Mass mortalities of the larval stage of the giant freshwater prawn, Macrobrachium rosenbergii, have been occurring in Bangladesh since 2011. Mortalities can reach 100% and have resulted in an 80% decline in the number of hatcheries actively producing M. rosenbergii. To investigate a causative agent for the mortalities, a disease challenge was carried out using infected material from a hatchery experiencing mortalities. Moribund larvae from the challenge were prepared for metatranscriptomic sequencing. De novo virus assembly revealed a 29 kb single‑stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. Primers were designed against the novel virus and used to screen cDNA from larvae sampled from hatcheries in the South of Bangladesh from two consecutive years. Larvae from all hatcheries screened from both years were positive by PCR for the novel virus, including larvae from a hatchery that at the point of sampling appeared healthy, but later experienced mortalities. These screens suggest that the virus is widespread in M. rosenbergii hatchery culture in southern Bangladesh, and that early detection of the virus can be achieved by PCR. The hypothesised protein motifs of Macrobrachium rosenbergii golda virus (MrGV) suggest that it is likely to be a new species within the Nidovirales order. Biosecurity measures should be taken in order to mitigate global spread through the movement of post-larvae within and between countries, which has previously been linked to other virus outbreaks in crustacean aquaculture.
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.
In shrimp culture, various diseases, white spot syndrome virus (WSSV) in particular, has been emerging as a serious constraint affecting shrimp culture in Bangladesh. In this study, effort has been given assessing the risk factors for WSSV outbreak under which 72 farms were investigated belonging 4 Upazilas of Bagerhat (Kochua, Rampal, Fakirhat) and Khulna (Paikgacha) district of Bangladesh from January to June 2011. Major scenario depicted improved traditional culture method. PCR test has been carried out to confirm WSSV infection. About 20 factors were considered in assessing the association of WSSV outbreak. Study revealed significant correlation with some factors like accessibility of cattle (r=0.630, p≤.01) and linked up with other ghers (r=0.754, p≤.01) within a cluster (Spearman’s rho correlation coefficient test). Pearson Correlation coefficient for salinity found to have significant correlation with the risk of WSSV infection (r= -0.727, p≤.01), followed by temperature (0.624, p≤.01) and average depth (-0.618, p≤.01), however, feeding kept 30.6% farms away from the outbreak followed by sludge removal (26.39% farms). On the contrary, uses of river water directly into the ghers pose 38.9% risk of being attacked which is absolutely nill and 1.4% for the underground and rain water respectively. Disease prone months found in March to June and out of the four upazilas, Fakirhat found to be less infected due to better management. Therefore, ensuring proper gher management practice, virus free Pl, awareness buildup at the farmer level and community based farm management development can act as preventive measures in reducing the risk of WSSV infection.
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