Study On The Molecular Epidemiology And Pathogenicity Of Pasteurella Multocida From Swine

Pasteurella multocida is an important pathogenic bacterium, which is responsible for various infections in humans and animals. The main diseases associated with this organism in pigs are pneumonic pasteurellosis and atrophic rhinitis. P. multocida is also a contributory agent in porcine respiratory disease complex, a multifactorial disease state increasingly problematic for swine producers. Although the primary disease is important, what's more significant is the fact that this bacteria predisposes to colonization and disease with other viral and bacterial pathogens, resulting in higger morbidity and mortality. This study focused on the pathogen epidemiology, bio-characteristics, antibiograms, virulence genetypes, the pathogenicity of P. multocida, immunogenicity genes, diagnostic method, and two fragments of recombinant subunit P. multocida toxin (rsPMT) were constructed for evaluation as candidate vaccines against progressive atrophic rhinitis (PAR) of swine. The results are as follows.1. Isolation and identification of P. multocida from pigs and pathogen epidemiology of pasteurellosis in ChinaThe presence of P. multocida in clinical specimens from diseased pigs with pneumonia or atrophic rhinitis from 16 provinces in China between 2003 to 2007 were analyzed, and a total of 233 isolates of Pasteurella multocida were obtained from 2,912 cases of clinical respiratory disease in pigs, giving an isolation rate of 8.0%. The isolation rate at different time points (years) were ranged from 6.4 to 10.2%. Serogroup A P. multocida was isolated from 92 cases (39.5%) and serogroup D isolates from 128 cases (54.9%); twelve isolates (5.2%) were untypable. P. multocida was the fourth most frequent pathogenic bacterium recovered from the respiratory tract, after Streptococcus suis, Haemophilus parasuis and Escherichia coli. The pathogenicities of 49 porcine P. multocida strains isolated from different regions of China were evaluated in mice. Based on mortalities occurring during a 7-day postinoculation period,36.7%,49.0% and 14.3% of 49 isolates were characterized as high, intermediate and low in pathogenicity, respectively. Among them, eleven toxigenic strains of P. multocida of serogroup D isolated from 37 samples of nasal swabs and lesioned lungs from pigs with typical clinical signs of atrophic rhinitis were characterized as high in pathogenicity. These results reveal that P. multocida are widely prevalent in China, and suggest such pathogenic bacteria may play an important role in causing respiratory disease of swine. 2. Phenotypic and genotypic characterization of antimicrobial resistance in P. multocida isolatesAntibiograms and relevant genotypes of porcine P. multocida isolates (n=233) recovered between 2003 and 2007 in China were assessed via broth microdilution test, PCR and sequencing. Among the 20 antimicrobials tested, the most prevalent resistance was to amoxicillin, lincomycin, clindamycin, tetracycline and sulfonamides (ranging from 58.0-96.6%), followed by tilmicosin (28.3%) and aminoglycosides (ranging from 12.0-14.2%). The frequency of antimicrobial resistance among P. multocida isolates from swine in China was higher than that reported from other countries, and 93.1% of the isolates showed multiple resistances,54.7% isolates were resistant to more than five. The most common resistance pattern observed among the isolates was multi-resistance to AMX+LIN+CLI+TET+SDM+STX. There was a progressive increase in multiresistance to more than seven antibiotics, from 16.2% in 2003 to 62.8% in 2007. Resistance profiles suggested that cephalosporins, florfenicol and fluoroquinolone were the drugs most likely to be active against P. multocida. For each antimicrobial agent, amoxicillin resistance was primarily mediated by blaTEM (80.7%), lincosamides resistance by ermA (36.7%), ermC (49.8%) and lnuA (24.0%), trimethoprims resistance by dfrA5 (18.5%), dfrA7 (32.9%) and dfrA13 (29.5%), tetracycline resistance by tet(H), tet(B) and tet(G), and sulfadimidine resistance by sull and sul2. Strains that harbored several genes that conferred resistance to the same antimicrobial agent were often significantly (P< 0.01) more multiresistant than others, and there was positive relation between drug resistance and genetypes. These results provide novel insights into the epidemiological characteristics of porcine P. multocida strains in China, and suggest the need for the prudent use of antimicrobial agents in food animals.3. The presence and distribution of virulence genes of P.multocida strains from swineAmong the 233 porcine P. multocida isolates, the 19 virulence gene regions ranged in prevalence from 4.7%(toxA) to 100%(ompA). Multiple adhesions (including ptfA, fimA and hsf-2),all iron acquisition factors (exbB, exbD, tonB, hgbA and fur), nanH, and various outer membrane proteins (ompA, ompH, oma87 and plpB) were found each to occur in over 90% of porcine strains. The VFs pfhA, tadD, toxA and pmHAS were present in<50% of strains each. The various VFs exhibited