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Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP) a
b
M.A. Wisselink , J.P. Koeman , T. S. G. A. M. van den b
Ingh & A. Willemse
a
a
Department of Clinical Sciences of Companion Animals , University of Utrecht , PO Box 80 154, Utrecht, 3508 TD, The Netherlands b
Department of Veterinary Pathology , University of Utrecht , The Netherlands Published online: 01 Nov 2011.
To cite this article: M.A. Wisselink , J.P. Koeman , T. S. G. A. M. van den Ingh & A. Willemse (1990) Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP), Veterinary Quarterly, 12:1, 21-28, DOI: 10.1080/01652176.1990.9694237 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694237
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP)
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M. A. Wisselinkl, J. P. Koeman2, T. S. G. A. M. van den Ingh2, and A. Willemsel SUMMARY Skin reaction patterns to the intradermal injection of a whole-body flea extract were examined in five physically healthy dogs and in 24 dogs with German Shepherd dog Pyoderma (GSP) at 15 and 30 minutes and at 1, 2, 4, 8, 24, 48 and 72 hours after the injection. In 10 out of 24 GSP dogs a positive skin reaction was observed macroscopically after 15 minutes. Delayed reactions at 24 or 48 hours were not observed. In the control group neither immediate nor delayed reactions were observed. The histopathologic skin changes were basically the same in both groups: an initial polymorphonuclear reaction followed by a mononuclear cell reaction. In the GSP dogs, however, these changes occurred earlier and were more prolonged than in the normal dogs. No flea-antigen-specific IgGd antibodies could be demonstrated by enzymelinked immunosorbent assay. It is concluded that delayed type hypersentitivity to flea antigen does not play a role in the pathogenesis of GSP Immediate type hypersensitivity may contribute to the disease in some cases.
INTRODUCTION
German Shepherd dog Pyoderma (GSP) has been defined as a chronic deep pyoderma in pure-bred and cross-bred German shepherds. The disease is
characterised by multiple deep-seated skin lesions with a typical morphology and distribution. Coagulase-positive staphylococci (CPS) are commonly found upon bacteriological examination (22). The primary lesions of GSP are pruritic and
located in the dorsolumbosacral region. This location is also known as the
preferred area for flea bites and flea-bite alleigy (5, 6, 8, 9, 10, 13, 14). In addition, more than half of the cases of GSP showed immediate skin-test reactivity to flea antigen. These findings have led to the hypothesis that flea-bite hypersensitivity (FBH) is a predisposing factor for GSP (22). In this study, the role of flea antigen in the pathogenesis of GSP was investigated by examining the macroscopic and histopathologic changes at skin-test sites at different time intervals following intradermal injections with flea antigen. The studies were conducted in GSP dogs and control dogs. MATERIALS AND METHODS
Animals
Twenty-three German shepherds and one mixed-bred German shepherd dog, 15 males (two castrated) and nine females (one ovariohysterectomised), with the clinical characteristics of GSP (22) were examined. Their ages ranged from 1 to 11 years, the mean being 6.1 years. Three female and two male healthy dogs without skin disease were used as controls: two mongrel dogs, one boxer, one beagle and one German shepherd. Their ages ranged from I to 8 years, the mean being 6 years. I
2
Department of Clinical Sciences of Companion Animals, University of Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands. Department of Veterinary Pathology, University of Utrecht, The Netherlands.
THE VETERINARY QUARTERLY, VOL.
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21
Intradermal allergy test Skin-test reactivity to a 0.1% whole-body flea extract (ARTU Biologicals N.V., Lelystad, The Netherlands) was tested according to the method described by Willemse and Van den
Brom (20). The test sites were inspected at 15 and 30 minutes and 1, 2, 4, 8, 24, 48 and 72 hours after the skin test. The wheals were considered positive if their diameter exceeded the average diameter of the wheals of the histamine and the diluent control.
Histopathology
Punch biopsies (6 mm 0) from skin-test sites of both GSP and control dogs were taken under local anaesthesia using a 1% lidocain solution. Biopsies were taken at t = 0, at 15 and 30 minutes and 1, 2, 4, 8, 24, 48 and 72 hours after the injection (Tables 1 and 2).
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In eight clinical cases biopsies were obtained from acute GSP lesions, which were
characterised by small papules and some surrounding erythema. All biopsies were fixed in 10% neutral buffered formalin and embedded in paraffin. Six-micrometer sections were cut and a hematoxylin-eosin (HE) stain was performed. Some specimens were also stained Table I. Histologic reaction pattern in five control dogs to flea antigen. The dermal reaction at different times (hours) was graded as mild (mi), moderate (mo) or severe (se). Time after injection hours 00.15
Grade
Intravascular gra
Perivascular gra
lym
Interstitial
his
gra
lym
his
2
2
mi
mo se
00.30
mi
mo se
01.00
mi
mo se
02.00
ph
2
mo
1
3
3
1
1
se
04.00
mi
3
2
2
mo
1
2
2
1
1
1
.,
se
08.00
I
mi
2
1
mo
2
se
24.00
mi
1
1
mo
1
se
48.00
mi
2
3
1
1
3
;
2
s
1
mo
2
3
4
1
3
3
2
2
se
72.00
mi
1
mo
2
2
1
1
2
2
se
(gra) granulocytes, (lym) lymphocytes, (his) histiocytes
22
THE VETERINARY QUARTERLY, VOL.
