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Bluetongue virus: European Community
inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods
C.A. Batten, K. Bachanek-Bankowska, A. Bin-Tarif, L. Kgosana, A.J. Swain, M. Corteyn, K. Darpel, P.S. Mellor, H.G. Elliott, C.A.L. Oura
To cite this version:
C.A. Batten, K. Bachanek-Bankowska, A. Bin-Tarif, L. Kgosana, A.J. Swain, et al.. Bluetongue virus:
European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods. Veterinary Microbiology, Elsevier, 2008, 129 (1-2), pp.80. �10.1016/j.vetmic.2007.11.005�.
�hal-00532356�
Accepted Manuscript
Title: Bluetongue virus: European Community
inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods
Authors: C.A. Batten, K. Bachanek-Bankowska, A. Bin-Tarif, L. Kgosana, A.J. Swain, M. Corteyn, K. Darpel, P.S. Mellor, H.G. Elliott, C.A.L. Oura
PII: S0378-1135(07)00546-9
DOI: doi:10.1016/j.vetmic.2007.11.005
Reference: VETMIC 3882
To appear in: VETMIC Received date: 14-8-2007 Revised date: 2-11-2007 Accepted date: 6-11-2007
Please cite this article as: Batten, C.A., Bachanek-Bankowska, K., Bin-Tarif, A., Kgosana, L., Swain, A.J., Corteyn, M., Darpel, K., Mellor, P.S., Elliott, H.G., Oura, C.A.L., Bluetongue virus: European Community inter-laboratory comparison tests to evaluate ELISA and RT-PCR detection methods,Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.11.005
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Accepted Manuscript
Bluetongue virus: European Community inter-laboratory comparison tests to 1
evaluate ELISA and RT-PCR detection methods.
2
Batten, C.A.1, Bachanek-Bankowska, K.1, Bin-Tarif, A.1, Kgosana, L.2, Swain, A.J.1, 3
Corteyn, M.1, Darpel, K.1, Mellor, P.S1, Elliott, H.G.3 and Oura, C.A.L1. 4
1Institute for Animal Health, Ash Road, Pirbright, Woking, GU24 ONF, UK.
5
2 Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK.
6
3Department of the Environment, Food and Rural Affairs (DEFRA), 1A Page Street, 7
London, SW1P 4PQ.
8 9
Corresponding Author:
10
Dr Carrie A Batten, 11
Institute for Animal Health, 12
Ash road, 13
Pirbright, 14
Woking, 15
GU24 0NF, 16
UK.
17
Tel: +44 (0) 1483 231146 18
Fax : +44 (0) 1483 235745 19
Email: carrie.batten@bbsrc.ac.uk 20
21 22 23 24 25 Manuscript
Accepted Manuscript
Abstract:
26
European Community national reference laboratories participated in two inter- 27
laboratory comparison tests in 2006 to evaluate the sensitivity and specificity of their 28
‘in-house’ ELISA and RT-PCR assays for the detection of bluetongue virus (BTV) 29
antibodies and RNA. The first ring trial determined the ability of laboratories to detect 30
antibodies to all 24 serotypes of BTV. The second ring trial, which included both 31
antisera and EDTA blood samples from animals experimentally infected with the 32
northern European strain of BTV-8, determined the ability of laboratories to detect 33
BTV-8 antibodies and RNA, as well as the diagnostic sensitivity of the assays. A total 34
of six C-ELISAs, six real-time RT-PCR and three conventional RT-PCR assays were 35
used. All C-ELISAs were capable of detecting the BTV serotypes currently 36
circulating in Europe (BTV-1, 2, 4, 8, 9 and 16), however some assays displayed 37
inconsistencies in the detection of other serotypes, particularly BTV-19. All C- 38
ELISAs detected BTV-8 antibodies in cattle and sheep by 21 dpi, while the majority 39
of assays detected antibodies by 9 dpi in cattle and 8 dpi in sheep. All the RT-PCR 40
assays were able to detect BTV-8, although the real-time assays were more sensitive 41
compared to the conventional assays. The majority of real-time RT-PCR assays 42
detected BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. These two ring trails 43
provide evidence that national reference laboratories within the EC are capable of 44
detecting BTV antibodies and RNA and provide specificity and sensitivity 45
information on the detection methods currently available.
