Feline subcutaneous pythiosis/Pitiose subcutanea em um felino.
The objective of this report was to describe the macroscopic, histopathological, and molecular characteristics of a localized invasive subcutaneous P. insidiosum infection in a 2-year-old female cat, undefined breed, which was taken to the Veterinary Hospital of the Federal University of Mato Grosso, Campus Cuiaba, with a history of volume increase near the anal region since it was younger. Ten months before the cat had clinical signs of dystocia and consequently cesarean section was performed. A few months later, the mass evolved reaching approximately 10cm in diameter. Clinical examination showed that the mass involved the lateral and dorsal portions of the anus and that its consistency was soft in the peripheral region and firm in the central region. Sonographic examination of the perianal region showed that the mass was heterogeneous, with anechoic content and a cavitary surface, positive with color Doppler, with indefinite contour and shape, suggestive of neoplasia. During the surgical mass excision, the tumor was found to involve the rectum bilaterally and externally and was adhered to adjacent structures, which hindered its removal with surgical safety margins. Mass fragments were sent for histopathological analysis in 10% formalin solution.
The animal died in the postoperative period and was subsequently necropsied. Macroscopic examination showed fragments of brownish, friable, and lobulated brown masses on the inner surface of the pelvic region, as well as hematomas in the subcutaneous tissue surrounding the surgical incision. Microscopic examination showed that the fragments removed during the surgery revealed multifocal areas of necrosis and eosinophilic material (similarly to the Splendore-Hoeppli reaction) with negative images, morphologically suggestive of hyphae (Figure 1A) in the dermis, panicle, and adjacent musculature. These structures were surrounded by a severe inflammatory infiltrate composed of macrophages with large eosinophilic and foamy cytoplasm (Figure 1B). There were also multinucleated giant cells, neutrophils, and eosinophils, as well as proliferation of connective tissue with marked collagen deposition, characterizing the lesion as dermatitis, panniculitis, and granulomatous myositis. The Grocott's methenamine silver (GMS) staining (Figure 1C), showed hyphae with irregular ramifications and rare septations.
For differential diagnosis of pythiosis, lagenidiosis, and zygomycosis, two paraffin-embedded sections measuring 10pm of each subcutaneous lesion were sent for molecular characterization. The DNA was extracted from the paraffin sample according to SHI et al. (2004), and PCR was performed according to GROOTERS & GEE (2002), using the primers PI-1: 5-TTCGTCGAAGCGGACTGCT-3'; and PI21 5>GCCGTACAACCCGAGAGTCATA-3' to amplify a 105-bp section that encoded a sequence of the P. insidiosum rDNA ITS1 gene (Figure 1E). The amplified product was analyzed through 2% agarose gel electrophoresis, and observed on the ChemiDoc[TM] XRS documentation system using ImageLab[TM] software, with a molecular weight marker of 100 bp. The obtained amplicon was purified using a GFX PCR DNA GelBand purification kit and sequenced in the ABI 3500 Genetic Analyzer (Applied Biosystems[R]), using specific oligonucleotides. The sequence was analyzed using the BLAST (NCBI) software, obtaining 97% identity with the P. insidiosum sequence (GenBank KP842848).
Immunohistochemistry technique was used in subcutaneous tissue sections with minor modifications, selected according to UBIALI et al. (2013), with the primary anti-P insidiosum antibody (1:500) and incubated at 37[degrees]C for 2 hours. The secondary antibody used was the alkaline phosphatase conjugate system (MACH 4 Universal AP Polymer Kit - Biocare Medical) and the samples were incubated for 30min at room temperature. The chromogen used was Warp Red (Biocare Medical). Sections were counterstained with hematoxylin. The hyphae were marked positive for P insidiosum (Figure 1D).
