Molting incidents of Hyalomma spp. carrying human pathogens in Germany under different weather conditions.

BACKGROUND: Hyalomma marginatum and H. rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa to central Asia but may also be found in Central and Northern Europe through introduction by migratory birds. METHODS: Ticks were collected while feeding or crawling on animals and humans, or from the environment, in different regions in Germany, between 2019 and 2021 in a citizen science study and from 2022 to 2023 in the wake of this study. RESULTS: From 2019 to 2023, a total of 212 Hyalomma adult ticks were detected in Germany. This included 132 H. marginatum and 43 H. rufipes ticks sent to research institutions and 37 photographic records that were only identified to genus level. The number of detected ticks varied over the years, with the highest number of 119 specimens recorded in 2019, followed by 57 in 2020. Most of the specimens were collected from horses, while some were collected from other animals, humans or found crawling on human clothes or other objects inside or outside houses. The screening of 175 specimens for Crimean-Congo hemorrhagic fever virus and of 132 specimens for Babesia/Theileria spp. by PCR gave negative results, while human-pathogenic Rickettsia were detected in 44% (77/175) of the total samples. Subsequent amplicon sequencing and phylogenetic analysis of representative samples determined the species of 41 Rickettsia aeschlimannii and one R. slovaca sequences. CONCLUSIONS: Analysis of climatic factors indicated a significantly higher probability of Hyalomma occurrence at locations with higher average spring temperature during the years 2019 and 2020 compared to randomly generated pseudo-absence locations. Dry and hot conditions probably facilitated Hyalomma nymphs' survival and molting into adults during these years.

Exotic ticks removed from German travelers.

We report the finding of five nymphs and three adult ticks attached to German tourists while traveling the American continents. All eight specimens were morphologically identified and confirmed genetically using the 16S rRNA gene and screened for Rickettsia spp. infections. Five tick species were identified: one Amblyomma mixtum nymph from Ecuador, one Amblyomma varium nymph from Colombia, three Amblyomma coelebs nymphs from Costa Rica, one Amblyomma americanum male from the USA, one Dermacentor andersoni female and one D. andersoni male from Canada. Tick-borne microorganisms screening using the pan-Rickettsia-PCR resulted in two positive and six negative ticks. The A. mixtum nymph was positive for Rickettsia amblyommatis, while the D. andersoni female was positive for Rickettsia peacockii.

Morphological and molecular phylogenetic characterization of Sarcocystis kani sp. nov. and other novel, closely related Sarcocystis spp. infecting small mammals and colubrid snakes in Asia.

We investigated the morphology and phylogenetic relationships of novel and previously recognized Sarcocystis spp. infecting small mammals and colubrid snakes in Asia. The nuclear 18S rRNA and mitochondrial cox1 of Sarcocystis sp.1 from mangrove snakes (Boiga dendrophila) in Thailand and Sarcocystis sp.2 from a ricefield rat (Rattus argentiventer) in Sumatra were partially sequenced. Sporocysts of Sarcocystis sp.1 induced development of sarcocysts in experimentally infected rats, which showed a unique ultrastructure that was observed previously by S.P. Kan in rats from Malaysia; therefore, we describe this species as Sarcocystis kani sp. nov. Its integration into the 18S rRNA phylogeny of Sarcocystis spp. cycling between small mammals and colubrid snakes helped clarify relationships among the so-called S. zuoi-complex of molecularly cryptic species: Sarcocystis kani sp. nov., S. sp.2, S. attenuati, S. scandentiborneensis, and S. zuoi were all included in this clade. Tree topology was resolved into dichotomies congruent with the morphological disparities between the taxa. However, cox1 gene sequencing (including newly sequenced S. singaporensis and S. zamani) revealed that Sarcocystis kani, S. attenuati, and S. scandentiborneensis were identical suggesting a recent, common ancestry. To identify other distinctive features, lineage-specific molecular patterns within both genes were examined revealing that all 18S rRNA sequences of the S. zuoi - complex possess a unique, 7-nt long motif in helix 38 of domain V7 that was different in S. clethrionomyelaphis which branched off basally from the complex. Three-dimensional homology modelling of COX1 protein structure identified amino acid substitutions within the barcode area specific for the S. zuoi-complex and substantial divergence in structurally important amino acids between Sarcocystis species of snakes as definitive hosts and other lineages of the Sarcocystidae. We discuss the utility of selected genes for species delimitation of the Sarcocystis spp. under investigation, which probably evolved during recent radiations of their intermediate and definitive hosts.

