American Canine Hepatozoonosis Research Articles

Efficacy of a topical combination of esafoxolaner, eprinomectin and praziquantel against Amblyomma maculatum infestations in cats.

Amblyomma maculatum, the Gulf Coast tick, infests a wide range of vertebrate species including livestock, dogs, cats, and humans. It is a species of significant veterinary and public health importance, especially as a vector of diseases, for instance American canine hepatozoonosis or tidewater spotted fever. An experimental study was conducted to evaluate the efficacy of NexGard® Combo, a topical endectoparasiticide product for cats combining eprinomectin, praziquantel and esafoxolaner, against induced infestations of A. maculatum in cats. This Good Clinical Practice (GCP) study used a randomized, negative controlled, masked design. Ten cats were allocated to an untreated group and ten to a treated group, dosed once on Day 0 at the minimum label dose. On Days -2, 7, 14, 21, 28, 35, and 42, cats were infested with ~50 unfed adult A. maculatum. On Days 3, 10, 17, 24, 31, 38, and 45, i.e., 72h after treatment and subsequent infestations, ticks were removed, counted and the numbers of live attached tick in each group were used for efficacy calculations. At each time-point, all untreated cats were adequately infested, demonstrating a vigorous tick population and an adequate study model. The curative efficacy after a single application against existing tick infestation, 72h after treatment, was 98.7%. The preventive efficacy, 72h after weekly infestations, over the following five weeks ranged from 93.8% to 99.4%.

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Documentation of the Expansion of the Gulf Coast Tick ( Amblyomma maculatum) and Rickettsia parkeri : First Report in Illinois

The Gulf Coast tick, Amblyomma maculatum, is of public and veterinary health concern, as it is the primary vector of Rickettsia parkeri and Hepatozoon americanum, causative agents of Rickettsiosis and American canine hepatozoonosis. The Gulf Coast tick's range has expanded over the last 50 yr into the mid-Atlantic states, and its expansion is expected to continue northward. We are reporting the presence of A. maculatum for the first time in Illinois, including a total of 18 specimens collected at 6 different sites during surveys in 2013 and 2019. Fourteen of these specimens were screened for Rickettsia parkeri, which resulted in the detection of this bacteria in 8 samples from 4 counties. By depositing these specimens in scientific collections, we provide materialistic evidence of their establishment in 2 counties. We urge health officials to rely on and use scientific collections to document the expansion of these and other vectors across the country. Additionally, we recommend that health practitioners become aware of the clinical similarities between Rocky Mountain Spotted Fever (caused by Rickettsia rickettsii) and "tidewater" fever (caused by R. parkeri).

The Evolving Medical and Veterinary Importance of the Gulf Coast tick (Acari: Ixodidae).

Amblyomma maculatum Koch (the Gulf Coast tick) is a three-host, ixodid tick that is distributed throughout much of the southeastern and south-central United States, as well as several countries throughout Central and South America. A considerable amount of scientific literature followed the original description of A. maculatum in 1844; nonetheless, the Gulf Coast tick was not recognized as a vector of any known pathogen of animals or humans for >150 years. It is now identified as the principal vector of Hepatozoon americanum, the agent responsible for American canine hepatozoonosis, and Rickettsia parkeri, the cause of an emerging, eschar-associated spotted fever group rickettsiosis identified throughout much of the Western Hemisphere. Coincident with these discoveries has been recognition that the geographical distribution of A. maculatum in the United States is far more extensive than described 70 yr ago, supporting the idea that range and abundance of certain tick species, particularly those with diverse host preferences, are not fixed in time or space, and may change over relatively short intervals. Renewed interest in the Gulf Coast tick reinforces the notion that the perceived importance of a particular tick species to human or animal health can be relatively fluid, and may shift dramatically with changes in the distribution and abundance of the arthropod, its vertebrate hosts, or the microbial agents that transit among these organisms.

Alternate Pathway of Infection with Hepatozoon americanum and the Epidemiologic Importance of Predation

The range of American canine hepatozoonosis (ACH) is expanding from the southern USA northward. Transmission of Hepatozoon americanum occurs by ingestion of infected Gulf Coast ticks, Amblyomma maculatum. The source of the protozoan for the tick remains undetermined; infected dogs are unusual hosts for the tick. Compare possible sources of infection by field investigations of 2 multiple-dog outbreaks of ACH. Twenty-eight privately owned dogs (Canis familiaris), 1 coyote (Canis latrans), 31 wild-trapped cotton rats (Sigmodon hispidus), 24 wild-trapped field mice (Peromyscus leucopus), and 9 wild-caught rabbits (Sylvilagus spp.) from sites in eastern Oklahoma were monitored for hepatozoonosis. Six laboratory-raised cotton rats (S. hispidus), 6 Sprague-Dawley rats (Rattus norvegicus), 6 C57BL/6J-Lystbg-J/J mice (Mus musculus), 6 outbred white mice (M. musculus), 6 New Zealand white rabbits (Oryctolagus cuniculus), and 2 dogs were acquired through commercial vendors for experimental transmission trials of H. americanum. Four of 15 dogs in a rural neighborhood and 5/12 hunting Beagles were confirmed to be infected by blood smear examination, muscle biopsy, and polymerase chain reaction assay of the 18S rRNA gene of Hepatozoon species. Histories and tick host preferences led to field collections of common prey of canids and experimental transmission trials of H. americanum to selected prey (M. musculus, S. hispidus, R. norvegicus, and O. cuniculus). Dogs with ready access to prey (4/15 dogs) or that were fed prey retrieved from hunts (5/12 hunting Beagles) became infected, providing evidence that predation is an important epidemiologic component of ACH infection. Experimental transmission studies identified a quiescent, infectious stage (cystozoite) of the parasite that provides an alternate mode of transmission to canids through predation, demonstrating that cotton rats, mice, and rabbits but not brown rats may act as paratenic hosts of H. americanum. Predation of prey harboring infected A. maculatum or containing cystozoites of H. americanum in their tissues provide 2 modes of transmission of ACH to dogs, putting unconfined dogs at increased risk of infection in endemic areas.

