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A broad-range 16S rRNA gene PCR assay followed by partial sequencing of the 16S rRNA gene was used for the detection of members of the family Anaplasmataceae in ticks in North Africa. A total of 418 questing Ixodes ricinus ticks collected in Tunisia and Morocco, as well as 188 Rhipicephalus ticks from dogs and 52 Hyalomma ticks from bovines in Tunisia, were included in this study. Of 324 adult I. ricinus ticks, 16.3% were positive for Ehrlichia spp., whereas only 3.4 and 2.8% of nymphs and larvae, respectively, were positive. A large heterogeneity was observed in the nucleotide sequences. Partial sequences identical to that of the agent of human granulocytic ehrlichiosis (HGE) were detected in I. ricinus and Hyalomma detritum, whereas partial sequences identical to that of Anaplasma platys were detected in Rhipicephalus sanguineus. However, variants of Anaplasma, provisionally designated Anaplasma-like, were predominant in the I. ricinus tick population in Maghreb. Otherwise, two variants of the genus Ehrlichia were detected in I. ricinus and H. detritum. Surprisingly, a variant of Wolbachia pipientis was evidenced from I. ricinus in Morocco. These results emphasized the potential risk of tick bites for human and animal populations in North Africa.

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.

To date, human granulocytic ehrlichiosis (HGE), the causative agent of which is likely transmitted by ticks in the Ixodes ricinus-Ixodes persulcatus complex, has not been diagnosed with certainty in patients outside the United States. The presence of a closely related vector tick, I. ricinus, as well as the occurrence of similar Ehrlichia spp. of veterinary importance, suggests that this disease is likely to be present in Europe. The aim of the present study was to compare the prevalence of antibodies against the HGE agent in sera collected from patients in groups at high risk for exposure to I. ricinus with that of a control population. Risk groups consisted of 150 forestry workers and 105 patients with an established diagnosis of Lyme disease. The control group was 103 healthy blood donors without a history of tick bites. We used a patient isolate of the HGE agent from Minnesota (J. L. Goodman, C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh, N. Engl. J. Med. 334:209-215, 1996) propagated in HL60 cells as the source of antigen for a specific immunofluorescence assay (IFA). Elevated IFA titers (> or = 1:80) were present in 21 of 150 (14%) serum samples from forestry workers and in 12 of 105 (11.4%) serum samples from Lyme disease patients, but in only 2 of 103 (1.9%) serum samples from blood donors (P < or = 0.01 for either of the at-risk groups versus blood donors). The results of this study suggest that the HGE agent or a closely related organism exists in southern Germany and that seroconversion to it is common among groups exposed to Ixodes ticks. Final proof that HGE occurs in Germany will require the isolation of the causative agent from patients. HGE should be considered in the differential diagnosis of febrile illnesses in individuals exposed to Ixodes ticks in Europe as well as in North America.

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.

Ticks act as vectors of many pathogens of domestic animals and humans. Anaplasma phagocytophilum in Europe is transmitted by the ixodid tick vector Ixodes ricinus. A. phagocytophilum causes a disease with diverse clinical signs in various hosts. A great genetic diversity of the groESL operon of A. phagocytophilum has been found in ticks elsewhere. In Slovenia, the variety of the groESL operon was conducted only on deer samples. In this study, the prevalence of infected ticks was estimated and the diversity of A. phagocytophilum was evaluated. On 8 locations in Slovenia, 1924 and 5049 (6973) I. ricinus ticks were collected from vegetation in the years 2005 and 2006, respectively. All three feeding stages of the tick's life cycle were examined. The prevalence of ticks infected with A. phagocytophilum in the year 2005 and in the year 2006 was 0.31% and 0.63%, respectively, and it did not differ considerably between locations. The similarity among the sequences of groESL ranged from 95.6% to 99.8%. They clustered in two genetic lineages along with A. phagocytophilum from Slovenian deer. One sequence formed a separate cluster. According to our study, the prevalence of A. phagocytophilum in ticks is comparable to the findings in other studies in Europe, and it does not vary considerably between locations and tick stages. According to groESL operon analysis, two genetic lineages have been confirmed and one proposed. Further studies on other genes would be useful to obtain more information on genetic diversity of A. phagocytophilum in ticks in Slovenia.

