An Infection-Tolerant Mammalian Reservoir for Several Zoonotic Agents Broadly Counters the Inflammatory Effects of Endotoxin.

Animals that are competent reservoirs of zoonotic pathogens commonly suffer little morbidity from the infections. To investigate mechanisms of this tolerance of infection, we used single-dose lipopolysaccharide (LPS) as an experimental model of inflammation and compared the responses of two rodents: Peromyscus leucopus, the white-footed deermouse and reservoir for the agents of Lyme disease and other zoonoses, and the house mouse Mus musculus Four hours after injection with LPS or saline, blood, spleen, and liver samples were collected and subjected to transcriptome sequencing (RNA-seq), metabolomics, and specific reverse transcriptase quantitative PCR (RT-qPCR). Differential expression analysis was at the gene, pathway, and network levels. LPS-treated deermice showed signs of sickness similar to those of exposed mice and had similar increases in corticosterone levels and expression of interleukin 6 (IL-6), tumor necrosis factor, IL-1ß, and C-reactive protein. By network analysis, the M. musculus response to LPS was characterized as cytokine associated, while the P. leucopus response was dominated by neutrophil activity terms. In addition, dichotomies in the expression levels of arginase 1 and nitric oxide synthase 2 and of IL-10 and IL-12 were consistent with type M1 macrophage responses in mice and type M2 responses in deermice. Analysis of metabolites in plasma and RNA in organs revealed species differences in tryptophan metabolism. Two genes in particular signified the different phenotypes of deermice and mice: the Slpi and Ibsp genes. Key RNA-seq findings for P. leucopus were replicated in older animals, in a systemic bacterial infection, and with cultivated fibroblasts. The findings indicate that P. leucopus possesses several adaptive traits to moderate inflammation in its balancing of infection resistance and tolerance.IMPORTANCE Animals that are natural carriers of pathogens that cause human diseases commonly manifest little or no sickness as a consequence of infection. Examples include the deermouse, Peromyscus leucopus, which is a reservoir for Lyme disease and several other disease agents in North America, and some types of bats, which are carriers of viruses with pathogenicity for humans. Mechanisms of this phenomenon of infection tolerance and entailed trade-off costs are poorly understood. Using a single injection of lipopolysaccharide (LPS) endotoxin as a proxy for infection, we found that deermice differed from the mouse (Mus musculus) in responses to LPS in several diverse pathways, including innate immunity, oxidative stress, and metabolism. Features distinguishing the deermice cumulatively would moderate downstream ill effects of LPS. Insights gained from the P. leucopus model in the laboratory have implications for studying infection tolerance in other important reservoir species, including bats and other types of wildlife.

Mating system is correlated with immunogenetic diversity in sympatric species of Peromyscine mice.

The number of reproductive partners per individual varies markedly across animal mating systems. This variation may be an important determinant of patterns of immunogenetic diversity, particularly at Major Histocompatibility Complex (MHC) Class I and II loci. To compare immunogenetic variation in taxa with markedly different mating systems, we used RNAseq-generated data to quantify genotypic diversity in three species of Peromyscine rodents: the monogamous California mouse (Peromyscus californicus) and the polygynandrous deer mouse (P. maniculatus) and brush mouse (P. boylii). By sampling populations of these species from multiple localities in California, we were able to conduct replicated analyses of the relationship between mating system and immunogenetic variation. Across the four localities sampled, diversity at MHC Class I and II genes was consistently higher in the two polygynandrous species. We found no evidence that sampling location (i.e., variation in habitat conditions) contributed to observed differences in MHC variation among populations or species. Collectively, our data indicate that immunogenetic variation in Peromyscine mice is associated with reproductive behavior, rather than geographic locality or habitat type. The consistently greater variability detected in the polygynandrous species examined suggests that balancing selection imposed by behaviorally-mediated pathogen exposure is important in maintaining variation at MHC genes in these animals.

Evolution of the rodent Trim5 cluster is marked by divergent paralogous expansions and independent acquisitions of TrimCyp fusions.

