The effect of a topical repellent containing permethrin
on the number of Culicoides midges caught near horses
with and without insect hypersensitivity in the
Netherlands.
Department of Equine Sciences, Faculty of Veterinary Medicine,
Utrecht University, the Netherlands
Drs. I.J. de Raat (No. 0352578)
July - December 2007
Supervisors:
Dr. Robin van den Boom
Dr. Marianne M. Sloet van Oldruitenborgh-Oosterbaan
Summary
Insect hypersensitivity in horses is most likely caused by Culicoides spp., although other
insects may also play a role. Until now no effective cure has been found for this condition.
There are numerous therapeutic and preventive measures used in the control of insect
hypersensitivity. One of the preventive methods which can be used to prevent insects biting
horses is a topical insecticide.
In this study the effect of a topical repellent containing permethrin was examined on
horses. Seven pairs of horses were used in this experiment. During two subsequent evenings
the Culicoides spp. and other flying insects attracted to the horses were captured. The horses
were placed inside a tent trap to collect Culicoides spp. and other insects attracted to the
horses. The first evening both horses of the pair were untreated. After the first capture one of
the horses was treated with the pour-on repellent. The capture session was repeated the next
day. This method seems suitable for testing insecticides on horses.
Similar percentages of Culicoides were trapped as in earlier studies in the Netherlands (C.
obsoletus 95.3% and C. pulicaris 4.5%). A reduction in numbers of Culicoides spp.,
percentage of bloodfed Culicoides obsoletus and total number of insects attracted to horses 24
hours after treatment with permethrin, did not reach a statistically significant difference.
During the study no negative side effects following the administration of permethrin were
found.
2
Samenvatting
Staart- en maneneczeem bij het paard wordt waarschijnlijk veroorzaakt door Culicoides spp.,
hoewel andere insecten ook een rol kunnen spelen. Tot nu toe is er geen effectieve
genezingsmethode gevonden voor deze aandoening. Er zijn verschillende therapeutische en
preventieve maatregelen die gebruikt worden om staart- en maneneczeem onder controle te
houden. Een van de preventieve methoden is een topicaal insecticide.
In dit onderzoek is de werking van een permethrin bevattend topicaal repellent onderzocht
bij het paard. In dit experiment zijn 7 paar paarden gebruikt. Gedurende twee opeenvolgende
avonden werden de Culicoides spp. en andere vliegende insecten die op de paarden afkwamen
gevangen. De paarden werden in een tentconstructie geplaatst om zo de insecten in de
omgeving van de paarden te verzamelen. Op de eerste avond waren beide paarden van het
paar onbehandeld. Na de eerste vangsessie werd één van de paarden behandeld met de pouron repellent. De vangsessie werd op de tweede dag herhaald. De bovenstaande methode lijkt
geschikt te zijn om de werking van insecticiden bij paarden te beoordelen.
De percentages van Culicoides die werden gevangen waren vergelijkbaar met eerdere
studies in Nederland (C. obsoletus 95.3% en C. pulicaris 4.5%). Er werden geen statistisch
significante verschillen gevonden in aantallen Culicoides spp., percentage Culicoides
obsoletus volgezogen met bloed en totale aantallen insecten aangetrokken tot paarden 24 uur
na behandeling met permethrin. Er zijn gedurende het onderzoek geen negatieve effecten
geconstateerd na de behandeling met permethrin.
3
Introduction
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). Insect hypersensitivity
is caused by bites of insects. Culicoides species are likely to be the most important causative
agent, although other insects, such as Simulium, Stomoxys and Haematobia, may also play a
role (Fadok and Greiner 1990, McMullen 1982). The condition 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 1988; Broström and Larsson 1987; Halldórsdóttir and Larsen 1991).
Until now no effective cure has been found for this condition. There are numerous
therapeutic and preventive measures used in the control of insect hypersensitivity. One of the
preventive methods which can be used to prevent insects biting horses is a topical insecticide.
In this study the effect of a topical repellent containing permethrin was examined on
horses. Permethrin is a synthetic pyrethroid and it is a moderately persistent insecticide that
has been used in veterinary medicine to control Stomoxys and other biting insects (Kenneth
1990).
In order to evaluate the effect of the repellent, the number of midges and other insects
attracted to horses was counted. A tent trap containing a bait horse was used to collect the
Culicoides spp. Also, the percentage of blood feeding Culicoides was assessed.
