Gravity Probe B: final results of a space experiment to test general relativity.

Gravity Probe B, launched 20 April 2004, is a space experiment testing two fundamental predictions of Einstein's theory of general relativity (GR), the geodetic and frame-dragging effects, by means of cryogenic gyroscopes in Earth orbit. Data collection started 28 August 2004 and ended 14 August 2005. Analysis of the data from all four gyroscopes results in a geodetic drift rate of -6601.8±18.3 mas/yr and a frame-dragging drift rate of -37.2±7.2 mas/yr, to be compared with the GR predictions of -6606.1 mas/yr and -39.2 mas/yr, respectively ("mas" is milliarcsecond; 1 mas=4.848×10(-9) rad).

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 (leer 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.

Susceptibility of red and gray foxes to infection by Ehrlichia chaffeensis.

Red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) were evaluated for their susceptibility to experimental infection with Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis. Two red foxes and three gray foxes were inoculated intravenously with E. chaffeensis (15B-WTD-GA strain) and were monitored at 7, 14, 21, and 28 days post inoculation (DPI) for evidence of infection using an indirect fluorescent antibody (IFA) assay, light microscopy, polymerase chain reaction (PCR), and cell culture methods. One red fox and one gray fox served as negative controls. Red foxes were susceptible to infection based on reisolation of E. chaffeensis from blood at 7 and 14 DPI, seroconversion by 7 DPI, and positive PCR assays on spleen and lymph nodes at 28 DPI. Morulae were not found in circulating leukocytes and clinical signs or lesions of ehrlichiosis were not observed. In contrast, gray foxes were refractory to infection based on negative results on all culture, PCR, serologic, and microscopic examinations. These findings imply that red foxes, but not gray foxes, are potential vertebrate reservoirs for E. chaffeensis. These findings also illustrate the need to verify serologic evidence of E. chaffeensis infection among wild animals.

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.

Evaluation of C3H/HeJ mice for xenodiagnosis of infection with Ehrlichia chaffeensis.

Because mice are experimentally susceptible to infection with Ehrlichia species, C3H/HeJ mice were evaluated as a potential xenodiagnostic model for detection of infection with and isolation of E. chaffeensis. Intraperitoneal inoculation of mice with E. chaffeensis-infected DH82 cell cultures produced seroconversion, with peak serum antibody titers of 1:256, at high dosages (>1.9 x 10(4) infected cells) but not at low dosages (1.9 or 1.9 x 10(2) infected cells). Ehrlichia chaffeensis was not reisolated from blood samples collected from inoculated mice on postinoculation day 21. Nested polymerase chain reaction (PCR), using primers specific for E. chaffeensis, was positive for only 2/70 (2.9%) tissue samples. A field evaluation in which C3H/HeJ mice were inoculated with blood and lymph node suspensions from 5 seropositive white-tailed deer, including 3 deer that were PCR positive for E. chaffeensis, failed to produce seroconversion in mice. The lack of seroconversion at low dosages, the failure to reisolate at any dosage, and the inability to confirm infection in PCR-positive field samples suggests C3H/HeJ mice are not a sensitive model for xenodiagnosis or detection of E. chaffeensis.

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.

Lack of seroreactivity to Ehrlichia chaffeensis among rodent populations.

A retrospective serosurvey for antibodies to Ehrlichia chaffeensis was conducted on eight species of wild rodents (Mus musculus, Oryzomys palustris, Peromyscus leucopus, Rattus norvegicus, Reithrodontomys humulis, Sciurus carolinensis, Sciurus niger, and Sigmodon hispidus) from the southeastern United States. Serum samples (n = 281) collected between 1973 and 1993 were evaluated using an indirect fluorescent antibody test. All samples, screened at a dilution of 1:32, were negative for antibodies to E. chaffeensis. Sixty-three percent of the rodents tested were from areas where E. chaffeensis has been confirmed or is strongly suspected to be endemic. These data suggest limited or no involvement of rodents in the epidemiology of E. chaffeensis.

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.

Isolation of Ehrlichia chaffeensis from wild white-tailed deer (Odocoileus virginianus) confirms their role as natural reservoir hosts.

