The Influence of Landscape Structure on the Occurrence of Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in Free-Living Neotropical Primates.

PURPOSE: Habitat fragmentation is the main threat to primate survival in the world. Additionally, changes in the environments in which they live can also contribute to exposure to pathogens. To investigate some pathogens that free-living primates may be exposed to in Rio Grande do Sul State (RS; southern Brazil) and characterize the forest remnants in which they live, we investigated anti-Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. antibodies in the serum of the animals. METHODS: We analyzed 105 serum samples from 63 black howler monkeys (Alouatta caraya), 39 southern brown howler monkeys (Alouatta guariba clamitans), and 03 capuchin monkeys (Sapajus nigritus cucullatus), which were captured in forest fragments of RS. Indirect fluorescence antibody test (IFAT) and indirect hemagglutination assay (IHA) were used to detect antibodies to the agents. We then characterized the landscapes in a multiscale approach in radii from 200 to 1400 m to investigate the relationship of the presence of the agents with landscape elements. RESULTS: In the IFAT-IgG, 13.3% (14/105) of the samples were seropositive for N. caninum, 4.8% (5/105) for T. gondii, and 5.7% (6/105) for Sarcocystis spp. In the IHA-IgM/IgG, 24.8% (26/105) were seropositive for T. gondii. The metrics that best explained exposure to agents were edge and patch density, forest cover, urban cover, and average Euclidean distance to the nearest patch. CONCLUSIONS: This study indicated that the primates were exposed to the agents studied, demonstrating that some landscape features are associated with exposures to the investigated pathogens.

Experimental Pythium aphanidermatum Infection in Rabbits.

Pythium aphanidermatum is a common plant pathogen, reportedly capable of human infection. This study investigated whether P. aphanidermatum can infect domestic rabbits. Four rabbits were inoculated with P. aphanidermatum subcutaneously and evaluated for 30 days. Blood and tissue samples were collected from all animals. The animals developed palpable nodules 5-10 days after inoculation. Necropsy revealed caseous nodular lesions characterized histopathologically by pyogranulomatous inflammation containing hyphae in the necrotic areas. P. aphanidermatum was isolated from the caseous material and anti-P. aphanidermatum antibodies were detected by serological examination. This is the first report of experimental P. aphanidermatum infection in mammals. The results demonstrate that this oomycete can produce infection at the inoculation site and induce an immune response. The results suggest that P. aphanidermatum may be undergoing adaptation to vertebrate hosts.

Information technology by mobile communication for the notification of canine visceral leishmaniasis / Tecnologia da informação por comunicação móvel para notificação de leishmaniose visceral canina

Visceral leishmaniasis is the most severe form of a human and animal disease complex entitled leishmaniasis, which is endemic to 70 countries. It is imperative to develop and offer technologies capable of increasing the resolution ability of control programs of this zoonosis. In the search for technological innovations in health, especially in environmental surveillance, the objective is to develop a mobile application (App) for smartphones in order to facilitate and systematize the notification of positive cases of canine visceral leishmaniasis (CVL) by veterinarians working in clinics for assisting the municipal health surveillance in the management of this zoonosis. Thus, we developed an App, C7 LVC - Canine Visceral Leishmaniasis Notification System, with formatting based on the CR Campeiro 7® software. The technology created enables the filling of important gaps in information systems, facilitating the transmission of data and the use of this data by public management bodies to take CVL prevention and control actions.(AU)

A leishmaniose visceral é a forma mais grave de um complexo de doenças humanas e animais, denominado leishmaniose, endêmica em 70 países. É imprescindível desenvolver e oferecer tecnologias capazes de aumentar a capacidade de resolução dos programas de controle desta zoonose. Na busca por inovações tecnológicas em saúde, principalmente na vigilância ambiental, o objetivo é desenvolver um aplicativo móvel (App) para smartphones a fim de facilitar e sistematizar a notificação de casos positivos de leishmaniose visceral canina (LVC) por médicos veterinários que atuam em clínicas auxiliando a vigilância sanitária municipal, na gestão desta zoonose. Para tanto, foi desenvolvido um App, C7 LVC - Sistema de Notificação da Leishmaniose Visceral Canina, com formatação baseada no software CR Campeiro 7®. A tecnologia criada possibilita o preenchimento de lacunas importantes nos sistemas de informação, facilitando a transmissão de dados e a utilização desses dados pelos órgãos da gestão pública para a tomada de ações de prevenção e controle da LVC.(AU)

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil

ABSTRACT: Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p 0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.

