Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan

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 sequences of groESL operon of Anaplasma phagocytophilum among human patients in Slovenia

Anaplasma phagocytophilum is an emerging tick-borne pathogen. Great genetic diversity of A. phagocytophilum has been described in animals and ticks. The present study is focused on the genetic variability of the groESL operon of A. phagocytophilum in human patients in Slovenia. During 1996–2008, there were 66 serologically confirmed patients with human granulocytic anaplasmosis. Of these, 46 were tested with a screening PCR for a small part of the 16S rRNA gene of A. phagocytophilum and 28 (60.9%) were positive. Positive samples were additionally tested with a PCR targeting the groESL operon and a larger fragment of the 16S rRNA gene. All amplicons were further sequenced and analyzed. The homology search and the alignment of the groESL sequences showed only one genetic variant. Sequence analysis of the 16S rRNA gene revealed 100% identity among amplicons. Slovenia is a small country with diverse climate, vegetation, and animal representatives. In previous studies in deer, dogs, and ticks, great diversity of the groESL operon was found. In contrast, in wild boar and in human patients from this study, only one genetic variant was detected. The results suggest that only one genetic variant might be pathogenic for humans or is competent enough to replicate in humans. To support this theory, other genetic markers and further studies need to be performed.     

Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands

Ixodes ticks harbour several human pathogens belonging to the order Rickettsiales, including Anaplasma phagocytophilum, the agent of human anaplasmosis. When ticks feed on A. phagocytophilum-infected mice, the pathogen enters the ticks' gut. The bacteria then migrate from the gut to infect the salivary glands of the ticks and are transmitted to the next host via the saliva. The molecular mechanisms that enable the migration of A. phagocytophilum from the gut to the salivary glands are poorly understood. Here we show that a secreted tick protein, P11, is important in this process. We show that P11 enables A. phagocytophilum to infect tick haemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands. Silencing of p11 impaired the A. phagocytophilum infection of tick haemocytes in vivo and consequently decreased pathogen infection of the salivary glands. In vitro experiments showed that P11 could bind to A. phagocytophilum and thus facilitate its infection of tick cells. This report provides new insights into A. phagocytophilum infection of ticks and reveals new avenues to interrupt the life cycle of Anaplasma and related Rickettsial pathogens.     

Study of the heterogeneity of 16s rRNA gene and groESL operone in the dna samples of Anaplasma phagocytophilum, Ehrlichia muris, and "Candidatus Neoehrlichia mikurensis" determined in the Ixodes persulcatus ticks in the area of Urals, Siberia, and far east of Russia

A total of 3552 Ixodes persulcatus from Sverdlovsk, Chelyabinsk, Novosibirsk, Irkutsk regions and Khabarovsk Territory were examined on the Ehrlichia and Anaplasma presence by nested PCR based on the 16S rRNA gene. Both Anaplasma phagocytophilum and Ehrlichia muris DNA were found in I. persulcatus in all studied regions. A. Phagocytophilum was detected in 1.3-6.3% of ticks and E. muris - in 2.0-14.1% of ticks. Moreover, "Candidatus Neoehrlichia mikurensis" DNA was found in 8 ticks collected in Novosibirsk, Irkutsk Regions and Khabarovsk Territory. Partial nucleotide sequences of 16S rRNA gene and groESL operone (1240-1300 bp) were determined for 65 samples of A. Phagocytophilum, 17 samples of E. muris and 4 samples of "Candidatus Neoehrlichia mikurensis". Nucleotide sequences of 16S rRNA gene and groESL operone of E. muris and "Candidatus Neoehrlichia mikurensis" were shown to be highly conservative, and nucleotide sequences of groESL operone of both E. muris and "Candidatus Neoehrlichia mikurensis" differed from the sequences found previously in other species of Ixodid tick. On the basis of analysis of the 16S rRNA gene and groESL operone sequences it was concluded that all revealed samples A. Phagocytophilum could be divided into 2 groups. GroESL operone sequences of A. Phagocytophilum from the first group were identical to each other but significantly differed from the known groESL operone sequences (less than 98.2% of similarity), whereas their 16S rRNA gene sequences were identical to the sequence of widely distributed and pathogenic for human A. Phagocytophilum genetic variant (CAHU-HGEl, GenBank AF093788) or differed from it by a single nucleotide substitution. The nucleotide sequences of groESL operone of A. Phagocytophilum from the second group differed from each other by 1-4 nucleotides and were closely related (99.2-99.4% of similarity) to the sequences of groESL operone ofA. phagocytophilum isolates found in Europe in Ixodes ricinus and roe deer. The nucleotide sequences of the 16S rRNA gene of A. Phagocytophilum from the second group were most similar to the sequence of the rare A. Phagocytophilum genetic variant previously found only in China (GenBank DQ342324).     

