Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

A Statovirus-like virus from respiratory tracts of patients, China.

The emerging evidence of human infections with emerging viruses suggests their potential public health importance. A novel taxon of viruses named Statoviruses (for stool-associated Tombus-like viruses) was recently identified in the gastrointestinal tracts of multiple mammals. Here we report the discovery of respiratory Statovirus-like viruses (provisionally named Restviruses) from the respiratory tracts of five patients experiencing acute respiratory disease with Human coronavirus OC43 infection through the retrospective analysis of meta-transcriptomic data. Restviruses shared 53.1%-98.8% identities of genomic sequences with each other and 39.9%-44.3% identities with Statoviruses. The phylogenetic analysis revealed that Restviruses together with a Stato-like virus from nasal-throat swabs of Vietnamese patients with acute respiratory disease, formed a well-supported clade distinct from the taxon of Statoviruses. However, the consistent genome characteristics of Restviruses and Statoviruses suggested that they might share similar evolutionary trajectories. These findings warrant further studies to elucidate the etiological and epidemiological significance of the emerging Restviruses.

Epidemiological behaviour and interventions of malaria in Niger, 2010-2019: a time-series analysis of national surveillance data.

BACKGROUND: Malaria remains a significant public health concern in Niger, with the number of cases increasing from 592,334 in 2000 to 3,138,696 in 2010. In response, a concerted campaign against the disease has been initiated. However, the implementation of these malaria interventions and their association with epidemiological behaviour remains unclear. METHODS: A time-series study was conducted in Niger from 2010 to 2019. Multiple data sources concerning malaria were integrated, encompassing national surveillance data, Statistic Yearbook, targeted malaria control interventions, and meteorological data. Incidence rate, mortality rate, and case fatality ratio (CFR) by different regions and age groups were analysed. Joinpoint regression models were used to estimate annual changes in malaria. The changes in coverage of malaria interventions were evaluated. RESULTS: Between 2010 to 2019, the incidence rate of malaria decreased from 249.43 to 187.00 cases per 1,000 population in Niger. Niamey had a high annual mean incidence rate and the lowest CFR, while Agadez was on the contrary. Joinpoint regression analysis revealed a declining trend in malaria incidence for all age groups except the 10-24 years group, and the mortality rate and the CFR initially decreased followed by an increase in all age groups. Niger has implemented a series of malaria interventions, with the major ones being scaled up to larger populations during the study period. CONCLUSIONS: The scale-up of multi-interventions in Niger has significantly reduced malaria incidence, but the rise in mortality rate and CFR addresses the challenges in malaria control and elimination. Malaria endemic countries should enhance surveillance of malaria cases and drug resistance in Plasmodium, improve diagnosis and treatment, expand the population coverage of insecticide-treated bed nets and seasonal malaria chemoprevention, and strengthen the management of severe malaria cases.

Genomic Characteristics of Emerging Intraerythrocytic Anaplasma capra and High Prevalence in Goats, China.

Anaplasma capra is an emerging tickborne human pathogen initially recognized in China in 2015; it has been reported in ticks and in a wide range of domestic and wild animals worldwide. We describe whole-genome sequences of 2 A. capra strains from metagenomic sequencing of purified erythrocytes from infected goats in China. The genome of A. capra was the smallest among members of the genus Anaplasma. The genomes of the 2 A. capra strains contained comparable G+C content and numbers of pseudogenes with intraerythrocytic Anaplasma species. The 2 A. capra strains had 54 unique genes. The prevalence of A. capra was high among goats in the 2 endemic areas. Phylogenetic analyses revealed that the A. capra strains detected in this study were basically classified into 2 subclusters with those previously detected in Asia. Our findings clarify details of the genomic characteristics of A. capra and shed light on its genetic diversity.

Continuous evolution and emerging lineage of seasonal human coronaviruses: A multicenter surveillance study.

