Download FAO Collaborative Studies for FMD Standardisation: Phase XIX - Virological Assays

Appendix 56
FAO Collaborative Studies for FMD Standardization : Phase XIX - Virological Assays
N.P. Ferris1, D.P. King1, G.H. Hutchings1, Y. Li, N. Goris2 and D.J. Paton1
1
BBSRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, UK
GU24 0NF
2
Veterinary and Agrochemical Research Centre, Groeselenberg 99, 1180 Ukkel, Belgium
Abstract:
The aim of Phase XIX was to complete a proficiency testing study for virology and serology. The
virological aim was to evaluate the ability of laboratories to discriminate between FMD positive and
negative samples by testing two proficiency test panels (one infectious and one non-infectious) for
the presence of FMD virus by means of virus isolation (VI), RT-PCR and antigen ELISA. Panel 1
consisted of 20 coded samples prepared from 11 ‘vesicular’ epithelia, eight of which were derived
from submissions of suspect cases of FMD or swine vesicular disease (SVD), while another three
were derived from epithelia from naïve animals. Fifteen samples were derived from six FMD virus
positive epithelia representing four different serotypes (two each of types O and A and one each of
types SAT 2 and Asia 1), one from a sample which had been found to be negative by antigen ELISA
and VI but positive by FMD RT-PCR and another from SVD positive epithelium. Some of the FMD
virus positive samples were prepared from 10-fold serial dilutions of two of the initial suspensions.
The participants were invited to test the samples by their available RT-PCR procedures and to
inoculate cell cultures that they routinely use for FMD diagnosis in attempts to isolate virus and to
confirm the specificity of any isolated virus. Panel 2 consisted of 12 coded samples prepared from
inactivated, cell culture supernatant fluids. Ten were derived from the propagation of four different
FMD virus serotypes and SVD virus and a further two prepared from uninoculated cell cultures.
Dilutions were prepared to yield moderate and weak concentrations of antigen of FMD virus types
O, A (two different strains) and Asia 1 and also moderate concentrations of FMD virus type SAT 2
and SVD virus. The participants were invited to test the samples by antigen ELISA. By the middle
of July, packages had been distributed to 35 laboratories in 34 countries in different parts of the
world. Twenty four laboratories received both panels, ten others received panel 2 only, while
another laboratory solely received panel 1. Template data sheets for result entry and for providing
information on test procedure were also supplied to each participant by email and for return to the
FAO World Reference Laboratory for FMD.
Introduction:
The aim of Phase XIX was to complete a proficiency testing study for serology, antigen detection
and detection of live FMD virus by RT-PCR and VI to gain some insight into whether individual
laboratory procedures are suitable for diagnostic use. To facilitate the study, the FAO World
Reference Laboratory for FMD, Pirbright Laboratory agreed to formulate and distribute identical
sample sets of three panels to participating laboratories - panel 1: a panel of infectious materials to
be tested by VI and RT-PCR, panel 2: a panel of non-infectious materials to be tested by antigen
detection ELISA and panel 3: a panel of non-infectious sera for serological testing. This report
summarises the methodology used for the preparation and dispatch of panels 1 and 2 and the
results achieved from the virological investigations subsequently undertaken by participating
laboratories.
Prior to commencement of Phase XIX a pilot study had been undertaken by five laboratories in
2004/2005 to evaluate the sensitivity and specificity of their routinely employed RT-PCR tests and
cell cultures for the detection and isolation of FMD virus using identical sets of 20 coded samples
prepared from epithelia derived from suspect cases of FMD or SVD. The objectives being: to test
the fitness for purpose of the prototype panel, to evaluate the logistics of sample preparation and
dispatch and to generate comparative data for publication. These objectives were achieved and the
findings published (Ferris et al. 2006) and concluding that the prototype panel was more or less
suitable for purpose but that it needed to be modified to include more and true negative sample
suspensions and to extend the serial dilution range of one of the titration series.
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Materials and Methods:
The preparation and composition of Panel 1 was carried out as previously described (Ferris et al.
2006; enough stocks of the initial epithelial suspensions having been prepared and stored at -80oC
to allow further panels to be composed) with revisions. A further 10-fold dilution step was added to
each of the titration series for O BHU 39/2004 and A IRN 5/2003 and three epithelial suspensions
derived from naïve animals (two bovine and one porcine) were included at the expense of one of
the SVD virus samples (UKG 76/74), one NVD sample (no virus detected; IRN 19/2003) and three
10-fold dilution steps from the Asia 1 PAK 20/2003 titration series. Thirty identical sets of 20
samples of 5 ml volumes were prepared and each sample was uniquely coded to ensure ‘blind’
testing and labelled with the suffix ‘V’.
Panel 2 consisted of 12 samples prepared from inactivated, cell culture supernatant fluids. Ten
samples were derived from four FMD virus serotypes (O, A, SAT 2 and Asia 1) and one SVDV and a
further two samples were derived from uninoculated cell cultures. Dilutions were prepared to yield
moderate and weak concentrations of FMD virus types O (strain O1 BFS 1860), A (A5 Allier and A22
IRQ 24/64) and Asia 1 (CAM 9/80) plus moderate concentrations of SAT 2 (K 183/74) and SVD
virus (ITL 1/92). Fifty identical sets of 4 ml sample volumes were prepared and each sample was
uniquely coded and labelled with the suffix ‘A’.
