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Background Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe. Methods Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene. Results Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozoon canis was detected in samples of 178 animals (3 Artiodactyla, 173 Carnivora, 1 Eulipotyphia, 1 Lagomorpha), H. martis in 125 (3 Artiodactyla, 122 Carnivora), H. sciuri in 13 (all Rodentia), Hepatozoon sp. in 47 (among which Hepatozoon sp. Vole isolate, all Rodentia) and H. ayorgbor in 4 (all Rodentia). Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon (4.2%; 32/754), most often H. canis ( n  = 22). Conclusions Hepatozoon canis is most present in mammals (especially in Carnivora such as gray wolves and golden jackals) and ticks, followed by H. martis , which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H. canis in ticks other than R. sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon . Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs. Graphical Abstract

Background Hippoboscid flies (Diptera: Hippoboscidae), also known as louse flies or keds, are obligate blood-sucking ectoparasites of animals, and accidentally of humans. The potential role of hippoboscids as vectors of human and veterinary pathogens is being increasingly investigated, but the presence and distribution of infectious agents in louse flies is still unknown in parts of Europe. Here, we report the use of molecular genetics to detect and characterize vector-borne pathogens in hippoboscid flies infesting domestic and wild animals in Austria. Methods Louse flies were collected from naturally infested cattle ( n  = 25), sheep ( n  = 3), and red deer ( n  = 12) across Austria between 2015 and 2019. Individual insects were morphologically identified to species level and subjected to DNA extraction for molecular pathogen screening and barcoding. Genomic DNA from each louse fly was screened for Borrelia spp., Bartonella spp., Trypanosomatida, Anaplasmataceae, Filarioidea and Piroplasmida. Obtained sequences of Trypanosomatida and Bartonella spp. were further characterized by phylogenetic and haplotype networking analyses. Results A total of 282 hippoboscid flies corresponding to three species were identified: Hippobosca equina ( n  = 62) collected from cattle, Melophagus ovinus ( n  = 100) from sheep and Lipoptena cervi ( n  = 120) from red deer ( Cervus elaphus ). Molecular screening revealed pathogen DNA in 54.3% of hippoboscids, including infections with single (63.39%), two (30.71%) and up to three (5.90%) distinct pathogens in the same individual. Bartonella DNA was detected in 36.9% of the louse flies. Lipoptena cervi were infected with 10 distinct and previously unreported Bartonella sp. haplotypes, some closely associated with strains of zoonotic potential. DNA of trypanosomatids was identified in 34% of hippoboscids, including the first description of Trypanosoma sp. in H. equina . Anaplasmataceae DNA ( Wolbachia spp.) was detected only in M. ovinus (16%), while < 1% of the louse flies were positive for Borrelia spp. and Filarioidea. All hippoboscids were negative for Piroplasmida. Conclusions Molecular genetic screening confirmed the presence of several pathogens in hippoboscids infesting domestic and wild ruminants in Austria, including novel pathogen haplotypes of zoonotic potential (e.g. Bartonella spp.) and the first report of Trypanosoma sp. in H. equina , suggesting a potential role of this louse fly as vector of animal trypanosomatids. Experimental transmission studies and expanded monitoring of hippoboscid flies and hippoboscid-associated pathogens are warranted to clarify the competence of these ectoparasites as vectors of infectious agents in a One-Health context. Graphical Abstract

