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.     

Detection of tick‐borne Anaplasma bovis,Anaplasma phagocytophilum and Anaplasma centrale in Spain

The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes species of medical and veterinary importance. The presence of Anaplasma spp. in ticks from birds, as well as in Haemaphysalis punctata (Ixodida: Ixodidae) specimens collected from cattle and vegetation in northern Spain was investigated. A total of 336 ticks from birds [174 Ixodes frontalis (Ixodida: Ixodidae), 108 H. punctata, 34 Hyalomma marginatum (Ixodida: Ixodidae), 17 Ixodes ricinus (Ixodida: Ixodidae) and three Ixodes spp.], and 181 H. punctata specimens collected from cattle (n = 71) and vegetation (n = 110) were analysed. Anaplasma bovis was detected in five H. punctata, including two from birds (1.9%) and three from vegetation (2.7%). Four I. frontalis (2.3%) (one co‐infected with ‘Candidatus Midichloria mitochondrii’) and one I. ricinus (5.9%) removed from birds, as well as four H. punctata (5.6%) collected from cattle showed Anaplasma phagocytophilum infection. In addition, Anaplasma centrale was found in two H. punctata, one from a cow (1.4%) and the other from vegetation (0.9%). This study represents the first evidence of the presence of A. bovis in European ticks, and reports the first detection of A. bovis and A. centrale in H. punctata, and the first finding of A. phagocytophilum and ‘Ca. Midichloria mitochondrii’ in I. frontalis.     

Anaplasma phagocytophilum in Questing Ixodes ricinus Ticks: Comparison of Prevalences and Partial 16S rRNA Gene Variants in Urban,Pasture, and Natural Habitats

Urban, natural, and pasture areas were investigated for prevalences and 16S rRNA gene variants of Anaplasma phagocytophilum in questing Ixodes ricinus ticks. The prevalences differed significantly between habitat types, and year-to-year variations in prevalence and habitat-dependent occurrence of 16S rRNA gene variants were detected.     

Comparative Experimental Infection Study in Dogs with Ehrlichia canis,E. chaffeensis,Anaplasma platys and A. phagocytophilum

Dogs acquire infections with the Anaplasmataceae family pathogens, E. canis, E. chaffeensis, E. ewingii, A. platys and A. phagocytophilum mostly during summer months when ticks are actively feeding on animals. These pathogens are also identified as causing diseases in people. Despite the long history of tick-borne diseases in dogs, much remains to be defined pertaining to the clinical and pathological outcomes of infections with these pathogens. In the current study, we performed experimental infections in dogs with E. canis, E. chaffeensis, A. platys and A. phagocytophilum. Animals were monitored for 42 days to evaluate infection-specific clinical, hematological and pathological differences. All four pathogens caused systemic persistent infections detectible throughout the 6 weeks of infection assessment. Fever was frequently detected in animals infected with E. canis, E. chaffeensis, and A. platys, but not in dogs infected with A. phagocytophilum. Hematological differences were evident in all four infected groups, although significant overlap existed between the groups. A marked reduction in packed cell volume that correlated with reduced erythrocytes and hemoglobin was observed only in E. canis infected animals. A decline in platelet numbers was common with E. canis, A. platys and A. phagocytophilum infections. Histopathological lesions in lung, liver and spleen were observed in all four groups of infected dogs; infection with E. canis had the highest pathological scores, followed by E. chaffeensis, then A. platys and A. phagocytophilum. All four pathogens induced IgG responses starting on day 7 post infection, which was predominantly comprised of IgG2 subclass antibodies. This is the first detailed investigation comparing the infection progression and host responses in dogs after inoculation with four pathogens belonging to the Anaplasmataceae family. The study revealed a significant overlap in clinical, hematological and pathological changes resulting from the infections.     

Presence of a novel Ehrlichia sp. in Ixodes granulatus found in Okinawa, Japan

Ehrlichia -specific DNA fragments of Ehrlichia omp-1 and groEL genes were found in two I. granulatus ticks which had been collected from wild small mammals in a subtropical zone in Japan. The DNA sequences of groEL and 16SrDNA of the suspected Ehrlichia were clustered into a group of E. chaffeensis , E. muris , and Ehrlichia sp. HF565 found in I. ovatus, but were distinctly different. Therefore the Ehrlichia strain was designated as a novel Ehrlichia sp. 360. The Ehrlichia sp. 360 was detected in I. granulatus but not in any other ticks. This suggests that I. granulatus is a probable vector of Ehrlichia sp. 360 in Japan.     

