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TRANSCRIPT
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Development of a competitive ELISA for the detection of antibodies against the 3B 1
protein of foot-and-mouth disease virus 2
3
Ming Yang,a Satya Parida,
b Tim Salo,
a Kate Hole,
a Lauro Velazquez-Salinas,
c, * Alfonso 4
Clavijoa,#,
† 5
6
National Centre for Foreign Animal Disease, Winnipeg, Manitoba, Canadaa ; The 7
Pirbright Institute, Ash Road, Pirbright, Surrey, UK b; Comision Mexico-Estados Unidos 8
para la Prevencion de la Fiebre aftosa y otras Enfermedades Exoticas de los Animales, 9
Cuajimalpa, Mexico c; 10
11
12
Running Head: Detection of antibody against 3B protein of foot-and-mouth disease virus 13
14
#Address correspondence to Alfonso Clavijo, [email protected] 15
16
* Present address: Foreign Animal Disease Research Unit, Agricultural Research Service, 17
U.S Department of Agriculture, Plum Island Animal Disease Center, Orient Point, NY, 18
USA. 19
†Present address: Institute for Infectious Animal Diseases, College Station, TX, USA 20
21
This is a contribution from the National Centre for Foreign Animal Disease, Canada 22
23
CVI Accepted Manuscript Posted Online 4 February 2015Clin. Vaccine Immunol. doi:10.1128/CVI.00594-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract 24
Foot-and-mouth disease (FMD) is one of the most highly contagious and economically 25
devastating diseases, that severely constrains the international trade of animals. 26
Vaccination against FMD is a key element in the control of FMD. However, vaccination 27
of susceptible animals raises critical issues, such as the differentiation of infected animals 28
from vaccinated animals. The current study developed a reliable and rapid test to detect 29
antibodies against the conserved, non-structural proteins (NSP) of the FMD virus 30
(FMDV) to distinguish infected animals from vaccinated animals. A monoclonal 31
antibody (mAb) against the FMDV NSP 3B was produced. A competitive enzyme-linked 32
immunosorbent assay (cELISA) for FMDV/NSP antibody detection was developed using 33
a recombinant 3ABC protein as the antigen and the 3B-specific mAb. Sera collected from 34
naïve, FMDV experimentally infected, vaccinated carrier and non-carrier animals were 35
tested using the 3B cELISA. The diagnostic specificity was 99.4% for naïve animals 36
(cattle, porcine and sheep) and 99.7% for vaccinated non-carrier animals. The diagnostic 37
sensitivity was 100% for experimentally inoculated animals and 64% for vaccinated 38
carrier animals. The performance of this 3B cELISA was compared to four commercial 39
ELISA kits using a serum panel established by the World Reference Laboratory for FMD 40
at Pirbright. The diagnostic sensitivity of the 3B cELISA for the panel of FMDV/NSP 41
positive bovine serum was 94%, which was comparable or better than commercially 42
available NSP antibody detection kits. This 3B cELISA is a simple, reliable test to detect 43
antibodies against FMDV non-structural proteins. 44
45
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Introduction 47
Foot-and-mouth disease (FMD) is one of the most highly contagious and economically 48
devastating diseases of cloven-hoofed animals and it severely constrains the international 49
trade of animals and animal products. Vaccination against FMD, in addition to the 50
slaughter and movement restrictions of infected animals, is a key element in the control 51
of FMD. However, countries that vaccinate in the event of an outbreak must re-establish 52
their FMD-free status to the satisfaction of their trading partners (1, 2). Vaccination of 53
susceptible animals raises critical issues, such as the differentiation of infected animals 54
from vaccinated animals and the development of carrier status because of subclinical 55
infection in vaccinated animals. 56
FMD is caused by the FMD virus (FMDV), which is a member of the genus 57
Aphthovirus and the family Picornaviridae (3) and it exhibits seven serotypes, O, A, Asia 58
1, C, SAT 1, SAT 2 and SAT 3. FMDV has a positive sense, single-stranded ribonucleic 59
acid (RNA) genome of 8,400 nucleotides that code for twelve proteins. Four structural 60
proteins (VP1, VP2, VP3 and VP4) compose the viral capsid, and eight proteins are non-61
structural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C and 3D). All 12 proteins allow the virus to 62
replicate in infected cells.(4-6). Antibodies to the 3ABC non-structural proteins (NSPs) 63
are a reliable indicator of infection regardless of the FMDV serotype. ELISAs for the 64
