Large Scale Spatial Risk and Comparative Prevalence of Borrelia miyamotoi and Borrelia burgdorferi Sensu Lato in Ixodes pacificus

Borrelia miyamotoi is a newly described emerging pathogen transmitted to people by Ixodes species ticks and found in temperate regions of North America, Europe, and Asia. There is limited understanding of large scale entomological risk patterns of B. miyamotoi and of Borreila burgdorferi sensu stricto (ss), the agent of Lyme disease, in western North America. In this study, B. miyamotoi, a relapsing fever spirochete, was detected in adult (n = 70) and nymphal (n = 36) Ixodes pacificus ticks collected from 24 of 48 California counties that were surveyed over a 13 year period. Statewide prevalence of B. burgdorferi sensu lato (sl), which includes B. burgdorferi ss, and B. miyamotoi were similar in adult I. pacificus (0.6% and 0.8%, respectively). In contrast, the prevalence of B. burgdorferi sl was almost 2.5 times higher than B. miyamotoi in nymphal I. pacificus (3.2% versus 1.4%). These results suggest similar risk of exposure to B. burgdorferi sl and B. miyamotoi from adult I. pacificus tick bites in California, but a higher risk of contracting B. burgdorferi sl than B. miyamotoi from nymphal tick bites. While regional risk of exposure to these two spirochetes varies, the highest risk for both species is found in north and central coastal California and the Sierra Nevada foothill region, and the lowest risk is in southern California; nevertheless, tick-bite avoidance measures should be implemented in all regions of California. This is the first study to comprehensively evaluate entomologic risk for B. miyamotoi and B. burgdorferi for both adult and nymphal I. pacificus, an important human biting tick in western North America.     

Relative importance of lizards and mammals as hosts for ixodid ticks in northern California

Lizards and mammals were trapped and examined for ticks from August 1992 to June 1993 in two habitat types, chaparral and woodland-grass, in northern California. Five tick species were collected from mammals (Dermacentor occidentalis, Haemaphysalis leporispalustris, Ixodes pacificus, I. spinipalpis, I. woodi), but only I. pacificus was found on lizards. Dermacentor occidentalis, I. pacificus, and I. woodi occurred in both habitats, whereas H. leporispalustris and I. spinipalpis were found only on animals trapped in chaparral. The tick species most commonly encountered on mammals was D. occidentalis in chaparral and I. pacificus in woodland-grass. Peak infestation of mammals occurred in spring for I. pacificus immatures and H. leporispalustris, summer for D. occidentalis immatures, fall through spring for I. woodi immatures, and fall through winter for I. spinipalpis. The primary aim of the study was to quantify the relative importance of the western fence lizard (Sceloporus occidentalis), which is reservoir-incompetent for Borrelia burgdorferi sensu lato (s.l.), and mammalian B.burgdorferi s.l.-reservoirs as hosts for the immature stages of I. pacificus in spring. The estimated relative utilization by I. pacificus of the western fence lizard versus mammals was 88% for larvae and 99% for nymphs in chaparral in May. When tick infestation data were corrected for a two-fold lower efficiency of field examinations for rodents than for lizards, the western fence lizard still accounted for 78% of larval and 98% of nymphal feedings. In woodland-grass, 46% of 100 I. pacificus larvae and 100% of 52 nymphs recovered from mammals or western fence lizards during May-June were collected from the lizards. However, this may represent an underestimate of the importance of the western fence lizard as a larval host in this habitat because inclement weather during the late May sampling period doubtless resulted in significantly decreased lizard activity. In conclusion, the western fence lizard was more heavily utilized by I. pacificus immatures, especially nymphs, than were rodents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.     

