First record of the pandemic fungus Batrachochytrium dendrobatidis in wild amphibians of the Mauritius Island

Amphibian species are facing numerous threats, including habitat destruction, deforestation, over-collection, agricultural chemicals, environmental pollution, predator species increase, and parasitic diseases. These factors have led to a decline in population and the extinction of amphibian species worldwide. Infectious diseases caused by parasitic, bacterial, fungal, and viral agents are also a significant threat to frogs. Chytridiomycosis is an infectious epidemic disease caused by Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), which have led to a decline in the global amphibian population due to their high death rate in infected regions. Additionally, ranavirosis is another emerging infectious disease (EID) that has caused the decline and extinction of amphibian populations. Studies on many amphibian species in Turkey have revealed the presence of chytridiomycosis, ranavirosis, and co-infections, adding to the known threats to amphibian species worldwide. This highlights the need for further research on the prevalence, transmission routes, prevention, and treatment of these pathogens, as well as the impacts of dual infections.

The recent emergence of the pathogen Batrachochytrium salamandrivorans (Bsal) is associated with rapid population declines of salamanders in Europe and its arrival to new areas could cause dramatic negative effects on other amphibian populations and species. Amphibian species, present in areas with high amphibian diversity such as Mexico, could be highly threatened due to the arrival of Bsal, particularly salamander species which are more vulnerable to chytridiomycosis caused by this pathogen. Thus, immediate surveillance is needed as a strategy to efficiently contend with this emerging infectious disease. In this study, we analyzed 490 wild and captive amphibians from 48 species across 76 sites in the North, Central, and South of Mexico to evaluate the presence of Bsal. Amphibians were sampled in sites with variable degrees of amphibian richness and suitability for Bsal according to previous studies. From the 76 sampling sites, 10 of them were located in areas with high amphibian richness and potential moderate to high Bsal habitat suitability. We did not detect Bsal in any of the samples, and no signs of the disease were observed in any individual at the time of sampling. Our results suggest that Bsal has not yet arrived at the sampled sites or could be at low prevalence within populations with low occurrence probability. This is the first study that evaluates the presence of Bsal in different regions and amphibian species in Mexico, which is the second most diverse country in salamander species in the world. We highlight the risk and the importance of continuing surveillance of Bsal in Mexico and discuss control strategies to avoid the introduction and spread of Bsal in the country.

Batrachochytrium fungi: stealth invaders in amphibian skin

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (β=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

As globalization lowers geographic barriers to movement, coinfection with novel and enzootic pathogens is increasingly likely. Novel and enzootic pathogens can interact synergistically or antagonistically, leading to increased or decreased disease severity. Here we examine host immune responses to coinfection with two closely related fungal pathogens: Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Both pathogens have had detrimental effects on amphibian populations, with Bd now largely enzootic, while Bsal is currently spreading and causing epizootics. Recent experimental work revealed that newts coinfected with Bd and Bsal had significantly higher mortality than those infected with either pathogen alone. Here we characterize host immunogenomic responses to chytrid coinfection relative to single infection. Across several classes of immune genes including pattern recognition receptors, cytokines, and MHC, coinfected host gene expression was weakly upregulated or comparable to that seen in single Bd infection, but significantly decreased when compared to Bsal infection. Combined with strong complement pathway downregulation and keratin upregulation, these results indicate that coinfection with Bd and Bsal compromises immune responses active against Bsal alone. As Bsal continues to invade naïve habitats where Bd is enzootic, coinfection will be increasingly common. If other Bd-susceptible species in the region have similar responses, interactions between the two pathogens could cause severe population and community-level declines.

Development of a multiplex real-time PCR surveillance assay for monitoring the health status of Ecuadorian amphibians at risk of extinction

Amphibian populations worldwide are experiencing significant declines, highlighting a critical aspect of the broader biodiversity crisis. Approximately 43% of all amphibian species are facing extinction due to factors such as habitat loss, pollution, climate change, and emerging diseases. The chytrid fungus Batrachochytrium salamandrivorans (Bsal) represents one of the major threats, because it is particularly dangerous for European salamanders. Southern Europe is especially vulnerable due to the presence of numerous endemic salamander species. Despite the risks, few studies have screened Italian salamanders for Bsal. We conducted a Bsal screening on 44 Golden Alpine Salamanders (Salamandra atra aurorae) from the Vezzena plateau in the Trentino-Alto Adige region (Northern Italy). Our molecular analysis of skin swabs revealed no presence of Bsal in any of the 44 specimens examined. Additionally, no macroscopic signs of Bsal-related skin damage were observed. The absence of Bsal in our samples is encouraging, suggesting that the investigated sites are currently unaffected by this pathogen. This finding aligns with other studies reporting no evidence of Bsal in Italy. Future research should explore the factors contributing to the absence of Bsal and the effectiveness of current conservation practices. While our findings are reassuring, the threat of Bsal remains a critical concern. Continued vigilance and enhanced conservation efforts are essential to protect salamander diversity and ensure the long-term survival of these important amphibian populations.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander's biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

