Download 42. Improved FMD diagnosis using real-time RT-PCR

Appendix 42
Prospects for improved laboratory diagnosis of FMD using real-time
RT-PCR
Nigel Ferris*, Scott Reid, Donald King, Geoff Hutchings and Andrew Shaw
Pirbright Laboratory, Institute for Animal Health, Ash Road, Woking, Surrey GU24 0NF, UK
Abstract:
Definitive diagnosis of FMD requires the detection of virus, antigen or genome in clinical material. The
aim was to evaluate the performance of an automated real-time RT-PCR procedure for this purpose.
Vesicular epithelia from eighteen countries were examined by ELISA, VI and RT-PCR. Retrospective
analysis by RT-PCR was also performed on available material of two sample subsets collected from
‘confirmed’ cases during the 2001 UK FMD outbreak : firstly, samples which were negative by both
ELISA and VI and secondly, others which were negative by ELISA on epithelial suspension but
positive by VI. There was broad agreement between RT-PCR and VI for 79% and VI/ELISA combined
for 82% of the overseas epithelial samples tested. There were no false negative results obtained with
RT-PCR since all samples assigned negative by RT-PCR were also negative by VI/ELISA. However, the
RT-PCR was able to detect FMDV in an additional 18% of the samples tested. Additionally, there was
good agreement between the RT-PCR and ELISA/VI for the UK outbreak samples save for a group of
related virus isolates from Wales. These viruses had evidently evolved during the epidemic and had a
nucleotide substitution in the RT-PCR probe site, which prevented detection by RT-PCR using the
routine diagnostic probe.
The ELISA and VI are deficient for specimens of poor quality where concentrations of infectious FMDV
may either be low or absent. The features that influence sample quality appear to be less important
for the RT-PCR as it can detect a small fragment of FMDV genomic RNA, not just live virus. Real-time
RT-PCR provides an extremely sensitive and rapid procedure that contributes to improved laboratory
diagnosis of FMD. However, the failure to detect the mutant FMDVs from the UK 2001 epidemic
illustrates the importance of constant monitoring of representative field FMDV strains by nucleotide
sequencing to ensure that the primers/probe set selected for the diagnostic RT-PCR is fit for purpose.
Introduction:
Control of outbreaks of foot-and-mouth disease (FMD) is dependent upon a system of monitoring and
early detection, which requires basic familiarity with clinical signs and the ability to characterise the
strain of virus responsible by laboratory tests. Definitive diagnosis of FMD requires the detection of
virus, antigen or genome in clinical material. Ideally, the sample of choice should be vesicular
epithelium from clinically affected animals since, during the acute stage of the disease, it is rich in
virus. The World Reference Laboratory (WRL) for FMD typically receives between 400 and 700
samples annually from overseas countries (Ferris and Donaldson, 1992; Table I), including other
sample types besides epithelia, e.g. epithelial suspensions, cell culture antigens, blood, throat swab
(probang) and milk samples. For almost twenty years, the WRL for FMD has used an indirect,
sandwich enzyme-linked immunosorbent assay (ELISA) (Roeder and Le Blanc Smith, 1987; Ferris and
Dawson, 1988; OIE, 2004) to identify FMDV. However, the ELISA is not 100% sensitive.
Consequently, suspensions of each specimen are also propagated in sensitive cell cultures (Ferris and
Dawson, 1988) and the specificity of any isolated virus confirmed by the ELISA. Whilst such virus
isolation (VI) methods are highly sensitive, they require four days before a negative result can be
concluded (and reported as ‘no virus detected’ [NVD]). It is evident from Table I that FMDV antigen
cannot be detected by ELISA and VI in approximately half the submitted samples. This has given
cause for concern as to the efficiency of sample collection and dispatch and also with respect to the
adequacy of the laboratory test procedures employed for their examination.
In emergencies, speed of diagnosis (clinical and laboratory confirmation) is of paramount importance
to control spread and eradicate disease. Approximately, 90% of positive epithelial samples received
during the 2001 UK FMD outbreak were so defined by the antigen ELISA on prepared suspensions,
the remainder being serotyped after amplification and isolation of virus following cell culture passage.
Negatives could only be classified following double passage in cell culture, which took 4 to 5 days.
