The interface between research and the diagnosis of an emerging tick-borne disease, human ehrlichiosis due to Ehrlichia chaffeensis.

Two new ehrlichial species that cause human disease have recently been identified: Ehrlichia chaffeensis and the currently unnamed agent of human granulocytic ehrlichiosis. Our objective was to review data on the clinical presentation, laboratory and epidemiological findings, therapy, and diagnostic procedures of patients with human ehrlichiosis due to E chaffeensis. From 1986 through 1994, 400 case patients were identified from 30 US states. Most patients had a nonspecific illness, characterized by fever and headache. Severe illness and death occurred, primarily in the elderly. Laboratory findings most commonly included leukopenia, thrombocytopenia, and elevated liver function test results. Antibody response was the basis for diagnosis, although polymerase chain reaction testing has been useful in research settings. Empirical treatment with tetracycline or its analogues should be begun as soon as possible after the onset of symptoms. Clinicians need to be alert for this illness when evaluating febrile patients whose history includes possible recent tick exposure.

Dose effects of seeds placement deviations from pre-planned positions in ultrasound guided prostate implants.

The effects of random (drifting) errors in actual seed placement during prostate implants have been evaluated by using geometrically optimized implants of I-125 and Pd-103 seeds. Results indicate that small random deviations in the seeds placement from the preplant position may affect the planned dose and beam profiles significantly.

Human ehrlichiosis: hematopathology and immunohistologic detection of Ehrlichia chaffeensis.

Human ehrlichiosis is a recently described zoonosis caused by a rickettsia that infects leukocytes. Most patients have fever, headache, chills, and myalgias and develop leukopenia, thrombocytopenia, anemia, and elevations in serum hepatic aminotransferases. The cause of the peripheral leukopenia and thrombocytopenia is not known. We studied peripheral blood smears, bone marrow aspirates, and bone marrow biopsy specimens from patients with serologically proven ehrlichiosis to characterize the pathologic changes associated with leukopenia or thrombocytopenia, to detect the presence of immunohistologically demonstrable ehrlichiae, and to establish the infected host target cell(s). Specimens were obtained from 12 patients, and immunohistology for Ehrlichia chaffeensis was performed on tissue sections, aspirated bone marrow, and peripheral blood smears. Mean leukocyte and platelet counts available for nine patients were white blood cell count 3,300/microL (range, 1,100 to 10,300/microL) and platelets 61,000/microL (range, 40,000 to 82,000/microL). Findings included myeloid hyperplasia (eight cases), megakaryocytosis (seven cases), granulomas (eight cases), marrow histiocytosis (one case), myeloid hypoplasia (one case), pancellular hypoplasia (one case), and normocellular marrow (two cases). Morulae of E chaffeensis were detected in four of 10 cases examined by immunohistology. Most ehrlichiae were detected within histiocytes, although morulae were rarely present within lymphocytes. Leukopenia, thrombocytopenia, or pancytopenia apparently most often results from peripheral sequestration or destruction; however, hypoplasia of marrow elements is present occasionally. Immunohistologic demonstration of E chaffeensis offers a direct means for establishing the etiologic diagnosis. These observations show the relatively frequent occurrence of bone marrow granulomas and suggest that infection of cells of the reticuloendothelial system may participate in the pathogenesis of human ehrlichiosis.

A single tissue culture system for the propagation of the agents of the human ehrlichioses.

Two newly emergent human diseases found in the United States, human monocytotropic ehrlichiosis (HME) and human granulocytotropic ehrlichiosis (HGE), are caused by pathogens of the genus Ehrlichia. The causative agent of HGE can be propagated in HL-60 human promyelocytic leukemia cells. Herein, we report the development of a method to propagate E. chaffeensis, the causative agent of HME, in HL-60 cells, thus providing a common system for the study of both species. The continuous propagation of E. chaffeensis requires the induction of HL-60 differentiation along the monocytic pathway toward phenotypically mature macrophages by the addition of 25-OH vitamin D3 to the growth medium.

