used as fumigants and contact insec-
ticides. The disadvantages of essen-
tial oils are their possible toxicity to
non-target organisms and low per-
sistence under field conditions (re-
viewed in Balandrin et al., 1985;
Isman, 2006, 2008). Botanical es-
sential oils are therefore represent-
ing potential alternatives to syn-
thetic chemical repellents. These
include essential oils of eucalyptus,
cedar wood, citronella, quelling
from lemon grass eucalyptus and
many others. These oils or their ac-
tive ingredients had variable degrees
of efficacy against mosquito vectors
and other arthropod-vectors.

3.2. Pesticidal action:
Piperitenone oxide (PO) has mul-
tiple effects as larvicidal, ovicidal,
oviposition-deterrent, developmen-
tal toxicity and repellent properties
against various stages of An. ste-
phensi (Tripathi et al., 2004). The
lethal action of PO was higher than
crude oil against the 4
th
instar larvae
(LD
50
61.64 & 82.95g/ml, respec-
tively). Exposure to PO completely
inhibited oviposition and egg hatch-
ing. Essential oil of the Indian
Railway Creeper Ipomoea cairica
caused complete morality of Ae.
aegypti, An. stephensi, Cx. traitae-
niorhynchus and Cx. quinquefascia-
tus at 120, 120, 100 & 170 ppm,
respectively after 24 hr-exposure. I.
cairica is an abundant indigenous
ornamental plant with broad medi-
cinal properties and has been used
in fencing domestic and peri-
domestic areas (Thomas et al.,
2004). The essential oils of Anacar-
dium occidentale, Copaifera
langsdorffii, Carapa guianensis, C.
winterianus and Ageratum con-
yzoides showed high insecticidal
activities against Ae. aegypti larvae
with LC
50
values of 14.5, 41, 57, 98
& 148 g/l, respectively. Treated
8

larvae and pupae were implicated in
completing development, with ab-
normal pigmentation and reduction
in adults wing size and fecundity
(De Mendonca et al., 2005). Ca-
shew nut oil will be a cheap agent in
mosquito control as it is a by-
product residue of processing indus-
trial cashew nuts and has a very low
commercial value. Together with
larvicidal activity and effects on
insect metamorphosis, plant essen-
tial oils seriously implicated the re-
productive efficiency and popula-
tion structure in mosquitoes. The
essential oils of Conyza newii and
Plectranthus marruboides had high
fumigant toxicity against An. gam-
biae s.s.; oil of C. newii, perillalde-
hyde and perillyl alcohol exhibited
fumigant toxicity higher than the
parent oil (Omolo et al., 2005). Oils
of the Indian juniper Juniperus ma-
cropoda fruits and anise P. anisum
seeds were highly effective as both
larvicidal and ovicidal against An.
stephensi, Ae. aegypti and Cx. quin-
quefasciatus (LD
95
s: 115.7-
149.7g/ml) (Prajapati et al., 2005).
Anees et al. (2008) reported high
larvicidal activity of Ocimum sanc-
tum oil against Ae. aegypti and Cx..
quinquefasciatus. In contrast, low to
moderate efficacies of O. basilicum
were reported against An. stephensi,
Ae. aegypti and Cx. quinquefascia-
tus (Prajapati et al., 2005; Pavela,
2009).
Carum carvi, A. graveolens, F.
vulgare, Za. limonella and C. ze-
doaria oils had significant larvicidal
activity against An. dirus and Ae.
aegypti after 24-hr exposure (Pita-
sawat et al., 2007). Oil of Za. limo-
nella was effective against Ae. ae-
gypti (LC
50
24.61 & LC
95
55.81
ppm), while An. dirus was highly
susceptible to C. zedoaria (zedoary)
oil (LC
50
29.69 & LC
95
40.23 ppm).
The larvae suffered behavioral and
pathological symptoms: restless-
ness, sluggishness, tremors and
convulsions and general paralysis
and subsequently died within 24 hr.
High larvicidal activity of Th. vul-
garis oils the larvae of Lucilia seri-
cata (Morsy et al., 1998b), Th. satu-
reoides and Satureja hortensis was
reported against Cx. quinquefascia-
tus (LC
50s
32.9, 43.6 & 36.1 g/ml,
respectively) (Pavela, 2009). Low
efficacy of N. sativa seed oil was
reported against An. stephensi, Ae.
aegypti and Cx. quin-quefasciatus
(Prajapati et al., 2005). In contrast,
high larvicidal efficacy of N. sativa
seed water-extract was obtained
against Cx. quinquefasciatus larvae
from Saudi Arabia (Fallatah, 2009a,
in press).

3.3. Insect repellents:
There are many formulations of
insect repellents (IRs) including
sprays, lotions, oils, powders for
skin and clothing (Isman, 2006,
2008; Katz et al., 2008). Oils active
ingredients as 2-phenethylepropion-
ate from peanut oil and p-menthane-
3,8-diol from mint are safe to use on
humans, but the protection time is
very short, usually less than 1hr.
Citronella or its active ingredient
citronellal is used in mosquito coils
9

to repel mosquitoes from outdoor
areas. The efficacy of IRs could
significantly be increased with hu-
man behaviour, i.e. avoid being out-
doors at the peak of mosquito biting
activity, wearing protective clothing
or when used with a contact insecti-
cide as permethrin on clothes
(Fradin and Day, 2002). Identifica-
tion of chemoreceptors and olfac-
tory-binding protein genes in se-
quenced genomes of important
mosquitoes (Holt et al., 2002; Nene
et al., 2007) will help the production
of potent and more specific repel-
lents that can be sprayed in space
thus alleviating the need for direct
and repeated application on human
skin (Katz et al., 2008).
The commercial chemical product
DEET (N,N diethyl-m-toluamide or
N,N-diethyl-3-methylbenzamide)
has been one of the most effective
and commonly used repellents
against biting insects and mosqui-
toes in terms of protection level and
persistence on human skin. Most
efficacy studies on botanical-based
repellents are conducted in compari-
son to the efficacy of DEET and
other chemical repellents in the
market. A soybean-oil-based repel-
lent protected human volunteers
against Ae. aegypti for ~1.6 hr com-
pared to ~5 hr by 23.8% DEET
(Fradin and Day, 2002). Barnard
and Xue (2004) compared repellent
efficacy of Bite Blocker (2% soy-
bean oil), ByGone, GonE!, Natrapel
(10% citronella), Neem Aura,
Sunswat, MosquitoSafe (25% gera-
niol) and Repel (oil of lemon euca-
lyptus)(26% p-menthane-3,8-diol)
to the synthetic repellents Off! (15%
DEET) & Skinsations (7% DEET).
The highest mean protection time
(eMPT) against Ae. albopictus, Cx.
nigripalpus and Ochlerotatus trise-
ria in the laboratory were by Autan,
Bite Blocker, Off! and Repel; they
prevented biting for 7.2 hr; com-
pared to protection time of 0.9-4.8
hr for the other compounds. Oil of
Za. piperitum (Japanese pepper tree)
exerted the highest protection
against Ae. aegypti, with median
complete protection time of 1 hr.
The protection time was increased
significantly by incorporating 10%
vanillin and reached 2.5 hr (Choo-
chote et al., 2007). Neem oil had
highly repellent effect against labo-
ratory and field populations of An.
stephensi (with ED
50
s 0.156 &
0.191 mg/cm
2
, respectively). How-
ever, efficacy was 22- & 27-fold
lower than DEET and permethrin,
(Vatandoost and Hanafi-Bojd,
2008). Different formulations con-
taining 10% dodecanoic acid (DDA)
strongly repelled nymphs of I. rici-
nus tick with 81-100% protection.
The commercial formulation Con-
traZeck
(10% DDA) was the high-

est effective and was as effective as
the coconut oil-based reference
product; eMPT of both preparations
was 8 hr. ContraZeck applied by
(~1.67mg/cm
2
) on human volun-
teers strongly prevented the attach-
ment of I. ricinus nymphs and
adults for at least 6 hr (Schwantes et
al., 2008). BioUD is a repellent
based on 7.75% 2-undecanone an
10

active ingredient isolated from the
stem and leaves of the wild tomato
L. hirsutum, which confers plants
resistance to insect herbivores.
BioUD
was approved by the US

Environmental Protection Agency
for use against mosquitoes and
ticks. Witting-Bissinger et al.
(2008) obtained a mean repellency
of BioUD comparable to 15%
DEET against Ae. albopictus over a
6-hr period in the laboratory. While
for Ae. aegypti, repellency by Bio-
UD was comparable to 7% DEET
over the same period. In human sub-
ject field trials (repellents applied at
1 ml/600 cm
2
), BioUD spray pro-
vided the same repellency or higher
than the two products, OFF! Active
IV
(25% DEET) and Deep Woods

OFF!
(30% DEET). Laboratory

trials showed that BioUD repelled
the American dog tick D. variabilis
from human skin (BioUD lotion)
and cloth (BioUD spray) at least for
2.5 hr after skin application. On
cloth, the mean repellency of Bio-
UD was comparable to 7% DEET
through 8-day period after applica-
tion. In a two-choice test, ticks spent
significantly more time on the 15%
DEET-treated filter paper than on
the BioUD-treated paper. BioUD
provided repellency similar to or
higher than DEET (98.1%) against
the ticks, D. variabilis, A. ameri-
canum and I. scapularis (Witting-
Bissinger et al., 2009). In head-to-
head assays, undiluted and 50% di-
lutions of BioUD were more repel-
lent than undiluted DEET against all
three tick species tested. The BioUD
concentration of 29.7%-39.5% is
equivalent in repellency to 98.11%
DEET against the three species of
ixodid ticks under laboratory condi-
tions. These results show that IRs
have strong repellent effect against
mosquitoes comparable or better
than DEET and other synthetic re-
pellents in the market. DDA-based
ContraZeck
, coconut oil-based and

BioUD formulations provided im-
portant data to test their efficacy
against mosquitoes.

4. Biological Effects
4.1. Histopathological effects:
Medicinal plants active com-
pounds have lipophilic nature,
which facilitate the differential
binding to cellular membranes and
membrane-bound structures and
proteins. Few studied the effect of
extracts, essential oils or active in-
gredients on insect biology to de-
termine histopathological & physio-
logical effects and molecular targets
of plant extracts or their products.
Studies using different mosquito
species and stages treated with ex-
tracts or active ingredients of medi-
cinal plants showed that the midgut
tissue is the most affected site. His-
topathological symptoms include
hypertrophy and lysis of the gut epi-
thelium, the brush border and mi-
crovilli, vacillations and protrusion
of cell contents in the gut lumen
with complete degeneration and
death of cells. The other tissues af-
fected include gastric caeca, Mal-
pighian tubules, muscles, nerve
ganglia and fat tissue. These studies
11

were done on An. gambiae larvae
treated with water extracts of Per-
sea americana (Koua et al.,

1998),
Cx. pipiens larvae treated with tan-
nic acid (a component of pomegra-
nate and many medicinal plants)
(Ray et al., 2001) or with myrrh oil
and oleo-resin (Massoud and Labib,
2000) or Cx. quinquefasciatus lar-
vae treated with myrrh oil and
powder (Ndione et al., 2006) or wa-
ter extracts of myrrh, pomegranate
and black seed (Fallatah, 2009b).
The symptoms are similar to those
reported in Cx. sitiens and the black
fly Simulium pertinax larvae treated
with the bacterial biopestcicide Ba-
cillus thuringiensis toxins (Weiser
and Zizka, 1994; Cavados et al.,
2004). Females of An. stephensi
given NeemAzal (NA) (neem-
commercial formulation with 34%
azadirachtin A) in artificial blood or
sucrose implicated in blood intake,
oviposition and oocyte development
(Lucantoni, et al., 2006). Ultra-
structural analysis of ovarian tissues
showed that females suffered severe
damage; complete blockage of
oogenesis and degradation of fol-
licular cells. These damaging effects
are due to potential interference
with hormonal control of egg devel-
opment and oviposition, which
could seriously implicate mosquito
fecundity and population growth.
Water preparation of dillapiol of the
long pepper, Piper aduncum, signif-
icantly reduced egg production in
Ae. aegypti and interfered with lar-
val survival. It also caused chromo-
somal aberrations and micronuclei
formation, symptoms that are simi-
lar to those caused by synthetic in-
secticides (Rafael et al., 2008). The
apparent different modes of action
might be due to different cell mem-
branes or proteins are targeted by
plant active components. In general
disruption of midgut epithelium and
cellular integrity will certainly af-
fect its vital functions in absorption
and ion active transport, which will
implicate insect developmental
processes and survival. Plant ex-
tracts might also have effects on ion
channels controlling ion transport
across membranes and mechano-
sensing proteins that provide resis-
tance to mechanical stress on the
midgut during feeding. This could
have significant effect on adult
mosquito midgut stretching during
blood feeding. Affecting the mos-
quito ability to ingest blood or ma-
nipulate it after ingestion could
block the development of the blood-
borne pathogens and successful
trans-mission to human or animal
hosts. Targeting the mosquito mid-
gut could also affect gut endosym-
bionts as Wolbachia proteobacteria,
which play critical roles in mosquito
longevity and development (Cook et
al., 2008). The effect on the nervous
system could be very important in
possible interference with nerve
impulse transmission to the brain
and thus implicates the mosquito
response to internal and external
stimuli. For example, it disturbs
adult mosquito ability to find the
right host for feeding or the right
oviposition site. The damaging ef-
12

fect of these extracts on muscles
could affect larval movement in wa-
ter for respiration or feeding or af-
fect adult mosquito ability to fly. In
all cases, there will be an overall
detrimental effect on mosquito de-
velopment and dispersal, which
represent essential elements for dis-
ease transmission process. But for
these studies to be complete, they
must test the effects of medicinal
plant products on infected mos-
quitoes as with malaria parasite and
how treatment affects parasite de-
velopment and mosquito vectorial
competence for disease trans-
mission.

4.2. Modes of action and molecular
targets:
The medicinal plant active ingre-
dients have major targets of action
that include, enzymes, hormones,
protein kinases and other targets yet
to be identified that play important
roles in cellular functions. Azadi-
rachtin and neem products exhibit
various modes of action against in-
sects such as anti-feedant, insect
growth regulator (IGR), fecundity
suppression and sterilization, ovipo-
sition repellency or attraction, af-
fecting biological fitness and block-
ing development of vector-borne
pathogens. These bioactivities of
neem products have been investi-
gated on many important disease
vectors including mosquitoes, flies
and triatomine bugs. Azadirachtin
mode of action as an IGR is block-
ing synthesis and release of moult-
ing hormones (ecdyosteroids) from
the prothoracic gland. This causes
incomplete ecdysis of immature
stages and sterility in adults. The
second effect is anti-feedant, which
was originally recognized as a
strong feeding-deterrent against de-
sert locusts (Bernays, 1990). Melia
trees contain salannins and volken-
sin isolated from the seeds of M.
volkensii from East Africa. Salannin
and volkensin function as anti-
feedants and stomach poisons
against chewing insects and mos-
quito larvae. Seeds of other Melia
species are rich in triterpenoids
(melicarpins) but also contain toxic
substances as meliatoxins from the
Chinaberry tree M. azadarcha in
Asia. Interestingly, the seeds of M.
azadarcha from Argentina, do not
contain meliatoxins, but contain
meliaretenins instead, which act as
strong anti-feedants against chewing
insects (Isman, 2006).
Proteins of some medicinal plants
or essential oils have strong anti-
oxidant activities and can attack cell
membranes and organelles as mito-
chondria. Extracts of myrrh, pome-
granate and milkweed (C. procera)
have potent anti-oxidant and free
radical scavenger bioactivity (Yo-
shimora et al., 2005; Ramos et al.,
2006; Rout and Banejee, 2007; Frei-
tas et al., 2007). Crude latex of C.
procera contains proteins with ap-
parent molecular mass ranging from
5 to 95 KD (Ramos et al., 2006;
Freitas et al., 2007). The identified
proteins include superoxide dismu-
tase (SOD), ascorbate peroxidase
(APO) and chitinases. The anti-
13

oxidative enzymes SOD and APO
are involved in regulation of pro-
duction of superoxides such as reac-
tive oxygen species (ROS) and hy-
drogen peroxide (H
2
O
2
) in response
to stress or pathogen infection. Su-
peroxides limited pathogen growth
and induction of pathogenesis-
related proteins as phytoalexins and
anti-oxidant enzymes. The latex
also has strong proteolytic activities
due to cysteine proteinases. The
broad enzymatic activities of C.
procera latex proteins had potential
roles in plant defense mechanisms
against phytopathogens and insects
and resistance to stress (Freitas et
al., 2007). Treatment of Cx. quin-
quefasciatus larvae with water ex-
tracts of myrrh, pomegranate and
black seed clearly changed larval
protein profiles in different patterns
compared to normal larvae (Falla-
tah, 2009b). Provision of Eurygas-
ter integriceps adults (a wheat pest)
with food and water containing A.
annua extract significantly de-
creased the digestive enzymes activ-
ity (Zibaee and Bandani, 2009), and
decreased the insect cellular imunity
(hemocyte numbers, phagocytosis,
nodule formation and phenoloxidase
activity) to entomopathogenic fun-
gus Beauveria bassiana.
There are two important issues
regarding the possible application of
medicinal plant-based pesticides in
mosquito control. Medicinal plant
chitinases could interfere with insect
chitin in the integument or the gut
lining and PTM, which could on
one hand seriously impair insect
development, moulting and blood
digestion. On the other hand, chiti-
nases enhanced the pathogen infec-
tivity as malaria to mosquito. Para-
site uses mosquito trypsins to acti-
vate his own chitinases to invade
mosquito midgut and PTM tissues
and ensure successful transmission
to host (Huber et al., 1991, Vinetz et
al., 2000; Dessens et al., 2001). The
anti-oxidant enzymes and free radi-
cal scavenging bioactivity of medi-
cinal plants could affect nitric oxide
(NO) synthesis, and have important
immune defense mechanism against
infection (Luckhart et al., 1998).
The down-regulation or blocking of
such mechanisms will increase pa-
rasite infectivity to the mosquito
and enhance subsequent transmis-
sion to the host. It is therefore, very
important to test the effect of medi-
cinal plants on parasite-mosquito
interactions and the transmission
process. This especially very useful
if botanical extracts are to be used
against adult mosquitoes. To apply
these to mosquito vectors, blocking
proteases could seriously implicate
blood-meal digestion and pathogen
infectivity to the mosquito and
transmission to the host. Despite the
down-regulation of immune res-
ponses could increase mosquito
susceptibility to pathogenic fungi
(important tools of biocontrol), it
could in the same time increase in-
fectivity of pathogens as malaria
parasite to the mosquito.
Functional genomics studies have
shown that many immune peptides,
neuropeptides and enzymes such as
14

protein kinases are highly conserved
at structural and functional levels
between mosquitoes and other or-
ganisms such as mice and humans
(Holt et al., 2002; Hafen, 2004).
Therefore, it is highly likely that
medicinal plants target these pro-
teins in the mosquito and interfere
with its developmental, lifespan and
anti-pathogen immune reactions.
The results of these studies could
lead to the development of novel
control strategies against mosqui-
toes and the diseases they transmit.

4.3. Resistance development and
field applications:
There are no reports on the possi-
bility of resistance develop-ment in
insect pests to botanical-based pes-
ticides. It is interestingly, that there
are a number of lepidopteran larvae
are naturally-feeding on Artemisia
plants, which indicates the presence
of mechanisms of tolerance or resis-
tance of these larvae to Artemisia
despite the fact that it is lethal to
other insects. Identifying such genes
responsible for larval resistance to
Artemisia could help the avoidance
of future resistance development in
insects targeted for control by Arte-
misia- or other botanical-derived
pesticides.
Despite the use of artemisinins as
antimalarial, their effectiveness was
hampered by low production, high
costs and the widespread of malaria
parasite resistance to the drug. The
main reason for resistance is the use
of artemisinin-based combination
therapies (ACTs) as a monotherapy
of malaria and the uncontrolled use
and sale of substandard ACTs con-
taining inadequate levels of the ac-
tive ingredient of the drug or fake
drugs. The development of parasite
resistance to the artemisinins is con-
sidered one of the most serious
problems in malaria control (New-
ton et al., 2001; Jamou et al., 2005,
Noedl, 2005; Noedl et al., 2008).

5. Disadvantages of botanical-
based pesticides and repellents

The majority of these botanical
compounds were used against agri-
cultural pests, and must be tried
against vectors. But, there are some
drawbacks that might shake con-
sumer confidence in botanical pesti-
cides and hamper wider use. These
include their perceived slow action
and the short persistence, sustain-
ability of the source plant, possible
resistance (desensitization) of in-
sects, expensive production registra-
tion and intellectual property protec-
tion or rights (IPP or IPRs) and
ethical considerations.

5.1. Safety to non-target organisms
and the environment:
An important issue in studying the
efficacy of botanical pesticides is
the determination of sub-lethal
doses effects on the beneficial ar-
thropods (Desneux et al., 2007)
which represent the large majority
of organisms co-inhabiting mos-
quito larval habitats (or agricultural
pests). Desneux et al. (2007) de-
fined a sub-lethal dose or concentra-
15

tion as that inducing no apparent
mortality in the experimental popu-
lation, and the sub-lethal effects as
effects (physiological or behav-
ioural) on individuals that survive
exposure to a pesticide (under ex-
perimental conditions).
These studies should focus on the
effects of both the sub-lethal and
median lethal doses on behaviour
and physiology of the target arthro-
pod. Such studies include the effects
on learning behaviour and neuro-
physiology of natural enemies and
pollinators. Many effects could ap-
pear in later periods after the actual
bioassay, and therefore, some re-
sults on safety to non-targets could
be unintentionally misleading. The
studies should be considered for
IPM programmes and as require-
ments on judging on the efficacy
and registration of botanicals and
subsequent commercialization.
Myrrh extracts proved effective
against a water beetle and a bug co-
inhabiting mosquito larval breeding
sites (Massoud and Labib, 2000).
Boeke et al. (2004) found that wa-
ter-extracts and crude oils are the
least toxic compared to organic-
solvent extracts on humans and
laboratory mice. The estimated safe
doses were 0.26-3 & 0.002-12.5mg/
Kg/day, respectively. The purified
azadirachtin has low toxicity of 12.5
mg/Kg/day in mice. The major con-
cern was the effect of exposure to
sub-acute or chronic doses on male-
female reproductive potential. The
authors concluded that there is no
specific trend for the toxic effects of
each type of the assessed neem
products. The effects will depend on
the plant species and origin, prepa-
ration and extraction processes,
post-production handling and stor-
age, the type and the concentration
of active ingredients and the level of
exposure or intake by humans or
animals. Quassinoids of P. sprucei
had neither toxicity to the brine
shrimp Artemia franciscana nor
cytotoxicity to mouse erythrocytes,
in spite of their high efficacy against
Ae. aegypti larvae and malaria para-
site (Silva et al., 2009). Botanical
extracts or crude oils should be safer
to non-targets in the control of
aquatic mosquito larvae or outdoor
adults, where minimal or no contact
with humans or animals.

5.2. Bioavailability and the role of
biotechnology and genomics:
Many medicinal plants are only
available for short periods per year
or possibly disappear as for wild
and rare plants. Tissue culture and
genetic engineering techniques
could help to overcome the problem
of plant source availability. Plant
secondary metabolites accumulate
in specific cells at specific devel-
opmental stages and physiological
conditions. Tissue culture and bio-
transformation (phytofarming) tech-
nologies helped to culture specific
plant tissues or genotypes or engi-
neering plants to produce chemicals
from their precursors to increase the
yield of specific compounds such as
azadirachtin. The flowers of pyre-
thrum were cloned by tissue culture
16

and millions of trees were cultivated
and yielded large amounts of pyre-
thrins. Identification of important
genes and mechanisms regulating
biosynthetic pathway of plant active
ingredients will significantly help
develop strategies to increase the
yield of the active ingredient. Aquil
et al. (2009) had successfully trans-
formed A. annua by gene HMGR
(3-hydroxy-3-methylglutaryl CoA
reductase) from the plant Catharan-
thus roseus. The resultant transgenic
plants contained significantly higher
amounts of artemisinin compared to
wild-type plants. Liu et al. (2009)
identified a number of highly ex-
pressed genes and transcriptional
factors that are involved in the bio-
synthetic pathway of artemisinin in
A. annua and were correlated with
density and production of artemisi-
nin. Davies et al. (2009) adopted a
nutrition-manipulation approach to
increase the yield of A. annua natu-
ral plants; maintaining nitrogen in-
take at certain levels such as 160
mg/L in irrigation water signifi-
cantly increased the plant biomass
and artemisinin amounts. It is also
possible that the plants can be engi-
neered to produce their own chemi-
cals or protectants directly, thus al-
leviating the need of purification or
periodical application. The ability to
culture or breed specific plants in
the laboratory is also very important
to preserve plants and make them
available for research and analysis
all over the year even outside the
plant native environment.
The establishing good culture sys-
tem is therefore requires detailed
knowledge of plant taxonomy, biol-
ogy, genomics, and cellular local-
ization of important secondary me-
tabolites. So, biotechnology offers a
great promise to increase the yield
of important natural compounds and
thus will reduce both the time and
costs of extraction of products. The
availability of large amounts of spe-
cific products and reduced costs of
production and registration will sig-
nificantly reduce the costs to con-
sumers and increase the market.

5.3. Standardization of efficacy bio-
assays and registration:
There are considerable variations in
chemical products even from the
same plant prepared by similar
methods. These variations are due to
the geographical distribution of the
plant and season of harvest and
many experimental reasons. There-
fore, for better assessment of effi-
cacy and side-effects of phyto-
chemical pesticides and repellents,
data should be documented and
methods should be standardized.
Trials on mosquito susceptibility
to botanicals have increased in vari-
ous countries and mainly on labora-
tory-reared mosquitoes. The meth-
ods used, test conditions and re-
ported median lethal values vary
considerably between different
laboratories. Thus, comparing the
results is difficult and could be mis-
leading in evaluating the efficacy of
botanicals on the target insect at
different geographical areas. To
17

achieve an acceptable level of com-
parison between efficacies of simi-
lar botanicals from different geo-
graphic regions is the standardiza-
tion of the active ingredients iso-
lated rather than crude preparations.
This requires standardization of ex-
traction and analytical methods,
equipment, storage facilities and
physicochemical properties of ac-
tive ingredients isolated. A strategy
is needed to evaluate bioassays and
clinical efficacy of botanicals
(Gertsch, 2009).
Despite large number of medici-
nal plants and their derivatives have
been tested against mosquitoes, few
compounds were further developed
for commercial uses. This means
that mis- or over-interpretation of
results have been made. False
claims of efficacy would lead to
serious health risks to people and
economic loses by producers
(Gertsch, 2009).
Many European countries and the
USA facilitate the process of regis-
tration of new botanical pesticides
or repellents, or even thinking of
complete exemption of such prod-
ucts from official approval. Com-
plete waiving of registration could
signifinclaty increase the market
size of botanicals and reduce the
cost to consumers. However, one
disadvantage is that low-quality and
unsafe products could pass to the
market without approval of the con-
cerned authorities. This is particu-
larly important for poor countries
with high levels of illiteracy and
may be unable to understand prod-
uct labels or safety remarks (Isman,
2006, 2008).

5.4. Ethical issues for the production
and application of botanicals in pest
control:
With increased interest in medici-
nal plants for research and commer-
cial drug discoveries, many ethical
concerns are raised. Such concerns
include people`s safety, preserving
plant biodiversity and the environ-
ment and IPRs of ethnic groups who
own the plant material or on whom
efficacy studies are carried out. The
Convention on Biological Diversity
(CBD) and the Bonn guidelines
have increased the awareness of the
importance to preserve the biodiver-
sity and the knowledge or IPRs re-
lated to medicinal plants (Gertsch,
2009). A very important issue is that
many medicinal plants are com-
monly used in everyday life of peo-
ple and thus, there must be a moni-
toring mechanism that increase the
production of a given product and
keep the market price unaffected.
Legal framework is also needed to
prevent the risk of holding patent
licences by companies to control the
use or commercialization of certain
plant products or knowledge or es-
tablish seed banks. The use of ge-
netically-modified crops and its im-
pact on wild life should be thor-
oughly investigated. These issues
could protect consumers and in-
digenous people rights to benefit
from their traditional knowledge,
products and benefiting from re-
search outcomes.
18

Conclusion

Medicinal plants are new sources
for control of mosquitoes and other
vectors, with aspects that must be
considered: 1. the optimal dose and
form of application, i.e. the effect of
the whole plant versus single consti-
tuents, 2. the molecular targets and
modes of action in the target mos-
quito stage and period of applica-
tion, the biological processes and
tissue specificity, 3. toxicity studies,
especially to non-targets organisms,
4. the effect on disease pathogens
development and infectivity to the
mosquito vector, this is particularly
important in the case of adulticides,
5. the possibility of resistance de-
velopment and the underlying me-
chanism, 6. the effects on mosquito-
associated microflora and fauna,
especially those in the midgut. In
addition, the efficacy of plant ex-
tracts should also be determined
empirically on various species of
mosquitoes, especially if more than
one vector species to be targeted for
control. In the less developed or
poor countries, in Africa, Asia and
Latin America, botanicals are avail-
able in large quantities at low cost
and therefore, they represent cheap
and low-tech methods for pest con-
trol, with minimal health and envi-
ronmental risks compared to chemi-
cal pesticides.
The botanical pesticides are valu-
able due to their perceived low
mammalian toxicity and environ-
mental hazards, particularly for the
control of public health pests. Me-
dicinal plants are important source
for novel pesticidal compounds and
must be fully exploited through har-
nessing the power of modern bio-
technology, genomics, proteomics
and chemistry. The development
and application of pesticides from
medicinal plants should be eva-
luated from economic and ethical
perspectives to improve human and
animal health and preserving biodi-
versity and the environment.

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integriceps (Heteroptera: Scutellari-
dae), against Beauveria bassiana.
Bull. Entomol. Res., 12:1-11.

25

Table 1: Efficacy of medicinal plants against different stages of mosquitoes.

Plant/Part/Active ingredient/Extract Species Efficacy/biological effect References
Azadirachta (neem), Melia spp.
Triterpenoids/leaves methanol An. step. larvae LC50:43-60ppm Siddiqui et al.,
2002.
Fruit/ethanol Ae. aeg. larvae LC99:0.133-0.189 g% Wandscheer et
al., 2004
limonoids An. step. larvae
An. step pupae &
adults
1pmm:91-96.7%, 1pmm: ~96%
0.05ppm: increased larval-pupal pe-
riods decreased adult longevity/ fecun-
dity
Nathan et al.,
2005.

wood and bark chippings/water An. gam. ss adult IE50: 0.4 gm/L, adult emergence at 1
day, and %100 pupation at 9 day
Howard et al.,
2009.
neem oil (0.03% w/v azadirachtin) An. gam larvae
pupae and adults
LC50:11ppm/8 days, prolonged larval
& pupation period, IE50: 6ppm/9 days
Okumu et al.,
2007.
cakes of neem oil and karanja
(Pongamia glabra)
Ae. aeg., An.
step., Cx. quinq.
larvae
LC95: 0.54, 0.77, 0.93%. Shanmugasudar-
am et al., 2008.
neemarin An. step., Cx.
quinq. larvae
LC90: 1.18, 3.18 mg/L Field: 2
L/hectare, max. efficacy at 7 days,
pupation & adult emergence affected
Vatandoost &
Vaziri, 2004.

crude neem seed powder, sprinkled
twice weekly on larval habitats
An. gam. adults

Field: 50% reduction in emergence Gianotti et al.,
2008.
emulsifiable concentrate (EC) of
neem oil
Ae. aeg., An.
step., Cx. quinq.
larvae
LC50: 0.54,0.77,0.93ppm, LC90: 3.8,
3.7, 3.7ppm reduced 100-80% in week
2-3
Dua et al., 2009.
Calotropis procera The milkweed or Apple of Sodom (ushaar),
leaves and flowers An. labr. larvae High efficacy Markouk et al.,
2000.
leaves and flowers An. step., Cx.
quinq. larvae
High efficacy Singh et al.,
2005.
whole latex in water Ae. aeg. larvae diluted 1:1: 100% mortality at 5 min,
diluted 1:1000: high mortality at 24 hr
Ramos et al.,
2006.
water-soluble dialyzable (DF) non-
dialyzable (NDF) rubber-free
fraction
Ae. aeg. larvae 50 or 10 mg/ml:: 100% mortality
within 24 hr , Partial prevention of egg
hatching & arrested larvae growth
Ramos et al.,
2006.

Plant/Part/Active ingredient/Extract Species Tested Efficacy/biological effect References
Commiphora molmol Myrrh or murr
oil extract Ae. casp.,
Cx. pip. Larvae
LC50: 0.2x10
2
g/l (instar3)
LC50: 0.016-1.6x10
2
g/l (instar 1-3)
Massoud &
Labib, 2000.
oleo-resin extract Ae. casp.,
Cx. pip. Larvae
LC50: 0.08x10
2
g/l (instar 3)
LC50: 0.06-0.5x10
2
g/l (instar 1-3)
Massoud &
Labib, 2000.
water extract Cx. quinq. larvae LC?? highly effective against Fallatah, 2009a
Punica, Pomegranate , water extract Cx. quinq. larvae LC?? highly effective against Fallatah, 2009a
Feronia limonia Swingle Indian
wood-apple, acetone extract of
dried leaves (n-hexadecanoic acid)
Ae. aeg., An.
step., Cx. quinq.
larvae
LC50s: 57.23, 79.58, 129.24 ppm Rahuman et al.,
2000
Quercus lusitania var. infectoria,
ethyl acetate fractions, gallotannins

Cx. pip larvae
LC50: 60-253 ppm (instar II)
LC50: 293-529 ppm (instar IV)
Redwane et al.,
2002
Cordia curassavica, roots naphtho-
quinones
Ae. aeg. larvae LC100: 25-12.5 g/ml after 24-hr
exposure
Ioset et al.
(2002)
Annona glabra, Stem, ethanol Ae. aeg. larvae LC50: 27 g/l De Mendonca et
al., 2005
Xanthopappus subacaulis Photo-
activated thiophene xanthopappins

Ae. albo. larvae

The LC50s
Tian et al.,
2006
Hugonia busseana, root bark,
sesquiterpenoid Hugonianene A,

An. gam. larvae

LC100: 0.01369 mg/mL, 48-72 hr,
Baraza et al.,
2007
Ficus racemosa, acetone, bark/
leaves, glunal acetate
Ae. aeg., An.
step., Cx. quinq.
larvae

LC50: 14.55, 28.5 and 41.42 ppm
Rahuman et al.,
2008
Abutilon indicum, beta-sitosterol, Ae. aeg., An. Abdul-Rahuman
26

petroleum ether step., Cx. quinq.
larvae
LC50: 11.49, 3.58 and 26.67 ppm et al., 2000
Picrolemma sprucei, quassinoids Ae. aeg. larvae LC50: 3.2-4.4 mg/L Silva, et al.,
2009
Trigonostemon reidioides, roots,
rediocides, methanol

C. felis fleas

LD90: 0.25-0.5 ppm.
Jayasuriya et al.,
2004
AkseBio2: (Thymbra spicata,
Origanum syriacum, Pimpinella
anisum, sesame oil, maize oil,
carvacrol, thymol, anethole).

Cx. pip larvae
LD90: 25 ppm (instar I-II)
LD90: 50 ppm (instar III-IV)
at 24 hr
Cetin et al.,
2004

Table 2: Active ingredients isolated from medicinal plants essential oils.

Active ingredients (plant part) Plant essential oil
thymoquinone, thymol, carvacrol, carvone, p-cymeme
juvacemene
juvabiones
chromenes and farnesol
black seed Nigella sativa
basil Ocimum. basilicum
Abies balsamea
Ageratum huostonianum
Piperitenone oxide mint Mentha spicata viridis
Eudesmane sesquiterpenoids, trans-chrysanthemyl monoter-
penoid contain p-menthane aldehyde eucamalol
Santolina insularis
Precocenes I and II, (anti-juvenile hormone activity) citronella grass Cymbopogon
winterianus
1,8-cineole

eugenol
thymol
menthol
rosemary Rosemarinus offici-
nale & Eucalyptus globus
clove Syzygium aromaticum
garden thyme Thymbra vul-
garis mint Mentha spp.
2-undecanone (from the stem and leaves and provides plant
resistance to insect herbivores)
wild tomato Lycopersicon hir-
sutum glabratum
limonene, sabinene and beta-myrcene (fruit oil)

Japanese pepper Zanthoxylum
piperitum
thymol (seeds), sabinene (leaves),
carvacrol
anethole (seeds)
linalool (fruits)
parsley Petroselinum crispum
thyme Th. vulgaris
anise Pimpinella anisum
coriander Coriander sativum
carvone (seeds)
limonene (seeds; fruit)

anethole (fruit)
1,8-cineole, p-cymene and -phellandrene (rhizome)
caraway Carum carvi
celery Apium graveoles;
mullilam Za. limonella
fennel Foeniculum vulgare
zedoary Curcuma zedoaria
Perillaldehyde and perillyl alcohol Conyza newii
2-phenethylepropionate,
p-menthane-3,8-diol
Peanut Arachis hypogaea
mint Mentha spp.
dillapiol long pepper Piper aduncum
Pyrethrum an oleo-resin of dried flowers (chrysanthemic acid
& pyrethric acid incorporating alcohol pyrethrolone, pyre-
thrin I & II)
Pyrethrum daisy (Chrysanthe-
mum) Tanacetum cinerariae-
folium

27



A SURVEY ON DIPTEROUS FLIES AND THEIR DENSITIES IN
ALEXANDARIA AND HURGADA, Egypt
By
AZZA S. ABD EL-HALIM
Research Institute of Medical Entomology, the General Organization for
Institutes and Teaching Hospitals Dokki, Giza, Egypt.

Abstract

The present study was performed from January to December 2009 to identify
the dipterous flies associated with human and animal diseases in Alexandaria
and Hurgada. The results indicated that 10233 flies belonging to 10 families,
18 genera and 21 species were trapped from Alexandaria and 41669 flies be-
longing to 9 families, 14 genera and 16 species were trapped from Hurgada .M.
domestica L. and Coproica vagans (Haliday) were the most abundant species
in the two studied areas. Statistical analysis showed that species of the two
families Muscidae and Sphaeroceridae were significantly higher in Hurgada
than Alexandaria due to the spread of garbage, fermented fruits and human &
animal excreta.
Key words: Dipterous flies, Alexandaria and Hurgada, Egypt.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Dipterous flies are among the
most important insect that affect the
health of human and animals (Wil-
son, 1991). They act as vectors of
pathogenic organisms either me-
chanically or biologically (Smith,
1973). The majority of species
breed in Carrion and decaying vege-
table and they may carry pathogens
to human food or drink or directly
to the human body, others attack the
human body producing different
types of myiasis (Smart, 1965).
This work aimed to survey and
identifies dipterous flies of medical
and veterinary importance in Alex-
andria and Hurgada localities.

Materials and Methods

This study was conducted for one
year (January till December 2009).
Flies were collected from Alexanda-
ria and Hurgada. The collections
were done by using standard insect
net or sticky traps from areas around
garbage accumulation and garbage
boxes (El-Bashier et al., 2006). Al-
so infested fruits, vegetables, fishes
excreta were obtained and kept in
small cages at laboratory, till emer-
gency of adults. Identifications were
28

carried out using keys given by
Zumpt (1965); Steyskal and El- Bai-
ly (1967); Soliman and Morsy
(1976); Shaumer et al. (1977, 1985,
1989); Shaumer and Kamal (1982,
1983); Mohamed and Shoukry
(1991); Morsy et al. (1991, 2001)
and Amin et al. (1997a, b).

Results

Table 1: Flies collected from Alexandaria and Hurgada (Min. Max.).
Species Alexandaria XSEM Hurgada XSEM
Calliphora vicina R.O. 0.0 0.0 ( 0 0 ) 0.08 0.08 ( 0 1 )
Chrysomyia albiceps (wied.) 7.3 2.5 ( 0 27 ) 8.9 2.7 ( 0 28 )
Lucilia sericata (Meig.) 13.8 10.8 ( 0 132) 0.08 0.08 ( 0 1 )
Musca domestica L. 506 .3 + 240 .1 ( 99 3066) 2333.0 258.4 ( 1118 3865 )
Musca Sorbans wied. 0.4 0.2 ( 0 - 2 ) 3.4 0.9 ( 0 9 )
Muscina stabulans (Fallen) 0.7 0.7 ( 0 8 ) 0.0 0.0 ( 0 - 0 )
Stomoxys calcitrans (L.) 0.3 0.3 ( 0 4 ) 0.08 0.08 ( 0 - 1 )
Hydrotaea meteorica (L.) 0.1 0.1 ( 0 1) 0.3 0.2 ( 0 - 2 )
Synthesiomyia nudiseta (Van.) 4.9 4.4 ( 0- 53 ) 0.0 0.0 ( 0 - 0 )
Limnophora variegata (stein) 0.0 0.0 ( 0 0 ) 0.08 0.08 ( 0 - 1 )
Limmophora multipunctata (s) 0.6 0.4 ( 0 4 ) 0.0 0.0 ( 0 -0 )
Physiphora demandata (Fabr.) 1.0 0.5 ( 0 5 ) 0.8 0.3 ( 0 - 3 )
Piophila casie (L.) 1.6 0.9 ( 0 -11) 0.7 0.3 ( 0 3 )
Parasarcophaga hirtipes Wied. 0.2 0.1 (0 1 ) 0.3 0.2 ( 0 - 2 )
Coproica vagans (Haliday) 290.7 276.7 ( 0 - 3333 ) 1010.8 815.5 ( 0 - 9886 )
Cop. Ferruginata (Stenh.) 1.5 0.8 ( 0 -10 ) 0.0 0.0 ( 0 - 0 )
Cop. digitata (Duda) 0.0 0.0 ( 0 0 ) 1.0 1.0 ( 0 - 12 )
Copromyza costalis Zetter. 1.8 1.3 ( 0 - 15 ) 0.0 0.0 ( 0 0 )
Copromyza marginalis (Adams) 0.8 0.5 ( 0 6 ) 0.0 0.0 ( 0 0 )
Meoneura vagans (Falln) 0.6 0.5 ( 0 6 ) 9.3 4.7 ( 0 56 )
Hippelate pusio Low 10.8 4.5 ( 0 -50 ) 3.0 1.5 ( 0 18 )
Drosophila melanogaster Meig. 8.2 7.1 ( 0 86 ) 0.3 0.3 ( 0 4 )
Sepsis thoracica (Rob.- Des.) 0.3 0.3 ( 0 3 ) 0.0 0.0 ( 0 0 )
Sepsis lateralis Wied. 1.0 0.3 ( 0 3 ) 0.0 0.0 ( 0 - 0 )
29

Table 2: Dipterous flies from Alexandaria and Hurgada

Families
Alexandaria Hurgada
Genus Species Specimens Genus Species Specimens
Calliphoridae 2 2 253 3 3 109
Muscidae 6 7 6160 4 5 28043
Otitidae 1 1 12 1 1 10
Piophilidae 1 1 19 1 1 8
Sarcopagiedae 1 1 2 1 1 4
Sphaeroceridae 2 4 3537 1 2 13343
Milichiidae 1 1 7 1 1 112
Chloropidae 1 1 129 1 1 36
Drosophilidae 1 1 98 1 1 4
Sepsidae 1 2 16 0 0 0
Total 17 21 10233 14 16 41669

Discussion

The survey yielded a total of
10233 flies from Alexandria belong-
ing to 21 species, 17 genera and 10
families namely Calliphoridae,
Muscidae, Otitidae, Piophilidae,
Sarcophagidae, Sphaeroceridae,
Milichiidae, Chloropidae, Drosophi-
lidae, and Sepsidae. From Hurgada
41669 flies were collected belong-
ing to 16 species, 14 genera and 9
families: Calliphoridae Muscidae,
Otitidae, Piophilidae, Sarcophagi-
dae, Sphaeroceridae, Milichiidae,
Chloropidae, and Drosophilidae.
Among the collected species
Musca domestica L. was the most
abundant in the two studied areas.
The results agreed with those of
Amin et al. (1997a, b; 1998). Mus-
cina stabulance (Fallen), Hydrotaea
meteorica (L.). Limnophora multi-
punctata (S.) physiphora demanda-
ta (Fab), Coproica ferruginata
(Stemh.), Copromyza costalis zetter,
Copromyza marginalis (Adams)
Sepsis thoracica (Rob.- Dec.) and S.
lateralis Wied. were found only in
Alexandaria city.
Concerning the total number of
species of each family in discending
order in Alexandaria was: Muscidae
(6160) > Sphaeroceridae (3537) >
Calliphoridae (253) > Chloropidae
(129) > Drosophilidae (98) > Pio-
philidae (19) > Sepsidae (16) > Oti-
tidae (12) > Milichiidae (7) > Sar-
cophagidae (2). However, in Hurga-
da the total number of species of
each family was: Muscidae (28043)
> Sphaeroceridae (13343) > Mili-
chiidae (112)> Calliphoridae (109)
> Chloropidae (36) > Otitidae (10)
30

>Piophilidae (8) > Drosophilidae
(4) > Sarcophagidae (4).
The total number of Musca do-
mestica L. was significantly higher
in Hurgada city than in Alexandaria
(t = 4.1, P < 0.01). This may be at-
tributed to the accumulation of gar-
bage, decaying fishes, human and
animal excreta which are the most
attractive breeding medium of this
species. M. domestica L. was the
most abundant in the two localities.
This agreed with Morsy et al.
(1991); Amin et al. (1997a, b) and
Abdel Halim et al. (2005).
Generally speaking, most zoonot-
ic protozoa and pathogenic bacteria
are transmitted by the fecal-oral
route in man and animal by arthro-
pod-borne vector. They exist in the
environment as oocysts, cysts or
spores as well as micro-organisms,
the infective were found in human
excreta, water, soil, green vegeta-
bles and food as well as being infec-
tive stages to subsequent generation
of hosts (Strickland, 2000).

Conclusion

The out come of this survey of the
dipterous flies in the two ecological-
ly different Egyptian localities pave
the way for the good understanding
of the zoonotic diseases transmis-
sion and prevalence in both Alexan-
dria and Hurgada cities. Conse-
quently, a feasible, specific and eco-
logically safe control measures can
be proposed.

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Saunders, Philadelphia, London,
Toronto, Montreal, Sydney, Tokyo.
Taha, M.A.; Kamal, S., 1984: Sur-
vey of the insect fauna in certain
areas of Southern Sinai. J. Fac.
Educ., Ain Shams Univ., 7:287-300.
Tantawi, M.; Greenberg, B.,
1993: Morphometric discrimination
of the sibling species Drosophila
melanogaster (Meigen) and D. sim-
lans. Syst. Entomol., 18:231-6.
Telford, H.S., 1970: Eristalis (Dip-
tera: Syrphidae) from America, No-
rth of Mexico. Ann. Entomol. Soc.
Amer., 63:1201-10.
Wilson, M.E., 1991: A World
Guide to Infection: Diseases, Distri-
bution, Diagnosis: Oxford Universi-
ty Press, Oxford.
Zumpt, F., 1965: Myiasis in Man
and Animals in the Old World: A
Textbook for Physicians, Veterina-
rians and Zoologists. Butterworth &
Co., London.
Zumpt, F.; Heinz, H.J., 1950: A
contribution to the study of the
morphology and homology of the
male terminalia of Calliphora and
Sarcophaga (Diptera: Calliphorid-
ae). Entomol. Month Mag., 86:207-
17.

33

Table 3: Flies Collected from Alexandria.
Species 1 2 3 4 5 6 7 8 9 10 11 12 13
C. vicina 0 0 0 0 0 0 0 0 0 0 0 0 0
C. albiceps 1 0 0 11 27 18 9 4 14 0 1 2 87
L. sericata 1 0 2 6 132 14 3 2 0 3 2 1 166
M. domestica 99 111 124 3066 118 101 103 373 413 724 523 321 6076
M. sorbans 0 0 0 2 0 1 0 0 1 0 1 0 5
M. stabulance 0 0 8 0 0 0 0 0 0 0 0 0 8
S. calcitrans 0 0 0 0 0 0 0 0 0 0 4 0 4
H. meteorica 1 0 1 0 53 1 2 0 1 0 0 0 59
S. nudiseta 0 0 1 0 0 0 0 0 0 0 0 0 1
L. variegata 0 0 0 0 0 0 0 0 0 0 0 0 0
L. multipunctata 1 0 0 0 0 0 0 0 0 0 4 2 7
P. demandata 1 0 0 0 2 1 0 0 0 0 5 3 12
P. casie 0 0 2 11 5 1 0 0 0 0 0 0 19
P. hirtipes 0 0 0 0 0 0 0 0 1 0 1 0 2
C. vagans 26 0 0 1 3 5 4 1 2 1 3333 112 3488
C. Ferruginata 2 0 0 0 0 1 0 0 0 2 10 3 18
C. Digitata 0 0 0 0 0 0 0 0 0 0 0 0 0
C. costalis 0 0 0 0 0 0 0 0 0 2 15 4 21
C. marginalis 0 0 0 0 0 0 0 0 0 2 6 2 10
M. vegans 0 0 6 1 0 0 0 0 0 0 0 0 7
H. pusio 10 1 7 2 3 1 0 1 1 31 50 22 129
D. melanogaster 2 0 0 0 1 0 0 0 0 0 86 9 98
S. thoracica 0 0 0 0 3 1 0 0 0 0 0 0 4
S. lateralis 0 0 0 0 10 2 0 0 0 0 0 0 12

34

Table 4: Flies Collected from Hurgada
Species 1 2 3 4 5 6 7 8 9 10 11 12 13
C. vicina 0 0 0 1 0 0 0 0 0 0 0 0 1
C. albi-
ceps
0 0 1 14 2 5 12 2 6 28 24 13 107
L.
sericata
0 0 0 0 0 1 0 0 0 0 0 0 1
M. dome-
stica
1411 1122 2681 3050 2888 2215 2637 1118 1348 3865 3110 2551 27996
M. sor-
bans
2 0 1 7 6 6 9 0 6 3 1 0 41
M. stabul-
ance
0 0 0 0 0 0 0 0 0 0 0 0 0
S. calci-
trans
0 0 0 1 0 0 0 0 0 0 0 0 1
H. meteo-
rica
0 0 0 0 0 0 0 0 0 0 0 0 0
S. nudise-
ta
0 0 0 0 1 1 0 0 2 0 0 0 4
L. varie-
gata
0 0 0 0 0 0 0 0 1 0 0 0 1
L.. multi-
p unctata
0 0 0 0 0 0 0 0 0 0 0 0 0
P. dema-
ndata
1 0 0 1 2 2 0 0 0 0 3 1 10
P. casie 1 0 0 0 1 0 0 0 3 0 1 2 8
P. hirtipes 0 0 0 0 2 2 0 0 0 0 0 0 4
C. vagans 1 0 2 5 9 1 0 68 1064 1212 1083 9886 13331
C. ferru-
ginata
0 0 0 0 0 0 0 0 0 0 0 0 0
C. digit-
ata
0 0 0 0 0 0 0 0 0 0 12 0 12
C. costalis 0 0 0 0 0 0 0 0 0 0 0 0 0
C. margi-
nalis
0 0 0 0 0 0 0 0 0 0 0 0 0
M. vegans 0 0 1 2 0 0 0 7 56 18 16 12 112
H. pusio 0 0 0 2 2 2 8 0 4 0 18 0 36
D. mela-
nogaster
0 0 0 0 0 0 0 0 4 0 0 0 4
S. thora-
cica
0 0 0 0 0 0 0 0 0 0 0 0 0
S.lateralis 0 0 0 0 0 0 0 0 0 0 0 0 0


35



SUSCEPTIBILITY OF BROMADIOLONE ANTICOAGULANT RODENTICIDE
IN TWO RODENT SPECIES AND ITS HAEMATOLOGIC EFFECT

By
MICHEAL.W. MIKHAIL AND YOUSRYA M. ABDEL- HAMID
Research Institute of Medical Entomology, Ministry of Health; Dokki,
Giza, Egypt

Abstract

Susceptibility levels of the Norway rat, Rattus norvegicus and the roof rat,
Rattus rattus to bromadiolone anticoagulant rodenticide by bioassay and bio-
chemical methods were studied. Animals were trapped from Giza and Qua-
lyobia Governorates in which the anticoagulant rodenticides were used to con-
trol rodents for long periods. Complete mortality was obtained for both species
and sexes within standard no-choice feeding test period (4 days) indicating
bromadiolone susceptibility. Treatment of rats with LD
50
showed high proth-
rombin times which also indicate the susceptibility of the tested animals. In
treated rats, bromadiolone caused significant decrease in the total erythrocytic
counts and increase in the total leucocytic counts. In survivors, RBCs, WBCs
approximately reached the control levels at day 43 post treatment. Also, treat-
ment decreased neutrophils, eosinophils, basophils and monocytes but in-
creased the lymphocytes in dead and survived animals more than in controls.
Keyword: Bromaliolone, Rodenticides, Rattus norvegicus, Rattus rattus.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction
Rodents are very common ani-
mals in many Egyptian Governo-
rates (Shoukry et al., 1986a, Morsy
et al., 1988). Besides their economic
hazard causing damage to agricul-
ture and contamination of stored
food materials, they also play an
important role as reservoir host for
many zoonotic diseases such as pla-
que and murine typhus (Abdon and
Samaan, 1962), Leishmaniasis (Mo-
rsy et al., 1982; El-Kady et al.,
1998), toxoplasmosis (Rifaat et al.,
1972) and trichinosis (Morsy et al.,
2000). They act as reservoir host for
parasites such as hymenolepiasis,
giardiasis, amoebiasis and schisto-
somiasis (Morsy et al., 1981, El Na-
hal et al., 1982).
In Egypt, the anticoagulant roden-
ticide was used on a large scale to
control rodents in agriculture &
public health purposes. They act by
preventing the normal mechanism
that control blood-clotting in tar-
geted rodents. Death resulting from
internal haemorrhage does not occur
before the elapse of 3-10 days after

36

ingestion. The toxicity symptoms
take some days to develop. They
had a number of potential harmful
histological changes in tissues of
some organs (Haram et al., 1993).
The development of resistance to
anticoagulants was recorded in sev-
eral parts of the world, for exam-
ples;(1) with the first generation
anticoagulants; Lund (1964) for
Norway rats in Denmark, Drum-
mond and Bentley (1967) in Eng-
land and Wales, Jackson et al.
(1971) in North Carolina, USA, (2)
with the second generation anticoa-
gulants: Brooks and Rowe (1987)
showed that difenacoum resistance
in Norway rats has been found in
England and Denmark, (3) resis-
tance of roof rat to difenacoum has
been reported in France and United
Kingdom, and (4) resistance to
bromadiolone has been found in
Norway rats in Denmark and United
Kingdom and in house mice in Can-
ada, Denmark, Sweden and United
Kingdom. In Egypt, the continued
use of the anticoagulant rodenticides
developed a sort of tolerance or re-
sistance in some rodent species in
several localities.
The present work was planned
and objected to investigate the sus-
ceptibility of bromadiolone anticoa-
gulant rodenticide to commensal
rodents; Rattus norvegicus and Rat-
tus rattus in Giza and Qualyobia
Governorates. The haematological
changes as a result of treatment with
LD50 of bromadilone and the time
required to survived rats to recover to
normal state were also investigated.

Wire box traps were used (WHO,
1982) to trap R. norvegicus and R.
rattus from Giza and Qualyobia
Governorates. The traps were deo-
dorized by cleaning with hot water
and soap before use. Traps were
baited, distributed in selected hous-
es at sunset, collected next morning
and transported to the laboratory.
Rats were kept for two weeks before
being tested and during this period
they were caged individually and
given water and a suitable diet. Two
days before testing, the weight and
sex of each rat were determined.
Immature ones (less than 150 gm
for R. norvegicus & 100 gm for R.
rattus), pregnant females and un-
healthy rats were excluded from the
experiment.
Susceptibility of rat under labora-
tory conditions was carried out by a
bioassay method. Bromadiolone
0.005% was used over 4-days by
no-choice feeding test. The amount
of anticoagulant bait eaten was rec-
orded daily and food pots were rep-
lenished daily with fresh poison
bait. After completion of feeding,
rats were fed on normal laboratory
diet. The
1
"3-[3-(4-bromobiphenyl-
4-yl)-3-hydroxy-1-phenylpropyl]-4-
hydroxy-coumarin" deaths day was
recorded. Resistant rats were those
survived for 24 days after 4-days
feeding. The susceptibility was de-
tected biochemically as prothrombin
times were measured (Quick et al.,
1935). A tablet of thromboplastin
with calcium was dissolved in 2ml

37

of distilled water and mixed gently
to have a homogeneous suspension.
Thromboplastin suspension (0.2 ml)
was reactivated by incubation at
37C for 15 min., then 0.1ml of rat
plasma (incubated for 3 minutes at
37C) was added quickly to suspen-
sion and time in seconds required
for clot formation was noted. When
the treated rats have little or no
change in prothrombin time, indi-
cating resistant but when showing
high prothrombin time, they were
susceptible. Total RBCs, WBCs &
differential leucocytes for rat spe-
cies were determined after oral
treatment with LD50 of bromadi-
olone (1.125 mg/kg for rats) at day
3 post treatment and every 10 days
till rat died or recovered (Brooks
and Rowe, 1987). Untreated rats
were used as control. RBC and
WBC counts were determined by
haemocytometer (Briton, 1963).
WBCs in thin leishman's stained
films were examined and the per-
cent of different types were counted.
Means and SD were calculated.
All data were analyzed using analy-
sis of variance (one-way ANOVA).
Means were separated into signifi-
cant ranges using Tukeys test when
a significant F test was obtained.
The relation of the mean blood
cell counts to survival days of ani-
mal was examined by regression
analysis. The computerized pro-
grams: SPSS version 8 and EzA-
NOVA version 0.96 were used for
such statistical analysis. Whatever
the level of significance, the P<0.05
was considered indicative of statis-
tical significance.

Results

Table 1: Criteria for R. norvegicus
(R.n.) and R. rattus (R.r.) fed for 4-
days on 0.005% bromadiolone.

R
e
s
i
s
t
a
n
c
e
%

M
o
r
t
a
l
i
t
y
%

Mean / Range
S
e
x

(
n
=
2
0

r
a
t
)

S
p
e
c
i
e
s

A
r
e
a

Death
day

Dose
consumed
(mg/kg)
Bait
consumption
(g)

Weight of rat
(g)
0
100
6.5
4.0 -10.0
6.4
3.4 8.1
27.7
20.0 34.0
226.6
157.0 -311.0
R
.

n

0 100 6
4.0 9.0
5.6
4.6 7.8
23.8
20.0 -36.0
198.9
166.0 357.0
G
i
z
a

0 100 9.6
7.0 16.0
6.7
2.5 9.8
18.4
7.0 25.0
136.5
107.0 -168.0
R
.

r
.

0 100 9.2
6.0 16.0
6.3
3.6 8.3
18.1
8.0 -26.0
128.2
110.0 -177.0
0 100 5.7
4.0 9.0
6.8
4.4 9.9
29.5
22.0 36.0
218.7
154.0-281.0
R
.

n
.

Q
u
a
l
u
b
i
y
a

0 100 5.8
4.0 9.0
6.6
3.9 8.5
28
21 - 38
217.3
155.0 283.0
0 100 8.2
5.0 13.0
7.4
4.2 11.1
20.5
9.0 27.0
140
107.0 184.0
R
.

r
.

0 100 7.7
6.0 14.0
7.2
3.1- 8.3
25
8.0 31.0
171.0
129.0 202.0

38

Table 2: Prothrombin time (seconds) after treatment of Norway and roof rats
with LD
50
bromadiolone.

A
r
e
a

Rat
condition
(N)
Prothrombin time (Mean S.D.)

Rattus norvegicus Rattus rattus
Male Female Male Female
G
i
z
a

Control (10) 012.30.5
aA
012.40.5
aA
012.40.5 a
A
012.400.5
aA

Dead (3) 130.503.5
A
125.005.7
A
118.505.0
A
130.008.5
A

Alive for 03 days(8) 059.005.3
A
064.907.4
A
052.1219.0
A
051.713.2
A

13 days(8) 022.403.1
A
026.804.7
B
023.805.7
A
023.007.2
A

23 days (8) 018.004.3
A
017.502.4
A
016.805.7
A
015.002.7
B

33 days (8) 014.001.1
A
013.801.4
A
013.301.8
A
013.300.8
A

43 days (8) 012.300.5
aA
012.300.5
aA
012.300.5
aA
012.300.5
aA

Q
u
a
l
u
b
i
y
a

Control (10) 012.300.5
aA
012.400.5
a A
012.300.5
aA
012.300.5
aA

Dead (3) 127.006.0
A
130.704.5
A
112.019.6
B
114.329.1
B

Alive for 03 days(7) 066.607.8
A
063.302.6
A
047.710.5
B
046.408.3
B

13 days(7) 024.002.1
A
021.902.4
A
023.006.7
A
023.406.1
A

23 days (7) 016.002.3
A
015.601.7
A
015.102.8
A
015.702.2
A

33 days (7) 012.900.9
aA
013.401.0
A
012.900.9
aA
013.001.0
aA

43 days( 7) 012.100.4
aA
012.300.5
aA
012.300.5
aA
012.300.5
aA

Each governorate: means with same letters (small vertically & capital horizontally) insignifica-
ntly different (one-way ANOVA, P>0.05).

