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.

Lipophilic components of diesel exhaust particles induce pro-inflammatory responses in human endothelial cells through AhR dependent pathway(s).

BACKGROUND: Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. RESULTS: Exposure-relevant concentrations of DEP (0.12 µg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 µg/cm2) induced low to moderate increases in IL-1α, IL-1ß, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. CONCLUSION: Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

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.

Molecular investigations of the bat tick Argas vespertilionis (Ixodida: Argasidae) and Babesia vesperuginis (Apicomplexa: Piroplasmida) reflect "bat connection" between Central Europe and Central Asia.

Argas vespertilionis is a geographically widespread haematophagous ectoparasite species of bats in the Old World, with a suspected role in the transmission of Babesia vesperuginis. The aims of the present study were (1) to molecularly screen A. vespertilionis larvae (collected in Europe, Africa and Asia) for the presence of piroplasms, and (2) to analyze mitochondrial markers of A. vespertilionis larvae from Central Asia (Xinjiang Province, Northwestern China) in a phylogeographical context. Out of the 193 DNA extracts from 321 A. vespertilionis larvae, 12 contained piroplasm DNA (10 from Hungary, two from China). Sequencing showed the exclusive presence of B. vesperuginis, with 100% sequence identity between samples from Hungary and China. In addition, A. vespertilionis cytochrome oxidase c subunit 1 (cox1) and 16S rRNA gene sequences had 99.1-99.2 and 99.5-100% similarities, respectively, between Hungary and China. Accordingly, in the phylogenetic analyses A. vespertilionis from China clustered with haplotypes from Europe, and (with high support) outside the group formed by haplotypes from Southeast Asia. This is the first molecular evidence on the occurrence of B. vesperuginis in Asia. Bat ticks from hosts in Vespertilionidae contained only the DNA of B. vesperuginis (in contrast with what was reported on bat ticks from Rhinolophidae and Miniopteridae). Molecular taxonomic analyses of A. vespertilionis and B. vesperuginis suggest a genetic link of bat parasites between Central Europe and Central Asia, which is epidemiologically relevant in the context of any pathogens associated with bats.

Impact of a freeway on the dispersal of ticks and Ixodes ricinus-borne pathogens: forested resting areas may become Lyme disease hotspots.

Man-made barriers are well known for their effects on ecosystems. Habitat fragmentation, for instance, is a recognised consequence of modern-day infrastructure. The aim of the present study was to investigate the diversity and abundance of tick species, as well as the risks of acquiring tick-borne infections in habitats adjacent to a freeway. Therefore, ixodid ticks were collected from the vegetation at two-week intervals (in the main tick season, from March to June) in eight habitats of different types (forest, grove, grassland) along both sides of a freeway. Ixodes ricinus females were molecularly screened for three species of tick-borne bacteria. In the study period, 887 ixodid ticks were collected. These included 704 I. ricinus (79.4%), 51 Dermacentor reticulatus (5.7%), 78 D. marginatus (8.8%), 35 Haemaphysalis inermis (3.9%) and 19 H. concinna (2.1%). There was no significant difference in the abundance of tick species between similar habitats separated by the freeway, except for the absence of Dermacentor spp. on one side. In I. ricinus females, the overall prevalence of Anaplasma phagocytophilum was low, and (in part due to this low rate) did not show significant difference between the two sides of the freeway. Rickettsia helvetica had significantly different overall prevalence between two distant habitats along the same side of the freeway (12.3% vs. 31.4%), but not between habitats on the opposite sides. Borrelia burgdorferi s.l. showed significantly different overall prevalence between habitats both on the same and on the opposite sides of the freeway (8.6-35.9%), and the difference was higher if relevant habitats were also separated by the freeway. Importantly, the prevalence rate of the Lyme disease agent was highest in a forested resting area of the freeway, and was significantly inversely proportional to the prevalence of A. phagocytophilum (taking into account all evaluated habitats), apparently related to deer population density. Prevalence rates of these bacteria also differed significantly on single sampling occasions between: (1) closely situated habitats of different types; (2) distant and either similar or different habitat types; and (3) habitats on the opposite sides of the freeway. In conclusion, the findings of the present study show that a fenced freeway may contribute to differences in tick species diversity and tick-borne pathogen prevalence along its two sides, and this effect is most likely a consequence of its barrier role preventing deer movements.

Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue.

BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.

Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer.

