Comparison Of Pathogenicity Characteristics Of Pasteurella Multocida Serotype A And D In Swine

Pasteurella muliocida（Pm）is one of the important pathogenic agents of porcine respiratory diseases.There are five capsular serotypes（A,B,D,E,F）,and the main porcine Pm isolates are serotype A and serotype D,which can cause progressive atrophic rhinitis（PAR）and pneumonia.Seriously affect the sustainable development of the world pig industry.In general,serotype A Pm is mainly isolated from the lungs of pigs with pneumonic Pasteurella disease,while serotype D Pm is mostly isolated from pigs with PAR.However,it is not absolute.Currently,the isolation rate of serotype D Pm from pigs with pneumonic Pasteurella disease has increased significantly,suggesting that different symptoms may correspond to any seroserotype.Pasteurella disease caused by pneumonia serotype Pm of serotype A and serotype D strains in the aspect of etiology characteristics whether there is any difference or even how differences,this research is using from pneumonia pasteurella sick pigs of serotype A and serotype D Pm has carried out A comparative study,To provide technical reserve and scientific basis for effective prevention and control of porcine pasteurellosis.In this study,9 clinical isolates of serotype A and 7 clinical isolates of serotype D Pm were selected as research subjects,and 2 standard Pm strains（The numbers are serotype A-CVCC403 and serotype D-CVCC399）were used as reference.The following tests were carried out:（1）morphological,culture,biochemical characteristics and ST type were identified by conventional bacteriological method and multi-locus sequence typing（MLST）technique;（2）PCR was used to detect 23 virulence genes.According to the pathogenicity test of mice,the representative strains with the strongest and weakest lethality were selected.The median lethal dose(LD₅₀)of mice was measured by accumulative method,pathological changes by HE staining method,the number of bacteria in tissues was measured and the adhesion to He La cells was measured by viable plate counting method and crystal violet staining method.（3）The ability of biofilm formation（BF）was determined by crystal violet staining;（4）PCR was used to detect 18 drug resistance genes;The drug resistance to 24 drugs was measured by agar diffusion and broth dilution.（5）Bacterial whole gene sequencing and compara tive genomics were used to analyze the differences in genome structure and gene function;（6）Transcriptome sequencing（RNA-seq）was used to compare gene expression differences..The results showed that:（1）Nine strains of serotype A,seven strains of serotype D,serotype A-CVCC403 and serotype D-CVCC399 grew barren on ordinary agar medium.On the medium containing blood or serum,they grew into light gray,round and moist dew like small colonies without hemolysis.The colony diameter of serotype A Pm was smaller than that of serotype D Pm;It is in the shape of ball rod or short rod,and arranged in single or double;Both have obvious logarithmic growth period and s Tablele growth period.The peak values of the highest viable bacteria of serotype A and D Pm are in the same order of magnitude,and there is no significant difference;33.3%（3/9）serotype A Pm and 85.7%（6/7）serotype D Pm had the same biochemical characteristics,and the other strains had different reactions to lactose,galactose,mannitol and xylose;Nine strains of serotype A Pm were ST133 and one strain of serotype A standard strain was ST42;Seven strains of serotype D Pm and one standard strain of serotype D were ST11.