Description of an intramonocytic haemoparasite, Hepatozoon lainsoni sp. nov. (Apicomplexa: Adeleorina: Hepatozoidae), infecting Ameiva ameiva lizard (Reptilia: Squamata: Teiidae) in northern Brazil.

Haemogregarine (Apicomplexa: Adeleorina) parasites are considered to be the most common and widespread haemoparasites in reptiles. The genus Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) can be found parasitizing a broad range of species and, in reptiles, they infect mainly peripheral blood erythrocytes. The present study detected and characterized a haemogregarine isolated from the lizard species, Ameiva ameiva, collected from the municipality of Capanema, Pará state, north Brazil. Blood smears and imprints from lungs, brain, heart, kidney, liver, bone marrow and spleen were observed using light microscopy and the parasite was genetically identified by molecular analysis. Morphological, morphometric and molecular data were obtained. Parasite gamonts were found in 49.5% (55/111) of the blood smears from A. ameiva, and were characterized as oval, averaging 12.0 ± 0.8 × 5.9 ± 0.6 µm2 in size, which displaced the nuclei of parasitized monocytes laterally. Parasite forms resembling immature gamonts were observed in the spleen and bone marrow of the lizards. Furthermore, phylogenetic analyses of 18S rRNA sequences did not reveal gene similarity with other Hepatozoon spp. sequences from reptiles. Thus, morphological and molecular analyses have identified a new species of Hepatozoon parasite, Hepatozoon lainsoni sp. nov., which infects monocytes of the A. ameiva lizard.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Island hitchhikers: pathogen agents of Madeira and Azores ticks.

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.

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.

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

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

Clinical, diagnostic and epidemiological implications of Hepatozoon spp., Babesia spp. and Leishmania infantum infection in cats and dogs in a Mediterranean periurban setting.

Hepatozoon spp., Babesia spp. and Leishmania infantum are common parasites of dogs in Mediterranean countries and are less frequent in cats, particularly Babesia spp. and L. infantum. Moreover, there is limited information on coinfections between these parasites and on L. infantum's distribution in blood, skin and lymphoid tissue in cats. We used PCR and DNA sequencing to investigate the prevalence of these parasites and the aetiology of Hepatozoon spp. and Babesia spp., in blood, skin, spleen and lymph node samples from up to 212 stray cats and 82 abandoned dogs in southeast Spain. All except 2 dogs were healthy; instead, 112 cats had clinical signs. The estimated PCR prevalences (95% confidence interval) were 25% (19-31%) Hepatozoon felis in cats, 13% (6-21%) Hepatozoon canis in dogs, 1% (0-4%) Babesia vogeli in dogs, 0% Babesia spp. in cats and 21% (15-26%) and 44% (33-55%) L. infantum in cats and dogs, respectively, and infections were not associated with each other. Leishmania infantum prevalence in lymphoid tissue was significantly higher in dogs than in cats (p < 0.001), and dogs had higher parasite loads than cats (p = 0.012). Moreover, L. infantum prevalence was significantly higher in the skin and lymphoid tissue compared to blood in infected, asymptomatic animals but it was similar in cats with clinical signs, which also had higher parasite loads compared to infected, asymptomatic cats (p < 0.05). The study highlights significant differences between sympatric dogs and cats with respect to the parasite infections investigated, as well as the need to examine both lymphoid tissue and skin samples to maximise the sensitivity of L. infantum infection diagnosis.

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.

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.

Molecular discrimination and genetic diversity of three common tick-borne pathogens in dogs in Thailand.

There was little information regarding the occurrence of canine vector-borne disease (CVBDs) in shelter dogs in Thailand. This work is the first report regarding a molecular method used to determine the occurrence and genetic diversity of three canine tick-borne pathogens (TBPs) (Hepatozoon canis, Anaplasma platys and Ehrlichia canis) in blood samples from 275 shelter dogs in the north and central areas of Thailand. The PCR results based on the 18S rRNA and 16S rRNA genes showed that 71 (25.82%) dogs were positive for at least a TBP. The overall occurrence rates of H. canis, A. platys and E. canis infections were 1.81, 16.36 and 7.64%, respectively. For the phylogenetic analysis, A. platys 16S rRNA gene was genetically diverse, while H. canis 18S rRNA and E. canis 16S rRNA genes were conserved. The haplotype diversity exhibited 12 and 2 haplotypes as well as 78 and 178 polymorphic sites of A. platys and E. canis 16S rRNA genes, respectively. Our findings could be used to improve the understanding of phylogeny and genetic diversity of TBP rRNA genes and used to ameliorate the diagnosis and control programmes for the diseases in Thailand.

