Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples.

Introduction: Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited. Methods: To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity. Results: We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries. Discussion: Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

A Multidisciplinary Approach to the First Autochthonous Case of Tularemia Reported in Portugal.

Francisella tularensis, a Gram-negative coccobacillus, is a highly virulent pathogen responsible for several zoonotic outbreaks in Europe in the last few decades. The authors report the case of a 46-year-old male who developed fever, myalgias and headache a week after having contact with animal feed contaminated by rodents. Serological tests were positive for Francisella tularensis. This first case of autochthonous tularemia in Portugal led to an intensive investigation involving several healthcare services and national governmental authorities. The authors address the possible underdiagnosis of this infection in the country.

Francisella tularensis, um cocobacilo Gram-negativo, é um microrganismo infecioso altamente virulento, responsável por vários surtos de doença na Europa nas últimas décadas. Os autores apresentam o caso de um homem de 46 anos com febre, mialgias e cefaleias cerca de uma semana após contacto com ração de animais contaminada por roedores. O estudo serológico foi positivo para Francisella tularensis. Este caso, o primeiro caso de tularémia autóctone notificado em Portugal, originou uma intensa investigação envolvendo diversas autoridades de saúde e governamentais portuguesas. Os autores alertam para a possibilidade de subdiagnóstico desta doença no país.

Diversity of microorganisms in Hyalomma aegyptium collected from spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia.

Ticks carry a diverse community of microorganisms including non-pathogenic symbionts, commensals, and pathogens, such as viruses, bacteria, protozoans, and fungi. The assessment of tick-borne microorganisms (TBM) in tortoises and their ticks is essential to understand their eco-epidemiology, and to map and monitor potential pathogens to humans and other animals. The aim of this study was to characterize the diversity of microorganisms found in ticks collected from the spur-thighed tortoise (Testudo graeca) in North Africa and Anatolia. Ticks feeding on wild T. graeca were collected, and pathogens were screened by polymerase chain reaction using group-specific primers. In total, 131 adult Hyalomma aegyptium ticks were collected from 92 T. graeca in Morocco (n = 48), Tunisia (n = 2), Algeria (n = 70), and Turkey (n = 11). Bacteria and protozoa detected included Hemolivia mauritanica (22.9%), Midichloria mitochondrii (11.4%), relapsing-fever borreliae (8.4%), Ehrlichia spp. (7.6%), Rickettsia spp. (3.4%), Borrelia burgdorferi s.l. (0.9%), Francisella spp. (0.9%), and Wolbachia spp. (0.8%). The characterization of Rickettsia included R. sibirica mongolitimonae (Algeria), R. aeschlimannii (Turkey), and R.africae (Morocco). Hemolivia mauritanica and Ehrlichia spp. prevalence varied significantly with the sampling region/country. We did not detect significant associations in microorganism presence within ticks, nor between microorganism presence and tick mitochondrial DNA haplogroups. This is the first report of Francisella persica-like, relapsing fever borreliae, M. mitochondrii, and Wolbachia spp. in H. aegyptium ticks collected from wild hosts from the South and Eastern Mediterranean region, and of R. sibirica mongolitimonae and R. africae in H. aegyptium from Algeria and Morocco, respectively. Given that T. graeca is a common species in commercial and non-commercial pet trade, the evaluation of the role of this species and its ticks as hosts for TBM is particularly relevant for public health.

Bactericidal activity of avian complement: a contribution to understand avian-host tropism of Lyme borreliae.

Complement has been considered as an important factor impacting the host-pathogen association of spirochetes belonging to the Borrelia burgdorferi sensu lato complex, and may play a role in the spirochete's ecology. Birds are known to be important hosts for ticks and in the maintenance of borreliae. Recent field surveys and laboratory transmission studies indicated that certain avian species act as reservoir hosts for different Borrelia species. Nevertheless, our current understanding of the molecular mechanisms determining host tropism of Borrelia is still in its fledgling stage. Concerning the role of complement in avian-host tropism, only a few bird species and Borrelia species have been analysed so far. Here, we performed in vitro serum bactericidal assays with serum samples collected from four bird species including the European robin Erithacus rubecula, the great tit Parus major, the Eurasian blackbird Turdus merula, and the racing pigeon Columba livia, as well as four Borrelia species (B. afzelii, B. garinii, B. valaisiana, and B. burgdorferi sensu stricto). From July to September 2019, juvenile wild birds were caught using mist nets in Portugal. Racing pigeons were sampled in a loft in October 2019. Independent of the bird species analysed, all Borrelia species displayed an intermediate serum-resistant or serum-resistant phenotype except for B. afzelii challenged with serum from blackbirds. This genospecies was efficiently killed by avian complement, suggesting that blackbirds served as dead-end hosts for B. afzelii. In summary, these findings suggest that complement contributes in the avian-spirochete-tick infection cycle and in Borrelia-host tropism.

