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Technical note: Use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices

K. Supré^*,1, S. De Vliegher^*, O. C. Sampimon, R. N. Zadoks^,2, M. Vaneechoutte, M. Baele^#, E. De Graef^#, S. Piepers^* and F. Haesebrouck^#

^* Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium
Animal Health Service, Deventer, the Netherlands
Quality Milk Production Services, Cornell University, Ithaca, NY
Laboratory Bacteriology Research, Department of Clinical Chemistry, Microbiology, and Immunology, Faculty of Medicine, Ghent University, Belgium
^# Department of Pathology, Bacteriology, and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Belgium

¹ Corresponding author: Karlien.Supre{at}UGent.be

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in milk samples from cows with and without mastitis. Elucidating their relevance in bovine udder health is hampered because identification at the species level, if done at all, used to be performed based on phenotypic features. To provide a rapid, cheap, and easy-to-use genotypic technique that can be used to identify CNS species from milk and teat apices from cows, the performance of transfer RNA-intergenic spacer PCR (tDNA-PCR) in combination with capillary electrophoresis was evaluated. After updating the tDNA library with CNS reference strains, 288 field isolates were identified with tDNA-PCR and gene sequencing, and the latter was used as the reference method. The field isolates were divided in 2 groups of 144. Isolates of the first group were identified with tDNA-PCR with a typeability of 81.9% and an accuracy of 94.1%. Peak patterns of these isolates were then added to the tDNA library with species identity as determined by DNA sequencing. The second group was identified with the updated tDNA library, resulting in 91.0% typeability and 99.2% accuracy. This study showed that the updated tDNA-PCR in combination with capillary electrophoresis was almost as accurate as gene sequencing but faster and cheaper (only $3 per isolate), and is a useful tool in observational studies concerning the epidemiology of bovine CNS species.

Key Words: bovine coagulase-negative staphylococci • milk • teat apex • transfer RNA-intergenic spacer polymerase chain reaction