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Immunologic aspects of German Shepherd dog Pyoderma (GSP)
Veterinary Immunology and Immunopathology, 19 (1988) 67-77 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
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Immunologic Aspects of German Shepherd Dog Pyoderma (GSP) M.A. WISSELINK l, W.E. BERNADINA 2, A. WILLEMSE 1and A. NOORDZIJ 2
1Department of Dermatology, Small Animal Clinic, State University of Utrecht, P.O. Box 80154, 3508 TD Utrecht (The Netherlands) 2Department of Veterinary Immunology, State University o/Utrecht, P.O. Box 80165, 3508 TD Utrecht (The Netherlands) (Accepted 23 December 1987)
ABSTRACT Wisselink, M.A., Bernadina, W.E., Willemse, A. and Noordzij, A., 1988. Immunologic aspects of German Shepherd dog Pyoderma (GSP). Vet. Immunol. Immunopathol., 19: 67-77. In 21 dogs with clinical features of German Shepherd dog Pyoderma (GSP) parameters of the specific and aspecific immune system have been examined. Chemotaxis and killing capacities of neutrophilic leucocytes were undisturbed, whereas in skin biopsies no specific immunoglobulinor complement deposits were found with immunofluorescence. With double immunodiffusion, antibodies against Gram-positive bacteria were found. In a laser nephelometric assay significantly elevated levels of IgG, IgGab, IgGd, IgM and bacterial components, associated and non-associated with circulating immune complexes, were detected. However, no relation was found with the disease state. It is concluded that dogs with GSP are immunologically normal reactors. A bacterial hypersensitivity reaction is hypothesized as a possible initiating factor in the pathogenesis of GSP.
1 INTRODUCTION
German Shepherd dog Pyoderma (GSP) is a chronic skin disease of middleaged German Shepherd dogs, characterized by multiple deep-seated skin lesions with a typical morphology and distribution, and associated with coagulase-positive staphylococci (CPS) (Wisselink et al., 1985). Response to antibiotics a n d / o r glucocorticoids is only temporary. So far, the pathogenesis of GSP remains unclear. Genetic a n d / o r immunologic abnormalities have been discussed as possible causative or predisposing factors. In addition, flea allergy has been hypothesized as being important for the pathogenesis of GSP (Wisselink et al., 1985). The aim of this study was further characterization of GSP. In a group of 21 0165-2427/88/$03.50
© 1988 Elsevier Science Publishers B.V.
68 dogs with GSP, parameters of the specific and nonspecific immune system have been examined. 2 MATERIALSAND METHODS 2.1 Animals Twenty German Shepherd dogs and one mixed-bred German Shepherd dog referred to the Utrecht University Small Animal Clinic, with the clinical characteristics of GSP (Wisselink et al., 1985) became available for this study. Twelve dogs were males, and 10 were females. Two of the female dogs had been ovariohysterectomized. Their ages ranged from 3 to 11 years, the median age being 7 years. In these dogs the deep pyoderma lesions had been present from 3 months to 6 years, with a median of 1.5 years. In 17 dogs the lesions had started in the dorsal lumbosacral region and on the thighs. In 3 dogs the first lesions had been seen on the head and the front legs. In one dog no information about the primary localization of the lesions could be obtained. On admittance the most frequent affected areas were: the dorsal lumbosacral region (14 X ), the thighs (13 X ) and the interdigital skin of four feet (11 X ). In 11 dogs the lesions were generalized. 2.2 Bacterial cultures After disinfection with methyl alcohol, duplicate samples of skin exudate were collected with sterile swabs. Bacteria were cultured on horse blood (5%) agar plates. 2.3 Intradermal testing Immediate skin test reactivity against whole body flea extract (Veterinhal: H.A.L. Allergen Laboratories, Haarlem, The Netherlands) was tested according to Willemse and Van den Brom (1982). 2.4 Granulocyte function tests Chemotaxis and bacterial killing tests were performed in 16 dogs, according to the method described by Kroese et al. (1981). However, instead of Staphylococcus aureus strain Oxford, CPS cultured from each patient were used as a test organism in the killing assay. This modification was introduced to achieve a better reflection of the in-vivo situation. All values were determined in duplicate. As a control, a physically healthy German Shepherd dog was used, which had also been part of the control population used by Kroese et al. ( 1981 ).
69
As this German Shepherd dog showed consistently normal activity in the bactericidal assay compared to the figures obtained from four healthy control dogs, this German Shepherd dog was thus defined as our "standard dog".
2.5 Serum complement values Total haemolytic complement activity (CH~o) was determined by the method described by Mayer (1961). Third complement (C3) levels in serum were assayed by single radial immunodiffusion. Established reference values were: 68265 HU for CHso and 74-150% for C3 (Biewenga, 1983).
2.6 Immunofluorescence tests From 21 dogs the following skin punch biopsies were obtained: macroscopically unaffected skin, skin adjacent to lesions, and affected skin areas. From seven dogs multiple specimens from each area were quick-frozen and cut on a cryostat. Specimens from 14 animals were fixed in 4% buffered formaldehyde and embedded in paraffin. Sections were then studied for the presence of invivo-bound immunoglobulins and C3 molecules by using monospecific rabbit anti-dog IgGab 1, IgGd 1, IgG-whole 1, IgA 1, IgM 1 and fluoresceinated goat antirabbit IgG (H + L)2 (two-step procedure) or fluoresceinated rabbit anti-C:~~ (one-step procedure).
2. 7 Laser nephelometric assay and detection of bacterial antigens and antibacterial antibodies by double immunodiffusion 2. 7.1 Normal dog serum Blood was obtained by venepuncture from 30 healthy pet dogs (age 6 months to 12 years; median 3 years). After incubation at room temperature for 1-2 h the sera were removed from the clots by centrifugation at 4000 g for 10 min at 4 ° C. All sera were filtered through a 0.22/~m Millipore filter '~. Aliquots were stored at - 70 ° C until used to establish reference values for both immune complex-associated and the total amount of IgM, IgA, IgG, IgGab and IgGd, and bacterial component in dog serum. 2.7.2 GSP serum samples Paired serum samples, of at least 14 days apart, were obtained from 15 German Shepherd dogs with GSP. These serum samples were handled as described for the healthy pet dogs. ~Department of Veterinary Immunology, State University, Utrecht, The Netherlands. -'Miles, I.C.N. Biomedicals Ltd, Buckinghamshire, Great Britain. :~Millipore Corporation, Bedford, MA 07130, U.S.A.
