Prevalence and antimicrobial resistance of food safety related Vibrio species in inland saline water shrimp culture farms / Prevalencia y resistencia a los antimicrobianos de especies de Vibrio relacionadas con la inocuidad de los alimentos en granjas de cultivo de camarones de aguas saladas continentales

This study evaluated the potential pathogenicity and antimicrobial resistance (AMR) of Vibrio species isolated from inland saline shrimp culture farms. Out of 200 Vibrio isolates obtained from 166 shrimp/water samples, 105 isolates were identified as V. parahaemolyticus and 31 isolates were identified as V. alginolyticus and V. cholerae, respectively. During PCR screening of virulence-associated genes, the presence of the tlh gene was confirmed in 70 and 19 isolates of V. parahaemolyticus and V. alginolyticus, respectively. Besides, 10 isolates of V. parahaemolyticus were also found positive for trh gene. During antibiotic susceptibility testing (AST), very high resistance to cefotaxime (93.0%), amoxiclav (90.3%), ampicillin (88.2%), and ceftazidime (73.7%) was observed in all Vibrio species. Multiple antibiotic resistance (MAR) index values of Vibrio isolates ranged from 0.00 to 0.75, with 90.1% of isolates showing resistance to ≥ 3 antibiotics. The AST and MAR patterns did not significantly vary sample-wise or Vibrio species-wise. During the minimum inhibitory concentration (MIC) testing of various antibiotics against Vibrio isolates, the highest MIC values were recorded for amoxiclav followed by kanamycin. These results indicated that multi-drug resistant Vibrio species could act as the reservoirs of antibiotic resistance genes in the shrimp culture environment. The limited host range of 12 previously isolated V. parahaemolyticus phages against V. parahaemolyticus isolates from this study indicated that multiple strains of V. parahaemolyticus were prevalent in inland saline shrimp culture farms. The findings of the current study emphasize that routine monitoring of emerging aquaculture areas is critical for AMR pathogen risk assessment.(AU)

Fish immune responses to natural infection with carp edema virus (Koi sleepy disease): An emerging fish disease in India.

Emerging pathogen, carp edema virus (CEV) causes koi sleepy disease (KSD) in Koi and common carp causing severe mortalities worldwide. In the present study, a total of 150 fish species belonging to eight different families were sampled from the ornamental fish retailers and farms, located in Karnataka, India. The OIE protocol viz., level-I, II and III diagnoses confirmed the infection of CEV in 10 koi fish. Interestingly, other fish species belonging to different fish family including cyprinidae family were negative to CEV. Further, CEV infection was confirmed by sequencing (partial 4a gene); it showed the similarity with that of CEV reported from India and Germany strains with similarity of 97.4-99.94% and belonged to genogroup IIa. TEM analysis of purified CEV, in vivo cohabitation and tissue infection experiments confirmed the CEV infection. In addition, viral load was significantly higher (106-7 copies) in koi collected from Dakshina Kannada than of Bengaluru (103-4 copies). To understand the host-pathogen interaction, different organs such as gill, kidney, liver and spleen from naturally (CEV) infected koi were used to study the immune gene responses by using eight innate and one adaptive immune response. Results indicated that TNF-α, RohTNF-α, iNOS, IFN-γ and IL-10, and catalyze ß-2M of MHC class I pathway genes were upregulated in koi. Higher expression of immune genes during the CEV infection may have inhibited viral replication and mount an antigenic adaptive response. Similar to other viral infections, interferon-γ play an important role during poxvirus infections. Quantification of immune genes in infected fish will provide insights into the host responses and provide valuable information to devise intervention strategies to prevent and control disease due to CEV.

Complete genome sequencing and characterization of single-stranded DNA Vibrio parahaemolyticus phage from inland saline aquaculture environment.

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.

Identification of novel vaccine candidates in the whole-cell Aeromonas hydrophila biofilm vaccine through reverse vaccinology approach.

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.

Isolation and Characterization of Bacteriophages from Inland Saline Aquaculture Environments to Control Vibrio parahaemolyticus Contamination in Shrimp.

