History

A 10-year-old 27.5-kg (60.5-lb) spayed female Labrador Retriever was referred to a veterinary dermatologist for evaluation of nonpruritic truncal scaling, erythema, and alopecia of 1 to 2 months’ duration. The result of prior bacterial culture of a skin lesion swab was negative. The dog also had a > 1-year history of progressive nonregenerative anemia, hypoalbuminemia, hyperglobulinemia, mild azotemia, high serum alkaline phosphatase activity, poorly concentrated urine with proteinuria, mild generalized lymph node enlargement, and intermittent diarrhea. A diagnosis of adrenocortical insufficiency had been made 2 years earlier and was managed with daily oral administration of prednisone and once-monthly desoxycorticosterone pivalate injections. During the preceding 6 months, the prednisone dosage was increased from 2.5 to 5 mg once daily to 5 mg twice daily because of fever and weakness; as a result, clinical signs improved. Also, in the preceding year, a diagnosis of hypothyroidism was made, and treatment with levothyroxine was prescribed. Serologic assessments for tick-borne diseases performed 3 months prior to the evaluation yielded negative results. Cytologic examination of fine-needle aspirate specimens from enlarged lymph nodes submitted by the referring veterinarian 6 months prior to the evaluation revealed a lymphoid hyperplasia or reactive process with a high number of neutrophils and large amounts of hemosiderin and melanin. The dog had no history of travel outside of the United States or contact with dogs that originated from outside of the United States.

Clinical and Gross Findings

Physical examination revealed bilaterally symmetric strips of lumbar scaling, hair loss, easily epilated fur, and erythema (Figure 1); in general, the dog's skin was mildly erythematous, and the coat was dry and dull. There was mild periocular and pedal hypotrichosis, an enlarged second digit on the left forepaw, and moderate temporal muscle wasting. The dog did not appear overtly pruritic, and no ectoparasites were visible. There was mild generalized lymphadenopathy. Skin scrapings were obtained for microscopic examination; no mites were detected.

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Photographs of a 10-year-old Labrador Retriever that was evaluated because of nonpruritic truncal scaling, erythema, and alopecia of 1 to 2 months’ duration. The dog also had a > 1-year history of progressive nonregenerative anemia, hypoalbuminemia, hyperglobulinemia, mild azotemia, high serum alkaline phosphatase activity, poorly concentrated urine with proteinuria, mild generalized lymph node enlargement, and intermittent diarrhea. Diagnoses of hypothyroidism and adrenocortical insufficiency had been made 1 and 2 years earlier, respectively. A—On the right flank, notice the area of lumbar alopecia, scaling, and erythema. The dog was bilaterally and symmetrically affected. B—In a higher-magnification view of the hypotrichotic flank area, the skin has extensive adherent silvery scaling and superficial crusting with occasional erosions.

Citation: Journal of the American Veterinary Medical Association 254, 7; 10.2460/javma.254.7.813

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Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Additional Dermatologic Findings

Cytologic examination of impression smears of scaly areas revealed scattered RBCs, neutrophils, and macrophages that had numerous small intra- and extracellular, round to oblong organisms suspected to be protozoal amastigotes (Figure 2). Reexamination of a previously collected lymph node aspirate specimen was requested, and multiple biopsy specimens of affected skin were obtained for histologic examination.

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Photomicrographs of a cytologic preparation of an impression smear of inflamed skin from the dog in Figure 1. A—Red blood cells and extracellular protozoal amastigotes (arrows) are visible. Romanowski-type stain; bar = 20 μm. B—Numerous protozoal amastigotes (arrow) are present within a macrophage. Romanowski-type stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 254, 7; 10.2460/javma.254.7.813

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Histopathologic Findings

Skin biopsy samples were fixed in neutral-buffered 10% formalin. Tissues were embedded in paraffin, sectioned at a thickness of 4 to 6 μm, and stained with H&E stain. In all sections, there were multifocal and coalescing areas of deeply basophilic, granular to crystalline material (mineralized collagen) at all levels of the dermis (Figure 3). Throughout areas of the dermis that were not mineralized, there was reactive fibroblast hyperplasia and mixed inflammation as evidenced by the presence of macrophages mixed with fewer lymphocytes and plasma cells as well as scattered multinucleated giant cells. There was marked irregular hyperplasia of the epidermis (acanthosis) and follicular infundibula with mild inflammation of deeper regions of follicles and of adnexal glands in nonmineralized areas. There was moderate to marked, disorganized or densely compacted orthokeratotic hyperkeratosis of the epidermis and follicular infundibula (comedones). Also present in all sections were large numbers of protozoal organisms, which sometimes were arranged in clusters within the cytoplasm of macrophages and multinucleated giant cells (Figure 4). Distinct cyst walls were not identified. The organisms were basophilic, ovoid structures (approx 2 μm in length and < 1 μm in width) that were periodic acid–Schiff reaction negative. On the basis of their size and morphology, the organisms were classified as protozoal amastigotes and not as trypomastigotes, which are long, slender, and flagellated.

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Photomicrograph of a section of a formalin-fixed skin biopsy specimen obtained from the dog in Figure 1. Multifocal and coalescing areas of deeply basophilic, granular to crystalline material (mineralized collagen [arrows]) are present in all levels of the dermis. This finding is consistent with calcinosis cutis attributable to long-term steroid oversupplementation. H&E stain; bar = 500 μm.

Citation: Journal of the American Veterinary Medical Association 254, 7; 10.2460/javma.254.7.813

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Higher-magnification photomicrograph of a section of a formalin-fixed skin biopsy specimen obtained from the dog in Figure 1. Notice the large numbers of protozoal organisms (arrows), which sometimes appear in clusters within the cytoplasm of macrophages and multinucleated giant cells. H&E stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 254, 7; 10.2460/javma.254.7.813

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Cytologic and Serologic Findings

On reexamination of the previously collected lymph node aspirate specimen, scattered extracellular protozoal organisms were found. A serum sample obtained from the dog was assessed for antibodies against Leishmania spp; testing revealed a Leishmania-specific antibody titer of 1:6,400.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: dystrophic dermal calcification and granulomatous dermatitis with orthokeratotic hyperkeratosis, acanthosis, and large numbers of intrahistiocytic protozoa.

Case summary: leishmaniasis and calcinosis cutis that was secondary to iatrogenic hyperadrenocorticism in a dog.

Comments

Possible causes of alopecia, erythema, and scaling in dogs include bacterial or fungal skin infection, parasitic skin infection (eg, infection with fleas, mites, or Leishmania spp), hypersensitivity disorders (atopy, food allergy, or flea-bite hypersensitivity), keratinization disorders (sebaceous adenitis), and cutaneous epitheliotropic lymphoma. On the basis of clinical, clinicopathologic, and serologic findings, a diagnosis of leishmaniasis was made for the dog of the present report. Leishmania spp are obligate intracellular protozoal parasites that infect mammals including humans, dogs, and cats. Leishmaniasis is endemic in 88 countries with approximately 2 million new human cases identified each year in Europe, Africa, South America, and Asia.^1–3 In humans, clinical forms of the disease include visceral, cutaneous, and mucosal leishmaniasis. Visceral leishmaniasis is caused by parasites of the Leishmania donovani–Leishmania infantum complex.³ Dogs are the primary reservoir for L infantum³; in Mediterranean countries, it is estimated that 50% to 80% of the canine population is seropositive for L infantum (reflecting exposure or infection), and 2% to 5% of those dogs develop the clinical illness canine leishmaniasis (CanL).⁴

Leishmanial organisms are transmitted by sandflies in most areas of the world. There are several species of sandflies in North America including Lutzomyia shannoni, which is capable of transmitting leishmanial organisms from infected dogs to hamsters in experimental settings.^5,6 However, natural sandfly transmission of Leishmania spp has not yet been documented in the United States. In the United States, most cases (commonly involving Foxhounds) of CanL may not involve a vector, and there is documentation that transmission can occur vertically with occasional horizontal transmission.⁷ During the past several decades, the frequency of CanL detection among imported and native dogs in North America has increased, including an epizootic caused by L infantum in American Foxhound kennels.⁸ Other rare cases in the United States have involved dogs exposed to the disease transplacentally from infected dams imported from Europe or exposed to the blood of other infected dogs via fighting, licking of wounds, or blood transfusions.^7–9 Cases in Mexico and Central and South America have also been reported.

