Phagocytophilum Strains Research Articles

Detection of <i>Anaplasma phagocytophilum</i> from Wild Boars and Deer in Japan

This study was conducted to determine the prevalence and geographic distribution of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, in wild deer and boars in Japan. We analyzed the blood samples of the study animals using PCR-targeting the 16S rDNA followed by DNA sequencing. Wild deer infected with Anaplasma spp., including Anaplasma bovis and Anaplasma centrale were detected in the region from Hokkaido to Kyushu. The infection rates of A. phagocytophilum, A. bovis, and A. centrale in deer were 15.6, 21.9, and 37.5%, respectively, and the corresponding infection rates in wild boar were 3.6, 17.9, and 3.6%, respectively. However, p44/msp2 genes specific to A. phagocytophilum were not detected among the 16S rDNA-positive samples on PCR analysis. In addition, the 16S rDNA sequences of A. phagocytophilum were 100% similar to those detected previously in the deer in Japan and 98.6% similar to those of A. phagocytophilum detected in the United States and Europe, and from the tick, Ixodes ovatus and Ixodes persulcatus in Japan. These findings suggested that the A. phagocytophilum-related sequences detected from deer and wild boars in Japan were different from those of typical A. phagocytophilum strains.

Genetic Variability ofAnaplasma phagocytophiluminIxodes persulcatusTicks and Small Mammals in the Asian Part of Russia

The specimens of 3552 questing adult Ixodes persulcatus and 1698 blood/tissue samples of small mammals collected in Ural, Siberia, and Far East of Russia were assayed for the presence of Anaplasma phagocytophilum by nested PCR based on the 16S rRNA gene. Totally, A. phagocytophilum was detected in 112 tick and 88 mammalian samples. The nucleotide sequences of the 16S rRNA gene and groESL operon (1244-1295 bp) were determined for A. phagocytophilum samples from 65 ticks and 25 small mammals. Six different 16S rRNA gene variants differing by 1-5 nucleotide substitutions were detected, and only one variant matched the sequences deposited in GenBank. Analysis of groESL sequences allowed the A. phagocytophilum samples to be divided into three groups; moreover, the samples from different groups also differed in the 16S rRNA gene sequences. The A. phagocytophilum sequences from group I were detected in 11 Myodes spp. samples from West Siberia and Far East and in 19 I. persulcatus samples from all examined regions; from group II, in 10 samples of Myodes spp. and common shrews (Sorex araneus) from Ural; and from group III, in four samples of Asian chipmunks (Tamias sibiricus) from West Siberia and Far East; and in 46 I. persulcatus samples from all examined regions. The nucleotide sequences of A. phagocytophilum groESL operon from groups I and II were strictly conserved and formed with A. phagocytophilum groESL sequence from a Swiss bank vole (Myodes glareolus) (GenBank accession no. AF192796), a separate cluster on the phylogenetic tree with a strong bootstrap support. The A. phagocytophilum groESL operon sequences from group III differed from one another by 1-4 nucleotides and formed a separate branch in the cluster generated by European A. phagocytophilum strains from roe deer (Capreolus capreolus) and Ixodes ricinus ticks.

Anaplasmosis in two dogs in France and molecular and phylogenetic characterization of Anaplasma phagocytophilum

Two dogs in France were diagnosed with Anaplasma phagocytophilum infection by real-time PCR. The most remarkable hematologic and biochemical findings were severe thrombocytopenia, mild neutrophilia, morulae in neutrophils, and increased serum concentration of the α2-globulin fraction detected by agarose gel electrophoresis of serum proteins. Using sequencing of the partial 16S rRNA and ankA genes, molecular characterization of the A. phagocytophilum strains showed that the organisms from both dogs were identical to the European strains isolated from horses and people. Based on phylogenetic analysis, the ankA gene was more discriminating than the 16S rRNA gene in distinguishing the majority of European and American strains of A. phagocytophilum infecting people and animals. Three isolates of A. phagocytophilum, 1 from Spain (cow) and 2 from Norway (sheep and deer), were external to the European and American clades.

