Suscetibilidade in vitro e in vivo de Pythium insidiosum: estudo comparativo entre acetato de caspofungina e imunoterapia em coelhos

O oomiceto aquático  Pythium insidiosum, classificado no Reino Stramenipila, é o agente etiológico da pitiose, uma doença crônica, piogranulomatosa, que acomete eqüinos, caninos, felinos, bovinos, ovinos e humanos que habitam regiões tropicais e subtropicais. Diversos protocolos para o tratamento da enfermidade têm sido utilizados, incluindo terapia com antifúngicos, cirurgia e imunoterapia. O presente estudo objetivou avaliar a suscetibilidade in vitro de 27 isolados clínicos de Pythium insidiosum ao acetato de caspofungina, bem como correlacionar os resultados obtidos in vitro com a resposta da terapêutica in vivo e comparar a eficácia de dois tratamentos, acetato de caspofungina e imunoterapia, utilizando coelhos como modelo experimental. Vinte e seis isolados de Pythium insidiosum provenientes de casos clínicos de pitiose em animais no Brasil (24 eqüinos, 01 canino e 01 ovino) e um isolado ATCC (58637) foram avaliados neste estudo. Os testes in vitro foram desenvolvidos utilizando-se a  macrotécnica em caldo seguindo o protocolo internacional M38-A do CLSI. O inóculo consistiu de uma suspensão de 2-3x103 zoósporos de Pythium insidiosum diluído 1:10 em caldo RPMI. As concentrações finais do acetato de caspofungina variaram de 0,25 – 128 ?g/mL. A leitura dos CIMs foi visual, considerando-se o crescimento ou não de hifas em 24 horas de incubação a 370C, sendo adotados 3 critérios de leitura: CIM0; CIM1 e CIM2 (100%, 90% e 50% de inibição de crescimento, respectivamente), assim como também foi determinada a concentração fungicida mínima. No ensaio in vivo, 15 coelhos inoculados subcutaneamente com 20.000 zoósporos de Pythium insidiosum foram divididos em 3 grupos de 5 animais (grupo 1, controle; grupo 2, tratado com imunoterápico Pitium Vac® e grupo 3, tratado com acetato de caspofungina). Os tratamentos iniciaram-se 25 dias após a inoculação e consitiram de: 1) 8 doses de imunoterápico administradas em intervalos de 14 dias; 2) 1 mg/kg/dia de acetato de caspofungina durante 20 dias consecutivos. Dezoito semanas após o início do experimento, os animais foram necropsiados e fragmentos de lesões foram coletados para análise histopatológica e morfométrica. Quatorze isolados (51,8%) evidenciaram CIM0 de 64 ?g/mL e 24 (88,8%) CIM1 com variação de = 8?g/mL a 64 ?g/mL. Na determinação da concentração fungicida mínima, 17 (62,9%) amostras requereram 64 ?g/mL. Os animais de ambos os tratamentos apresentaram redução da área de lesões, quando comparados aos animais do grupo controle (

