Study Of Haemophilus Parasuis On Population Genome Sequencing,Molecular Typing And Resistance

Haemophilus parasuis is the etiological agent of of Gl?sser’s disease of swine,which causes seriously damage and death to pigs by single infection,co-infection and secondary infection with other primary porcine pathogens,it has been one of the most important causative agents that seriously harmed the global swine industry.Due to the difficulty of isolation and culture in vitro,the study of H.parasuis started late and its basic researches about its population structure,variation,typing and resistance is deficient.In order to determine the feature of the population structure of H.parasuis,we sequenced firstly the genomes of 50 H.parasuis strains on Illumina Hiseq 2000 platform,the resulting genomic size was 2.13 to 2.49 Mb,GC content 39.39% to 39.97% and predicted gene number 2078-2417 per strain.This results revealed that HPS has different genome size and stable GC content.Genetic variation analysis was performed on the basis of detection and annotation single nucleotide polymorphism(SNP),Small In Del and structure variation(SV)using SH0165 genome as the reference.SNP,Small In Del and SV were extensively identified from 50 sequenced H.parasuis strains,which displayed dominant distribution on coding sequence(CDS)region.Synonymous SNPs were found mainly at CDS region,which probably predict purify selection.Small In Del primarily cause gene variation of CDS region by frame-shift,and gene variation generated from SV was insertion,deletion,inversion and intra-chromosomal translocation.The number of deletion was greater than the number of inversion and insertion respectively,which showed deletion trend of HPS at the level of SV.Compare with SH0165 genome,non-synonymous SNP,Small In Del and SV in CDS region were likely to lead to gene variations,whose KEGG enrichment were mainly ABC transporter,biosynthesis of amino acids and carbon metabolism.Multiple approaches,including gel immune-diffusion(GID),multiple PCR(m PCR),multilocus sequence typing(MLST),were carried out to classify H.parasuis isolates and their potency of classification were discussed.Of the 100 isolate tested,73(73%)and 93(93%)were serotyped by the GID test and m PCR,respectively,with a concordance rate of 66%(66/100).m PCR reduced the number of non-typable(NT)isolates from 27(27%)for the GID testing,to seven(7%).The analysis of capsule locus indicated the In Del and unknown sequences emerged in the serotype-specific region may contribute to the production of NT strains in m PCR and inconsistent results between GID and m PCR.MLST of 53 H.parasuis isolates identified 36 sequence types(STs),14 new alleles and 23 new STs.Comparative analysis between m PCR and MLST displayed firstly one ST can be assigned to the only one serotype.It was confirmed at the genetic level that more than one H.parasuis strains can circulate in a farm,and these strains could be strains from different serotypes,even the various ST strains from the same serotype.Dominant ST was present at serotype 4,5,7 and 8 Chinese isolates.Genomics-based approach was performed to predict resistance genes,9 resistant genes were obtained from 11 sequenced H.parasuis strains.These predicted genes displayed resistance profile of aminoglycoside,tetracycline,sulfonamide,chloramphenicol and beta-lactamase.The antimicrobial susceptibility test against aminoglycoside were carried out with streptomycin,kanamycin and tobramycin.The test results demonstrated that most of the 11 sequenced strains posses consistent results between their predicted resistant genes and resistance profile.Genomics-based methods probably provide an valuable research measure for drug resistance study of H.parasuis.