Application of nervous necrosis virus capsid protein-based antigen-presenting particles for vaccine development.

Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses.

Investigating the impact of chlorine dioxide in shrimp-rearing water on the stomach microbiome, gill transcriptome, and infection-related mortality in shrimp.

AIMS: This study aimed to assess the effects of chlorine dioxide (ClO2) in water on whiteleg shrimp Penaeus vannamei, evaluating its impact on the stomach microbiota, gill transcriptome, and pathogens. METHODS AND RESULTS: ClO2 was added to the aquarium tanks containing the shrimp. The application of ClO2 to rearing water was lethal to shrimp at concentrations above 1.2 ppm. On the other hand, most of the shrimp survived at 1.0 ppm of ClO2. Microbiome analysis showed that ClO2 administration at 1.0 ppm significantly reduced the α-diversity of bacterial community composition in the shrimp stomach, and this condition persisted for at least 7 days. Transcriptome analysis of shrimp gill revealed that ClO2 treatment caused massive change of the gene expression profile, including stress response genes. However, after 7 days of the treatment, the gene expression profile was similar to that of shrimp in the untreated control group, suggesting a recovery to the normal state. This 1.0-ppm ClO2 significantly reduced shrimp mortality in artificial challenges with an acute hepatopancreatic necrosis disease-causing Vibrio parahaemolyticus and white spot syndrome virus, which were added to rearing water. CONCLUSIONS: The use of ClO2 at appropriate concentrations effectively eliminates a significant portion of the bacteria in the shrimp stomach and pathogens in the water. The results of this study provide fundamental knowledge on the disinfection of pathogens in water using ClO2 and the creation of semi germ-free shrimp, which has significantly decreased microbiome in the stomach.

Characterization of Photobacterium damselae subsp. damselae isolated from diseased Asian seabass (Lates calcarifer) and the preliminary development of a formalin-killed cell vaccine.

Asian seabass (Lates calcarifer) is an economically important fish species that is widely cultivated in Thailand. However, aquaculture of Asian seabass is limited by infectious diseases. One of the most serious diseases is photobacteriosis, caused by Photobacterium damselae. Vaccination is recognized as an efficient disease prevention and pathogen control method for strengthening the aquaculture industry. To promote vaccine development, the characterization of pathogenic bacteria and their pathogenesis is required. In this study, isolates of P. damselae were obtained from commercial aquaculture farms in Thailand during 2019-2021. Analyses of 16S rRNA and the urease subunit alpha genes identified the isolates as P. damselae subsp. damselae (Phdd). Antibiotic susceptibility analyses showed that all Phdd isolates were resistant to amoxicillin (10 µg). Haemolysis and phospholipase activities were used to categorize P. damselae into three groups based on their biological activities. The pathogenicity of four candidates (SK136, PD001, PD002 and T11L) was tested in Asian seabass. Isolate SK136 showed the highest virulence, with a lethal dose (LD50) of 1.47 × 105 CFU/fish, whereas isolate PD001 did not show any virulence. Genotypic characterization, based on multi-locus sequence typing analysis, demonstrated that all candidates were novel strains with new sequence types (64, 65, 66 and 67). Preliminary vaccination using formalin-killed cells (FKCs) protected Asian seabass from artificial challenges. Taken together, these results provide fundamental knowledge for vaccine development against Phdd infection in Asian seabass.

Systemic immune responses do not affect significant immune responses in the skin.

Fish skin plays an important role in defending against pathogens in water, primarily through the secretion of skin mucus containing various immune-related factors. Local immune responses in the skin activate systemic immune responses by inflammatory cytokines. However, it remains unclear whether immune responses in the skin occur after systemic immune responses caused by pathogen invasion into the fish body. This study aimed to clarify the relationship between systemic immune responses and skin responses after intraperitoneal injection of formalin-killed cells (FKC) of Vibrio anguillarum. Although systemic inflammatory responses were observed in the spleen after injection, expression changes in the skin did not show significant differences. In contrast, expression of hemoglobin subunit genes significantly increased in the skin after FKC injection, suggesting that erythrocytes infiltrate extravascularly.

De Novo Assembly and Annotation of the Siganus fuscescens (Houttuyn, 1782) Genome: Marking a Pioneering Advance for the Siganidae Family.

