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Emergence of Enterocytozoon hepatopenaei

(EHP) in farmed Penaeus (Litopenaeus)
vannamei in India

Article in Aquaculture · January 2016

Impact Factor: 1.88 · DOI: 10.1016/j.aquaculture.2015.12.034

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Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus
(Litopenaeus) vannamei in India

K.V. Rajendran, Saloni Shivam, P. Ezhil Praveena, J. Joseph Sahaya Ra-

jan, T. Sathish Kumar, Satheesha Avunje, V. Jagadeesan, S.V.A.N.V. Prasad
Babu, Ashish Pande, A. Navaneeth Krishnan, S.V. Alavandi, K.K. Vijayan

PII: S0044-8486(15)30304-5
DOI: doi: 10.1016/j.aquaculture.2015.12.034
Reference: AQUA 631974

To appear in: Aquaculture

Received date: 17 November 2015

Revised date: 30 December 2015
Accepted date: 31 December 2015

Please cite this article as: Rajendran, K.V., Shivam, Saloni, Ezhil Praveena, P., Joseph
Sahaya Rajan, J., Sathish Kumar, T., Avunje, Satheesha, Jagadeesan, V., Prasad Babu,
S.V.A.N.V., Pande, Ashish, Navaneeth Krishnan, A., Alavandi, S.V., Vijayan, K.K.,
Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) van-
namei in India, Aquaculture (2016), doi: 10.1016/j.aquaculture.2015.12.034

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 ACCEPTED MANUSCRIPT

Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus)

vannamei in India

K.V. Rajendran1, Saloni Shivam2, P.Ezhil Praveena, J. Joseph Sahaya Rajan1, T. Sathish

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Kumar1, Satheesha Avunje1, V. Jagadeesan1, S.V.A.N.V. Prasad Babu1, Ashish Pande1, A.
Navaneeth Krishnan1, S.V. Alavandi1, K.K.Vijayan1

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1
ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome High Road, Raja

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Annamalai Puram, Chennai- 600028, India
2
ICAR-Central Marine Fisheries Research Institute, Post Box No. 1603, Ernakulam North

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P.O., Kochi-682 018, India
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*Corresponding author: ICAR-Central Institute of Brackishwater Aquaculture, #75 Santhome

High Road, Raja Annamalai Puram, Chennai- 600028, India; Tel: +91-044-24618817; Fax:
+91-044-24610311; E-mail address: rajendrankv@hotmail.com
 ACCEPTED MANUSCRIPT

ABSTRACT

Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, has emerged as a

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serious pathogen reported to be associated with retarded growth in cultured shrimp in many

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of the shrimp growing countries in Asia. As a part of ongoing disease surveillance among the

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farmed shrimp, we have investigated Penaeus (Litopenaeus) vannamei cultured in the south-

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east coast of India for EHP infection using light and scanning electron microscopy,

histopathology, polymerase chain reaction (PCR) and in situ hybridisation. Squash

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preparation of hepatopancreas and white faecal strings showed large number of

microsporidian spores. Spores under scanning electron microscope appeared oval and
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measured 1.7 x 1.0 µm. Histology of infected animals showed severe degeneration of

hepatopancreatic tubules. Basophilic inclusions resembling the developmental stages of EHP

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were found in the epithelial cells and large number of spore aggregations was observed in the

tubular lumen. Enlargement of haemal sinuses and encapsulation of hepatopancreatic tubules

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were also observed in some cases. DNA extracted from hepatopancreas was subjected to PCR
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amplification using primers targeting microsporidian ssu rRNA gene. The PCR yielded an
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expected product of ~951 bp and the sequences showed 100% identity with the EHP reported

from Vietnam, Thailand and China. Further screening of field samples were carried out using

nested PCR employing EHP-specific primers. Of the 137 juvenile P. vannmei samples tested,

10 were found to be positive in the first step and 77 in the nested PCR. Overall prevalence of

EHP was estimated to be 63.5%. However, only first step PCR-positive samples showed

discernible number of spores in the hepatopancreas under light microscope. Post larvae of P.

vannamei collected from a hatchery were found to be PCR negative for EHP. In situ

hybridisation using EHP-specific DIG-labelled probe showed positive signals in infected

hepatopancreatic tissue. Animals collected from white faeces syndrome (WFS)-affected pond
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showed higher prevalence of EHP (96.4%) compared to those from the unaffected pond

(39.7%). On the contrary, slow growing animals showed low prevalence (58.5%) compared

to normally growing animals (80.8%). Although EHP could be detected from slow-growing

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as well as WFS-affected animals, the present study could not conclusively elucidate the

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association of EHP with these clinical signs through experimental infection trials. This report

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forms the first record of the emergence of EHP infection in cultured SPF P. vannameiin

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India.