Consequently, the introduction by the UK Government towards the end of March of a 24/48 hour
culling policy (all animals to be slaughtered on infected premises within 24 hours of diagnosis and
those on neighbouring premises within 48 hours) meant that confirmation of disease was
subsequently made by clinical judgement alone. This policy, although playing a critical role in
controlling disease, caused huge controversy and provoked much debate on the likelihood of many
animals being slaughtered unnecessarily, fuelled by the finding that neither virus nor antibody could
be detected in samples received from many of the confirmed cases.
261
Recently, the development of a real-time reverse transcription polymerase chain reaction (RT-PCR)
procedure has provided an additional tool which can be used for FMD diagnosis (Reid et al., 2002).
Furthermore, this real-time RT-PCR method can be automated allowing increased throughput of
samples with fewer user-dependent steps (Reid et al., 2003). The authors have compared the
performance of a fully automated real-time RT-PCR (Reid et al., 2003) with VI and ELISA for the
detection of FMDV on the majority of epithelium samples received at the WRL for FMD from overseas
during a recent eighteen-month period (August 2002 until January 2004) and on two subsets from
confirmed cases from the recent UK outbreak : firstly, samples which were negative by both ELISA
and VI and secondly, others which were negative by ELISA on epithelial suspension but positive by
VI.
Materials and Methods:
Three hundred and thirty four samples of vesicular epithelium were received from eighteen countries
during the period of the study and were examined by ELISA, VI and RT-PCR (Table II). Upon receipt,
the pH of the transport buffer containing epithelial tissue was estimated using phenol red indicator. A
suspension of the epithelium (ES) was made in 0.04 M phosphate buffer (ideally this should be done
using a 10% concentration (w/v), but during this study a lower concentration was frequently used
due to paucity of material). A 1.4 ml aliquot was taken for the antigen detection ELISA (Ferris and
Dawson, 1988; OIE, 2004) whilst 0.2 ml aliquots were used to inoculate cell cultures of primary
bovine thyroid (Snowdon, 1966) and IB-RS-2 cells (De Castro, 1964) (five tubes of each cell type per
specimen). Additionally, 0.2 ml of ES were added to a 1 ml aliquot of Trizol solution and stored at 80°C until assay. Real-time RT-PCR was usually performed on the ES within one to two days of
preparation, with a diagnostic result typically being obtained within a single working day.
Three hundred and eighty samples from 331 separate premises (‘confirmed’cases) in the UK, which
had been found to be negative by both ELISA and VI in 2001, and 199 samples from a further 188
separate premises, which had been found to be positive by VI but negative by ELISA on epithelial
suspension, were re-evaluated by RT-PCR. In the majority of cases, the original epithelial suspension
(which had been stored at -80oC) was used but new suspensions were prepared for others, while
aliquots of the transport buffer were examined in a few cases (in the absence of both stored
suspension and submitted epithelium).
The real-time RT-PCR assay used in this study has been described elsewhere (Reid et al., 2003).
Briefly, total nucleic acid was extracted from the solution of Trizol/ES using a fully automated robot
system. This robot was then also used to pipette viral nucleic acid into a reverse transcription mix for
reverse transcription and complementary deoxyribonucleic acid into PCR reaction mix (including an
oligonucleotide primers/probe set targeting the internal ribosomal entry site of the FMDV genome).
Thermal cycling and concurrent fluorescence detection were performed and a cycle detection
threshold (i.e. the cycle at which a target sequence is detected [CT]) was recorded for each test
sample (Oleksiewicz, Donaldson and Sorensen, 2001). Sequencing of viruses was performed as
described by Knowles and Samuel (2003).
Results:
Samples submitted to the WRL for FMD from overseas
The results achieved by ELISA, VI in cell culture and RT-PCR are summarised in Table II for
comparison of the performance of the three assays. FMDV was detected in 195 samples by VI
(58.4%), in 125 by antigen ELISA (37.4%) and in 204 samples by VI/ELISA combined (61.1%).
These viruses represented four out of the seven FMDV serotypes (O, A, SAT 2 and Asia 1), although
the majority (68.6%) were of serotype O.
