Sex-biased infections scale to population impacts for an emerging wildlife disease.

Demographic factors are fundamental in shaping infectious disease dynamics. Aspects of populations that create structure, like age and sex, can affect patterns of transmission, infection intensity and population outcomes. However, studies rarely link these processes from individual to population-scale effects. Moreover, the mechanisms underlying demographic differences in disease are frequently unclear. Here, we explore sex-biased infections for a multi-host fungal disease of bats, white-nose syndrome, and link disease-associated mortality between sexes, the distortion of sex ratios and the potential mechanisms underlying sex differences in infection. We collected data on host traits, infection intensity and survival of five bat species at 42 sites across seven years. We found females were more infected than males for all five species. Females also had lower apparent survival over winter and accounted for a smaller proportion of populations over time. Notably, female-biased infections were evident by early hibernation and likely driven by sex-based differences in autumn mating behaviour. Male bats were more active during autumn which likely reduced replication of the cool-growing fungus. Higher disease impacts in female bats may have cascading effects on bat populations beyond the hibernation season by limiting recruitment and increasing the risk of Allee effects.

Immunogenicity, Safety, and Anti-Viral Efficacy of a Subunit SARS-CoV-2 Vaccine Candidate in Captive Black-Footed Ferrets (Mustela nigripes) and Their Susceptibility to Viral Challenge.

A preliminary vaccination trial against the emergent pathogen, SARS-CoV-2, was completed in captive black-footed ferrets (Mustela nigripes; BFF) to assess safety, immunogenicity, and anti-viral efficacy. Vaccination and boosting of 15 BFF with purified SARS-CoV-2 S1 subunit protein produced a nearly 150-fold increase in mean antibody titers compared to pre-vaccination titers. Serum antibody responses were highest in young animals, but in all vaccinees, antibody response declined rapidly. Anti-viral activity from vaccinated and unvaccinated BFF was determined in vitro, as well as in vivo with a passive serum transfer study in mice. Transgenic mice that received BFF serum transfers and were subsequently challenged with SARS-CoV-2 had lung viral loads that negatively correlated (p < 0.05) with the BFF serum titer received. Lastly, an experimental challenge study in a small group of BFF was completed to test susceptibility to SARS-CoV-2. Despite viral replication and shedding in the upper respiratory tract for up to 7 days post-challenge, no clinical disease was observed in either vaccinated or naive animals. The lack of morbidity or mortality observed indicates SARS-CoV-2 is unlikely to affect wild BFF populations, but infected captive animals pose a potential risk, albeit low, for humans and other animals.

Evidence gaps and diversity among potential win-win solutions for conservation and human infectious disease control.

As sustainable development practitioners have worked to "ensure healthy lives and promote well-being for all" and "conserve life on land and below water", what progress has been made with win-win interventions that reduce human infectious disease burdens while advancing conservation goals? Using a systematic literature review, we identified 46 proposed solutions, which we then investigated individually using targeted literature reviews. The proposed solutions addressed diverse conservation threats and human infectious diseases, and thus, the proposed interventions varied in scale, costs, and impacts. Some potential solutions had medium-quality to high-quality evidence for previous success in achieving proposed impacts in one or both sectors. However, there were notable evidence gaps within and among solutions, highlighting opportunities for further research and adaptive implementation. Stakeholders seeking win-win interventions can explore this Review and an online database to find and tailor a relevant solution or brainstorm new solutions.

Protection Generated by Prior Exposure to Pathogens Depends on both Priming and Challenge Dose.

Free-living hosts encounter pathogens at a wide range of frequencies and concentrations, including low doses that are largely aclinical, creating a varied landscape of exposure history and reinfection likelihood. While several studies show that higher priming doses result in stronger immunological protection against reinfection, it remains unknown how the reinfection challenge dose and priming dose interact to determine the likelihood and severity of reinfection. We manipulated both priming and challenge doses of Mycoplasma gallisepticum, which causes mycoplasmal conjunctivitis, in captive house finches (Haemorhous mexicanus), to assess reinfection probability and severity. We found a significant interaction between priming and challenge doses on reinfection probability, with the likelihood of reinfection by a high but not a low challenge dose decreasing exponentially at higher priming doses. While this interaction was likely driven by lower average infection probabilities for low-dose versus high-dose challenges, even the highest priming dose provided only negligible protection against reinfection from low-dose challenges. Similarly, pathogen loads during reinfection were significantly reduced with increasing priming doses only for birds reinfected at high but not low doses. We hypothesize that these interactions arise to some degree from fundamental differences in host immune responses across doses, with single low doses only weakly triggering host immune responses. Importantly, our results also demonstrate that reinfections can occur from a variety of exposure doses and across diverse degrees of standing immunity in this system. Overall, our study highlights the importance of considering both initial and subsequent exposure doses where repeated exposure to a pathogen is common in nature.

