Abstract Book of EAVLD2012 - eavld congress 2012

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EVALUATION OF AN ELISA ASSAY AS AN ANCILLARY METHOD IN A HERD WITH A NATURAL
TUBERCULOSIS INFECTION
I. Arnaiz 1 , C. Eiras 1 , M.L. Luaces 2 , J. Fraga 3 , M. Rodríguez 3 , C. Calvo 1
1 Laboratorio de Sanidade e Produción Animal de Galicia, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España
2
Área Veterinaria de Lugo, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España
3
Área Veterinaria de Vilalba, Servicio Provincial de Ganadería de Lugo, Consellería do Medio Rural e do Mar, Xunta de Galicia, Lugo, España
Keyword: Tuberculosis, ELISA, IFN-g, SIT, isolation
Introduction
The single intradermal tuberculin (SIT) test and the Interferongamma
assay (IFN-g) are the tuberculosis diagnostic tools in
the tuberculosis eradication program currently used in Spain.
These tests based on cell-mediated response can detect
animals in the early stages of infection (1), although not all
affected animals are detected with these tests. Humoral
immune response is found in cattle with natural infection and
this response could be detected by the use of an ELISA assay.
ELISA testing is not routinely used in bovine tuberculosis
control programs mainly due to a reduced sensitivity (2)
although it has been suggested that it might be used as a
complement to the tuberculin test, especially for the detection
of anergic tuberculous cattle (3).
The aim of this study is the evaluation of the use of SIT test
and the IFN-g assay together with an ELISA assay in order to
obtain the most complete detection of tuberculosis infected
animals in a herd previously confirmed to have tuberculosis.
Material & methods
314 animals from a tuberculosis infected beef herd from
Galicia, in north-west Spain, were tested using the SIT test.
The IFN-g detection assay (BovigamTM, Prionics AG,
Switzerland) was used in parallel with the SIT test. The 16
positive SIT animals (of which 15 animals were also detected
by IFN-g assay) and 17 cattle that were only detected by IFN-g
assay were slaughtered and their tissues were collected for
Mycobacterium tuberculosis complex (MTC) isolation. At the
same time, 20 cows older than 10 years were slaughtered too.
Tissues from the 53 slaughtered animals were inoculated into
the selective media (4). The MTC was recovered and
confirmed by real-time PCR (5) in 18 cattle.
The plasma collected for the IFN-g assay was also used on
the ELISA assay for M. bovis antibody (IDEXX Laboratories).
18 cattle were detected with the ELISA assay.
The agreement and kappa statistic measurements were
calculated by comparing the MTC isolation with SIT test, IFN-g
and ELISA assays respectively.
References
1. Rua-Domenech R, Goodchild AT, Vordermeier HM, Hewinson RG,
Christiansen KH, Clifton-Hadley RS: Ante mortem diagnosis of
tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon
assay and other ancillary diagnostic techniques. Res Vet Sci 2006,
81:190-210.
2.Ritacco V, Lopez B, Barrera L, Nader A, Fliess E, de Kantor: Further
evaluation of an indirect enzyme-linked immunosorbent assay for the
diagnosis of bovine tuberculosis. Zentralbl Veterinarmed B 1990, 37:19-
27.
3. Plackett P, Ripper J, Corner LA, Small K, de Witte K, Melville L,
Hides S, Wood PR: An ELISA for the detection of anergic tuberculous
cattle. Aus Vet J 1989, 66:15-19.
4. Idigoras P., Beristain X., Iturzaeta A., Vicente D., Pérez-Trallero E.
2000. Comparison of the automated nonradiometric BACTEC MGIT
960 system with Löwestein-Jensen, Coletsos, and Middlebrook 7H11
solid media for recovery of mycobacteria. European Journal of Clinical
Microbiology Infections Diseases 19: 350-354.
5. Huard R.C., Oliveira-Lazzarini L.C., Butler W.R., van Soolingen D.,
Ho J.L. 2003. PCR-Basaed method to differentiate the subspecies of
the Mycobacterium tuberculosis complex on the basis of genomic
deletions. Journal of Clinical Microbiology, vol 41, n 4: 1637-1650.
Results
10 bovine with MTC recovered were SIT test positive. 16 were
positive in the IFN-g assay and the ELISA detected 13 of the
18 cattle with MTC recovered (one of them was negative in
SIT test and IFN-g assay). One bovine with MTC recovered
was negative in the three tests; this animal was slaughtered
because it was more than10 years old.
The kappa statistic shows a lower agreement for the SIT test
(0.39) and the IFN-g assay (0.36) with the MTC isolation than
the ELISA assay (0.58).
Discussion & conclusions
The results suggest that the use of IFN-g and ELISA assays
as ancillary techniques might help to detect a larger number of
tuberculosis infected animals in a TB-confirmed herd. These
assays complement each other because they may detect
animals in different stages of tuberculosis.
Acknowledgements
We thanks IDEXX Laboratories, Inc., for providing the ELISA
test for detection of M. bovis antibodies. We also would like to
thank the Official Veterinary Services from Vilalba for assisting
with abattoir sampling, the Regional Animal Health Veterinary
Services for their support and the Laboratory of Animal Health
of Galicia.