Screening of alkaloids and withanolides isolated from Solanaceae plants for antifungal properties against non-wild type Sporothrix brasiliensis.

Antifungal resistance has often been found in animal sporotrichosis in Southern Brazil. The biological potential of compounds from plants of the Solanaceae family against infectious diseases is known, however, it is still unknown against Sporothrix brasiliensis. This study evaluated the anti-Sporothrix brasiliensis activity, synergism, cytotoxicity, and action mechanism of steroidal lactones (withanolides) and alkaloids isolated from these plants. Pure compounds of withanolide D (WNOD), physalin F (PHYF), withanicandin (WNIC), nicandin B (NICB), solasonine (SSON), and solamargine (SMAR) were tested against 12 Sporothrix brasiliensis isolated from cats (n = 11) and dogs (n = 2) through M38-A2 CLSI. For the compounds with the best activity, a checkerboard assay for synergism, sorbitol protection, and ergosterol effect for action mechanism; and MTT test for cytotoxicity were performed. The withanolides WNOD, PHYF, WNIC, and NICB were not antifungal, but SSON (MIC 0.125-1 mg/mL) and SMAR (MIC 0.5-1 mg/mL) were both fungistatic and fungicidal (MFC 0.5-1 mg/mL for both) against wild-type (WT) and non-WT isolates. The activity of SSON and SMAR was indifferent when combined with itraconazole. In the mechanism of action, cell wall and plasma membrane by complexation with ergosterol seemed to be two target structures of SSON and SMAR. SSON was selected for cytotoxicity, whose cell viability in MDBK cells ranged from 28.85 % to 101.75 %, and was higher than 87.49 % at concentrations ≤0.0015 mg/ml. Only the steroidal alkaloids SSON and SMAR were active against non-WT isolates, being promising antifungal candidates for the treatment of feline and canine sporotrichosis with low susceptibility to itraconazole.

Are polar extracts and essential oil from Origanum vulgare Linn. (oregano) an alternative against itraconazole-resistant dermatophytes from veterinary cases?

Although the conventional therapy against dermatophytosis is based on antifungal drugs and environmental disinfection, the emergence of itraconazole(ITZ)-resistant dermatophytes has encouraged the search for active compounds, such as Origanum vulgare L. (oregano) essential oil (EO). However, little is known about its effect in polar extracts or the mechanism of action of these extracts and EO. We investigated the antifungal activity of four polar extracts and one EO from oregano against ITZ-susceptible and ITZ-resistant dermatophytes and their action mechanism. Polar extracts were prepared as infusions at 10 (INF10) and 60 (INF60) minutes, decoction (DEC) and hydroalcoholic extract (HAE); EO was purchased. All extracts and itraconazole were tested against Microsporum gypseum, M. canis, M. nanum, Trichophyton mentagrophytes and T. verrucosum isolated from cats, dogs and cattle (n = 28), and humans (n = 2) (M38-A2, CLSI). Among polar extract, DEC stood out as antifungal, followed by INF10 and INF60; HAE was little active. For EO, all isolates were susceptible, including ITZ-resistant dermatophytes. EO was selected for action mechanism assays, and acted in cell wall and plasmatic membrane by complexing with fungal ergosterol. By chromatographic analysis, 4-hydroxibenzoic acid was the most prevalent compound in all polar extracts, followed by syringic acid and caffeic acid; luteolin was present only in HAE. For EO, carvacrol was the major compound (73.9%); followed by γ-terpinene (3.6%) and thymol (3.0%). These findings showed that the extract type of oregano has influenced the antifungal action on dermatophytes, highlight EO and DEC, that are promising as antifungal agent, including against ITZ-resistant dermatophytes.

Subcutaneous phaeohyphomycosis due to Aureobasidium pullulans infection in a dog

A case of subcutaneous phaeohyphomycosis in a dog with an ulcerative lesion on the right limb during a post-operative period of castration was described for the first time. The macroscopic and microscopic characteristics of the fungal colonies growth on the Sabouraud­dextrose agar were detailed. The fungus was identified as Aureobasidium pullulans on the basis of the phenotypic analysis, which was confirmed by sequencing of the internal transcribed spacers (ITS) region of rDNA. The patient might have acquired the infection through traumatic inoculation by environmental contact, along with the immunological condition during the stressful period of postoperative. The spontaneous remission of the lesion was observed in five weeks without antifungal treatment. This work highlights the importance of considering the pathogenic potential of this environmental fungus and the need of including it in the differential diagnosis of cutaneous lesions in dogs.

