Amplicon sequencing (AmpSeq) is a high-throughput, PCR-based sequencing approach for high-resolution and inexpensive genotyping. We designed and validated a 42 locus AmpSeq system for strain-level Leptospira genotyping directly from complex clinical samples such as urine, blood, or animal tissues.

Early predictors of fatal leptospirosis and the role of pathogen lineages remain poorly defined, limiting clinical risk stratification, genomic surveillance, and public health response in leptospirosis-endemic settings. We conducted a multicenter prospective cohort study of hospitalized patients with suspected leptospirosis in Thailand during 2015-2024. Among 459 patients with laboratory-confirmed cases, 25 (5.4%) died during hospitalization. Older age, higher total bilirubin, and higher leptospiremia were independently associated with in-hospital death, and a combined model demonstrated good discriminatory performance. We performed targeted amplicon sequencing analysis directly on clinical samples and whole-genome sequencing on available isolates. Genomic analysis identified Leptospira interrogans as the predominant species; clonal group 272 sequence type 34 was the predominant lineage and was observed in all patients with fatal cases for whom genomic data were available. Our findings support integration of clinical predictors and pathogen load for early risk stratification and highlight the potential value of genomic surveillance in leptospirosisendemic settings.

In 2021, the Los Angeles County (LAC) Department of Public Health suspected a leptospirosis outbreak in LAC affecting over 200 client-owned dogs. We aimed to characterize the outbreak and describe microbiologic findings, risk factors, diagnostic test performance, and outcomes in dogs diagnosed with leptospirosis at two specialty practices. Leptospira culture isolates from four cases were subjected to serotyping and whole-genome sequencing (WGS); WGS was also performed on one enriched genome isolate. After the outbreak, data were gathered on 59 cases and compared to the background hospital population (controls, n = 15,536). All isolates were Leptospira interrogans serovar Canicola, but each was distinct based on WGS. Cases clustered in space and in time. Cases evaluated during the outbreak peak had increased odds of exposure to indoor congregate facilities (ICFs). None of the 47 dogs with known leptospirosis vaccination history were completely vaccinated. Leptospira real-time PCR on blood and urine was positive in 15/56 (27%) and 49/54 (91%) of dogs, respectively. Initial serologic testing using the microscopic agglutination test and point-of-care tests was positive in 22/29 (76%) and 27/35 (77%) of dogs, respectively. Fifty-four (92%) of 59 dogs survived to discharge; some remained azotemic. No associated human cases were identified. In conclusion, L. interrogans serovar Canicola was associated with a leptospirosis outbreak in unvaccinated dogs, which had public health implications given widespread dog ownership rates. Data analysis suggested multiple infection sources, including ICFs. Urine PCR was the most sensitive diagnostic test. Such outbreaks might be prevented through more widespread vaccination.IMPORTANCELeptospirosis is a zoonotic bacterial disease transmitted through the urine of infected animals. We characterized an outbreak of leptospirosis in unvaccinated dogs in Los Angeles County and showed that cases were associated with housing in dog daycare and boarding facilities. Culture of several Leptospira isolates from the post-peak period identified Leptospira interrogans serovar Canicola, supporting the potential for dogs to act both as reservoirs and incident hosts for this serovar. Across the entire study period, multiple independent infection sources seemed likely based on molecular and spatial epidemiologic analysis, which might have included exposure to other dogs in indoor congregate facilities, rodents, or rodent urine. Our findings support the need for vaccination of dogs to reduce the risk to dog and human health, a high index of suspicion for the disease in unvaccinated dogs, the combined use of molecular and serologic tests to optimize diagnosis, and early treatment to optimize outcomes.

Melioidosis is increasingly recognised in tropical and subtropical regions worldwide as a serious and potentially fatal bacterial infection affecting humans and animals, acquired from the environment. Until now, human cases of melioidosis had not been reported in Southern Africa. Over a four-year period, we identified three human and two animal cases of melioidosis in South Africa and Namibia. Burkholderia pseudomallei isolates were investigated by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS). Phylogenetic analysis demonstrated substantial diversity, suggesting long-term cryptic persistence of the bacterium in the Southern African region. Limited awareness of the disease and inadequate diagnostic capacity likely contribute to its apparent rarity in the region. These findings underscore the urgent need for increased surveillance, improved diagnostics, and greater awareness of melioidosis in Southern Africa to better understand its true epidemiological burden and prevent future cases.

