Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus.

The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.

Clinical and serological follow-up of patients with human granulocytic ehrlichiosis in Slovenia.

An evaluation of the clinical outcome and the duration of the antibody response of patients with human granulocytic ehrlichiosis (HGE) was undertaken in Slovenia. Adult patients with a febrile illness occurring within 6 weeks of a tick bite were classified as having probable or confirmed HGE based on the outcome of serological or PCR testing. Thirty patients (median age, 44 years) were enrolled, and clinical evaluations and serum collection were undertaken at initial presentation and at 14 days, 6 to 8 weeks, and 3 to 4, 6, 12, 18, and 24 months. An indirect immunofluorescence assay (IFA) was performed, and reciprocal titers of > or =128 were interpreted as positive. Patients presented a median of 4 days after the onset of fever and were febrile for a median of 7.5 days; four (13.3%) received doxycycline. Seroconversion was observed in 3 of 30 (10.0%) patients, and 25 (83.3%) showed >4-fold change in antibody titer. PCR results were positive in 2 of 3 (66.7%) seronegative patients but in none of 27 seropositive patients at the first presentation. IFA antibody titers of > or =128 were found in 14 of 29 (48.3%), 17 of 30 (56.7%), 13 of 30 (43.4%), and 12 of 30 (40.0%) patients 6, 12, 18, and 24 months after presentation, respectively. Patients reporting additional tick bites during the study had significantly higher antibody titers at most time points during follow-up. No long-term clinical consequences were found during follow-up.

Prospective assessment of the etiology of acute febrile illness after a tick bite in Slovenia.

A prospective study established the etiology of febrile illnesses in residents of Slovenia that occurred within 6 weeks after a tick bite. A combination of laboratory and clinical criteria identified 64 (49.2%) of 130 patients as having confirmed, probable, or possible cases of tickborne disease during 1995 and 1996. Of the 130 patients, 36 (27.7%) had laboratory evidence of tickborne encephalitis, all of whom had clinically confirmed disease. Evidence of infection with Borrelia burgdorferi sensu lato was identified in 26 patients; 10 (7.7%) had confirmed Lyme borreliosis. Of 22 patients with evidence of Ehrlichia phagocytophila infection, 4 (3.1%) had confirmed ehrlichiosis. Infection by multiple organisms was found in 19 (14.6%) of 130 patients. Patients with meningeal involvement (43 [72.3%] of 59) were more likely to have confirmed tickborne disease than were patients with illness of undefined localization (18 [26.5%] of 68; P<.0001). Tickborne viral and bacterial infections are an important cause of febrile illness in Slovenia.

Detection of antibodies reactive with Ehrlichia chaffeensis in the raccoon.

Antibodies reactive with Ehrlichia chaffeensis were detected in raccoon (Procyon lotor) serum samples by using an indirect immunofluorescence assay. Samples from 411 raccoons trapped in the southeastern United States from 1977 to 1999 were tested. Serologically reactive samples with reciprocal titers of > or =16 were detected from 83 raccoons (20%) from 13 of 16 counties in eight states, indicating that raccoons are commonly exposed to E. chaffeensis. Samples collected as early as 1977 were positive. A polymerase chain reaction assay specific for E. chaffeensis failed to detect the presence of ehrlichial DNA in serum samples from 20 representative seroreactive raccoons. Because of serologic cross-reactivity among antigens derived from different Ehrlichia spp., additional immunologic, molecular, or culture-based studies will be required to confirm E. chaffeensis infections of raccoons in the southeastern United States.

PCR amplification and phylogenetic analysis of groESL operon sequences from Ehrlichia ewingii and Ehrlichia muris.

Broad-range PCR primers were used to amplify part of the groESL operon of the canine pathogen Ehrlichia ewingii, recently recognized as a human pathogen, and the murine pathogen Ehrlichia muris. Phylogenetic analysis supported the relationships among Ehrlichia species previously determined by comparison of 16S rRNA gene sequences. These sequences provide additional PCR targets for species for which few gene sequences have been determined.

PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland.

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 ticks [corrected] 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.

Dusky-footed wood rats (Neotoma fuscipes) as reservoirs of granulocytic Ehrlichiae (Rickettsiales: Ehrlichieae) in northern California.

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.

Shared vector-borne zoonoses of the Old World and New World: home grown or translocated?

