RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

The 18S rRNA genes of Haemoproteus (Haemosporida, Apicomplexa) parasites from European songbirds with remarks on improved parasite diagnostics.

BACKGROUND: The nuclear ribosomal RNA genes of Plasmodium parasites are assumed to evolve according to a birth-and-death model with new variants originating by duplication and others becoming deleted. For some Plasmodium species, it has been shown that distinct variants of the 18S rRNA genes are expressed differentially in vertebrate hosts and mosquito vectors. The central aim was to evaluate whether avian haemosporidian parasites of the genus Haemoproteus also have substantially distinct 18S variants, focusing on lineages belonging to the Haemoproteus majoris and Haemoproteus belopolskyi species groups. METHODS: The almost complete 18S rRNA genes of 19 Haemoproteus lineages of the subgenus Parahaemoproteus, which are common in passeriform birds from the Palaearctic, were sequenced. The PCR products of 20 blood and tissue samples containing 19 parasite lineages were subjected to molecular cloning, and ten clones in mean were sequenced each. The sequence features were analysed and phylogenetic trees were calculated, including sequence data published previously from eight additional Parahaemoproteus lineages. The geographic and host distribution of all 27 lineages was visualised as CytB haplotype networks and pie charts. Based on the 18S sequence data, species-specific oligonucleotide probes were designed to target the parasites in host tissue by in situ hybridization assays. RESULTS: Most Haemoproteus lineages had two or more variants of the 18S gene like many Plasmodium species, but the maximum distances between variants were generally lower. Moreover, unlike in most mammalian and avian Plasmodium species, the 18S sequences of all but one parasite lineage clustered into reciprocally monophyletic clades. Considerably distinct 18S clusters were only found in Haemoproteus tartakovskyi hSISKIN1 and Haemoproteus sp. hROFI1. The presence of chimeric 18S variants in some Haemoproteus lineages indicates that their ribosomal units rather evolve in a semi-concerted fashion than according to a strict model of birth-and-death evolution. CONCLUSIONS: Parasites of the subgenus Parahaemoproteus contain distinct 18S variants, but the intraspecific variability is lower than in most mammalian and avian Plasmodium species. The new 18S data provides a basis for more thorough investigations on the development of Haemoproteus parasites in host tissue using in situ hybridization techniques targeting specific parasite lineages.

Enterocytozoon bieneusi in fecal samples from calves and cows in Austria.

Enterocytozoon bieneusi is an obligate intracellular pathogen that infects livestock, companion animals, and wildlife and has the potential to cause severe diarrhea especially in immunocompromised humans. In the underlying study, fecal samples from 177 calves with diarrhea and 174 adult cows originating from 70 and 18 farms, respectively, in Austria were examined for the presence of E. bieneusi by polymerase chain reaction targeting the Internal Transcribed Spacer 1 (ITS1) region. All positive samples were further sequenced for genotype determination. Overall, sixteen of the 351 (4.6%) samples were positive for E. bieneusi, two of the 174 samples from cows (1.2%) and 14 of the 177 samples from calves (7.9%). In total, four genotypes, J (n = 2), I (n = 12), BEB4 (n = 3), and BEB8 (n = 1), were identified. The uncorrected p-distance between the four ITS1 lineages (344 bp) ranges from 0.3% to 2.9%. The lineages differ by 1 bp (I and J), 2 bp (J and BEB4), and 3 bp (I and BEB4), respectively, and BEB8 differs by 7 to 10 bp from the latter three lineages. Two of the E. bieneusi-positive calves showed an infection with two different genotypes. E. bieneusi occurred significantly more often in calves > 3 weeks (8/59) than in calves ≤ 3 weeks (6/118), respectively (p = 0.049). Calves with a known history of antimicrobial treatment (50 of 177 calves) shed E. bieneusi significantly more often than untreated calves (p = 0.012). There was no statistically significant difference in E. bieneusi shedding in calves with or without a medical history of antiparasitic treatment (p = 0.881). Calves showing a co-infection with Eimeria spp. shed E. bieneusi significantly more often than uninfected calves (p = 0.003). To our knowledge, this is the first report of E. bieneusi in cattle in Austria. Cattle should be considered as a reservoir for human infection since potentially zoonotic E. bieneusi genotypes were detected.

