Establishment of the Micro-agglutination Test for Antibody Detection ofCitrobacter freundii from Sheep (Ovis aries)
LI Jing1,2, LIU Yi-Ting1,2, YANG Jin-Qian1,2, DING Lue-Xuan1,2, XUE Yun1,2, DING Ke1,2, SI Li-Fang1,2, ZHAO Zhan-Qin1,2*
1 Key Lab of Animal Bacterial Infectious Disease Prevention, Control Technology, College of Animal Science, Technology, Henan University of Science, Techology, Luoyang 471003, China; 2 Key-Disciplines Lab of Safety of Environment, Animal Product, College of Animal Science, Technology, Henan University of Science, Techology, Luoyang 471003, China
Abstract：Ovine Citrobacter freundii was an emerging infectious disease affecting sheep (Ovis aries), in order to establish a rapid detection method for the disease, this study used the inactivated whole-cell of C. freundii LK-1 strain as the antigen, establishing an antibody detection method of micro-agglutination test (MAT) byoptimizing the reaction conditions. The optimal antigen concentrations of inactivated whole-cell of C. freundiiLK-1 strain were 2.0×; 109 cfu/mL and the incubation temperature was 37 ℃ for 7~9 h. The MAT was specificity for C. freundii of sheep, demonstrating negative reactivity (titer≤; 1∶; 2) against serum samplespositive for 8 common ovine pathogens. The antibodies in sheep could be detected at 14 d post vaccinationwith inactivated C. freundii LK-1 vaccines. The values at day 21 and 35 were more than 1∶; 16 and 1∶; 64,respectively. The intra-assay coefficients of variation (CV) were consistently 0%, while the inter-assay CV ranged from 5.43% to 6.60%, indicating acceptable reproducibility. 374 serum samples, which were detected by the developed MAT and the ELISA with the same antigen. The results of tests showed that the total positive rates were 9.9% and 10.7%, respectively. The coincidence of the positive and negative results was good(Kappa value was 0.897), showing that the sheep were infected by C. freundii in a certain area of Henan Province. This study provides a basis for vaccine antibody evaluation and epidemiological investigation of ovine C. frendii.
[1] 公翠萍, 娄剑锋, 孙威, 等. 2024. 弗氏柠檬酸杆菌可视化 LAMP 检测方法的建立 [J/OL]. 水产学杂志 , 1-6. (Gong C P, Lou J F, Sun W, et al.2024. Establishment of visual loop-mediated isothermal amplification(LAMP)assay for detection of Citrobacter freundii[J/ OL]. Chinese Journal of Fisheries, 1-6.) [2] 李康, 贺佳. 2018. 医学统计学[M]. 北京: 人民卫生出版社. pp. 237-238 (Li K, He J.2018. Medical Statistics[M]. People Medical Publishing House, Beijing, China, pp.237-238) [3] 李静.2018. 羊源致病性柠檬酸杆菌灭活疫苗佐剂的筛选[D]. 硕士学位论文, 河南科技大学, 导师: 赵战勤, pp.1-63. (Li J.2018. Screening of adjuvants for inactivated vaccine against sheep-derived pathogenic Citrobacterium freundii[D]. Thesis for M. S., Henan University of Sci-ence and Technology, Supervisor: Zhao Z Q, pp. 1-63.) [4] 刘会胜.2015. 羊源弗氏柠檬酸杆菌的分离鉴定及其生物学特性研究[D]. 硕士学位论文, 河南科技大学, 导师: 赵战勤 , 汪洋 , pp. 1-48. (LIU H S.2015. Isolation and identification of Citrobacterium freundinii from sheep and its biological characteristics[D]. Thesis for M. S., Henan University of Science and Technology, Supervi-sor: Zhao Z Q, Wang Y, pp. 1-48.) [5] 刘会胜, 赵战勤, 薛云, 等. 2015. 羊源致病性弗氏柠檬酸杆菌的分离与鉴定[J]. 畜牧兽医学报 , 46(09): 1593-1599. (Liu H S, Zhao Z Q, Xue Y, et al.2015. Isolation and identification of pathogenic Citrobacter freundii orig-inating from sheep[J]. Journal of Animal Husbandry and Veterinary Medicine, 46(09): 1593-1599.) [6] 刘梦佳, 任嘉瑞, 张耀相, 等. 2017. 弗氏柠檬酸杆菌的分离与鉴定[J]. 畜牧兽医杂志, 36(4): 3-6. (Liu M J, Ren J R, Zhang Y X, et al.2017. Isolation and identification of Citrobacter freundii[J]. Journal of Animal Husbandry and Veterinary Medicine, 36(4): 3-6.) [7] 刘亚暖, 姜肇旭, 颜世敢. 2023. 弗氏柠檬酸杆菌的致病性及分型研究进展[J]. 工业微生物 , 53(04): 53-56. (Liu Y N, Jiang Z X, Yan S G.2023. Progress in pathogenicity and typing studies of Citrobacter freundii[J]. Industrial Microbiology, 53(04): 53-56.) [8] 唐海波, 陈凤莲, 饶桂波, 等. 2017. 竹鼠源弗氏柠檬酸杆菌的分离鉴定及致病性分析[J]. 畜牧兽医学报 , 48(5): 922-929. (Tang H B, Chen F l, Rao G B, et al.2017. Iso-lation, identification and pathogenicity analysis of Citro-bacterium freundii from bamboo rats[J]. Journal of Ani-mal Husbandry and Veterinary Medicine, 48(5): 922-929.) [9] 王姝然, 范厚勇, 徐嘉楠, 等. 2024. 