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Key words: Enterocytozoon hepatopenaei (EHP), Penaeus (Litopenaeus) vannamei,
Emerging disease, Shrimp disease, slow growth, White faeces syndrome (WFS)

Abbreviations: EHP, Enterocytozoon hepatopenaei; WFS, white faeces syndrome; PCR,

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polymerase chain reaction; ISH, in situ hybridization; HP, hepatopancreas; ATM, aggregated,
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transformed microvilli.
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1. Introduction

Shrimp aquaculture in India has witnessed remarkable growth, both in terms of area

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under culture and export production, especially during the past five years. This major

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transformation happened after the introduction of the non-native species, whiteleg shrimp,

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Penaeus (Litopenaeus) vannamei during 2009-10. According to the production statistics from

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Marine Products Export Development Agency, after the introduction of the species, export

production has grown from 1,731 metric tonne (mt) to 35, 3413.1mt in 2014-15. However,

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lately, as witnessed earlier in the case of native tiger shrimp, Penaeus monodon, diseases of
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infectious aetiology as well as morbidity associated with poor management have caused

serious concerns to P. vannamei culture. Production losses due to multiple disease conditions
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such as running mortality syndrome (RMS), retarded growth due to various causes, white
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faeces/white gut syndrome have frequently been reported in cultured P. vannamei.

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Recently, shrimp farms in Asia and other areas have been reporting heavy infection
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with a microsporidian parasite, Enterocytozoon hepatopenaei (EHP) in cultured P. vannamei

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impacting the production due to severe growth retardation (Newman, 2015). The parasite was

first recorded from growth retarded tiger shrimp, Penaeus monodon from Thailand and

reported as an undesignated microsporidian (Chayaburakul et al., 2004). Subsequently, the

parasite was identified and characterised from P. monodon from Thailand in 2009 (Tourtip et

al., 2009). Although the parasite was recovered from slow growing shrimp, the association

with the slow growth was not statistically correlated at that point in time. However, Ha et al.

(2010) reported that the microsporidian is one of the pathogenic agents associated with white

faeces disease in cultured P. monodon in Vietnam. Subsequently, though unpublished, Felgel

(2012) has indicated that white faeces syndrome (WFS) of P. vannamei found in Vietnam
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and Thailand was associated with severe infection with a microsporidian morphologically

similar to E. hepatopenaei. Further, Tangprasittipap et al. (2013) have reported widespread

infection of the microsporidian in cultivated P. vannamei exhibiting WFS in Thailand. They

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also revealed that the parasite can be transmitted directly through oral route. However,the

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study reported that EHP is not the cause of WFS in P. vannamei (Tangprasittipap et al. 2013).

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Though EHP has not been scientifically explained as the cause of mortality, field-level

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observations indicate that the infection is associated with severe growth retardation

(Sritunyalucksana et al., 2014). Moreover, they have reported that the outbreaks of EHP are

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prevalent in China, Indonesia, Malaysia, Vietnam and Thailand, and the parasite was detected
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by PCR in samples of slow growing shrimp collected from India.
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EHP infection does not cause any specific clinical signs. Diagnosis of infection can be
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carried out by microscopic examination of squash preparation of hepatopancreas and

identification of the spores. Other diagnostic methods reported include histopathology, in

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situ hybridisation, polymerase chain reaction (PCR), loop-mediated isothermal amplification

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(LAMP) and real-time PCR (Suebsing et al., 2013; Tangprasittipap et al., 2013; Tang et al.,
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2015; Liu et al., 2015).

Recently, occurrence of retarded growth and white faeces syndrome in cultured P.

vannamei has widely been observed in India. The present study was undertaken to examine

the role of EHP in P. vannamei, considering the involvement of these microsporidia in WFS

and growth retardation and the widespread occurrence of these syndromes in farmed shrimp

in India. Samples of P. vannamei from shrimp farms located in the south-east coast of India

were screened for EHP infection using light and electron microscopy, histopathology, PCR
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and in situ hybridisation. Post larvae of P. vannamei collected from a hatchery were also

tested for EHP using PCR.

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2. Materials and Methods

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2.1. Sample collection

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Juveniles and sub-adults of Penaeus vannamei at 84-91 days of culture (DoC) with

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average body weight of 11.0 g (2.5-28.5 g) were collected from 3 shrimp farms located in
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Andhra Pradesh and Tamil Nadu. These farms were experiencing size variation/growth

retardation and white faeces syndrome in shrimp at this stage of farming. The animals were
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collected live and transported to the laboratory on dry ice. Water samples and white, floating
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faecal strings from the same ponds were also collectedand transported on ice. P. vannamei

post-larvae (PL-7) were collected from a hatchery near Chennai.