Evolution of cellular innate immune genes in response to viral threats represents a rich area of study for understanding complex events that shape mammalian genomes. One of these genes, TRIM5, is a retroviral restriction factor that mediates a post-entry block to infection. Previous studies on the genomic cluster that contains TRIM5 identified different patterns of gene amplification and the independent birth of CypA gene fusions in various primate species. However, the evolution of Trim5 in the largest order of mammals, Rodentia, remains poorly characterized. Here, we present an expansive phylogenetic and genomic analysis of the Trim5 cluster in rodents. Our findings reveal substantial evolutionary changes including gene amplifications, rearrangements, loss and fusion. We describe the first independent evolution of TrimCyp fusion genes in rodents. We show that the TrimCyp gene found in some Peromyscus species was acquired about 2 million years ago. When ectopically expressed, the P. maniculatus TRIMCyp shows anti-retroviral activity that is reversed by cyclosporine, but it does not activate Nf-κB or AP-1 promoters, unlike the primate TRIMCyps. These results describe a complex pattern of differential gene amplification in the Trim5 cluster of rodents and identify the first functional TrimCyp fusion gene outside of primates and tree shrews.

Mice Against Ticks: an experimental community-guided effort to prevent tick-borne disease by altering the shared environment.

Mice Against Ticks is a community-guided ecological engineering project that aims to prevent tick-borne disease by using CRISPR-based genome editing to heritably immunize the white-footed mice ( Peromyscus leucopus) responsible for infecting many ticks in eastern North America. Introducing antibody-encoding resistance alleles into the local mouse population is anticipated to disrupt the disease transmission cycle for decades. Technology development is shaped by engagement with community members and visitors to the islands of Nantucket and Martha's Vineyard, including decisions at project inception about which types of disease resistance to pursue. This engagement process has prompted the researchers to use only white-footed mouse DNA if possible, meaning the current project will not involve gene drive. Instead, engineered mice would be released in the spring when the natural population is low, a plan unlikely to increase total numbers above the normal maximum in autumn. Community members are continually asked to share their suggestions and concerns, a process that has already identified potential ecological consequences unanticipated by the research team that will likely affect implementation. As an early example of CRISPR-based ecological engineering, Mice Against Ticks aims to start small and simple by working with island communities whose mouse populations can be lastingly immunized without gene drive. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.

Finding a model for the study of Leishmania (Leishmania) mexicana infection: The Yucatan Deer mouse (Peromyscus yucatanicus) as a suitable option.

For more than four decades, the murine model has been employed extensively to understand immunological mechanisms associated with Leishmania infection. Although the use of laboratory mice has been very informative, mainly for L. (L.) major infection, the extrapolation to other Leishmania species and more importantly to human disease has been limited. Particularly in the case of L. (L.) mexicana, most infected mouse strains are highly susceptible and never presented asymptomatic infection, which is the main outcome in human. Thus, we postulated the use of Peromyscus yucatanicus, a primary reservoir of L. (L.) mexicana in the Yucatan Peninsula of Mexico, as an experimental model to study Leishmania infection. This rodent species can produce both asymptomatic and clinical infections therefore they seem more appropriate for studying host-pathogen interactions. In this review, we recapitulate the immunological findings observed in the traditional murine model of L. (L.) mexicana highlighting the differences with humans' infection and demonstrate the pertinence of P. yucatanicus as the experimental model for studying L. (L.) mexicana infection.

Impact of Sylvatic Plague Vaccine on Non-target Small Rodents in Grassland Ecosystems.

