Home About Browse Search

Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis

more than a gut feeling

Herthnek, David (2009). Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis Agriculturae Sueciae, 1652-6880 ; 2009:20
ISBN 978-91-86195-67-0
[Doctoral thesis]

[img] PDF


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.

Authors/Creators:Herthnek, David
Title:Molecular diagnostic methods for Mycobacterium avium subsp. paratuberculosis
Subtitle:more than a gut feeling
Series Name/Journal:Acta Universitatis Agriculturae Sueciae
Year of publishing :2009
Number of Pages:68
ALLI. Herthnek, D., Englund, S., Willemsen, P.T.J. & Bölske, G. (2006). Sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR. J Appl Microbiol 100, 1095-102. II. Herthnek, D. & Bölske, G. (2006). New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis. BMC Microbiol 6, 87 III. Herthnek, D., Nielsen, S.S., Lindberg, A., Bölske, G. (2008). A robust method for bacterial lysis and DNA purification to be used with real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in milk. J Microbiol Methods 75, 335-40 IV. Salgado, M., Herthnek, D. Bölske, G., Leiva, S., Kruze, J. (2009). First isolation of Mycobacterium avium subsp. paratuberculosis from wild guanacos (Lama guanicoe) in Tierra del Fuego island (Accepted for publication in Journal of Wildlife Diseases) V. Herthnek, D. & Bölske, G. Validation of a new detection method for Mycobacterium avium subsp. paratuberculosis in faeces by real-time PCR (manuscript)
Place of Publication:Uppsala
ISBN for printed version:978-91-86195-67-0
Publication Type:Doctoral thesis
Full Text Status:Public
Agrovoc terms:cattle, mycobacterium avium subsp paratuberculosis, paratuberculosis, diagnosis, pcr, microbiology
Keywords:cattle, confirmation, detection, diagnosis, F57, IS900, Johne’s disease, microbiology, Mycobacterium avium subsp. paratuberculosis, real-time PCR
Permanent URL:
ID Code:1954
Department:(VH) > Dept. of Biomedical Sciences and Veterinary Public Health
Deposited By: David Herthnek
Deposited On:09 Mar 2009 00:00
Metadata Last Modified:02 Dec 2014 10:15

Repository Staff Only: item control page


Downloads per year (since September 2012)

View more statistics