Human sporotrichosis outbreak caused by Sporothrix brasiliensis in a veterinary hospital in Southern Brazil.

Sporotrichosis is a mycotic disease caused by Sporothrix spp., whose zoonotic transmission by sick cats is the main infection route in Brazil. The aim of the current study is to report a human sporotrichosis outbreak caused by zoonotic transmission from a feline, with emphasis on the importance of making differential diagnosis and of using personal protective equipment. A hospital team member presented injury in the arm after having handled a cat that had been hospitalized for being hit by a car. The animal presented skin lacerations, myiasis, and full tibial fracture - there were no other signs of skin lesions. Clinical samples were collected from both the human and the suspected cat, for mycological culture; results have shown Sporothrix sp. growth. A search was conducted to identify other hospital team members who also had contact with the animal. Other six individuals also had suspected lesions in their arms, hands and ocular area; they were all subjected to sample collection. Mycological results have also confirmed Sporothrix spp.; sequencing analysis has shown that all seven humans were infected with Sporothrix brasiliensis. Since Southern Brazil is endemic of this disease, it is worth emphasizing the importance of taking into consideration zoonotic risks at the time to provide emergency care to stray animals, mainly felines, as well as of using Personal Protective Equipment while handling them - regardless of whether they present, or not, typical clinical symptoms or history of the disease, given the potential zoonotic risk posed by Sporothrix brasiliensis.

Feline sporotrichosis: a case series of itraconazole-resistant Sporothrix brasiliensis infection.

The treatment of feline sporotrichosis is a challenge for veterinary clinicians since refractory cases may occur, due either to patient and/or to pharmacological management errors or due to the development of antifungal resistance. Thus, we aimed to describe the therapeutic history of feline cases infected by itraconazole-resistant Sporothrix brasiliensis in an endemic region of Southern Brazil. Medical records of cats attended at the Veterinary Clinic Hospital (Pelotas/RS, Brazil) between 2016 and 2017 were reviewed. Twelve cases of infection by S. brasiliensis with that showed high minimum inhibitory concentration (MIC) values (≥ 4 µg/mL) to itraconazole by M38-A2 of CLSI were selected. At the hospital consultation, disseminated (cats 1-l0, 12) and localized (cat 11) skin lesions remained in the cats, even after treatment with fluconazole, ketoconazole (02/12), and itraconazole (ITZ, 09/12) performed before this study. High doses (25-100 mg/kg/day) of ITZ for up to 4 months (03/12, cats 2, 6, 12) or over 12 months (05/12, cats 1, 5, 7, 8, 11) did not provide a clinical cure, except for the association of ITZ plus potassium iodide (01/12, cat 12) for 3 months, which proved useful in infections with itraconazole-resistant S. brasiliensis. However, the combined issues of abandonment of therapy by owners for financial reasons, difficulties surrounding therapy administration (03/12, cats 6, 11, 12), and the inappropriate choice of medication (01/12, cat 6), together reflect the reality of this endemic region, which greatly compromises clinical healing. This study highlighted the occurrence of refractory cases by itraconazole-resistant S. brasiliensis in cats from Southern Brazil, as well as the abandonment of treatment and therapeutic errors. We warn of the need for antifungal susceptibility tests to adapt therapeutic protocols in feline sporotrichosis.

Disseminated histoplasmosis in a cat rescued in Fortaleza, Brazil, and successfully treated with itraconazole - First case report identified molecularly.

An unneutered female cat of unknown age presented bloody lesions on the edematous face, and respiratory signs. Cytology and culture from the skin sample collected with fine-needle aspiration showed yeasts inside activated macrophages, and fungal growth characteristic of Histoplasma spp., which was molecularly confirmed that was Histoplasma capsulatum var. capsulatum. The cat was successfully treated with oral itraconazole (10 mg/kg/daily) for 120 days. This is the first case report of feline histoplasmosis confirmed molecularly in Brazil.

Plants from Lamiaceae family as source of antifungal molecules in humane and veterinary medicine.

