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Background Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. Methods The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. Results In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. Conclusions The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species. Graphical Abstract

Background Infection with the cardiopulmonary nematode Angiostrongylus vasorum may cause severe disease in dogs, therefore prophylactic treatments are necessary to prevent infection in dogs at risk. A clinical field study was conducted to demonstrate the efficacy and safety of an oral combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) for the prevention of A. vasorum infection in dogs (prevention study). A survey study was conducted concurrently to determine the infection pressure in the same areas. Methods Prevention and survey studies were both conducted at the same veterinary clinics in endemic hot spots for A. vasorum in Denmark and Italy. The prevention study was a randomized, placebo controlled, double masked study where 622 client-owned dogs were treated and tested at 30 days intervals for 10 months. In the survey study 1628 dogs that were at risk of infection and/or were suspected to be infected were tested by fecal and/or serological methods, and the percent of dogs positive for A. vasorum was calculated. Results In the prevention study, there were no adverse events related to treatment with Simparica Trio®. Two placebo-treated animals became infected with A. vasorum during the 10-month study period, while none of the dogs in the combination product-treated group became infected. In the survey study, 12.2% of the study dogs were found positive to A. vasorum, indicating high exposure to the parasite during the period of the prevention study. Conclusions Monthly oral treatment with the combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) was 100% effective in the prevention of natural infection with A. vasorum in dogs in highly endemic areas. In endemic areas, A. vasorum occurrence in dogs at risk is considerable.

Background In three randomized, controlled laboratory efficacy studies, the efficacy in the prevention of patent infections of a topical combination of imidacloprid 10%/moxidectin 1% (Advocate® spot-on formulation for cats, Bayer Animal Health GmbH) against larval stages and immature adults of Aelurostrongylus abstrusus, as well as the treatment efficacy of a single or three monthly treatments against adult A. abstrusus, were evaluated. Methods Cats were experimentally inoculated with 300–800 third-stage larvae (L3). Each group comprised 8 animals and the treatment dose was 10 mg/kg bodyweight (bw) imidacloprid and 1 mg/kg bw moxidectin in each study. Prevention of the establishment of patent infections was evaluated by two treatments at a monthly interval at three different time points before and after challenge infection. Curative efficacy was tested by one or three treatments after the onset of patency. Worm counts at necropsy were used for efficacy calculations. Results In Study 1, the control group had a geometric mean (GM) of 28.8 adult nematodes and the single treatment group had a GM of 3.4 (efficacy 88.3%). In Study 2, the control group had a GM of 14.3, the prevention group had a GM of 0 (efficacy 100%), while the treatment group had a GM of 0.1 (efficacy 99.4%). In Study 3, the GM worm burden in the control group was 32.6 compared to 0 in all three prevention groups (efficacy 100% for all of those groups). Conclusions The monthly administration of Advocate® reliably eliminated early larval stages and thereby prevented lung damage from and patent infections with A. abstrusus in cats. Regarding treatment, a single application of Advocate® reduced the worm burden, but it did not sufficiently clear the infection. In contrast, three monthly treatments were safe and highly efficacious against A. abstrusus.

Background The yellow fever mosquito Aedes aegypti transmits viral diseases that have plagued humans for centuries. Its ancestral home are forests of Africa and ~400–600 years ago it invaded the New World and later Europe and Asia, causing some of the largest epidemics in human history. The species was rarely detected in countries surrounding the Mediterranean Sea after the 1950s, but during the last 16 years it re-appeared in Madeira, Russia and in the eastern coast of the Black Sea. We genotyped Ae. aegypti populations from the Black Sea region to investigate whether this is a recent invasion (and if so, where it came from) or a remnant of pre-eradication populations that extended across the Mediterranean. We also use the Black Sea populations together with a world reference panel of populations to shed more light into the phylogeographical history of this species. Results Microsatellites and ~19,000 genome-wide single nucleotide polymorphisms (SNPs) support the monophyletic origin of all populations outside Africa, with the New World as the site of first colonization. Considering the phylogenetic relationships, the Black Sea populations are basal to all Asian populations sampled. Bayesian analyses combined with multivariate analyses on both types of markers suggest that the Black Sea population is a remnant of an older population. Approximate Bayesian Computation Analysis indicates with equal probability, that the origin of Black Sea populations was Asia or New World and assignment tests favor the New World. Conclusions Our results confirmed that Ae. aegypti left Africa and arrived in New World ~500 years ago. The lineage that returned to the Old World and gave rise to present day Asia and the Black Sea populations split from the New World approximately 100–150 years ago. Globally, the Black Sea population is genetically closer to Asia, but still highly differentiated from both New World and Asian populations. This evidence, combined with bottleneck signatures and divergence time estimates, support the hypothesis of present day Black Sea populations being remnants of older populations, likely the now extinct Mediterranean populations that, consistent with the historic epidemiological record, likely represent the original return of Ae. aegypti to the Old World. Electronic supplementary material The online version of this article (10.1186/s13071-018-2933-2) contains supplementary material, which is available to authorized users.

