Familial cutaneous vasculopathy ofGerman shepherds: Clinical, genetic and

preliminary pathological andimmunological studies

J.A. Malcolm Weir, Julie A. Yager, Jeff L. Caswell, Wendy M. Parker,I.B. Johnstone, P.K. Basrur, Cathy Emms

AbstractA genodermatosis affecting the German shepherdbreed has been recognized in 26 dogs in Ontariosince 1991. Clinical signs, first noted in young puppies,are manifested as pyrexia and lethargy. The maincutaneous lesions are footpad swelling and depig-mentation, but there is also crusting and ulcerationof ear tips and tail tips, and focal depigmentation ofthe nasal planum. Affected puppies show no con-sistent abnormalities in hematological or biochemi-cal parameters, and immunological tests (antinu-clear antibody and rheumatoid factor titer,immunoglobulin levels, and CD4+ and CD8+T-lymphocyte percentages) are normal. Bone marrowanalysis has shown myeloid hyperplasia in 5 of7 cases and myelofibrosis has been detected in1 case. All but 3 of the 19 clinical cases have beenstrongly positive for platelet factor-3; however, nor-mal puppies routinely develop positive plateletfactor-3 tests. Furthermore, affected pups all had nor-mal numbers of platelets on repeat complete bloodcounts.

Light microscopic examination of footpad biopsiesreveals a multifocal nodular dermatitis in whichneutrophils and mononuclear inflammatory cellssurround foci of dermal collagenolysis, and degen-erative and inflammatory vessel lesions. Depigmentedlesions have a mild, cell-poor, interface dermatitis,characterized by single cell necrosis of the basalcells, in addition to the nodular dermatitis.

Similarities and differences between this disease,a condition known as collagen disorder of the footpadsof German shepherds and other forms of cutaneous

Department of Pathology (Weir, Yager, Caswell), Departmentof Clinical Studies (Parker), Department of BiomedicalSciences (Johnstone, Basrur), Ontario Veterinary College,University of Guelph, Guelph, Ontario NIG 2W1, and BarrieVeterinary Hospital (Emms), 6 Sophia Street East, Barrie,Ontario L4M 1Y2.

Reprint requests to Dr. Julie A. Yager.

This research has been funded through the support of PetTrust, Ontario Veterinary College.

vasculitis in the dog are discussed. The cause and thepathogenesis of the disease are yet to be elucidated;however, pedigree analysis indicates an autosomalrecessive inheritance pattern. Hypersensitivityreactions, directed against normal or damagedself-collagen, may be involved. The role ofcell-mediated immunity against native or alteredcollagen is an area worthy of further investigation.

ResumeVasculopathie cutanee familiale chez le Bergerallemand: Etude des aspects clinique, gene-tique, pathologique et immunologiqueUne genodermatose a ete identifiee chez 26 chiens derace Berger allemand depuis 1991 en Ontario. Lesjeunes chiens presentent de la lethargie et une pyrexie.Les lesions cutanees se manifestent par une depig-mentation et une enflure des coussinets plantaires, uneulceration et des crofutes 'a l'extremite' des oreilles etde la queue et une depigmentation focale du museau.La formule hematologique et le profil biochimique nedtemontrent aucune anomalie specifique, et lesepreuves immunologiques (anticorps antinucleaires,facteur rhumatoide, taux d'immunoglobulines, pour-centage (%) de lymphocytes T, CD4, et CD8) sontnormales. Une aspiration de la melle osseuse a

detmontre la pre'sence d'une hyperplasie myeloidedans 5 cas sur 7 et une myelofibrose dans un autrecas. Seize (16) des dix-neuf (19) cas cliniques ontpresente un resultat positif marque pour le facteurplaquettaire 3. Les chiots normaux developpentcouramment une reaction positive pour cette epreuve.Dans cette etude, les chiots atteints ont eu un compteplaquettaire normal lors des evaluations repeteesde la formule sanguine. Les coussinets plantairespresentaient des lesions microscopiques de dermatitenodulaire multifocale. Au niveau du derme, on notaitdes foyers de collage'nolyse ceintures de neutrophileset de celiules inflammatoires mononucleaires ainsi quedes lesions vasculaires degeneratives et inflamma-toires. Les lesions depigmentees avaient, en plusd'une dermatite nodulaire, une legere dermatited'interface, peu cellulaire, caracterisee par unenecrose cellulaire unique des cellules basales. Les

Can Vet J Volume 35, December 1994 763Can Vet J Volume 35, December 1994 763

auteurs comparent cette pathologie aux autres formesde vasculites cutane'es et 'a la maladie connue commele desordre du collagene des coussinets plantairesdecrit chez le Berger allemand. Bien que les causes etla pathogenese ne soient pas encore elucidees, lagenealogie indique un mode de transfert par geneautosomal recessif.