Human granulocytic ehrlichiosis (HGE) was recently described in North America. It is caused by an Ehrlichia species closely related to Ehrlichia phagocytophila and Ehrlichia equi, recognized to infect mostly ruminants and horses, respectively. The vector in North America is the tick Ixodes scapularis, which is also the vector of the Lyme disease agent, Borrelia burgdorferi. Previous serologic studies in patients with a diagnosis of Lyme borreliosis indicate that HGE may exist in Europe. We report the first documented case of HGE in Europe. The diagnosis was established by seroconversion to E. equi and the HGE agent and by PCR with sequence analysis of the gene encoding the HGE agent 16S rRNA. Interestingly, the patient presented with a self-limited but moderately severe illness. Thus, European physicians need to be aware that HGE exists in Europe and that the diagnosis should be considered in febrile patients with tick bites in areas where Lyme disease is endemic.

We examined 11 naturally occurring isolates of Ehrlichia equi in horses and two human granulocytic ehrlichiosis agent isolates in California for sequence diversity in three genes. Ehrlichia equi isolates were from Sierra (n = 6), Mendocino (n = 3), Sonoma (n = 1), and Marin (n = 1) counties, and human granulocytic ehrlichiosis (HGE) agent isolates were obtained from Humboldt county. PCR with specific primers for 16S rRNA, 444 Ep-ank and groESL heat shock operon genes successfully produced amplicons for all 13 clinical samples. The 444 Ep-ank gene of the HGE agent and E. equi isolates from northern California is different from the eastern U.S. isolates BDS and USG3. The translated amino acid sequence of the groESL heat shock operon gene fragment is identical among E. equi, the HGE agent, and E. phagocytophila, with the exception of the northern Californian equine CASOLJ isolate. Microheterogeneity was observed in the 16S rRNA gene sequences of HGE agent and E. equi isolates from northern California. These results suggest that E. equi and the HGE agent found in California are similar or identical but may differ from the isolates of equine and human origin found in the eastern United States.

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.

The potential role of ticks as vectors of Bartonella species has recently been suggested. In this study, we investigated the presence of Bartonella species in 271 ticks removed from humans in Belluno Province, Italy. By using primers derived from the 60-kDa heat shock protein gene sequences, Bartonella DNA was amplified and sequenced from four Ixodes ricinus ticks (1.48%). To confirm this finding, we performed amplification and partial sequencing of the pap31 protein and the cell division protein FtsZ encoding genes. This process allowed us to definitively identify B. henselae (genotype Houston-1) DNA in the four ticks. Detection of B. henselae in these ticks might represent a highly sensitive form of xenodiagnosis. B. henselae is the first human-infecting Bartonella identified from Ixodes ricinus, a common European tick and the vector of various tickborne pathogens. The role of ticks in the transmission of bartonellosis should be further investigated.

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.

A total of 1,667 Ixodes ricinus ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the Ehrlichia phagocytophila group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of Ehrlichia. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in I. ricinus in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.

Background

Ticks of the species Ixodes ricinus are the main vectors of Lyme Borreliosis and Tick-borne Encephalitis – two rapidly emerging diseases in Europe. Repellents provide a practical means of protection against tick bites and can therefore minimize the transmission of tick-borne diseases. We developed and tested seven different dodecanoic acid (DDA)-formulations for their efficacy in repelling host-seeking nymphs of I. ricinus by laboratory screening. The ultimately selected formulation was then used for comparative investigations of commercially available tick repellents in humans.

Methods

Laboratory screening tests were performed using the Moving-object (MO) bioassay. All test formulations contained 10% of the naturally occurring active substance DDA and differed only in terms of the quantitative and qualitative composition of inactive ingredients and fragrances. The test procedure used in the human bioassays is a modification of an assay described by the U.S. Environmental Protection Agency and recommended for regulatory affairs. Repellency was computed using the equation: R = 100 - NR/N × 100, where NR is the number of non-repelled ticks, and N is the respective number of control ticks. All investigations were conducted in a controlled laboratory environment offering standardized test conditions.

