Harresh Adikesavalu

Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

Pathology of Edwardsiella tarda infection in African catfish, Clarias gariepinus (Burchell 1822), fingerlings

Abstract Edwardsiella tarda is one of the serious fish pathogens infecting both cultured and wild fish species. This study aimed to assess the phenotypic characterization and pathogenicity of E. tarda isolated from Clarias gariepinus (Burchell) with dropsy and histopathological alterations. The causative agent was identified with Vitek 2, and its pathogenicity was determined by intramuscular injection. The challenged catfish exhibited vertical hanging, frothing, excess mucus production, listing, swollen abdomen, anorexia, fin and tail rot, and reddish operculum. The LD50 of E. tarda PBB and PBP strains was found to be 8.52 × 106 and 1.68 × 107 cells fish-1, respectively. Histopathological observations on catfish infected naturally revealed lymphocyte infiltration in muscle and focal necrosis, hyperplasia, edema, and swelling of the gill lamellar epithelium. The kidney of diseased fish exhibited ischemic type tubulopathy, necrosis of nephritic tubules, hyperplastic hematopoietic tissue, rupture of the tubular basement membrane, hydropic dystrophy of nephritic cells, neutrophil infiltration, fibrinoid necrosis of nephretic tubules, hemosiderin deposition, and edema. The liver sections revealed lymphocyte infiltration, dilation of hepatic sinusoids, expansion of space between hepatic sinusoids, and focal necrosis. The inflammatory responses observed in kidney and liver in the present study were presumably suppuration and were attributed to the potential virulence factors of E. tarda.

Rapid Detection of Flavobacterium columnare Infection in Fish by SpeciesspecificPolymerase Chain Reaction

This study describes rapid detection of Flavobacterium columnare induced columnaris disease in cultured freshwater fish, viz., Labeo rohita, Ctenopharyngodon idella, Puntius sp. and Anabas testudineus by species-specific polymerase chain reaction. Gill discolouration, yellow necrotic areas, white patches on gill, saddle back and erosion of scales were the prominent clinical signs in all diseased fish, except Puntius sp., which had typical signs of ulcer at the base of dorsal fin. Of the nine disease cases, eight were found columnaris positive through culture independent species-specific PCR. The two sets of F. columnare specific primers such as ColF, ColR and Col72F, Col1260R yielded amplicons of around 675 bp and 1000 bp, respectively in all positive samples. Phylogenetically, the nucleotide sequences of the positive samples namely, C1 and RG1 formed monophyletic group with F. columnare, thus confirmed the infection as columnaris.

Immunomodulatory effect of Guavarine®, aqueous guava leaf extract, on ornamental Koi carp Cyprinus carpio var. koi L. 1758

ABSTRACT The leaves and bark of guava (Psidium guajava) have a long history of medicinal uses. The immunomodulatory effect of Guavarine®, an aqueous guava leaf extract, was investigated on Cyprinus carpio var. koi L. The fish (8.33 ± 1.02 g) were injected with guava leaf extracts (10 or 100 µg/fish) intraperitoneally, and the innate immune parameters were monitored for 28 days. In fish injected with guava leaf extracts, the lymphocyte proliferation, nitric oxide production, and respiratory burst activity increased until the 14th day and decreased thereafter. The results indicated an elevation in the innate immune status of fish, possibly due to the presence of flavonoids in the leaf extracts.

ANTIBIOTIC RESISTANT MOTILE AEROMONADS INDUCED SEPTICEMIA IN PHILIPPINE CATFISH Clarias batrachus (Linnaeus, 1758) FINGERLINGS

Philippine catfish, Clarias batrachus farming is receiving much attention in recent years so also the diseases in catfish aquaculture. During the disease surveillance in catfish farms, mortalities were observed in C. batrachus fingerlings in a nursery and this report describes the bacteriological and histopathological observations made on the diseased catfish. The gross and clinical signs observed were lethargy, anorexia, vertical movement, skin erosion, operculum erosion, pale gills, reddening of mouth, fin haemorrhage, red lateral line and distended abdomen. The bacteria isolated from the kidney were identified phenotypically as species belonging to classical motile aeromonad group (Aeromonas caviae, A. hydrophila, A. jandaei and A. sobria) and newly described aeromonad group (A. aquariorum, A. fluvialis and A. rivuli). Multiple antibiotic resistance (MAR) index was in the range of 0.3-0.8. These antibiotic resistant motile aeromonads caused septicaemia and 100% mortality. Histologically, haemocyte infiltration, necrosis, inflammation of epidermal tissue, rough epidermal layer and fibrosis in muscle tissue, and loss of typical tubular epithelial lining, necrosis of tubular tissue, inflammation of epithelial layer, cellular and nuclear hypertrophy, pycnotic nuclei, karyolysis and hypoplastic haematopoietic tissue in the kidney of diseased catfish were noted. The inflammatory responses observed in the kidney of C. batrachus were indicative of suppurative infection.