Background Aedes albopictus, a known worldwide vector of several mosquito-borne disease pathogens including dengue, chikungunya and Zika viruses, was introduced into Europe in the late 1970s through global trade. First recorded in northern Italy in 1990, this mosquito species has rapidly spread throughout the country, where it was responsible for an outbreak of chikungunya in 2007 that affected more than 200 people. As part of the VectorNet project, which is aimed at improving preparedness and responsiveness for animal and human vector-borne diseases in Europe, a mosquito targeted study was carried out on the three southernmost Italian islands. The objective was to verify the current European southern distribution limits of Ae. albopictus and the potential occurrence of other invasive mosquito species, in the light of the introduction of high risk for vector-borne disease pathogens into Europe via migration flows. Results In the summer 2015, six surveys for container-breeding mosquitoes were carried out by setting up a network of oviposition traps and BG Sentinel traps in selected areas on the islands of Pantelleria, Lampedusa and Linosa. Aedes albopictus was found on all three islands under investigation. The consequences on public health with regard to the presence of this mosquito vector and the migrant people entering the country from Africa and the Middle East are also discussed here. Conclusions The detection of the Asian tiger mosquito on these islands, which represent the last European strip of land facing Africa, has important implications for public health policy and should prompt the national authorities to implement tailored surveillance activities and reinforce plans for preparedness strategies in such contexts.

Background: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far.Results: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites.Conclusions: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.

Background In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. Results Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15 dogs infected with other parasites than A. vasorum was positive before but negative after treatment. Conclusion Although heat treatment improves A. vasorum antigen detection between 7 and 10 wpi by immune complex disruption, we do not recommend systematic pretreating sera because of reduced antigen detection between 3 and 6 wpi and impairment of antibody detection, if performed contemporaneously.

Background Angiostrongylus vasorum is a metastrongyloid nematode localized in the right heart and the pulmonary arteries of domestic dogs. The number of reports in Europe has recently increased, presumably as a consequence of a growing awareness among clinicians, animal owners and researchers, but also due to a growing incidence and territorial spread. So far, no studies have been conducted to assess the prevalence and distribution of A. vasorum in domestic dogs in Romania, and the awareness among veterinarians is limited or absent. The aim of the present study was to evaluate the countrywide seroprevalence of circulating antigens of A. vasorum and specific antibodies against A. vasorum in domestic dogs from Romania. Methods Between November 2016 and July 2017, blood was sampled from a total of 1545 domestic dogs from 23 counties of Romania. Details about their gender, age, breed, housing, use and origin were collected. All serum samples were tested for the presence of A. vasorum circulating antigens (AG) using monoclonal and polyclonal antibodies in a sandwich ELISA. Additionally, a sandwich ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies was used for specific antibody (AB) detection. Results A total of 33 dogs (2.14%, 95% CI: 1.82–3.56%) were seropositive for A. vasorum antigen or antibodies against the parasite. Three dogs were positive for antigen only (0.19%, 95% CI: 0.07–0.57%) and 30 dogs (1.94%, 95% CI: 1.36–2.76%) were positive for antibodies only. No dog was positive for both tests. The overall prevalence (AB or AG) and the AB prevalence were significantly higher in pure breed dogs compared to mixed breeds and mongrel dogs (P

Background The zoonotic parasite Taenia saginata transmits between humans, the definitive host (causing taeniosis), and bovines as the intermediate host (causing cysticercosis). Central and western Asia and the Caucasus have large cattle populations and beef consumption is widespread. However, an overview of the extent of human T. saginata infection and bovine cysticercosis is lacking. This review aims to summarize the distribution of T. saginata in this region. Methods A systematic review was conducted, that gathered published and grey literature, and official data concerning T. saginata taeniosis and bovine cysticercosis in central and western Asia and the Caucasus published between January 1st, 1990 and December 31st, 2018. Where no data were available for a country within this period, published data from 1985–1990 were also accessed. Results From 10,786 articles initially scanned, we retrieved 98 full-text articles from which data were extracted. In addition, two unpublished datasets were provided on the incidence of human taeniosis. Data for human taeniosis and bovine cysticercosis were found for all countries except Turkmenistan. Human taeniosis prevalence varied from undetected to over 5.3%, with regional variations. Where bovine cysticercosis was detected, prevalences varied from case reports to 25%. Conclusions The public health burden of T. saginata is assumed to be small as the parasite is of low pathogenicity to humans. However, this review indicates that infection continues to be widespread and this may result in a large economic burden, due to the resources utilized in meat inspection and condemnation or processing with subsequent downgrading of infected carcasses. Electronic supplementary material The online version of this article (10.1186/s13071-019-3438-3) contains supplementary material, which is available to authorized users.

