Progress 08/01/19 to 07/31/23
Outputs
Target Audience:The target audience for this project include animal and poultry scientists and poultry industry personnel with a focus on broilerhealth.A total of four presentations were conducted to present the progress of the research results. Three presentations were given at the 2020, 2021, and 2023 Conference of Research Workers in Animal Diseases and one presentation was given at the 2023 International Poultry Scientific Forum. The audience at these meetings were scientists and researchers with a focus on poultry health. A white paper was also completed to inform and discuss with the UMES administration a potential patent licensing opportunity and collaborations between UMES and poultry industry. Changes/Problems:No major changes/problems occurred during the last year. However, initially the project was delayed at the start due to closures to COVID. In addition, personnel changes at Morgan State resulted in the delay of making the protein and an outside source was found. What opportunities for training and professional development has the project provided?The post doc working on this project has worked with USDA collaborators to establish the assays needed to conduct these experiments. The following training was provided: how to conduct plate lysis assays and methods to grown and recover Cp. In addition, one undergraduate student worked with the post doc in the lab for one semester and was trained on various laboratory techniques. How have the results been disseminated to communities of interest?Four presentations were completed during the course of this project. Three CRWAD presentations (2020, 2021, and 2023) and one presentation at the Southern poultry science meeting (2023). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact Statement: The results of this project demonstrated positive potential for the use of phage endolysins, specifically PlyCP41 as a novel tool in the fight against of Cp, a persisten challenge for the poultry industry. Not only will this benefit poultry health and welfare, it also has the potential to reduce production costs for the industry. Lower production costs, will translate to a reduced cost for the consumer. This potential cost reduction will benefit consumers as they will spend less money to purchase chicken. Aim 1. Express, characterize and quantify PlyCP41 expression in plants and yeast. 1.1. Verify that PlyCP41 demonstrates species-specific lytic activity for Cp. The lab will use plate lysis assays and in-house stocks to demonstrate species-specific lysis of Cp. 1.2. Quantify lytic activity and optimal storage condition of PlyCP41 in yeast and plants. 1.3. Predicting the most stable formulation for delivery to the chicken lower gut. The goal of this Aim is primarily to rule out some of the formulations that lack cell wall protection from gut fluids (naked enzyme, or whole cell extracts) and thus the enzyme might degrade quickly. A study was conducted to evaluate the endolysin activity from yeast when it is stored in a cold environment. The yeast strains were induced with galatose/raffinose and after overnight growth were harvested in 8 x 6ml aliquots. Media was removed from cells and cells were resuspended in 50mM NaPO4 pH7.5, 100mM NaCl. Four tubes were processed immediately for proteins and the remaining four were frozen -80 after adding glycerol to 30%. Fresh cells were prepped via Fast Prep (speed 6/40sec) with 0.5mm zirconium/silica beads in 1ml buffer described above. The frozen cells were removed from -80 after 24hr, thawed on ice, and centrifuged 5000G/5min/4C. Glycerol buffer was removed and replaced with 1ml 50mM NaPO4 pH7.5, 100mM NaCl. Cells were then processed via Fast prep as described above. Equal amounts of protein were serially diluted for plating in the same 50mM NaPO4 pH7.5, 100mM NaCl buffer. There was not any detectable difference in yeast endolysin activity with protein extracted from fresh and frozen cells. Over 200 gm of Nicotiana benthamiana plant tissue in which the CP41 protein is expressed (between March 1 and April 30, 2020). Plant tissue was frozen for further processing and more material is currently being produced for the project. In vitro Results: An in vitro study was performed to determine the effect of transgenic N. bethamiana (nicotine plant) and transgenic S. cerevisiae (yeast) with the endolysin PlyCp41 to kill Cp 509. There were five treatments (1. Buffer, control, 2. Unmodified N. behamiana, 3. Transgenic N. behamiana (transgenic nicotine plant expressing PlyCp41), 4. Unmodified S. cerevisiae, and 5. Transgenic S. cerevisiae (transgenic yeast expressing PLYCp41). The transgenic N. behamina (2.06 log10 CFU/ml C. perfringens), and transgenic S. cerevisiae (0.06 log10 CFU/ml C. perfringens), significantly reduced Cp 509 compared to the control and unmodified N. bethamiana and S. cerevisiae (4.93, 4.72, and 5.08 log10 CFU/ml C. perfringens, respectively) Ex Vivo Results: Intestinal contents were collected from 21-day old broiler chickens. Contents were collected from the proximal region (crop, proventriculus and gizzard), medial (small intestines) and