Identification of common human infectious and potentially zoonotic novel genotypes of Enterocytozoon bieneusi in cavernicolous bats in Thailand.

Enterocytozoon bieneusi is a common cause of human microsporidiosis and can infect a variety of animal hosts worldwide. In Thailand, previous studies have shown that this parasite is common in domestic animals. However, information on the prevalence and genotypes of this parasite in other synanthropic wildlife, including bats, remains limited. Several pathogens have been previously detected in bats, suggesting that bats may serve as a reservoir for this parasite. In this study, a total of 105 bat guano samples were collected from six different sites throughout Thailand. Of these, 16 from Chonburi (eastern), Ratchaburi (western), and Chiang Rai (northern) provinces tested positive for E. bieneusi, representing an overall prevalence of 15.2%. Based on ITS1 sequence analysis, 12 genotypes were identified, including two known genotypes (D and type IV) frequently detected in humans and ten novel potentially zoonotic genotypes (TBAT01-TBAT10), all belonging to zoonotic group 1. Lyle's flying fox (Pteropus lylei), commonly found in Southeast Asia, was identified as the host in one sample that was also positive for E. bieneusi. Network analysis of E. bieneusi sequences detected in this study and those previously reported in Thailand also revealed intraspecific divergence and recent population expansion, possibly due to adaptive evolution associated with host range expansion. Our data revealed, for the first time, multiple E. bieneusi genotypes of zoonotic significance circulating in Thai bats and demonstrated that bat guano fertilizer may be a vehicle for disease transmission.

Six-Year Study on Cutaneous Leishmaniasis in Al-Muthanna, Iraq: Molecular Identification Using ITS1 Gene Sequencing.

BACKGROUND: The current study aimed to determine the prevalence of cutaneous leishmaniasis (CL) in Al-Muthanna province (Iraq) and to characterize the Leishmania species that cause cutaneous lesions through conventional polymerase chain reaction techniques in some patients during the first 7 months of the year 2020. MATERIALS AND METHODS: Medical information on patients with CL was obtained from archived records at the Al-Muthanna Health Office's Public Health Department (2015-2020). In the Al-Hussein Teaching Hospital laboratory, 95 CL samples were collected and examined microscopically for molecular characterization using Giemsa staining. RESULTS: Between 2015 and 2020, 2,325 patients (1,184 men and 1,141 women) were enrolled. Although CL occurred across all age groups, those aged range of 5-14 years had the highest proportion of infections (53.0%). This study found that most infections occurred between December and February, peaking in January. Only 63 of 95 CL samples were positive for the Internal Transcribed Spacer 1 region. L. tropica was found in 39 samples (61.9%), whereas L. major was found in 24 samples (38.1%), in CL patients. Although dermal lesions develop in all body regions, a single lesion is the most common. The upper limbs (13 of 16 samples, 33.3%)were infected with L. tropica, whereas the lower limbs (9 of 14 samples, 37.5%) were infected with L. major. In contrast to L. major, most L. tropica lesions occur in urban areas. CONCLUSION: Our study indicates that CL is endemic in the Al-Muthanna province and that two Leishmania spp. coexist in the province. Molecular diagnosis is a vital component in determining many clinical symptoms of the Leishmania parasite as well as implementing suitable therapeutic, epidemiological, and control strategies.

Gut Fungal Microbiota Alterations in Pulmonary Arterial Hypertensive Rats.

The gut microbiome's imbalance has been implicated in the pathogenesis of pulmonary arterial hypertension (PAH), yet the contribution of the gut mycobiome remains largely unclear. This study delineates the gut mycobiome profile in PAH and examines its interplay with the bacterial microbiome alterations. Fecal samples from monocrotaline-induced PAH rats and matched controls were subjected to internal transcribed spacer 1 (ITS1) sequencing for fungal community assessment and 16S ribosomal RNA (rRNA) gene sequencing for bacterial community characterization. Comparative analysis revealed no significant disparities in the overall mycobiome diversity between the PAH and control groups. However, taxonomic profiling identified differential mycobiome compositions, with the PAH group exhibiting a significant enrichment of genera such as Wallemia, unidentified_Branch02, Postia, Malassezia, Epicoccum, Cercospora, and Alternaria. Conversely, genera Xeromyces, unidentified_Plectosphaerellaceae, and Monilia were more abundant in the controls. Correlations of Malassezia and Wallemia abundance with hemodynamic parameters were observed. Indications of bidirectional fungal-bacterial community interactions were also noted. This investigation reveals distinct gut mycobiome alterations in PAH, which are intricately associated with concurrent bacterial microbiome changes, suggesting a possible contributory role of gut fungi in PAH pathophysiology. These findings underscore the potential for novel gut mycobiome-targeted therapeutic interventions in PAH management.