4
Materials and methods
Animals
Fourteen Icelandic horses were used in this experiment. Ten horses were affected with insect
hypersensitivity and four horses were unaffected. All the horses were kept under the same
conditions and were pastured 24 hours a day throughout the year, without access to an open
stable. Ten mares and four geldings were used, ranging from 9 to 25 years of age, with an
average of 16.2 years.
Repellent containing permethrin
A permethrin containing pour-on repellent was used on horses. The product (Tectonika)
contained 36g/l permethrin and is registered in the Netherlands for use on cattle.
Fig. 1. C. Pulicaris
Fig. 2. C. obsoletus non-bloodfed
Fig. 3. C. obsoletus bloodfed.
Experimental setting
Five pairs of horses affected with insect hypersensitivity and two pairs of unaffected horses
were formed. During two subsequent evenings the Culicoides spp. and other flying insects
attracted to the horses were captured. The first evening both horses of the pair were untreated.
After the first capture session one of the horses was treated with the pour-on repellent.
Twenty ml of the permethrin containing repellent was poured over the dorsal midline of the
horse, from behind the ears to the proximal part of the tail. The number of insects attracted to
six of the seven pairs of horses was assessed again after 24 hours. Only one pair of horses was
assessed for the second time after 48 hours, because bad weather conditions made it
impossible to perform the capturing procedure after 24 hours. The treated and untreated
horses were kept in different pastures, to prevent the permethrin being transferred from one
horse to another.
Collection of insects
In the current study the experimental setting designed by Van der Rijt et al. (2007) was used
in order to collect the Culicoides spp. and other flying insects. During sunset the horses were
each placed inside separate tent traps placed 1 m apart. The tent traps consisted of a strong
metal frame secured to the ground using metal pegs and covered with a large mosquito net
(mesh opening of about 200µm). The back of the tent was kept open and the rest of the
5
netting remained on the ground. During sampling the horses were held by a handler inside the
tent.
After 30 minutes the horses were taken from the traps and the tents were closed. The
trapped insects were collected using a small vacuum cleaner (1400W, lowest power setting)
with a small piece of mosquito net placed in the tubing as a filter. The lowest power setting
was used, to prevent the insects from being damaged. The insects were placed in test tubes
with 70% ethylethanol solution until further processing.
Figure 4. Setting of tent traps containing bait horses and collection of insects using a vacuum cleaner.
Sample processing
The insects were identified (Oosterbroek et al. 2005) and counted manually. The insects were
placed in a Petri dish and examined with a stereo microscope. Culicoides pulicaris, bloodfed
and non-bloodfed C. obsoletus, other Culicoides spp. and other insects were put in separate,
labelled eppendorf cups.
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).
6
Results
The number of Culicoides spp. and other insects caught is shown in Table 1. The large
majority of the Culicoides was found to be C. obsoletus (21133/22166 = 95.34%). A smaller
number was C. pulicaris (1007/22166 = 4.54%).
The mean number of insects caught and the percentage of engorged C. obsoletus was
compared between the treated and untreated (control) horses. The capture sessions of the
treated horses on day 2, were compared with the capture sessions of the same horses on day 1,
before they had been treated with permethrin (fig. 5). The same was done for the control
horses. The treated horses and untreated (control) horses were also compared to each other on
both evenings. This was done to rule out weather influences (fig. 6).
No statistically significant difference was found between the horses treated with permethrin
and the untreated horses.
In this study seven horses were treated with permethrin and no negative side effects
following the administration of the permethrin pour-on were found. Attention was paid to
local effects on the skin such as itching, erythema, swollen skin, alopecia, and lesions.
General effects like ataxia, tremors, etc. were also not observed during the study.
