Small mammals as carriers of zoonotic bacteria on pig and cattle farms - Prevalence and risk of exposure in an integrative approach.

To prevent foodborne infections from pigs and cattle, the whole food chain must act to minimize the contamination of products, including biosecurity measures which prevent infections via feed and the environment in production farms. Rodents and other small mammals can be reservoirs of and key vectors for transmitting zoonotic bacteria and viruses to farm animals, through direct contact but more often through environmental contamination. In line with One Health concept, we integrated results from a sampling study of small mammals in farm environments and data from a capture-recapture experiment into a probabilistic model which quantifies the degree of environmental exposure of zoonotic bacteria by small mammals to farm premises. We investigated more than 1200 small mammals trapped in and around 38 swine and cattle farm premises in Finland in 2017/2018. Regardless of the farm type, the most common species caught were the yellow-necked mouse (Apodemus flavicollis), bank vole (Clethrionomys glareolus), and house mouse (Mus musculus). Of 554 intestine samples (each pooled from 1 to 10 individuals), 33% were positive for Campylobacter jejuni. Yersinia enterocolitica was detected in 8% of the pooled samples, on 21/38 farm premises. Findings of Salmonella and the Shiga-toxin producing Escherichia coli (STEC) were rare: the pathogens were detected in only single samples from four and six farm premises, respectively. The prevalence of Campylobacter, Salmonella, Yersinia and STEC in small mammal populations was estimated as 26%/13%, 1%/0%, 2%/3%, 1%/1%, respectively, in 2017/2018. The exposure probability within the experimental period of four weeks on farms was 17-60% for Campylobacter and 0-3% for Salmonella. The quantitative model is readily applicable to similar integrative studies. Our results indicate that small mammals increase the risk of exposure to zoonotic bacteria in animal production farms, thus increasing risks also for livestock and human health.

Genomic signatures of climate adaptation in bank voles.

Evidence for divergent selection and adaptive variation across the landscape can provide insight into a species' ability to adapt to different environments. However, despite recent advances in genomics, it remains difficult to detect the footprints of climate-mediated selection in natural populations. Here, we analysed ddRAD sequencing data (21,892 SNPs) in conjunction with geographic climate variation to search for signatures of adaptive differentiation in twelve populations of the bank vole (Clethrionomys glareolus) distributed across Europe. To identify the loci subject to selection associated with climate variation, we applied multiple genotype-environment association methods, two univariate and one multivariate, and controlled for the effect of population structure. In total, we identified 213 candidate loci for adaptation, 74 of which were located within genes. In particular, we identified signatures of selection in candidate genes with functions related to lipid metabolism and the immune system. Using the results of redundancy analysis, we demonstrated that population history and climate have joint effects on the genetic variation in the pan-European metapopulation. Furthermore, by examining only candidate loci, we found that annual mean temperature is an important factor shaping adaptive genetic variation in the bank vole. By combining landscape genomic approaches, our study sheds light on genome-wide adaptive differentiation and the spatial distribution of variants underlying adaptive variation influenced by local climate in bank voles.

The impact of wildlife and environmental factors on hantavirus infection in the host and its translation into human risk.

Identifying factors that drive infection dynamics in reservoir host populations is essential in understanding human risk from wildlife-originated zoonoses. We studied zoonotic Puumala orthohantavirus (PUUV) in the host, the bank vole (Myodes glareolus), populations in relation to the host population, rodent and predator community and environment-related factors and whether these processes are translated into human infection incidence. We used 5-year rodent trapping and bank vole PUUV serology data collected from 30 sites located in 24 municipalities in Finland. We found that PUUV seroprevalence in the host was negatively associated with the abundance of red foxes, but this process did not translate into human disease incidence, which showed no association with PUUV seroprevalence. The abundance of weasels, the proportion of juvenile bank voles in the host populations and rodent species diversity were negatively associated with the abundance index of PUUV positive bank voles, which, in turn, showed a positive association with human disease incidence. Our results suggest certain predators, a high proportion of young bank vole individuals, and a diverse rodent community, may reduce PUUV risk for humans through their negative impacts on the abundance of infected bank voles.

Urban forest soils harbour distinct and more diverse communities of bacteria and fungi compared to less disturbed forest soils.

