Characterization of Brucella spp. and other abortigenic pathogens from aborted tissues of cattle and goats in Rwanda.

BACKGROUND: Abortions cause tremendous economic losses in food-producing animals and may lead to food insecurity. OBJECTIVES: This study aimed to characterize Brucella spp. and other abortigenic pathogens from aborted tissues of cattle. METHODS: For cattle, aborted tissues (n = 19) were cultured, and Brucella spp. were detected using the genus-specific 16S-23S ribosomal DNA interspacer region (ITS) assay and speciated using Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR assays. Brucella negative samples were screened using the eight abortigenic pathogens PCR panel. Samples from an abortion outbreak that occurred within a goat tribe were included in this investigation. Sera of females (n = 8) and males (n = 2) were analyzed using the Rose Bengal Test (RBT) and indirect enzyme-linked immunosorbent assay (i-ELISA), while vaginal swabs (n = 3) and aborted tissues (n = 1) were cultured and characterized. RESULTS: The ITS-PCR detected Brucella DNA in cultures from two aborted tissues of cattle (10.5%, [2/19]), which were identified as B. melitensis (n = 1), and B. abortus (n = 1) using AMOS and Bruce-ladder PCR assays. Campylobacter fetus (n = 7) and Leptospira spp. (n = 4) including co-infections (n = 2) of C. fetus and Leptospira spp. were identified from the Brucella negative samples of cattle. Goats (100.0%, 10/10) were brucellosis seropositive on RBT and i-ELISA. Mixed infections caused by B. melitensis and B. abortus were isolated from the vaginal swabs (n = 3) and aborted tissues (n = 1). DISCUSSION AND CONCLUSIONS: This is the first identification of abortion-associated pathogens in aborted cattle indicating the enormous financial losses and a threat to public health. It is therefore essential to include these identified pathogens in the surveillance scheme of veterinary and human services.

The variable presence of Leptospira in the environment; an epidemiological explanation based on serial analysis of water samples.

Human leptospirosis involves the classic epidemiological triad (agent, host and environment); hence the investigations should include the knowledge on Leptospira within the animals and the environment. The objectives of this study are to explore the abundance of Leptospira in different climate zones of Sri Lanka and to describe the presence of Leptospira in the same water source at serial time points. First, water and soil samples were collected from different parts of Sri Lanka (Component-1); second, water sampling continued only in the dry zone (Component-2). Finally, serial water sampling from ten open wells was performed at five different time points (Component-3). Quantitative PCR of water and metagenomic sequencing of soil were performed to detect Leptospira. Three replicates for each sample were used for PCR testing, and positive result of two or more replicates was defined as 'strongly positive,' and one positive replicate was defined as positive. In the water and soil sample analysis in the whole country (Component-1), two out of 12 water sites were positive, and both were situated in the wet zone. Very small quantities of the genus Leptospira were detected by 16 amplicon analysis of soil in all 11 sites. In the dry zone water sample analysis (Component-2), only samples from 6 out of 26 sites were positive, of which one site was strongly positive. In the serial sample analysis (Component-3), Six, five, four, five, and six wells were positive in serial measurements. All wells were positive for at least one time point, while only one well was positive for all five time points. Proximity to the tank and greater distances from the main road were associated with strong positive results for Leptospira (P<0.05). The presence of Leptospira was not consistent, indicating the variable abundance of Leptospira in the natural environment. This intermittent nature of positivity could be explained by the repetitive contamination by animal urine.

Leptospira ainlahdjerensis sp. nov., Leptospira ainazelensis sp. nov., Leptospira abararensis sp. nov. and Leptospira chreensis sp. nov., four new species isolated from water sources in Algeria.

Leptospira strains were isolated from freshwater sampled at four sites in Algeria and characterized by whole-genome sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The cells were spiral-shaped and motile. Phylogenetic and MALDI-TOF MS analyses showed that the strains can be clearly distinguished from the other described species in the genus Leptospira, therefore representing two novel species of the pathogen subclade P1 and two novel species of the saprophyte subclade S1. The names Leptospira ainlahdjerensis sp. nov. (type strain 201903070T=KIT0297T=CIP111912T), Leptospira ainazelensis sp. nov. (201903071T=KIT0298T=CIP111913T), Leptospira abararensis sp. nov. (201903074T=KIT0299T=CIP111914T) and Leptospira chreensis (201903075T=KIT0300T=CIP111915T) are proposed.

