Bayesian diagnostic accuracy validation of milk ELISA-Ab, blood qPCR, and peripheral blood lymphocyte count tests to determine bovine leukosis virus status in dairy cows

Abstract

We assessed the diagnostic accuracy of an adapted antibody ELISA (ELISA-Ab) test, originally designed for bulk milk samples but applied on individual DHI-collected milk samples, to identify the bovine leukosis virus infection status of individual cows. Secondary objectives included identifying a fit-for-purpose threshold for result interpretation and evaluating whether the test’s specificity could be influenced by the sampling technique (i.e., DHI-collected milk samples). Additionally, we evaluated whether the accuracy of each test varied with cow age, categorizing cows as young (2 to 4 yr old) or older (>4 yr old). In 2023, 8 dairy herds in Québec, Canada, were selected based on their historical within-herd leukosis prevalence, which was estimated to range from 10% to 75%. From all milking cows within these herds (n = 637), milk samples were collected during regular DHI, and blood samples were collected by the research team within one week of the DHI sampling. An optical density threshold of ≥0.5 for the ELISA-Ab provided an optimal control of the misclassification cost across various leukosis prevalence and, to a lesser extent, false negative to false positive cost ratio scenarios. With this threshold, the sensitivity and specificity estimates (95% Bayesian credible interval [BCI]) were 92% (BCI: 88%, 95%) and 99% (BCI: 96%, 100%), respectively. Sensitivity was higher in cows >4 yr old (99%, BCI: 96%, 100%) compared with cows 2 to 4 yr old (88%, BCI: 80%, 94%). We observed lower ELISA-Ab specificity in cows milked immediately after a positive cow (median: 82%, BCI: 72%, 97%) compared with those milked after a negative cow (median: 91%, BCI: 85%, 99%), suggesting a milk carryover effect due to the sampling technique. This carryover effect had a more pronounced impact on the false positive rate in herds with 30% to 50% leukosis prevalence, with the largest differences observed at the 30% prevalence scenario. However, the overall influence of the carryover effect remained limited. In conclusion, the adapted ELISA-Ab test appears suitable for individual cow testing using DHI-collected milk samples, with higher sensitivity in cows >4 yr old. Its integration into existing milk recording programs provides a practical opportunity for herd-level leukosis monitoring.