Characterization of Microbial Communities Across Disease States and Environmental Conditions in Kemp’s Ridley (Lepidochelys Kempii) and Green Sea Turtles (Chelonia Mydas)

All species of sea turtles are threatened or endangered, with various diseases and conditions affecting populations around the world. Understanding healthy populations as well as populations beset by disease conditions, such as fibropapillomatosis and cold-stunning, could lead to helpful tools in the conservation management and medical treatment needed to protect these species. Microbial communities, or the microbiome, at different body sites of sea turtles likely play important roles in the health of these animals, from aiding in digestion to immune system regulation. Disruption of these communities, either through disease and/or environmental factors, may play a role in disease processes and recovery in sea turtle species. Given the importance of microbial communities in health and disease, my dissertation sought to: 1) characterize the microbiome of two species of sea turtles, Kemp’s ridley and green turtles, from the same habitat in the wild, 2) characterize the microbiome of cold-stunned Kemp’s ridley turtles through rehabilitation, and 3) investigate the respiratory microbiome of Kemp’s ridley turtles in relation to radiographic lung abnormalities and diagnostic tools. To carry out these objectives, I used sequencing of the 16S rRNA gene to identify microbial community composition of various body sites from sea turtles for each experiment. In wild turtles, I identified distinct core microbes from the oral cavity and cloaca of two species of healthy, wild caught sea turtles. In stranded turtles, I characterized the same body sites, oral cavity and cloaca, throughout rehabilitation and found shifts in the microbial community composition throughout hospitalization, including alterations due to antibiotic therapy. I also found that the microbiome did not correlate with disease condition or physiological abnormalities in stranded cold-stunned turtles. Since lung abnormalities are prevalent in cold-stunned turtles, I also examined the respiratory microbiome through tracheal washes and necropsy samples. I found that lungs contained a diverse and variable microbial community and identified limitations of tracheal washes as a diagnostic tool. Taken together, these results contribute to understanding the microbiome of sea turtles across disease states and environmental conditions by identifying the microbial community composition at different body sites, through different methods, and based on different disease conditions

Assessing the Bacterial Zoonotic Risks Associated with the Pet Trade

Background. Zoonotic diseases can impart devastating consequences on social, medical, ecological and economical levels. An important driver of zoonotic disease is the pet trade; however, little is known about the presence of bacterial species across publicly accessible surfaces within pet shops. Furthermore, the increasing popularity of exotic pets can disproportionately affect some species over others; for example, Python regius, colloquially known as the Ball Python, is the most traded pet python species globally. The extent of the trade, along with the practice of ’ranching’, is potentially unsustainable. Aims/Objectives. This project aimed to i) develop a DNA extraction method suitable for retrieving data from low-biomass samples; ii) identify key bacterial zoonotic pathogens, referred to as target genera, within the literature to provide a foundation for microbial community analysis; iii) characterise bacterial communities associated with a range of surfaces - primarily publicly accessible - within a pet shop; iv) characterise bacterial microbiota associated with Python regius and feeder rodents within breeding farms in Africa; and v) use the information gathered from the wider literature to infer the presence of target genera within the pet shop and animal samples. Results. Phenol-chloroform extraction was found to be the most sensitive DNA extraction method, with a limit of detection of 28 cells. The systematic literature review found that: i) there were research biases at the genus and animal level, with Salmonella and fish and birds investigated the most respectively. Overall, nearly 10 % of genera isolated from the pet shop samples were target genera. Samples clustered based on animal type, inferring the presence of distinct animal-associated microbiomes. However, with one exception, these relationships became less distinct when clustering the target genera only. Multiple target genera were present across at least half of the samples, in addition to those accounting for more than 1 % of the sequencing reads. Finally, all of the sample types from Python regius and feeder rodents contained target genera, although feeder rodents contained a greater number of target genera for oral and rectal samples relative to the corresponding snake samples. Summary. The results are indicative of the presence of multiple potentially pathogenic genera within pet shops and the key animal species. More research is required using techniques capable of identification at the species and strain level to confirm the pathogenicity potential

Epidemiological Analysis of Biosecurity Practices and Associated Prevalence of Diseases in Non-Commercial Poultry Flocks

A cross-sectional study was conducted in backyard poultry flocks among nine counties of Maryland from May 2011 to August 2011. The objective of this study was to obtain baseline data from a survey on biosecurity practices and investigate risk factors associated with positive findings of avian influenza (AI), Newcastle disease (ND), infectious laryngotracheitis (ILT), Mycoplasma gallisepticum (MG), and Salmonella Enteritidis (SE). Serum, tracheal, and cloacal swabs were randomly collected from 262 birds among 39 registered premises. Analysis revealed flock prevalence and seroprevalence respectively for the following: AI (0%, 23%), ND (0%, 23%), ILT (26%, 49%), MG (3%, 13%), SE (0%, ND). Vaccine status could not be confirmed for ND, ILT, or MG. Premises positives were identified by partial nucleotide sequencing. No statistically significant associations were identified, however, AI seroprevalence was positively associated with exposure to waterfowl (Relative Risk [RR] = 3.14, 95% confidence interval [CI] 1.1-8.9) and absence of pest control (RR=2.5; 95% CI, 0.6-10.4)

Host-microbe interactions in wild vertebrate populations.

