UMD Theses and Dissertations
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Item PROBIOTIC, PREBIOTIC, AND SYNBIOTIC APPROACHES IN SUSTAINABLE POULTRY PRODUCTION THROUGH MICROBIOME MODULATION(2023) Tabashsum, Zajeba; Biswas, Debabrata; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter is one of the prominent causative agents of acute gastroenteritis in the US and more than 70% of Campylobacter infections, known as Campylobacteriosis, are occurred through raw or undercooked poultry consumption or improper handling of contaminated poultry products. Moreover, reports show that the antibiotic resistance pattern of Campylobacter is persistent and in the absence of sub-therapeutic antibiotic growth promoter, colonization of this bacterial pathogen in poultry gut or on skin and the potential risk of cross-contamination of the finished poultry products are increasing, Therefore, both conventional and organic/pasture poultry farmers are searching for sustainable alternative to synthetic antibiotics which can reduce colonization and cross-contamination of poultry products with poultry-borne bacterial pathogens specifically Campylobacter. On the other hand, due to the consumers’ demand, majority poultry farmers are growing their chicks without sub-therapeutic growth promotor which leads to slow growth and higher mortality rates. Therefore, to make the poultry farming sustainable, farmers need alternative feed or water supplement which can promote poultry health and growth. Probiotics, prebiotics, or a combination of these two referred to as synbiotics, have emerged as a promising natural and alternative approach to sustainable animal farming. Probiotics and their metabolites such as conjugated linoleic acids (CLAs) play a crucial role in improving host health and act as antimicrobials against enteric pathogens. Our lab developed a genetically engineered probiotic, LC+mcra that can convert more CLA by over-expressing the mcra (myosin-cross-reactive-antigen) in Lactobacillus casei (LC). Further, prebiotic-like components such as bioactive phenolic extracts (BPEs) from berry pomace can stimulate the growth of beneficial microbes including LC, competitively inhibit growth of enteric bacterial pathogens, and promote the growth of chickens in a concentration-dependent manner when applied throughout the growth period. In our previous study, we observed that LC+mcra effectively eliminated Campylobacter jejuni (CJ) in co-culture condition as well as the cell free culture supernatants (CFCS) of LC+mcra was effective in growth reduction of CJ. LC+mcra and its CFCSs also reduced the adherence and invasion ability of CJ to both HD-11 and HeLa cells. Physicochemical properties and gene expressions related to CJ virulence were also altered by CFCSs treatments. These findings suggested, LC+mcra can be an alternative in controlling CJ growth along with other beneficial attributes of LC. Then, we aimed to enhance the efficiency of antimicrobial/beneficial activities of LC+mcra by combining BPEs. In mixed culture condition, LC+mcra in the presence of BPE reduced the growth of CJ more efficiently as well as the CFCS of LC+mcra in the presence of BPE. Interaction of CJ with cultured DF-1, HD-11, and HeLa were altered significantly. Further, combined treatments altered the physicochemical properties and expression of multiple virulence genes such as ciaB, cdtB, cadF, flaA, flaB of CJ. This finding indicates that BPE and LC+mcra in combination might be able to prevent colonization of CJ in poultry. So, in our present study at simulated gut conditions, we evaluated combined effect of LC+mcra and BPE in reducing growth of Campylobacter in cecum contents. Cecum contents were collected from chickens pre-inoculated with kanamycin resistant CJ (CJ-Km), incubated over 48h time period, while being supplemented with either BPE, CFCS from LC+mcra, or their combination. It was found that combined treatments were able to reduce both inoculated and naturally colonized Campylobacter more effectively. Microbiome analysis using 16S rRNA sequencing also revealed that combined treatments were capable of altering natural microflora positively within chicken cecum contents. Then, the effect of sustainable probiotics on CJ colonization and gut microbiome composition was evaluated using chicken as a model. A total of 120 chickens were used in duplicate trials to investigate the effectiveness of LC+mcra in decreasing CJ colonization by means of CJ-Km compared to the control group. We observed that LC+mcra could efficiently colonize various parts of the chicken gut and competitively reduce colonization of natural and challenged Campylobacter. Furthermore, 16S rRNA compositional analysis revealed lower abundance of Proteobacteria, higher abundance of Firmicutes, along with enriched bacterial genus diversity in gut of LC+mcra fed chicken. Outcomes of this study reveal high potential of LC+mcra as sustainable approach to decrease colonization of Campylobacter in poultry gut along with other beneficial attributes. So, we further evaluated the combined effect of LC+mcra and a low dose of BPE on Campylobacter colonization in chicken gut using a day-old chick model. Colonization of CJ-Km as well as the natural colonization of Campylobacter was reduced by the combined effect of LC+mcra and BPEs significantly at all time points. In the cecum contents of the LC+mcra and BPEs treatment group, there