Estimation of Mycobacterium avium subsp. paratuberculosis load in raw bulk tank milk in Emilia‐Romagna Region (Italy) by qPCR

Abstract

Consumption of milk and dairy products is considered one of the main routes of human exposure to Mycobacterium avium subsp. paratuberculosis (MAP). Quantitative data on MAP load in raw cows’ milk are essential starting point for exposure assessment. Our study provides this information on a regional scale, estimating the load of MAP in bulk tank milk (BTM) produced in Emilia‐Romagna region (Italy). The survey was carried out on 2934 BTM samples (88.6% of the farms herein present) using two different target sequences for qPCR (f57 and IS900). Data about the performances of both qPCRs are also reported, highlighting the superior sensitivity of IS900‐qPCR. Seven hundred and eighty‐nine samples tested MAP‐positive (apparent prevalence 26.9%) by IS900 qPCR. However, only 90 of these samples were quantifiable by qPCR. The quantifiable samples contained a median load of 32.4 MAP cells mL −1 (and maximum load of 1424 MAP cells mL −1). This study has shown that a small proportion (3.1%) of BTM samples from Emilia‐Romagna region contained MAP in excess of the limit of detection (1.5 × 101 MAP cells mL −1), indicating low potential exposure for consumers if the milk subsequently undergoes pasteurization or if it is destined to typical hard cheese production.

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

“…Sung and Collins (1998) have suggested that a MAP concentration of 10 6 cfu/mL should be used when modelling MAP destruction for safety reasons. This is high, and the more conservative figure of 10 4 cfu/mL, which is still high, as suggested by Grant et al (1999) would appear to be justified from the work of Ricchi et al (2016) as a worst-case scenario. The number of microbial survivors, N2, following the heat treatment of a microbial population, N1, at a defined temperature for a time, T, can be calculated using Eqn 1 (Stumbo 1973) providing the D-value of the organism is known.…”

Improved Culture Medium (TiKa) for Mycobacterium avium Subspecies Paratuberculosis (MAP) Matches qPCR Sensitivity and Reveals Significant Proportions of Non-viable MAP in Lymphoid Tissue of Vaccinated MAP Challenged Animals

“…Previous comparative studies of qPCR and conventional culture have demonstrated good correlation in high sample loads present in feces (Douarre et al, 2010; Mita et al, 2016) but the inability of conventional culture methods to accurately culture low loads of viable organisms from clinical samples introduces problems. Culture sensitivity for MAP has only ever been as good as 2–3 log 10 (Ricchi et al, 2016), thus at low loads correlations to DNA presence are difficult to obtain and the true relationship between genome equivalent values and the demonstrable viable MAP count in these samples remains uncertain (Kralik et al, 2012; Plain et al, 2015). The underlying reasons for these discrepancies are probably multi-variant.…”

“…The ability of MAP to exist in a variety of phenotypes, some with a high resistance to killing (Grant and Rowe, 2004), has increased the importance of providing accurate quantitative estimates of viable counts when testing for the presence of this pathogen in food (Botsaris et al, 2016; Galiero et al, 2016; Ricchi et al, 2016), animal and human samples (Timms et al, 2016). MAP is widely accepted as a difficult organism to culture reproducibly and accurately, particularly at low loads (Hines et al, 2007).…”

Development and validation of a loop‐mediated isothermal amplification assay—a rapid and sensitive detection tool forMycobacterium aviumsubsp.paratuberculosisin small ruminants

“…Here, an amount of 15 CFU per ml was detected with IS 900 in a qPCR and 200 CFU per ml with f 57 in a qPCR (Ricchi et al . ). Due to the results of the LOD of MAP in LAMP, compared to those in the qPCR shows, that ISM ap02 is a trustworthy target gene with a high sensitivity level.…”

“…Losing one potency step compared to feces testing milk is not unusual (Ricchi et al . ). With this in mind we especially concentrated on the cream layer to not miss MAP bacteria.…”