Jacqueline E. Dawson

Human Ehrlichiosis in the United States, 1985 to 1990

In 1986, a new disease was recognized in the United States [1]. Two lines of evidence suggested that the disease was caused by a member of the genus Ehrlichia: Inclusions resembling organisms of the genus were found in a patients leukocytes and serologic evidence of a recent infection with an Ehrlichia organism was found in this patients serum and in sera from other patients with similar illness [1-5]. Most cases were found in the south central or southeastern United States, and casecontrol studies confirmed the association of the disease with a history of tick exposure [4, 6-8]. Fever, headache, myalgia, and nausea or vomiting were common but nonspecific clinical findings [4, 6, 7]. These same studies suggested that, in addition to recent tick exposure, a hallmark of the disease was leukopenia [4, 6, 7]. Thrombocytopenia and abnormal liver function test results, particularly increased hepatic transaminase levels, have also been noted [1-5]. The spectrum of human infection ranged from asymptomatic to severe, sometimes fatal illness [8, 9]. Limited data suggest that tetracycline may decrease the duration of fever; the efficacy of chloramphenicol is controversial [4, 8, 10]. Subsequently, a new species of Ehrlichia, E. chaffeensis, was isolated from a patient suspected of having ehrlichiosis [11]. This agent was confirmed as the probable etiologic agent of human ehrlichiosis by complete sequencing of the polymerase chain reaction-amplified 16S rRNA gene of the isolate and 16S rRNA in blood specimens from two other serologically confirmed patients [12]. Sequence comparisons indicated that E. chaffeensis was closely related to E. canis [12]. Patients who had serologic evidence of a recent infection with E. chaffeensis had similar titers in serologic tests using E. canis [11]. The relatively small number of patients (40 patients or fewer) in previous studies of human ehrlichiosis prevented the analysis of several potential risk factors, particularly those that affect the course of the illness [4, 6-8]. We describe the epidemiologic, clinical, and laboratory features and response to treatment of 237 serologically confirmed cases of human ehrlichiosis identified in laboratory-based surveillance systems from 1985 through 1990 [8]. Methods Since the recognition of human ehrlichiosis in 1986, the Centers for Disease Control and Prevention (CDC) has offered serologic testing for the disease. In 1988, CDC and several state health departments stimulated surveillance by publishing reports in their bulletins and sending information to physicians asking them to collect serum samples from potential case-patients. In addition, several large private laboratories that routinely did serologic testing for Rocky Mountain spotted fever were asked to send all acute and convalescent serum pairs submitted for Rocky Mountain spotted fever to the CDC [8]. Serum samples from patients suspected of having Rocky Mountain spotted fever or other rickettsial diseases were tested for antibodies to E. canis. About 50% of the patients in our series were previously reported [1, 2, 4-8]. Initially, the indirect fluorescence antibody test for antibodies to E. canis in canine serum was adapted for human serum [2]. Ehrlichia canis was used as the antigen for routine diagnostic tests until 1990, when the putative causative agent of human ehrlichiosis, E. chaffeensis, was isolated. The techniques used by the CDC and the Oklahoma State Department of Health are described elsewhere [13]. All specimens sent to the CDC were also tested for antibodies to Rickettsia rickettsii, R. typhi, and Coxiella burnetii and those sent to the Oklahoma State Department of Health were tested for R. rickettsii. The case definition for ehrlichiosis required a minimum antibody titer of 64, in addition to a fourfold change in antibody titer to E. canis or E. chaffeensis from the acute to the convalescent phase. Patients with ehrlichiosis were interviewed with a standardized questionnaire; information about presumptive clinical diagnosis, clinical and serial laboratory findings, and treatment was abstracted from medical records. When a response to a specific question was not available, that patient was excluded from analysis for that specific variable. Because almost all case-patients had laboratory tests done at different laboratories and normal values for many of these laboratories were not available, published normal laboratory values were used to determine abnormal values. Age- and state-specific rates for ehrlichiosis were calculated using mean population estimates for the 5-year period from 1985 to 1990. The relative clinical effectiveness of various antibiotics was evaluated by classifying the case-patients who were hospitalized according to the type of antibiotic they received and by analyzing the time (days) from when the antibiotic was first administered to the first day of defervescence (temperature, 37 C for at least 24 hours), the