Isolation and Identification of Pasteurella Multocida in Swine and Study Immunogenicity of PMT Toxin
|School||Huazhong Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Pasteurella multocida Isolation and identification of PMT toxin Immunogenicity trials|
Pasteurella multocida (Pasteurella multocida, Pm) is an important livestock pathogens belonging to Pasteurella Division, mainly animals infectious pneumonia or hemorrhagic septicemia. Swine pasteurellosis major swine infectious atrophic rhinitis (Swine infectious atrophic rthinitis, AR) and lung disease (Pneumonic pasteurellosis), the two diseases to the development of aquaculture and bring huge economic losses. Kill more sexual by toxigenic Pasteurella multocida (of toxigenic of Pasteurella multocida, T Pm) and bronchiseptica wave Bordetella (Bordetella bronchiseptica, Bb) swine infectious atrophic rhinitis is caused by infectious diseases of pigs is a common and serious harm . Vaccination is the primary means of prevention and control AR. Found Bb key inactivated vaccine made of purified toxin from the D-type T Pm vaccine, their immune effects compared to the conventional inactivated vaccine alone toxoid vaccine, but the extracted Pasteurella dermonecrotic toxin (Pasteurella multocida toxin, PMT) inactivation and purification has not been effectively addressed, it is difficult to achieve large-scale production. With the development of molecular biology, biotechnology means of Pasteurella multocida toxin fragment expression, expression products, vaccine production is feasible. In this study the production of toxins to kill more sex the Pasteurella strains HN-13 the toxA gene 1140bp of the N-terminal fragment was cloned into the prokaryotic expression vector pET-28a system successfully constructed the the pET28a-toxAN recombinant expression plasmid, expression induced about the size for 45kDa fusion protein rtoxAN, (?) estern blot confirmed rtoxAN protein has good immunogenicity. This protein and inactivated of Bb (HH-0809) and made of white oil emulsified vaccine, the vaccine on mice, piglets, the pregnant sows security and protective immunity on mice and piglets. Pasteurella multocida isolated and identified from the clinical illness such as lung, kidney, heart, liver, brain and spleen organ also carried out work. The main contents are as follows: 1. Pigs kill more sex the Pasteurella the separation identified in 2010 from 530 clinical diseased lung, kidney, heart, liver, brain, and spleen and other organs were isolated more than 148 sexual killed Pasteurella type A non-toxigenic Pasteurella multocida 68; D-type non-toxigenic Pasteurella multocida 74; the D type toxigenic Pasteurella multocida three; unclassified non-toxigenic The Pasteurella multocida three. The immunogenicity study 2.PMT toxin (1) to reference the the toxA gene sequence (NO.AF240778) design primers the toxA gene N-terminal, to construct the recombinant plasmid pET28a-toxAN its inducible expression, Western blot analysis shows expression product rtoxAN The protein has good immunogenicity, while the expression product was purified. (2) proliferation cultured on TSB medium the Bb (HH-0809), after the collection of bacteria, the bacteria count, inactivated and concentrated, and purified good rtoxAN protein is made with white oil emulsified vaccine, this vaccine BB content of 2.0 the on the × 1010CFU/ml rtoxAN content 100μg/ml. (3) the trial vaccine inoculated Balb / C mice per 0.2m1 dose and regularly observed after inoculation, the mice feed intake and growth were normal, no abnormal reaction of the vaccine in mice safety. Carry out a trial of the vaccine Balb / C mice protective immunity test, the 32-week-old Balb / c female mice were randomly divided into two groups, the first group of immune trial of Bb-toxAN vaccine, the second group was injected with sterile PBS, two weeks after the first immunization to strengthen the immune. Two weeks, four weeks after the first immunization antibody levels were detected. 2 weeks after the second free, each half of the mice with the extracted PMT toxin (6μg) challenge, the other half with 5.0 × 106CFU (10 × LD50) HH-0809 challenge observed death. The results show that the trial vaccine can stimulate mice to produce high IgG antibodies against Bb and (?) RtoxAN; challenge Bb and PMT toxin, this vaccine protection rate of 100% and 87.5%, respectively, while the control group all died. (4) the trial the vaccine inoculated piglets and pregnant sows per head 4m1 dose regularly observed after vaccination, feed intake and growth of piglets and pregnant sows are normal, no abnormal reaction, pregnant sows does not appear to premature delivery, miscarriage phenomenon. Description piglets and pregnant sows are safe vaccine. 15 7-day-old piglets were randomly divided into three groups, Group 1 immune prepared of Bb-toxAN vaccine, Group 2 immune imported swine atrophic rhinitis vaccine group was injected with sterile PBS 3, two weeks after the first immunization strengthening immune. Two weeks, four weeks after the first immunization antibody levels were detected. 2 weeks after the second free, first of all pigs with BB (HH-0809) (3.0 x 109CFU) intranasal infection, 3 days after the infection of all pigs PMT toxin (1.2mg/kg body weight) intramuscularly then observed 28 days. The results show both vaccines could piglets induced a high IgG antibody against Bb, but only the Bb-toxAN vaccine group can induce piglets generated of high for toxAN IgG antibody; neutralization test showed that both vaccines could induce piglets high neutralizing antibodies; Both vaccines can be found in the challenge to provide 80% protection force of piglets.