Rickettsial pathogen uses arthropod tryptophan pathway metabolites to evade reactive oxygen species in tick cells

Reactive oxygen species (ROS) that are induced upon pathogen infection plays an important role in host defence. The rickettsial pathogen Anaplasma phagocytophilum, which is primarily transmitted by Ixodes scapularis ticks in the United States, has evolved many strategies to escape ROS and survive in mammalian cells. However, little is known on the role of ROS in A. phagocytophilum infection in ticks. Our results show that A. phagocytophilum and hemin induce activation of l ‐tryptophan pathway in tick cells. Xanthurenic acid (XA), a tryptophan metabolite, supports A. phagocytophilum growth in tick cells through inhibition of tryptophan dioxygenase (TDO) activity leading to reduced l ‐kynurenine levels that subsequently affects build‐up of ROS. However, hemin supports A. phagocytophilum growth in tick cells by inducing TDO activity leading to increased l ‐kynurenine levels and ROS production. Our data reveal that XA and kynurenic acid (KA) chelate hemin. Furthermore, treatment of tick cells with 3‐hydroxyl l ‐kynurenine limits A. phagocytophilum growth in tick cells. RNAi‐mediated knockdown of kynurenine aminotransferase expression results in increased ROS production and reduced A. phagocytophilum burden in tick cells. Collectively, these results suggest that l ‐tryptophan pathway metabolites influence A. phagocytophilum survival by affecting build up of ROS levels in tick cells.     

Vertical vs. horizontal transmission of the microbiome in a key disease vector,Ixodes pacificus

Vector‐borne pathogens are increasingly found to interact with the vector's microbiome, influencing disease transmission dynamics. However, the processes that regulate the formation and development of the microbiome are largely unexplored for most tick species, an emerging group of disease vectors. It is not known how much of the tick microbiome is acquired through vertical transmission vs. horizontally from the environment or interactions with bloodmeal sources. Using 16S rRNA sequencing, we examined the microbiome of Ixodes pacificus, the vector of Lyme disease in the western USA, across life stages and infection status. We also characterized microbiome diversity in field and laboratory‐collected nymphal ticks to determine how the surrounding environment affects microbiome diversity. We found a decrease in both species richness and evenness as the tick matures from larva to adult. When the dominant Rickettsial endosymbiont was computationally removed from the tick microbial community, we found that infected nymphs had lower species evenness than uninfected ticks, suggesting that lower microbiome diversity is associated with pathogen transmission in wild‐type ticks. Furthermore, laboratory‐reared nymph microbiome diversity was found to be compositionally distinct and significantly depauperate relative to field‐collected nymphs. These results highlight unique patterns in the microbial community of I. pacificus that is distinct from other tick species. We provide strong evidence that ticks acquire a significant portion of their microbiome through exposure to their environment despite a loss of overall diversity through life stages. We provide evidence that loss of microbial diversity is at least in part due to elimination of microbial diversity with bloodmeal feeding but other factors may also play a role.     

The spatial distribution of Borrelia burgdorferi-infected Ixodes ricinus in the Connemara region of County Galway,Ireland

Studies were carried out in the Connemara area of County Galway in the west of Ireland in order to determine the abundance and distribution of the tick, Ixodes ricinus and the prevalence of its infection with Borrelia burgdorferi. The tick was very abundant locally, in particular when associated with cattle, sheep and enclosed red deer. Large numbers of ticks not only occurred on the pastures, but also on adjacent roadside verges. No infections with B. burgdorferi could be demonstrated when nymphal ticks were sampled from central areas of the pastures, suggesting that livestock and red deer are probably not significant reservoirs of the spirochaete. Small numbers of infected nymphal and adult ticks were associated with hedges, dry stone walls, the margins of woodland adjoining infested pastures and in woodland from which livestock were excluded. Woodmice (Apodemus sylvaticus) were most numerous in such habitats and the majority were infected with B. burgdorferi.     