Avian haemosporidian parasites of accipitriform raptors.

BACKGROUND: The order Accipitriformes comprises the largest group of birds of prey with 260 species in four families. So far, 21 haemosporidian parasite species have been described from or reported to occur in accipitriform birds. Only five of these parasite species have been characterized molecular genetically. The first part of this study involved molecular genetic screening of accipitriform raptors from Austria and Bosnia-Herzegovina and the first chromogenic in situ hybridization approach targeting parasites in this host group. The aim of the second part of this study was to summarize the CytB sequence data of haemosporidian parasites from accipitriform raptors and to visualize the geographic and host distribution of the lineages. METHODS: Blood and tissue samples of 183 accipitriform raptors from Austria and Bosnia-Herzegovina were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR, and tissue samples of 23 PCR-positive birds were subjected to chromogenic in situ hybridization using genus-specific probes targeting the parasites' 18S rRNAs. All published CytB sequence data from accipitriform raptors were analysed, phylogenetic trees were calculated, and DNA haplotype network analyses were performed with sequences from clades featuring multiple lineages detected in this host group. RESULTS: Of the 183 raptors from Austria and Bosnia-Herzegovina screened by PCR and sequencing, 80 individuals (44%) were infected with haemosporidian parasites. Among the 39 CytB lineages detected, 18 were found for the first time in the present study. The chromogenic in situ hybridization revealed exo-erythrocytic tissue stages of Leucocytozoon parasites belonging to the Leucocytozoon toddi species group in the kidneys of 14 infected birds. The total number of CytB lineages recorded in accipitriform birds worldwide was 57 for Leucocytozoon, 25 for Plasmodium, and 21 for Haemoproteus. CONCLUSION: The analysis of the DNA haplotype networks allowed identifying numerous distinct groups of lineages, which have not yet been linked to morphospecies, and many of them likely belong to yet undescribed parasite species. Tissue stages of Leucocytozoon parasites developing in accipitriform raptors were discovered and described. The majority of Leucocytozoon and Haemoproteus lineages are specific to this host group, but most Plasmodium lineages were found in birds of other orders. This might indicate local transmission from birds kept at the same facilities (raptor rescue centres and zoos), likely resulting in abortive infections. To clarify the taxonomic and systematic problems, combined morphological and molecular genetic analyses on a wider range of accipitriform host species are needed.

A citizen science-based survey of avian mortality focusing on haemosporidian infections in wild passerine birds.

BACKGROUND: Haemosporidioses are common in birds and their manifestations range from subclinical infections to severe disease, depending on the involved parasite and bird species. Clinical haemosporidioses are often observed in non-adapted zoo or aviary birds, whereas in wild birds, particularly passerines, haemosporidian infections frequently seem to be asymptomatic. However, a recent study from Austria showed pathogenic haemosporidian infections in common blackbirds due to high parasite burdens of Plasmodium matutinum LINN1, a common parasite in this bird species, suggesting that virulent infections also occur in natural hosts. Based on these findings, the present study aimed to explore whether and to what extent other native bird species are possibly affected by pathogenic haemosporidian lineages, contributing to avian morbidity. METHODS: Carcasses of passerine birds and woodpeckers were collected during a citizen science-based survey for avian mortality in Austria, from June to October 2020. Tissue samples were taken and examined for haemosporidian parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon by nested PCR and sequencing the mitochondrial cytb barcode region, histology, and chromogenic in situ hybridization applying genus-specific probes. RESULTS: From over 160 dead bird reportings, 83 carcasses of 25 avian species were submitted for investigation. Overall haemosporidian infection rate was 31%, with finches and tits prevailing species counts and infections. Sequence analyses revealed 17 different haplotypes (4 Plasmodium, 4 Haemoproteus, 9 Leucocytozoon), including 4 novel Leucocytozoon lineages. Most infected birds presented low parasite burdens in the peripheral blood and tissues, ruling out a significant contribution of haemosporidian infections to morbidity or death of the examined birds. However, two great tits showed signs of avian malaria, suggesting pathogenic effects of the detected species Plasmodium relictum SGS1 and Plasmodium elongatum GRW06. Further, exo-erythrocytic tissue stages of several haemosporidian lineages are reported. CONCLUSIONS: While suggesting generally little contribution of haemosporidian infections to mortality of the investigated bird species, the findings indicate a possible role of certain haemosporidian lineages in overall clinical manifestation, either as main causes or as concurrent disease agents. Further, the study presents new data on exo-erythrocytic stages of previously reported lineages and shows how citizen science can be used in the field of haemosporidian research.

