First report of fungal Sporothrix schenckii complex isolation from feline with possible zoonotic transmission in the city of Belém, Pará, Brazil: Case report

Feline sporotrichosis is a subcutaneous, infectious, and contagious mycosis, with zoonotic characteristics. Until now there are no reports on the isolation of Sporothrix sp. in cats in the state of Pará. The present work aimed to isolate Sporothrix spp. from a cat with possible zoonotic involvement in the city of Belém, Pará. Through the active search for suspected cases of sporotrichosis, carried out by members of the Center for the Control of Zoonoses of Belém (CCZ, Belém) the suspicion of the disease in a feline was reported, with probable transmission to its owner. Clinical evaluation was performed, and biological material was collected from the animal for identifying the agent. Microscopic evaluation of the collected sample and microbial culture confirmed the presence of Sporothrix sp. in the sample. This study is the first to report the microbiological isolation of Sporothrix sp. in a cat in the metropolitan region of Belém, Pará, with possible zoonotic transmission. This indicates a probable underreporting of cases, an unknowing regarding the reality of health conditions and the need for more accurate and comprehensive evaluation of sporotrichosis in this region.


Introduction
Feline sporotrichosis is a subcutaneous, infectious, and contagious mycosis with a zoonotic characteristic. It is caused by a dimorphic fungus belonging to the Sporothrix schenckii complex, and each species differs with respect to their geographical distribution. S. brasiliensis is an emerging species that is restricted to Brazil and with high potential for zoonotic transmission (Rodrigues et al., 2016). In cats and dogs, the clinical manifestations begin with a subclinical infection that can progress to multiple skin lesions and a disseminated systemic form (Gremião et al., 2017).
Epidemiological studies indicate that the endemic regions with the highest prevalence of sporotrichosis are Brazil (5,814 cases), China (3,299 cases), and South Africa (3,154 cases) (Zhang et al., 2015). S. brasiliensis is associated with cats fection, and its distribution in Brazil is restricted to the Southern and Southeastern states (Rodrigues et al., 2014). Since 2000, incidences of sporotrichosis have been increased exponentially in Rio de Janeiro, where the disease is endemic (Barros et al., 2008).
Although there are no reports on the isolation of this fungus from the cats in the state of Pará, cases have been diagnosed in humans, with one died (Falcão et al., 2019). Considering the climate of the Amazon region associated with the considerable of these animals (approximately 22,070,633.12), many have free access to the external environment or are in a situation of neglect, contributing to the possibilities of exposure of these animals to Sporothrix sp. and consequently the ability to spread and maintain this agent in the region in this region (Caldas, 2019).
According to Gutierrez-Galhardo et al., (2015), the probable reason underlying the increasing number of human cases is the increasing direct involvement with animals and is an extremely crucial factor. As required notification are occurs only in the state of Rio de Janeiro, and the cases in Pará are probably underreported and there is an unknowing regarding the actual situation of sporotrichosis. Therefore, studies on the occurrence of sporotrichosis in the human inhabitants and animals in Pará are essential. In this study aimed to isolate Sporothrix sp. in a cat with possible zoonotic involvement in the city of Belém, Pará.

Case report
Members of the Zoonoses Control Center of Belém (Centro de Controle de Zoonoses de Belém -CCZ, Belém) actively going in searched for the suspected cases of sporotrichosis and reported a case of feline sporotrichosis. The animal was an adult cat, male, non-neutered, immunized with rabies vaccine, and resident in the town of Belém, Pará ( Figure 1A).
The pet owner was requested to schedule a visit to the veterinarian for assessing condition and confirming the suspected disease. Clinical examination was performed and biological samples were collected.
For microbiological culture, samples were individually collected from the lesions by scarification with a swab ( Figure. 1B) and slides were prepared using an imprint ( Figure 1C) for cytological evaluation. The swabs were packed in sterile saline solution, and the temperature was maintained at 4ºC during transportation. The materials were immediately sent to the Veterinary Microbiology Laboratory at UFRA for further processing. The owner reported that he too had lesions in the abdominal region that looked similar to those of the animal.

