SALMONELLA GALLINARUM 9R STRAIN

AS VACCINE

by Edir N. Silva Campinas, SP. Brazil





Brief History on the SG 9R-Strain

The rough (R) strain of Salmonella Gallinarum (SG), named as 9R, was developed in England from a number 9
field virulent smooth strain, with a paper published by Smith in 1956, based on the knowledge that rough
mutants lose their virulence capability. Many publications have proved its safety as a vaccine, non reverse in
virulence, and ability to protect chickens against fowl typhoid (FT). Since then, it has been used extensively in
many countries where the FT is endemic, mainly in brown layers, but also in white egg layers and breeders.
One of the greatest advantages of the use of the SG 9R vaccine is that it gives good protection and does not
interfere with the tests used for pullorum-typhoid control. Besides controlling FT, the SG 9R vaccine has been
used in several countries as an additional tool in the control of Salmonella Enteritidis (SE) in commercial layer
flocks. Better immunity, lower cost in comparison with kill products, no local reactivity, are its strongest points.



Infection and Immune-Protection Mechanism of Salmonella

Fowl typhoid (FT) in chickens and turkeys is caused by Salmonella enterica, subspecie enterica, serovar
Gallinarum (S. Gallinarum) and, in its acute form, is almost exclusively a septicaemic disease, more observed in
the later growing period, and in mature stock; although age is not a limiting factor for disease occurrence
(Berchieri, et al. 2001). The severity of FT is highly genetically dependent. The brown egg layer breeds are very
susceptible, while the white leghorn types are quite resistant, but still carry the organism for a long period of
time (Berchieri Jr., et al. 2000).

A rough mutant (R) strain of S. Gallinarum (SG), named as 9R, was developed in England from a field virulent
smooth strain number 9 (Smith, 1956), and used as a vaccine worldwide.

It is known that the infection with 9R results in a mild systemic infection, not prolonged, leading to both cellular
and humoral immune responses, which peak soon after bacterial clearance. The bacterial clearance after
vaccination occurs at three weeks post infection, and coincides with increases in circulating anti-Salmonella
antibodies, increases in T cell proliferation and the expression of interferon gamma. There is no persistence of
the vaccine strain in the gastrointestinal tract (Silva, et al., 1981; Wigley, et al., 2005).

Although, the immune response to SG, either infection or live vaccination, is poorly characterized (Jones, et al.,
2001), it has been demonstrated that protection could be related to outer membrane proteins (OMP).
Subcutaneous inoculation with purified OMP from SG field strain, gave better protection results in chickens
experimentally challenged with the wild type of SG, in comparison with 9R subcutaneously (SC) vaccinated
ones (Table 1). The protection was measured by clearance of challenge organism from internal organs
(Bouzoubaa, et al. 1989).
Issue No.4 / August 2010


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Table 1. Outer membrane protein (OMP) of Salmonella Gallinarum and 9R strain as vaccines for fowl typhoid in chickens.

Isolation of challenge strain (+/survivors)
Vaccinated group
Liver + spleen Ovary Total

Protection**
OMP one dose SC 3/40 1/20 4/20 80 %
OMP two doses* SC 2/40 0/20 2/20 90 %
9R one dose SC 7/40 4/20 8/20 60%
9R two doses* SC 7/40 4/20 8/20 60%

(*) 4 wks after firs vaccination
(**) Measured by clearance of systemic infection
Adapted from: Bouzoubaa, et al., 1989.





THE SALMONELLA GALLINARUM 9R-STRAIN AS A VACCINE FOR CHICKENS


The SG 9R Vaccine

The SG 9R vaccine is a suspension of the live avirulent stable rough strain of SG. It is the most commonly used
vaccine for FT (Harbourne et al., 1963) presented in the freeze-dried form.

The 9R-strain does not contain the somatic antigen characteristics of the smooth forms of SG, but it shows the
same biochemical reactions characteristics of smooth virulent SG strains. However, 9R-strain suspensions,
from broth or agar culture, are rough when examined by the acriflavine slide test.

Table 2. Characteristics of the Salmonella Gallinarum strains

Features Wild type 9R strain
Colony appearance Smooth Rough
Biochemical reactions Typical Typical
Reaction to somatic O antiserum Pos. Neg.



