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We isolated 17 viral strains capable of causing cytopathic effects in mammalian cells and death in neonatal mice from sand flies in China. Phylogenetic analysis showed that these strains belonged to the genus Phlebovirus. These findings highlight the need to control this potentially emerging virus to help safeguard public health.

Ticks are hematophagous vectors that transmit a variety of pathogens, posing significant threats to the health of both humans and animals. Tick midgut proteins play essential roles in blood digestion, feeding, toxic waste processing, and pathogen transmission. Dermacentor nuttalli is the primary vector of tick-borne pathogens, including rickettsioses in the Qinghai-Tibet Plateau. However, there is a lack of genomic, transcriptomic, and proteomic information regarding the biology of D. nuttalli . In this study, we assembled and compared the midgut transcriptomes of female D. nuttalli ticks at 0, 24, 48, 72, and 96 h during blood feeding, identifying the genes with differentially regulated expression following feeding. The obtained data were compiled and annotated in multiple databases including Nr, NT, PFAM, KOG, KEGG, and GO. The high-quality clean readings of midgut tissue at the different blood-feeding times were recorded as 22,524,912, 23,752,325, 20,377,718, 21,300,710, and 20,378,658, respectively. The transcripts were classified into eight large categories, including immunogenic proteases (8.37%), protease inhibitors (0.85%), transporters (3.96%), ligand binding proteins (1.98%), ribosomal function proteins (0.94%), heat shock proteins (0.30%), other proteases and miscellaneous proteins (57.61%), and unknown proteins (26.00%). Significant differences were observed in the genes obtained at 0, 24, 48, 72, and 96 h during blood feeding. The differentially expressed genes include catalytic proteins that play an important role in accelerating biochemical reactions, binding activity proteins which are involved in various molecular interactions, and proteins that actively participate in multiple metabolic pathways and cellular processes. Notably, the gene expression in the midgut of D. nuttalli shows dynamic changes every 24 h throughout the blood-feeding process. This change may represent an equivalent strategy of antigenic variation for ticks, designed to protect their essential feeding function against the host’s immune system. The tick antigens identified in this study may serve as promising candidates for the development of effective vaccines or as drug targets for acaricides.

This study aimed to clone and express chitinase genes from Bacillus proteolyticus strains and characterize the enzymatic properties of recombinant enzymes. Bacillus proteolyticus was isolated from the body of Dermacentor nuttalli and renamed IMH/B-1. Chitin-degrading enzymes were screened via clear zone assay and PCR. The chitinase A gene ( ChiA ) was successfully cloned, and the recombinant plasmid pET28a-rChiA-DP (Dermatestor-derived Protein) was constructed. Recombinant chitinase protein (rChiA-DP) was expressed in Escherichia coli BL21 using IPTG induction and purified by nickel-nitrilotriacetic acid (Ni–NTA) affinity chromatography. Bioinformatic tools were used to predict the rChiA-DP protein sequences, analyse its enzyme family classification, and identify key amino acid residues in its catalytic domain. The enzymatic activity of rChiA-DP, along with its nematode resistance and antifungal effects on Caenorhabditis elegans ( C. elegans ) and fungi ( Aspergillus sp.), was assessed under varied temperatures, pH, metal ions, salt concentrations and substrates. The amino acid sequence of the rChiA-DP contains a chitin-binding domain (CBD) (substrate binding), a fibronectin type III domain (FN3)(structural stability), and a catalytic domain with a typical TIM-barrel molecular structure (catalytic scaffold). SDS-PAGE analysis revealed that the molecular weight of the rChiA-DP was approximately 74.6 kDa, which is consistent with the theoretical predictions. The optimal conditions for rChiA-DP enzyme activity were 40 °C and pH 7.0. Enzyme activity was significantly enhanced by 10 mM Ba 2+ , Tris, K + , and Li + . Organic solvents such as methanol, ethanol, isopropanol, and isoamyl alcohol (10% concentration) also increased the activity. Conversely, positive metal ions such as Cu 2+ , Ni 2+ , Fe 3+ , Zn 2+ and Mn 2+ as well as SDS, DMSO, Tween 20/80 significantly inhibited the activity of the rChiA-DP. rChiA-DPs demonstrated varying degrees of decomposition activity against substrates such as colloidal chitin, chitin powder, nematode eggs, nematodes, shrimp shells, and tick eggs, with the highest activity observed for colloidal chitin (7.53 ± 0.86 U/mL). However, it exhibited no degradation activity on chitosan and tick surface. Compared with the heat-inactivated control group‌ and the s-buffer group, the rChiA-DP treatment significantly reduced the survival rate of C. elegans by 50.4% vs. heat-inactivated control ( P  < 0.01), indicating a potential antiparasitic effect. However, it showed no significant antifungal activity against fungi such as Aspergillus niger or Aspergillus flavus, and the diameter of the inhibition zone was not significantly larger than that of the negative control ( P  > 0.05). This study successfully prepared tick-derived rChiA-DPs and evaluated their enzymatic activity and anti-nematodal activity, providing enzymatic basis for the design of biopesticides targeting insect cuticle.