Oral vaccination is an emerging management strategy to reduce the prevalence of high impact infectious diseases within wild animal populations. Plague is a flea-borne zoonosis of rodents that often decimates prairie dog (Cynomys spp.) colonies in the western USA. Recently, an oral sylvatic plague vaccine (SPV) was developed to protect prairie dogs from plague and aid recovery of the endangered black-footed ferret (Mustela nigripes). Although oral vaccination programs are targeted toward specific species, field distribution of vaccine-laden baits can result in vaccine uptake by non-target animals and unintended indirect effects. We assessed the impact of SPV on non-target rodents at paired vaccine and placebo-treated prairie dog colonies in four US states from 2013 to 2015. Bait consumption by non-target rodents was high (70.8%, n = 3113), but anti-plague antibody development on vaccine plots was low (23.7%, n = 266). In addition, no significant differences were noted in combined deer mice (Peromyscus maniculatus) and western harvest mouse (Reithrodontomys megalotis) abundance or community evenness and richness of non-target rodents between vaccine-treated and placebo plots. In our 3-year field study, we could not detect a significant positive or negative effect of SPV application on non-target rodents.

Peromyscus leucopus mouse brain transcriptome response to Powassan virus infection.

Powassan virus (POWV) is a tick-borne Flavivirus responsible for life-threatening encephalitis in North America and some regions of Russia. The ticks that have been reported to transmit the virus belong to the Ixodes species, and they feed on small-to-medium-sized mammals, such as Peromyscus leucopus mice, skunks, and woodchucks. We previously developed a P. leucopus mouse model of POWV infection, and the model is characterized by a lack of clinical signs of disease following intraperitoneal or intracranial inoculation. However, intracranial inoculation results in mild subclinical encephalitis from 5 days post infection (dpi), but the encephalitis resolves by 28 dpi. We used RNA sequencing to profile the P. leucopus mouse brain transcriptome at different time points after intracranial challenge with POWV. At 24 h post infection, 42 genes were significantly differentially expressed and the number peaked to 232 at 7 dpi before declining to 31 at 28 dpi. Using Ingenuity Pathway Analysis, we determined that the genes that were significantly expressed from 1 to 15 dpi were mainly associated with interferon signaling. As a result, many interferon-stimulated genes (ISGs) were upregulated. Some of the ISGs include an array of TRIMs (genes encoding tripartite motif proteins). These results will be useful for the identification of POWV restriction factors.

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses.

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1-30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.

Infection resistance and tolerance in Peromyscus spp., natural reservoirs of microbes that are virulent for humans.

The widely-distributed North American species Peromyscus leucopus and P. maniculatus of cricetine rodents are, between them, important natural reservoirs for several zoonotic diseases of humans: Lyme disease, human granulocytic anaplasmosis, babesiosis, erhlichiosis, hard tickborne relapsing fever, Powassan virus encephalitis, hantavirus pulmonary syndrome, and plague. While these infections are frequently disabling and sometimes fatal for humans, the peromyscines display little pathology and apparently suffer few consequences, even when prevalence of persistent infection in a population is high. While these Peromyscus spp. are unable to clear some of the infections, they appear to have partial resistance, which limits the burden of the pathogen. In addition, they display traits of infection tolerance, which reduces the damage of the infection. Research on these complementary resistance and tolerance phenomena in Peromyscus has relevance both for disease control measures targeting natural reservoirs and for understanding the mechanisms of the comparatively greater sickness of many humans with these and other infections.

Photoperiodic regulation of behavior: Peromyscus as a model system.

Winter and summer present vastly different challenges to animals living outside of the tropics. To survive and reproduce, individuals must anticipate seasonal environmental changes and adjust physiology and behavior accordingly. Photoperiod (day length) offers a relatively 'noise free' environmental signal that non-tropical animals use to tell the time of year, and whether winter is approaching or receding. In some cases, photoperiodic signals may be fine-tuned by other proximate cues such as food availability or temperature. The pineal hormone, melatonin, is a primary physiological transducer of the photoperiodic signal. It tracks night length and provokes changes in physiology and behavior at appropriate times of the year. Because of their wide latitudinal distribution, Peromyscus has been well studied in the context of photoperiodic regulation of physiology and behavior. Here, we discuss how photoperiodic signals are transduced by pineal melatonin, how melatonin acts on target tissues, and subsequent consequences for behavior. Using a life-history paradigm involving trade-offs between the immune and reproductive systems, specific emphasis is placed on aggression, metabolism, and cognition. We discuss future directions including examining the effects of light pollution on photoperiodism, genetic manipulations to test the role of specific genes in the photoperiodic response, and using Peromyscus to test evolutionary theories of aging.

