Detection of Pasteurella multocida in domestic ruminants by isolation and polymerase chain reaction

Abstract

A study was undertaken to examine the occurrence of P. multocida organisms causing HS in apparently healthy and clinically ill domestic ruminants in a specified area of Thrissur district viz., Ollukkara block, covering one per cent of total ruminant population in the area. Besides the samples collected from Ollukkara block area, those brought to the Department of Microbiology, College of Veterinary and Animal Sciences, Mannuthy from suspected cases of HS, were also included in the study. A total of 309 samples comprising of nasal swabs, pharyngeal swabs, lung samples and blood samples were processed for isolation of P. multocida and detection by PCR. Detection of P. multocida by PCR was carried out using species-specific (PM-PCR), type-B specific (HS-B PCR) and nested primers. Pasteurella multocida could not be isolated from any of the clinical samples cultured for isolation from Ollukkara block area. Five isolates could be obtained from the blood samples from Palakkad district. Isolates obtained were characterized as P. multocida using standard bacteriological procedures. A reference strain P52 obtained from IVRI was used for comparison. All the isolates were found to be pathogenic for mice. Based on the fermentation patterns of dulcitol, sorbitol, trehalose and xylose all isolates as well as P52 could be biotyped as P. multocida subsp. multocida. All isolates were uniformly sensitive to ten out of twelve antibiotics tested and uniformly resistant to metronidazole. All isolates were confirmed as type-B P. multocida using species-specific primer pairs, KMT1SP6 and KMT1T7 and HS-B specific primer pairs, KTSP61 and KTT72. Multiplex PCR was used to characterize all the serotype B isolates, which generated two bands of size 460 bp and 590 bp. Only 14 clinical samples including the isolates were positive for P. multocida by PM-PCR. The entire samples tested positive by PM-PCR were confirmed as type-B P. multocida by HS-B specific PCR. The 14 samples found positive by PM-PCR when subjected to nested PCR, gave an amplified product of size 214 bp. Since nested PCR could detect Pasteurella multocida DNA in a high proportion of clinical samples found previously negative by PM-PCR, a definite conclusion could not be arrived about the feasibility of using this PCR assay for enhanced detection of P. multocida as only a random number of negative clinical samples could be screened. All the isolates showed a single REP-PCR profile, indicating a high level of homogeneity among them. Among the five isolates examined, only two (BP2 and BuP1) harboured plasmids. The sequence similarity searches of HS-B PCR amplified product with BLAST network showed that the sequence had 99 per cent identity with Pasteurella multocida unknown protein 1 and protein 2 gene (Accession No AF016260).