Anaplasma phagocytophilumInfection in Ticks, China–Russia Border

To the Editor: Anaplasma phagocytophilum, an emerging human pathogen of public health importance, is transmitted to humans most commonly by tick bites (1). The agent has been detected in various species of Ixodes ticks around the world (2) and in Dermacentor silvarum ticks in northeastern People’s Republic of China (3), where 3 A. phagocytophilum strains were isolated from wild and domestic animals (4). In the Asiatic region of Russia adjacent to China, A. phagocytophilum was identified in Ixodes persulcatus ticks, and A. bovis in Haemaphysalis concinna ticks (5). Human granulocytic anaplasmosis was reported in the southern area of the Russian Far East that borders China (6). The objectives of this study were to investigate the prevalence of A. phagocytophilum in ticks collected from the China–Russia border and to characterize the agent by molecular biology techniques.

Comparative Strain Analysis ofAnaplasma phagocytophilumInfection and Clinical Outcomes in a Canine Model of Granulocytic Anaplasmosis

A pilot study was conducted to determine whether existing human or canine strains of Anaplasma phagocytophilum would reproduce clinical disease in experimentally inoculated dogs similar to dogs with naturally acquired granulocytic anaplasmosis. Six hounds were inoculated intravenously with one human and two canine strains of A. phagocytophilum that were propagated in vitro in HL-60 cells or in infected autologous neutrophils. Infected dogs were monitored for lethargy, anorexia, petechiae, lymphadenopathy, and fever. Dogs were assessed for complete blood count (CBC), serum chemistry, and serology (IFA and SNAP® 4Dx®); for A. phagocytophilum blood load by quantitative polymerase chain reaction; and for cytokine production. Prominent clinical signs were generalized lymphadenopathy and scleral injection; only one dog developed fever lasting 4 days. Notable laboratory alterations included sustained leukopenia and thrombocytopenia in all dogs. A. phagocytophilum morulae were noted in blood between days 10 and 11, although all dogs retained A. phagocytophilum DNA in blood through day 60. All dogs seroconverted by days 10-15 by IFA, and by days 17-30 by SNAP 4Dx; cytokine analyses revealed 10-fold increases in interleukin-2 and interleukin-18 in the neutrophil-propagated 98E4 strain-infected dog. All A. phagocytophilum strains produced infection, although canine 98E4 strain reproduced clinical signs, hematologic changes, and inflammatory cytokine elevations most consistent with granulocytic anaplasmosis when recognized clinically. Therefore, this strain should be considered for use in future studies of A. phagocytophilum canine infection models.

EXPERIMENTAL INFECTION IN LAMBS WITH A RED DEER (CERVUS ELAPHUS) ISOLATE OF ANAPLASMA PHAGOCYTOPHILUM

Anaplasma phagocytophilum transmitted by Ixodes spp. ticks is the causative agent of tick-borne fever (TBF) in domestic ruminants. TBF is widespread along the coast of southern Norway and may cause a severe problem for the sheep industry. Red deer (Cervus elaphus) are important hosts for ticks and have been found to be infected naturally with A. phagocytophilum. However, it is unclear whether red deer could serve as reservoir hosts for A. phagocytophilum infections in sheep. We infected lambs experimentally with a red deer and a sheep isolate, respectively. The 497 base pairs of the partial 16S rRNA gene sequences of both isolates were 100% identical to GenBank accession number M73220; the 3.8 kilobases of the total ank gene sequences were 99% identical. Sixteen lambs were used, four lambs in each group. Two groups were inoculated with the red deer isolate on day 0, and then challenged on day 42 with the ovine or the red deer isolate, respectively. The third group was inoculated with the sheep isolate on day 0 and challenged with the red deer strain on day 42. Four lambs were used as uninfected controls. Blood samples for hematology, bacteriology, and serology were collected regularly for 12 wk. Presence of A. phagocytophilum in blood was determined using blood smears. Serologic response was measured by indirect immunofluorescence. Although animals inoculated with the ovine strain showed more severe clinical manifestations, lambs infected with the red deer isolate reacted with typical signs of TBF such as fever, bacteremia, and neutropenia. We conclude that A. phagocytophilum strains causing TBF in sheep might circulate in the red deer population in Norway.