Transcriptome markers of viral persistence in naturally-infected andes virus (bunyaviridae) seropositive long-tailed pygmy rice rats.

Long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are principal reservoir hosts of Andes virus (ANDV) (Bunyaviridae), which causes most hantavirus cardiopulmonary syndrome cases in the Americas. To develop tools for the study of the ANDV-host interactions, we used RNA-Seq to generate a de novo transcriptome assembly. Splenic RNA from five rice rats captured in Chile, three of which were ANDV-infected, was used to generate an assembly of 66,173 annotated transcripts, including noncoding RNAs. Phylogenetic analysis of selected predicted proteins showed similarities to those of the North American deer mouse (Peromyscus maniculatus), the principal reservoir of Sin Nombre virus (SNV). One of the infected rice rats had about 50-fold more viral burden than the others, suggesting acute infection, whereas the remaining two had levels consistent with persistence. Differential expression analysis revealed distinct signatures among the infected rodents. The differences could be due to 1) variations in viral load, 2) dimorphic or reproductive differences in splenic homing of immune cells, or 3) factors of unknown etiology. In the two persistently infected rice rats, suppression of the JAK-STAT pathway at Stat5b and Ccnot1, elevation of Casp1, RIG-I pathway factors Ppp1cc and Mff, and increased FC receptor-like transcripts occurred. Caspase-1 and Stat5b activation pathways have been shown to stimulate T helper follicular cell (TFH) development in other species. These data are also consistent with reports suggestive of TFH stimulation in deer mice experimentally infected with hantaviruses. In the remaining acutely infected rice rat, the apoptotic pathway marker Cox6a1 was elevated, and putative anti-viral factors Abcb1a, Fam46c, Spp1, Rxra, Rxrb, Trmp2 and Trim58 were modulated. Transcripts for preproenkephalin (Prenk) were reduced, which may be predictive of an increased T cell activation threshold. Taken together, this transcriptome dataset will permit rigorous examination of rice rat-ANDV interactions and may lead to better understanding of virus ecology.

Expansion of zoonotic babesiosis and reported human cases, Connecticut, 2001-2010.

To document the expansion of human babesiosis in Connecticut, we analyzed reservoir host sera for seroreactivity to Babesia microti Franca and reviewed Connecticut human surveillance case data collected during 2001-2010. Sera from white-footed mice, Peromyscus leucopus Rafinesque, from 10 towns in 5 counties, collected at 4-7-yr periods between 2001 and 2010, were tested for total immunoglobulins. The prevalence of B. microti-positive mice was compared with confirmed and probable human case reports tabulated by the Connecticut Department of Public Health. The highest babesiosis and rodent seroprevalence rates were in New London County, where this protozoan disease was first documented in the state. However, human cases and reservoir host infection increased significantly from 2001-2005 to 2005-2010 and in other parts of the state. Clinicians should be aware that the disease is not confined to long-established endemic areas of the state.

Reservoir targeted vaccine against Borrelia burgdorferi: a new strategy to prevent Lyme disease transmission.

A high prevalence of infection with Borrelia burgdorferi in ixodid ticks is correlated with a high incidence of Lyme disease. The transmission of B. burgdorferi to humans can be disrupted by targeting 2 key elements in its enzootic cycle: the reservoir host and the tick vector. In a prospective 5-year field trial, we show that oral vaccination of wild white-footed mice resulted in outer surface protein A-specific seropositivity that led to reductions of 23% and 76% in the nymphal infection prevalence in a cumulative, time-dependent manner (2 and 5 years, respectively), whereas the proportion of infected ticks recovered from control plots varied randomly over time. Significant decreases in tick infection prevalence were observed within 3 years of vaccine deployment. Implementation of such a long-term public health measure could substantially reduce the risk of human exposure to Lyme disease.

Expression profiling of lymph node cells from deer mice infected with Andes virus.