Microbial successional pattern along a glacier retreat gradient from Byers Peninsula, Maritime Antarctica.

The retreat of glaciers in Antarctica has increased in the last decades due to global climate change, influencing vegetation expansion, and soil physico-chemical and biological attributes. However, little is known about soil microbiology diversity in these periglacial landscapes. This study characterized and compared bacterial and fungal diversity using metabarcoding of soil samples from the Byers Peninsula, Maritime Antarctica. We identified bacterial and fungal communities by amplification of bacterial 16 S rRNA region V3-V4 and fungal internal transcribed spacer 1 (ITS1). We also applied 14C dating on soil organic matter (SOM) from six profiles. Physico-chemical analyses and attributes associated with SOM were evaluated. A total of 14,048 bacterial ASVs were obtained, and almost all samples had 50% of their sequences assigned to Actinobacteriota and Proteobacteria. Regarding the fungal community, Mortierellomycota, Ascomycota and Basidiomycota were the main phyla from 1619 ASVs. We found that soil age was more relevant than the distance from the glacier, with the oldest soil profile (late Holocene soil profile) hosting the highest bacterial and fungal diversity. The microbial indices of the fungal community were correlated with nutrient availability, soil reactivity and SOM composition, whereas the bacterial community was not correlated with any soil attribute. The bacterial diversity, richness, and evenness varied according to presence of permafrost and moisture regime. The fungal community richness in the surface horizon was not related to altitude, permafrost, or moisture regime. The soil moisture regime was crucial for the structure, high diversity and richness of the microbial community, specially to the bacterial community. Further studies should examine the relationship between microbial communities and environmental factors to better predict changes in this terrestrial ecosystem.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Detection of Trypanosoma evansi in a naturally infected cat in Indonesia using bioassay and molecular techniques.

Background and Aim: The prevalence of surra in domestic cat is seldom and it is caused by Trypanosoma brucei and Trypanosoma evansi. However, molecular diagnostic approaches are required owing to similarities in their morphology. In Yogyakarta, a domestic cat was diagnosed with trypanosomiasis; however, the causative species was undetermined. Therefore, we aimed to molecularly and biologically identify the isolate. Materials and Methods: Approximately 1 mL of blood from an infected cat was collected into EDTA tube and separated for inoculation into donor mice, blood smear, and DNA isolation. Two donor mice was then used for increasing the number of parasite in order to infect 10 experimental mice. Parasitemia was monitored daily in each experimental mouse by preparing a wet mount and Giemsa-stained thin blood smear. The blood of experimental mice that reached the peak of parasitemia was then collected and used for DNA isolation. Each blood sample, which collected from infected cat and experimental mice, was then isolated and amplified the DNA by polymerase chain reaction using ITS-1. The parasitemia pattern and viability of the animals were observed to determine the biological characteristics of trypanosomatid, while to assess the molecular characteristics, the internal transcribed spacer (ITS)-1 amplification was used. Results: The prepatent period of this trypanosomatid is between 2 and 4 dpi, whereas the life span of mice is approximately 4-10 dpi. Morphologically, the trypomastigote in the cat blood smear had long slender and intermediate shapes. However, only the long slender form was detected. Among the total of 410 nucleotides (NT) of ITS-1 sequences, 25 NT substitutions differed between the cat and mouse isolates. Phylogenetic analysis revealed that both samples had a close genetic relationship with T. evansi. Conclusion: Trypanosoma evansi, a highly virulent trypanosomatid, was isolated from a cat in Yogyakarta.

Development of a new multiplex PCR to detect fecal coccidian parasite.

