Isolation, Identification And Biological Characteristic Research Of Pasteurella Multocida From Swine

Pasteurella multocida is an important pathogenic bacterium and can be classified into toxigenic P. multocida(T+Pm) and non-toxigenic P. multocida(TPm). Usually, TPm can cause pneumonic pasteurellosis in pig, fowl cholera in poultry, hemorrhagic septicemia in cattle and sheep. T^Pm is a major pathogen of swine infectious atrophic rhinitis.P. multocida strains are separated into serogroup A, B, D, E, F basing on capsular antigen and serotype 1 to 16 basing on somatic antigen. OmpH is a major outer membrane protein of P. multocida. The OmpHs of different serotype are highly homologous, and can stimulate high titers of protective antibodies.T+Pm generates P. multocida toxin(PMT) which is a dermonecrotic toxin about 145kDa and is encoded by toxA gene. TPm does not have this toxA gene and can not cause severe AR. But injection of purified PMT alone often results in typical nasal turbinate atrophy in gnotobiotic piglets. It is abvious that PMT plays a key role in the course of AR. Extensive research indicats that the N terminal of PMT possesses cell-binding activity and the C terminal has catalytic activity; the holotoxin of PMT is pathogenic while the properly truncated toxin may lose pathogenic activity and remain immunogenic activity. The truncated toxin can be easily obtained to use in vaccines.The focus of this research is Pm and PMT. The projects are as following:1. A PCR method is established to detect P. multocida from swine. With this method and biochemical reaction, 66 strains of P. multocida were isolated from clinic samples. Every isolate was then studied on sentivity to antibiotics.2. Based on literature, 5 sets of PCR primers were designed which are respectively specific to the capsular gene of serogroup A, B, D, E, F of P. multocida, then PCR typing method for P. multocida was established. PCR typing assay indicated that, among 66 isolates, forty-six were serogroup D(28 from nasal swabs and 18 from lung), eighteen were serogroup A(14 from nasal swabs and 4 from lung), one was serogroup B(from lung) and one was untypable(from lung).3. A PCR method is established to detect Tl'm. The PCR results indicated that, among 66 strains of P. multocida, 8 isolates are toxingenic. The 8 isolates are all serogroup D and from the nasal swabs of swine suffering atrophic rhinitis.4. Basing on the nucleotide sequences of ompH gene published in GenBank, a pairof primers was designed to amplify the ompti gene of P. multocida. This ompti gene was cloned into pMD18-T to get pMD-ompH and was then sequenced. The homology of nucleotide sequence with other seven published ompH sequences on GenBank was 96%-99%. The expression vector named pET-ompH was constructed and was then transformed into E.coli ~BLi\. SDS-PAGE and Western-blot indicated that the ompH gene is highly expressed in BL2i as a 35kDa protein which has biological activity.5. Basing on the nucleotide sequences of tax A published in GenBank, a pair of primers with restriction sites was designed to amplify toxA gene of T+Pm. The PCR product was then cloned into pET-28b to obtain pET28b-to;cA. Sequencing result revealed that its size was identical to the five published toxA sequences in GenBank and the homology of nucleotide sequence is higher than 99%. The expression vector of pET28b-/oxA was transformed into E.coli BL21. But SDS-PAGE showed no expression of the holotoxin. Based on the plasmid of pET28b-roxA, four subcloned expressing vectors were then constructed with restriction enzymes: (DpET28b-N1513; (2) pET28a-1514C; (3)pET28b-N3173; ﹑ET28b-N3389. All these vectors were used to express truncated PMT. They were transformed into E.coli BLai. But only (D pET28b-N1513 and (2)pET28a-1514C resulted expression in E.coli BL2i, whichseparately expressed 1513 base pairs of 5' terminal and 2345 base pairs of 3' terminal of toxA. The expressed proteins are 57kDa and 87kDa respectively. Western-blot indicated that both recombinant proteins have biological activity.In summary, in this study, we isolated 66 strains of P. multocida in which 8 isolates are tox...