Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

Molecular detection of Hepatozoon felis in cats from Maio Island, Republic of Cape Verde and global distribution of feline hepatozoonosis.

BACKGROUND: Vector-borne diseases are emerging worldwide and have an important zoonotic relevance. In the last few years, the interest in vector-borne pathogens in cats has increased. However, studies on feline vector-borne pathogens on tropical islands are lacking. Islands differ from continental countries because they have an enclosed population of animals, with all year presence of the vectors and, most often, without vector control measures. This study focused on the molecular identification and phylogenetic analysis of vector-borne pathogens in autochthonous cats with a mixed indoor-outdoor lifestyle from Maio Island, Cape Verde archipelago. METHODS: Blood samples were collected from 80 asymptomatic cats, representing almost a quarter of the total cat population of the island. The presence of DNA of protozoa of the genus Hepatozoon and bacteria belonging to family Anaplasmataceae and to genus Bartonella was assessed by PCR and phylogenetic analysis was conducted. Statistical analysis was performed to identify risk factors associated with infection. For feline hepatozoonosis, a worldwide dataset of Hepatozoon felis sequences retrieved from mammal species and vectors along with Hepatozoon spp. sequences retrieved from felids was generated, phylogenetically analyzed and the geographical and host distribution was assessed. RESULTS: Hepatozoon felis genotype I was identified in 12 (15%) cats from Maio Island whereas none of the cats were PCR positive for the other pathogens tested. No significant association of H. felis infection with age, sex, location or presence of vectors was observed by statistical analysis in Cape Verde's cats. Phylogenetic analysis on the worldwide dataset of feline Hepatozoon sequences showed two significant distinct clades for H. felis genotype I and II. Different geographical distributions were assessed: H. felis genotype I was the only genotype found in Africa and has been reported worldwide, with the exception of Japan and Brazil where only H. felis genotype II has been reported. CONCLUSIONS: The identification of H. felis genotype I in cats in Maio Island highlights the need to further investigate the significance of H. felis genotypes and to clarify the epidemiological aspects of this infection.

Hepatozoon martis n. sp. (Adeleorina: Hepatozoidae): Morphological and pathological features of a Hepatozoon species infecting martens (family Mustelidae).

Species of the genus Hepatozoon (Adeleorina: Hepatozoidae) are arthropod-transmitted protozoan parasites that infect a wide range of vertebrate hosts. In the present study, we describe a new species of Hepatozoon primarily infecting martens and propose the name Hepatozoon martis n. sp., based on its unique morphological, molecular and pathogenic features. The overall prevalence of infection with H. martis n. sp. assessed by PCR in European pine martens (Martes martes) from Bosnia and Herzegovina and stone martens (Martes foina) from Croatia was 100% and 64%, respectively. Gamonts were found in neutrophils and monocytes, and various developmental stages were described in tissue cross-sections. Hepatozoon martis n. sp. shows a high predilection for muscle tissue, and the heart was the most frequently affected organ among the tissues tested by histopathology. Microscopically, pyogranulomatous lesions associated with the presence of the parasitic forms were observed in the cardiac and skeletal muscles of all positive animals examined. Furthermore, the possible existence of alternative, non-vectorial routes of transmission is discussed.

Molecular detection of Anaplasma bovis, Ehrlichia canis and Hepatozoon felis in cats from Luanda, Angola.

BACKGROUND: Molecular identification of tick-borne pathogen infection in cats from Africa is scarce. The presence of bacterial (Anaplasma and Ehrlichia) and protozoal (Babesia and Hepatozoon) agents was investigated in blood samples from 102 domestic cats from Luanda, Angola, by polymerase chain reaction and DNA sequencing. RESULTS: Three cats (2.9%) were found infected with Ehrlichia canis, three (2.9%) with Hepatozoon felis and one (1.0%) with Anaplasma bovis. The prevalence of infections with one single agent was 4.9%, and that of infection with two agents (i.e. E. canis and H. felis) was 1.0%. In total, six cats (5.9%) were found infected with at least one of the detected tick-borne agents. CONCLUSIONS: This is the first report of A. bovis, E. canis and H. felis in cats from Angola. To the best of our knowledge, A. bovis is also being reported for the first time in domestic cats outside of Japan. Cats are at a low to moderate risk of being infected with tick-borne agents in Luanda.

