Host population dynamics influence Leptospira spp. transmission patterns among Rattus norvegicus in Boston, Massachusetts, US

Abstract

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus, is an important reservoir of Leptospira spp. in urban settings. We investigated the presence of Leptospira spp. among brown rats in Boston, Massachusetts and hypothesized that rat population dynamics in this urban setting influence the transportation, persistence, and diversity of Leptospira spp. We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira spp. We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira spp. isolates obtained from frozen and fresh tissue from some of the 59 positive rat kidneys. When isolates were not obtained, we attempted genomic DNA capture and enrichment, which yielded 14 additional Leptospira spp. genomes from rats. We also generated an enriched Leptospira spp. genome from a 2018 human case in Boston. We found evidence of high genetic structure among rat populations that is likely influenced by major roads and/or other dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats that were tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and its transportation is influenced by rat population dynamics. Finally, our genomic analyses of the Leptospira spp. detected in the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other similar urban settings.

Fig 1. Rattus norvegicus population assignment.

STRUCTURE population assignment of 311 R. norvegicus with collections color coded according to genetic group (S1 Table); likely dispersal barriers and major open spaces are indicated with black bars and green shading, respectively. A) Inferred migrants are represented by one-sided arrows; shared genetic groups among collections from sites 13, 14, and 16, and separately sites 9 and 10, are indicated by thin lines; and lipL32 PCR positivity at 12 of 17 sites is indicated with asterisks. Two inferred migrants (sites 2 and 6) were both lipL32 PCR positive and are denoted by asterisks on one-sided arrows. B) Each individual R. norvegicus (n = 311) is assigned a probability of membership (Q ≥ 0.75) to one of 12 genetic groups (S3 Table). Collection ID is indicated on the X-axis and probability (Q) on the Y-axis; each thin vertical line represents the Q value for an individual rat. The map in panel A was created using ArcGIS software by Esri. ArcGIS and Arc-Map are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com. Basemap: Light Gray Canvas Base https://www.arcgis.com/home/item.html?id=8b3d38c0819547faa83f7b7aca80bd76.

Fig 2. Leptospira interrogans serogroup Icterohaemorrhagiae whole genome phylogeny.

A maximum likelihood phylogeny of 28 Leptospira interrogans serogroup Icterohaemorrhagiae isolate genomes from rats, two publicly available L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni complete genomes (GenBank accession# GCA_002073495.2 and GCA_010978155.1), and 13 enriched genomes (12 rats and 1 human) based upon a concatenated SNP alignment of 127 positions out of a core genome size of 2,123,028 nucleotides (nts). The phylogeny was rooted with reference genome L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130 (GenBank accession# GCA_000007685.1). Four major clades were observed (1-4 in white text) and bootstrap values are indicated at specific nodes. The color of the genome ID corresponds to the rat genetic groups in Fig 1; black text indicates that no genetic group was assigned. One inferred migrant (indicated with a black arrow) from site 6c was trapped at site 2c and carried Leptospira interrogans identical to its source collection (see R335-Migrant in clade 1).

Fig 3. Rattus norvegicus trap locations.

Map indicating 64 trap locations (white circles) at 17 sites (numbers), likely dispersal barriers (black bars), and major open spaces (green shading). Circle size corresponds to the number of total rat samples collected from each trap location. This map was created using ArcGIS software by Esri. ArcGIS and Arc-Map are the intellectual property of Esri and are used herein under license. Copyright Esri. All rights reserved. For more information about Esri software, please visit www.esri.com. Basemap: Light Gray Canvas Base https://www.arcgis.com/home/item.html?id=8b3d38c0819547faa83f7b7aca80bd76.

Fig 4. Leptospira interrogans serogroup Icterohaemorrhagiae clade 2 whole genome phylogeny.

Maximum likelihood phylogeny of four R. norvegicus and one human derived L. interrogans genomes from clade 2 (see Fig 2) based upon the concatenated SNP alignment of 33 positions out of a core genome of 3,280,516 nucleotides. The color of the genome ID corresponds to the rat genetic group in Fig 1; black text indicates that no genetic group was assigned.