BackgroundThe microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS.ResultsGeneric primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS.ConclusionsThe microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

Vibrio harveyi infection was found to occur in pond‐reared black tiger prawn in Thailand. The diseased prawns with a hepatopancreatic infection of V. harveyi showed bacterial invasions and multiplication in the tubular lumens. This condition was followed by necrosis of hepatopancreatic cells and the thickened basal lamina, subsequent granulomatous encapsulation of the invaded tubules, and production of granulation tissue around granulomatous lesions. Heavy bacterial multiplication in the hepatopancreatic tubules caused systemic bacterial dissemination, which resulted in marked necrosis in the heart and lymphoid organ. On the other hand, two prawns with percuticular infection by V. harveyi had bacterial invasions in the subcuticular, spongy connective tissue in the telson, and systemic dissemination was followed by the occurrence of bacteriaphagocytizing hemocytes in the various tissues. Both types of isolates (chitinase positive and negative) were moderately pathogenic to prawn; intramuscular injection of 105‐106 colony‐forming units per prawn resulted in mortalities of 53–100%, and the chitinase‐negative isolate was more virulent than the chitinase‐positive isolate. The injected prawns exhibited systemic infection (as in prawns with a natural percuticular infection) in the moribund condition and granulomatous encapsulation in the injected sites on days 1–7 postinjection.

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White tail disease (WTD) of the freshwater prawn Macrobrachium rosenbergii has recently been the cause of high mortalities in Thai prawn farms. The causative agents of this disease in other countries are M. rosenbergii nodavirus (Mr NV) and extra small virus (XSV), which are usually detected using reverse transcriptase-polymerase chain reaction (RT-PCR) protocols. Using RT-PCR, most Thai post-larvae (PL) samples showing gross signs of WTD tested positive for Mr NV but only a few were positive for XSV. In contrast, all tested brooder samples were positive for both Mr NV and XSV. The possibility that brooders infected with Mr NV and XSV could transmit the viruses to larvae and PL should be examined. Cloning, sequencing and comparison of deduced amino acid sequences of RT-PCR amplicons of WTD samples from Thailand with those of Mr NV and XSV previously reported from the French West Indies and China revealed that the Mr NV were closely related but not identical while those from XSV were identical. This is the first report of Mr NV and XSV from Thailand. KEY WORDS: White tail disease · Macrobrachium rosenbergii nodavirus · Extra small virus · RT-PCR detection · Brooder Resale or republication not permitted without written consent of the publisherDis Aquat Org 69: [255][256][257][258] 2006 Recently, XSV and Mr NV have been purified .Detection methods for Mr NV include a double antibody sandwich enzyme-linked immunosorbent assay (DS-ELISA) (Romestand & Bonami 2003) and viral genome-based detection methods such as dot blot hybridization, in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR) amplification (Sri Widada et al. 2003). Similar genome-based detection methods are also available for XSV (Sri Widada et al. 2003. More recently a single-tube, duplex RT-PCR method has been developed for simultaneous detection of Mr NV and XSV (Yoganandhan et al. 2005).In the present study, farmed Macrobrachium rosenbergii showing gross signs of WTD and grossly normal brooders were tested for the presence of Mr NV and XSV by RT-PCR (Sahul Hameed et al. 2004a), and selected amplicons were sequenced and compared to those previously reported for Mr NV and XSV from other countries. MATERIALS AND METHODS PL and brooders.Infected PL with prominent signs of whitish muscle in the abdominal region were collected from different locations in Thailand (Table 1). In addition, 3 samples of grossly normal, pond-reared brooders were collected from culture ponds in Rachaburi, Thailand. These samples were transported to the laboratory on dry ice and stored at -20°C.Total RNA extraction. Whole PL (150 mg), hemolymph (150 µl) or tissue fragments (150 mg) from abdominal muscle tissue, tail muscle or pleopods were homogenized in 300 µl of TN buffer (20 mM Tris-HCl, 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 12 000 × g for 15 min at room temperature (27 to 30°C). The supernatant (150 µl) was extracted using 1 ml of TRIzol reagent (GIBCO-BRL) according to the manufacturer's protocol. RNA was precipitated...

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A survey of cultivated giant freshwater prawns Macrobrachium rosenbergii from Thailand revealed the presence of unusual spherical to ovoid inclusions in nuclei of hepatopancreas tubule epithelial cells. These began as small eosinophilic inclusions that became more basophilic as they increased in size. They were present in both R-cells and E-cells but were largest and deeply basophilic only in the E-cells. Confocal laser microscopy revealed that stained nucleic acid fluorescence from the inclusions was lost by treatment with DNase I specific for double-and single-stranded DNA and also lost or reduced by treatment with mungbean nuclease specific for single-stranded nucleic acids. Transmission electron microscopy (TEM) revealed that the inclusions contained tightly packed, unenveloped, viral-like particles of approximately 25 to 30 nm diameter, resembling those produced by shrimp parvoviruses. However, PCR, in situ hybridization and immunohistochemical tests for shrimp parvoviruses previously reported from Thailand were all negative. These results suggested that the inclusions contained a parvo-like virus, not previously reported from M. rosenbergii in Thailand. KEY WORDS: Macrobrachium rosenbergii · Hepatopancreas · Parvo-like virus · Histopathology · Electron microscopy Resale or republication not permitted without written consent of the publisherDis Aquat Org 85: [167][168][169][170][171][172][173] 2009 natural infections of M. rosenbergii is similar to the one known as hepatopancreatic parvovirus (HPV) in penaeid shrimp (Anderson et al. 1990, Lightner et al. 1994) based on histopathology (Bonami et al. 1995, Lightner 1996. A recent proposal to the International Committee on Taxonomy of Viruses (Tijssen 2008) recommends that these penaeid shrimp parvoviruses be included in a new genus, Hepanvirus, in the sub-family Densovirinae of the family Parvoviridae as Penaeus monodon densovirus (PmDNV), Penaeus merguiensis densovirus (PmergDNV) and Penaeus chinensis densovirus (PchinDNV). These differ in genome size and organization and target tissue from another penaeid shrimp parvovirus also included in the sub-family Densovirinae, but in the genus Brevidensovirus, as Penaeus stylirostris densovirus (PstDNV) (Tattersall et al. 2005). PstDNV was formerly called infectious hypodermal and hematopoeitic necrosis virus (IHHNV) (Lightner 1996). The newly recommended names (Tattersall et al. 2005, Tijssen 2008) will be used in this manuscript, as will the ICTV method of naming shrimp viruses with non-italicized host species names and abbreviations (Fauquet et al. 2005).For successful specific pathogen free (SPF) stock development, diagnostic methods would be required for all of the pathogens included in any specific list of pathogens for M. rosenbergii. For this species, highly sensitive RT-PCR methods are available for MrNV and XSV (Romestand & Bonami 2003, Sri Widada et al. 2003, Yoganandhan et al. 2005, Pillai et al. 2006. However, for other viral pathogens, such as the parvo-like virus described above, no molec...