A Retrospective Study of Cutaneous Equine Sarcoidosis and Its Potential Infectious Aetiological Agents (Pages 51–62)

8/21/2019 A Retrospective Study of Cutaneous Equine Sarcoidosis and Its Potential Infectious Aetiological Agents (Pages 5162)

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2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology 51

Veterinary Dermatology2006, 17, 5162

BlackwellPublishingLtd

A retrospective study of cutaneous equine sarcoidosisand its potential infectious aetiological agents

IAN B. SPIEGEL*, STEPHEN D. WHITE, JANET E. FOLEY, NICOLE L.DRAZENOVICH, PETER J. IHRKE and VERENA K. AFFOLTER

*Veterinary Medical Teaching Hospital, Department of Medicine and Epidemiology, Center for VectorborneDisease, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University

of California, Davis, California 95616 USA

(Received1 June2005; accepted1 November2005)

Abstract Nine horses from ages 5 to 21 years were diagnosed with cutaneous equine sarcoidosis (ES) over an18-year period. In addition to skin, the lungs were frequently involved, with other organ systems affected lesscommonly. A predisposition for thoroughbreds and geldings was noted. Cutaneous lesions and signs includedcrusts, scales, alopecia and pruritus. These were found at various sites, particularly the legs/thighs/elbows, thorax,neck, face and ventral abdomen. Three horses were euthanized shortly after hospitalization; others survived as

long as 12 years. Histopathologic stains, immunohistochemistry and polymerase chain reaction assays on paraffin-embedded cutaneous specimens from eight horses for Mycobacteriumspp., Coccidioides immitis, Cryptococcusneoformans, Corynebacterium pseudotuberculosis, and Borrelia burgdorferiwere all negative. The aetiology of ESis unlikely microbial and continues to be a diagnosis of exclusion. ES, when limited to the skin, is associated witha good prognosis, with either partial or complete response to glucocorticoid therapy in all the surviving horses.

INTRODUCTION

Equine sarcoidosis (ES), also known as equine idio-pathic systemic granulomatous disease,1 generalized

granulomatous disease,2systemic granulomatous dis-ease,3equine histiocytic disease2and equine histiocyticdermatitis,4 is a rare multisystemic noncaseating pri-marily granulomatous and lymphoplasmacytic diseaseof unknown aetiology. It is characterized by skin lesions,involvement of one or more internal organs and oftensevere wasting,2,5,6and has been reported in horses,cattle, and humans.1,7,8 In horses, the organs mostcommonly affected are the skin, lungs, lymph nodesand gastrointestinal tract.2,5,9,10Other organs or tissuereportedly affected include liver, spleen, kidney, theskeletal system, heart, adrenal gland, thyroid glands,

pancreas and the nervous system.6

Stannard classified the skin lesions of equine sar-coidosis into two forms: (a) scaling and crusting, and(b) nodular or tumour-like masses.3The former is morecommon and is associated with the classical presentationof a focal or multifocal exfoliative dermatitis withinitially local or generalized extensive scaling and crust-ing, and a variable degree of alopecia as the diseaseprogresses, especially on the legs and the face, oftensparing the mane and the tail.11In addition, a third formhas been proposed, that is characterized by hyperkeratotic,crusted, alopecic plaques, especially on the legs ( localized)in an otherwise systemically healthy horse.6,12Serum

exudation may be associated with these lesions.2Theonset of skin lesions is often insidious, but may berapid.13Other noncutaneous clinical signs commonlyseen are persistent low-grade fever, exercise intolerance,

mild respiratory distress, weight loss, diminished appetite,peripheral lymphadenopathy, diarrhoea, icterus andlameness.5,6,14

Currently, ES is a diagnosis of exclusion. The mostlikely differential diagnoses include dermatophytosis,dermatophilosis, pemphigus foliaceus, erythemamultiforme, drug eruptions, multisystemic eosinophilicepitheliotrophic disease, equine systemic lupus erythema-tosus-like syndrome and toxicoses from arsenic, iodine,aluminium, silicon or hairy vetch.3,6Clinical manage-ment is often problematic. In some horses, the diseasemay regress spontaneously, whereas in others it responds

to corticosteroid (e.g. dexamethasone or prednisolone)treatment.Although the aetiology of sarcoidosis in humans is

unknown, it is hypothesized to be the result of an exag-gerated immunologic response of the helper/inducerT-cell arm of the immune system to an antigenic stimulus,such as an exogenous infectious agent or allergen.8,15,16

This cell-mediated immune response is suspected inhorses.5,6Although controversial, association of sarcoido-sis with tuberculosis (Mycobacteriumspp.) in humanshas been postulated because of the similar histopathologyseen in both conditions.16Polymerase chain reaction(PCR) assays have been utilized by numerous investiga-tors of human sarcoidosis, and have enabled the detectionof multiple species of mycobacterial DNA in sarcoidgranulomas/lesions (e.g. skin and lungs).1721 Fungialincluding cryptococcal (Cryptococcus neoformans),

Correspondence: Ian B. Spiegel, Red Bank Veterinary Hospital,197 Hance Avenue, Tinton Falls, NJ, 07724 USA. Tel.: +732 7473636; Fax: +732 747 6562; E-mail: [email protected]


