Tracking potential Leptospira sources following human cases of leptospirosis: A One Health approach applied to an ecosystem in Brittany, France.

Pathogenic Leptospira can cause leptospirosis: a widespread, potentially fatal bacterial zoonosis whose risk is mediated by the soil and water features, animal host distributions, meaning the local ecosystem. When human cases of leptospirosis occur, it is challenging to track down their source because ecosystem-level epidemiological knowledge on Leptospira is needed. Between 2016 and 2019 in a focal riparian ecosystem, the human population experienced an outbreak and successive cases of leptospirosis attributable to L. kirschneri and L. interrogans. The epidemiological investigation was carried out using the One Health approach, as described in international health guidelines. As a first step in this process, we investigated leptospiral carriage in the main animal hosts found in the region. We sampled 143 nutrias, 17 muskrats, and 10 Norway rats using convenient trapping. DNA was extracted from their kidneys, lungs, and urine and subjected to real-time PCR (RT-PCR) targeting the Leptospira 16S rDNA and lfb1 genes. In the farms along the river's stretch of interest, we sampled serum from 439 cattle and used a microscopic agglutination test to detect the presence of antibodies against Leptospira. Urine samples were concomitantly obtained from 145 cattle and were used in two analyses: RT-PCR targeting the Leptospira 16S rDNA gene and Leptospira culturing. We found th, wt rodents were the most likely source of the L. interrogans behind the human cases. The cattle tested negative for Leptospira DNA but positive for antibodies against the serogroups implicated in the human cases. We failed to identify the potential source of the L. kirschneri responsible for several human cases of leptospirosis. Our results call for further clarification of the Leptospira maintenance community, which may comprise known maintenance hosts, such as rodents, as well as taxa not commonly considered to be maintenance hosts but that can still spread Leptospira. The resulting research network will collaboratively conduct future eco-epidemiological surveys to illuminate the leptospirosis risks faced by humans and animals within ecosystems.

Structural investigation of vitamin K epoxide reductase domain-containing protein in Leptospira species: a potential target for the development of new leptospirosis treatments as an alternative to antibiotics.

Leptospirosis is a worldwide zoonosis caused by the motile bacterium Leptospira. This disease can cause hemorrhagic symptoms, multi-visceral and renal failures, resulting in one million cases and approximately 60,000 deaths each year. The motility of Leptospira is highly involved in its virulence and is ensured by the presence of two flagella in the periplasm. Several proteins that require the formation of disulfide bridges are essential for flagellar function. In Leptospira, these redox reactions are catalysed by the vitamin K epoxide reductase domain-containing protein (VKORdcp). The aim of the present work was to study the conservation of VKORdcp among Leptospira species and its interactions with putative substrates and inhibitor. Our results evidenced the presence of ten amino acids specific to either pathogenic or saprophytic species. Furthermore, structural studies revealed a higher affinity of the enzyme for vitamin K1 quinone, compared to ubiquinone. Finally, characterisation of the binding of a potential inhibitor revealed the involvement of some VKORdcp amino acids that have not been present in the human enzyme, in particular the polar residue D114. Our study thus paves the way for the future development of Leptospira VKORdcp inhibitors, capable of blocking bacterial motility. Such molecules could therefore offer a promising therapeutic alternative to antibiotics, especially in the event of the emergence of antibiotic-resistant strains.Communicated by Ramaswamy H. Sarma.

Identification of Pathogenic Leptospira kirschneri Serogroup Grippotyphosa in Water Voles (Arvicola terrestris) from Ruminant Pastures in Puy-de-Dôme, Central France.

