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Summary

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.

Free access
in American Journal of Veterinary Research

Summary

Transstadial transmission of granulocytic Ehrlichieae in dogs was attempted using ticks, Amblyomma americanum and Dermacentor variabilis. Ticks were exposed by feeding as nymphs on acutely infected pups; adult ticks then fed to repletion on susceptible adult dogs that were monitored daily for signs of infection. Evidence of transmission was not observed in control dogs or in those exposed to D variabilis. In contrast, dogs exposed to A americanum developed serologic or clinical evidence of infection, but organisms were not seen in blood smears until corticosteroids were administered, causing recrudescence and accompanying parasitemia. At 12 days after subinoculation of blood obtained from donor adult dogs, before corticosteroids were administered, a febrile response, thrombocytopenia, and appearance of morulae in neutrophilic granulocytes were observed in 2 susceptible recipient dogs.

Free access
in American Journal of Veterinary Research

SUMMARY

The ionophore A23187 was used to facilitate release and continued development of Anaplasma marginale in short-term erythrocyte cultures. Addition of 10 μM 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 μM 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 μM 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 μM 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.

Free access
in American Journal of Veterinary Research

SUMMARY

The development of Anaplasma marginale was studied in Dermacentor andersoni nymphs after they had fed on a calf with ascending Anaplasma infection. Gut tissues were collected on day 4 of tick feeding, from newly replete (fed) nymphs and on postfeeding days (pfd) 5, 10, 15, 20, and were processed for light and electron microscopy to determine density of A marginale colonies. Homogenates of gut tissues were prepared from nymphs collected on the same days and inoculated into susceptible, splenectomized calves to test for infectivity. Anaplasma colonies were detected in gut cells on pfd 5, 10, 15, and 20. Although colony density appeared to be higher on pfd 10 and 15, differences were not significant. Nymphal type-1 colonies were detected in highest numbers on pfd 5 and 10, transitional colonies were seen in highest numbers at pfd 10 and 15, and nymphal type-2 colonies were observed only on pfd 20. Gut homogenates that were collected from ticks at 4 days of feeding, when newly replete, and on pfd 20 caused anaplasmosis when injected into susceptible calves, but homogenates made from ticks collected on pfd 5, 10, and 15 were not infective. The data indicate that of the colony types of A marginale that develop in replete nymphs, nymphal type-1 and transitional colonies may contain organisms that are not infective for cattle.

Free access
in American Journal of Veterinary Research

SUMMARY

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.

Free access
in American Journal of Veterinary Research

Abstract

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. (Am J Vet Res 1996;57:1594–1598)

Free access
in American Journal of Veterinary Research

SUMMARY

Development of the rickettsia, Anaplasmamarginale, 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 marginaleDNA 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.

Free access
in American Journal of Veterinary Research

Summary

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.

Free access
in American Journal of Veterinary Research