Abstract. Problem: In bio-shuttle programs to mitigate the damage done by infections of broilers with

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3 Abstract Problem: In bio-shuttle programs to mitigate the damage done by infections of broilers with coccidia, chickens are vaccinated with fully virulent coccidia immediately after hatch. Before the coccidia build up to high numbers, anticoccidials are added to the feed, minimizing negative side effects associated with live vaccines. In recent years, bio-shuttle programs have become increasingly popular, but systematic, peer-reviewed research is lacking. Objective: The objective of the project is to compare three anticoccidials in bio-shuttle programs for broilers using different-strength challenge models. Approach: The three selected anticoccidials are narasin, nicarbazin and zoalene each representing a different type of anticoccidial. The different-strength challenges will be done by placing (1) vaccinated chicks on fresh litter, (2) vaccinated chicks on litter seeded with coccidia two weeks before placement, (3) vaccinated chicks on litter seeded with coccidia three days before placement and (4) not vaccinated chicks on litter seeded with coccidia three days before placement. The study will comprise four trials, one for each challenge model. In every trial, there will be three groups, one for each anticoccidial. All trials will be floor pen tests. Tested parameters will be weight gain and feed conversion, coccidian counts shed in feces and intestinal lesion scores. Value to the industry: We hypothesize that depending on the strength of the coccidia challenge a different type of anticoccidial will provide the best results. This knowledge will allow producers to select an anticoccidial based on the anticipated infection pressure. We also hypothesize that placing unvaccinated birds on seeded litter will give similar results as placing vaccinated chicks on seeded litter. If this hypothesis is correct, it will save producers money by not vaccinating their birds in every cycle, but only as needed to replace emerging resistant coccidia. 1

4 Proposal 1. Title Systematic Optimization of Bio-Shuttle Programs 2. Investigators R. Hauck and K.S. Macklin Department of Pathobiology and Poultry Science Department Auburn University 3. Objectives The objective of the study is to allow producers to select an anticoccidial based on the anticipated infection pressure and thus optimizing the use of anticoccidials in bio-shuttle programs for broilers. We will compare how three anticoccidials affect weight gain and feed conversion, coccidian counts shed in feces and intestinal lesion scores after challenging the chickens with differentstrength models. 4. Justification The economic damaged caused by infections with coccidia, including the costs to control coccidiosis by feed additives and/or vaccines, has been estimated at 4.54% of the gross revenue from sales of live broilers (Williams, 1999). The cost can be decreased by selecting anticoccidial feed additives that will provide the best results in a given situation. In the last few years, the situation has been complicated by the trend towards organic and antibiotic-free production in which the use of various feed additives that were routinely used to maintain intestinal health has been disallowed or restricted. Anticoccidials fall in one of two categories: ionophores or chemicals. Ionophores are antibiotics. They act against coccidia, but are only coccidiostatic, allowing a low-level replication of the parasites, so the birds will build immunity against coccidia. Because they are antibiotics, they cannot be used in antibiotic-free or organic production. Chemicals are a heterologous group with various modes of action. Most but not all of them are coccidiocidal and will completely kill susceptible coccidia. Because they are not antibiotics, they are still allowable in antibiotic-free production; however, they have been banned in organic production. A major problem with all anticoccidials is that coccidia will become resistant after the prolonged use of a single compound. For this reason, the anticoccidials used on a farm must be rotated on a regular base. In addition to anticoccidials, live vaccines have been included in the rotation to restore drug-sensitive coccidia populations. However, the use of vaccines can be problematic as well, because the vaccines consist of fully virulent strains. They do not cause immediate damage, because they are given at a strictly controlled low dose, but like the field strains, they can 2

