ARS Animal Health National Program (103)

Dr. Cyril G. Gay, National Program Leader, Animal Health and Safety, joined the National Program Staff (NPS), Animal Production, Product Value and Safety (APPVS), November 2002. Dr. Gay holds a B.Sc. in Chemistry and a Doctor of Veterinary Medicine (DVM) from Auburn University, and a Ph.D. in Microbiology from George Washington University. Dr. Gay has held several positions with increasing levels of responsibility in the private sector, his last position being the Director of New Product Technical Development for livestock products worldwide at Pfizer Inc. Dr. Gay also brings experience working with an action government agency, having previously held the position of Chief, Biotechnology Section, Center for Veterinary Biologics, Animal and Plant Health Inspection Service (APHIS), where he conducted the risk analysis for the first live recombinant-vectored vaccine licensed by a government agency worldwide.

Dr. Robert A. Heckert, National Program Leader, Animal Health, joined NPS-APPVS in January 2002. Dr. Heckert’s professional experiences include serving in several positions in industry and Agriculture Canada, being the Chief of Foreign Animal Disease Diagnosis for the Canadian Food Inspection Agency and a Research Assistant Professor at the University of Maryland, Virginia-Maryland Regional College of Veterinary Medicine.

Together, Dr. Gay and Dr. Heckert will set strategic goals and action plans for the Animal Health National Program, with Dr. Gay taking the lead on domestic and endemic diseases of the United States, and Dr. Heckert taking the lead on foreign and emerging animal diseases that pose a threat to American agriculture.

The Animal Health National Program currently includes 44 research projects supported by 104 scientists located at 14 research sites throughout the country. Critical to the success of the research conducted at ARS is the ability to build and maintain the infrastructure of our laboratories to ensure the highest level of biosecurity and quality research. Accordingly, we are continuing to develop plans to upgrade the animal research facilities at the National Animal Disease Center (NADC) in Ames Iowa. The Ames Master Plan received $40 million in the 2002 budget and an additional $75 million in supplemental funding in 2002. An Ames Modernization Branch has been established within the Facilities Division, planning and design teams were established, and three major architect engineering firms and three major construction management companies were selected in 2002.

Several scientists received accolades again this past year. Dr. J.P. Dubey was honored by the Thomson/Institute for Scientific Information as one of the world’s most cited authors. Dr. Dubey was identified as one of the top 15 researchers in the discipline of Plant and Animal Science and the only U.S. veterinarian. Dr. Dubey has over 1000 cited papers. Dr. Richard Gast received the 2002 American Egg Board Award from Poultry Science Association for outstanding published research paper relating to egg science; Dr. Bailey Mitchell was elected as Fellow in American Society of Agricultural Engineers and received the engineer of the Year Award from the Georgia Section of the American Society of Agricultural Engineers; Dr. Terrence Tumpey was awarded the James H. Nakano Citation from the National Center for Infectious Diseases of the Centers for Disease Control and Prevention for an outstanding scientific paper.

Scientists within the National Animal Health Program have again been very active in their fields both Nationally and Internationally. Activities have included: producing numerous peer-reviewed publications in high ranking scientific journals, serving as editors for various scientific journals, being invited speakers for national and international conferences, being reviewers on a wide and extensive list of peer-reviewed journals, being reviewers on various scientific panels, serving as senior officials and or committee members on numerous national and international scientific organizations, developing collaborative research proposals with private companies, submitting patent proposals of scientific principles or techniques, obtaining funding from outside agencies to conduct scientific research within the animal health mandate, working with various commodity groups to clarify research priorities.

A new generation of diagnostic tests are needed to detect emerging disease pathogens and their toxins, identify new variants of known disease pathogens, control or eradicate zoonotic diseases, and control diseases that impact production and trade.

A new versatile test for the detection of Bovine Tuberculosis in cattle and wildlife. In the last seven years, Mycobacterium bovis has been isolated from free ranging white-tailed deer in 12 counties in the northern part of the lower peninsula of Michigan. About 5 percent of wild deer, in an area identified as the TB core area, have TB. Tuberculosis has also been found in coyotes, raccoons, a fox, bear, feral cat, elk and bobcat and in 16 beef cattle and two dairy cattle herds in northern Michigan. This is the first reservoir of TB in wildlife identified within the United States and presents significant obstacles to the eradication effort. ARS scientists at the Bacterial Diseases of Livestock Research Unit at the National Animal Disease Center (NADC), Ames, Iowa, have responded to the challenge by developing an indirect blood test that measures nitric oxide induced in mammals in response to Mycobacterium bovis infections. The test can detect human, avium, and bovine TB. The advantage of this test is that it can be used in most mammals and only requires one blood sample, and thus the handling of animals once, as opposed to the official standard skin test that requires animals to be handled twice.

