Progress 03/01/08 to 11/30/12
Outputs
OUTPUTS: The objective of this study was to investigate the effects of supplemental dietary antibiotics (bacitracin) and litter conditions (fresh vs. reused) on the abundance of Clostridium perfringens, Salmonella spp. and Campylobacter spp. in commercial broiler chickens. These species are food-borne enteric pathogens commonly associated with poultry. Specific quantitative PCR (qPCR) assays were used to quantify C. perfringens, virulent C. perfringens that carried the genes encoding alpha-toxin (cpa) and NetB-toxin (netB), Salmonella, and Campylobacter in samples of ileal mucosa, cecal content, and litter. A strong positive correlation was found between the abundance of all three measurements of C. perfringens. The abundance of Salmonella spp. and C. perfringens was also shown to be correlated. The objective of a second study were to describe the bacterial composition of the poultry intestinal microbiome. We conducted a barcoded pyrosequencing study of 16S ribosomal RNA genes (rDNAs) investigating the phylogenetic diversity of bacteria presenting in the cecal contents and ileal mucosa of commercial broiler chickens . The V3 region of the 16S rDNAs was amplified from samples collected from the ileal mucosa and cecal content and sequenced by the 454 pyrosequencing. The 338177 sequences represented on average 3401 and 125 operational taxonomy units (OTUs) in chicken cecal content and ileal mucosa, respectively. Noticeable variation was observed for the number of OTUs revealed by different sequencing runs and data processing pipelines. Comparison studies suggested each microbiome was distinct from others, however, microbiomes from the same gut origin shared greater community similarity then microbiomes from different gut origins within the same species. PARTICIPANTS: Drs. Lilburn and Yu were the prinicipal investigators on the project. Dr. Lilburn, through his familiarity with the commercial poultry industry and production associated "enteric" diseases conceived the ideas behind the project. Dr. Yu is a molecular microbiologist with the appropriate expertise needed to apply both existing molecular techniques and to develop new tools needed to understand the complexity of the avian intestinal microbiome. Dr. Taylor was an additional co-PI whose statistical expertise was utilized in the development of appropriate statistical models needed to analyze the data generated by the project. Over the course of the grant and after discussions with other scientists, we were able to utilize the unique "litter environment" and dietary treatments (+/- dietary antibiotics)to generate litter and intestinal materials that were used by other scientists to expand the scope of the project. These experiments were conducted by the Rajashekara and Selvaraj labs, respectively. The data from the completed studies contributed to one MS thesis (M.Cressman) and one PhD dissertation (S. Wei). TARGET AUDIENCES: The target audience for the research reported herein are poultry production are primarily those individuals involved in management decisions (new litter, reused litter)and veterinarians who need to balance the health and welfare benefits associated with supplemental antibiotics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Campylobacter was not detected in any of the samples collected. Neither supplemental bacitracin or litter condition influenced the abundance of Salmonella. Generic C. perfringens was only detected in the ileal mucosa at very low levels at 10 days of age and was much higher at 35 days. Chicks placed on reused litter tended to have a lower abundance of generic C. perfringens compared with those on fresh litter. In the ileal mucosa, neither cpa nor netB was detected at 10 days of age but were detected at 35 days in the when no supplemental bacitracin was fed. The inclusion of dietary bacitracin reduced the abundance of generic C. perfringens as well as the cpa and netB genes in the ileal mucosa, cecal content, and litter. This is the first study that examined the effect of dietary bacitracin and litter status on the prevalence of these three common enteric pathogens. Litter reuse does not appear to increase the risk of necrotic enteritis or Salmonella infection unless there is contamination from a previous flock and the litter is not windrowed or otherwise managed prior to a subsequent placement of chicks. The sequences generated from the pyrosequencing study suggested that within an anatomical region of the gut, there was significant coverage (> 95%) of the bacterial diversity in the poultry intestinal microbiome. The pyrosequencing study recovered 39 newly identified genera in chicken cecal microbiome, including Butyricimonas, Odoribacter, Hydrogenoanaerobacterium, Moryella, Parasporobacterium, and Ruminococcus when compared with the species identified in public databases.