5 predispose the birds for Necrotic Enteritis (Tsiouris et al., 2013). As a solution of this dilemma, bio-shuttle programs have become increasingly popular in recent years. In bio-shuttle programs, the chicks are vaccinated against coccidia directly after hatch to introduce sensitive coccidia strains into the flocks. Before their numbers build up to high numbers to cause damage, anticoccidials are added to the feed when the feed is changed from starter to grower feed. Bio-shuttle programs have been promoted by vaccine- and anticoccidial producing companies; however, there is an absence in independent and systematic research on these programs. Literature research on Pubmed and Agricola does not return results for coccidia poultry bio-shuttle, coccidia poultry bioshuttle, Eimeria poultry bio-shuttle or Eimeria poultry bio-shuttle. For the study, we selected three anticoccidials covering the different types of anticoccidials in use, namely narasin, a coccidiostatic ionophore, nicarbazin, a coccidiocical chemical, and zoalene, a coccidiostatic chemical. We hypothesize that there will be only minor differences between the anticoccidials after a weak challenge, but that after a moderate and especially a strong challenge the cocciocidal chemical will produce the best results. We also hypothesize that placing unvaccinated birds on seeded litter will give similar results as placing vaccinated chicks on seeded litter. If this hypothesis is correct, it will save producers money by not vaccinating their birds in every cycle, but only as needed to replace emerging resistant coccidia. The objective relates to US Poultry s research priority Determine optimum shuttle and rotation programs for anticoccidials and coccidiosis vaccines to improve and retain efficacy. 5. Procedures Experimental design The study will comprise four trials. In every trial, there will be three groups, one for each anticoccidial, with six replicates each. Trial 1 will apply a weak challenge, trial 2 a moderate challenge, trial 3 a strong challenge and trial 4 a vaccine-less challenge. The different-strength challenges will be done by placing (1) vaccinated chicks on fresh litter, (2) vaccinated chicks on litter seeded with coccidia two weeks before placement, (3) vaccinated chicks on litter seeded with coccidia three days before placement and (4) not vaccinated chicks on litter seeded with coccidia three days before placement. The challenge models are compared in Table 1; Table 2 shows the schedule of events in all four trials. 3

6 Table 1: Comparison of challenge models Trial Challenge Vaccination Litter 1 Weak Yes Fresh 2 Moderate Yes Seeded with coccidia 14 days before start 3 Strong Yes Seeded with coccidia 3 days before start 4 Vaccine-less No Seeded with coccidia 3 days before start Table 2: Schedule of events in all three trials Bird age Trial 2: day -45 Trials 3 and 4: day -34 Trial 2: day -35 Trials 3 and 4: day -24 Trial 2: days Trials 3 and 4: days Event Non-coccidiosis vaccinated seeder birds raised in batteries Seeder birds challenged with 250,000 oocysts and five placed per pen to be utilized in the main portion of the trial Seeder birds shedding coccidia in the pens; seeder birds removed at the end of the period Day 0 Placement of study chicks Day 13 Wingband six birds per pen Day 14 Weigh birds by pen and count Change and weigh feed per experimental design Count coccidia shed in feces Sacrifice 3 birds / pen and score intestinal lesions Day 17 Count coccidia shed in feces Day 20 Count coccidia shed in feces Sacrifice 3 birds / pen and score intestinal lesions Day 28 Weigh birds by pen and count Change from grower to withdrawal feed in all groups Day 42 Weigh birds by pen and count Weigh back final withdrawal feed 4