2) Waters, W.R., Sacco, R.E., Fach, S.J., Palmer, M.V., Olsen, S.C., Kreeger, T.J. Analysis of mitogen-stimulated lymphocyte subset proliferation and nitric oxide production by peripheral blood mononuclear cells of captive elk (Cervus elaphus). Journal of Wildlife Diseases. 2002. v. 38. p. 344-351.

Rapid detection and diagnosis of classical swine fever. Rapid identification and diagnosis of a foreign animal disease is critical for successfully controlling and eradicating the disease following introduction. Rapid field-based assays to detect classical swine fever virus (CSFV) are not yet available. A rapid real-time fluorogenic hydrolysis probe TaqMan assay was developed by ARS scientists at the Plum Island Animal Disease Center, New York, and evaluated in pigs experimentally infected with a moderately virulent CSF isolate, Haiti-96. The assay was highly sensitive and specific, detecting infected animals up to 5 days prior to the appearance of CSF clinical symptoms. This rapid, real-time RT-PCR assay for CSFV provides a new diagnostic tool that will redefine disease management and control strategies for this highly significant disease.

1) G. R. Risatti, J. D. Callahan, W. M. Nelson, and M. V. Borca. Rapid Detection of Classical Swine Fever Virus by a Portable Real-Time Reverse Transcriptase PCR Assay. Journal of Clinical Microbiology 41: 500-505, 2003.

Rapid detection of avian influenza virus. A need exists for a rapid and sensitive diagnostic test for the direct detection of both low pathogenic and highly pathogenic avian influenza to support control and eradication strategies during an outbreak in poultry. In collaboration with the National Veterinary Services Laboratory (NVSL), Animal and Plant Health Inspection Service (APHIS), a real time (RT)-PCR test was developed by ARS scientists at the Southeast Poultry Research Laboratory in Athens, GA, which was validated for the detection of any influenza virus and the subsequent sub-typing of the virus. The test was validated and adopted for clinical use by APHIS and three State veterinary laboratories (Virginia, Pennsylvania, and North Carolina). This diagnostic test has already been used to help control the recent H7N2 influenza outbreak in Virginia and will enhance our nation’s ability to rapidly control avian influenza in the event of future outbreaks.

1) Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, Lohman K, Daum LT, Suarez DL. Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Journal of Clinical Microbiology 40: 3256-3260, 2002.

2) Durham, S. A New, Rapid Test for Avian Influenza. USDA Agricultural Research Magazine: February, 2003.

New Test for Toxoplasmosis. Toxoplasmosis is the leading cause of blindness in underdeveloped countries. It is also one of the most common causes of parasitic diseases in humans and animals. Pig products are a major source of Toxoplasma gondii infection in the U.S. ARS scientists at Parasite Biology, Epidemiology, and Systematics Laboratory in Beltsville, Maryland, with funding from the National Pork Producers Council, have developed a sensitive DNA test for the parasite in pig or other animal tissues. The test is very sensitive and specific and will be an important tool for protecting America’s food supply and for epidemiological investigations to decrease the numbers of pigs infected with this parasite.

High-throughput DNA sequencing and gene array technology for screening potential vaccine candidates will provide new insights into microbial pathogenesis, evolution, and vaccination strategies. DNA sequence information will facilitate molecular epidemiologic analysis of disease outbreaks, identification of virulence and host range factors, and development of a new generation of diagnostic and therapeutic agents.

Johne’s Disease. ARS scientists at our Bacterial Diseases of Livestock Research Unit at NADC, Ames, Iowa, in collaboration with the University of Minnesota completed the sequencing of the entire genome of Mycobacterium avium spp. paratuberculosis (M. paratuberculosis), the pathogen responsible for Johne’s Disease. This bacterium is related to the tubercle bacillus and causes a chronic wasting and debilitating enteritis and eventual death in cattle, sheep, goats, llamas, deer, and other wild or domestic ruminants. Public health officials are also concerned about recently published medical reports suggesting a link between M. paratuberculosis and Crohn's disease, a chronic inflammatory bowel disease in humans. ARS scientist have identified 19 unique sequences that will be the basis for functional genomics studies that may eventually lead to the development of new effective diagnostics and preventive and therapeutics tools for the control and eradication of this disease in the United States.