There was broad agreement between RT-PCR and VI for 265/334 (79.3%) and VI/ELISA combined for
274 (82%) of the epithelial samples tested when a cut-off CT value of ‘<39’ was used to assign the
samples as either FMDV positive or negative (results not shown). In general, VI positive samples
corresponded to a real-time RT-PCR CT value of <35, with only eight VI positive samples producing a
CT value in the range ≥35-<39. Furthermore, there were no false negative results obtained with RTPCR since all samples assigned negative by RT-PCR were also negative by VI/ELISA. Of particular
interest was the finding that RT-PCR was able to detect FMDV in an additional sixty (18%) of the
samples tested. Fresh ribonucleic acid (RNA) was prepared from these epithelial suspensions and the
RT-PCR was repeated with similar results confirming the presence of FMDV genome in these samples.
In addition, there were seventeen ‘suspect positive’ samples that generated CT values in the range
≥39 to <50. Repeat procedures were also performed for these samples with similar results,
suggesting that FMDV was also present in these samples although the FMDV copy number was lower
than the diagnostic threshold. The majority (96.9%) of VI positive samples were detected on the first
passage in cell cultures (within two days). However, in order to ensure detection of infectious FMDV
262
and to define NVD samples, a second cell culture passage was required (in total, up to four days).
Indeed six samples required this second passage in cell culture before the presence of FMDV was
demonstrated.
Calculations of the relative sensitivity and specificity of the RT-PCR at unit incremental increases in CT
value in comparison to either VI alone or to VI/ELISA combined are recorded in Table III. It shows
that 100% relative sensitivity was achieved by the RT-PCR at a CT value of <39, which justifies using
this as the ideal diagnostic cut-off for the RT-PCR, since it is the lowest CT value that achieves this.
2001 UK FMD outbreak samples – ELISA/VI negative
It can be seen from Table IV that 367 samples produced a CT value of 50 (FMDV genome not detected
by RT-PCR). However, 7 sample suspensions yielded CT values of <35, and 2 in each of the ranges
>35-<40, >40-<45 and >45-50. Subsequent to these findings, attempts were made to isolate virus
from available epithelial suspensions, with success (i.e. positive type O FMDV) in 3 out 5 samples
from the first category (paucity of material precluded examination of 2 samples) and 0, 1 and 0
samples from the other categories, respectively.
2001 UK FMD outbreak samples – ELISA negative/VI positive
FMDV genome was detected in 179/199 samples by RT-PCR (for simplicity, using <40 as the CT value
cut-off point; Table V). A further 10 and 2 samples produced CT values in the range >40-<45 and
>45-<50, respectively, while the remaining 8 samples generated a CT value of 50. FMDV failed to be
isolated in 3/6, 0/2 and 4/5 of the samples which were available from each category for subsequent
cell culture passage. The resulting cell culture grown antigens were tested by RT-PCR with positive
results, except one virus (UKG 13795/2001, originating from Crickhowell, Powys, Wales), which
consistently yielded a CT value of 50 from replicate tests. This virus isolate was sequenced to see if
the analysis might indicate why this virus was undetected by RT-PCR.
It was previously known that the Pan Asia type O FMDVs, whilst detectable by the real-time RT-PCR
assay, contain a nucleotide substitution within the TaqMan® probe region (Fig.1). The causative
FMDV strain of the UKG outbreak contains an additional substitution resulting in two mis-matches
with the universal probe. The Welsh virus was found to have a further, additional nucleotide
substitution resulting in three mis-matches within the probe region. This was evidently sufficient to
make it unrecognised by the conventional diagnostic probe.
This prompted a mini-study to examine the reaction of other virus isolates from the Crickhowell
region (all of which had originally typed as positive by ELISA on epithelial suspension) in RT-PCR
procedures using either the conventional diagnostic probe or one (P282T) designed to match the
Welsh mutant virus. The results of these tests are shown in Table V1 and indicate that all these
isolates were related, failed to be detected by the RT-PCR using the conventional diagnostic probe but
were recognised by the new probe.
Discussion:
The ELISA and VI have been recommended laboratory procedures for FMD diagnosis for nearly twenty
years based on their suitability to detect the presence of FMDV antigen in tissue samples. It is evident
from the present study that these procedures are deficient for certain specimens. The results for the
overseas samples show that while all VI/ELISA positive FMDV samples were also positive by real-time
RT-PCR (100% sensitivity at a CT cut-off value of <39) (Table III), FMD viral genome was detected in
a significant proportion of the samples examined in which FMDV antigen was not.
The low relative specificity values (a range in value from 63.1%. down to 38.1% depending upon cutoff) of the RT-PCR result from the doubtful premise that the combination of VI and ELISA is 100%
efficient for detection of FMDV. The authors are confident that this is not the case, that the RT-PCR
procedure has specific reaction for FMDV genome and that the low value is actually related both to
the performance of the comparative assays and to the quality of the samples submitted.
If one considers what the VI and ELISA procedures actually measure then it is evident that their
effectiveness for diagnostic use is inherently compromised. Virus isolation is dependent upon the
presence of infectious virus in sample submissions and while the ELISA can detect both infectious and
non-infectious FMD viral antigen, it is dependent upon the antigen being present in sufficient
concentration (1-2 ng/ml of antigen or 5-6 log10/ml of live virus) to work (N.P. Ferris, unpublished
results). If neither of these two conditions is met then FMDV will not be recognised.
Ideally, vesicular epithelium should be collected from an animal during the acute stage of FMD when
the concentration of virus associated with the sample is high. Unfortunately, samples submitted to
the WRL for FMD from overseas are very often collected late in the course of disease when the
amount of virus may either be waning or indeed be absent after clearance. Delayed reporting of
263
disease and late sample collection can arise for a variety of reasons, e.g. a lack of resources,
communication difficulties in areas of rugged terrain or a low perceived importance of FMD. Delays
can be worse depending on who has responsibility for sample collection; in some countries it is local
staff and in others countries staff from a specialised laboratory (often many miles away) carry out the
collection (Ferris et al., 1992). Secondary bacterial infection is a common sequel to virus infection and
can lead to a reduction in FMDV infectivity. Additionally, samples may be in transit for lengthy periods
and subjected to physico-chemical stresses (e.g. elevated temperatures between collection and
laboratory receipt) with the result that on arrival only small amounts of infectious virus, at best, may
be present.
FMDV survival can be adversely affected by harsh environmental conditions, including excessive
temperature, extremes of pH, disinfectants and desiccation. It is therefore advantageous to protect
samples during the interval between collection and testing (especially for VI). Their dispatch to
reference laboratories should follow specific guidelines to ensure their security (Kitching and
Donaldson, 1987). These features, which influence sample quality, are likely to be less important for
the RT-PCR as it can detect a small fragment of FMDV genomic RNA, not just live virus.
There was broad correlation between the results achieved by RT-PCR examination of the UK outbreak
sample subsets with the original (2001) diagnostic results derived from ELISA/VI suggesting that
definitive sample classification of both positivity and negativity by RT-PCR is achievable within a
relatively short timescale. However, the failure to detect the group of related virus isolates from
Wales (using the routine diagnostic probe), which had evidently evolved during the epidemic and had
a further nucleotide substitution in the RT-PCR probe site, illustrates the importance of constant
monitoring of representative field FMDV strains by nucleotide sequencing to ensure that the
primers/probe set selected for the diagnostic RT-PCR is fit for purpose.
Conclusions:
•
The real-time RT-PCR currently used at the WRL for FMD provides an extremely sensitive and
rapid additional procedure for improved laboratory diagnosis of FMD
Recommendations:
•
No specific recommendations
Acknowledgements:
This work was funded by the Department for the Environment, Food and Rural Affairs, United
Kingdom (project number SE1119).
References:
De Castro. M.P. 1964. Behaviour of the foot-and-mouth disease virus in cell cultures: susceptibility
of the IB-RS-2 line. Arch. Inst. Biol., São Paulo, 31: 63-78.
Ferris, N.P. & Dawson, M. 1988. Routine application of enzyme-linked immunosorbent assay in
comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular
diseases. Vet. Microbiol., 16 (3): 201-209.
Ferris, N.P. & Doanaldson, A.I. 1992. The world reference laboratory for foot and mouth disease: a
review of thirty-three years of activity (1958-1991). Rev. Sci. Tech. Off. Int. Epiz., 11 (3): 657-684.
Ferris, N.P., Donaldson, A.I., Shrestha, R.M. & Kitching, R.P. 1992. A review of foot and mouth
disease in Nepal. Rev. Sci. Tech. Off. Int. Epiz., 11 (3): 685-698.
Kitching, R.P. & Doanldson, A.I. 1987. Collection and transportation of specimens for vesicular
virus investigation. Rev. Sci. Tech. Off. Int. Epiz., 6 (1): 263-272.
