Use of a PCR method on fecal samples for diagnosis of sheep paratuberculosis

Abstract

The high sensitivity of PCR compared to the difficulties of fecal culture in sheep prompted the development of PCR protocols for detection of Mycobacterium avium subsp. paratuberculosis DNA in sheep feces. Although the PCR itself is well developed, and does not pose large technical problems, concentrating the bacteria from samples that may contain low numbers of bacilli using practical methods is still the main difficulty for the use of this technique. In this study, we describe an extraction protocol for the concentration and purification of M. avium subsp. paratuberculosis DNA from fecal samples and we compare it with other methods. The diagnostic performance of the freeze-boiling method was evaluated using a reference method [Vet. Rec. 134 (1994) 95] on fecal samples from a group of selected sheep from different flocks of known individual serological, pathological, and cultural paratuberculosis status. Using, as a reference, a combination of results in those conventional methods, the freeze-boiling PCR protocol showed a sensitivity of 94.1%, and a specificity of 92.3%.

Introduction

Fecal culture has been considered for years as the most sensitive, specific and precocious method for detection of clinical and subclinical paratuberculosis in cattle (Chiodini et al., 1984; Whitlock et al., 1995, Whitlock et al., 1997). Although it is still a powerful tool for control of paratuberculosis in this species, its extensive use has been hampered by its high cost and by its slow outcome. In sheep, the situation is even worse since many ovine strains do not grow well on artificial media (Cranwell, 1988; Collins et al., 1990; Juste et al., 1991; Huchzermeyer and Bastianello, 1992), and the ratio of cost of analysis to individual sheep value is much higher (Juste and Aduriz, 1990). As a consequence, the development of PCR techniques was received with enthusiasm as a promising alternative for specific detection of Mycobacterium avium subsp. paratuberculosis in pathological samples within a shorter delay time. Although there are some reports on the use of PCR techniques for diagnosis of paratuberculosis (van der Giessen et al., 1992a; Collins et al., 1993; Gwóźdź et al., 1997; Lillini et al., 1997), its use has not become widespread. One of the reasons for this seems to be that the preparatory phase (bacteria concentration, DNA extraction and purification steps) does not perform well enough as to make PCR a reliable alternative to fecal culture.

In this study, departing from a bacteria concentration technique used for fecal culture, we have tested several modifications aimed to increase performance of a PCR method by improving the balance between DNA extraction efficacy and workability that, ultimately, could make PCR useful for routine diagnosis.