A winter outbreak of anaplasmosis in a nonendemic area of Oklahoma: a possible role for Dermacentor albipictus.
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Anaplasmosis↗
Biomedical subjects
S A Ewing
Publications and source records attributed to S A Ewing.
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.
Bacterial Typing Techniques↗
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.
Animals↗
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.
Animals↗
Attempted transmission of Ehrlichia canis by Rhipicephalus sanguineus after passage in cell culture.
OBJECTIVES: To compare the transmissibility by the brown dog tick, Rhipicephalus sanguineus, of a recent isolate of Ehrlichia canis (Ebony) with that of another isolate (Oklahoma) that had been passaged in cell culture, and to assess the genetic similarity of the 2 isolates as reflected in the nucleotide (NT) sequence of 16S rDNA. ANIMALS: 13 healthy dogs of various ages and breeds. PROCEDURE: Larval and nymphal ticks were acquisition fed on acutely infected dogs, and, after molting, they were transmission fed as nymphs and adults, respectively, on Ehrlichia-naive dogs. All dogs were monitored daily by blood smear evaluation for evidence of parasitized leukocytes and by physical examination for clinical signs of ehrlichiosis. Serologic and hematologic values were measured weekly. Using a nested polymerase chain reaction, the 16S rDNA was amplified, and the NT sequence of the template DNA was determined. RESULTS: The Ebony isolate of E canis was successfully transmitted to dogs by nymphal and adult ticks. In contrast, no ticks that fed on dogs harboring the cell-cultured isolate (Oklahoma) transmitted it to dogs. On the basis of 16S rDNA sequence, the 2 isolates were 99.9% similar, with only 1 NT difference. CONCLUSIONS: These results reconfirm the vector potential of R sanguineus for E canis. Passage of the Oklahoma isolate of E canis in cell culture apparently adversely affected its transmissibility by ticks, raising the possibility that cell-cultured isolates of this rickettsia may lose their affinity for ticks. Determination of 16S rDNA sequence suggests minor strain variation within the species E canis.
Animals↗
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.
Animals↗
Vaccination of cattle with outer membrane protein-enriched fractions of Pasteurella haemolytica and resistance against experimental challenge exposure.
Administration of an N-lauroylsarcosine-derived outer membrane protein fraction of Pasteurella haemolytica A1 (SCI-1) induced a protective response in calves against intrathoracic challenge exposure with the homologous serovar. Outer membrane proteins from heterologous serovars, A6 and A9, induced partial protection that was associated with their respective similarities to serovar A1 in outer membrane protein profiles derived by use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Calves vaccinated with SCI preparations did not have detectable neutralizing antibody to P haemolytica A1 leukotoxin. Antibodies to whole-cell antigens, carbohydrate-protein subunit antigen, and SCI-1 were associated with resistance, which indicates that protein antigens shared among cell surface, carbohydrate-protein subunit, and SCI preparations are immunogenic and enhance resistance to experimental challenge exposure.
Animals↗
Western immunoblot analysis of Ehrlichia chaffeensis, E. canis, or E. ewingii infections in dogs and humans.
Ehrlichia chaffeensis, E. canis, and E. ewingii are genetically closely related, as determined by 16S rRNA gene base sequence comparison, but they exhibit biologic differences. E. chaffeensis is the etiologic agent of human ehrlichiosis. E. canis and E. ewingii cause two distinctly different forms of canine ehrlichiosis and infect different types of leukocytes, monocytes and granulocytes, respectively. E. chaffeensis can also infect dogs. In the study, Western immunoblot analysis of sera from dogs inoculated with E. chaffeensis, E. canis, or E. ewingii was performed to determine antigenic specificity and the intensities of the reactions to purified E. chaffeensis and E. canis antigens. At 2 to 3 weeks postexposure, antisera from four dogs inoculated with E. chaffeensis reacted with 64-, 47-, 31-, and 29-kDa proteins of E. chaffeensis but reacted poorly with E. canis antigen. In contrast, at 2 to 3 weeks postexposure, antisera from four E. canis-inoculated dogs reacted strongly with the 30-kDa major antigen of E. canis but reacted poorly with proteins from E. chaffeensis. At 4 weeks postexposure, the sera from three E. ewingii-inoculated dogs showed weak binding to 64- and 47-kDa proteins of both E. chaffeensis and E. canis. Convalescent-phase sera from human ehrlichiosis patients and sera from dogs chronically infected with E. ewingii strongly reacted with similar sets of proteins of E. chaffeensis and E. canis with similar intensities. However, sera from dogs chronically infected with E. canis reacted more strongly with a greater number of E. canis proteins than with E. chaffeensis proteins. The protein specificity described in the report suggests that dogs with E. canis infections can be distinguished from E. chaffeensis-infected animals by Western immunoblot analysis with both E. canis and E. chaffeensis antigens.
Animals↗
Eosinophilic mediastinitis, myositis, pleuritis, and pneumonia of cattle associated with migration of first-instar larvae of Hypoderma lineatum.