Laboratory diagnostic, epidemiological, and clinical characteristics of human leptospirosis in Okinawa Prefecture, Japan, 2003-2020.

BACKGROUND: Leptospirosis is considered an endemic disease among agricultural workers in Okinawa Prefecture, which is the southernmost part of Japan and has a subtropical climate, but data on the current status and trend of this disease are scarce. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a retrospective study of clinically suspected leptospirosis patients whose sample and information were sent to the Okinawa Prefectural Institute of Health and Environment from November 2003 to December 2020. Laboratory diagnosis was established using culture, nested polymerase chain reaction (PCR), and/or microscopic agglutination test (MAT) with blood, cerebrospinal fluid, and/or urine samples. Statistical analyses were performed to compare the epidemiological information, clinical features, and sensitivities of diagnostic methods among laboratory-confirmed cases. Serogroups and the species of Leptospira isolates were determined by MAT using 13 antisera and flaB sequencing. A total of 531 clinically suspected patients were recruited, among whom 246 (46.3%) were laboratory confirmed to have leptospirosis. Among the confirmed cases, patients aged 20-29 years (22.4%) and male patients (85.7%) were the most common. The most common estimated sources of infection were recreation (44.5%) and labor (27.8%) in rivers. Approximately half of the isolates were of the L. interrogans serogroup Hebdomadis. The main clinical symptoms were fever (97.1%), myalgia (56.3%), and conjunctival hyperemia (52.2%). Headache occurred significantly more often in patients with Hebdomadis serogroup infections than those with other serogroup infections. The sensitivities of culture and PCR exceeded 65% during the first 6 days, while the sensitivity of MAT surpassed that of culture and PCR in the second week after onset. PCR using blood samples was a preferable method for the early diagnosis of leptospirosis. CONCLUSIONS/SIGNIFICANCE: The results of this study will support clinicians in the diagnosis and treatment of undifferentiated febrile patients in Okinawa Prefecture as well as patients returning from Okinawa Prefecture.

Genetic diversity of Leptospira isolates in Lao PDR and genome analysis of an outbreak strain.

BACKGROUND: Although Southeast Asia is one of the most leptospirosis afflicted regions, little is known about the diversity and molecular epidemiology of the causative agents of this widespread and emerging zoonotic disease. METHODOLOGY/PRINCIPAL FINDINGS: We used whole genome sequencing to examine genetic variation in 75 Leptospira strains isolated from patients in the Lao PDR (Laos) between 2006 and 2017. Eleven serogroups from 4 Leptospira species and 43 cgMLST-defined clonal groups (CGs) were identified. The most prevalent CG was CG272 (n = 18, 26.8%), composed of L. interrogans serogroup Autumnalis isolates. This genotype was recovered throughout the 12-year period and was associated with deaths, and with a large outbreak in neighbouring Thailand. Genome analysis reveals that the CG272 strains form a highly clonal group of strains that have, for yet unknown reasons, recently spread in Laos and Thailand. Additionally, accessory genes clearly discriminate CG272 strains from the other Leptospira strains. CONCLUSIONS/SIGNIFICANCE: The present study reveals a high diversity of Leptospira genotypes in Laos, thus extending our current knowledge of the pan- and core-genomes of these life-threatening pathogens. Our results demonstrate that the CG272 strains belong to a unique clonal group, which probably evolved through clonal expansion following niche adaptation. Additional epidemiological studies are required to better evaluate the spread of this genotype in Southeast Asia. To further investigate the key factors driving the virulence and spread of these pathogens, more intense genomic surveillance is needed, combining detailed clinical and epidemiological data.

Sero-prevalence of anti-Leptospira antibodies and associated risk factors in rural Rwanda: A cross-sectional study.

