The aims of this study were to evaluate the host-tick-pathogen interface of Babesia spp. and Anaplasma phagocytophilum in restored areas in both questing and host-attached Ixodes ricinus and Dermacentor reticulatus and their small mammalian hosts.
Questing ticks were collected from 5 sites within the city of Leipzig, Germany, in 2009. Small mammals were trapped at 3 of the 5 sites during 2010 and 2011. DNA extracts of questing and host-attached I. ricinus and D. reticulatus and of several tissue types of small mammals (the majority bank voles and yellow-necked mice), were investigated by PCR followed by sequencing for the occurrence of DNA of Babesia spp. and by real-time PCR for A. phagocytophilum. A selected number of samples positive for A. phagocytophilum were further investigated for variants of the partial 16S rRNA gene. Co-infection with Rickettsia spp. in the questing ticks was additionally investigated.
4.1% of questing I. ricinus ticks, but no D. reticulatus, were positive for Babesia sp. and 8.7% of I. ricinus for A. phagocytophilum. Sequencing revealed B. microti, B. capreoli and Babesia spp. EU1 in Leipzig and sequence analysis of the partial 16S RNA gene of A. phagocytophilum revealed variants either rarely reported in human cases or associated with cervid hosts. The statistical analysis revealed significantly less ticks infected with A. phagocytophilum in a city park in Leipzig as compared to the other sampling sites. A. phagocytophilum-DNA was detected in 2 bank voles, DNA of B. microti in 1 striped field-mouse and of Babesia sp. EU1 in the skin tissue of a mole. Co-infections were detected.
Our results show the involvement of small mammals in the natural endemic cycles of tick-borne pathogens. A more thorough understanding of the interactions of ticks, pathogens and hosts is the essential basis for effective preventive control measures.
Babesia spp; Anaplasma phagocytophilum; Ixodes ricinus; Dermacentor reticulatus; Bank vole; Yellow-necked mouse; Recreational area; Host survey; Vector-host relation
Anaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.
The presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.
DNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.
Four ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.
Electronic supplementary material
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Anaplasma phagocytophilum; Zoonoses; Ixodes ricinus; Wildlife; Epidemiology
Previous studies revealed that the Pacific Coast tick (Dermacentor occidentalis) is infected occasionally with the agents of Lyme disease (Borrelia burgdorferi) or human granulocytic anaplasmosis (Anaplasma phagocytophilum) and that it is an inefficient experimental vector of B. burgdorferi. The relationship of the pajahuello tick (Ornithodoros coriaceus) to each of these bacterial zoonotic agents has not been reported. The primary bridging vector of both bacterial zoonotic agents to humans is the western black-legged tick (Ixodes pacificus). Because of the spatial and temporal overlap of D. occidentalis and O. coriaceus populations with those of I. pacificus in natural foci of B. burgdorferi and A. phagocytophilum in northwestern California, we conducted field and laboratory studies to determine if the Pacific Coast tick or the pajahuello tick potentially may serve as secondary vectors of either bacterium. Our findings reconfirmed that wild-caught D. occidentalis ticks are infected infrequently with B. burgdorferi or A. phagocytophilum, but some adult ticks from dense woodlands or chaparral were found to contain 2 important veterinary pathogens for the first time (Anaplasma bovis, A. ovis). The high prevalence of A. bovis infection (4.3%, n=185 ticks) within chaparral-derived ticks suggests that D. occidentalis could be an efficient vector of this rickettsia. Experimental attempts to transmit borreliae or Anaplasma spp. that may have been present in >100 wild-caught D. occidentalis adults to naïve rabbits were unsuccessful. Anaplasma spp. were not detected in O. coriaceus, but one (4.3%) of 23 nymphs was infected with B. bissettii. This finding and an antecedent report of a B. burgdorferi-like spirochete from the same tick species demonstrate that O. coriaceus sometimes acquires and transstadially passes Lyme disease group spirochetes. I. pacificus nymphs inhabiting a woodland nidus of B. burgdorferi and A. phagocytophilum had a 5-fold higher prevalence of borreliae than adult ticks from the same generational cohort. In contrast to the results of preceding studies carried out at the same site, none of the nymphal or adult ticks was PCR-positive for A. phagocytophilum. This suggests that the distribution of this rickettsia is highly focal or variable from year-to-year within this particular woodland.
