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12 December 2020 Discovery and Surveillance of Tick-Borne Pathogens
Rafal Tokarz, W. Ian Lipkin
Author Affiliations +

Within the past 30 yr molecular assays have largely supplanted classical methods for detection of tick-borne agents. Enhancements provided by molecular assays, including speed, throughput, sensitivity, and specificity, have resulted in a rapid increase in the number of newly characterized tick-borne agents.The use of unbiased high throughput sequencing has enabled the prompt identification of new pathogens and the examination of tick microbiomes.These efforts have led to the identification of hundreds of new tick-borne agents in the last decade alone. However, little is currently known about the majority of these agents beyond their phylogenetic classification. Our article outlines the primary methods involved in tick-borne agent discovery and the current status of our understanding of tick-borne agent diversity.

Ticks are vectors of the greatest diversity of vertebrate pathogens worldwide, in large part due to their hematophagous ectoparasitic lifestyle and promiscuity in host selection (Jongejan and Uilenberg 2004, McCoy et al. 2013, Estrada-Peña and De La Fuente 2014). Since the discovery of the first tick-borne agent in the late 19th century, over 20 bacterial, viral, and protozoan human tick-borne pathogens have been identified in the United States alone (Eisen et al. 2017, Smith and Kilborne 1893, Center for Disease Control and Prevention Diseases 2019). The discovery of most of these agents was correlated with the rapid tick expansion for several important human-biting tick species that occurred during the last century as well as advances in detection methods that were aimed at identification of new agents (Telford et al. 1997, McMullan et al. 2012, Kosoy et al. 2015, Monzón et al. 2016, Sonenshine 2018, Eisen and Paddock 2020). Within the past 30 yr, the implementation of molecular assays for tick-borne pathogen surveillance and discovery have had a major impact on our understanding of the diversity of tick-borne agents and their contribution to human disease. In particular, the employment of next-generation sequencing (NGS) has identified a wide range of novel tick-borne pathogens and facilitated studies of the tick microbiome that resulted in the discovery and characterization of hundreds of new tick-associated microbes (Tokarz et al. 2014b, Li et al. 2015a, Shi et al. 2016a, Bouquet et al. 2017, Pettersson et al. 2017, Harvey et al. 2018, Tokarz et al. 2018b, Sameroff et al. 2019). In addition to enhancing our knowledge of tickborne microbial diversity, NGS studies have provided invaluable insight into evolutionary relationships among tick-associated agents, identified new targets for diagnostic assays, and provided new approaches to understanding tick-borne diseases (TBDs) (McMullan et al. 2012, Li et al. 2015a, Tokarz et al. 2019). Our article will illustrate the remarkable improvements in the identification of tick-borne agents achieved with molecular assays and outline the current state of our understanding of tick-borne agent diversity.

Early Strategies for Tick-Borne Agent Discovery

Prior to the advent of molecular biology, tick-borne agents were historically identified through microscopy, isolation in culture or serology (Cowdry 1925, Burgdorfer et al. 1982, Benach et al. 1983, La Scola and Raoult 1997, Raoult and Roux 1997, Mans et al. 2015). Among the initial tick-borne agents identified were the apicomplexan parasites Babesia and Theileria that were visualized in blood smears from infected cattle (Mans et al. 2015, Smith and Kilborne 1893). Studies of Howard Ricketts were integral in recognizing tick transmission of the causative agent of Rocky Mountain Spotted Fever (RMSF), subsequently named Rickettsia rickettsii (Ricketts 1906, 1991; Solheim 1949; Parola et al. 2005). Serologic methods such as complement fixation and immunofluorescence assays (IFA) supplemented microscopy studies while transmission of the presumed etiologic agents between vector ticks and naïve vertebrate hosts were used to fulfill proof of causation (Solheim 1949, Raoult and Roux 1997, Mans et al. 2015). By mid 20th century, advances in tissue culture methods facilitated the isolation of many of the most common tick-borne pathogens and enabled the study of tick-borne viruses. Multiple combined approaches were often used for identification of novel agents. Large scale global programs aimed at the discovery of new viruses led to the isolation of a wide range of tick-borne viruses, some of which were subsequently characterized through serology and electron microscopy (Downs 1982). Borrelia burgdorferi sensu stricto (s.s.) was identified as the tick-borne agent of Lyme borreliosis by visualizing spirochetes in blood from patients and vector ticks and demonstrating serologic reactivity to cultured spirochetes with sera from Lyme borreliosis patients (Burgdorfer et al. 1982, Benach et al. 1983).

Although these approaches were successful in the identification of the agents responsible for most well-known TBDs, the discovery of new tick-borne agents was often compromised by limitations inherent to non-molecular methods. For some agents, isolation in culture proved challenging (Anda et al. 1996, Hue et al. 2013, Tokarz et al. 2014b). This limitation particularly applied to the constituents of the tick virome, where very little was learned about viral diversity for even the most clinically relevant tick species. Until recently, only a single virus was isolated from Ixodes scapularis Say, whereas we now know this tick has a very diverse virome (Telford et al. 1997, Tokarz et al. 2014b, Tokarz et al. 2018b). In addition, tick-borne viruses that were isolated in viral surveillance studies were often not characterized further (Palacios et al. 2013, Walker et al. 2015). Cross-reactivity of serologic assays was often a problem for accurate agent identification. This was especially evident in work with Rickettsia, where nonpathogenic bacterial endosymbionts could be mistaken for closely related pathogenic species, and serologic cross-reactivity among spotted fever Rickettsia likely impeded the identification of new distinct species (Parola et al. 2005).

Although these classical methods are still in use today, by the early to mid-1990s, polymerase chain reaction (PCR) mostly supplanted them as the primary tool for detection of tick-borne agents. The advantages of PCR, including low cost, speed and high throughput, made it a more effective tool for tick-borne pathogen surveillance. Dideoxy sequencing of PCR products provided genomic sequence data that could be used to more clearly differentiate microbial species and improve taxonomic classification. Over time, the utility of PCR was further enhanced by additional improvements in assay design, experimental methods. Although initial tick-borne pathogen surveillance studies often utilized tick pools for PCR analysis, advances in nucleic acid extraction facilitated the study of individual ticks (Johnson et al. 1992, Steiner et al. 1999). Laborious manual nucleic acid extractions have been increasingly supplanted by automated extraction platforms that can rapidly provide nucleic acid template material from ≥24 individual tick samples (Exner and Lewinski 2003, Tokarz et al. 2010, Tokarz et al. 2018b, Sameroff et al. 2019). The implementation of multiplex PCR assays that utilize primer pairs targeting >1 agent further reduced the cost and speed of testing (Tokarz et al. 2010, Hojgaard et al. 2014, Tokarz et al. 2017, Reller and Dumler 2018, Buchan et al. 2019, Sanchez-Vicente et al. 2019). As a result of these modifications, tick-borne pathogen surveillance studies now often report the testing of hundreds to thousands of tick samples (Tokarz et al. 2010, Aliota et al. 2014, Prusinski et al. 2014, Edwards et al. 2015, Hutchinson et al. 2015, Johnson et al. 2018, Sanchez-Vicente et al. 2019). Numerous surveillance studies have been reported from different countries and continents (Chalada et al. 2018, Cull et al. 2018, Guo et al. 2019). In the United States, the majority of surveillance work has focused on I. scapularis due to its association with Borrelia burgdorferi s.s. as well as other human pathogens. These studies established B. burgdorferi as the most prevalent tick-borne pathogen, with infection rates of 15 to 25% in nymphs and 40–70% in adult ticks (Tokarz et al. 2010, Aliota et al. 2014, Prusinski et al. 2014, Tokarz et al. 2017). Other agents transmitted by I. scapularis, such as Anaplasma phagocytophilum and Babesia microti, are usually detected in up 20% of nymphs, while <5% are infected with B. miyamotoi and Powassan virus. Tick surveillance studies also revealed that approximately up to 5% of nymphs and up to 15% of adult ticks simultaneously carry multiple pathogens (Tokarz et al. 2010, Aliota et al. 2014, Prusinski et al. 2014, Tokarz et al. 2017, Sanchez-Vicente et al. 2019). These findings correlate with serologic data from patients with TBDs, where probable co-infections have been documented going back several decades (Filstein et al. 1980, Benach and Habicht 1981, Burgdorfer et al. 1982, Krause et al. 1996, Hilton et al. 1999, Horowitz et al. 2013, Curcio et al. 2016, Tokarz et al. 2018a).

PCR as a Tool for Tick-Borne Agent Discovery

Although PCR is typically employed to target and amplify a single nucleic acid sequence, modifications in primer design have enabled the use of PCR as a discovery tool. Instead of targeting a single sequence, alignments of multiple sequences from different agents are used to identify conserved genomic loci within a larger taxonomic group such as a genus or family (Tokarz et al. 2010, Toledo et al. 2010, Tadin et al. 2016, Sanchez-Vicente et al. 2019). PCR primers are then designed within the most conserved regions with the assumption that they will recognize multiple (or all) members within the targeted taxonomic unit. Where necessary, degeneracies can be incorporated into these primer sequences to include divergent sequences. Once amplified, the PCR product is typically sequenced by standard dideoxy sequencing and the agent is classified through homology searches in GenBank (Fukunaga et al. 1995, Pancholi et al. 1995, Tokarz et al. 2010, Sanchez-Vicente et al. 2019). New agents can also be uncovered by examination of melting temperature of PCR products that would indicate the presence of novel sequences, an approach that led to the discovery of novel pathogenic species of Ehrlichia and Borrelia in patients with TBDs (Bell and Patel 2005; Pritt et al. 2011, 2016).

The introduction of PCR has had a profound impact on the identification of tick-borne agents. Many of the previously isolated but uncharacterized tick-borne viruses could now be identified, while newly emerging pathogenic viruses were rapidly characterized (Telford et al. 1997, Honig et al. 2004). Phylogenetic analyses of genomic sequences obtained with PCR led to the reclassification of many known tick-borne agents. One prominent example is the bacterial agent of human granulocytic anaplasmosis that initially was identified by microscopy and assigned as a species of Ehrlichia. Through molecular analysis of multiple genetic loci, this agent was shown to be distinct from Ehrlichia and was eventually placed within another genus with a new name, Anaplasma phagocytophilum (Chen et al. 1994, Dumler et al. 2001). PCR was frequently employed to examine the complex etiology of rickettsiosis and to differentiate highly similar species that could not be resolved through serology (Shapiro et al. 2010). It also was used to determine the presence of Rickettsia in ticks, especially R. rickettsii, a species historically presumed to be the primary cause of spotted fever disease in the United States. These studies occasionally failed to detect R. rickettsii in areas endemic for spotted fever but detected other species of Rickettsia that have been linked with human disease, including R. ambylommatis and R. montanensis (Apperson et al. 2008, Mcquiston et al. 2012, Wood et al. 2016, Sanchez-Vicente et al. 2019). These results led to the hypothesis that these other species of Rickettsia may be responsible for a portion of spotted fever cases typically attributed to R. rickettsii (Stromdahl et al. 2011, Gaines et al. 2014). In addition, PCR detection of pathogens in field-collected ticks has been used to identify tick species potentially serving as vectors of TBD (Pancholi et al. 1995; Savage et al. 2013, 2017)

A limitation of PCR assays is that only closely related agents can typically be identified with this approach. Historically, PCR assays aimed at agent discovery were focused on a few microbial genera or families that included known vertebrate pathogens and this precluded the discovery of agents with divergent genomes. In addition, by targeting highly conserved genetic loci, PCR can provide limited genomic information and subsequent analyses could be insufficient to determine actual taxonomic relationships (La Scola et al. 2003).

