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A 14th century CE Brucella melitensis genome and the recent expansion of the Western Mediterranean clade [1]
['George S. Long', 'Department Of Biology', 'Mcmaster University', 'Hamilton', 'Mcmaster Ancient Dna Centre', 'Departments Of Anthropology', 'Biochemistry', 'Jessica Hider', 'Department Of Anthropology', 'Ana T. Duggan']
Date: 2023-08
Brucellosis is a disease caused by the bacterium Brucella and typically transmitted through contact with infected ruminants. It is one of the most common chronic zoonotic diseases and of particular interest to public health agencies. Despite its well-known transmission history and characteristic symptoms, we lack a more complete understanding of the evolutionary history of its best-known species—Brucella melitensis. To address this knowledge gap we fortuitously found, sequenced and assembled a high-quality ancient B. melitensis draft genome from the kidney stone of a 14 th -century Italian friar. The ancient strain contained fewer core genes than modern B. melitensis isolates, carried a complete complement of virulence genes, and did not contain any indication of significant antimicrobial resistances. The ancient B. melitensis genome fell as a basal sister lineage to a subgroup of B. melitensis strains within the Western Mediterranean phylogenetic group, with a short branch length indicative of its earlier sampling time, along with a similar gene content. By calibrating the molecular clock we suggest that the speciation event between B. melitensis and B. abortus is contemporaneous with the estimated time frame for the domestication of both sheep and goats. These results confirm the existence of the Western Mediterranean clade as a separate group in the 14 th CE and suggest that its divergence was due to human and ruminant co-migration.
By comparing the gene content of the ancient B. melitensis genome with modern isolates, we provide evidence that neither the B. melitensis phylogenetic groups nor their respective sequence types necessarily correspond with the genetic components of their strains. The accessory genome of B. melitensis appears to be influenced by its geographic location, more so than its sequence type. We propose splitting the Eastern Mediterranean clade into three smaller groups to better reflect these genomic differences.
We describe the reconstruction of a high-quality ancient B. melitensis genome isolated from the kidney stone of a 14 th C. CE Italian friar. We confirm the presence of a B. melitensis genome from the Western Mediterranean clade in Italy during this time period and refine previous estimates for the diversification of this phylogenetic group. In addition, we propose that the speciation event separating B. abortus and B. melitensis occurred during the domestication of sheep and goats approximately 9000 BCE.
Funding: We thank the Social Sciences and Humanities Research Council of Canada (20008499 to HNP), and the Canadian Institute for Advanced Research for the funding to HNP. The Natural Sciences and Engineering Research Council of Canada also provided funding for this work through a grant awarded to GBG (RGPIN-2020-05733). We are grateful for the support provided by the Joseph-Armand Bombardier Canada Graduate Scholarships Program Doctoral Scholarships to JH. EH is funded by an NHMRC Investigator Award (GNT2017197). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Data Availability: Code used to analyze the mapped data can be found at
https://github.com/longg2/AncientBrucella . The scripts used for quality control, the metagenomic analyses, mapping, and the phylogenetics can be found at
https://github.com/longg2/LongBioinformatics . The raw sequencing data can be found on NCBI under the ID PRJNA966239 while the assembled genome is GCF 030370715.
Introduction
Brucellosis is a zoonotic disease caused by the bacterium Brucella that is most often transmitted to humans by domestic livestock through the consumption of animal products and the interactions with infected animals [1]. It is one of the most common zoonotic diseases with an estimated 500,000 cases each year, although its incidence is hypothesized to be far higher (∼ 5 to 12.5 million) [2, 3]. Three main species of Brucella are pathogenic to humans. B. melitensis is carried by sheep and goats while B. suis and B. abortus are found in pigs and cows, respectively [4]. Brucellosis is a serious infection causing fever, chills, extreme muscle pain, and long-term osteoarticular changes that can be observed on bones [1]. As a chronic disease it is of particular importance to public health due to its indirect, yet significant economic impacts, by decreasing the workforce and reducing livestock reproduction and their associated products [5]. Brucellosis is common today in Asia, the Middle East, Africa, South America, and the Mediterranean [3]. Southern European countries make up the bulk of brucellosis infections in Europe, with Greece accounting for 20.3% of human cases while Italy has the highest prevalence of cattle herds, sheep and goat flocks testing positive for Brucella within the European Union as of 2020 [6].
Brucellosis is thought to have been a common scourge in the Mediterranean in the past, especially during the Roman and Medieval periods before the advent of pasteurization, and when pastoral practices such as transhumance were commonplace [7, 8]. However, little historical or archaeological evidence exists to support this claim [9]. Purported cases from the Roman period were identified in 16 skeletons from Herculaneum dating to 79 CE through the presence of non-specific vertebral lesions [8]. While skeletal lesions are a common manifestation of brucellosis, occurring in 10 to 85% of modern clinical cases, the lesions tend to occur in similar locations and with a similar appearance as those caused by tuberculosis [10], leading to potential confusion in differentiating between the two diseases in the past. If the pattern of lesion occurrence in brucellosis cases was the same as it is today, we would expect that more differential diagnoses of brucellosis would be reported from ancient remains [10]. However, there are several reasons why brucellosis may be under-reported in paleopathological analyses of archaeological samples; it is likely mistaken for other diseases that similarly impact bone like tuberculosis, the bacterium had not left skeletal changes by the time of death [7, 11, 12], or the non-specific nature of the lesions precluded a diagnosis [12].
