The genetic and morphological diversity of Haemogregarina infecting turtles in Colombia: Are mitochondrial markers useful as barcodes for these parasites?

Adeleorinid parasites commonly infect turtles and tortoises in nature. Currently, our knowledge about such parasites is extremely poor. Their characterization is based on morphological and molecular approaches using the 18S rDNA molecular marker. However, there is a limitation with the 18S rDNA due to its slow rate of evolution. For that reason, the goals of this study were to 1) design primers for new molecular mitochondrial markers to improve the phylogenetic reconstructions of adeleorinid parasites and 2) to determine the morphological and genetic diversity of Haemogregarina infecting turtles and tortoises in Colombia. Turtles from 16 species representing six families were examined for the presence of haemoparasites. We analyzed 457 samples using PCR, and 203 of them were also analyzed by microscopy. Using a mitochondrial genome of Haemogregarina sequenced in this study, we designed primers to amplify fragments of the cytochrome oxidase I (coxI), cytochrome oxidase III (coxIII), and cytochrome b (cytb) mitochondrial markers in adeleorinid parasites. Lineages obtained from nuclear and mitochondrial molecular markers clustered according to the turtle lineages from which they were isolated. It is noteworthy that we found different evolutionary lineages within the same morphotype, which may indicate heteroplasmy and/or cryptic diversity in Haemogregarina. Due to this situation, we could not make a species delimitation, even when integrating the different lines of evidence we had in this study. However, the primers presented here are useful for diagnosis and, moreover, according to the available information, all three genes retain phylogenetic signals; thereby fragments amplified can be used in reconstructing evolutionary relationships. This effort contributes to the knowledge of the diversity of these parasites infecting continental turtles from Colombia.

Absence of Hemolivia mauritanica (Apicomplexa: Haemogregarinidae) in natural populations of Testudo graeca in Morocco.

During spring 2018, we captured 101 spur-thighed tortoises, Testudo graeca, from seven localities in central Morocco. All tortoises were examined for the presence of blood parasites Hemolivia mauritanica and Hyalomma aegyptium ticks, the known vectors. We looked for H. mauritanica infection by examination of blood smears and by genetic analysis with PCR using extractions from both tortoises and ticks. On all tortoises collected, 71.29% were infested with at least one tick, with a mean infestation intensity of 7.12 ticks/tortoise and maximum of 15.55 ticks/tortoises in Had Draa locality (Essaouira region). Although the definitive host is present and abundant in all tortoise populations, all blood samples were Hemolivia-negative. Our results support and confirm the finding of studies previously conducted in other populations of Morocco and indicate that H. mauritanica has a narrower distribution range than its tick vector.

Identical 18S rRNA haplotypes of Hepatozoon canis in dogs and foxes in Brandenburg, Germany.

Hepatozoon canis is a blood parasite of the suborder Adeleorina infecting wild and domestic canids. Transmission occurs by oral uptake of Rhipicephalus sanguineus sensu lato vector ticks infected with H. canis, but vertical transmission is also assumed to be possible. In German foxes, a high prevalence of H. canis has previously been reported despite the fact that R. sanguineus s.l. is not endemic. In the absence of knowledge about local transmission pathways, foxes should be considered to be possible reservoirs of H. canis and contribute to infection of domestic dogs. The present study aimed to determine how often foxes and dogs are infected in Brandenburg (Germany) and if identical or different H. canis 18S rRNA haplotypes are found in these host species. Hepatozoon spp. were detected by PCR in 46/1050 (4.4 %) of dog blood and 176/201 (77.6 %) of fox spleen samples from Brandenburg. Sequencing of 19 dog and 56 fox samples identified all as H. canis. For nine positive dogs, owners stated that they had never left Germany suggesting that autochthonous transmission occurs not only in foxes but also in dogs. Sequences for seven of these possible autochthonous cases were obtained and six were identical to the predominant haplotype found in the foxes. Haplotype network analysis confirmed that many dogs, including some without travel history, carried the same or very similar 18S rRNA haplotypes as the foxes suggesting that both hosts participate in the same epidemiological cycle.

Hepatozoon canis in hunting dogs from Southern Italy: distribution and risk factors.

