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Atrophic rhinitis is a disease of high economic impact for pig production, caused by Pasteurella multocida (P.m.) strains able to produce dermonecrotoxin (DNT). In general the diagnosis of pigs’ infection with P.m. is based on the direct detection of bacteria from nasal swabs and their identification as well as on the detection of DNT specific antibodies by using an ELISA test. The aim of the study was the elaboration of a PCR test for the detection of dermonecrotic strains of P.m. directly in nasal swabs. The optimalization of the process includes: estimation of Mg²⁺ concentration, the temperature of primer hybridization and the number of cycles. The specificity and sensitivity of the test was estimated for both bacterial culture and nasal swabs. The optimal conditions were as follow: Mg²⁺ concentration - 2.5 m M/reaction; the temperature of primers hybridization - 56°C; and the number of cycles - 50. The sensitivity of the test for both the culture and nasal swabs was 2.5 × 10³ cfu/ml. The test was also highly specific - the product of 501 bp was detected only in the samples containing DNA of P.m. Summarizing, the elaborated test was specific and sensitive enough to be used for routine detection of P.m. DNT strains in clinical samples and for the elimination of the carriers from the herd.
Dermonecrotoxigenic (DNT) strains of Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bbr), etiological agents of atrophic rhinitis (AR) of swine, are usually detected by conventional microbiological methods. Currently the main tasks in the diagnosis of infectious diseases is to detect the specific genes of pathogens directly from clinical samples within the shortest possible period of time by using sufficiently specific and sensitive methods. In this study two PCR tests were developed for the detection of genes encoding DNT in Pm and Bbr directly in nasal swabs. They were used for examining 481 nasal swabs collected from pigs raised in 56 farms, suspected of AR or displaying clinical symptoms of PAR. Additionally, the usefulness of PCR in the routine diagnosis of AR was compared with standard microbiological and serological methods. DNT+ Pm were directly evidenced in 74 samples (15.4%) from 27 tested herds (48.2%) while DNT+ Bbr were detected in 196 swabs (40.7%) from 41 farms (73.2%). Using the standard microbiological examination, the presence of bacterial cultures of the morphology typical of Pm was observed in 114 tested samples (23.7%) and those typical of Bbr, in 156 samples (32.45%). In the PCR test performed to confirm the presence of the DNT- -encoding gene in Pm isolates, positive results were obtained in 26 samples (22.8%), while among isolated Bbr strains, 95 out of 156 (60.1%) possessed the gene encoding DNT. In the ELISA test, 22 out of 26 isolates (84.6%) were DNT+. In the case of DNT- isolates of PM, the results of ELISA were in full agreement with PCR results. In total, PAR was detected in 27 farms (48.2%), NPAR in 21 farms (37.5%), and 8 farms (14.8%) were diagnosed as free from AR since no positive results were obtained by either method. Summarizing, the results of serological, bacteriological and molecular examinations clearly demonstrated the usefulness of PCR in the routine diagnosis of AR directly from clinical material.
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