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.