Can the essential oil of rosemary (Rosmarinus officinalis Linn.) protect rats infected with itraconazole-resistant Sporothrix brasiliensis from fungal spread?

INTRODUCTION: Itraconazole is the first-choice option to treat human and animal sporotrichosis. However, the emergence of itraconazole-resistant strains has encouraged research on new active antifungals. Among them, the essential oil of rosemary (Rosmarinus officinalis Linn., Lamiaceae) has shown antifungal activity in vitro. OBJECTIVE: Assessing, for the first time, the effectiveness of rosemary essential oil in vivo in experimental cutaneous sporotrichosis, as well as its chemical composition and action mode. METHODS: Itraconazole-resistant Sporothrix brasiliensis was inoculated in the left foot pad of 30 Wistar rats, which were randomized (n=10) for treatment with saline solution (control, CONT), itraconazole (ITRA, 10 mg/kg) and rosemary oil (ROSM, 250 mg/kg) for 30 days at an oral dose of 1 mL, daily. Clinical evolution, histopathology and fungal burden were investigated. GC-MS was used for chemical analysis; sorbitol protection and ergosterol effect were used to evaluate the action mechanism of rosemary oil. RESULTS: ROSM was the only group evolving to skin lesion remission, lack of edema and exudate, and mild-to-absent yeast cells. Rosemary oil delayed fungal spreading and protected systemic organs, mainly liver and spleen. The ROSM group presented lower fungal load than that observed for the CONT and ITRA groups (p<0.05). Antifungal action took place at complexation level after ergosterol application. Most compounds were 1,8-cineole/eucalyptol (47.91%), camphor (17.92%), and α-pinene (11.52%). CONCLUSIONS: These findings have evidenced that rosemary oil is a promising antifungal to treat sporotrichosis, since it protects systemic organs from fungal spread.

Chemical and cytotoxic analyses of three varieties of Brazilian propolis (green propolis, jataí propolis and brown propolis) and its anti-Sporothrix brasiliensis in vitro activity / [Análises químicas e citotóxicas de três variedades de própolis brasileiras (própolis verde, própolis jataí e própolis marrom) e sua atividade in vitro anti-Sporothrix brasiliensis]

In this study, we described the antifungal activity of three Brazilian propolis extracts: brown, green and from jataí bees against Sporothrix brasiliensis. The extracts were obtained from ethanolic extraction and their chemical composition was determined by high-performance liquid chromatography coupled to mass spectrometry. The cellular toxicity was measured in MDBK (Madin-Darby Bovine Kidney) cells and quantified by the MTT assay (3- (4,5 dimethylthiazol-2yl -2,5-diphenyl-2H bromine tetrazolato). For antifungal activity, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by broth microdilution. The results showed that cell toxicity was not observed at lower concentrations (0.097 to 0.39μg/ml) for all extracts in comparison to cell control. Among the chemical compounds identified, caffeic acid, p-coumaric acid, chlorogenic acid, ferulic acid and rutin were quantified. In antifungal activity, green and jataí did not exhibit activity against the isolates (MIC and MFC greater than 0.78mg/ml). However, all isolates of S. brasiliensis were sensitive to brown propolis (MIC of 0.09 to 0.78mg/ml), including the standard strain (P<0.001). Among the Brazilian propolis studied, the brown propolis showed activity against the S. brasiliensis isolates and more studies should be undertaken in order to evaluate its promising use in the treatment of sporotrichosis.(AU)

Neste estudo, descreveu-se a atividade antifúngica de três extratos de própolis brasileiras: marrom, verde e de abelhas jataí (Tetragonisca angustula), contra Sporothrix brasiliensis. Os extratos foram obtidos de extração etanólica, e a sua composição química foi determinada por cromatografia líquida de alta eficiência, acoplada à espectrometria de massa. A toxicidade celular foi medida em células MDBK (Madin-Darby Bovine Kidney), avaliada por observação microscópica e quantificada pelo ensaio MTT (3- (4,5-dimetiltiazol-2-ilo -2,5-difenil-2H bromo tetrazolato). Para a atividade antifúngica, determinou-se a concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM) por meio de microdiluição em caldo. Os resultados mostraram que a toxicidade celular não foi observada em concentrações menores (0,097 a 0,39μg/ml). Entre os compostos químicos identificados, foram quantificados o ácido cafeico, ácido p-cumárico, ácido clorogênico, ácido ferúlico e a rutina. Na atividade antifúngica, as própolis verde e jataí não apresentaram atividade contra os isolados (CIM e CFM maior que 0,78mg/ml), porém todos os isolados de S. brasiliensis foram sensíveis à própolis marrom (CIM de 0,09 a 0,78mg/ml), incluindo a cepa padrão (P<0,001). Entre as própolis brasileiras estudadas, a marrom mostrou atividade contra S. brasiliensis, e mais estudos devem ser realizados para avaliar seu uso promissor no tratamento da esporotricose.(AU)

Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins / Aplicabilidade do Platelia EIA® Aspergillus como teste diagnostico da aspergilose em pinguins

