Intradermal testing of horses with and without insect
hypersensitivity, using an extract of Culicoides
species trapped in the Netherlands.
Department of Equine Sciences, Faculty of Veterinary Medicine,
Utrecht University, the Netherlands
Drs. Miriam van Poppel (No. 0352551)
July - December 2007
Supervisors:
Dr. Robin van den Boom
Dr. Marianne M. Sloet van Oldruitenborgh-Oosterbaan
Contents
Abstract
p. 3
Samenvatting
p. 4
Introduction
p. 5
Materials and Methods
p. 7
Results
p. 10
Discussion
p. 17
Acknowledgements
p. 19
References
p. 20
2
Abstract
Equine insect hypersensitivity is currently diagnosed on the basis of characteristic clinical
symptoms and a compatible history. Intradermal allergy tests using a Culicoides
nubeculosus extract (Greer Laboratories) proved not to be reliable in identifying horses
with insect hypersensitivity in the Netherlands (Sloet 2006). Using tent traps covered
with mosquito netting, C. obsoletus and C. pulicaris were identified as the most common
species in the vicinity of horses in the Netherlands (94.1 % and 5.8% respectively) (Van
der Rijt et al. 2007). Therefore, it was the aim of the present study to evaluate the
usefulness of an extract of these ‘Dutch’ Culicoides species for the diagnosis of equine
insect hypersensitivity.
Thirteen pairs of horses were tested: one horse from each pair was known to suffer
from Culicoides hypersensitivity and the other had never shown any compatible
symptoms. Three concentrations of the ‘Dutch’ Culicoides whole body extract (1:1000
w/v, 1:10.000 w/v and 1:25.000 w/v) were tested together with a histamine solution (10
mg/ml) and phosphate buffered saline as control substances. Five 0.1 ml injections were
given intradermally on the mid region of the neck. Skin test reactions were evaluated
after 30 minutes and 1, 4 and 24 hours.
At all time points the absolute wheal diameter to the Culicoides antigen 1:1000 w/v
was significantly different (p< 0.01) between affected and unaffected horses. For
Culicoides antigen 1:10.000 w/v such difference was observed at 1 and 4 hours (p<0.05)
and at 24 hours (p<0.01), and for the 1:25.000 w/v solution only at 24 hours (p< 0.05).
Similar results were obtained at 30 min, 1 and 4 hours if ‘positive’ skin test reactions
were defined as those wheals having a diameter equal to or greater than the average of the
diameters of the wheals produced by the histamine and phosphate buffer controls.
In conclusion: This study indicates that the intradermal allergy test can support the
clinical diagnosis of equine insect hypersensitivity by using environment-related species
of Culicoides at a 1:1000 w/v concentration.
3
Samenvatting
Op dit moment wordt de diagnose van staart- en maneneczeem gebaseerd op de
karakteristieke klinische verschijnselen van de aandoening en een passende
ziektegeschiedenis. Een intradermale allergietest waarbij gebruik werd gemaakt van een
Culicoides nubeculosus extract (Greer Laboratories) bleek niet bruikbaar voor de
identificatie van paarden met staart- en maneneczeem in Nederland (Sloet 2006). Met
behulp van een tentconstructie met hier overheen muskietengaas, waren C. obsoletus en
C. pulicaris de meest geïdentificeerde soorten in de nabijheid van paarden in Nederland
(respectievelijk 94.1% en 5.8 %) (Van der Rijt et al. 2007). Het doel van dit onderzoek
was het testen van de bruikbaarheid van een extract van deze Nederlandse Culicoides
soorten in een intradermale test voor de diagnose van staart- en maneneczeem.
Dertien paren paarden werden getest: één paard van elk paar had een geschiedenis
van staart- en maneneczeem, het andere paard had geen verschijnselen van de
aandoening. Drie concentraties van het Nederlandse Culicoides extract (1:1000 w/v,
1:10.000 w/v en 1:25.000 w/v) werden getest samen met een histamine oplossing (10
mg/ml) en een fosfaat buffer als controle substanties. Vijf 0.1 ml injecties werden
intradermaal toegediend in de middenregio van de hals. De huidreacties werden
geëvalueerd na 30 minuten, 1, 4 en 24 uur.