46 47
Key Words: Bluetongue Virus; Diagnosis; RT-PCR; ELISA; Ring Trial 48
49 50
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1. Introduction 51
Bluetongue virus (BTV) is the prototype member of the genus Orbivirus within the 52
family Reoviridae. The bluetongue (BT) virus particle is approximately 70nm in 53
diameter comprising a dsRNA segmented genome. The ten dsRNA segments encode 54
five structural proteins (VP1-VP5) and three non structural proteins (NS1-NS3). The 55
genome is contained within a core particle consisting primarily of VP3 and VP7, 56
which is in turn encased in an outer capsid consisting of VP2 and VP5. VP2 is 57
responsible for attachment of BTV to mammalian cells and contains epitopes 58
responsible for haemagglutination, virus neutralisation and serotype specificity.
59
BTV is transmitted by Culicoides biting midges. The distribution of BTV is 60
restricted to areas where competent Culicoides vectors are present and where climate 61
conditions are favourable for adult vector activity (Mellor and Boorman, 1995). The 62
disease has a global distribution of 35˚S and 40˚N, although in parts of North America 63
and China it has been reported as far as 50˚N (Dulac et al., 1988; Kirkland et al., 64
2002). BTV disease (i.e. BT) is non contagious and is considered able to infect all 65
species of ruminant, although clinical disease is limited to certain breeds of sheep, 66
particularly the European fine wool and mutton breeds and some species of deer (e.g.
67
white-tailed). Clinical signs include fever, hyperaemia, coronitis, oedema, erosions 68
and ulcerations of the dermis. In severe cases death may occur in 8-10 days.
69
To date, 24 serotypes of BTV (BTV-1 to BTV-24) have been identified by 70
serum neutralisation assays (SNT) (Mellor and Boorman, 1995; Mertens and Diprose, 71
2004; Roy, 1992). The distribution and prevalence of these serotypes differs within 72
each endemic area. From 1998 to 2005, five serotypes of BTV (1, 2, 4, 9 and 16) have 73
been circulating in the Mediterranean Basin including Turkey, Greece, Italy, Tunisia, 74
Algeria, Sardinia, Corsica and Sicily. This is the largest BTV outbreak on record and 75
Accepted Manuscript
has resulted in the loss of >1.8 million animals (Mellor and Boorman, 1995; Mellor 76
and Wittmann, 2002; Tatem, 2003).
77
In August 2006 clinical cases of BT were suspected in the Netherlands. The 78
CIDC – Lelystad identified the presence of BTV RNA in samples from suspect cases 79
by RT-PCR targeting genome segment 10, and detected BTV specific antibodies in 80
serum samples by competitive ELISA (C-ELISA). These findings were confirmed by 81
the Community Reference Laboratory (CRL) at Pirbright, UK, who went on to 82
confirm that the outbreak was caused by BTV serotype 8 (BTV-8), a serotype that had 83
never before been observed in Europe. By the end of 2006 BTV had spread and 84
infected ruminants in a wide area across northern Europe including the Netherlands, 85
Belgium, Germany, northern France and Luxembourg. In 2007, BTV-8 has continued 86
to spread in these countries and has also infected ruminants in the United Kingdom, 87
Denmark and Switzerland.
88
Traditionally laboratory diagnosis of BTV has relied upon the detection of 89
BTV antibodies by agar gel immunodiffusion or ELISA and the detection of BTV 90
antigen by virus isolation in embryonated eggs followed by cell culture and virus 91
neutralisation tests (VNT). Recently new molecular diagnostic tests based on different 92
segments of the genome have been developed (Anthony, 2007; Katz, 1993; Monaco et 93
al., 2006; Orru, 2004; Polci, 2007; Shaw. A.E., 2007; Toussaint, 2007; Wilson, 2004;
94
Zientara, 2004).
