Large scale mortality in cultured Nile tilapia (Oreochromis niloticus): natural co-infection with Aeromonas hydrophila and Streptococcus iniae.

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.

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.

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.

Isolation, characterization and genomic analysis of vB-AhyM-AP1, a lytic bacteriophage infecting Aeromonas hydrophila.

AIMS: Aeromonas hydrophila is a zoonotic pathogen displaying resistance to multiple antibiotics. Here, we aim to develop a candidate biocontrol agent against A. hydrophila. METHODS AND RESULTS: In this study, we isolated and characterized the phage vB-AhyM-AP1 from sewage. It showed lytic activity against A. hydrophila strains. One-step growth curve revealed that the latent period lasted for 40 min. The burst size of one lytic cycle was 1413 PFU per infected cell. Temperature stability studies showed that the phage vB-AhyM-AP1 was active over temperatures ranging from 4 to 45°C for 1 h. pH stability studies indicated that the phage remained active within a pH range of 5-10 after 24 h of incubation. Stability tests in salt solutions showed that the phage was stable at salinities ranging from 0·1 to 2%. The phage also showed stabilities in organic solvents when incubated for 10 min. The Illumina Hiseq sequencing of its genome indicated that the phage vB-AhyM-AP1was a jumbo phage with a genome size of 2, 54 490 bp and GC content of 40·3%. The phylogenetic analysis of the terminase large subunit and major capsid protein indicated that the phage closely clustered with other Tevenvirinae phages. The genome encoded 455 ORFs and 22 tRNAs. The phage resulted in a reduction of 0·8 log units of viable A. hydrophila cells in biofilms grown on PVC coupons maintained in a low nutrient medium for 10 days. CONCLUSIONS: The phage showed lytic activity against planktonic and biofilm cells of A. hydrophila. Genome-based prediction showed it to be a strictly lytic phage without any virulence or antibiotic resistance genes indicating safety for environmental and clinical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The multidrug-resistant strains of A. hydrophila pose a significant health risk to both cultured fishes and consumers leaving few options for treatment. Phage vB-AhyM-AP1 may be used as a candidate biocontrol agent against A. hydrophila strains.

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.

Pathogenic marine microbes influence the effects of climate change on a commercially important tropical bivalve.

There is growing evidence that climate change will increase the prevalence of toxic algae and harmful bacteria, which can accumulate in marine bivalves. However, we know little about any possible interactions between exposure to these microorganisms and the effects of climate change on bivalve health, or about how this may affect the bivalve toxin-pathogen load. In mesocosm experiments, mussels, Perna viridis, were subjected to simulated climate change (warming and/or hyposalinity) and exposed to harmful bacteria and/or toxin-producing dinoflagellates. We found significant interactions between climate change and these microbes on metabolic and/or immunobiological function and toxin-pathogen load in mussels. Surprisingly, however, these effects were virtually eliminated when mussels were exposed to both harmful microorganisms simultaneously. This study is the first to examine the effects of climate change on determining mussel toxin-pathogen load in an ecologically relevant, multi-trophic context. The results may have considerable implications for seafood safety.

Evaluation of a digoxigenin-labelled probe for detection of Aeromonas spp.

An outer membrane protein-based Digoxigenin (DIG)-labelled DNA probe was developed for the specific detection of Aeromonas sp. from food/environmental/clinical samples. Dot blot reaction answered for all the Aeromonas isolates and was negative for Escherichia coli, Pseudomonas sp., Klebsiella sp., Vibrio parahaemolyticus, V. harveyi, V. alginolyticus, V. vulnificus. Edwardsiella tarda and Staphylococcus sp. As this protein is highly conserved in various Aeromonas species, the probe has the potential for use as a rapid and reliable diagnostic tool.

Cloning and expression of an outer membrane protein ompTS of Aeromonas hydrophila and study of immunogenicity in fish.

The outer membrane proteins of the warm water fish pathogen, Aeromonas hydrophila have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein designated ompTS was amplified by PCR excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and expressed using induction with isopropyl thiogalactoside (IPTG). The size of the expressed protein was 37 kDa as estimated by migration in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyclonal antibodies were raised in mice and rabbit against the purified protein and the reaction of the antibody was confirmed by Western blotting using the purified protein and A. hydrophila cultures. The Indian major carp, Labeo rohita Hamilton was immunized using the purified protein and developed antibodies with mean titers of 1:4000 on day 14 and 1:12,000 on day 28 showing promise that the protein is highly immunogenic in fish.