1. Bannantine, J., Stabel, J. The Gene Product of a Mycobacterium Paratuberculosis Specific Sequence, Hspx, Is Present Within Infected Macrophages and Is Recognized by Sera Fromsome Infected Cattle. Veterinary Microbiology: Vet Microbiol 2000 Oct 20;76(4):343-58.

2. Bannantine, J., Stabel, J. Identification of Two M.PARATUBERCULOSIS Gene Products Differently Recognized by Sera from Rabbits Immunized with Live Mycobacteria But Not Heat-Killed Mycobacteria. Journal of Medical Microbiology: 2001 Sep;50(9):795-804.

Brucellosis. Brucellosis remains a critical zoonotic pathogen with impact on the reproductive health of cattle, swine, sheep, and goats, and effects on human health. ARS scientists were critical partners this year in supporting a multi-center effort for sequencing the genome of Brucella suis, the cause of swine brucellosis. Completing the sequencing of Brucella suis is a major accomplishment that will lead to comparative genomics studies that could provide precise targets for developing vaccines against Brucella strains that are currently affecting livestock and wildlife reservoir hosts in the United States.

Bovine Leptospirosis. Little is known about the genetic organization or content of Leptospira

borgpetersenii serovar hardjo, the major cause of bovine leptospirosis worldwide and an important zoonotic disease. The genome of this bacterium is being sequenced at our Bacterial Diseases of Livestock Research Unit at NADC, Ames, Iowa, to identify genes associated with virulence and potential vaccine candidates. In 2002, a genomic library was successfully constructed and our scientists completed the shotgun phase of sequencing, resulting in the determination of about 95% of the total sequence. These data resulted in the identification of several outer membrane protein genes and genes associated with virulence factors and will have an impact on future vaccine development initiatives.

New understanding of pox virus genes. ARS scientists at our Plum Island Animal Disease Research Center, New York, have sequenced pox virus genes from many different species of animals. The genes of lumpy skin disease, camel pox, avian pox, sheep pox, goat pox and others have been fully sequenced, analyzed and compared. By comparing the genes of many different pox viruses, it has been possible to determine which genes are tightly conserved across the pox virus family. This will enable scientists to learn more about how pox viruses invade the host and cause disease and to develop successful intervention strategies for the future.

1) Afonso, C. L., Tulman, R., Lu, Z., Zsak, L., Sandybaev, N. T., Kerembekova, U. Z., Zaitsev, V. L., Kutish, G. F., Rock, D. L. The Genome of Camelpox Virus. Virology 295: 1-9, 2002.

The emergence of a new disease or introduction of an exotic disease into the United States could jeopardize agricultural commodities and pose a health risk to humans. Effective disease monitoring and surveillance will provide a better understanding of the ecology and natural life cycles of emerging pathogens and will facilitate development of control strategies to prevent disease transmission.

Epidemiology of Neosporosis. Neospora caninum has been identified as a major cause of abortion and neonatal mortality in cattle worldwide. Losses in California dairies due to neosporosis-associated abortions are estimated to be $35 million annually. Epidemiological investigations indicate that up to 45 percent of abortions in dairy cows are caused by N. caninum. ARS scientists discovered that the dog was the definitive host and the source of new infections. ARS investigated the prevalence of N. caninum antibodies in dogs and established that levels were significantly higher in dogs from beef and dairy cattle farms than dogs from urban areas. However, recent reports suggest that congenital infection from reactivation of the parasite is the main mode of transmission and cause of abortions. Considerable work remains to understand the biology and epidemiology of N. caninum, including the survival of oocysts in the environment and elucidating the life-cycle in dogs. ARS will continue to conduct studies that will provide information to help dairy farmers develop biosecurity measures to control abortions due to Neosporosis.

