Clinical and pathological findings of Rhodococcus equi infection in foals

ABSTRACT: Infection by Rhodococcus equi is considered one of the major health concerns for foals worldwide. In order to better understand the diseases clinical and pathological features, we studied twenty cases of natural infection by R. equi in foals. These cases are characterized according to their clinical and pathological findings and immunohistochemical aspects. Necropsy, histologic examination, bacterial culture, R. equi and Pneumocystis spp. immunohistochemistry were performed. The foals had a mean age of 60 days and presented respiratory signs (11/20), hyperthermia (10/20), articular swelling (6/20), prostration (4/20), locomotor impairment (3/20) and diarrhea (3/20), among others. The main lesions were of pyogranulomatous pneumonia, seen in 19 foals, accompanied or not by pyogranulomatous lymphadenitis (10/20) and pyogranulomatous and ulcerative enterocolitis (5/20). Pyogranulomatous osteomyelitis was seen in 3 foals, one of which did not have pulmonary involvement. There was lymphoplasmacytic (4/20), lymphoplasmacytic and neutrophilic (1/20) or pyogranulomatous arthritis (1/20), affecting multiple or singular joints. Immunohistochemistry revealed to be a valuable tool for the detection of R. equi, confirming the diagnosis in all cases. Furthermore, pulmonary immunostaining for Pneumocystis spp. demonstrates that a coinfection with R. equi and this fungal agent is a common event in foals, seen in 13 cases.

RESUMO: Infecção por Rhodococcus equi é considerado um dos maiores problemas sanitários para potros em todo o mundo. Para melhor compreender a apresentação clínica e patológica da enfermidade, foram avaliados vinte casos de infecção natural por R. equi em potros. Os casos são caracterizados de acordo com seus achados clínicos e patológicos e aspectos imuno-histoquímicos. Foram realizados exames de necropsia, histologia, bacteriologia e imuno-histoquímica para R. equi e Pneumocystis spp. Os potros tinham idade media de 60 dias e apresentaram sinais respiratórios (11/20), hipertermia (10/20), aumento de volume articular (6/20), prostração (4/20), distúrbios locomotores (3/20) e diarreia (3/20), entre outros. As lesões mais importantes eram pneumonia piogranulomatosa, vista em 19 potros, acompanhada ou não por linfadenite piogranulomatosa (10/20) e enterocolite ulcerativa (5/20). Osteomielite piogranulomatosa foi constatada em três potros, um dos quais não apresentava envolvimento pulmonar. Artrites afetando uma ou múltiplas articulações eram caracterizadas por infiltrado linfoplasmocítico (4/20), linfoplasmocítico e neutrofílico (1/20) e piogranulomatoso (1/20). A imuno-histoquímica demonstrou ser uma ferramenta valiosa na detecção de R. equi, permitindo confirmar o diagnóstico em todos os casos avaliados. Além disso, a imuno-histoquímica para Pneumocystis spp. demonstra que a coinfecção por R. equi e o agente fúngico é um evento frequente em potros, constatado em 13 casos.

Clinical and pathological findings of Rhodococcus equi infection in foals / Achados clínicos e patológicos da infecção por Rhodococcus equi em potros

Infection by Rhodococcus equi is considered one of the major health concerns for foals worldwide. In order to better understand the disease's clinical and pathological features, we studied twenty cases of natural infection by R. equi in foals. These cases are characterized according to their clinical and pathological findings and immunohistochemical aspects. Necropsy, histologic examination, bacterial culture, R. equi and Pneumocystis spp. immunohistochemistry were performed. The foals had a mean age of 60 days and presented respiratory signs (11/20), hyperthermia (10/20), articular swelling (6/20), prostration (4/20), locomotor impairment (3/20) and diarrhea (3/20), among others. The main lesions were of pyogranulomatous pneumonia, seen in 19 foals, accompanied or not by pyogranulomatous lymphadenitis (10/20) and pyogranulomatous and ulcerative enterocolitis (5/20). Pyogranulomatous osteomyelitis was seen in 3 foals, one of which did not have pulmonary involvement. There was lymphoplasmacytic (4/20), lymphoplasmacytic and neutrophilic (1/20) or pyogranulomatous arthritis (1/20), affecting multiple or singular joints. Immunohistochemistry revealed to be a valuable tool for the detection of R. equi, confirming the diagnosis in all cases. Furthermore, pulmonary immunostaining for Pneumocystis spp. demonstrates that a coinfection with R. equi and this fungal agent is a common event in foals, seen in 13 cases.(AU)

