Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 66-71).

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 73-80).

The intestinal microbiome (IM) or the community of commensal and pathogenic microbes that inhabit the intestinal tract of fish has long been of interest in aquaculture because of its hypothesized role in nutrient digestion and fish immunity. Artificial improvement to the IM of fish with pre- and probiotics has been shown to have benefits in some studies; however, the mechanisms behind these supplements are poorly understood because of a lack of knowledge on the basic structure of the IM of fish. The research described here aims to characterize the IM of the highly studied and commercially important Icatlurid catfish, channel catfish Ictalurus punctatus and blue catfish I. furcatus. In this study we evaluated differences between the homeostatic IM, across genotype (Objective 1) and developmental ontogeny (Objective 2), using high-throughput sequencing of 16S rRNA gene amplicons. We compared the IM of four selected strains of blue catfish and three selected strains of channel catfish at 193 days post-hatch (DPH) in Objective 1 (n = 40); while the influence of developmental ontogeny was evaluated by observing the IM of a single family of channel catfish at 3, 65, 125, and 193 DPH Objective 2 (n = 20). The bacterial phyla Fusobacteria, Firmicutes, and Proteobacteria were detected in all fish strains and developmental time points; however, at the genus level the abundances of different bacteria varied among experimental units, as well as being rather variable between individuals. At 3 DPH (n = 5) the IM of channel catfish sac-fry larvae showed the most variation between individuals; with bacteria from the genus Bradyrhizobium dominating the IM of two sac-fry (84% and 88%), and Flavobacterium, Lactobacillus, Comamonadaceae dominating the IM of the remaining three sac-fry analyzed (86%, 99%, and 97% respectively). The dominant bacteria in the gut of all other fish analyzed (n = 55), was Cetobacterium somerae (mean ± SD, 41.4% ± 36%), a commensal microbe that is known to produce vitamin B12. Statistical differences in the abundance of three different bacteria within the order Clostridiales (P ≤ 0.039) were detected in Objective 1, with more of these OTUs inhabiting the gut of channel catfish than that of blue catfish. Statistical differences in the abundance of Edwardsiella, a genus of bacteria known to cause disease in catfish, were detected across the two species of Ictalurid catfish in Objective 1 (P = 0.038), and across developmental ontogeny in Objective 2 (P = 0.021); however, these bacteria were detected at low abundance (0.002% - 0.004%). Comparisons of beta diversity showed significant differences between many strains of Ictalurids, with a highly significant difference between channel catfish and blue catfish (P = 0.001); however, few differences were detected when comparing beta diversity across the four time points over the first 193 DPH in Objective 2, with only 3 DPH and 125 DPH showing significant differences (P = 0.022). Overall these data suggest host-genotype, and to a lesser extent developmental ontogeny, influence the structure of the IM of Ictalurid catfish. As the first study to be conducted on the IM of these fish species, these results have performance implications on the culture of these commercially important catfish, while also enhancing our basic knowledge on the dynamics of the fish microbiome.

Stress during fish culture can impact growth, physiology and fillet quality. Maintenance of high quality seafood is important to ensure the production of a highly marketable product. The present study assessed how sequential stressors affect the physical, physiological, sensory and quality characteristics of channel catfish (Ictalurus punctatus) fillets. Temperature (25°C or 33°C) and dissolved oxygen (DO, ~2 mg/L or >5 mg/L) were maintained for four weeks, followed by socking and transport stress for a total of 12 treatments. After each stage of stress (environment, socking and transport), physical (length, weight and feed consumption), physiological (hematocrit, plasma cortisol, pH, glucose, lactate, total protein, osmolality and ionic composition) and fillet quality (color, texture, sensory and pathogen load) attributes of the fish and fillets were evaluated. Fillet yield decreased with increasing severity of environmental stress. Overall, increasing stress resulted in decreased feed consumption, growth and fillet yields. A cumulative stress response was identified with regard to circulating cortisol and glucose, which increased with each sequential stress event. Under low oxygen conditions there was a suppression of the stress response. Handling imposed a more pronounced physiological response than environmental conditions. The sequential stressors resulted in a less intensely colored fillet that was less red, which suggests the quality changes imposed are beneficial to the marketability of the fillets. However, increased redness in fish reared under high temperatures and oxygen levels suggest that an increased prevalence of red fillets can be expected. Sensory analysis revealed that fillet flavor was acceptable in all treatments with the severe stress treatment preferred due to lower intensity of less favorable flavor attributes. The changes in flavor were presumably due to fasting of the stressed fish and a subsequent purging of fat stores. As fish progressed through the harvest event, cook loss decreased, tenderness increased and pH increased, indicating that stress induced positive textural changes. Proteomic analysis revealed mainly down-regulation of structural and metabolic proteins, which indicates higher proteolytic activity and an adjustment in energy metabolism in response to stress. The overall effects of chronic environmental conditions and handling highlight the importance of managing for stress in cultured channel catfish.