Abstract Even today, an effective diagnostic test for aspergillosis in penguins is unknown, being the gold standard post-mortem examinations. The fungal antigen galactomannan (GM) has been used as a biomarker of disease in humans and is detected by the Platelia Aspergillus EIA (BioRad)®, a commercial kit based on the sandwich ELISA technique. It is standardized for use in neutropenic patients, however studies have demonstrated its usefulness also possible for birds. The aim of our study was to evaluate the effectiveness of Platelia Aspergillus EIA® test (BioRad-US) in the diagnosis of aspergillosis in Magellanic penguins, determining sensitivity, specificity, and positive and negative predictive values for different cut-off points. Were included in the study, blood serum samples (n = 29) Magellanic penguins in captivity that died by aspergillosis. Detection of GM was performed following manufacturer's instructions and the GM index was obtained by dividing the average value of OD of the duplicate of the clinical sample by duplicate OD of the average value of the cut-off sample provided by the kit. Through information database results were obtained for the presence of anti-Aspergillus fumigatus antibodies detected by agar gel immunodiffusion (AGID) for all serum samples. Results were analyzed using chi-square test and Kruskal-Wallis from SPSS 20.0, IBM®. ROC curve was obtained and from this, rates of sensitivity, specificity, positive and negative predictive values were also calculated based on four different cutoff points (0.5, 1.0, 1.5 and 2.0). The serum GM index did not differ between animals of the case and control group (pkw =0.097). In determining the ROC curve for serum GM detection the value of area under the curve was 0.635. From the values ​​determined by the coordinate of the curve, four different cut points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2 to 34.5% % and specificity between 87% and 26.1%. By comparing the serum GM index in group case as the presence or absence of antibodies detected by AGID was found p=0.503. The detection of GM the Platelia Aspergillus EIA® test seems is not be useful for the diagnosis of aspergillosis in naturally infected penguins.

Resumo Ainda hoje, um teste diagnóstico eficaz para aspergilose em pinguins não é conhecido, sendo o padrão-ouro os exames post-mortem. O antígeno fúngico galactomanana (GM) tem sido utilizado como biomarcador da doença em humanos, sendo detectado pelo Platelia Aspergillus EIA (BioRad)®, um kit comercial que se baseia na técnica ELISA sanduíche. É padronizado para utilização em pacientes neutropênicos, no entanto estudos tem demonstrado sua possível utilidade também para aves.O objetivo de nosso estudo foi avaliar a eficácia do teste Platelia Aspergillus EIA® (BioRad-US) no diagnóstico da aspergilose em pinguins-de-Magalhães, determinando sensibilidade, especificidade e valores preditivos positivos e negativos em diferentes pontos de corte. Foram incluídas no estudo, amostras de soro sanguíneo (n=29) de pinguins-de-Magalhães em cativeiro que vieram a óbito por aspergilose. A detecção de GM foi realizada seguindo instruções do fabricante e o índice de GM foi obtido dividindo o valor da média da DO da duplicata da amostra clínica pelo valor da média da DO da duplicata da amostra de cut-off fornecida pelo kit. Através de informações em banco de dados foram obtidos resultados sobre a presença de anticorpos anti-Aspergillus fumigatus, detectada por Imunodifusão em gel de ágar (IDGA) em todas as amostras séricas. Os resultados foram analisados utilizando-se teste de qui-quadrado e Kruskal-Wallis a partir do programa estatístico SPSS 20.0, IBM®. Curva ROC foi obtida e a partir desta, taxas de sensibilidade, especificidade, valores preditivo positivo e negativo foram igualmente calculados considerando quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0). O índice de GM sérica não diferiu entre os animais do grupo caso e controle (pKW = 0.097). Na determinação da curva ROC para detecção de GM sérica o valor da área sobre a curva foi de 0.635. A partir dos valores determinados pelas coordenadas da curva, quatro diferentes pontos de corte (0.5, 1.0, 1.5 e 2.0) foram analisados, resultando em taxas de sensibilidade variando de 86.2% a 34.5%, e de especificidade entre 87% e 26.1%. Ao comparar o índice de GM sérica nos animais do grupo caso quanto a presença ou não de anticorpos detectados pela IDGA foi encontrado p=0.503. A detecção de GM pelo teste Platelia Aspergillus EIA® não parece ser útil para o diagnóstico da aspergilose em pinguins naturalmente infectados.

Atividade in vitro do óleo essencial de Origanum vulgare L. em isolados clínicos de Aspergillus spp / In vitro activity of essential oil of Origanum vulgare L. in clinical isolates from Aspergillus spp

Aspergillus fumigatus, A. flavus e A. niger são os mais importantes agentes etiológicos da aspergilose, relevante micose aviária, com tratamento ineficaz e altas taxas de mortalidade. Em vista da importância da aspergilose, da necessidade de prospectar novos fármacos e do potencial terapêutico do óleo essencial de Origanum vulgare L. (OEO), o orégano, objetivou-se avaliar a sensibilidade in vitro de isolados clínicos de Aspergillus spp. em relação ao OEO. O óleo foi obtido por hidrodestilação em Clevenger, e a análise química realizada por cromatografia de massa (GC/MS). Observaram-se 15 diferentes compostos ativos, sendo 4-terpineol, hidrato de sabinene e timol os majoritários. Nos testes de microdiluição em caldo (Reference..., 2008), todos os isolados (n= 23) foram sensíveis ao OEO: A. fumigatus teve CIM entre 28,125mg/mL (0,1875%) e 450mg/mL (3,0%), A. flavus entre 112,5mg/mL (0,75%) e 450mg/mL, e A. niger 112,5mg/mL. CFM variou de 112,5mg/mL a 450mg/mL nos isolados de A. fumigatus, de 225mg/mL (1,5%) a 450mg/mL em A. flavus, e foi de 450mg/mL em A. niger. CIM e CFM foram idênticos em 6/14 isolados, o que demonstra que o óleo com a mesma concentração pode ter capacidade fungistática e fungicida. CIM 90 correspondeu à CIM máxima. Os resultados demonstram a atividade anti-Aspergillus do OEO, com CIM 90 de 450mg/mL (3%).(AU)

