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Despite their evolutionary, molecular biology and biotechnological significance, relatively fewer numbers of single-stranded DNA (ssDNA) filamentous phages belonging to the family Inoviridae have been discovered and characterized to date. The present study focused on genome sequencing and characterization of an ssDNA Vibrio parahaemolyticus phage V5 previously isolated from an inland saline shrimp culture farm. The complete circular genome of phage V5 consisted of 6658 bp with GC content of 43.7%. During BLASTn analysis, only 36% of phage V5 genome matched with other Vibrio phage genomes in the NCBI database with a sequence identity value of 79%. During the phylogenetic analysis, phage V5 formed a separate branch in the minor clade. These features indicate the novel nature of the phage V5 genome. Among 10 predicted open reading frames (ORFs) in the phage V5 genome, 6 encoded for the proteins of known biological functions, whereas the rest were classified as hypotheticals. Proteins involved in replication and structural assembly were encoded by the phage genome. However, the absence of genes encoding for DNA/RNA polymerases and tRNAs signified that phage V5 is dependent on the host`s molecular machinery for its propagation. As per our knowledge, this is the first study describing the novel genome sequence of an ssDNA V. parahaemolyticus phage from the inland saline environment.

Diseases hamper the growth of aquaculture, which is caused mainly by opportunistic pathogens that leads to mild clinical signs with periodic mortality at farm level. Present study was conducted to check the causative agent responsible for the death of rohu fish (Labeo rohita), which were showing clinical signs of abdominal dropsy. The moribund fish showed haemorrhages on skin, reddening of gill and pure bacterial cultures were obtained from the abdominal fluid. The isolates were biochemically identified and genetically confirmed as Aeromonas hydrophila based on PCR assay. They showed cytotoxic and haemolytic activity and produced several exoenzymes, which were accountable for the pathogenicity of the isolates. The isolates A11-A17 (A. hydrophila) were found to be associated with 5 virulent genes such as enterotoxin act, alt, lip, haemolytic toxin ahh, type 3 secretion genes such as aex. Furthermore, multiple antibiotic resistance (MAR) index of 0.525 was associated with A16, resistant to infections. The study proves the virulence of A. hydrophila A16 and its resistance to 21 out of 40 tested antibiotics (52%), which is considered as multidrug resistance of A. hydrophila suggesting it to be the emerging major threat to aquaculture.

Among the various bacterial pathogens associated with the aquaculture environment, Vibrio parahaemolyticus the important one from shrimp and human health aspects. Though having been around for several decades, phage-based control of bacterial pathogens (phage therapy) has recently re-emerged as an attractive alternative due to the availability of modern phage characterization tools and the global emergence of antibiotic-resistant bacteria. In the present study, a total of 12 V. parahaemolyticus specific phages were isolated from 264 water samples collected from inland saline shrimp culture farms. During the host range analysis against standard/field isolates of V. parahaemolyticus and other bacterial species, lytic activity was observed against 2.3–45.5% of tested V. parahaemolyticus isolates. No lytic activity was observed against other bacterial species. For genomic characterization, high-quality phage nucleic acid with concentrations ranging from 7.66 to 220 ng/µl was isolated from 9 phages. After digestion treatments with DNase, RNase and S1 nuclease, the nature of phage nucleic acid was determined as ssDNA and dsDNA for 7 and 2 phages respectively. During transmission electron microscopy analysis of phage V5, it was found to have a filamentous shape making it a member of the family Inoviridae. During efficacy study of phage against V. parahaemolyticus in shrimp, 78.1% reduction in bacterial counts was observed within 1 h of phage application. These results indicate the potential of phage therapy for the control of V. parahaemoyticus in shrimp. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00934-6.

