A comprehensive view of the evolutionary history and the dynamics of Mccp strains is essential to understand the epidemiology of CCPP

Contagious caprine pleuropneumonia (CCPP), caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), is a devastating disease of domestic goats and of some wild ungulate species. The disease is currently spreading in Africa and Asia and poses a serious threat to disease-free areas. A comprehensive view of the evolutionary history and dynamics of Mccp is essential to understand the epidemiology of CCPP. Yet, analysing the diversity of genetically monomorphic pathogens, such as Mccp, is complicated due to their low variability. In this study, the molecular epidemiology and evolution of CCPP was investigated using a large-scale genomic approach based on next-generation sequencing technologies, applied to a sample of strains representing the global distribution of this disease. A highly discriminatory multigene typing system was developed, allowing the differentiation of 24 haplotypes among 25 Mccp strains distributed in six genotyping groups, which showed some correlation with geographic origin. A Bayesian approach was used to infer the first robust phylogeny of the species and to date the principal events of its evolutionary history. The emergence of Mccp was estimated only at about 270 years ago, which explains the low genetic diversity of this species despite its high mutation rate, evaluated at 1.3 × 10 substitutions per site per year. Finally, plausible scenarios were proposed to elucidate the evolution and dynamics of CCPP in Asia and Africa, though limited by the paucity of Mccp strains, particularly in Asia. This study shows how combining large-scale genomic data with spatial and temporal data makes it possible to obtain a comprehensive view of the epidemiology of CCPP, a precondition for the development of improved disease surveillance and control measures. Introduction Contagious caprine pleuropneumonia (CCPP) is a severe respiratory disease affecting goats and some wild ruminant species. The disease, listed by the World Organisation for Animal Health (OIE) [1] has a great economic impact on livestock production in fragile rural economies and poses a serious threat to disease-free areas. CCPP is caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp), a member of the Mycoplasma mycoides cluster [2]. This cluster comprises five mycoplasmas which are pathogenic for ruminants, including Mycoplasma capricolum subsp. capricolum (Mcc), the closest relative of Mccp, and Mycoplasma mycoides * Correspondence: virginie.dupuy@cirad.fr CIRAD, UMR CMAEE, F-34398 Montpellier, France INRA, UMR1309 CMAEE, F-34398 Montpellier, France © 2015 Dupuy et al. This is an Open Access a (http://creativecommons.org/licenses/by/4.0) medium, provided the original work is prope creativecommons.org/publicdomain/zero/1.0 subsp. mycoides (Mmm), the agent of contagious bovine pleuropneumonia (CBPP). Since it was first isolated in 1976 [3], Mccp has only been isolated in 17 countries, mainly because of its fastidiousness in culture. However, clinical descriptions have been published in nearly 40 countries in Africa and Asia, suggesting a much wider distribution [4]. The disease is present in the Arabian Peninsula, North, Central and East Africa and Asia, but its boundaries are still uncertain, particularly in western and southern Africa and in Asia. In the last decade, an increasing number of outbreaks have been reported in both domestic and wild ruminants [5]. New detections were often the result of improved diagnosis, confirming the presence of the disease in suspected regions [6–8], but in certain cases, they indicated that the disease had spread to new territories [9,10]. rticle distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any rly credited. The Creative Commons Public Domain Dedication waiver (http:// /) applies to the data made available in this article, unless otherwise stated. Dupuy et al. Veterinary Research (2015) 46:74 Page 2 of 11 A comprehensive view of the evolutionary history and the dynamics of Mccp strains is essential to understand the epidemiology of CCPP. Molecular epidemiology investigations require robust genotyping tools with sufficient resolution, while evolutionary analyses are also constrained by the need for a reliable molecular clock to infer evolutionary timescales. Various molecular methods have been developed for the analysis of Mccp strains. The first study on the molecular evolution of Mccp was based on the 16S rRNA gene [11], which provided a basis for the phylogeny and systematics of bacteria since the evolutionary studies of Woese [12]. Mccp strains showed a surprisingly high degree of polymorphism between their two 16S rRNA gene copies [13], which allowed the use of this housekeeping gene to combine epidemiological and evolutionary analyses. Still, the study was limited by the low discriminatory power of this molecular marker. Despite the heterogeneity observed in the sequence of its two rRNA operons, the Mccp genome has been shown to be rather monomorphic. Thus, a phylogenic study of the M. mycoides cluster based on five partial housekeeping gene sequences showed a very low distance between Mccp strains [14]. The low diversity of such a monomorphic bacterium precluded the use of standard multilocus sequence typing (MLST), which relies on housekeeping genes [15]. Approaches based on alternative sequences, independently of their coding capacity, were thus preferred. Analysis of the H2 locus allowed the discrimination of four groups showing a good correlation with geographic origin [16]. This system was improved by the addition of seven loci, increasing the discriminatory power and leading to the description of five groups [4]. Although this multilocus sequence analysis (MLSA) scheme targets polymorphic loci by focusing on non-housekeeping genes (e.g., intergene regions, pseudogenes) it interrogates less than 1% of the genome and therefore has limited discriminatory power for epidemiological investigations. Moreover, it is not reliable for phylogenetic analyses because of differences in the molecular evolutionary clock among target sequences. These limitations can now be overcome by the recent development of high-throughput methods [17]. Even though single nucleotide polymorphism (SNP) frequencies are low in monomorphic pathogens, their numbers can be dramatically increased by enlarging the scale of the analysis. Using high-throughput data can enhance genetic investigations by providing a way to disclose genome-wide variations. Combining large-scale genomic data with spatial and temporal data already enabled a comprehensive view of the molecular epidemiology and evolution of bacterial pathogens like Salmonella typhi [18], Yersinia pestis [19], Mmm [20] and Mycobacterium tuberculosis [21]. Following the increasing use and affordability of DNA sequencing with next-generation sequencing (NGS) technologies, complete annotated genomes of several Mccp strains have become available [22–24], thereby making large-scale genomic investigations possible. In this study, we investigated the molecular epidemiology and evolution of CCPP using a large-scale genomic approach based on NGS data, on a sample of strains representing the global distribution of this disease. Our main objective was to develop a discriminatory genotyping method to investigate the genetic diversity and population structure of Mccp. A robust phylogeny was also inferred from a large phylogenomic data set and divergence time of Mccp strains was estimated to reconstruct the evolutionary history of CCPP. Materials and methods