Detection of Lyme disease and anaplasmosis pathogens via PCR in Pennsylvania deer ked

Borrelia burgdorferi and Anaplasma phagocytophilum are obligate intracellular parasites that maintain their life cycles in enzoonotic vector‐host cycles with Ixodes scapularis as a vector. In addition to ticks, the hosts are commonly infested with insects from the Hippoboscidae family. This study confirms the presence of B. burgdorferi and A. phagocytophilum in deer keds (Lipoptena cervi) removed from white‐tailed deer using PCR. Detection of these pathogens in deer ked represents a potential novel susceptibility of wildlife and also suggests the risk of transmission of these pathogens to humans and animals alike through the bite of an infected ectoparasite. This study represents the first instance in the U.S. of detection of tick‐borne pathogens in a member of the Hippoboscid family.     

The importance of passerine birds as tick hosts and in the transmission of Borrelia burgdorferi,the agent of Lyme disease: a case study from Scotland

Borrelia burgdorferi sensu lato (s.l.) is the causative agent of Lyme borreliosis, the most common tick‐borne zoonosis of humans in Europe and North America. Here, we assessed the relative importance of different passerine bird species as tick hosts and their contribution to the B. burgdorferi s.l. transmission cycle in a rural residential area in Scotland. We caught 1229 birds of 22 species during the tick‐questing season. On average, 29% carried larval ticks (0.8 larvae per individual) and 5% carried nymph ticks (0.06 nymphs per individual). All attached ticks tested were Ixodes ricinus. Using a nested‐PCR, we found that 20% of nymphs tested positive to B. burgdorferi s.l. and all these were of the genospecies Borrelia garinii. We identified two new bird species carrying infected nymphs: Eurasian Siskin Carduelis spinus and European Greenfinch Carduelis chloris. Ground‐foraging species were more important than arboreal species in hosting I. ricinus nymphs and B. burgdorferi s.l. Common Blackbirds Turdus merula were the most common hosts, with Song Thrushes Turdus philomelos, Dunnocks Prunella modularis, European Greenfinches and Chaffinches Fringilla coelebs also hosting high rates of infection.     

Interstadial and Infestation Level-dependent Variation in the Transmission Efficiency of Borrelia Burgdorferi from Mice to Ixodes Ricinus Ticks

The efficiency with which the spirochaete Borrelia burgdorferi sensu stricto was transmitted from laboratory mice to larval and nymphal Ixodes ricinus ticks was assessed, using the polymerase chain reaction. The transmission efficiency to nymphs was significantly greater than to larvae when both fed together on the same host. Increased tick infestation levels of mice were correlated with significantly greater engorgement weights and higher B. burgdorferi transmission coefficients from mice to nymphs. These observations indicate that both the feeding success of ticks and the transmission coefficients from host to tick may be influenced by the tick infestation level of an infected host. The infestation level and the relative numbers of each life stage of the tick are factors which should be considered in the design of transmission experiments.     

Nymphal survival and habitat distribution of Ixodes scapularis and Amblyomma americanum ticks (Acari: Ixodidae) on Fire Island, New York, USA

The distribution and survival of Ixodes scapularis and Amblyomma americanum were studied in deciduous and coniferous wooded habitats and in open habitats on Fire Island, New York, USA. The survival of nymphal I. scapularis in field enclosures was greater in forests than in open habitats, suggesting that greater survival contributes to the higher tick population in the woods. The nymphs of each species were more common in deciduous thickets (predominantly Aronia arbutifolia and Vaccinium corynbosum) than in coniferous woods (mostly Pinus rigida) in most but not all years. Larval I. scapularis were more common in coniferous sites in 1994, while the same ticks, as nymphs, were more common in deciduous sites in 1995. The survival of the nymphs was not consistently greater in either the deciduous or coniferous woods. Therefore, factors other than nymphal survival (e.g. larval overwintering survival and tick movement on hosts) probably influenced the relative nymph abundance in different forest types. Overall, the survival of A. americanum was far higher than that of I. scapularis.     