RESUMO: A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene -giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p 0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.

Information technology by mobile communication for the notification of canine visceral leishmaniasis

ABSTRACT: Visceral leishmaniasis is the most severe form of a human and animal disease complex entitled leishmaniasis, which is endemic to 70 countries. It is imperative to develop and offer technologies capable of increasing the resolution ability of control programs of this zoonosis. In the search for technological innovations in health, especially in environmental surveillance, the objective is to develop a mobile application (App) for smartphones in order to facilitate and systematize the notification of positive cases of canine visceral leishmaniasis (CVL) by veterinarians working in clinics for assisting the municipal health surveillance in the management of this zoonosis. Thus, we developed an App, C7 LVC - Canine Visceral Leishmaniasis Notification System, with formatting based on the CR Campeiro 7® software. The technology created enables the filling of important gaps in information systems, facilitating the transmission of data and the use of this data by public management bodies to take CVL prevention and control actions.

RESUMO: A leishmaniose visceral é a forma mais grave de um complexo de doenças humanas e animais, denominado leishmaniose, endêmica em 70 países. É imprescindível desenvolver e oferecer tecnologias capazes de aumentar a capacidade de resolução dos programas de controle desta zoonose. Na busca por inovações tecnológicas em saúde, principalmente na vigilância ambiental, o objetivo é desenvolver um aplicativo móvel (App) para smartphones a fim de facilitar e sistematizar a notificação de casos positivos de leishmaniose visceral canina (LVC) por médicos veterinários que atuam em clínicas auxiliando a vigilância sanitária municipal, na gestão desta zoonose. Para tanto, foi desenvolvido um App, C7 LVC - Sistema de Notificação da Leishmaniose Visceral Canina, com formatação baseada no software CR Campeiro 7®. A tecnologia criada possibilita o preenchimento de lacunas importantes nos sistemas de informação, facilitando a transmissão de dados e a utilização desses dados pelos órgãos da gestão pública para a tomada de ações de prevenção e controle da LVC.

Occurrence and molecular characterization of Giardia duodenalis from naturally infected dogs in the municipality of Santa Maria, Rio Grande do Sul, Brazil / Ocorrência e caracterização molecular de Giardia duodenalis em cães naturalmente infectados no município de Santa Maria, Rio Grande do Sul, Brasil

Giardiasis is an important and prevalent zoonosis in dogs and humans caused by Giardia spp. The close relationship between pets and humans has physical, emotional and social benefits. The dogs have an important role in Giardia duodenalis cycle and transmission. This study aimed to verify the occurrence of the parasite in dogs from Central Region, in Santa Maria, Rio Grande do Sul State, Brazil, from April to October 2018. Dog feces (230) were submitted to Faust coproparasitological and molecular analyses. The positive samples in the nested-PCR (β-giardin gene) were sent for DNA sequencing and phylogenetic analyses (Neighbor-Joining). The occurrence of G. duodenalis, was 5.6% (13/230) and 4.3% (10/230) detected by coproparasitological technique and nested-PCR, respectively. There was no difference in the sensitivity of the tests used. From the faecal samples analyzed, there were no differences among the variables: diagnostic techniques, local, sex, and age of the animals (p>0.05). Only in the stool examination methodology a difference was observed between the ages (p<0.05). G. duodenalis assemblages were C and D, frequently reported in dogs. The close relationship between dogs and people may allow co-infections of circulating parasites in the population, including Giardia spp. and increasing the risk of transmission of zoonotic agents.(AU)

A giardíase é uma zoonose importante e prevalente em cães e humanos, sendo causada por Giardia spp. A estreita relação entre animais de estimação e seres humanos traz benefícios físicos, emocionais e sociais. Os cães têm um papel importante no ciclo e transmissão de Giardia duodenalis. Este estudo teve como objetivo verificar a ocorrência do parasita em cães da Região Central, em Santa Maria, RS, Brasil, de abril a outubro de 2018. As fezes de cães (230) foram submetidas a técnica coproparasitológica de Faust e análises moleculares. As amostras positivas no nested-PCR (gene β-giardin) foram enviadas para sequenciamento de DNA e posterior análise filogenética (Neighbor-Joining). A ocorrência de G. duodenalis foi de 5,6% (13/230) e 4,3% (10/230) detectados pela técnica coproparasitológica e nested-PCR, respectivamente. Não houve diferença na sensibilidade dos testes utilizados. Das amostras fecais analisadas, não houve diferenças entre as variáveis: técnicas de diagnóstico, local, sexo e idade dos animais (p>0,05). Somente na metodologia de exame de fezes observou-se diferença entre as idades (p<0,05). As assemblages de G. duodenalis encontradas foram C e D, frequentemente relatadas em cães. A estreita relação entre cães e pessoas pode permitir co-infecções de parasitas circulantes na população, incluindo Giardia spp. e aumentando o risco de transmissão de agentes zoonóticos.(AU)