Characterization of the ftsZ gene from Ehrlichia chaffeensis,Anaplasma phagocytophilum,and Rickettsia rickettsii,and use as a differential PCR target

Degenerate primers corresponding to highly conserved regions of previously characterized ftsZ genes were used to PCR amplify a portion of the ftsZ gene from the genomic DNA of Ehrlichia chaffeensis (ftsZ(Ech)), Anaplasma phagocytophilum (ftsZ(Ap)), and Rickettsia rickettsii (ftsZ(Rr)). Genome walking was then used to amplify the 5' and 3' termini of the genes. The DNA sequences of the resulting amplification products yielded open reading frames coding for proteins with molecular masses of 42.0, 45.7, and 48.3 kDa for A. phagocytophilum, E. chaffeensis, and R. rickettsii, respectively. These homologs are 20 to 70 amino acids longer than the FtsZ proteins characterized in bacteria such as Escherichia coli and Bacillus subtilis, but do not possess the large extended carboxyl-termini found in the FtsZ proteins of Bartonella, Rhizobium, and Agrobacterium species. The functional domains important for FtsZ activity are conserved within the ehrlichial and rickettsial FtsZ protein sequences. The R. rickettsii FtsZ sequence is highly homologous to the FtsZ protein previously described for Rickettsia prowazekii (89% identity), and identical to the FtsZ protein of Rickettsia conorii. The percent identity observed between the A. phagocytophilum and E. chaffeensis FtsZ proteins is only 79% and is particularly low in the carboxyl-terminal region (15.8% identity). Primers were designed to PCR amplify a portion of the variable carboxyl-terminal region of the ftsZ gene, and used to differentiate each agent based on the size of the amplicons: A. phagocytophilum, 278 bp; E. chaffeensis, 341 bp; and Rickettsia spp., 425 bp.     

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.     

Molecular survey of Anaplasma phagocytophilum and Ehrlichia canis in red foxes (Vulpes vulpes) from central Italy

During the 2007-2008 hunting season, 150 spleen samples were collected from free-ranging red foxes (Vulpes vulpes) in central Italy. The specimens were tested by two nested PCR assays to detect DNA of Anaplasma phagocytophilum, etiologic agent of granulocytic ehrlichiosis of animals and humans, and DNA of Ehrlichia canis, which causes the monocytic ehrlichiosis in canids. None of the foxes were PCR-positive for E. canis; 25 (16.6%) were positive for A. phagocytophilum. No specific gross alterations were detected at necropsy, and no histopathologic lesions found on PCR-positive spleen samples.     

Intra-leukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel

The two-component system (TCS) composed of a pair of a sensor histidine kinase and a response regulator, allows bacteria to sense signals and respond to changes in their environment through specific gene activation or repression. The present study examined TCS in the obligatory intracellular bacteria Ehrlichia chaffeensis and Anaplasma phagocytophilum, that cause human monocytic ehrlichiosis (HME) and human granulocytic anaplasmosis (HGA) respectively. The genomes of E. chaffeensis and A. phagocytophilum were each predicted to encode three pairs of TCSs. All six genes encoding three histidine kinases and three response regulators were expressed in both E. chaffeensis and A. phagocytophilum cultured in human leukocytes. Pretreatment of host cell-free E. chaffeensis or A. phagocytophilum with closantel, an inhibitor of histidine kinases, completely blocked the infection of host cells. Treatment of infected cells 1 day post infection with closantel cleared infection in dose-dependent manner. All six genes in E. chaffeensis were cloned, recombinant proteins were expressed, and polyclonal antibodies were produced. Double immunofluorescence labelling and Western blot analysis revealed that all six proteins were expressed in cell culture. Autokinase activities of the three recombinant histidine kinases from E. chaffeensis were inhibited by closantel in vitro. A number of E. chaffeensis genes, including the six TCS genes, were downregulated within 5-60 min post closantel treatment. These results suggest that these TCSs play an essential role in infection and survival of E. chaffeensis and A. phagocytophilum in human leukocytes.     

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.     

