Experimental infection of domestic cats (Felis domesticus) with Cytauxzoon manul from Pallas' cats (Otocolobus manul).

In a random, blind study, six domestic cats were assigned to two treatment groups that received either sterile water or dexamethasone by subcutaneous injection prior to intravenous inoculation with Pallas' cat (Otocolobus manul) blood infected with Cytauxzoon manul. A seventh domestic cat served as a control and was inoculated only with sterile water. Cats were monitored for clinical signs consistent with cytauxzoonosis, and periodically screened for hemoparasitemia. All domestic cats (6/6) that received Pallas' cat blood infected with C. manul developed a low but detectible parasitemia by 9 days post-inoculation, yet remained clinically healthy. All domestic cats (7/7) were subsequently challenged with Cytauxzoon felis and developed clinical signs typical of cytauxzoonosis within 5 days post-challenge. Affected animals were euthanized and cytauxzoonosis was confirmed by histopathology. While inoculation of domestic cats with Pallas' cat blood infected with C. manul induced a parasitemia, it did not cause disease or provide protection against challenge with C. felis. Further studies are warranted to determine the potential for interspecies transmission and disease with C. manul.

Sequence variation of the ribosomal DNA second internal transcribed spacer region in two spatially-distinct populations of Amblyomma americanum (L.) (Acari: Ixodidae).

Sequence analysis of the ribosomal DNA second internal transcribed spacer (ITS 2) region in 2 spatially distinct populations of Amblyomma americanum (L.) revealed intraspecific variation. Nucleotide sequences from multiple DNA extractions and several polymerase chain reaction amplifications of eggs from mixed-parentage samples from both populations of ticks revealed that 12 of 1,145 (1.0%) sites varied. Three of the 12 sites of variation were distinct between the 2 A. americanum populations, which corresponded to a rate of 0.26%. Phylogenetic analysis based on ITS 2 sequences provided strong support (i.e., bootstrap value of 80%) that wild A. americanum clustered into a distinguishable group separate from those derived from colony ticks.

Comparison of tissue stages of Hepatozoon americanum in the dog using immunohistochemical and routine histologic methods.

American canine hepatozoonosis is caused by Hepatozoon americanum, a recently described species of apicomplexan protozoan parasite. An immunohistochemical procedure using a polyclonal antibody to sporozoites of H. americanum clearly identified asexual stages of H. americanum in canine striated muscle. The method also detects hepatozoa present in naturally infected coyotes and raccoons and reacts with certain other apicomplexans. Use of this immunohistochemical procedure confirms the canine intermediate host-parasite relationships that were presumptively established using conventional histopathologic methods.

A genotypically unique Babesia gibsoni-like parasite recovered from a dog in Oklahoma.

A small Babesia gibsoni-like parasite was identified and isolated as the cause of clinical babesiosis in a dog from Oklahoma. Because this was potentially the first documented case of B. gibsoni infection in Oklahoma, further characterization was warranted, and the 18S nuclear small subunit ribosomal RNA gene was sequenced. Sequence comparison with other piroplasms from dogs showed significant nucleotide sequence differences between this isolate and both B. canis and B. gibsoni. These findings demonstrate that in domestic dogs in North America there are at least 2 "small" B. gibsoni-like organisms with distinct nucleotide sequences and that the geographic distribution of the "small" canine Babesia species may be wider than previously recognized.

An indirect enzyme-linked immunosorbent assay for diagnosis of American canine hepatozoonosis.

American canine hepatozoonosis (ACH), caused by Hepatozoon americanum, is an emerging tick-borne disease of dogs. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate diagnosis of infection and study of the epidemiology of ACH has been developed using H. americanum sporozoites as antigen. Efficacy of the new test as a diagnostic tool was compared with that of skeletal muscle biopsy, the current gold standard for confirming H. americanum infection. Results show that the test is sensitive (93%) and specific (96%) and that it is as reliable as histopathologic examination of skeletal muscle for detecting infection. The ELISA would be suitable as a routine laboratory test for diagnosis of ACH.

Intravenous pharmacokinetics of penicillin G and antipyrine in ostriches (Struthio camelus) and emus (Dromaius novaehollandiae).

Penicillin G and antipyrine, which served as model drugs to assess the relative capacities of renal and hepatic elimination pathways, respectively, were each administered intravenously to six ostriches (Struthio camelus) and to six emus (Dromaius novaehollandiae). Drug concentrations in blood samples collected over a period of 12 hr after administration were assayed, and elimination half-life, mean residence time, clearance, and steady-state volume of distribution were calculated. Mean values for elimination half-life and mean residence time of penicillin G were significantly higher in emus than in ostriches; no significant differences in antipyrine pharmacokinetics between species were demonstrated.

Muscular sarcocystosis in coyotes from Oklahoma.

In a recent survey in Oklahoma (USA), 52 free-ranging coyotes were examined for the presence of sarcocysts. Two of these coyotes were found infected with sarcocysts in skeletal muscle. By light microscopy, the cyst wall was thin and smooth. Ultrastructurally, the cyst wall had minute villar protrusions. The sarcocysts were 14.4 to 50.4 microm wide and 46.8 to 99 microm long. This is the first report of Sarcocystis sp. sarcocysts in the skeletal muscle of coyotes.