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
Day 1
Day 2
A-1
B-1
A-2
B-2
C-1
D-1
C-2
D-2*
E-1
F-1
E-2*
F-2
G-1
H-1
G-2*
H-2
Total # insects
718
775
666
1171
1263
1175
592
255
459
534
1206
977
598
1375
2203
637
C. obs Total
233
381
503
605
922
871
419
128
235
273
754
699
158
279
504
162
113
120
48%
265
116
70%
359
144
71%
346
259
57%
617
305
67%
404
467
46%
266
153
63%
47
81
37%
159
76
68%
173
100
63%
448
306
59%
430
269
62%
105
53
66%
207
72
74%
397
107
79%
120
42
74%
13
9
7
3
42
31
11
1
10
11
102
65
12
21
10
5
C. obs. bloodfed
C. obs. non bloodfed
C. obs. % bloodfed
C. pulicaris
Day 1
Total # insects
C. obs Total
C. obs. bloodfed
C. obs. non bloodfed
C. obs. % bloodfed
C. pulicaris
Day 2
I-1
J-1
I-2*
229
172
120
59
43
6
38
21
64%
29
14
67%
3
3
50%
11
6
0
J-2
Day 1
Day 2
Day 1
Day 2
K-1
L-1
K-2*
L-2
M-1
N-1
M-2
N-2*
930
556
355
517
4387
3189
2532
3899
0
676
327
119
264
3938
2785
2278
3512
0
0
0%
376
300
56%
147
180
45%
77
42
65%
180
84
68%
1438
2500
37%
1424
1361
51%
895
1383
39%
1926
1586
55%
11
2
0
0
235
203
47
141
75
0
Table 1. The total number of other insects, the total number of Culicoides obsoletus, the number of bloodfed and empty C.
obsoletus caught, the percentage of blood fed C. obsoletus and the number of C. pulicaris caught. Every character represents
one horse.
* Capture of horse treated with permethrin. A to J represent horses affected with insect hypersensitivity, K to N unaffected
horses. All horses were assessed 24 hours after treatment, except K and L which were assessed after 48 hours.
7
Mean Culicoides obsoletus caught
Mean Culicoides obsoletus caught
2500
2500
Untreated
Before treatment
2000
2000
After treatment
1500
1500
1000
1000
500
500
Treated
0
0
C. obs. Bloodfed
C. obs. NonBloodfed
C. obs. Total
Figure 5. The mean of bloodfed, nonbloodfed and total numbers of Culicoides
obsoletus caught before and after treatment
of the tested group of horses. ± s.d.
C. obs.
Bloodfed
C. obs. NonBloodfed
C. obs. Total
Figure 6. The mean of bloodfed, non-bloodfed
and total numbers of Culicoides obsoletus caught
near treated and non-treated (control) horses on
day 2. ± s.d.
Figure 7. Percentage of insects attracted to horses on day 2, compared to day 1 (= 100%).
8
Figure 8. Percentage of Culicoides obsoletus attracted to horses on day 2, compared to day 1 (=100%).
% of bloodfed Culicoides obsoletus
Day 1: before treatment
90%
Day 2: after treatment
80%
70%
60%
50%
40%
30%
20%
10%
0%
A
D
E
G
I
K
M
Horses
Figure 9. Percentage of bloodfed Culicoides obsoletus on day 1 (before treatment) and day 2 (after
treatment).
9
In figure 7 the total number of insects caught on day 1 of both horses of the pair was set at
100%. The number of insects caught on day 2 of the treated and untreated horses was
expressed as a percentage (related to day 1). Formula: ((100/ # insects day 1) * # insects day
2). Treatment had a positive effect on four pairs of horses (A-B, C-D, K-L and M-N). In these
pairs all of the treated horses had fewer insects on day 2 compared to day 1.
The same was done for the total number of Culicoides obsoletus. This is shown in figure
8. The treatment had a positive effect on three out of seven pairs of horses (C-D, K-L, M-N).
In figure 9 the percentage of bloodfed Culicoides obsoletus is shown on day 1 (before
treatment) and on day 2 (after treatment). In horses D, E and I lower percentages bloodfed C.
obsoletus were caught on the day after treatment with permethrin. In the other horses higher
percentages of bloodfed C. obsoletus were caught on the second day.
Mean C. obsoletus bloodfed
2500
*
2000
1500
Untreated
Treated
1000
500
0
All horses
Figure 10a. Mean number of Culicoides
obsoletus caught near treated and untreated horses
on day 2. *p < 0.05
Insect
hypersensitivity
No insect
hypersensitivity
Figure 10b. Mean number of bloodfed
Culicoides obsoletus caught near treated and
untreated horses on day 2.
In figures 10, 11 and 12 the horses with and without insect hypersensitivity were evaluated
separately.
Figure 10 shows the results between treated and non treated horses on day 2. In the horses
with insect hypersensitivity the mean number of Culicoides obsoletus caught was almost
equal. The horses without insect hypersensitivity attracted a larger number of Culicoides
obsoletus. In this group the treated horses attracted fewer C. obsoletus than the untreated
horses.
10
Mean C. obsoletus (total)
2500
Mean C. obsoletus bloodfed
Before treatment
After treatment
2500
Before treatment
After treatment
2000
2000
1500
1500
1000
1000
500
500
0
All horses
Insect
hypersensitivity
No insect
hypersensitivity
Figure 11a. Mean number of Culicoides
obsoletus caught before and after treatment.