total duration of hospitalization, and the time until complete recovery (return to normal activities). In univariate analyses, only case-patients treated with tetracycline, chloramphenicol, or another antibiotic (alone) for at least 48 hours were included in the latter analysis. Patients whose eventual treatment included tetracycline or chloramphenicol and another antibiotic were classified on the basis of their initial treatment as long as they were initially treated with either tetracycline, chloramphenicol, or another antibiotic alone for at least 48 hours. The epidemiologic and clinical characteristics of case-patients whose initial treatment consisted of tetracycline were compared with those treated with chloramphenicol and with those treated with one or more other antibiotics (without tetracycline or chloramphenicol). To study possible risk factors for illness severity, we divided case-patients into the following groups: those who did not require hospitalization, those who were hospitalized but did not develop complications, those who were hospitalized and had one or more serious complications, and those who died. The Wilcoxon rank-sum test was used to compare group medians of nonparametrically distributed data, and the Fisher exact test was used to compare differences in proportions when variables were dichotomous. A multiple logistic regression was done using PC-SAS SAS Institute Inc., Cary, North Carolina [14]. Variables that were biologically plausible and were associated with the outcome (complications or death) that had a P value 0.20 were allowed to enter into the model. These variables were as follows: the time (in 4-day intervals) between the onset of illness and the day of their illness that they initially saw a physician, age group (<50 years, 50 to 59, >60 years of age, and whether treatment with tetracycline or chloramphenicol was begun during the first 3 days of illness, between days 4 to 7, or after day 7 of illness. Because treatment with antibiotics other than tetracycline or chloramphenicol was associated with an unfavorable outcome, a variable was created (classifying patients by whether they were treated with these other antibiotics [within the first 3 days of illness, between days 4 to 7, or after day 7]) to control for confounding. A backward method of elimination was used. Results Epidemiologic Findings Between 1985 and 1990, paired serum specimens from 237 patients met the serologic case definition. One case-patient was included on the basis of a serologic response to E. chaffeensis; the other 236 had a fourfold increase or decrease to E. canis. Six of these patients were subsequently tested for E. chaffeensis and all had an increasing or decreasing titer to this antigen. Diagnoses were made for a median of 41 and a mean of 46.4 case-patients each year (excluding the 4 diagnoses made retrospectively from sera collected in 1985). Most (68.2%) case-patients developed their first symptoms in May, June, or July; 93.5% had onset in the 6-month period between April and September. Patients were exposed to ticks and resided in 139 counties in 21 states. The state with the most cases was Oklahoma (57 patients; rate, 0.36 per 100 000 population per year); other states with many cases included Missouri (47 patients; 0.18 per 100 000) and Arkansas (18 patients; 0.15 per 100 000). The county with the most cases was Cherokee County, Oklahoma (10 patients; 6.25 per 100 000). Other counties where 4 or more case-patients resided or reported tick exposure included Muskogee, Oklahoma (6 patients; 1.75 per 100 000); Crawford, Arkansas (4 patients; 1.94 per 100 000); Sebastian, Arkansas (4 patients; 0.81 per 100 000); and Glynn, Georgia (4 patients; 1.31 per 100 000). Information about location of residence was available for 175 case-patients; these residences were described as rural for 116 (66.3%), suburban for 46 (26.3%), and urban for 13 (7.4%). The median age of case-patients was 44 years; most (74.5%) were men. Ehrlichiosis was particularly common in the 60-years-and-older age group (26.6% of total). Age-specific incidence rates increased steadily with increasing age and were highest for persons 60 to 69 years old (Figure 1). The overall sex-specific rate for men (27 per 100 000 000) exceeded that for women (9 per 100 000 000), and rates for men exceeded those for women in all age groups. Figure 1. Age- and sex-specific rates of human ehrlichiosis in the United States from 1986 to 1990. Tick bites or attachments were reported by 67.9% of the case-patients; another 15.5% reported being in a tick-infested area but did not recall a tick bite or attachment. A single exposure date was reported by 47.6% of the patients; among these persons, the median incubation period was 9 days (Table 1). Table 1. Numbers of Patients Affected and Time Interval for Onset of Symptoms, Admission to Hospital, and Hospital Discharge Clinical Findings Medical care was first sought a median of 4 days (range, 0 to 35 days) after the onset of symptoms.