Role of Migratory Birds in Introduction and Range Expansion of Ixodes scapularis Ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada

During the spring in 2005 and 2006, 39,095 northward-migrating land birds were captured at 12 bird observatories in eastern Canada to investigate the role of migratory birds in northward range expansion of Lyme borreliosis, human granulocytic anaplasmosis, and their tick vector, Ixodes scapularis. The prevalence of birds carrying I. scapularis ticks (mostly nymphs) was 0.35% (95% confidence interval [CI] = 0.30 to 0.42), but a nested study by experienced observers suggested a more realistic infestation prevalence of 2.2% (95% CI = 1.18 to 3.73). The mean infestation intensity was 1.66 per bird. Overall, 15.4% of I. scapularis nymphs (95% CI = 10.7 to 20.9) were PCR positive for Borrelia burgdorferi, but only 8% (95% CI = 3.8 to 15.1) were positive when excluding nymphs collected at Long Point, Ontario, where B. burgdorferi is endemic. A wide range of ospC and rrs-rrl intergenic spacer alleles of B. burgdorferi were identified in infected ticks, including those associated with disseminated Lyme disease and alleles that are rare in the northeastern United States. Overall, 0.4% (95% CI = 0.03 to 0.41) of I. scapularis nymphs were PCR positive for Anaplasma phagocytophilum. We estimate that migratory birds disperse 50 million to 175 million I. scapularis ticks across Canada each spring, implicating migratory birds as possibly significant in I. scapularis range expansion in Canada. However, infrequent larvae and the low infection prevalence in ticks carried by the birds raise questions as to how B. burgdorferi and A. phagocytophilum become endemic in any tick populations established by bird-transported ticks.     

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.     

Ecological correlates of a tick-borne disease, Anaplasma phagocytophilum, in moose in southern Norway

As the distribution and abundance of ticks increase, so do the risks of tick-borne diseases. Anaplasma phagocytophilum, transmitted by Ixodes spp. ticks, is a widespread tick-borne infection causing tick-borne fever (TBF) in domestic ruminants and human granulocytic anaplasmosis. However, the role of wildlife in its epidemiology is poorly understood. Evidence of infection has been detected in wild cervids, but the pathogenicity and ecological consequences are unknown. We conducted a serological study of moose (Alces alces) in two populations in southern Norway, one where TBF was endemic (Telemark) and the other where sheep ticks (Ixodes ricinus) were essentially absent (Hedmark). Seroprevalence to A. phagocytophilum antibodies was 79 and 0 %, respectively. In Telemark, seroprevalence was significantly higher among females that calved successfully (85 %) than among others (50 %). Body mass and winter mass change were unrelated to serostatus. Relative abundance of questing ticks in Telemark was highest in deciduous forest and lowest in mature coniferous forest and higher at easterly aspects and altitudes below 350 m. Habitat factors associated with high tick abundance were risk factors for seropositivity among moose. Our findings were consistent with anaplasmosis causing a persistent subclinical infection in moose without population-level effects. Further work is needed to establish the importance of moose as a reservoir for the disease in sympatric domestic livestock.     

Dactylella chichisimensis, sp. nov. in the Bonin (Ogasawara) Islands, Japan

ADactylella isolate obtained from soil in the Bonin (Ogasawara) Islands, Japan is described and illustrated as a new species,D. chichisimensis. The fungus is characterized by single terminal multiseptate clavate or ellipsoidal conidia at the apex of simple conidiophores and mycelium that forms chlamydospores and sclerotia. A key is provided for sixDactylella species that produce primary clavate or ellipsoidal conidia at the apex of simple conidiophores.     

Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States.

We discuss the ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western U.S. These agents, while emerging in the eastern U.S., remain stable or rare in the west. In the western U.S., tick vectors and mammalian hosts for B. burgdorferi and A. phagocytophilum are distinct from those in the eastern U.S. and considerably more variable. Spatial complexity, local extinctions, and low levels of movement among foci may determine the distribution and prevalence of these agents. High-prevalence A. phagocytophilum patches may be transient, possibly as host individuals become immune. Thus, A. phagocytophilum in California could exist in a metapopulation of interacting patches. Local dynamics are sensitive to host population sizes and minimum tick infestation levels. Determining critical values for these key factors and their interactions will be important for predicting the level and distribution of future infections in the western U.S.     