Op alle tijdstippen was de absolute diameter van de reactie op het Culicoides antigeen
1:1000 w/v tussen paarden met en zonder staart- en maneneczeem significant
verschillend (p<0.01). Bij het Culicoides antigeen 1:10.000 w/v was er een significant
verschil op 1 en 4 uur (p<0.05) en op 24 uur (p<0.01). Bij het Culicoides antigeen
1:25.000 w/v was er alleen op 24 uur een significant verschil (p<0.05).
Vergelijkbare resultaten werden behaald op 30 minuten, 1 en 4 uur indien een
‘positieve’ reactie werd benoemd als de diameter van de reactie op het Culicoides extract
gelijk of groter is dan de gemiddelde diameter van de positieve en de negatieve controle.
Conclusie: Dit onderzoek geeft aan dat een intradermale allergietest de klinische
diagnose van staart- en maneneczeem kan ondersteunen door gebruik te maken van
omgevingsgerelateerde Culicoides soorten in een 1:1000 w/v concentratie.
4
Introduction
Equine insect hypersensitivity or allergic dermatitis is a common skin disorder in horses.
The disease is also known as Sweet itch, Summer eczema, Queensland itch or Kasen
(Broström and Larsson 1987; Anderson et al. 1988; Halldórsdóttir and Larsen 1991).
Currently, insect hypersensitivity is considered a multifactorial disease, with hereditary
an environmental factors involved in its pathogenesis (Steinman et al. 2003). It is
characterised initially by numerous papules or wheals, tufted hair and hyperaesthesia
which are followed by intense pruritis. Self inflicted trauma causes exfoliation, exudation
of serum and patchy alopecia (Anderson et al. 1988). If the disease becomes chronic it
may lead to hyperkeratosis, scaling and the formation of transverse ridges in the skin
(Riek 1953; Robinson 1983).
Insect hypersensitivity is caused by bites of insects. Culicoides species are likely to be the
most important causative agent (Riek 1954; Baker and Quinn 1978; Braverman et al.
1983; Quinn et al. 1983; Morrow et al. 1986; Greiner et al. 1988; Larsen et al. 1988;
Halldórsdóttir et al. 1989; Ungar-Waron et al. 1990; Perris 1995; Mullens et al. 2005),
although other insects, such as Simulium, Stomoxys and Haematobia, may also play a role
(Riek 1954; Baker and Quinn 1978; Braverman et al. 1983; Mullens et al. 2005; Perris
1995).
Insect hypersensitivity is mainly the result of a type I (immediate) hypersensitivity
reaction. Repeated exposure to antigen present in the biting insects’ saliva leads to an
immunoglobulin E (IgE) antibody response. Subsequent exposure to antigen leads to the
release of histamine by mast cells encountering IgE antigen complexes. However, there is
also evidence of a type IV (cell mediated) hypersensitivity component in insect bite
hypersensitivity, indicated by the formation of persistent papules lasting up to 72h
following the injection of antigen (Holmes 1990).
At present the diagnosis of insect hypersensitivity is based on history, clinical signs,
exclusion of other causes of pruritis and the response to therapy (Foster and Cunningham
1996; Rosenkrantz et al. 1998; Perris 1995; Fadok 1997). Important characteristics are
the seasonality of the condition and the recurrence in subsequent years.
In the past, several studies have been performed in order to produce a suitable
intradermal test to differentiate between horses with and without insect hypersensitivity.
Until now no uniform results were obtained. In a previous study in the Netherlands, an
intradermal test was performed on twenty horses. An extract produced by the Greer
Laboratories in the USA, containing Culicoides nubeculosis, was used for this test. In
this study no significant difference between horses with and without insect
hypersensitivity was found (Sloet 2006). A possible reason for this outcome could be the
fact that the Culicoides spp. used in this intradermal test is not the main cause of insect
hypersensitivity in the Netherlands.
In a preceding study (Van der Rijt et al. 2007) it was determined which species of
Culicoides are attracted to horses in the Netherlands. The vast majority of the Culicoides
5
spp. attracted to horses is C. obsoletus and a smaller number of C. pulicaris. These
species are most active during sunset and less so during sunrise.
The aim of the present study was to determine if an intradermal skin test based on these
specific Culicoides spp. is useful in the Netherlands. It was expected that horses with
insect hypersensitivity would display a stronger reaction to the native Culicoides antigen
than horses without insect hypersensitivity.
6
Materials and methods
Animals
Twenty-six horses were used in this experiment. Thirteen horses were affected with
insect hypersensitivity and 13 horses were unaffected.