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is one of the most important bacterial diseases causing significant economic losses in the US catfish industry. Understanding the virulence mechanisms of E. ictaluri plays a vital role to develop preventives, such as vaccines for the disease. Therefore, further research is necessary to discover the new virulence mechanisms of this pathogen. The long-term goal of our group is to determine the mechanism of E. ictaluri pathogenesis and to develop effective live attenuated vaccines against ESC. The overall goal of this project is to understand the role of Type 6 secretion system (T6SS) in E. ictaluri virulence and determine the safety and efficacy of T6SS mutants in the catfish host. The central hypothesis is that T6SS in E. ictaluri provide an ability to invade the host cells and survive inside of the channel catfish neutrophils and macrophages, and mutation of T6SS genes will cause attenuation of the bacterial virulence. The rationale for the proposed research is that characterization of the T6SS in E. ictaluri will enlighten its role in E. ictaluri virulence, and T6SS genes can be targeted to develop live attenuated vaccines. In this study, we first constructed mutants of individual T6SS genes and a double mutant. The persistence, virulence, and vaccine efficacy of T6SS mutants were determined in the catfish fingerlings and fry infection model. The T6SS mutants Ei?evpC, Ei?evpC?hcp2, Ei?evpD, Ei?evpE, Ei?evpG, Ei?evpJ, and Ei?evpK were significantly attenuated and provided better protection against E. ictaluri 93-146 in channel catfish fingerlings. The role of T6SS mutants in adhesion and invasion of in vitro catfish epithelial indicated that Ei?evpN, Ei?evpO, and Ei?evpP significantly were less adherent and invasive. The survival and replication of T6SS mutants in in vitro catfish peritoneal macrophages cell line showed that T6SS mutants could survive up to 6 hours after phagocyted by catfish macrophages. The survival and resistance of T6SS mutants to stress conditions present in macrophages phagosome showed that hydrogen peroxide could limit the growth of T6SS mutants in BHI and minimal medium. Ei?evpA, Ei?evpH, Ei?evpM, Ei?evpN, and Ei?evpO exhibited a significant growth decrease.

Channel catfish (Ictalurus punctatus) are raised for aquaculture in ponds characterized by dramatic swings in dissolved oxygen concentration. If morning dissolved oxygen concentration falls below approximately 3.0 mg/L catfish consume less feed, leading to a reduction in growth and production. Although the effects of low oxygen on channel catfish appetite have been described, the underlying mechanisms responsible for those effects are unknown. Increased production of the neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) are implicated in other fishes as neuropeptides that reduce appetite once an environmental stressor is detected. This project characterizes the hematological responses and transcriptional response of the hypothalamus to acute, chronic, and cyclical (repeating periods of hypoxia and normoxia) hypoxia. During acute (12 hours) hypoxia, venous PO2 decreased within 6 hours, coupled with an increase in hematocrit and decreased blood osmolality. These changes reversed within 12 hours after returning to normoxia but were not coupled with a change in transcription of the genes for CRF and UI. If this pattern of hypoxia and normoxia is repeated cyclically for 5 days, the same physiological responses repeat continually. During chronic hypoxia up to 5 days in duration, channel catfish have a similar hematological response, but did not recover to normoxic control values over the duration of the challenge. Likewise, no significant change in gene expression of CRF or UI were detected during chronic hypoxia. The hypothalamic transcriptome was analyzed during a 12-hour exposure to hypoxia followed by a 12-hour normoxic recovery. Across all time points, 190 genes were differentially expressed, with the greatest numbers occurring during periods of hypoxia. Differentially expressed genes were grouped into Gene Ontology biological processes and were most overrepresented by the term “response to hypoxia,†which included genes involved with angiogenesis, red blood cell production, and negative feedback to hypoxia-inducible factor proteins. Although this study did not find a change in hypothalamic transcription of CRF and UI, it did identify multiple adaptive responses that work together to reduce the severity of hypoxia along with several gene candidates for future hypoxia studies.

