An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence.

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

Estrogen contamination increases vulnerability of amphibians to the deadly chytrid fungus.

Aquatic contaminants and infectious diseases are among the major drivers of global amphibian declines. However, the interaction of these factors is poorly explored and could better explain the amphibian crisis. We exposed males and females of the Brazilian Cururu Toad, Rhinella icterica, to an environmentally relevant concentration of the estrogen 17-alpha-ethinylestradiol (an emerging contaminant) and to the chytrid infection (Batrachochytrium dendrobatidis), in their combined and isolated forms, and the ecotoxicity was determined by multiple biomarkers: cutaneous, hematological, cardiac, hepatic, and gonadal analysis. Our results showed that Cururu toads had many physiological alterations in response to the chytrid infection, including the appearance of cutaneous Langerhans's cells, increased blood leukocytes, increased heart contraction force and tachycardia, increased hepatic melanomacrophage cells, which in turn led to gonadal atrophy. The estrogen, in turn, increased the susceptibility of the toads to the chytrid infection (higher Bd loads) and maximized the deleterious effects of the pathogen: reducing leukocytes, decreasing the contraction force, and causing greater tachycardia, increasing hepatic melanomacrophage cells, and leading to greater gonadal atrophy, which were more extreme in females. The exposure to estrogen also revealed important toxicodynamic pathways of this toxicant, as shown by the immunosuppression of exposed animals, and the induction of the first stages of feminization in males, which corroborates that the synthetic estrogen acts as an endocrine disruptor. Such an intricate relationship is unprecedented and reinforces the importance of studying the serious consequences that multiple environmental stressors can cause to aquatic populations.

Coevolution of a generalist pathogen with many hosts: the case of the amphibian chytrid Batrachochytrium dendrobatidis.

Generalist pathogens maintain infectivity in numerous hosts; how this broad ecological niche impacts host-pathogen coevolution remains to be widely explored. Batrachochytrium dendrobatidis (Bd) is a highly generalist pathogenic fungus that has caused devastating declines in hundreds of amphibian species worldwide. This review examines amphibian chytridiomycosis host-pathogen interactions and available evidence for coevolution between Bd and its numerous hosts. We summarize recent evidence showing that Bd genotypes vary in geographic distribution and virulence, and that amphibian species also vary in Bd susceptibility according to their geographic distribution. How much variation can be explained by phenotypic plasticity or genetic differences remains uncertain. Recent research suggests that Bd genotypes display preferences for specific hosts and that some hosts are undergoing evolution as populations rebound from Bd outbreaks. Taken together, these findings suggest the potential for coevolution to occur and illuminate a path for addressing open questions through integrating historical and contemporary genetic data.

Global trends in antimicrobial resistance on organic and conventional farms.

The important hypothesis that organic livestock management reduces the prevalence of antimicrobial resistance is either fiercely supported or bitterly contested. Yet, empirical evidence supporting this view remains fragmentary, in part because relationships between antimicrobial use and drug resistance vary dramatically across contexts, hosts, pathogens, and country-specific regulations. Here, we synthesize global policies and definitions of 'organic' and ask if organic farming results in notable reductions in the prevalence of antimicrobial resistance when directly examined alongside conventional analogs. We synthesized the results of 72 studies, spanning 22 countries and five pathogens. Our results highlight substantial variations in country-specific policies on drug use and definitions of 'organic' that hinder broad-scale and generalizable patterns. Overall, conventional farms had slightly higher levels of antimicrobial resistance (28%) relative to organic counterparts (18%), although we found significant context-dependent variation in this pattern. Notably, environmental samples from organic and conventional farms often exhibited high levels of resistance to medically important drugs, underscoring the need for more stringent and consistent policies to control antimicrobial contaminants in the soil (particularly on organic farms, where the application of conventional manure could faciliate the spread antimicrobial resistance). Taken together, these results emphasize the challenges inherent in understanding links between drug use and drug resistance, the critical need for global standards governing organic policies, and greater investment in viable alternatives for managing disease in livestock.

Amphibian Hymenochirus boettgeri as an experimental model for infection studies with the chytrid fungus Batrachochytrium dendrobatidis.