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JANUARY 1990
with Giemsa. Biopsies of skin-test sites were interpreted using a grading scale to indicate the degree of cellular infiltration (mild, moderate or severe) for each cell type observed, i.e., neutrophils, eosinophils, histiocytes, lymphocytes, plasma cells and mast cells. Enzyme-linked immunosorbent assay (ELISA)
Blood was drawn by venapuncture, allowed to clot at room temperature and centrifugated at 4,000 g for 10 minutes at 4° C. If not used immediately, serum was stored at -70° C. The assay was performed as described by Willemse et al. (21). Twenty pg of flea antigen was used to coat the polystyrene 96-well microtitre plates.
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Table 2. Histologic reaction pattern in 24 German shepherd dogs with GSP to flea antigen. The dermal reaction at different times (hours) was graded as mild (mi), moderate (mo) or severe (se).
Time after injection hours 00.15
Number of dogs 14
Grade
Intravascular gra
gra
2
2
mi mo
Perivascular lym
his
Interstitial gra
lym
his
se
00.30
7
mi
3
1
mi
2
3
mo
1
1
mo se
01.00
7
se
02.00
04.00
08.00
7
14
7
mi mo se
5
3
1
1
mi
6
8
mo
3
3
1
se
1
1
1
mi
3
2
mo
4
9
5
2
2
1
1
1
1
4
4
1
se
24.00
48.00
14
7
mi mo se
8
8
1
2
6
3
4 6
1
2
mi
1
2
2
1
1
2 2
1
mo
2
3
1
1
4
2
2
5
4
5
4
se
72.00
7
mi
mo se
I
1 1
(gra) granulocytes, (lym) lymphocytes, (his) histiocytes
RESULTS
Intradermal test In 10 out of 24 dogs with GSP, a positive skin-test reaction to flea antigen was observed after 15 minutes. The wheals were well-circumscribed and ovalshaped, ranged from 13 to 15 mm in diameter and disappeared after one to two hours. THE VETERINARY QUARTERLY. VOL. 12, No. I, JANUARY 1990
23
Although 15 GSP dogs had a history with important features of FBH, only seven of these dogs had immediate skin-test reactivity to flea antigen.
The reactions to histamine ranged from 15 to 22 mm in diameter and also disappeared after one to two hours. Delayed reactions at 24 or 48 hours were not observed. In the control group neither immediate nor delayed reactions were observed.
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Histopathology I. Control dogs Beginning at 1 hour after injection, a mild intravascular and perivascular infiltrate of neutrophilic granulocytes was observed in the dermis at the skin-test sites (Table 1). This intravascular and perivascular reaction was most prominent at 4 hours after injection, waned considerably in the following hours and had almost disappeared at 24 hours after injection. An interstitial reaction, i.e., a granulocytic infiltration between the dermal collagen
bundles, was seen at 2 hours after injection. It reached a maximum at 24 hours after injection and had diminished at 72 hours after injection. A mild-to-moderate mononuclear reaction was present in most dogs at 24 and
48 hours after injection. This reaction declined at 72 hours after injection. A plasma-cell reaction was not found. Moderate numbers of mast cells were
present in all biopsies. 2. GSP dogs a. Experimental lesions A slight inflammatory reaction (predominantly mononuclear) was present in 10 of the 19 dogs from which biopsies were taken prior to antigen injection (t = 0). A mild intravascular and perivascular granulocytic infiltrate was observed in two dogs at 15 minutes after injection and in the majority of the dogs at 2 hours after injection (Fig. 1; Table 2). This exudative reaction reached a maximum at 4 hours .
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24
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after injection, but it was still present in most dogs at 24 hours after injection (Fig. 2). Eosinophilic granulocytes as well as neutrophilic granulocytes were present in varying quantities in 11 dogs. GSP dogs with a positive skin test could not be distinguished from GSP dogs with a negative skin test by the severity and type of the lesions. The number of eosinophils also did not differ significantly between these groups. A perivascular lymphocytic infiltrate mixed with histiocytes
was first seen at 4 hours after injection and was still present at 72 hours after injection (Fig. 2). After 24 hours a moderate interstitial mononuclear infiltrate was also seen. It was
composed of lymphocytes and histiocytes that persisted until 72 hours after injection. A plasmacytic reaction was not found. There was no difference in numbers of mast cells between control dogs and GSP dogs. b. Spontaneous lesions
In the biopsies of acute lesions a predominantly exudative inflammation (pyoderma) was present in the upper and mid dermis. In one case the subcutis was also involved. At the periphery of the pyogenic area histiocytes and lymphocytes prevailed. Perivascular infiltrates of mixed cell types (granulocytes, lymphocytes and histiocytes) were seen in the area surrounding the diffuse inflammation and often granulocytes had accumulated intravascularly. In half of the cases many of the granulocytes were eosinophils. The plasmacell reaction was very slight. In all cases the epidermis was acanthotic and in two cases intra-epidermal abcesses were found. ELISA
In the dogs tested (n = 13) no flea-antigen-specific IgGd antibodies could be demonstrated.
