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 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.     

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 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.     

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.     

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 studies on Babesia,Theileria and Hepatozoon in southern Europe. Part I. Epizootiological aspects

Molecular epizootiology of piroplasmids (Babesia spp., Theileria spp.) and Hepatozoon canis was studied in mammals from southern Europe (mainly from Spain, but also from Portugal and France). Partial amplification and sequencing of the 18s rRNA gene was used for molecular diagnosis. In some particular cases (B. ovis and B. bovis) the complete 18s rRNA gene was sequenced. Blood samples were taken from domestic animals showing clinical symptoms: 10 dogs, 10 horses, 10 cows, 9 sheep and 1 goat. In addition, DNA samples were isolated from blood of 12 healthy dogs and from spleen of 10 wild red foxes (Vulpes vulpes). The results of the survey were the following: Piroplasmid infections: Approximately from 50 to 70% of wild or domestic mammals (symptomatic) were infected.Piroplasmids detected in ruminants were:COW: B. bovis, T. annulata and Theileria sp. (type C). Sheep and goat: B. ovis. Piroplasmids present in canids were: Babesia canis vogeli, Babesia canis canis, Theileria annae and B. equi. The only piroplasmid found in asymptomatic dogs was B. equi. Piroplasmids found in horse were: B. equi and B. canis canis.H. canis infections in canids: H. canis was absent of domestic dog samples, whereas all foxes studied were infected by this protozoa.Genetic analysis showed that most of piroplasmid and Hepatozoon isolates from southern Europe matched unambigously with previously described species, as demonstrated by the high level sequence identity between them, usually between 99 and 100%. Minor differences, usually detected in hypervariable regions of 18s rRNA gene are probably due to strain variations or rare genetic polymorphisms. A possible exception was B. bovis, which shows a relatively lower degree of homology (94%) with regard to other B. bovis isolates from several countries. The same is true for B. ovis, that showed a 94% identity with regard to Babesia sp. from South African cow and a 92% with rapport to B. bovis from Portugal.     

Pandemic (H1N1) 2009 influenza A virus infection associated with respiratory signs in sloth bears (Melursus ursinus)

In 2009, a pandemic influenza A virus (pH1N1) spread globally in humans and infected a broad range of captive animals with close human contact. In February 2014, a pH1N1 virus was isolated from a sloth bear with respiratory signs at a US zoo, demonstrating that recurring epidemics present an ongoing threat to animals, including threatened species. This is the first report of pH1N1 infection in sloth bears. To understand the sloth bear virus within the global context of pH1N1, phylogenetic trees were inferred including full‐length sequences from available non‐human, non‐swine hosts, representing four families in the order Carnivora and one order of birds. A combination of phylogenetic and epidemiological evidence strongly suggests the sloth bear was infected with a human‐origin pH1N1 virus, supporting the implementation of biosecurity measures to protect human and animal health.     

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.     

The first report of Hepatozoon sp. (Apicomplexa: Hepatozoidae) in neotropical felids from Brazil

In order to investigate the occurrence of Hepatozoon infection in Neotropical felids from Brazil, blood from the jugular or cephalic vein was taken from 29 non-domestic felids including ocelot (Leopardus pardalis), little spotted cat (Leopardus tigrinus), margay (Leopardus wiedii), and jaguarondi (Puma yagouaroundi) from the Northeast region of Brazil. Hepatozoon infection was confirmed by light microscopy and molecular techniques. The results showed five naturally infected felids. Partial sequences of the 18S rRNA gene of the Hepatozoon sp. from these felids were further analyzed. Sequences revealed that the isolates found are closely related to Hepatozoon sp. from domestic cats in Spain. Hepatozoon species from Neotropical felids were identified molecularly and characterized for the first time. This is also the first report of Hepatozoon infection in a little spotted cat.     

Diversity and molecular characterization of novel hemoplasmas infecting wild rodents from different Brazilian biomes

Although hemoplasma infection in domestic animals has been well documented, little is known about the prevalence and genetic diversity of these bacteria in wild rodents. The present work aimed to investigate the occurrence of hemotrophic mycoplasmas in wild rodents from five Brazilian biomes, assessing the 16S rRNA phylogenetic position of hemoplasma species by molecular approach. Spleen tissues were obtained from 500 rodents, comprising 52 different rodent species trapped between 2000 and 2011. DNA samples were submitted to previously described PCR protocols for amplifying Mycoplasma spp. based on 16S rRNA, followed by sequencing and phylogenetic inferences. Among 457 rodent spleen samples showing absence of inhibitors, 100 (21.9%) were PCR positive to Mycoplasma spp. The occurrence of hemotropic mycoplasmas among all sampled rodents was demonstrated in all five biomes and ranged from 9.3% (7/75) to 26.2% (38/145). The Blastn analysis showed that amplified sequences had a percentage of identity ranging from 86 to 99% with other murine hemoplasmas. The ML phylogenetic analysis of 16S rRNA gene of 24 positive randomly selected samples showed the presence of ten distinct groups, all clustering within the Mycoplasma haemofelis. The phylogenetic assessment suggests the circulation of novel hemoplasma species in rodents from different biomes in Brazil.     

Molecular characterization of feline Hepatozoon species from Brazil

Feline Hepatozoon species from Brazil was molecular identified and characterized for the first time in S?o Paulo state, Brazil. Partial sequences of the 18S rRNA gene from the Hepatozoon from three naturally infected cats were analyzed. Sequences revealed that feline Hepatozoon was closely related to the canine Hepatozoon canis from Brazil.     

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 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.     

Molecular detection of a Hepatozoon species in stray cats from a feline colony in North-eastern Spain

Within the framework of a local animal health programme, the presence of ectoparasites and haemoparasites was investigated in a colony of 25 cats in Barcelona. Diagnosis was performed both by standard parasitological procedures and molecular techniques. All cats were negative to haematozoan infection by microscopic examination of blood smears. However, Hepatozoon spp. was found in four cats as shown by amplification and sequencing of the 18S rRNA gene. Cat isolates were 100% identical to the isolate Hepatozoon spp. (Spain 2) from Southern Spain. This is the first time that Hepatozoon spp. has been identified in cats from Northern Spain.     

Effects of body weight and season on serum lipid concentrations in sloth bears (Melursus ursinus ursinus)

Serum lipid levels were measured in 66 healthy sloth bears (Melursus ursinus ursinus) living under semicaptive conditions with access to natural food resources in the Bannerghatta Biological Park (Karnataka, India), a portion of their native habitat range in the Indian peninsula. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein cholesterol levels were analyzed. The effects of age, body weight, and season on these lipid parameters were statistically evaluated. There were no correlations between age and any of the serum lipid parameters analyzed. Positive correlations of body weight to both triglyceride and HDL cholesterol levels in these bears were identified. In addition, seasonal trends in physiological serum lipid values, potentially due to variations in the sloth bear diet, were identified. Serum triglyceride levels were higher during postmonsoon season and cholesterol levels were higher during winter compared to other seasons. Serum lipid values obtained from sloth bears in this study were also compared to previously published data on other members of the family Ursidae. This is the first report of serum lipid values as a reference for sloth bears. These values can be used as sensitive predictors of overall health and nutritional status to aid in the captive management and feeding of these bears.     

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.     

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.