Hepatozoon canis is a hemoprotozoan organism that infects domestic and wild carnivores throughout much of Europe. The parasite is mainly transmitted through the ingestion of infected ticks containing mature oocysts. The aims of the present survey were to determine the prevalence of H. canis in hunting dogs living in Southern Italy and to assess potential infection risk factors. DNA extracted from whole blood samples, collected from 1433 apparently healthy dogs living in the Napoli, Avellino, and Salerno provinces of Campania region (Southern Italy), was tested by a quantitative real-time polymerase chain reaction (qPCR) assay to amplify H. canis. Furthermore, the investigated dog population was also screened by qPCR for the presence of Ehrlichia canis, a major tick-borne pathogen in Southern Italy, in order to assess possible co-infections. Two hundred dogs were H. canis PCR-positive, resulting in an overall prevalence of 14.0% (CI 12.2-15.9). Breed category (P < 0.0001), hair coat length (P = 0.015), and province of residence (P < 0.0001) represented significant risk factors for H. canis infection. The presence of H. canis DNA was also significantly associated with E. canis PCR positivity (P < 0.0001). Hunting dogs in Campania region (Southern Italy) are frequently exposed to H. canis, and the infection is potentially associated with close contact with wildlife. Further studies are needed to assess the pathogenic potential of H. canis, as well as the epidemiological relationships between hunting dogs and wild animal populations sharing the same habitats in Southern Italy.

Molecular survey of tick-borne pathogens reveals a high prevalence and low genetic variability of Hepatozoon canis in free-ranging grey wolves (Canis lupus) in Germany.

Wild carnivores are important hosts for ixodid ticks and presumed reservoirs for several tick-borne pathogens (TBPs) of medical and veterinary importance. However, little is known about the role that the European grey wolf (Canis lupus) plays in the enzootic cycle of TBPs. The recent recolonization of Central European lowland, including some regions in Germany, by the grey wolf, opened up an excellent opportunity for studying the impact of the wolf population on pathogen diversity and transmission dynamics. Therefore, in the present study, we investigated spleen samples collected from 276 grey wolves in 11 federal states in Germany for common TBPs by molecular methods. In total, 127 grey wolves (46.0 %) were found to be positive for Hepatozoon canis. Only two genetic variants of this protozoan parasite (herein designated as G1 and G2) were found to circulate among the grey wolves, which can be potentially shared between populations of domestic and other wild carnivores in the country. Two grey wolves were positive for Anaplasma phagocytophilum (0.7 %), and both were co-infected with H. canis G1 genotype. The presence of other pathogens could not be confirmed by PCR and sequencing. This study represents the first one reporting H. canis in a grey wolf population worldwide, and it provides highly relevant information, which may contribute to a better understanding of the epidemiology of TBPs and the pathogen transmission dynamics among the reintroduced population of grey wolves and other carnivores.

Haemogregarina daviesensis sp. nov. (Apicomplexa: Haemogregarinidae) from South American lungfish Lepidosiren paradoxa (Sarcopterygii: Lepidosirenidae) in the eastern Amazon region.

Based on morphology and morphometry of gametocytes in blood and molecular phylogenetic analysis, we described a new species of hemoparasite from the genus Haemogregarina isolated from Lepidosiren paradoxa in the eastern Amazon region. Haemogregarina daviesensis sp. nov. is characterized by monomorphic gametocytes of varying maturity stage and their dimensions were 16 ± 0.12 µm (range 13-18) in length and 6 ± 0.97 µm (range 5-8) in width. The morphological and morphometric data were not identical with other haemogregarine species from fish. All specimens of L. paradoxa analyzed were infected by H. daviesensis sp. nov. and the parasitemia level was moderate (1-28/2000 blood erythrocytes). Two sequences were obtained from L. paradoxa, and these constituted a monophyletic sister clade to the Haemogregarina species. In addition, H. daviesensis sp. nov. detected here grouped with Haemogregarina sp. sequences isolated from chelonian Macrochelys temminckii, with 99% bootstrap support. This study provides the first data on the molecular phylogeny of an intraerythrocytic haemogregarine of freshwater fish and highlights the importance of obtaining additional information on aspects of the general biology of these hemoparasites in fish populations, in order to achieve correct taxonomic classification.

