Progress 09/01/04 to 03/08/07
Outputs
Progress Report Objectives (from AD-416) Optimize current Mycoplasma gallisepticum (MG) vaccination protocols for layer hens and determine the mechanisms by which MG vaccinations induce immunological changes. Design and produce an efficacious recombinant MG vaccine by molecular technologies for use in egg-type (layer hen) and potentially meat-type (broiler and turkey) production systems. Approach (from AD-416) A secondary vaccination with the F strain MG will be performed on layer chickens previously treated with the MG vaccines, ts-11 or 6/85. Efficacy of the secondary treatment will be determined and effects on layer hen productivity, physiology, and immunology will be quantitated. Effects of the secondary vaccination on eggs produced by treated layer hens will be investigated. The effect of MG vaccinations on B- and T- cell subpopulations will be determined via flow cytometry. Real-time RT- PCR will be used to determine changes in cytokine mRNA levels of interferon-gamma, interleukin-1, interleukin-6, interleukin-15, and interleukin-18 immediately preceding and following vaccinations. Transformation procedures will be developed for the commensal Mycoplasma gallinarum. Concurrently, surface proteins of M. gallinarum will be identified and their associative genes will be cloned. Putative MG genes (e.g. GapA and PvpA), will then be surface-expressed in recombinant strains of the M. gallinarum via protein fusions utilizing the C-terminal end of M. gallinarum surface proteins and the N-terminal end of MG antigens/colonization factors. The resulting recombinant M. gallinarum specie(s) will be evaluated for stability and pathogenicity, transmission, and protection against MG infection. Accomplishments A new product (SPRAY-VAC STABILIZER) was developed for use with poultry vaccines. The live, bacterial organism Mycoplasma gallisepticum (MG) is available as a vaccine for use in the table egg sector of the poultry industry to control losses of 16 eggs per hen. However the vaccine is killed by chlorine in water and also by conditions that deviate from isotonicity. A product was developed and is being commercially marketed which adjusts the water (regardless of source ie., distilled, municipal or well) used in reconstituting and administering live Mycoplasma gallisepticum vaccines to poultry such that the optimal pH of 7.8 is produced while simultaneously adjusting the osmolarity to effect isotonicity. This product will prolong the life of the organism as it is being administered to poultry which often requires up to 50 minutes in order to vaccinate a house of 75,000 chickens. This research addresses USDA-ARS Animal Health National Program 103 Component 4: Countermeasures to Prevent and Control Respiratory Diseases and specifically Problem Statement 4C. Poultry Respiratory Diseases. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 23
Impacts
(N/A)
Publications
- Pharr, G.T., Branton, S.L., Cooksey, A.M., Hanson, L.A., Burgess, S.C., Bottoms, C.C., Mccarthy, F.M., Collier, S.D. 2006. The Recognition of a vlhA Protein from the F-Strain of Mycoplasma gallisepticum with Monoclonal Antibody 6F10. International Journal of Poultry Science. 5(8):789-795, 2006.
- Collier, S. D., Pharr, G. T., Branton, S. L., Evans, J. D., Leigh, S. A., and Felfoldi, B. 2006. Initial Proteomics Analysis of Differentially Expressed Proteins from Mycoplasma gallisepticum Vaccine Strains ts-11 and F Detected by Western Blotting. Int. J. of Poultry Sci. 5(4): 330-336.