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2. 7.3 Antisera Rabbit antisera to dog immunoglobulin (sub) classes IgM, IgA, IgG, IgGab and IgGd, as well as polyvalent rabbit antisera to Gram-positive (Gr + ) and Gram-negative ( G r - ) bacteria were prepared (Willemse et al., 1985; Bernadina et al., 1986). The antisera to dog Ig (sub)classes were made monospecific by absorption to Sepharose-linked immunoabsorbents. Nephelometer grade IgG fractions from all rabbit-prepared antisera were obtained by subjection to protein A chromatography and subsequent treatment as described earlier (Goudswaard et al., 1978, 1980). Monospecificity of each antiserum to dog Ig (sub) classes was confirmed when only one precipitation band against whole dog serum was obtained in both immunoelectrophoresis and double immunodiffusion. The rabbit antiserum to Gram-positive bacteria (A Gr + ) reacted specifically with dog-derived Gram-positive bacteria, i.e. Streptococcus, Staphylococcus, Erysipelothrix, Listeria and Bacillus. The specificity of the reaction was demonstrated by indirect inhibition studies, using the above Gr + antigens in a laser nephelometric system. No reactions were ever found between A Gr + serum and normal serum components, commonly used viral vaccines (Distemper, Rabies, Adeno, Parainfluenza and Parva virus), common soluble parasite antigens ( Demodex canis, Toxocara canis, Ascaris suum, Ctenocephalides fells) and soluble Gram-negative bacteria-derived antigens (Salmonella, Escherichia coli, Leptospira and Pseudomonas ). Likewise, the rabbit antiserum to Gr- bacteria reacted specifically with G r - material and was never found to react with other substances (Bernadina et al., 1986). 2.7.4 Standards Three types of reference preparations were used in the study: (1) Reference preparation (pooled normal dog serum) for the determination of the total levels of dog Ig. Established values (both by Mancini and Laser nephelometry, using pure dog immunoglobulins in PBS as standards): IgG, 14.1 mgeq/ml; IgGab, 7.0 mgeq/ml; IgGd, 6.1 mgeq/ml; IgM, 1.3 mgeq/ml; and IgA, 2.7 mgeq/ml. (2) Reference preparation for the determination of Gram + bacterial components which might be present in dog serum as both antibody-associated and non-antibody-associated. Added amount of bacterial antigens to pooled bacteria-free puppy serum: 2.25 mgeq/ml Staphylococci soluble antigens. (3) Reference preparations for immune complexes: (a) 3.5% PEG/10 m M EDTA-precipitated heat-aggregated dog IgG-whole in 6% BSA. Established values: 3728 ~tgeq/ml IgG, 3270 ttgeq/ml IgGab and 564 #geq/ml IgGd. (b) 3.5% PEG/10 m M EDTA-precipitated IgM/IgA - Circulating Immune Complexes (CIC)-rich dog serum in 6% BSA. Established values (using aggregated IgM and IgA as CIC standards): 116 #geq/ml IgM-CIC and 30 ttgeq/ ml IgA-CIC.
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(c) 3.5% PEG/10 m M EDTA-precipitated serum sample in 6% BSA/ PBS. Established values 863/lgeq/ml IgGab-CIC and 86/lgeq/ml IgGd-CIC. For all the standards used in the study, suitability for use in the laser nephelometer assay system was assessed by comparing their titration curves with those obtained with eight test specimens. In all cases standard titration curves were parallel to those of the test specimens (Bernadina et al., 1986).
2.8 Protein quantitation The Lowry method with human serum albumin (HSA) as a standard was used to determine total serum protein concentrations.
2.9 Laser nephelometry The concentrations of dog IgG, IgGab, IgGd, IgM and IgA as well as the serum content of bacterial component were determined with a Hyland Laser Nephelometer PDQ (Hyland, Silverspring, MD, U.S.A.), according to the method described by Deaton et al. (1976). The test samples and antisera were diluted optimally in filtered (0.22/lm Millipore filter) phosphate-buffered saline (PBS) and 4% phosphate-buffered polyethylene glycol (PBPEG). Twenty-five/ll of the diluted test sample was added to 1 ml of the diluted relevant antiserum. The antiserum-antigen mixture was incubated for 1 h at room temperature before being read on the nephelometer, always starting with bacterial component readings. All determinations were performed in duplicate, allowing less than 10% of difference between the readings. Standard curves were made for reference solutions. The lower limit of sensitivity was 0.5 /lgeq/ml for the bacterial component, 0.9 /lgeq/ml for complexed IgA (IgA-CIC), 1.0/lgeq/ml for IgGab-CIC and 1.2 pgeqml for IgMCIC, IgG-CIC and IgGd-CIC.
2.9.1 Precipitation of circulating immune complexes (CIC) Polyethylene glycol (PEG) was used to precipitate selectively CIC from test samples, according to the method described by Gauci et al. (1978).
2.10 Double immunodiffusion assay (DID) Bacterial antigens and antibacterial antibodies in serum were detected by DID in 0.6% agarose, using respectively Gram-specific anti-bacterial antisera (A Gr +, A G r - ) and sonicated soluble bacterial antigens from dog-derived Salmonella, Escherichia coli, Streptococcus canis, Streptococcus equisimilis and Staphylococcus coagulase-positive and negative serotypes. In the DID assay, antigens were used at routine concentrations of 3 mg/ml.
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In all cases with negative test results, the experiment was repeated using increased concentrations of either the antibacterial antiserum or the antigen under study.
2.11 Statistical analysis Significance of differences between the first and the second samples was checked with the Student t-test for paired observations. Significance of differences between GSP dogs and the reference group was investigated with an approximative Student t-test, modified for possibly unequal variances (Dixon and Massey, 1969). P < 0.05 was chosen as a level of significance. 3RESULTS
3. I Bacterial cultures Bacterial culturing revealed CPS in all dogs. In addition fl-hemolytic streptococci were cultured in 11 dogs and Proteus species in three dogs.
3.2 Intradermal testing Immediate skin test reactivity to a 0.1% whole-body flea antigen was observed in 13 dogs.
3.3 Granulocyte function test Of the 16 dogs tested, spontaneous migration and chemotaxis of leucocytes were within the limits of normality as established by Kroese et al. (1981). The bactericidal capacity of leucocytes was reduced in four dogs after 60 min compared with the "standard dog". However, in each of these cases the standard dog also showed decreased capacities compared with the reference values of Kroese et al. (1981).
3.4 Serum complement values Out of 21 dogs only three animals revealed a lowered CH~o value: 42 HU, 66 HU and 55 HU respectively. C3 levels were all within the reference limits.
3.5 Immunofluorescence tests (IFT) In 13 out of 19 dogs no immunofluorescence was observed in skin punch biopsies obtained from macroscopically unaffected skin. The six dogs having positive IFT reactions contained section-bound IgGab (3 X,), IgGd (2 X ) and
73 C3 (1 X ) m o l e c u l e s , on t h e b a s e m e n t m e m b r a n e ( t w o d o g s ) a n d t h e i n t e r c e l l u l a r s u b s t a n c e of t h e p r i c k l e cell l a y e r ( f o u r d o g s ) . B i o p s i e s of a f f e c t e d s k i n a n d s k i n a d j a c e n t to l e s i o n s s h o w e d , in 17 o u t of 21 dogs, o n l y I g G d , I g G a b a n d / o r I g M - p r o d u c i n g p l a s m a cells. N o p e r i v a s c u l a r d e p o s i t s w e r e o b s e r v e d . N o d i f f e r e n c e s were o b t a i n e d b e t w e e n t h e f o r m a l i n fixed specimens and the quick-frozen specimens.
