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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code SE3008

2. Project title

Detection and enumeration of Mycobacterium bovis from clinical and environment samples

3. Contractororganisation(s)

Veterinary Laboratories Agency                         

54. Total Defra project costs £ 548,808

5. Project: start date................ 01 April 1999

end date................. 31 December 2004

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

The overall aim of this project was to develop methods for the detection and enumeration of Mycobacterium bovis in clinical samples, such as cattle tissues, nasal mucus, blood, badger excreta and environmental samples. The development of such assays would allow rapid screening of samples from infected cattle and monitoring of the environment and badger populations for the presence of M. bovis. PCR development was performed by Dr G M Taylor, Imperial College, London.

Work undertaken as part of this project has resulted in the development of a number of direct biomolecular methods for detection, quantitation and genotyping of Mycobacterium bovis strains sampled from cattle, badgers and small mammals. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle a variety of tissue samples and biological fluids. DNA extraction with the NucliSens isolation kit (bioMérieux) has been shown to fulfil nearly all of these criteria. A bonus is that mycobacteria are immediately attenuated so minimising the risk to laboratory personnel and the requirement for category three containment. NucliSens extraction has been coupled with a sensitive PCR screening method for the Mycobacterium tuberculosis complex (IS1081, 6 copies per genome, detection limit < 1 genome copy). A series of genotyping PCRs for specific deletion events or regions of difference (RDs) within the M. tuberculosis complex have also been developed. These PCRs identify the occurrence of RDs 4, 7, 8, 9, 10, 12 and 13 (Brosch et al, 2001). The sensitivity and specificity of these PCRs have been tested on reference strains, spiked samples and on clinical and environmental samples collected in the field. Additional methods for confirmation of species include the oxyR285, and pncA157 polymorphic loci. The detection limits for all these single copy methods is 10 genome copies. They can be used as confirmatory assays for classical M. bovis and variants (e.g. M. bovis caprae) and for distinguishing other members of the complex (M. africanum, M. microti). All the above methods can be readily adapted to quantitative PCR methods with the use of SYBR Green interchelating dye on the RotorGene 3000 platform (Corbett Research).

Testing of clinical samples (bovine lymph nodes, n = 109) highlighted two shortfalls of the molecular approach. These are 1. Comparison of IS1081 PCR with the “gold standard” of culture initially showed a sensitivity of 70%. When RD4 or RD7 PCRs were compared with culture, the

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detection rate dropped to 50%. Secondly, the success rate of spoligotyping applied directly to the same extracts was around 50% compared with cultures. Recent work indicates that this deficit was due to delays in processing of archival tissues as well as with difficulty with lysis of the tough mycobacterial cell wall (Takade et al, 2003), rather than to inhibition of PCR or efficiency of DNA recovery itself. Either of these factors may restrict application of other more discriminant typing methods e.g. VNTR. In the closing months of SE3008 a means of increasing the efficiency of mycobacterial lysis was devised and tested using a further pool of 95 cattle lymph nodes. After modification of the extraction protocol, detection rate of the IS1081 and RD4 methods increased to 90% and 58% respectively. The IS1081 PCR is therefore a realistic screening method for rapid identification of positive cases and warrants further development and evaluation. Single-tube nested PCR offers a means of increasing sensitivity of the RD methods. Direct molecular typing from VL’s would greatly facilitate contact tracing. Techniques for this approach have also been developed as part of SE 3008 but further optimisation is required to improve sensitivity above the current level of 50% from lesioned tissue. Three automated or semi-automated DNA extraction systems were evaluated; the NucliSens Extractor and Minimag (both from bioMerieux) and the Qiagen EZ1 Robot and the potential for future automation are discussed.

In addition, a mycobactreiophage-based detection system (PhaB) was optimised for use in badger clinical samples by colleagues at the Public Health Laboratory Service Mycobacterium Reference Unit. This assay met with limited success at detecting M. bovis from clinical samples from M. bovis-infected badgers. Moreover some problems with specificity of the assay were encountered due to the ability of the mycobacteriophage used in this assay (D29) to infect mycobacteria other than members of the Mycobacterium tuberculosis complex.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Background and Overview.

Understanding the cattle-to-cattle spread of tuberculosis and the part other wildlife vectors play in the transmission of Mycobacterium bovis are important issues in controlling dissemination of this disease. Rapid molecular diagnostic methods are recognised as having an important role to play in the development of an effective strategy to combat herd breakdown by identifying wildlife reservoirs infected with M. bovis. PCR-based techniques have the potential to rapidly identify infected individuals and environmental contamination so that risk assessments can be made for spread of M. bovis to cattle. Therefore the Krebs Review recommended the development of a Mycobacterium bovis specific PCR-based assay for the detection of bacteria in badger carcasses, excreta and environmental samples. The development of such an assay would allow rapid screening of samples from badger carcasses and monitoring of the environment and badger populations for the presence of M. bovis.

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The objective of this proposal was to develop PCR-based methods which would allow detection and genotyping of M. bovis from badger carcasses, clinical samples and excreta. The development of a range of molecular tools for screening and typing of M. bovis also has applications in the rapid confirmation of infection from clinical samples submitted from reactor cattle where PCR has the potential to reduce confirmation times for visibly lesioned animals from 6 weeks to 2 - 3 days. Sensitive detection tools would also facilitate investigations of bovine pathogenesis, i.e. the detection of the kinetics of nasal shedding of M. bovis and its role in cattle-to cattle transmission.

It is envisaged that such tests could be used to monitor local levels of infection and environmental contamination. The ability to detect and fingerprint M. bovis directly from e.g. badger excreta and environmental samples by PCR would allow accurate epidemiological surveillance and reduce the need for bacterial culture. Such a test, or tests, if sufficiently sensitive, would also be of value for rapid screening of samples from badger carcasses and clinical samples taken from reactor animals. The detection and fingerprinting of M. bovis from badger carcasses would be particularly useful for DEFRA to maximise epidemiological information from the randomized badger culling trial (RBCT).

PCR is a very sensitive technique that allows detection of DNA from M. tuberculosis complex organisms when applied to bacterial cultures and sputum samples. However, similar levels of detection are not observed for all clinical samples. This is due to the presence of inhibitors to the PCR reaction. The primary aim of this proposal was to develop suitable extraction techniques to remove such inhibitors from badger tissue, urine, faeces and from soil so that PCR may be used to detect and fingerprint M. bovis present in these samples.

Therefore, in preliminary experiments carried out for this project, a number of DNA extraction and enrichment techniques have been evaluated for the purification of M. bovis DNA from contaminating materials present in the clinical samples which inhibit the PCR assay. A two-stage approach to M. bovis detection has been applied to these enriched samples. A number of potential PCR targets have been considered and tested using spiked samples in the laboratory and in field samples collected from badger excreta, live trapping of known excretors of M. bovis, monitoring of cattle nasal aspirates from experimentally infected animals and in cattle lymph nodes with visible lesions highly suggestive of M. bovis infection. These will be detailed in the report below.

In the first stage of this process, a low cost sensitive screening test has been developed which is specific for the M. tuberculosis complex. In the second stage we have developed a number of M. bovis specific PCR assays for confirmation of species and genotyping. We have investigated the feasibility of applying typing methods, such as spoligotyping and VNTR analysis, directly to clinical samples without the need to first culture these.

Development of DNA extraction techniques (Objectives 01-02).

In initial experiments a number of possible DNA recovery methods were assessed. These included:

1. The processing and digestion of tissue homogenates with sodium dodecyl sulphate (SDS) followed by removal of proteins and contaminants using hexadecyl trimethyl ammonium bromide (CTAB) and 5M NaCl.

2. A variant of method 1 in which lysozyme and proteinase K are used to digest tissues followed by 10% CTAB and removal of proteins with phenol: chloroform-isoamyl alcohol (25:24:1.). This is followed with isopropanol precipitation of DNA and washing of the pellet with 70% ethanol. This method was extensively used in early experiments and in preparation of reference strains used for positive controls and for real-time quantitative PCR (RT-PCR) standards and so is detailed in Appendix A.

(3) The use of zirconium beads and beating in the presence of phenol-chloroform-isoamyl alcohol with subsequent enrichment using DNA sequence capture to reduce inhibition by excess of host DNA.