Knowles, N.J. & Samuel, A.R. 2003. Molecular epidemiology of foot-and-mouth disease virus. Virus
Res., 91: 65-80.
OIE (World Organisation for Animal Health) 2004. Manual of diagnostic tests and vaccines for
terrestrial animals. 5th Ed. Parts I and II. OIE, Paris, 1178 pp.
Oleksiewicz, M.B., Doanldson, A.I. & Alexandersen, S. 2001. Development of a novel real-time
RT-PCR assay for quantitation of foot-and-mouth disease virus in diverse porcine tissues. J. Virol.
Meth. 92 (1): 23-35.
264
Reid, S.M., Ferris, N.P., Hutchings, G.H., Zhang, Z., Belsham, G.J. & Alexandersen S. 2002.
Detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse
transcription polymerase
chain reaction assay. J. Virol. Meth., 105 (1): 67-80.
Reid, S.M., Grierson, S.S., Ferris, N.P., Hutchings, G.H. & Alexandersen, S. 2003. Evaluation of
automated RT-PCR to accelerate the laboratory diagnosis of foot-and-mouth disease virus. J. Virol.
Meth. 107 (2): 129-139.
Roeder, P.L. & Le Blanc Smith, P.M. 1987. Detection and typing of foot-and-mouth disease virus
by enzyme-linked immunosorbent assay: a sensitive, rapid and reliable technique for primary
diagnosis. Res. Vet. Sci. 43: 225-232.
Snowdon, W.A. 1966. Growth of foot-and-mouth disease virus in monolayer cultures of calf thyroid
cells. Nature, 210 (40): 1079-1080.
Table 1. Number of samples received by the WRL for FMD from overseas between 1994 and
2003 and the numbers (and percentages) found to be positive or negative for FMD virus by
passage in cell culture and antigen detection ELISA
Year
a
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
Total
number of
Countriesa
Number of samples
Total
Positive
29
655
348 (53%)
29
697
421 (60%)
31
535
238 (44%)
29
526
334 (63%)
27
441
248 (56%)
43
595
357 (60%)
29
434
209 (48%)
32
619
197 (32%)
27
390
162 (42%)
19
475
262 (55%)
5,367
2,776 (52%)
countries which submitted samples to the WRL for FMD
Negative
307 (47%)
276 (40%)
297 (56%)
192 (37%
193 (44%)
238 (40%)
225 (52%)
422 (68%)
228 (58%)
213 (45%)
2,591 (48%)
Fig. 1. Nucleotide sequence changes within the TaqMan® probe site of the Pan Asia type O
FMD virus, the causative UKG 2001 FMD virus and the related Welsh viruses compared to
the FMD virus consensus sequence.
FMD virus consensus sequence:
Pan Asia Strain:
UKG 2001 FMD virus:
Related Welsh UKG 2001 FMD virus:
TaqMan® Probe Site
5’-GGATGCCCTTCAGGTACCCCGAGG-3’
5’-GGATGCCCTTCAGGTACCCTGAGG-3’
5’-GGATGCCCTTTAGGTACCCTGAGG-3’
5’-GGATGCCCTTTAAGTACCCTGAGG-3’
Nucleotide substitutions are indicated X
265
Table II. Detection of FMD virus in suspensions of submitted epithelia achieved by ELISA,
passage in cell culture (VI), ELISA and VI combined and by RT-PCR (August 2002-January
2004)
Country
No
VIb
ELISA
FMDV
NVDa
FMDV
ELISA/VI
NVD
FMDV
Bhutan
58
2
56
17
41
Botswana
5
5
5
Burundi
7
4
3
5
2
Hong
7
1
6
3
4
Kong
Iran
43
17
26
31
12
Iraq
18
1
17
6
12
Laos
35
18
17
33
2
Lebanon
5
3
2
3
2
Libya
1
1
1
Malaysia
12
8
4
12
Nepal
4
4
4
Pakistan
90
36
54
41
49
1
1
1
PATd
Philippines
9
8
1
7
2
Thailand
7
6
1
7
Turkey
24
10
14
17
7
3
1
2
3
UAEe
Vietnam
5
5
4
1
Total
334
125
209
195
139
a
NVD, no virus detected
b