3.6 Levels of serum immunoglobulins and Gram-positive bacterial components (Table 1 ) G S P dogs as a g r o u p h a d , w h e n c o m p a r e d w i t h t h e n o r m a l c o n t r o l s , signifi c a n t l y ( P < 0.05) e l e v a t e d levels of IgG, I g G a b , I g G d , I g M a n d b a c t e r i a l c o m TABLE 1 Serum immunoglobulin and Gram-positive bacterial component levelsa Reference group
IgG IgGab IgGd IgM IgA BC
Patients
Difference
2
SEM
n
x
SEM
n
24 16 7 3 3 24
1 1 1 0.1 0.3 2
30 30 30 30 30 30
49 31 12 2 4 55
4 3 1 0.2 0.4 5
15 15 15 15 15 15
S S S S NS S
aExpressed in mgeq/ml for immunoglobulins and in ttgeq/ml for bacterial component. Abbreviations: 2= mean; SEM = standard error of mean; n = number; S = significant (P < 0.05 ); NS = non significant (P> 0.05 ); BC = bacterial component. TABLE 2 PEG-precipitated circulating immune complexes (CIC)a Reference group
IgG-CIC IgGab-CIC IgGd-CIC IgM-CIC IgA-CIC BC-CIC
Patients
Difference
x
SEM
n
x
SEM
n
69 59 6 12 2 20
5 9 1 1 0.3 2
30 12 30 30 30 30
195 175 47 24 4 47
22 41 9 4 1 10
15 15 15 15 15 15
aExpressed in mgeq/ml. Abbreviations: see Table 1.
S S S S NS S
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ponent, but not of IgA ( P > 0.05). The increased level of IgG was mainly due to a significant rise in the concentration of IgGab. No significant differences ( P > 0.05 ) in immunoglobulin and bacterial component levels were seen between paired GSP sera.
3.7 Levels o[ PEG-precipitated CIC (Table 2 ) Significantly raised mean levels of IgG-CIC, IgGab-CIC, IgGd-CIC, IgMCIC and BC-CIC were observed in sera of dogs with GSP (P < 0.05). IgA-CIC levels were not significantly raised ( P > 0.05).
3.8 Antibacterial antibodies and bacterial antigens (Table 3 ) Double immunodiffusion studies in 15 dogs with GSP indicated the presence of antibodies to Gram-positive bacteria only. Except for two dogs (nos. 5 and 17), antibodies to both streptococci and staphylococci were observed. However, in contrast with the laser nephelometric test results (Tables 1 and 2) TABLE
3
Double immunodiffusion assay Patient
Antigens and antibodies tested a
no.
S.c.
S.eq.
Sta +
Sta-
I
II
I
II
I
II
I
II
3
-
+
-
-
+
+
+
+
4
+
+
-
+
+
+
+
+
5
+
+
.
6
+
+
+
+
+
+
+
+
7
+
+
+
+
-
+
+
+
9
+
+
+
-
+
+
+
+
10
+
+
+
-
+
-
+
+
11
+
+
+
+
+
+
+
+
12
+
-
-
-
+
+
+
+
13
+
+
+
+
+
+
+
+
17
+
.
18
+
+
+
+
+
+
+
+
19
--
--
+
+
+
+
--
--
20
-
--
+
+
+
+
+
--
21
-
-
+
+
+
+
-
--
.
.
.
.
.
.
.
.
.
.
.
aS.c. = Streptococcus canis; S . e q . = Streptococcus equisimilis; S t a + = Staphylococcus c o a g u l a s e +; S t a = Staphylococcus c o a g u l a s e - . I = first sample; II = second sample.
75 bacterial components could not be detected by immunodiffusion with A Gr + and A Gr- rabbit anti-serum. DISCUSSION In a previous report the clinical characteristics of German Shepherd dog Pyoderma (GSP) have been described (Wisselink et al., 1985). The aim of this study was a further characterisation of GSP by investigating some immunologic parameters. Inflammation and phagocytosis are considered as crucial in the host's defense system against staphylococcal infections (Verbrugh, 1979). Serum complement is one of the principal effector systems of the inflammatory response, together with granulocyte functions such as chemotaxis and bacterial killing. In man several complement deficiencies and neutrophil dysfunction syndromes have been reported, associated with an enhanced susceptibility to pyogenic infections (Dahl, 1981). In the dog an intracellular killing defect has been described in the Grey Collie Syndrome (Chusid et al., 1975) and in the Canine Granulocythopathy Syndrome (Renshaw et al., 1975). Impaired invivo and in-vitro neutrophilic chemotaxis was mentioned in the Pelger-Hu~t anomaly and in the so-called "bacterial pyoderma syndromes" (Bowles et al., 1979; Latimer et al., 1983). In this study spontaneous migration and chemotaxis of circulating leucocytes was normal in all GSP dogs. Bactericidal capacities were reduced in four out of 16 dogs (25%) after 60 min. As in these cases the control dog showed decreased bactericidal capacities as well, it is hypothesized that different properties of the isolated bacteria may be responsible for this phenomenon. Further characteristion of the bacteria by phage-typing may be helpful, particularly as GSP dogs with or without reduced bactericidal capacities could not be distinguished on the basis of clinical features. However, the results suggest that disturbed killing capacities of neutrophils do not play a crucial role in the pathogenesis of all dogs with GSP. In man, recurrent pyodermas may also be associated with C3 deficiency or hypercatabolism of C3. In the latter case, total hemolytic complement and Ca levels are decreased and patients are unable to opsonise bacteria normally (Dahl, 1981 ). The present data indicate that there is no evidence for complement deficiencies in dogs with GSP. Direct immunofluorescence testing revealed no significant deposits indicating autoimmunity or Arthus-type reactions. The results of the laser nephelometric assay indicate that GSP dogs are reacting immunologically to the bacterial load by producing extremely high levels of IgG (mainly IgGab). However, these results should be interpreted with caution. It should be kept in mind that polyclonal activation of B cells leading to increased production of non-specific Ig and/or autoantibodies, is a phenom-
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enon often observed in chronic infection, including various viral and bacterial infections (Stites et al., 1987). Protein A for instance, found on the surface of Staphylococcus intermedius in dogs and cats (Cox et al., 1986) has been reported to be a polyclonal B cell activator, and mitogenic for both B and T cells (Chen et al., 1982 ). On the other hand, results of CIC determinations seem to point to specific immune reactions, since the increased IgGab levels were always found in association with increased levels of IgGab-CIC. This again, found in association with significantly raised levels of bacterial component associated CIC, IgM-CIC, and IgGd-CIC is reminiscent of immune reactions of the affected dogs directed against specific bacteria. The fact that CIC levels between paired sera of individual dogs varied substantially, indicates the dynamic immune response to the existing bacterial load. Circulating immune complexes may simply represent a secondary event during the course of the disease. However, they may also account for some of the pathological manifestations: for instance, by interfering with important cell functions or by modulating the immune response (Theofilopoulos and Dixon, 1979). A pathogenic role is suggested if there is a relation between the amount of complexes and the severity of the disease state (Theofilopoulos and Dixon, 1979 ). In this series of GSP dogs such a relation could not be found. In the double immunodiffusion studies, bacterial component could not be detected. This finding turned out to be falsely negative, as the results of the laser nephelometry revealed. The latter technique showed that the GSP serum samples contained less than 0.5 ~geq/ml of Gram-negative material. Thus in further experiments the demonstration of only Gram-positive material was attempted. Based on the parameters investigated in this study we consider this group of GSP dogs as immunologically competent. Yet it remains true that, although septicaemia does not occur, increased colonization of the skin with pathogenic staphylococci is present in all cases. One explanation for this phenomenon might be the presence of hypersensitivity reactions in the skin of these dogs, as has been described in man (Adlam and Easmon, 1983) and experimental animals (Scott et al., 1978). According to this theory, repeated contact with staphylococci, induced, for instance, by flea bite hypersensitivity, atopic dermatitis or repeated microtrauma, might induce various types of hypersensitivity reactions to staphylococcal allergens, eventually resulting in GSP.