detection of antibodies against NSPs are widely used to differentiate between vaccinated 65
and infected animals because purified vaccines are free of NSPs, thus elicit antibodies 66
against only structural proteins (7). However, not all manufactures produce purified FMD 67
vaccines, and the degree of purity among FMD vaccine manufacturers is not always 68
identical (8). 69
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Several tests for the detection of antibodies against NSPs were reported, and some of 70
these tests were made into commercially available kits. The tests produced by Svanova, 71
Bommeli, and UBI and several other published tests (9-15) are not ideal, because these 72
tests require species-specific conjugated antibodies. Separate assays are required to test 73
samples from different species (cattle, deer, goats, and sheep) and no reagents are 74
available for wildlife (2, 16). A wide range of animal species are susceptible to FMDV. 75
Therefore, a competitive ELISA (cELISA) would be advantageous because serum 76
samples from different species could be tested without changing reagents (17). cELISAs 77
are simple, easy to perform and species independent. Numerous cELISAs for the 78
detection of antibodies against NSPs were used to differentiate vaccinated animals from 79
infected animals (1, 18). However, polyclonal antibodies were used as the competitor in 80
these tests. The use of polyclonal antibodies cannot ensure consistent quality compared 81
to monoclonal antibodies (mAb) because of batch-to-batch variations. Sørensen et al. (17) 82
produced a mAb against NSP-3B and developed a cELISA using the same mAb (L74D5) 83
as the capture and detector antibody in a blocking ELISA. The disadvantage of this 84
ELISA system is that when the antigen binds to the capture antibody, the same epitope 85
that is recognized by the polyclonal or competition antibodies might be hidden, which 86
reduces the test sensitivity. The PrioCHECK NS uses a specific mAb against NSP and a 87
recombinant NSP protein in a cELISA format. One study demonstrated that the 88
PrioCHECK NS test is sensitive and very specific in the buffalo populations of eastern 89
Africa (16). However, the use of a commercial kit for regular diagnosis and surveillance 90
can be costly. The development of an effective in-house test for the detection of 91
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antibodies against FMDV/NSP regardless of the species is necessary to make daily tests 92
more affordable. 93
A mAb against a conserved epitope located on the 3B NSP was produced. A cELISA 94
for FMD 3B-NSP antibody detection was developed using this mAb and a recombinant 95
3ABC protein. The assay was validated using a serum panel and samples collected from 96
naïve, experimentally inoculated, vaccinated, and carrier and non-carrier animals. 97
Development of a reliable NSP antibody assay would enable successful disease diagnosis 98
and the establishment of disease-free zones, and acceleration of global FMD control. 99
100
Materials and methods 101
102
Non-structural protein production. Cloning and expression of His-tagged 3ABC 103
recombinant protein was performed as described previously (1, 19). Recombinant 104
bacterial antigen was produced in cultures that were incubated for 2-3 hours at 37oC with 105
shaking until an OD 600 of 0.6–0.7 was reached. Protein expression was induced by the 106
addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 107
mM, and the solution was incubated for an additional 3 hours at 37oC with shaking. The 108
culture was pelleted using centrifugation at 8000 ×g for 15 minutes at 4oC. The cell pellet 109
was completely resuspended at room temperature in BugBuster reagent (25 U/mL, 110
Novagen, Darmstadt, Germany) to extract the recombinant 6xHis-3ABC. Benzonase 111
nuclease (Novagen) was added, and the suspension was incubated at room temperature 112
with gentle shaking for 10-20 minutes. Insoluble cell debris was collected using 113
centrifugation at 10,000 xg for 20 minutes at 4oC. The pellet was completely resuspended 114
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in the same volume of BugBuster reagent and centrifuged at 10,000 xg for 20 minutes at 115
4oC to collect the inclusion bodies. The final pellet was resuspended in 50 ml of 8 M 116
UREA, 0.1 M sodium phosphate buffer, 0.01 M Tris-HCl pH 8.0 until the inclusion 117
bodies (3ABC recombinant protein) became soluble and the solution was transparent. 118