The chytrid fungus Batrachochytrium salamandrivorans [Bsal] is causing declines in the amphibian populations. After a decade of mapping the pathogen in Europe, where it is causing dramatic outbreaks, and North America, where its arrival would affect to the salamander’s biodiversity hotspot, little is known about its current status in Asia, from presumably is native. Japan has several species considered as potential carriers, but no regulation is implemented against Bsal spreading. Previous Bsal known presence detected various cases on the Okinawa Island, southwestern Japan. Previous studies on its sister species, B. dendrobatidis presented a high genomic variation in this area and particularly on Cynops ensicauda. Here, we have done the largest monitoring to date in Japan on the Cynops genus, focusing on Okinawa Island and updating its distribution and providing more information to unravel the still unknown origin of Bsal. Interestingly, we have provided revealing facts about different detectability depending on the used molecular techniques and changes in its Japanese distribution. All in all, the Bsal presence in Japan, together with its low variability in the sequenced amplicons, and the lack of apparent mortalities, may indicate that this part of Asia has a high diversity of chytrids.

Amphibian populations have been declining around the world for more than five decades, and the losses continue. Although causes are complex, major contributors to these declines are two chytrid fungi, Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, which both cause the disease termed chytridiomycosis. Previously, we showed that B. dendrobatidis impedes amphibian defenses by directly inhibiting lymphocytes in vitro and in vivo by release of soluble metabolites, including kynurenine (KYN), methylthioadenosine (MTA), and spermidine (SPD). Here, we show that B. salamandrivorans cells and cell-free supernatants also inhibit amphibian lymphocytes as well as a human T cell line. As we have shown for B. dendrobatidis, high-performance liquid chromatography (HPLC) and mass spectrometry revealed that KYN, MTA, and SPD are key metabolites found in the B. salamandrivorans supernatants. Production of inhibitory factors by B. salamandrivorans is limited to mature zoosporangia and can occur over a range of temperatures between 16°C and 26°C. Taken together, these results suggest that both pathogenic Batrachochytrium fungi have evolved similar mechanisms to inhibit lymphocytes in order to evade clearance by the amphibian immune system.

AimAmphibian populations are threatened globally by anthropogenic change and Batrachochytrium dendrobatidis (Bd), a fungal pathogen causing chytridiomycosis disease to varying degrees of severity. A closely related new fungal pathogen, Batrachochytrium salamandrivorans (Bsal), has recently left its supposed native range in Asia and decimated some salamander populations in Europe. Despite being noticed initially for causing chytridiomycosis‐related population declines in salamanders, Bsal can also infect anurans and cause non‐lethal chytridiomycosis or asymptomatic infections in salamanders. Bsal has not yet been detected in the United States, but given the United States has the highest salamander biodiversity on Earth, predictive assessments of salamander risk to Bsal infection will enable proactive allocation of research and conservation efforts into disease prevention and mitigation.LocationThe United States, Europe and Asia.MethodsWe first predicted the environmental suitability for the Bsal pathogen in the United States through an ecological niche model based on the pathogen's known native range in Asia, validated on the observed invasive range in Europe using bioclimatic, land cover, elevation, soil characteristics and human modification variables. Second, we predicted the susceptibility of salamander species to Bsal infection using a machine‐learning model that correlated life history traits with published data on confirmed species infections. Finally, we mapped the geographic ranges of the subset of species that were predicted to be susceptible to Bsal infection.ResultsIn the United States, the overlap of environmental suitability and susceptible salamander species was greatest in the Pacific Northwest, near the Gulf of Mexico, and along the Atlantic coast, and in inland states east of the Plains region.Main ConclusionsThe overlap of these metrics identify salamander populations that may be at risk of developing Bsal infection and suggests priorities for pre‐emptive research and conservation measures to protect at‐risk salamander species from an additional pathogenic threat.

Emerging infectious diseases (EIDs) of wildlife have characteristics that make them difficult to manage, leading to reactive and often ineffective management strategies. Currently, two fungal pathogens, Pseudogymnoascus destructans (Pd) and Batrachochytrium salamandrivorans (Bsal), are causing declines in novel host species. To improve the application of management strategies addressing the risk of these pathogens to North American wildlife, we queried wildlife managers about their concerns regarding managing populations of bats and amphibians potentially impacted by Pd and Bsal. Using these responses, we identified aspects of each decision problem that were shared across pathogens, regions and agencies – and found similarities in decision-problem elements for disease management. Reframing management problems as decisions can enable managers to identify similarities across EIDs, i.e. uncertainties within management actions, and improve reactive responses if proactive management is not possible. Such an approach recognizes context-specific constraints and identifies relevant uncertainties that must be reduced in developing a response.

