| Avian pasteurella disease(Avian Pastetirellosis)is also called the fowl cholera,caused by pasteurella multocida.It is An acute contact septic infectious disease that primarily affects birds such as chickens and turkeys.The main clinical manifestation of infected poultry is acute death with high mortality.Antibiotics such as sulfa drug and chloramphenicol have some effect on the prevention and treatment of avian pasteurellosis.However,with the long-term and extensive use of antibiotics,it leads to drug residues in poultry products such as eggs and meat.On the other hand,it also leads to the enhancement of bacterial resistance.Fifteen serovars of pasteurella multocida which typically have a range of different virulence potentials,have been classfied.There is no cross-protection between serotypes.At present,the vaccine used in China is inactivated vaccine,and The best commercial inactivated vaccine has less than 80% protection rate against the infection of homologous serotype strain short protection period.Therefore,it is urgent to improve the immune protection effect and duration of inactivated vaccines,or to develop new and efficient vaccines to meet the needs of industrial development.Some scholars found that a large number of immunogenic proteins were found in the bacterial solution of fe-limiting culture comparative proteomics analysis of some bacteria in fe-limiting culture and non-fe-limiting culture,which was significantly up-regulated compared with the bacterial solution of non-fe-limiting culture.We hypothesized that there might be a large number of immunogenicity related proteins in the ir-restricted culture of avian pasteurella multocida,which can be developed as safe and effective subunit vaccine.It is also possible that vaccines made from bacteria cultured in this way will produce better and more efficient immune effects than those made from non-fe-limiting cultures of avian pasteulobacter.1.Study on pasteurella multocida iron-limited cultured and inactivated vaccinePm was cultured in either iron restricted(PMR)or normal mediums(PMN),the OD600 values were tested every 2 hours to detect Pm growth(Figure 1).The Pm growth capability had no different under the two conditions in the first 2 hours.The Pm growth was into the logarithmic phase after 4 hours and reached at the platform period until 12 hours later in normal medium.However,the logarithmic phase and platform period of the Pm in iron restricted medium showed 2 hours delay as compared with that in normal medium.The OD600 values of Pm in the iron restricted medium was significantly lower than that in normal medium at 4,6 and 8 hours(P<0.05),these results suggested that the growth of Pm was inhibited by iron deficiency.The antibody titers of chickens immunized with the two kinds of inactivated vaccine reached at the peak level at fourth week post vaccination,which lasted two weeks,and then was slow down.The antibody titers of chickens immunized with PMR was higher than that of chickens immunized with PMN from forth to sixth week post vaccination.These data indicated that the inactivated vaccine which cultured in iron restricted medium induced higher antibody titers than that in normal medium.Chickens were inoculated with highly pathogenic Pm(C48-1)at 14 days post vaccination to measure the protection of PMR or PMN.The C48-1 led all the control chickens dead within 24 hours.However,80% and 100% of chickens immunized with PMN or PMR were survival respectively.These results indicated that PMR provided a better protective effect than the PMN.2.Discovery of immunogenicity related proteins in avian pasteurellaTo better understand gene expressions of Pm under the iron deficiency condition,two-dimensional polyacrylamide gel electrophoresis(2-DE)were performed.Total 262 proteins were significantly different in iron restricted group.99 proteins were up-regulated,42 of the up-regulated proteins were more than five times;163 proteins were down-regulated,55 of the down-regulated proteins were more than five times.Based on the higher ratio(≥2.5)of spot density and molecular weight(MW)range of 17–72 Kd,13 proteins(B42,B65,B66,B67,B71,B73,B82,B84,B85,B86,B87,B93,B99)which expressed significant higher were selected for MS analysis(Table 2).Silico analysis was used in Protein functions by bioinformatics tools(Mascot software,Matrixscience Company).Only 11 of the 13 proteins could be retrieved.The functional classifications of these.These proteins belong to 2 protein families of Pm.B65,B67,B71 and B73 were outer membrane proteins H(OMPH);B82,B85,B87,and B93 outer membrane proteins A(OMPA).All the 8 proteins belonged to outer membrane proteins(OMPs).The other three protein B42,B66,B99 belonged to secreted proteins.B99 was aspartate ammonia-lyase(aspA),B66 was hyper protein(H),B99 was 30 S ribosomal protein S6(S6).Iron-regulated OMPs had effects on iron acquisition and stimulating immunity However,they were not secreted proteins,so our study focused on the roles of the other three secreted proteins.aspA,H and S6 were expression and purification,and immunized chickens respectively.The antibody titers were measured using indirect ELISA.Antibody titers showed high levels at 28 days post vaccination,chickens immunized with aspA had a significant increase in antibody responses on day 27 post-vaccination(P < 0.05).The aspA,H,S6 developed 80%,66.67%,80%,immunity respectively.The aspA and S6 showed which was higher protection than H.All the three subunit vaccine could protect chickens against the infection of Pm.3.Studies on the construction and function of aspA gene deletion strain of Pm.Sequence comparison and analysis of aspA gene of avian pasteurella were conducted with the whole genome of avian pasteurella standard strain Pm70 in NCBI database as referenceAccording to the homologous recombination technology,first of all,through the fusion of PCR method will avian pasteurella aspA gene homologous arm around fusion,and then the fusion segment and the resistance genes box by double enzyme connected to suicide plasmid,recycling electrontransfer methods will recombinant plasmid into suicide C48-1,finally through the resistance selection and PCR identification,successful build gene deletion strains,compared with wild type parent strains results show that amino acid catabolism of aspA gene function on avian pasteurella growth play an important role;It can improve the acid-fast ability of the bacillus avium and improve the anaerobic survival ability and the survival ability of the bacillus avium in iron-restricted environment.AspA gene deletion did not alter the virulence of avian pasteurella. |
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