distal region (cecum) of the gastrointestinal tract. Buffered peptone solution was added to the intestinal contents (40% w/w). Three treatments were investigated in this study. The three treatments were 1. Buffer, control; 2. Unmodified S. cerevisiae; and 3. Transgenic S. cerevisiae (expressing PLYCp41). In each region of the gut, the Cp 509 population was significantly reduced by the transgenic S. cerevisiae treatment compared to the control and the unmodified S. cerevisiae treatment. The Cp 509 population in contents of the proximal region of the gut was reduced by 93.6% compared to the control. Similar results were found with Cp 509 population in the medial (99.9% reduction) and distal region of the gut (94.7%). It was concluded from these studies that transgenic PlyCp41 was successfully expressed in S. cerevisiae and N. benthamiana. In addition, it exhibited lytic activity against cultured Cp (CP509) in vitro and endogenous Cp ex vivo. Aim 2. Develop an animal model for Cp colonization in poultry. 2.1 Developing the model. 2.2. In vivo testing of yeast and plant expressing PlyCP41 in feed on Cp colonization and comparison to commercial antibiotics. 2.3 Presence of lytic enzymes in gut will be monitored and quantified by ELISA/Western Blot. The project was modified to evaluate the efficacy of yeast and plant expressed PlyCP41 against commensal Cp in the intestinal tract of the bird. In vivo results: A between-subjects design with negative and positive control groups was employed to determine the anti-Clostridial effect of PlyCp41 expressed in yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana) cells when administered to broiler chickens during an Eimeria challenge. Two replicate studies were conducted to test PlyCp41 in yeast (rep 1) and plant (rep 2) cells. Newly hatched Ross 708 males (N=24) were randomly distributed into 12-floor pens and reared from 0-28d of age. At 14d, all birds except those in the negative control group were challenged with an oral gavage containing 41,000 Eimeria oocysts to create physical damage to the gut mucosa. Birds in the transgenic yeast and plant groups received a daily oral gavage containing S. cerevisiae yeast (~450ng/ml), or N. benthamiana plant cells to reduce the quantity of Clostridium perfringens (Cp) in the gut. Birds in the unmodified yeast and plant groups received the same daily dose of unmodified yeast and plant cells from 16-21d to determine if the yeast or plant cells without enzyme present have an impact on Cp levels or bird health during the challenge. All birds were euthanized on 28d, and samples of gut contents were collected for Cp enumeration. Data were analyzed for a one-way ANOVA using the GLM procedure (SAS 3.8, SAS Institute Inc., Cary, NC) to determine the effect of PlyCp41 in yeast and plant cells on the abundance of Cp in the gut. The PlyCp41 in vivo study shows mixed results, including a ~1-log (86%) reduction with transgenic plant cells, but no differences with yeast cells. The Cp levels from the transgenic plant treatment was 4.54 log10 CFU/ml C. perfringens compared to the Cp levels measured from the positive control (5.4 log10 CFU/ml C. perfringens). Expression of PlyCP41 in yeast. A galactose inducible expression construct encoding a yeast-codon optimized gene was used to produce intracellular PlyCP41 in S. cerevisiae that was cultured in URA minus media (to maintain the ura3 auxotrophic, PlyCP41 inducible vector), harvested and stored in 20% glycerol. Analysis of total transgenic yeast protein by Western blot revealed a prominent signal at the predicted size (~38.5 KDa) for PlyCP41 (335 amino acids). The concentration of PlyCP41 in the yeast cells used for these studies is estimated from the Western Blot data to be ~4.7% of the total intracellular yeast protein or 390-510 μg/mL of yeast suspension by comparison to purified, bacterially expressed PlyCP41 on the same Western blot.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Timmons, J.R., M. Barnas, W. Attuquayefio, D.M. Donovan, C. Skory, and R. Hammond. 2023. Phage Endolysins as Alternative Antibiotics to Control Clostridia in Poultry: Measuring the lytic activity of PlyCP41 using an ex vivo method. 103rd Conference of Research Workers in Animal Diseases. Abstract V-016. https://crwad.org/wp-content/uploads/CRWAD-VIRTUAL-ONLY-LIST-2023_FINAL-PRINT-VERSION.pdf
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Siragusa, G., M. Barnas, W. Attuquayefio, J. Timmons, C. Skory, R. Hammond, D. Donovan, Measuring the in vitro anti-clostridial activity of a phage endolysin expressed in Saccharomyces cerevisiae. Abstract No. T193, page 61. International Poultry Scientific Forum Georgia World Congress Center, Atlanta, Georgia January 23-24, 2023. https://www.ippexpo.org/education-programs/IPSF/docs/2023-IPSF-Abstracts.pdf
- Type:
Journal Articles
Status:
Submitted
Year Published:
2024
Citation:
Barnas, M.R., W. Attuquayefio, D.M. Donovan, C.D. Skory, R.W. Hammond, G.R. Siragusa, and J.R. Timmons. Submitted November 2023. Yeast expressing a phage endolysin reduces endogenous Clostridium perfringens ex vivo in chicken gut fluids. Avian Diseases.