THE VETERINARY QUARTERLY. VOL.
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JANUARY 1990
.
25
DISCUSSION
In this study only seven out of 15 GSP dogs with the clinical characteristics of FBH had immediate type skin reactions to flea antigen. The value of the intradermal test is still a matter of debate. Whereas Kristensen
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and Kieffer (10) reported 81% sensitivity for the intradermal test in dogs with FBH,
Muller et al. (13) conclude that the intradermal test with flea antigen is at best supportive but not diagnostic. Van Winkle even states that the intradermal test is unreliable for the diagnosis of FBH (19). Reports on the type of skin response to flea bites or intradermal injections with flea antigen are also controversial. In our series of GSP dogs only immediate type skin reactions were observed macroscopically, which agrees with the findings of other researchers (1, 14, 16). Gross and Halliwell (4) observed immediate and delayed type reactions to flea bites in the skin of dogs made allergic by 10 weeks exposure to fleas. Benjamini et al. (2) defined five stages in the cutaneous reaction patterns to flea bites in the guinea pig, including delayed type reactions. The controversy regarding the type of skin reaction might be due to the methods employed. Benjamini et al. (2) and Gross and Halliwell (4) studied skin reactions
to flea bites. In the other studies, including ours, skin reactions to injections of whole-body flea extract were studied. The most appropriate approach might be
that of Feingold and Benjamini (3), who used flea saliva as a test antigen. In guinea pigs this induced only delayed reactions. Another explanation for the presence or absence of delayed reactions might be the difference in duration of the flea allergy. Whereas the flea allergy in our series existed for at least several months or even years, in the series of Gross and Halliwell (4) animals were investigated after an exposure to fleas of only 10 weeks. The longstanding exposure in our cases might have induced a fourth-stage hypersensitivity as described by Benjamini et al. for the guinea pig (2). In this late stage an immediate type reaction developed, whereas the delayed type reaction disappeared. It might even be that the dogs with a negative skin test were not allergic at all or went through several of the stages described by Benjamini et al. and finally became anergic (Stage V). In our control dogs the histologic changes following intradermal injections of flea antigen were largely characterised by early polymorphonuclear infiltration. A mild mononuclear infiltration subsequently occurred. These changes can be regarded as atypical inflammatory reactions (17, 18). In the GSP group basically the same cell patterns were observed, although the intravascular and perivascular neutrophilic infiltrations occurred earlier and were more prolonged. Mononuclear cell infiltrations were also observed earlier in the GSP group. These differences might be explained by the permanent immune response in GSP dogs, afflicted as they are with multiple skin lesions. This is associated with high levels of circulating immune complexes (23), resulting in a permanent activation of polymorphonuclear cells. The associated increase in vascular permeability leads to increased perivascular leakage of inflammatory cells, including lymphocytes. In contrast to
the results of Gross and Halliwell (4), in our series it was not possible to discriminate between flea-test-positive and flea-test-negative GSP dogs on the basis of tissue eosinophilia. However, it should be noted that these authors used
a different flea antigen and studied mixed-breed dogs and beagles without
concurrent skin disease. The histologic skin changes of chronic GSP lesions have been described in an earlier report (22). In the present study early lesions, i.e., nonulcerated small
papules, were biopsied in an attempt to define the primary GSP lesions. The histologic patterns of these early lesions were similar to the chronic GSP lesions and gave no clue to the nature of the primary GSP lesion. 26
THE VETERINARY QUARTERLY, VOL. 12, No 1, JANUARY 1990
Summarising the results of this study on the possible role of FBH in the
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pathogenesis of GSP, we may conclude that there is no evidence for a delayed type allergy. Immediate type responses to flea antigen may play a role in some cases. The arguments in favour of this theory are (1) the characteristic primary location of the skin lesions and (2) the positive skin test, although they were low in number. Additional studies, however, did not support the idea of immediate type FBH playing a pathogenetic role, because (1) no flea-antigen-specific IgGd was found, (2) there was no relation between the severity of the skin lesions and the results of the intradermal tests and (3) histologically there were no characteristics of an immediate type response. Although specific precipitating factors for GSP have not been found so far, the observation of the early and prolonged local neutrophilic reactions to injections
with flea antigen in GSP dogs is interesting. For instance, in humans with pyoderma gangrenosum, intense polymorphic infiltration itself is supposed to contribute to the development of skin lesions by the action of its secretory products (7, 11). The same mechanism might play a role in the development of GSP lesions.
