Role of Maternal Antibodies in the Protection of Broiler Chicks against Campylobacter Colonization in the First Weeks of Life.

Thermophilic Campylobacter species are the most common cause of bacterium-mediated diarrheal disease in humans globally. Poultry is considered the most important reservoir of human campylobacteriosis, but so far, no effective countermeasures are in place to prevent the bacterium from colonizing broiler flocks. This study investigated maternal antibodies' potential to offer protection against Campylobacter in broiler chicks via a field trial and an immunization trial. In the field trial, breeder flocks with high and low anti-Campylobacter antibody levels in the yolk were selected based on serological screening. Offspring were subsequently monitored for maternal antibodies and Campylobacter prevalence during early life. Although maternal antibodies declined rapidly in the serum of broilers, offspring from flocks with lower anti-Campylobacter antibody levels seemed to be more susceptible to colonization. In the immunization trial, breeders from a seropositive breeder flock were vaccinated with an experimental bacterin or subunit vaccine. Immunization increased antibody levels in the yolk and consequently in the offspring. Elevated maternal antibody levels were significantly associated with reduced Campylobacter susceptibility in broilers at 2 weeks old but not at 1 and 3 weeks old. Overall, the protective effect of maternal immunity should be cautiously considered in the context of Campylobacter control in broilers. Immunization of breeders may enhance resistance but is not a comprehensive solution.

Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Divergent population responses following salamander mass mortalities and declines driven by the emerging pathogen Batrachochytrium salamandrivorans.

Understanding wildlife responses to novel threats is vital in counteracting biodiversity loss. The emerging pathogen Batrachochytrium salamandrivorans (Bsal) causes dramatic declines in European salamander populations, and is considered an imminent threat to global amphibian biodiversity. However, real-life disease outcomes remain largely uncharacterized. We performed a multidisciplinary assessment of the longer-term impacts of Bsal on highly susceptible fire salamander (Salamandra salamandra) populations, by comparing four of the earliest known outbreak sites to uninfected sites. Based on large-scale monitoring efforts, we found population persistence in strongly reduced abundances to over a decade after Bsal invasion, but also the extinction of an initially small-sized population. In turn, we found that host responses varied, and Bsal detection remained low, within surviving populations. Demographic analyses indicated an ongoing scarcity of large reproductive adults with potential for recruitment failure, while spatial comparisons indicated a population remnant persisting within aberrant habitat. Additionally, we detected no early signs of severe genetic deterioration, yet nor of increased host resistance. Beyond offering additional context to Bsal-driven salamander declines, results highlight how the impacts of emerging hypervirulent pathogens can be unpredictable and vary across different levels of biological complexity, and how limited pathogen detectability after population declines may complicate surveillance efforts.

Reducing Campylobacter colonization in broilers by active immunization of naive broiler breeders using a bacterin and subunit vaccine.

Campylobacter is the main cause of human gastroenteritis worldwide, with 50 to 80% of the cases related to consumption of poultry products. Maternal antibodies (MAB) from commercial breeder flocks may protect their progeny against infection during the first few weeks of life. We here studied the prevalence of Campylobacter antibody titers in broiler breeder flocks and to which extent immunization of broiler breeders increases maternal anti-Campylobacter titers in their progeny and protects the offspring against Campylobacter colonization. Two vaccines were used: a bacterin mix of 13 Campylobacter strains and a subunit vaccine comprising 6 immunodominant Campylobacter antigens. All sampled on-farm breeder flocks were positive for anti-Campylobacter antibodies, yet in some breeder flocks only very low titers were detected. Vaccination of SPF broiler breeder flocks with both subunit and bacterin vaccines resulted in a prolonged presence of anti-Campylobacter antibodies in the serum and intestinal mucus of chicks. These bacterin- or subunit vaccine-induced MAB conferred protection against Campylobacter colonization in chicks until 7 and 21 d of age, respectively, but only at a low challenge dose (102.5 cfu). The concentration of MAB in the mucus is probably too low to sufficiently capture Campylobacter when higher challenge doses are used. In conclusion, vaccinating broiler breeders protects their offspring against Campylobacter colonization under low pathogen exposure conditions.

One Health Approach to Globalizing, Accelerating, and Focusing Amphibian and Reptile Disease Research-Reflections and Opinions from the First Global Amphibian and Reptile Disease Conference.