Table 3: Mean
(1)
counts of red- and white blood for R. norvegicus
and R. rattus treated with bromadiolone

Rodent
Condition
GIZA QUALUBIYA
R. norvogicus R. rattus R. norvogicus R. rattus

RBCs ( 10
6
/ ul)
Cont
(2)
06.8 06. 6
A
06.5
A
06.4
A
06.7 06.6
A
06.5
A
06.5
A

Dead
(3)
03.8 03.5 03. 6 03.1 03.4 03.8 03.6 03.4
Survived
03 days
(4)
05.4
A
05.0
B
05.3
B
05.4
B
04.9
A
04. 9
B
05.2
B
05.5
B

13 days
(4)
05.6
AB
05.2
BC
05.5
BC
05.6
BC
05.3
A
04.9
B
05.4
BC
05.7
BC

23 days
(4)
06.0
BC
05.5
CD
05.8
CD
05.9
CD
05.7
B
05.3
BC
05.7
CD
06.0
CD

33 days
(4)
06.2
CD
05.8
D
06.1
DE
06.1
DE
06.0
BC
05.8
C
06.0
DE
06.2
AD

43 days
(4)
06.4
D
06.5
A
06.2
AE
06.2
AE
06.3
C
06.3
A
06.2
AE
06.3
A

F
(5)
34.1 36.1 22.4 55.6 43.2 18.6 27.9 34.8
df 6,45 6,45 6,45 6,45 6,41 6,41 6,41 6,41
WBCs (10
3
/ ul)
Cont
(2)
07.3
A
07.1
A
07.1
A
07.0
A
07.3
A
07.2
A
07.3
A
07.2
A

Dead
(3)
17.9 17.1 17.7 17.9 19.2 16.0 16.2 16.7
Survived
03 days
(4)
12.4
B
11.8 11.3
B
12.1
B
13.0
B
12.2
B
11.9
B
11.7
13 days
(4)
11.0
B
10.9 10.2
BC
11.2
BC
12.4
BC
11.8
BC
10.7
BC
10. 5
B

23 days
(4)
09.4
C
09.8 09.2
CD
10.1
CD
10.9
C
10.4
C
09.6
CD
09.3
B

33 days
(4)
08.4
CD
08.7 08.2
DE
08.8
D
08.4 08.8 08.3
DE
08.0
43 days
(4)
07.6
AD
07.1
A
07.7
AE
07.5
A
07.2
A
07.2
A
07.5
AE
07.3
A

F
(5)
37.7 71.3 28.3 47.6 32.3 51.9 38.3 54.3
d.f 6,45 6,45 6,45 6,41 6,41 6,41 6,41 6,41
1-Vertically, means with same letters not significantly different (P>0.05, Tukey test), SD values omitted
from table. 2-N=10 animal, 3. N= 2-3 animal, 4. N= 7-8 animal, 5. Significant, P <0.001

39

Table 4: Differential white blood cells for Rattus novegicus and R. rattus
males and females treated with bromadiolone

1. Neut = Neutrophils, Eos = Eiosinophils, Bas = Basophils, Lym = Lymphocytes, Mon+ Monocytes. 2-
SD values omitted. 3- Con= Control animl and S3, S13, S23, S33, S43= Animal survived for 3, 13, 23, 33,
43 days, respectively.

Table 5: Regression analysis for relation of mean blood cell counts to days of
rat survival

Rodent
RBC WBC
Equation
1
R
2
F (1,3)
2
Equation
1
R
2
F (1,3)
2

Giza
R.n. y= 5.30+0.03x 0.99 50.91** y= 12.56-0.12x 0.98 17.23*
R.n. y= 4.78+0.04x 0.94 46.66** y= 12.32-0.12x 0.99 44.4**
R.r. y= 5.19+0.03x 0.99 23.72* y= 11.42-0.09x 0.99 21.06*
R.r. y= 5.29+0.02x 0.99 47.65** y= 12.56-0.11x 0.99 49.99**
Qualubiya
R.n. y= 4.78+0.04x 1.00 85.58** y= 13.95-0.16x 0.96 74.45**
R.n. y= 4.57+0.04x 0.93 39.14** y= 13.09-0.13x 0.97 83.89**
R.r. y= 5.14+0.03x 1.00 82.01** y= 12.22-0.11x 1.00 74.03**
R.r. y= 5.45+0.02x 0.95 61.02** y= 11.94-0.11x 0.99 31.76*
1. Y= Cell count, X = day, R
2
= Coefficient of determination. 2.P < 0.05 (*), P < 0.01 (**).

Discussion

In the present study, bromadi-
olone 0.005% in diet (under no
choice feeding test for 4 days)
caused complete mortality (Tab. 1)
of R. norvegicus both sexes and R.
rattus at Giza and Qualyobia Gs

Cells
1

Mean %
2,3

Male Female
Con D S3 S13 S23 S33 S43 Con D S3 S13 S23 S33 S43
Giza
Rattus norvogicus
Neut 26.7 08.0 20.8 21.4 24.1 25.3 26.5 27.0 10.0 17.9 19.4 21.6 23.9 26.4
Eos 01.5 00.0 00.4 00.5 01.0 01.3 01.5 01.6 00.0 00.5 00.9 01.1 01.4 01.6
Bas 00.3 00.0 00.0 00.1 00.3 00.3 00.3 00.4 00.0 00.0 00.1 00.3 00.3 00.3
Lym 64.7 90.5 77.5 73.5 69.4 66.3 65.3 64.2 88.5 78.0 76.0 72.3 69.1 65.3
Mon 07.0 01.5 03.6 04.5 05.3 05.8 06.5 06.8 01.5 03.6 03.6 04.8 05.4 06.6
Rattus rattus
Neut 26.4 10.0 20.4 24.0 25.8 26.0 26.4 26.2 09. 7 20.6 22.4 24.6 25.7 26.3
Eos 01.8 00.0 00.4 00.6 01.1 01.8 01.8 01.8 00.0 00.3 0.60 00.9 01.4 01.7
Bas 00.3 00.0 00.0 0.0 00.1 00.3 00.3 00.3 00.0 00.0 00.0 00.1 00.3 00.3
Lym 64.2 88.0 75.1 69.9 67.0 65.8 64.8 65.1 89.3 75.6 72.6 69.0 66.9 66.7
Mon 07.3 02.0 04.1 05.5 06.0 06.5 06.9 06.6 01.0 03.6 04.4 05.4 05.7 06.4
Qualubiya
Rattus norvogicus
Neut 26.9 15.3 16.1 19.7 22.1 24.7 26.6 26.7 11.7 16.1 17.3 20.3 23.3 26.4
Eos 01.6 00.0 00.6 01.0 01.1 01.3 01.6 01.5 00.0 00.7 00.9 01.0 01.4 01.6
Bas 00.2 00.0 00.0 00.0 00.3 00.3 00.3 00.3 00.0 00.0 00.1 00.3 00.3 00.3
Lym 64.7 85.3 80.4 75.7 73.6 67.4 65.0 64.7 86.0 79.6 78.1 73.4 69.6 65.0
Mon 06.9 02. 7 02.9 03.6 05.0 06.3 06.6 06.8 02.3 03.6 03.6 05.1 05.4 06.7
Rattus rattus
Neut 26.6 11.0 20.1 22.7 25.1 26.0 26.7 26.5 11.3 22.4 24.6 26.1 26.7 26.7
Eos 01.6 00.0 00.4 00.4 01.3 01.6 01.6 01.7 00.0 00.6 00.9 01.3 01.8 01.7
Bas 00.3 00.0 00.0 00.0 00.1 00.3 00.4 00.4 00.0 00.0 00.0 00.1 00.3 00.3
Lym 64.5 87.0 75.3 72.0 68.0 66.1 64.4 64.2 86.7 73.0 69.7 67.0 65.0 64.7
Mon 07.1 02.0 04.1 04.9 05.4 06.0 06.9 07.2 02.0 04.0 04.9 05.4 06.4 06.6

40

indicating that the two rat species
are still susceptible to this anticoa-
gulant rodenticide. This agreed with
Mikhail and Allam (2007) who
showed that R. norvegicus and R.
rattus (Qualyobia G.) were suscept-
ible to bromadiolone, like the case
for other rodenticides e.g., warfarin
and flocoumafen (Shoukry et al.,
1986b; El Bahrawy and Morsy,
1990; Zidan et al., 1997) and chlo-
rophacinone (Hussien, 2001). Sax-
ena and Sahni (2008) showed that
0.005% bromadiolone was very ef-
fective in controlling the population
of R. rattus in India. However, other
studies showed that R. norvegicus
(Pelz, 2007; Markussen et al., 2008)
and Apodemus agrarius (Cao et al.,
2008) showed resistance to broma-
diolone
The bait consumption and corres-
ponding active ingredient intake
were more in R. rattus than in R
.norvegicus. The mean intake for R.
rattus was 6.7 & 6.3 mg/kg at Giza
and 7.4 & 7.2 mg/kg in Qualyobia
for males and females, respectively,
while for R. norvegicus was 6.4 &
5.6 mg/kg at Giza and 6.8 & 6.6
mg/kg at Qualyobia for males and
females, respectively.
The time to death was taken as a
parameter for anticoagulant efficacy
on treated rats. In this respect, R.
rattus recorded higher mean values
(9.6 & 9.2 days at Giza and 8.7 &
8.2 days at Qualyobia for males and
females, respectively) than R. nor-
vegicus (6.5 & 6.0 days at Giza and
5.7 & 5.8 days at Qualyobia for
males and females, respectively).
On the other hand, response of R.
norvegicus and R. rattus to broma-
diolone (LD50) and measuring the
prothromin time provided a satisfac-
tory method to evaluate the efficacy
of anticoagulants, and very useful in
comparing tolerance or resistance of
rodent species to anticoagulants
(Haldler and Shadbolt, 1975; Ende-
pols et al., 2007). Prothrombin time
assessed post treatment in the ani-
mal's plasma of the two tested ro-
dent species.
The elongated prothrombin times
for the treated animals compared
with the control (Tab. 2) showed
that the means prothrombin time
were not significantly (P>0.05) for
the two sexes and species of the
control animals in the two Governo-
rates. It was higher (P<0.05) in dead
animals than in control or survived
ones, then gradually decreased
(P<0.05) in survived animals till
reaching the control level at 33 or
43 days, and were higher (P <0.05)
in dead R. norvegicus than in dead
R. rattus in Qualyobia. This proved
that the rat species from the two
Governorates are still susceptible to
bromadiolone. Triff et al. (2002)
reported that coumatetralyl LD50
had the most negative action in
white rats 24 hr prothrombin time,
but values returned to normal level
after 48 & 72 hr. Hussien (2005)
found that the higher effect of war-
farin on the prothrombn time was
obviously noticed in R. norvegicus
and R. rattus caught from Qualyobia
G, indicating the susceptibility of
these rats to warfarin.

41

RBC counts (Tab. 3) showed that
each animal sex, counts for control,
dead and survived rats were signifi-
cantly different (F = 18.6-55.6, P
<0.05), but in dead animals counts
were significantly less (P<0.05)
than the corresponding controls, and
RBC counts for survived animals (at
the examined periods) were signifi-
cantly less than the corresponding
controls but were significantly high-
er than those for dead animals (P
<0.05). Also, the leucocytic (WBC)
counts showed that for each animals
sex were significantly different (F =
28.3-71.3, P< 0.05), dead animals
counts were significantly higher (P
< 0.05) than for the corresponding
controls, and that the counts for sur-
vived animals (at the examined pe-
riods) were significantly less than
those for the corresponding dead
animals but were significantly high-
er (P< 0.05) than those of controls
except survived animals at 43 days
where mean counts were insignifi-
cant (P>0.05) than in controls.
Abdel-Raheem et al. (1986) found
that the increase in white blood cells
in treated rats with diphacinone can
be considered as defensive mechan-
ism. Mikhail and Abdel-Hamid
(2007) reported that warfarin toxica-
tion caused significantly different
blood cell counts in treated rats
compared to the control ones. War-
farin significantly decreased total
erythrocytic count and increased the
total leucocytic count of treated rats.
The differential WBCs (Tab. 4)
showed that bromadiolone caused
significant decrease in neutrophils,
eosinophils, basophils and mono-
cytes but significant increase in
lymphocytes in dead and survived
animals than in controls. Neutro-
phils and eosinophils started to in-
crease in survivals at day 3 till ap-
proximately reached the control lev-
el at 43 days. Basophils were absent
in dead and 3 days survived animals
and started to appear at day 13 and
reached equal level as controls at 33
and 43 days. Lymphocytes showed
higher percentages in survived rats
at any period than in control animals
but less than dead ones. Monocytes
started to increase in survived ani-
mals at day 3 but do not reach the
control level. Such results are in
agreement with the previous studies.
Herman and hombrecher (1962)
showed reduction in erythrocytic,
leucocytosis, lymphocytopennia, eo-
sinophils and thrombocytopenia in
rats treated with warfarin. Omar et
al. (1975) reported that warfarin
caused significant decrease in neu-
trophils, eosinophils, basophils and
monocytes, but caused significantly
increase in lymophocytes in dead
and survived rats than in control.
Triff et al. (2002) reported that
coumatetralyl increased neutrophils
and lymphocytes in white rats.
Mikhail and Abdel-Hamid (2007)
reported that warfarin caused a sig-
nificant decrease in neutrophils, eo-
sinophils, basophils and monocytes
while caused a significant increase
in lymphocytes of the treated R.
norvegicus and R. rattus. The rela-
tion of RBC & WBC counts to sur-
vival period of the two rodent spe-

42

cies were examined by regression
analysis (Tab. 5). Red blood cell
counts increased as survival period
of the animal increased (R
2
= 0.93-
1.00, F=23.72-85.58, P<0.05) till
reaching normal level at day 43
post-treatment (P.T). On the other
hand, white blood cell counts de-
creased as the survival period of the
animal increased (R
2
= 0.96-1.00,
F=17.23-83.89, P< 0.05) till normal
level at day 43 P.T.

Conclusion

Rattus norvegicus and Rattus rat-
tus trapped from Giza and Qualyo-
bia Governorates were susceptible
to bromadilone 0.005% anticoagu-
lant rodenticide, which caused hae-
motologic changes. This anticoagu-
lant agent must be carefully applied
in the field by a well trained tech-
nical staff to avoid the human risk.

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45



SUBCLINICAL AUTOIMMUNE THYROID DISORDERS IN EGYPTIAN
PATIENTS WITH UNTREATED CHRONIC HEPATITIS C VIRUS IN-
FECTION
By
ZAKARIA Y. MOHRAN
1
, NADIA A. ABDEL KADER
1
, AMAL T. AB-
DEL MOEZ
1
and AMAL A. ABBAS
2
Departments of Tropical Medicine
1
, and Clinical Pathology
2
, Faculty of
Medicine, Ain Shams University, Cairo 11566, Egypt

Abstract

The frequency of anti-thyroid antibodies and subclinical thyroid disorders in
Egyptian patients with untreated chronic hepatitis type C was estimated. In ad-
dition, it determines the correlation between the seropositivity of anti-thyroid
antibodies and serum thyroid stimulating hormone level in chronic HCV posi-
tive patients. Also, the impact of hepatic decompensation in inducing thyroid
autoimmunity in such patients was evaluated. This study included 56 untreated
chronic hepatitis C patients and 28 healthy subjects of the same local popula-
tion as a control group.
The results showed that the mean thyroid stimulating hormone levels were
significantly higher in patients with chronic hepatitis C than in controls. Pa-
tients with decompensated chronic hepatitis C had insignificantly higher sub-
sequent autoimmune hypothyroidism than the compensated patients. A signifi-
cant positive correlation between the level of thyroid stimulating hormone and
anti-thyroglobulin, but not with anti-thyroperoxidase, was found.
Therefore, there is an association between chronic hepatitis C virus infection
and subclinical autoimmune thyroid disorders. Thyroid stimulating hormone
and anti-thyroglobulin antibodies screening for all chronic HCV patients, even
if antiviral treatment will not be initiated, should be done.
Key words: Autoimmune thyroid disorders, anti-thyroid antibodies, chronic
hepatitis.
Correspondence: Dr. Nadia A. Abdel Kader, Tropical Medicine Department, Faculty
of Medicine, Ain Shams University, Cairo, Egypt. Email:elsersy_n_a@yahoo.com.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction

Hepatitis C virus (HCV) infection
is a global health problem, being the
second most common chronic viral
infection in the world

(Crax et al.,
2008). The prevalence of HCV in-
fection varies throughout the world.
It is estimated that 170 million per-
- 46 -

sons in the world are infected with
HCV with a global prevalence of
about 3% (Lauer and Walker,
2001). The highest estimated preva-
lence of HCV infection has been
reported in Egypt, nearly 12%,
mostly type 4 (El-Awady et al.,
2009). Abdel-Aziz et al. (2000) re-
ported a prevalence rate of 24.3% in
a rural Egyptian community in the
Nile Delta. Moreover, studies of
HCV infection in Egypt have shown
a high prevalence of anti-HCV
among blood donors (Arthur et al.,
1997), and residents of rural areas
endemic for schistosomiasis (Dar-
wish et al., 1993). The natural histo-
ry of HCV4 seems to be similar to
other HCV genotypes, but the sug-
gestion that it is associated with a
worse prognosis after liver trans-
plantation is questionable

(Esmat
and El Raziky, 2007).
Several extra-hepatic diseases have
been associated with chronic HCV
infection, and in most cases appear
to be directly related to the viral
infection

(Andrade et al., 2008). The
mechanism of auto-reactive manife-
stations of HCV may be due to B-
cell activation threshold, in directly
infected lymphocytes and in in-
duced self reaction through a me-
chanism of molecular mimicry (Fer-
ri et al., 2008). These extra hepatic
manifestations include: hematologic
diseases such as cryoglobulinemia,
autoimmune disorders such as thy-
roiditis and renal disease, and der-
matologic conditions (El-Serag et
al., 2002). The most frequent and
clinically important endocrine dis-
orders that can be encountered in
HCV positive patients are thyroid
disorders and type 2 diabetes melli-
tus (Antonelli et al., 2009).
The prevalence of various thyroid
disorders and serum antithyroid an-
tibodies was generally higher in pa-
tients with chronic type C hepatitis
disease than in those with type B
hepatitis, type D hepatitis or a con-
trol series of uninfected individuals
(Antonelli et al., 2006). The fre-
quency of abnormally high levels of
anti-thyroid antibodies

in HCV-
infected patients varies markedly in
different series,

ranging from 2% to
48% (Ganne-Carrie et al., 2000).
Besides, it was found that up to
40% of HCV patients on interferon
alpha develop clinical or subclinical
thyroid disease (Tomer and Menco-
ni, 2009). Generally, a higher preva-
lence of thyroid antibodies in HCV
infection was reported even before
interferon administration (Hass et
al., 2009).
The aim of this study was to as-
sess the frequency of anti-thyroid
antibodies and subclinical thyroid
disorders in Egyptian patients with
untreated hepatitis C infection, de-
termine the correlation between the
seropositivity of anti-thyroid anti-
bodies and serum thyroid stimulat-
ing hormone level in HCV positive
patients, and study the impact of
hepatic decompensation in inducing
thyroid autoimmunity.
- 47 -

Subjects, Materials and Methods

This study is a case-control study.
The patients group consisted of 56
patients with untreated chronic
HCV infection (i.e. positive
HCVAb for more than 6 months).
All of the patients were admitted in
the Tropical Medicine Department,
Ain Shams University Hospital.
Informed consent was obtained
from all participants before enroll-
ment in the study. Right to refuse
participation was emphasized.
Two groups of HCV patients were
identified on the basis of the pres-
ence of hepatic decompen-sation.
The clinical evidence of hepatic de-
compensation included symptoms
and signs such as jaundice, bleeding
tendency, hematemesis and/or me-
lena, and ascites. This clinical evi-
dence was supported by laboratory
investigations and abdominal ultra-
sound findings.
G1: 28 compensated HCV posi-
tive patients.

G2: 28 decompensated
HCV positive patients.

Exclusion
Criteria: 1. Co infection with hepati-
tis B virus (HBV). 2. History of an-
tiviral treatment. 3. Autoimmune
hepatitis. 4. Other liver diseases e.g.
Primary biliary cirrhosis. 5. HCV
positive patients with hepatocellular
carcinoma. 6. Any symptom sug-
gestive of thyroid dysfunction or
history of thyroid operation. 7. Res-
idence in iodine deficiency areas.
Twenty eight age and sex matched
healthy subjects of the same local
population were included as a con-
trol group.
All patients were subjected to full
medical history, thorough clinical
examination. Laboratory investiga-
tions included urine and stool analy-
sis, the hepatitis viral markers
(HCVAb, HBsAg and HBcAb),
complete blood picture (CBC) and
the erythrocyte sedimentation rate
(ESR). Liver function tests included
total and direct bilirubin, total pro-
teins and albumin, PT, PTT, INR,
and liver enzymes (ALT, AST, Al-
kaline phosphatase). Kidney func-
tion tests included serum creatinine
and BUN, serum Na and K, alpha
fetoprotein (FP).
Specific thyroid investigations were
done for both patients and controls.
a- Thyroid Stimulating Hormone
(TSH), if abnormal, free thyroxine
(FT
4
) and serum free triiodo-
thyronine (FT
3
) were done. Serum
TSH

(normal range 0.286.82 mU/l)
was determined by the immune-
enzymometric assay. Circulating
FT
3
(normal range 1.8-4.2pg/ml)
and FT
4
(normal range 0.801.90
ng/dl).
b- Anti-thyroid antibodies: Anti-
thyroglobulin antibodies (TgAb)
and anti-thyroperoxidase antibodies
(TPOAb) were measured. Blood
samples were obtained, immediately
centrifuged at 3000 rpm for 10 mi-
nutes then serum samples were
stored at 70 C till time of assay.
Sequential ELISA (Enzyme Linked
Immune-Sorbent Assay) technique
- 48 -

was done using kits supplied by
Monobind Ink.USA. In this proce-
dure, the immobilization takes place
during the assay at the surfaces of
micro plate well. The interaction of
streptavidin coated on the well and
exogenously added biotinylated thy-
roglobulin antigen in case of TgAb
and biotylinated thyroperoxidase
antigen in case of TPOAb. Values
of TgAb125 IU/ml & TPOAb 40
IU/ml were considered positive.
Abdominal Ultrasound was done
for evaluations of liver and spleen
size, liver echogenicity, ascites, por-
tal and splenic vein diameter.
Data was analyzed on an IBM
personal computer, using Statistical
Package for Special Science (SPSS)
software computer program version
15.Data were described as mean
standard deviation (SD) for quantit-
ative (Numerical) variables and as
frequency & percentage for qualita-
tive (Categorical) variables.
(1) Independent Student t test was
used for comparison of quantitative
variables among two independent
groups. (2) One-way ANOVA test
was used for comparison of quantit-
ative variables among more than
two independent groups. Least sig-
nificant difference test (LSD) test
was used as post-hoc test. (3) Chi-
square test (or Fishers exact test
when appropriate) was used for
comparison of distribution of qua-
litative variables among different
group. (4) Correlation between con-
tinuous variables was performed
using Pearson correlation coeffi-
cient. Significance level (P) value: P
> 0.05 is insignificant (NS). P
0.05 is significant (S).

Results

All patients had chronic HCV in-
fection as evident by a positive test
for HCVAb more than 6 months
before the study. Sixteen (57.2%)
and 20 (71.4%) patients in compen-
sated and decompensated groups
respectively were having past histo-
ry of receiving the anti-bilharzial
treatment, either by injection or
orally. Urine and stool analysis re-
vealed no parasites. The mean age
& sex distribution of patients groups
and controls were matched (Tab. 1).
Laboratory investigations showed
significant difference between com-
pensated & decompensa-ted HCV
groups regarding the platelets count,
serum albumin, total and direct bili-
rubin, PT, INR & FP (Tab. 2).
Laboratory Thyroid Tests: Num-
ber of HCV patients with elevated
TSH was significantly higher in de-
compensated group (n=13, 46.4%)
in comparison to the compensated
group (n=6, 21.4%) Among the19
patients with elevated TSH, 47.36%
of them were females. FT
3
and FT
4

were determined for these 19 pa-
tients. Hypothyroidism was detected
in 2 & 5 patients in the compensated
and decompensated groups respec-
tively without significant difference.
Compensated hypothyroidism (i.e.
TSH 6.82 mU/l & FT
3
& FT
4

- 49 -

within

normal range) was diagnosed
in 12 patients.
Patients percent with positive
anti-thyroid antibodies was insigni-
ficantly different between compen-
sated and decompensated groups
(Fig. 1). Control subjects showed
normal TSH values and none was
positive for anti-thyroid antibodies
(Tab. 3a). Mean values of TSH,
TgAb & TPOAb were significantly
higher in HCV decompensated pa-
tients than controls. Mean value of
TPOAb is significantly higher in
HCV decompensated than HCV
compensated ones (Tab. 3b).
Anti-thyroglobulin antibody was
positive in 12 &15 patients in com-
pensated and decompensated ones,
respectively. While TPOAb was
negative in all compensated HCV
patients and positive in 4 HCV de-
compensated patients, one of them
had elevated TgAb too. Totally,
positive anti-thyroid antibodies
were detected in 30 HCV patients
(30/56; 53.57%), 50% were fema-
les. According to thyroid hormones
level, the30 patients were classified
(Tab. 4). Prevalence of subclinical

autoimmune hypothyroidism was
insignificantly higher in HCV de-
compensated than in

HCV compen-
sated ones. HCV infected patients
showed subclinical autoimmune
hypothyroidism in 12/30 patients
(40%). In HCV patients, a highly
significant positive correlation was
between level of TSH and TgAb,
but without TPOAb (Tab. 5).

Table 1: Age and sex distribution among groups

HCV Compensated HCV Decompensated Controls P value
Age (MeanSD),yrs. 50.48.22 53.575.6 48.82 10.5 0.103
Sex M
(No., %) F
12 (42.9%)
16 (57.1%)
14 (50%)
14 (50%)
13 (46.4%)
15 (53.6%)
0.866
P > 0.05 insignificant (NS), P 0.05 significant (S).

Table 2: Laboratory investigations of HCV patients

HCV Compensated HCV Decompensated P value
Hemoglobin

14.8718.15 10.161.76 0.177
WBCs (10
9
/L) 6.122.46 7.74.5 0.103
Platelets (10
9
/L) 195.48 119.5 88.18 44.1 0.003
ESR 40.118.8 35.39 23.25 0.408
ALT 50.6 46.0 32.39 26.67 0.077
AST 59.5745.12 58.86 34.5 0.947
Alk.Phosphatase 221.1794.56 203.82 77.36 0.455
Albumin 3.49 0.52 2.15 0.36 0.000
Total Bilirubin 1.06 0.39 4.17 2.7 0.000
Direct Bilirubin 0.350.25 1.691.5 0.000
PT 14.3 3.7 22.1 6.9 0.000
INR 1.19 0.15 1.86 0.6 0.000
FP 2.042.48 5.58 5.02 0.002
P > 0.05 insignificant (NS), P 0.05 significant (S).
Table 3a: Laboratory thyroid investigations of HCV patients.

50

HCV-Compensated HCV-Decompensated
P value No. % No. %
TSH Normal
Elevated
22 78.6
6 21.4
15 53.6
13 46.4
0.048
FT3 Normal
Decreased
4 66.7
2
a
33.3
8 61.5
5 38.5
0.829
FT4 Normal
Decreased
5 83.3
1 16.7
11 84.6
2 15.4
0.943
Anti-thyroglobulin(TgAb)
Normal
Elevated

16 57.1
12 42.9

13 46.4
15 53.6

0.422
Anti-thyroperoxidase (TPOAb)
Normal
Elevated

28 100
0

24 85.7
4
b
14.3

0.056
a
a patient negative for TgAb and TPOAb.
b
a patient positive for both TgAb and TPOAb.

Table 3b: Laboratory thyroid investigations of groups

HCV Compensated HCV Decompensated Controls P value
TSH
(MeanSD)
(min.-max.)

6.769.28
0.9-38.75

8.16.37
2-29.75

2.851.48
0.9-5
a
p 0.027
b
p 0.004
c
p 0.458
Anti-thyroglobulin(TgAb)
(MeanSD)
(Min.-max.)

186.7281
50-1850

209.3289.5
20-1350

7722
45-120
a
p 0.083
b
p 0.037
c
p 0.719
Anti-thyroperoxidase(TPOAb)
(MeanSD)
(min.-max.)

12.35.6
4-22.5

2117.2
2.2-75

95
3-22.5
a
p 0.259
b
p 0.000
c
p 0.004
a
p = P value for HCV Compensated versus Controls.
b
p = P value for HCV Decompensated versusControls.
c
p = P value for HCV Compensated versus HCV Decompensated. Serums TSH normal range 0.286.82
mU/l. Values of TgAb 125 IU/ml and of TPOAb 40 IU/ml were considered positive.

Table 4: Thyroid hormonal state in HCV patients with positive thyroid au-
toimmunity.

HCV
Compensated with positive
anti-thyroid antibodies (n=12)
HCV
Decompensated with positive
anti-thyroid antibodies (n=18)
P value
No. % No. %
Hypothyroidism 1 8.3 5 27.8

0.329
Compensated Hypothyroi-
dism
a
2 16.7 4 22.2
Total hypothyroidism 3 25 9 50
Euothyroid 9 75 9 50

a
TSH 6.82 mU/l and FT3 and FT4 within

normal range

Figure 1: Levels of Anti-thyroglobulin antibody among groups

51

Table 5: Correlation between TSH level and anti-thyroid antibodies in HCV
patients

Anti-thyroid Antibodies TSH Level
r value P value
Anti-thyroglobulin 0.516 0.000
Anti-thyroperoxidase 0.039 0.77

Discussion

Several extra hepatic manifesta-
tions have been reported in the natu-
ral history of hepatitis C virus infec-
tion (HCV). Up to 40-74% of pa-
tients infected with HCV might de-
velop at least one extra hepatic ma-
nifestation during the course of their
disease (Galossi et al., 2007). Thy-
roid dysfunction is the most com-
mon endocrinopathy associated with
hepatitis C infection (Tran et al.,
2009).
The present results showed high
percentage of seropositivity for
TGAb in HCV compensated and
decompensated groups and none in
the controls. While positive TPOAb
was found in 14.3% of HCV de-
compensated patients. Subclinical
autoimmune hypothyroidism was
detected in 25% and 50% of HCV
compensated and decompensated
groups, respectively. None of the
control subjects had thyroid dys-
function. These results confirmed a
higher prevalence

of autoimmune
thyroid involvement in chronic
HCV patients than in controls. Also,
these findings support the fact that
HCV could be one of the environ-
mental factors responsible for the
breakdown of immunological toler-
ance (Testa et al., 2006).
52

Antonelli et al. (2004) reported
that both hypothyroidism and thyro-
id autoimmunity are more com-mon
in patients with chronic hepatitis C
even in the absence of cirrhosis,
hepatocellular carcinoma, or Interfe-
ron treatment than in normal con-
trols or those with chronic hepatitis
B infection.

They found 17% and
21% of patients with HCV infection
to be positive for TGAb and
TPOAb, respectively. Thyroid dis-
ease, primarily hypothyroidism, was
diagnosed in 13% of patients. Such
remarkable variation may be attri-
butable to the different methods
used and/or to the different geogra-
phy, race, age, sex, genetic makeup
and HCV genotype of the popula-
tions targeted in these reported stu-
dies.
Generally, 40% of the patients
with positive anti-thyroid antibodies
had subclinical thyroid dysfunction,
either confirmed or compensated
hypothyroidism. This goes with a
study by Huang et al. (2006) who
reported that thyroid auto-anti-
bodies, either occurred before or
during interferon-alpha (INF-) plus
ribavirin combination therapy, carry
a high prediction of subsequent thy-
roid dysfunction.
Sex distribution in HCV patients
regarding thyroid autoimmunity and
autoimmune thyroid dysfunction
was nearly equal. However, it was
reported that among chronic HCV
positive patients, females are at a
higher risk of developing thyroid
dysfunction (Antonelli et al., 2004).
Moreover, thyroid disorders are
more likely to be diagnosed in fe-
males following INF- and ribavirin
therapy (Kee et al., 2006; Nadeem
et al., 2009). The mechanisms lead-
ing to the development of anti-
thyroid antibodies and thyroid dis-
orders during chronic hepatitis C
infection, especially after interferon-
based antiviral therapy, are only
partially understood (Ferri et al.,
2008). Patients who develop an IFN
induced thyroid disease perhaps are
genetically susceptible (Paran et
al., 2000). Moreover, it is impor-
tant to note that IFN- treatment
itself is associated with the autoim-
mune manifestations (Krause et
al., 2003). Therefore, the gender
has only partial effect in develop-
ment of the thyroid dysfunction
with or without antiviral treatment
and this can explain our findings.
Many studies found that female
gender pretreatment positive anti-
thyroid antibodies in addition to
pretreatment TSH are the most im-
portant predictive factors for subse-
quent thyroid dysfunction (Kabbaj
et al., 2006; Andrade et al., 2008;
Friedrich-Rust et al., 2009). Al-
though, The previous positivity for
anti-thyroid antibodies is the prin-
cipal risk factor for developing thy-
roid disease in the course of antivir-
al therapy (Andrade et al., 2008).
The present results showed that
TSH screening and anti-thyroid an-
tibodies detection is the most impor-
53

tant steps for the detection of au-
toimmune thyroid dysfunction in
chronic HCV patients. As the posi-
tivity of anti-thyroid antibodies is
one of the situations requiring spe-
cial caution before initiation of anti-
viral treatment (Esmat and El Ra-
ziky, 2007). Thus, screening for
serum TSH and anti-thyroid antibo-
dies is strongly recommended be-
fore, during and after INF- treat-
ment, and patients should be in-
formed about the risk of thyroid
dysfunction (Andrade et al., 2008).
So, detection of TGAb and TPOAb
in all HCV patients, independently
of IFN therapy was suggested and
the utility of a screening for HCV in
autoimmune thyroid disorder is
stressed. The autoimmune form of
thyroid dysfunction seems to have
more severe consequences and
longer evolution than non-auto-
immune form indicating the impor-
tance of early detection, in order to
adapt the follow-up of thyroid func-
tion and therapy (Gelu-Simeon et
al., 2009).
There was a significant positive
correlation between TSH level and
TGAb among the patients of this
study, this added more support for
the association between the thyroid
autoimmunity and the development
of subsequent hypothyroidism.

In
this study the percentage of HCV
patients with elevated TSH was sig-
nificantly higher in the decompen-
sated than the compensated group.
No significant difference was ob-
served between both groups regard-
ing the positivity of anti-thyroid
antibodies. This emphasized the
direct relation between the autoim-
munity and the viral infection pure-
ly disregarding the degree of hepatic
decompensation.

Besides, HCV decompensated
patients had insignificantly higher
subsequent autoimmune hypothy-
roidism than the HCV compensated
patients. Therefore, hepatic decom-
pensation had no effect on preva-
lence of autoimmune thyroid dys-
function in chronic HCV patients.
Rodrguez-Torres et al. (2008) re-
ported that the baseline thyroid dys-
function (TD) in 20% of the severe
hepatic fibrosis group and 10% of
the mild hepatic fibrosis group

(Rodrguez-Torres et al., 2008).
Another recent study reported a
significant decrease of total T
3
in
decompensated cirrhotic patients
compared to both chronic and com-
pensated cirrhotic HCV patients
(Moustafa et al., 2009).
This discrepancy can be attributed
to the fact that in the study of
Rodrguez-Torres et al. (2008) TD
was defined as history of TD, or
abnormal TSH or positive TPOAb.
In the present study, the subclinical
autoimmune thyroid disorder was
only considered.

Conclusion

54

The study showed a fairly high
prevalence of thyroid autoimmunity
and subclinical thyroid dysfunction
in chronic HCV infection.
There are two clinical implica-
tions: (1) Thyroid status, TSH and
anti-thyroid antibodies, should be
systemically evaluated in patients
with HCV infection even if antiviral
treatment will not be initiated. (2)
Patients with chronic HCV disre-
garding the degree of hepatic de-
compensation, require careful atten-
tion to diagnose and manage au-
toimmune thyroid disorder. More
research is needed to clarify the re-
lationship between hepatic fibrosis
progression and autoimmune ma-
nifestations.

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57



A STUDY ON THE PREVALENCE OF HOUSE DUST MITES
IN AL-ARISH CITY, NORTH SINAI GOVERNORATE, EGYPT
By
GIHAD T. El-SHERBINY
1
, EMAN T. El-SHERBINI
2
, NAGLA MOS-
TAFA K. SALEH
3
, FOUAD M. HARIDY
4
AND AYMAN T.A. MORSY
5
Department of Parasitology, Faculty of Pharmacy, October 6 Universi-
ty
1
, 6
th
October (Formerly Sinai University, El Arish), Department of Zo-
ology, El Nahda University
2
, Beni Sweif, Department of Zoology, Faculty
of Science, South Valley University
3
, General Organization for Veteri-
nary Services (formerly)
4
, and Tropical Medicine Unit, The Ministry of
Interior Hospitals
5
, Egypt.

Abstract

Free living mites comprise a huge and various groups of tiny arthropods in
the class Arachida, mainly of the Pyroglyphidae family. Exposure to allergens
derived from house dust mite (HDM) feces is a postulated risk factor for aller-
gic sensitization, asthma development and asthma morbidity. However, practi-
cal and effective method to mitigate these allergens in low-income, urban home
environments remains elusive. It well known that (HDM) physiology is greatly
affected by hydrothermal microclimatic condition. El Arish has subtropical
climate and warm humid summer, such situation are favourable to proliferate
house dust mites. As no valid data are available for house dust mites fauna of
El Arish, this study was carried out to determine the prevalence and contamina-
tion rates of homes in El Arish city. Samples of house dust collected in 2008
from 50 houses in El Arish city were subjected to acarological examination.
Acri were found in (34.6 %) of the samples collected from these homes. Re-
sults indicated that dust mites were present in all humid environments. Also,
hypersensitivity to dust mites was common among patients with asthma.
Key words: House dust mites, North Sinai, Al Arish, mite allergens, asthma.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

The term house dust mite (HDM)
is generalized to all species of
mites which have a worldwide dis-
tribution (Korsgaard, 1998). HDMs
belong to the class Arachnida. Of
the at least 50 species of HDMs
that have been found in domestic
house dust, two of the family Py-
roglyphidae, namely D. petronyssi-
nus and D. farinae, are the most
important in temperate climates,
both in terms of numbers and of
clinical relevance ( Gamal-Eddin et
al., 1982). Clinically, the HDMs
58

are of relevance because most
asthma symptoms in children and
young adults are associated with
both an immediate hypersensitivity
to their inhaled allergens (Brown et
al., 1995) and a familial tendency
towards atopic dermatitis (IIIis et
al., 2001). D. petronyssinus pre-
dominates over most HDM species
worldwide and is the most frequent
in tropical and subtropical areas. D.
farinae is the most common spe-
cies in dry, continental climate and
is rare in costal climates (Jackson
et al., 2005). The tropical rat mite,
Ornithonyssus bacoti, is one of the
most common houses invading
species; it is an obligate, blood
feeding ecto-parasite with world-
wide distribution (Fox, 1982). Nat-
ural host of Ornithonyssus bacoti
include several species of rat and
mice, hamsters, gerbils, voles, and
other wild rodents (Baker, 2005;
Flynn, 1973). Although none of
these species of mite are truly para-
sitic on human or pets, they bite
people readily, often producing
dermatitis and itching. Rat mite
infestations occur in structures
where rat nets are located, infesta-
tion are sometimes first noticed
following examination or after the
natural host have died or left the
structure and may also occur where
heavy mite infestation have devel-
oped around a rodent nest (Ebeling,
1975). If a rat has a nest in an attic
or other site indoors and it dies or
vacates the nest these mites will
leaves the nest and body in search
of attack humans (Morsy et al.,
1982). The bite of these ectopara-
sites cause mild to severe irritation
and sometimes a painful dermatitis
leaving dermal red spots (Michael
et al., 2000).
Although dust mites were ob-
served in dust by a scientist in
1694, it was not until the 1960's
that they were associated with al-
lergies and were recognized in the
past 30 years as the most important
sources of allergens in the human
habitation (Mumcuoglu et al.,
1999). In the last three decades, an
interest for HDM's types allergy
especially asthma (Morsy et al.,
1995) particularly in children in the
vicinity of HDM (Sacco et al.,
2003). Free- living mites have long
been recognized as one of the most
important sources of allergen of
house dust responsible for origin of
atopic and bronchial asthma, rhini-
tis and dermatitis, eczema, hay fev-
er, sinusitis, and middle ear infec-
tion (Platts- Mills and Chapman,
1987). As a very ancient organism,
the mites is enormous diversified
and adapted to a wide variety of
environments including plants, an-
imals, human, soil, fresh and salty
water, organic rabbles, houses,
mattresses and old books. They
thrive in their thousands in worm,
moist places feeding on dead skin
scales; they live in soft furnishing
such as beds, bedding, carpets, and
soft toys. A great number of factors
play a role in exposure to mites, the
prevalence of different categories
of mites vary considerably among
geographical locations. The differ-
59

ent socioeconomic conditions in-
fluence the prevalence of the do-
mestic mites. The environment fac-
tors play an important role in the
control of house dust mites. Thus,
understanding of the environmental
factors influencing mite population
can be expelled in mite control.
Sinai Peninsula is the veritable ga-
teway to Egypt from the east. It is
triangular in shape, and stretched
for 400 Km from north to south
and 200 Km from east to west. It is
generally hot during the summer,
stormy and exposed to cold air cur-
rent during the winter. Average
daily maximum temperature ranged
from 29
o
C-17
o
C and minimum
from 21
o
C-9
o
C in summer and win-
ter respectively. Airy relative hu-
midity fluctuates between 65% in
daytime and 85% at night in sum-
mer and between 60 % and 80 % in
winter respectively (Sadaka et al.,
2000).
This study aimed at the clarifica-
tion the relation between human
allergy and the house dust mites
(HDM) in Al- Arish city, North
Sinai Governorate.

Subjects, Material and Methods

Al Arish is the capital and largest
city, with 114,900 inhabitants esti-
mated (2002), apart of the immi-
grant workers and governmental
employees. It lies on the Mediter-
ranean coast of Sinai Peninsula,
about 214 miles northeast of Cairo.
A descriptive cross-section study
was designed to investigate the
mite fauna in houses of Al Arish.
The city was divided into 10 areas
in each area 5 houses were random-
ly selected. The owners were in-
formed about the design of the sur-
vey and those who accepted to par-
ticipate were dealt with.
Criteria of house selection: A
short characteristic of each house
and one of the examined places is
presented: a wooden new houses, a
bed about 35 years old, it is used
only at weekend more frequently in
summer-time, room is not heated,
and temperature and RH depends
on changes of environmental RH
and temperature fluctuations. b- A
flat on the end of the block house,
the room is cold and damp in win-
ter and sunny and warm in sum-
mer. A bed 12 years old is used
every night. C. A stone house
heated with gas and stove, rather
damp room on the first floor. A 5
years old bed is sometimes used for
sleeping. D. A warm apartment on
the first floor of the block has aver-
age air temperature about 20
o
C, RH
60 % in summer time, and 68%.
An old cotton mattress is used
every night for about 20 years. The
houses are a new stone with her-
metic windows, or an old (about 40
years) coach used in living room,
or a low income home included
single family detached houses. Al-
so, apartments in a free standing
building with three or fewer units,
and apartments in complexes with
three or more units were selected.
General criteria: Patient with his-
tory suggestive of asthma together
60

with healthy individuals as control
was randomly selected. All patient
and control completed a question-
naire to take a psychosocial history
including que-stion about age,
gender, educational level, employ-
ment, family history of asthma,
information relevant to asthma risk
factors, including: socioeconomic
status, home life, school, age of the
mattress, signs of humidity in the
bedroom, number of occupants,
symptoms, severity of symptoms,
asthma treatment and understand-
ing of treatment. They were sub-
jected to skin prick (Allergy prick
test {Scratch test} Zheng-zhou
Concern Medical Supplies Co.,
Ltd), by placing a little amount of
the allergen on the skin (fore arm),
and then scratching or pricking the
skin so that the allergen is intro-
duced under the skin surface.
Working solution of 1:10 wt/vol
was purchased from VACSERA,
Agousa, Giza, Egypt. Positive and
negative controls were used (1%
histamine hydrochloride and phy-
siological saline respectively). A
prick was made via drop with a
fine hypodermic needle no. 20 Af-
ter 20 minutes, the reaction was
measured and graded (Stites and
Terri, 1991) as ve= neither wheal
nor erythema, 1+ve= no wheal but
erythma >20 mm in diameter, 2
+ve= no wheal but erythema
>20mm, 3 +ve=wheal & erythema,
4 +ve=wheals with pseu-dopods&
erythema. Skin was carefully ex-
amined for reaction; swelling and
redness of the skin; results were
obtained within about 20 minutes.
Cross matched control subjects
were selected which were clinically
& parasitological healthy, regard-
less, allergic status. Blood samples
were collected from patients and
control to measure specific IgE-
ELISA (Chalottesville, Virginia,
USA) Kits (Chapman et al., 1987).
Collection of dust samples: Dust
samples were obtained once a
month from major foci indoor
(floor, carpets, mattresses, bedding,
structures where rat nets are lo-
cated) during the period from Sep-
tember 2008 till August 2009. A
vacuum equipped with a dust trap,
one square meter of each place was
vacuumed for a period of 1 minute.
All samples were put in plastic
bags and stored at 4
o
C to avoid
proliferation (Morsy et al., 1994).
Isolation and identification: Ten
milliliters of 90% lactic acid were
added to 100-250 mg of dust sam-
ple. Mixture was boiled and diluted
with 90ml distilled water (Morsy et
al., 1994, 1995). Mites against con-
trasting blue colored field were
removed by a fine needle and iden-
tified. They were mounted in 2
drops of Hoyer's medium (gum
Arabic 30gm, chloral-hydrate 200
gm, glycerin 20 ml, water 500 ml)
for study (Colloff and Spieksma,
1992). Data were expressed as No
of mites/gm dust.
Statistical analysis: Data eva-
luated by Chi-square test & P<0.5
was significant (SPSS, 1999).

61

Results
The results are shown in tables (1, 2, 3, 4 & 5), figures (1 & 2).

Table 1- Characteristics of the studied population.

Non-asthmatic Asthmatic % No. Characters
% No. % No.
72.2
27.8
26
10
64.3
35.7
9
5
70
30
35
15
Male
Female
100.0 36 100.0 14 100 50 Total

19.4
25.0
13.9
41.7

7
9
5
15

35.7
14.3
28.6
21.4

5
2
4
3

24
36
18
22

12
18
9
11
Age
1-10
11-20
21-30
31 and over
100.0 36 100.0 14 100 50 Total

Table 2: Samples examined from indoors and mites isolated

Mites Samples
Means no. of mites No.
mites
isolated
Positive
%
Number of samples
Per positive
sample
Per examined
sample
Positive
for mites
No. examined
9.4 3.2 864 34.6 92 266

Table 3: Mite species found in dust samples n=266

Positive samples Mite species
% No
42.4
16.3
32.6
8.7
39
15
30
8
D. pteronyssinus
D. farinae
O. bacoti
Unidentified mites
100 92 Total

Table 4: Prevalence of mites during summer and winter seasons

Mites Dust sample Seasons
Un
identified
O.
bacoti
D
.farinae
D.
pteronyssinuss
Positive for mites Examined
sample % No.
6
2
24
6
10
5
30
9
44.8
20
70
22
156
110
Summer
Winter
8 30 15 39 34.6 92 266 Total

Fig.1- Mite species found in dust samples n=266
0
20
40
60
Mite species
%
D.pteronyssinus
D.farinae
O.bacoti
Unidentifi ed

Table 5: Prevalence and risks associated with asthma status. N=14
62

Negative cases Positive cases Risks
% No. % No.
85.7
85.7
92.9
28.6
50.0
28.6
42.9
12
12
13
4
7
4
6
14.3
14.3
7.1
71.4
50.0
71.4
57.1
2
2
1
10
7
10
8
Family history of asthma
Smoking in home
Pets ( dogs & cats )
Moist wall, carpet, furniture
Insects ( cockroach, ants, others)
Crowded homes
Low socio-economic level
P>0.050 Probability

Fig.2- Prick test in relation to age.

0
10
20
30
40
50
1-10year 11-20 y 21-30 y >31y
Age
%
positive
Negative

Fig.3- Two types of houses in Al-Arish

Fig.4- Prick test technique Fig.5- D. petronyssinus
63

Discussion

This study recorded the presence
of family pyrogyphidae in acri-
fauna in the majority of samples and
proportion of individual mites, also
O. bacoti and the unidentified spp
dont belong to family Pyrogyphi-
dae).
In the present study, HDM found
in house dust in El Arish City was
(34.6%). Surveys done in Egypt
(Gamal-elddin et al., 1982) in Tanta
city (39%), Morsy et al.(1994) in
Qualyobia, Bakr et al. (1995) in
Shebin El-Kom, Sadaka et al.
(2000) in Alexandria city with an
average of 83.79%, El Shazly et al.
(2006) and El-Nahas et al. (2007) in
Dakahlia governorate 59.9% in ur-
ban houses and 46.3% in rural one.
Feldman et al. (1985) in Israel
found that Pyroglyphidae mites,
especially D. pteronyssinus consti-
tuted 90% of isolated mites from
house dust samples. The results also
agreed with Solarz et al. (1998) who
carried out states in upper Silesia
both in new and old age dwelling ,
heated with stoves, found that the
occurrence and dominance of D.
farinae was associated with low
humidity (50-65%) and temperature
of
18-27
o
C whereas the dominance of
D. pteronyssinus with humidity of
65-84% and temperature of 10-
24
o
C. So, latter species was most
abundant (42.4%) among the spe-
cies of mites collected in houses in
Al Arish especially unmodernized
homes in old tenement building
showed signs of disrepair associated
with damp (Collof et al., 1987).
In the present study, approx-
imately the majority of patients with
inhalant allergy had serum IgE con-
centration above the mean value
plus two standard deviations for
corresponding healthy population.
Kosaka and Tazawa (1976) found
that serum IgE level correlated to
the severity of asthmatic attack.
Morsy et al. (1994b) in Egypt found
immunoglobulins in patients with
atopic dermatitis due to mites in-
festation. Also, Morsy et al.
(1995a) isolated four species of
house dust mites from houses of
patients with allergic respiratory
diseases. Katoh et al. (2004) re-
ported that patients with HDM-
atopic dermatitis had considerably
increased level of serum total IgE
than controls. Domingo et al. (2004)
reported that atopic dermatitis is a
64

chronic inflammatory skin disease
that frequently precedes the devel-
opment of asthma or other respirato-
ry allergies. Morsy et al. (1995b)
reported Serum TNF-, IgG, IgM &
IgE in Egyptian scabietic children.
Abou Gamra et al. (2005) in Egypt
found immunopathogenic role of
IgG antibody and Rantes in house
dust mite-induced chronic bronchi-
tis. Taketomi et al. (2006) and Er-
win et al. (2007) reported high
mean level of total serum IgE in ast-
hmatic patients-sensitized to HDM.
Apart from HDM, correlation be-
tween arthropods infestation and
elevation of IgE was found in sca-
bietic patients (Morsy et al., 1993)
and respiratory allergic children due
to chironomid potent allergens
(Morsy et al., 2000).
O. bacoti or tropical rat mite is
not categorized as a type of house
dust mite. It doesnt belong to fami-
ly Pyroglyphidae. It parasitized rats
and man allover the world, frequent-
ly attacks persons living in rodent
infesting buildings and its bite may
produce irritation and sometimes
painful dermatitis or mite allergy. It
is the commonest mite infesting rat
in Egypt.
In this study O. bacoti was found
in relatively high numbers in places
of rat foundation (32.6%), and re-
ported nearly allover Egypt by Mor-
sy et al.(1983) in Ismailia, Zeese et
al. (1990) in Sharkia, Shoukry et al.
(1993) in South Sinai, Younis et al.
(1995) in Suez, El Kady et al.
(1995) in Suez canal Zone. The
present results agreed with the re-
ports of several acarologists that
dominance of one or another Der-
matophagoides species in house
dust is caused rather by the micro-
climate (mainly relative humidity
and temperature) of mite habitats
within individual homes than by
regional climatic condition (Dusba-
bek, 1975; Lintner, 1993; Yassin,
1997). Comparison of results of
mite prevalence indoors showed
difference in Acari density annually.
Almost D. pteronyssinus (97.5%)
are found in dust samples collected
in period of relatively higher humid-
ity in houses rather than in period
when humidity decreases. A wide
range of a biotic factors in the do-
mestic environment have been in-
vestigated for their influence on
HDM populations of these factors,
the most important is air humidity.
In many studies, positive correla-
tions are found between mite num-
bers or allergen levels and relative
or absolute air humidity of the home
(Hart, 1990; Harving, 1993). The
seasonality and geographic distribu-
tion of HDMs can also be explained
by air humidity. HDMs osmo-
regulate through their cuticle and
therefore require a high ambient
humidity to prevent excessive water
loss (Arlian, 1992). Larger HDM
populations are found when the ab-
solute indoor air humidity is above
781 kg (45% RH & 20
o
C), however
they can survive at a wide range of
humidy, depending on the tempera-
ture (Arlian, 1992; Brown, 1995).
The protonymph stage of the life
cycle is resistant to desiccation, thus
65

enabling HDM populations to sur-
vive prolonged period of low RH
(Arlian et al., 1983).
The present patients were una-
ware of mite sensitization status,
which minimized the risk that pa-
tient in control group might- under-
takes allergen avoidance measures.
DM allergen level are characterized
by high variability dependent most-
ly upon humidity level, from less
than 2 g/g of dust in very cold or
dry climates to mean levels in the
range of 2-15 g/g in countries with
climate more suited to mite repro-
duction (costal area). The greater
number of mites in houses with
wooden roofs and floors may be
because this material keeps the en-
vironments warmer and wetter, con-
tributing, somewhat, to adequate
intra-domiciliary level of tempera-
ture and humidity. Cracks and cre-
vices in the roofs and floors are also
good shelters for mites. Houses with
practice of sunbathing mattresses in
summer had fewer mites a fact that
may be attributed to the heat and
consequent dehydration with its
negative influence on mite popula-
tion (Morsy et al., 1983).
Other factors may also influence
HDM populations; socio-economic
factors may have some indirect in-
fluence on mite populations. Wal-
shaw and Evans (1987) suggested a
weak relationship between decreas-
ing social class and increase density
of mites whereas in the USA, child-
ren of lower socio-economic groups
have a higher incidence of asthma
(Wissow et al., 1988). An incased
risk of current asthma in examined
cases associated with a self-reported
family history of asthma, current &
life time childhood environmental
tobacco smoke exposure, but none
of these result were statistically sig-
nificant. Pets are one of the most
common indoor allergens along
with DMs, cockroaches and mold. It
is difficult to compare the dust mite
and cat allergens level due to differ-
ent study population and dust sam-
pling setting and method (vacuum,
time, and area sampling). The dust
mites mean levels in this study were
higher in sample collected from
house hold of people with current
asthma, but different not statistically
significant due to the small baseline
sample size.
On the other hand, arthropods
causing different pictures of asth-
matic allergy and/or dermatitis in
Egyptian children were fleas (El-
Okbi et al., 1991), bed-bug (Abou-
Gamra et al., 1991), human lice
(Abou Gamra et al., 1992), honey-
bee-sting (Morsy and Lashen,
1996a), a small beetle, Paederus
alfierii Koch (Morsy et al., 1996b),
dipterous larvae causing skin and
respiratory myiasis (Morsy et al.,
1999) and chironomid (Morsy et al.,
2000) and ants (Sanad et al., 2002).
Correlation between arthropods
infestation and elevation of IgE was
reported in scabietic patients (Morsy
et al., 1993) and respiratory allergic
children due to chironomid potent
allergens (Morsy et al., 2000).

Conclusion

66

In Al-Arish city, D. pteronyssinus
is the main species, O. bacoti is also
common. Mite allergen levels can
also be considered risky. The major-
ity of HDM-patients had marked
clinical pictures. HDM atopic der-
matitis occurred in cases with the
elevated ELISA-IgE. The seasonal
variations in mite abandance were
also of considerable value for the
feasible control measure. Obvious
signs of humidity in homes and the
mattress quality and quantity
represented significant risk factor
associated with high mite numbers
and the allergen levels. So, the iden-
tification of the causative agent of
the allergy is a must to suggest feas-
ible control and treatment For the
climatic conditions and HDM, fur-
ther study is ongoing and will be
publish soon elsewhere.

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71



ANTIMICROBIAL RESISTANT BACTERIA AMONG HEALTH CARE
WORKERS IN INTENSIVE CARE UNITS AT AIN SHAMS UNIVERSI-
TY HOSPITALS
By
AMANY TH. ABDEL RAHMAN
1
, SHEREEN F. HAFEZ
1
, SARA M.
ABDELHAKAM
2
, ZAINAB A. ALI-ELDIN
3
, IBRAHIM M.E. ABD EL-
HAMID ESMAT
4
, MARWA S. ELSAYED
1
, AND AISHA ABOUL-
FOTOUH
5

Departments of Microbiology and Immunology
1
, Tropical Medicine
2
, Internal
Medicine
3
, Anesthesia and Intensive Care
4
, Community, Environmental & Occ-
upational Medicine
5
, Faculty of Medicine, Ain Shams University, Cairo 11566.

Abstract

Fifty HCWs in ICUs of Internal medicine, Chest, Neonatology and Burn
were included in prospective cohort study. Collection of nasal, hand and rectal
swabs, proper biochemical identification, culture media and antibiotic sensi-
tivity tests were used to detect Methicillin-resistant Staphylococcus aureus
(MRSA); vancomycin-resistant Enterococci (VRE) & extended spectrum -
lactamase producing gram -ve bacilli (ESBLs). S. aureus was isolated from
34% of HCWs; 28% were nasal carriers, 4% were hand carriers and 2% had S.
aureus at both sites. Nasal and hand carriage rates of MRSA were 20% & 4%
respectively, with an overall rate of 22%. Gram -ve bacilli were isolated from
8% of HCWs hand swabs & showed Citrobacter koseri, Escherichia coli,
Klebsiella pneumoniae and Pseudomonas aeruginosa. Hand carriage rate of
ESBLs was 2%. Hand contamination with gram -ve bacilli and S. aureus was in
14% of HCWs. VRE carriage rate was 9.5%. ESBLs carriage rate in rectal
swabs was 21.43%. K. pneumoniae was the most common ESBLs producing
isolate (33.3%), followed by E. coli (18.75%). In combined disc method, az-
treonam was the most sensitive (90%) in detecting ESBLs. Burn ICU had
highest % of MRSA & ESBLs carriage. Neonatal ICU showed highest % of
VRE carriage. An insignificant association was between infection control train-
ing or antimicrobial intake and carriage of antimicrobial resistant bacteria.
Key Words: Health care workers, intensive care units, methicillin-resistant S. aureus,
vancomycin-resistant Enterococci, extended spectrum -lactamase producing gram -ve
bacilli.
Abbreviations: HCWs: health care workers, ICUs: Intensive care units, S. aureus: Staphylo-coccus aureus,
MRSA: Methicillin-resistant S. aureus, VRE: vancomycin-resistant Enterococci, ESBLs: extended spectrum
-lactamase producing Gram -ve bacilli.
Correspondence: Sara Mahmoud Abdelhakam, 37 Dr Mohammed Kamel Hussein St., El Nozha El Gadida,
5
th
floor, Cairo, Egypt. Tele: (+2) 0101601548, E-mail: saratropical@yahoo.com

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
72

Introduction

Hospitals and particularly inten-
sive care units (ICUs) are an impor-
tant breeding ground for the devel-
opment and spread of antibiotic re-
sistant bacteria. The risk for health
care workers (HCWs) exposure to a
specific pathogen is probably re-
lated to the prevalence of the impli-
cated pathogen in patient popula-
tions or the environment. HCWs
represent a source of colonization
for hospitalized patients, their own
families or both, and all three
groups can develop a clinical infec-
tion with these pathogens (Carmeli
et al., 1998; Ott and Wirick, 2008).
The emergence of antibiotic resis-
tance is considered one of the most
important threats to human health.
Besides difficulties in treatment,
multiresistant pathogens showed a
remarkable ability to be transferred
from patient to patient causing hos-
pital acquired infections (Bonten et
al., 2001; Masterton et al., 2003).
The intensive care units (ICUs)
are particularly appropriate for the
rapid emergence and spread of these
pathogens, due to many causes;
which include frequent use of
broad-spectrum antibiotics, crowd-
ing of patients with high levels of
disease acuity in relatively small
area, and the presence of more
chronically and acutely ill patients
who require prolonged hospitaliza-
tion and often harbor antibiotic-
resistant bacteria. Similarly, the
presence of invasive devices, such
as endotracheal tubes, blood and
urinary catheters, seems to encour-
age such infection (Kollef and Fras-
er, 2001).
Methicillin-resistant Staphylococ-
cus aureus (MRSA) remains among
the most important nosocomial pa-
thogens because of both the diversi-
ty and the severity of the infection
caused by them (Cespedes et al.,
2002). HCWs exposed to an envi-
ronment with a high rate of endemic
MRSA infection had a high inci-
dence of either hand or nasal colo-
nization. Transmission to patients is
likely to occur during routine pa-
tient care (Opal et al., 1990). Air-
borne transmission appears to be
quantitatively related to the number
of S. aureus colonizing the anterior
nares (Sheretz et al., 2001).
In hospitals with high rates of
MRSA, the use of vancomycin in-
creases, which in turn may increase
the risk for selecting vancomycin-
resistant Enterococci (VRE) (Her-
waldt, 1999). The nosocomial
transmission of VRE may occur in-
directly via the hands of hospital
staff (Low et al., 2001).
Pseudomonas aeruginosa, Kleb-
siella and Escherichia coli that har-
bor chromosomal or plasmid me-
diated beta-lactamase enzymes are
the major resistant Gram negative
pathogens. These organisms can be
normal flora of human gastrointes-
tinal tract. They can cause a variety
of clinical syndromes including uri-
nary tract infection, pneumonia, and
bacteraemia. These organisms are
an infection control concern because
they might transfer resistance to
73

more common pathogenic organ-
isms, which would then express
multi-drug resistance (Weinstein,
1998).
This work aimed to describe the
pattern of colonization with antimi-
crobial resistant bacteria; MRSA,
VRE, and Extended Spectrum -
lactamases producing Gram nega-
tive bacilli (ESBLs) among HCWs
with direct contact with patients in
ICUs. This screening study allows
detection of any HCW reservoir,
which is an important part of infec-
tion control program aiming at care-
ful eradication and follow-up to
prevent subsequent transmission of
multi-drug resistant bacteria to pa-
tients.