Rodents are the primary reservoirs for pathogenic Leptospira species, which cause leptospirosis. Among the key potential carriers are water voles, whose population outbreaks can consequently pose a major threat to human and animal health. We studied the prevalence, prominence, and epidemiology of pathogenic Leptospira species in water voles in central France. First, 46 voles were captured, and DNA was extracted from kidney, lung, liver, blood, and urine and tested for the presence of Leptospira using three molecular methods: PCR, O-antigen typing, and variable number tandem repeat (VNTR) typing. We also attempted to culture leptospires from kidney and urine samples. In addition, we investigated leptospiral antibodies in serum samples from 60 sheep using microscopic agglutination testing. These animals co-occurred with the voles, so we sought to assess their degree of exposure and involvement in pathogen dynamics. The overall prevalence of infection was 76.1% (CI95% [61.2%, 87.4%]). The only strain found was L. kirschneri serogroup Grippotyphosa and a similar VNTR profile was acquired. Leptospires were successfully cultured from kidney and urine samples for four voles. Three sheep had low antibody titers against the Leptospira serogroup Grippotyphosa. Taken together, our results suggest the exclusive carriage of L. kirschneri serogroup Grippotyphosa among water voles in central France. Nevertheless, their ability to act as reservoir hosts that transmit the pathogen to co-occurring livestock remains unclear and merits further research.

Modeling of the combined dynamics of leptospirosis transmission and seroconversion in herds.

Leptospirosis is a zoonotic disease-causing illness in both humans and animals resulting in related economic impacts due to production loss as well as prevention and control efforts. Several mathematical models have been proposed to study the dynamics of infection but none of them has so far taken into account the dynamics of seroconversion. In this study, we have developed a general framework, based on the kinetic model for animal leptospirosis, that combines both the antibody (exposure marker) and infection dynamics to simultaneously follows both seroconversion and infection status of leptospirosis in a herd population. It is a stochastic compartmental model (for transition rates) with time delay (for seroconversion) which describes the progression of infection by a SEIRS (susceptible, exposed, infected, removed and susceptible) approach and seroconversion by four-state antibody kinetics (antibody negative and three antibody positive states of different antibody levels). The model shows that it is possible to assess and follow both seroconversion and infection status through the prism of diagnostic testing. Such an approach of combined kinetics could prove very useful to assist the competent authorities in their analyzes of epidemic situations and in the implementation of strategies for controlling and managing the associated risks.

Circulating serogroups of Leptospira in swine from a 7-year study in France (2011-2017).

BACKGROUND: Leptospirosis is a widespread zoonotic disease caused by pathogenic Leptospira and is responsible for significant economic porcine livestock losses. Knowledge of Leptospira serogroups and their distributions is important for evaluation of the relevance of leptospirosis management measures, including use of the prophylactic vaccine that was recently made available in France. A retrospective study was conducted to determine the relationships between different circulating Leptospira serogroups. Pigs from across France presenting clinical signs suggestive of leptospirosis were tested with the microagglutination test (MAT) between 2011 and 2017. We used weighted averages to determine serogroup distributions according to MAT results and considering cross-reactions. RESULTS: A total of 19,395 pig sera, mostly from Brittany, were tested, and 22.7% were found to be positive for at least one Leptospira serogroup. Analysis of the 4,346 seropositive results for which the putative infective serogroup could be defined, revealed that two out of ten serogroups were much more frequent than the others: Australis (48.5%) and Icterohaemorrhagiae (38.2%). Other serogroups, including Autumnalis, Panama, Ballum, Tarassovi, Sejroe, Grippotyphosa, Bataviae, and Pomona, were less common. CONCLUSIONS: Although diagnostic laboratory data cannot be extrapolated to infer the distribution of Leptospira serogroups at the nationwide scale in France, the analysis of such data can provide an overview of the relationship between circulating Leptospira serogroups in space and time. During the last decade, protection against the serogroups Australis and Icterohaemorrhagiae would have prevented most of the clinical porcine leptospirosis cases in the large number of farms that we studied. In the future, epidemiological information related to circulating Leptospira serogroups should be extracted from data with a standardized approach for use in nationwide or international surveillance and prophylactic strategy support.

Report of One-Year Prospective Surveillance of SARS-CoV-2 in Dogs and Cats in France with Various Exposure Risks: Confirmation of a Low Prevalence of Shedding, Detection and Complete Sequencing of an Alpha Variant in a Cat.

