Within-herd prevalence thresholds for herd-level detection of mastitis pathogens using multiplex real-time PCR in bulk tank milk samples

Abstract

The objective of the study was to assess the value of quantitative multiplex real-time PCR examination of bulk tank milk samples for bovine mastitis pathogens as a tool for herd level diagnosis. Using a logistic regression model, this study is aimed at calculating the threshold level of the apparent within-herd prevalence as determined by quarter milk sample cultivation of all lactating cows, thus allowing the detection of a herd positive for a specific pathogen within certain probability levels. A total of 6,335 quarter milk samples were collected and cultured from 1,615 cows on 51 farms in Germany. Bulk tank milk samples were taken from each farm and tested by bacterial culture as well as the commercial PCR assay Mastit 4A (DNA Diagnostic A/S, Risskov, Denmark) identifying Staphylococcus aureus, Streptococcus dysgalactiae, Streptococcus agalactiae, and Streptococcus uberis. In addition, PCR was performed on pooled herd milk samples containing milk aliquots from all lactating cows in each of the 51 herds. Only 1 out of the 51 herds was found PCR positive for Streptococcus agalactiae in bulk tank and pooled herd milk samples, and cultured quarter milk samples. Spearman's rank correlations between the cycle threshold value of bulk tank milk PCR and the apparent within-herd prevalence were calculated in regard to Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis. For these pathogens, significant correlations were found. If 1 bulk tank milk sample per herd was tested, the estimated within-herd prevalence thresholds for 90% probability of detection were 27.6% for Staphylococcus aureus, 9.2% for Streptococcus dysgalactiae, and 13.8% for Streptococcus uberis on the cow level. On the quarter level, the within-herd prevalence had to be at least 32.6% for Staphylococcus aureus, 1.7% for Streptococcus dysgalactiae, and 4.3% for Streptococcus uberis to detect a herd as positive using a single bulk milk sample. The results indicate that mastitis pathogens in bulk tank milk can be identified by the applied PCR assay. Bulk tank milk examination is not a reliable tool for the identification of the named pathogens by single testing, but might be a valuable monitoring tool when used frequently with repeated testing. Furthermore, this approach could be a useful monitoring tool for detecting new pathogen occurrence in the herd.

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

“…DNA of CNS), apart from the detection target of BC. This comes in agreement with the findings of previous studies (Mahmmod et al ; Soltau et al ). One of the main different aspects between the qPCR analysis and BC is that qPCR analysis detects DNA, whereas BC detects only viable bacteria.…”

“…DNA‐based molecular diagnostics such as qPCR assays are increasingly implemented as a routine method for mastitis control programmes in the last few years in several countries (Nyman et al ; Timonen et al ; Soltau et al ). However, conventional bacterial culture (BC) is still regarded as the gold standard for mastitis diagnosis and implemented in routine diagnostic by most laboratories worldwide (Persson Waller ).…”

Assessment of the prevalence of Streptococcus uberis in dairy cow feces and implications for herd health

“…The recovery rates for S. uberis using culture-based methods alone has been reported to be low, with one study reporting the correct identification of S. uberis for 43% of the colonies obtained using modified Rambach media (n = 304 out of 706 putative colonies; Zadoks et al, 2005) and may have limitations for calculating the overall prevalence Assessment of the prevalence of Streptococcus uberis in dairy cow feces and implications for herd health of S. uberis carriage within a herd. Identification based on PCR has been used for the detection of S. uberis in mixed IMI (Gillespie et al, 1997;Ashraf et al, 2017;Soltau et al, 2017), as well as for detection of other bacteria within bovine feces, including Mycobacterium paratuberculosis (Collins et al, 1993) and Escherichia coli (Sharma, 2002). Given advances in availability of genome sequence information for S. uberis (Ward et al, 2009;Hossain et al, 2015), unique species-specific coding sequences have been identified and may be used in combination with culture-based detection methods to provide a specific tool for detection of S. uberis in herd prevalence studies.…”

Comparison of the population structure of Streptococcus uberis mastitis isolates from Austrian small-scale dairy farms and a Slovakian large-scale farm

“…uberis prevalence in dairy herds include implementation of hygiene measures, enhancement of the immune status of cows, and pathogen-specific treatment concepts (Krömker et al, 2014;Ruegg, 2014;Mansion-de Vries et al, 2015). Risk assessment based on monitoring data (Samson et al, 2016;Soltau et al, 2017;Taponen et al, 2017), the implementation of novel diagnostic tools (Archer et al, 2017;Leimbach and Krömker, 2018), improved strain characterization (Hossain et al, 2015;Davies et al, 2016;Vélez et al, 2017;MLST, 2018c), and vaccination (Collado et al, 2018) are contributing to improved epidemiological understanding of Strep. uberis IMI and new approaches to control and prevent infections.…”