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.

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.

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.

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.

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.

Carrion crows (Corvus corone) of southwest Germany: important hosts for haemosporidian parasites.

BACKGROUND: Avian malaria parasites (Plasmodium spp.) and other Haemosporida (Haemoproteus and Leucocytozoon spp.) form a diverse group of vector-transmitted blood parasites that are abundant in many bird families. Recent studies have suggested that corvids may be an important host for Plasmodium spp. and Leucocytozoon spp. METHODS: To investigate the diversity of Haemosporida of resident carrion crows (Corvus corone) and Eurasian Magpies (Pica pica) in southwest Germany, 100 liver samples of corvids were examined using a nested PCR method to amplify a 1063 bp fragment of the haemosporidian mitochondrial cytochrome b gene. The phylogenetic relationship of parasite lineages obtained from these birds was inferred. RESULTS: Haemosporidian DNA was detected in 85 carrion crows (89.5%) and in all five Eurasian Magpies. The most abundant parasite genus was Leucocytozoon with a prevalence of 85.3% (n = 95). 65.3% of the samples (n = 62) contained multiple infections. Thirteen haemosporidian lineages were isolated from the corvid samples. Female carrion crows were more likely infected with haemosporidian parasites than males. DISCUSSION: This study provides the first insight into the diversity of haemosporidian parasites of corvids in Germany. Very high prevalences were found and based on the applied diagnostic method also a high amount of multiple infections could be detected. Due to the high diversity of haemosporidian parasites found in corvids, they seem to be excellent model organisms to test species deliminations in haemosporidian parasites.

Bacteria of the genera Ehrlichia and Rickettsia in ticks of the family Ixodidae with medical importance in Argentina.

The aim of this study was to get an overview about the occurrence of bacteria from the genus Ehrlichia and Rickettsia in ixodid ticks with medical importance in Argentina. Therefore, in 2013 and 2014, free-living ticks were collected in different provinces of northern Argentina. These ticks were determined as Amblyomma sculptum, Amblyomma neumanni, Amblyomma parvum, Amblyomma triste, Amblyomma ovale, Amblyomma tonelliae and Haemaphysalis juxtakochi. All samples were tested to determine the infection with Ehrlichia spp. and Rickettsia spp. by PCR assays. Rickettsial DNA was detected in all tested tick species, with the exception of A. tonelliae. 'Candidatus Rickettsia amblyommii', 'Candidatus Rickettsia andeanae', and Rickettsia parkeri were found in A. neumanni, A. parvum, and A. triste, respectively. Another rickettsial species, Rickettsia bellii, was found in A. sculptum, A. ovale and H. juxtakochi. None of the tested ticks showed infection with Ehrlichia. The results of the study demonstrate that Rickettsia species belonging to the spotted fever group are associated with various species of Amblyomma throughout a wide area of northern Argentina, where cases of Amblyomma ticks biting humans are common.

Cerebral and non-cerebral coenurosis: on the genotypic and phenotypic diversity of Taenia multiceps.

We characterised the causative agents of cerebral and non-cerebral coenurosis in livestock by determining the mitochondrial genotypes and morphological phenotypes of 52 Taenia multiceps isolates from a wide geographical range in Europe, Africa, and western Asia. Three studies were conducted: (1) a morphological comparison of the rostellar hooks of cerebral and non-cerebral cysts of sheep and goats, (2) a morphological comparison of adult worms experimentally produced in dogs, and (3) a molecular analysis of three partial mitochondrial genes (nad1, cox1, and 12S rRNA) of the same isolates. No significant morphological or genetic differences were associated with the species of the intermediate host. Adult parasites originating from cerebral and non-cerebral cysts differed morphologically, e.g. the shape of the small hooks and the distribution of the testes in the mature proglottids. The phylogenetic analysis of the mitochondrial haplotypes produced three distinct clusters: one cluster including both cerebral isolates from Greece and non-cerebral isolates from tropical and subtropical countries, and two clusters including cerebral isolates from Greece. The majority of the non-cerebral specimens clustered together but did not form a monophyletic group. No monophyletic groups were observed based on geography, although specimens from the same region tended to cluster. The clustering indicates high intraspecific diversity. The phylogenetic analysis suggests that all variants of T. multiceps can cause cerebral coenurosis in sheep (which may be the ancestral phenotype), and some variants, predominantly from one genetic cluster, acquired the additional capacity to produce non-cerebral forms in goats and more rarely in sheep.

Parasite airlines: mapping the distribution and transmission of avian blood parasites in migratory birds.

During their journeys, migratory birds encounter a wider range of parasites than residents, transporting them over vast distances. While some parasites are widely distributed, transmission is not inevitable and depends on the presence of competent arthropod vectors as well as parasite compatibility with native bird species. Distinguishing between parasite distribution and transmission areas is crucial for monitoring and assessing risks to native bird species, as distribution areas, with the appropriate conditions, could become potential transmission areas. In this study, blood samples from 455 reed-living birds of the genera Acrocephalus, Locustella, and Emberiza, collected in the nature reserve "Die Reit" in Hamburg, Germany were screened, targeting haemosporidian parasites, trypanosomes, and filarioid nematodes. Determination of migratory bird age was employed to ascertain the transmission area of the detected parasites. Transmission areas were determined, based on information provided by resident and juvenile birds as well as findings in competent vectors. Long-distance migratory birds of the genus Acrocephalus showed a higher prevalence and diversity of blood parasites compared with partially migratory birds such as Emberiza schoeniclus. Notably, an age-dependent difference in parasite prevalence was observed in Acrocephalus spp., but not in E. schoeniclus. Nematodes were absent in all examined bird species. Proposed transmission areas were identified for nine haemosporidian lineages, showing three different types of transmission area, either with limited transmission in Europe or Africa, or active transmission in both regions.

