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How directly transmitted pathogens benefit from harming hosts is key to understanding virulence evolution. It is recognized that pathogens benefit from high within-host loads, often associated with virulence. However, high virulence may also directly augment spread of a given amount of pathogen, here termed ‘spreadability’. We used house finches and the conjunctival pathogen Mycoplasma gallisepticum to test whether two components of virulence—the severity of conjunctival inflammation and behavioural morbidity produced—predict pathogen spreadability. We applied ultraviolet powder around the conjunctiva of finches that were inoculated with pathogen treatments of distinct virulence and measured within-flock powder spread, our proxy for ‘spreadability’. When compared to uninfected controls, birds infected with a high-virulence, but not low-virulence, pathogen strain, spread significantly more powder to flockmates. Relative to controls, high-virulence treatment birds both had more severe conjunctival inflammation—which potentially facilitated powder shedding—and longer bouts on feeders, which serve as fomites. However, food peck rates and displacements with flockmates were lowest in high-virulence treatment birds relative to controls, suggesting inflammatory rather than behavioural mechanisms likely drive augmented spreadability at high virulence. Our results suggest that inflammation associated with virulence can facilitate pathogen spread to conspecifics, potentially favouring virulence evolution in this system and others.

Individuals are often co-infected with several parasite species, yet measuring within-host interactions remains difficult in the wild. Consequently, the impact of such interactions on host fitness and epidemiology are often unknown. We used anthelmintic drugs to experimentally reduce nematode infection and measured the effects on both nematodes and the important zoonosis Sin Nombre virus (SNV) in its primary reservoir (Peromyscus spp.). Treatment significantly reduced nematode infection, but increased SNV seroprevalence. Furthermore, mice that were co-infected with both nematodes and SNV were in better condition and survived up to four times longer than uninfected or singly infected mice. These results highlight the importance of investigating multiple parasites for understanding interindividual variation and epidemiological dynamics in reservoir populations with zoonotic transmission potential.

1. The loss of intact forest via logging can influence vector-borne disease dynamics in part by altering the abundance or diversity of mosquito species. Using an experimental field approach, we characterised how two types of logging (clearcut and repeat-entry shelterwood) affected temperate forest mosquito abundance and diversity in southwestern Virginia. 2.From May to September in 2008-2010, infusion-baited gravid traps were used to collect ovipositing female mosquitoes across experimental forest plots that varied in logging treatment. Of the 29680 collected adult female mosquitoes, the three dominant taxa captured were Aedes triseriatus (55%), Aedes japonicus (21%), and Culex pipiens/restuans (20%). 3. Logging treatment had a significant effect on the overall number of female mosquitoes caught per trap night, with lower average abundance of females on both logged treatments relative to two types of unlogged, control plots. When the three most abundant mosquito species were examined separately, logging treatment significantly influenced the abundance of both Aedes species, but did not significantly affect C. pipiens/restuans abundance. 4. Logging treatment did not influence the richness or diversity of mosquito species captured in gravid traps. However, logging treatment significantly altered the multivariate community composition of captured mosquitoes, an effect probably mediated by differential species-specific impacts of logging on abundance. 5. Overall, the results of the present study suggest that the risk of arboviruses transmitted by container-breeding Aedes species may be lower following a logging event in Appalachian forests because of reduced A. japonicus and A. triseriatus abundance with logging. Virginia Tech; NIH F31 Ruth L. Kirschstein National Research Service AwardUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1F31AI080160-01A1]; Sigma Xi We thank Nate Lambert, Allen Patton, R. J. Wilding, Jake Bova, Bonnie Fairbanks, Cari Lynn Squibb, Noah Thrope, Kylie Perkins, Lindsey McAlexander, Genevieve Dudzinsky, Jennifer Miller, and Laila Kirkpatrick for field and laboratory assistance. We thank James Adelman for statistical analysis assistance. We thank Jesse Overcash, Travis Belote, Bob Jones, and Carola Haas for assistance with SASAB logistics. Finally, we thank Tara Craig and Jennifer Smith for assistance with Figure 1 and multiple anonymous reviewers for helpful comments on the manuscript. Research funding was supported in part by Virginia Tech, Sigma Xi and an NIH F31 Ruth L. Kirschstein National Research Service Award for Individual Pre-doctoral Fellows [1F31AI080160-01A1]. The authors declare that they have no conflicts of interest related to this work. Public domain – authored by a U.S. government employee

