关键词: 马流产沙门氏菌, 分离鉴定, 微量凝集, 应用, 马

Abstract:

【Objective】Salmonella abortus equi was isolated and identified from the tissue of aborted foal. The aim for the study was to establish a sensitive, specific, easy to operate and high throughput microagglutination method by using the isolated Salmonella abortus equi, so as to realize the rapid diagnosis of Salmonella abortus equi antibody. 【Method】Selective medium for Salmonella was used for bacteria reproduction from aborted foal organs, and the purple colonies were selected and identified by Gram staining. The whole genome of bacteria was extracted, and 16S rRNA was used for PCR amplification and sequencing identification. Biochemical identification and serotype identification of the bacteria were carried out to make a comprehensive diagnosis of the etiology. The obtained positive bacteria was used as agglutinating antigen after inactivation, and the standard positive serum of Salmonella was used as positive control. The microagglutination assay was established by optimizing the reaction conditions, and then the specificity, sensitivity and repeatability of the method were verified. Meanwhile, a total of 151 serum samples from three different locations in north and northwest of China were detected with microagglutination. And 30 of these samples were randomly selected to be detected with both microagglutination and test tube agglutination, and the sensitivities of these two methods were compared and analyzed.【Result】A large number of purple target colonies could be obtained from the coloration culture medium of Salmonella. The results of gram staining showed that the isolated strains were gram-negative short bacillus, and 16S rRNA sequencing confirmed that the isolated strains were Salmonella. The biochemical identification results showed that all the 17 isolated strains were in line with the biochemical characteristics of Salmonella abortus equine, but could not ferment rhamnosus compared with C77-1, a strong strain of Salmonella abortus equi identified in China in the last century. Serotype identification confirmed that all isolated strains were Salmonella abortus equi, and the 17 isolated strains were named as ES-1-17 respectively. Therefore, it was confirmed that the abortion of horses was caused by Salmonella abortus equi infection. The agglutination antigen of Salmonella abortus equi was prepared by inactivating E.S-1 strain with formaldehyde, and a rapid and sensitive method of microagglutination was established for the detection of agglutination, in which the optimal concentration of agglutination antigen was 400 million mL^-1. The sensitivity was 1 titer higher than that of test tube agglutination, and the specificity was good without any cross reaction with sera against other equine infectious pathogens, and it had good repeatability. Equine sera collected from three epidemic areas in North China were detected with microagglutination for antibodies against Salmonella abortus equi, and the positive rates were 28.6%, 42.9% and 27.3%, respectively, while the positive rate was 0% in three non-epidemic areas in Northwest China. In addition, 30 samples of the above clinical serum were detected by these two methods, 10 of 33 were positive for microagglutination and 3 of 33 were positive for test tube agglutination with positive rates of 33% and 10%, respectively. The sensitivity and specificity of microagglutination were 100% and 74.1%, respectively, and the total coincidence rate was 76.7%.【Conclusion】Seventeen strains of Salmonella equi abortion were isolated and identified in this study. A microagglutinating antibody detection method for Salmonella abortus equi was established. This method was of high sensitivity, specificity and good repeatability, simple, rapid, high throughput, could be widely used in the horse abortion antibody detection of Salmonella in the animal, and was one of the important basis for evaluation of the disease diagnosis and vaccine.

Key words: Salmonella abortus equi, separation, microagglutination, application, horse