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1.  PCR Detection of Granulocytic Ehrlichiae in Ixodes ricinus Ticks and Wild Small Mammals in Western Switzerland 
Journal of Clinical Microbiology  2000;38(3):1002-1007.
The presence of granulocytic ehrlichiae was demonstrated by PCR in Ixodes ricinus ticks and wild small mammals in Switzerland in two areas of endemicity for bovine ehrlichiosis. Six ticks (three females and three nymphs) (1.4%) of 417 I. ricinus ticks collected by flagging vegetation contained ehrlichial DNA. A total of 201 small mammals from five species, wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), earth vole (Pitymys subterraneus), bank vole (Clethrionomys glareolus), and common shrew (Sorex araneus), were trapped. The analysis of I. ricinus mammals collected on 116 small mammals showed that nine C. glareolus voles and two A. sylvaticus mice hosted infected tick larvae. In these rodents, granulocytic ehrlichia infection was also detected in blood, spleen, liver, and ear samples. Further examinations of 190 small mammals without ticks or with noninfected ticks showed the presence of ehrlichial DNA in spleen and other tissues from six additional C. glareolus, three A. flavicollis, and one S. araneus mammals. This study suggests that A. sylvaticus, A. flavicollis, S. araneus, and particularly C. glareolus are likely to be natural reservoirs for granulocytic ehrlichiae. Partial 16S rRNA gene sequences of granulocytic ehrlichiae from ticks and rodents showed a high degree of homology (99 to 100%) with granulocytic ehrlichiae isolated from humans. In contrast, groESL heat shock operon sequence analysis showed a strong divergence (approximately 5%) between the sequences in samples derived from rodents and those derived from samples from questing ticks or from other published ehrlichia sequences. Dual infections with granulocytic ehrlichia and Borrelia burgdorferi were found in ticks and small mammals.
PMCID: PMC86323  PMID: 10698987
2.  Citrate Synthase Gene Sequence: a New Tool for Phylogenetic Analysis and Identification of Ehrlichia 
Journal of Clinical Microbiology  2001;39(9):3031-3039.
The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.
PMCID: PMC88292  PMID: 11526124
3.  Anaplasma phagocytophilum in ticks in Slovenia 
Parasites & Vectors  2010;3:102.
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.
PMCID: PMC2988007  PMID: 21050436
4.  Human granulocytic ehrlichiosis in southern Germany: increased seroprevalence in high-risk groups. 
Journal of Clinical Microbiology  1997;35(12):3244-3247.
To date, human granulocytic ehrlichiosis (HGE), the causative agent of which is likely transmitted by ticks in the Ixodes ricinus-Ixodes persulcatus complex, has not been diagnosed with certainty in patients outside the United States. The presence of a closely related vector tick, I. ricinus, as well as the occurrence of similar Ehrlichia spp. of veterinary importance, suggests that this disease is likely to be present in Europe. The aim of the present study was to compare the prevalence of antibodies against the HGE agent in sera collected from patients in groups at high risk for exposure to I. ricinus with that of a control population. Risk groups consisted of 150 forestry workers and 105 patients with an established diagnosis of Lyme disease. The control group was 103 healthy blood donors without a history of tick bites. We used a patient isolate of the HGE agent from Minnesota (J. L. Goodman, C. Nelson, B. Vitale, J. E. Madigan, J. S. Dumler, T. J. Kurtti, and U. G. Munderloh, N. Engl. J. Med. 334:209-215, 1996) propagated in HL60 cells as the source of antigen for a specific immunofluorescence assay (IFA). Elevated IFA titers (> or = 1:80) were present in 21 of 150 (14%) serum samples from forestry workers and in 12 of 105 (11.4%) serum samples from Lyme disease patients, but in only 2 of 103 (1.9%) serum samples from blood donors (P < or = 0.01 for either of the at-risk groups versus blood donors). The results of this study suggest that the HGE agent or a closely related organism exists in southern Germany and that seroconversion to it is common among groups exposed to Ixodes ticks. Final proof that HGE occurs in Germany will require the isolation of the causative agent from patients. HGE should be considered in the differential diagnosis of febrile illnesses in individuals exposed to Ixodes ticks in Europe as well as in North America.