95
The CRL for BT is responsible for sending out inter-laboratory comparison 96
tests (ring trials) to compare diagnostic methods for BTV. In June 2006, prior to the 97
BTV-8 outbreak in northern Europe, the CRL sent out a ring trial in order to 98
investigate the ability of laboratories to detect all 24 serotypes of BTV by ELISA.
99
When BTV-8 was confirmed in the Netherlands in August 2006 the majority of 100
Accepted Manuscript
diagnostic labs across northern Europe were not prepared for an outbreak of BTV.
101
Many commercial kits for the detection of BTV antibodies were on the market, but 102
validation data was limited. Also, in August 2006, very few real-time RT-PCR assays 103
had been published and many labs in northern Europe were using unpublished PCR 104
assays for BTV antigen detection. Thus the European Community (EC) instructed the 105
CRL to send out another ring trial in September 2006 in order to determine the ability 106
of reference laboratories across Europe to detect the BTV-8 strain circulating in 107
northern Europe in both blood and serum samples. This paper reports the findings of 108
both of these studies.
109 110
2. Materials and Methods 111
2.1 Samples 112
2.1.1 Ring trial 1 113
Reference antisera to the 24 BTV serotypes raised in sheep were sent to all national 114
BT reference laboratories in the EC. Serially diluted samples of BTV-16 antiserum 115
were prepared in normal sheep serum (Sigma, UK). Four samples diluted 1/25, 1/50, 116
1/100 and 1/200 were prepared to give an indication of test sensitivity. In addition 117
three BTV negative antisera (sheep, goat and cattle) and five positive antisera from a 118
related orbivirus, Epizootic Haemorrhagic Disease virus (EHDV-1, 2, 5, 7 and 318) 119
were prepared to test specificity. In total thirty-six randomly coded samples were 120
dispatched to each of twenty-nine participating EC national reference laboratories.
121 122
2.1.2 Ring trail 2 123
Four sheep and four cattle were infected with the northern European BTV-8 strain in 124
animal facilities at IAH, Pirbright (Darpel, 2007). Serum and blood samples were 125
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collected at intervals throughout the 35 day experiment. Blood samples were analysed 126
by RT-PCR and serum samples by C-ELISA within the CRL. Selections of negative 127
and positive samples were chosen to measure the diagnostic sensitivity of the test 128
(Table 1). In total 12 randomly coded blood samples and eleven serum samples were 129
dispatched to each of 23 participating EC national BT reference laboratories.
130 131
2.2 C-ELISA 132
The presence of BTV antibodies in the serum samples supplied in ring trails 1 and 2 133
were assessed with the ‘in-house’ ELISA being used for routine BT diagnosis in the 134
participating laboratories. A total of six different commercially available ELISAs 135
were used (Table 2).
136 137
2.3 RT-PCR 138
The presence of BTV RNA in the 12 blood samples (Table 1) supplied in the ring trial 139
was assessed using the ‘in-house’ RT-PCR assays being used for routine BT diagnosis 140
in the participating laboratories. Seven different real-time RT-PCR assays and three 141
conventional gel-based RT-PCR assays were used between the laboratories. In some 142
of the laboratories the samples were tested by more than one assay.