REFERENCES Adlam, C. and Easmon, C.S.F., 1983. Immunity and hypersensitivity to staphylococcal infection. In: C. Adlam and C.S.F. Easmon (Editors), Immunity and Hypersensitivity to Staphylococcal Infections, Vol. 1. Academic Press, London, pp. 275-316. Bernadina, W.E., Van Gestel, M., Willemse, A., Noordzij, A. and Van den Brom, W.E., 1986. Circulating immune complexes as detected by laser nephelometry after precipitation with poly-
77 ethylene glycol in sera of dogs with polyarthritis may contain antigens of bacterial origin. In: G.H.A. Borst et al. (Editors), Abstracts of the IVth International Symposium of Veterinary Laboratory Diagnosticians, Amsterdam, The Netherlands, pp. 892-896. Biewenga, W.J., 1983. Proteinuria in the Dog, Thesis. State University of Utrecht, Utrecht, The Netherlands, 174 pp. Bowles, C.A., Alsaker, R.D. and Wolfe, T.L., 1979. Study of the Pelger-Hu~t anomaly in foxhounds. Am. J. Pathol., 96: 237-247. Chen, W.-Y., Sager, S., Tung, E. and Fudenberg, H.H., 1982. Human peripheral blood lymphocyte activation by Protein A from Staphylococcus aureus. Infect. Immun., 36: 59-65. Chusid, M.J., Bujak, J.S. and Dale, D.C., 1975. Defective polymorphonuclear leucocytes metabolism and function in Canine Cyclic Neutropenia. Blood, 46: 921-930. Cox, H.U., Schmeer, N. and Newman, S.S., 1986. Protein A in Staphylococcus intermedius isolates from dogs and cats. Am. J. Vet. Res., 47: 1881-1885. Dahl, M.V., 1981. Clinical Immunodermatology. Year Book Publishers Inc., Chicago, IL, 261 pp. Deaton, D.D., Maxwell, K.W., Smith, R.S. and Greveling, R.L., 1976. Use of laser nephelometry in the measurement of serum proteins. Clin. Chem., 22: 1465-1471. Dixon, W.J. and Massey, F.J., 1969. Introduction to Statistical Analysis, 3rd Edn. McGraw-Hill Kogakushka, Tokyo, 638 pp. Gauci, L., Ursule, F. and Serrou, B., 1978. Semi-automated system permitting analysis of immune complex components. In: H. Peeters (Editor), Protides of Biological Fluids, Proceedings of the 26th Colloquium. Pergamon Press, New York, NY, pp. 29-32. Goudswaard, J., Van der Donk, J.A., Noordzij, A., Van Dam, R.H. and Vaerman, J.P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8: 21-28. Goudswaard, J., Verdouw-Chamalaun, C.V.M. and Noordzij, A., 1980. Quantitation of immunoglobulins in ovine sera and secretions by laser nephelometry. Comparison with the radial immunodiffusion (RID) technique. Vet. Immunol. Immunopathol., 1: 163-177. Kroese, F.G.M., Willemse, A. and Slappendel, R.J., 1981. Granulocyte function test in canine infectious diseases: methods and preliminary clinical results. Vet. Immunol. Immunopathol., 2: 455-466. Latimer, K.S., Prasse, K.W., Mahaffey, E.A. Dawe, D.L., Lorenz, M.D. and Duncan, J.R., 1983. Neutrophil movement in selected canine skin diseases. Am. J. Vet. Res., 44: 601-606. Mayer, M.N., 1961. Complement and complement fixation. In: E.A. Kabat and M.M. Mayer (Editors), Experimental Immunochemistry, 2nd Edn. Charles C. Thomas, Springfield, IL, pp. 133-235. Renshaw, H.W., Chatburn, C., Bryan, G.M., Bartsch, R.C. and Davis, W.C., 1975. Canine granulocytopathy syndrome: neutrophil dysfunction in a dog with recurrent infections. Am. J. Vet. Med. Assoc., 166: 443-447. Scott, D.W., MacDonald, J.M. and Schultz, R.D., 1978. Staphylococcal hypersensitivity in the dog. J. Am. Anim. Hosp. Assoc., 14: 766-799. Stites, D.P., Stobo, J.D. and Wells, J.V., 1987. Basic and Clinical Immunology, 6th Edn. Lange Medical Publications, Los Altos, CA, 734 pp. Theofilopoulos, A.N. and Dixon, F.J., 1979. The biology and detection of immune complexes. In: F.J. Dixon and H.G. Kunkel (Editors), Advances in Immunology, Vol. 28. Academic Press, New York, NY, pp. 89-189. Verbrugh, H.A., 1979. The phagocytic response in host resistance against Staphylococcal infections. Thesis, State University of Utrecht, Utrecht, The Netherlands, 148 pp. Willemse, A. and Van den Brom, W.E., 1982. Evaluation of the intradermal allergy test in normal dogs. Res. Vet. Sci., 32: 57-61. Willemse, A., Noordzij, A., Rutten, V.P.M.G. and Bernadina, W.E., 1985. Induction of non-IgE anaphylactic antibodies in dogs. Clin. Exp. Immunol., 59: 351-358. Wisselink, M.A., Willemse, A. and Koeman, J.P., 1985. Deep pyoderma in the German Shepherd dog. J. Am. Anim. Hosp. Assoc., 21: 773-777.