Anthropogenic changes to land use drive concomitant changes in biodiversity, including that of the soil microbiota. However, it is not clear how increasing intensity of human disturbance is reflected in the soil microbial communities. To address this issue, we used amplicon sequencing to quantify the microbiota (bacteria and fungi) in the soil of forests (n = 312) experiencing four different land uses, national parks (set aside for nature conservation), managed (for forestry purposes), suburban (on the border of an urban area) and urban (fully within a town or city), which broadly represent a gradient of anthropogenic disturbance. Alpha diversity of bacteria and fungi increased with increasing levels of anthropogenic disturbance, and was thus highest in urban forest soils and lowest in the national parks. The forest soil microbial communities were structured according to the level of anthropogenic disturbance, with a clear urban signature evident in both bacteria and fungi. Despite notable differences in community composition, there was little change in the predicted functional traits of urban bacteria. By contrast, urban soils exhibited a marked loss of ectomycorrhizal fungi. Soil pH was positively correlated with the level of disturbance, and thus was the strongest predictor of variation in alpha and beta diversity of forest soil communities, indicating a role of soil alkalinity in structuring urban soil microbial communities. Hence, our study shows how the properties of urban forest soils promote an increase in microbial diversity and a change in forest soil microbiota composition.

High Toxocara cati prevalence in wild, free-ranging Eurasian lynx (Lynx lynx) in Finland, 1999-2015.

In Finland, free-ranging Eurasian lynx (Lynx lynx) population has grown from 30 to 40 individuals to 2800 individuals since the species became partly protected in 1962. Changes in host population size are known to have an impact on host-parasite dynamics, and the Eurasian lynx population in Finland provides a unique opportunity for studying the potential effects of dramatic population increase and expansion of a solitary apex predator on their parasite prevalence and abundance. Toxocara cati is a zoonotic gastrointestinal parasite infecting domestic cats and wild felids worldwide. We studied T. cati infection prevalence and worm burden in 2756 Eurasian lynx individuals from Finland, covering the years 1999-2015. Toxocara cati worms that had been collected from intestinal contents were identified based on morphology. We performed regression analyses to investigate possible associations of age, sex, and host population density with T. cati infection. We found T. cati from 2324 (84.3%, 95% confidence interval 82.9-86.0) of the examined lynx. Each year, the infection prevalence was higher than 75% and not density dependent. The parasites were strongly aggregated, with older individuals harboring fewer T. cati than younger ones did. Old females aged 9-15 years had higher T. cati abundance than males of the same age group. Our results indicate that T. cati was a common and abundant parasite of Eurasian lynx throughout the study period, regardless of the changing population size and density.

Zoonotic Cryptosporidium spp. in Wild Rodents and Shrews.

There has been a significant increase in the number of reported human cryptosporidiosis cases in recent years. The aim of this study is to estimate the prevalence of Cryptosporidium spp. in wild rodents and shrews, and investigate the species and genotype distribution to assess zoonotic risk. Partial 18S rRNA gene nested-PCR reveals that 36.8, 53.9 and 41.9% of mice, voles and shrews are infected with Cryptosporidium species. The highest prevalence occurred in the Microtus agrestis (field vole) and Myodes glareolus (bank vole). Interestingly, bank voles caught in fields were significantly more often Cryptosporidium-positive compared to those caught in forests. The proportion of infected animals increases from over-wintered (spring and summer) to juveniles (autumn) suggesting acquired immunity in older animals. Based on Sanger sequencing and phylogenetic analyses, Apodemus flavicollis (yellow-necked mouse) is commonly infected with zoonotic C. ditrichi. Voles carry multiple different Cryptosporidium sp. and genotypes, some of which are novel. C. andersoni, another zoonotic species, is identified in the Craseomys rufocanus (grey-sided vole). Shrews carry novel shrew genotypes. In conclusion, this study indicates that Cryptosporidium protozoan are present in mouse, vole and shrew populations around Finland and the highest zoonotic risk is associated with C. ditrichi in Apodemus flavicollis and C. andersoni in Craseomys rufocanus. C. parvum, the most common zoonotic species in human infections, was not detected.

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.

Host-Dependent Clustering of Campylobacter Strains From Small Mammals in Finland.

