Zoonotic ecotype-I of Anaplasma phagocytophilum in sympatric wildcat, pine marten and red squirrel - Short communication.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, dogs, cats, horses and tick-borne fever in ruminants. In Europe, its main vector is the tick species Ixodes ricinus. In this study, spleen and liver samples, as well as ticks from 18 wild-living mammals (belonging to seven species) were analysed for the presence of A. phagocytophilum with molecular methods. The zoonotic ecotype-I of A. phagocytophilum was identified in a European wildcat (Felis silvestris) and its tick, a European pine marten (Martes martes) and a Eurasian red squirrel (Sciurus vulgaris). All PCR-positive samples were collected in 2019 and originated in the same geographic area. These results indicate that taxonomically diverse mammalian species can maintain the local enzootic cycle of the same genotype of A. phagocytophilum. To the best of our knowledge, this is the first report of the zoonotic variant of A. phagocytophilum in the wildcat and in the European pine marten in a broad geographical context, as well as in the red squirrel in Hungary. Since all these host species are well known for their urban and peri-urban presence, the results of this study verify their role in the synanthropic enzootic cycle of granulocytic anaplasmosis and tick-borne fever.

Anaplasmataceae closely related to Ehrlichia chaffeensis and Neorickettsia helminthoeca from birds in Central Europe, Hungary.

Increasing amount of data attest that (in the context of vector-borne infections) birds are not only important as hosts of blood-sucking arthropod vectors, but also as reservoirs of vector-borne pathogens. From 2015 to 2019 cadavers of 100 birds (from 45 species, nine orders) were collected in Hungary, and their organs were screened for DNA from a broad range of vector-borne bacteria with PCR and sequencing. Molecular analyses revealed the presence of Anaplasmataceae, and sequencing identified bacteria closely related to Neorickettsia helminthoeca and Ehrlichia chaffeensis in a Eurasian teal (Anas crecca) and a song thrush (Turdus philomelos), respectively. All samples were PCR negative for rickettsiae, borreliae, Francisella and Coxiella spp., as well as for piroplasms. To our knowledge, this is the first report of a Neorickettsia and an Ehrlichia sp., which belong to the phylogenetic groups of N. helminthoeca and E. chaffeensis, respectively, from Europe. The potential presence of these two vector-borne bacteria needs to be taken into account during future studies on the eco-epidemiology of Anaplasmataceae in Europe.

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.

DNA of free-living bodonids (Euglenozoa: Kinetoplastea) in bat ectoparasites: potential relevance to the evolution of parasitic trypanosomatids.

Kinetoplastids are flagellated protozoa, including principally free-living bodonids and exclusively parasitic trypanosomatids. In the most species-rich genus, Trypanosoma, more than thirty species were found to infect bats worldwide. Bat trypanosomes are also known to have played a significant role in the evolution of T. cruzi, a species with high veterinary medical significance. Although preliminary data attested the occurrence of bat trypanosomes in Hungary, these were never sought for with molecular methods. Therefore, amplification of an approx. 900-bp fragment of the 18S rRNA gene of kinetoplastids was attempted from 307 ixodid and 299 argasid ticks collected from bats, and from 207 cimicid bugs collected from or near bats in Hungary and Romania. Three samples, one per each bat ectoparasite group, were PCR positive. Sequencing revealed the presence of DNA from free-living bodonids (Bodo saltans and neobodonids), but no trypanosomes were detected. The most likely source of bodonid DNA detected here in engorged bat ectoparasites is the blood of their bat hosts. However, how bodonids were acquired by bats, can only be speculated. Bats are known to drink from freshwater bodies, i.e. the natural habitats of B. saltans and related species, allowing bats to ingest bodonids. Consequently, these results suggest that at least the DNA of bodonids might pass through the alimentary mucosa of bats into their circulation. The above findings highlight the importance of studying bats and other mammals for the occurrence of bodonids in their blood and excreta, with potential relevance to the evolution of free-living kinetoplastids towards parasitism.

Molecular-phylogenetic investigation of trichomonads in dogs and cats reveals a novel Tritrichomonas species.

