Phylogeography and molecular epidemiology of Yersinia pestis in Madagascar

Abstract

Background: Plague was introduced to Madagascar in 1898 and continues to be a significant human health problem. It exists mainly in the central highlands, but in the 1990s was reintroduced to the port city of Mahajanga, where it caused extensive human outbreaks. Despite its prevalence, the phylogeography and molecular epidemiology of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We examine island-wide geographic-genetic patterns based upon whole-genome discovery of SNPs, SNP genotyping and hypervariable variable-number tandem repeat (VNTR) loci to gain insight into the maintenance and spread of Y. pestis in Madagascar.

Methodology/principal findings: We analyzed a set of 262 Malagasy isolates using a set of 56 SNPs and a 43-locus multi-locus VNTR analysis (MLVA) system. We then analyzed the geographic distribution of the subclades and identified patterns related to the maintenance and spread of plague in Madagascar. We find relatively high levels of VNTR diversity in addition to several SNP differences. We identify two major groups, Groups I and II, which are subsequently divided into 11 and 4 subclades, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga, where there is evidence for multiple transfers both from and to the central highlands.

Conclusions/significance: The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity.

Figure 1. SNP phylogeny of 262 Malagasy isolates.

Nodes were named as in Morelli et al. (lower case letters) and belong to the 1.ORI3 group described there . Previously identified nodes that were expanded in this analysis (h, l and q) have additional number designations (e.g., q1) given to each new node in the expansions. The one entirely new node was assigned a new letter, “r.” Previously identified nodes that were not represented by any isolates in this study are represented by gray outlines. Colored nodes correspond to MLVA-identified subclades and are colored the same as their matching MLVA subclades in Figure 2A–B. The number of isolates in nodes with >1 isolate are indicated as are the number of SNPs on branches (red numbers) with >1 SNP. The nodes containing the two sequenced Malagasy strains, MG05-1020 and IP275, are labeled with the strain names.

Figure 2. Neighbor-joining dendrograms based upon MLVA data.

Dendrograms for Group I (A) and Group II (B) are indicated. The SNP phylogeny from Figure 1 is also indicated (C) for comparison. Subclades within Groups I and II are collapsed in the full phylogenies (dotted boxes) for those groups (colored triangles) and are then individually expanded to show the structure within each subclade. The expanded subclades are labeled based upon their membership in Group I or II and by a capital letter (e.g., I.A) and are indicated by colored bars. Bootstrap values ≥50 supporting individual subclades are indicated on the expanded subclade phylogenies. SNP locations are indicated by vertical red lines. These red lines are labeled with the SNP ID numbers presented in Table S2 on the full phylogenies for unaffiliated isolate-specific SNPs and on the expanded phylogenies for all other SNPs. The years of isolation for isolates within each full and expanded phylogeny are indicated beside the panel label and underneath the individual phylogeny, respectively. The gray subcluster marked by the gray arrow in subclade I.A represents the “Mahajanga I.A subcluster,” a subcluster containing most of the isolates from the Mahajanga plague focus. Seven isolates from the central highlands that also fell within this subcluster are labeled with a “CH.” Five Mahajanga isolates that did not belong in this subcluster are labeled with a gray “M” (A). Black stars indicate the locations of the two sequenced Malagasy strains, MG05-1020 in subclade I.B and IP275 in subclade I.H.

Figure 3. Geographic distribution of MLVA subclades in Madagascar.

The MLVA phylogenies for Groups I and II from Figure 2A-B are presented with labeled subclades. Light gray shaded districts indicate Madagascar districts where Y. pestis isolates used in this study were obtained. Colors within the mapped circles and squares correspond to the subclade color designations in the MLVA phylogenies. Divisions within those circles and squares indicate that multiple subclades were found at that location. Circles represent isolates where the city/commune of origin is known. Squares represent isolates where only the district of origin is known and are placed within their corresponding districts near to cities/communes containing the same subclade(s) where possible. Six isolates had unknown districts of origin and were not mapped. Unaffiliated Group I and II isolates are indicated by an “*” and a “+,” respectively; these symbols surrounded by a square indicate unaffiliated isolates where only the district of origin is known. The dark gray shaded area indicates the geographic area where Group II subclades are found. Note that some Group I subclades are also found in this area.

Figure 4. Geographic distribution of SNP-defined nodes in the strain MG05-1020 lineage.

The strain MG05-1020 lineage portion of the SNP phylogeny from Figure 1 is indicated as well as an enlarged cutout of the map from Figure 3 showing the geographic distribution of isolates from this lineage. For an explanation of the mapped circles and squares see the figure legend for Figure 3. Circles, squares and pie chart slices in the map are numbered based upon the node number in the SNP phylogeny for the isolates represented by those shapes. The isolate in node “q7” is not mapped due to its geographic origin being unknown.