- Peebles, E.D., Branton, S.L., Burnham, M.R., Whitmarsh, S.K., Gerard, P.D. 2007. Effects of Supplemental Dietary Phytase and 25- Hydroxycholecalciferol on the Blood Characteristics of Commercial Layers Innoculated Before or at the Onset of Lay with the F-Stain of Mycoplasma gallisepticum. Poultry Science. 86:768-774.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned to National Program (NP) 103 Animal Health. Fully 90% of commercial egg laying chickens are believed infected with Mycoplasma gallisepticum (MG) which results in decreased egg production, increased pharmaceutical costs, and mortality at an estimated cost of $140 million dollars/year. MG infection of broiler breeders is sporadic and results in either condemnation and slaughter of infected hens and/or infection of progeny which in turn, results in increased mortality, feed costs and slaughter house condemnation. "Backyard" flocks exist as primary reservoirs of infection. Numerous other species of Mycoplasma have been found in commercial poultry. Fully 20% of the approximately 140 known mycoplasmas can be found in birds and of these about one-half (12) are found in
poultry. Of these 12, only four have been investigated to any depth. At least five of the 12 mycoplasmas found in poultry can be isolated in commercial layer chickens. We do not know the effect(s) of these other mycoplasmal species on poultry and, coupled with the fact that mycoplasmal infections in poultry are not cleared with antibiotics (once infected, a chicken remains infected for the duration of its life) and mycoplasmal infections together with poultry respiratory viral pathogens are extremely deleterious; mycoplasmal infection in commercial chickens continues to present a dreaded scenario for the poultry industry. 2. List by year the currently approved milestones (indicators of research progress) Research Study Component: 1. Novel MG Vaccine Regiments a. 12 months - Complete 6/85 - F strain study (trial 1) and initiate 6/85 - F strain study (trial 2). b. 24 months - Complete 6/85 - F strain study (trial 2) and initiate ts- 11 - F strain study (trial 1). c. 36 months -
Complete ts-11 - F stain study (trial 1) and initiate ts- 11 - F strain (trial 2). d. 48 months - Complete ts-11 - F strain study (trial 2) and assess data to discern most efficacious vaccination regimen. 2. Novel Recombinant MG Vaccine(s) a. 12 months - Establish gene transfer in M. galllinarum and clone M. gallinarum membrane proteins. b. 24 months - Express reporter protein in M. gallinarum via protein fusions. c. 30 months - Express MG antigens in M. gallinarum via single protein fusions and assess efficacy of product. d. 36 months - Express multiple efficacious proteins in single M. gallinarum and assess effects on cell. e. 48 months - Complete in vivo experiments via laying trials to determine efficacy of recombinants as MG vaccines and determine immunological impact of efficacious vaccines. 4a List the single most significant research accomplishment during FY 2006. This accomplishment addresses the NP 103 Animal Health Component 4: "Counter Measures to Prevent and
Control Respiratory Diseases." Optimizing Vaccine Pressurization: Research into live Mycoplasma gallisepticum (MG) vaccine administration to layer chickens has shown nozzles used to administer live MG vaccines are extremely variable and in fact, in many instances the droplet size distribution are not known at all. Spray administration is extremely important in impacting subsequent seroconversion (positive blood tests). Research conducted consisted of utilizing the Carden-Prisock-Johnson (CPJ) Vaccinator to determine the optimum nozzle to dispense live MG vaccine. Live MG vaccine was mixed and then dispensed with coarse, medium or fine nozzles at 40psi. Dramatic increases in MG colony counts resulted from using the coarse spray nozzles as compared to the medium or fine spray nozzles. This accomplishment is important as it explains one factor (coarse spray nozzles) that can impact the administration of live MG vaccines which may, in turn, result in poor vaccination blood test
results. As a result of poor vaccine test results, re-vaccination must occur which entails costs of additional vaccine (approximately $1500/75,000 bird house) and labor. 5. Describe the major accomplishments to date and their predicted or actual impact. A commercial manufacturer for the CPJ vaccinator was identified and fabrication/production of the vaccinator was initiated in December 2005. Mycoplasma gallisepticum (MG) vaccination of table egg (layer) chickens has been labor intensive and costly (vaccine costs alone of approximately $1500/75,000 chickens) since the advent of the first live MG vaccine in 1988. In addressing problems of the table egg industry with MG vaccines, four major accomplishments have developed over the life of the project: 1) development of a self-propelled, constant speed spray vaccinator capable of vaccinating 75,000 chickens in 712 minutes with a 60% savings in labor and a concommitant 30% increase in seroconversion rate; 2) determination that the addition
of concentrated phosphate buffered saline to water used for live MG vaccine reconstitution is advantageous and important in maintaining the titer of the vaccine during administration and thereby positively impacting seroconversion; 3) determination of the optimal vaccine delivery pressure (40 psi + 5 psi) for live MG vaccine; and 4) determination of the optimal (coarse) spray nozzle for administration of live MG vaccine. In addition, a video was produced for instruction/direction for the CPJ vaccinator in the administration of live MG vaccine. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The CPJ vaccinator was turned over to a Mississippi-based manufacturing company for commercial manufacture which was initiated in December 2005. In addition,
information pertaining to use of the CPJ vaccinator for administering live MG vaccines as well as Newcastle and injections of bronchitis vaccines to layer chickens was provided to producers through both formal scientific venues as well as via one-on-one instruction in addition to a video. The technology is currently being utilized by the table egg (layer chicken) industry. The only constraint to adoption and durability of the technology is knowledge of its application through dissemination of the research which will be forthcoming in additional manuscripts and a workshop in mid-August 2006. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Branton, S. L., Evans, J. D., Leigh, S. A., Collier, S. D., Dozier III, W. A., Roush, W. B., Purswell, J. L., and Olanrewaju, H. A. 2006. Effects of overlaying F strain Mycoplasma gallisepticum onto commercial
layer hens previously vaccinated with 6/85 strain Mycoplasma gallisepticum. 143rd Annual American Veterinary Medical Association Annual Convention. Park, S. W., Peebles, E. D., Branton, S. L., Kidd, M. T., Whitmarsh, S. K., and Gerard, P. D. 2006. Effects of F-strain Mycoplasma gallisepticum inoculation at twelve or twenty two weeks of age and diet supplementation on the digestive and reproductive organ characteristics of commercial egg laying hens housed in a caged layer facility. Poultry Science. 85(Suppl. 1):40. Viscione, K. A., Peebles, E. D., Branton, S. L., Vance, A. M., Whitmarsh, S. K., Keirs, R. W., and Gerard, P. D. 2006. Effects of pre-lay 6/85 strain Mycoplasma gallisepticum inoculation on the digestive and reproductive organ characteristics of commercial laying hens when given alone or in conjunction with F-strain Mycoplasma gallisepticum inoculations during lay. Poultry Science. 85(Suppl. 1):40. Westmoreland, S. L., May, S. B., Gracey, M. I., Hawkins, D. L., Peebles, E.