Infecção por Rhodococcus equi é considerado um dos maiores problemas sanitários para potros em todo o mundo. Para melhor compreender a apresentação clínica e patológica da enfermidade, foram avaliados vinte casos de infecção natural por R. equi em potros. Os casos são caracterizados de acordo com seus achados clínicos e patológicos e aspectos imuno-histoquímicos. Foram realizados exames de necropsia, histologia, bacteriologia e imuno-histoquímica para R. equi e Pneumocystis spp. Os potros tinham idade media de 60 dias e apresentaram sinais respiratórios (11/20), hipertermia (10/20), aumento de volume articular (6/20), prostração (4/20), distúrbios locomotores (3/20) e diarreia (3/20), entre outros. As lesões mais importantes eram pneumonia piogranulomatosa, vista em 19 potros, acompanhada ou não por linfadenite piogranulomatosa (10/20) e enterocolite ulcerativa (5/20). Osteomielite piogranulomatosa foi constatada em três potros, um dos quais não apresentava envolvimento pulmonar. Artrites afetando uma ou múltiplas articulações eram caracterizadas por infiltrado linfoplasmocítico (4/20), linfoplasmocítico e neutrofílico (1/20) e piogranulomatoso (1/20). A imuno-histoquímica demonstrou ser uma ferramenta valiosa na detecção de R. equi, permitindo confirmar o diagnóstico em todos os casos avaliados. Além disso, a imuno-histoquímica para Pneumocystis spp. demonstra que a coinfecção por R. equi e o agente fúngico é um evento frequente em potros, constatado em 13 casos.(AU)

Clinical and pathological findings of Rhodococcus equi infection in foals / Achados clínicos e patológicos da infecção por Rhodococcus equi em potros

Infection by Rhodococcus equi is considered one of the major health concerns for foals worldwide. In order to better understand the disease's clinical and pathological features, we studied twenty cases of natural infection by R. equi in foals. These cases are characterized according to their clinical and pathological findings and immunohistochemical aspects. Necropsy, histologic examination, bacterial culture, R. equi and Pneumocystis spp. immunohistochemistry were performed. The foals had a mean age of 60 days and presented respiratory signs (11/20), hyperthermia (10/20), articular swelling (6/20), prostration (4/20), locomotor impairment (3/20) and diarrhea (3/20), among others. The main lesions were of pyogranulomatous pneumonia, seen in 19 foals, accompanied or not by pyogranulomatous lymphadenitis (10/20) and pyogranulomatous and ulcerative enterocolitis (5/20). Pyogranulomatous osteomyelitis was seen in 3 foals, one of which did not have pulmonary involvement. There was lymphoplasmacytic (4/20), lymphoplasmacytic and neutrophilic (1/20) or pyogranulomatous arthritis (1/20), affecting multiple or singular joints. Immunohistochemistry revealed to be a valuable tool for the detection of R. equi, confirming the diagnosis in all cases. Furthermore, pulmonary immunostaining for Pneumocystis spp. demonstrates that a coinfection with R. equi and this fungal agent is a common event in foals, seen in 13 cases.(AU)

