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ThesisItem Open Access ASSESSMENT OF IMMUNE RESPONSES TO RABIES VACCINATION IN FREE RANGING DOGS IN BENGALURU(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) VINAY, C. P.; VINAY, C. P.The present study was undertaken to assess the effectiveness of ongoing MDV programme in stray dogs of Bengaluru. In all, 260 sera samples from stray dogs in 26 wards from 8 zones of BBMP were collected for the study and tested by RFFIT and inhouse iELISA for HI response and Interferon-γ ELISA for CMIR. Overall, 71 per cent (171/240) samples from 8 zones of BBMP and 65 per cent (13/20) samples from 2 NC wards revealed adequate IR by RFFIT. By iELISA, 87 per cent (209/240) samples from 8 zones of BBMP and 90 per cent (18/20) samples from 2 NC wards revealed adequate IR. Overall, the iELISA revealed higher percentage of seroconversion than RFFIT as it detects the non-neutralizing antibodies in addition to the neutralizing antibodies. Among 8 zones of BBMP, the samples from B. west had highest seroconversion of 86.36 and 100 per cent by RFFIT and iELISA respectively. The Spearman’s rank correlation (r value ≠ 0 and p value <0.05) indicated some correlation and kappa value of 0.55 suggested a moderate agreement between the results of RFFIT and iELISA. The sensitivity and specificity of iELISA was found to be 100 per cent and 63.30 per cent respectively. The In-house iELISA can be used in large population surveys instead of RFFIT to overcome the disadvantages associated with it. The IFN–γ ELISA employed to study CMIR revealed 50 per cent (120/240) samples from 8 zones of BBMP and 90 per cent (18/20) samples from 2 NC wards with adequate CMIR. The highest CMIR of 81.82 per cent was observed in B. west zone. The adequate CMIR in serum samples with RFFIT titres < 0.5 IU/mL indicates the effect of vaccination being done under the ABC-ARV programme before initiation of MDV in BBMP. A sustained MDV programme enhanced to cover 70 per cent of the dog population will surely aid in elimination of dog-mediated rabies.ThesisItem Open Access EVALUATION OF PROTECTIVE EFFICACY OF FORMALIN INACTIVATED KYASANUR FOREST DISEASE VACCINE IN MICE(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR., 2022) ULLAS GOWDA, K. S.; B. M. CHANDRANAIKThe present study was undertaken with the objectives of evaluating safety and efficacy of Kyasanur Forest Disease (KFD) vaccine inactivated with different concentration of formalin in mice, quantification of Kyasanur Forest Disease virus (KFDV) by Real time PCR and its comparison with in-vivo titration in mice and to study the sequential experimental pathology of KFDV in mouse brain. In this study, KFD vaccine prepared in Chicken Embryo Fibroblast was inactivated with 0.04%, 0.06%, and 0.08 % concentrations of formalin. KFD vaccine inactivated with 0.04% and 0.06 % formalin failed the safety test as mice inoculated with the vaccine developed disease symptoms and/or death. KFD vaccine inactivated with 0.08% formalin passed the safety test since none of the mice inoculated with this vaccine showed symptoms and/or death. 0.08% formalin inactivated KFDV vaccine passed the potency test in mice with log protective index of was 5.678. Since the formalin content is relatively lower (0.08%) than in the currently available KFD vaccine (0.1%), this should induce no or lesser reactions of pain/swelling at the site of inoculation, which may increase the vaccine acceptance and vaccination coverage. The real time PCR on individual harvests yielded CT values of less than 20 on all five harvests (H1 to H5). The real time PCR on tenfold dilutions of the pooled harvests yielded CT values of 22.05, 24.13, 26.09, 29.34, 31.18, 34.02, 0.0 and 0.0 for dilutions of 10-1, 10-2,10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 respectively. Inoculations of these tenfold dilutions in mice by conventional mice inoculation test revealed that the titre (MLD50) of the virus of in the pooled harvest was at 10-6. Based on these findings, it could be concluded that when the CT value of individual vaccine harvests in real time PCR is less than 20, the KFD vaccine will have sufficient viral particles to pass the potency test. Real time PCR on tenfold dilutions of the vaccine harvests indicated that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 to 34. The sequential pathology of KFD virus in mice brain by intra cerebrally inoculating 1MLD50 of the virus showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indications of hind quarter paralysis on day 4 pi. These symptoms got aggravated with complete paralysis of hind quarters, inability to move followed by death on 5th and 6th dpi. Microscopically, brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. Cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected. Apoptosis of neurons was a consistent