The seasonal human coronaviruses (HCoVs) have zoonotic origins, repeated infections, and global transmission. The objectives of this study are to elaborate the epidemiological and evolutionary characteristics of HCoVs from patients with acute respiratory illness. We conducted a multicenter surveillance at 36 sentinel hospitals of Beijing Metropolis, China, during 2016-2019. Patients with influenza-like illness (ILI) and severe acute respiratory infection (SARI) were included, and submitted respiratory samples for screening HCoVs by multiplex real-time reverse transcription-polymerase chain reaction assays. All the positive samples were used for metatranscriptomic sequencing to get whole genomes of HCoVs for genetical and evolutionary analyses. Totally, 321 of 15 677 patients with ILI or SARI were found to be positive for HCoVs, with an infection rate of 2.0% (95% confidence interval, 1.8%-2.3%). HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 infections accounted for 18.7%, 38.3%, 40.5%, and 2.5%, respectively. In comparison to ILI cases, SARI cases were significantly older, more likely caused by HCoV-229E and HCoV-OC43, and more often co-infected with other respiratory pathogens. A total of 179 full genome sequences of HCoVs were obtained from 321 positive patients. The phylogenetical analyses revealed that HCoV-229E, HCoV-NL63 and HCoV-OC43 continuously yielded novel lineages, respectively. The nonsynonymous to synonymous ratio of all key genes in each HCoV was less than one, indicating that all four HCoVs were under negative selection pressure. Multiple substitution modes were observed in spike glycoprotein among the four HCoVs. Our findings highlight the importance of enhancing surveillance on HCoVs, and imply that more variants might occur in the future.

Profile and dynamics of infectious diseases: a population-based observational study using multi-source big data.

BACKGROUND: The current surveillance system only focuses on notifiable infectious diseases in China. The arrival of the big-data era provides us a chance to elaborate on the full spectrum of infectious diseases. METHODS: In this population-based observational study, we used multiple health-related data extracted from the Shandong Multi-Center Healthcare Big Data Platform from January 2013 to June 2017 to estimate the incidence density and describe the epidemiological characteristics and dynamics of various infectious diseases in a population of 3,987,573 individuals in Shandong province, China. RESULTS: In total, 106,289 cases of 130 infectious diseases were diagnosed among the population, with an incidence density (ID) of 694.86 per 100,000 person-years. Besides 73,801 cases of 35 notifiable infectious diseases, 32,488 cases of 95 non-notifiable infectious diseases were identified. The overall ID continuously increased from 364.81 per 100,000 person-years in 2013 to 1071.80 per 100,000 person-years in 2017 (χ2 test for trend, P < 0.0001). Urban areas had a significantly higher ID than rural areas, with a relative risk of 1.25 (95% CI 1.23-1.27). Adolescents aged 10-19 years had the highest ID of varicella, women aged 20-39 years had significantly higher IDs of syphilis and trichomoniasis, and people aged ≥ 60 years had significantly higher IDs of zoster and viral conjunctivitis (all P < 0.05). CONCLUSIONS: Infectious diseases remain a substantial public health problem, and non-notifiable diseases should not be neglected. Multi-source-based big data are beneficial to better understand the profile and dynamics of infectious diseases.

Age-dependent Gender Differences in COVID-19 in Mainland China: Comparative Study.

BACKGROUND: The ongoing pandemic of novel coronavirus disease 2019 (COVID-19) is challenging the global public health system. Sex differences in infectious diseases are a common but neglected problem. METHODS: We used the national surveillance database of COVID-19 in mainland China to compare gender differences in attack rate (AR), proportion of severe and critical cases (PSCC), and case fatality rate (CFR) in relation to age, affected province, and onset-to-diagnosis interval. RESULTS: The overall AR was significantly higher in females than in males (63.9 vs 60.5 per 1 million persons; P ˂ .001). In contrast, PSCC and CFR were significantly lower among females (16.9% and 4.0%) than among males (19.5% and 7.2%), with odds ratios of 0.87 and 0.57, respectively (both P ˂ .001). The female-to-male differences were age dependent, and were significant among people aged 50-69 years for AR and in patients aged 30 years or older for both PSCC and CFR (all P ≤ .001). The AR, PSCC, and CFR varied greatly from province to province. However, female-to-male differences in AR, PSCC, and CFR were significant in the epicenter, Hubei province, where 82.2% confirmed cases and 97.4% deaths occurred. After adjusting for age, affected province, and onset-to-diagnosis interval, the female-to-male difference in AR, PSCC, and CFR remained significant in multivariate logistic regression analyses. CONCLUSIONS: We elucidate an age-dependent gender dimorphism for COVID-19, in which females have higher susceptibility but lower severity and fatality. Further epidemiological and biological investigations are required to better understand the sex-specific differences for effective interventions.

An integrated data analysis reveals distribution, hosts, and pathogen diversity of Haemaphysalis concinna.

