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ACCESSION NO: 0168532 SUBFILE: CRIS
PROJ NO: ARK01611 AGENCY: CSREES ARK
PROJ TYPE: HATCH PROJ STATUS: EXTENDED MULTISTATE PROJ NO: NE-60
START: 01 OCT 1998 TERM: 30 SEP 2003 FY: 2002

INVESTIGATOR: Erf, G. F.

PERFORMING INSTITUTION:
POULTRY SCIENCES
UNIVERSITY OF ARKANSAS
FAYETTEVILLE, ARKANSAS 72703

GENETIC BASES FOR RESISTANCE AND IMMUNITY TO AVIAN DISEASES

OBJECTIVES

 Identify and characterize environmental, dietary and physiologic factors that modulate immune system development, optimal immune function and disease resistance in poultry genetic stocks.

APPROACH: Contributing research will include the Smyth line chickens which develop spontaneous post-hatch, autoimmune vitiligo. Three MHC-matched lines of chickens, all homozygous for the MHC B101 haplotype will be used in this project. Included are the autoimmune vitiliginous Smyth line (SL), the parental Brown line (BL), and the normally pigmented Light Brown Leghorn. Special emphasis will be placed on identifying environmental factors required for the expression of vitiligo in genetically susceptible SL chickens and on the immune mechanisms involved in autoimmune destruction of pigment cells in SL vitiligo. Additionally, immunomodulatory effects of dietary supplements on the avian immune system will be examined in broilers and in turkeys. Scientific methods used will include in vitro culture systems and flow cytometry.

PROGRESS: 2002/01 TO 2002/12
Mutant Smyth line chickens spontaneously develop post-hatch loss of eye and feather pigment. This loss of pigment is due to the destruction of pigment cells by the immune system. The similarities between the autoimmune loss of pigment cells in Smyth line chickens and the pigment loss observed in human vitiligo have lead to the acceptance of the SL chicken as the best animal model to study autoimmune vitiligo. During the last calendar year, we completed a study (funded by the National Vitiligo Foundation), on the role of environmental factors such as turkey herpesvirus (HVT) vaccine and other live virus vaccines (Newcastle disease virus), in the development of Smyth line vitiligo. We have shown that only live HVT vaccine can be associated with a higher incidence of vitiligo compared to control treatments. The fact that gluteraldehyde-fixed (dead HVT) did not increase the incidence of vitiligo above controls, points towards the importance of the HVT infection in triggering vitiligo expression. As HVT translocates to the feather tissue, where melanocytes are located, we hypothesize that the local immune response to HVT infection may lead to the destruction of already inherently defective melanocytes and the development of autoimmune vitiligo. In another vitiligo incidence study (funded by the National Vitiligo Foundation), we examined the effect of recombinant chicken interferon gamma (IFN-g) administered to SL chicks during the first 6 weeks of life compared to vehicle-injected chicks. The incidence of vitiligo by 20 weeks of age in IFN-g-injected chicks was 0 % for males and 87.5 % for females, whereas in vehicle-injected chickens the incidence of vitiligo at 20 weeks of age was 0 % for males and 25 % for females (none of the chickens were vaccinated at hatch - hence the low spontaneous incidence of vitiligo). The ability of IFN-g to induce expression of vitiligo in vitiligo susceptible SL chickens together with the observed gender difference in the effects of IFN-g on the expression of SL vitiligo adds to the value of this animal model for human autoimmune vitiligo and autoimmune disease in general. Currently, graduate students Amber Austin, Becky Lockhart, and Dilshika Wijesekera are all working on the melanocyte defect in Smyth line chickens that may lead to the recognition of the melanocytes by the immune system. Aspects examined include antioxidant state, extent of lipid-peroxidation, and oxidation of proteins in the target tissue, as well as the response of melanocytes to inflammatory agents and oxidative intermediates. These studies are conducted using ex vivo feather tissue and melanocyte cultures and are funded by the NIH-AREA grant (R15). Research on Smyth line chickens was presented at the International Meeting of the Pigment Cell Research Society at Egmond an Zee, the Netherlands and the Workshop on Molecular Pathogenesis of Marek's Disease and Avian Immunology, Limassol, Cyprus.

IMPACT: 2002/01 TO 2002/12

The use of an animal model that is genetically susceptible to development of autoimmune vitiligo provides an excellent opportunity to study the cause and effect relationship between genetic susceptibility and the factors leading to the onset and expression of autoimmune disease. Knowledge gained from these studies will find direct application in the management and prevention of autoimmune disease. Additionally, these studies on immune system dysfunction and mechanisms of pathogenesis will yield important new knowledge regarding immune system development and function in avian species.

PUBLICATIONS: 2002/01 TO 2002/12
1. Erf, G. F. 2002. Smyth line autoimmune vitiligo - similar to human autoimmune vitiligo. Pages 316-332 in Modern Concepts of Immunology in Veterinary Medicine-Poultry Immunology. Mathew, T., editor. Advances in Medical and Veterinary Virology, Immunology and Epidemiology, Thajema Publishers, Vineland, NJ.
2. Wang, W., R. F. Wideman, Jr., T. K. Bersi, and G. F. Erf. 2003. Pulmonary and hematological immune responses to intravenous cellulose micro-particles in broilers. Poult. Sci. in press.
3. Erf, G. F., T. K. Bersi, and H. S. Lillehoj. 2002. A role of interferon gamma in autoimmune vitiligo of Smyth line chickens. FEMS in press.
4. Wang, W., G. F. Erf, and R. F. Wideman. 2002. Effect of cage vs floor litter environments on the pulmonary hypertensive response to intravenous endotoxin and on blood-gas values in broilers. Poult. Sci. 81:1728-1737.
5. Wang, W., R. F. Wideman, and G. F. Erf. 2002. Pulmonary hypertensive response to endotoxin in cellulose-primed and unprimed broiler chickens. Poult. Sci. 81:1224-1230.
6. Wideman R. F., G. F. Erf, M. E. Chapman, W. Wang, N. B. Anthony, and L. Xiaofang. 2002. Intravenous micro-particle injections and pulmonary hypertension in broiler chickens: acute post-injection mortality and ascites susceptibility. Poult. Sci. 81:1203-1217.
7. Iqbal, M., J. D. Freiburger, G. F. Erf, and W. G. Bottje. 2002. Immunohistochemical evidence of cytochrome c oxidase subunit II involvement in pulmonary hypertension syndrome (PHS) in broilers. Poult. Sci. 81:1231-1235.
8. Wideman, R. F., and G. F. Erf. 2002. Intravenous microparticle injection and pulmonary hypertension in broiler chickens: Cardio-pulmonary hemodynamic responses. Poult. Sci. 81:877-886.

PROJECT CONTACT:

Name: Erf, G. F.
Phone: 501-575-8664
Fax: 501-575-3026
Email: gferf@comp.uark.edu

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ACCESSION NO: 0098242 SUBFILE: CRIS
PROJ NO: CA-V*-PHR-4652-AH96 AGENCY: CSREES CALB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1996 TERM: 30 SEP 2001 FY: 2000

INVESTIGATOR: Lam, K. M.

PERFORMING INSTITUTION:
POPULATION HEALTH & REPRODUCTION
UNIV OF CALIFORNIA (VET-MED)
DAVIS, CALIFORNIA 95616  

NEWCASTLE DISEASE VIRUS STRAINS

OBJECTIVES: A. To determine that Newcastle disease virus (ND V) can cause apoptosls in heterophils and macrophage of chickens. B. To determine that chicken heterophils and macrophage are capable of inducing oxidative burst, and the suppression of oxidative burst by Newcastle disease virus.

APPROACH: A. Heterophils and macrophages will be infected with ND V in vitro. Gel electrophoresis, electron microscopy, flow cytometry, and in situ hybridization will be used to confirm the presence of apoptosis in the infected cells. B. The ability of heterophils and macrophages to produce hydrogen peroxide will be determined by the stimulation of cells with dichclorofluorescein (DCF) and phorbol myristate acetate (PMA), and followed by flow cytometric examination. The effect of ND V on hydrogen peroxide production will also be determined. C. Heterophils and macrophages will be pre-treated with various recombinant human cytokines and then determine for their oxidative burst by DCF and PMA.

PROGRESS: 1996/10 TO 2001/09
The efforts in this year have been concentrated on chicken heterophils and thrombocytes and the effect of Newcastle disease virus on their functions. 1. It is widely believed that chicken heterophils and adherent cells do not have myeloperoxidase activity. However, this laboratory provides ample evidence showing that these cells do have myeloperoxidase activity. 2. Chicken thrombocytes are capable of chemotaxis and phagocytosis. These functions, however, are greatly reduced after a short-term incubation in vitro with Newcastle disease virus.

IMPACT: 1996/10 TO 2001/09
The goal of this project was to A. determine that Newcastle disease virus (ND V) can cause apoptosls in heterophils and macrophage of chickens. B. To determine that chicken heterophils and macrophage are capable of inducing oxidative burst, and the suppression of oxidative burst by Newcastle disease virus.

PUBLICATIONS: 1996/10 TO 2001/09
1. Lam KM. 1997. Myeloperoxidase activity in chicken heterophils and adherent cells. Vet. Immunol. Immunopathol. 57:327-335.
2. Lam KM. 1997. Activation, adhesion, migration and death of chicken thrombocytes. Comp. Haematol. Intl. 1:81-87.

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ACCESSION NO: 0182013 SUBFILE: CRIS
PROJ NO: CALV-AH-176 AGENCY: CSREES CALV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 1998 TERM: 30 SEP 2003 FY: 2001

INVESTIGATOR: Gardner, I. A.

PERFORMING INSTITUTION:
MEDICINE & EPIDEMOLOGY
UNIV OF CALIFORNIA (VET-MED)
DAVIS, CALIFORNIA 95616

QUANTITATIVE METHODS TO CERTIFY FREEDOM OF ANIMALS FROM PATHOGENS

OBJECTIVES: 1. Develop a Bayesian approach to certify disease freedom of a country/region that incorporates uncertainty in probability estimates. 2. Compare frequentist and Bayesian approaches to certify disease freedom using common data sets and to compare sample size requirements for surveys with both approaches.

APPROACH: 1. The Bayesian approach will be implemented with the Gibbs sampler, an interactive Markov-chain Monte Carlo method. The mathematical calculations will incorporate the prior probability that a country is free of disease, the uncertainty in sensitivity and specificity estimates and the possible clustering of positive test results at a herd level. The output will be a probabilistic estimate of disease freedom. 2. Frequentist and Bayesian estimates will be compared with common published data sets on porcine reproductive and respiratory syndrome and Newcastle Disease. The effect of selected prior distributions for the Bayesian approach will be evaluated. Sample sites used in frequentist calculations for surveys will be compared with estimates that we will derive using Bayesian approaches.

NON-TECHNICAL SUMMARY: If countries and regions are able to "certify" freedom from important animal pathogens, trade opportunities may increase and product export costs may decrease. To develop a Bayesian statistical approach (using Gibbs sampling) to quantification of disease freedom. The output from the model will be probability distributions that can be used to make inferences about the proportion of diseased herds, within-herd prevalence, and the probability that a country is free of disease. The research will be involve collaboration with others in the US, Australia and Switzerland.

PROGRESS: 2002/01 TO 2002/12
Quantitative approaches are needed to allow scientifically-valid inferences about freedom of animals from important pathogens that affect animal trade. Freedom in the context of these inferences includes a pathogen prevalence less than a threshold (e.g. <0.2% of infected herds). We developed a hierarchical Bayesian statistical model that uses herd-level test results from multiple herds in a region or country or zone, and adjusts for uncertainty in the sensitivity and specificity of tests and the prior probability of infectious agent. The model allows inferences about the post-test probability of freedom from infection, the proportion of infected herds, and the within-herd prevalence. Using published survey data for porcine reproductive and respiratory syndrome and Newcastle Disease in poultry, we have shown that inferences from our Bayesian approach are similar to those from an alternate simulation modeling approach. The Bayesian model is superior to previous methods because it allows inferences about the proportion of infected herds and within-herd prevalence which are important inputs into risk assessment models. The model has been modified to include the possibility of different sample sizes in each of the herds, and the use of additional tests in animals that are positive on the first screening test.

IMPACT: 2002/01 TO 2002/12
The new method has potential to be used internationally as a tool in substantiating a country's claim of freedom from animal pathogens.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

PROJECT CONTACT:

Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

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ACCESSION NO: 0177509 SUBFILE: CRIS
PROJ NO: CALV-CAHFS95CDFA6601 AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 JUL 1994 TERM: 30 JUN 2004 FY: 2001

INVESTIGATOR: Ardans, A. A.

PERFORMING INSTITUTION:
ADMINISTRATION
UNIV OF CALIFORNIA (VET-MED)
DAVIS, CALIFORNIA 95616

CALIFORNIA ANIMAL HEALTH AND FOOD SAFETY SYSTEM

OBJECTIVES: To provide laboratory diagnostic support of the highest quality for the surveillance and control of diseases and the enhancement of health of livestock and poultry in California, including programs designed to protect people from animal diseases transmissible to humans.

APPROACH: The CVDLS is composed of a full service, central reference laboratory at UC Davis and four laboratories located at Turlock (poultry), Fresno (poultry and regulatory services), Tulare (mammalian services) and San Bernardino (general services for poultry, mammalian and regulatory). These laboratories are linked by a computer based Management Information and Surveillance System to function as a single entity.

PROGRESS: 2002/01 TO 2002/12
Avian influenza, H6N2, reoccurred in Southern California with many flocks experiencing significant mortality and egg production drops of considerable duration. In contrast to previous years, numerous flocks in central California also became infected. Chickens in active egg production were most severely affected with little disease in younger pullets. Severe reproductive tract disease presenting with necrotic salpingitis and yolk peritonitis were consistent observations. Laboratory support included postmortem examination, virus isolation, and serology for commercial flocks. USDA and CDFA approved the use of vaccine in commercial flocks for which laboratory monitoring was provided. A trial is underway to compare commercially available influenza antigen rapid detection kits with virus isolation. The CAHFS was federally approved to participate in bovine tuberculosis slaughter plant surveillance, which has resulted in an increased sample submission and improved surveillance. Through this program three bovine tuberculosis infected dairy herds were discovered resulting in extensive testing and postmortem examination of over 200 cattle. One dairy, 6,400 cows, has been depopulated and another two herds are being scheduled. An entire herd test (2,496 cows) using the gamma interferon test, was conducted and results are being compared with caudal fold and comparative cervical testing along with postmortem findings. In late September exotic Newcastle disease was diagnosed in Southern California game birds by the San Bernardino branch, CAHFS. The disease was found to be rapidly spreading. Widespread infection was found among game birds, which unfortunately spread to commercial egg producing facilities. Late on December 24 the disease was diagnosed on a commercial premise with over one million birds and to date over three million birds have been euthanized along with over 110,000 game birds. The massive laboratory support required in support of the eradication has suspended many of the ongoing creative investigative efforts. Significant effort has been dedicated to the development and validation of rapid molecular based assays and is being coordinated with efforts at Southeastern Poultry Research Laboratory and the National Veterinary Services Laboratory (NVSL). Considerable personnel support has been provided by laboratories throughout the country, including NVSL who have provided pathologists, virology technicians, and clerical assistance. Milk is routinely screened for beta-lactam antibiotics using highly sensitive, rapid screening tests, which are known to have considerable false positive results, which result in the dumping of an entire tank truckload of milk. CAHFS developed a quantitative method using liquid chromatography with tandem mass spectrometry for beta-lactam antibiotics, which has been used to screen potentially contaminated large amounts of cheese. The assay was also used in a highly visible antibiotic contamination that occurred in a large bay area milk processing plant, which resulted in the recall of over 800,000 gallons of milk and milk products.

IMPACT: 2002/01 TO 2002/12
The resources of the California Animal Health and Food Safety Laboratory System (CAHFS) in concert with School of Veterinary Medicine researchers continue to demonstrate and investigate naturally occurring conditions of economic significance to California's production animal agriculture. During 2002 much of the system's resources has been dedicated to major animal health issues affecting California livestock and poultry.