Morphology and Phylogeny of Calyptospora paranaidji n. sp. (Eimeriorina: Calyptosporidae), an Apicomplexan Parasite of the Hepatic Tissue of Cichla piquiti Kullander & Ferreira, 2006, From a Reservoir in the Brazilian Amazon Region.

The coccidians of the family Calyptosporidae are parasites of the tissue and organs of fish and aquatic invertebrates, in particular in the tropical region. In contrast with other apicomplexans of the suborder Eimeriorina, the diversity and ecology of the species of the genus Calyptospora have been poorly investigated, resulting in a lacuna that restricts the understanding of the distribution and prevalence of this group of eukaryote microparasites in the Amazon region. In the present study, the integrated comparative analysis of morphological characteristics, histological and structural traits, and the sequences of a fragment of the 18S rRNA gene, provides support for the identification of a new species of Calyptospora, found parasitizing the hepatic tissue of the piscivorous blue peacock bass, Cichla piquiti, captured in the reservoir of the Estreito hydroelectric dam on the middle Tocantins River in northern Brazil. This new species was named Calyptospora paranaidji n. sp.

Haemogregarine (Apicomplexa: Adeleorina) infection in Vanderhaege's toad-headed turtle, Mesoclemmys vanderhaegei (Chelidae), from a Brazilian Neotropical savanna region.

Knowledge of blood parasites in Brazilian chelonians is limited, since they have been recorded in only six species. Mesoclemmys vanderhaegei (Bour) is a freshwater turtle with a wide geographic distribution in Brazil, but there is little information about its natural history. This paper reports on a study of the prevalence and infection intensity of a haemogregarine in two subpopulations of M. vanderhaegei. The study was conducted in two areas of Cerrado in the Upper Paraguay River basin in the state of Mato Grosso, Brazil, between November 2010 and August 2013. Ninety-five (53%) of the 179 turtles captured were positive for haemogregarine parasites. The parasitic forms observed were two morphotypes of intraerythrocytic gametocytes. The prevalence differed between size classes, increasing significantly according to the animals' body size. There was no significant difference between prevalence and sex, or between sampling periods. The mean parasite intensity was 9 parasites/2,000 erythrocytes (0.45%) and the parasite population presented an aggregated distribution, with an aggregation index of 19 and discrepancy of 0.772. This is the first record of a hemoparasite in the freshwater turtle M. vanderhaegei.

Two new species of Hepatozoon (Apicomplexa: Hepatozoidae) parasitising species of Philothamnus (Ophidia: Colubridae) from South Africa.

To date, only a few species of Hepatozoon Miller, 1908 have been described from amphibians and reptiles of South Africa, including two species from anuran hosts, three from saurians, one from chelonians, and two from ophidians. Hepatozoon bitis (Fantham, 1925) and Hepatozoon refringens (Sambon et Seligmann, 1907), parasitising Bitis arientans (Merrem) and Pseudoaspis cana (Linnaeus), respectively, were described in the early 1900s and since then there have been no further species of Hepatozoon described from snakes in South Africa. Blood smears, used in peripheral blood haemogregarine stage morphometrics, and whole blood used in molecular characterisation of haemogregarines were collected from the caudal vein of six snakes of three species, namely Philothamnus hoplogaster (Günther), Philothamnus semivariegatus (Smith) and Philothamnus natalensis natalensis (Smith). For comparison, a comprehensive table summarising available information on species of Hepatozoon from African snakes is presented. Haemogregarines found infecting the snakes from the present study were morphologically and molecularly different from any previously described from Africa and are thus here described as Hepatozoon angeladaviesae sp. n. and Hepatozoon cecilhoarei sp. n. Both haemogregarine species were observed to cause considerable dehaemoglobinisation of the host cell, in case of infection with H. angeladaviesae resulting in a characteristic peripheral undulation of the host cell membrane and karyorrhexis. To the authors' knowledge, these are the first haemogregarines parasitising snakes of the genus Philothamnus Smith described using both morphological and molecular characteristics in Africa.

Genetic diversity of Hepatozoon spp. in Hydrochoerus hydrochaeris and Pecari tajacu from eastern Amazon.

