The study on the effect of foot-and-mouth disease virus titer to the result of detected...

The study on the effect of foot-and-mouth disease virus titer to

the result of detected neutralization antibody titer

Hog Cholera Research DivisionLin 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 cover

IntroductionMaterials and MethodsResultConclusion


Introduction


Foot-and-Mouth disease

An acute contagious disease Cause severe economic lossNatural hosts :cloven-hoofed animals1514 in Italy19 centuries to now in the world


Foot-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


FMD 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)


FMD in Taiwan

The blanked vaccination against FMDV is implemented since 1997.

Taiwan becomes a FMD free country with vaccination since May 22, 2003.


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


(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



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



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



The amount of virus used per well is in the range log10 1.5–2.5 TCID50.

Valid Test of the VN



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



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.


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 TCID50


Serum neutralization test

Two-fold dilutions of test sera in 50 ul volumes.Mixed with 50 ul variant TCID50 O/TAW/97 FMD

V 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 o

f BHK cells under a microscope.


Result



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


The geomean of 1:4 sera

5.0

1.62.5

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


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



101.6

50.8

25.416 10.1 16 10.1 6.3

0102030405060708090100110

25 50 100 150 200 250 300 350



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


The geomean of 1:64 sera128

71.850.8 50.8

25.4 3218 22.6

0102030405060708090100110120130140150

25 50 100 150 200 250 300 350



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



445.7

337.8

256

194

128168.9 168.9

111.4100120140160180200220240260280300320340360380400420440

25 50 100 150 200 250 300 350

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.


Acknowledgements

Dr. Jong Ming-HwaMr. Lin Yu-FengMiss Li Hui-QiMiss Cai Shu-HuiMrs. Li Bao-ZhuMr. Huang Guan-Hwa


The study on the effect of foot-and-mouth disease virus titer to the result of detected...

Documents

Transcript of The study on the effect of foot-and-mouth disease virus titer to the result of detected...