(4) The use of Chelex 100 resin (BioRad) (IC).

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(5) The use of immunomagnetic separation using specific monoclonal antibodies linked to magnetic beads. This strategy has been used to isolate mycobacteria from faecal samples and has been successfully applied at VLA Weybridge to detect M. bovis in spiked badger faecal samples using a monoclonal antibody against the surface antigen MPB83.

(6) The use of Nuclisens guanidinium/silica kit for detection of M. tuberculosis from bone material and a variety of clinical samples (IC). This is a modification of the Boom method (Boom et al, 1990 and 1999). (The final NucliSens protocols arrived at for biological fluids and tissues are given in Appendices H and I respectively).

Comparison of methods

Six samples of badger faecal material, each approximately 400 mgs in weight, were spiked with purified M. bovis DNA from reference strain AF 2122/97. Duplicate samples were extracted with methods 2, 3 and 6. The recovery of DNA and presence of PCR inhibitors was assessed using a cyt b PCR specific for Meles meles DNA and the recovery of M. bovis DNA was checked using a PCR for contig. 816. The primers for this flanked a 12.7 Kb deletion in strains of M. bovis. This is now recognised as region of difference 7 (RD7) following comparative genomics with H37Rv.

Results. PCR inhibition was noted in all samples; this was successfully overcome by dilution (1/10) of extracts and addition of extra Taq polymerase (2.5 U cf. 0.5 U)Both badger DNA and M. bovis DNA were amplified and the two methods could be multiplexed. (Figure 1). From other pilot experiments (not shown) and a larger study of 50 samples spikes at VLA, it was soon apparent that the NucliSens kit was a far more realistic and convenient means of DNA extraction from difficult material such as faeces. Method 2 was more expensive, labour intensive and not considered to be suitable for transfer to a routine laboratory setting. The Chelex 100 resin extraction was effective in these pilot recovery experiments but DNA quality has been shown by others to be poor for downstream applications like short numbers of tandem repeat (sntr) typing, which was an issue here if the technique was to be used for VNTR typing (Hoff-Olsen et al, 1999). The NucliSens kit provides options for small (2ml) and large (9ml) sample tubes, and can be used with pellet-pestles (Anachem) for macerating tissues. This is essential for faeces and lymph node samples. Extra wash steps can be incorporated for dealing with co-purified polymerase inhibitors and the convenience of the kits modular form assists in minimising chances for cross-contamination. Additionally, the 5M GUSCN lysis buffer can deal with a range of diverse samples and attenuates M. bovis, reducing the infection risk for front-line and laboratory personnel. This was subsequently shown in a collaborative experiment with VLA Weybridge (Appendix B).

PCR methods and validation. (Objectives 01 - 03, 06, 08).

Milestone 08 / 01. An examination of M. bovis strains for deletion regions subsets likely to improve specificity of detection. The material used was genomic DNA prepared from M. bovis cultures: the aims were 1). To ensure detection of the greatest possible number of M. bovis isolates and 2). To compare deletion region polymorphisms with conventional genotyping methods. The material examined was DNA prepared from VLA culture banks and included the most common spoligotypes.

At the onset of the proposal it was envisaged that PCR targets for M. bovis would include IS6110 for rapid screening followed by an M. bovis - specific PCR using primers JB21 and 22. Rodriguez et al, (1995). This region is now known as RvD1, due to its deletion from H37Rv and other strains of M. tuberculosis. However, it soon became clear that this method detected some strains of M. tuberculosis in which RvD1 had not been deleted, including the sequenced strain CDC1551 (Taylor, unpublished observations; Metaxa-Mariatou et al, 2004) and also M. africanum strains from West Africa (Brosch et al, 2001). Therefore, an alternative strategy was adopted in which primers were designed to a region flanking a 12.7 Kb deletion from strains of M. bovis (Zumarraga et al, 1999). This approach was chosen to provide specificity for M. bovis in problematical samples collected from a variety of clinical sources and the environment. This deletion is now known as RD7 and will be referred to as such in the report. At this time the universality of this deletion was not known. Therefore, the specificity of this method was

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assessed using a number of reference and clinical samples held at Imperial College, VLA Weybridge and with collaborators at CSL and Oxford University. The method was shown to detect M. bovis strain AF2122/97, M. bovis BCG Pasteur, six animal isolates (including spoligotypes 9, 22 and 46) one strain of M. microti (spoligotype 32), and five clinical isolates of. M. bovis cultured from older patients with reactivation of tuberculosis acquired in early life (Sales et al, 2001 – publication attached).

Further checks on specificity were undertaken on VLA isolates. The RD7 method detected all M. bovis isolates in a 10 x 10 panel of common spoligotypes, 30 clinical isolates of M. microti from diverse animal species (spoligotypes 18, 28, 32 & 34), three reference strains of M. microti (NCTC NC08712-04, NC8710-04 and NC8337-01) and M. bovis from a fur seal, now recognised as a distinct member of the complex, M. pinnipedii (Cousins et al, 2003). Figure 2 shows multiplexing of RDs 4, 7 and 9 applied to the panel of common spoligotypes present in the UK. All RD methods were found to provide specific methods for this panel of M. bovis spoligotypes. No PCR product was detected in CDC 1551, H37Rv, M. canetti (Somalia), M. paratuberculosis (NCTC 8578), M. xenopi (NCTC 10042), (M. gordonae (NCTC 10267), M. fortuitum (NCTC 10394), M. intracellulare (NCTC 13950), M. marinum (NCTC2275) and M. avium (NCTC 8559). To further validate the PCR and extraction, spiked samples of badger faeces were prepared at VLA Weybridge and sent to Imperial College for assay. The RD7 method correctly identified 48/50 of these isolates of the M. tuberculosis complex (Appendix C). The two discrepant results were caused by positives in samples spiked with H37Rv and strongly suggest an M. bovis contaminant somewhere in the system. The samples spiked with water were all correctly negative.

Completion of the M. bovis genome sequence (SE3206) revealed that, based on current knowledge of the M. tuberculosis complex, the RD7 method would be expected to detect all M. bovis isolates (including those from exotic species of animals), all M. microti strains and a sub-group of M. africanum strains from West Africa (Brosch et al, 2002). Therefore, a PCR was developed for identification of “classical” M. bovis; this method used primers flanking the RD4 region of difference. This was followed by methods using flanking primers for all the key RD regions outlined in the Brosch paper as well as methods for the oxyR285 and pncA169 polymorphisms for species confirmation, flanking and internal primers for the TBD1 deletion event and a theortically more sensitive screening method, for insertion sequence IS1081, of which there are six copies in most strains of members of the M. tuberculosis complex compared to one copy of IS6110 (Dziadek et al, 2001). In an analogous way to the badger (Meles meles) housekeeper method for cyt b, a PCR was developed for verifying extraction of cattle DNA. The primer sequences, cycling conditions and amplicon sizes for all these methods are shown in Appendix D. Annealing temperatures were confirmed using a gradient block on a PCR Express thermal cycler (Hybaid Ltd). Magnesium optima were determined using the RotorGene 2000 or 3000 real-time PCR platforms (Corbett Research). This machine was evaluated as part of project SE3015 and conditions for quantitative PCR developed for M. bovis DNA using the SYBR Green minor groove binding dye. The original machine (RG 2000) was transferred to VLA Weybridge and upgraded to the 3000 model in 2002. Appendix E gives details of typical PCR conditions and reagents and a protocol for quantitative PCR, which can be applied to any individual method.

Minimum detection limits.

The minimum detection limits for the RD7, RD4 and the IS1081 methods were compared by testing serial dilutions of an M. bovis standard diluted in TE buffer, pH 7.0. The results are shown in Table 1 below. As expected, the IS1081 method was the most sensitive, detecting less than I genome equivalent.

Table 1.

PCR method Minimum detection fg Genome copiesRD7 47 10RD4 23.5 5

IS1081 2.35 0.5

Validation of the RD4 method.

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Testing against reference strains of M. bovis 2122/97, M. bovis BCG Pasteur and 10 M. bovis strains with the 10 most commonly encountered spoligotypes in GB confirmed that this method did detect M. bovis DNA (Figure 2). The M. tuberculosis strains H37Rv and CDC 1551 were not detected nor were any of the M. microti lineages used to validate the RD7 method. The original 50 spiked samples prepared at VLA were also assayed using the RD4 method. The pattern was identical to the RD7 results (see Appendix C). This again raised the possibility that contamination of the same two H37Rv spiked samples had occurred as these were positive by this second method as were some of the microti spiked samples.

