Abstract Book of EAVLD2012 - eavld congress 2012

S3 - P - 14
DEVELOPMENT OF MULTISPECIES SEROLOGICAL ASSAYS TO DETECT ANTIBODIES SPECIFIC
FOR Mycobacterium bovis IN SERUM SAMPLES
Lisset López 1 , Ana Ranz 1 , Angel Venteo 1 , Teresa Pérez 1 , Tamara Ruiz 1 , L DeJuan 2 , Beatriz Romero 2 , Mariana
Boadella 3 , Christian Gortázar 3 , Beatrice Boniotti 4 , Paolo Pasquali 5 , Paloma Rueda 1
1 1 INGENASA, Madrid Spain; 2 VISAVET, Madrid, Spain; 3 IREC, Barcelona, Spain; 4 I.Z.S. della Lombardia e dell'Emilia, Brescia, Italy ; 5 Istituto Superiore di
Sanità di Roma, Roma, Italy
Tuberculosis, PEN-SIDE TEST, DIAGNOSTIC, ELISA DR
Introduction
Bovine tuberculosis (TB) is a chronic bacterial disease of
animals and humans caused by M. bovis. In a large number of
countries bovine tuberculosis is a major infectious disease
among cattle, other domesticated animals, and certain wildlife
populations. Transmission to humans constitutes a public health
problem. For years eradication programmes have been carried
out. Traditional mycobacterium culture remains the gold
standard method for routine confirmation of infection but
Delayed Hypersensitivity test and gamma-interferon test are the
ones used in these programmes. Detection of antibodies
specific could be a useful alternative for TB diagnosis. Different
antibodies detection assays have been described although all of
them present sensitivity problems
The aim of this study has been to develop and validate
multispecies assays based on Double Recognition ELISA
(ELISA-DR) and Immunochromatography (IC) techniques for
detection of antibodies specific of M. bovis. using the
recombinant protein MPB83, expressed in insect cells.
Material & methods
Recombinant proteins ESAT6, CFP10, MPB64, MPB70 y MPB
83 were obtained, expressed and purified. Finally, MPB83 was
selected as the antigen for serological assays (ELISA and IC
assays)
To validate these assays, sera from different species previously
catalogued by gamma interferon assay (72 bovine sera), by
culture (46 buck, 205 wild boar) or by necropsy (205 deer and
138 wild boar experimental sera) were used. Moreover, cross
reactivity with M. avium subsp. paratuberculosis was
determined. For this propose sera and plasma of 9 cows and 9
goats positive to Paratuberculosis and negative to TB were
used.
Samples from different wild species such as alpaca, elephant
and American bison are going to be tested. In addition sera
collected from wild boar and deer are being evaluated.
Table 2.
Ref: ELISA DR
SPECIES N Accuracy
CATTLE 13 77%
WILD BOAR 200 90%
BUCK 46 89%
DEER 22 77%
Discussion & conclusions
INGENASA has developed two multispecies assays based on
the use of the recombinant protein MPB83 expressed in insect
cells. More than 300 samples from different species have been
analyzed giving high percentages of sensitivity and specificity.
Although higher number of samples is necessary, preliminary
results suggest that both diagnostic approaches are very useful
methods for control and surveillance of TB infection.
The immunochromatographic assay can become a useful tool in
situations where laboratory support and skilled personnel are
limited.
References
1. Schiller et al. Transboundary and Emerging Diseases. (2010),
57:205-220
2. Boadella et al. Preventive Veterinary Medicine (2012), 104:160-164
3. Waters et al. Clinical and Vaccine Immunology (2011), 18:1882-1888
(KBBE-2007-1-3-04) Strategies for the eradication of bovine tuberculosis
(TB-STEP)
Results.
Preliminary results show that both assays detect antibodies
specific of TB. None of them showed cross-reaction with
antibodies to PTB. Depending on the species analyzed,
sensitivity and specificity of ELISA-DR range between 70-100%
and 92-97%, respectively (Table I). Concerning to IC, accuracy
with respect to ELISA DR ranged between 77 and 90%
depending on the species studied (TABLE II).
Table 1
ELISA-DR
SPECIES N Sensitivity Specificity REFERENCE
CATTLE 72 88% 96% Bovigam®
WILD BOAR 138 100% 97% Necropsy
WILD BOAR 200 82% 92% Culture
BUCK 46 70% 94% Culture
DEER 205 93% 92% Necropsy