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The long-term goal is to develop diagnostic assays capable of rapid detection of pathogens important in food animal production systems. Recent bioterrorism events in the United States and outbreaks of foreign animal diseases in Europe (foot and mouth disease and swine vesicular disease) has heightened the awareness of need for developing strategies to protect the nations livestock from biological threats. In addition, pathogens endemic in the livestock industry continue to cost millions of dollars and innovative ways to control these pathogens are needed. Regardless of their nature and source, one of the keys to pathogen control is rapid detection so that appropriate control practices can be implemented in a timely manner. Electronic biosensors have shown promise as a rapid, cost effective and sensitive detection format. Recently, a conductimetric biosensor has been developed for detecting bacterial pathogens of food safety importance. As a model to detect viral pathogens, we propose to determine the ability of this biosensor to detect bovine viral diarrhea virus (BVDV) in both laboratory media as well as biological samples (blood and nasal swabs). Bovine viral diarrhea virus is one of the most important viral pathogens of cattle and is of worldwide importance. The major source of BVDV spread is cattle that are carriers of the virus, referred to as being persistently infected with BVDV. Detecting and eliminating cattle persistently infected with BVDV is a key component to the control and prevention of this virus. With the development of this biosensor using BVDV as a model, further adaptation could occur making it useful for detecting other pathogens important in the livestock industry. <P>
The specific aims of this project are the following: <OL> <LI> Construct a conductimetric biosensor specific for BVDV <LI> Determine the ability of the conductimetric biosensor to detect BVDV in culture media <LI> Determine the lower limit of the conductimetric biosensor for detecting BVDV in cell culture <LI> Determine the ability of the conductimetric biosensor to detect BVDV in serum and nasal swabs collected from cattle persistently infected with BVDV
NON-TECHNICAL SUMMARY: Rapid diagnosis of infectious diseases of livestock is essential for their control. This purpose of this project is to develope a rapid diagnositic test for viral pathogens of cattle.
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APPROACH: Specific Aim #1 A biosensor and detection method developed by Dr. Alocilja\'s will be used. The biosensor is designed with three disposable membrane pads for sample application, antigen capture, and sample absorption. The membranes in the biosensor are constructed in the following order. First is the fiberglass membrane for sample application, next is the nitrocellulose (NC) membrane with silver electrodes to capture the virus, and finally, the cellulose membrane is added to absorb the sample. The three-membrane strip is 5 mm wide and 50 mm long and attached to a re-usable silicon (Si) wafer platform. Polyaniline-labeled monoclonal antibody directed against the E1 protein of BVDV (15c5, Ed DuBovi, Cornell University) will be placed on the sample application pad following a standard procedure of oxidative polymerization of aniline monomer in the presence of ammonium persulfate. A silver paste pen is used to make the electrodes on the capture NC membrane. The silver paste is applied liberally to ensure consistency in the flow of electricity between the electrodes. The distance between the two electrodes is the capture zone. Swine anti-BVDV polyclonal antibody (USDA:NADL, Ames Iowa) will be directly immobilized on the NC membrane between the two silver electrodes. Specific Aim #2 and #3 The biosensor will first be tested for its ability to detect BVDV in a controlled laboratory setting. Two characterized strains of noncytopathic BVDV (SD-1 and 890) will be grown in bovine turbinate cells in EMEM containing 10% fetal equine serum at 37 C and 5% CO2. Concentration of virus in culture supernate will be determined by making ten-fold serial dilutions of the supernate and then inoculating in quadruplicate onto bovine turbinate cells in 96-well plates. After 5 days at 37 C and 5% CO2, cells will be stained for viral infection using the immunoperoxidase assay14 and virus titer calculated by standard methods. Undiluted cell culture supernate will be used to initially test the biosensor. Subsequently, serial 10-fold dilutions of the known concentration of culture supernate will be used to determine the detection limit of the device. Specific Aim #4 Serum samples will be collected from each of three calves known to be persistently infected with BVDV and housed at the MSU Veterinary Research Farm. Each calf is persistently infected with a different strain of BVDV, thus representing the antigenic diversity of the virus. Serum virus titer will be determined by making 10-fold dilutions, inoculating in quadruplicate onto bovine turbinate cells in 96-well plates, incubated for 5 days at 37 C and 5% CO2, and then stained for viral infection using the immunoperoxidase assay. Virus titer will be calculated by standard methods.
PROGRESS: 2002/10 TO 2007/09
With the growing threat of terrorism, safeguarding the nation\'s livestock industry has become an important component of homeland security. Foreign animal disease outbreaks can be economically devastating. The cost of controlling the disease can be staggering and includes costs for surveillance, biosecurity, animal disposal and producer compensation. Other indirect costs include loss of business for support industries and the reduction or cessation of international trade. Bovine viral diarrhea virus (BVDV) is one of the most insidious and economically devastating viral pathogens of cattle. The unique development of calves persistently infected with BVDV maintains the virus in the cattle population. Detection and removal of cattle persistently infected with BVDV is essential in controlling BVDV related clinical problems. The significance of this project is two fold. First, a biosensor is being developed that can be used for the rapid detection of pathogens that may be economically devastating to animal agriculture if not detected and dealt with quickly. Secondly, in the sequence of developing a tool capable of being used for detecting foreign animal diseases or agroterrorism agents, we are also developing a diagnostic assay that can be applied immediately to controlling BVDV, a disease of current importance in the USA. Initially we developed a sensitive, real-time biosensor for on-site detection of animal pathogens, such as foreign disease-causing viruses and bacteria. This biosensor was designed using BVDV as a model pathogen. The electrochemical biosensor design uses a conductive polymer molecule to convert the binding event between the antibody and antigen into an electrical signal. Following development of the architecture and experiments to optimize the reagents, the biosensor was successfully used to detect BVDV in pure culture and in artificially inoculated blood samples at a concentration 102CCID50/ml. The next phase of testing involved using the biosensor to detect BVDV in real field samples. Initially, the biosensor performance was assed in various samples known to contain BVDV including blood, nasal swab and ear-notches. Initial testing on known samples from BVDV PI animals showed that ear-notch elute samples produced the best sample matrix for detecting BVDV. The second objective was to test for BVDV infection in field samples. Thirty ear-notch samples that BVDV had previously been detected by antigen detection ELISA were tested. Twenty-seven of the thirty samples (90%) were classified correctly. The biosensor was then used to detect BVDV in 70 unknown ear-notch samples and compared to results obtained by the Diagnosis Center for Animal Population and Health using an antigen-capture ELISA (AC-ELISA). Unfortunately, the biosensor was unable to detect BVDV in three positive samples but was specific enough to produce a negative response in all 67 negative samples. The inability of the biosensor to detect the presence of BVDV in these samples may be due to the high variability observed in the signal response. Current research is trying to resolve variability in the biosensor response that may be affecting accuracy.
IMPACT: 2002/10 TO 2007/09
Cattle persistently infected with bovine viral diarrhea virus (BVDV) are the major source of virus spread within and between farms. Detecting and eliminating these animals is critical for the efficient control of this important virus. An inexpensive and rapid conductometric biosensor platform for detecting BVDV has been developed and initially tested. Rapid and cost effective detection of cattle persistently infected with BVDV will significantly enhance BVDV control programs both national and globally. Additionally, the developed technology can potentially be adapted to many important pathogens including foreign animal diseases those targeted as potential agroterroism agents.