Objective The aim of the study was to determine the prevalence of antibodies against hepatitis E virus (HEV), tick‐borne encephalitis virus (TBEV), West Nile virus (WNV), Crimean–Congo haemorrhagic fever (CCHF) virus, rabies virus, Echinococcus spp., Brucella spp., as well as the colonisation with methicillin‐resistant Staphylococcus aureus (MRSA) among Austrian veterinarians in dependence on their field of occupation, age, gender and, if applicable, the vaccination status. Methods and results A total of 293 Austrian veterinary practitioners were included in the study. All study participants had to fill in a questionnaire regarding personal details, field of occupation and vaccination status against TBEV and rabies. Furthermore, a nasal swab for detection of colonisation with MRSA and a blood sample were taken. Antibodies against HEV, TBEV, WNV, CCHF virus and Echinococcus spp. were analysed by ELISA tests. Antibodies against Brucella spp. were measured by complement fixation test. Rabies antibodies were analysed by virus neutralisation test. Most study participants were vaccinated against rabies (93.2%) and TBEV (94.3%). Accordingly, a high prevalence of TBEV antibodies was found (95.9%). HEV antibodies were detected in 11.3% of the study participants, with a significantly higher seroprevalence in the oldest age group (23.3%). MRSA colonisation was age‐depended and significantly higher in study participants with occupational livestock exposure (10.3% vs. 2.2%). Hygienic measures such as hand washing and disinfection led to less MRSA colonisation. Antibodies against WNV were found in 3.1% of the study participants. No antibodies against Brucella spp., Echinococcus spp. and CCHF were detected. Conclusions Despite the limitations of the study design, the included veterinarians represent the Austrian average veterinarians regarding gender, age and field of occupation. The vaccination rate against TBEV and rabies was high. HEV seroprevalence was lower than in other studies, whereas MRSA colonisation rates and WNV seroprevalence were comparable to results of other European studies. • Vaccination rates against rabies and TBEV and accordingly the respective seroprevalence rates exceed 90% among Austrian veterinary practitioners. • The prevalence of antibodies against HEV and the colonisation with MRSA increases with the age of the study participants from 6.2%/6.2% in the youngest age group to 23.3%/21.9% in the oldest group. • MRSA colonisation in Austrian veterinarians is significantly higher in those with livestock occupational exposure (10.3% vs. 2.2%) and is lower among those that wash and/or disinfect their hands (5.2% vs. 66.3%).

BACKGROUND: Both Dirofilaria repens and recently D. immitis are known to be endemic in Hungary. As one of several recent cases, the fatal case of a dog infested with D. immitis in Szeged, Southern Hungary, received attention from the media. Hence it was decided to catch mosquitoes in the garden where the dog lived to screen for filarioid helminths and Plasmodium spp. using molecular tools. METHODS: Mosquitoes were caught in Szeged, in the garden where the infected dog was kept, in July 2013 with M-360 electric mosquito traps and were stored in ethanol until further procedure. Female mosquitoes were classified to genus level by morphology. Each mosquito was homogenized and analyzed for filarioid helminths and avian malaria using standardized PCR techniques. Positive mosquito samples were further identified to species level by comparing a section of the mitochondrial COI gene to GenBank® entries. RESULTS: In this study, 267 blood-fed mosquitoes were caught in July 2013 in Szeged. Subsequent molecular screening revealed that not only D. immitis was present in the analyzed specimens but also DNA of D. repens, Setaria tundra and Plasmodium spp. was confirmed. CONCLUSIONS: The analysis of blood-fed mosquitoes for the diagnosis of Dirofilaria spp. and other mosquito-borne pathogens seems to be an adequate technique to evaluate if filarioid helminths are present in a certain area. Usually only unfed female mosquitoes are analyzed for epidemiological studies. However, blood-fed mosquitoes can only be used for screening if a pathogen is present because the role of the mosquito as vector cannot be classified (blood of bitten host). Furthermore, Setaria tundra was confirmed for the first time in Hungary.