Next-Generation Sequencing

Unlike standard dideoxy sequencing, which generates a single DNA sequence, NGS generates thousands to hundreds of millions of sequences, or reads, that are present in a sample (Liu et al. 2012, Goodwin et al. 2016). As a result, NGS sequencing generates large datasets that require a bioinformatic ‘pipeline’ for quality control of sequencing reads, subtraction of host reads, contig assembly, and homology searches that assign taxonomic identification to reads and contigs (Chevreux et al. 1999, Andrews and Bittencourt a 2010, Schmieder and Edwards 2011, Langmead and Salzberg 2012, Scholz et al. 2012, Li et al. 2015b). Often, microbial reads represent only a fraction of the total reads, and in the case of novel viral agents, these can have minimal homology to known sequences (Tokarz et al. 2014b, Li et al. 2015a, Tokarz et al. 2018b).

Two methods are used for metagenomic examination by NGS: amplicon sequencing and shotgun sequencing (Goodwin et al. 2016, Greay et al. 2018). Amplicon sequencing is a highly targeted approach that uses a consensus primer pair to amplify a specific, highly conserved region of a single gene that is further characterized through NGS. Because of its high sequence conservation in bacteria, the 16S rRNA has been the optimal target gene used for amplicon sequencing. The 16S rRNA gene consists of multiple overlapping constant and hypervariable regions. Primers designed to bind to constant regions are used to amplify all 16S rRNA sequences present in a sample. The diversity in the sample is determined by aligning the 16S rRNA PCR product to datasets comprising known rRNA sequences (Wu et al. 2008, Schloss et al. 2009, Caporaso et al. 2010, Quast et al. 2013, Cole et al. 2014).

The first metagenomic NGS study of tick-borne agents was published in 2011 (Andreotti et al. 2011). Numerous studies have been performed thereafter on a wide range of tick species worldwide (Carpi et al. 2011, Ponnusamy et al. 2014, Gofton et al. 2015, Rynkiewicz et al. 2015, van Treuren et al. 2015, Trout Fryxell and DeBruyn 2016, Gurfield et al. 2017). Although the 16S rRNA gene is an effective tool for the assessment of bacterial diversity, it does not detect viral or eukaryotic agents. In addition, 16S rRNA primers can be biased for one taxonomic group over another, whereas in other cases, because of the high sequence conservation, phylogenetic resolution may not be sufficient for discrete taxonomic assignments (Kim et al. 2011, Bonk et al. 2018).

An alternative approach, shotgun sequencing, uses a randomized set of short oligonucleotides for priming and amplification of all genomic material present in a sample (Bouquet et al. 2017, Greay et al. 2018, Tokarz et al. 2019). The primary advantage of this approach is that it enables the detection and identification of all bacterial, eukaryotic, and viral agents present and facilitates the assembly of complete genomes. This results in a more comprehensive characterization of all the agents present and improved taxonomic resolution. A disadvantage is that it tends to be more laborious and expensive, primarily due to the additional sample preparation required to enrich for microbial template through reduction of host nucleic acids (Lim et al. 2014, Tokarz et al. 2014b, Carpi et al. 2015, Sameroff et al. 2019). Shotgun sequencing is also less sensitive than 16S rRNA PCR. In addition, the datasets generated by shotgun sequencing are much larger and more complex leading to increased computational needs for the bioinformatic analyses.

Molecular Assay Confounds

Accurate NGS assessment of the tick microbiome can be heavily dependent on experimental factors (Narasimhan and Fikrig 2015). The surface of ticks can harbor a broad range of transiently acquired microbes that are likely not part of their internal microbiome. Unless removed prior to nucleic acid extraction, sequences of these agents will be amplified, and can erroneously be included as a portion of the microbiome (Binetruy et al. 2019). Reports of metagenomic analyses of ticks often include bacteria that likely represent environmental contaminants. Protocols for removal of environmental contaminants may include multiple washes of ticks with alcohol, hydrogen peroxide, or bleach. Of these, bleach treatment has been reported to have the best results (Hoffmann et al. 2020). Other confounds may include the presence of sequences that originate from reagent or laboratory contaminants (Salter et al. 2014). Occasionally, entire viral genomes have been recovered by NGS from clinical specimens, only to determine that they originated from laboratory reagents (Xu et al. 2013, Asplund et al. 2019). Bacterial sequences, and in particular ribosomal sequences, often can be found in water, enzymes, and buffers used for amplification (Salter et al. 2014, Gruber 2015). Ultraviolet irradiation of plasticware and enzymatic digestion of buffers may be helpful in minimizing the presence of nucleic acid contaminants (Woyke et al. 2011).

Sub-optimal PCR assay design also may contribute to misleading results. Ribosomal genes have often been used as targets for PCR because of the availability of genetic sequences for primer design, high degree of conservation, and the presence of multiple copies in the bacterial genome. However, designing agent-specific assays can be challenging, particularly within ribosomal genes. All sequences of genetic near-neighbors must be considered in primer design to minimize the potential for amplification of closely related bacteria (Tokarz et al. 2019). One solution is to design primers within other less conserved non-ribosomal genes. Another approach is to perform a secondary assay targeting an alternative genetic locus followed by dideoxy sequencing of PCR products for confirmation (Tadin et al. 2016).

Tick-Borne Bacterial Pathogens

Bacterial species within the genera Anaplasma, Borrelia, Coxiella, Ehrlichia, Francisella, and Rickettsia are responsible for the majority of reported human TBD worldwide (Parola and Raoult 2001, Eisen et al. 2017). Anaplasma phagocytophilum, the primary species of Anaplasma linked with human disease, is rapidly increasing in incidence in the United States (Dumler et al. 2001, Rosenberg et al. 2018). Borrelia species cause two distinct human diseases. Whereas species transmitted by ixodid (hard) ticks, cause Lyme borreliosis, argasid (soft) ticks transmit agents of relapsing fever (Steere et al. 2016, Talagrand-Reboul et al. 2018). The most common tick-borne disease in the Northern hemisphere is Lyme borreliosis. In North America, it is caused by B. burgdorferi and B. mayonii. Borrelia afzelli, B. garinii, and B. burgdorferi are the primary correlate agents of Lyme borreliosis in Eurasia. Some species of relapsing-fever Borrelia are also found in ixodid ticks, including the pathogenic B. miyamotoi (Fukunaga et al. 1995, Armstrong et al. 1996). Despite a wide array of Coxiella-like bacteria identified in ticks, Coxiella burnetti, the agent of Q fever, is the only known tick-borne pathogen from this genus (Zhong 2012). Ehrlichiosis is a disease of human and domestic animals caused by infection with species of Ehrlichia, with E. chaffeensis the primary agent of human disease in the United States (Ismail and McBride 2017). Francisella tularensis is the cause of tularemia, a rare but potentially fatal disease (Telford and Goethert 2020). A wide range of Rickettsia species have been identified in ticks worldwide. Although the majority are presumed to be nonpathogenic, several species can cause spotted fever, a potentially severe and lethal disease (Parola et al. 2005, Eisen and Paddock 2020).

The diagnostic methods employed for detection of these agents in clinical samples vary. Although molecular assays may be useful for some, during the early acute stages, serologic assays, typically IFA or enzyme-linked immunoadsorbent assays, are most frequently used for diagnosis (Wormser et al. 2006, Biggs et al. 2016, Connally et al. 2016). For Lyme borreliosis, the exceedingly low bacterial burden in blood makes molecular assays impractical, and serology is used almost exclusively for laboratory diagnosis (Hinckley et al. 2014, Connally et al. 2016).

In addition to pathogenic bacteria, PCR and NGS studies revealed that ticks often harbor nonpathogenic endosymbiotic bacteria. These mainly belong to four genera, including Coxiella, Francisella, Rickettsia, and Midichloria mitochondrii (Ahantarig et al. 2013). All have an obligate intracellular lifecycle and are highly abundant in ticks. In NGS tick microbiome studies, sequences attributed to endosymbionts often dominate those from other bacteria. The endosymbionts are passed transovarially and transtadially, and are usually highly prevalent in their hosts (Socolovschi et al. 2009). The Coxiella endosymbiont of Amblyomma americanum (L.) has been detected in 100% of examined ticks (Jasinskas et al. 2007). For other endosymbionts, the prevalence can vary depending on the geographic region and sex of the ticks examined (Cross et al. 2018, Tokarz et al. 2019). Although the roles the endosymbionts play in the tick life cycle are still unclear, their presence may be beneficial to the tick hosts. Endosymbionts have been shown to be essential for survival and fitness of Dermacentor andersoni Stiles and A. americanum (Zhong et al. 2007, Clayton et al. 2015). Some rickettsial endosymbionts synthesize essential nutrients that can supplement the tick host and also inhibit colonization with pathogens (Hunter et al. 2015; Bodnar et al. 2018, 2020).

16S rRNA studies of ticks have reported a variety of bacterial species at a substantially lower abundance compared to endosymbionts and pathogens. Although these may represent commensal bacteria, it is unclear if the detection of these agents represents stable or transient components of the microbiome or if these sequences originate from environmental or reagent contaminants (Salter et al. 2014, Tokarz et al. 2019). Most of these bacteria are extracellular and it's been suggested that intracellular bacteria may be the only real stable component of the microbiome for some ticks (Ross et al. 2018). Errors introduced during sequencing and insufficient bioinformatic analyses can overestimate the numbers of species identified by NGS (Tijsse-Klasen et al. 2010). In addition, some agents that are detected in ticks by molecular methods may not be a part of the actual microbiome, but rather represent the remains of a past bloodmeal (Allan et al. 2010, Landesman et al. 2019). Therefore, the presence of sequences from atypical agents at a low abundance needs to be verified by additional assays.

Tick Virome

Historically, the identification and characterization of tick-borne viruses has lagged considerably behind the discovery of cellular agents. The lack of a highly conserved consensus gene, such as 16S rRNA in bacteria, limited the utility of consensus PCR assays for discovery of tick-borne viruses (Lwande et al. 2013, Matsuno et al. 2015). Reverse transcriptase (RT)–PCR assays aimed at viral discovery were typically designed to target the viral RNA-dependent RNA polymerase (RdRp) (Maher-Sturgess et al. 2008, Zlateva et al. 2011). Despite the ubiquitous presence of genes encoding RdRp in RNA viruses, the poor sequence conservation, even among viruses within the same genus, limited the utility of this approach for discovery. In addition, these assays were based on alignments of the relatively few available sequences of members of a given genera or family, and overall this limited success in uncovering novel viral agents. The employment of tissue culture for virus isolation, although more costly and laborious, was more successful (McLean et al. 1962, Taylor et al. 1966, Downs 1982, St. George et al. 1984, Topolovec et al. 2003, Pinto Da Silva et al. 2005). An added benefit of this approach was the isolation of viruses that were capable of replication outside the vector host, suggesting the potential for transmissibility and vertebrate infection, especially when isolated in vertebrate cell lines such as Vero cells. However, virus isolation can frequently be unsuccessful, and viruses closely associated with their arthropod hosts were refractory to isolation in vertebrate cell lines. In retrospect, only a fraction of the components of tick viromes were isolated in tissue culture. In addition, after isolation, tick-borne viruses were often insufficiently characterized or misclassified due to the lack of adequate tools for molecular characterization (Taylor et al. 1966, Takahashi et al. 1982).

