Cutaneous Neosporosis During Treatment of Pemphigus Foliaceus in a Dog

Introduction

Neosporosis is a protozoal disease caused by the coccidial parasite Neospora caninum (N. caninum).¹ Common clinical presentations of the disease include progressive ascending paralysis of the hind limbs in young dogs, polymyositis, and multifocal central nervous system (CNS) signs that can lead to neonatal death.^1–3 Less frequently, the infection is diagnosed in adult or senior dogs presenting with neuromuscular and multifocal CNS signs.²³ Nonneuromuscular clinical presentations such as myocarditis, pneumonia, hepatitis, and dermatitis have been described as well.^2–4 In natural infection, the only known route for transmission of the protozoan is transplacental, and clinical signs in adult dogs are likely an exacerbation of a congenital infection rather than a postnatal acquisition of infection.²³ Presumptive diagnosis of neosporosis is based on clinical signs and elevated titers on indirect immunofluorescence assay (IFA).³ Definitive diagnosis is based on visualization of N. caninum tachyzoites in tissue or cytopathological samples.³ The recommended treatment of neosporosis is a combination therapy consisting of trimethoprim-sulfadiazine plus clindamycin or clindamycin alone.³

Canine cutaneous neosporosis is a rare dermatitis, which has been reported in five dogs to date.^15–8 Because Neospora-associated dermatitis has been described only in middle-aged or older dogs, it has been hypothesized that dermal neosporosis might be due to age-related immunodeficiency.⁵⁸

This report describes cutaneous neosporosis in a middle-aged dog during immunosuppressive treatment for pemphigus foliaceus.

Case Report

A 4-year-old, 45-kg, intact male rottweiler was presented to the Veterinary Teaching Hospital of the Universitat Autònoma de Barcelona (Spain) with a 10-day history of multiple papulonodular skin lesions on the trunk. No history of pruritus or pain associated with these lesions was reported.

At the time of presentation, the dog had been receiving prednisone^a (1 mg/kg body weight, q 48 hours) and azathioprine^b (1 mg/kg body weight, q 48 hours) on alternate days for the preceding 9 months to treat pemphigus foliaceus that was diagnosed based on evaluation of histopathological skin biopsy specimens.

No abnormalities other than dermatological were detected on general physical examination. Dermatological examination revealed multiple papules and nodules on the right lateral trunk. Lesions ranged from 0.5 to 2 cm in diameter, and some had an alopecic and crusted surface [Figure 1]. No lesions attributable to pemphigus foliaceus were detected. Differential diagnoses for this papulonodular dermatitis included infectious granulomas, neoplasia (i.e., mastocytoma and cutaneous lymphoma), immune-mediated diseases (i.e., sterile nodular panniculitis, sterile granuloma and pyogranuloma), and cutaneous/systemic reactive histiocytosis.

The initial diagnostic workup included a complete blood count (CBC), serum biochemical profile, serum protein electrophoresis (as the authors’ hospital is in a Leishmaniosis-endemic area), cytopathological examination of a fine-needle aspiration from a nodule, and multiple skin biopsies. No abnormal findings were detected in the blood tests. Cytopathological examination revealed a pyogranulomatous inflammation; bacterial or protozoan organisms were not observed. Histopathological examination of biopsy specimens stained with hematoxylin and eosin (H&E) revealed periadnexal pyogranulomatous dermatitis and intraluminal folliculitis [Figure 2]. Numerous, small, intracellular basophilic round bodies resembling protozoal tachyzoites were seen in the cytoplasm of epithelial cells [Figure 3] and in the cytoplasm of dermal macrophages. A presumptive diagnosis of pyogranulomatous dermatitis due to protozoan organisms was made. Specific immunohistochemical stainings for Leishmania infantum (L. infantum),⁹ Toxoplasma gondii (T. gondii),¹⁰ and N. caninum¹⁰ were performed, and serum samples were obtained for serological examination to detect antibodies against L. infantum,^c T. gondii,^d and N. caninum.¹¹ A diagnosis of Neospora-associated dermatitis was confirmed by positive immunohistochemical staining [Figure 4] and a 1:1600 serum antibody titer to N. caninum on the Neospora agglutination test (positive if higher than 1:200¹¹). Immunohistochemical and serological tests for L. infantum and T. gondii were both negative.

To further investigate the protozoan structural characteristics, the formalin-fixed skin specimens embedded in paraffin wax were deparaffinized in three successive xylene solutions. After thorough dewaxing, tissue was fixed in 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer (pH, 7.2) and processed for transmission electron microscopy examination.^e Ultrastructurally, numerous N. caninum tachyzoites were observed within keratinocytes as well as within macrophages. Tachyzoites were located in parasitophorous vacuoles in host cell cytoplasm. Tachyzoites were 2.25 to 4 μm × 1.7 to 2.8 μm in size and contained electron-dense rhoptries and numerous micronemes [Figure 5]. Protozoa divided by endodyogeny.

The dog was treated with clindamycin hydrochloride^f (12.5 mg/kg body weight, q 12 hours), and azathioprine therapy was discontinued. In order to maintain the pemphigus foliaceus in remission, prednisone therapy was maintained (1 mg/kg body weight, q 48 hours). Clinical improvement was evident after 21 days of treatment, but the cutaneous lesions did not completely resolve. Clindamycin therapy was continued, and prednisone therapy was gradually tapered and stopped after 3 more weeks. After 81 days of treatment with clindamycin, cutaneous lesions completely resolved. Despite the findings of a normal dermatological examination, clindamycin treatment was maintained for an additional month.