[Background] Aedes japonicus is an invasive vector mosquito from Southeast Asia which has been spreading across central Europe since the year 2000. Unlike the Asian Tiger mosquito (Aedes albopictus) present in Spain since 2004, there has been no record of Ae. japonicus in the country until now., [Results] Here, we report the first detection of Ae. japonicus in Spain, at its southernmost location in Europe. This finding was triggered by the citizen science platform Mosquito Alert. In June 2018, a citizen sent a report via the Mosquito Alert app from the municipality of Siero in the Asturias region (NW Spain) containing pictures of a female mosquito compatible with Ae. japonicus. Further information was requested from the participant, who subsequently provided several larvae and adults that could be classified as Ae. japonicus. In July, a field mission confirmed its presence at the original site and in several locations up to 9 km away, suggesting a long-time establishment. The strong media impact in Asturias derived from the discovery raised local participation in the Mosquito Alert project, resulting in further evidence from surrounding areas., [Conclusions] Whilst in the laboratory Ae. japonicus is a competent vector for several mosquito-borne pathogens, to date only West Nile virus is a concern based on field evidence. Nonetheless, this virus has yet not been detected in Asturias so the vectorial risk is currently considered low. The opportunity and effectiveness of combining citizen-sourced data to traditional surveillance methods are discussed., Mosquito Alert is currently promoted by the ‘la Caixa’ Banking Foundation and Dipsalut from the Diputació de Girona, and sponsored by the Company Lokimica S.A.

Background Cytauxzoonosis is an emerging tick-borne disease of domestic and wild felids. Cytauxzoon felis induces severe and often fatal disease in domestic cats. In Europe, clinical and subclinical infections caused by Cytauxzoon sp. are described. We report the first cases of Cytauxzoon sp. infection in domestic cats in Switzerland. Methods Clinical and laboratory data and results of PCR analyses were collected from Cytauxzoon sp. PCR-positive cats and the cats followed for up to 851 days. Results The cases were three two-month old kittens from the same litter (Cases 1–3) and two adult domestic shorthair cats (Cases 4 and 5). The cats originated from the north-west and west of Switzerland. Cases 1–3 presented with moderate to severe regenerative anaemia and intraerythrocytic inclusions. Cytauxzoon sp. was confirmed by PCR and sequencing. The kittens made a clinical and haematological recovery after blood transfusion and/or treatment with azithromycin and atovaquone, but erythroparasitaemia persisted. Case 4 presented with severe non-regenerative anaemia. Case 5 was healthy and used as a blood donor for Case 4. Following blood transfusion, Case 4 showed intraerythrocytic inclusions, and Cytauxzoon sp. was confirmed in both Cases 4 and 5 using PCR and sequencing. Case 4 achieved clinical and haematological remission after treatment with azithromycin, atovaquone and immunosuppressive drugs. Eight months later, Case 4 was presented again with anaemia but tested Cytauxzoon sp. PCR-negative. Sequencing of 1637 bp of the 18S rRNA gene of Cytauxzoon sp. revealed 100% nucleotide sequence identity among isolates of Cases 1–3 and between isolates of Cases 4 and 5, and 99% sequence identity between isolates of all cases. Phylogenetic analysis revealed the closest relationship of the Swiss isolates to Cytauxzoon sp. isolates from domestic cats and wild felids from France, Spain and Romania and to Cytauxzoon manul from a Pallas’s cat. Conclusions This is the first report of Cytauxzoon sp. infection in domestic cats in Switzerland. It is also the first report of infection in very young kittens and transmission of Cytauxzoon sp. to an adult cat by transfusion of blood from an asymptomatic cat. The cats recovered but some developed chronic asymptomatic erythroparasitaemia for up to 28 months. Domestic cats may act as reservoirs for Cytauxzoon sp. in Europe and blood donor cats should be screened for this agent by PCR.

Background In Europe the principal definitive host for Echinococcus multilocularis, causing alveolar echinococcosis in humans, is the red fox (Vulpes vulpes). Obtaining reliable estimates of the prevalence of E. multilocularis and relevant risk factors for infection in foxes can be difficult if diagnostic tests with unknown test accuracies are used. Latent-class analysis can be used to obtain estimates of diagnostic test sensitivities and specificities in the absence of a perfect gold standard. Samples from 300 foxes in Switzerland were assessed by four different diagnostic tests including necropsy followed by sedimentation and counting technique (SCT), an egg-PCR, a monoclonal and a polyclonal copro-antigen ELISA. Information on sex, age and presence of other cestode species was assessed as potential covariates in the Bayesian latent class models. Different Bayesian latent-class models were run, considering dichotomized test results and, additionally, continuous readings resulting in empirical ROC curves. Results The model without covariates estimated a true parasite prevalence of 59.5% (95% CI: 43.1–66.4%). SCT, assuming a specificity of 100%, performed best among the four tests with a sensitivity of 88.5% (95% CI: 82.7–93.4%). The egg-PCR showed a specificity of 93.4% (95% CI: 87.3–99.1%), although its sensitivity of 54.8% was found moderately low (95% CI: 48.5–61.0%). Relatively higher sensitivity (63.2%, 95% CI: 55.3–70.8%) and specificity (70.0%, 95% CI: 60.1–79.4%) were estimated for the monoclonal ELISA compared to the polyclonal ELISA with a sensitivity and specificity of 56.0% (95% CI: 48.0–63.9%) and 65.9% (95% CI: 55.8–75.6%), respectively. In the Bayesian models, adult foxes were found to be less likely infected than juveniles. Foxes with a concomitant cestode infection had double the odds of an E. multilocularis infection. ROC curves following a Bayesian approach enabled the empirical determination of the best cut-off point. While varying the cut-offs of both ELISAs, sensitivity and specificity of the egg-PCR and SCT remained constant in the Bayesian latent class models. Conclusions Adoption of a Bayesian latent class approach helps to overcome the absence of a perfectly accurate diagnostic test and gives a more reliable indication of the test performance and the impact of covariates on the prevalence adjusted for diagnostic uncertainty. Electronic supplementary material The online version of this article (10.1186/s13071-017-2562-1) contains supplementary material, which is available to authorized users.

