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ThesisItem Open Access ANTIBIOTIC RESISTANCE PROFILING OF BACTERIA ISOLATED FROM CORNEAL ULCERS IN DOGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-28) ASHTAMY M. G.; Dr. Binu K. ManiCorneal ulcers are one of the most frequent and painful ophthalmic conditions in dogs. Companion animals act as a source for transfer of AMR genes among bacteria between animals and humans. Antimicrobial resistance is a significant public health threat, which is mainly due to the indiscriminate use of antibiotics for a prolonged period of time. The present study deals with the isolation and identification of bacteria from corneal ulcers in dogs and their assessment of antibiotic resistance pattern by phenotypic and genotypic methods.The conjunctival swab samples collected from 15 dogs tested positive for corneal ulcers, were inoculated on to brain heart infusion agar and blood agar and incubated aerobically at 37 °C for 24 h. Out of the 15 samples, 18 isolates could be obtained with 12 as pure culture and three as mixed cultures, each with two types of colonies. The isolates were identified by their cultural characteristics on selective media, morphology, staining reactions and biochemical tests. The predominant isolates were Gram positive bacteria of the genera Staphylococcus (five), Corynebacterium (five) and Streptococcus (two). The rest of the six solates were Gram negative bacteria, viz. Pseudomonas aeruginosa (two), Klebsiella pneumoniae (two), Escherichia coli (one) and Neisseria spp. (one). Antibiotic susceptibility testing by disc diffusion method revealed that most of the isolates were resistant to quinolone group while only three of them were resistant to chloramphenicol. Polymerase chain reaction was performed to detect the genes,mecA, ermC, aac(6’)-aph(2”) and cat for resistance against methicillin, erythromycin, aminoglycoside and chloramphenicol, respectively and mutation in gyrA for resistance against quinolones, among the bacterial isolates. The mecAcould be detected in four, ermC in three and aac(6’)-aph(2”) in seven isolates.Out of seven isolates which amplified gyrA, the representative sample which was sequenced, revealed single point mutation. The mutation detected was silent, since it did not change any amino acid pattern. The cat gene could not be detected in any of the isolates.ThesisItem Open Access MOLECULAR DETECTION AND CHARACTERISATION OF CHICKEN ANAEMIA VIRUS FROM KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-01-28) VIDYA P.; Dr. Surya SankarChicken anaemia virus (CAV) is the aetiological agent of chicken infectious anaemia (CIA), an immunosuppressive disease which brings huge economic burden to poultry industry globally. Epidemiology of the disease and virulence of the circulating strains is to be known, while formulating prevention strategies to any infectious disease. So far, there are no reports regarding the presence of CAV among the poultry flocks in Kerala. In this scenario, the present is envisaged for the detection of CAV, employing polymerase chain reaction (PCR) using primers targeting the VP1, VP2 and VP3 genes of the virus, and its characterisation by nucleotide sequencing followed by phylogenetic analysis. Out of 100 pooled tissue samples collected from suspected cases, 29 were found to be positive for CAV by PCR. Isolation of virus from tissue homogenate of PCR-positive samples in embryonated chicken eggs through yolk ac route inoculation was carried out. Embryos were harvested 14 days post inoculation and PCR using same primers was carried out to confirm the presence of virus, but none of the samples turned positive. All the 29 samples were positive PCR targeting VP1 and VP3 gene. The representative amplicons from direct PCR targeting VP1, VP2 and VP3 genes were sequenced, analysed and compared with sequences in GenBank. The isolates from Kerala exhibited variations of about one per cent among each other and about two per cent variations were noted with other Indian isolates. Phylogenetic analysis based on VP1 gene revealed that samples clustered each other and also with isolates from different parts of India and with the vaccine strains, Del Ros, 26P4 and Cux-1. On amino acid analysis of the three genes, the profile of VP2 and VP3 is conserved in nature, while VP1 exhibited similarities with rapidly spreading and highly pathogenic strains of CAV. Histopathologic examination of thymus, bursa of Fabricius and bone marrow of PCR positive samples, lesions indicating apoptosis of thymic cortex, lymphocytic depletion in bursa of Fabricius and atrophy and aplasia involving all haematopoietic lineages of bone marrow were observed.ThesisItem Open Access ASSESSMENT OF IMMUNOGENIC POTENCY OF INACTIVATED VACCINE AGAINST RIEMERELLOSIS IN BREEDER DUCKS(COLLEGE OF VETERINARY AND ANIMALS SCIENCES MANNUTHY, THRISSUR , KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-09) LINA KUNJAMMA MATHEWS; Dr. Priya P. M.Riemerellosis or new duck disease, is an infectious disease in ducks caused by the bacterium Riemerella anatipestifer, which primarily causes an acute septicaemic infection in young ducklings. A subunit vaccine developed against the disease was compared to an inactivated vaccine in an earlier study, which proved the superiority of the latter in terms of efficacy. No studies have been carried out on maternally transferred antibodies (MTA) in ducklings born to the vaccinees. Hence, this study was undertaken. A total of 200 laying Kuttanad ducks of breeder stock maintained in the University Poultry and Duck Farm (UPDF), Mannuthy were grouped into two, comprising of 100 birds each, with T1 being the control group and T2, the treatment (vaccination) group. The oil-adjuvanted inactivated vaccine, prepared as per previously standardised protocol, was administered to T2group in two doses at weekly intervals. Blood samples, 20 each from T1 and T2, were collected on days 0, 14, 28, 56 and 90 post-immunisation (PI), whereas 10 eggs each from both the groups were collected on days 14, 28, 56 and 90 PI, to assess antibody titre in adult sera and egg yolk respectively, by ELISA. Fertile eggs collected between days 28 and 35 PI from each group were marked separately, incubated for hatching and the hatched-out day-old ducklings (n=24) from each group were selected randomly and reared. To evaluate MTA, blood was collected from six ducklings each from both the groups, on days 1, 3 and 10 to estimate antibody titre in the sera. The transfer of maternal antibodies was assessed by challenge studies upon inoculating 100 LD50 of RA1 subcutaneously to six ducklings each from both the groups on days 3 and 10. Statistical analysis of the ELISA titre of the adult duck sera revealed significant difference (p<0.001)between the titres of both the groups on all the days assessed except the 0th, confirming the potency of the vaccine in adult ducks. However, the assessment of MTA revealed some level of protective antibody in the ducklings in terms of mortality and gross pathological scoring, but not in levels detectable by ELISA.ThesisItem Open Access ISOLATION AND CHARACTERISATION OF INFECTIOUS BURSAL DISEASE VIRUS FROM KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-09-24) AKHILA JOY; Dr. Sreeja R. NairInfectious bursal disease (IBD), caused by the infectious bursal disease virus (IBDV), is one of the top five infectious diseases of poultry that discernibly affects the commercial poultry industry. The mutating viral genome of IBDV accounts for many field outbreaks even after following stringent biosecurity measures. In this study, for the detection of IBDV in Kerala, 51 bursa of Fabricius samples were collected from three to six-week-old chicken, with clinical signs or lesions suggestive of IBD. Reverse transcriptase-polymerase chain reaction (RT PCR) targeting the hypervariableVP2 region detected six positive samples, and one out of six samples was successfully isolated in 10-11 day-old embryonated chicken eggs (ECE) through the chorio-allantoic membrane (CAM) route. The virus isolated in ECE was genetically characterised by targeting the VP1, VP2 and VP3 genes. The sequence analysis revealed that the deduced amino acid sequence of VP1 gene of the isolate was homologous with an attenuated very virulent vaccine strain of Israel, mb and the VP2 gene was homologous with mb and Ventri IBDV plus vaccine strain of India. The analysis of VP3 gene also revealed the similarity of the isolate with vaccine strains except for a single variation S745N in its deduced amino acid sequence. The phylogenetic analysis of the isolate revealed the close relation of the isolate with mb, Ventri IBDV plus and a very virulent strain of Israel, ks. On sequence analysis of amino acids, the characteristic virulent marker amino acid motifs "SWSASGS" and "TDN" were present in the VP2 and VP1, respectively. Hence, the study revealed that the virus isolate has emerged from an attenuated very virulent vaccine strain and the change in virulence of the strain might be due to S745N in the VP3 gene or any change in the VP4 or VP5 gene, which is beyond the scope of the current study. The present study is the first attempt in Kerala in which analysis of the coding sequence of VP1, VP2 and VP3 genes are employed for characterisation.ThesisItem Open Access CHARACTERIZATION OF PLASMIDS OF Escherichia Koli ISOLATED FROM MASTITIS(COLLEGE OF VETERINARY AND ANIMAL SCIENCE,MANNUTHY, 1993) AVINASH GANPATRAO KARPE; Punnoose, K TEscherichia coli were isolated in 15.33 per cent cases of mastitis. Of the 46 E. coli isolated 43 were resistant to one to nine antibiotics and three were sensitive to all the 13 antibiotics tested. The organisms were resistant to rifampicin (78.26%) followed by oxytetracycline (50%), tetracycline (37.78%), nalidixic acid (19.56%), co-trimoxasole (8.69%) and gentamicin (6.52%). All the organisms were susceptible to kamamycin and norfloxacin. Among the multiple drug resistance oxytetracycline - rifampicin (OR) resistance was noticed in 76.2% cases. Twenty-six different patterns of antibiotic resistance were noticed among 43 E. coli