Milestone 07 / 01.Testing of clinical and field samples by conventional PCR (c PCR). Testing of clinical and field samples.

1.The first samples tested with the new PCR methods were 42 miscellaneous samples collected from badgers at Woodchester Park, Nympsfield. Of these, 16 were tracheal aspirates, 15 were urines, 4 were faeces and 7 were irrigations of bite wounds from a variety of anatomical sites. The samples were centrifuged and both pellets and supernatants assayed for badger and Mycobacterium bovis DNA (RD7). Badger DNA failed to amplify from a number of cases and there were no unequivocal positives for M. bovis (Appendix F). It was later learned that all these samples had been treated with oxalic acid to decontaminate them prior to culture and this probably explains the failure to detect any positives since oxalic acid may degrade DNA. With hindsight, relative insensitivity of the RD7 assay, limited extraction efficiency and co-purification of PCR inhibitors in faecal samples may also have contributed to these findings.

Collection of field samples was prevented in 2001 due to the UK outbreak of foot-and mouth virus which began in late February and remained a problem until the latter part of the year.

2. In 2002, samples of badger faeces were collected from the study site of Woodchester Park, Gloucester in collaboration with Drs. Gavin Wilson and Richard Delahay, CSL, Nympsfield, Gloucestershire. A total of 50 faecal samples were collected. Of these 30 were known “fresh” overnight samples, collected after dusting latrine sites with talcum the night before. Ten more were described as “recent looking”, deposited within previous 2 days. The last 10 samples were “old looking” apparently deposited several days previously. The 50 samples were collected from 14 latrine sites in use by 4 social groups of badgers known to be infected with M. bovis. The sites selected were scattered throughout the study site.The DNA extraction method was followed and faecal extracts were tested for badger DNA (cyct b PCR) and for M. bovis DNA using the RD4 assay multiplexed. Although badger DNA was recovered in 40/50 extracts, none were positive for M. bovis (Figure 3). In case this was due to sensitivity or fragment size of the persisting DNA, extracts were re-examined using IS1081 PCR. Again, no positives were seen. This showed the feasibility of recovering DNA from difficult environmental samples and of overcoming PCR inhibitors. It is concluded this was a true negative finding.

3. CSL also provided faecal samples collected from live trapping of badgers. These 4 animals were previous known excretors of M. bovis, determined by culture. Badger DNA was recovered from 3/4 samples, the fourth case (H43) was assayed at several dilutions but PCR inhibition remained problematical, probably due to the large size of the specimen provided. Two of the 4 badger faecal samples were clearly positive for M. bovis DNA using the IS1081 PCR. Figure 4 shows results from one of these, badger X49. It was found that a range of extract dilutions is necessary to overcome inhibition, particularly in large or recent faecal samples but that mycobacterial DNA can be successfully detected. The results of this small study are summarised in Table 2.

Table 2.

Badger Cyt b PCR IS1081 PCR CultureQ39 + + +H43 - - +X49 + + +X35 + - +

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4. Blood.The feasibility of testing animal blood for Mycobacterium bovis was also examined. Badger blood was collected from 3 animals at VLA Weybridge. Each sample was divided into two aliquots of 1ml. One of each of these was spiked with 10, 20 or 30 l of M. bovis DNA AF2122/97. DNA was extracted from all samples using the NucliSens kit and assayed for amplification of the housekeeper gene (Meles meles cyt b) and M. bovis recovery by real-time PCR using IS1081 for quantitation of the recovered DNA against a set of standards. This showed that a mean of 89% of added DNA was recovered and samples could be correctly ranked in terms of DNA spiking. However, additional steps were necessary at the GUSCN wash buffer, 70% ethanol and acetone wash stages to remove haemolysed components of the red blood cells from the silica.These studies were continued with blood samples from experimentally infected badgers kindly made available by Dr. Eamon Gormley, Division of Clinical Veterinary Sciences, University College Dublin.

Table 3 - Dublin Badgers.

Sample No. Badger ID Infection Cyt b PCR IS1081 PCR1 61 Yes + -2 76 Yes + -3 69 yes + -4 63 No + -5 64 No + -6 1 No + -7 72 No + -

Problems were again encountered with haemolysed blood components, requiring extra silica wash steps and additional Taq polymerase in the PCR reaction. As no evidence of circulating M. bovis DNA was found, this suggests blood assays may not be a productive avenue of future research for molecular detection or genotyping.

5. Cattle Nasal Aspirates.

Cattle nasal aspirates from calves experimentally infected by intratracheal administration of 4 x 105 M. bovis organisms were provided by Dr. Adam Whelan, VLA, Weybridge. After centrifugation, the supernatants (250l) from about every 2nd sample in the time course were assayed for cattle DNA and for M. bovis using the RD7 assay as a multiplex. No M. bovis positive samples were found in aspirates from animals 2104, 2183 or 2157 although all but 2 samples were positive for cyt b DNA.

Calf 2327 did have two samples positive for M. bovis when the IS1081 PCR was used; both were also culture positive. These results are listed in Table 4. The episodic nature of shedding was reflected in both the molecular and conventional analyses.

Table 4 –calf 2327 timepoints

Sample Sample date

Cyt b PCR M. bovis PCRIS1081

Culture(CFU/sample)

1 4/9/ 01 + - -/c2 18/9/01 + - -/c3 1/10/01 + + +(80)4 15/10/01 + - + (212)5 22/10/01 + - -/c6 25/10/01 + + + (312)7 12/11/01 + - + (24)8 15/11/01 + - -/c9 19/11/01 + - + (4)10 13/12/01 - - + (12)11 17/12/01 + - -/c

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12 27/12/01 + - -/c13 10/01/02 - - + (8)14 18/2/02 + - -/c

-/c = negative by culture.

6. Recovery of Mycobacterial DNA from fixed archival blocks.

Following several requests from VLA for a method for DNA extraction from paraffin-embedded fixed sections, one was developed. A combination of standard proteinase K digestion and guanidine / silica extraction was found to give the most satisfactory results for PCR amplification. The final protocol is shown in Appendix G. The method detected M. bovis DNA in fixed lung sections taken from experimentally infected mice (n=4) but was not extensively applied at IC during this project but the technology was transferred to VLA for their use.

7. Cattle lymph nodes with visible lesions. Two large studies were performed using a backlog of lymph nodes with visible lesions (VLN), material which had accumulated at VLA. This had been stored for several months at –20oC. The first study took place over 2003. Study 1. One hundred and nine samples were extracted using the procedures shown in Appendices H and I. Cultures were set up and compared with DNA assayed using the cyt b (Bos taurus) method and the M. bovis PCR methods for IS1081 and RD4. In this study, 98/109 samples were found to be culture positive and grew M. bovis. Of these, 67 were positive for IS1081, a sensitivity of 68%. Figure 5 shows typical findings using the IS1081 assay. Three of the 11 culture negative samples were IS1081 PCR positive. The RD4 PCR detected 49 of the 98 culture positives (50%). The reasons for the low PCR sensitivity was investigated. A modified hemi-nested IS1081 PCR method was used to re-amplify DNA from all 109 samples. The sensitivity rose by only 5% to 73% detected, suggesting that first round PCR was efficient. Cytochrome b housekeeper gene was successfully amplified from all tissues. These observations suggested that the level of DNA recovery from mycobacteria was probably the cause for low pick-up rate. A number of experiments were performed to find the cause. Spiking of control cattle tissue with purified DNA and QPCR showed good recovery (>90%). However, when spiked with different numbers of BCG cells it transpired that DNA recovery from these, again measured by real-time PCR, was as low as 22% of the theoretical value expected from cfu determined by culture. Three modifications were tried to improve the extraction efficiency. These were bead-beating (Ribolyser, Hybaid), sonication, or addition of 3 freeze-thaw cycles of extracts in liquid nitrogen. Sonication improved recovery in some samples but resulted in PCR failures of known positives and was therefore abandoned. This may have been due to variability between tissue homogenates. Bead beating was effective but slow, more expensive and tubes were prone to leakage leading to an unacceptable risk of cross-contamination. Freeze-thawing improved recovery of added DNA by 3 -fold and could be achieved without opening the tubes.

