title: Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk--sheep are not small cows.
abstract: Mastitis, inflammation of the mammary gland, is an important cause of disease, mortality, and production losses in dairy and meat sheep. Mastitis is commonly caused by intramammary infection with bacteria, which can be detected by bacterial culture or PCR. PathoProof (Thermo Fisher Scientific Ltd., Vantaa, Finland) is a commercially available real-time PCR system for the detection of bovine mastitis pathogens. Sheep differ from cattle in the bacterial species or bacterial strains that cause mastitis, as well as in the composition of their milk. The aim of this study was to evaluate whether the PathoProof system was suitable for detection of mastitis pathogens in sheep milk. Milk samples were collected aseptically from 219 udder halves of 113 clinically healthy ewes in a single flock. Aliquots were used for bacteriological culture and real-time PCR-based detection of bacteria. For species identified by culture, the diagnosis was confirmed by species-specific conventional PCR or by sequencing of a housekeeping gene. The majority of samples were negative by culture (74.4% of 219 samples) and real-time PCR (82.3% of 192 samples). Agreement was observed for 138 of 192 samples. Thirty-four samples were positive by culture only, mostly due to presence of species that are not covered by primers in the PCR system (e.g., Mannheimia spp.). Two samples were positive for Streptococcus uberis by culture but not by PCR directly from the milk samples. This was not due to inability of the PCR primers to amplify ovine Streptococcus uberis, as diluted DNA extracts from the same samples and DNA extracts from the bacterial isolates were positive by real-time PCR. For samples containing Staphylococcus spp., 11 samples were positive by culture and PCR, 9 by culture only, and 20 by PCR only. Samples that were negative by either method had lower bacterial load than samples that were positive for both methods, whereas no clear relation with species identity was observed. This study provides proof of principle that real-time PCR can be used for detection of mastitis pathogens in ovine milk. Routine use in sheep may require inclusion of primer sets for sheep-specific mastitis pathogens.
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK; Institute for Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G61 1QH, UK. Electronic address: ruth.zadoks@moredun.ac.uk.
authors.forename: Ruth N
authors.initials: RN
authors.lastname: Zadoks
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Tassi
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK.
authors.forename: Elena
authors.initials: E
authors.lastname: Martin
authors.affiliation: Thermo Fisher Scientific, Microbiology Division, Ratastie 2, PO Box 100, FI-01621 Vantaa, Finland.
authors.forename: Jani
authors.initials: J
authors.lastname: Holopainen
authors.affiliation: Biobest Laboratories Ltd., 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge, EH26 0PY, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: McCallum
authors.affiliation: The Royal (Dick) School of Veterinary Studies, Easter Bush Campus, Midlothian, EH25 9RG, UK.
authors.forename: James
authors.initials: J
authors.lastname: Gibbons
authors.affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK.
authors.forename: Keith T
authors.initials: KT
authors.lastname: Ballingall
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods / Q000662:veterinary*; D005260:Female; D008321:Mammary Glands, Animal / Q000382:microbiology; D008413:Mastitis / Q000382:microbiology / Q000662:veterinary*; D008892:Milk / Q000382:microbiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012756:Sheep; D012757:Sheep Diseases / Q000382:microbiology*; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D013291:Streptococcus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
keywords: PCR; culture; mastitis; sheep