First description of freshwater mite Unionicola sauerensis sp. nov. infesting thick-shelled river mussel Unio crassus.

A sample of 30 thick-shelled river mussels Unio crassus Philipsson (Unionida: Unionidae) was collected from the River Sauer in Luxembourg to acquire data on parasitic infestations of the mussels. Among other parasites, different development stages of freshwater mites were collected from the gills and the mantle of the mussels and were documented with bright-field, stereo, and confocal laser scanning microscopy and microscopic X-ray computed tomography. The retrieved data allowed a morphological description of larvae and female adults of the mites and assigning them to the genus Unionicola Haldeman (Trombidiformes: Unionicolidae) and the subgenus Pentatax Thor. Additionally, adult stages and larvae were barcoded by sequencing a section of the mitochondrial COI and 18S rRNA genes. This resulted in 4 new, similar Unionicola lineages from the adult stages, which differ in at least 14.7% (uncorrected p distance) from those already published. Barcoding of larval DNA was not successful. The comparison with known European species of the genus Unionicola and analysis of the barcoding results allowed the proposal of a new species of the genus Unionicola. The species was named Unionicola sauerensis sp. nov. after the River Sauer in Luxembourg, where the infested mussels were collected.

DNA barcoding of rumen flukes (Paramphistomidae) from bovines in Germany and Austria.

Rumen flukes have received growing veterinary attention in western and central Europe during the past two decades because of an increase in prevalence of infection in cattle and sheep, including cases of severe clinical disease. Historically, rumen fluke infections in Europe were assumed to be caused mainly by Paramphistomum cervi (or species, which were later considered to be synonymous with P. cervi), but more recently molecular studies demonstrated Calicophoron daubneyi to be the predominating species. For the present investigation, adult rumen flukes isolated from 23 cattle originating from ten farms in Germany (Saxony [1], Baden-Württemberg [4], Bavaria [5]) and one farm in Austria (Tyrol) were analyzed to establish partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and the complete sequence of the nuclear internal transcribed spacer 2 (ITS2). Flukes of five animals (dairy cows from three farms in Bavaria) were determined as P. leydeni, and flukes of 18 animals (dairy cows or cattle from cow-calf operations from eight farms in Saxony [1], Baden-Württemberg [4], Bavaria [2], and Tyrol [1]) were identified as C. daubneyi. Based on the molecular analysis of adult rumen flukes collected from cattle, the results of this investigation confirm the common occurrence of C. daubneyi in Germany and reveal the first definitive findings of P. leydeni in Germany and C. daubneyi in Austria.

Geographic and host distribution of haemosporidian parasite lineages from birds of the family Turdidae.

