Analytical specificity and sensitivity of a real-time polymerase chain reaction assay for identification of bovine mastitis pathogens

Abstract

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 beta-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 beta-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.

Somatic cell count and mastitis pathogen detection in composite and single or duplicate quarter milk samples

“…Moreover, some bacteria may be present in the teat canal without causing significant inflammation, and others may be present as contaminants (Pyörälä 2003, Nunes et al 2008. Accordingly, some authors have isolated mastitis pathogens from milk samples with very low SCCs, whereas others have found high proportions of microbiologically negative milk samples with high SCCs (Makovec & Ruegg 2003, Koskinen et al 2009, Oliveira et al 2013, as found in this study. Therefore, one problem regarding the diagnosis of mastitis is that indirect tests are compared with the "golden standard" of bacteriology, although mastitis does not always require the presence of infection (Pyörälä 2003, Nunes et al 2008.…”

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

“…Similar ranges were obtained when PCR-based molecular methods were compared with culture (Paradis et al, 2012;Spittel and Hoedemaker, 2012). However, molecular methods have been shown to higher detection potential than culture (Koskinen et al, 2009;Koskinen et al, 2010). We also show that the rapid detection of barely cultivatable and slow-growing mastitis pathogens, such as M. bovis, by PCR/ESI-MS represents a major advantage for veterinarians in preventing the spread of the disease within respective herds (Aebi et al, 2012).…”

Discrimination betweenMycoplasmaandAcholeplasmaspecies of bovine origin using digitonin disc diffusion assay, nisin disc diffusion assay, and conventional polymerase chain reaction

“…Although PCR-based platforms for the detection of mastitis pathogens directly in milk have been evaluated, 17,21 an extensive study of the ability of PCR assays to distinguish between Acholeplasma and Mycoplasma species using fieldbased milk samples has not been made. The results reported herein are consistent with previous findings that identified 2 PCR products for Acholeplasma species and 1 PCR product for all Mycoplasma species as described.…”