Abstract Book of EAVLD2012 - eavld congress 2012

S1 - P - 15
ACCURACY OF PARATUBERCULOSIS ANTIBODY ELISA AT PREDICTING FECAL SHEDDING OF
MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS IN CATTLE
Rakel T. Fernández 1 , Carmen Eiras 2 , Carmen Calvo 2 , Ignacio Arnaiz 2 , Fco. Javier Diéguez 1,3
1
Santiago de Compostela University (Veterinary Faculty), Department of Anatomy and Animal Production, Lugo, Spain
2
Animal Health and Production Laboratory, Lugo, Spain
3
Santiago de Compostela University (Veterinary Faculty), Institute of Food Analysis and Research, Lugo, Spain
Paratuberculosis, bovine, ELISA, fecal culture
Introduction
Paratuberculosis, also called Johne's disease, is chronic
granulomatous enteritis caused by Mycobacterium avium subsp.
paratuberculosis (MAP). This infectious disease has a worldwide
distribution affecting mainly ruminants, both domestic and wild,
but has also been isolated from other animal species. It causes
great economic losses in cattle farming 1 .
ELISA is an essential tool in paratuberculosis control programs,
although bacterial culture remains the reference confirmatory
test. In this line, the aim of this study was to determine the
accuracy of the ELISA test as a predictor of the MAP fecal
elimination "status" (determined by means of fecal culture) of
individual animals.
Materials & methods
The study was carried out in Galicia (north-west Spain). Galicia is
the major cattle-farming region of Spain. It was responsible for
35% of the milk and 12% of the beef produced in Spain,
constituting approximately 1.7% of the milk and 1.3% of the beef
produced in the European Union.
Bacteriological culture was performed in fecal samples from 239
animals that were collected during the period 2007-2010. 181
animals were negative to fecal culture and 58 were culture
positive animals. Serological analysis (ELISA) was also
performed in serum samples from the same animals.
Bacterial culture was performed as described by the World
Organization for Animal Health (OIE) (2008) 2 . Briefly, 1 g of feces
was added to 20 mL of sterile distilled water, and tubes were
shaken for 30 min and then allowed to stand undisturbed for 30
min. Five millilitres of the supernatant were added to 0.75%
hexadecylpyridinium chloride (HPC) (Sigma). Tubes were
inverted several times and allowed to stand undisturbed for 18 h
at room temperature for decontamination. Triplicate Herrold’s egg
yolk medium (HEYM) culture slopes containing amphotericin B,
vancomycin and nalidixic acid were inoculated with 0.1 mL of the
undisturbed sediment, incubated at 37º C and observed at 2-
week intervals for 16 weeks. Suspect colonies were evaluated for
mycobactin dependence along with morphology and acid-fast
staining. Positive-staining colonies were confirmed by PCR. For
every nine fecal samples a positive control (a positive field
sample) was included.
The ELISA used was “PARATUBERCULOSIS ANTIBODY
SCREENING” (Institute Pourquier, France). False-positive results
were reduced by pre-absorbing the samples with sonicates of the
environmental mycobacterium Mycobacterium phlei. Samples
were considered positive at a % sample:positive ratio of 55% or
more.
Receiver operating characteristic (ROC) procedure was used to
evaluate the overall diagnostic accuracy to estimate the antibody
titer that was the best cut-off point in terms of sensitivity and
specificity as a predictor of the bacteriological "status" of the
animals (culture positive/negative).
Results
The ROC curve -as measure of the accuracy of ELISA antibody
titers to predict the bacteriological “status”- indicated a good
diagnostic discrimination (Figure 1). The area under the curve
was 0.878. The best overall diagnostic precision was in the cutoff
point 155.7 with sensitivity of 84.5% and specificity of 78.9%
(Table 1).
Figure 1: ROC curve for overall accuracy of antibody titers as a
predictor of MAP bacteriological “status”
Table 1: Sensitivities and specificities obtained from ROC
procedure
Cut off point Sensitivity 1-Specificity
71.4 1.00 0.81
133.1 0.91 0.34
147.2 0.88 0.26
155.7 0.84 0.22
160.1 0.83 0.20
164.2 0.81 0.18
167.7 0.78 0.17
309.2 0.00 0.01
Discussion & conclusions
Traditionally the interpretation of ELISA is dichotomous (positive
or negative) based on the cut-off designed to optimize the
sensitivity and specificity to detect animals with antibodies. The
use of antibody titers on a continuous scale can improve the
amount of diagnostic information obtained by this technique. The
ELISA evaluated in this study, as is the case of previous studies
with different ELISAs 3 could allow a more accurate estimate of
the situation of the animal, in this case related to the
bacteriological “status”.
References
1. Kennedy, DJ, Benedictus, G (2001). Control of Mycobacterium avium
subsp. paratuberculosis infection in agricultural species. Scientific and
Technical Reviews, 20, 151-79.
2. World Organization for Animal Health (OIE) (2008). Manual of diagnostic
test & vaccines for terrestrial animals, Chapter 2.1.11.
3. Collins, MT (2002). Interpretation of a commercial bovine
paratuberculosis enzyme-linked immunosorbent assay by using likelihood
ratios. Clinical and Diagnostic Laboratory Immunology, 9, 1367-71.