BACKGROUND: Cryptosporidium spp., Cystoisospora belli and Cyclospora cayetanensis are common intestinal coccidian parasites causing gastroenteritis. The clinical presentation caused by each parasite is indistinguishable from each other. Uniplex polymerase chain reaction (PCR) for these three groups of intestinal coccidian parasites was developed by us in our laboratory. Thereafter, we planned to develop a single-run multiplex polymerase chain reaction (mPCR) assay to detect Cryptosporidium spp., C. belli and C. cayetanensis simultaneously from a stool sample and described it here as coccidian mPCR. METHODS: New primers for C. belli and C. cayetanensis were designed and uniplex PCRs were standardized. The coccidian mPCR was standardized with known positive DNA control isolates. It was validated with 58 known positive and 58 known negative stool samples, which were previously identified by uniplex PCR. RESULTS: The coccidian mPCR was standardized with earlier primers designed by us for Cryptosporidium spp. and C. cayetanensis, and a newly designed primer for the internal transcribed spacer-1 (ITS-1) gene for C. belli. The coccidian mPCR was 92.1% sensitive for Cryptosporidium spp., and 100% sensitive for C. belli and C. cayetanensis each, when tested on 116 known samples. It was 100% specific for all intestinal coccidian parasites. Two representative PCR products of the newly designed ITS-1 primer for C. belli were sequenced and submitted to the GenBank, which best match with the sequences of C. belli. CONCLUSION: A highly sensitive, specific, cost-effective, indigenous, single-run coccidian mPCR has been developed, which can simultaneously detect Cryptosporidium spp., C. belli and C. cayetanensis.

Molecular phylogenetics of the sucking louse genus Lemurpediculus (Insecta: Phthiraptera), ectoparasites of lemurs, with descriptions of three new species.

Sucking lice live in intimate association with their hosts and often display a high degree of host specificity. The present study investigated sucking lice of the genus Lemurpediculus from six mouse lemur (Microcebus) and two dwarf lemur (Cheirogaleus) species endemic to the island of Madagascar, considered a biodiversity hotspot. Louse phylogenetic trees were created based on cytochrome C oxidase subunit I (COI), elongation factor 1α (EF1α) and internal transcribed spacer 1 (ITS1) sequences. While clustering according to host species was generally observed for COI and ITS1, suggesting high host specificity of the examined lice, EF1α sequences alone did not distinguish between lice of different Microcebus species, possibly due to rather recent divergence. As bootstrap support for basal tree structure was rather low, further data are necessary to resolve the evolutionary history of louse-mouse lemur associations. Three new species of sucking lice are described: Lemurpediculus zimmermanni sp. Nov. From Microcebus ravelobensis, Lemurpediculus gerpi sp.nov. from Microcebus gerpi, and Lemurpediculus tsimanampesotsae sp. nov. from Microcebus griseorufus. These new species are compared with all known congeneric species and identifying features are illustrated for all known species of Lemurpediculus.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

Root-associated fungal microbiota of the perennial sweet sorghum cultivar under field growth.

Root-associated fungal microbiota, which inhabit the rhizosphere, rhizoplane and endosphere, have a profound impact on plant growth and development. Sorghum bicolor (L.) Moench, also called broomcorn or sweet sorghum, is a multipurpose crop. The comparison between annual and perennial sweet sorghum cultivars in terms of plant growth, as well as their interactions with belowground fungal microbiota, is still poorly understood, although there has been growing interest in the mutualism between annual sweet sorghum and soil bacteria or bacterial endophytes. In this study, the perennial sweet sorghum cultivar N778 (N778 simply) and its control lines TP213 and TP60 were designed to grow under natural field conditions. Bulk soil, rhizosphere soil and sorghum roots were collected at the blooming and maturity stages, and then the fungal microbiota of those samples were characterized by high-throughput sequencing of the fungal ITS1 amplicon. Our results revealed that the alpha diversity of the fungal microbiota in rhizosphere soil and root samples was significantly different between N778 and the two control lines TP213 and TP60 at the blooming or maturity stage. Moreover, beta diversity in rhizosphere soil of N778 was distinct from those of TP213 and TP60, while beta diversity in root samples of N778 was distinct from those of TP213 but not TP60 by PCoA based on Bray-Curtis and WUF distance metrics. Furthermore, linear discriminant analysis (LDA) and multiple group comparisons revealed that OTU4372, a completely unclassified taxon but with symbiotroph mode, was enriched in sorghum roots, especially in N778 aerial roots at the blooming stage. Our results indicate that Cladosporium and Alternaria, two fungal genera in the rhizosphere soil, may also be dominant indicators of sorghum yield and protein content in addition to Fusarium at the maturity stage and imply that the perennial sweet sorghum N778 can primarily recruit dominant psychrotolerant bacterial taxa but not dominant cold-tolerant fungal taxa into its rhizosphere to support its survival below the freezing point.

