Browsing by Author "Maurer, John J."

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    Alternative Analytical and Experimental Procedures to Explore Rumen Fermentation as Driven by Nutrient Supplies
    Imaduwa Wickrama Acharige, Sathya Sujani (Virginia Tech, 2023-06-29)
    Ruminant livestock play a vital role in fulfilling the nutrient requirements of humans by providing protein, energy, and essential microminerals. With the increasing demand for meat and dairy products, the ruminant industry must continue to improve the productivity and efficiency of ruminant animals with limited resources while minimizing the environmental impact. Rumen fermentation is the focal point of the productivity and efficiency of the animal and numerous chemical, physical and biochemical interactions make the rumen a complex ecosystem. Therefore, improving the understanding of fermentation dynamics in a holistic manner and characterizing how fermentation varies in response to different nutrient supplies can greatly expand our knowledge on rumen fermentation to develop better engineered rumen manipulation strategies. The central aim of these investigations was to employ alternative analytical strategies for holistic exploration of complex relationships among rumen, animal, and dietary variables and to estimate rumen volatile fatty acid (VFA) dynamics under different nutrient supplies. The objective of the first study was to explore the strengths and limitations of mixed-model meta-analysis, recursive feature elimination (RFE), and additive Bayesian networking (ABN) in identifying relationships among diet, rumen, and milk performance variables. Both mixed-models and ABN agreed upon most of the variables and relationships identified while RFE failed to capture interactions. Given the capacity of mixed models for quantitative inquiry and the potential of ABN to illustrate complex associations in a more intuitive way, future investigations combining both approaches hold potential to explore intercorrelated data in a holistic manner. Followed by the successful use of ABN in the first study, the goal of a follow up study was to investigate the potential of two different network approaches to explore rumen level interactions using data generated in continuous culture experiments. Two network analysis approaches, EBIC-LASSO network (ELN) and Bayesian learning network (BLN) were leveraged to explore the relationships among rumen fermentation parameters in continuous culture experiments. Unidirectional ELN illustrated prominent variables while BLN, which produces a directed acyclic graph, identified directional relationships implying causality. Overall, both networking approaches demonstrate strengths in capturing connectedness and directionality of rumen fermentation variables. In a complementary line of work, the next experiment focused on developing an alternative method for iso-tope based assessments to produce less expensive, and more efficient screening of fermentation conditions driven by diet. Cannulated wethers were used in this study and 4 dietary treatments combining lowly and highly degradable fiber (timothy hay and beet pulp, respectively) and protein (heat-treated soybean meal and soybean meal, respectively) were tested. Results indicated that fluid volume of the rumen and the rate of passage were influenced by protein, but not fiber, source. Higher rumen volumes and lower passage rates were associated with heat-treated soybean meals. The effect of dietary treatments on VFA absorption dynamics was prominent compared to the minimal changes in production dynamics. Overall, heat-treated soybean meal appears to influence VFA disappearance resulting in low concentrations within the rumen, but greater flux of VFA disappearance. In conclusion, this method demonstrated the capacity to estimate VFA dynamics beyond concentrations and molar proportions while being cost effective and more physiologically relevant. In a fourth study, we sought to investigate the growth performance and rumen VFA profile in response to different planes of nutrients and naturally occurring coccidiosis. Coccidiosis infection altered rumen isobutyrate concentrations and tended to alter major VFA concentrations suggesting the need of future work to explore coccidiosis effects on rumen fermentation. The first two investigations highlighted the potential and strength of leveraging alternative analytical tools to complement statistical approaches generally used in ruminant nutrition while concurrently improving ability to explain complex associations in the rumen. The third and fourth projects characterized the rumen VFA dynamics and profile in response to the different nutrient degradability and health status, respectively. Collectively, these investigations contribute to better understanding of rumen dynamics through novel analytical and experimental approaches.
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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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    Change in the Structure of Soil Microbial Communities in Response to Waste Amendments
    Buckley, Elan (Virginia Tech, 2020)
    Soil microbial communities are affected extensively by addition of amendments to their environment. Of particular concern is the addition of poultry litter, which contains a substantial C, energy, and nutrient supply, but also antibiotic resistance genes (ARG), antimicrobials, and a multitude of microbial species. This project seeks to primarily assess if there is a change in bacterial community structure in response to poultry litter amendments to pasture land across geographically independent land across northern Georgia. It may be that changes in the relative abundance of bacterial communities also result in alteration in ARGs, and the community resistance to antibiotics (“resistome”) which in turn increases the potential threat of antibiotic resistance genes. While another part of this study will determine changes in integrons and specific ARGs, this project will focus on changes in bacterial communities and the potential functional changes in the community, which in turn have consequences for ARG levels and its horizontal transfer to various members of the soil community. Addition of waste from livestock is a historical method for increasing nutrients needed in the soil for the cultivation of crops, and in turn causes pronounced shifts in soil microbial communities due to the addition of large amounts of carbon, nutrients, foreign microbes, and other material. This study is unique because it utilizes a novel and relatively large landscape-scale to determine if there are discernable and repeatable patterns of bacterial community structure change in response to amendment regardless of exact soil type or source of chicken litter amendment. In the future, these data can also provide insight into the changes in the relative abundance antibiotic related genes associated with community change.
