Pascale Van Rooij

Waterfowl: potential environmental reservoirs of the chytrid fungus Batrachochytrium dendrobatidis.

Infections with Batrachochytrium dendrobatidis (B. dendrobatidis), the causal agent of chytridiomycosis, have been shown to play an important role in the decline of amphibians worldwide. Spread of the fungus is poorly understood. Bird movement might possibly contribute to the spread of B. dendrobatidis in the environment. Therefore, 397 wild geese in Belgium were screened for presence of B. dendrobatidis on their toes using real-time quantitative PCR (qPCR). In addition, chemotaxis towards, adhesion, survival after desiccation and proliferation of B. dendrobatidis on keratinous toe scales from waterfowl were examined in vitro. qPCR revealed that 76 geese (15%) were positive for B. dendrobatidis. Results of the in vitro tests showed that B. dendrobatidis is attracted to the keratinous toes of aquatic birds on which they can adhere and even proliferate. However, desiccation is poorly tolerated. This suggests waterfowl are potential environmental reservoirs for B. dendrobatidis.

Amphibian chytridiomycosis : a review with focus on fungus-host interactions

Amphibian declines and extinctions are emblematic for the current sixth mass extinction event. Infectious drivers of these declines include the recently emerged fungal pathogens Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans (Chytridiomycota). The skin disease caused by these fungi is named chytridiomycosis and affects the vital function of amphibian skin. Not all amphibians respond equally to infection and host responses might range from resistant, over tolerant to susceptible. The clinical outcome of infection is highly dependent on the amphibian host, the fungal virulence and environmental determinants. B. dendrobatidis infects the skin of a large range of anurans, urodeles and caecilians, whereas to date the host range of B. salamandrivorans seems limited to urodeles. So far, the epidemic of B. dendrobatidis is mainly limited to Australian, neotropical, South European and West American amphibians, while for B. salamandrivorans it is limited to European salamanders. Other striking differences between both fungi include gross pathology and thermal preferences. With this review we aim to provide the reader with a state-of-the art of host-pathogen interactions for both fungi, in which new data pertaining to the interaction of B. dendrobatidis and B. salamandrivorans with the host’s skin are integrated. Furthermore, we pinpoint areas in which more detailed studies are necessary or which have not received the attention they merit.

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines. We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea. Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus. In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.

Resistance to Chytridiomycosis in European Plethodontid Salamanders of the Genus Speleomantes

North America and the neotropics harbor nearly all species of plethodontid salamanders. In contrast, this family of caudate amphibians is represented in Europe and Asia by two genera, Speleomantes and Karsenia, which are confined to small geographic ranges. Compared to neotropical and North American plethodontids, mortality attributed to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has not been reported for European plethodontids, despite the established presence of Bd in their geographic distribution. We determined the extent to which Bd is present in populations of all eight species of European Speleomantes and show that Bd was undetectable in 921 skin swabs. We then compared the susceptibility of one of these species, Speleomantes strinatii, to experimental infection with a highly virulent isolate of Bd (BdGPL), and compared this to the susceptible species Alytes muletensis. Whereas the inoculated A. muletensis developed increasing Bd-loads over a 4-week period, none of five exposed S. strinatii were colonized by Bd beyond 2 weeks post inoculation. Finally, we determined the extent to which skin secretions of Speleomantes species are capable of killing Bd. Skin secretions of seven Speleomantes species showed pronounced killing activity against Bd over 24 hours. In conclusion, the absence of Bd in Speleomantes combined with resistance to experimental chytridiomycosis and highly efficient skin defenses indicate that the genus Speleomantes is a taxon unlikely to decline due to Bd.

Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin

Global amphibian declines are in part driven by the chytrid fungus Batrachochytrium dendrobatidis, causing superficial dermatomycosis with epidermal hyperplasia and hyperkeratosis in infected amphibians. The susceptibility to chytridiomycosis and the severity of epidermal lesions in amphibians with chytridiomycosis are not consistent across species or even among individuals. Severe infections cause death of the animal most likely through disturbance of ion homeostasis. The mechanism by which this superficial skin infection results in epidermal lesions has so far eluded precise definition. It was the aim of this study to unravel how B. dendrobatidis causes alterations that affect skin integrity. Exposure of Xenopus laevis skin to B. dendrobatidis zoospore supernatant using skin explants and Ussing chambers caused rapid disruption of intercellular junctions, demonstrated using histology and transmission electron microscopy. The loss of intercellular junctions led to detachment-induced cell apoptosis, or anoikis. The zoospore supernatant induced neither apoptosis nor necrosis in isolated primary keratinocytes of X. laevis. This supports the idea that the loss of cell contacts triggered apoptosis in the skin explants. Mass spectrometric analysis of the protein composition of the supernatant revealed a complex mixture, including several new virulence associated proteins, such as proteases, biofilm-associated proteins and a carotenoid ester lipase. Protease and lipase activity of the supernatant was confirmed with a protease and lipase assay. In conclusion, B. dendrobatidis zoospores produce a complex mixture of proteins that quickly disturbs epidermal intercellular junctions leading to anoikis in the anuran skin. The role of the identified proteins in this process remains to be determined.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus’s egg?

BackgroundThe establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated.ResultsFlorfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 μg/ml for florfenicol and 8.0 μg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 μg/ml florfenicol or 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 μg/ml but not to 10 μg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 μg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection.ConclusionsWe thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Clinically healthy amphibians in captive collections and at pet fairs : a reservoir of Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis is the cause of the fungal disease chytridiomycosis, a potentially lethal skin disease of amphibians. Asymptomatically infected amphibians may pose a risk for environmental pathogen pollution. This study therefore assessed the role of healthy, captive amphibians as a reservoir of Batrachochytrium dendrobatidis. Samples were collected from captive amphibians in Belgium, the Netherlands, Germany and France (559 from anurans, 330 from urodelans and 4 from gymnophians) from private owners, zoos, and laboratories. In addition to which, 78 anurans from 19 living collections were sampled during a pet fair in the Netherlands. Nearly 3% of the captive amphibians were infected by B. dendrobatidis, and 13.6% of the collections yielded at least one positive result. At the fair, 7 out of 78 anurans, representing 2 collections were positive. None of the animals that tested positive showed any obvious health problems at the time of sampling. Our results demonstrate the potential of the amphibian pet trade as a vehicle for the spread of B. dendrobatidis.

Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further studies would be required to draw a causal link.

Road-killed Common Toads (Bufo bufo) in Flanders (Belgium) Reveal Low Prevalence of Ranaviruses and Batrachochytrium dendrobatidis

Road-killed common toads (Bufo bufo; n=1,740) from Flanders, Belgium, were tested for ranavirus and Batrachochytrium dendrobatidis (Bd) using polymerase chain reaction. Both infections were present at a very low prevalence (<0.2% with a confidence interval of 95% for ranavirus and 0.63% for Bd).

Recommendations on diagnostic tools for Batrachochytrium salamandrivorans

Batrachochytrium salamandrivorans (Bsal) poses a major threat to amphibian, and more specifically caudata, diversity. Bsal is currently spreading through Europe, and mitigation measures aimed at stopping its spread and preventing its introduction into naïve environments are urgently needed. Screening for presence of Bsal and diagnosis of Bsal-induced disease in amphibians are essential core components of effective mitigation plans. Therefore, the aim of this study was to present an overview of all Bsal diagnostic tools together with their limitations and to suggest guidelines to allow uniform interpretation. Here, we investigate the use of different diagnostic tools in post-mortem detection of Bsal and whether competition between Bd and Bsal occurs in the species-specific Bd and Bsal duplex real-time PCR. We also investigate the diagnostic sensitivity, diagnostic specificity and reproducibility of the Bsal real-time PCR and show the use of immunohistochemistry in diagnosis of Bsal-induced chytridiomycosis in amphibian samples stored in formaldehyde. Additionally, we have drawn up guidelines for the use and interpretation of the different diagnostic tools for Bsal currently available, to facilitate standardization of execution and interpretation.