Despite the probable zoonotic origin of SARS-CoV-2, only limited research efforts have been made to understand the role of companion animals in SARS-CoV-2 epidemiology. According to recent serological prevalence studies, human-to-companion animal transmission is quite frequent, which led us to consider that the risk of SARS-CoV-2 transmission from animal to human, albeit negligible in the present context, may have been underestimated. In this study, we provide the results of a prospective survey that was conducted to evaluate the SARS-CoV-2 isolation rate by qRT-PCR in dogs and cats with different exposure risks and clinical statuses. From April 2020 to April 2021, we analyzed 367 samples and investigated the presence of SARS-CoV-2 RNA using qRT-PCR. Only four animals tested positive, all of them being cats. Three cats were asymptomatic and one presented a coryza-like syndrome. We describe in detail the infection in two cats and the associated clinical characteristics. Importantly, we obtained SARS-CoV-2 genomes from one infected animal and characterized them as Alpha variants. This represents the first identification of the SARS-CoV-2 Alpha variant in an infected animal in France.

Serological and molecular survey of Leptospira spp. infections in wild boars and red foxes from Southeastern France.

BACKGROUND AND AIM: Leptospirosis is a zoonotic disease. Information on the recent prevalence of Leptospira in hunted wild animals is limited, particularly in southeastern France. A cross-sectional survey was conducted to assess the prevalence and diversity of Leptospira spp. among wild boars (Sus scrofa) and red foxes (Vulpes vulpes) from two military camps in Southeastern France. MATERIALS AND METHODS: Serological analyses were performed using microscopic agglutination tests and polymerase chain reaction (PCR) assays were used to demonstrate Leptospira spp. infection from boar kidney DNA extracts. RESULTS: According to the species, the positive sera were obtained from 18% of 358 boars and 6 % of 64 foxes tested. The prevalence rate is significantly higher (p≤0.02) in boars than in foxes. In wild boar, Australis represents the most recorded serogroup (15.9%), followed by Sejroe (2.8%) and icterohaemorhagiae (2.8%). In red fox, icterohaemorhagiae represents the most recorded serogroup (6.25%), followed by Sejroe (1.57%) and Hebdomadis (1.57%). PCR-based detection of Leptospira DNA was positive in 6/62 (9.6%) of the wild boars tested. CONCLUSION: The results of this study confirmed the importance of wild boar in the epidemiology of leptospirosis among wildlife in Southeastern France. Due to their predatory behavior and their varied diet, mainly composed of small mammals, red foxes could be considered sentinel animals of environmental contamination with leptospires.

High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households.

In a survey of household cats and dogs of laboratory-confirmed COVID-19 patients, we found a high seroprevalence of SARS-CoV-2 antibodies, ranging from 21% to 53%, depending on the positivity criteria chosen. Seropositivity was significantly greater among pets from COVID-19+ households compared to those with owners of unknown status. Our results highlight the potential role of pets in the spread of the epidemic.

Autochthonous human and animal leptospirosis, Marseille, France.

Autochtonous leptospirosis is an emerging zoonotic disease in Europe, particularly in France. We report a case of leptospirosis in a 36 year-old man, who is a recently arrived migrant from Tunisia and lives in a squat. He suffered from pulmonary and neurological involvement as well as hepatitis. Seven rats (Rattus norvegicus) were trapped in the squat where the patient lived. Leptospira spp. DNA was detected in the kidney of one rat, highlighting the most likely source of contamination. In addition to the classic recreational or professional exposure to fresh water and practice of outdoor sports as a source of leptospirosis contamination, unhealthy living conditions (homeless or squatting) and therefore frequent exposure to rats, are another risk factor for leptospirosis in Europe.

Molecular Detection of Pathogenic Leptospira in Grasscutters (Thryonomys swinderianus) from Côte d'Ivoire.

The geographical and climatic conditions, hot and humid in Côte d'Ivoire, are favorable to the prolonged survival of leptospira in the environment. In this country, cases of human leptospirosis are underestimated and the wild reservoirs unknown. In this study, 16S rDNA PCR-sequencing and variable number of tandem repeats typing investigations were performed in kidneys collected from 60 grasscutters (Thryonomys swinderianus) around the city of Yamoussoukro, including 10 bred grasscutters and 50 bush meat grasscutters. One sample was positive for Leptospira borgptersenii and another one for Leptospira interrogans; both collected from wild animals. Our study suggests that grasscutters, which are abundant wild rodents hunted and bred for culinary preparations in this region, can be healthy carriers of leptospira. Thus, hygiene measures should be taken, particularly by hunters and cooks.

Successful Leptospira genotyping strategy on DNA extracted from canine biological samples.

