Prevalence and Genetic Diversity of Bartonella Spp. in Northern Bats (Eptesicus nilssonii) and Their Blood-Sucking Ectoparasites in Hokkaido, Japan.

We investigated the prevalence of Bartonella in 123 northern bats (Eptesicus nilssonii) and their ectoparasites from Hokkaido, Japan. A total of 174 bat fleas (Ischnopsyllus needhami) and two bat bugs (Cimex japonicus) were collected from the bats. Bartonella bacteria were isolated from 32 (26.0%) of 123 bats. Though Bartonella DNA was detected in 79 (45.4%) of the bat fleas, the bacterium was isolated from only one bat flea (0.6%). The gltA sequences of the isolates were categorized into genotypes I, II, and III, which were found in both bats and their fleas. The gltA sequences of genotypes I and II showed 97.6% similarity with Bartonella strains from a Finnish E. nilssonii and a bat flea from a E. serotinus in the Netherlands. The rpoB sequences of the genotypes showed 98.9% similarity with Bartonella strain 44722 from E. serotinus in Republic of Georgia. The gltA and rpoB sequences of genotype III showed 95.9% and 96.7% similarity with Bartonella strains detected in shrews in Kenya and France, respectively. Phylogenetic analysis revealed that Bartonella isolates of genotypes I and II clustered with Bartonella strains from Eptesicus bats in Republic of Georgia and Finland, Myotis bats in Romania and the UK, and a bat flea from an Eptesicus bat in Finland. In contrast, genotype III formed a clade with B. florencae, B. acomydis, and B. birtlesii. These data suggest that northern bats in Japan harbor two Bartonella species and the bat flea serves as a potential vector of Bartonella transmission among the bats.

Detection of Zoonotic Bartonella Pathogens in Rabbit Fleas, Colorado, USA.

We detected 3 Bartonella species in wild rabbit fleas from Colorado, USA: B. vinsonii subsp. berkhoffii (n = 16), B. alsatica (n = 5), and B. rochalimae (n = 1). Our results support the establishment of the zoonotic agent B. alsatica in North America.

Ticks and tick-borne pathogens of dogs along an elevational and land-use gradient in Chiriquí province, Panamá.

Systematic acarological surveys are useful tools in assessing risk to tick-borne infections, especially in areas where consistent clinical surveillance for tick-borne disease is lacking. Our goal was to identify environmental predictors of tick burdens on dogs and tick-borne infectious agents in dog-derived ticks in the Chiriquí Province of western Panama to draw inferences about spatio-temporal variation in human risk to tick-borne diseases. We used a model-selection approach to test the relative importance of elevation, human population size, vegetative cover, and change in landuse on patterns of tick parasitism on dogs. We collected 2074 ticks, representing four species (Rhipicephalus sanguineus, R. microplus, Amblyomma ovale, and Ixodes boliviensis) from 355 dogs. Tick prevalence ranged from 0 to 74% among the sites we sampled, and abundance ranged from 0 to 20.4 ticks per dog with R. sanguineus s.l. being the most commonly detected tick species (97% of all ticks sampled). Whereas elevation was the best single determinant of tick prevalence and abundance on dogs, the top models also included predictor variables describing vegetation cover and landuse change. Specifically, low-elevation areas associated with decreasing vegetative cover were associated with highest tick occurrence on dogs, potentially because of the affinity of R. sanguineus for human dwellings. Although we found low prevalence of tick-borne pathogen genera (two Rickettsia-positive ticks, no R. rickettsia or Ehrlichia spp.) in our study, all of the tick species we collected from dogs are known vectors of zoonotic pathogens. In areas where epidemiological surveillance infrastructure is limited, field-based assessments of acarological risk can be useful and cost-effective tools in efforts to identify high-risk environments for tick-transmitted pathogens.

Ecological Traits Driving the Outbreaks and Emergence of Zoonotic Pathogens.

