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ThesisItem Open Access SCREENING OF BACTERIAL PATHOGENS ASSOCIATED WITH DISEASED FRESHWATER ORNAMENTAL FISHES(KARNATAKA VETERINARY, ANIMAL & FISHERIES SCIENCES UNIVERSITY, BIDAR, 2021) AMIT KUMAR SOREN,; S.K.GIRISHABacterial diseases are the major constraints in each and every fish culture system of ornamental fishery sector. Bacteria are considered as ubiquitous and are always in contact with the fish. Hence the risk of catching a disease is high when the fish falls under stressful conditions. Screening for major pathogenic bacteria associated with ornamental fish diseases will aid in knowing the probable reason for the untimely death in many ornamental cultures and rearing systems. In the present study, 50 isolates of bacteria were screened from 30 freshwater ornamental fish samples. The fishes exhibited clinical signs like exophthalmia, dropsy, erratic movement, dermal ulceration, haemorrhages, etc. The isolates were subjected to series of biochemical tests and polymerase chain reaction (PCR) assay for identifying their respective genus/species. The tests revealed that the isolates were dominated by the Gram-negative bacteria with Aeromonas spp., followed by Citrobacter spp. and Pleiosomonas spp. Virulence factors like haemolysis, gelatinase and starch hydrolysis were performed. Haemolytic assay revealed that 48% of the isolates were α-haemolytic and 26% were β-haemolytic. Amylase and gelatinase activity was present in 46% and 50% of the isolates respectively. Further, selected 4 isolates (positive for all the virulence factors) were used for 16S rRNA sequencing. The obtained sequences indicated the 4 selected isolates as Aeromonas hydrophila (F3), A. veronii (D2), A. sobria (F7) and Citrobacter freundii (G9-1A); two of them were deposited in GenBank [MN594480.1 (A. hydrophila F3) and MN593732.1 (A. veronii D2)]. The phylogenetic tree analysis found that A. hydrophila, A. sobria, A. veronii. and C. freundii were closely related to reference strain of A. hydrophila (FN997612.1), A.sobria (KM516017.1), A.veronii (EU770280.1) and C. freundii EnZ-4 (MN220573.1) respectively. The antibiotic susceptibility test of the 4 isolates revealThesisItem Open Access EVALUATION OF OUTER MEMBRANE PROTEIN K (OMPK) IN PROTECTING LITOPENAEUS VANNAMEI AGAINST VIBRIO HARVEYI INFECTION(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2020) SAPTAMI. R. JOGALEKAR; M.N.VENUGOPALOuter membrane proteins (OMPs) are present in many prokaryotes and in some organelles of eukaryotic cells. In Gram-negative bacteria, they are considered as the important molecules as they play various roles in bacterial adaptation including pathogenicity of bacterium. As OMPs are present on the outer surface of the bacterial cell and are the first line of contact between the bacterium and its surroundings, they are considered as good vaccine candidates. Studies have showed that vaccines consisting of immunogenic fractions can induce higher protection than inactivated whole-cell bacteria in fish and other vertebrates. Research has shown OMPs extracted from several bacterial fish pathogens viz., Edwardsiella ictaluri, E. tarda, Vibrio vulnificus, Aeromonas salmonicida, and A. hydrophila to be protective antigens in fish. As no study is currently available for the evaluation of the effect of OmpK on shrimp, present work aims to study the immunogenic potential of OmpK, and its suitability as a protective candidate against Vibrio harveyi infection in the shrimp, Litopenaeus vannamei. In the present study, preliminary investigation has been undertaken into existing OmpK sequences in GenBank database engaging bioinformatics tools to understand the suitability of OmpK as a vaccine candidate. Primers were designed for the amplification of OmpK gene of V. harveyi, and the gene was later cloned and expressed using E. coli SG cells. Further, the protein was purified through Ni-NTA affinity chromatography after successful confirmation by SDS-PAGE, and its effectiveness against V. harveyi was evaluated which showed RPS 75 respectively. Our results suggest that OmpK of V. harveyi could be used as a potential protective candidate for L. vannamei against V. harveyi infection.