Cellulosimicrobium is an emerging but still rare pathogen that has exhibited a predilection for immunocompromised patients and those with indwelling medical devices. A case of an immunocompetent 89-year-old man with a history of hypertension who presented with 1 day of subjective fever and right-sided flank pain and was found to be bacteremic secondary to cholecystitis is presented in the present study. Piperacillin-tazobactam was initiated, and a percutaneous cholecystostomy tube was placed the following day. The blood-cultured pathogen was originally identified as Cellulomonas, but whole-genome sequencing (WGS) was used to reidentify the organism as a new clade of Cellulosimicrobium. The patient successfully completed treatment with piperacillin-tazobactam, which resulted in clinical improvement and cholecystostomy tube removal. Therefore, this case highlights the need for accurate microbial identification and the potential of WGS in distinguishing between closely related species to diagnose and treat patients appropriately. Despite its promise, barriers to widespread WGS use include cost and turnaround time, highlighting the need for advances to fully integrate this technology into routine clinical practice.

BACKGROUND:Amblyomma variegatum threatens the Caribbean cattle industry owing to its role as a vector for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater disease, an economically important and potentially fatal ruminant disease. Amblyomma variegatum is also a public health concern as a vector for Rickettsia africae, the causative agent of African tick-bite fever. Efforts to eradicate A. variegatum on Caribbean islands are ongoing to protect cattle from disease and prevent the spread of the vector and diseases to the American mainland. However, reinfestations often occur, possibly owing to the maintenance of ticks by non-cattle hosts that escape treatment. St. Croix in the US Virgin Islands has experienced such eradication challenges. METHODS:To determine whether persistence of A. variegatum populations on non-cattle hosts contributes to cattle reinfestation on St. Croix, we analyzed 1 non-attached A. variegatum adult female collected from a human and 14 questing adult females collected via cloth dragging along vegetation transects in Lower Love, St. Croix, during 2023. We conducted host bloodmeal analysis by obtaining vertebrate cytochrome c oxidase subunit I (COI) sequences from A. variegatum DNA extracts using COI-targeted DNA capture and enrichment to identify the most recent host bloodmeal. We also screened tick DNA extracts for E. ruminantium and Rickettsia DNA using pCS20 Sol1 and PanR8 qPCR, respectively. Rickettsia species identification was determined using ompA polymerase chain reaction (PCR) and Sanger sequencing, followed by phylogenetic analysis. RESULTS:We identified vertebrate COI sequences for the genera Capra and Canis in two A. variegatum ticks. Although E. ruminantium was not detected, DNA from R. africae was present in all 15 ticks. CONCLUSIONS:These results suggest that goats, and possibly canines, may serve as alternative hosts for A. variegatum on St. Croix, US Virgin Islands, which complicates eradication efforts focused entirely on the treatment of cattle. Fortunately, E. ruminantium was not identified in any ticks. However, R. africae was ubiquitous, which may be of concern for public health.

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

Importance:Accurate species identification and antifungal susceptibility testing are essential for effective aspergillosis treatment. However, non-Aspergillus fumigatus species, such as Aspergillus tubingensis, are often misidentified and understudied, potentially compromising proper prognosis and treatment. Objective:To examine the species and prevalence of triazole resistance among clinical Aspergillus isolates in Southern California. Design, Setting, and Participants:This cross-sectional study collected clinical Aspergillus cultures from September 1, 2019, to June 30, 2023, at Kaiser Permanente Southern California, an integrated health system serving a diverse regional population. Triazole susceptibility testing and whole genome sequencing were performed on selected isolates. A total of 2421 consecutive Aspergillus cultures were included. Eighty putative Aspergillus niger isolates were selected for sequencing, including 44 with positive growth in the presence of at least one clinically relevant triazole. Main Outcomes and Measures:The primary outcome was the genome-based species identification of A tubingensis. The secondary outcome was triazole susceptibility above the A niger epidemiological cut-off values. The hypothesis that A tubingensis is a prevalent, underrecognized, triazole-resistant pathogen was developed during data collection. Results:Of 2421 cultures, 1835 were successfully cultured for Aspergillus. After purification and deduplication, 1505 isolates were screened for triazole resistance. A substantial fraction of putative A niger isolates grew at the A niger epidemiological cut-off for itraconazole (110 of 664 [15.1%]). DNA sequencing revealed that 59 of 80 putative A niger isolates (73.8%) were actually A tubingensis. Elevated triazole minimum inhibitory concentrations were not strongly associated with any known cyp51 mutations among the A tubingensis isolates. Conclusions and Relevance:In this cross-sectional study of Aspergillus isolates from Kaiser Permanente Southern California, A tubingensis was a prevalent but underrecognized cause of aspergillosis in Southern California. Its frequent misidentification, association with invasive infections, and triazole resistance underscore the need for improved diagnostics and species-specific epidemiological investigations.