Infecção por Rhodococcus equi é considerado um dos maiores problemas sanitários para potros em todo o mundo. Para melhor compreender a apresentação clínica e patológica da enfermidade, foram avaliados vinte casos de infecção natural por R. equi em potros. Os casos são caracterizados de acordo com seus achados clínicos e patológicos e aspectos imuno-histoquímicos. Foram realizados exames de necropsia, histologia, bacteriologia e imuno-histoquímica para R. equi e Pneumocystis spp. Os potros tinham idade media de 60 dias e apresentaram sinais respiratórios (11/20), hipertermia (10/20), aumento de volume articular (6/20), prostração (4/20), distúrbios locomotores (3/20) e diarreia (3/20), entre outros. As lesões mais importantes eram pneumonia piogranulomatosa, vista em 19 potros, acompanhada ou não por linfadenite piogranulomatosa (10/20) e enterocolite ulcerativa (5/20). Osteomielite piogranulomatosa foi constatada em três potros, um dos quais não apresentava envolvimento pulmonar. Artrites afetando uma ou múltiplas articulações eram caracterizadas por infiltrado linfoplasmocítico (4/20), linfoplasmocítico e neutrofílico (1/20) e piogranulomatoso (1/20). A imuno-histoquímica demonstrou ser uma ferramenta valiosa na detecção de R. equi, permitindo confirmar o diagnóstico em todos os casos avaliados. Além disso, a imuno-histoquímica para Pneumocystis spp. demonstra que a coinfecção por R. equi e o agente fúngico é um evento frequente em potros, constatado em 13 casos.(AU)

Employment of the 18s rRNA screening PCR technique in the detection of Equine Piroplasmosis, in horses of sports and military operations, of the Brazilian Army / Emprego da técnica de PCR para triagem da região do genoma 18s rRNA, detecção de Piroplasmose em equinos de desporto e operações militares do Exército Brasileiro

The present work had the objective of detecting the occurrence of Equine Piroplasmosis in horses housed in the 3rd Guards Cavalry Regiment (GCR) - Brazilian Army (BA) ˗ Porto Alegre, RS-Brazil, as well as to demonstrate the proactivity of PCR (Polymerase Chain Reaction) technique, aiming at the judicious use of the resources involved in the training and employment of Equines in the Brazilian Army. Fifty horses of the 3rd GCR - Porto Alegre ˗ RS, which are employed for Sport, Military Ceremonial, Law and Order Guarantee Operations (LOGO), were evaluated by means of the 18s r RNA screening with PCR technique, thirty eight horses with Babesia Caballi and Theileria Equi were detected, which corresponds to an incidence of 76% of the horses effective analyzed at the time. In this way, it can be verified that the Military activity have its "performance and effectiveness" factors threatened in case the health of the principal of his means employed, that is the horse, is compromised. The PCR technique then offers a reliable and feasible tool for the detection of Equine Piroplasmosis in BA horses.(AU)

O presente trabalho teve como objetivo detectar a ocorrência de Piroplasmose equina em cavalos alojados no 3º Regimento de Cavalaria de Guarda (RCG) - Exército Brasileiro (EB) - Porto Alegre, RS, Brasil, bem como demonstrar a forma proativa do método da PCR (reação em cadeia de polimerase), objetivando o uso criterioso dos recursos envolvidos no treinamento e emprego de equinos no Exército Brasileiro. Foram avaliados 50 cavalos da 3ª GCR-Porto Alegre, RS, empregados nas modalidades de: esporte, cerimonial militar e operações de garantia da lei e da ordem (GLO), por meio da triagem da região do genoma 18S rRNA mediante a aplicação do método da PCR. Foram positivas as amostras de 38 equinos para Babesia caballi e Theileria Equi, o que corresponde a uma incidência de 76% dos cavalos efetivos analisados na época. Dessa forma, verifica-se que as atividades militares tem seus fatores de "desempenho e efetividade" ameaçados no caso da saúde do principal de seus meios empregados, o Cavalo, estar comprometida. A técnica de PCR, então, oferece uma ferramenta confiável e viável para a detecção de Piroplasmose em equinos do EB.(AU)

