Ponnerassery S. Sudheesh

Pathogenicity of Vibrio parahaemolyticus in tiger prawn Penaeus monodon Fabricius: possible role of extracellular proteases

Abstract Tiger prawn, Penaeus monodon, were experimentally challenged with a virulent strain of Vibrio parahaemolyticus 25C, originally isolated from Penaeus orientalis in an outbreak of red leg disease at a shrimp farm in the Peoples Republic of China. The strain was found to be highly virulent to P. monodon with an LD50 value of 1×105 colony forming units (CFU)/prawn. The extracellular products (ECP) of the bacteria were also found to be toxic with an LD50 value of 8 μg protein/prawn. The strain was found to be a poor producer of hemolysins. Extracellular proteases present in the ECP were purified by Sephadex G-150 gel filtration and further analyzed by SDS-polyacrylamide gel electrophoresis. Two fractions viz., F I and F II, were obtained and both were found toxic to tiger prawns, but heat-inactivated fractions were not toxic.

Random amplified polymorphic DNA-PCR typing of Vibrio parahaemolyticus and V. alginolyticus isolated from cultured shrimps

Abstract The genomic diversity within Vibrio parahaemolyticus and V. alginolyticus was assessed by using random amplified polymorphic DNA (RAPD)-PCR. Fingerprinting of genomic DNA was carried out on 25 bacterial strains. Cluster analysis revealed the conglomeration of 15 strains of V. parahaemolyticus and 9 strains of V. alginolyticus into two distinct groups. Distinct clusters were observed within each species indicating genomic diversity in these bacteria.

Comparative Pathogenomics of Bacteria Causing Infectious Diseases in Fish

Fish living in the wild as well as reared in the aquaculture facilities are susceptible to infectious diseases caused by a phylogenetically diverse collection of bacterial pathogens. Control and treatment options using vaccines and drugs are either inadequate, inefficient, or impracticable. The classical approach in studying fish bacterial pathogens has been looking at individual or few virulence factors. Recently, genome sequencing of a number of bacterial fish pathogens has tremendously increased our understanding of the biology, host adaptation, and virulence factors of these important pathogens. This paper attempts to compile the scattered literature on genome sequence information of fish pathogenic bacteria published and available to date. The genome sequencing has uncovered several complex adaptive evolutionary strategies mediated by horizontal gene transfer, insertion sequence elements, mutations and prophage sequences operating in fish pathogens, and how their genomes evolved from generalist environmental strains to highly virulent obligatory pathogens. In addition, the comparative genomics has allowed the identification of unique pathogen-specific gene clusters. The paper focuses on the comparative analysis of the virulogenomes of important fish bacterial pathogens, and the genes involved in their evolutionary adaptation to different ecological niches. The paper also proposes some new directions on finding novel vaccine and chemotherapeutic targets in the genomes of bacterial pathogens of fish.

Systemic and mucosal immune response of rainbow trout to immunization with an attenuated Flavobacterium psychrophilum vaccine strain by different routes

Teleosts possess three immunoglobulin (Ig) heavy chain isotypes viz., IgM, IgT and IgD and all three isotypes are reported in rainbow trout. The expression of these Ig isotypes in response to different immunization routes was investigated and results provide a better understanding of the role these Igs in different tissues. Rainbow trout (Oncorhynchus mykiss) were immunized with an attenuated Flavobacterium psychrophilum strain, 259-93-B.17 grown under iron limiting conditions, by intraperitoneal, anal intubation and immersion routes. Serum, gill mucus, skin mucus and intestinal mucus samples were collected at 0, 3, 7, 14, 28, 42 and 56 days post immunization by sacrificing four fish from each treatment group and the unimmunized control group, and the IgM levels were estimated by an enzyme linked immunosorbent assay (ELISA). In addition, blood, gill, skin and intestinal tissue samples were collected for Ig gene expression studies. The secretory IgM, IgD and IgT gene expression levels in these tissues were estimated by reverse transcription quantitative real time PCR (RT-qPCR). Levels of IgM in serum, gill and skin mucus increased significantly by 28 days after immunization in the intraperitoneally immunized group, while no significant increase in IgM level was observed in fish groups immunized by other routes. Secretory IgD and IgT expression levels were significantly upregulated in gills of fish immunized by the immersion route. Similarly, secretory IgT and IgD were upregulated in intestines of fish immunized by anal intubation route. The results confirm mucosal association of IgT and suggest that IgD may also be specialized in mucosal immunity and contribute to immediate protection to the fish at mucosal surfaces.

