Construction And Biological Characteristics Of An Attenuated Strain Of Serotype A Avibacterium Paragallinarum

Avibacterium paragallinarum(Apg)belongs to the family Pasteurellaceae,genus Avium,which cause infectious rhinitis(IC)in chickens.IC leads to poor growth of breeder chickens and decreased egg production of laying hens,resulting in severe economic losses.The commonly used IC vaccines are whole bacteria inactivated vaccines.But such vaccines tend to have more serious side reactions after chicken vaccinations due to the bacteria components such as lipopolysaccharide(LPS).Therefore,it is urgent to study the live attenuated vaccine.In this study,a natural transformation method applied to Apg was established,and a attenuated serotype A Apg mutant was obtained by using natural transformation method and transposon technology,which provided the basis for the further development of live attenuated vaccine of Apg.1.Establishment of a method of natural transformation of ApgIn this study,we analyzed the genomes of Apg isolates,and compared with foreign APG reference strains and Haemophilus influenzae and Haemophilus parasuis in the pasteurellaceae family.We found that there were 1537～1622 copies of DNA sequence 5’-ACCGCACTT-3’ in the tested genome.This sequence was.predicted to be the uptake signal sequence(USS)for Apg.There were 16 related regulatory genes were identified in the genome of Apg.The binding site of cAMP receptor protein(CRP)can be identified in the upstream region of 11 genes.These results confirmed that Apg has the ability of natural transformation at the gene level.In this study,the shuttle plasmid of E.coli-Apg(pEA-KU,pEA-K)was constructed.The shuttle plasmid was successfully introduced into Apg through natural transformation.Through further experiments,it was also found that,the plasmid carrying USS was more easily absorbed by Apg during the natural transformation;at the same time,there are great differences in the ability of natural transformation between different Apg strains.Among the tested strains,the transformation efficiency of 2019/HB64 is significantly higher than that of other strains.2.Screening and biological characteristics of attenuated strainsThe transposon Tn5-Kan was introduced into Apg by natural transformation,the serotype A strain 2019/HB64,was used as the parent to construct a random mutation library.In order to find a candidate strain suitable for IC live attenuated vaccine,we screened a transposon mutation library.In a comparison of the biofilm forming ability as well as the hemagglutination ability of the mutant strains,the strain 2019/HB64-40 were found to have significantly lower correlation traits than the wild type,further studies using this strain revealed a slower growth rate and a shorter growth plateau,indicating that their growth performance was greatly affected.The transposon insertion site of the mutant was determined to be rsmA by chromosome walking technique.Animal experiments were conducted with to evaluate the pathogenicity of the strain 2019/HB64-40.45 experimental chickens were divided into three groups(2019/HB64 challenge group,2019/HB64-40 challenge group and blank control group)The experimental chickens were challenged at the age of 28 d,the clinical symptom score,pathological changes and respiratory tract bacterial excretion were evaluated.The results showed that all the test chickens in the 2019/HB64 challenge group had clinical symptoms within 3 days,and the score was 2.06,which showed severe facial swelling.On 7 days of autopsy,there were yellow cheese like substances in the infraorbital sinus and laryngeal bleeding.In the 2019/HB64-40 challenge group,only part of the test chickens(4/15)had clinical symptoms.The score on the third day was 0.40,and the infected chickens only showed slight facial swelling.7 d after challenge,no pathological changes were found in 2019/HB64-40 challenge group.Moreover,the amount of bacteria excreted in the 2019/HB64-40 challenge group lower than that in the 2019/HB64 challenge group.These results showed that the mutant 2019/HB64-40 was significantly weaker than the original strain 2019/HB64.The immune protective effect of attenuated strain 2019/HB64-40 was evaluated by animal test.Thirty experimental chickens were divided into three groups(immune group,no immune group and blank control group).The attenuated strain 2019/HB64-40 was used to immunize the experimental chickens by nasal drip at the age of 28 days.The results showed that a high level of HI antibody titer could be detected in the serum of the immune group 21 d after inoculation.The experimental chickens were challenged at the age of 49 days.In the no immunized group,all of them developed severe symptoms within 3 d,and the clinical symptom score was 2.5.1/10 of the immunized group had mild clinical symptoms,and the symptoms had subsided by 5 d.The excretion of Apg in the immunized group was significantly lower than that in the no immunized group.The attenuated strain 2019/HB64-40 has a good protective effect according to the clinical symptoms,the pathological changes of dissect and bacterial excretion.In conclusion,the random mutation library of serotype A Apg was constructed by transposon technology,and an attenuated strain was obtained through screening,which has the possibility of being a candidate strain of live attenuated vaccine,which provides a reference and basis for the further development of live attenuated vaccine of Apg.