Variation in Local and Systemic Pro-Inflammatory Immune Markers of Wild Wood Mice after Anthelmintic Treatment.

The immune system represents a host's main defense against infection to parasites and pathogens. In the wild, a host's response to immune challenges can vary due to physiological condition, demography (age, sex), and coinfection by other parasites or pathogens. These sources of variation, which are intrinsic to natural populations, can significantly impact the strength and type of immune responses elicited after parasite exposure and infection. Importantly, but often neglected, a host's immune response can also vary within the individual, across tissues and between local and systemic scales. Consequently, how a host responds at each scale may impact its susceptibility to concurrent and subsequent infections. Here we analyzed how characteristics of hosts and their parasite infections drive variation in the pro-inflammatory immune response in wild wood mice (Apodemus sylvaticus) at both the local and systemic scale by experimentally manipulating within-host parasite communities through anthelmintic drug treatment. We measured concentrations of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) produced in vitro in response to a panel of toll-like receptor agonists at the local (mesenteric lymph nodes [MLNs]) and systemic (spleen) scales of individuals naturally infected with two gastrointestinal parasites, the nematode Heligmosomoides polygyrus and the protozoan Eimeria hungaryensis. Anthelmintic-treated mice had a 20-fold lower worm burden compared to control mice, as well as a four-fold higher intensity of the non-drug targeted parasite E. hungaryensis. Anthelmintic treatment differentially impacted levels of TNF-α expression in males and females at the systemic and local scales, with treated males producing higher, and treated females lower, levels of TNF-α, compared to control mice. Also, TNF-α was affected by host age, at the local scale, with MLN cells of young, treated mice producing higher levels of TNF-α than those of old, treated mice. Using complementary, but distinct, measures of inflammation measured across within-host scales allowed us to better assess the wood mouse immune response to changes in parasite infection dynamics after anthelmintic treatment. This same approach could be used to understand helminth infections and responses to parasite control measures in other systems in order to gain a broader view of how variation impacts the immune response.

Linking community assembly and structure across scales in a wild mouse parasite community.

Understanding what processes drive community structure is fundamental to ecology. Many wild animals are simultaneously infected by multiple parasite species, so host-parasite communities can be valuable tools for investigating connections between community structures at multiple scales, as each host can be considered a replicate parasite community. Like free-living communities, within-host-parasite communities are hierarchical; ecological interactions between hosts and parasites can occur at multiple scales (e.g., host community, host population, parasite community within the host), therefore, both extrinsic and intrinsic processes can determine parasite community structure. We combine analyses of community structure and assembly at both the host population and individual scales using extensive datasets on wild wood mice (Apodemus sylvaticus) and their parasite community. An analysis of parasite community nestedness at the host population scale provided predictions about the order of infection at the individual scale, which were then tested using parasite community assembly data from individual hosts from the same populations. Nestedness analyses revealed parasite communities were significantly more structured than random. However, observed nestedness did not differ from null models in which parasite species abundance was kept constant. We did not find consistency between observed community structure at the host population scale and within-host order of infection. Multi-state Markov models of parasite community assembly showed that a host's likelihood of infection with one parasite did not consistently follow previous infection by a different parasite species, suggesting there is not a deterministic order of infection among the species we investigated in wild wood mice. Our results demonstrate that patterns at one scale (i.e., host population) do not reliably predict processes at another scale (i.e., individual host), and that neutral or stochastic processes may be driving the patterns of nestedness observed in these communities. We suggest that experimental approaches that manipulate parasite communities are needed to better link processes at multiple ecological scales.

The Immune and Non-Immune Pathways That Drive Chronic Gastrointestinal Helminth Burdens in the Wild.

Parasitic helminths are extremely resilient in their ability to maintain chronic infection burdens despite (or maybe because of) their hosts' immune response. Explaining how parasites maintain these lifelong infections, identifying the protective immune mechanisms that regulate helminth infection burdens, and designing prophylactics and therapeutics that combat helminth infection, while preserving host health requires a far better understanding of how the immune system functions in natural habitats than we have at present. It is, therefore, necessary to complement mechanistic laboratory-based studies with studies on wild populations and their natural parasite communities. Unfortunately, the relative paucity of immunological tools for non-model species has held these types of studies back. Thankfully, recent progress in high-throughput 'omics platforms provide powerful and increasingly practical means for immunologists to move beyond traditional lab-based model organisms. Yet, assigning both metabolic and immune function to genes, transcripts, and proteins in novel species and assessing how they interact with other physiological and environmental factors requires identifying quantitative relationships between their expression and infection. Here, we used supervised machine learning to identify gene networks robustly associated with burdens of the gastrointestinal nematode Heligmosomoides polygyrus in its natural host, the wild wood mice Apodemus sylvaticus. Across 34 mice spanning two wild populations and across two different seasons, we found 17,639 transcripts that clustered in 131 weighted gene networks. These clusters robustly predicted H. polygyrus burden and included well-known effector and regulatory immune genes, but also revealed a number of genes associated with the maintenance of tissue homeostasis and hematopoiesis that have so far received little attention. We then tested the effect of experimentally reducing helminth burdens through drug treatment on those putatively protective immune factors. Despite the near elimination of H. polygyrus worms, the treatment had surprisingly little effect on gene expression. Taken together, these results suggest that hosts balance tissue homeostasis and protective immunity, resulting in relatively stable immune and, consequently, parasitological profiles. In the future, applying our approach to larger numbers of samples from additional populations will help further increase our ability to detect the immune pathways that determine chronic gastrointestinal helminth burdens in the wild.