Aspergillus fumigatus, A. flavus and A. niger are the most important etiological agents of aspergillosis, a relevant avian mycosis, with innefective treatment and high mortality rates. Due the importance of aspergillosis, the necessity of prospection of new drugs and the therapeutic potential of the essential oil of Origanum vulgare L. (OEO), popularly known as oregano, aims to evaluate the in vitro sensitivity of Aspergillus spp. opposing to OEO. The oil was obtained by hydrodistillation in Clevenger, and the chemical analysis performed by mass chromatography (GC/MS). 15 different active compounds were observed, being 4-terpineol (18.4%), sabinene hydrate (15.6%) and thymol (13.6%), the majority components. In the in vitro susceptibility test, all strains showed sensitivity to OEO, MIC of Aspergillus fumigatus ranged from 28,125mg/mL (0,1875%) to 450mg/mL (3,0%), A. flavus 112,5mg/mL (0,75%) to 450mg/mL, and A. niger 112,5mg/mL. MFC ranged from 112,5mg/mL to 450mg/mL in the A. fumigatus isolates, 225mg/mL (1,5%) to 450mg/mL in A. flavus, and 450mg/mL for A. niger. The MIC and FMC values were identical in 6/14 of the isolated subjects, demonstrating that the oil with the same concentration can have fungistatic and fungicidal capacity. The results demonstrates anti-Aspergillus activities of OEO with CIM90 de 450mg/mL (3%).(AU)

Applicability of the Platelia EIA® Aspergillus test for the diagnosis of aspergilosis in penguins.

Even today, an effective diagnostic test for aspergillosis in penguins is unknown, being the gold standard post-mortem examinations. The fungal antigen galactomannan (GM) has been used as a biomarker of disease in humans and is detected by the Platelia Aspergillus EIA (BioRad)®, a commercial kit based on the sandwich ELISA technique. It is standardized for use in neutropenic patients, however studies have demonstrated its usefulness also possible for birds. The aim of our study was to evaluate the effectiveness of Platelia Aspergillus EIA® test (BioRad-US) in the diagnosis of aspergillosis in Magellanic penguins, determining sensitivity, specificity, and positive and negative predictive values for different cut-off points. Were included in the study, blood serum samples (n = 29) Magellanic penguins in captivity that died by aspergillosis. Detection of GM was performed following manufacturer's instructions and the GM index was obtained by dividing the average value of OD of the duplicate of the clinical sample by duplicate OD of the average value of the cut-off sample provided by the kit. Through information database results were obtained for the presence of anti-Aspergillus fumigatus antibodies detected by agar gel immunodiffusion (AGID) for all serum samples. Results were analyzed using chi-square test and Kruskal-Wallis from SPSS 20.0, IBM®. ROC curve was obtained and from this, rates of sensitivity, specificity, positive and negative predictive values were also calculated based on four different cutoff points (0.5, 1.0, 1.5 and 2.0). The serum GM index did not differ between animals of the case and control group (pkw =0.097). In determining the ROC curve for serum GM detection the value of area under the curve was 0.635. From the values ​​determined by the coordinate of the curve, four different cut points (0.5, 1.0, 1.5 and 2.0) were analyzed, resulting in sensitivity rates ranging from 86.2 to 34.5% % and specificity between 87% and 26.1%. By comparing the serum GM index in group case as the presence or absence of antibodies detected by AGID was found p=0.503. The detection of GM the Platelia Aspergillus EIA® test seems is not be useful for the diagnosis of aspergillosis in naturally infected penguins.

Antifungal susceptibility profile of diferent yeasts isolates from wild animals, cow's milk with subclinical mastitis and hospital environment / Perfil de suscetibilidade antifúngica de diferentes leveduras isoladas da microbiota de animais silvestres, leite com mastite subclínica e ambiente hospitalar

Abstract Yeast infections have acquired great importance due to increasing frequency in immunocompromised patients or patients undergoing invasive diagnostic and therapeutic techniques, and also because of its high morbidity and mortality. At the same time, it has been seen an increase in the emergence of new pathogenic species difficult to diagnose and treat. The aim of this study was to determine the in vitro susceptibility of 89 yeasts from different sources against the antifungals amphotericin B, voriconazole, fluconazole and flucytosine, using the VITEK® 2 Compact system. The antifungal susceptibility was performed automatically by the Vitek® 2 Compact system. The origin of the yeasts was: Group 1 - microbiota of wild animals (W) (26/89), 2 - cow's milk with subclinical mastitis (M) (27/89) and 3 - hospital enviorment (H) (36/89). Of the 89 yeasts submitted to the Vitek® 2 test, 25 (20.9%) were resistant to fluconazole, 11 (12.36%) to amphotericin B, 3 (3.37%) to voriconazole, and no sample was resistant to flucytosine. Regarding the minimum inhibitory concentration (MIC), fluconazole showed an MIC between 1 and 64 mg/mL for the three groups, voriconazole had an MIC between 0.12 and 8 mg/mL, amphotericin B had an MIC between 0.25 and 4 mg/mL for group H and group W respectively, between 0.25 and 16 mg/mL for group M and flucytosine had an MIC equal to 1μg/mL for all groups. The yeasts isolated from the H group showed the highest resistance to fluconazole 12/89 (13.49%), followed by group W (7.87%) and group M (5.62%). The more resistant group to voriconazole was followed by the M and H groups, the W group showed no resistance to this antifungal. Group H was the least resistant (2.25%) to amphotericin.

