Hepatozoon (Eucoccidiorida: Hepatozoidae) in wild mammals of the Americas: a systematic review.

BACKGROUND: The study of parasites provides insight into intricate ecological relationships in ecosystem dynamics, food web structures, and evolution on multiple scales. Hepatozoon Eucoccidiorida: Hepatozoidae) is a genus of protozoan hemoparasites with heteroxenous life cycles that switch infections between vertebrates and blood-feeding invertebrates. The most comprehensive review of the genus was published 26 years ago, and currently there are no harmonized data on the epizootiology, diagnostics, genotyping methods, evolutionary relationships, and genetic diversity of Hepatozoon in the Americas. METHODS: Here, we provide a comprehensive review based on the PRISMA method regarding Hepatozoon in wild mammals within the American continent, in order to generate a framework for future research. RESULTS: 11 out of the 35 countries of the Americas (31.4%) had data on Hepatozoon, with Carnivora and Rodentia orders having the most characterizations. Bats, ungulates, and shrews were the least affected groups. While Hepatozoon americanum, H. americanum-like, H. canis, H. didelphydis, H. felis, H. milleri, H. griseisciuri, and H. procyonis correspond to the identified species, a plethora of genospecies is pending for a formal description combining morphology and genetics. Most of the vectors of Hepatozoon in the Americas are unknown, but some flea, mite, and tick species have been confirmed. The detection of Hepatozoon has relied mostly on conventional polymerase chain reaction (PCR), and the implementation of specific real time PCR for the genus needs to be employed to improve its diagnosis in wild animals in the future. From a genetic perspective, the V4 region of the 18S rRNA gene has been widely sequenced for the identification of Hepatozoon in wild animals. However, mitochondrial and apicoplast markers should also be targeted to truly determine different species in the genus. A phylogenetic analysis of herein retrieved 18S ribosomal DNA (rDNA) sequences showed two main clades of Hepatozoon: Clade I associated with small mammals, birds, and herpetozoa, and Clade II associated with Carnivora. The topology of the tree is also reflected in the haplotype network. CONCLUSIONS: Finally, our review emphasizes Hepatozoon as a potential disease agent in threatened wild mammals and the role of wild canids as spreaders of Hepatozoon infections in the Americas.

Mercury bioaccumulation and Hepatozoon spp. infections in two syntopic watersnakes in South Carolina.

Mercury (Hg) is a ubiquitous environmental contaminant known to bioaccumulate in biota and biomagnify in food webs. Parasites occur in nearly every ecosystem and often interact in complex ways with other stressors that their hosts experience. Hepatozoon spp. are intraerythrocytic parasites common in snakes. The Florida green watersnake (Nerodia floridana) and the banded watersnake (Nerodia fasciata) occur syntopically in certain aquatic habitats in the Southeastern United States. The purpose of this study was to investigate relationships among total mercury (THg) concentrations, body size, species, habitat type and prevalence and parasitemia of Hepatozoon spp. infections in snakes. In the present study, we sampled N. floridana and N. fasciata from former nuclear cooling reservoirs and isolated wetlands of the Savannah River Site in South Carolina. We used snake tail clips to quantify THg and collected blood samples for hemoparasite counts. Our results indicate a significant, positive relationship between THg and snake body size in N. floridana and N. fasciata in both habitats. Average THg was significantly higher for N. fasciata compared to N. floridana in bays (0.22 ± 0.02 and 0.08 ± 0.006 mg/kg, respectively; p < 0.01), but not in reservoirs (0.17 ± 0.02 and 0.17 ± 0.03 mg/kg, respectively; p = 0.29). Sex did not appear to be related to THg concentration or Hepatozoon spp. infections in either species. We found no association between Hg and Hepatozoon spp. prevalence or parasitemia; however, our results suggest that species and habitat type play a role in susceptibility to Hepatozoon spp. infection.

New insights on the phylogeography of Hepatozoon canis in Brazil.

