The objectives of the current study were 1) to compare sample preparation workflows and quantitative real-time polymerase chain reaction assays (qPCR) as currently used in veterinary diagnostic laboratories with a study protocol utilizing commercially available reagents for individual Tritrichomonas foetus testing, 2) to assess the accuracy of pooling cultured smegma samples followed by extraction and qPCR testing as used in the study laboratory, and 3) to assess the specificity of the currently used primers and probes by sequencing all positive and presumptive positive samples identified in the study laboratory in an attempt to capture any nucleotide variability between T. foetus isolates and to rule out false-positive results possibly due to Simplicimonas moskowitzi. Eight hundred three cultured smegma samples were collected from different regions of the United States with the collaboration of 5 veterinary testing laboratories. The samples were processed individually by the respective laboratories, and then sent to the study laboratory and retested using the study protocol. Comparison testing showed an overall agreement of 95.89% between the veterinary testing laboratories and the study laboratory. One hundred seventy-six positive or presumptive positive samples plus 625 negative qPCR samples were combined and retested using a pooling protocol. Pools consisted of 1 positive sample and 4 negative samples (1/5). These pools were processed using the same study laboratory protocols, and 96% of the positive samples were detected in these pools. Nested PCR followed by sequencing confirmed 175 of the 178 samples classified as positive or presumptive positive in the study laboratory as containing T. foetus–specific DNA.

Brucellosis (caused by the bacterium Brucella abortus) is a zoonotic disease endemic in wild elk (Cervus canadensis) of the Greater Yellowstone Ecosystem, US. Because livestock and humans working with elk or livestock are at risk, validated tests to detect the B. abortus antibody in elk are needed. Using the κ-statistic, we evaluated the buffered, acidified plate antigen (BAPA) assay for agreement with the results of the four serologic tests (card test [card], complement fixation test [CF], rivanol precipitation plate agglutination test [RIV], standard plate agglutination test [SPT]) that are approved by the US Department of Agriculture for the detection of the B. abortus antibody in elk. From 2006 to 2010, serum samples collected from elk within B. abortus-endemic areas (n = 604) and nonendemic areas (n = 707) and from elk culture-positive for B. abortus (n = 36) were split and blind tested by four elk serum diagnostic laboratories. κ-Values showed a high degree of agreement for the card (0.876), RIV (0.84), and CF (0.774) test pairings and moderate agreement for the SPT (0.578). Sensitivities for the BAPA, card, RIV, CF, and SPT were 0.859, 0.839, 0.899, 1.00, and 0.813, whereas specificities were 0.986, 0.993, 0.986, 0.98, and 0.968, respectively. The positive predictive values and the negative predictive values were calculated for 2.6%, 8.8%, and 16.2% prevalence levels. These findings suggest the BAPA test is a suitable screening test for the B. abortus antibodies in elk.