Phylogenetic analysis of the origin and spread of plague in Madagascar

Background Plague is a zoonotic disease caused by the bacterium Yersinia pestis, highly prevalent in the Central Highlands, a mountainous region in the center of Madagascar. After a plague-free period of over 60 years in the northwestern coast city of Mahajanga, the disease reappeared in 1991 and caused several outbreaks until 1999. Previous research indicates that the disease was reintroduced to the city of Mahajanga from the Central Highlands instead of reemerging from a local reservoir. However, it is not clear how many reintroductions occurred and when they took place. Methodology/Principal findings In this study we applied a Bayesian phylogeographic model to detect and date migrations of Y. pestis between the two locations that could be linked to the re-emergence of plague in Mahajanga. Genome sequences of 300 Y. pestis strains sampled between 1964 and 2012 were analyzed. Four migrations from the Central Highlands to Mahajanga were detected. Two resulted in persistent transmission in humans, one was responsible for most of the human cases recorded between 1995 and 1999, while the other produced plague cases in 1991 and 1992. We dated the emergence of the Y. pestis sub-branch 1.ORI3, which is only present in Madagascar and Turkey, to the beginning of the 20th century, using a Bayesian molecular dating analysis. The split between 1.ORI3 and its ancestor lineage 1.ORI2 was dated to the second half of the 19th century. Conclusions/Significance Our results indicate that two independent migrations from the Central Highlands caused the plague outbreaks in Mahajanga during the 1990s, with both introductions occurring during the early 1980s. They happened over a decade before the detection of human cases, thus the pathogen likely survived in wild reservoirs until the spillover to humans was possible. This study demonstrates the value of Bayesian phylogenetics in elucidating the re-emergence of infectious diseases. Author summary In 1991 human cases of plague were reported in the city of Mahajanga, located in the west coast of Madagascar, after 60 years without human cases. Existing evidence suggests that Yersinia pestis, the causal agent of the disease, was reintroduced to the city from a mountainous region known as the Central Highlands. We performed a phylogeographic analysis on 300 Y. pestis genome sequences to determine how many migrations of the pathogen between the two locations were related to the reappearance of the disease in Mahajanga. The results revealed that two migrations from the Central Highlands were the cause of the outbreaks of plague in the west coast of the country during the 1990s. We also aimed to date the emergence of the Y.pestis variant that circulates in Madagascar and is also present in Turkey. To do this, we conducted a molecular dating analysis using an extended data set of 445 sequences, which contained sequences from Turkey and India (the country from which the pathogen was exported to Madagascar for the first time). The analysis indicated that this particular variant emerged in the first decade of the 20th century.


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Our results indicate that two independent migrations from the Central Highlands caused 48 the plague outbreaks in Mahajanga during the 1990s, with both introductions occurring during 49 the early 1980s. They happened over a decade before the detection of human cases, thus the 50 pathogen likely survived in wild reservoirs until the spillover to humans was possible. This study 51 demonstrates the value of Bayesian phylogenetics in elucidating the re-emergence of infectious 52 diseases.

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Author summary 54 In 1991 human cases of plague were reported in the city of Mahajanga, located in the 55 west coast of Madagascar, after 60 years without human cases. Existing evidence suggests that 56 Yersinia pestis, the causal agent of the disease, was reintroduced to the city from a mountainous 57 region known as the Central Highlands. We performed a phylogeographic analysis on 300 Y. 58 pestis genome sequences to determine how many migrations of the pathogen between the two 59 locations were related to the reappearance of the disease in Mahajanga. The results revealed that 60 two migrations from the Central Highlands were the cause of the outbreaks of plague in the west 61 coast of the country during the 1990s. We also aimed to date the emergence of the Y.pestis 62 variant that circulates in Madagascar and is also present in Turkey. To do this, we conducted a 63 molecular dating analysis using an extended data set of 445 sequences, which contained 64 sequences from Turkey and India (the country from which the pathogen was exported to Introduction 70 The gram-negative bacterium Yersinia pestis is the causal agent of plague, a zoonotic 71 disease transmitted by hematophagous vectors (fleas). Despite being primarily a rodent disease, 72 plague has infected humans for thousands of years [1,2], and has been responsible for at least 73 three severe pandemics throughout human history: the first pandemic or Justinian plague (541-74 750 CE), the second pandemic that started with the Black Death (14 th -18 th Century CE) and the 75 third pandemic that originated in China in 1855 and spread globally from 1894 [3]. Altogether, 76 plague claimed millions of lives during the course of these pandemics. 77 Currently, over 90% of the plague cases occur in the African continent [4]. The most 78 affected country is Madagascar, an island located on the eastern coast of southern Africa, which 79 accounts for 75% of all infections worldwide [5]. The strains of Y. pestis circulating in 80 Madagascar belong to the 1.ORI3 phylogenetic sub-branch, which is part of the Y. pestis lineage 81 responsible for the third plague pandemic [3]. According to historical records, plague was 82 introduced in Madagascar when steamboats coming from India arrived at the port city of 83 Toamasina in November 1898 [6,7]. Phylogenetic evidence suggests that plague was exported 84 from Madagascar to the Anatolian peninsula (modern day Turkey) [3].

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In decades following its introduction to Madagascar, plague spread through the country 86 establishing three infection foci. Two are located in the Northern and Central Highlands, regions 87 with an altitude of 800 meters above sea level, while the third one is found in the city of 88 Mahajanga, located on the west coast of the country [8,9]   rodents, fleas and shrews, were recovered from the online database EnteroBase (S1 Table) [18]. repeats and interspersed repeats in the CO92 genome using PILER-CR, TRF and a self-138 comparison by minimap2, respectively. Any SNPs in the repeat regions, or in a region that was 139 shared by ≤95% of genomes were discarded. The resulting core genomic, non-repetitive SNP 140 matrix contained 783 sites (S2 Table). 141 While the genotype of the Indian sequences had been determined by a previous study [3], 142 the presence in the matrix of 226 previously described SNPs related to 1.ORI3 (S3 Table) 143 confirmed the genotype of the remaining sequences. This entire matrix was used to generate a  Phylogeographic analysis of Y. pestis 159 The phylogeographic analysis was done with the BEAST 2 package bdmm [23], which 160 uses a structured (also called "multi-type") birth-death model for the tree-generating process.

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Under this approach each sequence is assigned to a "type" or subpopulation and the type changes reproductive number (R) in epidemiological contexts, are also estimated for each type. 168 We performed three replicate analyses with a MCMC of 300 million steps using a relaxed 169 lognormal molecular clock and the HKY model of nucleotide substitutions. Sequences from 170 Mahajanga were assigned to the type "0" and sequences from the Central Highlands to the type MCC tree files are provided as supporting information (S1-S6 Appendix).

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Migration dynamics of Y. pestis 216 We conducted a phylogeographic analysis based on the 300 human sequences from  The MAP tree used to summarize the migration history displayed five migration events, 231 four of which were from the Central Highlands to Mahajanga (Fig 2). The first migration    for Madagascan strains, although using only 33 genomes [32].