The study presents the first report of the ISKNV whole genome sequence isolated from angel fish (Pterophyllum scalare) in India, offering insights into gene composition, function, phylogeny, and potential vaccine development. The complete genome of AK-ISKNV was found to be of 110,460 bp with the GC content of 54.79 %. The complete genome has 122 open reading frames (ORFs) and 48 ORFs were predicted as functional proteins while the rest were hypothetical proteins. Phylogenetic tree constructed based on the major capsid protein and ATPase genes revealed AK-ISKNV in this study belong to the genus Megalocytivirus of the ISKNV-Genotype I. The whole genome exhibits a 99.81 % similarity, and the phylogenetic analysis indicates a close association with Angel Fish Iridovirus (AFIV), specifically identified as MK689685.1, originating from Australia. Further, phylogenetic analysis confirms the ISKNV genotype and its distinction from RSIV. The present study provides the first report of the complete genome sequence and annotation of the ISKNV strain (AK-ISKNV) isolated from India. Four proteins were selected as prospective vaccination candidates based on their high B-cell epitope score and antigenic locations. These proteins include myristylated membrane protein (ORF 105), main capsid protein (ORF 104), and two hypothetical proteins (ORF 11 and ORF 14). In conclusion, this research contributes vital genomic information essential for understanding and mitigating the impact of ISKNV, providing a foundation for targeted disease control measures in aquaculture.

Abstract White spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), and Acute hepatopancreatic necrosis disease (AHPND) are the major threats to the whiteleg shrimp, Litopenaeus vannamei. This study was conducted to develop multiplex polymerase chain reaction (M-PCR) for the detection of shrimp pathogens. This study was conducted between January 2020 to July 2022. Total of 328 samples were collected from shrimp farms of Karnataka and they were screened for WSSV, EHP, and AHPND by M-PCR. Several parameters were optimized for the standardization of M-PCR. The specificity and sensitivity of the reaction were evaluated. The results showed that this technique can detect WSSV, AHPND, and EHP in a single reaction with high specificity. Sensitivity result showed it can detect WSSV (0.3 pg/µl), AHPND (0.1 pg/µl), and EHP (0.2 pg/µl). Out of 328 samples, 72 (21.9%) samples were found to be positive for EHP, 16 (4.8%) samples were found to have been infected by WSSV and 5 (1.5%) samples were found to have multiple infections with WSSV, and EHP. In consideration of the specificity and sensitivity of this technique, we conclude that M-PCR could be used instead of a conventional PCR assay targeting individual genes for the rapid detection of WSSV, AHPND, and EHP simultaneously.

Litopenaeus vannamei, commonly known as the Whiteleg shrimp, emerges as one of the most economically lucrative species in India's commercial aquaculture sector. The primary objective of this study is to detect common pathogens affecting cultured shrimp in Karnataka using polymerase chain reaction (PCR) analysis. Over the period spanning from January 2020 to March 2022, a total of 278 samples were meticulously collected from shrimp farms in Karnataka. The screening process targeted both Office of Internationale des Epizootics (OIE) listed and non-OIE listed shrimp pathogens. The PCR analysis revealed the presence of white spot syndrome virus and Enterocytozoon hepatopenaei in the collected samples, with no detection of other pathogens. Out of the 278 samples, 63 (22.6%) were confirmed positive for EHP, while 14 (5%) showed infection by WSSV. Intriguingly, three samples were concurrently infected by both EHP and WSSV. Shrimp samples infected with WSSV exhibited distinctive white spots on the carapace, whereas EHP-infected shrimps displayed symptoms such as size variation and a white gut. Further histopathology analysis of gill tissue infected with WSSV revealed basophilic intra-nuclear inclusion bodies, while hepatopancreas tissue infected with EHP exhibited basophilic plasmodium. Notably, the study underscored a higher prevalence of EHP in shrimp farms across Karnataka. In light of these findings, the study strongly advocates for the implementation of stringent biosecurity measures and robust management practices in shrimp culture areas, emphasizing their critical role in sustaining the cultivation of L. vannamei in India.

Abstract Motile Aeromonas Septicaemia (MAS) is the most common bacterial fish illness. This is a opportunistic microorganisms that produce hemorrhagic septicaemia and ulcerative diseases in a wide range of aquatic and terrestrial animals, including humans. Antibiotics have been used as a most prevalent control option for MAS in both hatcheries and farms, despite the fact that it not only kill target bacteria but might harm host’s natural habitat and water quality parameters. So urge of using phage therapy among researchers is now increasing. Phages or bacteriophages are viruses that infect and replicate within bacteria and help to decline bacterial growth. In the present study Aeromonas phage GomatiRiver_11 phage has been isolated for the first time from various water bodies of Tripura, India. The whole genome sequence of the phage was found to be 10,390 bp with a total of 9 ORFs. The phylogenetic analysis of the species revealed that this phage is closely related to Aeromonas phage Asfd_1 (MK577502.1). Moreover, the electron microscopy analysis revealed Podoviridae characteristics of characterized phage genome. This new phage genome and its analysis will be essential for phage therapy against MAS affected fish species of Northeast India and associated water bodies.