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2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

infections have also been associated with humansarcoidosis,22 and other fungi, such as Coccidioidesimmitis, are potential causative agents, as the cutaneouslesions, systemic signs and histologic changes of thediseases are similar.6C. immitishas been reported inindividual human patients, including women treated

with corticosteroids for sarcoidosis.23

Although reference to cutaneous ES is commonin the literature of equine skin diseases,26,14 peer-reviewed reports are rare. Available data suggest thatthe syndrome is the result of persistent antigenic driveor immunologic response to an unknown trigger.5Inaddition to suspected infectious aetiologies, othercauses have been proposed, such as toxicosis with Viciavillosa(hairy vetch), and other related plant species.24,25

Hairy vetch toxicosis has been more commonly reportedin cattle in certain regions of the United States 26,27andother areas of the world, including South America.28

However, as ES is also observed in geographic regionsand pastures where hairy vetch is absent, hairy vetchtoxicosis is unlikely to be a common cause of ES.

Mycobacteria spp., C. immitis, C. neoformans,Corynebacterium pseudotuberculosisand Borrelia burg-dorferi, chosen for a variety of reasons, were investigatedas possible aetiologies for ES. In addition to controversialassociation with human sarcoidosis, Mycobacteriumspp. have rarely been implicated as the cause of cutaneouslesions in horses similar to those reported with ES.29

C. immitisis commonly present in geographical areaswith low rainfall and high temperature such as in northernCalifornia where this study took place.30C. neoformans

was included because it has been reported in humansarcoidosis.31,32It has been suggested that both C. immitisand C. neoformansmay cause secondary infectionsin human sarcoidosis because of immune suppressiveconditions associated with either the disease or immuno-suppressive treatment.23,33C. pseudotuberculosis isa well-recognized pathogen in horses.34 Because somespecies of Corynebacteriumproduce clinical syndromesin humans such as granulomatous lymphadenitis, pneu-monitis, pharyngitis, cutaneous infections and endo-carditis35(thus potentially mimicking the granulomatousreaction of sarcoidosis), they have been implicated in

the pathogenesis of human sarcoidosis.

36

B. burgdorferiwas included because of the presenceof BorreliaDNA in one horse out of three with ES withpositive titres to the spirochete.37However, although 30of 55 (54.6%) human sarcoidosis patients had antibodiesto B. burgdorferi, no organism DNA was found in thegranulomatous tissues.38

In horses, there has been minimal success in invest-igating possible aetiological agents of ES using cultures(for fungi, aerobic, anaerobic and acid-fast bacteria),special histologic stains (acid-fast, periodic acid-Schiff,Gomoris methenamine silver, auramine O), electronmicroscopy, animal inoculation studies, direct immuno-

fluorescence and immunoperoxidase testing.5,10,39The objective of this retrospective study was to

identify potential infectious agents that might beresponsible for ES in horses using special histological

stains, immunohistochemistry and PCR from paraffin-embedded biopsy samples of the skin and other availabletissues. In addition, this study reports and summarizesthe signalment, initial presenting clinical signs andhistopathologic changes seen with equine ES, the effec-tiveness of treatment modalities used and the clinical

outcome.

MATERIALS AND METHODS

Case materialThe data from 15 horses diagnosed with ES at theVeterinary Medicine Teaching Hospital of the Univer-sity of California at Davis (UCD-VMTH) and IDEXXLaboratories Incorporated in West Sacramento, Californiawere retrieved using a computer-based search for equinebiopsy samples between 1981 and 2004. The diagnosisof systemic granulomatous disease included the termsequine sarcoidosis, chronic granulomatous disease,idiopathic systemic granulomatous disease, sterile gran-ulomatous disease and/or hairy vetch poisoning. ESwas established primarily on histopathologic examina-tion of affected organs but also on history, laboratoryresults, clinical signs and elimination of other causes ofgranulomatous disease. Cases included were chosen onthe basis of history and physical examination, histologicalconfirmation of lesions consistent with ES and availa-bility of medical records and paraffin-embedded tissuesamples.

Clinical dataClinical data for horses with cutaneous ES were collectedfrom the medical records, and additional informationwas obtained from telephone interviews with the referringveterinarians and/or owners. The data included signalment(age, breed, gender), age of onset, age at diagnosis, priorhistory of skin disease, presenting complaints, clinicalsigns at presentation, onset of clinical signs, diseaseprogression, results of clinical pathology, imaging,histopathologic examination, treatment and outcome.

Histopathology and immunohistochemistry

Two of the authors (IBS and VKA) evaluated archivedhaematoxylin and eosin (H&E) sections of formalin-fixedparaffin-embedded tissues (skin and other organs, suchas lung and lymph nodes if available) from all 15 cases.These were reviewed for the presence of: multifocalnodular to diffuse noncaseating granulomatous der-matitis with histiocytes, multinucleated giant cells andfewer lymphocytes and specimens from nine horsesmet all inclusion criteria (Table 1). The paraffin blockswere no longer available for horse 2, but the specialstains performed at the time of biopsy submission werereviewed. In the remaining eight cases, special stainsincluding Gomoris methenamine silver (GMS);

Fites modified acid-fast (Fite-Faraco); and Brown andBrenn (B&B) were repeated and evaluated. Positivecontrol tissue was included for each special stainand processed simultaneously. Immunostaining with


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Equine Sarcoidosis 53

anti-Bacille Calmette-Guerin (BCG) antibodies wasused for the identification of microorganisms.40