- Type:
Other
Status:
Published
Year Published:
2023
Citation:
Barnas, M.R., W. Attuquayefio, D.M. Donovan, C.D. Skory, R.W. Hammond, G.R. Siragusa, and J.R. Timmons. 2023. PlyCP41 for treating necrotic enteritis in chickens. [White Paper]. February 2023, University of Maryland Eastern Shore.
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Progress 08/01/21 to 07/31/22
Outputs
Target Audience:A presentation was presented to particpants of the 2021 Conference of Research Workers in Animal Diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The post doc working on this project has worked with USDA collaborators to establish the assays needed to conduct these experiments. The following training was provided: how to conduct plate lysis assays and methods to grow and recover CP. How have the results been disseminated to communities of interest?An abstract was submitted for the 2022 CRWAD conference and the 2023 Southern Poultry Science meeting. What do you plan to do during the next reporting period to accomplish the goals?The next goals for this project are to determine the inhibitory effect in vivo by administering yeast cells containing the endolysin to broilers via feed additives.
Impacts
What was accomplished under these goals?
Specific objectives achieved for the reporting period include: 1.Determine the lytic activity of PlyCP41 (naked enzyme)using a plate lysis assay. 2. Measure the abundance of commensal CP in the crop, small intestine and ceca of healthy broiler chickens. 3. Measure of the efficacy of PlyCP41 to reduce CP in the intestinal contents from 14 day and 28 day old broiler chickens. The following was accomplished under these objectives: It was determined that the PlyCP41 enzymes all exhibited lytic activity against C. perfringens in vitro. In addition, it was found that commensal CP content was the lowest in the crop, followed by the small intestines, and the ceca. It was also determined thatPlyCP41 enzyme reduced C. perfringens in broiler chicken intestinal content. From these results, we were also able to accomplish the following goals:A study was conducted tomeasurethe in vitro anti-CP activity of PlyCP41 when expressed in transgenic Saccharomyces cerevisiae. PlyCP41 expressed inyeast reduced CP in vitro by 99.997% compared to CP levels in the negative control (CP alone, no enzyme).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Timmons, J.R., M. Barnas, B. Khatabi, P. Sardaru, D.M. Donovan, G. Hoffman, C. Skory, and R. Hammond. 2021. Phage Endolysins As Alternative Antibiotics To Control Clostridia In Poultry. 102nd Conference of Research Workers in Animal Diseases. Abstract V-002. https://crwad.org/wp-content/uploads/CRWAD-2021.Virtual-Oral-and-Poster-Program.17NOV2021.pdf
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Progress 08/01/20 to 07/31/21
Outputs
Target Audience:A presentation was presented to participants ofthe 2020 Conference of Research Workers in Animal Diseases. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has provided training for one post-doc and one Ph.D student. These individuals were successful in growingPlyCP41 using a plate lysis assay. How have the results been disseminated to communities of interest?An abstract was submitted for the 2021 CRWAD conference. What do you plan to do during the next reporting period to accomplish the goals?The issues with personnel and methodology have been resolved. There should not be any issue with completing the goals of the project.
Impacts
What was accomplished under these goals?