Publications:

1) Jenkins M, Baszler T, Bjorkman C, Schares G, Williams D. Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion.. Int J Parasitol 2002 May;32(5):631-6

2) Walsh CP, Vemulapalli R, Sriranganathan N, Zajac AM, Jenkins MC, Lindsay DS. Molecular comparison of the dense granule proteins GRA6 and GRA7 of Neospora hughesi and Neospora caninum.. Int J Parasitol 2001 Mar;31(3):253-8

Sheep Scrapie. Scrapie is a member of a group of diseases caused by prions that are called Transmissible Spongiform Encephalopathies (TSEs), which includes bovine spongiform encephalopathy (BSE). Scrapie is efficiently transmitted during lambing but other modes of transmission are poorly understood. Scientists at our Virus and Prion Diseases of Livestock Research Unit at NADC, Ames, Iowa, and our Animal Research Unit in Pullman, Washington, initiated transmission studies by describing the distribution and biochemical characteristics of the prion precursor in placenta and fetal tissues and fluids. It was discovered that if the fetus is genetically resistant to disease, the fetal placental cells do not support prion accumulation and it is therefore probable that transmission by the infected ewe does not occur. These findings will directly impact control and eradication procedures for sheep scrapie in the U.S.

Publications:

1) Tuo W, O'Rourke KI, Zhuang D, Cheevers WP, Spraker TR, Knowles DP. Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep. Proc Natl Acad Sci U S A 2002 Apr 30;99(9):6310-5

2) Herrmann LM, Baszler TV, Knowles DP, Cheevers WP. PrP(Sc) is not detected in peripheral blood leukocytes of scrapie-infected sheep: determining the limit of sensitivity by immunohistochemistry. Clin Diagn Lab Immunol 2002 Mar;9(2):499-502.

3) Suszkiw, J. Lost Lambs: Lessons Learned From Scrapie Outbreak. USDA Agricultural Research Service Magazine: November, 2002.

Chronic Wasting Disease (CWD). CWD is another member of a group of diseases called TSEs, which includes BSE and Scrapie. CWD is associated with serious losses to the captive elk industry and the wildlife management industries through loss of hunting revenues. The only choice currently available to animal health officials for controlling this disease is extensive depopulation of captive and free ranging animals exposed to the disease. Moreover, the interspecies transmission barriers of these diseases are not understood. CWD is reported in several areas of the U.S. but the true extent of the epidemic is not known because of the considerable cost of diagnostic testing. Using samples submitted by cooperating state wildlife management agencies, ARS scientists at the Animal Research Unit in Pullman, Washington, developed an inexpensive high throughput diagnostic test for early detection of infection in hunter-killed deer. The test has been transferred to private industry and is being evaluated for licensing by the USDA Center for Veterinary Biologics. The impact of this work will be facilitate increased surveillance within and outside the endemic areas.

Publications:

1) Spraker TR, Zink RR, Cummings BA, Sigurdson CJ, Miller MW, O'Rourke KI. Distribution of protease-resistant prion protein and spongiform encephalopathy in free-ranging mule deer (Odocoileus hemionus) with chronic wasting disease. Vet Pathol 2002 Sep;39(5):546-56

2) Hamir, A.N., Miller, J.M. Experimental cross-species transmission of CWD at NADC, Ames, Iowa. Chronic Wasting Disease Symposium. 2002. p. 14.

3) Spraker, T.R., O'Rourke, K.I., Balachandran, A., Zink, R.R., Cummings, B.A., Miller, M.W., Powers, B.E. Validation of monoclonal antibody F99/97.6.1 for immunohistochemical staining of brain and tonsil in mule deer (Odocoileus hemionus)with chronic wasting disease. Journal of Veterinary Diagnostic Investigation. 2002. 14:3-7.

Epidemiology of Avian Influenza. Avian influenza, H7N2, has been circulating in the live bird markets (LBMs) of the Northeast since 1994. This virus has the potential for becoming highly pathogenic. ARS scientists at the Southeast Poultry Research Laboratory in Athens, Georgia, in collaboration with the National Veterinary Services Laboratory (NVSL), Animal Plant Health Inspection Service (APHIS), and state officials from New York, New Jersey, and Pennsylvania, have conducted molecular epidemiology studies of viruses coming from the LBMs and have developed a Real Time (RT)-PCR test to rapidly detect birds infected with the influenza virus. The molecular epidemiology studies showed that a single family of viruses has remained in the markets for the last seven years. The RT-PCR test was compared with virus isolations for specificity and sensitivity as part of a validation study for deploying this diagnostic test. The molecular epidemiology data have increased our understanding of this virus and the RT-PCR test when validated will support multi-State influenza eradication efforts in the LBM system.

Publications:

1) Suarez,D.L., Woolcock,P.R., Bermudez,A.J., Senne,D.A. Isolation from turkey breeder hens of a reassortant H1N2 influenza virus with swine, human and avian lineage genes. Avian Diseases. 2002. 46:111-121.