PCR assays and DNA sequencingDNA was extracted from a total of 57 tissue samples(including skin samples for horses 1 and 3 9, and non-skin tissue samples from horses 1 and 57) and testedby PCR.41Two 50 m sections from paraffin-embeddedformalin-fixed cutaneous tissue samples and otheravailable lesional tissues (lung, lymph node andgastrointestinal tract), were placed in 1.5-mL tubes,deparaffinized with xylene (Merck, Rahway, NJ) and

washed with ethanol. They were then suspended inBuffer ATL (liquid-proprietary compound mixturecontaining edetic acid and sodium dodecyl sulphate)and proteinase K from a kit (Qiagen DNeasy tissuekit, Valencia, CA), mixed with 100 L of 0.1 mm glassbeads (Cole Parmer, Vernon Hills, Illinois), mixed ata high vortex setting for 5 min (Disrupter Genie, USAScientific, Ocala, FL), and incubated overnight at55 C. The tissue was then extracted with a commercialkit (Qiagen, Valencia, CA).

Controls. In all PCR methods, a nested PCR with a

target sequence within the glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene was used to provethe presence of amplifiable DNA. Results of all PCRassays were considered positive if the threshold cycle(Ct) value was equal to 40.

For Mycobacterium spp., amplification of the 16SrRNA gene region was performed as previouslydescribed,42using primers 246 (5-AGAGTTTGATC-CTGGCTCAG) and 247R (5-TTTCACGAACAAC-GCGACAA) for the first round, and M1 (5-AGTGGC-GAACGGGTGAGTAAC) and R7 (5-TTACGCCC-AGTAATTCCGGACAA) for the second round, in athermal cycler (MJ Research, Watertown, MA). DNA

extraction and PCR were performed in separate roomswith separate equipment including plugged injectortips. The products were separated by electrophoresisthrough 1% agarose gel and visualized with ethidium

bromide. All PCRs were run with a positive and nega-tive control. Similar protocols were used with the otherinfectious agents. For C. pseudotuberculosis, the phos-pholipase D (PLD) toxin gene was used. Amplificationwas performed with an ABI 7700 Prism SequenceDetector (Applied Biosystems, Foster City, CA) andthe products were analysed with the accompanyingsoftware.43Each 12-L reagent contained 1X TaqmanUniversal Master Mix (Applied Biosystems), 2 nmoleach primer, 400 pmol probe, and 1 L DNA. Thethermocycling conditions consisted of 50 C for 2 min,95 C for 10 min, and 40 cycles at 95 C for 15 s, followed

by 60 C for 1 min. For B. burgdorferi, a TaqMan assaywas used44 and for the subtyping of C. neoformans,DNA extraction and PCR restriction fragment lengthpolymorphism analysis of the phospholipase B (PLB1)gene was performed.45The DNA extraction and PCRprotocols followed for C. immitiswere those describedby Greene and colleagues.46

Statistical analysisData were maintained in Excel 2002 (Microsoft,Redmond, WA) and analysed in R (The R-DevelopmentCore Team, www.r-project.org). Summary statistics were

compiled for signalment, clinical findings and onset/ timeof diagnosis. Values of P< 0.05 were considered sig-nificant. Association of equine breed, age and genderwith ES was analysed with the chi-squared or Fishersexact test. Results and common trends in laboratory valueswere reported for horses affected with cutaneous ES.

RESULTS

Tissue sample analysisOf the 15 horses initially evaluated, clinical data werecollected from only nine that exhibited histologic

changes typical for ES (Table 1). The diagnostic skinsamples were obtained in winter (5/9), spring (1/9),summer (1/9) and autumn (2/ 9). Onset of clinical signswas reported in eight of the horses.

Horsenumber

Age at timeof diagnosis(years) Breed* Sex

Duration ofclinical signs priorto diagnosis(estimated in months) Organs involved

1 17 TB G 3 S, LG, LV, LN, GI, K2 10 QH M 1 S, LG, LN, MG, BM3 21 AAX G 1 S4 13 TB G 2 S5 10 TW G 2 S, LG, B (cervical vertebrae)6 5 TB G 6 S, LG7 13 TB G 1 S, LG, GI, B

(lateral femoral condyle)8 20 QH S Unknown S9 30 TB G 23 S

*TB, thoroughbreds; QH quarter horses; AAX, Arabian-Appaloosa cross; TW, Tennesseewalker.G, gelding; S, stallion; M, mare.S, skin; LG, lung; LV, liver; LN, lymph node; GI, gastrointestinal tract; K, kidney; BM, bonemarrow; B, bone (suspected involvement); MG, mammary gland.

Table 1. Signalment (age of onset, breed,gender), duration clinical signs prior todiagnosis, and organs involved


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Clinical dataSignalment. Details regarding the signalment (age,breed and gender) are listed in Table 1. A signifi-cant gender predilection was observed (P= 0.019).The mean age was 13.6 years and median age was13 years.

General clinical findings. All nine horses presentedwith skin lesions; five had other organs affected(Table 1). Radiographs suggested the presence of bonelesions with periosteal reaction and possible bonemarrow involvement of the lateral femoral condyle inhorse 7 and an osteopenic lesion in the cervical verte-

brae in horse 4, but these were not confirmed by histologicexamination. The five horses with pulmonary lesions(1, 2, and 57) had diffuse interstitial pulmonaryopacity/density on radiographs.

