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Title: Culicoides obsoletus extract relevant for diagnostics ofinsect bite hypersensitivity in horses
Authors: Nathalie M.A. van der Meide, ChantalMeulenbroeks, Christine van Altena, Anouk Schurink, Bart J.Ducro, Bettina Wagner, Wolfgang Leibold, Jens Rohwer,Frans Jacobs, Marianne M. Sloet vanOldruitenborgh-Oosterbaan, Huub F.J. Savelkoul, EdwinTijhaar
PII: S0165-2427(12)00270-XDOI: doi:10.1016/j.vetimm.2012.07.007Reference: VETIMM 8857
To appear in: VETIMM
Received date: 4-6-2012Revised date: 12-7-2012Accepted date: 17-7-2012
Please cite this article as: van der Meide, N.M.A., Meulenbroeks, C., vanAltena, C., Schurink, A., Ducro, B.J., Wagner, B., Leibold, W., Rohwer, J.,Jacobs, F., Oldruitenborgh-Oosterbaan, M.M.S., Savelkoul, H.F.J., Tijhaar,E., Culicoides obsoletus extract relevant for diagnostics of insect bitehypersensitivity in horses, Veterinary Immunology and Immunopathology (2010),doi:10.1016/j.vetimm.2012.07.007
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Culicoides obsoletus extract relevant for diagnostics of 1
insect bite hypersensitivity in horses2
Nathalie M.A. van der Meidea, Chantal Meulenbroeksb3
Christine van Altenaa, Anouk Schurinkc, Bart J. Ducroc, Bettina 4
Wagnerd, Wolfgang Leibolde, Jens Rohwere, Frans Jacobsf, 5
Marianne M. Sloet van Oldruitenborgh-Oosterbaang, Huub F.J. 6
Savelkoula, Edwin Tijhaara7
8
a) Cell Biology and Immunology Group, Wageningen University, 9
Wageningen, The Netherlands.10
b) Department of Infectious Diseases and Immunology, Faculty of 11
Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.12
c) Animal Breeding and Genomics Centre, Wageningen University, 13
Wageningen, The Netherlands.14
d) Population Medicine and Diagnostic Sciences, Cornell University, 15
Ithaca, New York, United States.16
e) Immunology unit, University of Veterinary Medicine Hannover, 17
Hannover, Germany.18
f) Laboratory of Entomology, Wageningen University, Wageningen, 19
Netherlands20
g) Department of Equine Sciences, Faculty of Veterinary Medicine,21
Utrecht University, Utrecht, the Netherlands22
23
Corresponding author. Tel.: +31 317 483967; Fax: +31 31724
483962; E-mail address: [email protected]
Correspondence address. Edwin Tijhaar PhD, Cell Biology and 26
Immunology Group, P.O. Box 338 6700 AH Wageningen, The 27
Netherlands; [email protected]
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Abstract29
Insect bite hypersensitivity (IBH) is an allergic dermatitis in 30
horses caused by the bites of Culicoides species. 31
The aim of the present study was to evaluate the applicability 32
of whole body extracts of C. obsoletus (the main species found 33
feeding on horses in the Netherlands), C. nubeculosus (rarely 34
found in The Netherlands) and C. sonorensis (typical for North 35
America) for diagnosis of IBH in horses in The Netherlands. 36
Blood and serum samples of 10 clinically confirmed IBH 37
affected and 10 healthy control horses were used to evaluate the 38
IgE titers (ELISA) against the Culicoides whole body extracts39
of the three Culicoides species. Basophil degranulation was 40
assessed by histamine release test (HRT) after stimulation with 41
these extracts at 5, 0.5 and 0.05 µg/ml.42
IBH affected horses had significantly higher IgE titers against 43
C. obsoletus than against C. nubeculosus and C. sonorensis.44
Furthermore, C. obsoletus induced significantly higher 45
histamine release in whole blood of IBH affected horses46
compared to the other extracts at 0.5 µg/ml. Western blot data 47
revealed IgE binding to many proteins in C. obsoletus extract.48
This interaction was absent or weak in C. nubeculosus and C. 49
sonorensis extracts for IBH affected horses.50
Results on individual level indicate that the HRT is more 51
sensitive than ELISA in diagnosing IBH. However ELISA is 52
more practical as a routine test, therefore the ELISA was 53
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further evaluated using C. obsoletus extract on 103 IBH 54
affected and 100 healthy horses, which resulted in a test 55
sensitivity and specificity of 93.2 % and 90.0 %, respectively. 56
The IgE ELISA readings enabled the analysis of the predicted 57
probability of being IBH affected. From an Optical density 45058
nm value of 0.33 onwards, the probability of IBH affected was 59
more than 0.9. The results presented in this paper show that the 60
use of native Culicoides spp that feed on horse, is important for 61
improved diagnosis and that the described ELISA based on C. 62
obsoletus can be used routinely to diagnose IBH in countries 63
where this species is the main Culicoides feeding on horses.64
65
Keywords: Horse (Equine) IgE, Insect Bite Hypersensitivity, 66
ELISA, Culicoides obsoletus, Culicoides nubeculosus, 67
Culicoides sonorensis68
69
Abbreviations: ASR, allergen specific release; AUC, area under 70
the curve; C. nubeculosus, Culicoides nubeculosus; C. 71
obsoletus, Culicoides obsoletus; C. sonorensis, Culicoides 72
sonorensis; HRT, histamine release test, IBH, Insect Bite 73
Hypersensitivity; WBE, whole body extract74
75
1. Introduction76
Insect bite hypersensitivity (IBH), also called ‘sweet itch’ or 77
‘summer eczema’, is a seasonal recurrent allergic dermatitis in 78
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horses caused by an allergy against the bites of midges 79
(Culicoides spp), or sometimes black flies (Simulium spp) and 80
to an even lesser extend other insects (Anderson et al., 1988; 81
Braveman et al., 1983; Broström et al., 1987; Mullens et al., 82
2005; Wilson et al., 2008). IBH is found in many countries of 83
the world with a prevalence ranging from 3 - 11.6% in areas in 84
the UK (McCaig, 1973); (Littlewood, 1998), 10 - 60% in areas 85
of Queensland, Australia (Riek, 1954) and 0 -71.4% in regions 86
of The Netherlands (Van Grevenhof et al., 2007). 87
Insect Bite Hypersensitivity is clinically characterized by 88
strong pruritus and irritation, leading to alopecia and even89
secondary lesions due to scratching and rubbing. These 90
symptoms are particularly found along the preferred feeding 91
sites of the insect, which is the ventral midline, mane and tail 92
region of the horse (Anderson et al., 1988; Braverman, 1988).93
Several studies indicate that the allergic reaction is 94
predominantly IgE-mediated (Hellberg et al., 2006; Wilson et 95
al., 2001). However IgG (T) also seems to be involved (Wagner 96
et al., 2006)97
Intradermal injections with Culicoides extracts often induce 98
immediate and delayed type skin reactions in allergic horses99
(Ferroglio et al., 2006; Sloet van Oldruitenborgh-Oosterbaan et 100
al., 2009). In Iceland, where Culicoides spp do not occur, IBH 101
has never been reported (Bjornsdottir et al., 2006; Wilson et al., 102
2006). Several Culicoides species have been associated with 103
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IBH, including C. sonorensis, C. nubeculosus, C. imicola, C. 104
obsoletus and C. pulicaris (Halldorsdottir et al., 1989; Mellor 105
