Tick-borne viruses and their risk to public health in the Caribbean: Spotlight on bats as reservoirs in Cuba.

In recent decades, tick-borne diseases (TBDs) have surged and expanded globally due to factors like changes in human activities, land use patterns, and climate change, and it have been associated with the emergence of zoonotic diseases. Cuba faces the impact of ticks on human health and the economy. Although Cuba has studied TBDs extensively for the past 50 years, focus on tick-borne viral pathogens affecting humans remains scant. Despite TBDs not currently being a major health concern in Cuba, factors like inadequate clinician awareness, climate conditions, global tick emergence, and evidence of zoonotic pathogens in ticks underscore the importance of enhanced TBD surveillance in the country. Here we revised the available information on ticks as vectors of pathogenic viruses to humans, spotlighting bats as potential reservoirs of tick-borne viruses (TBVs). Ticks on bats have gained interest as potential reservoirs of pathogenic viruses to humans in Cuba and worldwide. Understanding their role in maintaining viruses and their potential transmission to humans is crucial for the implementation of surveillance and control programs to reduce the risk of tick-borne viral diseases and public health management.

Season and host-community composition inside roosts may affect host-specificity of bat flies.

Bat flies are one of the most abundant ectoparasites of bats, showing remarkable morphological adaptations to the parasitic habit, while the relationship with their hosts is characterized by a high level of specificity. By collecting bat flies from live hosts, our intention was to elucidate the seasonal differences in bat fly occurrence and to describe factors regulating the level of incipient host specificity. Our results indicate that the prevalence and the intensity of infestation is increasing from spring to autumn for most host species, with significant differences among different fly species. Males showed higher infestation levels than females in autumn, suggesting a non-random host choice by flies, targeting the most active host sex. Bat-bat fly host specificity shows seasonal changes and host choice of bat flies are affected by the seasonal differences in hosts' behavior and ecology, the intensity of infestation and the species composition of the local host community. Nycteribiid bat flies showed lower host specificity in the swarming (boreal autumn) period, with higher prevalence recorded on non-primary hosts. Choosing a non-primary bat host may be an adaptive choice for bat flies in the host's mating period, thus increasing their dispersive ability in a high activity phase of their hosts.

Detection of bacterial and protozoan pathogens in individual bats and their ectoparasites using high-throughput microfluidic real-time PCR.

Among the most studied mammals in terms of their role in the spread of various pathogens with possible zoonotic effects are bats. These are animals with a very complex lifestyle, diet, and behavior. They are able to fly long distances, thus maintaining and spreading the pathogens they may be carrying. These pathogens also include vector-borne parasites and bacteria that can be spread by ectoparasites such as ticks and bat flies. In the present study, high-throughput screening was performed and we detected three bacterial pathogens: Bartonella spp., Neoehrlichia mikurensis and Mycoplasma spp., and a protozoan parasite: Theileria spp. in paired samples from bats (blood and ectoparasites). In the samples from the bat-arthropod pairs, we were able to detect Bartonella spp. and Mycoplasma spp. which also showed a high phylogenetic diversity, demonstrating the importance of these mammals and the arthropods associated with them in maintaining the spread of pathogens. Previous studies have also reported the presence of these pathogens, with one exception, Neoehrlichia mikurensis, for which phylogenetic analysis revealed less genetic divergence. High-throughput screening can detect more bacteria and parasites at once, reduce screening costs, and improve knowledge of bats as reservoirs of vector-borne pathogens. IMPORTANCE The increasing number of zoonotic pathogens is evident through extensive studies and expanded animal research. Bats, known for their role as reservoirs for various viruses, continue to be significant. However, new findings highlight the emergence of Bartonella spp., such as the human-infecting B. mayotimonensis from bats. Other pathogens like N. mikurensis, Mycoplasma spp., and Theileria spp. found in bat blood and ectoparasites raise concerns, as their impact remains uncertain. These discoveries underscore the urgency for heightened vigilance and proactive measures to understand and monitor zoonotic pathogens. By deepening our knowledge and collaboration, we can mitigate these risks, safeguarding human and animal well-being.