Support for the presence of brucellosis during the Medieval period includes an ancient genome identified from a pelvic nodule of an individual in Sardinia dating to c. 1300 CE; two individuals from Albania (c. 900—c. 1200 CE) with PCR products arising from lesioned ribs and vertebrae; and two French individuals from the 14th and 18th C. CE where an infection was identified via paleopathology [13–15]. This limited DNA evidence compared to other infectious diseases could be due to improper sample selection [16], poor DNA preservation [12, 17, 18], or its mistaken diagnosis [18]. It is also possible that a focus on other diseases such as tuberculosis has meant that researchers have neglected possible evidence for brucellosis in the archaeological record.
The Mediterranean region is thought to have been a common geographic location for the origin and divergence of all Brucella species (especially B. melitensis) due to the diversity of strains in this area and the basal position of the Western Mediterranean clade in the global Brucella phylogeny [19, 20]. Despite this, the evolutionary history of the disease in the region is poorly understood. Due to the widespread movement of infected animals across regional, national, and international borders, the phylogeography of Brucella on both local and larger scales has been challenging to elucidate using modern isolates [19]. Sheep and goats are the preferred hosts for B. melitensis, and while spillover infections can occur, these alternative hosts tend to result in dead-end infections [4, 21]. This is especially the case in some animals that require constant exposure to B. melitensis to sustain an infection [22]. Sheep and goat husbandry extended across the Mediterranean littoral zone (Cyprus to Atlantic Portugal) by 5000 BCE [23–25]. Based on the dates of expansion of ruminants into the region during the Neolithic and dates of divergence for B. melitensis, it is hypothesized that the bacterium entered the region with the introduction of ruminants and subsequently diversified [4, 26, 27]. Paleopathological diagnoses of brucellosis in human skeletal remains from Jordan and Palestine (2100–1550 BCE), and Bahrain (3000–1200 BCE) support this hypothesis [7, 28, 29].
Archaeological and genetic data on the spread of sheep into the Mediterranean illustrate the complexity of repeated expansions and contractions over multiple periods of domestication. Subsequent selective breeding, trade, and geographic expansion have impacted the genetic signatures of sheep causing them to have little resemblance to their early progenitors [25, 30, 31]. Wild sheep and goats were domesticated approximately 10,000–8000 BCE in the Fertile Crescent [25, 32–34]. First domesticated for meat, sheep were later kept for their secondary products such as milk and wool [33]. The first expansion of domesticated sheep and goats from the Zagros mountains (northwestern Iran to southeastern Türkiye) spread to the borders of Europe and Africa by 8000 BCE [25, 33] following two routes of expansion; the Danubian route ran from Türkiye through the Balkans to the interior of Europe, while a southern route went through North Africa (North; Türkiye, Calabria, Corsica, and France; South—Iraq, Jordan, Egypt, Morocco) [25, 34, 35].
Subsequent expansions related to secondary features (e.g., fat tailed sheep, wool sheep) occurred between 4000 to 3000 BCE in Europe [32, 33, 35]. Wool sheep likely originated in Southwest Asia, spread west and then across Europe from the Iberian Peninsula [30, 35]. Wool producing sheep partially replaced the initial domesticated sheep (hair sheep) and earlier breeds derived from previous introductions via different routes (Mediterranean versus Danubian). Evidence for this replacement is supported by a larger number of ancestral SNPs in modern domesticated sheep from southeastern to northwestern Europe (i.e., the proportion of ancestral genetic components decrease when one is further away from the Fertile Crescent) [30, 32]. Italy and the Balkans were hubs for migrations of early and late domesticates such as wool sheep [32, 35].
An acute lack of genome-scale data concerning B. melitensis has impeded our understanding of its origin, evolution, and spread. Until recently, public databases contained few B. melitensis genomes, limiting inferences on epidemiological history. This lack of data has since changed due to the recent focus on the Western Mediterranean clade of the bacterium in several studies [20, 27, 36]. Although it was hypothesized that this clade has a long-standing presence in the Mediterranean it was underrepresented in genomic databases [26]. A recent study increased the representation of the diversity within Western Mediterranean clade by adding 339 Italian strains from humans and ruminants to public databases [26]. A lack of public health support for genome surveillance may also explain in part why other regions, such as Africa, are similarly under-sampled [37, 38].
In contrast to the paucity of genome-scale data, the distribution of sequence types (ST)—the allelic profile of core housekeeping genes in a bacterial species—within B. melitensis is well defined [39, 40]. Sequence types provide a more nuanced view of the diversity within the Brucella genus. These STs can be used to quickly identify the phylogenetic group of an unknown B. melitensis strain as they are relatively well conserved. For example, there are five STs (ST 9, 11, 87, 88, and 89) present in the Western Mediterranean clade [40]. Biovars—groups of isolates divided by their biochemical properties—were previously used to categorize Brucella strains. The Western and Eastern Mediterranean phylogenetic groups, however, contain genomes arising from all three known B. melitensis biovars, indicating that STs are superior for discriminating between strains [40].
Only one partial ancient B. melitensis genome has been reconstructed to date, although its coverage is unfortunately too low to allow for genome scale analyses and comparison here (see S1 Appendix) [13]. The lack of ancient genomes impedes analyses attempting to determine the most recent common ancestor of extant B. melitensis as well as its overall phylogeographic history and gene content. Here, we describe the successful reconstruction of a high quality B. melitensis genome from the kidney stone of a beatified 14th CE friar, Sante Brancorsini (1343—1394 CE). The high resolution and precise date of this new genome along with its geographic location and associated time period make it particularly useful for addressing questions about the evolution of B. melitensis in the Mediterranean.
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[1] Url:
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011538
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