2) Swayne,D.E., Suarez,D.L. Evolution and Pathobiology of Avian Influenza Virus Virulence in Domestic Birds. Dodet,B., Vicari,M., editors. John Libbey Eurotext, Paris. Emerging Diseases. Emergence and Control of Zoonotic Ortho-and Paramyxovirus Diseases. 2001. p.35-42.

Revolutionary system for disease surveillance and epidemic management. The recent outbreaks of foot and mouth disease virus in the United Kingdom and Taiwan highlight the devastating and far reaching effects of foreign animal diseases. The impact on the economy, military, law enforcement community, social structure and the environment are well documented. ARS scientists at Plum Island, New York, Athens, Georgia, Laramie, Wyoming, Beltsville, Maryland, Frederick, Maryland, and Ames, Iowa, working in collaboration with the Department of Defense, have developed rapid on-site tests that detect and identify important animal, plant and food borne pathogens. These tests are designed for use at the farm gate or market place. The system is designed to communicate real-time data over the Internet to those who can take immediate action. Assays have or are being developed to protect animal health (foot and mouth disease virus, hog cholera virus), plant health (soybean rust, Karnal bunt), food (E.coli O157:H7, Listeria, Salmonella), water (cryptosporidium) and the environment. This unique system of detection, identification, and communication will enable the U.S. to better protect its valuable animal, crop, and food resources.

Publications:

1) Johnny D. Callahan, MS; Fred Brown, PhD; Fernando A. Osorio, MV, PhD, DACVM; Jung H. Sur, DVM, PhD; Ed Kramer; Gary W. Long, PhD; Juan Lubroth, DVM, PhD; Stefanie J. Ellis, BS; Katina S. Shoulars, MS; Kristin L. Gaffney, MS; Daniel L. Rock, PhD; William M. Nelson, MD. Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus. JAVMA, Vol 220, No. 11, June 1, 2002.

Host/Pathogen Interactions

A better understanding of the molecular and cellular mechanisms responsible for disease requires studies of disease agents, the host's response, and the environment. The genetic basis for differences in the pathogenicity among microbial organisms affecting livestock is needed to define the molecular factors that promote the emergence of highly virulent pathogens. Comprehensive studies on the immunological and genetic basis for host-pathogen interactions in diseases are needed to develop novel strategies for pathogen control.

Porcine Respiratory and Reproductive Syndrome (PRRS). PRRS is a highly infectious disease caused by an RNA virus. Infections in the United States account for up to 15-20% of the yearly economic losses in the swine industry. The vaccines currently on the market have been reported to be relatively ineffective. It is important to understand the reasons for these vaccine failures and the impact on the incidence of disease so that better control strategies can be developed. Scientists at our U.S. Meat Animal Research Center (MARC) in Clay Center, Nebraska, have obtained 18 different PRRS virus isolates from disease outbreaks in the Midwest and sequenced the NSP2 genes of these viruses to examine variations that may correlate with disease expression. Recombinant antigens have been developed and immunity to individual PRRS virus proteins evaluated in sera from naturally infected swine from around the country. The research team at MARC has conducted kinetic analysis studies of host-cell gene expression in response to PRRS virus infection using cDNA microarrays. Studies to date indicate that there are a number of responsive genes that increase in expression while others decrease in expression as a result of PRRS virus infection. In combination with the sequence analysis of a number of PRRS virus isolates, this research project will lead to an understanding of the host-virus interactions in the field and identification of viral genes responsible for induction of a protective immune response.

1) Fox, J.M., Chitko-McKown. Gene profiling of MARC-145 cells infected with porcine reproductive and respiratory syndrome virus using cross-species microarray hybridization. Research Workers in Animal Diseases Conference. 2001. Abstract No. 178.

Marek’s Disease Virus (MDV). Marek's disease is a lymphocyte proliferation (a type of cancer) and immune suppressive disease of chickens caused by a herpesvirus. Infections with this virus are nearly universal among commercial chicken flocks worldwide. The virus is relatively stable in the environment and is easily spread through the air. Despite widespread use of vaccines, economic losses from mortality of layers and breeders and condemnation of broilers continue to plague producers. Losses tend to be sporadic and unpredictable. Moreover, the virus continues to mutate to greater virulence, potentially reducing the effectiveness of many existing vaccines and causing concerns that vaccines currently considered effective will not protect in the future. Scientists at our Avian Disease and Oncology Laboratory (ADOL) in East Lansing, Michigan, have been conducting functional genomics studies of selected Marek's disease viral genes to better understand the mechanism by which the virus causes changes in infected chickens. Several virus preparations with specific deletions or changes in selected genes were developed and the effects compared to unaltered virus. Results to date indicate that the 132 base pair nucleic acid repeats are nonessential for MDV replication in cell culture and chickens and viruses lacking these repeat sequences are still pathogenic. Furthermore, the putative MDV oncogene called “meq’ could be deleted but was found to be essential for the transformation of lymphocytes in infected chickens. It was also found that the pp38 gene in the vaccine virus, Rispens, functions identically to the pp38 gene in the very virulent Md5 virus. Together, these studies are enhancing our knowledge of the mechanisms of virus replication and virulence, which is essential for developing a new class of vaccines against this very important disease.