Leptospirosis is an emerging worldwide zoonosis with a changing epidemiology responsible for an acute disease in humans and dogs. A better knowledge of the responsible bacterium Leptospira and in particular its various serovars and serogroups prevalence is essential for better diagnosis and prevention of the disease. The gold standard for leptospirosis diagnosis is the Microscopic Agglutination Test (MAT) but it requires long and fastidious laboratory work and sometimes results in controversial data. For these reasons, PCR-based techniques for detection of pathogenic leptospiral DNA in biological samples are currently replacing the MAT. However, these strategies do not provide any information regarding the infecting serovar or serogroup. In this study, an optimized genotyping method is described to allow the identification of Leptospira ssp. directly at serovars level using DNA extracted from canine blood and urine. 16S rDNA, Variable Number Tandem Repeat (VNTR) and Multispacer Sequence Typing (MST) protocols were adapted to biological samples. Eighty-eight DNA samples were analyzed from 72 different European canine clinical cases of leptospirosis confirmed by real-time PCR. 92% of DNA samples with Ct values below 34 were fully typed, and typing success decreased to about 30% for the other samples. Typing failure also showed a specie-specific correlation, with 63% of complete typing for L. interrogans and only 40% for L. kirschneri. Additionally, an exact match was observed between serological and molecular data for the few investigated cases where MAT data were available. This methodology is a suitable alternative to the MAT for determining the infecting serovar when Leptospira DNA from blood or urine is detected at Ct values below 34, contributing to clinical surveillance of leptospirosis.

Muskrats are greater carriers of pathogenic Leptospira than coypus in ecosystems with temperate climates.

Knowledge on the possible sources of human leptospirosis, other than rats, is currently lacking. To assess the distribution pattern of exposure and infection by Leptospira serogroups in the two main semi-aquatic rodents of Western France, coypus (Myocastor coypus) and muskrats (Ondatra zibethicus), results of micro-agglutination testing and renal tissue PCR were used. In coypus, the apparent prevalence was 11% (n = 524, CI95% = [9% - 14%]), seroprevalence was 42% (n = 590, CI95% = [38% - 46%]), and the predominant serogroup was Australis (84%). In muskrats, the apparent prevalence was 33% (n = 274, CI95% = [27% - 39%]), seroprevalence was 57% (n = 305, CI95% = [52% - 63%]), and the predominant serogroup was Grippotyphosa (47%). Muskrats should therefore be considered an important source of Grippotyphosa infection in humans and domestic animals exposed in this part of France.

Resistance to anticoagulant rodenticides in Martinique could lead to inefficient rodent control in a context of endemic leptospirosis.

Leptospirosis is a re-emergent worldwide zoonosis. It is endemic in Martinique where transmission conditions are favourable. Humans are usually infected through contact with water contaminated with urine of rodents. Recent human leptospirosis outbreaks in Martinique require today effective rodent management to prevent leptospirosis transmission. Nowadays, use of anticoagulant rodenticides (AR) is the main method implemented to control rodent populations. Nevertheless, intensive use of these AR has selected worldwide many VKORC1-based resistant rodent strains to AR. Our aim was to characterize the sensitivity of Martinique commensal rodents to AR to better prevent leptospirosis transmission. Resistance of house mice to first-generation and in rare cases even to second-generation ARs were clearly demonstrated in Martinique with the detection of the Y139C mutation with a very high allelic frequency of 40% and the A26T/Y139C double-mutation with an allelic frequency of 0.9%. In black rat, the most prevalent rodent in Martinique, 3 new Vkorc1 coding mutations were detected, the H68N, A115T and S149N mutations associated with moderate resistance to first generation AR. Therefore, rodent management in Martinique must be carried carefully to avoid resistance diffusion and maintain long-term effective rodent management, to be able to efficiently prevent leptospirosis transmission.

House Mice as a Real Sanitary Threat of Human and Animal Leptospirosis: Proposal for Integrated Management.

