Bovine TB: Appendix A - Summary of Standard Operating Procedures
1. Introduction
1.1 Standard operating procedures are prescribed for each stage of the trial. They are reviewed by the Independent Scientific Group in the light of experience to improve and streamline practices where possible. For reasons of staff safety and security the full text of procedures has not been published but the following summary sets out the main features of how the trial is being conducted. Other procedures, mainly relating to administration, have also been prescribed but are not summarised in this appendix.
1.2 The standard operating procedures relating to surveying, trapping, social group territory delineation and humane despatch have been subject to independent audit.
2. Triplet selection
2.1 The centre point of candidate trial areas (grouped into threes to form triplets) are identified by the ISG on the basis of specific criteria. These criteria include surface area, a minimum number of cattle holdings and, in particular, the immediate and past history of cattle TB breakdowns, for which there are reliable, current data.
3. Delineation of trial area boundaries
3.1 The centre points (described in paragraph 2) are developed into trial areas and buffer zones in three stages, as follows:
- candidate trial areas of 100 km² are mapped drawing a circle of radius 5.64 km about the centre point.
- proposed trial areas are mapped by adjusting candidate trial area boundaries to take account of natural features and known farm boundaries. Once proposed trial areas have been ratified by the ISG a list of occupiers of land in those areas is drawn up and used as the basis for visiting and enrolling participants in the trial.
- trial areas are formed by adapting proposed trial area boundaries to take account of survey information and are subject to ratification by the ISG.
In delineating boundaries care is taken to maintain, as far as possible, the circular shape of trial areas and the balance of key characteristics used to determine centre points.
3.2 Buffer zones are defined as follows:
- Inner buffer zone: an area extending 1 km from a trial area boundary which may not overlap any other buffer zones.
- Outer buffer zone: an area extending 1 km beyond an inner buffer zone boundary which may touch or overlap other outer buffer zones.
4. Visiting occupiers of land
4.1 Visits to occupiers of land are made to explain and request participation in the trial. The importance of the trial to the strategy to combat TB in cattle is emphasised and its voluntary and confidential nature explained. Formal consent to participation is sought by signature to a standard agreement which incorporates a map recording the boundaries of the occupier’s land. Occupiers are asked to signify if they are required to obtain consent from a third party. Participation may extend to culling and/or surveying and also auditing and research projects associated with the trial being conducted on an occupier’s land.
5. Surveying for badger activity
5.1 Surveys are undertaken to identify and record the location of main and subsidiary setts and other signs of badger activity including latrines, boundaries and visible runs. Such information is used to delineate badger social group territories, where present, finalise the boundaries of treatment areas and identify areas where traps may be sited. Surveys may also provide information on illegal interference with badgers and/or setts.
5.2 Types of surveys undertaken are as follows:
- initial - full surveys, undertaken prior to randomisation, of all three treatment areas in a triplet.
- post cull - a survey to assist in assessing the effectiveness of operations (particularly in proactive areas).
- reactive - limited surveys in response to confirmed cattle TB breakdowns. The aim of such surveys is to record all signs of badger activity and identify social groups using the breakdown premises.
- follow-up - restricted to proactive treatment areas. Follow up surveys are of setts only and are normally undertaken 5-9 months after completion of culling and annually thereafter. The aim is to identify badger activity and sites for placing cage traps.
- at 3 years - full surveys of samples of each treatment area (2 years after the initial survey in Year 1). Such surveys are carried out independently of earlier surveys.
- at 5 years - as for 3 year survey, but 4 years after the initial survey in year 1.
- spot surveys of setts to check for illegal interference.
6. Social group territory delineation
6.1 Badger social group territories are delineated to identify proactive and reactive treatment areas and so that trapped badgers may be attributed to social groups for analysis purposes. It is recognised that in some cases social group territories cannot be fully delineated; this may be because of the lack of survey data or because clear social group structures and territories may not exist due to low population densities or distribution of populations resulting from past badger removal operations.
6.2 The first step in the delineation process is to plot main setts on maps and construct hypothetical territories using Dirichlet tessellations. Secondly, tessellated boundaries are adjusted to take account of topographic features and field signs recorded during the surveying stage of the trial. Finally, setts are categorised according to the certainty with which they can be allocated to territories.
7. Randomisation
7.1 In the presence of two members of the ISG and an independent person, treatments are (by the role of a die) allocated at random to the three trial areas within a triplet. The allocation of treatments is only disclosed, on ISG advice, when surveying in the triplet concerned has ceased and culling operations are ready to begin. Trial participants are notified by letter of the treatment which has been allocated to their land.
8. Trapping
8.1 Pre-baiting is undertaken prior to trapping to familiarise badgers with bait and to maximise trapping efficiency. Trapping of badgers is by cage trap only and a closed season operates from February to April, to reduce the chance of catching lactating sows with dependent cubs. The maximum trapping period is 12 days but may be shortened if, for example, badger activity ceases. Trapping would be suspended if, due to bad weather, there was a risk that setts could be flooded or trapped badgers would suffer extreme exposure.