Shotgun sequencing enabled the identification of a wide range of tick-borne viruses, including the highly pathogenic Heartland virus in 2012 and Bourbon virus in 2015 (Mihindukulasuriya et al. 2009, McMullan et al. 2012, Kosoy et al. 2015). NGS also provided a platform for the investigation of tick viromes. Aside from gaining insight into viral diversity, the primary aim of tick virome studies was the discovery of novel viral pathogens. These studies were abetted by improvements in NGS, including decreased costs of sequencing, longer reads, and enhanced sequencing depth (Reuter et al. 2015). The introduction of dual labeled barcodes enabled pooling of samples, while minimizing read mis-assignments during bioinformatic analyses (MacConaill et al. 2018). NGS protocol modifications were also introduced, such as a ‘viral enrichment’ that included filtration and nuclease treatment prior to extraction to enhance the recovery of viral particles (Hall et al. 2014).

The first report that demonstrated the utility of NGS for tick virome discovery was published in 2014 (Tokarz et al. 2014b). It revealed the existence of a wide range of highly diverse viral sequences present in ticks within the United States. Subsequently, virome characterization analyses of a wide range of tick species from across the globe confirmed that ticks possess a very rich and diverse virome that includes representatives of Chuviridae, Phenuiviridae, Flaviviridae, Orthomyxoviridae, Reoviridae, Rhabdoviridae, Nairoviridae, Nyamiviridae, Peribunyaviridae, Nairoviridae, and Asfarviridae. (Tokarz et al. 2014b, Xia et al. 2015, Moutailler et al. 2016, Shi et al. 2016a, Pettersson et al. 2017, Harvey et al. 2018, Tokarz et al. 2018b, Meng et al. 2019, Sameroff et al. 2019, Gómez et al. 2020, Zhao et al. 2020, Wang et al. 2020). In I. scapularis alone, 18 distinct viral sequences have been identified by NGS (Tokarz et al. 2014b, Tokarz et al. 2018b, Tokarz et al. 2019).

The majority components of tick viromes are presumed to be arthropod-specific viruses and tick endosymbionts (Li et al. 2015a, Bouquet et al. 2017, Tokarz et al. 2018b, Sameroff et al. 2019). Both include viruses classified within viral orders Bunyavirales and Jingchuvirales, which are among the most frequent and abundant tick-borne viruses identified by NGS (Tokarz et al. 2014b, Li et al. 2015a, Pettersson et al. 2017, Tokarz et al. 2018b). A wide range of distinct Bunyavirales-like sequences have been found in individual tick species (Pettersson et al. 2017, Tokarz et al. 2018b). The majority of the tick-associated bunyaviruses identified by NGS consist of vertically transmitted viruses found in several Ixodidae tick species that lack a genomic segment encoding the glycoprotein required for receptor binding (Spiegel et al. 2016, Tokarz et al. 2018b, Vandegrift and Kapoor 2019). The Jingchuvirales order currently contains only the family Chuviridae and genus Mivirus (Li et al. 2015a). After their discovery through NGS studies of ticks, miviruses were identified in a wide range of arthropods (Tokarz et al. 2014a, Tokarz et al. 2014b, Li et al. 2015a, Pettersson et al. 2017, Harvey et al. 2018, Souza et al. 2018, Tokarz et al. 2018b, Meng et al. 2019, Sameroff et al. 2019, Temmam et al. 2019). Other, less frequently detected presumed arthropod-specific viruses and viral endosymbionts include rhabdovirus-like viruses, tetraviruses, tymoviruses, picorna/sobemo-like viruses, and pestivirus-like viruses (Tokarz et al. 2014b, Li et al. 2015a, Shi et al. 2016a, Pettersson et al. 2017, Tokarz et al. 2018b, Sameroff et al. 2019).

Outside the Americas, the primary causes of viral TBDs are Crimean Congo hemorrhagic fever virus, severe fever with thrombocytopenia syndrome virus, and tick-borne encephalitis virus (Ergönül 2006, Lindquist and Vapalahti 2008, Lei et al. 2015). In the United States, five tick-borne viruses have been implicated with human disease, including Heartland virus, Bourbon virus, Colorado tick fever virus and two distinct genotypes of Powassan virus (McLEAN and DONOHUE 1959, Telford et al. 1997, Pesko et al. 2010, McMullan et al. 2012, Kosoy et al. 2015, Yendell et al. 2015). Reports of symptomatic disease with these viruses are rare, which correlates with their infrequent detection in vector ticks. Infection rates of adult I. scapularis with Powassan virus are typically <5% and <2% of A. americanum ticks are usually infected with Heartland or Bourbon virus (Tokarz et al. 2010, 2017; Savage et al. 2013, 2016, 2017; Sanchez-Vicente et al. 2019).

Recent NGS virome studies have also identified several viruses that may require further exploration. Two virome studies identified divergent quaranjaviruses (Cholleti et al. 2018, Wille et al. 2020) classified within the family Orthomyxoviridae. These viruses have been isolated from the blood of children with febrile illness, with a follow-up study reporting neutralizing antibodies in about 8% of the endemic population (Mohammed et al. 1970). Another group, Jingmen tick viruses (JTVs), are genetically related to viruses belonging to the genus Flavivirus (Qin et al. 2014, Shi et al. 2016b). Originally identified within Rhipicephalus microplus (Canestrini) ticks, these viruses have since been identified in arthropods and mammals on five continents (Qin et al. 2014, Ladner et al. 2016, Emmerich et al. 2018, Souza et al. 2018, Meng et al. 2019, Pascoal et al. 2019, Sameroff et al. 2019, Vandegrift et al. 2020). Recent evidence suggests that these viruses are associated with febrile illness within China (Jia et al. 2019, Wang et al. 2019).

Eukaryotic Microbiome

The primary tick-borne eukaryotic microbes are species of apicomplexan piroplasms in the genera Babesia and Theileria that cause babesiosis and theileriosis, respectively (Homer et al. 2000, Mans et al. 2015). Over 100 species within each genus have been described in ixodid ticks worldwide. Babesiosis is among the oldest described TBD with a long history of being a major scourge of cattle. In late 19th century, studies intended to gain insight into the cause of this disease identified Babesia as the very first infectious agent transmitted by an arthropod vector (Smith and Kilborne 1893) In the United States, the majority of human infections are caused by B. microti. Rarely have infections with other species have been reported, with B. duncani occurring on the Pacific Coast and B. divergens-like species in the mid-South (Gray and Herwaldt 2019). Infections with Theileria spp. primarily affect horses and cattle and have no established link to human disease (Mans et al. 2015). Ticks can also harbor other apicomplexan parasites, mainly Hepatozoon spp., some of which may cause disease in domestic canines through ingestion of infected ticks (Allen et al. 2011, Austen et al. 2011).

Filarial nematodes have often been observed in microscopy studies of ixodid tick homogenates (Ko 1972, Londono 1976, Beaver and Burgdorfer 1984). The phylogenetic classification of the majority of these agents have remained obscure until recent PCR and NGS analyses characterized several North American species within a subgroup of Filaroidea (Henning et al. 2016; Cross et al. 2018; Tokarz et al. 2019, 2020). Little is currently known about the life cycles of tick-borne nematodes, although human disease has not been documented (Tokarz et al. 2020).

Other, less frequently reported tick-associated eukaryotic agents include trypanosomes and fungi (Greengarten et al. 2011, Tokarz et al. 2019). One study of I. scapularis identified 27 primarily entomopathogenic species of fungi at a single site (Greengarten et al. 2011). The majority of fungi are presumably acquired through environmental exposure and are likely present only on the surface of the tick. However, the presence of fungi can influence the tick virome, as certain novel viral sequences uncovered in tick shotgun sequencing studies appear to be of fungal origin (Tokarz et al. 2019).

Analyses of eukaryotic diversity in ticks have been partially impeded by the unavailability of amplicon sequencing studies analogous to bacterial 16S rRNA. Nonetheless, shotgun sequencing studies have been successful in detecting eukaryotic agents in ticks (Cross et al. 2018, Tokarz et al. 2019). These studies revealed that in comparison to viruses and bacteria, the eukaryotic component of the tick microbiome appears to be substantially less diverse.

Beyond the Microbiome

One of the primary questions that arose from the tick microbiome studies is the transmissibility of the discovered agents. This question is especially relevant to viruses, because they comprise the majority of the newly discovered and taxonomically classified tick-borne microbes. Many represent previously unknown, genetically diverse viral lineages, and therefore, no parallels can be drawn regarding their life cycle. Another challenge is that the majority appear to be refractory to common methods of tissue culture isolation, precluding studies beyond phylogenetic classification. Serologic methods could be helpful in examining the transmissibility of these agents. However, the rate of discovery of new microbes far outpaces the current capacity for such studies. NGS efforts aimed at demonstrating the presence of tick-borne agents in vertebrate hosts may be beneficial (Vandegrift et al. 2020). It has been presumed that aside from the known pathogens, the primary viral and bacterial components of the tick microbiome are non-transmissible or nonpathogenic endosymbionts. For some of these agents, studies have begun to question this assumption (Apperson et al. 2008, Bonnet et al. 2017, Vandegrift et al. 2020). It is also plausible that endosymbionts and pathogens can directly or indirectly interact within the tick, and as a result influence pathogen transmission (Bonnet et al. 2017). Therefore, in addition to agent discovery, future studies will need to focus on the interactions between the viral, bacterial and eukaryotic components of the tick microbiome and identify new approaches to examine their role in pathogen transmission.


Improvements of molecular and serologic assays for detection of TBD agents have largely replaced microscopy and cultivation. PCR assays have become the mainstay for molecular detection of tickborne agents. However, advances in sequencing technologies hold extraordinary promise. Capture sequencing, for example, uses agent-specific probes to selectively capture the template of interest before sequencing. Employment of capture probes results in an increase in yield of relevant reads compared to unbiased sequencing, and achieves a sensitivity to equivalent to real-time PCR (Briese et al. 2015, Allicock et al. 2018). Serology also has improved with the introduction of multiplex assays that employ short linear immunodominant peptides (Dessau et al. 2015, Lahey et al. 2015, Embers et al. 2016, Arumugam et al. 2019). The TBD-Serochip, for example, incorporates a wide range of specific immunodominant peptides from multiple agents. This allows simultaneous serologic detection and discrimination of antibodies to multiple TBD in a single assay (Tokarz et al. 2018a). Metabolomic analyses have identified metabolic signatures unique to early Lyme borreliosis (Molins et al. 2015, Fitzgerald et al. 2020). These studies have been extended to Southern Tick Associated Rash Illness, an illness of unknown etiology (Molins et al. 2017). Further development and refinement of these platforms will likely enable even more robust methods of diagnosis of TBD and facilitate the discovery of new agents.