Resumo As infecções por leveduras têm adquirido grande importância, devido ao aumento da sua frequência em pacientes imunocomprometidos ou pacientes submetidos a técnicas diagnosticas e terapêuticas agressivas, e devido sua alta morbidade e mortalidade. Paralelamente tem-se observado um incremento na aparição de novas espécies patógenas difíceis de diagnosticar e tratar. O objetivo desse estudo foi avaliar a suscetibilidade in vitro de 89 leveduras de diferentes origens frente aos antifúngicos Anfotericina B, Voriconazol, Fluconazol e Fluocitocina pelo Sistema Vitek® 2. O antifungigrama foi realizado automaticamente pelo Vitek® 2 Compact. A origem das leveduras foi: Grupo 1- Microbiota de Animais Silvestres (S) (26/89), 2- Leite com mastite bovina subclínica (L) (27/89) e 3- Ambiente Hospitalar (H) (36/89). Das 89 leveduras submetidas à carta Vitek®, 25 (20.09%) foram resistentes ao fluconazol, oito (8.99%) à anfotericina B, três (3.37%) ao voriconazol, e nenhuma amostra mostrou-se resistente a fluocitosina. O grupo três (H) foi mais resistente ao fluconazol que os demais, já o dois (L) foi mais resistente ao voriconazol e a anfotericina B que os outros dois. O fluconazol pode ter apresentado maior número de resistências devido ser um fármaco comumente usado principalmente em humanos. As leveduras isoladas de humanos apresentaram maior número de resistências aos fármacos testados do que as leveduras isoladas de animais silvestres. O que pode ocorrer devido a uma maior exposição dos humanos aos fármacos em relação aos animais que vivem isolados em ambientes selvagens e na maioria dos casos nunca teve contato com fármacos de qualquer origem.

Antifungal susceptibility profile of diferent yeasts isolates from wild animals, cow's milk with subclinical mastitis and hospital environment.

Yeast infections have acquired great importance due to increasing frequency in immunocompromised patients or patients undergoing invasive diagnostic and therapeutic techniques, and also because of its high morbidity and mortality. At the same time, it has been seen an increase in the emergence of new pathogenic species difficult to diagnose and treat. The aim of this study was to determine the in vitro susceptibility of 89 yeasts from different sources against the antifungals amphotericin B, voriconazole, fluconazole and flucytosine, using the VITEK® 2 Compact system. The antifungal susceptibility was performed automatically by the Vitek® 2 Compact system. The origin of the yeasts was: Group 1 - microbiota of wild animals (W) (26/89), 2 - cow's milk with subclinical mastitis (M) (27/89) and 3 - hospital enviorment (H) (36/89). Of the 89 yeasts submitted to the Vitek® 2 test, 25 (20.9%) were resistant to fluconazole, 11 (12.36%) to amphotericin B, 3 (3.37%) to voriconazole, and no sample was resistant to flucytosine. Regarding the minimum inhibitory concentration (MIC), fluconazole showed an MIC between 1 and 64 mg/mL for the three groups, voriconazole had an MIC between 0.12 and 8 mg/mL, amphotericin B had an MIC between 0.25 and 4 mg/mL for group H and group W respectively, between 0.25 and 16 mg/mL for group M and flucytosine had an MIC equal to 1µg/mL for all groups. The yeasts isolated from the H group showed the highest resistance to fluconazole 12/89 (13.49%), followed by group W (7.87%) and group M (5.62%). The more resistant group to voriconazole was followed by the M and H groups, the W group showed no resistance to this antifungal. Group H was the least resistant (2.25%) to amphotericin.

Effects of essential oils of Rosmarinus officinalis Linn. and Origanum vulgare Linn. from different origins on Sporothrix brasiliensis and Sporothrix schenckii complex / Efeitos dos óleos essenciais de Rosmarinus officinalis Linn. e Origanum vulgare Linn. de diferentes origens em Sporothrix brasiliensis e complexo Sporothrix schenckii

Rosmarinus officinalis L. (rosemary) and Origanum vulgare L. (oregano) are known to have antimicrobial properties, but studies on sporotrichosis are scarce. This study aimed to evaluate the anti-Sporothrix spp. activity of essential oils from commercial products and oils extracted from aerial parts of these plants and analyze their chemical constituents. S. schenckii complex and S. brasiliensis (n: 25) isolated from humans, cats, dogs, and environmental soil were tested through M27-A3 guidelines of CLSI with modification for phytotherapics. The essential oils of R. officinalis L. were similar for MIC50 and MFC50 ≤2.25mg/mL for extracted oil; and 4.5mg/mL and 9mg/mL, respectively, for commercial oil. Both products showed MIC90 of 18mg/mL and MFC90 of 36mg/mL. In O. vulgare L., the extracted oil had better activity with MIC50 and MFC50 ≤2.25mg/mL, and MIC90 and MFC90 of 4.5mg/mL, whereas the commercial oil showed MIC50 and MFC50 of 9mg/mL and MIC90 18mg/mL, respectively, and MFC90 of 36mg/mL. Through gas chromatography (CG/FID), thymol and α-terpinene were majority for extracted oil of O. vulgare L., and carvacrol and γ-terpinene made up the majority of the commercial oil. Both essential oils of R. officinalis L. showed 1,8-cineole and α-pinene as major. The fungal isolates were susceptible to all tested essential oils, including in itraconazole-resistant S. brasiliensis isolates. The extracted and commercial oils of the plants presented in vitro anti-Sporothrix spp. activity, and they are promising for treatment of sporotrichosis, including in cases refractory to itraconazole. More studies should be performed about toxicity and in vivo efficacy for its safe use.(AU)

