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20114
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Subject: Veterinary Microbiology × End date: 2011 × Start date: 2010 ×
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    ThesisItemOpen Access
    ISOLATION AND MOLECULAR CHARACTERIZATION OF Pasteurella multocida FROM SWINE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) SREEDEVI. T. K; Siju Joseph
    A total of 193 samples from swine were collected and processed for isolation of P.multocida, from which six isolates could be obtained in pure form. All the isolates were characterized using cultural, morphological and biochemical tests and were identified to be P.multocida. The antibiogram pattern of the isolates revealed that the isolates were resistant to metronidazole, oxacillin and penicillin G. All the isolates were sensitive to chloramphenicol, amoxycillin, ampicillin, cefotaxim, ciprofloxacin, co-trimoxazole, doxycycline, enrofloxacin, erythromycin, gentamicin, nitrofurantoin, norfloxacin and oxytetracycline. The isolates were subjected to PM-PCR and nested PCR for confirming them as P.multocida, which were then tested in a multiplex PCR to identify that all the six isolates belonged to P.multocida serogroup A. These isolates were further characterized genotypically using REP-PCR and REA and observed that REP-PCR and REA using Hpa II generated two profiles which had correlation with phenotypic grouping employing the sugar fermentation tests. Thus in the present study it was found that P.multocida serogroup A was the predominant serovar infecting swine causing pneumonic as well as septicaemic pasteurellosis. It is also concluded that REP-PCR and REA are appropriate tools for conducting molecular analysis of swine P.multocida isolates.
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    ThesisItemOpen Access
    DETECTION OF ANTIGENIC VARIANTS OF CANINE PARVOVIRUS BY POLYMERASE CHAIN REACTION AND SEQUENCING
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) MEENU BASHEER; M. Mini
    A study was conducted to detect the antigenic variants of canine parvovirus from faecal samples of clinically suspected dogs by PCR and sequencing. The samples were screened for the presence of CPV by HA test and the results were confirmed by HI test. Antigenic characterisation of field strains and vaccine strains was performed by employing PCR with differential pairs of primers and PCR-RFLP analysis. The partial sequences obtained from the amplified CPV VP2 gene fragment of nine samples were analysed to identify the nucleotide variations. A total of 78 faecal samples were collected from dogs suspected for CPV infection that were brought to veterinary hospitals attached to KAU and District Veterinary Centre, Thiruvananthapuram. Twenty eight (35.90 per cent) out of 78 samples were found to be positive to CPV infection by HA test and the results were further confirmed by HI test. Among 78 faecal samples tested by PCR, none of the samples were found positive for CPV-2, while 57 (73.08 per cent) were found positive with CPV-2ab primers. Out of 57 CPV-2ab positive samples, 15 were identified as CPV-2a types while remaining 42 were CPV-2b types. The specificity of PCR was further confirmed by RE digestion using Hha I enzyme. Thus, CPV-2b was identified as the predominant CPV type in this study. Among the four vaccines tested, Megavac-6, Nobivac® Puppy DP and Vanguard® 5 vaccines were found to be classical CPV-2 based vaccines while Duramune® max vaccine was found to contain the CPV-2b variant. All the 57 CPV-2ab positive samples, when subjected to PCR with CPV555 primer set, yielded a specific amplicon of 583 bp. On PCR-RFLP analysis with Mbo II enzyme, none of these products showed specific CPV-2c cleavage pattern of 500 and 83 bp. This indicated the absence of CPV-2c in this area. Sequence analysis of the CPV VP2 gene from the PCR products of nine samples revealed that seven were CPV-2b types and the remaining two were “new CPV-2a” types. No CPV-2c types were detected among the sequenced samples. Additional mutations at nucleotide 4104 of residue 440 and at nucleotide 4121 of residue 445 were also observed in the CPV sequences under study. Sequence similarity searches of the CPV sequences using BLAST analysis revealed that the sequences were highly specific to CPV, as indicated by the maximum identity (99-100 per cent) obtained with VP2 gene sequences of other CPV strains available in the GenBank. Phylogenetic analysis revealed that most of the CPV samples under study were related to variants typical to the country. Hence the Indian isolates were found to be genetically close to Asian isolates suggesting the migration of variants crossing the borders of different countries.
