Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk

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

Factors affecting phage D29 infection: a tool to investigate different growth states of mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay

Role of RNA synthesis in recovery of phage-sensitivity.

<p>Graph showing the number of MAP cells detected by the RapidMAP assay, after uninfectable MAP cells were treated with RIF (blue bars) and without RIF (red bars) before exposure to oxygen (t – 0) and after exposure to oxygen (t – 1). Error bars represent the standard deviations of the means of number of plaques recovered from the phage assay performed in triplicate.</p

Detection of <i>M. smegmatis</i> using D29 and TM4.

<p>Graph showing the number of plaques recovered following the phage assay when using phage TM4 and D29. The <i>M. smegmatis</i> cells tested were either grown with limiting oxygen (blue bars) or after these cells had been grown with aeration (red bars). In addition to the phage assay the viable count (CFU ml⁻¹) of both cultures was determined. Error bars represent the standard deviations of the means of number of plaques and colonies recovered from the phage assay and viable count, respectively, performed in triplicate.</p

Recovery of phage D29 infectivity by three strains of MAP.

<p>Graph showing the number of MAP cells detected using the RapidMAP assay (PFU ml⁻¹). Prior to dilution into fresh medium the MAP cells were either grown under self-limiting oxygen conditions and then samples were taken from the fresh cultures over a 7 d period. The three strains of MAP used were K10 (blue), DVL 453 (purple) and ATCC 19851 (red). Error bars represent the standard deviations of the means of number of plaques recovered from the phage assay performed in triplicate.</p

Difference in infectivity of MAP cells by D29 and TM4.

<p>Graph showing the number of plaques recovered following the phage assay when using phage TM4 and D29. The MAP cells tested were either grown with limiting oxygen (blue bars) or after these cells had been exposure to air for 9 days (red bars). Error bars represent the standard deviations of the means of number of plaques recovered from the phage assay performed in triplicate.</p

Effect of stationary phase bacteria on the attachment of phage D29 to MAP cells.

<p>Graph showing the number of unbound phage particles to MAP cells that are infectable (aerobic) and uninfectable (hypoxic) after 0 min (blue bars), 30 min (red bars) and 60 min (green bars). Error bars represent the standard deviations of the means of number of bacteriophage detected after each time point in triplicate.</p

Comparison of <i>M. smegmatis</i> cells detected by phage and viable count under growth limiting and aerobic conditions.

<p>Panel A Graph showing the results of the phage assay (PFU ml⁻¹; green and purple) and viable count (CFU ml⁻¹; blue and red) for <i>M. smegmatis</i> cultured over a 10 d period under self-limiting oxygen conditions (red and purple) or under conditions were free oxygen exchange occurred (blue and green). T = 0 are values recorded for initial cultures before incubation. Error bars represent the standard deviations of the means of number of plaques recovered from the phage assay performed in triplicate. Panel B Graph showing the number of <i>M. smegmatis</i> cells detected using the phage amplification assay (PFU ml⁻¹). Prior to dilution into fresh medium the <i>M. smegmatis</i> cells were either grown under self-limited oxygen conditions (red bars) or aerobic conditions (blue bars). Samples were taken from the fresh cultures over a 3 d period. Error bars represent the standard deviations of the means of number of plaques recovered from the phage assay performed in triplicate.</p