BACKGROUND: Haemaphysalis concinna, carrying multiple pathogens, has attracted increasing attention because of its expanded geographical range and significant role in disease transmission. This study aimed to identify the potential public health risks posed by H. concinna and H. concinna-associated pathogens. METHODS: A comprehensive database integrating a field survey, literature review, reference book, and relevant websites was developed. The geographical distribution of H. concinna and its associated pathogens was illustrated using ArcGIS. Meta-analysis was performed to estimate the prevalence of H. concinna-associated microbes. Phylogenetic and geographical methods were used to investigate the role of birds in the transmission of H. concinna-associated microbes. The potential global distribution of H. concinna was predicted by ecological niche modeling. RESULTS: Haemaphysalis concinna was distributed in 34 countries across the Eurasian continent, predominantly in China, Russia, and Central Europe. The tick species carried at least 40 human pathogens, including six species in the Anaplasmataceae family, five species of Babesia, four genospecies in the complex Borrelia burgdorferi sensu lato, ten species of spotted fever group rickettsiae, ten species of viruses, as well as Francisella, Coxiella, and other bacteria. Haemaphysalis concinna could parasitize 119 host species, with nearly half of them being birds, which played a crucial role in the long-distance transmission of tick-borne microbes. Our predictive modeling suggested that H. concinna could potentially survive in regions where the tick has never been previously recorded such as central North America, southern South America, southeast Oceania, and southern Africa. CONCLUSIONS: Our study revealed the wide distribution, broad host range, and pathogen diversity of H. concinna. Authorities, healthcare professionals, and the entire community should address the growing threat of H. concinna and associated pathogens. Tick monitoring and control, pathogen identification, diagnostic tools, and continuous research should be enhanced.

Genomic characters of Anaplasma bovis and genetic diversity in China.

The emergence of Anaplasma bovis or A. bovis-like infection in humans from China and the United States of America has raised concern about the public health importance of this pathogen. Although A. bovis has been detected in a wide range of ticks and mammals in the world, no genome of the pathogen is available up to now, which has prohibited us from better understanding the genetic basis for its pathogenicity. Here we describe an A. bovis genome from metagenomic sequencing of an infected goat in China. Anaplasma bovis had the smallest genome of the genus Anaplasma, and relatively lower GC content. Phylogenetic analysis of single-copy orthologue sequence showed that A. bovis was closely related to A. platys and A. phagocytophilum, but relatively far from intraerythrocytic Anaplasma species. Anaplasma bovis had 116 unique orthogroups and lacked 51 orthogroups in comparison to other Anaplasma species. The virulence factors of A. bovis were significantly less than those of A. phagocytophilum, suggesting less pathogenicity of A. bovis. When tested by specific PCR assays, A. bovis was detected in 23 of 29 goats, with an infection rate up to 79.3% (95% CI: 64.6% ∼94.1%). The phylogenetic analyses based on partial 16S rRNA, gltA and groEL genes indicated that A. bovis had high genetic diversity. The findings of this study lay a foundation for further understanding of the biological characteristics and genetic diversity of A. bovis, and will facilitate the formulation of prevention and control strategies.

Genomic characterization of an emerging Rickettsia barbariae isolated from tick eggs in northwestern China.

The continual emergence of tick-borne rickettsioses has garnered widespread global attention. Candidatus Rickettsia barbariae (Candidatus R. barbariae), which emerged in Italy in 2008, has been detected in humans from northwestern China. However, the lack of Candidatus R. barbariae genome and isolated strains limits the understanding of its biological characteristics and genomic features. Here, we isolated the Rickettsia for the first time from eggs of Rhipicephalus turanicus in northwestern China, and assembled its whole genome after next-generation sequencing, so we modified the proposed name to Rickettsia barbariae (R. barbariae) to conform to the International Code of Nomenclature of Prokaryotes. Phylogenetic analysis based on the whole genome revealed that it was most closely related to the pathogenic Rickettsia parkeri and Rickettsia africae. All virulence factors, present in the pathogenic spotted fever group rickettsiae, were identified in the R. barbariae isolate. These findings highlight the pathogenic potential of R. barbariae and the necessity for enhanced surveillance of the emerging Rickettsia in the human population.

Distribution and diversity of ticks determined by environmental factors in Ningxia, China.