Background Tick-borne infections resulting from regular tick infestation in dogs are a common veterinary health problem all over the world. The application of repellent and acaricidal agents to prevent transmission of pathogens is a major protection strategy and has been proven to be highly effective in several trials under laboratory and natural conditions in dogs. Despite such promising results, many dog owners still report tick infestation in their dogs although acaricidal agents are used. Information about the current infection status and changes of the infection status regarding tick-borne diseases (TBD) in dogs treated by the owner’s controlled acaricide application is lacking. Methods In this study 30 dogs were each treated with permethrin, fipronil + S-methoprene, or served as untreated controls. Application of the acaricide was performed by the owner who decided when and how often to use the spot on preparation. Over a period of 11 months, dogs were clinically examined and sampled for antibody responses against Babesia canis , Anaplasma phagocytophilum , Borrelia burgdorferi s. l., and TBE virus before the study started, 6 months later and at the end of the investigation period. Results The permethrin acaricide was applied on average 3.40 times within the examination period, whereas the fipronil + S-methoprene medication was applied 3.03 times. Approximately 2/3 of all dogs, independent of the group, had a positive immune response to one or more pathogens. Three dogs developed clinical symptoms of canine babesiosis, all other dogs remained healthy. Individual number of ticks per dog or number of infections per dog did not correlate with the application rate, and the number of ticks per dog did not influence the number of infections per dog. As owners did not apply the acaricides regularly no influence on the number of infections could be documented although the number of ticks was clearly reduced by the application of the spot-on drugs. Conclusions Clinical disease in dogs exposed to tick-borne pathogens is rare, although a humoral immune response reflecting infection is common. More educational training for dog owners is necessary to make the application of acaricides effective regarding the prevention of tick-borne diseases.

BACKGROUND: In Europe animal and human infections due to Dirofilaria repens are increasing. FINDINGS: In a nationwide screening for filarioid parasites in Austria, 7,632 mosquitoes were collected from June till October 2012 and divided into 437 pools according to same trapping date and sight and mosquito species. For the molecular detection, a real-time PCR approach was followed by conventional PCR. D. repens was detected in the villages Moerbisch and Rust, Burgenland in one Anopheles maculipennis group and one Anopheles algeriensis species pool, respectively. CONCLUSIONS: The geographical distribution of the two positive pools points to the invasion of D. repens from Eastern neighboring countries. The finding of D. repens in mosquito vectors suggests the occurrence of the causative agent for cutaneous dirofilariosis in Austria.

Information on mosquito-borne filarioid helminths in Austria is scarce, but recent discoveries of Dirofilaria repens indicate autochthonous distribution of this parasite in Eastern Austria. In the current xenomonitoring study, more than 48,000 mosquitoes were collected in Eastern Austria between 2013 and 2015, using different sampling techniques and storage conditions, and were analysed in pools with molecular tools for the presence of filarioid helminth DNA. Overall, DNA of D. repens, Setaria tundra, and two unknown filarioid helminths were documented in twenty mosquito pools within the mitochondrial cox1 gene (barcode region). These results indicate that S. tundra, with roe deer as definite hosts, is common in Eastern Austria, with most occurrences in floodplain mosquitoes (e.g., Aedes vexans). Moreover, DNA of D. repens was found in an Anopheles plumbeus mosquito close to the Slovakian border, indicating that D. repens is endemic in low prevalence in Eastern Austria. This study shows that xenomonitoring is an adequate tool to analyse the presence of filarioid helminths, but results are influenced by mosquito sampling techniques, storage conditions, and molecular protocols.

Tick-borne diseases pose a serious threat to human health in South-Eastern Europe, including Kosovo. While Crimean–Congo hemorrhagic fever (CCHF) is a well-known emerging infection in this area, there are no accurate data on Lyme borreliosis and tick-borne encephalitis (TBE). Therefore, we sampled and tested 795 ticks. Ixodes ricinus ( n  = 218), Dermacentor marginatus ( n  = 98), and Haemaphysalis spp. ( n  = 24) were collected from the environment by flagging (all from Kosovo), while Hyalomma marginatum ( n  = 199 from Kosovo, all from Kosovo) and Rhipicephalus bursa ( n  = 130, 126 from Albania) could be collected only by removal from animal pasture and domestic ruminants. Ticks were collected in the years 2014/2015 and tested for viral RNA of CCHF and TBE viruses, as well as for DNA of Borrelia burgdorferi sensu lato by real-time PCR. In Kosovo, nine ticks were positive for RNA of Crimean–Congo hemorrhagic fever virus and seven for DNA of B. burgdorferi s. l. None of the ticks tested positive for TBEV. CCHF virus was detected in one H. marginatum male specimen collected while feeding on grazing cattle from the Prizren region and in eight R. bursa specimens (five females and three males collected while feeding on grazing sheep and cattle) from the Prishtina region (Kosovo). B. burgdorferi s. l. was detected in seven questing ticks (four male and one female D. marginatus , two I. ricinus one female and one male) from the Mitrovica region (Kosovo). Our study confirmed that CCHF virus is circulating in Kosovo mainly in H. marginatum and R. bursa in the central areas of the country. B. burgdorferi s. l. was found in its major European host tick, I. ricinus , but also in D. marginatus , in the north of the Kosovo. In order to prevent the spread of these diseases and better control of the tick-borne infections, an improved vector surveillance and testing of ticks for the presence of pathogens needs to be established.