7 Experimental procedures Animals and housing Seven hundred and forty four male chicks will be acquired and placed randomly into 24, 24 ft 2 pens, so 31 birds will be placed into each pen. After euthanizing cumulatively six birds per pen on days 14 (3 birds) and 20 (3 birds), this will result in 25 birds per pen or 0.96 ft 2 /bird, a final stocking density that is comparable to commercial conditions. Softwood shavings will be used as bedding material. Feed and anticoccidials The birds will be fed a standard three feed diet consisting of corn and soybean meal that will be formulated to commercial poultry industry standards. These feeds as well as water will be provided to the birds ad libitum for the duration of the trial. Starter and withdrawal feed will contain no anticoccidials. Per experimental design, the grower feed will contain narasin at a concentration of 72 gram of active compound per ton of feed (72 ppm), nicarbazin at a concentration of 125 gram of active compound per ton of feed (125 ppm) or zoalene at a concentration of gram of active compound per ton of feed (113.5 ppm). The feed will be mixed at the Auburn University Poultry Research Farm s Feed Mill. Seeding the litter in trials 2, 3 and 4 For seeding the litter, 120 non-coccidiosis vaccinated, mixed sexed chicks will be raised in battery cages being fed a non-medicated feed. When 10 days old, they will be challenged with a 1 ml oral gavage of the same vaccine to be utilized in trials 1, 2 and 3 at a dose of approximately 250,000 oocysts per ml. Five birds will be placed into each of the pens and raised for 21 days, prior to being euthanized. Additional challenged birds will be kept in the battery cages as replacements for any mortality that may occur in the birds placed into the pens. The main trial will begin after 14 days in trial 2 and after 3 days in trials 3 and 4. Coccidia vaccination in trials 1, 2 and 3 All chicks will be vaccinated against coccidia at the commercial hatchery shortly after the chicks hatch using a commercial vaccine containing E. acervulina, E. maxima and E. tenella. Investigated parameters Zootechnical parameters: Cumulative body weight and feed consumption will be recorded for every pen at every change of feed and will then be tabulated into weight gain and feed conversion. Coccidial counts: On days 14, 17 and 20 fresh feces will be collected from at least three sites per pen. The samples of each pen will the thoroughly mixed and the number of oocysts per gram will be determined using a McMaster chamber. 5

8 Scoring of intestinal lesions: On days 14 and 20, three birds per pen will be randomly selected based upon their wingband and then euthanized. Their intestinal coccidial lesions in duodenum, jejunum and cecum will be scored on a scale between 0 and Literature review As stated above, literature research on Pubmed and Agricola does not return results for coccidia poultry bio-shuttle, coccidia poultry bioshuttle, Eimeria poultry bio-shuttle or Eimeria poultry bio-shuttle. To the best of our knowledge, this will be the first study to compare the three types of anticoccidials in a bio-shuttle program in floor pen tests. Narasin Narasin is a monovalent ionophore. It probably acts against the late stages of coccidia (Guyonnet et al., 1991) and, given at 80 ppm in the feed, it moderately suppresses development of immunity against coccidiosis (Karlsson and Reid, 1978). Narasin was tested against various Eimeria spp. at dose levels of ppm in floor pen studies (Ruff et al., 1980; Jeffers et al., 1988; Long et al., 1988). The studies by Ruff et al. (1980) and by Jeffers et al. (1988) employed various challenge techniques, including seeder birds removed three days before the start of the study and suspended seeder birds. However, because only Narasin was tested, there are no results of the comparative efficacy of different anticoccidials by the challenge method. The study by Long et al. (1988) used seeder birds. In all studies, the birds were medicated from day one and the chicks were not vaccinated. Narasin was also effective in the prevention of experimentally induced necrotic enteritis in broiler chickens (Brennan et al., 2001). Nicarbazin Nicarbazin is an equimolar mix of two compounds. It seems to have the greatest effect against second generation schizonts, but also acts against other stages (McLoughlin and Wehr, 1960). Given at 125 ppm in the feed, it slightly suppresses development of immunity against coccidiosis (Karlsson and Reid, 1978). Nicarbazin was tested against various Eimeria spp. at dose levels of ppm in the feed in floor pen studies. Challenge methods were direct inoculation (Cuckler et al., 1956) or seeder birds (Long et al., 1988). In both studies, the birds were medicated from day one and the chicks were not vaccinated. Zoalene Zoalene is also known as dinitolmide. It acts against Eimeria merozoites (Joyner, 1960). Given at 125 ppm in the feed, it does not suppress development of immunity against coccidiosis (Karlsson and Reid, 1978). Zoalene at a dose level of 125 ppm in the starter feed was tested against a mixed infection with E. acervulina, E. maxima and E. tenella in a floor pen study (Conway et al., 2001). 6