This work aimed to review the main plants of Lamiaceae family with activity against pathogenic fungi of medical and veterinary interest. Published studies in the main international databases between January 2002 and June 2016 showed that 55 botanical species belonging to 27 genus presented antifungal activity in different forms of extractions, mainly essential oils. Pathogenic fungi of Aspergillus spp., Candida spp., Malassezia spp., Cryptococcus spp., Sporothrix spp., Microsporum spp., Trichophyton spp. and Epidermophyton spp. genus were in vitro sensitive to several plants of Lamiaceae family. Chemical molecules isolated were described as promising use as antifungals in mycoses, highlighting estragole, 1,8-cineole, terpineol-4, γ-terpinene, among others. However, it should be alert to need of universal standardization in the laboratories tests with natural products.

Susceptibility of dermatophytic fungi to commonly used disinfectants / Suscetibilidade de fungos dermatófitos a desinfetantes comumente utilizados

This study aimed evaluate the antidermatophytic activity of three commercial disinfectants commonly used for environmental control of microorganisms in veterinary medicine. Sodium hypochlorite at 40 µL/mL, chloro-phenol derived at 30 µL/mL and chlorhexidine digluconate at 66.7 µL/mL were tested against 14 strains of dermatophytes, identified as Microsporum canis (n: 3) and Microsporum gypseum (n: 11). The tests was performed in accordance with guidelines of the Clinical and Laboratory Standards Institute (CLSI), documents M38-A2 and M51-A, adapted to disinfectants. In the microdilution broth test, chlorhexidine digluconate had MIC values (Minimum Inhibitory Concentration) of 4.16 µL/mL and MCF (Minimum Fungicidal Concentration) from 4.16 to 8.33 µL/mL, while chloro-phenol derived obtained MIC and MCF of 1.87 µL/mL, and both disinfectants had fungicidal activity at concentrations below the recommended. Sodium hypochlorite obtained MIC from 10 to 80 µL/mL and MFC of 40 to 80 µL/mL, requiring at most isolates twice the recommended concentration to achieve same activity. In the disc diffusion test, the mean inhibition zones for chlorhexidine digluconate was 10.53 mm, for chloro-phenol of 9.9 mm and for sodium hypochlorite was 6.2 mm. Chlorhexidine digluconate and chloro-phenol presented a significant reduction in the growth of dermatophytes, while sodium hypochlorite in concentration recommended showed a low antifungal activity against tested isolates.

O objetivo do estudo foi avaliar a atividade antidermatofítica de três desinfetantes comerciais frequentemente utilizados no controle ambiental de micro-organismos em medicina veterinária. Hipoclorito de sódio a 40 µL/mL, derivado de clorofenol a 30 µL/mL e digluconato de clorexidine a 66.7 µL/mL foram testados contra 14 cepas de dermatófitos, identificados como Microsporum canis (n: 3) e Microsporum gypseum (n: 11). Foram utilizadas as diretrizes do Clinical and Laboratory Standard Institute (CLSI), documentos M38-A2 e M51-A, com adaptações para desinfetantes. Na microdiluição em caldo, digluconato de clorexidine apresentou valores de CIM (Concentração Inibitória Mínima) de 4.16 µL/mL e CFM (Concentração Fungicida Mínima) entre 4.16 a 8.33 µL/mL; derivado de clorofenol obteve CIM e CFM de 1.87 µL/mL, demonstrando que ambos os desinfetantes apresentaram atividade fungicida em concentrações inferiores às recomendadas. O hipoclorito de sódio demonstrou CIM entre 10 a 80 µL/mL e CFM de 40 a 80 µL/mL, requerendo duas vezes a concentração recomendada pelo fabricante para obter atividade fungicida frente a maioria dos isolados fúngicos testados. No teste de disco-difusão, a média das zonas de inibição do digluconato de clorexidine foi de 10.53 mm; do derivado clorofenol 9.9 mm e do hipoclorito de sódio 6.2 mm. O digluconato de clorexidine e o derivado cloro-fenol apresentaram redução significante no crescimento dos dermatófitos testados, enquanto o hipoclorito de sódio, na concentração recomendada, demonstrou baixa atividade antifúngica contra os dermatófitos testados.

