In general practice in an rbST-authorized country, it is expected that at least 65 % of the combined milk in a tank load originates from treated and responding cows (see section 2.3), but the pooled tank milk obtained might still yield an overall positive assay result. To proof this we did a simulation experiment, where milk samples from two positively responding rbST-treated cows were mixed with one milk sample from a non-responding rbST-treated cow, i.e., simulating 33 % physiological non-responders and non-treated animals in the mixture. Twenty-four different mixtures were prepared, whereof 96 % showed responses above the assay decision limit, which complies with the <5 % false- negative screening criterion for veterinary drugs in animal products [14]. The advantage of analysing tank milk also applies to the milk of stables where cows were not treated. The very few false-positive responses will be “diluted away” by the many correctly
negatively responding milk samples. This was demonstrated by the analysis of 34 tank milk samples from different Dutch farms, of which none responded above the decision limit. Furthermore, analysing tank milk and obtaining an overall positive antibody response for milk of rbST-treated cows offers the future opportunity to develop fast, cheap and easy to use testing devices for on-farm use. The developed FCIA screening method features a
long detection window of at least two weeks after the last rbST treatment. Unfortunately, LC/MS confirmation methods for rbST in blood can only detect rbST for a short period following administration [15] and in milk, no time course studies were done in rbST- treated animals by these authors. Because of this, high frequency blood sampling would be necessary to ensure confirmation of rbST abuse by this LC/MS method. Therefore, for future veterinary control according to Commission Decision 2002/657/EC, we suggest a tiered screening method, which consists of three steps: First, a fast tank milk screening for anti-rbST antibodies using the present FCIA. Second, in case of suspicious findings, a more detailed individual bovine serum biomarker profiling is envisaged, which will provide additional evidence, since that will be based on more biomarkers and data of individual cows. And third, a sufficiently sensitive instrumental method is used for final confirmation of rbST itself in serum samples of the individual cows.
4 Conclusion
An FCIA screening method for anti-rbST biomarker in milk was successfully developed and applied to raw milk, pasteurized milk and raw milk pools (simulating tank milk analysis). The sample pretreatment known from serum analysis was adjusted and shortened for milk analysis, which facilitates the assay procedure and saves time. A preliminary validation of the screening method was performed by testing milk samples from untreated dairy cows, which allowed the calculation of an absolute decision limit. Sixty-seven percent of the tested milk samples from animals treated with rbST showed physiological antibody responses higher than the decision limit, whereas 94 % of untreated animals did not. This resulted in a false-positive rate of only 6 % and a false-negative rate of 33 %. The detection window ranged at least until two weeks after the last rbST treatment, where a positive antibody biomarker response still was found in 63 % of the cows. Antibody responses in milk were specific for rbST and similar to serum responses. Pasteurization did not fully obstruct the detectability of this biomarker, so even processed milk might be tested. In 34 tested tank milk samples from Dutch farms, no single false-positive result was found. Moreover, more than 95 % of simulated tank milk samples from rbST-treated cows showed a positive assay response, which is an adequate true-positive screening rate according to veterinary drug screening regulations.
Acknowledgements
This project was financially supported by the Dutch Ministry of Economic Affairs, Agriculture and Innovation (project 97202901). We kindly thank J. van Hende from the University of Gent for performing the animal experiment and M.J. Groot, M.H. Blokland and J.S. Ossenkoppele for their help with collecting sample materials.
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