Pathology in Practice

Jacob T. Ambridge Department of Veterinary Pathology, School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland

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Sei Ming Lee Vets4Pets Lancaster, Morecambe, England

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Christopher J. Palgrave Idexx Laboratories UK, Grange House, Sandbeck Way, Wetherby, England

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 BVM&S, PhD
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Jenny McKay Idexx Laboratories UK, Grange House, Sandbeck Way, Wetherby, England

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Pamela A. Kelly Department of Veterinary Pathology, School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland

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Alan T. Stevens Idexx Laboratories UK, Grange House, Sandbeck Way, Wetherby, England

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 BVetMed, MSc

History

A 5.5-year-old 24.4-kg spayed female Boxer was referred because of lumps on the ears and multifocal areas of alopecia over the elbows, hind limbs, ears, and flanks. The dog had a 4-year history of ongoing allergic dermatitis managed with either oclacitinib or, more recently, prednisolone (0.4 to 0.6 mg/kg, PO, q 24 h) prescribed by the referring veterinarian. During flare-ups in which secondary bacterial infection was considered a feature, adjunctive antimicrobial treatment (cephalexin; 15 to 25 mg/kg, PO, q 12 h) successfully resolved clinical signs; however, microbial cultures to confirm complete resolution were not pursued. The dog was up to date with vaccinations and had never traveled outside of the UK.

Clinical and Gross Findings

On examination, the dog had foci of alopecia over the elbow joints and thinning of the coat along the trunk and dorsum. There was also alopecia of the ear pinnae, primarily affecting the pinna margins. The hairs on the ears were noticeably fragile and easily epilated. Bilateral, multifocal, raised, hard, ulcerated nodules were on the internal and external aspects of the pinnae. No signs of otitis externa were detected on otoscopic examination. Cytologic evaluation of impression smear samples of the aural nodules was unrewarding. The prednisolone dosage was increased to 0.6 mg/kg, PO, every 24 hours, and treatment with cephalexin (20 mg/kg, PO, q 12 h) was started. At the follow-up examination 2 weeks later, the pinnal nodules had large central areas of ulceration and the ear pinnae were diffusely thickened (Figure 1).

Figure 1
Figure 1

Photograph of multiple raised, ulcerated nodular lesions on the inner aspect of the ear pinna of a 5.5-year-old 24.4-kg spayed female Boxer with a 4-year history of ongoing allergic dermatitis that had been managed with either oclacitinib or, more recently, prednisolone.

Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.01.0061

Histopathologic and Cytologic Findings

Cytologic assessment of an impression smear of an ulcerated pinnal nodule revealed mild blood contamination in a proteinaceous background with a low to moderate cellular yield. A direct smear of a fine-needle aspirate collected from one of the pinnal dermal nodules revealed abundant cocci and rods alongside numerous oval to crescentic cells (Figure 2). These cells were 2 to 4 × 5 to 7 μm in size and had lightly basophilic cytoplasm and small metachromatic nuclei, consistent with protozoal tachyzoites. There were also moderate numbers of variably degenerate neutrophils, rarer macrophages, and intracellular tachyzoites and cocci. A punch biopsy was performed and, following fixation in neutral-buffered 10% formalin solution, submitted for histologic examination.

Figure 2
Figure 2

Photomicrograph of a direct smear of a fine-needle aspirate collected from one of the pinnal dermal nodules of the dog described in Figure 1. The smear contains numerous 2- to 3-μm-wide protozoal tachyzoites (arrowheads) mixed with neutrophils (arrow). Wright-Giemsa stain; bar = 25 μm.

Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.01.0061

Histopathologic evaluation of this sample demonstrated infiltration of the dermis by large numbers of macrophages, neutrophils, plasma cells, and fewer lymphocytes and eosinophils, which encircled blood vessels and disrupted the dermal collagen. There were multifocal areas of hemorrhage, moderate edema, and scant fibrin. Numerous macrophages contained abundant intracytoplasmic, 2 × 3-μm, oval to crescentic protozoal tachyzoites that often caused margination of the nucleus. Similar tachyzoites were in the cytoplasm of keratinocytes and endothelial cells (Figure 3). The epidermis displayed moderate diffuse acanthosis, moderate spongiosis, multifocal neutrophil exocytosis with subcorneal pustule formation associated with colonies of coccoid bacteria, multifocal areas of necrosis, and multifocal areas of ulceration. Multifocally, blood vessels showed neutrophilic and fibrinoid vasculitis and, in areas of epidermal ulceration, were partially occluded by fibrin thrombi. There was also mild fibrosis of the superficial dermis.