Madagascar is the most plague-affected country globally, yet the phylogenetic diversity of Yersinia pestis in this country remains insufficiently characterized. In this study, we analyzed whole-genome sequences of 614 Y. pestis strains, with 141 strains newly sequenced, collected over 96 years across Madagascar. All isolates emerged in 1898 and belong to the phylogroup 1.ORI3, supporting a single introduction event to Madagascar, followed by local diversification. We identified 23 distinct subgroups, including eight previously undescribed. Although most novel subgroups were rarely detected or rapidly extinguished, the ɣ subgroup circulated between 2016 and 2019 in southeastern Madagascar-an area historically plague-free for 64 years. Our analysis revealed extensive strain diversity and subgroup persistence, with some subgroups, such as α and β, persisting silently for decades before reemerging during the 2017 pneumonic plague epidemic. These findings uncover deep, previously underestimated phylogenetic diversity and long-term dynamics of Y. pestis in Madagascar, providing critical insights for understanding transmission patterns and informing future plague surveillance and control efforts.

Valley fever, a disease caused by Coccidioides spp., is a fungal respiratory disease with an expanding range. Methods to culture the pathogen from soil, especially Coccidioides posadasii, are very challenging, limiting the genomics knowledge of environmental strains. In this study, we designed and tested a targeted DNA capture and enrichment system for the characterization of Coccidioides genomes without the need to culture. In this system, RNA probes are hybridized to Coccidioides DNA in a complex sample, followed by DNA amplification, sequencing, and analysis. Our enrichment system was targeted toward coding region sequences in C. posadasii str. Silveira and tested on control DNA spiked into soil; DNA hybridized to probes was then sequenced and correctly placed into a reference phylogeny, based on the known placement of the whole-genome sequence. We then applied the enrichment system to a range of sample types (soil, air filters, rodent tissue) from a site in Mesa, Arizona, USA. The enriched samples were sequenced and placed into the C. posadasii phylogeny to understand the phylogenetic diversity within the Mesa site over time. The results demonstrate that low DNA signal in most sample types was boosted after enrichment. Enriched sequences from air filters collected at multiple time points from the Mesa site linked two different isolates collected from fatal cases of Coccidioidomycosis in a pig-tailed macaque colony housed at the Mesa site. This represents the first time that environmental C. posadasii DNA was directly linked to Coccidioidomycosis and demonstrates the power of this approach for genomic epidemiology.IMPORTANCEAll human cases of Valley fever are acquired through environmental exposure, so surveillance and characterization of the pathogen in soil are critical for risk mitigation efforts. Current databases are biased toward human clinical isolates, and little is known about the genomics of environmental strains of Coccidioides posadasii. In this study, we designed, tested, and validated a probe enrichment system that amplifies trace DNA in a complex sample. Sequenced DNA can be used to link environmental exposure with human cases, directing public health agencies to interventions that limit human exposure. This use case was demonstrated in this study, as trace DNA trapped on air filters was linked to a fatal case of primate Coccidioidomycosis at a site in Arizona. The probe enrichment system described in this study represents a powerful tool to better understand the genomic composition of environmental C. posadasii strains, which can aid in public health investigations.

Human excrement composting (HEC) is a sustainable strategy for human excrement (HE) management that recycles nutrients and mitigates health risks while reducing reliance on freshwater, fossil fuels, and fertilizers. A mixture of HE and bulking material was collected from 15 composting toilets and composted as 15 biological replicates in modified 19-liter buckets under mesophilic conditions with weekly sampling for one year. We hypothesized that (i) the microbiome of 1 year old compost would resemble that of a soil and/or food and landscape waste compost microbiome more closely than the original HE; and (ii) the human fecal indicators, Escherichia coli and Clostridium perfringens, would be undetectable after 52 weeks using qPCR and culturing. This investigation identified unique successional trajectories within buckets (i.e. biological replicates) and significant shifts in microbial communities around 25 weeks across buckets, with reductions in fecal-associated taxa and increases in environmental taxa indicating effective composting. We present a comprehensive microbial time series analysis of HEC and show that the initial gut-like microbiome of HEC systems transitions to a microbiome similar to soil and traditional compost but that pathogen risk assessment is important if thermophilic temperatures are not achieved. This study also produced the highest resolution composting microbiome data to date, establishing a baseline for HEC optimization and thermophilic composting studies while serving as a resource for bioprospecting for enzymes and organisms relevant to upcycling waste.

Bovine leptospirosis can result in infertility, abortion, placentitis, weak offspring, stillbirths, and decreased milk production. The leading cause of bovine leptospirosis globally is Leptospira borgpetersenii serovar Hardjo. Asymptomatic cattle with leptospirosis serve as reservoir hosts of infection that can shed infectious leptospires for months to years. Sites of bacterial colonization include the kidney or the genital tract, or both, in both cows and bulls. In this observational study, we investigated leptospirosis in a herd of asymptomatic bulls; 24.1% (14/58) were shedding leptospires via urine as confirmed by lipL32 real-time PCR (rtPCR). The viability of leptospires in selected rtPCR-positive bull urine samples was confirmed by culture. Given that bovine semen may also act as a vector of disease transmission, we investigated frozen semen samples for evidence of leptospires; 1.5% (4/268) were lipL32 rtPCR-positive, and, of these, one was culture-positive. All isolates cultured from bull urine or semen were classified as L. borgpetersenii serogroup Sejroe serovar Hardjo. The viability and infectivity of the semen isolate were confirmed in the hamster model of leptospirosis. Our results report for the first time the ability to culture serovar Hardjo directly from frozen semen and highlight the potential role for bull urine and semen in the transmission of bovine leptospirosis.

BACKGROUND:Rhipicephalus (Boophilus) microplus causes significant cattle production losses worldwide because it transmits Babesia bovis and B. bigemina, the causative agents of bovine babesiosis. Control of these ticks has primarily relied on treatment of cattle with chemical acaricides, but frequent use, exacerbated by the one-host lifecycle of these ticks, has led to high-level resistance to multiple classes of acaricides. Consequently, new approaches for control, such as anti-tick vaccines, are critically important. Key to this approach is targeting highly conserved antigenic epitopes to reduce the risk of vaccine escape in heterologous tick populations. METHODS:We evaluated amino acid conservation within 14 tick proteins across 167 R. microplus collected from geographically diverse locations in the Americas and Pakistan using polymerase chain reaction (PCR) amplicon sequencing and in silico translation of exons. RESULTS:We found that amino acid conservation varied considerably across these proteins. Only the voltage-dependent anion channel (VDAC) was fully conserved in all R. microplus samples (protein similarity 1.0). Four other proteins were highly conserved: the aquaporin RmAQP1 (0.989), vitellogenin receptor (0.985), serpin-1 (0.985), and subolesin (0.981). In contrast, the glycoprotein Bm86 was one of the least conserved (0.889). The Bm86 sequence used in the original Australian TickGARD vaccine carried many amino acid replacements compared with the R. microplus populations examined here, supporting the hypothesis that this vaccine target is not optimal for use in the Americas. By mapping amino acid replacements onto predicted three-dimensional (3D) protein models, we also identified amino acid changes within several small-peptide vaccines targeting portions of the aquaporin RmAQP2, chitinase, and Bm86. CONCLUSIONS:These findings emphasize the importance of thoroughly analyzing protein variation within anti-tick vaccine targets across diverse tick populations before selecting candidate vaccine antigens. When considering protein conservation alone, RmAQP1, vitellogenin receptor, serpin-1, subolesin, and especially VDAC rank as high-priority anti-tick vaccine candidates for use in the Americas and perhaps globally.

BackgroundBurkholderia pseudomallei, causative agent of melioidosis, is a One Health concern as it is acquired directly from soil and water and causes disease in humans and agricultural and wild animals. We examined B. pseudomallei in soil and goats at a single farm in the Northern Territory of Australia where >30 goats acquired melioidosis over nine years.Methodology/principal findingsWe cultured 45 B. pseudomallei isolates from 35 goats and sampled soil in and around goat enclosures to isolate and detect B. pseudomallei and evaluate characteristics associated with its occurrence; 33 soil isolates were obtained from 1993-1994 and 116 in 2006. Ninety-two goat and soil isolates were sequenced; mice were challenged with six soil isolates to evaluate virulence. Sampling depth and total N/organic C correlated with B. pseudomallei presence. Twelve sequence types (STs) were identified. Most goat infections (74%) were ST617, some with high similarity to 2006 soil isolates, suggesting ST617 was successful at persisting in soil and infecting goats. ST260 and ST266 isolates were highly virulent in mice but other isolates produced low/intermediate virulence; three of these were ST326 isolates, the most common soil ST in 2006. Thus, virulent and non-virulent lineages can co-occur locally. Three genes associated with virulence were present in ST260 and ST266, absent in most ST326 isolates, and present or variably present in ST617.Conclusions/significanceAgricultural animals can influence B. pseudomallei abundance and diversity in local environments. This effect may persist, as B. pseudomallei was detected more often from soil collected inside and adjacent to goat enclosures years after most goats were removed. Following goat removal, the low virulence ST326, which was not isolated from soil when goats were present, became the predominant ST in soil by 2006. Although multiple diverse lineages of B. pseudomallei may exist in a given location, some may infect mammals more efficiently than others.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >= 20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