BACKGROUND: Leptospirosis is a zoonotic disease transmitted through the urine of wild and domestic animals, and is responsible for over 50,000 deaths each year. In East Africa, prevalence varies greatly, from as low as 7% in Kenya to 37% in Somalia. Transmission epidemiology also varies around the world, with research in Nicaragua showing that rodents are the most clinically important, while studies in Egypt and Chile suggest that dogs may play a more important role. There are no published studies of leptospirosis in Rwanda. METHODS & FINDINGS: We performed a cross-sectional survey of asymptomatic adults recruited from five occupational categories. Serum samples were tested using ELISA and Microscopic Agglutination Test (MAT). We found that 40.1% (151/377) of asymptomatic adults had been exposed to Leptospira spp. Almost 36.3% of positive subjects reported contact with rats (137/377) which represent 90.7% among positive leptospira serology compared with 48.2% of negative subjects (182/377) which represent 80.5% among negative leptospira serology (OR 2.37, CI 1.25-4.49) and 1.7 fold on prevalence ratio and 2.37 of odd ratio. Furthermore, being a crop farmer was significantly associated with leptospirosis (OR 2.06, CI 1.29-3.28). We identified 6 asymptomatic subjects (1.6%) who met criteria for acute infection. CONCLUSIONS: This study demonstrates a high prevalence of leptospiral antibodies infection among asymptomatic adults in rural Rwanda, particularly relative to neighboring countries. Although positive subjects were more likely to report rat contact, we found no independent association between rats and leptospirosis infection. Nonetheless, exposure was high among crop farmers, which is supportive of the hypothesis that rats together with domestic livestock might contribute to the transmission. Further studies are needed to understand infecting Leptospira servers and elucidate the transmission epidemiology in Rwanda and identify means of host transmitters.

Epidemiology of leptospirosis in Tanzania: A review of the current status, serogroup diversity and reservoirs.

BACKGROUND: Tanzania is among the tropical countries of Sub-Saharan Africa with the environmental conditions favorable for transmission of Leptospira. Leptospirosis is a neglected zoonotic disease, and although there are several published reports from Tanzania, the epidemiology, genetic diversity of Leptospira and its host range are poorly understood. METHODS: We conducted a comprehensive review of human and animal leptospirosis within the 26 regions of the Tanzanian mainland. Literature searches for the review were conducted in PubMed and Google Scholar. We further manually identified studies from reference lists among retrieved studies from the preliminary search. RESULTS: We identified thirty-four studies describing leptospirosis in humans (n = 16), animals (n = 14) and in both (n = 4). The number of studies varied significantly across regions. Most of the studies were conducted in Morogoro (n = 16) followed by Kilimanjaro (n = 9) and Tanga (n = 5). There were a range of study designs with cross-sectional prevalence studies (n = 18), studies on leptospirosis in febrile patients (n = 13), a case control study in cattle (n = 1) and studies identifying novel serovars (n = 2). The most utilized diagnostic tool was the microscopic agglutination test (MAT) which detected antibodies to 17 Leptospira serogroups in humans and animals. The Leptospira serogroups with the most diverse hosts were Icterohaemorrhagiae (n = 11), Grippotyphosa (n = 10), Sejroe (n = 10), Pomona (n = 9) and Ballum (n = 8). The reported prevalence of Leptospira antibodies in humans ranged from 0.3-29.9% and risk factors were associated with occupational animal contact. Many potential reservoir hosts were identified with the most common being rodents and cattle. CONCLUSION: Leptospirosis is prevalent in humans and animals in Tanzania, although there is regional and host variation in the reports. Many regions do not have information about the disease in either humans or their animal reservoirs. More studies are required to understand human leptospirosis determinants and the role of livestock in leptospirosis transmission to humans for the development of appropriate control strategies.

Application of simplified MLST scheme for direct typing of clinical samples from human leptospirosis cases in a tertiary hospital in the Philippines.