This study aimed to molecularly characterize the Hepatozoon spp. infecting domestic and wild dogs in Brazil. A total of 22 whole blood samples tested positive for Hepatozoon spp., and five samples were sequenced for the 18S rDNA gene from H. canis after PCR amplification with four primer sets. Phylogenetic analysis using Bayesian inference showed that the three H. canis isolates from domestic dogs were not monophyletic; however, they were more closely related to each other than to other H. canis sequences. The isolate from the hoary fox (Lycalopex vetulus) was phylogenetically more distant. Two haplotype networks were constructed, identifying 10 haplotypes of H. canis in Brazil, with H10 constituting the largest group. It contains nine isolates, including three from domestic dogs. The H5 haplotype grouped the sequence of L. vetulus with two additional sequences from hosts Tapirus terrestris and L. vetulus, representing the sole haplotype with wild hosts. Bayesian analysis suggested the possible existence of two genetic groups of H. canis in Brazil, indicating gene flow of this agent within the country. These findings contribute valuable insights for a more comprehensive understanding of the molecular diversity of Hepatozoon spp. in Brazil and may help in the development of effective control measures.

First molecular detection and genetic diversity of Hepatozoon sp. (Apicomplexa) and Brugia sp. (Nematoda) in a crocodile monitor in Nakhon Pathom, Thailand.

The crocodile monitor (Varanus salvator) is the most common monitor lizard in Thailand. Based on habitat and food, they have the potential to transmit zoonoses, with a high possibility of infecting ectoparasites and endoparasites. Diseases that could infect crocodile monitors and be transmitted to other animals, including humans. This research aims to identify and evaluate the phylogenetic relationships of Hepatozoon sp. and sheathed microfilaria in crocodile monitors. The phylogenetic analyses of Hepatozoon, based on 18S rRNA, and sheathed microfilaria, based on the COX1 gene, revealed that the Hepatozoon sp. were grouped with H. caimani, while sheathed microfilaria were grouped together with B. timori. This study provides insights into the genetic diversity and host-parasite interactions of hemoparasites in crocodile monitors in Thailand.

CORRECTING THE NAMES OF TWO HAEMOGREGARINA SPP. FROM LIZARDS IN EGYPT.

Two haemogregarine "species" names, Haemogregarina tarentannulari and Haemogregarina rawashi, were cited by Saoud et al. (1995) as having been described by Mohammed and Ramadan (1996, in press). However, the paper by Mohammed and Ramadan (1996) was never published and, therefore, these names and their authorities must be suppressed because they violate Chapter 3 (Criteria for Publication), Article 8 (What Constitutes Published Work) of the International Code of Zoological Nomenclature. The following new names are introduced to replace them based on the Principle of Priority (Chapter 6, Article 23, 23.1, and Chapter 11, Article 51, Recommendation 51E): Hepatozoon rawashi (Mohammed and Ramadan in Saoud, Ramadan, Mohammed and Fawzi, 1995) n. comb., with gamonts in the erythrocytes and meronts in the lungs and liver of the fan-footed gecko, Ptyodactylus hasselquisiti (Donndorff, 1798) from Egypt, and Haemogregarina tarentannulari (Mohammed and Ramadan in Saoud, Ramadan, Mohammed and Fawzi, 1995), with gamonts in the erythrocytes and meronts in the lungs and liver of the white-spotted wall gecko, Tarentola annularis (St. Hilaire, 1827) also from Egypt. This latter species was determined to be a junior synonym of Haemogregarina annularis El-Naffar, Mandour, and Mohammed 1991, which was later reassigned to the genus Hepatozoon based on their phylogenetic analysis of 18S rDNA gene sequences.

Phylogenetic analysis and haplotype networking of Hepatozoon felis infecting wild animals in Gir National Park, Gujarat, India.