The samples were stored at -80oC prior to distribution to each of the participating laboratories by
airfreight in dry ice. Templates for the return of the results from the proficiency testing were also
supplied and which included prompts designed to elicit information on individual laboratory test
methodology. The identities of the samples and virus serotypes are listed in Figures 1, 2 and 3.
Results and Discussion:
The results achieved for VI in cell culture are summarised in Figure 1. All laboratories used their
own in-house procedures for carrying out VI. A wide range of cell culture systems were employed
overall and included primary bovine thyroid and kidney cells, primary and secondary lamb kidney
cells, foetal goat tongue and BHK cells and other cells of porcine origin, including primary and
secondary kidney cells, PK-15 and SK-6 cells plus those of the IB-RS-2 cell line. Additionally, there
were differences in both how the cell monolayers used for sample analysis were grown (e.g. in
tubes, flasks or plates of different sizes) and the manner in which VI was performed. This detail is
not presented here but will be described in a full publication. The majority of the laboratories used
the antigen ELISA to confirm the specificity of any isolated virus.
It can be seen that there was a wide difference in the ability of participating laboratories to detect
infectious virus and was most probably caused by differences in the sensitivity of cell cultures used
for VI. Primary bovine thyroid cells were the most sensitive cell culture system for the detection of
FMD virus, followed by primary lamb kidney, foetal goat tongue and certain IB-RS-2 cells. It is also
tempting to speculate that the methodology for cell culture preparation and sample inoculation and
propagation influenced the result outcome. However, at least one laboratory (lab T) faced problems
in clearing its proficiency panels from customs and 8 days passed before their arrival in the
laboratory, by which time the samples had thawed and the retention of sample infectivity thus
compromised. The status of the sample integrity once received therefore may have influenced the
VI result outcome
The results of RT-PCR analysis are shown in Figure 2. The majority of RT-PCR tests were specific,
although there were several instances of false-positive results from testing the three negative
samples. However, there were differences in assay sensitivity as indicated by the ability to detect
FMD virus genome both in serially diluted samples and in the ‘PCR-positive but ELISA/VI negative’
sample, BOT 1/2003. Several laboratories used an SVD virus RT-PCR test procedure and in all
instances these were demonstrated to be specific for the detection of the SVD virus sample. In
general, laboratories had sufficiently useful RT-PCR test procedures to overcome any deficiency in
VI.
The results of antigen detection ELISA are shown in Figure 3 and indicate that the majority of
laboratories have workable assays, although of variable sensitivity, and the performance of which
is a reflection of the antisera employed for assay. The majority of laboratories employed polyclonal
reagents obtained from Pirbright, although antiserum stocks, serotypes and strains varied – for
example, labs 11 and 31 used reagents against only three FMD virus types (O, A and Asia 1; lab 31
using an A22 antiserum) plus SVD virus, lab 12 and 16 used reagents to four FMD virus serotypes
(O, A, C and Asia 1), while lab 21 did not use Asia 1 antiserum in their regime. Consequently,
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certain samples went undetected because the relevant type-specific antiserum was not included in
the particular ELISA. Labs 5, 7, 10, 13, 19, 21 and 26 used their own ‘in-house’ prepared reagents,
lab 10 a combination of Pirbright and ‘in-house reagents, lab 19 an unspecified ELISA kit while lab
13 used monoclonal antibodies.
Conclusions:
•
The wide variation in the VI results was most probably due to a range in sensitivity of the
cell cultures employed and which might also have been influenced by test methodology
•
The RT-PCR test procedures were generally specific and, although varying in sensitivity,
largely off-set any deficiencies in VI
•
Antigen ELISA’s were also generally workable but varied in sensitivity and were often
limited by using a panel of reagents of incomplete serotypic range
•
Phase XIX has provided a valuable opportunity to compare the diagnostic procedures which
are being used in National FMD Laboratories and how they performed on the proficiency set
of samples provided
•
Participating laboratories have recognised that taking part in proficiency testing schemes
such as this is necessary to gain confidence that diagnostic procedures are fit for purpose
in order to take remedial action if deficiencies are identified and to meet the requirements
of external quality assurance
Recommendations:
•
More detailed analysis of the results should be carried out with a view for paper submission
for publication
•
Efforts should be made to standardise VI procedures between laboratories
•
A panel of non-infectious samples for RT-PCR evaluation should be made available for
laboratories without the facilities (BSL3) to handle live FMD virus
•
Panel 2 would benefit from the inclusion of (inactivated) contemporary field FMD viruses
•
Laboratories should keep in stock reagents against all seven serotypes of FMD virus and
SVD virus for antigen ELISA
Acknowledgements:
We thank all the laboratories for participating in Phase XIX. This work was funded by the UK
Department of the Environment, Food and Rural Affairs (Defra projects SE1120 and SE1121) and
by FAO.
Reference:
Ferris, N.P., King, D.P., Reid, S.M., Hutchings, G.H., Shaw, A.E., Paton, D.J., Goris, N.,
Haas, B., Hoffmann, B., Brocchi, E., Bugnetti, M., Dekker, A. & De Clerq, K. 2006. Foot-andmouth disease virus: A first inter-laboratory comparison trial to evaluate virus isolation and RT-PCR
detection methods. Vet. Microbiol., 117, 130-140.
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Figure 1
P1 (2 or 3), positive on first (second or third) passage
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Figure 2
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Figure 3
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