1. Diagnosis of swine virus diseases and establishment of disease preventive and control measures for pig farms: In Taiwan, there are so many swine diseases and complication cases that always caused the confusion for clinician and pathologist to make diagnosis. The project is trying to combine clinician, pathologist and laboratory to identify swine virus disease in the field, and trying to collect disease preventive and control measures for pig farms. 2. Development and application of recombinant proteins of porcine circovirus type 2 Viral DNA of Taiwanese PCV2 was extracted and the ORF2-encoding region was amplified by PCR. The PCR product was inserted into the different vectors, and the expressed proteins in eukaryocytes were identified further with anti-PCV2 polyclonal antibody by Western blotting. Recombinant ORF2 protein will be purified with HisTrap column, and coated on the surface of 96-well ELISA plates. The antibodies titers of the sera from different farms and age groups will be determined by ELISA. We have designed different peptide-based antigen for PCV2 effective in swine. The peptide immunogen have various epitope from the ORF2 capsid protein of PCV2. The safe chemically-defined product would have considerable advantages for immuno-dot assays to detect antibody titers of serums. On the other hand, real-time PCR will be applied to detect antigens of PCV2 in serums. 3. Serosurveillance and diagnostic services for rinderpest and African swine fever: Previous serological investigation revealed that Taiwan is still free of rinderpest and African swine fever. Continuous nationwide surveillances on ruminants, mainly cattle, are essential to confirm the free status. Sera for detecting antibodies to rinderpest virus and African swine fever virus will be sampled by local disease diagnostic centers and submitted to the Animal Health Research Institute for detection of antibodies to specific pathogen by enzyme-linked immunosorbent assay (ELISA). 4. Investigation of the possible vector of bluetongue virus in Taiwan: Bluetongue virus (BTV) infection is prevalent among the cattle popolation in Taiwan and two BTV strains were isolated from goat in Kinmen County and from dairy cattle in Pingtung County in 2003. To date, the Culicoides vector responsible for BTV transmission in Taiwan has not remained unknown. The purpose of the study is to collect the insect samples from farms with high BTV serological prevalence. Through the way of BTV-specific nucleic acid detection, the competent vector for BTV transmission could be found. 5. Development of chromatographic strip test for the pen-side detection of foot and mouth disease virus antigen and antibody (2/2): This plan will develop the pen-side diagnosis kits for rapid detection of blood samples in vitro. The structure and non-structure protein genes of FMD/O/TAW/97 and FMD/TAW/99 were amplified with the RT-PCR method, and the RT-PCR products were inserted into pET and pPICZ expression vector, which were cloned into the expression system of prokaryotic and eukaryotic cell to acquire the recombinant protein. Subsequently, the monoclonal antibodies was produced by immune the mice with the recombinant protein and used to make the pen-side diagnosis kit for FMDV test according to the principle of chromatographic strip test methods. The pen-side diagnosis kits will be applied to the field samples for detecting the structure and non-structure protein of FMDV from FMDV infected animals. This methods could be used quickly and accurately to detect the latent infection of FMDV in animals. As a result it would be helpful to shorten the times of diagnosis and fast to control the diseases. 6. Strengthen the Veterinary Service of Animal Disease Diagnostic Center and Establish the Surveillance and Diagnosis of Important Animal Diseases:
Taiwan is now a member of WTO, animals and their products will be traded freely. Therefore, the risk of disease invasion increases tremendously and jeopardizes our public health. Actually, numerous infectious animal and zoonotic diseases have been introduced to Taiwan during the past decades. For the sustainable operation of animal production industry, we propose to apply clinical pathology, histopathology, immunochemistry, electron microscopy, etiological identification, epidemiology, etc. for animal disease diagnosis and to provide veterinary services to farmers, practitioners and authorities to take necessary preventive approaches. We also conduct disease monitoring and surveillance scheme to develop national animal disease control measures. 7. Study on the Etiology and Control Measures of Porcine Respiratory Diseases Complex: Recently, the respiratory diseases of swine have managed to emerge as a significant economic problem and present a substantial challenge to both veterinarians and producers. The porcine respiratory disease complex is most often due to the interaction of multi-factorial etiologies. The purpose of this study is to investigate important etiologic agents associated with porcine respiratory diseases and to provide control strategies for such diseases. We propose applying clinical pathology, histopathology, microbiology, and epidemiology methods to diagnose the porcine diseases and useful preventive approaches will be hopefully provided. The result of this study could improve the diagnostic abilities and strengthen the prevention network for the sustainable operation of animal production industry. 8. Establishment of the genomic databases of Taiwan poultry viral diseases: This project has the potential not only to improve the scientific understanding of H6 serotype of influenza virus, but also to uncover potential targets for the development of new vaccines, preventive measures and diagnostics, and to accumulate the knowledge of the overall molecular evolution of influenza viruses and other genetic factors that may determine their virulence. 9. National Accreditation of Diagnostic Laboratories for Bovine Spongiform Encephalopathy and Rabies: The international standard (ISO17025) is used to develop a quality system for rabies diagnosis by direct fluorescent antibody staining method to meet the requirement issued by Chinese National Laboratory Accreditation (CNLA). The biosafety level of Bovine Spongiform Encephalopathy (BSE) laboratory will be upgraded to BSL3 by this project simultaneously. Diagnosis and surveillance of transmissible spongiform encephalopathy (TSE) and rabies are routinely performed to accumulate necessary epidemiological data for declaring Taiwan is free from TSE and rabies. 10. Investigation and isolation of avian Clostridium botulinum: The goal of the study is to collect avian botulism cases by both of active and passive ways and to isolate avian Clostridium botulinum. The isolated strain will be used for production of botulism antiserum which may benefit all of avian species. 