Molecular investigation and clinical management of Hepatozoon Canis infection in an Indian jackal - a case report.

BACKGROUND: Hepatozoonosis is a common tick-borne illness reported from all over the world. The infection has been well documented in dogs and cats, and has also been identified in wild canids and felids. India is home to many canid species; however, the incidence of Hepatozoonosis in wild canids is rarely reported. A wide variety of protocols have been discussed for the clinical management of the infection in companion animals; however, the suitability of treatment protocols in wild canids is understudied. The current case report highlights the clinical management of Hepatozoonosis in an Indian jackal and molecular investigation to provide vital insights into the epidemiology of the disease. CASE PRESENTATION: A paraplegic Indian jackal was rescued from Melghat Tiger Reserve, Maharashtra, India. The animal had extensive decubital ulcers on the left pin bone and could not walk; however, the animal was active and dragged the hindlimb during locomotion. The vital parameters, blood and serum investigations were normal. Post physiotherapy, massage and infrared therapy, the animal could walk but started knuckling, resulting in injuries. Eight weeks into rehabilitation, the animal had a steep fall in haemoglobin concentration, platelet count, weight loss and was diagnosed with Hepatozoonosis. Considering the altered vital parameters, the jackal was rationally treated with Doxycyclin @ 20 mg/Kg O.D. (Once Daily) for 45 days along with supportive therapy. The jackal recovered after the treatment and led a normal life. CONCLUSION: Mono-drug regime using Doxycycline was effective in the alleviation of H.canis infection in jackal. The drug was effective in alleviating the clinical presentation without alteration of vital parameters. The molecular investigation provided qualitative inputs in understanding the epidemiology of Hepatozoon in wild canids.

Morphological detection and molecular characterization of Hepatozoon spp. from venomous terrestrial snakes in Iran.

Hepatozoon spp. are blood protozoans that can infect snakes. Infections with these parasites are found in more than 200 snake species and are considered to be the most frequent hemogregarines in snakes. In this study, a total of 73 dead snakes in captivity conditions, belonging to six different species, which were collected between June 2018 to October 2020 from different parts of Iran, were examined for the presence of blood parasites with microscopy and molecular methods. DNA was extracted from the removed heart, and PCR was done with two pairs of primers targeting the 18S rRNA gene. By microscopy, Hepatozoon spp. were detected in the heart blood of 29 out of the 73 (39.72%) snakes. From these 29 positive samples, eight were found to be positive using the PCR method. This study aimed to report for the first time the molecular characteristics of Hepatozoon spp. infecting venomous terrestrial snakes in Iran.

Apicomplexans in small mammals from Chile, with the first report of the Babesia microti group in South American rodents.

Small mammals play an essential role as disseminators of pathogens because they reach high population densities and have ubiquitous distributions. In the Northern Hemisphere rodents are well recognized as reservoirs for tick-borne bacteria of the Anaplasmataceae family and also apicomplexan protozoans. In contrast, South American rodents hosting these microorganisms have been rarely identified. In this study, we collected blood from rodents and marsupials in northern Chile and screened for Anaplasmataceae bacteria and apicomplexan protozoa. Overall, 14.7% of the samples were positive for Babesia, Hepatozoon, and Sarcocystidae using conventional PCR assays targeting the structural 18S rRNA locus (18S). Phylogenetic analyses performed with amplicons derived from 18S and cytochrome c oxidase (COI) gene provided evidence of a Babesia sp. belonging to the Babesia microti group in Phyllotis darwini, and a novel Babesia genotype in P. darwini and Abrothrix jelskii. Furthermore, four novel genotypes of Hepatozoon retrieved from Abrothrix olivacea, P. darwini, and Oligoryzomys longicaudatus, formed independent lineages within a clade that includes additional Hepatozoon spp. detected in South American rodents. Moreover, an incidental finding of a previously detected apicomplexan, herein designated as Sarcocystidae sp., was recorded in Thylamys opossums with a high prevalence, indicating a possible specific association with these mammals. Phylogenetic analysis of Sarcoystidae sp. clearly demonstrated its relatedness to apicomplexans detected in Australian marsupials. Our results expand the range of mammals hosting tick-borne apicomplexans in South America, highlight a novel clade consisting of South American babesias, and report for the first time the B. microti group infecting rodents in the region.