Experimental challenge of a North American bat species, big brown bat (Eptesicus fuscus), with SARS-CoV-2.

The recently emerged novel coronavirus, SARS-CoV-2, is phylogenetically related to bat coronaviruses (CoVs), specifically SARS-related CoVs from the Eurasian bat family Rhinolophidae. As this human pandemic virus has spread across the world, the potential impacts of SARS-CoV-2 on native North American bat populations are unknown, as is the ability of North American bats to serve as reservoirs or intermediate hosts able to transmit the virus to humans or to other animal species. To help determine the impacts of the pandemic virus on North American bat populations, we experimentally challenged big brown bats (Eptesicus fuscus) with SARS-CoV-2 under BSL-3 conditions. We inoculated the bats both oropharyngeally and nasally, and over the ensuing three weeks, we measured infectivity, pathology, virus concentrations in tissues, oral and rectal virus excretion, virus transmission, and clinical signs of disease. We found no evidence of SARS-CoV-2 infection in any examined bat, including no viral excretion, no transmission, no detectable virus in tissues, and no signs of disease or pathology. Based on our findings, it appears that big brown bats are resistant to infection with the SARS-CoV-2. The potential susceptibility of other North American bat species to SARS-CoV-2 remains to be investigated.

House finches with high coccidia burdens experience more severe experimental Mycoplasma gallisepticum infections.

Parasites co-infecting hosts can interact directly and indirectly to affect parasite growth and disease manifestation. We examined potential interactions between two common parasites of house finches: the bacterium Mycoplasma gallisepticum that causes conjunctivitis and the intestinal coccidian parasite Isospora sp. We quantified coccidia burdens prior to and following experimental infection with M. gallisepticum, exploiting the birds' range of natural coccidia burdens. Birds with greater baseline coccidia burdens developed higher M. gallisepticum loads and longer lasting conjunctivitis following inoculation. However, experimental inoculation with M. gallisepticum did not appear to alter coccidia shedding. Our study suggests that differences in immunocompetence or condition may predispose some finches to more severe infections with both pathogens.

Differential house finch leukocyte profiles during experimental infection with Mycoplasma gallisepticum isolates of varying virulence.

Leukocyte differentials are a useful tool for assessing systemic immunological changes during pathogen infections, particularly for non-model species. To date, no study has explored how experimental infection with a common bacterial pathogen, Mycoplasma gallisepticum (MG), influences the course and strength of haematological changes in the natural songbird host, house finches. Here we experimentally inoculated house finches with MG isolates known to vary in virulence, and quantified the proportions of circulating leukocytes over the entirety of infection. First, we found significant temporal effects of MG infection on the proportions of most cell types, with strong increases in heterophil and monocyte proportions during infection. Marked decreases in lymphocyte proportions also occurred during infection, though these proportional changes may simply be driven by correlated increases in other leukocytes. Second, we found significant effects of isolate virulence, with the strongest changes in cell proportions occurring in birds inoculated with the higher virulence isolates, and almost no detectable changes relative to sham treatment groups in birds inoculated with the lowest virulence isolate. Finally, we found that variation in infection severity positively predicted the proportion of circulating heterophils and lymphocytes, but the strength of these correlations was dependent on isolate. Taken together, these results indicate strong haematological changes in house finches during MG infection, with markedly different responses to MG isolates of varying virulence. These results are consistent with the possibility that evolved virulence in house finch MG results in higher degrees of immune stimulation and associated immunopathology, with potential direct benefits for MG transmission. RESEARCH HIGHLIGHTS House finches show a marked pro-inflammatory response to M. gallisepticum infection. Virulent pathogen isolates produce stronger finch white blood cell responses. Among birds, stronger white blood cell responses are associated with higher infection severity.

Host exposure history modulates the within-host advantage of virulence in a songbird-bacterium system.