The emergence of multidrug resistant (MDR) cancer phenotypes dramatically attenuates the efficiency of antineoplastic drug treatments often leading to the failure of chemotherapy. Therefore there is an urgent need to engineer new therapeutically useful agents and propose innovative approaches able to defeat resistant cancer cells. Although the remarkable anti-cancer features of silver nanoparticles (AgNPs) have already been delineated their impact on MDR cancer has never been investigated. Herein, we report that AgNPs have notable anti-proliferative effect and induce apoptosis mediated cell death both in drug sensitive and in MDR cancer cells. Furthermore we show evidence that AgNPs exert an inhibitory action on the efflux activity of MDR cancer cells which feature could be exploited to enhance drug accumulation. We verified synergistic interactions of AgNPs with six different antineoplastic agents on drug resistant cells which emphasizes the excellent potential of AgNPs as combinational partners in the chemotherapy of MDR cancer. FROM THE CLINICAL EDITOR: The treatment of cancer often fails due to the development of multidrug resistant (MDR) cancer cells. Hence, novel approaches are being investigated to combat drug resistant cancer cells. One particular method studied here uses silver nanoparticles (AgNPs). The authors showed that AgNPs had anti-proliferative effect and ?exerted an inhibitory action on ABC transporter. The findings could suggest the possible use of AgNPs in combination with other chemotherapeutic agents in the clinical setting.

First record of Ixodes ariadnae in Germany - Short communication.

A long-legged tick was collected from a hibernating greater mouse-eared bat (Myotis myotis) in Baden-Württemberg, Germany. Based on morphological characteristics as well as on partial COI and 16S rDNA gene sequences the tick was identified as an engorged female of Ixodes ariadnae. The greater mouseeared bat is a new host record for this tick species. Taking into account the geographical position of the collection site and the known migration distance of the greater mouse-eared bat, the present data suggest the autochthonous occurrence of I. ariadnae in Germany. This is the first record of I. ariadnae in Germany, and in any country other than Hungary, where this species has been recently discovered.

DNA of Theileria orientalis, T. equi and T. capreoli in stable flies (Stomoxys calcitrans).

BACKGROUND: From a veterinary-medical point of view, the stable fly, Stomoxys calcitrans, is perhaps the economically most important blood-sucking muscoid fly species (Diptera: Muscidae), owing to its worldwide occurrence, frequently high local abundance, direct harm caused to livestock, pet animals and humans, as well as its vector role. Considering the latter in the context of protozoan parasites, the stable fly is a mechanical vector of trypanosomes and Besnoitia besnoiti. However, its role as a vector of piroplasms appears to be seldom studied, despite old data suggesting mechanical transmission of babesiae by dipteran flies. METHODS: In this study 395 stable flies (and one Haematobia stimulans) were collected at a cattle farm with known history of bovine theileriosis, and at further nine, randomly chosen locations in Hungary. These flies were separated according to sex (30 of them also cut into two parts: the head with mouthparts and the thorax-abdomen), followed by individual DNA extraction, then screening for piroplasms by PCR and sequencing. RESULTS: In stable flies, Theileria orientalis and T. capreoli were identified at the cattle farm and T. equi was identified in three other locations. At the cattle farm, significantly more male stable flies carried piroplasm DNA than females. There was no significant difference between the ratio of PCR-positive flies between the stable (void of cattle for at least two hours) and the pen on the pasture with cattle at the time of sampling. Among dissected flies (29 S. calcitrans and 1 H. stimulans), exclusively the thoracic-abdominal parts were PCR-positive, whereas the head and mouthparts remained negative. CONCLUSIONS: Theileria DNA is detectable in stable flies, in the case of T. orientalis at least for two hours after blood-feeding, and in the case of T. capreoli also in the absence of infected hosts (i.e. roe deer). Male flies rather than females, and thoracic-abdominal (most likely crop) contents rather than mouthparts may pose a risk of mechanical transmission. These data suggest that it is worth to study further the vector role of stable flies in the epidemiology of theilerioses, in which not the immediate, but rather the delayed type transmission seems possible.

Synergistic Radiosensitization by Gold Nanoparticles and the Histone Deacetylase Inhibitor SAHA in 2D and 3D Cancer Cell Cultures.

Radiosensitizing agents are capable of augmenting the damage of ionizing radiation preferentially on cancer cells, thereby increasing the potency and the specificity of radiotherapy. Metal-based nanoparticles have recently gathered ground in radio-enhancement applications, owing to their exceptional competence in amplifying the cell-killing effects of irradiation. Our aim was to examine the radiosensitizing performance of gold nanoparticles (AuNPs) and the chromatin-modifying histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alone and in combination. We observed that the colony-forming capability of cancer cells decreased significantly and the DNA damage, detected by γH2AX immunostaining, was substantially greater after combinational treatments than upon individual drug exposures followed by irradiation. Synergistic radiosensitizing effects of AuNPs and SAHA were proven on various cell lines, including radioresistant A549 and DU-145 cancer cells. 3D cultures often manifest radio- and drug-resistance, nevertheless, AuNPs in combination with SAHA could effectively enhance the potency of irradiation as the number of viable cells decreased significantly when spheroids received AuNP + SAHA prior to radiotherapy. Our results imply that a relaxed chromatin structure induced by SAHA renders the DNA of cancerous cells more susceptible to the damaging effects of irradiation-triggered, AuNP-released reactive electrons. This feature of AuNPs should be exploited in multimodal treatment approaches.

