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.     

A parasitological, molecular and serological survey of Hepatozoon canis infection in dogs around the Aegean coast of Turkey

Canine hepatozoonosis is caused by the tick-borne protozoon Hepatozoon spp. The prevalence of the infection in the Aegean coast of Turkey was investigated by examination of blood smear parasitology and polymerase chain reaction (PCR) using blood samples from 349 dogs collected from Central Aydin, Kusadasi, Selcuk, Central Manisa, Bodrum and Marmaris within the Aegean coast of Turkey. The indirect fluorescent antibody test (IFAT) for the detection of Hepatozoon canis antibodies was also used to detect the exposure rate to H. canis. PCR amplifying a 666bp fragment of 18S rRNA gene of Hepatozoon spp. was used in the epidemiological survey. The prevalence of Hepatozoon spp. infection was 10.6% by blood smear parasitology and 25.8% by PCR. IFAT revealed that 36.8% of serum samples were positive for antibodies reactive with Hepatozoon spp. The PCR products of 18S rRNA gene of Hepatozoon spp. isolated from six infected dogs, one isolate originating from each of the six different locations, were sequenced. The results of sequence analysis indicate that they are closely related to Indian and Japanese isolates of H. canis. This is the first epidemiological study on the prevalence of H. canis infection in the dog, in Turkey.     

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.     

Hepatozoon spp.: report of some cases in dogs in Brasília, Brazil

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon spp. It has been reported in the United States, southern Europe, the Middle East, Africa and the Far East. In Brazil, canine hepatozoonosis is an emerging protozoal tick-borne disease, and is characterized by distinct clinical signs. The objective of this study was to analyze the laboratory findings of some hepatozoonosis cases in dogs in Brasília, Brazil, and their clinical signs. The animals of this experiment showed low parasitemia, similar to H. americanum, but the clinical signs presented were similar to H. canis. According to our observations and in agreement with O'Dwyer et al. [Vet. Parasitol. 94 (2001) 143], the Brazilian Hepatozoon appears more to resemble the species found in the eastern Hemisphere than with H. americanum of North America, or could be caused by a new species. Our data revealed that hepatozoonosis could be considered endemic in Brasília.     

Dogs with Hepatozoon canis respond to the oxidative stress by increased production of glutathione and nitric oxide

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon spp. It has been reported in the United States, southern Europe, the Middle East, Africa and the Far East. In Turkey, canine hepatozoonosis was reported for the first time in 1933. In the present study, serum glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO) and ceruloplasmin levels were analysed in 14 dogs infected with Hepatozoon canis as well as in 10 healthy dogs. Blood smears were prepared from peripheral blood and ticks were collected for identification in the laboratory. Rhipicephalus sanguineus was found only on diseased dogs. No ticks were observed on healthy dogs. The diagnosis of H. canis is made mainly by the detection of gametocytes within neutrophils and monocytes. The haematological diagnosis was confirmed using PCR analyses by amplifying a partial 18S rRNA gene sequence of Hepatozoon spp. Infection was detected in 14 animals. Compared to controls, the serum GSH, MDA and NO levels in infected animals increased significantly (p<0.05, <0.01 for MDA), whereas the concentrations of ceruloplasmin in diseased animals remained unaltered. The results of the present study suggest that in dogs infected with H. canis increased levels of GSH, MDA and NO may be related to host's defences against parasitic infection.     

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.     

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.     

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.     

A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria

The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.     

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.     

Prevalence and molecular characterization of Hepatozoon canis in dogs from urban and rural areas in Southeast Brazil

The objective of this survey was to investigate the prevalence of Hepatozoon infection in dogs in the rural and urban areas of Uberlândia, Brazil by PCR and molecular characterization. DNA was obtained from blood samples collected from 346 local dogs from both genders and various ages. Seventeen PCR products from positive blood samples of urban dogs and 13 from the rural dogs were sequenced. Partial sequences of the 18S rRNA gene indicated that all 30 dogs were infected with Hepatozoon canis similar in sequence to H. canis from southern Europe. Four local dog sequences were submitted to GenBank (accessions JN835188; KF692038; KF692039; KF692040). This study indicates that H. canis is the cause of canine hepatozoonosis in Uberlândia and that infection is similarly widespread in rural and urban dogs.     

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.     