and McCraig, 1974; Mullens et al., 2005; Sloet van 106
Oldruitenborgh-Oosterbaan et al., 2009; Townley et al., 1984).107
Intradermal tests on allergic and healthy control horses in 108
Northern Germany and British Columbia with extracts and 109
saliva of native and exotic Culicoides species, showed no 110
difference between the native and exotic Culicoides species, 111
indicating the presence of species-shared allergens (Anderson 112
et al., 1993; Langner et al., 2008). However, intradermal skin 113
tests in The Netherlands, with a commercial extract of C. 114
nubeculosus and wild-caught C. obsoletus, showed the lack of 115
cross reactivity between these Culicoides species (Sloet van 116
Oldruitenborgh-Oosterbaan, 2006; Sloet van Oldruitenborgh-117
Oosterbaan et al., 2009).118
The aim of the present study was to evaluate three Culicoides119
species for their applicability in diagnostic tests for IBH of 120
horses in The Netherlands: C. obsoletus which is most121
frequently found on horses in The Netherlands (De Raat et al., 122
2008; Van der Rijt et al., 2008), C. nubeculosus, widely 123
distributed in Europe, but only occasionally detected in The 124
Netherlands (Takken et al., 2008) and not found to be attracted 125
to horses (Van der Rijt et al., 2008) and C. sonorensis which is 126
only present in North America. Currently, C. sonorensis and C. 127
nubeculosus are often used in studies about IBH, because they 128
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can be successfully maintained in laboratory bred colonies 129
(Boorman, 1974). C. obsoletus however, are not available from 130
laboratory bred colonies and have to be collected from the wild. 131
An attempt to breed C. obsoletus has been made, but was not 132
very efficient (Boorman, 1985). Results presented in this report133
show that the use of native Culicoides spp that feed on horse, is 134
important for improved diagnosis and possibly, for future 135
immunotherapy development. A diagnostic ELISA for IBH 136
based on C. obsoletus is described that can be used routinely 137
and has a high specificity and sensitivity. 138
139
2. Material and methods140
141
2.1. Animals142
A total of 223 horses and horses located in different regions of 143
The Netherlands were included in this study. Pairs of clinically 144
confirmed IBH affected and healthy control horses kept at the 145
same location were formed (Schurink et al., 2009). Ten 146
clinically confirmed IBH affected and ten healthy control 147
Shetland ponies were used to compare the different Culicoides148
whole body extracts in different in vitro diagnostic tests. The 149
remaining 203 horses (76 Icelandic horses and 127 Shetland 150
ponies) were used to evaluate the predictive value and test 151
sensitivity and specificity of an ELISA using C. obsoletus152
whole body extract. 153
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Blood samples were taken from all horses and serum was 154
frozen in aliquots not later than 24 hours after blood sampling 155
and stored at -20 o C until use. Blood sampling was approved 156
by the Board on Animal Ethics and Experiments from 157
Wageningen University and Utrecht University.158
159
2.2. Collection of Culicoides insects160
C. obsoletus insects were captured during spring and summer 161
months using a pooter (aspirator to collect insects)162
(Supplementary Fig. 1) or an “Onderstepoort” suction light trap 163
kindly provided by Laboratory of Entomology, Wageningen 164
University.165
Horses wearing an anti-insect blanket were put outside around 166
dawn hours on warm ( > 20 °C), dry and low wind days and 167
Culicoides insects were collected directly from the horses using 168
the pooter. The insects were collected and completely frozen169
alive at -80 °C and stored at that temperature until preparation 170
of the extracts. A small fraction (5 %) of the insects collected 171
with the pooter was checked under a stereo microscope to 172
confirm the species. Identification of C. obsoletus was based on 173
size and wing patterns (Campbell and Pelham-Clinton, 1960)174
(Supplementary Fig. 2).175
The “Onderstepoort” suction light trap was operated from 176
before dusk to far after dawn near a horse stable for 19 days at 177
different locations in The Netherlands in the summer months of 178
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2009. Insects were captured in 100% alcohol and frozen the 179
next day in alcohol at -80 oC until determination. Culicoides 180
obsoletus insects were selected and separated from the other 181
captured insects using a stereo microscope as described above.182
Separated Culicoides obsoletus insects were used for the 183
preparation of extracts. 184
Three-day-old laboratory bred Culicoides sonorensis were a 185
kind gift from Arthropod Borne Animal Diseases Research 186
Unit Center for Grain and Animal Health, Manhattan, US. C. 187
nubeculosus insects were kindly donated by the Institute for 188
Animal Health, Pirbright, UK. All insects were kept frozen 189
(without alcohol) at -80 °C until preparation of the extracts. 190
191
2.3. Preparation of Culicoides protein extracts192
Whole body extracts (WBE) were prepared from about three193
hundred insects that were transferred to a 2 ml Eppendorf tube 194
with 1 ml of PBS containing a protease inhibitor cocktail195
(Sigma-Aldrich, P8849) and crushed with a micro pestle. The 196
insoluble material was removed by centrifugation at 13000g for 197
10 min at 4 °C. Supernatant was collected and filtered through 198
sterile Millex-GV filters (Millipore) with a pore diameter of 199
0.22 µm and protein content of the filtrate was determined by 200
OD280nm measurement on a Nanodrop spectrophotometer 201
(NanoDrop 1000, Thermo Scientific). Samples were aliquoted, 202
directly frozen in liquid nitrogen and stored at -80 °C until use. 203
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Quality of the protein WBE was checked by protein staining 204
with Gelcode Coomassie blue staining (Thermo Scientific) 205
after proteins were separated by 15% SDS-PAGE. 206
207
2.4. SDS PAGE and Western blotting208
Whole body protein extract samples, 20 µg/lane (Western 209
blotting) or 60 µg/lane (Coomassie staining) were heated at 96 210
°C for 5 min with sample buffer containing dithiotreitol (DTT)211
and separated by SDS-PAGE (15% gel). These separated 212
proteins were transferred to a nitrocellulose membrane213
(Protrans, Schleicher & Schuell, Bioscience GmbH) by means 214
of electrophoresis. Membranes were blocked with 5% non-fat 215
cow’s milk in Tris buffered saline (TBS)-Tween (10 mM Tris, 216
150 mM NaCl, pH 7.5, 0.05% (v/v) Tween 20) for 1 h at room 217
temperature (RT) and then incubated overnight with horse sera218
from allergic or control horses diluted 1:10 in 5% non-fat cow’s 219
milk in TBS-Tween. Membranes were then incubated for 1.5 220
hour with a mAb against horse IgE (αIgE-176) (Wagner et al., 221
2003) followed by goat anti-mouse IgG horseradish peroxidase 222