PUBLICATIONS: 2002/01 TO 2002/12
1. Adaska JM, Munoz-Zanzi CA, Hietala SK. 2002. Evaluation of result variability using a commercial Johne's disease ELISA kit and repeat testing of samples. Journal of Veterinary Diagnostic Investigation. 14:423-426.
2. Chin RP. 2002. Isolation of an unidentified, nonfermentative, gram-negative bacterium from turkeys and chickens: 38 cases (1995-2001). Avian Diseases, 46:447-452.
3. Colagross-Schouten AM, Mazet JA, Gulland FM, Miller MA, Hietala SK. 2002. Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California. Journal of Wildlife Diseases, 38:7-17
4. Cramer G, Kelton D, Duffield TF, Hobson JC, Lissemore K, Hietala SK, Peregrine AS. 2002. Neospora caninum serostatus and culling of Holstein cattle. Journal of the American Veterinary Medical Association, 221:1165-1168.
5. Crespo R, Ghazikhanian GY, Hall CI. 2002. Avulsion of the common retinaculum in meat turkeys. Avian Diseases, 46:245-248.
6. Crespo R, Stover SM, Shivaprasad HL, Chin RP. 2002. Microstructure and mineral content of femora in male turkeys with and without fractures. Poultry Science, 81:1184-1190.
7. Crespo R, Woolcock PR, Fadly AM, Hall C, Shivaprasad HL. 2002. Characterization of T-cell lymphomas associated with an outbreak of reticuloendotheliosis in turkeys. Avian Pathology, 31:355 -361.
8. Daft BM, Barr BC, Gardner IA, Read D, Bell W, Peyser KG, Ardans A, Kinde H, Morrow JK. 2002. Sensitivity and specificity of western blot testing of cerebrospinal fluid and serum for diagnosis of equine protozoal myeloencephalitis in horses with and without neurologic abnormalities. Journal of the American Veterinary Medical Association, 221:1007-1013.
9. Driessen B, Zarucco L, Steffey EP, McCullough C, Del Piero F, Melton L, Puschner B, Stover SM. 2002. Biochemical and histopathological changes associated with prolonged sevoflurane anaesthesia in horses. Journal of Veterinary Medicine, A 49:1-11.
10. Fosgate GT, Adesiyun AA, Hird DW, Hietala SK, Ryan J. 2002. Isolation of Brucella abortus biovar 1 from cattle and water buffalo on Trinidad. Veterinary Record, 151:272-273.
11. Fosgate GT, Adesiyun AA, Hird DW, Johnson WO, Hietala SK, Schurig GG, Ryan J. 2002. Comparison of serologic tests for detection of Brucella infections in cattle and water buffalo (Bubalus bubalis). American Journal of Veterinary Research, 63:1598-1605.
12. Fosgate GT, Hird DW, Read DH, Walker RL. 2002. Risk factors for foals developing Clostridium piliforme infection (Tyzzer's Disease) on a California Thoroughbred breeding farm. Journal of the Veterinary Medical Association, 220:785-790.
13. Gordus AG, Shivaprasad HL, Swift P. 2002. Salt toxicosis in ruddy ducks that winter on an agricultural evaporation basin in California. Journal of Wildlife Diseases, 38:124-131.
14. Haqshenas G, Huang FF, Fenaux M, Guenette DK, Pierson FW, Larsen CT, Shivaprasad HL, Toth TE and Meng XJ. 2002. The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and the chicken big liver and spleen disease virus. Journal of General Virology, 83:2201-2209.
15. Hobson JC, Duffield TF, Kelton D, Lissemore K, Hietala SK, Leslie KE, McEwan B, Cramer G, Peregrine AS. 2002. Neospora caninum serostatus and milk production of Holstein cattle. Journal of the American Veterinary Medical Association, 221:1160-1164.
16. Holstege DM, Puschner B, Whitehead G, and Galey FD. 2002. Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS. Journal of Agriculture Food Chemicals, 50:406-411.
17. Huang FF, Haqshenas G, Shivaprasad HL, Guenette DK, Woolcock PR, Larsen CT, Pierson FW, Elvinger F, Toth TE and Meng XJ. 2002. Heterogeneity and Seroprevalence of the Newly Identified Avian Hepatitis E Virus from Chickens in the United States. Journal of Clinical Microbiology, 40:4197-4202.
18. Munoz-Zanzi CA, Thurmond MC, Johnson WO, Hietala SK. 2002. Predicted age of dairy calves when colostrum-derived BVDV antibodies would no longer offer protection against disease or interfere with vaccination. Journal of the American Veterinary Medical Association, 221:678-685.
19. Nieto JE, Spier S, Pipers FS, Stanley SD, Smith D, and Snyder JR. 2002. Comparison of paste suspension formulated omeprazole in the healing of gastric ulcers in racehorses in active training. Journal of the American Veterinary Medical Association, 221:1139-1143
20. Peroni DL, Stanley S, Kollias-Baker C, Robinson NE. 2002. Prednisone per os is likely to have limited efficacy in horses. Equine Veterinary Journal, 34:283-287.
21. Puschner B, Booth MC, Tor ER, Odermatt A. 2002. Diterpenoid alkaloid toxicosis in cattle in Switzerland. Veterinary and Human Toxicology, 44:8-10.
22. Ridpath JF, Hietala SK, Sorden S, Neil JD. 2002. Evaluation of the reverse transcription-polymerase chain reaction/probe test of serum samples and immunohistochemistry of skin sections for detection of acute bovine viral diarrhea infections. Journal of Veterinary Diagnostic Investigation, 14:303-307.
23. Riggs SM, Puschner B, Tell LA. 2002. Management of an ingested lead foreign body in an Amazon Parrot. Veterinary and Human Toxicology, 44:345-348.
24. Shilton CM, Smith DA, Woods LW, Crawshaw GJ, Lehmkuhl HD. 2002. Adenoviral infection in captive moose (Alces alces) in Canada. Journal of Zoo and Wildlife Medicine, 33:73-79.
25. Shivaprasad HL, Crespo, R, Puschner B, Lynch S, Wright L. 2002. Myopathy in brown pelicans (Pelicanus occidentalis) associated with rancid feed. Veterinary Record, 150: 307-311.
26. Shivaprasad HL, Kim TJ, Woolcock PR, Tripathy DN. 2002. Genetic and antigenic characterizations of a poxvirus isolate from ostriches. Avian Diseases, 46:429-436.
27. Shivaprasad HL, Woolcock PR, McFarland MD, Curtis M, Karabatsos N. 2002. Turlock-like bunyavirus associated with encephalomyelitis and myocarditis in an ostrich chick. Journal of Veterinary Diagnostic Investigation, 14:363-370.
28. Shivaprasad HL and Droual R. 2002. Pathology of an atypical strain of P. gallinarum in chickens. Avian Pathology, 31:399-406.
29. Stamm LV, Bergen HL, Walker RL. 2002. Molecular typing of papillomatous digital dermatitis associated Treponema isolates based on analysis of the 16S-23S rDNA intergenic spacer regions. Journal of Clinical Microbiology, 40:3463-3469. Stott JL, Blanchard MT, Anderson M, Mass J, Walker RL, Kennedy PC, Norman BB, BonDurant RH, Oliver MN, Hanks D, Hall MR. 2002. Experimental transmission of epizootic bovine abortion (foothill abortion). Veterinary Microbiology, 88:161-173.
30. Suarez DL, Woolcock PR, Bermudez AJ, Senne D. 2002. Isolation from turkey breeder hens of a reassortant H1N2 influenza with swine, human and avian lineage genes. Avian Diseases, 46:111-121.
31. Taduesz FM, and Stanley SD. 2002. Improved sythesis of 13C,2H3- and 2H3-salmeterol by Cs2CO3-mediated monoalkylation of a primary amine. Journal of Labeled Compounds and Radiopharmaceuticals, 45:755-762.
32. Tegzes J, Puschner B. 2002. Amanita mushroom poisoning - efficacy of aggressive treatment in 2 dogs. Veterinary and Human Toxicology, 44:96-99.
33. Tegzes JH, Smarick SD, Puschner B. 2002. Coma and apnea in a dog with hydroxyzine toxicosis. Veterinary and Human Toxicology, 44:24-26.
34. Thurmond MC, Wesley OJ, Munoz-Zanzi CA, Chun-Lung S, Hietala SK. 2002. A method of probability diagnostic assignment that applies Bayes theorum for use in serologic diagnostics, using an example of Neospora caninum infection in cattle. American Journal of Veterinary Research, 63:318-325.
35. Turay HO, Caldwell A, Barr BC, Branson KR, Cockrell MKR, Marsh AE. 2002. Sarcocystis neurona reacting antibodies in Missouri feral domestic cats (Felis domesticus) and their role as an intermediate host. Parasitology Research, 88:38-43.
36. Van Hoogmoed LM, Harmon FA, Stanley SD, White J, Snyder J. 2002. In vitro investigation of the interaction between nitric oxide and cyclo-oxygenase activity in the equine ventral colon smooth muscle. Equine Veterinary Journal, 34:510-515.
37. Walker RL, Read DH, Hayes DC, Nordhausen RW. 2002. Equine abortion associated with the Borrelia parkeri-B. turicatae tick-borne relapsing fever spirochete group. Journal of Clinical Microbiology, 40:1558-1562.
38. Webby RJ, Woolcock PR, Krauss SL, Webster RG. 2002. Reassortment and influenza transmission of North American H6N2 influenza viruses. Virology, 295:44-53.
39. Woolcock PR, McFarland MD, Lai S and Chin RP. 2002. Enhanced Recovery of Avian Influenza Virus Isolates using a Combination of Chicken Embryo Inoculation Methods. Avian Diseases, 45:1030-1035.
40. Zakhartchouk A, Bout A, Woods LW, Lehmkuhl HD, Havenga MJE. 2002. Odocoileus hemionus deer adenovirus is related to the members of Atadenovirus genus. Archives of Virology, 147:841-847.

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ACCESSION NO: 0181168 SUBFILE: CRIS
PROJ NO: CONS-9802281 AGENCY: CSREES CONS
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 98-35204-6954
START: 01 DEC 1998 TERM: 31 OCT 2001 GRANT YR: 1998
GRANT AMT: $180,000

INVESTIGATOR: Sekellick, M. J.; Marcus, P. I.

PERFORMING INSTITUTION:
MOLECULAR AND CELL BIOLOGY
UNIV OF CONNECTICUT
STORRS, CONNECTICUT 06268

RECOMBINANT CHICKEN INTERFERONS AS ANTIVIRAL AGENTS

OBJECTIVES: 9802281. Our goal is to develop chicken interferons singly, or in synergistic mixtures, as novel biological response modifiers for the prevention or cure of viral diseases. Specific objectives include: (1) Determine the spectrum and degree of sensitivity of avian viruses of economic and public health importance to the action of Types I and II recombinant chicken interferons in vitro, in ovo, and in the chicken, acting singly, and in mixtures that display synergy; (2) Determine the nature of the heterogeneity in avian influenza virus sensitivity to recombinant chicken interferon; (3) Determine the antiviral effects of recombinant chicken interferon administered orally thorugh novel means and/or intranasally, as an effector of the humoral and mucosal system; and (4) develop a line of chickens with genetically enhanced sensitivity to the action of interferon.

APPROACH: The recently cloned and expressed genes of Types I and II chicken interferons will be produced as glycosylated recombinant molecules in stably transfected COS cells or in E. coli., purified, and tested for their in vitro, in ovo and in vivo activity against avian viruses of economic importance.

PROGRESS: 2000/01 TO 2000/12
Interferons (IFN) are components of the innate immune system and constitute the first and immediate line of defense against virus infection. They are produced rapidly by virus-infected cells, are released into the surrounding milieu within hours, and act within minutes following binding to specific cellular receptors on uninfected cells. Subsequent signal transduction and activation of transcription factors result in the activation of over 100 IFN-stimulated genes. The multiple intracellular modes of action that result from expression of these IFN-stimulated genes, and their efficacy against a broad spectrum of virus families, including those subject to antigenic changes that mute the effectiveness of vaccines, make IFNs novel biological modifiers worthy of tests to determine the range of their protective and curative properties. Double-stranded RNA (dsRNA) is a second biological response modifier of equally formidable activity. This class of molecules has emerged as singularly important in both the induction and action phases of the IFN system, and as an activator of many genes capable of producing multiple effects on cells and the immune system. Interestingly, many viruses have evolved mechanisms to prevent activation of cellular proteins designed to sense, and counteract, the presence of dsRNA. These include production of viral gene products which sequester dsRNA, and small helical RNAs. These molecules potentially prevent activation of dsRNA-dependent pathways of interferon action, or block expression of cellular genes activated exclusively by dsRNA that may contribute to the antiviral state. Not surprisingly, these means have provided viruses with highly effective mechanisms against IFN action. One of the most successful of the anti-IFN mechanisms is exemplified by the almost absolute resistance to the action of IFN displayed by avian reovirus (ARV). This resistance is attributed to the dsRNA-binding capacity of the sigma alpha core protein. Thus, dsRNA could be rate limiting in an ARV infected cell providing a means of preventing the development of an IFN- or dsRNA-mediated antiviral state. In support of this hypothesis, we have shown that dsRNA added exogenously to IFN-treated cells in the form of poly(rI):poly(rC) is effective in establishing in a dose-dependent manner an antiviral state against ARV as well as Newcastle disease virus, another virus that is otherwise highly resistant to interferon action. In order to abrogate IFN resistance, dsRNA must be added after, but not before, an IFN-mediated latent antiviral state is established. The combined sequential treatment with IFN and dsRNA may be useful in overcoming the anti-IFN activity of viruses of clinical interest, or in other clinical conditions.

IMPACT: 2000/01 TO 2000/12
The combined sequential application of interferon and double-stranded RNA may be useful in overcoming the anti-interferon activity of viruses of clinical interest, and even find relevance in other clinical conditions where interferon by itself is marginally, if at all, effective.

PUBLICATIONS: 2000/01 TO 2000/12
1. Sekellick, M.J., Carra, S.A., Bowman, A., Hopkins, D.A. and Marcus, P.I. 2000. Transient resistance of influenza virus to interferon action attributed to random multiple packaging and activity of NS genes. Journal of Interferon and Cytokine Research 20:963-970.
2. Marcus, P.I. and Sekellick, M.J. 2000. Combined action of interferon and dsRNA enhances antiviral effects. European Cytokine Network 11:186.

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ACCESSION NO: 0007173 SUBFILE: CRIS
PROJ NO: CONS00122 AGENCY: SAES CONS
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 AUG 1964 TERM: 30 JUN 2004 FY: 2001

INVESTIGATOR: Van Kruiningen, H.

PERFORMING INSTITUTION:
PATHOBIOLOGY
UNIV OF CONNECTICUT
STORRS, CONNECTICUT 06268

PULLORUM DISEASE CONTROL

OBJECTIVES: Pullorum-Typhoid Eradication.

APPROACH: This program involves the serologic testing of 500,000 to 700,000 avian blood samples per year for the presence of Salmonella pullorum and S. gallinarum. Reacting birds are called to the laboratory for bacteriological examination. IfS. pullorum or S. gallinarum is isolated, the reacting flock is retested at 21 day intervals until two successive negative flock tests are obtained. Control and regulatory action are administered by the Commissioner of Agriculture through the State Veterinarian.

PROGRESS: 2002/01 TO 2002/12
This is a collaborative project with the Connecticut Department of Agriculture. The purpose is to monitor and diagnose infectious diseases of poultry including Salmonella pullorum, Salmonella enteritidis, Mycoplasma gallisepticum and synoviae, Newcastle disease and Avian Influenza. A total of 35,340 agglutination, plate agglutination and hemagglutination tests were done. These included: 15,174 microtiter tests for S. pullorum (1 positive), 8,177 agglutination tests for M. gallisepticum (71 positive), 8,366 plate agglutination tests for M. synoviae (112 positive), 3 for Newcastle disease (O positive) and 3,620 for Avian Influenza (0 positive).

IMPACT: 2002/01 TO 2002/12
This monitoring program for infectious disease of Connecticut poultry identified several important disease agents, including Mycoplasma gallisepticum and synoviae.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

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ACCESSION NO: 0086778 SUBFILE: CRIS
PROJ NO: CONS00541 AGENCY: CSREES CONS
PROJ TYPE: HATCH PROJ STATUS: TERMINATED MULTISTATE PROJ NO: NE-138
START: 01 OCT 1996 TERM: 30 SEP 2002 FY: 2001

INVESTIGATOR: Khan, M. I.

PERFORMING INSTITUTION:
PATHOBIOLOGY
UNIV OF CONNECTICUT
STORRS, CONNECTICUT 06268

EPIDEMIOLOGY AND CONTROL OF EMERGING STRAINS OF POULTRY DISEASE RESPIRATORY AGENTS

OBJECTIVES: Develop and evaluate rapid diagnostic capabilities for the identification of emerging IBV, ILTV, mycoplasmas, and IBDV.

APPROACH: Infectious bronchitis virus (IBV) specific RT-PCR. IBV strains Massachusetts (Mass 41), Connecticut (Conn 46), Arkansas (Ark 99), and Delaware variant (072) will be initially grown in chicken embryo kidney cells (CEK), plaque-purified and characterized by oligonucleotide fingerprinting. The viruses will then be grown in 10-to-11-day-old chicken embryos, genomic viral RNA isolated by standard procedures, copied into cDNA by reverse transcription and cloned into the plasmid pUC18. The screening and identification of IBV specific segments of the cDNA will be done by cross-hybridization (DNA:RNA) against a panel of IBV field, and variant strains, and other avian pathogenic viruses of Newcastle disease, avian influenza, infectious laryngotracheitis, reovirus, fowlpox and infectious bursal disease. Clones containing unique sequences will be selected as strain-specific probes. Sequence analysis of the cDNA fragments will be performed in order to identify DNA primers specific for strains of IBV. Efficacy of the cDNA probes as well as RT-PCR's will be tested for their sensitivities and specificities in experimental infections. Eight-week-old SPF chickens will be inoculated intratracheally in IBV strains, Mass, Conn, Ark, and Delaware variant.