Borrelia burgdorferi; Anaplasma spp.; Dermacentor occidentalis; Ixodes pacificus; Ornithodoros coriaceus
The bacterium Anaplasma phagocytophilum has for decades been known to cause the disease tick-borne fever (TBF) in domestic ruminants in Ixodes ricinus-infested areas in northern Europe. In recent years, the bacterium has been found associated with Ixodes-tick species more or less worldwide on the northern hemisphere. A. phagocytophilum has a broad host range and may cause severe disease in several mammalian species, including humans. However, the clinical symptoms vary from subclinical to fatal conditions, and considerable underreporting of clinical incidents is suspected in both human and veterinary medicine. Several variants of A. phagocytophilum have been genetically characterized. Identification and stratification into phylogenetic subfamilies has been based on cell culturing, experimental infections, PCR, and sequencing techniques. However, few genome sequences have been completed so far, thus observations on biological, ecological, and pathological differences between genotypes of the bacterium, have yet to be elucidated by molecular and experimental infection studies. The natural transmission cycles of various A. phagocytophilum variants, the involvement of their respective hosts and vectors involved, in particular the zoonotic potential, have to be unraveled. A. phagocytophilum is able to persist between seasons of tick activity in several mammalian species and movement of hosts and infected ticks on migrating animals or birds may spread the bacterium. In the present review, we focus on the ecology and epidemiology of A. phagocytophilum, especially the role of wildlife in contribution to the spread and sustainability of the infection in domestic livestock and humans.
Anaplasma phagocytophilum; ecology; epidemiology; distribution; hosts; vectors
Anaplasma phagocytophilum is an intracellular tick-borne rickettsial pathogen, which causes granulocytic anaplasmosis in various species of livestock and companion animals and also in humans. Previously A. phagocytophilum has been isolated and propagated in cell lines derived from the tick Ixodes scapularis and in the human promyelocytic cell line HL60. In this study we used the Ixodes ricinus-derived cell line IRE/CTVM20 to isolate and propagate two new canine strains of A. phagocytophilum.
Blood samples were collected by veterinarians from two dogs, one from Germany and the other from Austria. Suspicion of clinical canine granulocytic anaplasmosis was raised by the treating veterinarians and after confirmation of A. phagocytophilum infection by real-time PCR, buffy coat cells were isolated and co-cultivated with IRE/CTVM20 cells maintained at 28 °C in L15/L15B medium.
In the tick cells, rickettsial inclusions were first recognised after 86 days of incubation. Electron microscopic examination of tick cells infected with one of the isolates revealed cytoplasmic vacuoles containing pleomorphic organisms with individual bacteria enveloped by a bilayer membrane. Sequencing of 16S rRNA genes confirmed the isolation of A. phagocytophilum and showed the highest identity to the A. phagocytophilum human HZ strain. The two A. phagocytophilum isolates were passaged several times in IRE/CTVM20 cells and transferred to the I. scapularis cell line ISE6. This confirms for the first time the successful establishment and continuous cultivation of this pathogen in I. ricinus cells as well as infectivity of these canine strains for I. scapularis cells.