(Dako, 1:1000 in milk powder/TBS-Tween). Between each 223
incubation step, membranes were washed three times with 224
TBS-Tween.225
Signal was detected by development with an enhanced 226
chemiluminescence (ECL) western blotting detection reagent227
(Amersham, GE Healthcare) according to the manufacturer's 228
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protocol and visualized by the use of Lumni-fil 229
chemiluminescent Detection Film (Roche, Woerden, The 230
Netherlands).231
232
2.5. Histamine release test (HRT)233
The histamine release by basophils was determined by a 234
modified method of Kaul (Kaul, 1998). Blood samples were 235
collected in anticoagulant tubes (EDTA) and kept at RT in the 236
dark until further use within 24 hours. The total blood cells237
were washed twice (500g for 10 min) with PBS to remove non 238
cell bound antibodies. Supernatant was discarded and the cell 239
pellet was resuspended in PBS to its original blood sample 240
volume. Endogenous histamine from whole body Culicoides 241
extracts was removed by PD-10 Desalting columns (GE 242
Healthcare) according to manufacturer’s recommendations. The 243
antigen induced histamine release was obtained by incubating 244
250 µl of washed blood cells with 250 µl of PIPES buffer (110245
mM NaCl, 5 mM KCl, 40 mM NaOH, 2 mM CaCl2, 25 mM 246
PIPES, 2 mM MgCl2) containing the histamine depleted 247
Culicoides whole body extracts at final concentrations of 5248
µg/ml, 0.5 µg/ml and 0.05 µg/ml at 37 °C for 60 min. 249
Spontaneous release was obtained by incubating 250 µl of 250
PIPES B buffer with 250 µl of washed blood cells at 37 °C for 251
60 min. Physical maximum release was obtained by boiling 200252
µl of washed blood cells with 800 µl of PIPES buffer for 10 253
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minutes. After incubation, all samples were chilled on ice for 5254
min and pelleted by spinning down at 700g for 10 min. The 255
cell-free supernatants were collected and stored at -20 °C. 256
Subsequently, competitive RIA was carried out as per the 257
manufacturer’s instructions (LDN Nordhorn, Germany) to 258
determine the histamine content of the supernatants. 259
The maximum amount of histamine obtained by boiling was set 260
to 100%. The histamine content of each test sample was 261
calculated from this maximum histamine release. The allergen 262
specific release (ASR) is calculated as: ASR = (sample induced 263
release- spontaneous release)/(maximum release – spontaneous 264
release) x 100 %. Net-histamine releases that were equal or 265
greater than 10 % of the maximum release were considered as 266
positive.267
268
2.6. Culicoides-specific IgE ELISA269
Specific IgE levels in sera of 10 IBH affected and 10 healthy 270
control Shetland ponies, binding the different Culicoides WBE, 271
were measured by ELISA. Optimal coating concentration, 272
serum dilution and antibody concentrations were determined 273
prior to the experiment by titration of the different components. 274
Costar 96-well microtiter plates were coated with 100 µl/well 275
of 10 µg/ml C. obsoletus, C. nubeculosus or C. sonorensis276
extract, diluted in PBS, and incubated overnight at 4 °C and 277
afterwards blocked with 150 µl of a 1.5% casein buffer (SDT, 278
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Germany) for 1.5 hour at RT. Plates were washed 5 times with 279
PBS containing 0.05% Tween20, followed by incubation for 280
1.5 hour at RT with 100 µl of horse serum samples diluted 1:5 281
in a 1.5% casein buffer. After washing, wells were incubated 282
for 1 hour at RT with 100 µl of 2.5 µg/ml mouse monoclonal283
anti-equine IgE-176 (Wagner et al., 2003) diluted in casein 284
buffer. After washing the plates 5 times with PBS containing 285
0.05% Tween20, goat anti-mouse peroxidase conjugate 286
(multispecies adsorbed, Serotec) diluted 1000 times in casein 287
buffer, was applied to the wells and incubated for 1 hour at RT.288
After 5 washes with PBS/0.05% Tween20, 100 µl 289
tetramethylbenzidine (high sensitivity, SDT, Germany) was 290
added to the wells and incubated for 10 min at room 291
temperature. The reaction was stopped with 100 µl/well of 1% 292
HCl. Absorbance was measured with a multi-mode microplate 293
reader (SpectraMax M5, Molecular Devices) at a wave length 294
of 450 nm corrected for 650 nm. Based on the preliminary 295
experiments a standard serum dilution of 1:5 was selected as 296
suitable for comparison of OD450nm values in the IgE ELISA. 297
The cut off level was assigned as the mean + 3 times the 298
standard deviation (SD) of the IgE levels of the healthy control 299
horses.300
An additional 203 horse serum samples, 103 IBH affected and 301
100 healthy control horses, were evaluated for C. obsoletus302
specific IgE (OD450nm) values in a 384 wells plate to determine 303
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the sensitivity and specificity of this Culicoides-specific 304
ELISA. The same conditions as described for the 96-wells plate 305
were used, with 20 µl volumes per well. Distribution plots of 306
the healthy and IBH affected horses were obtained by 307
categorizing horses according to their OD450nm values. The first 308
category ranged from 0 to 0.01, the second from 0.01-0.02 and 309
the following categories each increased with 0.02 up to 0.32. 310
Then the categories ranged from 0.32-0.35, 0.35-0.4 and 311
subsequent categories each increased with 0.2 until the 312
maximum OD450nm of 2.2 was reached. 313
Accuracy of diagnostic tests is often determined from so-called 314
Receiver-Operating Characteristic (ROC) curves. ROC-curves 315
represent the trade-off between sensitivity (i.e. true positive 316
rate) of a test and (1-specificity) (i.e. false positive rate) at all 317
possible positivity cut-off points. The area under the curve 318
summarizes the overall diagnostic accuracy. It takes values 319
from 0 to 1, where a value of 0 indicates a perfectly inaccurate 320
test and a value of 1 reflects a perfectly accurate test. A good 321
first choice for a test cut-off value that results in a balanced 322
optimal sensitivity and specificity is that value which 323
corresponds to a point on the ROC-curve nearest to the upper 324
left corner of the ROC graph.325
326
327
2.7. Statistical analysis328
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Analysis of variance was performed on log transformed data329
obtained by either HRT or ELISA to determine influence of 330
Culicoides species on the outcome. Factors included in the 331
model were, Culicoides-species (C. nubeculosus, C. sonorensis332
and C. obsoletus), IBH-status of the horse (yes/no) and 333
interaction between these two factors. Additionally, WBE 334
concentration (0.05, 0.5, 5 µg/ml) was included in the analysis 335
of HRT results. IBH-status within individual horses was 336
included as random factor since the same set of horses were 337
used in testing the three Culicoides types. Analysis was 338
