Batrachochytrium salamandrivorans

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By Fiona Ellsworth

Introduction

In vitro culture of Bs. (A) Monocentric thalli, with a few colonial thalli (designated by the black arrow). Sporangia's discharge tubes for zoospores (designated by the white arrow). (B) Scanning electron microscopic image. Mature sporangium with discharge tubes (designated by the letter D). [1].

Amphibians world-wide have recently come under threat from a pathogenic fungus of the chytrid family. First documented in South American amphibians, Batrachochytrium dendrobatidis (Bd) has now been documented as causing chytridiomycosis in a variety of different amphibians in different parts of the world. Batrachochytrium salmandrivorans (Bsal), of the same family as Bd, is the fungal pathogen that causes chytridiomycosis in salamanders[1]. Bsal, like Bd, has aquatic zoospheres that infect its host through their skin The diseased salamander subsequently develops deadly skin lesions. One key difference between Bsal and Bd is the low thermal tolerance of Bsal. The pathogenic fungus likely originated in Asia, and then traveled to Europe via the international pet trade[1]. Because of the great extent of the international salamander pet trade, it is likely that Bsal will eventually make its way to North America, and the rest of the world. Once it does, it is important for scientists to know as much as possible about the fungus, the disease that it causes, and what can be done to stop its spread. Scientists are currently working to answer these questions, in an effort to mitigate the mass extinction of amphibians worldwide which is currently underway.

Phylogeny

Maximum likelihood tree from analysis of partial 18S + 28S rRNA genes. Bsal in bold. Bsal forms clade with Bd. [1].

Batrachochytrium salamandrivorans and Batrachochytrium dendrobatidis are the two members in a clade of Chytridiomycota, characterized as parasites that infect amphibian hosts with a mostly lethal outcome. Martel et al (2014) used Bayesian estimates of divergence time to suggest that Bsal diverged from Bd 67.3 million years ago, in the late Cretaceous or early Paleogene[3]. For much of this time, Bsal was confined to Asia, in several reservoir species likely including Cynops pyrrhogaster, Cynops cyanurus, and Paramesotriton deloustali. These three species are unfortunately actively involved in the international pet trade[6]. Martel et al estimate that the potential for being a reservoir evolved in the ancestors of these modern Asian newts between 55 to 34 million years ago, in the Paleogene[3]. While the timeline for the arrival of Bsal in Asia and the evolution of resistance are relatively close on a scale of millions of years, there is still the difference of multiple millions of years between arrival and resistance, a length of time currently unavailable to amphibians in the rapidly changing environment of today’s planet.

Transmission: Pathogen Host Interactions

Stegen et al (2015) found that one of the factors that contribute to the extreme vulnerability of salamander populations to Bsal is the fast rate of host population collapse, once Bsal has been introduced[7]. This result is largely due to the high susceptibility of adults to the fungal pathogen, whose mass die offs then bring about a shift in the demographics of the population, which inhibits recovery. Adults are more susceptible than juveniles because of their increased interaction with potential pathogen hosts, through sexual intercourse, territorial disputes, or trips back and forth to where eggs are laid. Because Bsal is highly infectious through physical contact, this places adults at a much higher risk of infection than juveniles. Stegen et al further found that the effect of infection on a host is dependent on the concentration of the pathogen and the temperature. Furthermore, there appeared to be no immune defense mounted against the pathogen, leading to rapid succumbing of the host. Through experimentation, Stegen et al found that this lack of an immune response did not change depending on the concentration of the pathogen. Even at low concentrations, host were unable to erect any sort of defense, thereby eliminating vaccination as a potential method of pathogen elimination in populations. The only difference that low concentrations of pathogens seemed to cause was a slower buildup of infection. Ultimately, however, the infected individual died.

History

A Fire Salamander, one of the first species of salamanders to be observed as a host for Bsal. Prevelent in The Netherlands..