Cutaneous lesions and signs. Skin lesion distributionand the affected regions are listed in Table 2. Thesewere generalized except for horse 2, that had lesionslocalized to the dorsal thorax. Crusts were present ineight of the horses, and scales and alopecia/partialalopecia in five. Pruritus was reported to be present infive horses. There was associated pain in two pruritichorses and in one nonpruritic horse. For horse 9, theclinical record was incomplete. Figures 14 show the

alopecia and typical crusts and scales of ES lesions. Inhorse 6, generalized cutaneous ES was reported, butthe legs and pectoral areas remained unaffected. Theduration interval from initial clinical signs to diagnosisranged from approximately 16 months with a medianof 2 months and mean of 2.3 months. Three of thehorses had a record of previous skin disease distinctfrom their presenting complaint: an undiagnosedcutaneous nodule in the saddle region, a histologicallyconfirmed nongranulomatous exfoliative dermatosisand a subcutaneous C. pseudotuberculosisabscess present4 years previously.

Systemic clinical signs. Other clinical signs, recordedin Table 2, included weight loss, lymphadenopathy,peripheral oedema, depression and diarrhoea. Twohorses reportedly had an excellent appetite in the faceof weight loss; anorexia was reported in only three.An elevated rectal temperature was noted in one(40.4 C; horse 2).

Laboratory information. Laboratory information (Table 3)including complete blood cell count (CBC) and bio-chemical profiles were available for seven of the ninehorses.

Erythrocyte morphology abnormalities reportedincluded anisocytosis and rouleaux formation in sixand five horses, respectively. The creatinine kinase (CK)was mildly elevated in four horses. The electrolyte

Table 2. Clinical signs

Horsenumber

Alopeciacrusting*scaling

Pain orprurituslocation

Anatomicalskin lesions

Systemiclymph signsreported

Enlarged nodesor oedema

1 A, C, S Pr MRF, L, E, N, T (thighs) Anorexia weight lossdepression

Oedema(submandibular)

2 C Pn T (localized) Mild anorexia feverdepression Not reported

3 A, C, S Not reported L, VA Weight loss Oedema (leg)4 C Not reported MRF, L, BK, T (thigh) Weight loss Lymphadenopathy5 A, C, S

(scarring alopecia)Pr, Pn MRF, L, BK, N, VA Not reported Lymphadenopathy

oedema (leg)6 A, C Pr, Pn MRF, VA, T, N, BK, E

(not pectoral)Weight loss Lymphadenopathy

7 A, S Pr L (elbow, inguinal), VA(caudal area/sheath)diarrhoea depression

AnorexiaWeight loss

Lymphadenopathy

8 C, S no Pr N (and other not reported) Unknown Unknown9 C Pr L, T, N None Not reported

*A, alopecia; C, crusting; S, scaling (based on available information).Pn, pain; Pr, pruritus (based on available information).

MRF, mandibular region/face; L, legs (including thighs and elbows); VA, ventral abdomen; T, thorax; N, neck; BK, back; E, ears(all had diffuse cutaneous lesions except for horse # 2).

Figure 1. Areas of alopecia, with some scaling and crusting of ahorse with chronic granulomatous disease. (C, crusts; S, scales).


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(potassium, sodium and chloride) profiles of the horses

were unremarkable. Other tests infrequently performedand reported included: antinuclear antibody test (1 nega-tive), equine infectious anaemia Coggins (1 negative),C. pseudotuberculosistitre (two horses; 1:8 (negative)

and 1:320 (positive)) and coccidioidomycosis serology/titre (two negative immunodiffusion and complementfixation tests). No dermatophytes were cultured fromthe four horses tested.

Treatment. The treatment received by each of the ninehorses is shown in Table 4. Exact doses for medicationswere unavailable in some cases as body weights werenot always recorded. Frequency of use and techniquesused for topical treatments were unclear. Table 5 listsrecorded dosages.

Clinical follow-up/outcome. Horses 1 and 2 were euth-anized less than 1 month after presentation as bothhad multiple organ involvement and did not respond totreatment. Horse 7 had several organ systems involved,including the femoral bone, was unresponsive to oralprednisolone, and was euthanized 3 months after dis-charge from the hospital.

The remaining six horses survived, and the relevantcase-specific information is outlined in Table 3. Horses3, 4, 8 and 9 had only skin involvement. Horse 6 wasthe only horse with lung disease in which all lesions

completely resolved.There was either partial or complete response in allof the horses treated with glucocorticoids that survivedthe past 3 months. Information regarding exact courseor use of glucocorticoids was difficult to assess, as follow-up information was not always available. No horse withskin lesions only was euthanized. Follow-up was avail-able for four of the horses (horses 3, 5, 8 and 9) alivelonger than 3 months after the diagnosis. The diseasein horse 5 regressed after a 6-month tapered course oforal prednisolone and the horse was still alive for over8 years with alopecic scarring in previous lesional areasand orthopaedic age-related changes. The disease in

horse 8 spontaneously regressed, recurred in the sameseason (autumn) the following year, and then regressedagain. Horse 9 responded to oral corticosteroids andantibiotics and subsequently moved and was lost to

Figure 2. Prominent alopecia with some scaling of the face and ears.(A, alopecia; C, crusts; S, scales).