Rosmarinus officinalis L. (alecrim) e Origanum vulgare L. (orégano) são conhecidos pelas propriedades antimicrobianas, entretanto seus estudos na esporotricose são escassos. Este trabalho objetivou avaliar a atividade anti-Sporothrix spp. de óleos extraídos e comerciais dessas plantas e analisar seus constituintes químicos. Isolados do complexo S. schenckii e S. brasiliensis (n: 25) de humanos, gatos, cães e solo, foram testados pela diretriz M27-A3 do CLSI com modificações para fitoterápicos. Os óleos de R. officinalis L. foram similares com CIM50 e CFM50 ≤2.25mg/mL para extraído; e 4.5mg/mL e 9mg/mL, respectivamente, para comercial. Ambos os produtos demonstraram CIM90 de 18mg/mL e CFM90 de 36mg/mL. Em O. vulgare L., o óleo extraído apresentou melhor atividade com CIM50 e CFM50≤2.25mg/mL e CIM90 e CFM90 de 4.5mg/mL, ao passo que o óleo comercial mostrou CIM50 e CFM50 de 9mg/mL; e CIM90 de 18mg/mL e CFM90 de 36mg/mL. Por meio da cromatografia gasosa (CG/FID), timol e α-terpineno foram majoritários para o óleo extraído de O. vulgare L., e carvacrol e γ-terpineno para o comercial. Ambos os óleos de R. officinalis L. apresentaram 1,8-cineol e α-pineno como prevalentes. Os isolados foram sensíveis a todos os óleos essenciais testados, inclusive S. brasiliensis, resistentes ao itraconazol. Os óleos extraídos e comerciais de R. officinalis L. e O. vulgare L. apresentaram atividade anti-Sporothrix spp. in vitro e são promissores para o tratamento da esporotricose, inclusive em casos refratários ao itraconazol. Mais estudos devem ser realizados sobre toxicidade e eficácia in vivo para seu uso seguro.(AU)

Blood parameters and measurements of weight in the rehabilitation of Magellanic penguins (Spheniscus magellanicus, Foster 1781) / Parâmetros sanguíneos e medidas de peso na reabilitação de pinguins-de-Magalhães (Spheniscus magellanicus, Foster 1781)

During the austral winter the appearance of the Magellanic penguin (Spheniscus magellanicus) is common on the southern coast of Brazil. The oil pollution constitutes a major cause of death of these birds. The monitoring of the weight and blood variables is important to make decisions during the rehabilitation and release of these animals. Thus, the aim of this study was to evaluate the relationship between the penguins' survival during rehabilitation and the values of hematocrit (Hct), total plasma protein (TPP) and body weight (BW) of the birds received at Centro de Recuperação de Animais Marinhos (Recovery Centre for Sea Animals) between 2006 and 2009. The analysis took place weekly, and penguins were classified according to the outcome of the rehabilitation process, when 101 specimens were rehabilitated and released, and 67 died. Released animals showed a decrease in the Hct in the second and third weeks (41±6% and 40±5% respectively) followed by recovery of the values in the fourth and fifth week (41±4% and 43±4% respectively). TPP levels increased steadily over the collections, stabilizing at 7.3±0.8g/dL in the fourth week. On the other hand, animals that died, although following the same response for TPP, showed no significant difference in Hct between collections, and showed a tendency for loss of BW in the last weeks of rehabilitation. The curves of BW and Hct during rehabilitation were considered a potential prognostic indicator of penguins in rehabilitation. Animals that arrived at the recovery centre with BW, Hct and TPP above 2.700g, 45% and 5.6 g / dL, respectively, have a greater chance of recovery, making these parameters crucial points in decision making regarding the treatment to be conducted during rehabilitation.

Durante o inverno austral, é comum o aparecimento do pinguim-de-Magalhães (Spheniscus magellanicus) no litoral sul do Brasil. A poluição por petróleo constitui a maior causa de morte dessas aves. O acompanhamento do peso e de variáveis sanguíneas é importante para a tomada de decisões durante a reabilitação e liberação desses animais. O objetivo deste estudo foi avaliar a relação entre a sobrevivência dos pinguins durante a reabilitação e os valores de hematócrito (Ht), proteínas plasmáticas totais (PPT) e peso corpóreo (PC) das aves recebidas no Centro de Recuperação de Animais Marinhos entre 2006 e 2009. As análises ocorreram semanalmente, e os pinguins foram classificados em função do desfecho do processo de reabilitação, quando 101 espécimes foram reabilitados e liberados e 67 foram a óbito. Animais liberados apresentaram queda no Ht na segunda e terceira semanas (41±6% e 40±5%, respectivamente), seguida de recuperação dos valores na quarta e quinta semanas (41±4% e 43±4%, respectivamente). Os níveis de PPT aumentaram progressivamente ao longo das coletas, estabilizando na quarta semana em 7,3±0,8g/dL. Em contrapartida, animais que foram a óbito, apesar de seguirem a mesma resposta para as PPT, não apresentaram diferença significativa do Ht, entre as coletas, e demonstraram tendência à perda de PC nas últimas semanas de reabilitação. As curvas de PC e Ht foram consideradas indicadoras potenciais de prognóstico durante a reabilitação dos pinguins. Animais que chegaram ao centro de recuperação com PC, Ht e PPT acima de 2.700g, 45% e 5,6g/dL, respectivamente, apresentaram maior chance de recuperação, fazendo desses parâmetros pontos críticos na tomada de decisão quanto ao tratamento a ser conduzido durante a reabilitação.

In vitro susceptibility of Brazilian Pythium insidiosum isolates to essential oils of some Lamiaceae family species.

