Progress 07/01/23 to 06/30/24
Outputs
Target Audience:The target audiences reached during this reporting period were scientists in the field of animal infectious disease by sharing our research work in CRWAD 2024. We shared our work with the broader college community in the CFANS Research Symposium. The PI presented the work in two international meetings, reaching out to the broader animal scientists' community and researchers in the biological areas. Also, we presented the progress of this project in the Department of Animal Science showcase, that is open to the general public, with most attendees being department alumni and industry partners, representatives of commodity groups and other stakeholders. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The PhD student (Caitlin Klaeui) participating in this project has passed their preliminary examination and is expected to graduate in the early summer of 2025. A Master student (Jiayi Peng) has completed her research work and is expected to defend and graduate in the summer of 2024. One undergraduate student (Matthew Stone) worked in the project for the last 2 years and has completed their undergraduate program. We have recruited a new undergraduate student (Rachel Antonutti) to participate in this project from May 2024 to May 2025. How have the results been disseminated to communities of interest?The results of the work performed during the period reported have been disseminated to the different target audiences by oral and poster presentations in the CRAWD meeting (January 2024, Chicago, IL). The findings are also included in the NC1202 "Enteric Diseases of food animals: enhanced prevention, control and food safety" project report (March 2024). There were two different posters presented to the general public attending the Department of Animal Science Showcase on April 25, 2024, and in the CFANS Research Symposium (March 14th, 2024). What do you plan to do during the next reporting period to accomplish the goals?Specific aim 1 is completed and we are working on drafting the publication to disseminate findings. One of the abstracts presented in CRWAD 2024 presented this part of the project. Aim 2 is the aim that will be completed during this last period of the project. We have revisited the in vitro model to include different cell types to better represent what L. intracellularis infection process is in vivo. Our drug candidate tested in Aim 3 was unsuccessful to control the infection. We are preparing the report of our findings and will use the samples collected to pursue a new analysis to identify new candidate treatments for the infection.
Impacts
What was accomplished under these goals?
The specific aims of this project are to: 1. Identify the specific cell type(s) invaded by L. intracellularis 2. Define the cell proliferation-invasion cycle of L. intracellularis in intestinal epithelial cells 3. Evaluate epigallocatechin gallate (EGCG) as a potential strategy to ameliorate effects of L. intracellularis infection in pigs On aim 1, identifying the specific cell type(s) invaded by L. intracellularis, we have infected pig enteroids with Lawsonia intracellularis and collected samples at different times after infection. Those times are between 30 minutes and 2 days. We analyzed to identify what cells the bacteria are invading. We have identified that L. intracellularis was closely associated with cells that are actively proliferating and not with any differentiated cells. Specifically, we did not find any L. intracellularis in mucus-producing goblet cells, which are the cells that are most significantly decreased in L. intracellularis lesions. These results suggest that the changes that L. intracellularis induce in the cells of the intestine may be a consequence of infecting proliferating cells and not a direct effect of the infection. Our study has also revealed that L. intracellularis does not infect epithelial cells as the first target. Apparently, L. intracellularis first invades cells in the submucosa layer and it's only later on that the bacterium is found in the epithelial cells. To complete this aim, we are currently evaluating the short term infection process in vitro and in experimentally infected pigs at very early times after infection (7 days). Aim 2 focuses on defining the cell proliferation-invasion cycle. We are evaluating the time it takes for L. intracellularis to infect and expand in one host to be shed and infect a new host in an in vitro model by exposing uninfected enteroids to the medium in which infected enteroids are at different times after infection. This aim has been very technically challenging and is the main focus of the remainder of the project. After learning in aim 1 that the presence of mesenchymal (submucosa) cells may be needed to model the infection as it happens in animals, a complex culture system is being evaluated to answer the question of the time of dissemination. In our