BD in Salamanders

During routine sampling of tiger salamanders in 1999 in Arizona, USA, researchers noted little black spots on the trunk of the animal. Analysis under the microscope worryingly revealed it was BD.

This was the first time the disease had been identified in a species that wasn’t a frog. Increasing evidence is now being reported that the disease is very prevalent within salamanders.

What does this mean? Unfortunately, even less is known about how salamanders are affected compared to frogs. The need for research in both frogs and salamanders is astronomical and we are at a critical stage in combating this devastating disease

Culturing BD

One way of analyzing BD  is to grow it on a petri dish filled with agar. However, the fungi is always out-competed by faster growing bacteria, and one bacterial spore will ruin a sample.

Therefore it is crucial to work aseptically (minimizing contamination)  and in labs that have high biosecurity – to prevent BD escaping outside.

By storing the the fungi at 4 degrees Celsius, you can even trick BD to produce zoospores which can be harvested and analysed as well.

Ecology of BD

Chytrid fungi typically live in water or soil, however a minority are parasites of plants and insects. Only BD is known to infect vertebrate species

Chytridiomycosis mostly affect amphibian species associated with permanent water, such as streams, moist bogs and ponds. However, much is still unknown about the fungus and the disease in the wild.

Interactions between the fungus and environmental factors are known to be important. For example, Australian upland frog populations have suffered the greatest number of declines and extinctions. It has been suggested this is due to environmental stress, such as increased ultraviolet light exposure. However the ecology of BD is remains one of the most unexplored areas of the disease.

 

Does BD kill all types of amphibians?

Frogs which live or breed in permanent water at higher latitudes are more susceptible. Confirmed extinctions due to BD include the Northern gastric brooding frog, the Souther gastric brooding frog and the sharp-snouted day frog – all in Australia.

The hardest hit has been the Genus Atelopus – stream brooding toads of Central and South America – of 113 described species 30 are now extinct. Other frogs are far less susceptible, like the Bullfrog and the African clawed frog, but have been suggested to be carriers of disease. Bullfrogs are native to North America but exported worldwide as a laboratory and food species.

It appears that once BD is introduced into a frog population, one of two outcomes can occur. Either the infection rapidly spreads through the habitat killing the species. Or, if the frog is resistant, the fungi lives within the skin producing zoospores that further enhance the spread of disease – locally or globally.

Effect of season and temperature on mortality

BD offers an odd paradox to scientists: the pathogen is more virulent at lower temperatures, but outbreaks of disease are often seen in the summer months.

So what could be happening here? Current ideas suggest that the amphibian immune systems may be impaired by extremely temperatures, and that sheltering from the heat in cracks will increase spread of disease as many frogs may hide together. Additionally, the summer months is when frogs tend to breed, thus spreading the disease from one individual to the next.

Yet the idea that BD increases mortality at lower temperatures explains why massive frog population declines have been observed at high altitudes, particularly in South America.

Ecological studies are needed to determine mortality rates and impacts on abundance of frogs in different habitats.

 

Detection of BD

The most commonly used method for diagnosis is Polymerase Chain Reaction (PCR). This relies on the amplification of tiny amounts of specific regions of BD‘s DNA. However this has disadvantages, such as contamination, bias and PCR failure.

Recent advances in molecular technologies have resulted in the development of real-time Taqman assays. These are much faster and more sensitive than conventional PCR, whilst allowing the quantification of the amount of DNA present. It can detect as little as 1 zoospore in infected frog tissue and 7 to 14 (or more) days faster than classical histological methods.

Where did BD originate?

Trying to sort out the molecular epidemiology of BD is proving to be very difficult. As the disease quickly rose to attention from all corners of the map, trying to establish where the pathogen first came from is no easy task.

Localised endemic strains have been found in Asia and Africa in old museum samples. These samples are over 100 years and yet no large scale amphibian declines have been observed up until the late 1990’s. Conclusively, it is suspected this is where BD originated, as local frog populations may have acquired natural resistance to the local strains over many decades. Yet the introduction of BD-GPL has changed this.

Much more research is required here, as many of the ideas are only speculative and based on few samples. For more information see Fisher et al. (2009)

Emergence of Amphibian Chytridiomycosis in Britain

In the summer of 2004, 14 North American bullfrogs were extracted from ponds on the East Sussex/Kent border, the only place in Britain where the species is known to naturally exist in the wild. Two of the bullfrogs tested positive for BD and was the first instance in which it had been detected in the wild in Britain. An analysis of 170 native species between 1992-1997 did not find any evidence of the disease.

BD had been reported in captive, exotic frogs but never in the environment. Subsequently, in order to limit the spread of the disease, the organisation English Nature has removed tens of thousands of bullfrogs and tadpoles of other frog species from the area. Considering the widespread damage BD has inflicted to several South American countries, this emergence is of significant concern to the authorities.

How can humans limit disease transmission?

When a researchers enters the field to study BD, the risk of accidental contamination of clothing and equipment is rather large. Inexpensive disinfectants, such as bleach solution, should be used treat boots and field equipment, in addition to wearing disposable gloves, should be utilized to minimize spread.

On a larger scale, the two most common ways of spreading BD to new sites is the use of amphibians as fishing bait or their collection for pets. The former means that infected frogs may be placed in a previously unexposed habitat and transmitting the disease there, and thus this should be avoided. Once been taken into captivity, frogs should not be reintroduced into the wild.

After initial capture frogs should be placed in quarantine and examined before further action is taken. It is to be noted that cage soil/water will likely be contaminated and should not be dumped back into the environment.

These measures would dramatically reduce BD transmission but implementing them on a global scale is expensive and near impossible.

 

 

Life cycle

There are two distinctly different life stages for BD: the motile, flagellated zoospore and the reproductive thallus

The zoospore is free living and swims through water with its tail-like flagellum. Once in contact with the amphibians skin, the zoospore enters a keratinized skin cell. The exact mechanism of entry into the cell is unknown to date.

Once inside, the development of the thallus begins. The thallus is the form in which the fungus lives in the frog and is what ultimately kills it. It develops zoosporangium, zoospore factories. Via discharge tubes these zoospores are released into the environment.

It is not fully understood how BD disperses, but it is presumed it is via skin to skin contact between infected frogs during mating, fresh zoospores in the environment, or a combination of both.

Rosenblum et al. (2008) PNAS, 104 (44), 17034-17039