Ehrlichia-infected ticks on migrating birds.

During the spring of 1996, an estimated 581,395 Ehrlichia-infected ticks were imported into Sweden by migrating birds. Ehrlichia gene sequences found in ticks collected from these migrating birds were identical to those of granulocytic ehrlichiosis found in domestic animals and humans in Sweden. These findings support the idea that birds may play a role in dispersing Ehrlichia.

Identification of a p28 gene in Ehrlichia ewingii: evaluation of gene for use as a target for a species-specific PCR diagnostic assay.

PCR was used to amplify a 537-bp region of an Ehrlichia ewingii gene encoding a homologue of the 28-kDa major antigenic protein (P28) of Ehrlichia chaffeensis. The E. ewingii p28 gene homologue was amplified from DNA extracted from whole blood obtained from four humans and one canine with confirmed cases of infection. Sequencing of the PCR products (505 bp) revealed a partial gene with homology to outer membrane protein genes from Ehrlichia and Cowdria spp.: p30 of Ehrlichia canis (< or =71.3%), p28 of E. chaffeensis (< or =68.3%), and map1 of Cowdria ruminantium (67.3%). The peptide sequence of the E. ewingii partial gene product was deduced (168 amino acids) and the antigenicity profile was analyzed, revealing a hydrophilic protein with < or =69.1% identity to P28 of E. chaffeensis, < or =67.3% identity to P30 of E. canis, and < or =63.1% identity to MAP1 of C. ruminantium. Primers were selected from the E. ewingii p28 sequence and used to develop a species-specific PCR diagnostic assay. The p28 PCR assay amplified the expected 215-bp product from DNA that was extracted from EDTA-treated blood from each of the confirmed E. ewingii infections that were available. The assay did not produce PCR products with DNA extracted from E. chaffeensis-, E. canis-, or E. phagocytophila-infected samples, confirming the specificity of the p28 assay for E. ewingii. The sensitivity of the E. ewingii-specific PCR assay was evaluated and determined to detect as few as 38 copies of the p28 gene.

Epidemic typhus meningitis in the southwestern United States.

A patient residing in New Mexico had murine typhus diagnosed. A novel molecular assay was performed at the Centers for Disease Control and Prevention, and Rickettsia prowazekii, the agent of epidemic typhus, was found, rather than R. typhi. To our knowledge, this is the first reported case of epidemic typhus confirmed by means of polymerase chain reaction--based testing of cerebrospinal fluid, and it introduces a novel assay for the molecular diagnosis of both epidemic and murine typhus.

Human granulocytic ehrlichiosis--a clinical case in Scandinavia.

A clinical case of human granulocytic ehrlichiosis in Scandinavia is presented. The patient developed high fever, myalgia, headache and dyspnoea. Doxycycline treatment resulted in a dramatic improvement. Laboratory confirmation included a fourfold change in anti-Ehrlichia equi IFA titre and a positive PCR confirmed by gene sequence analysis.

Detection and differentiation of old world orthopoxviruses: restriction fragment length polymorphism of the crmB gene region.

A restriction fragment length polymorphism (RFLP) assay was developed to identify and differentiate Old World, African-Eurasian orthopoxviruses (OPV): variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and taterapox viruses. The test uses amplicons produced from virus genome DNA by PCR with a consensus primer pair designed from sequences determined for the cytokine response modifier B (crmB) gene of 43 different OPV strains of known taxonomic origin. The primer pair amplified a single specific product from each of the 115 OPV samples tested. Size-specific amplicons identified and differentiated ectromelia and vaccinia virus strains, which contain a truncated crmB gene, and enabled their differentiation from other OPV species. Restriction digests of amplified products allowed the identification and differentiation of variola, monkeypox, camelpox, vaccinia, and cowpox virus species and strains.

Survival of Ehrlichia chaffeensis in refrigerated, ADSOL-treated RBCs.

BACKGROUND: The purpose of this study was to investigate the persistence of viable Ehrlichia chaffeensis in ADSOL-treated RBCs stored at 4 to 6 degrees C. STUDY DESIGN AND METHODS: The continuous monocytic cell lines THP-1 and DH82 were infected with E. chaffeensis (St. Vincent isolate). Packed RBC units were inoculated in separate experiments with E. chaffeensis-infected cells as final concentrations of 8.02 x 10(4) (DH82) and 1.43 x 10(4) (THP-1) infected cells per mL. Aliquots were stored at 4 to 6 degrees C for 1 to 42 days. At selected intervals, nucleated cells from the RBC aliquots were obtained by using a ficoll-isopaque separation procedure. Uninfected DH82 cell cultures were inoculated with the harvested nucleated cells or supernatant. The cell cultures were evaluated for infection by weekly examination of Wright's (Diff-Quik) stained cytocentrifuged slides. PCR amplification was also used to test the harvested nucleated cells or supernatant for the presence of E. chaffeensis DNA. RESULTS: In both types of infected cell lines, E. chaffeensis was reisolated in DH82 cells for as long as 11 days from the cellular fraction and for up to 5 days from the supernatant fraction. PCR results were positive throughout the 42-day testing period. CONCLUSION: Cell-associated E. chaffeensis remains viable in ADSOL-treated RBCs stored at 4 to 6 degrees C for at least 11 days. These data suggest that transfusion-acquired infection is possible. Successful reisolation was achieved from the supernatant fraction, which suggests that RBC products treated with a WBC-reduction procedure may still present a risk for transfusion transmission. No correlation between PCR positivity and viability of bacteria was noted.