Bsal was first observed in fire salamander populations of East Europe, causing unexplained mass die-offs[1]. An Martel was the first researcher to investigate, document, and publish about Bsal, and she remains at the forefront of research. Bd was previously known to be causing high rates of mortality among amphibian populations worldwide, but Bsal appeared to be occupying a different niche, affecting species only of the order Urodela[2]. After the outbreak among Eastern European salamanders was made known, first documented by Martel et al in 2013, further study by Martel demonstrated that this disease likely originated in Asia and then spread to Europe[3]. Martel ascertained this by performing skin scrapes and qPCR to test for the fungal pathogen DNA in the amphibian DNA among populations from four continents. Bsal was detected only in populations from Asia and Europe. In the Asian populations, Bsal was observed among non-outbreak populations, suggesting that these populations had acquired immunity after long (millions of years) exposure to the fungus. Bsal was observed among outbreak European populations, suggesting that the fungus had recently arrived and was wreaking havoc among these naive populations. While it is encouraging that evolved immunity is possible, as illustrated by the Asian amphibian populations, the huge length of time over which this immunity arose and the high rates of mortality seen in populations lacking such immunity suggest that Bsal will have a highly negative impact on worldwide amphibian populations. Martel suggests that a likely method of transport of the disease, from Asia to Europe, is the international salamander pet trade, which is currently flourishing. The thriving of this pet trade will likely eventually spread Bsal to all seven continents, a highly worrisome reality. Bsal, and Bd, the two species of chytridiomycosis ravaging amphibian populations worldwide, are at the forefront of the agents involved in the sixth mass extinction currently underway on the planet. While immunity might have arose over millions of years in Asian populations, such a scenario is highly unlikely in today's global environment. The rampant lack of biosecurity and high levels of global interconnectedness create an ideal opportunity for Bsal and Bd to spread across the planet with little to stop them[3]. Scenarios like these are part of what has brought about the current sixth extinction and suggest a precarious future for this planet and its biodiversity.

Global Spread of Bsal

Microscopy of the skin of a Bsal infected salamander. A) Immunohistochemical stain. Numerous Bsal colonial thalli, particularly around lesion. B) Transmission electron microscopy of colonial thallus inside keratinocyte cell. [1].

Martel et al (2014) demonstrated the likelihood of the international pet trade serving as the main means of transportation of Bsal from Asia to Europe, and a projected means of eventual transportation from Europe to the Americas[3]. There is no geographic continuity between Asia and Europe that would adequately allow salamanders carrying Bsal to travel to Europe on their own. Therefore, human intervention likely played a role in this transportation. Martel et al suggested that this human intervention took the form of the international pet trade and supported this conclusion by testing skin scrapes from salamanders in European pet shops, London Heathrow Airport, and from a Hong Kong pet salamander exporter. Martel et al observed the presence of Bsal in several of the tested individuals, suggesting that the pet trade is a viable method of transport for this fungal pathogen. Furthermore, they saw transmission of the pathogen between members of different species via direct contact, another worrisome example of the ease of travel of Bsal.

While Bsal has not as yet been documented in the Americas, work by Martel et al (2014) has illustrated that this is a very real possibility [3]. The great extent of the international pet trade and its relative lack of biosecurity suggest that the spread of Bsal to the west is imminent. Another worrisome development for the western salamander is its unusually high susceptibility. This susceptibility was demonstrated in an experiment by Martel et al. Martel et al exposed 44 Western Palearctic salamanders (families Salamandridae and Plethodontidae) to Bsal and witnessed the rapid death of 41 of them. Western salamanders are little prepared to face the impending arrival of Bsal in the Americas. Not only is this lack of resistance worrisome in itself, but this is further concerning in light of the fact that salamander populations in the Americas are some of the most rich and diverse in the world [6].

While there is much evidence that Bsal will eventually make its way to Western salamander populations, as of June 2017 there is no evidence that the pathogen has already arrived in the New World. Klocke et al performed a citizen science study in response to the institution of the Lacey Act in January 2016 which attempts to restrict the transportation of 20 genera of salamanders deemed potentially “injurious” to environments due to the likelihood of them carrying diseases. Klocke et al asked pet salamander owners to performed skin swabs on their pets and return them to the researchers for analysis. Pet owners were provided with instructions and a swab kit. The researchers received back 639 swabs from 56 pet owners from 29 US states. The swabs included samples from 65 species, 22 genera, and 3 families (Ambystomatidae, Plethodontidae, and Salamandridae). Upon analysis of the samples, Klocke et al found evidence of Bd in 1.3% of the samples, and no evidence of Bsal. While this study found no evidence of Bsal already present in America, the low sample size and limited sample range of the study indicate that its results might not be entirely reliable. Furthermore, 99% of salamanders imported to the US in the past five years came through Asia [6]. Nevertheless, Bsal is evidently not yet rampant in the New World, despite the fact that its arrival is likely imminent.