Occurrence and diversity of arthropod-transmitted pathogens in red foxes (Vulpes vulpes) in western Austria, and possible vertical (transplacental) transmission of Hepatozoon canis.

Red fox (Vulpes vulpes) is the most abundant wild canid species in Austria, and it is a well-known carrier of many pathogens of medical and veterinary concern. The main aim of the present study was to investigate the occurrence and diversity of protozoan, bacterial and filarial parasites transmitted by blood-feeding arthropods in a red fox population in western Austria. Blood (n = 351) and spleen (n = 506) samples from foxes were examined by PCR and sequencing and the following pathogens were identified: Babesia canis, Babesia cf. microti (syn. Theileria annae), Hepatozoon canis, Anaplasma phagocytophilum, Candidatus Neoehrlichia sp. and Bartonella rochalimae. Blood was shown to be more suitable for detection of Babesia cf. microti, whilst the spleen tissue was better for detection of H. canis than blood. Moreover, extremely low genetic variability of H. canis and its relatively low prevalence rate observed in this study may suggest that the parasite has only recently been introduced in the sampled area. Furthermore, the data presented here demonstrates, for the first time, the possible vertical transmission of H. canis from an infected vixen to the offspring, and this could explain the very high prevalence in areas considered free of its main tick vector(s).

A redescription of Haemogregarina damiettae Ramadan et al. 1996 naturally infecting the Acanthodactylus boskianus from Egypt, with new merognic data.

Haemogregarina damiettae was previously described form Acanthodactylus boskianus in Egypt, and the description was base only on the blood stages with no data on the tissue stages. In the present study, both blood and tissue stages were encountered. The prevalence of infection was found to be 60 % (30/50). The blood stages were found to be confined to the erythrocytes and none in the leucocytes. The erythrocytic stages could be differentiated into small and large forms. The small form, trophozoite, measured 10 × 3 µm, while the large form, mature gamontocyte, measured 18 × 5 µm. The cytoplasm of the mature gamontocyte was finely granular and faintly stained with Giemsa, while the nucleus was relatively large and occupied about one third of the body length and was formed of a network of chromatin filaments. Infected erythrocytes were hypertrophied and their nuclei either longitudinally stretched or split into two fragments. The tissue stages were observed in the lungs and hearts of the infected lizards. The merogonic stages were evident as multinucleated ovoid to subspherical bodies enclosed by parasitophorous vacuoles. Mature meronts measured 16 × 12 µm and estimated to produce 15-25 merozoites.

Molecular characterization of Hepatozoon species in a Leopard Cat (Prionailurus bengalensis) from Thailand.

Hepatozoon gamonts were observed by light microscopy in neutrophils of a male, wild-caught Leopard Cat. Complete blood counts at presentation and 6 months later were unremarkable. Serologic tests were negative for both FIV and FeLV. A partial sequence of the 18S rRNA gene from the Hepatozoon found in the cat indicated that, compared with all species examined, the protozoan had the closest relationship (99.2% sequence similarity) with the Hepatozoon of the water python (Stegonotus cucullatus). The cat was clinically healthy at last report. Although Hepatozoon has been found in another wild cat in Thailand, this is the first report in a Leopard Cat. The pathogenicity of Hepatozoon in these cats remains uncertain.

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.

Prevalence and intensity of haemogregarine blood parasites and their mite vectors in the common wall lizard, Podarcis muralis.

We describe the general field population biology of haemogregarines and mites in the wall lizard, Podarcis muralis, examining the factors that determine the prevalence and intensity of infection. The intensity of infection by haemogregarines in females was slightly lower in summer than in spring, whereas males maintained similar levels of intensity among all the seasons, probably due to immunosuppressive effects of testosterone. This is also supported because the prevalence and the infection intensity by mites were higher in males than in females. Parasite load was higher in lizards with better body condition, which could be reflecting the mortality of infected lizards with poor condition.

Prevalence and intensity of blood and intestinal parasites in a field population of a Mediterranean lizard, Lacerta lepida.

We describe the blood and intestinal parasites in the Ocellated lizard, Lacerta lepida, examining the factors that determine the prevalence and intensity of infection of haemogregarines, and the prevalence of coccidia and nematodes. In relation to haemogregarines, no juveniles were detected as being infected, whereas 71.7 % of adults were infected. The prevalence of infection was positively related to the size of the adults. There were no differences between seasons or sexes in the prevalence or intensity of infection in adults. There were no significant differences in the prevalence of infection by nematodes between ages or sexes, nor in relation to the size of adult lizards, but adult lizards excreting coccidian oocysts tend to be smaller. During the mating period, reproductive activities lead to a decrease in the body condition. However, neither the intensity of haemogregarine's infection nor the prevalence of intestinal parasites was related to the lizards' body condition.