Anaplasmaspp. in Wild Mammals andIxodes ricinusfrom the North of Spain

Our objectives were to investigate the presence of Anaplasma spp. infection in red deer, wild boars, and Ixodes ricinus removed from deer surveyed in La Rioja, as well as to analyze the presence of Anaplasma spp. in I. ricinus from different Spanish regions--ours included. A total of 21 deer and 13 wild boar blood samples as well as 295 I. ricinus removed from deer, vegetation, or asymptomatic people were tested by polymerase chain reaction targeting Anaplasma spp. 16S rRNA gene and groESL heat shock operon. Twelve deer blood samples were found to be infected with Anaplasma centrale (n = 7) or Anaplasma phagocytophilum (n = 5). No wild boar blood samples gave positive polymerase chain reaction results. Further, A. phagocytophilum was detected in 12 out of 89 I. ricinus removed from deer and in 18 out of 168 I. ricinus collected over vegetation in the North of Spain. Anaplasma spp. was not detected in any of the 38 I. ricinus removed from people. Nucleotide sequences for 16S rRNA gene showed substancial heterogeneity. The etiological agent of human anaplasmosis was found in two deer blood samples, an adult tick from deer, and a nymph from vegetation. The 16S rRNA sequences for 12 out of 35 samples matched the sequence of the Ap-variant 1 strain previously described in the United States, and the remaining 19 positive samples (deer blood and I. ricinus) showed variations with unknown significance. Although the groEL DNA sequences varied among analyzed strains, the deduced amino acid sequences did not change for any of them. This study suggests that deer population from La Rioja harbors strains of A. phagocytophilum similar to that pathogen for humans and other of unknown pathogenicity. Further, it seems that the Ap-variant 1 is circulating among I. ricinus ticks from the North of Spain more frequently than the A. phagocytophilum strain associated to human anaplasmosis.

Potential infectivity of Anaplasma phagocytophilum strains in Ixodes ricinus ticks from Serbia

Anaplasma phagocytophilum strains from different geographical regions are characterised by diverse potential infectivity for humans and domesticated animals. We investigated the potential pathogenicity of A. phagocytophilum strains in Ixodes ricinus ticks from 11 geographically different localities in Serbia. Sequences obtained in this study showed a high variability of p44 paralogues. Some of them, however, formed groups with similarities greater than 86% ('similarity groups'). Previous studies showed that 'similarity groups' were nearly always country specific. Our results correlated with this observation, and we also observed significant clustering of paralogues according to vector and reservoir origin of A. phagocytophilum strains. According to the high genetic similarity of sequences isolated from ticks collected in four localities, namely Avala, Batrovci, Hajducka cesma and Ljubovija, with paralogues with proven pathogenicity isolated from human granulocytic anaplasmosis (HGA) patients and A. phagocytophilum infected sheep, we could assume that strains with potential infectivity for humans and domestic animals were present in Serbia.

Equine granulocytic anaplasmosis in the Czech Republic

Twelve confirmed cases of equine granulocytic anaplasmosis (EGA) and five additional suspected cases, showing a compatible clinical history and specific IgG titres of 1280 or above, were recorded in the...

Characterization of Anaplasma phagocytophilum and A. ovis infection in a naturally infected sheep flock with poor health condition

Anaplasma species are transmitted by ticks and cause diseases in humans and animals. These pathogens infect sheep, an economically important domestic animal worldwide. The current study was designed to characterize in 200 animals the infection with Anaplasma phagocytophilum and Anaplasma ovis and the genetic diversity of A. ovis strains collected from a naturally infected sheep flock with poor health condition. Sheep had 98% seroprevalence to Anaplasma spp. antibodies. PCR results confirmed the presence of A. phagocytophilum and A. ovis DNA in 11.5% and 37% of the sheep, respectively. Concurrent infections were detected in 6.5% of the sheep. Seventy-one adult ticks were collected from 45 sheep with infestations ranging from one to 15 ticks per animal. The analysis of A. ovis msp4 sequences demonstrated a previously unreported polymorphism for this pathogen with 17 different haplotypes in infected sheep. These results demonstrated that, although A. ovis msp4 haplotypes may be less variable when compared with Anaplasma marginale and A. phagocytophilum strains on a global scale, genetic polymorphisms occur in this locus in strains obtained from an infected sheep flock with poor health condition.