This prospective cohort study was
conducted on 50 apparently healthy
HCWs who have direct patient con-
tact in ICUs of Internal medicine
(17 HCWs), Chest (13 HCWs),
Neonatology (11 HCWs) and Burn
(9 HCWs), at Ain Shams University
Hospitals (ASUH). They were 6%
physicians, 74% nurses and 20%
cleaners. Eight percent of them were
males and 92% were females. Their
ages ranged from 16 to 50 years
(mean 26.94 years) and their dura-
tion of work in ICU ranged from 2
months to 18 years (mean 5.03
years).
All HCWs were subjected to: Inter-
viewing questionnaire which in-
cluded: personal data, occupational
experience, antimicrobial intake and
infection control training.
Nasal, hand, and rectal swabs were
collected from all the participating
HCWs.
The materials used were:
(1) Antibiotic sensitivity discs:
They were supplied from Oxoid
(CLSI, 2005).
A. Discs used for detection of
MRSA: Cefoxitin (FOX): (30 g per
disc). B. Discs used for detection of
ESBLs producing Gram negative
bacilli: Cefotaxime (CTX), Ceftazi-
dime (CTZ), Ceftriaxone (CRO)
and Aztreonam (ATM): (30 g per
each disc). C. For detection of VRE:
Vancomycin (VA): 500 mg/vial.
(2) Culture media (Collee and Marr,
1996): A. Media for isolation and
identification of Staphylococci:
Blood agar and mannitol salt agar
media. B. Media for isolation and
identification of Gram negative ba-
cilli: Mac-Conkey's agar medium.
C. Media for isolation and identifi-
cation of Enterococci: Aesculin bile
agar medium. D. Media and rea-
gents used for biochemical identifi-
cation: Sugar fermentation test me-
dia, Peptone water medium, Xylol
and Erlich's solutions, Glucose
phosphate medium, Methyl red in-
dicator solution, 40% KOH, 5%
solution of -naphthol in absolute
ethanol, Citrate agar medium,
Urease agar medium, Ferrous chlo-
ride gelatin medium, H
2
O
2
and Hu-
man plasma. E. Media for antibiotic
sensitivity test: Mueller-Hinton agar
medium.
74

For isolation of Staphylococci:
nasal (anterior nares) and hand
swabs were collected from each
HCW by sterile disposable swabs
and inoculated onto blood and man-
nitol salt agar plates.
For isolation of Enterococci and
Gram negative bacilli: rectal swabs
were collected from accepting par-
ticipants (42 HCWs) and inoculated
onto Aesculin bile agar and Mac-
Conkey's agar plates respectively.
The plates were incubated aerobi-
cally at 37C for 24 hrs (up to 48
hrs for mannitol salt agar plates).
Bacteriological examination of the
growth:
A. S. aureus isolates (CLSI,
2005):
Identification: Colonial morphology
on mannitol salt agar (1 mm yellow
colonies surrounded by yellow me-
dium) and blood agar (beta haemo-
lytic colonies). Gram stained
smears, Catalase positive test, Coa-
gulase (tube) positive test.
Detection of susceptibility of iso-
lates to methicillin: This was done
by Cefoxitin disc diffusion test.
B. Enterococcus isolates:
Identification: Gram stained
smears: Gram positive cocci ar-
ranged in pairs, short chains, or
singly, blackening around the colo-
nies due to hydrolysis of aesculin in
the presence of 40% bile in the me-
dium, Catalase negative test.
Detection of susceptibility of iso-
lates to vancomycin: It was done by
Vancomycin screen agar method
(CDC, 1999; NCCLS, 2000).
C. Gram negative bacilli:
Identification: By biochemical
reactions according to the conven-
tional methods (Collee et al., 1996).
Detection of ESBLs producing
Gram negative bacilli: All isolates
were tested for the production of
ESBLs by Combined disc method
with cefotaxime, ceftazidime, cef-
triaxone, aztreonam (30g per disc
for each drug), alone and in combi-
nation with clavulinic acid (10g)
(NCCLS, 1999, 2002).
Statistical Methodology: Data
were expressed as number and per-
centage and analyzed by SPSS ver-
sion 13. Ranked Spearman correla-
tion test was used.

Results

Nasal carriage rate of S.aureus among the participating HCWs was (15/50;
30%). 66.6% (10/15) of S. aureus isolates were MRSA strains. Nasal carriage
rate of MRSA among the participating HCWs was (10/50; 20%).

75

Table 1: Distribution of bacterial isolates in nasal swabs of 50 HCWs.

Isolated organism No. of isolates %
Gram positive
cocci
CONS 32 64
CONS+MSSA 5 10
CONS+MRSA 10 20
Gram positive
cocci +Gram negative
bacilli
CONS+ Citrobacter koseri 2 4
CONS+ E. coli 1 2
ESBL 0 0
Total no. of swabs 50 100
CONS (coagulase negative staphylococci), MSSA (methicillin sensitive S. aureus)
MRSA (methicillin resistant S. aureus), ESBL (extended spectrum lactamase).

Hand carriage rate of S.aureus in HCWs was 3/50 (6%). 66.6% (2/3) of
S.aureus isolates were MRSA strains. Hand carriage rate of MRSA in HCWs
was 2/50 (4%).

Table 2: Distribution of bacterial isolates in hand swabs of 50 HCWs.

Isolated organism No. of isolates %
Gram
positive
cocci
CONS 43 86
CONS+MSSA 1 2
CONS+MRSA 2 4
Gram
positive
cocci +Gram
negative
bacilli

CONS+ Citrobacter koseri (ESBL) 1 2
CONS+ E. coli 1 2
CONS+ Klebsiella pneumoniae 1 2
CONS+ Pseudomonas aeruginosa 1 2
Total no. of swabs 50 100

Table 3: Correlation between carriage of antimicrobial resistant bacteria and
infection control training, frequent antimicrobial intake & work duration.

Infection control training Bacterial strains
Sensitive Resistant
No. % No. %
Total

No. %
No 5 55.56 4 44.44 9 100.00
Yes 24 58.54 17 41.46 41 100.00
Total 29 58.00 21 42.00 50 100.00
Frequent antibiotic intake
No 27 60.00 18 40.00 45 100.00
Yes 2 40.00 3 60.00 5 100.00
Total 29 58.00 21 42.00 50 100.00
Duration of work

<= 10 years 27 62.79 16 37.21 43 100.00
> 10 years 2 28.57 5 71.43 7 100.00
Total 29 58.00 21 42.00 50 100.00
76

The number of S. aureus isolates
was 36% (15+3 /50). S. aureus was
isolated from 17/50 HCWs (34%),
of those, 14/50 (28%) were nasal
carriers, 2/50 (4%) were hand carri-
ers and 1/50 (2%) carried S. aureus
(MRSA) at both sites. The overall
MRSA carriage rate among HCWs
was 11/50 (22%). One HCW carried
MRSA at both nasal and hand
swabs. The highest rate of MRSA
carriage was in burn ICU 3/9
(33.3%) followed by chest ICU
(4/13; 30.8%), medical ICU (3/17;
17.6%) and Neonatal ICU 1/11
(9.1%).
Gram negative bacilli were isolated
from hand swabs of 4/50 (8%) of
HCWs. Hand carriage rate of ESBLs
was 1/50 (2%). Hand contamination
with pathogenic bacteria (gram -ve
bacilli and S.aureus) were in 7/50
(14%) of HCWs.
Rectal swab was done in 42
HCWs. 38/42 (90.5%) of isolated
Enterococcus species were vanco-
mycin-sensitive Enterococci (VSE),
while 4/42 (9.5%) were vancomy-
cin-resistant Enterococci (VRE).
Highest rate of VRE carriage was in
Neonatal ICU 2/9 (22.2%) followed
by Medical ICU 2/17 (11.8%). No
VRE carriers were detected in Chest
or Burn ICU.
Of Enterobacteriacea isolated
from rectal swabs, 32 were E. coli;
six of them were ESBLs producing
6/32 (18.75%) and nine were Kleb-
siella pneumoniae; three of them
were ESBLs producing 3/9 (33.3%).
Three isolates were Citrobacter ko-
seri without any detected ESBLs
production (N.B: Two HCWs found
colonized by two different species).
ESBLs carriage rate among rectally
swabbed HCWs was 9/42 (21.43%).
The overall ESBLs carriage rate
among HCWs was 10/42 (23.8%) [9
rectal & 1 hand swabs]. The highest
rate of colonization was in Burn
(3/7; 42.9%) followed by Chest
(3/9; 33.3%), Neonatal (2/9; 22.2%)
and Medical ICU (2/17; 11.8%). In
the combined disc method, Aztreo-
nam was the most sensitive (90%)
in detecting ESBLs followed by ce-
fotaxime (80%). The sensitivity of
ceftazidime and ceftriaxone was
60% each.
The questionnaire revealed that 41
(82%) of the participating HCWs
had lectures on infection control
[six (14.6%) before work 25
(61%) after work 10 (24.4%) be-
fore and after work]. As regard an-
timicrobials intake, only (5/50;
10%) of the HCWs used to take an-
tibiotic without doctor prescription
while (45/50; 90%) took it only
when indicated. There was insigni-
ficant association between infection
control training (P = 0.870), fre-
quent antimicrobial intake (P =
0.390) or duration of work (P =
0.089) and carriage of antimicrobial
resistant bacteria.

Discussion

Endemic antimicrobial resistance
is common in acute care facilities in
developing countries (WHO, 2001).
The continuous use of antimicrobial
agents increases selection pressure
77

favoring the emergence, multiplica-
tion, and spread of resistant strains.
In health care settings, the spread of
resistant organisms is facilitated
when hand washing, barrier precau-
tions, and equipment cleaning are
not optimal. HCWs are contami-
nated during patient care (hands,
clothes, nose and throat) and be-
come transient or permanent carri-
ers, subsequently transmitting bacte-
ria to other patients by direct contact
during care (WHO, 2002).
Persistent colonization of hospital
workers

may be a key factor contri-
buting to an epidemic; these

workers
are in close contact with patients
and they are in and

out of the hos-
pital every day (Smith et al., 2004).
S. aureus remains one of the most
frequently encountered nosocomial
pathogens. Human carriers are pre-
dominantly colonized by S. aureus
in the nares and may contaminate
their hands (Blok et al., 2003).
MRSA has become the predominant
form of clinically significant S. au-
reus within ICUs (Warren et al.,
2004).
In this study, nasal carriage rate of
S. aureus among the participating
HCWs was 30% & 66.6% of these
isolates were MRSA strains. These
results concerning MRSA carriage
are nearly similar to those reported
by Opal et al. (1990) who found
high rates (56%) of staphylococcal
colonization among nurses, 65% of
which were methicillin-resistant.
Other studies reported similar
rates of nasal carriage of S. aureus
but much lower rates of MRSA car-
riage. Badawi et al. (2001)

reported
rates of 26% and 5% of nasal S. au-
reus and MRSA carriages, respec-
tively, among HCWs in Urosurgery,
Surgery Departments and ICU of
Theodor Bilharz Medical Research
Institute in Egypt. Kampf et al.
(2003) reported rates of 33.8% and
0.7% of S. aureus and MRSA car-
riages, respectively, among HCWs
from three hospitals in Germany.
Hand carriage rate of S.aureus
among the participating HCWs in
this study was 6%. Hand carriage
rate of MRSA among the participat-
ing HCWs was 4%.
In previous studies, it was found
that the rate of S.aureus carriage on
hands of staff of the Department for
Thoracic and Cardiovascular Sur-
gery at the University Hospital of
Uppsala, Sweeden was 8.8% and all
isolates were methicillin susceptible
(Tammelin et al., 2003). The hand
carriage rate of S. aureus among
medical personnel in a Medical
Centre, New York City, was 10%;
one hand isolate was methicillin
resistant (Cespedes et al., 2002).
The evidenced hand bacterial con-
tamination pointed out in this study
emphasizes the fact that HCWs'
hands increase the risk of bacterial
transmission that is sometimes re-
sistant to antimicrobial agents. This
is a two-way hazard that could be
noxious to both patients and HCWs,
and which depends on the nature
and frequency of contact with infec-
tious materials, inoculum and preva-
lence of susceptible patients. Sever-
al obstacles to appropriate hand hy-
78

giene were reported such as incon-
veniently located or insufficient
numbers of sinks, limited available
resources for hand hygiene and pro-
tective barriers and the belief of
HCWs that glove use obviates need
for hand hygiene (Nijssen et al.,
2003).
The high prevalence of MRSA
carriage among HCWs in the
present study can be attributed to
high prevalence of MRSA among
patients which increases the expo-
sure potential among the participat-
ing HCWs. Rates of MRSA isola-
tion from patients ranging from
24% to 76% had been reported in
different studies in Egypt (El Hef-
nawy and El Wakil, 2000; El Kholy
et al., 2003). Suboptimal infection
control practices have a strong in-
fluence on the possibility of trans-
mission between patients and
HCWs. HCWs compliance with
hand hygiene and use of protective
barrier equipments may be incom-
plete, increasing rate of MRSA car-
riage (Boyce et al., 2004).
This study showed that the burn
ICU had highest percentage of
MRSA carriage among HCWs fol-
lowed by chest ICU. These results
were nearly similar to those re-
ported by Preetha et al. (1999) who
found that among the 34 hospital
personnel screened in the burns unit
of a tertiary care hospital in India,
76% were found to be carriers of S.
aureus. Of these, 50% were carriers
of MRSA. Cesur and Cokca (2004)
found that the highest rate of MRSA
carriage was in chest diseases de-
partment (25%) in Ankara Universi-
ty Hospital.
The burn unit is a particularly fer-
tile environment for MRSA because
of open wounds, frequent dressing
changes requiring handling by mul-
tiple HCWs, use of intraluminal
devices, and immuno-compromised
state of burn patients. Several mod-
es of transmission exist, including
transient colonization of hospital
staff and contact with heavily con-
taminated fomites and environmen-
tal surfaces around infected patients
(Cook, 1998).

In the current study, VRE carriage
rate among HCWs was 9.5%. In
contrast, Carmeli et al. (1998) found
that of the 55 stool specimens
processed, none contained VRE. The
discrepancy in results could be at-
tributed to different methodology;
vancomycin screening agar versus
colistin and nalidixic acid agar with
vancomycin 10 mg/ml. Because the
prevalence of VRE is rising, unsus-
pected exposure among HCWs in
both high risk units and general
wards is probably increasing. Tran-
sient hand carriage is certainly poss-
ible, but these organisms may even-
tually reach the gastrointestinal
tract, allowing a short or prolonged
colonization (Baran et al., 2002).
This study showed that the neo-
natal ICU had highest percentage of
VRE carriage among HCWs. Vari-
ous factors influence neonates' co-
lonization including method of deli-
very, invasive procedures such as
enteral feeding using nasogastric
tubes, hands of HCWs and antibiot-
79

ic use. High risk babies are more
likely to have antimicrobial resistant
microorganisms as the predominant
flora (Berthelot et al., 2001). High
density stool colonization is asso-
ciated with contamination of envi-
ronment with VRE when patients
are incontinent of stool (Donskey et
al., 2000). The hands of HCWs can
be contaminated during changing
the diapers or after touching conta-
minated environmental surfaces be-
cause colonized patients are often
unrecognized and cleaning of the
hospital environment may be inade-
quate (Ott and Wirick, 2008).

These
organisms are increasingly isolated
from non hospitalized individuals,
as they can be transmitted from the
mothers to their neonates during
nursing and HCWs acquire them by
subsequent contact with the neo-
nates (Baran et al., 2002).

In this study, among 42 rectally
swabbed HCWs, ESBL carriage rate
was 21.43% and overall ESBL car-
riage rate was 23.8%. This agreed
with Chambers et al. (1987) who
isolated several multiresistant spe-
cies of Klebsiella, Citrobacter and
Pseudomonas from ICU nurses. An-
tibiotic resistance in isolates of En-
terobacteriaceae is emerging in
many parts of the world. The in-
creased use of third generation ce-
phalosporins has led to changes in
patterns of bacterial -lactamases
and the emergence of ESBLs (Kaye
et al., 2000).
Omar et al.

(2001) reported that
the prevalence of ESBLs -producing
isolates among Enterobacteriaceae
members was 25.8% with the high-
est frequency found in K. pneumo-
niae. This reflects the rapid disse-
mination of ESBLs encoding plas-
mids with subsequent increase in
prevalence by time (Parasakthi et
al., 2000). Teaching hospitals have
been shown to have a higher preva-
lence of ESBLs mediated resistance
(Kaye et al., 2000). In contrast,
Carmeli et al.

(1998) found one
stool culture (1.8%) yielded ceftazi-
dime-resistant Citrobacter freundii
on MacConkey's agar supplemented
with ceftazidime. The higher results
in the present study can be ex-
plained by utilization of different
screening technique which was
combined disk method. This was
more reliable for detection of ESBLs
(NCCLS, 2002). ESBLs were in-
itially reported mainly in Klebsiella
pneumoniae

and to a lesser extent in
E. coli. A wide range of

organisms
e.g. Enterobacter, Serratia, Proteus,
Morganella, Salmonella, pseudo-
monas

and Citrobacter spp. have
been reported worldwide to harbor

such a resistance mechanism (De
Gheldre et al., 2003).
In the current work, the burn
ICU had highest percentage of
ESBL carriage among HCWs fol-
lowed by chest ICU. Again the burn
and chest ICU workers have the
highest carriage rate of ESBLs as
well as MRSA because the risk fac-
tors for acquiring antibiotic resistant
organisms are common for all anti-
biotic resistant pathogens. There
was no statistical significant differ-
ence between HCWs who received
80

infection control lectures and those
who didn't as regard the carriage
rate of resistant strains. Initial edu-
cational programs need to be fol-
lowed by reinforcement and infec-
tion control staff should evaluate
intrahospital compliance and identi-
fy lapses for further measures and
education. There was no statistical
significant difference between those
who received antibiotics frequently
and those who didn't. Also there
was no statistical significant differ-
ence with the duration of work
which can be explained by the small
sample size. The increasing carriage
rate of antimicrobial resistant bacte-
ria by HCWs is primarily related to
cross transmission from patients to
HCWs and vice versa. If medical
personnel are identified as vectors
of transmission, their isolates are
likely to reflect the antibiotic sus-
ceptibility profile prevalent in the
hospital setting.
Rahban et al. (2003) found no
association between gender, age, or
years of service and nasal carriage
of MRSA. In contrast, Eveillard et
al. (2004) found higher prevalence
of MRSA carriage in health care
providers when their length of ser-
vice exceeded 5 years. This obser-
vation could reflect a longer expo-
sure to patients colonized or in-
fected with MRSA.

Conclusion

The rate of antimicrobial resis-
tance among nosocomial pathogens
is steadily increasing. HCWs are at
high risk of acquisition and coloni-
zation with antimicrobial resistant
bacteria. Transient hand coloniza-
tion is the primary mean of cross
transmission. Judicious use of anti-
biotics and implementation of infec-
tion control measures are necessary
to limit prevalence of such emerg-
ing resistance.

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85



EIMERIA KHOBAHENSIS SP. N. (APICOMPLEXA: EIMERIIDAE)
FROM GUINEA FOWL, NUMIDA MELEAGRIS IN SAUDI ARABIA
By

MOHAMED S. ALYOUSIF* AND YASER R. AL-SHAWA
Department of Zoology, College of Science, King Saud University, P.O.
Box 26213, Riyadh 11486 Saudi Arabia. Fax 4678514 E-mail: myou-
sif@ksu.edu.sa

Abstract

Eimeria khobahensis sp.n. is described from the intestine of the guinea
fowl, Numida meleagris (Galliformes: Phasianidae) collected at and around
Khobah area near the Southern borders of Saudi Arabia. Sporulated oocysts are
ellipsoid, 2519.4 (23.3-27.6 17.8-20.6)m, with a smooth, greenish-yellow
bi-layered wall 1.9 (1.5-2.4)m. Micropyle and polar granule are present. An
oocyst residuum is present, consisting of several globules that are irregular in
shapes and sizes. Sporocysts are ellipsoid, 13.5 7.5 (11.9-14.2 6.7-8.8) m,
with a smooth single-layered wall and a prominent Stieda body, but no appar-
ent substieda body. The sporocyst residuum is present, composed of numerous
small granules. Sporozoites are comma-shaped, 10 3 (8-11 2.6-3.8) m,
each contains two refractile bodies.
Key words: Eimeria khobahensis sp. n. Guinea fowl, Numida meleagris, Saudi
Arabia.
Correspondence: Dr Mohamed S. Alyousif, Fax 4678514. email:myousif@ksu.edu.sa
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Eimeria infection is common as
parasites of Saudi Arabian birds
(Amoudi, 1987; Alyousif, and Al-
Shawa, 1998, 1999). Although sev-
eral species of Eimeria have been
described from members of the fam-
ily Phasianidae Pellrdy, 1974, but
only three species were reported
from genus Numida, Eimeria go-
rakhpuri Bhatia and Pande,1967; E.
grenieri Yvor and Aycardi, 1967
and E. numida Pellrdy, 1962. Dur-
ing the present survey of the parasit-
ic fauna of Saudi Arabian birds,
family Phasianidae, 20 Guinea fowl,
Numida meleagris were found to be
infected with one new species of
Eimeria. The present study de-
scribes the oocysts of this new spe-
cies of Eimeria and compares it
morphologically with previously
described species of Eimeria infect-
ing the same host species.


86


Between November, 2008 and
July, 2009, 100 adult guinea fowls,
were randomly chosen out of ap-
proximately 300 birds brought to
the bird market. Birds were caged
separately and their feces were col-
lected individually and mixed in 2.5
% (w/v) aqueous potassium dich-
romate solution to retard bacterial
action. Unsporulated oocysts were
allowed to develop for 6 days at
room temperature (26 2C) in Pe-
tri dishes containing a thin layer of
potassium dichromate and examined
daily. Sporulated oocysts were con-
centrated by flotation with sugar
solution (sp. gr. 1.30). 50 sporulated
oocysts and 50 sporocysts were ex-
amined under oil immersion with a
Zeiss Universal photomicroscope III
equipped with a 100x objective
lenses. Measurements were made
using a calibrated ocular micro-
meter. The number of layers of the
oocyst wall and its thickness and
detailed structure of the sporocysts
were determined by crushing the
sporulated oocysts under cover slip.
All measurements are in microme-
ters (m) with the mean followed
by the range in parentheses. Two
infected bird was sacrificed and tis-
sue samples of liver, gall bladder
small and large intestine were fixed
in 10% neutral buffered formalin
and processed for light microscopy
using standard histological method.
Paraffin sections were stained with
haematoxylin and eosin (H&E) and
examined for the presence of endo-
genous coccidian stages.

Results

During a survey of the parasitic
fauna of Saudi Arabian birds. Total
of 100 adult Guinea fowl, Numida
meleagris were examined for cocci-
dian infection. Twenty of them were
infected with new species of Eime-
ria. The present study describes the
morphological characteristic of this
new species of Eimeria.
Eimeria khobahensis (Figs. 1-5):
Description: Oocysts are ellipsoid
(n = 50), 2519.4 (23.3-27.6 17.8-
20.6) m, shape index (L/W) 1.29
(1.13-1.39) m. Oocyst surface
smooth, oocyst wall is greenish-
yellow, 1.9 (1.5-2.4) thick and bi-
layered by light microscopy. The
micropyle is present, 3.2 (2.5-3.7)
wide. Oocyst residuum present con-
sists of several globules, irregular in
shapes and sizes. One or more polar
granules are present. Sporocysts are
ellipsoid, 13.57.5 (11.9-14.26.7-
8.8) m, length/ width ratios, 1.8
(1.68-2.8) with smooth, single-
layered wall and a prominent Stieda
body; but no apparent substieda
body. Sporocyst residuum is pre-
sent, composed of many scattered
small granules. Sporozoites are
comma-shaped, 103 (8-112.6-
3.8), arranged head-to-tail within
sporocysts; each sporozoite contains
a large, sub-spherical, posterior re-
fractile body, 4.54.0 (3.7-4.93.5-
4.6) and a small, spherical anterior
refractile body 1.9 (1.6-2.7).

87

Table 1: Comparative data of Eimeria species described from Guinea fowls
Eimeria species Oocyst Sporocyst
shape mean size
(m)
micr-
opyle
resid-
uum
polare
granule
Mean size
(m)
E. gorakhpuri ellipsoid or
ovoid
20x14 - - + 10x6
E. grenieri ovoid 24x16 + - + 11x9
E. numidae ellipsoid 19x15 - - - No data
E. khobahensis sp.n. ellipsoid 25.0 x19.4 + + + 13.5 x 7.5

Taxonomic summary:

Type host: Numida meleagris (Ga-
lliformes: Phasianidae).
Type locality: Riyadh, Khobah,
Southern region, Saudi Arabia.
Prevalence: 20/100 (20%) Guinea
fowls passing oocysts.
Site of infection: Histological ex-
amination revealed the presence of
endogenous stages developed within
the epithelial cells of the small in-
testine. Sporulation time: Majority
of oocysts examined had completed
sporulation within 48 h. at 262
o
C.
Etymology: Species name khoba-
hensis is derived from the host habi-
tat name.
Type specimens: Oocysts in 10%
formalin and a photo-type are depo-
sited in the Parasitological Collec-
tion, Zoology Department, College
of Science, King Saud University,
Riyadh both as KSUC, 485.

Discussion

The present information on eime-
rian parasites of the genus Numida
is limited to reports of Eimeria go-
rakhpuri (Bhatia and Pande, 1967);
E. grenieri (Yvor and Aycardi,
1967) and E. numidae (Pellrdy,
1962). Eimeria khobahensis sp. n.
was distinguished from E. gorakh-
puri and E. numidae in having mi-
cropyle and oocyst residuum and in
having much larger oocysts. It dif-
fers from E. grenieri in being wider,
having oocyst residuum, possessing
longer sporocysts and in having
sporocyst residuum (Tab. 1). E.
khobahensis differs from all other
hitherto described eimerian species
from members of the family Phasia-
nidae in possessing oocyst resi-
duum.
The description of the new species
of Eimeria as a distinct form is
based primarily on structural fea-
tures, sporulation time, and geo-

88

graphic distribution. By comparing
E. khobahensis to other eimerian
species previously described from
guinea fowl, it is clear that E. kho-
bahensis is a distinct form which
leads the authors to consider it a
new species.

Conclusion

The present study should in new
species, Eimeria Khobahensis in
Guinea Fowl in Saudi Arabia.
This indicates that more study is a
must to identify the Eimeria fauna
in Saudi Arabia.

Acknowledgements

This study was supported by the
College of Science- Research Cen-
ter, Project No (Zoo/2009/41). The
authors would like to thank Mr. Ali
Al-Sawadi, Microtechnique Unit,
Department of Zoology, for kind
preparing histological specimens.

References
Alyousif, M.S.; Al-Shawa, Y.R.,
1998: Two new coccidia (Apicom-
plexa: Eimeriidae) from the green
peacock (Pavo muticus) from Saudi
Arabia. Parasitol. Int., 47:301-6.
1999: Coccidian parasites of the
green peacock (Pavo muticus L.) in
Saudi Arabia. Saudi J. Biol. Sci.,
6:111-7.
Amoudy, M.A., 1987: Eimeria ta-
hamensis n. sp. (Apicomplexa: Ei-
meriidae) from the Arabian quail
(Coturnix delegorguei arabica). J.
Protozool., 34: 455-6.
Bhatia, B.B.; Pand, B.P., 1967:
New eimerian species from guinea
fowl. Acta Vet. Acad. Sci. Hung.,
17:359-67.
Pellrdy, L., 1962: Agyongytyuk
coccidiosisa Eimeria numidae n.sp.
Magy. Allatorv. Lap., 17:18-9.
Pellrdy, L., 1974: Coccidia and
coccidiosis, 2
nd
ed. Verlag Paul Pa-
rey, Berline.
Yvor, P.; Aycardi, J., 1967: Une
nouvelle coccidie Eimeria grenieri
n. sp. (Protozoa: Eimeriidae) para-
site de la pintade Numida meleagris.
C. R. Acad. Sci. (Paris), 264:73-6.

Explanation of Figures

Photomicrographs of Eimeria khobahensis sp. n. from Numida meleagris. Scale bars = 10 m.
Fig. 1: Sporulated oocyst showing Stieda body (arrow) and refractile body (arrowhead).
Fig. 2: Sporulated oocyst showing micropyle (arrow) and oocyst residuum (arrowhead).
Fig. 3: Segmented meront with mature merozoites (arrow).
Fig. 4: Mature microgamont*.
Fig. 5: Mature macrogamont (arrow).


89

J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No. 7


89


J. Egypt. Soc. Parasitol., 40 (1), 2010: 89 92

HUMAN INFECTION WITH BERTIELLA STUDERI (CESTODE:
ANOPLOCEPHALIDAE) IN AN EGYPTIAN WORKER RETURNING
BACK FROM SAUDI ARABIA
By
EBTESAM M. Al-MATHAL
1
, NAGLA MOSTAFA K. SALEH
2

AND TOSSON A. MORSY
3

Department of Zoology, Girls College of Science, King Faisal Universi-
ty
1
, Dammam, Saudi Arabia, Dhahran 31311, P.O. Box: 10185, Email:
mathalem@hotmail.com, Department of Zoology, Faculty of Science,
South Valley University
2
, Department of Parasitology, Faculty of Medi-
cine, Ain Shams University
3
, Cairo 11566, Egypt

Abstract

Perhaps this is the first case of bertiellosis studeri record in Egyptian worker
returning back from Saudi Arabia. The patient was resistant to Niclosamide but
successfully treated with Commiphora molmol extract.
Key words: Bertiella studeri, Egyptian worker, Saudi Arabia, Commiphora
molmol.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Bertiella species is a frequent pa-
rasite in animals, particularly in
nonhuman primates. The definitive
hosts are monkeys including Maca-
ca radiate, M. syrichta syrichta, M.
syrichta fasviolaris, Cercopithecus
aethiops pygerythrus, C. nititans
schmidti and Hylobates hoolock
(Stunkard, 1940). Also, infection in
dogs was reported in Philippines
(Africa and Garcia (1935). Crockett
(1985) reported that Mona monkey
(Cercopithecus mona mona) is the
reservoir host of B. studeri in Nige-
ria. Gillespie et al. (2004) reported
gastrointestinal parasite of the gue-
nons of western Uganda.
Denegri and Perez-Serrano, 1997)
gave a review of bertiellosis in man.
Human infection occurs by the ac-
cidental ingestion of the interme-
diate host, an Acarus containing the
cysticercoid larva of Bertiella stude-
ri or B. mucronata (Achir et al.,
2008). The diagnosis is based on the
morphology of the gravid segments
and the small eggs with pyriform
embryo, characteristic of the Anop-
locephalinae (Galan-Puchades et al.,
2000).
This work aimed to report the first
record of Bertiella studeri (Blan-
chard, 1891), Stiles and Hassall,
1902, and to pay attention to this
small neglected zoonotic cestode.


90

Subject, Materials and Methods

The patient was a male immigrant
worker, 23 years old returning back
from Saudi Arabia in a short visit to
his family in Sharkia Governorate.
He was working in Jizan District on
the Saudi-Yemeni border. Some-
times, he shed gravid segment in
groups of about 12 to 24. He suf-
fered non specific digestive distur-
bances with constipation and/or
mild diarrhea.
Clinically, he was more or less
normal except the digestive distur-
bances. The segments on examina-
tion were gravid with the uterus
filled the entire segment. Stool ex-
amination showed eggs (45-47x49-
51microns) which were more or less
irregular, ovoid, with a pyriform
embryo.
Result

The case was diagnosed bertiello-
sis studeri. The patient did not re-
spond to Niclosamide treatment in
the usual dose. So, he was given
Mirazid
as two tablets on an empty

stomach two hours before breakfast
for six successive days. He stopped
passing segments and Kato thick
smear was negative in follow-up.

Discussion

This may be the first case of ber-
tiellosis studeri in Egypt acquired
from the Saudi-Yemeni border. Ga-
lan-Puchades et al. (2000) stated
that the morphological differences
reported in various earlier descrip-
tions of the species suggest that B.
studeri should be regarded as a "B.
studeri species complex" Improve-
ments are required in the descrip-
tions of new future findings in order
to clarify the specific diagnosis of
human bertiellosis. Evidence sug-
gests that a generalized diagnosis
exclusively based on egg size and
geographical distribution is insuffi-
cient to differentiate B. studeri and
B. mucronata (Meyner, 1895), or
other additional species may be af-
fecting humans.
Shoura and Morsy (1974) in Saudi
Arabia reported B. studeri in an
immigrant Yemeni. Achir et al.
(2008) in Algeria reported Bertiella
sp. in a man originating from Ye-
men. Apart from Yemen and Saudi
Arabia, Galan-Puchades et al.
(1997a) in Spain, reported infection
in a 33-year-old pregnant Spanish
woman probably of African origin
(Kenya) as she spent the six months
preceding this discovery in Kenya.
On the other hand, human cases of
zoonotic bertiellosis have been re-
ported worldwide. Human bertiello-
sis have been reported in Philip-
pines (Africa and Garcia, 1935), in
Malaya (Desowitz et al., 1961), Pa-
raguay (DAlessandro et al., 1963),
Minnesota (Stunkard et al., 1964),
Sri Lanka (Edirisinghe and Cumara-
rajan, 1976), Malaysia (Dissanaike
et al., 1977), Japan (Ando et al.,
1996), Thailand (Bhaibulaya, 1985),
Nigeria (Crockett, 1985), Equatorial
Guinea (Galan-Puchades et al.,
1997b), Vietnam (Xuan et al.,
2003), Mauritania (Bhagwant,
2004) and China (Sun et al., 2006).

91

Panda and Panda (1994) stated
that children were more susceptible
to Bertiella studeri infection than
adult. Bandyopadhyay and Manna
(1987) reviewed the pathogenic and
zoonotic potentiality of Bertiella
studeri. Pettifer (1984) identified
Bertiella studeri among helminth
fauna of the digestive tracts of the
Chacma baboons, Papio ursinus,
from different localities in the
Transvaal.
In the present study, the patient
was resistant to Niclosamide, but
responded well to Mirazid (Tonkol
and Morsy, 2008). Galan-Puchades
et al. (1997a) reported that Meben-
dazole failed to remove the parasite
but Niclosamide was effective. Gal-
lella et al. (2004) reported that hu-
man Bertiella studeri infection was
resistant to Niclosamide. Achir et
al. (2008) found that Niclosamide
was effective in one single dose.

Conclusion

The demonstration of Bertiella
studeri in an Egyptian immigrant
worker who probably acquired the
infection in Saudi Arabia, pave the
way to keep this non-human zoonot-
ic cestode in mind when dealing
gastro-intestinal troubles in both
Egypt and Saudi Arabia.

References

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93


J. Egypt. Soc. Parasitol., 40 (1), 2010: 93 106

EFFECT OF TREATMENT WITH NEEM (AZADIRACHTA INDICA)
COMPARED WITH BAYCOX DRUG ON THE CAECUM OF CHICKEN
EXPERIMENTALLY INFECTED WITH EIMERIA TENELLA
By
FAWZIA H. TOULAH, HAYAT A. ISMEEL AND SAMAR KHAN
Department of Zoology, Faculty of Science for Girl, King Abdulaziz Uni-
versity, Jeddah, Saudi Arabia, Lylak_s@hotmail.com;
dr.Hayat786@hotmail.com; Ftoulah@kau.edu

Abstract

The study evaluated the therapeutic efficacy of Neem herb in chicken expe-
rimentally infected with E. tenella compared to Baycox as a reference anticoc-
cidial drug. 120 broiler chicks were enrolled, randomly divided to 4 groups, (A,
B, C & D) non-infected non-treated (nave control), (B) infected with 10
4
E.
tenella oocysts (infected control), (C) infected and treated with Baycox (7
mg/kg b.w. for 2 days) and (D) infected and treated with Neem leaves water
extract (100 mg/kg b.w. for 9 days). Evaluation was by clinical signs, perfor-
mance data (gain weigh, food consumption oocyst shed/gram feaces (OPG)), in
addition to histopathological changes in all chickens. The results revealed that
chicks of GA had the best performance data compared to GB, GC & GD. In
GC & GD there were a remarkable improvement in the data performance, clin-
ical signs, gross and microscopically cecal lesions compared to GB. The effi-
cacy of Baycox (GC) was shown to be superior to that of Neem (GD) com-
pared to GB but an additive histopathological toxic effect besides those pro-
duced by E. tenella infection could be recorded. In contrast, Neem appeared to
have a remarkable improvement on cecal integrity.
Key words: Saudi Arabia, E. tenella, experimental infection, chicken, anticoc-
cidial drug, Neem, Baycox.
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Poultry industry represents one of
the major sources of animal proteins
for human requirements. Many
poultry farms have been raised in
Saudi Arabia. Outbreaks of some
detrimental and lethal managemen-
tal diseases were recorded all over
the country which could be referred
to the characteristic high tempera-
ture with high humidity climate, in
addition to the lack of hygienic con-
dition suitable for poultry rearing.
Chicken coccidiosis represents one
of the serious diseases of poultry
(McDougald, 2003). It is caused by
obligate intestinal protozoan para-
sites belonging to species of Eime-
94

ria. E. tenella primarily invades and
resides in the lining of the cecum of
exposed chickens. Infective sporo-
zoits enter the cecal mucosa by pe-
netrating villus epithelial cells, re-
sulting in extensive destruction of
the cecal epithelium, hemorrhagic
feces, reduction in body weight gain
and decrease in feed efficiency and
even mortality (Bould et al., 2009)
which lead to serious economic
consequences (Raymond and Oatru-
cua, 2000). The loss due to chickens
coccidian out breaks suggested to be
one 1000 million US$/ year (Augus-
tine, 2000; Biu et al., 2006).
The feed manufactures are using
synthetic feed additive anticoccidial
in the feed to combat infection. The
prolong use of synthetic anticocci-
dials often develop resistant in birds
to these drugs. On the other hand,
commercial coccidiostats led to in-
crease poultry rations cost. Under
these circumstances, it seems that
ultimate economical ways and
means be explored to avoid the use
of expensive coccidiostats (Jeuris-
sen et al., 1996).
New drugs have been developed
and administered on a rotational
basis with existing drugs. However,
this resulted in the increased cost of
poultry products. Furthermore, drug
or antibiotic residue in the poultry
products is potentially annoyance to
consumer. So, the regulation of an-
ticoccidial drugs should be streng-
thened gradually. Many workers
recommended the herbal medication
in poultry intestinal coccidiosis
caused by E. tenella as Artemisia
annua (Allen et al., 1997), sugar-
cane extract (El-Abasy et al., 2003),
Glycyrrhizae (Du and Hu, 2004),
Ganoderma lucidum (Dipeolu et al.,
2004) and Neem (Biu et al., 2006).
Neem (Azadirachta indica) widely
distributed in the western region in
Saudi Arabia and other countries as
Nigeria, Sudan and Borma (Lus-
combe and Taha, 1974). Khalifa et
al. (1998) and Toulah (2000) re-
ported a therapeutic efficacy of
dried Neem leaves in rabbits in-
fected with E. stieda in a dose 100
mg/kg without toxic effects. Tipu et
al. (2002) in study between salino-
mycin sodium and Neem fruit as
feed additive anticoccidials in broi-
lers reported that Neem fruit 150
gm/50kg had excellent performance
in terms of increasing food con-
sumption, gain weight and lowering
of oocyst out put.
Baycox (Toltrazuril) is a common
anticoccidial product and consi-
dered as reference drug in control-
ling coccidial species of chickens
(Mehlhorn et al., 1984). Laczay et
al. (1995), Lakkundi et al. (2002)
and El-Banna et al. (2005) reported
that Toltrazuril completely elimi-
nated coccidial lesions & minimized
oocyst shedding. The performance
data, lesion scores, and oocyst
counts showed that a 2-day treat-
ment with Toltrazuril successfully
controlled coccidiosis without re-
lapse of infection. They added that
Toltrazuril were used for supple-
mental control with in-feed anticoc-
cidials or as a primary anticoccidial
with non-medicated feed.
95

The purpose of the present study
was planned to asses the anticoc-
cidial efficacy of Neem green leaves
water extract compared with Baycox
as a reference drug on experimental
coccidiosis in broiler chickens.


Baycox (toltrazuril solution
(2.5%) was obtained from Bayer
(leverkusen, Germany). It was ad-
ministered in a dose 7mg/kg B.w
(0.3ml) for two successive days
(11
th
& 12
th
day post-infection=PI).
Neem fruit (Azadirachta mdica):
Fresh leaves were collected from
growing fields in Jeddah City. The
aqueous extract of the green leaves
was prepared through pouring of
100 ml of distilled water on 2gm of
leaves and grinding in electric mix-
er. The dose was adjusted at 100
mg/kg w.B (5 ml) and given for 9
successive days (11
th
till 19
th
day PI)
according to Toulah (2000).
The challenge oocyst of a pure
strain of E. tenella was allowed to
sporulate in potassium dicromate
solution (2.5%) at incubation with
28C supplied with glass of water.
The sporulated oocyst were cleared
and counted per 1.0 ml of the solu-
tion by McMaster technique (Coles,
1967). The sporulated oocysts were
used for oral induction of experi-
mental infection directly into the
chicken' crop by means of a plastic
tube attached to a calibrated syringe
in a dose of 1x10
4
oocysts of 21-day
old chickens (Saif et al., 2003).
A total of 120, 2 weeks old Arbor
Acr broiler chicks were purchased
from a private poultry farm (Alfakih
farms), where the chicks were
reared in a well-cleaned shed under
standard hygienic conditions. Vac-
cination was carried out against
New castle disease and periodical
fecal examinations were done. The
birds were weighted, randomly di-
vided into 4 main groups (30/group)
and allocated in (4) metallic cages
each with 3 floors supplemented
with movable beds for easily collec-
tion of fecal droppings. Each pen
was equipped with a feeder and wa-
ter where anticcocidial free com-
mercial broiled chicken feed and tap
water were provided for consump-
tion ad libitum. Standard hygienic
measures were applied. Four chick-
en groups were divided as: GA:
Non-infected non treated group
(nave control). GB: Infected non
treated (infected control). GC: In-
fected treated with oral dose of 7
mg/kg B.w for 2 successive day of
Baycox. GD: Infected and treated
with oral dose of 100 mg/kg B.w. of
Neem extract for 9 successive days
after blood appearance in bird feces
on 11
th
day.
Evaluation of the anticcocidial
efficacy of both Neem and Baycox
was based on research parameters
which included: A- Body weight of
birds was recorded at the beginning
(day 0) and at the end of the expe-
riment and weight gaining percent
was calculated (19 days PI). B-
Food consumption by determination
of offered and residual food amount/
96

week/ group. *Average food con-
sumption/ each group =offered food
amount- residual food. *Average
food consumption/ bird=
N
group n/ consumptio food Average

N = 30 birds/group. C- Mortality
rate and clinical signs after parasite
inoculation (morphological appear-
ance of bird listlessness, appetite,
loss of pigmentation, bloody diarr-
hea, and congested cloaca.
Fresh fecal dropping were col-
lected on 5
th
, 7
th
& 9
th
day PT to
find out mean number of oocysts
per gram faeces (OPG) for each
group (B, C and D) using McMaster
chamber (Coles, 1967). Dosage ef-
ficacy (percent of oocyst reduction)
was carried out for each drug (Javed
et al., 1994).
Dosage efficacy =
1
2 1
n
100 x n n

Where: n
1
= oocyst/gram of infected
non treated, n
2
= oocyst/gm of in-
fected and treated with either Neem
or Baycox.
Three birds each group were se-
quentially sacrificed for evaluating
patho-morphological changes at 5
th
,
7
th
& 9
th
days post-treatment (PT)
corresponding to 15
th
, 17
th
&19
th

days post infection, fixed pieces of
caecum were preserved in neutral
buffered 10% formalin for 24 hours.
The pieces were processed by paraf-
fin embedding technique, sectioned
and stained with haemtoxylin and
eosin (H & E) and examined micro-
scopically (Bancroft and Stevens,
1977).
Statistical analysis: Data was
analysis by one-way analysis of va-
riance (ANOVA) using the means
(M) and standard error of means
(SEM), with P<0.05 by the SPSS.10
statistical analysis program. The
data were recorded as mean + SEM
(Campbell and Machine, 1993).

Results

The results are shown in tables (1, 2 & 3) and figures (1-16).

Table 1: Effect of Baycox and Neem on weight gain in infected broiler chick-
ens.

Chicken group Weight on day 0 Weight on day 19
th
Weight gain %
A 131+5.56 670 + 11.40 80.44 %
B *176 + 19.13 606 + 28.03 70.95 %
C *228 + 9.69 *756 + 49.85 69.84 %
D 195 + 18.16 *1070 + 64.42 78.69 %

97

Table 2: Effect of Baycox and Neem on food consumption in infected broiler.

Group Offered food/week Residual food/week Avg. food consumption/ bird/week (gm)
A 15050+895.82 3025+351.48 400.83+27.59
B 14800+734.84 3950+628.49 386.67+32.10
C 14800+734.84 4662+924.15 337.92+53.79
D 14800+734.84 4112+552.40 356.25+36.28
Avg. = Average, gm. = gram

Table 3: Effect of Baycox and Neem on number of oocysts/ gram (OPG) feces
after 5, 7 & 9 days PT.

G Dosage
Number of Oocysts per gram of fecees (OPG) Percent of reduce in OPG
5
th
day 7
th
day 9
th
day 5
th
day 7
th
day 9
th
day
B -- 39550 + 2350 21800 + 1400 20350 + 350 --
C 7 m/kg *5350 + 450 *2700 + 300 *1300 + 300 86.47% 87.61% 93.61%
D 100mg/kg *22050 + 3350 *10200 + 1800 *5500 + 1000 44.25% 53.21% 72.97%
* Significant (P < 0.05).

Pathomorphological changes: The
effect of E. tenella in a dose of (10
4
)
on cecum was compared to the
structure of the control group.
Chicken cloaca was edematous and
inflamed, sometimes accompanied
with bleeding. Gross morphological
picture showed that the cecum was
two blind sacs append to the junc-
tion of ileum and colon, looked pale
and edematous at the beginning of
infection, became dark red and con-
gested by the days 17
th
&19
th
PI
(Fig. 1,2) on longitudinal incision,
cecum content was offensive dark
solid residual material with perfuse
mucus. On removing the residual
material the interior of the cecum
seemed sough and granulated with
some hemorrhagic areas especially
at the infection late stages.
Light microscopic of infected con-
trol group (GB) 15 days PI with E.
tenella compared to GA showed
structural changes of all layers of
cecum in form of disarrangement of
muscle layer (M) with many va-
cuoles in between.
Submucosa (Sm) was narrow and
atrophied compared to muscularis
mucosa (mm) which was thick and
hypertrophied (Fig. 3). Mucosa (m),
the favorite layer for parasite inva-
sion and completion of its life cycle,
was the most affected, layer 15 days
PI. There was total loss of columnar
cells covering villi (Vi), complete
destorsion of villious architecture
with goblet cells hyperplasia as well
as massive leukocytic infiltration,
mostly lymphocytes, and presence
of several stages of E. tenella; me-
rozoites, () schizonts () and
trophozoites (*) which moved to the
crypts (C) submucosa and musculo-
sa (Fig. 4), at day 17
th
PI. The spac-
98

es between muscle fibres were nar-
rowed while the submucosa was
still reduced with few connective
tissue elements, blood and lymphat-
ic vessels. Musclaris mucosa was
still very thick and extended. The
mucosal villi showed apical atrophy
and most of its columnar cells
showed cytoplasmic degeneration
with presence of the parasite differ-
ent stages either inside or outside
cells at intervillus spaces accompa-
nied by lymphocytic infiltration
(Figs. 5,6), on day 19
th
, muscle layer
was still thick with decreased inter-
fiberal spaces. Submucosa was ab-
sent and muscularis mucosa was
thinner compared to days 15
th
& 17
th

PI. The villi were more or less nor-
mal although some of their colum-
nar cells lining showed apical atro-
phy and disintegration of the mu-
cosal lining with distortion of the
nuclei positions and thickening of
their membranes. The villus core
showed plenty of different stages of
the parasite. However, they were
less than those of days 15 and 17 of
infection as well as the presence of
lymphotic cells aggregations and
scattered heamorragic foci mainly
of red blood cells (RBCs) (Figs. 7,
8).
Infected and treated with Baycox
(GC) for two successive days in a
dose of 7 mg/kg showed that the
longitudinal muscle layer appeared
within normal with no apparent
changes were detected in the sub-
mucosa (Fig. 9), the intercellular
spaces between the columnar cells
were destroyed with some villous
goblet cells hyperplasia, and haemo-
lysis of red blood cells (RBCs) with
lymphocytic infiltration in between.
Muscularis mucosa intermingled
with the villus core of the mucosa 5
days PT with Boycox (Fig. 10). Af-
ter 7 days of treatment by Boycox;
increased inter-digetation between
the submucosa and muscularis mu-
cosa was apparent. The villi were
closely packed with disappearance
of most of parasitic stages with mild
cytoplasmic degerative changes of
the columnar cells of the mucosa
and the muscularis mucosa layers
(Fig. 11). 9 days PT with Boycox
submucosa still showed interdigita-
tion with fibrotic changes while the
mucosa showed apical degeneration
changes in most of villi.
Infected and treated with Neem
(GD): On examining the transverse
sections of chicken cecum infected
with E. tenella and treated with wa-
ter extract of fresh Neem extract in
a dose of (100 mg/kg) for 5 succes-
sive days, musculosa was of normal
thickness and structure while sub-
mucosa showed less fibrotic chan-
ges compared to infected G. Mucosa
looked healthy in some areas; but
some villi showed normal appear-
ance with presence of sporadic
damage in other parts (Fig.13).
Some stages of E. tenella in inter
villus area, and crypts were seen
(Fig. 14). After 7-9 days PT with
Neem extract muscle layer almost
normal with some spaces between
muscle fibres, submucosa showed
clear structure and contents. Mucosa
was more or less normal but with
99

some sporadic degerative changes,
most of mucosa villi showed normal
columnar cells. There were still
lymphocytic and plasma cell infil-
tration in between villi (Fig. 15)
with presence of some stages (Fig.
16) parasite.

Discussion

Coccidians are obligatory intracel-
lular parasites infecting nearly all
vertebrates. Among Coccidia, the
genus Eimeria shows a wide distri-
bution in host species of wild ani-
mals (Abdel-Wasae and El Garhy,
2009).
Outbreaks of coccidiosis are re-
ported from various parts of the
country despite the usage of time
tested anticoccidial drugs in the
poultry feed. Besides, new Eimeria
species are emerging in Saudi Ara-
bia (Alyousif and Al-Shawa, 1998;
1999; 2005; 2006; Alyousif et al.,
2003; 2004; Amoudi, 2006; Toulah
and Al-Rawi, 2007; Alyousif et al.,
2009). All the Eimeria n. species
were identified in different domestic
and wild animals in Saudi Arabia.
So, relentless search for more po-
tent anticoccidials continues.
The initial hope underlying the
present experiment was to find a
natural product with anticoccidial
properties that could be used as a
feed additive with minimal process-
ing. Water extract of Neem extract
in a dose of 100 mg/kg b.w. for 9
day as herbal medication in E. tenel-
la infected chickens was evaluated
compared with Baycox in a dose of
7 mg/kg b.w. for 2 successive days
as reference drug to treat poultry
coccidiosis. Chickens infected with
E. tenella (10
4
) showed severe clini-
cal signs such as anorexia, depres-
sion, hypo-pigmentation severe,
continuous hemorrhages in feces
and hemorrhagic feces around the
cloaca. These agreed with Allam
(1989) and Saif et al. (2003).
As to gain weigh and food con-
sumption the present results showed
that GA had better gain weight
(80.44%) and consumed more food
(400.83+27.59) as compared to oth-
er GB (70.95% & 386.67+32.10),
GC (69.84% & 337.92+53.79) and
GD (78.69% & 356.25+36.28gm
food/bird). This agreed with Bui et
al. (2006) and Zulpo et al. (2007).
McDougal (2003) and Tipu et al.
(2002) attributed the decrease in
gain weight and impaired feed con-
version in E. tenella infected chick-
ens to anorexia. Hogg (1992) de-
clared that appetite was a major fac-
tor causing decreased performance.
Klasing et al. (1987) declared that
the body weight gain decreased af-
ter infection and the reduced body
weight gain is a major contributor to
production of loss that accompanies
coccidial infection in young chick-
ens the authors stated that the in-
flammatory immune responses di-
vert energy from growth which may
affect the weight gain while logan et
al. (1993) added that all Eimeria
isolates cause significant weight
suppression and impaired food con-
version rate (FCR). The reason for
this impairment is that the organism
100

destroys the absorptive mucocsal
surface, competes for micro nu-
trients resulting into metabolic im-
balances and adversely effects nu-
trient utilization.
The present result showed signifi-
cant increase in gain weight in chic-
kens treated with Baycox and Neem
with better gaining in those received
herbal medication (Neem). The data
in accordance with El-Deghidy and
El-Askalany (1989), and Mathis et
al. (2003) they found that the use of
Toltrazuri (7 mg/kg) for two con-
secutive days in drinking water be-
tween days 10 & 14 was the best
time for good coccidiosis control
allowing full performance. The re-
sults agreed with Tipu et al. (2002)
and Abbas et al. (2006), their result
revealed that the birds of non-
infected non-medicated group had
better weight gain as compared to
medicated ones. The birds of sali-
nomycin sodium treated group have
better weight gain and feed efficien-
cy as compared to other treated
groups but the difference was non
significant. Also, Neem fruit 150gm
/50kg feed had excellent perfor-
mance in terms of oocyst count and
lower mortality as compared to oth-
er treated groups. The curative ca-
pacity of Baycox solution and Neem
green leaves water extract were also
determined by comparing the mean
number of oocyst/gm feces (OPG).
Both medications significantly re-
duced the oocyst shedding on 5, 7,
& 9
th
days PT in GC & GD with
reduction of 86.47, 87.61 & 93.61%
respectively in group treated with
Baycox, while reduction in GD
treated with Neem was 44.25, 53.21
& 72.97%. Data showed that Bay-
cox had better reduction rates than
Neem and agreed with Mehlhon et
al. (1984), Safwat et al. (1988), Ma-
this et al. (2003) and El-Banna et al.
(2005). Toulah (2000) reported that
Neem dried leaves in a dose 100
mg/ kg B.w. in treatment of hepatic
coccidiosis in rabbits resulted in a
significant reduction in fecal oocyst
shedding without any toxic manife-
station on the host.
The current study revealed that
coccidiosis-related mortality was
prevented by both drugs and these
are in accordance with the studies
carried out by Toulah (2000), Tipu
et al. (2002) who used Neem fruit as
anticcocidial in broilers with a dose
of 150 g/50kg food supplement, and
Gowda et al. (1998) used Neem
kernel meal in diet of white leghorn
layers with a dose of 100 g/kg food
supplement. These studies declared
the safety of Neem medication.
Abbas et al. (2006) found that the
therapeutic doses of Neem on intes-
tinal coccidiosis in broiler chickens
depended on the methods of Neem
extraction, the authors reported that
dried Neem leaves were better than
alcoholic extract and the latter was
more potent than water extract in
reducing the fecal oocyst shed, yet
the authors recommended the water
extract of Neem leaves for its lowest
toxicity side effects.
The gross morphology of infected
ceca appeared congested and dark
red in color, edematous with thick-
101

ened wall, signs exaggerated with
increase in infection intensity (Wit-
lock, 1982; Witlock and Fetterer,
1983).
Lesions induced by E. tenella of a
dose (10
4
) in infected controls (GB)
were characterized principally by
severe villous atrophy, marked pro-
liferation of epithelial cells of intes-
tinal crypts, dilation and necrosis of
submucosal glands, multifocal areas
of severe inflammatory infiltrate,
foci of discrete hemorrhage asso-
ciated with various parasite intrale-
sional forms.
In some cases, parasites in various
stages of development were trans-
murally located throughout the mu-
cosa, with necrosis being more se-
vere where there were massive ac-
cumulations of schizonts with me-
rozoites. This reached peak by day
15
th
then tended to decrease by day
17
th
& 19
th
with less oocysts shed-
ding and milder cecal inflammation
compared to 15
th
day. The variances
could be related to acute or chronic
stages.
During acute stage, the sporulated
oocyst after being ingested under-
goes the process of excystation, the
released motile sporozoites actively
penetrate the intestinal epithelium
mainly at the villus tips, site of in-
vasion, while others travel within
the mucosa to the crypt epithelial
cells where they develop further. In
the crypt epithelium, sporozoites
become rounded and transform into
trophozoites that undergo schizogo-
ny or merogony (merozoites forma-
tion), asexual proliferation phase.
The merozoites are released in the
gut lumen, where they reinfected
epithelial cells close to those from
which they emerge and form addi-
tional schizont generations. The rap-
id multiplications are usually com-
pared by severe cecal tissues de-
structions (Jeurissen et al., 1996).
Following that, a short quiescent
period of parasitic activity and the
process developing chronic stage
occur where the host can modulate
its defensive mechanisms through
enhancing local mucosal immune
responses (Saif et al., 2003). The
lack of parasite development in cec-
al tissues, enhanced immune res-
ponses and improved relative pro-
portions of CD4
+
cells of chickens
orally infected with oocysts, birds
had mounted protective immune
responses which may prevent inva-
sion and development of sporozoites
in the cecal tissue (Allen 1999). Al-
so, the capacity of cecal mucosa in
repairing it and this could be done
through keeping the balance be-
tween the mitotic activity of crypt
enteric cells and desquamation pro-
duced principally by external ag-
gressors (Nabuurs, 1995).
Based on histopathological evalu-
ation of Baycox given in a dose 7
mg/kg B.w. for two successive days
(GC), and Baycox was found to de-
stroy the majority of all develop-
mental stages of E. tenella, breaking
its life cycle and resulting in a de-
crease in oocysts shedding with a
reduction rate 93.61%, however it is
worthy to mention that the decrease
in the oocyst output was accompa-
102

nied by apparent toxic histopatho-
logical effects on ceca of infected
chickens manifested by the atrophic
and degenerative changes of the
covering epithelial cells, villi and
muscularis mucosa layer. These
gave positive correlation with ex-
tensive drug administration. These
agreed with Jeurissen et al. (1996);
Adegbol (2004) and Dipeolu et al.
(2004).
In chickens of GD received Neem
extract (100 mg/kg B.w) for succes-
sive 9 days, the study showed that
although the therapeutic efficacy in
eliminating E. tenella stages was
lower than Baycox, as reduction rate
was 72.97% compared with 93.61%
yet, Neem was superior in improv-
ing histopathological changes ac-
companied E. tenella, normal thick-
ness, muscular layer arrangement,
submucosa and more or less normal
villi in both height and covering
epithelium that agreed with Tipu et
al. (2002) and Abbas et al. (2006).
In GD were the apparent lympho-
cytic, leukocytes and plasma cells
infiltration between the villi, the
same data was recorded by Biu et
al. (2006). The importance of lym-
phocyte in immune responses to
coccidian had been reported in
chicken by Rose et al. (1992), while
Allen (1999) reported that avain
humoral immunity in the intestinal
tract is mediated via secretion of
antibody by plasma cells located
within the gut lamina propria into
the intestinal lumen. Vervelde et al.
(1996) reported the major role of
intestinal leukocytes in protective
immunity following E. tenella in-
fection.
Conclusion

Evaluating the therapeutic efficacy
of Neem fresh leaves in comparison
with Baycox in experimentally in-
fected chickens with E. tenella, the
results revealed that Baycox is still
considered the most affected drug in
treatment of chicken coccidiosis and
shown to be superior to that of
Neem in the performance data (gain
weight, food consumption ext.) and
in decreasing oocyst shed, but an
additive histopathological toxic ef-
fects besides those produced by the
infection could be recorded. In con-
trast Neem appeared to have a re-
markable improvement on cecal
integrity, suggesting that Neem may
have immune-protective effects. So,
evaluation of drugs depending on
performance criteria and/or the de-
crease of oocyst shed are insuffi-
cient and other researching parame-
ters are of must.