Ticks are important vectors of zoonotic pathogens, and represent an increasing threat for human and animal health. Considering the complex natural environments of Ningxia Hui Autonomous Region, China, we expect the diverse tick species in this region. Here, we conduct a field survey on parasitic and host-seeking ticks. A total of 10,419 ticks were collected, which belonged to nine species of four genera. There were significant differences in terms of vegetation index, altitude, and seven climatic factors among the four tick genera -Hyalomma, Dermacentor, Haemaphysalis, and Ixodes, except between Haemaphysalis and Ixodes, where no significant differences were observed in these factors. The ecological niche modelling revealed that the suitable habitats for Hyalomma asiaticum was in the northwest Ningxia, with annual ground surface temperature as the most important factor. The suitable area for Dermacentor nuttalli was in the southwest and eastern regions of Ningxia with elevation as the highest contribution. D. silvarum was best suited to the southern Ningxia also with elevation as the most important factor. The four tick species including Haemaphysalis longicornis, Hae. qinghaiensis, Hae. japonica, and Ixodes persulcatus were best suited to the southernmost Ningxia with annual precipitation as the main factors for Hae. longicornis and elevation for the other three ticks. The results of predicted potential distribution of different tick species provide a scientific basis for the prevention and control of ticks and tick-borne diseases in the region. Furthermore, the subsequent impacts of the Greening Program to regain forests and grasslands from former agricultural lands in Ningxia on tick population dynamics deserve further investigation.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

Virome diversity shaped by genetic evolution and ecological landscape of Haemaphysalis longicornis.

BACKGROUND: Haemaphysalis longicornis is drawing attentions for its geographic invasion, extending population, and emerging disease threat. However, there are still substantial gaps in our knowledge of viral composition in relation to genetic diversity of H. longicornis and ecological factors, which are important for us to understand interactions between virus and vector, as well as between vector and ecological elements. RESULTS: We conducted the meta-transcriptomic sequencing of 136 pools of H. longicornis and identified 508 RNA viruses of 48 viral species, 22 of which have never been reported. Phylogenetic analysis of mitochondrion sequences divided the ticks into two genetic clades, each of which was geographically clustered and significantly associated with ecological factors, including altitude, precipitation, and normalized difference vegetation index. The two clades showed significant difference in virome diversity and shared about one fifth number of viral species that might have evolved to "generalists." Notably, Bandavirus dabieense, the pathogen of severe fever with thrombocytopenia syndrome was only detected in ticks of clade 1, and half number of clade 2-specific viruses were aquatic-animal-associated. CONCLUSIONS: These findings highlight that the virome diversity is shaped by internal genetic evolution and external ecological landscape of H. longicornis and provide the new foundation for promoting the studies on virus-vector-ecology interaction and eventually for evaluating the risk of H. longicornis for transmitting the viruses to humans and animals. Video Abstract.

Epidemiological and phylogenetic characteristics of emerging Anaplasma capra: A systematic review with modeling analysis.

Anaplasma capra, an emerging tick-borne pathogen, has caused a lot of concern since initially recognized in goats and patients in China in 2015, and has been reported in a wide range of domestic and wild animals as well as ticks worldwide, posing a threat to public health. In this systematic review, we established a comprehensive database to acquire the distribution and prevalence status of this pathogen, and collected all sequences of A. capra to summarize the details of genetic diversity by phylogenetic analysis. We also predicted the possible global distribution of A. capra by using ecological niche model. A. capra has been known to distribute in 18 countries across Asia, Europe and Africa. A total of 19 species of mammals from seven families have been reported as hosts, and domestic ruminants including goats, sheep and cattle were the major hosts. At least 8 tick species of 4 genera have been reported to carry A. capra, and Haemaphysalis longicornis was most commonly infected. Sheep and Rhipicephalus microplus had the highest positive rates among animals and ticks. Phylogenetic analysis based on gltA and groEL genes revealed that A. capra could primarily be divided into two clusters related to geographic location and animal hosts. The predictive model showed that the most suitable habitats for presence of A. capra were mainly located in Asia and eastern Europe. These cumulative data regarding A. capra of our study lay a foundation for the subsequent exploration of this emerging tick-borne pathogen.

Systematic review and integrated data analysis reveal diverse pangolin-associated microbes with infection potential.

