New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.     

Monozoic cysts of Hepatozoon canis

Small monozoic cysts found in the spleen of dogs infected with Hepatozoon canis are described from naturally and experimentally infected dogs. These forms of H. canis resemble cysts formed by other Hepatozoon species that infect frogs, lizards, and grey squirrels as intermediate hosts. The H. canis cyst stage differs in size and morphology from the large cysts of H. americanum, the second Hepatozoon species known to infect dogs.     

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa,Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of S o Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.     

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.     

Morphology is not a reliable tool for delineating species within Cryptosporidium

Within the coccidia, morphological features of the oocyst stage at the light microscope level have been used more than any other single characteristic to designate genus and species. The aim of this study was to conduct morphometric analysis on a range of Cryptosporidium spp. isolates and to compare morphological data between several genotypes of C. parvum and a second species C. canis, as well as a variation within a specific genotype (the human genotype), with genetic data at 2 unlinked loci (18S ribonucleic deoxyribonucleic acid and HSP 70) to evaluate the usefulness of morphometric data in delineating species within Cryptosporidium. Results indicate that morphology could not differentiate between oocysts from C. parvum genotypes and oocysts from C. canis, whereas genetic analysis clearly differentiated between the two. The small size of the Cryptosporidium spp. oocyst, combined with the very limited characters for analysis, suggests that more reliance should be placed on genetic differences, combined with biological variation, when delineating species within Cryptosporidium.     

Transmission of hepatozoon canis to dogs by naturally-fed or percutaneously-injected Rhipicephalus sanguineus ticks.

Hepatozoon canis is an apicomplexan protozoan parasite of dogs, prevalent in Asia, Africa, and southern Europe. Experimental transmission of H. canis to dogs was performed with laboratory-reared Rhipicephalus sanguineus nymphs that fed on a naturally infected dog or were percutaneously injected with canine blood containing H. canis gamonts. Dogs were inoculated by oral ingestion of adult ticks containing H. canis oocysts. Transstadial transmission of H. canis was recorded, whereas transovarial transmission could not be demonstrated. Oocysts were detected in 85% of the adult ticks that had engorged as nymphs on an infected dog and in 61% of the adult ticks resulting from nymphs injected percutaneously with blood from the same dog. Nine of 12 dogs (75%) inoculated with naturally fed or percutaneously injected ticks became parasitologically positive, and all showed seroconversion. Meronts were initially detected in the bone marrow 13 days postinoculation and gamonts 28 days after infection. The variation in the time of initial detection of parasitemia among infected dogs and the rapid appearance of gamonts in dogs immunosuppressed with corticosteroids suggest that immune mechanisms play an important role in controlling H. canis parasitism. The artificial acquisition of Hepatozoon parasites by percutaneous injection of ticks, demonstrated here for the first time, may serve as a useful tool for studies on transmission, vector-host relationships, and the immunology of infection with Hepatozoon species.     

A Hepatozoon species genetically distinct from H. canis infecting spotted hyenas in the Serengeti ecosystem, Tanzania

Health monitoring of spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, revealed Hepatozoon infection in all of 11 immature individuals examined following death from natural causes. Hepatozoon infection was probably an important factor contributing to mortality in two cases that exhibited clinical signs of ataxia, lethargy, ocular discharge, retching, and labored breathing before death. Whether Hepatozoon infection contributed to six deaths from fire, probable lion predation and unknown causes could not be determined. Four deaths from infanticide and starvation were unlikely to be associated with Hepatozoon infection. Histologic examination revealed lung tissue infected with cyst-like structures containing protozoan stages in all eight cases examined and interstitial pneumonia in most cases. Systemic spread of infection to several organs was found in three cases. Alignment of a 426 bp sequence from the parasite's 18s rRNA gene revealed a Hepatozoon species identical to that recently described from two domestic cats in Spain and only 7 bp substitutions when a 853 bp sequence was aligned to this cat Hepatozoon species. Previous reports of infection of wild carnivores in eastern and southern Africa with an unspecified Hepatozoon species similar in appearance to H. canis may have involved the species described in this study.     