Mares and geldings were equally distributed. The average age of the horses was 12 years
(5 to 25 years).
There were 20 Icelandic horses, four Shetland pony’s, one Friesian horse and one Dutch
Warmblood horse. The Icelandic horses and the Shetland pony’s were pastured 24 hours
a day throughout the year, without access to an open stable. The Friesian horse and the
Dutch Warmblood were stabled and turned out for a few hours a day.
The animals were kept on four different premises and on each location pairs of affected
and healthy horses were formed and tested.
The affected animals were categorized in two groups according to the severity of the
signs and the number of years during which they had displayed signs of insect
hypersensitivity (Stevens 1988).
Group 1
Initial signs
Mild to moderate
Group 2
Severe
Description
Mild to moderate signs:
rubbing, hair loss, skin
unbroken or broken. 2-7
years affected.
Severe signs: extensive
rubbing, extensive hair
loss, broken skin with
crusting, extreme
discomfort. > 7 years
affected.
Number of horses
8
5
Total
13
Table 1. Horses suffering insect hypersensitivity divided into two groups, according to the severity of the
signs.
Antigen preparation
An extract containing Culicoides obsoletus en C. pulicaris was produced in a laboratory
in Wageningen University by prof. H.F.J. Savelkoul. The Culicoides species were caught
in the Netherlands during the summer period of 2005 and 2006 and confirmed by dr. Yde
Jongema (Entomology, Wageningen University, The Netherlands). The trapping sessions
were carried out according to Van der Rijt et al. (2007) using a tent construction in which
bait horses were placed.
Since the Culicoides spp. had been stored in alcohol they had to be dried first. All flies
were collected in one big tube and they were centrifuged (5 min, 180 g). Most of the
alcohol was removed by pipetting. The remaining alcohol was removed by putting the
tube in a vacuum incubator for 2,5 hours at room temperature. 90 mg of dry Culicoides
spp. were obtained. To this, 6 ml of cold PBS was added, to which 4.5 μl of
phenylmethanesulfonyl fluoride solution (Sigma) was added. The flies were grinded in a
cold mortar with a pestle. The whole sample was transferred to a 50 ml tube and this was
7
spun down for 10 min at 4°C at 180 g. The supernatant was removed and sterilized by
filtration. After filtration, the concentration was measured using a Nanodrop
spectrophotometer at 280 nm. The concentration obtained was 0.95 mg/ml. The sample
was aliquoted in portions of 500 μl and frozen at -20°C.
The alcohol in which the Culicoides spp. were stored was also used to make an extract, to
test whether proteins from the flies did dissolve in the alcohol. The alcohol was
condensed by using a rotavapor. The sample was put in a round erlenmeyer, to which
four glass beads were added to prevent boiling. The erlenmeyer was put in a water bath
of 30ºC to prevent cooling of the sample. The system was placed under vacuum, and in 4
to 5 hours the alcohol was condensed. The precipitate that had formed was dissolved with
1 ml of PBS. The mixture obtained was filtrated and the concentration was measured on
the nanodrop spectrophotometer. The concentration was 2.89 mg/ml. This extract was
also stored at -20°C.
Intradermal test
The study was approved by the Dutch Animal Experiment Committee (DEC). The
intradermal skin test was performed from September to November 2007, during the
active season of Culicoides spp.. Skin testing was performed on unsedated horses in the
middle region of the neck, about 10 cm below the base of the mane of both healthy and
hypersensitive horses. The hair of the horses was clipped using an electric clipper blade,
the skin was cleaned with 70% alcohol and a permanent marker was used to indicate the
injection sites which were placed approximately five cm apart.
8
Five injections of 0.1 ml were placed intradermally using a 25Gx5/8” needle. Histamine
(Artuvetrin® Test) was used as a positive reference at a concentration of 1:1000 w/v. A
phosphate buffer (Artuvetrin® Test) was used as a negative reference. The Culicoides
extract was injected in three different concentrations: 1:1000 w/v, 1:10.000 w/v and
1:25.000 w/v.
The reaction was evaluated 30 minutes, 60 minutes, 4 hours and 24 hours after injection.
The wheal diameter was measured in millimetres (mm).
The firmness of the wheal was evaluated using a scale from 0 to +++ (Lebis et al. 2002).
