[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody (MAb)-based antigen capture enzyme-linked immunosorbent assay (ELISA) was compared with the routine culture methodology for the detection of Campylobacter fetus subspecies from bovine and ovine field samples inoculated into Clark's transport enrichment medium (TEM). The work was a collaboration between two different diagnostic laboratories, one in Canada and the other in England. In both labs, TEM samples were incubated for 4 days at 35 degrees C and then tested by culture and ELISA. The ELISA consisted of initial screening with MAb M1825 against C. fetus subspecies core lipopolysaccharide (LPS). All samples positive on ELISA screening were then retested by ELISA with MAb M1825 and MAbs M1177, M1183, and M1194, which recognize serotype A- and/or serotype B-specific C. fetus subspecies LPS epitopes. The Canadian samples consisted of 1,060 preputial washings from 529 bulls, of which 18 were positive by both culture and ELISA and 1,042 were negative by both methods. The English samples consisted of 321 tissue specimens, mostly stomach contents and placentas, from 190 aborted ovine and bovine fetuses. A total of 262 samples were negative by culture and ELISA, 52 samples were positive by culture and ELISA, and 7 samples were culture negative but ELISA positive. The results for all 70 culture-positive isolates were confirmed by conventional biochemical methods as C. fetus subsp. fetus, with 39 presumptively identified by the ELISA as serotype A and 30 presumptively identified as serotype B and with one sample containing isolates presumptively identified as serotype A and serotype B. A receiver operating characteristic analysis of the combined ELISA data from both countries resulted in an area under the curve of 0.997, with a sensitivity of 100% and a specificity of 99.5% relative to the results of culture. The data confirm that this ELISA method can be used as an excellent test for the screening of field samples in TEM for the presence of C. fetus subspecies.
[Show abstract][Hide abstract] ABSTRACT: A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was described and evaluated for use as a presumptive screening test for detection of Campylobacter fetus in bovine preputial washing and vaginal mucus samples. A total of 725 diagnostic samples collected in the field and submitted in Clark's transport enrichment medium (TEM) were analyzed. Cultural isolation of C. fetus was used as the standard for comparison. After incubation of the TEM vials for 4-5 days, fluid was removed for culture and ELISA testing. A sandwich ELISA format was used and the target antigen was C. fetus lipopolysaccharides (LPS). A rabbit anti-C. fetus polyclonal antiserum was used as the capture antibody. Murine monoclonal antibodies (MAbs) to C. fetus serotype A and B LPS core and O-polysaccharides and a goat anti-mouse horseradish peroxidase conjugate were used as detection antibodies. ELISA and culture results for the diagnostic samples were in complete agreement. Seven hundred and eight samples were negative by both tests. All 17 culture positive samples were positive by ELISA with a MAb to LPS core. The ELISA with MAbs to LPS O-polysaccharides detected all culture positive samples with the homologous C. fetus serotype. Sixty-six preputial wash samples from three known C. fetus culture positive bulls were also analyzed. Forty-nine of these samples were positive by both ELISA and culture, 16 were positive by ELISA only, and one was negative by both ELISA and culture. The results indicate that this ELISA is useful as a screening test for the detection of C. fetus in diagnostic samples.