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Subject: Veterinary Microbiology × End date: 2005 × Start date: 2005 × Type: Thesis × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    Nucleic Acid based detection of salmonellae in poultry
    (Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Muthuramalingam, M; KAU; Mini, M
    In the present study detection of salmonellae by Polymerase Chain Reaction in avian bio-materials was carried out. Isolation of salmonellae from avian bio-materials was also done. Differentiation of salmonellae based on molecular methods was carried out. Thirteen isolates from chicken and two from quails were characterized as Salmonella gallinarum using standard bacteriological procedures. With regard to the fermentation of the sugars all isolates fermented dulcitol, maltose, arabinose, trehalose, and mannitol. Variation in fermentation pattern was observed with xylose and sorbitol. All isolates were uniformly sensitive to chloramphenicol, ciprofloxacin, enrofloxacin, and pefloxacin, while all were resistant to tetracycline, furazolidone and cloxacillin. Two isolates were serotyped as Salmonella gallinarum by the National Salmonella and Escherichia Center, Kasauli. Forty-six samples were positive by both genus specific as well as serovar specific PCR. The genus specific and serovar specific PCR were used to confirm the identity of the isolates. Performing PCR on template DNA prepared from RV broth enriched sample was found to be an extremely rapid method for detection of Salmonella. Restriction enzyme analysis of the amplicon from the rfbS gene with enzyme Tfi І of all isolates revealed the expected 235 bp digestion. All the isolates carried plasmids. Two plasmid profiles were observed among the isolates examined. A multiplex PCR for virulence plasmid was carried out. The expected 571 bp level amplification, which is specific for Spv virulence region and 284 bp level amplification, which is specific for genus Salmonella, were obtained in all the isolates. An allele-specific PCR method was developed for serotype-specific detection of S. gallinarum. The expected 187 bp level amplicons were obtained in all the isolates. The sequence of the rfbS gene product has been submitted to the Genbank and has been assigned the accession No AF 442573 ATCC 9184 . The sequence showed 99 per cent identity with Salmonella gallinarum.
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    ThesisItemOpen Access
    Detection of pathogenic haemolytic bacteria in respiratory tract infections of livestock
    (Department of Veterinery Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Aparna, S; KAU; Krishnan Nair, G
    A study was undertaken to elucidate the role of haemolytic bacteria in respiratory tract infections of livestock. This envisaged the isolation and identification of the haemolytic bacteria, determining their antibiogram patterns and testing the pathogenicity of the isolates in mice. The study also envisaged the detection of Mannheimia haemolytica by polymerase chain reaction, determining the genetic relationship of Staphylococcus aureus isolates from different animal hosts using RAPD-PCR technique and also analysis of plasmid profiles of Escherichia coli isolates. Samples were collected from clinically ill livestock and at random from apparently healthy animals from in an around Thrissur district. A total of 309 samples were taken which consisted of nasal swabs, tracheal swabs, lung samples and blood samples. Samples were cultured on blood agar and on Mannheimia haemolytica selective medium. Mannheimia haemolytica could not be isolated from any of the samples. But pooled nasal swabs when cultured on blood agar gave an isolate with characteristics almost similar to Mannheimia haemolytica, but showed variations for ornithine decarboxylase activity and utilization of sugars like trehalose and salicin. As, no reference strain was available it was not possible to make a comparison and confirm the isolate as Mannheimia haemolytica. From the samples cultured on ordinary blood agar medium a total of 20 haemolytic bacterial isolates could be obtained. The different bacterial isolates were Staphylococcus aureus (40 per cent), Staphylococcus epidermidis (10 per cent), Escherichia coli (30 per cent), Klebsiella pneumoniae (5 per cent), Streptococcus pyogenes (5 per cent), Streptococcus agalactiae (5 per cent) and Arcanobacterium pyogenes (5 per cent). The haemolytic bacteria were identified based on morphology, cultural characteristics and biochemical tests. Antimicrobial sensitivity pattern of the isolates showed that almost all the isolates had high sensitivity to pefloxacin and ampicillin. Antimicrobial resistance was shown maximum to erythromycin. The three Staphylococcus aureus isolates, all the E. coli isolates and Klebsiella isolate caused death of mice. Rest of the five Staphylococcus aureus isolates, Streptococcus pyogenes , Streptococcus agalactiae and Arcanobacterium pyogenes did not cause death of mice but produced internal lesions and could be re-isolated. Staphylococcus epidermidis isolates could neither cause death, nor produce internal lesions and could not be re-isolated. None of the nasal and tracheal swabs, lung samples and blood samples gave a positive result for Mannheimia haemolytica specific PCR. A pooled sample of nasal swabs from cattle with respiratory infection gave a positive result for it. But PCR of the culture could not yield a positive result. Moreover, no reference strain was available to make a comparison and confirm the result. RAPD-PCR of the Staphylococcus aureus isolates showed that there was considerable genetic relationship between Staphylococcus aureus isolates of different species and also there was noticeable genetic diversity of the isolates within the host species. Plasmids could be isolated only from two of the six isolates of Escherichia coli studied. Plasmid profile analysis of the isolates could not ascertain any correlation between the virulence, antibiotic resistance and presence of plasmids.
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    ThesisItemOpen Access
    Detection and identification of pathogenic leptospires in bio-meterials
    (Department of Veterinary Microbiology, College of Veterinary and Animal Science, Mannuthy, 2005) Dhannia, A; KAU; Jayaprakashan, V
    In the present study an attempt has been made to detect and differentiate leptospires in bio-materials employing molecular techniques such as genus specific PCR, multiplex PCR, nested PCR, AP-PCR, LS-PCR and PCR-REA. Isolation trials for Leptospira were also made in the study and the isolates were tried to be differentiated employing the molecular methods mentioned above. The genus specific primers A and B were used to detect leptospires in clinical samples and samples from rodents. Out of the 147 samples only nine were positive for leptospiral DNA. Out of the nine positive samples eight were serum samples (four from cattle and four from dogs) and one was kidney of a bandicoot. Multiplex PCR, using the primers G1/G2 and B64-I/B64-II could differentiate leptospires into pathogenic and non-pathogenic ones. Among the pathogenic leptospires it could differentiate the species L. kirschneri, from other six pathogenic species viz L . interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. inadai and L. weillii. All the six isolates were found to be belonging to any of these six species. Nested PCR using the primers designed based on the sequence of L. borgpetersenii and L. interrogans amplified DNA from all the ten reference strains including the non-pathogenic serovar patoc and rachmati of L. kirschneri species. All the six isolates were amplified giving PCR products of expected sizes, 571 bp and 370 bp.