Migrating first-instar larvae of Hypoderma lineatum are a frequent cause of focal inflammatory lesions in connective tissues of the mediastinum, parietal and visceral pleura, peritoneum, lungs, diaphragm, and other loci. The lesions are characterized grossly by foci of yellowish or greenish gelatinous edema and microscopically by infiltration of the edematous tissue by a dense array of eosinophils. Lesions were recognized during a period of several weeks in late spring; the timing was attributable to events in the life cycle of the fly. The larvae, which were small (approximately 1 x 4.5 mm), transparent, and unobtrusive, were recovered from lesions in 12 of 20 cattle in which careful parasitologic examination was made.
Animals↗
Cryptosporidiosis in a cotton rat (Sigmodon hispidus).
Cryptosporidiosis, previously unreported in cotton rats (Sigmodon hispidus), was observed in one of nine cotton rats from Pryor, Oklahoma (USA). Infection was confined to the large intestine. Microscopically, numerous cryptosporidians measuring 2 to 3 microns in diameter were intimately associated with the luminal surface of colonic and rectal mucosae. The affected lamina propria of the large intestine was diffusely infiltrated by eosinophils, lymphocytes and macrophages. Ultrastructurally, numerous trophozoites and a single schizont were observed. Microvilli were displaced by the presence of cryptosporidians at the attachment site.
Animals↗
Effect of 4-bromo-calcium ionophore A23187 on release of Anaplasma marginale from bovine erythrocytes in vitro.
The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 microM A23187 to the cultures resulted in significant decrease in percentage of parasitized erythrocytes (PPE) by 24 hours after treatment; further development and increase in PPE was not observed. In contrast, the PPE of untreated cultures, those treated with dimethyl sulfoxide (DMSO) only and with 1 microM A23187 increased slightly during that time. Total erythrocyte count decreased in treated cultures in excess of that expected after samples of the medium were taken for analysis. The greatest cell loss and increased hemoglobin concentration in culture medium was observed in cultures treated with 10 microM A23187 and with an equivalent volume of DMSO. The DMSO appeared to cause hemolysis of some erythrocytes, but not of infected cells selectively. Release of A marginale inclusion bodies was seen by electron microscopy in samples from the 10 microM A23187-exposed cultures. At 30 minutes after treatment, free initial bodies were frequently seen. Inclusion body membranes and individual A marginale were associated with membranes of adjacent erythrocytes. Individual rickettsiae were seen in cell depressions and appeared to be entering erythrocytes. However, neither further invasion nor development of the parasite in erythrocytes was observed. Ionophore A23187 appeared to promote release of A marginale from erythrocytes, but did not enhance infection of erythrocytes or development of organisms in vitro.
Anaplasma↗
Development of Anaplasma marginale in salivary glands of male Dermacentor andersoni.
Development of the rickettsia, Anaplasma marginale, in salivary glands of male Dermacentor andersoni exposed as nymphs or adult ticks, was studied indirectly by inoculation of susceptible calves with homogenates and directly by examination, using light microscopy and a DNA probe; some unfed ticks were incubated before tissues were collected. Salivary gland homogenates made from ticks in every treatment group caused anaplasmosis when injected into susceptible calves; prepatent periods decreased as the time that ticks had fed increased. Colonies of A marginale were seen only in salivary glands of ticks exposed as adults and not in those exposed as nymphs; the percentage of salivary gland acini infected in these ticks increased linearly with feeding time. However, the probe detected A marginale DNA in salivary glands of ticks from both groups; the amount of DNA detected increased as feeding time was extended. The amount of A marginale DNA appeared to remain constant in gut tissues, but to increase in salivary glands. Salivary glands of adult-infected male ticks that were incubated, but did not feed a second time, became infected with A marginale, and the pattern of infection of acini varied with incubation temperature. Development of A marginale in salivary glands appears to be coordinated with the tick feeding cycle; highest infection rate was observed in ticks exposed as adults.
Anaplasma↗
Preliminary attempts to infect bovine erythrocytes in vitro with a tick-derived stage of Anaplasma marginale.
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Anaplasma↗
Persistence of Anaplasma marginale (Rickettsiales: Anaplasmataceae) in male Dermacentor andersoni (Acari: Ixodidae) transferred successively from infected to susceptible calves.
The persistence of Anaplasma marginale Theiler in male Dermacentor andersoni Stiles ticks exposed to the organism as adults was studied as the ticks were successively transferred to five susceptible calves. All calves fed upon by these ticks rapidly developed clinical anaplasmosis; incubation periods of infection ranged from 19 to 26 d and did not change significantly with successive feedings. Development of A. marginale in tick midgut and salivary glands was followed daily during tick feeding (total, 35 d) with light microscopy and DNA hybridization. With microscopy, A. marginale colonies persisted in midgut cells throughout the experiment. Large colonies were observed in gut muscle cells on days 8 through 35 and were the predominant infected cell type during this part of feeding. Colonies were seen in salivary gland acini from day 2 throughout the 35-d experiment. The DNA probe confirmed the presence of Anaplasma DNA in midgut and salivary glands throughout the experiment. Quantitative estimates of infection intensity in tissues of individual ticks approximated 10(7) initial body equivalents, confirming heavy infections. A marginale in midgut tissues decreased with feeding time, whereas the estimated number of organisms in salivary glands remained constant. These data demonstrate that D. andersoni males are efficient vectors of A. marginale and may be potential reservoirs of infection for ruminants for extended periods.
