The Performance of Three Immune Assays to Assess the Serological Status of Cattle Experimentally Exposed to Mycoplasma bovis

Abstract

Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16-98%), compared to the BIO K302: 47% (95% CI: 10-87%) and BIO K260: 28% (95% CI: 1-92%). However, for Sp, the BIO K302: 96% (95% CI: 87-99%) and the BIO K260: 100% (95% CI: 93-100%) out-performed Western blotting: 88% (95% CI: 56-98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29-86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21-54%) and 8% for BIO K260 (95% CI: 0-87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle.

Keywords: ELISA; Mycoplasma bovis; Western blotting; sensitivity; serology; specificity.

Figure 1

Reactivity of four monoclonal antibodies (MAb) with the S-vspA_R_A1 polypeptide expressed in this study. The monoclonal antibodies bind to the variable surface lipoproteins of Mycoplasma bovis. Lane 1, MAb_1E5; Lane 2, MAb_9F1; Lane 3, MAb_4D7; Lane 4, MAb_6E5. The 100 kDa, 50 kDa, 37 kDa, 25 kDa, and 20 kDa molecular markers (kDa) are shown.

Figure 2

Western blot analysis of the reactivity of sera from English cattle with the S-vspA_R_A1 recombinant polypeptide. The results for two exposed animals, AHVLA304 and AHVLA339, that were exposed to Mycoplasma bovis are shown. The serum samples were collected prior to M. bovis exposure, Day 0 (D0) and after exposure, Day 7 (D7), Day 14 (D14) Day 21 (D21), and Day 28 (D28). Lane 1, AHVLA370_D0; Lane 2, AHVLA370_D7; Lane 3, AHVLA370_D14; Lane 4, AHVLA370_D21; Lane 5, AHVLA370_D28; Lane 6, AHVLA304_D0; Lane 7, AHVLA304_D7; Lane 8, AHVLA304_D14; Lane 9, AHVLA304_D21; Lane 10, AHVLA304_D28; Lane 11, AHVLA339_D0; Lane 12, AHVLA339_D7; Lane 13, AHVLA339_D14; Lane 14, AHVLA339_D21; Lane 15, AHVLA339_D28; Lane 16, AHVLA ELISA positive control; Lane 17, AHVLA ELISA cut-off control. The 100 kDa, 50 kDa, 37 kDa, 25 kDa, and 20 kDa molecular markers (kDa) are shown.