Any insoluble material was removed using centrifugation at 10,000 xg for 20 minutes at 119
4oC. The collected supernatant containing the recombinant 3ABC NSP was aliquoted and 120
stored at -70oC. 121
122
Peptides, mouse immunization and monoclonal antibody production. The peptide, 123
H2N-CGPYAGPLERQKPLK –OH (20) derived from 3B1, was synthesized for 124
immunization and its purity was assessed using HPLC (New England Peptide, MA, 125
USA). The peptide was conjugated to the carrier protein-Keyhole limpet hemocyanin 126
(KLH) by New England Peptide. 127
The Canadian Science Centre for Human and Animal Health Animal Care Committee 128
approved animal use and study procedures (Animal use document #C-02-002). The 129
procedures for mouse immunization and mAb production were performed as previously 130
described (21). Hybridoma supernatants were screened using the recombinant 3ABC NSP 131
as the antigen in an indirect ELISA. The positive clone (F8-3Bp) was subcloned and the 132
isotype was determined using a mouse monoclonal antibody isotyping kit (Roche, 133
Indianapolis, IN, USA). 134
135
Development of the 3B cELISA. The optimal concentrations for recombinant 3ABC 136
protein, mAb and conjugated antibody dilutions were determined using checkerboard 137
titrations of known strongly positive, weakly positive and negative sera. The 3B cELISA 138
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was performed by coating microtiter plates (Nunc-Immunoplate, Roskilde, Denmark) 139
with 100 μl/well of recombinant 3ABC antigen that was diluted based on the titration of 140
each batch in 0.06 M carbonate/bicarbonate buffer, pH 9.6, and incubated overnight at 141
4°C. After five washes with the washing buffer (0.05% Tween20 in 0.01 M phosphate-142
buffered saline (PBS-T)), 100 µL of saturation buffer (50 mM Tris-Cl, 150 mM NaCl, 143
0.05% NaN3, 0.2% BSA, 6% D-Sorbitol) was added to each well and incubated 144
overnight at 4°C. The saturation buffer was discarded, and the plates were dried at room 145
temperature for 5 hours, heat-sealed under vacuum and stored up to 12 months at 4°C. 146
Plates were washed 3 times with PBS-T for testing, followed by the addition of 50 μl of 147
heat-inactivated test sera (final 1:5 dilution) in duplicate and an equal volume of 148
hybridoma culture supernatants (1:1000) in PBS-T. The plates were incubated at 37°C for 149
1 hour with agitation. After washing five times, 100 μl of horseradish peroxidase (HRP)-150
conjugated goat anti-mouse IgG (1:2000) (Jackson ImmunoResearch Laboratories Inc.) 151
in PBS-T was added and incubated for 1 hour at 37 °C with subsequent washing. The 152
3,3’,5;-tetramethyl benzidine dihydrochloride substrate (TMB, Sigma-Aldrich, St Lucia, 153
MO, USA) was added, and color development was stopped after 15 minutes with the 154
addition of 50 μl/well of 2 M sulfuric acid. The optical density (OD) was determined at 155
450 nm using an automated plate reader (Photometer Multiskan Reader, Labsystems, 156
Foster, VA, USA). 157
Results were calculated based on strong positive (Q1), weak positive (Q2) and negative 158
reference sera (Q3). Test results (mean of serum tested in duplicate wells) were derived 159
using the following formula: Percentage of inhibition (%) = [(Q3 OD - test sample OD) / 160
(Q3 OD – Q1 OD)] X 100%. 161
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Negative sera from bovine, porcine and ovine. Negative serum samples were collected 163
from naïve cattle (n = 503), pigs (n=508) and sheep (n=507) in Canada. Sera from naïve 164
cattle (n=353) and sheep (n=436) were also included in the evaluation in Mexico. 165
166
Raising of sera against the seven serotypes of FMDV in cattle, sheep, pigs and deer. 167
The FMDV strains (O1/UKG11/2001, A22 Iraq, A24/Cruzeiro/Br/55, C1 Noville, Asia 168
1/Shamir, SAT 1/BOT 1/68, SAT 1 KEN/4/98, SAT 2 SAU 1/2000 and SAT 3 ZIM 169
4/81) are reference strains that were obtained from the World Reference Laboratory for 170
FMD (WRLFMD) at the Pirbright Institute, UK. Baby hamster kidney clone 21 cells 171
(BHK-21) or lamb kidney primary cells (LK) were used to prepare viruses for the seven 172
FMDV serotypes. The cells were infected with FMDV in Glasgow’s MEM supplemented 173
with 2 mM L-glutamine and 50 µg/ml gentamycin. Viruses were harvested upon 174
observation of a complete cytopathic effect (CPE) and clarified using centrifugation at 175
2000 xg for 20 minutes. 176
All procedures involving experimental animal inoculations and care were performed 177
according to the Canadian Council of Animal Care guidelines. The local animal care 178
