Water and mosquitoes as key components of the infective cycle of Francisella tularensis in Europe: a review.

Francisella tularensis is the pathogen of tularemia, a zoonotic disease that have a broad range of hosts. Its epidemiology is related to aquatic environments, particularly in the subspecies holarctica. In this review, we explore the role of water and mosquitoes in the epidemiology of Francisella in Europe. F. tularensis epidemiology has been linked to natural waters, where its persistence has been associated with biofilm and amebas. In Sweden and Finland, the European countries where most human cases have been reported, mosquito bites are a main route of transmission. F. tularensis is present in other European countries, but to date positive mosquitoes have not been found. Biofilm and amebas are potential sources of Francisella for mosquito larvae, however, mosquito vector capacity has not been demonstrated experimentally, with the need to be studied using local species to uncover a potential transmission adaptation. Transstadial, for persistence through life stages, and mechanical transmission, suggesting contaminated media as a source for infection, have been studied experimentally for mosquitoes, but their natural occurrence needs to be evaluated. It is important to clear up the role of different local mosquito species in the epidemiology of F. tularensis and their importance in all areas where tularemia is present.

Exploring the influence of density-dependence and weather on the spatial and temporal variation in common vole (Microtus arvalis) abundance in Castilla y León, NW Spain.

BACKGROUND: The common vole has invaded the agroecosystems of northwestern Spain, where outbreaks cause important crop damage and management costs. Little is yet known about the factors causing or modulating vole fluctuations. Here, we used 11 years of vole abundance monitoring data in 40 sites to study density-dependence and weather influence on vole dynamics. Our objective was to identify the population dynamics structure and determine whether there is direct or delayed density-dependence. An evaluation of climatic variables followed, to determine whether they influenced vole population peaks. RESULTS: First- and second-order outbreak dynamics were detected at 7 and 33 study sites, respectively, together with second-order variability in periodicity (2-3 to 4-5-year cycles). Vole population growth was explained by previous year abundance (mainly numbers in summer and spring) at 21 of the sites (52.5%), by weather variables at 11 sites (27.5%; precipitation or temperature in six and five sites, respectively), and by a combination of previous abundance and weather variables in eight sites (20%). CONCLUSIONS: We detected variability in vole spatiotemporal abundance dynamics, which differs in cyclicity and period. We also found regional variation in the relative importance of previous abundances and weather as factors modulating vole fluctuations. Most vole populations were cyclical, with variable periodicity across the region. Our study is a first step towards the development of predictive modeling, by disclosing relevant factors that might trigger vole outbreaks. It improves decision-making processes within integrated management dealing with mitigation of the agricultural impacts caused by voles. © 2023 Society of Chemical Industry.

Vole outbreaks may induce a tularemia disease pit that prevents Iberian hare population recovery in NW Spain.

Iberian hare populations have suffered severe declines during recent decades in Spain. Between 1970 and 1990s, a rapid increase in irrigation crop surface in NW Spain (Castilla-y-León region) was followed by a common vole massive range expansion and complete colonization of lowland irrigated agricultural landscapes from mountainous habitats. The subsequent large cyclic fluctuations in abundance of colonizing common voles have contributed to a periodic amplification of Francisella tularensis, the etiological agent that causes human tularemia outbreaks in the region. Tularemia is a fatal disease to lagomorphs, so we hypothesize that vole outbreaks would lead to disease spill over to Iberian hares, increasing prevalence of tularemia and declines among hare populations. Here we report on the possible effects that vole abundance fluctuations and concomitant tularemia outbreaks had on Iberian hare populations in NW Spain. We analysed hare hunting bag data for the region, which has been recurrently affected by vole outbreaks between 1996 and 2019. We also compiled data on F. tularensis prevalence in Iberian hares reported by the regional government between 2007 and 2016. Our results suggest that common vole outbreaks may limit the recovery of hare populations by amplifying and spreading tularemia in the environment. The recurrent rodent-driven outbreaks of tularemia in the region may result in a "disease pit" to Iberian hares: at low host densities, the rate of population growth in hares is lower than the rate at which disease-induced mortality increases with increased rodent host density, therefore, keeping hare populations on a low-density equilibrium. We highlight future research needs to clarify tularemia transmission pathways between voles and hares and confirm a disease pit process.

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.

From pattern to process? Dual travelling waves, with contrasting propagation speeds, best describe a self-organised spatio-temporal pattern in population growth of a cyclic rodent.

