Description of Rhipicephalus hibericus sp. nov. (Ixodoidea: Ixodidae), a species of the Rhipicephalus sanguineus group in southwestern Europe.

We describe all the life stages of Rhipicephalus hibericus n. sp., provide the types, and present molecular support for a new species of the Rhipicephalus sanguineus sensu lato group, present in southwestern Europe, that has been historically confused with Rhipicephalus turanicus Pomerantzev, 1940. A new name is proposed for this taxon because it was impossible to ascribe to types of already described species in the group, deposited for more than 100 years in natural history institutions. The males have a dorsum showing deep and coarse punctations (absent in Rhipicephalus sanguineus sensu stricto) and adanal plates with large punctations (absent in R. sanguineus s.s.); the tail of the spiracular plate is as wide as the closest festoon (half the width in R. sanguineus s.s.). Females have large punctations in dorsal fields, a wide spiracular plate, and a "V" shaped genital opening; such a combination of characters cannot be found in other species of the group. Immatures are described from specimens collected on hosts (Rodentia and Eulipotyphla). Both larvae and nymphs are markedly smaller than R. sanguineus s.s. Nymphs display long, backward-projected auriculae; larvae are almost half the size of R. sanguineus s.s. The new species can hybridize with R. sanguineus s.s. in laboratory colonies producing an unfertile F2, laying brown and dry eggs that did not hatch. Phylogenetic analysis of partial coxI gene sequences placed R. hibericus in a well-supported clade with other sequences of R. sanguineus s.l. from Portugal, as a sister clade of R. sanguineus s.s. The new species does not belong to the R. turanicus group of species. Both 12S and 16S partial gene sequences were not as precise in the correct phylogenetic placement of R. hibericus, in part probably due to the existence of erroneously identified sequences in GenBank©. This description, together with the previous reinstatement of Rhipicephalus secundus and Rhipicephalus rutilus, and the description of the neotypes of R. sanguineus s.s. should help researchers to adequately identify their collections. Our findings demonstrate that R. turanicus is absent in southwestern Europe. Old collections should be re-examined to provide the actual range of the new species.

High diversity, novel genotypes, and vertical transmission of hemotropic Mycoplasma in micromammals.

Hemotropic mycoplasmas (hemoplasmas) are emerging zoonotic pathogens. Micromammals have received little attention as hosts for hemoplasmas despite their ubiquitous presence, high population abundances, and close association with humans. A PCR protocol targeting a fragment of the 16 S rRNA gene and direct sequencing in blood samples of 189 adult specimens and 35 fetuses belonging to three species of Eulipotyphla (shrews) and seven species of Rodentia, captured in three ecologically diverse habitats in North-Eastern Spain (Steppe, High Mountain, Mediterranean) yielded and occurrence of 26%, including 36% of 39 shrews and 23% of 150 rodents. Sequencing revealed the presence of 14 nucleotide sequence types (ntST) among the 56 readable sequences. In general, each ntST was associated with a given host species, although in some cases, the same ntST was sequenced in different species (chiefly rodents). Most ntST were closely related to rodent and/or bat hemoplasmas, but one was identical with Mycoplasma haemocanis/haemofelis, and others can be considered novel genotypes. High sequence diversity was detected in rodents, whereas in the white-toothed shrew (Crocidura russula), 9/11 sequences from two distant areas were identical. Phylogenetic and network analyses classified our sequences in different clades including hemoplasmas of rodents, carnivores, bats, and humans. Twelve of the fetuses (34.2%) of 9/12 litters (75.0%) of shrews and rodents were hemoplasma-positive, indicating frequent vertical transmission. Our study contributes to expanding our knowledge about the distribution, diversity, and transmission of hemoplasmas.

Bartonella infections are prevalent in rodents despite efficient immune responses.

