Signalment:  

A 6-year-old, female Labrador cross dog (Canis familiaris).This dog has a ~3y history of atopy which has been managed using allergen specific immunotherapy and symptomatic anti-pruritic therapy. At the time of biopsy the dog was receiving cyclosporin 30mg q 24hrs and ketoconazole 250mg q 24 hrs; prednisolone 6.25mg q 48hrs, Episoothe shampoo baths once a week.

Gross Description:  

On clinical examination, there were multiple erythemic dermal papular to nodular lesions located on the head, trunk and limbs ranging from 0.5cm to 4cm in diameter.

Histopathologic Description:

Ten tissue sections were examined from the biopsies submitted, only one was submitted as our Wednesday Slide Conference submission. In all sections there was moderate to marked hyperplasia of the epidermis and outer root sheath of the follicular infundibulum. Within basal and spinous layer keratinocytes of predominately the infundibulum, but also the epidermis were frequent, scattered protozoal pseudocysts containing numerous (~5 to 100) rectangular to banana-shaped zoites measuring ~2 x 5μm (Fig. 3-2). There were occasional nodular aggregates of neutrophils and eosinophils within the follicular epithelium and epidermis associated with the protozoal pseudocysts. Follicular infundibulae were distended with keratin, aggregates of degenerate neutrophils and sheets of free protozoal zoites. There was also occasional follicular rupture (Fig. 3-1). Within the dermis was a diffuse interstitial infiltrate of neutrophils, macrophages, lymphocytes and plasma cells. Associated with the inflammatory infiltrate were modest numbers of free and phagocytosed protozoal zoites, which in some areas may have been within vascular endothelium.

Morphologic Diagnosis:  

1. Moderate to severe, chronic, follicular and epidermal hyperplasia and hyperkeratosis with intra-keratinocyte protozoal pseudocysts DDx: Neospora,
2. Moderate to marked, diffuse, interstitial pyogranulomatous dermatitis with intrahistiocytic, intraendothelial and free protozoal zoites: DDx: Neospora
3. Moderate to marked, multifocal, neutrophilic folliculitis and pyogranulomatous furunculosis with numerous intrakeratinocyte and free protozoal zoites:DDx Neospora

Lab Results:  

Anti-Toxoplasma IgG Indirect Immunofluorescence (IFAT) > 1:512
Anti-Toxoplasma IgM Indirect Immunofluorescence (IFAT) < 1:32

Anti-Neospora Indirect Immunofluorescence (IFAT) > 1:25600
Indirect Immunohistochemistry: Anti-Neospora Antibody (monoclonal): positive;
Anti-Toxoplasma Antibody (polyclonal). 
Microbiology: Negative for fungal and bacterial growth

The two samples from the skin of the dog were strongly positive for Neospora caninum with a Neospora qPCR and a nested Neospora PCR.

Condition:  

Neospora caninum

Contributor Comment:  

Although indirect immunohistochemistry was positive using a monoclonal primary antibody against Neospora and a polyclonal primary antibody against Toxoplasma, the very high anti-Neospora titre supports a diagnosis of cutaneous Neosporosis in this case. Further work using pcr to further discriminate between Neospora and Toxoplasma is underway.

Cutaneous Neosporosis has been reported in dogs before, although it is more commonly recognized as a cause of neuromuscular disease in young dogs and abortion in cattle². The predominately cutaneous manifestation of Neosporosis demonstrated in this case may be associated with the concurrent immunotherapy this dog was receiving. CD4+ T-cells have been shown to play an important role in mice in protecting them against N. caninum infection⁸. Although cyclosporine is recognized to be an effective treatment for canine atopy with minimal side effects⁹, the combination of prednisolone and cyclosporine treatment in this dog would have had a suppressive effect upon cell mediated immunity which is important in protecting dogs against N. caninum infection⁸. Neosporosis has been previously reported in an adult dog receiving prednisone and azathioprine⁶. 

JPC Diagnosis:  

Haired skin: Dermatitis and furunculosis (Fig. 3-1), pyogranulomatous, multifocal, moderate, with neutrophilic folliculitis, and intraepithelial intrahistiocytic and free protozoa, Labrador cross (Canis familiaris), canine.

Conference Comment:  

Neospora caninum is an apicomplexan that up until 1988 was often misdiagnosed as Toxoplasma gondii.³ There are three infective stages: oocysts, tachyzoites, and tissue cysts. Canids, in addition to acting as an intermediate host, are considered the primary definitive host.5 Oocysts are only found in and shed by the definitive host.⁵ Tissue cysts, 110_m diameter with a 1-4_m thick cyst wall, are usually found in the brain, spinal cord, and rarely muscle, and contain numerous 2 X 8 _m bradyzoites.¹ Tachyzoites are 4-7_m X 1.5-5 _m and may be located within macrophages, keratinocytes, neutrophils, endothelial cells, or fibroblasts.⁵

Neosporosis affects a variety of species including sheep, goats, and deer, but most importantly, dogs and cattle.⁴ It is reported to be one of the most important causes of bovine abortion, and transplacental transmission can occur.⁴ In dogs, neurological disease in puppies is common, and cutaneous manifestations have been reported in immunosuppressed animals.⁴ Histologically, cutaneous lesions are composed of pyogranulomatous and eosinophilic to necrotizing and hemorrhagic dermatitis.⁷ Diagnosis primarily relies on serologic testing.⁷

N. caninum can be distinguished from T. gondii based on the following ultrastructural features.¹

Neospora caninum	Over 11 rhoptries Tachyzoites often not within a parasitophorous vacuole Tissue cysts are relatively uncommon
Toxoplasma gondii	Few rhoptries Always found in membrane-bound vacuole in cytoplasm

We thank Dr. C. H. Gardiner, PhD, veterinary parasitology consultant to the AFIP, for his review of this case.

References:

1. Brown CC, Baker DC, Barker IK: Alimentary system. In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., vol. 2, pp. 272-273. Elsevier Saunders, Philadelphia, PA, 2007
2. Dubey JP: Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 41:1-16, 2003
3. Gardiner CH, Fayer R, Dubey JP: An Atlas of Protozoan Parasites in Animal Tissues, 2nd ed., pp. 59-60. Armed Forces Institute of Pathology, Washington, DC, 1998
4. Ginn PE, Mansell JEKL, Rakich PM: Skin and appendages. In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., vol. 1, p. 711. Elsevier Saunders, Philadelphia, PA, 2007
5. Maie MG, Youssef S: Nervous system. In: Jubb, Kennedy and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., vol. 1, pp. 436-437. Elsevier Saunders, Philadelphia, PA, 2007
6. Ordeix L, Lloret A, Fondevila D, Dubey JP, Ferrer L, Fondati A: Cutaneous neosporosis during treatment of pemphigus foliaceus in a dog. J Am Anim Hosp Assoc 38:415-419, 2002
7. Scott DW, Miller WH, Griffin CE: Viral, rickettsial, and protozoal skin diseases. In: Muller and Kirks Small Animal Dermatology, 6th ed., pp. 532-533. Saunders, Philadelphia, PN, 2001
8. Steffan J, Favrot C, Mueller R: A systematic review and meta-analysis of the efficacy and safety of cyclosporin for the treatment of atopic dermatitis in dogs. Vet Dermatol 17:3-16, 2006
9. Tanaka T, Hamada T, Inoue N, Nagasawa H, Fujisaki K, Suzuki N, Mikami T: The role of CD4+ or CD8+ T cells in the protective immune response of BALB/c mice to Neospora caninum infection. Vet Parasitol 90:183-191, 2000

A virtual slide is not available for this case.

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Fig. 3-1 Haired skin

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