International audience; Background: In Europe, hard ticks of the subgenus Pholeoixodes (Ixodidae: Ixodes) are usually associated with burrow-dwelling mammals and terrestrial birds. Reports of Pholeoixodes spp. from carnivores are frequently contradictory, and their identification is not based on key diagnostic characters. Therefore, the aims of the present study were to identify ticks collected from dogs, foxes and badgers in several European countries, and to reassess their systematic status with molecular analyses using two mitochondrial markers. Results: Between 2003 and 2017, 144 Pholeoixodes spp. ticks were collected in nine European countries. From accurate descriptions and comparison with type-materials, a simple illustrated identification key was compiled for adult females, by focusing on the shape of the anterior surface of basis capituli. Based on this key, 71 female ticks were identified as I. canisuga, 21 as I. kaiseri and 21 as I. hexagonus. DNA was extracted from these 113 female ticks, and from further 31 specimens. Fragments of two mitochondrial genes, cox1 (cytochrome c oxidase subunit 1) and 16S rRNA, were amplified and sequenced. Ixodes kaiseri had nine unique cox1 haplotypes, which showed 99.2-100% sequence identity, whereas I. canisuga and I. hexagonus had eleven and five cox1 haplotypes, respectively, with 99.5-100% sequence identity. The distribution of cox1 haplotypes reflected a geographical pattern. Pholeoixodes spp. ticks had fewer 16S rRNA haplotypes, with a lower degree of intraspecific divergence (99.5-100% sequence identity) and no geographical clustering. Phylogenetic analyses were in agreement with morphology: I. kaiseri and I. hexagonus (with the similar shape of the anterior surface of basis capituli) were genetically more closely related to each other than to I. canisuga. Phylogenetic analyses also showed that the subgenus Eschatocephalus (bat ticks) clustered within the subgenus Pholeoixodes. Conclusions: A simple, illustrated identification key is provided for female Pholeoixodes ticks of carnivores (including I. hexagonus and I. rugicollis) to prevent future misidentification of these species. It is also shown that I. kaiseri is more widespread in Europe than previously thought. Phylogenetic analyses suggest that the subgenus Pholeoixodes is not monophyletic: either the subgenus Eschatocephalus should be included in Pholeoixodes, or the latter subgenus should be divided, which is a task for future studies.

Background In Europe, the red fox (Vulpes vulpes) is the main definitive host of Echinococcus multilocularis, the aetiological agent of a severe disease in humans called alveolar echinococcosis. The distribution of this zoonotic parasite among the fox population is remarkably aggregated with few heavily infected animals harbouring much of the parasite burdens and being responsible for most of the environmental parasitic egg contamination. Important research questions explored were: (i) spatial differences in parasite infection pressure related to the level of urbanization; (ii) temporal differences in parasite infection pressure in relation to time of the year; (iii) is herd immunity or an age-dependent infection pressure responsible for the observed parasite abundance; (iv) assuming E. multilocularis infection is a clumped process, how many parasites results from a regular infection insult. Methods By developing and comparing different transmission models we characterised the spatio-temporal variation of the infection pressure, in terms of numbers of parasites that foxes acquired after exposure per unit time, in foxes in Zurich (Switzerland). These included the variations in infection pressure with age of fox and season and the possible regulating effect of herd immunity on parasite abundance. Results The model fitting best to the observed data supported the existence of spatial and seasonal differences in infection pressure and the absence of parasite-induced host immunity. The periodic infection pressure had different amplitudes across urbanization zones with higher peaks during autumn and winter. In addition, the model indicated the existence of variations in infection pressure among age groups in foxes from the periurban zone. Conclusions These heterogeneities in infection exposure have strong implications for the implementation of targeted control interventions to lower the intensity of environmental contamination with parasite eggs and, ultimately, the infection risk to humans. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1951-1) contains supplementary material, which is available to authorized users.