The disease may be regarded as an excessive neutrophilic reaction to aspecific stimuli. In addition, there are other interesting similarities between GSP and pyoderma gangrenosum. Pyoderma gangrenosum (7, 12, 15) is an uncommon idiopathic skin disease characterised by distinct chronic ulcerations. The macroscopic appearance of skin lesions in pyoderma gangrenosum and GSP is similar. Since there are no specific clinical or laboratory features, the diagnosis of pyoderma gangrenosum is, like that of GSP, based on the appearance of the typical ulcers and the exclusion of other possibilities. Most cases occur between the ages of 30 and 50, with multiple painful lesions usually located on the lower extremities and the trunk. The most commonly described primary lesion is a pustule, but a considerable clinical variation exists. In 40% of the patients new skin lesions develop at sites of previous trauma. These observations apply to GSP as well (22). GSP might therefore serve as a model for the study of pyoderma gangrenosum in man. Further studies are needed to substantiate the validity of this model. REFERENCES I. Baker KP. Intradermal tests as an aid to the diagnosis of skin disease in dogs. J. Small Anim 2. 3. 4. 5.
6.
Pract 1971; 12: 445-52. Benjamini E, Feingold BF, and Kartman L. Skin reactivity in Guinea pigs sensitised to Ilea bites: The sequence of reactions. Proc Soc Exp Biol Med 1961; 108: 700-2. Feingold BF and Benjamini E. Allergy to flea bites. Ann Allergy 1961; 19: 1275-89. Gross TL and Halliwell REW. Lesions of experimental flea bite hypersensitivity in the dog. Vet Pathol 1985; 22: 78-81. Halliwell REW. Flea bite dermatitis. Compend Cont Ed 1979; 367-71. Halliwell REW. Flea bite hypersensitivity. RW Kirk (Editor) Current Veterinary Therapy VIII. Ch. 6 WB Saunders Co, Philadelphia, USA, 1983.
7. Hickman JG. Pyoderma gangrenosum. Clin Dermatol 1983; 1: 102-13. 8. Kieffer M and Kristensen S. Flea hypersensitivity in dogs and cats. Int J Dermatol 1979; 18: 70712.
9. Kristensen S, Haarlov N and Mourier H. A study of skin diseases in dogs and cats, IV. Patterns of flea infestation in dogs and cats in Denmark. Nord Vet Med 1978; 30: 401-13.
10.
Kristensen S and Kieffer M. A study of skin diseases in dogs and cats, V. The intradermal test in the diagnosis of flea allergy in dogs and cats. Nord Vet Med 1978; 30: 414-23. 11. Malech HL and Gallin JI. Current concepts: neutrophils in human diseases. New Engl J Med 1987; 317: 687-94.
12.
Moschella SL and Hurley UK, 1985.
13.
HI Dermatology, vol I and 2, 2nd ed. WB Saunders Co, London,
Muller GH, Kirk RW, and Scott DW. Small Animal Dermatology, 3rd ed. WB Saunders Co, Philadelphia, USA, 1983.
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27
14.
Nesbitt GH and Schmitz JA. Fleabite allergic dermatitis: a review and survey of 330 cases. J
Am Vet Med Assoc 1978; 173: 282-8.
15. Newell LM and Malkinson FD. Commentary: Pyoderma Gangrenosum. Arch Dermatol 1982;
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118: 769-73.
16. Schwartzman RM and Orkin M. A comparative study of skin diseases of dog and man. CC Thomas Springfield, IL, USA, 1962. 17. Sell S. Immunology, Immunopathology and Immunity. 4th ed. Elsevier, Amsterdam, The Netherlands, 1987. 18. Slauson DO and Cooper BL. Mechanisms of disease: A textbook of comparative general pathology. 1st ed., Williams and Wilkins, Baltimore, USA, 1982. 19. Van Winkle KA. An evaluation of flea antigens used in intradermal skin testing for flea allergy in the canine. J Am Anim Hosp Assoc 1981; 17: 343-54. 20. Willemse A and Van den Brom WE. Evaluation of the intradermal allergy test in normal dogs. Res Vet Sci 1982; 32: 57-61. 21. Willemse A, Noordzij A, Rutten VPMG, and Bernadina WE. Induction of non-IgE anaphylactic antibodies in dogs. Clin Exp Immunol 1985; 59: 351-8. 22. Wisselink MA, Willemse A, and Koeman JP. Deep pyoderma in the German Shepherd Dog. J Am Anim Hosp Assoc 1985; 21: 773-6. 23. Wisselink MA, Bernadina WE, Willemse A, and Noordzij A. Immunologic aspects of German Shepherd Dog Pyoderma. J Vet Immunol Immunopathol 1988; 19: 67-77.
28
THE VETERINARY QUARTERLY. VOL. 12, No. I, JANUARY 1990
Veterinary Quarterly Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tveq20
Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP) a
b
M.A. Wisselink , J.P. Koeman , T. S. G. A. M. van den b
Ingh & A. Willemse
a
a
Department of Clinical Sciences of Companion Animals , University of Utrecht , PO Box 80 154, Utrecht, 3508 TD, The Netherlands b
Department of Veterinary Pathology , University of Utrecht , The Netherlands Published online: 01 Nov 2011.
