Comparative Genomic Analysis and Evaluation of Control Strategies for Hypervirulent Aeromonas hydrophila in Channel Catfish (Ictalurus punctatus)
Type of DegreePhD Dissertation
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This dissertation is a reflection of the insight gained through the genetic analyses of a hypervirulent pathotype of the Gram-negative pathogen, Aeromonas hydrophila, (vAh) in the context of enzymatic, challenge, growth, and population-level data. After characterizing this pathogen of warmwater fishes through disease challenges, by histological descriptions, and by associations between genotype and phenotype, genetic profiles unique to vAh were evident. One significant example was the myo-inositol catabolism pathway, which provided an additional support for the use of biological control agents that aim to inhibit vAh proliferation by reducing the abundance of this carbon source in the environment. To quantify the resultant microbiome shifts of introducing these agents, foundational research was performed with the aim of developing a novel tool (provisionally referred to as ‘quantitative polybacterial polymerase chain reaction’ or ‘qpPCR’) to evaluate mixed cultures of bacteria at the species or subspecies level, while providing quantitative data on microbial abundance without the biases associated with culture-based and common culture-independent methods. While qpPCR was designed to be the first technique capable of defining the composition of any microbiome, unresolved biases associated with any method reliant on the polymerase chain reaction and/or with the purification of low abundance amplicons prevents the application of this method from being a practical replacement for existing microbial quantification methods. Therefore, the inclusion of this chapter is intended to serve as an experimental foundation for the application of the conserved and divergent sequences validated within this research for use in future research based on this method that uses real-time sequencing. Chapter one presents an overview of known virulence factors, amidst the phylogenetic reshuffling of what was considered the dominant research strain for A. hydrophila, a primer on the use of Bacillus spp. probiotics and the enzyme phytase in aquaculture, and a discussion of methods for the quantification of mixed cultures, with an emphasis on the biases surrounding these methods. Chapter two presents a genetic and phenotypic characterization of the pathogen vAh within the species A. hydrophila. Chapter three describes the preliminary development of the qpPCR method and proposes an alternate approach to circumvent pitfalls identified through extensive experimental evaluation. Finally, chapter four describes experiments in which the enzyme phytase and the probiotic Bacillus velezensis AP193 were used as feed additives to reduce disease due to vAh through reductions of the anti-nutrient myo-inositol. Collectively, this research has mediated a separation of knowledge by discussing what remaining literature on virulence factors is applicable within A. hydrophila, characterized a hypervirulent pathotype that has significant economic relevance to the aquaculture industry within the United States as well as other countries with farmed fishes, formed a foundation for the development of new quantitative methods for complex sample analyses, and explores the significance of shifts in gut microbiota based on the introduction of the phytase enzyme and the phytase-producing probiotic to collectively provide a deeper understanding of the effects these agents have when introduced to aquaculture systems.