distinctive associations with serogroups, tadD was significantly associated with serogroup A, hsf-1 and nanB were significantly associated with serogroup D, and hgbA and fur were associated with serogroups A and D. The genes pfhA and pmHAS were less common in serogroup D than in other serogroups. It was noted that the toxA gene, which is involved in the pathogenesis of progressive atrophic rhinitis in pigs, was found in only eleven strains, and it was strictly restricted to strains belonging to capsular serogroup D.4. Molecular diversity of P.multocida isolates from diseased swine in ChinaA total of 233 porcine P. multocida strains isolated from cases of pneumonia and progressive atrophic rhinitis (PAR) were evaluated for the genotypes and genetic diversity using several DNA fingerprinting methods:RAPD, (GTG) 5-PCR and Eric-PCR. The discriminatory index (CI) for RAPD was 39.48%, and 73.96% distinct bands were identified as polymorphic bands. Analysis of DNA banding patterns generated by RAPD revealed the presence of 92 different genotypes, among which 11 genotypes (12.0%) were preponderant profiling. All isolates could be grouped into 32 clusters at a similarity of 60% after dendrogram analysis. The largest lineage contained 44 strains belonging to serogroup A (20 strains), serogroup D (22 strains), serogroup B (1 strain) and untypable (1 strain), respectively. The discriminatory index for (GTG) 5-PCR was 49.36%, and 88.13% distinct bands were identified as polymorphic bands. These isolates could be divided into 115 different genotypes by their (GTG) 5-PCR fingerprints, among which 7 genotypes (6.08%) were preponderant fingerprints. All isolates could be grouped into 68 clusters at a similarity of 60%. The discriminatory index for Eric-PCR profiling was a relatively low value (CI=24.46%), and 73.46% distinct bands were identified as polymorphic bands. These isolates could be divided into 57 different genotypes by their Eric-PCR fingerprints, among which 11 genotypes (19.03%) were preponderant fingerprints. All isolates could be grouped into 36 clusters at a similarity of 60%. In summary, the results revealed there was significant diversity among analysed strains of porcine P. multocida strains, and suggested that (GTG) 5-PCR and RAPD are highly discriminatory techniques for detecting genetic variation and in epidemiological studies of P. multocida.5. Molecular characteristics and the pathogenicity of porcine toxigenic P. multocidaIn order to analysis the molecular characteristics, genetic background and the pathogenicity of toxigenic P. multocida isolates from swine in China, toxA gene from eleven strains were investigated. Their sequences analyzed here indicated that the P.multocida toxin (PMT) coded by toxA gene is a high conservatism protein. The homology of toxA gene nucleotide sequences of all strains hit above 99%, and the homology of amino acid sequences reached to 98.5% above. However, the nucleotides of toxA gene of five strains,have genesised mutation,in the site 911-912 and 2323-2324. The prevalence and genetic evolution were analyzed based on the toxA gene. We found that there are two highly diverse subgroups among all T+Pm isolates in China. By virulence tests, we found that the HN-13 strain of T+Pm showed high virulence, and the 50% lethal dose (LD50) of HN-13 in mice have been tested to be 4.25×103 CFU intra-abdominal infection. Further, the HN-13 intranasal infection model was also established using piglets. The results showed that there testing piglets not only have the clinical symptom of PAR, but also could cause pneumonia. Immunohistochemistry confirmed that neutrophil could be mobilized to counteracting incursive pathogenic bacteria during acute stage, and macrophagocyte could also participate in the mechanism, inducing cellular necrosis and pulpy pathological changes.6. Characteristics and immunogenicity of the N-terminal and C-terminal re-combinants of P.multocida toxinIn this study, five fragments of recombinant subunit P.multocida toxin (PMT) were constructed. Only pET28a-N1518 and pET28b-C2115 could be expressed efficiently in Escherichia coli. The molecular weight of the fusion proteins was 57 kDa and 78 kDa. Western blot confirmed that the two proteins could specifically react with an-tiserum against P.multocida toxin. No mice died after the intraperitoneal administration of these two proteins with the dose of 200μg, but Vero cell was pathologically changed after administration of 896ng/mL rPMT-N. The fusion protein of rPMT-N and rPMT-C was purified, and emulsified with Freund's adjuvant in equal volumes to get subunit vaccine. Mice immunized with rPMT-N, rPMT containing rPMT-N and rPMT-C, crude PMT only developed high titers of neutralizing antibodies. However, all piglets were challenged intranasally with 1×1010CFU CFU HN-13 strain of T+Pm. The protection efficiency of rPMT-N, rPMT and crude PMT against HN-13 strain were higher than the group immunized with rPMT-C. The data revealed that the fusion protein of rPMT-N had immunogenicity and potential for developing a subunit vaccine against PAR in pigs.