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.

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.

Rocky Mountain spotted fever (RMSF) is a deadly tick-borne disease caused by the bacterium Rickettsia rickettsii. An ongoing epidemic of RMSF is affecting tribal communities in Arizona, with nearly 500 cases and 28 deaths since 2003. The San Carlos Apache Tribe has been consistently working to prevent RMSF using tick collars on dogs, pesticide treatments around homes, and increasing education for nearly a decade. Besides monitoring human disease levels and tick burden on dogs, we have little understanding of the long-term impact of prevention practices on tick abundance and infection rates in the peridomestic environment. We evaluated risk factors associated for tick infestation at home sites across the San Carlos Indian Reservation as well as R. rickettsii and Rickettsia massiliae prevalence in off-host ticks. Although the presence of fencing appears protective, the number of nearby structures is the most important risk factor associated with increased adult and nymphal tick abundance, highlighting the impact of a free-roaming dog population.

Brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.), have spread globally and are an important vector of multiple pathogens affecting both dogs and humans. The control of these ticks on dogs and human dwellings often relies on synthetic pyrethroids, a chemical class of acaricides that targets the voltage-gated sodium channel (VGSC) protein of arthropod nerve cells, causing influxes of sodium and ultimately, paralysis. Invasive Rhipicephalus sanguineus s.l. ticks in the Americas can be broadly grouped into two main lineages: temperate and tropical (Rh. sanguineus sensu stricto (s.s.) and Rh. linnaei, respectively). Phenotypic resistance to synthetic pyrethroids in a verified Rh. sanguineus s.s. population has only been reported in the state of Rio Grande do Sul, Brazil. The objectives of our study were to 1) screen a small number (n = 10) of Rh. sanguineus s.s. from Brazil to check for VGSC gene mutations known to be associated with resistance to synthetic pyrethroids in ticks and other arthropods, and 2) provide molecular confirmation that all ticks were Rh. sanguineus s.s. We used next-generation DNA sequencing methods to analyze the VGSC gene and mitochondrial loci (12S, 16S, COI) of 10 brown dog ticks sampled from a stray dog in the Restinga sub-district of Porto Alegre, Rio Grande do Sul, Brazil. The progeny of other ticks from this animal had previously been shown to display a low level of resistance to deltamethrin. Analysis of mitochondrial genes confirmed these ticks were Rh. sanguineus s.s. We identified two known resistance mutations in domain II segments 4 and 5 of the VGSC gene (C190A and G215T). These specific mutations have not been reported previously in any brown dog tick lineages from the Americas, and this is the first case of VGSC mutations described from ticks confirmed to be Rh. sanguineus s.s. using genetic analyses. The discovery of these mutations in Rh. sanguineus s.s. is important for the effective management of ticks on dogs in Brazil and other countries where brown dog tick infestations occur.

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.

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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.

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.

Background Rhipicephalus (Boophilus) microplus causes significant cattle production losses worldwide because it transmits Babesia bovis and B. bigemina causative agents of bovine babesiosis. Control of these ticks primarily has relied on treatment of cattle with chemical acaricides, but frequent use, exacerbated by the one-host life cycle 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 PCR amplicon sequencing and in silico translation of exons. Results We found that amino acid conservation varied considerably across these proteins. Only one target, the voltage-dependent anion channel, was fully conserved in all 167 R. microplus samples (protein similarity 1.0). Five 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 protease 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 to 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 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 the voltage-dependent anion channel rank as high priority anti-tick vaccine candidates for use in the Americas and perhaps globally.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.), is an important vector for Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever. Current public health prevention and control efforts to protect people involve preventing tick infestations on domestic animals and in and around houses. Primary prevention tools rely on acaricides, often synthetic pyrethroids (SPs); resistance to this chemical class is widespread in ticks and other arthropods. Rhipicephalus sanguineus s.l. is a complex that likely contains multiple unique species and although the distribution of this complex is global, there are differences in morphology, ecology, and perhaps vector competence among these major lineages. Two major lineages within Rh. sanguineus s.l., commonly referred to as temperate and tropical, have been documented from multiple locations in North America, but are thought to occupy different ecological niches. To evaluate potential acaricide resistance and better define the distributions of the tropical and temperate lineages throughout the US and in northern Mexico, we employed a highly multiplexed amplicon sequencing approach to characterize sequence diversity at: 1) three loci within the voltage-gated sodium channel (VGSC) gene, which contains numerous genetic mutations associated with resistance to SPs; 2) a region of the gamma-aminobutyric acid-gated chloride channel gene (GABA-Cl) containing several mutations associated with dieldrin/fipronil resistance in other species; and 3) three mitochondrial genes (COI, 12S, and 16S). We utilized a geographically diverse set of Rh sanguineus s.l. collected from domestic pets in the US in 2013 and a smaller set of ticks collected from canines in Baja California, Mexico in 2021. We determined that a single nucleotide polymorphism (T2134C) in domain III segment 6 of the VGSC, which has previously been associated with SP resistance in Rh. sanguineus s.l., was widespread and abundant in tropical lineage ticks (>50 %) but absent from the temperate lineage, suggesting that resistance to SPs may be common in the tropical lineage. We found evidence of multiple copies of GABA-Cl in ticks from both lineages, with some copies containing mutations associated with fipronil resistance in other species, but the effects of these patterns on fipronil resistance in Rh. sanguineus s.l. are currently unknown. The tropical lineage was abundant and geographically widespread, accounting for 79 % of analyzed ticks and present at 13/14 collection sites. The temperate and tropical lineages co-occurred in four US states, and as far north as New York. None of the ticks we examined were positive for Rickettsia rickettsii or Rickettsia massiliae.

BACKGROUND:Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene.METHODOLOGY/PRINCIPAL FINDINGS:We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles.CONCLUSIONS/SIGNIFICANCE:Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation (tcdB, tcdA), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB- isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes (n=136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile. This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.