The dynamics of cyclic populations distributed in space result from the relative strength of synchronising influences and the limited dispersal of destabilising factors (activators and inhibitors), known to cause multi-annual population cycles. However, while each of these have been well studied in isolation, there is limited empirical evidence of how the processes of synchronisation and activation-inhibition act together, largely owing to the scarcity of datasets with sufficient spatial and temporal scale and resolution. We assessed a variety of models that could be underlying the spatio-temporal pattern, designed to capture both theoretical and empirical understandings of travelling waves using large-scale (>35,000 km2 ), multi-year (2011-2017) field monitoring data on abundances of common vole (Microtus arvalis), a cyclic agricultural rodent pest. We found most support for a pattern formed from the summation of two radial travelling waves with contrasting speeds that together describe population growth rates across the region.

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.

Linking Zoonosis Emergence to Farmland Invasion by Fluctuating Herbivores: Common Vole Populations and Tularemia Outbreaks in NW Spain.

The expansion and intensification of agriculture are driving profound changes in ecosystems worldwide, favoring the (re)emergence of many human infectious diseases. Muroid rodents are a key host group for zoonotic infectious pathogens and frequently invade farming environments, promoting disease transmission and spillover. Understanding the role that fluctuating populations of farm dwelling rodents play in the epidemiology of zoonotic diseases is paramount to improve prevention schemes. Here, we review a decade of research on the colonization of farming environments in NW Spain by common voles (Microtus arvalis) and its public health impacts, specifically periodic tularemia outbreaks in humans. The spread of this colonizing rodent was analogous to an invasion process and was putatively triggered by the transformation and irrigation of agricultural habitats that created a novel terrestrial-aquatic interface. This irruptive rodent host is an effective amplifier for the Francisella tularensis bacterium during population outbreaks, and human tularemia episodes are tightly linked in time and space to periodic (cyclic) variations in vole abundance. Beyond the information accumulated to date, several key knowledge gaps about this pathogen-rodent epidemiological link remain unaddressed, namely (i) did colonizing vole introduce or amplified pre-existing F. tularensis? (ii) which features of the "Francisella-Microtus" relationship are crucial for the epidemiology of tularemia? (iii) how virulent and persistent F. tularensis infection is for voles under natural conditions? and (iv) where does the bacterium persist during inter-epizootics? Future research should focus on more integrated, community-based approaches in order to understand the details and dynamics of disease circulation in ecosystems colonized by highly fluctuating hosts.

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.

Population cycles and outbreaks of small rodents: ten essential questions we still need to solve.

Most small rodent populations in the world have fascinating population dynamics. In the northern hemisphere, voles and lemmings tend to show population cycles with regular fluctuations in numbers. In the southern hemisphere, small rodents tend to have large amplitude outbreaks with less regular intervals. In the light of vast research and debate over almost a century, we here discuss the driving forces of these different rodent population dynamics. We highlight ten questions directly related to the various characteristics of relevant populations and ecosystems that still need to be answered. This overview is not intended as a complete list of questions but rather focuses on the most important issues that are essential for understanding the generality of small rodent population 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.

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.

Unintentional effects of environmentally-friendly farming practices: Arising conflicts between zero-tillage and a crop pest, the common vole (Microtus arvalis).

Common voles are a main European facultative, fossorial, farmland rodent pest that can greatly reduce crop yields during population outbreaks. Crop protection against common voles is a complex task that requires the consideration of a set of preventive and control measures within an integrated pest management strategy. A possible option could be to modify farming practices to reduce the availability of refuges for rodents and the damage to crops that they subsequently cause. Farming, however, must simultaneously meet multiple goals including the reduction of the carbon (C) emissions, soil erosion and water use, and the improvement of soil quality. Crop establishment through conservation agriculture strategies, like zero-tillage, would reduce crop management investment, but is also promoted in many regions to reduce C emissions and increase soil organic matter. It could, however, create favourable refuge habitats for fossorial rodent crop pests, like common voles, benefitting from reduced soil disturbance between crop rotations and thus increasing burrow persistence. Assessing the impact that tillage practices, their interaction with different crops and the influence of proximity to potential common vole sources, have on common vole occupancy could provide a valuable tool within an integrated management strategy. Using a 2-ha experimental field with 62 plots 180 m2 (each roughly matching common vole home range size) located experimental plots in north-western Spain, we tested how tillage practices, crop type (wheat, barley, vetch, Narbonne vetch, pea and fallow) and distances from possible colonization sources affect field use by common vole during low population density conditions. Our results show that tillage practices have more influence on common vole occurrence (zero tillage > reduced and conventional tillage) than other aspects such as crop type thus supporting the hypothesis that tillage practices play a key role in common vole habitat use.

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.

Spatial capture-recapture design and modelling for the study of small mammals.

