Slush-ice chilling has been applied to catfish fillets by processors for several years. However, little is known about the effect of this system on fillets’ safety and quality. Salmonella counts were reduced (P≤0.05) between 0.55 - 0.83 log CFU/g by slush ice treatments (0% - 4.5% salt), regardless of salt concentration. Salmonella counts for slush ice treated fillets were less (P≤0.05) than for untreated fillets during refrigerated storage. However, Salmonella reduction was similar (P>0.05) among all treatments after 12 days of storage. Sensory evaluation showed that a 24 h-slush-ice treatment negatively affected (P≤0.05) the texture, drip, and odor of fillets during storage at 2 ± 2°C. Lightness (61.7), hue (80.6), and chroma (10.1) values were similar (P>0.05) between slush-ice fillets and water-chilled fillets. Psychotrophs, coliforms, and E. coli counts (5.1, 1.6 and /g, respectively) were similar (P>0.05) among fillets collected before and after 24 h in slush ice.

The prevalence of Salmonella within poultry environments and on poultry products has been well documented. However, there has not been a study documenting the effect of utilizing used litter in newly constructed commercial broiler houses on Salmonella status or on the rate and source of Salmonella contamination within new houses. Objectives of this study are to 1) determine environmental source and rate of Salmonella spp. colonization in two newly constructed broiler houses 2) to evaluate the effect of mixing used broiler litter with clean litter in a new broiler house. Results of this study suggest that Salmonella contamination of the poultry house environment occurred within the first 2-4 weeks of bird placement and that the source of contamination may have been the chicks themselves. Litter inoculation may be beneficial in reducing Salmonella levels within the first flock if it is known that the chicks are already contaminated with Salmonella spp.

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Bacteriophage Felix O1, also known as enterobacteria phage O1, has been used to type Salmonella Typhi and is an excellent candidate for use in bioremedial and therapeutic applications. It has extremely high intra-species specificity and is strictly virulent in nature, unable to undergo lysogeny. To facilitate the development of the bacteriophage for use in these areas, the full sequence of the genome had been elucidated previously. In this work, identification and classification of functional coding sequences via reverse transcriptase-polymerase chain reaction was performed. All of the 115 putative open reading frames (ORFs) studied were found to be functional. 53.0%, 9.6%, and 18.3% of the ORFs investigated were found to initiate expression early, middle or late in the lytic cycle, respectively. Expression of the remaining 19.1% ORFs was evident when the amount of total RNA was increased and when samples were taken at a later time point. Comparisons between bacteriophage Felix O1 and the phage with the most shared homologs, phage T4, revealed many similarities in times of gene expression. / Master of Science

Tomato fruit have been associated with numerous outbreaks of salmonellosis in recent years. Trace back suggests tomato fruit may become contaminated during pre-harvest, however exact routes are unclear. The objective of this study was to determine the potential for Salmonella enterica serotype Newport to be associated with the roots, leaves, stems, and ultimately fruit of red round tomato plants through contaminated irrigation water, at various stages of plant development, when grown under controlled environmental conditions. Tomato plants were individually root irrigated with 250 or 350 ml (depending on growth stage) of 7 log CFU/ml S. Newport contaminated irrigation water every seven days. Presence of the pathogen in plant tissue was evaluated at five growth stages; 14 days post transplant, early fruit, mid fruit, full fruit, and terminal stages. At each stage, roots, stems, leaves, and two tomato fruit, if present, from four S. Newport and four water irrigated (negative control) tomato plants were sampled for S. Newport contamination. Association of S. Newport was detected in tomato roots and stems using both conventional plating and molecular techniques. Twenty-four samples were confirmed positive for S. enterica using PCR. Sixty-five percent of the roots, 40% of the stems, 5% of the leaves and 5% of the fruit sampled were confirmed to contain S. enterica. Overall, there was significant difference in the presence of S. Newport according to tissue sampled (roots > stems > leaves and fruit) (P > 0.05). There was no correlation between growth stage and presence of S. Newport in tissues (P > 0.05). Ultimately, irrigation with S. Newport has a low probability of contaminating tomato fruit. / Master of Science