Germany-wide citizen science study reveals spread of Babesia canis-infected Dermacentor reticulatus ticks by dogs travelling within the country.

The ornate dog tick Dermacentor reticulatus, vector of Babesia canis, has shown a considerable range expansion in several European countries. Previously, only few areas in Germany were recognised as endemic for B. canis, but a marked increase in autochthonous canine babesiosis cases and spread to new areas has been noted recently. To better assess the current risk for dogs, the present study screened 5913 specimens of D. reticulatus from all over Germany, collected in the frame of a Citizen Science study during 2019-2023. Moreover, 343 Dermacentor marginatus ticks were also included. Babesia detection was achieved by quantitative real-time PCR (qPCR). Positive samples were confirmed by sequencing. Moreover, a MGB-probe-based triplex qPCR was established to detect and distinguish between the canine Babesia spp. relevant in Europe, i.e. B. canis, Babesia vogeli and Babesia gibsoni. Overall, B. canis DNA was detected in five D. reticulatus specimens (0.08%). Two of the B. canis-positive ticks originated from areas previously known as endemic for canine babesiosis, namely from the area of Freiburg im Breisgau, federal state of Baden-Wuerttemberg, and from the district St. Wendel, federal state of Saarland. Three further B. canis-positive ticks were detected in districts not yet recognised as endemic, one each in the district of Mansfeld-Suedharz, federal state of Saxony-Anhalt, the district of Ravensburg, federal state of Baden-Wuerttemberg and in the city of Fürth, federal state of Bavaria. However, the tick in Fürth was found on a dog who had returned from a trip to the Breisgau region on the previous day, indicating translocation of the specimen out of this well-known endemic focus. The geographical distribution of the positive samples shows that B. canis is currently spreading in Germany, particularly via dogs travelling within the country, increasing the infection risk throughout the country. Important measures to contain a further spread of the pathogen include comprehensive year-round tick prophylaxis with licensed acaricides, not only to protect the individual pet, but also the entire dog population. Moreover, screening of dogs entering Germany from B. canis-endemic countries is required and any treatment should aim at pathogen elimination by use of appropriate imidocarb dosages.

Raising the bar: genus-specific nested PCR improves detection and lineage identification of avian haemosporidian parasites.

Avian haemosporidian parasites are useful model organisms to study the ecology and evolution of parasite-host interactions due to their global distribution and extensive biodiversity. Detection of these parasites has evolved from microscopic examination to PCR-based methods, with the mitochondrial cytochrome b gene serving as barcoding region. However, standard PCR protocols used for screening and identification purposes have limitations in detecting mixed infections and generating phylogenetically informative data due to short amplicon lengths. To address these issues, we developed a novel genus-specific nested PCR protocol targeting avian haemosporidian parasites. The protocol underwent rigorous testing utilizing a large dataset comprising blood samples from Malagasy birds of three distinct Passeriformes families. Furthermore, validation was done by examining smaller datasets in two other laboratories employing divergent master mixes and different bird species. Comparative analyses were conducted between the outcomes of the novel PCR protocol and those obtained through the widely used standard nested PCR method. The novel protocol enables specific identification of Plasmodium, Haemoproteus (Parahaemoproteus), and Leucocytozoon parasites. The analyses demonstrated comparable sensitivity to the standard nested PCR with notable improvements in detecting mixed infections. In addition, phylogenetic resolution is improved by amplification of longer fragments, leading to a better understanding of the haemosporidian biodiversity and evolution. Overall, the novel protocol represents a valuable addition to avian haemosporidian detection methodologies, facilitating comprehensive studies on parasite ecology, epidemiology, and evolution.

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.

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.

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.

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.

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.

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.

A real-time multiplex-nested PCR system for coprological diagnosis of Echinococcus multilocularis and host species.

A hybridization probe-based real-time multiplex-nested PCR system was developed for the simultaneous detection of Echinococcus multilocularis and host species directly from faecal samples. Species identification was determined by melting curve analysis. Specificity was assessed by using DNA extracted from various cestodes (E. multilocularis, Echinococcus granulosus (G1), Echinococcus ortleppi, Echinococcus canadensis (G6, G7), Taenia crassiceps, Taenia hydatigena, Taenia mustelae, Taenia pisiformis, Taenia serialis, Taenia taeniaeformis, Mesocestoides leptothylacus), carnivores (Vulpes vulpes, Vulpes corsac, Vulpes ferrilata, Canis familiaris, Felis catus, Martes foina), Microtus arvalis and Arvicola terrestris. The analytical sensitivity was 10 fg, evaluated with serially diluted DNA of E. multilocularis to 10 µl total DNA solution from E. multilocularis-negative canid faeces. Based on a comparison of 47 dog samples from China, the proportion of the E. multilocularis-positive-tested samples by the real-time multiplex-nested PCR was moderately higher (38% vs. 30%) as when tested with a previously evaluated nested PCR with a sensitivity of 70-100%, depending on the number and gravidity status of worms present in the intestine (Dinkel et al., J Clin Microbiol 36:1871-1876, 1998). To assess the epidemiological applicability of this method, 227 canid faecal samples collected in the field were analysed. This newly developed real-time multiplex-nested PCR system is a specific, sensitive and reliable method for the detection of E. multilocularis and host species in faecal samples for epidemiological purposes.

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.