Aims Manufacturing process used in preparation of probiotic products may alter beneficial properties of probiotics. The effect of different growth media and inactivation methods on the protective properties of canine-originated probiotic bacteria against adhesion of canine enteropathogens was investigated. Methods and Results Three established dog probiotics, Lactobacillus fermentumVET9A, Lactobacillus plantarumVET14A and Lactobacillus rhamnosusVET16A, and their mixture were assessed using the dog mucus pathogen exclusion model. The pathogens used were Enterococcus canis, Salmonella enterica serovar Typhimurium and Clostridium perfringens. The effect of growth media, one reflecting laboratory and the other manufacture conditions, and viability (viable and heat inactivated, 80 degrees C per 30min) on the pathogen exclusion properties of probiotics were characterized. Greater pathogen exclusion percentages were noted for probiotics growing in conditions reflecting manufacture when compared to laboratory (P<0 center dot 05). Inactivation of probiotics by heat (80 degrees C per 30min) increased pathogen exclusion compared with their viable forms (P<0 center dot 05). Conclusions Manufacturing process conditions such as growth media, incubation temperature and pretreatment methods may significantly affect the protective properties of the tested strains. Significance and Impact of the Study Growing conditions and pretreatment methods should be carefully considered when designing new probiotics to reduce the risk of common infections in dogs. The studied probiotics are promising potential feed additives for dogs.