PMCID: PMC230155  PMID: 9399527
5.  High Diversity of ankA Sequences of Anaplasma phagocytophilum among Ixodes ricinus Ticks in Germany 
Journal of Clinical Microbiology  2003;41(11):5033-5040.
In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were ≥99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.
PMCID: PMC262509  PMID: 14605135
6.  Detection and Identification of Ehrlichia spp. in Ticks Collected in Tunisia and Morocco 
Journal of Clinical Microbiology  2005;43(3):1127-1132.
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.
PMCID: PMC1081246  PMID: 15750072
7.  Prevalence of Borrelia burgdorferi and Granulocytic and Monocytic Ehrlichiae in Ixodes ricinus Ticks from Southern Germany 
Journal of Clinical Microbiology  1999;37(11):3448-3451.
A total of 287 adult Ixodes ricinus ticks, collected in two regions of southern Germany (Frankonia and Baden-Württemberg) where Borrelia burgdorferi infections are known to be endemic, were examined for the presence of 16S ribosomal DNA specific for the Ehrlichia phagocytophila genogroup, E. chaffeensis, E. canis, and B. burgdorferi by nested PCR. Totals of 2.2% (6 of 275) and 21.8% (65 of 275) of the ticks were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Two ticks (0.7%) were coinfected with both bacteria. Of 12 engorged I. ricinus ticks collected from two deer, 8 (67%) were positive for the E. phagocytophila genogroup and one (8%) was positive for B. burgdorferi. There was no evidence of infection with E. canis or E. chaffeensis in the investigated tick population. The nucleotide sequences of the 546-bp Ehrlichia PCR products differed at one or two positions from the original sequence of the human granulocytic ehrlichiosis (HGE) agent (S.-M. Chen, J. S. Dumler, J. S. Bakken, and D. H. Walker, J. Clin. Microbiol. 32:589–595, 1994). Three groups of sequence variants were detected; two of these were known to occur in other areas in Europe or the United States, whereas one has not been reported before. Thus, in the German I. ricinus tick population closely related granulocytic ehrlichiae are prevalent, which might represent variants of E. phagocytophila or the HGE agent.
PMCID: PMC85664  PMID: 10523532
8.  Characterization of an immunoreactive protein from the agent of human granulocytic ehrlichiosis. 
Journal of Clinical Microbiology  1997;35(5):1172-1178.
A gene that is homologous to the Ehrlichia chaffeensis groEL operon was recovered and characterized by broad-range PCR amplification of whole blood from patients with human granulocytic ehrlichiosis (HGE) and from infected HL60 cell cultures. Sequence analysis of an 820-bp DNA fragment recovered directly from human blood showed 76.5 and 76.3% identity with cognate sequences from E. chaffeensis and Cowdria ruminantium, respectively. Analysis of a 1.6-kb DNA fragment derived from an HGE agent-infected HL60 cell culture indicated a near-complete open reading frame that contained 75.6 and 75.2% sequence identity with the E. chaffeensis and C. ruminantium groEL sequences, respectively. Phylogenetic analysis of this fragment showed that the HGE agent-derived sequence was related to, but distinct from, the sequences of E. chaffeensis and C. ruminantium. Polyvalent antibody responses to a recombinant fusion protein based on the HGE agent groEL homolog were detected in three of three BALB/c mice that were infected by syringe inoculation with a Wisconsin strain of the HGE agent (WI-1) and nine of nine mice infected by Ixodes scapularis (Ixodes dammini) tick inoculation of an isolate from Nantucket Island, Mass. (NCH-1). No response was detected in mice infected with Borrelia burgdorferi or in control BALB/c mice. Further characterization of the sensitivity and specificity of immune responses to this protein will be facilitated by the use of recombinant fusion proteins or peptides based on the HGE agent-specific groEL homolog.
