Browsing by Author "Lee, Margie D."

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    Bacterial composition of a competitive exclusion product and its correlation with product efficacy at reducing Salmonella in poultry
    Lee, Margie D.; Pedroso, Adriana A.; Maurer, John J. (Frontiers, 2023-01-09)
    The mature intestinal microbiome is a formidable barrier to pathogen colonization. Day-old chicks seeded with cecal contents of adult hens are resistant to colonization with Salmonella, the basis of competitive exclusion. Competitive exclusion products can include individual microbes but are commonly undefined intestinal communities taken from adult animals and in commercial production is amplified in fermentator and sold commercially in freeze dried lots. While superior to single and multiple species probiotics, reducing Salmonella colonization by multiple logs, undefined products have limited acceptance because of their uncharacterized status. In this study, the bacterial composition of the master stock, preproduction seed stocks and commercial lots of a poultry competitive exclusion product, was defined by 16S rRNA sequence analysis, targeting the 16S rRNA variable region (V1-V3). The samples contained a diversity of genera (22–52 distinct genera) however, the commercial lots displayed less diversity compared to the seeds and the master stock. Community composition varied between seeds and the master stock and was not a good predictor of potency, in terms of log10 reduction in Salmonella abundance. While there was significant correlation in composition between seeds and their commercial lots, this too was a not a good predictor of potency. There was linear correlation between unclassified Actinobacteria, Peptococcus, and unclassified Erysipelotrichaceae, and Salmonella abundance (r2 > .75) for commercial seeds. However, upon review of the literature, these three genera were not consistently observed across studies or between trials that examined the correlation between intestinal community composition and Salmonella prevalence or abundance.
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    Conditions Necessary for the Transfer of Antimicrobial Resistance in Poultry Litter
    Oxendine, Aaron; Walsh, Allison A.; Young, Tamesha; Dixon, Brandan; Hoke, Alexa; Rogers, Eda Erdogan; Lee, Margie D.; Maurer, John J. (MDPI, 2023-06-03)
    Animal manures contain a large and diverse reservoir of antimicrobial resistance (AMR) genes that could potentially spillover into the general population through transfer of AMR to antibiotic-susceptible pathogens. The ability of poultry litter microbiota to transmit AMR was examined in this study. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 μg/mL), chloramphenicol (Cm; 25 μg/mL), streptomycin (Sm; 100 μg/mL), and tetracycline (Tc; 25 μg/mL). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA1 and sul1, respectively, in the poultry litter community. AMR gene load was determined relative to total bacterial abundance using 16S rRNA qPCR. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (<104 CFU/g). There was high abundance of resistance to Sm (106 to 107 CFU/g) and Tc (106 to 107 CFU/g) and a sizeable antimicrobial-resistance gene load in regards to gene copies per bacterial genome (aadA1: 0.0001–0.0060 and sul1: 0.0355–0.2455). While plasmid transfer was observed from Escherichia coli R100, as an F-plasmid donor control, to the Salmonella recipient in vitro, no AMR Salmonella were detected in a poultry litter microcosm with the inclusion of E. coli R100. Confirmatory experiments showed that isolated poultry litter bacteria were not interfering with plasmid transfer in filter matings. As no R100 transfer was observed at 25 °C, conjugative plasmid pRSA was chosen for its high plasmid transfer frequency (10−4 to 10−5) at 25 °C. While E. coli strain background influenced the persistence of pRSA in poultry litter, no plasmid transfer to Salmonella was ever observed. Although poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low under conditions commonly used to assess plasmid conjugation.
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    Differentiating Vaccine-Related Fowl Cholera from Naturally Occurring Disease
    Hutcheson, Anna R.; Thompson, Kasey; Maurer, John J.; Ferguson, Naola; Grogan, Karen; Roney, Stephen; Seahorn, Harmony; Lobsinger, Chris; Lee, Margie D. (2020-12)
    Vaccine-related fowl cholera must be considered when flock mortality increases after use of a live Pasteurella multocida vaccine product. All registered live vaccines serotype as Heddleston 3,4; however, in some regions this is also the most common scrotype of outbreak isolates in broiler breeders and turkeys. Therefore, serotyping may not be useful for diagnosing vaccine-related fowl cholera. This project sought to apply a vaccine-specific test to differentiate vaccine-related disease from naturally occurring outbreaks. Results indicate that vaccine strains were commonly isolated from broiler breeders exhibiting signs of fowl cholera postvaccination, but some of these isolates exhibited only serotype 4 antigenicity. The isolates' lipopolysaccharides, the target antigen for serotyping, contained compositional changes that may explain the varying serotype results and virulence of the commercial preparations. These results suggest that vaccine-related disease may be common in broiler breeders, and live commercial vaccine preparations need to be assessed for serotype and titer prior to use in order to reduce vaccine-related fowl cholera.
