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Use of serological techniques for diagnosis of Mycobacterium bovis infection in a llama herd
  1. G. S. Dean, PhD,
  2. H. M. Vordermeier, PhD1,
  3. T. R. Crawshaw, BVetMed, MSc, MRCVS,
  4. D. F. Twomey, MVB, MRCVS2,
  5. R. de la Rua-Domenech, DVM, PhD, DipECVPH, MRCVS3,
  6. L. Farrant, BVetMed, MRCVS4,
  7. R. Greenwald, MSc,
  8. K. Lyashchenko, PhD5 and
  9. R. J. Higgins, BVM&S, MSc, MRCVS6
  1. 1 Veterinary Laboratories Agency (VLA) — Weybridge, New Haw, Addlestone, Surrey KT15 3NB
  2. 2 VLA — Starcross, Staplake Mount, Starcross, Exeter EX6 8PE
  3. 3 TB Programme, Defra, Nobel House, 17 Smith Square, London SW1P 3JR
  4. 4 Animal Health, Clyst House, Winslade Park, Clyst St Mary, Devon EX5 1DY
  5. 5 Chembio Diagnostic Systems, 3661 Horseblock Road, Medford, NY 11763, USA
  6. 6 VLA — Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ
  1. g.dean{at}vla.defra.gsi.gov.uk

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Mycobacterium bovis is increasingly being identified in domestic species other than cattle in Great Britain (Defra 2008). Amendments to the legislation first introduced in 2006 and later incorporated into the current Tuberculosis (TB) Order (Anon 2007) resulted in the obligation to notify all tuberculous lesions in carcases of farmed and pet mammals, and has contributed to an increased number of submissions of nonbovine animals for mycobacterial culture to the Veterinary Laboratories Agency (VLA) over the past three years (Monies and others 2006, Defra 2008). Between 1999 and 2007, several cases of M bovis infection were recorded in Great Britain involving South American camelids, including llamas (Lama glama) and alpacas (Vicugna pacos) (Barlow and others 1999, Twomey and others 2007, Defra 2008).

Antemortem diagnosis of TB is difficult in South American camelids. The clinical signs are non-specific and include wasting, respiratory distress and sudden death (Barlow and others 1999, Twomey and others 2007). Immunological diagnostic methods are also problematic. Directive 92/65/EEC (Anon 1992) states that the tuberculin skin test is the official method for certification of TB freedom in South American camelids intended for international trade in the EU. The comparative tuberculin skin test in the postaxillary site is the recommended diagnostic test in the UK and many other countries, although this test has not been fully validated and is technically more difficult to perform in camelids than in cattle. Some small studies have reported reasonable sensitivity and specificity for this test (Thoen and others 1988, Johnson and others 1989, Stevens and others 1998, Cousins and Florisson 2005); however, others have suggested that it has a low predictive value (Bleem and others 1993, Hesketh and others 1994).

There are at present no in vitro diagnostic methods for TB in South American camelids that have been properly validated in a large number of animals of known infection status. Serological tests have been in development since the 1980s, including ELISA tests based on M bovis purified protein derivative (PPD) deoxycholate extract and the MPB70 antigen, but these have not convincingly discriminated between natural infection and vaccination, and issues of cross-reactivity with other mycobacteria have not been addressed (Thoen and others 1980, 1988, Johnson and others 1989, Hesketh and others 1994, Sugden and others 1997, Stevens and others 1998). In some animal species, for example, cattle (Lyashchenko and others 1998, 2004) and white-tailed deer (Odocoileus virginianus) (Waters and others 2004), there is variation between individual animals in the serological response to certain antigens following M bovis infection. Accordingly, assays have been developed that use multiple antigens in an attempt to improve immunodiagnosis.