Field and experimental studies have implicated white-tailed deer (Odocoileus virginianus) as probable reservoir hosts for Ehrlichia chaffeensis, the causative agent of human monocytic ehrlichiosis, but natural infection in deer has not been confirmed through isolation of E. chaffeensis. Thirty-five white-tailed deer collected from three Amblyomma americanum-infested populations in Georgia were examined for evidence of E. chaffeensis infection by serologic, molecular, cell culture, and xenodiagnostic methods. Twenty-seven deer (77%) had E. chaffeensis-reactive indirect fluorescent-antibody assay titers of > or = 1:64; and the blood, spleens, or lymph nodes of seven (20%) deer were positive in a nested PCR assay with E. chaffeensis-specific primers. E. chaffeensis was isolated in DH82 cell cultures from the blood of five (14%) deer, including two deer that were PCR negative. Combination of culture and PCR results indicated that six (17%) deer were probably rickettsemic and that nine (26%) were probably infected. Restriction digestion of PCR products amplified from deer tissues and cell culture isolates resulted in a banding pattern consistent with the E. chaffeensis 16S rRNA gene sequence. The sequences of all PCR products from deer tissues or cell culture isolates were identical to the sequence of the Arkansas type strain of E. chaffeensis. Xenodiagnosis with C3H mice inoculated intraperitoneally with deer blood, spleen, or lymph node suspensions was unsuccessful. When viewed in the context of previous studies, these findings provide strong evidence that E. chaffeensis is maintained in nature primarily by a tick vector-vertebrate reservoir system consisting of lone star ticks and white-tailed deer.

Development and use of specific polymerase reaction for the detection of an organism resembling Ehrlichia sp. in white-tailed deer.

The role of white-tailed deer (Odocoileus virginianus) in the epidemiology of Ehrlichia chaffeensis and the agent of human granulocytic ehrlichiosis (HGE) is not fully understood, and diagnostic procedures may be complicated by the recent detection of 16S rDNA sequence from an Ehrlichia sp.-like organism in wild deer. A specific forward primer (DGA) and an Ehrlichia spp. reverse primer (GA1UR) were constructed to amplify this new, distinct Ehrlichia sp.-like 16S rDNA. The DGA primer, a forward primer specific for E. chaffeensis (DCH), and a forward primer specific for the E. phagocytophila genogroup (GE9f) were each used with GA1UR in nested polymerase chain reactions to amplify 16S rDNA sequences from control samples containing the deer Ehrlichia sp.-like organism, E. chaffeensis, or the HGE agent. Primer pairs DGA/GA1UR and DCH/GA1UR specifically amplified 16S rDNA sequences from the corresponding target organism, whereas GE9f/GA1UR amplified 16S rDNA sequence from both the HGE agent and the deer Ehrlichia sp.-like organism. With a nested PCR using DGA/GA1UR and DCH/GA1IUR on DNA extracted from white blood cells from 62 deer from 10 populations in four U.S. states, we observed a high prevalence (65%) of 16S rDNA sequences of the deer Ehrlichia sp.-like organism, and a low prevalence (5%) of the E. chaffeensis sequence. In this field survey, E. chaffeensis-reactive antibodies detected by indirect fluorescence assays were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism, but not E. chaffeensis. Infestations of Amblyomma americanum also were associated (P < 0.001) with PCR evidence of the deer Ehrlichia sp.-like organism. The potential for serologic cross-reactions and non-specific PCR products arising from the deer Ehrlichia sp.-like organism should be considered when evaluating the role of deer and their ticks in the epidemiology of ehrlichial pathogens of humans.

A recombinant antigen from the heartwater agent (Cowdria ruminatium) reactive with antibodies in some southeastern United States white-tailed deer (Odocoileus virginianus), but not cattle, sera.