Despite the major threat of leptospirosis to public health in the Philippines, its epidemiologic data remain scarce. Multilocus sequence typing (MLST) is a method often used for identification of circulating Leptospira species and disease surveillance. Unfortunately, molecular typing of Leptospira isolates is not routinely done in most hospital settings. A simplified MLST scheme targeting three loci (adk, lipL41, mreA) was performed for rapid direct typing of Leptospira in clinical specimens. Blood samples from suspected or clinically diagnosed cases (n = 50) were initially screened via polymerase chain reaction (PCR) targeting 23S rRNA, 16S rRNA (rrs2), and lipL32 genes. From the nine positives, seven had interpretable data from MLST. Allelic profiles identified L. interrogans in all positive samples. Six were assigned to ST12 of serovar Manilae (serogroup Pyrogenes) while one sample cannot be clearly differentiated between two serovars/serogroups, Bataviae/Losbanos (serogroup Bataviae) or Australis (serogroup Australis), indicating possibility of a new ST. Phylogenetic analysis confirmed that the application of simplified MLST scheme produces consistent results with the seven-loci genetic profile of published Leptospira MLST schemes. Reduced scheme addressed the challenges often encountered in the amplification of full MLST genetic profile of Leptospira. The approach is a potential alternative to serological tests for rapid typing of clinical specimens and can also aid in investigations on disease epidemiology specifically to monitor occurrence, pathogen transmission, host specificity and susceptibility, and other factors that could lead to potential outbreaks.

Molecular and serological characterization of pathogenic Leptospira spp. isolated from symptomatic dogs in a highly endemic area, Brazil.

BACKGROUND: Leptospirosis is an endemic zoonosis in Brazil, with a great impact on human and animal health. Although dogs are frequently infected by pathogenic Leptospira, the current epidemiological understanding of canine leptospirosis is mainly based on serological tests that predict the infecting serogroup/serovar. Thus, the present study aimed at identifying the causative agent for severe cases of canine leptospirosis in a highly endemic area through the isolation and characterization of the isolated strains. RESULTS: Urine, serum and blood samples were collected from 31 dogs with suspected acute leptospirosis treated at the Veterinary Hospital Service of Santo Amaro University between 2018 and 2019. Acute infection was confirmed in 17 dogs (54.8%) by the associated use of Polymerase Chain Reaction (PCR), Microscopic Agglutination (MAT) and bacteriological culture. Eleven dogs (35.5%) had titers ≥800, with the most frequent serogroups being Autumnalis and Icterohaemorrhagiae (n = 4 each) and Canicola (n = 2). Leptospires were recovered from four dogs, and Multilocus Sequence Analysis (MLSA) revealed infection caused by L. interrogans, which were further characterized as serogroups Canicola (n = 1) and Icterohaemorrhagiae (n = 3). CONCLUSION: The identity of the isolates and serological pattern of MAT suggest that dogs are highly exposed to the serogroup Icterohaemorrhagiae and Canicola, also indicating possible circulation of serogroups not yet isolated in Brazil, notably serogroup Autumnalis. Our findings also reinforce the usefulness of using multiple diagnostic approaches to confirm acute canine leptospirosis.

Molecular epidemiology of Leptospira spp. among wild mammals and a dog in Amami Oshima Island, Japan.

Leptospirosis is a worldwide zoonosis caused by the pathogenic Leptospira spp. Canine and human leptospirosis sometimes occur on Amami Oshima Island, located in the Nansei Archipelago, southwestern Japan; however, information on the causative Leptospira spp. on this island is quite limited. This study aimed to investigate the molecular and serological characteristics of Leptospira spp. isolated from wild animals and a dog in Amami Oshima Island. We obtained seven Leptospira strains by culturing kidney tissues of wild animals, such as black rats (2), wild boars (3), and rabbit (1) as well as blood from a symptomatic dog. Using flaB sequencing and microscopic agglutination test with antisera for 18 serovars, the isolates were identified as Leptospira borgpetersenii serogroups Javanica (black rat), L. interrogans serogroup Australis (black rat and dog), and L. interrogans serogroup Hebdomadis (wild boar and rabbit). The sequence type (ST) of L. borgpetersenii serogroup Javanica was determined to be ST143 via multilocus sequence typing (MLST) using seven housekeeping genes. For L. interrogans, MLST and multiple-locus variable-tandem repeat analysis (MLVA) revealed identical ST and MLVA types in rat and canine isolates, whereas two STs and MLVA types were identified in wild boar isolates. The STs and MLVA types of rabbit and one of the wild boars were identical. Bacterial culture and flaB-nested polymerase chain reaction demonstrated a high rate of Leptospira infection in wild boars (58.3%, 7/12), whereas Leptospira spp. were detected in 4.8% of black rats (2/42). This study revealed diverse Leptospira genotype and serotype maintenance in wild mammals on Amami Oshima Island. MLST and MLVA indicated that black rats were a source of canine infection. Wild boars carry L. interrogans and are considered an important maintenance host because antibodies against serogroup Hebdomadis were detected in human and canine leptospirosis patients on this island.