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF VIBRIO MIMICUS AND VIBRIO (GRIMONTIA) HOLLISAE FROM SEAFOOD(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2020) PAVANA, V.,; M.N.VENUGOPALhealthy diet. Major health risks are involved in the consumption of raw or undercooked seafood that may be naturally contaminatedby foodborne pathogens present in the marine environment. Group of Vibrios are associated with live seafood, asthey are the indigenous microflora of the marine environment. Seafood contaminated withpathogenic Vibrios not only play an important role in the transmission of diseases but also act as areservoir in the marine realms. As the foodborne infections of Vibrio spp. are common in seafood, it is necessary to survey thepresence of toxigenic and non-toxigenic Vibrios from various seafood and their environment. In the present study, 50 different seafood samples were collected from Mangaluru (Dakshina Kannada District), Mulki, Udupi and Kundapura (Udupi District), Bhatkal, Ankola, and Karwar (Uttara Kannada District). A total of 365 isolates were suspected as Vibrios from the collected samples that were plated on different isolation media. Out of 365 suspected isolates, 3 isolates of V. mimicus and 1 isolate of V. hollisae were confirmed phenotypically by performing battery of biochemical tests. These isolates were further confirmed by performing Polymearse Chain Reaction (PCR) using specific primers, in which AraC gene was targeted to V. mimicus (488 bp) and gyrB gene to V. hollisae (363 bp).Phenotypically confirmed V. mimicus showed negative result as they failed to amplify at specific basepair and V. hollisae showed positive result by amplifying at its specific base pair. For further confirmation, 16S rRNA sequence analysis was done by out sourcing for the genotypically confirmed V .hollisae. The BLAST sequence analysis, revealed that V.hollisae strain matches with 91 V. alginolyticus, but 16S rRNA is closely related to Grimontia hollisae sequences present in NCBI gene database. Haemolyitc assay was also performed for the phenotypically confirmed V. mimicus and V. hollisae to test their virulence.V. mimicus showed no haemolysis on blood agar plate while V. hollisae showed a clear zone of haemolysis (β-hemolysis). This present study reveals that the samples collected from the study area did not show the presence of V.mimicus.ThesisItem Open Access STUDY OF COMBINED EFFICACY OF SYNTHESIZED DOUBLE STRANDED RNA (dsRNA) SPECIFIC TO STRUCTURAL AND NON-STRUCTURAL GENES OF WSSV IN PROTECTING LITOPENAEUS VANNAMEI FROM WSSV INFECTION(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2019) SHAIK THAHUR AHAMED; M.N.VENUGOPALRNA interference (RNAi) is a potential strategy to control shrimp viral diseases, including the white spot disease (WSD) caused by White Spot Syndrome Virus (WSSV). Although a significant progress has been achieved on dsRNA mediated anti-virus strategy development, there is still no treatment available to control spread of the disease. This investigation has mainly focused on utilizing viral structural and non-structural proteins subunit vaccines to protect the host. In the present study the protective and therapeutic efficacy of double-stranded RNAs (dsRNAs) targeting structural (VP281) and nonstructural gene (RR2) of WSSV singly and in combination in controlling viral multiplication in Litopenaeus vannamei was investigated by intramuscular injection. The suppression effects of shrimp mortality by either combined dsRNAs of RR2 and VP281 or dsRNA-RR2 & VP281 alone was monitored for 15 days after viral challenge in both protective and therapeutic experiments. Targeting the combination of RR2 and VP281 genes provided better protection (90% survival) compared to RR2 (80%) and VP281 (70%) in protective experiment indicating that targeting a combination of RR2 and VP281 would be effective in limiting WSSV multiplication. In therapeutic experiment, 80% of shrimp survivals were detected from combined dsRNAs treatment, while RR2, VP281 showed survival rates of 70% and 60 % respectively, suggesting that the combination of dsRNAs can therapeutically clear existing WSSV in shrimp after a relatively short time of injecting.ThesisItem Open Access IMPACT OF SHRIMP FARM EFFLUENTS ON WATER QUALITY IN THE COASTAL WATER OF UTTAR KANNADA(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2019) MORE RUTUJA.,; LAKSHMIPATHI M.T)Impact of shrimp farm effluent on the receiving coastal waters was studied from stocking to harvesting period for one complete culture cycle (120 days). The present study was conducted on three shrimp farms in the Uttar Kannada district at Ankola and Kumta, an area representative for the impacts of shrimp culture. Fortnightly sampling was carried out at eight stations from March 2019 to June 2019. During this study period shrimp farm effluent was subjected to detailed analysis and its possible impact was recorded. Physico-chemical and biological characteristics in relation with hydrographical parameters were studied to determine the water quality of inlet and outlet area of adjacent creek waters. The present study revealed that the shrimp farm directly release effluent into creek water during water exchange without any treatment. This discharge of effluent made an impacts on water and sediment quality (Temperature, dissolved oxygen, pH, salinity, ammonia-nitrogen, nitrite-nitrogen, nitrate-nitrogen, phosphate-phosphorous, organic carbon, plankton and chlorophyll-a). The values of Do (< 3 mg/L), ammonia (< 40.26 μg-at/l), BOD (˃ 18 mg/L), COD (˃ 30 mg/L) and sediment organic