Sándor Szekeres

Circulation of four Anaplasma phagocytophilum ecotypes in Europe

BackgroundAnaplasma phagocytophilum is the etiological agent of granulocytic anaplasmosis in humans and animals. Wild animals and ticks play key roles in the enzootic cycles of the pathogen. Potential ecotypes of A. phagocytophilum have been characterized genetically, but their host range, zoonotic potential and transmission dynamics has only incompletely been resolved.MethodsThe presence of A. phagocytophilum DNA was determined in more than 6000 ixodid ticks collected from the vegetation and wildlife, in 289 tissue samples from wild and domestic animals, and 69 keds collected from deer, originating from various geographic locations in The Netherlands and Belgium. From the qPCR-positive lysates, a fragment of the groEL-gene was amplified and sequenced. Additional groEL sequences from ticks and animals from Europe were obtained from GenBank, and sequences from human cases were obtained through literature searches. Statistical analyses were performed to identify A. phagocytophilum ecotypes, to assess their host range and their zoonotic potential. The population dynamics of A. phagocytophilum ecotypes was investigated using population genetic analyses.ResultsDNA of A. phagocytophilum was present in all stages of questing and feeding Ixodes ricinus, feeding I. hexagonus, I. frontalis, I. trianguliceps, and deer keds, but was absent in questing I. arboricola and Dermacentor reticulatus. DNA of A. phagocytophilum was present in feeding ticks and tissues from many vertebrates, including roe deer, mouflon, red foxes, wild boar, sheep and hedgehogs but was rarely found in rodents and birds and was absent in badgers and lizards. Four geographically dispersed A. phagocytophilum ecotypes were identified, that had significantly different host ranges. All sequences from human cases belonged to only one of these ecotypes. Based on population genetic parameters, the potentially zoonotic ecotype showed significant expansion.ConclusionFour ecotypes of A. phagocytophilum with differential enzootic cycles were identified. So far, all human cases clustered in only one of these ecotypes. The zoonotic ecotype has the broadest range of wildlife hosts. The expansion of the zoonotic A. phagocytophilum ecotype indicates a recent increase of the acarological risk of exposure of humans and animals.

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.

Dicipivirus (family Picornaviridae) in wild Northern white-breasted hedgehog (Erinaceus roumanicus)

Using random amplification and high-throughput sequencing technology a novel picornavirus with dicistronic genome organization and genetically related to canine picodicistrovirus (genus Dicipivirus, family Picornaviridae) was identified and characterized in Northern white-breasted hedgehogs. Hedgehog dicipivirus (hedgehog/H14/2015/HUN, MF188967) was detected in 15 (75%) of 20 faecal specimens by RT-PCR with high viral loads (up to 8.2x108 genomic copies/ml faeces). Hedgehog dicipivirus RNA was also identified in blood, ear skin, abdominal muscle and liver tissues. While the general dicistronic genome organization of hedgehog/H14/2015/HUN is similar to canine picodicistrovirus (5’UTR-P1-IGR-P2/P3-3UTR) there are some unique genome characteristics within the untranslated regions, especially in the functional IRES elements. This study reports the putative second member of the genus Dicipivirus, in a novel host species.

Eco-epidemiology of Novel Bartonella Genotypes from Parasitic Flies of Insectivorous Bats

Bats are important zoonotic reservoirs for many pathogens worldwide. Although their highly specialized ectoparasites, bat flies (Diptera: Hippoboscoidea), can transmit Bartonella bacteria including human pathogens, their eco-epidemiology is unexplored. Here, we analyzed the prevalence and diversity of Bartonella strains sampled from 10 bat fly species from 14 European bat species. We found high prevalence of Bartonella spp. in most bat fly species with wide geographical distribution. Bat species explained most of the variance in Bartonella distribution with the highest prevalence of infected flies recorded in species living in dense groups exclusively in caves. Bat gender but not bat fly gender was also an important factor with the more mobile male bats giving more opportunity for the ectoparasites to access several host individuals. We detected high diversity of Bartonella strains (18 sequences, 7 genotypes, in 9 bat fly species) comparable with tropical assemblages of bat-bat fly association. Most genotypes are novel (15 out of 18 recorded strains have a similarity of 92–99%, with three sequences having 100% similarity to Bartonella spp. sequences deposited in GenBank) with currently unknown pathogenicity; however, 4 of these sequences are similar (up to 92% sequence similarity) to Bartonella spp. with known zoonotic potential. The high prevalence and diversity of Bartonella spp. suggests a long shared evolution of these bacteria with bat flies and bats providing excellent study targets for the eco-epidemiology of host-vector-pathogen cycles.