PhD Theses.Microbes are ubiquitous, and are often found in close associations with a host, where they affect its physiology, immune functions and even behaviours. In this thesis, I explored host-microbe interactions in wild vertebrate species to disentangle the role of genetic and environmental determinisms of those interactions. In Chapter Two, together with my collaborators, I brought evidence for the evolution of local adaptation of three-spined stickleback (Gasterosteus aculeatus) gut microbiomes through population-specific microbiomes, caused by localised environmental pressures and/or genetic determinism. In Chapter Three, I established the baseline relationship between parasites and stickleback diet, as both can impact host-microbe interactions. I confirmed that feeding ecology is not independent of host-parasite interactions and both parasite resistance and feeding ecology evolve under local adaptation. Chapter Four highlighted how the host’s microbiome is influenced by genotype-by-environment interactions, with changes in the microbiome correlating with interactions between host evolutionary lineage, local environment, and seasonal variation. Interestingly, microbial diversity decreased with increased parasite infections, suggesting intricate host-parasite-microbe interactions. Additionally, I found evidence that a host’s microbiome is linked to feeding ecology, but the direction of this relationship was context-dependent. Finally, in Chapter Five, I tested the generality of the conclusions obtained in the fish system by changing host species to the philopatric and locally-adapted loggerhead sea turtle (Caretta caretta) nesting at the Cabo Verde Archipelago. I found population-specific cloacal microbiomes among closely related nesting groups and as well as host-parasite-microbe interactions. Overall, this thesis focused on teasing apart the diverse determinisms of wild host-microbe interactions. It relied on a series of field experiments and sampling of wild individuals and ultimately shows how fundamental the role of the host microbiome is for species evolution

Health and disease status of Australia's most critically endangered mammal the Gilbert's potoroo (Potorous gilbertii)

The Gilbert's potoroo (Potorous gilbertii) is a small marsupial endemic to the Two Peoples Bay Nature Reserve in the south-west of Western Australia. The Gilbert's potoroo is classified as Australia's most critically endangered mammal (IUCN 2006) with an estimated population of only 35 individuals. This thesis examines the health and disease status of the Gilbert's potoroo, presenting a strong case for the relatively new concept of disease as a potential threatening factor and modifier of population decline. Specific diseases, including Cryptococcus, ectoparasitism, endoparasitism, haemoparasitism, Toxoplasma and a novel Treponema organism are extensively studied. An assessment of the clinical significance of these diseases is made, and management strategies are recommended to minimise the impact of these diseases on both the wild and captive population. The novel Treponema organism which clinically presents with tenacious, green discharge and an associated balanoposthitis in males is molecularly characterized. Epidemiological studies show the effects of this agent on reproductive function and a penicillin-based treatment regime is trialled in the analogous long-nosed potoroo (Potorous tridactylus) with a recommendation to then trial this treatment regime in the critically endangered Gilbert's potoroo. Standard haematological and urinalysis findings are tabulated to form reference ranges for this species. A treatment regime for Cryptococcus in the analogous long-nosed potoroo is reported and parasitological findings, including the identification of a novel tick species are discussed. This thesis addresses key health issues, which have subsequently been incorporated into the Recovery Plan of the Gilbert's potoroo. A document encompassing multiple disciplines and expertise to support the recovery of this critically endangered marsupial in its current environment. In addition, this thesis outlines a recommended health monitoring and treatment protocol for future translocation procedures and provides a working example of the emerging importance of health monitoring in threatened species recovery programs

Intrinsic and Extrinsic Factors Shape Cane Toad Gut Bacteria Across an Expanding Invasive Range

The cane toad (Rhinella marina) is one of the most successful invasive species worldwide. Since their introduction to Queensland in the 1930’s, Australian cane toads have expanded westward and now are present in Western Australia. My thesis examines the gut bacteria in Australian cane toads to determine how environmental factors (e.g., diet, climate) and intrinsic factors of hosts (e.g., genetics, body size, parasite infection) interact to maintain and influence the composition and stability of intestinal bacteria. I first investigated sampling methodologies to determine whether non-lethal (cloacal and faecal) sampling accurately represent gut bacteria. I found that cloacal swabs are better proxies for large intestinal bacteria than faeces in toads. I then tested whether behaviours associated with invasion are correlated with intestinal bacterial community assemblage and function. Behaviours thought to be linked to invasion ability differ in toads from the extreme ends of this range. Although behaviour has been linked to gut bacteria in other taxa, cane toad gut bacteria has not been investigated. I characterised gut bacteria composition and behaviour of wild-sampled cane toads across their northern Australian range and found significant difference in bacterial community and predicted functions between Western Australia and Queensland cane toads, based on 16S rRNA sequencing. Environmental factors including Isothermality, Annual Mean Temperature and the presence of co-introduced lungworms (Rhabdias pheudosphaerocephala) best explained bacterial community assemblage. These same factors, in addition to certain behaviours linked to invasion ability (righting reflex time and the presence of righting reflex movements) best explained bacterial function. I then used Next Generation Sequencing to characterize and compare cane toad genetic (single nucleotide polymorphism), epigenetic (DNA methylation), and gut bacteria differences across populations. I found no significant association between host heterozygosity and gut bacterial diversity within individuals. However, I did find that pairwise genetic diversity was positively associated with pairwise epigenetic diversity. Interestingly, the positive correlation between pairwise epigenetic diversity and bacteria diversity was greater in pairs with lower genetic diversity. Finally, I examined cane toad diet (taxonomy of stomach contents) and found that the presence of plant matter in cane toads’ stomachs was associated with gut bacteria variation, but that gut bacteria was not significantly associated with the main component of cane toad diet (insects). My thesis provides important methodological advances in the study of amphibian gut bacteria and suggests that in cane toads, gut bacteria variation is strongly linked to lungworm infection and to DNA methylation. These results highlight possible mechanisms through which cane toads could increase the plasticity of their response to novel environments encountered during invasion.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 202

