Green spaces contribute to structural resilience of the gut microbiota in urban mammals.

The gut microbiome of wild animals is subject to various environmental influences, including those associated with human-induced alterations to the environment. We investigated how the gut microbiota of a synurbic rodent species, the striped field mouse (Apodemus agrarius), change in cities of varying sizes, seeking the urban microbiota signature for this species. Fecal samples for analysis were collected from animals living in non-urbanized areas and green spaces of different-sized cities (Poland). Metagenomic 16S rRNA gene sequencing and further bioinformatics analyses were conducted. Significant differences in the composition of gut microbiomes among the studied populations were found. However, the observed changes were dependent on local habitat conditions, without strong evidence of a correlation with the size of the urbanized area. The results suggest that ecological detachment from a more natural, non-urban environment does not automatically lead to the development of an "urban microbiome" model in the studied rodent. The exposure to the natural environment in green spaces may serve as a catalyst for microbiome transformations, providing a previously underestimated contribution to the maintenance of native gut microbial communities in urban mammals.

Social and environmental transmission spread different sets of gut microbes in wild mice.

Gut microbes shape many aspects of organismal biology, yet how these key bacteria transmit among hosts in natural populations remains poorly understood. Recent work in mammals has emphasized either transmission through social contacts or indirect transmission through environmental contact, but the relative importance of different routes has not been directly assessed. Here we used a novel radio-frequency identification-based tracking system to collect long-term high-resolution data on social relationships, space use and microhabitat in a wild population of mice (Apodemus sylvaticus), while regularly characterizing their gut microbiota with 16S ribosomal RNA profiling. Through probabilistic modelling of the resulting data, we identify positive and statistically distinct signals of social and environmental transmission, captured by social networks and overlap in home ranges, respectively. Strikingly, microorganisms with distinct biological attributes drove these different transmission signals. While the social network effect on microbiota was driven by anaerobic bacteria, the effect of shared space was most influenced by aerotolerant spore-forming bacteria. These findings support the prediction that social contact is important for the transfer of microorganisms with low oxygen tolerance, while those that can tolerate oxygen or form spores may be able to transmit indirectly through the environment. Overall, these results suggest social and environmental transmission routes can spread biologically distinct members of the mammalian gut microbiota.

Zoonotic pathogen screening of striped field mice (Apodemus agrarius) from Austria.

The striped field mouse (Apodemus agrarius) is known to carry several zoonotic pathogens, including Leptospira spp. and Dobrava-Belgrade orthohantavirus (DOBV). Since its first detection in 1996 in south-east Austria, the striped field mouse has further expanded its range in Austria. Here, we screened 35 striped field mice collected in an Austrian region near the Hungarian border for DOBV, Leptospira spp. and seven vector-borne pathogens. Hantavirus RT-PCR screening and DOBV IgG ELISA analysis led to the detection of two DOBV-positive striped field mice. The complete coding sequences of all three genome segments of both strains were determined by a combination of target enrichment and next-generation sequencing. Both complete coding S segment sequences clustered within the DOBV genotype Kurkino clade with the highest similarity to a sequence from Hungary. In one of 35 striped field mice, Leptospira borgpetersenii sequence type (ST) 146 was detected. Bartonella spp., Borrelia miyamotoi and Neoehrlichia mikurensis DNA was detected in four, one and two of 32 mice, respectively. Babesia, Anaplasma, Ehrlichia and Rickettsia specific DNA was not detected. Future investigations will have to determine the prevalence and invasion of these pathogens with the ongoing range expansion of the striped field mouse in Austria.

First report of Borrelia burgdorferi sensu stricto detection in a commune genospecies in Apodemus agrarius in Gwangju, South Korea.

Lyme disease is a tick-borne infectious disease caused by the Borrelia burgdorferi sensu lato complex. However, the distribution of Borrelia genospecies and the tissue detection rate of Borrelia in wild rodents have rarely been investigated. Here, we studied 27 wild rodents (Apodemus agrarius) captured in October and November 2016 in Gwangju, South Korea, and performed nested polymerase chain reaction targeting pyrG and ospA to confirm Borrelia infection. Eight rodents (29.6%) tested positive for Borrelia infection. The heart showed the highest infection rate (7/27; 25.9%), followed by the spleen (4/27; 14.8%), kidney (2/27; 7.4%), and lungs (1/27; 3.7%). The B. afzelii infection rate was 25.9%, with the highest rate observed in the heart (7/27; 25.9%), followed by that in the kidney and spleen (both 2/27; 7.4%). B. garinii and B. burgdorferi sensu stricto were detected only in the spleen (1/27; 3.7%). This is the first report of B. burgdorferi sensu stricto infection in wild rodents in South Korea. The rodent hearts showed a high B. afzelii infection rate, whereas the rodent spleens showed high B. garinii and B. burgdorferi sensu stricto infection rates. Besides B. garinii and B. afzelii, B. burgdorferi sensu stricto may cause Lyme disease in South Korea.