Figure 3
Figure 3
Figure 3

Photomicrograph of sections of a nodular pinnal lesion of the dog described in Figure 1. A—The dermis is infiltrated by numerous neutrophils mixed with macrophages, many of which contain abundant intracytoplasmic, 2 × 3-μm, oval to crescentic protozoal tachyzoites (arrows) that cause margination of nuclei (arrowhead). H&E stain; bar = 25 μm. B—Protozoal tachyzoites are seen individually (arrow) and in large groups (arrowheads) within cells. Immunohistochemistry staining for Neospora caninum antibody with rabbit anti-Neospora (University of Liverpool; dilution, 1:4,000); bar = 25 μm.

Citation: Journal of the American Veterinary Medical Association 260, 2; 10.2460/javma.21.01.0061

To confirm the identity of the protozoal organisms, immunohistochemistry (IHC) for Toxoplasma gondii and Neospora caninum was performed by use of rabbit anti-human T gondii (MyBioSource.com; dilution, 1:80) and rabbit anti-Neospora (University of Liverpool; dilution, 1:4000). The protozoal organisms were diffusely immunopositive for N caninum antigens and immunonegative for T gondii antigens (Figure 3).

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe pyogranulomatous dermatitis and panniculitis with erosion and ulceration and intralesional N caninum tachyzoites.

Case summary: cutaneous neosporosis in a dog.

Comments

Neospora caninum is a cyst-forming apicomplexan protozoal parasite of the Sarcocystidae family. The definitive hosts of N caninum are domestic dogs and various wild canid species1; intermediate hosts include cattle, dogs, sheep, deer, goats, and horses. Transmission occurs vertically and horizontally. Vertical transmission occurs via the transplacental, and possibly transmammary, routes in intermediate and definitive host species. Herbivorous intermediate hosts most commonly acquire horizontal infection via ingestion of oocysts from definitive (carnivore) host feces. After oocyst ingestion, sporozoites are released into the intestinal tract, penetrate intestinal epithelial cells, and then disseminate to a wide variety of tissues, where they eventually form bradyzoite cysts (asexual reproduction). These bradyzoite cysts can reactivate during pregnancy, leading to tachyzoite release and consequent abortion. Carnivorous hosts (eg, dogs) acquire horizontal infection via ingestion of infected intermediate host tissue, such as placenta, muscle, liver, brain, and heart, that contains bradyzoite cysts. Sexual reproduction only occurs in the definitive host, resulting in the release of unsporulated oocysts within feces. Oocysts sporulate over a 24- to 72-hour period before becoming infective to intermediate hosts.

Neospora caninum is best known as a cause of bovine abortion, as well as bovine myositis, in the intermediate host. In the definitive host, infections are primarily associated with neuromuscular pathologies in puppies, specifically ascending hind limb paralysis, polymyositis, and multifocal CNS disease, as well as myocarditis, pneumonia, and hepatitis. However, subclinical infections can occur and may progress to clinical disease later in life. Reactivation of dormant infections usually occurs in immunosuppressed animals, particularly those with iatrogenic immunosuppression.2 In adult patients with reactivated dormant infections, the most common features of disease are polymyositis, meningoencephalomyelitis, or both, with a variety of neurologic signs.3 Neospora caninum can infect any organ system and occasionally manifests as a localized cutaneous infection.2,4,5

Cutaneous infection with N caninum results in multifocal to generalized, ulcerative, papulonodular dermatitis. Cutaneous neosporosis in adult dogs is associated with therapeutic immunosuppression2,6 but has been reported in apparently immunocompetent patients.4 Extremely high experimental dosages of methylprednisolone (40 mg/kg, IM, q 7 d) have reactivated experimentally induced canine neosporosis.8 The dog of the present report was undergoing chronic anti-inflammatory treatment with prednisolone (0.4 to 0.6 mg/kg, PO, q 24 h), and it was likely that this immunomodulation contributed to reactivation of a latent N caninum infection.