Employment of the 18s rRNA screening PCR technique in the detection of Equine Piroplasmosis, in horses of sports and military operations, of the Brazilian Army / Emprego da técnica de PCR para triagem da região do genoma 18s rRNA, detecção de Piroplasmose em equinos de desporto e operações militares do Exército Brasileiro

The present work had the objective of detecting the occurrence of Equine Piroplasmosis in horses housed in the 3rd Guards Cavalry Regiment (GCR) - Brazilian Army (BA) ˗ Porto Alegre, RS-Brazil, as well as to demonstrate the proactivity of PCR (Polymerase Chain Reaction) technique, aiming at the judicious use of the resources involved in the training and employment of Equines in the Brazilian Army. Fifty horses of the 3rd GCR - Porto Alegre ˗ RS, which are employed for Sport, Military Ceremonial, Law and Order Guarantee Operations (LOGO), were evaluated by means of the 18s r RNA screening with PCR technique, thirty eight horses with Babesia Caballi and Theileria Equi were detected, which corresponds to an incidence of 76% of the horses effective analyzed at the time. In this way, it can be verified that the Military activity have its "performance and effectiveness" factors threatened in case the health of the principal of his means employed, that is the horse, is compromised. The PCR technique then offers a reliable and feasible tool for the detection of Equine Piroplasmosis in BA horses.(AU)

O presente trabalho teve como objetivo detectar a ocorrência de Piroplasmose equina em cavalos alojados no 3º Regimento de Cavalaria de Guarda (RCG) - Exército Brasileiro (EB) - Porto Alegre, RS, Brasil, bem como demonstrar a forma proativa do método da PCR (reação em cadeia de polimerase), objetivando o uso criterioso dos recursos envolvidos no treinamento e emprego de equinos no Exército Brasileiro. Foram avaliados 50 cavalos da 3ª GCR-Porto Alegre, RS, empregados nas modalidades de: esporte, cerimonial militar e operações de garantia da lei e da ordem (GLO), por meio da triagem da região do genoma 18S rRNA mediante a aplicação do método da PCR. Foram positivas as amostras de 38 equinos para Babesia caballi e Theileria Equi, o que corresponde a uma incidência de 76% dos cavalos efetivos analisados na época. Dessa forma, verifica-se que as atividades militares tem seus fatores de "desempenho e efetividade" ameaçados no caso da saúde do principal de seus meios empregados, o Cavalo, estar comprometida. A técnica de PCR, então, oferece uma ferramenta confiável e viável para a detecção de Piroplasmose em equinos do EB.(AU)

Biofilm formation by Rhodococcus equi and putative association with macrolide resistance / Formação de biofilme por Rhodococcus equi e possível associação com resistência a macrolídeos

Rhodococcus equi is a facultative intracellular pathogen, which cause severe pyogranulomatous pneumonia in foals and tuberculosis-like lesions in humans. Its ability to form biofilm was described in strains isolated from chronic diseases associated to treatment failures in humans. This study aimed to verify the biofilm formation by 113 R. equi isolated from equine samples (clinical and fecal) using two different methods (biofilm-culturing with and without additional glucose and epifluorescence microscopy). We also aimed to determine the efficacy of azithromycin, clarithromycin and erythromycin on R. equi in established biofilm. We found 80.5% (26/41) and 63% (58/72) biofilm-positive isolates, in fecal and clinical samples, respectively. The additional glucose increased the biofilm formation by R. equi fecal samples, but not by clinical samples. The antimicrobials tested herein were not able to eradicate R. equi in biofilm even at higher concentrations. This is the first study showing the biofilm formation by R. equi isolated from equine samples. Our findings indicate that R. equi biofilm-producers may be more resistant to the antimicrobials evaluated. Further studies are warranted to test this hypothesis.(AU)

