Isolation,Identification And MLST Typing Of Clostridium Perfringens From Cattle In Heilongjiang Province

Clostridium perfringens(C.perfringens)are Gram-positive anaerobic bacteria that cause a range of important human and animal diseases,such as food poisoning in humans and(Cattle Sudden Death Syndrome.CSDS).CSDS has an acute onset,rapid mortality and high mortality,and its incidence has increased significantly in recent years,which has brought huge economic losses to the cattle industry.C.perfringens carries a wide variety of toxins,and the diversity of serotypes and genotypes makes the prevention and treatment of the disease more difficult.In order to understand the epidemiological characteristics of C.perfringens strains isolated from CSDS in Heilongjiang province,the bacteriology,ecology and multilocus sequence typing of C.perfringens were performed on CSDS cases in Heilongjiang province,and provide theoretical basis for the prevention and control of CSDS.In this study,120 bovine intestinal tissue samples of CSDS cases from 43 cattle farms in 8cities of Heilongjiang Province were collected from September 2019 to September 2022.C.perfringens from the small intestine were isolated,purified,identified by biological characteristics,biochemical identification,PCR identification,mouse pathogenicity test and typing.To determine the type of toxin secreted by C.perfringens isolates,Sixteen major and minor toxin genes including cpa,cpb,etx,i A,cpe,net B,lam,pfo A,tpe L,cad A,col A,nag H,ibp,nan H,nan I and nan J in the isolates were detected by PCR.Results A total of 93 C.perfringens strains were isolated and identified,including 26 highly pathogenic strains and 67 low pathogenic strains.According to the main toxin typing results of C.perfringens bovis,the isolates were mainly type A strains,accounting for 56.99%(53/93),followed by type C strains,accounting for 38.71%(36/93),and the newly classified type F strains,accounting for 4.30%(4/93).A total of 26 strains(27.96%)of highly pathogenic C.perfrgens were identified,of which41.67%(15/36)were highly pathogenic C.perfrgens,20.75%(11/53)were highly pathogenic C.perfrgens,and 55.56%(10/18)were highly pathogenic C.perfrgens in Daqing area.Much more than other regions;Six secondary toxins were detected,the detection rate of col A was91.40%(85/93),pfo A was 88.17%(82/93),nan H was 74.19%(69/93),nan J was 55.91%(52/93),nan I was 43.01%(40/93).cad A was 3.23%(3/93);The virulence gene spectrum of pathogenic C.perfringens isolates could be divided into six types,of which 76.92%(20/26)of the virulent strains carried pfo A,col A,nan H,nan I and nan J minor toxins.In order to investigate the drug resistance of C.perfringens and to guide the clinical prevention and treatment of CSDS,Kirby-Bauer disk diffusion method was used to test the drug sensitivity of the isolates.The results showed that the isolates had a high resistance rate to commonly used antibiotics in clinical practice,among which the high resistance rates were tetracycline(94.62%),lincomycin(82.03%),erythromycin(70.92%)and spectinomycin(60.65%).The resistance rates to cefotaxime(21.73%),ciprofloxacin(35.05%),ampicillin(41.53%)and amoxicillin(42.71%)were relatively low.In order to identify C.perfringens more comprehensively,34 C.perfringens isolates were screened according to the characteristics of host source,serotype and pathogenicity,and then Multilocus Sequence Typing(MLST)analysis was performed.The results of MLST showed that the 34 isolates were divided into 34 STs,with an average of 12.5 alleles in each locus.The Simpson diversity index of STs was 0.9706,indicating that the collected samples had high genetic diversity.In the minimum spanning tree,two clonal complexes(CCs)were formed,and most of the clonal complexes contained strains from the same field,indicating that dominant strains may have formed and spread widely in this field.Most of the other STs were scattered,indicating different farming patterns among the fields,which may alter the genetic diversity of C.perfringens.The minimum spanning tree showed that high pathogenic strains and low pathogenic strains were clustered into one branch of the minimum spanning tree,and CC1 and CC2 were all low pathogenic strains.Therefore,it was speculated that STs typing had a strong relationship with the pathogenicity of C.perfringens,which provided more possibilities for the typing and pathogenicity investigation of C.perfringens.In conclusion,C.perfringens type A is the predominant type in the CSDS cases caused by C.perfringens in Heilongjiang,while C.perfringens type C strains are more virulent and most of them carry A variety of minor toxin genes.This may be one of the reasons why the clinical efficacy of the current commercial bovine C.perfringens vaccine varies greatly in different cattle groups.The multilocus sequence typing of C.perfringens isolates showed diversity,and dominant epidemic strains and multi-drug resistance had formed in some areas,leading to difficulties in prevention and control.The above results provided a theoretical basis for the screening of vaccine strains and sensitive antibiotics of C.perfringens.