Figure 3. Higher magnification of the alopecia and extensive scalingof the neck. (A, alopecia; S, scales).

Figure 4. Areas of partial alopecia along the dorsum witha moderate degree of crusting and scaling.


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follow-up. Horse 3 resolved after receiving dexameth-asone for 2 weeks with minimal improvement and

2 months of testosterone supplementation. The latterhad been gelded several months before skin lesiondevelopment and was lesion free for an additional12 years.

Histopathologic and immunohistochemicalfindings

The skin of all nine horses had predominantly nodularto occasionally diffuse granulomatous dermatitis char-acterized by irregular foci of histiocytes (with variablyvacuolated cytoplasm) and multinucleated giant cells

Table 3. Blood screen findings

Horsenumber Clinical chemistry* Haematology

1 AST (796 IU L1; normal 138 409 IU L1) neutrophils (9425 neutrophils L1; normal 2600 6800 neutrophils L1)T-bili (42.3 mol L1; normal 8.5 39.3 mol L1) fibrinogen (7 g L1; normal < 4 g L1)BUN (12.9 mmol L1; normal 4.39.6 mmol L1)

GGT (65 IU L

1

; normal 822 IU L

1

)SD (12 IU L1; normal 08 IU L1)2 globulin (52 g L1; normal 1747 g L1) neutrophils (10 948 neutrophils L1; normal 2600 6800 neutrophils L1)

T-bili (42.3 mol L1; normal 8.5 39.3 mol L1) monocytes (1292 monocytes L1; normal 0500 monocytes L1)fibrinogen (6 g L1; normal < 4 g L1)lymphocytes (1156 lymphocytes L1; normal 16005800 lymphocytes L1)haematocrit (27%; normal 30 46%)

3 lymphocytes (1200 lymphocytes L1; normal 1600 5800 lymphocytes L1)4 T-bili (44.5 mol L1; normal 8.5 39.3 mol L1) leucocytes (10 400 leucocytes L1; normal 500011 600 leucocytes L1)

lymphocytes (1352 lymphocytes L1; normal 16005800 lymphocytes L1)5 globulin (55 g L1; normal 1747 g L1) neutrophils (13 376 neutrophils L1; normal 2600 6800 neutrophils L1)

GGT (23 IU L1; normal 822 IU L1) leucocytes (15 200 leucocytes L1; normal 500011 600 leucocytes L1)fibrinogen (5 g L1; normal < 4 g L1)

6 GGT (14 IU L1; normal 822 IU L1)7 globulin (68 g L1; normal 1747 g L1) neutrophils (10 744 neutrophils L1; normal 26006800 neutrophils L1)

fibrinogen (5 g L1

; normal < 4 g L1

)haematocrit (29.9%; normal 3046%)

Note: horses 8 and 9 did not have laboratory work available/performed. Key:= increased/above normal and = decreased/below normal*AST, aspartate aminotransferase; T-bili, total bilirubin; BUN, bloodurea nitrogen; GGT, gamma-glutamyl transpeptidase; SD, sorbitoldehydrogenase.

Table 4. Treatments used, outcomes, and other case-specific information

Horsenumber

Oralsteroids*

Injectablesteroids* NSAID Other treatments

Known outcome and case-specific information (survival)and other comments

1 No DEX (IV) No TMS selenium sulphide(topical)

Euthanized < 2 monthsafter diagnosis

2 No No PBZ TMS Euthanized < 2 months after diagnosis3 DEX No No Testosterone (oral) Alive 12 years after diagnosis,

dead whenCaptan (topical) Manuscript publishedCopper naphthenate(topical)

4 PRED No No Not reported Alive at discharge; lost to follow-up5 PRED DEX (IM) PBZ Not reported Alive > 8 years after diagnosis, alive when

Manuscript published6 PRED No No Mineral oil (topical)

Glycerine (topical)Alive at discharge (3 months improved),Lost to follow-up

Selenium sulphide(topical)

7 PRED No No Not reported Alive at discharge, euthanized 3 months later8 No No No No Alive when manuscript published; seasonal

9 DEX No No Hydroxyzine (oral) Alive when manuscript publishedTMS (oral)Povidone-iodine(topical)

*DEX, dexamethasone; PRED, prednisolone.NSAID, nonsteroidal anti-inflammatory drug.PBZ, phenylbutazone.TMS, sulfamethoxazole and trimethoprim.


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(Fig. 5). There were fewer lymphocytes and rarely neu-trophils. In most instances, the dermal inflammatorynodules were not associated with follicular or adnexal

structures. In horse 1, although the nodules appearedmore closely associated with hair follicles, there wasno evidence of folliculitis. In horses 3 and 6, increasedneutrophils were associated with the granulomas, whichillustrate the possibility of a slightly different presentationof the same condition. Horse 7 had an increased numberof lymphocytes throughout the sections, especiallyassociated with giant cells (Fig. 6). Other common butnondiagnostic findings included mild acanthosis,mild diffuse hyperkeratosis, mild spongiosis and layeredcrusts composed of parakeratosis, degenerating inflam-matory and epithelial cells and serum.

Special stains including GMS, Fite-Faraco, and B&B

and immunohistochemistry for BCG were applied ineight cases. All the special histological stains were neg-ative, with the exception of B&B stains, which revealedthe presence of large gram-positive narrow bacilli in

granulomatous (inflammatory) and noninflammatoryareas in tissues from six horses (75%) (Figs 7 and 8). Thesepathogens were most consistent with Clostridiumspp.