Closely-related Borrelia burgdorferi (sensu stricto) strains exhibit similar fitness in single infections and asymmetric competition in multiple infections.

BACKGROUND: Wild hosts are commonly co-infected with complex, genetically diverse, pathogen communities. Competition is expected between genetically or ecologically similar pathogen strains which may influence patterns of coexistence. However, there is little data on how specific strains of these diverse pathogen species interact within the host and how this impacts pathogen persistence in nature. Ticks are the most common disease vector in temperate regions with Borrelia burgdorferi, the causative agent of Lyme disease, being the most common vector-borne pathogen in North America. Borrelia burgdorferi is a pathogen of high public health concern and there is significant variation in infection phenotype between strains, which influences predictions of pathogen dynamics and spread. METHODS: In a laboratory experiment, we investigated whether two closely-related strains of B. burgdorferi (sensu stricto) showed similar transmission phenotypes, how the transmission of these strains changed when a host was infected with one strain, re-infected with the same strain, or co-infected with two strains. Ixodes scapularis, the black-legged tick, nymphs were used to sequentially infect laboratory-bred Peromyscus leucopus, white-footed mice, with one strain only, homologous infection with the same stain, or heterologous infection with both strains. We used the results of this laboratory experiment to simulate long-term persistence and maintenance of each strain in a simple simulation model. RESULTS: Strain LG734 was more competitive than BL206, showing no difference in transmission between the heterologous infection groups and single-infection controls, while strain BL206 transmission was significantly reduced when strain LG734 infected first. The results of the model show that this asymmetry in competition could lead to extinction of strain BL206 unless there was a tick-to-host transmission advantage to this less competitive strain. CONCLUSIONS: This asymmetric competitive interaction suggests that strain identity and the biotic context of co-infection is important to predict strain dynamics and persistence.

Concordance of bacterial communities of two tick species and blood of their shared rodent host.

High-throughput sequencing is revealing that most macro-organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field-collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species-specific endosymbionts, Francisella and Rickettsia, and the commonly flea-associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick-specific endosymbionts and environmental bacteria that could potentially affect arthropod-vectored disease dynamics.

An ecosystem approach to understanding and managing within-host parasite community dynamics.

Hosts are typically coinfected by multiple parasite species, resulting in potentially overwhelming levels of complexity. We argue that an individual host can be considered to be an ecosystem in that it is an environment containing a diversity of entities (e.g., parasitic organisms, commensal symbionts, host immune components) that interact with each other, potentially competing for space, energy, and resources, ultimately influencing the condition of the host. Tools and concepts from ecosystem ecology can be applied to better understand the dynamics and responses of within-individual host-parasite ecosystems. Examples from both wildlife and human systems demonstrate how this framework is useful in breaking down complex interactions into components that can be monitored, measured, and managed to inform the design of better disease-management strategies.

Tick community composition in Midwestern US habitats in relation to sampling method and environmental conditions.

The ranges of many tick species are changing due to climate change and human alteration of the landscape. Understanding tick responses to environmental conditions and how sampling method influences measurement of tick communities will improve our assessment of human disease risk. We compared tick sampling by three collection methods (dragging, CO2 trapping and rodent surveys) in adjacent forested and grassland habitats in the lower Midwest, USA, and analyzed the relationship between tick abundance and microclimate conditions. The study areas were within the overlapping ranges of three tick species, which may provide conditions for pathogen exchange and spread into new vectors. Dermacentor variabilis (American dog tick) was found using all methods, Amblyomma americanum (lonestar tick) was found by dragging and CO2 trapping and Ixodes scapularis (blacklegged deer tick) was found only on rodents. Proportion of each species differed significantly among sampling methods. More ticks were found in forests compared to open habitats. Further, more ticks were collected by dragging and from rodents in hotter, drier conditions. Our results demonstrate that multiple sampling methodologies better measure the tick community and that microclimate conditions strongly influence the abundance and activity of individual tick 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.