Rhodococcus equi é um patógeno intracelular facultativo, o qual causa pneumonia piogranulosa severa em potros e lesões semelhantes à tuberculose em humanos. A sua capacidade de formar biofilme foi descrita em cepas humanas, isoladas a partir de doenças crônicas associadas a falhas de tratamento. Este estudo teve como objetivo verificar a formação de biofilme por 113 cepas de R. equi, isoladas a partir de amostras de equinos (clínicas e fecais), utilizando-se dois diferentes métodos (biofilme em cultura - com e sem adição de glicose - e microscopia de epifluorescência). Além disso, buscou-se determinar a eficácia da azitromicina, claritromicina e eritromicina sobre biofilme consolidado de R. equi. Verificou-se 80,5% (26/41) e 63% dos isolados (58/72) positivos para formação de biofilme, em amostras fecais e clínicas, respectivamente. A adição de glicose amentou a formação de biofilme em amostras fecais, mas não em amostras clínicas. Os antimicrobianos aqui testados não foram capazes de erradicar R. equi em biofilme consolidado, mesmo em concentrações elevadas. Este é o primeiro estudo a demonstrar a formação de biofilme por cepas de R. equi isoladas a partir de amostras de equinos. Os resultados indicam que os isolados de R. equi produtores de biofilme podem ser mais resistentes aos antimicrobianos avaliados. Estudos adicionais são necessários para testar essa hipótese.(AU)

Fatal Rhodococcus equi infection in goats in Southern Brazil / Infecção fatal por Rhodococcus equi em caprinos no Sul do Brasil

Background: Rhodococcus equi is a Gram-positive, aerobic, cosmopolitan coccobacillus present in the soil and in feces of birds and a large variety of domestic mammals. It is the etiologic agent that commonly causes pneumonia in foals. This case report describes the clinical signs, lesions and the microbiological analysis of samples collected from three goats infected with Rhodococcus equi in a farm in southern Brazil.Cases: Three Boer goats died one week after presenting clinical signs that included: diarrhea, ruminal atony and anorexia (Goat 1); abortion, progressive weight loss, appetite loss, pale mucosas and tachycardia (Goat 2); and submandibular edema and anemia (Goat 3). Upon necropsy, it was shown that all goats presented multiple liver nodes between 0.5 and 3.0 cm in diameter. When sectioned these nodes presented yellowish contents that were not resistant. Additionally, Goat 2 presented nodes with caseous contents in mesenteric lymph nodes and lungs, which were similar to those observed in the liver. Upon necropsy, samples of organs were collected and fixed in 10% formaldehyde for histopathological analyses and kept under refrigeration for microbiological analyses. Histopathological analyses of liver, lung and lymph node of all goats revealed the presence of multifocal necrotic areas associated with the deposition of basophilic granular material (mineralization)...(AU)

Rhodococcus equi é um cocobacilo Gram- -positivo, aeróbico, cosmopolita, presente no solo [7] e nas fezes de pássaros e de uma grande variedade de mamíferos domésticos [5]. A infecção por R. equi comumente relaciona-se aos diagnósticos de broncopneumonia supurativa em potros, associada ou não a colite ulcerativa [2], peritonite, pleurite, pericardite e artrite [8]. Em caprinos a bactéria foi isolada de abscessos pulmonares e hepáticos [5] e, além disso, existem relatos de infecção determinando abscessos em tecido ósseo, costelas e vertebras cervical e torácica [4]. Este relato descreve os sinais clínicos, os aspectos anatomopatológicos e análise microbiológica da infecção por Rhodococcus equi em caprinos no Sul do Brasil...(AU)

Fatal Rhodococcus equi infection in goats in Southern Brazil / Infecção fatal por Rhodococcus equi em caprinos no Sul do Brasil

