Blood transfusion transmission of the tick-borne relapsing fever spirochete Borrelia miyamotoi in mice.

BACKGROUND: Borrelia miyamotoi, a recently discovered relapsing fever spirochete, occurs in hard-bodied ticks wherever Lyme disease is endemic. Human infection is associated with relapsing fever and can cause meningoencephalitis in immunocompromised patients. A few cases of transfusion transmission of other relapsing fever spirochete species have been reported but none for B. miyamotoi. Our objective was to determine whether B. miyamotoi transfusion transmission could occur in a murine transfusion model. Herein, we report transfusion transmission of B. miyamotoi through fresh or stored red blood cells (RBCs) in a mouse model. STUDY DESIGN AND METHODS: Inbred mice were transfused with B. miyamotoi-infected murine blood that was either freshly collected or stored for 7 days before transfusion. Recipient blood was then longitudinally examined after transfusion by smear and wet mount for evidence of spirochetemia. RESULTS: Motile spirochetes were observed in immunocompromised (SCID) mouse recipients for 28 days after transfusion of both fresh and stored murine B. miyamotoi-infected RBCs. Transient spirochetemia was observed in immunocompetent DBA/2 and C57BL/6 mice, with spirochete clearance occurring within 5 days after transfusion. CONCLUSION: These data demonstrate that transfusion transmission of B. miyamotoi can occur in mice and suggest that it also may occur in humans.

Monitoring human babesiosis emergence through vector surveillance New England, USA.

Human babesiosis is an emerging tick-borne disease caused by the intraerythrocytic protozoan Babesia microti. Its geographic distribution is more limited than that of Lyme disease, despite sharing the same tick vector and reservoir hosts. The geographic range of babesiosis is expanding, but knowledge of its range is incomplete and relies exclusively on reports of human cases. We evaluated the utility of tick-based surveillance for monitoring disease expansion by comparing the ratios of the 2 infections in humans and ticks in areas with varying B. microti endemicity. We found a close association between human disease and tick infection ratios in long-established babesiosis-endemic areas but a lower than expected incidence of human babesiosis on the basis of tick infection rates in new disease-endemic areas. This finding suggests that babesiosis at emerging sites is underreported. Vector-based surveillance can provide an early warning system for the emergence of human babesiosis.

Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States.

Borrelia miyamotoi sensu lato, a relapsing fever Borrelia sp., is transmitted by the same ticks that transmit B. burgdorferi (the Lyme disease pathogen) and occurs in all Lyme disease-endemic areas of the United States. To determine the seroprevalence of IgG against B. miyamotoi sensu lato in the northeastern United States and assess whether serum from B. miyamotoi sensu lato-infected persons is reactive to B. burgdorferi antigens, we tested archived serum samples from area residents during 1991-2012. Of 639 samples from healthy persons, 25 were positive for B. miyamotoi sensu lato and 60 for B. burgdorferi. Samples from ≈10% of B. miyamotoi sensu lato-seropositive persons without a recent history of Lyme disease were seropositive for B. burgdorferi. Our results suggest that human B. miyamotoi sensu lato infection may be common in southern New England and that B. burgdorferi antibody testing is not an effective surrogate for detecting B. miyamotoi sensu lato infection.

Host-Feeding Behavior of Mosquitoes in the Florida Everglades.

Background: West Nile virus (WNV), Everglades virus (EVEV), and five species of Orthobunyavirus were isolated from mosquitoes collected in the Everglades in 2016-2017. Prior studies of blood meals of mosquitoes in southern Florida have related findings to acquisition and transmission of EVEV, St. Louis encephalitis virus, and WNV, but not the Orthobunyavirus viruses associated with the subgenus Melanoconion of the genus Culex. Materials and Methods: In the present study, blood-fed mosquitoes were collected in the Everglades in 2016, 2017, 2021, and 2022, and from an industrial site in Naples, FL in 2017. Blood meals were identified to host species by PCR assays using mitochondrial cytochrome b gene. Results: Blood meals were identified from Anopheles crucians complex and 11 mosquito species captured in the Florida Everglades and from 3 species collected from an industrial site. The largest numbers of blood-fed specimens were from Culex nigripalpus, Culex erraticus, Culex cedecei, and Aedes taeniorhynchus. Cx. erraticus fed on mammals, birds, and reptiles, particularly American alligator. This mosquito species could transmit WNV to American alligator in the wild. Cx. nigripalpus acquired blood meals primarily from birds and mammals and frequently fed on medium-sized mammals and white-tailed deer. Water and wading birds were the primary avian hosts for Cx. nigripalpus and Cx. erraticus in the Everglades. Wading birds are susceptible to WNV and could serve as reservoir hosts. Cx. cedecei fed on five species of rodents, particularly black and hispid cotton rats. EVEV and three different species of Orthobunyavirus have been isolated from the hispid cotton rat and Cx. cedecei in the Everglades. Cx. cedecei is likely acquiring and transmitting these viruses among hispid cotton rats and other rodents. The marsh rabbit was a frequent host for An. crucians complex. An. crucians complex, and other species could acquire Tensaw virus from rabbits. Conclusions: Our study contributes to a better understanding of the host and viral associations of mosquito species in southwestern Florida.

Identification of Borrelia burgdorferi ospC genotypes in host tissue and feeding ticks by terminal restriction fragment length polymorphisms.

We developed a high-throughput method based on terminal restriction fragment length polymorphisms (T-RFLP) to identify ospC genotypes from field-collected samples of Borrelia burgdorferi. We first validated the method by analyzing B. burgdorferi ospC previously identified by sequencing. We then analyzed and compared ospC genotypes detected from ear biopsy tissue from natural populations of the white-footed mouse, a major B. burgdorferi reservoir host species in the eastern United States, and larval ticks feeding on those individual mice. The T-RFLP method enabled us to distinguish all 17 ospC genotypes tested, as well as mixed samples containing more than one genotype. Analysis costs compare favorably to those of alternative ospC identification methods. The T-RFLP method will facilitate large-scale field studies to advance our understanding of genotype-specific transmission patterns.

Everglades virus: an underrecognized disease-causing subtype of Venezuelan equine encephalitis virus endemic to Florida, USA.

Everglades virus (EVEV) is subtype II of the Venezuelan equine encephalitis virus (VEEV) complex (Togaviridae: Alphavirus), endemic to Florida, USA. EVEV belongs to a clade that includes both enzootic and epizootic/epidemic VEEV subtypes. Like other enzootic VEEV subtypes, muroid rodents are important vertebrate hosts for EVEV and certain mosquitoes are important vectors. The hispid cotton rat Sigmodon hispidus and cotton mouse Peromyscus gossypinus are important EVEV hosts, based on natural infection (virus isolation and high seropositivity), host competence (experimental infections), and frequency of contact with the vector. The mosquito Culex (Melanoconion) cecedei is the only confirmed vector of EVEV based upon high natural infection rates, efficient vector competence, and frequent feeding upon muroid rodents. Human disease attributed to EVEV is considered rare. However, cases of meningitis and encephalitis are recorded from multiple sites, separated by 250 km or more. Phylogenetic analyses indicate that EVEV is evolving, possibly due to changes in the mammal community. Mutations in the EVEV genome are of concern, given that epidemic strains of VEEV (subtypes IAB and IC) are derived from enzootic subtype ID, the closest genetic relative of EVEV. Should epizootic mutations arise in EVEV, the abundance of Aedes taeniorhynchus and other epizootic VEEV vectors in southern Florida provides a conducive environment for widespread transmission. Other factors that will likely influence the distribution and frequency of EVEV transmission include the establishment of Culex panocossa in Florida, Everglades restoration, mammal community decline due to the Burmese python, land use alteration by humans, and climate change.

A ticking time bomb hidden in plain sight.

The deer tick transmits nearly half of the known tick-borne pathogens in the United States, and its expanding geographic range increases the risk of human infection. To decrease the abundance of and infection risk from deer ticks, approaches that include vaccines for human use and for animal hosts are desired.

An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands.

Anaplasma phagocytophilum is the agent of human anaplasmosis, the second most common tick-borne illness in the United States. This pathogen, which is closely related to obligate intracellular organisms in the genera Rickettsia, Ehrlichia, and Anaplasma, persists in ticks and mammalian hosts; however, the mechanisms for survival in the arthropod are not known. We now show that A. phagocytophilum induces expression of the Ixodes scapularis salp16 gene in the arthropod salivary glands during vector engorgement. RNA interference-mediated silencing of salp16 gene expression interfered with the survival of A. phagocytophilum that entered ticks fed on A. phagocytophilum-infected mice. A. phagocytophilum migrated normally from A. phagocytophilum-infected mice to the gut of engorging salp16-deficient ticks, but up to 90% of the bacteria that entered the ticks were not able to successfully infect I. scapularis salivary glands. These data demonstrate the specific requirement of a pathogen for a tick salivary protein to persist within the arthropod and provide a paradigm for understanding how Rickettsia-like pathogens are maintained within vectors.

Phylogeography of Borrelia burgdorferi in the eastern United States reflects multiple independent Lyme disease emergence events.