Infectious diseases that are transmitted from wildlife hosts to humans, such as the Ebola virus and MERS virus, can be difficult to understand because the pathogens emerge from complex multifaceted ecological interactions. We use a wildlife-pathogen system-prairie dogs (Cynomys ludovicianus) and the plague bacterium (Yersinia pestis)-to describe aspects of disease ecology that apply to many cases of emerging infectious disease. We show that the monitoring and surveillance of hosts and vectors during the buildup to disease outbreaks are crucial for understanding pathogen-transmission dynamics and that a community-ecology framework is important to identify reservoir hosts. Incorporating multidisciplinary approaches and frameworks may improve wildlife-pathogen surveillance and our understanding of seemingly sporadic and rare pathogen outbreaks.

Variation in the Microbiota of Ixodes Ticks with Regard to Geography, Species, and Sex.

Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.

Lyme disease, Virginia, USA, 2000-2011.

Lyme disease, caused by the bacterium Borrelia burgdorferi and transmitted in the eastern United States by the black-legged tick (Ixodes scapularis), is increasing in incidence and expanding geographically. Recent environmental modeling based on extensive field collections of host-seeking I. scapularis ticks predicted a coastal distribution of ticks in mid-Atlantic states and an elevational limit of 510 m. However, human Lyme disease cases are increasing most dramatically at higher elevations in Virginia, a state where Lyme disease is rapidly emerging. Our goal was to explore the apparent incongruity, during 2000-2011, between human Lyme disease data and predicted and observed I. scapularis distribution. We found significantly higher densities of infected ticks at our highest elevation site than at lower elevation sites. We also found that I. scapularis ticks in Virginia are more closely related to northern than to southern tick populations. Clinicians and epidemiologists should be vigilant in light of the changing spatial distributions of risk.

Release the hens: a study on the complexities of guinea fowl as tick control.

Established tick control strategies often involve methods that can be damaging to existing environmental conditions or natural host ecology. To find more environmentally friendly methods, biological controls, like predators of ticks, have been suggested. There are natural predators of ticks, but most are generalists and not expected to control tick populations. Helmeted guinea fowl (Numida meleagris (L.) (Galliformes: Numididae)) have been suggested to be biological controls of ticks, and therefore, tick-borne pathogens, but their potential role as hosts for ticks complicates the relationship. A study was conducted to assess whether guinea fowl reduces the abundance of lone star ticks, Amblyomma americanum (L.) (Acari: Ixodidae), or whether they are hosts of ticks. Using mark-recapture techniques, painted lone star ticks were placed into 3 different treatments: penned, excluded, and free range. The recapture rates of painted ticks were compared. There was a significant difference between excluded and free-range treatments, but not between excluded and penned or between free range and penned. To investigate the role of guinea fowl as hosts of ticks, coop floors were examined for engorged ticks. Engorged lone star nymphs that had fed on guinea fowl were found. Lastly, ticks collected were tested to identify the potential reduction in risk of tick-borne pathogens. This study found no evidence that guinea fowl are an effective biological control of lone star ticks or tick-borne pathogens, but they are hosts of lone star nymphs. Future studies are needed to assess the complex ecology of a biological control of ticks that is also a host.

Use of mammalian museum specimens to test hypotheses about the geographic expansion of Lyme disease in the southeastern United States.

Lyme disease, caused primarily in North America by the bacterium Borrelia burgdorferi sensu stricto, is the most frequently reported vector-borne disease in North America and its geographic extent is increasing in all directions from foci in the northeastern and north central United States. Several southeastern states, including Virginia and North Carolina, have experienced large increases in Lyme disease incidence in the past two decades, with the biggest changes in incidence occurring in the western portion of each state. We tested the hypothesis that B. burgdorferi s.s. was present in western Virginia and North Carolina Peromyscus leucopus populations prior to the recent emergence of Lyme disease. Specifically, we examined archived P. leucopus museum specimens, sampled between 1900 and 2000, for B. burgdorferi s.s. DNA. After confirming viability of DNA extracted from ear punch biopsies from P. leucopus study skins collected between 1945 and 2000 in 19 Virginia counties and 17 North Carolina counties, we used qPCR of two species-specific loci to test for the presence of B. burgdorferi s.s. DNA. Ten mice, all collected from the Eastern Shore of Virginia in 1989, tested positive for presence of B. burgdorferi; all of the remaining 344 specimens were B. burgdorferi-negative. Our results suggest that B. burgdorferi s.s was not common in western Virginia or North Carolina prior to the emergence of Lyme disease cases in the past two decades. Rather, the emergence of Lyme disease in this region has likely been driven by the relatively recent expansion of B. burgdorferi s.s. in southward-moving ticks and reservoir hosts in the mountainous counties of these two states.