143 144
3. Results 145
3.1 Ring trial 1 146
In the 23 BT national reference laboratories participating in the trial a total of six 147
commercial ELISAs were used to detect BTV antibodies (Table 2). The results 148
(summarised in Table 3) illustrate that the BDSL C-ELISA and VMRD ELISA were 149
able to detect antibodies to all 24 BTV serotypes; these results were consistent in 150
Accepted Manuscript
eight laboratories. Only one laboratory used the Pourquier and the ID-Vet ELISA and 151
both these kits detected 23 out of the 24 serotypes failing to detect antiserum against 152
BTV-19. However, when the ring trial samples were tested in the CRL the Pourquier 153
and ID-Vet ELISAs detected antibodies to all 24 serotypes including BTV-19. Seven 154
laboratories used the Ingenasa BTV ELISA and inconsistencies were seen in detecting 155
antibodies against BTV serotypes 3, 7, 10, 11, 12, 19 and 20. Two laboratories used 156
the IZS ELISA which failed to detect BTV serotype 7 and 19, and produced 157
inconclusive results with BTV serotypes 4, 9, 10, 12 and 15. All six ELISAs were 158
specific for BTV with only two laboratories reporting cross-reactions with EHDV 159
serotype 2 with the BDSL ELISA and one laboratory reported cross-reactivity to 160
EHDV serotype 1 using the VMRD ELISA. All the ELISAs could detect a 1/25 161
dilution of BTV-16 serum, with the BDSL kit having the highest sensitivity level as 162
analysed by detecting antibodies in BTV-16 antiserum diluted 1/200 (data not shown).
163 164
3.2 Ring trial 2 165
3.2.1 ELISA 166
In the 19 BT national reference laboratories participating in the trial a total of six 167
commercial ELISAs were used to detect BTV antibodies (Table 2). The six ELISAs 168
exhibited varied sensitivities when tested on the ring trial samples (Table 4). The 169
BDSL ELISA detected antibodies in samples from 6 and 7 dpi in sheep and cattle 170
respectively. The Pourquier, ID-Vet and VMRD ELISAs detected BTV antibodies by 171
8 dpi in sheep and 9 dpi in cattle. Inconclusive results were recorded at 7 dpi in sheep 172
indicating this is the limit of detection of the assays. The limit of detection for BTV 173
antibodies using the IZS and Ingenasa ELISA appeared to be at 8 dpi in sheep 174
Accepted Manuscript
however only one and three laboratories respectively used these assays in the trial. All 175
of the ELISAs detected BTV antibodies by 21 dpi in cattle and sheep (Table 4).
176 177
3.2.2 RT-PCR 178
3.2.2.1 Real-time RT-PCR assays 179
The results of the real-time RT-PCR assays are outlined in Table 5. All of the RT- 180
PCR assays detected BTV-8; however the sensitivity of the assays, measured by their 181
ability to detect BTV RNA in samples collected at different time-points post- 182
infection, varied. Three of the published assays (Shaw et al., 2007, Toussaint et al., 183
2007, Polci et al., 2007) exhibited extremely high sensitivity detecting BTV RNA as 184
early as 2 dpi in cattle and 3 dpi in sheep. The unpublished assays developed in 185
Holland and Germany showed similar high levels of sensitivity. The real-time RT- 186
PCR assay developed by Jimenez-Clavero et al (2006) was less sensitive first 187
detecting BTV RNA at 5 dpi in sheep. This assay did not detect BTV RNA at later 188
time-points of infection when the alternative assays remained positive (Table 5).
189 190
3.2.2.2 Conventional gel-based RT-PCR assays 191
The results of the conventional RT-PCR assays are shown in Table 6. All assays were 192
capable of detecting BTV-8 however there was some intra-laboratory variation in 193
sensitivity, especially when the assay developed by Anthony et al (2007) was used. Of 194
the five laboratories that used this assay variable results were reported. In one lab the 195
sensitivity of the assay was comparable with the best real-time RT-PCR assays 196
detecting BTV RNA as early as 2 dpi in cattle and 3 dpi in sheep. However other labs 197
only detected BTV RNA at 5 dpi in sheep and were unable to detect BTV RNA at 198
later stages of infection. The nested RT-PCR assay recommended in the OIE manual 199
Accepted Manuscript
((OIE), 2004) showed comparable sensitivity to the most sensitive real-time RT-PCR 200
assays.