Since its first description in coastal Connecticut in 1976, both the incidence of Lyme disease and the geographic extent of endemic areas in the US have increased dramatically. The rapid expansion of Lyme disease into its current distribution in the eastern half of the US has been due to the range expansion of the tick vector, Ixodes scapularis, upon which the causative agent, Borrelia burgdorferi is dependent for transmission to humans. In this study, we examined the phylogeographic population structure of B. burgdorferi throughout the range of I. scapularis-borne Lyme disease using multilocus sequence typing based on bacterial housekeeping genes. We show that B. burgdorferi populations from the Northeast and Midwest are genetically distinct, but phylogenetically related. Our findings provide strong evidence of prehistoric population size expansion and east-to-west radiation of descendent clones from founding sequence types in the Northeast. Estimates of the time scale of divergence of northeastern and midwestern populations suggest that B. burgdorferi was present in these regions of North America many thousands of years before European settlements. We conclude that B. burgdorferi populations have recently reemerged independently out of separate relict foci, where they have persisted since precolonial times.

Seasonal Dynamics of Mosquito-Borne Viruses in the Southwestern Florida Everglades, 2016, 2017.

Mosquitoes were collected for 12 consecutive months beginning June 2016, from 11 locations in the Florida Everglades, Collier County, and tested for viruses by isolation in Vero cells and subsequent identification. One species complex and 31 species of mosquitoes were identified from 668,809 specimens. Ochlerotatus taeniorhynchus comprised 72.2% of the collection. Other notable species were Anopheles crucians complex, Culex nigripalpus, Cx. erraticus, and Cx. cedecei. Seven species of virus were identified from 110 isolations: Everglades, Gumbo Limbo, Mahogany Hammock, Pahayokee, Shark River, Tensaw, and West Nile viruses. Everglades, West Nile, Tensaw, and Mahogany Hammock viruses were most frequently isolated. Largest numbers of viruses were identified from Cx. cedecei, Cx. nigripalpus, and An. crucians complex. Five species of virus were isolated from Cx. cedecei. Viruses were isolated from mangrove, cypress swamp, hardwood hammock, and sawgrass habitats. West Nile virus was isolated August through October when Cx. nigripalpus was most abundant. Everglades virus was the most frequently isolated virus from nine species of mosquitoes collected from June through August. Tensaw virus was isolated primarily from Anopheles species. Isolations were made in July, August, January, February, and April, suggesting that this virus may be present in host-seeking mosquitoes throughout the year. Mahogany Hammock, Shark River, Gumbo Limbo, and Pahayokee viruses were isolated primarily from Cx. cedecei from June through December. Shotgun metagenomic sequencing was used to document that seven pools of Cx. cedecei were infected with two arboviruses. As communities expand into the Everglades, more humans will become exposed to arboviruses.

Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia.

Borrelia miyamotoi is distantly related to B. burgdorferi and transmitted by the same hard-body tick species. We report 46 cases of B. miyamotoi infection in humans and compare the frequency and clinical manifestations of this infection with those caused by B. garinii and B. burgdorferi infection. All 46 patients lived in Russia and had influenza-like illness with fever as high as 39.5°C; relapsing febrile illness occurred in 5 (11%) and erythema migrans in 4 (9%). In Russia, the rate of B. miyamotoi infection in Ixodes persulcatus ticks was 1%-16%, similar to rates in I. ricinus ticks in western Europe and I. scapularis ticks in the United States. B. miyamotoi infection may cause relapsing fever and Lyme disease-like symptoms throughout the Holarctic region of the world because of the widespread prevalence of this pathogen in its ixodid tick vectors.

The Lyme disease agent exploits a tick protein to infect the mammalian host.

The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick-mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.

MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi.

Lyme borreliosis, caused by the tick-borne bacterium Borrelia burgdorferi, has become the most common vector-borne disease in North America over the last three decades. To understand the dynamics of the epizootic spread and to predict the evolutionary trajectories of B. burgdorferi, accurate information on the population structure and the evolutionary relationships of the pathogen is crucial. We, therefore, developed a multilocus sequence typing (MLST) scheme for B. burgdorferi based on eight chromosomal housekeeping genes. We validated the MLST scheme on B. burgdorferi specimens from North America and Europe, comprising both cultured isolates and infected ticks. These data were compared with sequences for the commonly used genetic markers rrs-rrlA intergenic spacer (IGS) and the gene encoding the outer surface protein C (ospC). The study demonstrates that the concatenated sequences of the housekeeping genes of B. burgdorferi provide highly resolved phylogenetic signals and that the housekeeping genes evolve differently compared with the IGS locus and ospC. Using sequence data, the study reveals that North American and European populations of B. burgdorferi correspond to genetically distinct populations. Importantly, the MLST data suggest that B. burgdorferi originated in Europe rather than in North America as proposed previously.

Emergence potential of mosquito-borne arboviruses from the Florida Everglades.

