Description succincte
(Allemand)
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Am IVB Zürich ist in den letzten Jahren bei Schweinen aus verschiedenen Betrieben eine Infektion mit Lawsonia (L.) intracellularis post mortem oder intra vitam nachgewiesen worden. Berichten aus anderen Ländern zufolge können diese Erreger einen Krankheitskomplex mit erheblichen wirtschaftlichen Verlusten hervorrufen. Daher ist eine Abklärung der Bedeutung dieser Infektion im Schweizerischen Schweinebestand angezeigt. In diesem Zusammenhang müssen neue, molekularbiologische und serologische, diagnostische Methoden eingesetzt werden.
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Description succincte
(Anglais)
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Infections with Lawsonia intracellularis in pigs have been diagnosed sporadically in the last years at our Institute by use of modified Ziehl-Neelsen staining and on the basis of pathomorphological and histological criteria. The aim of this study is to determine the prevalence and the economical importamce of this infection in Swiss pigs by use of additional molecular (PCR) and serological (IFAT) methods. These methods will be checked for their ability as routine diagnostics. Moreover, attempt will be done to improve other methods for direct or indirect demonstration of L. intracellularis.
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Objectifs du projet
(Allemand)
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Ziele dieser, im Rahmen einer Dissertation am IVBZ geplanten Untersuchungen sind:
- Die Prävalenz von serologisch positiven Zucht- und Mastschweinen gegen L. intracellularis in der Schweiz zu ermitteln,
- Schweine von ausgewählten positiven und negativen Beständen diesbezüglich koprologisch und
serologisch zu untersuchen,
- Verendete Tiere aus diesen Beständen pathologisch-anatomisch, histologisch und bakteriologisch zu
untersuchen (Zusammenarbeit mit IPVZ).
- Referenzkulturverfahren zur Vermehrung des Erregers für die Gewinnung von diagnostischen Antigenen
und zur Herstellung von DNA zu etablieren.
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Résumé des résultats (Abstract)
(Anglais)
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A total of 21 pigs aged 7 to 17 weeks with clinical symptoms suggestive for Porcine Proliferative Enteropathy (PPE) were examined for Lawsonia (L.) intracellularis by analysing the following parameters: (i) presence of intestinal gross pathologic and histologic lesions characteristic for PPE, (ii) detection of comma-shaped bacteria in enterocytes of altered intestinal mucosa by Warthin-Starry- and a modified Ziehl-Neelsen-stain, (iii) PCR amplification of L. intracellularis DNA from intestinal mucosa using two different pairs of oligonucleotide primers targeted against a 255-bp DNA fragment of the 16S rRNA-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. For all 21 pigs, the L. intracellularis etiology was confirmed by histologic as well as bacterioscopic examinations. Specific PCR amplification products were obtained from 20 of 21 pigs (95.2 %). Taking PCR positivity as the definite criterion, L. intracellularis was diagnosed in 20 pigs from eleven herds located in seven Swiss cantons (Argovia, Berne, Fribourg, Grisons, Lucerne, Schwyz, Thurgovia). To grow L. intracellularis in vitro, the cell culture method of Lawson et al. (J. Clin. Microbiol. 1993, 31, 1136-42) 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 culturability, accordingly. Except for occasional specific PCR amplifications from cell cultures maximum up to the fourth passage, any indications for growth of L. intracellularis in IEC-18 cells were not found.
The diagnostic tests used in our work have limitations in clinical application. Thus, a major objective of our study was to grow L. intracellularis in vitro. Cultivation of a bacterium is undeniably the first obligatory step of any development of new diagnostic procedures. The findings generated through this study argue for apparent difficulties in cultivation of the agent by using documented methodologies. However, the reasons that in our opinion hinder endorsement of cell culture as a generally useful tool to grow L. intracellularis are clearly open to speculation at this point. Whatever reasons are involved in our failure to reproduce the cultivation of L. intracellularis in IEC-18 cells, we are continuing to explore this method and to analyze furthermore the minimal in vitro growth requirements of this bacterium.
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Publications / Résultats
(Anglais)
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Dittmar, M., Hoelzle, L.E., Hoelzle, K., Corboz, L., Sydler, T., Miserez, R., Wittenbrink, M.M. (2003) Diagnosis of porcine proliferative enteropathy: detection of Lawsonia intracellularis by pathologic examinations, Polymerase chain reaction, and cell culture inoculation. J. Vet. Med. B., submitted for publication.
Dittmar, M., Hoelzle, L.E., Corboz, L., Sydler, T., Wittenbrink, M.M. (2003) Porcine proliferative enteropathy in Switzerland. SSM, Annual Meeting 2003 (Poster).
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