Biofilm vaccine has been recognised as one of the successful strategy to reduce the Aeromonas hydrophila infection in fish. But, the vaccine contains the protective and non-protective proteins, which may lead to show altered heterologous adaptive immunity response. Moreover, cross protection and effectiveness of previously developed biofilm vaccine was not tested against different geographical A. hydrophila isolates. Therefore, in the present study, whole-cell A. hydrophila biofilm vaccine was evaluated in rohu, vaccinated group showed increased antibody titer and protection against the different geographical A. hydrophila isolates namely KAH1 and AAH2 with 78.9% and 84.2% relative percentage survival, respectively. In addition, by using the immune sera of biofilm vaccinated group, a total of six protective proteins were detected using western blot assay. Further, the same proteins were identified by nano LC-MS/MS method, a total of fourteen candidate proteins showing the immunogenic property including highly expressed OMP's tolC, bamA, lamb, AH4AK4_2542, AHGSH82_029580 were identified as potential vaccine candidates. The STRING analysis revealed that, top candidate proteins identified may potentially interact with other intracellular proteins; involved in ribosomal and (tricarboxylic acid) TCA pathway. Importantly, all the selected vaccine candidate proteins contain the B-cell epitope region. Finally, the present study concludes that, whole-cell A. hydrophila biofilm vaccine able to protect the fish against the different geographical A. hydrophila isolates. Further, through reverse vaccinology approach, a total of fourteen proteins were identified as potential vaccine candidates against A. hydrophila pathogen.

Background: Vibrio anguillarum is one of the major pathogen causing economic loss in the aquaculture industry. And, most of the microorganisms will use biofilm strategy for their survival in the host. Hence, less effectiveness of antibiotic and free cell (whole cell) vaccine may be observed in the aquaculture practice. Therefore the developed vaccine should be the mirror image of pathogen molecule, in regard; the present study was carried out to standardize the nutrient requirement for optimum biofilm production and antigen expression of biofilm and free cells of Vibrio anguillarum. Also, to identify the deferentially expressed protein in the biofilm mode. Methods: Culture conditions were optimized for the maximum biofilm production of V. anguillarum on chitin flakes as a substrate in nutrient restricted conditions. Through proteomic approach such as SDS PAGE was conducted to know the protein profile of biofilm and free cells of V. anguillarum and further, nanoLC-MS/MS was used to identify the differentially expressed protein in the biofilm of V. anguillarum. Result: Maximum biofilm production was observed on the 3rd day on 0.1% TSB concentration supplemented with 0.3% chitin flakes and 2% NaCl. Significant changes in the antigen expression of biofilm of V. anguillarum were observed in SDS PAGE and it revealed that in the biofilm mode, three new novel proteins were expressed and about ten proteins repressed as compared to that of free cell counterpart.

Red sea bream iridovirus (RSIV) is the causative agent of the iridoviral disease with high mortality rates in cultured fish. Our laboratory reported the first case of RSIV infection in India which resulted in mass mortalities of Asian seabass, Lates calcarifer. The RSIV-LC strain isolated from infected fish was subjected to complete genome sequencing and analysis. The complete genome of RSIV-LC was found to be of 111,557 bp in size having a G + C content of 53 %. The complete genome has 114 open reading frames (ORFs) of which 38 ORFs were predicted as functional proteins while the rest were hypothetical proteins. Among the ORFs 26 were found to be core genes reported earlier to be homologous in iridovirus complete genomes. Phylogenetic tree constructed based on the 26 core gene sequences, major capsid protein and ATPase genes revealed RSIV-LC in this study to belong to the genus Megalocytivirus of the RSIV-Genotype II. The present study provides the first report of the complete genome sequence and annotation of the RSIV strain isolated from India.

Motile Aeromonas Septicemia (Aeromonas hydrophila) has caused severe economic loss in several aquaculture fish species such as carps, tilapia, salmon, and catfishes. In the present study, mass mortality of zebrafish (Danio rerio) was observed in fish maintained in the hatchery cum rearing center, along with clinical signs including enlarged abdomen and haemorrhages on the body surface. The etiological agent was identified as Aeromonas hydrophila through biochemical and molecular methods. The isolated A. hydrophila, AhZ1K was positive for all the phenotypic virulence factors and cytopathic effect was observed in D. rerio gill (DRG) cell line. Further, A. hydrophila, AhZ1K was found to produce biofilm and was positive for eight virulence genes and five antibiotic resistance genes with a Multiple Antibiotic Resistance (MAR) index of 0.6 and 0.825 for free and biofilm cells respectively, indicating the property of multidrug resistance. Therefore, as a preliminary approach to develop bio-control measure, novel lytic bacteriophages (phages namely AhFM4 & AhFM5) were isolated from Western Ghats riverine water systems. Transmission electron microscopy (TEM) confirms that both the lytic phages (AhFM4 & AhFM5) belong to Myoviridae. Interestingly, AhFM4 & AhFM5 displayed 96% and 70%; 15 and 25 min; 152 ± 27 PFU/host cell and 112 ± 13 PFU/host cell of adsorption, latency and burst size respectively. The phages were stable over a broad array of physicochemical parameters (temperatures, salinity, and pH), indicating that AhFM4 and AhFM5 have an immense capability to be developed as a substitute for antibiotics to control motile Aeromonas septicemia in aquaculture.