Atividade acaricida de diferentes solventes sobre fêmeas ingurgitadas e larvas de Rhipicephalus microplus / Acaricidal activity of different solvents on engorged females and larvae of Rhipicephalus microplus

Os carrapatos causam perdas econômicas à indústria pecuária no Brasil. Dessa forma, o uso de fitoterápicos como acaricidas mostrou eficácia no controle desses parasitas. Neste estudo, foi avaliada a eficácia in vitro dos seguintes fármacos: dimetil sulfóxido (DMSO), etanol, metanol, propilenoglicol e monolaurato de sorbitan polietilenoglicol em diluições de 100%, 20%, 10%, 5%, 1% e 0,5%. O efeito tóxico destes solventes em fêmeas e larvas ingurgitadas de Rhipicephalus microplus foi analisado. O teste de biocarrapaticidograma, foi utilizado para definir a eficiência reprodutiva das teleóginas de R. microplus e a eficiência dos produtos testados. Para análise da susceptibilidade das larvas foi utilizado o teste por imersão de larvas em seringa modificado. O uso de propilenoglicol e DMSO em fêmeas ingurgitadas e o uso de DMSO, etanol, metanol e propilenoglicol em larvas, todos à concentração de 0,5%, apresentaram uma menor taxa de mortalidade, podendo ter seu uso indicado em baixas concentrações como solventes de acaricidas utilizados no controle do R. microplus.(AU)

Cattle ticks cause economic losses to the cattle industry in Brazil. The use of phytotherapics as acaricides has shown efficacy of the control of these parasites. In the present study, the in vitro efficacy of the following drugs was evaluated: dimethyl sulfoxide (DMSO), ethanol, methanol, propylene glycol and polyethylene glycol sorbitan monolaurate at dilutions 100%, 20%, 10%, 5%, 1% e 0.5%. The toxic effect of these solvents on engorged female and larvae of Rhipicephalus microplus was analyzed. The immersion test was used to define the reproductive efficiency of engorged R. microplus females and efficiency of the drugs. The immersion test larvae modified syringe was used to analyze of the susceptibility of tick larvae. Propylene glycol and DMSO was used on engorged females and DMSO, ethanol, methanol and propylene glycol, all at concentration of 0.5%, were use on larvae the, showed the lowest mortality rate; this indicate that its use at low concentrations as solvent acaricide in the control of R. microplus could be recommended.(AU)

Isolamento e caracterização de espécies de Pythium de ambientes aquáticos no Estado do Rio Grande do Sul e avaliação da patogenicidade em modelo experimental / Isolation and characterization of Pythium species from swampy areas in the Rio Grande do Sul, Brazil, and evaluation of pathogenicity in an experimental model

Foram coletadas 186 amostras de água de ambientes pantanosos em 13 municípios das regiões Sul, Central e Oeste do Rio Grande do Sul, Brasil, com o objetivo de isolar e caracterizar espécies de Pythium e avaliar a sua patogenicidade empregando coelhos como modelo experimental. Em 11,8% (n=22) das águas coletadas foram isoladas diferentes espécies de Pythium incluindo: P. insidiosum (n=1), P. catenulatum (n=3), P. pachycaule voucher (n=1), P. rhizo-oryzae (n=3), P. torulosum (n=4) e Pythium spp. (n=10). Zoósporos desses micro-organismos foram produzidos in vitro e inoculados por via subcutânea em coelhos, os quais foram avaliados durante 45 dias. Dentre os oomicetos testados, apenas P. insidiosum evidenciou patogenicidade, causando pitiose no modelo experimental, evidenciando que, em nossas condições, apenas esta espécie de Pythium é patógena para mamíferos.(AU)

One hundred and eighty-six water samples from swampy areas were collected in 13 municipalities of South, Central and West regions of Rio Grande do Sul, Brazil, in order to isolate and characterize Pythium species and assess their pathogenicity using rabbits as experimental model. Different Pythium species were isolated from 22 (11.8%) water samples, including P. insidiosum (n=1), P. catenulatum (n=3), P. pachycaule voucher (n=1), P. rhizo-oryzae (n=3), P. torulosum (n=4) e Pythium spp. (n=10). Zoospores of these microorganisms were produced in vitro and inoculated subcutaneously into rabbits, which were assessed over 45 days. Only P. insidiosum showed pathogenicity, causing pythiosis in the experimental model.(AU)

