Gábor Majoros

Investigation of the Ecology of Francisella tularensis During an Inter-Epizootic Period

A 1-year study of the ecological cycle of Francisella tularensis was performed in an enzootic area during an inter-epizootic period. The study was based on multiple sampling of all major constituents of the disease cycle. Seroprevalence of tularemia in the European brown hare (Lepus europaeus) population was 5.1% (10/197) with low antibody titers (1/10 and 1/20), and F. tularensis ssp. holarctica was isolated from four hares. F. tularensis was not detected in the 38 common voles (Microtus arvalis), 110 yellow-necked mice (Apodemus flavicollis), or 15 stripped field mice (Apodemus agrarius) trapped during the study, or the by-catch of 8 Eurasian pygmy shrews (Sorex minutus) or 6 common shrews (Sorex araneus). A total of 1106 Ixodes ricinus and 476 Haemaphysalis concinna ticks were collected from vegetation, and 404 I. ricinus, 28 H. concinna ticks, and 15 Ctenophtalmus assimilis and 10 Nosopsyllus fasciatus fleas were combed off small mammals. One H. concinna female and one nymph collected from the vegetation was found infected with F. tularensis ssp. holarctica by TaqMan polymerase chain reaction, thus resulting a 0.42% (2/476) prevalence. F. tularensis-specific DNA was not detected in environmental water samples, and the examined 100 sheep, 50 cows, and 50 buffalos grazed at the study area were all seronegative. During inter-epizootic periods, F. tularensis ssp. holarctica seems to persist only in the European brown hare--H. concinna cycle at the studied habitat. H. concinna may not serve exclusively as an arthropod vector, but it may also harbor bacteria for 3-4 years through multiple life stages and act as an important reservoir of F. tularensis. Rodent species probably do not serve as true reservoir hosts of tularemia.

Ticks and the city: Ectoparasites of the Northern white-breasted hedgehog (Erinaceus roumanicus) in an urban park

The European hedgehog (Erinaceus europaeus) is known to host several ectoparasites and also tick-borne pathogens, but there is scant information on its eastern relative, the Northern white-breasted hedgehog (Erinaceus roumanicus). We have studied an urban population of E. roumanicus in a city park of central Budapest, Hungary, for 2 years to investigate their tick and flea species. A total of 5063 ticks and 818 fleas were collected from 247 hedgehogs (including 46 recaptures). Ectoparasite prevalence and intensity differed significantly (p<0.001) between the 2 study years attributable to the enhanced tick removal rate due to anaesthesia used in the second year. The most common tick species was Ixodes ricinus (93.7%) followed by unidentified Ixodes larvae (5%). Only 57 hedgehog ticks (I. hexagonus) were removed from 22 hedgehogs. One I. acuminatus and one Hyalomma marginatum nymph were also collected. Mean intensity of tick infestation was 26.5 (range: 0-155 ticks/host) and mean intensity of flea infestation was 6.6 (range: 0-78 fleas/host). Most fleas (99.4%) collected were hedgehog fleas (Archaeopsylla erinacei), dog fleas (Ctenocephalides canis) were found on 2 hedgehogs. Hyalomma marginatum has previously not been found in Hungary, and I. acuminatus was only reported sporadically before. The large number of ectoparasites and the 2 imported tick species may thus survive in close proximity to humans if hedgehogs are present. This calls attention to the risk of possible tick-borne human infections that urban hedgehogs can pose.

