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Characterisation of pathogenic Leptospira in invasive raccoons (Procyon lotor) in northeast and southwest France

Published online by Cambridge University Press:  04 November 2024

Florence Ayral*
Affiliation:
USC 1233-RS2GP, VetAgro Sup, Université de Lyon, Marcy L’Etoile, France
Elena Harran
Affiliation:
USC 1233-RS2GP, VetAgro Sup, Université de Lyon, Marcy L’Etoile, France
Christine Fournier-Chambrillon
Affiliation:
Groupe de Recherche et d’Etude pour la Gestion de l’Environnement (GREGE), Villandraut, France
Manon Gautrelet
Affiliation:
Groupe de Recherche et d’Etude pour la Gestion de l’Environnement (GREGE), Villandraut, France Centre de Recherche et de Formation en Eco-éthologie (CERFE), Université de Reims Champagne-Ardenne, Boult-aux-Bois, France Groupe d’Etude sur les Géomatériaux et Environnements Naturels, Anthropiques et Archéologiques (GEGENAA), Université de Reims Champagne-Ardenne, Reims, France
Océane Tourniaire
Affiliation:
USC 1233-RS2GP, VetAgro Sup, Université de Lyon, Marcy L’Etoile, France
Alexis Peutot
Affiliation:
USC 1233-RS2GP, VetAgro Sup, Université de Lyon, Marcy L’Etoile, France
Karine Groud
Affiliation:
USC 1233-RS2GP, VetAgro Sup, Université de Lyon, Marcy L’Etoile, France
Nathan Thenon
Affiliation:
Groupe de Recherche et d’Etude pour la Gestion de l’Environnement (GREGE), Villandraut, France UMR 1225, INRAE, Ecole Nationale Vétérinaire de Toulouse, Toulouse, France
Pascal Fournier
Affiliation:
Groupe de Recherche et d’Etude pour la Gestion de l’Environnement (GREGE), Villandraut, France
Céline Richomme
Affiliation:
Anses, Nancy Laboratory for Rabies and Wildlife, French Agency for Food, Environmental and Occupational Health and Safety (ANSES), Malzéville, France
*
Corresponding author: Florence Ayral; Email: florence.ayral@vetagro-sup.fr
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Abstract

Leptospirosis is a widespread zoonosis caused by bacteria of the genus Leptospira. Although crucial to mitigate the disease risk, basic epidemiological information is lacking, such as the identities of Leptospira maintenance hosts. The raccoon (Procyon lotor), an alien invasive species in France, could pose a public health risk if it carries pathogenic Leptospira. We investigated the rate and type (selective vs. unselective) of Leptospira carriage in the two main raccoon populations in France. Out of the 141 raccoons collected, seven (5%) tested quantitative PCR positive, targeting lfb1 gene, based on kidney, lung, and urine samples. Phylogenetic analysis revealed the presence of three different L. interrogans clusters. The results suggest that raccoons were more likely accidental hosts and made only a limited contribution to Leptospira maintenance.

Type
Short Paper
Creative Commons
Creative Common License - CCCreative Common License - BYCreative Common License - NCCreative Common License - ND
This is an Open Access article, distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives licence (http://creativecommons.org/licenses/by-nc-nd/4.0), which permits non-commercial re-use, distribution, and reproduction in any medium, provided that no alterations are made and the original article is properly cited. The written permission of Cambridge University Press must be obtained prior to any commercial use and/or adaptation of the article.
Copyright
© The Author(s), 2024. Published by Cambridge University Press

Leptospirosis is a global and potentially fatal zoonotic disease that affects all mammals, including humans, and is caused by pathogenic species of the genus Leptospira. The genetic polymorphism diversity of eight pathogenic species was recently studied using the lfb1 gene, identifying 46 species groups [Reference Garcia-Lopez1]. Leptospira is mainly transmitted through soil or water contaminated by the urine of infected animals. The bacteria can survive for days in aquatic environments, which are the origin of most human cases of leptospirosis. However, the source of environmental contamination is a range of mammalian maintenance hosts: Leptospira colonizes the kidneys, where they remain over the long term and are shed in the urine [Reference Thiermann2].