PMCID: PMC232724  PMID: 9114402
9.  Comparison of the Nucleotide Sequences of 16S rRNA, 444 Ep-ank, and groESL Heat Shock Operon Genes in Naturally Occurring Ehrlichia equi and Human Granulocytic Ehrlichiosis Agent Isolates from Northern California 
Journal of Clinical Microbiology  2000;38(4):1364-1369.
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.
PMCID: PMC86446  PMID: 10747108
10.  Dusky-Footed Wood Rats (Neotoma fuscipes) as Reservoirs of Granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in Northern California 
Journal of Clinical Microbiology  1999;37(10):3323-3327.
Dusky-footed wood rats (Neotoma fuscipes) and Peromyscus sp. mice (P. maniculatus and P. truei) were collected from one site in Placer County, one site in Santa Cruz County, and two sites in Sonoma County in northern California. Serum or plasma samples from 260 rodents were tested for antibodies to the agent of human granulocytic ehrlichiosis. Of these, samples from 25 wood rats (34% of those tested) and 10 (8%) Peromyscus sp. mice were found to be seropositive, but only those from one site. PCR assays targeting the groESL heat shock operon were conducted on all seropositive specimens and a subset of seronegative blood specimens. Ehrlichial DNA was identified in 17 (68%) of the 25 seropositive wood rat blood samples and in 1 of the 10 (10%) Peromyscus sp. specimens. None of 40 seronegative blood samples was PCR positive. Both seropositive and PCR-positive animals were collected during each trapping period. One male tick out of 84 Ixodes pacificus adults collected was PCR positive; samples of Dermacentor occidentalis nymphs and adults were negative. Nucleotide sequences of amplicons from three wood rat blood specimens and from the single PCR-positive tick differed by one and two bases, respectively, from a sequence previously obtained from Ehrlichia equi. At one site in Sonoma County, wood rats had a concurrent high prevalence of seropositivity and PCR positivity, while other sigmodontine rodents collected at the site were only occasionally infected. We suggest that dusky-footed wood rats serve as reservoirs of granulocytic ehrlichial agents in certain areas of northern California. The tick species involved in the transmission of granulocytic ehrlichiae among wood rats remains unknown.
PMCID: PMC85556  PMID: 10488199
11.  PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species. 
Journal of Clinical Microbiology  1997;35(8):2087-2092.
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.
PMCID: PMC229908  PMID: 9230387
12.  Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin. 
Journal of Clinical Microbiology  1997;35(6):1465-1468.
Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.
PMCID: PMC229768  PMID: 9163463
13.  Invasion and Intracellular Development of the Human Granulocytic Ehrlichiosis Agent in Tick Cell Culture 
Journal of Clinical Microbiology  1999;37(8):2518-2524.
Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick’s next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.
PMCID: PMC85271  PMID: 10405394
14.  Sequence Analysis of the ank Gene of Granulocytic Ehrlichiae 
Journal of Clinical Microbiology  2000;38(8):2917-2922.
The ank gene of the agent of human granulocytic ehrlichiosis (HGE) codes for a protein with a predicted molecular size of 131.2 kDa that is recognized by serum from both dogs and humans infected with granulocytic ehrlichiae. As part of an effort to assess the phylogenetic relatedness of granulocytic ehrlichiae from different geographic regions and in different host species, the ank gene was PCR amplified and sequenced from a variety of sources. These included 10 blood specimens from patients with confirmed human granulocytic ehrlichiosis (three from New York, four from Wisconsin, two from Slovenia, and one from Sweden). Also examined was a canine granulocytic ehrlichia sample obtained from Minnesota, Ehrlichia equi from California, Ehrlichia phagocytophila from Sweden, and the granulocytic ehrlichia isolate USG3. The sequences showed a high level of homology (>95.5% identity), with the lowest homology occurring between a New York HGE agent and the Swedish E. phagocytophila. Several 3-bp deletions and a variable number of 51- and 81-bp direct repeats were noted. Although the North American HGE sequences showed the highest conservation (>98.1% identity), phylogenetic analyses indicated that these samples represent two separate clades, one including the three New York HGE samples and the USG3 strain and another with the Wisconsin HGE and Minnesota canine sequences. Two of the New York samples and the USG3 strain showed 100% identity over the entire 3,696-bp product. Likewise, three of the Wisconsin human samples and the Minnesota dog sample were identical (3,693 bp). Whereas phylogenetic analysis showed that the E. equi sequence was most closely related to the Upper Midwest samples, analysis of the repeat structures showed it to be more similar to the European samples. Overall, the genetic analysis based on the ank gene showed that the granulocytic ehrlichiae are closely related, appear to infect multiple species, and can be grouped into at least three different clades, two North American and one European.
