Diagnosis of canine Hepatozoon spp. infection by quantitative PCR

Hepatozoon (H.) americanum and H. canis are the etiological agents of canine hepatozoonosis, a disease that is found worldwide and is also prevalent in the southeastern United States. Current laboratory diagnosis of canine hepatozoonosis caused by H. americanum is usually dependent on visual identification of Hepatozoon "onion skin cysts" in muscle biopsies, an approach that requires invasive sampling and can result in false negatives. We have developed a diagnostic method for detection of Hepatozoon spp. DNA that integrates nucleic acid extraction with extensive agitation to maximize DNA extraction efficiency. The DNA extracted from canine EDTA-whole blood is subjected to real-time PCR, and fluorescence resonance energy transfer (FRET) probes detect a signature polymorphism in the amplified DNA. This PCR method amplifies a fragment of the Hepatozoon 18S rDNA gene, detects as few as 7 genomic copies of Hepatozoon spp. per ml of blood with high specificity, and differentiates between H. americanum and H. canis amplicons. A surprising 300-fold increase of H. americanum 18S rDNA targets occurred during 3-0 days of storage of positive blood specimens. Examination of 614 EDTA-blood samples submitted mostly from the southeastern Unites States from dogs with suspected hepatozoonosis identified H. americanum in 167 samples (27.2%). An additional 14 samples (2.3%) were positive for H. canis, and 14 samples (2.3%) were positive for both H. americanum and H. canis. These results suggest that the Hepatozoon spp. 18S rDNA quantitative PCR may be a valuable tool that can improve diagnosis and therapy of canine hepatozoonosis.     

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp

To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of Hepatozoon spp., whereas DNA of H. americanum sporozoites was amplified in the presence of either universal or Hepatozoon spp.-specific primer sets; the amplified products were approximately 600bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of H. americanum sequences, within a group comprising other Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is H. canis, supporting previous reports that were based on morphological and morphometric analyses.     

Characterization of stages of Hepatozoon americanum and 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 transmission of Hepatozoon americanum to rodents

Laboratory-raised cotton rats (Sigmodon hispidus), outbred white mice (Mus musculus), and C57BL/6J-Lystbg-J/J mice (M. musculus) that were administered approximately 50 sporulated oocysts of Hepatozoon americanum (AF176836) by gavage developed inflammatory lesions containing parasitic cystozoites in cardiac and skeletal muscle, kidney, and lung. Sprague-Dawley rats (Rattus norvegicus) similarly exposed showed no evidence of infection. Cystozoites were first detected by histopathologic examination four weeks after exposure to oocysts. Globular, PAS-positive material accumulated around the cystozoites as the duration of infection lengthened. Nested PCR analysis of tissues collected 16 weeks post-exposure was positive for the 18S rRNA Hepatozoon sp. gene and the DNA sequence of the fragment amplified was 99.6% and 99.8% identical to H. americanum sequences previously reported from naturally-infected dogs (AF176836 and AY864676, respectively). Merogonous and gamontogonous stages of the parasite were not detected in any of the cystozoite-infected rodents.     

Analysis of the 18S rRNA gene sequence of a Hepatozoon detected in two Japanese dogs

Partial sequences of the 18S rRNA gene (625 bp) from a Hepatozoon detected in two canine hepatozoonosis cases, one clinical and one subclinical, in Japan were analyzed. Both sequences were identical to each other and they were closely related to the Hepatozoon canis strain found in Israel with 99% (617/625) nucleotide identity. Both Hepatozoon americanum and Hepatozoon catasbianae were distantly related to the Japanese Hepatozoon with 94% (586/625) and 91% (566/625) identities, respectively. In a phylogenetic tree, the Japanese Hepatozoon was most closely related to H. canis from Israel but was significantly different than H. americanum and H. catasbianae. These results suggest that the Hepatozoon detected in the Japanese dogs might be a strain variant of H. canis, but is apparently a different species than H. americanum.     