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2.2. Wet mount preparation and light microscopy

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Animals were dissected out with sterile tools. A small portion of hepatopancreas (HP)

was used to make tissue smear/impression on clean glass slides. Another portion of the HP

was taken on a clean glass slide and used for making squash preparation by applying a cover

glass with slight pressure. The squash preparations were observed directly under a phase

contrast microscope (Nikon, Japan) for the presence of spores. The smear preparations were

allowed to dry at room temperature, fixed in methanol for 15 min and stained with Giemsa as

described by Vavra and Maddox (1976). Another set of dried smear preparations were fixed

in 5 % neutral buffered formalin for 15 minutes and stained with hematoxylin and eosin as
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described by Humason (1972). The stained smears were dehydrated, mounted in DPX

following standard procedure and observed under a microscope. The same procedure was

used in studying spores from faecal matter. Purified spores were also observed under

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microscope using agar-layer method as outlined for myxozoans by Lom and Dykova (1992).

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Briefly, 1.5% agar solution was spread onto a clean glass slide to form a 1.0-1.5 mm thick

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uniform layer. The slide was allowed to set on a horizontal surface. A drop of spore

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suspension was spread on a clean cover glass and the cover glass was inverted and carefully

placed over the agar layer and observed under a phase contrast microscope.

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2.3. Histology
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Live animals were kept on ice for a brief period before dissection. This was followed
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by quick injection of Davidson’s AFA fixative into the head portion of the shrimp. The soft

head tissues were dissected out and further kept in the fixative for 48 h and processed using
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routine histological techniques (Bell and Lightner 1988). Briefly, the tissues were dehydrated
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in graded alcohol series (70%, 90%, and 100%) for 60 min each. After dehydration, tissues
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were treated with xylene for 60 min (twice) and paraffin blocks were made using tissue

embedding system (Leica Microsystems, Germany). Tissue sections (4-5 µm) were taken and

further processed and stained with hematoxylin and counter-stained with eosin using standard

procedure. The stained tissue sections were mountedin DPX and observed under a

microscope.

2.4 Purification of microsporidian spores from faecal matter and hepatopancreas

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Purification of microsporidian spores from faecal matter was performed as described

by Bukhari and Smith (1995) with minor modifications. The white faecal strings collected

from the farms were homogenized in distilled water. Then, 500 µL of diethyl ether was added

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to the homogenate and the samples were vortexed for 20 s and centrifuged at 5000 rpm for 3

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minutes (Eppendorf 5810 R, Germany). Both the fat layer and the supernatant were

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discarded, and the pellet was resuspended in 500 µL of distilled water and centrifuged at

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5000 rpm for 3 minutes. This washing procedure was repeated twice, and the pellet was

resuspended in distilled water. Similarly, frozen hepatopancreatic tissues were homogenized

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in a microcentrifuge tube and coarse tissue debris were removed by centrifugation. The spore
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pellets were washed in phosphate buffered saline by repeated centrifugation. The spore

preparations were stored at -80oC until further use.

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2.4. Scanning electron microscopy

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Semi purified spores from hepatopancreas and faecal matter were subjected to scanning
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electron microscopy. The scanning was carried out using HR-SEM (FEI Quanta FEG 200,
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USA) at the Sophisticated Analytical Instrumentation Facility (SAIF), Indian Institute of

Technology, Chennai.

2.5. DNA extraction

The genomic DNA was extracted from hepatopancreas and faecal matter of shrimp. In

the case of larval stages, pooled whole larvae were used and DNA from all these samples was

extracted using standard procedure. Briefly, samples of hepatopancreas and faecal matter

(approximately, 25 mg) and pooled larval stages (10 larvae in one pool) were homogenised
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and digested for 10 min at 95oC in 500 µL of lysis buffer (50 mMTris, 1 m Methylene

diamine tetra-acetic acid (EDTA), 500 m MNaCl, 1% SDS) and 0.1 mg proteinase K. The

mixture was centrifuged at 12000 rpm (Eppendorf 5810 R, Germany) for 10 min at 4 oC.

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After centrifugation, supernatant was collected carefully and two volumes of 100% ethanol

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were added and kept at −20 oC for 1 h. The mixture was centrifuged at 12000 rpm for 10 min

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at 4 oC, and the DNA pellet obtained was washed with 70% cold ethanol, air-dried and re-

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suspended in nuclease-free water.

2.6. PCR analysis

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For amplifying the small subunit ribosomal RNA (ssu rRNA) gene fragment, PCR
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amplification was performed using primers targeting microsporidian ssu rRNA gene (MF1-
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5’- CCGGAGAGGGAGCCTGAGA -3’and MR1-5’- GACGGGCGGTGTGTACAAA -3’)

as reported by Tourtip et al. (2009). The PCR amplification was carried out in a 25 μL
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reaction mixture containing Ampliqon IIII Taq DNA Polymerase 2x mastermix RED
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(Ampliqon A/S, Denmark) (Tris-HCl pH 8.5, (NH4)2So4, 3 mM MgCl2, 0.2% Tween 20®,
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0.4 mM dNTPs, 0.2 units/µL AmpliqonTaq DNA polymerase, Inert red dye and stabilizer),

1.0 µL (10 pmol) of forward and reverse primer each, 1.0 µL (100 ng) of template DNA. The

PCR amplification followed 35 cycles of denaturation at 94°C for 20 sec, annealing at 64°C

for 20 sec, and extension at 72°C for 45 sec, followed by a 5 min final extension at 72°C.