弗氏柠檬酸杆菌 RPA-LFD 快速检测方法的建立及应用[J]. 大连海洋大学学报, 39(02): 241-249. (Wang S R, Fan H Y, Xu J N, et al.2024. Establishment and application of a method for rap-id detection of Citrobacter freundii by recombinase poly-merase amplification(RPA)combined with lateral flow strips(LFD)[J]. Journal of Dalian Ocean University, 39(02): 241-249.) [10] 厦世邦, 吴金华. 2006. Kappa 一致性检验在检验医学研究中的应用[J]. 中华检验医学杂志, 29(1): 83-84. (Xia S B, Wu J H.2006. Application of Kappa consistency test in laboratory medicine research[J]. Chinese Journal of Laboratory Medicine, 29(1): 83-84.) [11] 薛巧.2016. 羊源弗氏柠檬酸杆菌Ⅰ型菌毛 FimA 和 FimH蛋白免疫原性分析[D]. 硕士学位论文, 河南科技大学,导师: 董发明, 赵战勤, pp. 1-59. (Xue Q.2016. Immu-nogenicity analysis of FimA and FimH proteins in Citro-bacterium freundii type Ⅰ fimbria derived from Arcillus freundii[D]. Thesis for M. S., Henan University of Sci-ence and Technology, Supervisor: Dong F M, Zhao Z Q, pp. 1-59.) [12] 薛云, 李静, 王臣, 等. 2018a. 羊源致病性柠檬酸杆菌 4 种佐剂灭活疫苗的免疫效果比较[J]. 中国预防兽医学报,40(8): 726-731. (Xue Y, Li J, Wang C, et al.2018. Com-parison of four kinds of adjuvants for inactivated Citro-bacter freundii vaccine in sheep[J]. Chinese Journal of Preventive Veterinary Medicine, 40(8): 726-731.) [13] 薛云, 王乐, 王臣, 等. 2018b. 副猪嗜血杆菌新型微量凝集试验抗体检测方法的建立[J]. 中国预防兽医学报, 40(9): 806-811. (Xue Y, Wang L, Wang C, et al.2018. Estab-lishment of the micro-agglutination test for detection of antibody against Haemophilus parasuis[J]. Chinese Jour-nal of Preventive Veterinary Medicine, 40(9): 806-811. [14] 周昱.2013. 食品中四种致病菌的多重 PCR 检测方法的建立和应用[D]. 硕士学位论文, 福建农林大学, 导师: 吴德峰 , pp. 44-53. (Zhou Y.2013. Stabilishment and ap-plicaion of multiplex PCR for detecion of four food-borne bacterial pathogens[D]. Thesis for M. S., Fujian Agriculture and Forestry University, Supervisor: Wu D F, pp. 44-53.) [15] 中国兽医药品监察所, 农业部兽药评审中心. 2011. 兽用生物制品质量标准汇编(2011)[M]. 北京: 中国农业出版社, pp. 241-243. (China Veterinary Drug Supervision In-stitute, Veterinary Drug Evaluation Center of the Minis-try of Agriculture. 2011. Compilation of Quality Stan-dards for Veterinary Biological Products(2011)[M]. Ag-ricultural Press, Beijing, China, pp. 241-243.) [16] Cardoso P H M, Moreno L Z, de Oliveira C H, et al.2021.Main bacterial species causing clinical disease in orna-mental freshwater fish in Brazil[J]. Folia Microbiologi-ca, 66(2): 231-239. [17] Collins J W, Keeney K M, Crepin V F, et al.2014. Citrobacter rodentium: Infection, inflammation and the microbiota[J]. Nature Reviews Microbiology, 12(9): 612-623. [18] Delgado G, Souza V, Morales R, et al.2013. Genetic charac-terization of a typical Citrobacter freundii[J]. PLOS ONE, 8(9): e74120. [19] Isobe J, Shima T, Kanatani J, et al.2014. Serodiagnosis using microagglutination assay during the food-poisoning out-break in Japan causedby consumption of raw beef con-taminated with enterohemorrhagic Escherichia coli O111 and O157[J]. Journal of Clinical Microbiology, 52(4): 1112-1118. [20] Li Y, Mustapha A.2004. Development of a polymerase chain reaction assay to detect enteric bacteria in ground beef[J]. Food Microbiology, 21(3): 369-375. [21] Liu H, Zhao Z, Xue Y, et al.2018. Fatal cases of Citrobacter freundii septicemia and encephalitis in sheep[J]. Journal of Veterinary Diagnostic Investigation, 30(2): 245-248. [22] Mundy R, MacDonald T T, Dougan G, et al.2005. Citrobacter rodentium of mice and man[J]. Cell Microbiology, 7(12): 1697-1706. [23] Ortega J, Corpa J M, Orden J A, et al.2015. Acute death asso-ciated with Citrobacter freundii infection in an African el-ephant(Loxodonta africana)[J]. Journal of Veterinary Di-agnostic Investigation, 27(5): 632-636. [24] Padua S B, Marques D P, Sebastiao F A, et al.2014. Isolation, characterization and pathology of Citrobacter freundii in-fection in native brazilian catfish pseudoplatystoma[J]. Brazilian Journal of Veterinary Pathology, 7(3): 151-157. [25] Xue Q, Zhao Z, Liu H, et al.2015. First comparison of adju-vant for trivalent inactivated Haemophilus parasuis se-rovars 4, 5 and 12 vaccines against Glässer's disease[J]. Veterinary Immunology and Immunopathology, 168(3-4): 153-158.
