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.

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.

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.

Geographical Distribution of Ljungan Virus in Small Mammals in Europe.

Ljungan virus (LV), which belongs to the Parechovirus genus in the Picornaviridae family, was first isolated from bank voles (Myodes glareolus) in Sweden in 1998 and proposed as a zoonotic agent. To improve knowledge of the host association and geographical distribution of LV, tissues from 1685 animals belonging to multiple rodent and insectivore species from 12 European countries were screened for LV-RNA using reverse transcriptase (RT)-PCR. In addition, we investigated how the prevalence of LV-RNA in bank voles is associated with various intrinsic and extrinsic factors. We show that LV is widespread geographically, having been detected in at least one host species in nine European countries. Twelve out of 21 species screened were LV-RNA PCR positive, including, for the first time, the red vole (Myodes rutilus) and the root or tundra vole (Alexandromys formerly Microtus oeconomus), as well as in insectivores, including the bicolored white-toothed shrew (Crocidura leucodon) and the Valais shrew (Sorex antinorii). Results indicated that bank voles are the main rodent host for this virus (overall RT-PCR prevalence: 15.2%). Linear modeling of intrinsic and extrinsic factors that could impact LV prevalence showed a concave-down relationship between body mass and LV occurrence, so that subadults had the highest LV positivity, but LV in older animals was less prevalent. Also, LV prevalence was higher in autumn and lower in spring, and the amount of precipitation recorded during the 6 months preceding the trapping date was negatively correlated with the presence of the virus. Phylogenetic analysis on the 185 base pair species-specific sequence of the 5' untranslated region identified high genetic diversity (46.5%) between 80 haplotypes, although no geographical or host-specific patterns of diversity were detected.

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.

Yersinia spp. in Wild Rodents and Shrews in Finland.

Yersinia enterocolitica and Yersinia pseudotuberculosis are important zoonotic bacteria causing human enteric yersiniosis commonly reported in Europe. All Y. pseudotuberculosis strains are considered pathogenic, while Y. enterocolitica include both pathogenic and nonpathogenic strains which can be divided into six biotypes (1A, 1B, and 2-5) and about 30 serotypes. The most common types causing yersiniosis in Europe are Y. enterocolitica bioserotypes 4/O:3 and 2/O:9. Strains belonging to biotype 1A are considered as nonpathogenic because they are missing important virulence genes like the attachment-invasion-locus (ail) gene in the chromosome and the virulence plasmid. The role of wild small mammals as a reservoir of enteropathogenic Yersinia spp. is still obscure. In this study, the presence of Yersinia spp. was examined from 1840 wild small mammals, including voles, mice, and shrews, trapped in Finland during a 7-year period. We isolated seven Yersinia species. Y. enterocolitica was the most common species, isolated from 8% of the animals; while most of these isolates represented nonpathogenic biotype 1A, human pathogenic bioserotype 2/O:9 was also isolated from a field vole. Y. pseudotuberculosis of bioserotype 1/O:2 was isolated from two shrews. The ail gene, which is typically only found in the isolates of biotypes 1B and 2-5 associated with yersiniosis, was frequently (23%) detected in the nonpathogenic isolates of biotype 1A and sporadically (6%) in Yersinia kristensenii isolates. Our results suggest that wild small mammals, especially voles, may serve as carriers for ail-positive Y. enterocolitica 1A and Y. kristensenii. We also demonstrate that voles and shrews sporadically excrete pYV-positive Y. enterocolitica 2/O:9 and Y. pseudotuberculosis 1/O:2, respectively, in their feces and, thus, can serve as a contamination source for vegetables by contaminating the soil.

DISTRIBUTION AND SEASONAL VARIATION OF LJUNGAN VIRUS IN BANK VOLES (MYODES GLAREOLUS) IN FENNOSCANDIA.

Ljungan virus (LV) is a picornavirus originally isolated from Swedish bank voles ( Myodes glareolus ) in 1998. The association of LV with human disease has been debated ever since, but fundamental data on the ecology of the virus are still lacking. Here we present results of the first intensive study on the prevalence of LV in bank voles trapped in Fennoscandia (Sweden and Finland) from 2009-12 as determined by PCR. Using an LV-specific real-time reverse transcriptase PCR, LV was detected in the liver of 73 out of 452 (16.2%) individuals and in 13 out of 17 sampling sites across Sweden and Finland (mean per site prevalence 16%, SE 3%, range 0-50%). We found more infected animals in autumn compared to spring, and lighter and heavier individuals had a higher prevalence than those with intermediate body masses. The result that LV prevalence is also lower in heavier (i.e., older) animals suggests for the first time that LV infection is not persistent in rodents.

