Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk

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.

Are we closer to understanding why viable cells of Mycobacterium avium subsp. paratuberculosis are still being reported in pasteurised milk?

“…The concentration was low ranging from 1 to 2 cells to 32 per 50 mL. The most recent study using phage amplification (Gerrard et al ) reported that most positive samples contained 1–2 cells/50 mL, a concentration below the theoretical detection limits of traditional culture assays (10 cells/mL).…”

“…Further research is required to fully understand the survival of MAP in pasteurised milk. The finding by Gerrard et al () that somatic cells secreted in milk are a major source, perhaps the main source, of MAP in hygienically produced milk may provide at last an explanation for the survival of MAP following HTST pasteurisation. It would be helpful to obtain the D ‐ and z ‐values of a range of MAP strains in somatic cells, containing variable levels of MAP, over the temperature range of 70 to 80 °C to determine whether intracellular location in somatic cells is protective.…”

“…However, PCR assays while much more sensitive and faster than traditional culture methods are unable to differentiate between dead and living cells (Timms et al 2011), reducing their value when applied to heat-treated milk and milk products. Because of these difficulties, phage amplification has been developed for the enumeration of viable MAP particularly in heated foods (Stanley et al 2007;Foddai and Grant, 2017;Gerrard et al, 2018). Stanley et al (2007) modified a commercial bacteriophage (phage)-based assay for M. tuberculosis (FASTPlaqueTB assay) using the broad-spectrum Mycobacterium phage D29 to detect viable cells of MAP in milk.…”

“…Work on the detection and/or quantification of MAP in pasteurised milk from 2001 is summarised in Table 7. Viable MAP has been found in pasteurised milk in the UK (Grant et al 2002a(Grant et al , 2002bGerrard et al 2018), the United States (Ellingson et al 2005), the Czech Republic (Ayele et al 2005) and Argentina (Paolicchi et al 2012) at a frequency ranging from 1.6% to 10.3%. The concentration was low ranging from 1 to 2 cells to 32 per 50 mL.…”

“…There is now significant evidence that commercial HTST pasteurisation does not consistently enable the production of heat‐treated milk free of viable cells of Mycobacterium avium subsp. paratuberculosis (MAP) (Grant et al b; Ayele et al ; Ellingson et al ; Grant et al ; Paolicchi et al ; Gerrard et al ).…”