Background Increasing numbers of genetic variants are being recognized among piroplasms, but the precise taxonomical status, the tick vector and the geographical range of several species or genotypes are still unknown. Bovine piroplasmosis was reported to re-emerge in north-east Hungary. Because Theileria-infection was newly diagnosed in one cattle herd in the same region of the country, the aim of this study was to molecularly identify the relevant agent, to find its local vector tick species, and to examine the range of Babesia/Theileria spp. of ruminants in Haemaphysalis sp. ticks collected previously in Hungary. Findings Blood samples were drawn on two occasions from 90 dairy cattle in northern Hungary, and ticks were collected on their pastures. In addition, questing ticks (315 Haemaphysalis inermis, 259 H. concinna and 22 H. punctata), which originated mainly in the same region of the country from 2007, were included in the study. DNA was extracted from these samples, followed by molecular analysis for piroplasms. In the cattle Theileria orientalis was identified, with 100 % sequence homology to isolates from Japan, China, South-Africa and Australia. Based on GenBank data this genotype has not been previously reported in Europe. The prevalence of infection in the herd remained almost constant in the main tick season, suggesting exposure in previous years. Retrospective analysis of ticks revealed the presence of Babesia crassa in H. inermis, for the first time in Europe and in this tick species. On the other hand, H. concinna carried five different piroplasms, including B. motasi that was also newly detected in Central-Eastern Europe and in this tick species; whereas H. punctata harboured Theileria sp. OT3, hitherto known to occur in the Mediterranean region. Conclusions Results of this study broaden the range of piroplasms that are infective for ruminants in Central-Eastern Europe. Although bovine babesiosis and theileriosis was known to occur in Hungary, molecular evidence is provided here for the first time on the presence of Babesia and/or Theileria spp. of sheep, goats and cervids in Hungary.

International audience; Background: Rapid, accurate and high-throughput identification of vector arthropods is of paramount importance in surveillance programmes that are becoming more common due to the changing geographic occurrence and extent of many arthropod-borne diseases. Protein profiling by MALDI-TOF mass spectrometry fulfils these requirements for identification, and reference databases have recently been established for several vector taxa, mostly with specimens from laboratory colonies.Methods: We established and validated a reference database containing 20 phlebotomine sand fly (Diptera: Psychodidae, Phlebotominae) species by using specimens from colonies or field-collections that had been stored for various periods of time.Results: Identical biomarker mass patterns ('superspectra') were obtained with colony-or field-derived specimens of the same species. In the validation study, high quality spectra (i.e. more than 30 evaluable masses) were obtained with all fresh insects from colonies, and with 55/59 insects deep-frozen (liquid nitrogen/-80°C) for up to 25 years. In contrast, only 36/52 specimens stored in ethanol could be identified. This resulted in an overall sensitivity of 87 % (140/161); specificity was 100 %. Duration of storage impaired data counts in the high mass range, and thus cluster analyses of closely related specimens might reflect their storage conditions rather than phenotypic distinctness. A major drawback of MALDI-TOF MS is the restricted availability of in-house databases and the fact that mass spectrometers from 2 companies (Bruker, Shimadzu) are widely being used. We have analysed fingerprints of phlebotomine sand flies obtained by automatic routine procedure on a Bruker instrument by using our database and the software established on a Shimadzu system. The sensitivity with 312 specimens from 8 sand fly species from laboratory colonies when evaluating only high quality spectra was 98.3 %; the specificity was 100 %. The corresponding diagnostic values with 55 field-collected specimens from 4 species were 94.7 % and 97.4 %, respectively.Conclusions: A centralized high-quality database (created by expert taxonomists and experienced users of mass spectrometers) that is easily amenable to customer-oriented identification services is a highly desirable resource. As shown in the present work, spectra obtained from different specimens with different instruments can be analysed using a centralized database, which should be available in the near future via an online platform in a cost-efficient manner.

Background Dirofilaria immitis and Angiostrongylus vasorum are both important potentially fatal canine nematodes with overlapping endemic areas, especially in Europe. The preadult and adult stages of both species are living in the Arteria pulmonalis and the right heart, and diagnostically detectable circulating parasite antigens have been demonstrated for both species. For the detection of D. immitis infections, a variety of commercial tests have been developed, however, they have not been evaluated for cross-reactions against circulating antigens of A. vasorum. Methods In this study, potential cross-reactions of sera from 16 dogs, which were experimentally infected with A. vasorum and which had circulating antigens as confirmed by a species-specific ELISA, were evaluated for the detection of A. vasorum antigen in six commercially available D. immitis test kits. Results In three fast tests (Witness® Dirofilaria, SensPERT® Canine Heartworm, SNAP® 4Dx® Plus), all sera were negative. One fast membrane ELISA (SNAP® HTWM RT Test) was positive with four sera (25%), and one serum delivered a non-valid result twice. In the PetChek® HTWM PF Test, depending on the interpretation protocol, 5 or 8 dogs (31.2 – 50%) were positive. With the DiroCHEK®-ELISA, a single A. vasorum-infected dog (6.2%) tested positive. Conclusions Due to potential cross-reactions with A. vasorum in commercially available test kits for the detection of D. immitis antigen, the simultaneous use of highly specific diagnostic methods for the differentiation of these two canine heart worms is recommended.