We would like to thank Stephen Sameroff, Center for Infection and Immunity, Columbia University, for assistance with the manuscript and Dr. Jorge Benach, Stony Brook University, for helpful suggestions. We are grateful to Dr. William Reisen for his thorough review and helpful comments. This work was facilitated by grants from the Steven & Alexandra Cohen Foundation and Global Lyme Alliance.

References Cited


Ahantarig, A., W. Trinachartvanit, V. Baimai, and L. Grubhoffer. 2013. Hard ticks and their bacterial endosymbionts (or would be pathogens). Folia Microbiol. (Praha). 58: 419–428. Google Scholar


Aliota, M. T., A. P. Dupuis, 2nd, M. P. Wilczek, R. J. Peters, R. S. Ostfeld, and L. D. Kramer. 2014. The prevalence of zoonotic tick-borne pathogens in Ixodes scapularis collected in the Hudson Valley, New York State. Vector Borne Zoonotic Dis. 14: 245–250. Google Scholar


Allan, B. F., L. S. Goessling, G. A. Storch, and R. E. Thach. 2010. Blood meal analysis to identify reservoir hosts for Amblyomma americanum ticks. Emerg. Infect. Dis. 16: 433–440. Google Scholar


Allen, K. E., E. M. Johnson, and S. E. Little. 2011. Hepatozoon spp infections in the United States. Vet. Clin. North Am. Small Anim. Pract. 41: 1221–1238. Google Scholar


Allicock, O. M., C. Guo, A. C. Uhlemann, S. Whittier, L. V. Chauhan, J. Garcia, A. Price, S. S. Morse, N. Mishra, T. Briese, and W. I. Lipkin. 2018. BacCapSeq: a platform for diagnosis and characterization of bacterial infections. MBio. 9: e02007–02018. Google Scholar


Anda, P., W. Sánchez-Yebra, M. del Mar Vitutia, E. Pérez Pastrana, I. Rodríguez, N. S. Miller, P. B. Backenson, and J. L. Benach. 1996. A new Borrelia species isolated from patients with relapsing fever in Spain. Lancet. 348: 162–165. Google Scholar


Andreotti, R., A. A. P. De León, S. E. Dowd, F. D. Guerrero, K. G. Bendele, and G. A. Scoles. 2011. Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing. BMC Microbiol. 11: 6. Google Scholar


Andrews, S., and S. Bittencourt a. 2010. FastQC: a quality control tool for high throughput sequence data – ScienceOpen. Babraham Inst. Google Scholar


Apperson, C. S., B. Engber, W. L. Nicholson, D. G. Mead, J. Engel, M. J. Yabsley, K. Dail, J. Johnson, and D. W. Watson. 2008. Tick-borne diseases in North Carolina: is “Rickettsia amblyommii” a possible cause of rickettsiosis reported as Rocky Mountain spotted fever? Vector Borne Zoonotic Dis. 8: 597–606. Google Scholar


Armstrong, P. M., S. M. Rich, R. D. Smith, D. L. Hartl, A. Spielman, and S. R. Telford, 3rd. 1996. A new Borrelia infecting Lone Star ticks. Lancet. 347: 67–68. Google Scholar


Arumugam, S., S. Nayak, T. Williams, F. S. D. S. Maria, M. S. Guedes, R. C. Chaves, V. Linder, A. R. Marques, E. J. Horn, S. J. Wong, S. K. Sia, and M. Gomes-Solecki. 2019. A multiplexed serologic test for diagnosis of Lyme disease for point-of-care use. J. Clin. Microbiol. 57: e01142–19. Google Scholar


Asplund, M., K. R. Kjartansdóttir, S. Mollerup, L. Vinner, H. Fridholm, J. A. R. Herrera, J. Friis-Nielsen, T. A. Hansen, R. H. Jensen, I. B. Nielsen, et al. 2019. Contaminating viral sequences in high-throughput sequencing viromics: a linkage study of 700 sequencing libraries. Clin. Microbiol. Infect. 25: 1277–1285. Google Scholar


Austen, J. M., U. M. Ryan, J. A. Friend, W. G. Ditcham, and S. A. Reid. 2011. Vector of Trypanosoma copemani identified as Ixodes sp. Parasitology. 138: 866–872. Google Scholar


Beaver, P. C., and W. Burgdorfer. 1984. A microfilaria of exceptional size from the ixodid tick, Ixodes dammini, from Shelter Island, New York. J. Parasitol. 70: 963–966. Google Scholar


Bell, C. A., and R. Patel. 2005. A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Diagn. Microbiol. Infect. Dis. 53: 301–306. Google Scholar


Benach, J. L., and G. S. Habicht. 1981. Clinical characteristics of human babesiosis. J. Infect. Dis. 144: 481. Google Scholar


Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman, G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G. Barbour, W. Burgdorfer, et al. 1983. Spirochetes isolated from the blood of two patients with Lyme disease. N. Engl. J. Med. 308: 740–742. Google Scholar


Biggs, H. M., C. B. Behravesh, K. K. Bradley, F. S. Dahlgren, N. A. Drexler, J. S. Dumler, S. M. Folk, C. Y. Kato, R. R. Lash, M. L. Levin, R. F. Massung, R. B. Nadelman, W. L. Nicholson, C. D. Paddock, B. S. Pritt, and M. S. Traeger. 2016. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis - United States a practical guide for health care and public health professionals. MMWR Recomm. Reports. 65: 1–44. Google Scholar


Binetruy, F., M. Dupraz, M. Buysse, and O. Duron. 2019. Surface sterilization methods impact measures of internal microbial diversity in ticks. Parasites and Vectors 12: 268. Google Scholar


Bodnar, J. L., S. Fitch, A. Rosati, and J. Zhong. 2018. The folA gene from the Rickettsia endosymbiont of Ixodes pacificus encodes a functional dihydrofolate reductase enzyme. Ticks Tick. Borne. Dis. 9: 443–449. Google Scholar


Bodnar, J., S. Fitch, J. Sanchez, M. Lesser, D. S. Baston, and J. Zhong. 2020. GTP cyclohydrolase I activity from Rickettsia monacensis strain Humboldt, a rickettsial endosymbiont of Ixodes pacificus. Ticks Tick. Borne. Dis. 11: 101434. Google Scholar


Bonk, F., D. Popp, H. Harms, and F. Centler. 2018. PCR-based quantification of taxa-specific abundances in microbial communities: Quantifying and avoiding common pitfalls. J. Microbiol. Methods 153: 139–147. Google Scholar


Bonnet, S. I., F. Binetruy, A. M. Hernández-Jarguín, and O. Duron. 2017. The tick microbiome: Why non-pathogenic microorganisms matter in tick biology and pathogen transmission. Front. Cell. Infect. Microbiol. 7: 236. Google Scholar


Bouquet, J., M. Melgar, A. Swei, E. Delwart, R. S. Lane, and C. Y. Chiu. 2017. Metagenomic-based Surveillance of Pacific Coast tick Dermacentor occidentalis identifies two novel bunyaviruses and an emerging human rickettsial pathogen. Sci. Rep. 7: 12234. Google Scholar


Briese, T., A. Kapoor, N. Mishra, K. Jain, A. Kumar, O. J. Jabado, and W. Ian Lipkin. 2015. Virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis. MBio. 6: e01491–15. Google Scholar


Buchan, B. W., D. A. Jobe, M. Mashock, D. Gerstbrein, M. L. Faron, N. A. Ledeboer, and S. M. Callister. 2019. Evaluation of a novel multiplex high-definition PCR assay for detection of tick-borne pathogens in whole-blood specimens. J. Clin. Microbiol. 57: e00513–00519. Google Scholar


Burgdorfer, W., A. G. Barbour, S. F. Hayes, J. L. Benach, E. Grunwaldt, and J. P. Davis. 1982. Lyme disease-a tick-borne spirochetosis? Science. 216: 1317–1319. Google Scholar


Caporaso, J. G., J. Kuczynski, J. Stombaugh, K. Bittinger, F. D. Bushman, E. K. Costello, N. Fierer, A. G. Peña, J. K. Goodrich, J. I. Gordon, et al. 2010. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7: 335–336. Google Scholar


Carpi, G., F. Cagnacci, N. E. Wittekindt, F. Zhao, J. Qi, L. P. Tomsho, D. I. Drautz, A. Rizzoli, and S. C. Schuster. 2011. Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks. PLoS One 66: e25604. Google Scholar


Carpi, G., K. S. Walter, S. J. Bent, A. G. Hoen, M. Diuk-Wasser, and A. Caccone. 2015. Whole genome capture of vector-borne pathogens from mixed DNA samples: A case study of Borrelia burgdorferi. BMC Genomics 16: 434. Google Scholar


Center for Disease Control and Prevention Diseases, National Center for Emerging and Zoonotic Infectious Diseases, and Division of Vector-Borne Diseases. 2019. Scholar


Chalada, M. J., J. Stenos, G. Vincent, D. Barker, and R. S. Bradbury. 2018. A molecular survey of tick-borne pathogens from ticks collected in central queensland, Australia. Vector Borne Zoonotic Dis. 18: 151–163. Google Scholar


Chen, S. M., J. S. Dumler, J. S. Bakken, and D. H. Walker. 1994. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32: 589–595. Google Scholar


Chevreux, B., T. Wetter, and S. Suhai. 1999. Genome sequence assembly using trace signals and additional sequence information. Comput. Sci. Biol. Proc. Ger. Conf. Bioinforma.'99, GCB, Hann. Ger. 45–56. Google Scholar


Cholleti, H., J. Hayer, F. C. Mulandane, K. Falk, J. Fafetine, M. Berg, and A. L. Blomström. 2018. Viral metagenomics reveals the presence of highly divergent quaranjavirus in Rhipicephalus ticks from Mozambique. Infect. Ecol. Epidemiol. 8: 1478585. Google Scholar


Clayton, K. A., C. A. Gall, K. L. Mason, G. A. Scoles, and K. A. Brayton. 2015. The characterization and manipulation of the bacterial microbiome of the Rocky Mountain wood tick, Dermacentor andersoni. Parasites and Vectors 8: 632. Google Scholar


Cole, J. R., Q. Wang, J. A. Fish, B. Chai, D. M. McGarrell, Y. Sun, C. T. Brown, A. Porras-Alfaro, C. R. Kuske, and J. M. Tiedje. 2014. Ribosomal Database Project: Data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42(Database issue): D633–642. Google Scholar


Connally, N. P., A. F. Hinckley, K. A. Feldman, M. Kemperman, D. Neitzel, S. B. Wee, J. L. White, P. S. Mead, and J. I. Meek. 2016. Testing practices and volume of non-Lyme tickborne diseases in the United States. Ticks Tick. Borne. Dis. 7: 193–198. Google Scholar


Cowdry, E. V. 1925. A Group of microorganisms transmitted hereditarily in ticks and apparently unassociated with disease. J. Exp. Med. 41: 817–830. Google Scholar


