| Avibacterium paragallinarum（Apg）formerly known as Haemophilus paragallinarum is a gram-negative bacterium that can cause infectious coryza（IC）in chickens.It has A、B、C three serotypes and can be subdivided into 9 serotypes,including A1-4,B-1 and C1-4.The main virulence factors（VFs）and effective protective antigen components of infectious coryza are unclear.In this paper,the isolates of Avibacterium paragallinarum were isolated from the infraorbital sinus of infected chickens in some chicken farms in Jiangsu Province.The bacteria is a caustic bacteria,so the culture conditions are optimized and the suitable culture conditions are finally optimized.Secondly,the whole genome sequencing of the isolates was carried out,and the pan-genome of the three isolates was analyzed by bioinformatics analysis.Finally,the recombination protein of the B strain of Avibacterium paragallinarum was successfully expressed by prokaryotic expression system,and the immunogenicity of recombinant protein was analyzed by western blot.The results provide a basis for the further development and preparation of subunit vaccine for infectious coryza.1 Isolation and Identification of Avibacterium paragallinarum in Jiangsu ProvinceThis part of the study is based on the diagnosis of some chicken farms in Jiangsu Province and taking contents from different batches of chicken suborbital sinus with suspected infectious coryza.The isolated bacteria were identified by isolation,culture,purification,PCR identification,serotype method and 16S r DNA,a total of 6 strains,including three A-serotype,two B-serotype,one C-serotype.It shows that three serotypes of chicken farms with infectious coryza in Jiangsu have been prevalent.Among them,A-serotypes is more common.2 Optimal Selection of Culture Medium for Avibacterium paragallinarumBecause of the harsh growth conditions,in order to explore the growth characteristics of Apg and compare the growth characteristics of different serotype strains.From different basic medium,different animal serum,different serum concentration,different NAD concentration,by spectrophotometric determination of OD600value,finally optimizing the appropriate culture conditions.The growth curves of three isolates of serotype-A（JSA001）,serotype-B（JSB001）and serotype-C（JSC001）were analyzed.It turns out the three strains of IC were all well grown in TSB liquid medium containing 5%chicken serum and 0.001%NAD.In addition,the growth stability of the three strains was short,the growth peak time and the number of living bacteria of each strain were different.The number of living bacteria of B-serotype and C-serotype culture decreased rapidly after 10-11 hours,after 9-10 hours of culture,the number of living bacteria of A-serotype strain decreased rapidly.Therefore,the appropriate culture time is about 10-11 hours when a large number of living bacteria are needed.3 Pan-genome and genomics analysis of Avibacterium paragallinarumPan-genome is the entire gene of a bacterial species,including core-genome and dispensable genome.The core genome is the gene common to all strains which related to its basic biological function;the dispensable genome that is specific to some bacteria or a particular bacterium which it reflects the diversity of bacterial genomes.A total genome sequence of 3 isolates and 25 strains of Apg which downloaded from the Genbank and comparative genomics analysis of 28 strains.According to the results of the pan-genome analysis of the Apg genome,its have genome are 6384 and core genome 1149 genes,the proportion of the core genome to the total genome is17.9%,indicating a rich diversity of gene composition in the genome of Apg strains.Various virulence genes were detected in 3 isolates and 25 reference strains,a total of82 virulence-related genes were found.It can be divided into the following categories:adhesion,infective,iron uptake,antiserum defense,toxins and others.3 isolates have other virulence factors not found in other reference strains,including ctr A,lip A,neu B,sug C,hly B.Drug resistance was detected in 3 isolates and 14 reference strains,the main manifestations of anti-macrolides,fluoroquinolones,cephalosporins,aminoglycosides,carbapenems,tetracycline drugs,et al.Moreover,the whole genes of Avibacterium paragallinarum were used to PSORTb,Ex PASy,Vaxi Jen and TMHMM sites,6 eligible protective antigen genes were screened and the homology was above 95%.The purpose of the part was to find out the genetic characteristics and phylogenetic relationship of the bacteria,to establish a pan-genome model,to understand the information of virulence and drug resistance,and to select protective antigens through reverse vaccine screening.It provides the basis for further study of genomics and vaccine development of Avibacterium paragallinarum.4 Cloning expression and immunogenicity analysis of alternative protective antigen of multiple serotypes of Avibacterium paragallinarumAvibacterium paragallinarum is the causative agent of infectious coryza in chickens.Compared to its clinical importance,studies on its pathogenesis as well as subunit vaccines have been lacking.In this study,multiple strains of bacteria were isolated from the infraorbital sinus of diseased chickens and identified as Avibacterium paragallinarum.The isolates were subjected to whole gene sequencing,and the whole genes of the three serotype isolates were compared with other Apg-related genes through public databases as well as immunogenicity prediction and six possible common protective antigen genes were screened and predicted to have good homology.The six target genes were cloned into the p ET-28a vector and the prokaryotic expression recombinant plasmid p ET-Apg B1-6 was constructed and transformed into BL21 for induction of expression,and suitable expression conditions were obtained by choosing the compatible conditions such as IPTG concentration,temperature and time.The three recombinant proteins were successfully purified by His affinity chromatography column,and the three recombinant proteins were analyzed by western blot using Apg’s serotype-B recovery serum.The experimental results showed that the three recombinant proteins had good immune response and were one of the protective antigens of Apg.The protein prediction results showed that they might become common antigens of other serotypes of Apg,which provided the basis for the subsequent establishment of Apg subunit vaccine as well as detection methods. |