The present study aimed to evaluate the in vitro antimicrobial action of Origanum vulgare, Origanum majorana, Mentha piperita and Rosmarinus officinalis on Pythium insidiosum oomycete zoospores. The antimicrobial activity evaluation was performed by the broth microdilution method according to CSLI M38-A2 documentation adapted to phytopharmaceuticals. Twenty-two P. insidiosum isolates were evaluated, and the minimum inhibitory concentration was determined at 100% growth inhibition. All P. insidiosum isolates evaluated showed a minimum inhibitory concentration ranging from 0.05 to 1.75 mg/mL when O. vulgare oil was used and from 0.11 to 3.5 mg/mL for O. majorana, M. piperita and R. officinalis oils. The results obtained indicate that the essential oils tested showed antimicrobial activity on P. insidiosum, with O. vulgare essential oil showing the best performance. These findings emphasize the potential use of plant essential oils as control agents in P. insidiosum infections; further research, however, is needed so as the in vivo activity of these oils can also be evaluated.

Fungi in the respiratory tract of horses with recurrent airway obstruction / Presença de fungos no trato respiratório de equinos e sua associação com obstrução recorrente das vias aéreas

This study aims to compare the concentration of viable fungi, especially those of the genus Aspergillus in the respiratory tract of stabled horses with and without Recurrent Airway Obstruction (RAO). Thirty two housed horses from four equestrian centers in Brazil were included in the study. These animals were submitted to clinical examination and to a respiratory sample collection. They were categorized into two groups: healthy and RAO-affected horses. Samples obtained by tracheobronchial washes were evaluated for fungal microscopy, quantitative culture and Aspergillus spp. quantification. Eighteen healthy and 14 RAO-affected horses were studied. Fungi were more frequently recovered in the RAO group, in comparison to controls, for both fungal microscopy (P<0.0001), fungal culture (P<0.0001) and Aspergillus spp. quantitative culture (p=0.001). In conclusion, horses with RAO have significantly higher fungal load in the respiratory tract in comparison to healthy horses. The implications of these findings in terms of the pathogenesis of RAO deserve additional investigation...

Este estudo objetivou comparar a concentração de fungos viáveis, especialmente do gênero Aspergillus, no trato respiratório de equinos estabulados com e sem obstrução recorrente das vias aéreas (ORVA). Trinta e dois equinos provenientes de quatro centros de treinamento equestre do Brasil foram incluídos no estudo. Os animais foram submetidos a exame clínico e coleta de amostra respiratória, sendo categorizados em dois grupos: sadios e ORVA. Os lavados traqueobrônquicos obtidos foram avaliados por exame micológico direto e cultivo quantitativo. Ao todo, 18 equinos saudáveis e 14 com ORVA foram estudados. Fungos foram mais frequentemente detectados em amostras do grupo ORVA em comparação com o grupo controle, tanto no exame micológico direto (P<0,0001) e cultivo quantitativo (P<0,0001) quanto na concentração de unidades formadoras de colônias (UFC) de Aspergillus spp. isolada em cultivo (p=0,001). Equinos com ORVA têm maior concentração de propágulos fúngicos no trato respiratório em comparação com animais sadios. As implicações desses achados na patogenia da ORVA merecem maior atenção e investigação...

Atividade antifúngica do óleo essencial de Origanum vulgare frente a Malassezia pachydermatis / Antifungal activity of Origanum vulgare essential oil against Malassezia pachydermatis

Objetivou-se com este estudo avaliar a atividade antifúngica in vitro do óleo essencial de Origanum vulgare frente a isolados clínicos de Malassezia pachydermatis. As folhas secas de O. vulgare foram adquiridas de distribuidor comercial com certificado de qualidade e origem e encaminhadas para extração do óleo essencial e cromatografia. Para realização do teste in vitro, foi utilizada a técnica de microdiluição em caldo (CLSI M27A3) com modificações para fitofármacos e M. pachydermatis. O óleo essencial de orégano foi testado nas concentrações de 28 a 0,87mg/mL diluído em caldo Sabouraud com 1% de tween 80. Todos os isolados foram testados em duplicata. Na análise cromatográfica do óleo essencial, foram identificados 12 compostos, sendo timol, a-terpineno e 4-terpineol os compostos majoritários. A CIM e a CFM dos 42 isolados de M. pachydermatis variaram de <0,87 a 7mg/mL, com valores de CIM50 e CIM90 de 1,18 e 3,28mg/mL, respectivamente. Com este estudo foi possível concluir que M. pachydermatis é sensível ao óleo essencial de orégano mesmo em concentrações baixas. Dessa maneira, o óleo essencial de orégano apresenta-se como promissor na bioprospecção de novos fármacos para o tratamento das otites e dermatites na clínica de pequenos animais...

The aim of this study was to evaluate the in vitro antifungal activity of essential oil of Origanum vulgare against clinical isolates of Malassezia pachydermatis. The dried leaves of O. vulgare were purchased from a commercial distributor with certified quality and origin and referred for essential oil extraction and chromatography. The technique for in vitro testing was microdilution (CLSI M27A3) with modifications to phytochemicals and M. pachydermatis. The essential oil of O. vulgare was tested at concentrations from 28 to 0.87mg/mL in Sabouraud broth diluted with 1% of tween 80. All isolates were tested in duplicate. In the chromatographic analysis of the essential oil 12 compounds were identified, and thymol, α-terpinene, 4-terpineol were the major compounds. The MIC and the MFC of the 42 isolates of M. pachydermatis ranged from <0.87 to 7mg/mL with MIC50 and MIC90 values of 1.18 and 3.28 mg/mL, respectively. With this study it was concluded that M. pachydermatis is sensible to O. vulgare essential oil even at low concentrations. Thus, the essential oil of O. vulgare is presented as bioprospecting in the promising new drugs for the treatment of otitis and dermatitis in small animal clinic...