Background Aedes (Stegomyia) albopictus is currently one of the most notorious globally invasive mosquito species. Its medical importance is well documented, and its fast expansion throughout most continents is being monitored with concern. It is generally assumed that its expansion through the Western Pacific island countries has not progressed since its establishment in Fiji in 1989. However, the current status of Ae. albopictus in the Pacific region is largely unknown. Findings According to data from the literature and our own observations, Ae. albopictus is currently present in the following countries of the southern Pacific region: Papua New Guinea, Solomon Islands, Fiji, and the Kingdom of Tonga, where it was first detected in July 2011. It is absent from New Caledonia and French Polynesia where routine entomological surveillance is carried out, and was not detected during entomological work in 2007, either on the Cook Islands or on the Wallis and Futuna Islands. The species was not reported from American Samoa in 2004, but it is mentioned as probably present in Vanuatu. This is the first report of Ae. albopictus in Tonga. Conclusions The introduction and establishment of Ae. albopictus in Tonga was expected due to the geographical proximity of this country to Fiji where the species is strongly established. The pathway of introduction is unknown. The expansion of Ae. albopictus in the Pacific region poses an increasing threat to public health given the role this mosquito plays as primary vector of emerging infectious diseases such as Chikungunya fever.

Background Culicoides biting midges are incriminated as biological vectors of a number of viruses, e.g. bluetongue virus. In order to define vector-free periods/areas and to assess the vectorial role of the various Culicoides species, a comprehensive knowledge on their spatio-temporal occurrence is required. Methods Biting midges were monitored on farm sites with livestock in the defined climatic regions, including high altitudes, of Switzerland by overnight trapping at 12 locations once a week over three years using UV-light traps. Based on morphological features, they were separated into three groups (i.e. Obsoletus, Pulicaris, other Culicoides spp.), and identification to the species level was achieved by protein profiling using MALDI-TOF mass spectrometry. Results Around 550,000 biting midges in total were collected, revealing a dominance (82 to 99%) of the Obsoletus group species up to an altitude of 1,200 m and of the Pulicaris group species above 1,500 m (85% at the highest trapping site at 2,130 m). The maximum number of midges collected in a summer night (756 to 19,682) as well as the total number of midges caught over three years (from 6,933 to 149,439) varied highly among the sites, whereas the annual variation in total midge abundance at the locations was statistically insignificant. MALDI-TOF MS of 100 randomly selected individual biting midges per trapping site yielded high quality spectra for 1,187 of the 1,200 (98.9%) specimens of which 1,173 could be assigned to one of the 15 Culicoides species for which biomarker mass sets are available in the reference database. Conclusions There are no biting midge-free zones in all of the agriculturally utilized areas (including alpine summer pastures) of Switzerland. Annual variations of midge numbers at the sampled locations were low, indicating that monitoring of midges should preferably be done by investigating a large number of sites for one season instead of few locations for extended periods of time. High throughput species identification of midges by MALDI-TOF MS is feasible, and this technique adds to other recently developed methods for the identification of midges (PCRs in various formats, interactive identification keys), facilitating epidemiological and biological in-depth studies of these important insects.

Background Over the last few decades, canine and feline thelaziosis caused by Thelazia callipaeda eye worms has gained the attention of the veterinary community due to the spread of this ocular infestation in geographical areas previously regarded as non endemic. The therapeutic efficacy of milbemycin oxime/praziquantel tablets (Milbemax®) against T. callipaeda was tested in naturally infested dogs and cats. Methods From January 2009 to July 2011 a placebo controlled and randomized field study was conducted in T. callipaeda endemic areas of Switzerland (CH) and Italy (ITA) involving client-owned animals. Dogs (n = 56) and cats (n = 31) were physically examined at enrolment Day 0 (D0) and twice afterwards (D7 and D14). Infested animals were orally treated with Milbemax® or with placebo tablets on D0 and, if an animal was found still infested with T. callipaeda, also on D7. On D14 nematodes were flushed from the conjunctiva, identified and counted. Results Out of 56 dogs, 43 were included in the statistical analysis, whereas 13 were excluded because the products under investigation were not administered with food, as required by the label. On D7 and D14, 72.7% and 90.9% of treated dogs were eye worm free, whereas in the placebo group 95.2% and 76.2% still harbored nematodes, resulting in a mean percentage worm count reduction for the Milbemax® group of 86.1% and 96.8%, respectively. Both results were significantly higher (p = 0.0001) than the placebo group. Out of the 31 cats included in the study at D7 and D14, 53.3% and 73.3% treated with Milbemax® were free of T. callipaeda, while 81.3% and 73.3 in the placebo group were still harbouring eye worms, resulting in a mean percentage worm count reduction for the treated group of 62.2% and 80.0%, respectively. Both results were significantly higher (p = 0.0106 and p = 0.0043) than the placebo group. Conclusions The commercial formulation of milbemycin oxime at the minimal dose of 0.5 mg/kg and 2 mg/k in dogs and cats, respectively, showed a high therapeutic efficacy in curing T. callipaeda infestations. The advantages of an oral application are additionally increased by the large spectrum of activity of praziquantel and milbemycin oxime against Cestodes and Nematodes infesting dogs and cats.