CHARACTERIZATION OF THE ENVIRONMENTAL RESISTOME IN THE GALAPAGOS ISLANDS, ECUADOR: A ONE HEALTH PERSPECTIVE

Antibiotic resistance represents one of our generation’s most pressing public health challenges, with some exports warning of an approaching post-antibiotic era. Mitigating this threat requires an understanding of the evolutionary ecology of resistance, including the unique ability of microorganisms to move between humans, animals, and the environment. However, significant questions remain regarding the role of the environment as a source and reservoir for antibiotic resistance. Moreover, there few environments left on earth where we can study background antibiotic resistance in the absence of significant anthropogenic influence. The Galapagos Islands of Ecuador, where the human population is restricted to 3% of the landmass, represent a unique model system to study how human activity influences antibiotic resistance patterns in wildlife and the environment in a largely protected ecosystem. With samples from humans, animals, and the environment, we designed a One Health study aimed at answering what, where, and who: what antibiotic resistance genes are present, where are they located in regards to mobile genetic elements, and who may be the presumptive bacterial host? We employed shotgun metagenomic sequencing to achieve a broad characterization of 90 environmental, wildlife, and human resistomes and mobilomes, and paired this data with targeted detection of the class I integron-integrase gene using a novel ddPCR assay in > 250 Galapagos samples. Additionally, we used a combination of 16S rRNA amplicon sequencing and taxonomic inference from metagenomes to profile the microbial communities associated with these samples. Our results suggest that human, environmental, and wildlife reservoirs are characterized by distinct resistomes and mobilomes, with overall abundance and diversity of antibiotic resistance genes (ARGs) increasing along a gradient of anthropogenic influence. Overall, we found wildlife to harbor fewer ARGs than wastewater and humans, though some exceptions were noted among land iguanas. Differential abundance analysis revealed ARGs unique to each wildlife species with possible bacterial hosts identified in taxonomic assignments in some cases. We recorded overall agreement between resistome and mobilome data sets, and correlation between taxa, ARGs, and MGEs pointed to a key relationship with Enterobacteriaceae.Doctor of Philosoph

Associations between gut microbiota composition and production efficiency of broiler chickens

Intestinal microbiota plays a key role in nutrient digestion and utilization with a profound impact on feed efficiency of livestock animals. However, the intestinal microbes that are critically involved in feed efficiency remain elusive. To identify bacteria associated with production efficiency in chickens, male Cobb broiler chicks were individually housed from day 14 to day 35. Individual RFI values were calculated for 56 chickens. Luminal contents were collected from the ileum, cecum, and cloaca of each animal on day 35. Bacterial DNA was isolated and subjected to 16S rRNA gene sequencing. Intestinal microbiota was classified to the feature level using Deblur and QIIME 2. High and low RFI groups were formed by selecting 15 and 17 chickens with the most extreme RFI values for subsequent LEfSe comparison of the difference in the microbiota. Spearman correlation analysis was further performed to identify correlations between the intestinal microbiota composition and RFI of all 56 chickens. No significant difference in evenness, richness, and overall diversity of the microbiota in the ileum, cecum, or cloaca was observed between high and low RFI groups of chickens. However, LEfSe analysis revealed a number of bacterial features being differentially enriched in either high or low RFI chickens. Spearman correlation analysis further indicated many differentially enriched bacterial features were significantly correlated with RFI (P < 0.05). Importantly, not all short-chain fatty acid (SCFA) producers showed a positive association with efficiency. While two novel members of Oscillibacter and Butyricicoccus were more abundant in low-RFI, high-efficiency chickens, several other SCFA producers such as Subdoligranulum variable and two related Peptostreptococcaceae members were negatively associated with feed efficiency. Moreover, a few closely-related Lachnospiraceae family members showed a positive correlation with feed efficiency, while others displayed an opposite relationship. Our results highlight the complexity of the intestinal microbiota and a need to differentiate bacteria to the species, subspecies, and even strain levels in order to reveal their true association with feed efficiency. Identification of RFI-associated bacteria provides possibilities to manipulate the intestinal microbiota for improving production efficiency, profitability, and sustainability of poultry production