and important brain lesion noticed in KFD infected mice. Present study is the first report on apoptosis of neurons due to KFD virus infection in mice.ThesisItem Open Access NUCLEOPROTEIN AND GLYCOPROTEIN GENE-BASED MOLECULAR CHARACTERIZATION OF RABIES VIRUS IN DIFFERENT SPECIES OF ANIMALS IN INDIA(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR., 2022) TILAK CHANDAN, S.; SHRIKRISHNA ISLOOR)A study on 14 brain samples collected from the cases of rabies in animals including wild carnivores from four Indian states during 2020-2022 was undertaken to update the molecular epidemiology of circulating Indian rabies virus (RABV) and evolutionary pattern. This was based on the phylogenetic analysis of the RABV with respect to complete sequence of nucleoprotein (N) and glycoprotein genes (G). All the samples were tested by lateral flow assay (LFA), direct fluorescent antibody assay (DFA), and reverse transcription- polymerase chain reaction (RT-PCR) and confirmed positive for rabies. The complete nucleotide sequences of N and G genes of RABV from all the samples were analysed by Neighbour-Joining method. Majority of the RABV sequences that included those reported from Asian countries showed affiliation of these sequences to three lineages viz., the Arctic-like 1a lineage, Asian clade and Indian subcontinent lineage. The Asian Clade comprised of two subclades- South East Asian 3 (SEA3) and SEA5- that corresponded to the Taiwan and China isolates respectively. Whereas, Indian RABV either reported earlier by others or those studied in the present investigation were found to be identified either under Arctic like 1a or Indian subcontinent lineages. Interestingly, Arctic-like 1a lineage described the close association of North Indian isolates irrespective of cross-species transmission. Whereas, those from the Southern states of Tamil Nadu and Kerala exhibited close association with the isolates from Sri Lanka and Nepal under the Indian subcontinent lineage. No other Indian isolates were found in this clade.ThesisItem Open Access ISOLATION AND CHARACTERIZATION OF CAMPYLOBACTER IN POULTRY(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2021) SAHITHYA, N.G.; D. RATHNAMMAThe present study was conducted to isolate and characterize Campylobacters from poultry. A total of 158 poultry samples were collected from RMS, CPF and BYPF from different districts. All the samples were subjected for pre-enrichment followed by selective isolation of Campylobacters on mCCDA and Columbia blood agar. Seventyfive (47.46%) Campylobacter isolates were obtained and further confirmed by PCR. Genus specific PCR was standardised to identify Campylobacter isolates at genus level by amplifying 16S rRNA gene. Further species level identification was done by amplifying lpxA gene where 52 isolates were identified as C.jejuniand seven as C.coli. Remaining 16 isolates were unidentified campylobacter species. The prevalence of C.jejuni was more (76%) compared to C.coli (9.33%). Antibiotic sensitivity of all 75 Campylobacter isolates was tested by standard Kirby-Bauer disc diffusion method. All Campylobacter isolates showed 100 per cent resistance to Clindamycin and Nalidixic acid, followed by Erythromycin (73.33 %), Tetracycline (61.33%), Azithromycin and Doxycline (53.33%) and Ampicillin and Amoxicillin/Clavulanic acid (44%). The presence of AMR genes in 52 C.jejuni isolates was carried out by PCR. Where all 52 isolates (100%) showed amplification of 23S rRNAgene for Macrolides followed by Fluoroquinolone (86.54%) by Amplifying QRDR region of gyrA gene, Tetracycline (53.84%) by amplifying tet (O) gene, 50 per cent isolates showed amplification of blaOXA-61 gene for Beta-lactams and 69.23 per cent isolates were found to harbour cmeB efflux pump gene. Amplification of cad-F and flaA virulent genes was carried out by subjecting 52C.jejuni isolates to PCR, all the C.jejuni isolates revealed cad-F gene and 29C.jejuni isolates revealed flaA gene . Partial nucleotide sequencing of cad-F gene was done for 12 campylobacter isolates. The phylogenetic analysis revealed two groups where farm isolates formed one group and isolates from retail meat shops formed another group. The highest mean interspecific divergence was found between LH and K2 (10.42%) and it was lowest between TF and BBY (0.02%). Genotyping of 50 C.jejuni isolates was carried out by ERIC-PCR and intra-species variability of C. jejuni isolates was determined by dendrogram analysis of C. jejuni that revealed 12 distinct clades with discriminatory power (D value) of 0.87. Campylobacter jejuni isolates under study formed two main clusters (A and B) with 100% heterogeneity.ThesisItem Open Access STUDIES ON BACTERIAL FLORA IN BOVINE AND BUBALINE SEMEN OF BREEDING BULLS IN KARNATAKA(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR-585401, 2022) NITHYANANDA, V. C.; ShivarajThe