Source: Authors.
For fungal isolation and demonstration dimorphism, the samples were sown on Sabouraud dextrose agar with chloramphenicol and Mycosel ® agar, and subsequently incubated at 25ºC and 37ºC for 15 days. The microorganisms obtained from the cultures were subjected to macromorphological and micromorphological examinations (Rudramurthy & Chakrabarti, 2017).
The anamnesis revealed the animal improperly accessed the streets, hunting habitats, and had hyporexia. The symptoms emerged approximately one month before reporting, and the cat was kept in isolation since symptom onset.
Clinical examination revealed cachexia, apathy, infarcted submandibular lymph nodes, and multiple skin lesions in the cephalic regions, on the face, ears, and left side. A few lesions had a circular and elevated appearance, however, most lesions had an erythematous, ulcerated, flaky, serosanguinous character, devoid of purulent and non-pruritic contents ( Figure   1D-E). The owner had lesions in the abdominal region that looked similar to those of the animal ( Figure 1F). The transmission possibly occurred through scratching while handling the cat approximately two weeks before symptom onset. The pet owner was advised to seek dermatological care due to a suspected zoonotic transmission, for better evaluation of his condition.
Biological material was collected from the cat for direct examination. Panoptic staining revealed the predominance of macrophages, lymphocytes, intermediate erythrocytes, and abundant rounded, oval, pleomorphic, cigar-shaped structures located intracellularly and extracellularly, with characteristics similar to the yeasts of the S. schenckii complex (Figure 2A-B).
On slide stain by the Gram method, yeast cells with a morphological appearance similar to those described by Romanowsky were also observed, in addition to rare Gram positive cocci.
Microbial growth was assessed on a weekly basis for 15 days. The cultures showed cream-colored colonies, glabrous and creamy yeast at 37ºC, and initially white-colored filamentous colonies with cottony texture at 25ºC ( Figure 2C  Based on the confirmatory diagnosis of sporotrichosis in the cat, a therapeutic protocol was instituted, comprising the oral administration of 15 mg/kg itraconazole, twice daily, until the remission of clinical signs, following the recommendations by Larsson (2011). Home visits were arranged for evaluating the development of the clinical presentation and therapeutic response. After one month, the animal showed clinical improvement and weight gain, suggesting treatment continuation ( Figure 3A). At four months, the lesions persisted, but were significantly reduced ( Figure 3B), and were clinically cured in the fifth month ( Figure 3C).

Discussion
This study is the first to report a case of feline sporotrichosis in Belém do Pará (Brazil), adding to the other cases in Brazil. It highlights the contribution of domestic cats in transmitting skin sporotrichosis, which is frequently encountered in veterinary clinics. Finally, the care necessary for avoiding zoonotic transmission was established herein.
The widespread growth of S. schenckii lato sensu in the environment occurs in places with high relative humidity (above 80%), and temperatures of approximately 26-30ºC (Carrada-bravo & Olvera-macías, 2013). According to the data obtained by the National Institute of Metereology in the year 2020, the relative humidity of Belém was above 80%, and the average temperatures were 20-35ºC. in addition, this climatic conditions of the municipality provided the ideal conditions for the growth and environmental maintenance of the fungus, and could contributed to the infection reported herein.
The presence of the etiological agents of sporotrichosis in the nature (surface of branches, soil, and tree barks) can induce fluctuations in the number of transmissions (Rodrigues et al., 2016). Cats have habits that make them more susceptible to sporotrichosis, including sharpening nails on trees and digging the soil for burying feces. This facilitates the sheltering of the fungus in the nail, causing illnesses or making them asymptomatic carriers (Rudramurthy & Chakrabarti, 2017). These were observed in the present case, increasing the reliability of clinical suspicion and prophylactic protocol.
Clinical suspicion based on clinical manifestations and epidemiological data is essential for the diagnosis of any disease. According to Barros et al.,(2011), feline sporotrichosis primarily manifests as form of nodular or plaque, firm, alopecic, and painless skin lesions that fistulate or ulcerate, releasing serosanguineous fluid. The cat evaluated had the lesions described in literature. The presentation of nodules and ulcers is common to sporotrichosis, however, can be found in other infectious diseases that affect humans and animals, including leishmaniasis, cryptococcosis, histoplasmosis, and mycobacteriosis, requiring a differential laboratory diagnosis. Apart from neoplastic processes, feline eosinophilic granuloma, squamous cell carcinoma, and lymphomas should also be included in the differential diagnosis (Larsson, 2011).
Since 1993, sporotrichosis cases are treated with 10 mg/kg itraconazole, administered daily, for several months. In some cases, therapy may last for up to one year (Larsson, 2011). After complete remission of the signs, treatment should be extended for four weeks for avoiding relapse. The use of corticosteroids and other immunosuppressants should be avoided during treatment, owing to the risk of promoting fungal multiplication and worsening of the clinical situation (Larsson, 2011).
Therapeutic procedures were prescribed to the owner as indicated in literature, and cure was assessed after administration of the correct treatment, which was established by considering the choice of medications and managing the health and environment of the animal.

Conclusion
This study is the first to report the microbiological isolation of Sporothrix sp. in a feline at the metropolitan region of Belém, Pará, with possible zoonotic profile. This indicates a probable underreporting of cases, unknowing regarding the reality of health conditions, and the need for more accurate and comprehensive evaluation of sporotrichosis in this region.