A 1-year-old 32.5-kg (71.5-lb) sexually intact male foxhound-Treeing Walker Coonhound cross with no travel history outside of Colorado was admitted to the Colorado State University Veterinary Teaching Hospital in the fall of 2008. The dog was examined because of a 2.5-month history of diarrhea, progressive weight loss despite a good appetite, dermatologic lesions, and hind limb stiffness. In an effort to control the diarrhea, which appeared to commence after a food change, at least 2 diet trials (including 1 raw meat type) were fed over the 2.5 months prior to admission at the veterinary teaching hospital, with no notable change in the diarrhea. Fecal flotation testing in early October revealed Giardia spp cysts, and metronidazole (23 mg/kg [10.5 mg/lb], PO, q 12 h for 8 days) and pyrantel pamoate (10.8 mg/kg [4.9 mg/lb], PO, once) were administered. The diarrhea improved slightly after these treatments, but returned. After the raw food trial was finished, bacterial culture of feces was found to be negative for Salmonella spp. Additionally, prior to evaluation at the veterinary teaching hospital, serum biochemical profile revealed that the dog was hyperproteinemic (9.1 mg/dL; reference range, 5.2 to 8.2 mg/dL), was hyperglobulinemic (6.3 mg/dL; reference range, 2.5 to 4.5 mg/dL), and had a high BUN concentration (39 mg/dL; reference range, 7 to 27 mg/dL).

Dermatologic lesions were initially nonpruritic and began as an alopecic area on the crown of the head. Over approximately 8 weeks, the lesions progressed to become erythematous and pruritic, with scaling involving the face, muzzle, and axillary regions. Weeks prior to evaluation at the veterinary teaching hospital, a skin scraping was negative for Demodex mites on microscopic examination and had negative dermatophyte culture results. A 7-day trial treatment with oral administration of ivermectin once a day (day 1, 0.08 mg/kg [0.03 mg/lb]; day 2, 0.15 mg/kg [0.07 mg/lb]; days 3 through 7, 0.23 mg/kg [0.1 mg/lb]) did not elicit improvement in the skin lesions. Twelve days after completion of ivermectin administration, a skin biopsy specimen was submitted to the Colorado State University Veterinary Diagnostic Laboratory; histologic evaluation revealed severe chronic folliculitis, potentially of bacterial origin, but no organisms were observed. Cephalexin (23 mg/kg, PO, q 12 h) was administered for 30 days with minimal clinical response prior to referral to the veterinary teaching hospital.

At the time of referral, the dog was bright, alert, and responsive, but was thin with a body condition score of 2.5/9. There was mucopurulent discharge from the left eye and bilateral blepharitis. The dog had generalized muscle wasting, peripheral lymphadenopathy, and pruritic lesions of diffuse thickening, alopecia, moderate erythema, and follicular casting and crusting on the cranium, face, pinna, ventrum, axillary, and inguinal regions. Palpation suggested bilateral tarsal joint effusion, and the left tarsus seemed painful.

Initial diagnostic tests included a CBC; serum biochemical profile; urinalysis; cytologic examination of fine needle aspirates of the prescapular lymph nodes; serologic testing for Diroflaria immitis antigen and antibodies against Ehrlichia canis, Anaplasma phagocytophilum, and Borrelia burgdorferi^a; trypsin-like immunoreactivity assay^b; microscopic examination of a skin scraping from an affected area on the calvarium; dermatophyte culture of hair collected from multiple regions; and abdominal ultrasonography.

