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While some pathogens are limited to single species, others can colonize many hosts, likely contributing to the emergence of novel disease outbreaks. Despite this biodiversity threat, traits associated with host niche expansions are not well understood in multihost pathogens. Here, we aimed to uncover functional machinery driving multihost invasion by focusing on Batrachochytrium dendrobatidis (Bd), a pathogen that infects the skin of hundreds of amphibians worldwide. We performed a meta-analysis of Bd gene expression using data from published infection experiments and newly generated profiles. We analysed Bd transcriptomic landscapes across the skin of 14 host species, reconstructed Bd isolates phylogenetic relationships, and inferred the origin and evolutionary history of differentially expressed genes under a phylogenetic framework comprising other 12 zoosporic fungi. Bd displayed plastic infection strategies when challenged by hosts with different disease susceptibility. Our analyses identified sets of differentially expressed genes under host environments with similar infection outcome. We stressed nutritional immunity and gene silencing as important processes required to overcome challenging skin environments in less susceptible hosts. Overall, Bd genes expressed during amphibian skin exploitation have arisen mainly via gene duplications with great family expansions, increasing the gene copy events previously described for this fungal species. Finally, we provide a comprehensive gene data set that can be used to further examine eco-evolutionary hypotheses for this host-pathogen system. Our study supports the idea that host environments exert contrasting selective pressures, such that gene expression plasticity could be one of the evolutionary keys leading to the success of multihost pathogens.

Host-pathogen specificity can arise from certain selective environments mediated by both the host and pathogen. Therefore, understanding the degree to which host species identity is correlated with pathogen genotype can help reveal historical host-pathogen dynamics. One animal disease of particular concern is chytridiomycosis, typically caused by the global panzootic lineage of the amphibian chytrid fungus (Batrachochytrium dendrobatidis, Bd), termed the Bd-GPL. This pathogen lineage has caused devastating declines in amphibian communities around the world. However, the origin of Bd-GPL and the fine-scale transmission dynamics of this lineage have remained a mystery. This is especially the case in North America where Bd-GPL is widespread, but disease outbreaks occur sporadically. Herein, we use Bd genetic data collected throughout the United States from amphibian skin swab and cultured isolate samples to investigate Bd genetic patterns. We highlight two case studies in Pennsylvania and Nevada where Bd-GPL genotypes are strongly correlated with host species identity. Specifically, in some localities bullfrogs (Rana catesbeiana) are infected with Bd-GPL lineages that are distinct from those infecting other sympatric amphibian species. Overall, we reveal a previously unknown association of Bd genotype with host species and identify the eastern United States as a Bd diversity hotspot and potential ancestral population for Bd-GPL.

Outbreaks of emerging infectious diseases are trained by local biotic and abiotic factors, with host declines occurring when conditions favour the pathogen. Extinction of the Tanzanian Kihansi spray toad (Nectophrynoides asperginis) in 2004 was contemporaneous with the construction of a dam, implicating habitat modification in the loss of this species. However, high burdens of a globally emerging infection, Batrachochytrium dendrobatidis (Bd) were synchronously observed implicating infectious disease in this toads extinction. Here, by shotgun sequencing skin DNA from archived toad mortalities and assembling chytrid mitogenomes, we prove this outbreak was caused by the BdCAPE lineage and not the panzootic lineage BdGPL that is widely associated with global amphibian extinctions. Molecular dating showed an invasion of BdCAPE across Southern Africa overlapping with the timing of the extinction event. However, post-outbreak surveillance of conspecific species inhabiting this mountainous region showed widespread infection by BdCAPE yet no signs of amphibian ill-health or species decline. Our findings show that despite efforts to mitigate the environmental impact caused by dams construction, invasion of the pathogen ultimately led to the loss of the Kihansi spray toad; a synergism between emerging infectious disease and environmental change that likely heralds wider negative impacts on biodiversity in the Anthropocene.

Wild birds can carry avian influenza viruses (AIV), including those with pandemic or panzootic potential, long distances. Even though AIV has a broad host range, few studies account for host diversity when estimating AIV spread. We analyzed AIV genomic sequences from North American wild birds, including 303 newly sequenced isolates, to estimate interspecies transmission and geographic diffusion patterns among multiple co-circulating subtypes. Our results show high transition rates within Anseriformes and Charadriiformes, but limited transitions between these orders. Patterns of interspecies transmission were positively associated with breeding habitat range overlap, and negatively associated with host genetic distance. Distance between regions (negative correlation) and summer temperature at origin (positive correlation) were strong predictors of diffusion. Taken together, this study demonstrates that host diversity and ecology can determine evolutionary processes that underlie AIV natural history and spread. Understanding these processes can provide important insights for effective control of AIV.