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    ThesisItemOpen Access
    EVALUATION OF OUTER MEMBRANE PROTEIN BASED IgM DOT ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE RAPID DIAGNOSIS OF CANINE LEPTOSPIROSIS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) G. ABHINAY; Siju Joseph
    Leptospirosis is an important public health threat in many places of south India, including Kerala. Diagnosis of leptospirosis is mostly dependent on serological methods. The undercurrent antibodies caused due to prior infection or vaccination, complicate the confirmation of disease, especially in an endemic region. Therefore, tests based on detecting leptospiral IgM antibodies can serve as a useful approach in diagnosing acute canine leptospirosis. The potential of IgM dot ELISA for diagnosis of acute canine leptospirosis was evaluated in this study. A total of 114 canine serum samples collected from clinical cases during the period of September, 2009 to January, 2011, presented at University Veterinary Hospitals at Mannuthy and Kokkalai, Thrissur were employed in the study. The samples included those collected from 22 healthy vaccinated animals and 28 healthy unvaccinated animals. The rest of the samples were taken from cases suspected for acute leptospirosis. Among 114 samples screened with MAT, 86 were positive cases, out of which 81 gave positive reaction with LAT. Among the 28 cases shown as negative by MAT, 23 showed a negative reaction with LAT. Among 81 cases diagnosed as positive by both, MAT and LAT, 13 samples showed positive results with IgM dot ELISA, showing a prevalence of 16.04 per cent. The serum samples from the healthy vaccinated animals and the healthy unvaccinated animals were shown as negative, when tested with IgM dot ELISA, proving the specificity of the test. Convalescent sera, collected from 8 cases among the 13 positive reactors of IgM dot ELISA when screened with MAT showed a four-fold increase in MAT titers, proving the sensitivity of the test. Latex agglutination test used in the study could prove useful as a convenient screening test for leptospirosis, under field conditions. However, LAT failed to differentiate between acute and chronic carriers of the infection. The OMP based IgM dot ELISA reported in the study would thus be of great value in diagnosing acute cases of canine leptospirosis. The test being rapid and economical, not requiring any special equipment or technical expertise, makes it suitable as a field level test.
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    ThesisItemOpen Access
    ISOLATION AND CHARACTERIZATION OF Riemerella anatipestifer FROM DUCKS
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES-MANNUTHY,THRISSUR, 2011) SURYA. P. S.; M. MINI
    Six bacterial isolates from ducks were characterized as Riemerella anatipestifer using morphological, cultural and biochemical characters. Duck isolate of Pasteurella multocida serotype A was used for comparison. All the R. anatipestifer isolates showed similar biochemical characters except for urease reaction and sugar fermentation. Riemerella anatipestifer and P. multocida were differentiated using tests like indole production, gelatin liquefaction, ornithine decarboxylase utilization and fermentation of glucose. All the R. anatipestifer isolates were sensitive to chloramphenicol, ciprofloxacin, enrofloxacin, norfloxacin, gentamicin, clindamycin, doxycycline and cefuroxime. The R. anatipestifer isolates were found to be non-pathogenic to mice, but highly pathogenic to ducklings when inoculated subcutaneously and intramuscularly. The whole cell protein profiles of all the R. anatipestifer isolates were similar, which was different from that of P. multocida. Six protein bands of molecular weights 93, 70, 60, 52, 40 and 28 kDa were intensely stained in R. anatipestifer profile. Restriction endonuclease analysis of genomic DNA was carried out using Hinf I, EcoR I, Not I and Hha I. Hinf I revealed polymorphic profiles, EcoR I yielded indistinguishable smearing pattern, Hha I showed similar pattern for all the isolates and Not I revealed no digestion. The Hinf I was more discriminatory as it could group all the isolates in to two profiles. Pasteurella multocida showed a different restriction enzyme pattern for Hinf I and Hha I. The different molecular techniques used in this study showed homogeneity in protein profile and genetic heterogeneity among field isolates of R. anatipestifer. Among the restriction enzymes used in the DNA fingerprinting of R. anatipestifer by restriction enzyme analysis, Hinf I was found to have more discriminatory power