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Immunologic Aspects of German Shepherd Dog Pyoderma (GSP) M.A. WISSELINK l, W.E. BERNADINA 2, A. WILLEMSE 1and A. NOORDZIJ 2
1Department of Dermatology, Small Animal Clinic, State University of Utrecht, P.O. Box 80154, 3508 TD Utrecht (The Netherlands) 2Department of Veterinary Immunology, State University o/Utrecht, P.O. Box 80165, 3508 TD Utrecht (The Netherlands) (Accepted 23 December 1987)
ABSTRACT Wisselink, M.A., Bernadina, W.E., Willemse, A. and Noordzij, A., 1988. Immunologic aspects of German Shepherd dog Pyoderma (GSP). Vet. Immunol. Immunopathol., 19: 67-77. In 21 dogs with clinical features of German Shepherd dog Pyoderma (GSP) parameters of the specific and aspecific immune system have been examined. Chemotaxis and killing capacities of neutrophilic leucocytes were undisturbed, whereas in skin biopsies no specific immunoglobulinor complement deposits were found with immunofluorescence. With double immunodiffusion, antibodies against Gram-positive bacteria were found. In a laser nephelometric assay significantly elevated levels of IgG, IgGab, IgGd, IgM and bacterial components, associated and non-associated with circulating immune complexes, were detected. However, no relation was found with the disease state. It is concluded that dogs with GSP are immunologically normal reactors. A bacterial hypersensitivity reaction is hypothesized as a possible initiating factor in the pathogenesis of GSP.
1 INTRODUCTION
German Shepherd dog Pyoderma (GSP) is a chronic skin disease of middleaged German Shepherd dogs, characterized by multiple deep-seated skin lesions with a typical morphology and distribution, and associated with coagulase-positive staphylococci (CPS) (Wisselink et al., 1985). Response to antibiotics a n d / o r glucocorticoids is only temporary. So far, the pathogenesis of GSP remains unclear. Genetic a n d / o r immunologic abnormalities have been discussed as possible causative or predisposing factors. In addition, flea allergy has been hypothesized as being important for the pathogenesis of GSP (Wisselink et al., 1985). The aim of this study was further characterization of GSP. In a group of 21 0165-2427/88/$03.50
© 1988 Elsevier Science Publishers B.V.
68 dogs with GSP, parameters of the specific and nonspecific immune system have been examined. 2 MATERIALSAND METHODS 2.1 Animals Twenty German Shepherd dogs and one mixed-bred German Shepherd dog referred to the Utrecht University Small Animal Clinic, with the clinical characteristics of GSP (Wisselink et al., 1985) became available for this study. Twelve dogs were males, and 10 were females. Two of the female dogs had been ovariohysterectomized. Their ages ranged from 3 to 11 years, the median age being 7 years. In these dogs the deep pyoderma lesions had been present from 3 months to 6 years, with a median of 1.5 years. In 17 dogs the lesions had started in the dorsal lumbosacral region and on the thighs. In 3 dogs the first lesions had been seen on the head and the front legs. In one dog no information about the primary localization of the lesions could be obtained. On admittance the most frequent affected areas were: the dorsal lumbosacral region (14 X ), the thighs (13 X ) and the interdigital skin of four feet (11 X ). In 11 dogs the lesions were generalized. 2.2 Bacterial cultures After disinfection with methyl alcohol, duplicate samples of skin exudate were collected with sterile swabs. Bacteria were cultured on horse blood (5%) agar plates. 2.3 Intradermal testing Immediate skin test reactivity against whole body flea extract (Veterinhal: H.A.L. Allergen Laboratories, Haarlem, The Netherlands) was tested according to Willemse and Van den Brom (1982). 2.4 Granulocyte function tests Chemotaxis and bacterial killing tests were performed in 16 dogs, according to the method described by Kroese et al. (1981). However, instead of Staphylococcus aureus strain Oxford, CPS cultured from each patient were used as a test organism in the killing assay. This modification was introduced to achieve a better reflection of the in-vivo situation. All values were determined in duplicate. As a control, a physically healthy German Shepherd dog was used, which had also been part of the control population used by Kroese et al. ( 1981 ).
69
As this German Shepherd dog showed consistently normal activity in the bactericidal assay compared to the figures obtained from four healthy control dogs, this German Shepherd dog was thus defined as our "standard dog".
2.5 Serum complement values Total haemolytic complement activity (CH~o) was determined by the method described by Mayer (1961). Third complement (C3) levels in serum were assayed by single radial immunodiffusion. Established reference values were: 68265 HU for CHso and 74-150% for C3 (Biewenga, 1983).
2.6 Immunofluorescence tests From 21 dogs the following skin punch biopsies were obtained: macroscopically unaffected skin, skin adjacent to lesions, and affected skin areas. From seven dogs multiple specimens from each area were quick-frozen and cut on a cryostat. Specimens from 14 animals were fixed in 4% buffered formaldehyde and embedded in paraffin. Sections were then studied for the presence of invivo-bound immunoglobulins and C3 molecules by using monospecific rabbit anti-dog IgGab 1, IgGd 1, IgG-whole 1, IgA 1, IgM 1 and fluoresceinated goat antirabbit IgG (H + L)2 (two-step procedure) or fluoresceinated rabbit anti-C:~~ (one-step procedure).
2. 7 Laser nephelometric assay and detection of bacterial antigens and antibacterial antibodies by double immunodiffusion 2. 7.1 Normal dog serum Blood was obtained by venepuncture from 30 healthy pet dogs (age 6 months to 12 years; median 3 years). After incubation at room temperature for 1-2 h the sera were removed from the clots by centrifugation at 4000 g for 10 min at 4 ° C. All sera were filtered through a 0.22/~m Millipore filter '~. Aliquots were stored at - 70 ° C until used to establish reference values for both immune complex-associated and the total amount of IgM, IgA, IgG, IgGab and IgGd, and bacterial component in dog serum. 2.7.2 GSP serum samples Paired serum samples, of at least 14 days apart, were obtained from 15 German Shepherd dogs with GSP. These serum samples were handled as described for the healthy pet dogs. ~Department of Veterinary Immunology, State University, Utrecht, The Netherlands. -'Miles, I.C.N. Biomedicals Ltd, Buckinghamshire, Great Britain. :~Millipore Corporation, Bedford, MA 07130, U.S.A.