Vector-borne pathogens of zoonotic concern in dogs from a Quilombola community in northeastern Brazil.

Canine vector-borne pathogens (CVBPs) comprise a group of disease agents mainly transmitted by ticks, fleas, mosquitoes and sand flies. In this study, we assessed the presence of CVBPs in an Afro-descendent community (Quilombola) of northeastern, Brazil. Dog blood samples (n = 201) were collected and analyzed by rapid test for the detection of antibodies against Leishmania spp., Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi sensu lato (s.l.), and antigens of Dirofilaria immitis. In addition, polymerase chain reactions were performed for Anaplasmataceae, Babesia spp., Hepatozoon spp., Rickettsia spp. and B. burgdorferi s.l. Overall, 66.7% of the dogs scored positive to at least one pathogen at serological and/or molecular methods. Antibodies against Ehrlichia spp. were the most frequently detected (57.2%; n = 115/201), followed by Anaplasma spp. (8.5%; n = 17/201), Leishmania spp. (8.5%; n = 17/201) and B. burgdorferi s.l. (0.5%; n = 1/201). For D. immitis, 11 out of 201 (5.5%) animals scored positive. At the molecular analysis, 10.4% (n = 21/201) of the samples scored positive for Babesia spp./Hepatozoon spp., followed by Anaplasmataceae (5.0%; n = 10/201) and Rickettsia spp. (3.0%; n = 6/201). All samples were negative for B. burgdorferi s.l. Our data demonstrated the presence of CVBPs in the studied population, with a high seropositivity for Ehrlichia spp. In addition, considering the detection of zoonotic pathogens in dogs and their relationship with people from Quilombola communities, effective control strategies are advocated for minimizing the risk of infection in this socially vulnerable human population and their pets.

Molecular and morphological description of the first Hepatozoon (Apicomplexa: Hepatozoidae) species infecting a neotropical turtle, with an approach to its phylogenetic relationships.

Haemogregarines (Adeleorina) have a high prevalence in turtles. Nevertheless, there is only one Hepatozoon species described that infects Testudines so far; it is Hepatozoon fitzsimonsi which infects the African tortoise Kinixys belliana. Colombia harbours a great diversity of chelonians; however, most of them are threatened. It is important to identify and characterize chelonian haemoparasite infections to improve the clinical assessments, treatments and the conservation and reintroduction programs of these animals. To evaluate such infections for the Colombian wood turtle Rhinoclemmys melanosterna, we analysed blood from 70 individuals. By using the morphological characteristics of blood stages as well as molecular information (18S rRNA sequences), here we report a new Hepatozoon species that represents the first report of a hepatozoid species infecting a semi-aquatic continental turtle in the world. Although the isolated lineage clusters within the phylogenetic clades that have morphological species of parasites already determined, their low nodal support makes their position within each group inconclusive. It is important to identify new molecular markers to improve parasite species identification. In-depth research on blood parasites infecting turtles is essential for increasing knowledge that could assess this potential unknown threat, to inform the conservation of turtles and for increasing the state of knowledge on parasites.

Evaluation of hematological alteration of vector-borne pathogens in cats from Bangkok, Thailand.