Bovine Immune Response to Vaccination and Infection with Leptospira borgpetersenii Serovar Hardjo.

This study examined the humoral and cellular response of cattle vaccinated with two commercial leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4+ and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4+ and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic.IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.

Next-Generation Sequencing Analysis of Pathogenic Leptospira: A Way Forward for Understanding Infectious Disease Dynamics in Low/Middle-Income, Disease-Endemic Settings.

In the current genomic era, knowledge of diversity of Leptospira, the spirochetal agents of leptospirosis, is changing rapidly. Next-generation sequencing has decreased in price and increased in scale, with the potential to democratize large-scale analysis of pathogens in resource-limited, low/middle-income (LMIC) regions. Consequently, the molecular classification of Leptospira, a pathogen disproportionately affecting LMIC countries, has changed dramatically over the last decade. Leptospira classification and molecular understandings of pathogen diversity have rapidly evolved, now most precisely based on core genome analysis supplemented by new insights provided by culture-independent methods directly using body fluids such as blood and urine. In places where leptospirosis disease burden is highest, genomic technologies have not been available, and serology-based methods remain the mainstay of leptospiral classification. Understanding the epidemiology, pathogenesis, and ultimately new approaches to treating and preventing leptospirosis requires detailed knowledge of regionally circulating Leptospira in highly endemic settings. Next-generation sequencing-based, culture-independent typing overcomes the limitation of culture isolation of Leptospira from clinical samples, with promise of providing public health-actionable information applicable to leptospirosis-endemic LMIC settings.

12 Novel clonal groups of Leptospira infecting humans in multiple contrasting epidemiological contexts in Sri Lanka.

Leptospirosis is a ubiquitous zoonotic disease and a major clinical challenge owing to the multitude of clinical presentations and manifestations that are possibly attributable to the diversity of Leptospira, the understanding of which is key to study the epidemiology of this emerging global disease threat. Sri Lanka is a hotspot for leptospirosis with high levels of endemicity as well as annual epidemics. We carried out a prospective study of Leptospira diversity in Sri Lanka, covering the full range of climatic zones, geography, and clinical severity. Samples were collected for leptospiral culture from 1,192 patients from 15 of 25 districts in Sri Lanka over two and half years. Twenty-five isolates belonging to four pathogenic Leptospira species were identified: L. interrogans, L. borgpetersenii, L. weilii, and L. kirschneri. At least six serogroups were identified among the isolates: Autumnalis (6), Pyrogenes (4), Icterohaemorrhagiae (2), Celledoni (1), Grippotyphosa (2) and Bataviae (1). Seven isolates did not agglutinate using available antisera panels, suggesting new serogroups. Isolates were sequenced using an Illumina platform. These data add 25 new core genome sequence types and were clustered in 15 clonal groups, including 12 new clonal groups. L. borgpetersenii was found only in the dry zone and L. weilii only in the wet zone. Acute kidney injury and cardiovascular involvement were seen only with L. interrogans infections. Thrombocytopenia and liver impairment were seen in both L. interrogans and L. borgpetersenii infections. The inadequate sensitivity of culture isolation to identify infecting Leptospira species underscores the need for culture-independent typing methods for Leptospira.

Hantavirus-Leptospira coinfections in small mammals from central Germany.