This study presents the first draft genome of Siganus fuscescens, and thereby establishes the first whole-genome sequence for a species in the Siganidae family. Leveraging both long and short read sequencing technologies, i.e., Oxford Nanopore and Illumina sequencing, we successfully assembled a mitogenome spanning 16.494 Kb and a first haploid genome encompassing 498 Mb. The assembled genome accounted for a 99.6% of the estimated genome size and was organized into 164 contigs with an N50 of 7.2 Mb. This genome assembly showed a GC content of 42.9% and a high Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness score of 99.5% using actinopterygii_odb10 lineage, thereby meeting stringent quality standards. In addition to its structural aspects, our study also examined the functional genomics of this species, including the intricate capacity to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) and secrete venom. Notably, our analyses revealed various repeats elements, which collectively constituted 17.43% of the genome. Moreover, annotation of 28,351 genes uncovered both shared genetic signatures and those that are unique to S. fuscescens. Our assembled genome also displayed a moderate prevalence of gene duplication compared to other fish species, which suggests that this species has a distinctive evolutionary trajectory and potentially unique functional constraints. Taken altogether, this genomic resource establishes a robust foundation for future research on the biology, evolution, and the aquaculture potential of S. fuscescens.

Molecular characterization of a short-chained pentraxin gene from kuruma shrimp Marsupenaeus japonicus hemocytes.

Pentraxins (PTXs) are a family of pattern recognition proteins (PRPs) that play a role in pathogen recognition during infection via pathogen-associated molecular patterns (PAMPs). Here, we characterized a short-chained pentraxin isolated from kuruma shrimp (Marsupenaeus japonicus) hemocytes (MjPTX). MjPTX contains the pentraxin signature HxCxS/TWxS (where x can be any amino acid), although the second conserved residue of this signature differed slightly (L instead of C). In the phylogenetic analysis, MjPTX clustered closely with predicted sequences from crustaceans (shrimp, lobster, and crayfish) displaying high sequence identities exceeding 52.67 %. In contrast, MjPTX showed minimal sequence identity when compared to functionally similar proteins in other animals, with sequence identities ranging from 20.42 % (mouse) to 28.14 % (horseshoe crab). MjPTX mRNA transcript levels increased significantly after artificial infection with Vibrio parahaemolyticus (48 h), White Spot Syndrome Virus (72 h) and Yellow Head Virus (24 and 48 h). Assays done in vitro revealed that recombinant MjPTX (rMjPTX) has an ability to agglutinate Gram-negative and Gram-positive bacteria and to bind microbial polysaccharides and bacterial suspensions in the presence of Ca2+. Taken together, our results suggest that MjPTX functions as a classical pattern recognition protein in the presence of calcium ions, that is capable of binding to specific moieties present on the surface of microorganisms and facilitating their clearance.

Metagenome-assembled genomes of three Hepatoplasmataceae provide insights into isopod-mollicute symbiosis.

The digestive organs of terrestrial isopods harbour bacteria of the recently proposed mollicute family Hepatoplasmataceae. The only complete genome available so far for Hepatoplasmataceae is that of 'Candidatus Hepatoplasma crinochetorum'. The scarcity of genome sequences has hampered our understanding of the symbiotic relationship between isopods and mollicutes. Here, we present four complete metagenome-assembled genomes (MAGs) of uncultured Hepatoplasmataceae members identified from shotgun sequencing data of isopods. We propose genomospecies names for three MAGs that show substantial sequence divergence from any previously known Hepatoplamsataceae members: 'Candidatus Tyloplasma litorale' identified from the semiterrestrial isopod Tylos granuliferus, 'Candidatus Hepatoplasma vulgare' identified from the common pill bug Armadillidium vulgare, and 'Candidatus Hepatoplasma scabrum' identified from the common rough woodlouse Porcellio scaber. Phylogenomic analysis of 155 mollicutes confirmed that Hepatoplasmataceae is a sister clade of Metamycoplasmataceae in the order Mycoplasmoidales. The 16S ribosomal RNA gene sequences and phylogenomic analysis showed that 'Candidatus Tyloplasma litorale' and other semiterrestrial isopod-associated mollicutes represent the placeholder genus 'g_Bg2' in the r214 release of the Genome Taxonomy Database, warranting their assignment to a novel genus. Our analysis also revealed that Hepatoplasmataceae lack major metabolic pathways but has a likely intact type IIA CRISPR-Cas9 machinery. Although the localization of the Hepatoplasmatacae members have not been verified microscopically in this study, these genomic characteristics are compatible with the idea that these mollicutes have an ectosymbiotic lifestyle with high nutritional dependence on their host, as has been demonstrated for other members of the family. We could not find evidence that Hepatoplasmataceae encode polysaccharide-degrading enzymes that aid host digestion. If they are to provide nutritional benefits, it may be through extra-copy nucleases, peptidases, and a patatin-like lipase. Exploration of potential host-symbiont interaction-associated genes revealed large, repetitive open reading frames harbouring beta-sandwich domains, possibly involved with host cell adhesion. Overall, genomic analyses suggest that isopod-mollicute symbiosis is not characterized by carbohydrate degradation, and we speculate on their potential role as defensive symbionts through spatial competition with pathogens to prevent infection.