201 202
4. Discussion 203
This paper describes the first two ring trials carried out amongst EC BT national 204
reference laboratories to test the ability of ‘in-house’ RT-PCR and ELISA assays to 205
detect both BTV RNA and antibodies. In June 2006 the first ring trial was dispatched 206
and the results indicated that the EC national BT reference laboratories were capable 207
of detecting antibodies against BTV serotypes circulating in Europe. The unexpected 208
outbreak of BT in northern Europe in the summer of 2006 made it essential that 209
effective diagnostic assays were in place in affected countries. It became vital that the 210
affected country, neighbouring countries and the EC were confident in the ability of 211
the national reference laboratory to detect BTV in affected animals. The unexpected 212
outbreak of BTV-8, a serotype that had never previously been active in Europe, 213
emphasised the importance of laboratories having in place assays that could detect not 214
only BTV serotypes present in Europe, but also serotypes that are circulating further 215
afield. Although the first ring trial indicated that the laboratories were capable of 216
detecting antibodies to BTV-8, a second ring trial, was set up as a direct result of the 217
BTV-8 outbreak in northern Europe, aimed to assess the diagnostic capabilities of EC 218
national BT reference laboratories to detect BTV-8 in sera and blood samples 219
collected directly from BTV-8 infected cattle and sheep.
220
For many years the detection of BTV specific antibodies has been carried out 221
using a C-ELISA (Afshar et al., 1992; Afshar et al., 1989; Afshar et al., 1987b) or a 222
blocking ELISA (Afshar et al., 1987a; Anderson, 1984). However, in the last few 223
years a number of commercially available BT antibody detection ELISAs have 224
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become available. These commercial assays are based on recombinant technology and 225
are very quick and easy to use. The ring trials described here were set up to determine 226
whether these commercial assays are able to detect antibodies to all 24 BTV serotypes 227
and whether they are capable of detecting BTV-8 antibodies in field samples. A total 228
of six commercially available ELISAs were used by the participating laboratories in 229
the ring trials. In the first ring trial, which was sent out before the northern European 230
BT outbreak, the majority of laboratories were routinely using the BDSL, the 231
Ingenasa and the VMRD ELISAs, however by the second ring trial many labs had 232
swapped to using the ID-Vet and Pourquier ELISAs. The reason for the reduced use 233
of the BDSL ELISA was that, due to the BTV-8 outbreak and the necessity for high 234
throughput testing, laboratories needed fast, robust assays that were easy to perform.
235
The participating laboratories found the BDSL ELISA laborious to perform (taking up 236
to four hours to complete) and lacking in robustness (some plates failed on control 237
parameters). In contrast the ID-Vet and Pourquier ELISAs were rapid to perform 238
(taking one hour to complete), readily available in Europe and plates consistently 239
passed on control parameters.
240
Results revealed some inconsistencies between kits in their ability to detect 241
antibodies to the 24 serotypes, however all six ELISAs were capable of detecting 242
antibodies to the serotypes currently circulating in Europe. Since the ring trial 243
Ingenasa has developed a new ELISA for the detection of BTV antibodies (INGEZIM 244
BTV DR 12.BTV.K0). When the panel of samples from the first ring trial were tested 245
by the CRL this assay detected antibodies to all 24 BTV serotypes.
246
The second ring trial investigated the diagnostic sensitivity of ‘in-house’ RT- 247
PCR and ELISAs in EC national BT reference laboratories. Relevant blood and serum 248
samples from experimentally infected cattle and sheep were incorporated in the trial 249
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in order to measure the limit of detection of the assays. The BDSL ELISA kit was 250
shown to be the most sensitive detecting BTV antibodies in sheep at 6 dpi and cattle 251
at 7 dpi. The Pourquier, ID-Vet and VMRD ELISA first detected BTV antibodies a 252
day later in both sheep and cattle. The sensitivity of the IZS and Ingenasa ELISAs 253
were slightly lower, however, few laboratories used these assays in the trial. When the 254
panel of sera from the second ring trial was tested by the CRL using the new 255
INGEZIM BTV DR 12.BTV.K0 ELISA for BTV antibody detection (Ingenasa), the 256
assay showed very good diagnostic sensitivity being able to detect antibodies in sheep 257
and cattle at 6 and 7 dpi in line with the BDSL ELISA. As expected all of the ELISAs 258
were capable of detecting BTV antibodies by 21 dpi in cattle and sheep. It is 259
important to note that the majority of the samples in the second ring trial were taken 260
from animals that had been experimentally infected with BTV-8 and that antibody 261
levels in cattle and sheep at these time points post-infection in the field may be 262
different.