Although leptospirosis is a major public health burden, management strategies are limited due to a lack of basic epidemiological knowledge, such as the role of various animal hosts in Leptospira maintenance across ecosystems. In order to assess the ability of a given mammal species to maintain Leptospira and to design better disease prevention approaches, it is crucial to determine the prevalence of Leptospira in target animal populations, as well as any host–pathogen adaptations that may be present (i.e., whether the target animals exclusively carry a particular Leptospira strain) [Reference Thiermann2]. The raccoon (Procyon lotor) is a North American species that has become invasive worldwide, notably in Europe and different regions of mainland France [Reference Larroque3]. Leptospira may circulate endemically in raccoons in North America or sporadically, as previously suggested in France [Reference Helman4, Reference Ayral5]. Therefore, the raccoons make different contributions to Leptospira epidemiology across ecosystems and it is important to explore the potential variability within populations in the country.

The aim of this study was to assess Leptospira infections in the two main raccoon populations in France. In addition, we aimed to determine whether selective carriage occurs in raccoons by genetically characterising any Leptospira DNA retrieved.

This work used a sub-sample of a wider study related to raccoon’s ecology, in northeastern and southwestern France, between 2019 and 2021 [Reference Umhang6]. The raccoons were found dead on the roads or trapped by duly licensed trappers in the context of invasive population management and sacrificed in accordance with the regulations on alien invasive species (French decree of 2 September 2016) and animal welfare guidelines (Directive 2010/63/EU). Therefore, an application to the Ethics Committee was not required.

All the animals were frozen immediately after collection. They were later thawed for necropsy to obtain kidney, lung, and urine samples (if available) from each animal. Kidney tissue and urine are the preferred biological materials for the detection of Leptospira. The lung was also included because a previous study showed lung colonisation in rats [Reference Zilber7]. The samples were then stored at –20°C until further analysis. DNA extraction was performed using the Nucleospin Tissue Kit (Macherey Nagel, Hoerd, France) according to the manufacturer’s instructions, and DNA samples were stored at –20°C until the molecular analyses could be performed. The presence of pathogenic Leptospira DNA in the kidney, lung, and urine samples was assessed by quantitative PCR (qPCR) targeting the 16S rRNA (rrs) gene and the AgPath-ID™ One-Step qPCR Reagents (Applied Biosystems, Austin, United States), as described elsewhere [Reference Waggoner8]. DNA samples with a cycle threshold (Ct) of less than 40 were considered to be positive and were further amplified by conventional PCR (cPCR) targeting the lfb1 gene as described previously [Reference Garcia-Lopez9]. The amplified products were verified by 1% agarose gel electrophoresis and subjected to Sanger sequencing (Genoscreen, Lille, France). Sequencing of the lfb1 gene provides information on the species group, which is related to both the bacterial species and the genogroup [Reference Garcia-Lopez1]. This approach could increase typing success when working with wildlife samples that may contain damaged DNA or low quantity of Leptospira DNA. A nucleotide BLAST search was performed (NCBI: http://blast.ncbi.nlm.nih.gov) to identify the Leptospira species. A phylogenetic tree was then generated using the Leptospira spp. lfb1 partial gene polymorphism in raccoon samples and reference strains provided elsewhere [Reference Garcia-Lopez1].

We included a total of 141 raccoons (100 and 41 from northeastern and southwestern France, respectively). Seven raccoons were qPCR positive based on kidney and/or urine samples. All the lung samples were qPCR negative. Among the tested raccoons, seven were infected (5%, CI95% [Reference Thiermann2%, Reference Tamura, Nei and Kumar10%]), including five from northeastern France and two from southwestern France. L. interrogans was detected in six samples coming from five of the seven qPCR-positive raccoons (two Leptospira typing failed). Phylogenetic analysis identified three distinct lfb1 species groups: the first (three amplicons coming from two raccoons) was described in reference strains belonging to the Autumnalis, Bataviae, and Australis serogroups; the second (one amplicon) was described in a reference strain belonging to the Djasiman serogroup; and the third (two amplicons) was described in reference strains belonging to the Icterohaemorrhagiae serogroup (Figure 1).

Figure 1. Phylogenetic tree of Leptospira DNA extracted from raccoons and characterized based on the lfb1 gene sequences (bootstrap analysis involving 1 000 replicates and 34 nucleotide sequences). The evolutionary distances (unit = base substitutions per site) were computed using the maximum composite likelihood method [Reference Tamura, Nei and Kumar10] and MEGA11 software. The pink circles represent the amplicons from the samples. The contigs are identified using a coding system with the format ‘RL NN M’: where NN indicates sample geographical origin (northeastern France = A and U, southwestern France = G) plus ID number and M indicates sample tissue type (urine = U, kidney = R).

