Infectious disease in wild animals has historically been a challenge that is difficult to overcome, primarily because isolating a disease outbreak to prevent further transmission in these types of populations is nearly impossible. Wild animals are free to roam, and humans often have limited means of tracking infection in populations. Vaccines and treatments can be formulated but are often somewhat impractical for wild populations because it is not feasible to vaccinate or treat every member in a susceptible community. One such pathogen, Batrochochytrium dendrobatidis (Bd) is infecting amphibian populations around the world to the point where many species are already extinct. Even though finding an effective preventative for the fungal pathogen may not mean that I am able to reach every member in a population, it may mean the difference between extinction and eventual release back into the wild for threatened populations.
In this study I hoped to create an attenuated version of Batrochochytrium dendrobatidis, by using a novel laser technology: SEPHODIS. This laser technology disrupts hydrogen bonds between proteins in the lumen of the cell while simultaneously preserving the membrane and associated proteins on the outside of the cell. This process ultimately affects the pathogenicity of the target but leaves identity markers intact so that the host immune system may recognize the pathogen and create antibodies against it. The laser was ultimately effective at killing Bd fungal cells, and I did observe a significant change in the appearance of the cells. However, samples obtained after exposure to the laser were contaminated and more research is needed to determine if SEPHODIS could be a feasible method for vaccine production.

Amphibians around the world are suffering the effects of the chytrid fungus, Batrachochytrium dendrobatidis (Bd). Whenever amphibians are housed in captivity, they must go through a decontamination protocol to ensure they are not infected with diseases such as Bd. Itraconazole is the most commonly used fungicide used in these protocols. This study set out to determine if Bd could develop resistance or tolerance to itraconazole. Two 24 well plates were prepared with different concentrations of itraconazole with Bd zoospores added. Plate 1 had concentrations similar to what animals are currently being treated with in decontamination protocols. Plate 2 had concentrations at and below the published minimum inhibitory concentration values (MIC). Plate 1 displayed the ability of itraconazole to kill Bd sporangia with higher concentrations and Plate 2 showed that even under published MIC values, Bd still struggled to complete its reproductive cycle. I find the evolution of a resistant/tolerant strain of Bd unlikely given the efficacy of this drug, the sensitivity of Bd to itraconazole, and the lack of evidence of the completion of Bd’s reproductive cycle under the conditions used in this study.

One way pathogen prevalence is maintained is by persistence within reservoir host species. Reservoir hosts are species that do not show any signs of disease when a pathogen infects them. As a result, the pathogen survives and is able to remain in the host population. Batrachochytrium dendrobatidis (Bd) is a chytrid fungus that has caused extensive amphibian declines. It has been suspected that reservoir hosts are a key to Bd remaining in certain amphibian populations. I studied dragonfly naiads (Anisoptera spp.), the aquatic life cycle stage immediately following hatching and preceding the emergence of wings, as potential reservoir hosts for Bd on the Mogollon Rim in Arizona. On the Mogollon Rim winter temperatures fall below the optimal thermal range for Bd. Boreal chorus frogs (Pseudacris maculata), the most common amphibian species on the Rim, maintain subzero body temperatures to survive the winter. Since the optimal thermal range for Bd is between 4°C and 25°C, it is unlikely that Bd can grow on the skin of these frogs during winter. As a result, it is unknown how Bd prevalence is maintained in the area. Recent studies showed that Bd can grow in non-amphibian hosts. I hypothesized that Bd could grow within the digestive tracts of dragonfly naiads, since they stay in the water and don’t maintain subzero body temperatures during the cold winters on the Rim. Non-native and native naiads were both included in this study; the non-native naiads were purchased from a company in California while the native naiads were captured from ponds on the Mogollon Rim. The digestive tracts of the naiads were then dissected, and the DNA was extracted using an animal tissue spin-column protocol. The extracted DNA was analyzed by qPCR. The qPCR analysis of the native and non-native dragonfly naiads revealed that the samples were either Bd-negative or very weakly Bd-positive, with most being the former. Based on these results, it does not appear that naiads are biologically significant reservoir hosts for Bd.

Amphibians have been experiencing a worldwide decline that is in part caused by an infectious disease, chytridiomycosis, specific to frogs and salamanders. Globally many species have declined or gone extinct because of the pathogenic fungus Batrachochytrium dendrobatidis, also known as the amphibian chytrid or Bd. By the time Bd was discovered it was too late to stop the spread and it has now been found on almost every continent. The trade of captive amphibians, used as pets, bait, and educational animals provides an opportunity to spread Bd. Because some amphibians can carry Bd without experiencing symptoms, it is possible for even healthy looking amphibians to spread the amphibian chytrid if they are moved from one location to another. Recently, a new species Batrachochytrium salamandrivorans (Bsal) was found on salamanders. Bsal was identified before it reached the United States, prompting concern regarding its spread and a call for regulation regarding the trade of captive amphibians. There are some regulations in place controlling the trade of amphibians, but they are insufficient to stop the spread of amphibian chytrid in captive populations. A 2016 law prohibits the importation of 201 salamander species. However, there is no central organization to sample or certify if amphibians are free from Bd or Bsal. Although some stores say they test for these pathogens the tests are unregulated and not reported to any central body. If the captive amphibian trade is to go disease free, there would need to be a significant push to coordinate testing efforts. To estimate Bd's prevalence in Arizona captive amphibian populations, I contacted pet stores, bait stores, and sanctuary or educational organizations to ask if I could sample their amphibian collections. My research built on the 2008 work of Angela Picco, who sampled for the amphibian chytrid in Arizona bait shops. I found that amphibian owners were often hesitant and unwilling to participate in this research opportunity. There are multiple reasons for this hesitancy including a fear of increased regulation, the potential for reporting to a government agency (USDA), or the eventual cessation of amphibian trade. The lack of willing participants suggests there may be difficulties in coordinating future sampling efforts for Bd and Bsal.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has captured human attention because it is a pathogen that has contributed to global amphibian declines. Despite increased research, much is still unknown about how it develops. For example, the fact that Bd exhibits phenotypic plasticity during development was only recently identified. In this thesis, the causes of phenotypic plasticity in Bd are tested by exposing the fungus to different substrates, including powdered frog skin and keratin, which seems to play an important role in the fungus's colonization of amphibian epidermis. A novel swelling structure emerging from Bd germlings developed when exposed to keratin and frog skin. This swelling has not been observed in Bd grown in laboratory cultures before, and it is possible that it is analogous to the germ tube Bd develops in vivo. Growth of the swelling suggests that keratin plays a role in the phenotypic plasticity expressed by Bd.