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Doctoral thesis, 2009

Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis

Herthnek, David


Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the chronic enteric disease paratuberculosis in ruminants that causes considerable economic losses worldwide. Due to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, import-related outbreaks have occurred. Diagnostic surveillance and outbreak investigations are mainly carried out by very slow culture methods. Faster and equally reliable molecular methods are needed for detection of MAP in several clinical matrixes. MAP is primarily shed in faeces, the most important testing material. The abundance of PCR inhibitory substances in faeces constitutes a diagnostic challenge. Semen, imported for breeding purposes, may contain MAP if the donor bulls are asymptomatic carriers. MAP has been suggested as the causative agent of Crohn's disease and its presence in consumers' milk might be of concern. In this thesis, the development and sensitivity assessment of protocols for detection of MAP in ruminant faeces, semen and milk by real-time PCR are described. The analytical sensitivities were assessed to 104 MAP/g faeces, 10 MAP/100 µl semen and 100 MAP/ml milk. The faeces direct PCR was validated on 202 proficiency test samples. MAP was detected in 97% of previously frozen positive samples - better than culture. Pellet and cream fractions of milk were pooled before cell lysis and DNA extraction by automated magnetic bead separation. In a study of 56 dairy herds, tank milk PCR was compared to culture of environmental faecal samples for herd prevalence testing. By the latter, 68% of the herds were positive, while 30% were positive by PCR. Due to the concluded low abundance of MAP in milk tanks, milk PCR would be more useful for testing of MAP in consumers’ milk, than for herd prevalence testing. Three real-time PCR systems were designed for confirmation of PCR positives and validated on 267 strains and 58 positive faecal and tissue samples. The system based on the gene F57 was the most specific. A faecal culture screening of 501 wild guanacos in Chile yielded MAP colonies from 21 guanacos (4.2%), representing the first isolation of MAP from wild animals in the Chilean Patagonia. Confirmation was done by PCR and typing was performed by PCR-REA. All strains proved to be of C type.


cattle; mycobacterium avium subsp paratuberculosis; paratuberculosis; diagnosis; pcr; microbiology

Published in

Acta Universitatis Agriculturae Sueciae
2009, number: 2009:20
ISBN: 9789186195670
Publisher: Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences

Authors' information

Herthnek, David
Swedish University of Agricultural Sciences, Department of Biomedical Science and Veterinary Public Health

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