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107



RODENT BORNE DISEASES AND THEIR FLEAS IN MENOUFIA
GOVERNORATE, EGYPT

By
MOHAMED ISMAIL SOLIMAN, AZZA S. ABD EL-HALIM AND
MICHEAL W. MIKHAIL
Research Institute of Medical Entomology, Ministry of Health, Dokki,
Giza, Egypt

Abstract
A preliminary survey of domestic rodent borne diseases and their fleas was
carried out in ten centers of Menofiya (Quesna, Shebeen El-Kom, Berka El-
Saabe, El-Bagour, El-Shohada, Tala, Menoff, Searth El-Lian, Ashmon and El-
Sadat) Governorate, Egypt. Rodent index (number of rodent / trap) and percen-
tage frequency of different rodent species were recorded in spring (2009). The
main species was Norway rat, Rattus norvegicus, the grey-bellied rat, R. rattus
alexandrinus, the white- bellied rat, R. rattus frugivorus and the house mouse,
Mus musculus. Searth El-Lian center showed the highest existing rodent- in-
dex, while Quesna center showed the lowest existing rodent-index. The Nor-
way rat, R. norvegicus showed higher frequency at Shebeen El-Kom, Berka El-
Saabe, El-Baguur, Searth El-Lian and El-Sadat. R. rattus alexandrinus showed
higher frequency at Tala center, while Rattus rattus frugivorus showed higher
frequency at El-Shohada, Menoff and Ashmon. M. masculus showed the lower
frequency at all centers. The common flea species attacking rodents at all cen-
ters were: the oriental rat flea, Xenopsylla cheopis, the mouse flea, Lyptopsylla
segnis and the dog flea, Ctenocephalides canis. The flea index (number of
flea/rodent) at Searth El-Lian was the highest, while Shebeen El- Kom showed
the lowest index. The oriental rat flea, X. cheopis was the highest frequency
distribution for all domestic rodent species studied while, the dog flea, C.s ca-
nis was the lowest. The adult rodents showed the higher frequency with fleas
than juveniles.
Key Words: Menoufia Governorate, Rodent borne diseases, Fleas.
________________________________________________________________________________________________________________________

Introduction

Rats and mice are very common
animals in many Egyptian Governo-
rates (Morsy, 1982, 84, 86; Shoukry
et al., 1986). Rodents cause severe
damage to agriculture and its prod-
ucts (Brooks and Rowe, 1987).
They find their way into houses and
building and feed on almost any
108

human or animals food materials.
Poltzer (1954), classified rodents to
domestic,commensal, field and wild
species. Baltzer (1966) showed that
most of murine rodents follow man
to almost all inhabited areas owing
to their ability for survival, multipli-
cation, reproduction and adaptation
to varieties of local situation. Three
species of commensal rodents are
distributed worldwide: brown rat
(Rattus norvegicus), black rat (Rat-
tus rattus) and house mouse (Mus
musculus) (Brooks and Rowe,
1987). The Norway or brown rat is a
gregarious species and under very
favorable conditions colonies of
several hundred strong may devel-
op. They are the common urban and
rural rats of temperate regions of
North America and Europe, living
both indoors and outdoors. In tropi-
cal areas, they are the mainly exist
in coastal seaports and irrigated
agriculture areas. In towns and vil-
lage they occur in garbage dumps
and other refuse, around cesspits, in
sewer systems and storm drains.
Rodents in addition to being de-
structive pests they have been to
serve as either carriers or reservoirs
for many parasitic and bacterial
which bean be spread to man and
animals by their habits (Cameron,
1949), their dropping and by their
arthropod, ectoparasite. Therefore,
studies of ectoparasite of rodents are
very important from epidemiologi-
cal point of view being considered
as potential vectors of various caus-
al agents of diseases. Fleas are re-
spectively ectoparasites of rat, man;
cat, mouse and poultry. Each flea
has its preferred host or related
group of hosts. However, the host
parasite relationship by its true
meaning is not absolute and fleas
pass from one host to another (Mor-
sy et al., 1982). Most rat fleas for
examples feed on several kinds of
animals and even man (Rumreich
and Wynn, 1945). No doubt, the
absence of host parasite relationship
in group fleas has its epidemiologi-
cal importance in the transmission
of plague and murine typhus fever a
well as other bacterial, viral parasit-
ic diseases. In fact, Xenopsylla
cheopis is widely disseminated on
various species of rodents in differ-
ent parts of the world. In Egypt
records were given by Abdou and
Smaan (1962), Hoogstraal (1956)
Arafa et al. (1973), Rifaat et al.
(1969), Morsy et al. (1982).
The aim of this work is to study
the rodent species and their role as
reservoirs of fleas in an attempt to
elucidate their medical importance
in relation to man and animal at
Menoufia Governorate, Egypt.


The method descried by Rifaat et
al. (1969) for capturing and trans-
porting of rodent animals was fol-
lowing throughout the present in-
vestigation. The studies were done
in spring 2009. One hundred and
fifty wire box traps were baited and
distributed in some selected residen-
tial houses at sunset in two villages
for each center in Minofiya Gover-
109

norate, Egypt. Distributed traps will
be collected next morning and en-
closed in separate white bag to
avoid escape ectoparasites and
transported to laboratory. In labora-
tory each animal was identified to
species and subspecies. The average
number of rodents per traps was
counted. Rats and mice were killed
with chloroform and fleas were col-
lected on white sheet by using a stiff
hard brush. Fleas were preserved of
70% alcohol in separate label spe-
cimen tubes. Identification of fleas
was done according to the key given
by Hoogstraal (1956), Arafa et al.
(1973). Data subjected to analysis
for variance and the method of least
significant differences (L.S.D.), the
method of Duncan (1955) was used.

Results

The results are shown in Tables
(1, 2, 3 & 4).

Discussion

The total number of rat catches,
rodent index (average number of
rodent/trap) and percentage fre-
quency of males to females of dif-
ferent rodent species which were
dominated at studied centers of Mi-
noufia Governorate are given (tab.
1). As regards the indoor result
showed that four species of rodents
were identified, Norway rat, R. nor-
vegicus, the grey- bellied rat, R. r.
alexandrinus, white- bellied rat, R.
r. frugivorus and house mouse, Mus
musculus. Searth El-Lian center
showed the higher frequency of ex-
isting rodent- index, i.e. 0.26, while
Quesna center showed the lower
frequency which rodent- index rec-
orded 0.09. Statistical analysis re-
vealed that, there are highly signifi-
cant difference between the fre-
quency distribution of rodent spe-
cies between centers, where F=
258.63 & LSD=1.57 for R. norvegi-
cus, F= 206.50 & LSD= 0.92 for R.
r. alexandrinus, F=477.9 &LSD=
0.99 for R. r. frugivorus and F=
818.09 & LSD= 0.63 for M. muscu-
lus. Rodent species showed highly
frequencies distribution percentage
in and between centers, which R.
norvegicus recorded highly frequen-
cy distribution percentage at She-
been El-Kom (56.8%), Berka El-
Saab (72.7), El-Bagour (53.1),
Searth El- Lian (76.9) and El- Sadat
(86.6%) centers. The highly fre-
quency distribution percentage for
R.r. alexandrinus was recorded at
Tala center (38.2%) and for R. r.
frugivorus was recorded at El- Sho-
hada center (40.9%), Menoff center
(86.95%) and Ashmon center
(51.6%). On the other hand M. mus-
culus showed the lower frequency
distribution percentage at all cen-
ters, but it recorded 18.2% at El-
Shahada. The domestic rodent spe-
cies in Egypt was reported by sever-
al investigators; Mahdi et al. (1970)
reported that R. norvegicus was the
dominant species at Suez area. Mor-
sy et al. (1986) showed that five
species of rodents were identified at
Suez Governorate they were M.
musculus, R. rattus, R. norvegicus,
A. cahirinus and Sekeetamys calu-
110

rus. Morsy et al. (1988) showed that
in Alexandria city the presence of R.
norvegicus was the more common
rats than R. r. alexandrinus. It is
worthy to refer to the role played by
these species in the transmission
and dissemination of serious patho-
gens to man. It has been proved that
the usual infestation of plague in
rats occurs in the Norway rat and
less frequently in roof rat and to the
house mouse. Allam et al. (2002)
showed that the common domestic
rodents identified at Damietta and
Qualybia Governorates were R. r.
alexandrinus, R. r. frugivorus and
R. norvegicus. Shoukry et al. (2006)
studied the distribution of rodent in
three different habitats (domestic,
peridomestic and wild rodents). It
was found seven rodent species
were identified, R. norvegicus, R. r.
alexandrines, R.r. frugivoru, M.
musculus, A. russattus, Meriones
sacramenti and Gerbillus pyrami-
dum. Mahmoud et al. (2008) re-
ported that R. norvegicus, R.r. alex-
andrinus, R.r. frugivorus, M. mus-
culus and A. cahirinus were record-
ed in Suez, Menoufia, Giza, De-
miatta and Beni-Suef. Norway rat,
R. norvegicus showed higher fre-
quency at Suez, Giza and Demiatta
Governorates (G). On the other
hand, R.r. alexandrinus showed the
high number at Menoufia G, while
M. musculus showed the highest
species at Beni-Suef G. They also
indicated that the frequency of
males to R. norvegicus was higher
than females in five governorates.

The overall flea index (for male,
female, adult and juvenile) and fre-
quency distribution percentage of
flea species at all centers (tab. 2, 3,
& 4). The common fleas attacking
rodents (tab. 2) were the oriental rat
flea, Xenopsylla cheopis, the mouse
flea, Lyptopsylla segnis and the dog
flea, Ctenocephalides canis. The
flea index at Searth El-Lian showed
the highest flea population density
(18.15), while the lowest density
was recorded at Menoff center (2.4)
Data also showed that, there is high-
ly significant difference of the three
flea species associated with the dif-
ferent rodent species, where
F=146.99 & LSD= 0.29 for X.
cheopis, F=443.92 &LSD= 0.09 for
Lyptopsylla segnis and F=1180.28
& LSD= 0.69 for C. canis. Data also
showed that oriental rat flea, X.
cheopis was the highest frequency
distribution for all domestic rodent
species at all centers. Which the
frequency distribution represented
between 100% at El-Sadat to 76.2%
at Shebeen El-kom centers. While L.
segnis represented between 0.0% at
El-Sadat to 23.8% at Shebeen El-
Kome centers. On the other hand, C.
canis showed the lowest which
represented between zero% at She-
been El-Kom, Berka El-Saab, Ash-
mon and El-Sadat centers to 28% at
Menoff centers. There was highly
frequency (tab. 3) of existing flea
index to males and females between
different rodent species and also
between different centers. On the
other hand, the prevalence of fleas
was higher among adult different
111

rodent species (tab. 4) compared
with juveniles. The density of flea
infesting domestic rodents was re-
ported by several investigators. Ri-
faat et al. (1969) reported that the
oriental rat-flea X. cheopis was the
most predominant species parasitiz-
ing commensal and domestic rodent
in Egypt. Morsy et al. (1986) stu-
died the fleas attacking the different
rodent species (Mus musculus, Rat-
tus rattus, R. norvegicus, A. cahiri-
nus & Sekeetamys culurus) at Suez
G. were fleas, X. cheopis, Pulex irri-
tans, C.felis, Ctenocephalides segnis
and Echidnophaga gallinacea.
Shoukry et al. (1987) found that
fleas were the most predominated
ecto-parasites in Ismailia G. The
number of collected fleas was Xe-
nopsylla cheopis (682), Echidno-
phaga gallinacea (667) and C. seg-
nis (41). Zeese et al. (1990) found
that five species of fleas were col-
lected from domestic rodent species
in Sharkia G. Fleas were L. segnis,
C. felis and Pulex irritans. Khalid et
al. (1992) showed that X. cheopis
the main parasitism species on the
rodent host's over the year, the
highest number of fleas was on R.
norvegicus. Allam et al. (2002)
showed that flea infestation varied
according to rodent species and to
bioclimatic conditions. Mahmoud et
al. (2008) reported that the common
fleas species attacking rodents at
Suez, Menofiya, Giza, Demiatta and
Bani-Suef Gs, were: X. cheopis, C.
felis, L. segnis and P. irritans. Abu-
Madi et al. (2001) showed that there
was no overall difference in abun-
dance of fleas between the two sex-
es. Davis (1951) reported that the
higher of flea infestations of fe-
males rat tended to remain in their
burrows for longer periods than
males. On the other hand in the rat
populations examined by Linardi et
al. (1985), males were more heavily
infested with X. cheopis than fe-
males and this was arguably attri-
buted to larger home range of male
rats. Morsy et al. (1988) showed
that the flea index and the infesta-
tion rate were directly related to
host's body size.

Conclusion

Menoufia Governorate is distin-
guished by the presence of high
population density of rats and mice
which more heavily infested with
fleas. Their intimate association
with man is of great potential dan-
ger as disseminators of serious pa-
thogens. Control program should be
embarked to rodents and ectopara-
sites. In addition, proper environ-
mental sanitation and health educa-
tion are essential to achieve success.

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114

Table 1: Frequency distribution % of domestic rodent species at Menoufia Governorate spring 2009.

LSD F
Frequency distribution

Rodent
Index
Ani-
mal
No.

Trap
No.

Area
Mus musculus R. r. Frugivorus R. r. alexandrines R. norvegicus
% F. M. T. % F. M. T. % F.
M
.
T. % F. M. T.
1.63
115.92
***
- - - - 28.6 3 1 4 35.7 1 4 5 35.7 3 2 5 0.09 14 150 Quesna
0.96
997.07
***
- - - - 43.2 5 11 16 - - - - 56.8 13 8 21 0.246 37 150
Shebeen
ElKom
2.32
216.62
***
3.0 1 - 1 21.3 4 3 7 3.0 - 1 1 72.7 13 11 24 0.22 33 150
Berka
ElSaabe
0.56
101.78
***
12.5 1 3 4 28.1 3 6 9 6.3 - 2 2 53.1 7 10 17 0.21 32 150
El
Bagour
1.11
106.17
***
18.2
1

3 4 40.9 4 5 9 22.7 4 1 5 18.2 4 - 4 0.15 22 150
El
Shohada
0.64
485.30
***
5.9 1 1 2 26.5 5 4 9 38.2 5 8 13 29.4 3 7 10 0.226 34 150 Tala
0.62
227.44
***
4.35 - 1 1 86.95 13 7 20 4.35 1 - 1 4.35 - 1 1 0.15 23 150 Menoff
1.21
899.08
***
- - - - 7.7 1 2 3 15.4 2 4 6 76.9 12 18 30 0.26 39 150
Searth
El-lian
1.16
363.37
***
- - - - 51.6 7 9 16 19.4 - 6 6 29.0 5 4 9 0.207 31 150 Ashmon
0.60
496.09
***
6.7 - 2 2 6.7 - 2 2 - - - - 86.6 12 14 26 0.2 30 150 El-Sadat
- -
818.09
***
- - -
488.9
***
- - -
206.5
***
- - -
258.63
***
- - - - - - F
- - 0.63 - - - 0.99 - - - 0.92 - - - 1.57 - - - - - - LSD
P
D
F

C
r
e
a
t
e
d

w
i
t
h

d
e
s
k
P
D
F

P
D
F

W
r
i
t
e
r

-

T
r
i
a
l

:
:

h
t
t
p
:
/
/
w
w
w
.
d
o
c
u
d
e
s
k
.
c
o
m
115

Table 2: Frequency distribution % of flea species at Menoufia Governorate spring 2009.

LSD F
Frequency Distribution%
Flea
Index
Rodent
Index
No. of
Fleas
Trapped
Animals
Trap
No.

Area
Ctenocephalides
canis
Lyptopsylla
segnis
Xenopsylla
cheopis
0.28
305.50
***
2.1 12.8 85.1 6.33 0.09 89 14 150 Quesna
1.17
131.88
***
- 23.8 76.2 4.05 0.246 150 37 150
Shebeen
El- Kome
0.47
106.12
***
- 15.6 84.4 7.57 0.22 250 33 150
Berka El-
Saabe
1.24
115.71
***
1.6 22 76.4 5.78 0.21 185 32 150
El- Ba-
gour
0.19
553.00
***
1.1 16.8 82.1 4.32 0.15 95 22 150
El- Sho-
hada
0.71
447.54
***
3 13.6 83.4 9.94 0.226 338 34 150 Tala
0.19
631.00
***
2.8 11.1 86.1 2.4 0.15 55 23 150 Menoff
1.64
134.49
***
0.3 3 96.7 18.15 0.26 708 39 150
Searth
El-Lian
1.24
217.55
***
- 5.6 94.4 10.5 0.21 326 31 150 Ashmon
- - - - 100 9.4 0.2 282 30 150 El - Sadat
- -
1180.28
***
443.92
***
146.99
***
- - - - - F
- - 0.69 1.09 0.29 - - - - - LSD
P
D
F

C
r
e
a
t
e
d

w
i
t
h

d
e
s
k
P
D
F

P
D
F

W
r
i
t
e
r

-

T
r
i
a
l

:
:

h
t
t
p
:
/
/
w
w
w
.
d
o
c
u
d
e
s
k
.
c
o
m
116

Table 3: Number of flea and flea-index (FI) for male (M) and female (F) of different rodent species.
LSD F M. musculus R. r. frugivours R. r. alexandrinus R. norvegicus Param
-eter
Area
F M Total F M Total F M Total F M Total F M Total F M Total
- - - - - -
- - - 3 1 4 1 4 5 3 2 5
rats
Quesna
- - - - - - - - - 9 2 11 5 16 21 42 18 60 fleas
2.29 1.99 2.66
57.22
***
28.89 ** 30.62 **
- - - 3 2 2.75 5 4 4.2 14 9 12
FI
- - - - - - 1 - - 5 11 16 - - - 13 8 21 rats
Shebeen
El Kom
- - - - - -
- - - 11 62 73 - - - 51 26 77
fleas
0.78 - - 28.9* 2.59 ns
0.94
ns
- - - 2.2 5.6 4.56 - - - 3.9 3.25 3.66
FI
- - - - - -
1 - 1 4 3 7 - 1 1 13 11 24
rats
Berka El
Saab
- - - - - -
- - 0 11 7 18 - 1 1 138 93 231
fleas
1.12 0.78 0.25 271.5**
225.2
***
310.6 ***
- - 0 2.75 2.33 2.57 - 1 1 10.6 8.45 9.62
FI
- - - - - - 1 3 4 3 6 9 - 2 2 7 10 17 rats
El
Bagoure
- - - - - - 1 4 5 12 21 33 - 11 11 48 88 136 fleas
1.36 1.05 1.08 40.17***
96.78
***
73.91 ***
1 1.33 1.25 4 3.5 3.66 - 5.5 5.5 6.85 8.8 8
FI
- - - - - -
1 3 4 4 5 9 4 1 5 4 - 4
rats
El
Shohada
- - - - - -
1 4 5 7 7 14 16 4 20 56 - 56
fleas
1.35 1.49 1.35
210.0
***
9.12 * 207.8***
1 1.33 1.25 1.75 1.4 1.55 4 4 4 14 - 14
FI
- - - - - -
1 1 2 5 4 9 5 8 13 3 7 10
rats
Talla
- - - - - - 0 0 0 46 36 82 31 41 72 46 138 184 fleas
0.38 1.15 0.48
259.9
***
558.6**
*
279.4***
0 0 0 9.2 9 9.1 6.2 5.1 5.5 15.3 19.7 18.8
FI
- - - - - -
- 1 1 13 7 20 1 - 1 - 1 1
rats
Menoff - - - - - -
- 2 2 32 15 47 1 - 1 - 5 5
fleas
- 1.49 1.11
4.79
ns
11.28* 24.93***
- 2 2 2.46 2.14 2.35 1 - 1 - 5 5
FI
- - - - - - - - - 1 2 3 2 4 6 12 18 30 rats
Searth
El lian
- - - - - -
- - - 11 13 24 12 21 33 248 403 651
fleas
0.81 1.87 1.99 199.9***
135.0**
*
169.0***
- - - 11 6.5 8 6 5.25 5.5 20.66 22.38 21.7
FI
- - - - - - - - - 7 9 16 - 6 6 5 4 9 rats
Ashm-
oon
- - - - - - - - - 30 48 78 - 33 33 118 97 215 fleas
2.22 0.86 0.67 466.5 **
143.1
***
226.2 ***
- - - 4.28 5.33 4.87 - 5.5 5.5 23.6 24.25 23.88
FI
- - - - - - - 2 2 - 2 2 - - - 12 14 26 rats
El Sadat
- - - - - -
- 0 0 - 4 4 - - - 119 195 278
fleas
- 0.84 0.66 1.0 ns
456.1
***
633.2 ***
- 0 0 - 2 2 - - - 9.9 11.45 10.69
FI
- - - - - - 1.0
ns
5.69* 13.33 *** 86.95 *** 55.76 *** 98.75 *** 4.8 * 10.86 *** 29.14 *** 264.8 *** 363.3 *** 175.5 *** - F
- - - - - - - 0.91 0.57 1.07 0.99 0.76 2.33 1.24 0.97 1.08 1.13 1.52 - LSD
P
D
F

C
r
e
a
t
e
d

w
i
t
h

d
e
s
k
P
D
F

P
D
F

W
r
i
t
e
r

-

T
r
i
a
l

:
:

h
t
t
p
:
/
/
w
w
w
.
d
o
c
u
d
e
s
k
.
c
o
m
117

Table 4: Number of flea and flea-index (FI) for adult and juvenile of different rodent species

LSD F M. musculus R. r. frugivours R. r. alexandrinus R. norvegicus Para-
meter
Area
Juvenile Adult Juvenile Adult Juvenile Adult Juvenile Adult Juvenile Adult Juvenile Adult
- - - - - - 0 4 2 3 1 4 rats
Quesna
- - - - - - 0 11 1 20 1 59 fleas
0.35 0.19 17.24 ** 833.56 *** - - 0 2.75 0.5 6.66 1 14.75 FI
- - - - - - 1 15 - - 8 13 rats
Shebeen
El Kom
- - - - - - 1 72 - - 21 56 fleas
- - 1.20
ns
1.00
ns
- - 1 4.8 - - 2.6 4.8 FI
- - - -
0
1 4 3 1 - 8 16 rats
Berka El Saab - - - -
0 0
11 7 1 - 65 166 fleas
0.26 0.59 198.47 *** 744.12 ***
0 0
2.75 2.33 1 - 8.12 10.37 FI
- - - - - 4 3 6 - 2 2 15 rats
El Bagoure - - - - - 5 9 24 - 11 18 118 fleas
- 1.07 9.10
ns
70.12 *** - 1.25 3 4 - 5.5 9 7.86 FI
- - - - - 4 1 8 - 5 - 4 rats
El Shohada - - - - - 5 6 7 - 20 - 56 fleas
- 1.21 1.00
ns
272.48 *** - 1.25 6 0.87 - 4 - 14 FI
- - - - - 2 1 8 1 12 2 8 rats
Talla
- - - - - 0 3 79 4 68 11 137 fleas
- 0.57 4.27
ns
276.59*** -
0 3
9.87 4 5.66 5.5 21.6 FI
- - - - - 1
3
17
1
- - 1 rats
Menoff - - - - - 2
3
44
1
- - 5 fleas
- 1.88 1.00
ns
6.29* - 2 1 2.58
1
- - 5 FI
- - - - - - - 3 1 6 6 24 rats
Searth El lian
- - - - - - - 24 2 31 47 604 fleas
- 1.36
16.6
ns

556.92*** - - - 8
2
5.16 7.8 25.16 FI
- - - - - - 1 15 1 5 2 7 rats
Ashmoon
- - - - - - 5 73 0 33 44 171 fleas
2.96 0.95
133.0
***
124.26 *** - -
5
4.86
0
6.6 22 24.4 FI
- - - - - 2 - 2 - - - 26 rats
El Sadat - - - - - 0 - 4 - - - 278 fleas
- 0.67 1.0
ns
621.18 *** -
0
- 2 - - - 10.69 FI
- - - - -
4.44
*
16.64 ***
99.35
***
8.61
**
4.75
*
81.43
***
648.12 *** - F (flea index)
- - - - - 0.99 1.34 0.83 1.23 1.04 1.92 0.87 - LSD (flea index)
P
D
F

C
r
e
a
t
e
d

w
i
t
h

d
e
s
k
P
D
F

P
D
F

W
r
i
t
e
r

-

T
r
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119



MIRAZID IN TREATMENT OF THREE ZOONOTIC TREMATODES
IN BENI-SWEIF AND DAKHALIA GOVERNORATES
By
AHMAD M.A. MASSOUD
1
, EMAN T. El-SHERBINI
2
, NAGLA MOS-
TAFA KAMEL SALEH
3
, MOHAMED FATHY ABOUEL-NOUR
4
and
AYMAN T.A. MORSY
5

Department of Tropical Medicine, Faculty of Medicine, Al-Azhar Univer-
sity
1
, Nasr City, Cairo, Departments of Zoology
2,3,4
, Faculty of Pharma-
cy, El Nahda University
2
, Beni-Sweif, Faculty of Science, South Valley
University
3
and Faculty of Science, Mansoura University
4
, and Tropical
Medicine Unit, The Ministry of Interior Hospitals
4
, Egypt.

Abstract

A total of 60 patients with schistosomiasis (40), fascialiosis (15) and hetero-
phyiasis (5) were selected Beni-Sweif and Mansoura Districts and subjected to
history taking, clinical examination, Kato thick smear, sedimentation and
hatching test (for schistosomiasis cases) at the beginning of the study, 2 & 3
months after treatment with Oleo-resin of Myrrh (Mirazid
) in a dose of
10mg/kg/day for 6 consecutive days an hour before breakfast. The results
showed a significant improvement in symptoms with minimal negligible or no
side effects. The cure rates, 2 & 3 months after treatment were 80.7%% &
11.8%% for schistosomia-sis, 93.3% & 6.6% for fascioliasis, and 100% for
heterophyiasis. The clinical picture of schistosomiasis before treatments were
easy fatigability, anorexia, nausea, vomiting, epigastria pain, abdominal disten-
tion, right upper guardant pain, colicky abdominal pain, left upper and/or lower
guardant pain, abdominal rumbling, dysentery, diarrhea, rectal bleeding, con-
stipation, and alternating bowel habit. Those of fascioliasis were abdominal
distention, dripping of saliva, right upper guardant, colicky abdominal pain,
weight loss, easy fatigability, intermittent jaundice, anorexia, nausea, vomiting,
epigastria, left upper and/or lower quadrant pain, right layer quadrant pain, loin
pain, abdominal rumbling, diarrhea, constipation, and alternating bowel habit
The safety and efficacy of C. molmol extract in treating heterophyiasis
(100%), fascioliasis (100%) and schistosomiasis (92.5%) were documented.
Key words: Schistosomiasis, fascialiosis, heterophyiasis, Mirazid, clinical,
parasitological follow-up.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

120

Introduction

Schistosomiasis is a very old en-
countered disease (Castellani and
Chalmers, 1913; Scott, 1937), but
remains a public health problem in
Egypt despite the continuous cont-
rolling efforts (El Alamy and Cline,
1977; El-Khoby et al., 2000).
Fasciola was diagnosed in nearly
all the Egyptian governorates with
prevalence rate varied between 2%
& 17% (Haseeb et al., 2002; El Sha-
zly et al., 2009). Besides, most of
the anti-schistosomal drugs and the
anti-fascioliasis have side effects or
low efficacy (WHO, 1995).
Previous surveys conducted in
five Governorates in 1993 in the
Nile Delta; Beheira, Gharbia, Meno-
ufia, Qalyoubia and Sharkia showed
that anoverall prevalence of S. man-
soni and S. haematobium was 39%
& 5% respectively (Michelson et
al., 1993). Most of anti-schisto-
somal drugs in use had either
deleterious side effects or low effi-
cacy (Ismail et al., 1994; 1999; Ros-
enkran et al., 1995; Renganthn and
Coli, 1998; Raso et al., 2004).
Safety and effectiveness of the
oleo-resin extract derived from
Myrrh, Commiphora molmol extract
as anti-schistosomiasis was reported
in laboratory and clinical studies
(Massoud et al., 1989, 1996; 1999
a,b,c, 2000; Gaballah et al., 2001;
El-Baz et al., 2003; Al-Ashry et al.,
2003).
Fascioliasis is an important world-
wide zoonotic disease. In Egypt,
human fascioliasis has been diag-
nosed in all provinces of the Delta
and in some provinces of Upper
Egypt. Studies in some villages in
the Delta have revealed prevalence
rates varied between 2%-17%
(WHO, 1995). Clinically, the dis-
ease occurs in acute and chronic
phases with complication particular-
ly in children (Farid and Amin,
1986). Haseeb et al. (2003) found
that major complications of fasci-
oliasis were bleeding and biliary
cirrhosis beside ectopic lesions. El-
Sefi et al. (1994) reported several
hepatobiliary complications of fas-
cioliasis among Egyptian patients as
recurrent attacks of cholecystitis,
gall bladder perforation, obstructive
jaundice, biliary lithiasis, periductal
fibrosis, cholangitis, segmental bile
duct stricture and hepatic granulo-
mas with abscess formation. Wahib
et al. (2006) correlated between fas-
cioliasis and hepatitis C. Most of the
anti-fascioliasis drugs were ineffec-
tive or unsafe, toxic, with many side
effects and were contra-indications
(Chen and Mott, 1990; Merino et
al., 1998; Coles et al., 2000).
Myrrh Arabian (Mecca Myrrh) or
Somalia is one of the oldest known
medicines widely used by the an-
cient Egyptians and Chinese (Che-
valier, 1996).
This work aimed to study the effi-
cacy, safety and side effects of the
Oleo-resin of Myrrh (Mirazid
) in
the treatment of schistosomiasis,
fascioliasis and heterophyiasis un-
der field conditions.

Patients, Materials and Methods
121

This study was carried out from
May 2009 to September 2009. Stool
analysis was carried out to select
patients with schistosomiasis (40),
fascioliasis (15) and heterophyiasis
(5). They were diagnosed by the
cross sectional study using direct
smears, flotation and Kato thick
smear technique. All patients agreed
to participate in this controlled trial,
and a questionnaire sheet was filed
out on each case.
Oleo-resin extract from Myrrh C.
molmol (Mirazid) was given in daily
as 10 mg/kg for 6 days. Every case
received capsules on empty stomach
an hour before breakfast. Stool was
exami-ned by Kato thick smears
(Katz et al., 1972) and sedimenta-
tion at 2
nd
& 3
rd
month post treat-
ment and intensity of positive cases
was calculated. Every patient was
asked to give information about any
complaint at time of the end of fol-
low up. At the end of 3 months fol-
low-up post treatment, all patients
were subjected to history taking
with stress on compliance with re-
gimen. By clinical examination, the
post treatment findings were com-
pared with pretreatment ones.
Statistical analysis: Data entry
processing analysis was done by
using the SPSS program version.
10.1999. Chi-square test signific-
ance was compared between differ-
ent groups and paired t test com-
pared between means (SPSS, 1999).
Results

The results are shown in tables (1, 2, 3, 4, 5 & 6).

Table 1: Patients groups personal data.

Variability Schistosomiasis (n=40) Fasciohasis (n=15) Heterophyiasis (n=5)

No. (%) No. (%) No. -%
Age group 11 -20 6 (15.0) 1(6.7) 3 (60.0)
21 -30 15 (37.5) 4 (26.7)
2 (40.0)
31 -40 10 (25.0) 7 (46.7)
----
41-50 6 (15.0) 2 (13.3)
----
51- 70 3 (12.5) 1 (6.7)
----
Male 28 (70.0) 8 (53.3)
2 (40.0)
Female 12 (30.0) 7 (46.7)
3 (60.0)
Education: Illiterate 15 (37.5) 10 (66.7)
2 (40.0)
Preparatory 12 (30.0) 3 (20)
1 (10.0)
Secondary 7 (17.5) 2 (13.3)
2 (20.0)
University 6 (15.0) ----
----
Occupation: House wife 8 (20.0) 7(48.6)
1 (20.0)
Farmers 10 (25.0) 5 (33.3)
1 (20.0)
Students 20 (50.0) 2 (6.7)
2 (40.0)
Manual workers 2 (5.0) 2(6.7)
1 (20.0)
122

Table 2: Stool examination of patients groups.

Parasites
No. Percent
Schistosomiasis alone 35/40 (80.7)
Schistosomiasis mixed
5/40 (19.3)
Heterophyes heterophyes 5/5 (100.0)
Fascioliasis alone 13/15 (85.0)
Fascioliasis mixed 2/15 (15.0)

Table 3: Schistosomiasis patients (n=40) before and after 3 months treatment.

Variabilty
Before treatment After treatment
Significant
No. (%) No. (%)
Asymptomatic 8 (20.0) --- --- P< 0.001
Symptomatic 32 (80.0) 7 (10.8) P< 0.001
Easy fatigability 20 (50.0) 2 (3.1) P< 0.001
Anorexia 25 (62.5) 3 (4.6) P< 0.001
Nausea 7 (17.5) --- --- P< 0.007
Vomiting 8 (20.0) 1 (1.5) P< 0.007
Discomfort After meal 20 (50.0) 1 (1.5) P< 0.001
Epigastria pain 28 (70.0) 2 (3.1) P< 0.001
Abdominal distention 30 (75.0) 1 (1.5) P<0.001
Right upper guardant pain 21 (52.5) 3 (4.6) P< 0.001
Colicky abdominal pain 25 (62.5) 2 (3.2) P< 0.001
Left upper guardant pain 13 (32.5) 1 (1.5) P< 0.001
Left lower guardant pain 20 (50.0) 2 (3.1) P<0.001
Loin pain 10 (25.0) 1 (1.5) P= .34
Rumbling in abdomen 20 (50.0) 3 (4.6) P< 0.001
Diarrhea 7 (17.5) 1 (1.6) P = .035
Dysentery 18 (45.0) 1 ( 1.6) P= .002
Rectal Bleeding 4 (10.0) --- --- P= .065
Constipation 3 (7.5) --- --- P=.13
Alternating bowel habit 7 (17.5) --- --- P= .007

Table 4: Parasitological and clinical cure rate.

Items
Schistosomiasis (n=40) Fascioliasis (n=15) Heterophyiasis (n=5)
No. Percent No. Percent No. Percent
After two months 35 80.7% 14 93.3% 5 100
After three months 2 11.8% 1 6.6% --- ---
Total cured 37 92.5 15 100 5 100
123

Table 5: Fascioliasis patients (n=15) before and after 3 months treatment.

Variability
Before treatment After treatment
Significant
No. (%) No. (%)
Asymptomatic 2 (13.3) 14 (93.3) P < .001
Symptomatic 13 (86.7) 1 (6.6) P < .001
Abdominal distention 13 (86.7) 1 (6.6) P <.001
Right upper guardant pain 12 (80) --- --- P <.001
Colicky abdominal pain 10 (66.7) 1 (6.6) P <.001
Weight loss 11 (73.3) 1 (6.6) P < .001
Easy fatigability 13 (86.6) 1 (6.6) P <.001
Dripping of saliva 12 (80) 1 (6.6) P < .001
Intermittent jaundice 1 (6.6) --- --- P = .30
Anorexia 1 (6.6) --- --- P = .30
Nausea 2 (12.7) --- --- P = .24
Vomiting 1 (6.6) --- --- P = .30
Discomfort to meal 10 (66.7) --- --- P <.001
Epigastria pain 13 (86.7) 1 (6.6) P <.001
Left upper quadrant pain 5 (33.3) --- --- P = .08
Left lower quadrant pain 4 (6.1) --- --- P = .099
Right layer quadrant pain 3 (4.6) --- --- P=.11
Loin pain 5 (33.3) --- --- P=.021
Rumbling in abdomen 10 (66.7) --- --- P <.001
Diarrhea 3 (20) --- --- P= .11
Constipation 2 (13.3) --- --- P= .24
Alternating bowel habit 5 (33.3) --- --- P= .021

Table 6: Side effects of treated patients after 3 months of treatment.

Side effects
Schistosoniasis Fascioliasis Heterophyiasis Total
No. % No. % No. % No. %
None 32 90.8 14 93.3 5 100 51/60 85.0
Giddiness 3 4.6 1 6.7 --- --- 4/60 6.6
Somnolence 1 1.5 --- --- --- --- 2/60 1.65
Heart burn 1 1.5 --- --- --- --- 1/60 1.65
Fleeting aches 2 1.5 --- --- --- --- 2/60 5.0
Itching 2 3.1 I 6.6 --- --- 3/60 5.0

Discussion

In the present study, the schisto-
somiasis patients (tab. 1) were in
age groups 21-30 years (37.5%) 31-
40 (25.0%), 11-20 & 41-50 (15%
124

each group) and 50-70 (7.5%). Fas-
cioliasis patients were in age group
31-40 (46.7%), 21-30 (26.7%), 41-
50 (13.3%) and 11-20 & 51-70
(6.7% each group). Heterophyiasis
patients were age groups 11-20
(60.0%) and 21-30 (40.0%) In the
schistosomiasis patients, the males
represented (70.0%), in fascioliasis
cases were 53.3%, and in hetero-
phyiasis cases were 40.0%. Illite-
rates represented 37.5%, 66.7% and
40.0% respectively. Students were
50.0%, 6.7% and 40.0% respective-
ly. The schistosomiasis (tab. 2)
alone (80.7%), mixed (19.7%), fas-
cioliasis alone (85%), mixed (15%),
and heterophyiasis. There was clini-
cal improvement in nearly all the
cases (tab. 3) post treatment.
Asymptomatic cases arise from
(20%) pretreatment up to (100%)
post- treatment. Also, the sympto-
matic cases showed significant im-
provement nearly in all manifesta-
tions. The improvement of symp-
toms in fascioliasis cases 3 month
after treatment. Three month after
treatment, there is a significant in-
crease in the percentage of asymp-
tomatic cases (13.3% vs. 93.3%).
Furthermore, among the sympto-
matic patients, there was a signifi-
cant improvement regarding the
abdominal distention pain, easy fa-
tigability discomfort after meal as
well as rambling in the abdomen.
Five cases were positive by Kato
but negative by hatching at 2
nd

month, and two were positive by
Kato but negative by hatching at 3
rd

month.
The repeated stool Kato thick
smear examination (tab. 5) showed
that the cure rate for schistosomiasis
cases were (80.7%) and (11.8%) at
2
nd
& 3
rd
months post treatment
respectively and totaled 92.5%, but
for fascioliasis cases the cure rate
was 93.3% and 6.6% at 2
nd
& 3
rd

months respectively and totaled
100. The cure rate for heterophyia-
sis was 100% at 2 months post-
treatment. The side effects reported
(tab. 6) by schistosomiasis and fas-
cioliasis 3
rd
month post treatment
were giddiness (6.6%), itching and
fleeting aches (5.0% for each sign)
and somnolence & heart burn
(1.65% for each sign).Among those
that still positive after single course
of treatment, there was marked re-
duction of the intensity of egg ex-
cretion with a statistical significance
difference at follow-up. However, a
second course was indicated.
The present results showed that all
the fascioliasis (100%) and hetero-
phyiasis (100%) and the schistoso-
miasis patients (92.5%) responded
well to C. molmol extract (Mirazid).
No doubt, the health significance
of schistosomiasis was due to the
fact that most of cases had more or
less silent symptoms or mild infec-
tion and also due to unexpected ac-
climatization of the community to a
very long standing health problem
(Younis and Khalil, 1998). Undoub-
tedly, zoonotic fascioliasis is an in-
creasingly recognized public health
problem in Egypt. However, the
disease treatment overcome certain
difficulties, as the usual known
125

drugs such as emetine hydrochloride
and praziquantel did not cure fasci-
oliasis by a single dose (Hammouda
et al., 1995). Morsy (2009) by the
parasitological and histopathologi-
cal studies reported that schistoso-
miasis mansoni infected mice and
treated with Praziquatel and/or Olti-
praz, both drugs gave unsatisfactory
results particularly the latter drug.
In treatment of fascioliasis using
several drugs the patient clinical
well being is one of the criteria of
drug efficacy (Kabil et al., 1994).
The clinical improvement of the
symptoms and signs present before
treatment has been emphasized to
be one of the assessment criteria for
the effectiveness of the antischisto-
somal therapy (Younis and Khalil,
1998). The present study showed an
overt clinical improvement of the
symptoms after treatment in agreed
with Massoud et al. (1996) and Ga-
ballah et al. (2001). The drug was
well tolerated by all patients and
safe as reported by Massoud et al.
(1997), El-Gohary et al., (1999),
Motawea et al. (2001) and El-Baz et
al. (2003). The present study also
revealed that cure rate of schistoso-
miasis 2 and 3 months post treat-
ment were 80.7%% and 92.5 % re-
spectively. The other remaining
positive cases were negative by hat-
ching. This results agreed with that
of Massoud et al. (1998; 1999b) and
Gaballah et al. (2001). Regarding
parasitological examination of fas-
cioliasis cases the cure rate was
93.3% and 100% at 2
nd
& 3
rd

months post treatment. Motawea et
al. (2001) reported that parasitolog-
ical cure rates of Fasciola cases
post-treatment was 95.9 % 97.6%,
98.3% & 98.6% after 1.2.3 & 4
weeks respectively. Surprisingly,
the cure rate of Fasciola patients
mixed with other parasites was
higher 98.4% after one week and
100% after 2 weeks. This agreed
with El-Gohary et al. (1999) who
found marked reduction of the in-
tensity of the deposited Fasciola
eggs with a statistical significant
difference that decreased the trans-
mission of the disease in the com-
munity.
Comparing the result with other
clinical trials using other drugs, Mi-
razid (C. molmol extract) proved
more effective and safer than other
drugs. Apt et al. (1995) reported 24
asymptomatic individuals in Chile
with the chronic hepatic fascioliasis
confirmed parasitologically were
treated with a single oral dose of
Triclabendazole (10mg/kg body wt.)
after an overnight fast.
However, 19/24 patients (79.2%)
were egg-negative 2 months post
treatment. Merino et al. (1998) re-
ported that Triclabendazole proved
to be a veterinary drug and that its
use was disapproved in humans to
this date, beside, its use, cost and
development of resistance (Kusel
and Hagan, 1999).
The Egyptian Ministry of Health
till now contraindicates the use of
this drug in children below 5 years.
El-Karaksy et al. (1999) reported
failure of 8 cases to treatment with
praziquantel. Resistance to treat-
126

ment with praziquantel was ob-
served at dose of 75 mg/day for 2
days, being repeated 15 days later
with no response. El-Morshedy et
al. (2000) treated 134 cases of fas-
cioliasis with Triclabendazole, 68
patients received a single dose of 10
mg/kg and 66 patients received 2
doses of 10mg/kg on 2 consecutive
days. Cure was assessed 5 weeks
after treatment. The cure rate was
79.4%of the first group and 93.9%
of the second group. One patient
developed biochemical cholestasis
on the 3
rd
day post treatment. El-
Karaksy et al. (1999) diagnosed
human fascioliasis in 40 Egyptian
children. All children were treated
with triclabendazole in a dose of 10
mg/kg as a single oral dose within
two months, 31 children (78%) were
cured as evidenced clinically, nor-
malization of eosinophil counts,
Fasciola antibody titers and absence
of Fasciola eggs in stools.
Generally speaking, Mirazid
is a
natural drug formed of the myrrh
extract that derived from the
Commiphora molmol tree, family:
Burseraceae (Wallis, 1967). Much
extract was obtained by spont-
aneously exudation from the cracks
and fissures that commonly form in
bark. Myrrh consists of 7-17%
volatile oil, 25-40% resin, 5761%
gum & 3-4% impurities (Marshall,
2004). Myrrh was approved by the
U.S. Food and Drug Administration
for food usage (21 Code of Federal
Registration-CFR 172.510) and was
given generally recognized as safe
(GRAS) status and as a flavor
ingredient (No. 2765) by the Flavor
Extract Manufacturers Association
(FEMA) (Ford et al., 1992). The
Council of Europe (1981) included
myrrh in the list of plants and parts
thereof acceptable for use in foods
and it is present in the French and
British Pharmacopoeias (Michie and
Copper, 1991).
Besides, Nomicos (2007) reported
that since antiquity, the genus
Commiphora is composed of more
than 200 species, and exploited as a
natural drug to treat pain, skin
infections, inflammatory conditions,
diarrhea, and periodontal diseases.
He added that in more recent
history, products derived from C.
myrrha and various other species of
Commiphora are becoming recog-
nized to have significant antiseptic,
anesthetic, and antitumor properties.
Traditional practice and evidence-
based research have supported that
these properties are directly attribu-
table to terpenoids (especially fura-
noses-quiterpenes), the active comp-
ounds present in myrrh essential oil.
Recently, current studies have focu-
sed on applying clinical trial metho-
dologiy to validate its use as an anti-
neoplastic, an antiparasitic agent,
and an adjunct in healing wounds.
C. molmol proved non toxic to
albino mice (Rao et al., 2001), and
safe for the male reproductive
organs and did not affect the bile
flow (Massoud et al., 2002). Omar
et al. (2005) tested Mirazid
safety
on adult male albino rats by the
assessment of serum levels of ALT,
AST & bilirubin, histopathology of
127

liver and the cytogenesis of bone
marrow cells. They reported an
insignificant increase in AST, ALT
& bilirubin, a normal hepatic tissue,
and a non significant increase in the
incidence of the chromosomal
aberrations.
Mirazid safety and effectiveness
was also, proved in treatment of
human schistosomiasis (Massoud et
al., 1996; 2000a; Gaballah et al.,
2001; Sheir et al., 2001; Badria et
al., 2001; El Baz et al., 2003; Soli-
man et al., 2004), the human
fascioliasis (El-Gohary et al., 1999;
Motawea et al., 2001; Massoud et
al., 2001b; Hassan et al., 2004;
Massoud et al., 2004a; Abo-Madyan
et al., 2004) the heterophyiasis sp.
(Massoud et al., 2001a;2007a;
Fathy et al., 2005) and the animal
fascioliasis (Haridy et al., 2003),
human and animal dicrocoeliasis
dendriticum (Massoud et al., 2003),
sheep monieziasis (Haridy et al.,
2004). Mirazid
was successfully
used in treatment of Fasciola sp., D.
dendriticum and Paramphistomum
sp. in farm animals (Haridy et al.,
2006), human Stronyloides sterco-
ralis (Massoud et al., 2006) and
human hymenolepiasis (Massoud et
al., 2007b).
Regarding protozoal infection
Mirazid in the combination with
Paromomycin suceessfully treated
cryptosporidiosis parvum in Egyp-
tian immunocompetent patients
(Massoude et al., 2008) and vaginal
trichomoniasis (Al-Zanbagi, 2007;
El-Sherbini et al., 2009).
Also, Myrrh has a potent mollu-
scicidal effect on the snails interme-
diate hosts of both species of the
schistosomiasis and the fascioliasis
(Massoud et al., 2000b;2004b; 2007
c; Allam et al., 2001; El Shazly et
al., 2001; Massoud and Habib,
2003), and against the medically
important Bithynia connollyi snail
(Shoukry, 2006) as well as the
larvicidal action against mosquitoes
borne-diseases; Culex pipiens and
Aedes caspius (Massoud and Labib,
2000), on the cotton leaf-worm,
Spodoptera littoralis (Shonouda et
al., 2000), and the fowl tick, Argas
persicus (Massoud et al., 2005).
In Saudi Arabia, "Mecca Mur or
Myrrha" successfully treated the
sheep liver fluke, dicrocoeliasis
dendriticum and animal fascioliasis
(Abo-Zenadah, 1999; 2005), the
human fascioliasis (Al Mathal and
Fouad, 2005), dicrocoeliasis dendri-
ticum in man and animals (Al-
Mathal and Fouad, 2004) and as
molluscicide against the snails
intermediate host of Schistosoma
mansoni in Saudi Arabia, Biom-
phalaria arabica adults, egg masses
and affected fecundity (Al-Mathal
and Fouad, 2006).

Conclusion

In Egypt, schistosomiasis haema-
tobium and mansoni, fascioliasis
species and heterophyiasis hetero-
phyes are more or less encountered
zoonotic diseases not only in Egypt
but also in many neighboring coun-
tries. On the other hand, the out-
128

come of the present results showed
that myrrh is a safe oleoresin of the
tree Commiphora molmol. It has a
dramatic efficacy (100%) in treating
fascioliasis and heterophyiasis. Re-
garding schistosomiasis the efficacy
was 92.5%. No doubt, the clinical
pictures of both schistosomiasis and
fascioliasis overlap one another, and
parasitologically diagnosis is a must
whenever possible.
Besides, a second course of treat-
ment was indicated for some schis-
tosomiasis. There was clinical im-
provement in most of cases. Also,
there was marked parasitological
cure in the majority of schistoso-
miasis and fascioliasis cases.

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135



DETECTION OF SOME INTESTINAL PROTOZOA
IN COMMERCIAL FRESH JUICES
By
SHEREEN F. MOSSALLAM
Parasitology Department, Faculty of Medicine,
Alexandria University, Egypt

Abstract
Fresh fruit juices are popular, but not always safe. For assessing the likelih-
ood of infection with newly emerging intestinal protozoa, commercial fresh
orange, lemon, sugar cane, strawberry, and mango juices were screened by wet
mounts, Weber's modified trichrome and modified Ziehl-Neelsen stains. Proto-
zoa viability was done by fluorescein-diacetate/propidium-iodide staining, and
infectivity was performed in Swiss albino mice. Results showed that 35.43%
were contaminated with one or more of Cryptosporidia, Microsporidia, and
Cyclospora, as well as Giardia spp. Strawberry was the most contaminated
juice (54.28%), while orange was the slightest (22.86%). Cryptosporidia was
the highest contaminant (61.29%), and Cyclospora was the least (14.52%). Mi-
crosporidia spp. was the most robust contaminant which retained its viability
and infectivity in juices in which it was detected. Moderately acidic strawberry
and mango juices and alkaline sugar cane juice pose a possible threat, due to
harboring the highest viable and infectious protozoa. Regarding highly acidic
juices, viability and infectivity decreased in lemon, yet was not still risk free.
Orange juice was comparatively safe, as viability dramatically declined, while
infectivity was completely abolished. Hence consumers, especially high risk
group, are placed at hazard of contracting intestinal protozoa infections, espe-
cially through moderately acidic and alkaline juices.
Key words: Fresh fruit juices, Cryptosporidia, Microsporidia, Cyclospora and
Giardia sp., Trichrome, Ziehl-Neelsen, fluorescein-diacetate/propidium-iodide.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Fruit-derived juices have high
consumer preference both in terms
of taste and nutritive value. Com-
mercial juices are pasteurized,
treated or fresh. Pasteurized juices
are thermally processed at 71C-
73C for 15-20 seconds then quickly
cooled down, and are found in refri-
gerated sections of stores. Treated
juices are exposed to a much higher
temperature, preservative is added
to which are not refrigerated but are
packed in special airtight shelf-
stable containers. Fresh juices are
commonly defined as un-

138

pasteurized untreated juices, having
relatively short refrigerated shelf
life, that are expressed from whole
fresh fruits by grounding, pressing,
squeezing, or crushing in pressers
(Parish, 1997). Microbial quality of
commercial fresh juices, served
through restaurants, cafes, or
vended in roadside shops and recre-
ational areas (beaches, parks, clubs)
and busy market places (shopping
malls, bus stations), remains ques-
tionable (Sandeep et al., 2001; Lues
et al., 2006). While a single piece of
contaminated fruit may infect one
person, a contaminated fruit which
is juiced may infect many individu-
als, and this implicates fresh juices
as one of the causes of food-borne
illness (Parish, 1997; Garcia et al.,
2006).
Food-borne diseases have a major
public health impact (Dawson,
2005). The debilitating costs in-
curred on countries' economy from
food-borne diseases are becoming
of increasing concern because of the
expanding sector of susceptible
people (Cimerman et al., 1999). The
epidemiology of food-borne diseas-
es is rapidly changing as newly rec-
ognized pathogens emerge and well-
recognized pathogens increase in
prevalence or become associated
with new food vehicles (Hedberg et
al., 1994). The commonly involved
pathogens include bacteria, viruses,
and fungi, whereas, parasites do not
cause as many outbreaks of food
borne illness as the others (Hedberg
et al., 1994; Parish, 1997; Dawson,
2005). Newly emerging protozoa,
having potential significance in ca-
tering practices in which ready-to-
eat foods are served without heat
treatment, can cause acute self-
limiting enteritis in the immuno-
competent individuals. In the very
young, the elders, pregnant women,
and in immunocompromised pa-
tients, such protozoa can lead to
severe protracted diarrhea (Cimer-
man et al., 1999; Dillingham et al.,
2002; Calvo et al., 2004; Jedrze-
jewski et al., 2007). Although infec-
tive doses of these contaminating
protozoa in fruit juices have not
been well established, yet based on
the standards provided for drinking
water, numbers required to cause
illness is very low, denoting the risk
they represent to public health, es-
pecially to risky consumers.
This study aimed to assess the
possibility of drinking commercial
fresh juices could serve as a route of
transmission of newly emerging
intestinal protozoa.

Material and Methods

One hundred seventy five com-
mercial un-pasteurized un-preserved
fresh orange, lemon, sugar cane,
strawberry, and mango juice sam-
ples were randomly purchased from
different small roadside market
vendors, restaurants, and cafes in
Alexandria; Egypt. Thirty five sam-
ples of each juice type were trans-
ferred from the presenting 200ml
cup to a labeled sterile container,
and were transported to the labora-
tory at 4C (Sandeep et al., 2001).

139

Each individual juice sample was
thoroughly mixed by shaking, and
pH estimation was performed for
every one (Brown et. al., 2006).
Juices were concentrated by centri-
fugation at 5,000Xg for 10 minutes,
and by Sheathers sugar flotation
technique (Markel et al., 1992).
Upon screening, ten microscopic
fields from each individual concen-
trate were randomly examined by
wet mounts (Garcia and Bruckner,
2001-a), Weber's modified trich-
rome (WMT) stain (Weber et al.,
1992), and modified Ziehl-Neelsen
(MZN) stain (Casemore et al.,
1985). Recovered protozoa were
measured using an ocular micro-
meter calibrated against a stage
micrometer slide (Fleck and Moody,
1993), and were cautiously differen-
tiated from the detected artifacts
(Garcia and Bruckner, 2001b). Pro-
tozoa co-infections were estimated
by determining samples harboring
single or mixed infections. Each
positive concentrate was stored in
2.5% potassium dichromate at 4C
(Current, 1985).
Stored concentrates, of each type
of juice, were pooled together, and
were washed several times in PBS,
each at 5,000 Xg for 15 minutes
(Negm, 2003). Supernatants were
decanted, juice pellets were used for
viability and infectivity assays. Via-
bility estimation was performed by
fluorogenic dyes: the fluorescein-
diacetate/propidium-iodide (FDA-
PI); 0.1 ml of stained protozoal sus-
pensions were allowed to settle for
60 seconds. Viable protozoa stained
with FDA, while dead (non-viable)
ones were stained with PI. Viable
and dead protozoa were counted,
and percentage viability was calcu-
lated (Viable/Total Viable+Non-
viable X100) (Jones and Senft,
1985). Prior to animal inoculation,
protozoa were counted in pooled
juice concentrates to make sure that
their infective doses were present
(Youssef et al., 1992). These were
Microsporidia spp. (2000 spores/
ml) (Awadalla et al., 2000), Cryp-
tosporidia (10
4
oocysts/ml) (Yous-
sef et al., 1992), Cyclospora (10
4

oocysts/ml) (Khalifa et al., 2001),
Giardia spp. (1000 cyst/ml) (Awa-
dalla et al., 1995). Infectivity assay
was performed on 36 locally bred
Swiss albino mice, 4-5 weeks,
weighing 20-25 gm each. They were
caged in the animal house at the
same laboratory conditions, fed on
standard pellet food and water in
addition to lipitum. Before inocula-
tion, stools of mice were examined
by wet mounts, WMT and MZN
stains to exclude the presence of any
parasite. Mice were divided into 2
groups; non-infected control (6
mice), each receiving 0.2 ml of PBS
and experimental group (30 mice).
The later was subdivided into five
subgroups (6 mice each); mice of
each subgroup were orally inocu-
lated with 0.2 ml of pooled concen-
trate of each juice type. On the se-
venth day post inoculation, stools
were collected from each mouse,
and were examined using wet
smears, WMT, and MZN stains. On
the same day, animals were sacri-

140

ficed by over dosage of diethyl eth-
er, and parts of their small intestines
were dissected, preserved in 10%
formalin to be stained with H&E,
and the different parasitic stages
were examined in five random mi-
croscopic fields by oil immersion
(El Mansoury et al., 2004; Allam et
al., 2004).

Results

The mean juice pH (tab. 1) ranged
between 2.9 & 7.5. Sugar cane juic-
es were alkaline, (mean pH, 7.5),
otherwise, the remaining juices were
acidic. Strawberrys mean pH was
4.5; mango was 4, while both lemon
and orange juices were more acidic
(mean pH, 3.2 & 2.9 respectively).
Upon screening the total 175 sam-
ples, three newly emerging intestin-
al protozoa spp. were Cryptospori-
dia, Microsporidia, and Cyclospora.
Besides, Giardia spp. was frequent-
ly recognized as well thus was in-
cluded in the study. Overall, 62/175
(35.43%) of all juice samples were
contaminated with protozoa, while,
149/175 (85.14%) contained arti-
facts. The juice of strawberry
showed (Tab. I, Fig 1) the highest
contaminated drink 19/35(54.28%),
while orange juice was the slightest
8/35(22.86%). The most frequently
detected contaminant within overall
positive juices was Cryptosporidia
spp. 38/62(61.29%), and the least
was Cyclospora spp. 9/62(14.52%),
while Microsporidia and Giardia
spp. were encountered in
14(22.58%) & 28(45.16%) of the
positive samples, respectively (Fig.
2). Co-existence of contaminants
differed markedly among various
juice samples; none of the positive
juices contained the four protozoa,
while the majority, 38/62, harbored
just one protozoan (61.29%), 20/62
contained two protozoa (32.26%),
whereas just 4/62 contained three
(6.45%). The most frequent proto-
zoa co-infection was Cryptosporidia
and Giardia spp. were in 15 sam-
ples (24.19%).
Detected protozoa were carefully
differentiated from artifacts present
in various samples; Fig.3 demon-
strates that in wet mounts, some
samples showed refractile spheres
simulating oocysts of Cryptospori-
dia spp. or fungi. MZN stain diffe-
rentiated between them, as cryptos-
poridial oocysts stained pink, and
appeared rounded (3-3.5m) con-
taining four sporozoites against a
greenish background (Fig.4), while
fungi stained green because they are
not acid fast, and were slightly larg-
er (4-6m) (Fig.5). By MZN stain,
seeds, appearing in strawberry juice
samples, looked like cryptosporidial
oocysts both in size and staining
color, but the seeds thick outer-
membrane and lack of its internal
structure suggested its plant origin
(Fig. 6). In wet sears, oocysts of
Cyclospora spp. appeared as large
refractile spheres (Fig.7). By MZN,
Cyclospora spp. oocysts were
spherical 8-10 m, showing staining
variability, as some were light pink
having intracytoplasmic vacuoles
(Fig.8), while others were deeply

141

stained against a greenish back-
ground (Fig.9). Stained fruit ele-
ment resembled Cyclospora spp.
Oocysts (Fig.10). Differentiation
between which was based on the
uneven cell wall of the fruit ele-
ment, the lack of its internal gra-
nules, and its larger size (10-12m).
Cysts of Giardia spp. appeared in
wet smears as ovoid bodies, measur-
ing from 10-14 m, having 2 to 4
nuclei. (Fig.11). By MZN, Giardia
spp. cysts were pink within a green-
ish background. It was possible to
identify their nuclei, axonemes, and
median bodies (Fig.12). Regarding
Microsporidia spp., they could not
be identified in wet smears, but by
WMT stain, small (1.5X1m) and
large (3X1.5m) spores appeared
ovoid having pinkish red walls, and
their cellular contents showed a po-
lar nucleus with a vacuole. Micro-
sporidial spores and rod-like bacilli
looked alike, as the later contained
terminal spores and large vacuole
that mimic Microsporidia spores.
Staining color was rarely helpful,
but contours and lengths were dif-
ferent as bacilli were rod shaped
with straight borders and were
slightly longer (4.5X1m) (Fig.13).
Fruit fibers were frequently present,
though not simulating protozoa, yet
they resembled nematode larvae, but
the former showed no definite inter-
nal structure (Fig.14).
Viability estimation performed by
FDA-PI dyes revealed that viable
protozoa fluoresced intensely green
with FDA (Figs 15-18), and dead
(non-viable) ones fluoresced bright
orange with PI (Figs 19-22). As
shown in the table, the only viable
protozoan isolated from orange
juice was Cryptosporidia spp. The
remaining juices retained viable Mi-
crosporidia, Cryptosporidium, and
Giardia spp., while viable Cyclos-
pora was encountered in strawberry
juice only. The drinks harboring
least viable protozoa were orange
(11.11%) and lemon juices (12.50 to
21.43%). Mango juice revealed
83.33 to 91.43% viable protozoa.
More protozoa survived in strawber-
ry (87.50- 92.59%), while survival
increased in sugar cane juice (94.12-
96.66%). Microsporidia spp. had
the highest viability percentages
among all juices which retained its
spores.
Infectivity assay showed that
stools and intestinal sections of all
mice orally inoculated with pooled
concentrates of strawberry, sugar
cane, and mango juices were posi-
tive for Microsporidia Cryptospori-
dium, and Giardia spp. (100% in-
fectivity), but not with Cyclospora
spp., despite being viable in straw-
berry juice concentrates. Mice given
orange juice, containing viable
Cryptosporidia spp., were not in-
fected with this protozoan. Like-
wise, viable Giardia and Cryptos-
poridia spp. in lemon concentrate
failed to induce infection in mice,
unlike Microsporidia spp. which
appeared in 16.67% of stools of
mice inoculated with lemon. Micro-
sporidia was the only protozoan that
retained its infectivity in all juice
types in which it has been detected.