Co-phylogenetic analysis of Anaplasma phagocytophilum and its vectors, Ixodes spp. ticks

The coevolutionary history of Ixodes spp. ticks, the obligately tick-transmitted bacterial pathogen Anaplasma phagocytophilum, and its various rodent reservoir hosts world-wide is not known. According to coevolution theory, the most recently evolved of tick-bacterial complexes could have difficulty maintaining A. phagocytophilum in nature, because transmissibility has not been efficiently maximized. This study was intended to examine the phylogeographic history of I. ricinus-subgroup ticks and A. phagocytophilum, provide an estimate for the date of the divergence of A. marginale and A. phagocytophilum, and evaluate whether there is correspondence between tick and Anaplasma spp. trees. Analysis of Ixodes spp. ticks showed a New World clade consisting of I. scapularis and I. pacificus, European I. ricinus as a sister group to this clade, and Asian I. persulcatus as basal. Of the three A. phagocytophilum genes evaluated, the most resolution was provided by the ankA gene. ankA sequences formed an Old World clade with eastern North America strains as a sister clade. California strains were highly diverse and did not form a clade. Base substitution rates were very comparable along both A. marginale and A. phagocytophilum lineages. Based on 16S rDNA analysis, maximum and minimum divergence times of A. phagocytophilum and A. marginale were calculated to be 78,296,703 and 43,415,708 years, respectively. If A. phagocytophilum did closely coevolve with specific I. ricinus-subgroup tick species, then A. phagocytophilum strains could have specialized on local tick species and optimized local infectivity in the Old World and eastern US. However, lack of absolute resolution of tick trees and conflicting prevalence data (with low prevalence in Asia and western North America) preclude us from inferring a tight coevolutionary relationship of tick species from this phylogeographic analysis.     

Clinical, epidemiologic, and environmental surveillance for ehrlichiosis and anaplasmosis in an endemic area of northern California.

Two forms of tick-borne leukocytotropic rickettsioses have been recognized in California since the mid-1990s: human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis and human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum. Between 1997 and 1999, two cases of HME and four cases of HGA were diagnosed in residents of southern Humboldt County, California. Environmental followup at case-patients' residences revealed dense populations of Ixodes pacificus ticks, particularly in grassy roadside areas. PCR evidence of A. phagocytophilum was detected in approximately 2.0% of I. pacificus; E. chaffeensis was not detected in any of 625 ticks tested. Serologic antibody to A. phagocytophilum was detected in two of 54 participants in a community epidemiologic study; one of these also had antibody to E. chaffeensis. Over 85% of study participants reported finding a tick on themselves in the preceding 12 mo. Residents of southern Humboldt County are at significant risk of tick bites and should take appropriate prevention measures to avoid infection with rickettsia and other tick-transmitted pathogens.     

Longitudinal analysis of tick densities and Borrelia, Anaplasma, and Ehrlichia infections of Ixodes ricinus ticks in different habitat areas in The Netherlands

From 2000 to 2004, ticks were collected by dragging a blanket in four habitat areas in The Netherlands: dunes, heather, forest, and a city park. Tick densities were calculated, and infection with Borrelia burgdorferi and Anaplasma and Ehrlichia species was investigated by reverse line blot analysis. The lowest tick density was observed in the heather area (1 to 8/100 m2). In the oak forest and city park, the tick densities ranged from 26 to 45/100 m2. The highest tick density was found in the dune area (139 to 551/100 m2). The infection rates varied significantly for the four study areas and years, ranging from 0.8 to 11. 5% for Borrelia spp. and 1 to 16% for Ehrlichia or Anaplasma (Ehrlichia/Anaplasma) spp. Borrelia infection rates were highest in the dunes, followed by the forest, the city park, and heather area. In contrast, Ehrlichia/Anaplasma was found most often in the forest and less often in the city park. The following Borrelia species were found: Borrelia sensu lato strains not identified to the species level (2.5%), B. afzelii (2.5%), B. valaisiana (0.9%), B. burgdorferi sensu stricto (0.13%), and B. garinii (0.13%). For Ehrlichia/Anaplasma species, Ehrlichia and Anaplasma spp. not identified to the species level (2.5%), Anaplasma schotti variant (3.5%), Anaplasma phagocytophilum variant (0.3%), and Ehrlichia canis (0.19%) were found. E. canis is reported for the first time in ticks in The Netherlands in this study. Borrelia lusitaniae, Ehrlichia chaffeensis, and the human granylocytic anaplasmosis agent were not detected. About 1.6% of the ticks were infected with both Borrelia and Ehrlichia/Anaplasma, which was higher than the frequency predicted from the individual infection rates, suggesting hosts with multiple infections or a possible selective advantage of coinfection.     