performed using PROC MIXED of SAS (SAS Inc, V9.2). 339
The relation of OD450nm value in the IgE-ELISA to the IBH-340
status (negative or positive) was analysed with a logistic 341
regression. The analysis was performed with the PROC 342
LOGISTIC of SAS (SAS Inc, V9.2). 343
3. Results344
345
3.1. Collection of Culicoides obsoletus346
Two different collection methods (“Onderstepoort” light trap 347
and pooter) were used to determine the most selective and 348
efficient way of collecting C. obsoletes from the wild. 349
Determination of insects collected with the “Onderstepoort” 350
light trap revealed that many different insect species were 351
collected in this manner, from which only a small fraction 352
(<1%) belonged to Culicoides species. A total of 766 353
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Culicoides midges were collected during these 19 days. The 354
large majority of these Culicoides were identified as C. 355
obsoletus (82 %), followed by C. dewulfii (6,5 %) and C. 356
punctatus (5,0 %).357
Using the pooter hundreds of Culicoides insects were easily 358
collected within an hour from a horse wearing an anti-insect 359
blanket. A small part (± 5 %) of the collected insects was used 360
for identification and these were all identified as Culicoides 361
obsoletus based on size and wing patterns. 362
363
3.2. Quality of extracts of Culicoides captures by 364
different methods365
WBE were prepared from C. obsoletus insects collected alive 366
with the pooter system, as well as insects collected in alcohol 367
with the Onderstepoort light trap. Insects that were stored in 368
alcohol were excluded in the following experiments due to 369
substantial degradation of the proteins revealed on SDS-PAGE 370
(Supplementary Fig. 3).371
The extracts from laboratory bred C. sonorensis and C. 372
nubeculosus and wild-caught C. obsoletus prepared in the same 373
manner from freshly frozen insects, showed some differences 374
in the lower molecular weight regions (indicated by arrows in 375
Fig. 1) on SDS-PAGE (15 % gel), but overall pattern and 376
intensity of the protein bands were similar, with no obvious 377
degradation, indicating similar quality of the different extracts. 378
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379
3.3. IgE-specific antibodies in horse sera specific for 380
Culicoides proteins as determined by western blotting381
Western blotting was performed to evaluate sera of clinically 382
confirmed IBH affected and healthy control Shetland ponies for 383
the presence of IgE specific for proteins from C. obsoletus, C. 384
sonorensis and C. nubeculosus. Typical examples of 5 allergic385
(upper panel) and 5 healthy horses (lower panel) are shown 386
(Fig. 2).387
The IgE in sera of all allergic horses reacted strongly to a 388
number of proteins from C. obsoletus extract, but much weaker389
with proteins from C. sonorensis and C. nubeculosus extract390
despite the similar quality of the extracts. The antigen 391
recognition pattern for each individual horse was different, but 392
most IBH affected horses reacted with a protein(s) around 20393
kDa. IgE from the healthy horses, except for one, hardly 394
recognized any proteins from any of the 3 Culicoides species. 395
Proteins that did bind to the IgE in serum of healthy horses all 396
had a Mw of 25 kDa or higher (Fig. 2).397
398
3.4. Basophil degranulation induced by Culicoides whole 399
body extracts determined by histamine release test400
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For each of the 3 different Culicoides species the allergen 401
specific release (ASR) of histamine from basophils was tested 402
at 3 WBE concentrations on whole blood samples of 10 IBH 403
affected and 10 healthy control Shetland ponies (Fig. 3),404
including the horses that were used for the western blot 405
analysis.406
At the highest WBE concentration of 5 µg/ml 10 out of 10 407
(100%) of the IBH affected horses scored positive on the C. 408
obsoletus WBE, while this was 8 out of 10 (80 %) for C. 409
nubeculosus and C. sonorensis. However, at this highest WBE 410
concentration some of the healthy horses also scored positive 411
on all Culicoides species. At a concentration of 0.5 µg/ml none 412
of the healthy control horses scored positive on any of the 413
extracts, but at this concentration only 20% of the IBH affected 414
horses scored positive with C. nubeculosus and C. sonorensis415
extract. In contrast, 100% of the IBH affected horses scored 416
positive when the C. obsoletus extract was used. At this 417
concentration the reactivity towards C. obsoletus was 418
significantly higher than to C. nubeculosus (p< 0.01) and C. 419
sonorensis (p< 0.001) (Fig. 3). At the lowest WBE420
concentration of 0.05 µg/ml 40% of the IBH affected horses421
were still found to be positive with C. obsoletus, whereas for C. 422
nubeculosus and C. sonorensis this was only 10% of the IBH 423
affected horses. Also at this concentration all healthy control 424
horses had a negative test results for all Culicoides extracts. 425
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426
3.5. IgE measurements in horse sera against Culicoides 427
whole body extracts by ELISA428
Specific IgE serum levels against WBE of the three different 429
Culicoides species were determined in an indirect ELISA (Fig.430
4).431
IgE levels expressed as OD450nm values of the clinically 432
confirmed IBH affected Shetland ponies against C. obsoletus433
extract were significantly higher compared to OD450nm values 434
of the same IBH affected horses against the other extracts (both 435
p < 0.0001). With C. obsoletus extract only, specific IgE serum 436
levels of IBH affected horses were significantly higher than437
specific IgE levels of healthy control horses (p < 0.0001). 438
439
3.6. Comparison of ELISA and HRT data for individual 440
horses441
Individual HRT (Fig. 5 upper panel) and ELISA (Fig 5 lower 442
panel) responses to the WBE of the different Culicoides species 443
were compared for the same Shetland ponies used in Figure 3444
and 4. The values at a concentration of 0.5 µg/ml per extract 445
were chosen to analyze the horses on individual level for the 446
HRT, because at this concentration the best distinction could be 447
made between IBH affected and healthy control horses (section 448
3.5). On individual level, for the C. obsoletus extract the 449
histamine release of all IBH affected horses was higher than 10 450
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% and therefore positive (Kaul et al., 1998), whereas the 451
maximum histamine release of all healthy control horses was 452
below 10 % of the maximum histamine release. For the ELISA 453
6 out of 10 IBH affected horses had OD450nm values against C. 454
obsoletus extract above the set cut-off level (mean + 3 times the 455
standard deviation SD of the OD450nm values of the healthy 456
control horses). With this cut-off level, all healthy horses were 457
negative. In 6 out of 10 IBH cases (horse 2, 3, 4, 5, 6, and 7) 458
