Host dispersal shapes the population structure of a tick-borne bacterial pathogen.

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick-borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies "Candidatus Borrelia aligera" was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick-borne pathogens are shaped by their host associations and the movement patterns of these hosts.

Tick- and fly-borne bacteria in ungulates: the prevalence of Anaplasma phagocytophilum, haemoplasmas and rickettsiae in water buffalo and deer species in Central Europe, Hungary.

BACKGROUND: Hunting constitutes an important industry in Europe. However, data on the prevalence of vector-borne bacteria in large game animal species are lacking from several countries. Blood or spleen samples (239 and 270, respectively) were taken from red, fallow and roe deer, as well as from water buffaloes, mouflons and wild boars in Hungary, followed by DNA extraction and molecular analyses for Anaplasma phagocytophilum, haemoplasmas and rickettsiae. RESULTS: Based on blood samples, the prevalence rate of A. phagocytophilum infection was significantly higher in red deer (97.9%) than in fallow deer (72.7%) and roe deer (60%), and in all these compared to mouflons (6.3%). In addition, 39.2% of the spleen samples from wild boars were PCR positive for A. phagocytophilum, but none of the buffalos. Based on blood samples, the prevalence rates of both Mycoplasma wenyonii (Mw) and 'Candidatus M. haemobos' (CMh) infections were significantly higher in buffaloes (Mw: 91.2%; CMh: 73.3%) than in red deer (Mw: 64.6%; CMh: 45.8%), and in both of them compared to fallow deer (Mw: 30.3%; CMh: 9.1%) and roe deer (Mw: 20%; CMh: 1.5%). The prevalence of Mw and CMh infection significantly correlated with the body sizes of these hosts. Furthermore, Mw was significantly more prevalent than CMh in buffaloes, red and roe deer. Mycoplasma ovis was detected in mouflons, M. suis in wild boars, R. helvetica in one fallow deer and one mouflon, and an unidentified Rickettsia sp. in a fallow deer. CONCLUSIONS: Forest-dwelling game animal species were found to be important carriers of A. phagocytophilum. In contrast, animals grazing grassland (i.e. buffaloes) were less likely to get infected with this Ixodes ricinus-borne pathogen. Water buffaloes, deer species, mouflons and wild boars harbored haemoplasmas that may affect domestic ungulates. Evaluated animals with larger body size had significantly higher prevalence of infection with haemoplasmas compared to smaller deer species. The above host species rarely carried rickettsiae.

Evidence for host specificity of Theileria capreoli genotypes in cervids.

Data on the prevalence of piroplasms in buffaloes and large game animal species are lacking from several central European countries. Therefore, to investigate the presence of Babesia/Theileria DNA in these hosts, 239 blood and 270 spleen samples were taken from cervids (red, fallow, and roe deer), as well as from water buffaloes, mouflons, and wild boars in southwestern Hungary, followed by DNA extraction and molecular analysis for piroplasms. All samples from buffaloes and wild boars were PCR negative. Based on spleen samples, the prevalence of piroplasms was significantly higher in red deer (41.7%) than in fallow deer (23.5%). Two genotypes of Theileria capreoli were identified, which showed significant association with their host species (i.e. genotype "capreoli-CE1" was exclusively found in roe deer, whereas red and fallow deer harbored only genotype "elaphi-CE1"). Genotype "elaphi-CE1" of T. capreoli was also detected in one mouflon. No Babesia spp. were identified. In conclusion, in the evaluated region, genotypes of T. capreoli show host-associations among cervids, and at least one of these genotypes may infect mouflons.

Babesia genotypes in Haemaphysalis concinna collected from birds in Hungary reflect phylogeographic connections with Siberia and the Far East.