70
2. 7.3 Antisera Rabbit antisera to dog immunoglobulin (sub) classes IgM, IgA, IgG, IgGab and IgGd, as well as polyvalent rabbit antisera to Gram-positive (Gr + ) and Gram-negative ( G r - ) bacteria were prepared (Willemse et al., 1985; Bernadina et al., 1986). The antisera to dog Ig (sub)classes were made monospecific by absorption to Sepharose-linked immunoabsorbents. Nephelometer grade IgG fractions from all rabbit-prepared antisera were obtained by subjection to protein A chromatography and subsequent treatment as described earlier (Goudswaard et al., 1978, 1980). Monospecificity of each antiserum to dog Ig (sub) classes was confirmed when only one precipitation band against whole dog serum was obtained in both immunoelectrophoresis and double immunodiffusion. The rabbit antiserum to Gram-positive bacteria (A Gr + ) reacted specifically with dog-derived Gram-positive bacteria, i.e. Streptococcus, Staphylococcus, Erysipelothrix, Listeria and Bacillus. The specificity of the reaction was demonstrated by indirect inhibition studies, using the above Gr + antigens in a laser nephelometric system. No reactions were ever found between A Gr + serum and normal serum components, commonly used viral vaccines (Distemper, Rabies, Adeno, Parainfluenza and Parva virus), common soluble parasite antigens ( Demodex canis, Toxocara canis, Ascaris suum, Ctenocephalides fells) and soluble Gram-negative bacteria-derived antigens (Salmonella, Escherichia coli, Leptospira and Pseudomonas ). Likewise, the rabbit antiserum to Gr- bacteria reacted specifically with G r - material and was never found to react with other substances (Bernadina et al., 1986). 2.7.4 Standards Three types of reference preparations were used in the study: (1) Reference preparation (pooled normal dog serum) for the determination of the total levels of dog Ig. Established values (both by Mancini and Laser nephelometry, using pure dog immunoglobulins in PBS as standards): IgG, 14.1 mgeq/ml; IgGab, 7.0 mgeq/ml; IgGd, 6.1 mgeq/ml; IgM, 1.3 mgeq/ml; and IgA, 2.7 mgeq/ml. (2) Reference preparation for the determination of Gram + bacterial components which might be present in dog serum as both antibody-associated and non-antibody-associated. Added amount of bacterial antigens to pooled bacteria-free puppy serum: 2.25 mgeq/ml Staphylococci soluble antigens. (3) Reference preparations for immune complexes: (a) 3.5% PEG/10 m M EDTA-precipitated heat-aggregated dog IgG-whole in 6% BSA. Established values: 3728 ~tgeq/ml IgG, 3270 ttgeq/ml IgGab and 564 #geq/ml IgGd. (b) 3.5% PEG/10 m M EDTA-precipitated IgM/IgA - Circulating Immune Complexes (CIC)-rich dog serum in 6% BSA. Established values (using aggregated IgM and IgA as CIC standards): 116 #geq/ml IgM-CIC and 30 ttgeq/ ml IgA-CIC.
71
(c) 3.5% PEG/10 m M EDTA-precipitated serum sample in 6% BSA/ PBS. Established values 863/lgeq/ml IgGab-CIC and 86/lgeq/ml IgGd-CIC. For all the standards used in the study, suitability for use in the laser nephelometer assay system was assessed by comparing their titration curves with those obtained with eight test specimens. In all cases standard titration curves were parallel to those of the test specimens (Bernadina et al., 1986).
2.8 Protein quantitation The Lowry method with human serum albumin (HSA) as a standard was used to determine total serum protein concentrations.
2.9 Laser nephelometry The concentrations of dog IgG, IgGab, IgGd, IgM and IgA as well as the serum content of bacterial component were determined with a Hyland Laser Nephelometer PDQ (Hyland, Silverspring, MD, U.S.A.), according to the method described by Deaton et al. (1976). The test samples and antisera were diluted optimally in filtered (0.22/lm Millipore filter) phosphate-buffered saline (PBS) and 4% phosphate-buffered polyethylene glycol (PBPEG). Twenty-five/ll of the diluted test sample was added to 1 ml of the diluted relevant antiserum. The antiserum-antigen mixture was incubated for 1 h at room temperature before being read on the nephelometer, always starting with bacterial component readings. All determinations were performed in duplicate, allowing less than 10% of difference between the readings. Standard curves were made for reference solutions. The lower limit of sensitivity was 0.5 /lgeq/ml for the bacterial component, 0.9 /lgeq/ml for complexed IgA (IgA-CIC), 1.0/lgeq/ml for IgGab-CIC and 1.2 pgeqml for IgMCIC, IgG-CIC and IgGd-CIC.
2.9.1 Precipitation of circulating immune complexes (CIC) Polyethylene glycol (PEG) was used to precipitate selectively CIC from test samples, according to the method described by Gauci et al. (1978).
2.10 Double immunodiffusion assay (DID) Bacterial antigens and antibacterial antibodies in serum were detected by DID in 0.6% agarose, using respectively Gram-specific anti-bacterial antisera (A Gr +, A G r - ) and sonicated soluble bacterial antigens from dog-derived Salmonella, Escherichia coli, Streptococcus canis, Streptococcus equisimilis and Staphylococcus coagulase-positive and negative serotypes. In the DID assay, antigens were used at routine concentrations of 3 mg/ml.
72
In all cases with negative test results, the experiment was repeated using increased concentrations of either the antibacterial antiserum or the antigen under study.
2.11 Statistical analysis Significance of differences between the first and the second samples was checked with the Student t-test for paired observations. Significance of differences between GSP dogs and the reference group was investigated with an approximative Student t-test, modified for possibly unequal variances (Dixon and Massey, 1969). P < 0.05 was chosen as a level of significance. 3RESULTS
3. I Bacterial cultures Bacterial culturing revealed CPS in all dogs. In addition fl-hemolytic streptococci were cultured in 11 dogs and Proteus species in three dogs.
3.2 Intradermal testing Immediate skin test reactivity to a 0.1% whole-body flea antigen was observed in 13 dogs.
3.3 Granulocyte function test Of the 16 dogs tested, spontaneous migration and chemotaxis of leucocytes were within the limits of normality as established by Kroese et al. (1981). The bactericidal capacity of leucocytes was reduced in four dogs after 60 min compared with the "standard dog". However, in each of these cases the standard dog also showed decreased capacities compared with the reference values of Kroese et al. (1981).
3.4 Serum complement values Out of 21 dogs only three animals revealed a lowered CH~o value: 42 HU, 66 HU and 55 HU respectively. C3 levels were all within the reference limits.
3.5 Immunofluorescence tests (IFT) In 13 out of 19 dogs no immunofluorescence was observed in skin punch biopsies obtained from macroscopically unaffected skin. The six dogs having positive IFT reactions contained section-bound IgGab (3 X,), IgGd (2 X ) and
73 C3 (1 X ) m o l e c u l e s , on t h e b a s e m e n t m e m b r a n e ( t w o d o g s ) a n d t h e i n t e r c e l l u l a r s u b s t a n c e of t h e p r i c k l e cell l a y e r ( f o u r d o g s ) . B i o p s i e s of a f f e c t e d s k i n a n d s k i n a d j a c e n t to l e s i o n s s h o w e d , in 17 o u t of 21 dogs, o n l y I g G d , I g G a b a n d / o r I g M - p r o d u c i n g p l a s m a cells. N o p e r i v a s c u l a r d e p o s i t s w e r e o b s e r v e d . N o d i f f e r e n c e s were o b t a i n e d b e t w e e n t h e f o r m a l i n fixed specimens and the quick-frozen specimens.