Tick infestation risk and <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> s.l. infection-induced increase in host-finding efficacy of female <Emphasis Type="Italic">Ixodes ricinus</Emphasis> under natural conditions

An investigation of the risk of human tick infestation, together with the prevalence of Borrelia burgdorferi s.l. infection, was conducted in a sylvatic habitat in western Germany to provide data needed for future risk-benefit evaluations of acaricides used for clothing impregnation. Additionally, data were collected on behavioural changes in Borrelia burgdorferi s.l.-infected adult female I. ricinus ticks and the possible impact of such changes on host-finding efficacy. The risk of I. ricinus-infestation was determined by collecting from the protective clothing of volunteers and by dragging in known tick-infested sites in the Kühkopf Mountain area, Koblenz, Germany, from June through October 2006. The overall tick infestation rate per person per hour was 7.4 ± 5.5, with the following sex- and stage-specific differences: males 0.32 ± 0.37, females 1.1 ± 1.2, nymphs 3.6 ± 4.4, larvae 2.4 ± 3.5. Concurrent dragging revealed an average 19.4 ± 16.2 times higher infestation rate as well as a markedly lower infection rate with borreliae in adult I. ricinus ticks when compared to ticks collected from exposed human volunteers. Although the difference in infection rates was statistically significant (P < 0.023) only in adult female ticks, our data indicate that B. burgdorferi s.l. infection may increase host-finding efficacy in adult I. ricinus. The overall exposure risk was 1.0 B. burgdorferi s.l.-infected ticks per person per hour of exposure, or 0.25 ticks per 100 m walking distance in the study area.     

Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Differences in the Transmissibility of Two Anaplasma phagocytophilum Strains by the North American Tick Vector Species, Ixodes Pacificus and Ixodes Scapularis (Acari: Ixodidae)

The etiologic agent of granulocytic anaplasmosis, Anaplasma phagocytophilum, has a circum-global distribution within the northern hemisphere and shows a host species predilection that varies by the geographic region in which the disease is found. Adaptation by the bacterium to a host species potentially contributes to the variation found worldwide but this is confounded by the bacterium's relationship with its tick vectors, all of which belong to the Ixodes ricinus group. We tested the hypothesis that tick vector species collected from geographic regions sympatric with particular A. phagocytophilum strains will show evidence of a higher degree of vector competence than will tick species and allopatric A. phagocytophilum strains. A reciprocal cross-transmission experiment was performed using an eastern and a western North American strain of A. phagocytophilum (Webster and MRK, respectively) and the two tick species, I. scapularis and I. pacificus, most commonly associated with human and animal transmission of the bacteria in the United States. The western tick, I. pacificus, showed a significantly higher vector competence for A. phagocytophilum than I. scapularis and the eastern isolate, Webster, was more transmissible than its western counterpart, MRK. These results indicate that geographic variation in host susceptibility to A. phagocytophilum strains may play a more important role in the epidemiology of granulocytic anaplasmosis than does the competence of its tick vectors to transmit the pathogen.     

Invasion of the Lyme Disease Vector <Emphasis Type="Italic">Ixodes scapularis</Emphasis>: Implications for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> Endemicity

Lyme disease risk is increasing in the United States due in part to the spread of blacklegged ticks Ixodes scapularis, the principal vector of the spirochetal pathogen Borrelia burgdorferi. A 5-year study was undertaken to investigate hypothesized coinvasion of I. scapularis and B. burgdorferi in Lower Michigan. We tracked the spatial and temporal dynamics of the tick and spirochete using mammal, bird, and vegetation drag sampling at eight field sites along coastal and inland transects originating in a zone of recent I. scapularis establishment. We document northward invasion of these ticks along Michigan’s west coast during the study period; this pattern was most evident in ticks removed from rodents. B. burgdorferi infection prevalences in I. scapularis sampled from vegetation in the invasion zone were 9.3% and 36.6% in nymphs and adults, respectively, with the majority of infection (95.1%) found at the most endemic site. There was no evidence of I. scapularis invasion along the inland transect; however, low-prevalence B. burgdorferi infection was detected in other tick species and in wildlife at inland sites, and at northern coastal sites in years before the arrival of I. scapularis. These infections suggest that cryptic B. burgdorferi transmission by other vector-competent tick species is occurring in the absence of I. scapularis. Other Borrelia spirochetes, including those that group with B. miyamotoi and B. andersonii, were present at a low prevalence within invading ticks and local wildlife. Reports of Lyme disease have increased significantly in the invasion zone in recent years. This rapid blacklegged tick invasion—measurable within 5 years—in combination with cryptic pathogen maintenance suggests a complex ecology of Lyme disease emergence in which wildlife sentinels can provide an early warning of disease emergence.     