Study 2. A formal test with this addition to the extraction procedure was begun in 2004 with assay of a further 95 lymph-node extracts from tissues with visible lesions. Of these, 86 were subsequently shown to be culture positive for M. bovis, nine were negative. Seventy-eight of the culture positives were IS1081 PCR positive (91%). Seven of the 9 culture negatives were also IS1081 PCR positive (Figure 6). The RD4 method detected 51 (59.3%) of culture positives and 4 of the culture negatives. In both studies all RD4 positives were also positive by IS1081 PCR.

The IS1081 method therefore represents a viable alternative to culture in situations where a rapid diagnosis is required or in cases where non-viable mycobacteria may be encountered.

Milestone 07 / 02. A quantitative PCR assay will be used to study the mycobacterial burden in animal tissues and environmental samples, especially those resulting from the Krebs field trial areas.

Quantitation of Visible Lesioned Lymph Node (VLN) samples.Real-time QPCR was used to measure M. bovis DNA in extracts prepared in study 1, of 109 cattle VLN samples (VLN material from badgers was not available at this stage as the samples from the RBCT were too precious to use for experimental optimisation processes). Ten VLN extracts were measured

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using the protocol shown in Appendix E. The results are shown in Table 5 below. A wide variation in mycobacterial burden was apparent. The units shown are genome equivalents / extract but values could be related to a fixed weight of tissue for prospective studies e.g. genome copies / 100 mg wet weight of tissue. In this experiment the IS1081 PCR method was used, but any of the RD and other genotyping PCRs (Appendix D) may be used with SYBR Green at a final dilution of 1/55,000. The use of IS1081 PCR for quantitation assumes that field strains of M. bovis contain the same copy number of IS1081 elements as reference strain used in preparation of the standards. This is the case for UK isolates and the greater majority of M. bovis strains likely to be encountered (Dziadek et al, 2001).

Table 5.

VLA samplesAF number

M. bovisGenome equivalents.

61/3188/03 2.54 x 106

61/3461/03 104

61/3462/03 91061/3609/03 48661/3689/03 3,50061/3692/03 1,11461/3694/03 91061/3709/03 3.606 x 105

61/3712/03 2,40561/3714/03 2.393 x 104

Spoligotyping and VNTR analysis of VLN extracts from studies 1 and 2.

Spoligotyping was undertaken at both VLA and Imperial College. Forty-seven VLN extracts from the first study of 109 cases were spoligotyped. Twenty-four of these generated recognisable patterns. In all cases these were PCR positive for both RD4 and IS1081. A further 5 produced recognisable spoligotypes but with missing spacers, probably reflecting a form of allele “drop-out”. The remaining 18 samples failed to give more than a few spacers and no type could be determined. Better results were obtained by modifying the standard spoligotyping protocol from 35 to 43 cycles of amplification, addition of extra Taq polymerase and by first finding optimal dilution of the extracts. All these findings were suggestive of poor DNA quality recovered in study 1. Spoligotyping of samples from study 2 is still ongoing at VLA.

VNTR typing. Twelve positive samples from study 2 were VNTR typed using the Rv1917c locus, (a typing system developed at Imperial College as part of SE3017). This requires PCR amplification of 4 polymorphic loci within Rv1917 (VNTR 1-4) and analysis on 3% agarose gels to find the numbers of variable repeats. VNTR 1 never amplified from any VLN extract; VNTR 4 was present in 5/12 samples and VNTRs 2 and 3 were always found. In M. bovis isolates the typical numbers of VNTRs 1 and 4 range from 8-10 copies, with PCR product sizes of 702-936 bp respectively. This suggests DNA fragment sizes were below these values. VNTR typing with this method was therefore not successful as no product longer than 700 bp could be amplified. Subsequent VNTR typing of these samples using the standard VNTR assays used at VLA (see Frothingham and Meeker-O’Connell, 1996 and Smith et al., 2003) confirmed that DNA extracted in this way was not suitable for VNTR typing.

Milestone 07 / 03. A quantitative reverse transcriptase real-time PCR method (QRT- real-time PCR) will be developed to assay cPCR positive badger field trial samples (n=50) for viable mycobacteria persisting in animal tissues.

Viability of Mycobacteria detected in archival VLN extracts.Several attempts were made to look for viable mycobacteria, as judged by the extracation of RNA from these. The NucliSens kit has been used for RNA as well as DNA extraction (bioMerieux technical literature) and should have yielded both. Reverse transcription was applied to DNAse 1–treated PCR positives from studies 1 and 2. A specific primer for reverse transcription was used ( IS1081) together with the Qiagen RN Easy RT-PCR kit, according to the Manufacturers’ instructions. All such

SID 5 (2/05) Page 11 of 29

experiments were unsuccessful. Denaturing (formaldehyde) agarose gels run with MOPS buffer showed no evidence of any RNA in the VLN homogenates.

Together with evidence from attempts at VNTR and spoligotyping, this further suggests degradation of RNA (and DNA) had occurred before transfer to the testing laboratory. The viability of mycobacteria in clinical and environmental samples remains to be established. This would require a prospective study of freshly gathered samples taken directly into a more suitable RNA buffer such as TriAzol. Long-term storage at – 20C should be avoided as should repeated freeze-thaw cycles and heat denaturation. A suitable method for rapid lysis and extraction would be bead beating in TriAzol RNase inhibiting buffer followed by immediate RT-PCR.

1. Milestone 09 / 01. Working in collaboration with VSD, Stormont it is planned to compare the requirements for sequence capture of mycobacterial DNA with silica extraction and direct cPCR developed at Imperial College. This would be studied using both “seeded” and field study samples.

2. Milestone 09 / 02. Evaluation of paramagnetic silica in the NucliSens extraction procedure using spiked samples.

3. Milestone 09 / 03. The potential for automation and scaling up the cPCR will be considered. The possibility for automation exists at the extraction stage. The manual silica extraction procedure may be automated using the NucliSens Extractor from Organon Teknika, Boxtel, The Netherlands (now bioMérieux).

Milestone 09/01. VSD Stormont switched to a standard Qiagen kit for extraction of DNA, this is generically very similar to the NucliSens kit from bioMerieux and so no comparison was made with sequence capture. However the PCR technology used at Stormont was transferred to VLA through project SE3015.

Milestones 09/02 and 09/03.

Three possible automated or semi-automted extraction systems have been evaluated as part of SE3008.

1. The NucliSens Extractor from bioMérieux. This is a large machine incorporating silica within disposable cartridges mounted within 10 workstations. The spun homogenate is added manually to the cartridges and then a series of automated wash and elution steps provide DNA in TE in a variable final volume ranging from 50 –200 ul. Ten samples can in theory be processed in about 40 minutes, realistically allowing 2-3 runs per day. An Extractor was leased from the company for a trial period with the intention of running 25 VLN samples through followed with PCR assays for IS1081, RD4 and bovine cyt b. It was soon apparent that the cartridges could not deal with supernatants from cattle lymph node extracts and the silica always blocked, almost certainly due to the lipid component of the tissues. Similar outcomes might be expected of any difficult tissue extract and therefore the Extractor cannot be recommended for applications

2. The Minimag from bioMerieux was also tried. This is a semi-automated device with 12 adjacent workstations using paragmagnetic silica to trap DNA. A moveable magnetic bar on the machine retains the washed silica whilst the liquid phase is removed by pipette, effectively replacing the need for centrifugation steps. This is a labour intensive protocol requiring many manual pipetting operations with wash buffers and finally wiith elution buffer. The operator is prompted through these steps with a built-in timer in the keypad console. As the extraction tubes are close together, there is considerable potential for cross-contamination, although this problem was not encountered with the 24 samples processed. The Minimag was able to cope with the character of lymph node extracts and PCR was successfully applied to demonstrate good recovery of cattle cytochome b DNA in all samples. However, the homogenate sample volume must be restricted to around 500 l, (cf. 9 mls for manual method) making it unlikely to detect weak positives. Again, this product is not recommended for routine processing of many samples.