Results

All test formulations strongly repelled nymphs of I. ricinus (100-81% protection) as shown by the MO-bioassay. The majority of ticks dropped off the treated surface of the heated rotating drum that served as the attractant (1 mg/cm2 repellent applied). The 10% DDA-based formulation, that produced the best results in laboratory screening, was as effective as the coconut oil-based reference product. The mean protection time of both preparations was generally similar and averaged 8 hours.

Repellency investigations in humans showed that the most effective 10% DDA-based formulation (~1.67 mg/cm2 applied) strongly avoided the attachment of I. ricinus nymphs and adults for at least 6 hours. The test repellent always provided protection (83-63%) against I. ricinus nymphs equivalent to the natural coconut oil based reference product and a better protection (88-75%) against adult ticks than the synthetic Icaridin-containing reference repellent.

Conclusion

We found that the 10% DDA-based formulation (ContraZeck®) is an easily applied and very effective natural repellent against I. ricinus ticks. By reducing the human-vector contact the product minimises the risk of transmission of tick-borne diseases in humans.

White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.

The purpose of the present study was to investigate the transmission of a human isolate of the agent of human granulocytic ehrlichiosis (HGE agent) from infected mice to larval ticks and to examine the population kinetics of the HGE agent in different stages of the tick life cycle. The HGE agent was quantitated by competitive PCR with blood from infected mice and with Ixodes scapularis ticks. The median infectious dose for C3H mice was 104 to 105 organisms when blood from an infected severe combined immunodeficient mouse was used as an inoculum. Uninfected larval ticks began to acquire infection from infected mice within 24 h of attachment, and the number of HGE agent organisms increased in larval ticks during feeding and after detachment of replete ticks. Molted nymphal ticks, infected as larvae, transmitted infection to mice between 40 and 48 h of attachment. Onset of feeding stimulated replication of the HGE agent within nymphal ticks. These studies suggest that replication of the HGE agent during and after feeding in larvae and during feeding in nymphs is a means by which the HGE agent overcomes inefficiencies in acquisition of infection by ticks and in tick-borne transmission to mammalian hosts.

The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.

Adult Ixodes ricinus ticks were collected in Switzerland and tested for the presence of coinfection with Borrelia burgdorferi sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for B. burgdorferi and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for B. burgdorferi.

A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.

Ixodes scapularis ticks were collected in 2000 and 2001 from two areas in Pennsylvania and tested for the presence of Anaplasma phagocytophilum and Borrelia burgdorferi by PCR and DNA sequencing. Of the ticks collected from northwestern and southeastern Pennsylvania, 162 of 263 (61.6%) and 25 of 191 (13.1%), respectively, were found to be positive for B. burgdorferi. DNA sequencing showed >99% identity with B. burgdorferi strains B31 and JD1. PCR testing for A. phagocytophilum revealed that 5 of 263 (1.9%) from northwestern Pennsylvania and 76 of 191 (39.8%) from southeastern Pennsylvania were positive. DNA sequencing revealed two genotypes of A. phagocytophilum, the human granulocytic ehrlichiosis (HGE) agent and a variant (AP-Variant 1) that has not been associated with human infection. Although only the HGE agent was present in northwestern Pennsylvania, both genotypes were found in southeastern Pennsylvania. These data add to a growing body of evidence showing that AP-Variant 1 is the predominant agent in areas where both genotypes coexist.