was notable change at phylum level microbiome compared to the control group. At genus level colonization of Lactobacillus was significantly higher (1.7 folds), Campylobacter colonization was reduced significantly (6.3 folds), and other microflora remained balanced due to the combined treatment of LC+mcra and BPEs. Therefore, LC+mcra with BPEs could be an alternative to improve the safety of poultry products and reduce campylobacteriosis in humans sustainably. The application period of this synbiotic compositions could be extended to improve the poultry growth rate as an additional benefit of the LC+mcra and BPEs.Item The Impact of Private Drinking Water Wells and Animal Feeding Operations on the Incidence of Acute Gastroenteritis in Maryland: A Mixed Methods Approach(2019) Murray, Rianna Teresa Frederika; Wilson, Sacoby M; Maryland Institute for Applied Environmental Health; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)More than 44.5 million people in the United States (approximately 14% of the population) rely on private domestic wells as their primary source of drinking water. The water quality of private wells is not regulated at the state or federal level, leaving homeowners with wells responsible for the safety of their water. Meanwhile, each year, an estimated 48 million people in the U.S. are sickened, 128,000 are hospitalized, and 3,000 die of gastrointestinal (GI) illnesses caused by microorganisms that are typically transmitted through contaminated food. Given that the potential of private wells as a non-foodborne transmission pathway for these microorganisms is not well understood, my dissertation evaluated these relationships, as well as characterized the quality of private well water in Maryland. My objectives were to: 1.) Investigate the water quality of private wells in Maryland and the effect of animal feeding operations on this water quality using fecal indicator bacteria; 2.) Investigate associations between the prevalence of private wells and the incidence of campylobacteriosis in Maryland at the zip code level using data from the FoodNet active surveillance System; and 3.) Investigate associations between the prevalence of private wells and the incidence of salmonellosis in Maryland at the zip code level using FoodNet data. My findings demonstrated that 43.2% of private wells tested in Maryland did not meet at least one federal health-based drinking water standard. Additionally, my data showed that the prevalence of private wells in Maryland is a risk factor for the incidence of campylobacteriosis and salmonellosis in the coastal counties of the state. The presence of broiler chicken operations in a zip code is also a risk factor for campylobacteriosis and salmonellosis in coastal counties. These findings provide evidence for the strengthening of private well water regulations and for improving education and outreach to private well owners on proper maintenance and testing for their wells.Item BIOACTIVE LACTOBACILLUS CASEI IN REDUCING GROWTH AND COLONIZATION OF CAMPYLOBACTER JEJUNI(2018) Tabashsum, Zajeba; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter jejuni (CJ) is one of the pre-dominant causative agents of acute gastroenteritis in the US and occurs commonly through handling/consumption of contaminated poultry products. Probiotics with enhanced bioactive metabolites such as conjugated linoleic acids (CLAs) play crucial role in improving host health and act as antimicrobials. Further, prebiotic like components such as bioactive phenolics from berry pomace extract (BPE) can stimulate growth of beneficial microbes including Lactobacillus casei (LC) and inhibit bacterial pathogens in vitro. In this study, we aimed to assess efficiency of CLA overproducing LC (LC+mcra) alone or in presence of BPE against CJ. LC+mcra alone or LC+mcra with BPE reduced CJ growth, adhesion and invasion efficiency to cultured cells and also altered physicochemical properties, gene expressions related to virulence. These findings suggest, BPE and LC+mcra in combination may able to prevent CJ colonization in poultry and reduce cross-contamination, hence control foodborne infections with CJ in human.Item REDUCED CAMPYLOBACTER INFECTION AND ENHANCED PERFORMANCE IN POULTRY WITH BIOACTIVE PHENOLICS THROUGH EPIGENETIC MODULATION OF THE GUT MICROBIOME(2017) Salaheen, Serajus; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter jejuni, a major enteric pathogen and a natural resident in the poultry gut, causes gastrointestinal illness followed by severe post-infection complications, including Guillain-Barré syndrome, reactive arthritis, myocarditis, and ulcerative colitis in humans. Risk assessment studies have projected a 30-fold reduction in human campylobacteriosis cases with only a 100-fold reduction in the number of C. jejuni colonizing the poultry gut. Current commercial poultry production practices involve use of antibiotic growth promoters (AGP); modulation of gut microbiota with AGPs for food safety and enhanced performance in poultry can be justified until acquisition of antibiotic resistance in zoonoses through inter-bacterial transfer of antibiotic resistance genes (ARGs) in a complex microbial community is considered. As an alternative, natural phenolics extracted from by-products of berry juice industry, with antimicrobial, anti-inflammatory, anticarcinogenic, antioxidant and vasodilatory activities, demonstrate promising