Ehrlichiosis in a Golf-Oriented Retirement Community

BACKGROUND Ehrlichiosis due to Ehrlichia chaffeensis usually occurs sporadically or in small clusters, with an annual incidence estimated at 3 to 5 cases per 100,000 population in areas of endemic disease. The putative principal vector is the Lone Star tick (Amblyomma americanum). We investigated an outbreak of ehrlichiosis that occurred in June 1993 among members of a golf-oriented retirement community (community A) in Tennessee. The community is densely wooded and borders a wildlife-management area where deer are numerous. METHODS We conducted a case-control study, using medical-history reviews, serologic testing, and testing with the polymerase chain reaction for E. chaffeensis infection. We also surveyed a sample of 10 percent of the households in community A and in another golf-oriented community (community B) more than 20 miles (32 km) from the wildlife-management area. Survey participants completed a questionnaire and provided specimens for serologic testing. In both communities, searches for ticks were undertaken. RESULTS Eleven cases of symptomatic ehrlichiosis were identified in the case-control study, 10 of which were in community A (attack rate, 330 per 100,000). Of 311 surveyed residents of community A, 12.5 percent had serologic evidence of past E. chaffeensis infection, as compared with 3.3 percent of 92 in community B (relative risk in community A as compared with community B, 3.9; 95 percent confidence interval, 1.2 to 12.2). The risk of infection was associated with tick bites, exposure to wildlife, golfing, and among golfers, retrieving lost golf balls from the rough. Persons who never used insect repellent were more likely to have had infection than persons who did. In community A, thousands of Lone Star ticks were found; in community B, only three ticks were found. CONCLUSIONS The high rate of E. chaffeensis infection in community A resulted from its proximity to a wildlife reserve. When outdoor recreational activities are common and concentrations of ticks are high, outbreaks of arthropod-borne zoonoses can be anticipated.

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.

WHITE-TAILED DEER AS A POTENTIAL RESERVOIR OF EHRLICHIA SPP.

We determined the antibody prevalence to Ehrlichia spp., in white-tailed deer (Odocoileus virginianus) and the geographic distribution of seropositive animals in 84 counties in Alabama, Arkansas, Florida, Georgia, Illinois, Kentucky, Louisiana, Maryland, Massachusetts, Mississippi, Missouri, North Carolina, South Carolina, Tennessee, Texas, Virginia, and West Virginia (USA). Using an indirect fluorescent antibody test we detected antibodies (≥ 1:128) to this bacterium in 544 (43%) of 1269 deer. Presence of antibodies to Ehrlichia spp. was related to a southerly latitude, low elevation, and resulting milder climatic conditions. It appears that white-tailed deer were naturally infected with Ehrlichia spp.; the infection was widely distributed throughout the southeastern United States. Based on these data, we propose that white-tailed deer play a role in the natural history of Ehrlichia spp. infection in the United States.

Immunohistochemical and In Situ Hybridization Studies of Influenza A Virus Infection in Human Lungs

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.

