| Swine pasteurellosis is a significant bacterial infectious disease caused by Pasteurella multocida,which is a Gram-negative facultative anaerobic bacterium of the Pasteurella genus of the Pasteurella family.P.multocida strains used to be dominated by serogroups B and A in pigs in China,and serogroup D was rare.However,in recent years,studies have found that serogroups A and D are the main prevalent serotypes,while serogroup B is rare.The prevention and control technology of swine pasteurellosis is relatively backward in China,the main use is still the traditional vaccine developed in the mid-20th century,swine pasteurellosis inactivated vaccine（serogroup B strain C44-1 strain）and swine pasteurellosis live vaccine（serogroup B strain EO630）.The serogroup of the traditional vaccine strain does not match the current epidemic serotype.Our latest study shows that it does not provide effective cross-protection against serogroup A and serogroup D strains.Therefore,research and development of a bivalent inactivated P.multocida vaccine for the current epidemic serogroup A and serogroup D is of great importance for the prevention and control of swine pasteurellosis.In this study,13 strains of P.multocida serogroup A（A1–A13）and 13 strains of P.multocida serogroup D（D1–D13）were selected as research objects and their basic biological characteristics,virulence,infection model and immunogenicity were systematically investigated.The vaccine strains were screened and obtained,and the bivalent inactivated vaccine（A7strain+D8 strain）of swine pasteurellosis was prepared.The safety and protective efficacy of the vaccine were objectively evaluated,which laid the foundation for the research and development of the inactivated swine pasteurellosis vaccine.The specific research contents are as follows.1.Basic biological characteristics of candidate strains of P.multocida from porcine sourcesThirteen candidate strains of type A（A1-A13）and type D（D1-D13）were systematically studied for their culture characteristics,growth curves,drug resistance and virulence characteristics.The results of growth curves showed that all 26 strains had obvious logarithmic growth and stable phase,and the time to reach the peak of viable bacteria content was between 8 and 12 h,and the peak of viable bacteria content was between 1.3×109 and 5.8×109 CFU/m L.Susceptibility testing of 19antibacterial drugs showed that all 26 strains were highly sensitive to 8 drugs,including ciprofloxacin,flufenicol and ceftazidime,and they were completely resistant to lincomycin.Multiple drug resistance is predominant in capsular D strains.The results of the virulence test in mice showed that the intraperitoneal injection was more pathogenic than the subcutaneous injection.The LD50 of 13 serogroup A strains was between 7-238 CFU,and that of type D strains was between 4.0×104–9.7×105CFU,indicating that the virulence of type A strains was generally higher than that of type D strains.This study provides basic experimental data for screening porcine-derived P.multocida vaccine strains and for high-density fermentation of antigens in late mass production.2.Immunogenicity of candidate P.multocida vaccine strains from porcine sourcesUsing P.multocida A strain A1-A13 and D strain D1-D13 as antigens,monovalent inactivated aluminium hydroxide gel adjuvanted vaccines were prepared.Immunological protection tests were performed in mice.The results showed that all strains of genotype A:L6 could not provide effective homologous protection,but some strains（2/13）could provide heterologous protection against A:L3.All A:L3strains could provide homologous protection and the protection rate was between 50%and 100%,but they could not provide heterologous protection against A:L6 strains.All type D strains can provide homologous protection and the protection rate is between 60%and 100%,and almost all type D strains have cross-protection effect.According to the growth characteristics,virulence characteristics and immunogenicity of the 26 strains,serogroup A virulent strains A5 and A7 and serogroup D virulent strains D5 and D8 were selected as experimental strains to compare the challenge between subcutaneous and venous routes of piglets.The results showed that with the same amount of challenge bacteria,subcutaneous and auricular vein inoculation of P.multocida can cause typical clinical signs and pathological changes of pasteurellosis,but auricular vein injection can cause more severe clinical manifestations.The A7 strain was more pathogenic to piglets than the A5 strain.The D8 strain was more pathogenic to piglets than the D5 strain.The results showed that the lethal dose of P.multocida strain A7 was 2.1×107 CFU and that of strain D8 was5.6×109 CFU.Disease severity in piglets depended on challenge dose.The results indicated that the infection models of A7 strain and D8 strain were successfully constructed.The results of the immune protection test of piglets