For the detectionof EHP infection in shrimp, PCR was performed with two sets of

EHP-specific primers (ENF779-5’-CAGCAGGCGCGAAAATTGTCCA-3’ and ENR779-5’-

AAGAGATATTGTATTGCGCTTGCTG-3’;ENF176-5’-CAACGCGGGAAAACTTACCA-

3’and ENR176-5’-ACCTGTTATTGCCTTCTCCCTCC-3’) in a nested PCR format as

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reported by Tangprasittipap et al. (2013). The PCR amplification was carried out in 25 μL

reaction mixture as described earlier except for the inclusion of EHP-specific primers. The

cycling condition consisted of initial denaturation at 94°C for 3 min followed by 35 cycles of

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denaturation at 94°C for 20 sec, annealing at 58°C for 20 sec and extension at 72°C for 45 sec

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with a final extension at 72°C for 5 min. The second step nested PCR reaction contained the

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same constituents as that of the first step PCR except for the nested primers and 1.0 μL of the

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first step product as template. The PCR reaction conditions consisted of initial denaturation at

94°C for 3 min followed by 35 cycles of denaturation at 94°C for 20 sec, annealing at 64°C

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for 20 sec and extension at 72°C for 20 sec with an additional extension at 72°C for 5 min.
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The PCR was carried out on a thermal cycler (Eppendorf, USA). An aliquot of PCR product

was resolved on 2.0 % agarose-Tris-acetate-EDTA (TAE) gels containing 0.5 µg/mL

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ethidium bromide alongside a 1 kb DNA ladder (SRL Pvt. Ltd., India)) and the amplified
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DNA was visualised under UV illumination using a gel documentation system (Bio-rad

Laboratories, USA).
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2.7. Cloning and sequencing

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The amplified PCR product was purified using GenElute TM gel extraction kit

(Sigma, Germany) according to the manufacturer’s instructions. The purified PCR product

was cloned in pTZ57R/t vector (Thermo Fisher Scientific, USA) and transformed into E. coli

DH5α competent cells. The recombinant clones were identified as the white colony on an

Xgal and IPTG plate. The plasmid DNA was purified using GenEluteTM Plasmid Miniprep

Kit (Sigma, Germany). Sequencing of plasmid DNA as well as PCR products was carried out

at First Base Laboratories, Malaysia. Sequence analysis was carried out using BLASTN and

Clustal Omega.
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2.8. In situ hybridisation (ISH)

The first step PCR product (778 bp) was purified using GenEluteTM gel extraction kit

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(Sigma, Germany) before labelling with digoxigenin. Gene probe for EHP was prepared by

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labelling the purified product labelled with digoxigenin-11-dUTP using DIG DNA labelling

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and detection Kit (Roche, Germany). Briefly, 15 µL of purified first step PCR product (1.0

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µg) was denatured for 10 min. The denatured DNA product was chilled on ice for 2 min. To

the denatured DNA, 2.0 µL of hexanucleotide mix (10x), 2.0 µL of dNTP labelling mix and

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2.0 µL of klenow enzyme were added, and gently mixed and briefly centrifuged. The reaction
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mixture was incubated at 37°C overnight and the reaction was stopped by heating at 65°C for

10 min. The labelling efficiency of the DIG-labelled EHP probe was analysed by comparing
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the intensity of colour development of the experimental probe with the control labelled DNA
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provided in the kit.

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For in situ hybridisation, the Davidson’s AFA-fixed tissues were embedded in

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paraffin and tissue sections were prepared as per the standard procedure. The deparaffinised
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and rehydrated tissue sections were treated with 100 µg/mL proteinase K followed by post-

fixation in pre-cooled 0.4% formaldehyde for 5 min at 4°C. The slides were incubated in 500

µL of 1 x pre-hybridization buffer (Merck, India) containing DIG labelled EHP probe (100

ng/mL). The slides were heated at 95°C for 6 min and immediately kept on ice for 5 min

followed by hybridisation at 42°C overnight. The final detection was carried out using anti-

DIG alkaline phosphatase (AP)-conjugated antibody (Roche, Germany) and visualised by

treating with 500 µL of colour substrate nitroblue tetrazolium and 5-bromo-4-chloro-3-

indolyl phosphate (NBT/BCIP) solution. The tissue sections were counter stained with 0.5%
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aqueous Bismarck Brown Y (Sigma, Germany), mounted in DPX and observed under a

microscope.

