Isolation And Identification Of Streptococcus Equi Subsp.equi From Donkey And Study On Some Biological Characteristics

Objectives:Streptococcus equi subsp equi are members of group C of Streptococcus spp.,Gram-positive,?-hemolytic streptococci.It can cause strangles disease in equines,an acute upper respiratory tract infectious disease,and is recognized as one of the most common infectious diseases of equines worldwide.In recent years,with the increase of people's demand for donkey meat,donkey milk and other products,the scale of donkey feeding in China has been increasing year by year.The lack of knowledge of scientific breeding and safety prevention and control has led to a gradual increase in cases of donkey infection with S.equi subsp.equi.In 2019,an epidemic situation of suspected equine disease occurred in donkey farms in Shihezi District,Xinjiang.In order to identify the pathogen,samples were collected from animals with clinical morbidity and mortality to isolate and identify the pathogen,analyze the genetic evolution,and study the biological characteristics,pathogenicity,antibiotic sensitivity of the pathogen and bactericidal effect of commonly used disinfectants,so as to provide scientific guidance and suggestions for the treatment and prevention of the disease.Methods:The affected donkey farms were clinically and pathologically observed and samples were collected from clinically infected animals to identify the isolate species by pathogen isolation,biochemical identification and PCR identification;the isolate genotype was determined by Se M gene sequencing,and the isolates were compared for homology and genetic evolution using DNAstar and MEGA7.0.The growth characteristics and hemolytic characteristics of the isolates under different culture conditions were determined,the carriage of virulence genes was detected by PCR,and the virulence of donkey source isolates was detected by mouse pathogenicity.K-B agar diffusion method was used to determine the susceptibility of isolates to 16 antibiotic drugs,and the national disinfectant detection criteria were used to determine the killing effect of a total of four disinfectants,neorvolin,iodophor,lysol,and 84 disinfectant,on donkey isolates.Results:In April 2019,donkeys in a donkey farm in Shihezi area developed elevated body temperature,depression,and multiple abscesses in the jaw,with an incidence of 50.9%and a mortality rate of 3.8%in adult donkeys.The incidence of donkey foals was 46.5%and the mortality rate was 9.3%.Pathological examination revealed significant abscesses in the nasal cavity,lungs,liver,and kidneys.Histopathological changes showed hepatic congestion,disappearance of hepatic lobular structure,hepatic cord disintegration,severe renal fibrosis and a large number of suppurative foci.The bacteria were isolated and identified,and 4 strains of Streptococcus equi subspecies equi(SEE)were obtained,numbered XJ201901-201904,with the genotype of se M-136,which was the most similar to the donkey isolate in Shandong,China in July 2018(99.4%).The isolate was beta hemolytic on sheep blood plates and grew best at 37?,p H7,and 1%Na Cl.It is sensitive to acidic and hypersaline environments.Five virulence genes,hyaluronic acid,peptidoglycan,fibronectin binding protein,recombinant enzyme,and hemolysin S,could be detected in donkey derived SEE isolates,and the virulence was different in intraperitoneally inoculated mice,with an LD₅₀between 3×10³and1×10⁶,and liver and kidney abscesses occurred in artificially infected mice,with histopathological changes consistent with naturally infected donkeys.Four donkey source SEE isolates were resistant to norfloxacin,kanamycin,and cotrimoxazole,and sensitive to six drugs including chloramphenicol,penicillin,gentamicin,ampicillin,sulfamethoxazole,and cephalothin and the recommended doses of neolamine,iodophor,lysol,and 84 disinfectants.Conclusions:In April 2019,the suspected epidemic situation of SEE in donkey farms in Shihezi,Xinjiang was caused by S.equi subspecies with genotype se M-136,which was highly homologous to the isolate of S.equi from donkey farms in Shandong,China,suggesting that SEE in donkey sources in Xinjiang may have originated in Shandong,China and invaded through introduction and animal transportation.All four donkey derived SEE isolates were weakly resistant to commonly used disinfectants and were not resistant to acid and high salt environments.It was resistant to norfloxacin,kanamycin,and cotrimoxazole,and sensitive to six drugs including chloramphenicol,penicillin,gentamicin,ampicillin,penicillin,and cephalothin.It is also highly virulent in mice with an LD₅₀up to 3×10³.It provides a scientific basis for the treatment and daily prevention and control of donkey source SEE.