PMCID: PMC87147  PMID: 10921951
15.  Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture. 
Journal of Clinical Microbiology  1996;34(3):664-670.
The equine granulocytic ehrlichiosis agent, Ehrlichia equi, is closely related or identical to the human granulocytic ehrlichiosis (HGE) agent. Both are suspected of being transmitted by ticks. We have successfully isolated E. equi in a cell line, IDE8, derived from a putative vector, the tick Ixodes scapularis. Peripheral blood leukocytes from an experimentally infected horse were inoculated onto IDE8 monolayers. Cultures were incubated in a candle jar at 34 degrees C in tick cell culture medium with NaHCO3 and an organic buffer [3-(N-morpholino)-propanesulfonic acid] (MOPS). Within 2 weeks, infected cells were detected in Giemsa-stained culture samples, and the organisms subsequently spread to uninfected cells in the cultures. E. equi was passaged serially by transferring a portion of an infected culture to new cell layers every 2 to 3 weeks. The identity of the organisms was confirmed by PCR using oligonucleotide primers specific for E. equi and the HGE agent and by immunocytology. Homologous equine antibodies and human anti-HGE convalescent serum recognized E. equi grown in tick cell culture. Electron microscopy revealed electron-lucent and -dense ehrlichia-like forms developing within host cell endosomes. E. equi passaged twice in tick cell culture retained infectivity and pathogenicity for the equine host, as demonstrated by intravenous inoculation of a suspension of infected tick cells and subsequent reisolation from peripheral blood, in fulfillment of Koch's postulates. The horse developed severe clinical signs, i.e., fever, inappetence, thrombocytopenia, icterus, and limb edema, typical of granulocytic equine ehrlichiosis, within 1 week.
PMCID: PMC228866  PMID: 8904434
16.  Prevalence of Neoehrlichia mikurensis in ticks and rodents from North-west Europe 
Parasites & Vectors  2012;5:74.
Neoehrlichia mikurensis s an emerging and vector-borne zoonosis: The first human disease cases were reported in 2010. Limited information is available about the prevalence and distribution of Neoehrlichia mikurensis in Europe, its natural life cycle and reservoir hosts. An Ehrlichia-like schotti variant has been described in questing Ixodes ricinus ticks, which could be identical to Neoehrlichia mikurensis.
Three genetic markers, 16S rDNA, gltA and GroEL, of Ehrlichia schotti-positive tick lysates were amplified, sequenced and compared to sequences from Neoehrlichia mikurensis. Based on these DNA sequences, a multiplex real-time PCR was developed to specifically detect Neoehrlichia mikurensis in combination with Anaplasma phagocytophilum in tick lysates. Various tick species from different life-stages, particularly Ixodes ricinus nymphs, were collected from the vegetation or wildlife. Tick lysates and DNA derived from organs of wild rodents were tested by PCR-based methods for the presence of Neoehrlichia mikurensis. Prevalence of Neoehrlichia mikurensis was calculated together with confidence intervals using Fisher's exact test.
The three genetic markers of Ehrlichia schotti-positive field isolates were similar or identical to Neoehrlichia mikurensis. Neoehrlichia mikurensis was found to be ubiquitously spread in the Netherlands and Belgium, but was not detected in the 401 tick samples from the UK. Neoehrlichia mikurensis was found in nymphs and adult Ixodes ricinus ticks, but neither in their larvae, nor in any other tick species tested. Neoehrlichia mikurensis was detected in diverse organs of some rodent species. Engorging ticks from red deer, European mouflon, wild boar and sheep were found positive for Neoehrlichia mikurensis.