We analysed the genomes and codon usage patterns of seven small (DNA and RNA) shrimp viruses. Effective number of codon (ENC) values indicated moderate (35 < ENC < 50) codon usage bias in shrimp viruses. Correlation analysis between GC compositions at non-synonymous codon and synonymous codon positions (GC1, 2 and GC3) as well as GC3 versus ENC curves indicated varying influences of mutational pressure on codon usage. The presence of deoptimized codons and host-antagonistic codon usage trends in shrimp viruses suggested the adaptation of a slow replication strategy by these viruses to avoid host defences. Low CpG frequencies indicated that shrimp viruses have evolved with underrepresentation of CpGs to avoid the host’s immune response.

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Aeromonas hydrophila is an important finfish pathogen, besides being an opportunistic human pathogen. In the present study, the genomes of three A. hydrophila-specific phages, CF8, PS1, and PS2, were isolated, characterized and sequenced. Transmission electron microscopy showed that all three phages had typical Myoviridae morphology. The linear dsDNA genomes of CF8, PS1, and PS2 were 238,150 bp, 237,367 bp, and 240,447 bp in length, with a GC content of 42.2%, 38.8%, and 38.8%, respectively. The low sequence similarity (67.6% - 69.8% identity with 27.0% - 29.0% query coverage) to other phage genomes in the NCBI database indicated the novel nature of the CF8, PS1, and PS2 genomes. A total of 244, 247, and 250 open reading frames (ORFs) were predicted in the CF8, PS1, and PS2 genome, respectively. During the annotation process, functional predictions were made for 28-31 ORFs, while the rest were classified as "hypothetical proteins" with yet unknown functions. Genes for tRNAs were also detected in all phage genomes. As all three phages in the present study had a very narrow host range with lytic activity against only one strain of A. hydrophila, these phages could be good candidates for phage typing applications. Moreover, the endolysin- and lytic-transglycosylase-encoding genes could be used for recombinant cloning and expression of anti-microbial proteins.

The present study evaluated the biofilm (BF) of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer. An 80-day experiment was carried out in circular fiber-reinforced plastic (FRP) tanks using free cell (FC) and BF of Vibrio anguillarum with triplicate in each. Heat-inactivated FC and BF cells at 107, 1010 and 1013 CFU/g fish/d were fed to fish for 20 days, agglutination antibody titer estimated at each 10 days interval up to 60-day post vaccination. As compared to FC and control there was a significant increase in agglutinating antibody titer in the biofilm vaccinated fishes. Among the 3 doses, BF at 1010 cfu/g fish/d was considered the ideal dose for vaccination. Relative percentage survival (RPS) was higher in biofilm vaccinated fish (85.4%) compared to that with free cells (27.0%). The study demonstrated the better performance of V. anguillarum biofilm oral vaccine compared that with free cell vaccine in L. calcarifer. The study further supports better performance of biofilm vaccine model with one more bacterial pathogen in a high carnivore fish.

In the present study, moribund Asian seabass (Lates calcarifer) fish samples were collected from open estuarine cages and observed for clinical signs followed by molecular diagnosis using primers targeting DNA polymerase gene of RSIV reported from OIE. The 568 bp amplicon confirmed the 100% sequence similarity with the reported sequences of RSIV from other countries. It formed the four clusters (RSIV, ISKNV, TBIV, and SDDV) with showing the close relation to Korean RSIV isolate than the RSIV isolate of Japanin the phylogenetic tree and multiple alignment analysis. The histopathogical examinations revealed, necrosed cellular material, melanomacrophage centers, RBC proliferation, lymphocytic infiltration, increased vacuoles and irregular intracytoplasmic viral inclusion bodies were observed in infected fish gill and spleen tissues. The challenge test with tissue homogenate reproduced 100% mortality in the healthy seabass by 6th day post infection (dpi). The first report of the virus in cultured Asian seabass from the region has been notified to OIE.