Hard ticks infesting dogs in Hungary and their infection with Babesia and Borrelia species

A survey was carried out in Hungary to investigate the occurrence of hard tick species (Acari: Ixodidae) collected from dogs and Borrelia and Babesia spp. detected in them. In total, 1,424 ticks were removed from 477 dogs appearing for clinical consultation in veterinary practices and clinics countrywide. Ixodes ricinus and Dermacentor reticulatus were the most common species occurring in most of the studied areas. Females of these two species were selected for molecular analyses. One to twelve specimens were used in each sample for DNA extraction. Polymerase chain reactions were performed with BSLF/BSL-R primers for detecting Borrelia spp. in I. ricinus and with PIRO-A1/PIRO-B primers to amplify Babesia spp. DNA in D. reticulatus. Randomly selected PCR products were sequenced to identify the pathogens’ species or subspecies. DNA of Borrelia spp. could be detected in six (5.6%) from 108 I. ricinus samples and 43 (29.9%) from 144 D. reticulatus samples were PCR-positive for Babesia spp. Sequencing revealed the highest similarity with Borrelia afzelii, Borrelia garinii and Babesia canis canis, respectively. Babesia and Borrelia spp. were identified in ticks with molecular methods for the first time in Hungary, and a high prevalence of B. canis canis in D. reticulatus females collected from dogs was detected.

Dermacentor reticulatus: a vector on the rise

Dermacentor reticulatus is a hard tick species with extraordinary biological features. It has a high reproduction rate, a rapid developmental cycle, and is also able to overcome years of unfavourable conditions. Dermacentor reticulatus can survive under water for several months and is cold-hardy even compared to other tick species. It has a wide host range: over 60 different wild and domesticated hosts are known for the three active developmental stages. Its high adaptiveness gives an edge to this tick species as shown by new data on the emergence and establishment of D. reticulatus populations throughout Europe. The tick has been the research focus of a growing number of scientists, physicians and veterinarians. Within the Web of Science database, more than a fifth of the over 700 items published on this species between 1897 and 2015 appeared in the last three years (2013–2015). Here we attempt to synthesize current knowledge on the systematics, ecology, geographical distribution and recent spread of the species and to highlight the great spectrum of possible veterinary and public health threats it poses. Canine babesiosis caused by Babesia canis is a severe leading canine vector-borne disease in many endemic areas. Although less frequently than Ixodes ricinus, D. reticulatus adults bite humans and transmit several Rickettsia spp., Omsk haemorrhagic fever virus or Tick-borne encephalitis virus. We have not solely collected and reviewed the latest and fundamental scientific papers available in primary databases but also widened our scope to books, theses, conference papers and specialists colleagues’ experience where needed. Besides the dominant literature available in English, we also tried to access scientific literature in German, Russian and eastern European languages as well. We hope to inspire future research projects that are necessary to understand the basic life-cycle and ecology of this vector in order to understand and prevent disease threats. We conclude that although great strides have been made in our knowledge of the eco-epidemiology of this species, several gaps still need to be filled with basic research, targeting possible reservoir and vector roles and the key factors resulting in the observed geographical spread of D. reticulatus.

Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in natural rodent and tick communities in Southern Hungary

The aim of this study was to investigate the natural cycle of the new human pathogenic bacteria Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum in Southern Hungary. We collected rodents with live-traps (2010-2013) and questing ticks with flagging in 2012. Small mammals were euthanized, tissue samples were collected and all the ectoparasites were removed and stored in 70% alcohol. We found relatively low overall prevalence of tick infestation (8%). Samples were analysed for A. phagocytophilum and Candidatus N. mikurensis with multiplex quantitative real-time PCR targeting a part of major surface protein 2 (msp2) and the heat shock protein groEL genes, respectively. The overall prevalence in tissue samples was 6.6% (skin) and 5.1% (spleen) for A. phagocytophilum and 1.7% (skin) and 3.4% (spleen) for Candidatus N. mikurensis. Candidatus N. mikurensis was only detected in Apodemus flavicollis and Apodemus agrarius, while A. phagocytophilum was found in A. flavicollis, A. agrarius, Myodes glareolus, Microtus arvalis and Mus musculus samples. Prevalence of A. phagocytophilum in skin samples of A. flavicollis was significantly higher than prevalence of N. mikurensis (p<0.05). Among questing Ixodes ricinus ticks we found three (8.8%) individuals (female, male, nymph) infected with Candidatus N. mikurensis. Five (3.1%) questing ticks had A. phagocytophilum infection (one I. ricinus male, two Dermacentor reticulatus females and two Haemaphysalis concinna females). We found one I. ricinus nymph removed from a male A. flavicollis with A. phagocytophilum infection. Our study provides new data on the occurrence of these pathogens in rodent tissue samples, questing ticks and engorged ticks in Southern Hungary.