Population genetic structure of the prairie dog flea and plague vector, Oropsylla hirsuta.

Oropsylla hirsuta is the primary flea of the black-tailed prairie dog and is a vector of the plague bacterium, Yersinia pestis. We examined the population genetic structure of O. hirsuta fleas collected from 11 prairie dog colonies, 7 of which had experienced a plague-associated die-off in 1994. In a sample of 332 O. hirsuta collected from 226 host individuals, we detected 24 unique haplotype sequences in a 480 nucleotide segment of the cytochrome oxidase II gene. We found significant overall population structure but we did not detect a signal of isolation by distance, suggesting that O. hirsuta may be able to disperse relatively quickly at the scale of this study. All 7 colonies that were recently decimated by plague showed signs of recent population expansion, whereas 3 of the 4 plague-negative colonies showed haplotype patterns consistent with stable populations. These results suggest that O. hirsuta populations are affected by plague-induced prairie dog die-offs and that flea dispersal among prairie dog colonies may not be dependent exclusively on dispersal of prairie dogs. Re-colonization following plague events from plague-free refugia may allow for rapid flea population expansion following plague epizootics.

Regional variation in immature Ixodes scapularis parasitism on North American songbirds: implications for transmission of the Lyme pathogen, Borrelia burgdorferi.

Borrelia burgdorferi, the etiological agent of Lyme disease, is transmitted among hosts by the black-legged tick, Ixodes scapularis, a species that regularly parasitizes various vertebrate hosts, including birds, in its immature stages. Lyme disease risk in the United States is highest in the Northeast and in the upper Midwest where I. scapularis ticks are most abundant. Because birds might be important to the range expansion of I. scapularis and B. burgdorferi, we explored spatial variation in patterns of I. scapularis parasitism on songbirds, as well as B. burgdorferi infection in bird-derived I. scapularis larvae. We sampled birds at 23 sites in the eastern United States to describe seasonal patterns of I. scapularis occurrence on birds, and we screened a subset of I. scapularis larvae for presence of B. burgdorferi. Timing of immature I. scapularis occurrence on birds is consistent with regional variation in host-seeking activity with a generally earlier peak in larval parasitism on birds in the Midwest. Significantly more I. scapularis larvae occurred on birds that were contemporaneously parasitized by nymphs in the Midwest than the Northeast, and the proportion of birds that yielded B. burgdorferi-infected larvae was also higher in the Midwest. We conclude that regional variation in immature I. scapularis phenology results in different temporal patterns of parasitism on birds, potentially resulting in differential importance of birds to B. burgdorferi transmission dynamics among regions.

No evidence for enzootic plague within black-tailed prairie dog (Cynomys ludovicianus) populations.