To cite this article: M.A. Wisselink , J.P. Koeman , T. S. G. A. M. van den Ingh & A. Willemse (1990) Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP), Veterinary Quarterly, 12:1, 21-28, DOI: 10.1080/01652176.1990.9694237 To link to this article: http://dx.doi.org/10.1080/01652176.1990.9694237
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Investigations on the role of flea antigen in the pathogenesis of German Shepherd dog Pyoderma (GSP)
Downloaded by [Fondren Library, Rice University ] at 17:49 20 November 2014
M. A. Wisselinkl, J. P. Koeman2, T. S. G. A. M. van den Ingh2, and A. Willemsel SUMMARY Skin reaction patterns to the intradermal injection of a whole-body flea extract were examined in five physically healthy dogs and in 24 dogs with German Shepherd dog Pyoderma (GSP) at 15 and 30 minutes and at 1, 2, 4, 8, 24, 48 and 72 hours after the injection. In 10 out of 24 GSP dogs a positive skin reaction was observed macroscopically after 15 minutes. Delayed reactions at 24 or 48 hours were not observed. In the control group neither immediate nor delayed reactions were observed. The histopathologic skin changes were basically the same in both groups: an initial polymorphonuclear reaction followed by a mononuclear cell reaction. In the GSP dogs, however, these changes occurred earlier and were more prolonged than in the normal dogs. No flea-antigen-specific IgGd antibodies could be demonstrated by enzymelinked immunosorbent assay. It is concluded that delayed type hypersentitivity to flea antigen does not play a role in the pathogenesis of GSP Immediate type hypersensitivity may contribute to the disease in some cases.
INTRODUCTION
German Shepherd dog Pyoderma (GSP) has been defined as a chronic deep pyoderma in pure-bred and cross-bred German shepherds. The disease is
characterised by multiple deep-seated skin lesions with a typical morphology and distribution. Coagulase-positive staphylococci (CPS) are commonly found upon bacteriological examination (22). The primary lesions of GSP are pruritic and
located in the dorsolumbosacral region. This location is also known as the
preferred area for flea bites and flea-bite alleigy (5, 6, 8, 9, 10, 13, 14). In addition, more than half of the cases of GSP showed immediate skin-test reactivity to flea antigen. These findings have led to the hypothesis that flea-bite hypersensitivity (FBH) is a predisposing factor for GSP (22). In this study, the role of flea antigen in the pathogenesis of GSP was investigated by examining the macroscopic and histopathologic changes at skin-test sites at different time intervals following intradermal injections with flea antigen. The studies were conducted in GSP dogs and control dogs. MATERIALS AND METHODS
Animals
Twenty-three German shepherds and one mixed-bred German shepherd dog, 15 males (two castrated) and nine females (one ovariohysterectomised), with the clinical characteristics of GSP (22) were examined. Their ages ranged from 1 to 11 years, the mean being 6.1 years. Three female and two male healthy dogs without skin disease were used as controls: two mongrel dogs, one boxer, one beagle and one German shepherd. Their ages ranged from I to 8 years, the mean being 6 years. I
2
Department of Clinical Sciences of Companion Animals, University of Utrecht, P.O. Box 80.154, 3508 TD Utrecht, The Netherlands. Department of Veterinary Pathology, University of Utrecht, The Netherlands.
THE VETERINARY QUARTERLY, VOL.
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JANUARY 1990
21
Intradermal allergy test Skin-test reactivity to a 0.1% whole-body flea extract (ARTU Biologicals N.V., Lelystad, The Netherlands) was tested according to the method described by Willemse and Van den
Brom (20). The test sites were inspected at 15 and 30 minutes and 1, 2, 4, 8, 24, 48 and 72 hours after the skin test. The wheals were considered positive if their diameter exceeded the average diameter of the wheals of the histamine and the diluent control.
Histopathology
Punch biopsies (6 mm 0) from skin-test sites of both GSP and control dogs were taken under local anaesthesia using a 1% lidocain solution. Biopsies were taken at t = 0, at 15 and 30 minutes and 1, 2, 4, 8, 24, 48 and 72 hours after the injection (Tables 1 and 2).
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In eight clinical cases biopsies were obtained from acute GSP lesions, which were
characterised by small papules and some surrounding erythema. All biopsies were fixed in 10% neutral buffered formalin and embedded in paraffin. Six-micrometer sections were cut and a hematoxylin-eosin (HE) stain was performed. Some specimens were also stained Table I. Histologic reaction pattern in five control dogs to flea antigen. The dermal reaction at different times (hours) was graded as mild (mi), moderate (mo) or severe (se). Time after injection hours 00.15
Grade
Intravascular gra
Perivascular gra
lym
Interstitial
his
gra
lym
his
2
2
mi
mo se
00.30
mi
mo se
01.00
mi
mo se
02.00
ph
2
mo
1
3
3
1
1
se
04.00
mi
3
2
2
mo
1
2
2
1
1
1
.,
se
08.00
I
mi
2
1
mo
2
se
24.00
mi
1
1
mo
1
se
48.00
mi
2
3
1
1
3
;
2
s
1
mo
2
3
4
1
3
3
2
2
se
72.00
mi
1
mo
2
2
1
1
2
2
se
(gra) granulocytes, (lym) lymphocytes, (his) histiocytes
22
THE VETERINARY QUARTERLY, VOL.