Chytridiomycosis is an infectious disease in amphibians caused by two chytrid fungi, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), and is the worst infectious disease known in wildlife so far. Worldwide spread of the disease has caused unprecedented loss of global amphibian diversity. Although some lineages of Bd are enzootic and are not as deadly as the pandemic lineage, nearly 40% of amphibian species are still declining globally due to chytridiomycosis. Efficient surveillance and monitoring of chytridiomycosis are the immediate safeguard against rapid declines or extinctions of amphibian populations. Previous studies showed that existing diagnostic assays were not sensitive to certain Bd haplotypes like those from Korea, China, India, Japan, and Brazil and thereby, there is a need for a universal, sensitive, specific, reproducible, and affordable diagnostic assay. We designed a one-step SYBR green-based quantitative polymerase chain reaction (nSYBR qPCR) for robust detection of Bd. It amplifies an 82 base-pair segment between the 5.8S rRNA and ITS2 of the Bd genome. The primer pair was tested in-silico on 40 isolates from four known Bd lineages. Using skin swab samples of wild amphibians and cultured zoospores from Australia and Panama, we compared the clinical specificity and sensitivity of the newly described primers to the existing TaqMan-based qPCR assay. From India, we used samples which had been previously tested with Nested PCR to validate the new primer pairs. The newly described primer pair was then tested on swab samples from Anura, Caudata and Gymnophiona from India. We report widespread chytridiomycosis with varying infection loads on them. The new assay showed comparable efficiency to the TaqMan-based qPCR assay. This diagnostic assay can facilitate widespread surveillance of chytridiomycosis where it has been previously absent, which may reveal several reservoirs of the pathogen and can improve our understanding of this important wildlife disease.

Chytridiomycosis, caused by the fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, is associated with massive amphibian mortality events worldwide and with some species’ extinctions. Previous ecological niche models suggest that B. dendrobatidis is not well‐suited to northern, temperate climates, but these predictions have often relied on datasets in which northern latitudes are underrepresented. Recent northern detections of B. dendrobatidis suggest that these models may have underestimated the suitability of higher latitudes for this fungus. We used qPCR to test for B. dendrobatidis in 1,041 non‐invasive epithelial swab samples from 18 species of amphibians collected across 735,345 km2 in Ontario and Akimiski Island (Nunavut), Canada. We detected the pathogen in 113 samples (10.9%) from 11 species. Only one specimen exhibited potential clinical signs of disease. We used these data to produce six Species Distribution Models of B. dendrobatidis, which classified half of the study area as potential habitat for the fungus. We also tested each sample for B. salamandrivorans, an emerging pathogen that is causing alarming declines in European salamanders, but is not yet detected in North America. We did not detect B. salamandrivorans in any of the samples, providing a baseline for future surveillance. We assessed the potential risk of future introduction by comparing salamander richness to temperature‐dependent mortality, predicted by a previous exposure study. Areas with the highest species diversity and predicted mortality risk extended 60,530 km2 across southern Ontario, highlighting the potential threat B. salamandrivorans poses to northern Nearctic amphibians. Preventing initial introduction will require coordinated, transboundary regulation of trade in amphibians (including frogs that can carry and disperse B. salamandrivorans), and surveillance of the pathways of introduction (e.g., water and wildlife). Our results can inform surveillance for both pathogens and efforts to mitigate the spread of chytridiomycosis through wild populations.

ABSTRACTAmphibian populations around the world are declining in part due to diseases from infection with the chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bs). While declines in more charismatic megafauna are common sources of public awareness and concern, such as the loss of elephants to poaching or polar bears to climate change, amphibians have been suffering a dramatic decline due to the outbreak of deadly fungal diseases with relatively little public attention. Various amphibian advocacy groups work to raise awareness of the issue, but given the limited funding and resources allocated to this cause, there remains a general lack of momentum to tackle the growing conservation threats to this group of animals and to examine policy weaknesses that may need to be adapted to help ensure their conservation. The international trade in live amphibians certainly contributes towards the global spread of these pathogens, but the true extent of spread remains unknown. To determine the degree to which the importation of amphibians into the United States was correlated with presence of known vectors of Bd spread, we compared US Fish and Wildlife Service wildlife trade records for all commercially traded live animals imported to the US from 2006 to 2014 against known species-level infection susceptibility. Approximately 26,859,034 live amphibians were imported into the US for commercial purposes between January 2006 and December 2014., Of these, 59.8% were specimens of species known to be susceptible to Bd infection and therefore may have introduced Bd into the country. Our findings demonstrate significant declines in the annual import quantities of 14 Bd-susceptible species between 2006 and 2014. These reductions could be due to a variety of factors, ranging from possible increased domestic production and a reduced need for foreign-sourced animals to reduced demand from changing market behaviors to the potential disease-driven decline of wild populations and greater difficulty in supplying these specimens. Our research supports the need for continued implementation of US policy, particularly the Lacey Act, to closely regulate wildlife imports to reduce the spread of highly virulent pathogens that threaten native species. Additionally, a rapid response mechanism is needed to control the introduction and spread of wildlife disease vectors when emergencies arise. Although the impact of the wildlife trade is just one facet of the overall amphibian conservation landscape, the information we present herein provides reason to develop increasingly robust rapid-response policies to protect wild amphibian populations in the midst of an emerging global disease crisis.