American canine hepatozoonosis.

American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy.

Larval Gulf Coast ticks (Amblyomma maculatum) [Acari: Ixodidae] as host for Hepatozoon americanum [Apicomplexa: Adeleorina].

Laboratory-reared larval Gulf Coast ticks (GCTs) (Amblyomma maculatum) were exposed experimentally and found to acquire Hepatozoon americanum infection while feeding on parasitemic dogs. These ticks supported gamogonic and sporogonic development of the apicomplexan, and oocysts from newly molted nymphs were infectious for a dog. Other nymphs from this cohort that were allowed to feed on a blood-parasite naive sheep molted normally; the resulting adult ticks contained oocysts that were infectious for another dog. Merogonic development of H. americanum in the dogs and the resulting lesions/disease appeared similar, irrespective of whether infectious oocysts were derived from nymphal or adult ticks that acquired infection as larvae. In the system previously known, nymphal ticks acquire infection and adults harbor infective oocysts, which vertebrate hosts ingest. Given that larval A. maculatum can acquire infection and nymphs can harbor viable oocysts as demonstrated by this study, the potential variety of vertebrate hosts that can alternate with GCTs in maintaining an endemic cycle is considerably expanded.

Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals.

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.

Giardia, Cryptosporidium, and Cyclospora and their impact on foods: a review.

While the risk from pathogenic microorganisms in foods has been recognized for hundreds of years, bacterial agents are generally implicated as the contaminants. Although many outbreaks of gastroenteritis caused by protozoan pathogens have occurred, it is only in the last 3 years that attention has focused on protozoan association with foodborne transmission. Recognized as waterborne parasites, Giardia, Cryptosporidium, and Cyclospora have now been associated with several foodborne outbreaks. The oocysts and cysts of these organisms can persist and survive for long periods of time both in water and on foods. While Cyclospora oocysts require a maturation period, Cryptosporidium oocysts and Giardia cysts are immediately infectious upon excretion from the previous host. As a result, these parasites have emerged as public health risks and have become a concern to the food industry. More than 200 cases of foodborne giardiasis (seven outbreaks) were reported from 1979 to 1990. Four foodborne Cryptosporidium outbreaks (with a total of 252 cases) have been documented since 1993. Cyclospora caused a series of sporadic outbreaks of cyclosporasis throughout North America that have affected over 3,038 people since 1995. Control and prevention of protozoan foodborne disease depends upon our ability to prevent, remove, or kill protozoan contaminants. This review will address the biology, foodborne and waterborne transmission, survival, and methods for detection and control of Giardia, Cryptosporidium, and Cyclospora.

Canine hepatozoonosis: comparison of lesions and parasites in skeletal muscle of dogs experimentally or naturally infected with Hepatozoon americanum.

We report previously undescribed, early lesions in skeletal muscle of dogs experimentally infected with Hepatozoon americanum by ingestion of laboratory-reared, infected Amblyomma maculatum. The earliest muscle lesion was recognized at the first interval of examination 3 weeks following exposure. The lesion consisted of a large, modified host cell whose cytoplasm frequently contained a demonstrable parasite. In skeletal muscle, the cell was consistently located between muscle fibers or in loose connective tissue adjacent to those fibers. Evidence suggesting that the parasite arrives in muscle and other tissue within the host cell cytoplasm is presented. Mucopolysaccharide encystment of the host cell, absent at this early stage, was acquired gradually and approached maximal development 26 weeks post exposure. Completion of the asexual cycle as evidenced by the presence of parasites entering vascular lumens within granulomas and also by the presence of gamonts in peripheral blood leukocytes, occurred within 28-32 days postexposure. Progression of the parasite cycle from meront to passage of zoites into vessel lumens of granulomas can occur in 11 or fewer days. The density with which parasitic lesions occur in one named skeletal muscle compared to other named muscles, although somewhat variable, was not significantly different in either experimentally induced or natural infections. The distribution of developmental stages of the parasite/lesion in four experimental infections (969 lesions) is compared with those in eight dogs with natural infections (557 lesions).