Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture

Abstract

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Bacteriological etiology and treatment of mastitis in Finnish dairy herds

“…The samples with DNA of some target species were largely easy to interpret because in 49.0% of the samples DNA of only one target species or group of species was detected. This agrees with previous reports where the same PCR test was used with smaller materials [24, 28]. In samples with DNA of two bacterial species (25.2%), a dominant species, being the likely causal agent of IMI, was found in 66.2% of the samples.…”

“…Thus, in 75% of all samples, the causal pathogen could be identified and the result was unequivocal. These results can be considered diagnostically valid [24]. In 13.4% of the samples, DNA of more than two bacterial species was detected, up to ten species in one sample.…”

“…Finland is to our knowledge the only country where PCR testing has largely replaced conventional culturing in routine microbiological diagnostics of quarter milk samples from mastitic cows [28, 29]. The PCR method used here identifies a wider range of target pathogens than commonly revealed by routine conventional culturing [24]. The uniform diagnostic method used and the large numbers of samples allowed us to make confident inferences on the microbiological etiology of mastitis in Finland.…”

“…In Finland, milk samples for bacteriology are routinely taken in most cases of mastitis [24]. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes the majority of these samples: 78% of Finnish dairy producers send milk samples to this laboratory (Personal communication Kristiina Sarjokari, Valio Ltd., 2016).…”

Mastites em ruminantes no Brasil

“…Em relação ao diagnóstico dos agentes causadores de mastites, ao nível internacional existe uma tendência crescente de utilização de ferramentas moleculares para o diagnóstico de patógenos, complementando, assim, os resultados obtidos na microbiologia clássica (McDonald et al 2005, Cremonesi et al 2006, Yamagishi et al 2007, Santos et al 2008, Onni et al 2010, Shome et al 2011, Ajitkumar et al 2012, Keane et al 2013, Rovai et al 2014, Hiitiö et al 2015. No Brasil, a utilização das ferramentas moleculares no diagnóstico dos agentes causadores de mastite ainda é escassa; dos 22 trabalhos apresen-…”