Tick cell lines; Anaplasma phagocytophilum; IRE/CTVM20; Dog; Electron microscopy
Lyme borreliosis (LB), tick-borne encephalitis (TBE) and human granulocytic anaplasmosis (HGA) are endemic in central part of Slovenia. We tested the hypothesis that patients with erythema migrans (EM) from this region, who have leukopenia and/or thrombocytopenia (typical findings in HGA and in the initial phase of TBE but not in patients with LB) are coinfected with Anaplasma phagocytophilum and/or with TBE virus, i.e. that cytopenia is a result of concomitant HGA or the initial phase of TBE. Comparison of clinical and laboratory findings for 67 patients with EM who disclosed leukopenia/thrombocytopenia with the corresponding results in sex- and age-matched patients with EM and normal blood cell counts revealed no differences. In addition, patients with typical EM and leukopenia and/or thrombocytopenia tested negative for the presence of IgM and IgG antibodies to TBE virus by ELISA as well as for the presence of specific IgG antibodies to A. phagocytophilum antigens by IFA in acute and convalescent serum samples. Thus, none of 67 patients (95% CI: 0 to 5.3%) with typical EM (the presence of this skin lesion attests for early Lyme borreliosis and is the evidence for a recent tick bite) was found to be coinfected with A. phagocytophilum or had a recent primary infection with TBE virus. The findings in the present study indicate that in Slovenia, and probably in other European countries endemic for LB, TBE and HGA, patients with early LB are rarely coinfected with the other tick-transmitted agents.
Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.
Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test.
The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.
Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.
Vector-borne disease; Emerging zoonoses; Candidatus N. mikurensis; I. ricinus; Anaplasma phagocytophylum
Ecological changes are recognized as an important driver behind the emergence of infectious diseases. The prevalence of infection in ticks depends upon ecological factors that are rarely taken into account simultaneously. Our objective was to investigate the influences of forest fragmentation, vegetation, adult tick hosts, and habitat on the infection prevalence of three tick-borne bacteria, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Rickettsia sp. of the spotted fever group, in questing Ixodes ricinus ticks, taking into account tick characteristics. Samples of questing nymphs and adults were taken from 61 pastures and neighboring woodlands in central France. The ticks were tested by PCR of pools of nymphs and individual adults. The individual infection prevalence was modeled using multivariate regression. The highest infection prevalences were found in adult females collected in woodland sites for B. burgdorferi sensu lato and A. phagocytophilum (16.1% and 10.7%, respectively) and in pasture sites for Rickettsia sp. (8.7%). The infection prevalence in nymphs was lower than 6%. B. burgdorferi sensu lato was more prevalent in woodlands than in pastures. Forest fragmentation favored B. burgdorferi sensu lato and A. phagocytophilum prevalence in woodlands, and in pastures, the B. burgdorferi sensu lato prevalence was favored by shrubby vegetation. Both results are probably because large amounts of edges or shrubs increase the abundance of small vertebrates as reservoir hosts. The Rickettsia sp. prevalence was maximal on pasture with medium forest fragmentation. Female ticks were more infected by B. burgdorferi sensu lato than males and nymphs in woodland sites, which suggests an interaction between the ticks and the bacteria. This study confirms the complexity of the tick-borne pathogen ecology. The findings support the importance of small vertebrates as reservoir hosts and make a case for further studies in Europe on the link between the composition of the reservoir host community and the infection prevalence in ticks.
Serologic and molecular evidence of Anaplasma phagocytophilum has been demonstrated in white-tailed deer (WTD; Odocoileus virginianus), and deer are an important host for the tick vector Ixodes scapularis. In this study, we describe experimental infection of WTD with A. phagocytophilum. We inoculated four WTD with a human isolate of A. phagocytophilum propagated in tick cells. Two additional deer served as negative controls. All inoculated deer developed antibodies (titers, ≥64) to A. phagocytophilum, as determined by an indirect fluorescent antibody test, between 14 and 24 days postinfection [p.i.]), and two deer maintained reciprocal titers of ≥64 through the end of the 66-day study. Although morulae were not observed in granulocytes and A. phagocytophilum was not reisolated via tick cell culture of blood, 16S reverse transcriptase nested PCR (RT-nPCR) results indicated that A. phagocytophilum circulated in peripheral blood of three deer through at least 17 days p.i. and was present in two deer at 38 days p.i. Femoral bone marrow from one deer was RT-nPCR positive for A. phagocytophilum at 66 days p.i. There was no indication of clinical disease. These data confirm that WTD are susceptible to infection with a human isolate of A. phagocytophilum and verify that WTD produce detectable antibodies upon exposure to the organism. Because adults are the predominant life stage of I. scapularis found on deer and because adult I. scapularis ticks do not transmit A. phagocytophilum transovarially, it is unlikely that WTD are a significant source of A. phagocytophilum for immature ticks even though deer have a high probability of natural infection. However, the susceptibility and immunologic response of WTD to A. phagocytophilum render them suitable candidates as natural sentinels for this zoonotic tick-borne organism.