First isolation of the Stephanoascus ciferrii in feline otitis in Brazil.

Ear infections in cats are uncommon, especially involving yeasts. This report describes the first isolation of the Stephanoascus ciferrii, teleomorph of the Candida genus, in a case of feline otitis in Brazil. The identification and characterization of Stephanoascus ciferrii were confirmed by the Vitek2 System (BioMerieux ®).

First isolation of the Stephanoascus ciferrii in feline otitis in Brazil

Ear infections in cats are uncommon, especially involving yeasts. This report describes the first isolation of the Stephanoascus ciferrii, teleomorph of the Candida genus, in a case of feline otitis in Brazil. The identification and characterization of Stephanoascus ciferrii were confirmed by the Vitek2 System (BioMerieux ®).

Aflatoxicose em cães na região Sul do Rio Grande do Sul / Aflatoxicosis in dogs in Southern Rio Grande do Sul

Descrevem-se os aspectos clinicopatológicos de casos de aflatoxicose em cães no Sul do Rio Grande do Sul. Foi realizado um estudo retrospectivo dos casos diagnosticados como aflatoxicose em cães necropsiados no Laboratório Regional de Diagnóstico (LRD) da Faculdade de Veterinária da Universidade Federal de Pelotas (UFPel) no período de 1978 a 2012. Em quatro casos o diagnóstico foi confirmado pela detecção de níveis de 89 a 191 ppb de aflatoxinas B1 e G1 no alimento dos cães. De um total de 27 cães com cirrose hepática, em seis havia suspeita de aflatoxicose pelas lesões macro e microscópicas e pelo tipo de alimentação que os cães recebiam. Os sinais clínicos nos casos confirmados e nos suspeitos caracterizaram-se por apatia, diarreia, icterícia e ascite, com evolução para morte em 8 a 30 dias nos casos confirmados e em 15 a 60 dias nos casos suspeitos. A dieta era à base de derivados de milho ou arroz, farelo de amendoim e, em um caso suspeito, a dieta era ração comercial. As alterações macroscópicas caracterizaram-se por ascite, icterícia, fígado aumentado de tamanho, com ou sem nódulos, hemorragia nas serosas, conteúdo intestinal hemorrágico. Os casos foram classificados de acordo com o padrão histológico principal, caracterizado por vacuolização difusa no citoplasma de hepatócitos nos casos agudos, por proliferação de ductos biliares e discreta fibroplasia nos casos subagudos e por fibrose acentuada nos casos crônicos. Aparentemente, a enfermidade não é importante como causa de morte em cães na região, no entanto, alerta-se para a possibilidade de casos com diagnóstico de cirrose hepática sem causa determinada serem causados por aflatoxicose.

Clinical pathological aflatoxicosis in dogs is described in southern Rio Grande do Sul. It was conducted a retrospective study of cases diagnosed as aflatoxicosis in dogs necropsied at the Regional Diagnostic Laboratory (LRD) of the Veterinary School of the Federal University of Pelotas (UFPel) in the period 1978-2012. In four cases the diagnosis was confirmed by detection of levels of aflatoxins B1 and G1, with the finding of 89-191ppb in the feed. The macroscopic and histologic lesions and the diet observed in six of 27 dogs with liver cirrhosis led to suspicion of aflatoxicosis. Clinical signs evidenced in confirmed or suspected cases were lethargy, diarrhea, jaundice and ascites, progressing to death within 8 to 30 days in confirmed cases, and within 15 to 60 days in suspected cases. The diet was corn and rice byproducts and peanut meal, and one of the dogs received commercial ration. Gross changes were characterized by ascites, jaundice, enlarged liver, with or without regenerative nodules, hemorrhages in serous membranes and bloody intestinal content. The cases were classified according to the main histological pattern, characterized by diffuse vacuolation of the cytoplasm of hepatocytes in acute cases, by proliferation of bile ducts, and mild fibrosis in subacute cases, and by severe fibrosis in chronic cases. Apparently the disease is not important as a cause of death in dogs in the region, nevertheless the possibility of cases of cirrhosis of unknown etiology would be caused by aflatoxicosis.

