Unresolved haemosporidia of the Australian skink, Egernia stokesii.

The Australian skink Egernia stokesii had been recognised as a host of two species of Plasmodium, Plasmodium mackerrasae and P. circularis; nevertheless, molecular data are available for only a single haemosporidian species of this host. Its sequences are labelled as "Plasmodium sp." or "Plasmodium mackerrasae", but morphological characteristics of this isolate are unavailable. Phylogenetic analyses of these sequences placed them into the clade of the genus Haemocystidium. In this study, blood samples of six E. stokesii were analysed by both, molecular and microscopic methods to clarify the haemosporidia of this lizard. Application of these approaches offered discordant results. Whereas sequence analysis clustered our isolates with lizard species of Haemocystidium, morphology of blood stages is more akin to Plasmodium than Haemocystidium. However, limited sampling, indistinguishable nuclei/merozoites and risk of possible hidden presence of mixed infection prevent reliable species identification of detected parasites or their description as new species of Haemocystidium.

Identification of potentially zoonotic parasites in captive orangutans and semi-captive mandrills: Phylogeny and morphological comparison.

Cysts and trophozoites of vestibuliferid ciliates and larvae of Strongyloides were found in fecal samples from captive orangutans Pongo pygmaeus and P. abelii from Czech and Slovak zoological gardens. As comparative material, ciliates from semi-captive mandrills Mandrillus sphinx from Gabon were included in the study. Phylogenetic analysis of the detected vestibuliferid ciliates using ITS1-5.8s-rRNA-ITS2 and partial 18S ribosomal deoxyribonucleic acid (rDNA) revealed that the ciliates from orangutans are conspecific with Balantioides coli lineage A, while the ciliates from mandrills clustered with Buxtonella-like ciliates from other primates. Morphological examination of the cysts and trophozoites using light microscopy did not reveal differences robust enough to identify the genera of the ciliates. Phylogenetic analysis of detected L1 larvae of Strongyloides using partial cox1 revealed Strongyloides stercoralis clustering within the cox1 lineage A infecting dogs, humans, and other primates. The sequences of 18S rDNA support these results. As both B. coli and S. stercoralis are zoonotic parasites and the conditions in captive and semi-captive settings may facilitate transmission to humans, prophylactic measures should reflect the findings.

Diversity of Karyolysus and Schellackia from the Iberian lizard Lacerta schreiberi with sequence data from engorged ticks.

Apicomplexan haemoparasites of the genera Schellackia Reichenow, 1919, and Karyolysus Labbé, 1894, seem to be common in lizards and widespread across the world. For decades, their identification has been based on morphological descriptions and life cycle patterns, with molecular characterizations, applied only recently. We used molecular characterization to confirm the identification of haemoparasites detected by microscopy in blood smears of Lacerta schreiberi Bedriaga, 1878, a lizard of the Iberian Peninsula. Since blood samples other than blood smears were not available from the studied lizards, 264 engorged ticks Ixodes ricinus (Linneaus, 1758) collected from them were used as an alternative non-invasive source of haemoparasite DNA for molecular genetic analyses. Of the 48 blood smears microscopically examined, 31 were positive for blood parasites (64.6% prevalence). We identified trophozoites and gamonts similar to Karyolysus lacazei (Labbé, 1894) (24/48; 50%) and Schellackia-like sporozoites (20/48; 41.7%). Mixed infections with both species occurred in 13 blood smears (27.1%). Sequence data were obtained for both parasites from engorged ticks. Phylogenetic analyses placed our unique haemogregarine sequence within the Karyolysus clade, nevertheless, within substantial polytomy. Thus, according to its morphology and effect on the host cell, we refer to this haemogregarine as Karyolysus cf. lacazei. Besides the Schellackia sequences being identical to a previously identified haplotype, we also obtained sequences of three new closely related haplotypes.

Haemogregarines of the Genera Haemogregarina, Hemolivia, and Hepatozoon Infecting Vietnamese Freshwater Turtles, with Additional Notes on Primer Specificity and Primer-template Mismatches Affecting Diagnostic Success.

