Diagnóstico de brucelose em amostras coletivas de leite bovino

Abstract

The dissertation was divided in three studies. Our first study determined the correlation between high somatic cell counts and positive milk ring tests (MRT). The milk ring test was performed on 181 milk samples from expansion tanks located at various rural sites in the regions of Brazil. Somatic cell counts (SCC) via flow cytometry were also performed. MRT identified 11 positive samples (6%) where the majority were reactive and 5 (2.8%) were found in the 6x105 cel/mL range. The results showed that there was no statistical relationship between high somatic cell counts and positive milk ring tests. Our second study evaluated the efficiency of an enzyme linked immunosorbent assay kit (ELISA) in diagnosing brucellosis in collective milk samples. The indirect ELISA test was used on 181 samples of fresh milk and identified 42 positive, 11 suspect and 128 negative samples. These results were compared to the MR test, the only test approved for collective milk samples by the Brazilian Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura Pecuária e Abastecimento). The ELISA test had sensitivity of 72.7%, specificity of 78.6% and a Kappa concordance index of 0.22. Subsequently, random samples from lactating animals in nine herds were tested individually by the rose Bengal test (RBT) and the 2-mercaptoethanol 2(ME) tests. Positive animals were found only in herds that tested positive in both ELISA and MRT. The ELISA test was neither as sensitive nor as specific as reported in other countries where the ELISA test is used and where brucellosis outbreaks have been confirmed in dairy herds. Our third study used the Polymerase Chain Reaction (PCR) technique to detect Brucella abortus in samples of fresh milk. Ninety samples were preselected for the PCR test including 45 that tested positive in ELISA and/or MRT, 11 suspect in ELISA and 34 negative in both tests. Extraction was carried out by enzymatic lysis followed by amplification using BAB and IS 711 primers, which amplify fragments of 498 base pairs. Visualization was accomplished with 1% agarose gel and an Alpha Digidoc Photo Documentation system. Brucella abortus DNA was not amplified in any of the samples, demonstrating that PCR is not the best screening technique for brucellosis in milk samples at the herd level, given that these microorganisms do not always escape into the milk.