Light and transmission electron microscopic characteristics of a novel Hepatozoon spp. in naturally infected cotton rats (Sigmodon hispidus)

A novel species of Hepatozoon was recently reported in cotton rats (Sigmodon hispidus) collected from an area of Oklahoma where American canine hepatozoonosis is endemic. In this study, the various stages of merogony of the parasite were characterized by light and electron microscopy. Meronts occurred within parasitophorous vacuoles in hepatocytes and ranged from mononucleated spherical forms to large, mature forms in vacuoles that contained approximately 50 peripherally arranged merozoites. Developing merozoites had characteristic apicomplexan organelles, including anterior and posterior polar rings, a conoid, microtubules, rhoptries, micronemes, and a trilaminar membrane. As the meronts matured, numerous curvilinear merozoites budded from a residual body. This morphologic characterization extends our understanding of this novel Hepatozoon and adds information about the hepatozoa, apicomplexan parasites that infect numerous species.

Experimental transmission of Hepatozoon americanum to New Zealand White rabbits ( Oryctolagus cuniculus) and infectivity of cystozoites for a dog

Inflammatory lesions containing parasitic cystozoites developed in multiple organs and tissues of laboratory-raised Oryctolagus cuniculus that were administered approximately 100 sporulated oocysts of Hepatozoon americanum (Oklahoma isolate, GenBank accession AF176836) orally. The predominantly granulomatous inflammatory lesions were detected histologically 8 weeks after exposure to oocysts. Cystozoites, recognized by cresent-shaped, uninucleated bodies surrounded by an accumulation of globular, PAS-positive polysaccharide material, were evident within macrophages as monozoic and dizoic cysts. Neither meronts nor gamonts were detected in any of the laboratory-raised lagomorphs during the 24-week observation period. Nested PCR assay of rabbit tissues for a 488 bp fragment of the 18S rRNA Hepatozoon spp. gene was positive at 8 and 24 weeks post-exposure. The sequence was 97.1% similar with sequence from the H. americanum carrier used to infect ticks. A Hepatozoon-free dog fed tissues from the 24-week post-exposure rabbit principal developed American canine hepatozoonosis. Gamonts were first detected 5 weeks after the dog ingested the rabbit tissues containing cystozoites. PCR assay of blood from the dog was positive for the Hepatozoon spp. gene fragment. Sequencing confirmed that the parasite in the dog was H. americanum.

Diversity of Hepatozoon species in naturally infected dogs in the southern United States

Hepatozoon americanum is a protozoan that causes American canine hepatozoonosis (ACH) in the southern United States; Hepatozoon canis, the causative agent of canine hepatozoonosis in Africa, Asia, Europe, and South America, has not previously been definitively identified in dogs in the United States. To characterize the diversity of Hepatozoon spp. in domestic dogs from Oklahoma, blood samples collected from dogs residing in an endemic area of the state, clinical cases presented to veterinarians with symptoms of ACH, and dogs housed at a local shelter were evaluated by a nested PCR designed to amplify a variable region of the 18S rRNA gene of blood ampicomplexa, including Hepatozoon spp. Hepatozoon sequences recovered from a dog from an area where ACH is endemic, from clinically ill dogs, and from one shelter dog most closely resembled H. americanum. However, two other shelter dogs had evidence of infection with H. canis or a closely related organism. A subsequent review of real-time PCR results from the Molecular Diagnostics Laboratory at Auburn University revealed that the majority of samples submitted from dogs from across the United States which tested positive for Hepatozoon spp. had H. americanum. However, some submissions were also found which contained DNA sequence of H. canis. Mixed H. americanum and H. canis-like infections also were detected. Our data suggest that H. americanum, H. canis, as well as H. canis-like organisms are present and may cause disease in dogs in the southern U.S.