0
+/+
++
+++
No palpable reaction
Very flat reaction with a badly defined outline
Reaction with just palpable thickness
Reaction with obvious thickness
Reaction with same thickness as histamine reference
Table 2. Meaning
of the firmness scale: -, +/-, +, ++ and +++.
Statistical
analysis
Statistical analysis
Results are given as mean ± sd. The results were statistically evaluated using the paired
Student-t test (p ≤ 0.05 was considered to be statistically significant).
The sensitivity and specificity of the test were explored for all used Culicoides extract
dilutions. The reaction in the horse was designated as positive, when the wheal diameter
evoked by the Culicoides extract minus the mean reaction to histamine and phosphate
buffered saline (PBS) was greater than zero.
9
Results
In all horses a visible and palpable wheal was noticeable immediately after injection. At
0.5 hour and 1 hour the wheals provoked by histamine were evident enough in all horses
to function as a positive reference value. After 4 hours the histamine wheals remained
clear in most horses, but in some horses the reaction to histamine had almost disappeared.
At 24 hours the wheals had disappeared in all horses, except one. Because at 24 hours the
histamine wheals had disappeared, it was impossible to compare the reactions to the
Culicoides antigen according to the positive reference.
The mean reaction to histamine was compared between horses with and without insect
hypersensitivity and they were not significantly different (Fig.1). The reactions to the
phosphate buffer were also equal in both affected and unaffected horses (Fig.2).
Mean histamine reactions
25,0
Insect hypersensitivity
Diameter (mm)
20,0
Control
15,0
10,0
5,0
0,0
0.5
1
4
24
Tim e
Fig 1. Mean histamine reactions in horses with and without insect
hypersensitivity. ± s.d.
Mean Phosphate buffered saline (PBS) reactions
14,0
Insect hypersensitivity
Control
12,0
Diameter (mm)
10,0
8,0
6,0
4,0
2,0
0,0
0.5
1,0
4,0
24,0
Time
Fig 2. Mean phosphate buffer saline (PBS) reactions in horses with and
without insect hypersensitivity. ± s.d.
10
1:1.000
1:1.000 Culicoides - avg. (histamine : buffer)
40
35
Control
30
**
25
**
25
***
Diameter (mm)
Diameter (mm)
**
Insect hypersensitivity
35
Control
30
20
40
**
Insect hypersensitivity
15
**
20
15
***
***
10
10
5
5
0
0
-5
0.5
0.5
1
4
24
1
4
24
-10
Time
Time
Fig 4. Wheal diameter of the reaction to
Culicoides antigen of 1:1000 w/v minus the
mean of the reaction to histamine and the
phosphate buffer in all horses. n=13. ± s.d.
** p < 0.01, *** p < 0.001.
Fig 3. Mean reaction to Culicoides antigen
1:1000 w/v in all horses. n=13. ± s.d.
** p < 0.01, *** p < 0.001.
At 0.5 hour, 1 hour, 4 hours and 24 hours the reaction to the Culicoides antigen 1:1000
w/v showed statistical difference between the horses affected and unaffected by insect
hypersensitivity (Fig.3).
When comparing the skin reaction to the Culicoides extract to the mean skin reaction
evoked by histamine and the phosphate buffer (PBS), a statistically significant difference
was present at all time points when using the Culicoides antigen concentration of 1:1000
w/v (Fig. 4).
Subsequently the results were analysed after dividing the horses into two different
categories according to the severity and duration of the signs of insect hypersensitivity.
Group 1 contained the mild to moderately affected horses and group 2 contained the
horses severely affected by insect hypersensitivity. At 1:1000 w/v the results of group 1
were statistically different between healthy and affected horses at 0.5 and 1 hour (Fig. 5)
and in group 2 at all time points (Fig. 6).
1:1.000 Group 2
1:1.000 Group 1
30
20
50
Insect hypersensitivity
45
Insect hypersensitivity
Controle
40
Controle
**
*
Diameter (mm)
Diameter (mm)
25
15
10
25
**
*
35
30
*
*
20
15
10
5
5
0
0
0.5
1
4
0.5
24
1
4
24
Time
Time
Fig 5. Mean reaction of group 1 (mildmoderately affected) to Culicoides antigen
1:1000 w/v. n=8. ± s.d.
* p < 0.05, ** p < 0.01.
Fig 6. Mean reaction of group 2 (severely
affected) to Culicoides antigen 1:1000 w/v.
n=5. ± s.d.