D., Park, S. W., and Branton, S. L. 2006. Determination of eggshell microstructural characteristics and associated physiological profiles in MG-vaccinated egg-laying chickens. Poutlry Science. 85(Suppl. 1):138. Vance, A., Peebles, E., Branton, S., Whitmarsh, S., and Gerard, P. 2006. Effects of time specific F-strain Mycoplasma gallisepticum inoculation on performance characteristics of commercial laying hens. Poultry Science. 85(Suppl. 1):148. Basenko, E., Peebles, E., Branton, S., Whitmarsh, S., and Gerard, P. 2006. Digestive and reproductive organ characteristics of commercial egg laying hens inoculated with S5-Strain Mycoplasma gallisepticum at either 10, 22, or 45 weeks of age. Poultry Science. 85(Suppl. 1)148. Viscione, K., Peebles, E., Branton, S., Vance, A., Whitmarsh, S., and Gerard, P. 2006. Effects of pre-lay 6/85-strain Mycoplasma gallisepticum inoculation on performance characteristics of commercial laying hens when given alone or in conjunction with F-strain Mycoplasma
gallisepticum inoculations during lay. Poultry Science. 85(Suppl. 1)148-149. Evans, J. D., Leigh, S. L., Collier, S. D., and Branton, S. L. 2006. Serological response and in vivo persistence of Mycoplasma gallisepticum vaccine strain 6/85 in layers and broilers. Poultry Science. 85(Suppl. 1) 164. Branton, S., Evans, J., Leigh, S., Dozier, W., Roush, W., Collier, S., Purswell, J., and Olanrewaju, H. 2006. Effects of pressurization on aerosol application of F-strain Mycoplasma gallisepticum. Poultry Science. 85(Suppl. 1)186. Collier, S. D., Pharr, G. T., Branton, S. L., Evans, J. D., and Leigh, S. A. 2006. Initial proteomics analysis of differentially expressed proteins from ts-11 and F strain detected by Western blotting. Poultry Science. 85(Suppl. 1)192. Leigh, S. A., Evans, J. E., Branton, S. L., and Collier, S. D. 2006. Effects of salt concentration on Mycoplasma gallisepticum vaccine FVAX-MG survival in solution. Poultry Science. 85(Suppl. 1)196. Vance, A. M., E. D. Peebles, S. L.
Branton, S. K. Whitmarsh, and P. D. Gerard, 2006. Effects of time specific F-strain Mycoplasma gallisepticum inoculation overlays on pre-lay ts-11-strain Mycoplasma gallisepticum inoculation on performance characteristics of commercial laying hens. Proc. SPSS, Poultry Sci. 85(Suppl. 1):4(Abstr. #11), Certificate of Merit- Graduate student (Vance) research paper competition, 27th Annual Meeting of the Southern Poultry Science Society, 2006. Cagle, S. B., T. A. Lenarduzzi, E. D. Peebles, R. W. Keirs, S. L. Branton, and P. D. Gerard, 2006. Association of serum total bile acid concentrations with incidence of fatty liver hemorrhagic syndrome and F- strain Mycoplasma gallisepticum infection in commercial laying hens. J. Miss. Acad. Sci. 51(1):65. Basenko, E. Y., E. D. Peebles, S. L. Branton, S. K. Whitmarsh, and P. D. Gerard, 2005. Effects of S6-strain Mycoplasma gallisepticum inoculation at ten, twenty-two, or forty-five weeks of age on the performance characteristics of commercial egg
laying hens. Poultry Sci. 84:1663-1670.