Helminths in Invasive Raccoons (Procyon lotor) from Southwest Germany.

As hosts of numerous zoonotic pathogens, the role of raccoons needs to be considered in the One Health context. Raccoons progressively expand their range as invasive alien species in Europe. This study aimed to investigate the intestinal helminth fauna of raccoons in Baden-Wuerttemberg, Germany, as no such screening had ever been conducted there. In total, we obtained 102 animals from hunters in 2019 and 2020. Intestinal helminths were retrieved using the SSCT (segmented sedimentation and counting technique) and identified morphologically and by PCR-based Sanger sequencing. Fecal samples were assessed using the ELISA PetChekTM IP assay (IDEXX, Germany) and flotation technique. The artificial digestion method was employed for analyzing muscle tissue. We detected species of four nematode genera (Baylisascaris procyonis, Toxocara canis, Capillaria spp., and Trichuris spp.), three cestode genera (Atriotaenia cf. incisa/procyonis, Taenia martis, and Mesocestoides spp.), and three trematode genera (Isthmiophora hortensis/melis, Plagiorchis muris, and Brachylaima spp.). Echinococcus spp. and Trichinella spp. were not found. The invasive behavior and synanthropic habits of raccoons may increase the infection risk with these helminths in wildlife, domestic and zoo animals, and humans by serving as a connecting link. Therefore, it is crucial to initiate additional studies assessing these risks.

Increased Vaccination Diversity Leads to Higher and Less-Variable Neutralization of TBE Viruses of the European Subtype.

Tick-borne encephalitis (TBE) is an infectious disease of the central nervous system. The causative agent is the tick-borne encephalitis virus (TBEV), which is most commonly transmitted by tick bites, but which may also be transmitted through the consumption of raw dairy products or, in rare instances, via infected transfusions, transplants, or the slaughter of infected animals. The only effective preventive option is active immunization. Currently, two vaccines are available in Europe-Encepur® and FSME-IMMUN®. In Central, Eastern, and Northern Europe, isolated TBEV genotypes belong mainly to the European subtype (TBEV-EU). In this study, we investigated the ability of these two vaccines to induce neutralizing antibodies against a panel of diverse natural TBEV-EU isolates from TBE-endemic areas in southern Germany and in regions of neighboring countries. Sera of 33 donors vaccinated with either FSME-IMMUN®, Encepur®, or a mixture of both were tested against 16 TBEV-EU strains. Phylogenetic analysis of the TBEV-EU genomes revealed substantial genetic diversity and ancestry of the identified 13 genotypic clades. Although all sera were able to neutralize the TBEV-EU strains, there were significant differences among the various vaccination groups. The neutralization assays revealed that the vaccination using the two different vaccine brands significantly increased neutralization titers, decreased intra-serum variance, and reduced the inter-virus variation.

High species diversity of Echinococcus spp. in wild mammals of Namibia.

An opportunistic survey for Echinococcus spp. in wild mammals was conducted in seven distinct study areas throughout Namibia, representing all major ecosystems, between 2012 and 2021. In total, 184 individually attributable faeces and 40 intestines were collected from eight species of carnivores, and 300 carcasses or organs of thirteen species of ungulates were examined for Echinococcus cysts. Nested PCR and sequencing of the mitochondrial nad1 gene led to the identification of five species of the Echinococcus granulosus sensu lato complex. Echinococcus canadensis G6/7 was found throughout Namibia at low frequency in lions, cheetahs, African wild dogs, black-backed jackals and oryx antelopes. Echinococcus equinus was present only in northern Namibia, locally at high frequency in lions, black-backed jackals and plains zebras. Echinococcus felidis was found only in one small area in the north-east of Namibia, but with high frequency in lions and warthogs. Echinococcus granulosus sensu stricto was identified only in two African wild dogs in the north-east of Namibia, and Echinococcus ortleppi occurred in central and southern Namibia in black-backed jackals and oryx antelopes. The development of fertile cysts indicated active intermediate host roles of oryx antelopes for E. canadensis and E. ortleppi, of warthogs for E. felidis, and of plains zebras for E. equinus. Our data support earlier hypotheses of exclusive or predominant wildlife life-cycles for E. felidis involving lions and warthogs, and - in Namibia - for E. equinus involving lions and/or black-backed jackals and plains zebras. Our data further support an interlink of wild and domestic transmission for E. ortleppi. A possible involvement of livestock and domestic dogs in transmission of E. canadensis G6/7 and E. granulosus s.s., the two parasite species with highest zoonotic potential, is uncertain for Namibia and needs further investigation.