BACKGROUND: Trichomonosis is a common infection in small animals, mostly manifesting in gastrointestinal symptoms such as diarrhea. Although oral trichomonads are also known, the species found colonizing the large intestine are more frequently detected protozoa. METHODS: In the present study, four wildcats, 94 domestic cats, and 25 dogs, originating from 18 different locations in Hungary, were investigated for the presence of oral and large intestinal trichomonads based on the 18S rRNA gene and ITS2. RESULTS: All oral swabs were negative by polymerase chain reaction (PCR). However, Tritrichomonas foetus was detected in a high proportion among tested domestic cats (13.8%) and dogs (16%), and Pentatrichomonas hominis only in two domestic cats. In addition, a novel Tritrichomonas genotype was identified in one cat, probably representing a new species that was shown to be phylogenetically most closely related to Tritrichomonas casperi described recently from mice. All positive dogs and half of the positive cats showed symptoms, and among cats, the most frequent breed was the Ragdoll. CONCLUSIONS: With molecular methods, this study evaluated the prevalence of oral and intestinal trichomonads in clinical samples of dogs and cats from Hungary, providing the first evidence of T. foetus in dogs of this region. In contrast to literature data, P. hominis was more prevalent in cats than in dogs. Finally, a hitherto unknown large intestinal Tritrichomonas species (closely related to T. casperi) was shown to be present in a cat, raising two possibilities. First, this novel genotype might have been a rodent-associated pseudoparasite in the relevant cat. Otherwise, the cat was actually infected, thus suggesting the role of a predator-prey link in the evolution of this trichomonad.

A screening of wild bird samples enhances our knowledge about the biodiversity of avian adenoviruses.

Wild birds are threatened by anthropic effects on a global scale, and their adenoviruses might contribute to their endangerment. Thus, it is important to reveal the real biodiversity of avian adenoviruses, as, unfortunately, this research topic is far from being prioritized. The turkey hemorrhagic enteritis is an economically important disease causing high mortalities, and its causative siadenoviral agent is only distantly related to other avian siadenoviruses in phylogenetic analyses. Both to enhance our knowledge about the biodiversity of wild bird adenoviruses and to possibly trace back the origin of the turkey hemorrhagic enteritis virus, numerous Hungarian wild bird samples were screened for adenoviruses using PCR, and the detected strains were typed molecularly. The screening revealed numerous new adenovirus types, several of which represent novel adenovirus species as well, in the genera Atadenovirus, Aviadenovirus and Siadenovirus.

Screening of Domestic Cats from North-Eastern Hungary for Hepatozoon felis and Cytauxzoon europaeus That Cause Infections in Local Wildcat Populations.

Among vector-borne protozoa Hepatozoon felis and Cytauxzoon europaeus are considered emerging species in felids in Europe. To investigate the presence of these two protozoa 127 domestic cats and 4 wildcats were screened by PCRs targeting the 18S rRNA gene of Hepatozoon spp. and piroplasms, as well as the cytb gene of Cytauxzoon spp. The samples were collected inside and outside a region of Hungary, where both protozoan groups are endemic in wildcats. Among domestic cats, one proved to be infected with H. felis. Furthermore, spleen samples of four wildcats were also examined, among which three tested positive for H. felis, and one had co-infection with C. europaeus. Importantly, H. felis from the co-infected wildcat belonged to genogroup II, similarly to H. felis from the positive domestic cat. Based on phylogenetic evidence, this genogroup probably represents a separate species from genogroup I of H. felis, which was hitherto reported from Mediterranean countries in Europe. The two other wildcats also harbored H. felis from genogroup I. Neither Hepatozoon nor Cytauxzoon infections were detected outside the recently discovered endemic region. In conclusion, this study demonstrates for the first time in Europe that H. felis from genogroup II may emerge in free-roaming domestic cats in regions where this protozoan parasite is endemic in wildcats.

Detection and genetic characterization of circoviruses in more than 80 bat species from eight countries on four continents.