Clinical diagnosis of N caninum can be challenging. Fecal flotation can demonstrate the presence of oocysts; however, clinically infected dogs typically do not shed oocysts. Serum biochemical analyses and hematologic abnormalities are usually determined by the organs affected, rather than specific to the causative agent. In cases with CNS involvement, CSF sampling can occasionally demonstrate tachyzoites; however, this is a rare finding, and the absence of tachyzoites does not eliminate N caninum infection as a differential diagnosis. In patients with cutaneous infection, tachyzoites are often plentiful in fine-needle aspirate samples of the lesions,7 allowing for rapid differentiation from other potential causes of nodular, ulcerative dermatitis, such as sterile nodular dermatitis, juvenile cellulitis, neoplasia (eg, mast cell tumors), dermatophytosis, leishmaniasis, and mycobacterial infection. In the present case, substantial numbers of identifiable tachyzoites were recovered on impression smear evaluation of the lesion. Cytologically and histologically, it is not possible to differentiate tachyzoites of N caninum from T gondii; therefore, IHC staining, PCR assay, or both are required for a definitive diagnosis. It is generally recommended to perform IHC staining for both pathogens in tandem, as cross-reactivity between these species can occur. Due to the relatively high prevalence of seropositivity against N caninum in apparently healthy dogs,9 PCR assay and serology results must be interpreted contextually, alongside clinical signs, to avoid false-positive diagnoses.

Treatment of N caninum infection usually involves a long course (> 4 weeks) of clindamycin with or without trimethoprim-sulfadiazine or pyrimethamine. Treatment should be continued for as long as clinical signs persist. Unfortunately, N caninum infections are often refractory to treatment, as bradyzoites in tissue cysts appear to be resistant to clindamycin treatment. Investigations into ponazuril as a potential therapeutic agent against N caninum show promise in mice10 and cows11 but require further study. Prognosis varies depending on the progression of disease at the time of intervention; advanced infections in puppies have an inherently poor prognosis. Treatment appears to be most effective in localized cutaneous infections. In our case, following cytologic confirmation of cutaneous protozoal parasitism, toxoplasmosis was considered the most likely differential diagnosis, and the dog was started on clindamycin (12.5 mg/kg, PO, q 12 h for 6 weeks). On receipt of the histology report, an adjustment in dosage and treatment regimen was considered to target N caninum; however, the dog was showing rapid improvement, and treatment with clindamycin was continued at the originally prescribed dosage. The owners reported the dog to be free of skin lesions 6 weeks after initiation of clindamycin treatment. However, 7 months after clinical resolution of skin lesions, the dog was presented with clinical signs consistent with neurologic disease; cerebral neosporosis was suspected. Financial constraints prohibited further investigation, and the dog was euthanized on welfare grounds; unfortunately, the owners declined a postmortem examination.

References

  • 1.

    Dubey JP, Jenkins MC, Rajendran C, et al. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet Parasitol. 2011;181(2-4):382387. doi:10.1016/j.vetpar.2011.05.018

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    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. 2002;38(5):415419. doi:10.5326/0380415

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Garosi L, Dawson A, Couturier J, et al. Necrotizing cerebellitis and cerebellar atrophy caused by Neospora caninum infection: magnetic resonance imaging and clinicopathologic findings in seven dogs. J Vet Intern Med. 2010;24(3):571578. doi:10.1111/j.1939-1676.2010.0485.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Poli A, Mancianti F, Carli MA, Stroscio MC, Kramer L. Neospora caninum infection in a Bernese Cattle Dog from Italy. Vet Parasitol. 1998;78(2):7985. doi:10.1016/S0304-4017(98)00135-6

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    McInnes LM, Irwin P, Palmer DG, Ryan UM. In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia. Vet Parasitol. 2006;137(3):355363. doi:10.1016/j.vetpar.2006.01.018

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Legnani S, Pantchev N, Forlani A, et al. Emergence of cutaneous neosporosis in a dog receiving immunosuppressive therapy: molecular identification and management. Vet Dermatol. 2016;27(1):49e14. doi:10.1111/vde.12273

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Mann TR, Cadore GC, Camillo G, Vogel FS, Schmidt C, Andrade CM. Canine cutaneous neosporosis in Brazil. Vet Dermatol. 2016;27(3):195197. doi:10.1111/vde.12294

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Dubey JP, Lindsay DS. Neosporosis in dogs. Vet Parasitol. 1990;36(1-2):147151. doi:10.1016/0304–4017(90)90103-I

  • 9.

