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.

A 90-day feeding trial was conducted to evaluate the effects of formic acid (FA) and astaxanthin (AX) on growth, survival, immune parameters, and tolerance to Vibrio infection in Pacific white shrimp. The study was divided into two experiments. In experiment 1, postlarvae-12 were randomly distributed into six groups and then fed four times daily with six experimental diets contained 0.3 % FA, 0.6 % FA, 50 ppm AX, 0.3 % FA + 50 ppm AX, 0.6 % FA + 50 ppm AX, or none of these supplements (control diet). After 60 days of the feeding trials, the body weight of all treatment groups was not significantly different from the control group, although shrimp fed formic acid had significantly lower body weight than shrimp fed 50 ppm AX. However, the 0.6 % FA + 50 ppm AX group had a significantly higher survival rate (82.33 ± 8.32 %) than the control group (64.33 ± 10.12 %). In experiment 2, Vibrio parahaemolyticus was added to each tank to obtain a final concentration of 104 colony-forming units/mL. Each treatment group received the aforementioned diets for another 30 days. At the end of this experiment, there was no difference in the weight gain among all experimental groups. However, the survival rate of shrimps whose diet included FA, AX, and their combination (in the range of 45.83–67.50 %) was significantly higher than the control group (20.00 ± 17.32 %). FA-fed shrimps also had significantly lower total intestinal bacteria and Vibrio spp. counts, while immune parameters [total hemocyte count (THC), phagocytosis activity, phenoloxidase (PO) activity, and superoxide dismutase (SOD) activity] of AX-fed groups were significantly improved compared with the other groups. In conclusion, FA, AX, and their combination are useful in shrimp aquaculture.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1234-x) contains supplementary material, which is available to authorized users.

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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...