The world's reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards.

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

Widespread triazole pesticide use affects infection dynamics of a global amphibian pathogen.

The sixth mass extinction is a consequence of complex interplay between multiple stressors with negative impact on biodiversity. We here examine the interaction between two globally widespread anthropogenic drivers of amphibian declines: the fungal disease chytridiomycosis and antifungal use in agriculture. Field monitoring of 26 amphibian ponds in an agricultural landscape shows widespread occurrence of triazole fungicides in the water column throughout the amphibian breeding season, together with a negative correlation between early season application of epoxiconazole and the prevalence of chytrid infections in aquatic newts. While triazole concentrations in the ponds remained below those that inhibit growth of Batrachochytrium dendrobatidis, they bioaccumulated in the newts' skin up to tenfold, resulting in cutaneous growth-suppressing concentrations. As such, a concentration of epoxiconazole, 10 times below that needed to inhibit fungal growth, prevented chytrid infection in anuran tadpoles. The widespread presence of triazoles may thus alter chytrid dynamics in agricultural landscapes.

Zoonotic pathogens linked with hedgehog diphtheric disease.

Hedgehog diphtheric disease (HDD), an ulcerative skin disease with a high fatality rate, is an emerging threat to European hedgehogs (Erinaceus europaeus). We explored the potential role of a panel of zoonotic pathogens in the presumed multifactorial nature of HDD in 188 hedgehogs from 3 wildlife rescue centres in Belgium. As expected, and with a prevalence of 67% in 57 hedgehogs with skin lesions, characteristic of HDD, the occurrence of Corynebacterium ulcerans was strongly associated with the disease. Remarkably, with a prevalence of 42% in affected animals, infections with Borrelia burgdorferi sensu lato were 3.92 times more likely to be detected in HDD (95% confidence interval: 1.650-9.880; p = .0024). Overall, 40 hedgehogs tested positive for the B. burgdorferi sensu lato complex, including Borrelia afzelii (n = 30), Borrelia bavariensis (n = 7) and Borrelia spielmanii (n = 7). Other widely occurring pathogens included Salmonella (prevalence of 19%, with three pulsed-field gel electrophoresis profiles) and Leptospira sp. (prevalence of 11%, including Leptospira interrogans and Leptospira borgpetersenii), but these were not associated with the occurrence of HDD. These findings show that hedgehogs in Belgium represent a significant reservoir of multiple zoonotic bacteria, of which toxigenic C. ulcerans and B. burgdorferi sensu lato are associated with widespread hedgehog skin pathology and mortality.

Tree Species Diversity and Forest Edge Density Jointly Shape the Gut Microbiota Composition in Juvenile Great Tits (Parus major).

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.

Impact of heavy metal exposure on biological control of a deadly amphibian pathogen by zooplankton.

Despite devastating effects on global biodiversity, efficient mitigation strategies against amphibian chytridiomycosis are lacking. Since the free-living pathogenic zoospores of Batrachochytrium dendrobatidis (Bd), the infective stage of this disease, can serve as a nutritious food source for components of zooplankton communities, these groups may act as biological control agents by eliminating zoospores from the aquatic environment. Such pathogen-predator interaction is, however, embedded in the aquatic food web structure and is therefore affected by abiotic factors interfering with these networks. Heavy metals, released from both natural and anthropogenic sources, are widespread contaminants of aquatic ecosystems and may interfere with planktonic communities and thus pathogen elimination rates. We investigated the interaction between zooplankton communities and chytridiomycosis infections in a Flemish agricultural region. Moreover, we also investigated the impact of heavy metal contamination, that was previously investigated in the region and presented in recent work, on zooplankton assemblages and chytridiomycosis infections. Finally, we tested the effect of sublethal concentrations of copper and zinc on Bd removal rates by Daphnia magna in a laboratory assay. Although zinc, copper, nickel and chromium were widely abundant pollutants, heavy metals were no driving force for zooplankton assemblages at our study locations. Moreover, our field survey did not reveal indirect effects of zooplankton assemblages on chytridiomycosis infections. However, sampling occasions testing negative for Bd showed a higher degree of copper contamination compared to positive sampling occasions, indicating a potential inhibitory effect of copper on Bd prevalence. Finally, whereas D. magna significantly reduced zoospore densities in its environment, sublethal concentrations of copper and zinc showed no interference with pathogen removal in the laboratory assay. Our results provide perspectives for further research on such a biological control strategy against chytridiomycosis by optimizing environmental conditions for pathogen predation.