Hematologic and serum biochemical analyses revealed that the dog had a nonregenerative anemia (PCV, 32%; reference range, 40% to 55%), monocytosis (1.4 × 10³ cells/μL; reference range, 0.2 × 10³ cells/μL to 1.0 × 10³ cells/μL), mild hypercalcemia (11.8 mg/dL; reference range, 9.2 to 11.7 mg/dL), hyperproteinemia (9.0 g/dL; reference range, 5.3 to 7.2 g/dL), and hyperglobulinemia (6.3 mg/dL; reference range, 2.0 to 3.8 g/dL). Urinalysis revealed that the dog had proteinuria and a urine specific gravity of 1.015. A cytologic examination of fine needle aspirates of the prescapular lymph nodes revealed reactive lymphocytes. Trypsin-like immunoreactivity was 9.6 μg/L (reference range, 5.7 to 45.2 μg/L). Results of a serologic test for detection of D immitis antigen were negative, and serum antibodies against E canis, A phagocytophilum, and B burgdorferi were not detected. Results of fungal culture and microscopic examination of skin scrapings were negative. Findings on abdominal ultrasonography included mild hepatomegaly, mild splenomegaly, diffuse abdominal lymphadenomegaly, a possible retained testis in the right side of the abdomen, and a thickening of the wall of the cranioventral portion of the urinary bladder.

Despite the fact that Leishmania spp amastigotes were not found on microscopic examination of multiple skin scrapings, on cytologic examination of fine-needle aspirates of the prescapular lymph nodes, or on histologic examination of skin biopsy specimens, the breed, clinical findings, and hematologic abnormalities were consistent with leishmaniasis. Thus, a serum sample was submitted for detection of antibodies against Leishmania spp.^c While awaiting results, the dog was discharged from the hospital and prescribed tylosin (10.6 mg/kg [4.8 mg/lb], PO, q 8 h) for 14 days and a probiotic,^d in an effort to ameliorate the diarrhea, in addition to the previously prescribed cephalexin.

After 10 days of treatment, the diarrhea had not resolved, and the dog continued to lose weight despite a good appetite. Serum antibodies against the Leishmania recombinant antigen K39, which is specific for Leishmania donovani complex, were detected.^c Whole blood and popliteal lymph node aspirates were submitted for a PCR assay of Leishmania DNA, and a serum sample was submitted for indirect fluorescent antibody testing for antibodies against Leishmania spp.^e The dog had a high serum indirect fluorescent antibody titer (1:1,024) against Leishmania infantum, and DNA specific for Leishmania spp was amplified in a whole blood sample and a popliteal lymph node aspirate via PCR assay, confirming the diagnosis of leishmaniasis. The owners declined treatment because of the dog's working status, the severity of the disease, and the expense of treatment with little chance of a cure.

The dog was euthanized by IV administration of an overdose of thiopental and pentobarbital, and a necropsy was performed. Histologic examination of skin specimens revealed chronic perifolliculitis, which was lymphohistiocytic and plasmacytic with multifocal to coalescing lesions. Intrahistiocytic amastigotes were observed, consistent with Leishmania organisms found in the haired skin. Membranoproliferative mesangial glomerulonephritis, with moderate to marked lymphoplasmacytic interstitial nephritis, portal hepatitis, and diffuse bone marrow hyperplasia, was reported. Although the Leishmania amastigotes were found in the skin, they were not found in other tissues.