Commercial catfish production is an inveterate industry within the southeastern United States. Bacterial disease is a significant detriment to global aquaculture, including the United States catfish industry. Among them, an atypical strain of the bacterium Aeromonas hydrophila has plagued the industry since the late 2000s. Atypical A. hydrophila (aAh) outbreaks are largely acute, resulting in catastrophic losses. The disease ecology, prevalence, and genetic distribution are poorly understood. Atypical Aeromonas hydrophila displays a rapid onset with few warning signs of the impending disease, making it difficult for early detection. At present there are two recognized haplotypes of aAh. This project aimed to investigate changes in the spatial and temporal distributions of these haplotypes. The analysis of clinical isolates from different geographic regions across multiple years revealed complete supplanting by the younger haplotype in the Mississippi Delta within 5 years of first isolation. Comparative genomics demonstrated distinct divergences in specific virulence components between the two strains, specifically the Type VI Secretion System, which may explain putative differences in outbreak dynamics and recent displacement of one strain by the other. Also, a rapid, non-lethal screening method was validated that can detect aAh within the catfish host. This method affords data collection regarding infection severity prior to onset of disease and, can predict aAh prevalence at the fish- and pond-levels. The occupancy model indicates more than half the population within a pond may be infected with aAh despite no overt signs of disease. Additionally, aAh is commonly present in approximately 10% of the population, providing the first evidence of a carrier state in this disease. Lastly, a compartmental SLIR model was used to investigate disease dynamics of aAh in catfish aquaculture ponds. Simulations suggest the introduction hypothesis does affect estimated pond profit and antibiotic intervention is an economical treatment for aAh. Routine monitoring was less economical and could dramatically reduce profit in some scenarios. Overall, this work expands our current knowledge of aAh in catfish aquaculture and lays the foundation for future studies investigating aAh management and mitigation of bacterial disease in catfish aquaculture.

This study was designed to develop a method of rapidly evaluating dietary proteins for channel catfish (Ictalurus punctatus) and to gain basic knowledge of its nitrogen metabolism with respect to dietary protein quantity and protein quality. Experiments l, 2 and 3 were short-term (7-week) growth trials with fingerling catfish in aquaria under controlled laboratory conditions. Parameters measured each week included instantaneous daily gain of the fish (IDG) and the activities of selected enzymes - glutamate dehydrogenase (GDH), aspartate aminotransferase (GOT), alanine aminotransferase (GPT) and alkaline phosphatase (AP). Fish were fed purified diets differing in protein quantity and protein quality in experiments 1 and 2, respectively. GDH, GOT, GPT and AP activity and IDG increased with increased dietary protein quantity. The consistency of the relationship between enzyme activity and IDG decreased as follows: GOT, GDH, GPT and AP. GOT activity and IDG increased, and GDH activity decreased, with increased dietary protein quality. IDG was positively correlated to GOT activity and negatively correlated to GDH activity. GPT activity was unrelated to either dietary protein quality or IDG. GDH and GOT activity responded to changes in dietary proteins within 3 weeks in experiments 1 and 2. The ratio of GOT activity to GDH activity was positively correlated to dietary protein quality. The growth and enzyme activity data from experiments 1 and 2 were used to develop indices of growth based on enzyme activity. The growth indices were tested as a method of rapidly evaluating dietary proteins in experiment 3 where fish were fed natural ingredient diets differing in protein source. The proteins included fish meal and four types of seafood processing wastes. GDH, GOT and GPT activity, the GOT/GDH ratio, IDG and the growth indices were similar among treatments in catfish fed different protein sources at 10 percent of the diet in experiment 3. The results of experiment 3 could not be used to adequately assess the growth indices and the GOT/GDH ratio but did indicate the feasibility of using seafood processing wastes as fish meal substitutes at 10 percent of the diet. / Ph. D.

Thesis (M.S.)--Mississippi State University. Department of Basic Sciences. / Title from title screen. Includes bibliographical references.

Since being first reported in the late 1980ies, the Enterobacterium Edwardsiella ictaluri has rose in prevalence to become one of the two most damaging pathogens affecting the channel catfish industry. Despite this significance of the pathogen, understanding of the development of the disease, especially its route of entry into the host and the earlier stages of the infection, is still incomplete. A series of challenges were conducted using bioluminescent E. ictaluri either by infecting fish through immersion or topical application of the bacteria directly on the intact or abraded epithelium. This showed that abraded fish developed septicemia and died faster than non-abraded ones. Furthermore, results from a co-habitation challenge suggested that the bacterium induced septicemia through the skin instead of becoming water-borne. Finally, a histological technique was developed allowing the determination that the bacteria radiated from the initial skin infection site and penetrated deeper into the tissue as the challenge progressed. These results all suggest that site of abrasion on the skin can act as a route of entrance for the pathogen into the fish, a fact never previously reported. Transposon mutagenesis was also performed to construct a library of 1728 mutants. Screening of this library allowed us to identify 16 genes which inactivation lead to a decrease in the bacterium ability to colonize the epithelium or cause mortality. Sequencing of these genes allowed the identification of RstA/B, a regulator of invasion genes in Salmonella enterica Typhimurium, a putative ribonuclease, similar to a Shigella protein regulating the expression of adhesin and a protein that constitutes the second member of a newly discovered adhesin family. Finally, to investigate the development of the infection, fish were infected by bioluminescent E. ictaluri and sampled at various time points. At each time point, nine organs (gills, muscles, intestine, spleen, liver, stomach, heart, head kidney and trunk kidney) were sampled, and their bioluminescence was measured and half of these organs were homogenized, serial diluted, and plate counts determined. This allowed confirmation of a complex disease pathogenesis during ESC involving a period of intense reproduction in the spleen, anterior and posterior kidneys followed by a sharp increase in the levels of bacteria in the blood.