committee approved all animal procedures prior to initiation of the study. Cattle, sheep, 179
pigs and deer (1, 22) were inoculated with one of the FMDV strains. Cattle and sheep 180
were infected via intradermal/subdermal injections on the dorsal aspect of the tongue and 181
pigs were inoculated in their heel bulb with a total viral dose of 106 50% tissue culture 182
infectious doses (TCID50) at five sites. Two white-tailed deer (D16, D20) were inoculated 183
intranasally with 104 TCID50 of FMDV OUKG 11/2001 diluted in 5 ml of culture 184
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medium. Blood samples were collected before (0 days post-inoculation (dpi)) and after 185
virus inoculation until 28-31 dpi. 186
187
Positive bovine serum panel for evaluation of the 3B cELISA. The bovine serum 188
panel (23) was established by the WRLFMD at the Pirbright Institute. This panel 189
evaluates new tests and provides quality control for new batches of existing tests. Thirty-190
six bovine serum samples were selected from a series of vaccination-challenge 191
experiments. The details of the experiments used to generate these samples were reported 192
previously (23). 193
194
Sera from control, vaccinated and carrier cattle. These sera were raised by the 195
WRLFMD at the Pirbright Institute. Cattle were vaccinated with O1 Manisa and 196
challenged with O UKG 34/2001 in two experiments, UV and UY (24, 25). The 197
vaccination dose in the second experiment (UY), was increased 10-fold compared to the 198
first (UV). Cattle were grouped into control (non-vaccinated), vaccinated non-carriers 199
and vaccinated carriers. The carrier state was confirmed by either positive virus isolation 200
or quantitative RT-PCR in the probang samples taken from individual animals (24, 25). 201
Serum samples were collected from these animals seven days before vaccination and at 202
seven days intervals until 168 days post challenge (dpc) (26, 27). However, not every 203
animal was sampled at all-time points. 204
205
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Statistical analysis. Correlation analyses for comparisons of the two test methods (the 206
3B cELISA versus PrioCHECK NS) were performed using Microsoft Excel. The 207
statistical significance was set at P<0.05. 208
209
Results 210
211
Production of a monoclonal antibody against the 3B peptide. The KLH-conjugated 212
FMDV 3B peptide (H2N-CGPYAGPLERQKPLK–OH) was used as the immunogen to 213
produce the peptide-specific mAb (F8-3Bp). This mAb is an IgG1 isotype with kappa 214
light chains. The mAb was examined for its reactivity and specificity against the 215
recombinant 3ABC NSP and the 3B peptide using an indirect ELISA. Results indicated 216
that the mAb reacted with the recombinant 3ABC protein and the 3B peptide in the 217
ELISA with an OD higher than 1.0 after background subtraction. The mAb F8-3Bp 218
competed with the serum from a FMDV-infected animal. 219
220
Development and evaluation of the 3B cELISA. A cELISA to detect antibodies against 221
FMDV NSP was developed using the newly generated mAb (F8-3Bp) and the 222
recombinant 3ABC protein as the antigen. The optimal concentrations for the 223
recombinant 3ABC protein, mAb and conjugated antibody dilutions were optimized 224
using checkerboard titrations of known strong positive, weak positive and negative sera. 225
A total of 1518 negative sera (bovine = 503, porcine=508 and ovine=507) were 226
collected from Canada and tested using the 3B cELISA. The frequencies of the 227
percentage inhibition (PI) generated from these sera were normal distributed (Fig 1a). 228
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The mean PIs for the negative sera were 4.43% (standard deviation (SD) =14.03), - 229
8.63% (SD=14.0) and -9.6% (SD=9.59) for bovine, porcine and ovine, respectively. 230
Similar results were obtained using sera collected from Mexico (bovine =353, 231
ovine=436) (Fig 1b). The mean PIs were -6.4 (SD=17.35) and -15.76 (SD=18.92) for 232
bovine and ovine, respectively. The negative cut-off values, which were calculated by the 233
addition of 3SD to the mean PIs of the negative serum, equaled 19-46% of the inhibition 234
for all animal species. Therefore, the cut-off value was set at <50% of inhibition (28). 235
Less than 1% of the tested negative samples collected from bovine, porcine and ovine 236
exceeded this cut-off value, which produced a diagnostic specificity of 99.4%. 237
A bovine serum panel for the evaluation of antibodies against FMDV NSP was tested 238