While the dog in this report was in utero, its foxhound dam had clinical manifestations consistent with leishmaniasis; in the summer of 2007, the foxhound dam was pregnant and had been brought to the veterinary teaching hospital for evaluation of epistaxis. On physical examination, lymphadenopathy was found, and approximately 11 fetuses were seen ultrasonographically. Serum biochemical profile and CBC revealed that the dam had eosinophilia (1.7 × 10³ cells/μL; reference range, 0.1 × 10³ cells/μL to 1.2 × 10³ cells/μL), regenerative anemia (PCV, 22%; reference range, 40% to 55%), hyperproteinemia (9.3 g/dL; reference range, 5.3 to 7.2 g/dL), and hyperglobulinemia (7.0 g/dL; reference range, 2.0 to 3.8 g/dL). Prothrombin and partial thromboplastin times were within reference limits. Results of a serologic test for D immitis antigen were negative, and serum antibodies against E canis, A phagocytophilum, and B burgdorferi^a were not detected. Fenbendazole (51.4 mg/kg [23.4 mg/lb], PO, q 24 h) was administered for 3 days and doxycycline (9.2 mg/kg [4.2 mg/lb], PO) was administered once prior to the determination that the dam was seronegative for E canis. At whelping, 4 puppies were stillborn, 1 puppy died within a week, and 1 died within a few weeks, and 4 puppies, which included the dog of this report, were seemingly healthy. The 4 stillborn puppies were necropsied at Colorado State University Veterinary Diagnostic Laboratory and found to be full-term males with lungs that appeared to have never been expanded with air. Severe autolysis was found in all stillborn puppies with the addition of splenomegaly, hepatomegaly, and renomegaly in 1 puppy; blood clots on the dorsal surface of the brain in 1 puppy; and petechial hemorrhages throughout the thymic parenchyma of 1 puppy. Histologic evaluation of affected tissues was not performed because of the degree of autolysis; therefore, no paraffin-embedded tissues were available for subsequent PCR assay of Leishmania DNA.

The dam was from a foxhound kennel club in Kansas and was bred in Colorado with a previously healthy Treeing Walker Coonhound from Colorado. The foxhound dam had lived primarily in Kansas, but had also traveled to foxhound kennel clubs in Michigan and South Carolina, all 3 states of which are included in the 21 states where the CDC has found serologic evidence of Leishmania exposure in hunting dogs.^1,2 Colorado, however, is not one of the states where either the CDC or a recent United States and Canadian seroprevalence study³ has found Leishmania spp in the canine population. The foxhound dam lived in Colorado for 8 months in 2007. Although her epistaxis reportedly resolved, she was euthanized about 8 months after returning to Kansas because of a high serum antibody titer for Leishmania spp.

Of the 4 puppies that survived, 3 (all male) remained in Colorado and 1 (female) returned with the dam to Kansas. The female puppy was euthanized at 1 year of age when a high serum antibody titer against Leishmania spp was detected. Of the 3 male littermates that never left Colorado, the clinically ill dog described in this report lived with 1 unaffected male littermate, and the other unaffected male littermate lived in a separate residence in the same neighborhood. The affected and unaffected littermates that lived together in Colorado also lived with their Treeing Walker Coonhound sire and an unrelated Treeing Walker Coonhound female. After the diagnosis of leishmaniasis was confirmed in the dog of this report, tests, which included PCR assays for detection of Leishmania DNA in whole blood samples and lymph node aspirates, indirect fluorescent antibody testing for detection of serum antibodies against Leishmania spp, and flow cytometry^f for determination of lymphocyte subset distributions (CD4+ and CD8+) in blood samples, were performed for all 4 dogs of the household. Flow cytometry results revealed a low CD4+:CD8+ lymphocyte ratio in the clinically affected male (420 CD4+ and 406 CD8+ [1:1 ratio]), compared with the unaffected littermate (649 CD4+ and 317 CD8+ [2:1 ratio]) and the 2 older Treeing Walker Coonhounds (309 CD4+ and 98 CD8+ cells/μL [3:1 ratio] and 693 CD4+ and 139 CD8+ [5:1 ratio]). On the basis of the negative serologic test results and PCR assay results, the sire, unaffected male littermate, and unrelated bitch did not appear to be infected by Leishmania spp at that time. However, it is important to note that because of the latent period, serum antibodies may not be detectable in Leishmania-infected dogs prior to the onset of clinical disease.⁴ This may have occurred in the unaffected 2.75-year-old male littermate and housemate that developed weight loss despite a good appetite and end-stage renal disease leading to euthanasia nearly 2 years after the dog of this report was determined to have leishmaniasis. However, diagnosis of leishmaniasis in this dog is unconfirmed, as necropsy and PCR testing were not performed.