Epidemiological, clinical and anatomopathological characterization of nasal rhinosporidiosis in a horse

The objective of this study is to describe the clinical, epidemiological and anatomopathological aspects of a case of nasal rhinosporidiosis in a horse in the Northeast of Brazil. A 15-year-old female horse of mixed breed presented a nodule in the mucosa of the right nasal septum. Surgical excision was performed and sent for histopathological examination. Macroscopically, the nodule had an irregular rhinosporidiosis was made based on macroscopic aspects and confirmed through histopathological examination. The presence of myriads of sporangia measuring 115 to 220 µm, containing endospores in its interior allowed the diagnosis. Although rhinosporidiosis is considered rare in Brazil, in the Northeast region there are descriptions of the disease in humans, suggesting that the disease in animals occurs and has possibly not been diagnosed. Therefore, it is important to include it as a differential diagnosis of diseases that affect the nasal cavity of horses in the region and that present similar lesions.

Rhinosporidiosis is endemic in India, Sri Lanka and Argentina, and isolated cases have been described in Australia, South Africa, United States and Brazil (Pereira & Meireles, 2007). In Brazil, although the disease occurs sporadically, there are reports of the disease in the southern region in cattle (Riet-Correa et al., 1983) and horses (Argenta et al., 2018;Bernardo et al., 2016;Sampaio et al., 2018;Santos et al., 2014), and in the southeast region in dogs (Neves et al., 2014) and horses (Zibordi et al., 2020). In the Northeast, the disease is frequent in humans (Abud & Pereira, 2007;Silva et al., 1975), but there are no descriptions in animals. Therefore, the objective is to describe the clinical, epidemiological and anatomopathological aspects of a case of nasal rhinosporidiosis in a horse in the Northeast region of Brazil.

Methodology
A 15-year-old female horse of mixed-breed, during a routine clinical evaluation, was observed to have a nodule inside the nostril. It was raised together with another horse on a farm in the semiarid region of Paraíba. It fed on native pasture and drank water from a dam. On clinical examination, the animal was active, with a present appetite, heart rate of 32 bpm, and respiratory rate of 16 bpm. The nodule was adhered to the mucosa of the right nasal septum, and was pink in color, with a verrucous appearance, irregular surface and soft consistency (Figure 1. A). A cytological examination of the nodule was performed using the fine needle aspiration technique (FNA), with a diagnosis suggestive of cryptococcosis. Nodulectomy with surgical margins measuring one centimeter in diameter was chosen, using local anesthetic block in the right infraorbital foramen, with 2% lidocaine.
The collected material was fixed in 10% buffered formalin and sent for histopathological processing, according to the protocol described by Michalany (1998). The slides were stained with hematoxylin and eosin (HE), in addition to special histochemical stains such as periodic acid Schiff (PAS), Grocott methenamine silver (GMS) and toluidine blue.

Results
Macroscopically, the nodule measured approximately 0.5x0.5x0.2 cm in size, with an irregular and reddish surface ( Figure 1. A, detail). When cut, it was soft, whitish and polypoid in shape. Microscopically, a discrete inflammatory infiltrate consisting of plasmocytes, lymphocytes, intact and degenerated neutrophils, and occasional macrophages associated with myriads of sporangia with endospores in their interior with morphology similar to Rhinosporidium seeberi in various stages of maturation was observed in lamina propria (Figure 1. B).
Young sporangia, measuring 7 to 65 µm, had a basophilic unilaminar wall, sometimes with a central nucleus surrounded by eosinophilic granular material (Figure 1. B). Mature sporangia were in greater number and measured from 115 to 220 µm, characterized by being rounded with a basophilic bilaminar wall, with immature endospores in the periphery and rounded mature endospores measuring up to 5 µm in the center. There were also sporangia with an intact cell wall and no internal structures, and others with a broken wall and free endospores. It was also observed proliferation of epithelial cells into the lamina propria, forming digitiform projections, in addition to acanthosis. The walls of the mature and immature sporangia, in addition to the wall and the internal granular bodies of the endospores, were strongly stained in pink (magenta) in the PAS (Figure 1. E). The GMS staining showed black impregnation of the walls of mature and ruptured sporangia and brown impregnation of endospores and granular bodies contained within ( Figure   1. D). Young sporangia were not marked. The toluidine blue staining showed blue impregnation of the walls of mature, ruptured and young sporangia, and purple impregnation of the endospores (Figure 1. F). After surgical removal, the animal received monthly follow-up for two months and no recurrence was reported.