Prevalence of Eimeria species, detected by ITS1-PCR, in broiler poultry farms located in seven provinces of northeastern Algeria.

Coccidiosis is an important global chickens' disease which can cause serious economic losses in the poultry industry worldwide. Little is known about the extent of infection or diversity, of the causative agent Eimeria spp., in Algeria. A priority, therefore, is to determine the prevalence and species composition to inform strategies on treatments and control measures. Samples were collected from 187 broiler farms, located in 7 Northeastern Algerian provinces (Jijel, Constantine, Skikda, Mila, Setif, Batna, Bordj bou-Arreridj), and Internal Transcribed Spacer 1 PCR (ITS1-PCR) was used to determine the prevalence and composition of Eimeria species in chickens. The survey revealed the presence of all seven species of Eimeria at different prevalences (E. maxima (69%), E. acervulina (68.4%), E. necatrix (11.2%), E. tenella (8%), E. praecox (4.3%), E. mitis (2.1%), E. brunetti (2.1%). Multiple infections, with up to 4 different Eimeria species present on a single farm, were the most frequent situation in our samples (51.9% mixed infections versus 47.6% single infections). All farms revealed infected samples, and we conclude that this parasite is a significant problem in these provinces.

Development of Multiplex PCR-based Protocols for Simultaneous Caterpillar Diagnosis of Three Spodoptera and One Mamestra Species (Lepidoptera: Noctuidae).

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.

Population genetic structure of diphyllobothriid tapeworms (Cestoda: Diphyllobothriidea) parasitising fish in the Baikal Rift Zone.

Tapeworms of the genus Dibothriocephalus are widely distributed throughout the world, and some are agents of human diphyllobothriasis, one of the most important fish-borne zoonoses caused by a cestode parasite. Until now, the population genetic structure of diphyllobothriid tapeworms in the Baikal Rift Zone (BRZ) has remained unexplored. The major aim of this study was to analyse the population genetic structure of D. dendriticus and D. ditremus parasitising fish in the BRZ based on internal transcribed spacer 1 (ITS1) and mitochondrial gene cytochrome oxidase subunit I (cox1) sequences. We found that both species had complex population genetic structures. Each species formed 2 clades (D. dendriticus: Clade 1 & 2; D. ditremus Clade A & B) that differed in genetic diversity. D. dendriticus haplotypes in Clade 1 formed a star-like sub-network with a main haplotype, whereas the haplotypes in Clade 2 formed a diffuse network. We assumed that the complex population genetic structure of D. dendriticus was a consequence of populations evolving under different palaeoecological conditions during the Last Glacial Maximum. In contrast to D. dendriticus, both clades in the D. ditremus samples formed a diffuse network. Our findings revealed hypothetical pathways in the formation of the population genetic structure of diphyllobothriids in the BRZ. On one hand, isolation by distance played an important role; on the other hand, lake recolonisation from refugia and a genetic bottleneck after the end of the Last Glacial Maximum had a possible influence.

Deep sequencing across multiple host species tests pine-endophyte specificity.

PREMISE: Foliar fungal endophytes vary in their distributions across landscapes or plant host taxa, indicative of specialized ecologies and host specific adaptations. Accounts of specialization, however, depend on the taxonomic breadth and geographic range of the host plants included in each study. A broad region-scale study or deep sampling of diverse potential host species still remains relatively rare but is becoming increasingly possible with high-throughput sequencing. METHODS: Amplicon sequencing was used to rapidly identify the fungal endophytic community among six pine (Pinus, Pinaceae) species co-occurring across northeastern United States and to test for site and host specialization. We focused on the endophytic genus Lophodermium (Rhytismataceae), whose species members are thought to specialize on different pine species, to test if amplicon sequencing could rapidly verify previously implied or discover new patterns of host specificity. RESULTS: While amplicon sequencing could analyze more samples at greater depths and recover greater numbers of unique Lophodermium taxa than when endophyte communities were surveyed with traditional culturing methods, patterns of specialization were not better supported. This may be because amplicon sequencing can indiscriminately capture non-host specific organisms found incidentally from plant tissues or because we have overestimated host-specificity in the past with biased culturing techniques. CONCLUSIONS: Amplicon sequencing can quickly identify patterns of host specificity by allowing large-scale surveys but has limitations in quantifying the level of intimacy of these relationships.