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

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

Getting under the birds' skin: tissue tropism of Borrelia burgdorferi s.l. in naturally and experimentally infected avian hosts.

Wild birds are frequently exposed to the zoonotic tick-borne bacteria Borrelia burgdorferi sensu lato (s.l.), and some bird species act as reservoirs for some Borrelia genospecies. Studying the tropism of Borrelia in the host, how it is sequestered in different organs, and whether it is maintained in circulation and/or in the host's skin is important to understand pathogenicity, infectivity to vector ticks and reservoir competency.We evaluated tissue dissemination of Borrelia in blackbirds (Turdus merula) and great tits (Parus major), naturally and experimentally infected with Borrelia genospecies from enzootic foci. We collected both minimally invasive biological samples (feathers, skin biopsies and blood) and skin, joint, brain and visceral tissues from necropsied birds. Infectiousness of the host was evaluated through xenodiagnoses and infection rates in fed and moulted ticks. Skin biopsies were the most reliable method for assessing avian hosts' Borrelia infectiousness, which was supported by the agreement of infection status results obtained from the analysis of chin and lore skin samples from necropsied birds and of their xenodiagnostic ticks, including a significant correlation between the estimated concentration of Borrelia genome copies in the skin and the Borrelia infection rate in the xenodiagnostic ticks. This confirms a dermatropism of Borrelia garinii, B. valaisiana and B. turdi in its avian hosts. However, time elapsed from exposure to Borrelia and interaction between host species and Borrelia genospecies may affect the reliability of skin biopsies. The blood was not useful to assess infectiousness of birds, even during the period of expected maximum spirochetaemia. From the tissues sampled (foot joint, liver, spleen, heart, kidney, gut and brain), Borrelia was detected only in the gut, which could be related with infection mode, genospecies competition, genospecies-specific seasonality and/or excretion processes.

First Case of Tularemia Reported in Portugal: Probably of Imported Origin.

The authors report the case of a 47-year-old man who walked in the countryside on the island of Bornholm, during the summer period. Three days later, fever, myalgias and adynamia began. The serological tests, Real-time PCR and isolation of the bacteria from the culture of lymph biopsy confirmed the presence of Francisella tularensis subsp. holarctica.

Bridging of cryptic Borrelia cycles in European songbirds.

The principal European vector for Borrelia burgdorferi s.l., the causative agents of Lyme disease, is the host-generalist tick Ixodes ricinus. Almost all terrestrial host-specialist ticks have been supposed not to contribute to the terrestrial Borrelia transmission cycles. Through an experiment with blackbirds, we show successful transmission by the widespread I. frontalis, an abundant bird-specialized tick that infests a broad range of songbirds. In the first phase of the experiment, we obtained Borrelia-infected I. frontalis (infection rate: 19%) and I. ricinus (17%) nymphs by exposing larvae to wild blackbirds that carried several genospecies (Borrelia turdi, B. valaisiana, B. burgdorferi s.s.). In the second phase, pathogen-free blackbirds were exposed to these infected nymphs. Both tick species were able to infect the birds, as indicated by the analysis of xenodiagnostic I. ricinus larvae which provided evidence for both co-feeding and systemic transmission (infection rates: 10%-60%). Ixodes frontalis was shown to transmit B. turdi spirochetes, while I. ricinus transmitted both B. turdi and B. valaisiana. Neither species transmitted B. burgdorferi s.s. European enzootic cycles of Borrelia between songbirds and their ornithophilic ticks do exist, with I. ricinus potentially acting as a bridging vector towards mammals, including man.

Ticks and tick-borne pathogens in wild birds in Greece.

Wild birds are common hosts of ticks and can transport them for long distances, contributing to the spreading of tick-borne pathogens. The information about ticks on birds and tick-borne pathogens in Greece is limited. The present study aimed to evaluate the prevalence and species of ticks infesting wild resident birds (mostly small passerines) in Greece, and to assess Borrelia and Rickettsia infection in the collected ticks. Detection of Borrelia burgdorferi s.l. was performed by nested PCR targeting the flaB gene. Rickettsia spp. were detected by PCR targeting the gltA and ompA genes. Seven (2 %) out of 403 birds examined in northern Greece in 2013 were infested with 15 ticks, identified as Ixodes frontalis, Ixodes acuminatus, Hyalomma marginatum, Hyalomma aegyptium and Hyalomma sp. All ticks were negative for Borrelia spp. while four of them were positive for rickettsiae (Rickettsia aeschlimannii in H. aegyptium and Rickettsia sp. in I. frontalis, H. aegyptium and H. marginatum). Ixodes acuminatus is reported for the first time in Greece and Sylvia borin is reported as a new host record for I. acuminatus.