BACKGROUND: Haemosporidians (Apicomplexa, Protista) are obligate heteroxenous parasites of vertebrates and blood-sucking dipteran insects. Avian haemosporidians comprise more than 250 species traditionally classified into four genera, Plasmodium, Haemoproteus, Leucocytozoon, and Fallisia. However, analyses of the mitochondrial CytB gene revealed a vast variety of lineages not yet linked to morphospecies. This study aimed to analyse and discuss the data of haemosporidian lineages isolated from birds of the family Turdidae, to visualise host and geographic distribution using DNA haplotype networks and to suggest directions for taxonomy research on parasite species. METHODS: Haemosporidian CytB sequence data from 350 thrushes were analysed for the present study and complemented with CytB data of avian haemosporidians gathered from Genbank and MalAvi database. Maximum Likelihood trees were calculated to identify clades featuring lineages isolated from Turdidae species. For each clade, DNA haplotype networks were calculated and provided with information on host and geographic distribution. RESULTS: In species of the Turdidae, this study identified 82 Plasmodium, 37 Haemoproteus, and 119 Leucocytozoon lineages, 68, 28, and 112 of which are mainly found in this host group. Most of these lineages cluster in the clades, which are shown as DNA haplotype networks. The lineages of the Leucocytozoon clades were almost exclusively isolated from thrushes and usually were restricted to one host genus, whereas the Plasmodium and Haemoproteus networks featured multiple lineages also recovered from other passeriform and non-passeriform birds. CONCLUSION: This study represents the first attempt to summarise information on the haemosporidian parasite lineages of a whole bird family. The analyses allowed the identification of numerous groups of related lineages, which have not been linked to morphologically defined species yet, and they revealed several cases in which CytB lineages were probably assigned to the wrong morphospecies. These taxonomic issues are addressed by comparing distributional patterns of the CytB lineages with data from the original species descriptions and further literature. The authors also discuss the availability of sequence data and emphasise that MalAvi database should be considered an extremely valuable addition to GenBank, but not a replacement.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden.

BACKGROUND: Passerine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects. METHODS: For molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin-eosin-stained sections. RESULTS: By PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses. CONCLUSION: This study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Molecular phylogeny and trait evolution of Madeiran land snails: radiation of the Geomitrini (Stylommatophora: Helicoidea: Geomitridae).

The Geomitrini is the most species-rich group of land snails in the Madeiran Archipelago. The phylogeny of the group is reconstructed based on mitochondrial and nuclear genetic markers. The timing of diversification, the colonisation history of the islands of the Madeiran Archipelago and the evolution of characters of the dart apparatus are studied. The results of the phylogenetic analyses confirm the sister group relationship of Geomitrini and Cochlicellini, but also show that several previously accepted genus-group taxa are not monophyletic. A new classification for the Geomitrini is proposed, including the description of two new genera, Domunculifex Brozzo, De Mattia, Harl & Neiber, n. gen. and Testudodiscula Brozzo, De Mattia, Harl & Neiber, n. gen. The onset of diversification of Geomitrini was dated in our analysis at 13 Ma, which largely coincides with the emergence of the present-day islands. The ancestral state estimation recovered the presence of two appendiculae in the reproductive system as the ancestral state in Geomitrini. One appendicula was lost three times independently within the tribe and is even missing completely in one group. The ancestral area estimation suggests recurrent colonisations of Madeira (and the Ilhas Desertas) from the older island Porto Santo.

Hybridization and recurrent evolution of left-right reversal in the land snail genus Schileykula (Orculidae, Pulmonata).

The land snail genus Schileykula Gittenberger, 1983 is distributed in arid limestone areas from western Turkey to north-western Iran. It comprises eight species, which display high variation in shell size and morphology. The cylindrical shells are 5-12 mm in height and the last shell whorls bear several inner lamellae and plicae. Two taxa differ in their chirality having sinistral shells, while all the others are dextrals such as the vast majority of orculids. The aim of this study was to establish a molecular genetic phylogeny of Schileykula and to test whether it conforms to the current morphology-based classification. Furthermore, we were interested in the phylogenetic position of the two sinistral forms in order to assess whether one or two reversals happened in the evolution of the genus. Nine out of ten species, including all four subspecies of Schileykula trapezensis and three of six subspecies of Schileykula scyphus, were investigated. A section of the mitochondrial cytochrome c oxidase subunit I gene was analyzed in 54 specimens of Schileykula and from a subsample, partial sequences of the mitochondrial genes for the 12S rRNA and the 16S rRNA, and a section of the nuclear H4/H3 histone gene cluster were obtained. The phylogenetic trees based on the mitochondrial sequences feature high support values for most nodes, and the species appear well differentiated from each other. The two chiral forms evolved independently and are not sister lineages. However, some groupings disagree with the present morphology-based classification and taxonomical conclusions are drawn. Schileykula trapezensis is polyphyletic in the molecular genetic trees; therefore, three of its subspecies are elevated to species level: Schileykula acampsis Hausdorf, 1996 comb. nov., Schileykula neuberti Hausdorf, 1996 comb. nov., and Schileykula contraria Neubert, 1993 comb. nov. Furthermore, Schileykula sigma is grouped within S. scyphus in the mitochondrial and nuclear trees and consequently treated as a subspecies of the latter (Schileykula scyphus sigma Hausdorf, 1996 comb. nov.). Schileykula nordsiecki, whose shell morphology is indistinguishable from that of the neighboring Schileykula scyphus lycaonica, but who differs in its genital anatomy, was confirmed to represent a distinct lineage. The phylogenies produced by the mitochondrial and nuclear data sets are to some extent conflicting. The patterns differ concerning the grouping of some specimens, suggesting at least two independent hybridization events involving S. contraria, S. scyphus and S. trapezensis. The results exemplify the importance of integrating both mitochondrial and nuclear sequence data in order to complement morphology-based taxonomy, and they provide further evidence for hybridization across distantly related lineages in land snails.

Acanthocephalan parasites collected from Austrian fishes: molecular barcoding and pathological observations.

Acanthocephalan parasites were collected from the intestinal tracts of 137 predominantly wild fish (1 barbel Barbus barbus, 3 European chub Squalius cephalus, 13 rainbow trout Oncorhynchus mykiss and 120 brown trout Salmo trutta) from 12 localities. The condition factor, intensity of acanthocephalan infection and pathological lesions, if applicable, were documented. Routine bacteriology and virology were performed, and the brown trout were additionally tested for the presence of the myxozoan parasite Tetracapsolioides bryosalmonae by PCR. In total, 113 acanthocephalans were barcoded by sequencing a section of the mitochondrial cytochrome oxidase subunit I (COI) gene. Barcoding of the acanthocephalan tissues resulted in 77 sequences, of which 56 were assigned to Echinorhynchus truttae (3 genotypes), 11 to Pomphorhynchus tereticollis (9 genotypes), 9 to Acanthocephalus sp. (5 genotypes) and 1 to Neoechinorhynchida. Most of these genotypes were detected for the first time. Statistically, the acanthocephalan infection did not have an impact on the condition factor of the brown trout. Infection with P. tereticollis caused more severe pathological changes in the digestive tract than E. truttae. The present study provides new data regarding the distribution of acanthocephalan species in Austria and their impact on individual fish. In addition, new barcoding data from acanthocephalan parasites are presented, and the occurrence of P. tereticollis in European chub in Austria and in brown and rainbow trout in general was confirmed for the first time.

Cryptosporidium parvum, Cryptosporidium ryanae, and Cryptosporidium bovis in samples from calves in Austria.

Fecal samples of 177 calves of up to 180 days of age with diarrhea from 70 farms in Austria were examined to obtain information on the occurrence of Cryptosporidium species. Initially, all samples were examined by phase-contrast microscopy. Cryptosporidium-positive samples (55.4%; n = 98) were screened by gp60 PCR, resulting in 68.4% (n = 67) C. parvum-positive samples. The remaining 31 gp60-PCR-negative and the phase-contrast microscopy negative samples (n = 79) were screened by PCR targeting a 700 bp fragment of the 18S rRNA gene. Sequencing of the PCR products revealed the presence of C. parvum (n = 69), C. ryanae (n = 11), and C. bovis (n = 7). The latter two species have never been described in Austria. C. parvum-positive samples were genotyped at the gp60 gene locus, featuring four subtypes (IIaA15G2R1, IIaA21G2R1, IIaA19G2R1, IIaA14G1R1). The most frequently detected subtype IIaA15G2R1 (n = 52) was present in calves from 30 different farms. IIaA14G1R1 (n = 5) occurred on a single farm, subtype IIaA21G2R1 (n = 4) on two farms, and subtype IIaA19G2R1 (n = 4) on three farms. The results confirm the widespread occurrence of zoonotic C. parvum in diarrheic calves.

