Heat-killed Malassezia pachydermatis suspension modulates the activity of macrophages challenged with Encephalitozoon cuniculi

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging ncephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internal- ized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Micro-bicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-g secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachyder-matis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-gama and tumor necrosis factor [TNF]-a) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-a and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

Dichotomous response of Malassezia-infected macrophages to Malassezia pachydermatis and Malassezia furfur

Malassezia pachydermatis and Malassezia furfur are lipophilic yeasts of the cutaneous microbiome, although these organisms are occasionally responsible for serious invasive infections in neonates. Since phagocytosis is an important mechanism mediating the adaptive immune response, here we evaluated the phagocytosis capacity and production of nitric oxide and cytokine by macrophages after challenged with M. furfur CBS-1878 and M. pachydermatis CBS-1696. The phagocytic indexes was determined using RAW 264.7 cultivated or not with M. furfur or M. pachydermatis in the concentrations of 5:1 or 2:1 (yeasts:macrophages ratio) for 6 h, 24 h, and 48 h following the challenges. Evaluation of nitric oxide and pro- and anti-inflammatory cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, IL-17A, interferon (IFN)-gama and tumor necrosis factor [TNF]-a) by Griess method and flow cytometry, respectively, were performed in the different intervals by collecting the cell culture supernatant. Results showed a higher phagocytic index in the 5:1 ratio in 24 h for both species. Malassezia pachydermatis-infected macrophages had superior phagocytic indexes than M. furfur-infected macrophages. Phagocytosis evaluation at 48 h showed significant microorganisms proliferation and macrophages death, particularly in macrophages infected with M. pachydermatis, suggesting yeast evasion mechanism. Significant variations in the nitric oxide production were observed in macrophages infected with both species. Levels of TNF-a and IL-4 cytokines have increased in M. furfur and M. pachydermatis macrophage-infected cultures, respectively. The low microbicidal activity and the presence of pro- and anti-inflammatory cytokines reinforce the dichotomous character of the relation of these yeasts with the host, acting as a commensal in the cutaneous microbiome or causing infection.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/277%), and iss (1/274%) in one isolate. The highest resistance rates were observed with amoxicillin (22/2782%), ampicillin (21/2779%), streptomycin (18/2767%), tetracycline (11/2741%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)′-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/27-7%), and iss (1/27-4%) in one isolate. The highest resistance rates were observed with amoxicillin (22/27-82%), ampicillin (21/27-79%), streptomycin (18/27-67%), tetracycline (11/27-41%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

Is Malassezia nana the main species in horses' ear canal microbiome?

ABSTRACT The objective of this study was to characterize genotypically Malassezia spp. isolated from the external ear canal of healthy horses. Fifty-five horses, 39 (70.9%) males and 16 (29.1%) females, from different breeds and adults were studied. External ear canals were cleaned and a sterile cotton swab was introduced to collect cerumen. A total of 110 samples were cultured into Dixon medium and were incubated at 32 °C for up to 15 days. Macro- and micromorphology and phenotypic identification were performed. DNA was extracted, strains were submitted to polymerase chain reaction technique, and the products obtained were submitted to Restriction Fragment Length Polymorphism using the restriction enzymes BstCI and HhaI. Strains were sent off to genetic sequencing of the regions 26S rDNA D1/D2 and ITS1-5.8S-ITS2 rDNA. Malassezia spp. were isolated from 33/55 (60%) animals and 52/110 (47%) ear canals. No growth on Sabouraud dextrose agar was observed, confirming the lipid dependence of all strains. Polymerase chain reaction-Restriction fragment length polymorphism permitted the molecular identification of Malassezia nana - 42/52 (81%) and Malassezia slooffiae - 10/52 (19%). Sequencing confirmed RFLP identification. It was surprising that M. nana represented over 80% of the strains and no Malassezia equina was isolated in this study, differing from what was expected.