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with Giemsa. Biopsies of skin-test sites were interpreted using a grading scale to indicate the degree of cellular infiltration (mild, moderate or severe) for each cell type observed, i.e., neutrophils, eosinophils, histiocytes, lymphocytes, plasma cells and mast cells. Enzyme-linked immunosorbent assay (ELISA)
Blood was drawn by venapuncture, allowed to clot at room temperature and centrifugated at 4,000 g for 10 minutes at 4° C. If not used immediately, serum was stored at -70° C. The assay was performed as described by Willemse et al. (21). Twenty pg of flea antigen was used to coat the polystyrene 96-well microtitre plates.
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Table 2. Histologic reaction pattern in 24 German shepherd dogs with GSP to flea antigen. The dermal reaction at different times (hours) was graded as mild (mi), moderate (mo) or severe (se).
Time after injection hours 00.15
Number of dogs 14
Grade
Intravascular gra
gra
2
2
mi mo
Perivascular lym
his
Interstitial gra
lym
his
se
00.30
7
mi
3
1
mi
2
3
mo
1
1
mo se
01.00
7
se
02.00
04.00
08.00
7
14
7
mi mo se
5
3
1
1
mi
6
8
mo
3
3
1
se
1
1
1
mi
3
2
mo
4
9
5
2
2
1
1
1
1
4
4
1
se
24.00
48.00
14
7
mi mo se
8
8
1
2
6
3
4 6
1
2
mi
1
2
2
1
1
2 2
1
mo
2
3
1
1
4
2
2
5
4
5
4
se
72.00
7
mi
mo se
I
1 1
(gra) granulocytes, (lym) lymphocytes, (his) histiocytes
RESULTS
Intradermal test In 10 out of 24 dogs with GSP, a positive skin-test reaction to flea antigen was observed after 15 minutes. The wheals were well-circumscribed and ovalshaped, ranged from 13 to 15 mm in diameter and disappeared after one to two hours. THE VETERINARY QUARTERLY. VOL. 12, No. I, JANUARY 1990
23
Although 15 GSP dogs had a history with important features of FBH, only seven of these dogs had immediate skin-test reactivity to flea antigen.
The reactions to histamine ranged from 15 to 22 mm in diameter and also disappeared after one to two hours. Delayed reactions at 24 or 48 hours were not observed. In the control group neither immediate nor delayed reactions were observed.
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Histopathology I. Control dogs Beginning at 1 hour after injection, a mild intravascular and perivascular infiltrate of neutrophilic granulocytes was observed in the dermis at the skin-test sites (Table 1). This intravascular and perivascular reaction was most prominent at 4 hours after injection, waned considerably in the following hours and had almost disappeared at 24 hours after injection. An interstitial reaction, i.e., a granulocytic infiltration between the dermal collagen
bundles, was seen at 2 hours after injection. It reached a maximum at 24 hours after injection and had diminished at 72 hours after injection. A mild-to-moderate mononuclear reaction was present in most dogs at 24 and
48 hours after injection. This reaction declined at 72 hours after injection. A plasma-cell reaction was not found. Moderate numbers of mast cells were
present in all biopsies. 2. GSP dogs a. Experimental lesions A slight inflammatory reaction (predominantly mononuclear) was present in 10 of the 19 dogs from which biopsies were taken prior to antigen injection (t = 0). A mild intravascular and perivascular granulocytic infiltrate was observed in two dogs at 15 minutes after injection and in the majority of the dogs at 2 hours after injection (Fig. 1; Table 2). This exudative reaction reached a maximum at 4 hours .
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24
THE VETERINARY QUARTERLY, VOL. 12, No. I, JANUARY 1990
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after injection, but it was still present in most dogs at 24 hours after injection (Fig. 2). Eosinophilic granulocytes as well as neutrophilic granulocytes were present in varying quantities in 11 dogs. GSP dogs with a positive skin test could not be distinguished from GSP dogs with a negative skin test by the severity and type of the lesions. The number of eosinophils also did not differ significantly between these groups. A perivascular lymphocytic infiltrate mixed with histiocytes
was first seen at 4 hours after injection and was still present at 72 hours after injection (Fig. 2). After 24 hours a moderate interstitial mononuclear infiltrate was also seen. It was
composed of lymphocytes and histiocytes that persisted until 72 hours after injection. A plasmacytic reaction was not found. There was no difference in numbers of mast cells between control dogs and GSP dogs. b. Spontaneous lesions
In the biopsies of acute lesions a predominantly exudative inflammation (pyoderma) was present in the upper and mid dermis. In one case the subcutis was also involved. At the periphery of the pyogenic area histiocytes and lymphocytes prevailed. Perivascular infiltrates of mixed cell types (granulocytes, lymphocytes and histiocytes) were seen in the area surrounding the diffuse inflammation and often granulocytes had accumulated intravascularly. In half of the cases many of the granulocytes were eosinophils. The plasmacell reaction was very slight. In all cases the epidermis was acanthotic and in two cases intra-epidermal abcesses were found. ELISA
In the dogs tested (n = 13) no flea-antigen-specific IgGd antibodies could be demonstrated.