Integrase-associated niche differentiation of endogenous large DNA viruses in crustaceans.

IMPORTANCE: Crustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Red sea bream iridovirus infection downregulates inflammation-related genes in the spleen of rock bream (Oplegnathus fasciatus).

This study investigated the kinetics of red sea bream iridovirus and host gene expression during infection in rock bream (Oplegnathus fasciatus), a species highly sensitive to the virus. After intraperitoneal injection of the viral solution at 104 TCID50/fish, the viral genome copy number in the spleen was 104.7 ± 0.2 and 105.9 ± 0.4 copies/µg DNA at 3 and 5 days post-injection (dpi), respectively. Using transcriptomic analyses via MiSeq, viral gene transcripts were detected at 3 and 5 dpi. Six genes including RING-finger domain-containing protein and laminin-type epidermal growth factor-like domain genes were significantly expressed at 5 dpi. Further, 334 host genes were differentially expressed compared with those before infection. Genes were clustered into four groups based on their expression profiles. Interferon-stimulated genes were more prevalent in groups showing upregulation at 5 dpi and 3 and 5 dpi. In contrast, the group showing downregulation at 3 dpi included inflammation-related genes, such as granzyme and eosinophil peroxidase genes. Downregulation of certain inflammation-related genes may contribute to the susceptibility of this fish to the virus.

Insights Into a Chitin Synthase of Kuruma Shrimp Penaeus japonicus and Its Role in Peritrophic Membrane and Cuticle Formation.

Synthesis of chitin is a subject of great interest in the fields of physiology and immunology of crustaceans. Chitinous tissues include not only the carapace, but also an acellular membrane in the intestine called the peritrophic membrane (PM). Here, we describe the first report of chitin synthase (CHS) of a penaeid shrimp, kuruma shrimp Penaeus japonicus. Histological observations showed that fecal matter in the midgut of kuruma shrimp was wrapped with a PM, which physically separated it from the midgut epithelium. Subsequently, the chitin synthase transcript was amplified from the midgut of the shrimp. The chitin synthase gene of kuruma shrimp (MjCHS) encodes 1,523 amino acid residues. Structural prediction analysis showed that the N-terminal region of MjCHS protein included nine transmembrane helices, the middle region included the catalytic region with several conserved motifs which are found in CHSs from other arthropods, and the C-terminal region included seven transmembrane helices. Although insects have distinct exoskeletal and intestinal chitin synthases, the phylogenetic analysis suggested that crustaceans have a single CHS. MjCHS mRNA was constantly detected in the digestive tract, including the midgut and hepatopancreas of both juvenile and adult kuruma shrimp, suggesting a stable synthesis of chitin in those organs. In contrast, MjCHS mRNA was also detected in the hindgut and uropod of juvenile shrimp. After molting, the mRNA levels of MjCHS in the stomach and uropod were higher than other molting cycles. These results suggest that MjCHS contributes to chitin synthesis in both the digestive tract and the epidermis, providing fundamental insights into chitin synthesis of crustaceans.

A novel liver-specific immunoglobulin heavy chain-like gene in a cartilaginous fish.

We identified a novel immunoglobulin (Ig) heavy chain-like gene (tsIgH) expressed in the liver of the banded houndshark Triakis scyllium by preliminary transcriptomic analysis. The tsIgH gene showed less than 30% of amino acid identities to Ig genes of the shark. The gene encodes one variable domain (VH) and three conserved domains (CH1-CH3) with a predicted signal peptide. Interestingly, this protein has only one cysteine residue in a linker region between VH and CH1 other than those required for the formation of the immunoglobulin domain. Genome sequencing revealed that each of the domains was encoded by a corresponding single exon, and the exon-intron structures of the homologues are conserved in the other cartilaginous fishes. By RT-qPCR analysis, the transcript of the tsIgH gene was observed only in the liver, while that of the IgM was mainly detected in the epigonal organ, liver, and spleen. The novel Ig-heavy chain-like gene in cartilaginous fish may provide new clues to the evolution of immunoglobulin genes.

scRNA-seq Analysis of Hemocytes of Penaeid Shrimp Under Virus Infection.