SID 5 (2/05) Page 12 of 29

3. Qiagen supplied their EZ 1 Robot for trial period of 3 days. This machine processes 6 samples within a 30 minute period and can be left once set up with tips and tubes and reagents which are in sealed plastic strips. The set-up process is easily completed within 5 minutes. The EZ1 coped well with small volumes of VLN homogenates and human blood. Six whole blood samples (human) were processed and all were PCR positive for amelogenin, confirming sex of the individual. Twelve cattle VLN were also extracted and all produced cyt b PCR bands (359 bp). Eleven of these 12 were also positive for M. bovis DNA using the IS1081 method. The EZ 1 therefore has potential for automating the processing of tissue homogenates and the Qiagen silica wash reagents were compatible with the bioMerieux silica and GUSCN initial stages. It is envisaged that with modification of the front-end processing of the VLN and other tissues, samples could be extracted on the EZ 1 with an excellent chance of success. This modification should take the form of scaling down the amount of tissue currently extracted, possibly to a biopsy needle sample. This could then be extracted in 2ml of GUSCN buffer (bioMerieux or Qiagen) with maceration using disposable pestles. After centrifugation the supernatant (200 l) could be transferred directly to the EZ 1 extraction tubes. Dilution of original sample would only be 1:10, with minimal loss in sensitivity. Currently a relatively large sample is extracted and then must be optimally diluted (range 1/5 –1/40) to remove PCR inhibition. Overall verdict: The EZ 1 is worth pursuing as an automated extraction system with modified procedures discussed above.

Future Work:

1. Direct typing and PCR detection of M. bovis from Visible Lesions

Direct detection of M. bovis from visible lesions (VL) by PCR has the potential to confirm M. bovis infection within a few days compared to at least 6 weeks for culture. Assays developed as part of this project, based on the detection of IS1081 in visible lesions from cattle, showed a 73% sensitivity compared to culture and therefore warrants further development and evaluation. In addition, direct molecular typing of strains by spoligotyping and VNTR from VL’s would greatly facilitate contact tracing and outbreak control by providing the SVS with typing data in real time rather than three months after the reactor is slaughtered. Techniques for this approach have been developed as part of SE3008 however further optimisation is required to improve sensitivity above the current level of 50% from lesioned tissue.

2. A cost-benefit analysis for the incorporation of PCR-based assays into routine bovine TB testing.

SID 5 (2/05) Page 13 of 29

Milestones 05/01-05/05 (Carried out by the Public Health Laboratory Service Mycobacterium Reference Unit.)Development of the PhaB assay for badgers: The PhaB (phage amplified biologically) assay was developed by Wilson and colleagues (1997) and is a sensitive alternative diagnostic technology for detecting live mycobacteria. The whole procedure is extremely rapid and simple to perform taking as little as 48 hours compared to the 2-4 weeks of conventional liquid-based culture. The sensitivity of the assay has been calculated to be as few as 10-100 mycobacteria per milliliter of sample, which is more sensitive than microscopy and at least as sensitive as conventional culture. In the first step of the assay, a test sample is incubated with mycobacteriophage (viruses which are specific for mycobacteria) and if there are any viable M. tuberculosis-complex organisms in the sample they become infected with phage. In the next step the phage remaining outside the mycobacteria are inactivated by a specific chemical treatment whereas any infecting phage that have entered the bacteria are protected. The protected phages replicate in the host and eventually cause the mycobacteria to lyse and release this new generation of replicated phage. The released phages are then plated onto a lawn of the rapidly growing M. smegmatis in which they can also replicate. After an overnight incubation, the cycles of infection, replication, and lysis of the phage are detected as clear areas of lysis or plaques in the turbid growth of the M. smegmatis lawn. The number of plaques in the lawn is related to the number of viable mycobacteria in the original sample. Development of the assay: Assay conditions were optimized to produce a final protocol as follows: 100l of sample is incubated with 10l of CaCl2 (1mM) and 10l of mycobacteriophage D29 (109 plaque forming units, [pfu]) for 2 h at 37oC. Extracellular bacteriophage are neutralized with the addition of 12 ml of 100 mM ferrous ammonium sulphate and 100l of the samples mixed in triple vented 90mm petri dishes with 1 ml M. smegmatis (ATCC607) and 1ml oleic acid albumindextrose catalase (OADC) enrichment media and 9 ml of 7H9with 1.5% agar (w/v; at ca. 54oC). Results are read the following day and in some cases at 48 hours, and recorded as pfu/ml. Evaluation of the sensitivity of the assay: Tracheal aspirates and urine from uninfected badgers held at the Natural Environmental Center at VLA Weybridge were spiked with serial dilutions of M. bovis in 7H9/10% OADC/1mM CaCl2. 100l of each sample was processed in the PhaB assay as described above. The spiked urine samples gave a titration curve that would be expected for the assay and confirmed the sensitivity of the assay to be between 10-100 live M. bovis per milliliter of sample. The number of plaques did not decrease with increasing dilution of M. bovis for the spiked tracheal aspirate perhaps indicating contamination of the sample with environmental mycobacteria that are susceptible to D29 infection.Evaluation of the assay on clinical samples from badgers experimentally infected with M. bovis: Tracheal aspirates, bronchial-alveolar lavage (BAL) and urine samples were obtained from badgers experimentally infected via the tracheal route in the Republic of Ireland with 10, 100 or 1000 cfu M. bovis as well as from control, uninfected badgers. 100l of each sample (blinded) was processed in the PhaB assay as described above. All negative control badgers were negative at 6 weeks post infection. One control badger was positive at 9 weeks (but pfu were low). At six weeks the PhaB assay was positive for 4 tracheal aspirates from 1/3 badgers infected with 1000 cfu, 2/3 badgers infected with 100 cfu and 1/3 badgers infected with 10 cfu M. bovis. At nine weeks the PhaB assay was positive for 4 urine samples from 2/3 badgers infected with 1000 cfu, 1/3 badgers infected with 100 cfu and 1/3 badgers infected with 10 cfu of M. bovis.Specificity of the assay: One draw back of the PhaB assay is the ability of the mycobacteriophage D29 to infect a variety of mycobacterial species thus limiting its specificity for the detection of M. bovis. It may be possible to develop a modified PhaB assay that exploits the selective inhibitory effect of the compound p-nitro--acetylamino--hydroxy propiophenone (NAP) against members of the M. tuberculosis complex to differentiate between the tubercle bacillus and other mycobacterial species (Riska et al., 1999)Evaluation of the assay on clinical field samples: Evaluation of the assay on field samples proved impossible due to the lack of availability of clinical samples either from the badger culling trial or Woodchester Park. This was caused by the outbreak of the Foot and Mouth epidemic in GB, which prevented badger trapping for much of the final year of this aspect of the project. Without a large number of samples for evaluation the results were not suitable for submission for publication in a peer reviewed journal.

SID 5 (2/05) Page 14 of 29

APPENDICES.

Appendix A - Protocol for genomic DNA extraction from M. tuberculosis cultures.

1. Scrape M. tuberculosis cultures from LJ slopes and suspend in 500 l TE buffer (0.01M Tris – HCl. Containing 0.001M EDTA, pH 8.0).

2. Heat kill cells for 5 minutes at 100 C.

3. Centrifuge in 1.5 ml Eppendorf tubes for 20 mins at full speed.

4.Resuspend pellet in 500 l TE buffer. (Available from Sigma T 9285 if required).

5. Add lysozyme (Sigma, L6876) to a final concentration of 1mg/ml. (50l of 10 mg/ml) Incubate 1 hr at 37 C.

6. Add 70 l of 10% SDS Sigma, L 4390) followed by 6 l of Proteinase K (stock 10mg/ml, Boehringer Mannheim) and incubate at 65 C for 10 minutes.

7. Add 80 l of 10% CTAB (hexadecyltrimethyl ammonium bromide, Sigma H 6269, made up in 0.7M NaCl). Vortex mix tubes briefly and incubate at 65 C for further 10 minutes.

8. Remove proteins with equal volume of phenol: chloroform-isoamyl alcohol (25:24:1. Sigma P 3803) and vortex for 10s. Centrifuge the tubes for 5 minutes in the Eppendorf centrifuge. Carefully transfer supernatant to fresh set of 1.5 ml tubes. Avoid transferring interface.

9. Precipitate DNA with addition of approximately equal volumes of isopropanol (Merck product 296945G) leave at –20C for 15 minutes and centrifuge for 15 minutes.