Comparison of salivary gland and midgut microbiome in the soft ticks Ornithodoros erraticus and Ornithodoros moubata.

Introduction: Ornithodoros erraticus and Ornithodoros moubata are the main vectors of African swine fever virus (ASFV) and the human relapsing fever spirochetes Borrelia hispanica and Borrelia crocidurae in the Mediterranean region and Borrelia duttoni in continental Africa. Manipulation of the tick microbiome has been shown to reduce vector fitness and competence in tick vectors, suggesting that the identification of key microbial players associated with tick tissues can inform interventions such as anti-microbiota vaccines to block pathogen development in the midgut and/or salivary glands. Methods: In this study, we analyzed and compared the microbiome of the salivary glands and midgut of O. erraticus and O. moubata. For the taxonomic and functional characterization of the tissue-specific microbiome, we used 16S rRNA amplicon sequencing and prediction of metabolic profiles using PICRUSt2. Co-occurrence networks were built to characterize the community assembly and identify keystone taxa in each tick species. Results: Our results revealed differences in the composition, diversity, and assembly of the bacterial microbiome of salivary glands and midgut within each tick species, but differences were more noticeable in O. moubata. Differences were also found in the microbiome of each tissue, salivary gland and midgut, between species. However, the 'Core Association Networks (CAN)' analysis revealed conserved patterns of interacting taxa in tissues within and between tick species. Different keystone taxa were identified in O. erraticus and O. moubata tissues, but Muribaculaceae and Alistipes were found as keystone taxa in the salivary glands of both tick species which justifies their use as anti-microbiota vaccine candidates to alter the microbiome and reduce tick fitness and/or block pathogen transmission. The high similarity of predicted metabolic pathways profiles between tissues of the two tick species suggests that taxonomic variability of the microbiome is not associated with significant changes in microbial functional profiles. Conclusion: We conclude that the taxonomic structure of the microbiome in O. erraticus and O. moubata is tissue-specific, suggesting niche partitioning of bacterial communities associated to these soft ticks. However, shared keystone taxa and conserved patterns of interacting taxa between tissues and tick species suggest the presence of key microbial players that could be used as anti-microbiota vaccine candidates to affect tick physiology and/or pathogen colonization.

Structural differences in the gut microbiome of bats using terrestrial vs. aquatic feeding resources.

Bat gut microbiomes are adapted to the specific diets of their hosts. Despite diet variation has been associated with differences in bat microbiome diversity, the influence of diet on microbial community assembly have not been fully elucidated. In the present study, we used available data on bat gut microbiome to characterize the microbial community assembly of five selected bat species (i.e., Miniopterus schreibersii, Myotis capaccinii, Myotis myotis, Myotis pilosus, and Myotis vivesi), using network analysis. These bat species with contrasting habitat and food preferences (i.e., My. capaccinii and My. pilosus can be piscivorous and/or insectivorous; Mi. schreibersii and My. myotis are exclusively insectivorous; while My. vivesi is a marine predator) offer an invaluable opportunity to test the impact of diet on bat gut microbiome assembly. The results showed that My. myotis showed the most complex network, with the highest number of nodes, while My. vivesi has the least complex structured microbiome, with lowest number of nodes in its network. No common nodes were observed in the networks of the five bat species, with My. myotis possessing the highest number of unique nodes. Only three bat species, My. myotis, My. pilosus and My. vivesi, presented a core microbiome and the distribution of local centrality measures of nodes was different in the five networks. Taxa removal followed by measurement of network connectivity revealed that My. myotis had the most robust network, while the network of My. vivesi presented the lowest tolerance to taxa removal. Prediction of metabolic pathways using PICRUSt2 revealed that Mi. schreibersii had significantly higher functional pathway's richness compared to the other bat species. Most of predicted pathways (82%, total 435) were shared between all bat species, while My. capaccinii, My. myotis and My. vivesi, but no Mi. schreibersii or My. pilosus, showed specific pathways. We concluded that despite similar feeding habits, microbial community assembly can differ between bat species. Other factors beyond diet may play a major role in bat microbial community assembly, with host ecology, sociality and overlap in roosts likely providing additional predictors governing gut microbiome of insectivorous bats.

