Repeatability of a Commercially Available ELISA Test for Determining the Herd-Level Salmonella enterica subsp. enterica Serovar Dublin Status in Dairy Herds Using Bulk Milk

Abstract

An Enzyme-Linked Immunosorbent Assay (ELISA) is currently available for detection of antibodies against Salmonella Dublin in bovine milk. However, when used in a surveillance program, samples may undergo various storage conditions. The objective of this study was to estimate the repeatability of an ELISA test when used on fresh and frozen samples. Each of 845 bulk milk collected samples was subdivided into 3 aliquots and analyzed using PrioCHECK TM Salmonella Ab Bovine Dublin. ELISA percent positivity results (PP%) were compared between aliquots submitted to the initial analysis and a second analysis conducted 24 h later. The third aliquots were either preserved for 13-14 days (n = 413) or 25-28 days (n = 432) at −20 • C prior to analysis and results were compared to the initial analysis. There was excellent concordance between the two initial values and with values obtained after 13-14 and 25-28 days-freezing. The corresponding concordance correlation coefficients were 0.96, 0.97, and 0.94, respectively. Bland-Altman plots showed differences of PP% of 0.1 percentage points on average between the initial and second fresh samples. Freezing for 13-14 and 25-28 days led to overestimation of the initial values by 0.1, and 0.4 percentage points, respectively. Regarding the classification of samples, greater disagreement was observed between 25 and 28 days-frozen and initial samples when using the cutoff 15% (kappa = 0.76) compared to 35% (kappa = 0.90). Our study showed that PrioCHECK TM has good repeatability and that frozen bulk milk samples could generate reliable results. However, the larger variability at lower PP% should be considered when setting up a threshold.

Variation in the Performance of Different Batches of Two Mycobacterium avium Subspecies paratuberculosis Antibody ELISAs Used for Pooled Milk Samples

“…Therefore, such effects cannot completely be neglected. The freezing of bulk milk samples for 25–28 days at −20 °C led to an overestimation of the percent positivity values of 0.4 percentage points compared to fresh samples using an ELISA for antibodies against Salmonella Dublin [ 19 ]. The results of others, however, indicate that stressors, such as freezing for up to 8 months, thawing and re-freezing, result in biologically negligible differences in the S/P values or OD ratios [ 20 , 21 ].…”