The present study was aimed to determine the phylogenetic relationship, haplotype network, and demographic dynamics of H. felis infecting the endangered Asiatic lions in Gir National Park, Gujarat, India, on the basis of partial 18S rRNA gene. The phylogenetic analysis based on the partial 18S rRNA gene sequences of H. felis exhibited the presence of two distinct genotypes of H. felis (HfG1 and HfG2) infecting the Indian wild felids, viz., the Asiatic lion, Royal Bengal tiger, and Indian leopard. The HfG1 and HfG2 genotypes exhibited 97.6-100% and 99.7-100%, and 96.9-98.7% nucleotide identity within and between themselves, respectively. The HfG1 genotype exhibited a higher genetic diversity as compared to HfG2. A total of 22 molecular signatures were identified in the 18S rRNA gene between these genotypes. Further, analysis of a total 67 sequences of H. felis (13 different host species from 13 countries of Africa, South America, Asia, and Europe) that were downloaded from GenBankTM, generated 30 haplotypes. Among all the haplotypes, Hap_17 (h=12) was the most frequent followed by Hap_12 (h=09) and Hap_4 (h=05). Out of 13 location-wise populations, India (h=12) shared the highest number of haplotypes followed by Japan (h=08), and the least number of haplotypes were found in Hungary (h=02). Population dynamics study involving neutrality tests and mismatch distribution, and genetic differentiation indices, revealed the presence of phylogeographic population structure and a constant population size indicating a uniform gene flow among the populations worldwide. In conclusion, a high genetic diversity along with the presence of two distinct genotypes of H. felis were observed on the basis of 18S rRNA gene sequence analysis.

Vector-borne parasites in dogs from Ukraine translocated to Poland following Russian invasion in 2022.

INTRODUCTION: Since 24 February 2022, the day the Russian aggression against Ukraine began, millions of refugees and thousands of pets crossed the Polish-Ukrainian border. Additionally, an unknown number of shelter and stray dogs and cats were rescued and translocated to Poland by private persons and non-profit organizations. The aim of the present study was to examine rescued dogs and cats for presence of canine vector-borne parasites to determine the role of armed conflict in spreading these parasites. METHODS: In July 2022 blood samples were collected from two animal shelters in central Poland hosting dogs and cats rescued from Ukraine. Animals were imported from various regions of Ukraine, including eastern and southeastern Ukraine (military conflict area). Fifty-three dogs (51 from two shelters and two owned ones) and one shelter cat were examined by molecular methods (PCR and sequencing) for the presence of Babesia/Theileria spp., Dirofilaria spp. and Hepatozoon spp. DNA. RESULTS: We detected Dirofilaria immitis, a parasite species non-endemic in Poland, in two dogs translocated from Ukraine (2/53 = 3.8% [95% CI 0.5-13.0%]). One dog had a history of previous heartworm infection. High prevalence of Hepatozoon canis (51% [95% CI 36.8-64.9%]) was noted among translocated dogs. Prevalence of Dirofilaria repens in Ukrainian dogs (18.9% [95% CI 9.4-32.0%) was similar to prevalence in dogs from central Poland (12%). Co-infection of D. repens and D. immitis was found in two dogs and six dogs were co-infected by D. repens and H. canis. Hepatozoon canis infection was also identified in an imported cat. CONCLUSION: We confirmed that this military conflict has facilitated the spread of canine vector-borne parasites, including zoonotic species.

Hepatozoon spp. infection in wild canids in the eastern United States.

BACKGROUND: Hepatozoon spp. are apicomplexan parasites known to cause musculoskeletal disease in a variety of animals. Two species are known to infect wild and domestic canids in the US: Hepatozoon canis and H. americanum. METHODS: In this study, blood, heart, and/or spleen samples were collected from 278 wild canids (180 coyotes, 93 red foxes, and 5 gray foxes) in the eastern US and tested via PCR for Hepatozoon. Histology slides of heart and skeletal muscle were assessed for Hepatozoon cysts and associated inflammation when fresh tissue was available (n = 96). RESULTS: Hepatozoon spp. were found in 24.2% (59/278) of individuals, with Hepatozoon canis in 14.0% (34/278) and H. americanum in 10.7% (26/278). One coyote was positive for both H. canis and H. americanum. Foxes were more likely to be positive for H. canis than coyotes (23% and 7% respectively, P = 0.0008), while only coyotes were positive for H. americanum. Of the eight sampled states, H. canis was present in six (Louisiana, North Carolina, Pennsylvania, South Carolina, Tennessee, and Virginia) while H. americanum was found in two southern states (South Carolina and Louisiana). Infection status was positively correlated with myositis and myocarditis, and heart or muscle cysts were found in 83% (5/6) of H. americanum-positive coyotes. CONCLUSION: This survey showed a moderate prevalence of H. canis and H. americanum in states where the parasite was previously unrecorded including South Carolina and Pennsylvania.