Autochtonous infection of dogs and slugs with Angiostrongylus vasorum in Hungary

On the course of a helminthological survey of the dogs of Baranya County, Hungary Angiostrongylus vasorum infection was detected in two asymptomatic dogs. Identification of the parasite was based on morphology of the first-stage larvae (L1) isolated from droppings, and successful experimental infection with first stage larvae to laboratory reared Discus rotundatus and Lissachatina fulica snails, in order to exclude species of the family Filaroididae that have similar larvae to A. vasorum. While angiostrongylosis is widespread among foxes, this is the first report of A. vasorum infection in housedog in Hungary. In gardens, where infected dogs were being kept 91 specimens of 6 species of limacid and arionid slugs were collected of which 5 specimens of Arion lusitanicus were found to carry larvae of A. vasorum. Dogs usually do not ingest such large slugs willingly. Frogs are known to act as paratenic hosts in the life cycle of A. vasorum. Since one of the infected dogs harboured also infection with the intestinal trematode Alaria alata, of which frogs certainly play the role of the second intermediate host, therefore it is assumed that in this case the dog became infected with A. vasorum by eating frogs.

Sarcocystis-infection of cattle in Hungary

BackgroundReports on Sarcocystis-infection of cattle are outdated or lacking in many European countries, including those in the Central-Eastern part of the continent. Therefore, to assess the prevalence of Sarcocystis spp. among bovids in Hungary, a countrywide survey was initiated. In addition, fulminant deaths of four cattle, that showed clinical signs and post mortem lesions resembling acute sarcocystiosis (“Dalmeny disease”), were investigated.MethodsDuring the countrywide survey individual heart and oesophagus samples were collected at slaughterhouses from 151 beef cattle and from 15 buffalo, kept in 31 places of Hungary. Analysis for Sarcocystis spp. was carried out with conventional PCRs for the 18S rDNA gene and gel electrophoresis, followed by sequencing of 36 strongly positive samples. Mortality cases were evaluated by histological, molecular, bacteriological and virological analyses of samples from various organs.ResultsAmong slaughtered cattle the rate of Sarcocystis-infection was 66%. S. cruzi was identified as the most prevalent species in aurochs-like breed, and the zoonotic S. hominis in Hungarian grey cattle. Concerning the sudden deaths of cattle, Sarcocystis-infection could not be demonstrated in organs showing haemorrhages, but S. cruzi cysts were present in the muscles. In one case “S. sinensis” was molecularly identified in the blood (indicating sarcocystaemia). Results of analyses for bacterial/viral pathogens were negative.ConclusionsS. cruzi appears to be the most prevalent Sarcocystis sp. in cattle in Hungary, followed by the zoonotic S. hominis. However, the rate of infection with both species was shown to differ between cattle breeds. The suspected role of Sarcocystis spp. as causative agents of the fatal cases could not be confirmed.

Studies on Coccidiosis of Calves in Hungarian Dairy Farms

In most Hungarian dairy farms clinical coccidiosis of calves had been misdiagnosed because of the lack of information on the presence of Eimeria sp. in local animals, causing inestimable animal health problems and economical losses. For this reason the first countrywide study on coccidiosis of calves was carried out in 86 dairy farms. Faecal samples were collected from 743 calves aged between two and four months old in 2005 and 2006. The number of animals enrolled per farm ranged between two and ten. During the qualitative ovoscopy, 100 oocysts per sample were examined for species identification. Eimeria oocysts were found in 245 (33%) samples. Of the seven species identified, the most prominent species were E. auburnensis (19.6%), E. ellipsoidalis (15.9%) and E. bovis (14.9%). Eimeriazuernii, E. cylindrica, E. pellita or E. subsphaerica oocysts were found in less than 20 samples each. Multiple infections with two or more species occurred in 83 (33.9%) calves. The oocysts of at least one of the two pathogenic species, E. bovis and/or E. zurnii, were found on every farm studied, which infected 118 (48.2%) animals, however, clinical coccidiosis was not observed. Coccidia of Eimeria were present in 74 (86.0%) out of 86 farms, occurring countrywide, where the number of Eimeria sp. ranged between one and six. Based on these preliminary results the authors emphasise that veterinarians and cattle owners should be familiar with clinical signs and the potential implications of cattle coccidiosis. Management procedures and application of anti-coccidial compound are needed for minimising the impact of coccidiosis.

Identification of Hepatozoon erhardovae Krampitz, 1964 from bank voles (Myodes glareolus) and fleas in Southern Hungary.

In order to investigate the prevalence and life cycle of apicomplexan parasites, small mammals were live-trapped with modified Sherman traps in Southern Hungary between 2010 and 2012. Altogether, 528 rodents (Apodemus flavicollis Melchior, 1834, Apodemus agrarius Pallas, 1771, Myodes glareolus Schreber, 1780, Microtus agrestis Linnaeus, 1761, Mus musculus Linnaeus, 1758 and Micromys minutus Pallas, 1771) were collected and four shrews (Sorex spp.) were by-catched. Captured animals belonging to non-protected species were euthanized, and spleen samples were preserved for histological and molecular analyses. During the examination of spleen smears, Hepatozoon parasites were observed in eight out of 48 bank voles (M. glareolus). DNA was isolated from altogether 221 spleen samples, and 18S rDNA was amplified using two different PCR protocols. The eight bank vole samples were positive with PCR, but none of the other M. glareolus spleen samples or any of the tissue samples from other species were found to be infected. Sequenced amplicons were very similar to Hepatozoon spp. detected in M. glareolus in Spain and Poland. Ectoparasites were collected from the small mammal carcasses and from the vegetation. Hepatozoon DNA was not found in the 181 ticks removed from the small mammals or in the 162 ticks collected with flagging, but was detected in all three flea species (4/43 Megabothris turbidus Rothschild, 1909, 3/10 Ctenophthalmus assimilis Taschenberg, 1880 and 7/78 Ctenophthalmus agyrtes Heller, 1896). Based on gamont morphology, vertebrate and arthropod host species and DNA sequences, the parasites in our study can be identified as Hepatozoon erhardovae.

A foreign invader or a reclusive native? DNA bar coding reveals a distinct European lineage of the zoonotic parasite Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum (Dutt and Srivastava, 1955))

Natural foci of Schistosoma turkestanicum (syn. Orientobilharzia turkestanicum) has been identified in the Gemenc Forest regions of Hungary utilising red deer as the definitive host. In order to identify the origins of this parasite in Europe standard DNA bar coding techniques were employed to sequence fragments of the cytochrome oxidase 1 (cox1) and the nuclear ribosomal internal transcribed region (ITS) from 10 individual adult male worms. Phylogenetic reconstruction using maximum likelihood phylogenetic reconstruction and haplotype networks of the cox1 showed all the worms to be of a distinct unique Hungarian lineage although some ITS haplotypes were shared with worms from populations in China and Iran. Molecular clock analysis suggests an early divergence event around 270,000years before present (YBP) between all S. turkestanicum populations giving rise to the Chinese, Iranian and Hungarian lineages. However, divergence of the sequences within the Hungarian population appears to have occurred approximately 63,000 YBP suggesting a long established population of S. turkestanicum in Europe. This suggests that the Hungarian population of S. turkestanicum has been native since the Ice Age and probably established itself during the last interglacial period as red deer moved into Europe from North Africa and the Middle East. This may also indicate that the parasite may have unknown populations established in several other countries in Eastern, Central and Southern Europe.