Isolation And Identification Of Salmonella Abortus Equi From Donkey And Preliminary Study On The Function Of SPI-12 Virulence Island

In recent years,donkey breeding has developed rapidly with the strong support of national policies for the development of herbivorous animal husbandry.With the continuous development of donkey industry,there are some problems worthy of our attention,for example,in the process of large-scale,due to the lack of scientific breeding and management methods,the overall production level of donkey is low and the occurrence of disease,mainly manifested in the emergence of disease and low fertility.The biological characteristics of donkey,such as long gestation period,low reproductive rate and slow growth,have become the main factors causing the slow development of donkey industry and the decline of donkey population.This is also one of the main factors restricting the development of the donkey industry.Salmonella abortus equi is the pathogen of equi parathphoid,which is mainly related to the abortion of female livestock,neonatal septicemia and multiple arthritis.In this paper,through the investigation of main pathogens in donkey farms in recent three years,it was found that the main pathogen causing abortion was Salmonella abortus equi.In order to further study the function of SPI-12,especially the pathogenic mechanism of SPI-12,a strain of Salmonella abortus equi was discovered and isolated from donkey in 2019,by studying the characteristics and genome analysis of the strain.In this study,deletion and complement strains of two virulence genes in SPI-12 were constructed to study the main pathogenic functions of SPI-12.To lay a foundation for further research on Salmonella abortus equi.From January 2019 to December 2021,a total of 742 samples of diseased dead donkeys（foals）were collected from 9 large-scale donkey farms（over 100 breeding females）in Liaocheng,Dezhou and Binzhou,Shandong Province.Using PCR and pathogen bacteria isolation method,common equine bacteria and viruses were detected in the diseased tissues of large-scale donkey farms,and brucella pathogen was detected in serum isolated from blood samples by amber detection method.The results showed that the detection rate of Escherichia coli was 48.89%（132/270）in diarrhea samples.In the pneumonia samples,the detection rates of Escherichia coli,Klebsiella pneumoniae and Streptococcus equis were 24.10%（80/332）,29.82%（99/332）and 22.29%（74/332）,indicating that the three kinds of bacteria were the main pathogens causing donkey pneumonia.The highest salmonella detection rate was 44.29%（62/140）in the aborted samples.At present,the main epidemic disease of large-scale donkey farm is bacterial disease.The results showed that the positive rate of brucellosis was 0,indicating that there was no brucellosis infection in the large-scale donkey farms in Shandong.In April 2019,there was an outbreak of female donkey abortions in Shandong Province.Of the 200 donkeys in a small donkey farm,45 female donkeys suffered embryo abortion between 6 and 11 months of gestation.According to the survey,all the pregnant donkeys were pregnant for the first time.From 45 a donkey miscarriage embryo of mediastinal lymph nodes,heart,lung,kidney,liver,spleen,brain and tissue samples collected in the colon tissue,a donkey source abortion salmonella strains was isolated,named AD19,using biochemical identification,credits,drug susceptibility testing,mice serum pathogenicity test,pathological and immunofluorescence and whole genome sequencing method to research of the plant,MUMmer and LASTZ tools were used to compare the genome sequences between the sample strain（Salmonella abortus equi AD19）and the reference strain（Salmonella typhimurium LT2）（or between two or more sample genome sequences）,and the comparative genome analysis was performed by CD-HIT rapid clustering analysis of core genes and specific genes.The results showed that AD19 strain showed moderate to high sensitivity to 8 antibiotics.In addition,all samples tested negative for Escherichia coli,equine herpesvirus and equine arteritis virus.This suggests that the miscarriage was caused by Salmonella abortus equi.In the pathogenicity study of Kunming mice,the Results showed that the LD₅₀ of AD19 strain was 1.88×10⁸ CFU,which could also cause abortion in pregnant kunming mice,and the half stream yield was1.22×10⁸CFU.It was found that kunming mice and donkeys had similar clinical symptoms and pathological changes through pathological sections and immunofluorescence,and the animal model of Salmonella abortus equi in kunming mice was established successfully for the first time.By analyzing the whole genome of AD19 strain and comparing it with LT2 strain,we found that the number of pseudogenes increased,the presence of salmonella pathogenic island（SPI-12）and pilus gene cluster（ste）,and the decrease of phage tail protein.These may be related to the host pathogenesis of Salmonella abortus equi.The SPI-12 of Salmonella abortus equi AD19,which caused mass donkey abortion,was further studied.The ccm F and ccm E genes in SP-12 of AD19 were knocked out byλRed homologous recombination technique,and the complement strain was constructed by p BR322 vector.After that,biochemical test,biofilm qualitative test,BHK-21 cell adhesion and invasion test,RAW264.7 intracellular survival test and mouse virulence test were compared to explore the functions of ccm F and ccm E genes encoding virulence factors and analyze the pathogenic process involved.The results showed that ccm F and ccm E gene deletion strains and complement strains were successfully constructed.Through comparative analysis of biological characteristics of wild strains AD19,deletion strains AD19Δccm F,AD19Δccm EandcomplementarystrainsAD19Δccm F/pccm F,AD19Δccm E/pccm E,the ccm F gene significantly influence the adhesion,invasion ability of Salmonella abortus equi and virulence and survival and reproduction,in macrophages company gene significantly influence the adhesion,the ccm E gene significantly affected the adhesion,invasion and virulence of Salmonella abortus equi,but had little effect on the survival and reproduction in macrophages.The ccm F and ccm E genes had no effect on miscarriage related virulence.In conclusion,Ccm F protein is involved in the regulation of virulence factors related to salmonella abortus adhesion,invasion and survival,and the Ccm E protein is involved in the regulation of virulence factors related to Salmonella abortus equi adhesion and invasion,but its specific regulatory mechanism needs to be further clarified.It is predicted that the main functions of SPI-12 in Salmonella abortus equi are adhesion,invasion,and survivorship related virulence.In conclusion,at the present stage,the large-scale donkey farms are mainly caused by bacterial diseases,and the donkey farms in Shandong are in the state of brucellosis for the time being.An AD19 strain of Salmonella abortus equi was isolated from an outbreak in a donkey farm in 2019,and the animal model of Salmonella abortus equi in kunming mice was successfully established for the first time.The main functions of Salmonella abortus equi SPI-12 were adhesion,invasion,and survivorship related virulence.This study provides an important theoretical basis for further study on the pathogenic mechanism of Salmonella abortus equi SPI-12 on equine.