Epidemiological Characteristics of Rodents and Chiggers with Orientia Tsutsugamushi in the Republic of Korea.

A survey of rodents and chiggers associated with Orientia tsutsugamushi was conducted in a rural region of the Republic of Korea (Korea) between 2014 and 2018. Overall Apodemus agrarius 15.2% had the highest seropisitive for O. tsutsugamushi, followed by Myodes regulus 11.4%. Monthly risk factors using logistic regression analysis were not associated with O. tsutsugamushi infections in rodents. The overall prevalence rate of O. tsutsugamushi among chiggers was 0.3%. The chigger (Leptotrombidium scutellare) and monthly (October) risk factors were associated with O. tsutsugamushi human infections (P<0.05). Orientia tsutsugamushi infections are endemic in rodents in Korea and people, for example, soldiers who are active outdoors, must employ preventive measures, especially during October (P<0.05). When there are many reports of O. tsutsugamushi infections in Korea. The Boryong strain 85.7% (2/14) was the most common strain detected in chiggers, followed by the Shimokoshi 7.1% (1/14) and Karp 7.1% strains.

Pathogenic Leptospira species in rodents from Corsica (France).

Leptospirosis is a worldwide emerging zoonotic disease caused by Leptospira species, that in some patients develop severe forms with high mortality. In France, Corsica is the area where the highest incidences have been reported. The present study was focused on the analysis of pathogenic Leptospira species in rodents of Corsica, as these micromammals are the main natural reservoirs of the bacteria, in order to identify the circulating species and to locate possible risk focuses of transmission, as no previous study on the presence of Leptospira species has been carried out in the island. Rattus rattus, Rattus norvegicus, Apodemus sylvaticus and Mus musculus domesticus were captured in the proximity of water sources along Corsica, the detection of pathogenic Leptospira species was carried out by amplification of the LipL32 gene. The bacteria were found in all the rodent species analyzed and widely. The general prevalence was 10.4%, reaching the maximum value in Bastia (45%). Leptospira interrogans and Leptospira borgpetersenii were identified by phylogenetic analysis, but also two sequences which corresponded to an unnamed Leptospira species, only previously found in rodents of New Caledonia. The high incidence of human leptospirosis in Corsica could be partially explained by the wide distribution of pathogenic Leptospira species identified in this study. Also, the presence of an unknown pathogenic species of Leptospira in an area with high prevalence, may be involved in the higher incidence of Leptospirosis in this island, however, the zoonotic capacity of this species remains unknown. The results obtained are interesting for public health since all positive samples were found near water sources and one of the routes of transmission of leptospirosis is contact with contaminated water. This information could help the competent entities to take preventive measures, reducing the incidence of human leptospirosis in Corsica.

Host Competency of the Multimammate Rat Mastomys natalensis Demonstrated by Prolonged Spirochetemias with the African Relapsing Fever Spirochete Borrelia crocidurae.

African multimammate rats, Mastomys natalensis, are widely distributed in sub-Saharan Africa and live in close association with humans. In West Africa, numerous field studies have shown these animals may be naturally infected with the relapsing fever spirochete Borrelia crocidurae, the primary cause of tick-borne relapsing fever in this region of the continent. However, naturally infected individual rats have never been examined over time; therefore, the true host competency of these rats for this spirochete is unknown. Therefore, using animals from an established laboratory colony of M. natalensis, rats were experimentally infected with B. crocidurae and their blood examined to 28 days postinoculation. These animals were highly susceptible to infection and displayed prolonged and cyclic spirochetemias. Our results demonstrate these peridomestic rodents are likely competent hosts for infecting argasid tick vectors and play a primary role in the enzootic cycle for B. crocidurae in West Africa.

Prevalence of Orientia tsutsugamushi, Anaplasma phagocytophilum and Leptospira interrogans in striped field mice in Gwangju, Republic of Korea.

