Dittmar M, Hoelzle L E, Hoelzle K, Sydler T, Corboz L, Miserez R, Wittenbrink M M
Institute of Veterinary Bacteriology, University of Zurich, Winterthurerstrasse 270, CH-8057 Zurich, Switzerland.
J Vet Med B Infect Dis Vet Public Health. 2003 Sep;50(7):332-8. doi: 10.1046/j.1439-0450.2003.00691.x.
A total of 21 pigs aged 7-17 weeks with clinical symptoms suggestive for Porcine Proliferative Enteropathy were examined for Lawsonia intracellularis by analysing the following parameters: (i) intestinal gross and histological lesions, (ii) presence of comma-shaped bacteria in enterocytes by Warthin-Starry and a modified Ziehl-Neelsen stain, (iii) PCR amplification of L. intracellularis DNA from intestinal mucosa by using two oligonucleotide primer pairs targeting a 255-bp DNA fragment of the 16S rDNA-gene and a 319-bp DNA fragment of the L. intracellularis chromosome. Specificity of PCR reactions was confirmed by using DNA extracted from the L. intracellularis reference strain N343 (ATCC 55672) as well as by DNA sequence comparisons of PCR amplification products with data bank entries. Intestinal gross lesion indicative for PPE were observed in 20 pigs (95.2%). For all 21 pigs, the L. intracellularis aetiology was confirmed by histological as well as bacterioscopical examinations. Specific PCR amplification products were obtained from 20 pigs (95.2%). Taking PCR positivity as the definite criterion, L. intracellularis was diagnosed in 20 pigs from 11 herds in seven Swiss cantons (Argovia, Berne, Fribourg, Grisons, Lucerne, Schwyz, Thurgovia). To grow L. intracellularisin vitro, the cell culture method of Lawson et al. (J. Clin. Microbiol. 1993: 31, 1136-1142) was adopted. Inocula prepared from heavily infected fresh and frozen ileal mucosa of 15 pigs were cultured in rat enterocytic IEC-18 cells (ATCC CRL 1589). Six cell culture passages of 10 days each were completed. The reference strain N343 was examined for cultivability, accordingly. Except for occasional specific PCR amplifications from cell cultures up to the second passage, any indications for growth of L. intracellularis in IEC-18 cells were not found.
对21头7至17周龄、有猪增生性肠炎临床症状的猪进行了检查，通过分析以下参数来检测细胞内劳森菌：(i)肠道大体和组织学病变；(ii)用Warthin-Starry染色法和改良齐尔-尼尔森染色法检测肠上皮细胞中是否存在逗号状细菌；(iii)使用两对寡核苷酸引物，从肠道黏膜中PCR扩增细胞内劳森菌DNA，一对引物靶向16S rDNA基因的255 bp DNA片段，另一对引物靶向细胞内劳森菌染色体的319 bp DNA片段。通过使用从细胞内劳森菌参考菌株N343（ATCC 55672）提取的DNA以及将PCR扩增产物的DNA序列与数据库条目进行比较，证实了PCR反应的特异性。在20头猪（95.2%）中观察到了指示猪增生性肠炎的肠道大体病变。通过组织学和细菌学检查，证实了所有21头猪的细胞内劳森菌病因。从20头猪（95.2%）中获得了特异性PCR扩增产物。以PCR阳性为明确标准，在瑞士七个州（阿尔高州、伯尔尼州、弗里堡州、格劳宾登州、卢塞恩州、施维茨州、图尔高州）11个猪群的20头猪中诊断出细胞内劳森菌。为了在体外培养细胞内劳森菌，采用了Lawson等人（《临床微生物学杂志》1993年：31卷，1136 - 1142页）的细胞培养方法。用15头猪严重感染的新鲜和冷冻回肠黏膜制备接种物，在大鼠肠上皮IEC - 18细胞（ATCC CRL 1589）中培养。完成了6次每次10天的细胞培养传代。相应地，对参考菌株N343的可培养性进行了检测。除了在细胞培养传至第二代时偶尔出现特异性PCR扩增外，未发现细胞内劳森菌在IEC - 18细胞中生长的任何迹象。