In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals.

OBJECTIVES: The purpose of this study was to compare the in vitro susceptibilities of 22 Brazilian isolates of Pythium insidiosum to antifungals using a standardized inoculum of zoospores and a proposed novel inoculum prepared from cultured mycelia (hyphae) of P. insidiosum. METHODS: A zoospore suspension of P. insidiosum was obtained by the zoosporogenesis technique. The hyphal inoculum was prepared from a suspension of P. insidiosum mycelium. Susceptibility to each drug was evaluated using the CLSI M38-A2 method. RESULTS: Of the 88 MIC comparisons performed, 36 (41%) showed the same MIC value for the two inocula. The agreement (differences not greater than one dilution) between MICs obtained with both types of inocula was 39.8% (35/88). In other MIC comparisons analysed, 17 (19.3%) showed differences of two or three dilutions. CONCLUSIONS: We conclude that the use of hyphal inocula of P. insidiosum for in vitro susceptibility tests could be a suitable method for evaluating antimicrobial susceptibility, particularly when it is not possible to obtain a standardized zoospore inoculum.

Wild animals as sentinels of Paracoccidioides brasiliensis in the state of Rio Grande do Sul, Brazil.

Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20%), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.

In vitro activity of disinfectants against Aspergillus spp.

Fungi of the Aspergillus genus are widespread and contaminate the environment. Thousands of conidia are released from each phialide and dispersed in the air every day. These fungi are considered important mycose-causing agents in hospitals. Due to this, research to determine prevalent fungi from the Aspergillus genus in hospital environments, and an adequate disinfection program in these areas is are needed. This study evaluated the susceptibility of Aspergillus spp. isolated from a veterinary environment against four disinfectants. Successive dilutions of disinfectants (log2) were used according to CLSI M38-A2 microdilution technique adapted to chemical agents against 18 isolates of this genus. After 72 hours of incubation, the Minimum Inhibiting Concentration and Minimum Fungicidal Concentration capable of inhibiting 50% and 90% of the isolates were determined. Chlorexidine-cetrimine, benzalconium chloride and a chlorophenol derivative proved to be effective against all isolates with a lower MIC than that suggested by the manufacturer, except for the A. flavus strain. Sodium hypochlorite was ineffective against three A. fumigatus, three A. flavus and one A. niger isolate. These results demonstrated that all studied disinfectants were effective against environmental isolates, with the exception of sodium hypochlorite, which showed lower effectiveness.

Study of bacteria isolated from the foot pad of Spheniscus magellanicus with and without bumblefoot / Estudo de bactérias isoladas de coxins plantares de Spheniscus magellanicus com e sem bumblefoot

The bumblefoot or pododermatitis is among the diseases with the highest morbidity in Magellanic penguins, sometimes evolving to septicemia and death. Therefore, this study aimed to relate the main species involved in the disorder, as well as the in vitro susceptibility profile of the microorganisms against routine antimicrobial usage in Veterinary Medicine. During two years in vivo material was harvested from 200 footpads (n=100 animals) for microbiological analysis and in vitro susceptibility tests against the Antibiotic enrofloxacin, streptomycin, penicillin and cephalosporin. Bacteria have been identified both as part of permanent and transient microbiota, also being associated to 100% of the pododermatitis cases. The most prevalent genus were Staphylococcus and Corynebacterium. The antibiograms of all the isolated bacteria resulted in greater susceptibility of the strains facing cephalosporin, followed by enrofloxacin, streptomycin and penicillin.

O bumblefoot ou pododermatite está entre as afecções de maior morbidade em pinguins-de-magalhães, podendo evoluir para septicemia e óbito. Portanto, o presente estudo objetivou relacionar as principais espécies bacterianas envolvidas na afecção, bem como o perfil de susceptibilidade in vitro destes microrganismos frente a antimicrobianos de uso rotineiro em medicina veterinária. Durante o período de dois anos, foi realizada colheita de material in vivo de 200 coxins plantares (n=100 animais) para análise microbiológica e testes de susceptibilidade in vitro frente aos antibióticos enrofloxacina, estreptomicina, penicilina e cefalosporina. Bactérias foram identificadas tanto como parte da microbiota permanente quanto da transitória, bem como estiveram associadas a 100% dos casos de pododermatite. Os gêneros mais prevalentes foram Staphylococcus e Corynebacterium. Os antibiogramas de todas as bactérias isoladas resultaram em maior sensibilidade das cepas frente à cefalosporina, seguida de enrofloxacina, estreptomicina e penicilina.

In vitro activity of disinfectants against Aspergillus spp

Fungi of the Aspergillus genus are widespread and contaminate the environment. Thousands of conidia are released from each phialide and dispersed in the air every day. These fungi are considered important mycose-causing agents in hospitals. Due to this, research to determine prevalent fungi from the Aspergillus genus in hospital environments, and an adequate disinfection program in these areas is are needed. This study evaluated the susceptibility of Aspergillus spp. isolated from a veterinary environment against four disinfectants. Successive dilutions of disinfectants (log2) were used according to CLSI M38-A2 microdilution technique adapted to chemical agents against 18 isolates of this genus. After 72 hours of incubation, the Minimum Inhibiting Concentration and Minimum Fungicidal Concentration capable of inhibiting 50% and 90% of the isolates were determined. Chlorexidine-cetrimine, benzalconium chloride and a chlorophenol derivative proved to be effective against all isolates with a lower MIC than that suggested by the manufacturer, except for the A. flavus strain. Sodium hypochlorite was ineffective against three A. fumigatus, three A. flavus and one A. niger isolate. These results demonstrated that all studied disinfectants were effective against environmental isolates, with the exception of sodium hypochlorite, which showed lower effectiveness.