prospects. In this study, we adopted mass-spectrometry, microbiological, phylogenetic, and metagenomic approaches to evaluate bioactive phenolic extracts (BPE) from blueberry (Vaccinium corymbosum) and blackberry (Rubus fruticosus) pomaces as AGP alternative. We detected that major phenolics in BPE included, but were not limited to, apigenin, catechol, chlorogenic acid, cinnamic acid, coumarin, ellagic acid, eugenols, flavan, gallic acid, gingerol, glucosides, glucuronides, myricetin, phenols, quercetin, quinones, rhamnosides, stilbenol, tannins, triamcinolone, and xanthine. BPE reduced C. jejuni growth and motility in vitro, resulting in lower adherence and invasiveness to chicken fibroblast cells. Anti-inflammatory effects of BPE significantly reduced the expression of pro-inflammatory cytokine genes in chick macrophage cell line ex vivo. Furthermore, BPE reduced the colonization of C. jejuni in broiler cecum by 1 to 5 logs while increasing broiler weight by 6% compared to 9.5% with commercial AGPs. Metagenomic analysis of broiler gut indicated that BPE caused an AGP-like pattern in bacterial communities with a comparative increase of Firmicutes and a concomitant reduction of Bacteroidetes in broiler ceca. AGP supplementation clearly caused phage induction and a richer resistome profile in the cecal microbiome compared to BPE. Functional characterization of cecal microbiomes revealed a significant variation in the abundance of genes involved in energy and carbohydrate metabolism. Our findings established a baseline upon which mechanisms of plant based antimicrobial performance-enhancers in regulation of animal growth can be investigated.Item Campylobacter spp. in bulk tank milk and milk filters from US dairy farms(2015) Del Collo, Laura P; Biswas, Debabrata; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter spp. are a common cause of foodborne outbreaks associated with raw or unpasteurized milk, and Campylobacter spp. have also been detected on most dairies in the US. An estimate of the prevalence of thermophilic Campylobacter spp. in bulk tank milk (BTM) on US dairy operations was determined as part of the National Animal Health Monitoring System’s Dairy 2014 study. Campylobacter spp. were detected in the BTM and milk filters from 34.2% of the 234 dairies. Isolates were obtained from 18.4% of the dairies. C. jejuni was the most frequently isolated species, and this species is also the most common cause of human infection. When resistance to a panel of nine antimicrobials was tested, 68.4% of C. jejuni isolates were resistant to tetracycline. This survey suggests that BTM from US dairies can be contaminated with pathogenic Campylobacter spp., and the consumption of unpasteurized, raw milk represents a human health risk.Item Campylobacter jejuni/coli - Host Intestinal Epithelial Cell Interaction(2006-08-15) zheng, jie; meng, jianghong; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Campylobacter jejuni/coli have been known to be major bacterial causes of human gastroenteritis worldwide for decades. Regarding its pathogenicity, little is known yet. A better understanding of the pathogenic mechanisms will provide important information, not only for generating molecular markers to differentiate pathogenic strains versus non-pathogenic ones; but also for developing rational strategies to prevent and control Campylobacter-caused disease. The objectives of this study were to characterize the pathogenic abilities of various C. jejuni/coli retail meat isolates, including their abilities to adhere to, invade into and transmigrate across human epithelial cells, to examine the role of NF-κB pathway in IL-8 secretion induced by Campylobacter, and to identify C. jejuni-specific adherence/invasion genes during host pathogen interaction. It was found that the adherence and invasiveness of total 43 Campylobacter retail meat isolates in human intestinal epithelial T84 cell model indicated that C. jejuni/coli present in retail meat were considerably diverse in their ability to adhere to and invade human epithelial cells. Meanwhile, eight putative virulence genes, determined by PCR, were shown to be widespread among the Campylobacter isolates. C. jejuni /coli-induced proinflammatory cytokine Interleukin (IL)-8 secretion in polarized human colonic epithelial cells T84 was examined, and the role of NF-κB pathway in Campylobacter-induced IL-8 secretion was determined. Data suggested that C. jejuni/coli induce basolateral-polarized secretion of IL-8 in human intestinal epithelial cells, and C. jejuni-induced IL-8 secretion is NF-κB-dependent. The effort to identify C. jejuni-specific adherence/invasion genes during host pathogen interaction by using restriction fragment differential display PCR (RFDD-PCR) has been made. As a result, it was not successful. However this study still provides useful information and experience on the application of this technique for prokaryotic gene expression analysis during host pathogen interaction, which remains an unexplored area. In summary, Campylobacter retail meat isolates exhibited wide diversity in cell culture model in the ability of adherence, invasion and transmigration. As the first line defense, intestinal epithelium activates NF-κB and secretes proinflammatory cytokine IL-8 in response to Campylobacter infection. Multiple virulence factors have roles in Campylobacter-intestinal epithelial cell interaction.