Recipient Organization
UNIVERSITY OF GEORGIA
200 D.W. BROOKS DR
ATHENS,GA 30602-5016
Performing Department
Poultry Science
Non Technical Summary
The emergence of antimicrobial or antibiotic resistant pathogens from food animal production has restricted the application of antimicrobial compounds in the poultry industry. Both European Union as well as USA has banned the application of medically important antibiotic growth promoters in poultry production. Removing antimicrobial compound application has increased the incidences of poultry gut pathogen infections, namely coccidiosis, necrotic enteritis, salmonellosis and campylobacteriosis. Probiotics stimulate the immune responses, competitively exclude pathogenic microbes, stimulate digestive enzymes, and produce antibacterial substances. Prebiotics, which are often supplemented in conjunction to probiotics, are non-digestible carbohydrates that stimulate the growth of beneficial bacteria within the intestines of the host. Supplementation of prebiotics has been shown to modulate the gut microbiota by increasing specific beneficial bacteria as well as mimicking attachment sites of pathogens to reduce pathogenic bacteria adherence to the intestinal wall. Nanoparticle vaccines designed to deliver antigens against intestinal pathogens can deliver vaccine payload through an oral route and are ideally suited elicit protective mucosal immunity against gut pathogens. This project will apply commercially available nutritional supplements or nanoparticle vaccine to control coccidiosis, necrotic enteritis, Salmonella, Campylobacter infections of poultry.
Animal Health Component
25%
Research Effort Categories
Basic
50%
Applied
25%
Developmental
25%
Goals / Objectives
Manipulation of the gut microbiota by supplementations with prebiotics and probiotics have shown promising results in controlling bacterial and protozoalinfections in poultry. Interest in probiotic microorganisms and prebiotic oligosaccharides has increased in the poultry community due to the restriction of antibiotic growth promotors as well as the emergence of antibiotic resistant pathogens, such as Salmonella. Due to the size similarities to cellular components, nanoparticles can enter living cells using the cellular endocytosis mechanism, in particular by pinocytosis, and hence are an ideal way to deliver antigen loads to immune cells through an oral route. Applying prebiotics/probiotics/nanoparticle vaccine has potential to replace antimicrobial compoundsGoals / Objectives:Replace antimicrobial products in poultry with Prebiotics/probiotics/nanoparticle vaccines to control coccidiosis, necrotic enteritis, Salmonella, and Campylobacter infections of poultry
Project Methods
Design of Chitosan and Polyanhydride Nanoparticle vaccines: Chitosan (ionic crosslinking) and polyanhydride (solvent displacement) nanoparticles will be prepared as described earlier (Dhakal, et al., 2018) and stored after lyophilization using 5% sucrose as cryoprotectant at -20°C until use. Briefly, for polyanhydride vaccine, 100mg polyanhydride dissolved in acetone will be mixed with 5mg of extracellular protein with span-80 under magnetic stirring. Ethanol will be added dropwise to form nanoparticles entrapped with CP extracellular protein. Subsequently, CP extracellular protein will be added to preformed particles and stirred for 1 hour for surface conjugation. Organic solvent will be removed under reduced pressure and the surface bound particle will be stabilized using the ionic crosslinker, 1.3-diaminopropane.Chitosan nanoparticles will be prepared by using the ionic crosslinking method, where nanoparticles form by intra- and inter-molecular crosslinking between the positively charged chitosan and the negatively charged sodium tripolyphosphate (TPP), as described previously (Dhakal, et al., 2018). Briefly, 50 mg of chitosan dissolved in water will be mixed with 2.5 mg CP extracellular proteins dissolved in PBS. 25 mg of TPP will be added under magnetic stirring to form the nanoparticles. The nanoparticles will be collected by centrifugation (10,000 ×g; 10 min).Both nanoparticles will be characterized for cytotoxicity, morphology, protein loading efficiency and pH stability as described previously (Dhakal, et al., 2018).Effect of nanoparticle vaccine on decreasing necrotic enteritisOne day old SPF broiler chicks (n=126) will be procured and will be distributed into one of the three treatment groups: a) no infection, (b) no vaccine and challenged with necrotic enteritis, (c) chitosan+polyanhydride vaccine and challenged with NE. The amount of CP in chitosan and polyanhydride nanoparticles used in this study will be based on preliminary studies. Birds in the vaccination groups will be inoculated on d1, 7, and 14 with chitosan or polyanhydride nanoparticle vaccine. Broilers in challenged group will be challenged with 10,000 oocysts of Eimeria on d14, and inoculated 5,000 CFU C. perfringens on day 17, and birds in non-challenged group will be inoculated with same amount of PBS. On Day 14 and 21, growth performance will be measured, and on Day 21, lesion score and C. perfringens levels will be measured. Six birds from each group will be euthanized at d24, 27, and 32. Serum, cloacal swabs, and bile samples will be collected for analyzing anti-CP specific IgA and IgG antibodies. Splenocytes will be analyzed for recall response, and liver and cecal tonsils will be analyzed for cytokines. Feed consumption and body weight gain will be measured at d21 and d31 to ensure that the nanoparticle vaccines do not have adverse effects on production performance.Effect of Synbiotic supplementation on Salmonella or Necrotic enteritisWe earlier showed that supplementing a commercially available synbiotic decreases Salmonella load in poultry [5, 6]. This study will quantify the ability of a synbiotic that contains Lactobacillus reuteri, Pediococcus acidilactici, Bifidobacterium animalis and Enterococcus faecium. L. reuteri on necrotic enteritis severity.Approach: There will be five treatments: 1) non-challenge control, 2) challenge control, 3) 0.05% synbiotic, 4) 0.10% synbiotic, and 5) 0.15% synbiotic. Synbiotics will be mixed in a basal diet. 300 Cobb 500 birds will be used in the study. There will be 10 birds per replicated battery cage and 6 replicate cages per treatment. Broilers in challenged group will be inoculated with 10,000 oocysts of Eimeria on d14, and inoculated 5,000 CFU C. perfringens on day 17, and birds in non-challenged group will be inoculated with same amount of PBS. On Day 14 and 21, growth performance will be measured, and on Day 21, lesion score and C. perfringens levels will be measured. Proinflammatory (IL-1, LITAF), anti-inflammatory (IL10) cytokine mRNA levels, anti-CP specific bile IgA will be measured. Similar approach will be conducted to study the effects of symbiotic on Salmonella where birds will be challenged with 1 X 109 CFU on d 21 in the above model.Effect of acidifier supplementation on Salmonella shedding in poultryThe overall objective of this proposal is to determine the effects of a commercially available acidifier (Biotronic Top3, Biomin) on performance, cecal Salmonella load, and Salmonella contamination in meat of broilers subject to a Salmonella enterica serovar Enteritidis. On day of hatch (DOH), chicks will be randomized into five treatments: 1) non-challenge control, 2) challenge control, 3) 0.1% acidifier, 4) 0.2% acidifier, and 5) 0.4% acidifier treatment groups based on body weight. All groups will be fed their respective diets throughout the 42 day period. At 21 days of age, birds will be individually challenged with 1 ml of 1 x 108 CFU field strain of Salmonella Enteritidis via oral gavage. On days 24, 28, 35, and 42, ceca will be collected for enumeration and incidence of Salmonella. Performance parameters to be measured