Metagenomic Evaluation of Bacteria from Voles.

Voles (Arvicolinae, Rodentia) are known carriers of zoonotic bacteria such as Bartonella spp. and Francisella tularensis. However, apart from F. tularensis, the bacterial microbiome of voles has not previously been determined in Finland and rarely elsewhere. Therefore, we studied liver samples from 61 voles using 16S ribosomal RNA gene PCR analysis, followed by Sanger sequencing. Twenty-three of these samples were also studied with tag-encoded pyrosequencing. The samples originated from 21 field voles (Microtus agrestis), 37 tundra voles (Microtus oeconomus), and 3 bank voles (Myodes glareolus). With the more conventional 16S rDNA PCR analysis, 90 (33%) of the recovered 269 sequence types could be identified to genus level, including Bartonella, Francisella, Mycoplasma, Anaplasma, and Acinetobacter in 31, 15, 9, 9, and 9 sequences, respectively. Seventy-five (28%) matched best with sequences of uncultured bacteria, of which 40/75 could be classified to the order Clostridiales and, more specifically, to families Lachnospiraceae and Ruminococcaceae. Pyrosequencing from 23 samples revealed comparable and similar results: clinically relevant bacterial families such as Mycoplasmataceae, Bartonellaceae, Anaplasmataceae, and Francisellaceae were recognized. These analyses revealed significant bacterial diversity in vole livers, consisting of distinct and constant sequence patterns reflecting bacteria found in the intestinal gut, but including some known zoonotic pathogens as well. The molecular bacterial sequence types determined with the two different techniques shared major similarities and verified remarkable congruency between the methods.

Microevolution of bank voles (Myodes glareolus) at neutral and immune-related genes during multiannual dynamic cycles: Consequences for Puumala hantavirus epidemiology.

Understanding how host dynamics, including variations of population size and dispersal, may affect the epidemiology of infectious diseases through ecological and evolutionary processes is an active research area. Here we focus on a bank vole (Myodes glareolus) metapopulation surveyed in Finland between 2005 and 2009. Bank vole is the reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE, a mild form of hemorrhagic fever with renal symptom) in humans. M. glareolus populations experience multiannual density fluctuations that may influence the level of genetic diversity maintained in bank voles, PUUV prevalence and NE occurrence. We examine bank vole metapopulation genetics at presumably neutral markers and immune-related genes involved in susceptibility to PUUV (Tnf-promoter, Tlr4, Tlr7 and Mx2 gene) to investigate the links between population dynamics, microevolutionary processes and PUUV epidemiology. We show that genetic drift slightly and transiently affects neutral and adaptive genetic variability within the metapopulation. Gene flow seems to counterbalance its effects during the multiannual density fluctuations. The low abundance phase may therefore be too short to impact genetic variation in the host, and consequently viral genetic diversity. Environmental heterogeneity does not seem to affect vole gene flow, which might explain the absence of spatial structure previously detected in PUUV in this area. Besides, our results suggest the role of vole dispersal on PUUV circulation through sex-specific and density-dependent movements. We find little evidence of selection acting on immune-related genes within this metapopulation. Footprint of positive selection is detected at Tlr-4 gene in 2008 only. We observe marginally significant associations between Mx2 genotype and PUUV genogroups. These results show that neutral processes seem to be the main factors affecting the evolution of these immune-related genes at a contemporary scale, although the relative effects of neutral and adaptive forces could vary temporally with density fluctuations. Immune related gene polymorphism may in turn partly influence PUUV epidemiology in this metapopulation.

Temporal dynamics of Puumala hantavirus infection in cyclic populations of bank voles.

Understanding the dynamics of zoonotic pathogens in their reservoir host populations is a prerequisite for predicting and preventing human disease epidemics. The human infection risk of Puumala hantavirus (PUUV) is highest in northern Europe, where populations of the rodent host (bank vole, Myodes glareolus) undergo cyclic fluctuations. We conducted a 7-year capture-mark-recapture study to monitor seasonal and multiannual patterns of the PUUV infection rate in bank vole populations exhibiting a 3-year density cycle. Infected bank voles were most abundant in mid-winter months during years of increasing or peak host density. Prevalence of PUUV infection in bank voles exhibited a regular, seasonal pattern reflecting the annual population turnover and accumulation of infections within each year cohort. In autumn, the PUUV transmission rate tracked increasing host abundance, suggesting a density-dependent transmission. However, prevalence of PUUV infection was similar during the increase and peak years of the density cycle despite a twofold difference in host density. This may result from the high proportion of individuals carrying maternal antibodies constraining transmission during the cycle peak years. Our exceptionally intensive and long-term dataset provides a solid basis on which to develop models to predict the dynamic public health threat posed by PUUV in northern Europe.

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.

Life-long shedding of Puumala hantavirus in wild bank voles (Myodes glareolus).

The knowledge of viral shedding patterns and viraemia in the reservoir host species is a key factor in assessing the human risk of zoonotic viruses. The shedding of hantaviruses (family Bunyaviridae) by their host rodents has widely been studied experimentally, but rarely in natural settings. Here we present the dynamics of Puumala hantavirus (PUUV) shedding and viraemia in naturally infected wild bank voles (Myodes glareolus). In a monthly capture-mark-recapture study, we analysed 18 bank voles for the presence and relative quantity of PUUV RNA in the excreta and blood from 2 months before up to 8 months after seroconversion. The proportion of animals shedding PUUV RNA in saliva, urine and faeces peaked during the first month after seroconversion, but continued throughout the study period with only a slight decline. The quantity of shed PUUV in reverse transcription quantitative PCR (RT-qPCR) positive excreta was constant over time. In blood, PUUV RNA was present for up to 7 months but both the probability of viraemia and the virus load declined with time. Our findings contradict the current view of a decline in virus shedding after the acute phase and a short viraemic period in hantavirus infection - an assumption widely adopted in current epidemiological models. We suggest the life-long shedding as a means of hantaviruses to survive over host population bottlenecks, and to disperse in fragmented habitats where local host and/or virus populations face temporary extinctions. Our results indicate that the kinetics of pathogens in wild hosts may differ considerably from those observed in laboratory settings.

Hantaviruses in Finnish soricomorphs: evidence for two distinct hantaviruses carried by Sorex araneus suggesting ancient host-switch.

Hantaviruses are emerging viruses carried by rodents, soricomorphs (shrews and moles) and bats. In Finland, Puumala virus (PUUV) was for years the only hantavirus detected. In 2009, however, Seewis virus (SWSV) was reported from archival common shrew (Sorex araneus) samples collected in 1982 in Finland. To elucidate the diversity of hantaviruses in soricomorphs in Finland, 180 individuals were screened, representing seven species captured from 2001 to 2012: hantavirus RNA was screened using RT-PCR, and hantaviral antigen using immunoblotting with polyclonal antibodies raised against truncated SWSV nucleocapsid protein. The overall hantavirus RNA prevalence was 14% (26/180), antigen could be demonstrated in 9 of 20 SWSV RT-PCR positive common shrews. Genetic analyses revealed that four soricomorph-borne hantaviruses circulate in Finland, including Boginia virus (BOGV) in water shrew (Neomys fodiens) and Asikkala virus (ASIV) in pygmy shrew (Sorex minutus). Interestingly, on two study sites, common shrews harbored strains of two different hantaviruses: Seewis virus and a new distinct, genetically distant (identity 57% at amino acid level) virus (Altai-like virus) which clusters together with viruses in the basal phylogroup I of hantaviruses with 62-67% identity at amino acid level. This is the first evidence of coexistence of two clearly distinct hantavirus species circulating simultaneously in one host species population. The findings suggest an ancient host-switching event from a yet unknown host to S. araneus. In addition, phylogenetic analyses of partial S and M segment sequences showed that SWSV in Finland represents a unique genotype in Europe.

Detection of Francisella tularensis in voles in Finland.