11. Prevalence of bluetongue viruses of serotypes 2 and 12 infections in cattle in Taiwan: The serological survey for Bluetongue virus (BT) will be performed of serotypes 2 and 12. The results will show the different serotype infection rate of BT in Taiwan. The specificity of the SN and the C-ELISA tests will be evaluated and effort on searching any new sertype will be continued. We expect to provide disease information to government for policy making on disease control and import-export trade. 12. Establishment the Surveillance and Diagnostic Laboratory of West Nile Virus Infection in Avian: West Nile virus infection is an important zoonosis. To prevent and control this emerging disease, the wild bird surveillance system is the most sensitive method. The aim of the project is to cooperate with the avian influenza surveillance project to create the West Nile virus infection surveillance system performing in the standard negative-pressured necropsy lab and biosafety lab. The result of this study could improve the diagnostic abilities and strengthen the prevention network for the sustainable operation of animal production industry. 13. Applied Detection Platforms on Infectious Pathogens and Intoxications of Domestic and Aquatic Livestock: The goal of the project is to establish analytical laboratory of drug and toxicant detection platform, and as well to install detection-related instruments and equipments. With the enhanced detection capability and process throughput, the preparedness laboratory shall receive cases from disease control institutions and clinical veterinarians for providing quality detection services with ensured accuracy and speed. The action items of the project include collecting and renovating domain knowledge on analytical technology and as well compiling analytical methodology of drug and toxicant detection in order for both practical laboratory applications and technician skillfulness advances. 14. The pharmacokinetics of aquatic antimicrobials in cobia: The purpose of this project is to establish the pharmacokinetic model of macrolide antibiotics in pretreated cobia. We will also set up the experimental animal model, sample collection, extraction and analysis techniques in live cobia. According to the analytic results, we will establish the pharmacokinetic model to evaluate the macrolides residues in the tissues in cobia. The final results will provide the reference to the government for defining the standard macrolides withdrawal period in the guideline. 15. The improvement of preparation of animal oil-adjuvant vaccines: The purpose of improvement of preparation of animal oil-adjuvant vaccines is to increase the stability of appearance and quality. This year an effort will make to seek the best formula and protocol in the preparation of oil-adjuvant vaccine. The anticipared successful improvement of oil-adjuvant vaccine will highly raise the benefit of animal vaccine industrial in Taiwan. 16. Production, Storage and Supply of Animal Vaccines and Diagnostic Reagents: This proposal is to perform the routine work in producing efficacy vaccines and diagnostic reagents used in the fields and diagnostic stations in Taiwan. In the context, we will perform: a, inviting experts to speak the quality control of vaccines; b, performing the medical examination for staffs; c, producing two kinds of inactivated vaccines; d, producing 4 kinds of diagnostic reagents; e, supplying enough products used in Taiwan. These products produced in this division will supply the highly qualified vaccines and diagnostic reagents to reduce the financial loss and to control the disease caused by pathogens. 17. Development of porcine bordetella bronchiseptica and actinobacillus pleuropneumoniae combined inactivated bacterins: The purpose of this study is to develop porcine bordetella bronchiseptica and actinobacillus pleuropneumoniae combined inactivated bacterins against these two diseases. To begin with, selecting seed strains, preparing for sera of identification, establishing growth curve. Two bacterins are cultured, harvested and adjusted to a final concentration, then inactivated using formalin and supplemented with preservative thimerosal, subsequently, mixed with water or oil soluble adjuvants. These suspensions are then used as bacterins for the immunization of mice and pigs in laboratory and in field. Eventually, the serum samples are collected from inoculating animals after weeks. The preliminary potency and safety assessments of trial bacterins are carried out in this year. 18. Study on the Pathogenesis and Development of Vaccine for Porcine Teschovirus: Porcine teschoviruses are widely distributed in pig, the disease was initially observed in Czecho Slovakia in 1929, this porcine teschovirus disease is also known as Teschen disease, Talfan disease, Porcine encephalomyelitis, Porcine poliomyelitis.Teschen disease virus was previously classified as enterovirus In 1979. Until 2000, the sequences were aligned and compared.Teschovirus to divided from enterovirus to become a new genus. In 2000, our institute studied a clinical case from a pig farm in Nantou district, this case turned out to be infected by serotype PTV-1; we will do serological surveillance test by VN (virus neutralization) test and ELISA test. Also we try to Identify of the PTV agent by virus isolation and RT-PCR. And try to reconstruct the evolutionary past based on the levels of sequence homology. By comparing sequences of Taiwanese isolates with the others, we may know epidemiology of porcine teschovirus in Taiwan. We will chose 3 months old pig PTV-negative by serum test, the challenge achieve via the oral or nasal cavities. Then sacrifice according to schedule. We will observe gross lesions and collect the specimens, especially from brain, spinal cord, intestines for Histopathology, serology, Isolation of virus, inmunohistochemistry (IHC), RT-PCR. We may know the Pathogenesis of porcine teschovirus, and compared with what is already known. In an area, such as Taiwan with high incidence, vaccine should be an important means for the control of this infection. In development for PTV vaccine, we will Chose suitable PTV-1 strains and pass in the PK-15 cell to obtain attenuated virus, verify virus titer and test efficacy of vaccine. 19. Maintaining and Upgrading of Veterinary Information Systems: The aims of this project are to strengthen and to maintain four databases and their related websites for extension of main knowledge of veterinary sciences to veterinary practitioners and animal raisers. It is expected that, through establishment of these information and extension to public, the services of veterinarians will be upgraded and the knowledge of animal raisers on animal health will be promoted. Therefore, the accomplishment of this project will provide the benefits to all animal industry as well.