人工感染羊嗜流产衣原体豚鼠流产模型的建立

Experimental Infection of Two Virulent Strains of Chlamydophila abortus in Guinea Pigs:an Animal Model for the Study of Chlamydiosis

目的通过人工感染怀孕豚鼠建立嗜流产衣原体发病模型,观察流产病理学变化的差异,确定豚鼠是否可以作为嗜流产衣原体的动物模型。方法35只清洁级怀孕40 d的豚鼠随机分为7组,每组5只。将羊嗜流产衣原体分离株B10011、CG1株分别以三个剂量组(10-1,10-2,10-3)腹腔攻毒1次,每只0.5 mL,以灭菌生理盐水为阴性对照。攻毒后观察豚鼠的临床体征、流产和死亡数量。攻毒后20 d安乐死处理剩余的大小豚鼠,无菌摘取组织,分成2份。一份-20℃冷冻保存,PCR检测病原在组织中的分布;另外一份固定于10%中性甲醛溶液,组织切片和免疫组化观察。结果(1)羊嗜流产衣原体分离株B10011和CG1均可造成怀孕豚鼠流产、产弱胎、畸形胎或死胎。2种分离株感染不同剂量造成豚鼠平均流产时间、流产数量、豚鼠死亡数量均有显著差异,且CG1株敏感性高于B10011株。(2)2个分离株均可造成肾脏、肝脏肿大,肺脏呈局灶性出血,卵巢出血坏死,胎盘子宫阜水肿;病理组织学显示肾小管上皮细胞变性、坏死,单核细胞大量增生;肝细胞颗粒样病变。脾髓质弥漫性出血,皮质区淋巴小结生发中心萎缩。(3)免疫组化结果显示衣原体包涵体主要分布于肾脏近曲小管、远曲小管,以及脾脏的髓质区。(4)PCR检测显示分离株感染后,肾脏、脾脏、肺脏、肝脏中均呈阳性。在卵巢组织中,高剂量组呈阳性。结论以B10011和CG1分离株人工感染怀孕豚鼠均可导致豚鼠出现流产,且CG1分离株敏感性较高,呈剂量-效应关系;感染豚鼠病理学变化与临床羊流产相似。因此,人工感染豚鼠流产模型可以用于反刍动物嗜流产衣原体病的研究。

Objective To investigate the pathogenicity of Guinea pigs infected with Chlamydia abortus B11001strain and CG1 strain and to determine the potential animal models for sheep Chlamydia abortus. Methods Thirty-five 40-day pregnant clean guinea pigs were randomly assigned to seven groups and inoculated intraperitoneally with：1 ） 10^-1 dilution of B10011; 2） 10^-2 dilution of B10011; 3） 10^-3 dilution of B10011; 4） 10^-1 dilution of CG1; 5）10-2 dilution of CG1; 6） 10^-3 dilution of CGI. Control group were administered intraperitoneally with sterile physiological saline. All animal were given with 0.5 mL above preparations. Each group was recorded daily and sacrificed by diethyl ether at day 20 postinoculation. The gross lesions were recorded and main organs including livers, spleens, ovary and uterus were collected and divided into two samples, one sample was kept at - 20~C and examined by PCR based on omp-1 gene. Another sample was fixed in a 10% formaldehyde solution for observing pathological lesions and immunohistochemical changes. Results （1） Guinea pigs showed the typical abortion by Chlamydia abortus B10011 strain and CG1 strain. Weak fetus, stillbirth or fetal malformation were also observed post inoculation. As for average the abortion time, the abortion number and numbers of died fetus, there was a significant difference between Chlamydia abortus CG1 strain and Chlamydia abortus B10011 strain. （2） Typical gross changes were seen in the organs ,such as swollen kidney, hemorrhagic liver, focal hemorrhage in lung, ovarian and placenta uterus. Renal tubule presented extensive hemorrhage, necrosis and mononuclear cell infiltration. Spleen exhibited atrophied in white pulps, and even lost of white pulps in some areas. The pulp was markedly congested and hemorrhagic. （3） Chlamydia inclusion bodies were identified in proximal convoluted tubule,distal convoluted tubule of the kidney and red pulp of spleen under microscopic examination. （4） omp-1 gene was found positive in lungs, livers, spleens and kidneies post inoculation. However, positive was only detected in ovaries with 10^-1 dilution of B10011 strain and 10^-1 dilution of CG1 strain while the other group proved to be negative. Conclusion The guinea pig infected by two isolated Chlamydophila abortus could develop an abortion, stillbirth and dead fetus similar to those in the aborted sheep. This study demonstrated that the Chlamydia abortus CG1 strain might be a virulent pathogen for guinea pigs. These guinea pigs infected with Chlamydia abortus CG1 strain may provide a useful animal model of sheep Chlamydial aborts.