Recombinant baculovirus techniques were used to express the 260 amino acid carboxyterminal portion of the 32 kilodalton (kDa) major antigenic protein (MAP 1) of Cowdria ruminantium, the heartwater agent, as a fusion protein. The recombinant MAP 1 was fused to an aminoterminal independently antigenic octapeptide sequence (FLAG peptide). Recombinant MAP 1 was used as an immunoblotting antigen to evaluate numerous reference antisera against organisms of the tribe Ehrlichieae. Monoclonal and polyclonal C. ruminantium antibodies, monoclonal anti-FLAG ascites, and antisera to Ehrlichia canis and Ehrlichia chaffeensis reacted with this antigen. Twelve of 79 sera collected 1980 to 1992 from southeastern U.S. white-tailed deer (Odocoileus virginianus) were also unexpectedly immunoblot-positive to MAP 1. These 12 deer sera had, as a group, significantly (P < 0.01) greater anti-E. chaffeensis titers (previously determined) than the sera from MAP 1 immunoblot-negative deer living in the same areas. None of the 262 sera from cattle living in the same areas were immunoblot-positive to MAP 1. All of an additional 50 cervine sera from Michigan (USA), 72 bovine sera from northern U.S. cattle, and 72 sera from Puerto Rican cattle were also immunoblot-negative to MAP 1. Sera from African sheep which were falsely seropositive to authentic MAP 1 were also immunoblot-positive to the recombinant MAP 1. Unidentified Ehrlichia spp. capable of serologic crossreactivity with the heartwater agent appear to be present in some southeastern U.S. white-tailed deer but not cattle. These or related Ehrlichia spp. may also be found elsewhere in the world in non-cervine species.

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.

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.

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.

Susceptibility of white-tailed deer (Odocoileus virginianus) to infection with Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis.

Although more than 320 cases of human ehrlichiosis have been diagnosed in 27 states since 1986, the reservoir host or hosts remain unknown. Since antibodies reactive to Ehrlichia chaffeensis, the etiologic agent of human ehrlichiosis, have been found in white-tailed deer (Odocoileus virginianus), we experimentally evaluated the susceptibilities of four white-tailed deer to infection with E. chaffeensis and Ehrlichia canis, a closely related species. A fifth deer served as a negative control. Isolation and nested PCR amplification results from peripheral blood indicated that E. chaffeensis circulated for at least 2 weeks. The deer developed antibodies to E. chaffeensis by day 10 after inoculation, but there was no indication of clinical disease. Immunohistochemical staining identified E. chaffeensis within macrophage-type cells in lymph nodes. The deer inoculated with E. canis did not become infected and did not seroconvert. These results indicate that white-tailed deer can support an E. chaffeensis infection with resulting rickettsemia of at least 2 weeks. The resistance to infection and the absence of seroconversion upon exposure to E. canis indicate that antibody responses previously detected among wild deer are not E. canis cross-reactions. The role of deer as competent reservoirs in the life cycle of E. chaffeensis remains to be explored with suspected tick vectors.

Severe moniliformiasis (Acanthocephala: Moniliformidae) in a gray squirrel, Sciurus carolinensis, from Arkansas, USA.

Three hundred and seventy-five acanthocephalans, Moniliformis clarki, were removed from the small and large intestines of a gray squirrel from Arkansas County, Arkansas (USA). This is the first report of M. clarki from Arkansas. Enteric lesions, including distension, perforating ulcers, enteritis, crypt hypertrophy, goblet cell hyperplasia, and occlusions of the intestinal tract were observed, indicating the pathogenic potential of this parasite.

Diagnostic studies for systemic necrotizing vasculitis. Sensitivity, specificity, and predictive value in patients with multisystem disease.

Forty patients with multisystem disease and suspected systemic necrotizing vasculitis were evaluated with a protocol designed to confirm the diagnosis with sequential testing. All patients underwent initial laboratory testing. Subsequent studies were individualized to the patient starting with "safe" tests (skin, muscle, rectal biopsies) and progressing to "invasive" tests (arteriography, kidney and lung biopsies). No single laboratory study was found to have adequate predictive value. Skin biopsy, rectal biopsy, and arteriography were insensitive, nonspecific, or had poor predictive values. Muscle biopsy was the most valuable safe procedure (sensitivity, 50%; specificity, 100%; predictive value, 100%; predictive value of negative biopsy, 76%; efficiency, 64%). A diagnostic approach to the patient with possible systemic necrotizing vasculitis is described.