A Review of Studies on the Transmission of Anaplasma phagocytophilum from Sheep: Implications for the Force of Infection in Endemic Cycles

We review the findings of a longitudinal study of transmission of the intracellular tick-borne bacterium Anaplasma phagocytophilum from sheep to Ixodes ricinus ticks under natural conditions of tick attachment in the UK. In this study, sheep-to-tick transmission efficiency varied in a quadratic relationship with the number of adult ticks that were feeding on the sheep. We raise the hypothesis that this relationship may be due to conflicting effects of the density of ticks on bacterial survival and target cell (neutrophil) fluxes at the tick-host interface: in the same sheep at the same time, resistance to ticks was progressively inhibited with increasing number of feeding adult ticks, and investigation of serological responses to tick antigens suggesting loss of resistance may be associated with polarisation of host Th1 to Th2 type responses to ticks. We also raise the hypothesis that these properties, with superimposed effects on tick survival, may mean that variation in tick density is an important causal factor of observed variations in the force of A. phagocytophilum infection amongst different geographic foci. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection of Borrelia burgdorferi and Anaplasma phagocytophilum in the black‐legged tick,Ixodes scapularis,within southwestern Pennsylvania

Prevalence studies of Borrelia burgdorferi and Anaplasma phagocytophilum have been rare for ticks from southwestern Pennsylvania. We collected 325 Ixodes scapularis ticks between 2011 and 2012 from four counties in southwestern Pennsylvania. We tested for the presence of Borrelia burgdorferi and Anaplasma phagocytophilum using PCR. Of the ticks collected from Pennsylvania, B. burgdorferi (causative agent of Lyme disease) was present in 114/325 (35%) and Anaplasma phagocytophilum (causative agent of Human Granulocytic Anaplasmosis) was present in 48/325 (15%) as determined by PCR analysis.     

Physiological Age of Field-collected Female Taiga Ticks, Ixodes persulcatus (Acari: Ixodidae), and their Infection with Borrelia burgdorferi Sensu Lato

In some studies the prevalence of tick infection (infection rate) and the intensity of infection are negatively correlated with unfed tick age (in the broad sense of this term). However, no special research has been carried out to consider the phenomenon thoroughly. The infection indices of the female taiga ticks, Ixodes persulcatus, infected with Borrelia burgdorferi s.l. were related to tick physiological age, an index that more precisely reflects tick physiological state than the time of tick collection in the field or the duration of tick survival under laboratory conditions. A novel quantitative technique of physiological age determination based on the evaluation of the ratios between sizes of the stable (scutum) and the changing (alloscutum) structures of the tick body was used. The age was estimated in accordance with the classical age-grade scale introduced by Balashov and a more fractional scale determined by the new technique. In total, 131 female ticks were examined for their infection and physiological age, 46 of which were infected with B. burgdorferi s.l. (mean infection rate 35.1%). The minimal intensity of infection was 0.4 bacterial cells per 100 fields of view whereas the maximal infection was 172 cells. There was no difference between the prevalence of infection in ticks of different physiological age. The intensity of infection obviously differed between ticks of different age groups in the scale introduced by Balashov but did not significantly differ between ticks of different age groups according to the fractional age-grade scale. The data concerning the relationships between Borrelia burgdorferi and unfed Ixodes ticks are considered.