Yersinia pestis, causative agent of plague, occurs throughout the western United States in rodent populations and periodically causes epizootics in susceptible species, including black-tailed prairie dogs (Cynomys ludovicianus). How Y. pestis persists long-term in the environment between these epizootics is poorly understood but multiple mechanisms have been proposed, including, among others, a separate enzootic transmission cycle that maintains Y. pestis without involvement of epizootic hosts and persistence of Y. pestis within epizootic host populations without causing high mortality within those populations. We live-trapped and collected fleas from black-tailed prairie dogs and other mammal species from sites with and without black-tailed prairie dogs in 2004 and 2005 and tested all fleas for presence of Y. pestis. Y. pestis was not detected in 2126 fleas collected in 2004 but was detected in 294 fleas collected from multiple sites in 2005, before and during a widespread epizootic that drastically reduced black-tailed prairie dog populations in the affected colonies. Temporal and spatial patterns of Y. pestis occurrence in fleas and genotyping of Y. pestis present in some infected fleas suggest Y. pestis was introduced multiple times from sources outside the study area and once introduced, was dispersed between several sites. We conclude Y. pestis likely was not present in these black-tailed prairie dog colonies prior to epizootic activity in these colonies. Although we did not identify likely enzootic hosts, we found evidence that deer mice (Peromyscus maniculatus) may serve as bridging hosts for Y. pestis between unknown enzootic hosts and black-tailed prairie dogs.

Genotypic diversity of Borrelia burgdorferi strains detected in Ixodes scapularis larvae collected from North American songbirds.

We genotyped Borrelia burgdorferi strains detected in larvae of Ixodes scapularis removed from songbirds and compared them with those found in host-seeking I. scapularis nymphs sampled throughout the eastern United States. Birds are capable of transmitting most known genotypes, albeit at different frequencies than expected based on genotypes found among host-seeking nymphs.

Vector-Borne Pathogens in Ectoparasites Collected from High-Elevation Pika Populations.

The American pika, Ochotona princeps, is projected to decline throughout North America as climate change reduces its range, and pikas have already disappeared from several locations. In addition to climate, disease spillover from lower elevation mammalian species might affect pikas. We sampled pika fleas in Colorado and Montana across elevations ranging from 2896 to 3612 m and screened them for the presence of DNA from rodent-associated bacterial pathogens (Bartonella species and Yersinia pestis) to test the hypothesis that flea exchange between pikas and rodents may lead to occurrence of rodent-associated pathogens in pika ectoparasites. We collected 275 fleas from 74 individual pikas at 5 sites in Colorado and one site in Montana. We found that 5.5% of 275 pika fleas in this study tested positive for rodent-associated Bartonella DNA but that variation in Bartonella infection prevalence in fleas among sites was not driven by elevation. Specifically, we detected DNA sequences from two loci (gltA and rpoB) that are most similar to Bartonella grahamii isolates collected from rodents in Canada. We did not detect Y. pestis DNA in our survey. Our results demonstrate evidence of rodent-associated flea-borne bacteria in pika fleas. These findings are also consistent with the hypothesis that rodent-associated pathogens could be acquired by pikas. Flea-borne pathogen spillover from rodents to pikas has the potential to exacerbate the more direct effects of climate that have been suggested to drive pika declines.

Factors affecting the microbiome of Ixodes scapularis and Amblyomma americanum.

The microbial community composition of disease vectors can impact pathogen establishment and transmission as well as on vector behavior and fitness. While data on vector microbiota are accumulating quickly, determinants of the variation in disease vector microbial communities are incompletely understood. We explored the microbiome of two human-biting tick species abundant in eastern North America (Amblyomma americanum and Ixodes scapularis) to identify the relative contribution of tick species, tick life stage, tick sex, environmental context and vertical transmission to the richness, diversity, and species composition of the tick microbiome. We sampled 89 adult and nymphal Ixodes scapularis (N = 49) and Amblyomma americanum (N = 40) from two field sites and characterized the microbiome of each individual using the v3-v4 hypervariable region of the 16S rRNA gene. We identified significant variation in microbial community composition due to tick species and life stage with lesser impact of sampling site. Compared to unfed nymphs and males, the microbiome of engorged adult female I. scapularis, as well as the egg masses they produced, were low in bacterial richness and diversity and were dominated by Rickettsia, suggesting strong vertical transmission of this genus. Likewise, microbiota of A. americanum nymphs and males were more diverse than those of adult females. Among bacteria of public health importance, we detected several different Rickettsia sequence types, several of which were distinct from known species. Borrelia was relatively common in I. scapularis but did not show the same level of sequence variation as Rickettsia. Several bacterial genera were significantly over-represented in Borrelia-infected I. scapularis, suggesting a potential interaction of facilitative relationship between these taxa; no OTUs were under-represented in Borrelia-infected ticks. The systematic sampling we conducted for this study allowed us to partition the variation in tick microbial composition as a function of tick- and environmentally-related factors. Upon more complete understanding of the forces that shape the tick microbiome it will be possible to design targeted experimental studies to test the impacts of individual taxa and suites of microbes on vector-borne pathogen transmission and on vector biology.

