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Alternative testing methods

 Added by  FrankJ (Guest)
 1 Oct 2009, 7:57 PM

This forum strand debates the various testing methods.

BOVINE TB AND THE USE OF PCR: SUMMARY OF 12 JULY MEETING, (Department for Environment, Food and Rural Affairs)
1. The group agreed that:
a. for PCR it is necessary to consider both analytical sensitivity and sampling techniques alongside methods of sample preparation;
b. there is potential in the faecal and air approaches but these are not yet at a stage where they can be recommended to Ministers for use; and
c. further work could be done on faecal sampling and also air sampling (see paragraphs 23 – 24).
2. Overall, the expert group concluded that PCR was not a test that could be usefully used for detecting TB in badgers based on the current state of
knowledge, particularly in the field. If the further research at 1c) was pursued, PCR might have some use for testing badgers for TB in the future. However, the group emphasised that there are no guarantees and that the current uncertainties may not be resolved and a practical (or low cost) test regime developed.
3. If the uncertainties surrounding sample extraction and handling could be resolved, and an integrated assay procedure validated, an automated portable test might be feasible in the longer-term (i.e. over several years). The timescale for this would depend on the outcome of an appropriate experimental programme and the resources applied.
Info from: http://www.scoop.it/t/bovine-tb-badgers-and-cattle/p/3340706774/bovine-tb-and-the-use-of-pcr-summary-of-12-july-meeting
PCR TB test – more a liability than an asset, says Badger Trust in its Press Release (see below) dated 14/11/12.
DEFRA Secretary Owen Paterson told a Commons committee that he is exploring the possibility of using PCR (Polymerase chain reaction) tests to identify infected badger setts as part of a future badger culling strategy. However the Badger Trust warns that the use of such a test to detect the presence of bovine tuberculosis (bTB) cannot tell whether the infection came from cattle, badgers or any other wildlife. In addition, use of this test on its own carries with it huge risks of precipitate and counter-productive action. Finding the bTB bacillus close to a sett would not in itself prove where a particular sample had come from and localised culling also raises the risk of perturbation. Consequently the possible range of error in the field could be massive.
Mr Paterson told the Environment, Food and Rural Affairs Committee last week [1] that it would make an enormous difference if only diseased animals could be traced. He claimed he had met ‘one of the world’s experts’ and concluded there was ‘potential’ for PCR to confirm disease in badger setts, and so target only infected badgers. However, the Badger Trust says there is no guarantee that traces of bTB in or near a sett would mean all or even any of the animals in it would be infected. Furthermore, killing them all would run the risk of spreading the disease by stirring up the badger population.
David Williams, chairman of the Badger Trust, said: “PCR is a relatively crude tool at present. Further investigation must address many issues of concern before expectations about PCR are raised any more. Mr Paterson’s interest in PCR is just another extension of the Coalition’s unreasonable and blinkered preoccupation with badgers at a time when the urgent need is for progress with an effective cattle vaccine and an acceptable DIVA test to distinguish between vaccinated and infected cattle”.
Mr Williams summarised difficulties for a practical field test:
· Did the sample come from cattle, badgers, foxes or other wildlife that could have walked through a farmyard, field or cow pat, any of them carrying infection?
· Did the sample come from a live badger or a long-dead one?
· If taken from soil did it come from a badger?
If a positive sample was found in a sett:
· Would you catch and kill all the badgers in that sett?
· Would you catch and then test beside setts where an infected sample was found?
· A positive sample from inside a sett could be from a badger not normally living in that sett or a fox using it temporarily.
· If you don't identify and test all the social group, a massive task, selective removal becomes impossible.
· Infected badgers do not necessarily excrete the TB bacillus.
· For a selective cull expensive trapping is going to be required unless you gas or poison the sett indiscriminately.
The PCR test is highly sensitive and can detect small amounts of genetic material (DNA and Ribo Nucleic Acid - RNA) in samples of blood, tissue or body fluids. A team at Warwick University has been working with the PCR test for several years, and a recent paper [2] described its use but concluded, crucially: “Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife”.
This need for further study echoes the report of a meeting of Defra experts and advisers on July 12 [3]. It said: “If the uncertainties surrounding sample extraction and handling could be resolved, and an integrated assay procedure validated, an automated portable test might be feasible in the longer-term (i.e. over several years). The timescale for this would depend on the outcome of an appropriate experimental programme and the resources applied”.
[1] http://www.farmersguardian.com/home/hot-topics/bovine-tb/paterson-to-explore-pcr-badger-sett-tb-test-potential/51166.article
[2] http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0027369
[3] http://archive.defra.gov.uk/foodfarm/farmanimal/diseases/atoz/tb/documents/pcr-meeting100712
The tuberculin test. Veterinary Microbiology 40, 111–124
M.L. Monaghan, a, M.L. Dohertya, J.D. Collinsa, J.F. Kazdab and P.J. Quinna
aFaculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland
bInstitut für Experimentelle Biologie und Medizin, Parkalee 1 40, 2061 Borstel, Germany
Accepted 6 January 1994. Available online 12 November 2002.
Tuberculin tests in general use today rely on the response to intradermal injections of tuberculin with assessment of the injection site for swelling at 72 hours post injection. Estimates of the sensitivity of tuberculin test range from 68–95% while specificity is estimated to be 96–99%. The sensitivity of the test is affected by the potency and dose of tuberculin administered, the interval post-infection, desensitisation, deliberate interference, post-partum immunosuppression and observer variation. Specificity is influenced by sensitisation as a result of exposure to M. avium, M. paratuberculosis and environmental mycobacteria and by skin tuberculosis.
Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses
Michael Coad, Derek Clifford, Shelley G. Rhodes, R. Glyn Hewinson, H. Martin Vordermeier and Adam O. Whelan
TB Research Group, Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom
Received 12 August 2009; accepted 16 October 2009; published online 20 October 2009
Abstract - The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon- (IFN-) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1 (IL-1) whilst confirming stable IFN- and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1 and elevated IL-10 responses, but importantly, does not influence antigen specific IFN- responses.
Ruth (Guest)
Re: the new test for TB which is claimed to be a field friendly device that relies on readily available and relatively low-cost components and can find the lethal pathogen in blood in just 20 minutes. www.scientificamerican.com/article.cfm?id=tuberculosis-detector
Yes this test does look interesting. I do think this is the way to go if possible. I feel very enthusiastic about this test.
By looking for proteins (antigens) that exit live Mycobacteria tuberculosis or bovis cells, that are 'soluble' and found in the fluid medium or serum, from culture or infected bodies (animals and people) respectively the same test can be used on any species. Thus different tests would not have to be developed and evaluated for badgers, cattle, cats and alpaca or humans as in detecting the immune response, whether antibodies, gamma interferon or cells etc.
Such a test for secreted mycobacteria proteins would detect active infection or and the presence of viable organisms that could reactivate.
In the case of animals if the policy is to kill all infected animals then the test is useful. Negative animals could be vaccinated if held until the test result is available, which could be within 45 minutes according to the article, and I know this to be possible. This could be very useful for badgers when it is useless to vaccinate with BCG animals already infected. Only infected animals would be culled. (Whether BCG vaccination would give a positive result would have to be sorted out before using the vaccine as an oral bait if this test were to be applied to trapped badgers in order to reduce the burden of infection) .
In the case of humans a test that does not distinguish between latently infected and active infection is not so useful, as true latent infections are refractory to treatment.
As referred to in the article, in humans the test for antibodies is complicated by the fact of treatment so the organism may be killed but the immune response from memory cells remain, both the cell mediated immune response and antibodies. This would not occur in animals because they are not treated. (I have just read that one of the reasons for treating people for 6 to 9 months may be to kill the latent TB bacteria which re-activate as well as the active disease causing bacteria - hence the success of a full course of therapy for TB).
Both the proteins used in the test described in the article are major mycobacterial antigens, the alpha crystallin is heat shock protein 16 and may have a function for longterm viability during latency, and antigen 85B is a secrotome protein and is a mycolyl transferase.
It is an interesting phenomenon of bacteria that they do secrete proteins out of the cell into the surrounding environment and these have been studied in detail for mycobacteria, molecular biology leading to much interesting information in the 2 tomes I have purchased on Mycobacterium tuberculosis!
The monoclonal antibodies used in the test must clearly distinguish these proteins of M tuberculosis and M bovis from other mycobacteria and environmental and avian bacteria that will have versions of these though perhaps not identical. I would guess the sequencing of the DNA of the different mycobacteria species, together with experimental work evaluating different mycobacteria species will answer this question of specificity. The monoclonal antibodies would be chosen for their specificity. Perhaps a few more such proteins could be detected and the fact that several are individually recognised in a micro-array would confirm the positive result as a true positive. Such a test might be more specific and sensitive than present tests, such as the skin test or the gamma interferon test.
If it is licensed in the USA I do hope that we use it over here in the UK and do not have to wait for years for DEFRA to reinvent the wheel as we have to do for FMD virus RNA detection, when we refused to use the USA test over her. Also, in the case of PCR detection of M bovis to be both specific and detect viable bacteria DEFRA has insisted on starting again and not using the test developed by Professor Wellington at Warwick University. It is all to do with money patents and bureaucracy.
The other point to make about BCG is that it multiplies for a certain length of time, say about 3 months, but then as I understand, unless the person in severely immuno-compromised, it is controlled and does not remain latent, so it leaves an immune footprint but would not be expected to give rise to these antigens detected in the proposed test after that. However, if it is out as bait and one does not know when the animal was infected by eating the bait, or whether oral re-infection may take place, I am not sure how useful the test would be in the situation of the badger and freely available oral BCG bait. However, the BCG vaccine by injection will be ready for use in the badger several years before, if not actually now, so therefore injecting a trapped badger and micro-chipping it, is a present or imminent possibility if the badger tests negative for TB. Again knowing when cattle are vaccinated by injection could be adjusted to screening by this new test, ie more than 6 months after vaccination. It will be important to check the clearance of BCG in the vaccinated animal with the test for antigens, 85B and alpha-crystallin and any others used in the new test.


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