semen samples were collected from three semen collection centres located in and around Bangalore. Eight HF bulls and five bubaline bulls from Farm-A, twelve HF bulls from Farm-B, eight HF bulls and eleven bubaline bulls from Farm-C, were selected for the study. Three ejaculates from each bull at fortnightly intervals were collected and subjected for the estimation of total bacterial load in the neat, extender and frozen semen to evaluate the level of contamination occurring during handling, processing and storage of semen. The bacterial load of the semen was found to be within the prescribed guidelines of BIS suggestive of the hygienic measures followed during semen collection, processing and preservation. The isolates were subjected to the genus level typing using cultural, staining characteristics and molecular methods. The isolates isolated in the present study were Staphylococcus spp., Bacillus spp., Klebsiella spp., Corynebacterium spp., Serratia spp. and Proteus spp., etc. The antibiogram of Staphylococcus isolates were highly sensitive to Imipenem, followed by Piperacillin Tazobactam and Ofloxacin. Bacillus, Klebsiella, Corynebacterium and Proteus isolates were highly sensitive to Imipenem, Piperacillin Tazobactam. Serratia isolates were highly sensitive to Piperacillin Tazobactam. The study revealed that the conventional antibiotics used in the semen stations were sufficient to reduce the bacterial load in the semen along with strict hygienic measures during the collection and processing of semen. But it was found that the conventionally used antibiotics like Penicillin G and Streptomycin were resistant to many bacterial isolates obtained in the present study.ThesisItem Open Access STUDIES ON MAJOR BACTERIAL-VIRAL AGENTS ASSOCIATED WITH DIARRHOEA IN CALVES(KARNATAKA VETERINARY, ANIMAL ANDFISHERIES SCIENCES UNIVERSITY, BIDAR, 2022) VEENAKUMARI A. N.; ARUN S. J.)Calf diarrhoea is a commonly reported disease of calves caused by multietiological agents leading to high morbidity and mortality. The present study was carried out to isolate and characterize predominant bacteria such as E. coli and Salmonella, and to detect bovine rotavirus and bovine coronavirus in the faecal samples of diarrhoeic and non-diarrhoeic calves. For this a total of 162 faecal samples were collected from diarrhoeic and non-diarrhoeic calves under six months of age. In total, 155 and 8 faecal samples were positive for E. coli and Salmonella respectively. Duplex PCR was performed to confirm E. coli by targeting the uidA and uspA genes and uniplex PCR was performed to confirm Salmonella by targeting the invA gene. The virulence genes of E. coli, namely, fimH, stx1 and stx2 were detected in 94.19% (146/155), 25.16% (39/155) and 32.25% (50/155), respectively. All the Salmonella isolates in our study were found to be S. Typhimurium 100% (8/8) and none were S. Enteritidis. The stn gene was detected in 87.5% (7/8) of Salmonella isolates. All 162 faecal samples were also screened for the presence of bovine rotavirus and bovine coronavirus by RT-PCR by targeting VP6 and N genes, respectively. Bovine rotavirus was detected in 47.53% (77 out of 162) samples, whereas none of the faecal samples were positive for bovine coronavirus. The antibiogram study by disc diffusion method revealed that the bacterial species were 100% resistant to metronidazole and polymyxin-B, but susceptible to ciprofloxacin and co-trimoxazole. The study gave an insight into the major bacterial and viral etiological profile, antibiogram and virulence potential of bacterial agents associated with calf diarrhoea. Key words: Calf diarrhoea, E. coli, Salmonella serovars, bovine rotavirus, bovine coronavirus, antibiogramThesisItem Open Access ISOLATION AND MOLECULAR CHARACTERIZATION OF E. coli FROM DIARRHOEIC CASES OF PIGS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2023) ANAND; BASAVARAJ AWATIE. coli is a common and commensal in the pig’s intestinal tract. However, some strains cause illness in both humans and animals and survive in the intestinal tract which leads to diarrhea and antibiotic resistance. Therefore, it is necessary to identify E. coli which causes diarrhea in pigs. A quick diagnosis of pathogenic E. coli is now achievable because of the development of PCR, which makes it possible to find pathogenic E. coli in bacterial isolates. The goal of the current research was to study the isolation, identification, biochemical characterization, antibiogram profile and molecular characterization of E. coli isolates by PCR. In the present study a total of 102 faecal samples were collected from suspected diarrhoeal cases of swine from different pig farms of Karnataka during the period July 2022 to January 2023 were analysed. The collected samples were subjected for cultural isolation, morphological identification and biochemical