Multi-locus sequence analysis of Mycoplasma capricolum subsp. capripneumoniae for the molecular epidemiology of contagious caprine pleuropneumonia

Abstract

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease of domestic goats. The exact distribution of CCPP is not known but it is present in Africa and the Middle East and represents a significant threat to many disease-free areas including Europe. Furthermore, CCPP has been recently identified in Tajikistan and China. A typing method with an improved resolution based on Multi-Locus Sequence Analysis (MLSA) has been developed to trace new epidemics and to elucidate whether the recently identified cases in continental Asia were due to recent importation of Mccp. The H2 locus, a polymorphic region already in use as a molecular marker for Mccp evolution, was complemented with seven new loci selected according to the analysis of polymorphisms observed among the genome sequences of three Mccp strains. A total of 25 strains, including the two new strains from Asia, were analysed by MLSA resulting in the discrimination of 15 sequence types based on 53 polymorphic positions. A distance tree inferred from the concatenated sequences of the eight selected loci revealed two evolutionary lineages comprising five groups, which showed good correlation with geographic origins. The presence of a distinct Asian cluster strongly indicates that CCPP was not recently imported to continental Asia. It is more likely that the disease has been endemic in the area for a long time, as supported by historical clinical descriptions. In conclusion, this MLSA strategy constitutes a highly discriminative tool for the molecular epidemiology of CCPP.

Figure 1

Tree derived from distance analysis of the eight concatenated MLSA loci. The tree was constructed using the unweighted neighbour-joining algorithm (Darwin 5.0). A single strain representing each of the 15 MLSA ST is displayed (see Table 1 for strain details). The two sequences presenting a large 960 nt deletion (09018 and C550/1) were grafted at their respective positions after tree construction (discontinuous lines) in order to avoid their influence during tree inference. The root of the tree is represented as a bold dot. Bootstrap percentage values were calculated from 1000 resamplings and values over 80% are displayed. The scale bar shows the equivalent distance to 1 substitution per 1000 nucleotide positions. The ST were numbered according to the group in which they clustered, followed by a three digit code that should allow intercalating additional ST as new sequences are obtained. Note that the two letter code following the strain name represents the corresponding country of isolation: AE, United Arab Emirates; CD, Chad; CH, China; ET, Ethiopia; NR, Niger; OM, Oman; SD, Sudan; TJ, Tajikistan; TN, Tunisia; UG, Uganda.

Figure 2

Geographic origins of the strains analysed in this study. Each strain is represented by a symbol corresponding to its MLSA group and by its ST. Note that when the exact location was not known the symbols were barred and were positioned arbitrarily in the corresponding country. The arrowed lines represent main routes of trade and other animal movements in the different areas.

Figure 3

Probable distribution of CCPP. The countries in which the disease has been described, those in which the etiologic agent has been detected using molecular tests and those in which it has been isolated are indicated. The arrow indicates the presence of the disease in Mauritius, where Mccp was isolated in 2009.