Microsporose mista canina - relato e caso / Microporosis mixta canina ? relato de caso / Canine mcrosporose mixed - case report

Dermatofitoses são micoses causadas por fungos como: Epidermophyton, Microsporum e Trichophyton. M. canis e M. gypseum são os principais responsáveis pelas infecções dermatofíticas em pequenos animais e acometem principalmente animais jovens. Essas micoses cursam com lesões nas camadas queratinizadas da pele e pelos e em infecções mistas, a intensidade das lesões pode ser aumentada, pois os agentes causais atuam potencializando o quadro clínico. Descreve-se um caso de microsporose mista canina em um filhote de cão da raça Rottweiler. Um canino da raça Rottweiller atendido em uma clínica particular, pois apresentava lesões alopécicas e descamativas em diferentes sítios do corpo. Foi realizada coleta por meio de raspado cutâneo e o material foi encaminhado para exame micológico. O exame direto do pelo revelou estruturas características de dermatofitose com hifas artrosporadas e aglomerados de artroconídeos. O cultivo micológico resultou em crescimento de colônias fúngicas sugestivas de M. gypseum e M. canis nos meios de cultura semeados com confirmação das características macro e micromorfológicas das duas diferentes espécies fúngicas. Assim, mediante processamento micológico estabeleceu-se, juntamente com o histórico do caso, o diagnóstico de microsporose mista canina. Desse modo, conclui-se a necessidade de exames laboratoriais para a confirmação do diagnóstico definitivo de dermatofitose, sendo a profilaxia, prevenção, controle e potencial zoonótico dessa micose dependente da espécie infectante, tendo importância o seu controle também por seu impacto em saúde pública.

Dermatophytosis mycoses are caused by fungi such as Epidermophyton, Microsporum and Trichophyton. M. canis and M. gypseum are the main responsible for dermatophytic infections in small animals and affect mainly young animals. These mycoses occur with lesions in the keratinized layers of the skin and the mixed infections and the intensity of the lesions can be increased because the causative agents act enhancing the clinical picture. We describe a case of mixed microsporose dog in a puppy dog breed Rottweiler. A canine breed Rottweiller served in a private clinic because he had alopecia, scaly lesions at different body sites. It was collected through skin scrapings and the material was sent for mycological examination. Direct examination of the structures revealed by the characteristics of dermatophytosis with of hyphae artrosporadas and arthroconidia clusters. The mycological cultivation resulted in growth of fungal colonies suggestive of M. canis and M. gypseum in culture media seeded with confirmation of macro and micromorphological characteristics of the two different fungal species. Thus, by processing mycological established together with the case history, the diagnosis of canine microsporose mixed. Through the described case is concluded the need for laboratory tests to confirm the definitive diagnosis of dermatophytosis and prophylaxis, prevention, control and zoonotic potential of this mycosis dependent on the infecting species, taking control of the same importance also for their impact on public health.

Dermatofitosis son micosis causadas por hongos como: Epidermophyton, Microsporum y Trichophyton. M.canis y M. gypseum que son los principales responsables en las infecciones por dermatofitos en animales pequeños y afectan principalmente animales jóvenes. Esas micosis se producen con lesiones en las capas queratinizadas de la piel, pelos y en infecciones mixtas, la intensidad de las lesiones puede ser aumentada, pues los agentes causantes actúan potencializando el cuadro clínico. Se describe un caso de microsporosis mixta canina en un cachorro de perro de la raza Rottweiller. Un canino de la raza Rottweiller atendido en una clínica privada que presentaba lesiones de alopecia y lesiones escamosas en diferentes partes del cuerpo. Se realizó recolección por medio de raspado cutáneo y el material fue enviado para examen micológico. El examen directo del pelo reveló estructuras características de dermatofitosis hifas artrosporadas y aglomerados de artroconidios. El cultivo micológico resultó en crecimiento de colonias fúngicas sugestivas de M. gypseum y M canis en los medios de cultura sembrados, con confirmación de características macro y micro morfológicas de dos especies fúngicas. Así, mediante procesamiento micológico establecido junto con el histórico del caso, el diagnóstico de microsporosis mixta canina. Se concluye la necesidad de exámenes de laboratorio para confirmación del diagnóstico definitivo de dermatofitosis, siendo que la profilaxis, prevención, control y potencial zoonótico de esa micosis depende de la especie infectante, siendo importante su control por el impacto que causa a la salud pública.