Cranioesophageal Pythiosis in a Horse

Background: Pythiosis is a chronic inflammatory disease that is caused by oomycete Pythium insidiosum. This illness affects several species including humans and horses. Equine is the most affected species, having no predisposition for breed, gender, or age. It is usually shown in cutaneous and subcutaneous forms, and the lesions, which grow quickly and are hard to treat, are located mainly in the extremities. The diagnosis is made via epidemiology, clinical signs, and macroscopic and microscopic aspects of the lesion. This study describes a case of cranioesophageal pythiosis in a horse, examining the epidemiological, clinical and pathological characteristics.Case: A 12-year-old male quarter horse, weighing 515 kg was taken to the Veterinary Hospital at the University Center of Espírito Santo (UNESC). The horse had an increase in volume in the cranioesophageal region, coughing, difficulty breathing, and a runny nose. On clinical examination, the horse showed an enlargement in the submandibular and retropharyngeal lymph nodes, subcutaneous edema in the larynx region, and a temperature of 38.2ºC. According to the owner, cough was recurrent and had lasted about 12 months even after treatment with different kinds of antimicrobials. On radiographic exam, there was a marked decrease in the tracheal lumen and increased soft tissue radiopacity in the region adjacent to the narrowing. The animal was taken to surgery to remove the mass, but he died because of complications during surgery. The animal’s owner did not allow necropsy, but a fragment of the mass in the cranioesophageal was removed and sent for histological examination. The fragment was fixed in 10% formalin and processed using routine histological analysis. Macroscopically, the mass was light yellowish and ulcerated, and it measured 7.0 × 5.0 × 5.0 cm. In the middle of the ulcerated areas, there were yellow and firm granular structures that were consistent with kunkers. Histologically, extending from the tracheal adventitia to the thyroid, there was a large number of lymphocytes, macrophages, neutrophils, eosinophils, and multinucleated cells (foreign body type) and a well-defined focus of coagulative necrosis, which was surrounded by a thin border of macrophages. Within the necrotic areas, there were negative images of tubuliform hyphae. Grocott’s silver methanamine staining showed hyphae that had irregular branches, rare septa, smooth and parallel walls, and was impregnated by silver. Histological sections of the mass were subjected to immunohistochemistry. Hyphae were positive for Pythium insidiosum.Discussion: The diagnosis of pythiosis was based on macroscopic, histological findings and positive immunostaining for Pythium insidiosum. This report shows the unusual location of the disease in the horse, which made the clinical diagnosis of the disease complex. Extracutaneous forms of pythiosis in horses are less frequent than cutaneous forms. The etiopathogenesis of these forms is still unclear, but it has been suggested that previous lesions in the intestinal mucosa caused by plant material or pathogens may be predisposing factors for the appearance of the enteric form of the disease. It was not possible to observe if the animal’s other organs were affected because a necropsy could not be performed. The agent probably penetrated the esophageal epithelium and spread throughout the trachea and thyroid, but its origin cannot be determined. The radiographic findings in this study are compatible with neoplasms. However, inflammatory processes such as those caused by pythiosis should be included in the differential diagnosis of horses with swelling in the cranial portion of the esophagus

Genotyping of South American clinical isolates of Pythium insidiosum based on single nucleotide polymorphism-based multiplex PCR

We aimed to genotype the South American clinical isolates of Pythium insidiosum using the single nucleotide polymorphisms (SNP) of the ribosomal DNA sequences (rDNA). Previously, an SNP-based multiplex-PCR was able to distinguish three different clades of P. insidiosum isolates. Thus, we used this assay to evaluate South American clinical isolates of P. insidiosum (n=32), standard strains from Costa Rica (n=4), Thailand (n=3), Japan (n=1), and India (n=1), a standard strain of Pythium aphanidermatum, and Brazilian environmental isolates of Pythium torulosum, Pythium rhizo-oryzae and Pythium pachycaule voucher (n=3). It was possible to allocate each American P. insidiosum isolate to clade I, the isolates of India, Japan, and Thailand to clade II, and the Thai isolate to clade III. P. aphanidermatum, P.torulosum, P.rhizo-oryzae and P.pachycaule voucher isolates were not amplified. For the first time, a P. insidiosum isolate from Uruguay, South America, was included in molecular analyzes. By SNP-based multiplex-PCR, it was possible to perform the identification and genotyping of the South American isolates of P. insidiosum, demonstrating similar genetic characteristics of these isolates. Key words: Pythium insidiosum, Pythiosis, molecular detection, genotype, single nucleotide polimorphisms.O objetivo deste estudo foi genotipar isolados clínicos de Pythium insidiosum da América do Sul utilizando polimorfismos de nucleotídeo único (SNP) de sequências de rDNA. Anteriormente, um multiplex-PCR baseado em SNP foi capaz de distinguir P. insidiosum em três diferentes clados. Dessa forma, utilizamos este método para avaliar isolados clínicos de P. insidiosum da América do Sul (n=32), cepas padrão da Costa Rica (n=4), Tailândia (n=3), Japão (n=1) e Índia (n=1), uma cepa padrão de Pythium aphanidermatum e isolados ambientais brasileiros de Pythium torulosum; Pythium rhizo-oryzae e Pythium pachycaule voucher (n=3). Os isolados analisados foram alocados aos clados: I (americanos), II (isolados da Índia, Japão e Tailândia), e III (um isolado tailandês). P. aphanidermatum, P.torulosum, P.rhizo-oryzae e P.pachycaule voucher não foram amplificados. Pela primeira vez, um isolado de P. insidiosum do Uruguai foi incluído em análises moleculares. Através da multiplex-PCR baseada em SNP, foi possível realizar a identificação e genotipagem dos isolados sulamericanos de P. insidiosum, demonstrando características genéticas semelhantes entre esses isolados

