Isolation,Identification And Biological Characteristics Of Haemophilus Parasuis And Streptococcus Suis In Large-scale Pig Farms

In recent years,Haemophilus parasuis（HPS）and Streptococcus suis（SS）have become important pathogens affecting the health of pigs,which can cause contact respiratory infectious diseases in pigs and cause huge losses to the pig industry.HPS and SS mainly cause multiple serositis,arthritis,meningitis and sepsis in pigs.There are currently 15 and 35 known HPS and SS serotypes,respectively.The prevalent serotypes of HPS in China are mainly serotypes 1,4,5 and 13,and the prevalent serotypes of SS are mainly serotypes 1,2,7 and 9.Although pigs can be vaccinated to prevent HPS and SS,there are many serotypes of the two bacteria,and the existing commercial vaccine can not fully cover all serotypes.Therefore,after pigs are infected with HPS or SS,the common treatment method is still to use antibiotics.However,with the extensive use of antibiotics,many strains have drug resistance,which makes the clinical treatment effect poor.Therefore,serotyping HPS and SS and studying their drug resistance and potential virulence factors can provide ideas for the prevention and control of this kind of bacterial diseases.In this study,the serotypes,drug sensitivity,virulence factors and NAD dependence of all strains were studied.The main contents and results are as follows:1.Isolation and identification of HPS and SSFrom March 2021 to January 2022,322 diseased materials such as brain tissue,lung and joint effusion of diseased pigs were collected from 3 large-scale pig farms in Hainan.After bacterial culture characteristic experiment,biochemical experiment and PCR typing identification,32 strains of HPS and 38 strains of SS were successfully identified.The results showed that HPS in Hainan was mainly epidemic type 4（50%）,non separable type（40.6%）and type 1（9.3%）;SS is mainly prevalent in type 21（34.2%）,non separable（26.3%）,type 9（21.1%）and type 8/21（18.4%）.2.Study on the culture characteristics of HPS and SSHPS grew well on TSA solid medium（serum+NAD）and chocolate agar medium.The growth curve of HPS was measured.Type 1 and type 4 HPS were inoculated into TSB（serum+NAD）,TSB（serum）and TSB（without any addition）medium respectively.The growth rate and NAD dependence of bacteria were reflected by OD_600nm value.The results showed that the HPS isolates in this experiment were strictly NAD-dependent,had strong adaptability to the culture environment,and grew faster.The OD_600nm value of type 1 and type 4 HPS peaked at 11 hour and 10 hour,respectively.SS grew well on blood agar medium,chocolate agar medium and TSA solid medium（serum+NAD）,and obvious growth was observed on blood agar mediumβHemolytic ring.The growth curve of SS was measured,and the results showed that the growth rate of SS isolates was faster,and the OD_600nm value reached the peak value at about 7 hour.3.Drug sensitivity of HPS and SSThirty-two strains of HPS and 38 strains of SS were tested for drug sensitivity by using drug sensitive paper method.The results showed that all HPS were resistant to kanamycin,neomycin,streptomycin and sulfonamides,and the resistance rate was 100%;it is highly sensitive toβ-lactams,quinolones,macrolides,chloramphenicol,florfenicol,doxycycline and spectinomycin,and the resistance rate is basically 0;moderately sensitive to gentamicin and amikacin.The results of SS drug sensitivity test showed that all SS were resistant to sulfonamides,macrolides,kanamycin,neomycin,streptomycin,gentamicin and amikacin,and the resistance rate was 100%;it is highly sensitive toβ-lactam drugs and spectinomycin,and the resistance rate is 0;it was moderately sensitive to chloramphenicol,florfenicol,doxycycline and quinolones,and the medium sensitivity rate was more than 60%.The drug resistance genes of 32 HPS and 38 SS isolates were detected by PCR.It was found that only quinolones（gyr A,gyr B,par C and par E）resistance genes were detected in type 1 HPS,and the carrying rates were 100%,66.6%,100%and 100%respectively;sulfonamides（SulⅠand sulⅡ）,tetracyclines（Tet A and Tet B）,florfenicol（folk）,quinolones（gyr A,gyr B,par C,par E）resistance genes were detected in type 4 and non genotypable HPS.the carrying rate of drug resistance genes in the two HPS was more than 75%.The results of PCR detection of SS resistance genes showed that sulfonamides,tetracyclines,quinolones,aminoglycosides and macrolides resistance genes were detected in type 9 and8/21 SS,and the carrying rate of resistance genes was more than 70%,especially for sulfonamides,the carrying rate of resistance genes was 100%.Among them,florfenicol resistance gene was also detected in type 9 SS,and the carrying rate was 75%;Type 21 SS detected quinolones（par E）,aminoglycosides（aph A1,str A and str B）and macrolides（erm B）resistance genes,and the carrying rates were 84.6%,100%,92.3%,84.6%and 92.3%respectively;the SS carrying rate of non separable type was similar to that of type 21.4.Detection of virulence genes of HPS and SSThe virulence genes of 32 strains of HPS and 38 strains of SS were detected by PCR.It was found that 11 virulence genes were detected in type 1 HPS strains,8 in type 4 HPS strains and 7 in non genotypable HPS.Among them,the virulence genes vta2,vta3,hhd A,hhdb,cdt B and cdtc were highly conserved in the three serotypes of HPS,and the carrying rate was almost 100%.Eight virulence genes were detected in type 9 and 8/21 SS,and six virulence genes were detected in type 21 and non separable SS.Among them,the virulence genes orf2,Znu A,adpb,Str A,Gapdh and Fbps were highly conserved in SS of four serotypes,the carrying rate of Gapdh and Fbps was 100%,and the carrying rate of the other four virulence genes was more than 70%.