Species identification and phylogenetic analysis of canine Leishmania in Diebu County, Gansu Province / 中华地方病学杂志

Objective:To investigate the species types and phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province, so as to provide a basis for exploring new methods of prevention and control of canine visceral leishmaniasis. Methods:DNA was extracted from blood samples of eight asymptomatic Leishmania-infected dogs in Luoda administrative village in Diebu County, Gansu Province. Ribosomal internal transcribed spacer 1 (ITS-1) gene fragments were amplified and isolated by PCR, and then the amplified target fragments were sequenced. The MEGA 7.0 software was used for multiple sequence alignment, and a phylogenetic tree was constructed by neighbor-joining method to analyze the phylogenetic relationship of canine Leishmania in Diebu County, Gansu Province. Results:Fragments of about 320 bp corresponding in size to the target sequence ITS-1 were isolated from all of the eight asymptomatic Leishmania-infected dogs blood samples. ITS-1 sequence alignment showed that the sequence homology between 8 samples and Leishmania infantum MG969403, MN648755 strains was 99.1% - 100.0%; phylogenetic tree showed that all 8 samples were clustered into one branch with Leishmania infantum. Conclusion:Leishmania infantum is identified from all of the eight asymptomatic Leishmania-infected dogs blood samples in Diebu County, Gansu Province.

[Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania].

OBJECTIVE: To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. METHODS: Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. RESULTS: After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 µmol/L and 0.08 µmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/µL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/µL Leishmania genomic DNA. CONCLUSIONS: A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.

Comparative Study of Oral Bacteria and Fungi Microbiota in Tibetan and Chinese Han Living at Different Altitude.

Knowledge about the impact of altitude and ethnicity on human oral microbiota is currently limited. To obtain the baseline of normal salivary microbiota, we analyzed the bacteria and fungi composition in Tibetan (HY group) and Han population (CD group) living at different altitudes by using next-generation sequencing (NGS) technology combined with PICRUSt and FUNGuild analyses. There were significant differences in oral microbiota composition between the two groups at phylum and genus levels. At the phylum level, the HY group had higher relative abundances of Firmicutes and Ascomycota, whereas the Bacteroidetes and Basidiomycota in the CD group were richer. These changes at the phylum level reflected different dominant genus compositions. Compared with the Han population, Candida, Fusarium, Zopfiella, Streptococcus, Veillonella and Rothia in Tibetan were higher. Surprisingly, the Zopfiella was found almost exclusively in the Tibetan. The PICRUSt and FUNGuild analysis also indicated that the function of the bacterial and fungal communities was altered between the two groups. In conclusion, our results suggest that there are significant differences in oral microbial structure and metabolic characteristics and trophic modes among Tibetan and Han population living at different altitudes. We first established the oral microbiota framework and represented a critical step for determining the diversity of oral microbiota in the Tibetan and Han population.

Molecular Characterization of an Emerging Root-Knot Nematode Meloidogyne enterolobii in North Carolina, USA.

