EXPLORING ANTIMICROBIAL ALTERNATIVES FOR CONTROLLING INTESTINAL INFECTIONS IN POULTRY.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 1018495
• Project Start Date: Dec 17, 2018
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Veterinary and Biomedical Sciences

Non Technical Summary
The use of antibiotics to treat animal diseases and for growth promotion is a concern and is increasingly being scrutinized. The new impetus to decrease antibiotic usage has increased the focus on antibiotic alternatives. The overall goal of this project is to decrease the use of antibiotics in poultry by improving the bird's ability to protect itself from intestinal diseases through modulation and conditioning of the developing immune system either prior to, or at the time of, immune stimulation to achieve an enhanced vaccination response and/or response to challenge from enteric disease pathogens. We propose to provide substances either in ovo or in a starter feed and/or the drinking water that will enhance or potentiate immune development during the crucial early development period of a bird's life. We will formulate an adjuvant / immune potentiator using 1,3 beta glucan and administer it either in ovo or in the drinking water. Additionally, we will formulated a diet that is high in digestible protein by using cricket powder, containing ingredients that will enhance immune development (beta glucans) and that will provide for the early establishment of an intestinal microbiota. We will assess the potential of immunizing and protecting birds during this period for various intestinal pathogens. The expected outcome would include the establishment of protocols whereby certain immune adjuvants, modulating diets and/or water preparations could be provided to birds prior to, or at the time of, vaccination to provide enhanced protection and combat Salmonella and Campylobacter infections without using antimicrobials.

Animal Health Component

40%

Research Effort Categories

Basic

10%

Applied

40%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1090 - Immunology;

Keywords

antimicrobial alternatives

immune potentiation

beta glucans

Goals / Objectives
Our goal is to improve the bird's ability to protect itself from enteric diseases by modulating the developing immune system either prior to, or at the time of, immune stimulation to achieve an enhanced vaccination response and/or response to challenge from intestinal disease pathogens (including Salmonella spp. and Campylobacter jejuni). We hypothesize that we can provide substances either in ovo or in a formulated feed and/or a drinking water preparation that will enhance or potentiate immune development (i.e., condition the immune system) during the crucial early development period of a bird's life. The overall long-term goal of this project is to decrease the use of antibiotics in poultry by improving the bird's ability to protect itself from intestinal diseases.Objective 1: To explore the effect that beta-glucans have on the early development of the bird's immune system by assessing the acquired and innate immune responses to in ovo and spray administered poultry vaccines.Objective 2: Develop a prestarter/starter feed that will promote the enhanced development of the bird's immune system and assess the development of acquired and innate immune responses to poultry vaccines.Objective 3: Determine the effectiveness of beta-glucan administration (objective 1) and prestarter/starter feed (objective 2) on the prevention of Salmonella spp and Campylobacter jejuni infections of poultry.Our expected outputs include having a better understanding of the development of the young bird's immune responsiveness and how we can potentiate these responses to better protect the bird from enteric infections and thus decrease the use of antimicrobials.