Diversity ofAnaplasma phagocytophilumStrains, USA

We analyzed the structure of the expression site encoding the immunoprotective protein MSP2/P44 from multiple Anaplasma phagocytophilum strains in the United States. The sequence of p44ESup1 had diverged in Ap-variant 1 strains infecting ruminants. In contrast, no differences were detected between A. phagocytophilum strains infecting humans and domestic dogs.

Distinct Ecologically Relevant Strains ofAnaplasma phagocytophilum

Distinct Ecologically Relevant Strains of<i>Anaplasma phagocytophilum</i>

Possible Differential Host Tropism in Anaplasma phagocytophilum Strains in the Western United States

Despite organization into a single species, Anaplasma phagocytophilum, of bacteria from diverse hosts, there is evidence that different hosts vary in susceptibility to local strains of this bacterial pathogen. In particular, there is evidence that clinical hosts (humans, horses, and dogs) in the western part of the United States may be infected with strains of bacteria that differ from those in the reported wildlife reservoir, the dusky-footed woodrat. There is a lack of spatial overlap between clinical cases with woodrat exposure, genetic studies show highly distinctive strains in woodrats compared with those from clinical hosts, and experimental studies failed to transfer infection from woodrats into horses. These data suggest that multiple distinct A. phagocytophilum strains could circulate in western U.S. ecosystems. Host barriers to different bacterial strains would suggest the need for careful discrimination of A. phagocytophilum strains and further research to understand the host-pathogen interactions that result in differential outcomes in infection.

Characterization of Anaplasma Infections in Sicily, Italy

This study aimed to characterize infection with Anaplasma marginale, A. phagocytophilum, A. ovis, and A. platys in humans, animals, and ticks in Sicily, Italy, during 2003-2006. Serologic (competitive ELISA [cELISA]) and indirect immunofluorescence antibody [IFA]; N= 1990) and DNA (polymerase chain reaction [PCR]; N= 2788) tests were conducted on horse, donkey, cattle, sheep, goat, pig, dog, cat, roe deer, wild boar, human, and tick samples. The results reported herein suggested that in Sicily cattle are a major reservoir for A. marginale, dogs for A. platys, and sheep and goats for A. ovis. Domestic animals, such as cattle, horses, donkeys, sheep, dogs, and cats, may serve as reservoir for A. phagocytophilum, but different strains may infect ruminants and humans. All Anaplasma spp. characterized in Sicily had some distinctive genotypes for this region. Low genetic diversity was observed in A. ovis and A. platys, whereas A. marginale and A. phagocytophilum strains showed high genetic diversity. These results expanded our knowledge about the prevalence of Anaplasma spp. in Sicily and provided information to understand the epidemiology of these infections and implement measures to diagnose, treat, and control transmission to humans and animals in this region.

Prevalence of Anaplasma phagocytophilum and its coinfection with Borrelia afzelii in Ixodes ricinus and Ixodes persulcatus ticks inhabiting Tver Province (Russia) – a sympatric region for both tick species

Human granulocytic anaplasmosis (HGA) and Lyme borreliosis (LB) are tick-borne infectious diseases caused by Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato species, respectively. In this study, p44/msp2 paralogues specific to A. phagocytophilum and 5S-23S rRNA gene-intergenic spacers specific to B. burgdorferi sensu lato species were detected by PCR in ticks collected in two regions, Tver (Kalinin) and Konakovo, of the Tver (Kalinin) Province located 150 km north-west of Moscow. The PCR amplicons obtained were further characterized by sequencing and RFLP analysis. In the total of 199 ticks collected, 8.8 % (7/80) and 33.8 % (27/80) of Ixodes ricinus, and 2.5 % (3/119) and 45.4 % (54/119) of Ixodes persulcatus, were found to be infected with A. phagocytophilum and B. burgdorferi sensu lato spp., respectively. Of those 199 ticks, 5 (2.5 %) were coinfected with A. phagocytophilum and Borrelia afzelii. Phylogenetic analysis revealed unique p44/msp2 paralogous genes in A. phagocytophilum-infected Russian ticks. The sequence similarities with those of A. phagocytophilum in the United States, UK and Japan ranged from 42 % to 80.4 %, and there were no sequences showing more than 90 % similarity with those sequences from the other countries. The results showed that the p44/msp2 sequence similarity groups may provide an index of adaptation of A. phagocytophilum strains to specific vector ticks or reservoir hosts in different countries and areas. These findings suggest that there is a public health threat from HGA and LB in Tver Province surrounding Moscow.