Cryptosporidium canis n. sp. from domestic dogs.

Oocysts of Cryptosporidium, from the feces of a naturally infected dog and from an HIV-infected human, were identified as the previously reported canine genotype of Cryptosporidium parvum, hereafter referred to as Cryptosporidium canis n. sp. Also among the oocysts from the dog, a trace amount of C. parvum bovine genotype was detected. Cryptosporidium canis oocysts from both the dog and human were infectious for calves. Oocysts excreted by calf 1 (dog source) were approximately 90% C. canis and 10% C. parvum, whereas those excreted by calf 3 (human source) were 100% C. canis. Oocysts from calf 1 infected calf 2 resulting in excretion by calf 2 of oocysts approximately 90% C. parvum and 10% C. canis. Oocysts of C. canis were not infectious for BALB/c neonatal mice or immunosuppressed C57 juvenile mice, although all control mice became infected with the C. parvum Beltsville isolate. Oocysts of C. canis from calf 1 and the human were structurally indistinguishable from oocysts of the C. parvum Beltsville isolate (bovine). However, C. canis oocysts differed markedly at the molecular level from all known species of Cryptosporidium based on sequence data for the 18S rDNA and the HSP 70 gene. The differences in genetics and host specificity clearly differentiate C. canis as a new species.     

Hepatozoon sauritus: a polytopic hemogregarine of three genera and four species of snakes in north Florida, with specific identity verified from genome analysis

Hemogregarines from Thamnophis s. sirtalis, Coluber constrictor priapus, Elaphe obsoleta quadrivittata, and E. g. guttara in northern Florida appeared to be conspecific on the basis of similar gamonts from all the hosts and sporogonic stages obtained from 3 hosts. The resemblance of gamonts to those of Hepatozoon sauritus, described from T. sauritus sackenii in southern Florida, justified comparison of DNA isolates from the type infection of H. sauritus with samples from each of the northern Florida hosts and with a morphologically distinct species, H. sirtalis, from northern Florida. A nucleotide sequence (530 bp) alignment of the 18S ribosomal RNA gene revealed 2 hemogregarine haplotypes that varied at 15 sites (p distance = 2.8%), which included 10 transitions and 5 transversions. Two well-supported clusters (100% bootstrap support) were revealed by a neighbor-joining tree topology. One cluster included the type infection of H. sauritus and all 4 of the other samples from the northern Florida hosts, with samples of H. sirtalis comprising a second cluster. Hepatozoon sauritus, therefore, is a polytopic species in contrast to the 8 other Hepatozoon species thus far described from snakes in Florida, each of which appears to parasitize a single host species.     

Naturally occurring hepatozoonosis in coyotes from Oklahoma

Nine of 16 free-ranging coyotes (Canis latrans) from central Oklahoma (USA) had naturally acquired infections of Hepatozoon americanum. Infections were confirmed by recognition of tissue stages closely resembling H. americanum in skeletal and cardiac muscle. At the time coyotes were collected they were infested with a variety of ticks, including adult Gulf Coast ticks (Amblyomma maculatum). We propose that the high prevalence of H. americanum in this small sample of free-ranging coyotes and the ability of these same animals to harbor adult populations of A. maculatum is an important component of the epizootiology of canine hepatozoonosis in North America.     

Experimental infection of adult and juvenile coyotes with domestic dog and wild coyote isolates of Hepatozoon americanum (Apicomplexa: Adeleorina)

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

Hepatozoon sp. in wild carnivores in Texas

Twelve coyotes (Canis latrans), three bobcats (Lynx rufus) and six ocelots (Felis pardalis) from the Gulf Coast of Texas were infected with Hepatozoon sp. The geographic distribution of infected wild animals coincides with the highest prevalence of Hepatozoon canis infection in domestic dogs for which the wild species may act as a reservoir.     

