Hepatozoon Sp Research Articles

Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Presence of Ehrlichia sp and Hepatozoon sp in dogs infested with ticks or with previous exposure attended at a veterinary centre in Trujillo, Peru

The present study determined the prevalence of Ehrlichia sp morulae and Hepatozoon sp gamonts in dogs treated at a veterinary medical centre in the city of Trujillo. Blood samples were taken from dogs infested with ticks or exposed in the three months prior to the study. Blood samples (3 mL) were collected from the cephalic vein of 95 dogs in EDTA tubes. The samples were centrifuged. A drop of the phlogistic layer of each sample was placed on a slide, which was extended with a coverslip and subjected to Wright staining. The smears were observed under an optical microscope at 40X and 100X. A prevalence of Ehrlichia sp of 31.6 ± 9.4% was found. No presence of Hepatozoon sp gamonts was observed.

Molecular Diagnostic Confirmation and Haemato-Biochemical Changes of Clinical Hepatozoon canis Infection in a Rhipicephalus sanguineus Tick-Infested Dog from Colima, Mexico

Canine hepatozoonosis is a tick-borne disease caused by Hepatozoon canis (James, 1905) and Hepatozoon americanum (Vincent-Johnson, 1997). The disease has been reported infrequently in Mexico. The aim of this report was to describe haemato-biochemical changes and confirm diagnosis by molecular-based methods of clinical infection by H. canis in a dog. A 9-year-old female, boxer dog, Canis familiaris (Linnaeus, 1758), was taken to a veterinary clinic at Colima, Mexico. The dog showed depression and more than 2 weeks of gradual weight loss without decreased appetite. Rhipicephalus sanguineus (Latreille, 1806) ticks were found when the dog was physically examined. Blood samples were collected for smear, hematological, and biochemical analyses, and PCR assays. Hematological analysis revealed an apparently non-regenerative normochromic normocytic moderate anemia, mild thrombocytopenia, left shift without neutrophilia, and mild lymphopenia associated with possible stress. Biochemical analysis showed hyperbilirubinemia, hyperamylasemia, hypoalbuminemia, hyperglobulinemia, increased ALT, and hypocalcemia. Microscopic examination of Diff-Quick-stained blood smears showed capsule-shaped structures consistent with gametocytes of Hepatozoon sp. Clinical history, hematological changes, and molecular assays described in the report confirmed the first case of canine hepatozoonosis caused by H. canis in the Pacific region of Mexico.

Candidatus Neoehrlichia mikurensis and Hepatozoon sp. in voles (Microtus spp.): occurrence and evidence for vertical transmission

Candidatus Neoehrlichia mikurensis (CNM) and Hepatozoon spp. are important vector-borne parasites of humans and animals. CNM is a relatively recently discovered pathogen of humans. Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide. The present study aimed to determine the prevalence of CNM and Hepatozoon spp. in three species of Microtus and to assess the occurrence of vertical transmission in naturally-infected voles. Molecular techniques were used to detect pathogen DNA in blood and tissue samples of captured voles and their offspring. The prevalence of CNM in the vole community ranged 24–47% depending on Microtus species. The DNA of CNM was detected in 21% of pups from three litters of six infected Microtus dams (two Microtus arvalis and one M. oeconomus) and in 3/45 embryos (6.6%) from two litters of eight CNM-infected pregnant females. We detected Hepatozoon infection in 14% of M. arvalis and 9% of M. oeconomus voles. Hepatozoon sp. DNA was detected in 48.7% of pups from seven litters (6 M. arvalis and 1 M. oeconomus) and in two embryos (14.3%) obtained from one M. arvalis litter. The high prevalence of CNM infections in the Microtus spp. community may be a result of a relatively high rate of vertical transmission among naturally infected voles. Vertical transmission was also demonstrated for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.

