Effect of carryover and presampling procedures on the results of real-time PCR used for diagnosis of bovine intramammary infections with Streptococcus agalactiae at routine milk recordings

Yasser S. Mahmmod, Marshal M. Mweu, Søren S. Nielsen, Jørgen Katholm, Ilka C. Klaas

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


The use of PCR tests as diagnostics for intramammary infections (IMI) based on composite milk samples collected in a non-sterile manner at milk recordings is increasing. Carryover of sample material between cows and non-aseptic PCR sampling may be incriminated for misclassification of IMI with Streptococcus agalactiae (S. agalactiae) in dairy herds with conventional milking parlours. Misclassification may result in unnecessary costs for treatment and culling. The objectives of this study were to (1) determine the effect of carryover on PCR-positivity for S. agalactiae at different PCR cycle threshold (Ct) cut-offs by estimating the between-cow correlation while accounting for the milking order, and (2) evaluate the effect of aseptic presampling procedures (PSP) on PCR-positivity at the different Ct-value cut-offs.The study was conducted in four herds with conventional milking parlours at routine milk recordings. Following the farmers' routine pre-milking preparation, 411 of 794 cows were randomly selected for the PSP treatment. These procedures included removing the first streams of milk and 70% alcohol teat disinfection. Composite milk samples were then collected from all cows and tested using PCR. Data on milking order were used to estimate the correlation between consecutively milked cows in each milking unit. Factors associated with the PCR-positivity for S. agalactiae were analyzed using generalized estimating equations assuming a binomially-distributed outcome with a logit link function. Presampling procedures were only significant using cut-off 37. A first-order autoregressive correlation structure provided the best correlation between consecutively milked cows. The correlation was 13%, 11%, 9% at cut-offs <40, 37, and 34, respectively. PSP did not reduce the odds of cows being PCR-positive for S. agalactiae.In conclusion, carryover and non-aseptic sampling affected the PCR results and should therefore be considered when samples from routine milk recordings are used. In relative terms, higher cut-offs resulted in higher between-cow correlation, but the absolute amount of carryover may not be affected although this was not tested.

Original languageEnglish (US)
Pages (from-to)512-521
Number of pages10
JournalPreventive Veterinary Medicine
Issue number4
StatePublished - Mar 1 2014

Bibliographical note

Funding Information:
This research work was funded by Faculty of Health and Medical Sciences, University of Copenhagen . The authors gratefully wish to acknowledge staff of Eurofins Steins Laboratory for providing us the lab space to conduct this research and for their technical and logistic support. They would like to thank Knowledge Centre for Agriculture, Cattle for funding of PCR testing and also, the technicians for their support in samples collection. Thanks to the Danish farmers for their help and making their cows available for our study.


  • Carryover
  • Milking order correlation
  • PathoProof™ PCR
  • Presampling preparations
  • Teat disinfection
  • Test misclassification


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