Standardization of Fluorescence Polarization Assay on milk samples for immunological diagnosis of Brucellosis in dairy cattle

ALEJANDRA CAPOZZO; NICOLA, ANA; ELENA, SEBASTIÁN; ESTÉVEZ MADERO

Buenos Aires

Conferencia; Brucellosis 2011, International Research Conference; 2011

Milk sampling is a low labor-intensive method for testing dairy cattle, and allows to more consistently screening herds for infection without the additional scheduling of serum collection. The aim of this study was to develop and validate a protocol to apply Fluorescence Polarization Assay (FPA) on milk samples, in an area were vaccination with B.abortus S19 is applied. Milk was used untreated or processed by a standardized protocol. The assay was validated using 102 negative and 76 positive paired serum-milk samples. Milk samples were analyzed by milk ring test (MRT) and indirect ELISA (iELISA). Serum samples were evaluated by Competitive ELISA (cELISA), conventional FPA, and Seroagglutination test. Isolation was also performed. Animals were considered infected when the bacteria could be isolated or when they had positive outcomes by both serum-FPA and cELISA, assuming that antibodies induced by B. abortus S19 vaccine were excluded by these assays. When processed milk was used, ROC analysis gave off a cut off value of 79 milipolarization Units (mPU) that yielded 93.5% sensitivity and 91.3 % specificity. For untreated milk, a threshold of 74.5 mPU was obtained, with both sensitivity and specificity around 90% (Sensitivity= 90.7%; specificity= 90.2 %). When a range of undetermined samples was considered (73-78 mPU), and cut off value fixed in 79mPU for positive, and below 72mPU for negative samples, both sensitivity and specificity raised to 97,22% and 97,85% for raw milk, and to 95.7% and 96.08% for treated milk, respectively. Results from the evaluation of individual milk samples using this criterion were concordant to determinations performed by iELISA on milk samples (=0.911), and also by cELISA and FPA on matched serum samples (=0.962 and 0.94, respectively). Animals with positive Brucella isolation were in all cases positive to FPA-milk, even when the outcome was negative by other methods. The status of a tested raw or processed milk sample by FPA can be considered negative with values lower than or equal to 72 mPU, and positive when mPU value is equal or greater to 79mPU. A sample with mPU values between 73 and 78 is considered to be doubtful, and has to be confirmed with a second test. In this conditions, the application of milk-FPA can be used as a screening method for the individual diagnosis of Brucellosis in areas where Brucella S19 vaccine is applied.