BACKGROUND: Pathogens face strong selection from host immune responses, yet many host populations support pervasive pathogen populations. We investigated this puzzle in a model system of Bartonella and rodents from Israel's northwestern Negev Desert. We chose to study this system because, in this region, 75-100% of rodents are infected with Bartonella at any given time, despite an efficient immunological response. In this region, Bartonella species circulate in three rodent species, and we tested the hypothesis that at least one of these hosts exhibits a waning immune response to Bartonella, which allows reinfections. METHODS: We inoculated captive animals of all three rodent species with the same Bartonella strain, and we quantified the bacterial dynamics and Bartonella-specific immunoglobulin G antibody kinetics over a period of 139 days after the primary inoculation, and then for 60 days following reinoculation with the same strain. RESULTS: Contrary to our hypothesis, we found a strong, long-lasting immunoglobulin G antibody response, with protective immunological memory in all three rodent species. That response prevented reinfection upon exposure of the rodents to the same Bartonella strain. CONCLUSIONS: This study constitutes an initial step toward understanding how the interplay between traits of Bartonella and their hosts influences the epidemiological dynamics of these pathogens in nature.

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.

Viral Zoonoses in Small Wild Mammals and Detection of Hantavirus, Spain.

We screened 526 wild small mammals for zoonotic viruses in northwest Spain and found hantavirus in common voles (Microtus arvalis) (1.5%) and high prevalence (48%) of orthopoxvirus among western Mediterranean mice (Mus spretus). We also detected arenavirus among small mammals. These findings suggest novel risks for viral transmission in the region.

A road map for in vivo evolution experiments with blood-borne parasitic microbes.

Laboratory experiments in which blood-borne parasitic microbes evolve in their animal hosts offer an opportunity to study parasite evolution and adaptation in real time and under natural settings. The main challenge of these experiments is to establish a protocol that is both practical over multiple passages and accurately reflects natural transmission scenarios and mechanisms. We provide a guide to the steps that should be considered when designing such a protocol, and we demonstrate its use via a case study. We highlight the importance of choosing suitable ancestral genotypes, treatments, number of replicates per treatment, types of negative controls, dependent variables, covariates, and the timing of checkpoints for the experimental design. We also recommend specific preliminary experiments to determine effective methods for parasite quantification, transmission, and preservation. Although these methodological considerations are technical, they also often have conceptual implications. To this end, we encourage other researchers to design and conduct in vivo evolution experiments with blood-borne parasitic microbes, despite the challenges that the work entails.

Patterns of flea infestation in rodents and insectivores from intensified agro-ecosystems, Northwest Spain.

BACKGROUND: Fleas frequently infest small mammals and play important vectoring roles in the epidemiology of (re)emerging zoonotic disease. Rodent outbreaks in intensified agro-ecosystems of North-West Spain have been recently linked to periodic zoonotic diseases spillover to local human populations. Obtaining qualitative and quantitative information about the composition and structure of the whole flea and small mammal host coexisting communities is paramount to understand disease transmission cycles and to elucidate the disease-vectoring role of flea species. The aims of this research were to: (i) characterise and quantify the flea community parasiting a small mammal guild in intensive farmlands in North-West Spain; (ii) determine and evaluate patterns of co-infection and the variables that may influence parasitological parameters. METHODS: We conducted a large-scale survey stratified by season and habitat of fleas parasitizing the small mammal host guild. We report on the prevalence, mean intensity, and mean abundance of flea species parasitizing Microtus arvalis, Apodemus sylvaticus, Mus spretus and Crocidura russula. We also report on aggregation patterns (variance-to-mean ratio and discrepancy index) and co-infection of hosts by different flea species (Fager index) and used generalized linear mixed models to study flea parameter variation according to season, habitat and host sex. RESULTS: Three flea species dominated the system: Ctenophthalmus apertus gilcolladoi, Leptopsylla taschenbergi and Nosopsyllus fasciatus. Results showed a high aggregation pattern of fleas in all hosts. All host species in the guild shared C. a. gilcolladoi and N. fasciatus, but L. taschenbergi mainly parasitized mice (M. spretus and A. sylvaticus). We found significant male-biased infestation patterns in mice, seasonal variations in flea abundances for all rodent hosts (M. arvalis, M. spretus and A. sylvaticus), and relatively lower infestation values for voles inhabiting alfalfas. Simultaneous co-infections occurred in a third of all hosts, and N. fasciatus was the most common flea co-infecting small mammal hosts. CONCLUSIONS: The generalist N. fasciatus and C. a. gilcolladoi dominated the flea community, and a high percentage of co-infections with both species occurred within the small mammal guild. Nosopsyllus fasciatus may show higher competence of inter-specific transmission, and future research should unravel its role in the circulation of rodent-borne zoonoses.