Aeromonas species is ubiquitously present in aquatic habitats, can cause motile Aeromonas septicemia (MAS). However, it is very complex to identify Aeromonas spp. based on its phenotypes. In the present study in mid -November 2018, we investigated a case of severe mortality of wild caught mascara barb (Dawkinsia assimilis) endemic to the Western Ghats, India. No cytopathic effect was observed in DRG and CSK fish cell line after inoculating filtrate of the tissue homogenates and found negative for all major viral pathogens. Here, aetiological agent was identified as bacteria, presumed as Aeromonas hydrophila, and Escherichia coli based on biochemical tests. Later, PCR assay was performed for the molecular confirmation by gyrB gene resulted in confirmation of Aeromonas as A. dhakensis with 99.63% similarity with A. dhakensis from SA1 of diseased pacu. Multiple anti-biotic resistance (MAR) index of A. dhakensis was 0.9112. Further, A. dhakensis was positive for ten antibiotic resistance and seven virulence genes with beta-hemolysis colonies. The LD50 value was found to be 3.42 x 105 CFU/mL in experimental trails. In the present study, a tetracycline, colistin, and ESBL resistant hypervirulent A. dhakensis was isolated from the diseased endemic mascara barb. Ten natural ingredients and five disinfectants found to be effective against A. dhakensis. To the best of our knowledge, this is the first evidence of extensively drug-resistant (XDR) virulent A. dhakensis associated with disease outbreak in endemic mascara barb in India.

Gut microbiota is known to influence the health, nutrient absorption, reproduction, and other metabolic activities in both terrestrial and aquatic animals and can be used as biomarker to predict the performance in selective breeding programs. Several studies have generated information on microbial community structures associated with, developmental stages, dietary modifications, osmotic stress, and wild and captive production conditions. The analysis of wild and captive reared P. indicus gut microbiota revealed that Proteobacteria, Fusobacteria, Tenericutes, and Bacteroidetes, were the dominant phyla, indicating host is the major determinant factor in theformation of gut microbiota. Microbes associated with the developmental stages (egg, nauplii, zoea, mysis, PL1, PL6 and PL12) showed that Proteobacteria and Bacteroidetes, were the two dominant phyla. To study the effects of the probiotics on the gut microbiota, a combination of Bacillus spp. were added into the rearing environment of shrimp larvae. The analysis showed that the abundance of Proteobacteria was higher in probiotic treated group compared to control, indicating the possibilities for microbiota modulation at early stages. Further, understanding how environmental factors influence the shaping of gut microbiota will aid in using gut microbiota as a tool to prevent and control diseases in aquaculture. Intestine of shrimps adapted to two different salinities showed that the dominant phyla recorded was Proteobacteria followed by Bacteroidetes and Fusobacteria, though the abundance of these microbes varied significantly due change in salinity. These observations provide valuable information to device intervention strategies for health and production.

In India, culture of tilapines has been gaining importance over last few decades and it is the second widely farmed fish species in the World. There have been increased large-scale mortalities observed in last few years due to DNA and RNA virus associated with coinfection of bacteria, fungi, parasites etc., were considered as a major global threat to the tilapia and aquaculture industry. In this study, large scale mortality of tilapia (Oreochromis mossambicus) was observed in wild populations located in Krishna Raja Sagara reservoir in Mysuru, Thonnur Lake and Erode Lake in Pandavapura, Mandya. A detailed investigation was performed from these regions which reported mass mortality in these water bodies. Tissues from the fishes (brain, gill, kidney, spleen and liver) were collected for examination of parasites, histology and molecular detection (virus and bacteria). The results of polymerase chain reaction showed that all the samples were found negative for DNA viruses and for one RNA virus namely VNN but all the samples from 3 regions (reservoir and lakes) were positive for TiLV. Syncytial hepatitis lesion along with glial cells were observed in the TiLV-infected tilapia. TiLV isolated from this study (OM1 and OM2) shared 94.30% and 95.52% nucleotide identity with the TiLV isolated from West Bengal, India (MF502419.1) and Israel (KJ605629.1) respectively. NCBI-BLAST search results using gyrB gene revealed that the isolates G 1 & H 2 were identified and confirmed as Aeromonas veronii with 99.52% and 99.42% nucleotide similarity to the A. veronii isolate XX-57 (JX025939.1) from diseased Chub and A. veronii isolate JH-12 (JX025895.1) from diseased gold fish from China respectively. To the best of our knowledge, this is the first report of concurrent infection TiLV and A. veronii associated with mass mortality in Oreochromis mossambicus in Karnataka, India. In vitro study showed the cytopathic effects on OniL cell lines with initially elongated cells followed by rounding and finally detachment leading to death of the cells.