PCR analysisNested PCR for the target sequence within the house-keeping gene, GAPDH, revealed amplifiable DNA in100% of the tissues in the eight horses evaluated. However,PCR analysis for mycobacterial, coccidioidal, crypto-coccal, corynebacterial or B. burgdorferi DNA werenegative in all horses, including horse 3 with a positiveserological titre to C. pseudotuberculosis.

DISCUSSION

Cutaneous sarcoidosis in horses presents with skinlesions different from those reported in the humandisease. Generalized exfoliative lesions may be seenbut the most typical signs are crusting, scaling and

Table 5. Medication dosages

Horsenumber Dose information* (where available)

1 Corticosteroids (dexamethasone 20 mg IV b.i.d.) and antibiotics (sulfamethoxazole and trimethoprim, 12 tablets orally b.i.d.;tablet size unreported).

2 Phenybutazone (2 g in AM and 1 g in PM orally) and antibiotics (sulfamethoxazole and trimethoprim 15 tablets orally b.i.d.;

tablet size unreported).3 Testosterone supplementation orally (dose unavailable) for two months after a 2-week course of dexamethasone (20 mg orallyb.i.d. and taper).

4 Oral prednisolone (unknown dose), 0.04 mg kg1of dexamethasone intramuscularly once daily for 5 days, then prednisolone1.7 mg kg1orally once a day for 2 weeks, then 1 mg kg1orally once a day for 2 weeks, and finally tapered to once every otherday.

5 This horse also received 2 mg kg1of phenylbutazone orally twice daily as needed for neck stiffness.6 Oral prednisolone (1.3 mg kg1orally b.i.d. for 3 weeks, then 1.3 mg kg1orally s.i.d. with a taper of 50 mg week1for at least

2 months.7 Oral prednisolone (1.3 mg kg1orally s.i.d. for 5 weeks then e.o.d.).8 No treatment reported.9 Dexamethasone orally (0.02 mg kg1q.i.d. for 3 days, then 0.01 mg kg1q.i.d. for 3 days, then the same dose e.o.d. for 5 days)

along with a course of hydroxyzine pamoate (0.9 mg/kg orally b.i.d. for 2 weeks) and antibiotics (sulfamethoxazole andtrimethoprim 30 mg kg1orally b.i.d. for 14 days).

*b.i.d., twice daily; s.i.d., once daily; e.o.d., every other day.

Figure 5. Horse 1: dermal nodule composed of predominantlyepithelioid macrophages (M) and multinucleated giant cells (G).Connective tissue is prominent (CT). (H&E. Magnificationbar = 100 m).

Figure 6. Horse 7: a predominant marginal infiltration oflymphocytes (L) is associated with the histiocytic nodule andgiant cells (G). Some blood vessels are also noted (BV).(H&E. Magnification bar = 50 m).


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alopecia occurring on the face, girth area, ventralabdomen, axillary and inguinal region, neck, shoulder,legs, prepuce and scrotum.1,9,10,13,37In one case report,eventual involvement of over 60% of the skin surface ofa miniature pony was described,1whereas others describethe typical clinical signs, and occasional nodules.1,9,10,13,37

The majority of lesions in the present study conformedto the general pattern and were observed on extremities,including legs, thighs and elbows, neck and thorax, aswell as the ventral abdomen, mandibular region, face,ears), and back. More than half of the horses studied

were pruritic. Previous retrospective studies and casereports did not mention this finding,1,9,10,13,37and it ispossible that pruritus was present and not reported, asone text states that horses with cutaneous sarcoidosisare often pruritic.47Pruritus has been reported as a clinicalsign for human cutaneous sarcoidosis.48

Noncutaneous clinical signs of ES, indicating systemicinvolvement, include weight loss, anorexia, respiratorydistress, peripheral lymphadenopathy, fever and exerciseintolerance.1,9,10,37These are similar to observations inhumans with sarcoidosis.8,49,50In this study, over halfof the horses had weight loss, possibly the result of anorexia

or, alternatively, granulomatous inflammation of thegastrointestinal tract with subsequent malabsorption ofnutrients. Gastrointestinal lesions, which have been reportedpreviously in horses9and in humans,51were confirmedin one horse and suspected in another, with nonremittingdiarrhoea. These two horses were euthanized within 1 and3 months respectively, suggesting a poor prognosis asso-ciated with gastrointestinal tract involvement and ES.

More than half of the horses had lung involvementwith a diffuse interstitial pulmonary opacity/densitydepicted on radiographs. Pulmonary lesions are com-monly reported in ES1,5,9and human sarcoidosis.5255

Lymphadenopathy, a common characteristic of

human sarcoidosis50,53,55and ES,1,9,10was detected inthree of the horses in this study. Peripheral oedema,seen in three horses, has previously been reported in ES,10

and is an unusual presentation of human sarcoidosis.56

Additional clinical signs depended on the organ systeminvolved. Radiographs showed lesions on the lateralfemoral condyle and cervical vertebrae of two horses.Although not reported previously in horses, vertebralsarcoidosis occurs in human sarcoidosis57 where thebone is commonly affected.55The lesions in liver, kidneyand mammary gland tissues were similar to earlierfindings.9 Liver and kidney involvement occurs inhuman sarcoidosis.58,59

Other organs reported to be affected in humansinclude spleen,15 the musculoskeletal system,60 car-

diac,61,62neurological system63and the eyes.64Ocularinvolvement in the horse has not been reported,11butinvolvement of the spleen, heart and neurologicalsystem has been reported in ES,6 including a case ofencephalitis in the absence of concurrent skin involve-ment.65Although these were not specifically identifiedor investigated in the present study, the range of potentialorgan involvement needs consideration in ES.