This study aimed to identify and characterize genetically species of the genus Hepatozoon detected in Hydrochoerus hydrochaeris (capybaras) and Pecari tajacu (collared peccaries) from two localities from the Eastern Amazon. Blood samples from 196 free-living H. hydrochaeris from Marajó Island and 109 P. tajacu kept in captivity in Belém, Pará, were collected and analyzed for the presence of Hepatozoon spp. Partial sequences of the 18S rRNA gene were obtained and analyzed in comparison to others available in the NCBI database. Our results demonstrated a high prevalence of Hepatozoon canis in both mammals and the existence of four haplotypes of Hepatozoon spp., three of Hepatozoon canis and one of Hepatozoon cuestensis, found only in H. hydrochaeris. In addition, these data increase the genetic diversity of H. canis from the Eastern Amazon, as well as reporting, for the first time, the infection of mammals by H. cuestensis and P. tajacu by H. canis.

Transstadial Transmission of Hepatozoon canis by Rhipicephalus sanguineus (Acari: Ixodidae) in Field Conditions.

This study investigated possible transovarial and transstadial transmission of Hepatozoon canis by Rhipicephalus sanguineus (Latreille) ticks collected from naturally infected dogs in a municipal dog shelter and the grounds of the shelter. Four hundred sixty-five engorged nymphs were collected from 16 stray dogs that were found to be infected with H. canis by blood smear and PCR analyses and maintained in an incubator at 28 °C for moulting. Four hundred eighteen nymphs moulted to adults 14-16 d post collection. Unfed ticks from the shelter grounds comprised 1,500 larvae, 2,100 nymphs, and 85 adults; were sorted according to origin, developmental stage, and sex into 117 pools; and screened by 18S rRNA PCR for Hepatozoon infection. Of 60 adult tick pools examined, 51 were infected with H. canis. The overall maximum likelihood estimate (MLE) of infection rate was calculated as 21.0% (CI 15.80-28.21). Hepatozoon canis was detected in 31 out of 33 female pools (MLE 26.96%, CI 17.64-44.33) and 20 out of 27 male pools (MLE 14.82%, CI 20.15-46.41). Among 42 unfed nymph pools collected from the shelter, 26 were infected with H. canis, and MLE of infection was calculated as 1.9% (CI 1.25-2.77). No H. canis DNA was detected in any of the gDNA pools consisting of larva specimens. Partial sequences of the 18S rRNA gene shared 99-100% similarity with the corresponding H. canis isolates. Our results revealed the transstadial transmission of H. canis by R. sanguineus, both from larva to nymph and from nymph to adult, in field conditions. However, there were no evidence of transovarial transmission.

Rhipicephalus turanicus, a new vector of Hepatozoon canis.

The distribution of Hepatozoon canis mainly encompasses areas where its main tick vector, Rhipicephalus sanguineus sensu lato, is present. However, the detection of this pathogen in dogs, foxes and golden jackals well outside the areas inhabited by this tick species reinforced the hypothesis that additional ixodids are involved in the life cycle and transmission of this protozoon. The present study provides, for the first time, data supporting the sporogonic development of H. canis in specimens of Rhipicephalus turanicus collected from a naturally infected fox from southern Italy. The epidemiological role of R. turanicus as a vector of H. canis is discussed, along with information on the potential use of cell cultures for the experimental infection with H. canis sporozoites. The in vitro infection of canine leucocytes by sporozoites from ticks is proposed as a potential tool for future in-depth studies on the biology of H. canis.

The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa).

Endemic Sicilian pond turtles Emys trinacris Fritz, Fattizzo, Guicking, Tripepi, Pennisi, Lenk, Joger et Wink were examined for the presence of haemogregarine parasites. The presence of haemogregarines, occurring mainly in the microgametocyte stage (13.2 ± 0.12 µm in length and 6.4 ± 0.52 µm in width), was observed in approximately 9% of the sampled E. trinacris. Based on the observed morphology and on the sequencing of nuclear 18S rDNA, we identified the parasite as Haemogregarina stepanowi Danilewsky, 1885. Morphometric study of uninfected and infected red blood cells has shown that H. stepanowi induces different changes in erythrocyte shape depending on the infective stage. The differential count of leukocytes in specimens infected with H. stepanowi showed no significant difference compared with healthy specimens. However, considering the health problems which might be induced by H. stepanowi in the closely related European pond turtle Emys orbicularis (Linneaus), monitoring of the health status of the infected Sicilian populations of E. trinacris is desirable. The restricted distribution of populations of Emys infected with haemogregarines in Sicily is quite puzzling and the possible human-mediated introduction of the parasite in Sicily is briefly discussed.

