Abstract
A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.
Highlights
A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented
CBPP was eradicated from Europe in the beginning of the 20th century [3] but has reemerged in every decade since [4]
The two currently available tests for serological diagnosis of CBPP recommended by the Organization for Animal Health (OIE), the complement fixation test [15] and a competitive ELISA [16], are based on whole cell M. mycoides SC
Summary
A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against onethird of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. LppQ has been used to develop an indirect ELISA [25], and Vmm, a variable surface protein, has recently been studied along with five novel putative variable surface proteins as Analysis of Humoral Immune Responses to M. mycoides SC recombinant proteins expressed in Escherichia coli [26]. That study demonstrated the feasibility of producing recombinant surface proteins from M. mycoides SC in E. coli and screening for antibodies in sera from CBPP-affected bovines by Western and dot blotting
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