The use of singleplex and nested PCR to detect Batrachochytrium dendrobatidis in free-living frogs

Many microorganisms are able to cause diseases in amphibians, and in the past few years one of the most reported has been Batrachochytrium dendrobatidis. This fungus was first reported in Brazil in 2005; following this, other reports were made in specimens deposited in museum collections, captive and free-living frogs. The aim of this study was to compare singleplex and nested-PCR techniques to detect B. dendrobatidis in free-living and apparently healthy adult frogs from the Brazilian Atlantic Forest. The sample collection area was a protected government park, with no general entrance permitted and no management of the animals there. Swabs were taken from the skin of 107 animals without macroscopic lesions and they were maintained in ethanol p.a. Fungal DNA was extracted and identification of B. dendrobatidis was performed using singleplex and nested-PCR techniques, employing specific primers sequences. B. dendrobatidis was detected in 61/107 (57%) and 18/107 (17%) animals, respectively by nested and singleplex-PCR. Nested-PCR was statistically more sensible than the conventional for the detection of B. dendrobatidis (Chi-square = 37.1; α = 1%) and the agreement between both techniques was considered just fair (Kappa = 0.27). The high prevalence obtained confirms that these fungi occur in free-living frogs from the Brazilian Atlantic Forest with no macroscopic lesions, characterizing the state of asymptomatic carrier. We concluded that the nested-PCR technique, due to its ease of execution and reproducibility, can be recommended as one of the alternatives in epidemiological surveys to detect B. dendrobatidis in healthy free-living frog populations.

Susceptibility variation of Malassezia pachydermatis to antifungal agents according to isolate source

Malassezia pachydermatisis associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) animals to azole and polyene antifungals using the M27-A3 protocol. Isolates from G1 animals were less sensitive to amphotericin B, nystatin, fluconazole, clotrimazole and miconazole.

Aspergilose em cães - revisão / Aspergillosis in dogs - review

Aspergillus spp. são fungos ubíquos no ambiente e as espécies de maior importância na clínica veterinária são A. fumigatus, A. terreus e A. deflectus. A. terreus é mais comumente associado à aspergilose sistêmica e A. fumigatus a infecções localizadas. A ocorrência da aspergilose está ligada à queda de imunidade por diversas causas como uso de imunossupressores, corticóides e doenças debilitantes intercorrentes. Pode-se suspeitar de aspergilose com base no histórico e exame físico e a doença é confirmada através de rinoscopia, radiografia, tomografia computadorizada, cultura de fungos, histopatologia e achados sorológicos. O tratamento efetivo da doença é difícil, sendo a terapia tópica uma alternativa viável e com resultados positivos na aspergilose nasal, mas não é suficiente quando o agente atingiu os tecidos moles extra-cavidade, sendo necessária terapia sistêmica conjunta; o protocolo sistêmico requer terapia prolongada, a qual nem sempre é eficaz. Embora se disponha de diversos registros dessa doença na literatura mundial, ainda são escassas suas descrições em nosso país. A aspergilose canina é uma doença de relevada importância na clínica veterinária e o rápido diagnóstico resulta em melhor resposta à terapia e prognóstico para os animais.

Aspergillus are ubiquitous in environment and the most species involved in veterinary diseases are A. fumigatus, A. terreus and A. deflectus, A. terreus is usually related to systemic diseases, and A. fumigatus with no disseminated infections. Aspergillosis is associated with immunodeficiency, caused by immunosuppressive drugs, corticosteroids and debilitated diseases. Suspicion of aspergillosis is based on the historic and physical examination of the animal, and diagnosisis confirmed using rhinoscopy, radiography, computed tomography, culture, histopathology and sorology tests. The effective treatment of the disease is really hard; topic therapy is an alternative on nasal aspergillosis, but is not enough when the fungus has reached the soft tissues extra cavity, becoming necessary systemic therapy, which is longer and presents low efficacy. Although it has many descriptions of these diseases in available literature, in Brazil there are a few references about that. Concluding, canine aspergillosis could not forget by veterinary clinicians, because the fast diagnosis result in better response to therapy and prognosis for the animals.