Cystic echinococcosis in donkeys in eastern Africa.

Cystic echinococcosis (CE) is endemic in humans and domestic animals in eastern Africa. All the species of the Echinococcus granulosus sensu lato complex have been reported in this region except for E. equinus, possibly due to the small number of studies involving equids. This study reports the frequency of different Echinococcus species in donkeys from eastern Africa. A total of 5961 donkeys were examined during meat inspection in 3 slaughterhouses in Kenya. Identification of Echinococcus spp. was achieved through polymerase chain reaction-restriction fragment-length polymorphism and sequencing of the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 gene. The prevalence of CE was 5.7% (337/5961). The 263 genotyped cysts belonged to E. equinus (n = 163), E. granulosus sensu stricto (n = 70), E. canadensis (G6/7) (n = 26) and E. ortleppi (n = 4). One donkey harboured a metacestode of Spirometra theileri. All E. equinus cases, except 2, originated from southern Ethiopia, whereas the other species were more evenly distributed across the study area. Most of the cysts belonging to E. equinus were fertile (111/163), while those of the other species were non-fertile. This is the first report of Echinococcus spp. in donkeys from sub-Saharan Africa and the first confirmation of E. equinus in East Africa. The frequent fertility of E. equinus cysts in donkeys affirms their suitability as intermediate hosts of this species, while low frequency and cyst fertility suggest a marginal role of donkeys in the transmission of E. granulosus s. s., E. canadensis (G6/7) and E. ortleppi.

Update and prognosis of Dermacentor distribution in Germany: Nationwide occurrence of Dermacentor reticulatus.

A considerable range expansion of Dermacentor reticulatus has been observed in several European countries, which is concerning in the light of its vector function for several pathogens, including Babesia canis and tick-borne encephalitis virus (TBEV). The present study provides an update on the distribution of Dermacentor ticks in Germany, using a citizen science approach. Ticks were collected by citizens from March 2020 to May 2021, and submitted along with information on the date and location of collection, potential hosts and details about the circumstances of discovery. In total, 3,292 Dermacentor specimens were received, of which 76.4% (2,515/3,292) were identified as D. reticulatus and 23.0% (758/3,292) as D. marginatus, while 0.6% (19/3,292) were too damaged for species-level identification. Dermacentor reticulatus was received from all federal states of Germany. Maxent species distribution models predicted suitable environmental conditions for D. reticulatus throughout Germany. Findings on the vegetation or on pastured animals without travel history confirmed the occurrence of this tick species as far north as the most northern German federal state Schleswig-Holstein. In contrast, the distribution of D. marginatus still appears to be limited to southwestern Germany, although the northward shift of the distribution limit observed in the preceding citizen science study, as compared with previous published distributions, was confirmed. This shift was also predicted by Maxent species distribution models, reflecting the broader distribution of the tick occurrence data contributed by citizens. Most D. reticulatus ticks were found on dogs (1,311/1,960, 66.9%), while D. marginatus was mainly discovered on hoofed animals (197/621, 31.7%) and humans (182/621, 29.3%). Human tick bites were reported in 0.7% (14/1,960) of host-assigned D. reticulatus and 3.4% (21/621) of host-assigned D. marginatus. Further studies to investigate an increasing endemisation of Babesia canis in Germany as well as the relevance of D. reticulatus for TBEV spread throughout the country, e.g., by traveling dogs, are urgently needed. In view of the activity of D. reticulatus during winter or the colder months, which complements that of Ixodes ricinus, a year-round tick protection of at least dogs is strongly recommended.