BACKGROUND: Cats can be carriers of infected arthropods and be infected with several vector-borne pathogens (VBPs) but there is limited knowledge about their pathogenic role in cats. This study aimed to assess the prevalence of some feline vector-borne agents by molecular technique and to characterize the hematological findings associated with these infections in a cat population from Bangkok Thailand. RESULTS: PCR was positive with at least one pathogen in 237 out of 372 subjects (63.7%), with prevalence of 39.5% (147/372) for Babesia spp., 36.9% (137/372) for hemoplasmas and 3.2% (12/372) for Hepatozoon spp. The cats older than 1 year were at significantly greater risk for VBPs infection (P = 0.001; OR = 1.43; 95% CI: 1.12 - 1.81) and hemoplasmas infection (χ2 = 10.8, df = 1; P < 0.0001; OR = 2.45; 95% CI: 1.49 - 4.01). A significant association between hematological findings and hemoplasma infection were identified in the present study. Besides, VBPs infection revealed more frequent in male cats (χ2= 6.38, df = 1, P = 0.01). Macrocytic hypochromic type of anemia was observed in cats infested with blood-sucking arthropods compared to the non-infested cats presented. CONCLUSIONS: The current study confirmed that Babesia, Hepatozoon and hemoplasmas had infected semi-domesticated cats in Bangkok, Thailand, with Babesia and hemoplasmas being dominant in prevalence. Some hematological findings were significantly associated with cats infected with vector-borne pathogens in this study including leukocyte count and platelets count that may help support veterinary technicians in diagnosis and appropriate treatment. Campaigns of VBPs monitoring in Bangkok emphasizing on the investigation of vectors and possible routes of the infection in animals should be conducted to prevent the transmission of the pathogens.

Prevalence and distribution of pathogen infection and permethrin resistance in tropical and temperate populations of Rhipicephalus sanguineus s.l. collected worldwide.

The brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.) Latreille (Acari: Ixodidae), is a peridomestic, cosmopolitan parasite of dogs known to vector numerous pathogens of veterinary and medical importance. Recent phylogenetic analyses separate this tick into temperate and tropical lineages. Populations of Rh. sanguineus s.l. have been reported to exhibit sodium channel target site insensitivity to permethrin and etofenprox, which is likely due to the prolonged use of pyrethroids against many pests in and around the home. In this study, populations collected in the Caribbean, Africa, Asia, Europe and North America, were tested to identify the distribution of a known resistance mechanism, pathogen-vector interactions and phylogeny in relation to latitude. Using molecular assays, populations from 29 distinct locations were simultaneously geographically typed and screened for bacterial infection by Rickettsia, Ehrlichia, Babesia and Hepatozoon species, and for the presence of a sodium channel single nucleotide polymorphism known to confer permethrin resistance. Implications of these results on Rh. sanguineus s.l. management in association with geographical distribution will be discussed.

An Hepatozoon americanum-like protozoan in crab-eating (Cerdocyon thous) and grey pampean (Lycalopex gymnocercus) foxes from Uruguay.

In South America, apicomplexan parasites of the genus Hepatozoon have been sporadically detected in mammals. Previous studies in wild canids from Brazil and Argentina demonstrated infections by species genetically related to Hepatozoon americanum. The aim of the present work was to detect the presence of Hepatozoon in road-killed foxes encountered in Uruguayan highways. Blood samples from 45 crab-eating (Cerdocyon thous) and 32 grey pampean (Lycalopex gymnocercus) foxes were analyzed by PCR for Hepatozoon 18S rRNA gene. Eight foxes (10.4%) were found to be infected with an H. americanum-like protozoan, an Hepatozoon closely related to H. americanum. Bayesian and maximum-likelihood phylogenetic analyses revealed that the sequences obtained in this study cluster with H. americanum from the United States, and with an H. americanum-like species from dog and foxes from Brazil and Argentina. In the Unites States, H. americanum causes severe disease in dogs. In addition to this, an increasing habitat overlap between dogs and foxes makes the presence of H. americanum-like protozoan in foxes acquires veterinary relevance. This work represents the first report of L. gymnocercus infected with an H. americanum-like protozoan, and of wild canids infected with Hepatozoon in Uruguay.

Hepatozoon in Eurasian red squirrels Sciurus vulgaris, its taxonomic identity, and phylogenetic placement.