The SG is a non mobile serovar (they do not have flagellum), belonging to the Salmonella serogroup D1, with
somatic antigens 1, 9 and 12, the same as in other pathogenic strains for chickens such as S. Enteritidis (SE)
and S. Pullorum (SP). So, cross protection is expected between them (Table 3). The SG 9R vaccine has being
used in several countries as an additional tool in the control of SE in commercial layer flocks (Feberwee et al.,
2001).

Table 3. Classification of the most important poultry pathogenic Salmonella.

Flagellum H antigens
Serovar

Serogroup
Somatic O
antigens
Phase 1 Phase 2
S. Gallinarum D1 1, 9, 12 Non mobile Non mobile
S. Pullorum D1 1, 9, 12 Non mobile Non mobile
S. Enteritidis D1 1, 9, 12 g,m [1,7]
S. Typhimurium B 1, 4, [5], 12 i [1,2]





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Use of the SG 9R-Strain Vaccine in Chickens

The use of the SG 9R vaccine for FT must be done before field challenge occurs, and it should be considered in
a FT endemic area. Due to it cross reactivity it has been used as a tool to control SE infection in layers and
breeders.

Vaccination reduces flock losses due to FT, contributes to the reduction of SE infection and egg transmission in
infected flocks, but still the field infection can still occur (Table 4 and 5).

There are some indications that the use of the 9R vaccine can reduce the shedding of non related serogroup as
S. Typhimurium (Table 6).

Vaccination should be considered as part of an FT and SE eradication program.


Table 4. Morbidity and mortality of chickens vaccinated with 9R and challenged with a pathogenic strain of S. Gallinarum four
weeks later.

Vaccination Liver culture of survivors Mortality (%)
Chicken
Age Route Vaccin. Control Vaccin. Control
Meat-type 8 wks SC 0 % 20% 5 25
Brown-egg 1 day SC 33% NS 50 100
W.Leghorn 1 day SC 14% 24% 0 6
W.Leghorn 10 wks SC 5% 58% 0 5
W.Leghorn 10 wks Oral 15% 58% 0 5
Meat-type 8 wks Oral 0% 20% 20 25
Adapted from: Silva, et al., 1981.



Table 5. Mortality following challenge of one-year-old meat-type chickens vaccinated subcutaneously with 9R and oil-emulsion
(OE) vaccine.

Weeks post-challenge Mortality
Vaccine
1 2 3 4 5 6 7 Total %
9R 10 4 0 1 0 0 0 15/42 36
SG OE 11 12 0 0 0 1 0 24/44 55
Control 20 10 5 5 1 0 0 41/44 93
Adapted from: Silva, et al., 1981.




Vaccination: Doses, Age and Route of Administration

The vaccine titer is important. The dose used per bird is between 10
6
to 10
7
colony forming units CFU (OIE,
2004).

Injections, either SC or intramuscular, are the preferable vaccination routes. Two doses protects better than one
single vaccination. Water vaccination has been used in many situations, but it is less protective. See Table 4
(Bouzoubaa, et al., 1989; Silva, et al., 1981).

It is usual to vaccinate at 6-8 weeks and again at 14-16 weeks of age. Field data has demonstrated that broiler
chicks vaccinating at day-old at the hatchery plant level, does not interfere with production parameters.
Antimicrobials should be avoided before and after vaccination. A booster dose should be required, and
vaccination should be avoided during laying period.




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Based on the effect on body weigh gain, 4 weeks of age was recommended as the minimum vaccination age of
the 9R vaccine considering the safety and efficacy of the vaccine (Lee, et al,. 2005).


Table 6. Protection of 10 wks-old W.Leghorn subcutaneously vaccinated (10
7
CFU/dose) with 9R vaccine and orally challenged six
wks later with Salmonella Typhimurium (10
9
CFU/bird).

Fecal droppings (Number of CFU per gram)
Time post-challenge
Vaccinated Control
24 hours 10
7
10
7

7 days 10
3
10
7

28 days 10
4
10
7

35 days 10
4
10
7

Adapted from: Silva, et al., 1981.



Transmission of the SG 9R-Strain

Vaccinated chickens can harbor the strain for a short period of time, and the length depends on the age and
breed (Tables 7 and 8), as is seen both in experimental SG infection and vaccination (Berchieri, et al., 2005;
Silva, et al., 1981).