Humans inhabiting the Old World and New World share a wide variety of pathogens. Processes that result in the disjunct biogeographic distribution of pathogens with common vertebrate reservoirs or vectors are more difficult to unravel than those influencing the distribution of infections spread only through human-to-human transmission. The origins of species and complexes of tick-borne bacteria are unclear. The agent of Lyme borreliosis may have speciated in the New World following geographical isolation of ticks harboring ancestral spirochetes; the subsequent spread to Europe of B. burgdorferi sensu stricto may have occurred within historical times. Other tick-borne agents, such as the ehrlichiae causing human granulocytic ehrlichiosis, are genetically very similar in the Old World and New World. As the taxonomic distinctions among these related agents of human and veterinary importance appear increasingly blurred, the processes leading to the current discontinuous geographic distributions will also become the source of continuing speculation. Accumulating data suggest an Old World origin for a group of bacteria that include B. elizabethae, a human pathogen first identified from the New World. The potential public health significance of these newly described organisms is undefined, but of international interest as their vertebrate reservoir has been introduced throughout the world.

Outcome of diagnostic tests using samples from patients with culture-proven human monocytic ehrlichiosis: implications for surveillance.

We describe the concordance among results from various laboratory tests using samples derived from nine culture-proven cases of human monocytic ehrlichiosis (HME) caused by Ehrlichia chaffeensis. A class-specific indirect immunofluorescence assay for immunoglobulin M (IgM) and IgG, using E. chaffeensis antigen, identified 44 and 33% of the isolation-confirmed HME patients on the basis of samples obtained at initial clinical presentation, respectively; detection of morulae in blood smears was similarly insensitive (22% positive). PCR amplifications of ehrlichial DNA targeting the 16S rRNA gene, the variable-length PCR target gene, and the groESL operon were positive for whole blood specimens obtained from all patients at initial presentation. As most case definitions of HME require a serologic response with compatible illness for a categorization of even probable disease, PCR would have been required to confirm the diagnosis of HME in all nine of these patients without the submission of a convalescent-phase serum sample. These data suggest that many, if not most, cases of HME in patients who present early in the course of the disease may be missed and underscore the limitations of serologically based surveillance systems.

Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species.

A phylogenetic investigation was done on the members of the genus Bartonella, based on the DNA sequence analysis of the groEL gene, which encodes the 60 kDa heat-shock protein GroEL. Nucleotide sequence data were determined for a near full-length fragment (1368 bp) of the groEL gene of the established Bartonella species and used to infer intraspecies phylogenetic relationships. Phylogenetic trees were inferred from multiple sequence alignments by using both distance and parsimony methods, which demonstrated an architecture composed of six well-supported lineages. The results are consistent with relationships deduced from recent sequence analysis studies based upon citrate synthase (gItA) and previously observed genotypic and phenotypic characteristics; however, they showed greater statistical support at the intragenus level. This suggests that groEL may be a more robust tool for phylogenetic analysis of Bartonella lineages.

Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis.

BACKGROUND: Human ehrlichiosis is a recently recognized tick-borne infection. Four species infect humans: Ehrlichia chaffeensis, E. sennetsu, E. canis, and the agent of human granulocytic ehrlichiosis. METHODS: We tested peripheral-blood leukocytes from 413 patients with possible ehrlichiosis by broad-range and species-specific polymerase-chain-reaction (PCR) assays for ehrlichia. The species present were identified by species-specific PCR assays and nucleotide sequencing of the gene encoding ehrlichia 16S ribosomal RNA. Western blot analysis was used to study serologic responses. RESULTS: In four patients, ehrlichia DNA was detected in leukocytes by a broad-range PCR assay, but not by assays specific for E. chaffeensis or the agent of human granulocytic ehrlichiosis. The nucleotide sequences of these PCR products matched that of E. ewingii, an agent previously reported as a cause of granulocytic ehrlichiosis in dogs. These four patients, all from Missouri, presented between May and August 1996, 1997, or 1998 with fever, headache, and thrombocytopenia, with or without leukopenia. All had been exposed to ticks, and three were receiving immunosuppressive therapy. Serum samples obtained from three of these patients during convalescence contained antibodies that reacted with E. chaffeensis and E. canis antigens in a pattern different from that of humans with E. chaffeensis infection but similar to that of a dog experimentally infected with E. ewingii. Morulae were identified in neutrophils from two patients. All four patients were successfully treated with doxycycline. CONCLUSIONS: These findings provide evidence of E. ewingii infection in humans. The associated disease may be clinically indistinguishable from infection caused by E. chaffeensis or the agent of human granulocytic ehrlichiosis.

