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.
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.
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.
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.
Equine Granulocytic Anaplasmosis (EGA) is caused by Anaplasma phagocytophilum, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by Ixodes ricinus. A large number of genetic variants of A. phagocytophilum circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent A. phagocytophilum of 14 cases of EGA in naturally infected horses with molecular methods on the basis of 4 partial genes (16S rRNA, groEL, msp2, and msp4).
All DNA extracts of EDTA-blood samples of the horses gave bands of the correct nucleotide size in all four genotyping PCRs. Sequence analysis revealed 4 different variants in the partial 16S rRNA, groEL gene and msp2 genes, and 3 in the msp4 gene. One 16S rRNA gene variant involved in 11 of the 14 cases was identical to the "prototype" variant causing disease in humans in the amplified part [GenBank: U02521]. Phylogenetic analysis revealed as expected for the groEL gene that sequences from horses clustered separately from roe deer. Sequences of the partial msp2 gene from this study formed a separate cluster from ruminant variants in Europe and from all US variants.
The results show that more than one variant of A. phagocytophilum seems to be involved in EGA in Germany. The comparative genetic analysis of the variants involved points towards different natural cycles in the epidemiology of A. phagocytophilum, possibly involving different reservoir hosts or host adaptation, rather than a strict species separation.
Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.
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
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.
We report the isolation and partial genetic characterization of two equine strains of granulocytic Ehrlichia of the genogroup Ehrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. Granulocytic Ehrlichia was isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, the groESL heat shock operon gene, and the ank gene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, the groESL gene, and the ank gene, the DNA sequences of the two equine Ehrlichia isolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an American Ehrlichia strain of the E. phagocytophila genogroup, differences were found in the groESL gene and ank gene sequences.
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.
Portugal; Anaplasma phagocytophilum; Polymerase chain reaction (PCR); Ticks; Ixodes ricinus; Ixodes ventalloi; research
The first tissue culture isolates of the unique Anaplasma phagocytophilum strain, Ap-Variant 1, were obtained in the Ixodes scapularis tick-derived cell line ISE6. Two isolates were from goat blood samples: one from a goat infected with I. scapularis ticks from Rhode Island and a second from a goat infected by serial passage of blood from the first infected goat. Eight isolates were made directly from I. scapularis ticks collected from white-tailed deer in Minnesota and represent the first isolations of an Anaplasma species directly from ticks. Each of the 10 isolates had a 16S rRNA gene sequence identical to that previously described for Ap-Variant 1, but differences within the ank gene were found that suggest natural variation. Prevalence of Anaplasma in the Minnesota ticks was 63.9%; 23 of 36 ticks tested by PCR were positive. Six of the tick-derived isolates were obtained from a set of 18 PCR-positive ticks, for a 33.3% isolation success rate. The conservation of host tropism among the Rhode Island and Minnesota isolates of Ap-Variant 1 was examined by use of experimental infections of mice and a goat. A Minnesota tick-derived isolate (MN-61-2) was used to inoculate naïve animals, and this isolate was able to infect a goat but unable to infect each of five mice, confirming that the Minnesota isolates have the same host tropism as Ap-Variant 1 from the northeastern United States. Light and electron microscopy of the Ap-Variant 1 isolate MN-61-2 in ISE6 cells showed cytoplasmic inclusions characteristic of A. phagocytophilum with pleomorphic bacteria in membrane-bound vacuoles and both electron-dense and electron-lucent forms.
Analysis of several nucleotide polymorphisms in polymorphic genes (ankA, gltA, and groESL) from 16S rRNA gene-based genetic variants of Anaplasma phagocytophilum from dogs in the western United States defined at least two sets of multigene polymorphisms to further characterize these variants. The multigene polymorphism approach holds promise for development of a genotyping scheme for this important pathogen.
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
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.
Anaplasma phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) in humans and tick-borne fever (TBF) in ruminants. The bacterium invades and replicates in phagocytes, especially in polymorphonuclear granulocytes.
In the present study, skin biopsies and ticks (Ixodes ricinus) were collected from tick feeding lesions on 38 grazing lambs between two and three weeks after access to pastures. The histopathological changes associated with tick bites and A. phagocytophilum infection, were described. In addition the skin biopsies were examined by immunohistochemistry. Furthermore, samples from blood, skin biopsies and ticks were examined by serology, PCR amplification of msp2 (p44), genotyping of rrs (16S rRNA) variants, and compared with the results obtained from histological and immunohistochemical investigations.
Tick bites were associated with chronic and hyperplastic inflammatory skin lesions in this study. A. phagocytophilum present in skin lesions were mainly associated with neutrophils and macrophages. Bacteria were occasionally observed in the Tunica media and Tunica adventitia of small vessels, but were rarely found in association with endothelial cells. PCR and genotyping of organisms present in blood, ticks and skin biopsies suggested a haematogenous and a local spread of organisms at the tick attachment sites.
The present study describes different aspects of A. phagocytophilum infection at the site of tick bite, and indicates that A. phagocytophilum rarely associates with endothelium during the early pathogenesis of infection.
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.
Five-month-old lambs were experimentally infected with two 16S rRNA genetic variants of Anaplasma phagocytophilum, variants 1 (GenBank accession no. M73220) and 2 (GenBank accession no. AF336220). Additional sequencing of the groESL heat shock operon gene indicated that these variants differ in three nucleotides at positions 782, 824, and 890. The variants were obtained by blood sampling of A. phagocytophilum-infected lambs from one sheep flock in Norway and were stored at −70°C with 10% dimethyl sulfoxide as a cryoprotectant before being inoculated intravenously into susceptible lambs. The infectious blood contained, per ml, approximately 0.5 × 106 neutrophils infected with either of the variants. Six weeks after the primary inoculation, the lambs were challenged with the same infectious dose of the heterologous variant. The results of the study indicate a marked difference in clinical manifestation, neutropenia, antibody response, and cross-protection after experimental infection with the two variants of A. phagocytophilum.
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.
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.
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.
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.
Human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellusundulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D. silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.
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.
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.
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.
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.