To confirm the presence of Mycobacterium avium subsp. paratuberculosis(MAP) in commercially pasteurized cow's milk, several studies were carried out in European and American countries. In our research we examined 22 samples of retail HTST milk , collected from supermarkets in 3 Italian regions (Lombardia, Emilia-Romagna and Lazio) to verify the presence and the vitality of MAP.

The cultures from the pasteurised milk were monitored for growth in Herrold's medium with Mycobactin J and PANTA antibiotics, with previous chemical decontamination on HPC 0,75%.

The comparative evaluation of method DNA extraction between Adiapure-Adiagene and Qiagen kit was carried out. The first is based on the capture bacteria by means of magnetic uncoated beads and lysis by chemical-mechanical destruction, whereas with the second method bacteria were destroyed only by chemical lysis. The Quiagen kit results were less sensitive than the Adiapure-Adiagene.

The extracted DNA from milk samples was assayed by magnetic uncoated beads (Adiagene-Adiapure) and after the lysis of bacteria obtained by chemical and grinding destruction with glass beads (Q-Biogen, FAST-PREP).

To detect the presence of MAP genetic material, we developed IS 900 PCR multiplex (Adiavet-Adiagene) and IS 900 PCR nested home-made using the following primers: P90/P91 and P24/P25. The PCR products were analysed by agarose gel electrophoresis (1,5% agarose). The integrated procedure combining optimal DNA isolation and IS 900 PCR methods detection had a reproducibile detection limit of about 10 MAP cells per ml when a starting sample volume of 10 ml of MAP-spiked milk was analyzed. A sample was considered positive when amplified product was noted at 229 bp for the IS 900 primers.

All 22 cartons samples of pasteurized cow's milk, produced in Italy and purchased in different supermarkets, have been found culture negative.

Only one milk sample resulted positive to commercial PCR kit and PCR home-made too. This milk sample from raw milk was collected from small dairy farms in the province of Rieti, which has the highest herd prevalence of paratuberculosis in the Latium region ( 52% , ±5%-CI 95% ).

This is the first report of MAP detection in retail milk in Italy, but now it is necessary to verify the vitality in dairy food (milk and cheese). For this reason further investigations are in progress relating to recovery of MAP from milk using more sensitive cultural method as non-radiometric MIGIT system without chemical decontamination of milk sample and successive typing by molecular techniques.