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Real-time PCR analysis of a groESL heat shock operon segment showed the presence of two genetic lineages of Anaplasma phagocytophilum in Ixodes ricinus ticks as well as one variant in wild red and roe deer, the latter supposedly representing the natural reservoir of one variant of A. phagocytophilum.

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

Genetically distinct subpopulations have adapted to different niches

The emerging tick-borne pathogen Anaplasma phagocytophilum is under increasing scrutiny for the existence of subpopulations that are adapted to different natural cycles. Here, we characterized the diversity of A. phagocytophilum genotypes circulating in a natural system that includes multiple hosts and at least 2 tick species, Ixodes ricinus and the small mammal specialist I. trianguliceps. We encountered numerous genotypes, but only 1 in rodents, with the remainder limited to deer and host-seeking I. ricinus ticks. The absence of the rodent-associated genotype from host-seeking I. ricinus ticks was notable because we demonstrated that rodents fed a large proportion of the I. ricinus larval population and that these larvae were abundant when infections caused by the rodent-associated genotype were prevalent. These observations are consistent with the conclusion that genotypically distinct subpopulations of A. phagocytophilum are restricted to coexisting but separate enzootic cycles and suggest that this restriction may result from specific vector compatibility.

In Europe, ixodid ticks are important arthropod vectors of human and animal pathogens, but comprehensive studies of the prevalence of all relevant pathogens in Central Europe are scarce. As a result of ecological changes, the incidences of tick-borne infections are expected to increase. In this study, 1,394 nymphal and adult Ixodes ricinus ticks sampled monthly during the active season from 33 ecologically distinct collection sites throughout Luxembourg were screened for all human tick-borne pathogens relevant in Central Europe. Species were identified by sequence analysis of detection PCR amplicons. Mean infection rates of ticks were 11.3% for Borrelia burgdorferi sensu lato, 5.1% for Rickettsia sp., 2.7% for Babesia sp., and 1.9% for Anaplasma phagocytophilum. No tick was found to be infected with Coxiella sp., Francisella tularensis subsp., or Tick-borne encephalitis virus (TBEV). A total of 3.2% of ticks were infected with more than one pathogen species, including mixed Borrelia infections (1.5%). Seasonal variations of tick infection rates were observed for Borrelia, Babesia, and Anaplasma, possibly reflecting a behavioral adaptation strategy of questing ticks. A positive correlation between the grade of urbanization and Borrelia infection rate of ticks was observed, suggesting an established urban zoonotic cycle. We also found Hepatozoon canis (0.1%) and Bartonella henselae (0.3%), which so far have not been found in questing Ixodes ricinus ticks in Central Europe.

Background

The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.

Results

Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.

Conclusion

At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

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.

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.

The importance of Ixodes ricinus in the transmission of tick-borne pathogens is well recognized in the United Kingdom and across Europe. However, the role of coexisting Ixodes species, such as the widely distributed species Ixodes trianguliceps, as alternative vectors for these pathogens has received little attention. This study aimed to assess the relative importance of I. ricinus and I. trianguliceps in the transmission of Anaplasma phagocytophilum and Babesia microti among United Kingdom field voles (Microtus agrestis), which serve as reservoir hosts for both pathogens. While all instars of I. trianguliceps feed exclusively on small mammals, I. ricinus adults feed primarily on larger hosts such as deer. The abundance of both tick species and pathogen infection prevalence in field voles were monitored at sites surrounded with fencing that excluded deer and at sites where deer were free to roam. As expected, fencing significantly reduced the larval burden of I. ricinus on field voles and the abundance of questing nymphs, but the larval burden of I. trianguliceps was not significantly affected. The prevalence of A. phagocytophilum and B. microti infections was not significantly affected by the presence of fencing, suggesting that I. trianguliceps is their principal vector. The prevalence of nymphal and adult ticks on field voles was also unaffected, indicating that relatively few non-larval I. ricinus ticks feed upon field voles. This study provides compelling evidence for the importance of I. trianguliceps in maintaining these enzootic tick-borne infections, while highlighting the potential for such infections to escape into alternative hosts via I. ricinus.