Ticks and fleas that have been feeding on infected dogs can harbor leishmanial organisms.¹⁰ In laboratory studies,^11,12 after fleas and ticks obtained from infected dogs were macerated and injected IP or given orally to hamsters, some of the hamsters had positive test results for Leishmania infection; however, natural transmission of leishmaniasis to dogs by these ectoparasites has not been determined, to our knowledge.

Once a dog is bitten by an infected sandfly, protozoal promastigotes are engulfed by host macrophages and distributed throughout the body; intracellular amastigotes develop, and clinical signs may develop after an incubation period of 1 month to 7 years.¹³ Most Leishmania-exposed dogs do not develop clinical illness¹⁴; the initial host response has a major role in development of disease. Dogs that develop a strong helper T lymphocyte type 1 response and have high circulating concentrations of associated helper T lymphocyte type 1 cytokines (eg, interferon γ) and increased cellular immunity are most likely to resist infection. Certain dog breeds (Boxer, German Shepherd Dog, Cocker Spaniel, and Rottweiler) appear predisposed to develop CanL, as are dogs < 3 years or > 8 years of age. Concurrent immunosuppressive illnesses contribute to development of clinical disease.¹⁵ As a systemic disease, CanL can affect any tissue or organ and causes nonspecific signs including weight loss, lymphadenopathy, lethargy, splenomegaly, fever, uveitis, and lameness. Skin lesions can include exfoliative, erosive, nodular, papular, or pustular dermatitis. Common clinicopathologic abnormalities include nonregenerative anemia, hyperglobulinemia, hypoalbuminemia, azotemia, proteinuria, and high circulating liver enzyme activities.^2,4 A diagnosis of leishmaniasis in a dog is made on the basis of clinical signs, clinicopathologic findings, results of cytologic or histologic examination of specimens obtained from lymph nodes and skin lesions, and detection of serum Leishmania-specific IgG via immunofluorescent antibody testing or ELISA. Polymerase chain reaction assays can also detect leishmanial DNA in tissue and fluid samples.^2,4 In endemic areas, positive test results for Leishmania infection are common among dogs and may or may not indicate that the dogs have CanL, which is a clinical and clinicopathologic diagnosis.¹³

Treatment of CanL typically involves administration of a combination of allopurinol and a pentavalent antimonial drug (meglumine antimoniate) or miltefosine.^4,15 In the past, meglumine and miltefosine have been unavailable in the United States, other than by importation on a case-by-case basis through FDA compassionate-use protocols. In 2016, miltefosine^a was approved in the United States by the FDA for the treatment of human leishmaniasis. Other less commonly used or second-line treatments for CanL include administration of marbofloxacin, liposomal or lipid-emulsified amphotericin B, aminosidine, pentamidine isethionate, sodium stibogluconate, and paromomycin sulfate.^1,4,15,16 In a limited early clinical trial in dogs, domperidone appeared to have potential as an immunostimulant of the cell-mediated helper T lymphocyte type 1 immune response for treatment and prevention of CanL.¹⁷ The goal of treatment is to reduce severity of clinical signs, improve organ function, and decrease parasite load to reduce disease transmission risk; it is very difficult to achieve a parasitological cure, and short-term treatments are usually followed by relapse within a year.¹⁸ For this reason, parasitostatic drugs such as allopurinol are usually combined with a leishmanicidal treatment and administration of the drug combination is continued for several months or years after clinical cure.^18,19 Allopurinol administration is not discontinued unless there is complete clinical and clinicopathologic recovery and the dog is negative for serum anti–Leishmania antibody.¹⁵ Clinicopathologic testing, including urinalysis and assessment of the urine protein-to-creatinine ratio, should be performed at the end of the first month of treatment, then every 3 to 4 months until complete recovery, and every 6 to 12 months thereafter. In addition, serum anti–Leishmania antibody titer should be measured every 6 months; detection of a > 2-fold increase in the titer between consecutive assessments is suggestive of relapse.¹⁵