Threat of Bsal to North America. A) Map of Bsal habitat suitability. B) Map of salamander species richness. C) Areas vulnerable to Bsal outbreak. Black squares represent major port cities for the international salamander pet trade. [12].

Characteristics of Bsal: Habitat, Morphology, Host

Chytridiomycosis is the disease previously known to be caused by Batrachochytrium dendrobatidis and since 2013 known to be caused by Batrachochytrium Salamandrivorans as well[1]. Bd and Bsal, both fungal pathogens of the genus Batrachochytrium, infects amphibians and causes high mortality rates. These two species, however, occupy slightly different niches and thereby allows both of them to coexist[3]. Bsal only infects salamanders and newts, the two species of the family Urodela. It is however, extremely deadly for these species. Midwife toads, the organisms most susceptible to Bd, are known to be entirely immune to Bsal, further outlining the separate niches of these to fungal pathogen species[6]. No members of the anuran and caecilian orders, the two other major orders of amphibians, were susceptible to Bsal Bsal therefore has a much narrower host range than Bd, potentially limiting its worldwide influence.

Skin scrapes of infected salamanders illustrate the effects of Bsal on its host[1]. Colonial thalli are seen throughout all epidermal layers, and cause skin lesions and deep ulcerations on the infected organism. Around the skin erosions were damaged keratinocytes, keratin producing epidermal cells, each containing a Bsal thallus. Many of these epidermal cells showed signs of hyperplasia (abnormal enlargement) and hyperkeratosis (thickening of the outer layer of skin) [1]. No signs of Bd were found in any of the Bsal infected organisms, further exemplifying each of the strains’ very specific niches. Individuals infected with Bsal experienced death around 7 days after infection, after going through a series of health problems including anorexia, apathy, and ataxia (the loss of control over body movement) [4]. Not only has Bsal been experimentally shown to be extremely deadly to its amphibian host, but it is also highly infectious. Housing infected and uninfected individuals together has demonstrated infection rates of previously uninfected individuals of around 25 days[#References|[1]]]. Furthermore, deceased infected individuals retain the virus for a brief period after death, increasing the possibility of transmission. Death soon follows infection.

The morphology of the fungal pathogen Bsal includes motile zoospores which, in vitro, are released from a monocentric thallus[#References|[1]]]. A thallus is a plant body without stem, roots, or other appendages, and are typical of fungi. A monocentric thallus produces a single reproductive organ, a zoospore in the case of Bsal. Occasionally a colonial thallus is seen, which is a thallus that produces more than a single reproductive organ. Zoospores are generally spherical in shape, with a diameter on average 4.6 um, an uneven surface dotted with protrusions, and a single flagellum for facilitation of movement[#References|[1]]]. Sporangium form (on average with a diameter of 27.9 um) at the tip of discharge tubes as the mechanism to release zoospores. Bd does not form these tubes in vitro, and neither does it produce as many colonial thalli either in vitro or in vivo. Another morphological difference between these two strains is their thermal tolerance. Bsal has a distinctly lower thermal tolerance than Bd. Bsal has an optimal temperature range for growth between 10℃ and 15℃[4]. Contrastingly, Bd grows best at temperatures between 17℃ and 25℃. Bsal on the other hand has been known to grow at temperatures as low as 5℃, and dies at temperatures greater than 25℃. Thus the growth temperature ranges of these two strains are distinctly different. While both Bd and Bsal have narrow temperature ranges, it is suspected that both would exhibit temperature adaptations to the environments in which they find themselves[#References|[2]]]. Therefore the temperature ranges detailed here might not be absolute. This is worrisome for efforts to limit the spread and impact of these fungal pathogens on worldwide amphibian populations.