PROGRESS: 1996/10 TO 2002/09
Development of recombinant DNA vaccine that expresses S1 of IBV and Immunogenicity studies A recombinant fowlpox virus (rFPV) containing a cDNA copy of S1 gene of IBV (rFPV-S1) was constructed and its immunogenicity and vaccine potential were evaluated. Initially, rFPV-S1 was shown to express the S1 protein in vitro by using indirect immunofluorescence staining and Western blot analysis. Later, in vivo expression was demonstrated by the detection of IBV-specific serum IgG and neutralization antibodies in the sera of chickens immunized with rFPV-S1. That the recombinant virus elicited anti-IBV protective immunity was indicated by the manifested, relatively mild clinical signs of disease, decreased titers of recovered challenge virus, and less severe histological changes of the tracheas in virulent IBV-challenged chickens previously receiving rFPV-S1 as compared to parental FPV vaccinated control birds. In contrast, chickens immunized with either recombinant or parental FPV were resistant to a subsequent, virulent FPV challenge. As to a preferred method of immunization, wing web inoculation appeared to be superior to the subcutaneous route since a greater percentage of birds vaccinated by the former protocol exhibited an anti-IBV humoral immune response. Thus, rFPV-S1 has potential as a poultry vaccine against both fowlpox and infectious bronchitis.

IMPACT: 1996/10 TO 2002/09
Impact: 1. Recombinant Fowlpox virus containing S1 gene has potential for a poultry vaccine against both fowlpox and infectious bronchitis. 2. DNA vaccine containing whole S gene instead of S1 of IBV in pCMV plasmid vector could provide better protection against IBV infection.

PUBLICATIONS: 1996/10 TO 2002/09
1. Wang, X., W. M. Schnitzlein, D. N. Tripathy and M. I. Khan. 2002. Construction and immunogenicity studies of recombinant fowlpox containing the S1 gene of Infectious bronchitis virus. Avian Dis.46: 831-834.
2. Khan, M. I. 2002. Avian Pathogenic Mycoplasmas. PCR detection of Microbial Pathogens. Methods in Molecular Biology. eds. J. Frey and K. Sachse. Humana Press Inc. Totowa, NJ. November, 2002.

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ACCESSION NO: 0406071 SUBFILE: CRIS
PROJ NO: AP-511 AGENCY: ERS MTED
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 15 OCT 2001 TERM: 31 DEC 2003

INVESTIGATOR: Hahn, W.; Salin, D.; Harvey, D.

PERFORMING INSTITUTION:
Economic Research Service
USDA/ERS
1800 M STREET NW
WASHINGTON, DISTRICT OF COLUMBIA 20036

AP SPECIAL TOPICS: THE ECONOMIC EFFECT OF CHANGES IN SANITARY REGULATIONS ON BROILER TRADE IN THE AMERICAS

OBJECTIVES: Sanitary and phytosanitary (SPS) measures are impediments to trade and affect both the flow and the magnitude of trade. Newcastle Disease (ND) and highly pathogenic avian influenza (HPAI), included in List A of the International Organization for Epizootics (OIE) classification of transmissible animal diseases, are two highly infections diseases that restrict poultry trade. The END-free status gives the United States an advantage in poultry trade. However, changes in END status of potential large poultry suppliers could have a major impact in U.S poultry exports, especially in the high-value white meat. For Instance, since 1999 Mexico (the sixth world largest broiler importer) has intensify efforts to gain more END free states and eligibility to export fresh, chilled, and frozen poultry to the United States. Effective August 1, 2002 Canada recognized Brazils poultry inspection system. Eight Brazilian states were recognized free of END by the Canadian Food Inspection Agency (CFIA). Brazil is the second largest world exporter. The three objectives are (1) Analyze the impact on broiler markets and trade due to changes in sanitary regulations affecting trade in the Americas; (2) Measure how broiler prices, production, and trade will change (over the intermediate/long run) as a result of allowing Mexicos and Brazils END-free regions to ship poultry to the United States and Canada; and (3) Measure the sensitivity of these results to alternative estimates of supply and demand elasticities.

APPROACH: Objective 1: Analyze price differentials of poultry products between the countries in the Americas to determine the potential trade flows between countries. We will be collecting primary data on the structure of the Mexican broiler marketing system. Secondary price and quantity data will be collected for all four countries. Objectives 2 and 3: Production, export, price, and population estimates will be obtained from the other three countries and added to import data from the ERS. These will be combined to describe the broiler supply and demand conditions in the Americas. The U.S.-Mexico broiler trade model will be expanded to include Canada and Brazil. This model takes a static baseline and replicates it if there are no policy changes. The policy changes evaluated are changes in END-free-status for regions in Mexico and Brazil, and the subsequent allowance by APHIS/FSIS for these END-free regions to ship product to the United States.

NON-TECHNICAL SUMMARY: This project evaluates the potential economic impact of changes in sanitary restrictions on broiler trade in the Americas.

PROJECT CONTACT:

Name: Hahn, W.
Phone: 202-694-5175
Email: WHAHN@ers.usda.gov

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ACCESSION NO: 0181427 SUBFILE: CRIS
PROJ NO: FLA-VME-03777 AGENCY: CSREES FLA
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 04 MAR 1999 TERM: 30 SEP 2003 FY: 2001

INVESTIGATOR: Spalding, M. G.; Forester, D. J.

PERFORMING INSTITUTION:
COLLEGE OF VETERINARY MEDICINE
UNIVERSITY OF FLORIDA
GAINESVILLE, FLORIDA 32610

SURVEILLANCE FOR DISEASES OF WILDLIFE WHICH ARE TRANSMISSIBLE TO LIVESTOCK AND POULTRY IN FLORIDA

OBJECTIVES: The objectives are to determine the distribution and prevalance of wildlife diseses that are transmissible to livestock and pountry in Florida. These diseases include parasitic and infectious diseases such as Newcastle Disease in double-crested cormrants, eastern equine encephalitis in sandhill cranes and whooping cranees, mycoplasmal conjunctivitis in songbirds, and fascioloidosis, haemonchosis, dictyocaulosis, tuberculosis, and chronic wasting disease in white-tailed deer, and others.

APPROACH: Serologic studies and virus isolations will be conducted on eggs, nestlings, and adult double-crested cormorants from several colonies in Florida to identify Newcastle disease virus. Sandhill crane adults and chicks of various ages will be captured and tested serologially for antibodies to eastern equine encephalitis (EEE) virus. Sentinel studies will be conducted using pen-reared bobwhites to determine seasonal transmission of EEE virus. Mosquitoes will be collected, identified, pooled, and tested for EEE virus. Other species of wildlife including songbirds and white-tailed deer will be examined to detect mycoplasmal conjunctivitis, chronic wasting disease and other diseases.

PROGRESS: 2001/10 TO 2002/10
We continue to receive specimens, especially from the Florida Fish and Wildlife Conservation Commission and monitor them for exposure to eastern equine encephalitis and West Nile virus. Additional specimens, especially of native doves had been tested for exposure to pigeon paramyxovirus 1, and virus that is very similar to Newcastle Disease virus and of concern to the poultry farmers in Florida.

IMPACT: 2001/10 TO 2002/10
Pigeon paramyxovirus 1, though very similar to Newcastle Disease virus, does not appear to be virulent in domestic poultry.

PUBLICATIONS: 2001/10 TO 2002/10
1. Forrester, D. J. and M. G. Spalding. 2001. Salmonellosis in a wild turkey from Florida. Florida Field Naturalist 29(2): 51-53.
2. Spalding, M.G., S.A. Nesbitt, S.T. Schwidert, and R.J. Dusek. 2001. The use of radio transmitters to monitor survival of sandhill crane chicks. Proc. North Ammerican Crane Workshop 8: 213-215.
3. Nesbitt, S.A., M.J. Folk, K.A. Sullivan, S.T. Schwikert, and M.G. Spalding. 2001. An update of the whooping crane release project through June 2001. Proc. N. Am. Crane Worksh. 8: 62-73.
4. Frederick, P. C., M. G. Spalding, and R. Dusek. 2002. Wading birds as bioindicators of mercury contamination in Florida, USA: Annual and geographic variation. Environmental Toxicology and Chemistry 21(1): 163-167.
5. Spalding, M. G., C. A. Yowell, D. S. Lindsay, E. C. Greiner, and J. B. Dame. 2002. Sarcocystis meningoencephalitis in a northern gannet (Morus bassanus). Journal of Wildlife Diseases 38(2): 432-437.
6. Varela, A., J.M. Kinsella, and M.G. Spalding. 2002. Presence of encysted immature nematodes in a released whooping crane (Grus americana). Journal of Zoo and Wildlife Medicine 32: 523-525.
7. Buergelt, C.D., B.L. Homer, and M.G. Spalding. 2002. Causes of mortality in the Florida panther (Felis concolor coryi). Annals of the New Your Academy of Sciences 969: 350-353.

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ACCESSION NO: 0178041 SUBFILE: CRIS
PROJ NO: FLAV-CO-00204 AGENCY: CSREES FLAV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 16 APR 1998 TERM: 30 MAR 2001 FY: 2001

INVESTIGATOR: Romero, C. H.; Butcher, G. D.

PERFORMING INSTITUTION:
PATHOBIOLOGY
UNIVERSITY OF FLORIDA
GAINESVILLE, FLORIDA 32610

DEVELOPMENT OF RECOMBINANT POULTRY VACCINES

OBJECTIVES: To validate existing stocks of recombinant herpes virus of turkeys (HVT) as to gene content and expression in chicken embryo fibroblasts (CEFs). To prepare stocks of recombinant HVT and maintain the stocks in liquid nitrogen for future validation and preparation of vaccine. To develop assays for identifying and testing gene expression in the recombinant vaccines. To identify new insertion sites in the HVT genome for cloning of immunogenic genes obtained from avian viruses of economic impact. To identify genes of avian pathogens that code for immunogenic proteins that confer disease resistance in vaccinated chickens. To develop new recombinant vaccines based on HVT as a vector and to perform potency and protection tests in chickens.

APPROACH: Cultures are prepared from chicken embryos on the 11th day of incubation . Large stocks of primary cells, chicken embryo fibroblasts (CEFs) are aliquoted and maintained frozen in liquid nitrogen tanks. Only these cell stocks guaranteed of extraneous viral or bacterial contamination are used in the validation or development of new assays on recombinant viruses and DNA molecules. Recombinant and wild-type viral RNA and DNA is extracted from infected CEFs and purified in order to locate and identify the inserted foreign genes in the viral genome utilizing reverse transcription and polymerase chain reaction (RT-PCR) amplification. The functionality of these genes is determined utilizing techniques such as Western blots, immunoprecipitation and enzymatic assays. New genomic insertion sites are found by cloning viral fragments in bacterial plasmids and by looking for unique restriction sites in these fragments. Then, by a process of homologous recombination in CEFs utilizing infectious HVT DNA and recombinant plasmid DNA recombinant viruses are screened for as proof of the non-essentiality of the insertion site. Genes of avian pathogens known to code for immunogenic proteins are then generated by PCR or RT-PCR and tested for protein expression before being cloned into the HVT vector or eukaryotic expression vectors.

PROGRESS: 1998/04 TO 2001/03
Specific-pathogen-free chickens injected with 200ug of naked DNA molecules expressing the glycoprotein B of serotypes 1 and 3 of Marek's disease virus and the MEQ protein were not protected from challenge with the virulent RB-1B strain. The chicken interferon gamma and interleukin-2 genes have been amplified and cloned in order to test them as immunological adjuvants when administered with DNA molecules that express Marek's disease virus genes. The capsid protein VP2 of infectious bursal disease virus has been re-created and injected into chickens in order to evaluate its expression in vivo and its fate and distribution in lymphod tissues. Final transfer vectors for generating recombinant vaccines based on the herpes virus of turkeys and the Rispens strain of Marek's disease virus are now constructed and being tested in transfection experiments. Assays based on the RT-PCR have been developed in order to test the transcription of foreign genes expressed from recombinant vaccines based on the herpes virus of turkeys. These assays test for the transcription of the full gB,gC and gD genes of serotypes 1, 2 and 3 of Marek's disease virus. Similarly, primers have been developed for the differential amplification of the complete gB, gC and gD genes of serotypes 1, 2 and 3 of Marek's disease virus. Restriction fragment polymorphism of the DNA products with selected enzymes add to the specificity of the assay. Several non-essential regions in the genome of the herpes virus of turkeys and the Rispens strain have been cloned and characterized. The nucleotide and deduced amino acid sequence of turkey interleukin-2 has been determined. Restriction analysis of genomic DNA extracted from fowl poxvirus vaccines and from two fowl poxvirus recombinants used by the poultry industry in the United States allows for the differentiation of mild from hotter strains. Similarly, restriction analysis of DNA and probing of recombinant fowl poxvirus vaccines against Newcastle disease from different commercial sources allows for the distinction between these vaccines.

IMPACT: 1998/04 TO 2001/03
The ability to generate functionally active genes of avian pathogens has been established. The RT-PCR technique can be used for the validation and testing of recombinant vaccines.

PUBLICATIONS: 1998/04 TO 2001/03
1. ROMERO,C.H. and CHUNG,H.Y. Restriction fragment length polymorphism of classical and recombinant fowlpox virus vaccines used by the poultry industry in the USA. XII International Poxvirus Symposium, June 6-10, 1998. St. Thomas, Virgin Islands, USA.
2. ROMERO,C.H. and CHUNG,H.Y. Amplification and restriction analysis of a highly conserved gene of Marek's disease virus to differentiate starins of serotypes 1,2 and 3. American Association of Avian Pathologists Meeting, July 25-29, 1998. Baltimore, Maryland, USA.
3. ROMERO, C.H., Cai, X., Elyar, J.S. and Evans, K. Cloning, sequence, and expression of turkey interleukin-2. Avian Diseases, 2000 (submitted).

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ACCESSION NO: 0400221 SUBFILE: CRIS
PROJ NO: 6612-32000-019-00D AGENCY: ARS 6612
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 09 APR 1996 TERM: 08 APR 2001 FY: 2001

INVESTIGATOR: TUMPEY T; MITCHELL B W; HOLT P S; SWAYNE D E

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
ATHENS, GEORGIA 30613

STIMULATION OF MUCOSAL IMMUNITY IN CHICKENS TO PROTECT AGAINST ENTERIC AND RESPIRATORY PATHOGENS

OBJECTIVES: Examine the development of local humoral immune response at mucosal surfaces in chickens and compare this response with systemic immunity. Develop vaccines for mucosal immunity against intestinal and respiratory pathogens in poultry and diagnostic tests that will predict effectiveness. Determine the mechanisms for generation of airborne pathogens. Develop controls to improve poultry health and enhance mucosal vaccine effectiveness by reducing airborne pathgens and dust.

APPROACH: Birds will be orally infected with salmonella enteritidis (SE) and serum and intestinal anti-SE antibody levels will be ascertained over time. The birds will be re-infected to determine the development of serum and intestinal immunological memory. Immune recognition of different components of SE in serum and the intestinal tract will be compared. The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds and following the progression of the infection. The development of immunity in the intestinal tract will be dilineated by immunoassay of intestinal contents and elispot analysis of purified lamina propria lymphocytes. Dust and bacterial counts will be measured in hatching cabinets and other poultry production areas. Dust reduction techniques studied will include lowering air velocity and using an electrostatic space charge with a grounded collection system. Experiments will be conducted to characterize airborne transmission of SE and to explore treatments for reducing it.