The Greater Everglades Region of South Florida is one of the largest natural wetlands and the only subtropical ecosystem found in the continental United States. Mosquitoes are seasonally abundant in the Everglades where several potentially pathogenic mosquito-borne arboviruses are maintained in natural transmission cycles involving vector-competent mosquitoes and reservoir-competent vertebrate hosts. The fragile nature of this ecosystem is vulnerable to many sources of environmental change, including a wetlands restoration project, climate change, invasive species and residential development. In this study, we obtained baseline data on the distribution and abundance of both mosquitos and arboviruses occurring in the southern Everglades region during the summer months of 2013, when water levels were high, and in 2014, when water levels were low. A total of 367,060 mosquitoes were collected with CO2-baited CDC light traps at 105 collection sites stratified among the major landscape features found in Everglades National Park, Big Cypress National Preserve, Fakahatchee State Park Preserve and Picayune State Forest, an area already undergoing restoration. A total of 2,010 pools of taxonomically identified mosquitoes were cultured for arbovirus isolation and identification. Seven vertebrate arboviruses were isolated: Everglades virus, Tensaw virus, Shark River virus, Gumbo Limbo virus, Mahogany Hammock virus, Keystone virus, and St. Louis encephalitis virus. Except for Tensaw virus, which was absent in 2013, the remaining viruses were found to be most prevalent in hardwood hammocks and in Fakahatchee, less prevalent in mangroves and pinelands, and absent in cypress and sawgrass. In contrast, in the summer of 2014 when water levels were lower, these arboviruses were far less prevalent and only found in hardwood hammocks, but Tensaw virus was present in cypress, sawgrass, pinelands, and a recently burned site. Major environmental changes are anticipated in the Everglades, many of which will result in increased water levels. How these might lead to the emergence of arboviruses potentially pathogenic to both humans and wildlife is discussed.

Langerhans cell deficiency impairs Ixodes scapularis suppression of Th1 responses in mice.

Ixodes scapularis ticks transmit a number of human pathogens, including the Lyme disease spirochete Borrelia burgdorferi. I. scapularis suppresses host immunity in the skin to promote feeding and systemically skew T-helper (Th)-cell differentiation toward Th2 cells in secondary lymphoid organs. Although components of tick saliva are known to influence Th-cell polarization, the mechanism whereby tick feeding in the skin modulates regional and systemic Th-cell responses is unknown. In this study, the role of the epidermal Langerhans cell (LC) subset of skin dendritic cells in tick-mediated Th1/Th2-cell immunomodulation was assessed. Mice deficient in LCs (Langerin-DTA mice) exhibited enhanced lymph node (LN) concanavalin A (ConA)-induced Th1 responses after tick infestation in comparison to results for uninfested Langerin-DTA or wild-type (WT) mice, whereas effects on Th2-cell production of interleukin 4 were more variable. Nonetheless, the altered T-cell response did not impact tick feeding or refeeding. Gamma interferon production by ConA-stimulated LN cells of both WT and LC-deficient mice was enhanced by as much as fourfold after B. burgdorferi-infected-tick feeding, indicating that immunomodulatory effects of tick saliva were not able to attenuate the Th1 immune responses induced by this pathogen. Taken together, these findings show a requirement for LCs in the tick-mediated attenuation of Th1 responses in regional lymph nodes but not in the spleens of mice and show that the presence of a pathogen can overcome the Th1-inhibitory effects of tick feeding on the host.

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks.

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.

Climate and tick seasonality are predictors of Borrelia burgdorferi genotype distribution.

The blacklegged tick, Ixodes scapularis, is of significant public health importance as a vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The timing of seasonal activity of each immature I. scapularis life stage relative to the next is critical for the maintenance of B. burgdorferi because larvae must feed after an infected nymph to efficiently acquire the infection from reservoir hosts. Recent studies have shown that some strains of B. burgdorferi do not persist in the primary reservoir host for more than a few weeks, thereby shortening the window of opportunity between nymphal and larval feeding that sustains their enzootic maintenance. We tested the hypothesis that climate is predictive of geographic variation in the seasonal activity of I. scapularis, which in turn differentially influences the distribution of B. burgdorferi genotypes within the geographic range of I. scapularis. We analyzed the relationships between climate, seasonal activity of I. scapularis, and B. burgdorferi genotype frequency in 30 geographically diverse sites in the northeastern and midwestern United States. We found that the magnitude of the difference between summer and winter daily temperature maximums was positively correlated with the degree of seasonal synchrony of the two immature stages of I. scapularis. Genotyping revealed an enrichment of 16S-23S rRNA intergenic spacer restriction fragment length polymorphism sequence type 1 strains relative to others at sites with lower seasonal synchrony. We conclude that climate-associated variability in the timing of I. scapularis host seeking contributes to geographic heterogeneities in the frequencies of B. burgdorferi genotypes, with potential consequences for Lyme borreliosis morbidity.