142

The histopathological examination
showed that Cryptosporidia oocysts
and Giardia trophozoites were
found on the brush borders of ileal
sections and intestinal lumens of
sacrificed animals (Fig. 23). Micro-
sporidial spores were identified in-
side villous enterocytes just beneath
the brush border (Fig. 24), whereas,
Cyclospora spp. oocysts were not
detected in ileal sections of mice fed
with any type of juice. Stools and
intestinal sections of mice inocu-
lated with PBS were negative.

Discussion

Over the past couple of years,
questions regarding the microbial
safety of fresh juices have been on
the forefront. Street-vended fresh
juices present an integral part of
many countries' economy. However
in view of their ready consumption,
quick methods of cleaning, handling
and extraction they could often
present a public health threat (Pa-
rish, 1997). Thus, it was needed to
establish the likelihood of newly
emerging intestinal protozoa within
some commercial fresh juices. Mi-
crosporidia, Cryptosporidia, Cyc-
lospora, and Giardia spp. were de-
tected in 35.43% of the chosen fresh
juices. None of the selected types of
juices was completely devoid of
contamination, with one or more of
these intestinal protozoa, in different
mixtures. The highest detected con-
taminant among all juices was Cryp-
tosporidiaum spp., which was pre-
viously ranked second to rotavirus
as the most prevalent pathogenic
cause of diarrhoeal outbreaks (Dil-
lingham et al., 2002). Reports of
contaminated fruit juices exist in the
literature, most of which deled with
fresh ones (Krause et al., 2001;
Sandeep et al., 2001).
Strawberry juice presented the
highest contaminated drink in this
study (54.28%). Various berries
have been formerly accused as poss-
ible vehicles of Cyclospora, Micro-
sporidia, and Cryptosporidia spp.
(Herwaldt and Beach, 1999; Calvo
et al., 2004). Several factors poten-
tially account for this; remarkable
among which is the pores on the
surface of strawberries that are more
likely to harbor soil, with the possi-
ble consequence of increased num-
bers of microorganisms, rather than
would smooth surfaced fruits. Being
too fragile, organisms may gain
access through tissue insults (Kniel
et al., 2002). Washing and sanitiz-
ing strawberries reduce native sur-
face microflora, thereby resulting in
flourishing of pathogenic contami-
nants due to the reduction in compe-
tition for space and nutrients
(Brackett, 1992). Once polluted,
removal of organisms by routine
cleansing practices of strawberries
is particularly difficult (Kniel et al.,
2002). Other juices examined in this
study were derived from fruits hav-
ing peels, and that is probably why
they were less polluted than straw-
berries. Fruit peels have waxy layer
in their cuticles, which diminishes
contamination of the interior flesh
(Hill and Wenzel, 2009). Tough

143

orange and lemon walls and the
hard sugar cane rind might have
reduced their contamination, but
still didn't make them risk-free.
Mangoes have thin walls, thus are
prone to creasing during washing or
packing. It was suggested that on
washing creased mangoes, gases
within which contract causing in-
ward hydrostatic potential that
draws in water from the exterior of
fruit, allowing entry of microorgan-
ism (Penteado et al., 2004). Juices
derived from fruits having peels
have been occasionally identified as
pathogen vehicles (Castillo et al.,
2006; Oliveira et al., 2006).
Another notable contaminating fac-
tor is water addition (Friedman et
al., 1997).
Unlike orange juice, water enters
in the reconstitution of strawberry,
mango, and lemon juices, in differ-
ent dilutions, while crunched ice is
commonly added to sugar cane. It
was noted that orange was the least
contaminated juice, followed by the
sugar cane. The association between
this and the presence of Giardia
spp. as a second common contami-
nant to Cryptosporidium spp. in
juices diluted with water strongly
suggests that water might have been
a major source of contamination.
Cryptosporidia, Giardia, Microspo-
ridia and Cyclospora spp. were
commonly isolated from water
(Steiner et al., 1997; Dowd et al.,
2003), and the coexistence of the
former two is the most known (Le
Chevallier et al., 1991). The high
degree of environmental contamina-
tion with these protozoa, their sta-
bility in the environment, their rapid
rate of dispersal, and finally their
small size which enable them to es-
cape filtration, all account for their
persistence in water (Steiner et al.,
1997).
While pathogen contamination
routes have not been definitively
confirmed in any juice outbreak,
reoccurring themes appear to be
linked to all. These include conta-
mination with untreated manure, the
use of non potable water, containing
untreated sewage and sludge, for
crop irrigation, for produce or uten-
sil washing (Steiner et al., 1997;
Garcia et al., 2006). Additionally,
cross contamination during
processing conditions may provide
opportunities for microorganism to
infiltrate any juice. A common pic-
ture showed the unhygienic sur-
roundings, often with swarming
houseflies particularly at the vendor
sites. Other potential source of con-
tamination is through handlers pre-
paring juices, due to their ignorance
towards proper hygienic practices.
Vendors usually do not wash the
juicers or the storage containers,
each time the fresh lot of juice gets
mixed with a small amount of pre-
vious lot of juice. The later may
contain some pathogens which can
serve as inoculums for the fresh lot,
especially that robust organisms can
survive on stainless steel surfaces
for several hours (Lues et al., 2006;
Oliveira et al., 2006).
Once polluted

by whatever means,
contaminant survival is further de-

144

pendent on intrinsic pH of the juice,
and its storage temperature (Santil-
lana-Hayat et al., 2002; Brown et
al., 2006). Fortunately, not all pro-
tozoa detected in this study were
viable. The least viables were de-
tected in highly acidic orange and
lemon juices (11.11- 21.43%). Sur-
vival increased in strawberry and
sugar cane, while alkaline sugar
cane retained the highest viables
(96.66%). Most fruits were general-
ly believed to be unusual vehicles of
transmission for human pathogens
due to the presence of natural organ-
ic acids, which are relied upon to
retard the growth of microorgan-
isms. Thes acids depressed the in-
ternal pH of microbial cells by alter-
ing their membrane permeability
(Ryu and Beuchat, 1998). However,
isolation of potentially viable proto-
zoa from fresh produce (Calvo et
al., 2004; Robertson and Gjerde,
2000; Jedrzejewski et al., 2007),
together with the reported juice-
related outbreaks (Millard et al.,
1994; Parish, 1997) challenge this
supposition. According to Friedman
et al. (1997), the high acidity of
orange juice caused 85% loss of
viability of Cryptosporidia spp. oo-
cysts, while this value dramatically
decreased to 35% in drinks with
higher pH, and agreed with Brown
et al. (2006). While, moderately
acidic berry juices (Herwaldt and
Beach, 1999), and apple juice (Mil-
lard et al., 1994), as well as alkaline
sugar cane juice (Oliveira et al.,
2006) harbored pathogenic organ-
isms long enough to cause food-
borne illness. Survival of protozoa
is also dependent on juice tempera-
ture (Santillana-Hayat et al., 2002).
Orange juice is usually served at
room temperature; sugar cane is
frequently cooled by crunched ice,
while others are stored at 4C-8C
before consumption. Moreover, ice
cubes are commonly added to juices
to be chilly, yet this should not be of
concern as juices are usually drunk
before ice melting. Even if the juice
is consumed after ice melting,
which is uncommon in sugar cane
due to its quick fermentable nature,
this does not present a hazard (Oli-
veira et al., 2006). It has been prov-
en that freezing remarkably declines
protozoa viability. Whereas, at 4C-
8C or at room temperature, organ-
isms viability could be retained
long enough to cause food-borne
illness (Fayer and Nerad, 1996; San-
tillana-Hayat et al., 2002; Li et al.,
2003; El Mansoury et al., 2004).
In the current study, infectivity as-
say indicated that not all viable pro-
tozoa turned out to be infectious.
Microsporidia, Cryptosporidia, and
Giardia spp. in strawberry, sugar
cane, and mango juices retained
their infectivity. In agreement with
Negm (2003), Cyclospora spp. oo-
cysts were not able to complete their
life cycle and produce infections in
mice fed with strawberry concen-
trates, as being un-sporulated (non-
infectious). On the other hand, via-
ble Microsporidia spores found in
lemon were infectious to mice, de-
noting that this was the most robust
protozoan which retained both its

145

viability and infectivity in various
juice in which it was detected. This
could be explained by Li et al.
(2003), and Jedrzejewski et al.
(2007), who related the considerable
longevity and tolerance of Micro-
sporidia spores to the protective
effect of their tough chitinous walls,
which allow their stability in the
environment and keep them infec-
tive for days to weeks outside their
hosts. Viable Cryptosporidia and
Giardia spp. encountered in lemon
and orange juices turned out to be
non infective, although it was do-
cumented that less than ten organ-
isms could induce infection by ei-
ther protozoa (Mandell et al., 1995;
Dillingham et al., 2002). The lack of
infectivity of some viable protozoa
could be because fluorogenic stains
are known to overestimate viability
compared with infectivity estimates
(Castro-Hermida et al., 2006). Oth-
erwise, probably membrane integri-
ties of such protozoa were not af-
fected by juice acidity, yet pH in-
duced ultra-structural changes re-
sulting in the prevention of their
invasion or attachment to the host
cells. Alternatively, DNA alteration
would have occurred, thus prevent-
ing replication of these organisms,
and so preventing animal infectivity
(Castro-Hermida et al., 2006), and
these assumptions entail future EM
studies.

Conclusion

The moderately acidic strawberry,
mango juices, and the alkaline sugar
cane juice pose a possible health
threat, due to harboring the highest
viable and infectious intestinal pro-
tozoa. These could be responsible
for diarrhoea in healthy people, and
devastating presentations in high
risk consumers. In highly acidic
juices, viability and infectivity de-
creased in lemon, reflecting that it is
still not risk free, while in orange,
viability dramatically declined, and
infectivity was completely ab-
olished, denoting its comparative
safety. Like other countries, we
need to implement measures that
would critically minimize and con-
trol the spread of juice-borne infec-
tions through tracing the point of
contamination. In view of the threat
posed by fresh fruit juices, juices to
which milk is added, and other pop-
ular oriental Egyptian beverages,
including hibiscus (karkadee), euca-
lyptus (carob), doum, licorice juice
(erkesous), tamarind juice (tamr
hendy) and sobya, deserve future
evaluation as well..

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Legend to figures

Fig 1: Protozoa contamination percentages within each juice type.
Fig 2: Frequency of protozoa contaminants within overall positive juices

Plate I:
Fig 3: Saline smear of juice concentrate showing refractile spheres resembling Cryptosporidia
spp. oocysts or fungi (X400).
Fig 4: MZN-stained juice concentrate smear illustrating pink Cryptosporidia spp. oocyst
(X1000).
Fig 5: MZN-stained juice concentrate smear revealing greenish fungi simulating Cryptosporidia
spp. oocysts (X1000).
Fig 6: MZN-stained juice concentrate smear demonstrating a seed resembling Cryptosporidia
spp. oocyst (X1000).
Fig 7: Saline smear of juice concentrate showing refractile Cyclospora spp. oocysts (X400).
Fig 8: MZN-stained juice concentrate smear revealing faintly stained Cyclospora spp. oocyst
with intracytoplasmic vacuole (X1000).
Fig 9: MZN-stained juice concentrate smear demonstrating deeply stained Cyclospora spp. oo-
cyst (X1000).
Fig 10: MZN-stained juice concentrate smear showing fruit element resembling Cyclospora spp.
oocyst (X1000).
Fig 11: Saline smear of juice concentrate showing Giardia spp. cysts (X400).
Fig 12: MZN-stained juice concentrate smear demonstrating Giardia spp. cysts (X1000).
Fig 13: WMT-stained juice concentrate smear illustrating difference between small (m) and large
(M) Microsporidia spp. spores and rod-like bacilli (b) (X1000).
Fig 14: MZN-stained juice concentrate smear demonstrating fruit fiber (X1000).

Plate II:
Fig 15: Viable Cryptosporidia spp. oocyst fluorescing bright green (FDA-PI, X1000).
Fig 16: Viable Cyclospora spp. oocyst fluorescing bright green (FDA-PI, X1000).
Fig 17: Viable Giardia spp. cyst fluorescing bright green (FDA-PI, X1000).
Fig 18: Viable Microsporidia spp. spores fluorescing bright green (FDA-PI, X1000).
Fig 19: Non-viable Cryptosporidia spp. oocyst fluorescing bright orange (FDA-PI, X1000).
Fig 20: Non-viable Cyclospora spp. oocyst fluorescing bright orange (FDA-PI, X1000).
Fig 21: Non-viable Giardia spp. cyst fluorescing bright orange (FDA-PI, X1000).
Fig 22: Non-viable Microsporidia spp. spores fluorescing bright orange (FDA-PI, X1000).
Fig 23: Giardia spp. trophozoites (G) and Cryptosporidia spp. (C) oocysts along brush border of
ileal sections and intestinal lumen (H&E, X1000).
Fig 24: Microsporidia spp. spores inside villous enterocytes just beneath brush border of ileal
sections (H&E, X1000).


140


141

Table I: Protozoa contamination, viability and infectivity in juices

Juice

Protozoa
spp.
Contamination
of
Individual Juices
Pooled Juices
Viability Infectivity
N. of
samples
% %
N. of
mice
%
S
t
r
a
w
b
e
r
r
y

Positive
samples
19
(54.28%)
Microsporidia 7 36.84 92.59 6 100
Cryptosporidia 10 52.63 87.50 6 100
Mean pH 4.5
Cyclospora 9 49.37 90.00 0 0
Giardia 7 36.84 88.89 6 100
S
u
g
a
r

c
a
n
e

Positive
samples
9
(25.71%)
Microsporidia 1 11.11 96.66 6 100
Mean pH 7.5
Cyclospora 0 0 UD 0 0
Giardia 4 44.44 94.12 6 100
M
a
n
g
o

Positive
samples
12
(34.28%)
Microsporidia 2 16.67 91.43 6 100
Mean pH 4
Giardia 6 50.00 83.33 6 100
L
e
m
o
n

Positive
samples
14
(40.00%)
Microsporidia 4 28.57 21.43 1
16.6
7
Mean pH 3.2
Giardia 8 57.14 12.50 0 0
O
r
a
n
g
e

Positive
samples
8
(22.86%)
Microsporidia 0 0 UD 0 0
Mean pH 2.9
Giardia 3 37.50 0 0 0
35 samples were collected from each juice type. UD: Undetermined.


142

Fig 1

Fig 2

J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No. 12 P.I

J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No. 12 P.II


151

J. Egypt. Soc. Parasitol., 40 (1), 2010: 151 -158

EFFECTS OF RICINUS COMMUNIS, BRASSICA NIGRA AND MIN-
ERAL OIL KEMESOL ON SOME BIOCHEMICAL ASPECTS OF
LARVAE STAGE OF SPODOPTERA LITTORALIS (BOISD)
(LEPIDOPTERA: NOCTUIDAE)
By
NAJAT A. KHATTER AND FATEN F. ABULDAHB
Department of Biology, Faculty of Science, Girls Colleges Branch, King
Abdul-aziz University Jeddah, Saudi Arabia

Abstract

The third instars larvae of Spodotera littoralis were topically treated with two
plant oils, Ricinus communis and Brassica nigra and one mineral oil, Kemesol
95% dissolved in petroleum ether and acetone at concentrations of 0.8, 1.6, 2.0,
3.0 & 4 %. The results revealed that the mean values of the total haemolymph
and fat body protein was reduced in larvae treated with B. nigra and Kemesol
95 %. A significant decrease was observed in haemolymph and fat body pro-
tein contents in larvae treated with all tested compound, the remarked decrease
was noticed at the highest dose (4%) in both two solvents.
Key words: Spodotera littoralis, Ricinus communis, Brassica nigra, Kemesol.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

No doubt, insects of medical, vete-
rinary or agricultural important play
destructive role on human welfare
(Morsy et al., 2001). The develop-
ment of modern ecological friend
compounds received much attention
(Al-Mathal and Fouad, 2006). Botan-
ical molluscicides (Shoukry, 2006;
Massoud et al., 2007) or insecticide
(Abdel Halim and Morsy, 2005) and
mineral oils are one of the effective
agents for insect control (Reddy et
al., 2002). They affect the synthesis
of protein, lipids and carbohydrates;
consequently any balance of these
agents induces confusion in the se-
quence of metamorphosis and meta-
bolism (Morsy et al., 2000; Scott et
al., 2003, Seth et al., 2004; Cespedes
et al., 2005; Pavela, 2005).
This study aimed at investigation of
biochemical effects of two plant oils;
Ricinus communis and Brassica nigra
and one mineral oil; Kemesol 95% on
larvae of the cotton leaf worm, Spo-
dotera littoralis (Boisd). Also, the
biochemical changes on haemo-
lymph, fat body, protein, carbohy-
drate and lipid content were measured
as a marker for insecticidal activity.


S. littoralis pupae were obtained
from a laboratory culture, maintained

152
in the biology Department, Faculty of
Science for Girls, Jeddah.
The plant oils used were: Ricinus
communis and Brassica nigra. The
mineral oil was Kemesol 95%. All
tested compounds were dissolved in
petroleum ether and acetone at con-
centrations of 0.8, 1.6, 2, 3, & 4%.
The different concentrations of each
compound were applied topically us-
ing automatic pipette.
Two groups of the third larval in-
stars of S. littoralis were collected
from the stock culture; the first group
was topically treated with different
doses of the tested compounds dis-
solved in petroleum ether and the
second one treated with the same
doses of the three tested compounds
dissolved in acetone.
Treated and untreated larvae were
incubated at 272C & 702% R.H.
Control group was maintained and
treated only with the solvent used.
Samples of haemolymph and fat bo-
dies were collected from the treated
and check groups and values of hae-
molymph and fat body content of pro-
tein, carbo-hydrates and lipids were
estimated within 48 hrs after treat-
ments (Singh and Bhathal, 1992).
Statistical analysis: All data were
corrected according to Abbotts For-
mula (1925), and expressed as mean
standard deviation. Significant dif-
ferences between individual means
were determined by student t " test
for paired observations. Level of sig-
nificance of each experiment was
stated to be non significant (p>0.05),
significant (p<0.05) and highly sig-
nificant (p<0.05).
Results and Discussion

The main values of the haemo-
lymph and fat body, protein content
during the 3
rd
instar of larval stage S.
littoralis are presented in table 1 and
2. Statistical analysis of results indi-
cated that the haemolymph and fat
body protein contents of 3
rd
larval
instars treated with B. nigra and Ke-
mesol 95% were significantly de-
creased as compared with the check
group (P< 0.05). The effect was dose
dependant, i.e. as a dose increased the
protein content decreased. On the
other hand, R. communis treatments
caused a significantly higher total
protein than that of untreated control
group (P<0.05), haemolymph, and fat
body protein content in all treated and
untreated group tended to decrease as
a result of extraction of botanical oils
by solvents and dissolving the miner-
al oil.
The carbohydrate content:
Statistical analysis of data in
tables 3 and 4 revealed the following
significant increase in the haemo-
lymph carbohydrate content which
was observed at dose of 0.8%, it
reached 49.1 & 48.6 mg/ml. Haem.
for B. nigra and Kemesol 95% as
compared with 23.2 mg/ml Haem. In
the control group the dose of 4% of R.
communis increased the level of hae-
molymph carbohydrate content as
compared with dose obtained by other
doses when it increased from 23.4 to
41.5 mg/ml Haem. The dose of 0.8%
of B. nigra and R. communis in-
creased the level of carbohydrate con-
tent in fat body as compared with

153
other doses, where reached 24.5 and
68.5 mg/g fat body.
The lipid content:
Statistical analysis of data in tables
(5 & 6) revealed that the following
treatments with all tested compounds
significantly increased the haemo-
lymph lipid content. Treatments with
Kemesol 95% with dosage of 0.8%
induced pronounced increase in hae-
molymph lipid content; it was 26.4,
while it was 30.5 mg/ml Haem. when
3
rd
larval instars treated with Kemesol
95% at dosage of 4%. The highest
lipid content for R. communis treat-
ments was 25.3 mg/ml Haem. at 4%.
In the present study an increase in
haemolymph and fat body lipid con-
tents of treated larval stage of S. litto-
ralis was observed. These observa-
tions may be explained that botanicals
and mineral oil increased the conver-
sion rate of carbohydrate to lipid
leading to the haemolymph and fat
body of the treated larvae. The tested
compounds affected mainly the fat
body and this also led to a strong ac-
cumulation of carbohydrates in tis-
sues.
The achieved results were in
agreement with Mesbah et al. (2007)
who found that plant flavonoids had
been shown by many investigators to
have an effect on insect behaviour,
growth, and development. Quercetin
is one of many bioflavonoids that ex-
ist in several fruits and vegetables.
Results indicated that the botanicals
and mineral oil inhibited the anabol-
ism of the treated insects. The meta-
bolic activity is mostly of catabolic
pattern. Results indicated that petro-
leum ether was more effective than
acetone when used as solvent for both
plant oils and mineral oil.
The present data agreed with that of
Hegazy et al. (1992), Hashem (1994),
Shonouda et al. (2000), Reddy et al.
(2002), Scott et al. (2003), Pineda et
al. (2004), Seth et al. (2004), Nathan
et al. (2005). Also, the data agreed
with that of Pavela (2005) who tested
thirty-four essential oils against lar-
vae of S. littoralis, found that these
oils were highly toxic of the 3
rd
larval
instars of S. littoralis after topical
application. The high degree of bio-
degradation exhibited by most phyto-
cemicals is what makes them eco-
friendly and attractive as replace-
ments of synthetic chemicals in the
first place. Although the evaluation of
phytochemicals is yet in its infancy
and much research aims to further
characterize promising agents and
discover new agents in insect control
programs. The present work showed a
strong efficiency of the botanical ex-
tracts which could be used alone or in
combination with sub-lethal doses of
certain insecticides to control the cot-
ton leaf worm. Stringer et al. (2008)
found that successful trapping of fe-
male Thysanoplusia orichalcea (F.) in
either a lure-and-kill or mass trapping
system may offer an effective way to
manage its population size and Kostic
et al. (2008) were tested the toxicity
and anti-feedant activity of Osmium
basilicum against second instars gyp-
sy-moth larvae in the laboratory bio-
assay, they found that all tested solu-
tions showed low to moderate larvi-
cidal effect in both residual toxicity

154
test and in chronic larval mortality
bioassay.

Conclusion

No doubt, the larval stage of the co-
tton leaf worm, Spodotera littoralis
(Boisd) hardly affected the human
welfare. The outcome results proved
that the botanicals extraction (Ricinus
communis and Brassica nigra) and
the mineral oil (Kemesol 95%) inhi-
bited the anabolism of the treated in-
sects.

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156
Table 1: Haemolymph protein content of 3rd instar larvae of Spodoptera
littoralis (Boisd) treated with different doses of Brassica nigra, Ricinus
communis and Kemesol 95% dissolved in petroleum ether and acetone.

Haemolymph protein content (mg / ml Haem.) S.D

Solvent
used
Dose
Kemesol 95% Ricinus communis Brassica nigra Conc %
21.5 0.24** 71.5 0.36** 15.2 0.73** Pet.ether
4. 0
19.3 0.63** 68.4 0.45** 14.2 0.86** Acetone
23.7 0.45** 63.9 0.63** 16.1 1.01** Pet.ether
3. 0
21.5 0.60** 61.5 1.82** 15.2 1.71** Acetone
28.1 0.67** 55.3 1.68** 26.3 1.01** Pet.ether
2. 0
24.3 0.86** 51.2 0.46* 22.1 0.41** Acetone
31.5 0.16* 48.7 0.86* 29.5 0.68* Pet.ether
1. 6
26.2 0.46* 44.3 0.46*** 27.1 0.60* Acetone
33.4 0.56* 38.4 0.92*** 32.4 0.99* Pet.ether
0. 8
31.6 0.34* 36.9 0.91*** 30.1 0.86* Acetone
38.4 0.32 42.1 0.36 40.1 0.90 Pet.ether
control
36.4 0.16 40.2 0.84 39.03 0.86 Acetone

Table 2: Fat body protein content of 3rd instar larvae of S. littoralis treated
with different doses of B. nigra, R. communis and Kemesol 95% dissolved in
petroleum ether and acetone.

Fat body protein content (mg / g Fat body) S.D
Solvent
used
Dose
Kemesol 95% Ricinus communis
Brassica
nigra
Conc %
10.7 1.53** 48.1 0.18** 5.9 0.96** Pet.ether
4. 0
8.2 0.71** 32.5 0.72** 4.7 0.18** Acetone
11.7 0.84** 28.9 0.02** 6.8 0.96** Pet.ether
3. 0
9.2 0.72** 22.1 0.35** 5.4 0.45** Acetone
12.0 1.39* 19.5 .0.45* 9.01 1.36* Pet.ether
2. 0
10.8 0.45* 16.1 0.40* 7.9 0.52** Aceton
12.4 0.63* 19.3 0.17* 10.3 0.72* Pet.ether
1. 6
11.0 0.46*** 16.5 0.89* 9.1 1.82* Aceton
13.1 0.47*** 14.1 0.86*** 12.1 0.53* Pet.ether
0. 8
12.1 0.69*** 11.5 0.54*** 9.3 072* Aceton
14.2 1.35 14.1 1.72 13.2 0.86 Pet.ether
control
11.9 0.41 11.5 0.96 11.2 0.16 Aceton


157
Table 3: Haemolymph-carbohydrate content of 3rd instar larvae of S. littoralis
treated with different doses of B. nigra, R.communis and Kemesol 95%
dissolved in petroleum ether and acetone.

Haemolymph-carbohydrate content (mg / ml Haem. ) S.D
Solvent
used
Dose
29.4 0.53* 41.51 0.13** 34.2 1.23* Pet.ether
4. 0
28.4 0.89* 36.1 0.19* 30.1 0.15* Acetone
32.9 1.32** 31.9 0.54** 37.5 0.16* Pet.ether
3. 0
30.7 1.52*** 28.5 0.31** 36.1 1.32* Acetone
38.5 0.71** 32.9 0.98** 45.4 0.56** Pet.ether
2. 0
36.1 0.81* 31.1 1.32* 41.3 0.72** Acetone
44.5 1.21** 36.5 0.45* 47.2 0.15** Pet.ether
1. 6
41.7 1.65** 34.2 0.69** 42.3 1.38** Acetone
48.6 0.72** 35.7 1.62* 49.1 0.16** Pet.ether
0. 8
43.2 1.36** 32.3 0.91* 45.4 1.23** Acetone
25.6 0.72 25.1 1.32 25.1 0.96 Pet.ether
control
23.7 1.36 23.4 1.86 23.2 0.72 Acetone

Table 4: Fat body carbohydrate content of 3rd instar larvae of S. littoralis
treated with different doses of B. nigra, R. communis and Kemesol 95%
dissolved in petroleum ether and acetone

Fat body carbohydrate content (mg/g Fat body) S.D
Solvent
used
Dose
15.4 1.5** 22.1 0.72** 25.1 0.12** Pet.ether
4. 0
13.2 0.45** 18.1 1.32* 23.2 0.12** Acetone
8.1 0.2* 52.1 0.71** 42.5 1.32** Pet.ether
3. 0
7.9 0.5* 49.9 1.36** 37.7 0.69** Acetone
15.0 1.36** 82.3 0.96** 38.18 1.32** Pet.ether
2. 0
13.2 1.5** 79.5 1.32** 36.12 0.92** Acetone
9.1 0.878* 78.5 0.92** 36.1 1.52** Pet.ether
1. 6
9.1 0.86* 71.7 0.83** 34.4 0.91** Acetone
7.5 0.53* 68.5 0.32** 24.5 0.13** Pet.ether
0. 8
5.3 1.4* 61.7 0.82** 14.2 0.86** Acetone
4.6 0.89 4.4 1.32 4.5 1.56 Pet.ether
control
4.1 1.5 4.3 0.94 4.2 0.31 Acetone


158

Table 5: Haemolymph Lipid content of 3rd instar larvae of S. littoralis
treated with different doses of B. nigra, R. communis and Kemesol 95%
dissolved in petroleum ether and acetone.

Haemolymph Lipid content ( mg / ml Haem. ) S.D
Solvent
used
Dose
30.5 1.23** 25.3 0.75** 15.2 1.23* Pet.ether
4. 0
29.1 0.15** 24.1 0.31* 13.1 0.13* Acetone
22.13 0.53*** 20.5 0.64*** 18.7 0.16* Pet.ether
3. 0
20.9 0.89*** 19.3 0.86*** 15.2 1.32* Acetone
24.1 1.51* 22.9 1.32* 20.9 0.56*** Pet.ether
2. 0
22.9 0.81* 21.0 0.96*** 18.7 0.72* Acetone
26.5 0.96* 20.2 1.63*** 28.5 0.15* Pet.ether
1. 6
17.5 0.72* 17.5 0.91* 26.1 0.14** Acetone
28.3 0.71** 20.2 1.63*** 26.4 0.96* Pet.ether
0. 8
22.1 1.32* 19.1 1.86*** 22.3 0.72* Acetone
20.15 0.71 20.2 1.32 20.1 0.71 Pet.ether
control
19.2 0.72 19.1 1.86 18.7 1.36 Acetone

Table 6: Fat body Lipid content of 3rd instar larvae of S. littoralis treated with
different doses of B. nigra, R. communis and Kemesol 95% dissolved in
petroleum ether and acetone.

Fat body Lipid content (mg / g Fat body ) S.D)
Solvent
used
Dose
136.5 1.5** 160.5 0.72** 150.2 0.12** Pet.ether
4. 0
122.3 0.45** 154.3 1.32** 146.7 0.12** Acetone
111.6 0.2** 131.5 0.71* 142.0 1.32** Pet.ether
3. 0
106.5 0.5** 116.7 1.36* 138.5 0.69** Acetone
78.5 1.36* 301.0 0.96** 77.2 1.32*** Pet.ether
2. 0
72.4 1.5** 277.5 1.32** 68.5 0.92*** Acetone
101.7 0.87* 365.3 0.92** 106.4 1.52** Pet.ether
1. 6
99.2 0.86** 311.4 0.83** 101.2 0.91** Acetone
92.7 0.53* 163.4 1.32** 82.2 0.13* Pet.ether
0. 8
86.1 1.4** 116.5 0.92* 76.3 1.02* Acetone
62.7 0.53 63.4 1.11 62.1 0.13 Pet.ether
control
36.1 1.4 61.2 0.92 57.8 1.32 Acetone

135

159



CHOLEOEIMERIA RIYADHAE SP.N. (APICOMPLEXA: EIMERIIDAE)
FROM THE LIZARD, SCINCUS SCINCUS (SAURIA: SCINCIDAE)
IN SAUDI ARABIA

By
MOHAMED S. ALYOUSIF* AND YASER R. AL-SHAWA
Department of Zoology, College of Science, King Saud University,
P.O.Box 26213, Riyadh 11486, Saudi Arabia.

Abstract

Choleoeimeria riyadhae species new was described from the gall bladder of
the lizard, Scincus scincus (Sauria: Scincidae) collected from east of Riyadh
City, Central Region. Sporulated oocysts are broadly ellipsoid, 36.8x30.5
(33.4-39.1x28.7-32.5) m, with a smooth, brownish-yellow bilayered wall; the
shape index (L/W) is 1.21 (1.19-1.23) m. Micropyle, polar granule and oocyst
residuum are absent. Sporocysts are elongate-ellipsoid, 14.8x9.1 (13.7-
15.5x8.1-10.4) m with shape index (L/W), 1.63 (1.52-1.74) m. Sporocyst
residuum is present. The sporocysts lack a Stieda body. Sporozoites are bana-
na-shaped blunt at one end and slightly tapered at the other. Sometimes, free
sporozoites were seen within oocysts.
Keywords: Choleoeimeria riyadhae species new, lizard, Scincus scincus, Saudi
Arabia.
*Corresponding author: Department of Zoology, College of Science, King Saud Uni-
versity P.O.Box 26213, Riyadh 11486, Saudi Arabia, Fax 4678514.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Genus Choleoeimeria was pro-
posed by Paperna and Landsberg
(1989) as a new genus to comprise
some tetrasporocystic and dizoic
Eimerialike coccidia infecting the
epithelial cells of gall bladders of
reptiles. Phylogenetic analysis based
on nucleotide sequence of sub-unit
ribosomal RNA gene confirmed the
status of the genus Choleoeimeria
(Jirku et al., 2002). Species in genus
Choleoeimeria have ellipsoid oo-
cysts, infecting the epithelial cells of
gall bladders of reptiles, lack a Stie-
da body on the apical part of sporo-
cyst, sporulated in the lumen of the
gall bladder and digestive tract and
undergo endogenous development
in the epithelium of the gall bladder
(Paperna and Landsberg, 1989).
Modry and Jirku (2006) provided a
revision of coccidia possessing te-
trasporocystic oocysts with dizoic
sporocysts known from lizards of
160

the family Scincidae and placed
some of them into the genera Cho-
leoeimeria and Acroeimeria. Only,
one Choleoeimeria saqanqouri has
been described from Scincus scincus
(Abdel-Baki et. al., 2008).
This paper described a new spe-
cies of Choleoeimeria from the gall
bladder of S. scincus from Saudi
Arabia.


During a survey of coccidian
parasites of lizards, a total of 12
adult Scincus scincus were captured
alive in August 2008 from the east
side of Riyadh City, the Capital of
Saudi Arabia. Animals were caged
separately and their feces were col-
lected individually and placed sepa-
rately in a thin layer of 2.5%
aqueous (w. /v) potassium dichro-
mate solution and examined micro-
scopically for the incidence of coc-
cidian oocysts after concentration
by sheather's sugar flotation tech-
nique. To identify site of infection,
within the host, smears of the muco-
sa throughout the whole length of
the gut and wall of the gall bladder
were prepared in the usual technique
for histological studies. The infected
lizards were found to have cloudy
gall bladders content and contained
mass of oocysts in all stages of spo-
rulation. A small aliquot of gall
bladder fluid was pipetted onto a
clean slide, covered with coverslip,
sealed with petroleum jelly and in-
cubated at 262
o
C. The progress of
sporulation was microscopically
observed hourly. 50 sporulated oo-
cysts and 50 sporocysts were ex-
amined and measured by using
1000x light microscopy. The num-
ber of layers of the oocyst wall, its
thickness and detailed structure of
the sporocysts were determined by
crushing the oocysts under a cover-
slip. All measurements are in mi-
crometers (m) with the mean fol-
lowed by the range in parentheses.
The tissue samples of liver, gall
bladder, small and large intestine
from one infected lizard were fixed
in 10% neutral buffered formalin
and processed for light microscopy
examination using standard histo-
logical methods. The paraffin sec-
tions were stained with the haemox-
ylin and eosin (H&E) and giemsa
stain (Garcia and Bruckner, 2001)
and examined for the presence of
endogenous coccidian stages.

Results

Choleoeimeria riyadhae (Figs. 1-4):
Description: The sporulated oo-
cysts (N=50) were broadly ellipsoid
with rounded ends. They measured,
36.8x30.5 (33.4-39.1x28.7-32.5)
m; shape index (L/W), 1.21 (1.19-
1.23) m. The oocyst wall was
smooth and light brownish yellow
0.9 (0.8-1.0) thick and composed of
two layers of approximately equal
thickness. Micropyle, polar granule
and oocyst residuum were absent.
The sporocysts were elongate-
ellipsoid with a few slightly irregu-
lar in shape and size. They meas-
ured 14.8x 9.1 (13.7-15.5x8.1-10.4)
161

m, with shape index (L/W) ratio,
1.63 (1.52-1.74) m. The sporocyst
wall was smooth, colourless, and
single layered, and composed of two
valves joined by a longitudinal su-
ture. The sporocyst residuum con-
sisted of a cluster of large granules.
The sporocysts lacked a Stieda
body. Sporozoites were banana-
shaped, measuring 16.5x3.2 (15.5-
17x2.7-3.8), blunt at one end and
slightly tapered at the other. Some-
times free sporozoites could be seen
in the oocysts which may be due to
the oocysts age.

Taxonomic summary

Type host: Scincus scincus (Sauria: Scincidae).
Type locality: East of Riyadh city, Central Region, Saudi Arabia.
Prevalence: Two of twelve (17%) hosts examined.
Site of infection: Histological examination revealed endogenous stages devel-
oped within cytoplasm of epithelial cells lining the gall bladder (Figs. 3-4). No
endogenous stages were reported in intestine.
Speculation time: 1-4 hrs. at 262
C.
Etymology: The new species name riyadhae is derived from the name of
Riyadh region where the hosts were collected.
Type specimens: Oocysts in 10% formalin and a phototype were deposited in
the Parasitological Collection, Zoology Department, College of Science, King
Saud University, Riyadh both as KSUC, 461.

Table 1: Measurements of Choleoeimeria riyadhae sp. n., and Choleoeimeria
species found in other reptiles (family: Scincidae).

Choleoeimeria
species
Oocyst Sporocyst
Shape Mean
size(m)
Shape
index
Shape Mean
size(m)
Shape
index
C. auratae ellipsoid 27.7x18.5 1.5 ellipsoid 11.8 x 8.5 no data
C. chalcides cylindrical 35x18.6 1.88 broadly ellipsoid 11.9 x 8.9 1.35
C. egerniae elongate
ellipsoid
30.3x16.1 no data ellipsoid 10.3 x 8.3 no data
C. egregia oval 27.6x17.4 1.59 ovoid 10.2 x 6.6 1.24
C. fasciatus
C. hemprichii
cylindrical
ellipsoid
34.9x16.2
34.6x21.4
2.2
1.62
ellipsoid
ellipsoid
12x8.7
11.6 x 8
1.4
1.43
C.pellopleuris cylindrical 31x14 2.21 ellipsoid 7 x 9 1.26
C. sadlieri
C.saqanqouri
C. scincellae
cylindrical
ellipsoid
cylindrical
35.2x16.7
34.8x23.4
29.8x15.9
2.12
1.5
1.89
irregular
ellipsoid
ovoid
12x10.4
11.5x8.9
10.9 x 8
1.16
1.3
1.36
C. scinci ellipsoid 36x25 no data ellipsoid 14 x 10 no data
C. riyadhae
sp. n.
broadly
ellipsoid
36.8x30.5 1.21 elongate ellipsoid 14.8 x 9.1 1.63
162

Discussion

Eimeriid coccidia of reptiles
represent a diverse assemblage of
species differing both in the basic
morphology of the sporulated oo-
cysts and endogenous development.
Modry and Jirku (2006) proposed
species in the genus Choleoeimeria
have ellipsoid oocysts infect epi-
thelial cells of the gall bladder of
reptiles, sporulate in the lumen of
the gall bladder and digestive tract,
undergo endogenous development
in the epithelium of the gall bladder,
lack a Stieda body on the apical part
of sporocyst, and have a wall com-
posed of two plates joined by meri-
dian suture. The genus Choleoeime-
ria was erected by Paperna and
Landsberg, 1989 to comprise Eime-
rialike coccidia infecting the epi-
thelial cells of gall bladders of rep-
tiles. Phylogenetic analysis based on
nucleotide sequence of sub-unit ri-
bosomal RNA gene confirmed the
status of the genus Choleoeimeria
(Jirku et. al. 2002). Modry and Jirku
(2006) provided revision of cocci-
dian possessing tetrasporocystic
oocysts with dizoic sporocysts from
lizards of the family Scincidae and
placed some of them into genus
Choleoeimeria. In the present study,
we demonstrated that the gall blad-
der was the only site for the endo-
genous development of Choleoei-
meria riyadhae sp. n. and no endo-
genous stages were detected in in-
testine.
Several species of Choleoeimeria
have been described from members
of the family Scincidae (Modry and
Jirku, 2006; Abdel-Baki et al.,
2008). However, only three species
of Choleoeimeria were reported
from genus Scincus. They were
Choleoeimeria scinci (Phisalix,
1923) from Scincus officinalis from
Tunis; C. hemprichii (Alyousif and
Al-Shawa, 2005) from Scincus
hemprichii from Saudi Arabia and
C. saqanqouri (Abdel-Baki et. al.,
2008) from Scincus scincus from
Egypt. Eleven species of Choleoei-
meria, inhabit the endothelium of
the gall bladder of the scincid hosts
(Tab. 1). These were C. auratae
(Alyousif and Al-Rasheid, 2001)
from Mabuya aurata in Saudi Ara-
bia; C. chalcides (Probert et al.,
1988) from Chalcides ocellatus in
Egypt; C. egerniae (Cannon, 1967)
from Egernia whitti in Australia; C.
egregia (Telford, 1997) from Eu-
meces egregious in USA; C. fascia-
tus (Upton et al., 1991) from Eu-
meces fasciatus also inUSA; C.
hemprichii (Alyousif and Al-Shawa,
2005) from Scincus hemprichii in
Saudi Arabia; C. pellopleuris (Bo-
vee, 1971) from Lygosoma pellop-
leurum in Japan; C. sadlieri (Modry
and Jirku, 2006) from Marmoros-
phax tricolor in New Caledonia; C.
saqanqouri (Abdel-Baki et al.,
2008) from Scincus scincus from
Egypt; C. scincellae (Telford 1997)
from Scincellae lateralis in USA;
and C. scinci (Phisalix 1923) from
Scincus officinalis in Tunisia.
Choleoeimeria riyadhae sp.n. can
be easily distinguished from C. au-
ratae, C. egerniae, C. egregia, C.
163

pellopleuris, and C. scincellae, in
having much larger oocysts and
sporocysts, having much smaller
shape index of oocyst, and much
larger shape index of sporocysts and
in the shape of oocysts, and differs
markedly from C. fasciatus, C. pel-
lopleuris, and C. sadlieri in having
broadly ellipsoid oocyst rather than
cylindrical shape in the oocysts of
these three species. Besides, it dif-
fers from C. fasciatus and C. sad-
lieri in lacking polar granule. Cho-
leoeimeria riyadhae resemble those
of C. chalcides, C. hemprichii C.
saqanqouri and C. scinci in size of
oocysts, but differ from them in
having much wider oocysts, much
smaller shape index of the oocysts,
in the shape and size of sporocysts,
and in having much larger length to
width ratio of sporocysts. This is in
addition to the difference in the
geographical locations and the in-
fected host species. By comparing
Choleoeimeria riyadhae to other
eimerid species previously de-
scribed from scincid hosts, it is clear
that C.riyadhae is a distinct form,
which leads us to consider it as a
new species.

Conclusion

The identification of the new spe-
cies named Choleoeimeria riyadhae
pave the way for the extensive field
surveys for other new species of
Apicomplexa: Eimeriidae, not only
in Saudi Arabia but also in other
countries where reptiles are com-
mon

References

Abdel-Baki, A.S.; El-Fayomi, H.
M.; Sakran, Th.; Abdel-Haleem,
H.M., 2008: Choleoeimeria saqan-
qouri sp. nov (Apicomplexa: Eime-
riidae) infect-the gall bladder of
Scincus scincus scincus (Reptelia:
Scincidae) from Tunis. Acta Proto-
zool., 47:143-7.
Alyousif, M.S.; Al-Rasheid, A.S.,
2001: Eimeria auratae n. sp. (Api-
complexa: Eimeriidae) infecting the
lizard Mabuya aurata in Saudi Ara-
bia. Parasitol. Inter., 50:27-32.
2005: Eimeria hemprichii n.sp.
(Apicomplexa: Eimeriidae) from the
lizard, Scincus hemprichii (Sauria:
Scincidae) in Saudi Arabia. J.
Bovee, E.C., 1971: New species of
Eimeria from lizards of Japan.
Trans. Amer. Microsc. Soc., 90: 336
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Cannon, L.R.G., 1967: New cocci-
dia from Australian lizards: II- Ei-
meria. Parasitol., 57:237-50.
2001: Artifacts that can be confused
with parasitic organisms. In: Diag-
nostic Medical Parasitology. 4
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Ed., American Society for Microbi-
ology Press, Washington, D.C.
Jirku, M.; Modry, D.; Slapeta, J.
R.; Koudela, B.; Lukes, J., 2002:
The phylogeny of Goussia and Cho-
leoeimeria (Apicomplexa: Eimeri-
orina) and the evolution of excysta-
tion structures in coccidian. Protis-
tol., 135: 379-90
164

Modry, D.; Jirku, M., 2006: Three
new species of coccidia (Apicom-
plexa: Eimeriidae) from the Marble-
throated scink, Marmorosphax tri-
color Bavay, 1869 (Reptelia: Scin-
cidae), endemic to New Caledonia
with taxonomic revision of Eimeria
spp. from scincid hosts. Parasitol.
Res., 99:419-28.
Paperna, I.; Landsberg, J.H.,
1989: Description and taxonomic
discussion of eimerian coccidian
from African and Levantine geck-
oes. S. Afr. J. Zool., 24: 345-55.
Phisalix, M., 1923: Development
sporogonique du Coccidium scinci
nov. sp.,
parasite des voies biliares du Scin-
cus officinalis Laur. Bull. Mus.
Natl. Hist. Nat. Paris, 29:446-7.
Telford, S.R., 1997: Two new spe-
cies of Eimeria Schneider, (Api-
complexa: Eimeriidae) from scinks
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Explanation of Figures

Figs. 1-2: Photomicrographs of living oocysts of Choleoeimeria riyadhae sp. n. Scale
bar= 10m.
Fig.1: Sporulated oocyst showing four elongate ellipsoid sporocysts (arrow).
Fig.2: Sporulated oocyst containing free sporozotes (*).
Figs. 3-4: Photomicrographs of endogenous stages of Choleoeimeria riyadhae infecting
gall blad-der epithelium of Scincus scincus. Scale bar = 10m.
Fig. 3: Microgamont (arrow) and host nucleus (arrowhead).
Fig. 4: Macrogamont (arrow) and host nucleus (arrowhead).

160

J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No.14

J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No.15
J. Egypt. Soc. Parasitol., 40 (1), 2010, Article No. 15 P2


165



DISINFECTION EFFICACY OF SODIUM DICHLOROISOCYANURATE
(NADCC) AGAINST COMMON FOOD-BORNE
INTESTINAL PROTOZOA
By
LOBNA A. EL ZAWAWY,

DOAA EL-SAID, SAFIA M. ALI
AND FOUAD M. FATHY
Department of Parasitology, Faculty of Medicine, Alexandria University,
Alexandria, Egypt.

Abstract

The present study was conducted to investigate the efficacy of sodium dich-
loroisocyanurate (NaDCC) on the infective stages of common food-borne in-
testinal protozoa; Entamoeba histolytica (E. histolytica), Giardia lamblia (G.
lamblia), Cryptosporidium, Cyclospora and Microsporidia; beside its effect on
raw green vegetables and fruits. Parasites, isolated from stool of patients with
diarrhea or dysentery, were exposed to NaDCC solution (1g/l) for one and two
hours. Disinfection effect of NaDCC was assessed by in-vitro viability, using
trypan blue stain, and infectivity bioassay in laboratory animals as indicated by
fecal and intestinal parasitic counts. Raw vegetables and fruits were dipped in
NaDCC solution in the same concentration and exposure time as used for
treatment of the parasites. Results revealed statistically significant reductions in
viability and infectivity of all examined parasites indicating their susceptibility
to NaDCC. Relative variations in susceptibility were revealed; E. histolytica
and G. lamblia were most susceptible (100% reduction) followed by Microspo-
ridia then Cryptospridium and Cyclospora. NaDCC did not affect the consis-
tency, color, taste or flavor of raw green vegetables and fruits. The proved effi-
cacy of NaDCC, in cheap and convenient dry tablet form, makes it a promising
tool in decontaminating raw vegetables and fruits from food-borne protozoan
parasites at household and restaurant levels as well as in catering and fresh
produce industry. It is also recommended for disinfection of food preparation
surfaces and equipment.
Keywords: Food-borne protozoa, Sodium dichloroisocyanurate, disinfections,
viability, infectivity, experimental animals.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

The food-borne parasitic diseas-
es are generally under recognized,
however, they are becoming more
common and their impact on health
have been established (Dorny et al.,

166

2009). Although food-borne proto-
zoan parasites have been found re-
sponsible for many outbreaks of
gastroenteritis, recently the attention
has focused on them (Rose and Slik-
fo, 1999). Because of inadequate
systems for routine diagnosis and
monitoring or reporting for many of
the food-borne protozoan parasites,
the incidence of human disease and
parasite occurrence in food is unde-
restimated (Dorny et al., 2009).
Recognized as waterborne para-
sites, Giardia, Cryptosporidium,
and Cyclospora have now been as-
sociated with several food-borne
outbreaks. These parasites emerged
as public health risks and have be-
come a concern to the food industry
(Rose and Slifko, 1999). Beside to
the previously mentioned protozoa,
E. histolytica and Microsporidia
infections have been also linked to
food consumption. The shedding of
cysts, oocysts or spores into faeces
which may then, directly or indirect-
ly (via sewage or irrigation water),
contaminate raw vegetables and
fruits occurs on a global scale and it
may be common in countries where
the hygienic conditions (especially
water quality) are compromised
(WHO, 1999). Some data are avail-
able to indicate that animal wastes
remain an important source of con-
tamination (Slifko et al., 2000). Pa-
rasitic protozoa do not multiply in
foods but can persist and survive for
long periods of time both in water
and on foods in cool and damping
environment (Rose and Slifko,
1999; Dawson, 2005). Consump-
tion of raw and undercooked vege-
tables, to retain the natural taste and
preserve heat-labile nutrients, can
increase the risk of transmission of
food-borne parasites, while none of
these organisms has been shown to
be a problem for heat processed
food (Slifko et al., 2000; Dawson,
2005).
Numerous surveys have been car-
ried out in many countries and re-
vealed the presence of protozoan
parasites on raw vegetables and
fruits. Straw and raspberries, lettuce
and basil have been the implicated
vehicles of transmission in out-
breaks of Cyclospora cayetanensis
infection (CDC, 1996c; 1997b,c,d;
Abou El Naga, 1999). A survey of
vegetables has revealed the presence
of Cryptosporidium oocysts on let-
tuce, radish, tomato, cucumber, ci-
lantro and carrot (Monge and Chin-
chilla, 1996). Microsporidia has
also been isolated from the similar
vegetables and fruits (Calvo et al.,
2004). In addition, raw sliced vege-
tables were reported to be a vehicle
of transmission in an outbreak of
giardiasis and amoebic dysentery
(Mintz et al., 1993).
The ecology of these parasites
makes their control difficult. Pre-
vention of contamination at all
points of the food chain from prima-
ry production to the final consumer
is preferred over the different con-
trol procedures (WHO, 1999). For
general control of parasitic protozoa
in the food chain, the first important
step is to follow good hygienic prac-
tice in food service and catering in-

167

dustries (Dawson, 2005). Vigorous-
ly washing vegetables and fruits
with safe running tap water reduces
the number of microorganisms, but
not totally eliminate the risk of in-
fections especially when use tank
water in this process as it may act
per se as a source of infections. Ad-
ditional 10 to 100 fold reductions
can sometimes be achieved by
treatment with disinfectants (WHO,
1999). However, such chemicals or
disinfectants must be safe, effective
in complete eradication of infections
with no direct or indirect side ef-
fects (Gilbert, 1970). The mechan-
ism of action of many disinfectants
on microbial cells is poorly unders-
tood. In addition, little is known
about the efficacy of disinfectants
on food-borne protozoan parasites.
The sodium dichloroisocyanurate
(NaDCC) is a kind of organic chlo-
rine disinfectant. It contains about
62% of available chlorine (Martin-
dale, 1993). When it dissolves in
water it hydrolysis gives hypochlor-
ous acid (HOCl) that is the active
moiety, isocyanurate and isocyanu-
rate chlorine. These compounds
form a chlorine protein that carries
out microbicidal activity (D'Auria et
al., 1989). The latter protein carrier
forms a balancing reservoir of stabi-
lized chlorine, rapidly compensating
for in-action depletion of free avail-
able chlorine (FAC) (Clasen and
Edmondson, 2006). The antimi-
crobial activity of NaDCC was re-
ported against 29 gram- negative, 29
gram- positive bacteria and 66 fungi
(D'Auria et al., 1989) and also
against viruses (Dychdala, 2001).
Furthermore it was found to be ef-
fective in killing and destruction of
metacercariae of Fasciola gigantica
(El Zawawy et al., 2002). In addi-
tion, its broad spectrum activity ex-
tended to protozoa as Trichomonas
vaginalis, Acanthamoeba castellanii
cysts and it may also extend to in-
testinal protozoa (D'Auria et al.,
1989; Khunkitti et al., 1996).
The present study conducted to
investigate the potential effect of
NaDCC on the infective stages of
the most common food-borne proto-
zoan parasites; E. histolytica, G.
lamblia, Cryptosporidium, Cyclos-
pora and Microsporidia. In addition
its use in washing raw green vege-
tables was studied.


Disinfectant: NaDCC disinfectant
tablets supplied by Coreline Chemi-
cals Limited Company (U.K) were
used in the present study. Each tab-
let (1g) was dissolved in 500 ml
water to form a colorless solution.
Then the disinfectant solution was
mixed with an equal amount of each
investigated parasite suspension so
that the final concentration of the
disinfectant was 1g/l, according to
the manufacturer specifications.
Parasites: Fifty stool samples were
collected from patients suffering
from diarrhea or dysentery and ad-
mitted to out patients clinics of
Alexandria University Hospital.
Each sample was concentrated by
saline sedimentation concentration

168

and Sheather's sugar floatation tech-
niques, then examined by wet
mount iodine smears, modified
Ziehl-Neelsen acid fast stain (MZN)
(Garcia and Bruckner, 1997) and
modified trichrome stain (MTS)
(Weber et al., 1992). Positive sam-
ples for cysts of E. histolytica and
G. lamblia, oocysts of Cryptospori-
dium and Cyclospora and spores of
Microsporidia were pooled sepa-
rately and the parasites in each
pooled sample were isolated accord-
ing to Lump et al. (1993), sus-
pended and stored in 2.5% potas-
sium dichromate at 4

C until used
(Uga et al.,2002). In case of Cyclos-
pora, positive samples were placed
in 2.5% potassium dichromate in
covered Petri-dish and incubated at
room temperature for two weeks.
Daily changes in the appearance of
the organisms were observed by
phase contrast microscopy till spo-
rulation (Soave and Johnson, 1995).
Before use, the isolated parasites
were washed three times in distilled
water to remove the potassium dich-
romate and pellet the parasites by
centrifugation for five minutes each
at 1500 rpm (Youssef et al.,1992).
Each parasite was counted with a
haemocytometer and adjusted to the
required concentration for the expe-
rimental study by dilution in appro-
priate amount of distilled water to
form a suspension. Each parasite
suspension was mixed with an equal
amount of disinfectant (NaDCC)
solution. Non-treated control for
each parasite (parasite control) was
prepared by mixing equal amounts
of distilled water and each parasite
suspension in a concentration simi-
lar to that used with the disinfectant.
Another control formed of disinfec-
tant solution alone (disinfectant con-
trol) was prepared in the same way
as for treatment of the parasites
(1g/l water).
Each of the five disinfectant-
parasite suspensions was divided
into two portions; the first one was
exposed to the disinfectant for one
hour (hr.) while the second one was
exposed for two hours (hrs.) at room
temperature. After exposure, the
suspension tubes were centrifuged
at 1500 rpm for five minutes to pel-
let the parasites. Disinfectant solu-
tion was discarded and the parasites
in the sediment were washed twice
in distilled water by centrifugation
and finally re-suspended in distilled
water.
In vitro viability assay: By stain-
ing 500l of each suspension
(NaDCC-treated parasites and non-
treated parasite control) with a vital
stain; 0.2% trypan blue stain (Ox-
ford Lab Reagent, 23850, Mumbai-
400 002) (Powell et al.,2003). The
number of viable parasites/100 or-
ganisms in each treated suspension
was counted in ten fields under a
light microscope and the mean
counts were calculated for each pa-
rasite and compared with that of the
corresponding control and the per-
centage reduction (% R) was calcu-
lated (Penido et al., 1994).
Infectivity bioassay in experimen-
tal animals: Laboratory reared ani-
mals, free from parasites, were used

169

to assess the infectivity of the para-
sites. They were divided into two
main groups: GI: Control group was
further subdivided into two sub-
groups (SG): SG Ia: Treated non-
infected SG (six Swiss albino mice
and six white rats). Each animal in
this SG was inoculated orally with
0.1ml of disinfectant control solu-
tion. SG Ib: Non-treated infected
SG (24 Swiss albino mice and six
white rats). SG was further subdi-
vided into five SG. Each of the first
four SG, six mice each (Ib1-Ib4)
was inoculated orally with 0.1
ml/mouse of one of the non-treated
parasite control suspension so that
the infective dose for each mouse
was 5x10
3
cysts for E. histolytica
(Sadaka et al., 2001), 1x10
6
oocysts
for Cryptosporidium (Heine et al.,
1984), 1x10
6
sporulated oocysts for
Cyclospora (Soave and Johnson,
1995), and 1x10
5
spores for Micro-
sporidia (Allam et al., 1999). While
the fifth SG (Ib5), which was
formed of six white rats, was inocu-
lated orally with 0.1 ml of non-
treated G. lamblia suspension con-
taining 2 x 10
5
cysts/ rat (Youssef et
al., 1996).
GII: Experimental group: (24
Swiss albino mice and six white
rats). It was further subdivided into
ten SG. Each of the first eight SG
(six mice each); IIa 1&2 (E. histoly-
tica-treated SG), IIb 1&2 (Cryptos-
poridium-treated SG), IIc 1&2 (Cyc-
lospora-treated SG) and IId 1&2
(Microsporidia-treated SG) was
inoculated orally with 0.1 ml/mouse
of one of the parasite suspension
treated with NaDCC for one and
two hrs. respectively. While the re-
maining two SG (IIe 1&2), six
white rats each, were inoculated
orally with 0.1 ml/rat of G. lamblia
suspension treated with NaDCC for
one and two hrs. respectively. The
infective dose for each animal was
similar to that of the corresponding
non-treated infected control (Ib).
Scarification of animals was
done at time of maximum infection
and colonization of intestine by the
parasites which was 7
th
day post
infection (P.I) for all SG (Youssef et
al., 1994; Tzipori et al., 1997; Al-
lam et al., 2004) except E. histolyti-
ca infected SG (Ib1 and IIa1&2)
which were sacrificed three weeks
P.I (Sadaka et al., 2001).
Stool was collected from each
animal in each infected SG on sacri-
fice day and wet mount iodine
smears were done from stool sample
of each animal infected with E. his-
tolytica and G. lamblia. While
smears were done from stool sample
of each animal infected with Cryp-
tosporidia, Cyclospora and Micro-
spridia and stained with MZN (Gar-
cia and Bruckner, 1997) and MTS
(Weber et al., 1992) respectively.
Ten high power field (HPF) were
examined for each sample except
for those of Microspridia in which
ten oil immersion fields (OIF) were
examined. Infectivity % in each an-
imal SG was estimated. the mean
number of cysts or oocysts /HPF or
spores/OIF in the ten fields was
counted and the mean count was
calculated for each animal SG. %R

170

in the mean fecal parasitic count in
each animal SG infected with
treated parasites was also calculated
in comparison with the correspond-
ing non-treated infected control.
After scarification of animal by
overdose of diethyl-ether, parts of
colon were dissected from each
mouse infected with E. histolytica
and parts of small intestine were
removed from each animal infected
with the other parasites. Then they
were fixed in 10% formalin and
processed for histopathological ex-
amination using MTS for Microspo-
ridia (Weber et al., 1992) and hae-
matoxylin and eosin (H&E) stain
for the remaining parasites (Current
and Reese, 1986). Intestinal sections
of each animal were examined mi-
croscopically for parasites and the
infectivity% in each animal SG was
calculated. Counting of different
parasitic stages was done under oil
immersion lens except G. lamblia
trophozoites which were counted
using high power magnification.
Ten microscopic fields for each sec-
tion were examined, the mean was
taken and the mean count of para-
sites/HPF or OIF was calculated for
each animal SG. The % R in the
mean intestinal parasitic count in
each animal SG infected with
treated parasites was also calculated
in comparison with the correspond-
ing non-treated infected control.
Effect of NaDCC on vegetables
and fruits: Some fresh green leaves
of lettuce, parsley and celery, raw
eaten vegetables as cucumbers and
pepper and non peel able fruits as
strawberries and tomatoes were
used in the present study, as sam-
ples of vegetables and fruits, to in-
vestigate the effect of NaDCC on
them. They were divided into two
portions; the first one was soaked in
NaDCC solution for one hr., while
the second portion was dipped in
NaDCC solution for two hrs. at the
same concentration used for treat-
ment of the parasites (1g/l water).
After exposure to the disinfectant
solution, the vegetables and fruits
were removed and washed several
times with clean running water, then
examined as regards; color, consis-
tency, taste and flavor.
Statistical analysis: Data were sta-
tistically analyzed by using student
t-test. P values 0.05 were consi-
dered statistically significant (Casle,
1977).

