Proceedings of the 10th International Colloquium on Paratuberculosis

different procedures: Hexadecylpyridinium Chloride (HPC) solution and Herrold´s Yolk Agar
medium (HEYM) with Mycobactin J and ANV and 4% NaOH and 5% oxalic acid and
Lowestein-Jensen medium (LJ) with Mycobactin J and PACT. Slants <strong>of</strong> <strong>the</strong> two media were
incubated for maximum 20 weeks and checked at 1-2-week interval. Colonies with
mycobacterial morphology were stained by using <strong>the</strong> Ziehl-Neelsen staining method and
were sub-cultivated on HEYM and on LJ, respectively. Bacteria were also fur<strong>the</strong>r analyzed
using <strong>the</strong> PCR methods described above. MAP negative bacteria were analyzed to
determine its identity using a PCR to amplify <strong>the</strong> 16S rRNA gen using universal primers.
PCR products were sequenced. Sequences were compared with <strong>the</strong> public sequence
database RIDOM for similarity-based species identification.
RESULTS
Districts, herds’ sizes and number <strong>of</strong> animals sampled in every herd, as well as serological
and PCR results are presented in Table 1 and Table 2, respectively. Ten percent (31/315),
268 (87%) and 8 (2.6%) <strong>of</strong> <strong>the</strong> samples were positive, negative and doubtful, respectively
with ELISA A (Table 1). Seventy percent (10/14) <strong>of</strong> <strong>the</strong> herds were considered positive, when
having at least one ELISA A-seropositive animal. From 39 positive and doubtful samples in
ELISA A, two animals (5.1%) belonging to two different herds (herds 9 and 10) and two
different districts (districts E and F) were also positive with ELISA B, 37 (94%) were negative
and none was doubtful (Table 1). All doubtful results in ELISA A were negative with ELISA B.
Six fecal samples from <strong>the</strong> 31 serological positive animals with ELISA A were positive
in <strong>the</strong> nested PCR (Table 2). One positive animal in <strong>the</strong> real-time PCR were also positive in
<strong>the</strong> nested PCR (Table 2). Only 16% and 6.5% <strong>of</strong> <strong>the</strong> ELISA A-positive animals were positive
in <strong>the</strong> nested PCR and real-time PCR, respectively. Samples from herds in which just one
positive animal were detected with ELISA A, were always negative in <strong>the</strong> PCR tests (Table
2). Cultivation <strong>of</strong> feces was negative for MAP in all inoculated samples in all inoculated
media. However atypical mycobacteria were isolated from LJ medium. HEYM showed lower
contamination compared to LJ medium. Atypical mycobacteria isolates were confirmed as
acid-fast rod-shape bacteria in <strong>the</strong> Ziehl-Neelsen stain and were identified as Mycobacterium
engbakii (99.78% <strong>of</strong> similarity in RIDOM) by sequencing <strong>of</strong> <strong>the</strong> amplified 16S rRNA gen.
Table 1. Paratuberculosis ELISA A-test results <strong>of</strong> herds from a dairy region in Colombia
Herd District
Herd
size
Samples Elisa A Elisa B1
positive negative doubtful positive negative doubtful
1 A 102 20 3 17 0 0 3 0
2 B 75 19 0 19 0 nd 2 nd nd
3 C 146 21 3 17 1 0 4 0
4 274 29 1 28 0 0 1 0
5
6
D
108
81
19
25
0
3
18
19
1
3
0
0
1
6
0
0
7
116 23 1 22 0 0 1 0
8
9
E
96
144
20
22
4
6
16
13
0
3
0
1
4
8
0
0
10 F 226 23 5 18 0 1 4 0
11
G
80 20 1 19 0 0 1 0
12
76 20 0 20 0 nd nd nd
13 H 77 21 4 17 0 0 4 0
14 I 40 25 0 25 0 nd nd nd
307 31 268 8 2 37 0
1
Performed only to positive and doubtful samples with Elisa A
2
Not determined
98