Life history characteristics of birds influence patterns of tick parasitism.

Introduction: Birds serve as reservoirs for tick-borne pathogens as well as hosts for multiple tick species of public health relevance.  Birds may perpetuate life cycles of vectors and vector-borne pathogens and disperse disease vectors over long distances, supplementing populations at range margins or seeding invading populations beyond the edges of current tick distributions.  Our goal for this study was to identify life history characteristics of birds that most strongly affect tick parasitism. Materials and Methods: We collected 6203 ticks from 5426 birds from two sites in eastern North America and used field-derived parasitism data and published literature to analyze impacts of life history factors on tick parasitism in birds. Results and Discussion: We identified body size and nest site to have the strongest impact on tick prevalence and abundance in the songbird species included in this study.  Our findings reveal site-independent patterns in tick parasitism on birds and suggest that physical more than behavioral characteristics may influence a bird species' suitability as a host for ticks. Conclusions: The data and results published here will contribute to a growing body of literature and information on bird-tick interactions and will help elucidate patterns of tick and tick-borne pathogen geographic expansion.

Using Landscape Analysis to Test Hypotheses about Drivers of Tick Abundance and Infection Prevalence with Borrelia burgdorferi.

Patterns of vector-borne disease risk are changing globally in space and time and elevated disease risk of vector-borne infection can be driven by anthropogenic modification of the environment. Incidence of Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, has risen in a number of locations in North America and this increase may be driven by spatially or numerically expanding populations of the primary tick vector, Ixodes scapularis. We used a model selection approach to identify habitat fragmentation and land-use/land cover variables to test the hypothesis that the amount and configuration of forest cover at spatial scales relevant to deer, the primary hosts of adult ticks, would be the predominant determinants of tick abundance. We expected that land cover heterogeneity and amount of forest edge, a habitat thought to facilitate deer foraging and survival, would be the strongest driver of tick density and that larger spatial scales (5-10 km) would be more important than smaller scales (1 km). We generated metrics of deciduous and mixed forest fragmentation using Fragstats 4.4 implemented in ArcMap 10.3 and found, after adjusting for multicollinearity, that total forest edge within a 5 km buffer had a significant negative effect on tick density and that the proportion of forested land cover within a 10 km buffer was positively associated with density of I. scapularis nymphs. None of the 1 km fragmentation metrics were found to significantly improve the fit of the model. Elevation, previously associated with increased density of I. scapularis nymphs in Virginia, while significantly predictive in univariate analysis, was not an important driver of nymph density relative to fragmentation metrics. Our results suggest that amount of forest cover (i.e., lack of fragmentation) is the most important driver of I. scapularis density in our study system.

Borrelia miyamotoi, Other Vector-Borne Agents in Cat Blood and Ticks in Eastern Maryland.

We collected blood and tick samples in eastern Maryland to quantify vector-borne pathogen exposure and infection in healthy cats and to assess occupational disease risk to veterinary professionals and others who regularly interact with household pets. Thirty-six percent of healthy cats parasitized by ticks at time of examination (9/25) were exposed to, and 14% of bloods (7/49) tested PCR-positive for, at least one vector-borne pathogen including several bloods and ticks with Borrelia miyamotoi, a recently recognized tick-borne zoonotic bacterium. There was no indication that high tick burdens were associated with exposure to vector-borne pathogens. Our results underscore the potential importance of cats to human vector-borne disease risk.