The host immune response can exert strong selective pressure on pathogen virulence, particularly when host protection against reinfection is incomplete. Since emerging in house finch populations, the bacterial pathogen Mycoplasma gallisepticum (MG) has been increasing in virulence. Repeated exposure to low-doses of MG, a proxy for what birds likely experience while foraging, provides significant but incomplete protection against reinfection. Here we sought to determine if the within-host, pathogen load advantage of high virulence is mediated by the degree of prior pathogen exposure, and thus the extent of immune memory. We created variation in host immunity by experimentally inoculating wild-caught, MG-naïve house finches with varying doses and number of exposures of a single pathogen strain of intermediate virulence. Following recovery from priming exposures, individuals were challenged with one of three MG strains of distinct virulence. We found that the quantitative pathogen load advantage of high virulence was strongly mediated by the degree of prior exposure. The greatest within-host load advantage of virulence was seen in hosts given low-dose priming exposures, akin to what many house finches likely experience while foraging. Our results show that incomplete host immunity produced by low-level prior exposure can create a within-host environment that favors more virulent pathogens.

Incomplete host immunity favors the evolution of virulence in an emergent pathogen.

Immune memory evolved to protect hosts from reinfection, but incomplete responses that allow future reinfection may inadvertently select for more-harmful pathogens. We present empirical and modeling evidence that incomplete immunity promotes the evolution of higher virulence in a natural host-pathogen system. We performed sequential infections of house finches with Mycoplasma gallisepticum strains of various levels of virulence. Virulent bacterial strains generated stronger host protection against reinfection than less virulent strains and thus excluded less virulent strains from infecting previously exposed hosts. In a two-strain model, the resulting fitness advantage selected for an almost twofold increase in pathogen virulence. Thus, the same immune systems that protect hosts from infection can concomitantly drive the evolution of more-harmful pathogens in nature.

Differing House Finch Cytokine Expression Responses to Original and Evolved Isolates of Mycoplasma gallisepticum.

The recent emergence of the poultry bacterial pathogen Mycoplasma gallisepticum (MG) in free-living house finches (Haemorhous mexicanus), which causes mycoplasmal conjunctivitis in this passerine bird species, resulted in a rapid coevolutionary arms-race between MG and its novel avian host. Despite extensive research on the ecological and evolutionary dynamics of this host-pathogen system over the past two decades, the immunological responses of house finches to MG infection remain poorly understood. We developed seven new probe-based one-step quantitative reverse transcription polymerase chain reaction assays to investigate mRNA expression of house finch cytokine genes (IL1B, IL6, IL10, IL18, TGFB2, TNFSF15, and CXCLi2, syn. IL8L). These assays were then used to describe cytokine transcription profiles in a panel of 15 house finch tissues collected at three distinct time points during MG infection. Based on initial screening that indicated strong pro-inflammatory cytokine expression during MG infection at the periorbital sites in particular, we selected two key house finch tissues for further characterization: the nictitating membrane, i.e., the internal eyelid in direct contact with MG, and the Harderian gland, the secondary lymphoid tissue responsible for regulation of periorbital immunity. We characterized cytokine responses in these two tissues for 60 house finches experimentally inoculated either with media alone (sham) or one of two MG isolates: the earliest known pathogen isolate from house finches (VA1994) or an evolutionarily more derived isolate collected in 2006 (NC2006), which is known to be more virulent. We show that the more derived and virulent isolate NC2006, relative to VA1994, triggers stronger local inflammatory cytokine signaling, with peak cytokine expression generally occurring 3-6 days following MG inoculation. We also found that the extent of pro-inflammatory interleukin 1 beta signaling was correlated with conjunctival MG loads and the extent of clinical signs of conjunctivitis, the main pathological effect of MG in house finches. These results suggest that the pathogenicity caused by MG infection in house finches is largely mediated by host pro-inflammatory immune responses, with important implications for the dynamics of host-pathogen coevolution.

Development and validation of a house finch interleukin-1ß (HfIL-1ß) ELISA system.

