Establishment Of Infection Model Of Streptococcus Equi Subspecies Equi And Identification Of Its Phage

Strangles is an acute infectious disease caused by Streptococcus equi subspecies equi(S.equi),horses,donkeys,mules are susceptible.Strangles was classified as the third category in the list of animal diseases,which was published by Ministry of Agriculture,but there were no regular official reports of the outbreak.Our research group had made the surveillance of Strangels' epidemic situation in China,and found that the incidence of strangles has increased gradually in recent years,as the result of the increase of horse and donkey circulation.Strangles has become one of the most infectious diseases damaging horses and donkeys breeding industry.since the research started late and lack of attention,there is still no effective ways for Strangles prevention and treatment.Studies on Strangles vaccines in the world indicated that there were no effective vaccines that could provide long term protection(more than 6 months),and the safety of vaccine was also a problem needed to be solved.Antibiotics were still the main treatment for Strangles,and would inevitably lead to the increase of drug-resistant bacteria and the decrease of animal immune function and other problems.In order to develop vaccines and treatment products of Strangles,A pathogenic S.equi strain(HLJ2018)was isolated,and the biological characteristics were studied.Horses and mice S.equi infection models were established with the use of HLJ2018.One phage strain(SP2019)was also isolated,this is the first report of the S.equi phage since the 1980 s in the world.Main works are as follows:Study on biological characteristics of S.equi clinical isolate and establishment of artificial infection model: Strangles pathogen was isolated from a horse farm in Heilongjiang province.The following work has been carried out: 1.Gram staining method and microscopy;2.Biochemical identification;3.Sequencing identification of se M protein's gene;4.Sequencing identification of 16 S rRNA gene;5.Antibiotic resistance;6.Lethality to mice and infectivity to organs.7.Dose and way for artificial infected horses.The clinical isolate was identified as S.equi and named as HLJ2018;HLJ2018was sensitive to ceftriaxone,erythromycin,teicolanine,benzoxicillin and rifampicin,but resistant to doxycycline,amoxicillin and clindamycin.Mice were infected with different doses of HLJ2018 by intraperitoneal injection,and it was found that the minimum complete lethal dose of this strain on mice was 1×105CFU.After the mice were anatomized,HE staining was performed on each organ,and it was found that HLJ2018 mainly proliferated in the spleen,resulting in the broken of the spleen structure,with little impact on the heart,liver,lung,kidney,brain and other parts.In infected group,1011 CFU bacteria were feed to each horse,and horses showed 100% morbidity.Compared with the control group,the infected horses showed typical symptoms of Strangles,such as mandibular lymph node swelling and festering.Biological characteristics of S.equi phage,and treatment of Strangle with phage: After screening thousands of environmental samples by using HLJ2018 as the host bacteria,the phage of S.equi was successfully isolated,and named as SP2019.The following work has been carried out: 1.Observation of phage morphology by electron microscope.2.Determination of the optimal multiplicityof infections.3.Lysis spectrum of different S.equi strains.4.A preliminary study of phage therapy for Strangles.The results showed that SP2019 was a short tailed phage,and the optimal multiplicity of infections was 0.01.SP2019 was intolerance to high temperature and sensitive to acid and alkali.SP2019 could also lyse some S.equi strains besides HLJ2018.It can not improve infected mice survival rate by intraperitoneal injection of SP2019.By intravenous injection of SP2019 suspension to Strangles horses,the swelling of mandibular lymph node disappeared faster than Strangles horses without treatment.In this study,the biological characteristics of the S.equi clinical isolated stain HLJ2018 were studied,and the bacterial suspension prepared by HLJ2018 was used to infect mice and horses,the infection models of S.equi were initially established,which laid a foundation for further studies on vaccine and treatment of Strangles.Meanwhile,the phage SP2019 against HLJ2018 was isolated.This was the only report of S.equi phage in recent 40 years,the previous report was in the United States in the 1977.The basic biological characteristics of SP2019 were studied.The discovery of phage SP2019 would open up a new direction for the treatment of Strangles,and provide an important tool for the study of S.equi evolution and host specificity.This work was important for the vaccine and treatment of Strangles.