9 In this study, the birds were infected via their feed. Treatment started on day one and the chicks were not vaccinated. Combination of anticoccidials and vaccines For a long time, vaccines have been included in the rotation of anticoccidials to restore drugsensitive coccidia populations by replacing resistant field isolates with susceptible vaccine strains (Jeffers, 1976; Chapman, 1994; Mathis and Broussard, 2006; Peek and Landman, 2006). It has also been suggested that interbreeding between field and vaccine parasites results in susceptible progeny (Williams, 2002). Bio-shuttle programs as described above are a more recent trend, but follow the same logic (Kimminau, 2017; Newman, 2017). While there seem to be no peer-reviewed publications evaluating bio-shuttle programs, a few presentations at scientific meetings have dealt with the bio-shuttle programs in recent years. The topics were the effects of various anticoccidials as bio-shuttle alternatives for broilers after a necrotic enteritis challenge (Da Costa, 2017), determining the extent of coccidia drug restoration when broilers are grown with bio-shuttle programs (Kimminau, 2017) and the combination or bioshuttle programs with a plant product (Wang, 2016), so our project clearly differs from these approaches. Cited references Brennan, J., Bagg, R., Barnum, D., Wilson, J., Dick, P., Efficacy of Narasin in the prevention of Necrotic Enteritis in broiler chickens. Avian Dis. 45, Chapman, H.D., Sensitivity of field isolates of Eimeria to monensin following the use of a coccidiosis vaccine in broiler chickens. Poult. Sci. 73, Conway, D.P., Mathis, G.F., Johnson, J., Schwartz, M., Baldwin, C., Efficacy of diclazuril in comparison with chemical and ionophorous anticoccidials against Eimeria spp. in broiler chickens in floor pens. Poult. Sci. 80, Cuckler, A.C., Malanga, C.M., Ott, W.H., The antiparasitic activity of nicarbazin. Poult. Sci. 35, Da Costa, M., Effects of various anticoccidials as bio-shuttle alternatives for broilers under a Necrotic Enteritis challenge. Presentation at the AAAP meeting 2017 in Indianapolis, IN. Guyonnet, V., Johnson, J.K., Long, P.L., Activity of lasalocid, monensin, narasin, and salinomycin against late stages of development of Eimeria spp. in broiler chickens. Poult. Sci. 70, 162. Jeffers, T.K., Reduction of anticoccidial drug resistance by massive introduction of drugsensitive coccidia. Avian Dis. 20,

10 Jeffers, T.K., Tonkinson, L.V., Callender, M.E., Schlegel, B.F., Reid, W.M., Anticoccidial efficacy of narasin in floor pen trials. Poult. Sci. 67, Joyner, L.P., The coccidiostatic activity of 3, 5-dinitroorthotoluamide against Eimeria tenella. Res. Vet. Sci. 1, Karlsson, T., Reid, W.M., Development of immunity to coccidiosis in chickens administered anticoccidials in feed. Avian Dis. 22, Kimminau, E., Determining the extent of coccidia drug restoration when broilers are grown with bioshuttle program. Presentation at the AAAP meeting 2017 in Indianapolis, IN. Long, P.L., Johnson, J., McKenzie, M.E., Anticoccidial activity of combinations of narasin and nicarbazin. Poult. Sci. 67, Mathis, G.F., Broussard, C., Increased level of Eimeria sensitivity to diclazuril after using a live coccidial vaccine. Avian Dis. 50, McLoughlin, D.K., Wehr, E.E., Stages in the life cycle of Eimeria tenella affected by nicarbazin. Poult. Sci. 39, Newman, L.J., Coccidiosis Control with a Hybrid ( Bio-shuttle ) Program. Presentation at the AAAP meeting 2017 in Indianapolis, IN. Peek, H.W., Landman, W.J.M., Higher incidence of Eimeria spp. field isolates sensitive for diclazuril and monensin associated with the use of live coccidiosis vaccination with paracox-5 in broiler farms. Avian Dis. 50, Ruff, M.D., Reid, W.M., Rahn, A.P., McDougald, L.R., Anticoccidial activity of narasin in broiler chickens reared in floor pens. Poult. Sci. 59, Tsiouris, V., Georgopoulou, I., Batzios, C., Pappaioannou, N., Diakou, A., Petridou, E., Ducatelle, R., Fortomaris, P., The role of an attenuated anticoccidial vaccine on the intestinal ecosystem and on the pathogenesis of experimental necrotic enteritis in broiler chickens. Avian Pathol. 42, Wang, Y.-T., Evaluation of the efficacy of different coccidiosis bioshuttle programs and dietary capsicum-turmeric oleoresins on broiler performance. Presentation at the AAAP meeting 2016 in San Antonio, TX. Williams, R.B., Anticoccidial vaccines for broiler chickens: pathways to success. Avian Pathol. 31, Williams, R.B., A compartmentalised model for the estimation of the cost of coccidiosis to the world s chicken production industry. Int. J. Parasitol. 29,