Table 7. Persistence of 9R strain in meat-type chickens vaccinated at day of age by subcutaneous (SC) and oral routes

Blood Liver Spleen Droppings Intestine Time
PI
SC Oral SC Oral SC Oral SC Oral SC Oral
3 h + - ++ - + - Pos Pos Pos Pos
5 h + - ++ + ++ + Pos Pos Pos Pos
10 h - - ++ + ++ - Pos Pos Pos Pos
24 h ++ + +++ + ++ + Pos Pos Pos Pos
48 h +++ - +++++ ++ ++++ + Pos Pos Pos Pos
7 d ++++ +++ +++ +++ +++ ++ Neg Neg Pos Pos
14 d ++++ +++ ++++ + ++ ++ Pos Neg Pos Neg
21 d ++ - - - +++ - Pos Neg Pos Neg
28 d - - ++++ +++ ++ ++ Neg Neg Neg Neg
35 d + - ++ + + + Neg Neg Neg Neg
Adapted from: Silva et al., 1981.



Table 8. Persistence of 9R strain in two different breeds of chickens vaccinated at day of age by subcutaneous route

White Leghorn Brown-egg producing Time
PI
Spleen Feces Spleen Feces
12 hours - - - -
24 hours - - - -
48 hours + +++++ - -
7 days - ++ + +++
14 days - - ++ ++
21 days ++ - +++++ +
28 days - - ++ -
35 days - - + -
Adapted from: Silva et al., 1981.


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The 9R-strain can be egg transmitted in a few cases if applied by injection during egg production period, but it is
rare. The 9R-strain was detected only in small numbers in the first weeks post-vaccination, at a prevalence level
of 1% with 95% CI (Feberwee, et al., 2001). In another study, the vaccine strain could not be isolated from any
of the 450 pools of 10 eggs, confirming the indication of its rare spread to the egg content (Feberwee, et al.
2001a). Even in experimentally FT infected flocks, egg contamination is rarely found (Berchieri, et al., 2005).

The horizontal transmission of the SG-9R strain has not been proved. In a field study, the potential spread of the
vaccine strain from vaccinated flocks to non-vaccinated flocks has been studied after both the primary and the
booster injection at four different rearing farms and at one layer farm. The vaccinated and the non-vaccinated
flocks were monitored at regular intervals by bacteriologic and serologic examination. No evidence was found
for the fecal spread of the vaccine strain (Feberwee, et al. 2001). Experimentally, meat type chickens can
excrete the 9R-strain by the feces for up to five weeks after day-old vaccination; excretion was reduced to 24h
post-vaccination when chicks were vaccinated at 8 wks of age, but was not detected in 10 weeks old white
Leghorns, nor in one-year old meat type vaccinated by SC (Silva, et al., 1981).



Vaccination with SG 9R in Layer-Chickens

Some simple measures can be taken in FT infected flocks to help control the disease. For example, the removal
of dead birds quickly from the chicken house seems to prevent and reduce the FT in the flock (Oliveira, et al.
2005).

Besides controlling FT, the SG 9R vaccine has been used as an additional tool in the control and in the
reduction of SE in commercial layer flocks, but is not expected to confer complete protection against infection in
the field (Feberwee et al., 2001).

In a field study in the Netherlands, 80 commercial layer flocks from an area of increased risk of SE infection
were vaccinated with a SG 9R. The efficacy study was done by assessment of the flock level occurrence of SE
infections in the vaccinated groups compared with that of a non-vaccinated groups of 1,854 flocks hatched in
the same period. The occurrence of SE infections in the vaccinated group was 2/80 (2.5%), and significantly
lower (P = 0.01) than that of the nonvaccinated group (214/1854 = 11.5%). Table 9 (Feberwee, et al. 2001a).


Table 9. Field trial with the occurrence of SE infections in commercial layer flocks

9R vaccinated groups* (80 flocks) Non-vaccinated groups* (1,854 flocks)
2/80 (2.5%)a 214/1,854 (11.5%)b
(*) Flocks hatched in the same period from an area of increased risk of SE infection
Source: Feberwee, et al. 2001. Statistic analysis (P=0.01).