Dermatite multifatorial em um canino / Multifactorial dermatitis in a dog

Relatou-se um caso clínico de dermatite multifatorial em cão, envolvendo Demodex canis, Sporothrix schenckii e bactérias dos gêneros Staphylococcus e Streptococcus. O animal, macho, da raça Pit Bull, com um ano e três meses de idade, apresentou lesões úmidas, hiperêmicas, descamativas e ulcerativas nas regiões do pescoço e do tórax. Exames laboratoriais confirmaram o diagnóstico de infecção concomitante por fungo, bactéria e parasito. A terapia antimicrobiana com moxidectina, itraconazol e amoxicilina foi efetiva, com remissão dos sinais clínicos após 30 dias de tratamento. Este relato salienta a importância do diagnóstico de certeza, que foi determinante para estabelecimento da terapêutica adequada e recuperação do animal.

A clinical case of dermatitis multifactorial in a dog was reported, involving Demodex canis, Sporothrix schenckii, and bacteria of the genera Staphylococcus and Streptococcus. The animal, male, Pit Bull, with a year and three months of age had soaked lesions, was hyperemic, scaly and had an ulcer in the neck and chest. Laboratory tests confirmed the diagnosis of concomitant infection by fungi, bacteria and parasites. Antimicrobial therapy with moxidectin, itraconazol and amoxicillin was effective, with remission of clinical signs after 30 days of treatment. This report emphasizes the importance of an accurate diagnosis, which was crucial to establish the correct treatment and recovery of the animal.

Perfil de suscetibilidade de leveduras do gênero Candida isoladas de animais ao óleo essencial de Rosmarinus officinalis L / Susceptibility profile of Candida spp. isolated from animals to the essential oil of Rosmarinus officinalis L

Neste trabalho foram avaliadas a atividade antifúngica e composição química do óleo essencial de Rosmarinus officinalis (alecrim). O óleo essencial foi obtido, através da hidrodestilação, das partes aéreas da planta, o qual foi analisado por cromatografia gasosa com detectores de ionização em chama (GC/FID) e espectrometria de massas (GC/MS). Esse óleo foi testado em isolados de C. albicans da mucosa vaginal de fêmeas caninas, casos clínicos de candidíase em animais cepas padrões e espécies não-albicans, usando a técnica de microdiluição em caldo (CLSI-M27A2). A análise cromatográfica do óleo permitiu identificar como principais compostos cânfora, verbenona e 1,8-cineol. Os valores de CIM do óleo para os isolados padrões de C. albicans variaram entre 1,25 e 2,5 µL mL-1 e de 2,5 a 5,0 µL mL-1 para CFM, enquanto que para os isolados não-albicans observou-se CIM entre 1,25 e 5,0 µL mL-1 e CFM de 2,5 a 10,0 µL mL-1. C. albicans isolada de animais apresentou valores de 2,5 a >10,0 µL mL-1 para CIM e de 5,0 a >10,0 µL mL-1 para CFM. Os resultados demonstraram atividade fungicida e fungistática do óleo essencial de alecrim em isolados de Candida spp. provenientes de animais.

In this work the antifungal activity and chemical composition of the Rosmarinus officinalis (rosemary) essential oil against Candida spp. were evaluated. The essential oil was obtained by hydrodistillation of aerial parts, and analyzed by gas chromatography with a flame ionization detector (GC/FID) and gas chromatography coupled with mass spectrometry (GC/MS). This oil was tested against fourteen isolates of Candida sp. (ten C. albicans and four no-albicans species) by the microdillution broth assay (CLSI-M27A2). Chromatography analyses of the essential oil showed compounds, as camphor, verbenone and 1,8-cineole as major constituents. MIC and MFC values for C. albicans standard strains were 1.25 - 2.5 µL mL-1 and 2.5 - 5.0 µL mL-1, respectively. MIC and MFC for the non-albicans species were 1.25 - 5.0 µL mL-1 and 2.5 - 10.0 µL mL-1, respectively. C. albicans isolates obtained from animals exhibited MIC and MFC values of 2.5 - > 10.0 µL mL-1 and 5.0 - > 10.0 µL mL-1, respectively. According to the results, the essential oil of rosemary presented fungicidal and fungistatic activities against Candida spp.

Atividade inibitória do óleo essencial de orégano em fungos de importância médica e veterinária / Inhibitory activity of origanum essential oil against important fungus in veterinary

The in vitro activity of Origanum vulgare essential oil against fungal isolates was evaluated. A total of 27 clinical isolates were used, including: C. albicans, S. schenckii, M. pachydermatis, Aspergillus flavus, and A. fumigatus. Microdilution in broth technique (NCCLS M27-A2 and M-38) was used and susceptibility was expressed as Minimum Inhibitory Concentration (MIC). Essential oil was obtained by hydrodistillation in Clevenger and analyzed by gas chromatography, showing the presence of 4-terpineol, alpha-terpineol, 4-terpinene, thymol and carvacrol, as the main compounds. Origanum oil MIC for C. albicans varied from 125 to 500mL/mL; for S. schenckii, from 250 to 500mL/mL; for M. pachydermatis, from 15 to 30mL/mL; and for Aspergillus, from 30 to 60mL/mL. Isolates sensitivity showed to the origanum oil stimulates the accomplishment of new studies, including in vivo tests, contributing to the search of alternative treatments to mycosis.