Progress 10/01/14 to 09/30/18
Outputs
Target Audience:This is a basic research project aimed at improving immunity, health and performance of piglets, without use of antibiotics. The target audiences are researchers with interest in pig health and pig producers. Pork production is a vital to the US and global economies. Maintaining healthy hogs and increasing production is important. Weaning piglets early is a common practice aimed at increasing production, but has negative consequences because the digestive and immune systems are not mature and passive immune protection is removed. Thus, early weaned piglets are susceptible to gastrointestinal issues and infections. One such condition, post-weaning diarrhea (PWD), is caused by gut colonization by pathogenic bacteria and leads to growth inhibition and death of piglets. Including low levels of antibiotics in the feed or piglets was used to prevent PWD. Because of the link between overuse of antibiotics in animal production and the rise in antibiotic resistant bacterial infections there are new restrictions on antibiotics. Therefore, natural ways to promote immunity and healthy guts is essential. One way to promote health and immunity is through positive gut bacteria. Segmented filamentous bacteria (SFB) bacteria promote mucosal immunity and help block growth of pathogenic bacteria. The goal this project is to investigate SFB colonization in the intestine of weaned piglets and determine whether it positively influences development of intestinal immunity and incidence PWD. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In total there have been 5 graduate students and 6 undergraduate students participating on this research project. During the reporting year, this project provided direct support to three undergraduates, two graduate students (MS), and one High School student. Three other Undergraduate students working on this project were supported via a different training mechanism. Students gained animal handling, necropsy, immunology/molecular biology, microbiology and other general and widely applicable laboratory skills as a part of this project. All students are involved in assisting with optimizing the protocols that are being used to address the experimental goals of the project and are responsible for presenting their results to the lab as well as train others in the technique. The work was also presented in professional conferences by the PI and the students. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?This project ended Sept 2018.
Impacts
What was accomplished under these goals?
For this reporting period Oct. 2017- Sept 2018 while we were still working to optimize two other protocols to detect SFB, RT PCR and Fluorescent in situ Hybridization (FISH) We made significant progress on Specific Aim Progress on Specific Aim 2 (Investigate the production of IgA and Th17 associated cytokines in piglet intestine of SFB+ and SFB - piglets). For this aim, we analyzed fecal samples at various ages, (birth, weaning and post-weaning) for the presence of IgA and IL-17 by ELISA. These analyses showed that IgA expression as expected is highest in young piglets due to passive immunity through milk consumption and decreases after weaning due to cessation of milk consumption. After weaning we observed an increase in IgA production by the piglet and that the piglets that expressed SFB had the highest levels of IgA. ELISA evaluation of IL-17 levels was consistent with these data, in that higher levels of IL-17 were detected in the fecal samples SFB+ animals as compared to SFB-. Unfortunately, we have too few piglets that are SFB+ to be able perform statistical analysis on these data. It is difficult to make any solid conclusions from this data however the trend is in keeping with our hypothesis and what has been reported in the literature. We were unsuccessful in getting usable data to evaluate the levels of IL-17 and other Th17 related cytokines in the intestinal tissues by q-RT-PCR as planned, these assays needs to be done later. We were also unable to make headway on Specific Aim 3 (Compare incidences of PWD in SFB+ and SFB- piglets). So far the data we have does not indicate a significant difference in the incidence of PWD in SFB+ and SFB- negative animals, but the numbers of animals we have are low and therefore we cannot make definitive conclusions.