Les auteurs considerent qu'un mecanisme d'hyper-sensibilite a mediation cellulaire dirige contre le col-lagene normal et degenere pourrait etre en cause etserait un volet de recherche a envisage.

(Traduit par docteur Thirkse Lanthier)

Can Vet J 1994; 35: 763-769

Introductionkin diseases of genetic origin are of increasing con-cern to the owners and breeders of purebred dogs.

These diseases, known as genodermatoses, are manifestedby a wide range of clinical expressions and are among themost prevalent of all skin conditions. Furthermore, newgenodermatoses are being recognized at an ever increas-ing frequency.We have recently identified, in Ontario, a new geno-

dermatosis affecting purebred German shepherds. It ismanifested in young puppies as a cutaneous vascu-lopathy, and likely represents a disease of immune dys-function. This paper outlines our current knowledgeof the pathological, clinical, immunological, and geneticbasis of the disease, with the purpose of alerting vet-erinary practitioners to the existence of this new andpotentially serious inherited disease in German shepherds.

Materials and methodsDogs included in the study were derived from 3 breed-ing kennels in Ontario, with 1 additional case from anowner in Kentucky, USA. Patients included in thisstudy were purebred German shepherd puppies, identi-fied clinically as having lesions consistent with thedisease in question. Twenty-six affected dogs (12 male,14 female) have been recorded to date. Of these, 7(4 male, 3 female ) have been donated to the OntarioVeterinary College for detailed clinical evaluation andstudy. The ages of the dogs ranged from 4 to 7 wk of ageat first presentation.

Clinicopathological tests performed on the subjectsincluded complete blood count (CBC) (13/26), bio-chemical profile (12/26), urinalysis (2/26), bone marrowaspirate and core biopsy (7/26), Coombs' tests at 4°C(9/26) and 37°C (13/26), plasma tyrosine levels (1/26),and joint aspiration and cytology (2/26). Radiographs oflong bones, chest, and abdomen were performed on4/26 dogs. Ophthalmological, neurological, and elec-trocardiographic examinations were performed on the7 dogs donated to the Ontario Veterinary College fordetailed study.

Routine immunological analysis included antinu-clear antibody (ANA) titer (11/26), rheumatoid factortiter (11/26), radial immunodiffusion for immunoglob-ulin levels (11/26), and platelet factor-3 (PF-3) analysis(19/26). Serum was stored at 4°C for 24 h in order toobserve the development of a cryoprecipitate.

Percentages of CD4+ and CD8+ T-lymphocyteswere measured on 2 dogs by flow cytometry, using

Figure 1. Firm swelling on dorsum of nose (short arrow) andfocal area of depignientation on the nasal septum (long arrows)on a 5-month-old affected male.

functionally-tested monoclonal antibodies to canineCD antigens. Using peripheral blood lymphocytes at aconcentration of 1 X 107 cells/mL (separated by aFicoll-Hypaque density gradient (1), 100 pL of cellsuspension were added to the well on a round-bottomed, 96-well plate, sitting in ice. The sample waswashed by centrifuging it at 250 X g for 5 min, followedby aspiration of the supernatant and resuspension ofthe cell pellet in an equal volume of buffer solution(0.1 M phosphate buffered saline (PBS) with 5mMEDTA, 0.1% sodium azide, and 10% heat-inactivatedhorse serum). The plate was centrifuged again (250 x gfor 5 min), and the supernatant was removed andreplaced by 20 pL of primary antibody. The plate wasthen incubated for 45 min at 4°C and washed twice, aspreviously described. One hundred microliters of rabbitantimouse IgG-fluorescein isothiocyanate (FITC) (ZymedLaboratories Inc., San Francisco, California, USA) at a1:100 concentration in buffer solution were added andthe plate incubated for a further 45 min at 4°C. The sam-ple was then washed 2X with PBS containing 0.1%sodium azide. After the final wash, 100 pL of cold0.5% paraformaldehyde solution (pH 7.2) were mixedwith the sample cell pellet and transferred to a tubewrapped in aluminum foil and containing 400 pL of0.5% paraformaldehyde. Flow cytometry was then per-formed on the sample using a flow cytometer (FACScan,Becton Dickinson Canada Inc, Mississauga, Ontario) todetermine percentages of CD4+ and CD8+ peripheralblood T-lymphocytes.Footpad biopsies were taken from 10/26 clinical