Human Granulocytic Ehrlichiosis in Massachusetts

Two new tick-borne zoonoses have recently emerged as threats to public health in North America. Both are caused by infection with Ehrlichia species, obligate intracellular bacteria that localize within the phagosomes of leukocytes. The spectrum of illness varies from mild to severe; about one third of patients require hospitalization. Persons exposed to ticks may present with a spotless spotted fever or a disease similar to Lyme disease that does not feature erythema and is accompanied by fever, chills, severe headache, myalgia, malaise, and nausea. A maculopapular or petechial rash, however, may be present [1, 2]. Laboratory findings include thrombocytopenia, leukopenia, and abnormal hepatic function test results. Treatment with tetracyclines induces defervescence within 48 hours. Ehrlichia first attracted attention in 1935 as pathogens of dogs [3] and shortly thereafter as potential zoonotic agents. An infection similar to mononucleosis was reported in western Japan [4], and the ehrlichial cause of Sennetsu fever was subsequently confirmed by subinoculating mice with patient blood and observing the characteristic inclusions (morulae) within mononuclear cells. The index case of human ehrlichiosis in North America was discovered in a tick-exposed Arkansas resident in 1986 [5]. Morulae were seen within the patients monocytes, and his serum reacted against antigens of E. canis, the agent of tropical canine pancytopenia. In 1990, a new species, closely related to E. canis, was cultivated from a febrile Arkansas patient and designated E. chaffeensis [6, 7]. About 400 illnesses caused by this agent have since been confirmed in 30 states. Nine of these cases were fatal. Since 1990, morulae have been discovered in the granulocytes of 12 febrile patients from northern Minnesota and Wisconsin [8]. The serum of these patients did not react with E. chaffeensis antigen. In addition, an analysis of sequences obtained from a polymerase chain reaction (PCR) assay using universal eubacterial 16S ribosomal DNA (rDNA) primers indicated that the pathogen was closely related to, if not conspecific with, E. phagocytophila and E. equi [9], agents of ovine tick-borne fever and equine ehrlichiosis, respectively. Two of the 12 reported cases were fatal. This infection has been designated human granulocytic ehrlichiosis because morulae are restricted to polymorphonuclear leukocytes. The public health importance, especially the geographic distribution, of human granulocytic ehrlichiosis remains undescribed because of the novelty of the disease and because confirmatory diagnostic tests are not widely available. We describe the definitive identification of this ehrlichia organism from a patient in New England, a discovery that suggests that human granulocytic ehrlichiosis may infect residents of certain sites outside the upper midwestern United States. Case Report A 68-year-old woman presented to the Nantucket Cottage Hospital in Massachusetts on 1 November 1994 with a 5-day history of fever, confusion, headache, and fatigue. She had been in her usual state of good general health and had no significant medical history other than a previous tick bite in June 1994. She took no medications routinely and had no allergies. The patient had removed an unidentified tick from her left flank approximately 5 days before symptoms developed. The tick had not been saved. She had noted no rashes or other signs or symptoms after the tick bite. At presentation, the patient appeared flushed and was somewhat confused as to the time and place, but was otherwise alert and neurologically intact. Her oral temperature was 40 C, her pulse rate was 88 beats/min, and her blood pressure was 110/60 mm Hg. Physical examination showed no evidence of rash or cellulitis at the site of the tick bite and no other remarkable findings. No organomegaly was noted. An automated complete blood count showed an abnormal leukocyte count (3200 109/L) and platelet count (40 000 109/L). A manual differential count showed 32% segmented neutrophils, 27% bands, 34% lymphocytes, 6% monocytes, and 1% metamyelocytes. The hematocrit was 0.38. A chemical analysis of the blood showed low potassium and calcium levels and slightly elevated urea nitrogen levels (22 mg/dL). Liver enzyme levels were elevated: alanine aminotransferase level, 0.07 kat/L; aspartate aminotransferase level, 0.12 kat/L; and lactate dehydrogenase level, 7.80 kat/L. Infection with Babesia microti was suspected, and a Giemsa-stained thin peripheral blood smear was carefully scrutinized. No intraerythrocytic organisms were noted, but an inclusion was noted in a band neutrophil. The patient was suspected of having Lyme disease or ehrlichiosis, and doxycycline (100 mg orally twice a day for 10 days) was started pending confirmatory analysis at the Harvard School of Public Health. The patient defervesced within 24 hours and had an uneventful recovery. An anticoagulated venous blood sample (ethylenediaminetetracetate, 10 mM) obtained at presentation and kept refrigerated for 2 days was examined for evidence of ehrlichiae. Buffy-coat smears stained with the Wolbach Giemsa variant for rickettsiae [10] showed rare inclusions (12 on 5 slides) that were morphologically consistent with ehrlichial morulae within bands and metamyelocytes (Figure 1). The patients serum contained borderline IgM (titer, 1:64) against E. chaffeensis antigen by the indirect fluorescent antibody test [6] but did not contain IgG. No reactivity was noted to antigens of Babesia microti (indirect fluorescent antibody test [11]) or Borrelia burgdorferi (immunoblot [12]). Figure 1. Morula (arrow) within a metamyelocyte from the patients blood (buffy-coat smear, Wolbach Giemsa stain; original magnification, 1250). In a room in which intact Ehrlichia species or their DNA had never been manipulated, an aliquot of the anticoagulated blood was pipetted into a sterile cryotube using a barrier-filtered micropipettor. This sample was sent to the Centers for Disease Control and Prevention for analysis by PCR and automated cycle sequencing. By using a modification of the previously described nested PCR procedure with ehrlichia-specific primers [13] for the 16S rRNA gene, an 1158-base pair amplification product was obtained and sequenced. The sequence of this amplification product (GenBank accession number U23038) did not differ from the sequence of the 16S rRNA gene amplified from the persons in Minnesota and Wisconsin who had diagnoses of human granulocytic ehrlichiosis [9]. Convalescent serum samples obtained on 17 November 1994 and 10 January 1995 continued to show borderline IgM reactivity to E. chaffeensis antigen. Specific IgG to E. chaffeensis was first detected in the 10 January sample (titer, 1:128). Immunoglobulin G reactivity to antigen of E. equi, which has been used as a surrogate for the agent of human granulocytic ehrlichiosis, was not seen in the serum sample obtained at the original presentation (1 November). However, IgG reactivity was present at a titer of 1:64 in the 17 November sample and at a titer of 1:256 in the 10 January sample. No reactivity to antigens of Rickettsia rickettsii, R. typhi, or Coxiella burnetii was seen in the acute and convalescent serum samples (indirect fluorescent antibody test). Seroconversion for Lyme disease or babesiosis was not observed. Discussion An elderly tick-exposed patient from Nantucket, Massachusetts, presenting with a febrile illness and nonspecific symptoms, was initially suspected of having Lyme disease or babesiosis. Laboratory test results indicated both thrombocytopenia and leukopenia, which we use as markers for B. microti infection (unpublished data), and a Giemsa-stained thin blood smear was carefully scrutinized. A suspicious inclusion detected within a granulocyte led to a careful search for ehrlichiae, and classic morulae were detected by examining buffy-coat smears. Ehrlichial DNA was amplified and sequenced, and the identity of the agent was confirmed by comparing this sequence with those amplified from patients from the upper midwestern United States who were accessioned in GenBank. Our report describes the first definitive identification of this pathogen in a New England resident. A case of human ehrlichiosis was reported from nearby Cape Cod in 1992 [14]; in retrospect, this case may be considered to have been caused by the agent of human granulocytic ehrlichiosis [15]. Serum from that patient was reported to strongly react (IgG titer >1:512) to antigens of E. chaffeensis. In contrast, serum samples from the original Minnesota and Wisconsin cases of human granulocytic ehrlichiosis have no reported reactivity with E. chaffeensis [8]. However, convalescent serum samples from several cases of human granulocytic ehrlichiosis that have recently been diagnosed and confirmed by molecular analysis in Minnesota apparently react with antigens of E. chaffeensis (Persing DH, Mayo Foundation. Personal communication). The degree of cross-reactivity between E. chaffeensis and the agent of human granulocytic ehrlichiosis may eventually be described as more cases of human granulocytic ehrlichiosis are analyzed. However, the presence of granulocytic morulae and the fact that the agent has the same molecular identity as the pathogen identified in the upper midwestern United States establish the diagnosis of human granulocytic ehrlichiosis in our patient. Whether the agent constitutes an antigenic variant remains to be determined. The agent of human granulocytic ehrlichiosis may frequently infect residents of sites where deer ticks are abundant but may be diagnosed as a rashless Lyme disease. Human granulocytic ehrlichiosis may be successfully treated with tetracyclines, and a response to treatment might affirm a presumptive diagnosis of Lyme disease. In 1991, we found that 11% of serum samples obtained from residents of a well-studied Lyme-disease focus in Cape Cod reacted with antigen of E. chaffeensis [16], and