Publications
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:This is a basic research project aimed at improving immunity, health and performance of piglets, without use of antibiotics. The target audiences are researchers with interest in pig health and pig producers. Pork production is vital to the U.S. and global economies. Maintaining healthy hogs and increasing production is important. Weaning piglets early is a common practice aimed at increasing production, but has negative consequences because the digestive and immune systems are not mature and passive immune protection is removed. Thus, early weaned piglets are susceptible to gastrointestinal issues and infections. One such condition, post-weaning diarrhea (PWD), is caused by gut colonization by pathogenic bacteria and leads to growth inhibition and death of piglets. Adding low levels of antibiotics in the feed or piglets was used to prevent PWD. Because of the link between overuse of antibiotics in animal production and the rise in antibiotic resistant bacterial infections, there are new restrictions on antibiotics. Therefore, natural ways to promote immunity and healthy guts is essential. One way to promote health and immunity is through positive gut bacteria. Segmented filamentous bacteria (SFB) bacteria promote mucosal immunity and help block growth of pathogenic bacteria. The goal of this project is to investigate SFB colonization in the intestine of weaned piglets and determine whether it positively influences development of intestinal immunity and incidence of PWD. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the reporting year, this project provided direct support to two undergraduates and two masters' students. Four other undergraduates working on this project were supported via a different training mechanism. Students are gaining laboratory skills in animal handling, necropsy, immunology/molecular biology, microbiology and other general and widely applicable experience as a part of this project. All students are involved in assisting with optimizing the protocols used to address the experiment goals of the project and are responsible for presenting their results to the lab as well as to train others in the technique. Several of the students have traveled with the PI to student centered conferences to present their work. This project supported travel for the PI and one of the MS students to receive training on how to isolate lymphocytes from intestine and Peyer's patches in the laboratory of Mark R. Boothby, M.D. and Ph. D, in the Department of Microbiology and Immunology at Vanderbilt University Medical Center. The PI was also able to update skills by attending a Flow cytometrytraining from the Flow Cytometry Core at UNC Chapel Hill. The PI and several students (4 UG and 1 MS students) attended and presented research at the 2017 MANRRS National Conference. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?During the next reporting year (extension) we will focus on the following: 1) Continue optimizing the Fluorescent in situ Hybridization FISH protocols to detect SFB. 2) Optimize qPCR analysis of samples for SFB and PWD causing bacteria. 3) continue to assess the incidence of PWD by scoring the fecal samples of the piglets that are SFB+ and SFB-. 4) Establish protocols and train students in the isolation and stimulation of intraepithelial lymphocytes (IEL) and Peyer's Patch lymphocytes. If we have the time, money and personnel we would also like to begin to optimize a technique called flow FISH and evaluate samples with this technique. Using this technique will allow us to use flow cytometry instead of florescent microscopy to detect SFB and would be a great tool to quantitate the level of SFB and determine its size and viability in the fecal sample. No one has reported this novel approach. Finally, we would also like to begin working on establishing the protocols for freezing the white blood cells of pigs and culturing SFB in the lab using the published papers that were successful in doing this. This would be a great tool for asking questions about the immune response to SFB using in vitro models and would be helpful for the grant submission.
Impacts
What was accomplished under these goals?
For this reporting period, Oct. 2016- Sept 2017, we have accomplished the following: made significant progress on Specific Aim 1, to determine when SFB begins to colonize the gut of young piglets. For this aim we; 1) collected and analyzed fecal samples and intestinal tissues from piglets at various ages, (birth, weaning and post-weaning), 2) submitted fixed intestinal tissues for histological processing and began analysis, and 3) began analysis of fecal samples via staining, PCR and Fluorescence In Situ Hybridization (FISH). As a control, many samples were sent to IDEXX, a company that tests samples for the presence of SFB. Following our submission, the company confirmed that our protocols were working properly and that we are, indeed, detecting SFB in our samples. It was verified that many of the samples for which we obtained positive results were also determined to be positive by IDEXX. We have concluded from our analysis thus far that PCR analysis of fecal samples detected SFB in piglets of all ages, not just after weaning. However, PCR analysis of intestinal tissues suggested that SFB is primarily present after weaning, which is more consistent with expectations and the literature. Therefore, we hold that to determine colonization of SFB testing of intestinal tissues is more accurate than fecal sampling. We are still working to optimize two other protocols to detect SFB, RT PCR and Fluorescent in situ Hybridization (FISH). Progress on Specific Aim 2 (Investigate the production of IgA and Th17 associated cytokines in piglet intestine of SFB+ and SFB - piglets) is ongoing. We have detected IgA in the fecal samples of piglets and determined that the IgA expression is high in young piglets and decreases after weaning and then begins to increase. This finding is consistent with what is known and is encouraging. We are now in the process of evaluating the level of IL-17 protein and mRNA in the fecal and intestinal tissues, respectively. It will be difficult to make any conclusions from this data however, because we do not have a high number of SFB+ and SFB- animals to conduct statistical analysis. This fact also complicates specific aim 3 (Compare incidences of PWD in SFB+ and SFB- piglets). So far, the data we have does not indicate a significant difference in the incidence of PWD in SFB+ and SFB- negative animals, but the numbers of animals we have are low and therefore we cannot make definitive conclusions. We will continue to work toward increasing the numbers and hope to collect more samples this summer.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audiences for this project are researchers with interest in pig health directly and pig producers indirectly. Pork production is a vital to the US and global economies. Maintaining healthy hogs and increasing production is important to the pork industry and consumers. Weaning piglets early is a practice that helps to increase pork production, but is challenging because the digestive and immune systems are not fully mature. Therefore, piglets are more susceptible to gastrointestinal (GI) issues such as post-weaning diarrhea (PWD). PWD leads to anorexia, growth inhibition and death of piglets. To promote the health of weaned pigs and protect against PWD, it is important that the gut be colonized by positive bacteria and that strong intestinal immunity develops. Segmented filamentous bacteria (SFB) are non-pathogenic bacteria that promote the maturation of mucosal immunity in animals and help block colonization of the gut by pathogenic bacteria. The goal this project is to investigate SFB colonization in the intestine of weaned piglets and determine whether SFB colonization positively influences development of intestinal immunity and incidence of post-weaning diarrhea. This project will provide basic scientific evidence that could be used in future research aimed at improving mucosal immunity and health and performance of weaned piglets, ultimately helping the industry provide safe and nutritious pork products for consumers, without use of antibiotics. Changes/Problems:Although staff have made gains this last year we are behind schedule because over 100 samples were lost when a freezer failed. These samples were collected the first summer (2015, YR1) of the project and therefore there were few samples to evaluate in the subsequent fall and spring. The freezer has been fixed allowing us to collect more samples during the second summer (2016 YR2) of the project. In addition, there was a malfunction in a major piece of equipment (needed to isolate DNA) that took a few months to fix. This equipment is once again in working order and work continues optimizing protocols and evaluating these samples for the presence of SFB. To accomplish the goals set for this project for the next reporting period (final year) we will use continue to apply these protocols to reaching the experimental goals. What opportunities for training and professional development has the project provided?To date in total there have been four graduate students (3 Master's and 1 PhD) and nine undergraduate students participating on this research project. During the reporting year, this project provided direct support to two undergraduates and 1 PhD student while four undergraduate and 1 MS student were supported via an additional training mechanism. Students are gaining molecular biology, microbiology and other laboratory skills as a part of this project. All students are involved in assisting with optimizing the protocols that are being used to address the experimental goals of the project and are responsible for presenting their results to the lab as well as train others in the technique. Several of the students have traveled with the project director to student centered conferences to present their work, while several of the students have attended conferences. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project will provide basic scientific evidence that could be used in future research aimed at improving mucosal immunity and health and performance of weaned piglets, ultimately helping the industry provide safe and nutritious pork products for consumers, without use of antibiotics. For this reporting period we have accomplished the following; 1) collected fecal samples and intestinal tissues from piglets at various ages, (birth, weaning and post-weaning), 2) submitted fixed intestinal tissues for histological processing and begun analyzed on those that have been returned, others are still in process 2) stored intestinal tissues for further processing and DNA analysis. 3) Begun analysis of fecal samples via staining, PCR and Fluorescence In Situ Hybridization (FISH)., analysis is ongoing. 4) As a control, many samples have been submitted to IDEXX, a company that tests samples for the presence of SFB, this is being done so that we have confirmation that we our protocols are working properly and that we are indeed detecting SFB in our samples. In addition, the PI has contacted a lab that works with SFB in rodents to provide fecal samples so that we have an internal positive control for our assays. From our analysis so far we have preliminary data that SFB can be detected in the swine at the A&T unit. So far though we have detected it in piglets of all ages not just those at or after weaning. This has prompted us to ask additional questions that we will seek to address and has slightly postponed us moving onto the last aim which is to compare PWD and immune responses in SFB and SFB- negative animals, we will continue collecting samples and data and may reevaluate the way we approach this aim. In summary, we have made gains in optimizing protocols, but are not yet at the point where we have complete confidence that we are detecting SFB and have not yet collected enough data to address our hypothesis.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:The target audience for this project are: science/research community, health professionals, pork producers and industry personnel, and consumers. Pork production in the United States is a vital part of the economy. Maintaining healthy hogs and increasing production is important to the pork industry, consumers, and the economy. Overall, this project will provide basic scientific evidence that will be used in future investigations aimed at improving mucosal immunity and overall health and performance of weaned piglets. The long-term impact of this work may lead to changes in the industry that will help shift from the use of antibiotic growth promoters in swine production (and potentially production of other animals). This ultimately will help the industry provide safe and nutritious food for consumers. Changes/Problems:This summer researchers collected tissue and fecal samples from 100 piglets as was outlined in the project proposal for specific Aim 1. Unfortunately the freezer that was being used to store these samples, until they could be further processed for analysis, failed. ALL of the samples were lost. This loss severely put the progress for the project behind as researchers will have to spend this upcoming summer re-collecting samples for Specific Aim 1 and may or may not also be able to move onto Specific Aim #2 as was planned. This may cause significant deviations from the proposed schedule and timeline. What opportunities for training and professional development has the project provided?This project has provided experiential learning opportunities and training in experimental techniques in the area of animal science, immunology, microbiology, and molecular biology, for a total of nine trainees (4 graduate and 5 undergraduate) in the Department of Animal Sciences. Professional development activities for trainees and the PI have also been provided. The students have participated in journal clubs and watched several webinars to increase their understanding of the project and the experimental techniques used. Also the PI, one grad student and the Research Technician have had the opportunity to connect with the Kenneth Simpson lab at Cornell University Vet School to gain insight and visit the Gookin lab at NC State Vet School for training in Florescence In situ Hybridization (FISH) a technique that we will be incorporating into this study. How have the results been disseminated to communities of interest?To date the results of the work efforts have been disseminated via poster presentations, by graduate and undergraduate students. What do you plan to do during the next reporting period to accomplish the goals?This first year has been dedicated to optimizing protocols and confirming that the lab is able to detect SFB. For the next reporting period we intend to keep pressing forward as was outlined in the proposal and working toward asking important questions and gathering data that is publishable. Because of some findings researchers have chosen to pursue a couple of new techniques that will help us answer a question that was not originally proposed. Researchers have all of the necessary equipment for these techniques and have received training from an expert at NC State in August of 2015 that will help in completing these experiments.