Diversity, multifaceted evolution, and facultative saprotrophism in the European Batrachochytrium salamandrivorans epidemic.

While emerging fungi threaten global biodiversity, the paucity of fungal genome assemblies impedes thoroughly characterizing epidemics and developing effective mitigation strategies. Here, we generate de novo genomic assemblies for six outbreaks of the emerging pathogen Batrachochytrium salamandrivorans (Bsal). We reveal the European epidemic currently damaging amphibian populations to comprise multiple, highly divergent lineages demonstrating isolate-specific adaptations and metabolic capacities. In particular, we show extensive gene family expansions and acquisitions, through a variety of evolutionary mechanisms, and an isolate-specific saprotrophic lifecycle. This finding both explains the chytrid's ability to divorce transmission from host density, producing Bsal's enigmatic host population declines, and is a key consideration in developing successful mitigation measures.

Epidermal galactose spurs chytrid virulence and predicts amphibian colonization.

The chytrid fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans cause the skin disease chytridiomycosis in amphibians, which is driving a substantial proportion of an entire vertebrate class to extinction. Mitigation of its impact is largely unsuccessful and requires a thorough understanding of the mechanisms underpinning the disease ecology. By identifying skin factors that mediate key events during the early interaction with B. salamandrivorans zoospores, we discovered a marker for host colonization. Amphibian skin associated beta-galactose mediated fungal chemotaxis and adhesion to the skin and initiated a virulent fungal response. Fungal colonization correlated with the skin glycosylation pattern, with cutaneous galactose content effectively predicting variation in host susceptibility to fungal colonization between amphibian species. Ontogenetic galactose patterns correlated with low level and asymptomatic infections in salamander larvae that were carried over through metamorphosis, resulting in juvenile mortality. Pronounced variation of galactose content within some, but not all species, may promote the selection for more colonization resistant host lineages, opening new avenues for disease mitigation.

Diet diversity and environment determine the intestinal microbiome and bacterial pathogen load of fire salamanders.

Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection Is Species Specific and Varies between Strains.

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level (B. innocens, B. pilosicoli, B. murdochii, B. hampsonii, and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O-glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Widespread Disease in Hedgehogs (Erinaceus europaeus) Caused by Toxigenic Corynebacterium ulcerans.

Toxin-producing Corynebacterium ulcerans, a causative agent of diphtheria in humans, was isolated from 53 hedgehogs in Belgium during the spring of 2020. Isolates showed low levels of acquired antimicrobial drug resistance. Strain diversity suggests emergence from an endemic situation. These findings stress the need for raising public awareness and improved wildlife disease surveillance.

Batrachochytrium salamandrivorans Threat to the Iberian Urodele Hotspot.

The recent introduction of the chytrid fungus Batrachochytrium salamandrivorans into northeastern Spain threatens salamander diversity on the Iberian Peninsula. We assessed the current epidemiological situation with extensive field sampling of urodele populations. We then sought to delineate priority regions and identify conservation units for the Iberian Peninsula by estimating the susceptibility of Iberian urodeles using laboratory experiments, evidence from mortality events in nature and captivity and inference from phylogeny. None of the 1395 field samples, collected between 2015 and 2021 were positive for Bsal and no Bsal-associated mortality events were recorded, in contrast to the confirmed occurrence of Bsal outbreak previously described in 2018. We classified five of eleven Iberian urodele species as highly susceptible, predicting elevated mortality and population declines following potential Bsal emergence in the wild, five species as intermediately susceptible with variable disease outcomes and one species as resistant to disease and mortality. We identified the six conservation units (i.e., species or lineages within species) at highest risk and propose priority areas for active disease surveillance and field biosecurity measures. The magnitude of the disease threat identified here emphasizes the need for region-tailored disease abatement plans that couple active disease surveillance to rapid and drastic actions.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions.