Molecular analysis of microbial communities identified in different developmental stages of Ixodes scapularis ticks from Westchester and Dutchess Counties, New York

Ixodes scapularis ticks play an important role in the transmission of a wide variety of pathogens between various mammalian species, including humans. Pathogens transmitted by ticks include Borrelia, Anaplasma and Babesia. Although ticks may harbour both pathogenic and non-pathogenic microflora, little is known about how the diversity of the microflora within ticks may influence the transmission of pathogens. To begin addressing this question, we examined the composition of bacterial communities present in Ixodes scapularis collected from Westchester and Dutchess Counties, New York State, at different developmental and nutritional stages. Genetic fingerprints of bacterial populations were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis for bacterial identification. The fingerprints of the TTGE bands were grouped into five clusters. The most abundant DNA sequence found in all the samples was Rickettsia, followed by Pseudomonas and Borrelia. Ralstonia, Anaplasma, Enterobacterias, Moraxella, Rhodococcus and uncultured proteobacterium were present as well. We also determined the prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi by PCR and DNA sequence analysis. Statistical analyses indicated significant variations in the bacterial communities depending on tick developmental stage and degree of engorgement. We suggest that these two elements affect microbial diversity within the tick and may in turn influence pathogen transmission to humans and animals after tick bite.     

Dog tick-borne diseases in Sicily

In Sicily many tick borne diseases are endemic, in particular way those that see like main carrier ticks that prefer, for their vital cycle, climatic conditions characterized by high temperatures and a warmth-humid atmosphere. The more important pathologies transmitted by ticks causing diseases in dogs are babesiosis and ehrlichiosis. Borrelia burgdorferi, Anaplasma phagocytophilum, Rickettsia conorii, Coxiella burnetii and tick transmitted encephalitis virus assume particular relevance because they are agents of zoonosis. Our centre, C.R.A.Ba.R.T, have conducted many researches and carried out many tests for diagnostic aim in order to estimate the spread of the main tick borne diseases in Sicilians' dogs. A study lead on 342 dogs has evidenced seroprevalence for Babesia canis, Ehrlichia canis and Rickettsia respective of 5.17%, 21.70% and 53.43%. A study on zoonotic agent seroprevalences in dogs gave the following percentages: C. burnetii 31.50%, R. conorii 73.60% and A. phagocytophilum 32.80%. The data carried out from IZS Sicily diagnostic service on 5,634 tests done in 2004-2005, confirm the experimental results on the presence of B. canis, E. canis, R. conorii, A. phagocytophilum, C. burnetii, Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in all the Sicilian areas.     

Tick-borne rickettsial pathogens in ticks and small mammals in Korea

In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.     

Factors driving the abundance of ixodes ricinus ticks and the prevalence of zoonotic I. ricinus-borne pathogens in natural foci

Environmental factors may drive tick ecology and therefore tick-borne pathogen (TBP) epidemiology, which determines the risk to animals and humans of becoming infected by TBPs. For this reason, the aim of this study was to analyze the influence of environmental factors on the abundance of immature-stage Ixodes ricinus ticks and on the prevalence of two zoonotic I. ricinus-borne pathogens in natural foci of endemicity. I. ricinus abundance was measured at nine sites in the northern Iberian Peninsula by dragging the vegetation with a cotton flannelette, and ungulate abundance was measured by means of dung counts. In addition to ungulate abundance, data on variables related to spatial location, climate, and soil were gathered from the study sites. I. ricinus adults, nymphs, and larvae were collected from the vegetation, and a representative subsample of I. ricinus nymphs from each study site was analyzed by PCR for the detection of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum DNA. Mean prevalences of these pathogens were 4.0% ± 1.8% and 20.5% ± 3.7%, respectively. Statistical analyses confirmed the influence of spatial factors, climate, and ungulate abundance on I. ricinus larva abundance, while nymph abundance was related only to climate. Interestingly, cattle abundance rather than deer abundance was the main driver of B. burgdorferi sensu lato and A. phagocytophilum prevalence in I. ricinus nymphs in the study sites, where both domestic and wild ungulates coexist. The increasing abundance of cattle seems to increase the risk of other hosts becoming infected by A. phagocytophilum, while reducing the risk of being infected by B. burgdorferi sensu lato. Controlling ticks in cattle in areas where they coexist with wild ungulates would be more effective for TBP control than reducing ungulate abundance.     