10. Wash the pellets in 900l 70% ethanol and air dry. DNA is taken up in 50 – 100l 0.1 X TE buffer and stored at –20 C before PCR.

SID 5 (2/05) Page 15 of 29

Appendix B.

Heat Inactivation Trial of M. bovis

This experiment was carried out to determine the efficacy of Nuclisens lysis buffer with or without heat treatment in killing any M.bovis isolated from a VL sample.

MethodA confirmed VL tissue sample (AF61/2834/02) was homogenised using a pestle and mortar and 18ml of Nuclisens lysis Buffer (at Room temperature) added. The suspension was then pipetted back equally into two 9ml tubes labelled “Heated” and “Unheated”.A 1ml aliquot of M.bovis AF61/2122/97 (6 x 107 CFU/ml) was added to two tubes of 9ml Nuclisens lysis buffer labelled “Heated” and “Unheated” respectively.The “Heated” samples were then heated in a water bath at 95C for 5 minutes. Three hundred microlitres of each treated sample was then inoculated onto 4 labelled 7H11 slopes. Four 300l aliquots of 61/2122/97 (untreated) were also inoculated onto 7H11 slopes as a positive control. The slopes were incubated at 37C for 6 weeks.

ResultsHeated Unheated Control

61/2122/97 Neg Neg Neg Neg Neg Neg Pos Pos Pos61/2834/02 Neg Neg Neg Neg Neg Neg Pos Pos Pos

Neg = No Growth ObservedPos = M. bovis Growth

Conclusion

The Nuclisens lysis buffer inactivated over 12 hours at 4C the M. bovis organism completely without the need for heat inactivation. Samples from VL tissues treated in this way can be safely used outside of a Cat III area.

SID 5 (2/05) Page 16 of 29

Appendix C. Validation of the DNA extraction method and RD7-based PCR on spiked samples of badger faeces. The RD7 method correctly identified 48/50 of isolates of the M. tuberculosis complex.

SID 5 (2/05) Page 17 of 29

Sample Spiked with Faeces (mgs)

Cyt b PCR

Expected result

RD7 PCR RD4 PCR

1 H37Rv 370 ++ - - -2 Bovis AN5 180 + + + +3 Microti ? 340 + + + +4 Bovis AN5 450 + + + +5 Bovis AN5 360 + + + +6 Water 300 + - - -7 AF 2122/97 320 + + + +8 AF 2122/97 230 + + + +9 AF 2122/97 280 + + + +

10 AF 2122/97 280 + + ++ +11 Water 350 + - - -12 Bovis AN5 300 + + + +13 Bovis AN5 390 + + ++ +14 Water 550 + - - -15 Bovis AN5 410 ++ + ++ +16 Microti ? 400 ++ + ++ +17 AF 2122/97 460 ++ + ++ +18 AF 2122/97 340 ++ + + +19 AF 2122/97 300 ++ + + +20 AF 2122/97 410 + + + +21 AF 2122/97 340 ++ + + +22 Bovis AN5 310 + + + +23 Bovis AN5 280 + + + +24 Water 450 ++ - - -25 Bovis AN5 630 ++ + + +26 Bovis AN5 520 + + + +27 H37Rv 270 + - + +28 Microti ? 440 + + ++ +29 Bovis AN5 440 + + + +30 AF 2122/97 520 + + + +31 AF 2122/97 380 ++ + + +32 AF 2122/97 310 ++ + + +33 Water 230 ++ - - -34 Water 220 + - - -35 Bovis AN5 250 + + + +36 Bovis AN5 370 + + + +37 Water 430 + - - -38 Water 370 + - - -39 AF 2122/97 470 + + + +40 Water 370 ++ - - -41 Water 250 + - - -42 Microti ? 400 + + + +43 Water 600 + - - -44 H37Rv 410 + - - -45 Bovis AN5 340 + + ++ +46 Microti ? 390 + + + +47 AF 2122/97 470 ++ + + +48 H37Rv 460 ++ - + +49 AF 2122/97 540 + + + +50 Water 310 + - - -

Appendix D.

Diagnostic and Genotyping PCR assays developed during SE 3008.

PCR Primer Sequence [Mg] Anneal

temp

Amplicon

size (bp)

RvD1 JB21JB22

5-TCGTCCGCTGATGCAAGTGC-35'-CGTCCGCTGACCTCAAGAAG-3

1.5 54 500

IS1081 F2R2 R3

5-CTGCTCTCGACGTTCATCGCCG-35-GGCACGGGTGTCGAAATCACG-35-TGGCGGTAGCCGTTGCGC-3

1.5

2.0

58

58

135

113RD4flanking

F1F2R1

5-AATGGTTTGGTCATGACGCCTTC-35-TGTGAATTCATACAAGCCGTAGTC-3G5-CCCGTAGCGTTACTGAGAAATTGC-3

2.0 58176

142

RD7flanking

F1F2Reverse

5-ATCTTGCGGCCCAATGAATC-35-TCGGTCAGCAAGACGTTGAAG-35-ACTTCAGTGCTGGTTCGTGG-3

2.0 58211105

RD8flanking

For.Rev.

5GAGTCTATATAGTGTGCTCATGGGGCTAGC-35-GCTTGCTGGCGATCATTGGTCT-3

2.0 64 178

RD9flanking

For.Rev.

5-TCGGCGGTGACGGTATCGTC-35-CGGAACAAGCCTTATCTACCGTCCC-3

2.0 58 104

RD 10flanking

For.Rev.

5-CCGCACTGACCATGCCATTTACC-35-GCGTTGAAGCGCTACATCGC-3

2.0 54 140

RD12flanking

For.Rev.

5-CACACCTGGTTCGTGTTGCG-3GGTGAAGAACGTCGTCAAGCACAT-3

2.0 58 123

RD13flanking

For.Rev.

5-ATCGCTCGTTCGTCGGCTTC-35-GGCAAGACCGGGCCTTTGAC-3

2.0 60 136

TBD1flanking

For.Rev.

5-ATCGAAAGGCTAACGGGTGC-35-GTCCAAGGTTACGGTCACGCT-3

2.0 58 139

MBD1internal

For.Rev.

5-TTCTGCCTCGACAAGTCCTCAT-35-GGGAGCTCTGCGACGTT-3

2.0 56 180

Meles meles

Cyt b FCyt b R

5-CCATCCAATATCTCAGCATGATGAAA-35-GCTCCCAAAAAGACATTTGTCCTCA-3

2.0 64 359

Bos taurus

Cyt b FCyt b R

5-CCATCGAACATTTCATCATGATGGAA-35-GCTCCTCAGAATGATATTTGTCCTCA-3

2.0 64 359

pncA For.Reverse

5-ATCAGCGACTACCTGGCCGA-35-GGAGTACCGCTGACGCATG-3

2.0 64 140

OxyR For.Rev.

5-CGCGCTGTCAGAGCTGACTTT-35-TCTGCGGAATCAGTGTCACC-3

2.0 62 150

SID 5 (2/05) Page 18 of 29

Appendix E.

Standard PCR and quantitative (QPCR) protocol.

“Hot-start” PCR is performed in a final volume of 25 l using the Corbett Research RotorGene 3000 real-time platform and an Excite Core kit (BioGene) according to the Manufacturer’s instructions. This is a convenient UNG-ready kit which permits restriction of any carryover amplicons with uracil glycosylase, should this be required. All primers were used at a concentration of 25 pmoles / tube. SYBR Green dye (BioGene) was included in the PCR master mix at a final dilution of 1/55,000 of the stock. This dilution was found optimal in preliminary experiments and allowed the reactions to be followed on the Corbett platform. After an initial denaturation step (8 min at 95oC), 45 cycles of amplification were performed as follows: denaturation at 95oC for 10 s, annealing at optimal temperature (range 54- 64oC) for 30 s, extension at 72oC for 20 s and acquisition of fluorescent signal at 85oC. At this temperature, most primer-dimers are not seen during the run. A final extension was performed at 72°C for 2 min. At the end of the run, a melt analysis was determined to screen for positive samples using the RotorGene software. PCR products were also always analysed by electrophoresis on 3% (wt/vol) agarose gels.