Results

Effects of NaDCC on the para-
sites' viability (Tab. I) was assessed
by using 0.2% trypan blue stain.
Living parasites showed dye exclu-
sion activity and appeared clear with
a very light blue colour, while dead
organisms looked dark blue in co-
lour and the internal structure could
not be detected.
After exposure to NaDCC for
one hr., the mean number of viable
cysts/100 E. histolytica cysts was
4.5, while in the corresponding non-
treated parasite control it was 94.6
(%R= 95.2%). This difference was
statistically significant (p<0.001).
Extension of exposure up to two hrs

171

added a significant extra-reduction
in the viability reaching 98.8%. The
viability difference between the two
exposure times was statistically sig-
nificant (p<0.001). Figures (1 & 2)
display viable and dead cysts of E.
histolytica respectively.
The mean number of viable oo-
cysts /100 Cryptosporidium and that
for Cyclospora oocysts was 21.5 &
23.1 respectively after one hr. expo-
sure to NaDCC while in the corres-
ponding non-treated parasite control
it was 97.2 and 95.5 respectively
(reduced by 77.9% &75.8% respec-
tively). This difference was statisti-
cally significant (p<0.001). Further
significant reduction in the mean
number of viable oocysts (%
R=96.9% & 96.2% respectively)
could be obtained after contact with
NaDCC for two hrs. The difference
in the mean number of viable oo-
cysts between the two exposure
times was statistically significant
(p<0.001). Viable and dead oocysts
of Cryptosporidium are shown (figs.
3, 4 respectively), those of Cyclos-
pora shown in (figs. 5,6 respective-
ly).
Microsporidia spores treated
with NaDCC for one hr. showed
reduction by 77.8% in their viability
as the mean number of viable spores
/100 organisms was 21.3 compared
with 96.0 in the corresponding non-
treated parasite control, with signifi-
cant difference (p<0.001). 97.1%
reduction was reached after two hrs
exposure to NaDCC. Difference of
mean number of viable spores be-
tween two exposure times was sig-
nificant (p<0.001). (Figs. 7, 8 viable
and dead spores respectively),
After exposure to NaDCC for an
hr., the mean number of viable
cysts/100 G. lamblia cysts was 5.2,
while in the corresponding non-
treated parasite control it was 95.2
(%R= 94.5%). This difference was
Extension of exposure to two hrs
induced a further significant reduc-
tion in the mean number of viable
cysts and % R reached 98.3%. The
viability difference between the two
exposure times was statistically sig-
nificant (p<0.001). (Figs. 9,10 via-
ble and dead cysts respectively).
Effects of NaDCC on the para-
sites' infectivity (Tab. II): No deaths
were observed either in the control
SG (Ia &Ib) or in the experimental
SG (IIa,b,c,d & e) [mortality rate
was 0%]. All animals (100%) inocu-
lated with non-treated parasite con-
trol (SG Ib1- Ib5) were infected
whether by detection of the parasites
in stool or in intestinal sections.
In mice infected with E. histoly-
tica cysts exposed to NaDCC for
one hr. (SG IIa1), the infectivity rate
was 16.6%. Mean number of cysts
in stool of mice was 0.1/HPF com-
pared with 3.5 in non-treated in-
fected control mice (SGIb1) (%R=
97.1%) with significant difference
(p<0.001). Fig (11) showed E. histo-
lytica cyst in stool. Mean number of
trophozoites/OIF in colonic sections
was 0.2 compared to 5.9 in non-
treated infected control (%R=
96.6%) with significant difference
(p < 0.001).

172

Table I: Parasites' viability as indicated by 0.2%trypan blue stain, in non-
treated parasite control and NaDCC-treated parasites after
exposure for one and two hrs.

Parasites

Mean no (X) of
viable parasites/ 100
organisms SD in
non-treated parasite
control (%)
Parasites' viability in NaDCC-treated parasites
Test of sig.
After one hr. exposure After two hrs. exposure
Mean no (X) of
viable parasites / 100
organisms SD (%)
%R
Mean no (X) of
viable parasites / 100
organisms SD (%)
%R
E.histolytica 94.6 2.3 (94.6%) 4.5 0.9 (4.5%) 95.2 1.1 0.2 (1.1%) 98.8
t1= 364.8
*
, p
<0.001
t2= 32.199
*
,
p <0.001
Cryptospori-
dium
97.2 1.9 (97.2%) 21.52.4 (21.5%) 77.9 2.9 1.4 (2.9%) 96.9
t1= 242.73
*
,
p<0.001
t2= 71.262
*
,
p <0.001
Cyclospora 95.5 1.2 (95.5%) 23.11.9 (23.1%) 75.8 3.6 1.6 (3.6%) 96.2
t1= 321.73
*
, p
<0.001
t2= 53.813
*
,
p <0.001
Microspori-
dia
96.0 0.6 (96%) 21.31.1 9 (21.3%) 77.8 2.7 0.3 (2.7%) 97.1
t1= 596.17
*
, p
<0.001
t2= 71.258
*
,
p <0.001
G. lamblia 95.2 1.7 (95.2%) 5.20.3 (5.2%) 94.5 1.6 0.41 (1.6%) 98.3
t1= 521.36
*
, p
<0.001
t2= 29.667
*
,
p <0.001

%R: % reduction.
t1: Student t-test and its significance between mean no of viable parasites/100 organisms after exposure to
NaDCC for one hr., for each treated parasite, and those of the corresponding non-treated parasite control.
t2: Student t-test and its significance between mean no of viable parasites/100 organisms after exposure to
NaDCC for one hr. and those after exposure for two hrs for each treated parasite.
*: Statistically significant (p <0.001).

E. histolytica trophozoite was
detected intraluminal with apparent
nucleus in colonic sections of in-
fected mouse whether inoculated
with non-treated or treated parasites
(Fig. 12). After exposure for two hrs
to NaDCC (SG IIa2) no mouse was
found to be infected since no cysts
or trophozoites were detected in
stool or in colonic sections respec-
tively (%R =100%).
The infectivity% of Cryptospori-
dium and Cyclospora in mice in-
fected with each parasite exposed to
NaDCC for one hr. (SG IIb1 &IIc1
respectively) was 33.3% each. The
mean number of oocysts in stool of
these mice was 2.2 and 2.6/HPF
compared with 9.8 and 11.6 in the
corresponding non-treated infected
control respectively. %R was 77.6%
& 76.7% respectively, with signifi-
cant difference (p< 0.001). Cryptos-
poridium and Cyclospora oocysts in
stool are shown (figs. 13,14) respec-
tively. Mean number of oocysts/OIF
in intestinal sections of mice was
1.4 & 1.8 compared with 6.2 & 7.6
oocysts in corresponding to non-
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173

treated infected control respectively.
%R =77.4%& 76.3% respectively.
This difference was statistically
significant (p<0.001). Cryptospori-
dium oocysts was small rounded on
the brush border of the mucosal epi-
thelium of ilial sections of mice in-
oculated with treated or non-treated
parasites (Fig. 15). Cyclospora oo-
cysts were rounded in the mucosal
villi of ilial sections of mice inocu-
lated with treated or non-treated
parasites (Fig. 16). In mice inocu-
lated with oocysts exposed to
NaDCC for two hrs (SG IIb2 &IIc2
respectively), infectivity was 16.6%
for each one, mean number of oo-
cysts in stool was 0.4& 0.6/HPF and
%R was 95.9& 94.8% respectively.
Difference in the mean fecal parasit-
ic counts between the two exposure
times was statistically significant
(p<0.001). In intestinal sections the
mean number of oocysts/OIF= 0.3
&0.4 & %R= 95.2% &94.7% re-
spectively. The difference in mean
intestinal parasitic counts between
two exposure times was statistically
significant (p<0.001).
Table II: Mean fecal and intestinal parasitic count and percentage reduction
in different groups of animals.

Test of sig. %R
Mean intestinal
parasitic count
SD
Test of sig. %R
Mean fecal para-
sitic count SD
Infectiv-
ity %
No of
animals

Animal groups
t1= 8.709
*
,
p <0.001
-

96.6
100%
5.9 1.6(OIF)
0.20.1(OIF)
0.00.0
t1= 20.509
*
,
p <0.001
-

97.1%
100%
3.5 0.4(/HPF)
0.10.07(/HPF)
0.00.0
100%
16.6%
0%
6 mice
6 mice
6 mice
Ib1
IIa1
IIa2
E.histolytica
infected SG
t1= 6.443
*
,
p <0.001
t2 =12.321*,
p <0.001

77.4%
95.2%
6.2 1.8(OIF)
1.40.3(OIF)
0.30.05(OIF)
t1= 16.327
*
,
p <0.001
t2 = 14.412
*
,
p <0.001

77.6%
95.9%
9.8 1.1(/HPF)
2.20.3(/HPF)
0.40.06(/HPF)
100%
33.3%
16.6%
6 mice
6 mice
6 mice
Ib2
IIb1
IIb2
Cryptospori-
dium infected
SG
t1= 12.460
*
,
p <0.001
t2 = 4.897
*
,
p = 0.001

76.3%
94.7%
7.6 0.9(OIF)
1.80.7(OIF)
0.40.02(OIF)
t1= 15.097
*
,
p <0.001
t2 = 6.643
*
,
p <0.001

76.7%
94.8%
11.6 1.3(/HPF)
2.60.7(/HPF)
0.60.03(/HPF)
100%
33.3%
16.6%
6 mice
6 mice
6 mice
Ib3
IIc1
IIc2
Cyclospora
infected SG
t1= 25.402
*
,
p <0.001
t2 =7.924
*
,
p <0.001

82.0%
95.7%
32.3 2.2(OIF)
5.81.3(OIF)
1.40.4(OIF)
t1= 18.939
*
,
p <0.001
t2 = 11.631
*
,
p <0.001

80.1 %
96.9%
22.6 2.2(/OIF)
4.5 0.8(OIF)
0.7 0.02(OIF)
100%
16.6%
16.6%
6 mice
6 mice
6 mice
Ib4
IId1
IId2
Micropsporidia
infected SG
t1= 30.435
*
,
p <0.001
-

96.4%
100%
24.81.9(/HPF)
0.90.3(/HPF)
0.00.0
t1= 20.381
*
,
p <0.001
-

96.2%
100%
7.8 0.9(/HPF)
0.30.05(/HPF)
0.00.0
100%
16.6%
0%
6 rats
6 rats
6 rats
Ib5
IIe1
Ie2
G. lamblia
infected SG

SGIb1: Mice infected with non-treated E.histolytica cysts, SGIIa1, 2: Mice infected with E.histolytica treated cysts for 1 hr.,
2 hrs. SGIb2: Mice infected with non-treated Cryptosporidium oocysts, SGIIb1, 2: Mice infected with Cryptosporidium
treated oocysts for 1 hr., 2 hrs. SGIb3: Mice infected with non-treated Cyclospora oocysts, SGIIc1,2: Mice infected with
Cyclospora treated oocysts for 1 hr., 2 hrs. SGIb4: Mice infected with non-treated Micropsporidian spores, SGIId1, 2: Mice
infected with Micropsporidia treated spores for 1 hr., 2 hrs., SGIb5: Rats infected with non-treated Giardia cysts, SGIIe1,2:
Rats infected with Giardia treated cysts for 1 hr., 2 hrs. %R: % reduction.
t1: Student t-test and its significance between experimental SGII1 and the corresponding non-treated control SG.
t2: Student t-test and its significance between two experimental SG for each parasite.
*: Statistically significant (p <0.001).
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174

Mice inoculated with microspori-
dian spores treated with NaDCC for
one hr. (SGIId1) showed infectivi-
ty% of 16.6% and the mean number
of spores in stool of these mice was
4.5/OIF compared with 22.6 in the
corresponding control and the %R
was 80.1%. This difference was sta-
tistically significant (p<0.001). Fig.
(17) display microsporidian spores
in stool. While the mean number of
spores in intestinal sections of these
mice was 5.8/OIF compared with
32.3 in the corresponding non-
treated infected control (%R was
82.0%). This difference was statisti-
cally significant (p<0.001). Spores
of Microsporidia were seen inside
the villous enterocytes and inside
the crypts of ilial sections of mice
whether infected with treated or
non-treated spores (Fig. 18). After
exposure for two hrs, (SGIId2) the
infectivity% was still 16.6% but the
mean number of spores in stool was
reduced to 0.7 spores/OIF and the
%R was 96.9%. The difference in
the mean fecal parasitic counts be-
tween the two exposure times was
The mean number of spores in intes-
tinal sections was also reduced to
1.4/OIF (%R was 95.7%) and the
difference in the mean intestinal
parasitic count between the two ex-
posure times was statistically signif-
icant (p<0.001).
The infectivity % among rats
inoculated with G.lamblia cysts
treated with NaDCC for one hr. (SG
IIe1) was 16.6% and the mean fecal
parasitic count was 0.3 cysts /HPF
compared with 7.8 in the corres-
ponding non-treated infected control
and the %R was 96.2%. This differ-
ence was statistically significant
(p<0.001). G.lamblia cysts in stool
are clarified in Fig. (19).The mean
number of trophozoites/HPF in the
intestinal sections was 0.9 compared
with 24.8 in non-treated infected
rats (%R was 96.4%). This differ-
ence was statistically significant
(p<0.001). Giardia trophozoites
were observed as groups of pear
shaped organisms in between the
intestinal villi and along the mucos-
al surfaces of intestinal sections of
rats infected whether with treated or
non-treated cysts (Figs. 20&21).
After exposure for two hrs, (SGIIe2)
no rat was found to be infected as
no cysts or trophozoites were de-
tected in the stool or in the intestinal
sections respectively denoting
%R=100.
Effect of NaDCC on raw vegeta-
bles and fruits: Leaves of green
vegetables, raw eaten vegetables
and fruits exposed to NaDCC ap-
peared normal and fresh. No
changes occurred in their colour,
consistency, taste or flavor. They
were easily rinsed with water after
being removed from the disinfectant
without leaving any trace of chemi-
cal on them. The two investigated
exposure times (1 & 2 hrs) gave the
same results.

Discussion


175

Isolation of intestinal protozoa
from different vegetables and fruits
available on the market has been
reported (Monge and Arias, 1996;
Calvo et al., 2004) and human in-
fection could be attributed to their
consumption (Robertson et al.,
2005). These important emerging
and re-emerging intestinal protozoa
are difficult to diagnose or treat
(Dorny et al., 2009; Didier et al.,
2004). Therefore, their control de-
pends primarily on the ability to
prevent, remove or kill protozoal
contaminants (Rose et al., 1999).
NaDCC, present a dry convenient
form of stable long-acting chlorine
(Clasen and Edmondson, 2006). Its'
wide biocidal spectrum have been
reported (D'Auria et al., 1989;
Khunkitti et al., 1996; El Zawawy et
al., 2002). Chlorine concentration
allowed for life-time consumption
in drinking water is not sufficient to
kill the resistant infective stages of
most intestinal protozoa, however
higher concentrations can be used
for disinfection of contaminated
vegetables and fruits.
In the present study, NaDCC at a
concentration of 1 gm/l and expo-
sure times of one and two hrs was
chosen to investigate its effect on
infective stages of the most common
food-borne protozoan parasites that
their impact on health have been
established namely; E. histolytica,
G. lamblia, Cryptosporidium, Cyc-
lospora and Microsporidia. Parasite
inactivation after exposure to the
NaDCC was assessed by in-vitro
viability assay by vital stain (trypan
blue) and in-vivo infectivity bioas-
say in laboratory bred animals indi-
cated both by fecal and intestinal
parasitic counts.
Considering E. histolytica, high
significant reduction in cyst viabili-
ty was observed after one hr. expo-
sure to 1gm/l NaDCC (95.2%), con-
firmed by parallel high significant
reductions of mean parasitic counts
(97.1%, 96.6% in fecal and intestin-
al parasitic counts respectively). E.
histolytica cysts were no more in-
fective to mice after two hrs expo-
sure to NaDCC (R=100%) and via-
bility reached 98.8%. Such figures
reflect the relative high efficacy of
NaDCC against E. histolytica at the
selected concentration and exposure
times. Also, Dychdala (2001) re-
ported that susceptibility to hypoch-
lorous acid (HOCl), the active moie-
ty in NaDCC, has been established
in bacteria, helminths and protozoa
including E. histolytica and G. lam-
blia. Stringer et al.(1975) reported
low CT value ( = conc. in mg x ex-
posure time in min.) of 20 (2mg/l
for 10 min), using in-vitro excysta-
tion only as index of viability. Hoff
and Akin (1986), reported a CT val-
ue of 90 (5mg/l for 18 min.) for in-
activation of E. histolytica by free
chlorine without specifying the ex-
act reduction percentages.
As regards G. lamblia, cyst via-
bility was also effectively reduced
by NaDCC after one hr. exposure
(94.5%), together with high reduc-
tion of mean parasitic counts
(96.2% and 96.4% in fecal and in-
testinal parasitic counts respective-

176

ly). Cysts were completely non in-
fective after two hrs. exposure to
NaDCC (%R=100%) and vitality
reached 98.3%. These high figures
prove the effective inactivation of
G. lamblia cysts by NaDCC which
was comparable to that observed
with E. histolytica. In accordance,
Jarroll et al.(1981), stated that chlo-
rine cysticidal concentrations and
dynamics of disinfection are almost
similar for G. lamblia and E. histo-
lytica. Sinski (2003) reported a CT
value of 93-121 for inactivation of
G. lamblia by free chlorine, which
exceeds that of E. histolytica, denot-
ing higher relative resistance. Rice
(1982) reported over 90% reduction
in viability of G. lamblia cysts as
indicated by excystaion- at a drink-
ing water chlorine concentration of
2.5mg/l.
However, Khalifa et al. (2001)
found only 0.15% infectivity reduc-
tion after the exposure to 8ppm
(=8mg/l) chlorine for extended con-
tact duration of 24 hrs.
Regarding Cryptosporidium oo-
cysts, although significant reduc-
tions in vitality (77.9%) and mean
fecal and intestinal parasitic counts
(77.6% and 77.4% respectively)
were observed after one hr. expo-
sure, high significant reductions
could be obtained after extension of
contact time up to two hrs. (Vitality:
96.9% -mean parasitic counts:
95.9% and 95.2% respectively).
This difference reflects the relative
resistance of Cryptosporidium oo-
cysts to the shorter treatment unlike
the case with E. histolytica and G.
lamblia which were more suscepti-
ble to NaDCC. In agreement, Khali-
fa et al. (2001) reported reduced
Cryptosporidium infectivity of 44%
using 8mg/l chlorine for 24 hrs.
Meanwhile, Korich et al. (1990)
found that 80ppm of free chlorine
for 90 min. was necessary to pro-
mote inactivation of Cryptospori-
dium oocysts with CT value of
7.200 which also reflects the ob-
served parasite higher relative resis-
tance. The authors concluded that
chlorine disinfection alone would
not be sufficient in controlling
Cryptosporidium in drinking water,
where chlorine should not exceed 5
mg/l, according to WHO guidelines
(World Health organization, 1993).
The same authors also found that
Cryptosporidium was 30 times more
resistant to ozone and 14 times to
chlorine dioxide than G. lamblia
cysts. Pereira et al. (2008) using
HOCl acid at 2ppm, obtained a
maximal Cryptosporidium inactiva-
tion rate of 49.04% after 120 min.
On the contrary, Fayer (1995)
treated the oocysts with aqueous
solution of hypochlorite bleach at
conc.1.35% for two hrs. without
eliminating infectivity. Moreover,
Cryptosporidium oocysts can sur-
vive up to 12 months in these
aqueous suspensions (Current and
Haynes, 1984). Its resistance could
be attributed to the thick oocyst wall
acting as a barrier for diffusion of
the disinfectant.
Cyclospora oocysts, showed sig-
nificant reduced vitality (75.7%)
and mean fecal and intestinal para-

177

sitic counts (76.7% and 76.3% re-
spectively), after one hr. comparing
with the corresponding control.
High significant reductions could be
obtained after two hrs exposure (vi-
tality: 96.2%- mean parasitic counts:
94.8% and 94.7% in fecal and intes-
tinal counts respectively). These
figures reflect a relative resistance
to the shorter disinfection exposure
which is comparable to that ob-
served with Cyptosporidium. In ac-
cordance, Calvo et al. (2004) stated
that Cryptosporidium and Cyclospo-
ra were particularly resistant to dis-
infecting agents. Gaafar (2007), re-
ported 0.02% infectivity reduction
of Cyclospora at 4mg/l chlorine for
24 hrs indicating virulence of this
parasite in drinking water. Similar-
ly, Khalifa et al. (2001 reported
0.03% infectivity reduction after
chlorine treatment at 8ppm for 24
hrs. These authors further observed
that exposure to 1ppm ozonated wa-
ter for 9 min. resulted in less infec-
tivity reduction of Cyclospora rela-
tive to Cryptosporidium (81% and
100% respectively). The resistance
of Cyclospora oocysts may be ex-
plained by the presence of thick oo-
cyst wall in addition to the sporo-
cyst wall inside.
Microsporidia, after one hr. ex-
posure to NaDCC, showed signifi-
cant reductions in both vitality
(77.8%) and mean fecal and parasit-
ic counts (80.1% and 82% respec-
tively). High significant reductions
were possible after two hrs. duration
of disinfection (vitality: 97.1%-
mean parasitic counts: 96.9% and
95.7% in fecal and intestinal spore
counts respectively). These figures
indicated a relative resistance mid-
way between Cryptosporidium and
Giardia. Also, Khalifa et al. (2001)
reported 54% infectivity reduction
of Microsporidia spores by chlorine
at 8ppm for 24 hrs and 100% reduc-
tion by 1ppm ozone for 9 min.
Johnson et al. (2003) found that
chlorination of water at 2mg/l for 8
min., was an effective means for
inactivation of spores of Encephali-
tozoon spp. inoculated in cell cul-
ture using monolayer of rabbit kid-
ney cells. Cotte et al.(1999) reported
an outbreak of microsporidiosis in
France and attributed it to potential
factors including the non-use of
chlorine in the water treatment facil-
ities providing the locality, the
spores small size helped to escape
filtration and finally to unknown
possible resistance to physical
agents or disinfectants. Also, Venc-
zel et al. (1997) found that Clostri-
dium perfringens spores were more
susceptible for inactivation by chlo-
rine than Cryptosporidium oocysts.
But, Gaafar (2007) found that mi-
crosporidial spores were more resis-
tant to solar disinfection than Cryp-
tosporidium and Cyclospora, with
infectivity remaining unreduced
after 24 hrs exposure. Microspori-
dia and G. lamblia as well were re-
ported to be more resistant to UV
rays than to chlorine both are con-
trary to Cryptosporidium (Sinski,
2003).
Comparing vital stain figures
with those of animal infectivity, it is

178

important to notice that the in-vitro
assay provides an indication of via-
bility by measuring changes in
cyst/oocyst wall permeability whe-
reas in-vivo infectivity is a measure
of the ability of an oocyst to excyst,
invade and multiply within the host
(Bukhari et al, 2000); therefore it is
a more sensitive indicator of para-
site inactivation. For instance, an
incomplete disinfection damage
may not be sufficient enough to
overcome membrane impermeabili-
ty to vital stain (the oocyst then ap-
pears intact, pale blue and inter-
preted as morphologically living),
but still can cause some functional
change in the more sensitive sporo-
zoites sufficient to prevent the at-
tachment to or invasion of the host
cell. On the other hand, the relative-
ly low number of parasites counted
in vital stain (usually 100), when
extrapolated to the large parasite
population may not be sensitive
enough, thus causing some discre-
pancy between dye viability and
mice infectivity reduction percen-
tages.
Considering the mechanism of
action of NaDCC and the differenc-
es between protozoa in the degree of
resistance to disinfection; the active
moiety HOCl is a powerful oxidiz-
ing agent. It is a multi-target reactor,
acting on cell wall and amino
groups in proteins including en-
zymes, leading to; progressive oxi-
dation of thiol groups into sulphides
and sulphoxides, and deleterious
effect on DNA synthesis via forma-
tion of chlorinated derivatives of
nucleotides. The end result is inhibi-
tion of protein synthesis and cell
membrane damage (Russell, 2003).
The un-dissociated HOCl was neu-
trally charged molecule which
should easily penetrate a bacterial
cell wall. It is the diffusion barrier
of the cyst/oocyst wall limiting the
access of chlorine to its target sites,
together with susceptibility of pro-
teins including those of cell respira-
tion to oxidation and denaturation
by HOCl, which ultimately deter-
mines the susceptibility of individu-
al parasites. The cell wall with its
composition, thickness and relative
porosity represents a classical in-
trinsic type of resistance (McDon-
nell and Russell, 1999). Cryptospo-
ridium oocyst type excreted in stool
is known to have a particularly thick
wall, Cyclospora oocyst has an ad-
ditional sporocyst wall inside and
microsporidial spores have multi-
layered spore wall (Roberts and Ja-
novy, 2005) and these may be the
cause of the reported resistance of
these protozoan parasites to the
NaDCC, especially after short time
exposure.
The chosen organic chlorine
compound NaDCC, has advantages
over sodium hypochlorite bleach
NaOCl, which releases all its free
chlorine in solution resulting in
short action, being also unstable and
decomposes with storage. NaDCC
releases about 60% of its chlorine as
FAC, while the rest remains in equi-
librium as stabilized reservoir chlo-
rine in form of chlorinated isocya-
nurate; when FAC is depleted, the

179

reservoir releases to gain balance.
Therefore its action is maintained
for longer duration with increased
efficacy (Bloomfield and Miles,
1997). The stabilizing FAC reser-
voir promotes biocidal action when
subjected to increased organic loads
or pH changes (Dychdala, 2001).
NaDCC dry tablets are acidic due to
the effervescent base, thus keeping
the active moiety HOCl, which is a
weak acid, un-dissociated unlike
NaOCl which is alkaline. The
unique feature of isocyanurate is the
cyanuric acid carrier which contains
chlorine in a solid, stable and dry
form. Effervescent NaDCC tablets
are self-dissolving and more conve-
nient to use than NaOCl bleach
which is a corrosive liquid subject
spillage and may damage skin or
eye. NaDCC also, exhibit less pro-
duction of toxic substance, trihalo-
methanes (THM), and denoting
lower level of toxicity (Clasen and
Edmondson, 2006). In the present
study, none of the animals died
when inoculated with the disinfec-
tant control solution indicating non
toxic effect of NaDC. Mazzola et al.
(2003) compared NaDCC with vari-
ous chemical disinfectants, includ-
ing 10% NaOCl in a variety of bac-
teria- relevant hospital settings and
they recommended NaDCC, due to
its biocidal effectiveness, slow de-
composition and liberation of HOCl
to maintain appropriate level of
FAC, its low level of toxicity and
low corrosiveness against metal,
plastic or rubber (unlike chlorine
dioxide). In comparison, ozone
which acts at low dosage and short
time is expensive, corrosive and not
available at household level. Pereira
et al. (2008) reported that pretreat-
ment of water with ozone can result
in formation of byproducts as THM,
together with bromates known to
have a carcinogenic potential. In
addition, concentration of organic
matter, changes in pH and tempera-
ture found in water in nature de-
range its potency (Finch et al.,
1993). Solar (UV) disinfection is
slow, hindered by turbidity (Joyce et
al 1996), and radiation intensity is
much reduced beyond 10 cm depth
in transparent water containers
(Caslake et al., 2004). So, parasites
like Cryptosporidium may regain
activity following exposure to UV
(Rochelle et al., 2005). Such draw-
backs of ozone and UV radiations
make them particularly unsuitable
for disinfection of vegetables.
As regards the effect of NaDCC
on the physical properties of vege-
tables eaten raw, vegetable leaves
and soft fruits, no change in color,
consistency, taste or flavor was ob-
served after one and two hrs of dip-
ping in NaDCC concentration,
1gm/l.
The disinfectant could be easily
rinsed in running water leaving no
traces. This agreed with El Zawawy
et al. (2002). Nicoll and Prendergast
(2009) reported a superior effect for
NaDCC over hypochlorite in disin-
fection of shredded salad ingre-
dients. It could reduce microbial
numbers, retard enzymatic activity

180

and improve shelf-life of sensory
quality.
Nascimento et al. (2003) found
that a concentration of 200 ppm
(0.2gm/l) of NaDCC, yielded supe-
rior results compared to NaOCl and
other agents used to sanitize fresh
vegetables against bacteria and fun-
gi. Parkinson et al. (1996) found
that NaDCC was effective for steri-
lization of plants from the field us-
ing concentrations ranging from
300mg up to 5 gm /l for 48 hrs
without minimal phytotoxicity.

Conclusion

The disinfection efficacy of
NaDCC at concentration of 1gm/l
and exposure time two hrs has been
proved in the current study to cover
cysts/oocysts/spores of all examined
intestinal protozoa, namely: E. his-
tolytica, G. lamblia, Microsporidia,
Cryptospridium and Cyclospora,
arranged in order of relative suscep-
tibility. The potent dry tablet form
of NaDCC is safe, rapid-dissolving
and cheap which makes it accepta-
ble and affordable.
It also retains the natural proper-
ties of vegetables and fruits, which
meets the recent popular tendency to
consume fresh natural foods where
both normal taste and heat-labile
nutrients are preserved. It is recom-
mended for disinfection of salad,
vegetables and non-peelable fruits
together with food preparation sur-
faces and equipment at household
and restaurant levels. It can also be
used in catering and fresh produce
industry before preparation.

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185

Legend to figures

Fig 1: Viable cyst of E. histolytica stained with 0.2% trypan blue. (x1000)
Fig 2: Dead cyst of E. histolytica stained with 0.2% trypan blue. (x1000)
Fig 3: Viable oocysts of Cryptosporidium stained with 0.2% trypan blue. (x1000)
Fig 4: Dead oocysts of Cryptosporidium stained with 0.2% trypan blue. (x1000)
Fig 5: Viable oocyst of Cyclospora stained with 0.2% trypan blue. (x1000)
Fig 6: Dead oocyst of Cyclospora stained with 0.2% trypan blue. (x1000)
Fig 7: Viable spores of Microsporidia stained with 0.2% trypan blue. (x1000)
Fig 8: Dead spores of Microsporidia stained with 0.2% trypan blue. (x1000)
Fig 9: Viable cysts of G. lamblia stained with 0.2% trypan blue. (x1000)
Fig 10: Dead cysts of G. lamblia stained with 0.2% trypan blue. (x400)
Fig 11: Iodine stained stool smear showing E. histolytica cyst. (x1000)
Fig 12: Section in colon of mice demonstrating trophozoites of E. histolytica. (H&E x 1000)
Fig 13: Stool smear stained with MZN showing oocysts of Cryptosporidium. (x1000)
Fig 14: Stool smear stained with MZN showing oocysts of Cyclospora. (x1000)
Fig 15: Section in the small intestine of mice displaying oocyst of Cryptosporidium. (x1000)
Fig 16: Section in the small intestine of mice demonstrating oocyst of Cyclospora. (x1000)
Fig 17: Stool smear stained with MTS showing microsporidian spores (x 1000)
Fig 18: Section in the small intestine of mice displaying microsporidian spores (MTS x1000)
Fig 19: Iodine stained stool smear showing G. lamblia cysts. (x 1000)
Fig 20: Section in small intestine of rat demonstrating trophozoites of G. lamblia. (H&E x 400)
Fig 21: Higher magnification of G. lamblia trophozoites in small intestine of rat (H&E x 1000)

186


195



EFFECT OF PLANT MOLLUSCICIDES ON SELECTED ENZYMES
RELATED TO ENERGY METABOLISM IN BIOMPHALARIA
ARABICA SNAILS MOLLUSCAN HOSTS TO SCHISTOSOMA
MANSONI IN SAUDI ARABIA
By

SOOAD AL-DAIHAN
Department of Biochemistry, Science College, King Saud University,
P.O Box 22452, Zip code 11495, Saudi Arabia

Abstract

Schistosomiasis is one of the most important human parasitic diseases. One
of the possible methods for the control is through the molluscan intermediate
host of the parasite. Biomphalaria arabica,molluscan hosts to Schistosoma
mansoni in Saudi Arabia were treated with sublethal concentrations (LC
25
) of
dry powdered leaves Solanum nigrum. Effect of plant on ectonucleotidases
(NTPdases) (ADPase & ATPase), sodium/potassium adenosine triphosphatase
(Na
+
/K
+
ATPase) and creatine kinase (CK) was traced. The plant molluscicide
was potent in inhibiting the four investigated enzymes giving a percentage in-
hibition range between 45-55%. The effect of the inhibited enzymes on the
compatibility of the snail hosts to schistosome parasite was discussed. In con-
clusion, the use of sublethal concentration of S. nigrum to disturb the biochem-
ical profile of the snail hosts could be a promising and safe strategy to control
the disease.
Key words: Schistosome, Biomphalaria arabica, Solanum nigrum, ecto-
nucleotidases, Na
+
/K
+
ATPase, Creatine kinase
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------.

Introduction

Schistosomiasis or bilharzias is
primary tropical parasitic disease
that first described in 1851 by
Theodor Bilharz. It caused by
blood-dwelling fluke worms of the
genus Schistosoma that reside in the
abdominal veins of their vertebrate
definitive hosts (Chitsulo et al.,
2000). Among human parasitic dis-
eases, schistosomasis is the second
most prevalent tropical diseases,
after malaria, affecting 500-600 mil-
lion people and is a leading cause of
severe morbidity in many parts of
the world (Ahmed et al., 2007. The
prevalence of schistosomiasis is
clustered in the Eastern and South-
western provinces, due to the pre-

195

ferable environmental conditions
(Arfaa, 1976; Habib et al., 1977).
Other factors may contribute to the
increase in prevalence of the infec-
tion including the large number of
expatriates, many from countries
with higher prevalence of schisto-
somiasis, and hence the possibility
of parasitic infection among them
(Abahussain, 2005). Human infec-
tion with both intestinal and urinary
schistosomiasis has a wide distribu-
tion in the kingdom of Saudi Arabia
(Morsy et al., 1974; Ashi et al.,
1989) as well as the snail-vectors
(Al Mathal and Fouad, 2006; Abdu,
2009).
The strategies of schistosomiasis
control have been shifted fundamen-
tally over the past few decades,
since the introduction of modern
schistosomicides, particularly Mec-
ca Myrrh or Mirazid (Massoud et
al., 2007; Tonkol and Morsy, 2008).
Besides, schistosomiasis can in
principle be eliminated by behavior-
al changes, sanitation, and safe wa-
ter supply, as has been shown in
Japan (Minai et al., 2003).
Schistosome parasite has a com-
plex lifecycle with two hosts, a mol-
luscan intermediate host, freshwater
snails and a mammalian final host.
The snail host represents the weak-
est point in the parasite lifecycle.
The dynamic interaction between
snail hosts and their schistosome
parasites leads either to a state of
coexistence in which the schisto-
some parasite thrives and produces
subsequent stages of it's life cycle
(sporocysts and cercariae), or to in-
compatibility, where the trematode
is either destroyed and eliminated
by the snail defensive responses or
fails to develop because the snail
host is physiologically unsuitable
(Bayne and Yoshino, 1989; Van der
Knaap and Loker, 1990; El-Ansary
and Qurashy, 1994).
B. arabica is the molluscan host
for S. mansoni in Saudi Arabia. As
intermediate hosts, molluscs play a
major role in the transmission of
schistosomes; they are the sites of
an intense multiplication of the
parasites. Thus, the snail control
strategies are considered a priority
for the reduction of schistosomiasis
(Lardans and Dissous, 1998).
There is considerable evidence
that trematodes vary in their energy-
obtaining metabolism. Fasciola he-
patica produces succinate and pro-
pionate (Lloyd, 1986) while Schis-
tosoma mansoni produces mainly
lactate and under certain condition
in addition to lactate it produces
succinate (Van Hellemond et al.,
1997).
As many parasitic helminthes,
schistosomes have portion of their
life cycle in different habitats in
which the availability of oxygen
vary frequently, also the type and
availability of nutrients may also
vary and both being controlled by
the host. In addition, helminthes
depend on their host for removing
wastes since it does not have a sys-
tem for circulating oxygen or re-
moving wastes (Saz, 1981). Moreo-
ver, the growth and multiplication
of the parasites is an increasing bur-

195

den on the host's metabolism, in-
volving provision of food for
growth and energy and the need to
process the parasite waste products.
In addition it is well documented
that schistosome parasites are very
sensitive to any change in the ATP
level. These initiate our interest to
study the possibility of impairing
energy metabolism of B. arabica
snails using sublethal concentration
of S. nigrum plant molluscicide in a
trial to render them less compatible
for the development of the parasite.


Snail collection: Specimens of
Biomphalaria arabica were col-
lected from a farm near Riyadh. The
snails were left in the lab for 45
days and was examined to be sure
that they were free from parasitic
infection. They were fed with let-
tuce leaves ad lib. A sample of the
snails was randomly chosen and
dissected.
Preparation of tissue homogenate:
One gram of snail soft tissue was
homogenized in 5ml distilled water
and then centrifuged at 3000 rpm,
the supernatant was used for the
biochemical analyses (Nabih et al.,
1989).
Molluscicide-treatment: This was
performed according to the toxicity
study of S. nigrum plant on B. ara-
bica snails previously done by El-
Ansary and El-Daihan (2007). Four
groups of 10 snails each were ex-
posed to 3 ppm concentration of S.
nigrum (LC
25
) dissolved in dechlo-
rinated water in 1 liter capacity tank.
Snails were fed fresh lettuce leaves
ad lib during the 24 hours contact
period. Dead snails were discarded
and the remaining snails were used
for the biochemical analysis. Un-
treated control groups were estab-
lished.
Measurement of ectonucleotides:
ADPase and NTPase were measured
according to the method of (Pennial,
1966) in which the colorimetric de-
termination of liberated inorganic
phosphate was measured according
to Fisk and Subbarrow (1925).
Measurement of ATPase: Na
+
/K
+

ATPase was assessed according to
the method of Bettowski et al.
(1998) in which the colorimetric
determination of liberated inorganic
phosphate was measured according
to Fisk and Subbarrow (1925) and
auabain was used as inhibitor.
Measurement of Creatine kinase
(CK): CK was done by using a di-
agnostic Kit, a product of Al-Mota-
hida Company, KSA.
Statistical analysis: The data was
carried out using Student t-test
(Graph Pad Prism Computer Pro-
gram, SPSS, 1999).

Results

The results are shown in table (1) and figure (1)

195

Table 1: Activities of ADPase, NTPase, ATPase, and Creatine kinase in control
and Solanum nigrum- treated B. arabica.

Snail
Parameter

Control Treated-Snail P<
ADPase Range moles/gm

Mean S.D
0.084-0.11

0.0980.0053
0.044-0.063

0.1610.008
S(0.0006)
NTPase Range moles /gm

Mean S.D
0.155-0.186

0.1740.00667
0.084-0.123

0.1050.0086

S(0.0008)
ATPase Range moles /gm

Mean S.D
0.100-0.134

0.1140.00733
0.055-0.071

0.05950.0041
S(0.0007)

CK Range moles /gm

Mean S.D

0.0403-0.0408

0. 0400.0001

0.0120-0.0163

0.01510.0010
S(0.0001)

Figure 1: Percentage changes induced by molluscicide of energy parameters


195

Discussion

Exploitation of the snail host is
effectively accomplished by an in-
tegration of host and parasite physi-
ologies accompanied by dramatic
and dynamic changes in host sur-
vival, behaviour, defence-immune
function, nutrition, metabolism and
reproduction. A reasonable infe-
rence from this integration is that
the infected snail host represents an
'extended- phenotype' of the parasite
(Thompson, 1997). These 'extended
phenotype' symbolize the essence of
parasitism (Thompson, 1990,1991,-
1993). Within the spectrum of com-
patibility, these host-parasite associ-
ations are generally characterized by
a high rate of infection, relatively
low host mortality, a short prepatent
period and high cercarial output
(Brown, 1994).
Homolactate fermentation is sup-
posedly well exemplified by schis-
tosomes. Strictly speaking, in homo-
lactate fermentation glucose is con-
verted exclusively into lactic acid by
glycolysis (Saz, 1981). Energy, as
ATP, is trapped in the reactions cat-
alyzed by phosphoglycerate kinase
and pyruvate kinase. The pathway
remains in redox balance as the re-
ducing equivalents (NADH) gener-
ated in the early stages of the path-
way are reoxidised by the terminal
step, catalysed by lactate dehydro-
genase.
Energy demand and supply are
balanced and tightly regulated for
economy and efficiency of energy
use. Cells with high and fluctuating
energy requirements may increase
the rate of ATP hydrolysis within
seconds by several orders of magni-
tude. The Na
+
/ K
+
ATPase as an
electrogenic pump transports three
Na
+
out of the cell and two K
+
in-
side the cell by using the energy
derived from the hydrolysis of one
molecule of ATP and thereby plays
a critical role in maintaining these
ions gradients across the plasma
membranes (Hamada et al., 2003).
The gradient produced by this en-
zyme, is coupled to physiological
functions such as cell proliferation,
volume regulation and maintenance
of electrogenic potential of different
types of cells. It is well known, that
energy metabolism of S. mansoni
miracidia is pre-adapted to the occa-
sional hypoxic conditions within
their molluscan hosts (Boyunaga et
al., 2001), and that schistosome pa-
rasite is very sensitive to any change
in ATP levels within molluscan tis-
sues.
Cell membrane ectonucleotidases
such as NTPDases are low specifici-
ty enzymes that hydrolyze all tri-
phosphates to produce either ADP
or adenosine as their main products
associated with in organic phos-
phate (Plesner, 1995; Zimmermann,
1999).
Creatine kinase catalyzes the re-
versible transphosphorylation of
creatine by ATP, which suggests
that it plays a significant role in case
of energy depletion. Impairment in
the creatine kinase/ phosphocreatine
system of transphosphorylation for
the generation of readily- available

195

energy led to deterioration in the
energy metabolism (Andres et al.,
2002).
In the present study, ADPase,
NTPase, ATPase and creatine ki-
nase of B. arabica snails were sig-
nificantly inhibited by sublethal
concentration of S. nigrum plant
molluscicide. Inhibition of these
enzymes might disturb the ability of
these snail species to utilize ATP as
a critical high energy compound
needed for reproduction, locomotion
and support of the developing para-
site post exposure of B. arabica to
schistosome infection. Importance
of ATP reserves for the develop-
ment of intramolluscan schistosome
parasite was previously ascertained.
S.mansoni-infected B. glabrata
snails usually reduced their locomo-
tory and reproductive activities to
safe ATP to maintain parasite sur-
vival (Gerard, 1996). Reduced lo-
comotion coincides with significant-
ly decreased phosphoarginine/ATP
phosphate exchange rates in the foot
of infected snails.
In the present study, inhibition of
Na+/K+ ATPase of the host could
affect the active transport of nu-
trients such as amino acids, fatty
acids and glucose from the host to
the parasite. Moreover inhibition of
this enzyme could leads to swelling
and death of the schistosome para-
sites (Smith and Strout, 1980).
Moreover, the significant inhibi-
tion of ADPase and NTPdase with
50% and 45% respectively, could
dramatically affect the adenine nuc-
leotide levels and induce lower ade-
nylate energy charge (AEC) to be in
the stressed range which could af-
fect the survival of the snail host
and/or the developing schistosome
parasite. The importance of the sta-
bilization of the AEC of the infected
snail hosts for the developing para-
site was previously reported by El-
Ansary (1999) who recorded that
reduction of locomotors activity
together with the stimulation of the
glycolytic pathway could be easily
correlated to the stabilization of the
AEC within the non-stressed range
in S. mansoni- parasitized B. alex-
andrina snails, molluscan hosts of
schistosome in Egypt.
The outcome results agreed with
El-Ansary and Al-Daihan (2007) in
which they recorded that sublethal
concentration of s.nigrum was effec-
tive in inhibiting lactate dehydroge-
nase aspartate and the alanine ami-
notransferases as enzymes which
could be easily correlated to aero-
bic/ anaerobic energy generation of
B. arabica.
Generally speaking, the medical
plants and herbs are more or less
worldwide distributed and available
(Chevalier, 1996; Borrelli and Izzo,
2000). Besides, the use of plants
(Sherif and El-Sawy, 1977; Borrelli
and Izzo, 2000), their extracts (Per-
rett and Whitefield, 1996) or latex
(Schall et al., 1998) or even herbs
(Al Mathal and Fouad, 2006; Mas-
soud et al., 2007) for snails control
received many considerable atten-
tion worldwide.

Conclusion

195

The outcome data ascertained the
possibility of use sublethal concen-
trations of plant molluscicide (dry
powdered leaves Solanum nigrum)
to affect the physiology of the schis-
tosome molluscan host (Biomphala-
ria arabica) in order to render them
unsuitable for the developing para-
site

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197



EFFECTS OF SCHISTOSOMA MANSONI EXPERIMENTAL
INFECTION ON SOME INORGANIC ELEMENTS IN THE SNAIL
HOST BIOMPHALARIA ALEXANDRINA
By
OSAMA M. S. MOSTAFA* and SAAD M. BIN DAJEM
Department

of Biology, Faculty of Science, King Khaled University, Abha,
P.O Box 9004, Saudi Arabia, Fax: 096672289300 ext.1762,
E-mail: Osamamostafa@hotmail.com

Abstract

The alteration in the concentrations of metallic ion Pb, Zn, K, Na, Co, Fe,
and Cu in the soft parts of the Biomphalaria alexandrina snails shedding Schis-
tosoma mansoni cercariae was detected by flame atomic absorption spectrome-
try. Six elements Pb, Zn, K, Na, Co, and Cu were found to be present at signif-
icantly higher concentrations in cercariae-shedding snails compared with unin-
fected snails. The concentration of Fe ion showed non-significant decrease in
the tissues of cercariae-shedding snails. Variation in the present results com-
pared with related previous studies lead to the suggestion that the effect of tre-
matode parasitism on fresh-water snails should not be considered universal and
might be varies according to the trematode-snail combination, the organs or the
tissues analyzed and the analytical method used.
Key words: Biomphalaria, Schistosoma, Gastropoda, Trematoda, metallic ion,
spectrometry.
Permanent address: *Department

of

Zoology, Faculty of Science, Ain Shams
University, Cairo 11566, Egypt.

Introduction

The pathobiochemical effects of
larval trematodes on naturally or
experimentally infected freshwater
snails were studied. The effects on
proteins and enzymes were reported
(Loker and Hertel, 1987; Nabih et
al.1990; Zelck et al.1995; el-Ansary
et al.2000; Mostafa et al.2001;
Mostafa 2002; Mahmoud et al.,
2002; El-Dafrawy et al.2006). The
influence of trematode infection on
the lipid composition of Biomphala-
ria glabrata was demonstrated
(Thompson 1987; Fried et al. 1989),
as well as the changes in the sugar
content (Crews and Yoshino, 1990;
Perez et al., 1994).
Crews and Yoshino (1991) found
that Schistosoma mansoni affected
the translatable levels of mRNA and
polypeptide synthesis in the ovo-
testis and albumen gland of B. gla-
brata.
198

On other hand, the alterations in
the metallic ion concentrations
(conc.) in different snail-trematode
models were recorded. Gabrashans-
ka et al. (1991) analyzed Ca, Na,
Rb, Sb, Ce, Cr, Cs, Cu, Fe & Zn in
the hepato-pancrease of Lymnaea
stagnalis infected with Echinostoma
revolutum using neutron activation
analysis. Layman et al.(1996) using
flame and graphite-furnace atomic
absorption spectrometry and induc-
tively coupled plasma atomic emis-
sion spectrometry (ICP-AES) de-
tected the conc. of some metallic
ions in the digestive gland-gonad
complex (DGG) of Helisoma trivol-
vis infected with E. trivolvis. Ong et
al. (2004) using ICP-AES investi-
gated the amounts of sixteen ele-
ments in the soft parts of B. glabra-
ta infected with S. mansoni. Mosta-
fa (2008) determined the alteration
in the conc. of metallic ion Ca, Pb,
Zn, K, Na, Fe, Cu & Co in the soft
parts of the Lymnaea natalensis
shedding Fasciola gigantica cerca-
riae using flame atomic absorption
spectrometry.
All these authors proved the inor-
ganic metabolism alterations in the
snails due to trematode infection.
This study aimed at the determina-
tion of the alterations in some inor-
ganic elements in the soft parts of
Biomphalaria alexandrina snails
infected with Schistosoma mansoni.


Lab-bred B. alexandrina (5-8 mm
in shell diameter) were individually
placed in wells of a 24-well plate,
along with freshly hatching five to
seven S. mansoni miracidia and 0.5
ml dechlorinated tap water. S. man-
soni miracidia were obtained from
Schistosome Supply biological Pro-
gram (SBSP) at Theodor Bilharz
Research Institute (TBRI). The
snails were left overnight at room
temperature to ensure maximum
penetration, and then removed from
the wells and reared in culture. The
total number of exposed snails was
125. Snails were placed in 5 plastic
trays with a holding capacity of 1.5
liters of water. Each tray contained
25 snails at a time in dechlorinated
water and was supplied with lettuce
leaves (fresh or boiled and dried) for
snail feeding. Trays were loosely
covered with a glass plate to reduce
evaporation, and maintained at
room temperature 27-29C. Water
was changed every three day. From
the 4
th
to 6
th
week post-exposure,
snails were examined for cercarial
shedding which were isolated and
used in the study. Most of the shed-
ding snails were collected in 5
th
week

post-exposure. About 110
clean, non exposed snails (nearly of
same age and size of shedding ones)
were used as control.
Snails were dissected in deionized
water to separate soft parts from
shells. Soft parts were rinsed, at
least, three times with deionized
water. Excess water was removed
from the soft parts by using filter
papers. Soft tissues were grounded
with mortar and pestle, pooled to
achieve a weight of 500mg for each.
199

About 35 snails were used to
achieve a single pool of soft parts.
Three pools of soft parts from snails
shedding S. mansoni cercariae and
clean, non-exposed snails were pre-
pared for analysis. Wet-weighted
samples were digested in 10 ml of
concentrated nitric acid by boiling
to dryness. The residue from each
digested sample was diluted to 25
ml with deionized water in a volu-
metric flask.
Elemental analysis by flame atom-
ic absorption spectrometry using
Perkin-Elmer model 3100 AAS was
performed to determine the concen-
tration of metallic ion Pb, Zn, K,
Na, Fe, Cu & Co in the soft parts of
the snails. The flam wavelength and
sample aspiration rate were opti-
mized according to the manufactur-
er's recommendations, and four
aqueous standards having analyte
concentrations within the linear re-
sponse range of the instrument and
containing the same concentration
of nitric acid as the samples were
used for calibration. Each sample,
standard and blank, was analyzed
using three 10-s integrations. The
reagent blank was prepared and its
value was subtracted to give the
final concentration. The final ele-
ment concentration (C) was calcu-
lated as follows:
1000
=
wt
V F
C where
F=standard factor calculated from
the standard curve, V= volume of
sample and wt= wet weight of sam-
ple.
Data are expressed in micrograms
of element per gram of wet tissue.
Results were subjected to Student's
t- test using SPSS program version
8 to determine the significant of
data.

Results

Of seven elements analyzed by the
flame atomic absorption spectrome-
try, six detectable elements Pb, Zn,
K, Na, Co, and Cu were found to be
present at significantly higher con-
centrations in S. mansoni cercariae-
shedding snails compared with un-
infected ones. The conc. of element
Fe showed non-significant decrease
in soft tissues of cercariae-shedding
snails (Table 1).

Table 1: Contents of seven metals (mean SE) in micrograms /gram of wet
soft parts of clean, non-exposed and cercariae-shedding B. alexandrina.
Metal clean, non-exposed snails shedding snails P-value
Pb 0.0770.004 0.1640.005 0.001
***
Zn 0.2140.003 0.3960.006 0.001
***

K 4.9840.29 8.8240.36 0.001
***

Na 6.5420.42 16.3710.62 0.001
***

Co 0.0390.002 0.0990.005 0.001
***

Fe 5.8030.32 4.5860.34 0.58
NS
Cu 0.0510.003 0.1540.005 0.001
***

***very highly significant,
NS
non- significant
200

Discussion

Despite the differences in snail-
trematode models examined, the
methods of analysis applied and the
degree of significance, the present
results were discussed with that of
Gabrashanska et al. (1991), Layman
et al. (1996), Ong et al. (2004) and
Mostafa (2008). In contrast to the
findings of Ong et al. (2004) and
Mostafa (2008) in which the trema-
tode infection lowered the amount
of Pb ion, the present investigation
declared a significant elevation in
this metallic ion. In the present
work, the significant increase in the
concentration of metallic ion Zn in
the infected snails was agreed with
Layman et al. (1996) and Mostafa
(2008) and disagreed with Gabra-
shanska et al. (1991) and Ong et
al.(2004), but Ong et al. (2004) and
Mostafa (2008) found that the con-
centration of K ion showed signifi-
cant increase in the infected snails.
In the present investigation, the sig-
nificant increase in Na ion in S.
mansoni cercariae-shedding snails
was agreed with Gabrashanska et al.
(1991), Layman et al. (1996), and
Ong et al.(2004) but disagreed with
Mostafa (2008). Co ion was de-
tected in both infected and non-
infected snails and its concentration
was significantly increased in the
soft tissues of S. mansoni cercariae-
shedding snails, in contrast to the
finding of Mostafa (2008) who re-
ported that Co ion was found to be
present at concentration level below
the detection limits of the analytical
method used in non-infected L. na-
talensis snails and F. gigantica-
cercariae shedding snails. The sig-
nificant increase in the content of
Cu in the infected snails was corre-
lated with Layman et al. (1996),
Ong et al. (2004) and Mostafa
(2008) but uncorrelated with Gabra-
shanska et al. (1991). The lower
concentration of Fe in the infected
snails observed in the present inves-
tigation was agreed with Gabra-
shanska et al. (1991) but disagreed
with Layman et al. (1996), Ong et
al. (2004) and Mostafa (2008).
Similar variations in the results
were reported (Kaufer et al., 2002)
in the comparison with the effect of
Euhaplorchis californiensis on me-
tallic ions in the host snail Cerithi-
dea californica with other investiga-
tions; they referred such variations
to intrinsic differences in the larval
trematode-snail system used.
The reasons of such alterations in
the metallic ion concentrations in
snails as a result of infection with
trematode larvae are still unknown.
Kaufer et al. (2002) and Ong et al.
(2004) reported that they did not
know why the concentrations of
certain metals are significantly in-
creased, whereas others are signifi-
cantly decreased in infected versus
uninfected snails. However, few
authors gave some explanations for
such changes. Layman et al. (1996)
reported that the changes in metallic
ions as the results of larval parasit-
ism in the snails reflect alterations
in ionic balance and an influx of
certain ions and an out-flux of other
201

ions from the larval trematodes to
the snail hosts. In addition, Evans et
al. (2001) referred the changes in
the concentration of certain ele-
ments in the snails infected with
larval trematodes to the pathologic
effect of these larvae on the diges-
tive gland cells of the infected
snails. An important function of the
digestive gland cells is the storage
of various elements, where the ele-
ments are held in the membrane-
insoluble granules in the cells; de-
struction of the digestive gland cells
by larval trematodes probably re-
duces the storage volume and hold-
ing capacity of elements in the in-
fected snails (Evans et al., 2001). In
addition, the disturbance occurred in
the metallic ion concentrations in
the snails infected with trematode
larvae must be considered as one of
the causes of alterations occurred in
the biological activities and the in-
crease in the mortality of the in-
fected snails recorded by various
authors (Mohamed et al.1998; Cruz-
Mendoza et al.2006; Dreyfuss et
al.1999; Gutirrez et al.2000; Sala-
zar et al. 2006).
Generally speaking, human schis-
tosomiasis is complex of acute and
chronic diseases with widely differ-
ing signs and symptoms and wide-
spread infection in the tropics. It is
believed that there are 120 million
symptomatic cases, of which 20
million are suffering from severe
disease (WHO 1999). S. mansoni is
endemic in 55 countries mainly in
sub-Saharan Africa, including the
Arabian Peninsula, Egypt, Libya
and Sudan, but also in some coun-
tries and territories of South Amer-
ica (Brazil, Venezuela, Surinam and
some Caribbean islands). S. haema-
tobium is currently found in 53
countries in the Middle East and
most of the African continent in-
cluding the islands of Madagascar
and Mauritius (Strickland, 2000).
Historically, in Egypt both S. man-
soni and S. haematobium were
highly prevalent in the Nile Delta in
many rural areas (Scott, 1937).
However, schistosomiasis and their
snail-vectors are still common in
certain foci (Khoby et al., 2000; El
Baz et al., 2003; Abo-Madyan et al.,
2005). On the other hand, schisto-
somiasis and their snail-vectors
were identified in Saudi Arabia
(Morsy et al., 1974; Arafa, 1976;
Habib et al., 1977; Brown et al.,
1980; Al-Mathal and Fouad, 2006;
Bin Dajem, 2009). The Statistical
Year Book of Ministry of Health
(2006) showed that the overall
prevalence rate of bilharziasis in the
Kingdom of Saudi Arabia was
2.2/100,000. Urinary schistosomi-
asis was 33.4% and intestinal one
was 66.6% without any double in-
fection.

Conclusion

The effect of trematode parasitism
on fresh-water snails should not be
considered universal and might be
varied according to the trematode-
snail combination, the organs or the
tissues analyzed and the analytical
method used.
202

No doubt, controlling of the snail-
vectors of zoonotic trematodes is
the main means of minimizing dis-
ease transmission. The present out-
come results of the disturbance oc-
curred in the metallic ion concentra-
tions in the snails infected trema-
tode must be considered as one of
the causes of alterations occurred in
the biological activities and the in-
crease in the mortality of the in-
fected snails.

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205



THE EFFECT OF A SUBLETHAL CONCENTRATION OF SOLANUM
NIGRUM ON SOME ANTIOXIDANTS IN BIOMPHALARIA ARABICA
By

SOOAD K.AL-DAIHAN, J.S. N.KAGGWA AND
AFAF K. EL- ANSARY
Department of Biochemistry, College of Science, King Saud University,
P. O. Box 22452, Riyadh 11495, Kingdom of Saudi Arabia.