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.

Sialyl-Lewis x-independent infection of human myeloid cells by Anaplasma phagocytophilum strains HZ and HGE1.

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that infects neutrophils and neutrophil precursors. Bacterial recognition of P-selectin glycoprotein ligand-1 (PSGL-1) and the alpha2,3-sialylated- and alpha1,3-fucosylated-moiety sialyl-Lewis x (sLe(x)), which modifies the PSGL-1 N terminus, is important for adhesion to and invasion of myeloid cells. We have previously demonstrated that A. phagocytophilum organisms of the NCH-1 strain that utilize an sLe(x)-modified PSGL-1-independent means of entry can be enriched for by cultivation in undersialylated HL-60 cells that are unable to construct sLe(x). Because it was unknown whether other A. phagocytophilum isolates share this ability, we extended our studies to the geographically diverse strains HZ and HGE1. HL-60 A2 is a clonal cell line that is defective for sialylation and alpha1,3-fucosyltransferase. HL-60 A2 cell surfaces, therefore, not only lack sLe(x) but also are virtually devoid of any other sialic acid- and/or alpha1,3-fucose-modified glycan. By cultivating HZ and HGE1 in HL-60 A2 cells, we enriched for bacterial subpopulations (termed HZA2 and HGE1A2) that bind and/or infect myeloid cells in the absence of sialic acid and alpha1,3-fucose and in the presence of antibody that blocks the N terminus of PSGL-1. Thus, multiple A. phagocytophilum isolates share the ability to use sLe(x)-modified PSGL-1-dependent and -independent routes of entry into myeloid cells. HZA2 and HGE1A2 represent enriched bacterial populations that will aid dissection of the complexities of the interactions between A. phagocytophilum and host myeloid cells.

Sequence analysis of the msp4 gene of Anaplasma ovis strains

Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep ( Ovis canadensis) and mule deer ( Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovis msp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovis msp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.

Reservoir Competency of Goats for Anaplasma phagocytophilum

The susceptibility of goats to infection by Anaplasma phagocytophilum (A. phagocytophilum) strains Ap-Variant 1 and Ap-ha was assessed by infestation of goats with field-collected Ixodes scapularis (I. scapularis) ticks. Both strains were infectious in the goat model. These results demonstrate that goats can be used in a laboratory setting to propagate A. phagocytophilum. Transmission from an infected goat to a naïve goat by I. scapularis tick feeding was used to demonstrate that goats are reservoir-competent for the Ap-Variant 1 strain.

New Findings on Members of the Family Anaplasmataceae of Veterinary Importance

Members of the family Anaplasmataceae are obligate intracellular Gram-negative bacteria that naturally infect a variety of wild and domestic animal species, the spillover of which may lead to zoonosis. I discuss new findings on members of the family Anaplasmataceae of veterinary importance and therefore, I will describe the recent findings on Neorickettsia risticii in the trematode and related Neorickettsia species. I also will review the recent progress on Aegyptianella pullorum and other Aegyptianella sp., "Candidatus Neoehrlichia mikurensis" and Anaplasma phagocytophilum strains in various hosts. The whole genome sequences of two important veterinary pathogens-Anaplasma marginale, the bovine anaplasmosis agent, and Ehrlichia (formerly Cowdria) ruminantium, the agent of heartwater of ruminants-have been published. Taken together, these advances in research of the family Anaplasmataceae in the veterinary field provide us with insights into the evolution, reservoir, and transmission of these organisms in nature and their pathogenesis in natural and accidental hosts. It is through this work that surveillance, diagnosis, preventive measures, and treatment of ehrlichioses of both animals and humans can be improved.