Field survey of rodents for Hepatozoon infections in an endemic focus of American canine hepatozoonosis

Eighteen of 31 (58%) cotton rats ( Sigmodon hispidus) and 8 of 24 (33.3%) white-footed mice ( Peromyscus leucopus) that were wild-trapped from 4 American canine hepatozoonosis endemic sites in Oklahoma were infected with Hepatozoon species. The predilection organ for merogony of the Hepatozoon species in cotton rats was the liver. Meronts were not detected in any of the white-footed mice. A 488 bp DNA fragment that includes a variable region of the 18S rRNA Hepatozoon gene amplified from blood or tissue of these infected animals. Sequences from eight cotton rats were 100% identical to each other as were sequences from three white-footed mice 100% identical to each other. The cotton rat sequence and the white-footed mouse sequence were 98.8% identical, differing in 6 bp of the 488 bp fragment. The DNA sequence from cotton rats was 97.7% identical to a Hepatozoon sp. described in a large bandicoot rat from Thailand and 97.5% identical to a Hepatozoon sp. in a bank vole from Brazil. The sequence from white-footed mice was 98.6% identical to the bandicoot rat sequence and 98.4% identical to the bank vole sequence. However, the sequences were only 90.6% (cotton rat) and 91.4% (white-footed mouse) identical to H. americanum. These findings suggest that the rodents are obligate intermediate hosts for distinct Hepatozoon spp., but not H. americanum.

Characterization of Stages ofHepatozoon americanumand of Parasitized Canine Host Cells

American canine hepatozoonosis is caused by Hepatozoon americanum, a protozoan parasite, the definitive host of which is the tick, Amblyomma maculatum. Infection of the dog follows ingestion of ticks that harbor sporulated H. americanum oocysts. Following penetration of the intestinal mucosa, sporozoites are disseminated systemically and give rise to extensive asexual multiplication in cells located predominantly in striated muscle. The parasitized canine cells in "onion skin" cysts and in granulomas situated within skeletal muscle, as well as those in peripheral blood leukocytes (PBL), were identified as macrophages by use of fine structure morphology and/or immunohistochemical reactivity with macrophage markers. Additionally, two basic morphologic forms of the parasite were observed in macrophages of granulomas and PBLs. The forms were presumptively identified as merozoites and gamonts. The presence of a "tail" in some gamonts in PBLs indicated differentiation toward microgametes. Recognition of merozoites in PBLs supports the contention that hematogenously redistributed merozoites initiate repeated asexual cycles and could explain persistence of infection for long periods in the vertebrate host. Failure to clearly demonstrate a host cell membrane defining a parasitophorous vacuole may indicate that the parasite actively penetrates the host cell membrane rather than being engulfed by the host cell, as is characteristic of some protozoans.

Experimental Infection of Adult and Juvenile Coyotes with Domestic Dog and Wild Coyote Isolates of Hepatozoon americanum (Apicomplexa: Adeleorina)

Each of five adult and four juvenile coyotes (Canis latrans) was exposed to an oral dose of 50 Hepatozoon americanum oocysts recovered from Amblyomma maculatum ticks that previously fed on either naturally infected domestic dogs (Canis familiaris) or naturally infected wild coyotes. All coyotes exposed to H. americanum became infected, regardless of isolate source, and all exhibited mild to moderate clinical disease that simulated American canine hepatozoonosis in naturally infected dogs. At 100 days postexposure, parasitemia was greater in juvenile than adult coyotes (0.9% and 0.3%, respectively); radiographic imaging of femurs revealed moderate exostosis in all juveniles and mild to moderate new bone growth in four of five (80%) adult coyotes. Gross postmortem analysis of bone lesions demonstrated variation between age groups of coyotes but not between isolates of H. americanum. Microscopic evaluation of skeletal muscle revealed that parasite-induced lesions were significantly more numerous (t = 5.0, df = 7, P = 0.001) in juvenile than adult coyotes. Results of this study indicate that juvenile and adult coyotes are equally susceptible to experimental infection with H. americanum isolated from domestic dog and wild coyote sources. The age of coyotes at the time of exposure, and possibly the number of H. americanum oocysts ingested, might influence morbidity and mortality, but it appears that both adult and juvenile coyotes could be reservoirs of H. americanum.

Transmission of Hepatozoon americanum (Apicomplexa: Adeleorina) by ixodids (Acari: Ixodidae).

American canine hepatozoonosis (ACH) caused by Hepatozoon americanum Vincent-Johnson, Macintire, Lindsay, Lenz, Baneth, and Shkap is an emerging, often fatal, tick-borne protozoal disease of dogs in the United States of America. Dogs acquire infection by ingesting ticks that contain oocysts. To understand the invertebrate (definitive) host range of H. americanum, experiments were carried out using four ixodids, Rhipicephalus sanguineus (Latreille), Dermacentor variabilis Say, Amblyomma americanum (L.), and Amblyomma maculatum Koch. Laboratory-reared nymphal ticks were fed on dogs that were either naturally or experimentally infected with H. americanum; when these ticks molted to the adult stage they were either fed to susceptible dogs or were dissected and examined for the presence of oocysts. Mature H. americanum oocysts were found in >90% of A. maculatum (both males and females), whereas oocysts were not found in any of the other three species. These results confirm that A. maculatum is an excellent host and vector for H. americanum and also suggest that this apicomplexan may have a narrow invertebrate host range, at least among ixodid ticks that are likely candidate vectors in the United States.

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.

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.

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.