Several bat-associated circoviruses and circular rep-encoding single-stranded DNA (CRESS DNA) viruses have been described, but the exact diversity and host species of these viruses are often unknown. Our goal was to describe the diversity of bat-associated circoviruses and cirliviruses, thus, 424 bat samples from more than 80 species were collected on four continents. The samples were screened for circoviruses using PCR and the resulting amino acid sequences were subjected to phylogenetic analysis. The majority of bat strains were classified in the genus Circovirus and some strains in the genus Cyclovirus and the clades CRESS1 and CRESS3. Some strains, however, could only be classified at the taxonomic level of the order and were not classified in any of the accepted or proposed clades. In the family Circoviridae, 71 new species have been predicted. This screening of bat samples revealed a great diversity of circoviruses and cirliviruses. These studies underline the importance of the discovery and description of new cirliviruses and the need to establish new species and families in the order Cirlivirales.

Molecular epidemiological study on ticks and tick-borne protozoan parasites (Apicomplexa: Cytauxzoon and Hepatozoon spp.) from wild cats (Felis silvestris), Mustelidae and red squirrels (Sciurus vulgaris) in central Europe, Hungary.

BACKGROUND: Among live wild mammals adapted to urban and peri-urban habitats in Europe, members of the families Felidae, Mustelidae and Sciuridae deserve special attention as pathogen reservoirs because all of these families include members that are kept as pets. We report here the results of our study on two important groups of tick-borne protozoan parasites in ticks and tissues of wild cats, mustelids and red squirrels. METHODS: DNA was extracted from the tissues of carnivores (wild cats, mustelids; n = 16) and red squirrels (n = 4), as well as from ixodid ticks (n = 89) collected from these hosts. These DNA extracts were screened for piroplasms and Hepatozoon spp. using conventional PCR analysis and sequencing. In addition, 53 pooled samples of 259 questing Haemaphysalis concinna ticks were evaluated for the presence of Hepatozoon DNA, followed by phylogenetic analyses. RESULTS: One wild cat was found to be coinfected with Cytauxzoon europaeus and a new genotype of Hepatozoon felis, and two additional wild cats were infected with H. felis from a different phylogenetic group. In mustelids, Hepatozoon martis and two further Hepatozoon genotypes were detected. The latter clustered separately, close to others reported from eastern Asia. In addition, Hepatozoon sciuri was detected in red squirrels. Morphologic and molecular analyses verified eight tick species. One wild cat was infected with a H. felis genotype that was significantly different from that in Ixodes ricinus females infesting this cat. Only three pools of questing H. concinna nymphs tested positive for Hepatozoon, one of which contained H. martis. CONCLUSIONS: This study provides the first evidence of the occurrence of any Cytauxzoon species and of three Hepatozoon species in Hungary. In addition to H. martis, two further mustelid-associated Hepatozoon genotypes were detected, one of which was new in terms of phylogenetic and broader geographical contexts. This may be the first indication that H. felis genotypes from both of its phylogenetic groups occur in Europe. This also appears to be the first evidence of H. felis and C. europaeus coinfection in felids in Europe, and of autochthonous H. felis infection in wild cats north of the Mediterranean Basin. New tick-host associations were also observed in this study. Based on the results, H. felis and H. martis might survive transstadially in I. ricinus and H. concinna, respectively.

Isolation of infectious Lloviu virus from Schreiber's bats in Hungary.

Some filoviruses can be transmitted to humans by zoonotic spillover events from their natural host and filovirus outbreaks have occured with increasing frequency in the last years. The filovirus Lloviu virus (LLOV), was identified in 2002 in Schreiber's bats (Miniopterus schreibersii) in Spain and was subsequently detected in bats in Hungary. Here we isolate infectious LLOV from the blood of a live sampled Schreiber's bat in Hungary. The isolate is subsequently sequenced and cultured in the Miniopterus sp. kidney cell line SuBK12-08. It is furthermore able to infect monkey and human cells, suggesting that LLOV might have spillover potential. A multi-year surveillance of LLOV in bats in Hungary detects LLOV RNA in both deceased and live animals as well as in coupled ectoparasites from the families Nycteribiidae and Ixodidae. This correlates with LLOV seropositivity in sampled Schreiber's bats. Our data support the role of bats, specifically Miniopterus schreibersii as hosts for LLOV in Europe. We suggest that bat-associated parasites might play a role in the natural ecology of filoviruses in temperate climate regions compared to filoviruses in the tropics.