Ehrlichia schotti is similar, if not identical, to Neoehrlichia mikurensis. Neoehrlichia mikurensis is present in questing Ixodes ricinus ticks throughout the Netherlands and Belgium. We propose that Ixodes ricinus can transstadially, but not transovarially, transmit this microorganism, and that different rodent species may act as reservoir hosts. These data further imply that wildlife and humans are frequently exposed to Neoehrlichia mikurensis-infected ticks through tick bites. Future studies should aim to investigate to what extent Neoehrlichia mikurensis poses a risk to public health.
PMCID: PMC3395572  PMID: 22515314
Vector-borne disease; Emerging zoonoses; Candidatus N. mikurensis; I. ricinus; Anaplasma phagocytophylum
17.  Molecular Cloning and Sequencing of Three Granulocytic Ehrlichia Genes Encoding High-Molecular-Weight Immunoreactive Proteins 
Infection and Immunity  1998;66(4):1356-1363.
Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.
PMCID: PMC108060  PMID: 9529053
18.  Serologic Testing for Human Granulocytic Ehrlichiosis at a National Referral Center 
Journal of Clinical Microbiology  1999;37(3):558-564.
An indirect immunofluorescence assay (IFA) was used to identify patients with antibodies reactive to the human granulocytic ehrlichiosis (HGE) agent. Serum samples collected from clinically ill individuals were submitted to the Centers for Disease Control and Prevention by physicians via state health departments from throughout the United States and tested against a panel of ehrlichial and rickettsial pathogens. Antibodies reactive to the HGE agent were detected in 142 (8.9%) of 1,602 individuals tested. There were 19 confirmed and 59 probable (n = 78) cases of HGE as defined by seroconversion or a fourfold or higher titer to the HGE agent than to the Ehrlichia chaffeensis antigens. The average age of patients with HGE was 57 years, and males accounted for 53 (68%) of the patients. Cases of HGE occurred in 21 states; 47 (60%) of the cases occurred in Connecticut (n = 14), New York (n = 18), and Wisconsin (n = 15). Onset of HGE was identified from April through December, with cases peaking in June and July. The earliest confirmed cases of HGE occurred in 1987 in Wisconsin and 1988 in Florida. No fatalities were reported among the 78 patients with confirmed or probable HGE. Reactivity to the HGE agent and to either Coxiella burnetii, Rickettsia rickettsii, or Rickettsia typhi was infrequent; however, 74 (52%) of the 142 individuals who were positive for HGE had at least one serum sample that also reacted to the E. chaffeensis antigen. Thirty-four persons with confirmed or probable human monocytic ehrlichiosis due to E. chaffeensis also had antibodies to the HGE agent in at least one serum sample. The specific etiologic agent for 30 patients was not ascribed because of similarity of titers to both ehrlichial antigens. The use of both antigens may be required to correctly diagnose most cases of human ehrlichiosis, especially in geographic regions where both the HGE agent and E. chaffeensis occur.
PMCID: PMC84468  PMID: 9986812
19.  Intracellular Infection by the Human Granulocytic Ehrlichiosis Agent Inhibits Human Neutrophil Apoptosis 
Infection and Immunity  2000;68(3):1125-1133.
In patients with human granulocytic ehrlichiosis (HGE), the HGE agent has been seen only in the peripheral blood granulocytes, which have a life span too short for ehrlichial proliferation. To determine if the HGE agent delays the apoptosis of human peripheral blood neutrophils for its advantage, peripheral blood granulocytes consisting mostly of neutrophils were incubated with freshly freed host cell-free HGE agent in vitro. The HGE agent induced a significant delay in morphological apoptosis and the cytoplasmic appearance of histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the HGE agent freshly freed from the host cells, noninfectious purified HGE agent stored frozen and thawed also had antiapoptotic effect, which was lost with proteinase K treatment but not with periodate treatment. Treatment of neutrophils with a transglutaminase inhibitor, monodansylcadaverine, blocked the antiapoptotic effect of the HGE agent. Addition of oxytetracycline, however, did not prevent or reverse the antiapoptotic effect of the HGE agent. These results suggest that binding of a protein component(s) of the HGE agent to neutrophils and subsequent cross-linking and/or internalization of the receptor and ehrlichiae are required for antiapoptotic signaling, but ehrlichial protein synthesis and/or proliferation is not required. MG-132, a proteasome inhibitor, and cycloheximide accelerated the apoptosis of neutrophils and overrode the antiapoptotic effect of the HGE agent. Studies with specific inhibitors suggest that protein kinase A, NF-κB, and interleukin 1β are not involved in the antiapoptotic mechanism of the HGE agent.
