Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA.
Vet Microbiol. 2013 Feb 22;162(1):265-9. doi: 10.1016/j.vetmic.2012.08.002. Epub 2012 Aug 13.
Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.
已通过细胞培养多次传代获得非致病性细胞内劳森菌变异体，但尚无关于使致病性分离株减毒所需传代次数的信息。本研究评估了在体外传代 10、20 和 40 次后猪对细胞内劳森菌的易感性。三组（每组六只动物）分别用细胞内劳森菌的纯培养物在传代 10、20 或 40 时接种，一组用安慰剂接种。在接种后 28 天内监测动物的临床症状、粪便排出和血清 IgG 反应。在每组的两只动物中，于第 14、21 和 28 天评估基于免疫组化鉴定的肠黏膜中细胞内劳森菌特异性抗原量的大体和组织学病变以及感染程度。接种传代 10 和 20 的动物表现出与猪增生性肠病相关的增生性病变，其特征是肠黏膜中存在 Lawsonia 特异性抗原。在整个研究过程中，任何时间点接种传代 40 的猪均未显示增生性病变或 Lawsonia 抗原的存在。在感染传代 10 和 20 的猪中观察到相似的粪便排出模式，但感染传代 40 的猪仅在短时间内排出。在感染传代 10 和 20 的猪中可从感染后第 14 天检测到血清 IgG 反应，但在感染传代 40 的动物中则完全没有。这些结果表明，细胞内劳森菌在体外传代 20 至 40 次之间毒力特性减弱。这些信息对于设计未来的实验模型以及研究细胞内劳森菌毒力减弱的机制将非常有价值。