Isolation of Microsporum gypseum from the haircoat of health wild felids kept in captivity in Brazil

Os dermatófitos são os fungos mais comumente associados às micoses superficiais em animais e homem. O estado de portador assintomático do gato doméstico o caracteriza como importante fonte de infecção, entretanto, até então, tal aspecto não foi pesquisado em relação a seus parentes selvagens. O objetivo desta pesquisa foi determinar a presença de dermatófitos no pelame de felídeos selvagens sadios, mantidos em cativeiro na Fundação Parque Zoológico de São Paulo. A amostragem foi constituída de 130 animais adultos de ambos os sexos, sendo 25 leões (Panthera leo), 12 tigres (Panthera tigris), 6 onças-pintadas (Panthera onca), 4 leopardos (Panthera pardus), 2 leopardos-das-neves (Panthera uncia), 2 suçuaranas (Puma concolor), 2 guepardos (Acinonyx jubatus), 1 jaguatirica (Leopardus pardalis), 28 gatos-do-mato-pequenos (Leopardus tigrinus), 10 gatos-maracajás (Leopardus wiedii), 8 gatos-do-mato-grandes (Leopardus geoffroyi), 22 gatos-mouriscos (Herpaylurus yaguaroundii) e 8 gatos-palheiros (Oncifelis colocolo). As amostras foram obtidas a partir da fricção de quadrados de carpete estéreis no pelame dos animais. Os carpetes foram então semeados em placas de Petri contendo ágar Mycobiotic (Difco®) e incubadas a 25ºC por 4 semanas. As colônias obtidas foram repicadas em tubos contendo ágar Sabouraud dextrose com cloranfenicol (100mg/L) e submetidas a microcultivo em lâmina, para posterior identificação através de suas características macro e microscópicas. Microsporum gypseum foi isolado de duas leoas (1,6 por cento) aparentemente saudáveis, ambas mantidas em terrários. Os contaminantes mais prevalentes foram dos gêneros Penicillium (27,9 por cento); Cladosporium (24,5 por cento); Acremonium (12,1 por cento); Scopulariopsis e Chrysosporium (9,8 por cento); e Aspergillus (5,3 por cento). A ocorrência de dermatófitos no pelame de felídeos selvagens sadios, mantidos em cativeiro, confirma seu estado de portador assintomático e os caracteriza como fontes de infecção par...

Benefícios advindos da interação homem-cão / Man-dog interaction and their benefits

A integração homem-animal, principalmente com o cão, tem sido descrita como benéfica, tanto para a saúde física como para a saúde mental do ser humano. Esses benefícios podem ser observados desde o momento em que se tem um animal de estimação em casa, propiciando relaxamento e fornecendo carinho à pessoa, até a zooterapia e os serviços prestados pelos cães aos deficientes físicos. Porém, dentro dessa relação, é necessário manter a saúde do animal, para que este não transmita nenhuma zoonose e também não seja prejudicado. Para isso, é necessária a avaliação do animal pelo veterinário, o qual também tem uma importância social indispensável neste contexto

Isolamento de actinomicetales aerobios do genero Nocardia de processos infecciosos dos animais domésticos / Isolation of aerobic actinomicetales genus Nocardia from infections of domestic animals

Descreveu-se o isolamento de Actinomicetales do gênero Nocardia associados a patologia veterinária, principalmente a mastite bovina. Foi isolado um total de 28 amostras; destas, 24 identificadas como Nocardia asteroides, todas provenientes de leite de vaca cocm mastite; as outras 04 amostras classificadas como Nocardia brailiensis foram isoladas de "swab" vaginal de suino, muco traqueal de bovino da raça Holsteinfriesian P. O. com comprometimento pulmonar e as outras duas do leite de vacas com mastite