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.

Assessing bat droppings and predatory bird pellets for vector-borne bacteria: molecular evidence of bat-associated Neorickettsia sp. in Europe

In Europe, several species of bats, owls and kestrels exemplify highly urbanised, flying vertebrates, which may get close to humans or domestic animals. Bat droppings and bird pellets may have epidemiological, as well as diagnostic significance from the point of view of pathogens. In this work 221 bat faecal and 118 bird pellet samples were screened for a broad range of vector-borne bacteria using PCR-based methods. Rickettsia DNA was detected in 13 bat faecal DNA extracts, including the sequence of a rickettsial insect endosymbiont, a novel Rickettsia genotype and Rickettsia helvetica. Faecal samples of the pond bat (Myotis dasycneme) were positive for a Neorickettsia sp. and for haemoplasmas of the haemofelis group. In addition, two bird pellets (collected from a Long-eared Owl, Asio otus, and from a Common Kestrel, Falco tinnunculus) contained the DNA of a Rickettsia sp. and Anaplasma phagocytophilum, respectively. In both of these bird pellets the bones of Microtus arvalis were identified. All samples were negative for Borrelia burgdorferi s.l., Francisella tularensis, Coxiella burnetii and Chlamydiales. In conclusion, bats were shown to pass rickettsia and haemoplasma DNA in their faeces. Molecular evidence is provided for the presence of Neorickettsia sp. in bat faeces in Europe. In the evaluated regions bat faeces and owl/kestrel pellets do not appear to pose epidemiological risk from the point of view of F. tularensis, C. burnetii and Chlamydiales. Testing of bird pellets may provide an alternative approach to trapping for assessing the local occurrence of vector-borne bacteria in small mammals.

Prevalence of Borrelia miyamotoi and Borrelia burgdorferi sensu lato in questing ticks from a recreational coniferous forest of East Saxony, Germany

The hard tick Ixodes ricinus is the most important vector of tick-transmitted pathogens in Europe, frequently occurring in urban parks and greenbelts utilized for recreational activities. This species is the most common vector of the causative agents of Lyme borreliosis in Europe. Similarly, the species spreads Borrelia miyamotoi, causing a relapsing-fever like illness. A total of 1774 Ixodes ricinus (50 females, 68 males, 840 nymphs and 818 larvae) were collected with flagging between March and September 2014 in a coniferous forest patch in Niederkaina near the town of Bautzen in Saxony, Germany. To measure questing tick density a time-based density estimating method was utilized. From each month, a total of 100 adults and nymphal ticks and all larvae (pools of 10 individuals per tube/month) were selected for the molecular analyses. For simultaneous detection of B. burgdorferi s.l. and B. miyamotoi a duplex real-time PCR targeting the flaB locus was performed. Prevalence of B. burgdorferi s.l. was 9.4% (female: 6%, male: 2.9%, nymph: 12.2%, larva: 0%) and minimum prevalence of B. miyamotoi was 1.2% (female: 0%, male: 4.3%, nymph: 2.8%, larva: 0.1%) in the 714 samples with real-time polymerase chain reaction. A real-time PCR reaction was utilized first to target the histone-like protein gene (hbb) of B. burgdorferi s.l., a hemi-nested outer surface protein (ospA) gene conventional PCR was then performed followed by a restriction enzyme analysis to distinguish B. burgdorferi s.l. genospecies. Seven B. afzelii, one B. burgdorferi s.s., one B. bavariensis and four B. miyamotoi infections were confirmed. Prevalence of Lyme borreliosis spirochetes was significantly higher in nymphs than in adults (p<0.01, Fisher exact test) probably due to the diluting effect of the local roe deer population. Our data highlight the potential risk of human infection with the emerging pathogen B. miyamotoi within the study area.

