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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
Areas to coverAreas to cover
IntroductionMaterials and MethodsResultConclusion
行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan
IntroductionIntroduction
行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan
Foot-and-Mouth diseaseFoot-and-Mouth disease
An acute contagious disease Cause severe economic lossNatural hosts :cloven-hoofed animals1514 in Italy19 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
Foot-and-Mouth diseaseFoot-and-Mouth disease
Aphthovirus, Picoranviridae ssRNASeven serotypes (O, A, C, SAT 1, SA
T 2, SAT 3 and Asia 1 )No cross protection between each ser
otypes
行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan
FMD in TaiwanFMD in Taiwan
Invasion of FMD in TaiwanThe first : 1913-1916 The second : 1924-1929FMD new outbreak in March 1997Swine adapted FMDV (O/TAW/97)The second FMDV invasion occurred in 1999A 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
FMD in TaiwanFMD 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
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)
Two types To detect the antibodies to viral structural pr
oteins (SP) To detect the antibodies to viral nonstructura
l 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)
Serotype-specific Detect antibodies elicited by vaccinatio
n 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)
Detecting the neutralization antibodies of sera
Requires cell culture facilities The use of live virusTakes 2–3 days to provide resultsConfirming 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)
The amount of virus used per well is in the range log10 1.5–2.5 TCID50.
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)
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)
ObjectiveObjective
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
Materials and MethodsMaterials 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 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
Serum neutralization testSerum neutralization test
Two-fold dilutions of test sera in 50 ul volumes.Mixed with 50 ul variant TCID50 O/TAW/97 FM
DV in 96 wells microplate.One hundred microliter mixtures were co-cultured
with 100 ul BHK-21 cells (3 x 105/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
ResultResult
行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan
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
TCID50
BT 127 28 40 93 142 200 266 282 347
PD 100 25 50 100 150 200 250 300 350
4 4 ≦ 3 4 ≦ 3 ≦ 3 ≦ 3 ≦ 3 ≦ 3
4 4 ≦ 3 ≦ 3 ≦ 3 ≦ 3 ≦ 3 ≦ 3 ≦ 3
Ab
titer
4 8 4 4 4 ≦ 3 ≦ 3 ≦ 3 ≦ 3
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
5.0
1.6
2.5
1.6
0.0 0.0 0.0 0.00.00.51.01.52.02.53.03.54.04.55.05.56.0
25 50 100 150 200 250 300 350
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
TCID50
BT 127 28 40 93 142 200 266 282 347
PD 100 25 50 100 150 200 250 300 350
16 32 32 32 16 4 8 8 4
16 256 128 32 16 32 32 8 8
Ab
titer
16 128 32 16 16 8 16 16 8
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
101.6
50.8
25.416
10.116
10.1 6.3
0102030405060708090100110
25 50 100 150 200 250 300 350
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
TCID50
BT 127 28 40 93 142 200 266 282 347
PD 100 25 50 100 150 200 250 300 350
64 128 32 32 32 16 16 16 8
64 512 256 128 128 32 64 32 32
64 128 64 32 128 32 64 16 16
64 64 32 32 16 32 32 16 32
Ab
titer
64 64 64 64 16 16 16 8 16
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
128
71.8
50.8 50.8
25.4 3218 22.6
0102030405060708090100110120130140150
25 50 100 150 200 250 300 350
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
TCID50
BT 127 28 40 93 142 200 266 282 347
PD 100 25 50 100 150 200 250 300 350
256 512 512 128 128 64 64 64 128
256 512 256 128 128 256 256 256 128
256 256 128 256 128 64 64 128 64
256 512 512 512 256 256 256 128 128
Ab
titer
256 512 512 512 512 128 512 512 128
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
445.7
337.8
256
194
128168.9 168.9
111.4
100120140160180200220240260280300320340360380400420440
25 50 100 150 200 250 300 350
ConclusionConclusion
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
AcknowledgementsAcknowledgements
Dr. Jong Ming-HwaMr. Lin Yu-FengMiss Li Hui-QiMiss Cai Shu-HuiMrs. Li Bao-ZhuMr. 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
行政院農業委員會家畜衛生試驗所 Animal Health Research Institute, Council of Agriculture, Executive Yuan