Project Methods
Eggs and Birds. Specific pathogen free (SPF) eggs will be procured from a commercial company (Valo; Adel, IA) and used as our source of chickens. Eggs will be incubated in small capacity, modern incubators with temperature and humidity digital controls and automatic turners. Just prior to hatch the eggs will be transferred to hatching units. It is at this time that in ovo vaccination using a 20 gauge hypodermic needle and injecting the vaccine preparations into the embryo's amnionic fluid.Housing and rearing. Following hatch, birds will be removed from the hatchers and transferred to BL2 containment rooms. The birds will be housed on the floor with conventional electric brooders and typical commercial wood shaving litter. Care of the birds will be performed by UNL laboratory animal care-takers under the supervision of the facility supervisor. If expertise or advice is needed the PI will lend assistance.Feed and water. Reverse osmosis water will be provided ad libitum using conventional poultry (jug-type) waterers. An organic, antibiotic-free starter/grower diet obtained from Purina will be our control diet. This diet contains 18% crude protein. Our prestarter diet will be formulated by adding a highly digestible protein (i.e. food grade Aketta cricket protein powder; Aspire Foods, Austin, TX), to provide a final diet containing 28% crude protein. We will also add beta-glucan (BioGlena EG®; Indianapolis, IN) at 0.4% of the diet to provide a final amount of approximately 250ppm beta-glucan. Fecal material from mature SPF chickens provided an antibiotic-free, organic poultry feed will be collected and mixed into the prestarter feed at a 2% wgt / wgt basis (i.e. 2 g feces per 100 g feed). The prestarter feed will be prepared within 24 hours of hatch and a sample sent for feed analysis (Midwest Laboratories, Omaha, NE). The diets (control and prestarter) will be made available immediately upon placement in the brooding facility.Vaccines. Coccidiosis vaccines: Inovocox (Zoetis, Inc.) will be used for in ovo vaccination. Coccivac-B52 (Merck Animal Health) will be used as a spray vaccine at day of age. Infectious bursal disease vaccine (IBDV): Bursaplex (Zoetis) will be used for in ovo vaccination. Bursine-2 (Zoetis) will be used for drinking water administration. Note: Infectious Bursal Disease is a viral disease that causes immunosuppression in the growing bird.Antibody response to vaccines. ELISA assays will be used to determine acquired humoral immune responses to vaccination.Innate immunity assay. The OxiSelectTM In Vitro ROS/RNS assay kit (green fluorescence), (Cell Biolabs, Inc., San Diego, CA) will be used for assaying plasma. The assays will be performed in the laboratory of faculty member Dr. Jay Reddy (coinvestigator).Objective 1. We will evaluate the adjuvant effect of beta-glucans on in ovo vaccination using IBDV (Bursaplex) vaccine. The adjuvant dose of beta-glucan is based on a previous report (8). There will be twelve eggs / embryos per group. The following groups will be used: group 1 = nonvaccinated controls, group 2 = control vaccinated with sterile diluent, group 3 = Bursaplex vaccine as per label directions, group 4 = Bursaplex vaccine + beta-glucan (BioGlenaPure®, Algaeon Inc., Indianapolis, IN) at 2% solution, group 5 = 2% beta-glucan in sterile diluent / no vaccine. Eggs / embryos will be in ovo inoculated at 18 days of incubation and transferred to the hatcher. Following hatch birds will be placed in the brooding facility and placed on the control antibiotic-free starter/grower diet (see above). At 3 weeks of age the trial will be terminated and samples collected for antibody and innate immunity assessment along with body weights. Our facilities will not allow for replication within the same trial therefore, the trial will be repeated in the same manner. We will repeat this trial using the same design but substitute Inovocox vaccine (a live coccidiosis vaccine) for Bursaplex. We will evaluate the adjuvant effect of beta-glucans on oral vaccination using IBDV (Bursine-2) vaccine administered in the drinking water. Twelve birds will be used per group. Group 1 = non vaccinated controls receiving no beta-glucans. Group 2 = birds receiving Bursine -2 in the drinking water at 7 days of age. Group 3 = birds will receive 2% beta-glucan in their drinking water 24 hours prior (i.e. 6 days of age) to drinking water vaccination with Bursine-2 which will also contain 2% beta-glucan. At 3 weeks of age the trial will be terminated and samples collected for antibody and innate immunity assessment along with body weights. Our facilities will not allow for replication within the same trial therefore, the trial will be repeated in the same manner. We will repeat this trial using the same design but substitute Coccivac vaccine (a live coccidiosis vaccine) for Bursine-2. The only exception is that the Coccivac vaccine will be administered by the intranasal / intraocular route to emulate spray vaccination.Objective 2. Our prestarter diet will be formulated and prepared as described above. We will have 4 groups of birds having 12 birds per group. Group 1 = nonvaccinated control birds on the control diet. Group 2 - nonvaccinated control birds on the prestarter diet. Group 3 = vaccinated birds on the control diet. Group 4 = vaccinated birds on the prestarter diet. Birds will be placed immediately after hatch into separate brooding facilities. Groups 1 and 2 will be housed separately in the same room and provided the control and prestarter diets respectively. Similarly, groups 3 and 4 will be housed separately in the same room (but different from groups 1 and 2) and provided control and prestarter diets respectively. Groups 1 and 2 will be sham inoculated with sterile water by the intraocular / intranasal route prior to placement in the brooding facility. Groups 3 and 4 will be vaccinated with IBD vaccine (i.e. Bursin-2) by the intraocular / intranasal route prior to placement in the brooding facility. Groups 1 and 3 will receive the control diet for the duration of the trial. Groups 2 and 4 will receive the prestarter diet for the first 5 days following hatch and then receive the control diet for the remainder of the trial. At 3 weeks of age the trial will be terminated and samples collected for antibody and innate immunity assessment along with body weights. Our facilities will not allow for replication within the same trial therefore, the trial will be repeated in the same manner. We will repeat this trial using the same design but substitute Coccivac vaccine (a live coccidiosis vaccine) for Bursine-2.Objective 3. For experiments to determine protection against Salmonella spp we will use a protocol similar to that described previously.(43) Briefly, groups of birds will have received beta-glucan by in ovo delivery (objective 1) or in the prestarter feed (objective 2). On day 3 post hatch birds will be orally inoculated with a nalidixic acid resistant strain of Salmonella spp (NRSal). On days (post hatch) 1, 3, 5, 7, 10 and 14, 2-3 birds in each group will be euthanized and their ceca aseptically collected for NRSal enumeration. Depending on the results, variation of this experimental design will include using seeder birds and comingling negative control birds and treated birds. Studies to determine the protective effect on Campylobacter jejuni infections will be performed as previously described (44) using Campylobacter jejuni as the challenge organism and a similar experimental design as described above for Salmonella spp infections.

Progress 12/17/18 to 09/30/21

Outputs
Target Audience:The target audience for this project are scientists that have an interest in poultry health and poultry producers along with allied groups. Changes/Problems:We had difficulties during the pandemic with personnel changes and scheduling. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been disseminated to communities through conference presentations and poster presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We hypothesized that a never before recognized way in which beta glucans enhance the innate and adaptive immune responses is through avian thrombocytes (AVTs). We proposed to prove this hypothesis by using both in vitro and in vivo approaches with AVTs and chickens. When AVTs were induced with 2% beta glucan, 30% of the cells were found positive for ROS production as compared to 0.8% of untreated control AVTs. These results indicate that AVTs have the capacity to produce ROS and the potential to participate in innate immunity. It also demonstrates that beta glucan can induce an innate immune response in AVTs. The results of the in vivo studies in which beta glucan was incorporated into the diets of birds did not demonstrate any differences in responses of AVTs between beta glucan diet fed birds and birds on negative control diets. These results are not totally unexpected because it has been reported that the immune enhancing effects of beta glucan occurs in the face of a pathogen challenge and may involve adaptive immune responses.

Publications

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience is the poultry industry and poultry producers in all poultry sectors (i.e. meat chickens, egg laying chickens, turkeys, etc.). There is great interest by the broiler companies located in Nebraska. Changes/Problems:The COVID-19 pandemic has seriously interupted our work and work plan. Not only was our laboratory temporarily shut down als the animal facilities were temporarily closed. Recently, we have been able to function but not yet at full capacity. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Through conference presentaions and meetings with other scientists and poultry producers. What do you plan to do during the next reporting period to accomplish the goals?Due to the COVID-19 pandemic our work has been interrupted. We plan on addressing the work listed under objective 3.

Impacts
What was accomplished under these goals? When avian thrombocytes (AVTs) were induced with 2% beta glucan, 30% of the cells were found positive for ROS production as compared to 0.8% of untreated control AVTs. These results indicate that AVTs have the capacity to produce ROS and the potential to participate in innate immunity. It also demonstrates that beta glucan can induce an innate immune response in AVTs. The results of the in vivo studies in which beta glucan was incorporated into the diets of birds did not demonstrate any differences in responses of AVTs between beta glucan diet fed birds and birds on negative control diets. These results are not totally unexpected because it has been reported that the immune enhancing effects of beta glucan occurs in the face of a pathogen challenge and may involve adaptive immune responses. We conducted two trials in which we placed birds into conventional brooding facilities with a hatch-brood feed or conventional feed. We also had a group that was kept in the hatcher for three days with access to hatch-brood feed (i.e. supplemented with cricket protein, beta-glucans and fecal material from adult birds) and water. We assessed the ability of birds to respond to Newcastle disease (ND) vaccine via the adaptive humoral response (i.e. serologic antibody). We found that vaccinated birds in both trials responded equally well independent of the diet or hatch-brood conditions.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Reynolds, D., Reddy, J. Arumugam, R. The effects of beta glucan on thrombocytes and innate immunity. International Nutritional Science and Food Chemistry Conference (OLCNSFC-2019). Venice, Italy. November 11-12, 2019. Poster presentation.

