Incidence of Mycobacterium avium subspecies paratuberculosis in bulk-tank milk samples from different regions in Switzerland.

Mycobacterium avium subspecies paratuberculosis (MAP) is a pathogen causing chronic inflammation of the intestine in many animals, including primates, and is considered to be implicated in Crohn's disease in human beings (HermonTaylor and others 2000). Crohn's disease is a chronic granulomatous ileocolitis of humans, with significant similarity to paratuberculosis. High herd prevalences of MAP have been found in dairy cattle in particular (Thorne and Hardin 1997). A recent USDA survey indicated that 41 per cent of dairy herds in the USA have at least one MAP ELISA-positive animal. Roughly 22 per cent of all dairy herds in the USA have at least 10 per cent of animals testing positive (Collins 1998). It has long been known that MAP can be cultured from the milk of cows clinically infected with paratuberculosis (Taylor and others 1981). Recent work has shown that MAP can also be cultured from the milk of apparently healthy, subclinically infected cows (Sweeney and others 1992, Streeter and others 1995). Moreover, thermal tolerance data indicate that this organism is more heat resistant than other Mycobacterium species and is capable of surviving the pasteurisation of raw milk (Chiodini and Hermon-Taylor 1993, Grant and others 1998, Sung and Collins 1998). A resistance to pasteurisation and a possible causal connection to Crohn's disease means that MAP is a significant food-safety issue. The aim of this study was to collect prevalence data of MAP in bulk-tank milk samples from different regions in Switzerland and to compare these results with existing data from the literature. A total of 501 raw milk samples originating from three different regions in Switzerland (north-east, central and north-west) were screened for the presence of MAP. The samples were collected between February and April 2000, and February and March 2001, and were kept frozen until investigation. Samples of milk (10 ml) were mixed with 100 111 Triton X-100 (Calbiochem) and centrifuged at 1600 g for 30 minutes. The resulting pellet was then transferred into blue ribolyser tubes (Hybaid), resuspended in 400 111 mycobacterial lysis buffer (2mM ethylenediamine tetraacetic acid [ EDTA], 400mM sodium chloride, 10mM TrisHCl [ pH 8-0],0-6 per cent sodium dodecylsulphate, 331 t1/ml proteinase K) and incubated overnight at 37°C. The samples were centrifuged at 6-6 m/second for 45 seconds in a ribolyser (Hybaid) to facilitate DNA extraction. DNA was then extracted with phenol/chloroform/isoamylalcohol (25:24:1) and precipitated with 3M potassium acetate for 30 minutes at -70°C, centrifuged (13,000 g for 15 minutes), washed in 70 per cent ethanol, dried and resuspended in TriS-EDTA buffer. A sample of the resuspended DNA (5 111) was used for an isgoo nested PCR specific to MAP. The primers p90 5'-GAAGGG TGTTCGGGGCCGTCGCTTAGG-3' and p915'-GGCGTTGAGGTCGAT CGCCCACGTGAC-3' were used for the first 30 amplification cycles in accordance with Hermon-Taylor and others (2000).