Fingerprinting of Ehrlichia species by repetitive element polymerase chain reaction.

To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.

Tissue diagnosis of Ehrlichia chaffeensis in patients with fatal ehrlichiosis by use of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

In the United States, human ehrlichiosis is a complex of emerging tick-borne diseases caused by 3 distinct Ehrlichia species: Ehrlichia chaffeensis, Ehrlichia ewingii, and the human granulocytotropic ehrlichiosis agent. Ehrlichioses are characterized by a mild to severe illness, and approximately 4% of cases are fatal. Because these obligate intracellular bacteria are difficult to resolve with routine histologic techniques, their distribution in tissues has not been well described. To facilitate the visualization and detection of ehrlichiae, immunohistochemistry (IHC), in situ hybridization (ISH), and polymerase chain reaction (PCR) assays were developed by use of tissues from 4 fatal cases of E. chaffeensis infection. Evidence of E. chaffeensis via IHC, ISH, and PCR was documented in all 4 cases. Abundant immunostaining and in situ nucleic acid hybridization were observed in spleen and lymph node from all 4 patients. Significantly, in 2 of these patients, serologic evidence of infection was absent. Use of IHC, ISH, and PCR to visualize and detect Ehrlichia in tissues can facilitate diagnosis of ehrlichial infections.

Evaluation of indapamide 1.25 mg once daily in elderly patients with mild to moderate hypertension.

The objective of this study was to evaluate the safety and efficacy of indapamide 1.25 mg once daily as monotherapy in elderly patients (65 years and older) with mild to moderate essential hypertension. Two hundred and seventy-nine (279) elderly patients were enrolled in a washout period, during which patients received single-blind placebo for 4 weeks. Patients demonstrating supine diastolic pressures between 95 mm Hg and 114 mm Hg at the end of the 4-week placebo washout period were entered into the 8-week double-blind treatment period. Two hundred and four (204) patients qualified for the study and were randomized to the double-blind treatment; 103 patients received indapamide 1.25 mg and 101 patients received placebo for 8 weeks. Overall, 177 patients (92 indapamide and 85 placebo) completed the study. The primary efficacy criterion was the mean change in supine diastolic blood pressure (DBP) from double-blind baseline to the end of 8 weeks of therapy. By week 8 of the double-blind treatment period, indapamide 1.25 mg produced a statistically significant (P = 0.0037) decrease in supine DBP of 8.2 mm Hg compared to a decrease of 5.3 mm Hg produced in the placebo group. Additionally, indapamide 1.25 mg was statistically (P = 0.0028) more effective than placebo in reducing supine systolic BP (SBP) (-10.1 vs -4.2 mm Hg). The incidence of drug-related adverse events during the double-blind treatment period was similar between the two treatment groups. A low dose of indapamide, 1.25 mg, given once daily for 8 weeks was effective as monotherapy with respect to BP reduction in an elderly population with mild to moderate hypertension. Indapamide 1.25 mg was safe and generally well tolerated in this elderly patient population.

Persistent infection of C3H/HeJ mice by Ehrlichia chaffeensis.

Description of the pathobiology of the recently described zoonotic agent of human ehrlichiosis (Ehrlichia chaffeensis) would be greatly facilitated by the availability of a convenient experimental animal model of infection. We determined whether C3H/HeJ mice could sustain persistent infection by this predominantly monocyte-inhabiting rickettsia. Such mice rapidly produced an intense specific IgG response upon inoculation of ehrlichiae, and high titers were demonstrable for more than 6 months thereafter. Ehrlichiae were reisolated from the peripheral blood and spleen of 1 mouse at day 11 after inoculation. DNA of E. chaffeensis was more frequently detected within these tissues by polymerase chain reaction. Other candidate rodent models appeared to be poor hosts for this pathogen. About half of intact and virtually all splenectomized white-footed mice that were inoculated seroconverted. Sera from inoculated voles and hamsters did not react to antigens of E. chaffeensis. The C3H/Hej mouse becomes persistently infected by this rickettsia, and may serve as a useful model for studies of the immune response to the agent of human ehrlichiosis.