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    Informal nutrition symposium: leveraging the microbiome (and the metabolome) for poultry production
    Lee, Margie D.; Ipharraguerre, Ignacio R.; Arsenault, Ryan J.; Lyte, Mark; Lyte, Joshua M.; Humphrey, Brooke; Angel, Roselina; Korver, Douglas R. (Elsevier, 2022-02)
    Knowledge of gut microbiology of poultry has advanced from a limited ability to culture relatively few microbial species, to attempting to understand the complex interactions between the bird and its micro biome. The Informal Nutrition Symposium 2021 was intended to help poultry scientists to make sense of the implications of the vast amounts of information being generated by researchers. This paper represents a compilation of the talks given at the symposium by leading international researchers in this field. The symposium began with an overview of the historical developments in the field of intestinal microbiology and microbiome research in poultry. Next, the systemic effects of the microbiome on health in the context of the interplay between the intestinal microbiota and the immune system were presented. Because the microbiome and the host communicate and influence each other, the novel field of kinomics (the study of protein phosphorylation) as used in the study of the poultry microbiome was discussed. Protein phosphorylation is a rapid response to the complex of signals among the microbiome, intestinal lumen metabolites, and the host. Then, a description of why an understanding of the role of microbial endocrinology in poultry production can lead to new understanding of the mechanisms by which the gut microbiota and the host can interact in defined mechanisms that ultimately determine health, pathogenesis of infectious disease, and behavior was given. Finally, a view forward was presented underscoring the importance of understanding mechanisms in microbiomes in other organ systems and other species. Additionally, the importance of the development of new-omics platforms and data management tools to more completely understand host microbiomes was stressed.
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    Peeling back the many layers of competitive exclusion
    Maurer, John J.; Cheng, Ying; Pedroso, Adriana; Thompson, Kasey K.; Akter, Shamima; Kwan, Tiffany; Morota, Gota; Kinstler, Sydney; Porwollik, Steffen; McClelland, Michael; Escalante-Semerena, Jorge C.; Lee, Margie D. (Frontiers, 2024-03-21)
    Baby chicks administered a fecal transplant from adult chickens are resistant to Salmonella colonization by competitive exclusion. A two-pronged approach was used to investigate the mechanism of this process. First, Salmonella response to an exclusive (Salmonella competitive exclusion product, Aviguard®) or permissive microbial community (chicken cecal contents from colonized birds containing 7.85 Log₁ₒ Salmonella genomes/gram) was assessed ex vivo using a S. typhimurium reporter strain with fluorescent YFP and CFP gene fusions to rrn and hilA operon, respectively. Second, cecal transcriptome analysis was used to assess the cecal communities’ response to Salmonella in chickens with low (≤5.85 Log₁ₒ genomes/g) or high (≥6.00 Log₁ₒ genomes/g) Salmonella colonization. The ex vivo experiment revealed a reduction in Salmonella growth and hilA expression following co-culture with the exclusive community. The exclusive community also repressed Salmonella’s SPI-1 virulence genes and LPS modification, while the anti-virulence/inflammatory gene avrA was upregulated. Salmonella transcriptome analysis revealed significant metabolic disparities in Salmonella grown with the two different communities. Propanediol utilization and vitamin B12 synthesis were central to Salmonella metabolism co-cultured with either community, and mutations in propanediol and vitamin B12 metabolism altered Salmonella growth in the exclusive community. There were significant differences in the cecal community’s stress response to Salmonella colonization. Cecal community transcripts indicated that antimicrobials were central to the type of stress response detected in the low Salmonella abundance community, suggesting antagonism involved in Salmonella exclusion. This study indicates complex community interactions that modulate Salmonella metabolism and pathogenic behavior and reduce growth through antagonism may be key to exclusion.
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    Pioneer colonizers: Bacteria that alter the chicken intestinal morphology and development of the microbiota
    Lee, Margie D.; Pedroso, Adriana A.; Lumpkins, Brett; Cho, Youngjae; Maurer, John J. (Frontiers, 2023-03)
    Microbes commonly administered to chickens facilitate development of a beneficial microbiome that improves gut function, feed conversion and reduces pathogen colonization. Competitive exclusion products, derived from the cecal contents of hens and shown to reduce Salmonella colonization in chicks, possess important pioneer-colonizing bacteria needed for proper intestinal development and animal growth. We hypothesized that inoculation of these pioneer-colonizing bacteria to day of hatch chicks would enhance the development of their intestinal anatomy and microbiome. A competitive exclusion product was administered to broiler chickens, in their drinking water, at day of hatch, and its impact on intestinal morphometrics, intestinal microbiome, and production parameters, was assessed relative to a control, no treatment group. 16S rRNA gene, terminal restriction fragment length polymorphism (T-RFLP) was used to assess ileal community composition. The competitive exclusion product, administered on day of hatch, increased villus height, villus height/width ratio and goblet cell production similar to 1.25-fold and expression of enterocyte sugar transporters 1.25 to 1.5-fold in chickens at 3 days of age, compared to the control group. As a next step, chicks were inoculated with a defined formulation, containing Bacteroidia and Clostridia representing pioneercolonizing bacteria of the two major bacterial phyla present in the competitive exclusion product. The defined formulation, containing both groups of bacteria, were shown, dependent on age, to improve villus height (jejunum: 1.14 to 1.46fold; ileum: 1.17-fold), goblet cell numbers (ileum 1.32 to 2.51-fold), and feed efficiency (1.18-fold, day 1) while decreasing Lactobacillus ileal abundance by onethird to half in birds at 16 and 42 days of age, respectively; compared to the phosphate buffered saline treatment group. Therefore, specific probiotic formulations containing pioneer colonizing species can provide benefits in intestinal development, feed efficiency and body weight gain.