Fungal microbiota isolated from healthy pig skin

Background:   Researches have been developed to observe the normal microbiota of different animal species. This subject is of major importance for the control of potential infection risks. Fungi can be found in various substrates, foodstuffs (cereals, meat, milk, vegetables) and also in the skin, mucosae, respiratory and gastrointestinal tracts of animals. With the dissemination of immunosuppressive diseases in swine herds over the last years, the number of concomitant diseases caused by opportunist microorganisms is gradually increasing in literature. The objective of this study was to determine the microbiota of pig skin with no apparent lesions. Materials, Methods and Results: A number of 261 pigs from 11 swine farms located in six municipalities of the State of Rio Grande do Sul, in Southern Brazil, were used for the study, in the period from April 2005 to April 2006. After being cleaned with water and 70% ethanol, skin samples were collected by friction of circular and sterile hair brushes against the posterior ventral region of the animals, on an area of no more than 10 cm. After sample collection, the brushes were wrapped with the same aluminium foil used in the sterilization process. Within the next 24 hours, the material was streaked onto agar and incubated at 25°C to 30ºC for up to four weeks. Micromorphology was used for mold identification purposes, and the process employed lactophenol cotton blue staining. Whenever an initial identification was not possible due to the absence of characteristic structures, the isolate would be picked onto Potato agar to stimulate the development of reproductive structures. Yeasts and yeast-like fungi were characterized by physiological routine assays and differential tests, such as chlamydoconidia production and germ tube tests, and also by cultivation in HiCrome Agar. Isolates that produced arthroconidia were classified into the genera Geotrichum or Trichosporon. A number of 501 isolates were obtained, of which 297 were molds and 204 yeasts. Among the molds, the hyalohyphomycetes prevailed with 211 isolates, followed by 53 pheohyphomycetes and 33 zygomycetes. Two hundred and four yeast samples were identified as Candida albicans and Trichosporon spp., in addition to other far less frequent species, such as C. glabrata. Discussion: The varied range of species isolated from the skin of pigs in this study can be explained by a number of factors, such as type of management, swine farm installations and environmental variations. The diversity of the microbiota found in relation to other studies demonstrates the necessity of this kind of research, because knowledge of the prevailing microbiota in a determined region facilitates the evaluation of potential impacts of sporadic or emerging new fungal diseases in herds, particularly in immunosuppressed animals. The observation of 17 Scopulariopsis brevicaulis isolates is worth pointing out, as its presence, associated with environmental and host factors, may favor the infection of pigs and the clinical development of the Dermatitis in the species. Knowledge of the diversity of mycological agents that are in direct contact with healthy animals may assist the diagnosis of exotic etiologies, particularly in animals with immunosuppressive diseases, considering that these are being diagnosed in swine with an increasing frequency

Soroprevalência da infecção por pythium insidiosum em equinos no estado do Rio Grande do Sul