previous report we indicated the possibility of exploring if L. intracellularis survives in protozoans in the environment before infecting other animals. With more and more research reports utilizing the attenuated vaccine as inoculum source for experimental infection, we have decided not to focus on the identification of the environmental reservoir of Lawsonia until there is some clarity on whether vaccination may be a potential for infection. A limitation we had identified to succeed in this aim was the detection of L. intracellularis in the in vitro samples. We have successfully optimized the DNA extraction method and PCR detection of the bacterium to proceed with this aim. The optimization of the DNA extraction and PCR is being reported as method report currently in preparation for submission. Aim 3 evaluated the potential of a chemical compound extracted from green tea, epigallocatechin gallate (EGCG) to ameliorate the effects of L. intracellularis infection in pigs. We did not find EGCG to be active in reducing or controlling the infection. In the process of evaluating EGCG, we gathered enough evidence to challenge the principle that L. intracellularis may not activate Wnt signaling as it has been suggested in the literature. We took advantage of the animal experiment we have set up to test EGCG and collected samples of early infection to perform a RNAseq analysis that will provide more information on the mechanisms of early infection and pathogenesis. This information will also allow to identify new compounds as potential management or treatment strategies.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Peng Y, Klaeui C, Gebhart CJ, Saqui-Salces M. 2024. Goblet cells are not the entry point for Lawsonia intracellularis during 24 hours infection in swine enteroids. CRWAD, Chicago, Illinois. January 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Klaeui C, Peng Y, Gebhart CJ, Saqui-Salces M. 2024. (-)-Epigallocatechin-3-gallate does not affect Lawsonia intracellularis infection of McCoy cells or swine enteroids. CRWAD, Chicago, Illinois. January 2024.
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:The target audience reached by our work in this reporting period was the scientific community, particularly researchers that are focused on the proliferative enteropathy induced by Lawsonia intracellularis. This audience was reach by our presentations of results in the CRWAD meeting, the NC1202 annual meetings, the Gordon Lawson IleitisSymposium, and the . Further scientific fellows, industry and students were exposed to our work results presented in the ASAS Midwest meeting.The general public and overall University population were reached by our dissemination of results in the Animal Science Department Showcase and by invited presentations to industry partners and in the advances in genome biology and technology (AGBT) - Ag workshop. Changes/Problems:We have faced manychallenges in the study that have delayed our productivity. Some of those challenges were due to supply chain issues that have been mostly resolved by now. We have also faced technical challenges related to novel technology application (micro-fluidics system use for tridimensional cultures;limited antibodies that work with swine; lack of specific detection methods for Lawsonia other than qPCR, which limits our capacity to make immediate decisions while experiments are in progress). The major challenge, however, has been with aim 3, as our preliminary data werenot reproducible. We suggested this may the case in our previous report and now we have confirmed that is the case. We are still preparing a report to share these findings. The rejection of our initial hypothesis opened the opportunity to challenge the suggested idea by the literature that Wnt signaling is activated in Pe lesions. After analyzing archive samples and doing Wnt activations studies invitro, we found no evidence of Wnt signaling activation afterLawsonia intracellularisinfection. This is novel information and we are preparing a manuscript with our findings. We are changing our aim 3 to identify molecular mechanisms associated withL. intracellularisinfection in pigs. To do that, we experimentally infected pigs withL. intracellularisand will collect tissues at 7, 14 and 21 days post-infection (n=3 per time) for RNA sequencing analysis (sample collection will occur in August 2023). This analysis will provide information on genes and pathways changing early in the infection and leading to lesion development. There is only one RNAseq analysis published that was done by extracting RNA from proliferating,Lawsoniahighly-infected crypts at 21 days post infection. The crypts were laser dissected from fixed tissue. Our approach will provide information about earlier molecular changes in the intestine and will guide the selection of compounds that could control the development of disease. We foresee a request of no-cost extension