Isolation,Identification And Biological Characteristics Research Of Haemophilus Parasuis Guizhou Strain

Haemophilus parasuis(HPS)is a conditional pathogen that colonizes the upper respiratory tract of pigs.The disease caused by Haemophilus parasuis was named Gl?sser's disease also.It is characterized by multiple cellulosic serositis and arthritis and is distributed worldwide.In recent years,under the influence of PCV2,PRRSV,mycotoxins,etc.,which causes immunosuppression,the incidence and mortality of Haemophilus parasuis continues to increase,and its prevalence is on the rise,with increasing harm.With the gradual ban of antibiotics,vaccination and enhanced feeding management will be the main measures to prevent and control HPS infection in the future.Because HPS has multiple serotypes and has obvious local characteristics,the cross-protection between strains of different serotypes is low,so the isolation and identification of HPS strains infected in different regions is not only conducive to a comprehensive understanding of the epidemiology of the disease.The characteristics can also provide suitable candidate strains for vaccine research of the disease.Therefore,in this study,14 HPS positive samples from some districts and counties in Guizhou Province from 2018 to 2019 were tested for HPS isolation and identification,and some biological characteristics of the isolates identified and purified strains were studied in order to enrich Guizhou.The epidemiological data of the epidemic strains of HPS infection in the province laid the foundation for further research on the etiology and vaccines of HPS.Related research contents are as follows:1.The separation and identification of HPSIn the experiment,124 samples(blood,nasal swabs,spleen,lungs,etc.)sent from some districts and counties in Guizhou Province from 2018 to 2019 were enriched with TSA medium contain 5 ?g/m L NAD.Mixed bacterial nucleic acid,which extracted by bacterial DNA Extraction Kit,were PCR amplified and sequenced using HPS 16 S r RNA specific primers.The results shows that 14 samples were positive for HPS(all of which were lung-enriched bacteria),its nucleotide similarity was 98.1% to 100%,andbelonged to 5 different evolutionary branches.The detection results of mixed infection with viruses in positive samples showed that HPS and PCV2,CSFV and PRRSV mixed infection rates were 42.9%;HPS and PCV2,CSFV and PRRSV four-way mixed infection rate reached 7.1%(1/14),the single infection rate of HPS is only 14.2%(2/14).By picking a single colony from the positive bacterial solution,after multiple generations of separation and purification,three HPS isolates were finally isolated.The three HPS strains showed translucent needle-like colonies with smooth and neat surfaces on TSA solid medium and blood agar medium,and Gram staining was Gram-negative bacilli.Among them,HPS/CN/Guizhou/426 and HPS/CN/Guizhou/478 strains are all short rod-shaped,and the HPS/CN/Guizhou/449 strains are short-rod and filament-shaped,which conforms to the polymorphic characteristics of HPS.Biochemical identification results showed that the three HPS strains had negative reactions to mannose,mannitol,xylose,urea,tannose,etc.,and positive reactions to sucrose,fructose,glucose,maltose,nitrate reduction,novobiocin,lactose,etc.It also meets the biochemical reaction characteristics of HPS.The satellite test results showed that the three HPS isolates could be combined with Staphylococcus aureus in the blood agar medium.The satellite phenomenon was not obvious.After purification,the three HPS strains were amplified by PCR with 16 S r RNA specific primers and sequenced.The comparison of the obtained sequence with the corresponding sequence before showed that the nucleotide similarity was 100%.2.Biological characteristics of HPS isolatesIn order to understand the biological characteristics of the three strains of HPS isolates,the main biological characteristics of the culture characteristics,NAD tolerance,serotype PCR identification and drug sensitivity tests were studied.The results show that the three HPS strains are strictly NAD dependent.When the fetal bovine serum content is5% and the NAD concentration is less than 16?g/m L,the OD600 nm value of the bacterial solution in TSB liquid culture medium is proportional to the NAD concentration.Therefore,the optimal amount of NAD determined by experiment is 16?g/m L.Three HPS isolation and purification strains were cultured in SS medium,LB solid medium,TSA solid medium(without fetal bovine serum and NAD),modified Y medium,Mac Conkey medium,Eosin blue and Chua's agar There was no growth on the base,and they were grown on LB solid medium and TSA medium containing 16?g/m L NAD and5% fetal bovine serum,respectively.After 24 hours of cultivation on NAD-free blood plates,small white needle-like colonies appeared.Using HPS serotype PCR identification primers reported by Howell and Jia,etc.,three HPS isolates were serotype PCR identified.Only serum type 5 specific primers amplified the target bands that match the expected size,indicating that The three HPS isolation and purification strains were all serotype 5.The detection results of virulence genes related to the three HPS isolation and purification strains showed that the HPS/CN/Guizhou/XYPA/2018426 strain and the HPS/CN/Guizhou/BJXXW/2018478 strain contained the virulence gene MPB,which amplified the expected amplification strip.With fragments of the same size,the HPS/CN/Guizhou/QNDS/2018449 strain contains the virulence gene E30.The drug sensitivity test results showed that the three HPS isolates were all sensitive to enrofloxacin and florfenicol,and were sensitive to ciprofloxacin,neomycin,streptomycin,kanamycin,gentamicin,sulfonamide Antibiotics such as isoxazole,trimethoprim,co-trimoxazole,clindamycin,augmentin,and ceftazidime have all been resistant.