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3. Results

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3.1. Light and scanning electron microscopy

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Juveniles and sub-adults of Penaeus vannamei at 84-91 days of culture were

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randomly collected from farms, and the samples included slow growing as well as normally
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growing animals (Fig. 1A). These animals did not show any other external abnormalities.

Samples were also collected from pond which showed animals with white/empty gut and
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obvious floating white faecal strings in water (Fig. 1B, C). Smear/squash preparation of
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hepatopancreas (HP) of infected shrimp showed large number of microsporidian spores. The

spores appeared oval and measured 1.2 x 0.8 µm in hepatopancreatic smear stained with
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Giemsa and haematoxylin-eosin (Fig. 2A, B). Faecal material including floating white faecal
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strings collected from shrimp farms also showed heavy infection with the spores. The spores
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isolated from faecal matter showed similar morphology and morphometry as that observed in

the HP. The isolated spores were first observed under a light microscope using agar method

(Fig.2C). Smear preparations of the semi purified spore suspension were further stained with

haematoxylin and eosin (Fig.2D). Spores isolated from infected hepatopancreas as well as

faecal matter were further observed under a scanning electron microscope and the spores

measured 1.7x 1.0 µm. Under electron microscope, spores appeared oval with smooth surface

showing dent-like curvature on the spore wall and ridge-like elevations (Fig. 3A, B).

3.2. Histology and in situ hybridisation

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Histologically, severe necrotic changes were noticed in the hepatopancreas. Large

eosinophilic to basophilic inclusions indicating presumptive developmental stages of the

microsporidian could be noticed in the tubular epithelium. These stages were predominantly

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seen in the distal ends of hepatopancreatic tubules and most of the tubular epithelium in this

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region showed detachment from the basal membrane (Fig. 4A). The basal part of the tubular

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epithelium showed granular material and spore-like structures (Fig. 4B). Abnormally

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enlarged haemal sinuses were also noticed in the inter-tubular spaces (Fig. 4C). In some of

the sections, the spores were noticed in vacuolated structures (Fig. 4D). Sloughing of the

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tubular epithelial cells was pronounced in heavily infected HP and large spore aggregations
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were noticed in the tubular lumen (Fig. 4E). Host response in the form of encapsulation of

the tubule along with enlarged haemal sinuses were noticed in heavily infected HP. In some
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of the HP sections, large number of rod-shaped bacterial cells was also noticed in the tubular
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lumen indicating secondary infection. Positive ISH signals could be detected in the infected

hepatopancreatic tubular epithelium (Fig. 4F). Heavily infected HP showed ISH positive
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spore aggregations in the degenerated tubular epithelium as well as in the lumen, confirming
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the EHP infection.

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3.3. PCR analysis

Firstly, DNA isolated from hepatopancreas and faecal matter was subjected to PCR

amplification using primers designed from ssu rRNA of microsporidians. Some of the heavily

infected HP samples showed discernible amplification with the primer pair. The PCR

amplification yielded a product size of ~951bp (Fig.5A). The PCR products were sequenced

and 920 nt sequence was obtained.

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Subsequently, for screening the shrimp samples the nested PCR specific for EHP was

employed. In the first step PCR, of the 137 juvenile P. vannamei samples collected from 3

farms (2 from Andhra Pradesh and 1 from Tamil Nadu),10 animals were found to be positive

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showing a PCR product of 778 bp (Fig. 5B). All these samples were also found to be heavily

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infected, as evidenced by the observation of spores in HP smear preparation under light

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microscope. A total of 77 samples, excluding the samples tested positive in the first step

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PCR, showed positive amplification in the nested PCR with a very prominent band of 176 bp

(Table 1; Fig.5C). The samples which were tested positive in the first step were not

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subjected to nested PCR. The estimated prevalence was found to be 63.5% in the tested
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samples (Table 1). Interestingly, 10 pools (10 animals in one pool) of post larvae of P.

vannamei tested were found to be nested PCR negative for EHP (Table 1).
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To find out the prevalence of EHP infection, shrimp samples were collected from a

farm which experienced WFS and other one without the syndrome. The shrimp thus collected
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were classified into slow growing and normal animals based on body weight. PCR analysis
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showed that of the 30 normally growing animals collected from ponds without WFS, 18
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samples were found to be nested PCR positive for EHP (60% prevalence). However, only 5

out of 28 animals (17.9%) were tested positive in the slow growing animals. Similarly, 43

normally growing animals collected from WFS-affected pond showed 41 EHP-positive

animals (95.3%), whereas all the 13 slow growing animals collected form the same pond

were found to be PCR positive for EHP (Table 2). The overall prevalence of EHP infection

in WFS-affected pond was found to be higher (96.4%) than that detected in animals without

WFS (39.7%). However, 59 of 73 normally growing animals (80.8%) were found to be EHP

positive, whereas only 24 of 41 slow growing animals (58.5%) showed EHP infection in

nested PCR (Table 3).