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

Abstract Background Pathogenic Leptospira species are globally important zoonotic pathogens capable of infecting a wide range of host species. In marine mammals, reports of Leptospira have predominantly been in pinnipeds, with isolated reports of infections in cetaceans. Case presentation On 28 June 2021, a 150.5 cm long female, short-beaked common dolphin (Delphinus delphis delphis) stranded alive on the coast of southern California and subsequently died. Gross necropsy revealed multifocal cortical pallor within the reniculi of the kidney, and lymphoplasmacytic tubulointerstitial nephritis was observed histologically. Immunohistochemistry confirmed Leptospira infection, and PCR followed by lfb1 gene amplicon sequencing suggested that the infecting organism was L. kirschneri. Leptospira DNA capture and enrichment allowed for whole-genome sequencing to be conducted. Phylogenetic analyses confirmed the causative agent was a previously undescribed, divergent lineage of L. kirschneri. Conclusions We report the first detection of pathogenic Leptospira in a short-beaked common dolphin, and the first detection in any cetacean in the northeastern Pacific Ocean. Renal lesions were consistent with leptospirosis in other host species, including marine mammals, and were the most significant lesions detected overall, suggesting leptospirosis as the likely cause of death. We identified the cause of the infection as L. kirschneri, a species detected only once before in a marine mammal – a northern elephant seal (Mirounga angustirostris) of the northeastern Pacific. These findings raise questions about the mechanism of transmission, given the obligate marine lifestyle of cetaceans (in contrast to pinnipeds, which spend time on land) and the commonly accepted view that Leptospira are quickly killed by salt water. They also raise important questions regarding the source of infection, and whether it arose from transmission among marine mammals or from terrestrial-to-marine spillover. Moving forward, surveillance and sampling must be expanded to better understand the extent to which Leptospira infections occur in the marine ecosystem and possible epidemiological linkages between and among marine and terrestrial host species. Generating Leptospira genomes from different host species will yield crucial information about possible transmission links, and our study highlights the power of new techniques such as DNA enrichment to illuminate the complex ecology of this important zoonotic pathogen.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Escherichia coli is a diverse pathogen, causing a range of disease in humans, from self-limiting diarrhea to urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) is the most frequently observed uropathogen in UTIs, a common disease in high-income countries, incurring billions of dollars yearly in treatment costs. Although E. coli is easily grown and identified in the clinical laboratory, genotyping the pathogen is more complicated, yet critical for reducing the incidence of disease. These goals can be achieved through whole-genome sequencing of E. coli isolates, but this approach is relatively slow and typically requires culturing the pathogen in the laboratory. To genotype E. coli rapidly and inexpensively directly from clinical samples, including but not limited to urine, we developed and validated a multiplex amplicon sequencing assay, called ColiSeq. The assay consists of targets designed for E. coli species confirmation, high resolution genotyping, and mixture deconvolution. To demonstrate its utility, we screened the ColiSeq assay against 230 clinical urine samples collected from a hospital system in Flagstaff, Arizona, USA. A limit of detection analysis demonstrated the ability of ColiSeq to identify E. coli at a concentration of ~2 genomic equivalent (GEs)/mL and to generate high-resolution genotyping at a concentration of 1 × 105 GEs/mL. The results of this study suggest that ColiSeq could be a valuable method to understand the source of UPEC strains and guide infection mitigation efforts. As sequence-based diagnostics become accepted in the clinical laboratory, workflows such as ColiSeq will provide actionable information to improve patient outcomes.IMPORTANCEUrinary tract infections (UTIs), caused primarily by Escherichia coli, create an enormous health care burden in the United States and other high-income countries. The early detection of E. coli from clinical samples, including urine, is important to target therapy and prevent further patient complications. Additionally, understanding the source of E. coli exposure will help with future mitigation efforts. In this study, we developed, tested, and validated an amplicon sequencing assay focused on direct detection of E. coli from urine. The resulting sequence data were demonstrated to provide strain level resolution of the pathogen, not only confirming the presence of E. coli, which can focus treatment efforts, but also providing data needed for source attribution and contact tracing. This assay will generate inexpensive, rapid, and reproducible data that can be deployed by public health agencies to track, diagnose, and potentially mitigate future UTIs caused by E. coli.