Hepatozoon ellisgreineri n. sp. (Hepatozoidae): description of the first avian apicomplexan blood parasite inhabiting granulocytes.

Blood parasites of the genus Hepatozoon (Apicomplexa, Hepatozoidae) infect all groups of terrestrial vertebrates, and particularly high prevalence and species diversity have been reported in reptiles and mammals. A few morphologically similar species, in which gamonts inhabit mononuclear leukocytes and red blood cells, have been described in birds. Here, we report a new Hepatozoon species, which was found in wild-caught secretary birds Sagittarius serpentarius, from Tanzania. Hepatozoon ellisgreineri n. sp. can be readily distinguished from all described species of avian Hepatozoon because its gamonts develop only in granulocytes, predominantly in heterophils, a unique characteristic among bird parasites of this genus. Additionally, this is the first reported avian apicomplexan blood parasite, which inhabits and matures in granulocytes. We describe H. ellisgreineri based on morphological characteristics of blood stages and their host cells. This finding broadens knowledge about host cells of avian Hepatozoon spp. and other avian apicomplexan blood parasites, contributing to the better understanding of the diversity of haematozoa. This is the first report of hepatozoonosis in endangered African birds of the Sagittariidae.

First report and description of a Cyrilia sp. (Apicomplexa: Haemogregarinidae) from a freshwater Cururu Stingray Potamotrygon cf. histrix (Elasmobranchii: Potamotrygonidae), from the Amazon Region, Brazil.

A haemogregarine is described in 12 cururu stingray (Potamotrygon cf. histrix), from Mariuá Archipelago, Negro River, in the Brazilian Amazon Basin. All animals, both male and female, were parasitized by the haemogregarine and parasitaemia varied between 0.8% and 10% of erythrocytes. The stages observed included trophozoites or merozoites, suspected meronts, and gamonts presumed to be of two types, macrogamonts and microgamonts. Most stages were observed inside mature erythrocytes, while others were extracellular. The stages observed were most similar to those characteristics of the genus Cyrilia, than to any other fish haemogregarine and may represent a new Cyrilia species.

Characterization of Hepatozoon spp. in Leptodactylus chaquensis and Leptodactylus podicipinus from two regions of the Pantanal, state of Mato Grosso do Sul, Brazil.

Hepatozoon sp. are parasites that commonly infect frogs and arthropod vectors. This species has variability in the morphological and morphometric characteristics. Due to these variations, the naming of the species is thus impaired and only by visualizing the sporogonic cycle in vector and by molecular studies this problem can be solved. Recently, the use of molecular genetics has helped the species denomination. In this work, we collected 145 frogs (68 Leptodactylus chaquensis and 77 Leptodactylus podicipinus) in different sampling sites, where were found 18 (26.47%) L. chaquensis and 24 (31.17%) L. podicipinus parasitized; besides of gamonts, schizogonic forms were also seen in animals organs. The positivity difference between the collection sites for both frog species was not significant (p = 0.958). Comparing gamonts found in each species of anuran, we observed differences in morphology. The comparison in the molecular level for L. podicipinus was not possible due to small amount of blood obtained, just L. chaquensis had their parasites DNA sequenced. The amplified and sequenced samples, named HEP1 to HEP10, are presented in the phylogenetic tree as a different branch from other haemogregarines described on other hosts. Therefore, we have seen that, although the morphology and morphometry of the collected parasites at each site showed differences, the sequencing of these samples revealed identical species of Hepatozoon, and different compared to those from GenBank, thereby demonstrating that the species of Hepatozoon in L. chaquensis observed in this study probably represent a new species.

Prevalence and diversity of Hepatozoon in native and exotic geckos from Brazil.

