Transcript PowerPoint-presentasjon

Detection of Mycobacterium avium subsp. paratuberculosis in
caprine feces using Adiavet® Paratb real time kit
Johansen TB1*, Nilsen S1, Lindheim D2, Sølverød I2, Olsen I1and Djønne B1
1Norwegian Veterinary Institute, PO box 750 Sentrum, N-0106 OSLO, Norway
2 Healthier Goats, project for eradication of CAE, CLA and Johne’s disease in Norwegian goats, TINE Norwegian Dairies BA, Ås, Norway
*[email protected]
Introduction
Mycobacterium avium subsp. paratuberculosis (Map) is the
causal agent of paratuberculosis or Johnes disease in
ruminants. In Norway, the disease has created problems
for the goat industry [1]. This study was initiated as a part
of a project; ”Healthier Goats, project for eradication of
CAE, CLA and Johne’s disease in Norwegian goats”
(http://geithelse.tine.no/English). A problem in the
follow-up of the herds after eradication is the long time
needed for culture of Map. There is therefore a need for
rapid diagnosis.
The aim of this study was to validate a commercial real
time PCR kit for detection of Map in goat feces under
Norwegian conditions.
Materials and Methods
One hundred and twenty two fecal samples from goats
were analyzed by real time PCR and culture (Table 1). PCR
was performed with Adiavet® Paratb real time kit
(Adiagène, Saint-Brieuc, France), using the company’s new
protocol for DNA isolation, where 3-10 g of feces can be
analyzed, increasing the sensitivity of the method.
Results
The summarized results for the Adiavet® Paratb real time
PCR are given in table 1. Map was detected in all 20
samples from the four naturally infected goats by PCR, but
only in 15 by culture. Real time PCR was able to detect
Map in 22 out of 24 previously culture positive samples.
These samples had been stored at -20°C in up to three
years, providing an explanation for the reduced sensitivity
compared to culture for these samples. 18 previously
culture negative samples from positive herds were also
analyzed, and Map was detected in three samples. The
specificity was confirmed as all 60 samples from goats in
known paratuberculosis free areas were both PCR and
culture negative. Table 2 compares ct values from the real
time PCR assay and culture results for each of the 20 fecal
samples from four naturally infected goats.
Table 1. Results real time PCR and culture on goat fecal samples
Material
PCR + PCR - Culture + Culture - Total
Naturally infected goats *
Goats from
paratuberculosis free
areas
Previously culture
positive samples**
Previously culture
negative samples, known
positive herds**
20
0
15
5
20
0
60
0
60
60
22
2
24
0
24
3
15
0
18
18
Table 1: Real time PCR and culture of 122 caprine fecal samples.
*Four goats naturally infected with Map, sampled five times each.
**Stored at -20°C, originating from 2008-2010.
Table 2. Culture vs PCR
Goat no.
Culture CT value
7029 day 1
+
32,60
7029 day 2
-
34,88
7029 day 3
+
32,27
7029 day 4
-
34,33
7029 day 5
+
35,03
7037 day 1
+
17,94
7037 day 2
+
16,44
7037 day 3
+
17,15
7037 day 4
+
18,49
7037 day 5
+
18,60
7041 day 1
+
31,66
7041 day 2
+
34,86
7041 day 3
-
29,80
7041 day 4
+
33,96
7041 day 5
+
35,13
7057 day 1
+
38,10
7057 day 2
-
34,90
7057 day 3
-
38,55
7057 day 4
+
33,33
7057 day 5
+
34,76
Table 2: Results from culture and
real time PCR analysis of feces
from four goats natuarlly
infected with Map. Each goat was
sampled five times.
Photo: Birte Graeber, NVI
Conclusions
Goat with Johne’s disease. Photo: Nils Leine
Acknowledgement
Funded by: Adiagène, member of the AES Laboratory Group, and
“Healthier Goats, project for eradication of CAE, CLA and Johne’s
disease in Norwegian goats” and the Norwegian Veterinary Institute
Reference
1. Djonne B: Paratuberculosis in goats--a special focus on the
Nordic countries. Acta Vet Scand 2003, 44: 257-259.
• The Adiavet® Paratb real time kit (Adiagène) worked
well for analysis of goat feces in Norway.
• The sensitivity was better than culture in the tested
material, but testing of more samples is required for
statistical interpretation. The possibility to analyse 3 g
or more feces is valuable for diagnosis.
• None of the samples from paratuberculosis free herds
were positive in the real time PCR, confirming the
specificity of the test.
• Culture still is the gold standard for diagnosis of Map,
however real time PCR decreases the time of diagnosis
in goats from 12-16 weeks to two days. This is of great
importance in an eradication programme like “Healthier
Goats.”