Diagnostic Manual Akkreditiertes Prüflaboratorium Register-Nr. AKS-P-20312-EU

Staatlic he Akkred itierun g sstelle Hann o ver

State of the Art of CSF Diagnostic Methods Irene Greiser-Wilke Institute of Virology Department of Infectious Diseases EU Reference Laboratory for CSF University of Veterinary Medicine Hannover 30559 Hannover, Germany

Content of the Diagnostic Manual

Laboratory Diagnosis of CSF

• Description of the disease • Guidelines to recognise suspect holdings

INDIRECT

DIRECT

• Checking and sampling procedures Virus isolation

• Collection and transport of samples • Virological tests • Serological tests

.1 m 01 o r f as

02 1.20

• Genetic typing of virus isolates

Antigen detection - FAT - ELISA (Ag)

Antibody detection - Neutralisation test - ELISA (Ab)

Nucleic acid - RT-PCR

• Safety requirements for CSF laboratories Depner, 2003

Depner, 2003

Primary Outbreak

Secondary Outbreak

clinical signs of CSF

clinical signs, epidemiology

AgELISA, FAT, PCR one test positive FAT and PCR or FAT and AgELISA or AgELISA and PCR positive

AgELISA, FAT, PCR one test positive Virus isolation positive

Virus isolation positive

Serology positive AbELISA, VNT

Virus isolation Confirmation of CSF

Confirmation of CSF Depner, 2003

Depner, 2003

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CSF Antigen ELISA Diagnostic Manual, 2002

New Developments

Each NSFL is in charge for the licensing of different batches of commercial CSF antigen ELISAs. The CRL does not recommend a specific antigen ELISA. At present, CSF antigen ELISAs can be obtained from: Bommeli, CH Synbiotics, F ID-Lelystad, NL IDEXX, USA and S

• Antigen ELISAs • Antibody-ELISAs • RT-PCR methods

• Low sensitivity • Allowed for herd screening only

Positive samples

Evaluation of 5 Commercially available AbAb-ELISAELISA-Kits* Kits*

(from animal experiments)* experiments)* ELISA

14 d.p.i. n pos

• Ceditest CSFV (Cedi-Diagnostics)(E2) • Ceditest CSFV 2.0 (Cedi-Diagnostics)(E2) – Currently not on the market • Herdchek CSFV/Ab (IDEXX)(E2) • Chekit-CSF-Sero (Bommeli, now IDEXX)(E2) • Chekit-CSF-Marker (Bommeli, now IDEXX)(Erns)

21 d.p.i.

%

n

pos

%

n pos %

VNT

81

57

70.4

63

56

88.9

68 63 92.6

Ceditest CSFV

118 38

32.2

75

66

88.0

75 67 89.3

Ceditest CSFV 2.0

118 44

37.3

75

65

86.7

75 67 89.3

Herdchek CSFV/Ab

118 39

33.1

75

67

89.3

75 68 90.7

Chekit-CSF-Sero

118 63

53.4

75

70

93.3

75 71 94.7

Chekit-CSF-Marker

117 57

48.7

74

65

87.8

75 67 89.3

* Willie Loeffen, Sjaak Quak, 2005

Negative Field Samples (finishing pigs)* pigs)* ELISA

28 d.p.i.

* Willie Loeffen, Sjaak Quak, 2005

PanPan-pesti Positive Field Samples*

n

Positive

Specificity

Ceditest CSFV

792

2

99.7%

Ceditest CSFV 2.0

792

0

100%

Herdchek CSFV/Ab

792

0

Chekit-CSF-Sero

792

Chekit-CSF-Marker

792

n

Positive

% positive

Ceditest CSFV

173

16

9.2%

Ceditest CSFV 2.0

156

0

0%

100%

Herdchek CSFV/Ab

166

0

0%

34

95.7%

Chekit-CSF-Sero

156

3

1.9%

8

99.0%

Chekit-CSF-Marker

173

78

45.1%

* Willie Loeffen, Sjaak Quak, 2005

ELISA

* Willie Loeffen, Sjaak Quak, 2005

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Summary* Summary*

Preliminary Conclusions* Conclusions*

Sensitivity „ VNT most sensitive at 14 d.p.i., compares to ELISAs after that „ Chekit-CSF-Sero consistently most sensitive ELISA „ Chekit-CSF-Marker compares to other ELISAs - More sensitive at 14 d.p.i. - More sensitive with certain strains (Paderborn)

• ELISA’s overall performance more or less comparable • Higher sensitivity goes together with lower specificity • In the test system ELISA-VNT, sensitivity of VNT will limit overall sensitivity

Specificity (random CSFCSF-negative samples) „ Chekit-CSF-Sero least specific ELISA, followed by Chekit-CSF-Marker „ Other ELISA’s comparable with very few false positives (Herdchek none at all) * Willie Loeffen, Sjaak Quak, 2005

RTRT-PCR: detection of viral genomic fragments

* Willie Loeffen, Sjaak Quak, 2005

PCR: Theory

• The DNA-polymerase used in PCR can only amplify DNA • As pestiviruses are RNA viruses, the first step to be performed is Reverse Transcription

2n

GelGel-based RTRT-PCR protocols

Evaluation Agarose gel electrophoresis

Real-time (qPCR)

• most diagnostic RT-PCRs amplify a region in the UTR • several different protocols are being used, e.g.