Cross, S. T., M. L. Kapuscinski, J. Perino, B. L. Maertens, J. Weger-Lucarelli, G. D. Ebel, and M. D. Stenglein. 2018. Co-infection patterns in individual ixodes scapularis ticks reveal associations between viral, eukaryotic and bacterial microorganisms. Viruses. 10: 388. Google Scholar


Cull, B., M. E. Pietzsch, K. M. Hansford, E. L. Gillingham, and J. M. Medlock. 2018. Surveillance of British ticks: an overview of species records, host associations, and new records of Ixodes ricinus distribution. Ticks Tick. Borne. Dis. 9: 605–614. Google Scholar


Curcio, S. R., L. P. Tria, and A. L. Gucwa. 2016. Seroprevalence of Babesia microti in Individuals with Lyme Disease. Vector Borne Zoonotic Dis. 16: 737–743. Google Scholar


Dessau, R. B., J. K. Møller, B. Kolmos, and A. J. Henningsson. 2015. Multiplex assay (Mikrogen recomBead) for detection of serum IgG and IgM antibodies to 13 recombinant antigens of Borrelia burgdorferi sensu lato in patients with neuroborreliosis: the more the better? J. Med. Microbiol. 64: 224–231. Google Scholar


Downs, W. G. 1982. The Rockefeller Foundation virus program: 1951–1971 with update to 1981. Annu. Rev. Med. 33: 1–29. Google Scholar


Dumler, J. S., A. F. Barbet, C. P. J. Bekker, G. A. Dasch, G. H. Palmer, S. C. Ray, Y. Rikihisa, and F. R. Rurangirwa. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and “HGE agent” as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51: 2145–2165. Google Scholar


Edwards, M. J., L. A. Barbalato, A. Makkapati, K. D. Pham, and L. M. Bugbee. 2015. Relatively low prevalence of Babesia microti and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in the Lehigh Valley region of eastern Pennsylvania. Ticks Tick. Borne. Dis. 6: 812–819. Google Scholar


Eisen, R. J., and C. D. Paddock. 2021. Tick and tickborne pathogen surveillance as a public health tool in the United States. J. Med. Entomol 58: 1490–1502. Google Scholar


Eisen, R. J., K. J. Kugeler, L. Eisen, C. B. Beard, and C. D. Paddock. 2017. Tick-borne zoonoses in the United States: persistent and emerging threats to human health. ILAR J. 58: 319–335. Google Scholar


Embers, M. E., N. R. Hasenkampf, M. B. Barnes, E. S. Didier, M. T. Philipp, and A. C. Tardo. 2016. Five-antigen fluorescent bead-based assay for diagnosis of Lyme disease. Clin. Vaccine Immunol. 23: 294–303. Google Scholar


Emmerich, P., X. Jakupi, R. von Possel, L. Berisha, B. Halili, S. Günther, D. Cadar, S. Ahmeti, and J. Schmidt-Chanasit. 2018. Viral metagenomics, genetic and evolutionary characteristics of Crimean-Congo hemorrhagic fever orthonairovirus in humans, Kosovo. Infect. Genet. Evol. 65: 6–11. Google Scholar


Ergönül, Ö. 2006. Crimean-Congo haemorrhagic fever. Lancet Infect. Dis. 6: 203–214. Google Scholar


Estrada-Peña, A., and J. De La Fuente. 2014. The ecology of ticks and epidemiology of tick-borne viral diseases. Antiviral Res. 108: 104–128. Google Scholar


Exner, M. M., and M. A. Lewinski. 2003. Isolation and detection of Borrelia burgdorferi DNA from cerebral spinal fluid, synovial fluid, blood, urine, and ticks using the Roche MagNA Pure system and real-time PCR. Diagn. Microbiol. Infect. Dis. 46: 235–240. Google Scholar


Filstein, M. R., J. L. Benach, D. J. White, B. A. Brody, W. D. Goldman, C. W. Bakal, and R. S. Schwartz. 1980. Serosurvey for human babesiosis in New York. J. Infect. Dis. 141: 518–521. Google Scholar


Fitzgerald, B. L., C. R. Molins, M. N. Islam, B. Graham, P. R. Hove, G. P. Wormser, L. Hu, L. V. Ashton, and J. T. Belisle. 2020. Host metabolic response in early Lyme disease. J. Proteome Res. 19: 610–623. Google Scholar


Fukunaga, M., Y. Takahashi, Y. Tsuruta, O. Matsushita, D. Ralph, M. McClelland, and M. Nakao. 1995. Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan. Int. J. Syst. Bacteriol. 45: 804–810. Google Scholar


Gaines, D. N., D. J. Operario, S. Stroup, E. Stromdahl, C. Wright, H. Gaff, J. Broyhill, J. Smith, D. E. Norris, T. Henning, et al. 2014. Ehrlichia and spotted fever group Rickettsiae surveillance in Amblyomma americanum in Virginia through use of a novel six-plex real-time PCR assay. Vector Borne Zoonotic Dis. 14: 307–316. Google Scholar


Gofton, A. W., C. L. Oskam, N. Lo, T. Beninati, H. Wei, V. McCarl, D. C. Murray, A. Paparini, T. L. Greay, A. J. Holmes, M. Bunce, U. Ryan, and P. Irwin. 2015. Inhibition of the endosymbiont “Candidatus Midichloria mitochondrii” during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia. Parasites and Vectors 8: 345. Google Scholar


Gómez, G. F., J. P. Isaza, J. A. Segura, J. F. Alzate, and L. A. Gutiérrez. 2020. Metatranscriptomic virome assessment of Rhipicephalus microplus from Colombia. Ticks Tick. Borne. Dis. 11: 101426. Google Scholar


Goodwin, S., J. D. McPherson, and W. R. McCombie. 2016. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 17: 333–351. Google Scholar


Gray, E. B., and B. L. Herwaldt. 2019. Babesiosis surveillance - United States, 2011–2015. MMWR Surveill. Summ. 68: 1–16. Google Scholar


Greay, T. L., A. W. Gofton, A. Paparini, U. M. Ryan, C. L. Oskam, and P. J. Irwin. 2018. Recent insights into the tick microbiome gained through next-generation sequencing. Parasites and Vectors 11: 12. Google Scholar


Greengarten, P. J., A. R. Tuininga, S. U. Morath, R. C. Falco, H. Norelus, and T. J. Daniels. 2011. Occurrence of soil- and tick-borne fungi and related virulence tests for pathogenicity to Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 48: 337–344. Google Scholar


Gruber, K. 2015. Here, there, and everywhere: From PCRs to next-generation sequencing technologies and sequence databases, DNA contaminants creep in from the most unlikely places. EMBO Rep. 16: 898–901. Google Scholar


Guo, H., P. F. Adjou Moumouni, O. Thekisoe, Y. Gao, M. Liu, J. Li, E. M. Galon, A. Efstratiou, G. Wang, C. Jirapattharasate, et al. 2019. Genetic characterization of tick-borne pathogens in ticks infesting cattle and sheep from three South African provinces. Ticks Tick. Borne. Dis. 10: 875–882. Google Scholar


Gurfield, N., S. Grewal, L. S. Cua, P. J. Torres, and S. T. Kelley. 2017. Endosymbiont interference and microbial diversity of the Pacific coast tick, Dermacentor occidentalis, in San Diego County, California. PeerJ. 2017. 5:e3202. Google Scholar


Hall, R. J., J. Wang, A. K. Todd, A. B. Bissielo, S. Yen, H. Strydom, N. E. Moore, X. Ren, Q. S. Huang, P. E. Carter, et al. 2014. Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery. J. Virol. Methods 195: 194–204. Google Scholar


Harvey, E., K. Rose, J.-S. Eden, N. Lo, T. Abeyasuriya, M. Shi, S. L. Doggett, and E. C. Holmes. 2018. Extensive diversity of RNA viruses in Australian ticks. J. Virol. 93:e01358–18. Google Scholar


Henning, T. C., J. M. Orr, J. D. Smith, J. R. Arias, J. L. Rasgon, and D. E. Norris. 2016. Discovery of filarial nematode DNA in Amblyomma americanum in Northern Virginia. Ticks Tick. Borne. Dis. 7: 315–318. Google Scholar


Hilton, E., J. DeVoti, J. L. Benach, M. L. Halluska, D. J. White, H. Paxton, and J. S. Dumler. 1999. Seroprevalence and seroconversion for tick-borne diseases in a high-risk population in the northeast United States. Am. J. Med. 106: 404–409. Google Scholar


Hinckley, A. F., N. P. Connally, J. I. Meek, B. J. Johnson, M. M. Kemperman, K. A. Feldman, J. L. White, and P. S. Mead. 2014. Lyme disease testing by large commercial laboratories in the United States. Clin. Infect. Dis. 59: 676–681. Google Scholar


Hoffmann, A., V. Fingerle, and M. Noll. 2020. Analysis of tick surface decontamination methods. Microorganisms. 8: 1–16. Google Scholar


Hojgaard, A., G. Lukacik, and J. Piesman. 2014. Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays. Ticks Tick. Borne. Dis. 5: 349–351. Google Scholar


Homer, M. J., I. Aguilar-Delfin, S. R. Telford, 3rd, P. J. Krause, and D. H. Persing. 2000. Babesiosis. Clin. Microbiol. Rev. 13: 451–469. Google Scholar


Honig, J. E., J. C. Osborne, and S. T. Nichol. 2004. The high genetic variation of viruses of the genus Nairovirus reflects the diversity of their predominant tick hosts. Virology. 318: 10–16. Google Scholar


Horowitz, H. W., M. E. Aguero-Rosenfeld, D. Holmgren, D. McKenna, I. Schwartz, M. E. Cox, and G. P. Wormser. 2013. Lyme disease and human granulocytic anaplasmosis coinfection: impact of case definition on coinfection rates and illness severity. Clin. Infect. Dis. 56: 93–99. Google Scholar


Hue, F., A. Ghalyanchi Langeroudi, and A. G. Barbour. 2013. Chromosome sequence of Borrelia miyamotoi, an uncultivable tick-borne agent of human infection. Genome Announc. 1:e00713–13. Google Scholar


Hunter, D. J., J. L. Torkelson, J. Bodnar, B. Mortazavi, T. Laurent, J. Deason, K. Thephavongsa, and J. Zhong. 2015. The Rickettsia endosymbiont of Ixodes pacificus contains all the genes of de novo folate biosynthesis. PLoS One 10: e0144552. Google Scholar


Hutchinson, M. L., M. D. Strohecker, T. W. Simmons, A. D. Kyle, and M. W. Helwig. 2015. Prevalence Rates of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae) in Host-Seeking Ixodes scapularis (Acari: Ixodidae) from Pennsylvania. J. Med. Entomol. 52: 693–698. Google Scholar


Ismail, N., and J. W. McBride. 2017. Tick-Borne Emerging Infections: Ehrlichiosis and Anaplasmosis. Clin. Lab. Med. 37: 317–340. Google Scholar


Jasinskas, A., J. Zhong, and A. G. Barbour. 2007. Highly prevalent Coxiella sp. bacterium in the tick vector Amblyomma americanum. Appl. Environ. Microbiol. 73: 334–336. Google Scholar