Impacts
(N/A)
Publications
- Evans, J.D., Leigh, S.A., Branton, S.L., Collier, S.D., Pharr, G.T., Bearson, S.M. 2005. Mycoplasma gallisepticum: current and developing means to control the avian pathogen. Journal of Applied Poultry Research. 14:757- 763.
- Peebles, E.D., Basenko, E.Y., Branton, S.L., Whitmarsh, S.K., Gerard, P.D. 2006. Effects of s6-strain mycoplasma gallisepticum inoculation at ten, twenty-two, or forty-five weeks of age on the blood characteristics of commercial egg laying hens. Poultry Science. 85:2012-2018, 2006.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Fully 90% of commercial egg laying chickens are believed infected with Mycoplasma gallisepticum (MG) which results in decreased egg production, increased pharmaceutical costs, and mortality at an estimated cost of $140 million dollars/year. MG infection of broiler breeders is sporadic and results in either condemnation and slaughter of infected hens and/or infection of progeny which in turn, results in increased mortality, feed costs and slaughter house condemnation. "Backyard" flocks exist as primary reservoirs of infection. Numerous other species of Mycoplasma have been found in commercial poultry. Fully 20% of the approximately 140 known mycoplasmas can be found in birds and of these about one-half (12) are found in poultry. Of these 12, only four have been investigated to any depth. At least
five of the 12 mycoplasmas found in poultry can be isolated in commercial layer chickens. We do not know the effect(s) of these other mycoplasmal species on poultry and, coupled with the fact that mycoplasmal infections in poultry are not cleared with antibiotics (once infected, a chicken remains infected for the duration of its life) and mycoplasmal infections together with poultry respiratory viral pathogens are extremely deleterious; mycoplasmal infection in commercial chickens continues to present a dreaded scenario for the poultry industry. 2. List the milestones (indicators of progress) from your Project Plan. Research Study Component: 1. Novel MG Vaccine Regiments a. 12 months - Complete 6/85 - F strain study (trial 1) and initiate 6/85 - F strain study (trial 2). b. 24 months - Complete 6/85 - F strain study (trial 2) and initiate ts- 11 - F strain study (trial 1). c. 36 months - Complete ts-11 - F stain study (trial 1) and initiate ts- 11 - F strain (trial 2). d. 48
months - Complete ts-11 - F strain study (trial 2) and assess data to discern most efficacious vaccination regimen. 2. Novel Recombinant MG Vaccine(s) a. 12 months - Establish gene transfer in M. galllinarum and clone M. gallinarum membrane proteins. b. 24 months - Express reporter protein in M. gallinarum via protein fusions. c. 30 months - Express MG antigens in M. gallinarum via single protein fusions and assess efficacy of product. d. 36 months - Express multiple efficacious proteins in single M. gallinarum and assess effects on cell. e. 48 months - Complete in vivo experiments via laying trials to determine efficacy of recombinants as MG vaccines and determine immunological impact of efficacious vaccines. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Research Study Component: Novel MG Vaccine Regimens Complete 6/85 F strain study (trial 2)
and initiate ts-11 F strain study (trial 1). Milestone Substantially Met 2. Research Study-Component: Novel Recombinant MG Vaccine(s) Express reporter protein in Mycoplasma gallinarum via protein fusions. Milestone Not Met Other 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY 2006 Complete ts-11 F strain study (trial 1) and initiate ts-11 F strain study (trial 2). Also, to continue attempts at transformation of Mycoplasma gallinarum via conjugation using Enterococcus faecalis as well as to initiate the use of Mycoplasma gallinaceum (contingency plan) as a vehicle for expression of Mycoplasma gallisepticum antigens/colonization factors. FY 2007 Complete ts-11 F strain study (trial 2) and assess data to discern the most efficacious vaccination regimen. Also, assuming a method of transforming Mycoplasma gallinarum has been developed,
express reporter protein in Mycoplasma gallinarum via protein fusions. FY 2008 Milestones will be developed with new Project Plan. 4a What was the single most significant accomplishment this past year? Optimizing Vaccine Pressurization: Research into live Mycoplasma gallisepticum (MG) vaccine administration to layer chickens has shown that the pressure (psi) utilized for spray administration is extremely important in impacting subsequent seroconversion (positive blood tests). A survey of the layer chicken industry showed that pressure settings used for the administration of live MG vaccine varied from 35-70 psi. Research conducted consisted of utilizing the CPJ Vaccinator to determine the optimum pressure setting to dispense live MG vaccine. Live MG vaccine was mixed and then dispensed at either 40 or 60 psi. Dramatic increases in MG colony counts resulted from using the lower (40 psi) setting as compared to the higher 60 psi setting while subsequent tests showed that 40 psi + 5