High mitochondrial sequence divergence in synanthropic flea species (Insecta: Siphonaptera) from Europe and the Mediterranean

BackgroundAdult fleas are haematophagous ectoparasites of warm-blooded vertebrates, particularly mammals. Among them, the cat flea (Ctenocephalides felis) and the human flea (Pulex irritans) have high veterinary-medical significance, owing to their cosmopolitan distribution and role in the transmission of important vector-borne pathogens. While the taxonomy of Ct. felis has been investigated on a morphological basis during the past decades, its molecular-phylogenetic analyses have been only recently conducted. This study expands the knowledge on Ct. felis from hitherto less studied geographical regions, and includes representatives from additional flea families, less investigated with molecular approaches.MethodsFleas were collected in four countries of the Mediterranean Basin (Croatia, Italy, Malta and Israel), as well as in Hungary, from domestic and wild carnivores, rodents and humans. The DNA extracts of representative fleas (n = 148), belonging to ten species of eight genera, were used for PCR amplification of part of their cytochrome c oxidase subunits 1, 2 (cox1, cox2) and 18S rRNA genes, followed by sequencing and phylogenetic analyses.ResultsThe majority (65.6%) of Ct. felis felis cox2 sequences showed 99.4–100% similarity to each other (haplogroup A), whereas those from Malta and Israel had 98.1–98.7% sequence similarity (haplogroup B), and a third sequence from Israel (haplotype C) had as low as 96.3% sequence similarity in comparison with a reference sequence from group “A”. Except for the shape of the head, no consistent morphological differences (e.g. in chaetotaxy) were found between haplogroups “A” and “C”. Haplotypes of Ct. canis were genetically more homogenous, with 99.6–100% sequence similarity to each other. However, when P. irritans collected from humans was compared to those from three species of wild carnivores, these only had 96.6% cox2 similarity. The mouse flea, Leptopsylla segnis and the northern rat flea, Nosopsyllus fasciatus were both shown to have haplotypes with low intraspecific cox2 similarities (96.2 and 94.4%, respectively). Taken together, differences between mitochondrial lineages within four flea species exceeded that observed between two Chaetopsylla spp. (which had 97.3% cox2 similarity). The topologies of cox1 and cox2 phylogenetic trees were in line with relevant sequence comparisons. Conversely, 18S rRNA gene analyses only resolved differences above the species level.ConclusionsCtenocephalides felis felis, P. irritans, L. segnis and N. fasciatus were shown to have such a high level of mitochondrial gene heterogeneity, that the uniformity of these flea taxa should be reconsidered. Although the present results are limited (especially in the case of L. segnis and N. fasciatus), there appears to be no geographical or host restriction, which could explain the divergence of these genetic lineages.

Borrelia miyamotoi: egy újabb, humán patogén, kullancs által terjesztett, visszatérő lázat okozó baktérium

Absztrakt: A Borrelia miyamotoi-t 1995-ben fedeztek fel. Ez egy, emberben visszaterő lazat (relapsing fever, febris recurrens) okozo bakterium, amelyet az Ixodes ricinus fajcsoportba tartozo kullancsok terjesztenek. Ez a korokozo genetikailag, jarvanytanilag es az altala okozott korkep tekinteteben is kulonbozik a szinten kullancsok altal terjesztett Borrelia burgdorferi sensu lato (Lyme spirochaeta) bakteriumoktol. Eddig vilagszerte tobb mint 50 heveny lazas megbetegedesben szenvedő paciensből mutattak mar ki, ezenfelul harom tumoros betegben meningoencephalitist okozott ez a korokozo. A kulonboző előhelyeken talalhato kullancsok es gazdaik fertőzottsegenek merteke, eloszlasa es a fertőzes mechanizmusa nem tisztazott meg. A B. miyamotoi elsősorban lazat okoz, ami miatt mas, kullancsok altal terjesztett fertőzesekkel is osszeteveszthető. Az utobbi evek intenziv vizsgalatai alapjan nemcsak egyre tobb foldrajzi regiobol mutatjak ki ezt a bakteriumot kullancsokbol, de folyamatosan novekszik a publikalt human...Borrelia miyamotoi is a recently described relapsing fever spirochete transmitted by ticks of the Ixodes ricinus complex. This pathogen is different from Borrelia burgdorferi sensu lato (the Lyme borreliosis spirochetes) in its epidemiology, ecology and also genetics. Over 50 patients have been described worldwide with Borrelia miyamotoi disease, and three immunocompromised patients were reported with neurological symptoms. Our knowledge about Borrelia miyamotoi infection in ticks and its distribution in different habitats and also the mechanism of the infection is limited. The most common symptom is fever; thus it can be easily confused with other tick-borne diseases. Due to the intensive research in recent years, Borrelia miyamotoi infection in ticks and hosts has been reported from different regions and also the number of patients is increasing, thus this bacterium is considered as an emerging pathogen. In this literature review we would like to summarize the available knowledge about this spirochete. Orv Hetil. 2017, 158(29): 1124-1130.