Epidemiological survey of tick-borne pathogens in dogs in Anhui Province, China.

Tick-borne diseases have continued to increase worldwide in both developing and many developed countries due to the widespread of different tick species and tick's adaptability to different climatic weather. In order to investigate the prevalence of the tick-borne pathogens, EDTA-anticoagulated whole blood samples were aseptically collected from 765 pet dogs in twenty veterinary clinics located in sixteen prefecture-level cities in Anhui Province, China, and the samples were examined and analyzed for tick-borne pathogens using both microscopy and PCR. Our result analysis revealed 17(2.22%) positive samples to Babesia spp and 4(0.52%) positive samples to Hepatozoon spp, of which case of co-infection was recorded in Lu'An and Chuzhou. The BLAST analysis results of the 18S rRNA gene revealed that the dogs were infected with Babesia gibsoni and Hepatozoon canis. All samples were negative for Anaplasma spp., Ehrlichia spp., and Rickettsia spp. This is the first molecular report of B. gibsoni and H. canis in dogs in Anhui, China.

The first report on Hepatozoon canis in dogs and wolves in Poland: clinical and epidemiological features.

BACKGROUND: Canine hepatozoonosis caused by Hepatozoon canis is a common infection in dogs, with frequent case reports from the Mediterranean region and more recently from several Central European countries, such as Hungary and Germany. Despite the high prevalence of H. canis in red foxes, no infections have been reported to date in dogs in Poland. We describe here the first autochthonous cases of H. canis infection in dogs, including their clinical features, and report the prevalence of H. canis in grey wolves from different regions of Poland. METHODS: Thin smears prepared from blood samples collected from dogs were evaluated by microscopic examination. A total of 60 wolves and 47 dogs were tested. Infections were confirmed by PCR and sequencing. RESULTS: Gamonts of H. canis were found in > 50% of the neutrophils of two dogs and in < 10% of the neutrophils in another five dogs. Molecular typing by PCR sequencing of the 18S ribosomal RNA gene fragment confirmed infections in 11 dogs from different regions of Poland, in 2.7% of dogs attending veterinary practices in central Poland and in 35% of wolves from various geographical regions of Poland. Clinical features manifested mostly in older dogs, and the most common signs were anaemia and apathy. Young dogs usually remained asymptomatic. CONCLUSIONS: This is the first report of H. canis infection in dogs and wolves in Poland. Although the exact vector of the parasite is not known, veterinary practitioners should be aware of this new parasitosis and should consider appropriate diagnostics to confirm/exclude this infection. Further studies are needed to understand the transmission routes of H. canis in domestic and wild canids in Poland.

A novel Babesia sp. of the "Western Babesia group", detected in opossums from Guatemala.

Babesia spp. are tick-borne protozoans that involve birds and mammals in their transmission cycles and cause babesiosis, a severe hemolytic malaria-like disease. Opossums of the genus Didelphis are recognized hosts of tick-borne pathogens. Therefore, exploring tick-borne agents in Didelphis species is important to understand the circulation of pathogens in areas where opossums occur. In this study, we targeted Anaplasmataceae, Babesia, Borrelia and Hepatozoon DNA in ticks, blood and organ samples collected from three hunted Didelphis marsupialis specimens in eastern Guatemala. While the samples were negative for Hepatozoon and bacterial DNA, sequences of Babesia 18S rDNA, cox1 and cytb genes were retrieved from two opossums. Ticks collected on the animals included Amblyomma parvum and an undetermined Ornithodoros sp. The Babesia sp. detected in this study (Babesia sp. THB1-2) clusters phylogenetically within the "Western Babesia group", which includes pathogenic species such as Babesia conradae, Babesia duncani, and Babesia negevi. Our results represent the first record of a Babesia sp. in Guatemala and highlight the importance of D. marsupialis as potential spreaders of ticks and pathogens in Central America.

Molecular prevalence and genetic diversity of Hepatozoon spp. in stray cats of Izmir, Türkiye.