Detecção de proteínas imunorreativas de Rickettsia sp. cepa Mata Atlântica / Detection of immunoreactive proteins of Rickettsia sp. Atlantic Forest strain

A Febre Maculosa Brasileira (FMB) é uma doença infecciosa, transmitida por carrapatos ao homem. Uma nova riquetsiose humana foi descrita como causadora de Febre Maculosa no Estado de São Paulo, sendo denominada de Rickettsia sp. cepa Mata Atlântica. O presente trabalho teve como objetivo detectar e identificar proteínas com potencial de estimular o sistema imune de hospedeiro mamífero, desta nova cepa descrita. Para tanto, foi realizado a extração proteica total de Rickettsia sp. cepa Mata Atlântica. As proteínas extraídas foram fracionadas por eletroforese. As bandas proteicas foram transferidas para membranas de nitrocelulose por migração elétrica e submetidas à técnica de Western-blot, para detecção proteica. Ao todo sete proteínas imunorreativas foram detectadas. Duas proteínas apresentaram maior abundancia, com peso molecular, de 200 e 130 kDa respectivamente. Através da comparação de mapas proteômicos existentes e pelo peso molecular que estas proteínas apresentaram, sugere-se que as duas proteínas detectadas representem rOmpA (200 kDa) e rOmpB (130 kDa). As demais proteínas detectadas apresentaram menor ocorrência e peso molecular inferior a 78 kDa, podendo representar membros da família de antígenos de superfície celular (Sca - Surface cell antigen). As proteínas detectadas poderão servir como base de estudo na elaboração de métodos diagnósticos sensíveis e específicos, no desenvolvimento de vacinas, além de possibilitarem novos estudos para terapias mais eficazes.(AU)

Brazilian Spotted Fever (BSF) is an infectious disease transmitted by ticks to humans. A new human rickettsial infection was reported to cause spotted fever in the State of São Paulo and was named Rickettsia sp. Strain Atlantic Forest. This study aimed to detect and identify proteins with potential to stimulate the immune system of mammalian host of this new strain described. Therefore, we performed total protein extraction Rickettsia sp. Strain Atlantic Forest. The extracted proteins were fractionated by electrophoresis. The protein bands were transferred to nitrocellulose membrane by electrical migration and subjected to Western blot for protein detection. In all, seven immunoreactive proteins were detected. Two proteins showed higher abundance, with molecular weight of 200 and 130 kDa respectively. By comparing existing proteomic maps and the molecular weight of these proteins showed that, it is suggested that the two proteins detected representing rOmpA (200 kDa) and rOmpB (130 kDa). The other detected proteins had lower occurrence and molecular weight less than 78 kDa, which may represent members of the cell surface antigens Family (Sca - Surface cell antigen). The detected proteins may serve as a study based on the development of sensitive and specific diagnostic methods in the development of vaccines and they enable further studies to more effective therapies.(AU)

In vitro interaction of antifungal and antibacterial drugs against Cryptococcus neoformans var. grubii before and after capsular induction.

This study evaluated the synergistic interactions between amphotericin B (AMB) and azithromycin (AZM), daptomycin (DAP), linezolid (LNZ), minocycline (MINO), fluconazole (FLZ), flucytosine (5FC), linezolid (LZD), or tigecycline (TIG) against clinical isolates of Cryptococcus neoformans var. grubii before and after capsule induction. High synergism (>75%) was observed for the combinations, AMB+5FC, AMB+TIG, AMB+AZM, AMB+LZD and AMB+MINO but only in the strains after capsule induction. The results show that the presence of the capsule may lower the minimum inhibitory concentrations (MICs) of antifungal agents, but antimicrobial activity can be improved by combining antifungal and antibacterial agents.

Treatment of experimental pythiosis with essential oils of Origanum vulgare and Mentha piperita singly, in association and in combination with immunotherapy.