Kinetics of indomethacin degradation I: Presence of alkali.

The kinetics of indomethacin degradation were followed in alkaline aqueous solutions at various temperatures between 20.1 and 40.7 degrees. The apparent first-order rate constants were evaluated from log absorbance versus time plots at lambda max 318 nm. The primary salt effect was positive. The rate constant-hydroxide-ion concentration profile was linear with a positive slope, suggesting the following rate law: kobs = k1 [OH-]. The experimental data fit the proposed reaction of degradation, I- + OH k1 leads to products, where I- = mono-dissociated indomethacin species. Activation energies and other related parameters were calculated from Arrhenius-type plots.

Kinetics of indomethacin degradation II: Presence of alkali plus surfactant.

The kinetics of indomethacin were studied in the presence of the surfactants ethoxylated lanolin, polysorbate 80, and cetrimonium bromide under alkaline conditions at 30.3 degrees. The degradation followed apparent first-order kinetics. Plots of kobs versus surfactants; concentrations were curved with negative slopes for nonionic surfactants; but with the ionic surfactant, the plots showed a marked positive change in kobs as the surfactant concentration passed through the critical micelle concentration. Literature model systems adequately explained the data for nonionic surfactants but not for the ionic surfactant. A new set of equations was derived for each case using electrochemical potentials. The experimental data for all three surfactants fit the derived equations quite well.

Site-specific geographic association between Amblyomma americanum (Acari: Ixodidae) infestations and Ehrlichia chaffeensis-reactive (Rickettsiales: Ehrlichieae) antibodies in white-tailed deer.

Serum samples from white-tailed deer, Odocoileus virginianus Zimmermann, collected from 1982 through 1992 from the southeastern United States were tested for antibodies reactive to Ehrlichia chaffeensis Anderson, Dawson, Jones, & Wilson, the causative agent of human ehrlichiosis. Results were compared between areas based on known infestations of the lone star tick, Amblyomma americanum L., a suspected vector of E. chaffeensis. One hundred and twenty-five of 300 (41.7%) deer tested positive (> or = 1:128) for E. chaffeensis-reactive antibodies by fluorescent antibody analysis. Thirty of 30 (100%) collection areas known to be lone star tick infested contained deer that tested positive for E. chaffeensis-reactive antibodies, corresponding to 121/150 (80.7%) of deer examined. A few deer, 4/150 (2.7%) of those examined, from 2 of 30 (6.7%) areas where lone star ticks were not detected were positive for E. chaffeensis-reactive antibodies. This site-specific geographic association between A. americanum and the presence of E. chaffeensis-reactive antibodies in deer provides strong evidence that A. americanum is a natural vector of E. chaffeensis or a closely related species among white-tailed deer.

Novel Ehrlichia organism (Rickettsiales: Ehrlichieae) in white-tailed deer associated with lone star tick (Acari: Ixodidae) parasitism.

Polymerase chain reaction (PCR) evidence of a novel Ehrlichia organism was found recently in wild white-tailed deer, Odocoileus virginianus Zimmermann, and lone star ticks, Amblyomma americanum L., from the southeastern United States. To evaluate whether lone star tick parasitism was associated with the presence of this novel Ehrlichia organism in deer, 2 retrospective studies were conducted using specific nested PCR to test archived deer serum samples. The 1st study of 150 serum samples collected from a single deer population over a 15-yr period examined the temporal association between the presence of the Ehrlichia organism in deer and parasitism by lone star ticks. The deer Ehrlichia was not detected in serum samples collected before 1986, when lone star ticks were absent or rare, but was detected in samples collected in 1986 and every year thereafter, when lone star ticks became increasingly abundant. In the 2nd study, serum samples from 120 deer from 24 sites in 14 southeastern states were tested to evaluate if a site-specific, spatial association existed between the presence of the deer Ehrlichia and lone star ticks. All 60 serum samples from the 12 deer populations without evidence of lone star tick infestation were negative for the deer Ehrlichia, whereas 83% of the 12 populations infested by lone star ticks had PCR evidence of infection. These data suggest that lone star ticks may be a vector of the deer Ehrlichia; however, they do not preclude the involvement of other arthropods in maintaining infection with this organism in deer populations.