PMCID: PMC97257  PMID: 10678916
20.  Transovarial Transmission of Francisella-Like Endosymbionts and Anaplasma phagocytophilum Variants in Dermacentor albipictus (Acari: Ixodidae) 
Journal of medical entomology  2009;46(3):625-632.
Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D. albipictus ticks collected from Minnesota white-tailed deer (Odocoileus virginianus) for bacteria of the genera Anaplasma, Ehrlichia, Francisella, and Rickettsia using polymerase chain reaction (PCR) gene amplification and sequence analyses. We detected Anaplasma phagocytophilum and Francisella-like endosymbionts (FLEs) in nymphal and adult ticks of both sexes at 45 and 94% prevalences, respectively. The A. phagocytophilum and FLEs were transovarially transmitted to F1 larvae by individual ticks at efficiencies of 10–40 and 95–100%, respectively. The FLEs were transovarially transmitted to F2 larvae obtained as progeny of adults from F1 larval ticks reared to maturity on a calf, but A. phagocytophilum were not. Based on PCR and tissue culture inoculation assays, A. phagocytophilum and FLEs were not transmitted to the calf. The amplified FLE 16S rRNA gene sequences were identical to that of an FLE detected in a D. albipictus from Texas, whereas those of the A. phagocytophilum were nearly identical to those of probable human-nonpathogenic A. phagocytophilum WI-1 and WI-2 variants detected in white-tailed deer from central Wisconsin. However, the D. albipictus A. phagocytophilum sequences differed from that of the nonpathogenic A. phagocytophilum variant-1 associated with Ixodes scapularis ticks and white-tailed deer as well as that of the human-pathogenic A. phagocytophilum ha variant associated with I. scapularis and the white-footed mouse, Peromyscus leucopus. The transovarial transmission of A. phagocytophilum variants in Dermacentor ticks suggests that maintenance of A. phagocytophilum in nature may not be solely dependent on horizontal transmission.
PMCID: PMC2751609  PMID: 19496436
Ixodid tick; Anaplasma; Francisella-like; transovarial transmission
21.  Prevention of infectious tick-borne diseases in humans: Comparative studies of the repellency of different dodecanoic acid-formulations against Ixodes ricinus ticks (Acari: Ixodidae) 
Ticks of the species Ixodes ricinus are the main vectors of Lyme Borreliosis and Tick-borne Encephalitis – two rapidly emerging diseases in Europe. Repellents provide a practical means of protection against tick bites and can therefore minimize the transmission of tick-borne diseases. We developed and tested seven different dodecanoic acid (DDA)-formulations for their efficacy in repelling host-seeking nymphs of I. ricinus by laboratory screening. The ultimately selected formulation was then used for comparative investigations of commercially available tick repellents in humans.
Laboratory screening tests were performed using the Moving-object (MO) bioassay. All test formulations contained 10% of the naturally occurring active substance DDA and differed only in terms of the quantitative and qualitative composition of inactive ingredients and fragrances. The test procedure used in the human bioassays is a modification of an assay described by the U.S. Environmental Protection Agency and recommended for regulatory affairs. Repellency was computed using the equation: R = 100 - NR/N × 100, where NR is the number of non-repelled ticks, and N is the respective number of control ticks. All investigations were conducted in a controlled laboratory environment offering standardized test conditions.