Abundance patterns of two Oropsylla (Ceratophyllidae: Siphonaptera) species on black-tailed prairie dog (Cynomys ludovicianus) hosts.

Behavioral, genetic, and immune variation within a host population may lead to aggregation of parasites whereby a small proportion of hosts harbor a majority of parasites. In situations where two or more parasite species infect the same host population there is the potential for interaction among parasites that could potentially influence patterns of aggregation through either competition or facilitation. We studied the occurrence and abundance patterns of two congeneric flea species on black-tailed prairie dog (Cynomys ludovicianus) hosts to test for interactions among parasite species. We live-trapped prairie dogs on ten sites in Boulder County, CO and collected their fleas. We found a non-random, positive association between the two flea species, Oropsylla hirsuta and O. tuberculata cynomuris; hosts with high loads of one flea species had high loads of the second species. This result suggests that there is no interspecific competition among fleas on prairie dog hosts. Host weight had a weak negative relationship to flea load and host sex did not influence flea load, though there were slight differences in flea prevalence and abundance between male and female C. ludovicianus. While genetic and behavioral variation among hosts may predispose certain individuals to infection, our results indicate apparent facilitation among flea species that may result from immune suppression or other flea-mediated factors.

Geographic Expansion of Lyme Disease in the Southeastern United States, 2000-2014.

Background. The majority of Lyme disease cases in the United States are acquired on the east coast between northern Virginia and New England. In recent years the geographic extent of Lyme disease has been expanding, raising the prospect of Lyme disease becoming endemic in the southeast. Methods. We collected confirmed and probable cases of Lyme disease from 2000 through 2014 from the Virginia Department of Health and North Carolina Department of Public Health and entered them in a geographic information system. We performed spatial and spatiotemporal cluster analyses to characterize Lyme disease expansion. Results. There was a marked increase in Lyme disease cases in Virginia, particularly from 2007 onwards. Northern Virginia experienced intensification and geographic expansion of Lyme disease cases. The most notable area of expansion was to the southwest along the Appalachian Mountains with development of a new disease cluster in the southern Virginia mountain region. Conclusions. The geographic distribution of Lyme disease cases significantly expanded in Virginia between 2000 and 2014, particularly southward in the Virginia mountain ranges. If these trends continue, North Carolina can expect autochthonous Lyme disease transmission in its mountain region in the coming years.

Population genetic structure of the Lyme disease vector Ixodes scapularis at an apparent spatial expansion front.

Modeling and empirical evidence suggests that Lyme disease is undergoing geographic expansion from principal foci in the midwestern and northeastern United States. Virginia is at the southern edge of the current expansion zone and has seen dramatic rise in human Lyme disease cases since 2007, potentially owing to a recent increase in vector abundance. Ixodes scapularis is known throughout the eastern US but behavioral or physiological variation between northern and southern lineages might lead northern-variant ticks to more frequently parasitize humans. We hypothesized that recent spatial and numerical increase in Lyme disease cases is associated with demographic and/or spatial expansion of I. scapularis and that signals of these phenomena would be detectable and discernable in population genetic signals. In summer and fall 2011, we collected nymphal I. scapularis by drag sampling and adult I. scapularis from deer carcasses at hunting check stations at nine sites arranged along an east-west transect through central Virginia. We analyzed 16S mtDNA sequences data from up to 24 I. scapularis individuals collected from each site and detected a total of 24 haplotypes containing 29 segregating sites. We found no evidence for population genetic structure among these sites but we did find strong signals of both demographic and spatial expansion throughout our study system. We found two haplotypes (one individual each) representing a lineage of ticks that is only found in the southeastern United States, with the remaining individuals representing a less genetically diverse clade that is typical of the northern United States, but that has also been detected in the American South. Taken together, these results lead us to conclude that I. scapularis populations in Virginia are expanding and that this expansion may account for recent observed increases in Lyme disease.