1. VILCEK et al. 1994 (PanPesti) 5‘ UTR 288bp -----------------------------------------------------------------------------------------------2. KATZ et al. 1993 (CSFV) E2 308bp/172bp 3. DIAZ DE ARCE et al. 1998 (CSFV)

NS5B

174bp

4. GREISER-WILKE et al. 1998 (CSFV) 5’ UTR

402bp

5. AGÜERO et al. 2004 (CSFV)

133bp

5’ UTR

3

Specificity

What is realreal-time PCR?

KATZ protocol CSFV 0518 / neg / BDV D27/99 Chemnitz

• Technique that allows direct monitoring of a PCR reaction as it occurs • It measures and quantifies fluorescent signals

„nested PCR“

• Machines: integrated thermocyclers allowing computer-assisted evaluation (fluorescence signals; photomultipliers)

308bp

172bp

• Fluorescent label in the reaction needed • Labels can be direct or indirect

Evaluation of realreal-time PCR • Increase of fluorescence proportional to increase of product (linear correlation) • Fluorescence is monitored during each PCR cycle providing an amplification plot

Evaluation Methods: Methods: Direct Fluorescent Labelling Use of dsDNA intercalating dyes (SYBR Green 1)

Fluorescence

Baseline

dR threshold

Ct

Cycle #

PestivirusPestivirus-specific RTRT-PCR (SybrGreen) SybrGreen) • PCR with pestivirus-specific primers V324/V326 (Vilcek et al., 1994) • RNA was isolated from PK15(A) cells infected with Alfort/187

Advantages/disadvantages Advantages/disadvantages of intercalating dyes + Universal applicability – You can use it for any PCR (in theory) + High signals – Every DNA molecule binds many dye molecules + Specificity can be checked by melting curve analysis - But: no real differentiation between correct product and artefacts! + Low reagent costs - Cannot multiplex - Low specificity

4

Evaluation Methods: Methods: TaqMan probes (2)

Evaluation Methods: Methods: TaqMan probes (1)

Taq DNADNA-Polymerase (5' nuclease activity) activity)

CSFVCSFV-specific TaqMan Nested RTRT-qPCR 5`

CSFVCSFV-specific TaqMan RTRT-qPCR

UTR

Primer 324

UTR

PANPESTI AMPLICON

Primer 326 Position 395395- 375

Position 108 - 128

Primer A11

3`

PANPESTI AMPLICON

5`

UTR

UTR

Primer A11

PANPESTI AMPLICON

Position 161161-186

Primer A14 Position 353353-373

210 bp

Primer A14

CSFV probe

Position 353353-373

Position 161161-186

3`

286 - 310

CSFV Probe 286 - 310

McGoldrick et al., 1998

Specificity

Sensitivity Alfort/187 Alfort/187

CSFV 422 CSFV 657

107,6

106,6 105,6 104,6 103,6 102,6

BVDV-NADL BDV-Moredun BDV-Chemnitz

101,6

5

Advantages/disadvantages Advantages/disadvantages of TaqMan Probes

Advantages of realreal-time PCR - evaluation is performed during PCR („real time“)

+ Multiplexing capability – Useful for quantification (internal control) + High specificity – Target specific due to complementary probes + High reagent costs (probe) - No size determination of the amplicon - No exclusion of contamination / probe degradation - Size of the target to be amplified limited to about 150 base pairs to obtain a maximum efficiency

Comparative Performance of Diagnostic Tests*

- electrophoresis not necessary - high sensitivity (1-20 pg) - high specificity (hybridisation, melting curves) - high sample throughput in short time - reduced contamination risk („closed tube“ PCR) - allows quantification of PCR products - fully automised procedure - expensive equipment - amplicons can not be sequenced

Conclusions • According to EU regulations, both antigen and antibody tests have to be performed in the national reference laboratories • Performance is monitored in yearly ring tests • Virus isolation and virus neutralisation are still the „gold standards“ • Due to low sensitivity antigen-ELISAs are only useful for herd testing • They are continously being replaced by RT-PCR

Reference test: virus isolation

* Dewulf et al., 2004

• Performance of commercially available antibody-ELISAs has increased

References

Any questions??? questions???

Aguero, M., J. Fernandez, L.J. Romero, M.J. Zamora, C. Sanchez, S. Belak, M. Arias, and J.M. Sanchez-Vizcaino, 2004. A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples. Vet Res 35:551-563. Depner, K., 2003. In: Workshop on CSF: Clinical signs, epidemiology and control. Hannover, October 6 - 9 Diaz-de-Arce H., Nunez J.I., Ganges L., Barreras M., Frias M.T., and F. Sobrino, 1998. An RTPCR assay for the specific detection of classical swine fever virus in clinical samples. Vet. Res. 29:431-440. Greiser-Wilke I., Depner K., Fritzemeier J., Haas L., and V. Moennig, 1998. Application of a computer program for genetic typing of classical swine fever virus isolates from Germany. J. Virol. Meth. 75:141-150. Katz J.B., Ridpath J.F., and S.R. Bolin, 1993. Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach. J. Clin. Microbiol. 31:565-568. Loeffen W., and S. Quak, 2005. In: Annual Meeting of National Swine Fever Laboratories, Brussels, Belgium, May 11-12 Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Part 2, Section 2.1., Chapter 2.1.13, Classical Swine Fever http://www.oie.int/eng/normes/mmanual/A_00036.htm McGoldrick A., Bensaude E., Ibata G., Sharp G., and D.J. Paton, 1999. Closed one-tube reverse transcription nested polymerase chain reaction for the detection of pestiviral RNA with fluorescent probes. J. Virol. Meth. 79:85-95. Vilcek S., Herring A.J., Nettleton P.F., Lowings J.P., and D.J. Paton, 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 136:309-323.

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