There has been increasing global concern about the spillover transmission of pangolin-associated microbes. To assess the risk of these microbes for emergence as human pathogens, we integrated data from multiple sources to describe the distribution and spectrum of microbes harbored by pangolins. Wild and trafficked pangolins have been mainly recorded in Asia and Africa, while captive pangolins have been reported in European and North American countries. A total of 128 microbes, including 92 viruses, 25 bacteria, eight protists, and three uncharacterized microbes, have been identified in five pangolin species. Out of 128 pangolin-associated microbes, 31 (including 13 viruses, 15 bacteria, and three protists) have been reported in humans, and 54 are animal-associated viruses. The phylogenetic analysis of human-associated viruses carried by pangolins reveals that they are genetically close to those naturally circulating among human populations in the world. Pangolins harbor diverse microbes, many of which have been previously reported in humans and animals. Abundant viruses initially detected in pangolins might exhibit risks for spillover transmission.

Genomic Characterization of Theileria luwenshuni Strain Cheeloo.

Theileria, a tick-borne intracellular protozoan, can cause infections of various livestock and wildlife around the world, posing a threat to veterinary health. Although more and more Theileria species have been identified, genomes have been available only from four Theileria species to date. Here, we assembled a whole genome of Theileria luwenshuni, an emerging Theileria, through next-generation sequencing of purified erythrocytes from the blood of a naturally infected goat. We designated it T. luwenshuni str. Cheeloo because its genome was assembled by the researchers at Cheeloo College of Medicine, Shandong University, China. The genome of T. lunwenshuni str. Cheeloo was the smallest in comparison with the other four Theileria species. T. luwenshuni str. Cheeloo possessed the fewest gene gains and gene family expansion. The protein count of each category was always comparable between T. luwenshuni str. Cheeloo and T. orientalis str. Shintoku in the Eukaryote Orthologs annotation, though there were remarkable differences in genome size. T. luwenshuni str. Cheeloo had lower counts than the other four Theileria species in most categories at level 3 of Gene Ontology annotation. Kyoto Encyclopedia of Genes and Genomes annotation revealed a loss of the c-Myb in T. luwenshuni str. Cheeloo. The infection rate of T. luwenshuni str. Cheeloo was up to 81.5% in a total of 54 goats from three flocks. The phylogenetic analyses based on both 18S rRNA and cox1 genes indicated that T. luwenshuni had relatively low diversity. The first characterization of the T. luwenshuni genome will promote better understanding of the emerging Theileria. IMPORTANCE Theileria has led to substantial economic losses in animal husbandry. Whole-genome sequencing data of the genus Theileria are currently limited, which has prohibited us from further understanding their molecular features. This work depicted whole-genome sequences of T. luwenshuni str. Cheeloo, an emerging Theileria species, and reported a high prevalence of T. luwenshuni str. Cheeloo infection in goats. The first assembly and characterization of T. luwenshuni genome will benefit exploring the infective and pathogenic mechanisms of the emerging Theileria to provide scientific basis for future control strategies of theileriosis.

Metavirome of 31 tick species provides a compendium of 1,801 RNA virus genomes.

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses.

Coinfection of Two Rickettsia Species in a Single Tick Species Provides New Insight into Rickettsia-Rickettsia and Rickettsia-Vector Interactions.

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses. There is an ongoing debate as to whether established infections by one Rickettsia species preclude the maintenance of the second species in ticks. Here, we identified two Rickettsia species in inoculum from Haemaphysalis montgomeryi ticks and subsequently obtained pure isolates of each species by plaque selection. The two isolates were classified as a transitional group and spotted fever group rickettsiae and named Rickettsia hoogstraalii str CS and Rickettsia rhipicephalii str EH, respectively. The coinfection of these two Rickettsia species was detected in 25.6% of individual field-collected H. montgomeryi. In cell culture infection models, R. hoogstraalii str CS overwhelmed R. rhipicephalii str EH with more obvious cytopathic effects, faster plaque formation, and increased cellular growth when cocultured, and R. hoogstraalii str CS seemed to polymerize actin tails differently from R. rhipicephalii str EH in vitro. This work provides a model to investigate the mechanisms of both Rickettsia-Rickettsia and Rickettsia-vector interactions. IMPORTANCE The rickettsiae are a group of obligate intracellular Gram-negative bacteria that include human pathogens causing an array of clinical symptoms and even death. There is an important question in the field, that is whether one infection can block the superinfection of other rickettsiae. This work demonstrated the coinfection of two Rickettsia species in individual ticks and further highlighted that testing the rickettsial competitive exclusion hypothesis will undoubtedly be a promising area as methods for bioengineering and pathogen biocontrol become amenable for rickettsiae.