cases. Tissue samples were placed in 10% bufferedformalin solution and processed routinely for hema-toxylin and eosin staining. Special stains, such asMasson's trichrome, periodic acid-Schiff (PAS), Martiusscarlet blue (MSB), phosphotungstic acid-hematoxylin(PTAH), Alcian blue, and Wolbach's reticulin, wereapplied to selected sections. Postmortem examination wascarried out on 3 clinical cases (2 female, 1 male ), euth-anized for humane reasons. Tissues were processed asabove.The pattern of inheritance was examined by analyzing

the pedigrees of 5 generations of affected dogs. Throughthe chi-square test, the mode of inheritance wasdetermined.

764Can Vet J Volume 35, December 1994764 Can Vet J Volume 35, December 1994

Figure 2. Swollen footpads with depigmentation on an8-week-old, affected female. Note the small ulcer on the cen-tral footpad.

ResultsClinical findingsThe first clinical signs are noted between 4 and 7 wk ofage, in most instances within 7 to 10 d of the 1st vacci-nation against distemper, hepatitis, parvovirus, andparainfluenza virus. Repeat vaccinations have resultedin worsening or recurrence of systemic signs and cuta-neous lesions, sometimes in as little as 24 h postvacci-nation. Affected puppies are usually lethargic andpyrexic, with varying degrees of lymphadenopathy.Some dogs exhibit transient lameness due to jointswelling and footpad pain. Some owners have notedthat exercise seems to exacerbate the lesions, and thatcold weather and snow seem to worsen the problem.

Cutaneous lesions include swelling on the bridge of thenose (Figure 1), along with crusting and ulceration of theear margins, nasal planum, and tips of the tail.Depigmentation of the nasal planum has also beennoted in several cases (Figure 1). Footpad lesions varyfrom mild to moderate swelling and depigmentation tosevere swelling and ulceration of the central portionof the footpad (Figure 2). Generally, all footpads areaffected, including the metacarpal and metatarsal pads.There is a general clinical impression that affected

dogs have dropped carpi and tarsi ("flat-footed") andhave stretchable, "loose-fitting," skin. The breedersreport "rubber puppies" among those affected in a litter.These dogs were destroyed, but no postmortem wasperformed. Owners also complain of the failure of theears to stand erect, and of the footpads being abnormallysoft and doughy in texture. A skin extensibility testwas performed on 3 dogs. The indices were 9.2%,12.0% and 16.7%, respectively. An index of less than17% is considered normal.

Superficial pyoderma has been noted in several dogs,correlating to episodes of footpad lesions. Bacterialculture of pyoderma pustules has yielded large numbersof hemolytic staphylococci.A variety of therapeutic regimes have been attempted,

including systemic antibiotic therapy using chloram-phenicol, amoxicillin, clindamycin, cephalexin, sys-temic glucocorticoids (prednisone PO at doses rang-ing from 1 mg to 2.5 mg/kg body weight (BW)), dapsone(Avlosulfon, Ayerst Laboratories, Montreal, Quebec) at

Figure 3. Low power photomicrograph of a typical footpadlesion showing nodular aggregation of inflammatory cells inthe mid-dermis. Hematoxylin and eosin. Bar = 250 pm.

1 mg/kg BW, ql2h, PO, and calcium and vitamin Esupplementation. Palliative treatment, including ban-daging of the feet, has also been utilized to minimize fur-ther trauma to the affected areas. To date, none of thesetherapies has been deemed satisfactory, although somedogs showed mild improvement (decrease in pyrexia andlessening of footpad swelling) when treated with systemiccorticosteroids for 2 to 4 wk.