The first report of Anaplasma phagocytophilum and a novel Theileria spp. co-infection in a South African giraffe

Organisms of the genera Anaplasma and Theileria are important intracellular bacteria and parasites that cause various tick-borne diseases, threatening the health of numerous animals as well as human beings. In the present study, a 12-month-old male wild South African giraffe (Giraffa camelopardalis giraffa) originating from South Africa, and living in Zhengzhou Zoo (located in the urban district of Zhengzhou in the provincial capital of Henan), suddenly developed an unknown fatal disease and died 1 day after the onset of the clinical signs. By microscopic examination of Giemsa-stained blood smears combined with nested PCR and DNA sequence analysis, Anaplasma phagocytophilum, Anaplasma bovis and a novel Theileria spp. were found in the blood of this giraffe. The six other Cervidae animals in the zoo and three ruminants living in the same colony house with them were found to be negative for both Anaplasma and Theileria in their blood specimens. We report on the first case of an A. phagocytophilum infection and the occurrence of a novel Theileria spp. in the blood of a giraffe. This is the first reported case of a multi-infection of A. bovis, A. phagocytophilum and Theileria spp. in a giraffe, as revealed by microscopic examination of blood smears and the results of nested PCR and DNA sequencing.     

Two species of Dasya (Ceramiales, Rhodophyta) from Bonin Islands, southern Japan, with the description of Dasya boninensis sp. nov.

Two species of Dasya in the Dasyaceae (Ceramiales, Rhodophyta) are reported from Bonin Islands, southern Japan. Dasya murrayana Abbott et Millar, new to Japan, is characterized by the following set of features: the tufted habit (up to 30 erect axes developing from a basal disc), small‐sized (6–10 mm high and 350–500 μm in diameter in the middle region), thinly but completely corticated axes, rigid and incurved pseudolaterals forming corymbose heads at the apices of axes and branches, the absence of adventitious monosiphonous filaments, a large number of tetrasporangial stichidia and spermatangial branches per fertile pseudolateral and slender spermatangial branches (35–45 μm in diameter). Dasya boninensis Masuda, Kurihara et Kogame, sp. nov. is characterized by short but thick (10–30 mm high and 600–1000 μm in diameter at the middle portion), heavily corticated axes, indistinct pericentral cells except for the upper portion in transverse sections, soft, straight pseudolaterals and adventitious monosiphonous filaments densely covering the axis and branches, a small number of tetrasporangial stichidia and spermatangial branches per fertile pseudolateral, thick spermatangial branches (65–90 μm in diameter), and short‐necked cystocarps. A dichotomous key to the taxa found in Japanese waters is given.     

Adaptations of the Tick-Borne Pathogen, Anaplasma marginale, for Survival in Cattle and Ticks

The tick-borne cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) multiplies within membrane-bound inclusions in host cell cytoplasm. Many geographic isolates of A. marginale occur that vary in genotype, antigenic composition, morphology and infectivity for ticks. A tick cell culture system for propagation of A. marginale proved to be a good model for study of tick-pathogen interactions. Six major surface proteins (MSPs) identified on A. marginale from bovine erythrocytes were conserved on A. marginale derived from tick cells. MSP1a and MSP1b were adhesins for bovine erythrocytes, while only MSP1a was found to be an adhesin for tick cells. The tandemly repeated portion of MSP1a was found to be necessary and sufficient for adhesion to both tick cells and bovine erythrocytes. Infectivity of A. marginale isolates for ticks was dependent on the adhesive capacity of the isolate MSP1a, which was found to involve both the adhesive properties and sequence of the repeated peptides. Cattle immunized with A. marginale derived from bovine erythrocytes or tick cells demonstrated a differential antibody response to MSP1a and MSP1b that resulted from the differential expression of these proteins in cattle and ticks cells. MSP2, derived from a multi-gene family, was found to undergo antigenic variation in cattle and ticks and may contribute to establishment of persistent A. marginale infections. MSP1a has been used as a stable genetic marker for geographic isolates because the molecular weight varies due to differing numbers of the tandem repeats. However, phylogenetic studies of A. marginale isolates from North America using MSP1a and MSP4 demonstrated that MSP4 was a good biogeographic marker, while MSP1a varied greatly among and within geographic areas. Infection and development of A. marginale in cattle and tick cells appears to differ and to be mediated by several surface proteins encoded from the small genome. This revised version was published online in July 2006 with corrections to the Cover Date.