263
EDTA blood samples from animals experimentally infected with BTV-8 were 264
included in ring trial 2 to determine the ability of EC Reference laboratories to detect 265
BTV RNA using in house RT-PCR assays. Fourteen laboratories used a total of six 266
different real-time RT-PCR assays. All six assays were capable of detecting BTV-8 267
RNA however they varied in their level of sensitivity. The real-time RT-PCR assays 268
developed in the UK, France, Belgium, Holland, Germany and Italy all proved 269
extremely sensitive detecting BTV RNA at 3 dpi in sheep. Some intra-laboratory 270
variation was seen at the limit of detection at 2 dpi in cattle, however all laboratories 271
detected BTV RNS by 5 dpi. The assay developed by Jimenez-Clavero et al (2006) 272
was found to have a reduced sensitivity, although it was able to detect BTV RNA at 273
the peak of infection at a time when the animals were likely to exhibit clinical signs of 274
Accepted Manuscript
disease. This assay however did not detect BTV RNA at later time-points of infection 275
when the alternative assays remained positive. The primers and probe in this assay 276
were positioned in a part of segment 5 of the BTV genome that had various 277
mismatches which reduced the sensitivity of the assay.
278
Ten laboratories tested the EDTA blood samples using conventional gel-based 279
RT-PCR assays. Three different protocols were used which included one nested and 280
two conventional procedures. There was considerable intra-laboratory variation in the 281
results reported from the five laboratories that used the RT-PCR assay developed by 282
Anthony et al (2007). These inconsistent results were most likely due to the fact that 283
different RNA extraction methods were used between the laboratories. In contrast the 284
four laboratories that used the OIE nested RT-PCR method found this assay to be as 285
sensitive as the real-time RT-PCR assays detecting viral RNA at 3 dpi in sheep and 286
with a limit of detection at 2 dpi in cattle. The PCR carried out with the nested OIE 287
primers proved very sensitive, however there are many drawbacks associated with 288
using conventional gel-based RT-PCR assays as opposed to real-time assays to 289
process multiple samples in an outbreak. The use of real-time RT-PCR avoids the 290
time consuming gel electrophoresis step making it faster to perform than conventional 291
RT-PCR assays, also real-time assays are easily adapted to a 96-well format, making 292
them well suited to high through-put diagnostic systems and process automation.
293
Additionally real-time assays amplify the target cDNA within a ‘closed-tube’ format, 294
significantly reducing risks of cross-contamination and false positives.
295
Regular proficiency tests are important to establish confidence in the 296
reliability of laboratory diagnostic procedures. In future ring trials we plan to address 297
both how sensitive the commercial ELISAs are in the detection of antibodies to the 24 298
Accepted Manuscript
serotypes and also the ability of national reference laboratories to detect BTV strains 299
currently circulating in Europe by RT-PCR.
300 301
Acknowledgements 302
The authors would like to thank Eugene van Rooij (CIDC-Lelystad) and Bernd 303
Hoffmann (FLI-Riems) for supplying blood samples from BTV infected animals.
304
They thank Don King, Scott Reid and Andrew Shaw for their help and advice with the 305
diagnostic assays. The authors are very grateful to Eva Veronesi, Malcolm Turner, 306
Barry Collins and Luca Colmari for their help with the animal experiments.
307 308
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samples. Vet Italia 43, 77-87.