the HRT and ELISA values against C. obsoletus WBE 459
correlated with each other. Interestingly, the IBH affected 460
horses with OD450nm values below the cut-off level in the 461
ELISA (horses 1, 4, 8 and 10) did have a high positive 462
histamine release with the HRT using C. obsoletus WBE. One 463
horse had a higher histamine release after stimulation with C. 464
nubeculosus and C. sonorensis WBE compared to C. obsoletus, 465
but did have a higher IgE level against C. obsoletus when 466
measured by ELISA. 467
468
3.7. ELISA test sensitivity and specificity469
The results described in paragraph 3.7 indicate that the HRT 470
outperforms the ELISA as a diagnostic test for IBH, but the 471
ELISA is more practical as a routine test. It is much less 472
laborious and can be performed on serum samples, while the 473
HRT requires fresh full blood. Therefore the ELISA using C. 474
obsoletus extract was further evaluated with sera of 103475
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clinically confirmed IBH affected horses and 100 healthy 476
control horses (76 Icelandic horses and 127 Shetland ponies).477
When categorizing the healthy and IBH affected horses478
according to their OD450nm values from the IgE ELISA, two 479
distributions with equal variance were observed which are 480
nearly baseline-separated (Fig. 6a). Although the distribution 481
curve of healthy and IBH affected horses overlapped 482
somewhat, the IBH-affected horses had higher serum IgE levels 483
against C. obsoletus WBE compared to healthy control horses484
(p < 0.0001) (Fig. 6b).485
The pattern of the ROC-curve (Fig. 6c) indicates that the 486
sensitivity sharply increases already at low false positive rates.487
The sensitivity of the test is therefore high over a large range of 488
cut-off points. The accuracy of the test as evaluated by the area 489
under the curve (AUC) is high and amounted to 0.97, indicating 490
that high sensitivity is achieved with a high specificity.The 491
point on the ROC-curve nearest to the upper left corner of the 492
curve corresponds with a sensitivity of 93.2% and a specificity 493
of 90.0% and is obtained at an OD450nm cut-off value of 0.2. 494
Logistic regression was performed to analyze the relation of 495
IgE OD450nm values to the IBH-status. The response IBH-496
affected or healthy was regressed on OD450nm values.497
Figure 6d shows the predicted probabilities of both IBH-498
outcomes (i.e. affected or healthy) related to OD450nm values. 499
With IgE-values close to zero the probability of being healthy 500
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is much higher than the probability of having IBH; up to an IgE 501
value (OD450nm value) of 0.07 (true for 70 % of the healthy 502
horses) the probability of being IBH negative is approximately 503
10 times higher than being IBH positive and 5 times higher for 504
a value of 0.12 (true for 85 % of the healthy horses). At the 505
inflection point at an OD450nm value of approximately 0.2 the 506
probability being IBH-positive or negative is equal. From an 507
OD450nm value of 0.33 onwards (true for 75 % of the IBH 508
affected horses) the probability of IBH-positive is more than 509
0.9. 510
511
512
4. Discussion513
In this study we evaluated three different Culicoides whole 514
body extracts for their applicability for in vitro diagnosis of 515
IBH horses in The Netherlands. We show that C. obsoletus (a 516
species found feeding on horses in The Netherlands) whole 517
body extract is much better for in vitro diagnosis of IBH by 518
ELISA and HRT, than C. nubeculosus and C. sonorensis (not 519
found feeding on horses in the Netherlands) whole body 520
extracts. An IgE-ELISA with C. obsoletus whole body extract 521
performed with 103 IBH affected horses and 100 healthy 522
horses located in the Netherlands demonstrated a high 523
sensitivity and specificity (93.2 % and 90.0 %, respectively) 524
and can thus be used as a valuable test to diagnose horses for 525
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IBH in countries where C. obsoletus is the main species found 526
feeding on horses.527
This study describes an easy and selective method of catching 528
Culicoides spp. attracted to horses by using a pooter. Although529
this method is initially more labor intensive than an 530
“Onderstepoort” suction trap, it is a selective way of collecting 531
preferentially those Culicoides species attracted to horses. This 532
obviates the need for the labor intensive selection of the desired 533
Culicoides species out of a large majority of unwanted insects 534
that is required when using a light trap. The biggest advantage 535
of the pooter method is that the insects are caught in a gentle 536
way that keeps them alive, preventing substantial protein537
degradation as is observed for insects captured in alcohol by the 538
“Onderstepoort” light trap. 539
The majority of Culicoides spp that were caught by both 540
capturing methods were found to be C. obsoletus: over 80% 541
when using the light trap and nearly 100% when directly 542
collected from the horse by the pooter. This is in agreement 543
with earlier studies performed in The Netherlands that also 544
found C. obsoletus to be the main Culicoides species attracted 545
to horses (De Raat et al., 2008; Van der Rijt et al., 2008). In 546
other countries, such as England (Wilson et al., 2008) Ireland 547
(Townley et al., 1984), Japan (Yamashita et al., 1957) and 548
United States (Mullens et al., 2005), C. obsoletus was also 549
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found to be the most important Culicoides species attracted to 550
horses. 551
Comparison of three Culicoides extracts demonstrates that in 552
The Netherlands horses with IBH have more specific IgE 553
directed against whole body extracts of C. obsoletus than 554
against Culicoides species that do not feed on these horses. 555
Although cross-reactivity between different Culicoides species 556
has been reported (Anderson et al., 1993; Langner et al., 2008)557
our study shows weaker IgE binding in Western blot and 558
ELISA to proteins of non-indigenous Culicoides species559
compared to native C. obsoletus, which was also observed 560
before (Wilson et al., 2008). This indicates the importance of 561
using extracts from native Culicoides species feeding on horses562
for reliable diagnostics of IBH. 563
The binding of IgE from allergic horses with C. sonorensis and564
C. nubeculosus proteins might be due to cross reactivity 565
between these proteins and the C. obsoletus antigens and 566
maybe also proteins of other insect species that the horses were 567
exposed to. The horses in this study might have been exposed 568
to the native C. nubeculosus, but our as well as another study 569
performed in The Netherlands (Van der Rijt et al., 2008) did 570
not find any C. nubeculosus insects to be attracted to horses. 571
Although C. sonorensis and C. nubeculosus antigens have 572
