S. A. Ewing

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.

The natural history of Anaplasma marginale.

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1alpha, provided information about the biogeography and evolution of A. marginale, and msp1alpha genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.

Canine hepatozoonosis: two disease syndromes caused by separate Hepatozoon spp.

Hepatozoonosis is caused by apicomplexan haemoparasites of the genus Hepatozoon, which are closely related to Plasmodium spp. and piroplasms. Recent research revealed that two tick-borne Hepatozoon spp. infect dogs and cause distinct syndromes. Comparisons of these related species illustrates that whereas Hepatozoon canis appears to be well adapted to its canine host, Hepatozoon americanum, an emerging pathogen producing severe and frequently fatal myositis, is highly virulent and might have recently crossed the species barrier from a wild host.

American Canine Hepatozoonosis

SUMMARY American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy.

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 canis (Rickettsiales: Ehrlichieae) by Dermacentor variabilis (Acari: Ixodidae)

Four trials were conducted in which laboratory-reared Dermacentor variabilis nymphs were exposed to Ehrlichia canis by feeding on experimentally infected dogs as soon as classical morulae were detected in peripheral blood monocytes. After molting 25, 50 or 90 adult tick pairs were permitted to feed on 7 Ehrlichia-naive dogs. Transmission occurred in trials 1 (1/1 dog), 3 (1/1 dog) and 4 (2/2 dogs) but not in trial 2 (0/3 dogs), with 4 of 7 dogs becoming infected. Successful transstadial transmission was demonstrated by detection of morulae in peripheral blood lymphocytes and by seroconversion to Ehrlichia canis 30 d post-exposure. Incubation periods ranged between 17 and 22 days (mean = 19). Clinical signs, typical of ehrlichiosis, included mucopurulent ocular discharge, lymphadenopathy and malaise with accompanying pyrexia, leukopenia and thrombocytopenia. Pyrexia, thrombocytopenia and erythrophagocytosis and vacuolization of the cytoplasm of monocytic cells were observed 1-4 d prior to detection of morulae. This is the first demonstration that a tick other than Rhipicephalus sanguineus is capable of transstadial transmission of this important pathogen of dogs.

Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30-Based PCR Assay

ABSTRACT Detection of vector-borne pathogens is necessary for investigation of their association with vertebrate and invertebrate hosts. The ability to detect Ehrlichia spp. within individual experimentally infected ticks would be valuable for studies to evaluate the relative competence of different vector species and transmission scenarios. The purpose of this study was to develop a sensitive PCR assay based on oligonucleotide sequences from the unique Ehrlichia canis gene, p30, to facilitate studies that require monitoring this pathogen in canine and tick hosts during experimental transmission. Homologous sequences for Ehrlichia chaffeensis p28 were compared to sequences of primers derived from a sequence conserved among E. canis isolates. Criteria for primer selection included annealing scores, identity of the primers to homologous E. chaffeensis sequences, and the availability of similarly optimal primers that were nested within the target template sequence. The p30-based assay was at least 100-fold more sensitive than a previously reported nested 16S ribosomal DNA (rDNA)-based assay and did not amplify the 200-bp target amplicon from E. chaffeensis, the human granulocytic ehrlichiosis agent, or Ehrlichia muris DNA. The assay was used to detect E. canis in canine carrier blood and in experimentally infected Rhipicephalus sanguineus ticks. Optimized procedures for preparing tissues from these hosts for PCR assay are described. Our results indicated that this p30-based PCR assay will be useful for experimental investigations, that it has potential as a routine test, and that this approach to PCR assay design may be applicable to other pathogens that occur at low levels in affected hosts.