Abstract

Schistosomisis is endemic in many rural areas of developing countries. The
life cycle of schistosomes is complex with two hosts, an intermediate snail host
and a definitive human host. Biomphalaria arabica is the intermediate host for
Schistosoma mansoni in Saudi Arabia. One method of controlling the disease is
to break the life cycle at the intermediate host snail stage using molluscicides.
Snails kill schistosomes by a mechanism involving production of reactive oxy-
gen species. In this study malondialdehyde (MDA), and the antioxidants gluta-
thione (GSH), catalase (CAT) and glutathione peroxidase (GP
x
) were deter-
mined in tissue homogenates of B. arabica treated with sublethal concentration
(LC
25
) of the plant molluscicide Solanum nigrum. MDA, GSH and CAT were
significantly increased in molluscicide-treated snails compared to con-
trols(p<0.000). GP
x
was decreased in treated snails. It therefore appears that a
sublethal concentration of S.nigrum increases both ability of snail tissue to
generate cytotoxic ROS and antioxidants for protection of the tissue against the
cytotoxicity. The increase in the level of ROS would decrease snail- schisto-
some compatibility.
Keywords: Biomphalaria arabica, Schistosoma mansoni, Solanum nigrum,
Glutathione, Reactive oxygen species, Catalase, Malondialdehyde, Glutathione
peroxidase.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Schistosomiasis or bilharziasis, is
a disease caused by parasitic trema-
todes, of which Schistosoma man-
soni, S. haematobium and S. japoni-
cum are specifically affect man.
The disease is endemic in some ru-
ral areas in developing countries,
however, due to increase in immi-
gration and tourism it now occurs in
all municipalities over the world
(Chitsulo et al., 2000). Despite the
numerous control efforts in a num-
ber of countries, about 200 million
people are infected worldwide, out
206

of these 120 million are symptomat-
ic and 20 million have severe debili-
tating disease (WHO, 1998).
Control efforts usually target the
life cycle of schistosomes which
have two hosts; the definitive host
or mammalians including man and
the intermediate one or fresh water
snails.
In the Kingdom of Saudi Arabia
(KSA) the snail Biomphalaria ara-
bica is the intermediate host of S.
mansoni and three snails, namely
Bulinus truncates, B. beccarii and
B. reticulates are intermediate hosts
for S. haematobium (Arafa, 1976;
Habib et al., 1977). The suscepti-
bility of the two former species was
confirmed in the laboratory. Uri-
nary and intestinal schistosomiases
were observed in most parts, mainly
rural areas, of the Kingdom with the
highest prevalence in Gizan Prov-
ince, ranging from 43% to 91%.
Schistosomiasis control programs
using treatment and snail control
started in 1974 have lead to signifi-
cant reduction in prevalence rates all
over KSA (Ahmed et al., 1990; Al-
Ghahtani and Amin, 2005). Howev-
er, the potential danger of a resur-
gence of the disease due to the de-
velopment of water resources and
presence of a great number of in-
fected foreign workers was signifi-
cant (Ashi et al., 1989).
The host-parasite compatibility is
a highly specific phenomenon in
schistosomes. The mechanisms by
which schistosomes escape the
hosts immune response in suscepti-
ble snails are unknown. However,
several reactive oxygen species
(ROS), the free radicals superoxide
ion (O
-
2
) and hydroxyl radical (OH
)
and hydrogen peroxide (H
2
O
2
) in
particular are produced in response
to a variety of insults including pa-
rasites, toxins or their metabolites.
These ROS are cytotoxic, and are
known to kill helminth parasites;
including sporocyts in incompatible
snail-trematode associations (Ba-
bior, 1978; Smith and Bryant, 1986;
Adema et al., 1994). The free radi-
cals also cause lipid peroxidation. In
an immuno response to schisto-
somes, snail hemocytes produced
ROS (Shozawa et al., 1989; Sulli-
van et al., 1995; Hahn et al., 2001;
Mahmoud and Rizk, 2004).
Several free radical scavenging
systems, including antioxidant en-
zymes and nonenzymatic antioxi-
dants, provide effective protection
against oxidative damage by ROS.
Antioxidant enzymes have been re-
ported in susceptible and non-
susceptible snails (Nabih and El-
Ansary, 1993; Farrag, 2000; Mah-
moud and Rizk, 2004).
One of the strategies to control
schistosomiasis is to break the life
cycle by reduction of the interme-
diate snail hosts. The control of
snail population with molluscicides
has been one of the most effective
methods for reducing the risk of
schistosomiasis transmission in en-
demic areas (McCullough, 1992).
The niclosamide, a synthetic mol-
luscicide was recommended for
large scale use in schistomiasis con-
trolling programs (Webbe, 1987;
207

WHO, 1998). Synthetic mollusci-
cides are expensive, toxic to non-
target organisms; snails have devel-
oped resistance to them and have
deleterious effects on the environ-
ment (Cooppan et al., 1986; Pointer
and Giboda, 1999).
On the other hand, plant mollusci-
cides have proved to be a cheaper
and effective alternative (Massoud
et al., 2007). Moreover, plant mol-
luscicides are locally produced and
biodegradable (Zidan et al., 1998),
even in KSA (Al Mathal and Fouad,
2006). The exact mode of action of
most molluscicides is not yet com-
pletely understood. However, some
of these compounds may affect
some vital enzyme activities in tis-
sues and lead to snail death.
Sublethal concentrations of plant
molluscicides have been reported to
affect both compatibility of B. alex-
andrina and S. mansoni (El-Ansary
et al., 2001) and some enzymatic
activities in B. alexandrina and B.
arabica (El-Ansary et al., 2002 ; El-
Ansary and Daihan, 2007). Solanum
nigrum was observed to have mol-
luscidal activity on some Egyptian
snail species (Ahmed and Ramzy,
1997) and affected some enzyme
activities in B. Arabica (El-Ansary
and Al-Daihan, 2007)
Many toxic compounds are known
to act through generation of ROS.
No information concerning the ef-
fect of sublethal molluscicide con-
centrations on antioxidants in B.
arabica snails is yet available.
This study aimed to analyze some
antioxidants in B. arabica and to
assess the effect of a sublethal con-
centration (LD
25
) of dry powdered
leaves of S. nigrum on the parame-
ters for exploring the possibility of
employing the plant to affect snail-
schistosome compatibility for feasi-
ble control of schistosomiasis in
KSA.


Snails: B. arabica were collected
from a farm nearby Riyadh City.
They were kept in the lab for 45
days, fed on fresh lettuce leaves ad
lib and examined to make sure they
were parasite-free. A sample of the
snails was randomly chosen and
dissected.
Toxicity study: Leaves of the wild
plant S. nigrum were collected, air-
dried and used as powder. The LC
25

(3ppm) was determined (El-Ansary
and Daihan, 2007). The plant powd-
er was dissolved in de-chlorinated
water. Snails were put in a one-liter
Pyrex jar filled with 3ppm S. ni-
grum and fed on fresh lettuce leaves
ad lib and kept in the tank for 24
hrs.
Preparation of tissue homogenate:
One gram of snail soft tissue, from
6-10 snails, was homogenized in
5ml bi-distilled water, the homoge-
nate centrifuged at 3000 rpm and
the supernatant was biochemical
analyzed (Nabih et al., 1989)
Malondialdehyde assay: MDA
was determined in the supernatant
by the thiobarbituric acid (TBA)
method described by Ruiz-Larrea et
208

al. (1994). The results were ex-
pressed as moles/g soft tissue.
Glutathione assay: The GSH con-
tent of the supernatant was deter-
mined as described by Beutler et al.
(196). The results were expressed as
moles/ g soft tissue.
Assay of enzyme activities: GP
x

activity was assayed by a kit (Ran-
dox Laboratories Ltd, UK) based on
a method described by Paglia and
Valentine (1967). Oxidation of GSH
by GP
x
was coupled to oxidation of
NADPH by glutathione reductase
and the decrease in absorbance at
340 nm at 37

C was determined.
The activity was calculated as
nmoles/ mg protein.
CAT activity was assayed as de-
scribed by Aebi (1984). The activity
was expressed as nmoles/mg pro-
tein.
Protein determination: Protein in
the supernatant was estimated ac-
cording to Bradford (1976).
Statistical analysis: The signific-
ance of differences between two
mean values was calculated using
the Student t- test. A probability
level (P) of 0.05 or less was consi-
dered significant (SPSS, 1999).

Results

The effect of a sublethal concen-
tration S.nigrum on levels of MDA
and GSH and activities of GP
x
and
CAT are shown in the table (1) and
figure (1). The levels of MDA and
GSH and the activity of CAT were
significantly increased (p < 0.001)
in treated snails.
However, GP
x
activity decreased
in treated snails although the de-
crease was not significant (p<
0.062). Details are given in table (1)
and figure (1).

Table 1: Antioxidants and Malondialdehyde (MDA) levels in tissue homog-
genate of control and S. nigrum-treated snails.

Group
(N = 4)
GSH
(moles/g tissue)
GPx
(nmoles/mg protein)
CAT
(nmoles/mg protein)
MDA
(moles/g tissue)
Control 0.103 0.004 0.9 0.23 0.522 0.073 0.068 0.0033
Treated 0.161 0.008 0.35 0.067 1.3 0.29 0.088 0.0017
P-value P < 0.0001 P < 0.062 P < 0.0001 P < 0.0001

Discussion

The biochemical equilibrium be-
tween the host defense system and
parasite is a delicate one and slight
disturbances can change the out-
come of such interactions (Zelek
and Von Janowsky, 2004). If the
balance shifts in the hosts favor, the
parasite was eliminated by the
hosts defense system. The snail-
schistosome compatibility was cor-
related with the ability of the snails
defense system to generate the cyto-
toxic ROS and mechanisms for de-
toxification of these species (Hahn
et al., 2001; Mahmoud and Rizk,
2004). Oxidative stress occurs
209

whenever more ROS are produced
than can be detoxified by antioxi-
dants. The free radicals kill the pa-
rasites in resistant snails but may
not be produced in enough quanti-
ties or may be detoxified at a faster
rate in susceptible snails.

0
50
100
150
200
250
GSH GPx CAT MDA
Antioxidants and Malondialdehyde(MDA)
P
e
r
c
e
n
t
a
g
e

C
h
a
n
g
e

i
n

l
e
v
e
l
s
Control
Treated

Figure 1: Percentage change of antioxidants and malondialdehyde (MDA) in control
( ) and S. nigrum- treated snail ( ). Controls =100%.

ROS have relatively short half-
lives and thus, determination of the
concentrations in tissues was diffi-
cult. However, levels of ROS in a
tissue can be assessed indirectly by
measurement of lipid peroxidation
products such as MDA, non-
enzymatic antioxidants such as GSH
or antioxidant enzymes such as
CAT and GP
x
.
the MDA and GSH levels and CAT
activity increased significantly
(p<0.001) in treated snail tissue.
However, GP
x
activity decreased
in treated snails although the de-
crease was not significant.
The main cellular targets for ROS
are membrane lipids, which upon
oxidation form lipid peroxides. In
this study, the detection of MDA
showed that B. arabica snail tissue
generates cytotoxic free radicals.
The significant increase of MDA in
treated snails suggested that S. ni-
grum stimulates generation of ROS.
This finding was not surprising as
many toxic compounds are known
to function through mechanisms
210

involving production of ROS. In-
creased levels of cytotoxic free radi-
cals would be deleterious to snail
tissue. However, in presence of
schistosome parasites increased le-
vels of the cytotoxic radicals in snail
tissue may eliminate the parasite.
One of the radicals, the superoxide
ion, is rapidly scavenged by supe-
roxide dismutase and converted into
H
2
O
2
, which is also cytotoxic. The
antioxidants detected in this study,
namely GSH, GP
x
and CAT detoxi-
fy H
2
O
2
. The increased level of
GSH and CAT activity in treated
snails indicated that the snail tissue
is partly or fully protected against
the cytotoxic H
2
O
2
as the mollusci-
cide caused a simultaneous eleva-
tion in both ROS and the two anti-
oxidants. Although GSH plays a
central role in protection against
ROS in most organisms (Ross,
1988), yet there are little studies on
levels GSH with regards to schisto-
somes. Administration of the anti-
schistosomal compound oltipraz has
been observed to increase GSH le-
vels in host tissue whereas the levels
in S. mansoni in vivo were depleted
(Bennett and Depenbush, 1984).
GSH can detoxify H
2
O
2
directly or
in conjunction with GP
x
. Since the
activity of GP
x
was not increased in
treated snails it may be concluded
that GSH mainly detoxified H
2
O
2

directly.
CAT is a very potent enzyme for
scavenging H
2
O
2
, thus its high ac-
tivity in treated snails confirms that
S. nigrum stimulated formation of
cytotoxic free radicals, H
2
O
2
in par-
ticular, in snail tissue. In susceptible
snail tissue a high activity of CAT
was reported to reduce parasite kill-
ing (Hahn et al., 2001; Mahmoud
and Rizk, 2004). Moreover, Nabih
and El-Ansary (1993) found that
CAT in susceptible snail tissue ho-
mogenate had a higher affinity for
H
2
O
2
than that in resistant snail tis-
sue homogenate.
Although the concentration of the
substrate, H
2
O
2,
increased in treated
snails GP
x
activity decreased. Cor-
rocher et al. (1986) reported that the
increased production ROS inacti-
vated GP
x
. Therefore, it appeared
that in the current study the mollus-
cicide increased both the level of
H
2
O
2
and the CAT activity but the
activity of the enzyme and GSH
level were not high enough to de-
toxify all the H
2
O
2
produced, thus
the excess H
2
O
2
in treated snail tis-
sue inhibited GP
x
.
The excess H
2
O
2
produced in
treated snails may cause parasite
death even in susceptible snails.
Hahn et al. (2001) pointed out that
resistant snails contain more supe-
roxide dismutase than susceptible
ones and Mahmoud and Rizk (2004)
reported that the activity of superox-
ide dismutase in resistant snails was
twice that observed in susceptible
ones, thus resistant snails produce
more H
2
O
2
than susceptible ones. In
snails H
2
O
2
is the main ROS in-
volved in schistosome killing (Mko-
ji et al., 1988; Goodall et al., 2004).
Since the schistosomes didnt pro-
duce catalase (Nabih and El-Ansary,
1993) enhanced production of H
2
O
2

211

induced by S. nigrum increased the
efficacy of the free radical in elimi-
nating the parasite from snail tissue.
S. nigrum proved to be hepato-
protective (Hussein et al., 2005),
anticancer (Lee and Lim, 2006),
anti-ulcerogenic (Jainu and Devi,
2006) and anti-hyperglycemic (Val-
lassenor and Lamadrid, 2006). Con-
sequently, it can be used safely
without causing any side effects on
human population.

Conclusion

The outcome results of this study
showed that the sub-lethal concen-
tration of Solanum nigrum increased
both the ability of snail tissue to
generate cytotoxic ROS, H
2
O
2
, in
particular, and protection of the tis-
sue from the cytotoxicity through
the induction of the antioxidants
GSH and catalase. The increased
level of the cytotoxic radicals would
eliminate the schistosome immature
parasite. A sub-lethal concentration
of the molluscicide would lead to
cytotoxic killing of the schistosomes
without killing the snails, thus main-
taining the ecosystem.

Acknowledgement

The study was kindly funded by
the Applied National Research
Project Grant from the Deanship of
The Scientific Research, King Saud
University, Saudi Arabia.

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215



DISTRIBUTION AND SEASONAL ACTIVITY OF MOSQUITOES IN
AL MADINAH AL MUNWWRAH, SAUDI ARABIA
By
S.M.KHEIR; A.M. ALAHMED, M.A AL KURIJI, AND
SALEEM F. AL ZUBYANI
Department of Plant Protection, College of Food and Agricultural
Sciences, P. O. Box 2460, King Saud University, Riyadh 11451, King
Abdul Aziz City for Science and Technology, Riyadh, and Ministry of
Health, Al Madinah Al Munwwrah, Saudi Arabia.

Abstract

In this study, 2654 adults and mosquito larvae, which belong to 18 species
and 4 genera, were collected: Aedes (2 spp.), Anopheles (7 spp.), Culex (8 spp.)
and Culiseta (1sp.). They were Aedes caspius, Ae. aegypti, Anopheles. azaniae,
An. d'thali, An. multicolor, An. rhodesiensis, An. stephensi, An. Sub-pictus, An.
turkhudi, Culex laticinctus, Cx. perexiguus, Cx. pipiens, Cx. quin-quefasciatus,
Cx. simpsoni, Cx. theileri, Cx. tritaeniorhynchus, Cx. univittatus and Culiseta
longiareolata. A total of 2270 mosquito larvae were collected, and Culex spp.
were the most abundant, where 1629 (71.76%) larvae were collected, followed
by 499 (21.98%) Anopheles spp., 94 (4.14%) Aedes spp. and 48 (2.12%) Culi-
seta longiareolata. Of, 384 adult mosquitoes collected Culex spp. were the
most abundant and 328 (85.42%) were collected, followed by 22 (5.73%)
Aedes spp., 19 (4.94%) Anopheles spp. and 15 (3.91%) Culiseta longiareolata.
The physical properties of the water in the breeding sites of mosquito larvae
showed that pH of water varied between 6.9 & 9.9, the total dissolved salts
(TDS) varied between 378-9504 ppm and water temperature varied between
8.7C in winter to 29.9C in summer. There was no correlation between pH &
TDS of water in breeding site and distribution of larvae.
The population density started to increase in March, with a peak in August
when temperature was 36C. The activity started to decrease in October, and
minimum activity was in January, when temperature was below 5C. The sea-
sonal abundance of adult mosquitoes was not affected by rainfall.
A. aegypti, vector of Dengue fever virus, Cx. tritaeniorhynchus, vector of Rift
Valley fever and Cx. univittatus, vector of sindbis virus were reported for the
first time in Al Madinah Al Munawwrah Region. These vectors constituted a
major health problem, and every effort should be made for feasible control.
Key words: Saudi Arabia, Al Madinah Al Munawwrah, adults and mosquito
larvae, medical importance
216

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Introduction

Many reports are available on the
distribution of mosquitoes (Diptera:
Culicidae) in Saudi Arabia (Mat-
tingly and Knight, 1956; Zaher,
1973; Buttiker, 1981; Will et al.,
1985; Abdullah and Merdan, 1995;
Jupp et al. 2002, Miller et al., 2002;
Abdoon and Al Shahrani, 2003;
Alahmed et al., 2007 and Al Gham-
di et al., 2008), but very little in-
formation is available on mosquito
fauna of Al Madinah Al Munawwa-
raha Region.
During the past few decades, Sau-
di Arabia has witnessed tremendous
efforts in social development and
urbanization in all provinces, which
have affected insect fauna, particu-
larly mosquitoes. Expansion of
agricultural projects, development
of water resources and urbanization
led to creation of more permanent
and temporary breeding sites for
mosquitoes.
The present work was undertaken
to study the distribution of mosquito
fauna, as well as the seasonal abun-
dance of adult mosquitoes in Al
Madinah Al Munawwarah Region
in the western part of Saudi Arabia.


Study Area: The study area is an
upland plateau scored by numerous
valleys (wadis) covered with grasses
and scrub vegetations which are
used for pasture. Al Madinah Al
Munawwarah Region has a typical
desert climate, which is cold rainy
in winter and hot dry in summer. Al
Madinah Al Munawwarah is one of
the biggest oases in the region and
famous for growing dates. The
study area lies between Lat. 23.00,
26.00 N and Long. 37.51, 39.59
E. (Fig 1).
Larval Collection: During the
period March 2004 to Feb. 2006, a
mosquito survey (Diptera: Culici-
dae) was conducted in Al Madinah
Al Munawwarah Region, in the
western part of Saudi Arabia
(Fig.1). Weekly field trips were
made to collect mosquito larvae
from all potential breeding sites by a
standard mosquito larval dipper
with extendable handle; and three to
five scoops were taken from each
breeding site (350 ml each). Larvae
were extracted, preserved into 80%
ethyl alcohol in glass vials with
screw caps, labeled and sent to the
Entomology Laboratory, College of
Food and Agricultural Sciences,
King Saud University, Riyadh. Lar-
vae were mounted as described by
R.E. Harbach from Natural History
Museum, London (Personal Comm-
unication), and identified by stan-
dard keys (Hopkins, 1952; Matting-
ly and Knight, 1956; Harbach,
1988; Al-Tubiakh, 1995). Repre-
sentative samples were sent to Brit-
ish Natural History Museum, Lon-
don for confirmation.
In each larval breeding site, the
following information were record-
ed: coordinates of the breeding site,
date and time of larval collection,
current weather conditions, water
217

temperature, pH and total dissolved
salts (TDS), degree of water turbidi-
ty and motion, type of breeding site
(e.g. irrigation canals, rain water
collections, ponds, septic tank or
water storage tanks) and presence or
absence of shadow, algae or aquatic
plants.

Fig. 1: Collection sites of mosquito in Al Madinah Al Munwwrah, Saudi Arabia.

Adult Collection: Adult mosqui-
toes were collected from Al Madi-
nah Al Munawwarah Region, using
CDC and standard New Jersey (NJ)
light traps (Bioquip Company, Gar-
dena, CA, 90248-3602, USA).The
light traps were attached to a battery
that supplies power, and installed
permanently near suitable mosquito
breeding sites. The light traps were
218

operated once every two weeks
from sun-set to sun-rise the follow-
ing day throughout the study period.
The collected mosquitoes were
packed, labeled and transported to
the Entomology Laboratory in
Riyadh. Adult mosquitoes were
identified by using standard identi-
fication keys of Mattingly and
Knight (1956); Sebai et al. (1974)
Harbach (1988); Glick (1992).
Some representative samples of
identified mosquitoes were sent to
the British Natural History museum
in London for confirmation.

Results

During this study, 2270 mosquito
larvae, which belong to 4 genera
and 18 species, were collected.
These are Aedes (2 spp.), Anopheles
(7 spp.), Culex (8 spp.) and Culiseta
(1 sp.). The collected larvae were
identified as follows:
Ae. caspius, Ae. aegypti, An. aza-
niae, An. d'thali, An. multicolor, An.
rhodesiensis, An. stephensi, An.
subpictus , An. turkhudi, Cx lati-
cinctus, Cx. perexiguus, Cx. pipiens,
Cx. quinquefasciatus, Cx. simpsoni,
Cx. theileri, Cx. tritaeniorhynchus,
Cx. univittatus and Culiseta longia-
reolata.
Culex larvae were the most abun-
dant, and 1629 (71.76%0) were col-
lected, followed by 499 (21.98%)
Anopheles, 94 (4014%) Aedes and
487 (2.12%) Culiseta (Tab. 1).
Mosquito larvae were collected (tab.
2) from stagnant or slowly running
irrigation canals with submerged or
floating plants, grassy or slightly
shaded water pools and water sto-
rage tanks. Larvae were also en-
countered in temporary or perma-
nent brackish polluted water, drai-
nage water collections with some
algae and from abandoned wells.
The results showed that there is no
significant correlation between tem-
perature, pH and TDS of the water
in the breeding site and distribution
of mosquito larvae.
During this study, 384 adult mos-
quitoes were collected. Out of these,
148 (38.54%) were collected by
CDC light traps, while 236
(61.46%) were collected by NJ light
traps (Tab. 3). Adult Culex spp.
were the most abundant, and 328
(85.42%) were collected, followed
by 22 (5.73%) Aedes spp., 19
(4.94%) Anopheles spp. and 15
(3.91%) Culiseta sp.
The seasonal activity of adult mos-
quitoes was investigated. The popu-
lation density started to increase in
March, and a peak of activity was
attained in August when the temper-
ature was 36 C. The population
density started to decrease in Octo-
ber, and a minimum activity was
reached in January, when the tem-
perature was below 20 C (Fig 2).
No relationship was observed be-
tween rainfall and mosquito activity,
but the number of mosquitoes col-
lected was increased during rainy
season when the temperature was
between 25 C- 30 C (Fig 3).

2

0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
30
35
M
e
a
n

t
e
m
p
.

(
C
)
M
e
a
n

N
o
.

o
f

m
o
s
q
u
i
t
o
e
s

c
o
l
l
e
c
t
e
d
month
No. of mosquitoes Mean temp.

Fig. 2: Effect of temperature on seasonal abundance of mosquito in Al Madinah Al Munawwara
Region.

0
4
8
12
16
20
0
5
10
15
20
25
30
35
M
e
a
n

r
a
i
n
f
a
l
l

(
m
m
)
M
e
a
n

N
o
.

o
f

m
o
s
q
u
i
t
o
e
s

c
o
l
l
e
c
t
e
d
month
No. of mosquitoes Mean rainfall

Fig. 3: Effect of rainfall on seasonal abundance of mosquito in Al Madinah Al Munawwara Re-
gion.

Discussion

This study has shown that Culex
larvae were the most abundant
(71.76%), and were collected from
various habitats. The widespread of
Culex larvae might be due to the
fact that they can exploit a wide va-
riety of aquatic habitats for their
development and survival, and can
tolerate highly polluted aquatic en-
vironment and relatively saline wa-
ter. In fact, beside water quality,
221

there are some other factors which
determine the suitability of various
types of aquatic habitats for mosqui-
toes, like presence or absence of
shade and aquatic vegetation and
degree of water motion and turbidi-
ty.
Cx tritaeniorhynchus, the main
vector of Rift Valley fever virus in
southern part of Saudi Arabia (Mil-
ler et al., 2002; Jupp et al., 2002),
Ae. aegypti, the main vector of yel-
low fever virus, and Cx. univittatus,
a potential vector of sindbis virus in
the Eastern part of Saudi Arabia
(Will et al., 1985) were encoun-
tered. The presence of these mos-
quito vectors constitutes a great
health problem, and every effort
should be made to control them so
as to prevent the spread of Rift Val-
ley fever yellow fever and sindbis
viruses in this region
Culiseta longiareolata was also
reported in this region, but in few
numbers. Adults of this species nev-
er enter houses and rarely bite man
(Salit et al., 1994), so this species
appears to be of no medical impor-
tance, but its larvae may be canniba-
listic and prey on aquatic insects
and tadpoles (Blaustein and Marga-
lit, 1994).
The results showed that adult mos-
quitoes were collected from differ-
ent parts of the region, but in differ-
ent densities. In fact, the climate in
this region is conducive for devel-
opment and survival of mosquitoes,
which makes the control programs
very difficult. Agricultural expan-
sions and new irrigation canals,
pools and extensive farming may
also help in wide spread and occur-
rence of mosquitoes in this region
(Al Zahrani, 2007).
The number of mosquitoes col-
lected was very low, this might be
due to regular and extensive adulti-
cides and larvicides applications to
control malaria in the region.
In this study, some species of
Anopheles were collected as larvae
but not as adults, like An. multico-
lor, An. subpictus and An. turkhudi.
This might be due to some differ-
ences in the adult behavior, suggest-
ing that CDC and NJ light traps are
not suitable for collection of Ano-
pheles mosquitoes. This is true, be-
cause most of Anopheles mosqui-
toes are indoors feeders and they do
not come near light traps which
were placed outside houses. Since
malaria is an endemic disease in Al
Madinah Al Munawwarah, we sug-
gest the use of more efficient me-
thods for sampling Anopheles mos-
quitoes like spray sheet method.

Acknowledgement

The financial support was kindly
from King Abdul Aziz City for
Science and Technology is highly
appreciated. Thanks are also ex-
tended to Dr. Ralph Harbach (Natu-
ral History Museum, London) for
confirming the identification of the
mosquito specimens.

References

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2

3

229



EFFICACY OF PUNICA GRANATUM EXTRACT ON IN-VITRO AND
IN-VIVO CONTROL OF TRICHOMONAS VAGINALIS
By

GEHAD M. EL-SHERBINI
1
, KHADRA M. IBRAHIM
2
, EMAN T. EL
SHERBINY
3
, NEVEIN M. ABDEL-HADY
4
AND TOSSON A. MORSY
5

Department of Parasitology, Faculty of Pharmacy
1
, October 6 Uni-
versity, 6
th
October City, Department of Gynaecology and Obstetrics, Al-
Azahar Faculty of Medicine for Girls
2
, Department of Zoology, El Nahda
University
3
, Beni Sweif, Department of Pharmacognosy, Faculty of
Pharmacy, Al-Azhar University for Girls
4
, Department of Parasitology,
Faculty of Medicine, Ain-Shams University
5
, Cairo 11566, and, Egypt

Abstract

Trichomoniasis vaginalis is now an important worldwide health problem.
Metronidazole has so far been used in treatment, but the metronidazole-
resistant strains and unpleasant adverse effects have been developed. Treatment
of patients with metronidazole refractory vaginal trichomoniasis constitutes a
major therapeutic challenge and treatment options are extremely limited. The
last 7 years have seen over seven times as many publication indexed by Mid-
line dealing with pomegranate (Punica granatum) than in all the years preced-
ing them, because of this, and the virtual explosion of interest in pomegranate
as a medicinal and nutritional product that has followed, this work is accord-
ingly launched. Natural plant extract purified from Pomegranate (Roman) was
in-vitro investigated for its efficacy against T. vaginalis on Diamond media.
Besides, infection women (18/20) who accepted to be treated with P. granatum
juice were completely curedand followed-up for two months.
The anti-trichomoniasis vaginalis activity of P. granatum extract (in-vitro
and in-vivo) gave very promising results.
Key word: Trichomonas vaginalis, Punica granatum, in-vitro, in-vivo treat-
ment, Diamond media.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Although Trichomonas vaginalis
was first described by Donne (1836)
yet active studies did not begin until
the 20
th
century. The research has
been a progression of the phases
through-out the last sixty years and
has gone from developing axenic
culture and defining nutritional re-

230

quirements to finding an effective
treatment (Pooch et al., 1996). It
was considered either as a harmless
vaginal colonizer or simply a minor
nuisance (Pereira et al., 2007).
Trichomoniasis infection was ac-
counted to about half of all the cur-
able sexually transmitted diseases
worldwide (Hook et al., 1999). The
incidence of this sexually transmit-
ted parasite has reached the epi-
demic levels in many countries
(Lewis et al., 1997). Also, T. vir-
ginals survived in swimming pool,
where man may acquire infection
(Pereira et al., 2007). The general
annual adults infection was 180-200
millions and being higher than that
of gonorrhoea, syphilis, and the
Chlamydia infections all together
(Schwab and Burgess, 2004). In
many Arab countries trichomoniasis
was reported; Jordan (Morsy and El
Dasouki, 1979)., Iraq (Mahdi et al.,
2001), Egypt (Morsy et al., 1984;
Negm and Elhaleem, 2004), Saudi
Arabia (Al-Zanbagi, 2007; Al-
Zanbagi et al., 2005), Libya (Ka-
ssem and Majoud, 2006) and Tuni-
sia (Zribi et al., 2008). The wide
diversion in the subtypes of T.
vaginalis isolates caused different
clinical symptoms with diversity of
innate immune responses (Hussien
et al., 2005). The infection was al-
ways associated with other sexually-
transmitted diseases (STDs) and a
sensitive marker for high risk sexual
behaviour (James et al., 1995). Be-
sides, T. vaginalis in males caused
non-gonococcus urethritis (Wilson
et al., 1980), but serious complica-
tions (Benchimol et al., 2008),
Also, T. vaginalis adherence me-
diates differential gene expression
in human epithelial cells was re-
ported (Kucknoor et al., 2005). The
premature rupture of membranes,
low-birth weight, preterm labour
(Cotch et al., 1997), female infertil-
ity (El Shazly et al., 2004), and the
postpartum infection, even in as-
ymptomatic woman were associated
with the trichomoniasis (Schwab,
2002). T. vaginalis is a factor in
genesis and caused cervical neopla-
sia (Zhang and Begg, 1994), pro-
gression of the cervical carcinoma
(Sayed El-Ahl et al., 2002) and it
phagocytes sperm cells during the
sexual intercourse (Benchimolb et
al., 2008). Unlike other STDs, T.
vaginalis rate was more prevalent
among women of all ages (Bowden
and Garnett, 1999), since trichomo-
niasis was a curable infection by a
single dose metronidazole (Okun et
al., 2005), successful control of
STDs was aided by sensitive, sim-
ple, rapid test(s). WHO (1999) men-
tioned that more than 80% of the
world's population relies on tradi-
tional medicine for their primary
healthcare needs.
No doubt, treating patients low-
ered the overall disease prevalence
and morbidity. Metronidazole has
so far been the most widely used
drug for treating trichomoniasis
vaginalis (Houang et al., 1997).
But, metronidazole can lead to
drug resistance and potential risks
of mutagenesis and carcinogenicity

231

(El-Sherbini et al., 2009). In addi-
tion, its side effects such as head-
ache, dry mouth, glossitis, and urti-
caria caused by lenity treatment or
high doses have been described
(Klebanoff et al., 2001). However,
at least 5% of the clinical trichomo-
niasis was caused by strains resis-
tant to commonly used drugs (El-
Moamly and Rashad, 2008). Be-
sides, Hussien et al. (2005) reported
the presence of different strains of T.
vaginalis, and they added that the
lack of approved alternative thera-
pies for T. vaginalis treatment indi-
cated that the higher and sometimes
toxic doses of metronidazole were
used.
The use of herbal medicines
represents a long history of human
interactions with the environment
(Brown, 1995). Plants used for tra-
ditional medicine contain a wide
range of substances (Chevalier,
1996) that can be used to treat
chronic as well as infectious dis-
eases (Diallo et al., 1999). The
medical value of plants lies in some
chemical substances that produce a
definite physiological action on the
human body (Tonkol and Morsy,
2008; Abdel Hady et al., 2008).
The most important of these bio-
active compounds of plants are al-
kaloids, flavanoids, tannins, and
phenolic compounds (Edeoga et al.,
2005).
Natural products are not only the
basis for traditional or ethnic medi-
cine, but also screening natural plant
products provided highly successful
new regimens for human welfare
(Hussain et al., 1992). Many of the
new natural product groups of me-
dicinal and/or herbs have shown
anti-parasitic properties in-vitro and
in-vivo studies of surprising effi-
cacy and selectivity (Kayser et al.,
2003).
In the present study, the efficacy
of Punica granatum extract against
cultured Trichomonas vaginalis and
trichomoniasis infected women was
evaluated.


The institutional review boards of
all participating hospitals have ap-
proved all clinical studies. The
study is registered at the Ministry of
High Education and Scientific Re-
search, Academy of Scientific Re-
search and Technology No. 292473.
The participants were informed, to
allow for an attrition rate (i.e.
women who discontinue participa-
tion in the study entirely, including
failure to complete all follow-up).
Thus, 33 women will be available
for intention to the study. The pa-
tients were recruited from the out-
patient Clinic, Department of Gy-
naecology and Obstetrics, Al-
Zahraa University hospitals. Poten-
tially eligible patients were identi-
fied clinically and parasitologally.
They were asked to complete a brief
personal and medical questionnaire
to screen for the tri-chomoniasis
history and any gynaecological
complications. Upon completion of
the study, women assigned to con-
trol group were given the choice
232

undergoing the study program. Pa-
tients in whom treatment failed and
for whom re-infection from a sexual
partner was a possibility were ex-
cluded from the case definition.
"Failure to respond" was defined as
persistence or recurrence (within a
month) of symptoms and signs of
vaginitis together with the following
confirmatory laboratory feature of
vaginal trichomoniasis: high vaginal
pH increased the numbers of poly-
morphonclear leukocytes, and a
visualization of the motile tricho-
monads on the direct vaginal smear
microscopic examination.
In vitro Trichomonas cultures of
individual specimens were per-
formed using Dimoned's medium
Modified (Remel), and they were
examined at 24 hr and 48 hr for the
presence of motile trichomonads.
Two vaginal swabs were obtained
from childbearing period trichomo-
niasis infected women by sterile
vaginal swab. The first swab was
obtained from the lateral wall of
vagina and was used to make a wet
mount preparation on a glass slide
with a drop of normal saline and
looking for motile trichomonads
(Garcia, 2001). The second swab
was obtained from the posterior
fornix of the vagina and inoculated
immediately after collection in the
Diamond media at 32
o
C and exam-
ined for the motile trichomonads at
the 24, 48 and 96 hours of the incu-
bation period (Diamond, 1957).
Preparation of the extract: P.
granatum fruits were selected from
tree that was neither treated with
any insecticide nor with plant fertil-
izer. The fruits were carefully
washed with sterile distilled water.
The peels of pomegranate fruits
were manually removed, sun dried
and powdered. Powder was ex-
tracted with a Soxhlet extractor us-
ing methanol for 24 hr (Negi et al.,
2003). Extract was filtered through
Whatman No. 41 filter paper for
removal of peel particles. (Solvent
was removed under gentle pressure
in rotary evaporator till dryness, and
residue was stored at 4
o
C (Guo et
al., 2005).
Extract was tested at different
concentrations, diluted with sterile
normal saline against cultured T.
vaginalis. Control culture lacked
extract. All culture-media were in-
cubated at 37
o
C and examined for
living T. vaginalis. Twenty tricho-
moniasis vaginalis infected women
whose partners were trichomoni-
asis-free (Wilson and Ackers, 1980)
accepted to be treated by using
vaginal washing by the different
concentrations of the fresh P.
granatum juice.
Of the women three had candidi-
asis as well. Post-treatment, they
were followed up clinically and
parasitologically for two months to
ensure complete cure.
Statistical analysis: Data were ex-
pressed as means and values were
evaluated by the Chi-square test and
P<0.5 was considered significant
(SPSS, 1999).

233

Results

The mean age of the female pa-
tient was 37.2 year (range, 25-58
years). The duration of vulvovaginal
symptoms was (range, 4 months to
5 years). The in-vitro susceptibility
of isolates of T. vaginalis to P.
granatum extract was determined at
two pH values. At pH 4.65 P.
granatum showed an effect against
T. vaginalis, they dead immediately
in the tube containing 50 mg &100
mg extract, and within 0.5 hr in tube

with 20mg extract. At pH 6.0 the
extract had no effect (tab. 1). At
10% concentration, all trophozoites
died (Fig.1). The bars represented
the mean standard deviation from
three individual experiment of per-
centage reduction in trophozoites
viability compared to control
(100%). One of the women who did
not cure had candidiasis, the other
two women with candidiasis were
cured.
Table 1: Effect of pomegranate extract (PH 4.65& PH 6.00) on viability of T.
vaginalis in hours.

Viability of T .vaginalis in 0.1 ml Diamond medium per hour Extract conc. in
0.1 ml medium
24 1.0 0.5 0.0

Alive
Dead
Dead
Dead
Dead

Alive
Active flagella
Dead
Dead
Dead

Alive
A. flagella
Dead
Dead
Dead

Alive
A. flagella
A. flagella
Dead
Dead
(At PH 4.65)
0.0m ( control)
0.1ml (10 mg)
0.2ml (20 mg)
0.5ml ( 50 mg)
1.0ml (100 mg)

Alive
Alive
Alive
Alive
Active
A. flagella
A. flagella
A. flagella

Alive
Alive
Alive
Alive
Alive
Active flagella
Active flagella
Active flagella

Alive
Alive
Alive
Alive
Alive
A. flagella
A. flagella
A. flagella

Alive
Alive
Alive
Alive
Alive
Alive
Alive
Alive
(At PH 6.00)
0.0m ( control)
0.1ml (10 mg)
0.2ml (20 mg)
0.5ml ( 50 mg)
1.0ml (100 mg)
1.5ml (150 mg)
2.0ml (200 mg)
3.0ml 300 mg)

Table 2: Effect of pomegranate fresh juice as vaginal washes in treatment of
trichomoniasis-infected women, as evaluated clinically and parasitologically.

Pomegranate fresh juice No. treated No. cured Cure percent
Concentration (100%) 5 5 100
Total 20 18 90

234

Figure1: Effect of P. granatum extract on T. vaginalis trophozoites viability.
viability of trophozoite
0
50
100
150
C M 1% 5% 10%
conce. of extract
% Viability

C: control (Trophozoite without extract), M: Metronidazole (100ug/m) =+ve control
Discussion

The studies into other chemicals
and/or medicinal plants or herbs for
alternative regimens inexpensive,
effective, short course of treatment
and safe to use in pregnancy against
T. vaginalis is a must. Globally,
herbal remedies have been studied
under rigorous controls and was
technologically approved by authors
in many countries.
Successful determination of bio-
logically active compounds from
plant material is largely dependant
on the type of solvent used in the
extraction procedure properties of a
good solvent in plant extraction in-
duce ease of evaporation at low
heat, promotion of rapid physiologic
absorption of the extract, preserva-
tive action and inability to cause the
extract to complex or dissociate
(Michie and Cooper, 1991). The
choice depended on the targeted
compounds. The most commonly
used solvents for investigation of
microbial activity in plants are
methanol, ethanol, and water (Rojas
et al., 2006).
In the present study, P. granatum
extract on T. vaginalis in Diamond
media showed 100% efficacy in
dilution up to 10%. On the other
hand, extracts in the concentrations
of 5%, 1% & 0.5% killed 40%, 25%
& 10% of T. vaginalis respectively.
Generally speaking, metronidazole
was worldwide used within the 2
years of its introduction, but the
lack of surveillance data of vaginal
trichomoniasis and clinical and
microbiological response to treat-
ment, made incidence of Metroni-
dazole resistance spares. The devel-
opment of drug resis-tance in hu-
man against commonly used treat-
ment has necessitated a search for
new anti-agent substances from
other sources including plants (Er-
dogrul, 2002). The Pomegranate
fruit has been used for centuries in
ancient cultures for its medicinal
purposes. It is widely consumed
fresh and in beverage forms as juice
and wine (Longtin, 2003). Proper-
ties attributed to its high content of
polyphenols, including ellagic acid
in its free and bound forms, and

235

other flavouroids (Gil et al., 2000).
The last two decades many authors
dealt with Punica granatum (Pome-
granate) as a medicinal plant (Kris-
shna, 1999). It is a shrub or small
tree which several parts have been
used by old Indian physicians.
Nowadays, parts of pomegranate are
used as an astringent, anti-microbial
haemostatic, anti-diabetes, anti-
helminthes (Machado et al., 2002),
anti-prostate cancer (Retting et al.,
2008.), improved antioxidant and
anti-mutagenic activities (Negi et
al., 2003), anti-oxidant function in
elderly subject (Guo et al., 2008),
anti-fungal peptide (Guo et al.,
2009), and anti-Candida (Tayel and
El-Tras, 2009), mouth-anti-T. gin-
givalis (Disilvestro et al., 2009), as
heart-healthy juice (Basu et al.,
2009), prevention of the cardiovas-
cular diseases (Rosenblat et al.,
2009). Dried per carp was decocted
with other herbs and used to treat
colic, dysentery, leucorrhoea (Duke
et al., 1985). In the present study,
18/20 trichomoniasis women treated
with pomegranate juice as vaginal
washing completely cured. One of
the two untreated women has can-
didiasis. But, other two trichomoni-
asis and candidiasis women cured.
Generally speaking, vulvovaginal
candidiasis is a name given to C.
albicans infection of the vagina as-
sociated with avulva dermatitis most
common with Pregnancy. Vaginal
thrush and monilia are also names
for C. albicans. Most women no-
ticed from time to time that they
have a discharge from the vagina
which keeps the mucous lining of
the vagina moist. Overgrowth of C.
albicans causes a heavy white curd-
like vaginal discharge, a burning
sensation in the vagina and vulva
and/or an itchy rash on the vulva
and surrounding skin. Oestrogen
causes the lining of the vagina to
mature and to contain glycogen, a
substrate on which C. albicans
thrives. Lack of oestrogen in
younger and older women makes
vulvovaginal candidiasis much less
common (Metwally et al., 2006).
Abdelaziz and Ashour (1987) re-
ported microbial contamination of a
hexetidine mouth wash. Talaat et al.
(2010) in Egypt reported candidiasis
infection among catheter-associated
urinary tract infection in 4 intensive
care units at Alexandria university
hospitals and El-Nawawy et al.
(2006) reported infection among
hospitalized children intensive care
unit. Halawa et al. (2010) in USA
reported that cardiac rhythm man-
agement devices CRMD endocardi-
tis accounts for about 10% of all
device-related infections, and car-
diac infection caused by Candida
sp. is a rare event. They added that
from 1969 to 2009, a total of 15
male patients with CRMD-Candida
endocarditis (12 pacemakers & 3
implanted cardio-verterdefibrillator)
were found. All non-albicans Can-
dida sp. were frequently recovered,
a major fungal embolus occurred in
27% of patients and two of 10 pa-
tients who received defined antifun-
gal therapy and device explantation
expired. CRMD Candida endocardi-

236

tis is a rare and serious clinical
event. Detailed data concerning the
pomegranate juice and treatment of
candidiasis will be published later
on elsewhere
Also, the rind of fruit and flower,
combined with aromatics, such as
cloves, cinnamon, coriander, pepper
etc as bowel astringent in the diar-
rhea (Blatter et al., 2001). It was
used externally in treatment of the
vaginal discharge, mouth sores, and
throat infections (Brown, 1995).
Methanol extracts of P. granatum
fruit exhibited a higher degree of
antimicrobial activity (Prashanth et
al., 2001). The fruit was success-
fully used to treat the dysentery,
diarrhea and gastralgia (Warrier et
al., 2002). Voraavuthikunchai et al.
(2005) reported that P. granatum
contains 25% tannins which made it
an effective astringent. In old medi-
cine, pomegranate as a pharmacy
unto itself was used as an anti-
parasitic agent, a blood tonic, and
heal apathies, and ulcers (Jurenka,
2008). Mohan et al. (2009) found
that Pomegranate juice has anti-
hypertensive action in Ang II dia-
betic model. Gould et al. (2009)
reported the antimicrobial activities
of pomegranate rind extract (PRE)
in combination with Fe (II) and Cu
(II) salts against extended-spectrum
multidrug-resistant Pseudomonas
aeruginosa. Anti-microbial suspen-
sion assays were carried out using
aqueous extract of pomegranate
alone or in combination with metals
salts against P. aeruginosa.
The extract: metal salt combina-
tion enhanced with the addition of
vitamin C showed marked activities
shown for aqueous PRE/ Cu (II)
preparations were greatly enhanced
by addition of reductant vitamin C.
But, the aqueous PRE/Fe (II) prepa-
rations were inactive, regardless of
addition of vitamin C. Combination
of PRE and Cu (II) salts and vitamin
C showed greatest activity against
clinical isolates of P. aeruginosa.
Of great interest is the efficacy
pomegranate juice in cancer treat-
ment. Adhami et al. (2009) reported
that Pomegranate fruit from the tree
P. granatum has been dubbed as the
"nature's power fruit" dating back to
Biblical times, the tree itself is at-
tributed to possess extraordinary
medicinal properties. The geo-
graphical distribution of the tree,
being native to the Middle East and
some Asian countries, is generally
attributed to a lack of interest in its
medicinal properties by many west-
ern scientists. However, the unique
biochemical composition of the
pomegranate fruit being rich in an-
tioxidant tannins and flavonoids has
recently drawn attention of many
investigators to study its exceptional
healing qualities. Recent research
has shown that pomegranate ex-
tracts selectively inhibit the growth
of breast, prostate, colon and lung
cancer cells in culture. In preclinical
animal studies, oral consumption of
pomegranate extract inhibited grow-
th of lung, skin, colon and prostate
tumors. An initial phase II clinical
trial of pomegranate juice in pa-

237

tients with prostate cancer reported
significant prolongation of prostate
specific antigen doubling time. This
review focuses on recent investiga-
tions into the effects of pomegranate
fruit on cancer. Adams et al. (2010)
stated that the pomegranate fruit, a
rich source of ellagitannins (ET),
has anticancer and anti-athero-
sclerotic properties. On consump-
tion of the pomegranate ETs hydro-
lyze, releasing ellagic acid, which is
then converted to 3, 8-dihydroxy-
6H-dibenzo[b,d]pyran-6-one (uro-
lithin) derivatives by gut microflora.
They concluded that pomegranate
ET-derived compounds have poten-
tial for the prevention of estrogen-
responsive breast cancers.
Koyama et al. (2010) reported that
the IGF axis is critical for the regu-
lation of apoptosis in many human
cancer cell lines and potent anti-
tumorigenic effects of pomegranate
juice and extracts have been re-
ported. They concluded that the in-
teractions between IGF system and
pomegranate-induced apoptosis.
Sturgeon and Ronnenberg (2010)
clarified the effects of pomegranate
constituents on key hormones
known to be involved in breast can-
cer could result in important infor-
mation for consumers and shed fur-
ther light on the impact of diet on
breast cancer risk. Kasimsetty et al.
(2010) found that the ellagitannins
and urolithins released in the colon
upon consumption of pomegranate
juice in considerable amounts could
potentially curtail the risk of colon
cancer development, by inhibiting
the cell proliferation and inducing
apoptosis. Gugliucci (2010) found
that the pomegranate juice polyphe-
nols increased the hepatocyte para-
oxonase 1 secretion.
Bialonska et al. (2009) mentioned
that consumption of pomegranate
products leads to a significant ac-
cumulation of ellagitannins in the
large intestines, where they interact
with complex gut microflora. They
added that pomegranate by-products
and punicalagins inhibited the
growth of pathogenic clostridia and
Staphyloccocus aureus. The probi-
otic lactobacilli and bifidobacteria
were generally not affected by ella-
gitannins, while relatively small
growth inhibition by ellagic acid
likely resulted from decreasing me-
dia quality due to formation of tan-
nin-protein complexes. Growth of
Bifidobacterium animalis was mild
inhibited by the punicalagins, puni-
calins, and ellagic acid. POMx sup-
plementation significantly enhanced
growth of B. breve & B. infantis.
On the other hand, the pomegra-
nate extract was successfully used
in the controlling insects of medical
and veterinary importance as well as
pests. Morsy et al. (1998) used four
solvent extracts of each of Lemon-
grass (Symbopogon citratus), San-
tonica (Artemisia cinae) and Pome-
granate (Punica granatum) against
the 3
rd
instars larvae of the myiasis
producing Chrysomyia albiceps.
They found that pomegranate ex-
tracts showed larvicidal activity
with LC
50
ranging between 25 ppm
(acetone extract) and 280 ppm

238

(chloroform extract), Santonica
showed larvicidal activity with LC
50

between 48 ppm (ethanol extract)
and 380 ppm (acetone extract), and
Lemongrass showed activity with
LC
50
between 135 ppm (ethanol
extract) and 570 ppm (chloro-form
extract). They concluded that the
most effective was the acetone ex-
tract of pomegranate, followed by
ethanol extract of Santonica and
lastly ethanol extract of Lemon-
grass, and that the shift to insect
control by plant extracts pave the
way to a somewhat healthy envi-
ronment. Also, Mazyad et al. (1999)
used pomegranate juice as larvicide
against the myiasis producing3rd
stage larvae of Lucilia sericata.
Verghese and Sreedevi (2006) re-
ported that the trips have developed
resistance to insecticides of the or-
ganophosphates, carbamates, syn-
thetic pyrethroids, etc., which form
the important core of the recom-
mendation for trips management.
Scirtothrips dorsalis, the pest of
fruits is highly polyphagous and of
late has become serious on grapes;
Clothianidin 0.008% gave the best
control with a low mean of 0.26%
berry damage/bunch as compared to
4.42% in the unsprayed check. It
was on par with acephate and mo-
nocrotophos, but significantly supe-
rior to Clothianidin 0.006% and di-
methoate. Verghese and Sreedevi
(2007) reported that the Aphis puni-
cae (Homoptera: Aphididae) is a
very serious pest attacking pome-
granate and the major predators
preying on A. punicae in the pome-
granate ecosystem were Cheilo-
menes sexmaculata, Scymnus sp.,
Pseudaspidemerus circumflexo, Pa-
ragus serratus, Ischiodon scutellaris
and Chrysopa sp. The predators
were distributed uniformly among
different tree quadrants and fol-
lowed the same distributional pat-
tern of A. punicae during their peak
in January and February. The popu-
lation of predators started building
up along with aphid population and
reached maximum at high aphid
densities and declined as the prey
availability declined. This indicated
that predators followed the same
trend of their prey, A. punicae,
showing a clear numerical response.
Savant et al. (2010) reported a se-
lective and sensitive multiresidue
analysis method for the simultane-
ous determination of 50 pesticides
of different chemical classes in
three commercially important fruits
of different nature viz. grape, po-
megranate, and mango. Jarvis et al.
(2010) reported that pomegranate
juice inhibits cytochrome P450 en-
zymes involved in warfarin meta-
bolism, and that the potential inte-
raction between pomegranate juice
and warfarin, as pomegranate juice
inhibits cytochrome P450 enzymes
involved in warfarin metabolism.
They added that this did not defini-
tively prove the association between
the pomegranate juice consumption
and the increased warfarin bioactivi-
ty but highlights the importance of
taking a complete drug, food and
juice history when assessing pa-
tients with unstable anticoagulation.

239

Conclusion

No doubt, the studies into medici-
nal plants and/or herbs for alterna-
tive regimens inexpensive, effec-
tive, short course of treatment and
safe to use in pregnancy against T.
vaginalis is a must. The present
study showed statistically signifi-
cant effects on T. vagi-nalis strains.
These proposed benefits, however,
are in assays that are as yet invali-
dated, and further research is needed
to prove the validity of these tests.
The results proved that Punica
granatum extract proved safe and
effective regimen agents in treating
T. vaginalis infection. Besides, the
fruit itself is at least available in all
countries. This will be the basic
form the basis for further investiga-
tion in the potential discovery of
new natural bioactive compounds.
Besides, the authors approved the
final manuscript and clearly declare
that they have no competing inter-
ests. No doubt, the role played by
the pomegranate juice in controlling
trichomoniasis co-infected with
Candida cannot be neglected.

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J. Egypt. Soc. Parasitol., 40 (1), April 2010, Article No. 21




245



EVALUATION OF "MYRRH EXTRACT" AGAINST SCHISTOSOMA
MANSONI: A HISTOLOGICAL STUDY
By
AHMED M.A. MASSOUD
1
, FAIKA H. EL EBIARY
2
, SUZI H. IBRAHIM
2

HANAN A.A. SALEH
2
AND HAZEM H.M. KHALIL
3

Departments of Tropical Medicine
1
, Histology
2
, and Internal Medicine
3
,
Faculties of Medicine, Al Azhar University
1
, Masr City, and Ain Shams
University
2,3
, Cairo 11566, Egypt

Abstract

This study investigated the effect of myrrh extract on different developmental
stages of Schistosoma mansoni. Sixty albino mice were used and divided into
three main groups: GI (control group), GII (infected group) and GIII (infected-
treated group). The last group was further divided into 3 subgroups where the
drug was administered in a dose of 500mg/kg body weight for 5 days starting
on the 1
st
day PI for IIIA, on the 21
st
day PI for IIIB and on the 45
th
day PI for
IIIC. A morphometric study was performed for the mean number and perimeter
of granulomas. In GII, typical bilharzial granulomas were frequently encoun-
tered in the portal tracts with numerous eosinophils, collagen fiber deposition
and reticular fiber condensation. Hepatocytes revealed vacuolation, nuclear
affection and depletion of glycogen. In GIII, granulomas were less frequently
observed with apparent decrease of eosinophils. The maximum effect of the
drug was observed in SGs IIIB and IIIC as detected by significant decrease in
the mean number and size of granulomas, paucity of eosinophils, decreased
fibrosis and reticular fibers and the restoration of the glycogen content in the
hepatocytes. The present data proved that myrrh has a valuable schistosomicid-
al effect against different stages of S. mansoni. This chemotherapeutic effect
was more evident when the drug was given to infected mice on the 21
st
as well
as on the 45
th
day PI.
Key Words: Schistosoma mansoni, Myrrh extract, Histological study.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Schistosomiasis remains a public
health problem in Egypt, despite the
continuous control measures (El-
Khoby et al., 2000; Abo-Madyan et
al., 2004). Its major pathology, gra-
nulomatous inflammation is a cellu-
lar immune response to antigens
secreted by schistosome ova. The
chronic egg-induced granulomatous
response in the liver and intestine
may eventually cause extensive tis-
sue scarring and development of

246

portal hypertension (Wynn et al.,
2004).
Chemotherapy is the most effec-
tive method for the short term con-
trol of schistosomiasis. Although
praziquantel (PZQ) is the drug of
choice for treatment of schistoso-
miasis in most endemic areas, yet it
has many draw backs. The low effi-
cacy of PZQ (Renganthan and Coli,
1998) due to appearance of drug
tolerance or resistance (Ismail et al.,
1994, 1999; Fallon et al., 1997) to-
gether with the potential carcinoge-
nicity, genotoxicity (Rosenkranz et
al., 1995), mutagenicity (Montero et
al., 1993) and lethality in big dose
(Kheir et al., 1995) makes the ur-
gent need for a new effective safe
treatment a necessity and priority of
utmost public health importance.

Myrrh is an oleo-gum resin de-
rived from the stem of Commiphora
molmol (C. molmol) tree, family
Burseraceae (Wallis, 1967). Myrrh
was approved by the US Food and
Drug Administration for food use
(21 Code of Federal Registration-
CFR 172.510) and was generally
recognized as safe (GRAS) status as
a flavor ingredient (No. 2765) by
the Flavor Extract Manufacturer's
Association (FEMA) (Ford et al.,
1992). The council of Europe in-
cluded myrrh in the list of plants
and parts that are acceptable for use
in foods (Council of Europe, 1981).
It is one of the oldest medicinal
plants used by Ancient Egyptians
for medical purposes as well as in
mummification (Chevalier, 1996).
Myrrh contains a resin (Myrrhin), a
volatile oil (Myrrhol), gum and a
bitter principle (Massoud et al.,
2001b; Morsy et al., 2005).
Traditionally, myrrh has been
used by Ancient Egyptians, Sume-
rians and Greeks to treat worms
(Haridy et al., 2004), by Chinese to
relieve pain and swelling due to
traumatic injury (Massoud et al.,
2001c) and by Somalis to treat the
stomach complaints, diarrhea and
wounds (Massoud et al., 2001b).
Tincture of Myrrh has been used
for therapy of aphthous ulcer (Mas-
soud et al., 2000a), treatment of sore
throat and pharyngitis (Massoud et
al., 2001b). Studies proved that C.
molmol is effective as an anti-
inflammatory (Massoud et al.,
2000a), anti-tumor (Cox, 1993), and
anti-carcinogenic (Peters and War-
ren, 1969). Experimental, clinical
and laboratory studies (Tonkol and
Morsy, 2008) conducted to evaluate
the efficacy of Myrrh extract proved
the efficacy of the drug for schisto-
somiasis (El Baz et al., 2003; Soli-
man et al., 2004), heterophyiasis
(Massoud et al., 2001; 2007; Fathy
et al., 2005), fascioliasis (Motawea
et al., 2001; Massoud et al., 2001;
Hassan et al., 2004; Abo-Madyan et
al., 2004), dicrocoeliasis dendritium
(Massoud et al., 2003) for Cestoda

(Massoud et al.,2007) and for some
Nematoda (Massoud et al., 2006) as
well as Coccidia (Massoud et al.,
2008; Al-Mathal, 2010).
For Myrrh is a well-tolerated re-
medy with a high margin of safety
for the liver, kidney and heaemato-
poeitic system besides, its non-

247

mutagenicity despite its repeated
administration for relatively long
periods (Webb, 1976).

During the life cycle of Schisto-
soma mansoni, it is worth mention-
ing that, cercaria penetrates the skin
and transforms immediately into
Schistosomule. Then, the schisto-
somule migrates to the lung then to
the heart to reach hepatoportal cir-
culation to become immature worm
(>16 days). The pairing of male and
female occurs where they move
against blood stream to reach the
mesenteric veins where egg produc-
tion starts >35 days (Badria et al.,
2001).

The aim of the present work was
to evaluate the effect of Myrrh ex-
tract on the liver of mice infected
with the Egyptian strain of S. man-
soni. The study included its effect
on the different developmental stag-
es (Schistosomule, immature and
mature worms) by adjusting the
time of drug administration.


Sixty male albino mice (20-25 gm
weight) used in this study were pur-
chased from Theodore Bilharz Re-
search Institute; Imbaba (TBRI).
The animals were divided into three
groups: GI (Control group): In-
cluded 15 mice which were subdi-
vided into 3 subgroups; 5 mice
each. Subgroup IA: mice were left
as non-infected, non-treated control.
Subgroup IB: mice were treated
with myrrh extract dissolved in
cremophor EL (500mg/kg). Sub-
group IC: mice were treated with
the solvent of the drug (cremophor
EL) by the same dose.
GII (infected non-treated group):
included 15 mice. Each mouse was
infected sub-cutaneously (at chest
region) with 6010 cercariae of S.
mansoni Egyptian strain (Drury and
Wallington, 1980)

GIII (infected treated group): in-
cluded 30 mice. They were all in-
fected with S. mansoni as in G II.
They were further subdivided equal-
ly into 3 subgroups according to the
time of administration of myrrh ex-
tract. Subgroup IIIA: mice treated
with the drug starting on the 1
st
day
postinfection (PI) (to detect effect
on schistosomule). Subgroup IIIB:
mice treated with the drug starting
on the 21
st
day PI (effect on imma-
ture worm). Subgroup IIIC: mice
treated with the drug starting on the
45
th
day PI (effect on mature worm).
The animals were kept in the
Medical Research Center, Faculty of
Medicine, Ain-Shams University,
and were allowed food and water ad
libitum for 80 days from the start of
the experiment.
The Myrrh extract: (1gm of oleo-
resin+3 gm Cremophore EL+100cm
Distilled water) The emulsified
oleo-resin extract was kindly pre-
pared at Pharco Pharmaceutical
Company, Alexandria, Egypt, to-
gether with the emulsifier Cremo-
phor EL. Cremophor EL is a caster
oil derivative, which is mainly used
as an emulsifying and solubilizing
agent for the production of aqueous
preparations (Bancroft and Cook,

248

1994). The drug was given orally on
an empty stomach via stomach tube
after overnight fasting in a dose of
500mg/kg body weight per day
(Badria et al., 2001)

for five succes-
sive days.
At the experimental end, the ani-
mals were sacrificed and their livers
were dissected out. Specimens were
fixed in 10% formol saline. Paraffin
sections were prepared 5-7 m thick
and stained by: Haematoxylin and
Eosin, Periodic Acid Schiff's reac-
tion, Masson's trichrome stain
(Drury and Wallington, 1980), Car-
bol Chromotrope reaction and the
Wilder's technique (Bancroft and
Cook, 1994).