All test formulations strongly repelled nymphs of I. ricinus (100-81% protection) as shown by the MO-bioassay. The majority of ticks dropped off the treated surface of the heated rotating drum that served as the attractant (1 mg/cm2 repellent applied). The 10% DDA-based formulation, that produced the best results in laboratory screening, was as effective as the coconut oil-based reference product. The mean protection time of both preparations was generally similar and averaged 8 hours.
Repellency investigations in humans showed that the most effective 10% DDA-based formulation (~1.67 mg/cm2 applied) strongly avoided the attachment of I. ricinus nymphs and adults for at least 6 hours. The test repellent always provided protection (83-63%) against I. ricinus nymphs equivalent to the natural coconut oil based reference product and a better protection (88-75%) against adult ticks than the synthetic Icaridin-containing reference repellent.
We found that the 10% DDA-based formulation (ContraZeck®) is an easily applied and very effective natural repellent against I. ricinus ticks. By reducing the human-vector contact the product minimises the risk of transmission of tick-borne diseases in humans.
PMCID: PMC2362118  PMID: 18397516
22.  Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes Ticks in the Baltic Regions of Russia 
Journal of Clinical Microbiology  2001;39(6):2237-2242.
The presence and distribution of Ehrlichia spp. and Borrelia burgdorferi sensu lato was demonstrated among ixodid ticks collected in the Baltic regions of Russia, where Lyme borreliosis is endemic. A total of 3,426 Ixodes ricinus and 1,267 Ixodes persulcatus specimens were collected, and dark-field microscopy showed that 265 (11.5%) I. ricinus and 333 (26.3%) I. persulcatus ticks were positive. From these samples, 472 dark-field-positive and 159 dark-field-negative ticks were subjected to PCR and subsequent reverse line blot hybridization. Fifty-four ticks (8.6%) carried Ehrlichia species, and 4 (0.6%) carried ehrlichiae belonging to the Ehrlichia phagocytophila complex, which includes the human granulocytic ehrlichiosis agent. The E. phagocytophila complex and an Ehrlichia-like species were detected only in I. ricinus whereas Ehrlichia muris was found exclusively in I. persulcatus, indicating a possible vector-specific infection. Borrelia garinii was found predominantly in I. persulcatus, but Borrelia afzelii was evenly distributed among the two tick species. Only two I. ricinus ticks carried B. burgdorferi sensu stricto, while Borrelia valaisiana and a newly identified B. afzelii-like species were found in 1.7 and 2.5% of all ticks, respectively. Of the dark-field-positive ticks, only 64.8% yielded a Borrelia PCR product, indicating that dark-field microscopy may detect organisms other than B. burgdorferi sensu lato. These observations show that the agent of human granulocytic ehrlichiosis may be present in ticks in the Baltic regions of Russia and that clinicians should be aware of this agent as a cause of febrile disease.
PMCID: PMC88117  PMID: 11376063
23.  Inter- and Intralaboratory Comparison of Ehrlichia equi and Human Granulocytic Ehrlichiosis (HGE) Agent Strains for Serodiagnosis of HGE by the Immunofluorescent-Antibody Test 
Journal of Clinical Microbiology  1999;37(9):2968-2973.
Human granulocytic ehrlichiosis (HGE) is usually diagnosed by immunofluorescent antibody (IFA) serology with Ehrlichia equi-infected neutrophils or HGE agent-infected cultured HL60 cells. The HGE agent and E. equi are antigenically diverse, and interpretation of serologic results is also often variable. Thus, we investigated the sensitivity and specificity of various HGE agent and E. equi antigens used for IFA diagnosis by three different laboratories. Serum samples from 28 patients with well-characterized HGE and 9 patients with suspected HGE who were investigated by PCR, blood smear examinations, and serology were used, along with 9 serum samples from patients with other rickettsial and ehrlichial infections. Each serum sample was tested with up to 10 different antigen preparations. Overall, qualitative IFA results agreed in 70% of the samples. Titers among antigens were similar (r = 0.89 to 0.96), but titers of individual samples varied by fourfold or more in 5 of 81 (6%) of the serum samples. Sensitivity ranged from 100% to 82%, and specificity varied from 100% to 67%, but these differences were not significant, even among those tested in the same laboratory or between two different laboratories. Antibodies were detected in 14 to 44% of acute-phase sera from confirmed HGE patients. Most false-positive reactions resulted with Ehrlichia chaffeensis; when these sera were excluded, the specificity of most antigens was 91 to 100%. These data indicate that IFA results often agree and that IFA is useful for diagnosis of HGE in convalescence. However, without further standardization, variability among serologic tests using E. equi and HGE agent isolates for diagnosis of HGE will occasionally provide discrepant results and confound diagnosis.