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.

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.

Supplementation of nano-selenium in fish diet: Impact on selenium assimilation and immune-regulated selenoproteome expression in monosex Nile tilapia (Oreochromis niloticus).

Selenium (Se), a fundamental element of nutrigenomic science in fish nutrition, was used to investigate its impact on selenoproteome expression and Se regulation in tilapia. Different concentrations (T1 - 0, T2 - 0.5, T3 - 1.0 and T4 - 2.0 mg/kg of feed) of dietary nano-Se were incorporated in the diets of monosex Nile tilapia. A total of 180 tilapia fingerlings with initial weight (15.73 ± 0.05 g) were stocked in 150 L capacity FRP tanks categorized into four diet groups with triplicate each for a feeding trial of 90 days. At the end of first, second and third months of the feeding trial, gill, liver, kidney and muscle tissues were harvested to evaluate the effect on the kinetics of Se bioaccumulation and assimilation as well as immune-regulated selenoprotein transcripts (GPx2, SelJ, SelL, SelK, SelS, SelW and Sepp1a) and their synthesis factors (SPS1 and Scly). The findings depicted that significantly (p < 0.05) higher weight gain was found in the diet supplemented with 1.0 mg/kg of nano-Se. The theory of second-order polynomial regression supported the same. The liver showed significantly (p < 0.05) higher Se accumulation and concentration factor among the harvested tissues in a different timeline. All the selected immune-regulated selenoproteins and synthesis factors in different fish tissues showed significantly (p < 0.05) up-regulation in the diet supplemented with 1.0 mg/kg of nano-Se for the second month. Therefore, the present findings suggested that the supplementation of nano-Se could be more effective for improved growth, better selenium regulation and expression of immune-regulated selenoproteins in the fish model.

Complete genome analysis of a red seabream iridovirus (RSIV) isolated from Asian seabass (Lates calcarifer) in India.

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.

Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

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.

Correction to: Characterization and genome sequencing of three Aeromonas hydrophila-specific phages, CF8, PS1, and PS2.

Authors would like to correct the incorrect version.

Evaluation of biofilm of Vibrio anguillarum for oral vaccination of Asian seabass, Lates calcarifer (BLOCH, 1790).

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.

Genome dynamics and evolution of codon usage patterns in shrimp viruses.

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.

Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Channa striatus.

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).

Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field.

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.

Development of a monoclonal antibody-based flow-through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon.

A flow-through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 µg mL(-1) crude WSSV protein, respectively. The FTA could be completed in 8-10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1-step polymerase chain reaction (PCR) and in between that of the 1- and 2-step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post-larvae, WSSV was first detected 14, 16 and 18 h post-infection (hpi) by FTA, immunodot and one-step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one-step PCR, respectively. The FTA was more sensitive (25/27) than one-step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4-8 °C. The FTA is available as a rapid test kit called 'RapiDot' for the early detection of WSSV under field conditions.

Epitope analysis of white spot syndrome virus of Penaeus monodon by in vivo neutralization assay employing a panel of monoclonal antibodies.

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.

Vasovasostomy. A clinical study with 10 years' follow-up.

PIP: 36 cases of vas reconstruction after vasectomy are reported with 10 years of follow-up study. 83% of the vasectomies had been done for family planning purposes and the major reason for wanting vas reconstruction was the desire for more children. The majority of the patients were between 31 and 40 years of age and all were sexually active. After careful examination for active spermatogenesis and vas patency, end-to-end anastomosis was performed, usually using a splint. Bed rest for at least 5 days postoperatively was recommended. Semen samples were analyzed for motile sperm at 3-month intervals. 70% of the 30 patients undergoing vasovasostomy showed spermatozoa in their semen, usually within 1 year. Only 1 of the 6 epididymovasostomy patients showed spermatozoa and the poor results associated with this procedure may be due to previous postvasectomy inflammation. No correlation was found between the interval between vasectomy and anastomosis, age and splint use, and the success of the reconstruction. 5 of the 14 patients with normal sperm counts produced normal pregnancies.^ieng