Francisella tularensis is a highly virulent intracellular bacterium causing the zoonotic disease tularemia. It recurrently causes human and animal outbreaks in northern Europe, including Finland. Although F. tularensis infects several mammal species, only rodents and lagomorphs seem to have importance in its ecology. Peak densities of rodent populations may trigger tularemia outbreaks in humans; however, it is still unclear to which extent rodents or other small mammals maintain F. tularensis in nature. The main objective of this study was to obtain information about the occurrence of F. tularensis in small mammals in Finland. We snap-trapped 547 wild small mammals representing 11 species at 14 locations around Finland during 6 years and screened them for the presence of F. tularensis DNA using PCR analysis. High copy number of F. tularensis-specific DNA was detected in tissue samples of five field voles (Microtus agrestis) originating from one location and 2 years. According to DNA sequences of the bacterial 23S ribosomal RNA gene amplified from F. tularensis-infected voles, the infecting agent belongs to the subspecies holarctica. To find out the optimal tissue for tularemia screening in voles, we compared the amounts of F. tularensis DNA in lungs, liver, spleen, and kidney of the infected animals. F. tularensis DNA was detectable in high levels in all four organs except for one animal, whose kidney was F. tularensis DNA-negative. Thus, at least liver, lung, and spleen seem suitable for F. tularensis screening in voles. Thus, liver, lung, and spleen all seem suitable for F. tularensis screening in voles. In conclusion, field voles can be heavily infected with F. tularensis subsp. holarctica and thus potentially serve as the source of infection in humans and other mammals.

Analysis of complete Puumala virus genome, Finland.

Puumala virus causes nephropathia epidemica, a rodent-borne zoonosis that is endemic to Europe. We sequenced the complete Puumala virus genome that was directly recovered from a person who died and compared it with those of viruses from local bank voles. The virus strain involved was neither a unique nor rare genetic variant.

Co-circulation of two Puumala hantavirus lineages in Latvia: a Russian lineage described previously and a novel Latvian lineage.

Puumala hantavirus (PUUV) causes a mild form of haemorrhagic fever with renal syndrome in Europe. Seven genetic lineages of PUUV have thus far been recorded, which exhibit geographic structure within the distribution of its natural host, the bank vole (Myodes glareolus). This study presents evidence for two distinct PUUV lineages co-circulating in Latvia: one previously described from Russia and a novel one that appears to be endemic. The Latvian lineage (LAT) is considerably divergent and several amino acid markers make it easily distinguishable. Phylogenetic analysis suggested a possibility of different evolutionary histories for the PUUV genome segments of LAT.

Orthopox virus infections in Eurasian wild rodents.

The genus Orthopoxvirus includes variola (smallpox) virus and zoonotic cowpox virus (CPXV). All orthopoxviruses (OPV) are serologically cross-reactive and cross-protective, and after the cessation of smallpox vaccination, CPXV and other OPV infections represent an emerging threat to human health. In this respect CPXV, with its reservoir in asymptomatically infected wild rodents, is of special importance. In Europe, clinical cowpox has been diagnosed in both humans and animals. The main objective of this study was to elucidate the prevalence of OPV infections in wild rodents in different parts of Eurasia and to compare the performance of three real-time polymerase chain reaction (PCR) methods in detecting OPV DNA in wildlife samples. We investigated 962 wild rodents from Northern Europe (Finland), Central Europe (Germany), and Northern Asia (Siberia, Russia) for the presence of OPV antibodies. According to a CPXV antigen-based immunofluorescence assay, animals from 13 of the 17 locations (76%) showed antibodies. Mean seroprevalence was 33% in Finland (variation between locations 0%-69%), 32% in Germany (0%-43%), and 3.2% (0%-15%) in Siberia. We further screened tissue samples from 513 of the rodents for OPV DNA using up to three real-time PCRs. Three rodents from two German and one Finnish location were OPV DNA positive. The amplicons were 96% to 100% identical to available CPXV sequences. Further, we demonstrated OPV infections as far east as the Baikal region and occurring in hamster and two other rodent species, ones previously unnoticed as possible reservoir hosts. Based on serological and PCR findings, Eurasian wild rodents are frequently but nonpersistently infected with OPVs. Results from three real-time PCR methods were highly concordant. This study extends the geographic range and wildlife species diversity in which OPV (or CPXV) viruses are naturally circulating.