The dog remained in remission of Neospora-associated dermatitis 10 months after discontinuation of clindamycin therapy and in remission of pemphigus foliaceus 12 months after discontinuation of immunosuppressive therapy.

Discussion

Protozoal dermatitis is an unusual cause of nodular dermatitis, with the exception being cutaneous leishmaniosis in endemic areas.¹²

Differential diagnoses for protozoal dermatitides in dogs include L. infantum, Caryospora spp., Sarcocystis canis (S. canis), T. gondii, and N. caninum infections. Leishmania infantum is easily distinguished in tissue and cytopathological samples because of the morphological features of the amastigotes containing kinetoplast.¹² The presence of caryocysts and sexual stages of the parasite in dermal connective tissue cells characterize Caryospora spp. infection.¹³ Sarcocystis canis is distinguished by the presence of their schizonts. Sarcocystis canis divides by endopolygeny, whereas T. gondii and N. caninum divide by endodyogeny.¹³

Among these protozoal dermatitides, N. caninum and T. gondii dermatitides are the most difficult to differentiate. Due to the similar morphology in cytopathological smears and in H&E-stained sections, N. caninum has been misdiagnosed as T. gondii for many years;¹⁴ however, cases of cutaneous toxoplasmosis in dogs have not been confirmed.⁵ Immunohistochemical techniques and ultrastructural examination may aid in distinguishing N. caninum from T. gondii. In this case, immunohistochemical examination provided the definitive diagnosis, because tachyzoites were stained by N. caninum antibody but not by T. gondii antibody. Ultrastructurally, N. caninum has a thicker cyst wall and tachyzoites have more numerous micronemes and rhoptries than T. gondii, and, as observed in this dog, the rhoptries in N. caninum are electron-dense, whereas those of T. gondii are electron-lucent.¹

Middle-aged to older dogs seem to be more susceptible to the dermal form of neosporosis, suggesting an age-related immunodeficiency.⁵⁸ In this case, the affected dog was an adult dog (4 years of age), and it was receiving immunosuppressive drugs. Recently, it has been demonstrated in mice that interferon-γ(INF-γ) is an important T-helper 1 (Th1) cytokine for protection against N. caninum infection at the early stage of infection.¹⁵ Moreover, CD4+ T cells are essential to the production of specific antibodies and therefore play an important role in the host protection during the later stages of the infection.¹⁵ The suppressive effects on cell-mediated immunity due to glucocorticoids¹⁶ and the inhibition of proliferation and differentiation of both B and T lymphocytes due to azathioprine¹⁶ might have had a role in the development of cutaneous neosporosis in this dog. However, lymphopenia was not detected on CBC, and no tests to assess lymphocyte function were performed to conclude that this dog was immunosuppressed.

Transplacental transmission is the only proven natural route of N. caninum infection.²³ Some of the puppies transplacentally infected have clinical manifestations at young ages, whereas other puppies of the same litter may carry the infection subclinically for years. This subclinical stage is also suggested by the fact that seroprevalence of clinically normal dogs is much greater than the prevalence of clinical illness.² Acquired immunodeficiency due to immunosuppressive therapy in a transplacentally infected dog with subclinical disease might have caused the development of clinical signs of neosporosis.

Complete clinical remission was obtained after 81 days of clindamycin therapy. Clindamycin has been successfully used in two previous cases of canine cutaneous neosporosis.⁵⁷ However, in these two previous cases, the cutaneous lesions resolved 30 and 21 days after starting clindamycin therapy, respectively.⁵⁷ Whether the immunosuppressive therapy used in this case might have played a role in the need for longer therapy remains unclear.

Cutaneous lesions related to neosporosis were not present 10 months after clindamycin therapy was stopped, and lesions attributable to pemphigus foliaceus were not observed almost 1 year after discontinuing immunosuppressive therapy. The lack of recurrence of pemphigus foliaceus signs after discontinuation of immunosuppressive therapy suggests a drug-induced pemphigus foliaceus, which is clinically and histopathologically indistinguishable from spontaneous pemphigus foliaceus.¹⁷ To the authors’ knowledge, this represents the first reported case of canine cutaneous neosporosis with a long-term follow-up. Based on the lack of data about dogs recovered from cutaneous neosporosis, it is difficult to predict possible recurrences of the disease in this dog. In case of relapsing clinical signs attributable to pemphigus foliaceus, alternative immunomodulatory therapies, such as a tetracycline and niacinamide combination, should be considered.¹⁷ Restarting with immunosuppressive therapy could lead to a relapse of the clinical signs of neosporosis if the dog were clinically cured but still harbored the parasite.

Conclusion

Cutaneous neosporosis is a rare disease only recognized in older dogs.^15–8 This report documents a case of cutaneous neosporosis in a middle-aged dog receiving immunosuppressive drugs. The authors conclude that cutaneous neosporosis should be considered as a differential diagnosis of papulonodular dermatitis, especially in immunodeficient adult dogs.

Acknowledgments

The authors thank Xavier Roura and Mariona Camps for their scientific supervision of the manuscript.