Diversity of Hepatozoon species in wild mammals and ticks in Europe

BackgroundHepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe.MethodsSamples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene.ResultsOf the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozooncanis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H.martis in 125 (3 Artiodactyla, 122 Carnivora), H.sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H.ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H.canis (n = 22).ConclusionsHepatozooncanis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H.martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H.canis in ticks other than R.sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.Graphical

Novel Ehrlichia and Hepatozoon genotypes in white-eared opossums (Didelphis albiventris) and associated ticks from Brazil

White-eared opossums (Didelphis albiventris) are well adapted to anthropized areas. The increased contact with domestic animals and humans mediates the transmission of arthropod-borne pathogens. Despite the worldwide occurrence of tick-borne Anaplasmataceae and Hepatozoidae species in a variety of vertebrates, few studies reported serological evidence or molecular detection of theses agentes in marsupials. Up to now, while Ehrlichia/Anaplasma spp. have only been detected in marsupials from Brazil, Hepatozoon spp. have been reported in marsupials from Chile, Australia and Brazil. The present work aimed to investigate, using molecular techniques and blood smear analysis, the presence of Ehrlichia spp., Anaplasma spp., and Hepatozoon sp. in the blood and ticks collected from D. albiventris in urban forest fragments from midwestern Brazil. Between May and December 2017, 43 D. albiventris (27 males and 16 females) were captured for blood and tick collection in the city of Campo Grande, state of Mato Grosso do Sul, midwestern Brazil. Ticks (46 Amblyomma dubitatum nymphs and 24 Amblyomma spp. larvae) were collected from 14 out 43 (32.5%) of the white-eared opossums. Panoptic-stained blood smears were performed using peripheral blood (tail tip) of the captured opossums. DNA extracted from blood and tick samples were subjected to PCR/qPCR assays for Anaplasmataceae agents (rrs, gltA, groEL, sodB, and dsb genes, and 23S-5S intergenic region) and Hepatozoon spp. (18S rRNA gene), followed by Sanger sequencing, BLASTn and phylogenetic analyses. An inclusion resembling Ehrlichia morulae was found in a white-eared opossum's monocyte from a blood smear stained with Panoptic. Five (11.63% [5/43]) white-eared opossums’ blood samples and 7 (25% [7/28]) tick samples (2 pools of Amblyomma spp. larvae and 5 pools of A. dubitatum nymphs) were positive for Anaplasmataceae via a PCR assay targeting the conserved rrs gene. Phylogenetic analysis based on the rrs gene positioned three sequences obtained from opossums and ticks together as a subclade within the Ehrlichia canis clade. However, all samples were negative in a qPCR assay specific for E. canis based on the dsb gene. Phylogenetic analyses positioned the gltA and 23S-5S ITS sequences obtained from opossums’ blood samples in a separate clade from the other validated Ehrlichia species. One (2.3% [1/43]) opossum blood sample was positive for the 18S rRNA gene of Hepatozoon sp. The phylogenetic analysis positioned the Hepatozoon sp. sequence obtained from a D. albiventris specimen in a clade with a sequence previously detected in a black storm petrel (Oceanodroma melania) from Mexico. All the other sequences of Hepatozoon sp. previously detected in marsupials from Brazil were positioned in a separated clade. The present work showed the occurrence of putative novel genotypes of Ehrlichia sp. and Hepatozoon sp. in white-eared opossums and associated A. dubitatum ticks from midwestern Brazil.

Prevalence of Ehrlichia canis and Hepatozoon canis in sheltered dogs in southern Aburrá Valley, Colombia.

Background: Bacteria of the Anaplasmataceae family and canine hemoparasitic protozoans transmitted by ticks are common in Colombia due to circulation and biological adaptation of vector Rhipicephalus sanguineus sensu lato (s.l.). Objective: To detect the circulation of Ehrlichia canis and Hepatozoon canis in sheltered dogs in three municipalities in southern Aburrá Valley, province of Antioquia, Colombia. Methods: Primers were used to amplify the 16S rRNA associated with the Anaplasmataceae family, dsb for Ehrlichia sp. and 18S rRNA for Hepatozoon sp. Results: Of the 357 samples of venous blood obtained, representing all the sheltered dogs in the study zone, Ehrlichia canis DNA was detected in 2.2% of individuals, showing identity of 100% with previous sequences from the GenBank. Hepatozoon canis showed 8.7% (31/357) prevalence of infection, with 100% identity to genotypes from Japan, Brazil, and Spain. Only one sequence of H. canis exhibited a phylogenic divergence concerning H. canis previously reported in Brazil and the Old World. Conclusions: This study confirms the circulation of E. canis and H. canis in asymptomatic shelter dogs in the south-central zone of the Aburrá Valley, province of Antioquia, Colombia. The present study is the first molecular detection of H. canis in the Province of Antioquia and the third report of canine hepatozoonosis from Colombia, highlighting the importance of considering this agent in veterinary clinic.