Meningomyeloradiculitis as an Unusual Presentation of Neuroborreliosis in Childhood.

We report a pediatric case of Lyme neuroborreliosis-associated meningomyeloradiculitis with atypical manifestations and negative initial cerebrospinal fluid borrelial antibodies. Transverse myelitis and painful radiculoneuritis have rarely been described in pediatric neuroborreliosis. Clinical manifestations are wide ranging and nonspecific, and the serologic diagnosis is often delayed in the acute phase.

The importance of lizards and small mammals as reservoirs for Borrelia lusitaniae in Portugal.

Borrelia lusitaniae is a pathogen frequent in the Mediterranean area. Apart from lizards, evidence for birds and small mammals as competent reservoirs for this genospecies has been occasional. We collected questing ticks, skin biopsies and Ixodes sp. ticks feeding on lizards, birds and small mammals in a B. burgdorferi s.l. (sensu lato) enzootic area to assess their importance in the maintenance of B. lusitaniae. Borrelia lusitaniae was the most prevalent genospecies in questing ticks and was commonly found in larvae feeding on Psammodromus algirus. One biopsy infected with B. lusitaniae was collected from the tail of one Podarcis hispanica, which suggests systemic infection. Ixodes ricinus larvae feeding on Apodemus sylvaticus were infected with B. lusitaniae but with a lower prevalence. Our results reinforce the importance of lizards as reservoirs for B. lusitaniae, suggesting that P. algirus, in particular, acts as main reservoir for B. lusitaniae in Portugal.

Blackbirds Turdus merula as competent reservoirs for Borrelia turdi and Borrelia valaisiana in Portugal: evidence from a xenodiagnostic experiment.

To confirm that thrushes, such as blackbirds Turdus merula, play a role as reservoir for some Borrelia genospecies, we performed a xenodiagnostic experiment with blackbirds captured in a mixed wood located in Western Portugal where Borrelia turdi, an uncommon genospecies in Europe, was the most prevalent genospecies associated with birds. Two out of five birds harboured B. turdi infected Ixodes frontalis at the time of capture. Four out of five birds transmitted spirochaetes to Ixodes ricinus xenodiagnostic ticks: two birds transmitted Borrelia valaisiana to 25.7% and 10.5% of ticks, and two transmitted B. turdi to 6.4% and 5.4% of ticks. Our results showed that blackbirds transmit B. valaisiana and B. turdi to I. ricinus feeding larvae, acting as reservoir hosts for these genospecies in nature.

Portuguese hosts for Ornithodoros erraticus ticks.

The hematophagous soft tick Ornithodoros erraticus feeds nocturnally on multiple warm-blooded vertebrate hosts. This tick is often found living buried in the soil of traditional pigpens. O. erraticus is an important infectious disease vector both for humans and animals. In the Iberian Peninsula, this tick serves as the vector of human tick-borne relapsing fever caused by the spirochete Borrelia hispanica. The natural ecosystems maintaining this spirochete are not well understood, with details of competent vertebrate reservoirs and tick-host interactions poorly understood. Investigation of arthropod blood meal composition provides evidence linking the vector to specific hosts, providing insights into possible disease reservoirs. Ticks collected from two pigpens located in southern Portugal were subjected to blood meal analysis. PCR amplification of vertebrate cytochrome b was used to disclose the original host from which 349 ticks had derived their previous blood meal. Host origins for blood meal analysis from 79 of 349 ticks revealed that 46.8% had previously fed from pigs, 35.4% human, 13.9% bovine, 5.1% sheep, 1.3% rodent, and 1.3% from birds. Three samples revealed mixed blood meals, namely, human-pig (1.3%), sheep-pig (1.3%), and bovine-pig (1.3%). The major role of pigs as hosts is consistent with fieldwork observations and underlines the importance of pigs for maintaining O. erraticus tick populations. Humans serve as accidental hosts, frequently confirmed by reports from both producers and veterinarians. Other livestock species and wildlife prevalent in the region appear only to have a minor role in maintaining this tick. The results demonstrate the importance of blood meal analysis to determine tick hosts providing a tool for investigation of sylvatic cycle for Borrelia hispanica.