First investigation of pathogenic bacteria, protozoa and viruses in rodents and shrews in context of forest-savannah-urban areas interface in the city of Franceville (Gabon).

Rodents are reservoirs of numerous zoonotic diseases caused by bacteria, protozoans, or viruses. In Gabon, the circulation and maintenance of rodent-borne zoonotic infectious agents are poorly studied and are often limited to one type of pathogen. Among the three existing studies on this topic, two are focused on a zoonotic virus, and the third is focused on rodent Plasmodium. In this study, we searched for a wide range of bacteria, protozoa and viruses in different organs of rodents from the town of Franceville in Gabon. Samples from one hundred and ninety-eight (198) small mammals captured, including two invasive rodent species, five native rodent species and 19 shrews belonging to the Soricidae family, were screened. The investigated pathogens were bacteria from the Rickettsiaceae and Anaplasmataceae families, Mycoplasma spp., Bartonella spp., Borrelia spp., Orientia spp., Occidentia spp., Leptospira spp., Streptobacillus moniliformis, Coxiella burnetii, and Yersinia pestis; parasites from class Kinetoplastida spp. (Leishmania spp., Trypanosoma spp.), Piroplasmidae spp., and Toxoplasma gondii; and viruses from Paramyxoviridae, Hantaviridae, Flaviviridae and Mammarenavirus spp. We identified the following pathogenic bacteria: Anaplasma spp. (8.1%; 16/198), Bartonella spp. (6.6%; 13/198), Coxiella spp. (5.1%; 10/198) and Leptospira spp. (3.5%; 7/198); and protozoans: Piroplasma sp. (1%; 2/198), Toxoplasma gondii (0.5%; 1/198), and Trypanosoma sp. (7%; 14/198). None of the targeted viral genes were detected. These pathogens were found in Gabonese rodents, mainly Lophuromys sp., Lemniscomys striatus and Praomys sp. We also identified new genotypes: Candidatus Bartonella gabonensis and Uncultured Anaplasma spp. This study shows that rodents in Gabon harbor some human pathogenic bacteria and protozoans. It is necessary to determine whether the identified microorganisms are capable of undergoing zoonotic transmission from rodents to humans and if they may be responsible for human cases of febrile disease of unknown etiology in Gabon.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or 'dead' enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Allobodo chlorophagus n. gen. n. sp., a Kinetoplastid that Infiltrates and Feeds on the Invasive Alga Codium fragile.

A novel biflagellate protist that consumed chloroplasts inside material of the invasive marine green alga Codium fragile was reported from the U.S. east coast in 2003. We observed a similar association in C. fragile from five sites in Nova Scotia, Canada during 2013 and 2014. After incubating Codium fragments for 2-3 days, some utricles and filaments contained numerous chloroplast-consuming cells. Transmission electron microscopy (TEM) confirmed that these were kinetoplastids with a pankinetoplast, large electron-dense droplets in the cytoplasm and a connective between the paraxonemal rod bases, but no conspicuous para-cytopharyngeal rod, all consistent with U.S. material observed in 2003. The ITS1-5.8S rRNA-ITS2 sequences from 13 Nova Scotia isolates were identical. SSU rRNA gene phylogenies placed the Codium-associated kinetoplastid in neobodonid clade '1E'. Clade 1E likely contains no previously described species, and branches outside all other major neobodonid groups, either as their sister or as a separate lineage, depending on rooting. These results indicate that the kinetoplastid represents a single species that merits a new genus (and family), and we describe it as Allobodo chlorophagus n. gen., n. sp. The lack of evidence for food sources other than Codium is consistent with a parasitic association, but other possibilities exist (e.g. necrotrophy).

Use of AFLP for the study of eukaryotic pathogens affecting humans.

Amplified fragment length polymorphism (AFLP) is a genotyping technique based on PCR amplification of specific restriction fragments from a particular genome. The methodology has been extensively used in plant biology to solve a variety of scientific questions, including taxonomy, molecular epidemiology, systematics, population genetics, among many others. The AFLP share advantages and disadvantages with other types of molecular markers, being particularly useful in organisms with no previous DNA sequence knowledge. In eukaryotic pathogens, the technique has not been extensively used, although it has the potential to solve many important issues as it allows the simultaneous examination of hundreds or even thousands of polymorphic sites in the genome of the organism. Here we describe the main applications published on the use of AFLP in eukaryotic pathogens, with emphasis in species of the groups fungi, protozoa and helminths, and discuss the role of this methodology in the context of new techniques derived from the advances of the next generation sequencing.

Neobodonids are dominant kinetoplastids in the global ocean.

Kinetoplastid flagellates comprise basal mostly free-living bodonids and derived obligatory parasitic trypanosomatids, which belong to the best-studied protists. Due to their omnipresence in aquatic environments and soil, the bodonids are of ecological significance. Here, we present the first global survey of marine kinetoplastids and compare it with the strikingly different patterns of abundance and diversity in their sister clade, the diplonemids. Based on analysis of 18S rDNA V9 ribotypes obtained from 124 sites sampled during the Tara Oceans expedition, our results show generally low to moderate abundance and diversity of planktonic kinetoplastids. Although we have identified all major kinetoplastid lineages, 98% of kinetoplastid reads are represented by neobodonids, namely specimens of the Neobodo and Rhynchomonas genera, which make up 59% and 18% of all reads, respectively. Most kinetoplastids have small cell size (0.8-5 µm) and tend to be more abundant in the mesopelagic as compared to the euphotic zone. Some of the most abundant operational taxonomic units have distinct geographical distributions, and three novel putatively parasitic neobodonids were identified, along with their potential hosts.

Taming Parasites by Tailoring Them.

The next-generation gene editing based on CRISPR (clustered regularly interspaced short palindromic repeats) has been successfully implemented in a wide range of organisms including some protozoan parasites. However, application of such a versatile game-changing technology in molecular parasitology remains fairly underexplored. Here, we briefly introduce state-of-the-art in human and mouse research and usher new directions to drive the parasitology research in the years to come. In precise, we outline contemporary ways to embolden existing apicomplexan and kinetoplastid parasite models by commissioning front-line gene-tailoring methods, and illustrate how we can break the enduring gridlock of gene manipulation in non-model parasitic protists to tackle intriguing questions that remain long unresolved otherwise. We show how a judicious solicitation of the CRISPR technology can eventually balance out the two facets of pathogen-host interplay.

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts.

Members of the genus Paramoeba (including Neoparamoeba) (Amoebozoa) are single-celled eukaryotes of economic and ecological importance because of their association with disease in a variety of marine animals including fish, sea urchins, and lobster. Interestingly, they harbor a eukaryotic endosymbiont of kinetoplastid ancestry, Perkinsela sp. To investigate the complex relationship between Paramoeba spp. and Perkinsela sp., as well as the relationships between different Paramoeba species, molecular data was obtained for four novel isolates. We also acquired new data from the urchin pathogen P. invadens. Comprehensive molecular phylogenetic analyses were carried out using 33 newly obtained 18S rDNA sequences from the host amoebae and 16 new 18S rDNA sequences from their corresponding Perkinsela sp., together with all publicly available 18S molecular data. Intra-isolate 18S rDNA nucleotide diversity was found to be surprisingly high within the various species of Paramoeba, but relatively low within their Perkinsela sp. endosymbionts. 18S rDNA phylogenies and ParaFit co-evolution analysis revealed a high degree of congruence between the Paramoeba and Perkinsela sp. tree topologies, strongly suggesting that a single endosymbiotic event occurred in the common ancestor of known Paramoeba species, and that the endosymbionts have been inherited vertically ever since.

Isolation of Novel Trypanosomatid, Zelonia australiensis sp. nov. (Kinetoplastida: Trypanosomatidae) Provides Support for a Gondwanan Origin of Dixenous Parasitism in the Leishmaniinae.

The genus Leishmania includes approximately 53 species, 20 of which cause human leishmaniais; a significant albeit neglected tropical disease. Leishmaniasis has afflicted humans for millennia, but how ancient is Leishmania and where did it arise? These questions have been hotly debated for decades and several theories have been proposed. One theory suggests Leishmania originated in the Palearctic, and dispersed to the New World via the Bering land bridge. Others propose that Leishmania evolved in the Neotropics. The Multiple Origins theory suggests that separation of certain Old World and New World species occurred due to the opening of the Atlantic Ocean. Some suggest that the ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia evolved on Gondwana between 90 and 140 million years ago. In the present study a detailed molecular and morphological characterisation was performed on a novel Australian trypanosomatid following its isolation in Australia's tropics from the native black fly, Simulium (Morops) dycei Colbo, 1976. Phylogenetic analyses were conducted and confirmed this parasite as a sibling to Zelonia costaricensis, a close relative of Leishmania previously isolated from a reduviid bug in Costa Rica. Consequently, this parasite was assigned the name Zelonia australiensis sp. nov. Assuming Z. costaricensis and Z. australiensis diverged when Australia and South America became completely separated, their divergence occurred between 36 and 41 million years ago at least. Using this vicariance event as a calibration point for a phylogenetic time tree, the common ancestor of the dixenous genera Leishmania, Endotrypanum and Porcisia appeared in Gondwana approximately 91 million years ago. Ultimately, this study contributes to our understanding of trypanosomatid diversity, and of Leishmania origins by providing support for a Gondwanan origin of dixenous parasitism in the Leishmaniinae.

Heme pathway evolution in kinetoplastid protists.

BACKGROUND: Kinetoplastea is a diverse protist lineage composed of several of the most successful parasites on Earth, organisms whose metabolisms have coevolved with those of the organisms they infect. Parasitic kinetoplastids have emerged from free-living, non-pathogenic ancestors on multiple occasions during the evolutionary history of the group. Interestingly, in both parasitic and free-living kinetoplastids, the heme pathway-a core metabolic pathway in a wide range of organisms-is incomplete or entirely absent. Indeed, Kinetoplastea investigated thus far seem to bypass the need for heme biosynthesis by acquiring heme or intermediate metabolites directly from their environment. RESULTS: Here we report the existence of a near-complete heme biosynthetic pathway in Perkinsela spp., kinetoplastids that live as obligate endosymbionts inside amoebozoans belonging to the genus Paramoeba/Neoparamoeba. We also use phylogenetic analysis to infer the evolution of the heme pathway in Kinetoplastea. CONCLUSION: We show that Perkinsela spp. is a deep-branching kinetoplastid lineage, and that lateral gene transfer has played a role in the evolution of heme biosynthesis in Perkinsela spp. and other Kinetoplastea. We also discuss the significance of the presence of seven of eight heme pathway genes in the Perkinsela genome as it relates to its endosymbiotic relationship with Paramoeba.

Long-term spatiotemporal stability and dynamic changes in the haemoparasite community of spiny mice (Acomys dimidiatus) in four montane wadis in the St. Katherine Protectorate, Sinai, Egypt.

BACKGROUND: Long-term field studies of parasite communities are rare but provide a powerful insight into the ecological processes shaping host-parasite interactions. The aim of our study was to monitor long-term trends in the haemoparasite communities of spiny mice (Acomys dimidiatus) and to identify the principal factors responsible for changes over a 12 year period. METHODS: To this end we sampled four semi-isolated populations of mice (n = 835) in 2000, 2004, 2008 and 2012 in four dry montane valleys (wadis) located in the Sinai Massif, Egypt. RESULTS: Overall 76.2 % of spiny mice carried at least one of the five haemoparasite genera (Babesia, Bartonella, Haemobartonella, Hepatozoon, Trypanosoma) recorded in the study. Prevalence of haemoparasites varied significantly between the sites with the highest overall prevalence in Wadi Tlah and the lowest in W. El Arbaein, and this changed significantly with time. In the first two surveys there was little change in prevalence, but by 2008, when the first signs of a deepening drought in the region had become apparent, prevalence began to drift downwards, and by 2012 prevalence had fallen to the lowest values recorded from all four sites over the entire 12-year period. The overall mean species richness was 1.2 ± 0.03, which peaked in 2004 and then dropped by more than 50 % by 2012. Species richness was highest among mice from Wadi Tlah and peaked in age class 2 mice (young adults). Site was the most significant factor affecting the prevalence of individual parasite species, with Trypanosoma acomys and Hepatozoon sp. occurring mainly in two wadis (W. Tlah & W. Gharaba). In four of the five genera recorded in the study we observed a significant drop in prevalence or/and abundance since 2004, the exception being Hepatozoon sp. CONCLUSIONS: During the 12-year-long period of study in the Sinai, we observed dynamic changes and possibly even cycles of prevalence and abundance of infections which differed depending on parasite species. Although the exact reasons cannot be identified at this time, we hypothesize that the effects of a 15-year-long scarcity of rainfall in the local environment and a fall in host densities over the period of study may have been responsible for a drop in transmission rates, possibly by a negative impact on vector survival.

Selenoproteins of African trypanosomes are dispensable for parasite survival in a mammalian host.

The trace element selenium is found in polypeptides as selenocysteine, the 21(st) amino acid that is co-translationally inserted into proteins at a UGA codon. In proteins, selenocysteine usually plays a role as an efficient redox catalyst. Trypanosomatids previously examined harbor a full set of genes encoding the machinery needed for selenocysteine biosynthesis and incorporation into three selenoproteins: SelK, SelT and, the parasite-specific, Seltryp. We investigated the selenoproteome of kinetoplastid species in recently sequenced genomes and assessed the in vivo relevance of selenoproteins for African trypanosomes. Database mining revealed that SelK, SelT and Seltryp genes are present in most kinetoplastids, including the free-living species Bodo saltans, and Seltryp was lost in the subgenus Viannia from the New World Leishmania. Homology and sinteny with bacterial sulfur dioxygenases and sulfur transferases suggest a putative role for Seltryp in sulfur metabolism. A Trypanosoma brucei selenocysteine synthase (SepSecS) null-mutant, in which selenoprotein synthesis is abolished, displayed similar sensitivity to oxidative stress induced by a short-term exposure to high concentrations of methylglyoxal or H2O2 to that of the parental wild-type cell line. Importantly, the infectivity of the SepSecS knockout cell line was not impaired when tested in a mouse infection model and compensatory effects via up-regulation of proteins involved in thiol-redox metabolism were not observed. Collectively, our data show that selenoproteins are not required for survival of African trypanosomes in a mammalian host and exclude a role for selenoproteins in parasite antioxidant defense and/or virulence. On this basis, selenoproteins can be disregarded as drug target candidates.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Flagellar membrane proteins in kinetoplastid parasites.

All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. Although flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites, including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

Flagellar motility in eukaryotic human parasites.

A huge variety of protists rely on one or more motile flagella to either move themselves or move fluids and substances around them. Many of these flagellates have evolved a symbiotic or parasitic lifestyle. Several of the parasites have adapted to human hosts, and include agents of prevalent and serious diseases. These unicellular parasites have become specialised in colonising a wide range of biological niches within humans. They usually have diverse transmission cycles, and frequently manifest a variety of distinct morphological stages. The motility of the single or multiple flagella plays important but understudied roles in parasite transmission, host invasion, dispersal, survival, proliferation and pathology. In this review we provide an overview of the important human pathogens that possess a motile flagellum for at least part of their life cycle. We highlight recently published studies that aim to elucidate motility mechanisms, and their relevance for human disease. We then bring the physics of swimming at the microscale into context, emphasising the importance of interdisciplinary approaches for a full understanding of flagellate motility - especially in light of the parasites' microenvironments and population dynamics. Finally, we summarise some important technological aspects, describing challenges for the field and possibilities for motility analyses in the future.

Kinetoplastid flagellates overlooked by universal primers dominate in the oxygenated hypolimnion of Lake Biwa, Japan.

Kinetoplastid flagellates, microscopically often detected from various aquatic environments and considered ubiquitous are seldom reported in molecular diversity studies with universal eukaryote DNA primers. To investigate this inconsistency, we examined nanoflagellate diversity in Lake Biwa, Japan by 18S rRNA gene clone libraries using universal eukaryote and kinetoplastid-specific primers. We also examined the abundance of kinetoplastids by Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization. No, kinetoplastid sequences were detected in the universal eukaryote primers library from epilimnion and hypolimnion in different seasons. However, kinetoplastid flagellates were detected with kinetoplastid-specific probe from all of the seasons and contributed up to 11.9 and 36.0% of total eukaryotes in the epilimnion and hypolimnion, respectively. Thus, kinetoplastids probably are a significant, sometimes dominant, group in the hypolimnion, contributing up to 43.7% of the total flagellates. Using group-specific primers, kinetoplastid sequences were also obtained from both epilimnion and hypolimnion library. Therefore, we attributed the inconsistency to the divergent nature of 18S rRNA gene of kinetoplastids, which lead to their undetection in the universal eukaryote primer libraries. This study revealed that kinetoplastids have significant ecological importance in the hypolimnion of Lake Biwa, suggesting that these flagellates have been overlooked in other studies using universal eukaryote primers.

New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: an open resource.

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host-pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.

Phylogeny and Reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996.

Hemistasia phaeocysticola is a marine flagellate that preys on diatoms and dinoflagellates among others. Although its morphology and ultrastructure were previously observed and characterized, its phylogenetic position has not been analyzed using molecular sequence data. This flagellate was classified as a kinetoplastid on the basis of the presence of a kinetoplast in the mitochondrion. However, several morphological characteristics similar to those of diplonemids, a sister group of kinetoplastids, have also been noted. Herein, we report that H. phaeocysticola branches within the diplonemid clade in the phylogenetic tree reconstructed by analyzing 18S rRNA gene sequences. Its systematic placement based on this finding is also discussed.

Support for the coevolution of Neoparamoeba and their endosymbionts, Perkinsela amoebae-like organisms.

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.

Development of loop-mediated isothermal amplification targeting 18s ribosomal DNA for rapid detection of Azumiobodo hoyamushi (Kinetoplastea).

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.