Numerical response of a mammalian specialist predator to multiple prey dynamics in Mediterranean farmlands.

The study of rodent population cycles has greatly contributed, both theoretically and empirically, to our understanding of the circumstances under which predator-prey interactions destabilize populations. According to the specialist predator hypothesis, reciprocal interactions between voles and small predators that specialize on voles, such as weasels, can cause multiannual cycles. A fundamental feature of classical weasel-vole models is a long time-lag in the numerical response of the predator to variations in prey abundance: weasel abundance increases with that of voles and peaks approximately 1 yr later. We investigated the numerical response of the common weasel (Mustela nivalis) to fluctuating abundances of common voles (Microtus arvalis) in recently colonized agrosteppes of Castilla-y-Léon, northwestern Spain, at the southern limit of the species' range. Populations of both weasels and voles exhibited multiannual cycles with a 3-yr period. Weasels responded quickly and numerically to changes in common-vole abundance, with a time lag between prey and weasel abundance that did not exceed 4 months and occurred during the breeding season, reflecting the quick conversion of prey into predator offspring and/or immigration to sites with high vole populations. We found no evidence of a sustained, high weasel abundance following vole abundance peaks. Weasel population growth rates showed spatial synchrony across study sites approximately 60 km apart. Weasel dynamics were more synchronized with that of common voles than with other prey species (mice or shrews). However, asynchrony within, as well as among sites, in the abundance of voles and alternative prey suggests that weasel mobility could allow them to avoid starvation during low-vole phases, precluding the emergence of prolonged time lag in the numerical response to voles. Our observations are inconsistent with the specialist predator hypothesis as currently formulated, and suggest that weasels might follow rather than cause the vole cycles in northwestern Spain. The reliance of a specialized predator on a functional group of prey such as small rodents does not necessarily lead to a long delay in the numerical response by the predator, depending on the spatial and interspecific synchrony in prey dynamics.

Zoonotic Bacteria in Fleas Parasitizing Common Voles, Northwestern Spain.

We detected Francisella tularensis and Bartonella spp. in fleas parasitizing common voles (Microtus arvalis) from northwestern Spain; mean prevalence was 6.1% for F. tularensis and 51% for Bartonella spp. Contrasted vector-host associations in the prevalence of these bacteria suggest that fleas have distinct roles in the transmission cycle of each pathogen in nature.

Zoonotic pathogens in fluctuating common vole (Microtus arvalis) populations: occurrence and dynamics.

Diseases and host dynamics are linked, but their associations may vary in strength, be time-lagged, and depend on environmental influences. Where a vector is involved in disease transmission, its dynamics are an additional influence, and we often lack a general understanding on how diseases, hosts and vectors interact. We report on the occurrence of six zoonotic arthropod-borne pathogens (Anaplasma, Bartonella, Borrelia, Coxiella, Francisella and Rickettsia) in common voles (Microtus arvalis) throughout a population fluctuation and how their prevalence varies according to host density, seasonality and vector prevalence. We detected Francisella tularensis and four species of Bartonella, but not Anaplasma, Borrelia, Coxiella or Rickettsia. Bartonella taylorii and B. grahamii prevalence increased and decreased with current host (vole and mice) density, respectively, and increased with flea prevalence. Bartonella doshiae prevalence decreased with mice density. These three Bartonella species were also more prevalent during winter. Bartonella rochalimae prevalence varied with current and previous vole density (delayed-density dependence), but not with season. Coinfection with F. tularensis and Bartonella occurred as expected from the respective prevalence of each disease in voles. Our results highlight that simultaneously considering pathogen, vector and host dynamics provide a better understanding of the epidemiological dynamics of zoonoses in farmland rodents.

Density-Dependent Prevalence of Francisella tularensis in Fluctuating Vole Populations, Northwestern Spain.

Tularemia in humans in northwestern Spain is associated with increases in vole populations. Prevalence of infection with Francisella tularensis in common voles increased to 33% during a vole population fluctuation. This finding confirms that voles are spillover agents for zoonotic outbreaks. Ecologic interactions associated with tularemia prevention should be considered.

Tularemia Outbreaks and Common Vole (Microtus arvalis) Irruptive Population Dynamics in Northwestern Spain, 1997-2014.

During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain.