Direktnachweis von Mycobacterium avium subsp. paratuberculosis aus Lebensmitteln und klinischem Material und Charakterisierung von Isolaten verschiedener Herkunft mittels molekularbiologischer Methoden

• Forschungsstelle: BLV
• Projektnummer: 1.01.09
• Projekttitel Englisch: Direct detection and characterization of Mycobacterium avium subsp. paratuberculosis from food and clinical samples by means of molecular methods.

Schlüsselwörter (Englisch)

Mycobacterium avium subsp. paratuberculosis - paratuberculosis - Crohn's disease - PCR-based detection, molecular typing

Kurzbeschreibung (Englisch)

Paratuberculosis (Johne's disease) is a chronic disease caused by Mycobacterium (M.) avium subsp. paratuberculosis, which causes major financial losses to the livestock industry. Although paratuberculosis is a noti-fiable disease, the prevalence of this infection in Switzerland is not known. M. avium subsp. paratuberculosis has also been linked to Crohn's disease in humans, a chronic inflammatory disease of the lower bowel. One constraint on efforts to control the disease is the limitation in the sensitivity of currently available laboratory diagnostic techniques. The overall goal of this proposal is to develop and optimize a PCR method for the detection of the agent in food (milk, meat) and feces. This research will provide a new tool for the quick, accurate, and early identification of infected cattle, particularly those animals subclinically infected.
Another aim of the study is to collect M. avium subsp. paratuberculosis strains from different sources (animals, humans, food) and to compare them using molecular techniques as RFLP, PCR-REA, and PFGE.

Projektziele (Englisch)

- Development of a PCR assay for the direct detection of M. paratuberculosis in food (milk and meat prod-ucts) and clinical specimen; comparison to culture methods;
- Assessment of the prevalence of M. paratuberculosis in milk and meat products from infected and from disease-free herds;
- Collection of M. paratuberculosis strains from different origin;
- Comparative study of M. paratuberculosis strains using RFLP and PFGE.

Abstract (Englisch)

Two different diagnostic methods for the detection of Mycobacterium avium subspecies paratuberculosis, the etiological agent of Johne's Disease, were compared. Fecal samples were derived from 310 cows, representing 13 commercial dairy herds located in 8 cantons of Switzerland. Detection tests for Mycobacterium avium subspecies paratuberculosis were culture (gold standard) and a newly designed Real-time PCR. Real-time PCR identified 31 of 310 animals (10%) as positive whereas culture identified 20 (6.5%) as positive. The observed agreement of the two tests was 91.3% and the Kappa-value was 42%. Our Real-time PCR had a sensitivity of 60% and a specificity of 93.4% when culture was used as the gold standard. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5% to 10%. Since Real-time PCR and culture data were in good agreement, and furthermore, Real-time PCR generates data in a short time in contrast to culture we consider Real-time PCR as a reliable alternative method to culture for the detection of Mycobacterium avium subspecies paratuberculosis in a national surveillance program.

Publikationen / Ergebnisse (Deutsch)

Seitert, G. (2003) Entwicklung und Validierung molekularer Methoden für den Nachweis von Mycobacterium avium subsp. paratuberculosis in Gewebe von geschlachteten agemagerten Schweizer Kühen. 44. Arbeitstagung des Arbeitsgebietes Lebensmittelhygiene der Deutschen Veterinärmedizinischen Gesellschaft, Garmisch-Partenkirchen, Deutschland.Seitert,

Publikationen / Ergebnisse (Englisch)

G., Bissig-Choisat, B., Kohler, C., Wittwer, M., Wassenaar, T. M., Jemmi, T., Frey, J., and K. Bögli-Stuber (2004) Development and Validation of molecular methods for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues from slaughtered emaciated Swiss cows. Submitted.

Bögli-Stuber, K., C. Kohler, G. Seitert, B. Glanemann, M. C. Antognoli, M. D. Salman, M. M. Wittenbrink, M. Wittwer, T. M. Wassenaar, T. Jemmi, and B. Bissig-Choisat (2003) Detection of Mycobacterium avium subspecies paratuberculosis in Swiss Dairy Cattle by Real-time PCR, Culture and Serology. Submitted.

Bögli-Stuber, K., Kohler, C., Seitert, G., Glanemann, B., Antognoli, M.C., Salman, M.D., Wittenbrink, M.M., Wittwer, M., Wassenaar, T., Jemmi, T., Bissig-Choisat, B. (2005): Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays. Journal of Applied Microbiology, Vol. 99, p. 587-597