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372
Accepted Manuscript
Shaw. A.E., M., P., Alpar, H.O., Anthony, S., Darpel, K.E., Batten, C.A., Carpenter, 373
S., Jones, H., Oura, C.A.L., King, D.P., Elliott, H., Mellor, P.S. and Mertens, 374
P.P.C., 2007, Development and validation of a real-time RT-PCR assay to 375
detect genome bluetongue virus segment 1. Journal of virological methods.
376
Tatem, A.J., Baylis, M., Mellor, P.S., Purse, B.V., Capela, R., Pena, I. & Rogers, D.J.
377
, 2003, Prediction of bluetongue vector distribution in Europe and North 378
Africa using satellite imagery. . Veterinary Microbiology 97, 13-29.
379
Toussaint, J.F., Sailleau, C., Breard, E., Zientara, S., De Clercq, K., 2007, Bluetongue 380
virus detection by two real-time RT-qPCRs targeting two different genomic 381
segments. Journal of virological methods 140, 115-123.
382
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polymerase chain reaction detection of bluetongue and epizootic 384
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385
Zientara, S., Breard, E., Sailleau, C., 2004, Bluetongue diagnosis by reverse 386
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387
Accepted Manuscript
Table 1: Blood and serum samples sent to the European Community Bluetongue 1
national reference laboratories as part of ring trial 2.
2
3
dpi – days post infection 4
5
Ring trial 2
Blood Samples Serum Samples
Cattle Uninfected Sheep Uninfected
Sheep Uninfected Cattle Uninfected
Cattle 2 dpi Sheep 6 dpi
Sheep 3 dpi Cattle 7 dpi
Cattle 4/5 dpi Sheep 7 dpi
Sheep 5 dpi Cattle 9 dpi
Cattle 10 dpi Sheep 8 dpi
Sheep 8 dpi Cattle 21 dpi
Cattle 27 dpi Sheep 21 dpi
Sheep 27 dpi Cattle: Netherlands field sample Cattle: Netherlands field sample Sheep: German Field sample
Sheep: German Field sample Table 1
Accepted Manuscript
Table 2: Availability of commercial C-ELISAs for the detection of BTV antibodies.
Commercially available BTV ELISA for detection of BTV antibodies
Assay Available from
BDSL www.BDSL2000.com
VMRD www.vmrd.com
ID –Vet www.id-vet.com
Pourquier www.institut-pourquier.fr
Ingenasa www.ingenasa.es
IZS www.izs.it
Table 2
Accepted Manuscript
Table 3: Comparison of C-ELISA results for ring trial 1, generated from 23 participating laboratories.
1
Assay BDSL INGENASA* VMRD ID VET IZS Pourquier
No: Labs 8 7 8 1 2 1
P N Inc P N inc P N Inc P N inc P N inc P N inc
BTV1 8 7 8 1 2 1
BTV2 8 7 8 1 2 1
BTV3 7 1 1 6 8 1 2 1
BTV4 8 7 8 1 1 1 1
BTV5 8 7 8 1 2 1
BTV6 8 7 8 1 2 1
BTV7 7 1 2 2 3 5 2 1 1 1 1 1
BTV8 8 7 8 1 2 1
BTV9 8 6 1 8 1 2 1
BTV10 7 1 2 5 8 1 2 1
BTV11 7 1 4 2 1 8 1 2 1
BTV12 7 1 4 2 1 8 1 2 1
BTV13 8 7 8 1 2 1
BTV14 8 7 8 1 2 1
BTV15 8 7 8 1 2 1
BTV16 8 6 1 8 1 1 1 1
BTV17 8 7 8 1 2 1
BTV18 8 7 8 1 2 1
BTV19 7 1 7 7 1 1 1 1 1
BTV20 8 5 2 8 1 2 1
BTV21 8 7 8 1 2 1
BTV22 8 7 8 1 2 1
BTV23 8 7 8 1 2 1
BTV24 8 7 8 1 2 1
* Ingenasa has recently released a new BTV antibody detection ELISA (INGEZIM BTV DR 12.BTV.K0). This assay has been shown by 2 the CRL to detect antibodies to all 24 BTV serotypes
3 Table 3
Accepted Manuscript
Table 4: Comparison of C-ELISA results for ring trial 2, generated from 19 participating laboratories.