Molecular characterization of Hepatozoon spp. infection in endangered Indian wild felids and canids

Hepatozoon species are parasites that infect a wide variety of domestic and wild animals. The objective of this study was to perform the molecular detection and characterization of Hepatozoon spp. in Asiatic lion, Indian tiger, Indian leopard, Indian wild dog, Indian domestic dog and cat based on partial 18S rRNA gene sequences from Hepatozoon spp. in the naturally infected animals. Hepatozoon spp. could be detected in blood samples of 5 out of 9 Asiatic lions, 2 out of 5 Indian tigers, 2 out of 4 Indian leopards and 2 out of 2 Indian wild dogs and, 2 out of 4 domestic cats and 2 out of 3 domestic dog samples by PCR. Sequencing of PCR amplicon and BLAST analysis of partial 18S rRNA gene sequences indicated that the Hepatozoon spp. in Asiatic lion, Bengal tiger, Indian leopard and domestic cat was Hepatozoon felis (98-99% similarity) and in the Indian wild and domestic dog the phylogenetic neighbour was Hepatozoon canis (97-100% similarity). Presence of H. felis and H. canis in both domestic and wild animals suggested that they are not host specific and the same parasite causes infection in domestic and wild felids and canids in India and from different parts of the world. To our knowledge, this is the first report on detection and molecular characterization of H. felis infection in Asiatic lions, Indian tigers, Indian leopards and H. canis in Indian wild dog. Hepatozoon spp. may be a potential pathogen and an opportunistic parasite in immuno-compromised animals and could thus represent a threat to endangered Indian wild felids and canids.     

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.     

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.     

A case of Iriomote cat (Prionailurus bengalensis iriomotensis) with Hepatozoon felis parasitemia

We herein present clinical findings of an Iriomote cat with Hepatozoon felis parasitemia. A male Iriomote cat was captured for ecological analyses three times from January 2010 to January 2011. Although this cat did not show any hematological abnormalities at the time of the first capture, H. felis parasitemia and increased serum creatine kinase levels were detected at the second and third captures. H. felis infection was confirmed by polymerase chain reaction, and amplified 18S ribosomal RNA gene fragments were 100% identical to those of H. felis in leopard cats in Korea. Although the virulence of H. felis in this cat was suggested to be low, this is the first report of an H. felis-infected Iriomote cat with parasitemia.     

Treatment of dogs infected with Hepatozoon americanum: 53 cases (1989-1998)

OBJECTIVE: To determine clinical and pathologic findings before and after short-term (group 1) and long-term (group 2) treatment in dogs with Hepatozoon americanum infection. DESIGN: Retrospective study. ANIMALS: 53 dogs with H. americanum infection. PROCEDURE: Medical records of dogs that were treated for hepatozoonosis diagnosed on the basis of meront or merozoite stages in skeletal muscle were reviewed. RESULTS: Circulating gametocytes of H. americanum were identified in 12 of 53 dogs. Dogs were treated with various drugs, including toltrazuril, trimethoprim-sulfadiazine, clindamycin, pyrimethamine, and decoquinate. Mean WBC counts prior to treatment were 85,700 and 75,200 cells/microl in groups 1 and 2, respectively, and 1 month after initiation of treatment were 12,600 and 14,600 cells/microl, respectively. Initial response to treatment was excellent in all dogs. Twenty-three of 26 dogs in group 1 relapsed at least once and died within 2 years; mean (+/- SD) survival time was 12.6+/-2.2 months. Twenty-two of 27 group-2 dogs survived; 11 dogs had no clinical signs and were still receiving decoquinate (mean duration of treatment, 21 months), 11 dogs had no clinical signs after treatment for 14 months (range, 3 to 33 months; mean survival time, 39 months [range, 26 to 53 months]), 2 dogs were lost to follow-up, and 3 dogs were euthanatized because of severe disease. CONCLUSIONS AND CLINICAL RELEVANCE: Although no treatment effectively eliminated the tissue stages of H. americanum, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and excellent quality of life.     