Our results suggest that raccoons could potentially spread pathogenic Leptospira given that Leptospira DNA was found in the kidney and urine samples. However, raccoons seem more likely to serve as non-maintenance or accidental hosts (i.e., short-term infection and shedding) than as maintenance hosts (i.e., long-term infection and shedding). Indeed, maintenance hosts, namely Rattus species, have prevalence levels exceeding 20% in France [Reference Desvars-Larrive11] and in other countries as a result of chronic renal colonisation [Reference Thiermann2]. Populations with lower prevalence levels are therefore unlikely to maintain Leptospira for long periods. In this study, the Leptospira infection rate encompassed 5% (CI95% [Reference Thiermann2%, Reference Tamura, Nei and Kumar10%]) of the tested raccoons, which is lower than that found in rats, and this magnitude was strengthened by a recent study in Germany, a bordering country [Reference Reinhardt12]. In contrast to rats, Leptospira DNA was not detected in the lung, highlighting possible differences in the pathogeny or host adaptation between the two species.

In addition, we identified three species groups of Leptospira within the infected raccoons, a result that is consistent with the hypothesis that they are non-maintenance hosts. Indeed, Leptospira maintenance hosts appear to selectively carry specific strains; for example, rats are the primary hosts for the L. interrogans serogroup Icterohaemorrhagiae [Reference Thiermann2]. The diversity of Leptospira species-groups we found in raccoons supports the idea that carriage is not selective, as has been suggested elsewhere [Reference Ayral5, Reference Reinhardt12]. In other words, raccoons may be sporadically infected by strains present in the environment but not able to maintain particular strain for prolonged periods. However, only one sample (RLG57R) coming from the southwest population could be typed. Additional raccoons should be therefore analysed to further clarify the species group diversity in this subpopulation.

Lastly, the magnitude of the Leptospira infection rate was similar in raccoons sampled in northeastern and southwestern France, suggesting that raccoons could have similar epidemiological contributions in both regions and ecosystems.

In conclusion, our results suggest that, unlike rats, raccoons are unlikely to maintain Leptospira, although they may spread them somewhat in the environment. However, some raccoon populations are currently found in peri-urban areas, in close proximity to humans and dogs. The risk of transmission should not be neglected, especially if the raccoon densities increase. Under such conditions, it would be important to reassess the risk of Leptospira transmission associated with raccoons.

Data availability statement

The data that support the findings of this study are available on request.

Acknowledgements

We thank Fabien Egal (Association Départementale des Piégeurs Agréés de la Gironde [ADPAG]), Thibault Gritti (Office Français de la Biodiversité [OFB]), and Estelle Isère-Laoué (GREGE) for their technical support. We are grateful to Guillaume Le Loc’h, the veterinary students, and internship students at the Ecole nationale vétérinaire de Toulouse who helped with the necropsies. We thank the Association Nationale Recherche et Technologie, the Office Français de la Biodiversité, the MRRNP, the Direction Régionale de l’Environnement, de l’Aménagement et du Logement du Grand Est and de Nouvelle-Aquitaine, the Département de la Gironde, the Mairie de Villenave d’Ornon (Isabelle Maille), the FDC51, the GREGE, and the CERFE for funding this work.

Author contributions

Formal analysis: A.P., E.H., K.G., O.T.; Writing – review & editing: A.P., C.R., C.F., M.G., N.T., P.F.; Conceptualization: C.R., M.G., F.A.; Data curation: C.R., C.F., M.G., P.F.; Funding acquisition: C.R., F.A.; Project administration: C.R.; Investigation: C.F., N.T., P.F.; Software: E.H.; Visualization: E.H.; Writing – original draft: E.H., F.A.; Supervision: K.G., F.A.; Methodology: F.A.; Validation: F.A.

Footnotes

These authors contributed equally to this work

References

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Figure 0

Figure 1. Phylogenetic tree of Leptospira DNA extracted from raccoons and characterized based on the lfb1 gene sequences (bootstrap analysis involving 1 000 replicates and 34 nucleotide sequences). The evolutionary distances (unit = base substitutions per site) were computed using the maximum composite likelihood method [10] and MEGA11 software. The pink circles represent the amplicons from the samples. The contigs are identified using a coding system with the format ‘RL NN M’: where NN indicates sample geographical origin (northeastern France = A and U, southwestern France = G) plus ID number and M indicates sample tissue type (urine = U, kidney = R).