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Methods for the Detection of Mycobacterium Bovis within edible tissues fromTB -suspect Cattle following slaughter and salvage
Project Code: M01022
Health and Safety Lab (HSL)
This project was performed in order to investigate the extent of carcass infection by Mycobacterium bovis within TB-infected cattle. All animals used in the study were confirmed as bovine tuberculosis (TB) ‘reactor’ animals, i.e. the animals had been immunologically assessed by intradermal tuberculin test and confirmed as exposure-positive for the causative organism. The cattle used for the work were sourced from Over Thirty Months Scheme (OTMS) slaughterhouses in order to simplify accessibility to required tissues. The majority of the animals were dairy cattle of >30 months. Project objectives were as follows:
- To produce a protocol, approved by FSA and DEFRA, to achieve carcass sampling and microbiological analysis for 150 TB reactor cattle.
- To identify reactor herds within areas of the UK commonly associated with bovine TB. Close liaison with DEFRA regional personnel was undertaken to achieve this.
- To review scientific data from peer reviewed and Government publications, and produce a written report to include current opinion on probable routes of transmission and infectious dose for M. bovis acquisition by humans. This information assisted an informed choice to be made regarding the cattle tissues that would be used for subsequent microbiological analysis.
- To identify abattoirs involved in reactor slaughter, which were willing to allow on-site sampling; to then undertake site visits to obtain carcass samples for microbiological testing.
- To perform analyses to detect M. bovis and, where possible, identify different types of tissue regions affected by this bacterium for all sampled carcasses.
- To perform an agreed extension to the work to further evaluate primer specificity of the hsp65 PCR approach, to include Mycobacterium avium and Mycobacterium avium, subsp. paratuberculosis type strains. Additional PCR primers, specific for RD4 and IS1081 M. bovis gene targets, to be compared directly with the hsp65 PCR using archived material from main study.
FINDINGS - MAIN STUDY
Tissue material was successfully obtained from 153 cattle carcasses by HSL and Silsoe staff, then analysed by HSL using both culture and DNA-based methods. HSL culture-based results were obtained for all 153 carcasses. Of the 153, HSL has found that 34 (22.2%) gave culture-positive results for M.bovis in pooled Head and Respiratory Tract (HART) carcass tissues, and 7 (4.5%) for pooled Carcass and Edible Offal (CEO) tissues (see Introduction for composition). Four animals (2.6%) generated culture-based positive results for both HART and CEO pooled tissues. Of the 153 reactor animals sampled, 110 (71.9%) had no visible lesions (NVL) and 43 (28.1%) had visible lesions (VL); 25 of these had one lesion; 14 had 2 lesions; 4 had 3 lesions. PCR and DNA sequencing was used to confirmed the identity putative M. bovis colonies. Some HART tissues collected by HSL were also required by DEFRA for routine TB testing. Data for VLA’s culture analysis were made available for comparison with HSL’s culture data. Of the 153 carcasses sampled by HSL, HART tissue samples for 100 animals were also required by DEFRA. Of these 100 samples analysed jointly, VLA provided HSL with results for 96 samples, and of these, 10 (10.4%) were confirmed culture positive by VLA and a further 19 (19.8%) were classified positive because tissues were VL (29 in total – 30.2%). The remainder were culture negative. Of the 29 samples classified positive by VLA, 14 (48.3%) corresponded to positive results from HSL’s parallel HART tissue analyses. Fifteen VLA culture-positives corresponded to negative results from HSL’s culture work, although some of these samples had generated putative mycobacterial growth at HSL; these were later found to be M. bovis negative following colony PCR. Four positive result obtained by HSL were not detected by VLA. The remaining samples analysed by HSL, which generated culture positives from HART tissues, were not required by VLA. Of the 153 sets of pooled HART samples tested by HSL’s PCR method, 130 (84.9%) generated PCR positive results following hemi-nested PCR that targeted the mycobacterial hsp65 gene. Of the 153 sets of pooled CEO samples analysed, 123 (80.4%) gave a positive PCR result. DNA sequencing confirmed the identity of amplified products from PCR as M. bovis. Forty percent of PCR products obtained from culture-independent analysis were sequenced in this way to definitively confirm that PCR products represented members of the Mycobacterium tuberculosis complex. One hundred and six of the PCR-positive results were common for respective pooled HART and CEO tissues of the same carcasses. Analysis of an agreed 65 (42%) of the CEO carcass tissues - performed to resolve the region(s) of most commonly infected by M.bovis - applied PCR-based methods, because re-culturing of these individual samples after the initial pooled culture was not possible within the time course of the project. From these PCR data, at least one or more CEO tissue types produced a positive result in all cases. However, 10 of the 65 had previously failed to generate a PCR positive for pooled CEO tissue analysis. Possible reasons for this are discussed later in this document. Environmental samples were also obtained from MHS and abattoir staff and were analysed using PCR only. Knives used for lung and retropharyngeal dissection were both negative for M.bovis following cleaning in the knife bath, though a deep water sample from the knife bath itself was positive. The upper surface water sample from the bath was negative. The apron of the MHS inspector examining lungs was negative when soiled, but was found to be positive after jet washing and re-swabbing. Other samples were positive, including those from gutting staff apron; clean and dirty, and soiled knives used by the 2 other MHS staff. From the data it is evident that CEO, as well as HART carcass tissues contained M. bovis bacteria that are detectable using culture and PCR-based approaches. The incidence of detection differed considerably between the two approaches, although there was an overlap between culture and PCR-based positive data. Specifically, the hemi-nested PCR screening approach has indicated that M.bovis DNA was present in between 80 and 84% of all carcasses analysed, with close to 21% positive by culture-based analysis. Every carcass that produced a culture positive result at HSL also produced a PCR positive, though there were many PCR positive results that failed to generate cultivable M.bovis. Forty-three (28.1%) of the 153 samples were obtained from VL animals, and 41 of these carcasses produced a PCR positive result within one or more tissue regions. However, the majority of carcasses sampled were NVL, yet only 5 from 153 sets of samples failed to produce a PCR-positive result of any kind. The wider extent of such infections may therefore be difficult to monitor by visual analysis alone, and unless obvious tissue pathology is noted from these CEO (non-thoracic) carcass regions, samples from those regions are sent infrequently to VLA for further analysis. Of the VL category, the majority suffered only 1 lesion and may have passed MHS inspection if otherwise healthy. All evidence from culture and PCR-based tests suggests that M.bovis is widely detectable within the carcasses examined, from both VL and NVL animals.
FINDINGS – PCR METHODS COMPARISON
Optimisation of the IS1081 and RD4 PCR primers was achieved successfully. These two PCR
primer sets, and the hsp65 primer set developed at HSL, were evaluated against a series of control
DNA samples to establish their levels of specificity for Mycobacterium bovis. Control DNAs included Mycobacterium avium, Mycobacterium paratuberculosis, Mycobacterium bovis strains
and Mycobacterium microti. M. microti is a member of the M. tuberculosis complex. Both RD4
and IS1081 primer sets were found to have a higher degree of specificity with control DNAs than
the hsp65 primer set. IS1081 primers successfully amplified M. bovis 10772, M. bovis BCG, a
confirmed M. bovis isolate and Mycobacterium microti. IS1081 primers did not to amplify either
M. paratuberculosis or M. avium DNA. RD4 primers successfully amplified M. bovis 10772 and
M. bovis positive isolate DNA, but not M. microti or M. bovis BCG. RD4 primers also failed to
amplify M. paratuberculosis or M. avium DNA. Although the primer set for the hsp65 target is
known to have a perfect match only for M. tuberculosis complex members (M. bovis; M.
tuberculosis; M. microti; M. africanum), this evaluation found that both M. paratuberculosis and
M. avium generated PCR products from this primer set. The hsp65 primer set was capable of
successfully amplifying M. bovis 10772, M. bovis BCG and the positive M. bovis isolate DNA. It
also – as was expected – amplified M. microti DNA. Later amplification incorporating the newly
designed hsp65 reverse primer (but retaining the original forward primer) demonstrated much
improved specificity; with an amplification profile showing the same fidelity as that observed for
the IS1081 primer set; both M. paratuberculosis and M. avium were excluded.
PCR was completed using RD4, IS1081 and both original and modified hsp65 primer sets, using
non-nested PCR in all cases. Results for the IS1081 target gave positive results for 50% of HART and 43.5% of CEO samples. This demonstrated that the archived DNA template from bovine reactor tissues was of sufficient quality to allow non-nested PCR analysis. Results for RD4 amplifications were less successful, and despite excellent results from primer specificity tests and from reaction controls, none of the test DNA samples produced PCR products for HART or CEO DNA extracts. The original hsp65 PCR successfully amplified mycobacterial DNA from 11 (11.9%) HART and 13 (14%) CEO pooled sample extracts. The amended hsp65 primers produced positive results from 45 (49%) HART and 12 (13%) CEO pooled sample extracts. Nineteen (42%) of the 45 positive HART analyses detected with the amended hsp65 primers were commonly positive with IS1081. Four (33.3%) of samples were commonly positive between these primer sets for the CEO sample analyses. The failure of the RD4 primers appears unrelated to DNA template quality in view of the successful amplifications from other primer sets.
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