| 马流产沙门氏菌的分离鉴定及其微量凝集抗体检测方法的建立与应用 |
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| 引用本文： | 郭奎,王宁,王金慧,初晓雨,赵语婷,郭巍,刘荻萩,胡哲,王晓钧. 马流产沙门氏菌的分离鉴定及其微量凝集抗体检测方法的建立与应用[J]. 中国农业科学, 2020, 53(10): 2112-2121. DOI: 10.3864/j.issn.0578-1752.2020.10.017 |
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| 作者姓名： | 郭奎 王宁 王金慧 初晓雨 赵语婷 郭巍 刘荻萩 胡哲 王晓钧 |
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| 作者单位： | 中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨 150069 |
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| 基金项目： | “十三五”国家重点研发计划(2018YFD0502205) |
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| 摘 要： | 【目的】 对流产马驹组织进行马流产沙门氏菌的分离和鉴定,以分离株制备凝集抗原,旨在建立一种敏感、特异、操作简便和高通量的微量凝集方法,实现对马流产沙门氏菌抗体进行快速检测。【方法】 利用沙门氏菌显色培养基对流产的马驹脏器进行细菌分离,对紫色菌落进行革兰氏染色鉴定;提取细菌全基因组,利用16S rRNA 进行 PCR扩增和测序鉴定,并对菌株进行生化鉴定和血清型鉴定,对病因进行综合诊断。利用分离获得的阳性菌株进行灭活后作为凝集抗原,以马流产沙门氏菌标准阳性血清作为阳性对照,通过反应条件优化,建立微量凝集试验方法,并对该方法的特异性、敏感性、重复性进行了验证。与国家行业标准中的试管凝集试验方法进行对比,同时利用微量凝集对华北和西北各3个地方的共151份血清样品进行检测,并随机选取30份样品,利用微量凝集和试管凝集进行检测,并对两种方法检测的敏感性进行比较分析。【结果】 各流产马驹组织在沙门氏菌显色培养基上划线结果,均可以获得大量紫色目标菌落;革兰氏染色结果表明,分离的菌株为革兰氏阴性短杆菌,16S rRNA测序证实分离株为沙门氏菌属,生化鉴定结果表明,分离的17株菌均符合马流产沙门氏菌的生化特性,但相比上世纪强毒株马流产沙门氏菌C77-1,均不能发酵鼠李糖;血清型鉴定证实分离株均为马流产沙门氏菌,将分离的17株马流产沙门氏菌分别命名为E.S-1—17。因此证实马匹流产均为马流产沙门氏菌感染所致。利用甲醛对E.S-1菌株进行了灭活处理,成功制备了马流产沙门氏菌凝集抗原,并建立了一种快速敏感的微量凝集检测方法,其中优化了最佳反应凝集抗原浓度为4亿/mL。其敏感性比试管凝集高1个滴度,其特异性与其他常见马传染病病原阳性血清无交叉反应,并且具有良好的重复性。利用微量凝集方法,对华北3个疫情区进行检测,阳性率分别为28.6%、42.9%和27.3%,西北3个无疫情区阳性率均为0%。此外,利用两种方法对相同的30份临床血清进行检测,微量凝集和试管凝集方法阳性份数分别为10份和3份,阳性率分别为33.3%和10%。与试管凝集相比,微量凝集诊断敏感性为100%,诊断特异性为74.1%,总体符合率为76.7%。【结论】 分离鉴定获得了17株马流产沙门氏菌,建立了马流产沙门氏菌微量凝集抗体检测方法,该方法敏感性高、特异性强、重复性好,简便、快速、高通量,可以广泛应用于马属动物中马流产沙门氏菌抗体的检测,是该病诊断及疫苗评估的重要依据之一。
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| 关 键 词： | 马流产沙门氏菌 分离鉴定 微量凝集 应用 马 |
| 收稿时间： | 2019-04-29 |
Establishment and Preliminary Application of Microagglutination Detection Method for Salmonella Abortus Equi |
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| GUO Kui,WANG Ning,WANG JinHui,CHU XiaoYu,ZHAO YuTing,GUO Wei,LIU DiQiu,HU Zhe,WANG XiaoJun. Establishment and Preliminary Application of Microagglutination Detection Method for Salmonella Abortus Equi[J]. Scientia Agricultura Sinica, 2020, 53(10): 2112-2121. DOI: 10.3864/j.issn.0578-1752.2020.10.017 |
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| Authors: | GUO Kui WANG Ning WANG JinHui CHU XiaoYu ZHAO YuTing GUO Wei LIU DiQiu HU Zhe WANG XiaoJun |
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| Affiliation: | State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069 |
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| 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. |
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| Keywords: | Salmonella abortus equi separation microagglutination application horse |
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