European orthohantaviruses (Puumala orthohantavirus (PUUV); Dobrava-Belgrade orthohantavirus (DOBV), genotype Kurkino; Tula orthohantavirus (TULV)), and Leptospira spp. are small mammal-associated zoonotic pathogens that cause diseases with potentially similar symptoms in humans. We investigated the frequency of Leptospira spp. and hantavirus single and double infections in small mammals from 22 sites in Thuringia, central Germany, during 2017. TULV infections were detected at 18 of 22 sites (mean prevalence 13.8%, 93/674). PUUV infections were detected at four of 22 sites (mean prevalence 1.5%, 7/471), and respective PUUV sequences formed a novel phylogenetic clade, but DOBV infections were not detected at all. Leptospira infections were detected at 21 of 22 sites with the highest overall prevalence in field voles (Microtus agrestis) with 54.5% (6/11) and common voles (Microtus arvalis) with 30.3% (205/676). Leptospira-hantavirus coinfections were found in 6.6% (44/671) of common voles but only in two of 395 bank voles. TULV and Leptospira coinfection probability in common voles was driven by individual (age) and population-level factors. Coinfections seemed to be particularly associated with sites where Leptospira spp. prevalence exceeded 35%. Future investigations should evaluate public health consequences of this strong spatial clustering of coinfections.

Spatial autocorrelation and co-occurrence of six serovarieties of Leptospira in goat herds of the State of Guanajuato, Mexico.

The epidemiological behavior of six Leptospira serovarieties was analyzed by spatial autocorrelation and co-occurrence of leptospirosis, diagnosed in goat herds located in the State of Guanajuato, Mexico. A total of 1650 goat serum samples were analyzed by microscopic agglutination (MAT). True prevalence (Pv) and 95% confidence interval (CI95) were determined. Spatial autocorrelation was calculated using the spdep package, applying the global Moran's I and local Moran's I of Leptospira in Guanajuato. The probabilistic model of co-occurrence was applied using the co-occur package. Seroprevalence in the State was found to be 45.5% (CI95 42.96; 48.06%). The highest registered frequency was for the Icterohemorrhagiae serovar (Pv 34.16%; CI95 31.74, 36.65%), followed by the serovar Hardjo-prajitno (Pv: 6.77%; CI95 5.33, 8.40%). Other serovarieties showed a Pv < 5%. Global spatial autocorrelation, only for the Icterohemorrhagiae serovar, was I > 1, while local Moran's I revealed that five of the six Leptospira serovarieties were spatially correlated. The probabilistic model of co-occurrence detected negative associations between Icterohemorrhagiae and the other serovarieties. The current study demonstrates the presence of Leptospira in goat herds of the State of Guanajuato. The diagnosed serovarieties show an aggregation pattern associated to risk zones and disease-transmitting vectors. Antibody co-occurrence analysis revealed dominance of the Icterohemorrhagiae serovar. A multidisciplinary approach including spatial epidemiology, ecological analyses, and serological vigilance will generate useful information for the prevention and control of leptospirosis in caprine production units.

Leptospira in river and soil in a highly endemic area of Ecuador.

BACKGROUND: Leptospira are shed into the environment via urine of infected animals. Rivers are thought to be an important risk factor for transmission to humans, though much is unknown about the types of environment or characteristics that favor survival. To address this, we screened for Leptospira DNA in two rivers in rural Ecuador where Leptospirosis is endemic. RESULTS: We collected 112 longitudinal samples and recorded pH, temperature, river depth, precipitation, and dissolved oxygen. We also performed a series of three experiments designed to provide insight into Leptospira presence in the soil. In the first soil experiment, we characterized prevalence and co-occurrence of Leptospira with other bacterial taxa in the soil at dispersed sites along the rivers (n = 64). In the second soil experiment, we collected 24 river samples and 48 soil samples at three points along eight transects to compare the likelihood of finding Leptospira in the river and on the shore at different distances from the river. In a third experiment, we tested whether Leptospira presence is associated with soil moisture by collecting 25 soil samples from two different sites. In our river experiment, we found pathogenic Leptospira in only 4 (3.7%) of samples. In contrast, pathogenic Leptospira species were found in 22% of shore soil at dispersed sites, 16.7% of soil samples (compared to 4.2% of river samples) in the transects, and 40% of soil samples to test for associations with soil moisture. CONCLUSIONS: Our data are limited to two sites in a highly endemic area, but the scarcity of Leptospira DNA in the river is not consistent with the widespread contention of the importance of river water for leptospirosis transmission. While Leptospira may be shed directly into the river, onto the shores, or washed into the river from more remote sites, massive dilution and limited persistence in rivers may reduce the environmental load and therefore, the epidemiological significance of such sources. It is also possible that transmission may occur more frequently on shores where people are liable to be barefoot. Molecular studies that further explore the role of rivers and water bodies in the epidemiology of leptospirosis are needed.