An epidemiological survey was carried out by performing an Enzyme Linked Immuno Sorbent Assay (ELISA) test to determine the seroprevalence of Pythium insidiosum infection in equine in Rio Grande do Sul State (RS), Brazil. The serological study covered seven geographical regions of RS, classified according to the Instituto Brasileiro de Geografia e Estatística (IBGE). The samples were obtained from official veterinary service (Serviço Veterinário Oficial, SVO) linked to the Secretaria da Agricultura, Pecuária e Agronegócio of RS (SEAPA-RS) to proceed the investigation of equine infectious anemia in 2014. Samples were collected during the months of September and October of 2013, covering the seven geographical regions of RS, and totalized 1,002 serum samples. The seroprevalence for P. insidiosum in RS was 11.1% (CI95% 9.23 to 13.22). The relative risk (RR) of the presence of antibodies anti-P. insidiosum was in the regions Southeast 11.17 (CI95%, 4.65 to 26.8), Porto Alegre 4.62 (CI95%, 1.70 to 12.55), Southwest 11.17 (CI95%, 4.65 to 26.8) and Northwestern 3.72 (CI95%, 1.52 to 9.09). The highest prevalence (69.1%) was observed in females with RR of 1.59 (CI95%, 1.11 to 2.27). When the presence of dams was evaluated, the seropositivity was evident in 74.4%, presenting an association of 2.13 (CI95%, 1.16 to 3.91) compared to farms without dams. In properties with veterinary assistance, the frequency of 72.7% and RR of 3.04 (CI95%,, 1,85 to 4,98) of seropositivity were observed. Due to the importance of pythiosis in horse herds, this study highlights the presence of anti-P. insidiosum antibodies in horses in RS, Brazil.Um levantamento soroepidemiológico foi realizado através do teste de ELISA indireto para determinar a soroprevalência da infecção por Pythium insidiosum em equinos no estado do Rio Grande do Sul (RS), classificadas de acordo com o Instituto Brasileiro de Geografia e Estatística (IBGE). As amostras utilizadas eram provenientes do cadastro das propriedades do Serviço Veterinário Oficial (SVO), da Secretaria da Agricultura, Pecuária e Agronegócio do RS (SEAPA-RS), coletadas para o inquérito da anemia infecciosa equina de 2014. As coletas foram realizadas durante os meses de setembro e outubro de 2013, abrangendo as sete mesorregiões geográficas do RS, e totalizaram 1.002 amostras de soro. Do total das amostras testadas, 11.1% (CI95% 9.23 to 13.22) foram soropositivas para P. insidiosum. Constatou-se o risco relativo (RR) da presença de anticorpos anti-P. insidiosum nas regiões Sudeste 11,17(IC95%, 4,65- 26,8), Porto Alegre 4,62 (IC95%, 1,70-12,55), Sudoeste 11,17 (IC95%, 4,65-26,8) e Noroeste 3,72 (IC95%, 1,52-9,09). Observou-se a maior soroprevalência (69,1%) em fêmeas com RR de 1,59 (IC95%, 1,11- 2,27). Quanto à presença de açudes, evidenciou-se soropositividade em 74,4% das propriedades, apresentando associação de 2,13 (IC95%,1,16-3,91) em comparação com propriedades sem açude. Em propriedades com assistência veterinária, foi verificada a frequência de 72,7% e RR de 3,04 (IC95%,1,85-4,98). Diante da relevância da pitiose em rebanhos equinos, destaca-se a presença de anticorpos anti-P. insidiosum em equinos no estado do RS

Diagnóstico imuno-histoquímico de pitiose cutânea em equinos

Descrevem-se cinco casos de pitiose cutânea em equinos enviados ao Laboratório de Patologia Veterinária da Universidade Federal do Rio Grande do Sul, Brasil. Microscopicamente focos necróticos circundados por infiltrados de eosinófilos, neutrófilos, macrófagos e abundante tecido conjuntivo fibroso foi observado. Filamentos de hifas esparsamente septadas foram identificadas no centro das áreas necróticas. Na coloração de prata metenamina de Grocott hifas septadas, ramificadas foram também observadas no interior dos focos de necrose. O diagnóstico de pitiose foi confirmado em todos os casos através do teste de imuno-histoquímica em cortes de tecido usando um anticorpo policlonal anti- Pythium insidiosum. Imuno-histoquímica em tecidos fixados em formalina podem ser úteis no diagnóstico de pitiose quando o material submetido não pode ser mais utilizado para cultivo