Small mammals are known to carry Campylobacter spp.; however, little is known about the genotypes and their role in human infections. We studied intestinal content from small wild mammals collected in their natural habitats in Finland in 2010-2017, and in close proximity to 40 pig or cattle farms in 2017. The animals were trapped using traditional Finnish metal snap traps. Campylobacter spp. were isolated from the intestinal content using direct plating on mCCDA. A total of 19% of the captured wild animals (n = 577) and 41% of the pooled farm samples (n = 227) were positive for C. jejuni, which was the only Campylobacter species identified. The highest prevalence occurred in yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) which carried Campylobacter spp. in 66.3 and 63.9% of the farm samples and 41.5 and 24.4% of individual animals trapped from natural habitats, respectively. Interestingly, all house mouse (Mus musculus) and shrew (Sorex spp.) samples were negative for Campylobacter spp. C. jejuni isolates (n = 145) were further characterized by whole-genome sequencing. Core genome multilocus sequence typing (cgMLST) clustering showed that mouse and vole strains were separated from the rest of the C. jejuni population (636 and 671 allelic differences, 94 and 99% of core loci, respectively). Very little or no alleles were shared with C. jejuni genomes described earlier from livestock or human isolates. FastANI results further indicated that C. jejuni strains from voles are likely to represent a new previously undescribed species or subspecies of Campylobacter. Core-genome phylogeny showed that there was no difference between isolates originating from the farm and wild captured animals. Instead, the phylogeny followed the host species-association. There was some evidence (one strain each) of livestock-associated C. jejuni occurring in a farm-caught A. flavicollis and a brown rat (Rattus norvegicus), indicating that although small mammals may not be the original reservoir of Campylobacter colonizing livestock, they may sporadically carry C. jejuni strains occurring mainly in livestock and be associated with disease in humans.

Population fluctuations and spatial synchrony in an arboreal rodent.

Climatic conditions, trophic links between species and dispersal may induce spatial synchrony in population fluctuations. Spatial synchrony increases the extinction risk of populations and, thus, it is important to understand how synchrony-inducing mechanisms affect populations already threatened by habitat loss and climate change. For many species, it is unclear how population fluctuations vary over time and space, and what factors potentially drive this variation. In this study, we focus on factors determining population fluctuations and spatial synchrony in the Siberian flying squirrel, Pteromys volans, using long-term monitoring data from 16 Finnish populations located 2-400 km apart. We found an indication of synchronous population dynamics on a large scale in flying squirrels. However, the synchrony was not found to be clearly related to distance between study sites because the populations seemed to be strongly affected by small-scale local factors. The regularity of population fluctuations varied over time. The fluctuations were linked to changes in winter precipitation, which has previously been linked to the reproductive success of flying squirrels. Food abundance (tree mast) and predator abundance were not related to population fluctuations in this study. We conclude that spatial synchrony was not unequivocally related to distance in flying squirrels, as has been observed in earlier studies for more abundant rodent species. Our study also emphasises the role of climate in population fluctuations and the synchrony of the species.

Recent establishment of tick-borne encephalitis foci with distinct viral lineages in the Helsinki area, Finland.

Number of tick-borne encephalitis (TBE) cases has increased and new foci have emerged in Finland during the last decade. We evaluated risk for locally acquired TBE in the capital region inhabited by 1.2 million people. We screened ticks and small mammals from probable places of TBE virus (TBEV) transmission and places without reported circulation. The TBEV positive samples were sequenced and subjected to phylogenetic analysis. Within the study period 2007-2017, there was a clear increase of both all TBE cases and locally acquired cases in the Helsinki area. The surveillance of ticks and small mammals for TBEV confirmed four distinct TBEV foci in the Helsinki area. All detected TBEV strains were of the European subtype. TBEV genome sequences indicated that distinct TBEV lineages circulate in each focus. Molecular clock analysis suggested that the virus lineages were introduced to these foci decades ago. In conclusion, TBE has emerged in the mainland of Helsinki area during the last decade, with at least four distinct virus lineages independently introduced into the region previously. Although the overall annual TBE incidence is below the threshold for recommending general vaccinations, the situation requires further surveillance to detect and prevent possible further emergence of local TBE clusters.

Evolution and postglacial colonization of Seewis hantavirus with Sorex araneus in Finland.

Hantaviruses have co-existed with their hosts for millions of years. Seewis virus (SWSV), a soricomorph-borne hantavirus, is widespread in Eurasia, ranging from Central Siberia to Western Europe. To gain insight into the phylogeography and evolutionary history of SWSV in Finland, lung tissue samples of 225 common shrews (Sorex araneus) trapped from different parts of Finland were screened for the presence of SWSV RNA. Forty-two of the samples were positive. Partial small (S), medium (M) and large (L) segments of the virus were sequenced, and analyzed together with all SWSV sequences available in Genbank. The phylogenetic analysis of the partial S-segment sequences suggested that all Finnish SWSV strains shared their most recent common ancestor with the Eastern European strains, while the L-segment suggested multiple introductions. The difference between the L- and S-segment phylogenies implied that reassortment events play a role in the evolution of SWSV. Of the Finnish strains, variants from Eastern Finland occupied the root position in the phylogeny, and had the highest genetic diversity, supporting the hypothesis that SWSV reached Finland first form the east. During the spread in Finland, the virus has formed three separate lineages, identified here by correlation analysis of genetic versus geographic distance combined with median-joining network analysis. These results support the hypothesis that Finnish SWSV recolonized Finland with its host, the common shrew, from east after the last ice age 12,000-8000years ago, and then subsequently spread along emerging land bridges towards west or north with the migration and population expansion of its host.