Publications
- Effect of litter conditions on broiler chicken intestinal microbiome as revealed by a poultry intestinal tract chip (PITChip). S. Wei,* M. Cressman, M. Lilburn, and Z. Yu, 2012. The Ohio State University, Columbus. Poultry Science Association Annual Meeting.
- Prevalence and abundance of Clostridium perfringens in chickens as affected by Bacitracin and litter management. S. Wei,* A. Gutek, M. Lilburn, and Z. Yu, 2012. The Ohio State University, Columbus. Poultry Science Association Annual Meeting.
- Shanmugasundaram, R., M.S. Lilburn, and R.K. Selvaraj, 2012. Effect of recycled litter on immune cells in the cecal tonsils of chickens. Poultry Sci. 91:95-100.
- Wei, S., M.S. Lilburn and Z.T. Yu, 2012. Barcoded 16S rDNA V3 Pyrosequencing Amplicons Depicted the intestinal Microbiomes of Chicken and Turkey. (Submitted to FEMS Microbiology).
- Wei, S., A. Gutek, M.S. Lilburn, and Z.T. Yu, 2012. Abundance of pathogens in the gut and litter of broiler chickens as affected by bacitracin and litter management. (Submitted to Vet Immunol Immunopath).
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: The overreaching goal of this project has been to characterize some of the physiologic outcomes occurring within the intestinal environment of broiler chickens due to rearing environment, in this case the bedding material. It is very common within the broiler industry to reutilize broiler litter (bedding)for multiple flocks. In the experiment reported herein, we compared different aspects of the gut immune response in broiler chicks that were raised on fresh pine shavings or litter/shavings that had been used for two previous flocks. Half the chicks within each litter treatment (fresh, recycled)were fed diets with subtherapeutic antibiotic supplementation (with, without). There were four replicate pens per litter/antibiotic combination. At 10 and 35 d of age, the cecal tonsils were analyzed for intestinal immune measurements. The cecal tonsils of birds reared on recycled litter had increased IL-1 mRNA (P < 0.01) and a lower percentage of CD4+CD25+ cells at 10 and 35 d of age when compared with those of chicks reared on fresh litter. Birds fed diets supplemented with bacitracin had a reduction in CD4+ cells (P = 0.01) at 10 d of age when compared with that of chicks that were not fed the antibiotic. The combination of bacitracin supplementation and fresh litter resulted in an approximate 10-fold increase in IL-10 mRNA (P = 0.01) at 10 d of age compared with unsupplemented chicks reared on fresh litter. Within the unsupplemented treatment, the chicks reared on recycled litter had 25-fold (P = 0.01) and 39-fold (P = 0.02) increased in IL-4 mRNA levels at 10 and 35 d of age, respectively, when compared with chicks reared on fresh litter. The results suggest that in chicks reared on recycled litter, the intestinal immune response skewed toward an inflammatory response whereas fresh litter supports an anti-inflammatory intestinal immune response. There was no significant interaction between subtherapeutic antibiotic supplementation and the litter environment in terms of alterring the IL-1 mRNA levels in cecal tonsils. This suggests that there is a minimal effect of the antibiotic with respect to alleviating the inflammatory response induced by recycled litter. PARTICIPANTS: The PI, M. Lilburn, The Ohio State University, Department of Animal Science and Z. T. Yu, The Department of Animal Sciences, R. J. Taylor, The Ohio State University, Department of Entomology collaborated on this project. Training opportunities were provided to undergraduate interns and the data generated in this report was part of a summer undergraduate research internship project for the first author, A.H. Loudon. TARGET AUDIENCES: The target audience is the commercial broiler industry and those individuals, primarily veterinarians, that are responsible for both overall avian health but also food safety initiatives within their respective companies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The litter and antibiotic treatments utilized in the current experiment are common practices utilized by the broiler segment of the poultry industry. The practice of recycling litter is primarily done as a cost effective practice but it has also been reported to facilitate the development of a mature microbiome in young chicks. The practice of incorporating subtherapeutic dietary antibiotics is primarily done to enhance feed utilization but also to control the proliferation of select classes of intestinal microbes (i.e. Clostridia) can potentiate necrotic enteritis, a generic term for nonspecific intestinal inflammation and necrosis. The intestine is the largest immune tissue in the body in terms of total immune cells so it is important to understand the response of select aspects of the intestinal immune response to common industry practices that are known to influence the development of the intestinal microbiome. There are numerous cytokines (local cellular responses)that are known to reflect the intestinal immune response to its environment and that is what we determined in this study. The results suggest that at both younger and older ages, the microbial environment of recycled litter elicits a strong immune intestinal response in broiler chicks. The response observed in the young chicks is espcially important because the first two weeks is a critical period in intestinal development with respect to digestive maturation and the challenges resulting from native or induced (vaccination) coccidial oocyst shedding. The fact that the antibiotic treatment was without effect in alleviating the inflammatory response to recycled litter simply means that the particular antibiotic used (bacitracin) was ineffective against the particular microbes present in the litter that were contributing to the inflammatory response.