BACKGROUND: A unique clade of the bacterium Mycoplasma gallisepticum (MG), which causes chronic respiratory disease in poultry, has resulted in annual epidemics of conjunctivitis in North American house finches since the 1990s. Currently, few immunological tools have been validated for this songbird species. Interleukin-1ß (IL-1ß) is a prototypic multifunctional cytokine and can affect almost every cell type during Mycoplasma infection. The overall goal of this study was to develop and validate a direct ELISA assay for house finch IL-1ß (HfIL-1ß) using a cross-reactive chicken antibody. METHODS: A direct ELISA approach was used to develop this system using two different coating methods, carbonate and dehydration. In both methods, antigens (recombinant HfIL-1b or house finch plasma) were serially diluted in carbonate-bicarbonate coating buffer and either incubated at 4 °C overnight or at 60 °C on a heating block for 2 hr. To generate the standard curve, rHfIL-1b protein was serially diluted at 0, 3, 6, 9, 12, 15, 18, 21, and 24 ng/mL. Following blocking and washing, anti-chicken IL-1b polyclonal antibody was added, plates were later incubated with detecting antibodies, and reactions developed with tetramethylbenzidine solution. RESULTS: A commercially available anti-chicken IL-1ß (ChIL-1ß) polyclonal antibody (pAb) cross-reacted with house finch plasma IL-1ß as well as bacterially expressed recombinant house finch IL-1ß (rHfIL-1ß) in immunoblotting assays. In a direct ELISA system, rHfIL-1ß could not be detected by an anti-ChIL-1ß pAb when the antigen was coated with carbonate-bicarbonate buffer at 4°C overnight. However, rHfIL-1ß was detected by the anti-ChIL-1ß pAb when the antigen was coated using a dehydration method by heat (60°C). Using the developed direct ELISA for HfIL-1ß with commercial anti-ChIL-1ß pAb, we were able to measure plasma IL-1ß levels from house finches. CONCLUSIONS: Based on high amino acid sequence homology, we hypothesized and demonstrated cross-reactivity of anti-ChIL-1ß pAb and HfIL-1ß. Then, we developed and validated a direct ELISA system for HfIL-1ß using a commercial anti-ChIL-1ß pAb by measuring plasma HfIL-1ß in house finches.

Host Responses to Pathogen Priming in a Natural Songbird Host.

Hosts in free-living populations can experience substantial variation in the frequency and dose of pathogen exposure, which can alter disease progression and protection from future exposures. In the house finch-Mycoplasma gallisepticum (MG) system, the pathogen is primarily transmitted via bird feeders, and some birds may be exposed to frequent low doses of MG while foraging. Here we experimentally determined how low dose, repeated exposures of house finches to MG influence host responses and protection from secondary high-dose challenge. MG-naive house finches were given priming exposures that varied in dose and total number. After quantifying host responses to priming exposures, all birds were given a secondary high-dose challenge to assess immunological protection. Dose, but not the number of exposures, significantly predicted both infection and disease severity following priming exposure. Furthermore, individuals given higher priming doses showed stronger protection upon secondary, high-dose challenge. However, even single low-dose exposures to MG, a proxy for what some birds likely experience in the wild while feeding, provided significant protection against a high-dose challenge. Our results suggest that bird feeders, which serve as sources of infection in the wild, may in some cases act as "immunizers," with important consequences for disease dynamics.

Identification and functional characterization of the house finch interleukin-1ß.

Interleukin-1ß (IL-1ß), an inflammatory cytokine of the IL-1 family, is primarily produced as a precursor protein by monocytes and macrophages, then matures and becomes activated through proteolytic catalysis. Although the biological characteristics of avian IL-1ß are well known, little information is available about its biological role in songbird species such as house finches that are vulnerable to naturally-occurring inflammatory diseases. In this study, house finch IL-1ß (HfIL-1ß) was cloned, expressed, and its biological function examined. Both precursor and mature forms of HfIL-1ß consisting of 269 and 162 amino acids, respectively, were amplified from total RNA of spleen and cloned into expression vectors. HfIL-1ß showed high sequential and tertiary structural similarity to chicken homologue that allowed detection of the expressed mature recombinant HfIL-1ß (rHfIL-1ß) with anti-ChIL-1ß antibody by immunoblot analysis. For further characterization, we used primary splenocytes and hepatocytes that are predominant sources of IL-1ß upon stimulation, as well as suitable targets to stimulation by IL-1ß. Isolated house finch splenocytes were stimulated with rHfIL-1ß in the presence and absence of concanavalin A (Con A), RNA was extracted and transcript levels of Th1/Th2 cytokines and a chemokine were measured by qRT-PCR. The addition of rHfIL-1ß induced significant enhancement of IL-2 transcript, a Th1 cytokine, while transcription of IL-1ß and the Th2 cytokine IL-10 was slightly enhanced by rHfIL-1ß treatment. rHfIL-1ß also led to elevated levels of the chemokine CXCL1 and nitric oxide production regardless of co-stimulation with Con A. In addition, the production of the acute phase protein serum amyloid A and the antimicrobial peptide LEAP2 was observed in HfIL-1ß-stimulated hepatocytes. Taken together, these observations revealed the basic functions of HfIL-1ß including the stimulatory effect on cell proliferation, production of Th1/Th2 cytokines and acute phase proteins by immune cells, thus providing valuable insight into how HfIL-1ß is involved in regulating inflammatory response.