This study investigated the prevalence of Orientia tsutsugamushi, Anaplasma phagocytophilum, and Leptospira interrogans in wild rodents through molecular detection using organ samples and through serological assay using blood samples of mice collected from two distinct sites in Gwangju Metropolitan City, Republic of Korea (ROK). A total of 47 wild rodents, identified as Apodemus agrarius (A. agrarius), were captured from June to August 2016. The seroprevalence of antibodies against bacterial pathogens in A. agrarius sera was analyzed; 17.4% (8/46) were identified as O. tsutsugamushi through indirect immunofluorescence assay and 2.2% (1/46) were identified as Leptospira species through passive hemagglutination assay. Using polymerase chain reaction, the spleen, kidney and blood samples were investigated for the presence of O. tsutsugamushi, A. phagocytophilum, and L. interrogans. Out of the 47 A. agrarius, 19.1% (9/47) were positive for A. phagocytophilum and 6.4% (3/47) were positive for L. interrogans, while none were positive for O. tsutsugamushi. Four out of 46 (8.7%) blood samples, six out of 45 (13.3%) spleen samples, and one out of 47 (2.1%) kidney samples were positive for A. phagocytophilum. Three out of 47 (6.4%) kidney samples were positive for L. interrogans. The sequencing results of PCR positive samples demonstrated > 99% similarity with A. phagocytophilum and L. interrogans sequences. A. phagocytophilum was mostly detected in the spleen, whereas L. interrogans was mostly detected in the kidneys. Notably, A. phagocytophilum and L. interrogans were detected in A. agrarius living in close proximity to humans in the metropolitan suburban areas. The results of this study indicate that rodent-borne bacteria may be present in wild rodents in the metropolitan suburban areas of ROK.

Molecular identification of the source of an uncommon tularaemia outbreak, Germany, autumn 2016.

BackgroundIn 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.AimWe describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.MethodsWe incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case's lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.ResultsNo bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case's isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.ConclusionThe discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.

Colonization and pathology of Borrelia afzelii in its natural hosts.

Studies of Borrelia burgdorferi sensu lato in laboratory mice and humans have shown that spirochaetes disseminate from the site of infection (skin) to internal tissues, and cause various pathological effects. However, less is known about colonization and pathology of Lyme borreliosis spirochaetes in their natural hosts. In the present study, we assessed the colonization and manifestations during B. afzelii infection in reservoir hosts (yellow-necked mouse, Apodemus flavicollis; bank vole, Myodes glareolus; common shrew, Sorex araneus) infected in the wild. The infection prevalence and bacterial load was measured in skin (ear), joints and heart by quantitative PCR, and pathology in infected joints was evaluated by histology. The prevalence of B. afzelii was higher in skin than in joints and heart, but most animals that were positive in skin were also positive in internal tissues, and there was no difference between species in tissue-specific prevalence. Thus, spirochaetes disseminated from skin to other tissues in a similar way in all species. The bacterial load varied among host species and among different tissues within the same host species. In the case of skin and joints, bank voles and common shrews had higher bacterial loads than yellow-necked mice. In hearts, voles had higher bacterial loads than shrews and mice. Histological analyses showed no inflammation in joints of infected animals when compared to controls. We conclude that B. afzelii disseminates to internal tissues in natural hosts, but that levels of colonization vary between both species and tissues. There is as yet little evidence for pathological effects in natural hosts.

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe.

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.

Association of rodent-borne Leptospira spp. with urban environments in Malaysian Borneo.

Although leptospirosis is traditionally considered a disease of rural, agricultural and flooded environments, Leptospira spp. are found in a range of habitats and infect numerous host species, with rodents among the most significant reservoirs and vectors. To explore the local ecology of Leptospira spp. in a city experiencing rapid urbanization, we assessed Leptospira prevalence in rodents from three locations in Malaysian Borneo with differing levels of anthropogenic influence: 1) high but stable influence (urban); 2) moderate yet increasing (developing); and 3) low (rural). A total of 116 urban, 122 developing and 78 rural rodents were sampled, with the majority of individuals assigned to either the Rattus rattus lineage R3 (n = 165) or Sundamys muelleri (n = 100). Leptospira spp. DNA was detected in 31.6% of all rodents, with more urban rodents positive (44.8%), than developing (32.0%) or rural rodents (28.1%), and these differences were statistically significant. The majority of positive samples were identified by sequence comparison to belong to known human pathogens L. interrogans (n = 57) and L. borgpetersenii (n = 38). Statistical analyses revealed that both Leptospira species occurred more commonly at sites with higher anthropogenic influence, particularly those with a combination of commercial and residential activity, while L. interrogans infection was also associated with low forest cover, and L. borgpetersenii was more likely to be identified at sites without natural bodies of water. This study suggests that some features associated with urbanization may promote the circulation of Leptospira spp., resulting in a potential public health risk in cities that may be substantially underestimated.