Hepatozoon spp. are an apicomplexan protozoan parasites that infect vertebrates including mammals, marsupials, birds, reptiles, and amphibians. Among Hepatozoon species, H. canis and H. felis are causative agents of hepatozoonosis in dogs and cats, respectively and have veterinary importance. This study aimed to determine the prevalence of Hepatozoon spp. in stray cats living in Izmir and investigate genetic diversity among positive samples. To achieve this aim, the prevalence of Hepatozoon spp. 18S rRNA gene was screened by PCR in DNA samples extracted from blood samples of stray cats (n = 1012). Then, Hepatozoon-positive samples were sequenced and the generated data were used for species identification, phylogenetic and haplotype analyses. According to the results, among the samples screened, 2.37 % (24/1012) of them were found to be Hepatozoon-positive, and of these positive samples, 18 (18/24; 75 %) were successfully sequenced. BLAST and phylogenetic analyses revealed that all of these samples were H. felis. Also, phylogenetic analysis showed that H. felis samples were genotype I. Within H. felis samples isolated from cats living in different countries/regions, 9 haplotypes were detected and among these haplotypes, H-1 was found to be prevalent (n = 20 H. felis isolates). In conclusion, this study showed that the prevalence of Hepatozoon spp. was low in stray cats analyzed. Also, H. felis genotype I was predominant in comparison to other Hepatozoon species.

Host specificity of passerine Lankesterella (Apicomplexa: Coccidia).

Lankesterella parasites are blood coccidians that have recently gained attention as their records in common passerine species emerge. To date, their occurrence has been molecularly confirmed in several passerine genera, mainly among members of the families Paridae and Acrocephalidae. Despite their relatively high prevalence in some host populations, their life cycles remain unclear, mosquitoes or mites being the proposed vectors. The aim of this study was to reveal Lankesterella host specificity, focusing mainly on parasites of tit and warbler species (families Paridae and Acrocephalidae). We have determined the 18S rRNA gene sequences of Lankesterella from 35 individuals belonging to eight different host species. Phylogenetic analysis revealed that passerine Lankesterella are host-specific, with specificity at the host genus or species level. Besides Lankesterella, Isospora sequences were obtained from avian blood as well, pointing out the need for barcoding.

Discovery of a new Hepatozoon species namely Hepatozoon viperoi sp. nov. in nose-horned vipers in Türkiye.

Although Hepatozoon spp. remains the most prevalent intracellular protozoa infecting snakes, it was reported only in a few snake species of the Colubridae family in Türkiye. Moreover, studies on these hemoparasites are not available in venomous nose-horned vipers from Türkiye. In this study, we investigated Hepatozoon spp. in three individual Vipera ammodytes using morphological and molecular methods. Our results were positive for intraerythrocytic Hepatozoon spp. gamonts in all three snakes, exhibiting low parasitemia. The microscopic findings were further confirmed through molecular data. A genus-specific PCR assay targeting the 18S rRNA gene region of Hepatozoon spp., was performed using HemoF/HemoR and Hep300/Hep900 primers. The obtained sequences were concatenated and used in phylogenetic analyses in comparison with different Hepatozoon species. Although our (OP377741) isolate was separated into a different branch, it was clustered with the isolates of H. massardi (KC342526), H. cevapii (KC342525), and H. annulatum (ON262426) from Brazilian snakes. Moreover, gene similarity and pair-wise distance between our isolate and other Hepatozoon species infecting snakes were found to be 89.30-98.63% and 0.009-0.077, respectively. Hence, we reported a new species of Hepatozoon, namely Hepatozoon viperoi sp. nov. infecting V. ammodytes. Since the literature does not indicate the existence of such a Hepatozoon species in V. ammodytes in different countries, our data may contribute to the expanding knowledge of Hepatozoon species in snakes, providing new insights into the biodiversity of the haemogregarine protozoan parasite.

Molecular Survey and Microscopic Examination of Haemoparasites Infecting Lizards from Morocco.

