Source: UNIV OF CALIFORNIA (VET-MED) submitted to
A RAPID AND INEXPENSIVE KIT FOR ON-SITE DIAGNOSIS OF VESICULAR STOMATITIS AND FOOD AND MOUTH DISEASE.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0196642
Grant No.
(N/A)
Project No.
CALV-AH-206
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2007
Grant Year
(N/A)
Project Director
Yilma, T. D.
Recipient Organization
UNIV OF CALIFORNIA (VET-MED)
(N/A)
DAVIS,CA 95616
Performing Department
PATHOLOGY, MICROBIOLOGY & IMMUNOLOGY
Non Technical Summary
Vesicular stomatitis (VS) is an important disease of cattle, horses and pigs throughout the Americas. Food-and-mouth disease (FMD) is a highly contagious vesicular disease affecting up to 70 species of cloven-hoofed mammals, both domestic and wild. We would like to develop a rapid an dinexpensive penside diagnostic kit that can provide immediate on-site differentiation of VS from FMD.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113310109020%
3153410104040%
3153410110140%
Goals / Objectives
1: To develop chromatographic matrix strips coated with baculovirus-expressed VSV-N and FMDV-RdRp proteins and optimize the biochemical conditions and reaction sensitivity of such strips. 2: To test the efficiency and accuracy of the diagnostic kit using serum and whole blood from known VSV-infected and non-infected naive cattle.
Project Methods
The proposed diagnostic kit will determine the presence of anti-N or anti-RdRp antibodies in whole blood or serum samples. Serum samples will be obtained from known VSV-infected, FDMV-infected and uninfected calves (10/group). An internal control for the test will be incorporated in the final product to ensure the fidelity of the kit. Serum samples from the same animals will also be tested on the current VSV-specific and VMDV-specific diagnostic ELISA kits to evaluate accuracy.

Progress 10/01/02 to 09/30/07

Outputs
OUTPUTS: Foot and mouth disease and vesicular stomatitis cannot be distinguished clinically. Although vesicular stomatitis causes losses in animal productivity by itself, the biggest impact is the similarity to the truly devastating livestock disease, foot and mouth disease. Thus we have worked on a kit to rapidly diagnose and distinguish animals infected with the vesicular stomatitis virus from those infected with foot and mouth disease virus. Results from this project will be disseminated through presentations at national and international meetings or conferences and by publication in scientific journals. PARTICIPANTS: Dr. Tilahun Yilma (DVM, PhD), VM:ILMB, UC Davis. Principal investigator. Dr. Shabbir Ahmad, developed recombinant baculovirus high-level expression viruses expressing the VSV N protein and purified the recombinant protein for the test kits. Dr. Leslie Jones, VM:ILMB, UC Davis, co-investigator, designed animal studies, immune assays, trained and supervised graduate students, postdocs, and technicians. Francisco Monge-Navarro (DVM), VM:ILMB, graduate student, developed the recombinant P3D protein. Shirley Leung, VM:ILMB, UC Davis, laboratory supervisor trained and supervised graduate students, postdocs, technicians, and lab assistants in lab safety. Ensured lab equipment maintenance, supplies, and compliance with university regulations. Sandy Yan, VM:ILMB, UC Davis, laboratory technician. This project provided training for graduate students and postdoctoral scholars. TARGET AUDIENCES: Organizations and individuals involved in the control and prevention of reportable livestock diseases.

Impacts
Vesicular Stomatitis (VS) is an economically important disease of livestock throughout the Americas. Clinical signs in cattle and pigs are undistinguishable from foot-and-mouth disease (FMD), one of the most devastating viral diseases of livestock. Currently, outbreaks of FMD-free areas must be diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm whether it is VS, FMD, or another vesicular disease. To expedite the test and reduce expenses, we proposed to develop a rapid and simpler diagnostic tool which is needed to identify VS and FMD on site. The proposed diagnostic kit utilizes two different proteins from VSV and FMDV inexpensively produced in the baculovirus expression system to check for the presence of specific antibodies in animal sera. We have developed baculovirus expression systems producing large quantities of N protein of VSV. In addition, we have also developed a baculovirus expression system to produce a highly conserved recombinant FMDV protein (P3D) in large quantities. These baculovirus-produced antigens have been used to develop ELISA kits for detection of antibody. They have also been used to develop lateral flow chromatographic pen-side kits for detection of antibody to these two diseases. We are also developing monoclonal antibodies for development of similar pen-side kits to detect antigen in blood, sera or vesicular fluid.