isolates giving a reliability of 60.46 per cent in differentiating the isolates. Hence, antibiogram could only be used as an adjunct to plasmid profiling in epidemiological studies. The resistograms revealed cent per cent resistance to lead, followed by antimony (32.6%), copper (30.43%), silver (19.56%) and cetrimide (2.17%). All the isolates were sensitive to cadmium and mercury. Among the 46 E. coli isolates, 9 different resistogram patterns were obtained giving reliability of 19.56 per cent in differentiating the strains. A correlation between the antibiotics and heavy metal resistance such aacs; lleeaada , an+-imcny and copper, was observed in descending order. of the forty-six E. coli isolates three (6.52%) were hemolytic on sheep blood agar. Two of the three hemolytic strains were also enterotoxigenic. Thirteen of the 46 (28.26%) E. coli isolates were enterotoxigenic, when tested by rabbit ligated ileal loop assay. Two of the thirteen (15.38%) enterotoxigenic isolates were also hemolytic. Fourteen of the 24 (58.33%) drug resistant E. coli transferred drug resistance against one or more antibiotics to the recipient organism. In none of the cases the furazolidone resistance was transferred. All the three hemolytic E. coli isolates transferred the hemolytic character by conjugation indicating the plasmid borne nature of hemolysin production. None of the enterotoxin producing E. coli could transfer the character to recipient by conjugation. Of the 40 wild E. coli screened for plasmid profiles two were devoid of any plasmid. The number of plasmids varied from one to five with molecular size ranging from 1.47 to (ii i ) 128.8 Kb. Among the thirty-eight plasmid bearing E. coli isolates, altogether 35 different plasmid profile patterns were obtained as six E. coli isolates showed three different plasmid profiles. The isolates with similar plasmid profiles revealed similar antibiogram, resistogram and Ent character. The hemolytic strains contained large as well as small plasmids. Only large plasmids, along with hemolytic character and drug resistance were transferred to the recipient. It indicated that the plasmids carrying genes for hemolysin production also harboured antibiotic resistance genes. In case of enterotoxigenic E. coli (ETEC) the Ent character was not transferred by conjugation, which could be due to the location of the character on small plasmids which are nonconjugative. Two of the three ETEC strains which were hemolytic, transferred the Hly character, but not the Ent character to the recipient E. coli. Ethidium bromide was found to be the best curing agent among the chemicals used. The degree of curing increased with the time of exposure. Ethidium bromide cured R-factors, Hly, Ent and F'lac characters. Furazolidone resistance was neither transferred by conjugation nor cured. But nalidixic acid resistance was eliminated by EE. Elevated temperature (45°C) cured antibiotic resistance Hly, Ent, F'lac characters. The (iv) rate of curing increased from fifth day onwards. Large plasmids were eliminated in all the cases. The study of, transfer of plasmids by conjugation, their elimination by curing, and plasmid profiles of wild, transconjugant and cured strains of E. coli revealed correlation between plasmid and virulence characters. There was a definite correlation between Hly character and antibiotic resistance as they were co-transferred on conjugation and the transconjugants harboured only one large plasmid exhibiting these two characters. The photographic technique developed with ordinary single lens reflex camera, resulted in colour photographs with better resolution. This technique could serve as a substitute to Polaroid camera system. The results obtained from the present study led to the following conclusions. * Indiscriminate use of antibiotics should be dispensed with and antibiogram should be carried out prior to treatment. The drugs of choice were kanamycin, norfloxacin, gentamicin, co-trimoxazole, furazolidone and nalidixic acid. V, The antibiograrn and resistogram could serve as an adjunct to plasmid profile analysis in epidemiological studies. The role of plasmids in the production of hemolysin, enterotoxin and the antibiotic resistance was established. Ethidium bromide was found to be the best chemical curing agent, whereas elevated temperature (45^C) showed the best curing effect among physical and chemical methods of curing. Only larger plasmids could be transferred by conjugation. Plasmid profile analysis could be used in differentiating and identifying E. coli strains and thus serves as a better epidemiological tool. There was a correlation between hemolysin production and antibiotic resistance since both the characters were co-transferred and co-eliminated. Photographic technique of plasmid DNA with ordinary single lens reflex camera was found to serve as a substitute to polaroid camera system.ThesisItem Open Access PREVALENCE OF CHLAMYDIAL AGENTS IN LIVESTOCK IN KERALA(COLLEGE OF VETERINARY AND ANIMAL SCIENCES Mannuthy - Trichur, 1988) FRANCIS, REJI; James, P.C.