carbon (4.4%) and planktonic biomass (0.54 – 5.12 mg/m³)) the chlorophyll-‘a’ (˃ 0.33 mg/m³) was observed. The investigation showed increased load of receiving waters by direct release of shrimp farm effluent to surrounding coastal waters. The effluents contained high amount of solids and nutrients generated from uneaten food material, shrimp excreta, which increased the concentration during discharge period. The water and sediment values were found to be increasing with the end of culture period. Among this parameters water temperature, DO, BOD, salinity, pH and nutrients showed wide range of fluctuations at outlet in creek. The study revealed that the shrimp farm effluent discharge have negative impact on receiving coastal waters bodies.ThesisItem Open Access SCREENING OF FRESHWATER ORNAMENTAL FISHES FOR VIRAL DISEASES(KARNATAKA VETERINARY, ANIMAL & FISHERIES SCIENCES UNIVERSITY, BIDAR, 2019) KUSHALA, K. B; S.K.GIRISHAIn the international ornamental fish trade, more than 90% of the freshwater fishes are farm bred and about 4000 species of freshwater ornamental fishes were traded every year. Hence, the global ornamental fish trade had become the platform for spread of exotic pathogens especially viral pathogens. Major DNA viruses viz., cyprinidae herpes virus-2, koi herpes virus (KHV), carp edema virus(CEV), epizootic haematopoietic necrosis virus (EHNV), infectious spleen and kidney necrosis virus (ISKNV), and RNA viruses viz., spring viraemia of carp virus (SVCV), viral haemorrhagic septicemia (VHS) and viral nervous necrosis virus(VNN) are causing diseases and results in significant economic loss in ornamental fish trade. Therefore, to develop a strategy to controls the diseases, the present study has been undertaken to screen the viral pathogens present in ornamental fishes traded in Karnataka. In this present study, moribund and dead ornamental fishes were collected from three districts viz., Dakshina Kannada, Bengaluru Central and Ramanagar. Totally 146 different ornamental fishes were screened for seven viral pathogens which were showing different clinical signs like haemorrhages, sunken yes, body discoloration, scale erosion and swelling gills. Out of 146 ornamental fish, ISKNV was detected in 20 samples (13.6%) and CEV was detected in 4 samples (2.37%) by PCR targeting specific genes but there was no co-infection of the both virus in a single fish. None of the samples were found to be positive for DNA viruses’ viz., KHV, EHNV and RNA viruses viz., SVCV, VHS and VNN. NCBI-BLAST analysis of sequenced CEV strains were more similar (97.4-99.94%) to the India and Germany CEV strains whereas ISKNV strains were more similar (98.76%- 100%) to ISKNV strains from Japan and Australia. Phylogenetic tree revealed that our CEV strains belong to lineage 1 of koi carp whereas our ISKNV strains belong to genotype I of Megalocytivirus. This is the first report of ISKNV and second report of CEV from India.ThesisItem Open Access PHYTOPLANKTON DYNAMICS IN THE COASTAL WATERS OF PADUBIDRI, UDUPI DISTRICT(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2009-06) MEGHANA P.N.; H.R.V.REDDY; S.M.SHIVPRAKASSeasonal variation of phytoplankton was studied in the coastal waters of Padubidri with reference to hydrographic parameters for a period of one year (February 2008 to January 2009). The sampling was made at monthly intervals at 5, 10, 15m depts. The salinity values ranged from 30.64 to 35.11ppt and exhibited bimodal seasonal oscillation with maximum values during pre monsoon period. The dissolved oxygen content varied between 4.0 and 6.5 mg/l with moderate values during pre monsoon period. The pH values were observed during post monsoon period. Among the nutrients ammonia and nitrite showed high values during post monsoon period while nitrate, phosphate and silicate showed higher values during pre monsoon period. Total phytoplankton population varied from 100 to 10, 95,000 cells/m3. Diatoms dominated the phytoplankton and contributed about 92% of total phytoplankton population followed by dinoflagellate (6%) and cyanobacteria (2%). In total 22 species of diatoms were encountered. The diatoms comprised of Bacteriastrum, Biddulphia, Cerataulina, Chaetoceros, Coscinodiscus, Cyclotella, Ditylum, Eucamphia, Leptocylindrus, Mellosira, Planktoniella, Rhizosolenia, Skeletonema, Streptotheca, Tricerratium, Asterionella, Fragillaria, Navicula, Nitzschia, Pleurosigma, Thalassionema, and Thallassiothrix..The dinophyceae was represented by 5 genera such as Ceratium, Dinophysis, Ornithoceros, Peridinium and Preperidinium and blue green algae by Tricodesmium and Oscillatoria The chlorophyll- “a” content fluctuated from 1.28 to 4.81mg/l Overall, phytoplankton population density was high during summer periods and low during post monsoon periods. The statistical analysis showed strong relationship between temperature, salinity, nutrients and phytoplankton abundance.