Ancient enzootic associations between wildlife and their infections allow evolution to innovate mechanisms of pathogenicity that are counterbalanced by host responses. However, erosion of barriers to pathogen dispersal by globalization leads to the infection of hosts that have not evolved effective resistance and the emergence of highly virulent infections. Global amphibian declines driven by the rise of chytrid fungi and chytridiomycosis are emblematic of emerging infections. Here, we review how modern biological methods have been used to understand the adaptations and counteradaptations that these fungi and their amphibian hosts have evolved. We explore the interplay of biotic and abiotic factors that modify the virulence of these infections and dissect the complexity of this disease system. We highlight progress that has led to insights into how we might in the future lessen the impact of these emerging infections.

Application of Disinfectants for Environmental Control of a Lethal Amphibian Pathogen.

Chytridiomycosis is an emerging infectious disease threatening amphibian populations worldwide. While environmental disinfection is important in mitigating the disease, successful elimination of Batrachochytrium dendrobatidis (Bd) without excessively harming ecosystems is challenging. We selected peracetic acid (PAA) as the most potent of six commercially available products regarding their ability to inhibit growth of a highly virulent Bd strain. PAA killed Bd after 5 min of exposure to approximately 94.7 mg/L. We examined the toxicity of PAA against three invertebrate species and Discoglossus pictus tadpoles. 93% of invertebrates, but none of the tadpoles survived 5 min of exposure to 94.7 mg/L. Tadpoles showed no adverse effects after 5 min exposure to concentrations of approximately 37.9 mg/L or lower. Addition of PAA to aquatic microcosms decreased pH, while dissolved oxygen (DO) initially increased. Degradation of PAA reversed the pH drop, but caused a massive drop in DO, which could be remedied by aeration. As proof of concept, microcosms that were aerated and treated with 94.7 mg/L PAA sustained survival of tadpoles starting 48 h after treatment. Disinfecting aquatic environments using PAA could contribute to mitigating chytridiomycosis, while preserving at least some invertebrate diversity, but requires temporary removal of resident amphibians.

Landscape epidemiology of Batrachochytrium salamandrivorans: reconciling data limitations and conservation urgency.

Starting in 2010, rapid fire salamander (Salamandra salamandra) population declines in northwestern Europe heralded the emergence of Batrachochytrium salamandrivorans (Bsal), a salamander-pathogenic chytrid fungus. Bsal poses an imminent threat to global salamander diversity owing to its wide host range, high pathogenicity, and long-term persistence in ecosystems. While there is a pressing need to develop further research and conservation actions, data limitations inherent to recent pathogen emergence obscure necessary insights into Bsal disease ecology. Here, we use a hierarchical modeling framework to describe Bsal landscape epidemiology of outbreak sites in light of these methodological challenges. Using model selection and machine learning, we find that Bsal presence is associated with humid and relatively cool, stable climates. Outbreaks are generally located in areas characterized by low landscape heterogeneity and low steepness of slope. We further find an association between Bsal presence and high trail density, suggesting that human-mediated spread may increase risk for spillover between populations. We then use distribution modeling to show that favorable conditions occur in lowlands influenced by the North Sea, where increased survey effort is needed to determine how Bsal impacts local newt populations, but also in hill- and mountain ranges in northeastern France and the lower half of Germany. Finally, connectivity analyses suggest that these hill- and mountain ranges may act as stepping stones for further spread southward. Our results provide initial insight into regional environmental conditions underlying Bsal epizootics, present updated invasibility predictions for northwestern Europe, and lead us to discuss a wide variety of potential survey and research actions needed to advance future conservation and mitigation efforts.

Salamander loss alters litter decomposition dynamics.

Biodiversity loss affects ecosystem functioning. Top down effects of amphibian declines on the trophic food web of the forest floor are poorly understood. Here we quantify and explain the effects of disease-driven loss of salamanders on the dynamics of forest leaf litter. Using paired mesocosms, within a Belgian forest, we tested the effect of fire salamander (Salamandra salamandra) presence on the trophic cascade that results in the decomposition rate of good quality (maple - Acer pseudoplatanus) and poorer quality (oak - Quercus robur) leaf litter, over an 18 month period. The presence of salamanders reduced decomposition rate of Quercus litter up to 20%. This was associated with a significantly higher predation rate on detritivores, which altered the functional composition of the invertebrate community. Functional composition analysis of the litter microbiome showed less bacteria associated with leaf litter degradation on the Quercus litter in the presence of salamanders. Salamanders thus influence ecosystem functions through trophic cascades and promote the retention of the leaf litter fraction in poorer quality litter.