Improved detection of Mycobacterium avium subsp. paratuberculosis in milk by immunomagnetic PCR
Introduction
The possible link between Mycobacterium avium subsp. paratuberculosis (M. ptb) and Crohn’s disease in humans has been the subject of increasing debate in recent years (Chiodini, 1989; Thompson, 1994; Travis, 1995; Chiodini and Rossiter, 1996; McFadden and Fidler, 1996). Although a definite association is still not proven, the suggestion has been made that milk could be a possible vehicle of transmission of M. ptb from cattle with Johne’s disease to humans (Hermon-Taylor, 1993). Certainly several studies provide evidence that M. ptb is secreted into the milk of a proportion of cattle clinically and subclinically infected with Johne’s disease (Taylor et al., 1981; Sweeney et al., 1992; Streeter et al., 1995). Recent studies suggesting that M. ptb may survive pasteurisation of milk have provoked interest in testing milk supplies for the presence of this organism. However, there is currently no reliable, simple, rapid method to detect M. ptb infection of milk.
Molecular techniques such as PCR could potentially provide a rapid means of detecting M. ptb in milk. However, it is well documented that the PCR reaction can be inhibited by substances present in foods and clinical specimens, and milk is a substrate which is known to be inhibitory to the PCR reaction. The whey fraction of milk, which is high in Ca2+ ions, is particularly inhibitory to the PCR reaction, and the cream and pellet fractions also show some degree of inhibition (Bickley et al., 1996). Appropriate sample preparation prior to PCR detection of organisms in foods is crucial in order to ensure that the PCR reaction is able to proceed under optimal conditions to maximise sensitivity (Lantz et al., 1994). Previous approaches taken to facilitate the application of PCR to milk have included: enzymatic digestion followed by membrane filtration (Starbuck et al., 1992); dilution of the milk sample (Bickley et al., 1996); and extraction and purification of DNA from the milk sample (Hotzel et al., 1996; Millar et al., 1996). Most of these procedures significantly lengthen the time required to determine the presence/absence of an organism in a particular milk sample.
Recently, we described a novel immunomagnetic separation (IMS) method for the isolation of M. ptb from milk (Grant et al., 1998). IMS relies upon the interaction between cell surface antigens and antibodies attached to paramagnetic beads to effect the separation of the desired organism from a heterogeneous suspension of micro-organisms, such as are found in food or clinical samples. At the same time the desired organism is effectively separated from substances in milk which could inhibit the PCR reaction. In theory, this IMS method could maximise the sensitivity of IS900 PCR applied to milk. Use of IMS in conjunction with PCR (IMS–PCR) has previously been described for the detection of Salmonella spp. (Widjojoatmodjo et al., 1991, Widjojoatmodjo et al., 1992) and M. avium (Li et al., 1996) in faeces samples. This paper describes a series of experiments carried out to evaluate the potential use of IMS in combination with IS900 PCR as a novel IMS–PCR assay for screening milk samples for the presence of M. ptb.
Section snippets
Milk samples
In experiments to evaluate the potential use of IMS–PCR involving spiked milk, raw milk aseptically obtained from healthy Friesian cows was inoculated with M. ptb type strain NCTC 8578, or field strains B2 or B4 previously isolated from cattle in Northern Ireland. Culture conditions and inoculum preparation were as previously described (Grant et al., 1996).
Naturally infected milk was obtained from several sheep in a local flock with a history of Johne’s disease. Prior to lambing, the blood of
Minimum detection limits of direct IS900 PCR and IMS–PCR
The application of IMS to a milk sample improved the sensitivity of IS900 PCR compared to PCR applied directly (Fig. 1). IMS prior to PCR resulted in increased signal intensity compared to direct PCR, and enabled the detection of lower concentrations of M. ptb. The minimum detection limit of the IMS–PCR assay applied to 50 ml aliquots of milk, centrifuged and resuspended in 1 ml PBS-T prior to IMS, was estimated to be 103 CFU/50 ml on the basis of spiking experiments. The minimum detection limit of
Discussion
The IMS–PCR assay described in this report represents a novel, sensitive and specific method of detecting the presence of M. ptb in milk. Spiking experiments indicated that the minimum detection limit of the IMS–PCR assay was in the region of 103 CFU/50 ml of milk, which was considerably lower than for direct IS900 PCR (>105 CFU/50 ml). It is recognised that PCR will only detect the DNA of M. ptb, and therefore an IS900 PCR positive result does not necessarily indicate the presence of viable M. ptb
Acknowledgements
This research was funded by the Ministry of Agriculture, Fisheries and Food, United Kingdom. We are grateful to Dr. Jane Bickley and Mrs. Rajagopalan Swarnageetha at LGC for independently testing milk samples by IS900 PCR during the blind trial.
References (22)
- et al.
Paratuberculosis: a potential zoonosis?
Vet. Clin. N. Am.
(1996) Causation of Crohn’s disease: the impact of clusters
Gastroenterology
(1993)- et al.
Sample preparation methods in PCR-based detection of food pathogens
Trends Food Sci. Technol.
(1994) - et al.
Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions
Lett. Appl. Microbiol.
(1996) Crohn’s disease and the mycobacterioses: a review and comparison of two disease entities
Clin. Microbiol. Rev.
(1989)- et al.
Inactivation of Mycobacterium paratuberculosis in cow’s milk at pasteurization temperatures
Appl. Environ. Microbiol.
(1996) - et al.
Isolation of Mycobacterium paratuberculosis from milk by immunomagnetic separation
Appl. Environ. Microbiol.
(1998) - et al.
Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction
J. Appl. Bacteriol.
(1996) - et al.
Rapid detection of Mycobacterium avium in stool sample from AIDS patients by immunomagnetic PCR
J. Clin. Microbiol.
(1996) - et al.
Mycobacteria as possible causes of sarcoidosis and Crohn’s disease
J. Appl. Bacteriol.
(1996)
IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows’ milk in England and Wales
Appl. Environ. Microbiol.
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2016, Brazilian Journal of MicrobiologyCitation Excerpt :As a result, the detection limit of the IMS-IS1 PCR procedure reached 101 CFU/mL that is comparable to that described by Mundo et al.17 and Khare et al.2 by radiometric detection and real-time PCR, respectively. This limit of detection is higher than those described by Grant et al.,14 Metzger-Boddien et al.,28 and Stratmann et al.15 but slightly lower than those described by Foddai et al.29 who used specific antibodies or peptides to detect Map by immunomagnetic separation techniques. IMS-IS1 PCR procedure was assessed under field conditions, and the results were compared with those obtained by milk culture, fecal culture, and a serum in-house ELISA.