Evaluation of methods for detection of Campylobacter in raw milk: A multi-country study

Raw milk is considered a high-risk source of Campylobacter due to faecal contamination from healthy cattle and farm environments, thus linking raw milk consumption to global outbreaks. Detection of Campylobacter in raw milk poses challenges due to low contamination levels and antibacterial properties of the milk. Culture-based protocols for Campylobacter detection in milk vary, mainly with regard to pH adjustment and the choice of enrichment broth. This European collaborative study was organised by the EU Reference Laboratory (EURL) for Campylobacter together with eight EU National Reference Laboratories (NRL) for Campylobacter with the purpose to evaluate methods for culture-based detection of Campylobacter in raw cow's milk. The study was divided into two parts, an interlaboratory part and an intralaboratory part, both organised around the same two protocols. The aim of protocol 1 was to evaluate the impact of pH adjustment and storage of the milk on the culturability of Campylobacter over time. Aliquots of the spiked milk were adjusted either to pH 7.0 or pH 7.6 or left unadjusted. The milk was stored up to 48 h at refrigerated temperature and Campylobacter was quantified according to ISO 10272-2 on day 0, 1 and 2. The aim of protocol 2 was to evaluate which enrichment broth, Bolton broth (BB) or Preston broth (PB), showed highest sensitivity in detection of Campylobacter. The spiked milk was enriched in BB and PB as described in ISO 10272-1:2017 or ISO 10272-1:2017/Amd1.2023. In the interlaboratory part, each milk batch was collected locally by each participating NRL/EURL and inoculated with the same Campylobacter strain. In the follow-up intralaboratory part, the EURL-Campylobacter repeated the tests in protocol 1 and 2 but used different Campylobacter strains and strains subjected to thermal stress prior to inoculation. The results show that pH adjustment of raw milk has a negligible impact on culture-based detection of Campylobacter, regardless of strain and level of environmental stress. The composition of milk and properties of the inoculated strain influence culture-based detection of Campylobacter over storage time, and strains subjected to additional stress prior to inoculation in milk are reduced in culturability much faster than the same strains prepared under normal conditions. Finally, the study showed that PB without Campylobacter growth supplement is less effective than BB in detecting Campylobacter in raw milk.