using the 3B cELISA and compared with other commercially available tests. This serum 239
panel (n=36) was selected from a series of vaccination-challenge experiments (23, 29). 240
All samples were seropositive by the virus neutralization test (VNT) (23). Comparison of 241
the 3B cELISA with commercially available tests indicated that the 3B cELISA detected 242
the highest percentage of positive sera (94%) among the NSP antibody detection tests 243
(Table 1). PrioCHECK NS detected slightly lower positive percentage (92%) than the 3B 244
cELISA. Bommedi, UBI and Svanova demonstrated positive results of 61-81%. The 3B 245
cELISA detected positive NSP antibody in 34 of 36 serum samples, and it failed to detect 246
positive antibodies against NSP in two samples (UV10 and UV 19) that were also 247
negative in the Bommedi, UBI and Svanova tests. These two samples were from 248
vaccinated (O1/Manisa) and challenged (O1/UKG) carriers. Their VNT titers were 178 249
and 256, respectively. Three serum samples (2 from the vaccinated carrier group, 1 from 250
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the vaccinated non-carrier) with VNT titers > 512 exhibit a negative NSP antibody 251
response in the PrioCHECK NS (29). 252
Serum samples from vaccinated non-carriers in vaccination-challenge experiment #1 253
were tested using both the 3B cELISA and PrioCHECK NS to evaluate the 3B cELISA as 254
a test for differentiating vaccinated from infected animals (DIVA). The PrioCHECK NS 255
is the most commonly used test for FMD/NSP-specific antibody detection. Therefore, the 256
3B cELISA results were compared with the PrioCHECK NS. A total of 114 and 111 of 257
115 samples resulted in percentage inhibitions below (<) 50% for 3B cELISA and 258
PrioCHECK NS. These results indicate that there were no or little antibodies against NSP 259
present in these vaccinated non-carrier animals. One exceptional sample (UV8), which 260
was collected at 42 dpc showed a positive antibody response with a PI of 60%, which 261
produced a DIVA test specificity of 99.1% for the 3B cELISA (Fig 2a). Four samples 262
demonstrated positive antibody responses in the PrioCHECK NS with a test 263
specificity of 96.6% (Fig2b). All cattle in the control group (non-vaccinated, 264
challenged) exhibited a PI > 50% from 14 dpc, which indicates the presence of 265
antibodies to 3B NSP. The 3B cELISA results demonstrate a good correlation with the 266
PrioCHECK NS results with a correlation coefficient of 0.87 and 0.98 for the two 267
experiments, respectively (Fig 3). Moreover, the sera taken from vaccinated-carrier cattle 268
were tested using 3B cELISA and compared with previous PrioCHECK NS results (Fig 269
4). Seven of 11 cattle showed positive seroconversion in both 3B cELISA and 270
PrioCHECK NSs after 14 dpc. However, two carrier cattle (UV2 and UV14), which 271
were confirmed positive using reverse transcription PCR were negative in these two 272
tests. The other two carrier cattle (UV5 and UY76) showed positive antibody 273
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responses against NSP in the PrioCHECK NS, but negative antibody responses in the 274
3B cELISA. The correlation coefficient between the 3B cELISA and the PrioCHECK 275
NS for both experiments was 0.70 with p< 0.0001 (Fig4c). 276
277
Detection of seroconversion in FMDV inoculated animals using 3B cELISA. The 278
antibody responses to FMDV/NSP in experimentally inoculated animal species (15 cattle, 279
9 porcine, 18 sheep, and 2 deer) were examined using the 3B cELISA. Antibodies to NSP 280
were undetectable from 0-5 dpi for swine, cattle and deer (Table 2). One of 18 sheep 281
showed a positive antibody response against NSP on 5 dpi. A total of 89-100% of 282
samples from all tested species demonstrated positive antibody responses at 9 dpi. Two 283
samples, one pig and one sheep, demonstrated positive NSP antibody response after 9 284
dpi. Therefore seroconversions occurred mainly between 6-9 dpi. All animal samples, 285
except one pig, remained positive for antibody responses until the end of the experiment 286
(28 dpi). These results indicated that the 3B cELISA detected antibodies against NSP in 287
all tested animal species. 288
Serum samples from animals experimentally inoculated with all seven serotypes were 289
tested to determine whether the 3B cELISA detected NSP antibodies in sera of different 290
serotypes. The results demonstrated positive antibody response to NSP at 8-10 dpi, and 291