Haemaphysalis concinna is the second most common tick species attaching to birds in Hungary. Recently, Babesia genotypes, found in Siberia and the Far East, have been detected in this tick species collected from the vegetation in Hungary and Slovakia. The aim of this study was to molecularly investigate if these piroplasms also occur in H. concinna carried by migratory birds, which might explain their occurrence in the western Palaearctic. During a 2-year period, 321 H. concinna larvae and nymphs were collected from 121 passerine birds (of 19 species) in Hungary. These were molecularly investigated for the presence of piroplasm DNA with PCR and sequencing. The prevalence of PCR positive ticks was 15.9% (51 out of 321). Piroplasm PCR positivity of H. concinna ticks was significantly more frequent during the summer and autumn compared to spring, suggesting that migratory birds arriving in Hungary from the north or north east are the most important in the dispersal of H. concinna-associated piroplasms. Three genotypes, i.e. Babesia sp. "Irk-Hc133", "Irk-Hc130" (originally found in Irkutsk, Siberia) and "Kh-Hc222" (originally found in Khabarovsk, Far East) were detected. Phylogenetically all these belonged to the group formed by Babesia spp. of ruminants. Four bird species, which had 14-60% prevalence of PCR positive ticks, are known to be associated with northeast to southwest autumn migration. In conclusion, the presence of Central and East Asian Babesia genotypes in Central Europe are most likely related to bird species with known eastern migratory habit and/or phylogenetically substantiated connections between their eastern and western Eurasian populations.

An unexpected advantage of insectivorism: insect moulting hormones ingested by song birds affect their ticks.

Ecdysteroids are important hormones that regulate moulting in arthropods. Three-host ixodid ticks normally moult to the next stage after finishing their blood meal, in the off-host environment. Presumably, three-host ticks that feed on the blood of insectivorous vertebrate hosts can be exposed to high levels of exogenous ecdysteroids causing them to initiate apolysis (the first step of moulting) on the vertebrate host. The aim of the present study was to investigate whether ticks undergo apolysis on insectivorous song birds, and if this phenomenon is associated with the seasonal variation in the availability of moths and with the presence of naturally acquired ecdysteroids in avian blood. During a triannual survey, 3330 hard tick larvae and nymphs were collected from 1164 insectivorous song birds of 46 species. A noteworthy proportion of ticks, 20.5%, showed apolysis. The occurrence of apolytic ticks on birds was correlated with the known seasonality of lepidopteran caterpillars. In addition, 18 blood samples of tick-infested birds were analysed with liquid chromatography - tandem mass spectrometry. Eight samples contained ecdysteroids or their derivatives, frequently in high concentrations, and the presence of these was associated with tick apolysis. In conclusion, naturally acquired ecdysteroids may reach high levels in the blood of insectivorous passerine birds, and will affect ticks (feeding on such blood) by shortening their parasitism.

Diversity of Haemaphysalis-associated piroplasms of ruminants in Central-Eastern Europe, Hungary.

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.

Screening of bat faeces for arthropod-borne apicomplexan protozoa: Babesia canis and Besnoitia besnoiti-like sequences from Chiroptera.

BACKGROUND: Bats are among the most eco-epidemiologically important mammals, owing to their presence in human settlements and animal keeping facilities. Roosting of bats in buildings may bring pathogens of veterinary-medical importance into the environment of domestic animals and humans. In this context bats have long been studied as carriers of various pathogen groups. However, despite their close association with arthropods (both in their food and as their ectoparasites), only a few molecular surveys have been published on their role as carriers of vector-borne protozoa. The aim of the present study was to compensate for this scarcity of information. FINDINGS: Altogether 221 (mostly individual) bat faecal samples were collected in Hungary and the Netherlands. The DNA was extracted, and analysed with PCR and sequencing for the presence of arthropod-borne apicomplexan protozoa. Babesia canis canis (with 99-100% homology) was identified in five samples, all from Hungary. Because it was excluded with an Ixodidae-specific PCR that the relevant bats consumed ticks, these sequences derive either from insect carriers of Ba. canis, or from the infection of bats. In one bat faecal sample from the Netherlands a sequence having the highest (99%) homology to Besnoitia besnoiti was amplified. CONCLUSIONS: These findings suggest that some aspects of the epidemiology of canine babesiosis are underestimated or unknown, i.e. the potential role of insect-borne mechanical transmission and/or the susceptibility of bats to Ba. canis. In addition, bats need to be added to future studies in the quest for the final host of Be. besnoiti.