Publications
- Loudon, A.H., R. Shanmugasundaram, M.S. Lilburn and R.K.Selvaraj, 2011. Intestinal physiology and regulatory T cell response to immediate or delayed access to feed and water in Pekin ducklings. Poultry Sci 90:2041-2046.
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Progress 03/01/10 to 02/28/11
Outputs
OUTPUTS: The first data generated from this proposal was summarized in the past year. Chicks were reared on fresh pine shavings (litter) or litter that had accumulated for over a year (multiple flocks). Litter, intestinal mucosa, and cecal content samples were collected at multiple ages during the growing period. The microbial profiles of the litter and mucosa were characterized using PCR and denaturing gradient gel electrophoresis (DDGE). Fresh litter was characterized by a high proportion of environmental bacteria whereas the profile in the reused litter reflected a high proportion of intestinal bacteria. The intestinal bacteria from the chicks reared on fresh litter was predominantly Lactobaccillus spp. versus Clostridiales in chicks reared on reused litter. The influence of fresh litter on intestinal microbial profiles decreased with age of the chicks. PARTICIPANTS: The PI, M. Lilburn, The Ohio State University, Department of Animal Science and Z. T. Yu, The Department of Animal Sciences, R. J. Taylor, The Ohio State University, Department of Entomology collaborated on this project. Training opportunities were provided to undergraduate interns. TARGET AUDIENCES: The target audience for this project are poultry health professionals. This research would also be of interest to those involved in rearing broilers without the use of subtherapeutic antibiotics in the diet which would marginally negate the environmental effects reported herein. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Enhancing the protection and safety of the agriculture and food supply through Animal Management Systems is of national interest. This research is among the first studies that addresses the relationship between the bacterial profile in the rearing environment of broiler chicks and the subsequent bacterial profile in the intestine and ceca of chicks during growth. The ceca is the tissue/organ most commonly sampled with respect to profiling the microbiota populations in poultry because of its expansive bacterial population and its terminal location near the colon. The data reported herein suggest that the cecal microbiota are less affected by the environmental bacterial profiles (fresh, reused litter) than the intestinal mucosa populations. The data also suggests that with age, there is a merging in the intestinal microbiota patterns in chicks raised in the two environments.
Publications
- Cressman, M.D., Z.Yu, M.C. Nelson, S.J. Moeller, M.S. Lilburn, and H.N. Zerby, (2010). Appl. Environ. Micro. 76:6572-6582. Interrelations between the microbiotas in the litter and in the intestines of commercial broiler chickens.
