Pathology in Practice

Janice Y. Park 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Paula R. Giaretta 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Glaucia D. Kommers 3Departamen to de Patologia, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil.

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Roy Pool 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Sara Lawhon 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Caitlin E. Older 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Philippa Gibbons 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Rodolfo G. Madrigal 2Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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Raquel R. Rech 1Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843.

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History

An 18-month-old 55.5-kg (122.0-lb) Dorper ewe with a 2-week history of dyspnea that progressed to open-mouthed breathing was presented to the Texas A&M University Veterinary Medical Teaching Hospital. The referring veterinarian had previously treated the ewe with ceftiofur sodium and flunixin meglumine without improvement in its condition.

Clinical and Gross Findings

At the referral evaluation, the ewe had a high respiratory rate of 60 breaths/min and lung sounds were increased bilaterally, more severely on the right side. Physical examination revealed cervical lymphadenopathy and foamy to mucopurulent discharge from the left nostril that later progressed to bilateral discharge. The face was bilaterally swollen, and there was a 4 × 2-cm ulcerated and necrotic area on the dental pad (Figure 1). Computed tomography of the head revealed an expansile, heterogeneous, bilateral soft tissue mass that was slightly centered to the right of midline within the rostral aspect of the nasal cavity. The mass caused leftward deviation of the nasal septum, and there were areas of osteolysis in the rostral portions of the vomer and incisive bones. When drained, the mass contained a brown exudate admixed with necrotic tissue. Owing to the ewe's poor prognosis, the owner elected euthanasia (by IV injection of phenobarbital sodium and phenytoin solution) without necropsy.

Figure 1—
Figure 1—

Photographs of the right nostril (A) and dental pad (B) of an 18-month-old Dorper ewe that had been dyspneic for 2 weeks. Notice the rhinofacial swelling, the mucopurulent discharge from the nasal cavity, and a necrotic and ulcerated lesion on the dental pad.

Citation: Journal of the American Veterinary Medical Association 257, 11; 10.2460/javma.2020.257.11.1133

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

Histopathologic Findings

During clinical examination, a biopsy specimen of the nasal mucocutaneous junction was collected, microscopic examination of which revealed multifocal to coalescing eosinophilic granulomas that had expanded the dermis and deep submucosa. The inflammatory infiltrate was primarily composed of epithelioid macrophages, eosinophils, neutrophils, and multinucleated giant cells (Figure 2). The granulomatous inflammation was centered on areas of necrosis; the core of the granulomas contained 3- to 5-μm-wide, irregularly branching hyphae surrounded by degranulated eosinophils. Multifocally, the hyphae had invaded the wall of arteries and resulted in inflammation, necrosis, and thrombosis. The hyphae did not react with H&E or periodic acid–Schiff stain but did react with Gomori methenamine silver stain (Figure 3).

Figure 2—
Figure 2—

Photomicrograph of a section of a tissue sample from the nasal mucocutaneous junction of the sheep in Figure 1. Notice the central area of eosinophilic debris and eosinophils, which is surrounded by multinucleated giant cells (arrows), consistent with an eosinophilic granuloma. H&E stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 257, 11; 10.2460/javma.2020.257.11.1133

Figure 3—
Figure 3—

Photomicrographs of other sections of a tissue sample from the nasal mucocutaneous junction. The core of the granulomas contains nonstaining hyphae (arrows) surrounded by degranulated eosinophils and karyorrhectic debris. H&E stain; bar = 50 μm. Inset—Gomori methenamine silver stain highlights irregularly branching, nonparallel-walled hyphae. Gomori methenamine silver stain; bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 257, 11; 10.2460/javma.2020.257.11.1133

Additional Laboratory Findings

For identification of the hyphae, genomic DNA was extracted from frozen and formalin-fixed paraffin-embedded tissue samples of the nasal mucosa for a panfungal PCR assay. The panfungal PCR assay used internal transcribed spacer (ITS) primers (ITS3 and ITS4) that targeted the ITS2 region and yielded a single positive band.1 The obtained PCR assay product was sequenced in both directions, and 301 base pairs were aligned with every fungal species sequence in an online database.a The search resulted in a match to Pythium insidiosum with 100% identity. Tissue sections of the nasal mucosa underwent immunohistochemical analyses, performed as previously described,2 and the hyphae reacted positively with a polyclonal anti–P insidiosum antibody and negatively with a monoclonal anti-Rhizopus antibody. Rhizopus sp was cultured from nasal mucosa samples collected 12 hours after euthanasia of the ewe but was considered a contaminant.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe, chronic, multifocal to coalescing, granulomatous, and eosinophilic rhinitis and gingivitis.