First report of Hepatozoon sp. In a dog in the Western Amazon, Brazil

Canine hepatozoonosis is a worldwide disease, caused by protozoa of the genus Hepatozoon, whose transmission occurs when the dog ingests ticks or part of them containing sporulated oocysts. The objective was to report the identification of subclinical Hepatozoon sp. in a dog in the state of Rondônia, in the Western Amazon, Brazil. A female dog, two years old, mixed breed, resident in the urban area of the municipality of Rolim de Moura (Rondônia) was taken to a veterinary clinic due to lameness of the right hind limb. In the anamnesis, the animal did not present pain on palpation of the affected limb. There were ectoparasites in the animal's body (fleas and ticks). Blood was collected in a tube with EDTA to perform the complete blood count and blood smears. The blood count showed normocytic and normochromic anemia and thrombocytopenia. In blood smears, gametes were observed in neutrophils. Given the report, we concluded that Hepatozoon sp. circulates in dogs from the Western Amazon, emphasizing the importance of including the investigation of hemoparasites in the veterinary clinical routine, even when the animal is asymptomatic.

THE EFFECT OF HEPATOZOON SP.INFECTION ON SOME BIOLOGICAL PARAMETERS OF THE MOSQUITO CULEX PIPIENS COMPLEX

Hepatozoon is a very diverse genus with more than 300 species currently assigned to it, basedlargely on morphological characters, host specify and life cycle patterns. Peripheral blood smearsof naturally infected vipers Cerastes cerastes cerastes (hornless) showed intracytoplassmicbanana-shaped gametocytes in the erythrocytes. Upon initial examination in 12% out of 100 collectedvipers harbored Hepatozoon sp. Effect of Hepatozoon sp. infection on some biological parametersof Culex pipiens complex showed no significant difference between longevity of noninfectedand infected ones, and between mortality rate, and pre-oviposition period of both. Fertilityfemale mosquitoes were significantly affected. Infected females produced significantlyfewer eggs/female as compared to those deposited by non-infected ones, without significant differenceincubation periods of eggs deposition. The mean oviposition rate of fully engorged infectedCx. pipiens complex female was highly significant less than that of non-infected femaleones. Statistical analysis revealed a highly significant difference (P<0.0001) between the larvalmortality from infected and non-infected female ones.

Genetic diversity of Hepatozoon spp. in rodents from Chile.

This study aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from Valdivia, Chile. A total of 74 rodents (synanthropic n=38; wild n=36) were trapped in Valdivia. We performed conventional PCR assays for Apicomplexa organisms targeting two overlapping 18S rDNA gene fragments (600 bp and 900 bp) followed by sequencing of selected amplicons. Hepatozoon spp. occurrence was 82.43% (61/74). Twelve sequences obtained from the 600 bp and ten from the 900 bp 18S rDNA fragments were identified as Hepatozoon sp. Six sequences obtained from 18S rDNA-based overlapping PCR protocols were used for concatenated (1,400 bp) phylogenetic, haplotype and distance analyses. Hepatozoon spp. 18S rDNA concatenated sequences from the present study were detected in Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus, and Abrothrix longipilis grouped with Hepatozoon species earlier described in rodents and reptiles from Chile and Brazil. Nucleotide polymorphism of the six 18S rDNA sequences (1,400 bp) from this study, and other Chilean sequences from rodents and rodent's ticks, showed high diversity with a total of nine Chilean haplotypes. Three haplotypes from Valdivia were identified for the first time in this study, suggesting the circulation of novel haplotypes in rodents from southern Chile.

Post mortem protozoan hemoparasites detection in wild mammals from Mato Grosso state, Midwestern Brazil.

To a better insight into the epidemiology and genetic diversity of protozoan hemoparasites infections in wild mammals, this study aimed to the post mortem detection of DNA from species of the order Piroplasmida (Babesia sp., Cytauxzoon sp., and Theileria sp.) and suborder Adelorina (Hepatozoon sp.) using polymerase chain reaction based on the 18S rRNA gene followed by genetic sequencing of blood and spleen samples collected from carcasses of 164 free-ranging and captive wild mammals from Mato Grosso state. Among them, one Leopardus pardalis, three Panthera onca, two Puma concolor were positive for Cytauxzoon sp., and six Tapirus terrestris tested positive for Piroplasmida, while one L. pardalis was positive for Hepatozoon sp. Furthermore, an uncharacterized piroplasmid genetically related to Theileria sp. previously detected in cats from Brazil was described in lowland tapirs. Despite the controversy regarding the epidemiological threat of these protozoa, the detection of these tick-borne agents in wild free-living and captive mammals, even when asymptomatic, demonstrates the importance of monitoring, particularly in hotspots such as the state of Mato Grosso, to verify the circulation and genetic diversity, to anticipate the possible emergence of diseases, and even their consequences to other animals as well as humans.