Dogs infected with a human granulocytotropic Ehrlichia spp. (Rickettsiales: Ehrlichieae).

Dogs were found to be susceptible to human granulocytotropic Ehrlichia spp. Infection was produced through the bite of Ixodes scapularis Say (= dammini Spielman, Clifford, Piesman & Corwin) nymphs and adults that acquired infection while feeding as larvae on experimentally infected mice. Dogs were also infected by intravenous injection of mouse blood or dog blood from parasitemic donors. Parasites were demonstrable in neutrophils within 8 or 9 d after nymphs began feeding; prepatent periods were longer when infection was induced by adult tick feeding (18 d) or by transfusion of mouse blood (12 d). The shortest prepatent period observed was 5 d in a dog infected by transfusion of blood from a parasitemic dog. Infections in dogs were mild and apparently transient. Mild thrombocytopenia was the most commonly observed abnormality. Parasites could be detected by light microscopy during the acute phase of infection (4 or 5 d) and parasite DNA by polymerase chain reaction as early as 5 d after exposure but not at 6-9 d after morulae were first observed in neutrophils. Likewise, dog blood was infectious for mice at 2 d but not at 25 d, and for dogs at 3 d but not at 13 d after morulae were first observed in neutrophils. Seroconversion occurred as early as 11 d after onset of tick feeding and persisted until dogs were euthanatized. Gross and histopathologic lesions were similar to those observed in dogs with E. canis (Donatien & Lestoquard), E. chaffeensis Anderson, Dawson & Wilson, and E. ewingii Anderson, Greene, Jones & Dawson infections but were generally milder than any of these. The moderate enlargement of lymphoid organs observed grossly was reflected histologically as mild to moderate reactive hyperplasia, which was largely follicular (B cell).

Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae).

Ehrlichia chaffeensis Anderson, Dawson & Wilson, causative agent of human (predominantly monocytic) ehrlichiosis, was successfully transmitted experimentally by Amblyomma americanum (L.) to white-tailed deer, Odocoileus virginianus (Zimmerman). Deer were needle-exposed intravenously to E. chaffeensis in tissue-culture canine macrophage (DH82) cells, and 11 d later were exposed to laboratory-reared A. americanum larvae, nymphs, and adults for acquisition feeding. Three months after this feeding, naive deer and dogs were exposed to recently molted nymphs and adults. Attempted reisolation of the pathogen by way of tissue culture was successful from one needle-exposed deer but not from the tick-exposed deer or dogs. Based on serologic evidence and polymerase chain reaction data, both nymphal and adult ticks transmitted E. chaffeensis to naive deer but not to dogs.

Development and evaluation of a recombinant antigen, monoclonal antibody-based competitive ELISA for heartwater serodiagnosis.

Cowdria ruminantium is the etiologic agent of heartwater, a tick-transmitted foreign animal disease with considerable potential for entrance into the USA. A competitive enzyme-linked immunosorbent assay (cELISA) was developed to detect serologic responses to C. ruminantium infection. The cELISA utilized a recombinant form of the C. ruminantium major antigenic protein (MAP-1) as the antigen and an anti-MAP-1 monoclonal antibody as the competing indicator reagent. Experimental antisera to C. ruminantium and a wide variety of related ehrlichial organisms were used to evaluate cELISA reactivity. Only sera against C. ruminantium, Ehrlichia canis, E. chaffeensis, and a recently discovered cervine ehrlichia-like organism reacted positively in the cELISA. Specificity of the cELISA was > or = 99.5% in a survey of 1,774 southeastern US and Puerto Rican slaughter cattle sera but was only 85% in a group of 79 hunter-killed white-tailed deer (Odocoileus virginianus) from the southeastern USA. Reference true-positive and cELISA false-positive sera were further analyzed by end point titrations using the cELISA and by indirect fluorescent antibody (IFA) tests for reactivity with C. ruminantium, E. canis, and E. chaffeensis antigens. True heartwater-positive sera were significantly more reactive using the cELISA and C. ruminantium IFA procedures (P < 0.05), whereas false-positive sera were significantly more reactive with the antigens used in the E. chaffeensis IFA procedure (P < 0.05). A group of sera from 210 field-origin ruminants residing on known or potentially heartwater-endemic Caribbean islands revealed a substantial (12.4%) prevalence of cELISA-positive specimens. The cELISA is a relatively specific serodiagnostic test for heartwater in cattle and could be used to monitor for possible introduction of the disease into the USA. The cELISA may also be an excellent tool for monitoring the success of an ongoing Caribbean Amblyomma tick eradication program designed to eliminate the biological vector responsible for the perpetuation and spread of this dangerous foreign animal disease.