Adeleorid apicomplexan parasites of the genus Hepatozoon Miller, 1908 are broadly distributed among the rodents. Broader molecular data on Hepatozoon from Palaearctic squirrels are necessary for evaluation of diversity and origin of Hepatozoon in Eurasian red squirrel Sciurus vulgaris populations, considering ongoing invasion by Gray squirrel S. carolinensis. Our report brings a set of molecular data from a population of S. vulgaris in the Czech Republic, non-invaded by any invasive squirrel species. Cadavers of 41 Eurasian red squirrels were examined using nested PCR targeting 18S rRNA gene; 30 animals tested positive for the presence of Hepatozoon spp. DNA in at least one tissue. Phylogenetic analysis of obtained sequence types revealed relatedness to sequences of Hepatozoon sp. from S. vulgaris from Spain and the Netherlands, forming a sister clade to Hepatozoon isolates from other European rodents. The fact that all available 18S rRNA gene sequences form a monophyletic clade is interpreted as a presence of a single Hepatozoon species in S. vulgaris in continental Europe, most probably Hepatozoon sciuri. The presented molecular data on the Hepatozoon from European squirrels provides a basis for future studies on possible exchange of Hepatozoon species between Eurasian red and gray squirrels.

First molecular detection of Hemolivia and Hepatozoon parasites in reptile-associated ticks on Iriomote Island, Japan.

Hepatozoon and Hemolivia are members of the haemogregarines and are reported in reptiles and reptile-associated ticks. However, no studies have reported on Hepatozoon and Hemolivia in Japanese reptile-associated ticks. This study aimed to molecularly identify and to characterize Hepatozoon and Hemolivia in Japanese reptile-associated ticks, Amblyomma geoemydae (Cantor, 1847) and Amblyomma nitidum (Hirst & Hirst, 1910). A total of 41 and 75 DNA samples from A. geoemydae and A. nitidum ticks, respectively, were used for screening of Hepatozoon and Hemolivia with polymerase chain reaction targeting 18S rDNA. As a result, Hemolivia and Hepatozoon were detected in two A. geoemydae and one A. nitidum, respectively. The sequences of Hemolivia spp. showed a 99.5% (1,050/1,055 bp) identity with Hemolivia parvula (KR069083), and the Hemolivia spp. were located in the same clade as H. parvula in the phylogenetic tree. The sequences of Hepatozoon sp. showed a 98.4% (1,521/1,545 bp) identity with Hepatozoon colubri (MN723844), and the Hepatozoon sp. was distinct from validated Hepatozoon species in the tree. Our findings highlight the first molecular record of Hemolivia in Japan and present the first detection of Hepatozoon in A. nitidum. Further investigations on these tick-borne protozoa are required to understand their life cycle and pathogenicity.

Vector-borne pathogens in dogs of different regions of Iran and Pakistan.

Canine vector-borne diseases (CVBDs) are highly prevalent in tropical and subtropical countries, mainly due to favorable climate conditions and reduced adoption of preventive measures. This study aimed to provide a comprehensive overview on the prevalence of CVBDs in Iran and Pakistan where limited data are available. Blood samples were collected from 403 dogs from six provinces in Iran and Pakistan to assess the presence of pathogen DNA (i.e., Anaplasma spp., Coxiella burnetii, Ehrlichia spp., Rickettsia spp., Babesia spp., Hepatozoon spp., filarioids, and Leishmania spp.). Sera were also screened by an immunofluorescence antibody test for the detection of antibodies against Leishmania infantum. In total, 46.9% of dogs scored positive to Hepatozoon canis being the most frequently detected (41.4%), followed by Anaplasma platys (6.4%), Ehrlichia canis (3.4%), Rickettsia spp. (2.2%), Babesia vogeli (1.0%), and L. infantum (0.3%). A seroprevalence of 9.6% to anti-L. infantum IgG was also recorded. Data reported herein demonstrate that dogs from Iran and Pakistan are at a high risk of CVBDs, particularly of canine hepatozoonosis. Effective control strategies are advocated for minimizing the risk of infection in animals and humans, also in consideration of the zoonotic potential of some pathogens detected.