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3.4. Sequence analysis

After analysing the sequences (920 nt) generated by primers specific to ssu rRNA for

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its quality, the primer sequences were omitted and the remaining 896 nt sequence was

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subjected to BLATN search. The search revealed 100 hits in the database and all were found

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to be related to microsporidian sequence records showing 86-100% identities. The EHP

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sequence generated form P. vanammei from India showed 100% identity with that of 18S

rRNA gene of EHP sequences reported from P. vanammei from Vietnam (GenBank

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KP759285) and Thailand (GenBank KF362130). EHP reported from China (GenBank
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KP027539) also showed complete identity, however, the reported sequence was only 416 nt

long. Two more EHP 18S rRNA sequences reported from P. monodon (GenBank KF362129
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and FJ496356) showed 99 and 96% identities, respectively. Other microsporidians which
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showed high similarity (89-93%) with the present EHP sequence were Enterospora

nucelophila, Nucleospora salmonis and Enterocytozoon salmonis, all infecting fish.

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Nucleotide sequence of EHP generated in the present study was aligned with the EHP
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sequences reported from P. vannamei and P. monodon using Clustal Omega and was
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depicted in Fig. 6.

4. Discussion

Enetrocytozoon hepatopenaei (EHP), a microsporidian parasite, initially reported from

tiger shrimp (Penaeus monodon) and subsequently from whiteleg shrimp (Penaeus

vannamei), has become one of the most serious emerging pathogens in all the shrimp

growing countries in Asia.In the present investigation, EHP) could be identified and

characterised for the first time from farm-reared P. vannamei from India. Microsporidians are
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unicellular eukaryotes that are obligate intracellular parasites infecting wide range of

eukaryotic hosts, from protists to humans (Corradi, 2015). Several species of microsporidians

have been reported from penaeid shrimp (Lightner, 1996). In the present study, typical EHP

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spores could be identified in the smear preparation of hepatopancreatic tissues through light

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microscopy. However, only heavily infected HP showed the presence of spores in the smear

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preparation under the microscope. This indicates that light infection of EHP can go

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undetected if only microscopy is used for the detection. The morphology and morphometry of

the spores showed close similarity with the spores of EHP reported from P. monodon

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(Tourtip et al., 2009) and P. vannamei (Tangprasittipap et al., 2013). However, spores
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observed in the present study were found to be larger than the previously reported EHP and

the negligible variation in spore size could be due to the difference in the processing of the
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spores for microscopy.

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Histologically, heavily infected HP showed severe necrotic changes as evidenced by

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sloughing of tubular epithelial cells, degenerated cells and spore accumulation in the tubular
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lumen. Histological features observed in the present study are in accordance with the reports
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on the hepatopancreatic microsporidian infection in P. monodon and P. vannamei

(Chayaburakul et al., 2004; Tourtip et al., 2009; Tangprasittipap et al., 2013). Further, the

parasite’s affinity for the epithelial cells has been reported for the genus Enterocytozoon,

including the human-infecting species E. Bieneusi (Desportes et al., 1985). Some of the

histological changes observed in the infected HP of P. vannamei due to EHP also showed

similarity with Enterospora canceri reported from crab (Stentiford et al., 2007).

PCR screening of P. vannamei from 3 farms located in the south-east coast of India

showed relatively high prevalence of EHP infection (63.5%), as revealed by the nested PCR
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result. However, light microscopic observation and screening with first step PCR revealed

only a very low prevalence (7.3%) in P. vannamei. This suggests that the EHP can remain as

an undetectable chronic infection in shrimp. A significant observation made in the study is

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that the post-larval samplesof P. vannamei collected from a hatchery were tested negative for

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EHP in nested PCR. Tangprasittipap et al., 2013 and Thitamadee et al., 2016 have also

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recorded similar observation in which they reported that the post larvae originated from

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SPFstocks of P. vannamei were found to be negative for EHP. Though an extensive EHP

screening survey involving many hatcheries only would give a better picture about the

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presence of EHP in post larvae, this preliminary screening result and similar reports from
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other countries such as Thailand (Tangprasittipap et al., 2013) indicate that EHP could be a

farm-associated infection where the shrimp contract infection from the contaminated
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environment. Further, based on the earlier reports on similar hepatopnacreatic microsporidian

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in P. monodon from Malaysia (Anderson et al., 1989) and P. japonicus from Australia

(Hudson et al., 2001), it has been suggested that EHP is not exotic but an endemic parasite of
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Australasian region (Thitamadee et al., 2016). However, in India, it is yet to be ascertained