The diagnosis of P. insidiosum was based on clinical, histological, and molecular aspects. Pythiosis, lagenidiosis, and zygomycosis are caused by distinct etiological agents, but with similar clinical and histopathological characteristics (GROOTERS, 2003). Cutaneous pythiosis causes panniculitis, and nodular and ulcerative dermatitis, thus it is recommend the use of deep biopsies rather than superficial biopsies or punches (GROOTERS, 2003). In addition, a diagnosis based on oomycosis and zygomycosis has been historically problematic because the macroscopic lesions associated with pythiosis are easily confused with those caused by neoplasms or bacterial infection, so the tissue is completely fixed and specimens are not collected for culture (GROOTERS, 2003; RAKICH et al., 2005). In the present case, the initial clinical suspicion was a neoplastic process, so the entire sample was fixed in 10% formalin, making the microbiological examination impossible. With respect to microscopic features, RAKICH et al. (2005) described two cases of feline pythiosis with lesions in small intestine and mesentery. The lesions were composed of dense fibrous connective tissue stroma containing eosinophils, macrophages, and fewer lymphoid cells with foci of necrosis composed of bright eosinophilic material and nuclear debris. The GMS-stained sections confirmed the presence of few to moderate numbers of hyphae within areas of necrosis. In both cats, there was no formation of distinct granulomas. The morphological lesion observed in the present study was similar to that reported by RAKICH et al. (2005). However, microscopies finds also revealed the presence of granulomatous formations with macrophages with large eosinophilic and foamy cytoplasm and multinucleated giant cells.
The diagnosis in the present study was confirmed by molecular tools and immunohistochemistry procedure. Molecular techniques for diagnosis of infectious diseases in humans and animals have become increasingly important, since they offer greater specificity than the morphological characterization of culture isolates or immunohistochemical labeling of infected tissues (GROOTERS & GEE, 2002). The literature showed few reported cases of pythiosis in cats (GROOTERS, 2003; RAKICH et al., 2005; FORTIN et al., 2017). This case described the manifestation of pythiosis in subcutaneous tissues. Epidemiological data on this case of feline pythiosis were limited and it was uncertain whether the cat had a direct contact with pond water or not. The present cat was an indoor/ outdoor cat which is common in urban Cuiaba municipality environment. Pythiosis in cats is rare and when is diagnosed late tends to evolve in the animal's death. This case suggests that this disease should be included in the differential diagnosis of chronic infections and neoplasias associated with the presence of masses in feline subcutaneous tissues. It also highlighted the importance of tissue biopsy with appropriate diagnostic tools, such as histology, immunohistochemistry and molecular techniques for increasing frequency of diagnosis confirmation of pythiosis.
Returned by the author 02.14.19
The authors are grateful to Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) for financial support.
BIOETHICS AND BIOSSECURITY COMMITTEE APPROVAL
We authors of the article entitled "Feline subcutaneous pythiosis: A case report" declared, for all due purposes, the project that gave rise to the present data of the same has not been submitted for evaluation to the Ethics Committee of the Universidade Federal do Mato Grosso, but we are aware of the content of the Brazilian resolutions of the National Council for Control of Animal Experimentation--CONCEA "http://www.mct.gov.br/index.php/ content/view/310553.html" if it involves animals.
Thus, the authors assume full responsibility for the presented data and are available for possible questions, should they be required by the competent authorities.
DECLARATION OF CONFLICTING OF INTERESTS
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
The authors contributed equally to the manuscript.
BISSONNETTE, K.W. et al. Nasal and retrobulbar mass in a cat caused by Pythium insidiosum. Journal of Medical and Veterinary Mycology, v.29, n.1, p.39-44, 1991. Available from: <https://www.ncbi.nlm.nih.gov/pubmed/1648127>. Accessed: May, 14, 2018.
FORTIN, J.S. et al. Sublingual pythiosis in a cat. Acta Veterinaria Scandinavica, v.59, n.1, p.63, 2017. Available from: <https:// www.ncbi.nlm.nih.gov/pmc/articles/PMC5615467/>. Accessed: May, 14, 2018. doi: 10.1186/s13028-017-0330-z.
GAASTRA, W. et al. Pythium insidiosum: An overview. Veterinary Microbiology, v.146, n.1-2, p.1-16, 2010. Available from: <https:// www.ncbi.nlm.nih.gov/pubmed/20800978>. Accessed: May, 14, 2018. doi: 10.1016/j.vetmic.2010.07.01.
GALIZA, G.J.N. et al. Occurrence of mycoses and pythiosis in domestic animals: 230 cases. Pesquisa Veterinaria Brasileira, v.34, n.3, p.224-232, 2014. Available from: <https://dx.doi. org/10.1590/S0100-736X2014000300005>. Accessed: May, 14, 2018. doi: 10.1590/S0100-736X2014000300005.
GROOTERS, A.M. Pythiosis, lagenidiosis, and zygomycosis in small animals. The Veterinary Clinics Small Animal Pratice, v.33, n.4, p.695-720, 2003. Available from: <https://www.ncbi. nlm.nih.gov/pubmed/12910739>. Accessed: May, 14, 2018. doi: 10.1016/S0195-5616(03)00034-2.