The 18S rRNA gene is a widely used molecular marker for haemogregarines. In recent decades, many primers more or less specific to various haemogregarine genera have been designed. This study applied five commonly used primers targeting the 18S rRNA gene of haemogregarines to blood samples from 168 individuals of nine turtle species captured in Northern Vietnam. Three haemogregarine genera, Haemogregarina, Hemolivia, and Hepatozoon, were detected. Selective specificity of primers EF/ER, HemoFN/HemoRN, and Hemo1/Hemo2 to haemogregarine genera was observed and elucidated by primer-template mismatches. In total, 13 out of 168 turtles (prevalence 7.7%) were both microscopically and PCR positive for haemogregarines. Additionally, a single Heosemys grandis turtle was PCR positive but microscopically negative. Numerous turtles carried mixed infections by various haemogregarines; a single turtle was even coinfected by haemogregarines of all three studied genera: Haemogregarina, Hemolivia, and Hepatozoon. Among the detected haemogregarines, some provided sufficient molecular and morphological data for completing their species diagnosis. Two were described as new species: Haemogregarina cyclemydis sp. nov. from Cyclemys pulchristriata and Hemolivia cruciata sp. nov. from Cuora galbinifrons, so far the first Hemolivia from Southeast Asia. Haemogregarina cuorae Chai and Chen, 1990, required a redescription with reassignment to the genus Hepatozoon Miller, 1908.

Against growing synonymy: Identification pitfalls of Hepatozoon and Schellackia demonstrated on North Iranian reptiles.

The analyses of molecular data represent an effective tool for increasing the credibility of taxonomy and facilitate the description of species. Nevertheless, in haemoprotozoa, the growing amount of available sequential data is not matched by the still limited number of well-defined species. We identified four protistan haemoparasites in North Iranian reptiles: two Hepatozoon and two Schellackia species. Hepatozoon colubri and Hepatozoon ophisauri were morphologically identified in their type hosts, their partial 18S rDNA was analyzed, and thorough literature data were included in their redescription. The scarce data on the detected Schellackia spp. did not allow for their formal species description. Using an integrative approach, including morphological and geographical features, host specificity, molecular data, and the data published thus far, we face the following main difficulties hindering reliable diagnosis. (1) The lack of molecular data on well-described and named species. (2) The insufficiency of using only morphological and biological features, or only sequential data without morphology, to perform an absolutely reliable species diagnosis. (3) Typical morphological features are more substantial than metric means. (4) High risk of synonymy is present in taxonomy of blood Protista. (5) Artefacts caused by blood smear processing further complicate the correct morphological determination.

Landscape epidemiology of neglected tick-borne pathogens in central Europe.

Studies of tick-borne diseases (TBDs) in Europe focus on pathogens with principal medical importance (e.g. Lyme disease and tick-borne encephalitis), but we have limited epidemiological information on the neglected pathogens, such as the members of the genera Anaplasma, Rickettsia, Babesia and Candidatus Neoehrlichia mikurensis. Here, we integrated an extensive field sampling, laboratory analysis and GIS models to provide first publicly available information on pathogen diversity, prevalence and infection risk for four overlooked zoonotic TBDs in the Czech Republic. In addition, we assessed the effect of landscape variables on the abundance of questing ticks at different spatial scales and examined whether pathogen prevalence increased with tick density. Our data from 13,340 ticks collected in 142 municipalities showed that A. phagocytophilum (MIR = 3.5%) and Ca. Neoehrlichia mikurensis (MIR = 4.0%) pose geographically uneven risks with localized hotspots, while Rickettsia (MIR = 4.9%) and Babesia (MIR = 1.1%) had relatively homogeneous spatial distribution. Landscape variables had significant effect on tick abundance up to the scale of 1 km around the sampling sites. Questing ticks responded positively to landscape diversity and configuration, especially to forest patch density that strongly correlates with the amount of woodland-grassland ecotones. For all four pathogens, we found higher prevalence in places with higher densities of ticks, confirming the hypothesis that tick abundance amplifies the risk of TB infection. Our findings highlight the importance of landscape parameters for tick vectors, likely due to their effect on small vertebrates as reservoir hosts. Future studies should explicitly investigate the combined effect of landscape parameters and the composition and population dynamics of hosts on the host-vector-pathogen system.

Candidatus Neoehrlichia mikurensis is widespread in questing Ixodes ricinus ticks in the Czech Republic.

Candidatus Neoehrlichia mikurensis, the causative agent of tick-borne "neoehrlichiosis" has recently been reported in humans, mammals and ticks in Europe. The aim of this study was to map the distribution of this bacterium in questing ticks in the Czech Republic. A total of 13,325 Ixodes ricinus including 445 larvae, 5270 nymphs and 7610 adults were collected from vegetation by flagging in 140 Czech towns and villages from every region of the Czech Republic. The ticks were pooled into 2665 groups of 5 individuals respecting life stage or sex and tested for the presence of Ca. Neoehrlichia mikurensis by conventional PCR targeting of the groEL gene. The bacterium was detected in 533/2665 pools and 125/140 areas screened, showing an overall estimated prevalence of 4.4 % in ticks of all life stages. Phylogenetic analysis revealed only small genetic diversity among the strains found. Two pools of questing larvae tested positive, suggesting transovarial transmission. According to this study, Ca. Neoehrlichia mikurensis is another tick-borne pathogen widespread in I. ricinus ticks in the Czech Republic.