Serological study of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Tarassovi and Ballum in beef cattle, sheep and deer in New Zealand.

AIMS: To estimate animal-level seroprevalence of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovars Ballum and Tarassovi, in beef cattle, sheep and deer on New Zealand farms, and herd/flock-level seroprevalence of any serovar when existing same-sera data for serovars Hardjobovis and Pomona were included, and to determine associations between risk factors and animal-level seroprevalence. METHODS: Banked sera from sheep (n = 82), beef (n = 54) and deer (n = 62) herds/flocks (n = 3,878 animals) from seven regions were analysed using the microscopic agglutination test. Titres of ≥48 were designated positive. Herds/flocks were considered positive if either ≥1, ≥2 or ≥3 animals were positive. Existing same-sera data for serovars Hardjobovis and Pomona were included to establish farm-level any-serovar seropositivity. Factors associated with serological status were analysed using generalised estimating equations. RESULTS: Animal-level seroprevalence for serovars Ballum, Copenhageni, and Tarassovi, respectively, was 13.7 (95% CI = 11.7-16.0)%, 12.6 (95% CI = 10.6-14.7)% and 18.0 (95% CI = 15.7-20.5)% for beef cattle, 10.5 (95% CI = 9.0-12.1)%, 16.7 (95% CI = 14.9-18.6)% and 14.0 (95% CI = 12.4-15.8)% for sheep and 6.6 (95% CI = 5.3-8.2)%, 15.5 (95% CI = 13.5-17.7)% and 3.6 (95% CI = 2.7-4.8)% for deer, respectively. Herd/flock-level seroprevalence for Ballum was 86.6, 52.4 and 39.0% for sheep, 85.2, 52.7 and 33.3% for beef cattle and 50.8, 27.9 and 21.3% for deer at definitions ≥1, ≥2 and ≥3 seropositive animals per species, respectively. For Copenhageni, corresponding data were 95.1, 73.2 and 56.1% for sheep, 68.5, 48.2 and 29.6% for beef cattle and 73.8, 57.4 and 41.0% for deer, and for Tarassovi, 80.5, 59.7 and 45.1% for sheep, 83.3, 68.5 and 61.1% for beef cattle, and 42.6, 16.4 and 4.9% for deer. Seropositivity to all serovars was observed from all regions, with some differences in seroprevalence observed between species and regions, but not between islands. Combining with Hardjobovis and Pomona data, herd/flock-level seropositivity for all animal species and all five Leptospira serovars was 100% at definition ≥1 animal positive, and 97.5 and 96.3% for sheep flocks, 87.8 and 97.8% for beef cattle herds, and 89.3 and 75% for deer herds at ≥2 and ≥3 animals positive, respectively. CONCLUSIONS: Seropositivity to serovars Ballum, Copenhageni and Tarassovi is common in sheep, beef cattle and deer New Zealand and most, or all farms have ≥1 livestock species seropositive to ≥1 serovar. CLINICAL RELEVANCE: Serovars Ballum, Tarassovi and Copenhageni should be considered when clinical or subclinical signs of leptospirosis are observed in sheep, beef cattle or deer. Livestock sector workers are potentially at risk of exposure.

High incidence of asymptomatic leptospirosis among urban sanitation workers from Kota Kinabalu, Sabah, Malaysian Borneo.