* p < 0.05, ** p < 0.01.
11
1:10.000 Culicoides - avg. (histamine : buffer)
1:10.000
20
40
Insect hypersensitivity
**
Insect hypersensitivity
Control
15
Control
**
30
25
*
*
20
Diameter (mm)
Diameter (mm)
35
**
15
10
10
**
5
0
0.5
5
1
4
24
-5
0
0.5
1
4
24
-10
Time
Time
Fig 7. Mean reaction to Culicoides antigen
1:10.000 w/v in all horses. n=13. ± s.d.
* p < 0.05, ** p < 0.01.
Fig 8. Wheal diameter of the reaction to
Culicoides antigen of 1:10.000 w/v minus the
mean of the reaction to histamine and the
phosphate buffer in all horses. n=13. ± s.d.
** p < 0.01.
At 1 hour, 4 hours and 24 hours there was a statistically significant difference between
the healthy and affected horses to the reaction of the Culicoides antigen 1:10.000 w/v
(Fig. 7).
When comparing the skin reaction of the Culicoides extract to the mean skin reaction
evoked by histamine and the phosphate buffer (PBS), there was a statistical difference at
1 hour, 4 hours and 24 hours (Fig. 8).
When comparing the two groups at 1:10.000 w/v, there was no statistical difference
between affected and unaffected horses in group 1 (Fig. 9). In group 2 there was a
statistically significant difference at 1 hour, 4 hours and 24 hours (Fig. 10).
1:10.000 Group 2
1:10.000 Group 1
40
40
Insect hypersensitivity
Insect hypersensitivity
35
Control
Control
30
30
Diameter (mm)
Diameter (mm)
35
25
20
15
25
***
15
10
10
5
5
0
*
*
20
0
0.5
1
4
24
0.5
Time
1
4
24
Time
Fig 10. Mean reaction of group 2
(severely affected) to Culicoides antigen
1:1000 w/v. n=5. ± s.d.
* p < 0.05, *** p < 0.001.
Fig 9. Mean reaction of group 1 (mildmoderately affected) to Culicoides antigen
1:1000 w/v. n=8. ± s.d.
12
1:25.000
1:25.000 Culicoides - avg. (histamine : buffer)
25
10
15
10
5
*
Insect hypersensitivity
Control
Control
Diameter (mm)
Diameter (mm)
20
15
*
Insect hypersensitivity
*
*
5
0
0.5
1
4
24
-5
0
0.5
1
4
-10
24
Time
Time
Fig 11. Mean reaction to Culicoides antigen
1:25.000 w/v in all horses. n=13. ± s.d.
* p < 0.05.
Fig 12. Wheal diameter of the reaction to
Culicoides antigen of 1:25.000 w/v minus the
mean of the reaction to histamine and the
phosphate buffer in all horses. n=13. ± s.d.
* p < 0.05.
When using the Culicoides antigen at 1:25.000 w/v, there was only a statistically
significant difference at 24 hours (Fig. 11).
When comparing the Culicoides reactions to the mean of the histamine and the
phosphate buffer (PBS) for all horses, there was a statistically significant difference at 1
hour, 4 hours and 24 hours using the 1:25.000 w/v concentration.
There was no statistical difference between affected and unaffected horses in group 1
and 2, except at 24 hours in group 2 (Fig. 13 and 14).
1:25.000 Group 2
1:25.000 Group 1
30
25
Control
25
Diameter (mm)
Diameter (mm)
20
*
Insect hypersensitivity
Insect hypersensitivity
15
10
Control
20
15
10
5
5
0
0
0.5
1
4
0.5
24
1
4
24
Time
Time
Fig 13. Mean reaction of group 1 (mildmoderately affected) to Culicoides antigen
1:25.000 w/v. n=8. ± s.d.
Fig 14. Mean reaction of group 2
(severely affected) to Culicoides antigen
1:25.000 w/v. n=5. ± s.d.
* p < 0.05.
13
The sensitivity and specificity of the test were explored for all used concentrations of the
Culicoides extract. The reaction in the horse was designated as positive, when the wheal
diameter evoked by the Culicoides extract minus the mean reaction to histamine and
phosphate buffered saline (PBS) was greater than zero. Table 3 shows the number of
positive reactions at the Culicoides extract 1:1000 w/v dilution at all time points. Table 4,
5 and 6 show the sensitivity and specificity of the test for all Culicoides extract
concentrations at the different time points.