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that whether indigenous P. monodon had the EHP infection which might have been
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overlooked because of the focus on other pathogens. As the microsporidian spores are

expelled out through faecal matter, pond bottom could act as an ideal source of infection and

the spores could persist over a long period of time. It has been reported that spores of some of

the microsporidians can retain infectivity from 2-12 weeks in 30‰ artificial seawater

depending upon the temperature (Fayer, 2004). Further, as stated earlier, absence of any

obvious clinical signs of infection and difficulty in identifying the pathogen through visual

examination increase the risk of disease dissemination. In this scenario, it could be predicted

that EHP might remain as a serious impediment to shrimp culture in the coming years, unless

strict monitoring and management measures are implemented to control the dissemination of
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the infection. Further, recent reports from major shrimp farming regions of the country such

as Andhra Pradesh, where farmers are not adhering to the suggested better management

practices (BMP) such as pond preparation with crop intervals, indicate that the current culture

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practices could aggravate the EHP infection. Similar observations have been recorded from

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other shrimp growing countries such as Thailand where poor biosecurity measures resulted in

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the spread of EHP infection (Thitamadee et al., 2016).

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Light microscopic observation on white faecal matter revealed that the samples

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collected from different farms contained large number of microsporidian spores. Association
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of microsporidian with white faeces syndrome (WFS) had been reported previously (Ha et al.

2010) and, Felgel (2012) has indicated that severe infection with a microsporidian
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morphologically similar to E. hepatopenaei was associated with WFS of P. vannamei. On the

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contrary, later, Tangprasittipap et al., 2013 reported that EHP is not the cause of WFS and,

further, according to Sriurairatana et al. (2014) the syndrome arises due to transformation,
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sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies.

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Interestingly, recently, Vibrio bacteria and six species of fungi have been isolated from
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shrimp naturally infected with white faeces syndrome (Chaweepack et al., 2015). However,

none of these studies have recorded the presence of large number of EHP spores in the white

faecal matter, and this is in contrast to the present observation. Although the cause of the

aggregated, transformed microvilli (ATM) associated with the WFS is yet to be identified, it

has been reported that increase in the prevalence of ATM coincided with the increased

prevalence of acute hepatopancreatic necrosis disease (AHPND) and EHP (Sriurairatana et

al., 2014). As per the available information, investigation on WFS in India revealed no

association with Vibrio or fungi and the major pathogen recovered from the shrimp farms has

been only EHP. Therefore, it is reasonable to speculate that white faeces could be due to
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major hepatopancreatic dysfunction and the pathology might be due to either a single

pathogen or a combination of biotic and abiotic factors. However, the available data is not

sufficient enough to attribute EHP as the causative agent of retarded growth and production

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of white faeces. Though it is stated by Tangprasittipap et al.(2013) that severity of EHP

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infection might get exacerbated by the underlying causes of WFS, there is reason to believe

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that EHP infection might aggravate the WFS in shrimp.

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A significant observation made in the present study is that a high prevalence of EHP

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(96.4%) has been detected in the animals collected from pond which experienced WFS,
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compared to 39.7% prevalence observed in the animals collected from ponds without WFS.

When the PCR results were analysed by classifying the animals according to the body weight
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into normal and slow growing groups, it has been found that slow growing animals had low
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prevalence (58.5%) of EHP compared to normal animals which showed a prevalence of

80.8%. On the contrary, first step PCR revealed that 14.6% of the slow growing animals were
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EHP positive while EHP could be detected only in 5.5% of the normally growing animals.
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The present investigation suggests that a statistically well-designed study is required

to find out the correlation between the presence of EHP (PCR positivity) and farm-level

outcome of the crop, especially the slow growth and occurrence of white faeces. Also,

studies need to be undertaken on disease transmission, infectivity profile, potential role of

other micro and macro crustaceans as reservoirs of infection. Study on horizontal and

vertical transmission, physiological dysfunction due to hepatopancreatic pathology, and the

threshold of parasitic load required for shrimp mortality are also required to unravel the role

of the emerging EHP parasite in the production loss in farmed shrimp.The present study
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forms the first record of EHP in cultured shrimp from India and the first attempt to correlate

EHP, slow growth and WFS.

Acknowledgements

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This investigation was carried out under the National Surveillance Programme for

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Aquatic Animal Diseases (NSPAAD), coordinated by the National Bureau of Fish Genetic

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Resources (NBFGR), Lucknow. The authors acknowledge the Indian Council of Agricultural

Research (ICAR) and National Fisheries Development Board (NFDB), Govt. of India, for the

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financial support to carry out this work. Authors also thank the shrimp farmers of Andhra
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Pradesh and Tamil Nadu states forprovidingsamples for this study.
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Fig. 1. Farm-level observations made during the collection of samples of Penaeus vannamei.