11 7. Resume of investigators Ruediger Hauck Education Freie Universität Berlin DVM 2002 Veterinary Medicine Freie Universität Berlin PhD 2006 Veterinary Medicine Freie Universität Berlin Habilitation 2012 Poultry Diseases Career History Since February 2017: Assistant Professor at Auburn University with a shared appointment at the Department of Pathobiology and the Department of Poultry Science June 2015 February 2017: Postdoc at the School of Veterinary Medicine, University of California, Davis September 2013 May 2015: Member of the team Antimicrobial Resistance at the Federal Office for Consumer Protection and Food Safety, Berlin, Germany March 2008 February 2013 Postdoc at the Institute of Poultry Diseases, Department of Veterinary Medicine, Freie Universität Berlin June 2007 February 2008: Postdoc at the Poultry Diagnostic and Research Center, University of Georgia March 2006 May 2007: Postdoc at the Department of Poultry Science, University of Georgia Other Experience and Professional Memberships Diplomate of the European College of Poultry Veterinary Science Certified Veterinary Poultry Specialist (Veterinary Chamber Berlin) Certified Veterinary Microbiology Specialist (Veterinary Chamber Berlin) Member of the American Association of Avian Pathologists Member of the editorial board of Avian Diseases President of the Southern Conference on Avian Diseases (SCAD) Publication List 52 publications in peer reviewed scientific journals 70 publications in proceedings of scientific meetings 5 book chapters and 1 book 6 publications in non peer-reviewed journals 38 oral presentations at scientific meetings 4 active participations in continuing education and education events 9

12 Kenneth S. Macklin Education Northern Illinois University, DeKalb, Illinois B.S Biology Northern Illinois University, DeKalb, Illinois M.S Biology Auburn University, Auburn, Alabama PhD 2003 Poultry Science Career History 2015-present: Professor and Extension Specialist, Department of Poultry Science, Auburn University : Associate Professor and Extension Specialist, Department of Poultry Science, Auburn University : Assistant Professor and Extension Specialist, Department of Poultry Science, Auburn University Accomplishments Have worked with the US poultry industry in determining the probable causes and developing solutions to E. coli caused diseases in broiler chickens. Is involved with developing novel methods to extend the effective life of broiler litter by controlling ammonia and pathogens. Involved with determining methods to identify and control foodborne pathogens on poultry farms. Has performed research into the role of diet and gut microbiome on necrotic enteritis development. Honors and Awards College of Agriculture Dean s Grantsmanship Award, 2015 AU Open Source Intelligence Laboratory Affiliate Faculty, 2014 Southern Conference on Avian Disease Director, 2014 Poultry Science Association Outstanding Service Award, 2012 AU Food System Initiative Virtual Faculty, 2012 Poultry Science Association - Early Achievement Award in Extension, 2008 Professional Activities Southern Conference on Avian Disease President National 4-H Poultry and Egg Conference Committee Member present Poultry Science Association - Member of the Food Safety and Health Committee present Southern Conference on Avian Disease Director present Alabama Poultry and Egg Associations Annual Meeting Planning Committee present Alabama Avian Health Advisory Board Member present Publication Record Refereed Journal Articles: 52 Books/Book Chapters: 7 Symposia and Conference Proceedings: 67 Scientific Presentations/Abstracts: 123 Technical Presentations: 187 Popular Articles and Reports:

13 8. Current or previous research on the Subject Dr. Hauck has recently started his research program on coccidiosis. Besides molecular biological characterization of Eimeria maxima isolates, he has conducted a first animal experiment the testing the efficacy of alternative products against coccidia and investigating the changes in the microbiota after infection with coccidia. Dr. Macklin has a long history of working with coccidia and Necrotic Enteritis. Currently he is conducting Antimicrobial Sensitivity Tests of recent field isolates. 9. Facilities and Equipment Required and Available The trials require a broiler house with at least 24 pens and cages for the seeder birds under BSL 1 conditions. These as well as computers and office space are provided by the Department of Poultry Science of Auburn University. 10. Research timetable Date Event / milestone 09/01/2018 Begin of project October - November 2018 January 2019 February - March 2019 August 2019 September - October 2019 January 2020 February - March st trial Seeder birds for 2nd trial 2 nd trial Seeder birds for 3rd trial 3 rd trial Seeder birds for 4th trial 4 th trial 11

14 11. Personnel Support Provided by the University R. Hauck, Assistant Professor PI and management of bird trials K.S. Macklin, Professor co-pi and management of bird trials T. Dormitorio, Laboratory Technician Support for sample collection and parasitological investigations J.T. Krehling, Laboratory Technician Poultry Farm Personal Support for sample collection and parasitological investigations Facility maintenance 12. Financial Support from the university a. From University: None, except the salaries noted above will be supported by Auburn University. b. Other Funding Sources: No other pending funding sources have been identified for this research. 13. Institutional Units Involved All research activities will be performed in the Department of Pathobiology and the Department of Poultry Science at Auburn University. 14. Budget One Master student will commit 50% of their time conducting the animal studies, investigating the samples and help with the analysis of the data ($8,080 + $234 fringe benefits + $808 tuition each year). The amount for material and supplies include $6,000 for 4,000 sexed birds (800 per trial plus 200 seeder birds per trial; each bird $1.5) and $24,000 for 24 tons of feed; these costs are split evenly between the two years. In the first year, additionally $1,500 for laboratory material to determine the coccidia counts and for the coccidia vaccine used to infect the seeder birds are included. This amounts to costs for supplies of $16,500 in the first year and $15,000 in the second year. $2,000 for travel in the second year will cover the cost for presenting the results at one national scientific meeting. 15. Total funds requested: $59,505 over twenty four months. 12

15 16. Indirect Costs: 15% = $7, Receipt of Funds Needed Biannually: Make Check Payable to: Auburn University Contracts and Grants Accounting 208 M. White Smith Hall Auburn, AL Mail Check to: Auburn University Contracts and Grants Accounting 208 M. White Smith Hall Auburn, AL IACUC statement All activities in this proposal comply with the provisions of the Institutional Animal Care and Use Committee as specified by the Animal and Plant Inspection Service, USDA in 9 CFR Part 1 (1091). 19. Statement I The Department of Poultry Science of Auburn University agrees to provide the following to USPOULTRY: (a) Progress reports on the research project every six months until the project is completed; (b) Within three months following completion of the research funded, to provide the final project report (using the format for final reports) of the results; (c) The University understands that USPOULTRY will retain 25 percent of the approved funds until the final report has been provided to the association; and (d) give permission to the association to provide the information to the industry. 20. Statement II Progress reports will be submitted every six months. 21. Statement III USPOULTRY makes no claim on discoveries or invention patents made by scientist/institutions utilizing USPOULTRY research funds. USPOULTRY assumes no liability associated with either the conduct of research or the outcome or use of research findings acquired with USPOULTRY funds. 13