Time point
30 minutes
1 hour
4 hours
24 hours
Affected horses
10
12
13
10
Unaffected horses
1
1
10
7
Table 3. Number of positive reactions at the different time points at the Culicoides extract
dilution 1:1000 w/v.
Time points
30 minutes
1 hour
4 hours
24 hours
Sensitivity
77%
92%
100%
77%
Specificity
92%
92%
23%
46%
Table 4. Sensitivity en specificity of the test at all time points at the Culicoides extract dilution 1:1000 w/v.
Time points
30 minutes
1 hour
4 hours
24 hours
Sensitivity
38%
62%
78%
62%
Specificity
78%
92%
62%
100%
Table 5. Sensitivity en specificity of the test at all time points at the Culicoides extract dilution 1:10.000 w/v.
Time points
30 minutes
1 hour
4 hours
24 hours
Sensitivity
8%
38%
46%
46%
Specificity
92%
85%
85%
92%
Table 6. Sensitivity en specificity of the test at all time points at the Culicoides extract dilution 1:25.000 w/v.
14
The firmness of the reactions to the used Culicoides concentrations at the four different
time points is shown in table 5, 6 and 7.
Insect hypersensitivity
Horse nr. 0.5 hr 1 hr
1
+++
+++
2
++
++
3
++
+++
4
++
+++
5
+++
+++
6
+++
+++
7
+++
+++
8
+++
+++
9
+++
++
10
+++
+++
11
+
+
12
+++
+++
13
+++
+++
4 hr
++
+/++
+++
+++
+++
+++
++
+/+++
+/++
+
No insect hypersensitivity
0.5 hr 1 hr 4 hr 24 hr
++
++
+++ +/+++
+++ ++
+/+/+/+
+
+
+
+
+
++
++
+++ +/++
++
+++ +/++
+
+
+
+/+
+++
+++ +
+/++
+++ +/+/+++
+++ +
++
+
+/+/-
24 hr
+
+/+++
++
++
++
+
+/+
++
Horse nr.
14
15
16
17
18
19
20
21
22
23
24
25
26
Table 5. Firmness of the wheals in horses with and without insect hypersensitivity at 1:1000 w/v. For the
interpretation of the wheal firmness scale ( -, +/-, +, ++ and +++): see table 2.
Insect hypersensitivity
Horse nr. 0.5 hr 1 hr
1
+
+
2
+
+
3
+++
+++
4
+++
+++
5
+++
+++
6
+
++
7
+++
++
8
+++
+++
9
+
+/10
+++
+++
11
++
++
12
+++
+++
13
++
+++
4 hr
+/++
+++
+++
+/++
++
+/++
+/+
+
No insect hypersensitivity
0.5 hr 1 hr 4 hr 24 hr
++
++
+
+
+/+/+/+/+
+/+/+/+/+
++
+/+++
++
+
++
+
+/++
++
+
++
+
+/+++
+++ +/+++
+++ +
++
+
+/-
24 hr
+
+
+
+
+/+
++
Horse nr.
14
15
16
17
18
19
20
21
22
23
24
25
26
Table 6. Firmness of the wheals in horses with and without insect hypersensitivity at 1:10.000 w/v. For the
interpretation of the wheal firmness scale ( -, +/-, +, ++ and +++): see table 2.
15
Insect hypersensitivity
Horse nr. 0.5 hr 1 hr
1
+
+
2
+
+/3
++
++
4
+++
+++
5
+++
+++
6
++
++
7
+
+
8
+++
+++
9
+
+/10
++
+
11
+
++
12
+++
+++
13
+
+/-
4 hr
+
+
+++
+++
+/+
+
+/+
-
No insect hypersensitivity
0.5 hr 1 hr 4 hr 24 hr
++
+
+
+/++
+
+/+/+/++
+
+/+
+
++
++
+/+++
++
+
++
+
+/+/+/++
+
+/+
+
+/+++
+++ +/+
+/-
24 hr
+/+
+
+/+
Horse nr.
14
15
16
17
18
19
20
21
22
23
24
25
26
Table 7. Firmness of the wheals in horses with and without insect hypersensitivity at 1:25.000 w/v. For the
interpretation of the wheal firmness scale ( -, +/-, +, ++ and +++): see table 2.