Bartonellae of Synanthropic Four-Striped Mice (Rhabdomys pumilio) from the Western Cape Province, South Africa.

Bartonella is a species-rich bacterial genus that infects a wide variety of wild and domestic animals, including rodents. Despite high levels of murid rodent diversity in Africa, associated Bartonella prevalence and diversity remains understudied, particularly within the southern African subregion. To address this, we sampled endemic four-striped mice, Rhabdomys pumilio, from three rural and two urban localities in the Western Cape Province, South Africa. PCR screening and multilocus sequence analysis inclusive of five genome regions (gltA, nuoG, ribC, rpoB, and ITS), were respectively used to evaluate Bartonella status and diversity in these synanthropic rodent populations. An overall infection rate of 15% was recovered, ranging from 0% for an urban locality to 36.4% for a rural locality, consistent with the higher flea abundance recorded at the latter sites. Nucleotide sequencing and phylogenetic analyses confirmed the presence of three distinct Bartonella lineages (I-III), with lineages II and III grouping with bartonellae previously detected in R. pumilio from nature reserves in the Free State Province of South Africa, and lineage I being novel and sister to Bartonella strains identified previously in Micaelamys namaquensis. Our results indicate significant landscape effects on infection rates, highlight differential PCR assay performance, and identify three host-associated Bartonella lineages in Rhabdomys from South Africa.

Wild mice in and around the city of Utrecht, the Netherlands, are carriers of Clostridium difficile but not ESBL-producing Enterobacteriaceae, Salmonella spp. or MRSA.

Mice in buildings are a hygiene hazard because they harbour several zoonoses and animal diseases. The aim of this study was to gather information on specific bacteria in house mice caught in the urban environment. Mice caught in snap traps during pest control activities were collected in and around the city of Utrecht, the Netherlands, during May-June 2014, October-November 2015 and September-November 2016. The gut contents were analysed for ESBL/AmpC-producing Enterobacteriaceae, Salmonella spp., and Clostridium difficile and the buccal cavities were swabbed for methicillin-resistant S. aureus (MRSA). In total, 109 house mice (Mus musculus) and 22 wood mice (Apodemus sylvaticus) were examined. One mouse was found positive for Enterobacter spp. Salmonella spp. and MRSA were not found. Of n = 80 mice, 35·0% carried C. difficile (ribotypes in descending order of frequency: 014/020, 258, 002, 005, 013, 056, 081 and two unknown ribotypes). In conclusion, mouse droppings are a hazard for transmission of C. difficile to humans and their environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that mice in buildings can carry Clostridium difficile ribotypes that are associated with clinical disease in humans. Whether the mice are the source or whether they picked up these bacteria from the human environment has not been investigated. Either way, mouse droppings in the indoor environment are a hazard for transmission of C. difficile to humans.

The spleen microbiota of small wild mammals reveals distinct patterns with tick-borne bacteria.

BACKGROUND: Wild mammals serve as reservoirs for a variety of microbes and play an important role in the enzootic cycles of these microbes. Some of them are vector-borne bacteria in the genera Anaplasma, Ehrlichia and Rickettsia of the order Rickettsiales, which can cause febrile illnesses in human beings as well as animals. Anaplasma spp., Ehrlichia spp. and many spotted fever group (SFG) Rickettsia spp. are transmitted to mammalian hosts by tick vectors during blood meals. As a powerful sequencing method, the next generation sequencing can reveal the complexity of bacterial communities in humans and animals. Compared with limited studies on blood microbiota, however, much fewer studies have been carried out on spleen microbiota, which is very scarce in wild mammals. Chongming Island is the third biggest island in China. It was unclear whether there were any vector-borne bacteria in Chongming Island. In the present study, we explored the bacterial microbiota in the spleens of wild mice and shrews from the rural areas of Chongming Island and investigated the prevalence of vector-borne bacteria. METHODOLOGY/PRINCIPAL FINDINGS: Genomic DNAs were extracted from the spleen samples of 35 mice and shrews. The 16S rDNA V3-V4 regions of the DNA extracts were amplified by PCR and subjected to the 16S rDNA-targeted metagenomic sequencing on an Illumina MiSeq platform. All the 35 spleen samples obtained data with sufficient coverage (99.7-99.9%) for analysis. More than 1,300,000 sequences were obtained after quality control and classified into a total of 1,967 operational taxonomic units (OTUs) clustered at 97% similarity. The two most abundant bacterial phyla were Firmicutes and Proteobacteria according to the analysis of rarefied sequences. Among the bacterial communities detected in this study, Anaplasma, Rickettsia and Coxiella were adjacently clustered by hierarchical analysis. Significant differences in many bacterial features between Anaplasma-positive and Anaplasma-negative samples were identified by LEfSe analysis and Wilcoxon rank-sum test, suggesting that the Anaplasma-infection of small wild mammals was associated with a specific pattern of spleen microbiota. CONCLUSIONS/SIGNIFICANCE: Our study has comprehensively characterized the complex bacterial profiles in the spleens of wild mice and shrews from Chongming Island, Shanghai city. This work has revealed distinct spleen bacterial communities associated with tick-borne bacteria in wild animals. The detection of tick-borne bacteria highlights the risk of contracting pathogens with public health importance upon tick-exposure in the studied areas.

