The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: The 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. RESULTS: Generic 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. CONCLUSIONS: The 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.

Transmission of the highly pathogenic avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand.

This present study is the first to quantify the transmission of avian influenza virus H5N1 within flocks during the 2004 epidemic in Thailand. It uses the flock-level mortality data to estimate the transmission-rate parameter ( beta ) and the basic reproduction number (R(0)). The point estimates of beta varied from 2.26/day (95% confidence interval [CI], 2.01-2.55) for a 1-day infectious period to 0.66/day (95% CI, 0.50-0.87) for a 4-day infectious period, whereas the accompanying R(0) varied from 2.26 (95% CI, 2.01-2.55) to 2.64 (95% CI, 2.02-3.47). Although the point estimates of beta of backyard chickens and fighting cocks raised together were lower than those of laying hens and broiler chickens, this difference was not statistically significant. These results will enable us to assess the control measures in simulation studies. They also indicate that, for the elimination of the virus, a critical proportion of the susceptible poultry population in a flock (i.e., 80% of the population) needs to be vaccinated.

Comparison of four Taura syndrome virus (TSV) isolates in oral challenge studies with Litopenaeus vannamei unselected or selected for resistance to TSV.

Taura syndrome virus (TSV) infection in TSV-resistant (TSR) and TSV-susceptible (Kona) Litopenaeus vannamei (also called Penaeus vannamei) was investigated using histology, in situ hybridization (ISH), conventional reverse transcription polymerase chain reaction (RT-PCR) assays, and SYBR-Green real-time RT-PCR analysis. The shrimp were challenged by feeding with minced tissues of L. vannamei infected with 4 genotypic variants of TSV (Bz01, Th04, UsHi94, and Ve05). Survival probabilities of TSR shrimp were higher than those for Kona shrimp with all 4 variants. Th04, UsHi94, and Ve05 gave no Taura syndrome lesions with TSR shrimp. In contrast, TSR shrimp challenged with Bz01 and Kona shrimp with all 4 TSV variants exhibited severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phases of TSV infection, respectively. TSV was not detected by RT-PCR in TSR shrimp infected with Th04, UsHi94, and Ve05, or in Kona shrimp infected with Ve05 but was detected in TSR shrimp infected with Bz01 and in Kona shrimp infected with Bz01, Th04, and UsHi94. Real-time RT-PCR revealed that mean TSV copy numbers in TSR shrimp infected with Bz01, Th04, and UsHi94 were significantly (p < 0.0005) lower than those in Kona shrimp. In contrast, mean TSV copy numbers in TSR and Kona shrimp infected with Ve05 were not significantly different (p > 0.4). The results show that TSR L. vannamei are susceptible to infection but give high survival rates following challenge by all 4 variants of TSV.

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells.

Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV.

Experimental infection of Penaeus monodon with Taura syndrome virus (TSV).

Clinical signs and lesions of Taura syndrome virus (TSV) infection in Penaeus monodon have not been documented although the virus has been detected in this shrimp species by reverse transcription polymerase chain reaction (RT-PCR). This study provides the first evidence of TSV infection in P. monodon by histological and in situ hybridization (ISH) analyses. We performed experimental bioassays with groups of P. monodon using inocula of P. monodon and Litopenaeus vannamei (Th04PmTSV and Th04LvTSV, respectively), which were collected from Thailand in 2004 and found to be positive for TSV by RT-PCR. Samples of shrimp for histological and ISH analyses were collected on Days 2, 14, and 28 post-inoculation. Mortality among TSV-inoculated P. monodon appeared on Day 3, with 2 out of 10 shrimp dying. Severe necrosis of cuticular epithelial cells and lymphoid organ spheroids, indicative of acute and chronic phase lesions of TSV infection, respectively, were detected in the samples. Sequence analyses of the capsid protein 2 (CP2) gene showed that Th04PmTSV and Th04LvTSV isolates were different; however, both belonged to a phylogenetic family of Asian TSV isolates. The results of this study demonstrated that both mortality and histological lesions are associated with TSV infection in P. monodon.