An emerging threat to agriculture, Meloidogyne enterolobii Yang & Eisenback, 1983, is a tropical species and considered to be the most damaging root-knot nematode (RKN) in the world because of its wide host range, aggressiveness, and ability to overcome resistance to RKN in many crops. It was first detected in the United States on ornamental plants in Florida in 2001 but has since been identified in North Carolina, South Carolina, and Louisiana. Several thousand RKN populations were collected from North Carolina field crops, ornamental plants, and turfgrasses for species identification in the Nematode Assay Laboratory in the North Carolina Department of Agriculture & Consumer Services. From 2006 to 2019, root systems showing galling symptoms were dissected under the microscope, and females were obtained for DNA analysis. When only soil samples were submitted, the second-stage juveniles or males were used instead. Molecular characterization was performed via polymerase chain reaction with species-specific primers and DNA sequencing on the ribosomal DNA 18S-ITS1-5.8S and 28S D2/D3 and mitochondrial DNA CoxII-16S. One hundred thirty-five representative RKN populations from North Carolina were characterized and identified as M. enterolobii. Six populations from China where the species was originally described were included in this study for identity confirmation and comparison. As of December 2019, M. enterolobii has been confirmed from a limited number of fields in 11 North Carolina counties: Columbus, Craven, Greene, Harnett, Johnston, Lenoir, Nash, Pitt, Sampson, Wayne, and Wilson. Currently, M. enterolobii is the most important emerging RKN species in the United States and causes severe damage to agronomic and horticultural crops, especially sweetpotato in North Carolina.

Development of a fluorescent recombinase-aided isothermal amplification-based nucleic acid assay for detection of Leishmania / 中国血吸虫病防治杂志

Objective To develop a fluorescent recombinase-aided isothermal amplification (RAA)-based nucleic acid assay for detection of Leshimania. Methods Specific primers and probes were designed targeting Leishmania internal transcribed spacer 1 (ITS1) gene for RAA assay, and a fluorescent RAA assay was developed for detection of Leishmania following screening of primer pairs and optimization of primer and probe concentrations. The sensitivity of RAA assay for detection of Leishmania was evaluated using recombinant plasmid containing Leishmania ITS1 gene sequences at different copies and Leshimania genomic DNA at different concentrations as templates, and the specificity of RAA assay for detection of Leishmania was evaluated using the genomic DNA of transfusion-transmitted parasites, including Babesia microti, Toxoplasma gondii, Plamodium vivax, P. ovale, P. falciparum, P. malariae, L. donovani and L. infantum. Results After the optimal primer pair was screened from 9 pairs of primer combinations, the final primer and probe concentrations were optimized as 0.3 μmol/L and 0.08 μmol/L, respectively. Nucleic acid detection of Leishmania was completed by the fluorescent RAA assay at an isothermal temperature of 39 °C within 20 min. Remarkable florescent signals were seen within 5 min following RAA detection of genomic DNA of L. donovani and L. infantum, and no cross-reactions were observed with B. microti, T. gondii, P. vivax, P. ovale, P. falciparum or P. malariae. The lowest limitation of detection of the fluorescent RAA assay was 10 copies/μL recombinant plasmid containing Leishmania ITS1 gene sequences and 1 fg/μL Leishmania genomic DNA. Conclusions A rapid, simple, sensitive and specific fluorescent RAA assay is successfully developed for detection of L. donovani and L. infantum, which is effective for field screening of leishmaniasis.

Population structure of leishmania major in southeastern morocco.

Cutaneous leishmaniasis (CL) is an infectious disease caused by various Leishmania species. It is among the most neglected tropical diseases and has been considered a major health threat over the past decades in the country. Its zoonotic form caused by Leishmania (L) major is the most prevalent in Morocco. This study investigated the population structure of L. major in southeastern Morocco. Samples (n = 67) were collected from patients with CL in five different endemic areas located in three provinces (Ouarzazate, Tinghir, and Zagora). These samples were then sequenced using two nuclear markers: internal transcribed spacer 1 (ITS1) and a fragment of the virulence factor GP63. Next, the sequences were edited and analyzed. Molecular diversity indices showed a high population genetic diversity but an overall low haplotype diversity. Our results suggest small population differentiation, indicating a low geographic structure. Tajima's D and Fu's Fs tests both suggested recent population expansion based on the significant deviations from neutrality in both tests for all populations except Tinghir, which may be due to a small sample size. Based on our findings, the region is experiencing rapid population expansion caused by recent CL outbreaks, and one of them has been recently studied. In addition, analysis of molecular variance and FST suggested gene flow between Zagora and both Ouarzazate and Tinghir. Nonetheless, no gene flow was observed between Tinghir and Ouarzazate. To the best of our knowledge, this is the first analysis of the population structure of L. major in Morocco. The results of this study provide crucial background information for epidemiological studies by showing the presence of gene flow between populations and clonal expansion in cases of an outbreak. This will drive authorities to reconsider the implemented control strategies.