Proceedings of the 10th International Colloquium on Paratuberculosis

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Diagnosis <strong>of</strong> paratuberculosis in cattle from a dairy region in Columbia
Fernández-Silva JA 1,2 , Abdulmawjood A 1 , Akineden Ö 1 , Bülte M 1
1 Institut für Tierärztliche Nahrungsmittelkunde, Justus Liebig Universität Giessen; 2 Escuela de
Medicina Veterinaria, Universidad de Antioquia
ABSTRACT
In Colombia serum and fecal samples from 307 asymptomatic lactating Holstein cows over
three years <strong>of</strong> age from 14 herds with no previous diagnosis <strong>of</strong> paratuberculosis were taken.
All Serum samples were analyzed with a lipoarabinomannan based-ELISA (ELISA A).
Positive and doubtful samples in ELISA A were analyzed with a protoplasmic antigens
based-ELISA (ELISA B), including pre-absorption with Mycobacterium phlei. Fecal samples
from animals positive in ELISA A were analyzed using a nested IS900-PCR and a F57 /
ISMav2-real-time PCR. Fecal samples <strong>of</strong> animals from ELISA A-seropositive herds were
decontaminated with 0.75% Hexadecylpyridinium Chloride and cultured on Herrold´s Yolk
Agar medium. The same samples were decontaminated later with 4% NaOH and 5% oxalic
acid and cultured on Lowestein-Jensen medium. Ten percent (31/315) <strong>of</strong> <strong>the</strong> samples and
70% (10/14) <strong>of</strong> <strong>the</strong> herds were positive with ELISA A. Only two animals <strong>of</strong> two different herds
were positive with ELISA B. Six fecal samples were positive with PCR and only one was
simultaneously positive in <strong>the</strong> two PCR types. Serological and PCR results did not always
coincide. Cultivation was negative for paratuberculosis in all samples inoculated. However
Mycobacterium engbaekii was isolated from LJ medium. Results confirm <strong>the</strong> presence <strong>of</strong>
paratuberculosis in dairy herds in Colombia and demonstrate <strong>the</strong> limitations <strong>of</strong> available
diagnostic tests for detection <strong>of</strong> subclinical infections, <strong>the</strong> determinant influence <strong>of</strong> ELISA
type used, <strong>the</strong> low bacterial shedding in <strong>the</strong> cattle feces examined, <strong>the</strong> possible roll <strong>of</strong> o<strong>the</strong>r
mycobacteria and <strong>the</strong> effect <strong>of</strong> conservation on <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis.
INTRODUCTION
Paratuberculosis and <strong>the</strong> circulation <strong>of</strong> its causal agent Mycobacterium avium subsp.
paratuberculosis (MAP) among dairy herds and wild animals in South America have been
already demonstrated. However a consistent prevalence for <strong>the</strong> whole subcontinent or for
some countries has not been yet estimated. In Colombia updated information about <strong>the</strong>
presentation <strong>of</strong> paratuberculosis for dairy cattle is limited. Although <strong>the</strong> disease has been
diagnosed in sheep and in cattle, more information about this disease in Colombia is needed.
The objective <strong>of</strong> this study was <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis in dairy cattle from herds
with no previous diagnosis <strong>of</strong> paratuberculosis, using serological, cultural and molecular
methods in a dairy region in Colombia.
MATERIALS AND METHODS
Three hundred seven Holstein lactating cows over three years old from 14 herds belonging
to 9 districts <strong>of</strong> a municipality in a dairy region in Colombia were sampled. None <strong>of</strong> <strong>the</strong> herds
had a previous diagnosis <strong>of</strong> paratuberculosis and had no clinical cases at <strong>the</strong> moment <strong>of</strong>
sampling. Blood and fecal samples were taken and frozen at -20°C until analysis at <strong>the</strong>
Justus-Liebig-University Giessen (Germany). For screening all serum samples, a commercial
lipoarabinomannan (LAM) based indirect ELISA without pre-absorption (ELISA A) was used.
For verification <strong>of</strong> positive and doubtful ELISA A samples, a commercial test based on
detection <strong>of</strong> antibodies to protoplasmic MAP antigens with pre-absorption with
Mycobacterium phlei (ELISA B) was used. A herd was considered seropositive if at least one
animal tested positive with <strong>the</strong> ELISA A. DNA isolation from fecal samples <strong>of</strong> ELISA A
seropositive animals was carried out with a commercial DNA preparation kit (High Pure PCR
Template Preparation Kit, Roche). DNA samples were tested with an IS900 nested PCR
(Bull et al., 2003). Samples were also tested with a real-time PCR that amplify <strong>the</strong> F57 and
<strong>the</strong> ISMav2 molecular targets (Schonenbrucher et al., 2008). All fecal samples from herds
that tested positive in ELISA A and/or in PCR were decontaminated and cultivated using two
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