Piroplasms in farmed American bison, Bison bison from Romania.

The American bison (Bison bison) is the largest terrestrial mammal of North America, with around 350,000 individuals in the wild and in private herds but the knowledge regarding the presence of different vector-borne pathigens in these mammals is very poor. Babesia and Theileria spp. are tick-borne apicomplexan parasites which are considered to be among the most commonly found blood parasites of large ruminants, often with a high economic importance. However, the knowledge on piroplasms of bisons is extremely scarce. The aim of our study was to evaluate the presence of apicomplexan parasites in blood and tissues of farmed American bison from Romania. Overall, we tested 222 blood samples and 11 tissues samples (heart, liver, and spleen) from farmed B. bison raised for meat in Romania. All the samples were analyzed by nPCR targeting the 18SrRNA gene for piroplasmids. All positive samples were sequenced and analyzed phylogenetically. The overall prevalence of infection with piroplasmids in American bison was 1.65%, with Babesia divergens and Theileria sp. identified following sequencing. To our knowledge, this is the first report of piroplasms detected in blood and tissues of farmed B. bison from Europe. Further studies are necessary in order to obtain a better overview on the epidemiological status and clinical relevance of piroplasms in farmed American bisons.

The Presence of Ehrlichia Canis in Rhipicephalus Bursa Ticks Collected from Ungulates in Continental Eastern Europe.

INTRODUCTION: Rhipicephalus bursa is a common tick parasite of small-to-medium size ungulates, principally in warm, temperate, and subtropical areas. Although common in livestock and showing a wide geographic distribution, its epidemiological role in tick-borne bacterial disease is barely known. This study addressed the knowledge gap and aimed to screen for the presence of Anaplasmataceae and spotted fever group (SFG) Rickettsia species in R. bursa ticks collected from domestic animals in Romania, Eastern Europe. MATERIAL AND METHODS: A total of 64 pools of R. bursa ticks collected from small ungulates were tested by PCR for Anaplasmataceae DNA presence using group-specific primers. Specific testing was performed for Anaplasma marginale/A. centrale/A. ovis, A. platys, A. phagocytophilum, Ehrlichia canis, and SFG Rickettsia. The positive samples were purified and sequenced, and sequences analysis was used to identify the species and to confirm the PCR results. RESULTS: The only pathogen identified in this study was E. canis. The obtained sequences confirmed the PCR results. The presence of E. canis in R. bursa in Romania and in ticks from sheep was shown for the first time in this study. CONCLUSION: Based on these findings, it may be presumed that the E. canis DNA originated from ticks; however, the vectorial role of R. bursa (and other arthropod species) in the transmission of E. canis should be proved experimentally.

First record of Ixodes simplex found on a human host, with a review of cases of human infestation by bat tick species occurring in Europe.

Ixodes simplex is a bat tick species, a common parasite of the Schreibers' bent-winged bat, Miniopterus schreibersii. Its distribution is linked to the range of its host, free stages occurring exclusively inside the underground bat shelters. Here we present the first case of human infestation with I. simplex. An adult female tick was found attached to the upper limb after a visit to the underground shelter of a large bat colony. This unusual host selection is a likely consequence of the reduction of suitable hosts, as the number of bats was much lower at the time of the visit than in previous years. Bat ticks rarely feed on humans, with soft ticks (Argasidae) being more commonly involved. In the light of the potential vectorial capacity of I. simplex, the incidence and potential future risks are discussed.

Rickettsia spp. in bats of Romania: high prevalence of Rickettsia monacensis in two insectivorous bat species.