Hepatozoon is a genus of hemogregarines constituting the most widespread and common reptile hemoparasite. Although various molecular assessments of these parasites have been conducted in lizards from Africa and Europe, similar studies are needed for South American lizards. Through amplification and sequencing of fragments of the 18S rRNA gene, we assess the prevalence of Hepatozoon parasites in 230 geckos from South America, including the endemic species Hemidactylus agrius, Hemidactylus brasilianus, Lygodactylus klugei, Phyllopezus pollicaris, Phyllopezus periosus, and an exotic species, Hemidactylus mabouia . We found an overall low prevalence of Hepatozoon infection (7/230, 3%) with only 3 of the 6 host species infected with Hepatozoon ( Hemidactylus mabouia , P. pollicaris, and P. periosus). Within the 7 infected host samples, 5 genetically distinct lineages of Hepatozoon parasites were identified, only 1 of which was similar to previously published haplotypes. Thus, although prevalence is low, genetically based diversity of Hepatozoon in geckos from South America is very high. Three of these lineages appear basal to 1 of the major clades of Hepatozoon, suggesting that this clade might have originated in South America, and thereby indicating a potential phylogeographic pattern that had not been previously identified. Future studies should assess the distribution and competence of invertebrate hosts in the regions analyzed, and Hepatozoon diversity in other less well-known regions of the world.

Coccidial dispersion across New World marsupials: Klossiella tejerai Scorza, Torrealba & Dagert, 1957 (Apicomplexa: Adeleorina) from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) (Mammalia: Didelphimorphia).

Klossiella tejerai Scorza, Torrealba & Dagert, 1957 is a primitive coccidian parasite reported from the New World marsupials Didelphis marsupialis (Linnaeus) and Marmosa demerarae (Thomas). The current work describes K. tejerai from the Brazilian common opossum Didelphis aurita (Wied-Neuwied) in Southeastern Brazil, evidencing the coccidial dispersion across opossums of the same family. The sporocysts recovered from urine samples were ellipsoidal, 20.4 × 12.7 µm, with sporocyst residuum composed of scattered spherules and c.13 sporozoites per sporocyst, with refractile bodies and nucleus. Macrogametes, microgametes, sporonts, sporoblasts/sporocysts were identified within parasitophorous vacuoles of epithelial cells located near the renal corticomedullary junction. Didelphis marsupialis should not have transmitted K. tejerai to D. aurita because they are not sympatric; however M. demerarae is sympatric with D. marsupialis and D. aurita. Therefore, D. aurita becomes the third host species for K. tejerai in South America.

Does thermal ecology influence dynamics of side-blotched lizards and their micro-parasites?

Hosts and parasites form interacting populations that influence each other in multiple ways. Their dynamics can also be influenced by environmental and ecological factors. We studied host-parasite dynamics in a previously unexplored study system: side-blotched lizards and their micro-parasites. Compared with uninfected lizards, the infected lizards elected to bask at lower temperatures that were outside their range of preferred temperatures. Infected lizards also were not as precise as uninfected lizards in maintaining their body temperatures within a narrow range. At the ecological scale, areas with higher infection rates coincided with more thermally heterogeneous microhabitats as well as with the areas where lizards tended to live longer. Thermal heterogeneity of lizards' microhabitats may provide important clues to the spatial and temporal distribution of infections.

Fecundity reduction in the second gonotrophic cycle of Culex pipiens infected with the apicomplexan blood parasite, Hepatozoon sipedon.

Fecundity reduction is a well-recognized phenomenon of parasite infection in insects. Reduced production of eggs might increase longevity of a host and release nutrients to both host and parasite that would otherwise be used for oogenesis. The objective of this study was to assess effects on fecundity caused by Hepatozoon sipedon, an apicomplexan blood parasite of snakes, in its invertebrate host, the mosquito Culex pipiens. In the first gonotrophic cycle, the mean number of eggs laid by mosquitoes infected with H. sipedon did not differ significantly from those laid by uninfected mosquitoes. However, in the second gonotrophic cycle infected mosquitoes laid significantly fewer eggs than did uninfected mosquitoes, and fecundity was reduced by 100% in mosquitoes with parasite burdens of more than 60 oocysts. There was a significant negative correlation between parasite burden, or the number of oocysts, and the number of eggs produced in the second gonotrophic cycle. Significantly fewer viable larvae hatched from eggs laid by infected compared to uninfected mosquitoes in the second gonotrophic cycle. These data indicate that fecundity reduction occurs in this system, although the physiological mechanisms driving this phenotype are not yet known.