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* Finnzymes Diagnostics, Keilaranta 16A, 02150 Espoo, Finland
University of Helsinki, Faculty of Veterinary Medicine, Department of Production Animal Medicine, 04920 Saarentaus, Finland
Finnish Food Safety Authority Evira, 00790 Helsinki, Finland
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, T2N 4N1 Canada
# Microbiologia e Imunologia, CIISA/Faculdade de Medicina Veterinária, 1300-477 Lisboa, Portugal
|| Veterinary Medical Teaching Hospital, Department of Clinical Sciences, Kansas State University, Manhattan 66506
¶ TINE Produsentrådgiving, Mastittlaboratoriet, 6402 Molde, Norway
** Department of Animal Pathology, Hygiene and Health, University of Milan, 20133 Milan, Italy
CanWest DHI, Guelph, Ontario, N1K 1E5 Canada
1 Corresponding author: mikko.koskinen{at}finnzymes.fi
Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal β-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and β-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay’s diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.
Key Words: bovine intramammary infection • real-time PCR • validation
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