1 2
Assay BDSL INGENASA* VMRD ID VET IZS Pourquier
No: Labs 5 3 6 10 1 8
P N inc P N inc P N inc P N inc P N inc P N inc
Sheep Day 0 5 3 6 10 1 8
Cow Day 0 5 3 6 10 1 8
Sheep 6dpi 5 3 6 10 1 7 1
Cow 7dpi 5 3 6 10 1 8
Sheep 7dpi 5 3 3 2 1 5 4 4 1 6 2
Cow 9dpi 5 3 2 3 1 1 8 1 1 1 6 1
Sheep 8dpi 5 2 1 6 10 1 8
Cow 21dpi 5 3 6 10 1 8
Sheep 21dpi 5 3 6 10 1 8
Cow Holland 5 3 6 10 1 8
Sheep Germany 5 3 6 10 1 8
3
dpi – days post infection; P – Positive; N – Negative; inc – inconclusive 4
5
* Ingenasa has recently released a new BTV antibody detection ELISA (INGEZIM BTV DR 12.BTV.K0). This assay has been shown by 6
the CRL to show similar sensitivity to the BDSL ELISA.
7 8 Table 4
Accepted Manuscript
Table 5: Comparison of real-time RT-PCR results generated from 14 participating laboratories.
1
Real-time RT-PCR from different labs vs BTV-8 Cow
neg
Sheep neg
Cow 2 dpi
Sheep 3 dpi
Cow 5 dpi
Sheep 5 dpi
Cow 10 dpi
Sheep 8 dpi
Cow 27 dpi
Sheep 27 dpi
Cow NL
Sheep D
(Shaw. A.E., 2007) N N P P P P P P P P P P
(Shaw. A.E., 2007) N N P P P P P P P P P P
(Shaw. A.E., 2007) N N N P P P P P P P P P
(Shaw. A.E., 2007) N N P P P P P P P P P P
Holland
(unpublished) N N N P P P P P P P P P
Holland
(unpublished) N N inc P P P P P P P P P
Holland
(unpublished) N N P P P P P P P P P P
Germany
(unpublished) N N inc P P P P P P P P P
Germany (unpublished)
N N P P P P P P P P P P
(Toussaint, 2007) N N P P P P P P P P P P
(Toussaint, 2007) N N inc P P P P P P P P P
(Polci, 2007) N N P P P P P P P P P P
(Jimenez-Clavero
et al., 2006) N N N N N P P P P N P N
(Jimenez-Clavero
et al., 2006) N N N N N P P P N N N N
dpi – days post infection; P – Positive; N – Negative; inc – inconclusive 2
Table 5
Accepted Manuscript
Table 6: Comparison of gel-based RT-PCR results generated from nine participating 1
laboratories.
2
Gel-based RT-PCR from different labs vs BTV-8 Cow
neg Sheep neg Cow
2 dpi Sheep 3 dpi Cow
5 dpi Sheep 5 dpi Cow
10 dpiSheep 8 dpi Cow
27 dpiSheep 27 dpi Cow
NL Sheep D (Anthony,
2007)
N N N P P P P P P N P P
(Anthony,
2007) N N P P P P P P P P P P
(Anthony, 2007)
N N N N N P P P N N N P
(Anthony,
2007) N N N N N P P P P N P P
(Anthony, 2007)
N N P N P P P P P inc P P
((OIE), 2004)
single N N N N P P P P P N P P
((OIE), 2004)
nested N N P P P P P P P P P P
((OIE), 2004)
nested N N inc P P P P P P P P P
((OIE), 2004)
nested N N N P P P P P P P P P
((OIE), 2004) nested
N N P P P P P P P P P P
dpi – days post infection; P – Positive; N – Negative; inc – inconclusive 3
4 Table 6