Highly pathogenic avian influenza viruses H5N8 clade 2.3.4.4 caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade-2.3.4.4A in 2014/2015 and clade-2.3.4.4B since 2016 up to today. Both clades were highly virulent in chickens, but only clade-B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 2.3.4.4 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in-vitro experiments indicated that clade-2.3.4.4A and clade-2.3.4.4B viruses have a preference for NS1 of 237-aa and 217-aa, respectively over NS1 of 230-aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C-terminus of clade-B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade-A virus was observed. Remarkably, the NS1 of clade-A and clade-B, regardless of length, was efficient to block interferon induction in infected chickens and changes in the NS1 C-terminus reduced the efficiency for interferon antagonism. Together, the NS1 C-terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks.ImportanceThe panzootic H5N8 highly pathogenic avian influenza viruses of clade 2.3.4.4A and 2.3.4.4B devastated poultry industry globally. Clade 2.3.4.4A was predominant in 2014/2015 while clade 2.3.4.4B was widely spread in 2016/2017. Both clades exhibited different pathotypes in ducks. Virus factors contributing to virulence and transmission are largely unknown. The NS1 protein is typically composed of 230 amino-acids (aa) and is an essential interferon (IFN) antagonist. Here, we found that the NS1 protein of clade 2.3.4.4A preferentially evolved toward long NS1 with 237-aa, while clade 2.3.4.4B evolved toward shorter NS1 with 217-aa (exclusively found in Europe) due stop-codons in the C-terminus (CTE). We showed that the NS1 CTE of H5N8 is required for efficient virus replication, transmission and endotheliotropism in ducks. In chickens, H5N8 NS1 evolved toward higher efficiency to block IFN-response. These findings may explain the preferential pattern for short NS1 and high fitness of the panzootic H5N8 in birds.Subject categoryAnimal, RNA Viruses

Free-living eukaryotic microbes may reduce animal diseases. We evaluated the dynamics by which micrograzers (primarily protozoa) apply top-down control on the chytrid Batrachochytrium dendrobatidis (Bd) a devastating, panzootic pathogen of amphibians. Although micrograzers consumed zoospores (∼3 µm), the dispersal stage of chytrids, not all species grew monoxenically on zoospores. However, the ubiquitous ciliate Tetrahymena pyriformis, which likely co-occurs with Bd, grew at near its maximum rate (r = 1.7 d-1). A functional response (ingestion vs. prey abundance) for T. pyriformis, measured using spore-surrogates (microspheres) revealed maximum ingestion (Imax) of 1.63 × 103 zoospores d-1, with a half saturation constant (k) of 5.75 × 103 zoospores ml-1. Using these growth and grazing data we developed and assessed a population model that incorporated chytrid-host and micrograzer dynamics. Simulations using our data and realistic parameters obtained from the literature suggested that micrograzers could control Bd and potentially prevent chytridiomycosis (defined as 104 sporangia host-1). However, simulated inferior micrograzers (0.7 x Imax and 1.5 x k) did not prevent chytridiomycosis, although they ultimately reduced pathogen abundance to below levels resulting in disease. These findings indicate how micrograzer responses can be applied when modelling disease dynamics for Bd and other zoosporic fungi.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), have caused the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd infection in 1016 specimens collected throughout Costa Rica. We found Bd-infected hosts collected as early as 1964, and a infection prevalence average per decade of just 4%. The infection prevalence remained relatively low and geographically constrained until the 1980s when epizootics are hypothesized to have occurred. After that time, infection prevalence increased three-fold and Bd-infected hosts we collected throughout the entire country. Our results, suggest that Bd may either have invaded Costa Rica earlier than previously known, and spread more slowly than previously reported, or that an endemic lineage of the pathogen may exists. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine whether an endemic lineage of Bd or the Global Panzootic Lineage (identified from mass die off sites globally) was present in Costa Rica and responsible for the epizootics that caused amphibian communities to collapse.