8.2. Standard procedures require traps to be checked as early as possible during the day and set to trap as late as possible. These provisions seek to minimise the period for which trapped badgers remain in cages and to reduce catches of diurnal non-target species. These procedures may be varied in the interests of staff safety.
9. Humane despatch procedures
9.1 The despatch of badgers may only be undertaken by fully trained MAFF staff in accordance with strict procedures. Cage trapped badgers are despatched by shooting to the head. Immediately after shooting, checks are undertaken to ensure that the badger is dead and confirmation that such checks have been completed is indicated by the application of a coloured marker to the carcase. If there is any doubt that a badger is dead a second shot is administered immediately.
9.2 Non-target species are released unless injured to an extent that would make it inhumane to do so. For smaller species despatch is by one or more sharp blows to the head. Veterinary advice would normally be sought on the despatch of other species.
10. Submission of carcases for post-mortem examination
10.1 Procedures set out how carcases must be bagged, labelled and transported to the place of post-mortem. Particular attention is paid to health and safety issues to avoid possible aerosol transmission of bacteria from carcases.
11. Blood samples from despatched badgers
11.1 Blood samples are taken from despatched badgers for use in the development of blood-based diagnostic tests for M. bovis infection in live badgers, and for the establishment of a serum bank. Samples are taken only after the badger has been despatched and checks completed that it is dead.
12. Post-mortem procedures
12.1 Post-mortem examination of badgers is undertaken to determine M. bovis infection at the time of death. This involves examination for the presence or absence of suspected tuberculous lesions and the collection of a standard selection of lymph nodes and, where applicable, collection of suspicious lesions. Infection with M. bovis is determined by bacteriological examination of the specimens collected at post-mortem examination. Tubercle bacilli are quantitated in the respiratory tract and urine. Other material may be collected for collateral research projects approved by the ISG.
12.2 A check is made for cage related injuries which, if present, must be recorded for analysis.
13. Mycobacteria - cultural examination of badger tissues
13.1 Mycobacteria are slow growing organisms requiring specialized media for their cultivation. The medium must contain substances sufficient to suppress the growth of contaminants but allow the growth of Mycobacteria.
13.2 Culture tubes are examined weekly for up to 6 weeks for evidence of growth of Mycobacteria. Where the culture demonstrates growth suspected to be Mycobacteria, a further set of culture tubes, containing typing media for confirmation of M. bovis, are inoculated and incubated. These cultures are examined until sufficient growth occurs to enable a differential reading for identification to be made, usually about 3 weeks. A known positive culture of M. bovis is inoculated onto each new batch of media and an uninoculated tube used as a negative culture to act as controls.
14. Spoligotyping
14.1 Spoligotyping ("spacer oligonucleotide typing") is based on the detection of DNA polymorphisms present at one particular chromosomal locus, the “Direct Repeat” (DR) region, which is uniquely present in Mycobacterium tuberculosis complex bacteria. Spoligotyping represents the first polymerase chain reaction (PCR)-based fingerprinting technique for Mycobacterium bovis to be widely accepted and is the only currently available technique which allows cost effective fingerprinting of every M. bovis isolate from GB.
14.2 The DR region in M. bovis BCG consists of directly repeated sequences of 36 base pairs, which are interspersed by non-repetitive DNA spacers, each 35 to 41 base pairs in length. In M. bovis BCG there are 49 copies of the DR sequence whereas in other strains of M. bovis the number of DR elements vary significantly and this variation forms the basis for strain typing using spoligotyping.
14.3 Spacer sequences are initially amplified by PCR using primers targeted to the DR sequence. The presence or absence of spacers within the DR region is detected by hybridisation of the PCR product to a set of immobilised oligonucleotides each corresponding to one of 43 potential unique spacer DNA sequences within the DR and visualised using a chemiluminescence system. The variation in spacers is used to obtain different hybridisation patterns of the amplified DNA.
14.4 The value of spoligotyping for strain differentiation of M. bovis has been assessed critically in several studies and it is now recommended that spoligotyping be used for rapid screening of isolates, and followed up by a more discriminatory technique such as PGRS-RFLP if further discrimination is required. A further advantage of spoligotyping over the RFLP techniques is that, being PCR-based, it can potentially allow for simultaneous diagnosis and typing from clinical samples.
15. Identification of controls
15.1 Confirmed TB incidents (i.e. "case herds") in trial areas are subject to epidemiological investigations (using questionnaire TB 99) involving the selection of three control herds in the same trial area. One control herd will be contiguous to the case herd and the other two will be selected at random.
15.2 Data on potential risk factors is collected for individual reactor cattle in case herds. Procedures are prescribed for the collection of equivalent information for individual cattle in control herds and for the selection of those animals. Rules on sampling, defining which herds are eligible for selection as contiguous and non-contiguous herds and for managing farmer refusals are also prescribed.
16. Notification of TB herd breakdowns
16.1 Standard procedures are laid down so that breakdowns in reactive areas trigger reactive culling. All breakdowns in trial areas are recorded on a central database with account being taken of cattle movements into and out of those areas.
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B 
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Page last modified:
12 August 2003
Page published: 5 February 2003