Proceedings of the 10th International Colloquium on Paratuberculosis

Table 2. Paratuberculosis PCR results <strong>of</strong> herds from a dairy region in Colombia
Nested PCR a Real-Time PCR b
Herd District
Elisa A-
positive positive negative positive negative
1 A 3 1 2 1 2
2 B 0 nd nd nd nd
3 C 3 2 1 0 3
4 1 0 1 0 1
5
6
D
0
3
nd
0
nd
3
nd
0
nd
3
7
1 0 1 0 1
8 E 4 1 3 0 4
9 6 1 5 0 6
10 F 5 0 5 1 4
11 G 1 0 1 0 1
12 0 nd nd nd nd
13 H 4 0 4 0 4
14 I 0 nd nd nd nd
31 5 26 2 29
a
Performed only to positive samples with Elisa A
b Performed only to positive samples with Elisa A
c Not determined
DISCUSSION
Our preliminary hypo<strong>the</strong>sis was <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis in a higher proportion <strong>of</strong>
animals by using <strong>of</strong> indirect and direct methods. In <strong>the</strong> same way it was expected a higher
concordance between <strong>the</strong> different tests used. However confirmation <strong>of</strong> all ELISA Aseropositive
animals with ELISA B was not achieved. Differences in antigens LAM in ELISA
A and protoplasmic antigen in ELISA B is a possible explanation <strong>of</strong> <strong>the</strong> poor concordance
between both tests. In addition <strong>the</strong> use <strong>of</strong> <strong>the</strong> pre-absorption phase in <strong>the</strong> ELISA B could
reduce <strong>the</strong> number <strong>of</strong> false positives, but also <strong>the</strong> detection <strong>of</strong> true positives (McKenna et al.,
2005). On <strong>the</strong> o<strong>the</strong>r hand, a cross-reaction with o<strong>the</strong>r mycobacteria could have played an
important role in <strong>the</strong> positive results obtained with <strong>the</strong> ELISA A. The capacity <strong>of</strong> o<strong>the</strong>r
mycobacteria to interfere with pre-absorbed ELISA serological test in cattle has been
demonstrated (Muskens et al., 2007).
The poor agreement between PCR and serology contradicted <strong>the</strong> proposed idea that
<strong>the</strong> combined use <strong>of</strong> ELISA and fecal PCR has <strong>the</strong> potential to increase <strong>the</strong> overall sensitivity
for <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis infection in dairy cows. The low shedding rate <strong>of</strong> MAP
organisms in subclinical animals can make real time-PCR equal or less sensitive than ELISA
(Wells et al., 2006). On <strong>the</strong> o<strong>the</strong>r hand, although some primers use in <strong>the</strong> PCR systems have
shown adequate results (e.g. F57), o<strong>the</strong>rs have shown limitations (e.g. ISMav2). In addition,
single-round PCR has been evaluated as very reliable, but <strong>the</strong> used <strong>of</strong> nested PCR assays
could lead to high contaminations risks (Mobius et al., 2008).
Regarding <strong>the</strong> cultural results, negative samples from ELISA-positive herds could be
explained by <strong>the</strong> low or still absent MAP shedding rate in seropositive animals, by <strong>the</strong> low
sensitivity <strong>of</strong> culture or by <strong>the</strong> culture <strong>of</strong> samples from false positive animals in ELISA A. In
fact, in herds without previous diagnosis or clinical cases <strong>of</strong> paratuberculosis, <strong>the</strong> positive
predictive value <strong>of</strong> an ELISA test is expected to be low (Muskens et al., 2003). ELISA can
predict better <strong>the</strong> presence <strong>of</strong> culture positive animals when <strong>the</strong>se are heavy shedders. The
sensitivity <strong>of</strong> <strong>the</strong> ELISA is higher when a heavy instead a low shedder is tested. Negative
results or false positive results in a cross-sectional study have a low probability <strong>of</strong> deliver a
positive culture, if just a single sampled is planned (Sweeney et al., 2006).
Finally <strong>the</strong> conservation <strong>of</strong> serum and fecal samples at -20°C for several weeks could
have affected ELISA and culture results. Serum-ELISA results can change from positive to
negative (Alinovi et al., 2009) and fecal samples can undergo a significantly reduction <strong>of</strong>
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