Affected dogs appear to recover gradually by 5 to 6 moof age, although many have residual scarring of thefootpads (and nose) and the footpads remain abnor-mally soft. While no recurrence of the skin lesions hasbeen noted in previously affected dogs at the time of theirannual booster vaccinations, it would appear that manydogs experience pyrexia and lethargy for several days fol-lowing vaccination. Panosteitis has been associatedwith annual booster vaccinations in dogs from theaffected kennel.The intense discomfort and pain experienced by some

dogs with this condition has, on occasion, necessitatedeuthanasia for humane reasons. Immunosuppressivedoses of corticosteroids, treatment with dapsone, and theuse of narcotic analgesics failed to relieve the pyrexia,pain, and marked footpad and distal limb swelling of 2of the dogs (1 male, 1 female) donated to the OntarioVeterinary College. Both of these dogs had been vac-cinated while in the study, but the vaccines had beengiven 3 to 4 wk prior to exacerbation of the lesions.

Can Vet J Volume 35, December 1994 765

Figure 4. An area of dermal collagen fragmentation andlysis associated with the accumulation of neutrophils andmononuclear inflammatory cells. Hematoxylin and eosin.Bar = 25 pm.

Clinical pathologyAffected puppies have shown no significant abnormal-ities in the biochemical parameters examined. Completeblood counts were either normal or showed mild tomoderate neutrophilia. In general, the elevation inleukocyte numbers has correlated well with overt clin-ical signs, such as footpad lesions, pyrexia, or jointpain. Platelet counts have been in the high normalrange. Cytology performed on joint aspirates from 2 ofthe dogs revealed mild chronic degenerative joint diseasein 1 case and mild chronic active nonseptic inflammationin the other. Both aspirates were performed during peri-ods of overt clinical disease. Coombs' tests, at both4°C and 37°C, have been negative in all cases. Plasmatyrosine levels performed on 1 dog were 71.3 nmol/L,which is considered to be in the normal range for the dog(2). No significant abnormalities on ophthalmological,neurological, electrocardiographic, or radiographicexamination have been noted.Bone marrow aspirates and core biopsies did not

reveal any consistent abnormalities. In 5 of 7 cases,an elevated myeloid to erythroid ratio (between 4:1and 6:1) was noted. This generally corresponded toCBC values indicating mild to moderate leucocytosis anda mild shift to the left. In a 2-month-old female, the bonemarrow exhibited decreased cellularity, mild erythroidhypoplasia, mild myeloid hyperplasia, and moderatemegakaryocytosis. It also had mild neutrophilia and amild anemia. One other case, a 6-month-old male,exhibited bone marrow changes consistent with mildmyelofibrosis and a concurrent mild increase inmegakaryocytes. Core biopsy repeated 1 mo laterrevealed increased stroma, whereas a 3rd core biopsyafter another month was normal. This dog did not at anytime exhibit any abnormalities indicative of myelofi-brosis, such as nonregenerative normocytic, nor-mochromic anemia.

Clinical immunologyAntinuclear antibody (ANA) titers and rheumatoid fac-tor (RF) titers have been absent, and immunoglobulin lev-els have been normal in cases examined to date.Percentages of CD4+ and CD8+ T-lymphocytes inperipheral blood were 50.6% and 20.8%, respectively,in case 1, and 43.6% and 10.1%, respectively, in case 2.

Figure 5. Vasculitis affecting small dermal venules.Inflammatory cells within the vessel wall are a mixture of neu-trophils and mononuclear cells. Hematoxylin and eosin.Bar = 25 pm.

These values fall within the normal range (3). Serumstored at 4°C for 24 h has failed to develop acryoprecipitate.

All but 3 of the 19 cases tested have been strongly pos-itive on PF-3 test. The 3 negative cases were judged clin-ically to be only mildly affected (little footpad swellingor depigmentation and no systemic signs). One of thesepuppies was receiving immunosuppressive doses ofcorticosteroids, known to cause false negative results (4).All dogs have had normal numbers of platelets on repeatCBC. Results of the PF-3 test have consistently beennegative in cases that recovered and no longer exhibitedcutaneous or systemic signs. When the PF-3 test wasrepeated using normal puppy sera as controls, the nor-mal puppies also developed positive tests.