BACKGROUND: Spotted fever group rickettsiae represent one of the most diverse groups of vector-borne bacteria, with several human pathogenic species showing an emerging trend worldwide. Most species are vectored by ticks (Ixodidae), with many zoonotic reservoir species among most terrestrial vertebrate groups. While the reservoir competence of many different vertebrate species is well known (e.g. birds, rodents and dogs), studies on insectivorous bats have been rarely performed despite their high species diversity, ubiquitous urban presence and importance in harboring zoonotic disease agents. Romania has a high diversity and ubiquity of bats. Moreover, seven out of eight SFG rickettsiae species with zoonotic potential were previously reported in Romania. Based on this, the aim of this study was to detect Rickettsia species in tissue samples in bats. METHODS: Here we report a large-scale study (322 bats belonging to 20 species) on the presence of Rickettsia spp. in Romanian bat species. Tissue samples from insectivorous bats were tested for the presence of Rickettsia DNA using PCR detection amplifying a 381 bp fragment of the gltA gene. Positive results were sequenced to confirm the results. The obtained results were statistically analyzed by chi-squared independence test. RESULTS: Positive results were obtained in 14.6% of bat samples. Sequence analysis confirmed the presence of R. monacensis in two bat species (Nyctalus noctula and Pipistrellus pipistrellus) in two locations. CONCLUSION: This study provides the first evidence of a possible involvement of these bat species in the epidemiology of Rickettsia spp., highlighting the importance of bats in natural cycles of these vector-borne pathogens.

The heart microbiome of insectivorous bats from Central and South Eastern Europe.

Host associated microbiome not only may affect the individual health-status or provide insights into the species- or group specific bacterial communities but may act as early warning signs in the assessment of zoonotic reservoirs, offering clues to predict, prevent and control possible episodes of emerging zoonoses. Bats may be carriers and reservoirs of multiple pathogens such as viruses, bacteria and parasites, showing in the same time robust immunity against many of them. The microbiota plays a fundamental role on the induction, training and function of the host immune system and the immune system has largely evolved in order to maintain the symbiotic relationship of the host with these diverse microbes. Thus, expanding our knowledge on bat-associated microbiome it can be usefully in understanding bats' outstanding immune capacities. The aim of this study was to investigate the presence of different bacterial communities in heart tissue of insectivorous bats, Nyctalus noctula, Pipistrellus pipistrellus and Rhinoplophus hipposideros, from Central and Eastern Europe using high-throughput sequencing of variable regions of the 16S rRNA. In addition, species-specific PCRs were used to validate the presence of the vector-borne pathogens Bartonella spp. and Rickettsia spp. In this study we identified a wide variety of bacterial groups, with the most abundant phyla being Proteobacteria and Firmicutes. The results showed that at individual level, the year or location had no effect on the diversity and composition of the microbiome, however host species determined both structure and abundance of the bacterial community. We report the presence of vector-borne bacteria Bartonella spp. in samples of N. noctula and indications of Rickettsia spp. in R. hipposideros. Our results provide a first insight into the bacterial community found in heart tissue of bats from Central and South Eastern Europe.

Wide Distribution and Diversity of Malaria-Related Haemosporidian Parasites (Polychromophilus spp.) in Bats and Their Ectoparasites in Eastern Europe.

Malaria is responsible for major diseases of humans, while associated haemosporidians are important factors in regulating wildlife populations. Polychromophilus, a haemosporidian parasite of bats, is phylogenetically close to human-pathogenic Plasmodium species, and their study may provide further clues for understanding the evolutionary relationships between vertebrates and malarial parasites. Our aim was to investigate the distribution of Polychromophilus spp. in Eastern Europe and test the importance of host ecology and roost site on haemosporidian parasite infection of bats. We sampled bats and their ectoparasites at eight locations in Romania and Bulgaria. DNA was extracted from blood samples and ectoparasites and tested individually for the presence of DNA of Polychromophilus spp. using a nested PCR targeting a 705 bp fragment of cytB. Two species of Polychromophilus were identified: Po. melanipherus in Miniopterus schreibersii and associated ectoparasites and Po. murinus in rhinolophid and vespertilionid bats (6 species) and their ticks and nycteribiid flies. Only cave-dwelling bat species (and their ectoparasites) showed infections, and we found a strong correlation between infections with Polychromophilus parasites and Nycteribiidae prevalence. We report the high genetic diversity of Polychromophilus spp. in Eastern Europe, suggesting that the simultaneous presence of varied host and vector assemblages enhances bat haemosporidian parasite diversity.