Etiologia bacteriana da mastite bovina no Estado de Säo Paulo, Brasil / Bacterial etiology of bovine mastitis in the State of Säo Paulo

Exames microbiológicos em 2533 amostras de leite procedentes de 32 propriedades de exploraçäo leiteira, de 18 municípios do Estado de Säo Paulo, Brasil, sendo 11,21% de amostras negativas ao exame microbiológico. Nas amostras positivas foram isolados: Staphylococcus sp. (49,23%); Streptococcus sp. (27,08%); Corynebacterium sp. (30,67%); Enterobacteriaceae (4,26%); Pseudomonas sp. (1,06%) e 5,64% correspondente a várias bactérias (Actinobacillus sp.; Moraxella sp.; Brucella sp.; Listeria sp.; Aeromonas sp.; Kurthia sp.; Alcaligenes sp.; Lactobacillus sp.; Chromobacterium sp.; Neisseria sp.; Acinetobacter sp.; Nocardia sp.; Streptomyces sp.). Fungos filamentosos e leveduras representaram 11,68%. Predominaram os isolamentos em cultura pura, mas foram registradas culturas mistas tanto de fungos como bactérias

Microbiota fungica na pelagem de camundongos de linhagens isogênicas e congênicas mantidos em biotério de criaçäo / Fungal microbiota of isogenic and congenic strains on nice in animal holding rooms

Com o desenvolvimento da tecnologia e do manejo de animais de Laboratório, mantidos em biotérios que seguem diferentes normas de trabalho, de acordo com os objetivos a que se propöem, é mister que se avalie a microbiota dos ambientes de criaçäo, bem como dos animais que deles fazem parte. Estudou-se a freqüência de fungos na pelagem de 95 camundongos (47 machos e 48 fêmeas de Linhagem isogênicas (A/Sn (10), Balb/c (16), CBA (15), C57BL/10J (10) e congênitas (B10. A (14), B10.d2/o (15) e B 10.D2/n, com idades que variavam entre 37 55 dias. A coleta de material foi realizada pela fricçäo de um pedaço de tapete esterilizado, em toda regiäo corpórea dos camundongos, seguida de compressäo do material em placas de Petri, contendo agar Sabouraud dextrose acrescido de coranfenicol (100 microng/ ml) - ASD e Mycobiotic agar' (MA), para o isolamento dos fungos. Verificou-s, maior número de isolamentos no primeiro (224) do que na segunda (61). As freqüências de isolamento foram, respectivamente: Penicillium sp (93,68%), Trichoderma sp (92,63%), Cladosporium sp (38,95%), torulapsis sp (7,37%, Rhizapus sp (4, 21%), Aureobasidium sp (3, 16%), Absidia sp (1,05%), Asperigillus sp (1,05%), Mycellia sterilia (1,05%), Trichotecium sp (1,05%), Candida sp (1,05%) e Rhadotorula sp (1,05%). Maior número de gênero fúngicos foi isolado de Linhagens isogênicas e de camundongos do sexo feminino. Estes resultados foram estatisticamente significativos para p = 0,05 na prova dos sinais. Entre os fungos isolados nos ambientes de criaçäo incluiram-se os seguintes gêneros Absidia, Aureobasidium. Cladosporium, Cepnalosporium, Ebicoccum, Geotrichum, Mycelia sterilia, Panicillium e Trichoderma. ªNäo foram encontrados fungos considerados patogênicos para as Linhagens estudadas, tanto nos animais como nas diferentes salas de criaçäo

Dermatose por Candida albicans em cäo / Canine dermatosis by Candida albicans

Candidíase cutânea, em cadela mestiça Weimaraner de 4 anos de idade, com lesöes úmidas e pruriginosas, zonas de hiperqueratose e alopecia, localizadas na regizäo occipital e extremidade dorsal do membro anterior esquerdo. O exame do material revelou a presença de grande quantidade de células leveduriformes, näo se observando filamentos, o cultivo resultou em Candida albicans