3.6 Levels of serum immunoglobulins and Gram-positive bacterial components (Table 1 ) G S P dogs as a g r o u p h a d , w h e n c o m p a r e d w i t h t h e n o r m a l c o n t r o l s , signifi c a n t l y ( P < 0.05) e l e v a t e d levels of IgG, I g G a b , I g G d , I g M a n d b a c t e r i a l c o m TABLE 1 Serum immunoglobulin and Gram-positive bacterial component levelsa Reference group
IgG IgGab IgGd IgM IgA BC
Patients
Difference
2
SEM
n
x
SEM
n
24 16 7 3 3 24
1 1 1 0.1 0.3 2
30 30 30 30 30 30
49 31 12 2 4 55
4 3 1 0.2 0.4 5
15 15 15 15 15 15
S S S S NS S
aExpressed in mgeq/ml for immunoglobulins and in ttgeq/ml for bacterial component. Abbreviations: 2= mean; SEM = standard error of mean; n = number; S = significant (P < 0.05 ); NS = non significant (P> 0.05 ); BC = bacterial component. TABLE 2 PEG-precipitated circulating immune complexes (CIC)a Reference group
IgG-CIC IgGab-CIC IgGd-CIC IgM-CIC IgA-CIC BC-CIC
Patients
Difference
x
SEM
n
x
SEM
n
69 59 6 12 2 20
5 9 1 1 0.3 2
30 12 30 30 30 30
195 175 47 24 4 47
22 41 9 4 1 10
15 15 15 15 15 15
aExpressed in mgeq/ml. Abbreviations: see Table 1.
S S S S NS S
74
ponent, but not of IgA ( P > 0.05). The increased level of IgG was mainly due to a significant rise in the concentration of IgGab. No significant differences ( P > 0.05 ) in immunoglobulin and bacterial component levels were seen between paired GSP sera.
3.7 Levels o[ PEG-precipitated CIC (Table 2 ) Significantly raised mean levels of IgG-CIC, IgGab-CIC, IgGd-CIC, IgMCIC and BC-CIC were observed in sera of dogs with GSP (P < 0.05). IgA-CIC levels were not significantly raised ( P > 0.05).
3.8 Antibacterial antibodies and bacterial antigens (Table 3 ) Double immunodiffusion studies in 15 dogs with GSP indicated the presence of antibodies to Gram-positive bacteria only. Except for two dogs (nos. 5 and 17), antibodies to both streptococci and staphylococci were observed. However, in contrast with the laser nephelometric test results (Tables 1 and 2) TABLE
3
Double immunodiffusion assay Patient
Antigens and antibodies tested a
no.
S.c.
S.eq.
Sta +
Sta-
I
II
I
II
I
II
I
II
3
-
+
-
-
+
+
+
+
4
+
+
-
+
+
+
+
+
5
+
+
.
6
+
+
+
+
+
+
+
+
7
+
+
+
+
-
+
+
+
9
+
+
+
-
+
+
+
+
10
+
+
+
-
+
-
+
+
11
+
+
+
+
+
+
+
+
12
+
-
-
-
+
+
+
+
13
+
+
+
+
+
+
+
+
17
+
.
18
+
+
+
+
+
+
+
+
19
--
--
+
+
+
+
--
--
20
-
--
+
+
+
+
+
--
21
-
-
+
+
+
+
-
--
.
.
.
.
.
.
.
.
.
.
.
aS.c. = Streptococcus canis; S . e q . = Streptococcus equisimilis; S t a + = Staphylococcus c o a g u l a s e +; S t a = Staphylococcus c o a g u l a s e - . I = first sample; II = second sample.
75 bacterial components could not be detected by immunodiffusion with A Gr + and A Gr- rabbit anti-serum. DISCUSSION In a previous report the clinical characteristics of German Shepherd dog Pyoderma (GSP) have been described (Wisselink et al., 1985). The aim of this study was a further characterisation of GSP by investigating some immunologic parameters. Inflammation and phagocytosis are considered as crucial in the host's defense system against staphylococcal infections (Verbrugh, 1979). Serum complement is one of the principal effector systems of the inflammatory response, together with granulocyte functions such as chemotaxis and bacterial killing. In man several complement deficiencies and neutrophil dysfunction syndromes have been reported, associated with an enhanced susceptibility to pyogenic infections (Dahl, 1981). In the dog an intracellular killing defect has been described in the Grey Collie Syndrome (Chusid et al., 1975) and in the Canine Granulocythopathy Syndrome (Renshaw et al., 1975). Impaired invivo and in-vitro neutrophilic chemotaxis was mentioned in the Pelger-Hu~t anomaly and in the so-called "bacterial pyoderma syndromes" (Bowles et al., 1979; Latimer et al., 1983). In this study spontaneous migration and chemotaxis of circulating leucocytes was normal in all GSP dogs. Bactericidal capacities were reduced in four out of 16 dogs (25%) after 60 min. As in these cases the control dog showed decreased bactericidal capacities as well, it is hypothesized that different properties of the isolated bacteria may be responsible for this phenomenon. Further characteristion of the bacteria by phage-typing may be helpful, particularly as GSP dogs with or without reduced bactericidal capacities could not be distinguished on the basis of clinical features. However, the results suggest that disturbed killing capacities of neutrophils do not play a crucial role in the pathogenesis of all dogs with GSP. In man, recurrent pyodermas may also be associated with C3 deficiency or hypercatabolism of C3. In the latter case, total hemolytic complement and Ca levels are decreased and patients are unable to opsonise bacteria normally (Dahl, 1981 ). The present data indicate that there is no evidence for complement deficiencies in dogs with GSP. Direct immunofluorescence testing revealed no significant deposits indicating autoimmunity or Arthus-type reactions. The results of the laser nephelometric assay indicate that GSP dogs are reacting immunologically to the bacterial load by producing extremely high levels of IgG (mainly IgGab). However, these results should be interpreted with caution. It should be kept in mind that polyclonal activation of B cells leading to increased production of non-specific Ig and/or autoantibodies, is a phenom-
76
enon often observed in chronic infection, including various viral and bacterial infections (Stites et al., 1987). Protein A for instance, found on the surface of Staphylococcus intermedius in dogs and cats (Cox et al., 1986) has been reported to be a polyclonal B cell activator, and mitogenic for both B and T cells (Chen et al., 1982 ). On the other hand, results of CIC determinations seem to point to specific immune reactions, since the increased IgGab levels were always found in association with increased levels of IgGab-CIC. This again, found in association with significantly raised levels of bacterial component associated CIC, IgM-CIC, and IgGd-CIC is reminiscent of immune reactions of the affected dogs directed against specific bacteria. The fact that CIC levels between paired sera of individual dogs varied substantially, indicates the dynamic immune response to the existing bacterial load. Circulating immune complexes may simply represent a secondary event during the course of the disease. However, they may also account for some of the pathological manifestations: for instance, by interfering with important cell functions or by modulating the immune response (Theofilopoulos and Dixon, 1979). A pathogenic role is suggested if there is a relation between the amount of complexes and the severity of the disease state (Theofilopoulos and Dixon, 1979 ). In this series of GSP dogs such a relation could not be found. In the double immunodiffusion studies, bacterial component could not be detected. This finding turned out to be falsely negative, as the results of the laser nephelometry revealed. The latter technique showed that the GSP serum samples contained less than 0.5 ~geq/ml of Gram-negative material. Thus in further experiments the demonstration of only Gram-positive material was attempted. Based on the parameters investigated in this study we consider this group of GSP dogs as immunologically competent. Yet it remains true that, although septicaemia does not occur, increased colonization of the skin with pathogenic staphylococci is present in all cases. One explanation for this phenomenon might be the presence of hypersensitivity reactions in the skin of these dogs, as has been described in man (Adlam and Easmon, 1983) and experimental animals (Scott et al., 1978). According to this theory, repeated contact with staphylococci, induced, for instance, by flea bite hypersensitivity, atopic dermatitis or repeated microtrauma, might induce various types of hypersensitivity reactions to staphylococcal allergens, eventually resulting in GSP.