A. Retarded growth observed during ~90 days of culture; B. White/empty gut (arrow)

and discolouration of hepatopancreas; C. Floating white faeces in the pond water.

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Fig. 2. Light microscopic observations on the spores of Enterocytozoon hepatopenaei

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recovered from P. vannamei. A. Smear preparation of infected hepatopancreas stained

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with Giemsa showing EHP spores. B. Smear preparation stained with haematoxylin-
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eosin (arrows indicate spores); C. EHP spores purified from white faecal matter observed

on agar layer; D. Purified spores stained with haematoxylin-eosin.

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Fig. 3. Scanning electron microscopic observation of spores of Enterocytozoon hepatopenaei

A. Low magnification; B. High magnification. Note the smooth surface of the spore wall

with dent-like curvature.

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Fig. 4.Histological sections of hepatopancreas of P. vannamei infected with Enterocytozoon hepatopenaei. A. Tubule epithelium showing

presumptive developmental stages of EHP. Note the basophilic inclusions and granular bodies (arrows; H&E, x 400) B. Tubular

epithelium showing detachment from the basement membrane. Note the spore-like structures between the epithelium and basement

membrane (H&E, x 400); C. Completely detached tubule and enlarged haemal sinus (star mark; H&E,x 400); D. Spore-like bodies in

the vesicles (arrows; H&E, x 400); E. EHP spores in the highly degenerated hepatopancreatic tubule lumen (H&E, x 1000); F. In situ

hybridisation reaction with the EHP infected hepatopancreatic tissues showing signals in the epithelial cells (x 400).
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Fig. 5. Agarose gel electrophoresis of the PCR products amplified from the hepatopancreatic
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DNA of P. vannamei. A. PCR amplification product (~951 bp) obtained using primers

targeting ssu rRNA of microsporidians B. First step PCR amplification using

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Eneterocytozoon hepatopenaei-specific primers. Note- two samples (lane 5 & 6) showed

detectable amplification; C. Nested PCR amplification of the first step product. Note-
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one of the samples (lane 1) did not show any amplification. M- Molecular weight

marker; Lane numbers indicate field samples tested for EHP, +ve- Positive control; –ve-

Negative control.
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Fig. 6. Clustal Omega alignment of microsporidian small subunit rRNA sequences. The sequences generated from the Enterocytozoon

hepatopenaei infecting P. vannamei (EHP-India) was compared with the EHP sequences reported from P. vannamei from Vietnam

(GenBank KP759285) and Thailand (GenBank KF362130) showing 100% identity and EHP from P. monodon (EHP-Mon-GenBank

362129) showing 99% identity. Nucleotide variations are marked in grey.

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Table 1.
Details of the samples tested and estimated prevalence of EHP using nested PCR.

Number PCR result

S. Species Sample of Prevalence
No samples Positive Negative (%)

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. tested 1st step Nested Total

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1 Penaeus vannamei Juvenile 137* 10 77 87 50 63.5

Post 100 (10 0 0 0 100 0

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larvae pools)

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* This includes 23 animals for which body weight was not recorded during collection and in
that 10 animals were found to be positive for EHP.

Table 2. NU
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Comparative prevalence of EHP in Penaeus vannamei collected from ponds with and without
WFS.

Nature of Nature Weight Days of Number First Nested Total Total Prevalence
culture
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pond of (g) of step positive negative (%)

animals (DoC) animals
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Without Normal 9.0-28.5 84 30 3 18 18 12 60.0

WFS (15.9)
Slow 2.5-8.5 84 28 4 5 5 23 17.9
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growth (5.7)
Total prevalence 39.7
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With WFS Normal 9.5-26.5 91 43 1 41 41 2 95.3

(15.2)
Slow 5.0-8.5 91 13 2 13 13 0 100.0
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growth (7.2)
Total prevalence 96.4

Table 3.
Comparative prevalence of EHP in normal and slow growing shrimp, Penaeus vannamei.

Nature of Weight Number First Nested Total Total Prevalence

animals (g) of step positive negative (%)
animals
Normal 9.0-28.5 73 4 59 59 14 80.8
(15.6)
Slow growth 2.5-8.5 41 6 18 24 17 58.5
(6.5)
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Highlights

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Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, was identified and

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characterised from farmed Penaeus vannamei from India for the first time.
 The parasite was isolated from animals showing retarded growth and white faecal

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syndrome (WSF).

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 Severe necrosis of the hepatopnacreatic tissue was the major pathology.
 Overall prevalence of EHP estimated by nested PCR analysis was 63.5%.

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 High prevalence (96.4%) of infection detected in shrimp affected with WFS.
 ssu rRNA gene sequence showed 100% identity with the EHP reported from Vietnam,
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Thailand and China.
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