Attempted transmission of human granulocytotropic Ehrlichia (HGE) by Amblyomma americanum and Amblyomma maculatum.

Transstadial transmission of human granulocytotrophic Ehrlichia (HGE) was attempted in dogs using Amblyomma americanum (L.) and A. maculatum Koch, two species that, as adults, feed readily on human beings. Larvae and nymphs were acquisition-fed on a dog that was parasitemic with HGE. Two months later, following digestion of the blood meal and subsequent molting to nymphal or adult stage, these ticks were fed to repletion on HGE-naive dogs. None of the dogs developed clinical evidence of ehrlichiosis. Parasites were not observed in blood smears by light microscopy, HGE DNA was not detected by polymerase chain reaction, and none of the dogs seroconverted. Based on this trial, we conclude that, unlike E. chaffeensis, HGE is probably not transmitted from dog to dog by either A. americanum or A. maculatum.

The first isolation, in vitro propagation, and genetic characterization of Ehrlichia canis in Israel.

Ehrlichia canis, the etiologic agent of canine ehrlichiosis, was isolated in Israel from a naturally infected dog with acute signs of the disease. The organism designated E. canis 611, was passaged experimentally to a beagle, from which it was propagated in primary canine monocytes. The organism was then grown in vitro in a continuous canine cell line, DH82. Nine beagles subsequently injected with whole E. canis-infected blood all developed typical symptoms of ehrlichiosis. An indirect immunofluorescence antibody test to E. canis was developed and compared with a commercial kit, revealing a good correlation between the two assays. Transmission electron microscopy of DH82 cells infected with the Israeli strain of E. canis (611), revealed organisms similar to those described in the literature: two different forms of morulae appeared, one tightly, the other loosely, packed. The 16S rRNA gene sequence obtained from the Israeli Ehrlichia isolate was compared with other isolates, E. canis Oklahoma and E. canis Florida. The Israeli strain 16S rRNA had three nucleotide differences from the Oklahoma isolate, and four nucleotide differences from the Florida isolate, in addition to one nucleotide gap in each. The Israeli isolate was found to be 0.54% different from the Oklahoma strain, and 0.61% different from the Florida strain. There are the same magnitudes of differences displayed by the other most closely related group in the phylogenetic tree, namely Ehrlichia equi, Ehrlichia phagocytophilia and the human granulocytic ehrlichia.

Natural coinfection of a white-tailed deer (Odocoileus virginianus) population with three Ehrlichia spp.

