Development and Evaluation of IgM ELISA for the detection of FMDV specific IgM antibodies in

bovine and ovine sera

Usman Waheed1&2, Debi Gibson1, David J Paton1, Qaiser M Khan2

and Satya Parida1*

1-Institute for Animal Health, Pirbright, UK

2-National Institute for Biotechnology and Genetic Engineering (NIBGE),

Faisalabad, Pakistan.

Clinical Signs

Aims

• Whether IgM test has potential to discriminate between recent and historic infection in field out breaks, particularly in unvaccinated populations of sheep where lesions are very difficult to find out?

• Whether the IgM test has potential to identify infection in vaccinated populations as a DIVA screening or confirmatory test?

Design of experiment

• Assay development

• 270 negative serum (cattle+ sheep) samples were analysed

• Serum samples from two cattle vaccine challenge experiments were evaluated

• Serum samples from two sheep vaccine challenge experiments were evaluated

• 870 serum samples were analysed from 2001 outbreak

Frequency distribution

Frequency Distribution

0

20

40

60

80

100

120

0 ≥0<.1 ≥.1<.2 ≥.2<.3 ≥.3<.4 ≥.4<.5 ≥.5<.6

optical density

Fre

qu

en

cy

Results from vaccine challenge

expt-I (Parida et al., 2006, Vaccine)

VI positive-Black blocks; IgA positive (a) ; Animal dead ( …); RT-PCR positive-

Double lined rectangle; CEDI positive (c)

Results from vaccine challenge expt-II

VI positive-Black blocks; IgA positive (a) ; Animal dead ( …); RT-PCR positive-

Double lined rectangle; CEDI positive (c) (Parida et al., 2006, Vaccine)

IgM antibody detection in unvaccinated animals in vaccine I &

II experiments

Experimental Control Animals

0

0.2

0.4

0.6

0.8

1

1.2

0 D

PV

5 D

PV

14DP

V2

dpc

7 dp

c12

dpc

16 d

pc28

dpc

42 d

pc56

dpc

70 d

pc84

dpc

98 dpc

112

dpc12

6 dpc

140

dpc15

4 dpc

168

dpc

Days

OD

Va

lue

UV22

UV23

UV24

UV25

UV26

Experim ental Control Anim als

-0.2

0

0.2

0.4

0.6

0.8

1

OD

PV

5DP

V14

DP

V

2DP

C7D

PC

14D

PC

21D

PC

34D

PC

50D

PC

63D

PC

77D

PC

91D

PC

105D

PC

Days

OD V

alu

es

UY93

UY94

UY95

UY96

UY97

UnvaccinatedUnvaccinatedUnvaccinatedUnvaccinatedUnvaccinated Unvaccinated

Detection of IgM antibody in vaccinated carrier animals

Carrier Animals Vaccine I

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 D

PV

5 D

PV

14D

PV

2 dp

c7

dpc

12 d

pc16

dpc

28 d

pc42

dpc

56 d

pc70

dpc

84 d

pc98

dpc

112

dpc12

6 dpc

140

dpc15

4 dpc

168

dpc

Days

OD

Va

lue

s

UV2

UV14

UV5

UV10

UV13

UV11

UV19

UV9

UV17

Carrier animals vaccine II

0

0.2

0.4

0.6

0.8

1

1.2

OD

PV

5DPV

14D

PV

2DPC

7DPC

14D

PC

21D

PC

34D

PC

50D

PC

63D

PC

77D

PC

91D

PC

105D

PC

Days

OD

Va

lue

s

UY76

UY83

UY90

Detection of IgM in vaccinated and transiently infected cattle

No or transient Infection

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 D

PV

5 D

PV

14D

PV

2 dp

c7

dpc

12 d

pc16

dpc

28 d

pc42

dpc

56 d

pc70

dpc

84 d

pc98

dpc

112

dpc12

6 dpc

140

dpc15

4 dpc

168

dpc

Days

OD

Va

lue

s

UV6

UV7

UV8

UV15

UV3

UV4

UV18

UV16

UV20

UV21

UV12

Clinical and virological results of vaccinated and unvaccinated control sheep following

indirect aerosol challengeGroups Sheep

VR20v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR21v10 ─ ─ ─ ─ ─ ─ ─

VR22v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR23v10 ─ ─ ─ ─ ─ ─ ─ ─

VR24v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR25v10 ─ ─ ─ ─ ─ ─ ─ ─ ─