Laboratory Detection of Bsal

Effective methods of detection of Bsal included skin scrapes and subsequent PCR[1]. Martel designed species-specific PCR primers for the purpose of easily identifying Bsal infected amphibians, a much needed tool for combating the spread of the pathogen. These primers, STerF and STerR, amplify the 5.8S rRNA gene and the adjacent transcribed spacer regions, ITS1 and ITS2. With these primers, Martel et al was experimentally able to identify the presence of Bsal in all tested amphibian tissues, even several samples from already deceased individuals. Perhaps most importantly, this PCR method did produced only Bsal-positive results, not Bd-positive results, thereby offering researchers an effective way to test for Bsal alone and not Bd in amphibian populations. This reliable specificity is highly important to the containment and discouragement of Bsal.

Treatment

The established antimycotic (antifungal) treatment for Bd has failed in its use against Bsal[#References|[10]]]. These treatments include antimycotic agents such as Florfenicol, Voriconazole, Polymyxin E, Itraconazole, and Terbinafine. Blooi et al, in a series of experiments performed on fire salamanders from populations in the Netherlands, suggest that this is due to different minimum inhibitory concentrations (MIC) necessary for use against Bd versus Bsal. Furthermore, use of antimycotic agents in conjunction with one another, as opposed to separately, was documented to cure infected salamanders of Bsal. Blooi et al also established the ability of thermal treatments in eliminating Bsal from its host. However, the temperature range at which Bsal begins to die is also at the temperature limit for which most salamander species can survive, rendering thermal treatments useless in widespread combatting of the fungal pathogen. Blooi et al tested MIC levels for several antimycotic agents commonly used against Bd, including Florfenicol, Voriconazole, Polymyxin E, Itraconazole, and Terbinafine. They found some of the MIC levels to be higher for Bsal, and some to be lower, when compared to Bd. This explains the widespread failure of these agents, at the levels used for Bd, to effectively treat Bsal. They also found that while these treatments on their own and at the proper MICs, could inhibit Bsal growth, they could rarely eliminate the fungal pathogen. However, Polymyxin E used alongside Itraconazole or Voriconazole could actually clear the salamander host of infection. Furthermore, temperature in conjunction with these antimycotic agents played a key role in clearing of the pathogen from the host. Blooi et al found that raising the temperature to 20℃ substantially improved the efficacy of the antimycotic treatments, and was particularly useful for salamander species that could not survive temperatures greater than 25℃, the limit for Bsal survivorship [6].

Efforts to Address the Rise of Bsal

In January 2017, the US passed the Lacey Act which attempts to restrict listed “injurious” species from transport into and within the country. Several salamander genera were included on the injurious species list, due to their likelihood of spreading Bd and Bsal to other populations. The Lacey Act however was curtailed in April 2017 when the Supreme Court ruled that it only applied to cross-state transport, not transport of injurious species within states. This interpretation will likely limit the ability of the Lacey Act to prevent against the spread of Bsal[6].

Because of the length of time for which Bd has been known to the scientific community, more has been observed and written about Bd than Bsal. However, scientists are making an active effort to learn more about these fungal pathogens and their interactions with their host in an effort to mitigate the already extreme losses of the sixth mass extinction[1][3][6]. The more that is known, the better able conservationists will be able to protect these amphibian species. As has been mentioned here, An Martel has been at the forefront of identification and observation of Bsal[1][3].

Yap et al used salamander pet trade data, salamander hotspots, and habitat suitability for reservoir species to map projected regions of North America at high risk of a Bsal outbreak. They concluded that the North Western coast (along Washington State and leading into Canada), Northern Florida and the rest of the American South East, and Mexico City (likely because of the prevalence of the pet trade, particularly coming from Asia, here) were at the highest risk of an outbreak of the fungal pathogen.

After assessing the high risk North America, an invaluable hotspot for salamander biodiversity and species richness, faces from a Bsal epidemic, Yap et al advocates for preventative measures as opposed to reactive ones. Preventative actions would be more cost effective and have a higher likelihood of success. Yap et al specifically recommends that the US halt all pet trading activity. While this seems extreme, the potential for the arrival of Bsal in the Americas is incredibly high, and once here, the consequences are dire. This is further exacerbated by the fact that as of yet, there is no entirely successful method known to combat the spread of Bsal once it has gained a footing in a population. Until a cure or other form of management is found, prevention is likely the best mode of action. Yap et al note that there is little internationally agreed upon mandatory regulation on EID’s (emerging infectious diseases), particularly animal ones. Some such framework of international cooperation to detect and quarantine infected individuals is necessary in this current climate of frequent global travel and exchange.

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2018, Kenyon College.