Leptospirosis is a reemerging zoonosis and ranges in severity from benign to sometimes fatal. In cattle, infection may be responsible for abortion and infertility cases causing economic losses. Humans may be contaminated through direct contact with urine of infected animals or indirectly though interaction with urine-contaminated environment. Many wildlife species living close to cattle, especially commensal rodents may play a role in the transmission of leptospires. Because little is known on the epidemiology of nonmaintenance Leptospira serovars, appropriate management is still limited. On a French farm where human and cattle leptospirosis were detected, the transmission cycle was explored to propose appropriate mitigation measures. For that, commensal rodents present on the farm were trapped and their leptospires carriage was studied by molecular methods. Trapped mice were shown to carry two pathogenic Leptospira species (L. interrogans and L. kirschneri). Since these 2 serogroups were simultaneously detected in the trapped mice and in the cows of this farm, we suspected an initial Leptospira transmission from mice to cows requiring an effective management of mice on this farm. Because resistance to anticoagulant rodenticides due to Vkorc1 mutations has been largely described in rodents and first-generation anticoagulant rodenticides seemed to be inefficient in controlling mice on this farm, susceptibility of these mice to anticoagulants has been characterized by Vkorc1 sequencing. 50% of the trapped mice carried mutations in the Vkorc1 gene leading to severe resistance to first-generation anticoagulants. The management of such mice that are a real sanitary threat can be achieved only by using the most toxic second-generation anticoagulants or nonanticoagulant solutions.

Leptospirosis, one neglected disease in rural Senegal.

A serological study was carried out in two Senegalese villages located in the Sine-Saloum region in order to estimate the presence of anti-leptospiral antibodies in humans and animals, and to identify the predominant serogroups. Seven hundred and forty-nine serum samples were collected from humans (n = 545), dogs (n = 33), donkeys (n = 20), goats (n = 52), sheep (n = 43) and N'Dama cattle (n = 56), all originated from Dielmo and Ndiop villages. All samples were tested for different serovars of pathogenic Leptospira species by the microscopic agglutination test. Considering titres ≥ 1:100, 7.7% [CI 95:5.5 to 9.9] on the 545 human blood samples tested and 42.2% [CI95 :35.4 to 48.9] on the 204 animal blood samples tested were found to be positive to one or more serovars. The results obtained indicate that the Australis serogroup is the most prevalent serogroup in human (67.3%) and cattle (27.3%). Serogroup Icterohaemorhagiae is the most frequent serogroup in goat (55.6%) and donkey (37.5%). Canicola (23.4%), Icterohaemorhagiae (21.1%) and Australis (12.5%) serogroups are the most prevalent serogroups in dogs. This study shows that diverse Leptospira serovars occur in a wide range of wild and domestic mammal species, as well as in humans in Senegal. However, further studies are needed to better understand the complexity of Leptospira epidemiology in Africa, identify the reservoirs of different serogroups and estimate its impact on livestock. Understanding the multi-host epidemiology of leptospirosis is essential to control and prevent the disease.

Pet rodents as possible risk for leptospirosis, Belgium and France, 2009 to 2016.

Leptospirosis is an under-reported and emerging zoonotic disease which is potentially fatal in humans. Rodents are the main reservoirs for pathogenic Leptospira spp., but diagnosis in these animals is difficult, and their infection, which does not induce symptoms, usually goes unoticed. Although the exposures of most human cases of leptospirosis are poorly documented, we were able to identify six human cases of leptospirosis which were associated with direct contact with pet rodents (mice or rats) in Belgium and France between 2009 and 2016. All cases had severe disease and for all, the presence of Leptospira spp. DNA in the kidneys of their pet animals was confirmed, strongly suggesting that excretion of leptospires in urine was the way of transmission. Half of the cases shared the serogroup Icterohaemorrhagiae, which is usually associated with severe disease, with the pet rats which they were in contact with. With the popularity of rats and mice as pets, this study should contribute to raising awareness on asymptomatic pet rodents as a source of Leptospira infections.