Impacts
What was accomplished under these goals?
The goal of the research proposed here is to investigate when SFB colonize the small intestine of piglets weaned between 18-21 days old; and determine whether colonization of the gut with SFB, influences development of intestinal immunity and incidence of post-weaning diarrhea. For this reporting period the major activities completed include: developing and testing protocols for obtaining tissue and fecal samples for analysis, performing DNA extraction and PCR analysis, and histological preparation and analysis of samples. Using these protocols, we have been able to work toward specific objective # 1- Determine when SFB begins to colonize the gut of young piglets. Polymerase Chain reaction (PCR) and light microscopy have been used to detect SFB in intestine and fecal samples collected from piglets ranging in age from (Before weaning) 1 day old, (at Weaning) 18 days old and post-weaning and 32 days old. The significant preliminary results from these activities suggest that 1) SFB seems to be abundant in our swine facility (sequencing confirmation and mouse control studies to be assured that it is indeed SFB that we are detecting is needed and in process) and 2) more SFB DNA was detected in fecal samples of 18 day (weaned) animals than in 1 day old (pre weaning) or 32 d old (post-weaning) animals. However more accurate quantitative analysis is needed, but this finding is consistent with the published information that SFB is more abundant in the gut of animals around the time of weaning. From this data we have derived new questions that we are pursuing; 1) although maybe detecting differences in the DAN levels in the tissue and fecal samples, PCR does not allow us to determine if the bacteria are viable or distinguish morphology. This is relevant because we may detect SFB in fecal samples from all age groups, but is SFB alive (viable) and at what stage of development is it in. This will help researchers determine and distinguish whether the animal is just "passing" non-viable, non-colonized bacteria or if it the animal is actively colonized with active live organisms also we are interested to know Where does SFB may come from, ie can we find it in the environment (soil, dust, etc.). If it is in the environment, is it inhaled/ can we detect it in the lungs of pigs (this is relevant to a collaboration with Dr. Waterman who is studying respiratory disease induced from swine CAFOs. Researchers are still actively engaged in microscope analysis of the samples for detection of SFB, we are incorporating two new techniques in order to be confident in our visualizations. Researchers will use fluorescent in situ hybridization or (FISH) and FISH flow cytometry to help us determine the morphology and viability of the SFB in the tissues and Fecal and environmental samples collected. Although progress is being made, there have been no data collected that would cause a change in knowledge, action or condition beyond the lab at this point.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Lauren Blackwell, T. Herbert, K. Blair, J. Martin, D. Conklin and Radiah C. Minor. The Search for Segmented Filamentous Bacteria in the Dust of a Swine CAFO Poster presentation NC-LSAMP annual Research conference. Oct 2015. Pembroke, NC. ** Third place
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Maya Brooks, T. Herbert, K. Blair, J. Martin, D. Conklin and Radiah C. Minor Experimenting with Polymerase Chain Reaction Technology Poster presentation NC-LSAMP annual Research conference. Oct 2015. Pembroke, NC. **Second Place
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
Christina Bradshaw, L. Blackwell, T. Herbert, K. Blair, J. Martin, D. Conklin and Radiah C. Minor Detecting Segmented Filamentous Bacteria in Swine Using Grams Stain Poster presentation NC-LSAMP annual Research conference. Oct 2015. Pembroke, NC. ** First Place
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2015
Citation:
T. Herbert*, K. Blair*, J. Martin, D. Conklin and Radiah C. Minor Determining the Prevalence of Segmented Filamentous Bacteria (Candidatus Savagella) in Piglets Before, During, and After Weaning. Poster presentation ABRCMS Nov 2015
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