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The skin disease chytridiomycosis is linked to global amphibian declines but effective mitigation measures require improved understanding of the mechanisms underpinning the disease ecology. This study identifies key mediators of interactions between the fungal pathogen and amphibian skin, providing a marker of host colonization that can predict susceptibility between amphibian species.

The pathogen Batrachochytrium dendrobatidis (BD) associated with widespread amphibian declines is present in Europe but has not consistently caused disease-induced declines in that region. Here, the authors suggest that an endemic strain of BD with low virulence may protect the hosts upon co-infection with more virulent strains.

Abstract Rapid, early, and reliable detection of invasive pathogenic microorganisms is essential in order to either predict or delineate an outbreak, and monitor appropriate mitigation measures. The chytrid fungus Batrachochytrium salamandrivorans is expanding in Europe, and infection with this fungus may cause massive mortality in urodelans (salamanders and newts). In this study, we designed and validated species‐specific primers and a probe for detection of B. salamandrivorans in water. In a garden pond in close proximity to the B. salamandrivorans index site in the Netherlands, B. salamandrivorans‐infected newts had been detected in 2015 and have been monitored since. In 2016 and 2017, no B. salamandrivorans was detected at this site, but in 2018 B. salamandrivorans flared up in this isolated pond which allowed validation of the technique in situ. We here present the development of an environmental DNA technique that successfully detects B. salamandrivorans DNA in natural waterbodies even at low concentrations. This technique may be further validated to play a role in B. salamandrivorans range delineation and surveillance in both natural waterbodies and in captive collections.

Despite the microbiome’s key role in health and fitness, little is known about the environmental factors shaping the gut microbiome of wild birds. With habitat fragmentation being recognised as a major threat to biological diversity, we here determined how forest structure influences the bacterial species richness and diversity of wild great tit nestlings (Parus major). Using an Illumina metabarcoding approach which amplifies the 16S bacterial ribosomal RNA gene, we measured gut microbiota diversity and composition from 49 great tit nestlings, originating from 23 different nests that were located in 22 different study plots across a gradient of forest fragmentation and tree species diversity. Per nest, an average microbiome was determined on which the influence of tree species (composition and richness) and forest fragmentation (fragment area and edge density) was examined and whether this was linked to host characteristics (body condition and fledging success). We found an interaction effect of edge density with tree species richness or composition on both the microbial richness (alpha diversity: Chao1 and Shannon) and community structure (beta diversity: weighted and unweighted UniFrac). No significant short-term impact was observed of the overall faecal microbiome on host characteristics, but rather an adverse effect of specific bacterial genera on fledging success. These results highlight the influence of environmental factors on the microbial richness as well as the phylogenetic diversity during a life stage where the birds’ microbiota is shaped, which could lead to long-term consequences for host fitness.

To avoid being eaten, poisonous prey animals must rely on fast passage of toxins across a predator’s oral tissue, a major barrier to large molecules. Here, Raaymakers et al. show that antimicrobial peptides co secreted with frog toxins enhance intoxication of a snake predator by permeabilizing oral cell layers.

Batrachochytrium dendrobatidis and B. salamandrivoransare both important pathogens of amphibians, but they differ in their host ranges, infection strategies, and host immune responses. Here, Farrer and colleagues compare their genomes and transcriptomes to identify the genetic basis of these differences.

The largest current disease-induced loss of vertebrate biodiversity is due to chytridiomycosis and despite the increasing understanding of the pathogenesis, knowledge unravelling the early host-pathogen interactions remains limited. Batrachochytrium dendrobatidis (Bd) zoospores attach to and invade the amphibian epidermis, with subsequent invasive growth in the host skin. Availability of an in vitro assay would facilitate in depth study of this interaction while reducing the number of experimental animals needed. We describe a fluorescent cell-based in vitro infection model that reproduces host-Bd interactions. Using primary keratinocytes from Litoria caerulea and the epithelial cell line A6 from Xenopus laevis, we reproduced different stages of host cell infection and intracellular growth of Bd, resulting in host cell death, a key event in chytridiomycosis. The presented in vitro models may facilitate future mechanistic studies of host susceptibility and pathogen virulence.

Amphibians face many threats leading to declines and extinctions, but the chytrid fungal skin pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) have been identified as the causative factors leading to one of the greatest disease-driven losses of amphibian biodiversity worldwide. Infection may lead to different clinical outcomes, and lethal infections are commonly associated with unrestricted, exponential fungal growth in the amphibian epidermis. Mechanisms underpinning Bd and Bsal growth in the amphibian host are poorly understood. Here, we describe a quorum sensing mechanism that allows cell-to-cell communication by Bd and Bsal in order to regulate fungal densities and infection strategies. Addition of chytrid culture supernatant to chytrid cultures resulted in a concentration-dependent growth reduction and using dialysis, small metabolites were shown to be the causative factor. U-HPLC-MS/MS and in vitro growth tests identified the aromatic alcohol tryptophol as a key metabolite in regulating fungal growth. We determined tryptophol kinetics in both Bd and Bsal and confirmed the autostimulatory mode of action of this quorum sensing metabolite. Finally, we linked expression of genes that might be involved in tryptophol production, with in vitro and in vivo chytrid growth. Our results show that Bd and Bsal fungi use tryptophol to act as multicellular entities in order to regulate their growth.

