Explore the vast range of titles available.

Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results We designed FungiQuant, a TaqMan ® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r 2 -values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from <10% and <20%, respectively. We further showed that FungiQuant has a limit of quantification 25 copies and a limit of detection at 5 copies. Lastly, by comparing results from human-only background DNA with low-level fungal DNA, we showed that amplification in two or three of a FungiQuant performed in triplicate is statistically significant for true positive fungal detection. Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans.

Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology. We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup. Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics.

Background 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. Methods 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. Results 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. Conclusions 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. Graphical Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated. We collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp. B. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.

Melioidosis is an underreported human disease of tropical and sub-tropical regions caused by the saprophyte Burkholderia pseudomallei . Although most global melioidosis cases are reported from tropical regions in Southeast Asia and northern Australia, there are multiple occurrences from sub-tropical regions, including the United States (U.S.). Most melioidosis cases reported from the continental U.S. are the result of acquiring the disease during travel to endemic regions or from contaminated imported materials. Only two human melioidosis cases from the continental U.S. have likely acquired B . pseudomallei directly from local environments and these cases lived only ~7 km from each other in rural Texas. In this study, we assessed the risk of acquiring melioidosis from the environment within the continental U.S. by surveying for B . pseudomallei in the environment in Texas where these two human melioidosis cases likely acquired their infections. We sampled the environment near the homes of the two cases and at additional sampling locations in surrounding counties in Texas that were selected based on ecological niche modeling. B . pseudomallei was not detected at the residences of these two cases or in the surrounding region. These negative data are important to demonstrate that B . pseudomallei is rare in the environment in the U.S. even at locations where locally acquired human cases likely have occurred, documenting the low risk of acquiring B . pseudomallei infection from the environment in the continental U.S.

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. Graphical Abstract

Burkholderia 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. We 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. Agricultural 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.

Burkholderia thailandensis, an opportunistic pathogen found in the environment, is a bacterium closely related to B. pseudomallei, the cause of melioidosis. Human B. thailandensis infections are uncommon. We isolated B. thailandensis from water in Texas and Puerto Rico and soil in Mississippi in the United States, demonstrating a potential public health risk.

Background The American dog tick, Dermacentor variabilis , is an important vector of pathogens to humans, wildlife and domestic animals in North America. Although this tick species is widely distributed in the USA and Canada, knowledge of its range-wide phylogeographic patterns remains incomplete. Methods We carried out a phylogenetic analysis of D. variabilis using samples collected from 26 USA states and five Canadian provinces. Tick samples ( n = 1053 in total) originated from two main sources: existing archives (2000–2011), and new collections made from 2012 to 2013. We sequenced a 691 bp fragment of the cox 1 gene from a subset ( n = 332) of geographically diverse D. variabilis . DNA extracted from individual ticks ( n = 1053) was also screened for a Francisella -like endosymbiont, using a targeted 16S rRNA sequencing approach, and important pathogens ( Rickettsia spp. and Coxiella burnetii ), using species-specific quantitative PCR assays. Results Maximum parsimony analysis of cox 1 sequences revealed two major groups within D. variabilis with distinct geographical distributions: one from the eastern USA/Canada (Group 1) and one from the west coast states of the USA (California and Washington; Group 2). However, genetic subdivisions within both of these two major groups were weak to moderate and not tightly correlated with geography. We found molecular signatures consistent with Francisella -like endosymbionts in 257 of the DNA extracts from the 1053 individual ticks, as well as Rickettsia spp. and Coxiella burnetii in a small number of ticks ( n = 29 and 2, respectively). Phylogenetic patterns for Francisella -like endosymbionts, constructed using sequence data from the bacterial 16S rRNA locus, were similar to those for D. variabilis , with two major groups that had a nearly perfect one-to-one correlation with the two major groups within D. variabilis . Conclusions Our findings reveal a distinct phylogenetic split between the two major D. variabilis populations. However, high levels of genetic mixture among widely separated geographical localities occur within each of these two major groups. Furthermore, our phylogenetic analyses provide evidence of long-term tick-symbiont co-evolution. This work has implications for understanding the dispersal and evolutionary ecology of D. variabilis and associated vector-borne diseases.