PROGRESS: 2000/10 TO 2001/09
PROTECT AGAINST ENTERIC AND RESPIRATORY PATHOGENS 1. What major problem or issue is being resolved and how are you resolving it? The poultry industry is still threatened by avian influenza virus (AIV) and can result in substantial economic losses. Although the incidence of highly virulent AI strains are relatively rare, less pathogenic strains often circulate through chicken and turkey flocks and are responsible for significant morbidity, mortality and production losses throughout the world. Current parenteral vaccines and vaccine technologies provide protection against clinical signs and death from highly pathogenic AI virus challenge. However, prevention of highly and mildly pathogenic AI virus replication at mucosal sites is inconsistent and thus limits vaccines in preventing transmission. This is thought to be due in part to the observation that systemic vaccination is a poor inducer of mucosal immunity and therefore organisms can invade the host before the systemic immunity can impede the infection. Because AIV invade the mucosal surfaces, emphasis should be placed on vaccines that induce strong mucosal immunity. Thus, the main objective of this CRIS will be to develop mucosal vaccines for controlling AI in poultry and to characterize the immune effectors mediating vaccine protection. The development of new vaccine approaches for controlling such emerging or reemerging pathogens can be of great importance to the profitability of the poultry industry. In addition, effective mucosal vaccine strategies developed for AI will enable scientists to develop vaccine approaches for controlling other respiratory or gastrointestinal pathogens that affect poultry. 2. How serious is the problem? Why does it matter? It has been estimated that the next outbreak of a highly pathogenic AIV such as the 1983 outbreak in Pennsylvania will have an economic impact of up to $120 million to the poultry industries. Furthermore, the H5N1 outbreak in Hong Kong in 1997 has shown us that avian influenza subtypes, which were not thought to infect humans, are able to cross species barrier and cause disease to humans. During this outbreak, domestic chickens served as an intermediate host for the transmission of H5N1 virus from wild aquatic birds to humans. This realization has further strengthened the search for new and improved vaccines to protect against highly pathogenic AIV. Successful completion of experiments outlined in this CRIS could provide the next generation of vaccines capable of neutralizing these pathogens at the mucosal surfaces. 3. How does it relate to the National Program(s) and National Component(s)? National Program 103, Animal Health (100%) Vaccine Strategies, which prevent the initiation and dissemination of the organism within flocks, fit well with the animal health research component on Disease Control Strategies. 4. What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2000 year. The overall objective is the development of new vaccine approaches for controlling AI in poultry and to characterize the immune effectors mediating vaccine protection. This work at Southeast Poultry Research Laboratory identified a mucosal route of vaccination that results in protection against lethal AI virus challenge. This was done for purposes of improving the mucosal immune response(s) to AI in chickens. The specific accomplishment was the determination of significant antiviral antibody response that was detected after intratracheal vaccine administration via ELISA. This information will be used to develop efficacious vaccines for stimulating protective immunity against AI and possibly other respiratory pathogens that affect poultry. B. Other Significant Accomplishment(s), if any. Research was also conducted to characterize the immune responses to mucosal vaccination and develop improved methods for identifying cells and antibody harvested from mucosal sites. This work at Southeast Poultry Research Laboratory led to the development of a ELISPOT assay to detect specific antibody secreting cells in the spleen and other tissues. Relatively high levels of IgG, and lower IgA secreting cells were detected from chickens vaccinated by the intratracheal or subcutaneous route. This work is crucial for the identification of immune effectors mediating protection and might suggest ways of maximizing such responses for poultry. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. This project was redirected to avian influenza vaccine development during FY2000. The Salmonella vaccine development that preceded this work has laid a foundation for commencing mucosal vaccine studies with AIV. These accomplishments include; (1) improved biological reagents needed to measure IgG and IgA antibody responses in chickens and turkeys, (2) identification of mucosal adjuvants that improve protection of killed vaccines in the gastrointestinal tract of poultry. (3) identification of alternative routes of vaccine administration for the induction of optimal immune responses. 6. What do you expect to accomplish, year by year, over the next 3 years? The development of mucosal vaccines in poultry will continue through FY2004. This process will include development and testing of novel mucosal vaccines and directly comparing them to traditional parenteral AI virus vaccines. Our research will continue to determine the optimal route of mucosal vaccination and test the inclusion of mucosal adjuvants and immunomodulators to a vaccine formula in an attempt to improve the quality of the immune response. The vaccines that have been prepared for this study include inactivated whole virus vaccines and subunit vaccines. New vaccine technologies will also be included to test protective immunity against AIV in chickens and turkeys. We will also examine whether these vaccines can be adapted for in ovo vaccination protocols. For FY2003 we will then characterize the antibody and cellular immune responses in various locations of the respiratory and intestinal tract of vaccinated animals. 7. 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 & durability of the technology product? No activities were reported. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) No news articles or trade publications were reported.

PUBLICATIONS: 2000/10 TO 2001/09
1. Chaubal,L.H., Holt,P.S. Characterization of motility and identification of flagella proteins in the avian pathogen Salmonella pullorum. American Journal of Veterinary Research. 2000. v.60.p.1322-1327.
2. Katz,J.M., Lu,X., Frace,M. Morken,T., Zaki,S.R., Tumpey,T.M. Pathogenesis of and Immunity to Avian Influenza A H5 viruses. Biomedicine Pharmacotherapy. 2000. v.54.p.178-187.
3. Tumpey,T.M., Lu,X., Morken,T., Zaki,S.R., Katz,J.M. Depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza A virus isolated from humans. Journal of Virology. 2000. v.73.p.6105-6116.
4. Tumpey,T.M., Renshaw,M., Clements,J., Katz,J.M. Mucosal delivery of inactivated influenza vaccine induces B cell dependent heterosubtypic protection against a lethal influenza A H5N1 virus. Journal of Virology. 2001. v.75.p.5141-5150.

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ACCESSION NO: 0402463 SUBFILE: CRIS
PROJ NO: 6612-32000-021-00D AGENCY: ARS 6612
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 12 OCT 1998 TERM: 11 OCT 2003 FY: 2001

INVESTIGATOR: KING D J; SEAL B S; VACANT; KAPCZYNSKI D R; SWAYNE D E; MITCHELL B W

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
ATHENS, GEORGIA 30613

VIRULENCE DETERMINANTS IMPORTANT TO PATHOGENESIS OF NEWCASTLE DISEASE VIRUS AND AVIAN PNEUMOVIRUS

OBJECTIVES: Molecular and biological characterization of new Newcastle disease virus (NDV) and avian pneumovirus (APV) isolates for molecular epidemiology critical in tracking outbreaks and resolution of issues that impact international trade. Identification and further characterization of determinants important to avian paramyxovirus pathogenesis. Development of improved control strategies, including vaccination and diagnostics based on data from prior objectives. Determine the reservoirs & sources of APV.

APPROACH: Acquisition of new NDV and APV isolates from poultry oubtreaks in the U.S. and other countries and surveys of North American wild bird populations. Biological and molecular properties of isolates will be compared with those acquired in prior years for virulence and epidemiologic determinations. Antigenic characterization will be accomplished using monoclonal and polyclonal antibodies. Gene sequences of isolates will be identified by automated sequencing of DNA prepared from RNA by reverse transcriptase polymerase chain reaction or from cloned DNA prepared from purified RNA. Isolates with sequences typical of virulent strains, but with low virulence for chickens will be passaged in chickens to select or adapt strains for virulence. NDV persistence after clinical recovery will be assessed. APV vaccines will be developed and evaluated for the ability to protect turkeys. Athens, GA, SEPRL; Main Lab & Bldg. 1 BL-2; Bldg 34 BL-3; 1/07/99.

PROGRESS: 2000/10 TO 2001/09
1. What major problem or issue is being resolved and how are you resolving it? Newcastle disease (ND) is a viral disease of poultry and a worldwide problem first recognized in the U.S. in the 1930s. Newcastle disease virus (NDV), also identified as avian paramyxovirus type 1 (APMV-1), infects all known wild and domestic bird species. Different NDV strains vary in virulence and produce differing clinical forms of the disease that range from high mortality to mild or inapparent respiratory infections. The mild form is the predominant one seen in the U.S. Avian pneumovirus (APV) infections of turkeys in the U.S., in contrast to ND, are of recent concern. APV was isolated from commercial turkeys in Colorado in 1996. The disease was reported in the United Kingdom in 1985 and prior to 1996 the disease was exotic to North America. The virus causes a mild, but rapidly spreading, upper respiratory disease and adverse effect on weight gain and feed conversion. Secondary bacterial infections increase the severity of the disease. The initial diagnosis of the disease in the U.S. was delayed because the U.S. isolate was of a different subtype than had been isolated elsewhere and serological assays to detect the new subtype were not available. Research to resolve the problem includes characterization of new isolates, determining their virulence, and developing control strategies, including vaccination and diagnostics. 2. How serious is the problem? Why does it matter? The U.S. is free of virulent or exotic ND by successfully eradicating infections when they have occurred, most recently in 1998. Exotic ND is a reportable disease and its presence in commercial poultry will result in embargoes of poultry export from the effected country, a critical event for a major poultry exporter like the U.S. The threat of exposure of commercial poultry to virulent virus is constant and from several sources. It can be tracked from countries such as Mexico where the disease is indigenous, from the site of periodic ND outbreaks in migrating cormorants as occurred in 1992, 1997, and 1998 in the U.S., and from virus detected in smuggled birds or birds held in quarantine which has occurred in most years since the mid-1970s . Infections of commercial poultry with APMV-1 strains of low virulence also have economic impact due to reduced productivity from respiratory disease and reduced rate of weight gain. APV infections continue to cause productivity losses in turkeys in the Upper Midwest particularly in the state of Minnesota. 3. How does it relate to the National Program(s) and National Component(s)? The project contributes to National Program 103, Animal Health (100 percent). The research characterizes emergent domestic and exotic NDV and APV. The molecular and biological characterization of new virus isolates, the development of improved identification methods, and studies of the pathogenesis of those isolates are relevant to the Pathogen Detection and Diagnostics, the Microbial Genomics, and Mechanism of Disease components of the plan. The characterization of the immune response and development of improved vaccines are relevant to the Animal Immunology and the Strategies to Control Infectious and Non-Infectious Disease components of the plan. 4. What were the most significant accomplishments this past year? A. Most significant accomplishment. Existing diagnostic tests for APV were ineffective. Scientists on the project cloned the gene for the matrix protein of APV and collaborated with investigators at the University of Minnesota in development of a diagnostic test for APV antibodies. The test employs protein produced in a bacterial system from the cloned matrix gene as a target to capture antibodies in serum of infected turkeys. Preliminary tests of turkey serum from experimentally infected birds and field cases demonstrated high specificity of the new test. The problem of false positive reactions in the standard test were eliminated with the new test. B. Other significant accomplishments. The passage of domestic NDV isolates from birds species other than commercial poultry was evaluated as a method of identifying the risk of those isolates for poultry. One virus of moderate virulence initially became highly virulent after passage and demonstrated the potential risk of NDV infections of other birds for poultry. Existing APV diagnostic tests do not work in bird species other than chickens and turkeys. An additional APV serological method (competitive ELISA) was developed for testing serum of multiple bird species in ongoing surveillance studies. This test overcomes the limitations of previous tests that were designed for use in a single species, a severe limitation for surveillance studies. Effective vaccines against APV have limited protective ability. An inactivated APV vaccine was produced and limited protective immunity in turkeys was evaluated. This suggests that current technologies for producing killed APV vaccines will not be effective in producing an effective vaccine for control. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. The APV Colorado isolate (APV/CO) was the first U.S. isolate of APV and was previously found by sequence analysis of the virus genome to be a unique virus relative to the European APV types A and B strains. A mild disease was produced in turkeys inoculated with APV/CO and virus was recovered from the nasal cavity up to 5 days post-infection. Antiserum from the infected turkeys neutralized APV/CO and the Types A and B viruses from Europe but antisera against Types A and B did not neutralize APV/CO. Low and moderate virulence NDV strains are not known to cause overt disease but probes to identify virus genome in tissues of infected birds detected virus in heart muscle and air sac membranes of birds infected with those viruses. The lesions may predispose individuals to secondary infections and decreased meat and egg production even in the absence of obvious disease. Nucleotide sequence analysis of a low virulence NDV isolate from a live bird market in the Northeastern U.S. identified the virus as similar to isolates from other countries thought to be exotic to the U.S. Project scientists have continued to refine diagnostic tests utilizing nucleic acid amplification and sequencing for prediction of virulence of NDV isolates and for NDV epidemiology. Project scientists demonstrated that SPF white leghorns should be used for NDV pathogenicity evaluations because they are more susceptible to clinical disease and mortality than white rocks. 6. What do you expect to accomplish, year by year, over the next 3 years? Characterization of new domestic and exotic NDV and APV isolates to identify the diversity and risk of those isolates to poultry will continue through FY2006. Surveillance of wild bird populations in the Midwestern and Southeastern regions of the United States to obtain virus isolates of NDV and APV will continue through FY2003 and characterization of those isolates will continue through FY2005. A serological survey to detect birds positive to APV will also continue through FY2003. Identification of reservoirs of virus infections of wild birds that pose a risk to commercial poultry is the purpose of the surveillance studies. Antibodies specific to different proteins of NDV and APV will be prepared in FY2002 and will be evaluated as diagnostic reagents in virus characterization studies and as an adjunct to pathogenesis studies through FY2004. Pathogenesis studies, to identify tissue targets and virulence, of APV and NDV isolates will continue in chickens and turkeys through FY2006 for the purpose of identifying virulence markers that will lead to improved diagnostic test methods. Experimental models to test protective immunity against NDV and APV with newly formulated vaccines will be assessed by FY2003. The role of antibody and cell mediated immune response to NDV and APV in vaccinated and naive birds will be evaluated during the protective immunity studies and studies focusing on specific components of those responses will extend through FY2006. 7. 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 & durability of the technology product? Incumbent scientists on the project collaborated with investigators at the University of Minnesota in development of recombinant antigen in a diagnostic for serological identification of antibodies to avian pneumovirus. The test has been evaluated with serum from experimental infected turkeys and from field cases and was shown to have high specificity and eliminated the problems of false positives in the standard test. Transfer of current findings to other scientists and diagnosticians will continue by publication of research results and via direct communication through correspondence and short term laboratory visits. 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) King,D.J.,Seal,B.S. Current understanding of molecular correlates of virulence in Newcastle Disease Virus. International Hatchery Practice. 2000. v.14(7)(Suppl.):Vaccination at work in commercial broilers, p. 10-11. Gulati,B.R., Cameron,K.T., Seal,B.S., Goyal,S.M., Halvorson,D.A., Njenga, M.K. Development of a more sensitive and specific ELISA for detecting avian pneumovirus antibodies. Gobbles. 2000.v.57.p.16-18.

PUBLICATIONS: 2000/10 TO 2001/09
1. Cameron,K., Zhang,X., Seal,B., Rodriguez,M., Njenga,M.K. Antigens to viral capsid and non-capsid proteins are present in brain tissues and antibodies in sera of Theiler's virus-infected mice. Journal of Virological Methods. 2001.v.91.p.11-19.
2. Gulati,B.R., Cameron,K.T., Seal,B.S., Goyal,S.M., Halvorson,D.A., Njenga,M.K. Development of highly sensitive and specific enzyme-linked immunosorbent assay based on recombinant matrix protein for detection of avian pneumovirus antibodies. Journal of Clinical Microbiology. 2000.v.38. p.4010-4014.
3. Kapczynski,D.R., Koci,M., Kelley,L., Schultz-Cherry,S. Use of in vitro expressed capsid protein from turkey astrovirus to protect poults from PEMS-associated disease. Program of the Fifth International Congress of Veterinary Virology, Brescia, Italy. August, 2000.(Abstract)
4. Kapczynski, D.R.,Smith, C. Immune response of turkeys following intranasal vaccination with BPL-inactivated avian pneumovirus and live-virus challenge. Program of the American Association of Avian Pathologists at the American Veterinary Medical Association annual meeting. Boston, MA. July 14-18, 2001.(Abstract)
5. King,D.J. Efficacy of vaccine in protection against velogenic Newcastle Disease (ND). Proceedings of the United Animal Health Association.2000. p.591-593.
6. King,D.J.,Swayne,D.E. Newcastle disease update: International ND problems. Proceedings of the United Animal Health Association. 2000. p. 593-596.
7. King,D.J. Selection of Thermostable Newcastle Disease Virus from Reference and Vaccine Strains. Avian Diseases. 2001.v.45.p.512-516.
8. King,D.J., Kommers,G.D., Brown,C.C., Seal,B.S. Virulence of pigeon Newcastle disease virus isolates for chickens. Proceedings of the Fiftieth Western Poultry Disease Conference. 2001.p.15-16.
9. Kommers,G.D., King,D.J., Seal,B.S., Brown,C.C. Pathogenesis of five different pigeon-origin isolates of Newcastle disease virus for domestic chickens. Veterinary Pathology. 2000.v.37(5).p.546(Abstract No.94).
10. Locke,D.P., Sellers,H.S., Crawford,J.M., Schultz-Cherry,S., King,D.J., Meinersmann,R.J.,Seal,B.S. Newcastle disease virus phosphoprotein gene analysis and transcriptional editing in avian cells. Virus Research.2000. v.69.p.55-68.
11. Seal,B.S., Sellers,H.S. Avian paramyxoviruses evolve independently of their mammalian counterparts and deserve a genus designation among the subfamily Paramyxovirinae in the family Paramyxoviridae. Program of the Ninth Annual Meeting of the Society for Molecular Biology and Evolution, Athens, GA. July,7-10, 2001.(Abstract)
12. Seal,B.S. Molecular evolution of Newcastle disease virus and the application of molecular diagnostics. Program of the Respiratory Diseases of Poultry Symposium, American Association of Avian Pathologists at the American Veterianry Medical Association Annual Meeting in Boston, MA. July 14-18, 2001.(Abstract)
13. Seal,B.S. Avian Pneumoviruses and Emergence of a New Type in the United States. Animal Health Research Reviews.2000.v.1.p.67-72.
14. Seal,B.S., King,D.J. Newcastle Disease Virus. Available from: http://www.els.net Encyclopedia of Life Sciences[2000].
15. Sellers,H.S., Schultz-Cherry,S., Brown,C.C., Seal,B.S., King,D.J. Induction of apoptosis by Newcastle disease virus. Program of the Fifth International Congress of Veterinary Virology, Brescia, Italy. August,2000.(Abstract).
16. Suarez,D.L., Swayne,D.E., King,D.J. The ongoing threat of avian influenza and Newcastle disease to the U.S. poultry industry. Proceedings of the Fiftieth Western Poultry Disease Conference.2001.p.1-7.
17. Swayne,D.E., Suarez,D.L., King,D.J. Avian influenza (AI) and velogenic Newcastle disease (VND) update. Proceedings of 35th National Meeting of Poultry Health and Processing.2000.p.37-45.