The classification of cells in non-model organisms has lagged behind the classification of cells in model organisms that have established cluster of differentiation marker sets. To reduce fish diseases, research is needed to better understand immune-related cells, or hemocytes, in non-model organisms like shrimp and other marine invertebrates. In this study, we used Drop-seq to examine how virus infection affected the populations of hemocytes in kuruma shrimp, Penaeus japonicus, which had been artificially infected with a virus. The findings demonstrated that virus infection reduced particular cell populations in circulating hemolymph and inhibited the expression of antimicrobial peptides. We also identified the gene sets that are likely to be responsible for this reduction. Additionally, we identified functionally unknown genes as novel antimicrobial peptides, and we supported this assumption by the fact that these genes were expressed in the population of hemocytes that expressed other antimicrobial peptides. In addition, we aimed to improve the operability of the experiment by conducting Drop-seq with fixed cells as a source and discussed the impact of methanol fixation on Drop-seq data in comparison to previous results obtained without fixation. These results not only deepen our understanding of the immune system of crustaceans but also demonstrate that single-cell analysis can accelerate research on non-model organisms.

MicroRNA Regulation in Infectious Diseases and Its Potential as a Biosensor in Future Aquaculture Industry: A Review.

An infectious disease is the most apprehensive problem in aquaculture as it can lead to high mortality in aquatic organisms and massive economic loss. Even though significant progress has been accomplished in therapeutic, prevention, and diagnostic using several potential technologies, more robust inventions and breakthroughs should be achieved to control the spread of infectious diseases. MicroRNA (miRNA) is an endogenous small non-coding RNA that post-transcriptionally regulates the protein-coding genes. It involves various biological regulatory mechanisms in organisms such as cell differentiation, proliferation, immune responses, development, apoptosis, and others. Furthermore, an miRNA also acts as a mediator to either regulate host responses or enhance the replication of diseases during infection. Therefore, the emergence of miRNAs could be potential candidates for the establishment of diagnostic tools for numerous infectious diseases. Interestingly, studies have revealed that miRNAs can be used as biomarkers and biosensors to detect diseases, and can also be used to design vaccines to attenuate pathogens. This review provides an overview of miRNA biogenesis and specifically focuses on its regulation during infection in aquatic organisms, especially on the host immune responses and how miRNAs enhance the replication of pathogens in the organism. In addition to that, we explored the potential applications, including diagnostic methods and treatments, that can be employed in the aquaculture industry.

Comparative genome analyses of three serotypes of Lactococcus bacteria isolated from diseased cultured striped jack (Pseudocaranx dentex).

Lactococcosis, caused by members of the genus Lactococcus, represents a devastating disease inducing mass mortalities and economic losses in many fish species worldwide. The present work aimed to compare the whole genome sequences of three different serotypes of Lactococcus garvieae isolated from diseased cultured striped jack (Pseudocaranx dentex) in Ehime prefecture, Japan. The three serotypes showed different virulence in the challenge test using Japanese amberjack (Seriola quinqueradiata). The genome sequencing revealed that two of the strains (serotype I and serotype III) were identified as L. garvieae, while the third strain (serotype II) was identified as L. formosensis. The chromosome sizes of the three serotypes ranged from 1.9 to 2.0 Mb; the GC content ranges were 38.2 to 38.9%; and the numbers of predicted protein-coding sequences (CDSs) were from 1922 to 1959. Only the serotype II harbours two plasmids, sizes of around 14 kb and 9 kb. The detected virulence factors varied among the different serotypes with some shared factors like adherence, anti-phagocytosis, secretion system, toxin (haemolysin), serum resistance, antimicrobial resistance and others. The genomes also contained factors responsible for resistance to toxic compounds. The genome of the serotype III tended to encode more prophage regions than the other serotypes.

Type I interferon induced by polyinosinic-polycytidylic acid does not contribute to the efficacy of a formalin-killed cell vaccine against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus).