of the project to complete this analysis, as the Genomics Center may take longer than 12 weeks to complete the sequencing and the students in the project need to learn to perform this analysis. This change will generate new knowledge in the field but will also provide a new learning and development opportunity for the PhD student working on the project. What opportunities for training and professional development has the project provided?One PhD student (Ramya Lekha Medida) that completed their program in January 2022 worked on this project as part of their dissertation work. In January 2022, a MS student (JiayiPeng) joined our group and is working on Aim 2 of the project. This student is preparing to defend their thesis in December 2023 orJanuary 2024. We have currently a PhD student (Caitlin Kaleui) working on aims 1 and 3. This student started as a MS student but has decided to cointue as a PhD student. This student is the proposing the novel idea that L. intracellularis may use protozoans as a way of transfer among pigs and surviving in the environment. The student will take preliminary examination in August 2023 and the expectation is they will develop a proposal to explore this novel idea. Both the MS and PhD students in the lab have been supported by a undergraduate student (Matthew Stone) who has became the specialist in using the microfluidics system used for aim 2 of the project. Matthew also supports with enteroid culture and histology. How have the results been disseminated to communities of interest?The results of the work performed during the period reported have been disseminated to the different target audiences by presentation of oral presentations in CRAWD meeting (January 2023, Chicago, IL), a project progress report in the NC1202 "Enteric Diseases of food animals: enhanced prevention, control and food safety" project meeting (Januare 2023); poster presentations in the ASAS Midwest Meeting in Madison WI, and a poster presentation to the general public attending the Department of Animal Science Showcase on April 16, 2023. We also presented two posters, one keynote lecture and one oral presentation in the Gordon Lawson Ileitis Symposium on September 16, 2022. Our undergradaute student presented a poster with their work on the microfluidics system in the UMN Undergradaute Research Symposium on May 4th, 2023. What do you plan to do during the next reporting period to accomplish the goals?For specific aim 1, identify the cell types invaded by L. intracellularis, we will continue with the immunohistochemical analysis of the early infection times samples we have already collected and processed. The challenging part of this work is to co-localize cellular markers with the bacterium antibody as there are limited options of antibodies raised in different species to avoid false positives. If we find there are markers that do not work, we would then try in situhybridization. We were expecting to have completed this aim by the end of the second year but technical challenges have delayed our progress. For aim 2, defining the cell proliferation-invasing cycle of the bacterium, we have generated performed experiments that have resulted in non-detectable Lawsonia intracellularis. We are revising our methodological approach to define if this is because the bacterial load in the samples is below the sensitivity of our detection methods or if it is because of real absence of viable bacteria, and thus inability to infect other cells. We may re-direct some of the effort in this aim to explore the role of protozoans as ways of survival and transfer of Lawsonia in the environment and animals. Aim 3 is undergoing significant changes (see changes/problems section). We had already indicated in our last report that the preliminary data that had supported our hypothesis was not reproducible, and as indicated in this report, we have extended our analysis wihtout success. However, the challenge to the original hypothesis resulted in the production of evidence that Wnt signaling is not activated in the lesions in vivo or induced in vitro by the infection. We are now modifying this aim to identify other molecular mechanisms involved in the development of PE. To do that, we experimentally infected pigs with L. intracellularis and will collect tissues at 7, 14 and 21 days post-infection (n=3 per time) for RNA sequencing analysis. This analysis will provide information on genes and pathways changing early in the infection and leading to lession development. There is only one RNAseq analysis published that was done by extracting RNA from poliferating, Lawsonia highly-infected crypts at 21 days post infection. The crypts were laser dissected from fixed tissue. Our approach will provide information about earlier molecular changes in the intestine and will guide the selection of compounds that could control the development of disease.
Impacts
What was accomplished under these goals?
Minnesota ranks as the second in number of pigs raised in the U. S. with more than 3,000 pig farms. Porcine proliferative enteropathy (PE), caused by L. intracellularis, is an intestinal disease of world-wide concern. PE manifests in diarrhea and weight loss, sometimes leading to death because of the decreased capacity of the animals to absorb and utilize nutrients. According to the USDA swine health and health management survey of 2012, PE was reported in 28.7% of the wean-to-finish production sites, with about 30% of animals on site affected by the disease. A unique pathogenic characteristic of L. intracellularis is that the bacterium lives inside the cells of the intestinal lining. The infection makes those cells increase in number (proliferation) but lose function. Until recently, the understanding ofL. intracellularispathogenesis was limited by the lack of a laboratory model capable of reproducing changes observed in animals. We have developed pig mini-guts in the lab (enteroids, intestinal organoids) and have successfully established aL. intracellularisinfection modelthat reproduces most cell changes observed in animals. Using this model, we have discovered that the bacterium does not use the mechanism to make cells proliferate that was previously suggested and we are working in identifying how the bacteria induce those changes and what cells are the main target of infection. We have also identified that the infection causes changes in different cell types of the intestine, having a stronger effect in decreasing cells that produce mucus and cells that secrete intestinal hormones. This information will help us to develop treatments and management strategies to reduce the incidence of this disease in pigs. The goal of this proposal is to define mechanisms involved inL. intracellularispathogenesis and to evaluate management strategies forLawsonia intracellularisinfection. The specific aims of this project are to: 1. Identify the specific cell type(s) invaded byL. intracellularis 2. Define the cell proliferation-invasion cycle ofL. intracellularisin intestinal epithelial cells 3. Evaluate epigallocatechin gallate (EGCG) as a potential strategy to ameliorate effects ofL. intracellularisinfection in pigs On aim 1, identifying the specific cell type(s) invaded by L. intracellularis, we have infected pig enteroids with Lawsonia intracellularis and collected samples at different times after infection. Those times are between 30 minutes and 2 days. Those samples are currently being analyzed to detect the bacteria in them and identify what cells the bacteria are invading. This study is especially important because there is no information in the literature about the dynamics of infection. In farms, the infection is detected when the pigs show diarrhea or have lost significant weight. From animal studies, it is well known that PE lesions reach their peak at around 21 days after experimental infection. However, studies have not analyzed what happens in the early hours of infection and what cells are the target. Our preliminary results suggest that the first cells infected by Lawsonia intracellularis are transient amplifying cells (cells in the intestine that are proliferating and not yet fully functional) and as the infection progresses, the intestine loses cells that produce mucus and hormones, severly compromising intestinal function. Aim 2 focuses on defining the cell proliferation-invasion cycle. To achieve this aim, we are working on the premise that for Lawsonia intracellularis to spread from one animal to another and infect a herd, the bacterium needs to first invade the cells of the intestine of one pig, grow there and eventually bereleased in the feces. Fecal-oral contamination is considered the way of infection in pigs, however, this bactirum can't be cultured from feces. Our in vitro study evaluates these ideas by exposing uninfected enteroids to the medium in which infected enteroids are, at different times after infection. We then hope to detect infection in the uninfected samples to prove bacterial shedding is the way of transmission. Technically, this aim has been difficult to complete because it is difficult to detect the bacterium and we have not been able to detect infection of the uninfected enteroids afterexposing them to media. Those observations have suggested that in animals, Lawsonia intracellularismay usea different way to get in the environment and infect other animals. A newPhD student working on this has hypothesized Lawsonia may utilize protozoans as vehicle to spread in the environment and among animals. This is a significant, and exciting, change in condition that, if proven correct, may lead to a change of action in the management of the disease. Aim 3 evaluates the potential of a chemical compound extracted from green tea, epigallocatechin gallate (EGCG) to ameliorate the effects of L. intracellularis infection in pigs. We chose EGCG as a potential compound to use to manage the disease because the compound is natural and considered safe for humans, with potential to be accepted as safe for animals too. EGCG has been reported to reduce intestinal pathogen loads in some studies and it can interact with some of the molecules that participate in a mechanism of proliferation that was suggested in previous studies is responsible for the development of lesions. To evaluate EGCG effect on Lawsonia intracellularis infection, we performed a multiplicity of infection (MOI) analysis exposing McCoy cells and swine enteroids infected with Lawsonia intracellularis to different amounts of EGCG. One experiment showed a reduction in the amount of Lawsonia in the media collected from infected enteroids. However, there was no dose-response effect, and the cells were damaged. We then evaluated if the compound was toxic to the intestinal cells and found that intestinal cells exposed to levels of EGCG that would affect the bacterium, also affect the viability of cells after long-exposure (longer than 3 days). Because of the dose needed and the inability to know in the field when the infection happens, using a compound that has potential negative side-effects and a very narrow application time is not an option for further exploring this compound in animals. This aim has resulted in an undesirable outcome. However, in the process of evaluating if EGCG would affect the molecular pathway suggested in PE lesions, we could not find evidence of the pathway activation in our in vitro model. Moreover, we searched for evidence in archive samples of PE lesions of pigs experimentally infected and again observed no evidence of this pathway to be active. These observations can provide a reason for EGCG not having the effect we expected, but also have provided new information that challenges some assumptions in the field. As a change of action, we are in the process of experimentally infecting pigs to collect tissue samples and reevaluate the pathways that may be affected by Lawsonia intracellularis infection and identify new compounds as potential management or treatment strategies.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Klaeui C, Medida RL, Stone M, Marshall-Lund L, Gebhart CJ, Saqui-Salces M+. 2022. Lawsonia intracellularis increases cell proliferation in swine enteroids in vitro. Journal of Anim Sci 100 (Suppl 2):191-192
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Medida RL, Vasquez E, Lund LM, Gebhart CJ, Saqui-Salces M+. 2021. Evaluation of molecules involved in intestinal changes induced by Lawsonia intracellularis infection. FASEB Journal, 35;S1:03067
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Klaeui C, Resende T, Gebhart CJ, Saqui-Salces M+. 2023. Lawsonia intracellularis does not activate canonical Wnt signaling in cell lines in vitro or in swine intestine. CRWAD, Chicago, Illinois. January 2023
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Klaeui C, Resende T, Gebhart CJ, Saqui-Salces M+. 2022. Evaluation of canonical Wnt signaling in cell lines and swine intestines infected with Lawsonia intracellularis. Gordon Lawson Ileitis Symposium, St. Paul, Minnesota. September 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Klaeui C, Medida RL, Stone M, Marshall-Lund L, Gebhart CJ, Saqui-Salces M+. 2022. Lawsonia intracellularis increases cell proliferation in swine enteroids in vitro. ASAS Midwest Meeting, March, Omaha, Nebraska.
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:The main target audience reached by our work in this reporting period was the scientific community focused on the study of animal diseases. This audience was reach by our presentations of results in the CRWAD meeting and the NC1202 annual meetings. Further scientific fellows, industry and students were exposed to our work results presented in the ASAS Midwest Section Meeting. The general public and overall University population were reached by our dissemination of results in the Animal Science Department Showcase. Changes/Problems:During this first year of the project, we have experience two significant challenges: a) Supply chain issues: some of the materials used for the in vitro model, and a reagent (Matrigel) that is indispensable for the culture of enteroids, have been severely limited in availability. During 2021, Matrigel was out of stock for about 4 months, so once we used all the reagent we had in stock, we had to stop all experiments for 2 months until more reagent was available. Recovering culture to have enough material to perform experiments takes approximately 2.5 months, thus this supply chain issue generated a significant delay during the beginning of the project. The issues are still present, but we have generated larger and more frequent orders of reagents and supplies to avoid another experiment shut down due to supplies. If the supply chain issues continue this second year, we may consider a no-cost time extension, but at this date, we expect to be able to complete the studies on time. b) Contamination: Working with bacteria infecting mammalian cells is complex and delicate. We suffered the loss of two experiments because of undesirable bacterial contamination in our infection experiments. We have identified the source of contamination and the recovery of cell lines and culture system is in progress. We are now evaluating if previous experiments were free of contamination and once the evaluation is completed, we will decide if they need to be repeated. If that were the case, it would imply significant delay and additional effort. Scientifically speaking, our hypothesis for aim 3 developed from preliminary data was not supported by the extended experimental results. We have a preliminary finding that was not foreseen in our proposal, the potential effect of the chemical compound