psi made no difference in colony counts. This accomplishment is important as it explains one factor (pressure setting of the vaccinator) that can impact the administration of live MG vaccines which may, in turn, result in poor vaccination blood test results. As a result of poor vaccine test results, re-vaccination must occur which entails costs of additional vaccine (approximately $1500/75,000 bird house) and labor. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Mycoplasma gallispeticum (MG) vaccination of table egg (layer) chickens has been labor intensive and costly (vaccine costs alone of approximately $1500/75,000 chickens) since the advent of the first live MG vaccine in 1988. In addressing problems of the table egg industry with MG vaccines, three major accomplishments have developed over the life of the project: 1) the development of a self-propelled, constant speed spray vaccinator capable of vaccinating
75,000 chickens in 712 minutes with a 60% savings in labor and a concommitant 30% increase in seroconversion rate; 2) the determination that the addition of concentrated phosphate buffered saline to water used for live MG vaccine reconstitution is advantageous and important in maintaining the titer of the vaccine during administration and thereby positively impacting seroconversion; and 3) determination of the optimal vaccine delivery pressure (40 psi + 5 psi) for live MG vaccine. The above research is an offshoot of work currently being conducted on the 12 and 24 month milestones of the research study-component: Novel MG Vaccine Regimens. Further, it addresses the Strategies to Control Infectious and Non-Infectious Disease component of the NP 103 Animal Health National Program Action Plan and directly impacts Performance Measure 1.2.1 of the ARS Strategic Plan. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to
become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The journal article detailing the design of the CPJ Vaccinator was published in early 2005. The vaccinator was turned over to a local manufacturing company for commercial manufacture. In addition, information pertaining to both the inclusion of concentrated phosphate buffered saline to the water used for vaccine reconstitution as well as information pertaining to optimal pressure for dispensing live Mycoplasma gallisepticum (MG) vaccine was provided to producers through both formal scientific venues as well as via one-on-one instruction. The technology is currently being utilized by the table egg (layer chicken) industry. The only constraint to adoption and durability of the technology is knowledge of its application through dissemination of the research which will be forthcoming in additional manuscripts. 7. List
your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Park, S. W. 2005. The effects of F-strain Mycoplasma gallisepticum inoculation at twelve or twenty two weeks of age and diet supplementation on the performance and physiological characteristics of commercial layers. M. S. Thesis. Mississippi State, MS. 39762.
Impacts
(N/A)
Publications
- Branton, S.L., Lott, B.D., Evans, J.D., Collier, S.D., Roush, W.B., Bearson, S.M., Bearson, B.L., Pharr, G.T. 2005. A self-propelled, constant speed spray vaccinator for commercial layer chickens. Avian Diseases. 49:147-151.
- Peebles, E.D., Burnham, M.R., Walzem, R.L., Branton, S.L., Gerard, P.D. 2004. Effects of fasting on serum lipids and lipoprotein profiles in the egg-laying hen (gallus domesticus). Comparative Biochemistry and Physiology. Part A 138:305-311.
- Branton, S.L., Roush, W.B., Evans, J.D., Collier, S.D., Pharr, G.T. 2005. A self-propelled, constant speed spray vaccinator for commercial layer chickens. Southern Poultry Science Society Meeting. Abstract 210. p. 50.
- Collier, S.D., Pharr, G.T., Branton, S.L., Evans, J.D., Leigh, S.A. 2005. Proteomic analysis of mycoplasma gallisepticum vaccine strain f. American Society for Microbiology Annual Meeting. Abstract 0-022. p. 427.
- Collier, S.D., Pharr, G.T., Branton, S.L., Evans, J.D. 2005. Effects of mg vaccinations on b and t-cell populations in layer chickens. Southern Poultry Science Society Meeting. Abstract 206. p. 49.
- Evans, J.D., Collier, S.D., Pharr, G.T., Branton, S.L. 2005. In vivo persistence of mycoplasma gallisepticum vaccine strain 6/85 singly and following challence by mycoplasma gallisepticum f-strain. Southern Poultry Science Society Meeting. Abstract 84. p. 21.
- Park, S.W., Peebles, E.D., Branton, S.L., Kidd, M.T., Whitmarsh, S.K., Gerard, P.D. 2005. Effects of f-strain mycoplasma gallisepticum inoculation at twelve weeks of age and diet supplementation on the performance and egg characteristics of commercial layers. Southern Poultry Science Society Meeting. Abstract 84. p. 41.
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