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We evaluated serologic responses of cattle, bison, elk, and swine representing negative control, early vaccination (4–8 wk), late vaccination (21–28 wk) or booster vaccination, early after-experimental challenge (2–4 wk), and late after-experimental challenge (8–21 wk), in a brucellosis fluorescence polarization assay (FPA; n = 10 sera per species per treatment) using negative control sera from cattle, bison, elk, and swine ( n = 5 per species). Sera from cattle shedding Brucella abortus strain RB51 in milk were also evaluated against the 20 negative control sera. The species of negative control sera used in the FPA could increase ( p < 0.05) delta millipolarization (mP; delta mP = sample mP − negative control mP) results. In general, the species of negative control sera did not alter the interpretation of FPA results in control, vaccinated, or infected animals. Even after repeated RB51 vaccinations in bison, cattle, or elk, or in cattle shedding RB51 in milk, serologic results from the FPA remained negative. Species differences in FPA results were noted; elk developed robust humoral responses very quickly after infection that resulted in strong positive FPA results. In cattle and bison, humoral responses appeared to develop over a longer period of time, and greater delta mP values were detected at later times after infection. Sensitivity of the FPA for detecting infected animals was greatest for elk in early challenge samples and bison in late challenge samples. Our data suggest that species of origin of negative control sera does not influence interpretation of the FPA in natural hosts of Brucella abortus.

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M. bovis culturing and PCR. Paired comparisons of all data (n=92) demonstrated that ELISA and LST results compared to the culturing results did not present significant differences (P=0.27 on McNemar's test and P=0.12 on Fisher's exact test, respectively). Using culturing as the gold standard, the sensitivity and specificity of ELISA were 79.5% (95% CI: 64.5-89.2%) and 75.5% (95% CI: 62.4-85.1%), respectively, whereas LST demonstrated 100% sensitivity (95% CI: 91.03-100%) and 92.5% specificity (95% CI: 82.1-97.0%). The ELISA results did not reveal significant differences in relation to the LST results (P>0.99 on Fisher's exact test). Using the latter as the gold standard, the sensitivity and specificity of ELISA were 79.1% (95% CI: 64.8-88.6%) and 79.6% (95% CI: 66.4-88.5%), respectively. The use of ELISA with the recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen complements the diagnostic coverage provided by CITT and increases the removal of infected animals from herds.

The performance of a fluorescence polarization assay (FPA) that detects antibodies to Mycobacterium bovis in bovine sera is described. The FPA reported here is a direct binding primary screening assay using a small polypeptide derived from the M. bovis MPB70 protein. A secondary inhibition assay confirms suspect or presumed positive samples. Specificity studies involved five different veterinary laboratories testing 4461 presumed negative bovine samples. FPA specificity was 99.9%. The FPA was used to identify herd status as either M. bovis infected or non-infected. Herd surveillance studies (nine herds) were performed in Mexico and South Africa. The FPA had a specificity of 100% (two negative herds), and correctly identified six of seven infected herds. Finally, sera from 105 slaughter animals that had gross lesions in lymph nodes similar to those seen with bovine tuberculosis were tested by the FPA. Thin sections from the associated formalin-fixed paraffin-embedded samples of lymph nodes were stained using hematoxylin and eosin (H&E) for morphologic examination and using the Ziehl-Neelsen (ZN) method for detection of acid-fast bacilli. Of the 105 animals, 78 were classified as TB suspect based on lesion morphology, 21 were positive by ZN, 9 were positive by FPA and 13 were positive by PCR for the tuberculosis group of Mycobacterium. Among the 21 ZN positives, 11 (52.4%) were PCR positive. Among the 9 FPA positives, 8 (88.9%) were PCR positive. For the 13 PCR positives, 8 (61.5%) were FPA positive and 11 (84.6%) were ZN positives. These results show that use of the FPA for detection of M. bovis infection of cattle has value for bovine disease surveillance programs.

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum , a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a “gold standard” set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.