Introduction.

Own investigations and data from the literature strongly support the thesis, that small numbers of M. paratuberculosis survive high temperature short time pasteurization of milk. The aim of the studies reported here was to determine an end point for this type of heat treatment, at which surviving organisms are no longer detectable.

Materials and methods.

Inoculation of raw milk: three field-strains of M. paratuberculosis at 10⁴ cfu/ml each Heat treatment: pilot plant pasteurizer, sample volume 10-20 l, holder spiral shaped, heating at 72-90°C, flow rate 30-100 l/h, holding time 40-60 s. Detection of heat injured M. paratuberculosis: centrifugation of heated milk samples at 14,000 x g for 10 min without decontamination, inoculation of pellets simultane-ously into modified Dubos medium for resuscitation and directly onto HEYM. Confirmation of cultures by acid-fast staining and IS900 based PCR, viability stainings with iodo-nitro-tetrazolium chloride.

Results and discussion.

A 5 to 6 log reduction of initial counts was achieved by temperature-time combinations of 72, 75, 80, 90°C for 40 and 60 s. However, in 45 of 48 experiments surviving M. paratuberculosis cells were detected. All positive results were obtained within 8-12 weeks of incubation, which is much faster than in experiments with holding times below 30 s (data not shown). These results were obtained even without resuscitation. According to these observations a possible heat activation of M. paratuberculosis is supposed. This thesis is supported by viability stainings: in bacterial clumps from a fresh culture a uniform distribution of 1-10% active cells can be seen. After heating of this culture only a few clumps show active cells concentrated in active spots.