The Emergence and Dynamics of Tick-Borne Encephalitis Virus in a New Endemic Region in Southern Germany.

Tick-borne encephalitis (TBE) is the most important viral tick-borne infection in Europe and Asia. It is emerging in new areas. The mechanisms of emergence are fairly unknown or speculative. In the Ravensburg district in southern Germany, TBE emerged, mainly over the last five years. Here, we analyzed the underlying epidemiology in humans. The resulting identified natural foci of the causal TBE virus (TBEV) were genetically characterized. We sampled 13 potential infection sites at these foci and detected TBEV in ticks (Ixodes ricinus) at eight sites. Phylogenetic analysis spurred the introduction of at least four distinct TBEV lineages of the European subtype into the Ravensburg district over the last few years. In two instances, a continuous spread of these virus strains over up to 10 km was observed.

Untangling the actual infection status: detection of avian haemosporidian parasites of three Malagasy bird species using microscopy, multiplex PCR, and nested PCR methods.

The development of new molecular methods has significantly improved the detection and identification of avian haemosporidian parasites (Plasmodium, Haemoproteus and Leucocytozoon) compared to microscopic examination. Very large numbers of previously hidden Haemosporida species of a wide range of avian hosts have thus been discovered in the last two decades. However, test parameters of the various detection methods remain largely unevaluated. In this study, the merits of microscopy, multiplex PCR, and nested PCR were compared to identify the infection status of three Malagasy bird species. A total of 414 blood samples of Hypsipetes madagascariensis, Foudia omissa and F. madagascariensis, as well as 147 blood smears, were examined for haemosporidian infection. Thirty-four lineages of haemosporidian parasites could be identified, of which six have been detected for the first time. Microscopy, multiplex and nested PCR showed differences in detection rate, most likely due to low parasitemia of chronically infected birds. The combination of both PCR methods yielded the best results. In particular, detection of multiple infections could be greatly improved and will enable more precise prevalence estimates of individual haemosporidian species in wild birds in the future.

Tick-Borne Encephalitis Vaccination Protects from Alimentary TBE Infection: Results from an Alimentary Outbreak.

In May 2017, a hospitalized index case of tick-borne encephalitis (TBE) was confirmed by Serology. The case was linked to alimentary infection by raw milk from a goat farm in the region of Tübingen, Baden-Württemberg, Germany, where no previous TBE cases in the area had been reported before. The TBE focus was confirmed by isolation of the TBE virus from ticks and Serological confirmation of past infection in one of the five flock goats. Additional investigations by the local public health office identified 27 consumers of goat milk at the putative period of exposure. For 20/27 exposed persons, anamnestic information was gained by the local public health office. Twelve/fourteen exposed and non-vaccinated people developed clinical illness and were confirmed as TBE cases by Serology. Five/six vaccinated and exposed people did not develop the disease. The one exposed and vaccinated person had their last TBE vaccination booster more than 15 years ago, and therefore a booster was more than 10 years overdue. None of the regularly vaccinated and exposed persons developed clinical overt TBE infection. We report the first known TBE outbreak, during which, protection by TBE vaccination against alimentary TBE infection was demonstrated.

The Spatial Distribution of Dermacentor Ticks (Ixodidae) in Germany-Evidence of a Continuing Spread of Dermacentor reticulatus.