The neutrophilia, hyperfibrinogenaemia and hyper-globulinaemia found are commonly reported withES.1,5,9,11,37 These values are probably increased, atleast in part, because of inflammation. Additional data

reported are mild nonregenerative anaemia, hypercal-caemia and abnormal kidney and liver functiontests.1,5,9,11,37Elevated globulin levels are to be expectedwith prominent inflammatory lesions as seen withsarcoidosis. None of the nine horses in this study hadhypercalcaemia, but many had neutrophilia, hyperfi-brinogenaemia, hyperglobulinaemia, and two horseshad a mild nonregenerative anaemia.

The age range of 521 years is similar to previousreports of affected horses (3 months to over20 years),1,9,10,13,37 and suggests no age predilectionfor ES. Cutaneous ES may have a predilection forthoroughbreds. However, as breed distribution was not

compared with that of a reference population, and casenumbers were small, it was not possible to determinewhether or not thoroughbreds were statistically over-represented. Nevertheless, this observation is similar to

Figure 7. Horse 8: large gram-positive straight bacilli (B)in granulomatous (inflamed) areas (B&B. Magnificationbar = 500 m). These are likely the result of contaminationin sampling or processing.

Figure 8. Horse 7: large gram-positive straight bacilli (B)in noninflamed areas (B&B. Magnification bar = 500 m).These are likely resulting from contamination in samplingor processing.


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that of previous reports.9,10,37Given the small numbers,males and geldings in particular were significantlyover-represented, although a firm conclusion on genderpredilection for ES was not possible. This was also trueof two previous reports of cutaneous ES.13,37However, inhumans, women are affected two to eight times more

frequently than men.16

As geldings appear over-represented,it would be interesting to determine if there is a temporalassociation between ES and castration. One horse inthis study was healthy for 15 years, and according tothe owner, within 2 months of the castration, developedlesions that resolved after 2 months of oral testosteronetreatment. The horse then remained lesion free for 12 years.ES appears to be seasonally independent; only one horsepresented with repetitive transient seasonal clinicalsigns and other retrospective studies did not report aseasonal influence.13,24 However, the season seems toinfluence development of sarcoidosis in humans.6669

Previously, the prognosis given for recovery fromES has been poor.2,6This study indicates a more favourableprognosis, particularly if fewer organ systems are involved.Even horses with involvement of several organ systemssurvived for many years. Gastrointestinal involvementsuggests a poor prognosis, whereas ES limited to theskin suggests a good prognosis.

To the authors knowledge, this is the first attempt toinvestigate potential infectious aetiologies of ES usinga combination of multiple histological stains, immuno-histochemistry and PCR to detect intralesional DNAof Mycobacterium spp., C. immitis, C. neoformans,C. pseudotuberculosis, and B. burgdorferi. PCR is a

highly sensitive and specific technique when run withappropriate controls but false-negative results mayoccur as a result of (a) degraded target DNA, (b) PCR-inhibiting substances present in tissue specimens, or(c) insufficient extraction of target DNA.70The latter isparticularly a concern for mycobacteria as these bacteriaare difficult to lyse because of the lipid-rich cell wall.71

Degradation of the target DNA is also a concern, as manyof the current samples were greater than 510 years oldand had been fixed in formalin, which can diminish the PCRamplification signal.72However, good-quality GAPDHsignals were obtained with all of the samples, indicating

that the horse DNA had been efficiently extracted.The negative PCR results, immunohistochemistryand special histological stains were negative foracid-fast bacteria, all suggesting that ES is unlikelyto be caused by an active mycobacterial infection.The failure to detect mycobacterial DNA in any of theparaffin-embedded samples tested accords with theresults of some studies of human sarcoidosis usingPCR.73,74 Other PCR studies of human sarcoidosishave sometimes detected mycobacterial DNA (espe-cially Mycobacterium tuberculosisand Mycobacteriumavium complex) in granulomatous lesions in organssuch as skin and lungs.1721,7580Thus, there is evidence

for mycobacteria as a cause for sarcoidosis, but alsoevidence to the contrary. It also seems unlikely thatother bacteria are the cause of ES. Only one horse hada weak positive serology titre for C. pseudotuberculosis,

which likely indicated a previous exposure as the PCRwas negative. Histologic features of C. pseudotuberculosisinfections are quite different from ES and are mainlycharacterized by a pyogranulomatous inflammationthat may contain numerous eosinophils.81

The occasional large gram-positive narrow bacilli,

located in both granulomatous (inflamed) and noninflamedareas were most consistent with Clostridiumspp. Thisfinding is most likely to be caused by contamination asthese bacteria were not associated with granulomatouschanges. The organisms are also unusual in skin specimens.