Food limitation constrains host immune responses to nematode infections.

Trade-offs in the allocation of finite-energy resources among immunological defences and other physiological processes are believed to influence infection risk and disease severity in food-limited wildlife populations. However, this prediction has received little experimental investigation. Here we test the hypothesis that food limitation impairs the ability of wild field voles (Microtus agrestis) to mount an immune response against parasite infections. We conducted a replicated experiment on vole populations maintained in large outdoor enclosures during boreal winter, using food supplementation and anthelmintic treatment of intestinal nematodes. Innate immune responses against intestinal parasite infections were compared between food-supplemented and non-supplemented voles. Voles with high food availability mounted stronger immune responses against intestinal nematode infections than food-limited voles. No food effects were seen in immune responses to intracellular coccidian parasites, possibly owing to their ability to avoid activation of innate immune pathways. Our findings demonstrate that food availability constrains vole immune responses against nematode infections, and support the concept that spatio-temporal heterogeneity in food availability creates variation in infectious disease susceptibility.

Intraguild predation and competition impacts on a subordinate predator.

Intraguild (IG) predation and interspecific competition may affect the settlement and success of species in their habitats. Using data on forest-dwelling hawks from Finland, we addressed the impact of an IG predator, the northern goshawk Accipiter gentilis (goshawk), on the breeding of an IG prey, the common buzzard Buteo buteo. We hypothesized that the subordinate common buzzard avoids breeding in the proximity of goshawks and that interspecific competitors, mainly Strix owls, may also disturb common buzzards by competing for nests and food. Our results show that common buzzards more frequently occupied territories with a low IG predation threat and with no interspecific competitors. We also observed that common buzzards avoided territories with high levels of grouse, the main food of goshawks, possibly due to a risk of IG predation since abundant grouse can attract goshawks. High levels of small rodents attracted interspecific competitors to common buzzard territories and created a situation where there was not only an abundance of food but also an abundance of competitors for the food. These results suggest interplay between top-down and bottom-up processes which influence the interactions between avian predator species. We conclude that the common buzzard needs to balance the risks of IG predation and interference competition with the availability of its own resources. The presence of other predators associated with high food levels may impede a subordinate predator taking full advantage of the available food. Based on our results, it appears that interspecific interactions with dominant predators have the potential to influence the distribution pattern of subordinate predators.

De novo lipogenesis is suppressed during fasting but upregulated at population decline in cyclic voles.

Arvicolines are susceptible to the development of fatty liver during short-term fasting. We examined the potential role of de novo lipogenesis (DNL) (i) in the development of fasting-induced fatty liver and (ii) during a population cycle by measuring the mRNA expression of acetyl-CoA carboxylase-1 (ACC1) and fatty acid synthase (FAS). Laboratory voles (Microtus oeconomus and Microtus arvalis) were fed or fasted for 12 or 18 h and their liver mRNA levels were determined. Both species showed decreased mRNA expression of ACC1 and FAS during fasting. This suggests that DNL does not participate in the development of fatty liver in voles, different from human non-alcoholic fatty liver disease. In wild bank voles (Myodes glareolus), the mRNA levels of the genes of interest were higher during the population decline compared to the increase phase. In conclusion, DNL was suppressed during acute fasting but upregulated during a long-term population decline-a period of purported scarcity of high-quality food.

Food provisioning alters infection dynamics in populations of a wild rodent.