In Europe, two tick species of the genus Dermacentor occur, Dermacentor marginatus and Dermacentor reticulatus. When the spatial distribution of both species in Germany was studied comprehensively for the first time in 1976, D. marginatus populations were recorded along the Rhine and Main river valleys in southwestern Germany, while D. reticulatus was very rare. In the last 50 years, however, a considerable range expansion of D. reticulatus has been noted in several European countries. To assess the current distribution of Dermacentor spp. in Germany, citizens were asked to send in ticks suspected to belong to the genus Dermacentor or that were of "unusual" appearance. From February 2019 until February 2020, 3,902 Dermacentor ticks were received in total. Of those, 15.48% (604/3,902) were identified as D. marginatus and 84.24% (3,287/3,902) as D. reticulatus, while 11 specimens could not be identified to species level. The majority of D. reticulatus specimens was collected from dogs (1,212/2,535; 47.12%), while D. marginatus was mostly collected from horses (184/526; 34.98%). Our results confirm that the adults of both Dermacentor species are active all year round. D. reticulatus specimens were sent in from all federal states except the Free and Hanseatic City of Hamburg, while D. marginatus specimens were only received from locations in southwestern Germany. Overall, data obtained from this citizen-science study show that D. reticulatus has significantly expanded its range, especially in northern Germany. Regarding D. marginatus, new locations northwest of the previous range were detected, although the distribution has remained rather stable as compared to D. reticulatus. The spread of D. reticulatus, the vector of Babesia canis, is of major importance for veterinarians and dog owners in terms of canine babesiosis outbreaks or endemization in hitherto B. canis-free areas. Thus, veterinarians and veterinary students need to be informed about the new situation to be able to give adequate advice to dog owners on the extended D. reticulatus range and appropriate control measures.

Echinococcus ortleppi and Echinococcus canadensis G6/7 affect domestic animals in western Zambia.

Cystic echinococcosis (CE) is endemic in many parts of sub-Saharan Africa. In contrast to the eastern part of the continent, very little data exists on the current disease situation in southern Africa including Zambia. This study determined frequency and species identity of Echinococcus spp. circulating in livestock and dogs in the Western Province of Zambia. Cysts were collected in slaughterhouses at meat inspection (cattle) and during examination of home slaughtered pigs, while dog faecal samples were collected per-rectum and examined microscopically for the presence of taeniid eggs. Individual taeniid eggs from faecal samples and individual protoscoleces from cysts were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and/or sequencing of the NADH-dehydrogenase subunit 1 (nad1) and cytochrome C oxidase 1 (cox1) gene. Fifty-four of 2000 cattle (2.7%) were found infected with a total of 65 cysts, predominantly fertile lungs cysts; all cysts were identified as Echinococcus ortleppi. Two out of 52 home-slaughtered pigs (3.8%) were infected with a fertile lung cyst each; both cysts were also identified as E. ortleppi. Microscopic examination revealed 10/289 dog faecal samples to contain taeniid eggs, of which four samples (two each) contained Echinococcus canadensis (G6/7) or Taenia hydatigena, respectively. This is the first insight in the Echinococcus species circulating in Zambia providing premises for further studies into transmission dynamics of CE in the southern African region.

Spatial distribution of alpha-gal in Ixodes ricinus - A histological study.

Alpha-gal syndrome is a complex allergic disease in humans that is caused by specific IgE (sIgE) against the carbohydrate galactose-α-1,3-galactose (alpha-gal). Tick saliva contains alpha-gal, and tick bites are considered a major cause of the induction of alpha-gal-sIgE. The origin of alpha-gal in tick saliva remains unclarified. The presence of alpha-gal in tick tissue was visualized in this study to provide an overview of the spatial distribution of alpha-gal and to further elucidate the origin of alpha-gal in tick saliva. Fed and unfed Ixodes ricinus females were examined by histology, immunohistochemistry, immunofluorescence, transmission electron microscopy and immunoelectron microscopy using the alpha-gal-specific monoclonal antibody M86 and Marasmius oreades agglutinin (MOA) lectin. Alpha-gal epitopes were detected in the midgut, hemolymph and salivary glands, and the immunofluorescence analysis revealed signs of the endocytosis of alpha-gal-containing constituents during the process of hematophagy. Alpha-gal epitopes in endosomes of the digestive gut cells of the ticks were observed via immunoelectron microscopy. Alpha-gal epitopes were detected in dried droplets of hemolymph from unfed ticks. Intense staining of alpha-gal epitopes was found in type II granular acini of the salivary glands of fed and unfed ticks. Our data suggest that alpha-gal is not ubiquitously expressed in tick tissue but is present in both fed and unfed ticks. The findings also indicate that both the metabolic incorporation of constituents from a mammalian blood meal and endogenous production contribute to the presence of alpha-gal epitopes in ticks.

Ixodes frontalis: a neglected but ubiquitous tick species in Germany.