16
Discussion
In the past, several studies have been performed in order to produce a suitable
intradermal test to differentiate between horses with and without insect hypersensitivity.
Until now no uniform results were obtained (Halldórsdóttir et al. 1989; Kolm-Stark and
Wagner 2002; Fadok 1997; Ferroglio et al. 2006).
In a previous study in the Netherlands, an intradermal test was performed on twenty
horses. An extract produced by the Greer Laboratories in the USA, containing Culicoides
nubeculosis, was used. In this study no significant difference between horses with and
without insect hypersensitivity was found (Sloet 2006). A possible reason for this
negative outcome could be the fact that the Culicoides spp. used in the intradermal test is
not the main cause of insect hypersensitivity in the Netherlands. The negative results
could be attributed to a lack of cross-reactivity between Culicoides spp. (Sloet 2006;
Kolm-Stark and Wagner 2002) and the presence of a different antigenicity among
extracts of different Culicoides spp. Variations in results could also be caused by the
injection technique itself (Halldórsdóttir et al. 1989).
In a preceding study (Van der Rijt et al. 2007) it was determined that C. obsoletus and a
smaller number of C. pulicaris are the most common species of Culicoides attracted to
horses in the Netherlands.
When performing an intradermal skin test in the Netherlands using an extract
containing Culicoides obsoletus and C. pulicaris, it showed there was an increased
reactivity towards these antigens in horses affected with insect hypersensitivity,
compared with unaffected horses. This difference was most apparent when using the
extract at a concentration of 1:1000 w/v.
When using the extract 1:10.000 w/v there was no statistical difference between the
affected and control horses at 0.5 hour. An explanation could be that the concentration of
the antigen is too low to evoke a reaction after 0.5 hour in the affected horses to
differentiate them from the control horses.
When looking at the results of the Culicoides antigen 1:25.000 w/v the results were
only statistically different at 24 hours. However, the individual results showed that 6 out
of 15 affected horses showed a positive skin reaction and the other horses showed no
response. Concluding, this concentration was not useful in practice to differentiate
between affected and unaffected horses.
The sensitivity and specificity of the test were explored for all used concentrations of
the Culicoides antigen (table 4, 5 and 6). The reaction in the horses was designated as
positive, when the wheal diameter evoked by the Culicoides extract minus the mean
reaction to histamine and phosphate buffered saline (PBS) was greater than zero. The
horses which had a positive reaction according to the calculation above were designated
as horses affected with insect hypersensitivity (cut-off value). The concentration of the
extract at 1:1000 w/v at 1 hour seems to be the best cut-off value for this intradermal test,
because at this time point and at this concentration the greatest number of horses affected
with insect hypersensitivity could be designated as positive (12 out of 13 horses), while
only one of the unaffected horses was designated as positive. Using this cut-off value the
sensitivity of the test was 92% and the specificity was also 92%, which overall was
highest of all calculations (table 4, 5 and 6).
17
In a previous study in Norway 23 Icelandic horses were challenged with whole body
extracts of the following 4 species of Culicoides: C. pulicaris, C. chiopterus, C. obsoletus
and C. impunctatus. These midges were collected mainly in the south east and North
West of Norway. The tests were performed in January-February. Eight of nine unaffected
horses failed to respond to any of the four antigens, the remaining animal responded to
two of the four antigens. Ten of the 14 affected horses responded to at least three of the
four antigens, while two of the animals in this group failed to respond to any. The mean
responses to C. chiopterus, C. obsoletus and C. impunctatus, read after 30 min, 60 min
and four hours were significantly higher in the affected horses than in the unaffected
horses. A significant difference was also found in the mean respons to C. chiopterus and
C. impunctatus, read after 24h (Halldórsdóttir et al. 1989). These results are similar to the
results in our study. This seems to suggest that the particular Culicoides species used in
the intradermal test is important.
A study performed in the USA tested horses for insect, grass and mould
hypersensitivitiy. The Culicoides extract was prepared by the allergy laboratory (Greer
Laboratories) from a colony of C. variipennis. Less than 25% of normal horses reacted
tot Culicoides extracts at 250 PNU/ml. Approximately 80% of all hypersensitive horses
showed positive reactions to at least the highest dilution of the Culicoides extract (Fadok
1997).