Serological Response to SG 9R Vaccination

Although the 9R is a rough non-mobile strain, with a lack of somatic Oantigens, it still may stimulate the
production of transient antibodies (Ab) detectable depending on the test used. There is no direct relationship
between Ab titers and protection, although specific Ab may be involved (Bouzoubaa, et al, 1989; Silva, et al.,
1981). The serological tests could be detecting Ag against OMP and no to somatic antigens, since all chickens
vaccinated with the 9R-strain or with OMP developed antibodies detectable by the microagglutination (MAG)
test, and in some vaccinated groups as many as 100% of the birds developed antibody levels detected by
seroagglutination (Bouzoubaa, et al. 1989).

The MAG test is much more sensitive for Ab detection than the pullorum test. MAG test can detect Ab as early
as two weeks post-vaccination (PV), with 100% of positive birds by the 7
th
weeks after a single subcutaneous
(SC) dose. Some reactors to the pullorum test can be detected between 2 and 7 weeks post-vaccination. These
serological results suggest that a period of at least seven weeks should be allowed between vaccination with 9R
and testing for pullorum-typhoid (Silva, et al., 1981).


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However, SG 9R vaccinated flocks can be serologically differentiated from field SG, SP, SE or S. Typhimurium
(ST) infected flocks as shown in Table 10.

There are commercially available Elisa Kits to detect antibodies against Salmonella infection in chickens. The
indirect LPS-BD-Elisa (ID-Lelystad, Lelystad, The Netherlands) detects antibodies against Salmonella group B
and D antigens (O-antigens 1, 4, 5, 9 and 12). More specific Kits based on gm and i-DAS-Elisa are highly
specific and sensitive tests for detecting antibodies against the gm and i flagellum antigens of SE and ST.

Vaccinated layers with SG 9R showed 0% positive results in the pullorum test (rapid plate agglutination test for
detecting antibodies against SP/SG). When the LPS-BD-Elisa was used, the positive results were 59.0%. And,
the mean specificities of two blocking ELISAs (gm- and i-double antibody sandwich ELISAs) on the same sera
were 99.6% and 96.1%, respectively (Feberwee, et al. 2001a).


Table 10. Serological response of chicken to SG 9R vaccination and infected with the most prevalent avian pathogenic Salmonella
strains.

Elisa tests
Flock status
Pullorum
Test
LPS Flagellum
Micro-
agglutination test
S. Gallinarum 9R vac. - / + +++ - ++++
S. Gallinarum infected +++++ +++++ - ++++
S. Pullorum infected +++++ +++++ - ++++
S. Enteritidis infected ++ ++++ +++++ ++
S.Typhimurium infected + / - ++++ +++++ -
Pullorum test = Rapid plate agglutination test for detecting antibodies against SP/SG.
Elisa LPS = Lipopolysaccharide BD Elisa for detecting antibodies against Salmonella serogroups B and D.
Elisa flagellum = Blocking Elisa based on the flagellar antigens gm and i of SE and ST.
Adapted from: Feberwee, et al. 2001a; Silva, et al. 1981.




How to Test the Vaccine Protection in Chickens?

There is no satisfactory method of assessing the protection afforded by the vaccine in the field. However,
experience has shown that the vaccine can provide some benefit in situations where control cannot be achieved
by hygiene and management alone.

Laboratory evaluation is based on mortality and re-isolation of challenge strain (clearance rate). Vaccination of
young layer hens at age of 2, 4 and 6 week-old showed protection rate against challenge with the wild-type SG
observed 21 days after one-dose vaccination, as measured by mortality, was 0% to 5% in the vaccinated group,
while the control not vaccinated group was 95% to 100%. In addition, the control group demonstrated a 95% to
100% re-isolation rate of the challenge strain in internal organs and the caecum, while in the vaccinated group
only a 1% to 60% re-isolation rate was observed. Table 11 (Lee, et al. 2005).


Table 11. Laboratory test to evaluate 9R vaccine protection in layers hens vaccinated with one dose at age of 2, 4 and 6 weeks of
age

Challenged 21 days PV with a wild-type of SG
Features
Vaccinated group Non-vaccinated group
Mortality 0% to 5% 95% to 100%
Re-isolation from organs 1% to 60% 95% to 100%
Lee, et al. 2005.





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Safety and Post-Vaccination Reaction of SG 9R-Strain

The 9R strain is not known to be pathogenic to humans, and there are no special risks associated with the
manufacture or use of the vaccine.