The ornithophilic tick species Ixodes frontalis (Panzer) is spatially distributed in Europe, Asia and northern Africa. It can serve as a carrier of different bacteria and viruses, but little is known of its actual vector competence. In addition, adult females of this species are associated with the avian "tick-related syndrome" (TRS). Like most ornithophilic tick species, I. frontalis is usually collected from bird nests or directly from their hosts. Reports of I. frontalis being collected with the flagging method are scarce. In Germany, the species is considered as very rare. In the few reports that are available, the majority of ticks has been discovered on migrating birds. Therefore, knowledge of the actual distribution of this species in Germany was strictly limited. In this study, we report the finding of 1084 individuals of I. frontalis collected with the flagging method in urban areas and gardens in several regions of Germany. Furthermore, the species was discovered on 35 dead Eurasian blackbirds (Turdus merula) from all over Germany. Finally, we report of five new possible cases of TRS, three in free-ranging birds and two in captive birds, including a Harris's hawk (Parabuteo unicinctus) kept for falconry.

Fasciola spp. in Armenia: Genetic diversity in a global context.

Fasciolosis, a food- and waterborne infection caused by the trematodes Fasciola hepatica and F. gigantica, is recognized by WHO as a neglected zoonotic disease. Whereas F. hepatica is distributed worldwide in cooler climates, F. gigantica occurs mainly in the tropics of Africa and Asia. The southern Caucasus, with Armenia, is one of the most northern regions where both species occur and may produce hybrids. In this study, livestock in central Armenia was surveyed for fasciolosis, the causative species were determined and the genetic diversity of both species was estimated. Total prevalence in sheep (1794), cattle (324) and goats (9) was 21.2%, 15.7% and 44.4%, respectively. After morphological identification and sequencing of a mitochondrial (nad1) and a nuclear marker gene (28S rRNA), 62 collected specimens were allocated to F. hepatica (n = 55) and F. gigantica (n = 7). Intraspecific diversity was evaluated for the complete nad1 gene, resulting in 29 haplotypes of F. hepatica and six haplotypes of F. gigantica. Diversity was higher among F. gigantica than F. hepatica in the Armenian sample set, a difference that was confirmed analyzing available sequences for both species worldwide. Maximum genetic distance between haplotypes in global networks was 49 nucleotide steps for F. gigantica compared to 15 for F. hepatica. In the available sample sets, F. hepatica showed higher diversity in western Asia and the Middle East compared to Europe and eastern Asia, while for F. gigantica loosely structured clusters comprising mainly western/southern Asian and African haplotypes could be identified. A distinct clade comprising haplotypes from Zambia was basal in the phylogenetic tree. Biogeographical implications of these data are discussed.

Imported Hyalomma ticks in Germany in 2018.

BACKGROUND: Hyalomma marginatum and Hyalomma rufipes are two-host tick species, which are mainly distributed in southern Europe, Africa and middle-eastern Asia. They are well-known vectors of Crimean Congo hemorrhagic fever (CCHF) virus and other viruses as well as Rickettsia aeschlimannii. In recent years, these tick species have been found sporadically in Germany, but they do not belong to the autochthonous tick fauna in Germany. METHODS: Ticks with unusual morphology were collected and sent from private persons or public health offices to involve institutions for morphological identification and further testing. All ticks identified as Hyalomma spp. were tested using molecular detection methods for CCHF virus, Rickettsia spp., Coxiella burnetii and Coxiella-like organisms, Babesia spp. and Theileria spp. RESULTS: Thirty-five ticks with an unusual appearance or behaviour were reported to us during summer-autumn 2018. For 17 of them, the description or photos implied that they belong to the hard tick genus Hyalomma. The remaining 18 ticks were sent to us and were identified as adult Hyalomma marginatum (10 specimens) or adult Hyalomma rufipes (8 specimens). All ticks tested negative for CCHF virus, Coxiella burnetii, Coxiella-like organisms, Babesia spp. and Theileria spp. The screening for rickettsiae gave positive results in 9 specimens . The Rickettsia species in all cases was identified as R. aeschlimannii. CONCLUSIONS: These results show that exotic tick species imported into Germany were able to develop from the nymphal to the adult stage under appropriate weather conditions. Fifty percent of the ticks carried R. aeschlimannii, a human pathogen, while CCHF virus or other pathogens were not detected. Imported Hyalomma ticks may be the source of exotic diseases acquired in Germany.

