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Kerala Veterinary and Animal Sciences University, Wayanad

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2017 - 201922020 - 20232
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Subject: Veterinary Microbiology × Start date: 2010 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    COMPARATIVE EFFICACY OF DIFFERENT TESTS FOR ANTE-MORTEM DIAGNOSIS OF BOVINE TUBERCULOSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES, MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-11-07) SARIKA N; Dr. Binu K. Mani
    Blood and nasal swab samples, 50 each and 17 milk samples were collected from animals with symptoms suggestive of bTB, presented for slaughter, at Thrissur corporation slaughterhouse, Kuriachira. The samples were subjected to detection by ZN staining, IS6110 insertion element specific PCR to detect MTBC, 12.7 kb fragment specific mPCR to differentiate M. bovis and M. tuberculosis, real-time PCR specific for a region upstream of p34 gene to detect the presence of M. bovisand M. tuberculosis, bovine gamma interferon immuno assay of blood and isolation and further characterisation. Histopathology was also performed on post-mortem tissue samples. The presence of drug resistance genes (viz., rpoB for rifampicin, pncA for pyrazinamide and katG for isoniazid) were detected in the isolates obtained using mPCR. Further, comparison of the results of different ante-mortem tests to that of isolation from the tissue samples, which being the gold-standard for confirmative diagnosis of TB, was made and analysed statistically. Finally, in order to confirm efficacy of the various ante-mortem diagnostic methods assessed in the study, 118 apparently healthy dairy cattle, maintained in organised farms and households in and around Thrissur were screened for TB. In the present study, ZN staining on nasal swab/milk/tissue samples was found to be highly specific, but showed poor sensitivity. The sensitivity of the PCR (nasal swab), real-time PCR (nasal swab), isolation (nasal swab) and gamma interferon assay was 100, 100, 100 and 50 per cent, respectively. The specificity of PCR (nasal swab), PCR (blood), real-time PCR (nasal swab), isolation (nasal swab) and gamma interferon assay was 95.83, 85.42, 97.92, 100 and 91.67 per cent, respectively. The sensitivity and specificity of PCR and real-time PCR from tissue samples was 100. None of the molecular methods could detect mycobacteria in any of the milk samples. Visible lesions were absent in the tissue samples. The HP of PCR positive tissue samples revealed multiple areas of mononuclear infiltration, epitheloid cells and mildchanges in lung samples. Present study revealed that, nasal swabs were found to be a better choice for ante-mortem diagnosis of bTB, than milk. Isolation of bacteria from nasal swab and PCR of the nasal swab were found to have maximum sensitivity and specificity for diagnosing bTB. Screening of 118 cattle maintained in organised and unorganised farms in Thrissur district, revealed a positivity rate of 2.54, 7.63 and 2.54 respectively for PCR, gamma interferon immuno assay and isolation, respectively. Drug resistance genes were detected in five of the isolates obtained, of which, all the M. bovis isolates amplified all three genes namely rpoB, pncA and katG. However, the M. tuberculosis isolates showed discordant results and failed to amplify all genes. In conclusion, a single method alone did not specifically detect mycobacteria in the present study. Therefore, a combination of tests could be used for screening and accurate diagnosis of bTB, considering other factors like, availability of samples, cost of the test and diagnostic facilities available in the laboratory.