The ticks Amblyomma americanum and Ixodes scapularis, strongly implicated vectors of Ehrlichia chaffeensis and the human granulocytic ehrlichiosis (HGE) agent, respectively, commonly are found on white-tailed deer (Odocoileus virginianus). As deer can be infected with E. chaffeensis, the HGE agent, and another Ehrlichia-like organism, a deer population parasitized by both tick species in coastal Georgia was tested for evidence of Ehrlichia spp. infection using serologic, molecular, and culture techniques. Antibodies to both E. chaffeensis (geometric mean titer = 111) and Ehrlichia equi, surrogate antigen for the HGE agent, (geometric mean titer = 1,024) were detected by indirect fluorescent antibody testing. Nested polymerase chain reaction employing species-specific primers demonstrated sequence-confirmed 16S rDNA fragments of 3 distinct Ehrlichia spp. in this population: E. chaffeensis (1/5), the HGE agent (3/5), and an Ehrlichia-like organism previously described from white-tailed deer (5/5). Ehrlichia chaffeensis was isolated in culture from the inguinal lymph node of a single deer. An Ehrlichia-type morula was identified in a neutrophil of 1 deer on examination of blood smears. This work provides the first evidence of the HGE agent in a nonhuman host in the southeastern United States and documents infection with both E. chaffeensis and the HGE agent in a single deer population, thereby supporting the importance of white-tailed deer in the natural history of the human ehrlichioses agents.

Natural history of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in the piedmont physiographic province of Georgia.

The roles of wild mammals and ticks in the epidemiology of Ehrlichia chaffeensis at a suspected endemic site were investigated using serologic testing, culture, and polymerase chain reaction (PCR) supported by restriction endonuclease analysis and DNA sequencing. Antibodies reactive to E. chaffeensis (> or = 1:64) were detected in 92% of white-tailed deer (Odocoileus virginianus), 21% of raccoons (Procyon lotor), and 8% of opossums (Didelphis virginianus), but not in 8 other species of mammals. Of 7 species of ticks found by host and environmental sampling, Amblyomma americanum was the dominant species, accounting for greater than 99% of all ticks collected. Deer, raccoons, and opossums were the only species parasitized by all life stages of A. americanum, and A. americanum was the only tick parasitizing deer. A nested PCR protocol incorporating E. chaffeensis-specific primers detected E. chaffeensis DNA in blood, lymph nodes, or spleen from 54% of deer examined. The nested PCR detected E. chaffeensis DNA in 6 of 50 (12%) individual adult A. americanum collected from the environment, in 14 of 79 (18%) pools representing 402 adult A. americanum collected from the environment, and in 7 of 25 (28%) pools of mixed stages of A. americanum collected from deer. Although no Ehrlichia spp. were isolated in culture, sequencing of representative amplicons from deer and ticks confirmed PCR products as E. chaffeensis. These data provide strong evidence that white-tailed deer and lone star ticks are the primary reservoir and vector of E. chaffeensis, respectively. The same PCR protocol, incorporating primers specific for an Ehrlichia-like organism of white-tailed deer, detected this organism in blood, lymph nodes, or spleen from 96% of these deer. The Ehrlichia-like organism of deer was detected by PCR from 0 of 50 individual ticks, 7 of 79 (9%) pools, and 1 of 25 (4%) pools of A. americanum collected from deer. Sequencing of representative amplicons from deer and ticks confirmed PCR products as Ehrlichia-like organism of deer. These data suggest that the Ehrlichia-like organism of deer is present in both the deer and lone star ticks populations at this location.

Ehrlichia-like 16S rDNA sequence from wild white-tailed deer (Odocoileus virginianus).

The reservoir hosts of Ehrlichia chaffeensis, etiologic agent of human ehrlichiosis are unknown. Initially, white-tailed deer (WTD) were serologically implicated as possible reservoirs of E. chaffeensis. Subsequent studies showed that WTD were susceptible to infection with E. chaffeensis and that deer-to-deer transmission by a tick vector, Amblyomma americanum, is possible under experimental conditions. To determine if wild WTD were infected with E. chaffeensis, whole blood was collected from 10 deer from Oklahoma and Georgia. All 10 deer had antibodies reactive to E. chaffeensis. Whereas E. chaffeensis was not isolated, restriction enzyme mapping and sequencing of the 16S rDNA gene revealed that a unique Ehrlichia-like agent was present. All 10 deer appeared to be infected with the same agent. We suspect that A. americanum is the vector of this new agent based upon the previously published temporal association between the appearance of E. chaffeensis seropositive WTD and A. americanum. However, the taxonomic and antigenic relationships, geographic distribution, epidemiology, and zoonotic potential of this agent are yet to be determined.