Increased detection of mastitis pathogens by real‐time PCR compared to bacterial culture

Keane, Orla M.; Budd, Kathleen E.; Flynn, James; McCoy, Finola

doi:10.1136/vr.101598

Cited by 37 publications

(31 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Our findings showed that both tests are equally highly specific for detection of CNS from milk, whereas qPCR has a higher Se than BC at all the tested cut‐offs for qPCR and BC. This is consistent with previous studies, which found that PCR shows a higher Se than BC for detecting bacteria in milk samples (Taponen et al ; Shome et al ; Keane et al ).…”

Section: Discussionsupporting

confidence: 93%

See 1 more Smart Citation

Bayesian estimation of qPCR and bacterial culture accuracy for detection of bovine coagulase‐negative staphylococci from milk and teat apex at different test cut‐off points

Mahmmod

Svennesen

Katholm

et al. 2019

J of Applied Microbiology

View full text Add to dashboard Cite

Aim To primarily estimate the sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 quantitative PCR (qPCR) assay and bacterial culture (BC) for diagnosis of intramammary infections (IMI) and teat apex colonization (TAC) with coagulase‐negative staphylococci (CNS) at different cut‐offs for qPCR cycle threshold values using Bayesian latent class analysis. A secondary objective was to evaluate two cut‐offs of BC for diagnosis of IMI and TAC with CNS. Methods and Results We randomly selected 13–20 cows with subclinical mastitis from eight dairy herds. Teat skin samples and aseptically collected foremilk samples were collected from the right hindquarters (n = 149) for BC and qPCR analysis. The Se of qPCR was always higher than BCSe in diagnosis of IMI, however; the Sp of BC was higher than qPCRSp. BCSe and BCSp showed no substantial difference between the tested BC cut‐offs. In contrast to IMI, estimates of BC and qPCR in diagnosing TAC were different. BCSe was higher than qPCRSe at all tested cut‐offs, however; qPCRSp was higher than BCSp. Conclusion The overall performance of qPCR is higher than BC in the diagnosis of IMI; however, the performance of BC is better than qPCR in diagnosis of TAC. The qPCR and BC are valid diagnostics for bovine IMI with CNS. However, for TAC, both techniques require further investigation to reduce the uncertainty of the true status of the quarter and teat skin. Significance and Impact of the Study We reported, for the first time, the diagnostic performance of new mastitis technology (Mastit4 PCR) and culture for detection of CNS in milk and nonmilk samples in dairy herds with automatic milking systems. Our findings will improve the interpretation of the test results of culture and qPCR assay and subsequently, will strengthen the control of IMI with CNS in dairy cows.

show abstract

Section: Discussionsupporting

confidence: 93%

“…Shome et al 2011;Keane et al 2013).TheSe and Sp estimates of qPCR (94 and 77%) are comparable with estimates of Nyman et al (2016),…”

supporting

confidence: 68%

Bayesian estimation of qPCR and bacterial culture accuracy for detection of bovine coagulase‐negative staphylococci from milk and teat apex at different test cut‐off points

Mahmmod

Svennesen

Katholm

et al. 2019

J of Applied Microbiology

View full text Add to dashboard Cite

show abstract

“…This phenomenon did not seem to play an important role in the current study of ovine milk, possibly due to the fact that samples were from clinically healthy animals and to the short delay between sampling and culturing. Another explanation for real-time-PCR-positive, culture-negative results is higher sensitivity of real-time PCR compared with culture (Cederlöf et al, 2012;Keane et al, 2013). The number of positive samples in our study was too small for meaningful analysis of the comparative sensitivity and specificity of real-time PCR and culture.…”

Section: Discussionmentioning

confidence: 72%

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk—Sheep are not small cows

Zadoks

Tassi

Martín

et al. 2014

Journal of Dairy Science

View full text Add to dashboard Cite

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.

show abstract

“…The same interpretation system could be applied also in PCR diagnostics [31]. In general, PCR testing has proven to be a fast, sensitive and reliable test suitable for routine mastitis bacteriology [24, 28, 32]. An advantage of the PCR test is the comprehensive panel of pathogens, and uniform procedure and interpretation, in contrast to bacteriological culturing, where performance and procedures of laboratories may differ [33].…”

Section: Discussionmentioning

confidence: 99%

Bacteriological etiology and treatment of mastitis in Finnish dairy herds

et al. 2017

View full text Add to dashboard Cite

BackgroundThe Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010–2012.ResultsThe data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year.ConclusionsThe PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.

show abstract

Increased detection of mastitis pathogens by real‐time PCR compared to bacterial culture

Abstract: Rapid and accurate identification of mastitis pathogens is important for disease control. Bacterial culture and isolate identification is considered the gold-standard in mastitis diagnosis but is time-consuming and results in many culture-negative

Cited by 37 publications

References 30 publications

Bayesian estimation of qPCR and bacterial culture accuracy for detection of bovine coagulase‐negative staphylococci from milk and teat apex at different test cut‐off points

Bayesian estimation of qPCR and bacterial culture accuracy for detection of bovine coagulase‐negative staphylococci from milk and teat apex at different test cut‐off points

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk—Sheep are not small cows

Bacteriological etiology and treatment of mastitis in Finnish dairy herds

Contact Info

Product

Resources

About