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.

TEMPORAL ASSOCIATION OF AMBLYOMMA AMERICANUM WITH THE PRESENCE OF EHRLICHIA CHAFFEENSIS REACTIVE ANTIBODIES IN WHITE-TAILED DEER

From 1981 through 1993, tick infestations and serum antibodies reactive to Ehrlichia chaffeensis, the causative agent of human ehrlichiosis, were monitored among white-tailed deer (Odocoileus virginianus) at Whitehall Experimental Forest, Clarke County, Georgia (USA). Neither ticks nor E. chaffeensis antibodies were detected during the first two years of the study. Infestations of the lone star tick (Amblyomma americanum), a suspected vector of E. chaffeensis, first were noted on deer in 1983. Prevalence and intensity of A. americanum sharply increased from 1985 to 1989, and prevalence was 100% from 1990 to 1993. Antibodies reactive to E. chaffeensis were first detected in 7% of deer sampled in 1986. Antibody prevalence increased to 21% in 1987 and was 100% from 1988 to 1993. This temporal association between the establishment of A. americanum and the appearance of E. chaffeensis antibodies provides evidence to support the concept that A. americanum could be a natural vector of E. chaffeensis. The high prevalence of antibodies among all age classes of deer also reaffirms that white-tailed deer may be sensitive natural sentinels for monitoring the distribution of E. chaffeensis.

Persistent Ehrlichia chaffeensis infection in white-tailed deer.

Four white-tailed deer (Odocoileus virginianus) were inoculated intravenously with a deer-origin isolate (15B-WTD-GA) of Ehrlichia chaffeensis. The course of infection was monitored using indirect fluorescent antibody (IFA), polymerase chain reaction (PCR), and culture over a 9 m period. All deer became rickettsemic within 24 days post inoculation (DPI), and all developed antibody titers >1:64 to E. chaffeensis by 17 DPI. Titers in all deer fell below 1:64 during 87 to 143 DPI. One deer exhibited a second period of seropositivity (peak titer of 1:256) from 207 to 271 DPI but was culture and PCR negative during this period. Rickettsemia was confirmed by reisolation of E. chaffeensis as late as 73 to 108 DPI in three deer. Positive PCR results were obtained from femur bone marrow of one deer and from rumenal lymph node of another deer at 278 DPI. None of the deer developed clinical signs, hematologic abnormalities, or gross or microscopic lesions attributable to E. chaffeensis. Two uninoculated control deer were negative on all tests through 90 DPI at which time they were removed from the study. Herein we confirm that white-tailed deer become persistently infected with E. chaffeensis, have initial rickettsemias of several weeks duration and may experience recrudescence of rickettsemia, which reaffirm the importance of deer in the epidemiology of E. chaffeensis.

Intestinal intussusception associated with adenovirus infection in Mexican children

Formalin-fixed intestinal tissue specimens from 12 Mexican pediatric patients with intussusception were examined for the presence of adenovirus. Four patients (33%) had detectable adenovirus antigen in epithelial cells as determined by using immunohistochemical analysis. Two of the patients with positive immunohistochemical results had antigens in dendritic and mononuclear inflammatory cells, and 3 patients had positive results for species C adenovirus by in situ hybridization using adenovirus species-specific probes (A-F). A real-time polymerase chain reaction assay specific for species C (nonenteric) adenoviruses was used to confirm immunohistochemical results and to amplify adenovirus DNA for sequencing. A sequence similar to that for adenovirus serotype 1 was found in 1 patient, serotype 2 in another, and serotype 6 in a third; in the fourth patient, the sequence was indeterminate between serotypes 2 and 6. The assays used in this study proved useful for the identification of species C adenoviruses in formalin-fixed specimens from Mexican pediatric patients with intussusception.