THE VETERINARY QUARTERLY. VOL.
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JANUARY 1990
.
25
DISCUSSION
In this study only seven out of 15 GSP dogs with the clinical characteristics of FBH had immediate type skin reactions to flea antigen. The value of the intradermal test is still a matter of debate. Whereas Kristensen
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and Kieffer (10) reported 81% sensitivity for the intradermal test in dogs with FBH,
Muller et al. (13) conclude that the intradermal test with flea antigen is at best supportive but not diagnostic. Van Winkle even states that the intradermal test is unreliable for the diagnosis of FBH (19). Reports on the type of skin response to flea bites or intradermal injections with flea antigen are also controversial. In our series of GSP dogs only immediate type skin reactions were observed macroscopically, which agrees with the findings of other researchers (1, 14, 16). Gross and Halliwell (4) observed immediate and delayed type reactions to flea bites in the skin of dogs made allergic by 10 weeks exposure to fleas. Benjamini et al. (2) defined five stages in the cutaneous reaction patterns to flea bites in the guinea pig, including delayed type reactions. The controversy regarding the type of skin reaction might be due to the methods employed. Benjamini et al. (2) and Gross and Halliwell (4) studied skin reactions
to flea bites. In the other studies, including ours, skin reactions to injections of whole-body flea extract were studied. The most appropriate approach might be
that of Feingold and Benjamini (3), who used flea saliva as a test antigen. In guinea pigs this induced only delayed reactions. Another explanation for the presence or absence of delayed reactions might be the difference in duration of the flea allergy. Whereas the flea allergy in our series existed for at least several months or even years, in the series of Gross and Halliwell (4) animals were investigated after an exposure to fleas of only 10 weeks. The longstanding exposure in our cases might have induced a fourth-stage hypersensitivity as described by Benjamini et al. for the guinea pig (2). In this late stage an immediate type reaction developed, whereas the delayed type reaction disappeared. It might even be that the dogs with a negative skin test were not allergic at all or went through several of the stages described by Benjamini et al. and finally became anergic (Stage V). In our control dogs the histologic changes following intradermal injections of flea antigen were largely characterised by early polymorphonuclear infiltration. A mild mononuclear infiltration subsequently occurred. These changes can be regarded as atypical inflammatory reactions (17, 18). In the GSP group basically the same cell patterns were observed, although the intravascular and perivascular neutrophilic infiltrations occurred earlier and were more prolonged. Mononuclear cell infiltrations were also observed earlier in the GSP group. These differences might be explained by the permanent immune response in GSP dogs, afflicted as they are with multiple skin lesions. This is associated with high levels of circulating immune complexes (23), resulting in a permanent activation of polymorphonuclear cells. The associated increase in vascular permeability leads to increased perivascular leakage of inflammatory cells, including lymphocytes. In contrast to
the results of Gross and Halliwell (4), in our series it was not possible to discriminate between flea-test-positive and flea-test-negative GSP dogs on the basis of tissue eosinophilia. However, it should be noted that these authors used
a different flea antigen and studied mixed-breed dogs and beagles without
concurrent skin disease. The histologic skin changes of chronic GSP lesions have been described in an earlier report (22). In the present study early lesions, i.e., nonulcerated small
papules, were biopsied in an attempt to define the primary GSP lesions. The histologic patterns of these early lesions were similar to the chronic GSP lesions and gave no clue to the nature of the primary GSP lesion. 26
THE VETERINARY QUARTERLY, VOL. 12, No 1, JANUARY 1990
Summarising the results of this study on the possible role of FBH in the
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pathogenesis of GSP, we may conclude that there is no evidence for a delayed type allergy. Immediate type responses to flea antigen may play a role in some cases. The arguments in favour of this theory are (1) the characteristic primary location of the skin lesions and (2) the positive skin test, although they were low in number. Additional studies, however, did not support the idea of immediate type FBH playing a pathogenetic role, because (1) no flea-antigen-specific IgGd was found, (2) there was no relation between the severity of the skin lesions and the results of the intradermal tests and (3) histologically there were no characteristics of an immediate type response. Although specific precipitating factors for GSP have not been found so far, the observation of the early and prolonged local neutrophilic reactions to injections
with flea antigen in GSP dogs is interesting. For instance, in humans with pyoderma gangrenosum, intense polymorphic infiltration itself is supposed to contribute to the development of skin lesions by the action of its secretory products (7, 11). The same mechanism might play a role in the development of GSP lesions.