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ACCESSION NO: 0401966 SUBFILE: CRIS
PROJ NO: 6612-32000-021-01T AGENCY: ARS 6612
TYPE: PROJ USDA INHOUSE PROJ STATUS: TERMINATED
START: 01 SEP 1998 TERM: 11 FEB 2001 FY: 2001

INVESTIGATOR: SEAL B S

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
ATHENS, GEORGIA 30613

CONSTRUCTION OF A NEWCASTLE DISEASE VIRUS MINI-GENOME

OBJECTIVES: Newcastle disease continues to pose a threat to U.S. poultry production with continued outbreaks worldwide reported by the Office of International Epizootes. Evidence from our laboratory demonstrates these agents are evolving so they are becoming more distantly related from current live-virus vaccines. The overall goal is to use reverse genetics of NDV to develop rescue systems for the creation of genetically engineered infectious viral clones.

APPROACH: Since NDV genomic RNA is minus-sense and therefore noninfectious, several cloned proteins must be expressed along with a positive sense anti-genomic RNA. The nucleoprotein (NP), phosphoprotein (P) and the polymerase (L) are currently being cloned into expression vectors. A NDV mini-genome that contains the NP, P and L genes with the appropriate leader and trailer sequences will be constructed. A reporter gene will be inserted between the P and L genes. The NDV mini-genome will be coexpressed with viral NP, P and L genes and monitored for expression of the reporter gene to validate a functional NDV rescue system. Athens, Georgia - Southeast Poultry Research Laboratory, Building 4, B/L-3; 01/07/99. Scientists & technicians associated with project: Bruce Seal and Joyce Bennett.

PROGRESS: 2000/10 TO 2001/09
1. What major problem or issue is being resolved and how are you resolving it? 2. How serious is the problem? Why does it matter? 3. How does it relate to the National Program(s) and National Component(s)? 4. What were the most significant accomplishments this past year? D. Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the U.S. Poultry and Egg Association. Additional details of research can be found in the report for the parent project 6612-32000-021-00D Paramyxovirus Infections of Poultry. Complete nucleotide sequencing of the Newcastle disease virus (NDV) vaccine strain B1 is necessary to understand pathogenesis and develop NDV as a vaccine vector. Using nucleotide sequencing and mapping techniques, the full-length genomic sequence of the NDV vaccine strain B1 was determine. The sequence has been published in Genbank for use by all scientists. 5. Describe the major accomplishments over the life of the project including their predicted or actual impact. 6. What do you expect to accomplish, year by year, over the next 3 years? 7. 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 & durability of the technology product? 8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below) Sellers, H.S. and Seal, B.S. The full-length genomic sequence of NDV strain B1. GenBank accession number NC002617.

PUBLICATIONS: 2000/10 TO 2001/09
 None.
 

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ACCESSION NO: 0404751 SUBFILE: CRIS
PROJ NO: 6612-32000-028-00D AGENCY: ARS 6612
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 07 APR 2001 TERM: 31 JAN 2003 FY: 2001

INVESTIGATOR: TUMPEY T; SWAYNE D E; VACANT; SUAREZ D L; MITCHELL B W

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
ATHENS, GEORGIA 30613

STIMULATION OF MUCOSAL IMMUNITY IN CHICKENS TO PROTECT AGAINST ENTERIC AND RESPIRATORY PATHOGENS

OBJECTIVES: Examine the development of local humoral immune response at mucosal surfaces in chickens and compare this response with systemic immunity. Develop vaccines for mucosal immunity against intestinal and respiratory pathogens in poultry and diagnostic tests that will predict effectiveness. Determine the mechanisms for generation of airborne pathogens.

APPROACH: The protective role of serum and mucosal antibodies will be ascertained by passive administration of antibodies to naive birds and following the progression of the infection. The development of immunity in the intestinal tract will be dilineated by immunoassay of intestinal contents and elispot analysis of purified lamina propria lymphocytes.

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ACCESSION NO: 0405248 SUBFILE: CRIS
PROJ NO: 6612-32000-038-00D AGENCY: ARS 6612
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 15 NOV 2001 TERM: 14 NOV 2006

INVESTIGATOR: KING D J; KAPCZYNSKI D R; SEAL B S; VACANT; SWAYNE D E; MITCHELL B W

PERFORMING INSTITUTION:
AGRICULTURAL RESEARCH SERVICE
ATHENS, GEORGIA 30613

IDENTIFICATION OF VIRULENCE DETERMINANTS, PATHOGENETIC MECHANISMS, ...AVIAN PARAMYXOVIRUSES

OBJECTIVES: 1. Characterization of emergent Newcastle disease virus (NDV) and avian pneumovirus (APV) isolates to extend the capabilities for molecular epidemiology of the most important avian paramyxovirus (APMV) infections. 2. Further characterization of determinants important to APMV pathogenesis. 3. Development of improved control strategies, including vaccination, diagnostics and identification of NDV and APV reservoirs.

APPROACH: NDV and APV isolates will be acquired from outbreaks and from surveys of North American wild bird populations. Wild waterfowl will be surveyed for APV specific antibodies. Antigenic differentiation with monoclonal and polyclonal antibodies and nucleotide sequence analysis of NDV and APV genes will provide molecular characterization and epidemiologic determinations. Virulence, persistence, and pathogenesis will be evaluated by inoculation of chickens or turkeys. Sequence analysis in combination with results from pathogenesis studies of isolates or infectious clones rescued from cloned NDV will provide the basis for identification of virulence markers useful for diagnostic development. Immunity to APV and NDV infections and newly developed live, inactivated and subunit vaccines will be assayed for both antibody mediated and cellular immune responses. Serum and mucosal antibody will be quantitated and isotype determined. Cytokine regulators of the immune response will be assayed. BSL-2 and BSL-3Ag, 8/10/01.

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ACCESSION NO: 0189393 SUBFILE: CRIS
PROJ NO: GEOV-0456 AGENCY: CSREES GEOV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2001 TERM: 30 SEP 2004 FY: 2002

INVESTIGATOR: Sellers, H. S.

PERFORMING INSTITUTION:
COLLEGE OF VET MEDICINE
UNIVERSITY OF GEORGIA
110 RIVERBEND ROAD
ATHENS, GEORGIA 30602

DETECTION, ISOLATION AND CHARACTERIZATION OF AVIAN VIRUSES

OBJECTIVES: The objectives of this proposal are to provide diagnostic virology services for the U.S. poultry industry, conduct applied research on current avian disease isolates from the field, and improve detection and isolation methods for monitoring avian viruses. In addition to classical virus isolation techniques currently used in the diagnostic virology laboratory, we will incorporate molecular-based assays, which will have major benefits to the diagnostic laboratory. First, it will reduce the total number of SPF embryos required for diagnostic services, as they can sometimes be difficult to obtain and second, it will result decrease expenses incurred for the embryos, which have increased in price by approximately 44% since last year. Existing methods of viral detection and isolation will be improved with the addition of a repository of fluorescent-labeled antibodies for direct/indirect detection of viral antigens. Continued research on diagnostic applications will improve turn-around time, accuracy and client satisfaction. Specific research projects will involve all major avian viruses in collaboration with the clinicians, faculty, and students at PDRC, as dictated by field situations.

APPROACH: Objectives. The following objectives are broad as this project is long-term and continuing flexibility is needed to adjust for new situations in the field. To provide diagnostic virology services in an accurate and reliable manner for the U.S. poultry industry Improve methods of detection and monitoring of avian viruses Apply new monoclonal antibodies (Mab) as they become available to diagnostic cases (i.e. monoclonal antibodies to IBDV, J. Rosenberger) Utilize fluorescent (FITC)-labeled antibodies for direct detection of viral antigen, as in recent subclinical Infectious Laryngotracheitis (ILT) cases Apply PCR and nucleic acid probe technology for diagnostic applications (i.e. real-time PCR utilizing the light cycler) Maintain contacts and working relationships with other research and diagnostic facilities for the exchange of data and reagents Conduct applied research on current avian virus diseases isolated from the field in collaboration with clinicians, faculty, students at PDRC and other poultry professionals in the field

NON-TECHNICAL SUMMARY: Despite rigorous vaccination in commercial poultry, avian viruses continue to cause problems resulting in production losses for the poultry industry. This project provides methods for detection, isolation and characterization of avian viruses.

PROGRESS: 2001/10 TO 2002/09
The mission of the diagnostic virology laboratory is to provide accurate and timely diagnostic virology services for the U.S. poultry industry, conduct applied research on current avian disease isolates from the field, and improve detection and isolation methods for monitoring avian viruses. During 2001-2002, the diagnostic virology lab processed 578 cases and isolated 687 viruses. This past year virus isolation for avian leukosis was added as a full-time service. Six hundred and forty six whole blood samples were submitted for leukosis isolation and subsequent antigen capture ELISA. Several new molecular-based assays have been incorporated. PCR is now available in a multiplex format for enteric coronaviruses and astroviruses. We are currently evaluating the sensitivity and specificity of this assay directly from field samples. In addition, an RT-PCR is available for Reoviruses. Our goal is to enhance our diagnostic offerings for enteric viruses of poultry. A live avian adenovirus vaccine (serotype 8) is currently being evaluated both pathologically and serologically in its ability to protect against inclusion body hepatitis caused by different serotypes of adenoviruses that were isolated over the past two years. Recent outbreaks of inclusion body hepatitis have been classified as serotype 11 adenovirus. During the past year (fall 2000-present) we have gathered evidence that a mild infectious laryngotracheitis virus (ILTV) is circulating in broiler flocks in the southeast. The condition is characterized by mild tracheitis, swollen sinuses and conjunctivitis with no mortality and minimal serological response. We are investigating the spread of the disease in broilers, evaluation of laboratory isolation and diagnostic procedures, and farm clean-out.

IMPACT: 2001/10 TO 2002/09
Improved methods of virus detection and isolation will expedite control measures used in the field to control and in some cases eradicate viral diseases. An emphasis has been placed on molecular diagnostics in the past year and as a result the time required for positive identification has been minimized thus providing much needed information in less time.

PUBLICATIONS: 2001/10 TO 2002/09
Kapczynski, D.R., H.S. Sellers, V. Simmons, S. Schultz-Cherry. Sequence analysis of the S3 gene from a turkey reovirus. Accepted to Virus Genes 2/2002.

PROJECT CONTACT:

Name: Sellers, H. S.
Phone: 706-542-5647
Fax: 706-542-5630
Email: hsellers@arches.uga.edu
URL: http://www.avian.uga.edu

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ACCESSION NO: 0194953 SUBFILE: CRIS
PROJ NO: GEOV-0466 AGENCY: CSREES GEOV
PROJ TYPE: SPECIAL GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2002-30001-12128 PROPOSAL NO: 2002-04435
START: 15 JUN 2002 TERM: 14 JUN 2004 GRANT YR: 2002
GRANT AMT: $2,000,000

INVESTIGATOR: Prasse, K. W.; Dickerson, H. W.; Miller, D. M.; Glisson, J. R.

PERFORMING INSTITUTION:
COLLEGE OF VET MEDICINE
UNIVERSITY OF GEORGIA
110 RIVERBEND ROAD
ATHENS, GEORGIA 30602

CORE ANIMAL DIAGNOSTIC LABORATORY

OBJECTIVES: Principle objective is to develop a regional capability to accurately and rapidly diagnose eight specific foreign animal diseases. Secondly, to establish a secure communications network with the other designated laboratories so that data may be shared throughout the network and with federal authorities.

APPROACH: Personnel will be trained in diagnostic procedures in eight foreign animal diseases. New equipment will be purchased to perform the diagnostic tests to detect the eight diseases. Developing a computerized reporting system in collaboration with 11 other states for reporting foreign animal diseases.

NON-TECHNICAL SUMMARY: There is a critical need for a national animal health reporting system to detect and report foreign animal diseases. This project will contribute to the development of a network of detecting and reporting foreign animal diseases nationwide.

PROJECT CONTACT:

Name: Miller, D. M.
Phone: 706-542-5568
Fax: 706-542-5977
Email: miller@vet.uga.edu

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ACCESSION NO: 0152351 SUBFILE: CRIS
PROJ NO: IND073055V AGENCY: CSREES IND
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 15 DEC 1995 TERM: 30 DEC 1998 FY: 1999

INVESTIGATOR: Guo, P.

PERFORMING INSTITUTION:
MICROBIOL PATHOLOGY & PUB HLTH
PURDUE UNIVERSITY
WEST LAFAYETTE, INDIANA 47907

CONSTRUCTION OF ATTENUATED RECOMBINANT AVIAN INFECTIOUS LARYNGOTRACHEITIS VIRUS VACCINES

OBJECTIVES: Our long term objective is to develop a polyvalent vaccine which is both effective in stimulating a high level of mucosal immunity against several avian respiratory tract infections, and is easy to administer, such as via aerosal spray to facilitate vaccination on large-flock chicken farms. Our short term goal of this proposal is to construct recombinant avian infectious laryngotracheitis viruses (ILTV) expressing the Fusion (F) glycoprotein of Newcastle disease virus (NDV) and the hemagglutinin (HA) of avian influenza virus (AIV), individually or in combination. Expression of the non-ILTV proteins will be monitored, and pathogenicity, immunogenicity and stability of the recombinant viruses will be tested by in vivo experiments.

APPROACH: Genes coding for the F protein of NDV and HA protein of AIV will be cloned by PCR after reverse transcription. The genes will be introduced into the ILTV genome via homologous recombination using marker genes for selection. Expression of the foreign genes will be monitored by immunofluorescence and western blot. Vaccination experiments will be performed to determine the minimum dose for 100% protection against NDV, AIV, and ILTV; stability of the recombinant viruses will be tested through in vivo passaging. Pathogenicity of the recombinant ILT viruses will be assessed symptomatically as well as by screening for tracheal lesions. The best route of vaccination to stimulate mucosal immunity will be determined by administration of the recombinant ILT-HA virus via different inoculation routes.

PROGRESS: 1995/12 TO 2000/09
Our research has recently identified an hepatoma cell line for the cultivation of infectious laryngotracheitis virus (ILTV), elucidated the assembly pathway of this pathogen, constructed three dual viral promoters simultaneously recognized by both mammalian and E. coli cells, documented the transactivation of the early SV40 promoter by ILTV co-infection, developed a simple procedure for ILT diagnosis and constructed recombinant ILTV with pathogenic gene deletion and foreign gene insertion. The recombinant ILTV will be used as a vector to develop a polyvalent vaccine for mucosal immunity against multiple avian respiratory tract infections.

PUBLICATIONS: 1995/12 TO 2000/09
1. Guo, P., E.Scholz, B.Maloney, and E.Welniak. 1994. Construction of recombinant avian infectious laryngotracheitis virus expressing bata-gal gene and DNA sequencing of insertion region. Virology 202:771-781.
2. Scholz, E., R. E. Porter, and P. Guo. 1994. Differential diagnosis of infectious laryngotracheitis from other avian respiratory disease by a simplified PCR procedure. J Virol Meth. 50:313-322.
3. Guo,P., E.Scholz, J.Turek, R.Nordgren, and B.Maloney. 1993. Assembly pathway of avian infectious laryngotracheitis virus. Am J Vet Res 54:2031-2039.
4. Scholz, E., C. L. Zhang, and P. Guo. 1993. Transactivation of the early SV40 promoter by avian infectious laryngotracheitis virus in avian hepatoma cells. J Virol Meth 45:291-301.
5. Scholz,E., E.Welniak, T.Nyholm, and P.Guo. 1993. An avian hepatoma cell line for cultivation of infectious laryngotracheitis virus and for expression of foreign genes with mammalian promotor. J Virol Meth 43:273-286.
6. Scholz, E. and P. Guo. 1995. Construction of Recombinant Avian Infectious Laryngotracheitis Virus with TK Gene disrupted by Bata-gal Coding Sequence. In Imm Viral Inf. Proc. 3rd Intl Cong Vet. Virol, 379-384..
7. Huang, Q., Y. Mat-Arip and P. Guo. 1997. Sequencing of a 5.5-kb DNA fragment and identification of a gene for a subunit of helicase/primase complex of avian laryngotracheitis virus. Virus Gene 15:(2): 119-121.

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ACCESSION NO: 0184128 SUBFILE: CRIS
PROJ NO: IOW03599 AGENCY: CSREES IOW
PROJ TYPE: HATCH PROJ STATUS: NEW MULTISTATE PROJ NO: NC-228
START: 01 OCT 1999 TERM: 30 SEP 2004 FY: 2002

INVESTIGATOR: Reynolds, D. L.

PERFORMING INSTITUTION:

VETERINARY MEDICINE
IOWA STATE UNIVERSITY
AMES, IOWA 50011

AVIAN RESPIRATORY DISEASES: PATHOGENESIS, SURVEILLANCE, DIAGNOSIS AND CONTROL

OBJECTIVES: Objective #1. Determine the pathogenesis and interactions of specific agents. Objective #2. Surveillance, occurrence and consequences of agents and hosts on disease susceptibility. Objective #3. Develop new and improved methods for the diagnosis, prevention and control of avian respiratory diseases.