（2）The LD₅₀of 3 strains serotype A Pm（serotype A-HF11-1,serotype A-AHma21-6,serotype A-AHma21-4）and serotype A-CVCC403 ranged from 821 to 2.440×104 CFU/piece.The LD₅₀of 3 strains of serotype D Pm（serotype D HN-1,serotype D-JSLing19-10,serotype D-AHma21-3）and serotype D-CVCC399 ranged from 2.912×10⁴to 1.127×10⁶CFU/piece.Serotype A-HF11-1 with LD₅₀of 821CFU/mouse had the highest number of bacteria in tissues and the most obvious lesions,while serotype D-CVCC399 with LD₅₀of 1.127×10⁶CFU/piece had the lowest number of bacteria in tissues and the least lesions.Three strains of serotype A Pm adhered to He La cell surface significantly more than three strains of serotype D Pm.The positive rate of 16 virulence genes such as ptf A、fim A、hsf-1、hsf-2、exb B、exb D、ton B、hgb A、hgb B、Fur、omp H、oma87、plp B、nan H、sod A and sod C was 100%.Tox A and tbp A genes were not detected.The positive rate of omp A gene in serotype A and D Pm was 40%and 100%,respectively.Pfh A,tad D and pm HAS as are only detected in serotype A Pm,and nan B is only found in serotype D Pm.（3）The BF formation ability of seven strains of serotype D Pm was weaker（1+）than that of D-CVCC399（2+）.88.9%（8/9）of serotype A Pm and A-CVCC403 BF formation ability were above medium（2+/3+）,only one serotype A Pm BF formation ability was weak（1+）.（4）Nine strains of serotype A,seven strains of serotype D,A-CVCC403 and D-CVCC399 were resistant to clindamycin,insensitive to erythromycin,and sensitive to norfloxacin,ciprofloxacin and kanamycin.The detection rates of resistant genes sul1 and sul2 were 18.7%and 55.6%,respectively.The detection rates of tet A,tet B,tet G and tet R were 11.1%,22.2%,44.4%and 11.1%,respectively.The detection rates of bla TEM,bla OXA and bla SHV were 5.5%,11.1%and5.5%,respectively.Sul3,tet H,bla CTX,dfr A5,dfr A7,dfr A13,em A,em C,and lnu A were not detected.（5）The whole genome sequences of serotype A and serotype D Pm strains were well collinear,with a similarity of 98.65%.There were no insertion and deletion sequences,no plasmid and phage gene sequences,and there were 9 translocations,2inverted regions and 2 CRISPR elements.The two functional genes are mostly related to translation,ribosomal structure,biogenesis,carbohydrate transport and me Tableolism.They are mainly concentrated in the biosynthesis of antibiotics,the biosynthesis and me Tableolic pathway of secondary me Tableolites.（6）Compared with serotype D Pm,virulence related genes such as pgi,hem H,gal E,lpx H and ptf A were up-regulated in serotype A Pm.rcp B,rcp A,tad G,tad F,tad C,pil A,HF11＿1000827,csr A,cya A,hfq and other BF formation related genes were up-regulated in serotype A Pm strain.Drug-resistant genes such as Tae A,ugd,Lpx A,glp T,optr A,Aba F and Uhp T were down-regulated.HF11＿1001468,HF11＿1001462,HF11＿1001467,HF11＿1001738 in pentose and glucuronate conversion pathways,and HF11＿1000163,HF11＿1000168 in nod-like receptor signaling pathways.HF11＿1000559 in bacterial chemotactic signaling system was up-regulated in serotype A Pm strains.The results showed that:（1）The morphology and growth characteristics of serotype A and serotype D Pm were similar,but the colony diameter of serotype A Pm was larger than that of serotype D Pm.Physiological and biochemical characteristics are diverse,mainly reflected in the difference in glycolysis ability;The composition of ST serotype is diverse;There was no significant difference in drug resistance.The toxicity,BF formation and cell adhesion of serotype A Pm were stronger than those of serotype D Pm.（2）Translocation and inversion regions and CRISPR elements in the whole genome sequences of serotype A and serotype D Pm may cause differences in some microbial phenotypes between them.（3）Five differentially expressed virulence genes pgi,hem H,gal E,lpx H,ptf A and com EA were screened by RNA-seq.The ten differentially expressed genes rcp B,rcp A,tad G,tad F,tad C,pil A,HF11＿1000827,csr A,cya A,hfq related to BF formation ability and adhesion were composed of genes.These genes are up-regulated in serotype A Pm,which can contribute to the synthesis of pili protein,enhance cell adhesion and BF formation ability,and then affect its virulence.