PURPOSE: Understanding the factors influencing parasite diversification, their distribution and infection patterns among hosts provides valuable information on the impact of parasitism on natural populations and the dynamics and evolution of disease outbreaks. In this study, we investigated prevalence and intensity of infection by blood parasites, the diversity, distribution, host specificity and infection patterns of apicomplexan parasites in lizards of Morocco. METHODS: Samples from lizard specimens were collected between 2019 and 2021 from different prospected localities in Morocco. In total, 1500 lizards were collected, and their blood and tail tips were screened for blood parasites. RESULTS: Using a PCR approach, we detected two distinct apicomplexan parasite groups (haemogregarines and haemococcidians) and also Stramenopiles (Proteromonas). A significant difference was identified between hosts families, with no agamids or skinks infected and with particularly high infection rates in lacertids. Twenty-four samples were sequenced and included in a phylogenetic assessment. Two samples of Atlantolacerta andreanskyi were infected with unidentified species of Proteromonas. Three samples were infected with haemococcidians, including two samples with Schellackia and another sample with Lankesterella, more closely related to others isolated from bird hosts. These are the first reports of Lankesterella and Proteromonas in these lizards from Morocco. Nineteen different isolates of the complex of Hepatozoon/Karyolysus were identical or very similar to previously sequenced parasites from North Africa and Iberian Peninsula and included new host species. CONCLUSION: Our results highlight the need for future research on the vectors involved in transmission that may influence the ecology and distribution patterns of these parasites. Given that Morocco is a hot spot of endemicity and reptile richness, our results are relevant not only in the context of their taxonomy, but can also have implications for conservation.

Hepatozoon Miller, 1908 parasites in the Colubridae snakes Clelia clelia (Daudin, 1803) and Drymarchon corais (Boie, 1827) from the Eastern Amazonia.

Based on the genetic, morphological, and morphometric data of blood gamonts, we identified Hepatozoon parasites in colubrid snakes sampled in the Eastern Amazon region. Hepatozoon trigeminum was detected in the mussurana snake Clelia clelia and exhibited wide and elongated gamonts (mean dimensions: 14.25±0.65 × 4.31±0.43 µm) with an evident parasitophorous vacuole. Hepatozoon odwyerae sp. nov. was described in the indigo snake Drymarchon corais, whose gamonts have elongated and thin bodies (mean dimensions: 13.41±0.79 × 3.72±0.35 µm) with one end more tapered than the other. Phylogenetic analyses, based on the amplification of a 441 bp fragment of the 18S rRNA gene, revealed that the novel sequences of Hepatozoon spp. from our study were closely related to hemogregarine lineages found in lizards and snakes from Brazil, forming a well-supported monophyletic clade with them. The present study provides the first species description of Hepatoozon in D. corais and a new record of a host species for C. clelia using the integrated taxonomic data. We also highlight the importance of further investigations into the diversity of Hepatozoon in snakes, a rich but underestimated group of parasites, especially in the Amazonian biome.

Molecular characterization of Schellackia parasites in an urban population of sand lizards (Lacerta agilis) from Berlin, Germany.

Lizards are hosts of several taxa of unicellular parasites of the phylum Apicomplexa, including Karyolysus, Schellackia, Lankesterella, and Hepatozoon. Parasite prevalence and the impact of infections on lizard biology remain largely unexplored. In this study, blood parasite infections were investigated in sand lizards (Lacerta agilis) from Berlin, Germany. Eighty-three individuals were investigated, and the detected blood parasites were identified as Schellackia sp. The combination of microscopic and molecular screening revealed a prevalence of 14.5%. Parasitemia values were low and most infections were subpatent. Phylogenetic analysis recovered a close relationship of the Schellackia parasites of this study with Schellackia sp. parasites of different Lacerta and Podarcis lizard species from Spain. Monitoring of Schellackia parasite infections in free-ranging lizards contributes to a better understanding of the distribution, diversity, and phylogenetic relationships of the neglected parasite taxon.

Hepatozoon species infecting domestic cats from countries of the Mediterranean basin.