The msp2 and p44 genes encode polymorphic major outer membrane proteins that are considered unique to the intraerythrocytic agent of Anaplasma marginale and the intragranulocytic agent of Anaplasma phagocytophilum, respectively. In the present study, however, we found an msp2 gene in A. phagocytophilum that was remarkably conserved among A. phagocytophilum strains from human granulocytic anaplasmosis (HGA) patients, ticks, and a horse from various regions in the United States, but the gene was different in a sheep isolate from the United Kingdom. The msp2 gene in the A. phagocytophilum strain HZ genome was a single-copy gene and was located downstream of two Ehrlichia chaffeensis omp-1 homologs and a decarboxylase gene (ubiD). The msp2 gene was expressed by A. phagocytophilum in the blood from HGA patients NY36 and NY37 and by A. phagocytophilum isolates from these patients cultured in HL-60 cells at 37°C. The msp2 gene was also expressed in a DBA/2 mouse infected by attaching ticks infected with strain NTN-1 and in a horse experimentally infected by attaching strain HZ-infected ticks. However, the transcript of the msp2 gene was undetectable in A. phagocytophilum strain HZ in SCID mice and Ixodes scapularis ticks infected with strain NTN-1. These results indicate that msp2 is functional in various strains of A. phagocytophilum, and relative expression ratios of msp2 to p44 vary in different infected hosts. These findings may be important in understanding roles that Msp2 proteins play in granulocytic ehrlichia infection and evolution of the polymorphic major outer membrane protein gene families in Anaplasma species.
Anaplasma phagocytophilum is a Gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.
In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., “Candidatus Neoehrlichia mikurensis,” and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., “Candidatus Neoehrlichia mikurensis,” and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and “Candidatus Neoehrlichia mikurensis” were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.
Understanding the variation in prevalence of Borrelia burgdorferi sensu lato (Lyme Borreliosis Spirochaetes, LBS) and Anaplasma phagocytophilum (causing tick-borne fever in ruminants and human granulocytic ehrlichiosis) in ticks is vital from both a human and an animal disease perspective to target the most effective mitigation measures. From the host competence hypothesis, we predicted that prevalence of LBS would decrease with red deer density, while prevalence of A. phagocytophilum would increase.
Based on a sample of 112 adult and 686 nymphal Ixodes ricinus ticks collected with flagging during questing from 31 transects (4–500 m long) corresponding to individual seasonal home ranges of 41 red deer along the west coast of Norway, we tested whether there were spatial and seasonal variations in prevalence with a special emphasis on the population density of the most common large host in this area, the red deer (Cervus elaphus). We used a multiplex real-time PCR assay for detection of A. phagocytophilum and LBS.
Prevalence of LBS was higher in adult female ticks (21.6%) compared to adult male ticks (11.5%) and nymphs (10.9%), while prevalence was similar among stages for prevalence of A. phagocytophilum (8.8%). Only partly consistent with predictions, we found a lower prevalence of LBS in areas of high red deer density, while there was no relationship between red deer density and prevalence of A. phagocytophilum in ticks. Prevalence of both bacteria was much higher in ticks questing in May compared to August.
Our study provides support to the notion that spatial variation in host composition forms a role for prevalence of LBS in ticks also in a northern European ecosystem, while no such association was found for A. phagocytophilum. Further studies are needed to fully understand the similar seasonal pattern of prevalence of the two pathogens.