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Progress 03/01/09 to 02/28/10
Outputs
OUTPUTS: Sample preparation and DNA extraction: From the first experiment which comprised six consecutive flocks reared on either clean litter (each flock) or reused litter, 288 samples were collected for mucosal, digesta, and litter. These samples were pooled after careful mixing based on samples types for each age (day 10 and 35) within each growth cycle. 18 samples resulted from each age within each growth cycles. Community DNA was extracted from each of these composite samples. The quality of the DNA extract was assessed using agarose gel electrophoresis. For some of the samples, the DNA yield was too low to be further analyzed. The DNA extraction was repeated for those samples. Adequate community DNA was obtained eventually for all the samples. PCR and DGGE analysis:All the samples were subjected to community fingerprinting analysis using both polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Both PCR and DGGE have been done for all the composite samples. Some of the samples need to be repeated for DGGE analysis. After the repetition of the DGGE, the DGGE banding profiles will be converted to a binary matrix and subjected to statistical analysis (principal component analysis or non-metric multidimensional scaling) to infer relationship between litter status and intestinal microbiota in chickens. These samples will be subjected to analysis for pathogen load (i.e., C. perfringens, Salmonella, C. jejuni, and C. septicum) to examine the effect of litter status on bird health. After analysis of DGGE fingerprinting of all the samples, select samples will be chosen for microarray or pyrosequencing analysis to gain detailed information on the reciprocal effect of litter and intestinal microbiota. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: This project is of interest to numerous segments of the poultry industry. Poultry veterinarians are interested in the microbiology of the intestinal tract because of the prevalence of intestinal bacterial infections (enteritis) that can predispose birds to secondary bacterial challenges that can become systemic. A second target audience are poultry managers who are increasingly concerned with the environmental aspects of poultry production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The procedures utilized after the samples were collected were sufficient to generate the DDGE fingerprinting data that can now be utilized for more detailed microbiotia analysis. This will allow us to accurately determine the impact of differences in the environment (clean versus reuses litter) on the microbiome in young versus older broilers.
Publications
- No publications reported this period
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Progress 03/01/08 to 02/28/09
Outputs
OUTPUTS: This project did not begin until the fall of 2008 due to budget negotiations between Ohio State and USDA. From the fall of 2008 until the fall of 2009, 6 flocks of broilers were grown in pens with pine shavings for litter. At the end of each flock, the pen was either completely cleaned and rebedded with clean litter or the existing litter was piled in the center of the pen for two weeks prior to being redistributed for reuse. At the end of each flock, the litter in each pen was quantitatively weighed and approximately two pounds of a pooled litter sample from each pen was put into plastic bags and frozen. An equal quantity (two pounds) of clean litter was replaced in each pen. In each successive flock, samples of mucosa from the small intestine and cecal contents were collected from 3 birds per pen at 10 and 35 days of age for intestinal microbial community analysis. PARTICIPANTS: Michael Lilburn is the PI on the grant and designed the experimental protocol and organized the data collection. Zhontang Yu (Co-PI) facilitated the microbial DNA extraction and microbial community analysis in his lab. John Anderson, a research associate in my lab, did the actual intestinal and cecal sample collection and organized the collection and sampling of litter. TARGET AUDIENCES: This project is of interest to numerous segments of the poultry industry. Poultry veterinarians are interested in the microbiology of the intestinal tract because of the prevalence of intestinal bacterial infections (enteritis) that can predispose birds to secondary bacterial challenges that can become systemic. A second target audience are poultry managers who are increasingly concerned with the environmental aspects of poultry production. The use of litter over many flocks will result in a concentration of nitrogen and phosphorus within the litter and the potential for the volatilization of a percentage of the nitrogen as ammonia. A time course analysis of the components of litter via reuse may allow for the development of strategies to minimize nitrogen volatization prior to the litter being removed and applied to fields. PROJECT MODIFICATIONS: The quantification of litter build up over the course of the six flocks was not in the original proposal. It was added to the protocol because of the realization that litter reuse is a standard industry practive but there is not actual data on how it changes over time with respect to nutrient accumulation.
Impacts
The intestina microbial analysis is still to be completed. The weight of the litter in each pen increased approximately 90 lbs after each of the first three flocks (Start = 30 lbs fresh litter; Flock 1, 127 lbs; Flock 2, 213 lbs; Flock 3, 302 lbs). The absolute increase in litter weight for the next three flocks was less (Flock 4, 346 lbs; Flock 5, 385 lbs; Flock 6, 454 lbs). These represent the mean values of 8 replicate pens. The dry matter percentage and total litter phosphorus and nitrogen are still to be determined.
Publications
- No publications reported this period
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