This study investigated the in vivo antimicrobial activity of the essential oils of Origanum vulgare and Mentha piperita both singly, associated and in combination with immunotherapy to treat experimental pythiosis. The disease was reproduced in 18 rabbits divided into six groups (n=3): group 1, control; group 2, treated with essential oil of Mentha piperita; group 3, treated with essential oil of Origanum vulgare; group 4, treated with commercial immunotherapic; group 5, treated with a association of oils of M. piperita and O. vulgare and group 6, treated with a combination of both oils plus immunotherapy. Essential oils were added in a topical cream base formula, and lesions were treated daily for 45 days. The animals in groups 4 and 6 received a dose of immunotherapeutic agent every 14 days. The results revealed that the evolution of lesions in groups 5 and 6 did not differ from one another but differed from the other groups. The lesions of group 5 increased 3.16 times every measurement, while those of group 6 increased 1.83 times, indicating that the smallest growth of the lesions occurred when the combination of therapies were used. A rabbit from group 5 showed clinical cure at day 20 of treatment. This research is the pioneer in the treatment of experimental pythiosis using essential oils from medicinal plants and a combination of therapies. This study demonstrated that the use of essential oils can be a viable alternative treatment to cutaneous pythiosis, particularly when used in association or combination with immunotherapy.

Synergysm of voriconazole or itraconazole with other antifungal agents against species of Fusarium

Antecedentes: Las micosis causadas por Fusarium son difíciles de tratar porque tanto in vivo como in vitro estos hongos muestran resistencia a casi todos los fármacos antimicóticos disponibles, lo que explica las altas tasas de mortalidad. Una tentativa de resolver la resistencia fúngica es combinar los fármacos antifúngicos, en especial los preparados con mecanismos de acción diferentes. Objetivos: Valorar las interacciones in vitro de la combinación de voriconazol o itraconazol con otros fármacos antimicóticos frente a 32 aislamientos de Fusarium spp: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum y Fusarium solani. Métodos: Las interacciones farmacológicas se valoraron con el método de microdilución en tablero de ajedrez, que también incluyó la determinación de la concentración inhibitoria mínima de cada fármaco por separado de acuerdo con el documento M38-A2, 2008, del Clinical and Laboratory Standards Institute. Resultados: Las mejores combinaciones fueron voriconazol + terbinafina que mostró sinergia frente al 84% de los aislamientos de Fusarium. Otras combinaciones sinérgicas fueron: voriconazol + itraconazol (50%), voriconazol + fluconazol (50%), voriconazol + miconazol (38%), voriconazol + flucitosina (22%) y voriconazol + ketoconazol (25%). Las sinergias observadas con las combinaciones de itraconazol fueron itraconazol + terbinafina (25%), e itraconazol + flucitosina (9,37%). Los antagonismos observados fueron voriconazol + fluconazol (3%) e itraconazol + flucitosina (12,5%). Conclusiones: La sinergia observada con voriconazol + terbinafina fue extraordinaria. Para dilucidar mejor la utilidad potencial de los hallazgos del presente estudio, han de realizarse nuevos estudios tanto in vivo como in vitro (AU)

Background. Infections caused by Fusarium are difficult to treat because these fungi show in vitro and in vivo resistance to practically all the antifungal agents available, which explains the high mortality rates. An attempt to overcome fungal resistance is the combination of antifungal agents, especially those with different mechanisms of action. Aims. Evaluate the in vitro interactions of combinations of voriconazole or itraconazole with other antifungal agents against 32 isolates of Fusarium spp.: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum and Fusarium solani. Methods. Drug interactions were assessed by a checkerboard microdilution method that also included the determination of the MIC of each drug alone according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2, 2008. Results. The best combinations were voriconazole + terbinafine which showed synergism against 84% of Fusarium strains. Other synergistic combinations were voriconazole + itraconazole (50%), voriconazole + fluconazole (50%), voriconazole + miconazole (38%), voriconazole + flucytosine (22%) and voriconazole + ketoconazole (25%). The synergisms observed with itraconazole combinations were itraconazole + terbinafine (25%) and itraconazole + flucytosine (9.37%). The antagonisms observed were: voriconazole + fluconazole (3%) and itraconazole + flucytosine (12.5%). Conclusions. The synergism showed by voriconazole + terbinafine was remarkable. To better elucidate the potential usefulness of our findings, new in vivo and in vitro studies deserve be performed (AU)

Canine gastrointestinal pythiosis treatment by combined antifungal and immunotherapy and review of published studies.