characterization. Based on cultural, morphological and biochemical identification out of 102 samples, 79 samples were found positive for E. coli which accounted for 77.45 per cent. The positive samples identified by biochemical tests were subjected to PCR for confirmation of E. coli isolates. According to the PCR method, out of the 102 samples that were processed, 66 samples were found to be positive for E. coli which accounts for 64.70 per cent. The antibiotic sensitivity test was performed in accordance with the standard guidelines. E. coli isolates found highly sensitive to Gentamicin (HLG), Ceftriaxone (CTR) and Chloramphenicol (C). Intermediate sensitivity towards Streptomycin and Ampicillin (A/S). Highly resistance towards Cefepime (CPM) and Amoxycillin (AMC). While resistant towards Enrofloxacin (EX) and Tetracycline (TE).ThesisItem Open Access STUDIES ON AEROBIC BACTERIA ISOLATED FROM BOVINE SUBCLINICAL AND CLINICAL ENDOMETRITIS(KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR, 2021) PAVITHRA B.S.; B.E. SHAMBULINGAPPABacterial endometritis results in lower and delayed conception rates in affected cows. The present study was conducted to isolate and characterize aerobic bacteria from subclinical (SE) (n=82) and clinical endometritis (CE) (n=51) cases in cows. In addition, studies on antibiogram profile, MIC and major virulence genes were also carried out. Fifty eight of 82 uterine lavage samples showing 1-5% of PMN cells by endometrial cytology were considered as subclinical endometritis cases. Bacterial isolation and phenotypic characterization of the isolates revealed E. coli, S. aureus, S. uberis, P. aeroginosa, P. mirabilis, K. pneumoniae and B. cereus from SE and in addition to these, R. equi and C. bovis were also recovered in CE cases. PCR was used to confirm the bacterial species targeting uspA and uidA genes in E. coli and nuc gene in S. aureus. Major virulence genes in E. coli viz., fimH, fyuA, kpsMTII, csgA were detected by PCR in 100%, 47.82%, 17.39%, 26.08% of the SE isolates, and in100%, 73.07%, 38.46%, 34.61% of CE isolates respectively. Spa gene was detected in all S. aureus isolates and the spa type was t359. The antibiogram study by disc diffusion method using most commonly used 19 antibacterials revealed that most of the isolates were susceptible to ciprofloxacin, enrofloxacin, gentamicin and ceftriaxone, and resistant to penicillin and polymyxin B. MIC values for ceftiofur, ciprofloxacin and gentamicin were within the susceptibility range for most of the isolates. Out of 36 S. aureus isolates, phenotypic methicillin resistance was detected in two whereas mecA gene was detected only in one isolate by PCR. The study gives an insight into the etiological profile, antibiogram, virulence potential of the bacterial agents associated with bovine endometritis. Key words: Endometritis, aerobic bacteria, virulence genes, antimicrobial susceptibility, minimum inhibitory concentrationThesisItem Open Access PRASHANTH(KVAFSU,BIDAR, 2019) PRASHANTHA study on bacteriological investigation of canine pyoderma cases was conducted at the Veterinary College, Shivamogga. Exudate/pus/lesion swabs were collected from clinical cases of canine pyoderma (n=126) and subjected to isolation of bacteria, identification of staphylococcal isolates by molecular method and other bacterial isolates by phenotypic methods. The bacteriological processing of the samples resulted in the recovery of 95 staphylococcal isolates and 18 other bacterial isolates. On culture, staphylococci were the most predominantly (n=95, 75.39%) isolated organisms. Amongst staphylococci, S. pseudintermedius (n=82, 86.31%), coagulase positive staphylococci, was the most predominantly organism detected by species-specific nuc PCR. The S. pseudintermedius isolates were further subjected to detection of siet gene by PCR and 84.14% of the isolates were positive. The isolates obtained other than staphylococcal species were E. coli, Pseudomonas species, Klebsiella species, Proteus species. Subsequently in vitro antimicrobial sensitivity testing was carried out for all the isolates. S. pseudintermedius showed highest susceptibility to clindamycin and the highest resistance to enrofloxacin, and the other bacterial agents were found sensitive to co-trimoxazole and ceftriaxone and resistant to amoxicillin/sulbactam and enrofloxacin. Three of the four isolates which were phenotypically methicillin resistant were positive for mecA gene PCR. A study on the occurrence patterns of canine pyoderma revealed higher frequency of cases in male dogs, in the age group of 1-2 years and in Labrador breed. We report the S. pseudintermedius as the most predominant pathogen associated with the canine pyoderma in the study area. The emergence of methicillin resistance in S. pseudintermedius emphasises the need for devise of strategies for its control.