BACKGROUND: Deer mice (Peromyscus maniculatus) are the principal reservoir hosts of Sin Nombre virus (SNV), the cause of the great majority of hantavirus cardiopulmonary syndrome (HCPS) cases in North America. SNV, like all hantaviruses with their reservoirs, causes persistent infection without pathology in deer mice and appear to elicit a regulatory T cell response. Deer mice are also susceptible to Andes virus (ANDV), which causes the great majority of HCPS cases in South America, but they clear infection by 56 days post infection without signs of disease. RESULTS: We examined lymph node cell responses of deer mice infected with ANDV to determine expression profiles upon in vitro recall challenge with viral antigen. Because the deer mouse genome is currently unannotated, we developed a bioinformatics pipeline to use known lab mouse (Mus musculus) cDNAs to predict genes within the deer mouse genome and design primers for quantitative PCR (http://dna.publichealth.uga.edu/BlastPrimer/BlastPrimer.php). Of 94 genes examined, 20 were elevated, the plurality of which were Th2-specific, whereas 12 were downregulated. Other expressed genes represented Th1, regulatory T cells and follicular helper T cells, and B cells, but not Th17 cells, indicating that many cellular phenotypes participate in the host response to Andes virus. CONCLUSIONS: The ability to examine expression levels of nearly any gene from deer mice should allow direct comparison of infection with SNV or ANDV to determine the immunological pathways used for clearance of hantavirus infection in a reservoir host species.

Associations between innate immune function and ectoparasites in wild rodent hosts.

Immune function is an important component of host fitness, and high investment in immunity should occur when the benefits outweigh the costs, such as when risk of parasitism is high. We sampled two rodent hosts, white-footed mice (Peromyscus leucopus), and prairie voles (Microtus ochrogaster), and their tick, flea, and mite ectoparasites. A bacterial killing assay was used to measure the host's innate immune function. We hypothesized that classes of hosts (species, sexes, or age classes) with overall higher tick burdens would have a higher innate immune function as an evolutionary response to historically greater exposure. We hypothesized a weaker relationship between the fleas and mites and immune function because of high host specificity in fleas and the absence of known vector function in North American mites. Ectoparasites were significantly overdispersed on hosts. In accordance with our hypothesis, Peromyscus that had higher tick burdens also exhibited significantly higher bacterial killing ability compared to Microtus. There was no significant difference in total flea burden between rodent species and no relationship with bacterial killing ability. Microtus had higher burdens of mites in each order than Peromyscus, and female rodents had higher mite burdens than males. The benefits of maintaining high levels of innate immune factors appear to be greater than the energetic costs for Peromyscus compared to Microtus.

Kinetics of immune responses in deer mice experimentally infected with Sin Nombre virus.

Deer mice are the principal reservoir hosts of Sin Nombre virus, the etiologic agent of most hantavirus cardiopulmonary syndrome cases in North America. Infection of deer mice results in persistence without conspicuous pathology, and most, if not all, infected mice remain infected for life, with periods of viral shedding. The kinetics of viral load, histopathology, virus distribution, and immune gene expression in deer mice were examined. Viral antigen was detected as early as 5 days postinfection and peaked on day 15 in the lungs, hearts, kidneys, and livers. Viral RNA levels varied substantially but peaked on day 15 in the lungs and heart, and antinucleocapsid IgG antibodies appeared in some animals on day 10, but a strong neutralizing antibody response failed to develop during the 20-day experiment. No clinical signs of disease were observed in any of the infected deer mice. Most genes were repressed on day 2, suggesting a typical early downregulation of gene expression often observed in viral infections. Several chemokine and cytokine genes were elevated, and markers of a T cell response occurred but then declined days later. Splenic transforming growth factor beta (TGF-ß) expression was elevated early in infection, declined, and then was elevated again late in infection. Together, these data suggest that a subtle immune response that fails to clear the virus occurs in deer mice.

Development of a baited oral vaccine for use in reservoir-targeted strategies against Lyme disease.