Detection of DNA of Babesia canis in tissues of laboratory rodents following oral inoculation with infected ticks.

BACKGROUND: Babesia spp. are apicomplexan parasites which infect a wide range of mammalian hosts. Historically, most Babesia species were described based on the assumed host specificity and morphological features of the intraerythrocytic stages. New DNA-based approaches challenge the traditional species concept and host specificity in Babesia. Using such tools, the presence of Babesia DNA was reported in non-specific mammalian hosts, including B. canis in feces and tissues of insectivorous bats, opening questions on alternative transmission routes. The aim of the present study was to evaluate if B. canis DNA can be detected in tissues of laboratory rodents following oral inoculation with infected ticks. METHODS: Seventy-five questing adult Dermacentor reticulatus ticks were longitudinally cut in two halves and pooled. Each pool consisted of halves of 5 ticks, resulting in two analogous sets. One pool set (n = 15) served for DNA extraction, while the other set (n = 15) was used for oral inoculation of experimental animals (Mus musculus, line CD-1 and Meriones unguiculatus). Blood was collected three times during the experiment (before the inoculation, at 14 days post-inoculation and at 30 days post-inoculation). All animals were euthanized 30 days post-inoculation. At necropsy, half of the heart, lung, liver, spleen and kidneys were collected from each animal. The presence of Babesia DNA targeting the 18S rRNA gene was evaluated from blood and tissues samples. For histopathology, the other halves of the tissues were used. Stained blood smears were used for the light microscopy detection of Babesia. RESULTS: From the 15 pools of D. reticulatus used for the oral inoculation, six were PCR-positive for B. canis. DNA of B. canis was detected in blood and tissues of 33.3% of the animals (4 out of 12) inoculated with a B. canis-positive pool. No Babesia DNA was detected in the other 18 animals which received B. canis-negative tick pools. No Babesia was detected during the histological examination and all blood smears were microscopically negative. CONCLUSIONS: Our findings demonstrate that B. canis DNA can be detected in tissues of mammalian hosts following ingestion of infected ticks and opens the question of alternative transmission routes for piroplasms.

Bats and ticks: host selection and seasonality of bat-specialist ticks in eastern Europe.

BACKGROUND: Parasites may actively seek for hosts and may use a number of adaptive strategies to promote their reproductive success and host colonization. These strategies will necessarily influence their host specificity and seasonality. Ticks are important ectoparasites of vertebrates, which (in addition to directly affecting their hosts) may transmit a number of pathogens. In Europe, three hard tick species (Ixodidae: Ixodes ariadnae, I. simplex and I. vespertilionis) and at least two soft tick species (Argasidae: Argas transgariepinus and A. vespertilionis) are specialized for bats. METHODS: Here we report data on the host range of these ticks and the seasonality of tick infestation on wild caught bats in south-east Europe. We collected 1803 ticks from 30 species of bats living in underground shelters (caves and mines) from Romania and Bulgaria. On the basis of tick-host associations, we tested several hypotheses on host-parasite evolutionary adaptations regulating host specificity, seasonality and sympatric speciation. RESULTS: We observed significant differences in host specificity and seasonality of abundance between the morphologically different bat specialist ticks (I. simplex and I. vespertilionis) likely caused by their host choice and their respective host-seeking behavior. The two highly generalist, but morphologically similar tick species (I. ariadnae and I. vespertilionis) showed temporal differences in occurrence and activity, thus exploiting significantly different host communities while occurring in geographical sympatry. CONCLUSIONS: We conclude that bat-specialist ticks show a wide range of adaptations to their hosts, with differences in specificity, seasonality of occurrence, the prevalence and intensity of infestation and all these contribute to a successful division of temporal niches of ticks sharing morphologically similar hosts occurring in geographical sympatry.

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.

Bartonella DNA in heart tissues of bats in central and eastern Europe and a review of phylogenetic relations of bat-associated bartonellae.