reducing the amount of bacteria shed after infection. We will further test this in vitro. If the results are promising, then we will move forward with the animal trial indicated in the original project aim 3, although with an updated rationale. If the in vitro results of this reduction are not reproducible, there will not be a reason for the animal trial as originally proposed, although we may still perform one to collect samples for an exploratory analysis of changes in signaling pathways induced by the infection to guide a new selection of potential therapeutical targets. What opportunities for training and professional development has the project provided?We have a graduate student working on the project as thesis. Because of changes in staff in the laboratory of our collaborator, Dr. Gebhart, this graduate student has taken over the bacterial culture work, in addition to their original involvement with only the infection and enteroid part of the project. This change has resulted in new opportunities to learn and expand scientific skills. The use of themicrofluidics system required in aim 2 of the project is complicated. We have an undergraduate student supported by an Undergraduate Research Opportunity Program (UROP) grant being trained on the use of the system and working on the standardization of the experimental conditions needed for completion of aim 2. How have the results been disseminated to communities of interest?The results of the work performed during the period here reported have been disseminated to the different target audiences by presentation of an oral presentation in CRAWD meeting (December 4-7, 2021, Chicago, IL), a project progress report in the NC1202 "Enteroic Diseases of food animals: enhanced prevention, control and food safety" project meeting (December 4th, 2021, Chicago, IL); a poster presentation in the ASAS Midwest Meeting (March 14-16, 2022, Omaha, NE), and a poster presentation to the general public attending the Department of Animal Science Showcase on April 28, 2022. What do you plan to do during the next reporting period to accomplish the goals?We are currently working on repeating the sample collection for aim 1 of the project to be able to complete the analysis. We expect the sample collection will be completed by September 2022 and the analysis by the end of the second year of the project. On aim 2, we expect to have defined the experimental conditions for the study within the next 6 months to be able to complete sample and data collection within the second year of the project, and the analysis to be completed early in the third year of the project. On aim 3, we have achieved significant progress and new experiments are needed to define if EGCG reduced the bacterial load shed after infection. Those experiments will be completed during the second half of the second year of the project and if the results are positive, then animal trials would be expected during the third year of the project. We also expect the MS graduate student working on this project to be preparing their thesis for potential defense by the end of the second year or start of the project's third year. We will recruit a graduate student to take over the project before our current student's graduation.
Impacts
What was accomplished under these goals?
We did considerable progress on aim 3 of the project. Treatment of infected enteroids with ECGC resulted in no changes in any of the genes of interest that we evaluated. However, we observed a reduction of the amount ofLawsonia intracellularisdetected in the culture media after 3 and 5 days of infection, suggesting that the compound may have activity against the bacteria release and potentially helping to reduce its dissemination/shedding. Thein vitroevaluation of that effect is on progress. We did samples collections to start the analysis indicated in aim 1 of the project. Unfortunately, there were not enough infected cells in the samples to help with the determination of the specific cell types invaded byL. intracellularisand we are planning to repeat the experiment with higher infection MOI within the next couple of months. For the completion of aim 2, we need the microfluidics system to establish a culturesystem that simulates the infection - bacterial reproduction inside the cell - bacterial release - infection of new cells cycle. As there is no information in the literature on the time that each of this processes take, only suggested times based on observations in animal trials, there is a significant amount of work needed to define experimental conditions.The acquisition ofthe equipment necessary for establishing the system was completed and we are currently working on the standardization of the experimental conditions to perform the experiments and data generation.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Klaeui C, Medida RL, Stone M, Marshall-Lund L, Gebhart CJ, Saqui-Salces M. 2022. Lawsonia intracellularis increases cell proliferation in swine enteroids in vitro. ASAS Midwest Meeting, March , Omaha, Nebraska.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Medida RL, Vasquez E, Lund LM, Gebhart CJ, Saqui-Salces M. 2021. Swine enteroids reproduce gene expression changes observed in Lawsonia intracellularis- infected pig intestine, CRWAD, Chicago, Illinois. December 3rd to 7th, 2021.
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