with monovalent inactivated vaccines of P.multocida strains A7 and D8（6.0×109 CFU/m L）showed that there were no vaccine residues,disease symptoms or deaths after immunization.14 days after immunization,a lethal dose of each parental strain was used for auricular vein challenge.The results showed that all piglets in the control group became ill and died,characterized by severe cellulose pneumonia,whereas 8 piglets in the A7 monovalent vaccine group showed no obvious symptoms,and the protection rate was 100%（8/8）.Only 1 out of 8 piglets in the D8 monovalent vaccine group showed mild symptoms,and the protection rate was 87.5%（7/8）.This indicates that the A7 and D8monovalent vaccines have good safety and protective efficacy.3.Safety of the bivalent inactivated vaccine against swine pasteurellosis（strain A7+strain D8）A bivalent inactivated vaccine against swine pasteurellosis（strain A7+strain D8）was prepared using P.multocida strain A7 and strain D8 as antigens.Systematic safety studies were conducted on the vaccine,including single-dose vaccination,repeated single-dose vaccination,single overdose safety study in piglets and pregnant sows,and single overdose safety study in mice.The results showed that the mental status and appetite of piglets and pregnant sows vaccinated with the bivalent inactivated swine pasteurellosis vaccine（strain A7+strain D8）were normal.There were no clinical signs such as diarrhea,vomiting and death.There was a transient increase in body temperature in the vaccinated pigs.However,the mean body temperature did not exceed 1°C 24 h after vaccination and returned to normal within48 h.There were no significant local inflammations,tissue lesions or vaccine residues at the vaccination sites of the pigs in the vaccination group at the end of the 21-day observation period.The experimental results of the effect of vaccination on pig production performance showed that the bivalent inactivated vaccine against swine pasteurellosis（type A A7 strain+type D D8 strain）had no significant effect on growth,weight gain and feed conversion ratio of the target animal pigs in comparison with the blank control group（P>0.05）.This indicates that the vaccine is safe for both piglets and pregnant sows.The results of the mouse safety study show that it is safe to vaccinate non-target mice with a bivalent inactivated vaccine against swine pasteurellosis（strain A7+strain D8）at an excessive dose.4.Protective efficacy of bivalent inactivated swine pasteurellosis vaccine（A7strain+D8 strain）This study was designed to determine the minimum immune dose of a laboratory produced bivalent inactivated swine pasteurellosis vaccine（strain A7+strain D8）in mice and piglets.The results of the immune protection test in mice showed that all mice in the 0.4 m L inactivated vaccine group survived against the attack of the virulent strains of serogroups A and D and that no obvious symptoms or pathological changes occurred.More than 9/10 protection against two different serotypes of strains was achieved with the 0.2m L vaccine dose.There was a significant reduction in the protection rate（6/10 and 4/10）in the 0.1 m L vaccine group.This suggests that the minimum dose of inactivated vaccine required to provide more than 8/10 protection in mice is 0.2 m L per vaccine.This provides a reference for assessing immune protection efficacy in surrogate animals when testing vaccine efficacy.The results of the immune protection test in piglets showed that 2 m L/head of the bivalent inactivated vaccine against swine pasteurellosis could completely protect the piglets against the attack of the virulent strains of serogroups A and D.When the dose was reduced to 1 m L/head,the immune piglets could still resist the attack of the virulent strain.More than 80%（4/5）of the vaccinated piglets were protected.When the dose was reduced to 0.5 m L/head,the protection rate of the immunized piglets was significantly lower.The immunological efficacy could not provide sufficient protection to the immunized piglets（≤2/5）.These results indicated that the minimum immunogenic dose of this vaccine was 1 m L/head to provide more than 4/5 protection to piglets.The prepared bivalent inactivated vaccine（A7 strain+D8 strain）had strong immunoprotective efficacy against the currently circulating A and D strains.In conclusion,from 13 type A and 13 type D strains of P.multocida,a serogroup A virulent strain A7 and a serogroup D virulent strain D8 with strong immunogenic were selected in this study,respectively.The bivalent inactivated vaccine（strain A7+strain D8）produced with these two strains had a good safety profile in piglets and mice.And it has a strong immunological protection against the current epidemic strains A and D.This study lays the foundation for the development and application of a bivalent inactivated vaccine（A7+D8 strains）against swine pasteurellosis in the later phase. |
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