HistologyAt low magnification (2.5 X), the footpad lesions displaya pattern of nodular to diffuse dermatitis (Figure 3), withthe aggregates of inflammatory cells centered on foci ofdegenerating collagen. The inflammatory infiltrate iscomposed of mononuclear inflammatory cells and neu-trophils (Figure 4). Between the inflammatory cellaggregates, the dermis is edematous and lightly infiltratedwith mononuclear cells. The vessel lesions primarilyinvolve postcapillary venules (Figure 5), but, in a fewinstances, arterioles and small arteries have been affected(Figure 6). Lesions are generally subtle, including muralthickening, focal karyorrhexis, and pyknosis of nuclei,and infiltration with occasional mononuclear inflam-matory cells. Large numbers of neutrophils are occa-sionally observed within the vessel media. Fibrinoidinfiltrates have not been identified within vessel walls,but PAS-positive material has been a frequent finding.Depigmented nasal lesions have shown a mild focalinterface dermatitis with hydropic degeneration of basalcells and pigmentary incontinence.

Tissues from postmortem cases have revealed verysimilar findings. All showed evidence of extensive col-lagenolysis of the footpads, peritendinous sheaths, deepfascia and dermis of the distal limbs, and dermis ofthe ventral thorax and ventral neck. Vessel lesions havebeen observed in sections from the skin and associatedsoft tissues. The skeletal muscle sections were histo-logically normal. In 1, 12-week-old female, the

76 a etJVlme3,Dcebr19766 Can Vet J Volume 35, December 1994

Figure 6. Vasculitis affecting medium-sized dermal vessel.Mononuclear cells are beneath the intima (arrow) and kary-orrhectic nuclei are present in the media (arrowheads).Hematoxylin and eosin. Bar = 100 ,m.

thymus appeared grossly and histologically atrophic. Thispuppy also exhibited histological evidence of splenicatrophy, as indicated by a diminution of lymphoid tissue,a lack of germinal centers, and a paucity of lymphocytesin the T-cell dependent areas of the periarteriolar lym-phoid sheath. Gross hepatomegaly, deemed to be steroidhepatopathy on histological examination, was noted.This puppy had been on prednisone treatment up tothe time of euthanasia.

GeneticsThe pattern of occurrence of this affliction in over 5 gen-erations of the affected dogs suggests that the mode ofinheritance is in keeping with an autosomal recessivetrait. Carrier sires and dams have been identified for thepurpose of counselling the breeders in question, and toallow for the possibility of test matings. A pedigreechart based on information relevant to the inheritance pat-tern is shown in Figure 7.

DiscussionThis condition may be synonymous with the one that hasbeen described as collagen disorder of the footpads ofGerman shepherds (5), where a single litter of youngGerman shepherds all developed focal ulceration, ten-derness, and swelling of the footpads. Lesions regressedby 1 y of age, but affected dogs died by 2 to 3 y of agefrom renal amyloidosis. The histological lesions illus-Can Vet J Volume 35, December 1994

Pedigree Analysis

Figure 7. Pedigree chart of families of affected Germanshepherd puppies.

trated for collagen disorder of the footpads of Germanshepherds appear similar to those of our dogs. However,the two conditions differ in several respects. In the col-lagen disorder of the footpads, the authors did not reportnasal or pinnal lesions and vessel lesions were notnoted in the histological description. Dogs with thecollagen disorder of the footpads all died of renalamyloidosis: we have not seen any indication of renalamyloidosis in our dogs, although it may be too early toassume that amyloidosis will not develop.

Cutaneous vasculitis in the dog is an area of some con-troversy. The lesions considered classical for vasculitisin human patients, namely, fibrinoid degeneration of thevessel wall and leukocytoclasis (fragmentation) ofinvading leukocytes, are seldom seen in canine cutaneousvasculitis (6). Histologically, the vascular lesions inthe German shepherd puppies are subtle. While there areoccasional unequivocal lesions of vasculitis (the presenceof large numbers of inflammatory cells within the wallof an arteriole), the lesions are generally more degen-erative. For this reason, we are calling this disease a vas-culopathy, rather than a vasculitis.The most common pathogenesis suggested for vas-

culitis lesions is the deposition of circulating immunecomplexes, leading to a type III hypersensitivity reaction.Preliminary attempts to demonstrate immunoglobulin inthe vessels in naturally occurring lesions and at sites ofhistamine injection have been unsuccessful (unpub-lished results).The disease in a majority of our dogs has shown a tem-

poral association between the administration of rou-tine vaccines (for distemper, parvovirus, parainfluenzavirus, and hepatitis) and the subsequent development ofclinical signs, and lesions have worsened or recrudescedupon revaccination. It could be postulated that the vac-cine antigens, or their adjuvants, may be provoking anaberrant immune response. Focal cutaneous vasculitis hasbeen shown in some dogs to be associated with rabiesvaccination, via rabies-specific immunofluorescenceof dermal blood vessels (7). However, there have alsobeen cases in this study that exhibited clinical signsprior to, or without, the challenge from vaccination.