responses remained positive until the end of the experiment (Fig 5). Two sera from sheep 292
experimentally inoculated with SAT 3 showed a positive seroconversion after 10 dpi and 293
remained positive until 28 dpi. These results showed that 3B cELISA is serotype 294
independent for NSP-specific antibody detection. 295
296
Discussion 297
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The use of vaccines to FMD is an economic strategy to prevent and control this disease. 298
The differentiation between infected and vaccinated animals is also important in 299
surveillance efforts. An accurate test for detecting infection in susceptible animals is 300
needed, especially for infections after vaccination (30). ELISA for the detection of 301
antibodies against FMDV/NSP can distinguish between vaccinated animals and infected 302
animals. A good mAb that recognizes strain-independent epitopes on FMDV/NSP is 303
required to develop tests for FMDV/NSP antibody detection. Linear B-cell epitopes 304
located in the FMDV NSP were identified (31, 32). Höhlich et al. (20) identified six 305
linear B-cell epitopes in the NSPs. Three of six peptides were recognized by the sera 306
collected from all infected animals regardless of the infecting strains. None of the sera 307
from vaccinated animals contained antibodies against these peptides in their experiments. 308
Therefore, our study, used one (CGPYAGPLERQKPLK) of the 3B peptides (20) as the 309
immunogen for mAb production and the mAb produced against this peptide was used as 310
the basis for the 3B cELISA to differentiate vaccinated from infected animals. 311
The mAbs that are raised by peptides may not recognize original or recombinant 312
protein (33, 34). However, the mAb F8-3Bp that was produced in the current study 313
reacted with the 3B peptide and recombinant 3ABC. Similar results were also obtained 314
by Yang et al (35), who successfully produced mAbs against FMDV/NSP 3D using 315
immunization with recombinant 3D and boosted with a 3D peptide. Different species may 316
recognize different epitopes. Sera from FMDV-infected pigs failed to react with any 317
linear epitope located on the 3D protein (35). The peptide used for immunization in this 318
study was recognized by sera from all infected animals independent of animal species 319
(20). 320
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A 3B cELISA was developed using the recombinant 3ABC protein and the 3B-specific 321
mAb. A cut-off value of 50% inhibition was established based on 2307 negative sera, 322
which provided a clear distinction between positive and negative sera. Diagnostic 323
specificities using this cut-off value were 99.4% for all tested animal species (bovine, 324
porcine and ovine). The diagnostic specificity of a previously developed 3ABC indirect 325
ELISA for bovine was estimated at 96.4% (15). Some previously developed tests were 326
designed to detect NSP antibodies predominantly in cattle, but these tests are less useful 327
in sheep and pigs. Sheep, in particular, may fail to develop detectable levels of these 328
antibodies because of the frequently subclinical nature of FMD (30, 36). Immune 329
responses to infection may vary within individuals, virus serotypes and animal species, 330
but our 3B cELISA detected antibodies against FMDV/NSP in cattle, swine and sheep in 331
a serotype-independent manner. However, more deer and buffalo samples may need to be 332
tested. 333
Evaluations of the performance of a new test require comparisons of the performance to 334
standard samples. A serum panel (n=36) collected from FMD vaccine-challenged animals 335
(WRLFMD, Pirbright UK) was used to evaluate the 3B cELISA. The test results 336
indicated that the 3B cELISA detected the highest percentage of positive sera (94%) 337
among the NSP antibody detection tests (PrioCheck NS, Bommedi, UBI and Svanova). 338
The 3B cELISA failed to detect two positive samples (UV10 and UV 19) from this panel. 339
These samples consisted of sera collected from cattle that were vaccinated and challenged 340
by direct contact. The most reliable and consistent route of infection in experimental 341
studies on FMD is generally via intradermal/subdermal injection of virus (2). Therefore, 342
one possible explanation for this observation may be that vaccinated cattle challenged by 343
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contact with infected donors rather than by needle inoculation were generally 344
subclinically infected and produced low levels of NSP antibody because of limited viral 345