Polyinosinic-polycytidylic acid (poly I:C) is a type of pathogen-associated molecular pattern that can strongly induce the expression of type I interferon (I-IFN). Our previous study has demonstrated that the combination of poly I:C with a recombinant protein antigen not only stimulated the expression of I-IFN but also conferred protection against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus). In this study, our aim was to develop a better immunogenic and protective fish vaccine, for which we intraperitoneally coinjected P. olivaceus with poly I:C and formalin-killed cells (FKCs) of E. piscicida and compared the efficiency of protection against E. piscicida infection with that of FKC vaccine alone. Results showed that the expression levels of I-IFN, IFN-γ, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and the interferon-stimulated genes (ISGs) ISG15 and Mx were significantly increased in the spleen of fish inoculated with poly I:C + FKC. The results of ELISA showed that the levels of specific serum antibodies in the FKC and FKC + poly I:C groups were gradually increased until 28 days postvaccination and were significantly higher than those in the PBS and poly I:C groups. At 3 weeks after vaccination in the challenge test, the respective cumulative mortality rates of fish in the PBS, FKC, poly I:C, and poly I:C + FKC groups were 46.7%, 20.0%, 33.3%, and 13.3% under low-concentration challenge and 93.3%, 46.7%, 78.6%, and 53.3% under high-concentration challenge. This study showed that poly I:C may not provide an effective adjuvant effect with FKC vaccine for intracellular bacterial infections.

Impact of purine nucleotide on fatty acid metabolism and expression of lipid metabolism-related gene in the liver cell of rainbow trout Oncorhynchus mykiss.

Studies have suggested that dietary purine nucleotides (NT) affect the muscle and liver fatty acid composition of rainbow trout. To examine the direct regulation of liver fatty acid metabolism by purine NT in rainbow trout, the liver cells were cultured in the presence of 500 µmol/L inosine, adenosine, or guanosine monophosphate (IMP, AMP, or GMP). The expression of pparα was significantly decreased in the liver cells cultured with purine NT for 24 h, whereas the expression of fads2 (Δ5) was increased. Docosahexaenoic acid (DHA) content in the liver cells was significantly higher after culturing with GMP. To determine the dose-dependent effects of NT, 50, 100, and 500 µmol/L GMP was added to the liver cells cultured in L-15 medium. At 48 h, 20:4n - 6, 22:5n - 3, 22:6n - 3, Æ© PUFA, and Æ© n - 3 PUFA content in the 50 µM GMP-containing medium was significantly higher compared with the other medium. The expression of Δ5 fads2, elovl2, and elovl5 in the liver cells was significantly higher in the 500 µmol/L GMP-containing medium at 48 h along with increased srebp-1 expression. These results suggest that purine NT directly affect fatty acid composition through modification of fatty acid metabolism-related genes in the liver of rainbow trout.

Genome sequence of Chionoecetes opilio bacilliform virus, a nimavirus infecting the snow crab Chionoecetes opilio.

Nimaviridae (class Naldaviricetes) is a family of double-stranded DNA viruses infecting crustaceans, with the only officially recognized representative being white spot syndrome virus (WSSV). Chionoecetes opilio bacilliform virus (CoBV) was isolated as the causative agent of milky hemolymph disease in the snow crab Chionoecetes opilio, an economically important crustacean in the northwestern Pacific. Here, we present the complete genome sequence of CoBV and show that it is unambiguously a nimavirus. The CoBV genome is a 240-kb circular DNA molecule with 40% GC content that encodes 105 proteins, including 76 WSSV orthologs. Phylogenetic analysis based on eight naldaviral core genes established that CoBV is a member of the family Nimaviridae. The availability of the CoBV genome sequence provides a deeper understanding of CoBV pathogenicity and nimavirus evolution.

Genomic characterization and identification of virulence-related genes in Vibrio nigripulchritudo isolated from white leg shrimp Penaeus vannamei.

Vibrio nigripulchritudo causes vibriosis in penaeid shrimps. Here, we used Illumina and Nanopore sequencing technologies to sequence the genomes of three of its strains (TUMSAT-V. nig1, TUMSAT-V. nig2, and TUMSAT-V. nig3) to explore opportunities for disease management. Putative virulence factors and mobile genetic elements were detected while evaluating the phylogenetic relationship of each isolated strain. The genomes consisted of two circular chromosomes (I and II) plus one or two plasmids. The size of chromosome I ranged from 4.02 to 4.07 Mb with an average GC content of 46%, while the number of predicted CDSs ranged from 3563 to 3644. The size of chromosome II ranged from 2.16 to 2.18 Mb, with an average GC content of 45.5%, and the number of predicted CDSs ranged from 1970 to 1987. Numerous virulence genes were identified related to adherence, antiphagocytosis, chemotaxis, motility, iron uptake, quorum sensing, secretion systems, and toxins in all three genomes. Higher numbers of prophages and genomic islands found in TUMSAT-V. nig1 suggest that the strain has experienced numerous horizontal gene transfer events. The presence of antimicrobial resistance genes suggests that the strains have multidrug resistance. Comparative genomic analysis showed that all three strains belonged to the same clade.