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, Norway2 Healthier Goats, project for eradication of CAE, CLA and Johne’s disease in Norwegian goats, TINE Norwegian Dairies BA, Ås,

Norway *tone.johansen@vetinst.no

IntroductionMycobacterium 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 MethodsOne 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.

ResultsThe 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.

Conclusions• 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.”

Table 1. Results real time PCR and culture on goat fecal samples

MaterialPCR

+PCR -

Culture +

Culture -

Total

Naturally infected goats *

20 0 15 5 20

Goats from paratuberculosis free areas

0 60 0 60 60

Previously culture positive samples**

22 2 24 0 24

Previously culture negative samples, known positive herds**

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.Cultur

eCT

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

AcknowledgementFunded 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

Reference1. Djonne B: Paratuberculosis in goats--a special focus on the Nordic countries. Acta Vet Scand 2003, 44: 257-259.

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.

Goat with Johne’s disease. Photo: Nils Leine

Photo: Birte Graeber, NVI