previously been successfully used in different diagnostic tests 573
(Langner et al., 2009; Schaffartzik et al., 2010; Schaffartzik et 574
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al., 2011) our study clearly shows the importance of using an 575
extract of a Culicoides species to which horses have been 576
actually exposed to for diagnosis. Western blot data revealed577
many IgE binding proteins in C. obsoletus extract that were578
absent in C. nubeculosus and C. sonorensis extracts. 579
Interestingly, a protein of around 20 kDa from C. obsoletus580
extract was found to be bound by IgE from almost all clinically 581
confirmed IBH affected horses, whereas this was not observed 582
for C. nubeculosus and C. sonorensis extracts and also not for 583
IgE from healthy horses. This makes this protein an interesting 584
candidate allergen for further characterization. 585
The C. obsoletus insects collected from the wild using the 586
pooter were all female species, since females need blood to 587
reproduce and were trying to feed on the horse when captured. 588
Although WBE of laboratory-bred insects were of both sexes, it 589
is unlikely that this explains the lower allergen-reactivity by C. 590
nubeculosus and C. sonorensis in this study. Other studies 591
found Culicoides extracts made from males only, to be just as 592
effective in stimulating horse basophils (Marti et al., 1999) and 593
non-salivary antigens from the thorax of Culicoides spp. have594
shown IgE-reactivity with IBH-affected horses (Wilson et al., 595
2001). One of the IBH affected horses reacted even slightly 596
stronger in the HRT with C. nubeculosus and C. sonorensis597
than with C. obsoletus WBE, and therefore rules out a lack of 598
antigens of lower protein quality in the C. nubeculosus and C. 599
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sonorensis WBE. This was also demonstrated by the similar 600
pattern and intensity of the protein bands of all three WBE on 601
SDS-PAGE.602
Surprisingly, some horses had low IgE binding in the ELISA 603
but a high histamine release in response to Culicoides extract 604
stimulation in the HRT. IgG (T) has also been observed to bind 605
to skin mast cells and therefore might play an important role in 606
the histamine release reaction in IBH (Wagner et al., 2006). 607
Therefore, the observation that some horses have low IgE 608
binding in ELISA but a high histamine release in response to 609
Culicoides extract stimulation, might be due to cross-linking of610
allergen specific IgG(T) instead of IgE. 611
The results in this study indicate that the histamine release test 612
(HRT) might be more sensitive and reliable for diagnosis of 613
IBH than the ELISA. However, this comparison was made on a 614
relatively small number of horses (10 IBH affected and 10 615
healthy control horses). When tested with a large number of 616
horses, the IgE ELISA, resulted in a high specificity (90.0) and 617
sensitivity (93.2) and proved to be the method of choice for 618
routine screening, because it is more robust, easier to perform 619
and more economical than the HRT. The test clearly 620
discriminates between IBH-affected and healthy controls such 621
that there is little overlap of distributions. The accuracy of the 622
test as evaluated by the area under the curve (AUC) of the 623
ROC-graph is high (0.97), indicating that high sensitivity is 624
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achieved with a high specificity. At an OD450nm cut-off value 625
for positivity of 0.2 the test has a sensitivity of 93.2% and a 626
specificity of 90.0%. However, this cut-off value does not take 627
the actual OD450nm value of an individual horse into account, 628
apart from being lower or higher than the determined cut-off 629
value for positivity. Therefore logistic regression analysis was 630
performed to determine the relation of the IgE OD450nm values 631
to the IBH-status. For most horses the ELISA can determine 632
with 90-100% probability the correct IBH-status of the 633
individual tested horse. For those OD450nm values where 634
reliability is less, e.g. around the inflection point at OD450nm635
value of 0.2, the horse owner can choose for an additional HRT 636
to obtain a more conclusive diagnosis. Western blot analysis 637
on C. obsoletus whole body extracts using serum of healthy 638
horses showed some IgE reactivity of these horses against 639
proteins with Mw above 25 kD. Therefore, the use of selected 640
recombinant proteins from C. obsoletus might further improve 641
the sensitivity of the ELISA described in this study.642
At present, treatment of IBH is based on insect avoidance by 643
stabling, use of anti-midge blankets (pajamas) or insect 644
repellants and suppression of symptoms by the use of 645
corticosteroids. Specific immunotherapy might be possible and 646
some immunotherapy trials have been carried out with647
Culicoides whole body extracts, but with varying results 648
(Anderson et al., 1996; Barbet et al., 1990). The use of purified 649
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allergens could further improve diagnostics, but could also be a 650
benefit for immunotherapy. Collecting insects is very time 651
consuming and for immunotherapy over 10000 insects were 652
necessary per horse (Anderson et al., 1996), therefore 653
recombinant allergens from an infinite source would be a654
sensible alternative for whole body extracts.655
Currently, allergens have been identified and produced from 656
species that are not present or common in The Netherlands, e.g. 657
C. sonorensis and C. nubeculosus (Langner et al., 2009; 658
Schaffartzik et al., 2011). Implementation of future 659
immunotherapy in horses will depend on the availability of 660
correct allergens and therefore the use of allergens from 661
Culicoides spp to which horses have been exposed, which for 662
The Netherlands is mostly C. obsoletus, might be crucial. 663
In conclusion, our results show that horses with IBH in the 664
Netherlands have much more IgE antibodies against Culicoides 665
obsoletus proteins compared to Culicoides sonorensis and 666
Culicoides nubeculosus proteins which can be routinely 667
detected in different diagnostic tests. The developed ELISA to 668
identify sensitization against C. obsoletus allergens provides a 669
valuable diagnostic test to discriminate IBH affected from 670
healthy control horses in The Netherlands, but will also be 671
valuable in other countries were C. obsoletus is mostly found 672
feeding on horses. 673
674
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Conflict of interest675
None676
677
Acknowledgements678
The authors want to thank all horse owners for their 679
cooperation. We would like to thank Christian Plasschaert for 680
supplying monoclonal anti-horse IgE 176 and Marleen Scheer 681
for participating in the collection of blood samples. 682
This work is financially supported by the Dutch Technology 683
