B01003 (Formerly FS3104): Characterisation of the non-linear thermal inactivation kinetics observed for Mycobacterium paratuberculosis in milk

Study Duration: November 1996 to October 2000

Contractor: Queens' University of Belfast

Previous MAFF-funded research has found that Mycobacterium avium subsp. paratuberculosis (MAP) was able to survive laboratory heat treatments simulating commercial milk pasteurisation conditions (72°C for 15 seconds). This project was commissioned to clarify the mechanism(s) that aid survival of this organism and also identify what combination of holding times and temperatures would be necessary to ensure the complete inactivation of MAP if present in milk in high numbers.

The aims of the project were:

• To determine the mechanism(s) that enables MAP to survive pasteurisation by investigating a number of factors which may influence thermal inactivation characteristics. The factors to be investigated include the effect of suspending menstruum (whole milk v skim milk v UHT milk), source of inoculum (solid v liquid medium), declumping, macrophage engulfment, heat shock response, freezing and thawing, stage of growth and rate of heating.
• To identify a processing regime that will achieve the complete inactivation of high numbers of MAP in milk.
• To optimise decontamination protocols for the isolation of MAP and Mycobacterium bovis from milk.
• To modify the immunomagnetic PCR assay for application to different types of cheese.

Mycobacterium paratuberculosis (MAP) cells were found to be more resistant to heat if they were clumped, engulfed by macrophage, not previously frozen, or obtained from liquid culture. They were found to be more heat sensitive if heated in skim milk, after declumping, or if they had been frozen and thawed before heating.

Heating MAP cells was shown to produce a small number of extra intracellular proteins in response to a brief heat treatment (heated to 63°C and cooled immediately to 37°C) compared with unheated control MAP cells. These may have been potential heat shock proteins.

Circumstantial evidence was obtained that the natural tendency of MAP cells to exist as clumps of cells rather than single entities may contribute to its survival for extended periods during heating:

• de-clumped (i.e. single cells) were shown to be approximately half as heat resistant as clumped cells;
• inocula obtained from liquid culture were noticeably more clumped when viewed microscopically than inocula obtained from slope, and the former were shown to be more heat resistant;
• by the means of a acid-fast viability stain, viable cells were shown to be present in clumps of predominantly dead cells at heating times corresponding to the tail region of the thermal inactivation curve for MAP heated at 63°C.

Laboratory pasteurisation trials were carried out to determine the effect of higher pasteurisation temperatures (75, 78, 80, 85 and 90°C) for 15 seconds and extended holding times at 72°C of 20 seconds and 25 seconds on the thermal inactivation of high numbers of MAP in milk (106CFU/ml). Each of the heat treatments studied achieved a substantial (5-6 log_10,) reduction in numbers of viable MAP. However, small numbers of viable MAP were isolated from a proportion of the heated milk samples at each of the higher temperatures investigated. A longer holding time of 25 seconds at 72°C was shown to be more effective at inactivating MAP than a higher pasteurisation temperature. Only one of the three strains studied (B2) yielded small numbers of survivors after heating at 72°C for 20 seconds but it was completely inactivated by extending the holding time at 72°C by a further 5 seconds to 25 seconds. It was concluded that extending the holding time at 72°C was more likely to achieve the complete inactivation of MAP in milk than raising the pasteurisation temperature.

To identify the optimum chemical decontamination protocol to use to culture MAP and Mycobacterium bovis from raw and commercially pasteurised milk in a UK milk survey, a number of chemical decontamination protocols were compared with respect to percentage recovery of a spiked population from milk, minimum detection limit, and ease of application. The methods examined were: treatment with 0.2, 0.5 or 0.75% (w/v) hexadecylpyridinium chloride (HPC) for 1, 2 or 5 hours; modified Cornell methods employing brain/heart infusion broth containing 0.75% (w/v) and 0.9% (w/v) HPC; and several methods using C₁₈-carboxypropylbetaine (CB-18&#8482), to aid the sedimentation of mycobacteria from liquid samples.

Overall, treatment of the milk pellet obtained after centrifugation at 2500 x g for 15 minutes with 0.75% HPC for 5 hours was found to be superior to the other methods in all respects for the isolation of both MAP and M. bovis. This method resulted in the highest mean percentage recoveries of MAP (18.8%) and M. bovis (6.3%) and permitted detection of the lowest number of MAP and M. bovis cells in spiked milk (30CFU/40ml and 100CFU/40ml respectively). This method also contained fewer steps than the other protocols and so was considered most suitable for large-scale milk testing.

In contrast to milk, which is liquid, cheeses are semi-solid or solid. Therefore, in order to be able to successfully apply the immunomagnetic PCR (IMS-PCR) method to cheese samples, liquefaction and homogenisation was essential, otherwise the Dynabeads coated with anti-MAP antibody would not be able to interact with MAP cells present to achieve selective separation. Sample preparation generally involved dilution and stomaching the cheese in 2% tri-sodium citrate solution, liquefaction by heating in a 37°C waterbath, centrifugation and application of IMS to the resulting pellet. IMS-PCR protocols were successfully developed for both laboratory-produced soft and hard cheese, which permitted the sensitive detection of MAP by IMS-PCR in cheeses made from milk spiked with as few as 10CFU per 100ml milk.

An effective chemical decontamination protocol (involving CB-18&#8482 treatment, centrifugation and decontamination with 0.75% HPC for 5 hours) was identified for soft cheese but not for hard cheese. The latter has a much higher microbial load which created problems of overgrowth of slopes by contaminants.

During cheesemaking the majority of MAP cells in cheesemilk end up in the cheese rather than in the whey. Viable MAP cells were recoverable from soft cheese made from spiked raw milk for up to 35 days during storage at 4°C, although the percentage of MAP cells recovered decreased over time. A variety of retail unpasteurised cheeses were bought at a local supermarket and tested by both IMS-PCR and culture. MAP was detected in some cheese samples by IMS-PCR but no viable MAP were ever isolated due largely to problems with media contamination.

The final report is available from the Agency's Information Centre.

To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk)

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email science@foodstandards.gsi.gov.uk