Leptospirosis is a public health challenge in Sabah State of Malaysian Borneo. Rapid urbanization, rural-to-urban migration, and undocumented immigration in Sabah have increased the pressure on the urban garbage disposal system. Rodents and other small animals thrive under these conditions. We hypothesized that urban sanitation workers would be at risk of developing leptospirosis. In total, 303 urban sanitation workers with a mean age of 42.6 years were enrolled in this study. The serum samples collected from these workers were subjected to the microscopic agglutination test (MAT), PCR and nucleotide sequencing of the amplicons to confirm the presence of Leptospira. The phylogenetic analysis using the neighbor joining method was performed to assess whether they were pathogenic. In this study 43.8% (133/303) of the samples were MAT-seropositive and among them, 29 (21.8%) were positive by PCR. Nucleotide sequencing of the amplicons confirmed the presence of Leptospira. Phylogenetic analysis showed that our strains belonged to the pathogenic group of Leptospira. A high proportion of urban sanitation workers were seropositive for leptospirosis, and a considerable number were PCR positive for Leptospira, thereby indicating asymptomatic infections. Further research is needed to confirm whether this is a transient phenomenon or antibiotic therapy is required.

Multi-locus sequence analyses reveal a clonal L. borgpetersenii genotype in a heterogeneous invasive Rattus spp. community across the City of Johannesburg, South Africa.

BACKGROUND: Rattus spp. are frequently implicated as key reservoir hosts for leptospirosis, one of the most common, but neglected, bacterial zoonoses in the world. Although leptospirosis is predicted to be a significant public health threat in Africa, studies from the continent are limited. METHODS: Rattus spp. (n = 171) were sampled (January-May 2016) across the City of Johannesburg, South Africa's largest inland metropole. Rattus spp. genetic diversity was evaluated by full length (1140 bp) cyt b sequencing of 42 samples. For comparison, a further 12 Rattus norvegicus samples collected in Cape Town, South Africa's largest coastal metropole, were also genotyped. Leptospira infections were identified and genotyped using real-time PCR and multi-locus (lfb1, secY and lipL41) DNA sequencing. RESULTS: Five R. norvegicus haplotypes were identified across Johannesburg, four of which have not previously been detected in South Africa, and one in Cape Town. Across Johannesburg we identified a Leptospira spp. infection prevalence of 44% (75/171) and noted significant differences in the prevalence between administrative regions within the metropole. Multi-locus sequence analyses identified a clonal genotype consistent with L. borgpetersenii serogroup Javanica (serovar Ceylonica). DISCUSSION: The prevalence of infection identified in this study is amongst the highest detected in Rattus spp. in similar contexts across Africa. Despite the complex invasion history suggested by the heterogeneity in R. norvegicus haplotypes identified in Johannesburg, a single L. borgpetersenii genotype was identified in all infected rodents. The lack of L. interrogans in a rodent community dominated by R. norvegicus is notable, given the widely recognised host-pathogen association between these species and evidence for L. interrogans infection in R. norvegicus in Cape Town. It is likely that environmental conditions (cold, dry winters) in Johannesburg may limit the transmission of L. interrogans. Spatial heterogeneity in prevalence suggest that local factors, such as land use, influence disease risk in the metropole. CONCLUSIONS: In South Africa, as in other African countries, leptospirosis is likely underdiagnosed. The high prevalence of infection in urban rodents in Johannesburg suggest that further work is urgently needed to understand the potential public health risk posed by this neglected zoonotic pathogen.

Didelphis albiventris as a carrier of Leptospira sp. in the central nervous tissue in the semiarid region of Northeast, Brazil.

Leptospirosis has been investigated in several species of wild animals. The white-eared opossum (Didelphis albiventris) is a mammal common in the brazilian semi-arid, so, this study aimed to investigate its role in the occurrence of the leptospirosis in the region Northeast of Brazil. 12 animals were used, from which samples were collected for the attempt of isolation, molecular detection and serological examination. There was no microbial growth, nor were any anti-Leptospira sp. antibodies found in the serological samples. The PCR detected leptospiric DNA in the central nervous system (CNS) of five animals (41.7 %). The gene in one of the samples was sequenced and showed identity with Leptospira interrogans. The presence of Leptospira sp. in the CNS of Didelphis albiventris does not allow the characterization of the studied animals as reservoirs with potential for transmission of the pathogen in the region, however it represents a site that needs to be further investigated.