High prevalence and genetic diversity of Haemoproteus columbae (Haemosporida: Haemoproteidae) in feral pigeons Columba livia in Cape Town, South Africa.

In this study, we explore blood parasite prevalence, infection intensity, and co-infection levels in an urban population of feral pigeons Columba livia in Cape Town. We analyze the effect of blood parasites on host body condition and the association between melanin expression in the host's plumage and parasite infection intensity and co-infection levels. Relating to the haemosporidian parasite itself, we study their genetic diversity by means of DNA barcoding (cytochrome b) and show the geographic and host distribution of related parasite lineages in pigeons worldwide. Blood from 195 C. livia individuals was collected from April to June 2018. Morphometric measurements and plumage melanism were recorded from every captured bird. Haemosporidian prevalence and infection intensity were determined by screening blood smears and parasite lineages by DNA sequencing. Prevalence of Haemoproteus spp. was high at 96.9%. The body condition of the hosts was negatively associated with infection intensity. However, infection intensity was unrelated to plumage melanism. The cytochrome b sequences revealed the presence of four Haemoproteus lineages in our population of pigeons, which show high levels of co-occurrence within individual birds. Three lineages (HAECOL1, COLIV03, COQUI05) belong to Haemoproteus columbae and differ only by 0.1% to 0.8% in the cytochrome b gene. Another lineage (COLIV06) differs by 8.3% from the latter ones and is not linked to a morphospecies, yet. No parasites of the genera Leucocytozoon and Plasmodium were detected.

The nuclear 18S ribosomal DNAs of avian haemosporidian parasites.

BACKGROUND: Plasmodium species feature only four to eight nuclear ribosomal units on different chromosomes, which are assumed to evolve independently according to a birth-and-death model, in which new variants originate by duplication and others are deleted throughout time. Moreover, distinct ribosomal units were shown to be expressed during different developmental stages in the vertebrate and mosquito hosts. Here, the 18S rDNA sequences of 32 species of avian haemosporidian parasites are reported and compared to those of simian and rodent Plasmodium species. METHODS: Almost the entire 18S rDNAs of avian haemosporidians belonging to the genera Plasmodium (7), Haemoproteus (9), and Leucocytozoon (16) were obtained by PCR, molecular cloning, and sequencing ten clones each. Phylogenetic trees were calculated and sequence patterns were analysed and compared to those of simian and rodent malaria species. A section of the mitochondrial CytB was also sequenced. RESULTS: Sequence patterns in most avian Plasmodium species were similar to those in the mammalian parasites with most species featuring two distinct 18S rDNA sequence clusters. Distinct 18S variants were also found in Haemoproteus tartakovskyi and the three Leucocytozoon species, whereas the other species featured sets of similar haplotypes. The 18S rDNA GC-contents of the Leucocytozoon toddi complex and the subgenus Parahaemoproteus were extremely high with 49.3% and 44.9%, respectively. The 18S sequences of several species from all three genera showed chimeric features, thus indicating recombination. CONCLUSION: Gene duplication events leading to two diverged main sequence clusters happened independently in at least six out of seven avian Plasmodium species, thus supporting evolution according to a birth-and-death model like proposed for the ribosomal units of simian and rodent Plasmodium species. Patterns were similar in the 18S rDNAs of the Leucocytozoon toddi complex and Haemoproteus tartakovskyi. However, the 18S rDNAs of the other species seem to evolve in concerted fashion like in most eukaryotes, but the presence of chimeric variants indicates that the ribosomal units rather evolve in a semi-concerted manner. The new data may provide a basis for studies testing whether differential expression of distinct 18S rDNA also occurs in avian Plasmodium species and related haemosporidian parasites.