Model organisms are crucial in research as they can provide key insights applicable to other species. This study proposes the use of the amphibian species Hymenochirus boettgeri, widely available through the aquarium trade, as a model organism for the study of chytridiomycosis, a disease caused by the fungus Batrachochytrium dendrobatidis (Bd) and linked to amphibian decline and extinction globally. Currently, no model organisms are used in the study of chytridiomycosis, particularly because of the lack of availability and nonstandardized methods. Thus, laboratories around the world use wild local species to conduct Bd infection experiments, which prevents comparisons between studies and reduces reproducibility. Here, we performed a series of Bd infection assays that showed that H. boettgeri has a dose- and genotype-dependent response, can generalize previous findings on virulence estimates in other species, and can generate reproducible results in replicated experimental conditions. We also provided valuable information regarding H. boettgeri husbandry, including care, housing, reproduction, and heat treatment to eliminate previous Bd infections. Together, our results indicate that H. boettgeri is a powerful and low-ecological-impact system for studying Bd pathogenicity and virulence.

Coinfection with chytrid genotypes drives divergent infection dynamics reflecting regional distribution patterns.

By altering the abundance, diversity, and distribution of species-and their pathogens-globalization may inadvertently select for more virulent pathogens. In Brazil's Atlantic Forest, a hotspot of amphibian biodiversity, the global amphibian trade has facilitated the co-occurrence of previously isolated enzootic and panzootic lineages of the pathogenic amphibian-chytrid (Batrachochytrium dendrobatidis, 'Bd') and generated new virulent recombinant genotypes ('hybrids'). Epidemiological data indicate that amphibian declines are most severe in hybrid zones, suggesting that coinfections are causing more severe infections or selecting for higher virulence. We investigated how coinfections involving these genotypes shapes virulence and transmission. Overall, coinfection favored the more virulent and competitively superior panzootic genotype, despite dampening its transmission potential and overall virulence. However, for the least virulent and least competitive genotype, coinfection increased both overall virulence and transmission. Thus, by integrating experimental and epidemiological data, our results provide mechanistic insight into how globalization can select for, and propel, the emergence of introduced hypervirulent lineages, such as the globally distributed panzootic lineage of Bd.

Co-infecting pathogen lineages have additive effects on host bacterial communities.

Amphibian skin bacteria may confer protection against the fungus Batrachochytrium dendrobatidis (Bd), but responses of skin bacteria to different Bd lineages are poorly understood. The global panzootic lineage (Bd-GPL) has caused amphibian declines and extinctions globally. However, other lineages are enzootic (Bd-Asia-2/Brazil). Increased contact rates between Bd-GPL and enzootic lineages via globalization pose unknown consequences for host-microbiome-pathogen dynamics. We conducted a laboratory experiment and used 16S rRNA amplicon-sequencing to assess: (i) whether two lineages (Bd-Asia-2/Brazil and Bd-GPL) and their recombinant, in single and mixed infections, differentially affect amphibian skin bacteria; (ii) and the changes associated with the transition to laboratory conditions. We determined no clear differences in bacterial diversity among Bd treatments, despite differences in infection intensity. However, we observed an additive effect of mixed infections on bacterial alpha diversity and a potentially antagonistic interaction between Bd genotypes. Additionally, observed changes in community composition suggest a higher ability of Bd-GPL to alter skin bacteria. Lastly, we observed a drastic reduction in bacterial diversity and a change in community structure in laboratory conditions. We provide evidence for complex interactions between Bd genotypes and amphibian skin bacteria during coinfections, and expand on the implications of experimental conditions in ecological studies.

Response to Comment on "Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity".

Lambert et al question our retrospective and holistic epidemiological assessment of the role of chytridiomycosis in amphibian declines. Their alternative assessment is narrow and provides an incomplete evaluation of evidence. Adopting this approach limits understanding of infectious disease impacts and hampers conservation efforts. We reaffirm that our study provides unambiguous evidence that chytridiomycosis has affected at least 501 amphibian species.

