title: The effect of sampling technique on PCR-based bacteriological results of bovine milk samples.
abstract: The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis.
authors.affiliation: Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Mäntsälä, Finland. Electronic address: heidi.hiitio@helsinki.fi.
authors.forename: Heidi
authors.initials: H
authors.lastname: Hiitiö
authors.affiliation: Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Mäntsälä, Finland.
authors.forename: Heli
authors.initials: H
authors.lastname: Simojoki
authors.affiliation: Department of Clinical Medicine, Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, Kreutzwaldi 62, 51014, Tartu, Estonia.
authors.forename: Piret
authors.initials: P
authors.lastname: Kalmus
authors.affiliation: Thermo Fisher Scientific Ltd., Ratastie 2, 01620 Vantaa, Finland.
authors.forename: Jani
authors.initials: J
authors.lastname: Holopainen
authors.affiliation: Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Mäntsälä, Finland.
authors.forename: Satu
authors.initials: S
authors.lastname: Pyörälä
authors.affiliation: Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Mäntsälä, Finland.
authors.forename: Suvi
authors.initials: S
authors.lastname: Taponen
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D008321:Mammary Glands, Animal / Q000382:microbiology; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D013203:Staphylococcal Infections / Q000662:veterinary; D013210:Staphylococcus
keywords: Staphylococcus spp; bovine mastitis; milk sampling; real-time PCR