BACKGROUND: Bats are among the most widely distributed mammals worldwide and can represent hosts or reservoirs for a number of different pathogens. Bartonella spp. are opportunistic bacterial pathogens, which are transmitted by a large variety of arthropods. The aim of this study was to investigate the presence and host-associations of these Gram-negative bacteria in heart tissues of bats collected in four different countries from eastern and central Europe and to analyze their phylogenetic relationship with other bat-associated bartonellae. RESULTS: The results of this study show for the first time the presence of Bartonella spp. DNA in heart tissues of bats from central and eastern Europe. The overall prevalence of the infection was 1.38%. Phylogenetic analysis identified four new Bartonella spp. sequences, which were closely related with other Bartonella previously isolated from bats in Europe and North America. CONCLUSIONS: The gltA sequences of Bartonella spp. showed considerable heterogeneity in the phylogenetic analysis resulting in six different clades. Our study demonstrated the presence of Bartonella spp. only in heart tissues of bats from Romania, with two new bat species recorded as hosts (Myotis cf. alcathoe and Pipistrellus pipistrellus).

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.

Analyses of separate and concatenated cox1 and 18S rRNA gene sequences indicate that the bat piroplasm Babesia vesperuginis is phylogenetically close to Cytauxzoon felis and the 'prototheilerid' Babesia conradae.

Babesia vesperuginis is the only piroplasm known to infect bats. Unlike most members of the genus Babesia, it is probably transmitted by a soft tick species (i.e. Argas vespertilionis). Recently, two studies have been conducted to clarify the phylogenetic status of this species, and both agreed on placing it into a basal position among Babesia sensu stricto (s.s.). However, several important groups of piroplasms were not included in the already reported phylogenetic trees of B. vesperuginis isolates. Therefore, the aim of the present study was to amplify an approx. 950-bp fragment of the cytochrome c oxidase subunit 1 (cox1) gene of B. vesperuginis from A. vespertilionis specimens, and to compare its sequences with those from other piroplasmid groups in a broader phylogenetic context. Sequence comparisons focusing on either 18S rRNA or cox1 genes, as well as phylogenetic analyses involving separate and concatenated 18S rRNA and cox1 sequences indicate that B. vesperuginis is more closely related to the phylogenetic group of Theileriidae than to Babesia s.s. In particular, B. vesperuginis clustered closest to Cytauxzoon felis and the 'prototheilerid' B. conradae. The results of this study highlight that B. vesperuginis is a unique and taxonomically important species, which should be included in future studies aimed at resolving the comprehensive phylogeny of Piroplasmida.

New records for Anaplasma phagocytophilum infection in small mammal species.

BACKGROUND: Tick-borne diseases pose a major threat in public health. The epidemiological dynamics of these diseases depends on the tick vector species and their hosts, as well as the geographical distribution and ecology of both. Among many possible hosts for ticks, small mammals have a major role in the development of immature stages of several tick species. Small mammals are also important reservoir hosts for several pathogenic agents and possible reservoirs for Anaplasma phagocytophilum. In this context, the aim of our study was to evaluate the prevalence of A. phagocytophilum in small mammal species in Romania. RESULTS: A total of 791 small mammals of 31 species were tested by PCR, targeting the rrs gene for detection of A. phagocytophilum DNA. Positive results were obtained in 20 small mammals: five Apodemus flavicollis (6.49%), three Sorex araneus (9.09%), three A. uralensis (4.84%), two A. sylvaticus (3.92%), and one of each Spermophilus cittelus (7.14%), Microtus agrestis (3.85%), Sorex minutus (3.85%), Muscardinus avellanarius (3.13%), Crocidura suaveolens (2.44%), Mus spicilegus (2%) and M. arvalis (1.75%). CONCLUSIONS: Eleven small mammal species were found to be carriers of A. phagocytophilum, suggesting a possible involvement of these species in its epidemiology. To our knowledge, this is the first report of A. phagocytophilum in S. minutus, C. suaveolens, M. spicilegus, M. avellanarius and S. citellus.

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.