Background Hard ticks act as arthropod vectors in the transmission of human and animal pathogens and are widely distributed in northern China. The aim of this study is to screen the important tick-borne pathogens (TBPs) carried by hard ticks in Inner Mongolia using metagenomic next-generation sequencing (mNGS) and to estimate the risk of human infection imposed by tick bites. Methods The adult Dermacentor nuttalli ( n  = 203) and Ixodes persulcatus ( n  = 36) ticks feeding on cattle were collected. The pooled DNA samples prepared from these ticks were sequenced as the templates for mNGS to survey the presence of TBPs at the genus level. Individual tick DNA samples were detected by genus--specific or group-specific nested polymerase chain reaction (PCR) of these TBPs and combined with DNA sequencing assay to confirm the results of mNGS. Results R. raoultii (45.32%, 92/203), Candidatus R. tarasevichiae (5.42%, 11/203), Anaplasma sp. Mongolia (26.60%, 54/203), Coxiella- like endosymbiont (CLE) (53.69%, 109/203), and Babesia venatorum (7.88%, 16/203) were detected in D. nuttalli , while R. raoultii (30.56%, 11/36), Anaplasma sp. Mongolia (27.80%, 10/36), and CLE (27.80%, 10/36) were detected in I. persulcatus . The double- and triple-pathogen/endosymbiont co-infections were detected in 40.39% of D. nuttalli and 13.89% of I. persulcatus , respectively. The dual co-infection with R. raoultii and CLE (14.29%, 29/203) and triple co-infection with R. raoultii , Anaplasma sp. Mongolia, and CLE (13.79%, 28/203) were most frequent in D. nuttalli . Conclusions This study provides insight into the microbial diversity of D. nuttalli and I. persulcatus in Inner Mongolia, China, reporting for the first time that Candidatus R. tarasevichiae had been found in D. nuttalli in China, and for the first time in the world that Anaplasma sp. Mongolia has been detected in I. persulcatus . This study proves that various vertically transmitted pathogens co-inhabit D. nuttalli and I. persulcatus , and indicates that cattle in Inner Mongolia are exposed to several TBPs. Graphical Abstract

Background Ticks are known to transmit a wide range of diseases, including those caused by bacteria, viruses, and protozoa. The expansion of tick habitats has been intensified in recent years due to various factors such as global warming, alterations in microclimate, and human activities. Consequently, the probability of human exposure to diseases transmitted by ticks has increased, leading to a higher degree of risk associated with such diseases. Methods In this study, we conducted a comprehensive review of domestic and international literature databases to determine the current distribution of tick species in Inner Mongolia. Next, we employed the MaxEnt model to analyze vital climatic and environmental factors influencing dominant tick distribution. Subsequently, we predicted the potential suitability areas of these dominant tick species under the near current conditions and the BCC-CSM2.MR model SSP245 scenario for the future periods of 2021–2040, 2041–2060, 2061–2080, and 2081–2100. Results Our study revealed the presence of 23 tick species from six genera in Inner Mongolia, including four dominant tick species ( Dermacentor nuttalli , Ixodes persulcatus , Dermacentor silvarum , and Hyalomma asiaticum ). Dermacentor nuttalli , D. silvarum , and I. persulcatus are predominantly found in regions such as Xilin Gol and Hulunbuir. Temperature seasonality (Bio4), elevation (elev), and precipitation seasonality (Bio15) were the primary variables impacting the distribution of three tick species. In contrast, H. asiaticum is mainly distributed in Alxa and Bayannur and demonstrates heightened sensitivity to precipitation and other climatic factors. Our modeling results suggested that the potential suitability areas of these tick species would experience fluctuations over the four future periods (2021–2040, 2041–2060, 2061–2080, and 2081–2100). Specifically, by 2081–2100, the centroid of suitable habitat for D. nuttalli , H. asiaticum , and I. persulcatus was predicted to shift westward, with new suitability areas emerging in regions such as Chifeng and Xilin Gol. The centroid of suitable habitat for H. asiaticum will move northeastward, and new suitability areas are likely to appear in areas such as Ordos and Bayannur. Conclusions This study provided a comprehensive overview of the tick species distribution patterns in Inner Mongolia. Our research has revealed a significant diversity of tick species in the region, exhibiting a wide distribution but with notable regional disparities. Our modeling results suggested that the dominant tick species’ suitable habitats will significantly expand in the future compared to their existing distribution under the near current conditions. Temperature and precipitation are the primary variables influencing these shifts in distribution. These findings can provide a valuable reference for future research on tick distribution and the surveillance of tick-borne diseases in the region. Graphical Abstract