replication in the presence of high levels of structural antibodies produced against 346
vaccine (23). 347
The aim of this study was to develop a DIVA test, and samples collected from 348
vaccinated cattle in two vaccine-challenge experiments were analyzed. Theoretically, 349
antibody responses against FMDV/NSP in the vaccinated population should be similar to 350
the naive population. Serum samples collected from 11 vaccinated and challenged non-351
carrier cattle demonstrated positive virus isolation results from probang samples that 352
cleared by 28 days post-initial contact (24, 25) were tested using the 3B cELISA and 353
PrioCHECK NS. A total of 114 of the 115 samples from these animals (-7 dpv to168dpc) 354
showed negative results in the 3B cELISA, which indicated that no antibodies against 355
NSP were present in those serum samples. These cattle were challenged after vaccination, 356
but there was little or no FMDV replication because of induction of the protective 357
immune response. Therefore no antibodies against NSP were detected. 358
Samples collected from control cattle (non-vaccinated, challenged) were also tested 359
using the 3B cELISA and the results were comparable with the PrioCHECK NS results 360
(23). The PrioCHECK NS demonstrated the highest diagnostic specificity among the 361
commercial ELISA kits in a comparative study (37, 38). The 3B cELISA exhibits a good 362
correlation with the PrioCHECK NS results with a correlation coefficient of 0.87 and 363
0.98 for experiment 1 and 2, respectively. Clearly, the 3B cELISA can be used to detect 364
antibodies against FMDV/NSP and differentiate vaccinated from infected animals (39). 365
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Vaccinations in FMD endemic countries or during an outbreak situation result in a high 366
possibility that the clinical diseases will be masked in animals with partial immunity that 367
are exposed to live virus (36). The challenge for FMD diagnosis is to identify vaccinated 368
animals that have had contact with live virus and become carriers. The sera from 369
vaccinated-carrier cattle in two vaccine-challenge experiments were tested using the 3B 370
cELISA and compared with PrioCHECK NS test results. Seven of 11 cattle showed 371
seroconversion in both the 3B cELISA and PrioCHECK NS tests after 14 dpc. Two 372
vaccinated-carrier cattle (UV2 and UV14), which were confirmed positive results using 373
RT-PCR, showed negative results in both the 3B cELISA and PrioCHECK NS tests. The 374
other two vaccinated-carriers (UV5 and UY76) showed positive antibody responses 375
against FMDV/NSP in the PrioCHECK NS test, but were negative in the 3B cELISA. 376
The development of antibodies to NSP correlates with the extent of virus replication. 377
Therefore, the immune response induced by vaccination may reduce virus replication and 378
the amount of FMDV/NSP produced, which results in a low anti-NSP antibody response 379
(17, 26, 40). One outcome could be that, the antibody response may fall below the 380
detection limit of the 3B cELISA because of a decreased level of FMDV/NSPs. However, 381
some vaccinated carrier animals fail to develop antibodies against the NSPs (11). Present 382
tests for antibodies to NSPs cannot completely guarantee that a population of vaccinated 383
animals exposed to live virus contains no carriers (2). Caution must be taken when 384
analyzing data from vaccinated animals. The definitive identification of carrier status or 385
subclinically infected animals requires the recovery of live FMDV from these animals or 386
the potentially use of real time RT-PCR ((2, 36). 387
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In conclusion, the 3B cELISA that was developed using a 3B-specific mAb and a 388
recombinant 3ABC antigen is a simple and reliable diagnostic test for the detection of 389
FMDV 3B-specific antibodies. The 3B cELISA exhibited comparable performance to the 390
PrioCHECK NS test, and it out-performed other FMDV/NSP antibody detection tests. 391
The binding epitope of the mAb used in this 3B cELISA is well characterized and 392
conserved among the FMD seven serotypes. The test is serotype and species independent. 393
The advantages of the 3B cELISA over the PrioCHECK NS include (1) a reduced assay 394
time using pre-coated plates of under four hours for the 3B cELISA compared to an assay 395
time of 20 hours for the PrioCHECK NS and (2) a much smaller cost for the 3B cELISA 396
(< $1 per sample in duplicate) than the PrioCHECK NS test ($5-6 per sample). The 397