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

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.

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.

Anaplasma phagocytophilum is an emerging tick-borne pathogen that infects humans, domestic animals and wildlife throughout the Holarctic. In the far-western United States, multiple rodent species have been implicated as natural reservoirs for A. phagocytophilum. However, the presence of multiple A. phagocytophilum strains has made it difficult to determine which reservoir hosts pose the greatest risk to humans and domestic animals. Here we characterized three genetic markers (23S–5S rRNA intergenic spacer, ank and groESL) from 73 real-time TaqMan PCR-positive A. phagocytophilum strains infecting multiple rodent and reptile species, as well as a dog and a horse, from California. Bayesian and maximum-likelihood phylogenetic analyses of all three genetic markers consistently identified two major clades, one of which consisted of A. phagocytophilum strains infecting woodrats and the other consisting of strains infecting sciurids (chipmunks and squirrels) as well as the dog and horse strains. In addition, analysis of the 23S–5S rRNA spacer region identified two unique and highly dissimilar clades of A. phagocytophilum strains infecting several lizard species. Our findings indicate that multiple unique strains of A. phagocytophilum with distinct host tropisms exist in California. Future epidemiological studies evaluating human and domestic animal risk should incorporate these distinctions.

Background

Tick-borne Anaplasma phagocytophilum and Rickettsia spp. are considered to be emerging human pathogens, but only limited data are available on their occurrence in Sweden. Two real-time PCR assays followed by nested PCR and sequence analysis were carried out to investigate the prevalence of A. phagocytophilum and spotted fever rickettsiae in ticks from seven areas in Sweden.

Results

In 139 pooled samples, representing a total of 1245 Ixodes ricinus ticks (204 larvae, 963 nymphs, 38 males, 40 females), the overall positive mean infection prevalence was 1.3-15.0% for A. phagocytophilum and 1.5-17.3% for R. helvetica. A. phagocytophilum was only detected in nymphs (1.7-19.4%), whereas R. helvetica was detected in all three tick stages. Support for vertical and transstadial transmission was only obtained for R. helvetica. Both agents showed similar infection rates across study areas, although infection rates were greater in coastal areas.

Conclusions

The results show that both pathogens occurred in all seven locations, indicating that they are prevalent in Sweden and should be considered etiological agents in patients recently bitten by ticks.

Anaplasma phagocytophilum DNA is detected in Portuguese Ixodes ricinus and I. ventalloi ticks.

A total of 278 Ixodes ticks, collected from Madeira Island and Setúbal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setúbal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setúbal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

To estimate the likelihood of people coming into contact with the recently described tick-borne agent “Candidatus Neoehrlichia mikurensis,” we compared its prevalence to those of Lyme disease spirochetes and Anaplasma phagocytophilum in questing adult Ixodes ricinus ticks collected in various Central European sites and examined ticks, which had been removed from people, for the presence of these pathogens. Whereas spirochetes infected questing adult ticks most frequently (22.3%), fewer than a third as many ticks were infected by “Ca. Neoehrlichia mikurensis” (6.2%), and about a sixth harbored A. phagocytophilum (3.9%). On average, every twelfth encounter of a person with an I. ricinus tick (8.1%) may bear the risk of acquiring “Ca. Neoehrlichia mikurensis.” Although a fifth of the people (20%) had removed at least one tick infected by “Ca. Neoehrlichia mikurensis,” none displayed symptoms described for this pathogen, suggesting that its transmission may not be immediate and/or that immunocompetent individuals may not be affected. Because immunosuppressed patients may be at a particular risk of developing symptoms, it should be considered that “Ca. Neoehrlichia mikurensis” appears to be the second most common pathogen in I. ricinus ticks. In our survey, only Borrelia afzelii appears to infect Central European vector ticks more frequently.