The presence of granulocytic ehrlichiae was demonstrated by PCR in Ixodes ricinus ticks and wild small mammals in Switzerland in two areas of endemicity for bovine ehrlichiosis. Six ticks (three females and three nymphs) (1.4%) of 417 I. ricinus ticks collected by flagging vegetation contained ehrlichial DNA. A total of 201 small mammals from five species, wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), earth vole (Pitymys subterraneus), bank vole (Clethrionomys glareolus), and common shrew (Sorex araneus), were trapped. The analysis of I. ricinus mammals collected on 116 small mammals showed that nine C. glareolus voles and two A. sylvaticus mice hosted infected tick larvae. In these rodents, granulocytic ehrlichia infection was also detected in blood, spleen, liver, and ear samples. Further examinations of 190 small mammals without ticks or with noninfected ticks showed the presence of ehrlichial DNA in spleen and other tissues from six additional C. glareolus, three A. flavicollis, and one S. araneus mammals. This study suggests that A. sylvaticus, A. flavicollis, S. araneus, and particularly C. glareolus are likely to be natural reservoirs for granulocytic ehrlichiae. Partial 16S rRNA gene sequences of granulocytic ehrlichiae from ticks and rodents showed a high degree of homology (99 to 100%) with granulocytic ehrlichiae isolated from humans. In contrast, groESL heat shock operon sequence analysis showed a strong divergence (approximately 5%) between the sequences in samples derived from rodents and those derived from samples from questing ticks or from other published ehrlichia sequences. Dual infections with granulocytic ehrlichia and Borrelia burgdorferi were found in ticks and small mammals.

We compiled information on the distribution of ticks in the western Palearctic (11°W, 45°E; 29°N, 71°N), published during 1970–2010. The literature search was filtered by the tick’s species name and an unambiguous reference to the point of capture. Records from some curated collections were included. We focused on tick species of importance to human and animal health, in particular: Ixodes ricinus, Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, H. sulcata, Hyalomma marginatum, Hy. lusitanicum, Rhipicephalus annulatus, R. bursa, and the R. sanguineus group. A few records of other species (I. canisuga, I. hexagonus, Hy. impeltatum, Hy. anatolicum, Hy. excavatum, Hy. scupense) were also included. A total of 10,280 records was included in the data set. Almost 42 % of published references are not adequately referenced (and not included in the data set), host is reported for only 61 % of records and a reference to time of collection is missed for 84 % of published records. Ixodes ricinus accounted for 44.3 % of total records, with H. marginatum and D. marginatus accounting for 7.1 and 8.1 % of records, respectively. The lack of homogeneity of the references and potential pitfalls in the compilation were addressed to create a digital data set of the records of the ticks. We attached to every record a coherent set of quantitative descriptors for the site of reporting, namely gridded interpolated monthly climate and remotely sensed data on vegetation (NDVI). We also attached categorical descriptors of the habitat: a standard classification of land biomes and an ad hoc classification of the target territory from remotely sensed temperature and NDVI data. A descriptive analysis of the data revealed that a principal components reduction of the environmental (temperature and NDVI) variables described the distribution of the species in the target territory. However, categorical descriptors of the habitat were less effective. We stressed the importance of building reliable collections of ticks with specific references as to collection point, host and date of capture. The data set is freely downloadable.

Electronic supplementary material

The online version of this article (doi:10.1007/s10493-012-9600-7) contains supplementary material, which is available to authorized users.

In Wisconsin and Minnesota, Ixodes scapularis (Ixodes dammini) ticks are the vector of three microorganisms that may cause significant disease in humans and lower mammals. These diseases include Lyme borreliosis, which is caused by Borrelia burgdorferi, babesiosis, which is caused by Babesia microti, and human granulocytic ehrlichiosis (HGE), which is caused by an apparently new species in the genus Ehrlichia. Immunoserologic testing was performed on sera from patients with a diagnosis of one of these diseases to determine if there was evidence of coinfection with one or more of the other agents. Of 96 patients with Lyme borreliosis, 9 (9.4%) demonstrated immunoserologic evidence of coinfection: 5 (5.2%) with the agent of HGE, 2 (2.1%) with B. microti, and 2 (2.1%) with both microorganisms. Of 19 patients diagnosed with HGE, 3 (15.8%) showed immunoserologic evidence of coinfection: 1 (5.3%) with B. burgdorferi, 1 (5.3%) with B. microti, and 1 (5.3%) with both microorganisms. One patient diagnosed with babesiosis was also seropositive for ehrlichiosis. These results provide evidence for coinfection, perhaps explaining the variable manifestations and clinical responses noted in some patients with tick-transmitted diseases. In certain clinical settings, laboratory testing for coinfection is indicated to ensure that appropriate antimicrobial treatment is given.

The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.