First report of the infectious spleen and kidney necrosis virus (ISKNV) infection in ornamental fishes in India.

Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018-19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42%) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76% to 100%. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV-positive fish species, three species were reported positive to ISKNV for the first time in the world. Further, the in vitro experiment showed the growth of virus in Asian sea bass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures need to be taken to control these emerging pathogens.

Toxic Algae Silence Physiological Responses to Multiple Climate Drivers in a Tropical Marine Food Chain.

Research on the effects of climate change in the marine environment continues to accelerate, yet we know little about the effects of multiple climate drivers in more complex, ecologically relevant settings - especially in sub-tropical and tropical systems. In marine ecosystems, climate change (warming and freshening from land run-off) will increase water column stratification which is favorable for toxin producing dinoflagellates. This can increase the prevalence of toxic microalgal species, leading to bioaccumulation of toxins by filter feeders, such as bivalves, with resultant negative impacts on physiological performance. In this study we manipulated multiple climate drivers (warming, freshening, and acidification), and the availability of toxic microalgae, to determine their impact on the physiological health, and toxin load of the tropical filter-feeding clam, Meretrix meretrix. Using a structural equation modeling (SEM) approach, we found that exposure to projected marine climates resulted in direct negative effects on metabolic and immunological function and, that these effects were often more pronounced in clams exposed to multiple, rather than single climate drivers. Furthermore, our study showed that these physiological responses were modified by indirect effects mediated through the food chain. Specifically, we found that when bivalves were fed with a toxin-producing dinoflagellate (Alexandrium minutum) the physiological responses, and toxin load changed differently and in a non-predictable way compared to clams exposed to projected marine climates only. Specifically, oxygen consumption data revealed that these clams did not respond physiologically to climate warming or the combined effects of warming, freshening and acidification. Our results highlight the importance of quantifying both direct and, indirect food chain effects of climate drivers on a key tropical food species, and have important implications for shellfish production and food safety in tropical regions.

Influence of some environmental variables and addition of r-lysozyme on efficacy of Vibrio harveyi phage for therapy.

Bacteriophage therapy is a viable proposition for controlling luminous vibriosis caused by Vibrio harveyi in shrimp aquaculture. However, environmental factors influence the growth and activity of phage and affect its efficiency in controlling bacterial diseases. An essential problem in the use of vibrio phage as a therapeutic agent was the development of resistance to phage attachment, rendering them resistant to the lytic action of phage. This problem could be overcome by applying a cocktail of phages. This study aimed to evaluate the effect of salinity and pH on the phage activity and also to study the role of recombinant shrimp lysozyme on the performance of the V. harveyi phage. Out of three different levels of salinity (20, 25 and 30 ppt) and pH (6, 7 and 8) tested, optimum phage activity was observed at a salinity of 25 ppt and at neutral pH. Application of recombinant shrimp lysozyme in combination with V. harveyi phage significantly improved the activity of phage in in vitro assay as well as in microcosm study using seawater. The application of phage along with lysozyme can be a useful approach to overcome the inability of phage to enter the bacteria and thus eliminate or reduce fish/ shrimp pathogenic bacteria in aquaculture.

Occurrence of clinical genotype Vibrio vulnificus in clam samples in Mangalore, Southwest coast of India.

Vibrio vulnificus is an opportunistic human pathogen causing gastroenteritis, wound infection and primary septicemia. V. vulnificus population has been divided into subpopulations based on their phenotype and genotype characteristics. In this study, 38.5% (10/26) of clam (Meretrix meretrix) samples obtained from Mangalore markets were seen to harbor V. vulnificus. Biochemical characterization of V. vulnificus isolates showed the strains to belong to Biotype 1 phenotype. Genotyping of strains using the 16S rRNA and virulence correlated gene (vcg) typing methods identified the isolates to be of 16S rRNA typeB and vcgC type respectively. Analysis of representative 16S rRNA and vcg gene sequences further substantiated that the V. vulnificus associated with clams in the present study to be of clinical origin, implicated as virulent type responsible for causing infection in humans.

Pathogenic marine microbes influence the effects of climate change on a commercially important tropical bivalve.