Morphology of Pholeoixodes species associated with carnivores in the western Palearctic: Pictorial key based on molecularly identified Ixodes (Ph.) canisuga, I. (Ph.) hexagonus and I. (Ph.) kaiseri males, nymphs and larvae.

Three Palearctic members of the subgenus Pholeoixodes, i.e., Ixodes canisuga, Ixodes hexagonus and Ixodes kaiseri are frequently collected from dogs, cats, red foxes, badgers and other carnivorous/insectivorous hosts in Europe. While a pictorial identification key has been reported for female Pholeoixodes ticks, a similar work has not been done on their male, nymphal and larval specimens. This study was initiated in order to clarify and re-examine those morphological characters of these three tick species, which can be used relatively easily to identify/distinguish them. In the case of larvae the aims included finding alternatives to chaetotaxy, which is difficult to observe and its usefulness is also affected by uncertainties in literature data. For this, 609 Pholeoixodes ticks (males, nymphs and larvae) were collected from carnivores, hedgehogs and their environment in six European countries (representing Western, Central and Southeastern Europe), followed by detailed morphological examination and/or molecular analyses to confirm the identity of their species. Based on the morphology of 84 molecularly analyzed specimens and a new identification key compiled accordingly, altogether 116 I. canisuga, 277 I. hexagonus and 216 I. kaiseri males, nymphs and larvae were identified. Ixodes kaiseri was not found in Western Europe, where I. canisuga predominated. In Central Europe, all three Pholeoixodes species were collected, the largest number of specimens represented by I. hexagonus. On the other hand, in Southeastern Europe I. kaiseri had the highest abundance. In conclusion, the morphology of internal spur on the first coxae (as the traditionally used character to distinguish I. hexagonus from other Pholeoixodes species) is trustworthy to recognize males but is less informative in the case of nymphs and larvae. The latter can be identified more properly by observing the morphology of basis capituli. In particular, nymphs and larvae of I. canisuga have anteriorly flattened basis capituli, forming a plateau that surrounds the base of the hypostome. On the other hand, nymphs and larvae of I. hexagonus and I. kaiseri lack a similar plateau, but (unlike I. canisuga) have cornuae, which are either posterolaterally or caudally directed, respectively.

Molecular detection of vector-borne bacteria in bat ticks (Acari: Ixodidae, Argasidae) from eight countries of the Old and New Worlds.

BACKGROUND: Despite the increasingly recognized eco-epidemiological significance of bats, data from molecular analyses of vector-borne bacteria in bat ectoparasites are lacking from several regions of the Old and New Worlds. METHODS: During this study, six species of ticks (630 specimens) were collected from bats in Hungary, Romania, Italy, Kenya, South Africa, China, Vietnam and Mexico. DNA was extracted from these ticks and analyzed for vector-borne bacteria with real-time PCRs (screening), as well as conventional PCRs and sequencing (for pathogen identification), based on the amplification of various genetic markers. RESULTS: In the screening assays, Rickettsia DNA was only detected in bat soft ticks, whereas Anaplasma phagocytophilum and haemoplasma DNA were present exclusively in hard ticks. Bartonella DNA was significantly more frequently amplified from hard ticks than from soft ticks of bats. In addition to Rickettsia helvetica detected by a species-specific PCR, sequencing identified four Rickettsia species in soft ticks, including a Rickettsia africae-like genotype (in association with a bat species, which is not known to migrate to Africa), three haemotropic Mycoplasma genotypes in Ixodes simplex, and Bartonella genotypes in I. ariadnae and I. vespertilionis. CONCLUSIONS: Rickettsiae (from both the spotted fever and the R. felis groups) appear to be associated with soft rather than hard ticks of bats, as opposed to bartonellae. Two tick-borne zoonotic pathogens (R. helvetica and A. phagocytophilum) have been detected for the first time in bat ticks. The present findings add Asia (China) to the geographical range of R. lusitaniae, as well as indicate the occurrence of R. hoogstraalii in South Africa. This is also the first molecular evidence for the autochthonous occurrence of a R. africae-like genotype in Europe. Bat haemoplasmas, which are closely related to haemoplasmas previously identified in bats in Spain and to "Candidatus Mycoplasma haemohominis", are reported here for the first time from Central Europe and from any bat tick.

