Genetic and Serological Analysis of Lipoprotein LppA in Mycoplasma mycoides subsp. mycoides LC and Mycoplasma mycoides subsp. capri

Abstract

The genes encoding the 62-kDa lipoproteins from theMycoplasma mycoides subsp. mycoideslarge-colony type (LC) strain Y-goat and the M. mycoidessubsp. capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been namedlppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp. mycoidessmall-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplifiedlppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp. mycoidesLC and M. mycoides subsp. capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in allM. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the other members of the M. mycoidescluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.

Phylogeny of the Mycoplasma mycoides cluster based on analysis of five conserved protein-coding sequences and possible implications for the taxonomy of the group

“…Although early DNA-DNA hybridization studies by Askaa et al (1978) suggested that Mmc was closer to MmmSC than to MmmLC, analyses of 1D and 2D PAGE protein patterns proved the opposite (Costas et al, 1987;Leach et al, 1989;Olsson et al, 1990;Rodwell, 1982;Salih et al, 1983). Identification using a DNA probe (Taylor et al, 1992) and analysis of LppA sequence similarity and serological cross-reactions (Monnerat et al, 1999) also suggested a very close relatedness between MmmLC and Mmc, and two phylogenetic studies of the M. mycoides cluster concluded that they should be grouped into a single entity (Pettersson et al, 1996;Thiaucourt et al, 2000). However, the International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Mollicutes has indicated that additional comparative analysis is required in order to clarify this matter (Johansson & Bradbury, 2007).…”

“…Additional evidence has recently been presented by Vilei et al (2006) based on rpoB sequence comparisons. In the present work, comparisons have been extended to five genes using a similar sample of strains, though strain WK354, included as an Mmc strain in previous studies (Monnerat et al, 1999;Thiaucourt et al, 2000), was characterized in this work as MmmLC by both growth inhibition and dot immunobinding techniques. As in previous studies, the data obtained in this work show that MmmLC is closer to Mmc than to the biotype MmmSC and that MmmLC and Mmc are not sufficiently distant to constitute separate entities.…”

“…Another controversial issue is the distinction of MmmLC and Mmc as separate subspecies: numerous investigations, based on both genetic and antigenic analyses, suggest that they may be classed together as a single subspecies within the M. mycoides subcluster (Costas et al, 1987;Leach et al, 1989;Monnerat et al, 1999;Olsson et al, 1990;Rodwell, 1982;Salih et al, 1983;Taylor et al, 1992;Vilei et al, 2006).…”

Study on Seroprevalence and Risk Factors Contagious Bovine Pleuropneumonia in Southern Nationand Nationality People of Ethiopia Regional State in Amaro Special District

“…The main problem in eradication is the frequent occurrence of sub asymptomatic infections and the persistence of chronic carriers after the clinical phase. Serological analysis is the most important diagnostic tool for the control of CBPP, but it is significantly hampered by the relatively low sensitivity and specificity of the methods (Abdo et al al., 2003;Vilei et al, 2000;Monnerat et al Contagious Bovine Pleuropneumonia (CBPP) is both an epidemic and endemic disease of cattle that affects production through mortality and reduction in product It also retards genetic improvement and limits working ability of cattle (Tambi and Maina, 2004). Accordingly, cattle in CBPP-infected areas (epidemic and endemic) are divided into three classes namely, calves and yearlings below 3 years, adult males and reproductive females.…”

Removal of a subset of non-essential genes fully attenuates a highly virulentMycoplasmastrain

“…The first two regions (D1 and D2) encode enzymes and putative glycerol transporters involved in the production of hydrogen peroxide using the glycerol-dependent metabolism of mycoplasmas (10). The region D3 encodes major antigens (LppA/P72) in Mycoplasma mycoides (21) that induced T cell responses early in infection (22). The region D4 includes an integrative conjugative element (ICE), the MIB-MIP system (9) and the F1-likeX0 ATPase (15).…”