2) Reddy SM, Lupiani B, Gimeno IM, Silva RF, Lee LF, Witter RL. Rescue of a pathogenic Marek's disease virus with overlapping cosmid DNAs: use of a pp38 mutant to validate the technology for the study of gene function. Proc Natl Acad Sci U S A 2002 May 14;99(10):7054-9

3) Gimeno IM, Witter RL, Hunt HD, Lee LF, Reddy SM, Neumann U. Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression. Vet Pathol 2001 Sep;38(5):491-503

New functional genomic tools and molecular biological methods will facilitate the identification of host genes that control immunological and physiological responses to microbial agents. Novel information concerning the nature and function of genes will lead to a better understanding of the genetic basis of disease resistance, enhancing our ability to manipulate the host immune system, and the development of new, more effective control strategies.

Genetic diversity panel of beef cattle. Scientists at our U.S. Meat Animal Research Center (MARC) in Clay Center, Nebraska, have now characterized over 180 single nucleotide polymorphisms (SNPs) in more than 50 bovine genes. These have been used to estimate the genetic diversity in beef cattle with automated SNP assays developed for high-throughput MALDI-TOF MS genotyping. This is significant because DNA sequence variation provides the fundamental material for improving livestock through selection. Our results indicated that once the halotypes for a given gene locus are sufficiently characterized it is possible to correctly assign halotypes to the vast majority of the genotypes in commercial populations of cattle. This allows the evaluation of these alleles as risk factors for infection phenotypes in commercial populations of cattle.

1) Heaton MP, Harhay GP, Bennett GL, Stone RT, Grosse WM, Casas E, Keele JW, Smith TP, Chitko-McKown CG, Laegreid WW. Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle. Mamm Genome 2002 May;13(5):272-81

2) Stone RT, Grosse WM, Casas E, Smith TP, Keele JW, Bennett GL. Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers. Mamm Genome 2002 Apr;13(4):211-5.

Genetic resistance to virus infection in chickens. With Marek’s disease costing the poultry industry over $160 million a year, developing methods that augment current vaccinal control methods is a high priority. ARS, in collaboration with the University of Delaware, screened chickens that were disease resistant and susceptible by the new technology of DNA microarrays, which measures expression levels of many genes. Genetic mapping of the differentially expressed genes revealed many positional candidate genes for resistance to Marek’s disease. This approach of integrating global expression studies with mapping shows great promise to rapidly identify disease resistance genes, which can be quickly transferred to the poultry industry to enhance disease resistance.

Effective vaccination strategies are needed to overcome the inherent genetic variation of pathogens. New generations of vaccines using pathogen subunits, whole organisms, viral-vectors, deletion mutants, naked DNA, and peptide vaccines to prevent economically important livestock diseases that are endemic or exotic to the United States are needed to provide better disease prevention strategies. The development of new biosecurity management practices that incorporate these new prevention tools need to be explored.

New tools to control foot-and-mouth disease virus (FMDV). Current models and simulations have predicted devastating economical and environmental consequences if FMDV were introduced in the United States. Based on recent experiences with FMDV outbreaks in Europe, vaccination is now being considered as a first-line intervention tool in the containment of a potential outbreak in the U.S., rather than traditional depopulation strategies that would result in destruction of millions of animals. Scientist at our FMDV Research Unit at the Plum Island Animal Disease Center, New York, have developed adenoviral based constructs containing the structural proteins of the FMDV capsid and the viral protease required to process these protein molecules into empty capsids. Laboratory tests of these viral vaccines have demonstrated virus neutralizing activity and protection of animals as early as five days after a single inoculation, lasting for at least 42 days. The same adenoviral vectors have been engineered to contain antivirals such as Type I porcine interferon (Ad5-pIFNalpha) and have been shown to confer protection from viral challenge as early as 24 hours and for as long as four days after a single inoculation. The combination of empty capsid vaccines and antivirals have been tested in swine and shown to induce complete protection from infection in 24 hours and for as long as 42 days upon direct challenge with FMDV. This combined therapy provides for the first time a true outbreak response strategy to arrest the spread of FMDV and limit animal exposure to this highly contagious disease. Further work will be needed to research and develop these innovative control tools for other animal species, including cattle.