REFERENCES Adlam, C. and Easmon, C.S.F., 1983. Immunity and hypersensitivity to staphylococcal infection. In: C. Adlam and C.S.F. Easmon (Editors), Immunity and Hypersensitivity to Staphylococcal Infections, Vol. 1. Academic Press, London, pp. 275-316. Bernadina, W.E., Van Gestel, M., Willemse, A., Noordzij, A. and Van den Brom, W.E., 1986. Circulating immune complexes as detected by laser nephelometry after precipitation with poly-
77 ethylene glycol in sera of dogs with polyarthritis may contain antigens of bacterial origin. In: G.H.A. Borst et al. (Editors), Abstracts of the IVth International Symposium of Veterinary Laboratory Diagnosticians, Amsterdam, The Netherlands, pp. 892-896. Biewenga, W.J., 1983. Proteinuria in the Dog, Thesis. State University of Utrecht, Utrecht, The Netherlands, 174 pp. Bowles, C.A., Alsaker, R.D. and Wolfe, T.L., 1979. Study of the Pelger-Hu~t anomaly in foxhounds. Am. J. Pathol., 96: 237-247. Chen, W.-Y., Sager, S., Tung, E. and Fudenberg, H.H., 1982. Human peripheral blood lymphocyte activation by Protein A from Staphylococcus aureus. Infect. Immun., 36: 59-65. Chusid, M.J., Bujak, J.S. and Dale, D.C., 1975. Defective polymorphonuclear leucocytes metabolism and function in Canine Cyclic Neutropenia. Blood, 46: 921-930. Cox, H.U., Schmeer, N. and Newman, S.S., 1986. Protein A in Staphylococcus intermedius isolates from dogs and cats. Am. J. Vet. Res., 47: 1881-1885. Dahl, M.V., 1981. Clinical Immunodermatology. Year Book Publishers Inc., Chicago, IL, 261 pp. Deaton, D.D., Maxwell, K.W., Smith, R.S. and Greveling, R.L., 1976. Use of laser nephelometry in the measurement of serum proteins. Clin. Chem., 22: 1465-1471. Dixon, W.J. and Massey, F.J., 1969. Introduction to Statistical Analysis, 3rd Edn. McGraw-Hill Kogakushka, Tokyo, 638 pp. Gauci, L., Ursule, F. and Serrou, B., 1978. Semi-automated system permitting analysis of immune complex components. In: H. Peeters (Editor), Protides of Biological Fluids, Proceedings of the 26th Colloquium. Pergamon Press, New York, NY, pp. 29-32. Goudswaard, J., Van der Donk, J.A., Noordzij, A., Van Dam, R.H. and Vaerman, J.P., 1978. Protein A reactivity of various mammalian immunoglobulins. Scand. J. Immunol., 8: 21-28. Goudswaard, J., Verdouw-Chamalaun, C.V.M. and Noordzij, A., 1980. Quantitation of immunoglobulins in ovine sera and secretions by laser nephelometry. Comparison with the radial immunodiffusion (RID) technique. Vet. Immunol. Immunopathol., 1: 163-177. Kroese, F.G.M., Willemse, A. and Slappendel, R.J., 1981. Granulocyte function test in canine infectious diseases: methods and preliminary clinical results. Vet. Immunol. Immunopathol., 2: 455-466. Latimer, K.S., Prasse, K.W., Mahaffey, E.A. Dawe, D.L., Lorenz, M.D. and Duncan, J.R., 1983. Neutrophil movement in selected canine skin diseases. Am. J. Vet. Res., 44: 601-606. Mayer, M.N., 1961. Complement and complement fixation. In: E.A. Kabat and M.M. Mayer (Editors), Experimental Immunochemistry, 2nd Edn. Charles C. Thomas, Springfield, IL, pp. 133-235. Renshaw, H.W., Chatburn, C., Bryan, G.M., Bartsch, R.C. and Davis, W.C., 1975. Canine granulocytopathy syndrome: neutrophil dysfunction in a dog with recurrent infections. Am. J. Vet. Med. Assoc., 166: 443-447. Scott, D.W., MacDonald, J.M. and Schultz, R.D., 1978. Staphylococcal hypersensitivity in the dog. J. Am. Anim. Hosp. Assoc., 14: 766-799. Stites, D.P., Stobo, J.D. and Wells, J.V., 1987. Basic and Clinical Immunology, 6th Edn. Lange Medical Publications, Los Altos, CA, 734 pp. Theofilopoulos, A.N. and Dixon, F.J., 1979. The biology and detection of immune complexes. In: F.J. Dixon and H.G. Kunkel (Editors), Advances in Immunology, Vol. 28. Academic Press, New York, NY, pp. 89-189. Verbrugh, H.A., 1979. The phagocytic response in host resistance against Staphylococcal infections. Thesis, State University of Utrecht, Utrecht, The Netherlands, 148 pp. Willemse, A. and Van den Brom, W.E., 1982. Evaluation of the intradermal allergy test in normal dogs. Res. Vet. Sci., 32: 57-61. Willemse, A., Noordzij, A., Rutten, V.P.M.G. and Bernadina, W.E., 1985. Induction of non-IgE anaphylactic antibodies in dogs. Clin. Exp. Immunol., 59: 351-358. Wisselink, M.A., Willemse, A. and Koeman, J.P., 1985. Deep pyoderma in the German Shepherd dog. J. Am. Anim. Hosp. Assoc., 21: 773-777.