Phylogenetic analyses of bat-associated bugs (Hemiptera: Cimicidae: Cimicinae and Cacodminae) indicate two new species close to Cimex lectularius.

BACKGROUND: Bats are regarded as the primary (ancestral) hosts of bugs of the family Cimicidae. The historically and economically most important species in the family is the common bedbug (Cimex lectularius), because of its worldwide occurrence and association with humans. This molecular-phylogenetic study was initiated in order to expand the knowledge on the phylogeny of cimicid bugs of bats, by investigating samples from Hungary, Romania (representing central-eastern Europe) and two further countries (South Africa and Vietnam). RESULTS: Altogether 216 cimicid bugs were collected (73 Ci. lectularius, 133 Ci. pipistrelli, nine Cacodmus ignotus and one Ca. sparsilis). Members of the Cimex lectularius species group were found both in the environment of bats (only Myotis emarginatus, which is a cave/attic-dwelling species) and on three crevice-dwelling bat species (two pipistrelloid bats and M. bechsteinii). On the other hand, Ci. pipistrelli always occurred off-host (near M. myotis/blythii, which are cave/attic-dwelling species). In addition, two Cacodmus spp. were collected from Pipistrellus hesperidus. The morphological characters of these specimens are illustrated with high resolution pictures. Analysis of cytochrome c oxidase subunit 1 (cox1) sequences generated from 38 samples indicated relative genetic homogeneity of Ci. pipistrelli, while the Ci. lectularius group had two haplotypes (collected from pipistrelloid bats in Hungary and Vietnam) highly divergent from other members of this species group. These results were confirmed with molecular and phylogenetic analyses based on the internal transcribed spacer 2 (ITS2). Bat-associated bugs morphologically identified as Ca. ignotus and Ca. sparsilis were different in their cox1, but identical in their ITS2 sequences. CONCLUSIONS: Molecular evidence is provided here on the existence of two new genotypes, most likely new species, within the Ci. lectularius species group. The relevant specimens (unlike the others) were collected from pipistrelloid bats, therefore the association of Ci. lectularius with different bat host species (pipistrelloid vs myotine bats) should be evaluated further as a possible background factor of this genetic divergence. In addition, Ca. ignotus is reported for the first time in South Africa.

DNA of Piroplasms of Ruminants and Dogs in Ixodid Bat Ticks.

In this study 308 ticks (Ixodes ariadnae: 26 larvae, 14 nymphs, five females; I. vespertilionis: 89 larvae, 27 nymphs, eight females; I. simplex: 80 larvae, 50 nymphs, nine females) have been collected from 200 individuals of 17 bat species in two countries, Hungary and Romania. After DNA extraction these ticks were molecularly analysed for the presence of piroplasm DNA. In Hungary I. ariadnae was most frequently identified from bat species in the family Vespertilionidae, whereas I. vespertilionis was associated with Rhinolophidae. Ixodes ariadnae was not found in Romania. Four, four and one new bat host species of I. ariadnae, I. vespertilionis and I. simplex were identified, respectively. DNA sequences of piroplasms were detected in 20 bat ticks (15 larvae, four nymphs and one female). I. simplex carried piroplasm DNA sequences significantly more frequently than I. vespertilionis. In I. ariadnae only Babesia vesperuginis DNA was detected, whereas in I. vespertilionis sequences of both B. vesperuginis and B. crassa. From I. simplex the DNA of B. canis, Theileria capreoli, T. orientalis and Theileria sp. OT3 were amplified, as well as a shorter sequence of the zoonotic B. venatorum. Bat ticks are not known to infest dogs or ruminants, i.e. typical hosts and reservoirs of piroplasms molecularly identified in I. vespertilionis and I. simplex. Therefore, DNA sequences of piroplasms detected in these bat ticks most likely originated from the blood of their respective bat hosts. This may indicate either that bats are susceptible to a broader range of piroplasms than previously thought, or at least the DNA of piroplasms may pass through the gut barrier of bats during digestion of relevant arthropod vectors. In light of these findings, the role of bats in the epidemiology of piroplasmoses deserves further investigation.