1) Grubman MJ, Mason PW. Prospects, including time-frames, for improved foot and mouth disease vaccines. Rev Sci Tech 2002 Dec;21(3):589-600

2) Chinsangaram J, Moraes MP, Koster M, Grubman MJ. Novel viral disease control strategy: adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease. J Virol 2003 Jan;77(2):1621-5

3) Mayr GA, O'Donnell V, Chinsangaram J, Mason PW, Grubman MJ. Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirus-FMDV constructs. Vaccine 2001 Feb 28;19(15-16):2152-62

New vaccine to control avian coccidiosis. Novel vaccination strategies using antigen-encoding DNA plasmids have recently been shown to successfully induce protective cellular and humoral immune responses against a variety of infectious diseases. Scientists at our Parasite Biology, Epidemiology, and Systematics Laboratory in Beltsville, MD, have documented for the first time in chickens that immune responses induced by DNA vaccination can be enhanced by co-injecting recombinant cytokines or plasmids encoding these cytokines. Administration of recombinant IFN-g gene decreased oocyst production, enhanced body weight gain, and improved disease resistance following coccidia challenge infection. The results of this study clearly document that chicken cytokines can be powerful adjuvants providing additional impetus to further investigate their biological activities at the molecular and immunological levels.

1) Lillehoj HS, Lillehoj EP. Avian coccidiosis. A review of acquired intestinal immunity and vaccination strategies. Avian Dis 2000 Apr-Jun;44(2):408-25

2) Lillehoj HS, Choi KD, Jenkins MC, Vakharia VN, Song KD, Han JY, Lillehoj EP. A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis. Avian Dis 2000 Apr-Jun;44(2):379-89

Development of a marked vaccine for Newcastle disease virus. Newcastle disease is a foreign animal disease that has the potential to devastate the poultry industry. The capability to easily differentiate vaccinated from infected birds would enhance current Newcastle Disease control programs. At the Southeast Poultry Research Laboratory in Athens, GA, a vaccine was developed that is a shell of the whole virus and retains the important surface structures that induce immunity. The virus used in this vaccine cannot infect or be transmitted from bird to bird because the virus genome and internal proteins have been removed. This “virosome” Newcastle disease vaccine was administered intra-nasally and shown to protect chickens against a lethal Newcastle disease virus challenge. Because vaccinated birds can be differentiated from infected birds, this vaccine may provide an effective tool to control future outbreaks of Newcastle disease virus in the United States.

1) Kapczynski, D.R., Tumpey, T.M. Development of an inactivated Newcastle disease virosome vaccine that protects against challenge from velogenic Texas GB. Proceedings of the American Association of Avian Pathologists. American Veterinary Medical Association. 2002. CD-ROM. O'Fallon, IL: Veterinary Software Publishing. Abstract.

Understanding the early development of the immune system in each of our food animals will enable scientists to apply new approaches to vaccine delivery and to use novel bio-therapeutics for disease treatments. Adapting existing knowledge of rodent and human immunology to domestic animals will speed up the process and cost effectiveness of research. Fundamental research is urgently needed in mucosal immunology and the mechanisms involved in innate and acquired immunity in order to have a solid foundation to improved methods of targeting host/pathogen interactions. Understanding how a host responds to invasion by a pathogen will lead to development of a whole new series of methods to prevent primary infection and abrogate the development of disease.

Avian Immunology. The accurate assessment of the host immune response to poultry infectious agents such as bacteria, viruses, and parasites is critical for monitoring the health of the flocks. ARS scientists at Beltsville, Maryland, produced several monoclonal antibodies to study host immune responses in chickens and have transferred them to diagnostic firms for further development. Several of these monoclonal antibodies can now be used to assess the immune status of chickens vaccinated with different infectious agents. The results to date indicate that these monoclonal antibodies are useful in the identification of lymphocyte subpopulations and macrophages in the tissues and the peripheral blood from chickens infected with microbial agents or vaccinated with the viral vaccines.

1) Miyamoto, T.,Lillehoj,H.S.,Sohn,E.J.,Min,W. Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay. Veterinary Immunology and Immunopathology. 2001. 80:245-257.