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.

Hepatozoon Infecting Bats in the Southeastern Brazilian Rainforest.

Tick-borne protozoans of the genus Hepatozoon are obligate hemoparasites that can infect domestic and wild terrestrial vertebrates. Main hepatozoonosis affects canids and involves mainly Hepatozoon canis and Hepatozoon americanum. However, molecular studies revealed the capacity of H. canis to infect a wide range of wild mammals. In July 2018, we conducted an epidemiological survey for tick-borne pathogens in wild hosts, assaying Hepatozoon sp. occurrence in 34 bats captured in different habitats within a conservation unit in the state of Espírito Santo, southeastern Brazil. Blood and spleen tissue DNA samples were submitted to PCR amplifications of Babesia/Theileria and Hepatozoon 18S rRNA gene and 21% (7/34) were positive for Hepatozoon sp. Phylogenetic inferences grouped the obtained sequences from Seba's short-tailed bat (Carollia perspicillata) with the H. canis cluster, and from the great fruit-eating bat (Artibeus lituratus) with rodent-associated Hepatozoon cluster. Further studies are needed to characterize the epidemiological role of Seba's short-tailed bat and the great fruit-eating bat in the wild transmission cycle of these hemoparasites in Brazil.

A survey of hemoparasites and ectoparasites in Nasua nasua Linnaeus, 1766 with a redescription of Hepatozoon procyonis Richards, 1961 based on morphological and molecular data.

Hemoparasites are vector-borne parasites that infect wild carnivores worldwide. Since data on hemoparasite infections in Nasua nasua from Brazil are lacking, the aim of this study was to investigate the occurrence of hemoparasites and ectoparasites in N. nasua from different areas of Brazil. Blood samples and ectoparasites from 83 N. nasua were collected in Botucatu, Palmital, and São Paulo municipalities. Samples were screened via microscopy and molecular methods to detect hemoparasites. Tissues from two N. nasua were obtained for histopathological and molecular analyses. All 83 samples were negative for piroplasms on morphological and molecular examination. Thin blood smears of nine animals were positive for Hepatozoon gamonts. The gamonts shared morphological characteristics of Hepatozoon procyonis. Meronts were detected in the liver and spleen tissue of one animal. Twenty-one blood samples and four tissue samples were PCR positive for Hepatozoon sp. The sequences obtained were 97% identical to those of Hepatozoon felis, Hepatozoon ursi, and Hepatozoon sp. Based on searches for similarity and morphology, we identified the sequences as belonging to H. procyonis. This study provides epidemiological data on hemoparasite infections and redescribe H. procyonis based on morphological, morphometrical, and molecular analyses.

Molecular detection and phylogenetic analysis of Hepatozoon spp. in questing Ixodes ricinus ticks and rodents from Slovakia and Czech Republic

By amplification and sequencing of 18S rRNA gene fragments, Hepatozoon spp. DNA was detected in 0.08% (4/5057) and 0.04% (1/2473) of questing Ixodes ricinus ticks from Slovakia and Czech Republic, respectively. Hepatozoon spp. DNA was also detected in spleen and/or lungs of 4.45% (27/606) of rodents from Slovakia. Prevalence of infection was significantly higher in Myodes glareolus (11.45%) than in Apodemus spp. (0.28%) (P < 0.001). Sequencing of 18S rRNA Hepatozoon spp. gene amplicons from I. ricinus showed 100% identity with Hepatozoon canis isolates from red foxes or dogs in Europe. Phylogenetic analysis showed that at least two H. canis 18S rRNA genotypes exist in Slovakia of which one was identified also in the Czech Republic. The finding of H. canis in questing I. ricinus suggests the geographical spread of the parasite and a potential role of other ticks as its vectors in areas where Rhipicephalus sanguineus is not endemic. Sequencing of 18S rRNA gene amplicons from M. glareolus revealed the presence of two closely related genetic variants, Hepatozoon sp. SK1 and Hepatozoon sp. SK2, showing 99-100% identity with isolates from M. glareolus from other European countries. Phylogenetic analysis demonstrates that 18S rRNA variants SK1 and SK2 correspond to previously described genotypes UR1 and UR2 of H. erhardovae, respectively. The isolate from Apodemus flavicollis (Hepatozoon sp. SK3b) was 99% identical with isolates from reptiles in Africa and Asia. Further studies are necessary to identify the taxonomic status of Hepatozoon spp. parasitizing rodents in Europe and the host-parasite interactions in natural foci.

Molecular detection and characterization of Hepatozoon spp. in dogs from the Central part of Turkey

Canine hepatozoonosis is a tick-borne protozoal disease caused by Hepatozoon spp. Two species of Hepatozoon are currently known to infect dogs as Hepatozoon canis and H. americanum. Although H. canis generally causes a chronic infection with relatively mild clinical alterations compared to H. americanum, infection by H. canis can be life-threatening. The disease is widespread in USA, Africa, Europe, South America, and Asia. To determine the frequency of infection with Hepatozoon spp. in stray dogs from Central Anatolia Region of Turkey, a total of 221 blood samples collected over a three-year period were evaluated by using genus specific Polymerase Chain Reaction (PCR) designed to amplify a fragment of 666bp located in 18 S rRNA gene of Hepatozoon spp. Eight (3.61%) blood samples were positive for Hepatozoon spp. For the classification of species, all positive PCR products were purified with a PCR purification kit and sequenced. Sequencing results of eight representative amplicons indicated that 6 were 98–99% identical to the sequence of H. canis and the other 2 sequences were 95–97% identical to the sequence of Hepatozoon spp. So it was named Hepatozoon sp. MF. A phylogenetic tree was constructed from the sequences of the tick-borne agents identified previously and in this study using the neighbor-joining method. The nucleotide sequences were compared to the H. canis sequences reported in Turkey using the nucleotide Basic Local Alignment Search Tool (BLAST) program. The results of this study are significant in terms of the presence of a novel canine Hepatozoon genotype.

Arthropod-borne pathogens circulating in free-roaming domestic cats in a zoo environment in Brazil

Recently, tick and flea-borne pathogens have been detected in wild carnivores maintained in captivity in Brazilian zoos. Since free-roaming cats are frequently found in Brazilian zoos, they could act as reservoirs for arthropod-borne pathogens, which could be transmitted to endangered wild carnivores maintained in captivity in these institutions. On the other hand, stray cats in zoos may play a role as sentinels to pathogens that circulate among wild animals in captivity. The present work aimed to detect the presence of Anaplasmataceae agents, hemoplasmas, Bartonella species, piroplasmas, and Hepatozoon sp. DNA in blood samples of 37 free-roaming cats in a Brazilian zoo. Three (8%) cats were positive for Anaplasma spp. closed related to Anaplasma phagocytophilum; 12 (32%) cats were positive for hemoplasmas [two (5%) for Mycoplasma haemofelis, five (13.5%) for Candidatus Mycoplasma haemominutum, and five (13.5%) for Candidatus Mycoplasma turicensis]; 11 (30%) were positive for Bartonella spp., six (16%) were positive Babesia vogeli and one (3%) for Theileria sp. Coinfection with multiple arthropod-borne agentes was observed in sampled cats. None of sampled cats were positive for Ehrlichia spp., Cytauxzoon spp., or Hepatozoon spp. in PCR. This is the first molecular detection of Babesia vogeli and Theileria sp. in domestic cats in Brazil. The control of the population of free-roaming cats in these conservation institutions is much needed aiming to prevent the potential transmission to endangered wild animals maintained in captivity, such as wild neotropical wild felids, as well as to human beings visiting zoos.

Hepatozoon canis in German red foxes (Vulpes vulpes) and their ticks: molecular characterization and the phylogenetic relationship to other Hepatozoon spp.

In this study, the prevalence of Hepatozoon spp. in red foxes (Vulpes vulpes) and their ticks from Germany, as well as molecular characterizations and phylogenetic relationship to other Hepatozoon spp. were investigated. DNA extracts of 261 spleen samples and 1,953 ticks were examined for the presence of Hepatozoon spp. by a conventional polymerase chain reaction (PCR) targeting the 18S rRNA gene. The ticks included four tick species: Ixodes ricinus, Ixodes canisuga, Ixodes hexagonus and Dermacentor reticulatus. A total of 118/261 foxes (45.2%) and 148/1,953 ticks (7.5%) were Hepatozoon PCR-positive. Amplicons from 36 positive foxes and 41 positive ticks were sequenced. All sequences obtained from foxes and 39/41 from ticks had a 99% similarity to Hepatozoon canis, whereas two ticks' sequences had a 99% identity to Hepatozoon sp. The obtained Hepatozoon sequences in this study were phylogenetically related to other Hepatozoon sequences detected in other countries, which may represent strain variants. The high prevalence of H. canis DNA in red foxes in this study supports the suggested role of those animals in distribution of this parasite. Furthermore, detection of DNA of H. canis in foxes and all examined tick species collected from those foxes allows speculating about previously undescribed potential vectors for H. canis and suggests a potential role of the red fox in its natural endemic cycles.