VR26v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR27v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR28v10 ─ ─ ─ ─ ─ ─ ─ ─ ─

VR29v10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR30s10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR31s10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR32s10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR33s10 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR34v4 ─ ─ ─ ─ ─ ─ ─ ─ ─

VR35v4 ─ ─ ─ ─ ─ ─ ─

VR36v4 ─ ─ ─ ─ ─ ─ ─ ─

VR37v4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR38v4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR39v4 ─ ─ ─ ─ ─ ─

VR40v4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR41v4 ─ ─ ─ ─ ─ ─ ─ ─

VR42v4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR43v4 ─ ─ ─ ─ ─ ─ ─ ─ ─

VR44s4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR45s4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR46s4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR47s4 ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

VR48c ─ ─ ─ ─

VR49c ─ ─ ─ ─

VR50c ─ ─ ─ ─

VR51c ─ ─ ─

VR52c ─ ─ ─ ─ ─

VR53c ─ ─ ─ ─

VR54c ─ ─ ─ ─

VR55c ─ ─ ─ ─ ─

Clin

ical signs

Fever >39.5C

RNA / V

irus

NSP

Clin

ical signs

Fever >39.5C

RNA / V

irus

NSP

Clin

ical signs

Fever >39.5C

RNA / V

irus

NSP

4 d

ays vaccin

ation

Controls

8-28 dpc 35 to 39 dpc3-7 dpc

10 d

ays vaccin

ation

Parida et al., 2008, Vaccine

Detection of IgM in unvaccinated and clinically infected sheep

Control

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

-10 DPI 7 DPI 10 DPI 14 DPI 21 DPI 28 DPI 35 DPI 40 DPI

DPI

OD

va

lue

VR 48

VR49

VR50

VR51

VR52

VR53

VR54

VR55

Unvaccinated

Detection of IgM in 10 days vaccinated and aerosol challenged

sheep

10 Days vaccinated

0

0.2

0.4

0.6

0.8

1

1.2

0 DPV 9 DPV 7 DPC 10

DPC

14

DPC

21

DPC

28

DPC

35

DPC

39

DPC

Days

OD

va

lue

s

VR 20

VR21

VR22

VR23

VR24

VR25

VR26

VR27

VR28

VR29

Detection of IgM in 4 days

vaccinated and aerosol challenged

sheep

4 days vaccinated

0

0.2

0.4

0.6

0.8

1

1.2

0 DPV 3 DPV 7 DPC 10

DPC

14

DPC

21

DPC

28

DPC

35

DPC

40

DPC

Days

OD

va

lue

s

VR34

VR35

VR36

VR37

VR38

VR39

VR40

VR41

VR41

VR42

VR43

Detection of infection by IgM test in 2001 Outbreak serum samples

• 521 bovine and ovine serum samples originated from in and around the farms where clinical lesions were found-early submission- 16 samples were found positive

• 147 samples from originated from in and around the farms where clinical lesions were found-late submission- No positive in IgM test

• Out of 1600 serum samples ~200 were found positive in SP tests. They were retested for NSP and IgM antibodies-almost all are positive for NSP, but only 3 are positive for IgM

Conclusions

• After FMD vaccination or transient infection, IgM persists for approximately 2-3 weeks.

• IgM test has potential to discriminate between recent and historic infection in field out breaks, particularly in unvaccinated populations of sheep where lesions are very difficult to find out

• Persistence of IgM has been associated with persistence of other pathogens and now the same is shown for FMDV. This requires further study.

• Preliminary findings indicate that though emergency vaccine induces IgM, the antibody does not persist but reappears if animals become infected. Therefore the IgM test has potential to identify infection in vaccinated populations as a DIVA screening or confirmatory test.

Acknowledgements

• Paul Barnett

• Sarah Cox

• Nigel Ferris

Funding

• Defra, UK

• EU-IMPROCON

• HEC, Pakistan