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    ThesisItemOpen Access
    DETECTION AND CHARACTERISATION OF BACTERIAL AND VIRAL AGENTS ASSOCIATED WITH NEONATAL DIARRHOEA IN CALVES
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, WAYANAD , LERALA VETRINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-03-25) SARITHA BABY; Dr. Chintu Ravishankar
    Neonatal calf diarrhea (NCD) is a major threat to cattle farmersworldwide. It causes huge financial losses mainly due to morbidity andmortality of the calves. The etiology of NCD is complex disease in which both infectious agents and non infectious factors are involved. A comprehensiveanalysis was carried out to detect the major bacterial and viral agentsresponsible for NCD in Kerala and also to assess non-infectious factors thatcontribute to the condition. A total of 120 diarrhoeic faecal samples collected from different cattle farms in Thrissur and Wayanad districts of Kerala duringthe period from October 2019 to September 2022 were used for the study.Each farm has its own management practices that were found to influence the onset of diarrhoea. In the study it was observed that unhygienic conditions, overcrowding and changes in nutrition were the major predisposing factors for NCD. The incidence of diarrhoea was higher in farms housing large number of animals. Calves below one month of age were affected the most and the maximum number of diarrhoeic calves (26 per cent) was found in the 22-30 days age group. Among the infectious agents, E. coli, Salmonella, rotavirus and coronavirus were focused in the current investigation. The isolated bacteria were characterized by biochemical testing and E. coli was found to be the main infectious agent associated with the condition with a prevalence of 84.17 per cent. From the 120 samples, a total of 101 isolates of E. coli could be isolated. None of the samples were found to be positive for Salmonella. Antibiotic sensitivity testing revealed that the all E. coli. isolates were resistant to Penicillin G, Cefotaxime/Clavulanic acid and Cefpodoxime. The percentage of isolates resistant to Amoxicillin/sulbactam and Enrofloxacin, Ciprofloxacin, Co-Trimoxazole and Tetracycline, Gentamicin, and Nitrofuratonin were 92.08 per cent, 85.15 per cent, 80.2 per cent, 77.23 per cent, and 72.25 per cent respectively. Only 53.47 and 51.49 per cent of the isolates were resistant to chloramphenicol and streptomycin, respectively. When the E. coli isolates were subjected to phylogrouping using a quadruplex PCR, it was observed that B1 was the most prevalent group in this study with 28 isolates. Group A, D, C, E, F and clade I were also detected but with a lower prevalence. The fimbrial genes are responsible for virulence of E. coli and played a role in causing diarrhoea. The presence of fimbrial genes F5, F41 and F17 was also tested employing PCR. Of the isolates tested ten were detected as positive for F17 with a positivity of 16.13 per cent. Fimbrial genes F5 and F41 were not detected in any of the isolates. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed for the detection of the viral agents. For rotavirus and coronavirus, VP6 gene and N gene were targeted respectively. Out of the 120 samples, 14 (11. 67 per cent) were found to be positive for rotavirus. The diarrhoeic faeces of calves infected with rotavirus were gray or creamy white and watery and all the affected calves were above one week of age. Coronavirus could not be detected in any of the samples. The results of the study indicate that diarrhoea in calves in Kerala was caused by both bacterial and viral agents. Varying degrees of antibiotic resistance were also detected against the common antibiotics. Good care of the new born calves and hygienic practices in farms will go a long way in control of the condition
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    ThesisItemOpen Access
    DEVELOPMENT OF A MODIFIED INACTIVATED VACCINE AND ITS COMBINATION WITH A RECOMBINANT PROTEIN AGAINST LEPTOSPIROSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2017-12-30) MANJU SOMAN; M.Mini
    The study was taken up with an aim to develop a foolproof technique for prevention of leptospirosis. It involved the development and immunity evaluation of a modified whole cell inactivated vaccine incorporating the predominant leptospiral serovars, Australis, Autumnalis and Pomona in hamsters. The study also ascertained the genus specific immunoreactivity of a truncated recombinant leptospiral LigA protein and its combination with the modified inactivated vaccine, in hamsters. The immunomodulatory effect of incomplete Freund’s adjuvant (IFA) and the aluminium hydroxide gel adjuvant in hamsters was also assessed. Primers were designed for a highly immunodominant region of the ligA gene, spanning nucleotides from 1873 to 3363. The PCR amplified 1491 bp fragment of ligA DNA was cloned into pET -32a vector and expressed in E.coli BL21(DE3). The conditions optimum for expression of this recombinant protein were analysed. Maximum expression was obtained following induction with 2 mM IPTG, at an incubation temperature of 28o C following six hours of incubation at 200 rpm shaking speed. The Ni-NTA purified rLigA protein was used for immunization of hamsters. The optimum concentration of the rLigA protein and modified inactivated vaccine required for immunisation of hamsters, was determined by immunising four sets of hamsters with four different concentrations of the antigens, 14 days apart. It was revealed that the concentration of 80µg/ 40 µg of Lig A protein and 108 leptospires per millilitre gave the maximum IgG ELISA and MAT titres.Six vaccine groups were set up for six different vaccine combinations which included the modified inactivated vaccine, rLigA protein and a combination of the modified inactivated vaccine and rLigA protein. Adjuvants IFA and aluminium hydroxide were used in the study. The serum antibody titres on days 0, 7,14 and 27 were determined by MAT and recombinant IgG ELISA The virulence of laboratory strains of Leptospira interrogans serovars Pomona (homologous) and Icterohaemorrhagiae (heterologous) was enhanced by serial passage in hamsters and these were used as challenge organisms in the study. The LD50, of the serovars Pomona and Icterohaemorrhagiae, in hamsters was determined as 106.893 organisms and 107.38 organisms, respectively and the challenge was carried out with 100 LD50 (≈109 ) organisms, on 28th day post first immunization. Challenge studies revealed maximum protection levels of 80 to 100 per cent in groups immunised by modified inactivated vaccine alone and combination of rLigA and inactivated vaccine. Groups immunised with rLigA protein alone showed 60-70 per cent protection to both serovars. The highest MAT titres to homologous and heterologous serovars were presented by the groups immunized with a combination of rLigA protein and modified inactivated vaccine. These groups elicited higher MAT titres to heterologous serovar Icterohaemorrhagiae compared to whole cell vaccine alone, which indicated the genus specificity contributed by the partial rLigA protein. It also showed that the inactivated vaccine and recombinant protein compliment each other in increasing the respective immunogenicity. The study revealed that IFA adjuvanted rLigA protein could elicit the maximum ELISA titres in hamsters, followed by the group immunised by IFA adjuvanted rLigA protein combined with modified inactivated vaccine. The IFA adjuvanted vaccine groups showed higher ELISA titres compared to those adjuvanted with aluminium hydroxide but the aluminium hydroxide adjuvanted vaccine groups showed consistent increase in antibody titres.
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    ThesisItemOpen Access
    ASSESSMENT OF CELL MEDIATED AND HUMORAL IMMUNE RESPONSE TO SUBUNIT VACCINE AGAINST RIEMERELLOSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2019-12-30) RINSHA BALAN; Priya P. M.
    Riemerellosis is a bacterial disease among ducks, caused by Riemerella anatipestifer, which has been well documented as a cause of considerable economic loss to the duck production in Kerala. At least 21 serotypes of the organism have been identified globally. Since vaccination is the mainstay for the control of the disease, a research work was undertaken to prepare subunit vaccine employing recombinant OmpA of R. anatipestifer and assessment of cell mediated and humoral immune responses of the vaccine and also to evaluate the comparative efficacy with that of the developed inactivated vaccine. Broth culture of R. anatipestifer at a concentration of 2.5 OD values at 525 nm with a dose of 1 mL per bird subcutaneously was selected as LD50. L per bird subcutaneously was selected as LD50. A total of 52, day-old ducklings were divided into three treatment groups with ten birds each. They were injected with 0.5 mL of different types of vaccine subcutaneously. Group I (T1) served as control with 22 birds including six birds each for challenge control of inactivated and subunit vaccine. Group II (T2) was injected with an inactivated vaccine (7x109 cfu/mL), which was prepared as per the protocol standardised in the Department of Veterinary Microbiology and group III (T3) and group IV (T4) were administrated with different antigen concentration of subunit vaccine (equal quantity of the rompA protein (250µg and 500µg) and montanide, respectively). A booster dose was given at third week post-primary vaccination to T2, T3 and T4. It was observed that, by using both crude Omp and rOmpA based ELISA, inactivated vaccine birds (T2) produced higher antibody titre during early age while in the subunit vaccine group, the titre was higher during later stage. An early antibody response is required to lower the mortality rate in riemerellosis as the organism targets young ducklings. Thus, it could be inferred from this study that inactivated vaccine was more effective than subunit vaccine A significant CMI response was also shown by inactivated vaccine groups on 14th and 28th day post-vaccination by lymphocyte proliferation assay (LPA). Challenge studies to assess the protective response revealed 100 per cent protection for inactivated vaccine group (T2), 80 per cent protection for T4 group and 70 percent protection for T3 group. All the vaccinated birds were having significantly less gross lesion when compared to the challenge control groups. On analysing the cytokine mRNA expression levels using real-time PCR, the inactivated vaccine group showed significantly higher (p< 0.05) mRNA levels of IL-6, IL-12B and IFN-γ gene on day 28 than the two subunit vaccine groups. It was found that the inactivated vaccine was superior in terms of results obtained from the challenge study, antibody titre, CMI response and gene expression analysis than the subunit one. Hence, it is desirable to advocate the use of inactivated vaccine in field condition owing to its easiness to prepare and low cost.