Sequence analysis of the ank gene of granulocytic ehrlichiae.

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.

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.

Alastrim smallpox variola minor virus genome DNA sequences.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific.

Infection with agents of human granulocytic ehrlichiosis, lyme disease, and babesiosis in wild white-footed mice (Peromyscus leucopus) in Connecticut.

White-footed mice, Peromyscus leucopus, were captured in southern Connecticut during 1997 and 1998 to determine the prevalence of infections caused by granulocytic Ehrlichia sp., Borrelia burgdorferi, and Babesia microti. Of the 50 mice captured and recaptured, 25 of 47 (53.2%) and 23 of 48 (47.9%) contained antibodies to the BDS or NCH-1 Ehrlichia strains, respectively, as determined by indirect fluorescent antibody (IFA) staining methods. The majority (83.3%) of 48 mice also contained antibodies to B. burgdorferi, as determined by enzyme-linked immunosorbent assay. Moreover, 20 of 26 (76.9%) contained antibodies to B. microti by IFA staining methods. In nested PCR tests using the 16S rRNA gene, the DNA of the human granulocytic ehrlichiosis (HGE) agent was detected in 17 of 47 mice (36.2%), but only 4 (23.5%) of these 17 mice were PCR positive at each capture. Antibody-positive reactions to granulocytic Ehrlichia sp. organisms were detected in 17 of 23 (73. 9%) of the PCR-positive mice. The sequences from PCR products from nine positive blood samples were identical to the HGE agent. Ehrlichia spp. were cultured from three of five mice captured in April 1998, including one that was PCR positive in April 1997. In addition, 2 of 14 larval Ixodes scapularis pools, which were attached to two PCR-positive mice, contained DNA of the HGE agent. A high percentage of white-footed mice are infected or have been infected naturally by the HGE agent with low-level persistent infection or frequent reinfection in some individual mice. However, the changes noted in the presence of DNA and antibodies in repeated blood and serum samples from individual mice over several months of field collection suggests that infection with granulocytic Ehrlichia is transient in most wild P. leucopus.

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.

Serological evidence of Ehrlichia infection in Swedish Lyme borreliosis patients.

We studied sera from patients who had participated in a prospective study of borreliosis in Sweden and had acquired tick bites in areas of the country with a high prevalence of granulocytic ehrlichial infections in animals. The sera were examined for IgG anti Ehrlichia antibodies by an indirect immunofluorescence assay using a locally isolated bovine Ehrlichia antigen. Confirmation of the serological results was done at the Unité des Rickettsies, Marseille, France. Three out of 37 of the investigated patients and 1 out of 100 investigated healthy blood donors had significant antibody titres to granulocytotropic Ehrlichiae. No patient or blood donor had specific antibody titres to Ehrlichia chaffeensis. These data suggest that Scandinavian Ehrlichia species can infect and evoke immunological response in tick-exposed humans.

Feline granulocytic ehrlichiosis--a report of a new clinical entity and characterisation of the infectious agent.

A 14-month-old shorthaired cat was presented to the Animal Hospital in Skara, Sweden, with a two-day history of lethargy, anorexia and tachypnoea. Clinical examination and laboratory investigations revealed fever, dehydration, tick infestation, neutrophilia with left shift, lymphopenia, hyperglycaemia and intracytoplasmic neutrophilic Ehrlichia inclusions. After treatment with intravenous doxycycline and lactated Ringer's solution the temperature returned to normal. Oral treatment with doxycycline continued for 20 days. The ehrlichiosis diagnosis was confirmed by serology, polymerase chain reaction and DNA sequencing. No relapse was observed during the eight-month follow-up period. The granulocytotropic Ehrlichia strain found in the cat was later characterised by analysis of the 16S rRNA gene sequence which showed 100 per cent identity to DNA sequences found in Swedish canine and equine granulocytotropic Ehrlichia strains. This is, to the best of the authors' knowledge, the first reported case of granulocytic ehrlichiosis in a cat.

Nested PCR assay for detection of granulocytic ehrlichiae.

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

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.

Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island.

A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient's residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks.

Terminal region sequence variations in variola virus DNA.

Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.

PCR strategy for identification and differentiation of small pox and other orthopoxviruses.

Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.

Topography of variola smallpox virus inverted terminal repeats.

We examined the nucleotide sequences of the inverted terminal repeat (ITR) regions adjacent to the covalently closed hairpin end sequences of three variola major and four minor strains from smallpox outbreaks in Europe, Asia, Africa, and South America. The ITR regions ranged in size from 581 to 1051 base pairs (bp) and contained no apparent open reading frames. Two nonrepetitive sequence elements, NR1 and NR2, were conserved and resembled nonrepetitive elements in the ITRs of other orthopoxviruses. Depending on strain, the terminally positioned NR1 and the more internal NR2 flanked a direct repeat region containing from none to four copies of a 69-bp sequence and one copy of a 54-bp related sequence partial repeat. A distinctive pattern of ITR topography of NR1 and NR2 flanking a single copy of the 69-bp unit characterized each of three examined alastrim variola minor strains. A nonalastrim African minor strain from the last natural case of smallpox in Somalia in 1977 showed the largest ITR region of the examined viruses because of a second direct repeat cluster following NR2.