Pythium insidiosum is an oomycete, a fungal like microorganism, which infects mammals, causing pythiosis in animals and humans, especially in tropical and subtropical regions around the world. The treatment for this infection is very difficult, and therapeutic options commonly comprise surgery, immunotherapy and antimicrobial drugs. The present report describes the clinical healing of a dog with gastrointestinal pythiosis by treatment with a combination of antifungals and immunotherapy, as well as reviews the cases reported in the literature that used some type of therapy for canine pythiosis. A 2.5-year-old male beagle initially showed sporadic vomiting episodes, and this symptom became more frequent 5 months after the onset of clinical signs. Celiotomy procedure found thickness of the stomach wall extending to the pylorus and duodenum. A biopsy was performed, and the diagnosis of pythiosis was made by mycological, histopathological analyses and molecular identification. Therapy was based on an association of terbinafine plus itraconazole during 12 months and immunotherapy for 2.5 months. The healing of the dog reported here allows us to propose the use of immunotherapy associated with antifungal therapy to treat canine gastrointestinal pythiosis. However, additional studies should be performed on a larger number of patients to establish a standard treatment protocol for canine pythiosis.

Pitiose em ovinos nos estados de Pernambuco e Bahia / Pythiosis in sheep from Pernambuco and Bahia States, Brazil

A pitiose é uma doença infecciosa causada pelo oomiceto aquático P. insidiosum que acomete animais e o homem, especialmente habitantes de áreas úmidas. A enfermidade apresenta como característica principal a formação de lesões com aspecto granulomatoso nos hospedeiros. Neste trabalho, relatou-se a ocorrência da pitiose em ovinos nos estados de Pernambuco (PE) e Bahia (BA), Nordeste do Brasil, bem como foi avaliada a eficácia de um imunoterápico frente a esta enfermidade. Amostras de sangue de 53 ovinos foram coletadas, sendo 49 animais oriundos de propriedades localizadas em PE e quatro animais provenientes da BA. Sete ovinos demonstraram sinais clínicos de pitiose ovina. Um dos animais foi submetido à eutanásia e sua cabeça e linfonodo submandibular foram coletados e enviados para análises laboratoriais. Seis ovinos foram submetidos à imunoterapia, sendo mantidos nas instalações do setor de ovinocultura da Univasf/Petrolina-PE durante o tratamento. As técnicas de ELISA, cultura fúngica e reação em cadeia da polimerase (PCR) foram utilizadas como métodos diagnósticos da pitiose ovina, sendo eficientes para confirmação dos casos clínicos no rebanho. Ao exame microscópico do material coletado da cavidade nasal de um animal eutanasiado, observou-se uma área focalmente extensa de necrose com presença de infiltrado difuso de neutrófilos íntegros e degenerados margeando a cartilagem. Somente um animal apresentou cura clínica, indicando uma eficiência no tratamento da pitiose de 16,7% (1/6). O aumento de casos de pitiose tem sido denotado em diversos municípios de PE e da BA. Neste contexto, o emprego do imunoterápico pode ser uma alternativa a ser pesquisada. Portanto, estudos futuros devem ser realizados para investigar o efeito da imunoterapia aplicada à pitiose em ovinos.

Pythiosis is a devastating infectious disease caused by an aquatic oomycete, Pythium insidioum, and affects animals and humans that inhabit wetlands. The disease is characterized mainly by granulomatous lesions in the hosts. The purpose of this study was to report the occurrence of pythiosis in sheep in the states of Pernambuco (PE) and Bahia (BA), Northeastern Brazil, as well as to evaluate the efficacy of an immunotherapic against ovine pythiosis. Blood samples were collected from 53 sheep, 49 from flocks in counties located in PE and four from BA. Seven sheep showed clinical signs of ovine pythiosis; one of them was submitted to euthanasia and its head and submandibular lymph node was collected and sent for histopathologic and mycological analyses. Other six sheep were treated with an immunotherapic. During the treatment the animals were kept in the Sheep Industry Sector facilities at Univasf/Petrolina-PE. ELISA, fungal culture and polymerase chain reaction (PCR) methods were used to confirm the diagnosis of clinical ovine pythiosis in the sheep flock. At microscopic examination of the material collected from the nasal cavity of a sheep euthanized was observed a focally extensive area of necrosis with presence of diffuse infiltration of intact and degenerated neutrophils bordering the cartilage. Only one sheep showed clinical cure, indicating efficiency in the pythiosis treatment of 16.7% (1/6). Ovine pythiosis has been increasing in several municipalities of PE and BA. In this context, the immunotherapy may be an alternative to be searched. Therefore, further studies are needed to investigate the effect of immunotherapy on ovine pythiosis.

Synergysm of voriconazole or itraconazole with other antifungal agents against species of Fusarium.

