1. The study on the effect of foot- and-mouth disease virus titer to the result of detected neutralization antibody titer Hog Cholera Research Division Lin Yeou-liang ( 口蹄疫病毒力價對中和抗體力價檢測結果之影響研究 ) ( 豬瘟研究組 林有良 ) 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

2. Areas to cover  Introduction  Materials and Methods  Result  Conclusion 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

3. Introduction 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

4. Foot-and-Mouth disease  An acute contagious disease  Cause severe economic loss  Natural hosts :cloven-hoofed animals  1514 in Italy  19 centuries to now in the world 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

5. Foot-and-Mouth disease  Aphthovirus, Picoranviridae  ssRNA  Seven serotypes (O, A, C, SAT 1, SAT 2, SAT 3 and Asia 1 )  No cross protection between each serotypes 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

6. FMD in Taiwan  Invasion of FMD in Taiwan  The first ： 1913-1916  The second ： 1924-1929  FMD new outbreak in March 1997  Swine adapted FMDV (O/TAW/97)  The second FMDV invasion occurred in 1999  A Pan Asia topotype serotype O (O/TAW/99) 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

7. FMD in Taiwan  The blanked vaccination against FMDV is implemented since 1997.  Taiwan becomes a FMD free country with vaccination since May 22, 2003. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

8.  To certify individual animals prior to import or export (i.e. for trade).  To confirm suspected cases of FMD.  To substantiate absence of infection.  To demonstrate the efficacy of vaccination. Four purposes of serological test for FMD 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

9.  Two types  To detect the antibodies to viral structural proteins (SP)  To detect the antibodies to viral nonstructural proteins (NSPs). Serological tests for FMD 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

10.  Serotype-specific  Detect antibodies elicited by vaccination and infection  Virus neutralisation (VN) test  The solid-phase competition ELISA  The liquid-phase blocking ELISA The SP tests 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

11.  Detecting the neutralization antibodies of sera  Requires cell culture facilities  The use of live virus  Takes 2–3 days to provide results  Confirming the positive result of ELISA Virus Neutralization Test 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

12.  The amount of virus used per well is in the range log10 1.5–2.5 TCID 50. Valid Test of the VN 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

13.  Different interpretations of test results will be appropriate according to the different purposes.  The final serum dilution in the serum/virus mixture  A titre of 1:45 or more --- positive  A titre of less than 1:16 --- negative  Titres of 1:16 to 1:32 --- doubtful To Interpret the Result of VN test 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan (http://www.oie.int/eng/normes/mmanual/A_00024.htm)

14. Objective  The aim of this study is to know whether or not the different virus titer will cause the variation of detected result of neutralization antibody titer. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

15. Materials and Methods  Known titer sera: 1:4, 1:16, 1:64, 1:256  FMDV titers neutralized with sera: 25, 50, 100, 150, 200, 250, 300, 350 TCID 50 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

16. Serum neutralization test  Two-fold dilutions of test sera in 50 ul volumes.  Mixed with 50 ul variant TCID 50 O/TAW/97 FMDV in 96 wells microplate.  One hundred microliter mixtures were co-cultured with 100 ul BHK-21 cells (3 x 10 5 /ml).  Incubating in 37 ℃ incubator contained 3.5% CO 2 for 48 hours.  Observing the cytopathic effect (CPE) formation of BHK cells under a microscope. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

17. Result 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

18. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan A titer of 1:4 sera

19. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan The geomean of 1:4 sera

20. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan A titer of 1:16 sera

21. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan The geomean of 1:16 sera

22. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan A titer of 1:64 sera

23. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan The geomean of 1:64 sera

24. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan A titer of 1:256 sera

25. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan The geomean of 1:256 sera

26. Conclusion  The geomean value of detected NA titer will be within the acceptable range when the virus titer is in the range log10 1.5–2.5 TCID50.  The detected result of NA titer will be more stable, if the virus titer kept between log10 1.7 TCID50 and 2.2 TCID50. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

27. Acknowledgements  Dr. Jong Ming-Hwa  Mr. Lin Yu-Feng  Miss Li Hui-Qi  Miss Cai Shu-Hui  Mrs. Li Bao-Zhu  Mr. Huang Guan-Hwa 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan

28. 行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan