Journal of Invertebrate Pathology (Journal of Insect Pathology)

Not AvailableHepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment

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Not AvailablePenaeus (Litopenaeus) vannamei shrimp samples (hepatopancreas and faeces) collected from grow-out farms were evaluated for the presence of newly emerged microsporidian parasite Enterocytozoon hepatopenaei by concentration techniques using either water–ether sedimentation or Sheather’s sugar solution for spore recovery and subsequent microscopic detection by modified trichrome stain (Ryan-blue method). This improved selective staining protocol can easily resolve the identification of microsporidian spores in hepatopancreatic tubules compared to conventional stains. This method enables differential diagnosis of microsporidian spores by a characteristic staining pattern of pinkish-red, often with a belt-like diagonal stripe seen in the middle of the spore and a halo of unstained area at one end which could be easily distinguished from similarly staining particles or debris.Not Availabl

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Not AvailableThe emerging microsporidian parasite Enterocytozoon hepatopenaei (EHP), the causative agent of hepatopancreatic microsporidiosis, has been widely reported in shrimp-farming countries. EHP infection can be detected by light microscopy observation of spores (1.7 × 1 μm) in stained hepatopancreas (HP) tissue smears, HP tissue sections, and fecal samples. EHP can also be detected by polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene or the spore wall protein gene (SWP). In this study, a rapid, sensitive, specific, and closed tube visual loop-mediated isothermal amplification (LAMP) protocol combined with FTA cards was developed for the diagnosis of EHP. LAMP primers were designed based on the SSU rRNA gene of EHP. The target sequence of EHP was amplified at constant temperature of 5 °C for 45 min and amplified LAMP products were visually detected in a closed tube system by using SYBR™ green I dye. Detection limit of this LAMP protocol was ten copies. Field and clinical applicability of this assay was evaluated using 162 field samples including 106 HP tissue samples and 56 fecal samples collected from shrimp farms. Out of 162 samples, EHP could be detected in 62 samples (47 HP samples and 15 fecal samples). When compared with SWP-PCR as the gold standard, this EHP LAMP assay had 95.31% sensitivity, 98.98% specificity, and a kappa value of 0.948. This simple, closed tube, clinically evaluated visual LAMP assay has great potential for diagnosing EHP at the farm level, particularly under low-resource circumstances.Not Availabl

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Not AvailableShrimp aquaculture in India has made significant strides especially after the introduction of Pacific white shrimp in the year 2009.However,intensification of culture practice has exacerbated several disease issues. Apart from infectious diseases,the Indian shrimp aquaculture has been witnessing certain syndromes,affecting productivity.One such syndrome associated with significant morbidity and mortality is popularly termed as running mortality syndrome (RMS) by shrimp farmers. Since 2011, RMS has been widely prevalent in the shrimp farms in Andhra Pradesh (AP) and Tamil Nadu (TN). The affected shrimp show patches of whitish musculature in the abdominal segments as a clinical sign with continuous low-level mortalities, especially after about 35–40 days of shrimp culture. We tried to investigate if this kind of mortality is infectious in nature. Investigations conducted in 34 farms comprising 25RMS affected and 9 healthy farms were tested negative for all the major OIE listed and other pathogens such as white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), hepatopancreatic parvo virus (HPV), infectious myonecrosis (IMNV), Taurasyndromevirus (TSV), yellowheadvirus(YHV) ,and Penaeus vannamei noda virus(PvNV).Bacteriological examination of haemolymph and hepatopancreas of RMS affected shrimp showed the predominance of Vibrio spp., such as Vibrio parahaemolyticus and Vibrio azureus. Histopathological examination of the hepatopancreas was found to be largely normal, except for haemocytic infiltration in the abdominal segments. Hemolymph of affected shrimp was observed to have less concentration of major and trace minerals than healthy ones .Bioassay through feeding RMS affected shrimp tissue to healthy shrimp and co-habitation experiment of healthy shrimp with the affected animals failed to induce RMS. When maintained in water with optimal physico-chemical parameters,affected shrimps showed recovery within 6–7days. On the other hand,environmental parameters of pond waters such as total ammonia nitrogen, nitrite nitrogen and turbidity were relatively higher than the optimal values in RMS affected farms. Multiple correspondence statistical analysis of critical factors indicated running mortality to be associated with high stocking densities, high nitrite-N, and high turbidity. Though the study could not identify any specific known aetiological agent associated with RMS affected shrimp, failure to reproduce the syndrome by bioassay, the recovery of affected shrimp under the optimal environmental conditions and the positive correlation with critical environmental parameters and the stocking densities to mortality rates clearly suggest RMS to be a pond ecosystem or pond management associated syndrome rather than infectious in nature and thus can be overcome through best management practices.Not Availabl

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Not AvailableWe report here the genome sequence of Vibrio campbellii LB102, isolated from the broodstock rearing system of a shrimp hatchery in India. Sequence analysis revealed the presence of effector toxins of the type III (YopT, sharing 39% identity with Yersinia pestis) and type VI (VgrG-3 and hemolysin coregulated protein of V. cholerae) secretion systems.Not Availabl