Molecular cloning and characterization of the Ehrlichia chaffeensis variable-length PCR target: an antigen-expressing gene that exhibits interstrain variation.

A clone expressing an immunoreactive protein with an apparent molecular mass of 44 kDa was selected from an Ehrlichia chaffeensis Arkansas genomic library by probing with anti-E. chaffeensis hyperimmune mouse ascitic fluid. Nucleotide sequencing revealed an open reading frame (ORF) capable of encoding a 198-amino-acid polypeptide. The ORF contained four imperfect, direct, tandem 90-bp repeats. The nucleotide and deduced amino acid sequences did not show close homologies to entries in the molecular databases. PCR with primers whose sequences matched the sequences flanking the ORF was performed with DNA samples extracted from cell cultures infected with nine different isolates of E. chaffeensis, blood samples from seven patients with monocytic ehrlichiosis, and Amblyomma americanum ticks collected in four different states. The resulting amplicons varied in length, containing three to six repeat units. This gene, designated the variable-length PCR target, is useful for PCR detection of E. chaffeensis and differentiation of isolates.

Diagnosis of human ehrlichiosis by PCR assay of acute-phase serum.

A PCR assay of 43 acute-phase serum samples was evaluated as a method for early detection of human granulocytic ehrlichiosis (HGE) and determination of etiology when serologic testing is inconclusive. Sequence-confirmed products of the HGE agent were amplified from three individuals residing or having exposure history in Minnesota or Wisconsin, and similarly confirmed products from Ehrlichia chaffeensis were amplified from three individuals from Florida or Maryland. Etiology, as determined by PCR and serology, was the same whenever there was a fourfold difference between the maximum titers of antibodies to both antigens, indicating that presumptive determination of etiology may be based on fourfold differences in titers. PCR testing determined that E. chaffeensis was the etiologic agent for one individual who had similar titers of antibodies to both agents. PCR assay of acute-phase serum in the absence of whole blood specimens may be a useful method for early detection of human ehrlichiosis and determination of etiology when serologic testing is inconclusive.

Identity of ehrlichial DNA sequences derived from Ixodes ricinus ticks with those obtained from patients with human granulocytic ehrlichiosis in Slovenia.

Adult Ixodes ricinus (Acari: Ixodidae) ticks collected near Ljubljana, Slovenia, were tested for the agent of human granulocytic ehrlichiosis (HGE) by using PCR assays based on the 16S rRNA gene. Three (3.2%) of 93 ticks were found to contain granulocytic ehrlichiae. Nucleotide sequences of portions of the bacterial groESL heat shock operon amplified from these ticks were identical or nearly (99.8%) identical to those previously determined for human patients with HGE from Slovenia, providing additional evidence that the ticks were infected with the HGE agent. This study identified I. ricinus as the likely vector for these ehrlichial pathogens of humans in this part of Europe.

Human granulocytic ehrlichiosis in Europe: clinical and laboratory findings for four patients from Slovenia.

Febrile illnesses following a tick bite in patients from Slovenia were evaluated for an ehrlichial etiology. A case of acute human granulocytic ehrlichiosis (HGE) was confirmed by seroconversion to the HGE agent or molecular identification of ehrlichial organisms. Acute infection with the HGE agent was confirmed in four patients. None of the patients had detectable antibodies to the HGE agent at their first visit, but polymerase chain reaction analysis was positive for three patients. All four patients subsequently seroconverted to the HGE agent as shown by high titers of antibody. Clinical features and laboratory findings were similar to those in reports from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only two received antibiotic therapy (of whom only one was treated with doxycycline). HGE is an emerging tick-borne disease in the United States and should now be included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Serologic evidence of infection with Ehrlichia spp. in wild rodents (Muridae: Sigmodontinae) in the United States.

Rodent (Muridae: Sigmodontinae) blood and sera collected from 14 states were tested for seroreactivity to a cultured isolate of the human granulocytic ehrlichiosis (HGE) agent by using an indirect immunofluorescence assay. Of the 1,240 samples tested, 136 (11%) were found to be reactive at titers of > or = 32. Rodents with HGE agent-specific antibodies were found in New York (23% of 491 samples; geometric mean endpoint titer [GMT] = 441), Connecticut (11% of 100 samples; GMT = 481), California (9% of 32 samples; GMT = 323), Colorado (2% of 212 samples; GMT = 256), Florida (7% of 27 samples; GMT = 362), Maryland (7% of 15 samples; titer = 64), New Jersey (4% of 76 samples; titer = 256), and Wisconsin (13% of 8 samples; titer = 128). Samples from Georgia (n = 16), Illinois (n = 27), Nevada (n = 27), North Carolina (n = 52), Ohio (n = 57), and Utah (n = 100) were not reactive. The earliest seroreactive sample was from a Peromyscus leucopus mouse collected in June 1986 in Connecticut, and the majority of the seroreactive samples (68%) were from this species. Samples from other Peromyscus species (P. boylii, P. maniculatus, and P. gossypinus) were also found to be reactive, with a GMT for the genus of 410. Several species of Neotoma woodrats (N. fuscipes, N. lepida, N. albigula, and N. mexicana) from California and Colorado had antibodies that reacted with the HGE agent (genus GMT = 194), suggesting that enzootic cycles of Ehrlichia spp. exist outside of the areas of confirmed human disease. Attempts to amplify and detect ehrlichial DNA from the limited tissues available (n = 40 animals) were unsuccessful. Further studies are needed to determine the identity of the organisms inducing antibody production in these rodent species and to elucidate the epidemiology and public health importance of these agents.

Human ehrlichiosis in central Europe.

Ehrlichioses are tick-transmitted diseases associated with illnesses of animals for decades, but recently recognised to be emerging human diseases. In the last ten years increasing number of cases of human infections caused by Ehrlichia chaffeensis and granulo-cytic ehrlichia were described in the United States. Several reports also indicate the presence of infection with the human granulocytic ehrlichiosis (HGE) agent in Europe. The first confirmed acute human disease caused by HGE agent was reported from Slovenia. Until 1997, five patients have been discovered in a prospective study on the etiology of febrile illnesses occurring within six weeks following a tick bite, conducted at the Department of Infectious Diseases, University Medical Centre, Ljubljana, Slovenia. The diagnosis of acute HGE was established by seroconversion to the HGE agent and/or molecular identification of ehrlichial organisms. None of the patients had detectable morulae on blood smear examination. Clinical characteristics and laboratory findings were similar to those reported from the United States, although the disease course was relatively mild in the Slovenian cases. All patients recovered rapidly and without sequelae, although only three patients received antibiotic therapy (of whom only two were treated with doxycycline). Many ehrlichiosis cases could go undetected due to a lack of physician awareness, lack of public knowledge, or limited investigation. HGE should now be also included in the differential diagnosis of febrile illnesses occurring after a tick bite in Europe.

Isolation and characterization of Ehrlichia chaffeensis strains from patients with fatal ehrlichiosis.

Two new isolates of Ehrlichia chaffeensis (designated Jax and St. Vincent) were obtained from patients with fatal ehrlichial infections. Patients developed characteristic manifestations of severe disease due to E. chaffeensis, including marked thrombocytopenia, pulmonary insufficiency, and encephalopathy. Primary isolation was achieved in DH82 cells; the Jax and St. Vincent isolates were detected within 19 and 8 days postinoculation, respectively. The isolates were characterized by molecular evaluation of the 16S rRNA gene, the groESL heat shock operon, a 120-kDa immunodominant protein gene, and an incompletely characterized repetitive-motif sequence (variable-length PCR target [VLPT]). The sequences were compared with those of the corresponding molecular regions in the type isolate (Arkansas). St. Vincent contained one fewer repeat unit in both the 120-kDa protein gene and the VLPT compared with corresponding sequences of the Jax and Arkansas isolates. 16S rRNA gene sequences from the two new isolates had 100% identity to the corresponding sequences of the 91HE17 and Sapulpa isolates of E. chaffeensis, and to the corrected 16S rRNA gene sequence of the Arkansas isolate. The Jax isolate grew more slowly than the St. Vincent isolate in DH82 cells, and both of the new isolates grew more slowly than the extensively passaged Arkansas isolate. Although specific associations between ehrlichial pathogenicity and genotype were not identified from these comparisons, recovery of this organism from a spectrum of clinical presentations remains an integral step in understanding mechanisms of disease caused by E. chaffeensis.

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species.

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.