Jia, N., H. B. Liu, X. B. Ni, L. Bell-Sakyi, Y. C. Zheng, J. L. Song, J. Li, B. G. Jiang, Q. Wang, Y. Sun, et al. 2019. Emergence of human infection with Jingmen tick virus in China: a retrospective study. Ebiomedicine. 43: 317–324. Google Scholar


Johnson, B. J., C. M. Happ, L. W. Mayer, and J. Piesman. 1992. Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene. Am. J. Trop. Med. Hyg. 47: 730–741. Google Scholar


Johnson, T. L., C. B. Graham, S. E. Maes, A. Hojgaard, A. Fleshman, K. A. Boegler, M. J. Delory, K. S. Slater, S. E. Karpathy, J. K. Bjork, et al. 2018. Prevalence and distribution of seven human pathogens in host-seeking Ixodes scapularis (Acari: Ixodidae) nymphs in Minnesota, USA. Ticks Tick. Borne. Dis. 9: 1499–1507. Google Scholar


Jongejan, F., and G. Uilenberg. 2004. The global importance of ticks. Parasitology. 129(Suppl):S3–14. Google Scholar


Kim, M., M. Morrison, and Z. Yu. 2011. Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes. J. Microbiol. Methods 84: 81–87. Google Scholar


Ko, R. C. 1972. The transmission of Ackertia marmotae Webster, 1967 (Nematoda: Onchocercidae) of groundhogs (Marmota monax) by Ixodes cookei. Can. J. Zool. 50: 437–450. Google Scholar


Kosoy, O. I., A. J. Lambert, D. J. Hawkinson, D. M. Pastula, C. S. Goldsmith, D. C. Hunt, and J. E. Staples. 2015. Novel thogotovirus associated with febrile illness and death, United States, 2014. Emerg. Infect. Dis. 21: 760–764. Google Scholar


Krause, P. J., S. R. Telford, A. Spielman, V. Sikand, R. Ryan, D. Christianson, G. Burke, P. Brassard, R. Pollack, J. Peck, and D. H. Persing. 1996. Concurrent Lyme disease and babesiosis: Evidence for increased severity and duration of illness. J. Am. Med. Assoc. 275: 1657–1660. Google Scholar


La Scola, B., and D. Raoult. 1997. Laboratory diagnosis of rickettsioses: current approaches to diagnosis of old and new rickettsial diseases. J. Clin. Microbiol. 35: 2715–2727. Google Scholar


La Scola, B., Z. Zeaiter, A. Khamis, and D. Raoult. 2003. Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm. Trends Microbiol. 11: 318–321. Google Scholar


Ladner, J. T., M. R. Wiley, B. Beitzel, A. J. Auguste, A. P. Dupuis, 2nd, M. E. Lindquist, S. D. Sibley, K. P. Kota, D. Fetterer, G. Eastwood, et al. 2016. A multicomponent animal virus isolated from mosquitoes. Cell Host Microbe 20: 357–367. Google Scholar


Lahey, L. J., M. W. Panas, R. Mao, M. Delanoy, J. J. Flanagan, S. R. Binder, A. W. Rebman, J. G. Montoya, M. J. Soloski, A. C. Steere, et al. 2015. Development of a multiantigen panel for improved detection of Borrelia burgdorferi infection in early Lyme disease. J. Clin. Microbiol. 53: 3834–3841. Google Scholar


Landesman, W. J., K. Mulder, B. F. Allan, L. A. Bashor, F. Keesing, K. LoGiudice, and R. S. Ostfeld. 2019. Potential effects of blood meal host on bacterial community composition in Ixodes scapularis nymphs. Ticks Tick. Borne. Dis. 10: 523–527. Google Scholar


Langmead, B., and S. L. Salzberg. 2012. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9: 357–359. Google Scholar


Lei, X. Y., M. M. Liu, and X. J. Yu. 2015. Severe fever with thrombocytopenia syndrome and its pathogen SFTSV. Microbes Infect. 17: 149–154. Google Scholar


Li, C. X., M. Shi, J. H. Tian, X. D. Lin, Y. J. Kang, L. J. Chen, X. C. Qin, J. Xu, E. C. Holmes, and Y. Z. Zhang. 2015a. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Elife. 4:e05378. Google Scholar


Li, D., C. M. Liu, R. Luo, K. Sadakane, and T. W. Lam. 2015b. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics. 31: 1674–1676. Google Scholar


Lim, Y. W., M. Haynes, M. Furlan, C. E. Robertson, J. K. Harris, and F. Rohwer. 2014. Purifying the impure: Sequencing metagenomes and metatranscriptomes from complex animal-associated samples. J. Vis. Exp (94): 52117. Google Scholar


Lindquist, L., and O. Vapalahti. 2008. Tick-borne encephalitis. Lancet. 371: 1861–1871. Google Scholar


Liu, L., Y. Li, S. Li, N. Hu, Y. He, R. Pong, D. Lin, L. Lu, and M. Law. 2012. Comparison of next-generation sequencing systems. J. Biomed. Biotechnol. 2012: 251364. Google Scholar


Londoño, I. 1976. Transmission of microfilariae and infective larvae of Dipetalonema viteae (Filarioidea) among vector ticks, Ornithodoros tartakowskyi (Argasidae), and loss of microfilariae in coxal fluid. J. Parasitol. 62: 786–788. Google Scholar


Lwande, O. W., J. Lutomiah, V. Obanda, F. Gakuya, J. Mutisya, F. Mulwa, G. Michuki, E. Chepkorir, A. Fischer, M. Venter, et al. 2013. Isolation of tick and mosquito-borne arboviruses from ticks sampled from livestock and wild animal hosts in Ijara District, Kenya. Vector Borne Zoonotic Dis. 13: 637–642. Google Scholar


MacConaill, L. E., R. T. Burns, A. Nag, H. A. Coleman, M. K. Slevin, K. Giorda, M. Light, K. Lai, M. Jarosz, M. S. McNeill, et al. 2018. Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing. BMC Genomics 19: 30. Google Scholar


Maher-Sturgess, S. L., N. L. Forrester, P. J. Wayper, E. A. Gould, R. A. Hall, R. T. Barnard, and M. J. Gibbs. 2008. Universal primers that amplify RNA from all three flavivirus subgroups. Virol. J. 5: 16. Google Scholar


Mans, B. J., R. Pienaar, and A. A. Latif. 2015. A review of Theileria diagnostics and epidemiology. Int. J. Parasitol. Parasites Wildl. 4: 104–118. Google Scholar


Matsuno, K., C. Weisend, M. Kajihara, C. Matysiak, B. N. Williamson, M. Simuunza, A. S. Mweene, A. Takada, R. B. Tesh, and H. Ebihara. 2015. Comprehensive molecular detection of tick-borne phleboviruses leads to the retrospective identification of taxonomically unassigned bunyaviruses and the discovery of a novel member of the genus phlebovirus. J. Virol. 89: 594–604. Google Scholar


McCoy, K. D., E. Léger, and M. Dietrich. 2013. Host specialization in ticks and transmission of tick-borne diseases: A review. Front. Cell. Infect. Microbiol. 3: 57. Google Scholar


McLean, D. M., and W. L. Donohue. 1959. Powassan virus: isolation of virus from a fatal case of encephalitis. Can. Med. Assoc. J. 80: 708–711. Google Scholar


McLean, D. M., E. J. McQueen, H. E. Petite, L. W. Macpherson, T. H. Scholten, and K. Ronald. 1962. Powassan virus: field investigations in northern Ontario, 1959 to 1961. Can. Med. Assoc. J. 86: 971–974. Google Scholar


McMullan, L. K., S. M. Folk, A. J. Kelly, A. MacNeil, C. S. Goldsmith, M. G. Metcalfe, B. C. Batten, C. G. Albariño, S. R. Zaki, P. E. Rollin, et al. 2012. A new phlebovirus associated with severe febrile illness in Missouri. N. Engl. J. Med. 367: 834–841. Google Scholar


McQuiston, J. H., G. Zemtsova, J. Perniciaro, M. Hutson, J. Singleton, W. L. Nicholson, and M. L. Levin. 2012. Afebrile spotted fever group Rickettsia infection after a bite from a Dermacentor variabilis tick infected with Rickettsia montanensis. Vector Borne Zoonotic Dis. 12: 1059–1061. Google Scholar


Meng, F., M. Ding, Z. Tan, Z. Zhao, L. Xu, J. Wu, B. He, and C. Tu. 2019. Virome analysis of tick-borne viruses in Heilongjiang Province, China. Ticks Tick. Borne. Dis. 10: 412–420. Google Scholar


Mihindukulasuriya, K. A., N. L. Nguyen, G. Wu, H. V. Huang, A. P. da Rosa, V. L. Popov, R. B. Tesh, and D. Wang. 2009. Nyamanini and midway viruses define a novel taxon of RNA viruses in the order Mononegavirales. J. Virol. 83: 5109–5116. Google Scholar


Mohammed, Y. S., M. Gresikova, K. Adamyova, A. H. Ragib, and K. el-Dawala. 1970. Studies on arboviruses in Egypt: II. Contribution of arboviruses to the aetiology of undiagnosed fever among children. J. Hyg. (Lond). 68: 491–495. Google Scholar


Molins, C. R., L. V. Ashton, G. P. Wormser, A. M. Hess, M. J. Delorey, S. Mahapatra, M. E. Schriefer, and J. T. Belisle. 2015. Development of a metabolic biosignature for detection of early Lyme disease. Clin. Infect. Dis. 60: 1767–1775. Google Scholar


Molins, C. R., L. V. Ashton, G. P. Wormser, B. G. Andre, A. M. Hess, M. J. Delorey, M. A. Pilgard, B. J. Johnson, K. Webb, M. N. Islam, et al. 2017. Metabolic differentiation of early Lyme disease from southern tick-associated rash illness (STARI). Sci. Transl. Med. 9: eaal2717. Google Scholar


Monzón, J. D., E. G. Atkinson, B. M. Henn, and J. L. Benach. 2016. Population and evolutionary genomics of Amblyomma americanum, an expanding arthropod disease vector. Genome Biol. Evol. 8: 1351–1360. Google Scholar


Moutailler, S., I. Popovici, E. Devillers, M. Vayssier-Taussat, and M. Eloit. 2016. Diversity of viruses in Ixodes ricinus, and characterization of a neurotropic strain of Eyach virus. New Microbes New Infect. 11: 71–81. Google Scholar


Narasimhan, S., and E. Fikrig. 2015. Tick microbiome: the force within. Trends Parasitol. 31: 315–323. Google Scholar


Palacios, G., N. Savji, A. Travassos da Rosa, H. Guzman, X. Yu, A. Desai, G. E. Rosen, S. Hutchison, W. I. Lipkin, and R. Tesh. 2013. Characterization of the Uukuniemi virus group (Phlebovirus: Bunyaviridae): evidence for seven distinct species. J. Virol. 87: 3187–3195. Google Scholar


Pancholi, P., C. P. Kolbert, P. D. Mitchell, K. D. Reed, Jr, J. S. Dumler, J. S. Bakken, S. R. Telford, 3rd, and D. H. Persing. 1995. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis. J. Infect. Dis. 172: 1007–1012. Google Scholar