Foundation STW (STW-NWO), the Dutch Federation of horse 684
breeding ('s-Hertogenbosch, The Netherlands) and ALK-Abelló 685
/ Artu Biologicals (Almere, The Netherlands).686
687
References688
Anderson, G.S., Belton, P., Jahren, E., Lange, H., Kleider, N., 689
1996, Immunotherapy trial for horses in British 690
Columbia with Culicoides (Diptera: Ceratopogonidae) 691
hypersensitivity. Journal of medical entomology 33, 692
458-466.693
Anderson, G.S., Belton, P., Kleider, N., 1988, The 694
hypersensitivity of horses to Culicoides bites in British 695
Columbia. The Canadian Veterinary Journal 29, 718 -696
723.697
Anderson, G.S., Belton, P., Kleider, N., 1993, Hypersensitivity 698
of horses in British Columbia to extracts of native and 699
Page 29 of 47
Accep
ted
Man
uscr
ipt
29
exotic species of Culicoides (Diptera: 700
Ceratopogonidae). Journal of medical entomology 30, 701
657-663.702
Barbet, J.O.Y.L., Bevier, D., Greiner, E., 1990, Specific 703
immunotherapy in the treatment of Culicoides704
hypersensitive horses: A double blind study. Equine 705
Veterinary Journal 22, 232-235.706
Bjornsdottir, S., Sigvaldadottir, J., Brostrom, H., Langvad, B., 707
Sigursson, A., 2006, Summer eczema in exported 708
Icelandic horses: influence of environmental and 709
genetic factors. Acta Veterinaria Scandinavica 48.710
Boorman, J., 1974, The maintenance of laboratory colonies of 711
Culicoides variipennis (Coq.), C. nubeculosus (Mg.) 712
and C. riethi Kieff.(Diptera, Ceratopogonidae). Bulletin 713
of Entomological Research 64, 371-377.714
Boorman, J., 1985, Rearing Culicoides obsoletus (Diptera, 715
Ceratopogonidae) on agar cultures of nematodes. 716
Progress in clinical and biological research 178, 229.717
Braveman, Y., Ungar-Waron, H., Frith, K., Alder, H., Danieli, 718
Y., Baker, K.P., Quinn, P.J., 1983, Epidemiological and 719
immunological studies of sweet itch in horses in Israel. 720
The Veterinary Record 112, 521-524.721
Braverman, Y., 1988, Preferred landing sites of Culicoides 722
species (Diptera: Ceratopogonidae) on a horse in Israel 723
and its relevance to summer seasonal recurrent 724
Page 30 of 47
Accep
ted
Man
uscr
ipt
30
dermatitis (sweet itch). Equine Veterinary Journal 20, 725
426-429.726
Broström, H., Larsson, Å., Troedsson, M., 1987, Allergic 727
dermatitis (sweet itch) of Icelandic horses in Sweden: 728
an epidemiological study. Equine Veterinary Journal 19, 729
229-236.730
Campbell, J.A., Pelham-Clinton, E.C., 1960, A taxonomic 731
review of the British species of" Culicoides" Latreille 732
(Diptera, Ceratopogonidae). Royal Society of 733
Edinburgh.734
De Raat, I.J., van den Boom, R., van Poppel, M., Sloet van 735
Oldruitenborgh Oosterbaan, M.M., 2008, The effect of a 736
topical insecticide containing permethrin on the number 737
of Culicoides midges caught near horses with and 738
without insect bite hypersensitivity in the Netherlands. 739
Tijdschrift voor Diergeneeskunde 2008 133, 20.740
Ferroglio, E., Pregel, P., Accossato, A., Taricco, I., Bollo, E., 741
Rossi, L., Trisciuoglio, A., 2006, Equine Culicoides742
hypersensitivity: evaluation of a skin test and of 743
humoral response. Journal of Veterinary Medicine 744
Series A 2006 53, 30-33.745
Halldorsdottir, S., Larsen, H.J., Mehl, R., 1989, Intradermal 746
challenge of Icelandic horses with extracts of four 747
species of the genus Culicoides. Research in veterinary 748
science 47, 283-287.749
Page 31 of 47
Accep
ted
Man
uscr
ipt
31
Hellberg, W., Wilson, A.D., Mellor, P., Doherr, M.G., 750
Torsteinsdottir, S., Zurbriggen, A., Jungi, T., Marti, E., 751
2006, Equine insect bite hypersensitivity: immunoblot 752
analysis of IgE and IgG subclass responses to 753
Culicoides nubeculosus salivary gland extract. 754
Veterinary Immunology and Immunopathology 2006755
113, 99-112.756
Kaul, S., 1998, Type I allergy in the horse. Basic development 757
of a functional in vitro assay (FIT). Thesis. Veterinary 758
University, Hannover, Germany.759
Langner, K.F.A., Darpel, K.E., Drolet, B.S., Fischer, A., 760
Hampel, S., Heselhaus, J.E., Mellor, P.S., Mertens, 761
P.P.C., Leibold, W., 2008, Comparison of cellular and 762
humoral immunoassays for the assessment of summer 763
eczema in horses. Veterinary Immunology and 764
Immunopathology 122, 126-137.765
Langner, K.F.A., Jarvis, D.L., Nimtz, M., Heselhaus, J.E., 766
McHolland, L.E., Leibold, W., Drolet, B.S., 2009, 767
Identification, expression and characterisation of a 768
major salivary allergen (Cul s 1) of the biting midge769
Culicoides sonorensis relevant for summer eczema in 770
horses. International Journal for Parasitology 39, 243-771
250.772
Page 32 of 47
Accep
ted
Man
uscr
ipt
32
Littlewood, J., 1998, Incidence of recurrent seasonal pruritus 773
(‘sweet itch’) in British and German shire horses. 774
Veterinary record 142, 66-67.775
Marti, E., Urwyler, A., Neuenschwander, M., Eicher, R., Meier, 776
D., de Weck, A.L., Gerber, H., Lazary, S., Dahinden, 777
C.A., 1999, Sulfidoleukotriene generation from 778
peripheral blood leukocytes of horses affected with 779
insect bite dermal hypersensitivity. Veterinary 780
Immunology and Immunopathology 71, 307-320.781
McCaig, J., 1973, A survey to establish the incidence of sweet 782
itch in horses in the United Kingdom. The Veterinary 783
Record 93, 444-446.784
Mellor, P.S., McCraig, J., 1974, The probable cause of" sweet 785
itch" in England. The Veterinary Record 95, 411-415.786
Mullens, B.A., Owen, J.P., Heft, D.E., Sobeck, R.V., 2005, 787
Culicoides and other biting flies on the Palos Verdes 788
Peninsula of Southern California, and their possible 789
relationship to equine dermatitis. Journal of the 790
American Mosquito Control Association 21, 90-95.791
Riek, R., 1954, Studies on allergic dermatitis (Queensland itch) 792
of the horse: the aetiology of the disease. Australian 793
Journal of Agricultural Research 5, 109-129.794
Schaffartzik, A., Marti, E., Crameri, R., Rhyner, C., 2010, 795
Cloning, production and characterization of antigen 5 796
like proteins from Simulium vittatum and Culicoides 797
Page 33 of 47
Accep
ted
Man
uscr
ipt
33
nubeculosus, the first cross-reactive allergen associated 798
with equine insect bite hypersensitivity. Veterinary 799
Immunology and Immunopathology 137, 76-83.800
Schaffartzik, A., Marti, E., Torsteinsdottir, S., Mellor, P.S., 801
Crameri, R., Rhyner, C., 2011, Selective cloning, 802
characterization, and production of the< i> Culicoides 803
nubeculosus</i> salivary gland allergen repertoire 804
associated with equine insect bite hypersensitivity. 805
Veterinary Immunology and Immunopathology 139, 806
200-209.807
Schurink, A., van Grevenhof, E.M., Ducro, B.J., van Arendonk, 808
J.A.M., 2009, Heritability and repeatability of insect 809
bite hypersensitivity in Dutch Shetland breeding mares. 810
Journal of animal science 87, 484-490.811
Sloet van Oldruitenborgh-Oosterbaan, M.M., 2006. Advances 812
in diagnosis and management of Culicoides813
hypersensitivity. In: Proceedings of the Annual 814
Congress of the British Equine Veterinary Association, 815