Questing for the identity of Hepatozoon in foxes

Apicomplexan parasites of genus Hepatozoon invade the blood cells of many mammalian species, being transmitted by range of arthropods. In domestic carnivores, three species of Hepatozoon were described to date: H. canis and H. americanum in dogs and H. felis in cats. The classification of Hepatozoon in wild carnivores is still not complete due to lack of field and experimental data as well as phylogenetic studies. The aim of this study is to carry out a survey on the prevalence and diversity of Hepatozoon sp. in red foxes Vulpes vulpes. Samples of tissues were collected from dead foxes in 11 counties of Romania; 91 samples of liver tissue were examined in total. DNA extraction was performed with commercial kit according to the manufacture's protocol. Hepatozoon sp. DNA was detected by PCR using primers amplifying 400-600 bp long part of 18S rRNA gene. These primers are commonly used for diagnostic purposes in dogs and in some studies also for detection of Hepatozoon sp. in wild carnivores. PCR products were sequenced to validate positive results of reaction. DNA of parasite was confirmed in 55% of examined samples. Recent findings classified Hepatozoon sp. in foxes and other wild canids in Europe as H. canis. However, Rhipicephalus sanguineus, the only known vector of H. canis in Europe, is absent in most of our sampling sites. Moreover, this tick is typical for the dogs but rare or even absent in foxes. In order to clarify the identity of the parasite, the next step of our study is to focus on amplification of longer or full segment of 18s rRNA gene to allow more accurate phylogenetic analyses and comparison with H. canis sequences from dogs and Hepatozoon isolates from other carnivores. This study was supported by IGA UVPS Brno, project 115/2013/FVL.

A molecular survey on host specificity of feline and canine Hepatozoon in model site of northern Kenya

Species of the genus Hepatozoon are apicomplexan parasites transmitted by variety of hematophagous arthropods to a wide range of intermediate hosts. Hepatozoon infections are broadly distributed around the world and the presence of this parasite is confirmed both in cats and dogs. Until now, two species from canines (H. canis, H. americanum) and a single species from felines (H. felis) have been reported. While the canine Hepatozoon spp. are transmitted by ticks, the vector for H. felis remains unknown. The aim of presented study was to determine the prevalence and the diversity of Hepatozoon in population of domestic cats and dogs living in close contact in rural communities of Samburu pastoralists in northern Kenya and to evaluate the existence of possible cross-transmissions between both hosts. Between years 2007-2012 in total, 135 and 258 blood samples from cats and dogs from the area of Mt. Kulal (5 localities) was collected, respectively. The DNA from blood (preserved in ethanol) was extracted using phenol-chlorophorm method and followed by conventional PCR screening. First round of PCR was done by using Piroplasmid-F and Piroplasmid-R primers, amplifying 18S rRNA gene of Hepatozoon spp. (400 bp). A second PCR assay was performed in samples positive by Piroplasmid primers to amplify a larger fragment (1400 bp) of the 18S rRNA gene. All PCR amplicons comming from positive samples were sequenced and determined according to the BLAST match. In cats, this revealed 110 out of 135 (81.5%) positive samples, of which we obtained 104 sequences with an identity of 98% -100% to an existing GenBank accession. In total, 77 sequences were confirmed as Hepatozoon felis, 26 samples was proven to be Hepatozoon sp. and a single sample was determined as Hepatozoon canis with an identity of 96%. Regarding dogs, 121 out of 258 (47%) were positive and we gained 107 sequences of which 105 showed similarity to Hepatozoon canis and the remaining two proved to be Hepatozoon sp. Despite a close contact of hosts, obviously cats and dogs do not share the same parasite, which demonstrates a great majority of clearly identified Hepatozoon canis/Hepatozoon felis. Mentioned facts could be explained either by strict host specificity or by presence of different vector or by both statements.