Screening of wild ruminants from the Kaunertal and other alpine regions of Tyrol (Austria) for vector-borne pathogens.

Knowledge about vector-borne pathogens important for human and veterinary medicine in wild ruminants in Tyrol (Austria) is scarce. Blood samples from Alpine ibex (Capra ibex; n = 44), Alpine chamois (Rupicapra rupicapra; n = 21), roe deer (Capreolus capreolus; n = 18) and red deer (Cervus elaphus; n = 6) were collected over a period of 4 years (2015-2018) in four regions in North Tyrol, with a primary focus on the Kaunertal. Blood spots on filter paper were tested for the presence of DNA of vector-borne pathogens (Anaplasmataceae, Piroplasmida, Rickettsia and filarioid helminths). Anaplasma phagocytophilum and Babesia capreoli were detected in two of 89 (2.3%) blood samples. Rickettsia spp., Theileria spp. and filarioid helminths were not documented. One Alpine chamois was positive for A. phagocytophilum and B. capreoli. Moreover, an ibex from the Kaunertal region was positive for A. phagocytophilum. While the ibex was a kid less than 1 year old, the chamois was an adult individual. Further research is recommended to evaluate effects of climate change on infection rates of North Tyrolean wild ruminants by these pathogens and the distribution of their vectors.

Protozoan parasites in Culex pipiens mosquitoes in Vienna.

Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector-borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June-August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemosporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n = 2-10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species of protozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable.

Indication for selfing in geographically separated populations and evidence for Pleistocene survival within the Alps: the case of Cylindrus obtusus (Pulmonata: Helicidae).

BACKGROUND: Cylindrus obtusus is one of the most prominent endemic snail species of the Eastern Alps. It is restricted to alpine meadows and calcareous rocky habitats above 1500 m. Peculiar intraspecific differences have been observed in its genital tract in the eastern populations the two mucus glands associated with the love dart sac are highly variable, while almost no variation was observed in the western populations. This raises the question whether the mode and success of reproduction of the respective populations are different. To find out whether these anatomical differences reflect genetic differentiation, which might be an indication for distinct glacial refugia, we investigated a 650 bp fragment of the mitochondrial cytochrome oxidase subunit 1 gene (COI) (280 individuals) and 9 microsatellite loci from samples (487 individuals from 29 populations) covering the whole distribution range of the species. RESULTS: The COI sequences show a geographic differentiation between eastern, central and western populations. The westernmost localities, which were covered under ice sheets during glacial periods, are characterized by extreme low variability. Overall genetic distances among all individuals are small (max. 1.7% p-distance). The microsatellite analysis reveals a high differentiation between populations, implying restriction of gene flow. The highest genetic variability was found in the central populations. Remarkably, nearly all individuals from the eastern populations, which are more variable in their genital morphology, are homozygous in all microsatellite loci, although different alleles were found within populations. CONCLUSIONS: The most peculiar outcome of the study is the strong evidence for selfing in C. obtusus as indicated by the microsatellite data in the easternmost populations. This finding is supported by the deformation of the mucus glands in the same populations. Since mucus glands play an important role in sexual reproduction, it seems plausible that in selfing organisms these structures are reduced. The phylogeographic structure revealed by COI sequences implies that the species has survived the ice ages within the Calcareous Alps. The small genetic distances among all individuals (max. 1.7%) suggest that C. obtusus has experienced severe bottlenecks in the past.

Molecular identification and phylogenetic analysis of Dipetalonema evansi (LEWIS, 1882) in camels (Camelus dromedarius) of Iran.

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far and molecular studies are scarce compared to other livestock. In this study, we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa-stained blood smears, and blood was also spotted on Whatman FTA(®) cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8 %) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however, D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s), have become feasible with the provision of the first specific COI sequences in this study.