GROOTERS, AM.; GEE, M.K. Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum. Journal of Veterinary Internal Medicine, v.16, n.2, p. 147-152, 2002. Available from: <https://www.ncbi. nlm.nih.gov/pubmed/11899029>. Accessed: May, 14, 2018.
MENDONZA, L.; NEWTON, J.C. Immunology and immunotherapy of the infections caused by Pythium insidiosum. Medical Mycology, v.43, n.6, p.477-486, 2005. Available from: <https://www.ncbi.nlm.nih.gov/pubmed/16320491>. Accessed: May, 14, 2018. doi: 10.1080/13693780500279882.
RAKICH, P.M. et al. Gastrointestinal pythiosis in two cats. Journal of Veterinary Diagnostic Investigation, v.17, n.3, p.262-269, 2005. Available from: <https://www.ncbi.nlm.nih.gov/ pubmed/15945385>. Accessed: May, 14, 2018.
REIS JR., J.L. et al. Disseminated pythiosis in three horses. Veterinary Microbiology, v.96, n.3, p.289-295, 2003. Available from: <https:// www.ncbi.nlm.nih.gov/pubmed/14559176>. Accessed: May, 14, 2018. doi: 10.1016/j.vetmic.2003.07.005.
SANTURIO, J.M. et al. Granulomatous rhinitis associated with Pythium insidiosum infection in sheep. Veterinary Record, v.163, n.9, p.276-277, 2008. Available from: <https://www.ncbi.nlm.nih. gov/pubmed/18757907>. Accessed: May, 14, 2018. doi: 10.1136/ vr.163.9.276.
SHI, S.R. et al. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution. Histochemistry and Cell Biology, v.122, n.3, p.211-218, 2004. Available from: <https://www.ncbi.nlm.nih.gov/pubmed/15322858>. Accessed: May, 14, 2018. doi: 10.1007/s00418-004-0693-x.
UBIALI, D.G. et al. Pathology of nasal infection caused by Conidiobolus lamprauges and Pythium insidiosum in sheep. Journal of Comparative Pathology, v.149, n.2-3, p.137-145, 2013. Available from: <http://dx.doi.org/10.1016/jjcpa.2012.12.002>. Accessed: May, 14, 2018. doi: 10.1016/jjcpa.2012.12.002.
Luciana Maria Curtio Soares (1) (iD) Diego Montagner Schenkel (1) Janaina Marcela Assuncao Rosa (2) Ludmila Silva Azevedo (3) Tainara Renata Tineli (4) Valeria Dutra (2) (iD) Edson Moleta Colodel (1) Caroline Argenta Pescador (1) * (iD)
(1) Laboratorio de Patologia Veterinaria, Faculdade de Medicina Veterinaria (FAVET), Universidade Federal de Mato Grosso (UFMT), 78060-900, Cuiaba, MT, Brasil. E-mail: firstname.lastname@example.org. * Corresponding author.
(2) Laboratorio de Microbiologia, Faculdade de Medicina Veterinaria (FAVET), Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brasil.
(3) Setor de Diagnostico por Imagem, Faculdade de Medicina Veterinaria (FAVET), Universidade Federal de Mato Grosso (UFMT), Cuiaba, MT, Brasil.
(4) Universidade Federal de Santa Catarina (UFSC), Campus de Curitibanos, SC, Brasil.
Caption: Figure 1--Female cat, undefined breed, 2 years old. A. Area of necrosis showing negative images, morphologically suggestive of hyphae (arrow), surrounded by eosinophilic material (similar to the Splendore-Hoeppli reaction). HE staining (40x magnification). B. A severe inflammatory infiltrate composed of multinucleated giant cells and macrophages with large eosinophilic and foamy cytoplasm with connective tissue proliferation. Hematoxylin and eosin (HE) staining (40x magnification). C. Evidence of hyphae with irregular ramifications and rare septations. Grocott's methenamine silver (GMS) staining (40x magnification). D. Immunostaining of P insidiosum using the primary anti-P insidiosum antibody (1:500). IHC, Warp Red (40x magnification). E. Pythium insidiosum expression in a cat by Nested-PCR showing amplification of 105bp in lane 1. M: molecular weight marker; CP: Positive control; CN: Negative Control.
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|Author:||Soares, Luciana Maria Curtio; Schenkel, Diego Montagner; Rosa, Janaina Marcela Assuncao; Azevedo, Lu|
|Date:||Mar 1, 2019|
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