Chitin is one of the natural biopolymer found abundantly in the shells of crustaceans, insects and in cell walls of fungi. In this study, we determined the effect of dietary administration of 0.5, 0.75 and 1% chitin on the immune response and disease resistance in freshwater prawn, challenged against Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). We observed a significantly enhanced immune response, indicated as higher prophenoloxidase activity and respiratory burst of hemocytes, in 0.75% chitin-diet fed prawns compared to the chitin-free-diet fed prawns. Importantly, the relative percent survival (RPS) following challenge with white muscle disease (WTD) viruses was found relatively high in M . rosenbergii fed with diet containing 0.75% chitin (63.16%), suggesting an increased resistance to disease susceptibility. These results indicate that the incorporation of chitin in prawn diet would be beneficial in stimulating the immune response and thereby developing resistance against diseases.

A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p

The present study was conducted to find the effect of dietary brewer’s yeast on growth, survival, immune response, and resistance to white muscle disease in giant freshwater prawn, Macrobrachium rosenbergii. The brewer’s yeast was supplemented at graded levels 0, 5, 10, and 20 g per kg diet, and the experiment was conducted for 75 days. After the feeding trials, growth, survival, immune parameters like prophenoloxidase activity, respiratory burst, and total hemocyte count were assessed. The growth, specific growth rate, and survival were not found significantly different among the treatment and control groups “prophenoloxidase activity and respiratory burst” were found significantly different (p

A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1 x 10⁻⁶ resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100 μg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1 x 10⁻⁶ dilution) at 27 °C for 90 min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12 dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12 dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1 x 10⁻⁶ dilution) incubated with panel of the MAbs at 100 μg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72 hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48 hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24 hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12 hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.

Our laboratory has developed a biofilm oral vaccine of Aeromonas hydrophila, which has given significantly higher antibody agglutination titre and protection in herbivorous carps and omnivorous walking catfish compared to that with free cell vaccine. Against this background, in the present study A. hydrophila biofilm oral vaccine was evaluated in Channa striatus, a carnivorous fish model. The fish was fed with biofilm (BF) and free cell (FC) of A. hydrophila vaccine at 10(10) cells/g fish/day for 20 d. Serum antibody production monitored with a monoclonal antibody based ELISA for 60 day post vaccination. Significantly higher antibody titre was recorded with BF compared to that with FC. Furthermore, BF vaccinated fish upon challenge with A. hydrophila at 10(9) cfu/ml had significantly higher relative per cent survival (88) than that with FC (29.6).

Organophosphate based pesticides account for a major percentage of pesticides used in domestic, agricultural and industrial applications throughout the world. They are highly popular because they are effective, non persistent and relatively less expensive. Quinalphos is a synthetic organophosphate which has become a matter of concern because of its potentiality and hazardous effect. Hence, the present investigation was designed to assess the impact of quinalphos on total protein content in the selected tissues and growth rate of freshwater fish, Cyprinus carpio. The lethal toxicity of quinalphos to the fish exposed for 96 h was found to be 2.75 ppm. For sublethal toxicity study, the fish were exposed to two concentration viz., 1/5 th of LC 50 (0.550 ppm) and 1/10 th of LC 50 (0.275 ppm) along with control as reference for 4, 8, 16 and 32 days. Decrease in total protein concentration and growth rate were observed in 4 th and 8 th day of exposure, but on 16 th and 32nd day of exposure all the values reached nearer to normal condition. The decline in total protein levels and relative growth rate at initial exposure periods may indicates the high energy demand associated with imposed quinalphos stress. Restoration of protein fractions to normal, implies that after 8 days of exposure there seems to exist an oscillatory phase in protein turnover towards a more synthetic phase leading to the establishment of recuperation and adaptation phenomena.

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 μg/mL for A. invadans compared to 56 μg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10(-11) dilution compared to a 10(-8) dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8°C. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.