APPROACH: Iowa will contribute to those studies concerning avian pneumovirus. Iowa will develop rapid diagnostics, explore new vaccination methods and study the pathogenesis of avian pneumoviruses. This will be done by employing molecular techniques and nonconventional vaccination methods and by exploring the role of passive immunity.

NON-TECHNICAL SUMMARY: Respiratory diseases afflicting poultry in modern commercial production operations are complex entities. Numerous factors including infectious agents, non-infectious agents and environmental factors may contribute to the disease complex. The purpose of this project is to have a significant impact on the diagnosis, control and prevention of poultry respiratory diseases.

PROGRESS: 2002/01 TO 2002/12
A study was initiated to evaluate biosecurity related to composting of large amounts of animal carcasses. In order to achieve a large amount of animal tissue, cattle carcasses were used. Twelve cattle carcasses averaging approximately 1,000 lbs. each, were delivered to the research site. The carcasses were placed into three 20-ft long windrow segments (constructed end-to-end to produce a single 60-ft long windrow). Each segment utilized one of three different cover materials (silage, ground cornstalks, finished yard waste compost). Newcastle disease virus (NDV) and avian encephalomyelitis virus (AEV) were used to evaluate the degree of bio-containment provided by composting. Twenty dozen 10-day-old embryonating chicken eggs were inoculated with NDV vaccine strain. Similarly, 20 dozen 6-day-old embryonating chicken eggs were inoculated with AE vaccine strain. The carcasses placed into the composting windrows were contaminated with these eggs prior to covering so as to simulate composting of diseased animals and contaminated bedding and feed. Specific Pathogen Free (SPF) chickens were used as sentinels to evaluate the bio-containment provided by composting. These birds were housed under SPF conditions prior to the beginning of the study. Twenty-four of the 12-week-old chickens were wing banded, sampled (blood) and transferred to the project site one day after the construction of the windrows. Four chickens were placed in each of six cages surrounding the composting windrow. Blood samples were collected from each bird following transport to the field research site, at the end of weeks 1, 2, 3, 4, 6 and 8. All serum samples have been tested for specific NDV and AE antibodies. The hemagglutination-Inhibition (HI) test is done for NDV. All samples from the sentinel poultry have tested negative for NDV. The same NDV and AE vaccines were used to test the ability of the composting system to inactivate pathogenic viruses found within diseased animal carcasses. Twenty-four vials and 12 cassettes were prepared from each virus. Eight cryogenic vials and 4 dialysis cassettes were inserted in each test section of the windrow. The vials are retrieved at day 1, the end of weeks 1,2,3,4, and 6 (remaining two vials to be retrieved at the end of weeks 8 and 10). The dialysis cassettes were collected at the end of weeks 1, 2, 3, and 4 from corn stalk and silage piles test segments. Cassettes that had been buried in the yard waste could not be recovered due to unanticipated plugging of the access port. These will be removed at the end of the biosecurity evaluation for the initial trial. Embryonated chicken eggs were inoculated with material from the recovered samples (10 eggs used for each sample). Test results are pending at this time.

IMPACT: 2002/01 TO 2002/12
The findings indicate that composting may be a safe and environmental feasible way to dispose of massive amounts of animal carcasses that may occur in such catastrophic disease events as avian influenza, foot and mouth disease, etc.

PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period

PROJECT CONTACT:

Name: Good, C.
Phone: 515-294-4544
Fax: 515-294-2909
Email: cgood@iastate.edu

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ACCESSION NO: 0181438 SUBFILE: CRIS
PROJ NO: IOWV-400-63-17 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 MAY 1998 TERM: 31 DEC 1998 FY: 1999

INVESTIGATOR: Reynolds, D. L.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES, IOWA 50011

STUDIES ON NEWCASTLE DISEASE VACCINATION IN TURKEYS

OBJECTIVES: The objective of this project is to assess a new recombinant vaccine for use in turkeys for the treatment of Newcastle disease. This vaccine is a fowlpox virus that expresses some Newcastle disease virus proteins. It has been proven efficacious for both fowlpox and Newcastle disease control in chickens. We are exploring the potential of using this vaccine to control Newcastle disease in turkeys. This product offers some potential advantages over conventional vaccines by decreasing the vaccine reaction in vaccinated birds and thus lessening the potential of precipitating respiratory disease complications post-vaccination

APPROACH: Some poults will be vaccinated with rFP/NDV at the hatchery. Vaccination of these poults will be by subcutaneous injection. Some poults will be vaccinated orally at 3 weeks of age. Blood samples will be collected by the medial wing vein method. Birds will be challenged with Texas GB strain of velogenic Newcastle disease virus by the intramuscular route. Following challenge, 5 birds per group (20 in all) will be tracheal swabbed using cotton-tipped applicators. Protection and efficacy will be assessed by challenge results, tracheal viral shedding of the challenge virus and seroconversion using HI titers.

NON-TECHNICAL SUMMARY: Newcastle disease in turkeys. This project will assess a new recombinant vaccine for use in turkeys for the treatment of Newcastle disease.

PROGRESS: 1998/05 TO 1998/12
The fowlpox vectored recombinant Newcastle vaccine (rFPNDV) was evaluated in turkeys. Turkeys received the rFPNDV subcutaneously at a day of age and then some turkeys were boostered vaccinated at 3 weeks of age with conventional B1 ND vaccine by the intranasal method. Birds were challenged at 6 weeks of age. It was found that those birds receiving B1 ND vaccine were protected but those birds recieiving only rFPNDV were marginally protected when compared to nonvaccinated birds.

IMPACT: 1998/05 TO 1998/12
The rFPNDV used by itself will not convey strong protection against challenge with velogenic NDV.

PUBLICATIONS: 1998/05 TO 1998/12
None, 1999

PROJECT CONTACT:

Name: Reynolds, D. L.
Phone: 515-294-0914
Email: dlr@iastate.edu

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ACCESSION NO: 0181440 SUBFILE: CRIS
PROJ NO: IOWV-400-63-26 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 OCT 1998 TERM: 30 SEP 1999 FY: 1999

INVESTIGATOR: Reynolds, D. L.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES, IOWA 50011

STUDIES ON OASIS IN TURKEYS

OBJECTIVES: The objective of this trial is to determine if the nutritional supplement Oasis has any beneficial effect on enhancing the immune system of young turkeys.

APPROACH: Birds will be allowed access only to Oasis for 48 hours following hatch and prior to placing them into battery brooders. Corresponding control birds will not receive Oasis, food or water. Upon placing the birds they will be vaccinated with a commercial adjuvanated vaccine. Blood samples will be collected at weekly intervals for weeks. Antibody titers and total serum immunoglobulin will be assessed on the birds and the groups will be compared.

NON-TECHNICAL SUMMARY: Treatment/vaccine for Newcastle disease in turkeys. Assessing nutritional supplement prior to administration of a new recombinant vaccine for use in treating Newcastle disease in turkeys.

PROGRESS: 1998/10 TO 1999/09
Oasis is a commercial product that is used when transporting turkey poults or chick from the hatchery to remote locations. The product is a hydrated gel that is placed in the transport box. The hatchling birds pick and eat the material and is supplies nutrition and hydration during the transport period. The objective of this trial was to determine if Oasis had a positive influence on the immune response of the bird. Birds were hatched and one grup was placed on oasis for 48 hours while a control group received nothing. The birds were also vaccinated with IBD immediately following hatch to determine if birds receiving Oasis would mount a higher antibody response. The results indicated there was no difference in the immune response to vaccination between those birds receiving oasis and those that did not.

IMPACT: 1998/10 TO 1999/09
Hydrated gel material will not act to stimulate a better immune response in commercial poultry.

PUBLICATIONS: 1998/10 TO 1999/09
None, 1999

PROJECT CONTACT:

Name: Reynolds, D. L.
Phone: 515-294-0914
Email: dlr@iastate.edu

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ACCESSION NO: 0181442 SUBFILE: CRIS
PROJ NO: IOWV-400-63-88 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 SEP 1997 TERM: 31 AUG 1998 FY: 1999

INVESTIGATOR: Reynolds, D. L.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
S. AND 16TH ELWOOD
AMES, IOWA 50011

STUDIES ON NEWCASTLE DISEASE

OBJECTIVES: The objective of this proposal is to determine if intrayolk-sac (IYS) injection could be used to efficaciously vaccinate birds against Newcastle disease.

APPROACH: Groups of birds were vaccinated by the IYS route with commercial licensed Newcastle disease (ND) vaccine. These birds will be compared to birds that had been vaccinated in the coventional way (spray) or had not received vaccine. Seroconversion will be determined by weekly blood collection and performing HI titers. Protection will be assessed by challenging the birds with Texas GB strain of NDV.

NON-TECHNICAL SUMMARY: Newcastle disease in chickens. Objective is to determine if an intrayolk-sac injection could be used to vaccinate against Newcastle disesase.

PROGRESS: 1997/09 TO 1998/08
Immunoglobulins purified from the egg yolks of chickens vaccinated against Newcastle disease virus (NDV) were digested with pepsin. The resulting Fab' fragments were either injected in ovo or subcutaneously at the day of hatch. The absorption of the Fab' fragments and their biologic activity was assessed by serologic assay (HI titers) and challenge to the Texas GB strain of NDV. It was found that no biologic activity, i.e. HI titers or protection against challenge, occurred with Fab' vaccinated chickens.

IMPACT: 1997/09 TO 1998/08
Passive immunization may be an important strategy in controlling certain avian pathogens. The results of this study that passive immunity will require the entire immunoglobulin molecule.

PUBLICATIONS: 1997/09 TO 1998/08
Reynolds, D. L., S. Akinc and A. Ali. Studies on the passive immunity employing the Fab' fragment of chicken immunoglobulin. Oral presentation. AAAP/AVMA annual convention meeting. New Orleans, LA. July 10 - 14, 1999.

PROJECT CONTACT:

Name: Reynolds, D. L.
Phone: 515-294-0914
Email: dlr@iastate.edu

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ACCESSION NO: 0132087 SUBFILE: CRIS
PROJ NO: IOWV-701-23-10-0005 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 1986 TERM: 30 JUN 2001 FY: 2001

INVESTIGATOR: Reynolds, D. L.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
IOWA STATE UNIVERSITY
AMES, IOWA 50011

RESPIRATORY DISEASES OF POULTRY

OBJECTIVES: To develop diagnostic techniques to aid in the control of poultry respiratory diseases of economic importance. To improve current methods of management and prevention of poultry respiratory diseases.

APPROACH: To apply modern laboratory techniques and develop new products that relate to the above objectives.

PROGRESS: 1986/07 TO 2001/06
Newcastle disease (ND) is a highly contagious viral disease of poultry capable of causing high morbidity and mortality. The traditional strategy for controlling outbreaks of highly pathogenic ND is to eradicate exposed, or potentially exposed, flocks of birds. Although this strategy has proved successful, it typically results in large numbers of birds being euthanized. The environmental, economic and animal ethical issues of this strategy are of increasing concern. The objective of this study was to examine the potential for using passive immunization as an alternative strategy for controlling highly pathogenic outbreaks of ND. Here we determine the time interval between exposure and providing protection by administering anti-Newcastle disease virus (NDV) specific immunoglobulin subsequent to virulent NDV challenge. Different groups of chickens were passively immunized (i.e. received Anti-NDV antibody) at various times with respect to challenge corresponding to 24 hrs. prechallenge, day of challenge, 1, 2, 3, 4, 5, 6, 8 and 9 days post challenge. HI titers were evaluated prechallenge prior to antibody injection and 24 hours post passive immunization. Serologic results indicated that all birds passively immunized had titers between 10 and 12 log2. The results of the challenge indicated that all birds that received passive immunization by 3 days following challenge were protected. Protection began to wane by 4 days post challenge and little (if any) protection was afforded by 8 days post challenge. In general, if birds were administered immunoglobulins prior to clinical signs of ND, they were afforded protection.

IMPACT: 1986/07 TO 2001/06
The results of the passive immunity studies indicate that providing passive protection to birds at the time of, or subsequent to, challenge can provide protection. This method of protection may be an alternative to eradication.

PUBLICATIONS: 1986/07 TO 2001/06
Reynolds, Donald and Sevinc Akinc. Passive immunization protects birds following challenge with virulent NDV. Oral presentation. AVMA / AAAP annual meeting. Boston, MA. July 14-18, 2001.

PROJECT CONTACT:

Name: Reynolds, D. L.
Phone: 515-294-0914
Fax: 515-294-1401
Email: dlr@iastate.edu

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ACCESSION NO: 0175014 SUBFILE: CRIS
PROJ NO: MD-D-115 AGENCY: CSREES MD.
PROJ TYPE: HATCH PROJ STATUS: TERMINATED MULTISTATE PROJ NO: NE-138
START: 01 OCT 1996 TERM: 30 SEP 2002 FY: 2001

INVESTIGATOR: Vakharia, V. N.; Heckert, R. A.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
UNIV OF MARYLAND
COLLEGE PARK, MARYLAND 20742

EPIDEMIOLOGY AND CONTROL OF EMERGING STRAINS OF POULTRY RESPITORY DISEASE AGENTS

OBJECTIVES: Design and implement novel immune and genetic prophylactic strategies for effective control of respitory diseases caused by emerging IBV, ILTV, mycoplasmas, IBDV, and CAV.

APPROACH: Approach: The genomic DNA of chicken anemia vius (CAV) will be cloned and complete nucleotide sequence determined. The VPI and VP2 genes will be subcloned and expressed in a baculovirus expression system. The synthesized proteins will be characterized with monoclonal antibodies and then evaluated as immunogens serologically and via challenge.

PROGRESS: 1996/10 TO 2002/09
To develop an attenuated, multi-spectrum vaccine candidate that can protect against classical and variant strains of infectious bursal disease virus (IBDV), we constructed several full-length and chimeric cDNA clones of segments A and B of the D78 and GLS strains. Using the cRNA-based reverse genetics system developed for IBDV, we generated recombinant viruses after transfection in Vero cells. A panel of IBDV-specific monoclonal antibodies (MAbs) was used to characterize the recovered viruses and their replication kinetics was compared with that of the parental D78 strain in vitro. Viruses deficient in the expression of VP5 nonstructural protein (NS) grew to slightly lower titers than D78 virus and exhibited decreased cytotoxic and apoptotic effects in cell culture. To evaluate the efficacy of the recombinant IBDV vaccine, we inoculated 3-week-old chickens with this virus and challenged them with STC and variant GLS viruses. Based on histopathology and serology tests, we observed that the chickens inoculated with recombinant IBDV vaccine (containing a GLS-specific epitope) failed to induce any pathological lesions or clinical signs of disease, and were completely protected against both classic and variant IBDV strains. In another study, we show that dimethylsulfoxide (DMSO) enhances liposome-mediated transfection of nucleic acid in chicken macrophage cells and that this could be exploited for transcutaneous delivery of naked DNA through the intact skin of chickens. We found that DMSO enhanced transfection efficiencies of lipofectamine and polyethyleneimine in HD-11 chicken macrophage cells. Based on this principle, we showed that transcutaneous delivery of a DNA plasmid-dimethylsulfoxide mixture (1:1) to untreated skin of chickens result in a wide distribution of the plasmid in the body. Distribution studies were done using plasmids encoding enhanced green fluorescent protein (EGFP) reporter gene and a bivalent DNA vaccine coding for IBDV and Newcastle disease virus (NDV) immunogenic protein genes. This bivalent vaccine induced mucosal and systemic immune responses, as evidenced by IgA and IgM production in the tears and serum of vaccinated chickens. Mucosal immune responses in the tears after topical vaccination were significantly higher than after intramuscular delivery of the same DNA vaccine and were characterized by the absence of an IgG response. The biodistribution of plasmid indicated that topical delivery with DMSO resulted in a wide distribution and persistence of the plasmid until 15 weeks post-primary vaccination. Both delivery methods resulted in insert-specific message being made in several body tissues, but after topical delivery the virus-specific mRNA could be detected in the bone marrow of one out of three chickens until 15 weeks post-primary vaccination. Furthermore, transcutaneous delivery of this DNA vaccine using DMSO conferred protection from challenge with virulent IBDV (86% survival) and NDV (86% survival). This novel transcutaneous method of delivery of a DNA vaccine shows promise as being an easy and effective way to deliver nucleic acids through intact skin for vaccination or therapeutic purposes.

IMPACT: 1996/10 TO 2002/09
We have developed an attenuated, multivalent IBDV vaccine that can protect against classic and variant strains, and have developed methods for delivery of a DNA vaccine. These findings will aid in the development of cost-effective vaccines for viral pathogens and better equip the chicken farmers with tool necessary to combat viral diseases.