Tick-borne diseases (TBDs) are caused by pathogens of human and veterinary concern representing a major public health issue worldwide. Although feline medicine has progressed much in the recent decades, data on feline TBDs (FeTBDs) remain scant. Therefore, this study aimed to assess the prevalence of apicomplexan parasite infections, associated risk factors and clinical-hematological abnormalities in domestic feline populations from countries of the Mediterranean basin. Blood and serum samples from cats (n = 600) living in France, Greece, Israel, Italy, Portugal and Spain were collected along with animal data (i.e., age, sex, breed, housing conditions and geographical origin), clinical signs and laboratory blood test parameters. Cats were grouped according to their age as kitten (up to one year), young (between one and six years), mature (between seven and ten years) and senior (older than ten years). Blood samples were tested for Hepatozoon spp. and piroplasmids by conventional PCR targeting 18S rRNA gene. The overall prevalence of Hepatozoon spp. infection was 14.5%, being significantly higher in cats from Greece (30%) and Portugal (23%), followed by Spain (15%), Israel (15%) and France (4%). Cats from Italy scored negative. Hepatozoon felis was identified in 86 animals, with three different sequence types and H. silvestris was detected in one shelter cat from Portugal. No piroplasmid DNA was amplified. The risk of Hepatozoon spp. infection was related to feline geographical provenience, housing condition and age. No statistical correlation was reported with any clinical signs, while increased alanine aminotransferase (ALT) activity was the only laboratory abnormality significantly associated (p = 0.03) with the infection. Data suggest a high circulation of H. felis, and only occasionally of H. silvestris, within domestic feline populations in the Mediterranean basin, mainly in shelter or free roaming and young cats with asymptomatic or subclinical infection.

Molecular characterization of haemosporidian and haemogregarine diversity in southwestern Iberian amphibians and reptiles.

The knowledge of the diversity and geographic distribution of parasite species is the first step towards understanding processes of global epidemiology and species conservation. Despite recent increases in research on haemosporidian and haemogregarine parasites of reptiles and amphibians, we still know little about their diversity and parasite-host interactions, especially in the Iberian Peninsula, where a few studies have been conducted. In this study, the haemosporidian and haemogregarine diversity and phylogenetic relationships of the parasites in southwestern Iberian amphibians and reptiles were assessed using PCR approaches on blood samples of 145 individuals from five amphibian and 13 reptile species. The amphibians did not present any of both groups of parasites studied. Regarding reptiles, five Hepatozoon, one Haemogregarina, and one Haemocystidum haplotypes were found infecting four different species, revealing new host records for these parasites. Among them, we found one new Haemocystidium haplotype and three new and a previously reported Hepatozoon haplotype from a north African snake. The latter finding suggests that some Hepatozoon parasites may not be host-specific and have large geographic ranges even crossing geographical barriers. These results increased the knowledge about the geographic distribution and the number of known host species of some reptile apicomplexan parasites, highlighting the great unexplored diversity of them in this region.

Field survey and molecular characterization of apicomplexan parasites in small mammals from military camps in Afghanistan.

Small mammals are an important reservoir for causative agents of numerous infectious diseases, including zoonotic and vector-borne diseases. The occurrence of these pathogens represents a regional but permanent threat for humans and animals in general and might especially weaken military personnel and companion animals in abroad missions. In our study, small mammals collected in military camps in Afghanistan (Feyzabad, Mazar-e Sharif, and Kunduz) were investigated for the presence of apicomplexans using histopathology and molecular methods. For this purpose, well-established and newly developed real-time PCR assays were applied. A high prevalence was detected not only in house mice (Mus musculus), but also in shrews (Crocidura cf. suaveolens) and grey dwarf hamsters (Cricetulus migratorius). The molecular characterization based on the 18S rRNA gene revealed a close relationship to a cluster of Hepatozoon sp. detected in voles of the genus Microtus. Hepatozoon canis DNA was detected in one house mouse as well as in two Rhipicephalus ticks from a dog puppy. In addition, around 5% of the house mice were found to be infected with far related adeleorinids showing the highest sequence identity of 91.5% to Klossiella equi, the only published Klossiella sequence at present. For their better phylogenetic characterization, we conducted metagenomics by sequencing of two selected samples. The resulting 18S rRNA gene sequences have a length of about 2400 base pairs including an insertion of about 500 base pairs and are 100% identical to each other. Histopathology together with organ tropism and detection rates verified this sequence as of Klossiella muris. In conclusion, we documented naturally occurring protozoan stages and the additional taxonomic characterization of a well-known commensal in mice by applying a combination of different approaches. The study is of medical, social, and biological importance for ensuring human and animal health in military camps and also stresses the required awareness for the potential risk of zoonoses.