Case summary: rhinofacial pythiosis in a sheep.

Comments

Pythium insidiosum is an oomycete classified in the kingdom Stramenopila, phylum Oomycota, and order Pythiales (family Pythiaceae).3 This organism is often found in tropical and subtropical regions, with reports of pythiosis especially in South and Central America, the Gulf Coast region of North America, Southeast Asia, New Zealand, and eastern coastal Australia.4 The life cycle of P insidiosum begins when its hyphal form colonizes plant tissues and differentiates into sporangia.5 As they mature, sporangia create vesicles in which zoospores form.5 Zoospores break out of the vesicle walls into water and begin another cycle on a different plant. If an animal is present, motile zoospores come into contact with small cutaneous wounds or are ingested, and then they encyst, germinate, and further invade an animal or human host.5 Clinical signs of pythiosis depend on the site of entry.5 The disease is not transmitted among animals or between animals and humans.

In animals, P insidiosum infection is associated with a wide range of clinical signs and may result in cutaneous, nasal, gastrointestinal, or, rarely, systemic disease.4 Pythiosis is most common in horses, which more often develop the cutaneous form, followed by dogs, which more commonly develop gastrointestinal lesions.4 Pythiosis can also affect humans, cattle, sheep, goats, cats,4 and some exotic animal species such as dromedary camels,6 Bengal tigers,7 and white-faced ibis.8

As observed in the case described in the present report, a rhinofacial distribution of pythiosis is common in sheep.9 However, pythiosis in sheep can develop as cutaneous or gastrointestinal lesions.10,11 Dissemination to the lungs has been documented for sheep in northeastern Brazil.11 Vasocentric activity, commonly associated with infections by zygomycetes, is occasionally observed in cases of pythiosis.2,9,10 The main differential diagnoses for the gross lesions include neoplasia (eg, squamous cell carcinoma) and fungal infection (eg, conidiobolomycosis).9,12

In contrast to pythiosis, zygomycosis caused by Conidiobolus lamprauges most commonly affects the rhinopharyngeal region in sheep.9 However, differentiation between pythiosis and conidiobolomycosis requires additional testing, such as culture, PCR assay, or immunohistochemical analysis, of appropriate specimens from affected animals.9

For the sheep of the present report, pythiosis was diagnosed on the basis of hyphal morphology and results of panfungal PCR assay and immunohistochemical analysis of tissue samples of the nasal mucosa. The staining pattern of the hyphae (ie, positive Gomori methenamine silver staining and no periodic acid–Schiff staining [the latter owing to a lack of chitin]) supported the diagnosis of pythiosis.13 Although P insidiosum is not considered a fungus, the oomycete contains an ITS region in its genome that can be detected with a panfungal PCR assay.1 It must be noted that the sheep of the present report did not undergo necropsy; thus, the extension of lesions to other tissues was unknown. To the authors’ knowledge, the case described in the present report is the first case of rhinofacial pythiosis in a sheep in the United States. This case highlighted the importance of considering pythiosis as a differential diagnosis in sheep with chronic respiratory signs in conjunction with nasal masses, particularly if those sheep are located in the Gulf Coast region of the United States.

Footnotes

a.

Nucleotide BLAST, National Center for Biotechnology Information, Bethesda, Md. Available at: www.ncbi.nlm.nih.gov/nuccore/MT710696. Accessed Mar 15, 2016.

References

  • 1. Meason-Smith C, Edwards EE, Older CE, et al. Panfungal polymerase chain reaction for identification of fungal pathogens in formalin-fixed animal tissues. Vet Pathol 2017;54:640648.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Martins TB, Kommers GD, Trost ME, et al. A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle. J Comp Pathol 2012;146:122131.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Santurio JM, Argenta JS, Schwendler SE, et al. Granulomatous rhinitis associated with Pythium insidiosum infection in sheep. Vet Rec 2008;163:276277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Gaastra W, Lipman LJ, De Cock AW, et al. Pythium insidiosum: an overview. Vet Microbiol 2010;146:116.

  • 5. Mendoza L, Hernandez F, Ajello L. Life cycle of the human and animal oomycete pathogen Pythium insidiosum. J Clin Microbiol 1993;31:29672973.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Wellehan JF, Farina LL, Keoughan CG, et al. Pythiosis in a dromedary camel (Camelus dromedarius). J Zoo Wildl Med 2004;35:564568.

  • 7. Buergelt C, Powe J, White T. Abdominal pythiosis in a Bengal tiger (Panthera tigris tigris). J Zoo Wildl Med 2006;37:186189.