Occurrence of the gulf coast tick (Acari: Ixodidae) on wild and domestic mammals in north-central Oklahoma

Abstract Parasitic life stages of Amblyomma maculatum Koch were collected from domestic cattle and several species of wild mammals during a 3.5-yr study (May 1998–October 2001) in north-central Oklahoma. Adult ticks were the predominant life stage collected from cattle, white-tailed deer, coyotes, and raccoons, whereas only immature ticks were collected from cotton rats and white-footed mice. The prevalence of adult A. maculatum on white-tailed deer (n = 15) examined in June, July, and August 1998 was 80, 100, 100%, respectively. The prevalence of adult A. maculatum on cattle (n = 84) ranged from 52% in February 1999 to 100% in May 1999. The prevalence of adult A. maculatum on coyotes (n = 16) was 100% in April 1998 and 43% on coyotes (n = 7) examined in January 2001. The prevalence of adult A. maculatum on raccoons (n = 23) examined during May, June, and July 1999 was 13%. No A. maculatum of any life stage were recovered from opossums (n = 7). Nine hundred forty-five rodents were trapped over 294 trap-nights; prevalence of A. maculatum larvae and nymphs on cotton rats (n = 395) was 34 and 15%, respectively, whereas on white-footed mice (n = 517), prevalence was 1.5 and 1.4%, respectively. No A. maculatum were recovered from pack rats (n = 33). There were significant differences (P = 0.0001) in larval infestation prevalence between cotton rats and white-footed mice in the spring, summer, and fall and for nymphs in the spring and summer. Results of A. maculatum parasitism and seasonal occurrence on hosts in this study are compared with previous research conducted in Oklahoma and with collection records of A. maculatum in the Entomology Museum at Oklahoma State University.

Canine hepatozoonosis: comparison of lesions and parasites in skeletal muscle of dogs experimentally or naturally infected with Hepatozoon americanum

We report previously undescribed, early lesions in skeletal muscle of dogs experimentally infected with Hepatozoon americanum by ingestion of laboratory-reared, infected Amblyomma maculatum. The earliest muscle lesion was recognized at the first interval of examination 3 weeks following exposure. The lesion consisted of a large, modified host cell whose cytoplasm frequently contained a demonstrable parasite. In skeletal muscle, the cell was consistently located between muscle fibers or in loose connective tissue adjacent to those fibers. Evidence suggesting that the parasite arrives in muscle and other tissue within the host cell cytoplasm is presented. Mucopolysaccharide encystment of the host cell, absent at this early stage, was acquired gradually and approached maximal development 26 weeks post exposure. Completion of the asexual cycle as evidenced by the presence of parasites entering vascular lumens within granulomas and also by the presence of gamonts in peripheral blood leukocytes, occurred within 28-32 days postexposure. Progression of the parasite cycle from meront to passage of zoites into vessel lumens of granulomas can occur in 11 or fewer days. The density with which parasitic lesions occur in one named skeletal muscle compared to other named muscles, although somewhat variable, was not significantly different in either experimentally induced or natural infections. The distribution of developmental stages of the parasite/lesion in four experimental infections (969 lesions) is compared with those in eight dogs with natural infections (557 lesions).

New developments in canine hepatozoonosis in North America: a review.

Canine hepatozoonosis is caused by Hepatozoon canis and Hepatozoon americanum, apicomplexan parasites transmitted to dogs by ingestion of infectious stages. Although the two agents are phylogenetically related, specific aspects, including characteristics of clinical disease and the natural history of the parasites themselves, differ between the two species. Until recently, H. canis infections had not been clearly documented in North America, and autochthonous infection with H. americanum has yet to be reported outside of the southern United States. However, recent reports demonstrate H. canis is present in areas of North America where its vector tick, Rhipicephalus sanguineus, has long been endemic, and that the range of H. americanum is likely expanding along with that of its vector tick, Amblyomma maculatum; co-infections with the two organisms have also been identified. Significant intraspecific variation has been reported in the 18S rRNA gene sequence of both Hepatozoon spp.-infecting dogs, suggesting that each species may represent a complex of related genogroups rather than well-defined species. Transmission of H. americanum to dogs via ingestion of cystozoites in muscle of infected vertebrates was recently demonstrated, supporting the concept of predation as a means of natural transmission. Although several exciting advances have occurred in recent years, much remains to be learned about patterns of infection and the nature of clinical disease caused by the agents of canine hepatozoonosis in North America.