Bullfrog farms release virulent zoospores of the frog-killing fungus into the natural environment.

Bullfrog farming and trade practices are well-established, globally distributed, and economically valuable, but pose risks for biodiversity conservation. Besides their negative impacts on native amphibian populations as an invasive species, bullfrogs play a key role in spreading the frog-killing fungus Batrachochytrium dendrobatidis (Bd) in the natural environment. Bullfrogs are tolerant to Bd, meaning that they can carry high infection loads without developing chytridiomycosis. To test the potential of bullfrog farms as reservoirs for diverse and virulent chytrid genotypes, we quantified Bd presence, prevalence and infection loads across approximately 1,500 farmed bullfrogs and in the water that is released from farms into the environment. We also described Bd genotypic diversity within frog farms by isolating Bd from dozens of infected tadpoles. We observed individuals infected with Bd in all sampled farms, with high prevalence (reaching 100%) and high infection loads (average 71,029 zoospore genomic equivalents). Average outflow water volume from farms was high (60,000 L/day), with Bd zoospore concentration reaching approximately 50 million zoospores/L. Because virulent pathogen strains are often selected when growing in tolerant hosts, we experimentally tested whether Bd genotypes isolated from bullfrogs are more virulent in native anuran hosts compared to genotypes isolated from native host species. We genotyped 36 Bd isolates from two genetic lineages and found that Bd genotypes cultured from bullfrogs showed similar virulence in native toads when compared to genotypes isolated from native hosts. Our results indicate that bullfrog farms can harbor high Bd genotypic diversity and virulence and may be contributing to the spread of virulent genotypes in the natural environment. We highlight the urgent need to implement Bd monitoring and mitigation strategies in bullfrog farms to aid in the conservation of native amphibians.

Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.

Anthropogenic trade and development have broken down dispersal barriers, facilitating the spread of diseases that threaten Earth's biodiversity. We present a global, quantitative assessment of the amphibian chytridiomycosis panzootic, one of the most impactful examples of disease spread, and demonstrate its role in the decline of at least 501 amphibian species over the past half-century, including 90 presumed extinctions. The effects of chytridiomycosis have been greatest in large-bodied, range-restricted anurans in wet climates in the Americas and Australia. Declines peaked in the 1980s, and only 12% of declined species show signs of recovery, whereas 39% are experiencing ongoing decline. There is risk of further chytridiomycosis outbreaks in new areas. The chytridiomycosis panzootic represents the greatest recorded loss of biodiversity attributable to a disease.

Historical amphibian declines and extinctions in Brazil linked to chytridiomycosis.

The recent increase in emerging fungal diseases is causing unprecedented threats to biodiversity. The origin of spread of the frog-killing fungus Batrachochytrium dendrobatidis (Bd) is a matter of continued debate. To date, the historical amphibian declines in Brazil could not be attributed to chytridiomycosis; the high diversity of hosts coupled with the presence of several Bd lineages predating the reported declines raised the hypothesis that a hypervirulent Bd genotype spread from Brazil to other continents causing the recent global amphibian crisis. We tested for a spatio-temporal overlap between Bd and areas of historical amphibian population declines and extinctions in Brazil. A spatio-temporal convergence between Bd and declines would support the hypothesis that Brazilian amphibians were not adapted to Bd prior to the reported declines, thus weakening the hypothesis that Brazil was the global origin of Bd emergence. Alternatively, a lack of spatio-temporal association between Bd and frog declines would indicate an evolution of host resistance in Brazilian frogs predating Bd's global emergence, further supporting Brazil as the potential origin of the Bd panzootic. Here, we Bd-screened over 30 000 museum-preserved tadpoles collected in Brazil between 1930 and 2015 and overlaid spatio-temporal Bd data with areas of historical amphibian declines. We detected an increase in the proportion of Bd-infected tadpoles during the peak of amphibian declines (1979-1987). We also found that clusters of Bd-positive samples spatio-temporally overlapped with most records of amphibian declines in Brazil's Atlantic Forest. Our findings indicate that Brazil is post epizootic for chytridiomycosis and provide another piece to the puzzle to explain the origin of Bd globally.