Lesions with some clinical similarities to those inour dogs may be seen in cases of cryoglobulinemia,resulting from cold-reacting erythrocyte autoantibodies

767

-unknown sex * U *-dead*-Affected female U-Affected male

hemagglutinating intravascularly and causing vascu-lar insufficiency via obstruction, stasis, spasm, andthrombosis. Skin lesions in cryoglobulinemia affectthe extremities (paws, ears, nose, and tail) and are exac-erbated by exposure to cold temperatures, but footpadswelling is not a feature (8-10). None of the Germanshepherd puppies tested have exhibited cryoprecipitateformation or had a positive titer on Coombs' testat 40C.

Rheumatoid arthritis, an immune-mediated erosive pol-yarthritis, has been associated with polyarteritis nodosa,a collective term for severe necrotizing inflammation ofsmall and medium-sized vessels (11). With this in mind,rheumatoid factor tests, which assess the presence of hostautoantibody of the IgG or IgM subclass to altered self-IgG (12), were carried out. None of the 11 affecteddogs that were tested exhibited a positive titer on this test.This, in combination with lack of supporting histolog-ical, radiographic, and joint cytological evidence, rulesout rheumatoid arthritis.

Other, apparently hereditary, vasculopathies havebeen reported in the literature. One, referred to as idio-pathic cutaneous and renal glomerular vasculopathy ofgreyhounds, shares some of the characteristics of the con-dition we are investigating (13). It affects both sexes andis manifested as cutaneous and renal lesions. Cutaneouslesions are erythematous, cutaneous swellings locatedaround the tarsus, stifle, inner thigh, and occasionally onthe forelimb. Lesions are slow to heal and do not respondto systemic antimicrobials. Affected dogs may exhibit avariety of syndromes, including cutaneous signs only,cutaneous signs with signs of systemic illness (fever,lethargy), and cutaneous and systemic signs with con-current evidence of kidney disease, such as azotemia. Ahereditary component was proposed due to the restric-tion of this condition to 1 breed and the apparent ten-dency to affect litters or closely related dogs, whilesparing unrelated dogs living in the same kennel andmanagement group. The authors' hypothesis (13) that agenetic predisposition to react abnormally to an infec-tious agent, or to some exogenous factor, is associatedwith the disease in greyhounds may also apply to the dis-ease we are investigating in German shepherds.Another inherited vasculitis syndrome has been

reported recently in the Scottish terrier in Denmark(14). In this instance, 5 Scottish terriers exhibited eitherbilateral nasal discharge at 3 to 4 wk of age or bilateraland ulcerative destruction of the nasal philtrum, nostrils,and nasal mucosa at 5 to 6 mo of age. Histologicalexamination revealed a leukocytoclastic vasculitis. Dueto the close relationship of the affected dogs, a heredi-tary basis is suspected, with an autosomal dominantmode of inheritance having been proposed. Despite thelimitation of cutaneous lesions to the nose, it is interestingto note the similarities between this condition and ourcases. All dogs exhibited poor response to therapy (dap-sone, prednisone, various systemic and topical antimi-crobials and antifungals), and in 1 of the dogs, a temporalassociation with vaccination for distemper, hepatitis, par-vovirus, and kennel cough, with the development ofclinical signs 7 d postimmunization, was noted.The loose-fitting skin, dropped carpi and tarsi, and the

failure of ears to stand erect in many of our cases haveprompted the hypothesis that these dogs may be exhibit-

768

ing signs of biochemically or structurally abnormalcollagen. Known by a variety of synonyms such ascutaneous asthenia, rubber puppy disease, dominantcollagen dysplasia, and dermatosparaxis, collagendefects may be manifested in a variety of ways, includ-ing skin hyperextensibility, cutaneous fragility, or both(5). In humans, there are at least 19 forms of the diseaseclassified under the general name of Ehlers-Danlossyndrome (EDS) (15). Ehlers-Danlos syndrome andrelated animal syndromes can be the result of a varietyof underlying genetic abnornalities in collagen structure,fibril packing, or synthesis of associated matrixcomponents (16). While a collagen defect is unlikely inthese German shepherd puppies, investigation of possibleabnormalities in collagen structure is currently underwayusing electron microscopy and biochemical analysisof collagen from affected dogs.