Discussion
The diagnosis of rhinosporidiosis was made based on clinical and macroscopic aspects, and confirmed through histopathological examination. The presence of myriads of sporangia measuring 115 to 220 µm containing endospores in their interior, and the positive PAS staining for capsule and internal granules of the endospores allowed the diagnosis.
The epidemiology, infection mechanisms and pathogenesis of rhinosporidiosis are not fully elucidated. It is believed that the transmission of R. seeberi is related to sources of stagnant water and soil contaminated by spores (Rath et al., 2015), and the nasal mucosa is the main site of infection through inhalation of spores or direct contact with water, allowing for agent inoculation (Neves et al., 2014).
There are no reports of animal-to-animal transmission (Ferreira & Ferreira, 1990). In this case, the animal had access to a dam, which was probably the source of infection. The importance of diagnosing this case is highlighted, as it awakens to the existence of sources of infection, as well as cultural and environmental characteristics of the Northeast region, which include the accumulation of water in dams, high temperatures and humidity, which possibly favor the perpetuation and development of this etiological agent, increasing the possibility of occurrence of the disease. It is noteworthy that in the Northeast region there is a description of the disease in humans, mainly in the states of Maranhão, Piauí and Bahia (Abud & Pereira, 2007;Silva et al., 1975;Vallarelli et al., 2011), suggesting that the disease also occurs in animals, but possibly has not been diagnosed.
After inoculation, it's believed that the microorganism invades the subepithelial stroma of the nasal mucosa or skin and induces chronic inflammation, which consequently culminates in the formation of solitary or multiple polyps, papillomas or warts (Jones et al., 2000). Depending on the dimensions of the lesions, there may be occlusion of the nasal passages and associated respiratory symptoms (Abud & Pereira, 2007;Jones et al., 2000). In this case, the verrucous lesion was only a finding during the clinical evaluation and respiratory signs were not observed. Possibly the characteristics of solitary lesions and slow growth, associated with the possibility of this disease being self-limiting (Neves et al., 2014) have contributed to the low occurrence of rhinosporidiosis diagnoses and, consequently, difficulty in clarifying its epidemiology, infection mechanisms and pathogenesis. Therefore, the importance of reports of this pathology in different animal and human species is highlighted.
The macroscopic lesion observed in this case was similar to that described in other cases of rhinosporidiosis in horses (Argenta et al., 2018;Tizzano et al., 2021;Zibordi et al., 2020). It is noteworthy that, in addition to the nasal septum mucosa, rhinosporidiosis lesions in horses have also been described in the larynx (Santos et al., 2014;Zibordi et al., 2020).
Histologically, Coccidioides immitis, Emmonsia parva and E. crescens have morphological characteristics similar to R. seeberi, which include the size of the sporangia and the presence of endospores (Santos et al., 2014). However, C. immitis sporangia are typically smaller, more uniform in size and shape, and with beads measuring 20-200 µm in diameter. Emmonsia spp. organisms are larger and form single endospores, which are characterized by presenting uninucleate, spherical conidia, measuring 10-20 µm in diameter for E. parva and up to 300 mm for E. crescens (Caswell & Williams, 2016). PAS staining evidenced the capsule and internal granules of the endospores and has been used to identify the agent in nasal mycoses in humans (Boni et al., 2002) and horses (Sampaio et al., 2018), as well as to differentiate from C. immitis, which stain only the capsule of the endospores (Jones et al., 2000).
Other less frequent forms of diagnosis have been performed, including polymerase chain reaction (PCR) (Husain et al., 2020;Leeming et al., 2007) and antibody detection through the indirect immunofluorescence test (Crosara et al., 2009) and ELISA-dot (Sudasinghe et al., 2011), however in this case, due to the size of the fragment and clinical suspicion of cryptococcosis, they were not performed.
The treatment chosen was surgical excision of the nodule, considering that the main clinical suspicion based on cytological examination was cryptococcosis. In this case, complete removal of the nodule with a wide surgical margin resulted in complete healing without a history of recurrence. However, cases of recurrence after surgical resection have been observed in horses and it is postulated that during the surgical procedure there may be a release of endospores in adjacent tissues that culminate in the formation of lesions (Burgess et al., 2012;Zibordi et al., 2020), therefore, it has been recommended to perform surgical resection associated with cauterization in horses (Leeming et al., 2007;Zibordi et al., 2020). It's believed that cauterization contributes to the destruction of possible remaining structures of R. seeberi, reducing the chances of recurrence (Bernardo et al., 2016). Treatment with systemic chemical agents, such as antifungals or antimicrobials, has not been recommended, as they have low penetration into the sporangia membrane (Tiwari et al., 2015).
Considering that in this species the lesion occurs mainly in the nasal septum mucosa (Tiwari et al., 2015), it is important to carry out the differential diagnosis with other diseases that have a similar macroscopic presentation, such as nasal polyps and neoplasms (Trotte et al., 2008), in addition to granulomatous lesions caused by Aspergillus spp. (Caswell & Williams, 2016), Cryptococcus neoformans, Histoplasma spp., Pseudoallescheria boydii, Conidiobolus coronatus and Pythium insidiosum (Santos et al., 2014).

Conclusion
The occurrence of rhinosporidiosis in horses in the Northeast region of Brazil highlights the need to carry out research and publications in relation to its casuistry. It is believed that the macroscopic characteristics of the granulomatous lesion and the previously non-occurrence of this agent at the site influence the misdiagnosis, which is sometimes attributed to other infectious agents or inflammatory and neoplastic processes.