Lyme disease is a major human health problem which continues to increase in incidence and geographic distribution. As a vector-borne zoonotic disease, Lyme disease may be amenable to reservoir targeted strategies for control. We have previously reported that a vaccinia virus (VV) based vaccine expressing outer surface protein A (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease, protects inbred strains of laboratory mice against infection by feeding ticks and clears the ticks of infection when administered by gavage. Here we extend these studies to develop an effective bait formulation for delivery of the VV based vaccine and test its characteristics under simulated environmental conditions. We show that this vaccine is efficacious in decreasing acquisition of B. burgdorferi by uninfected larval ticks as well as in decreasing transmission from infected ticks to its natural reservoir, Peromyscus leucopus, when fed to mice in oral baits. Using live, in vivo imaging techniques, we describe the distribution of vaccinia virus infection after ingestion of the baited vaccines and establish the use of in vivo imaging technology for optimization of bait delivery. In summary, a VV based OspA vaccine is stable in an oral bait preparation and provides protection against infection for both the natural reservoir and the tick vector of Lyme disease.

Linking disease and community ecology through behavioural indicators: immunochallenge of white-footed mice and its ecological impacts.

1. Pathogens and immune challenges can induce changes in host phenotype in ways that indirectly impact important community interactions, including those that affect host-pathogen interactions. 2. To explore host behavioural response to immune challenge, we exposed wild white-footed mice (Peromyscus leucopus) to an immunogen from an endemic, zoonotic pathogen, the spirochete Borrelia burgdorferi. White-footed mice are a major reservoir host of Lyme disease (LD) spirochetes in northeastern USA and an abundant member of forest communities. The activity patterns, foraging behaviour, and space use of white-footed mice have implications for population growth rates of community members upon which mice incidentally prey (i.e. gypsy moths and native thrushes), as well as potentially determining host-vector encounter rates and human risk of LD. 3. Immunochallenge led to specific humoral (antibody) and cellular (i.e. elevated neutrophils and eosinophils) immune responses, supporting use of the immunogen as a surrogate for pathogenic infection. 4. Immunochallenged mice had reduced wheel-running activity early in the night when measured in the lab. However, mouse activity, as measured by track plates in natural field experiments, did not differ between mice exposed to the immunogen and unexposed mice. 5. Foraging behaviour of wild mice in the field - assessed with giving-up densities of seed at artificial feeding stations - was affected by exposure to the immunogen. Whereas immunochallenge did not influence whether foraging mice gained information on patch quality while foraging, it led to reductions in predator avoidance during foraging, suggesting that the proportion of space used by foraging mice may be greater as a result of immunochallenge. This increased space use is predicted to increase encounter rates with patchily distributed LD vectors (ticks) and with incidental prey items. 6. Thus, immunochallenge in white-footed mice, and potentially pathogenic infection, have the potential to indirectly impact community interactions, including those important for pathogen transmission.

A temporal dilution effect: hantavirus infection in deer mice and the intermittent presence of voles in Montana.

The effect of intermittently occurring, non-reservoir host species on pathogen transmission and prevalence in a reservoir population is poorly understood. We investigated whether voles, Microtus spp., which occur intermittently, influenced estimated standing antibody prevalence (ESAP) to Sin Nombre hantavirus (SNV, Bunyaviridae: Hantavirus) among deer mice, Peromyscus maniculatus, whose populations are persistent. We used 14 years of data from central Montana to investigate whether ESAP among deer mice was related to vole presence or abundance while controlling for the relationship between deer mouse abundance and ESAP. We found a reduction in deer mouse ESAP associated with the presence of voles, independent of vole abundance. A number of studies have documented that geographic locations which support a higher host diversity can be associated with reductions in pathogen prevalence by a hypothesized dilution effect. We suggest a dilution effect may also occur in a temporal dimension at sites where host richness fluctuates. Preservation of host diversity and optimization of environmental conditions which promote occurrence of ephemeral species, such as voles, may result in a decreased ESAP to hantaviruses among reservoir hosts. Our results may extend to other zoonotic infectious diseases.