PMCID: PMC85424  PMID: 10449483
24.  Natural infection of small mammal species in Minnesota with the agent of human granulocytic ehrlichiosis. 
Journal of Clinical Microbiology  1997;35(4):853-855.
The natural reservoirs for the agent of human granulocytic ehrlichiosis (HGE) are suspected to be the small mammals that host immature stages of Ixodes scapularis ticks. To determine if such small mammals are naturally infected, we collected blood and serum samples from small mammal species in rural and suburban areas of Minneapolis and St. Paul, Minn. Samples were collected from white-footed mice (Peromyscus leucopus), eastern chipmunks (Tamias striatus), southern red-backed voles (Clethrionomys gapperi), and insectivorous shrews (Blarina brevicauda and Sorex cinereus). Blood samples were tested by PCR for active infection with the HGE agent, and sera from P. leucopus mice were tested for serologic evidence of infection by indirect immunofluorescence. PCR analyses revealed the presence of HGE agent DNA in 20 of the 190 samples (10.5%) tested. Of the 119 P. leucopus mouse serum samples that were analyzed, 12 (10.1%) contained Ehrlichia equi antibodies. In 3 of 119 (2.5%) P. leucopus mice from which both blood and serum were collected. HGE agent DNA and antibodies against E. equi were present. Animals with evidence of infection with the HGE agent are widely distributed around the Minneapolis-St. Paul area in regions with known I. scapularis tick activity. Small mammals that are frequent hosts for larval I. scapularis ticks and that are found in areas where HGE occurs are likely to be a major reservoir from which infected ticks that bite humans are derived.
PMCID: PMC229689  PMID: 9157141
25.  Serologic Evidence of a Natural Infection of White-Tailed Deer with the Agent of Human Granulocytic Ehrlichiosis in Wisconsin and Maryland 
White-tailed deer participate in the maintenance of the Ixodes tick life cycle and are reservoirs for some tick-borne infectious agents. Deer may be useful sentinels for tick-transmitted agents, such as ehrlichiae. In order to determine whether white-tailed deer are markers of natural transmission or are reservoirs for the human granulocytic ehrlichiosis (HGE) agent, we performed indirect immunofluorescent-antibody (IFA) tests and immunoblotting with the HGE agent and Ehrlichia chaffeensis on sera from 43 and 294 deer captured in northwest Wisconsin during 1994 and 1995, respectively, and 12 deer from southern Maryland. According to IFA testing, 47% of 1994 Wisconsin sera, 60% of 1995 Wisconsin sera, and 25% of Maryland sera contained HGE agent antibodies. All IFA-positive deer sera tested reacted with the 44-kDa band which is unique to the Ehrlichia phagocytophila group. Serologic reactions to E. chaffeensis were detected by IFA testing in 15 of 337 (4%) Wisconsin deer and in 10 of 12 (83%) Maryland deer, while 60 and 80% of E. chaffeensis IFA-positive Wisconsin and Maryland deer sera, respectively, reacted with the E. chaffeensis 28- to 29-kDa antigens by immunoblotting. A total of 4% of deer from Wisconsin and 25% of deer from Maryland were found by IFA testing to have antibodies to both the HGE agent and E. chaffeensis; 75% of these were confirmed to contain E. chaffeensis antibodies by immunoblotting. These results suggest that white-tailed deer in diverse geographical regions of the United States are naturally infected with the HGE agent, E. chaffeensis, or both and that these animals, and potentially humans, are exposed to infected ticks at a high frequency in nature.
PMCID: PMC96198  PMID: 9801331

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