PUBLICATIONS: 1996/10 TO 2002/09
1. Elankumaran, S., Heckert, R.A., and Moura, L. 2002. Persistence and tissue distribution of a variant strain of infectious bursal disease virus in commercial broiler chickens. Avian Dis. 46:169-176.
2. Heckert, R.A., Elankumaran S, Oshop G, and Vakharia V.N. 2002. A novel transcutaneous plasmid-dimethylsulfoxide delivery technique for avian nucleic acid immunization. Vet Immunol Immunopathol. 89:67-81.
3. Oshop G, Elankumaran S, Heckert R.A. 2002. DNA vaccination in the avian. Vet Immunol Immunopathol. 89:1-12. Oshop, G.L., Elankumaran, S., Vakharia, V.N., and Heckert, R.A. 2002. In ovo delivery of DNA to the avian embryo. Vaccine (in press).
4. Vakharia, V.N. 2002. Molecular determinants of virulence in infectious bursal disease virus. 3rd European Concerted Research Action (COST 839) Meeting on Immunosuppressive Viral Diseases in Poultry, April 25-28, Leipzig, Germany.
5. Liu, M., Brandt, M., Liu, Y., Edwards, G.H., and Vakharia, V.N. 2002. Recombinant attenuated IBDV vaccine that protects against classic and variant strains. 138th American Veterinary Medical Association Annual Convention, July 13-17, 2002, Nashville, TN.
6. Liu, M., and Vakharia, V.N. 2002. Two amino acid residues in VP2 protein of IBDV are involved in cell entry and efficient replication in vivo. 21st Annual Meeting of American Society for Virology, July 20-24, Lexington, KY.

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ACCESSION NO: 0190633 SUBFILE: CRIS
PROJ NO: MD-VTMD-9201 AGENCY: CSREES MD.
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2002-35204-11601 PROPOSAL NO: 2001-02372
START: 15 DEC 2001 TERM: 31 DEC 2003 GRANT YR: 2002
GRANT AMT: $228,000

INVESTIGATOR: Samal, S.; Gelb, J.

PERFORMING INSTITUTION:
VETERINARY MEDICINE
UNIV OF MARYLAND
COLLEGE PARK, MARYLAND 20742

RECOMBINANT NEWCASTLE DISEASE VIRUS EXPRESSING IBV SPIKE PROTEIN.

OBJECTIVES: A recombinant Newcastle Disease Virus (NDV) containing the spike S1 glycoprotein gene of avian infectious bronchitis virus (IBV) will be recovered from cloned cDNAs. The level of expression, intracellular transport, and processing of the IBV S1 protein expressed from the recombinant NDV will be examined. The recombinant NDV expressing the S1 protein will be evaluated as a vaccine to control both of these economically important diseases.

APPROACH: A reverse genetic system developed by our laboratory will be used to recover a recombinant Newcastle disease virus (NDV) containing the S1 gene of infectious bronchitis virus (IBV). This system involves simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from co-transfected plasmids. The S1 gene of IBV strain Mass 41 will be inserted into the 3' proximal locus of the NDV strain LaSota full-length cDNA clone. The correct expression of the IBV S1 protein will be determined by immunoprecipitation and immunofluorescence assays. The pathogenicity and immunogenicity of recombinant NDV expressing the S1 protein of IBV will be evaluated in two-week-old chickens. The vaccinated chickens will be challenged with virulent NDV and IBV strains. For the vaccine to be satisfactory, 90% of the vaccinated chickens should be negative for virus recovery and survive challenge.

NON-TECHNICAL SUMMARY: IBV is the most common respiratory disease in poultry in the USA in recent years. Currently available IBV vaccines are unstable. In contrast, NDV strain LaSota is a safe and stable vaccine for both NDV and IBV. We have developed a system to produce live NDV strain LaSota from DNA. This system not only allows us to produce live NDV strain LaSota from DNA but also allows us to introduce foreign genes into the genetic material of NDV. We propose to insert the immunogenic and protective portion of the spike protein of Infectious Bronchitis Virus (IBV) into the genetic material of NDV strain LaSota.

PROGRESS: 2002/01 TO 2003/01
We have constructed two NDV cDNA constructs containing the S1 gene of IBV strain Mass 41. In one construct, the S1 gene was introduced into the first position, before the NP gene, in the full-length cDNA of NDV strain Beaudette C. In the other construct, the S1 gene of Mass 41 was introduced into the third position, after P gene, in the full-length cDNA of NDV strain LaSota. Recombinant NDV strains Beaudette C and LaSota containing the S1 gene were recovered after transfection of HEp-2 cells following our standard recovery protocol. Recombinant NDV strains containing the S1 gene of IBV were first examined by RT-PCR to confirm the presence of the S1 gene in the genome of recombinant NDV. To determine whether the recombinant NDV strains correctly expressed the IBV S1 protein in infected cells, Western blot analysis was performed. Our results showed that correct size S1 protein was expressed from recombinant NDV strains. Pathogenicity studies of these recombinant NDV strains in 10-day-old chicks showed that the ICPI values were slightly lowered. Studies are underway to determine whether the S1 protein is incorporated into NDV particles.

IMPACT: 2002/01 TO 2003/01
The use of the recombinant NDV vector to deliver the S protein of IBV should provide immunity against IBV and NDV strains. Unlike currently available IBV attenuated vaccines and field strains, NDV is highly stable. Thus, the recombinant NDV/IBV vaccine should not contribute to the evolution of new IBV variants that continue to plague the poultry industry. Our proposed vaccine will be highly beneficial to the poultry industry.

PUBLICATIONS: 2002/01 TO 2003/01
No publications reported this period

PROJECT CONTACT:

Name: Samal, S.
Phone: 301-314-6813
Fax: 301-314-6855
Email: ss5@umail.umd.edu

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ACCESSION NO: 0180471 SUBFILE: CRIS
PROJ NO: MDR-9802290 AGENCY: CSREES MD.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 98-35204-6427
START: 01 OCT 1998 TERM: 30 SEP 2001 FY: 2000 GRANT YR: 1998
GRANT AMT: $140,000

INVESTIGATOR: Samal, S. K.

PERFORMING INSTITUTION:
REGIONAL COLLEGE OF VET MED
UNIV OF MARYLAND
COLLEGE PARK, MARYLAND 20742

PRODUCTION OF INFECTIOUS NEWCASTLE DISEASE VIRUS FROM CDNA: POTENTIAL FOR VACCINE DEV. AND BASIC

OBJECTIVES: 9802290. 1. Construction of a full-length NDV cDNA clone. 2. Recovery and characterization of infectious NDV from cDNA. 3. Construction of NDV with mutations in the fusion protein cleavage site.

APPROACH: A full-length cDNA of the genomic RNA of NDV will be constructed using reverse-transcriptase polymerase chain reaction. The cDNA will be cloned into a plasmid flanked by a T7 promoter and ribozyme sequences. Infectious NDV will be produced by the intercelluler coexpression of T7 based plasmid cDNAs. One cDNA will encode the complete NDV genome and the other cDNAs will encode NDV proteins required for first round of virus specific mRNA synthesis. T7 polymerase will be supplied by a replication-deficient recombinant vaccinia virus. The recovered virus will be characterized using several invitro methods. Mutations will be introduced into NDV cDNA by site directed mutagenesis.

PROGRESS: 1999/10 TO 2000/09
A recombinant NDV strain, Beaudette C, was generated from cloned cDNAs. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. The sequence of the cleavage site of the fusion protein of the recombinant NDV were altered by site-directed mutagenesis. Recombinant NDV containing the mutation required trypsin activation for fusion and infectivity in cell culture. The virulence of the recombinant NDV with altered fusion protein cleavage site was also lowered. This result showed that the cleavage of the fusion protein plays an important role in the pathogenesis of NDV.

IMPACT: 1999/10 TO 2000/09
Recovery of infectious recombinant Newcastle Disease Virus from cloned DNA can produce better vaccines against Newcastle Disease in poultry. This new technology will also enable recombinant Newcastle Disease Virus to be used as vaccine vector for other avian pathogens. Thus, production of recombinant Newcastle Disease Virus has a great potential for the development of vaccines against avian pathogens and will significantly benefit the poultry industry.

PUBLICATIONS: 1999/10 TO 2000/09
Krishnamurthy, S., Huang, Z. and Samal, S.K. 2000. Recovery of a virulent strain of Newcastle Disease Virus from cloned cDNA: epression of a foreign gene results in growth retardation and attenuation. Virology 278: 168-182.

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ACCESSION NO: 0180895 SUBFILE: CRIS
PROJ NO: MICK-9803219 AGENCY: CSREES MICK
PROJ TYPE: SMALL BUSINESS GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 98-33610-6303
START: 01 SEP 1998 TERM: 31 AUG 2001 GRANT YR: 1998
GRANT AMT: $225,000

INVESTIGATOR: Reilly, J. D.

PERFORMING INSTITUTION:
ORIGEN, INC.
NATURA, INC.
3900 COLLINS ROAD
LANSING, MICHIGAN 48909

IMMORTAL CELL LINE FOR POULTRY VACCINE PRODUCTION & DIAGNOSTICS

OBJECTIVES: 9803219. There are two technical objectives for this SBIR Phase II proposal. The first technical objective is to develop methods for using OCLTM cells to make vaccines that are at least comparable to traditional methods in yield and efficacy. The viruses that will be used are SB1-OCLTM, CVI988-OCLTM, avian influenza virus, and vaccine strains of avian reovirus, Newcastle disease virus, fowlpox virus, cell culture-adapted infectious bursal disease virus, and duck enteritis virus. The second technical objective is to compare sensitivity and range of OCLTM-based VI assays to traditional methods. The viruses that will be used are avian influenza virus, avian reovirus, Newcastle disease virus, fowl pox virus, and duck enteritis virus.

APPROACH: The components of the first objective are: 1) One-step growth curves to determine yield of virus in OCLTM cells compared to traditional methods, 2) Modify various growth parameters including initial cell density, MOI, temperature, media composition to maximize the yield of each virus, 3) Compare immunogenicity of virus produced on OCLTM cells to virus produced by traditional methods, 4) Compare safety of virus produced on OCLTM cells to virus produced by traditional methods, and 5) Scale growth of each virus up to production levels. Specifically, the components of the second objectives are: 1) Compare sensitivity of OCLTM-based VI assays to traditional methods, and 2) Compare susceptibility of a panel of virus strains and field isolates for each virus tested to traditional methods of detection. The Immunogenicity and safety tests will be performed as described for each virus in 9 CFR PP113.

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ACCESSION NO: 0172971 SUBFILE: CRIS
PROJ NO: MISV-322090 AGENCY: CSREES MISV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1996 TERM: 30 JUN 2002 FY: 2002

INVESTIGATOR: Wang, C.; Magee, D.; Keirs, R. W.

PERFORMING INSTITUTION:
COLLEGE OF VETERINARY MEDICINE
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

INFECTIOUS BRONCHITIS VIRUS INFECTION IN COMMERCIAL BROILERSAND CHICKEN EMBRYOS

OBJECTIVES: Serotype IBV from selected Mississippi flocks experiencing respiratory problems by RT-PCR and IFA; Determine whether commonly used vaccine strains mutate at serial passage in chicken embryos, and Evaluate the persistence and the replication competition if IBV strains in various organs and tissues.

APPROACH: Trachea, cecal tonsils and cloacal contents will be collected from birds suffering from respiratory problems within Mississippi. The viruses isolated will be serotyped by IFA with monoclonal antibodies (MAbs) and PCR specific for Mass, Conn or Ark strains. SPF chicken embryos will be artificially inoculated with Mass, Ark or Mass/Ark combination respectively. The viruses will be passaged from chicken to chicken and examined for mutation by PCR and sequencing. The persistence of IBV in tissues or lymphocytes will be determined by IFA and RT-PCR.

NON-TECHNICAL SUMMARY: The project seeks to understand how infectious bronchitis virus infection occurs in and affects commercial broilers and chicken embryos.

PROGRESS: 1996/10 TO 2002/06
Infectious bronchitis (IB) is an acute, highly contagious viral respiratory disease and one of the most common and economically important diseases in the poultry industry. The objectives of this study were to 1) study the epidemiology of IB in Mississippi; 2) find the best method to detect and type infectious bronchitis virus (IBV); and 3) understand the pathogenesis of IBV. We have identified that Arkansas 99 (Ark 99) serotype was the predominate serotype that caused the 1988 IB outbreak. The results also indicate that within a 1 year period Ark type IBV in Mississippi was spread with little or no change in its genetic sequence. We also found that the reverse transcription polymerase chain reaction (RT-PCR) is more sensitive method to detect and type IBV than the indirect fluorescent antibody assay (IFA), especially when there is more than one strain of IBV involved, but the IFA is rapid and cheaper than the RT-PCR. Our study suggests fusion is the mechanism for IBV to enter the cells. Entry into susceptible cells of IBV seems to be more efficient at a slightly basic pH. The study also suggests that the feline aminopeptidase N molecule plays a role in IBV entry.

IMPACT: 1996/10 TO 2002/06
This study help to understand the pathogenesis of infectious bronchitis virus and to utilize the best method to diagnose the disease when there is an IB outbreak. The resulting information provided prevention and control strategies to save millions of dollars lost annually due the virus, consequently significantly enhancing profitability.

PUBLICATIONS: 1996/10 TO 2002/06
1. Wang C, Miguel B, Hong C, Austin FW, and Keirs RW. Comparision of the immunofluorescent assay and reverse transcription-polymerase chain reaction to detect and type infectious bronchitis virus. Avian Dis, 1999, 43:590-596.
2. Shi Q and Wang C. Genetic relationships of infectious bronchitis virus isolates from Mississippi broilers. Avian Dis, 2000, 44:66-73.
3. Miguel B, Pharr GT, and Wang C. The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Arch. Virol. 2002; 55:57-63.

PROJECT CONTACT:

Name: Wang, C. L.
Phone: 662-325-1205
Fax: 662-325-1031
Email: wang@cvm.msstate.edu

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ACCESSION NO: 0172972 SUBFILE: CRIS
PROJ NO: MISV-329040 AGENCY: CSREES MISV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1996 TERM: 30 JUN 2002 FY: 2002

INVESTIGATOR: Montgomery, R. D.; Maslin, W.; Boyle, C. R.

PERFORMING INSTITUTION:
COLLEGE OF VETERINARY MEDICINE
MISSISSIPPI STATE UNIV
MISSISSIPPI STATE, MISSISSIPPI 39762

THE HEAD-ASSOCIATED LYMPHOID OF CHICKENS AND ITS IMMUNOLOGICAL ROLE

OBJECTIVES: Improve the methodology used to evaluate the HALT; Effects of various stressors on the HALT; and (3) Effect of GH on pathogenesis of various pathogens.

APPROACH: An ELISA will be developed to quantitate the various classes of immunoglobulin (Ig) present in tears (IgA, IgG, IgM) of chickens exposed to a test antigen (B. abortus). Once developed, the ELISA will be used to re-evaluate the default GH B. abortus assay to determine which parameters can be modified to increase the sensitivity and specificity of the assay; Stressors known to have an impact on lymphoid systems and/or the respiratory tract will be given to various ages of SPF chicks. Following exposure, these chicks will be subjected to both structural and functional analysis to determine the effect of these agents on the HALT; and Respiratory tract-oriented microbial pathogens, including viruses and bacteria will be given to both GH-intact and GHx chicks of various ages. The pathogenesis of these stressors will be monitored in both types of subjects by conducting sequential microbial recoveries and histological analyses of targtet sites specific for the particular agent used. Optionally, if the pathogenesis of vaccine viruses are studied, the GH-intact and GHx chicks may be challenged at the end of the trial to determine the degree of protection in both.

NON-TECHNICAL SUMMARY: Defining the immunological role of the head-associated lymphoid tissues of chickens is the focus of this project. This work will aid us in understanding how to utilize the tissue to enhance immune responses in the chicken.

PROGRESS: 1996/10 TO 2002/06
Thirty-six modified-live virus vaccines, including 16 infectious bronchitis virus (IBV), 10 Newcastle Disease virus (NDV), and 10 NDV/IBV vaccines were evaluated for their effects on the gland of Harder (GH) and other head-associated lymphoid tissue (HALT) sites. Some of the IBV vaccines, either alone or in combination with NDV, were found to interfere with the GH/HALT's ability to respond to antigenic stimulation and to alter specific histological attributes. Two hundred and eight E. coli were collected from various lesions (respiratory, intestinal, yolk, joint, etc.). These isolates were characterized biochemically, analyzed for their plasmid content(s), analyzed for their sensitivity to antibiotics, and evaluated for their lethality in embryonated chicken eggs, which reputedly correlates with in vivo pathogenicity in young chickens. One of the E. coli, which repeatedly spared embryonated eggs was adapted to grow in the presence of nalidixic acid and evaluated in the chicks that hatched. Although overall hatchability was reduced, a number of infected embryos did hatch into viable and healthy-appearing chicks. However, those chicks had lowered body weights and increased early mortality. Consistently high levels of the nalidixic acid resistant-E. coli were recovered from the yolk of these chicks and moderate levels were detected in their lung and trachea. E. coli was also recovered from the respiratory tract of non-infected chicks that were hatched in contact with the E. coli chicks, indicating that E. coli can be transmitted vertically through the embryo and amplified horizontally to susceptible neonates at the time of hatching. Data from an extensive respiratory outbreak that occurred in Mississippi broilers during 1998-1999 was collected and analyzed. Arkansas and, to some extent, Connecticut IBV, were the principal agents detected in this outbreak. Epidemiological factors collected with these cases included date received, identity of growout company involved, age of birds, strain(s) of IBV in vaccination program, infectious bursal disease (IBD) vaccination status, condition of samples received, any respiratory lesions noted, and the geographic location of farm. In general, the number and distribution of 1) cases received, 2) cases positive for virus, and 3) viruses detected were proportional to the epidemiological factors collected.