Molecular characterization of Myxobolus catmrigalae (Myxosporea: Myxobolidae) infecting the gill lamellae of carp Cirrhinus mrigala (Hamilton)

The present study attempted sequencing the 18S rRNA gene of Myxoboluscatmrigalae infecting the gill lamellae of carp, Cirrhinusmrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30–50 spores each. The spore size was 17.90xa0±xa00.70xa0×xa07.40xa0±xa00.40xa0μm. The sporoplasm contained two large nuclei of size 0.57xa0±xa00.09xa0μm and no iodinophilous vacuole. The DNA sequence of M.catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77–87xa0% homogeneity. On the phylogenetic tree, M.catmrigalae (KC933944) was clustered with M.pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthysmolitrix. The species most closely related to M.catmrigalae in GenBank was M.pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthysnobilis with 87xa0% homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

Meningoencephalitis in farmed monosex Nile tilapia (Oreochromis niloticus L.) caused by Streptococcus agalactiae

Abstract Aquaculture of tilapia is a new research venture in India. With intensification in farming practices, tilapia are increasingly susceptible to bacterial infections. This article describes the isolation and identification of pathogenic bacteria from cultured monosex Nile tilapia, Oreochromis niloticus (L.), that experienced moderate to severe mortalities in West Bengal, India between September and August 2014 and histopathological alterations in various organs. Gram-positive diplococci, identified as Streptococcus agalactiae with Streptococcus identification kits and 16S rDNA sequencing analysis, were isolated from the brain, operculum, and kidney. Other bacteria from the kidney were identified as Aeromonas sobria, A. caviae, Klebsiella pneumoniae ssp. pneumoniae, Escherichia coli, and Enterobacter cloacae. Staphylococcus epidermis was isolated from opercular hemorrhages. Histological sections of the infected tilapia brain revealed meningoencephalitis and granulomatous lesions. Sections from other organs indicated congestion, hemorrhagic and hyperplastic cells, necrosis, vacuolation, hemosiderin deposition, hypertrophic nuclei, melanomacrophage aggregation, and ruptured veins. This report is the first description of S. agalactiae as a primary pathogen causing meningoencephalitis in cultured tilapia in India.

Polypeptide Profiling of Pangas Catfish (Pangasius pangasius) Serum Globulin Protein Fraction and Development of Anti-pangas Serum Globulin-HRPO Immunoconjugate for Rapid Detection of Bacterial Infection

Bacterial diseases continue to be a major economic factor for commercial catfish farming. Correct diagnosis of diseases is, therefore, important for effective treatment measures and to prevent production losses. The present study aimed at fractionating and characterizing the serum globulin proteins of pangas catfish, Pangasius pangasius by salt fractionation and SDS-PAGE analysis. The protein profile of fractionated serum globulin proteins of P. pangasius revealed a total of 12 bands, viz., 136.64, 120.58, 95.87, 79.92, 72.67, 63.04, 45.99, 43.32, 38.65, 34.82, 28.46 and 19.08 kDa. Anti-pangas serum globulin (ASG) horseradish peroxidase (HRPO) immunoconjugate (ASGHRPO immunoconjugate) was prepared following sodium periodate procedure and raising immune serum against it in rabbit. The sero-reactivity of ASG-HRPO immunoconjugate was assessed by dot-ELISA and direct ELISA. In dot-ELISA, development of intense colour spot was noted only when ASG-HRPO immunoconjugate was used either at neat or 1:10 dilution. Similarly, the direct ELISA results showed low sero-reactivity with a dilution titer value <200 against pangas serum globulin protein fraction as antigen. Dot-ELISA was also performed to detect the presence of specific antibodies against Aeromonas hydrophila and Edwardsiella tarda in P. pangasius separately by experimental inoculation. Development of intense colour spot was observed in both cases, which in turn indicated the specificity of ASG-HRPO immunoconjugate in detecting the anti- A. hydrophila and anti- E. tarda antibodies in P. pangasius serum. The development of colour spot upon the addition of sensitized fish immune serum and incubation suggested that the ASG-HRPO immunoconjugate can be used for the seromonitoring of specific antibodies and serodiagnosis of diseases caused by E. tarda and A. hydrophila.

Association of Aeromonas salmonicida subsp. achromogenes in the haemorrhagic blister of cultured carp Cyprinus carpio in West Bengal, India

Abstract Objective To study the association of Aeromonas salmonicida subsp. achromogenes in the haemorrhagic blister of cultured exotic carp Cyprinus carpio in West Bengal, India, its cellular fatty acid composition and antibiotic sensitivity. Methods The phenotypic characterization and antibiotic sensitivity of bacterial flora of diseased fish were performed. The whole cellular fatty acid composition of Aeromonas salmonicida ( A. salmonicida ) was determined by MIS Sherlock automatic identification system. Results Aeromonas hydrophila, A. salmonicida and Pseudomonas alcaligenes were isolated from the haemorrhagic blister. The fatty acid methyl esters pattern confirmed the A. salmonicida strain as A. salmonicida subsp. achromogenes . Unsaturated fatty acid 16:1 w7c/16:1 w6c (39.09%), saturated fatty acid 16:0 (26.84%) and the mono-unsaturated fatty acids 18:1 w7c (8.89%) and 16:1 iso I/14:0 3OH (8.49%) were the most common fatty acids, which accounted for 83.31% of the total fatty acids. A. salmonicida subsp. achromogenes was highly susceptible to broad spectrum antibiotics such as chloramphenicol, ciprofloxacin, co-trimoxazole, gentamycin, nitrofurantoin and oxytetracycline. Conclusions The finding on the presence of A. salmonicida in carps necessitates molecular level study on establishing the prevalence of this bacterium in Indian aquaculture systems especially on its free-living viable but non-culturable state.