In Italy intradermal tests were carried out on 18 horses with clinical signs of
Culicoides hypersensitivity and 23 horses without clinical signs. In this study an extract
of C. obsoletus, caught in the USA, was used. The results with a 25µg/µL of Culicoides
extract showed that, even in winter when clinical signs were absent, all affected horses
are characterized by the presence of, at least, a 1 cm welt and a skinfold thickness
increase >10% as cut-off at 24 h. A significant reaction was absent in the unaffected
horses. The conclusion made by the authors of this study was that the increase of skinfold
thickness at five and 24 hours and the fact that differences between Culicoides extract
and histamine were significant only at 24h, confirmed the presence of both immediate
(type I) and delayed (type IV) hypersensitivity reactions in affected horses and suggests
the validity of this skin test for diagnosis (Ferroglio et al. 2006). These findings are
similar to those found in the present study (especially those obtained in the severely
affected group).
A study in Austria tested a standardized extract of C. variipennis and 21 other
allergens relevant within Austria on 81 Icelandic horses. C. variipennis evoked a positive
cutaneous reaction in one of 38 normal and three of the 43 affected horses at the dilution
of 1:50.000 or 1:25.000 and in 24 of 38 normal and 13 of 43 affected horses at a dilution
of 1:10.000. No significant differences between the two groups were found in the
reactions of the other 21 allergens. A possible cause for the few responses in the present
study might be the lacking of cross-reactivity, as C. variipennis is not a species prevailing
in Austria (Kolm-Stark and Wagner 2002), or not using an optimal concentration of the
extract in this study, as we found the strongest reactions using a 1:1000 w/v dilution of
the Culicoides extract.
The reactivity of the skin depends basically on two variables; the allergen concentration
of the extract and the sensitivity of the individual (Halldórsdóttir et al. 1989). In the
current study the results were compared between two different groups. This was done to
18
find out if there was a relationship between the intensity of reaction to the Culicoides
extract and the severity and duration of insect hypersensitivity between the different
groups. It showed that group 2 (containing the most severe cases of insect
hypersensitivity) reacted at more time points than group 1 in all used concentrations of
the Culicoides extract. Group 2 had more significantly different reactions compared to
the control horses than group 1, especially at the later time points.
The firmness of the wheals provoked by the Culicoides extract was also assessed, but
these results were not statistically evaluated. However when looking at these results
(Table 5, 6 and 7) it seems evident that horses with insect hypersensitivity have an overall
higher firmness score at the different time points and Culicoides concentrations,
compared to horses without insect hypersensitivity.
In this study horses with and without insect hypersensitivity were compared during the
active season of Culicoides spp. Because the results found were promising, it would be
interesting to repeat the test during the inactive season of Culicoides spp. to establish if
the horses react in a similar way as during the active season of Culicoides spp.
Also in future studies the extract itself can be made more suitable for intradermal
testing in horses. The extract used in the current study contained C. obsoletus and C.
pulicaris. To improve the use of an intradermal skin test in horses, one could produce an
extract containing only one Culicoides species at a time. Substantial quantities of
different non-allergenic components present in the extract may act as irritants and give
false positive reactions (Halldórsdóttir et al. 1989). To minimize the influence of these
non-specific irritants, Culicoides engorged with mammal blood (presumably from horses)
can be left out of the extract, to minimize reaction to allergens not belonging to
Culicoides spp.
A few horses without insect hypersensitivity reacted more to the Culicoides antigen
dilutions than we had expected. A possible reason for the more than average reaction to
Culicoides extracts in some horses without known insect hypersensitivity, could be that
these horses are apparently asymptomatic cases of insect hypersensitivity, small airway
inflammatory disease or atopy (Evans et al. 1992; Kolm-Stark and Wagner 2002), which
have been suggested to give positive reactions to intradermal testing. In future studies
horses could be further examined for the prevalence of these asymptomatic diseases.
Concluding: this study shows that it is beneficial to adjust intradermal tests, used for the
diagnosis of insect hypersensitivity, to the specific species of Culicoides which cause
insect hypersensitivity in the concerning country. The authors of this study found the
Culicoides concentration of 1:1000 w/v most useful for intradermal testing in horses.
Acknowledgements
We would like to thank all horse owners for letting us use their horses for this
experiment, and their cooperation during the study. Further we would like to thank our
supervisors dr. R. van den Boom and dr. M.M. Sloet van Oldruitenborgh-Oosterbaan for
their help in performing the study and writing this report.
19
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