    Reichel MP, Ellis JT, Dubey JP. Neosporosis and hammondiosis in dogs. J Small Anim Pract. 2007;48(6):308312. doi:10.1111/j.1748-5827.2006.00236.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Gottstein B, Eperon S, Dai WJ, Cannas A, Hemphill A, Greif G. Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice. Parasitol Res. 2001;87(1):4348. doi:10.1007/s004360000306

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Kritzner S, Sager H, Blum J, Krebber R, Greif G, Gottstein B. An explorative study to assess the efficacy of toltrazuril-sulfone (ponazuril) in calves experimentally infected with Neospora caninum. Ann Clin Microbiol Antimicrob. 2002;1:4 doi:10.1186/1476-0711-1-4

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Figure 1

    Photograph of multiple raised, ulcerated nodular lesions on the inner aspect of the ear pinna of a 5.5-year-old 24.4-kg spayed female Boxer with a 4-year history of ongoing allergic dermatitis that had been managed with either oclacitinib or, more recently, prednisolone.

  • Figure 2

    Photomicrograph of a direct smear of a fine-needle aspirate collected from one of the pinnal dermal nodules of the dog described in Figure 1. The smear contains numerous 2- to 3-μm-wide protozoal tachyzoites (arrowheads) mixed with neutrophils (arrow). Wright-Giemsa stain; bar = 25 μm.

  • Figure 3

    Photomicrograph of sections of a nodular pinnal lesion of the dog described in Figure 1. A—The dermis is infiltrated by numerous neutrophils mixed with macrophages, many of which contain abundant intracytoplasmic, 2 × 3-μm, oval to crescentic protozoal tachyzoites (arrows) that cause margination of nuclei (arrowhead). H&E stain; bar = 25 μm. B—Protozoal tachyzoites are seen individually (arrow) and in large groups (arrowheads) within cells. Immunohistochemistry staining for Neospora caninum antibody with rabbit anti-Neospora (University of Liverpool; dilution, 1:4,000); bar = 25 μm.

  • 1.

    Dubey JP, Jenkins MC, Rajendran C, et al. Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum. Vet Parasitol. 2011;181(2-4):382387. doi:10.1016/j.vetpar.2011.05.018

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    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. 2002;38(5):415419. doi:10.5326/0380415

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Garosi L, Dawson A, Couturier J, et al. Necrotizing cerebellitis and cerebellar atrophy caused by Neospora caninum infection: magnetic resonance imaging and clinicopathologic findings in seven dogs. J Vet Intern Med. 2010;24(3):571578. doi:10.1111/j.1939-1676.2010.0485.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Poli A, Mancianti F, Carli MA, Stroscio MC, Kramer L. Neospora caninum infection in a Bernese Cattle Dog from Italy. Vet Parasitol. 1998;78(2):7985. doi:10.1016/S0304-4017(98)00135-6

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    McInnes LM, Irwin P, Palmer DG, Ryan UM. In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia. Vet Parasitol. 2006;137(3):355363. doi:10.1016/j.vetpar.2006.01.018

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6.

    Legnani S, Pantchev N, Forlani A, et al. Emergence of cutaneous neosporosis in a dog receiving immunosuppressive therapy: molecular identification and management. Vet Dermatol. 2016;27(1):49e14. doi:10.1111/vde.12273

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Mann TR, Cadore GC, Camillo G, Vogel FS, Schmidt C, Andrade CM. Canine cutaneous neosporosis in Brazil. Vet Dermatol. 2016;27(3):195197. doi:10.1111/vde.12294

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Dubey JP, Lindsay DS. Neosporosis in dogs. Vet Parasitol. 1990;36(1-2):147151. doi:10.1016/0304–4017(90)90103-I

  • 9.

    Reichel MP, Ellis JT, Dubey JP. Neosporosis and hammondiosis in dogs. J Small Anim Pract. 2007;48(6):308312. doi:10.1111/j.1748-5827.2006.00236.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Gottstein B, Eperon S, Dai WJ, Cannas A, Hemphill A, Greif G. Efficacy of toltrazuril and ponazuril against experimental Neospora caninum infection in mice. Parasitol Res. 2001;87(1):4348. doi:10.1007/s004360000306

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Kritzner S, Sager H, Blum J, Krebber R, Greif G, Gottstein B. An explorative study to assess the efficacy of toltrazuril-sulfone (ponazuril) in calves experimentally infected with Neospora caninum. Ann Clin Microbiol Antimicrob. 2002;1:4 doi:10.1186/1476-0711-1-4

    • Crossref
    • Search Google Scholar
    • Export Citation

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