BACKGROUND: Infections caused by Fusarium are difficult to treat because these fungi show in vitro and in vivo resistance to practically all the antifungal agents available, which explains the high mortality rates. An attempt to overcome fungal resistance is the combination of antifungal agents, especially those with different mechanisms of action. AIMS: Evaluate the in vitro interactions of combinations of voriconazole or itraconazole with other antifungal agents against 32 isolates of Fusarium spp.: Fusarium chlamydosporum, Fusarium oxysporum, Fusarium proliferatum and Fusarium solani. METHODS: Drug interactions were assessed by a checkerboard microdilution method that also included the determination of the MIC of each drug alone according to CLSI (Clinical and Laboratory Standards Institute) document M38-A2, 2008. RESULTS: The best combinations were voriconazole+terbinafine which showed synergism against 84% of Fusarium strains. Other synergistic combinations were voriconazole+itraconazole (50%), voriconazole+fluconazole (50%), voriconazole+miconazole (38%), voriconazole+flucytosine (22%) and voriconazole+ketoconazole (25%). The synergisms observed with itraconazole combinations were itraconazole+terbinafine (25%) and itraconazole+flucytosine (9.37%). The antagonisms observed were: voriconazole+fluconazole (3%) and itraconazole+flucytosine (12.5%). CONCLUSIONS: The synergism showed by voriconazole+terbinafine was remarkable. To better elucidate the potential usefulness of our findings, new in vivo and in vitro studies deserve be performed.

Pythium insidiosum: morphological and molecular identification of Brazilian isolates / Pythium insidiosum: identificação morfológica e molecular de isolados brasileiros

Pythium insidiosum is an oomycete belonging to the kingdom Stramenipila and it is the etiologic agent of pythiosis. Pythiosis is a life-threatening infectious disease characterized by the development of chronic lesions on cutaneous and subcutaneous, intestinal, and bone tissues in humans and many species of animals. The identification of P. insidiosum is important in order to implement a rapid and definitive diagnosis and an effective treatment. This study reports the identification of 54 isolates of P. insidiosum of horses, dogs and sheep that presented suspicious clinical lesions of pythiosis from different regions in Brazil, by using morphological and molecular assays. Throughout the PCR it was possible to confirm the identity of all Brazilian isolates as being P. insidiosum.

Pythium insidiosum é um oomiceto pertencente ao Reino Stramenopila e agente etiológico da pitiose, uma doença infecciosa com riscos de morte. A pitiose é caracterizada pelo desenvolvimento de lesões crônicas sobre os tecidos cutâneos, subcutâneas, intestinal e ósseo em humanos e muitas espécies de animais. A identificação de P. insidiosum é importante, a fim de se obter um diagnóstico rápido e definitivo, bem como um tratamento eficaz. Este estudo relata a identificação de 54 isolados de P. insidiosum de cavalos, cães e ovelhas que apresentavam lesões compatíveis e suspeita clínicas de pitiose, provenientes de diferentes regiões do Brasil, através de métodos morfológicos e moleculares. Através da PCR foi possível confirmar a identidade de todos os isolados brasileiros como sendo P. insidiosum.

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.

A retrospective search for bovine respiratory syncytial virus (BRSV) antigens in histological specimens by immunofluorescence and immunohistochemistry

Bovine respiratory syncytial virus (BRSV) has been only sporadically identified as a causative agent of respiratory disease in Brazil. This contrasts with frequent reports of clinical and histopathological findings suggestive of BRSV-associated disease. In order to examine a possible involvement of BRSV in cases of calf pneumonia, a retrospective search was performed for BRSV antigens in histological specimens submitted to veterinary diagnostic services from the states of Rio Grande do Sul and Minas Gerais. Ten out of 41 cases examined (24.4 percent) were positive for BRSV antigens by immunohistochemistry (IPX). Eight of these cases (19.5 percent) were also positive by indirect immunofluorescence (IFA), and 31 cases (75.6 percent) were negative in both assays. In the lungs, BRSV antigens were predominantly observed in epithelial cells of bronchioles and less frequently found in alveoli. In one case, antigens were detected only in the epithelium of the alveolar septae. The presence of antigen-positive cells was largely restricted to epithelial cells of these airways. In two cases, positive staining was also observed in cells and cellular debris in the exudate within the pulmonary airways. The clinical cases positive for BRSV antigens were observed mainly in young animals (2 to 12 month-old) from dairy herds. The main microscopic changes included bronchointerstitial pneumonia characterized by thickening of alveolar septae adjacent to airways by mononuclear cell infiltrates, and the presence of alveolar syncytial giant cells. In summary, the results demonstrate the suitability of the immunodetection of viral antigens in routinely fixed tissue specimens as a diagnostic tool for BRSV infection. Moreover, the findings provide further evidence of the importance of BRSV as a respiratory pathogen of young cattle in southeastern and southern Brazil.