0
All horses
Insect hypersensitivity
No insect
hypersensitivity
Figure 11b. Mean number of bloodfed
Culicoides obsoletus caught before and after
treatment.
In figure 11 the same horses were evaluated before and after treatment. Evident in this figure
is that the horses without insect hypersensitivity attracted fewer Culicoides obsoletus after
treatment and overall they attracted more C. obsoletus than the horses with insect
hypersensitivity.
Figure 12 shows the percentage of bloodfed Culicoides obsoletus in horses with and
without insect hypersensitivity. In horses without insect hypersensitivity a lower percentage
of Culicoides obsoletus had fed, although the absolute number of bloodfed C. obsoletus was
significantly lower in affected horses.
Percentage bloodfed Culicoides obsoletus
70%
60%
50%
40%
30%
Insect hypersensitivity
No insect hypersensitivity
20%
10%
0%
Figure 12. Mean percentage of bloodfed Culicoides
obsoletus in the group of horses with and without insect
hypersensitivity (both treated and untreated horses).
11
Discussion
In this study the effect of a (3.6%) permethrin containing pour-on repellent was tested for
usability on horses. The product used contains 36g/l permethrin and is registered in the
Netherlands for use on cattle. Permethrin is a synthetic pyrethroid and it is a moderately
persistent insecticide that has been used in veterinary medicine to control Stomoxys and
similar biting flies (Kenneth 1990). The rapid knock-down effect on flying insects possessed
by all natural and some synthetic pyrethroids strongly suggests that these insecticides have a
neurotoxic action. Several modes of action of pyrethroids have been suggested by different
authors (Kenneth 1990). The most accepted principle is that they bind to the voltage-gated
sodium channel in nerves. This protein opens causing stimulation of the nerve and closes to
terminate the nerve signal. Pyrethroids bind to this gate and prevent it from closing normally
which results in continuous nerve stimulation. This explains the tremors exhibited by
poisoned insects. They lose control over their nervous system and are unable to produce
coordinated movement (Valles and Koehler 2003).
In this study a tent trap was used to evaluate the effect of a repellent in horses. The advantage
of this method is that insect species which are specifically attracted to horses can be caught
without being damaged. The large majority of the Culicoides were found to be C. obsoletus
(95.34%). A smaller number was C. pulicaris (4.54%). These results are in agreement with
the results found by Van der Rijt et al. (2007) and Townley et al. (1984). Although the results
suggest that the repellent containing permethrin had a positive effect on repelling Culicoides
obsoletus, these differences were not statistically significant. In further research the tested
group of horses could be enlarged, to see if this has an effect on the results.
All horses were scored for the second time after 24 hours, except for K and L. These
horses were assessed after 48 hours. It seems that treatment with permethrin did have a
positive effect on repelling insects in these two horses. It could be that permethrin has not
fully spread over the horses’ body after 24 hours. Alternatively, the study could be repeated
after treating/spraying the whole body.
The treated horses without insect hypersensitivity seem to react better to the treatment with
permethrin, compared to the treated horses with insect hypersensitivity. Horses without insect
hypersensitivity attracted more Culicoides obsoletus than horses affected with insect
hypersensitivity (Fig. 10 and Fig. 11). This is in agreement with the study by Van der Rijt et
al. (2007). However, in the current study only two pairs of horses without insect
hypersensitivity were used. The relatively high number of Culicoides obsoletus caught near
the horses without insect hypersensitivity could be attributed to weather influences beneficial
for the Culicoides spp. on these days. In order to confirm a relationship between the
occurrence of insect hypersensitivity and the number of Culicoides attracted to horses, a
different experimental setup should be used. Pairs of horses with and without insect
hypersensitivity should be tested at the same time, to rule out weather influences.
In the past little research has been performed on the use of permethrin on horses. Previous
reports have shown that permethrin sprays protect horses against Culicoides and other biting
flies such as Stomoxys for up to 10 days (Blackman and Hodson 1977; Lang et al. 1981).
In a study by Schmidtmann (2001), the repellency of several commercially available
synthetic pyrethroid insecticides, with permethrin as the active ingredient, was tested against
the blood-feeding of mosquitoes and black flies from cattle and ponies. They used a similar
experimental setting as in the present study by comparing the blood-feeding rates of insects
between treated and non-treated animals. They also used an animal enclosure trap sample
12
system. Blood feeding of Simulium bivittatum from ponies treated with a permethrin fly wipe
was reduced significantly by 98% and 99% at one and seven days post treatment respectively.
