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Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis

Englund, Stina (2002). Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis. Diss. (sammanfattning/summary) Uppsala : Sveriges lantbruksuniv., Acta Universitatis agriculturae Sueciae. Veterinaria, 1401-6257 ; 123
ISBN 91-576-6366-1
[Doctoral thesis]

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Abstract

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of paratuberculosis, also known as Johne’s disease, a chronic intestinal infection in cattle and other ruminants. Paratuberculosis is characterised by diarrhea and weight loss that occurs after a period of a few months up to several years without any clinical signs. The considerable economic losses to dairy and beef cattle producers are caused by reduced milk production and poor reproduction performance in subclinically infected animals. Early diagnosis of infected cattle is essential to prevent the spread of the disease. Efforts have been made to eradicate paratuberculosis by using a detection and cull strategy, but eradication is hampered by the lack of suitable and sensitive diagnostic methods. This thesis, based on five scientific investigations, describes the development of different DNA amplification strategies for detection and characterisation of M. paratuberculosis. Various ways to pre-treat bacterial cultures, tissue specimens and fecal samples prior to PCR analysis were investigated. Internal positive PCR control molecules were developed and used in PCR analyses to improve the reliability and to facilitate the interpretation of the results. The sensitivity of the ultimate methods was found to be approximate that of culture and allowed detection of low numbers of M. paratuberculosis expected to be found in subclinically infected animals. Genomic DNA of a Swedish mycobacterial isolate, incorrectly identified by PCR as M. paratuberculosis was characterised. The isolate was closely related to M. cookii and harboured one copy of a DNA segment with 94% similarity to IS900, the target sequence used in diagnostic PCR for detection of M. paratuberculosis. This finding highlighted the urgency of developing or evaluating PCR systems based on genes other than IS900. A PCR-based fingerprinting method using primers targeting the enterobacterial intergenic consensus sequence (ERIC) and the IS900 sequence was developed and successfully used to distinguish M. paratuberculosis from closely related mycobacteria, including the above mentioned mycobacterial isolate. In conclusion, the molecular biology techniques developed in these studies have proved useful for accelerating the diagnostic detection and characterisation of M. paratuberculosis.

Authors/Creators:Englund, Stina
Title:Molecular biology techniques as a tool for detection and characterisation of Mycobacterium avium subsp. paratuberculosis
Year of publishing :May 2002
Volume:123
Number of Pages:63
Papers/manuscripts:
NumberReferences
ALLI. Englund, S., Ballagi-Pordány, A., Bölske, G. and Johansson, K.-E. 1999. Single PCR and nested PCR with a mimic molecule for detection of Mycobacterium avium subsp. paratuberculosis. Diagnostic Microbiology and Infectious Disease 33: 163-171. II. Englund, S., Bölske, G., Ballagi-Pordány, A. and Johansson, K.-E. 2001. Detection of Mycobacterium avium subsp. paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene. Veterinary Microbiology 81: 257-271. III. Halldorsdottir, S., Englund, S., Fredsvold Nilsen, S. and Olsaker, I. 2002. Detection of Mycobacterium avium subsp. paratuberculosis by buoyant density centrifugation, sequence capture PCR and dot-blot hybridisation. Vet. Microbiol. 87:327-340 IV. Englund, S., Bölske, G. and Johansson, K.-E. 2002. An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis. FEMS Microbiology Letters 209: 267-271. V. Englund, S. IS900/ERIC-PCR as a tool for distinguishing M. avium subsp. paratuberculosis from closely related mycobacteria. (Submitted).
Place of Publication:Uppsala
ISBN for printed version:91-576-6366-1
ISSN:1401-6257
Language:English
Publication Type:Doctoral thesis
Full Text Status:Public
Agris subject categories.:L Animal production > L73 Animal diseases
Subjects:Not in use, please see Agris categories
Agrovoc terms:cattle, paratuberculosis, animal diseases, mycobacterium avium, bacterioses, diagnosis, molecular biology, sweden
Keywords:cattle, mycobacteria, microbiology, PCR, Johne's disease, paratuberculosis
ID Code:272
Department:(VH) > Institutionen för veterinärmedicinsk mikrobiologi
Deposited By: Stina Englund
Deposited On:05 May 2003 00:00
Metadata Last Modified:02 Dec 2014 10:03

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