Parola, P., and D. Raoult. 2001. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin. Infect. Dis. 32: 897–928. Google Scholar


Parola, P., C. D. Paddock, and D. Raoult. 2005. Tick-borne rickettsioses around the world: emerging diseases challenging old concepts. Clin. Microbiol. Rev. 18: 719–756. Google Scholar


Pascoal, J. O., S. M. Siqueira, R. D. C. Maia, M. P. Juan Szabó, and J. Yokosawa. 2019. Detection and molecular characterization of Mogiana tick virus (MGTV) in Rhipicephalus microplus collected from cattle in a savannah area, Uberlândia, Brazil. Ticks Tick. Borne. Dis. 10: 162–165. Google Scholar


Pesko, K. N., F. Torres-Perez, B. L. Hjelle, and G. D. Ebel. 2010. Molecular epidemiology of Powassan virus in North America. J. Gen. Virol. 91: 2698–2705. Google Scholar


Pettersson, J. H. O., M. Shi, J. Bohlin, V. Eldholm, O. B. Brynildsrud, K. M. Paulsen, Å. Andreassen, and E. C. Holmes. 2017. Characterizing the virome of Ixodes ricinus ticks from northern Europe. Sci. Rep. 7: 10870. Google Scholar


Pinto Da Silva, E. V., A. P. A. Travassos Da Rosa, M. R. T. Nunes, J. A. P. Diniz, R. B. Tesh, A. C. R. Cruz, C. M. A. Vieira, and P. F. C. Vasconcelos. 2005. Araguari virus, a new member of the family Orthomyxoviridae: Serologic, ultrastructural, and molecular characterization. Am. J. Trop. Med. Hyg. 73: 1050–1058. Google Scholar


Ponnusamy, L., A. Gonzalez, W. Van Treuren, S. Weiss, C. M. Parobek, J. J. Juliano, R. Knight, R. M. Roe, C. S. Apperson, and S. R. Meshnick. 2014. Diversity of Rickettsiales in the microbiome of the lone star tick, Amblyomma americanum. Appl. Environ. Microbiol. 80: 354–359. Google Scholar


Pritt, B. S., L. M. Sloan, D. K. Johnson, U. G. Munderloh, S. M. Paskewitz, K. M. McElroy, J. D. McFadden, M. J. Binnicker, D. F. Neitzel, G. Liu, et al. 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365: 422–429. Google Scholar


Pritt, B. S., P. S. Mead, D. K. H. Johnson, D. F. Neitzel, L. B. Respicio-Kingry, J. P. Davis, E. Schiffman, L. M. Sloan, M. E. Schriefer, A. J. Replogle, et al. 2016. Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet. Infect. Dis. 16: 556–564. Google Scholar


Prusinski, M. A., J. E. Kokas, K. T. Hukey, S. J. Kogut, J. Lee, and P. B. Backenson. 2014. Prevalence of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and Babesia microti (Piroplasmida: Babesiidae) in Ixodes scapularis (Acari: Ixodidae) collected from recreational lands in the Hudson Valley Region, New York State. J. Med. Entomol. 51: 226–236. Google Scholar


Qin, X. C., M. Shi, J. H. Tian, X. D. Lin, D. Y. Gao, J. R. He, J. B. Wang, C. X. Li, Y. J. Kang, B. Yu, et al. 2014. A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors. Proc. Natl. Acad. Sci. U. S. A. 111: 6744–6749. Google Scholar


Quast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P. Yarza, J. Peplies, and F. O. Glöckner. 2013. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41(Database issue):D590–596. Google Scholar


Raoult, D., and V. Roux. 1997. Rickettsioses as paradigms of new or emerging infectious diseases. Clin. Microbiol. Rev. 10: 694–719. Google Scholar


Reller, M. E., and J. S. Dumler. 2018. Development and clinical validation of a multiplex real-time quantitative PCR assay for human infection by Anaplasma phagocytophilum and Ehrlichia chaffeensis. Trop. Med. Infect. Dis. 3: 14. Google Scholar


Reuter, J. A., D. V. Spacek, and M. P. Snyder. 2015. High-throughput sequencing technologies. Mol. Cell 58: 586–597. Google Scholar


Ricketts, H. T. 1906. The transmission of Rocky Mountain spotted fever by the bite of the wood-tick (Dermacentor occidentalis). J. Am. Med. Assoc. XLVII: 358. Google Scholar


Ricketts, H. T. 1991. Some aspects of Rocky Mountain spotted fever as shown by recent investigations. 1909. Rev. Infect. Dis. 13: 1227–1240. Google Scholar


Rosenberg, R., N. P. Lindsey, M. Fischer, C. J. Gregory, A. F. Hinckley, P. S. Mead, G. Paz-Bailey, S. H. Waterman, N. A. Drexler, G. J. Kersh, et al. 2018. Vital Signs: Trends in Reported Vectorborne Disease Cases — United States and Territories, 2004–2016. MMWR. Morb. Mortal. Wkly. Rep. 67: 496–501. Google Scholar


Ross, B. D., B. Hayes, M. C. Radey, X. Lee, T. Josek, J. Bjork, D. Neitzel, S. Paskewitz, S. Chou, and J. D. Mougous. 2018. Ixodes scapularis does not harbor a stable midgut microbiome. ISME J. 12: 2596–2607. Google Scholar


Rynkiewicz, E. C., C. Hemmerich, D. B. Rusch, C. Fuqua, and K. Clay. 2015. Concordance of bacterial communities of two tick species and blood of their shared rodent host. Mol. Ecol. 24: 2566–2579. Google Scholar


Salter, S. J., M. J. Cox, E. M. Turek, S. T. Calus, W. O. Cookson, M. F. Moffatt, P. Turner, J. Parkhill, N. J. Loman, and A. W. Walker. 2014. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12: 87. Google Scholar


Sameroff, S., R. Tokarz, R. A. Charles, K. Jain, A. Oleynik, X. Che, K. Georges, C. V. Carrington, W. I. Lipkin, and C. Oura. 2019. Viral diversity of tick species parasitizing cattle and dogs in Trinidad and Tobago. Sci. Rep. 9: 10421. Google Scholar


Sanchez-Vicente, S., T. Tagliafierro, J. L. Coleman, J. L. Benach, and R. Tokarz. 2019. Polymicrobial nature of tick-borne diseases. MBio. 10:e02055–19. Google Scholar


Savage, H. M., M. S. Godsey, A. Lambert, N. A. Panella, K. L. Burkhalter, J. R. Harmon, R. R. Lash, D. C. Ashley, and W. L. Nicholson. 2013. First detection of heartland virus (Bunyaviridae: Phlebovirus) from field collected arthropods. Am. J. Trop. Med. Hyg. 89: 445–452. Google Scholar


Savage, H. M., M. S. Godsey, Jr, N. A. Panella, K. L. Burkhalter, D. C. Ashley, R. R. Lash, B. Ramsay, T. Patterson, and W. L. Nicholson. 2016. Surveillance for Heartland virus (Bunyaviridae: Phlebovirus) in Missouri during 2013: first detection of virus in adults of Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 53: 607–612. Google Scholar


Savage, H. M., K. L. Burkhalter, M. S. Godsey, Jr, N. A. Panella, D. C. Ashley, W. L. Nicholson, and A. J. Lambert. 2017. Bourbon virus in field-collected ticks, Missouri, USA. Emerg. Infect. Dis. 23: 2017–2022. Google Scholar


Schloss, P. D., S. L. Westcott, T. Ryabin, J. R. Hall, M. Hartmann, E. B. Hollister, R. A. Lesniewski, B. B. Oakley, D. H. Parks, C. J. Robinson, et al. 2009. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75: 7537–7541. Google Scholar


Schmieder, R., and R. Edwards. 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics. 27: 863–864. Google Scholar


Scholz, M. B., C. C. Lo, and P. S. Chain. 2012. Next generation sequencing and bioinformatic bottlenecks: the current state of metagenomic data analysis. Curr. Opin. Biotechnol. 23: 9–15. Google Scholar


Shapiro, M. R., C. L. Fritz, K. Tait, C. D. Paddock, W. L. Nicholson, K. F. Abramowicz, S. E. Karpathy, G. A. Dasch, J. W. Sumner, P. V. Adern, et al. 2010. Rickettsia 364D: A newly recognized cause of eschar-assodated illness in California. Clin. Infect. Dis. 50: 541–548. Google Scholar


Shi, M., X. D. Lin, J. H. Tian, L. J. Chen, X. Chen, C. X. Li, X. C. Qin, J. Li, J. P. Cao, J. S. Eden, et al. 2016a. Redefining the invertebrate RNA virosphere. Nature. 540: 539–543. Google Scholar


Shi, M., X. D. Lin, N. Vasilakis, J. H. Tian, C. X. Li, L. J. Chen, G. Eastwood, X. N. Diao, M. H. Chen, X. Chen, et al. 2016b. Divergent viruses discovered in arthropods and vertebrates revise the evolutionary history of the Flaviviridae and related viruses. J. Virol. 90: 659–669. Google Scholar


Smith, T., and F. L. Kilborne. 1893. Investigations into the nature, causation, and prevention of Texas or southern cattle fever. U.S. Dept. of Agriculture, Bureau of Animal Industry. Google Scholar


Socolovschi, C., O. Mediannikov, D. Raoult, and P. Parola. 2009. The relationship between spotted fever group rickettsiae and ixodid ticks. Vet. Res. 40: 34. Google Scholar


Solheim, W. G. 1949. Studies on Rocky Mountain Fungi—I. Mycologia. 41: 623–631. Google Scholar


Sonenshine, D. E. 2018. Range expansion of tick disease vectors in North America: Implications for spread of tick-borne disease. Int. J. Environ. Res. Public Health 15: 478. Google Scholar


Souza, W. M., M. J. Fumagalli, A. O. Torres Carrasco, M. F. Romeiro, S. Modha, M. C. Seki, J. M. Gheller, S. Daffre, M. R. T. Nunes, P. R. Murcia, et al. 2018. Viral diversity of Rhipicephalus microplus parasitizing cattle in southern Brazil. Sci. Rep. 8: 16315. Google Scholar


Spiegel, M., T. Plegge, and S. Pöhlmann. 2016. The role of phlebovirus glycoproteins in viral entry, assembly and release. Viruses. 8: 202. Google Scholar


St George, T. D., D. H. Cybinski, A. J. Main, N. McKilligan, and D. H. Kemp. 1984. Isolation of a new arbovirus from the tick Argas robertsi from a cattle egret (Bubulcus ibis coromandus) colony in Australia. Aust. J. Biol. Sci. 37: 85–89. Google Scholar


Steere, A. C., F. Strle, G. P. Wormser, L. T. Hu, J. A. Branda, J. W. R. Hovius, X. Li, and P. S. Mead. 2016. Lyme borreliosis. Nat. Rev. Dis. Prim. 2: 16090. Google Scholar


Steiner, F. E., R. R. Pinger, and C. N. Vann. 1999. Infection rates of Amblyomma americanum (Acari: Ixodidae) by Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) and prevalence of E. chaffeensis-reactive antibodies in white-tailed deer in southern Indiana, 1997. J. Med. Entomol. 36: 715–719. Google Scholar


