| As an acute contact disease,Strangles is mainly caused by Streptococcus equi subsp.equi(S.equi),which has a high incidence in equine animals and can cause huge economic losses to equine animal breeding industry.S.equi infects equine animals of all ages and continues to harm the equine industry worldwide.At present,the pathogenesis and virulence factors of S.equi as well as the host interaction and pathogenesis of S.equi are not completely clear.In this study,we sequenced and compared the genomes of Streptococcus equis equine subspecies from donkey in Xinjiang,hoping to provide the pathogenic mechanism and genomic information of strangles.In this study,the growth characteristics,drug resistance characteristics and virulence of three S.equi strains in xinjiang(HT1112,HTP133 and HTP232)isolated and identified by our laboratory were analyzed.Results each strains had different growth curves.HT1112 was the fastest growing strain among the three strains.Challenged mice,the results showed that HTP133 challenged group had the strongest toxicity effect on mice,followed by HTP232 and HT1112.The bacterial load in lung of challenged mice was higher than that in spleen,and the bacterial load in HTP133 challenged group was the highest.HT1112 was resistant to cefoxitin,penicillin,clarithromycin,ciprofloxacin,sulfadiazine and clindamycin,and HTP133 and HTP232 were resistant to cefuroxime and penicillin.The genes of HT1112,HTP133 and HTP232 were extracted,and the whole genome of each strains was sequenced using IIumina and Pac Bio RS II sequencing platform.The results showed that(1)The gene size of HT1112 strain was 2.16 Mb,GC content was 41.36%,and 2 167 genes could be encoded.112 virulence genes and 134 drug resistance genes were compared in VFDB and CARD database.(2)The gene size of HTP133 strain was 2.15 Mb,GC content was 41.34%,which could encode 2 010 genes.Through comparison between VFDB and CARD database,HTP133 strain had 169 virulence genes and 124 drug resistance genes.(3)The gene size of HTP232 strain was 2.19 Mb,GC content was 41.3%,and 2 223 genes could be encoded.Through comparison between VFDB and CARD database,170 virulence genes and 172 drug resistance genes were found.(4)Based on 16 S r RNA phylogenetic analysis and watchman genetic analysis,HT1112 had the best homology with S.equi 4047.Collinear comparison of the genomes of three S.equi strains with those of the international strains showed that there were rearrangements among the strains.The collinearity of the three donkey strains HT1112,HTP133 and HTP232 was close,and that of XJ 5012 and S.equi 4047 was conversant.HTP232 strain had a unique gene expressing hyaluronic acid in collinearity comparison.The homology comparison of the four virulence factors Se M,Ide E,Ide E2 and EAG showed that the homology of Ide E,Ide E2 and EAG of the comparison strains was 99.9%-100%,and the homology of Se M was 97%-99.6%,which was consistent with the results of genomic collinearity comparison.Se M,Ide E,Ide E2 and EAG proteins were expressed and purified and mice were immunized respectively.The expression levels of IFN-γ,IL-4,IL-6 and IL-10 in Se M and EAG groups were significantly higher than those in Ide E and Ide E2 groups(P < 0.05).The results showed that the Ide E2 group had the highest protection rate of 80%,the EAG and Se M groups had 66.7%,and the Ide E group had50%.The results of all the immune groups showed that the Ide E2 group had the lowest bacterial charge,followed by Se M and Ide E group,and EAG group had the highest bacterial charge.The expression levels of cytokines in spleen of mice showed that the expression levels of IFN-γ,TCR,MHC I,RP105,TLR1,TLR2,TLR3,TLR4,TLR6 and TLR9 in Ide E group were significantly higher than those in other groups(P< 0.05);The expression level of IL-10 in Ide E2 group was significantly higher than that in other groups(P< 0.05);The expression levels of MHC II,IL-2,IL-4,IL-6,IL-12 and IL-17 A in EAG group were significantly higher than those in other groups(P < 0.05).In conclusion,this study compared and analyzed the biological characteristics,virulence and drug resistance of three S.equi strains from donkey in xinjiang.The whole genome of the isolates was sequenced and collinear comparison was performed.The virulence related factors of the three isolates were sequenced and their biological functions were analyzed.It provides a theoretical basis for understanding the pathogenic mechanism and prevention and control of this strain from genome level. |
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