Lymphocyte exocytosis, which has been describedin humans,82was seen in one horse. Otherwise, all ninehorses presented with virtually identical lesions, includingvariably sized aggregates of multinucleated histiocytic giantcells and epithelioid cells with small numbers of neu-trophils, lymphocytes and plasma cells within the superfi-cial and deep dermis. This is similar to previous reports.5,6

In conclusion, the failure to obtain positive resultsfrom the PCR assays and special histological stainsindicate that the granulomatous lesions associated withES are unlikely to be caused by persistent mycobacterial,coccidioidal, cryptococcal, corynebacterial or Borreliainfections. Failure to detect an infectious aetiologybecause of elimination of the infectious agent(s) bythe immune system is unlikely, as the granulomatousinflammation did not subside without treatment. Thisleads to the hypothesis that the tissue reaction associatedwith ES is the response to a persistent antigenic triggerthat is difficult to degrade by the host. The nature of thistrigger is still unknown and ES remains a disease of un-

determined aetiology and therefore, a diagnosis of exclusion.

ACKNOWLEDGEMENTS

The authors thank IDEXX Laboratories Inc., WestSacramento, California for access to case material, thelate Dr Tony Stannard for pioneering most of theinitial information regarding sarcoidosis in horses andfor Figs 3, 4 and 5, Dr Thelma Lee Gross for aidingthe acquisition of cases from IDEXX and the horseowners. Finally, the authors are thankful for resources

from the George H. Muller Fund for Research inVeterinary Dermatology at the School of VeterinaryMedicine, University of California, Davis.

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Rsum Neuf chevauxgs de 5 21 ans ont t diagnostiqus avec une sarcoidose cutane (ES) en 18 ans. Enplus des lsions cutanes, une atteinte pulmonaire tait frquemment note, les autres organes tant moinstouchs. Une prdisposition pour les animaux de race pure tait observe. Les lsions cutanes regroupaient descrotes, des squames, une alopcie et un prurit. Elles taient observes sur diffrentes zones, notamment les mem-bres, les cuisses, les coudes, le thorax, le cou, la face et labdomen ventral. Trois chevaux ont t euthanasis peuaprs lhospitalisation, dautres ont survcu jusqu 12 ans. La recherche de micro-organismes par histologie,immunohistochimie, et PCR (pour Mycobacteriaspp., Coccidioides immitis, Cryptococcus neoformans, Coryne-

bacterium pseudotuberculosis, et Borrelia burgdorferi) tait ngative. Ltiologie de lES est peu probablementdorigine microbienne et ce diagnostic reste un diagnostic dexclusion. LES limit la peau est de bon pronostic,avec une rmission partielle ou complte avec une corticothrapie.

Resumen Nueve caballos de entre 521 aos de edad se diagnosticaron de sarcoidosis equina cutnea (ES)durante un perodo de 18 aos. Adems de la piel, los pulmones aparecieron afectados con frecuencia, y otrosrganos con menor frecuencia. Se observ una predisposicin por caballos pura sangre y caballos castrados. Losndulos se presentaron en varios lugares, pero especialmente en las extremidades/muslos/codos, trax, cuello,cara y vientre. Tres caballos se sacrificaron poco despus de ser hospitalizados, otros sobrevivieron hasta 12 aos.Tinciones histolgicas e inmunohistoqumicas frente a Mycobacteria spp., Coccidioides immitis, Cryptococcusneoformans, Corynebacterium pseudotuberculosisyBorrelia burgdorferifueron negativas. La etiologa de ES posi-blemente no es microbiana y contina siendo un diagnstico por exclusin. Si esta limitada a la piel, el pronsticoes generalmente favorable, con respuesta parcial o completa a la terapia de glucocorticoides en todos los caballos

supervivientes.

Zusammenfassung Neun Pferde im Alter von 5 bis 21 Jahren wurden ber einen Zeitraum von 18 Jahren mitkutaner equiner Sarkoidose (ES) diagnostiziert. Zustzlich zur Haut war die Lunge hufig involviert, whrendandere Organsysteme weniger hufig betroffen waren. Eine Prdisposition fr Vollblter und Wallachen wurdefestgestellt. Kutane Vernderungen und Symptome beinhalteten Krusten, Schuppen, Alopezie und Juckreiz.Diese wurden in verschiedenen Lokalisationen gefunden, vor allem an den Extremitten/Oberschenkeln/Ellbgen,am Thorax, Hals, Gesicht und am ventralen Abdomen. Drei Pferde wurden kurz nach der Einweisung euthanasiert;andere berlebten bis zu 12 Jahre. Die histopathologischen Frbungen, die Immunhistochemie und die PolymeraseChain Reaction Assays, die an den in Paraffin eingebetteten Hautproben von acht Pferden auf der Suche nachMycobacteria spp., Coccidioides immitis, Cryptococcus neoformans, Corynebacterium pseudotuberculosis undBorrelia burgdorferidurchgefhrt wurden, waren alle negativ. Eine mikrobielle tiologie von ES ist unwahr-scheinlich und somit bleibt ES eine Ausschlussdiagnose. Wenn die ES auf die Haut beschrnkt war, bestand eine

gute Prognose, mit entweder teilweisem oder komplettem Ansprechen auf die Glukokortikoidtherapie bei allenberlebenden Pferden.

Documents

A Retrospective Study of Cutaneous Equine Sarcoidosis and Its Potential Infectious Aetiological Agents (Pages 51–62)