Stromdahl, E. Y., J. Jiang, M. Vince, and A. L. Richards. 2011. Infrequency of Rickettsia rickettsii in Dermacentor variabilis removed from humans, with comments on the role of other human-biting ticks associated with spotted fever group Rickettsiae in the United States. Vector Borne Zoonotic Dis. 11: 969–977. Google Scholar


Tadin, A., R. Tokarz, A. Markotić, J. Margaletić, N. Turk, J. Habuš, P. Svoboda, M. Vucelja, A. Desai, K. Jain, et al. 2016. Molecular survey of zoonotic agents in rodents and other small mammals in Croatia. Am. J. Trop. Med. Hyg. 94: 466–473. Google Scholar


Takahashi, M., C. E. Yunker, C. M. Clifford, W. Nakano, N. Fujino, K. Tanifuji, and L. A. Thomas. 1982. Isolation and characterization of Midway virus: A new tick-borne virus related to nyamanini. J. Med. Virol. 10: 181–193. Google Scholar


Talagrand-Reboul, E., P. H. Boyer, S. Bergström, L. Vial, and N. Boulanger. 2018. Relapsing fevers: neglected tick-borne diseases. Front. Cell. Infect. Microbiol. 8: 98. Google Scholar


Taylor, R. M., H. S. Hurlbut, T. H. Work, J. R. Kingston, and H. Hoogstraal. 1966. Arboviruses isolated from ARGAS TICKS IN Egypt: Quaranfil, Chenuda, and Nyamanini. Am. J. Trop. Med. Hyg. 15: 76–86. Google Scholar


Telford, S. R., 3rd, and H. K. Goethert. 2020. Ecology of Francisella tularensis. Annu. Rev. Entomol. 65: 351–372. Google Scholar


Telford, S. R., 3rd, P. M. Armstrong, P. Katavolos, I. Foppa, A. S. Garcia, M. L. Wilson, and A. Spielman. 1997. A new tick-borne encephalitis-like virus infecting New England deer ticks, Ixodes dammini. Emerg. Infect. Dis. 3: 165–170. Google Scholar


Temmam, S., D. Chrétien, T. Bigot, E. Dufour, S. Petres, M. Desquesnes, E. Devillers, M. Dumarest, L. Yousfi, S. Jittapalapong, et al. 2019. Monitoring silent spillovers before emergence: a pilot study at the tick/human interface in Thailand. Front. Microbiol. 10: 2315. Google Scholar


Tijsse-Klasen, E., M. Fonville, L. Van Overbeek, J. H. Reimerink, and H. Sprong. 2010. Exotic rickettsiae in Ixodes ricinus: Fact or artifact? Parasites and Vectors 3: 54. Google Scholar


Tokarz, R., K. Jain, A. Bennett, T. Briese, and W. I. Lipkin. 2010. Assessment of polymicrobial infections in ticks in New York state. Vector Borne Zoonotic Dis. 10: 217–221. Google Scholar


Tokarz, R., S. Sameroff, M. S. Leon, K. Jain, and W. I. Lipkin. 2014a. Genome characterization of Long Island tick rhabdovirus, a new virus identified in Amblyomma americanum ticks. Virol. J. 11: 26. Google Scholar


Tokarz, R., S. H. Williams, S. Sameroff, M. Sanchez Leon, K. Jain, and W. I. Lipkin. 2014b. Virome analysis of Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate viruses. J. Virol. 88: 11480–11492. Google Scholar


Tokarz, R., T. Tagliafierro, D. M. Cucura, I. Rochlin, S. Sameroff, and W. I. Lipkin. 2017. Detection of Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, Borrelia miyamotoi, and Powassan Virus in Ticks by a Multiplex Real-Time Reverse Transcription-PCR Assay. mSphere. 2:e00151–17. Google Scholar


Tokarz, R., N. Mishra, T. Tagliafierro, S. Sameroff, A. Caciula, L. Chauhan, J. Patel, E. Sullivan, A. Gucwa, B. Fallon, et al. 2018a. A multiplex serologic platform for diagnosis of tick-borne diseases. Sci. Rep. 8: 3158. Google Scholar


Tokarz, R., S. Sameroff, T. Tagliafierro, K. Jain, S. H. Williams, D. M. Cucura, I. Rochlin, J. Monzon, G. Carpi, D. Tufts, et al. 2018b. Identification of Novel Viruses in Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis Ticks. mSphere. 3:e00614–00617. Google Scholar


Tokarz, R., T. Tagliafierro, S. Sameroff, D. M. Cucura, A. Oleynik, X. Che, K. Jain, and W. I. Lipkin. 2019. Microbiome analysis of Ixodes scapularis ticks from New York and Connecticut. Ticks Tick. Borne. Dis. 10: 894–900. Google Scholar


Tokarz, R., T. Tagliafierro, W. Ian Lipkin, and A. R. Marques. 2020. Characterization of a Monanema nematode in Ixodes scapularis. Parasites and Vectors 13: 371. Google Scholar


Toledo, A., P. Anda, R. Escudero, C. Larsson, S. Bergstrom, and J. L. Benach. 2010. Phylogenetic analysis of a virulent Borrelia species isolated from patients with relapsing fever. J. Clin. Microbiol. 48: 2484–2489. Google Scholar


Topolovec, J., D. Puntarić, A. Antolović-Pozgain, D. Vuković, Z. Topolovec, J. Milas, V. Drusko-Barisić, and M. Venus. 2003. Serologically detected “new” tick-borne zoonoses in eastern Croatia. Croat. Med. J. 44: 626–629. Google Scholar


Trout Fryxell, R. T., and J. M. DeBruyn. 2016. The microbiome of Ehrlichiainfected and uninfected lone star ticks (Amblyomma americanum). PLoS One 11:e0146651. Google Scholar


Van Treuren, W., L. Ponnusamy, R. J. Brinkerhoff, A. Gonzalez, C. M. Parobek, J. J. Juliano, T. G. Andreadis, R. C. Falco, L. B. Ziegler, N. Hathaway, et al. 2015. Variation in the microbiota of Ixodes ticks with regard to geography, species, and sex. Appl. Environ. Microbiol. 81: 6200–6209. Google Scholar


Vandegrift, K. J., and A. Kapoor. 2019. The ecology of new constituents of the tick virome and their relevance to public health. Viruses. 11: 529. Google Scholar


Vandegrift, K. J., A. Kumar, H. Sharma, S. Murthy, L. D. Kramer, S. Ostfeld, P. J. Hudson, and A. Kapoor. 2020. Presence of segmented flavivirus infections in North America. Emerg. Infect. Dis. 26: 1810–1817. Google Scholar


Walker, P. J., C. Firth, S. G. Widen, K. R. Blasdell, H. Guzman, T. G. Wood, P. N. Paradkar, E. C. Holmes, R. B. Tesh, and N. Vasilakis. 2015. Evolution of Genome Size and Complexity in the Rhabdoviridae. PLoS Pathog. 11:e1004664. Google Scholar


Wang, Z. D., B. Wang, F. Wei, S. Z. Han, L. Zhang, Z. T. Yang, Y. Yan, X. L. Lv, L. Li, S. C. Wang, et al. 2019. A new segmented virus associated with human febrile illness in China. N. Engl. J. Med. 380: 2116–2125. Google Scholar


Wang, S., T. Zhao, X. Yu, Z. Lin, X. Hua, and L. Cui. 2020. Characterization of tick viromes collected from dogs in China. Biosaf. Heal. 2: 79–88. Google Scholar


Wille, M., E. Harvey, M. Shi, D. Gonzalez-Acuña, E. C. Holmes, and A. C. Hurt. 2020. Sustained RNA virome diversity in Antarctic penguins and their ticks. ISME J. 14: 1768–1782. Google Scholar


Wood, H., L. Dillon, S. N. Patel, and F. Ralevski. 2016. Prevalence of Rickettsia species in Dermacentor variabilis ticks from Ontario, Canada. Ticks Tick. Borne. Dis. 7: 1044–1046. Google Scholar


Wormser, G. P., R. J. Dattwyler, E. D. Shapiro, J. J. Halperin, A. C. Steere, M. S. Klempner, P. J. Krause, J. S. Bakken, F. Strle, G. Stanek, et al. 2006. The clinical assessments treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: Clinical practice guidelines by the Infectious Diseases Society of America. Clin. Infect. Dis. 43: 1089–1134. Google Scholar


Woyke, T., A. Sczyrba, J. Lee, C. Rinke, D. Tighe, S. Clingenpeel, R. Malmstrom, R. Stepanauskas, and J. F. Cheng. 2011. Decontamination of MDA reagents for single cell whole genome amplification. PLoS One 6: e26161. Google Scholar


Wu, D., A. Hartman, N. Ward, and J. A. Eisen. 2008. An automated phylogenetic tree-based Small subunit rRNA Taxonomy and Alignment Pipeline (STAP). PLoS One 3: e2566. Google Scholar


Xia, H., C. Hu, D. Zhang, S. Tang, Z. Zhang, Z. Kou, Z. Fan, D. Bente, C. Zeng, and T. Li. 2015. Metagenomic profile of the viral communities in Rhipicephalus spp. ticks from Yunnan, China. PLoS One 10: e0121609. Google Scholar


Xu, B., N. Zhi, G. Hu, Z. Wan, X. Zheng, X. Liu, S. Wong, S. Kajigaya, K. Zhao, Q. Mao, et al. 2013. Hybrid DNA virus in Chinese patients with seronegative hepatitis discovered by deep sequencing. Proc. Natl. Acad. Sci. U. S. A. 110: 10264–10269. Google Scholar


Yendell, S. J., M. Fischer, and J. E. Staples. 2015. Colorado Tick Fever in the United States, 2002–2012. Vector Borne Zoonotic Dis. 15: 311–316. Google Scholar


Zhao, T., H. Gong, X. Shen, W. Zhang, T. Shan, X. Yu, S. J. Wang, and L. Cui. 2020. Comparison of viromes in ticks from different domestic animals in China. Virol. Sin. 35: 398–406. Google Scholar


Zhong, J. 2012. Coxiella-like endosymbionts. Adv. Exp. Med. Biol. 984: 365–379. Google Scholar


Zhong, J., A. Jasinskas, and A. G. Barbour. 2007. Antibiotic treatment of the tick vector Amblyomma americanum reduced reproductive fitness. PLoS One 2: e405. Google Scholar


Zlateva, K. T., K. M. Crusio, A. M. Leontovich, C. Lauber, E. Claas, A. A. Kravchenko, W. J. Spaan, and A. E. Gorbalenya. 2011. Design and validation of consensus-degenerate hybrid oligonucleotide primers for broad and sensitive detection of corona- and toroviruses. J. Virol. Methods 177: 174–183. Google Scholar
© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.
Rafal Tokarz and W. Ian Lipkin "Discovery and Surveillance of Tick-Borne Pathogens," Journal of Medical Entomology 58(4), 1525-1535, (12 December 2020).
Received: 19 August 2020; Accepted: 27 October 2020; Published: 12 December 2020
next-generation sequencing
pathogen discovery
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