Abstract
A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.
Highlights
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic wasting disease of cattle and other ruminants, characterised by a reduction in milk yield, severe diarrhoea, weight loss and intermandibular oedema
The phage-PCR assay detected viable MAP in 10.3 % (37/368) of the pasteurised milk samples collected as defined by the presence of plaques which gave a positive IS900-PCR result
Since each plaque arising from the phage-PCR assay represents detection of one viable MAP cell, counting the plaques allows enumeration of MAP as well as absolute detection
Summary
Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), a chronic wasting disease of cattle and other ruminants, characterised by a reduction in milk yield, severe diarrhoea, weight loss and intermandibular oedema (commonly referred to as bottle jaw). Many studies have shown that the conditions used for pasteurisation are sufficient to inactivate MAP, some studies have shown that both low-temperature long-time (LTLT) and high-temperature short-time (HTST) pasteurisation did not totally inactivate MAP when present in milk at levels greater than 1 x cfu ml-1 (Chiodini and Hermon-Taylor, 1993; Grant et al, 1996). This finding led to dairy processing centres to adjust the holding time of HTST pasteurisation from 15 s (the legal minimum in the UK), to 25 s to increase the likelihood of totally inactivating MAP. MAP has been reported to survive the pasteurisation process and has been detected by culture in a number of surveys of retail pasteurised milk in different parts of the world with a prevalence ranging between
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