Progress 12/17/18 to 09/30/19

Outputs
Target Audience:The target audience is the poultry industry and poultry producers in all poultry sectors (i.e. meat chickens, egg laying chickens, turkeys, etc.). Additionally, other scientists involved in poultry health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been disseminated to colleagues in the poultry health field through conferences and meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. - Typically, immunity is enhanced by using vaccines and adjuvants to stimulate the adaptive component of the immune response leading to increased humoral (i.e. antibody) and/or cell mediated responses. However, an equally important component of the bird's immune system is the often unnoticed innate immune response. Innate immunity is characteristically nonspecific, rapid and the very first line of immune defense. There are several immune cells that contribute to innate immunity which include leukocytes such as neutrophils (heterophils in avian species), monocytes, macrophages and natural killer cells. These cells are involved in phagocytosis, antigen presentation, free radical and cytokine production. A cell type that has until recently been overlooked but has been increasingly recognized for its role in innate and adaptive immunity is the avian thrombocyte. Second only to red blood cells, thrombocytes / platelets are the most abundant cells in the circulatory system. Thrombocytes and platelets have long been recognized for their role in hemostasis and wound healing. Thrombocytes differ from platelets in that they have nuclei whereas platelets do not. Thrombocytes are present in lower vertebrates such as the avian species whereas platelets are present in mammalian species such as humans. The phagocytic ability of avian thrombocytes has been well established. However, more recent studies have demonstrated that avian thrombocytes are involved in the inflammatory response in a variety of ways which encompass both innate and adaptive immune responses to both bacterial and viral pathogens. Beta-glucans (β- 1,3 / 1,6 glucans) are derived from extracts of the cell walls of plant and yeast cells. Glucans (i.e., β-1,3 glucan) have been used for various health promoting effects in humans and other animals. When glucans are consumed orally they have been shown to have immune enhancing effects primarily on the innate immune system including improved macrophage, neutrophil and natural killer cell activity. Most of these immune enhancing effects have been attributed to boostering innate immunity by modulating cytokine production in monocytes, macrophages and epithelial cells. Studies in poultry have demonstrated that when glucans were included in the diet certain aspects of the immune system were increased. These effects were attributed to the production of cytokines and support the concept of enhancing the innate immune response. We hypothesized that a never before recognized way in which beta glucans enhance the innate and adaptive immune responses is through thrombocytes. We proposed to prove this hypothesis by using both in vitro and in vivo approaches with avian thrombocytes and chickens. Flow cytometry results identifying avian thrombocytes: Unlike other species, chicken immunology research is limited by the availability of antibodies for a few prototypic immune cell types. When such antibodies are available, they can be used as valuable tools to address various mechanistic details at a single cell level by flow cytometry. One major advantage of this approach is that expression of various immunological readouts (e.g., ROS, surface molecules etc.,) can be related to specific subset of cells. By using multi-color flow cytometric analysis, we made an attempt to characterize the chicken thrombocytes leading us to optimize conditions for their identification by using antibody for CD41/61 (marker of thrombocytes). When AVTs were induced with either TBHP or 2% beta glucan, 30% of the cells were found positive for ROS production as compared to 0.8% of untreated control AVTs. These results indicate that AVTs have the capacity to produce ROS and the potential to participate in innate immunity. It also demonstrates that beta glucan can induce an innate immune response in AVTs. The results of the in vivo studies in which beta glucan was incorporated into the diets of birds did not demonstrate any differences in responses of AVTs between beta glucan diet fed birds and birds on negative control diets. These results are not totally unexpected because it has been reported that the immune enhancing effects of beta glucan occurs in the face of a pathogen challenge and may involve adaptive immune responses. Additionally, the modulation of the immune response by beta glucan has been largely attributed to leukocytes in models in which beta glucans are administered parenterally. The mechanism of beta glucan reaching the circulatory system whereby AVTs would be exposed is yet to be delineated but it has been suggested that M cells of the Peyer's patches may play an important role. Objective 2 - The conventional approach of transferring newly hatched birds to the brooder facility as soon after hatch as practically possible is now being reexamined. An alternative approach is to leave the birds in the hatcher (or hatcher environment) for the first 2-4 days in order to alleviate stress, allow for better acclimation and provide for a healthier transition to the permanent brooding facility. During this time birds typically have access to water and sometimes feed. Our approach is to formulate a feed that is optimal for bird growth and development during this crucial early period in a bird's life. We will focus on formulating a diet that is high in digestible protein, contain ingredients that will enhance immune development and will provide for the early establishment of an intestinal and respiratory microbiota. We will assess the potential of immunizing and protecting the bird during this period for various respiratory pathogens. We conducted two trials in which we placed birds into conventional brooding facilities with a hatch-brood feed or conventional feed. We also had a group that was kept in the hatcher for three days with access to hatch-brood feed (i.e. supplemented with cricket protein, beta-glucans and fecal material from adult birds) and water. We assessed the ability of birds to respond to Newcastle disease (ND) vaccine via the adaptive humoral response (i.e. serologic antibody). We found that vaccinated birds in both trials responded equally well independent of the diet or hatch-brood conditions. Objective 3 - No work to this point in the project.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Reynolds, Don, Reddy, Jay and Rajkumar Arumugam. Enhancing Innate Immunity for Better Respiratory Disease Protection. In: The proceeding of The Western Poultry Disease Conference - Association of Poultry Science Specialists (ANECA) International meeting. Puerto Vallarta, Mexico. April 2-6, 2019. pp.212-214
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Modulating the Immune Response of the Bird. Nebraska Poultry Industries Annual Meeting. 2018. Columbus, NE.