Emerging evidence suggests that the immune system is vulnerable to disruption in response to a wide variety of chemical contaminants; thus, there is a need to test chemicals for immunotoxicity. To understand how chemicals impact the ability of the immune system to ward off infection, a model system featuring fathead minnows (FHM, a common toxicological model) infected with Yersinia ruckeri (a bacteria) has been used. Using this model system, the impacts of chemicals on immune system function can be evaluated via pathogen resistance challenges, where a fish is infected with a pathogen and their ability to defend against the pathogen and survive infection is determined. However, the use of Y. ruckeri is unfavorable given that fish must be injected with Y. ruckeri to develop an infection, a process that is time consuming and inconsistent with natural routes of exposure. Thus, the goal of this project was to develop a new host-pathogen system for FHMs by identifying a pathogen that induces infection via immersion. To do this, the ability of three bacterial pathogens, Flavobacterium columnare, Aeromonas sobria and Aeromonas allosaccharophila, to infect FHMs via immersion was evaluated. Results of this study revealed that of the three bacterium evaluated, only F. columnare was capable of inducing an infection via immersion in FHMs and infection was only successful following a fin-clip procedure, in which a small portion of the caudal fin is removed. Overall, this result establishes the potential for a FHM-F. columnare model system for future use in immunotoxicity testing.

Nickel is the most prominent heavy metal in the effluents associated with crude oil extraction and production. Given that these effluents are released into the ocean, investigating the toxicity of nickel on marine life is pertinent. A current method for evaluating the toxicity of oil effluents is the sheepshead minnow larval growth and survival (LGS) test, which exposes larval fish to varying concentrations effluents or associated single chemicals over a 7-day period. However, current legislation, like the Frank E. Lautenberg Act, requires that animal testing be refined whenever possible to enhance animal welfare. The fish embryo toxicity (FET) test, which investigates chemical toxicity using fish embryos over a 7-day exposure period, is a potential alternative method capable of meeting legislative needs related to animal welfare. The objective of this study was to determine if a sheepshead minnow FET test is a viable replacement for the sheepshead minnow LGS test. To accomplish this, the results of sheepshead minnow LGS and FET tests using nickel were compared. The results of this study show that the LGS test is more sensitive than the FET test. In addition, evidence suggests that it may be possible to improve the sensitivity of the FET test by including sublethal metrics as FET test endpoints.

Bacillus anthracis is a bacterial pathogen that causes the often lethal disease anthrax. This research aims to characterize the role of potential virulence genes in Bacillus anthracis. Virulence is a pathogen’s ability to damage the host. Studying virulence allows us to understand infection mechanisms and develop novel ways to target pathogens. Previous work identified a collection of potential virulence mutants (Franks et al, 2014) each containing a genetic disruption that renders a gene non-functional. These mutants were pulled out in initial screenings but were never characterized further. We confirmed that one mutant, TN2, also exhibits decreased virulence in a Galleria mellonella survival assay. We know that TN2 has a disruption in a promoter region that we hypothesize controls two genes: a putative BNR repeat domain protein (TN2A) and a glycosyl-like 2 transferase family protein (TN2B). For my project, I attempted insertional mutagenesis to inactivate these genes with the goal of confirming that the genes are linked to virulence, rather than unintended mutations elsewhere in the genome. After successfully creating insertional mutant 2B, through the disruption of the TN2B gene, I am working to further characterize the mutant to determine its role in immune evasion. Specifically, I will compare the ability of the wild-type and mutants to survive exposure to various antimicrobial defenses conserved in humans and waxworms. This research could help identify a novel bacterial virulence factor and its potential mechanisms of action thus expanding our understanding of bacterial pathogenesis.

The gram-positive bacterium, Bacillus anthracis, is responsible for the deadly disease Anthrax. B. anthracis is dangerous due to virulence factors, or defenses the bacteria uses to infect a host. We hope to better understand how this bacterium interacts with its hosts by studying the genes necessary for virulence. Bacterial mutants, which have a change in their genetic sequence, sometimes show reduced ability to cause disease in a host. Studying these mutants helps us understand the bacteria’s infection method. Previously our lab created a library of mutants using a technique called transposon mutagenesis and then screened these transposon mutants for phenotypes linked to decreased virulence. This resulted in the identification of 11 transposon mutants that were less effective at causing disease in the nematode Caenorhabditis elegans (Franks et al.). While all 11 mutants could be interesting for further characterization, it is necessary to prioritize them as this is still too many to study. In this project, we tested these mutants using a second infection model, the caterpillar Galleria mellonella. G. mellonella is an ideal model due to its optimal size for injection, conserved innate immune defenses, and previous success as an infection model for B. anthracis (Malmquist et al.). We found that only one of these 11 mutants, TN2, had reduced virulence in both C. elegans and G. mellonella. Future research will focus on confirming the genetic change in this mutant and determining the mechanism by which it contributes to infection. This could lead to new antibiotic targets in the future.

Mercury (Hg) is released into the environment by coal-burning powerplants and artisanal gold mines. Aquatic bacteria then convert the inorganic mercury into highly toxic methyl mercury. Turtles acquire mercury through their diet, and it bioaccumulates throughout their long lifetime. Toenail clippings can be used to determine Hg concentrations in turtles. Toenail samples were collected from Trachemys scripta elegans (red-eared sliders) in the Brazos River near Granbury and the Clear Fork of the Trinity River as it flows through Fort Worth. All toenails were dried in a 60℃ oven and put into a direct Hg analyzer that uses thermal decomposition, gold amalgamation, and atomic absorption spectrometry to determine total Hg. Toenails from the Brazos river had significantly more Hg on average than those in the Clear Fork, 658.302µg/kg and 400.146µg/kg respectively. The results were unexpected as the Brazos river near Granbury is considered less polluted than the Clear Fork of the Trinity, which is supported by observations of insect larvae such as hellgrammites which were common in the Brazos but absent in the Clear Fork of the Trinity. Our hypothesis is that red-eared sliders in the Brazos are feeding at a higher trophic level than those in the Clear Fork. Fecal samples and a lack of invertebrate prey in the Clear Fork suggest red-eared sliders primarily feed on algae. In the Brazos river we observed several species of insect larvae underneath rocks and hypothesize that the red-eared sliders are feeding on this abundant food source. Mercury is known to biomagnify and therefore red-eared sliders in the Brazos are likely ingesting more mercury than those in the Clear Fork.