Development of SYBR Green based real time PCR assay for detection of monodon baculovirus in Penaeus monodon.

Shrimp farming is one of the most important aquaculture activities. Expansion and intensification of shrimp farming has been accompanied with the outbreak of diseases, which threaten the development and sustainability of the industry. Viral diseases are the major challenges faced by shrimp farming industries. The prevention/control of such diseases have become critical in determining the viability of the shrimp farming. The disease caused by monodon baculovirus (MBV) is the major limiting factor especially in shrimp hatchery. There are no therapeutic measures to control the viral diseases. So the detection of the disease is crucial in the prevention and spread of the disease. Hence, in this study, SYBR Green based real time polymerase chain reaction (PCR) assay was developed for the detection of MBV. The sensitivity of the real time PCR was determined using 10-fold dilutions of purified plasmid DNA with the concentration in the range of 10(1)-10(5) copies per reaction. The assay could detect as low as 12 copies indicating that the assay was sensitive and could be effectively used for the quantification of MBV. The real time PCR assay was found to be specific and did not show any amplification with P. monodon infected with infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syndrome virus (WSSV) and hepatopancreatic parvo-like virus (HPV). The novelty of this assay is that it could be employed for diagnosis of low level MBV infection in broodstock using fecal matter of shrimp, a non-invasive diagnostic tool.

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus.

AIMS: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. METHODS AND RESULTS: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. CONCLUSIONS: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India.

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.

Evaluation of Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 on immune indices of tiger shrimp, Penaeus monodon.

Occurrence of widespread epizootics among cultured stock of shrimp has put research programmes on preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study two bacteria, Pseudomonas sp. PM 11 and Vibrio fluvialis PM 17 were selected as candidate probionts from a pool of bacteria isolated from gut of farm reared sub-adult shrimp and tested for their effect on the immunity indicators of tiger shrimp. Sub-adult shrimp, weighing 14 to 22 g were treated in separate experiments with Pseudomonas sp. PM 11 and V. fluvialis PM 17 at 10(3) bacterial cells ml(-1) in the experimental shrimp culture tanks. One set of experimental animals was treated every 3 days and another set of animals every 7 days with each of the candidate probionts. Estimation of immunological indicators such as haemocyte counts, phenol oxidase and antibacterial activity showed declining trends. The haemocyte counts dropped from 31 x 10(3) to 65 x 10(3) ml(-1) on the first day to 4-16 x 10(3) ml(-1) on the 45th day. Similarly, the phenol oxidase activity declined from 12-32 units on the first day to 11-14 units on 45th day of the experiment. Antibacterial activity of haemolymph reduced to 46-67 percent on the 45th day of the experiment. The results of the study suggest that, the criteria used for the selection of putative probiotic strains in the present study, such as predominant growth on primary isolation media, ability to produce extracellular enzymes and siderophores, did not bring about the desired effect in vivo and improve the immune system in shrimp. Hence, new protocols have to be evolved for selection of microbe(s) as putative probiotics and that, detailed understanding of proven probiotics, employed presently on empirical basis may provide a clue on the selection procedure.