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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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    Free-Living Aquatic Turtles as Sentinels of Salmonella spp. for Water Bodies
    Hernandez, Sonia M.; Maurer, John J.; Yabsley, Michael J.; Peters, Valerie E.; Presotto, Andrea; Murray, Maureen H.; Curry, Shannon; Sanchez, Susan; Gerner-Smidt, Peter; Hise, Kelley; Huang, Joyce; Johnson, Kasey; Kwan, Tiffany; Lipp, Erin K. (Frontiers, 2021-07-22)
    Reptile-associated human salmonellosis cases have increased recently in the United States. It is not uncommon to find healthy chelonians shedding Salmonella enterica. The rate and frequency of bacterial shedding are not fully understood, and most studies have focused on captive vs. free-living chelonians and often in relation to an outbreak. Their ecology and significance as sentinels are important to understanding Salmonella transmission. In 2012–2013, Salmonella prevalence was determined for free-living aquatic turtles in man-made ponds in Clarke and Oconee Counties, in northern Georgia (USA) and the correlation between species, basking ecology, demographics (age/sex), season, or landcover with prevalence was assessed. The genetic relatedness between turtle and archived, human isolates, as well as, other archived animal and water isolates reported from this study area was examined. Salmonella was isolated from 45 of 194 turtles (23.2%, range 14–100%) across six species. Prevalence was higher in juveniles (36%) than adults (20%), higher in females (33%) than males (18%), and higher in bottom-dwelling species (31%; common and loggerhead musk turtles, common snapping turtles) than basking species (15%; sliders, painted turtles). Salmonella prevalence decreased as forest cover, canopy cover, and distance from roads increased. Prevalence was also higher in low-density, residential areas that have 20–49% impervious surface. A total of 9 different serovars of two subspecies were isolated including 3 S. enterica subsp. arizonae and 44 S. enterica subsp. enterica (two turtles had two serotypes isolated from each). Among the S. enterica serovars, Montevideo (n = 13) and Rubislaw (n = 11) were predominant. Salmonella serovars Muenchen, Newport, Mississippi, Inverness, Brazil, and Paratyphi B. var L(+) tartrate positive (Java) were also isolated. Importantly, 85% of the turtle isolates matched pulsed-field gel electrophoresis patterns of human isolates, including those reported from Georgia. Collectively, these results suggest that turtles accumulate Salmonella present in water bodies, and they may be effective sentinels of environmental contamination. Ultimately, the Salmonella prevalence rates in wild aquatic turtles, especially those strains shared with humans, highlight a significant public health concern.
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    The Influence of Necrotic Enteritis, Environmental Factors, and Genetics on Intestinal Development Pathways and Disease Occurrence in Broiler Chickens
    Kinstler, Sydney Regan (Virginia Tech, 2023-08-03)
    Intensified poultry production to meet global food demands has faced challenges associated with the removal of in-feed antibiotics due to concerns over antibiotics resistance. The reduction of low-dose antibiotics in feed has allowed for reemergence of intestinal diseases that diminish animal welfare and producer economics. Alternative mechanisms to preventing disease are therefore required. The objective of this dissertation was to examine factors that contribute to chicken development and health including intestinal structure and function, environment, and genetic selection. Chapter 2 investigated the host response to infection of the parasite Eimeria maxima that predisposes chickens to a bacterial infection Clostridium perfringens. Intestinal structure, function, inflammatory response, and epithelial composition was examined during a mild subclinical infection. Analysis of E. maxima and C. perfringens as individual infections revealed how each pathogen contributes to a co-infection. E. maxima caused a more severe inflammatory response, increasing pathology scores, shortening intestinal villi, and elongating crypts in the jejunum at peak infection. C. perfringens was shown to manipulate intestinal epithelial composition by influencing stem cells to differentiate into secretory goblet cells. The most deleterious effects were observed when the pathogens were introduced together, increasing pathology scores further, damaging intestinal villi, and increasing crypt depth. The introduction of C. perfringens and E. maxima also increased signaling for the production of reactive oxygen species, stimulation of tumor necrosis factor- that is involved in innate immunity, and decreased transcription of Hes1, which is involved in Notch signaling towards absorptive cell differentiation. Hes1 has previously been shown to be involved in the inflammatory response and could be an area of interest in determining new treatments to prevent or relieve the effects of E. maxima and C. perfringens. Chapter 3 applied an environmental perspective to disease prevention and examined the properties of C. perfringens that allow it to persist in the poultry house environment. Spores resist treatments used to sanitize poultry houses and litter has been shown to be a reservoir for disease, potentially increasing occurrence in certain houses. The metabolic and physiological properties of C. perfringens