Background Ticks are vectors of numerous pathogens, with their bacterial composition, abundance, diversity, and interaction influencing both their growth and disease transmission efficiency. Despite the abundance of ticks in Inner Mongolia, China, comprehensive data on their microbial communities are lacking. This study aims to analyze the microbial communities within ticks from Inner Mongolia to inform innovative control strategies for interrupting pathogen transmission. Methods Tick samples were collected from animals and vegetation in multiple locations across Inner Mongolia and stored at − 80 °C. Ticks were identified using morphological keys and molecular biology methods. Full-length 16S rRNA gene sequencing was performed on collected samples. Bacterial community composition and diversity were mainly analyzed using bioinformatic tools such as QIIME, phyloseq, and DESeq2. Alpha diversity was assessed using Chao1, ACE, and Shannon indices, while beta diversity was evaluated using Bray-Curtis dissimilarity matrices. LEfSe analysis was applied to identify taxa associated with ecological and biological variables. Results A total of 5,048,137 high-quality read counts were obtained, forming an average of 789.3 OTUs per sample. Proteobacteria, Firmicutes, and Bacteroidetes were the most dominant phyla. Bacterial community composition varied significantly with geography, with Dermacentor nuttalli showing a higher abundance of Rickettsia in Xilingol League, while other regions had different dominant genera. The microbial community also differed based on the feeding status of ticks. Additionally, the microbiota of engorged ticks showed organ specificity. Pathogen detection efforts revealed the presence of nine pathogens across all three tick species. D. nuttalli was found to carry a significantly higher burden of pathogenic bacteria, making it the most potentially threatening tick species in Inner Mongolia. Conclusions The study highlights significant variations in tick microbiomes influenced by geographic location, feeding status, and tick species. It underscores the importance of enhancing tick and tick-borne disease surveillance in Inner Mongolia for early detection and control of emerging pathogens. Graphical Abstract

Background Bacteria of the order Rickettsiales ( Alphaproteobacteria ) are obligate intracellular parasites that infect species from virtually every major eukaryotic lineage. Several rickettsial genera harbor species that are significant emerging and re-emerging pathogens of humans. As species of Rickettsiales are associated with an extremely diverse host range, a better understanding of the historical associations between these bacteria and their hosts will provide important information on their evolutionary trajectories and, particularly, their potential emergence as pathogens. Results Nine species of Rickettsiales (two in the genus Rickettsia , three in the genus Anaplasma , and four in the genus Ehrlichia ) were identified in two species of hard ticks ( Dermacentor nuttalli and Hyalomma asiaticum ) from two geographic regions in Xinjiang through genetic analyses of 16S rRNA, gltA , and groEL gene sequences. Notably, two lineages of Ehrlichia and one lineage of Anaplasma were distinct from any known Rickettsiales, suggesting the presence of potentially novel species in ticks in Xinjiang. Our phylogenetic analyses revealed some topological differences between the phylogenies of the bacteria and their vectors, which led us to marginally reject a model of exclusive bacteria-vector co-divergence. Conclusions Ticks are an important natural reservoir of many diverse species of Rickettsiales. In this work, we identified a single tick species that harbors multiple species of Rickettsiales, and uncovered extensive genetic diversity of these bacteria in two tick species from Xinjiang. Both bacteria-vector co-divergence and cross-species transmission appear to have played important roles in Rickettsiales evolution.