There is growing evidence that climate change will increase the prevalence of toxic algae and harmful bacteria, which can accumulate in marine bivalves. However, we know little about any possible interactions between exposure to these microorganisms and the effects of climate change on bivalve health, or about how this may affect the bivalve toxin-pathogen load. In mesocosm experiments, mussels, Perna viridis, were subjected to simulated climate change (warming and/or hyposalinity) and exposed to harmful bacteria and/or toxin-producing dinoflagellates. We found significant interactions between climate change and these microbes on metabolic and/or immunobiological function and toxin-pathogen load in mussels. Surprisingly, however, these effects were virtually eliminated when mussels were exposed to both harmful microorganisms simultaneously. This study is the first to examine the effects of climate change on determining mussel toxin-pathogen load in an ecologically relevant, multi-trophic context. The results may have considerable implications for seafood safety.

Detection of koi herpesvirus in common carp, Cyprinus carpio L., by loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for koi herpes virus (KHV) detection in common carp was designed. A set of four primers, two inner and two outer, were designed based on the sequence of the thymidine kinase (tk) gene of KHV. Time and temperature conditions for detection of KHV were optimized for 60 min at 65 degrees C. The detection limit using LAMP was found to be similar to that by polymerase chain reaction. In this study, we have developed a highly sensitive and rapid diagnostic procedure for detection of KHV infection in common carp.

High genetic diversity in the coat protein and 3 untranslated regions among geographical isolates of Cardamom mosaic virus from south India.

A survey was conducted to study the biological and genetic diversity of Cardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3 terminal region consisting of the coat protein (CP) coding sequence and 3 untranslated region (3 UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3 UTR, making CdMV one of the highly variable members of Potyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.

Detection of Salmonella spp. in tropical seafood by polymerase chain reaction.

The incidence of Salmonella spp. in tropical seafood was studied using standard microbiological techniques and polymerase chain reaction (PCR). Six of 20 finfish (30%), 4 of 20 clams (20%) and 1 of 20 shrimp (5%) were positive by culture techniques and by PCR. In a comparative study of different selective enrichment broths and selective plating media, more than one enrichment broth and selective agar were found to be necessary for efficient detection of Salmonella from seafood. Selenite cystine broth (SCB) was found to be more efficient compared to tetrathionate broth (TTB) while both bismuth sulfite agar (BSA) and hektoen enteric agar (HEA) were equally effective as selective plating media for fish. In the case of clams, HEA was found to be more effective. The presence of Salmonella spp. could be detected by PCR amplification of DNA extracted directly from the enrichment broths. In two cases, enrichment broths that were positive by PCR did not yield Salmonella by conventional methods.

Role of chitin in the survival of Vibrio parahaemolyticus at different temperatures.

Washed cells of Vibrio parahaemolyticus declined in numbers when incubated in phosphate-buffered saline for 6 h at different temperatures. Addition of chitin flakes to phosphate-buffered saline not only helped the organism to survive, but also resulted in an increase in cell numbers, particularly at 10 degrees C. The effect of chitin could not be simulated by N-acetylglucosamine, yeast extract, starch, or casein.

Evaluation of methods for enumeration of Vibrio parahaemolyticus from seafood.

The efficiency of several enrichment broths in recovering Vibrio parahaemolyticus inoculated into fish homogenates was studied. Recovery by the most probable number technique was very low in all the broths, while direct plating on thiosulfate citrate bile salt sucrose agar yielded better recovery. A decrease in the enrichment time to 8 from 18 h did not improve recovery. At concentrations exceeding 2.5 micrograms/ml, polymyxin was inhibitory to V. parahaemolyticus.

Survival of Vibrio parahaemolyticus in cold smoked fish.

Fresh samples of mullet (Mugil cephalus) and oil sardines (Sardinella longiceps) obtained from a fish market were subjected to cold smoking. Some of the samples harboured low levels of Vibrio parahaemolyticus. After cold smoking, however, many samples showed relatively high levels of V. parahaemolyticus suggesting that a small population of naturally occurring organisms could multiply to significant levels during the process of cold smoking or during subsequent storage at room temperature. Nevertheless, smoke components were observed to exert an inhibitory effect on V. parahaemolyticus in broth. Salt concentration 1% appeared to increase the sensitivity of V. parahaemolyticus to smoke components.

Levels of Vibrio parahaemolyticus in Indian shrimp undergoing processing for export.

A study of 56 samples of raw prawns, 50 samples of processed prawns, and 57 samples of frozen prawns from five prawn-processing factories around Mangalore for the levels of Vibrio parahaemolyticus revealed that most of these samples carry a load of less than 10/g. None of the samples had counts greater than 10(2)/g, indicating that the prawn-processing factories are able to meet the International Commission on Microbiological Specifications for Foods (ICMSF) suggested limit of 10(2) V. parahaemolyticus per gram for frozen prawns.