The disease may be regarded as an excessive neutrophilic reaction to aspecific stimuli. In addition, there are other interesting similarities between GSP and pyoderma gangrenosum. Pyoderma gangrenosum (7, 12, 15) is an uncommon idiopathic skin disease characterised by distinct chronic ulcerations. The macroscopic appearance of skin lesions in pyoderma gangrenosum and GSP is similar. Since there are no specific clinical or laboratory features, the diagnosis of pyoderma gangrenosum is, like that of GSP, based on the appearance of the typical ulcers and the exclusion of other possibilities. Most cases occur between the ages of 30 and 50, with multiple painful lesions usually located on the lower extremities and the trunk. The most commonly described primary lesion is a pustule, but a considerable clinical variation exists. In 40% of the patients new skin lesions develop at sites of previous trauma. These observations apply to GSP as well (22). GSP might therefore serve as a model for the study of pyoderma gangrenosum in man. Further studies are needed to substantiate the validity of this model. REFERENCES I. Baker KP. Intradermal tests as an aid to the diagnosis of skin disease in dogs. J. Small Anim 2. 3. 4. 5.
6.
Pract 1971; 12: 445-52. Benjamini E, Feingold BF, and Kartman L. Skin reactivity in Guinea pigs sensitised to Ilea bites: The sequence of reactions. Proc Soc Exp Biol Med 1961; 108: 700-2. Feingold BF and Benjamini E. Allergy to flea bites. Ann Allergy 1961; 19: 1275-89. Gross TL and Halliwell REW. Lesions of experimental flea bite hypersensitivity in the dog. Vet Pathol 1985; 22: 78-81. Halliwell REW. Flea bite dermatitis. Compend Cont Ed 1979; 367-71. Halliwell REW. Flea bite hypersensitivity. RW Kirk (Editor) Current Veterinary Therapy VIII. Ch. 6 WB Saunders Co, Philadelphia, USA, 1983.
7. Hickman JG. Pyoderma gangrenosum. Clin Dermatol 1983; 1: 102-13. 8. Kieffer M and Kristensen S. Flea hypersensitivity in dogs and cats. Int J Dermatol 1979; 18: 70712.
9. Kristensen S, Haarlov N and Mourier H. A study of skin diseases in dogs and cats, IV. Patterns of flea infestation in dogs and cats in Denmark. Nord Vet Med 1978; 30: 401-13.
10.
Kristensen S and Kieffer M. A study of skin diseases in dogs and cats, V. The intradermal test in the diagnosis of flea allergy in dogs and cats. Nord Vet Med 1978; 30: 414-23. 11. Malech HL and Gallin JI. Current concepts: neutrophils in human diseases. New Engl J Med 1987; 317: 687-94.
12.
Moschella SL and Hurley UK, 1985.
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HI Dermatology, vol I and 2, 2nd ed. WB Saunders Co, London,
Muller GH, Kirk RW, and Scott DW. Small Animal Dermatology, 3rd ed. WB Saunders Co, Philadelphia, USA, 1983.
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14.
Nesbitt GH and Schmitz JA. Fleabite allergic dermatitis: a review and survey of 330 cases. J
Am Vet Med Assoc 1978; 173: 282-8.
15. Newell LM and Malkinson FD. Commentary: Pyoderma Gangrenosum. Arch Dermatol 1982;
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118: 769-73.
16. Schwartzman RM and Orkin M. A comparative study of skin diseases of dog and man. CC Thomas Springfield, IL, USA, 1962. 17. Sell S. Immunology, Immunopathology and Immunity. 4th ed. Elsevier, Amsterdam, The Netherlands, 1987. 18. Slauson DO and Cooper BL. Mechanisms of disease: A textbook of comparative general pathology. 1st ed., Williams and Wilkins, Baltimore, USA, 1982. 19. Van Winkle KA. An evaluation of flea antigens used in intradermal skin testing for flea allergy in the canine. J Am Anim Hosp Assoc 1981; 17: 343-54. 20. Willemse A and Van den Brom WE. Evaluation of the intradermal allergy test in normal dogs. Res Vet Sci 1982; 32: 57-61. 21. Willemse A, Noordzij A, Rutten VPMG, and Bernadina WE. Induction of non-IgE anaphylactic antibodies in dogs. Clin Exp Immunol 1985; 59: 351-8. 22. Wisselink MA, Willemse A, and Koeman JP. Deep pyoderma in the German Shepherd Dog. J Am Anim Hosp Assoc 1985; 21: 773-6. 23. Wisselink MA, Bernadina WE, Willemse A, and Noordzij A. Immunologic aspects of German Shepherd Dog Pyoderma. J Vet Immunol Immunopathol 1988; 19: 67-77.
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THE VETERINARY QUARTERLY. VOL. 12, No. I, JANUARY 1990