Morphometric and statistical
study: The mean number of granu-
lomatous reactions was counted in
10 low power fields (x10) in each
mouse in the infected and infected-
treated groups. The mean number of
granulomas in each mouse was con-
sidered as one variable (n=10). The
mean perimeter of the granulomat-
ous reactions was measured. This
was done in 25 granulomas random-
ly selected from each group or sub-
group. (n=25). These measurements
were done using the Image Analyzer
(Leica Q 500 MC). Student's "t" test
was used to compare the data and
the p value was calculated using
SPSS program. P<0.05 was consi-
dered significant.

Results

Examination of H&E stained sec-
tions of GI (sGs A, B & C) (non-
infected mice) revealed that the liver
parenchyma consisted of hepatic
lobules. Each lobule was formed of
cords of hepatocytes radiating from
the central vein and separated by
blood sinusoids. The hepatocytes
appeared polygonal in shape. They
had rounded nuclei with peripheral-
ly dispersed chromatin and promi-
nent nucleoli. At the periphery of
the hepatic lobules, portal tracts
could be detected (Fig. 1). PAS
stained sections demonstrated nor-
mal glycogen content in the hepato-
cytes (Fig. 2). Masson's trichrome
stained sections showed that there
was scanty and fine collagen fibers
mainly in the portal area and to a
lesser extent in between the hepato-
cytes (Fig. 3). Using Wilder's tech-
nique, there was a delicate reticular
network in the portal tract, in be-
tween the hepatocytes and surround-
ing the central vein (Fig. 4).
Examination of sections of GII
(infected group) stained by H&E
stain revealed that most of the portal
tracts appeared thickened and
showed schistosomal granulomas
consisted of many inflammatory
cells. They were variable in size,
mostly large with irregular outlines.
The ova were deposited close to
each other with subsequent fusion of
the granulomas and the inflammato-
ry reaction (Fig. 5). Despite the
presence of multiple granulomata,
the lobular architecture of the liver
was more or less preserved. The
hepatic parenchymal cells near or
around the granulomas were ob-
viously affected. Hepatocytes re-

249

vealed highly vacuolated cytoplasm.
Many nuclei appeared small and
some were deeply stained (Fig. 6).
Notably, bilharzial pigments were
frequently seen especially around
granulomas (Fig. 6). By using Car-
bol chromotrope, the granulomas
appeared very cellular with numer-
ous eosinophils which appeared
pink in color (Figs.7&8). There was
marked generalized depletion of
glycogen content of the hepatocytes
(Fig. 9). Sections stained by Mas-
son's Trichrome showed numerous
granulomas clearly delineated by
delicate concentric layers of loosely
arranged collagenous fibers giving it
the onion peel appearance. Fibrous
tissues appeared extending between
adjacent granulomas and tended to
link portal tracts (Fig. 10). Using
Wilder's technique, the reticular fi-
bers appeared condensed in the con-
fluent granulomas. The portal tracts
appeared markedly thickened with
heavy deposition of the reticular
fibers (Fig. 11).
In general, examination of sec-
tions of GIII revealed marked im-
provement as compared to GII.
sGIIIA revealed that the schisto-
somal granulomas were less fre-
quent in the portal tracts. Many he-
patocytes appeared acidophilic with
central vesicular nuclei while some
still showed vacuolation (Fig. 12).
Eosinophils were apparently less
in granulomas as compared to GII
(Fig. 13). Most PAS-stained hepato-
cytes were depleted from glycogen
while some of them showed a mod-
erate content (Fig. 14). Masson's
trichrome stain showed that the gra-
nulomas were well delineated by the
collagenous fibers. The scanty fibers
were detected between the hepato-
cytes (Fig. 15) but reticular fibers
were less in the portal areas as com-
pared to group II and appeared short
and rarified (Fig. 16).
Examination of sections from
sGIIIB revealed an apparent de-
crease in the number of granulomas
and large areas were granuloma
free. On the other hand, the few
granulomas present were less cellu-
lar as compared to GII (Fig. 17).
Most of the hepatocytes appeared
acidophilic with central vesicular
nuclei similar to the control group.
Bilharzial golden brown pigment
could also be detected (Fig. 18). In
the granulomas, eosinophils were
apparently decreased as compared
to GII (Fig. 19). The glycogen con-
tent was moderate in most cells
while some still showed depleted
glycogen content. The collagen fi-
bers were well circumscribed in the
granulomas and scanty fibers were
detected between the hepatocytes
(Fig. 20). The reticular fibers ap-
peared less condensed and rarified
in portal tracts.
Subgroup IIIC showed that the
schistosomal granulomas were rare-
ly present in the portal tracts. They
appeared small, discrete, less cellu-
lar and clearly delineated from the
surrounding liver tissue (Fig. 21).
The liver architecture was complete-
ly preserved.
The fusion between granulomas
and extension of inflammatory reac-

250

tion between portal tracts was hard-
ly seen in different sections. Most of
the hepatocytes were normal. They
showed acidophilic granular cytop-
lasm with rounded vesicular nuclei
(Fig. 22). The eosinophils were very
few in granulomas (Fig. 23).
The glycogen content was near
normal in many hepatocytes while
some showed moderate content. The
collagen fiber content was de-
creased in the granulomas and was
similar to the control group in be-
tween the hepatocytes. The reticular
network in the granulomas was de-
creased and rarified (Fig. 24)
Statistical analysis: A significant
decrease was detected in the mean
number of granulomas/10 LPF as
well as in their mean perimeter in
subgroups IIIA, B and C as com-
pared to GII (SPSS, 1999).

Table 1: Mean number of granulomas/10 fields (x10) and perimeter of granu-
lomas (m) in different groups.

GII (infected) GIII (infected treated)
Subgroup IIIA
(1
st
day PI)
IIIB
(21
st
day PI)
IIC
(45
th
day PI)
No of granulomas /10 LPF 31.45 7.31 *22.164.25 *17.344.06 *12.323.97
Perimeter of Granulomas 849.18 164.23 *754.2496.67 *634.1584.28 *589.5382.36
* Significant in comparison to GI
Discussion

In the present study, S. mansoni
experimental infected mice revealed
that most of the livers portal tracts
were occupied by schistosomal gra-
nulomas. They were of variable size
and irregular outlines. Bilharzial
granulomas were described in many
preliminary studies to be formed of
lymphocytes around the eggs. These
cells were rapidly replaced by eosi-
nophils, neutrophils and monocytes
(Alexis and Radovan, 1980). Bo-
loukere et al. (1993) stated that eggs
are brought into the liver by the por-
tal blood flow and they emblaze in
venous radiculi of the intrahepatic
portal system. Granulomas forma-
tion is mediated by a T cell response
to egg secreted soluble egg antigens
(SEAs), which induce a delayed
hypersensitivity response with tu-
mor necrosis factor (TNF) produc-
tion (Luckas et al., 1994). Moreo-
ver, in bilharzial granulomas forma-
tion, TNF was proved to induce in-
tercellular adhesion molecule type 1
(ICAM-1) expression that partici-
pates in mediating cellular recruit-
ment and adhesion, lymphocyte ac-
tivation and subsequent production
of other inflammatory lymphokines
that are implicated in the develop-
ment of granulomas (Shahen et al.,
1989).
The present study showed the pre-
servation of hepatic architecture and
lobular organization. This preserva-
tion of lobular architecture could be
attributed to the fact that the peri-
portal inflammation and granulo-
matous response to schistosomal
eggs are local phenomenon and con-
sequently it does not involve the


2
5
1


252

whole parenchyma (Jacob et al.,
1997)

In the present study, the hepatic
parenchymal cells were affected
near and around the granulomas.
Some hepatocytes showed the va-
cuolated cytoplasm, while others
showed deeply stained nuclei. Also,
generalized depletion of glycogen
content was detected. This was con-
sistent with the results of other stu-
dies (Chesney et al., 1988; Singh et
al., 2004). The toxins from adult
worms might be involved in antigen
antibody reaction that would selec-
tively injure hepatic cells (Alexis
and Radovan, 1980). Furthermore,
hepatic cells alteration might be in
part due to the local edema that oc-
curs around the inflammatory gra-
nulomas (Singh et al., 2004).
Bilharzial pigment in the present
study was frequently seen. This
could be explained by the fact that
Schistosoma pigment was a haem
derivative which increased by the
digestive breakdown of host eryt-
hrocytes in adult Schistosoma
worms (Hugues et al., 1997). Jacob
et al. (1997) proved that the co-
localization of Schistosoma pigment
with intracellular adhesion molecule
I (ICAM-1), Kupffer cells and pha-
gocytic cells were present in the
granulomas and portal tracts.
In the present study, fibrous tissue
appeared extending between adja-
cent granulomas and tended to link
portal tracts. This coincided with the
results of other studies (Singh et al.,
2004). During the liver injury, the
hepatic stellate cells are activated
and differentiate into myofibroblasts
that secrete extracellular matrix pro-
teins, including collagen, fibronectin
and glycosamino-glycans (Chesney
et al., 1988). It was proved that the
reduction in the liver parenchymal
collagen content of S. mansoni in-
fected mice after treatment by inter-
feron was due to strong inhibition
of the hepatic stellate cells activa-
tion (Hugues et al., 1997). Further-
more, the extracts of isolated egg
granulomata from liver of experi-
mentally infected mice with S. man-
soni stimulated in vitro proliferation
of fibroblasts (Luis et al., 1983).

The reticular fibers in group II
appeared condensed in the confluent
granulomas. Gay and Miller (1978)
explained that in several reparative
phenomena, collagen producing
cells underwent a shift in collagen
synthesis from type III to type I dur-
ing the evolution process. As re-
gards the origin of collagen there
were four proposed sources includ-
ing condensation of pre-existing
reticulin, production of new colla-
gen by proliferating fibroblasts,
production by perisinusoidal me-
senchymal cells or by the hepato-
cytes themselves (Grimaud and Bo-
rojevic, 1980).

In the infected treated group (all
subgroups), most of the hepatocytes
showed a normal histological pro-
file. The granulomas were small
discrete and clearly delineated from
the surrounding liver tissue. These
findings were confirmed by the
morphometric study. There was a
significant decrease in the mean

253

number as well as the mean perime-
ter of the granulomas in the infected
treated subgroups when compared
with the infected non-treated one.
Moreover, the observed granulomas
showed collapse of their reticular
fibers and paucity of eosinophils.
There was marked reduction of col-
lagen fibers deposition between the
hepatocytes.
Myrrh extract resulted in a signifi-
cant reduction of worm and egg
count in the liver and intestine when
administered to hamsters or mice
infected with S. mansoni (Massoud
et al., 2004). This result could ex-
plain the improvement in the histo-
logical picture of the liver which
was most evident in subgroups IIIB
and IIIC. These findings indicate
that the immature and mature adult
stages of S. mansoni are more sus-
ceptible to myrrh extract. This coin-
cided with a previous study where
in vitro adult worms were subjected
to 10 ml of 1 PPM concentration of
Myrrh extract and resulted in their
structural alteration within 30 mi-
nutes (Hassan et al., 2003). Besides,
it was reported that praziquantel has
little susceptibility on the immature
forms of S. mansoni than on the
adult forms (Botros et al., 2005).

Conclusion

Myrrh extract showed signifi-
cant efficacy against Schistoso-
ma mansoni infected mice. This
was most evident against imma-
ture and adult stages and to a
lesser extent against schistoso-
mula stage. The preventive role
of myrrh in hepatosplenic schis-
tosomiasis is ongoing and will
be published soon.

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Legends

Fig. 1: Liver section of a mouse in SG IA showing hepatic lobule formed of hepatocytes separated by blood
sinusoids, acidophilic granular cytoplasm and a central vesicular nucleus (C: central vein; P: portal area).
H&E X 400
Fig. 2: Liver section of a mouse in SG IA showing normal glycogen content in hepatocytes. PAS X 400
Fig. 3: Liver section of a mouse in SG IB showing scanty collagen fibers content in between hepatocytes and
in portal area. Masson's trichrome stain X 400
Fig. 4: Liver section of a mouse in SG IC showing delicate reticular network in portal area and in between
hepatocytes. Wilder's technique X 400
Fig. 5: Liver section of a mouse in G II showing many periovular granulomas appear very cellular and varia-
ble in size. Notice fusion between 2 granulomas (). H&E X 100
Fig. 6: Liver section of a mouse in GII showing highly vacuolated hepatocytes. Most hepatocytes reveal
small nuclei while others are deeply stained (). Presence of golden pigment at periphery of granuloma (G:
granuloma). H&E X 400
Fig. 7: Liver section of a mouse in GII showing a granuloma with numerous eosinophilic infiltration ().
Carbol chromotrope X 640
Fig. 8: Liver section of a mouse in GII showing numerous eosinophils with specific pink colour (). Carbol
chromotrope X 1000
Fig. 9: Liver section of a mouse GII showing a marked depletion of glycogen content in the hepatocytes. (G:
granuloma) PAS stain X 400
Fig. 10: Liver section of a mouse in GII showing fibrocellular granulomas with onion peels arrangement of
collagen fibers extended between adjacent granulomas and link portal tracts. Masson's trichrome stain X 400
Fig. 11: Liver section of a mouse in GII showimg portal tracts markedly thickened by deposition of reticular
fibers in periovular granulomas. Notice confluence and bridging between adjacent granulomas. Wilder's
technique X 400
Fig. 12: Liver section of a mouse in SG IIIA showing a granulomatous reaction occupying a portal tract.
Most hepatocytes appear acidophilic with central vesicular nuclei whereas some hepatocytes appear vacuo-
lated. H&E X 400
Fig. 13: Liver section of a mouse in SG IIIA showing cellular infiltration of a granuloma with less numerous
eosinophilic () infiltrations as compared to GII. Carbol chromotrope stain X 640
Fig. 14: Liver section of a mouse in SG IIIA showing the majority of hepatocytes with marked depletion of
glycogen content and sporadic hepatocytes with moderate glycogen content. PAS stain X 400
Fig. 15: Liver section of a mouse in SG IIIA showing part of a well circumscribed fibrocellular granuloma.
Scanty collagen fibers appear in between hepatocytes. Masson's trichrome stain X 400
Fig. 16: Liver section of a mouse in SG IIIA showing decreased reticular fibers in portal tract as compared to
GII. Wilder's technique X400
Fig. 17: Liver section of a mouse in SG IIIB showing fibrocellular granuloma with few inflammatory cells.
H&E X 100
Fig. 18: Liver section of a mouse in SG IIIB showing normal histological profile of most of hepatocytes and
mild cellular infiltration. Presence of bilharzial pigment. H&E X 400
Fig. 19: Liver section of a mouse in SG IIIB showing a fibrocellular granuloma with few scattered eosino-
phils. Carbol chromotrope stain X 640
Fig. 20: Liver section of a mouse in SG IIIB showing a decrease in density of collagen fiber content in fibro-
cellular granulomas. Masson's trichrome stain X400
Fig. 21: Liver section of a mouse in sG IIIC showing a granuloma with few inflammatory cells. H&E X 100
Fig. 22: Liver section of a mouse in SG IIIC showing most hepatocytes nearly similar to control. H&E X 400
Fig. 23: Liver section of a mouse in SG IIIC showing very few eosinophils infiltrating a granuloma. Carbol
chromotrope stain X 640
Fig. 24: Liver section of a mouse in subgroup IIIC showing decreased and rarified reticular fibers in portal
tract. Wilder's technique X400.
259



THE IMPACT OF EXAMS ANXIETY ON THE LEVEL OF TRIGLYCE-
RIDES IN UNIVERSITY FEMALE STUDENTS
By
TAHIA A. MAIMANEE
Department of Biology, Science Collage, King Abdel-Aziz University,
P.O.Box:11853 Jeddah 21463 KSA email: tmaimani@kau.edu.sa

Abstract
Anxiety affects the level of blood fats such as the triglycerides according to
several studies conducted in various conditions causing anxiety as exam for the
university students. The health experts suggested that the anxiety works to sti-
mulate the autonomic nervous system which in turn leads to the appearance of
a group of physiologic symptoms.
The current study showed the changes happened in the triglycerides levels in
the female university students before and after exams at the intermediate anxie-
ty level compared to other high and low levels of anxiety. In addition, there
was a difference in triglycerides levels in female students of college of Science
before and after exam. This difference did not appear in case of other colleges.
The exam type had an impact as the significant difference appeared in the trig-
lycerides levels during the periodical tests and these differences did not appear
in the final exam.
Key words: Saudi Arabia, University female students, Anxiety, Triglycerides.
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction
Many authors confirmed the rela-
tionship between anxiety and the
increase of triglycerides in humans.
A study by Kuczmierczyk et al.
(1996) on cholesterol and triglyce-
rides levels on thirty eight persons
who suffered from general anxiety,
showed a high rise in cholesterol
and triglycerides levels. In addition,
(Agarwal et al., 1997) rated the le-
vels of plasma cholesterol, triglyce-
rides, and total lipids in twelve stu-
dents exposed to different grades of
exams psychic exhaustion. The stu-
dents levels of plasma cholesterol
and triglycerides relates directly
with the extent of exams psychic
exhaustion. Sevincok et al. (2001)
concluded that persons suffering
from general anxiety and depression
have high levels of cholesterol, trig-
lycerides, and LDL. With regard to
HDL, they have low percents of it.
Those patients are vulnerable to
death caused by coronary arteries
diseases more than those who suffer
from only general anxiety or depres-
sion.
260

The present study aimed at the
clarification of the relation between
anxiety and the elevation of trigly-
cerides in female University stu-
dents.

Subjects Materials and Methods

Blood samples were taken ran-
domly from one hundred female
students in King Abdul Aziz Uni-
versity, Jeddah, around a year and a
half during exams periods. Sheets
were filled out on each participant
including personnel data and medi-
cal history. Those with parasitic
and/or microbial agents that may in
one way or another cause anxiety
were excluded. Venous blood sam-
ples collected were cared to be from
the students of different ages, col-
leges, social economic level, and
educational levels. In addition, it
was taken into account that student
answered the attitude list towards
exam. Two blood samples were tak-
en from each student to compare the
triglycerides levels before and after
the exam. The samples were given
serial numbers and centrifuged to
separate sera (Hennery, 1974). The
first sample was taken before carry-
ing out the exam whereas the
second one was taken 24 hours after
the exam from the same student and
given the same serial number. The
triglycerides levels were measured
by Reflotron apparatus (Lee et al.,
2007). Beck depression inventory
and beck anxiety inventory were
used to assess the level of depres-
sion and anxiety.

Results

The effect of Exam Anxiety on the
level of Triglycerides showed a sig-
nificant increase in the triglycerides
levels before exam (t= 2.782;
P<0.025) more than after exam at
medium degree of anxiety. No sig-
nificant difference were recorded in
the mean of triglycerides levels at
the low-degree of anxiety before
exam [t= 0.597; (n.s)] and after ex-
am. There were any significant dif-
ferences in the mean of triglycerides
levels at the high-degree of anxiety
before exam (t= 0.204; n.s) (tab.1,
fig.1). The results referred to the
rise of triglycerides mean before
exam and decreased after exam at
medium-degree of anxiety.
The detaile3d results are shown in
tables (1, 2, 3, 4, 5 & 6) and figures
(1, 2, 3 & 4).

Table 1: Triglycerides levels before and after exam in students with respect to
different degrees of anxiety.

Taking Sample Period
Anxiety Degree
Low Medium High
Before Exam 6.65 + 99.07 16.34* + 139.73 21.41 + 103.59
After Exam 6.48 +94.02 9.24 + 102.09 6.56 + 99.75
* A significant difference P <0.001 according to t.test when comparing medium degree of anxiety before and
after exam.
261

Fig. 1: Relation (Mean S.E) of triglycerides levels before and after exam at
different anxiety degrees.
Low Medium High Anxiety

M
e
a
n

o
f

T
r
i
g
l
y
c
e
r
i
d

m
g

/

d
l
0
20
60
80
100
120
140
160
180
200

* A significant difference P <0.001 according to t.test when comparing medium anxiety before exam to the
post exam period.

Table 2: Triglycerides level before and after exam with respect to different
age groups.

Ages groups
18-20 years 21-23 years 24-27 years
Before Exam 10.87 + 116.51 * 15.76 + 123.29 * 29.29 + 108.81 *
After Exam 8.03 + 101.19 6.24 + 93.25 9.17 + 100.52
* No significant differences according to t.test when comparing different age groups before and after exam.

Age effect on triglycerides level:
There was no significant differences
in relation to the mean of triglyce-
rides levels before or after exam (t=
1.823; n.s) in age from 18-20 years.
In addition, no significant differenc-
es appeared in relation to the mean
of triglycerides levels before or after
exam (t= 1.847; n.s) in age from 21-
23 years. Results also showed no
significant differences appeared in
relation to the mean of triglycerides
levels before or after exam (t=
0.323; n.s) in age from 24-27 years.
*
TG Before exam
TG After exam
262

There were a significant difference
of triglycerides level coefficient fac-
tor before exam (df = 56; p<0.001)
more than after exam in the age
from 18-20 years. No differences
were noticed for correlation coeffi-
cient in age groups from 21-23
years or 24-27 years when carrying
out the same exam (Tab. 2).
Effect of marital status on triglyce-
rides levels: Levels were not af-
fected by the students' marital sta-
tus. There was a significant increase
in the triglycerides levels before
exam (t = 2.315; P<0.05) compared
to after exam in the married stu-
dents.
There was a significant increase in
the level of the triglycerides before
exam (t = 2.230; P<0.05) compared
to the level after exam in a single
student (Tab. 3, Fig. 2). There was
an obvious increase in the mean of
triglycerides level before exam and
a decrease after exam in both
groups.

Table 3: Triglycerides level before and after exam with respect to students' ma-
rital status.

Marital Status
Married Single
Before Exam 9.17 + 118.60 * 17.98 + 114..46 *
After Exam 5.50 + 100.47 2.53 + 74.09
* A significant difference P <0.001 according to t.test when comparing single student before and after exam.
* A significant difference P <0.001 according to t.test when comparing the married students before and after
exam.
Fig. 2: Relation (Mean S.E) of triglycerides levels before and after exam with differ-
ent student marital status.

* A significant difference P <0.001 according to t.test when comparing single student before and after exam.
* A significant difference P <0.001 according to t.test when comparing married students before and after
exam
S i n g l e M a r r i e d S t a t u s
M
e
a
n
o
f T
rig
ly
c
e
rid
m
g
/ d
l
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
2 0 0
2 2 0
*
TG Before exam
TG After exam
*
263

Effect of exam type on triglyce-
rides level: The exam type affects
the mean of triglycerides levels as
there was a significant increase in
the triglycerides levels before exam
(t = 2.529; P<0.025) compared to
the level after exam and during the
periodical exam. There was signifi-
cant difference in the mean of trig-
lycerides levels during final exams
before exams (t = 0.860; n.s) com-
pared to after exam period (Tab. 4,
Fig. 3).
.
Table 4: Triglycerides levels before and after exams with respect to exams.

Exam Type
Periodical Final
Before Exam 15.43 + 132.85 * 8.64 + 109.15
After Exam 7.73 + 99.52 6.61 + 97.78
* A significant difference P <0.001 according to t.test when comparing period before and after exam.
Fig. 3: Level of triglycerides after and before exam with respect to exam types.

* A significant difference P <0.001 according to t.test when comparing period student before and
after exam.

P e r i o d F i n a l E x a m
M
e
a
n

o
f

T
r
ig
l
y
c
e
r
id

m
g

/

d
l
0
2 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
1 8 0
TG Before exam
TG After exam
*
264

Effect of educational (academic)
levels on triglycerides: There were
no significant differences in the
mean of triglycerides levels before
exam (t = 0.784; n.s) compared to
after exam in first class students.
There were any significant differ-
ences in the mean of triglycerides
levels before exam (t = 1.790; n.s)
compared to after exam in second
class students.
Besides, there were any signifi-
cant differences in the mean of trig-
lycerides levels before exam (t =
1.524; n.s) compared to after exam
in third class students. Also, there
were any significant differences in
the mean of triglycerides levels be-
fore exam (t = 1.066; n.s) compared
to after exam in fourth class stu-
dents (Tab. 5).

Table 5: Triglycerides levels before and after exam with respect to different
educational levels.

Taking Sample
Period
Academic Levels
First Second Third Fourth
Before Exam 34.28 + 132.69 * 8.96 + 112.84 * 14.45 + 118.55 * 34.36 + 116.98 *
After Exam 24.12 + 122.34 5.94 + 93.05 6.52 + 96.97
6.75 + 76.75
* No significant differences according to t.test when comparing different education levels before and after
exam.

Effect of different colleges on
triglycerides levels: Triglycerides
levels are affected by different col-
leges as the statistical analysis
showed a significant increase in the
triglycerides levels before exam
more than after exam in case of
Science Students College. There
were any noticed significant differ-
ences in the mean level of triglyce-
rides before exam (t = 0.625; n.s)
compared to after exam in Home
Economics students College. In ad-
dition, there were no significant dif-
ferences in the mean level of trigly-
cerides before exam (t = 1.615; n.s)
compared to after exam in case of
theoretical colleges students.
Also, there were no significant
differences in the mean level of trig-
lycerides before exam (t = 0.560;
n.s) compared to after exam in case
of Medicine College students (Tab.
6, Fig. 4).
The results showed the relation-
ship of the mean of triglycerides
levels after and before exam with
different colleges as we notice the
increase of triglycerides levels mean
before exam and the fall of its levels
after exam in case of Science Col-
lege students.

265

Table 6: Triglycerides levels before and after exam with respect to different
colleges.

Different Colleges
Science Medicine Home Econ. Theoretical
Before Exam 11.67 + 130.62 * 5.91 + 82.14 8.65 + 95.38 81.72 + 184.72
After Exam 5.58 + 100.05 11.47 + 89.00 8.47 + 90.30 53.92 + 138.33
* A significant difference P <0.025 according to t.test when comparing college of science students before and
after exam.

Fig. 4: Level relationship (Mean S.E) of triglycerides before and after exam
with different colleges.

* A significant difference P <0.025 according to t.test when comparing college of science students before and
after exam.

M
e
a
n

o
f

T
r
i
g
l
y
c
e
r
i
d

m
g

/

d
l
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
TG Before exam
TG After exam
Science Medicine Home Econ. Theoretical Colleges
*
266

Discussion

Researchers paid great attention to
the study of exams anxiety relation-
ships as well as the effect extent on
triglycerides levels. Exams anxiety
is a state of genenal anxiety that
most students suffer from during
exams period. Students who have
high anxiety degrees are more suf-
fering during exams because high
anxiety degrees make persons less
able to concentrate, the matter that
leads to breakdown of behavioral
system. In addition, solidarity and
integration levels decline leading to
the deterioration of performance in
exams and learning acquisition.
Anxiety happened during exams
varies from a student to another. For
example, the student who studies
lessons regularly, anxiety will be a
positive urge helping him / her to
study hard. On the other hand, the
student who spends his/her time
playing and roaming, anxiety will
have a strong effect on. Essawi
(1974) who examined 300 male and
female students of University of
Alexandria reported that the majori-
ty of the study samples were from
College of Arts. Besides, few stu-
dents did not feel anxiety absolutely
during exam which totals 11.1%.
The majority felt anxiety in different
degrees varied from low and high
anxiety in 88.89%, whereas 5%
feeling anxiety may reach the extent
of danger and breakdown.
the triglycerides level increased in
the students who suffered from me-
dium degree of anxiety where other
classes (high or low anxiety de-
grees) were not affected. This ex-
plains the impact of medium anxiety
on the mean level of triglycerides
which was discussed by previous
studies such as Othman (2001) who
showed that the relationship among
different anxiety levels and exam
performance, the students with high
anxiety degree needed to remove the
psychic stresses so that they could
improve their standards in exam
performance. Students with low-
anxiety degree needed to an extent
of stress to stimulate anxiety and
hence improve their performance.
The medium anxiety degree stu-
dents were those who have the abili-
ty of achievements as they were
characterized with stability and mo-
tivation which urged them to the
achievement.
With regard to the different age
groups, results showed that the trig-
lycerides level was not affected by
different ages. This may be due to
that students' ages are similar to
each other; they were about 18-27.
It is well-known that triglycerides
levels increased with aging. This
fact was proved by Charles (2001)
who studied the cholesterol center in
a hospital. The study assured that
heart attacks and arteries diseases
were the most common diseases
causing death to those who ex-
ceeded 65 years in percentage of
70%. He showed that triglycerides
and LDL increases in males at the
age of 60 years but in females at the
age of 85years. Studying the exam
267

type, results showed that triglyce-
rides levels were affected by the
exam type as there were significant
differences during the periodical
exam. This can be explained by the
fact that student during the periodi-
cal exam was in a state of fair and
anxiety because she does not know
how the exam will be. It also may
be because the unfamiliarity with
teaching staff members and the
questions method. The absence of
significant difference in the final
exam may be due to that student
familiarity with exams situations
and good training on such situations
as well as knowing her achievement
results. Current study revealed that
the effect of different colleges on
the mean level of triglycerides. A
change was noticed in the triglyce-
rides level mean in Science College
Students while no change was ap-
peared in other colleges students. A
recent study (Ahaneku et al., 2001)
performed on college of medicine
students showed that there is a
chance to know the alimentary re-
quirements of college of medicine
students during exams. In addition,
there is a chance to direct the aca-
demic program in drawing solution
to decrease anxiety during exams.
The study did not show any rela-
tionship between different academic
levels and triglycerides levels. The
results gained by this study refer
that there was a change in the trigly-
cerides levels mean with some va-
riables. That may be related to
psychic stresses accompanying the
exam period and their relation ex-
tent to triglycerides level. Most of
the previous studies, which tackled
the impact of psychic stresses and
especially during exams period, as-
sured these facts.
Generally speaking, triglyceride is
a liquid or neutral fat consisting of
glycerol combined with three fatty-
acid molecules. Triglycerides are
synthesized from the products of
digestion of dietary fat; they are the
form in which fat is stored in human
body (Martin, 1980).
On the other hand, Julius et al.
(1992) reported that home blood
pressure readings by self-monitoring
(14 readings in 7 days) were com-
pared to readings taken in the clinic
in 937 participants. Two hyperten-
sive groups emerged; one with both
clinic and home hypertension ("sus-
tained" N = 47) and one with high
clinic but normal home blood pres-
sure ("white coat" N = 50). Groups
with "white coat" and "sustained"
hypertension were very similar.
Both groups were overweight, had
faster heart rates, elevated choles-
terol, insulin, and triglyceride and
decreased HDL levels. Blood pres-
sure readings at previous exams
(age 5, 8, 21 & 22) were elevated in
both the "sustained" and white coat
hypertension group compared to the
normotensive controls. Because of a
higher risk of coronary heart disease
and a risk for late development of
sustained hypertension, the subjects
with white coat hypertension were
counselled on nonpharmacologic
methods to control the blood pres-
268

sure elevation and to ameliorate co-
ronary risk factors.
George et al. (2007) found corre-
lation between the hypertriglyceri-
demia and many physical and clini-
cal risk factors. Burton et al. (2009)
reported that depression and poor
social support were significant risk
factors for coronary heart disease
(CHD), and stress and anxiety can
trigger coronary events. People ex-
periencing such psychosocial diffi-
culties were more likely to be phys-
ically inactive, which is also an in-
dependent risk factor for CHD.
Friedberg et al. (2009) mentioned
that forgiveness was associated with
better psychological and physical
health and in particular cardiovascu-
lar functioning. Despite these find-
ings, most forgiveness studies in-
volve healthy participants. They
found that higher levels of forgive-
ness were associated with lower
levels of anxiety (p < 0.05), depres-
sion (p < 0.01), and perceived stress
(p < 0.005) as well as lower total
cholesterol to HDL and LDL to
HDL ratios (both at p < 0.05) after
controlling for age and gender. The
psychological indices did not me-
diate the relationship between for-
giveness and cholesterol ratios.
They concluded that the psycholog-
ical correlates of forgiveness are
similar in cardiac patients and
healthy individuals. Further, among
cardiac patients, forgiveness may be
associated with reduced risk for fu-
ture cardiovascular events.
Fragakis et al. (2010) mentioned
that parental history of coronary
artery disease (CAD) is considered
an important risk factor for early
atherosclerosis. The onset of the
inflammatory process of atheroscle-
rosis initiates early during childhood
in children with positive family his-
tory (PFH) of CAD. They found
that higher values of body mass in-
dex (BMI), total cholesterol, LDL,
cholesterol, TG, SDE, leucocytes,
and CRP were calculated in children
with PFH. They concluded that in
individuals with PFH of CAD the
inflammatory process of atheroma-
tosis appeared to begin early in
childhood. Except for triglycerides,
this inflammatory process appears
to occur independently of several
traditional cardiovascular risk fac-
tors.
Alvi et al. (2010) reported that
one third of students were found to
have anxiety and depression which
was associated with the sociodemo-
graphic and educational factors in-
cluded age, sex, birth order, number
of the siblings, monthly income,
monthly expenditure on education,
academic performance in the pro-
fessional examination, past medical
and past psychiatric history, sub-
stance abuse and family history of
psychiatric illness.

Conclusion

The outcome results showed that
nervous stress caused from exam
anxiety led to the increase of trigly-
cerides in the existence of some
variables included in the study af-
fecting personal general health.
269

Besides, one must keep in mind
the other risks that cause the eleva-
tion of the triglycerides with their
mild to fatal side effects.

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271



SCANNING ELECTRON MICROSCOPIC STUDY OF TROPHOZOITE
AND CYST STAGES OF NAEGLERIA FOWLERI
By
SANAA N. ANTONIOS
Department of Parasitology, Faculty of Medicine, Tanta University, Egypt.

Abstract

Whole trophozoites and cysts of axenically cultivated Naegleria fowleri were
prepared for study of their surface morphology by scanning electron micro-
scopy (SEM). Trophozoites and cyst stages were studied from Chang's culture
media. Some trophozoites were examined after animal inoculation and brain
isolation to compare the changes in surface features. Photomicrographs of
freeze-dried and critical point-dried organisms fixed with glutaraldehyde were
presented along with views of both isolates of trophozoites to compare the sur-
face features.
SEM revealed the surface of trophozoites to be undulating, wrinkled and
covered at irregular intervals by protruding vesicles. There were also surface
extensions which were long and thin in brain isolates which may help in the
contact and cytolysis of host cells at some distance from the trophozoite. Some
cysts appeared wrinkled while others smooth, and empty cysts were also seen
with many pores on the surface.
Key words: SEM, Naegleria fowleri, trophozoites and cysts.
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------

Introduction

Naegleria fowleri is a free-living
amoeboflagellate known to cause
fatal human disease-primary amoe-
bic meningo-encephalitis (PAM).
Although, it is a somewhat rare dis-
ease, cases of PAM have been re-
ported from a variety of countries
worldwide (Huang et al., 2010). It
occurs in water as well as in water-
hyacinth root (Ramirez et al., 2010).
The determinants of the virulence
and pathogenicity of N. fowleri are
mainly undefined (John et al.,
1984). In vitro, studies have impli-
cated toxins and cytopathic enzymes
(Wong et al., 1967; Chang, 1979;
Hysmith and Franson, 1982) and
phagocytosis (Brown, 1978; 1979).
The in vitro cytolysis of mammalian
cells following contact with living
trophozoites of N. fowleri was ob-
served (Marciano et al., 1982).
Besides, John et al. (1984)

pro-
posed that a sucker-like structure
was observed by scan electron mi-

272

croscope on amoebae of human iso-
lates of N. fowleri. The number of
suckers was related to virulence of
the strain.
The present study added know-
ledge of ultrastructure of surface of
N. fowleri kept in axenic medium
and after mouse-brain passage.


Naegleria fowleri amoebae were
grown axenically in Chang's me-
dium

(Chang, 1971). At 72 hours
of culture age 9early stationary
growth phase), amoebic culture
tubes were centrifuged and the se-
diment was removed from the
growth medium, rinsed in Page
amoeba saline (1967) and cover
slips were fixed at room temperature
23
o
C for 1/2 hour with 2.5%
(Vol/Vol) glutaraldehyde in phos-
phate buffer (pH 7.2).
Amoebae were isolated from
brains of infected mice 5 days post
infection and were rinsed in a solu-
tion of polylysine in water. After 30
minutes, the surface was thoroughly
washed with Page saline and the
adsorbed layer of polylysine was not
displased. The cell suspension was
placed on the surface and the amoe-
bae attach as soon as they settle.
Cover slips were fixed as before and
dehydrated in acetone and were crit-
ical point dried, mounted on stubs
and coated with gold-palladium.
All specimens were photographed
on Novascan 30 SEM (Hayat,
1978).

Results

The axenically cultivated tropho-
zoites: Trophozoites showed undu-
lating finely wrinked surface with
some protruding vesicles or blebs of
various sizes at irregular intervals
(Figs. 1, 2). Pseudopodia were also
found which were thick and short
(Fig. 3). Small disks similar to food
cups were seen on the surface of
some amoeba (Fig. 4).
Brain isolates: Many of the tro-
phozoites examined after mouse
brain passage showed surface exten-
sions which were quite long and
thin which appeared to have been
fixed while progressing motile (Fig.
5). The cytoplasm of them was in
continuity with the cytoplasm of the
cell and was bounded by surface
plasmalemma. Extra amoebic ve-
sicles were also found which may
represent portions of surface exten-
sions (Fig. 6).
Amoebic cysts: Some amoebic
cyst stage was found from culture
isolate. The precystic stage ap-
peared rounded-up with wrinkled
surface (Fig. 7). Neither vesicles
nor extensions were found on the
surface. The proper cyst appeared
round with smooth surface (Fig. 8).
Empty cyst was also found which
appeared as round disc full of pores
(Fig. 9).

Discussion

As a study of the surface exten-
sions of N. fowleri was of particular
interest, this work has permitted

273

visualization of these delicate struc-
tures over-most of their surface ex-
tent. SEM seems to have permitted
study of both trophozoite and cyst
stages in details. Numerous surface
extensions where noticed in this
work which were of normal occur-
rence on such amoebae. However,
the extensions in brain isolates were
much longer than culture isolates
which indicated their active role in
physical contact with brain tissue
cells. The function of surface ex-
tensions was controversy. Brown
(1978) found that N. fowleri may
destroy cultured mouse embryo
cells by aphagocytosis-like mechan-
ism. While Hysmith and Franson
(1982) reported that the pathoge-
nicity of N. fowleri may destroy cul-
tured mouse embryo cells by apha-
gocytosis-like mechanism, and that
the pathogenicity of N. fowleri was
due to toxins and enzyme.
So, no conclusive evidence has
been offered to indicate that either
of these factors play the important
role in tissue lysis and destruction.
As regards other surface structures,
a search was made during the
present work for suckers which
were described by John et al. (1984)
in human isolates. Few discs were
seen but there was no evidence of
well-organized suckers in N. tro-
phozoites.
Concerning the cyst stage, three
distinct morphological types were
noticed. The first appeared as spher-
ical mature seemed viable type with
corrugated surface. This could be
considered pre-cyst stage. The
second one was similar but having
smooth surface. The third type ap-
peared as empty disc-like type with
many pores on the surface indicat-
ing that it is non-viable. Carter
(1966) found that empty non-viable
cysts were constant finding in cul-
tures of N. fowleri, but pores and
plugs are not seen in the cysts. But,
Jonckheere and Van de Voorde
(1976)

showed empty pores of N.
fowleri cyst in side view using
phase contrast microscope.

Conclusion

Naegleria is a free-living amoebae
existing in soil and aquatic envi-
ronments worldwide. Besides, the
Naegleria spp. can serve as vehicles
for facultative pathogens, such as
Legionella. The present SEM study
showed the presence surface pores
in the cyst wall of N. fowleri which
helped in emptying the cell content.

Acknowledgement

The author would like to Dr. Wal-
ter E. Wilhelm; Professor of Biolo-
gy is due to his gracious contribu-
tion of time and practical effort to
help in the inoculation and mainten-
ance the experimented with Naegle-
ria fowleri.

274

Fig. 1: N. fowleri amoeba from axenic culture, surface wrinkled with protruding blebs (X 8000).
Fig. 2, 3: Trophozoites of N. fowleri with thick and short pseudopodia (X 4000).
Fig. 4: N. fowleri amoeba with surface wrinkles and a disk similar to food cup (X 8000).

275

Fig. 5: Micrograph of N. fowleri amoeba isolated from mouse brain shows long and thin surface
extension with multiple pseudopodia (X 8000).
Fig. 6: Micrograph of amoebae isolated from mouse brain showing pseudopodia and some vesi-
cles (X 4000).
Fig. 7: Amoebic cyst with wrinkled surface (X 4000).
Fig. 8: Amoebic cyst with smooth surface (X 4000).
Fig. 9: Empty cyst full of pores (X 4000).


276

References

Brown, T. 1978: Observations by
light microscopy on the cytopatho-
genicity of Naegleria fowleri in
mouse embryo-cell cultures. J. Med.
Microbiol., 11:249.
Brown, T., 1979: Observation by
immunofluorescence microscopy
and electron microscopy on the cy-
topathogenicity of Naegleria fowleri
in mouse embryo-cell cultures. J.
Med. Microbiol. 12:363-71.
Chang, S.L., 1971: Small free-
living amebas. Curr. Top. Comp.
Pathobiol., 1:201-54.
Carter, R.F., 1972: Primary amoe-
bic meningo-encephalitis. Trans. R.
Soc. Trop. Med. Hyg., 66:193-213.
Chang, S.L., 1979: Pathogenesis of
pathogenic Naegleria amoeba. Folia
Parasitol., 26:195-200.
De Jonckheere, J.; Van de Voor-
de, H. 1976: Differences in de-
struction of cysts of pathogenic and
nonpathogenic Naegleria and Acan-
thamoeba by chlorine. Appl. Envi-
ron. Microbiol., 31:294-7.
Hayat, M.A., 1978: Introduction to
Biology Scanning Electron Micro-
scopy. University Park Press, Bal-
timore, London, Tokyo.
Huang, S.W.; Hsu, B.M., 2010:
Survey of Naegleria and its resist-
ing bacteria-Legionella in hot spring
water of Taiwan using molecular
method. Parasitol. Res., March 20.
[Epub ahead of print].

Hysmith, R.M.; Franson, R.C.,
1982: Elevated levels of cellular and
extracellular phospholipases from
pathogenic Naegleria fowleri. Bio-
chem. Biophys. Acta 711:26-32.
John, D.T., 1982: Primary amoebic
meningoencephalitis and the biolo-
gy of Naegleria fowleri. Ann. Rev.
Mcrobiol. 36:101-23.
John, D.T., Cole, T.B.; Marciano-
Cabral, F.M., 1984: Sucker-like
structures on the pathogenic amoeba
Naegleria fowleri. Appl. Environ.
Microbiol. 47:12-4.
Marciano-Cabral, F.M.; Patter-
son, M.; John, D.T.; Bradley,
S.G., 1982: Cytopathogenicity of
Naegleria fowleri and Naegleria
gruberi for established mammalian
cell cultures. J. Parasitol. 68:1110-6.
Wong, M.M.; Karr, S.L.; Chow,
C.K., 1977: Changes in the viru-
lence of Naegleria fowleri main-
tained in vitro. J. Parasitol. 63:872-
8.
Page, F.C., 1967: Taxonomic crite-
ria for limax amoebae, with descrip-
tions of 3 new species of Hartman-
nella and 3 of Vahlkampfia. J. Pro-
tozool. 14:499-521.
Ramirez, E.; Robles, E.; Marti-
nez, B., 2010: Free-living amoebae
isolated from water-hyacinth root
(Eichhornia crassipes). Exp. Parasi-
tol., February 1. [Epub ahead of
print]


277


277

Medical Parasitology Department

Medical Parasitology Department Annual Conference 2010

Tuesday 30
th
March: A Workshop:

The New Advances in Laboratory Diagnosis of Intestinal and Blood Parasites.

Chair persons:
Prof. Dr. Ibrahim A. Abo El-Asaad, Head of Medical Parasitology Dep., Facu-
lty of Medicine, Tanta University.
Prof. Dr. Mohamed M. Eid, Medical Parasitology, Faculty of Medicine, Tanta
University.
Prof. Dr. Samy I. El-Kowrany, Medical Parasitology, Faculty of Medicine,
Tanta University
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Thursday: 1
st
of April.

1
st
Session: 10.00 a.m. 12.30 p.m.

Chair persons:
Prof. Dr. Ibrahim A. Abo El-Asaad, Head of Medical Parasitology Dep., Facu-
lty of Medicine, Tanta University.
Prof. Dr. Tosson A. Morsy, Medical Parasitology, Faculty of Medicine, Ain-
Shams University.
Prof. Dr. Nabeel S. Gaweesh, Medical Parasitology, Faculty of Medicine,
Tanta University.
Prof. Dr. Sanaa N. Antonios, Medical Parasitology, Faculty of Medicine, Tanta
University.
Moderator: Dr. Marwa A. Hasby, Medical Parasitology, Faculty of Medicine,
Tanta University.

Papers presented:
1-Rodents, Fleas, Plaque: Dr. Micheal W. Mikhail, Assistant Professor,
Research Institute of Medical Entomology, MOH.
2- Rodents borne diseases & their fleas in Menoufia Governorate, Egypt: Prof.
Dr. Mohamed I. Soliman, General Director, Research Institute of Medical
Entomology, MOH.
3- Efficacy of Zingiber officinale on 3
rd
stage larvae and adult fecundity of
Musca domestica & Anopheles pharoensis: Dr. Azza S. Abd-Elhamid, Assis-
tant Professor, Research Institute of Medical Entomology, MOH.
4- The impact of subchronic mercury exposure on the immune response and
pathogenesis of the experimental cryptosporidiosis: Prof. Dr. Howaida I.
Ismail, Medical Parasitology, Faculty of Medicine, Tanta University.

278

5- ELISA seroprevalence of Toxoplasma gondii in draught horses in Greater
Cairo, Egypt: Prof Dr. Fouad M. Haridy, Formerly, General Organization of
Veterinary Services.
6- A study of the prevalence of house dust mites in Al-Arish City, North Sinai
Governorate, Egypt: Prof. Dr. Gehad T. El-Sherbini, Faculty of Pharmacy,
October 6 University,
8- Stem cells & Liver fibrosis: Dr. Dina M. Abo-Raya, Assistant Lecturer,
Medical Parasitology, Faculty of Medicine, Tanta University.
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Coffee Break: 12.30- 01.00 p.m.
----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
2
nd
Session: 01.00- 03.00 p.m.
Chair persons:
Prof. Dr. Ahmed A. Daoud, Medical Parasitology, Faculty of Medicine, Tanta
University.
Prof. Dr. Soaad A. Salem, Medical Parasitology Faculty of Medicine, Tanta
University.
Prof. Dr. Sirria M. El- Marhoumy, Medical Parasitology Faculty of Medicine,
Tanta University.
Prof. Dr. Mohamed M. Eid, Medical Parasitology, Faculty of Medicine, Tanta
University.

Papers presented:

1-Cutaneous Leishmaniasis with special references in Egypt: Prof. Dr. Tosson
A. Morsy, Medical Parasitology, faculty of Medicine, Ain-Shams University.
2-Zoonotic Fascioliasis in the Eastern Nile Delta: Prof. Dr. Atef M. Al-Shazly,
Head of Medical Parasitology Dep., Faculty of Medicine, Mansoura
University.
3-Brucellosis in Egyptian female patients: Prof Dr. Ahmed A. Sabah, Medical
Parasitology, Faculty of Medicine, Al-Azhar University.
4-Food additive and Hymenolepis nana infection: An experimental study: Prof
Dr. Kholoud A. El-Nouby, Medical Parasitology, Faculty of Medicine, Tanta
university.
5-Health Risk Impact in Toshka, Upper Egypt: Dr. Nahla M. Shoukry, Faculty
of Science, Suez Canal University.
7- Human Fascioliasis and HCV: Major Dr. Ayman T. Morsy, Tropical
Medicine Unit, The ministry of Interior Hospitals, Egypt.
8-Congenital exposure to Schistosoma mansoni infection: Impact on the future
immune response and the future outcome: Dr. Ahmed A. Othman, Lecturer,
Medical Parasitology, Faculty of Medicine, Tanta University.
9-Games Parasites Play: Dr. Marwa A. Hasby, Assistant Lecturer, Medical
Parasitology, Faculty of Medicine, Tanta University.

279

='--' -= --- ---' '--` ',' --= _=- --=- /,--' '--` -,-'
--' ,-=' ,'' _ ---' -- ,= -'= -'= _'= -'=' '='
2004 :
:,-=-' '',-' : `
1 ( ',' --= _=- --=- : -`
2 ( : `,-' _' 16 --, 1934
3 ( -- ,= -'= '=' ='--' -= --- --- '-- : -,=,'
:'+,'= .'=' `,-' : ','`
1 ( --- -- ,= -= ,' =' -=' ,,''- 1957
2 ( --, -- ,= -= ,' '=' ='--' -= ,'- 1961
3 ( .- -- ,= -= ,' ,-='- - ,'- 1965
4 ( --, -- ,= -= ,' '=' ='--' -= -,- 1971
:'+'- _-' --'=,' : '`''`
1 ( _ =-' _ ,= 15 / 9 / 1959 _ -- _-''- ,-=' -=' ,=- -+- _
.'`-' '----` _-,'` ---' -
2 ( - ,-'-' ',- _------ .-= 4 / 1 / 1963 _' 4 / 9 / 1972
3 ( _ -- ,= -= ,'- '=' ='--' -= --- -- 20 / 7 / 1972 _'-- -='-- '-- `
25 / 7 / 1977 _'-- '-- ` 31 / 7 / 1982
4 ( ,--' ,,=--' '-' -,= ',- -=' ,-= --' ,-=' ,-=' '=- ,-=
._-'' -=-' ,-''-
5 ( - -=,--' -`' -='--' -'-` --'=,' ---' ,-'' -='' -- 2001 / 2004
- -=,--' -`' -'-` --'=,' ---' ,-'' -='' ,-= 2004 / 2007
:'-'+- : '-
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2 ( ',-'+'-' -=,--' -` --' _--+' '+=' ',-= ,-=
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.,-- . - -` , ,='
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.--
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7 ( -=, -, -- _ ',-='' _,-,'` ,=--' ,- _'= ,- ,-'= --- -'
110 .-----' ,'--' ,=-' '+-=- _ ,- ` -- _ ',- _-----
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9 ( .=- - --' '-' ,,-' ,'+' `'=- _ .=- '-=`' _-'- _ ,-=
.,'=' ''' _=- ,--' - --,-` -'-` ' ''=- --- ,-=' - -
'-'' '' - '=-- - , '--- _'' _ - ' ,- ) , ' - ' - -- (
" ' ','= - . ) ( '' .'=' =- = _-- ) "( .,=' - -


280

Al-Fayuom is a wonderful area of Egypt with a rich and interesting history. It
is an area where Egyptians often vacation and which is constantly growing
more popular among Europeans. This 692 sq. mile depression was a lush
paradise during prehistoric times. Its water level was eighty-five meters higher
than today (currently 45 meters below sea level) and the Nile regularly flooded
through the low mountains separating it from the Fayoum. At 215 square
km, the current lake Qaroun remains Egypt's largest salt water lake. The
prehistoric people who lived here were, at first, nomadic hunters and gatherers,
but later began harvesting plants near the lake. This developed into what is said
to be the earliest agricultural area in the world, where fences were erected and
guarded warehouses built. It has remained an agriculture center, well known
for its fruits, vegetables and chickens.

ZOONOSIS INFECTIOUS DISEASES AND HUMAN WELFARE
First Announcement for second announcement contact Secretary General

NOTICE TO CONTRIBUTORS

This is a regular journal published by the Egyptian Society of Parasitology and is
concerned with the publication of research papers in the field of Parasitology and
related subjects. Three volumes are published annually April, August and December.
Two types of communications appear regularly; papers and correspondence. Letter to
the editor is also published. Publication is not restricted to Fellows of the Society but
non-Fellows can also publish on their own expense. Manuscripts should be signed by
the author or authors concerned and sent to the Chief Editor. All the manuscripts are
peer reviewed. However, the authors alone are responsible about all the data and the
scientific views they expressed.

Instructions for preparation of manuscripts: Paper should be sent in type-
written, on one side of the paper (page size 17x24 cm, font 11, and tables on separate
file), all on a CD-R., or by E: mail. Papers should be as concise as possible and
provided with a brief abstract (only very short summary of your own results), key
words and a brief conclusion.

Illustrations (photographs, line drawings, graphs and tables) should be kept to the
minimum. Details of results presented in this way should not be repeated by anyhow
in the text. Original colored plates (12x18 cm) can be included only at the authors
expense. Line drawings, maps, graphs and tables should be approximately final size.
Pronouns such as I, my, we, our, should not be used and my or our results should be
replaced by in the present study or results. Discussion should be for your own results
in relation to others and not in any way, a review of literature or aim of the work.

References should be listed at the end of the paper and arranged in an alphabetical
order of authors, including: (i) author or authors' names and initials, (ii) year of
publication, (iii) full title of paper, (iv) Journals title, (v) volume number, (vi) issue
number, (vii) first and last page numbers. Titles of journals cited are to be abbreviated
as in the World List of Scientific Periodicals, 4
th
edition, 1964. The M.Sc. or Ph.D.
thesis is not accepted at all as a reference. The abbreviations throughout the text and
tables should conform to the Standard International Units as far as possible.

Letters to the Editor should be as possible not to exceed 400 words, graphs and
illustrations will not be accepted and references must be kept to a minimum.

Authors (except letters to the editor) will be supplied with 90 cover reprints free
of charge and requests for additional reprints at the authors expense should be sent to
the Secretary General together with the manuscript.

Abstracts of this journal are published in Helminthological Abstracts, Series A,
Protozoological Abstracts (Commonwealth Institute of Parasitology), Veterinary Bu-
lletin and Index Veterinarius (Commonwealth Bureau of Animal Health), Tropical
Diseases Bulletin, Review of Applied Entomology, Quarterly Bibliography of Major
Tropical Diseases, US Medline database (Index Medicus ISSN: 0253-5890) and
WHO Eastern Mediterranean Region Index Medicus (ww.emro.who.int/His/VHSL/
Imemr.htm).
--------------------------------------------------------------------------------------------------
Al-Taawen Press: Cairo, Egypt, Fax: + (202) 26970043 Mobile: 012/7338132

Consultant Board

Acarology,
Prof. Dr. Kawther M. El-Kammah, Faculty of Agriculture, Cairo University.
Community, Environmental and Occupational Medicine:
Prof. Dr. Aly Abdel-Hady Massoud, Faculty of Medicine, Ain Shams University.
Dermatology and Venaerology:
Prof. Dr. Mohammad Amin Amer, Faculty of Medicine, Zagazig University.
Entomology:
Prof. Dr. Bahira Mahmoud El-Sawaf, Faculty of Science, Ain Shams University.
Forensic Medicine and Toxicology:
Prof. Dr. Mohamed Safwat Soliman, Faculty of Medicine, Ain Shams University.
Gastroenterology, Hepatology and Infectious Diseases:
Prof. Dr. Ahmad Mohamed A. Massoud, Faculty of Medicine, Al-Azhar University.
Prof. Dr. Mamdouh M. El-Bahnasawy, Military Medical Academy.
Histology:
Prof. Dr. Prof. Dr. Ahmed Said El Morsy, Faculty of Medicine, Ain Shams University.
Internal Medicine:
Prof. Dr. Mohamed Safwat Seif-El Nasr, Faculty of Medicine, Al-Azhar University.
Ministry of Health:
Prof. Dr. Mohamad Mustafa, General Director Endemic Diseases and Schisto Control.
Prof. Dr. Zeinab Mohamed Youssef, Undersecretary for Endemic Diseases.
Pathology:
Prof. Dr. Amal Mohamed Mangoud, Faculty of Medicine, Zagazig University.
Prof. Dr. Badawiya Bayoumi Ibrahim, Faculty of Medicine, Cairo University.
Pediatrics:
Prof. Dr. Hamed Mahmoud Shatla, Faculty of Medicine, Ain Shams University.
Pharmaceutical Science:
Prod. Dr. Neiven M. Abdel-Hady, Faculty of Pharmacy, Al-Azhar University for Girls.
Plant Protection and Rodent Control:
Prof. Dr. Awad Farahat A. El Bahrawy, Faculty of Agriculture, Suez Canal University.
Public Health and Behavioral Medicine:
Prof. Dr. Saad Mohamed Motawea, Faculty of Medicine, Mansoura University.
Zoology and Malacology:
Prof. Dr. Fathy A. Abdel-Ghaffar, Faculty of Science, Cairo University.

*****
Past Presidents since the Foundation of the Society

1969-1987: Prof. Dr. Mahmoud Hafez, Faculty of Science, Cairo University.
1988-1996: Prof. Dr. Hamid M. Khalil, Faculty of Medicine, Ain Shams University.
1997-2002: Prof. Dr. Nabil Taha Nasr, Faculty of Medicine, Cairo University.
2003-2009: Prof. Dr. Mohsen M. Hassan, Faculty of Medicine, Zagazig University.

JOURNAL OF THE EGYPTIAN SOCIETY OF PARASITOLOGY
(WORLD FEDERATION OF PARASITOLOGISTS,
MEMBER AT LARGE, AFRICA)
http://www.parasitology.eg.net
* * * * *
THE EXECUTIVE AND ADVISORY BOARD 2010

President: Prof. Dr. Abdel Hameed Abdel Tawab Sabry
Vice President: Prof. Dr. Atef Mohamed Aly El Shazly
Treasurer: Assist-Prof. Dr. Nahla M. Shoukry
Secretary General: Prof. Dr. Tosson Aly Morsy
Members: Prof. Dr. Ahmad Aly Aly Sabah
Dr. Fouad Mohamed Sayed Haridy
Prof. Dr. Magdy Abdel Latif Saleh Arafa
Prof. Dr. Mohamed El-Husseiny Fayad
Prof. Dr. Sanaa Nageeb Antonios Boctor
* * * * *
Honorary Members: Prof. Dr. Ebtesam M. Al-Mathal (East Saudi Arabia),
Prof. Dr. Saeed A. Al-Harthi (West Saudi Arabia),
Prof. Dr. Gehan Gamal-Eldin El Fandy (Egypt),
Prof. Dr. Mary E. Wilson (USA),
Prof. Dr. Patrice Bouree (France),
Prof. Dr. Reda L. Roufael El-Gamal (Egypt),
Prof. Dr. Robert W. Ashford (United Kingdom),
Prof. Dr. Safiya S.M. Khalil (Egypt),
Prof. Dr. Santiago Mas-Coma (Spain),
Prof. Dr. Sayeda Aly Gafaar (Egypt).
* * * * *
The Chief Editor: Prof. Dr. Tosson Aly Morsy
Editors: Prof. Dr. Abdallah Michael Boghdadi (Cairo University),
Prof. Dr. Amany Helmy M. Lashin (Benha University),
Prof. Dr. Heba M. Abdel Aaty (Ain Shams University),
Prof. Dr. Faiza Hussein Mohammad (Al-Azhar University for Girls),
Prof. Dr. Manal Farouk El Garhy (Cairo University),
Prof. Dr. Manal Salah-Eddin Mahmoud (Ain Shams University),
Prof. Dr. Mervat Z. El-Azzouni (Alexandria University),
Prof. Dr. Mohammad El-Salahy M.M. Moneib (Assuit University),
Prof. Dr. Samy El-Sheikh Tayel (Al-Azhar University),
Assist-Prof. Dr. Gehad T. El-Sherbini (6
th
October University).

* * * * *
For Communications: Professor Dr. Tosson A. Morsy, Department of Parasitology,
Faculty of Medicine, Ain Shams University, Cairo 11566, Egypt.
Fax: (+202)24036497 Mobile: (+2) 0101331999
E-mail:morsyegypt2000@yahoo.com or morsy2000@hotmail.com
* * * * *
THE EGYPTIAN BOOK CENTER
Deposit No. 695/1978

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