Anaplasma; Borrelia; Dilution effects; Host competence hypothesis; Ixodes ricinus; Lyme Borreliosis Spirochaetes; Prevalence; Red deer; Rodents; Ticks
A total of 60 sheep were exposed to Anaplasma phagocytophilum infection on an enclosed area of Ixodes ricinus-infested pasture in North Wales, United Kingdom, and rapidly acquired acute A. phagocytophilum infections detectable by PCR and blood smear examination. Of the ticks that had engorged in the previous instar on infected sheep, 52% of adult ticks and 28% of nymphs were PCR positive; a significant, 10-fold increase in prevalence compared to that of ticks that engorged on sheep preinfection was observed (P = 0.015). The likelihood that ticks were PCR positive, after feeding on the sheep and molting to the next instar, increased marginally with increasing numbers of infected neutrophils per milliliter of blood of their sheep host (P = 0.068) and increased significantly when they were collected from sheep carrying higher numbers of adult female ticks (P = 0.017), but increasing numbers of feeding nymphs had a significant negative effect on transmission (P = 0.049). The numbers of circulating neutrophils and of infected neutrophils also varied significantly with the numbers of ticks feeding on the sheep when the blood was collected. Our study suggests that ruminants are efficient reservoirs of A. phagocytophilum during the acute and post-acute phases of infection. The risk of ruminant-derived infections may, however, be strongly affected by variations in tick densities, which may influence transmission from acutely infected animals via effects on the numbers of infected cells in the blood and possibly by within-skin modulation of infection.
Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F1 larvae by individual ticks at efficiencies of 10–40 and 95–100%, respectively. The FLEs were transovarially transmitted to F2 larvae obtained as progeny of adults from F1 larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission.
Ixodid tick; Anaplasma; Francisella-like; transovarial transmission
Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.
For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The OspA gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.
Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ2 = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ2 = 24.04, p < 0.0001); Strøm vs. Tjore (χ2 = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ2 = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ2 = 16.27, p = 0.0001); Strøm vs. Tjore (χ2 = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ2 = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ2 = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ2 = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.
The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.
Substantial exposure to Borrelia miyamotoi occurs through bites from Ixodes ricinus ticks in the Netherlands, which also transmit Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum. Direct evidence for B. miyamotoi infection in European populations is scarce. A flu-like illness with high fever, resembling human granulocytic anaplasmosis, has been attributed to B. miyamotoi infections in relatively small groups. Borrelia miyamotoi infections associated with chronic meningoencephalitis have also been described in case reports. Assuming that an IgG antibody response against B. miyamotoi antigens reflects (endured) infection, the seroprevalence in different risk groups was examined. Sera from nine out of ten confirmed B. miyamotoi infections from Russia were found to be positive with the recombinant antigen used, and no significant cross-reactivity was observed in secondary syphilis patients. The seroprevalence in blood donors was set at 2.0% (95% CI 0.4–5.7%). Elevated seroprevalences in individuals with serologically confirmed, 7.4% (2.0–17.9%), or unconfirmed, 8.6% (1.8–23%), Lyme neuroborreliosis were not significantly different from those in blood donors. The prevalence of anti-B. miyamotoi antibodies among forestry workers was 10% (5.3–16.8%) and in patients with serologically unconfirmed but suspected human granulocytic anaplasmosis was 14.6% (9.0–21.8%); these were significantly higher compared with the seroprevalence in blood donors. Our findings indicate that infections with B. miyamotoi occur in tick-exposed individuals in the Netherlands. In addition, B. miyamotoi infections should be considered in patients reporting tick bites and febrile illness with unresolved aetiology in the Netherlands, and other countries where I. ricinus ticks are endemic.