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 β-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.

Penaeus vannamei farming is one of the profitable businesses of the aquaculture sector in India. As a result of excessive farming, shrimps were susceptible to various exotic, transboundary, and emerging pathogens. The purpose of this study is to monitor the prevalence of shrimp pathogens in Karnataka. A total of 91 samples of P. vannamei post larvae and juveniles were collected from April 2022 to November 2022 in Karnataka. Samples were screened for pathogens as listed in Office of Internationale des Epizootics (OIE) or World Organisation for Animal Health (WOAH) which include infectious hypodermal and hematopoietic necrosis virus (IHHNV), Enterocytozoon hepatopenaei (EHP), acute hepatopancreatic necrosis disease (AHPND), hepatopancreatic parvovirus (HPV), infectious myonecrosis virus (IMNV), yellow head virus (YHV), taura syndrome virus (TSV), white spot syndrome virus (WSSV), and other diseases such as decapod iridescent virus-1(DIV-1), and monodon baculovirus (MBV). Out of 91 samples, 5 (5.5%) samples were positive for WSSV, 26 (28.6%) samples were positive for EHP, and 2 (2.2%) samples had co-infection caused by EHP and WSSV. In this study, we have reported a high prevalence of EHP than WSSV in all three coastal districts of Karnataka. Farmers were using specific pathogen-free (SPF) seeds for culture but still, their cultures are getting infected with the same pathogens which indicates poor pond preparation and bio-security. So we strongly recommend that farmers have to follow good management practices and bio-security to increase the productivity and sustainability of P. vannamei farming in India.

Background:Nile tilapia is a highly valuable fish in the aquaculture sector. A culture farm has reported heavy mortalities of tilapia. Aims:The present study aimed to identify the etiological agent responsible for the heavy mortality in cage cultured tilapia. Methods:The moribund and freshly dead fishes were analyzed for clinical signs. Biochemical and molecular characterizations were performed to identify the etiological agents of the disease. Also, polymerase chain reaction (PCR) assay was used to detect the presence of the virulence genes. The susceptibility of the isolates to various antibiotics was tested by the disk diffusion method. Results:The results of the biochemical tests and PCR assay confirmed that co-infection with Aeromonas hydrophila, and Streptococcus iniae was responsible for the disease severity. Phylogenetic analysis of the 16S rRNA gene showed that A. hydrophila and S. iniae isolates shared 99% and 98% sequence homology with A . hydrophila and S. iniae previously deposited in the Genbank database. The multiple antibiotic resistance (MAR) index of A. hydrophila was 0.16 and that of S. iniae was 0.71. The PCR test revealed that both pathogens harbored numerous virulence factors. The experimental infection study confirmed that the synergistic action of A. hydrophila and S. iniae led to increased mortality in tilapia. Histopathological changes were observed in the liver and spleen tissues of the co-infected fishes. Conclusion:These findings indicate that the disease outbreak in the tilapia culture farm occurred as a result of co-infection by A. hydrophila and S. iniae.

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.

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 (T-1 - 0, T-2 - 0.5, T-3 - 1.0 and T-4 - 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 im proved growth, better selenium regulation and expression of immune-regulated selenoproteins in the fish model.

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.

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.

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.

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.

Vibrio anguillarum has been described as a leading cause of disease in various species of aquatic animals from many parts of the world. Fish vaccination is a useful strategy for several disease problems in aquaculture. Outer membrane proteins of bacteria are considered ideal candidates for vaccine development. In the present study, OmpK gene of V. anguillarum was cloned and sequenced. The recombinant OmpK was overexpressed and purified by affinity chromatography on Ni-NTA Superflow resin. Immunoreactivity of the pure OmpK was studied by western blotting. The fresh water Indian major carp Labeo rohita (Hamilton, 1822) was well protected when challenged with V. anguillarum after vaccination with OmpK compared to non vaccinated fish. Specific antibody against OmpK and bacterial inhibition was detected in the vaccinated fish. Our results suggest OmpK as a candidate vaccine molecule against V. anguillarum.