In another study a four per cent permethrin pour-on (cis:trans = 80:20) was tested for the
control of insect hypersensitivity on horses (Stevens et al. 1988). Thirty to forty ml of the
permethrin solution was applied along the backline of the horses. Of the 43 animals which
had signs before treatment, 11 were healed and 26 were much improved. Overall the authors
claimed an 86% positive response to treatment. In those authors’ experiments, one to three
applications a week of permethrin pour-on gave good results.
In the current study, seven horses were treated with permethrin and no side effects occurred
after treatment. Known general side effects following the use of pyrethrines are:
hypersensitivity, tremors, ataxia, vomiting (not very likely in the horse) and convulsions
(Reigart and Roberts 1999). Also, topical adverse reactions have been reported in animals. In
dogs erythema, itching and alopecia on the side of application, but also on other skin areas,
were reported (Tjälve 1997). In an experimental setting two out of 50 horses reacted adversely
to treatment with permethrin (Stevens et al. 1988). The signs were itching and swollen skin
and those signs disappeared within two days of stopping treatment.
In an earlier study (Mullens et al. 2000) the persistence of permethrin (5%) applied to the
dorsum of calves in the field against Culicoides sonorensis was estimated using a hair-bloodfeeding bioassay in the laboratory. Hair clippings were taken before treatment and several
days after treatment from the dorsum, side and belly of treated and control calves. In the
laboratory insects were allowed to feed through thin hair layers and a parafilm membrane on
warmed sheep blood. Some intoxication after exposure to hair was noted up to 28 days after
treatment with permethrin. Hair from the dorsum caused more intoxication for a longer period
than hair from other body regions. Residues on belly hair did not significantly reduce feeding
at any time, suggesting that permethrin is not distributed to all parts of the body.
Field trials using treated and control calves and enclosure nets showed that dorsal
applications of five % permethrin were not effective in reducing engorgement, despite some
intoxication of the insects. A possible explanation for this outcome could be that the preferred
feeding sites of C. sonorensis on calves are the belly and upper legs. A 0.2% permethrin spray
application on the ventral abdomen (250 ml) did result in >80% reduction of C. sonorensis at
3 and 7 days after treatment, but activity had subsided by 10 days after treatment.
Culicoides spp. have different preferred feeding sites on animals. There are few references to
the preferential biting sites of midge species on animal hosts. Campbell and Pelham-Clinton
(1960) reported that collections from the back of horses and cattle consisted almost entirely of
C. pulicaris. In Ireland one horse was used as a bait animal to attract Culicoides species. Ten
species landed on the bait horse and seven species engorged. Culicoides obsoletus and C.
dewulfi were the species most commonly attracted, representing 89.8 per cent of those landing
and 90.05 per cent of midges engorging respectively. Of the midges which were attracted to
the bait, 4.69 per cent were C. pulicaris. Landing occurred on all areas of the body. Biting
occurred predominantly on the predilection sites of insect hypersensitivity lesions where 74.3
per cent of the midges engorged, 49.4 per cent engorging on the mane region (Townley et al.
1984). In other research it was found that C. obsoletus fed almost exclusively on the belly of
cattle and approaches the animal from below (Kettle 1977). Because results in the literature
are contradictory, more research needs to be done to establish the major feeding sites of C.
obsoletus.
Because the horses in this study were treated with a permethrin pour-on over the dorsal
midline of the horse, from behind the ears to the proximal part of the tail, it is possible that the
13
concentration of permethrin is not high enough at major feeding sites of C. obsoletus. This
may be the reason that the percentage of blood fed C. obsoletus in this study was not
significantly different to the percentages of the control horses.
A solution to this problem could be the application of permethrin spray, which can be
used on all body regions. This may contribute to a better distribution of the permethrin over
the horses’ body, preventing the Culicoides species from biting.
The Icelandic horses in this study were treated with 20 ml of the permethrin containing
repellent. This dosage was extrapolated from the dosage of this product, normally used for
cattle. Because there were no adverse effects observed at this dosage in horses, it may be
possible to use a slightly larger volume in future studies to find out if better results can be
obtained.
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 want to thank Barend Blankenstein for
taking the beautiful pictures of our Culicoides spp. and 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.
a
Virbac Animal Health, Barneveld, the Netherlands
14
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