Cats surviving natural infection with Cytauxzoon felis: 18 cases (1997-1998).

Eighteen cats surviving natural infection with Cytauxzoon felis were identified. All cats came from a limited geographic area in northwestern Arkansas and northeastern Oklahoma. Clinical signs in most cats were similar to those described for cytauxzoonosis; however, 4 cats were asymptomatic. All cases were initially diagnosed by microscopic identification of signet ring-shaped piroplasms in erythrocytes of peripheral blood smears. Four of 4 cats tested had detectable serum antibodies to C felis. Four different cats were positive by polymerase chain reaction (PCR). Partial sequencing of the PCR product from 1 cat revealed >99% homology with the reported sequence of C felis. Repeated examination of blood smears from 12 cats revealed that the erythroparasitemia was generally persistent for the duration of follow-up (3-154 days). Survival did not seem dependent on treatment, as only 1 cat was treated with a drug with potential antiprotozoal activity (imidocarb dipropionate), and 4 cats received no treatment. The findings of this study may indicate the existence of a less virulent strain of C felis.

Naturally occurring Ehrlichia chaffeensis infection in coyotes from Oklahoma.

A nested polymerase chain reaction assay was used to determine the presence of Ehrlichia chaffeensis, E. canis, and E. ewingii DNA in blood samples of free-ranging coyotes from central and northcentral Oklahoma. Of the 21 coyotes examined, 15 (71%) were positive for E. chaffeensis DNA; none was positive for E. canis or E. ewingii. Results suggest that E. chaffeensis infections are common in free-ranging coyotes in Oklahoma and that these wild canids could play a role in the epidemiology of human monocytotropic ehrlichiosis.

Attempted transmission of Ehrlichia chaffeensis among white-tailed deer by Amblyomma maculatum.

A deer was needle-exposed intravenously to Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in canine macrophage (DH82) cells and 7 days later was infested with laboratory-reared Amblyomma maculatum (Koch) (Acari:Ixodidae) nymphs for acquisition feeding. After molting, the adult ticks were allowed to feed on a naive deer. The organism was reisolated from the needle-exposed deer by cell culture and E. chaffeensis DNA was detected in the deer's blood by PCR. Similar isolation/recovery techniques were used for the tick-exposed deer and no evidence of infection was found. Although these findings must be considered as preliminary owing to inadequate controls, the data suggest that A. maculatum is probably not a suitable vector for E. chaffeensis.

Skeletal lesions of canine hepatozoonosis caused by Hepatozoon americanum.

Canine hepatozoonosis, caused by Hepatozoon americanum, is an emerging tick-borne disease of dogs in North America. In addition to the skeletal and cardiac myositis that are prominent features of the disease, there is disseminated periosteal bone proliferation in most dogs that manifest clinical disease. Each of six experimentally infected animals (four dogs and two coyotes) and seven of eight naturally infected dogs had gross or histopathologic osteoproliferative lesions. Experimental animals were 6-9 months of age when exposed. Naturally infected dogs were 8 months to 11 years old when subjected to necropsy. Lesions occurred primarily on the diaphysis of the more proximal long bones of the limbs; however, flat and irregular bones were frequently involved. Lesions involving metacarpals, metatarsals, and digits were infrequent. The earliest observed periosteal lesions were in an experimentally infected dog 32 days after exposure to sporulated oocysts of H. americanum. There were hypertrophy and hyperplasia of osteoprogenitor cells, and osteoblasts appeared in the cellular zone of the periosteum. Spicules of woven bone oriented perpendicularly to bone cortex followed. Later yet, periosteal new bone was remodeled and tended to become oriented parallel to the cortical bone. Horizontally oriented zones of remodeled, condensed bone sometimes occurred in multiple layers on the original cortex, forming "pseudocortices." The osseous lesions of American canine hepatozoonosis, with few variations, are remarkably similar to those of hypertrophic osteopathy in domestic dogs and other mammalian species, including humans.

Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma.

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.

American canine hepatozoonosis. An emerging disease in the New World.

Hepatozoon canis was first described from dogs in 1905 in India and Rhipicephalus sanguineus was identified as the vector. Dogs on the Texas Gulf Coast were recognized in 1978 to have hepatozoonosis, and it was thought that H. canis had entered the New World. Later, it was realized that American canine hepatozoonosis (ACH) is more debilitating than its Old World counterpart, often resulting in death. When the malady and parasite were characterized, a new species, H. americanum, was described, in 1997. Phylogenetic analysis, based on 18S rRNA gene sequence and classical taxonomic features, revealed that the two dog parasites are closely related. Amblyomma maculatum, the Gulf Coast tick (GCT), has been demonstrated to be an excellent vector; nymphal ticks were readily infected and oocysts from newly molted adults were uniformly infectious for dogs. The merogonic cycle of H. americanum in dogs and the sporogonic development in the invertebrate host have been described. ACH is diagnosed primarily by histologic examination of skeletal muscle. Curative therapy is not available, but anti-protozoal and anti-inflammatory drugs may prolong life. Naturally infected coyotes have been found in Oklahoma and Texas, and experimental infections have been produced in this canid. Additional effort is needed to determine the vertebrate host range of H. americanum and to define the enzootic cycle of which dogs have become a part; likewise, more work is required to determine whether larval GCTs can acquire infection and transmit it as nymphs.