The prognosis for a dog with CanL depends on presence and severity of renal disease and whether the dog is treated and monitored according to published guidelines.¹⁵ The mean survival time of dogs with L infantum infection that receive treatment is 4.7 years,²⁰ and it has been reported that 75% of dogs without chronic renal failure are likely to be alive at 4 years after diagnosis.²¹ Stringent parasite control (application of deltamethrin collars or permethrin spot-ons) is required for infected dogs to prevent them from being a source of infection for other dogs.⁴ There is a public health risk for Leishmania infection, and it is a so-called monitored condition that has to be reported by monthly summaries to the Washington State Department of Agriculture.²²

The dog of the present report was native to Washington State, and it was unknown how or when it was exposed to Leishmania spp; the owner was carefully questioned and stated that the dog was born in the United States, never traveled outside of the country, and was never exposed to dogs of European origin or Foxhounds. One of the authors (KSC) was aware of 2 other cases of leishmaniasis in the nearby area (Tacoma, Wash); one case involved an animal at the local zoo (no details available regarding species or geographic origin), and the other case involved a dog imported from Iraq by an American soldier who had adopted the dog while stationed there. The latter case leads to the concern that infected dogs without clinical signs of leishmaniasis that are imported from Leishmania-endemic countries could act as foci for infection for dogs in the United States via an unknown vector or bloodborne transmission.

The dog of the present report was initially treated with allopurinol (11 mg/kg [5 mg/lb], PO, q 12 h indefinitely) and marbofloxacin (1.8 mg/kg [0.8 mg/lb], PO, q 24 h for 4 weeks). Benazepril hydrochloride (0.37 mg/kg [0.17 mg/lb], PO, q 24 h) was prescribed for treatment of proteinuria. To address calcinosis cutis attributable to steroid oversupplementation, the prednisone dosage was reduced to 5 mg, PO, once daily for 2 weeks and then 2.5 mg, PO, once daily. Monthly desoxycorticosterone pivalate injections for management of hypoadrenocorticism and administration of levothyroxine for treatment of hypothyroidism were continued by the primary care veterinarian. The dog responded well to treatment for leishmaniasis and the prednisone dosage reduction; the truncal coat condition normalized and scaling resolved, as did prior muscle atrophy and toe swelling. The dog's activity level and attitude also improved remarkably. Reevaluation of clinicopathologic variables 1 month after initiation of treatment revealed resolution of prior anemia and azotemia, normalization of serum alkaline phosphatase activity, and improved serum albumin concentration. Hyperglobulinemia and proteinuria persisted; therefore, the benazepril dosage was increased to 0.37 mg/kg, PO, every 12 hours and miltefosine was added (2 mg/kg [0.9 mg/lb], PO, q 24 h for 4 weeks) to the treatment regimen. Allopurinol treatment was continued unchanged. Three months after treatment initiation, clinicopathologic analyses revealed continued improvement of serum albumin and globulin concentrations and normalization of Hct and BUN concentration; however, proteinuria was persistent and the dog had developed a urinary tract infection, which was treated with a 2-week course of amoxicillin–clavulanic acid. Owing to financial constraints, the owner declined follow-up culture of a urine sample, and further management of the case was taken over by the primary care veterinarian.

Six months after initiation of treatment, the dog's serum anti–Leishmania antibody titer was rechecked by the primary care veterinarian, and the result indicated a marked increase (titer of 1:25,600). A CBC and serum biochemical panel revealed persistently low-normal albumin concentration, decreased Hct, and increased BUN concentration, compared with findings after 3 months. Allopurinol administration was continued, and domperidone (1 mg/kg [0.45 mg/lb], PO, q 12 h for 4 weeks) was prescribed. Continued close monitoring of the dog's renal function, including determination of the urine-to-protein creatinine ratio and microbial culture of a urine sample as well as rechecking the serum anti–Leishmania antibody titer in 6 months, was recommended; however, the owner declined further diagnostic testing. Two months after completion of domperidone treatment, the owner indicated that the dog was continuing to do clinically well. However, 6 months later (ie, 15 months after diagnosis), azotemia and proteinuria worsened, and the dog developed uncontrollable epistaxis despite a platelet count and blood pressure measurements that were within reference intervals. The owner elected euthanasia; no necropsy was performed.