Anaplasmosis; Borrelia miyamotoi; Ixodes ricinus; relapsing fever; serology
During the spring in 2005 and 2006, 39,095 northward-migrating land birds were captured at 12 bird observatories in eastern Canada to investigate the role of migratory birds in northward range expansion of Lyme borreliosis, human granulocytic anaplasmosis, and their tick vector, Ixodes scapularis. The prevalence of birds carrying I. scapularis ticks (mostly nymphs) was 0.35% (95% confidence interval [CI] = 0.30 to 0.42), but a nested study by experienced observers suggested a more realistic infestation prevalence of 2.2% (95% CI = 1.18 to 3.73). The mean infestation intensity was 1.66 per bird. Overall, 15.4% of I. scapularis nymphs (95% CI = 10.7 to 20.9) were PCR positive for Borrelia burgdorferi, but only 8% (95% CI = 3.8 to 15.1) were positive when excluding nymphs collected at Long Point, Ontario, where B. burgdorferi is endemic. A wide range of ospC and rrs-rrl intergenic spacer alleles of B. burgdorferi were identified in infected ticks, including those associated with disseminated Lyme disease and alleles that are rare in the northeastern United States. Overall, 0.4% (95% CI = 0.03 to 0.41) of I. scapularis nymphs were PCR positive for Anaplasma phagocytophilum. We estimate that migratory birds disperse 50 million to 175 million I. scapularis ticks across Canada each spring, implicating migratory birds as possibly significant in I. scapularis range expansion in Canada. However, infrequent larvae and the low infection prevalence in ticks carried by the birds raise questions as to how B. burgdorferi and A. phagocytophilum become endemic in any tick populations established by bird-transported ticks.
A major challenge in sheep farming during the grazing season along the coast of south-western Norway is tick-borne fever (TBF) caused by the bacteria Anaplasma phagocytophilum that is transmitted by the tick Ixodes ricinus.
A study was carried out in 2007 and 2008 to examine the prevalence of A. phagocytophilum infection and effect on weaning weight in lambs. The study included 1208 lambs from farms in Sunndal Ram Circle in Møre and Romsdal County in Mid-Norway, where ticks are frequently observed. All lambs were blood sampled and serum was analyzed by an indirect fluorescent antibody assay (IFA) to determine an antibody status (positive or negative) to A. phagocytophilum infection. Weight and weight gain and possible effect of infection were analyzed using ANOVA and the MIXED procedure in SAS.
The overall prevalence of infection with A. phagocytophilum was 55%. A lower weaning weight of 3% (1.34 kg, p < 0.01) was estimated in lambs seropositive to an A. phagocytophilum infection compared to seronegative lambs at an average age of 137 days.
The results show that A. phagocytophilum infection has an effect on lamb weight gain. The study also support previous findings that A. phagocytophilum infection is widespread in areas where ticks are prevalent, even in flocks treated prophylactic with acaricides.
Although Ixodes spp. are the most common ticks in North-Western Europe, recent reports indicated an expanding geographical distribution of Dermacentor reticulatus in Western Europe. Recently, the establishment of a D. reticulatus population in Belgium was described. D. reticulatus is an important vector of canine and equine babesiosis and can transmit several Rickettsia species, Coxiella burnetii and tick-borne encephalitis virus (TBEV), whilst Ixodes spp. are vectors of pathogens causing babesiosis, borreliosis, anaplasmosis, rickettsiosis and TBEV.
A survey was conducted in 2008-2009 to investigate the presence of different tick species and associated pathogens on dogs and cats in Belgium. Ticks were collected from dogs and cats in 75 veterinary practices, selected by stratified randomization. All collected ticks were morphologically determined and analysed for the presence of Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia DNA.
In total 2373 ticks were collected from 647 dogs and 506 cats. Ixodes ricinus (76.4%) and I. hexagonus (22.6%) were the predominant species. Rhipicephalus sanguineus (0.3%) and D. reticulatus (0.8%) were found in low numbers on dogs only. All dogs infested with R. sanguineus had a recent travel history, but D. reticulatus were collected from a dog without a history of travelling abroad. Of the collected Ixodes ticks, 19.5% were positive for A. phagocytophilum and 10.1% for Borrelia spp. (B. afzelii, B. garinii, B. burgdorferi s.s., B. lusitaniae, B. valaisiana and B. spielmanii). Rickettsia helvetica was found in 14.1% of Ixodes ticks. All Dermacentor ticks were negative for all the investigated pathogens, but one R. sanguineus tick was positive for Rickettsia massiliae.