There are at least three genetically distinct small piroplasms from dogs.

The 18S nuclear subunit ribosomal RNA (18S rRNA) gene of small piroplasms isolated from dogs from Okinawa (Japan), Oklahoma, North Carolina, Indiana, Missouri, and Alabama, was isolated and sequenced. Phylogenetic analysis of these sequences and comparisons with sequences from other Babesia, Cytauxzoon, and Theileria species revealed that all canine small babesial isolates, with the exception of isolates from California and Spain, were placed in a group containing the Babesia spp. sensu stricto. Within the Babesia spp. sensu stricto, there was support for separating the small canine piroplasms from the large canine piroplasm, Babesia canis. The isolate from California was in a distinct phylogenetic clade, closely related to babesial isolates from wildlife and humans from the Western US. The canine isolate from Spain was closely related to Babesia microti. These results suggest that there are multiple small piroplasm species in dogs. The isolates from the Midwestern and Eastern US and the one from Japan probably represent a single species with wide geographic distribution.

Two Theileria cervi SSU RRNA gene sequence types found in isolates from white-tailed deer and elk in North America.

Two Theileria cervi SSU rRNA gene sequence Types, F and G, from white-tailed deer (Odocoileus virginianus) and elk (Cervus elaphus canadensis) isolates in North America were confirmed. Previously, nucleotide sequencing through a single variable (V4) region showed the presence of SSU rRNA gene Types F and G in T. cervi isolates from white-tailed deer and an elk. In this study, both sequence types were found in four T. cervi isolates (two from deer and two from elk). Microheterogeneity only appeared in the Type G gene, resulting in Subtypes G1, G2 and G3. Subtype G1 was found in two elk and one white-tailed deer T. cervi isolate; Subtypes G2 and G3 were found in a white-tailed deer T. cervi isolate. The Type F SSU rRNA genes were identical in nucleotide sequence in both elk and white-tailed deer T. cervi isolates. The high degree of conservation in the Type F variable regions may be exploited to design specific oligonucleotide primers for parasite detection by the polymerase chain reaction in cervine or tick hosts.

Naturally occurring hepatozoonosis in coyotes from Oklahoma.

Nine of 16 free-ranging coyotes (Canis latrans) from central Oklahoma (USA) had naturally acquired infections of Hepatozoon americanum. Infections were confirmed by recognition of tissue stages closely resembling H. americanum in skeletal and cardiac muscle. At the time coyotes were collected they were infested with a variety of ticks, including adult Gulf Coast ticks (Amblyomma maculatum). We propose that the high prevalence of H. americanum in this small sample of free-ranging coyotes and the ability of these same animals to harbor adult populations of A. maculatum is an important component of the epizootiology of canine hepatozoonosis in North America.

A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma.

Polymerase chain reaction and Southern hybridization were used to survey for the presence of Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii in blood samples of 65 dogs that harbored ticks from northcentral and northeastern Oklahoma. Dog blood samples were also examined for antibodies against E. canis and E. chaffeensis, using an immunofluorescent antibody test. Ten of 65 dogs (15.4%) examined were positive for Ehrlichia spp. by PCR. Four (6.2%) were positive for E. ewingii, 2 (3.1%) for E. canis, and 4 (6.2%) for E. chaffeensis. Seven dogs (10.8%) were seropositive for E. canis or E. chaffeensis. Ticks collected from PCR-positive dogs were examined by PCR for the presence of Ehrlichia DNA. Several groups of ticks were PCR-positive for E. ewingii or E. canis. E. canis was detected in Rhipicephalus sanguineus, which is considered the major vector for that organism. E. ewingii was detected in a larger variety of ticks, including the only known vector Amblyomma americanum, as well as in Dermacentor variabilis and R. sanguineus. Results suggest that Ehrlichia spp. which are canine and human pathogens circulate in dogs in Oklahoma and in several tick species that feed on dogs.

Observations on tissue stages of Hepatozoon americanum in 19 naturally infected dogs.

Lesions and associated tissue stages of Hepatozoon americanum in 19 naturally infected dogs are described. Schizogony takes place in an unidentified host cell which, during the early stages of the asexual cycle, is contained within a broad, multilamellar mucopolysaccharide 'cyst.' Material forming the cyst appears to be host-derived. An intense inflammatory response follows rupture of the schizont and disintegration of the cyst wall. There is unusually intense angiogenesis associated with the resulting granulomatous inflammation initiated by the freed merozoites. Phagocytized zoites enter the canine circulatory system through the walls of these vessels. Evidence is presented that suggests a single infecting episode can cause prolonged (> or = 9 months) infection, and further, that infection is perpetuated by repeated asexual cycles. Parasites in peripheral blood leukocytes include both those with and without a visible nucleus.

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.

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.