Entericidin Is Required for a Probiotic Treatment (Enterobacter sp. Strain C6-6) To Protect Trout from Cold-Water Disease Challenge

ABSTRACT Flavobacterium psychrophilum causes bacterial cold-water disease in multiple fish species, including salmonids. An autochthonous Enterobacter strain (C6-6) inhibits the in vitro growth of F. psychrophilum, and when ingested as a putative probiotic, it provides protection against injection challenge with F. psychrophilum in rainbow trout. In this study, low-molecular-mass (≤3 kDa) fractions from both Enterobacter C6-6 and Escherichia coli K-12 culture supernatants inhibited the growth of F. psychrophilum. The ≤3-kDa fraction from Enterobacter C6-6 was analyzed by SDS-PAGE, and subsequent tandem mass spectroscopy identified EcnB, which is a small membrane lipoprotein that is a putative pore-forming toxin. Agar plate diffusion assays demonstrated that ecnAB knockout strains of both Enterobacter C6-6 and E. coli K-12 no longer inhibited F. psychrophilum (P < 0.001), while ecnAB-complemented knockout strains recovered the inhibitory phenotype (P < 0.001). In fish experiments, the engineered strains (C6-6 ΔecnAB and C6-6 ΔecnAB) and the wild-type strain (C6-6) were added to the fish diet every day for 38 days. On day 11, the fish were challenged by injection with a virulent strain of F. psychrophilum (CSF 259-93). Fish that were fed C6-6 had significantly longer survival than fish fed the ecnAB knockout strain (P < 0.0001), while fish fed the complemented knockout strain recovered the probiotic phenotype (P = 0.61). This entericidin is responsible for the probiotic activity of Enterobacter C6-6, and it may present new opportunities for therapeutic and prophylactic treatments against similarly susceptible pathogens.

Optimization of efficacy of a live attenuated Flavobacterium psychrophilum immersion vaccine.

This study was aimed at optimizing the efficacy of a recently developed live attenuated immersion vaccine (B.17-ILM) as a promising vaccine against bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum in salmonids. Rainbow trout (RBT) fry were vaccinated by immersion, and different parameters affecting vaccination such as fish size, vaccine delivery time, dose, duration of protection, booster regimes and vaccine growth incubation time were optimized. Specific anti-F.xa0psychrophilum immune response was determined by ELISA. Protective efficacy was determined by challenging with a virulent strain of F.xa0psychrophilum (CSF-259-93) and calculating cumulative percent mortality (CPM) and relative percent survival (RPS). All vaccinated fish developed significantly higher levels of serum antibody titers by week 8 when compared to their respective controls. Immersion vaccination for 3, 6 and 30xa0min produced significant protection with comparable RPS values of 47%, 53% and 52%, respectively. This vaccine provided significant protection for fish as small as 0.5xa0g with an RPS of 55%; larger fish of 1xa0g and 2xa0g yielded slightly higher RPS values of 59% and 60%, respectively. Fish vaccinated with higher vaccine doses of ∼10(10) and 10(8) colony forming units mL(-1) (cfuxa0ml(-1)) were strongly protected out to at least 24 weeks with RPS values up to 70%. Fish vaccinated with lower doses (∼10(6) and 10(5)xa0cfuxa0mL(-1)) had good protection out to 12 weeks, but RPS values dropped to 36% and 34%, respectively by 24 weeks. Vaccine efficacy was optimum when the primary vaccination was followed by a single booster (week 12 challenge RPSxa0=xa061%) rather than two boosters (week 12 challenge RPSxa0=xa048%). Vaccination without a booster resulted in a lower RPS (13%) indicating the necessity of a single booster vaccination to maximize efficacy. This study presents key findings that demonstrate the efficacy and commercial potential for this live attenuated BCWD vaccine.

Effects of Diel Temperature Fluctuation on Growth, Stress Response, and Immune Function of Burbot

AbstractNorth American Burbot Lota lota maculosa occupy habitats from deep, cold lakes to prairie streams. Controlled laboratory trials were conducted to evaluate the growth, immune response, and thermal stress physiology of juvenile Burbot subjected to fluctuating, high diel temperatures. Age-0 Burbot from a captive propagation program associated with population restoration in the Kootenai River, Idaho, were tested in temperature cycles that imitated summer conditions observed in regional stream habitats. The diel temperature cycle for the treatment group varied from 18°C at night to 27°C during the day, whereas the control group was held at a constant temperature of 14°C. We evaluated survival, growth, feed efficiency, heat shock protein 70 (hsp70) levels, hsp70 gene expression, serum lysozyme levels, and proximate body composition in samples taken from fish before, during, and at the completion of the trials. Both treatments had high survival and similar growth rates, but the control group gained signi...