A serious outbreak of TB involving a large British llama herd (Twomey and others 2007) provided an opportunity to evaluate two of these serological assays, the VetTB STAT-PAK (rapid test [RT]) (Waters and others 2006) and the multiantigen print immunoassay (MAPIA) (Lyashchenko and others 2000), in naturally infected camelids. The RT is a portable lateral flow chromatographic assay that uses three Mycobacterium tuberculosis-complex specific antigens, MPB83, ESAT-6 and CFP-10, bound to a nitrocellulose membrane. Antibody present in serum binds to coloured latex beads and produces a coloured band on the membrane. The MAPIA utilises a range of antigens, shown in Fig 1, printed on to nitrocellulose strips that are incubated with serum samples. Antibody present in the serum of sensitised animals is visualised using conjugated anti-protein A. This short communication reports the results of these tests when used in a llama herd affected with TB caused by M bovis infection.

FIG 1

Antigen recognition patterns by antibody from 14 llamas (Lama glama) with visible lesions typical of bovine tuberculosis, and a negative control sample (Neg) in the multiantigen print immunoassay test

Fourteen of 15 animals with grossly visible lesions of TB at postmortem examination had been blood sampled antemortem and were included in the study. Thirteen of the animals were sampled postmortem for mycobacterial culture and Ziehl-Neelsen (ZN) staining to confirm their TB status: all 13 were positive by ZN and 12 yielded M bovis by culture. The results of serological testing, alongside the skin test, culture and ZN results, are summarised in Table 1. The time intervals between these tests for individual llamas are not given in this report; a full description of the outbreak, including a timescale over which the tests were performed, will be published separately. All 14 animals were positive by MAPIA, and 11 of them (78·6 per cent) were positive using RT. Two of the RT-positive animals showed a positive response only when blood samples were taken three weeks after a skin test. This suggested that the sensitivity of the RT was increased after a skin test, although this observation was not statistically significant. Only two of the 14 llamas showed positive results to the comparative postaxillary tuberculin skin test.

Fig 1 shows the MAPIA results for the 14 llamas, alongside a negative control sample. There were differences in the antigen responses and strength of responses between the animals. MPB83 was the serodominant antigen, with all animals responding to either the individual antigen or the Acr1/MPB83 fusion protein. This is similar to observations in cattle and white-tailed deer infected with M bovis (Lyashchenko and others 2004, Waters and others 2004), and in South American camelids infected with Mycobacterium microti (Lyashchenko and others 2007). Responses were detected with lower frequency to ESAT-6, CFP-10, MPB59, MPB70, Acr1 and PPD-B, as well as the fusion protein CFP-10/ESAT-6. There were no responses to the MPB64 or 38kDa antigens.

In the present study, MAPIA yielded more positive results overall than other antemortem tests; however, it was not shown to be statistically more sensitive than RT. A similar observation has been made in a study of South American camelids infected with M microti, where more animals responded to MAPIA than RT, although this was also based on a small number of animals (Lyashchenko and others 2007). Given the response to antigens in MAPIA that are not currently included in RT, it might be useful to incorporate some of these antigens into the RT cassette to improve its sensitivity, especially as RT is less complex to manufacture and easier to use than MAPIA for routine testing.

In conclusion, this study demonstrates the limitation of skin testing for detecting diseased animals in this herd of llamas, and shows that both MAPIA and RT have potential value as in vitro tests for TB in South American camelids. In order to support the routine use of these tests in diagnosis of TB in camelids, further studies are required using animals known to be uninfected to determine the specificity of the tests. This is particularly important to both policymakers and herd owners, given that a substantial number of seropositive llamas were removed from this herd but had no postmortem evidence of infection (data not shown). Additionally, it would be helpful to use an electronic reader for the assays so that a standardised cut-off point could be determined, rather than relying on the subjective interpretation of test read-outs that is currently used. The application of these tests in Great Britain is currently restricted to tuberculin skin test-negative animals in herds of South American camelids with confirmed M bovis infection, subject to the herd owner's consent, using blood samples taken between 10 and 30 days after the skin test.

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