Red fox (Vulpes vulpes) is the most abundant wild canid species in Austria, and it is a well-known carrier of many pathogens of medical and veterinary concern. The main aim of the present study was to investigate the occurrence and diversity of protozoan, bacterial and filarial parasites transmitted by blood-feeding arthropods in a red fox population in western Austria. Blood (n = 351) and spleen (n = 506) samples from foxes were examined by PCR and sequencing and the following pathogens were identified: Babesia canis, Babesia cf. microti (syn. Theileria annae), Hepatozoon canis, Anaplasma phagocytophilum, Candidatus Neoehrlichia sp. and Bartonella rochalimae. Blood was shown to be more suitable for detection of Babesia cf. microti, whilst the spleen tissue was better for detection of H. canis than blood. Moreover, extremely low genetic variability of H. canis and its relatively low prevalence rate observed in this study may suggest that the parasite has only recently been introduced in the sampled area. Furthermore, the data presented here demonstrates, for the first time, the possible vertical transmission of H. canis from an infected vixen to the offspring, and this could explain the very high prevalence in areas considered free of its main tick vector(s).

BACKGROUND: Effective control of tick infestation and pathogen transmission requires profound knowledge of tick biology in view of their vector function. The particular time of the year when the different tick species start to quest and the favoured sites on the canine host are of major interest. The efficacy of acaricides/repellents to control ticks in the field requires observation. METHODS: To address these issues, 90 dogs, grouped in “untreated”, “acaricide/repellent” (permethrin) and “acaricide only” (fipronil) animals and subjected to tick infestation under natural conditions in Burgenland (Eastern Austria), were examined. The number and species of ticks occurring during and outside the protection time was evaluated during a period of 11 months and the biting location on the dogs’ skin was recorded. RESULTS: Of the 700 ticks collected, the most common species in that particular walking area was Ixodes ricinus, followed by Dermacentor reticulatus and Haemaphysalis concinna. Regarding the on-host activity, D. reticulatus displayed more infestations in early spring and late autumn, whereas I. ricinus occurred almost one month later in spring and one month earlier in autumn. H. concinna followed a monophasic pattern of activity with a peak in summer. The preferred feeding sites of the ticks on the dogs were on the head, neck, shoulder and chest. This distribution over the dog’s body was not influenced by the use of the drugs, although on the whole fewer ticks (22.5% of all ticks) were found during the protection time. Interestingly, differences occurred with the use of drugs compared to non-protected dogs with regard to the infestation over the year. Acaricide-treated dogs displayed a higher prevalence in April, May and September, whereas dogs of the acaricide/repellent group showed a higher infestation in March, July, October and November. CONCLUSION: The different tick species display different on-dog activity peaks over the year, during which particular canine diseases can be expected and predicted, considering the specific incubation times for each pathogen. The tick species occurring in this study do not seem to choose particular sites on the dogs. Their arrival place seems to represent the attachment and consequently the feeding sites. The use of acaricides leads to a significantly (p<0.01) lower number of infesting ticks but no change of the distribution pattern on the dogs was observed.