Recent work in a human patient with EDS has revealeda possible link between collagen disorders and chronicvasculitis (16). A woman with type VIII EDS, which wasmanifested as resorptive osteolysis, cardiac valvulardisease, and cutaneous vasculitis, was shown to producea T-lymphocyte-derived cytokine with binding affinityfor type I collagen. The investigators theorized thatcellular autoimmunity to type I collagen might be theunderlying cause for the chronic vasculitis. There existsthe possibility that a similar type IV hypersensitivity tosome form of collagen exists in our affected dogs.Investigation for autoantibodies to collagen is presentlyunderway.The positive PF-3 tests in 16 of 19 affected dogs

tested was initially of considerable interest. This test,which is designed to detect antiplatelet antibody, isbased on the premise that (a) antiplatelet antibodycauses immunological injury to platelets and exposureof membrane procoagulant phospholipids (PF-3 activ-ity), and (b) the coagulation process is enhanced in thepresence of PF-3 (18). Platelets are sensitive to injury,and any modification of their surface membrane couldmake PF-3 available. A positive test, therefore, mayreflect immune-mediated damage to platelets, or maysimply be due to platelet activation as a result of expo-sure to a damaged vessel wall (4). The control for this testis normal dog serum. Initially encouraged by positivetests in 16 of the affected pups, we repeated the assayusing serum collected from normal pups, to rule outthe possibility that the positive result was age rather thandisease related. It was found to be the former. ThePF-3 test is, therefore, unreliable in young dogs.

Vasculitis is associated with some forms of systemiclupus erythematosus (SLE). A subpopulation of canineSLE patients that have a compensated thrombocyto-cytic state with no evident thrombocytopeniaexists (19). Since a breed predilection to SLE has beensuggested in German shepherds (20), a similar sce-nario of compensated thrombocytolysis may be occur-ring in our subjects, although there is no other evi-dence for SLE.

Arteritis has been described in some cases of der-matomyositis, a genodermatosis of collies and Shetlandsheepdogs (20). In fact, vessel lesions resembling thoseseen in the German shepherds are considered by some tobe central to the pathogenesis of this disease (21). Theskin lesions of dermatomyositis differ clinically from

Can Vet J Volume 35, December 1994

those of the German shepherd cutaneous vasculopa-thy, although similar anatomical sites seem predisposed(nose, ear tips, tail tips, and feet). The 2 diseases have ajuvenile onset and often both are ameliorated uponreaching maturity.The transient myelofibrosis apparent in 1 of the dogs

is difficult to explain in the context of this footpadcondition. Myelofibrosis itself is a poorly understoodcondition and is postulated to be the result of myelo-proliferative diseases, exposure to toxins, ionizing radi-ation, chemotherapeutic agents, or viruses, secondary tobone marrow damage or malignancies. Many cases aresaid to be idiopathic (23-25). A genetic basis for itsdevelopment has also been proposed in human studies(24,25). Other studies have implicated a role formegakaryocyte or platelet-derived growth factors inthe development of myelofibrosis, through the stimulationof fibroblast proliferation and collagen secretion (24,26).The affected dog in our study exhibited only a mildincrease in megakaryocytes on initial bone marrowanalysis. One paper reported that 5 dogs with myelofi-brosis exhibited a moderate to marked megakaryocytosis(24). Elevated levels of immune complexes in humanpatients suffering from idiopathic myelofibrosis havebeen suggested as evidence of an underlying immune-mediated mechanism for its occurrence (27-28). If ele-vated levels of immune complexes are responsible for thevasculitis lesions exhibited in our dogs, the possibilityalso exists that immune complexes are mediating thedevelopment of myelofibrosis in our dog.A great deal more work must be accomplished to

better understand this condition. Electron microscopy andimmunohistochemistry of selected tissues, in addition tobiochemical analysis of collagen and assays for collagenautoantibodies, should help to elucidate the pathogenesisof this disease. Identification of carrier sires and damswill enable us to provide genetic counselling to breed-ers, and their cooperation will allow us to control the dis-semination of this undesirable condition in Germanshepherds in Canada.

AcknowledgmentsWe thank Dr. Peter Moore, University of California,Davis, USA for the generous gift of monoclonalantibodies recognizing canine CD4+ and CD8+T-lymphocytes. cvi

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