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    The Role of the Salmonella spvB IncF Plasmid and Its Resident Entry Exclusion Gene traS on Plasmid Exclusion
    Oluwadare, Mopelola; Lee, Margie D.; Grim, Christopher J.; Lipp, Erin K.; Cheng, Ying; Maurer, John J. (2020-05-15)
    Salmonella enterica cause significant illnesses worldwide. There has been a marked increase in resistance to fluoroquinolones and beta-lactams/cephalosporins, antibiotics commonly used to treat salmonellosis. However, S. enterica serovars vary in their resistance to these and other antibiotics. The systemic virulence of some Salmonella serovars is due to a low copy number, IncF plasmid (65-100 kb) that contains the ADP-ribosylating toxin, SpvB. This virulence plasmid is present in only nine Salmonella serovars. It is possible that the spvB-virulence plasmid excludes other plasmids and may explain why antibiotic resistance is slow to develop in certain Salmonella serovars such as S. Enteritidis. The distribution of plasmid entry exclusion genes traS/traT and traY/excA are variable in Salmonella IncF and IncI plasmids, respectively and may account for differences in emergent antimicrobial resistance for some Salmonella serovars. The goal of this study is to determine the contribution of the Salmonella spvB-virulence plasmid in F-plasmid exclusion. From conjugation experiments, S. Typhimurium exhibited lower conjugation frequency with incFI and incFII plasmids when the spvB-virulence plasmid is present. Furthermore, introduction of cloned incFI traS into a "plasmidless" S. Typhimurium LT2 strain and Escherichia coli DH5 alpha excluded incFI plasmid. However, deletion of the virulence plasmid traS did not affect plasmid exclusion significantly compared to a spvB control deletion. In addition, differences in F plasmid conjugation in natural Salmonella isolates did not correlate with IncF or SpvB-virulence plasmid genotype. There appear to be other plasmid or chromosomal genes at play in plasmid exclusion that may be responsible for the slow development of antibiotic resistance in certain serovars.
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    Strength Lies in Diversity: How Community Diversity Limits Salmonella Abundance in the Chicken Intestine
    Pedroso, Adriana A.; Lee, Margie D.; Maurer, John J. (Frontiers, 2021-06-15)
    The transfer of the intestinal microbiota from adult to juvenile animals reduces Salmonella prevalence and abundance. The mechanism behind this exclusion is unknown, however, certain member species may exclude or promote pathogen colonization and Salmonella abundance in chickens correlates with intestinal community composition. In this study, newly hatched chicks were colonized with Salmonella Typhimurium and 16S rRNA libraries were generated from the cecal bacterial community at 21, 28, 35, and 42 days of age. Salmonella was quantified by real-time PCR. Operational taxonomic units (OTUs) were assigned, and taxonomic assignments were made, using the Ribosomal Database Project. Bacterial diversity was inversely proportional to the Salmonella abundance in the chicken cecum (p < 0.01). In addition, cecal communities with no detectable Salmonella (exclusive community) displayed an increase in the abundance of OTUs related to specific clostridial families (Ruminococcaceae, Eubacteriaceae, and Oscillospiraceae), genera (Faecalibacterium and Turicibacter) and member species (Ethanoligenens harbinense, Oscillibacter ruminantium, and Faecalibacterium prausnitzii). For cecal communities with high Salmonella abundance (permissive community), there was a positive correlation with the presence of unclassified Lachnospiraceae, clostridial genera Blautia and clostridial species Roseburia hominis, Eubacterium biforme, and Robinsoniella peoriensis. These findings strongly support the link between the intestinal bacterial species diversity and the presence of specific member species with Salmonella abundance in the chicken ceca. Exclusive bacterial species could prove effective as direct-fed microbials for reducing Salmonella in poultry while permissive species could be used to predict which birds will be super-shedders.