were utilized to separate the microbe from other poultry litter bacteria to enumerate spores within houses. A selective and differential medium combined with a heat treatment was developed to isolate C. perfringens spores from poultry litter samples. On average, houses that had histories of necrotic enteritis harbored a greater abundance of C. perfringens spores. Colonies that were isolated on the specialized medium were confirmed using PCR as C. perfringens. Lastly, Chapter 4 examined how genetic selection for multiple traits has influenced early intestinal development compared to divergently selected lines based on eight-week body weight. This study showed the morphological and gene expression differences between lines and revealed that most pathways involved in intestinal development are conserved through genetic selection. The major differences between lines were an increase in peptide transporter PepT1 on d5 and d7 in chicks selected for low eight-week body weight (LWS) compared to high weight selected (HWS) chicks and modern broiler Cobb500 chicks. In HWS chicks, the opposite mechanism was observed with an increase in expression of secretory goblet cell marker Muc2. The findings of these studies give multiple perspectives into poultry production and how major factors in management including nutrition, environment, and genetics can be used to increase efficiency while preventing disease.
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    Outlining a balance-point model of homeostasis in the small intestine of broiler chickens
    Cloft, Sara E. (Virginia Tech, 2022-07-01)
    Since the removal of in feed antibiotics in the past few years commercial poultry production is especially sensitive to the health of the small intestine. Healthy small intestines balance nutrient absorption and defensive barrier functions to ensure the chicken is able to meet the whole-body nutritional needs and is able to help prevent internalization of pathogens or potentially toxic components. This balance can only be maintained under stable conditions. When a disturbance event occurs the intestine imbalances until a new, and less efficient, balance can be achieved. The objective of this dissertation is to propose a novel model to understanding intestinal homeostasis in the face of various disturbance events. Chapter 2 investigated the effects of Runting Stunting Syndrome on broiler chickens in four different groups of chicks displaying clinical symptoms. The major finding in this study was that in two of the four groups the expression of stem cell gene Olfactomedin 4 was absent from the crypt though other functional genes were found to still be expressed there. Chapter 3 characterized intestinal gene expression following a single challenge of Eimeria acervulina in broiler chickens. During Eimeria infection gene expression of multiple host defense peptide genes were decreased compared to uninfected chickens. Further, Eimeria infected chickens increased cell proliferation within the crypt and post-peak infection showed signs of intestinal recovery. Additionally, chapter 3 developed a novel method for visualizing Eimeria as it infects the intestine. In chapters 4 and 5 cell type population changes during the peri-hatch intestinal maturation process were evaluated. Peri-hatch intestinal maturation is critical for the successful transition from embryonic to post-hatch life. Chapter 4 profiled changes in proliferative cells and gene expression of various stem cell marker genes during the peri-hatch period: the last three days of embryogenesis and the first week post-hatch. The stem cell marker gene Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) decreased during the post-hatch period while Olfactomedin 4 increased post-hatch. Both stem cell genes were expressed within the intestinal crypt, though prior to hatch Lgr5 was expressed in the lamina propria and villi as well. Additionally, the marker of proliferation Ki67 gene was expressed in cells throughout the intestine prior to hatch but became restricted to the crypts and along the center of the villi. Chapter 5 assessed the effect of providing probiotics to late term embryos via in ovo feeding (IOF). The effects of IOF were primarily observed on embryonic day 20 (e20), roughly 48 hours after IOF. On e20 the embryos in ovo fed probiotics in saline had increased expression in the ileum of Peptide Transporter 1 (PepT1) a marker gene for enterocytes and Mucin-2 (Muc2) a marker gene for goblet cells compared to non-injected control embryos. Also, on e20 the embryos in ovo fed saline only had numerically increased PepT1 and Muc2 compared to non-injected control embryos. The difference in responses between the probiotic and saline fed embryos on e20 suggests different routes of stimulation. These investigations illustrate various possible scenarios and means of investigating intestinal homeostasis during disturbance events.
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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 Poultry Litter Resistome in the Transmission of Antimicrobial Resistance to Salmonella
    Oxendine, Aaron Casey (Virginia Tech, 2021-05-17)
    There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï ­g/ml), chloramphenicol (Cm; 25 ï ­g/ml), streptomycin (Sm; 100 ï ­g/ml), and tetracycline (Tc; 25 ï ­g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter.
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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.