D. reticulatus was confirmed to be present as an indigenous parasite in Belgium. B. lusitaniae and R. helvetica were detected in ticks in Belgium for the first time.
Ticks; Dermacentor reticulatus; Dogs; Cats; Belgium; Borrelia; Anaplasma; Rickettsia
Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed.
From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C).
Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results.
Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.
Ixodes ricinus; tick-borne diseases; surveillance; economic evaluation; Italy.
Anaplasma phagocytophilum , the causative agent of granulocytic anaplasmosis, affects several species of wild and domesticated mammals, including horses. We used direct and indirect methods to compare and evaluate exposure to A. phagocytophilum in horses in northern Tunisia.
Serum from 60 horses was tested by IFA for antibodies to A. phagocytophilum , and whole blood was tested for A. phagocytophilum 16S rRNA gene using a nested-PCR. To examine the risk of A. phagocytophilum transmission, 154 ticks that had been collected from horses were examined for the presence of A. phagocytophilum by nested-PCR targeting 16S rRNA gene.
This is the first time that A. phagocytophilum has been detected in horses in Tunisia, with an overall seroprevalence of 40/60 (67%). Six of the seroreactive samples (10%) had an IFA titer of 1:80, 14 (23%) of 1:160, 8 (13%) of 1:320 and 12 (20%) a titer 1 ≥ 640. The seroprevalence revealed no significant regional and sex differences. In contrast, a significant difference was observed between breeds. Eight (13%) of the horses were positive for A. phagocytophilum in the PCR, with no significant breed and age differences. Hyalomma marginatum was a predominant tick species (130/154), and 3 were infected by A. phagocytophilum (a prevalence of 2.3%). The concordance rate of A. phagocytophilum detection between IFA and PCR had a k value of −0.07.
The results presented in this study suggest that horses infested by ticks in Tunisia are exposed to A. phagocytophilum.
Anaplasma phagocytophilum; Horses; Ticks; 16S rRNA gene; nPCR; IFA; Tunisia
Tick-transmitted rickettsial diseases, such as ehrlichiosis and spotted fever rickettsiosis, are significant sources of morbidity and mortality in the southern United States. Because of their exposure in tick-infested woodlands, outdoor workers experience an increased risk of infection with tick-borne pathogens. As part of a double blind randomized-controlled field trial of the effectiveness of permethrin-treated clothing in preventing tick bites, we identified tick species removed from the skin of outdoor workers in North Carolina and tested the ticks for Rickettsiales pathogens.
Ticks submitted by study participants from April-September 2011 and 2012 were identified to species and life stage, and preliminarily screened for the genus Rickettsia by nested PCR targeting the 17-kDa protein gene. Rickettsia were further identified to species by PCR amplification of 23S-5S intergenic spacer (IGS) fragments combined with reverse line blot hybridization with species-specific probes and through cloning and nucleotide sequence analysis of 23S-5S amplicons. Ticks were examined for Ehrlichia and Anaplasma by nested PCR directed at the gltA, antigen-expressing gene containing a variable number of tandem repeats, 16S rRNA, and groESL genes.