Spatial capture-recapture modelling (SCR) is a powerful analytical tool to estimate density and derive information on space use and behaviour of elusive animals. Yet, SCR has been seldom applied to the study of ecologically keystone small mammals. Here we highlight its potential and requirements with a case study on common voles (Microtus arvalis). First, we address mortality associated with live-trapping, which can be high in small mammals, and must be kept minimal. We designed and tested a nest box coupled with a classic Sherman trap and show that it allows a 5-fold reduction of mortality in traps. Second, we address the need to adjust the trapping grid to the individual home range to maximize spatial recaptures. In May-June 2016, we captured and tagged with transponders 227 voles in a 1.2-ha area during two monthly sessions. Using a Bayesian SCR with a multinomial approach, we estimated: (1) the baseline detection rate and investigated variation according to sex, time or behaviour (aversion/attraction after a previous capture); (2) the parameter sigma that describes how detection probability declines as a function of the distance to an individual´s activity centre, and investigated variation according to sex; and (3) density and population sex-ratio. We show that reducing the maximum distance between traps from 12 to 9.6m doubled spatial recaptures and improved model predictions. Baseline detection rate increased over time (after overcoming a likely aversion to entering new odourless traps) and was greater for females than males in June. The sigma parameter of males was twice that of females, indicating larger home ranges. Density estimates were of 142.92±38.50 and 168.25±15.79 voles/ha in May and June, respectively, with 2-3 times more females than males. We highlight the potential and broad applicability that SCR offers and provide specific recommendations for using it to study small mammals like voles.

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.

Avian predators as a biological control system of common vole (Microtus arvalis) populations in north-western Spain: experimental set-up and preliminary results.

BACKGROUND: Ecologically based rodent pest management using biological control has never been evaluated for vole plagues in Europe, although it has been successfully tested in other systems. The authors report on the first large-scale replicated experiment to study the usefulness of nest-box installation for increasing the breeding density of common kestrels (Falco tinnunculus) and barn owls (Tyto alba) as a potential biological control of common vole (Microtus arvalis) abundance in agricultural habitats in north-western Spain. RESULTS: The results show that: (1) population density of both predator species increased in response to both nest-site availability and vole density; (2) voles are a major prey for the common kestrels during the breeding period; (3) vole density during the increase phase of a population cycle may be reduced in crop fields near nest boxes. CONCLUSION: The installation of nest boxes provides nesting sites for barn owls and kestrels. Kestrel populations increased faster than in areas without artificial nests, and the common vole was one of their main prey during the breeding season. The results suggest that local (field) effects could be found in terms of reduced vole density. If so, this could be an environmentally friendly and cheap vole control technique to be considered on a larger scale.

Sperm competition differentially affects swimming velocity and size of spermatozoa from closely related muroid rodents: head first.

Sperm competition favours an increase in sperm swimming velocity that maximises the chances that sperm will reach the ova before rival sperm and fertilise. Comparative studies have shown that the increase in sperm swimming speed is associated with an increase in total sperm size. However, it is not known which are the first evolutionary steps that lead to increases in sperm swimming velocity. Using a group of closely related muroid rodents that differ in levels of sperm competition, we here test the hypothesis that subtle changes in sperm design may represent early evolutionary changes that could make sperm swim faster. Our findings show that as sperm competition increases so does sperm swimming speed. Sperm swimming velocity is associated with the size of all sperm components. However, levels of sperm competition are only related to an increase in sperm head area. Such increase is a consequence of an increase in the length of the sperm head, and also of the presence of an apical hook in some of the species studied. These findings suggest that the presence of a hook may modify the sperm head in such a way that would help sperm swim faster and may also be advantageous if sperm with larger heads are better able to attach to the epithelial cells lining the lower isthmus of the oviduct where sperm remain quiescent before the final race to reach the site of fertilisation.

Sperm competition, sperm numbers and sperm quality in muroid rodents.

Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An "overall sperm quality" parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and developmental pathways underlying processes of sperm formation, maturation, transport in the female reproductive tract, and preparation for fertilization must all evolve in concert.

Sexual selection halts the relaxation of protamine 2 among rodents.

Sexual selection has been proposed as the driving force promoting the rapid evolutionary changes observed in some reproductive genes including protamines. We test this hypothesis in a group of rodents which show marked differences in the intensity of sexual selection. Levels of sperm competition were not associated with the evolutionary rates of protamine 1 but, contrary to expectations, were negatively related to the evolutionary rate of cleaved- and mature-protamine 2. Since both domains were found to be under relaxation, our findings reveal an unforeseen role of sexual selection: to halt the degree of degeneration that proteins within families may experience due to functional redundancy. The degree of relaxation of protamine 2 in this group of rodents is such that in some species it has become dysfunctional and it is not expressed in mature spermatozoa. In contrast, protamine 1 is functionally conserved but shows directed positive selection on specific sites which are functionally relevant such as DNA-anchoring domains and phosphorylation sites. We conclude that in rodents protamine 2 is under relaxation and that sexual selection removes deleterious mutations among species with high levels of sperm competition to maintain the protein functional and the spermatozoa competitive.