The Separation And Identification Of Haemophilus Parasuis In Liaoning Region And The Drug-resistance Study

Haemophilus parasuis(HPS)disease was once considered as sporadic type disease caused by stress which could secondarily cause porcine multiple fibrinous serositis and multiple arthritis.It was separated by SCHERMER and EHRLICH in 1922 for the first time and proved as a pathogenic bacteria.HPS causes porcine fever,dyspnea,lameness,swelling joints and hind limb paralysis clinically and abundant pathologic changes including multiple fibrinous serositis,pericarditis,meningitis and arthritis.In Liaoning region,HPS disease not only acts as one of major causes of piglet morbidity and mortality,but also causes abundant infection in adult pigs which resulted in extremely severe economic loss in pig industry.Consequently,it is necessary to separate and identify HPS and conduct relevant research to establish basis for the prevention and treatment of this bacteria in Liaoning region.Major results are as follows:In this study,137 samples are collected from a portion of cities,districts and villages in Liaoning province from March,2015 to December,2016.Through the separation and culture of bacteria,gram stain and microscopic observation,biochemical test,PCR identification and 16S rRNA sequence analysis.Since HPS characterizes by difficulty to culture which affected the separation rate to some extent,a series of culture optimizing tests also conduct in this study by adding various concentrations of serum and NAD content and measuring the growth curves of separated strains every 6h.OD600 uses to reflect the quantity of alive bacteria and growth rate of different culture time and concentration.1.Nine strains of HPS were separated and named as HN1512,XM1601,HC1604A,HC1604B,DD1604,YK1603,HN1604,TL1607,LY1611.The serotypes were identified with multiple PCR technologies and results showes that one strain of serotype 1,three strains of serotype 7,two strains of serotype 9,one strain of serotype 11 and one strain of serotype 14 were separated.2.Culture optimizing tests shows that HPS grew well under the serum concentration of 5%and NAD final concentration of 10 ?g/mL.With the NAD concentration to 20 ?g/mL,the concentration of bacterial solution not significantly influences and with the serum concentration increased to 7%or 9%will increase the bacterial solution with slightly.3.In order to understand the sensitivity and drug-resistance of Liaoning separation strains,the drug sensitivity of Liaoning separation strains to 25 drugs was examined in this study.Results show:?These strains are highly sensitive to ampicillin,minocycline and cefazolin which could be used rationally to direct clinical treatment and avoid loss of pig farms.?Meanwhile,it also discoveres that Liaoning strains were severely drug-resistant.All separated strains were multiple drug-resistant,at least resistant to three drugs.One strain was even resistant up to ten drugs.?PCR technology uses to examine 23 drug-resistant genes of HPS separated strains and nine strains detect with drug-resistant genes.Seven drug-resistant genes and nine drug-resistant genes detect in both genomic DNA and plasmid DNA.It is reveales that the result of drug-resistant phonotype fundamentally in line with and positively correlate with the result of drug-resistant genes.This study lays a foundation for the study of Liaoning strains and the drug-resistant mechanisms.