Background Ticks (Arthropoda, Ixodida), after mosquitoes, are the second most prevalent vector of infectious diseases. They are responsible for spreading a multitude of pathogens and threatening the health and welfare of animals and human beings. However, given the history of tick-borne pathogen infections in the Inner Mongolia Autonomous Region of China, surprisingly, neither the genetic diversity nor the spatial distribution of haplotypes within ticks has been studied. Methods We characterized the haplotype distribution of Dermacentor nuttalli in four main pastoral areas of the Inner Mongolia Autonomous Region, by sampling 109 individuals (recovered from sheep) in April–August 2019. The 16S rRNA gene, cytochrome c oxidase subunit I (COI), and the internal transcribed spacer 2 region (ITS2) were amplified and sequenced from extracted DNA. Results Twenty-six haplotypes were identified using 16S rRNA sequences, 57 haplotypes were identified with COI sequences, and 75 haplotypes were identified with ITS2 sequences. Among the three genes, total haplotype diversity was greater than 0.7, while total nucleotide diversity was greater than 0.06. Neutrality tests revealed a significantly negative Tajima’s D result, while Fu's Fs was not significantly positive. Fixation index values (F ST ) indicated that the degree of genetic differentiation among some sampled populations was small, while for others it was moderate. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that among populations. The mismatch analysis of D. nuttalli exhibited double peaks. Conclusion The genetic diversity of D. nuttalli populations in our region can likely adapt to different geographical environments, thereby leading to genetic diversity, and creating genetic differentiation among different populations. However, genetic differentiation is cryptic and does not form a pedigree geographical structure. Graphical Abstract

Background The genus Rickettsia contains the lineages spotted fever group (SFG), typhus group (TG), and transitional group (TRG). The spotted fever group Rickettsia (SFGR) is transmitted by ticks. The tick species Dermacentor nuttalli is considered the main vector carrying SFGR in Inner Mongolia. Studying the genetic diversity and population structure of Rickettsia is essential for developing effective control strategies and predicting evolutionary trends of Rickettsia . Methods In 2019 we collected 408 D. nuttalli in the Inner Mongolia Autonomous Region, detected the percentage of Rickettsia -positive specimens, and characterized the haplotypes. From the Rickettsia -positive ticks, the gltA and ompA genes were extracted, amplified, and sequenced. Results Ten haplotypes of the gltA gene and 22 haplotypes of the ompA gene were obtained. The phylogenetic analysis showed that the haplotypes G1–G7 and G9 of the gltA gene cluster with Rickettsia raoultii , while G8 and G10 cluster with Rickettsia sibirica . Haplotypes O1–O15, O18 and O20–O22 of the ompA gene cluster with R. raoultii , while O16 and O19 cluster with R. sibirica . The average haplotype diversity was 0.3 for gltA and 0.7 for ompA . The average nucleotide diversity was greater than 0.05. Neutrality tests were nonsignificant for Tajima’s D results and Fu’s F s results. The fixation index values ( F ST ) showed that the degree of genetic differentiation between most sampled populations was small ( F ST  < 0.05), whereas some populations showed a medium ( F ST  > 0.05) or large ( F ST  > 0.15) degree of differentiation. Analysis of molecular variance (AMOVA) revealed that the variation within populations was greater than that between populations. The mismatch analysis of Rickettsia showed double peaks. Conclusions We found two Rickettsia spp. ( R. raoultii and R. sibirica ). The high genetic disparity of Rickettsia allows for easy adaption to different environments. Genetic differentiation between populations is small, and Rickettsia populations do not show a geographically differentiated structure. The high rates of retention and infection of Rickettsia in D. nuttalli together with the animal husbandry exchange in Inner Mongolia gradually led to the harmonization of genetic characteristics of Rickettsia across various regions. Overall, the significant genetic diversity and geographical structure of Rickettsia in D. nuttalli are critical for SFGR control. Graphical Abstract