2) Li, G.,Lillehoj,H.S.,Min,W. Production and characterization of monoclonal antibodies detecting the chicken interleukin-15 receptor alpha chain. Veterinary Immunology and Immunopathology. 2001. 82:215-227.

Treatment and prevention of mastitis in dairy cows. Three hundred thousand dairy cows suffer acute endotoxin shock and death annually due to mastitis caused by Escherichia coli, which occurs primarily during the early periparturient period. Scientists at our Immunology and Disease Resistance Laboratory in Beltsville, Maryland, have identified and characterized the gene for soluble CD14, which binds and neutralizes endotoxins responsible for mastitis. The gene was cloned and recombinant bovine (rbo)-CD14 protein was successfully produced and evaluated. Intraperitoneal injection of rboCD14 together with endotoxin reduced fatality in mice. Preliminary studies indicate that intramammary injection of soluble rboCD14 is 100% effective in preventing mastitis by E. coli in lactating dairy cows. This research will potentially lead to a product that for the first time can be used effective to treat and prevent mastitis caused by E. coli.

1) Wang,Y.,Zarlenga,D.S.,Paape,M.J.,Dahl,G.E. Recombinant bovine CD14 sensitizes the mammary gland to lipopolysaccharide. Veterinary Immunology and Immunopathology. 2002. 86:115-124.

The molecular and cellular basis of the host animal's response to disease is critical to our understanding of how virulence factors correlate with the pathologic manifestation of disease. The interaction of environmental factors such as the role of air quality and the incidence of air-borne pathogens need to be examined. Understanding the immunological responses to pathogens is needed to develop immune modulatory strategies for improved vaccine delivery. Mechanisms controlling vector-pathogen-host interactions need to be investigated. Molecular probes will aid in determining the method and frequency of pathogen persistence in recovered and subclinically-infected animals.

Pine Needle Abortion. Grazing needles of ponderosa pine, lodgepole pine, common juniper or Monterey Cypress by pregnant cattle results in abortion/premature parturition, retained placentas and endometritis. Substantial economic losses to cattle producers in the western U.S. and Canada occur annually from ponderosa pine needle-induced abortion. A significant accomplishment by ARS scientists has been the isolation and identification of isocupressic acid (ICA) as the abortifacient toxin in pine needles. Furthermore, two ICA derivatives, succinyl and acety ICA were also identified in pine needles and were active abortifacient toxins. ELISA’s were developed for ICA and it’s sera metabolites using polyclonal antibodies. Using this assay, the absorption into the serum and elimination into the urine of three ICA metabolites was measured in a cow dosed orally with common juniper. Other accomplishments have include screening twenty-three tree and shrub species from throughout the western and southern states for abortifacient levels of ICA. Seasonal changes in ICA levels were monitored in ponderosa pine populations in 5 locations. Field and pen studies determined that nutrient status influenced cows to graze pine needles. Similarly, weather patterns had significant impacts on cow’s propensity to graze pine needles. This better understanding of what toxins are involved, what species of plant is involved and when the toxin is produces will give ranchers better ability to control the poisoning.

1) Stegelmeier BL, Gardner DR, James LF, Panter KE, Molyneux RJ. The toxic and abortifacient effects of ponderosa pine. Vet Pathol 1996 Jan;33(1):22-8

2) Gardner DR, Panter KE, James LF, Stegelmeier BL. Abortifacient effects of lodgepole pine (Pinus contorta) and common juniper (Juniperus communis) on cattle. Vet Hum Toxicol 1998 Oct;40(5):260-3

Potential new prevention for Milk Fever in cows. Solanum glaucophyllum is a plant that contains the active form of vitamin D. Scientists at our Periparturient Diseases of Livestock Research Unit at the National Animal Disease Center (NADC), Ames, Iowa, have investigated the utility of this plant in the prevention of subclinical hypocalcemia in periparturient dairy cows. We have discovered that when used in combination with diets high in anions (chloride), we can significantly reduce the incidence of subclinical hypocalcemia in dairy cows. Availability of this plant will offer further opportunities to dairy farmers in avoiding complications due to hypocalcemia such as displaced abomasum, retained placenta, and mastitis.

1) Cheng, Y-J., Goff, J.P., Horst, R.L. The effect of Solanum glaucophyllum on calcium and phosphorus utilization in lactating cows. American Dairy Science Association meeting. 2002. Abstract p. 108.