IMPACT: 1996/10 TO 2002/06
This research indicated that by our use of 36 modified live virus vaccines which included 16 infectious bronchitis virus, 10 Newcastle Disease viruses, and 10 NDV/IBV vaccines, either alone or in combination, we were successful in interfering with the GH/HALT's ability to respond to antigenic stimulation and to alter specific histological attributes.

PUBLICATIONS: 1996/10 TO 2002/06
1. Montgomery, R. D., C. R. Boyle, W. R. Maslin, and D. L. Magee. Attempts to reproduce a runting/stunting-type syndrome using infectious agents isolated from affected Mississippi broilers. Avian Dis. 41:80-92. 1997.
2. Montgomery, R. D., W. R. Maslin, and C. R. Boyle Effects of Newcastle disease vaccines and Newcastle disease/infectious bronchitis virus combination vaccines on the head-associated lymphoid tissues of chickens. Avian Dis. 41:399-406. 1997.
3. Montgomery, R. D., C. R. Boyle, T. A. Lenarduzzi, and L. S. Jones. Chicks Hatched from Escherichia coli-infected Embryos. Avian Diseases. 43:553-563. 1999.

PROJECT CONTACT:

Name: Montgomery, R. D.
Phone: 662-325-1205
Fax: 662-325-1031
Email: montgomery@cvm.mssstate.edu

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ACCESSION NO: 0187676 SUBFILE: CRIS
PROJ NO: MO-ASAH0594 AGENCY: CSREES MO.
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM: 30 SEP 2001 FY: 2001

INVESTIGATOR: Ledoux, D. R.; Bermudez, A. J.; Rottinghaus, G. E.

PERFORMING INSTITUTION:
ANIMAL SCIENCES
UNIVERSITY OF MISSOURI
COLUMBIA, MISSOURI 65211

CHARACTERIZATION OF TOXICOLOGICAL EFFECTS OF MULTIPLE MYCOTOXINS IN POULTRY

OBJECTIVES: Determine the additive, synergistic, or antagonistic effects of low levels of multiple mycotoxins on performance, organ weights, hematology, serum chemistry, and immune function of poultry.

APPROACH: Chicks and poults will be fed combinations of low levels of mycotoxins (naturally occurring levels) for three to four weeks and the individual and combined effects of the mycotoxins will be assessed based on growth performance, organ weights, hematology, serum chemistry, and immune function. The primary and secondary antibody response to inactivated Newcastle disease virus (NDV) will be used to examine the humoral immune response of broilers and turkeys. A [3H]-thymidine incorporation assay will be used to assess the proliferation of chick and turkey lymphocytes in response to two mitogens, concanavalin A and pokeweed mitogen. An Escherichia coli challenge will be used to evaluate the ability of chicks and turkeys, fed combinations of mycotoxins, to clear the bacteria from the peripheral circulation. Additionally, birds will be fed mycotoxins, vaccinated against selected poultry diseases and vaccine titers determined. In addition, vaccinated birds will then be challenged with the disease organisms to determine if the efficacy of the vaccine has been reduced by the mycotoxins.

NON-TECHNICAL SUMMARY: U.S. poultry producers have a continuous problem with mycotoxin-contaminated feedstuffs causing poorly defined syndromes. It has been speculated that many of these syndromes might be caused by synergism between low levels of several mycotoxins. Results of these studies will demonstrate whether multiple toxins are responsible for previously reported syndromes.

PROGRESS: 2001/01 TO 2001/12
A 21-day experiment was conducted to determine if the turkey could be used as a model for evaluating the efficacy of adsorbents to ameliorate the toxic effects of aflatoxin (AF). Dietary treatments fed from day of hatch included: 0 ppb AF, 100 ppb AF, 200 ppb AF, 300 ppb AF, 400 ppb AF, 500 ppb AF, and 600 ppb AF. AF was supplied by A. parasiticus culture material that contained 986 ppm AFB1, 29 ppm AFB2, 464 ppm AFG1, and 9 ppm AFG2. Compared with controls, poults fed 200 ppb AF or higher had reduced (P < .001) feed intake and lower (P < .001) body weight gains. Significant mortality (14/20) occurred in poults fed 600 ppb AF. Compared with controls, poults fed 100 ppb AF or higher had lower (P < .001) relative liver weights, whereas poults fed 200 ppb AF or higher had increased (P < .001) relative kidney weights. Histopathological analysis indicated the presence of liver lesions in poults fed 100 ppb AF or higher. The primary hepatic lesions were biliary hyperplasia, hepatocellular hyperplasia, and hepatic necrosis with the severity of lesions increasing with increasing AF dose. Kidney lesions were noted in poults fed diets containing 400 ppb AF or higher with a mild to moderate membranous thickening of the glomerular capillary basement membrane noted in most specimens. Results confirm previous reports that suggest turkeys are very sensitive to the toxic effects of AF. The lowest level of AF (100 ppb) that caused toxic effects in poults in this study is 20 fold lower than the levels (2 ppm or higher) reported to cause toxic effects in broilers under laboratory conditions.

IMPACT: 2001/01 TO 2001/12
Results suggest that the turkey could be used as a more sensitive model to evaluate the efficacy of adsorbents to ameliorate the toxic effects of AF at levels that have been reported to cause toxicity under field conditions.

PUBLICATIONS: 2001/01 TO 2001/12
1. Broomhead, J. N., D. R. Ledoux, A. J. Bermudez, and G. E. Rottinghaus, 2002. Chronic effects of fumonisin B1 in broilers and turkeys fed dietary treatments to market age. Poultry Science 81:56-61.
2. Ledoux, D. R., G. E. Rottinghaus, and A. J. Bermudez, 2001. In vitro binding of mycotoxins by adsorbents does not always translate into in vivo efficacy. Pp. 279-287 In: Mycotoxins And Phycotoxins In Perspective At The Turn Of The Millenium. Proceedings of the Xth International IUPAC symposium on Mycotoxins and Phycotoxins.
3. Butkeraitis, P., J. N. Broomhead, E. A. Guaiume, D. R. Ledoux, A. J. Bermudez, and G. E. Rottinghaus, 2002. A turkey model for evaluating the efficacy of adsorbents to ameliorate the toxic effects of aflatoxin. Abstracts International Poultry Scientific Forum, January 14-15, Atlanta, Georgia, page 45.
4. Ledoux, D. R., J. Broomhead, A. J. Bermudez, and G. E. Rottinghaus, 2001. Mycotoxin-Nutrient interactions. pp. 82-94. Proceedings 62nd Minnesota Nutrition Conference & Minnesota Corn Growers Association Technical Symposium. Minneapolis, Minnesota, September 11-12.
5. Ledoux, D. R., G. E. Rottinghaus, A. J. Bermudez, and J. N. Broomhead, 2001. Is fumonisin B1 a threat to the poultry industry? Abstracts The World Mycotoxin Forum, 14-15 May 2001, Noordwijk, The Netherlands. Page 80.

PROJECT CONTACT:

Name: Ledoux, D. R.
Phone: 573-882-1140
Email: ledouxd@missouri.edu

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ACCESSION NO: 0088380 SUBFILE: CRIS
PROJ NO: OHO00740 AGENCY: CSREES OHO
PROJ TYPE: HATCH PROJ STATUS: TERMINATED MULTISTATE PROJ NO: NC-168
START: 01 OCT 1997 TERM: 30 SEP 2003 FY: 2002

INVESTIGATOR: Nestor, K. E.; Velleman, S. G.

PERFORMING INSTITUTION:
ANIMAL SCIENCES
OHIO STATE UNIV
WOOSTER, OHIO 44691

ADVANCED TECHNOLOGIES FOR THE GENETIC IMPROVEMENT OF POULTRY

OBJECTIVES: Develop, compare, and integrate emerging technologies with classical quantitative genetics for improvement of economic traits in poultry.

APPROACH: Long-term lines of turkeys selected for increased egg production and increased 16-week body weight and their corresponding randombred controls will be maintained. Also, Japanese quail lines divergently selected for 4-week body weight (HW, LW) and plasma yolk precursor (HP, LP) and lines (HW-HP, HW-LP) selected for a combination of these traits will be maintained. A randombred (R1) control population of Japanese quail will be maintained without selection. Attempts will be made to study genetic variation among the experimental turkey and Japanese quail lines by DNA fingerprinting and study of MHC haplotypes. The turkey lines will be studied for resistance to certain diseases including fowl cholera and Newcastle disease. Turkey proteoglycans during skeletal development will be characterized.

PROGRESS: 1997/10 TO 2003/09
Beltsville Small White (BSW) turkeys have been utilized as an experimental model in the study of bacterial, parasitic, and fungal diseases. Given the critical role of the major histocompatibility comple (MHC) antigens in the initial steps of immune response to specific pathogens, the MHC Class II of BSW turkeys was characterized. Southern blot analysis of PvuII-digested turkey DNA that was hybridized with a chicken Class II beta gene genomic clone revealed two restriction fragment length polymorphism profiles not previously identified in experimental or commercial breeder lines of turkeys. These fingerprint profiles differed in a single 6.0-kb gand that was present in approximately 38% of the birds examined. The DNA fragments of 5.0, 4.1, 3.3, and 3.1 were present in both profiles. Furthermore, no mixed lymphocyte reaction was observed between individuals within the BSW turkey line. The present results indicate that BSW turkeys represent a unique source of genetic diversity for MHC Class II haplotypes. Candidate male and female breeders from a number of genetic lines of turkeys that were reared intermingled, with the sexes housed in different buildings on the same farm, were vaccinated with a live Newcastle disease virus vaccine (type B1, strain B1, Lasota) just prior to the commencement of egg production. In 1999, an average mortality of 5.8 % occurred immediately following vaccination and the level of mortality varied among lines. Mortality was greater in large-bodied lines than in small-bodied lines. Affected birds exhibited multiple areas of focal necrosis in the liver and spleen and congestion of the heart and lungs. The percentage mortality occurring following similar vaccination in 2000 averaged 2.6 and mortality was greater in one line (F line) than the other genetic groups and higher in females than in males. Mortality in the F line, selected for increased body weight and known to be susceptible to various diseases, averaged 15.1% for both years. Attempts failed in both years to isolate Pasteurella multocida or other bacteria. There was a positive correlation between increased body weight and increased mortality following vaccination with the live LaSota vaccine.

IMPACT: 1997/10 TO 2003/09
The MHC has been shown to be involved in disease resistance in turkeys. Knowledge of variation in MHC haplotypes is important for turkey breeders when they select for disease resistance. Selection for increased growth rate reduces disease resistance in turkeys so commercial turkey breeders should include selection for disease resistance in their program.

PUBLICATIONS: 1997/10 TO 2003/09
1. Sacco, R. E., K. E. Nestor, and R. A. Kunkle, 2000. Genetic variation in response of turkeys to experimental infection with Bordetella avium. Avian Dis. 44:197-200.
2. Sacco, R. E., R. B. Rimler, X. Ye, and K. E. Nestor, 2001. Identification of new major histocompatibility complex Class II restriction fragment length polymorphisms in a closed experimental line of Beltsville Small White turkeys. Poultry Sci. 80:1109-1111.

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ACCESSION NO: 0166758 SUBFILE: CRIS
PROJ NO: VA-135458 AGENCY: CSREES VA.
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 JAN 1995 TERM: 31 DEC 1998 FY: 1999

INVESTIGATOR: Lee, J. C.

PERFORMING INSTITUTION:
COLLEGE OF VETERINARY MEDICINE
VIRGINIA POLYTECHNIC INSTITUTE
BLACKSBURG, VIRGINIA 24061

DISEASE, EPIDEMIOLOGIC AND TOXICOLOGIC INVESTIGATIONS IN VIRGINIA

OBJECTIVES: To identify and respond in a timely manner to sudden disease outbreaks and toxicoses affecting Virginia agricultural producers.  To determine the economic impact of these outbreaks.

APPROACH: Funds will be allocated to individual investigators or interdisciplinary teams for 3 purposes: To investigate disease entities of unknown etiology and with potential risk of significant economic loss to agricultural animals in Virginia.  To support epidemiologic studies of disease and impaired productivity of animals. These data will support economic analysis of the impact of specific diseases and conditions on Virginia producers. To supplement field investigations in which toxicologic agents are suspected and to develop analytical techniques for toxic substances determined to be of clinical importance in Virginia.

NON-TECHNICAL SUMMARY: Sudden disease outbreaks and toxicoses impact Virginia agricultural producers and require timely investigation and assessment. This project allocates funds to research disease outbreaks and toxicoses with potentially significant economic loss to agricultural animals in Virginia. Projects must be for one of three purposes: 1-Investigate disease entities of unknown etiology, 2-Epidemiologic studies of disease and impaired productivity, or 3-Field investigations where toxicologic agents are suspected.

PROGRESS: 1995/01 TO 1998/12
This project was designed to provide a mechanism for quick, initial response to disease and toxicity, particularly in Virginia. Several studies were conducted as need arose, including Equine Potomac Horse Fever, avian influenze, and Newcastle Disease. Recently the project funded a study on turkey enteritis syndrome (TES), a condition causing serious economic loss and producer changes in Virginia. Turkey Corona Virus (TVC) and Cochlosoma anatis (a protozoan parasite)are two agents frequently isolated from TES outbreaks. TVC is readily transmitted, but not so after four hours of infected animal removal. C. anatis alone causes enteritic disease. In combination, these agents result in a higher mortality rate and illness is more severe than with a single agent.

IMPACT: 1995/01 TO 1998/12
This project allows quick response to conditions of animal and related human health in the Commonwealth of Virginia. As concerns of disease, toxicology, or potential epidemics emerge, veterinarians are enabled to respond quickly and provide initial research to assess and resolve situations.

PUBLICATIONS: 1995/01 TO 1998/12
No publications reported this period

PROJECT CONTACT:

Name: Lee, J. C.
Phone: 540-231-4807
Fax: 540-231-7367
Email: jclee@vt.edu

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ACCESSION NO: 7000416 SUBFILE: ICAR
PROJ NO: W9601 AGENCY: OTHER FEDERAL
START: 01 APR 1997 TERM: 31 MAR 2000 FY: 1997

INVESTIGATOR: WRIGHT P

PERFORMING INSTITUTION:
CANADIAN FOOD INSPECTION AGENCY NATIONAL CENTRE FOR FOREIGN ANIMAL DISEASES
820 Elgin Avenue
Winnipeg, Manitoba R3E 3M2

DEVELOPMENT, STANDARDIZATION AND VALIDATION OF SEROLOGICAL TESTS FOR THE DIAGNOSIS OF FOREIGN ANIMAL DISEASES.

NARRATIVE: IMPACT: Oct 1996 Enhanced capability and confidence in the detection of: - E/WEE (Eastern/Western equine encephalitis), ND (Newcastle disease) and AI (avian influenza) in imported ostriches. - E/WEE, FMD and VS (Vesicular stomatitus) in imported llamas and alpacas. International recognition and confidence related to our proficiency in the detection of VS, HC (hog cholera), PR (pseudo rabies), ASF (African swine fever), ND, AI and TRT (turkey rhinotracheitis) in traditional species.

OBJECTIVES: Oct 1996 To develop, monitor and improve, on an ongoing basis, diagnostic reagents and protocols for the diagnosis of foreign animal disease, and ensure that they meet or exceed the standards for prescribed tests as set by the OIE Standards Commission.

PROGRESS:
Oct 1996 new project

NOTES: CONTACT: WRIGHT P; PHONE: 204-984-1007; FAX: 204-275-0402

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ACCESSION NO: 7000548 SUBFILE: ICAR
PROJ NO: NV9401 AGENCY: OTHER FEDERAL
START: 01 APR 1997 TERM: 31 MAR 1998 FY: 1997

INVESTIGATOR: VYDELINGUM S; TECH

PERFORMING INSTITUTION:
CANADIAN FOOD INSPECTION AGENCY CENTRE OF EXPERTISE FOR PLANT QUARANTINE PESTS
3851 Fallowfield Road R.R. #7
Nepean, Ontario K2H 7V2

VALIDATION OF PCR METHODS FOR THE DETECTION OF FAD VIRUSES

NARRATIVE: IMPACT: Oct 1996 Reliable, accurate, sensitive, rapid and easy to perform FAD tests is the anticipated benefit.

OBJECTIVES: Oct 1996 - Complete the validation of the PCR method for the detection of Hog cholera, Bluetongue and pseudorabies viruses. - Validate the PCR method for African swine fever, Newcastle disease and Avian influenza viruses.

PROGRESS:
Oct 1996 new project

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