  • 8. Pesavento PA, Barr B, Riggs SM, et al. Cutaneous pythiosis in a nestling white-faced ibis. Vet Pathol 2008;45:538541.

  • 9. Ubiali DG, Cruz RA, De Paula DA, et al. Pathology of nasal infection caused by Conidiobolus lamprauges and Pythium insidiosum in sheep. J Comp Pathol 2013;149:137145.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Pessoa CR, Riet-Correa F, Pimentel LA, et al. Pythiosis of the digestive tract in sheep. J Vet Diagn Invest 2012;24:11331136.

  • 11. Tabosa IM, Riet-Correa F, Nobre VM, et al. Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil. Vet Pathol 2004;41:412415.

  • 12. Riet-Correa F, Dantas AFM, Azevedo EO, et al. Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil. Pesq Vet Bras 2008;28:2935.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Mauldin EA, Peter-Kennedy J. Integumentary system. In: Maxie MG, ed. Pathology of domestic animals. 6th ed. St Louis: Elsevier, 2016;658659.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photographs of the right nostril (A) and dental pad (B) of an 18-month-old Dorper ewe that had been dyspneic for 2 weeks. Notice the rhinofacial swelling, the mucopurulent discharge from the nasal cavity, and a necrotic and ulcerated lesion on the dental pad.

  • Figure 2—

    Photomicrograph of a section of a tissue sample from the nasal mucocutaneous junction of the sheep in Figure 1. Notice the central area of eosinophilic debris and eosinophils, which is surrounded by multinucleated giant cells (arrows), consistent with an eosinophilic granuloma. H&E stain; bar = 20 μm.

  • Figure 3—

    Photomicrographs of other sections of a tissue sample from the nasal mucocutaneous junction. The core of the granulomas contains nonstaining hyphae (arrows) surrounded by degranulated eosinophils and karyorrhectic debris. H&E stain; bar = 50 μm. Inset—Gomori methenamine silver stain highlights irregularly branching, nonparallel-walled hyphae. Gomori methenamine silver stain; bar = 20 μm.

  • 1. Meason-Smith C, Edwards EE, Older CE, et al. Panfungal polymerase chain reaction for identification of fungal pathogens in formalin-fixed animal tissues. Vet Pathol 2017;54:640648.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Martins TB, Kommers GD, Trost ME, et al. A comparative study of the histopathology and immunohistochemistry of pythiosis in horses, dogs and cattle. J Comp Pathol 2012;146:122131.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Santurio JM, Argenta JS, Schwendler SE, et al. Granulomatous rhinitis associated with Pythium insidiosum infection in sheep. Vet Rec 2008;163:276277.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4. Gaastra W, Lipman LJ, De Cock AW, et al. Pythium insidiosum: an overview. Vet Microbiol 2010;146:116.

  • 5. Mendoza L, Hernandez F, Ajello L. Life cycle of the human and animal oomycete pathogen Pythium insidiosum. J Clin Microbiol 1993;31:29672973.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Wellehan JF, Farina LL, Keoughan CG, et al. Pythiosis in a dromedary camel (Camelus dromedarius). J Zoo Wildl Med 2004;35:564568.

  • 7. Buergelt C, Powe J, White T. Abdominal pythiosis in a Bengal tiger (Panthera tigris tigris). J Zoo Wildl Med 2006;37:186189.

  • 8. Pesavento PA, Barr B, Riggs SM, et al. Cutaneous pythiosis in a nestling white-faced ibis. Vet Pathol 2008;45:538541.

  • 9. Ubiali DG, Cruz RA, De Paula DA, et al. Pathology of nasal infection caused by Conidiobolus lamprauges and Pythium insidiosum in sheep. J Comp Pathol 2013;149:137145.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Pessoa CR, Riet-Correa F, Pimentel LA, et al. Pythiosis of the digestive tract in sheep. J Vet Diagn Invest 2012;24:11331136.

  • 11. Tabosa IM, Riet-Correa F, Nobre VM, et al. Outbreaks of pythiosis in two flocks of sheep in northeastern Brazil. Vet Pathol 2004;41:412415.

  • 12. Riet-Correa F, Dantas AFM, Azevedo EO, et al. Outbreaks of rhinofacial and rhinopharyngeal zygomycosis in sheep in Paraíba, northeastern Brazil. Pesq Vet Bras 2008;28:2935.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Mauldin EA, Peter-Kennedy J. Integumentary system. In: Maxie MG, ed. Pathology of domestic animals. 6th ed. St Louis: Elsevier, 2016;658659.

    • Search Google Scholar
    • Export Citation

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