Neutropenia associated with osteomyelitis due to Hepatozoon canis infection in a dog

A 4-year-old, intact male Shiba dog was referred to Yamaguchi University Animal Medical Center, Yamaguchi, Japan, for the following complaints: anorexia, lethargy, intermittent fever, gingival bleeding and abdominal purpura. The dog presented with persistent neutropenia. Histopathological examination of a bone marrow sample revealed round to oval structures that resembled Hepatozoon micromerozoites and formed a "wheel-spoke" pattern. Furthermore, mature neutrophils were observed around these structures. PCR and sequencing using bone marrow aspirate confirmed Hepatozoon canis (H. canis) infection. These findings suggest that the neutropenia observed in this case was associated with osteomyelitis due to H. canis infection. This is the first report of neutropenia associated with H. canis infection. H. canis infection can be included in the differential diagnosis in canine cases of neutropenia in areas where the disease is endemic.     

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).     

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.     

Experimental transmission of the Texas strain of Hepatozoon canis

Nine dogs were fed Hepatozoon canis-exposed Rhipicephalus sanguineus ticks; 6 showed clinical signs suggestive of canine hepatozoonosis, and the parasite was found in sections of muscle tissue from 2 of the dogs. Schizont-like cysts were found in the skeletal muscle of both infected dogs as were gametocytes in the circulating leucocytes. Periosteal new-bone lesions were seen in 1 dog. Attempts to infect a raccoon, 3 cats, 6 laboratory mice, and tick- or canine-cells in vitro, failed.     

Myocarditis and myositis due to infection with Hepatozoon species in pine martens (Martes martes) in Scotland

Postmortem examinations of four pine martens which had died as a result of road accidents in Scotland revealed focal, granulomatous lesions in the heart and skeletal muscles of three of them. An immunoperoxidase staining technique showed that the lesions were due to infection with Hepatozoon species. A PCR-based assay was used to confirm the presence of Hepatozoon DNA in the infected tissues. The nucleotide base sequence of the PCR products suggested that the infecting organism was probably a new species of Hepatozoon, most closely related to, but distinct from, Hepatozoon canis. The pine martens were in good physical condition and there was no indication that the infection was causing ill health.     

American canine hepatozoonosis.

Hepatozoon americanum infection is an emerging tickborne disease in the southern United States. This organism causes a very different and much more severe disease than does Hepatozoon canis, the etiologic agent of canine hepatozoonosis in the rest of the world. H americanum is transmitted through ingestion of the definitive host, Amblyomma maculatum (the Gulf Coast tick). Clinical signs of American canine hepatozoonosis tend to wax and wane over time and may include lameness, weakness, pain, muscle atrophy, fever, and mucopurulent ocular discharge. Radiographs typically reveal periosteal proliferation of various bones. Extreme leukocytosis is the most common laboratory finding, along with a mild elevation of serum alkaline phosphatase. Diagnosis is made by visualization of gamont-containing neutrophils or monocytes on examination of blood smears; observation of typical cysts, meronts or pyogranulomas on muscle biopsy; or detection of serum antibodies against H americanum sporozoites. Common complications of chronic infection include glomerulopathies, amyloidosis, and vasculitis. Although the prognosis for this disease in the past was guarded to poor, recent advances in treatment have increased the long-term survival rate of infected dogs.     

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.     

A serosurvey of Hepatozoon canis and Ehrlichia canis antibodies in wild red foxes (Vulpes vulpes) from Israel

A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.     

Hepatozoon americanum: an emerging disease in the south-central/southeastern United States

Objective – To review the clinical epidemiologic and pathophysiologic aspects of Hepatozoon americanum infection in dogs.
Data Sources – Data from veterinary literature were reviewed through Medline and CAB as well as manual search of references listed in articles pertaining to American canine hepatozoonosis.
Veterinary Data Synthesis – H. americanum is an emerging disease in endemic areas of the United States. It is vital that practitioners in these areas become familiar with the clinical syndrome of hepatozoonosis and the diagnostic modalities that can be utilized to document the presence of infection. Additionally, veterinarians must understand the epidemiology of the disease in order to better prevent infections in their veterinary patients. Recent data have been published that shed new light on transmission of H. americanum to dogs; however, much remains unknown regarding patterns of infection and the natural vertebrate host source.
Conclusions – While the prognosis for untreated H. americanum remains poor, for patients in which the disease is recognized and properly treated the outcome is favorable. Understanding the complex life cycle, numerous clinical symptoms, and treatment protocol will assist veterinarians who are treating patients with hepatozoonosis.