While pathogens are often assumed to limit the growth of wildlife populations, experimental evidence for their effects is rare. A lack of food resources has been suggested to enhance the negative effects of pathogen infection on host populations, but this theory has received little investigation. We conducted a replicated two-factor enclosure experiment, with introduction of the bacterium Bordetella bronchiseptica and food supplementation, to evaluate the individual and interactive effects of pathogen infection and food availability on vole populations during a boreal winter. We show that prior to bacteria introduction, vole populations were limited by food availability. Bordetella bronchiseptica introduction then reduced population growth and abundance, but contrary to predictions, primarily in food supplemented populations. Infection prevalence and pathological changes in vole lungs were most common in food supplemented populations, and are likely to have resulted from increased congregation and bacteria transmission around feeding stations. Bordetella bronchiseptica-infected lungs often showed protozoan co-infection (consistent with Hepatozoon erhardovae), together with more severe inflammatory changes. Using a multidisciplinary approach, this study demonstrates a complex picture of interactions and underlying mechanisms, leading to population-level effects. Our results highlight the potential for food provisioning to markedly influence disease processes in wildlife mammal populations.

Monitoring biothreat agents (Francisella tularensis, Bacillus anthracis and Yersinia pestis) with a portable real-time PCR instrument.

In the event of suspected releases or natural outbreaks of contagious pathogens, rapid identification of the infectious agent is essential for appropriate medical intervention and disease containment. The purpose of this study was to compare the performance of a novel portable real-time PCR thermocycler, PikoReal™, to the standard real-time PCR thermocycler, Applied Biosystems® 7300 (ABI 7300), for the detection of three high-risk biothreat bacterial pathogens: Francisella tularensis, Bacillus anthracis and Yersinia pestis. In addition, a novel confirmatory real-time PCR assay for the detection of F. tularensis is presented and validated. The results show that sensitivity of the assays, based on a dilution series, for the three infectious agents ranged from 1 to 100 fg of target DNA with both instruments. No cross-reactivity was revealed in specificity testing. Duration of the assays with the PikoReal and ABI 7300 systems were 50 and 100 min, respectively. In field testing for F. tularensis, results were obtained with the PikoReal system in 95 min, as the pre-PCR preparation, including DNA extraction, required an additional 45 min. We conclude that the PikoReal system enables highly sensitive and rapid on-site detection of biothreat agents under field conditions, and may be a more efficient alternative to conventional diagnostic methods.

Physiological condition of bank voles (Myodes glareolus) during the increase and decline phases of the population cycle.

The dynamics of animal populations are greatly influenced by interactions with their natural enemies and food resources. However, quantifying the relative effects of these factors on demographic rates remains a perpetual challenge for animal population ecology. Food scarcity is assumed to limit the growth and to initiate the decline of cyclic herbivore populations, but this has not been verified with physiological health indices. We hypothesized that individuals in declining populations would exhibit signs of malnutrition-induced deterioration of physiological condition. We evaluated the association of body condition with population cycle phase in bank voles (Myodes glareolus) during the increase and decline phases of a population cycle. The bank voles had lower body masses, condition indices and absolute masses of particular organs during the decline. Simultaneously, they had lower femoral masses, mineral contents and densities. Hemoglobin and hematocrit values and several parameters known to respond to food deprivation were unaffected by the population phase. There were no signs of lymphopenia, eosinophilia, granulocytosis or monocytosis. Erythrocyte counts were higher and plasma total protein levels and tissue proportions of essential polyunsaturated fatty acids lower in the population decline. Ectoparasite load was lower and adrenal gland masses or catecholamine concentrations did not suggest higher stress levels. Food availability seems to limit the size of voles during the decline but they can adapt to the prevailing conditions without clear deleterious health effects. This highlights the importance of quantifying individual health state when evaluating the effects of complex trophic interactions on the dynamics of wild animal populations.

Predator-vole interactions in Northern Europe: the role of small mustelids revised.

The cyclic population dynamics of vole and predator communities is a key phenomenon in northern ecosystems, and it appears to be influenced by climate change. Reports of collapsing rodent cycles have attributed the changes to warmer winters, which weaken the interaction between voles and their specialist subnivean predators. Using population data collected throughout Finland during 1986-2011, we analyse the spatio-temporal variation in the interactions between populations of voles and specialist, generalist and avian predators, and investigate by simulations the roles of the different predators in the vole cycle. We test the hypothesis that vole population cyclicity is dependent on predator-prey interactions during winter. Our results support the importance of the small mustelids for the vole cycle. However, weakening specialist predation during winters, or an increase in generalist predation, was not associated with the loss of cyclicity. Strengthening of delayed density dependence coincided with strengthening small mustelid influence on the summer population growth rates of voles. In conclusion, a strong impact of small mustelids during summers appears highly influential to vole population dynamics, and deteriorating winter conditions are not a viable explanation for collapsing small mammal population cycles.