When quantitation was required e.g. of bovine LN with visible lesions, standard curves were included with individual standards assayed in triplicate. Samples were assayed in duplicate using extracts diluted appropriately to overcome PCR inhibition. The optimal dilutions were determined during the course of IS1081 PCR assays. The standard used was DNA purified from strain AF 2122/97. Concentration of DNA in the stock was determined using a Gene Quant II UV spectrophotometer (Pharmacia). Standards were prepared from serial dilutions of the stock over the range 10 -1 to 10-8 and a working range found. Typically this was between 10-2 and 10 –6 of stock. The standards were diluted in TE buffer containing 245 ug/ml yeast tRNA as a carrier to minimise the effects of both freeze thawing and loss of DNA to the sides of plastic ware. For the latter reason "No-Stick” 0.2 ml micro-tubes (Alpha Labs) were used throughout for DNA standard preparation, PCR and storage. Two hundred l of each standard was prepared and sub-aliquoted into 10 x 20l lots. In this way, each tube was thawed once and then discarded. These measures largely prevented loss in sensitivity of standards seen when standard tubes were repeatedly frozen and thawed over a period of some weeks.

SID 5 (2/05) Page 19 of 29

Appendix F .

Woodchester Park samples.

Sample Badger AF No Sample type

Cyt b Pellet

Cyt b Super.

M.bovis Pellet

M.bovis Super.

culture

1 Z078 61/96/3325 BW - - - - -2 Z026 61/96/3316 BW - + - - M.bovis3 J031 61/99/3510 BW ++ ++ - - M.bovis4 D052 61/99/4175 BW ++ ++ - - -5 Z026 61/96/3316 BW ++ ++ - - M.bovis6 D034 61/98/3247 BW - - - -7 CO78 61/91/4438 Faeces - + - - -8 A074 61/98/3888 Faeces - - - - M.bovis9 D034 61/98/3246 Faeces - - - - M.bovis10 J031 61/99/3509 Faeces - - - - M.bovis11 J066 61/98/3328 TA + + - - M.bovis12 Z076 61/96/3322 TA ++ ++ - - -13 D004 61/99/0764 TA ++ ++ - - -14 A074 61/98/3886 TA ++ ++ - - M.bovis15 Z078 61/96/3324 TA ++ + - - -16 Z064 61/96/4916 TA ++ ++ - - -17 C083 61/97/4337 TA + ++ - - M.bovis18 A074 61/98/3737 TA ++ ++ - -19 D034 61/98/3244 TA + ++ - - M.bovis20 D074 61/98/3267 TA + ++ - - M.bovis21 Z071 ? TA ++ ++ - - -22 J031 61/99/3507 TA + - - - M.bovis23 Q047 61/99/3615 TA + ++ + - M.bovis24 D028 61/993477 TA + + - - -25 H015 61/99/4205 TA + ++ - - -26 D068 61/99/4189 TA + ++ + - -27 D052 61/99/0147 RSUBM - - - - M.bovis28 HO44 61/95/4129 urine - - - - -29 D052 61/99/4932 Urine - - - + M.bovis30 D074 61/98/3268 Urine - + - + -31 Z078 61/96/3327 Urine - + - + -32 Z064 61/96/4915 Urine - - - - -33 Z076 61/96/3321 Urine + - - - -34 Z071 61/96/3318 Urine + - - - -35 A074 61/98/3887 Urine + - - - M.bovis36 M115 61/97/4654 Urine - - - - Mbovis37 D028 61/99/3478 Urine - - - - -38 J031 61/99/3508 Urine - ++ - - -39 Q078 61/99/4321 Urine + + - - -40 J014 61/99/4228 Urine + + - + -41 H008 61/99/4200 Urine + ++ - - -42 Z026 61/96/3314 Urine - + - + -Blank - - - -Blank - - - -

TA = tracheal aspirate; BW = bite wound

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Appendix G.

DNA Extraction from Paraffin-embedded tissue sections.

1. Place 2 x 10 m tissue sections in a 2ml Eppendorf micro centrifuge tube.

2. Add 500 l of xylene, leave for 5 mins.

3. Centrifuge full speed (14,000 rpm) for 5 mins. at room temperature (RT).

4. Carefully remove supernatant by pipetting, leaving any pellet intact. (Dispose of waste xylene to

non-chlorinated solvent waste).

5. Repeat steps 2-4 once.

6. Add 500 l of 100% ethanol to remove xylene; vortexing gently over a period of 5 mins.

7. Centrifuge at full speed at RT for 5 mins.

8. Remove ethanol by pipetting, leaving the pellet behind.

9. Repeat steps 6-8 once.

10. Dry open micro centrifuge tube in a hot block at 40-500 C for 10-15 mins. until all traces of

ethanol have evaporated.

11. Resuspend the pellet in 90 l of 1 x TE buffer, pH 7.4. Add 10 l of proteinase K solution

(Qiagen Solution) mix by vortexing and incubate at 56 C on the hot block for 20 mins. (Some

robust sections may take longer to digest.)

12. Add 900l of lysis buffer (GUSCN) from the NucliSens kit (bioMérieux).

13. Vortex initially and place on mixing wheel for 30 mins at RT. Heat to 950C for 5 mins.

14. Centrifuge at 14,000 rpm for 5 mins.

15. Collect supernatant by pipetting into a fresh tube.

16. Add 50 l of the silica suspension from the bioMerieux kit, vortex briefly and leave horizontal on

bench at RT for 15 mins.

17. Centrifuge at 14,000 rpm for 3-5 mins.

18. Remove GUSCN supernatant but retain for safe disposal by incineration.

19. Wash silica with 900 l of wash buffer from the bioMérieux kit by vortexing 2-3 times over a 5-

minute period.

20. Centrifuge for 2-3 mins to pellet the silica.

21. Remove supernatant and repeat steps 19-20 once, pooling the waste washes containing

GUSCN for safe disposal by incineration.

22. Wash the silica pellet with 70% ethanol by vortexing.

23. Centrifuge for 5 mins.

24. Remove supernatant and repeat steps 22-23 once.

25. Wash pellet with 1 ml acetone, centrifuge and dispose of supernatant safely to non-chlorinated

solvent waste.

26. Dry silica pellet either on bench at RT or in hot-block set to about 500C. Silica pellet should

disaggregate on vortexing when it is dry.

27. Add 50 l elution buffer from bioMerieux kit.

SID 5 (2/05) Page 21 of 29

28. heat to 600C for 10 mins to maximise elution from silica.

29. Centrifuge at 14,000 rpm for 5 mins.

30. Collect supernatant into non-stick or low-retention tubes (Alpha Labs).

31. PCR s are best set up immediately upon fresh extracts but can be stored at –200C until use.

SID 5 (2/05) Page 22 of 29

Appendix H.

Standard DNA extraction method using the NucliSens kit from bioMérieux.

1. Add 9ml of Nuclisens Lysis buffer to the sample. Heat in a boiling water bath for 5 mins. Can be left overnight to allow the cells to lyse. Soft tissue samples, e.g. faeces or can be disrupted with disposable pellet-pestles (Anachem). Blood, urine, nasal aspirates or BAL can be dealt with directly.

2. “Snap-freeze” the tubes twice in liquid nitrogen to assist with lysis of mycobacteria.

3. When thawed, centrifuge samples at 2000 rpm for 5 mins. Pour the supernatants into 15 ml conical centrifuge tubes (Corning, orange cap). Add 50 l of silica suspension (Isolation reagents, NucliSens kit) to each tube and cap tightly. (Keep silica mixed regularly to ensure accurate dispensing).

4. Rotate on spinning wheel for 30 mins to allow DNA trapping onto silica beads.

5. Centrifuge at 2000 rpm for 5 mins, discarding supernatants into disposable Falcon or similar tube.

6. Wash silica twice by vortexing with 1ml of GUSCN wash buffer (Isolation reagents, NucliSens kit). Silica pellet should become a suspension. Discard supernatants after centrifugation into the waste tube. Dispose of this eventually into sharps bin for incineration. (Particularly dirty or clogged silica can be washed twice with 2ml of GUSCN).

7. Wash silica twice with 2ml of 75% ethanol. Supernatants after centrifugation can be discarded into the drain.

8. Wash silica once with 1ml of acetone. Centrifuge and dispose of supernatants into waste Falcon for incineration.

9. Air dry the tubes until silica will disperse upon vortexing. Add 100 l of TE elution buffer (Isolation reagents, NucliSens kit). Heat to 60oC for 10 mins, vortex, centrifuge and transfer the supernatants into 0.5ml low-retention flat-cap tubes (Alpha Labs). Minimise transfer of silica or repeat as necessary to avoid PCR inhibition.

Steps 3 –9. If any evidence of leakage of GUSCN buffer (dry white powder on tube exteriors) rinse off well with tap water to avoid carry-over contamination of other tubes, vortex mixer, centrifuge etc. It will be necessary to clean these pieces of equipment between batches.

SID 5 (2/05) Page 23 of 29

Appendix I.

DNA extraction method for bovine tissue samples.

1. Line the floor of a Class I cabinet with greaseproof paper. Add three autoclave bags, one to contain waste, one pestles & mortars and the other used instruments.

2. Place four tissue samples in the cabinet each with one large (110mm diameter) sterile pestle and mortar and one small (90mm diameter) sterile, acid washed pestle and mortar (Soak in 1% HCl for one hour to remove contaminating DNA then wash, dry and autoclave). Add four sets of scissors and forceps and wrap the equipment in a 30 x 30cm polythene bag to prevent contamination.

3. Label a sterile universal and a 9ml tube of Nuclisens Lysis buffer (one for each per sample) with the VLA reference number and place in the cabinet with bottles containing 5% oxalic acid and sterile sand. Add one container of buffered formalin for histology (label with reference number, species and tissue type) plus one quarter sheet of greaseproof paper per sample.

4. Have to hand outside the cabinet a high temperature autoclave bag plus TST strip and autoclave tin for disposal of discarded tissue.

5. Use the scissors and forceps on the greaseproof paper to sort through one tissue sample at a time for lesions, removing as much fat as possible.

6. Take a 1cm3 portion of lesioned material for histology and place one small piece into the small pestle and mortar for DNA extraction.

7. Cut the remainder into 3mm pieces and place in the large pestle and mortar for culture.

8. Discard any fat, unwanted tissue, packaging and the used quarter sheet of greaseproof paper into the waste autoclave bag.

9. Add 5cm3 of sterile sand (glass beads) to the mortars and using the pestle grind the tissue until it is a homogenate.

10. Discard the scissors and forceps into the instrument autoclave bag. Re-wrap each pestle and mortar in the 30 x 30cm polythene bag after each sample has been homogenised to avoid cross contamination.

11. Add 10ml of 5% Oxalic Acid to the large mortar, mix, pour into the labelled universal and leave for 10 minutes then centrifuge for a further 10 minutes at 1100g. Discard the acid supernatant, re-suspend in 20 ml of 0.85% saline and repeat the centrifugation. Then discard the saline supernatant and re-suspend in 10ml of 0.85% saline. Inoculate 300l of the final suspension onto six slopes of media (1 LJ base, 1 LJG, 1 LJP 3 7H11) and incubate for 6 weeks.

12. Add 9ml of Nuclisens Lysis buffer from the labelled tube to the small mortar and mix to a suspension. Then pipette the homogenate back into the tube, heat in a waterbath at 95oC for 5 minutes. Can be left overnight to allow the cells to lyse.

13. From this stage, the routine extraction method for DNA extraction onto silica and the washing and elution steps can be followed (Appendix H).

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FIGURES.

Figure 1. RD7 / cyt b multiplex in development.

Figure 2. RD multiplex assay applied to a panel of 10 different spoligotypes of M. bovis. RD7 product = 211 bp; RD4 product = 176 bp, RD9 product =

Figure 3. Woodchester Park faecal samples.

50 badger faecal samples collected from 14 latrine sites used by 4 social groups. Cyt b (Meles meles) and RD4 multiplex). IS1081 PCR was also negative on all 50.

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L 3 2.5 2 1.5 1 mM Mg Cl

RD7

Cyt b

1 2 3 4 5 6 7 8 9 10 bl + 1 = 61/3558/00 type 9 2 = 61/1121/01 type 17 3 = 61/2145/00 type 12 4 = 61/5415/00 type 11 5 = 61/0140/01 type 20 6 = 61/3979/00 type 13 7 = 61/0288/01 type 22 8 = 61/5448/00 type 10 9 = 61/0681/01 type 2510 = 61/1307/01 type 35

400 bp

100 bp

Cyt b 359 bp

RD4 176 bp

Figure 4. IS1081 PCR of badgers from the live trapping experiment.

Figure 5. Cattle lymph nodes with visible lesions assayed using IS1081 PCR with gel electrophoresis on 3% agarose.

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Lane 1 = nilLane 2 = 100 bp ladder.Lane 3 = +ve controlLane 4 =Water blankLane 5 = badger X49/10Lane 6 = badger H43/40Lane 7 = badger Q39/40Lane 8 = badger X49/40

1 2 3 4 5 6 7 8

IS1081 product135 bp

Figure 6. IS1081 PCR applied to cattle VLN, study 2.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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Boom, R, Sol, CJA, Salimans, MMM, Jansen, CL, Wertheim-van Dillen, PME and van der Noordaa, J. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28: 495–503, 1990.

Boom, R, Sol, C, Beld, M, Weel, J, Goudsmit, J, Wertheim-van Dillen, P. Improved silica-guanidinium thiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles. Journal of Clinical Microbiology, 37: 615–619, 1999.

Brosch, R, Gordon, SV, Marmiesse, M, Brodin, P, Buchrieser, C, Eiglmeier, K, Garnier, T, Gutierrez, C. Hewinson, G, Kremer, K, Parsons, LM, Pym, AS, Samper, S, van Soolingen, D, Cole, ST. A new evolutionary scenario for the Mycobacterium tuberculosis complex. 3684–3689 PNAS 99: 3684-3689, 2002.

Cousins, DV, Bastida, R, Cataldi, A, Quse, V, Redrobe, S, Dow, S, Duignan, P, Murray, A , Dupont, C, Ahmed, N, Collins, DM, Butler, WR, Dawson, D, Rodriguez, D, Loureiro J, Romano, MI, Alito, A, Zumarraga, M, Bernardelli, A. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov. Int J Syst Evol Microbiol. 53: 1305-1314, 2003.

Dziadek, J., Sajduda, A. & Borun, T. M. Specificity of insertion sequence-based PCR assays for Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis 5, 569–574, 2001.

Frothingham, R. & Meeker-O'Connell, W. A. Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144:1189-96, 1998.

Hoff-Olsen, P, Mevag, B, Staalstrøm, E, Hovde, B, Egeland, T, Olaisen, B. Extraction of DNA from decomposed human tissue: An evaluation of five extraction methods for short tandem repeat typing. Forensic Science International 105: 171–183, 1999.

Metaxa-Mariatou, V, Vakalis, N, Gazouli, M, Nasioulas, G. The 500-base-pair fragment of the putative gene RvD1-Rv2031c is also present in the genome of Mycobacterium tuberculosis. In Vivo 2004 18: 33-35, 2004.

Riska PF, Su Y, Bardarov S, Freundlich L, Sarkis G, Hatfull G, Carriere C, Kumar V, Chan J, Jacobs WR Jr. Rapid film-based determination of antibiotic susceptibilities of Mycobacterium tuberculosis strains by using a luciferase reporter phage and the Bronx Box. J Clin Microbiol. 37:1144-9, 1999.

Rodriguez, J G, Mejia, GA, Del Portillo, P, Patarroyo, ME, Murillo, LA. Species-specific identification of Mycobacterium bovis by PCR. Microbiology 141: 2131-2138, 1995.

Sales, MPU, Taylor, GM, Hughes, S, Yates, M, G. Hewinson, G, Young, DB, Shaw R. Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources. Journal of Clinical Microbiology 39:4558-4562, 2001.

Smith NH, Dale J, Inwald J, Palmer S, Gordon SV, Hewinson RG, Smith JM. The population structure of Mycobacterium bovis in Great Britain: Clonal expansion. Proc Natl Acad Sci U S A. 100:15271-5, 2003.

Takade, A, Umeda, A, Matsuoka, M, Yoshida, S-I, Nakamura, M, Amako, K. Comparative studies on the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique. Microbiol. Immunol. 47: 265-270, 2003.

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