Genetic analysis of Rhipicephalus sanguineus sensu lato ticks, parasites of dogs in the Canary Islands, Cyprus, and Croatia, based on mitochondrial 16S rRNA gene sequences.

The aim of this work was to perform an analysis based on mtDNA sequences of the 16S rRNA gene in order to determine the phylogenetic position of ticks belonging to the Rhipicephalus sanguineus group from the Canary Islands, Cyprus, and Croatia. All the haplotypes obtained from ticks collected in the Canary Islands and Croatia grouped with R. sanguineus sensu stricto from France, Portugal, Italy, Switzerland, Argentina, Chile, Uruguay, and the USA. The sequences of R. sanguineus sensu lato from Cyprus formed a clade with R. sanguineus s.l. from Egypt, Turkey, and Romania, which belongs to the "Rhipicephalus sp. morphotype I" or "southeastern European lineage." Ticks determined as R. turanicus s.l. from Cyprus clustered separately from the remaining clades of the R. sanguineus group, including R. turanicus s.s. The data show that R. sanguineus s.s. is present in the Canary Islands and Croatia, while R. sanguineus "southeastern lineage" is found in Cyprus.

Avian malaria on Madagascar: prevalence, biodiversity and specialization of haemosporidian parasites.

Previous studies about geographic patterns of species diversity of avian malaria parasites and others in the Order Haemosporida did not include the avian biodiversity hotspot Madagascar. Since there are few data available on avian malaria parasites on Madagascar, we conducted the first known large-scale molecular-based study to investigate their biodiversity. Samples (1067) from 55 bird species were examined by a PCR method amplifying nearly the whole haemosporidian cytochrome b gene (1063 bp). The parasite lineages found were further characterized phylogenetically and the degree of specialization was determined with a newly introduced host diversity index (Hd). Our results demonstrate that Madagascar indeed represents a biodiversity hotspot for avian malaria parasites as we detected 71 genetically distinct parasite lineages of the genera Plasmodium and Haemoproteus. Furthermore, by using a phylogenetic approach and including the sequence divergence we suspect that the detected haemosporidian lineages represent at least 29 groups i.e. proposed species. The here presented Hd values for each parasite regarding host species, genus and family strongly support previous works demonstrating the elastic host ranges of some avian parsites of the Order Haemosporida. Representatives of the avian parasite genera Plasmodium and Leucocytozoon tend to more often be generalists than those of the genus Haemoproteus. However, as demonstrated in various examples, there is a large overlap and single parasite lineages frequently deviate from this rule.

Phylogenetics of tick-borne encephalitis virus in endemic foci in the upper Rhine region in France and Germany.

OBJECTIVE: Tick-borne encephalitis (TBE) caused by the tick-borne encephalitis virus (TBEV) is the most important tick-borne arboviral disease in Europe and Asia. The Upper Rhine Valley is thought to be the very western border of TBEV distribution in Europe. The aim of our study was to identify natural foci and isolate TBEV from ticks, to determine the prevalence of TBEV in local tick populations and to study the phylogenetic relatedness of circulating TBEV strains in this region. MATERIAL AND METHODS: Ticks were collected between 2016, 2017 and 2018 by flagging. TBEV was isolated from collected ticks and phylogenetic analyses were performed. Minimal infection rates (MIR) of the collected ticks were calculated. RESULTS: At 12 sampling sites, a total of 4,064 Ixodes ticks were collected in 2016 and 2017 -(and one single collection 2018). 953 male, 856 female adult ticks and 2,255 nymphs were identified. The MIR rates were 0,17% (1/595) for Schiltach (Germany) and 0,11% (1/944) for Foret de la Robertsau (France), respectively. Overall, the three newly described TBEV strains, isolated in the years 2016 and 2017 from the Upper Rhine Valley have no close phylogenetic relation and show a genetic relationship with strains from eastern Europe. The 2018 TBEV strain from Aubachstrasse (Germany), however, is closely related to the TBEV found in Schiltach (Germany). CONCLUSION: In conclusion, we demonstrate, to our knowledge for the first time, the phylogenetic relations of the newly isolated TBEV strains on both sides of the upper Rhine river.