Anaplasma phagocytophilum subverts tick salivary gland proteins

Anaplasma phagocytophilum is a bacterium that is transmitted by Ixodes spp. ticks, in which it resides in salivary glands. Ticks inoculate the pathogen into hosts together with an array of salivary molecules that reduce host anti-tick inflammation. Sukumaran et al. recently showed that A. phagocytophilum uses a tick salivary protein, Salp16, to enhance its uptake from the host and into the salivary gland. Occupation and exploitation of tick salivary glands have implications for the maintenance and detection of A. phagocytophilum in its vector and early pathogen interactions with its hosts.     

Coexistence of emerging bacterial pathogens in Ixodes ricinus ticks in Serbia

The list of tick-borne pathogens is long, varied and includes viruses, bacteria, protozoa and nematodes. As all of these agents can exist in ticks, their co-infections have been previously reported. We studied co-infections of emerging bacterial pathogens (Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Francisella tularensis) in Ixodes ricinus ticks in Serbia. Using PCR technique, we detected species-specific sequences, rrf-rrl rDNA intergenic spacer for B. burgdorferi s.l., p44/msp2 paralogs for A. phagocytophilum, and the 17 kDa lipoprotein gene, TUL4, for F. tularensis, respectively, in total DNA extracted from the ticks. Common infections with more than one pathogen were detected in 42 (28.8%) of 146 infected I. ricinus ticks. Co-infections with two pathogens were present in 39 (26.7%) of infected ticks. Simultaneous presence of A. phagocytophilum and different genospecies of B. burgdorferi s.l. complex was recorded in 16 ticks, co-infection with different B. burgdorferi s. l. genospecies was found in 15 ticks and eight ticks harbored mixed infections with F. tularensis and B. burgdorferi s.l. genospecies. Less common were triple pathogen species infections, detected in three ticks, one infected with A. phagocytophilum / B. burgdorferi s.s. / B. lusitaniae and two infected with F. tularensis / B. burgdorferi s.s. / B. lusitaniae. No mixed infections of A. phagocytophilum and F. tularensis were detected.     

Anaplasma phagocytophilum: deceptively simple or simply deceptive?

Anaplasma phagocytophilum is an obligate intracellular rickettsial pathogen transmitted by ixodid ticks. This bacterium colonizes myeloid and nonmyeloid cells and causes human granulocytic anaplasmosis--an important immunopathological vector-borne disease in the USA, Europe and Asia. Recent studies uncovered novel insights into the mechanisms of A. phagocytophilum pathogenesis and immunity. Here, we provide an overview of the underlying events by which the immune system responds to A. phagocytophilum infection, how this pathogen counteracts host immunity and the contribution of the tick vector for microbial transmission. We also discuss current scientific gaps in the knowledge of A. phagocytophilum biology for the purpose of exchanging research perspectives.     

Major surface protein 1a effects tick infection and transmission of Anaplasma marginale

Anaplasma marginale, an ehrlichial pathogen of cattle and wild ruminants, is transmitted biologically by ticks. A developmental cycle of A. marginale occurs in a tick that begins in gut cells followed by infection of salivary glands, which are the site of transmission to cattle. Geographic isolates of A. marginale vary in their ability to be transmitted by ticks. In these experiments we studied transmission of two recent field isolates of A. marginale, an Oklahoma isolate from Wetumka, OK, and a Florida isolate from Okeechobee, FL, by two populations of Dermacentor variabilis males obtained from the same regions. The Florida and Oklahoma tick populations transmitted the Oklahoma isolate, while both tick populations failed to transmit the Florida isolate. Gut and salivary gland infections of A. marginale, as determined by quantitative PCR and microscopy, were detected in ticks exposed to the Oklahoma isolate, while these tissues were not infected in ticks exposed to the Florida isolate. An adhesion-recovery assay was used to study adhesion of the A. marginale major surface protein (MSP) 1a to gut cells from both tick populations and cultured tick cells. We demonstrated that recombinant Escherichia coli expressing Oklahoma MSP1a adhered to cultured and native D. variabilis gut cells, while recombinant E. coli expressing the Florida MSP1a were not adherent to either tick cell population. The MSP1a of the Florida isolate of A. marginale, therefore, was unable to mediate attachment to tick gut cells, thus inhibiting salivary gland infection and transmission to cattle. This is the first report of MSP1a being responsible for effecting infection and transmission of A. marginale by Dermacentor spp. ticks. The mechanism of tick infection and transmission of A. marginale is important in formulating control strategies and development of improved vaccines for anaplasmosis.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.     

Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland

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.