Publications

  • Development of rapid and inexpensive diagnostic kits for foot-and-mouth disease and Rift Valley fever. F. Monge-Navarro, JF Papin, PH Verardi, LA Jones, TD Yilma. Presented at the 26th Annual Meeting of the American Society for Virology, Oregon State University, July 14-18, 2007


Progress 01/01/06 to 12/31/06

Outputs
The P3D fragment of the FMDV genome was excised from pCRP3D with a Bgl II and Sfu II double digestion and gel purified. The DNA fragments were cloned into the baculovirus transfer vector pMelBacB, restriction enzyme analysis was performed, and sequencing was done to confirm the lack of mutations. Standard methods were utilized to transfect the plasmid and the baculovirus DNA into Sf9 insect cells to generate the recombinant baculoviruses. Plaque purification was used to isolate the recombinant viruses. Sequencing of the relevant regions of the recombinant baculovirus was done to confirm that no mutations occurred. A Ni-NTA column (Qiagen) was used to purify the protein from cell pellets and supernatants from infected Sf9 insect cells. Most of the protein was found in the cell pellets instead of the supernatant, thus the secretion signals from the pMelBacB system were not functioning optimally. A large quantity of the purified P3D protein has been made and will be used to generate an ELISA kit as well as being used for preparation of the chromatographic matrix strips. Using known positive and negative sera, the tests will be optimized. Once the FMD protein has been successfully used in a chromatographic test kit, the VSV N protein will be added on a separate line and the kit will be tested with sera positive for either virus.

Impacts
A rapid and inexpensive penside diagnostic kit specific for vesicular diseases will allow on-site identification of VS and FMD in case of a vesicular disease outbreak. Whether it is a bioterrorist attack or natural disease outbreak, such kit would enable veterinarians and authorities to react more quickly than if they have to depend on the current technology and wait for hours, and possibly days, before the infectious agent is identified.

Publications

  • No publications reported this period


Progress 01/01/05 to 12/31/05

Outputs
Vesicular stomatitis (VS) is an economically important disease of livestock throughout the Americas. Clinical signs in cattle and pigs are undistinguishable from foot-and-mouth disease (FMD), one of the most devastating viral diseases of livestock. Currently, outbreaks of FMD-free areas must be diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm whether it is VS, FMD, or another vesicular disease. To expedite the test and reduce expenses, we proposed to develop a rapid and simpler diagnostic tool is needed to identify VS and FMD on site. The proposed diagnostic kit utilizes two different proteins from VSV and FMDV inexpensively produced in the baculovirus expression system to check for the presence of specific antibodies in animal sera. We have developed baculovirus expression systems producing large quantities of N protein of morbiliviruses and VSV. Such proteins have proven to be excellent coating antigens for ELISAs for the diagnosis of rinderpest in cattle, peste-des-petits ruminants in goats, and VSV in cattle. In addition, we have also developed a baculovirus expression system to produce recombinant FMDV P3D proteins in large quantities. We have cloned the FMDV P3D gene into the pMelBac plasmid transfer vector. This vector has been used to transfect insect cells and we are currently isolating and characterizing the recombinant baculovirus expression vectors.

Impacts
A rapid and inexpensive penside diagnostic kit specific for vesicular diseases will allow on-site identification of VS and FMD in case of a vesicular disease outbreak. Whether it is a bioterrorist attack or natural disease outbreak, such kit would enable veterinarians and authorities to react more quickly than if they have to depend on the current technology and wait for hours, and possibly days, before the infectious agent is identified.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
Vesicular stomatitis (VS) is an economically important disease of livestock throughout the Americas. Clinical signs in cattle and pigs are undistinguishable from foot-and-mouth disease (FMD), one of the most devastating viral diseases of livestock. Currently, outbreaks of FMD-free areas must be diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm whether it is VS, FMD, or another vesicular disease. To expedite the test and reduce expenses, we proposed to develop a rapid and simpler diagnostic tool is needed to identify VS and FMD on site. The proposed diagnostic kit utilizes two different proteins from VSV and FMDV inexpensively produced in the baculovirus expression system to check for the presence of specific antibodies in animal sera. We have developed baculovirus expression systems producing large quantities of N protein of morbiliviruses. Such proteins have proven to be excellent coating antigens for ELISAs for the diagnosis of rinderpest in cattle and peste-des-petits ruminants in goats. In addition, we have also developed a baculovirus expression system to produce recombinant FMDV proteins in large quantities.

Impacts
A rapid and inexpensive penside diagnostic kit specific for vesicular diseases will allow on-site identification of VS and FMD in case of a vesicular disease outbreak. Whether it is a bioterrorist attack or natural disease outbreak, such kit would enable veterinarians and authorities to react more quickly than if they have to depend on the current technology and wait for hours, and possibly days, before the infectious agent is identified.

Publications

  • Yilma, T., F. Aziz, S. Ahmad, L. Jones, R. Ngotho, H. Wamwayi, B. Beyene, M. Yesus, B. Egziabher, M. Diop, J. Sarr, and P. Verardi. Global eradication of rinderpest with recombinant vaccines and rapid diagnostic kits produced in Africa under the auspices of AU/IBAR. The Global Rinderpest Eradication Programme Technical Consultation 2002, Rome, Italy, September 30-October 4, 2002.
  • Yilma, T., F. Aziz, S. Ahmad, L. Jones, R. Ngotho, H. Wamwayi, B. Beyene, M. Yesus, B. Egziabher, M. Diop, J. Sarr, and P. Verardi. Inexpensive vaccines and rapid diagnostic kits tailor-made for the global eradication of rinderpest. International Symposium on Vaccines for OIE List A and Emerging Animal Diseases, Ames, Iowa, September 16-18, 2002.
  • Yilma, T.D., S. Ahmad, P.H. Verardi, et al. A strategy for a successful end to the perpetual rinderpest eradication program: recombinant vaccines and diagnostic kits for rinderpest. In M. Jeggo (Ed.), Proceedings of the Third Research Co-ordination Meeting of the Co-ordinated Research Project on 'Rinderpest seromonitoring and surveillance in Africa using immunoassay technologies'. UN-FAO/International Atomic Energy Agency, Vienna, Austria, October 16-20, 2000.
  • Ismail T, Ahmad S, Saliki J, Mebus C, Yilma T: Differential diagnosis between rinderpest-virus vaccinated and infected animals using the nucleocapsid protein expressed in baculovirus. Proc. U.S. Ani. Hlth. Assn, 97th Annual Meeting, Las Vegas, Nevada, pages 107-117, 1994.
  • Ismail T, Ahmad S, D'Souza-Ault M, Bassiri M, Saliki J, Mebus C, Yilma T: Cloning and expression of the nucleocapsid gene of virulent kabete o strain of rinderpest virus in baculovirus: use in differential diagnosis between vaccinated and infected animals. Virology 198: 138-147, 1994.
  • Yilma, T., F. Aziz, S. Ahmad, L. Jones, R. Ngotho, H. Wamwayi, B. Beyene, M. Yesus, B. Egziabher, M. Diop, J. Sarr, and P. Verardi. Inexpensive vaccines and rapid diagnostic kits tailor-made for the global eradication of rinderpest, and technology transfer to Africa and Asia. Dev. Biol. 114:271-283, 2003.


Progress 01/01/03 to 12/31/03

Outputs
One of the most feared animal diseases is foot and mouth disease (FMD). The virus causing this disease is rapidly spread, causes great economic damage, and can persist in the environment, making outbreaks difficult and expensive to contain. Due to these qualities, the virus is also a major threat as an agricultural bioterrorist agent. Another virus that is endemic to the Americas, vesicular stomatitis virus (VSV), also causes a vesicular disease that is clinically indistinguishable from FMD. Thus, a diagnostic kit to rapidly and accurately diagnose the cause of an outbreak of a vesicular disease in livestock is greatly needed. We have previously cloned the VSV nucleocapsid (N) protein in a high-level baculovirus expression system. We are currently cloning the FMDV polymerase protein (RdRp) into this system. Once we have large quantities of the viral proteins purified, we will spot-coat the proteins and an internal control protein on a chromatographic matrix strip precoated with protein-G-colloidal gold. We will then optimize the chromatographic test to minimize background and maximize sensitivity. Antisera from animals infected with VSV or FMDV will be tested to validate the kit.

Impacts
Development of a rapid and inexpensive kit to diagnose the cause of vesicular diseases in livestock will greatly improve the identification and control of these diseases.

Publications

  • Ahmad, S., M. Bassiri, A. K. Banerjee, and T. Yilma. 1993. Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals. Virology 192:207.


Progress 01/01/02 to 12/31/02

Outputs
Funding for this project started October 1, 2002. There has not been adequate time to perform the work prior to the request for an end-of-year progress report. Progress will be reported on the 2003 report

Impacts
This information will be supplied in the 2003 report.

Publications

  • No publications reported this period