The endolysin PlyCP41 is known to exhibit lytic activity against Clostridium perfringens (CP) cells in vitro, and therefore could potentially be used in vivo to achieve similar results within the gastrointestinal tract (GIT). The objectives of this study were to confirm the lytic activity of PlyCP41 using a plate lysis assay and determine the abundance of commensal CP in the crop, small intestine, and ceca of healthy broiler chickens. Three different enzyme preparations of PlyCP41 were diluted to a starting concentration of 2 mg/ml then spot plated onto 11 ml of semisolid agar containing 1 ml of CP cells at 55 OD concentration. Each enzyme was serial-diluted then plated on a grid using 5 µl per spot in descending concentrations. The plates were incubated at 37°C for 2 h then observed for lytic activity. All three enzyme preparations lysed CP cells at 100 ng of enzyme per spot, and one purified protein preparation was effective at 10 ng per spot. Next, to measure CP levels in the gut, twenty newly hatched Cobb-500 broilers were raised on built-up litter from 0-14d. At 14d, three randomly selected birds were euthanized, then their crop, small intestine, and cecal contents were removed and immediately diluted 1:10 in PBS. Serial dilutions were conducted to 10-6, then 0.1 ml of each dilution was spread in duplicate onto Tryptose Sulfite Cycloserine (TSC) agar and incubated under anaerobic conditions for 24h at 37°C. Typical black colonies were counted, and CFU/g of contents was estimated. A 1 g aliquot of contents from each section was also reserved for DNA extraction and qPCR to determine CP abundance. The crop contained the fewest CP cells, with TSC plates averaging 7 x 104 CFU/g and qPCR averaging a Cq value of 24.9. The small intestine harbored approximately 8 x 105 CFU/g (Cq of 19.9), and the ceca had a similar population of 6 x 105 CFU/g and an average Cq of 18.8. Future studies will test these enzyme preparations in vivo to determine the ability of PlyCP41 to reduce CP cells in the GIT of broiler chickens.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Timmons, J.R., M. Barnas, D.M. Donovan, G. Hoffman, C. Skory, and R. Hammond. Phage Endolysins As Alternative Antibiotics To Control Clostridia In Poultry. Conference of Workers in Animal Diseases, page 62. 2020. Abstract No. 147.
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Progress 08/01/19 to 07/31/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:UMES was notified of funding for the project August 2019, however funding from NIFA was not received until October 2019. Due to closures due to COVID, we were not able to hire the post-doc in spring as planned. Work was halted from mid-March to mid-July. A post-doc was hired in August to start validating the animal model. What opportunities for training and professional development has the project provided?
Nothing Reported
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?A post-doc was hired in August to work on this project to develop the animal model. The team has "met" several times in August and September to discuss the progress of the project. Work has begun to validate the animal model for CP. It is expected this project will be complete by July 31, 2021.
Impacts
What was accomplished under these goals?
A prelimary study was conducted to evaluate the endolysin actitivy from yeast when it is stored in a cold environment. The yeast strains were induced with galatose/raffinose and after overnight growth were harvested in 8 x 6ml aliquots. Media was removed from cells and cells were resuspended in 50mM NaPO4 pH7.5, 100mM NaCl. Four tubes were processed immediately for proteins and the remaining four were frozen -80 after adding glycerol to 30%. Fresh cells were prepped via Fast Prep (speed 6/40sec) with 0.5mm zirconium/silica beads in 1ml buffer described above. The frozen cells were removed from -80 after 24hr, thawed on ice, and centrifuged 5000G/5min/4C. Glycerol buffer was removed and replaced with 1ml 50mM NaPO4 pH7.5, 100mM NaCl. Cells were then processed via Fast prep as described above. Equal amounts of protein were serially diluted for plating in the same 50mM NaPO4 pH7.5, 100mM NaCl buffer. There was not any detectable difference in yeast endolysin activity with protein extracted from fresh and frozen cells. Additionally, the ARS Beltsville collaborator produced over 200 gm of Nicotiana benthamiana plant tissue in which the CP41 protein is expressed (between March 1 and April 30,2020). Plant tissue has been frozen for further processing and more material is currently being produced for the project. The development of a plant virus-based vector that can be used to express CP41 in clover is also be explored.
Publications
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