Anaplasma↗
Analyses of Ehrlichia canis and a canine granulocytic Ehrlichia infection.
Ehrlichia canis and canine granulocytic Ehrlichia sp. (CGE) infect canine monocytes and granulocytes, respectively. E. canis has been cultured in vitro and used to develop an immunofluorescence assay. CGE has not been cultured, and a serologic assay is not available. The sera of dogs infected with CGE were reported to react with E. canis by immunofluorescence. In this study, the temporal response of immunoglobulin G (IgG) was determined by an enzyme-linked immunosorbent assay (ELISA) with purified E. canis antigen in four dogs experimentally infected with E. canis, in two dogs experimentally infected with CGE, and in one dog infected with E. canis and subsequently infected with CGE. E. canis-infected dogs developed an IgG ELISA result of 1.5 or greater for the optical density signal/noise ratio by 2 months postinfection. CGE challenge of a dog with a previous E. canis infection induced an anamnestic increase in the IgG ELISA result; however, CGE infection alone did not induce a significant IgG ELISA response. Western immunoblot analysis showed that dogs infected with E. canis developed antibodies initially that reacted with low-molecular-mass proteins (30, 24, and 21 kDa) and subsequently with higher-molecular-mass proteins (160, 100, 78, 64, 47, and 40 kDa). In contrast, CGE-infected dogs showed reactions with the same higher-molecular-mass proteins of E. canis but, unlike E. canis-infected dogs, not with the low-molecular-mass proteins of E. canis. Of 10 serum samples collected in the field of Indonesia from dogs with tropical canine pancytopenia, all had an optical density signal minus noise value of 2.54 or greater in the IgG ELISA and reacted with E. canis antigen in a pattern similar to that of serum samples from dogs experimentally infected with E. canis in Western immunoblotting. This study suggests that the IgG ELISA and Western immunoblotting with purified E. canis as the antigen are useful in distinguishing between E. canis and CGE infections in dogs.
Animals↗
Susceptibility of dogs to infection with Ehrlichia chaffeensis, causative agent of human ehrlichiosis.
Ehrlichia chaffeensis, the newly recognized agent of human ehrlichiosis, is closely related to E canis, the causative agent of canine ehrlichiosis. Eight pups were inoculated IV with E chaffeensis-, or with E canis-infected DH82 cells, or organisms released from these host cells. Two additional pups served as nonexposed controls. Marked thrombocytopenia was observed in the E canis-infected pups, but not in those infected with E chaffeensis. Homologous serologic response was observed in the E chaffeensis-exposed pups by postinoculation day (PID) 14 and in the E canis-exposed pups by PID 21. Ehrlichia chaffeensis and E canis were reisolated from the respective inoculated pups on each of 8 attempts from PID 7 to 26. One E chaffeensis-exposed pup that was challenge exposed with E canis via blood transfusion, developed fever, anorexia, and thrombocytopenia, suggesting lack of cross protection against E canis.
Animals↗
Development of Anaplasma marginale in male Dermacentor andersoni transferred from parasitemic to susceptible cattle.
The development and transmission of Anaplasma marginale was studied in Dermacentor andersoni males. Laboratory-reared male D andersoni were allowed to feed for 7 days on a calf with ascending A marginale parasitemia. The ticks were then held in a humidity chamber for 7 days before being placed on 2 susceptible calves. Anaplasmosis developed in the calves after incubation periods of 24 and 26 days. Gut and salivary glands were collected from ticks on each day of the 23-day experiment and examined with light and electron microscopy. Colonies of A marginale were first observed in midgut epithelial cells on the sixth day of feeding on infected calves, with the highest density of colonies found in gut cells while ticks were between feeding periods. The first colonies contained 1 large dense organism that subsequently gave rise to many reticulated organisms. Initially, these smaller organisms were electron-lucent and then became electron-dense. On the fifth day after ticks were transferred to susceptible calves for feeding, A marginale colonies were found in muscle cells on the hemocoel side of the gut basement membrane. A final site for development of A marginale was the salivary glands. Colonies were first seen in acinar cells on the first day that ticks fed on susceptible calves, with the highest percentage of infected host cells observed on days 7 to 9 of that feeding. Organisms within these colonies were initially electron-lucent, but became electron-dense.
Anaplasma↗
Serologic diagnosis of human ehrlichiosis using two Ehrlichia canis isolates.
Ehrlichia canis or a closely related rickettsial organism has been implicated serologically and morphologically as the causative agent of human ehrlichiosis in the United States. Although E. canis has been serially propagated in primary canine monocytes, only a limited quantity of antigen is obtained by this method. A continuous canine macrophage cell line, DH82, supports the growth of a new isolate of E. canis established from the whole blood of a carrier dog in Oklahoma. Serologic comparison of the Oklahoma isolate in the continuous canine cell line with a Florida isolate in commercial antigen slides revealed 100% specificity and 87.5% sensitivity.
Animals↗