Birmingham: BEVA, pp. 176-177.816
Sloet van Oldruitenborgh-Oosterbaan, M.M., Van Poppel, M., 817
De Raat, I.J., Van Den Boom, R., Savelkoul, H.F.J., 818
2009, Intradermal testing of horses with and without 819
insect bite hypersensitivity in the Netherlands using an 820
extract of native Culicoides species. Veterinary 821
Dermatology 20, 607-614.822
Page 34 of 47
Accep
ted
Man
uscr
ipt
34
Takken, W., Verhulst, N., Scholte, E.J., Jacobs, F., Jongema, 823
Y., van Lammeren, R., 2008, The phenology and 824
population dynamics of Culicoides spp. in different 825
ecosystems in The Netherlands. Preventive veterinary 826
medicine 87, 41-54.827
Townley, P., Baker, K.P., Quinn, P.J., 1984, Preferential 828
landing and engorging sites of Culicoides species 829
landing on a horse in Ireland. Equine Veterinary Journal830
16, 117-120.831
Van der Rijt, R., Van den Boom, R., Jongema, Y., Van Sloet 832
Oldruitenborgh-Oosterbaan, M.M., 2008, Culicoides833
species attracted to horses with and without insect 834
hypersensitivity. The Veterinary Journal 178, 91-97.835
Van Grevenhof, E.M., Ducro, B., Heuven, H.C.M., Bijma, P., 836
2007, Identification of environmental factors affecting 837
the prevalence of insect bite hypersensitivity in 838
Shetland ponies and Friesian horses in the Netherlands. 839
Equine Veterinary Journal 39, 69-73.840
Wagner, B., Miller, W.H., Morgan, E.E., Hillegas, J.M., Erb, 841
H.N., Leibold, W., Antczak, D.F., 2006, IgE and IgG 842
antibodies in skin allergy of the horse. Veterinary 843
Research 37, 813-825.844
Wagner, B., Radbruch, A., Rohwer, J., Leibold, W., 2003, 845
Monoclonal anti-equine IgE antibodies with specificity 846
for different epitopes on the immunoglobulin heavy 847
Page 35 of 47
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ted
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ipt
35
chain of native IgE. Veterinary Immunology and 848
Immunopathology 92, 45-60.849
Wilson, A., Harwood, L., Björnsdottir, S., Marti, E., Day, M., 850
2001, Detection of IgG and IgE serum antibodies to 851
Culicoides salivary gland antigens in horses with insect 852
dermal hypersensitivity (sweet itch). Equine Veterinary 853
Journal 33, 707-713.854
Wilson, A.D., Harwood, L., Torsteinsdottir, S., Marti, E., 2006, 855
Production of monoclonal antibodies specific for native 856
equine IgE and their application to monitor total serum 857
IgE responses in Icelandic and non-Icelandic horses858
with insect bite dermal hypersensitivity. Veterinary 859
Immunology and Immunopathology 2006 112, 156-170.860
Wilson, A.D., Heesom, K.J., Mawby, W.J., Mellor, P.S., 861
Russell, C.L., 2008, Identification of abundant proteins 862
and potential allergens in Culicoides nubeculosus 863
salivary glands. Veterinary Immunology and 864
Immunopathology 122, 94-103.865
Yamashita, J., Kitamura, Y., Nakamura, R., 1957, Studies on 866
"Kasen" of horses in Hokkaido: IV. Researches on the 867
punkies in Hokkaido with description of a new species. 868
Japanese Journal of Veterinary Research 5, 89-96.869
870
871
872
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872
Legends to the figures873
874
875
Fig. 1. Coomassie staining of proteins from C. obsoletus extract 876
(O), C. nubeculosus (N) and C. sonorensis (S) whole body 877
extracts demonstrating similar quality of the extracts. M 878
represents the molecular weight marker.879
880
Fig. 2. Immunoblot analysis of 5 IBH affected and 5 healthy 881
horses using whole body extracts of C. obsoletus (O), C. 882
nubeculosus (N) and C. sonorensis (S). Proteins were separated 883
on 15 % SDS-PAGE gels and transferred to nitrocellulose 884
membranes. Binding of IgE from horse sera was detected with 885
an anti-equine IgE mouse mAb and HRP goat anti-mouse IgG. 886
The molecular weight marker (M) is indicated on the left in 887
kDa. 888
889
Fig. 3. Culicoides induced histamine release as percentage of 890
maximum release of 10 IBH affected horses and 10 healthy 891
control horses. Whole blood samples were analyzed after 892
stimulation with three different Culicoides whole body extracts893
(O = C. obsoletus, N = C. nubeculosus, S = C. sonorensis) 894
tested at three different concentrations (µg/ml, x-axis) by a 895
histamine release test (HRT). Results are presented in box 896
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plots. The horizontal line near the middle of the box is the 897
median of the measurements. The bottom and top of the box are 898
the 25th and 75th percentile, respectively. The end of the 899
whiskers represent the minimum and maximum value. The 900
stars represent the outliers. Horses with histamine release 901
above the cut off value of 10 % (highlighted in gray) were 902
considered positive (Kaul et al., 1998).903
Statistical analysis was performed on log transformed data. The 904
p values account for unequal variance. 905
906
Fig. 4. IgE levels presented as OD450nm values against three 907
different Culicoides extracts sera diluted 1:5 of 10 IBH affected908
horses and 10 healthy control horses. Results are presented in 909
box plots, for a definition see legend of Fig. 3. 910
Statistical analysis was performed on log transformed data. The 911
p values account for unequal variance. 912
913
Fig. 5. Comparison between histamine release test (HRT) and 914
ELISA of individual horses. Numbers 1-10 represent IBH 915
affected horses, horses 11 – 20 represent healthy control horses. 916
IgE levels (ELISA) are presented as OD450nm values; Histamine 917
release after stimulation with whole body extract at a 918
concentration of 0.5 µg/ml, is presented as percentage of 919
maximum release. The cut-off of each test is highlighted in 920
light gray and corresponds to 10 % for the HRT and mean + 3 921
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times standard deviation of the C. obsoletus values of the 922
healthy control horses for the ELISA. 923
924
Fig. 6 Validation of the Culicoides obsoletus-specific IgE 925
ELISA on serum samples of 100 healthy and 103 IBH-affected 926
horses927
a. Distribution plots of the healthy and IBH-affected horses928
categorized for their OD450nm ELISA reading. 929
b. Box plot of IgE levels of the healthy and IBH-affected 930
horses presented as OD450nm values The horizontal line near the 931
middle of the box is the median of the measurements. The 932
bottom and top of the box are the 25th and 75th percentile, 933
respectively. The end of the whiskers represent the minimum 934
and maximum value. The stars represent the outliers. The p 935
value accounts for unequal variance.’936
c. ROC curve of Culicoides obsoletus-specific IgE ELISA on 937
sera samples of 100 healthy and 103 IBH-affected horses.938
The area under the curve, indicating diagnostic accuracy of the 939
test was 0.97.940
d. Estimated probability to be IBH-affected in relation to the 941
OD450450nm value. Determined by logistic regression analysis 942
of the obtained ELISA data.943
944
945
946
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