Serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP) with an indirect ELISA based on the specific lipoprotein LppQ of Mycoplasma mycoides subsp. mycoides SC

Abstract

An indirect ELISA, based on the specific and strongly antigenic recombinant peptide of the N′-terminal half of the lipoprotein LppQ from Mycoplasma mycoides subsp. mycoides small colony type (SC) was developed for the detection of antibodies to M. mycoides subsp. mycoides SC. It was evaluated for its suitability for serodiagnosis and monitoring of contagious bovine pleuropneumonia (CBPP). The recombinant peptide containing poly-histidine residue tails was expressed in Escherichia coli and subsequently purified by Ni²⁺ chelate affinity chromatography to be used as antigen to coat microtiter ELISA plates. The specificity of the antigen was tested against rabbit hyperimmune sera directed against related Mycoplasmas of the M. mycoides cluster and with sera from cattle that were either free of CBPP, but suffered from other mycoplasmal infections such as M. bovis, or showed cross-reactions in the complement fixation test. The sensitivity of the ELISA was assessed with sera from artificially infected animals and with sera from cattle originating from areas where CBPP was endemic at the time of blood sampling. The study revealed that the ELISA was both specific and sensitive for CBPP positive bovine sera and was shown also to be robust to harsh climatic conditions.

Introduction

Serodiagnosis plays a key role in survey and control programs to combat contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides SC. CBPP represents one of the major bacterial threats in raising cattle, particularly in Africa, and hence demands sensitive and reliable tests. The complement fixation test (CFT), which is currently the official and most widely used serodiagnostic test, is fairly sensitive in the acute phase of the disease. However, it was found that sensitivity lowers considerably after the acute phase and that the CFT becomes insensitive 3 months after infection (Poumarat et al., 1989, Abdo et al., 1998). The strong similarity of the components of the surface of M. mycoides subsp. mycoides SC with those of other, closely related, mycoplasmas causes serological cross-reactions which can lead to false positive diagnostic results (Etheridge and Buttery, 1976, Cheng et al., 1994, Stark et al., 1995). It is, therefore, important to characterize specific antigens of M. mycoides subsp. mycoides SC. Recently, a competitive ELISA was developed on the basis of a monoclonal antibody which specifically recognized an as yet uncharacterized 80 kDa antigen of M. mycoides subsp. mycoides SC (Le Goff and Thiaucourt, 1998). This c-ELISA significantly improved serodiagnosis of CBPP (Le Goff and Thiaucourt, 1998). Systematic genetic, biochemical and antigenetic analysis of surface exposed lipoproteins of M. mycoides subsp. mycoides SC led to the identification of an antigenetically highly specific lipoprotein named LppQ (Abdo et al., 2000). The N-terminal domain of the mature LppQ was shown to be surface exposed, inducing a strong, specific, early and persistent immune response in naturally and experimentally infected animals. In contrast, the C-terminal domain of LppQ possessed an integral membrane structure built up of repeated units, rich in hydrophobic and aromatic amino acids, a motif which is commonly found among mycoplasmal membrane proteins. We have used a recombinant peptide, based on the specific antigenic N-terminal half of LppQ, to develop an indirect ELISA intended to fulfil the needs for effective mass screening of animals for CBPP. The ELISA was designed for high sensitivity and specificity for CBPP and for an easy and accurate application under adverse conditions such as high temperatures during storage.