Overview of laboratory methods to diagnose Leptospirosis and to identify and to type leptospires

Leptospirosis is a virulent zoonosis with a global distribution. Pathogenic spirochetes of the genus Leptospira are responsible for this disease, and the primary animal reservoirs are rodentvvvs. Direct and indirect contact with infected urine constitutes the main route of transmission. Renal failure and advanced abortions are frequently observed in animals affected by leptospirosis, causing serious problems for farms. In humans, there is a high rate of mortality (10 percent), and farmers and persons in contact with water are frequently exposed. However, vaccines and strict prevention measures confer protection against leptospirosis. Serological tests facilitate the detection and identification of leptospire strains. Such tests are based on specific surface antigen recognition and are used for clinical analyses. To determine which serovars circulate in the environment, leptospires must be typed. Molecular methods, such as restriction enzyme-based techniques and the sequencing of specific regions, permit serovar identification. Unfortunately, although there are numerous techniques, they are not very efficient, and thus, new methods must be developed. With the advent of genomic sequencing, a substantial amount of information regarding leptospire genomes is now available, facilitating the selection of regions to discriminate between strains. Typing is important for both epidemiologic purposes and clinical analyses

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Overview of laboratory methods to diagnose Leptospirosis and to identify and to type leptospires.

Leptospirosis is a virulent zoonosis with a global distribution. Pathogenic spirochetes of the genus Leptospira are responsible for this disease, and the primary animal reservoirs are rodentvvvs. Direct and indirect contact with infected urine constitutes the main route of transmission. Renal failure and advanced abortions are frequently observed in animals affected by leptospirosis, causing serious problems for farms. In humans, there is a high rate of mortality (10 percent), and farmers and persons in contact with water are frequently exposed. However, vaccines and strict prevention measures confer protection against leptospirosis. Serological tests facilitate the detection and identification of leptospire strains. Such tests are based on specific surface antigen recognition and are used for clinical analyses. To determine which serovars circulate in the environment, leptospires must be typed. Molecular methods, such as restriction enzyme-based techniques and the sequencing of specific regions, permit serovar identification. Unfortunately, although there are numerous techniques, they are not very efficient, and thus, new methods must be developed. With the advent of genomic sequencing, a substantial amount of information regarding leptospire genomes is now available, facilitating the selection of regions to discriminate between strains. Typing is important for both epidemiologic purposes and clinical analyses.

First Observation of Leptospira interrogans in the Lungs of Rattus norvegicus.

We report the first two cases of pulmonary presence of leptospires in apparently healthy rats captured in a city park in Lyon (France). Only renal carriage of Leptospira has been described in the literature. Blood serology was performed in parallel with molecular and histological analyses of the kidney and lung samples. We isolated leptospires from the kidneys of two out of three seropositive wild rats. These results were confirmed by specific detection of pathogenic Leptospira by real-time PCR. Moreover, Leptospira DNA was detected in lung tissues. Immunohistochemistry and Warthin-Starry staining revealed that leptospires were present on the surface of the ciliated epithelium of the bronchi. Using PCR of the rrs (16S) gene and Multispacer Sequence Typing, DNA extracts of the kidney and lung were identified as belonging to Leptospira interrogans serovar Icterohaemorrhagiae "CHU Réunion." This first observation of the presence Leptospira in the lung with simultaneous renal carriage will require further study in future on several target organs to gain a better understanding of the Leptospira infection in wild rat.

Hedgehogs and Mustelid Species: Major Carriers of Pathogenic Leptospira, a Survey in 28 Animal Species in France (20122015).

Human leptospirosis is a zoonotic and potentially fatal disease that has increasingly been reported in both developing and developed countries, including France. However, our understanding of the basic aspects of the epidemiology of this disease, including the source of Leptospira serogroup Australis infections in humans and domestic animals, remains incomplete. We investigated the genetic diversity of Leptospira in 28 species of wildlife other than rats using variable number tandem repeat (VNTR) and multispacer sequence typing (MST). The DNA of pathogenic Leptospira was detected in the kidney tissues of 201 individuals out of 3,738 tested individuals. A wide diversity, including 50 VNTR profiles and 8 MST profiles, was observed. Hedgehogs and mustelid species had the highest risk of being infected (logistic regression, OR = 66.8, CI95% = 30.9-144 and OR = 16.7, CI95% = 8.7-31.8, respectively). Almost all genetic profiles obtained from the hedgehogs were related to Leptospira interrogans Australis, suggesting the latter as a host-adapted bacterium, whereas mustelid species were infected by various genotypes, suggesting their interaction with Leptospira was different. By providing an inventory of the circulating strains of Leptospira and by pointing to hedgehogs as a potential reservoir of L. interrogans Australis, our study advances current knowledge on Leptospira animal carriers, and this information could serve to enhance epidemiological investigations in the future.