Combined supplementary methods and results

Vertebrate ocular microbiomes are poorly characterized and virtually unexplored in wildlife species. Pathogen defense is considered a key function of microbiomes, but determining microbiome stability during disease is critical for understanding the role of resident microbial communities in infectious disease dynamics. Here, we characterize the ocular bacterial microbiome of house finches (Haemorhous mexicanus), prior to and during experimental infection with an inflammatory ocular disease, Mycoplasmal conjunctivitis, caused by Mycoplasma gallisepticum. In ocular tissues of healthy house finches, we identified 526 total bacterial operational taxonomic units (OTUs, 97% similarity), primarily from Firmicutes (92.6%) and Proteobacteria (6.9%), via 16S rRNA gene amplicon sequencing. Resident ocular communities of healthy female finches were characterized by greater evenness and phylogenetic diversity compared to healthy male finches. Regardless of sex, ocular microbiome community structure significantly shifted 11 days after experimental inoculation with M. gallisepticum. A suite of OTUs, including taxa from the genera Methylobacterium, Acinetobacter, and Mycoplasma, appear to drive these changes, indicating that the whole finch ocular microbiome responds to infection. Further study is needed to quantify changes in absolute abundance of resident taxa and to elucidate potential functional roles of the resident ocular microbiome in mediating individual responses to this common songbird bacterial pathogen.

Infectious diseases are ubiquitous in nature and can be strong selective forces for the evolution of wild populations. Individual hosts are commonly infected with multiple parasites simultaneously, and this pattern has been documented in many systems. Recent evidence suggests parasites can partition host resources as direct competitors or indirectly through resource- or immune-mediated interactions, and these interactions can have significant implications for host fitness and the health of the population. Nevertheless, much of this work has focused on single parasite infections, though in nature and in humans, most hosts are infected with multiple parasites. The consequences of ‘co-infection’ can be important for the immune response and host health by changing the ecology of host population dynamics, negatively affecting host fitness in the form of disease, and disrupting the normal host response to infection; however little research has focused on this area until relatively recently. Understanding the complex dynamics of co-infection can be difficult; yet, using a community ecological approach can provide a framework to make sense of these complicated interactions. Indeed, parasite infection was common in the Mountain Lake Biological Station populations of wild Peromyscus hosts, with over 90% of mouse captures over the course of 3 consecutive mouse breeding seasons (May – August: 2010-2012) infected with 3.4 different parasites on average. Using rigorous and controlled statistical models, I found that when co-infections were present, parasite associations were positive in nature and variable in strength from weak to strong. Most parasite associations were contemporary, but a few were time-lagged, and the combination of all associations created a complex within-host parasite community. Using a perturbation experiment, I found there was a decrease in nematode burden with ivermectin treatment, and a concomitant increase in coccidian protozoa. Importantly, mice treated with a single oral dose of anthelmintic had significantly lower survival than control mice, suggesting that treatment may be detrimental to survival and fitness because of unintentional increases in co-infected parasites. Furthermore, these results suggest that there are strong negative interactions between nematodes and coccidia. Additionally, effects of ivermectin treatment were not isolated to treated individuals only, as there were knock-on effects to untreated individuals within the population of a similar type and magnitude as seen in mice that received ivermectin treatment. Experimental perturbation of the within-host parasite community revealed that parasites interact in a reciprocal manner. To examine direct and indirect interactions between microparasites and macroparasites across differing host systems (i.e. blood and gastrointestinal tract), I applied a randomized four-level factorial treatment (control, ivermectin, fipronil, dual ivermectin and fipronil) to individuals in the host Peromyscus population designed to reduce either microparasites (via fipronil to reduce fleas, a vector of Bartonella), macroparasites (via ivermectin to reduce nematode infection), or both (via dual treatment). Again, ivermectin treatment reduced nematodes, and an interaction with coccidia resulted in reduced recapture. However, fipronil treatment had no effect on Bartonella infection, and there were unexpected effects of fipronil on coccidia. It has been suggested that fipronil also acts to reduce nematode infection, so the effect of fipronil on coccidia was also, possibly, an effect of nematode removal. These results suggest that microparasites may impose a heavier cost to their hosts than macroparasites, especially if infection intensities increase suddenly or rapidly. Overall, these data provide support for conducting manipulative field experiments, rather than relying on observations of non-random associations to determine that parasites are interacting with each other. However, if manipulative field experiments are not possible because of logistical or ethical constraints, then the most reliable way to detect the presence of strong parasite associations is to conduct a combined cross-sectional and longitudinal analysis, controlling for as many confounding individual host traits and environmental factors as possible, to look for associations that remain consistent across both tests. In addition, the data from these studies present strong evidence that within-host communities provide an arena for direct and indirect host and parasite interactions that apply strong selective forces to populations at larger scales.