The SG 9R-strain has been proved to be safe for chicken without reversion to virulence and spread to non-
target flocks, although mutant resistant to antibiotic has been shown to require a longer period of time to
establish systemic infection (Silva et al., 1981).

Mild lesions can be seen in the liver and spleen of brown-egg and meat type chicks vaccinated at day of age,
but not in white leghorn, similarly vaccinated (Silva, et al., 1981).

As the vaccine causes a transient systemic infection with replication in the liver and spleen during the first
weeks post-vaccination, some degree of growth depression could be expected, but not mortality, although this is
not a field observation (Silva, et al., 1981).

The vaccination at 2 weeks old of laying hens with one and 10 doses causes a small depression of body weight
gain at 5 weeks, but not with chickens vaccinated with one and 10 doses at the age of 4 and 6 weeks Table
12 (Lee, et al,. 2005), or in white leghorn (Silva, et al., 1981).



Table 12. Effect of one or 10 doses of the 9R vaccine on the body weight gain of laying hens

Age of vaccination Body weight gain Mortality
2 wks Small depression at 5 wks of age No
4 and 6 wks No depression No
Lee, et al,. 2005.
































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BIBLIOGRAPHY


Berchieri Jr., A., Oliveira, GH., Pinheiro, LAS., & Barrow, P. 2000. Experimental Salmonella gallinarum infection in light laying hen lines.
Braz. J. Microbiol. 31:50-52.
Bouzoubaa, K., Nagaraja, KV., Kabbaj, FZ., Newman, JA., Pomeroy, BS. 1989. Feasibility of using proteins from Salmonella gallinarum vs.
9R live vaccine for the prevention of fowl typhoid in chickens. Avian Dis. 33:385-391.
Feberwee, A., de Vries, TS., Hartman, EG., de Wi,t JJ., Elbers, AR., de Jong, WA. 2001a. Vaccination against Salmonella enteritidis in
Dutch commercial layer flocks with a vaccine based on a live Salmonella gallinarum 9R strain: evaluation of efficacy, safety, and
performance of serologic Salmonella tests. Avian Dis., 45:83-91.
Feberwee, A., Hartman, EG., de Wit, JJ., de Vries, TS. 2001. The spread of Salmonella gallinarum 9R vaccine strain under field conditions.
Avian Dis., 45:1024-1029.
Harbourne, JF., Williams, BM., Parker, WH. & Fincham, IH., 1963. The prevention of fowl typhoid in the field using a freeze-dried 9R
vaccine. Vet. Rec., 75:858-861.
Jones, MA., Wigley, P., Page, KL., Hulme, SD., Barrow, PA. 2001. Salmonella enterica Serovar Gallinarum requires th Salmonella
pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in
chickens. Infect. and Immun., 69:5471-5476.
Lee, YJ., Mo, IP., & Kang, MS. 2005. Safety and efficacy of Salmonella gallinarum 9R vaccine in young laying chickens. Avian Pathol.
34:362-366.
OIE. 2004. Fowl typhoid and pullorum disease. OIE terrestrial Manual. Chap. 2.7.5. p.868-877.
Oliveira, GH., Berchieri Jr., A., Fernandes, AC. 2005. Experimental infection of laying hens with Salmonella enterica serovar Gallinarum.
Braz. J. Microbiol. 36:51-56.
Ryll, M., & Hinz, KH. 1995. [Differentiation of Salmonella gallinarum rough and smooth strains using gas chromatography analysis of their
cell-bound fatty acids] [Article in German]. Berl Munch Tierarztl Wochenschr. 108:347-349.
Silva, EN., Snoeyenbos, GH., Weinack, OM., Smyser CF. 1981. Studies on the use of 9R strain of Salmonella gallinarum as a vaccine in
chickens. Avian Dis. 25:38-52.
Smith, HW. 1956. The use of live vaccine in experimental Salmonella gallinarum infection in chickens with observations on their interference
effect. J. Hyg. 54:419-432.
Wigley, P., Hulme, S., Powers, C., Beal, R., Smith, A., Barrow, P. 2005. Oral infection with the Salmonella enterica serovar Gallinarum 9R
attenuated live vaccine as a model to characterize immunity to fowl typhoid in the chicken. Vet. Res., 2:1-9.