VI, serotype of FMD virus isolated by passage (VI) in
c
CT, threshold cycle value
d
PAT, Palestinian Autonomous Territories
f
UAE, United Arab Emirates
NVD
17
5
3
41
5
2
4
31
7
33
5
1
12
4
44
1
8
7
18
3
5
204
12
11
2
46
1
6
130
RT-PCR for FMD virus
(CTc value)
<35 >35 >39 >45 50
<39 <45 <50
36
4
6
12
1
4
1
4
1
1
6
1
32
11
33
5
12
4
59
1
8
7
21
3
5
244
1
1
2
1
5
1
20
2
1
6
16
1
1
1
8
5
20
3
53
cell culture characterised by ELISA
Table III. Relative sensitivity of the real-time RT-PCR for FMD virus at successive threshold
cycle value cut-off points in comparison with VI in cell culture or VI plus ELISA combined
Threshold cycle
Value cut-off
<35
<36
<37
<38
<39
<40
<41
<42
<43
<44
<45
<46
<47
<48
<49
<50
a
VIa
Sensitivity
95.9
97.0
99.0
99.5
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
Specificity
59.0
56.1
54.0
51.1
50.4
46.0
43.2
42.4
40.3
40.3
38.8
38.8
38.8
38.1
38.1
38.1
VI/ELISA combined
Sensitivity
Specificity
96.1
63.1
98.0
60.0
99.0
57.7
99.5
54.6
100.0
53.8
100.0
49.2
100.0
46.2
100.0
45.4
100.0
43.1
100.0
43.1
100.0
41.5
100.0
41.5
100.0
41.5
100.0
40.8
100.0
40.8
100.0
40.8
VI, virus isolation in cell culture with specificity of cytopathic effect confirmed by ELISA
266
Table IV. Detection of FMD virus by RT-PCR in suspensions of submitted epithelia from
clinically confirmed cases found to be ELISA/VI negative after sample receipt in 2001
Animal
No of
samples
Sheep
253
Cattle
98
Pig
14
Cattle + Sheep
5
Deer
1
Not known
9
Total
380
a
CT, threshold cycle value
<35
2
5
7
RT-PCR for FMD virus (CTa value)
>35-<40
>40-<45
>45-<50
2
2
2
2
2
2
50
245
93
14
5
1
9
367
Table V. Detection of FMD virus by RT-PCR in suspensions of submitted epithelia from
clinically confirmed cases found to be ELISA negative/VI positive after sample receipt in
2001
Animal
No of
samples
Sheep
131
Cattle
60
Cattle+Sheep
2
Goat
3
Not known
3
Total
199
a
CT, threshold cycle value
<35
87
41
2
1
2
133
RT-PCR for FMD virus (CTa value)
>35-<40
>40-<45
>45-<50
33
6
1
12
3
1
1
1
46
10
2
50
4
4
8
Table VI. Detection of ‘mutant’ FMD virus by RT-PCR in suspensions of submitted epithelia
from clinically confirmed cases in Powys, Wales found to be positive after sample receipt in
2001
Sample ref
Animal
Origin
FMD
No.
UKG 13795/2001e
Sheep
Crickhowell, Powys
1861
RT-PCR for FMD
virus (CTb value)
P269Rc P282Td
50
35.81
UKG 13705/2001
Cattle
Crickhowell, Powys
1846
50
32.35
UKG 13708/2001
Cattle
Crickhowell, Powys
1848
50
31.52
UKG 13724/2001
Cattle
Powys
1852
50
29.75
UKG 13734/2001
Cattle
Powys
1857
50
27.6
UKG 13777/2001
Cattle
Powys
1860
50
40.91
Crickhowell, Powys
1868
50
31.06
UKG 13798/2001
NKf
UKG 13949/2001
Cattle
Abergavenny, Powys
1889
50
32.85
UKG 14004/2001
Cattle
Crickhowell, Powys
1896
50
31.46
UKG 14221/2001
Cattle
Abergavenny, Powys
1938
50
27.59
UKG 14339/2001
Cattle
Crickhowell, Powys
1945
50
31.08
a
OD, optical density value originally achieved on the epithelial suspension
b
CT, threshold cycle value
c
P269R, the normal diagnostic probe
d
P282T, ‘UK’ probe
e
UKG 13795/2001 serotyped on first passage cell culture antigen, all other virus isolates listed
serotyped on epithelial suspension
f
NK, not known
267