Detection of Cryptosporidium sp. in two new seal species, Phoca vitulina and Cystophora cristata, and a novel Cryptosporidium genotype in a third seal species, Pagophilus groenlandicus, from the Gulf of Maine

Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium.     

Antigenic analysis of gamonts of Hepatozoon canis purified from leukocytes

Hepatozoon canis is a tick-borne apicomplexan parasite of dogs that infects neutrophils and parenchymal tissues. To study the antigenic characteristics of this parasite, a technique was devised for the purification of gamonts from peripheral blood neutrophils. White blood cells were separated on Ficoll-Hypaque density gradients and the gamonts were released from the host neutrophils by nitrogen cavitation. The blood used for purification originated from dogs with natural or experimental infections of H. canis with a parasitemia of 1.4-33%. The number of parasites collected ranged from 1.5 X 10(6) to 4.2 X 10(7). Portions of purified gamonts were separated and examined under phase and scanning electron microscopy, and the remaining purified parasites were then used as a source of antigens to characterize the humoral immune response by western blot analysis. Serum antibodies from infected dogs recognized more than 15 gamont antigens, and the antigenic patterns observed with sera from naturally and experimentally infected dogs were nearly similar. Four immunodominant protein bands of relative molecular weights of 107, 88, 63, and 28 kDa were recognized by all of the sera examined. The technique applied here for the isolation of host cell-free gamonts will facilitate studies on antigenic composition and immune responses against H. canis and on antigenic relationships between Hepatozoon from different host species and geographic regions.     

Naturally occurring and experimentally transmitted Hepatozoon americanum in coyotes from Oklahoma

Twenty free-ranging coyotes (Canis latrans) in Oklahoma (USA) were examined for the presence of naturally occurring infections with Hepatozoon americanum and to determine if bone lesions attributable to H. americanum were present. Although eight of the 20 free-ranging coyotes were found to be naturally infected with H. americanum, no bone lesions were detected. In addition, two coyote pups were exposed to H. americanum oocysts collected from experimentally infected ticks and the course of the resulting infection was followed. Both experimentally infected coyotes developed hepatozoonosis detectable by specific muscle lesions beginning 4 wk after exposure. Bone lesions were detected grossly and histologically at necropsy. Histologic evidence of periosteal bone proliferation ranged from segmental areas of plump hypercellularity and thickening of the periosteum, with minor degrees of osteogenesis, to extensive proliferation of woven bone and periosteal hypercellularity and thickening. Nymphal Amblyomma maculatum that fed on one of the experimentally infected coyote pups became infected and mature H. americanum oocysts were recovered when the ticks molted to adults. These results demonstrate that coyotes in some parts of Oklahoma are naturally infected with H. americanum, that experimentally infected coyotes can develop clinical disease, including characteristic bone lesions, and that A. maculatum nymphs can acquire infections by feeding on them.     

A new species of Cryptosporidium (Apicomplexa: Cryptosporidiidae) from eastern grey kangaroos (Macropus giganteus)

Cryptosporidium macropodum n. sp is described. Oocysts of C. macropodum from the feces of kangaroos (Macropus spp.) are morphologically indistinguishable from other mammalian Cryptosporidium species, including C. parvum, C. hominis, C. suis, and C. canis. The oocysts are fully sporulated on excretion, lack sporocysts, and have an average width of 4.9 microm (4.5-6.0), a length of 5.4 microm (5.0-6.0), and a length:width ratio of 1.1. Phylogenetic analyses of the 18S ribosomal RNA, actin, and heat shock protein 70 (HSP70) loci demonstrate that C. macropodum is genetically distinct from all described Cryptosporidium species, including others found in marsupials. The parasite seems to be highly host-specific, because it has been found only in marsupials to date. Therefore, based on biological and molecular data, we consider C. macropodum a new species.     