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important human pathogen. The Centers for Disease Control and Prevention (CDC) estimates that 1,027,561 people become ill with nontyphoidal Salmonellosis annually, and S. Typhimurium is one of the most common disease causing serovars. Quantification of the true number of cases of salmonellosis is hampered by the presence of a carrier state. These carriers are animals and humans that carry the pathogens for a variable period of time without showing any clinical signs. One of the biggest barriers to controlling and preventing salmonellosis in a population is identification of these carriers. Identifying these latent carriers of chronic infections is vital to preventing such disease transmission and creating avenues for novel control and treatments. In my dissertation research, we developed a cell culture model to study latent Salmonella infections. By activating human monocytes with retinoic acid and vitamin D3, we were able to isolate Salmonella from such cells 45 days after inoculation. We subsequently used this model to identify genes that were upregulated in this chronic infection model. We found that aceA, a gene that codes for isocitrate lyase, is significantly upregulated on days 10 and 30 post infection. Isocitrate lyase is part of the glyloxylate cycle. Some bacterial species have developed a mechanism to utilize acetone as a carbon source to synthesize tricarboxylic acid (TCA) cycle intermediates. This anaplerotic reaction allows organisms to conserve carbon and use alternative carbon sources. This cycle is one way in which bacteria can adapt and survive in an intracellular environment. This intracellular survival is key to latent infections persisting within a host. It is biologically plausible that, in order to survive in a latent state, S. Typhimurium would up-regulate genes that would facilitate intracellular survival. After establishing the cell culture model, we tested the hypothesis that aceA is upregulated in latent infections of S. Typhimurium in a mouse model. We orally challenged mice that were resistant to Salmonella infection, collected their feces, and collected tissue specimens at several time points up to 135 days post-challenge. These samples were cultured and tested using quantitative polymerase chain reaction (qPCR). The qPCR results showed that tissue samples from inoculated mice had increased aceA expression 95 days after challenge. Finally, we examined whether aceA expression could be detected in cattle lymph node samples. Supra-mammary lymph nodes from 40 dairy cattle and mesenteric lymph nodes from 100 culled cattle were sampled and submitted for culture and qPCR. None of the supra-mammary lymph nodes were positive for Salmonella via culture or aceA qPCR; however, 11 mesenteric lymph nodes showed increased aceA expression in qPCR compared to 5 culture positive lymph nodes. Further research is necessary, but these results demonstrate some of the advantages of using genetic primers to identify latent Salmonella infections in clinically normal cattle. In addition, the assay may be able to differentiate between latent and active salmonellosis, and could be used to provide targeted drug delivery. / Doctor of Philosophy / Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important human pathogen. Determining the true number of cases of salmonellosis is made more difficult because of the presence of a carrier state. These carriers are animals and humans that carry the pathogens for a variable period of time without showing any clinical signs. Identifying these latent carriers of chronic infections is vital to preventing such disease transmission and creating avenues for novel control and treatments. In my dissertation research, we looked at genetic markers from an offshoot of the TCA cycle, the glyoxylate pathway. We used these markers to test the hypothesis that these glyoxylate pathway genes would be upregulated in latent S. Typhimurium infections. Our research involved developing a cell culture model, then using the results from the cell culture model to inform a mouse model, and then a cattle lymph node diagnostic study. The cell culture model indicated that the gene for isocitrate lyase, aceA, is significantly upregulated compared to housekeeping genes. We found the presence of aceA in chronically infected mice, as well as cattle lymph node samples. Further research is necessary, but these results demonstrate some of the advantages of using genetic primers to identify latent Salmonella infections in clinically normal cattle.

Recognizing that Salmonella is a leading cause of bacterial foodborne illness in the United States (US), the US government established disease reduction goals for salmonellosis incidence. Surveillance data are used to monitor progress toward these goals, and to characterize salmonellosis epidemiology (e.g., specific serotype causing disease, model national and regional trends in incidence). This study used data from 11,411 culture-confirmed salmonellosis cases among Virginia residents reported to Virginia Department of Health during 2012-2022 to characterize salmonellosis epidemiology in Virginia using incidence rate ratios (IRRs) and a negative binomial Bayesian splines model. Trends were modelled separately for salmonellosis overall and for individual sub-types at the state level. State-level modelled incidence ranged between 10.9-12.9 (per 100,000 people); the lowest and highest reported incidence occurred in 2020 and 2019, respectively. During the COVID-19 pandemic, incidence appears to have decreased; incidence has not returned to pre-pandemic levels. According to the splines model, there was a substantial decrease in state-level culture-confirmed salmonellosis incidence in 2021 compared to 2018, which is consistent with trends nationally. Of the187 total Salmonella serotypes reported in Virginia during 2012-2022, 64% of culture-confirmed cases were attributable to six serotypes: Typhimurium/I 4,[5],12:i:- (20%), Enteritidis (18%), Newport (11%), and Javiana (8%), Bareilly (4%), and Braenderup (4%). The incidence of illnesses to specific attributable serotypes appeared to change between 2012 and 2022 with some serotypes decreasing in incidence (e.g., the incidence of S. Typhimurium/I 4,[5],12:i:-) and others increasing (e.g., S. Braenderup). Opposing serotype-specific trends may be contributing to the apparent stability in overall salmonellosis incidence in Virginia during 2012 through 2022. Some serotypes had an overall high reported incidence across all months, while others had distinct peaks in incidence during summer months (June-August). Similar to national trends, children <4 years old had the highest reported incidence of all age groups across all serotypes, indicating that reducing incidence in this population should be prioritized for achieving the desired reduction in salmonellosis incidence. Analyses using whole genome sequencing data indicated that within each of the 6 most common serotypes, most isolates represented 1-3 subtypes. Overall, our analyses indicate that trends in reported culture-confirmed salmonellosis incidence in Virginia are largely consistent with national trends and support the use of analyses of salmonellosis incidence at the subtype level to inform intervention efforts to address incidence of infections caused by specific serotypes. / Master of Science in Life Sciences / Salmonellosis is a foodborne disease caused by infection with the bacteria Salmonella. Salmonellosis cases reported to health departments are used to monitor the levels of disease that are occurring in the population at state and national levels. However, barriers to reporting, such as limited access to health care or a patient specimen not being collected, mean that not all cases that occur within the population are reported. While reported cases do not represent all cases occurring, they can provide important information about the types of Salmonella infecting people in a specific population, such as people residing in a state. Among reported cases, those that have a Salmonella isolate obtained from a patient sample are considered culture-confirmed and can be used to study the reported levels of disease with additional insights provided about the type of Salmonella causing infection. Our study analyzed data from 11,411 culture-confirmed reported cases of salmonellosis to identify trends in the levels, or incidence, of salmonellosis in Virginia between 2012-2022. Incidence of reported culture-confirmed salmonellosis cases occurring in the population were calculated to identify the number of cases occurring per 100,000 persons in Virginia. Temporal analyses were also conducted to identify trends in salmonellosis incidence in Virginia. The reported incidence of the most common subtypes of Salmonella causing infection varied over time by year and month. Children under 4 years old were found to have the highest reported incidence, suggesting that reducing salmonellosis incidence in young children should be prioritized. Overall, the patterns in reported incidence from culture-confirmed salmonellosis cases in Virginia largely reflected those seen nationally, indicating that the same strategies for reducing the incidence of reported salmonellosis will be beneficial at regional and national levels.