Geographical distribution of Orientia tsutsugamushi strains in chiggers from three provinces in Korea.

Chiggers were collected from the central and southern parts of South Korea between April and November, 2009 with the aim of investigating the seasonal and geographical distribution of Or. A total of 1136 chiggers were identified. They included eight species belonging to four genera, as follows: Leptotrombidium scutellare (27.2%, n = 309), L. pallidum (54.6%, n = 621), L. orientale (6.25%, n = 71), L. palpale (1.59%, n = 18), L. zetum (2.0%, n = 23), Euschoengastia koreaensis (1.5%, n = 17), Cheladonta ikaoensis (0.08%, n = 1) and Neotrombicula japonica (1.05%, n = 12). The density of L. pallidum was high from April to May, whereas L. scutallare was not found in spring, being observed from October. Serotype-specific nested PCR targeting the 56 kDa protein gene and sequencing analysis identified that the strains of 1136 O. tsutsugamushi in the chiggers as Boryong (6.8%), Kanda (0.4%), Oishi (0.3%), Jecheon (0.1%), Youngworl (0.1%) and Wonju (0.1%). Our findings indicate that L. pallidum and L. scutellare are dominant species in Korea and have geographical and seasonal variations.

Reclassification of Borrelia spp. Isolated in South Korea Using Multilocus Sequence Typing.

Here, we used multilocus sequence typing (MLST) to evaluate 3 intergenic genes (16S rRNA, ospA, and 5S-23S IGS) in Borrelia isolated from South Korea to analyze the relationships between host, vector, and molecular background. We identified B. afzelii, B. yangtzensis, B. garinii, and B. bavariensis. This study is the first report for the identification of B. yangtzensis using MLST in South Korea.

Encephalitozoon cuniculi infection in Barbary striped grass mice (Lemniscomys barbarus).

INTRODUCTION: Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that commonly induces subclinical infections in rabbits, but occurs also in a range of other species, including various rodents, carnivores, humans and birds. The present report describes encephalitozoonosis in a group of captive Barbary striped grass mice (Lemniscomys barbarus) in a zoo collection. The aetiology was confirmed by immunohistochemistry and PCR with subsequent sequencing. The source of infection is not known.

Molecular Epidemiological Survey of Leptospira Infection of Wild Rodents in the Urban Settlement of Cambodia.

INTRODUCTION: Leptospirosis remains a major public health threat in Cambodia. In this study, we aimed at facilitating the development of preventive strategies against leptospirosis in Cambodia by conducting molecular epidemiological surveys of Leptospira infection among wild rodents. MATERIALS AND METHODS: One hundred sixty-three wild rodents were captured in the capital Phnom Penh and surrounding areas, and their kidneys and bladders were collected for analysis. Identification of wild rodent species was determined by using the cytochrome c oxidase I gene. TaqMan PCR of the flagellin B gene (flaB) was performed to detect Leptospira, and species of the isolates were identified by flaB sequencing analysis. RESULTS: The species and respective number of rodents collected were as follows: Rattus norvegicus, 80 (49.1%); Rattus argentiventer, 53 (32.5%); Rattus exulans, 6 (3.7%); Rattus indica, 15 (9.2%); Maxomys surifer, 3 (1.8%); and Rattus sp., 6 (3.7%). Leptospira was detected in 20 out of the 163 rodents (12.3%) and was categorized as either Leptospira interrogans or Leptospira noguchii. R. norvegicus had the highest prevalence of Leptospira (17.5%), and R. argentiventer and Rattus sp. showed infection rates of 9.4% and 16.7%, respectively. CONCLUSIONS: Based on these results, wild rodents living in urban areas of Cambodia were found to be important reservoirs of highly virulent Leptospira. The flaB sequence results of our study provided information regarding the prevalence of Leptospira species, which was dependent on the rodent species. This study is the first study on leptospirosis in wild rodents in the urban areas of Cambodia, where there is limited information on leptospirosis.