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.

3D bioprinted hydrogel model incorporating ß-tricalcium phosphate for calcified cartilage tissue engineering.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while-despite its key role in the osteochondral region-only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing ß-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

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.

Molecular identification of badger-associated Babesia sp. DNA in dogs: updated phylogeny of piroplasms infecting Caniformia.

BACKGROUND: Piroplasms are unicellular, tick-borne parasites. Among them, during the past decade, an increasing diversity of Babesia spp. has been reported from wild carnivores. On the other hand, despite the known contact of domestic and wild carnivores (e.g. during hunting), and a number of ixodid tick species they share, data on the infection of dogs with babesiae from other families of carnivores are rare. METHODS: In this study blood samples were collected from 90 dogs and five road-killed badgers. Ticks were also removed from these animals. The DNA was extracted from all blood samples, and from 33 ticks of badgers, followed by molecular analysis for piroplasms with PCR and sequencing, as well as by phylogenetic comparison of detected genotypes with piroplasms infecting carnivores. RESULTS: Eleven of 90 blood DNA extracts from dogs, and all five samples from badgers were PCR-positive for piroplasms. In addition to the presence of B. canis DNA in five dogs, sequencing identified the DNA of badger-associated "Babesia sp. Meles-Hu1" in six dogs and in all five badgers. The DNA of "Babesia sp. Meles-Hu1" occurred significantly more frequently in dogs often taken to forests (i.e. the preferred habitat of badgers in Hungary), than in dogs without this characteristic. Moreover, detection of DNA from this Babesia sp. was significantly associated with hunting dogs in comparison with dogs not used for hunting. Two PCR-positive dogs (in one of which the DNA of the badger-associated Babesia sp. was identified, whereas in the other the DNA of B. canis was present) showed clinical signs of babesiosis. Engorged specimens of both I. canisuga and I. hexagonus were collected from badgers with parasitaemia, but only I. canisuga contained the DNA of "Babesia sp. Meles-Hu1". This means a significant association of the DNA from "Babesia sp. Meles-Hu1" with I. canisuga. Phylogenetically, "Babesia sp. Meles-Hu1" belonged to the "B. microti" group. CONCLUSIONS: This is the first detection of the DNA from a badger-associated Babesia sp. in dogs, one of which also showed relevant clinical signs. Based on the number of dogs with blood samples containing the DNA of "Babesia sp. Meles-Hu1" in this study (i.e. exceeding the number of B. canis-positives), these findings should not be regarded as isolated cases. It is assumed that dogs, which are used for hunting or frequently visit forests, are more likely to be exposed to this piroplasm, probably as a consequence of infestation with I. canisuga from badgers or from the burrows of badgers. The above results suggest that "Babesia sp. Meles-Hu1" should be added to the range of piroplasms, which are naturally capable of infecting hosts from different families of Caniformia.

Molecular evidence of a badger-associated Ehrlichia sp., a Candidatus Neoehrlichia lotoris-like genotype and Anaplasma marginale in dogs.

The family Anaplasmataceae contains pathogenic and endosymbiotic bacteria of veterinary-medical importance. In this study, 90 blood samples from rural dogs, five blood samples from road-killed European badgers and 34 ticks, i.e. 27 Ixodes (Pholeoixodes) canisuga, six I. (Ph.) hexagonus and one Haemaphysalis concinna collected from the badgers were molecularly analysed for members of Anaplasmataceae. Apart from the molecular evidence of Anaplasma phagocytophilum in one dog and Wolbachia sp. associated with Dirofilaria repens in five dogs, four species/genotypes not yet known to occur in canine hosts have also been found. These included A. marginale in two dogs, a badger-associated Ehrlichia sp. in one dog, a Candidatus Neoehrlichia lotoris-like genotype in six dogs and the DNA of arthropod-associated wolbachiae in three dogs. In two badgers the DNA from the Candidatus N. lotoris-like genotype was identified. Among ticks, four I. canisuga carried the DNA of the above badger-associated Ehrlichia sp., one I. canisuga contained the Candidatus N. lotoris-like genotype, and in H. concinna Wolbachia DNA was present. In conclusion, results shown here should be interpreted as the first molecular evidence for exposure of dogs to three members of Anaplasmataceae, i.e. A. marginale, a badger-associated Ehrlichia sp. and a Candidatus N. lotoris-like agent. The presence of DNA in the blood of relevant animals may also indicate susceptibility to these bacteria, but in support of this, further studies are needed.