Molecular characterization of the 18S rDNA gene of an avian Hepatozoon reveals that it is closely related to Lankesterella

As a part of intensive study of blood parasite infections in a population of the passerine bird blue tit (Cyanistes caeruleus, Paridae), we detected a parasite species that, based on its morphological similarity, was tentatively identified as Hepatozoon parus, the only species of this parasite genus described from birds of this family. However, morphological measurements show that H. parus is slightly larger than the parasite detected in our population. A molecular characterization of the parasite species was conducted by amplification of the 18S rDNA gene, using primers that were reported previously to amplify in Hepatozoon sp. of water pythons. Additional primers were developed based on the new sequence obtained. The 1,484-bp fragment amplified reveals that the parasite from our bird population is more closely related to Lankesterella minima than to Hepatozoon species from other vertebrates according to analysis using the BLAST comparison method (93% identity). In addition, phylogenetic analyses using parsimony and Kimura procedures unequivocally related the parasite species detected by PCR with L. minima. The bootstrap values obtained were 97% and 100%, respectively. These results imply that this parasite is a species of a lankesterellid instead of Hepatozoon.     

Molecular and morphological description of a Hepatozoon species in reptiles and their ticks in the Northern Territory, Australia

Ticks, representing 3 species of Amblyomma, were collected from the water python (Liasis fuscus) and 3 additional reptile species in the Northern Territory, Australia, and tested for the presence of Hepatozoon sp., the most common blood parasites of snakes. In addition, blood smears were collected from 5 reptiles, including the water python, and examined for the presence of the parasite. Hepatozoon sp. DNA was detected in all tick and reptile species, with 57.7% of tick samples (n = 187) and 35.6% of blood smears (n=35) showing evidence of infection. Phylogenetic analysis of the 18S rRNA gene demonstrated that half of the sequences obtained from positive tick samples matched closest with a Hepatozoon species previously identified in the water python population. The remaining sequences were found to be more closely related to mammalian and amphibian Hepatozoon species. This study confirms that species of Amblyomma harbor DNA of the same Hepatozoon species detected in the water pythons. The detection of an additional genotype suggests the ticks may be exposed to 2 Hepatozoon species, providing further opportunity to study multiple host-vector-parasite relationships.     

Hepatozoon ursi n. sp. (Apicomplexa: Hepatozoidae) in Japanese black bear (Ursus thibetanus japonicus)

Morphological and genetic features of a new Hepatozoon species, Hepatozoon ursi n. sp., in Japanese black bear (Ursus thibetanus japonicus) were studied. Schizogonic developmental stages were observed in the lungs of Japanese black bears. The schizonts were sub-spherical in shape and 45.7+/-4.6 x 42.7+/-4.5 microm in size. Each mature schizont contained approximately 80-130 merozoites and 0-5 residual bodies. The merozoites were 7.0+/-0.7 x 1.8+/-0.3 microm in size. Intraleukocytic gametocytes were slightly curved, cigar-like in shape and had a beak-like protrusion at one end. The size of the gametocytes was 10.9+/-0.3 x 3.3+/-0.2 microm. The analyses of the18S rRNA gene sequences supported the hypothesis that H. ursi n. sp. is different from other Hepatozoon species. Mature Hepatozoon oocysts were detected in two species of ticks (Haemaphysalis japonica and Haemaphysalis flava) collected on the bears infected with H. ursi n. sp. Two measured oocysts were 263.2 x 234.0 microm and 331.8 x 231.7 microm, respectively. The oocysts contained approximately 40 and 50 sporocysts, respectively. The sporocysts were sub-spherical in shape and 31.2+/-2.5 x 27.0+/-2.9 microm in size. Each sporocyst contained at least 8-16 sporozoites, with the sporozoites being 12.2+/-1.4 x 3.5+/-0.5 microm in size. H. ursi n. sp. is the first Hepatozoon species recorded from the family Ursidae.