The hatching success of a bird's egg is one of the key determinants of avian reproductive success, which may be compromised by microbial infections causing embryonic death. During incubation, outer eggshell bacterial communities pose a constant threat of pathogen translocation and embryo infection. One of the parental strategies to mitigate this threat is the incorporation of maternal immune factors into the egg albumen and yolk. It has been suggested that habitat changes like forest fragmentation can affect environmental factors and life-history traits that are linked to egg contamination. This study aims at investigating relationships between microbial pressure, immune investment and hatching success in two abundant forest bird species and analyzing to what extent these are driven by extrinsic (environmental) factors. We here compared (1) the bacterial load and composition on eggshells, (2) the level of immune defenses in eggs, and (3) the reproductive success between great (Parus major) and blue (Cyanistes caeruleus) tits in Belgium and examined if forest fragmentation affects these parameters. Analysis of 70 great tit and 34 blue tit eggshells revealed a similar microbiota composition (Enterobacteriaceae, Lactobacillus spp., Firmicutes and Bacteroidetes), but higher bacterial loads in great tits. Forest fragmentation was not identified as an important explanatory variable. Although a significant negative correlation between hatching success and bacterial load on the eggshells in great tits corroborates microbial pressure to be a driver of embryonic mortality, the overall hatching success was only marginally lower than in blue tits. This may be explained by the significantly higher levels of lysozyme and IgY in the eggs of great tits, protecting the embryo from increased infection pressure. Our results show that immune investment in eggs is suggested to be a species-specific adaptive trait that serves to protect hatchlings from pathogen pressure, which is not directly linked to habitat fragmentation.

Seed-based pigeon diets could be expected to result in exposure of pigeons to mycotoxins such as deoxynivalenol (DON). Ingestion of low to moderate contamination levels of DON may impair intestinal health, immune function and/or pathogen fitness, resulting in altered host-pathogen interactions and thus different outcome of infections. Here we demonstrate that DON was one of the most frequently detected mycotoxins in seed-based racing pigeons feed, contaminating 5 out of 10 samples (range 177-1,466 μg/kg). Subsequently, a toxicokinetic analysis revealed a low absolute oral bioavailability (F) of DON in pigeons (30.4%), which is comparable to other avian species. Furthermore, semi-quantitative analysis using high-resolution mass spectrometry revealed that DON-3α-sulphate is the major metabolite of DON in pigeons after intravenous as well as oral administration. Following ingestion of DON contaminated feed, the intestinal epithelial cells are exposed to significant DON concentrations which eventually may affect intestinal translocation and colonization of bacteria. Feeding pigeons a DON contaminated diet resulted in an increased percentage of pigeons shedding Salmonella compared to birds fed control diet, 87 ± 17% versus 74 ± 13%, respectively. However, no impact of DON was observed on the Salmonella induced disease signs, organ lesions, faecal and organ Salmonella counts. The presented risk assessment indicates that pigeons are frequently exposed to mycotoxins such as DON, which can affect the outcome of a Salmonella infection. The increasing number of pigeons shedding Salmonella suggests that DON can promote the spread of the bacterium within pigeon populations.

It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health.

Background and aimsBoth deoxynivalenol (DON) and nontyphoidal salmonellosis are emerging threats with possible hazardous effects on both human and animal health. The objective of this study was to examine whether DON at low but relevant concentrations interacts with the intestinal inflammation induced by Salmonella Typhimurium.MethodologyBy using a porcine intestinal ileal loop model, we investigated whether intake of low concentrations of DON interacts with the early intestinal inflammatory response induced by Salmonella Typhimurium.ResultsA significant higher expression of IL-12 and TNFα and a clear potentiation of the expression of IL-1β, IL-8, MCP-1 and IL-6 was seen in loops co-exposed to 1 µg/mL of DON and Salmonella Typhimurium compared to loops exposed to Salmonella Typhimurium alone. This potentiation coincided with a significantly enhanced Salmonella invasion in and translocation over the intestinal epithelial IPEC-J2 cells, exposed to non-cytotoxic concentrations of DON for 24 h. Exposure of Salmonella Typhimurium to 0.250 µg/mL of DON affected the bacterial gene expression level of a limited number of genes, however none of these expression changes seemed to give an explanation for the increased invasion and translocation of Salmonella Typhimurium and the potentiated inflammatory response in combination with DON.ConclusionThese data imply that the intake of low and relevant concentrations of DON renders the intestinal epithelium more susceptible to Salmonella Typhimurium with a subsequent potentiation of the inflammatory response in the gut.

Salmonellosis caused by Salmonella Typhimurium is one of the most important bacterial zoonotic diseases. The bacterium persists in pigs resulting in asymptomatic 'carrier pigs', generating a major source for Salmonella contamination of pork. Until now, very little is known concerning the mechanisms used by Salmonella Typhimurium during persistence in pigs. Using in vivo expression technology (IVET), a promoter-trap method based on ΔpurA attenuation of the parent strain, we identified 37 Salmonella Typhimurium genes that were expressed 3 weeks post oral inoculation in the tonsils, ileum and ileocaecal lymph nodes of pigs. Several genes were expressed in all three analyzed organs, while other genes were only expressed in one or two organs. Subsequently, the identified IVET transformants were pooled and reintroduced in pigs to detect tissue-specific gene expression patterns. We found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes during Salmonella peristence in pigs. Furthermore, we compared the persistence ability of substitution mutants for the IVET-identified genes sifB and STM4067 to that of the wild type in a mixed infection model. The ΔSTM4067::kanR was significantly attenuated in the ileum contents, caecum and caecum contents and faeces of pigs 3 weeks post inoculation, while deletion of the SPI-2 effector gene sifB did not affect Salmonella Typhimurium persistence. Although our list of identified genes is not exhaustive, we found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes of pigs and we identified STM4067 as a factor involved in Salmonella persistence in pigs. To our knowledge, our study is the first to identify Salmonella Typhimurium genes expressed during persistence in pigs.