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

BACKGROUND: Adult 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. METHODS: Fleas 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. RESULTS: The 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. CONCLUSIONS: Ctenocephalides 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.

PLA short sub-micron fiber reinforcement of 3D bioprinted alginate constructs for cartilage regeneration.

In this study, we present an innovative strategy to reinforce 3D-printed hydrogel constructs for cartilage tissue engineering by formulating composite bioinks containing alginate and short sub-micron polylactide (PLA) fibers. We demonstrate that Young's modulus obtained for pristine alginate constructs (6.9 ± 1.7 kPa) can be increased threefold (up to 25.1 ± 3.8 kPa) with the addition of PLA short fibers. Furthermore, to assess the performance of such materials in cartilage tissue engineering, we loaded the bioinks with human chondrocytes and cultured in vitro the bioprinted constructs for up to 14 days. Live/dead assays at day 0, 3, 7 and 14 of in vitro culture showed that human chondrocytes were retained and highly viable (∼80%) within the 3D deposited hydrogel filaments, thus confirming that the fabricated composites materials represent a valid solution for tissue engineering applications. Finally, we show that the embedded chondrocytes during all the in vitro culture maintain a round morphology, a key parameter for a proper deposition of neocartilage extracellular matrix.

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.

Mitochondrial gene heterogeneity of the bat soft tick Argas vespertilionis (Ixodida: Argasidae) in the Palaearctic.

BACKGROUND: Recently, a high degree of mitochondrial gene heterogeneity was demonstrated between conspecific ixodid ticks of bats in Eurasia. Argas vespertilionis is a soft tick species of mainly vespertilionid bats, also with a wide distribution in the Old World. The aim of this study was to investigate the morphology, mitochondrial gene heterogeneity and host range of A. vespertilionis in the Old World. RESULTS: Altogether 318 soft tick larvae were collected from 17 bat species (belonging to six genera) in seven countries. Based on the general morphology (setal arrangement) of 314 A. vespertilionis larvae, and the detailed measurements of fifteen larvae, only minor morphological differences (in dorsal plate size and the type of serrate setae) were observed between specimens from Europe and Vietnam. On the other hand, cytochrome c oxidase subunit 1 (cox1) and 16S rRNA gene sequence analyses of 17 specimens showed that A. vespertilionis from Europe is genetically different (with up to 7.5% cox1 and 5.7% 16S rRNA gene sequence divergence) from specimens collected in Vietnam, and their phylogenetic separation is well supported. CONCLUSION: In its evaluated geographical range, no larval phenotypic differences justify the existence of separate species under the name A. vespertilionis. However, phylogenetic analyses based on two mitochondrial markers suggest that it represents a complex of at least two putative cryptic species. The broad host range of A. vespertilionis might partly explain its lower degree of mitochondrial gene heterogeneity in comparison with ixodid bat tick species over the same geographical region of Eurasia.

Duration of HPV-associated risk for high-grade cervical intraepithelial neoplasia.

OBJECTIVE: To evaluate the duration of high-risk HPV-associated cancer risk. STUDY DESIGN: Patients who had had a routine diagnostic Hybrid Capture Tube Test (HCT) due to squamous cell abnormalities of the uterine cervix were followed-up until the endpoint of histologically diagnosed cervical intraepithelial neoplasia (CIN). RESULTS: Six hundred and thirty-eight women were followed during a cumulative follow-up of 16,423 patient months. The adjusted relative risk associated with the positive HR-HCT test for high-grade CIN/52.0 (20.9-19.2)/ proved to be higher than that of the cytological atypia/5.44 (2.52-11.77)/. At the end of the 30 months of follow-up the crude and adjusted risks for CIN2+ were 214.3 (28.4-1615.7) and 196.7 (25.4-1525.2), respectively in the HPV 16/18 group, and after 30 months, the crude and adjusted RR decreased to 57.6 (10.4-318.9) and 29.2 (5.02-170.0). In the groups of other high-risk types and possibly high-risk types the general tendency was the same. However, new CIN2+ cases were not detected after the 30th month of follow-up in these later groups. CONCLUSIONS: HPV16/18 associated relative risk is nearly 200 times higher than that of the HPV negative population and an outstanding risk persists with duration of about 30 months. The risk is manifested in progression to high-grade CIN lesions mainly within a 2 years interval after the first detection of HPV 16/18 infection.