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

The redwood chipmunk contributes to the maintenance of tick-borne diseases in northern California. The range of redwood chipmunks overlaps that of western black-legged ticks and tick-borne disease, including granulocytic anaplasmosis and Lyme borreliosis. Chipmunks have high Anaplasma phagocytophilum PCR- and seroprevalence, are infested with a diversity of Ixodes spp. ticks, and are reservoir competent for Borrelia burgdorferi. We hypothesized that chipmunks could maintain tick-borne disease on the forest floor while also potentially bridging infection to arboreal sciurids as well. We used radio-telemetry to evaluate chipmunk movement and use of trees, characterized burrows, described prevalence of tick-borne disease, and identified ticks on these chipmunks. A total of 192 chipmunks from Hendy Woods, Mendocino County, California, USA, was evaluated between November 2005 and April 2009. The mean density was 2.26–5.8 chipmunks/ha. The longest detected life span was 3 years. Female weights ranged from 80–120 g and males from 80–180 g. The A. phagocytophilum and Borrelia spp. seroprevalence was 21.4% and 24.7%, respectively, and PCR prevalence for these pathogens was 10.6% and 0%, respectively. Ixodes spp. ticks included I. angustus, I. ochotonae, I. pacificus, and I. spinipalpis. The mean infestation level was 0.92 ticks/chipmunk. Based on telemetry of 11 chipmunks, the greatest distance traveled ranged from 0.14–0.63 km for females and 0.1–1.26 km for males. Areas occupied by chipmunks ranged from 0.005–0.24 km2 for females and 0.006–0.73 km2 for males. On 3 occasions, chipmunks were found in trees. Burrows were identified under a moss-covered redwood log, deep under a live redwood tree, under a Douglas fir log, in a clump of huckleberry, in a root collection from an overturned Douglas fir tree, and in a cluster of exposed huckleberry roots. The biology of the redwood chipmunk has multiple features that allow it to be an important reservoir host for tick-borne disease in northwestern California.

Background

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.

Methods

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).

Results

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.

Conclusions

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.

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.

Anaplasmosis, a tick-borne cattle disease caused by the rickettsia Anaplasma marginale, is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to both the dairy and beef industries worldwide. Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma, of which A. marginale is the type species, now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila group (which causes human granulocytic ehrlichiosis), respectively. Live and killed vaccines have been used for control of anaplasmosis, and both types of vaccines have advantages and disadvantages. These vaccines have been effective in preventing clinical anaplasmosis in cattle but have not blocked A. marginale infection. Thus, persistently infected cattle serve as a reservoir of infective blood for both mechanical transmission and infection of ticks. Advances in biochemical, immunologic, and molecular technologies during the last decade have been applied to research of A. marginale and related organisms. The recent development of a cell culture system for A. marginale provides a potential source of antigen for the development of improved killed and live vaccines, and the availability of cell culture-derived antigen would eliminate the use of cattle in vaccine production. Increased knowledge of A. marginale antigen repertoires and an improved understanding of bovine cellular and humoral immune responses to A. marginale, combined with the new technologies, should contribute to the development of more effective vaccines for control and prevention of anaplasmosis.

Anaplasma phagocytophilum, the agent of human anaplasmosis, persists in ticks and mammals. We show that A. phagocytophilum induces the phosphorylation of actin in an Ixodes ricinus tick cell line and Ixodes scapularis ticks, to alter the ratio of monomeric/filamentous (G/F) actin. A. phagocytophilum–induced actin phosphorylation was dependent on Ixodes p21-activated kinase (IPAK1)–mediated signaling. A. phagocytophilum stimulated IPAK1 activity via the G protein–coupled receptor Gβγ subunits, which mediated phosphoinositide 3-kinase (PI3K) activation. Disruption of Ixodes gβγ, pi3k, and pak1 reduced actin phosphorylation and bacterial acquisition by ticks. A. phagocytophilum–induced actin phosphorylation resulted in increased nuclear G actin and phosphorylated actin. The latter, in association with RNA polymerase II (RNAPII), enhanced binding of TATA box–binding protein to RNAPII and selectively promoted expression of salp16, a gene crucial for A. phagocytophilum survival. These data define a mechanism that A. phagocytophilum uses to selectively alter arthropod gene expression for its benefit and suggest new strategies to interfere with the life cycle of this intracellular pathogen, and perhaps other Rickettsia-related microbes of medical importance.

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.