Background Besnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries. Findings Clinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses. Conclusions This is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region represents a further example, when human activity (i.e. cattle trading) is the main factor involved in the geographical spread of an infectious disease.

Altogether 2004 Ixodes ricinus ticks, from 37 places in Hungary, were analysed in pools with a recently developed multiplex real-time PCR for the presence of Candidatus Neoehrlichia mikurensis and for other representatives of the genus. Ca. Neoehrlichia mikurensis was identified in nine sampling sites, indicating three separated endemic regions along the borders of Hungary. In addition, results of samples from seven places (except for the western part of the country) were positive in the genus-specific (Ca. Neoehrlichia sp.) PCR, but were negative for Ca. Neoehrlichia mikurensis.

Background The worldwide distributed hematophagous poultry red mite Dermanyssus gallinae (De Geer, 1778) is one of the most important pests of poultry. Even though 35 acaricide compounds are available, control of D. gallinae remains difficult due to acaricide resistances as well as food safety regulations. The current study was carried out to identify putative excretory/secretory (pES) proteins of D. gallinae since these proteins play an important role in the host-parasite interaction and therefore represent potential targets for the development of novel intervention strategies. Additionally, putative transmembrane proteins (pTM) of D. gallinae were analyzed as representatives of this protein group also serve as promising targets for new control strategies. Methods D. gallinae pES and pTM protein prediction was based on putative protein sequences of whole transcriptome data which was parsed to different bioinformatical servers (SignalP, SecretomeP, TMHMM and TargetP). Subsequently, pES and pTM protein sequences were functionally annotated by different computational tools. Results Computational analysis of the D. gallinae proteins identified 3,091 pES (5.6%) and 7,361 pTM proteins (13.4%). A significant proportion of pES proteins are considered to be involved in blood feeding and digestion such as salivary proteins, proteases, lipases and carbohydrases. The cysteine proteases cathepsin D and L as well as legumain, enzymes that cleave hemoglobin during blood digestion of the near related ticks, represented 6 of the top-30 BLASTP matches of the poultry red mite’s secretome. Identified pTM proteins may be involved in many important biological processes including cell signaling, transport of membrane-impermeable molecules and cell recognition. Ninjurin-like proteins, whose functions in mites are still unknown, represent the most frequently occurring pTM. Conclusion The current study is the first providing a mite’s secretome as well as transmembranome and provides valuable insights into D. gallinae pES and pTM proteins operating in different metabolic pathways. Identifying a variety of molecules putatively involved in blood feeding may significantly contribute to the development of new therapeutic targets or vaccines against this poultry pest., Parasites & Vectors, 6 (1), ISSN:1756-3305

Background The mosquito-borne filarial nematodes Dirofilaria immitis and Dirofilaria repens primarily affect dogs but also cats, causing heartworm disease or subcutaneous dirofilariosis, respectively, and both may also cause zoonotic diseases in humans. Several mosquito species have been reported as competent vectors for these nematodes, but no data are available for the invasive mosquito species Aedes japonicus (Theobald, 1901). The objective of this study was to describe the development of both D. immitis and D. repens under standardised experimental laboratory conditions in mosquitoes. Methods For this purpose, both a laboratory strain and field-collected individuals of the invasive mosquito species Ae. japonicus and, for comparative purposes, a laboratory strain of Aedes geniculatus, a rare indigenous species sharing habitats with Ae. japonicus, and of the tropical species Aedes aegypti were used. Anticoagulated microfilariaemic blood was fed at a density of 3000 mf/ml to mosquitoes with a hemotek system. Blood-fed mosquitoes were incubated at 27 °C and 85% relative humidity, and specimens were dissected under the microscope at pre-set time points to observe developmental stages of both Dirofilaria species. Additionally, real-time PCRs were carried out in some microscopically negative samples to determine the infection rates. Results In field-collected Ae. japonicus infectious L3 larvae of both D. immitis and D. repens developed, rendering this mosquito species an efficient vector for both filarial species. Additionally, Ae. geniculatus was shown to be an equally efficient vector for both filarial species. Aedes japonicus mosquitoes from a laboratory colony were refractory to D. immitis but susceptible to D. repens, whereas Ae. aegypti was refractory to both filarial species. Conclusions To our knowledge, Aedes japonicus was for the first time shown to be an efficient vector for both D. immitis and D. repens, indicating that this invasive and locally highly abundant species may contribute to a transmission of filarial worms. The data emphasize the necessity to perform vector competence studies with local mosquito populations as basis for risk assessments. We further demonstrated that detection of filarial DNA in a mosquito species alone does not allow to draw reliable conclusions with regard to its vector competence.