reduced time and cost to detect antibodies against NSP while maintaining diagnostic 398
sensitivity and specificity is critical for FMD diagnosis. 399
400
Acknowledgements 401
The authors gratefully thank the animal care staff, for expert animal services, and 402
Professor Soren Alexandersen and Dr. Charles Nfon for critical review of the manuscript. 403
This work was supported by funds from the Canadian Food Inspection Agency and 404
theDepartment for Environment, Food and Rural Affairs, UK (DEFRA). 405
406
Legends 407
FIG 1 Frequency distribution of the negative sera tested using the 3B cELISA. (a) 408
Serum samples collected from disease-free bovines (n = 503), porcine (n=508) and ovine 409
(n=507) in Canada and (b) bovines (n=353) and ovine (n=436) in Mexico. Recombinant 410
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3ABC protein was coated onto microtiter plates. Equal volumes of diluted sera and mAb 411
(F8-3Bp) were added to the plates and allowed to compete with antibodies in serum 412
samples. Results are expressed as a percentage of inhibition. 413
FIG 2 Evaluation of the 3B cELISA using sera from vaccinated, non-carrier cattle. 414
Serum samples from vaccine experiment #1(UV3,4,6,7,8,12,15,16,18,20,21) were 415
collected from vaccinated non-carrier cattle at 7 days intervals from -7 dpv to 168 dpc 416
during the course of experiments. Samples tested using (a) the 3B cELISA, and (b) the 417
PrioCHECK NS. 418
FIG 3 Correlations between the 3B cELISA and PrioCHECK NS results in sera 419
collected from the control group (un-vaccinated and challenged). Correlation 420
coefficients between the 3B cELISA and the PrioCHECK NS were determined for 421
vaccine experiment #1 (a) and vaccine experiment #2 (b). 422
FIG 4 Test results of the 3B cELISA and PrioCHECK NS using sera collected from 423
vaccinated-carrier cattle. Serum samples were collected from vaccinated-carrier cattle 424
at 7 days intervals from -7 dpv to 168 dpc during the course of the two vaccination-425
challenge experiments and assayed by (a) 3B cELISA and, (b) PrioCHECK NS. (c) 426
Correlations between the 3B cELISA and PrioCHECK NS test. 427
FIG 5 Detection of FMDV/NSP antibodies in animals inoculated with FMDV seven 428
serotypes using the 3B cELISA. Sera collected from animals experimentally inoculated 429
with seven FMDV serotypes (a) O, (b) A24, (c) A22, (d) C1, (e) Asia 1, (f) SAT 1, (g) 430
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SAT 2, (h) SAT 3 and tested using the 3B cELISA. C: cattle; S: sheep; P: porcine, and D: 431
deer. 432
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1 TABLE 1 Comparison of test results of FMDV NSP antibody evaluation panel (n = 36). All animals 2
(vaccinated and control group) were challenged and shown to be seropositive by VNT. Test results 3
except 3B cELISA have been previously reported by Parida et al (2007) 4
___________________________________________________________________________________ 5
Animal group 3B cELISA PrioCHECKa Bommedi
b UBI
c Svanova
d 6
Pos/total Pos/total Pos/total Pos/total Pos/total 7
___________________________________________________________________________________8
Vaccinated carrier 18/20 18/20 14/20 11/20 10/20 9
Vaccinated non-carrier 4/4 3/4 3/4 2/4 2/4 10
Control carrier 7/7 7/7 7/7 6/7 6/7 11
Control non-carrier 5/5 5/5 5/5 5/5 4/5 12
13
Total seropositive # 34/36 33/36 29/36 24/36 22/36 14
Total seropositive % 94% 92% 81% 67% 61% 15
___________________________________________________________________________________ 16
17 aPrioCHECK NS: Cedi Diagnostics B.V. Lelystad, The Netherlands. 18
bBommedi:CHEKIT-FMD-3ABC bo-ov, Bommeli Diagnostics, Liebefeld-Bern, Switzerland. 19
cUBI: UBI FMDV NS ELISA (CATTLE), United Biomedical Inc, Hauppauge, NY. 20
dSvanova: Svanovir FMDV 3ABC-Ab ELISA, Svanova Biotech AB, Uppsala, Sweden. 21
22
23
24
25
TABLE 2 Summary of the FMDV NSP specific antibody response from different animal 26
species inoculated with different serotypes of FMDV 27
___________________________________________________________________________________ 28
5 dpi 7 dpi 9 dpi 28 dpi 29
Pos # Pos % Pos # Pos % Pos # Pos % Pos # Pos % 30
___________________________________________________________________________________ 31
Porcine (n=9) 0 0% 1 12.5% 8 89% 8 89% 32
Sheep (n=18) 1 6% 14 82% 17 94% 18 100% 33
Cattle (n=15) 0 0% 8 53% 15 100% 15 100% 34
Deer (n=2) 0 0% 2 100% 2 100% 2 100% 35
___________________________________________________________________________________ 36
37
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