The lone star tick (Amblyomma americanum) accounted for 95.0 and 92.9% of ticks submitted in 2011 (n = 423) and 2012 (n = 451), respectively. Specimens of American dog tick (Dermacentor variabilis), Gulf Coast tick (Amblyomma maculatum) and black-legged tick (Ixodes scapularis) were also identified. In both years of our study, 60.9% of ticks tested positive for 17-kDa. “Candidatus Rickettsia amblyommii”, identified in all four tick species, accounted for 90.2% (416/461) of the 23S-5S-positive samples and 52.9% (416/787) of all samples tested. Nucleotide sequence analysis of Rickettsia-specific 23S-5S IGS, ompA and gltA gene fragments indicated that ticks, principally A. americanum, contained novel species of Rickettsia. Other Rickettsiales, including Ehrlichia ewingii, E. chaffeensis, Ehrlichia sp. (Panola Mountain), and Anaplasma phagocytophilum, were infrequently identified, principally in A. americanum.
We conclude that in North Carolina, the most common rickettsial exposure is to R. amblyommii carried by A. americanum. Other Rickettsiales bacteria, including novel species of Rickettsia, were less frequently detected in A. americanum but are relevant to public health nevertheless.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-014-0607-2) contains supplementary material, which is available to authorized users.
Ticks; Rickettsiales pathogens; Rickettsia; Ehrlichia; Reverse line blot hybridization
The natural life cycle of Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, consists of alternate infection of two distinct hosts, ticks and mammals, in which bacterial surface proteins are expected to have a critical role. The present study investigated regulation of A. phagocytophilum p44 genes, which encode the P44 major surface proteins. Quantitative real-time reverse transcription-PCR analysis revealed that the amount of p44 mRNA obtained from spleens of A. phagocytophilum-infected SCID mice was approximately 10-fold greater than the amount obtained from salivary glands of A. phagocytophilum-infected Ixodes scapularis nymphs. Similarly, the amount of p44 mRNA obtained from A. phagocytophilum-infected HL-60 cells per bacterium was significantly greater than the amount obtained from infected ISE6 tick cells. The relative amount of p44 mRNA was approximately threefold higher in A. phagocytophilum-infected HL-60 cells cultured at 37°C than in A. phagocytophilum-infected HL-60 cells cultured at 28°C. Although there are more than 100 p44 paralogs, we observed expression mainly from the p44 expression locus (p44E) in various host environments. Interestingly, transcription of the A. phagocytophilum gene encoding the DNA binding protein ApxR was also significantly greater in A. phagocytophilum-infected HL-60 cells than in infected ISE6 tick cells. Gel mobility shift and DNase I protection assays revealed recombinant ApxR binding to the promoter regions of p44E and apxR. ApxR also transactivated the p44E and apxR promoter regions in a lacZ reporter assay. These results indicate that p44 genes and apxR are specifically up-regulated in the mammalian host environment and suggest that ApxR not only is positively autoregulated but also acts as a transcriptional regulator of p44E.
Anaplasma phagocytophilum causes human granulocytic anaplasmosis (HGA) in humans, which has been recognized as an emerging tick-borne disease in the United States and Europe. Although about 65 cases of HGA have been reported in Europe, some of them do not fulfill the criteria for confirmed HGA. Confirmation of HGA requires A. phagocytophilum isolation from blood, and/or identification of morulae in granulocytes and/or positive PCR results with subsequent sequencing of the amplicons to demonstrate specific rickettsial DNA. Seroconversion or at least fourfold increase in antibody titers to A. phagocytophilum has been used as criteria for confirmed HGA also.
Infection with A. phagocytophilum was confirmed by PCR in a patient in Sicily, Italy, who had negative serology for A. phagocytophilum. A fragment of A. phagocytophilum 16S rDNA was amplified by two independent laboratories and sequenced from two separate patient's blood samples. The 16S rDNA sequence was identical in both samples and identical to the sequence of the A. phagocytophilum strain USG3 originally obtained from a dog.
Infection with A. phagocytophilum was confirmed in a patient without a detectable antibody response against the pathogen. The results reported herein documented the first case of confirmed HGA in Sicily, Italy. These results suggested the possibility of human infections with A. phagocytophilum strains that result in clinical symptoms and laboratory findings confirmatory of HGA but without detectable antibodies against the pathogen.
Human granulocytic anaplasmosis; Anaplasma phagocytophilum; 16S rDNA sequence