O Vírus Respiratório Sincicial Bovino (BRSV) tem sido raramente identificado como agente etiológico de doença respiratória em bovinos no Brasil. Isso contrasta com freqüentes relatos clínicos e histopatológicos sugestivos de enfermidade associada ao BRSV. Com o objetivo de investigar o possível envolvimento do BRSV em casos de pneumonia em bovinos jovens, realizou-se uma pesquisa retrospectiva para antígenos do BRSV em cortes histológicos de materiais submetidos a quatro serviços de diagnóstico nos Estados do Rio Grande do Sul e Minas Gerais. Dez entre 41 casos examinados (24,4 percent) foram positivos para antígenos do BRSV por imunohistoquímica (IPX). Oito desses casos (19,5 percent) também foram positivos por imunofluorescência (IFA) e 31 casos (75,6 percent) foram negativos em ambos os testes. Nos pulmões, antígenos virais foram detectados predominantemente nas células epiteliais dos bronquíolos e menos freqüentemente nos alveólos. Em um caso, antígenos virais foram detectados exclusivamente no epitélio alveolar. A presença de células positivas restringiu-se ao epitélio dessas vias aéreas. Em dois casos, antígenos virais foram detectados em células descamativas e em restos celulares no exsudato das vias aéreas. Os casos positivos para antígenos do BRSV eram oriundos principalmente de animais jovens (2 a 12 meses de idade) de rebanhos leiteiros. As principais alterações histológicas observadas foram pneumonia bronco-intersticial caracterizada por espessamento dos septos alveolares adjacentes às vias aéreas, infiltrados de células mononucleares e presença de células gigantes multinucleadas nos alveólos. Os resultados obtidos demonstram a utilidade dos métodos de detecção de antígenos em cortes histológicos para o diagnóstico da infecção pelo BRSV. Além disso, esses resultados confirmam evidências anteriores sobre a importância do BRSV como patógeno respiratório no sudeste e sul do Brasil.

Infecção aguda e latente em ovinos inoculados com o herpesvírus bovino tipo 5 (BHV-5) / Acute and latent infection in sheep inoculated with bovine herpesvirus type-5 (BHV-5)

Infecção experimental de ovinos com o herpesvírus bovino tipo 5 (BHV-5) reproduziu vários aspectos da infecção pelo BHV-5 em bovinos. Inoculação intranasal foi seguida de extensiva replicação viral na cavidade nasal, excreção e transmissão do vírus a outros animais, estabelecimento e reativação de latência, e o desenvolvimento de meningoencefalite clínica em um animal. Ovinos inoculados com a amostra brasileira EVI-88 apresentaram hipertermia transitória, hiperemia da mucosa nasal e corrimento nasal de seroso a muco-purulento. Os animais eliminaram vírus em secreções nasais em títulos de até 107,11DICC50/ml por até 16 dias. Um cordeiro apresentou sinais clínicos de encefalite no dia 10 pós-inoculação, sendo sacrificado in extremis no início do dia 13. Infectividade foi detectada em várias regiões do encéfalo desse animal, incluindo os hemisférios anterior e posterior, córtex dorso- e ventro-lateral, ponte, pedúnculo cerebral, cerebelo e bulbo olfatório. Alterações histológicas foram observadas em várias regiões do encéfalo, principalmente no hemisfério anterior, córtex ventro-lateral e pedúnculos cerebrais, e consistiram de meningite mononuclear, manguitos perivasculares, gliose focal, necrose e inclusões intranucleares em neurônios . Quatro ovinos mantidos como sentinelas adquiriram a infecção e eliminaram vírus a partir do final do segundo dia, até 7 dias. Ovinos inoculados com a amostra argentina A663 apresentaram apenas hiperemia e umidecimento da mucosa nasal, embora eliminassem vírus nas secreções nasais por até 15 dias. Tratamento dos animais com dexametasona a partir do dia 50 pós-inoculação provocou reativação da infecção latente e eliminação viral durante até 11 dias por 76,9% (10/13) dos animais inoculados e por 100% (3/3) dos animais sentinela. Esses resultados demonstram que ovinos são susceptíveis à infecção aguda e latente pelo BHV-5 e sugerem que infecções naturais de ovinos por este vírus podem potencialmente ocorrer. Ness sentido, uma possível participação da espécie ovina como reservatório natural desse vírus deve ser melhor investigada