Retrospective evaluation of laboratory data on canine vector-borne infections from the years 2004-2008

The detection and therapy of canine vector-borne diseases in imported dogs are of major importance in small animal practice. Over the last years, the import of dogs from the Mediterranean region and Southeast Europe has increased, countries often endemic for a variety of vector-borne diseases such as babesiosis, hepatozoonosis, leishmaniosis, dirofilariosis or ehrlichiosis. This retrospective study presents the evaluation of data from our diagnostic laboratory on vector-borne infections in imported dogs from the years 2004-2008. Specific antibodies were detectable in 20.5% of all samples with individual detection rates of 8.9%, 9.6% and 10.8% for Babesia canis ssp., Leishmania spp. and/or Ehrlichia canis. A total of 5.5% of all samples tested by direct methods were positive. Up to 1.1% of Giemsa-stained blood/buffy coat smears were positive for B. canis ssp., Rickettsia spp. or Hepatozoon spp. Microfilariae were detectable by the Knott's Test in 6.4% and heartworm antigen was detectable using the DiroChek-ELISA in 3% of the examined samples. EDTA-blood samples were positive for Leishmania spp.-(14.9%), E. canis- (5.3%) and A. phagocytophilum-DNA (5.0%) by PCR. Therewith, imported dogs have a high chance of being carriers of pathogens. As some diseases may also be of a zoonotic concern, in case of the availability of competent vectors, the key focus in the future should be aimed at the prevention of importing infected dogs or at compulsory diagnostic screening and treatment of infected dogs at the time of import.     

Infection with a Hepatozoon sp. closely related to Hepatozoon felis in a wild Pampas gray fox (Lycalopex -Pseudalopex -gymnocercus) co-infected with canine distemper virus

A species of Hepatozoon closely related to Hepatozoon felis found in the skeletal and cardiac muscle of a wild Pampas gray fox (Lycalopex gymnocercus) is described. The fox was euthanized after showing severe incoordination. On necropsy and histopathology there was bilateral, diffuse, severe, sub-acute, necrotizing bronchointerstitial pneumonia, with intracytoplasmic and intranuclear eosinophilic inclusion bodies. Canine distemper virus was detected by immunohistochemistry in the bronchiolar epithelium, syncytial cells, alveolar macrophages and pneumocytes. The skeletal muscle and myocardium contained multiple round to oval protozoan cysts ranging from 64 μm × 75 μm to 98 μm × 122 μm, with a central eosinophilic meront-like core surrounded by concentric rings of mucinous material resembling Hepatozoon americanum cysts but smaller in size. Macrophages within rare pyogranulomas and monocytes/macrophages in adjacent sinusoidal blood vessels in the skeletal muscle contained intracytoplasmic round protozoa consistent with merozoites or developing gamonts of Hepatozoon. Hepatozoon sp. infection was confirmed by PCR of skeletal muscle and the sequenced 18S rRNA PCR product was found to be 99% identical to H. felis by BLAST analysis and deposited in GenBank as accession number HQ020489. It clustered together in the phylogenetic analysis with published H. felis sequences and separately from H. canis, H. americanum and other Hepatozoon species. However, the close relatedness of the fox Hepatozoon to H. felis does not rule out infection with a different and possibly unknown Hepatozoon species.     

New advances in molecular epizootiology of canine hematic protozoa from Venezuela, Thailand and Spain

The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied. In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77 per cent of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied. In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30 per cent of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found. In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira 1 from Brazil.     

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.     

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.     

Molecular detection of Hepatozoon canis in dogs and ticks in Shaanxi province,China

Hepatozoon canis, transmitted by Rhipicephalus sanguineus, is a tick-borne pathogen and causes canine hepatozoonosis. Until now, only limited previous studies were conducted on the molecular detection and characterization of Hepatozoon sp. in dogs in China. Blood samples were collected from 93 sick dogs that were clinically diagnosed as babesiosis but tested negative for Babesia, and 103 apparently healthy dogs, as well as their infesting ticks in Xi’an and Hanzhong cities, Shaanxi province of China. PCR amplifying partial 18S rRNA gene was used to detect the DNA of Hepatozoon sp. Genetic and phylogenetic analysis were performed to determine the Hepatozoon species. Our results demonstrated that H. canis was identified from the sick dogs and the infested ticks in Hanzhong, with no significant differences of prevalence between both genders and ages. No positive blood or tick samples were found in Xi’an. Moreover, all the 18S rRNA gene sequences recovered from both dogs and the infested ticks showed a high genetic similarity with each other, and also presented a close relationship with other known sequences in and outside China. In conclusion, H. canis was identified in babesiosis-suspected dogs and ticks infesting them in Shaanxi, China, although the association between clinical signs and H. canis need further study.     

Detection of Ehrlichia canis by PCR in different tissues obtained during necropsy from dogs surveyed for naturally occurring canine monocytic ehrlichiosis

A molecular study for the detection of Ehrlichia canis was carried out on tissues obtained at necropsy from randomly selected dogs with the intention of investigating naturally-occurring canine ehrlichiosis. The tissues evaluated for the presence of E. canis included lymph nodes, spleen, liver, bone marrow, and blood. Eight of the 18 dogs included were found to be positive for E. canis by polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene. Two dogs were positive for Anaplasma platys of which one dog was co-infected with E. canis and A. platys. Blood (5/8) and lymph nodes (5/8) were the tissues found to yield the highest number of positive E. canis PCR results with 7/8 dogs positive in the blood or lymph node. E. canis and A. platys DNA could be amplified by PCR when tissue samples were obtained 72h after the time of death.