Background Small mammals are crucial for the life history of ixodid ticks, but their role and importance in the transmission cycle of tick-borne pathogens is mostly unknown. Candidatus Neoehrlichia mikurensis (CNM) and Anaplasma phagocytophilum are both tick-borne pathogens, and rodents are discussed to serve as main reservoir hosts for CNM but not for the latter especially in Germany. Analysing the prevalence of both pathogens in small mammals and their ticks in endemic regions may help to elucidate possible transmission paths in small mammal populations and between small mammals and ticks. Methods In 2012 and 2013, small mammals were trapped at three different sites in Germany. DNA was extracted from different small mammal tissues, from rodent neonates, foetuses and from questing and attached ticks. DNA samples were tested for CNM and A. phagocytophilum by real-time PCR. Samples positive for A. phagocytophilum were further characterized at the 16S rRNA gene locus. Results CNM was detected in 28.6% of small mammals and in 2.2% of questing and 3.8% of attached ticks. Altogether 33 positive ticks were attached to 17 different hosts, while positive ticks per host ranged between one and seven. The prevalences for this pathogen differed significantly within small mammal populations comparing sites (χ2: 13.3987; p: 0.0004) and between sexes. Male rodents had an approximately two times higher chance of infection than females (OR: 1.9652; 95% CI: 1.32-2.92). The prevalence for CNM was 31.8% (95% CI: 22-44) in rodent foetuses and neonates (23 of 67) from positive dams, and 60% (95% CI: 35.7-80.25) of positive gravid or recently parturient rodents (9 out of 15) had at least one positive foetus or neonate. Anaplasma phagocytophilum was detected at a low percentage in rodents (0-5.6%) and host-attached ticks (0.5-2.9%) with no significant differences between rodent species. However, attached nymphs were significantly more often infected than attached larvae (χ2: 25.091; p

Background Three laboratory studies were conducted to assess the repellent and insecticidal efficacy of a combination of fipronil and permethrin (Frontline Tri- Act®/Frontect®) against three mosquito species (Aedes albopictus, Aedes aegypti and Culex pipiens) on dogs. Methods In each study, 16 healthy adult dogs were allocated to two groups. Eight dogs were treated with the new topical spot-on combination of fipronil and permethrin on Day 0 and the other eight dogs served as untreated controls. Each dog was exposed to mosquitoes on Days 1, 7, 14, 21 and 28 (and also on Day 35 in the A. aegypti study). After a 1-h exposure period, all mosquitoes were counted and categorized as live or dead and fed or non-fed. Live mosquitoes were kept in an insectary and observed for mortality counts 4, 24 and 48 h post-exposure (PE) for Aedes spp. and 24 and 48 h PE for C. pipiens. Repellency and insecticidal efficacies were defined as the percent reduction in the number of fed and live mosquitoes, respectively, in the treated group as compared to the untreated control group. Results Repellency against A. albopictus was ≥93.4% through Day 21 and 86.9% on Day 28. It was ≥91.0% through Day 35 against A. aegypti and ≥90.4% through Day 28 against C. pipiens. Insecticidal efficacy against A. albopictus was ≥97.1% at 24 h PE from Day 7 to Day 28. It was ≥98.0% for the first 3 weeks and still 75.7% on Day 35 against A. aegypti at 24 h PE. For C. pipiens, insecticidal efficacy ranged from 93.8% (Day 7) to 30.9% (Day 28) at 48 h PE. Conclusions A single topical administration of the combination of fipronil and permethrin provides repellency against mosquitoes on dogs for at least 4 weeks. The product may therefore significantly reduce the potential for the transmission of vector-borne pathogens through the inhibition of mosquito feeding, as well as the discomfort associated with mosquito bites. Moreover, mosquito mortality was induced by contact with the treated dogs, which could aid in the control of mosquitoes, and hence the control of mosquito-borne diseases, in the local vicinity of treated dogs.

Background Despite their close association with human dwellings, the role of synanthropic rodents in the epidemiology of vector-borne infections is seldom studied. The aim of the present study was to compensate for this lack of information, by the molecular investigation of vector-borne bacteria in peridomestic rodents and their ectoparasites. Findings Fifty-two rodents (mainly house mice and brown rats) were caught alive in buildings and checked for blood-sucking ectoparasites; followed by molecular analysis of these, together with spleen samples, for the presence of vector-borne agents. Haemoplasma infection was significantly more prevalent among brown rats, than among house mice. A novel haemoplasma genotype (with only 92-93% similarity to Candidatus Mycoplasma turicensis and M. coccoides in its 16S rRNA gene) was detected in a harvest mouse and a brown rat. Sporadic occurrence of Rickettsia helvetica, Anaplasma phagocytophilum, Borrelia burgdorferi s.l. and Bartonella sp. was also noted in rodents and/or their ectoparasites. Conclusions These results indicate that synanthropic rodents, although with low prevalence, may carry zoonotic and vector-borne pathogens indoors.