Background: Rhodococcus equi is a Gram-positive, aerobic, cosmopolitan coccobacillus present in the soil and in feces of birds and a large variety of domestic mammals. It is the etiologic agent that commonly causes pneumonia in foals. This case report describes the clinical signs, lesions and the microbiological analysis of samples collected from three goats infected with Rhodococcus equi in a farm in southern Brazil.Cases: Three Boer goats died one week after presenting clinical signs that included: diarrhea, ruminal atony and anorexia (Goat 1); abortion, progressive weight loss, appetite loss, pale mucosas and tachycardia (Goat 2); and submandibular edema and anemia (Goat 3). Upon necropsy, it was shown that all goats presented multiple liver nodes between 0.5 and 3.0 cm in diameter. When sectioned these nodes presented yellowish contents that were not resistant. Additionally, Goat 2 presented nodes with caseous contents in mesenteric lymph nodes and lungs, which were similar to those observed in the liver. Upon necropsy, samples of organs were collected and fixed in 10% formaldehyde for histopathological analyses and kept under refrigeration for microbiological analyses. Histopathological analyses of liver, lung and lymph node of all goats revealed the presence of multifocal necrotic areas associated with the deposition of basophilic granular material (mineralization)...

Rhodococcus equi é um cocobacilo Gram- -positivo, aeróbico, cosmopolita, presente no solo [7] e nas fezes de pássaros e de uma grande variedade de mamíferos domésticos [5]. A infecção por R. equi comumente relaciona-se aos diagnósticos de broncopneumonia supurativa em potros, associada ou não a colite ulcerativa [2], peritonite, pleurite, pericardite e artrite [8]. Em caprinos a bactéria foi isolada de abscessos pulmonares e hepáticos [5] e, além disso, existem relatos de infecção determinando abscessos em tecido ósseo, costelas e vertebras cervical e torácica [4]. Este relato descreve os sinais clínicos, os aspectos anatomopatológicos e análise microbiológica da infecção por Rhodococcus equi em caprinos no Sul do Brasil...

Susceptibility profile of Brazilian Rhodococcus equi isolates against azithromycin, clarithromycin and erythromycin / Perfil de susceptibilidade de isolados brasileiros de Rhodococcus equi frente à azitromicina, claritromicina e eritromicina

Rhodococcus equi infection treatment is usually a macrolide (azithromycin - AZM, clarithromycin - CLR and erythromycin - ERY) and rifampicin combination. However, resistance cases have been reported, especially for ERY. In view of the need of a study about Brazilian isolates susceptibility profile, this study aimed to characterize the minimum inhibitory concentration (MIC) of the macrolides - AZM, CLR and ERY - against 44 R. equi isolates. It was found all isolates CLR and AZM sensitive; however, for ERY, 27% (12/44) were classified as intermediate sensitivity. R. equi Brazilian isolates used here showed a large susceptibility profile, except against ERY, for which it was observed some resistance evidence. In order to avoid failures in the equine rhodococcosis therapy it was highlighted the importance of microbiological culture and antimicrobial susceptibility testing in vitro before beginning treatment.(AU)

No tratamento de infecções por R. equi, usualmente emprega-se uma combinação de macrolídeos (azitromicina - AZI, claritromicina - CLARI e eritromicina - ERI) e rifampicina. Todavia, casos de resistência frente a esses antimicrobianos vêm sendo reportados, especialmente à ERI. Tendo em vista a necessidade de um estudo sobre o perfil de susceptibilidade de isolados brasileiros, buscou-se verificar a concentração inibitória mínima (CIM) de AZI, CLARI e ERI frente a 44 isolados de R. equi de diferentes origens. Todos os isolados analisados demonstraram perfil de sensibilidade à AZI e CLARI; em contraste, frente à ERI, 27% (12/44) apresentaram sensibilidade intermediária. A fim de evitar falhas no tratamento da rodococcse equina, destaca-se a importância da cultura microbiológica e a realização de testes in vitro de susceptibilidade antimicrobiana de R .equi antes do início do tratamento.(AU)

Atividade de nanoformulações de Melaleuca alternifolia e terpinen-4-ol em isolados de Rhodococcus equi / Melaleuca alternifolia activity in nanoformulations and terpinen-4-ol against Rhodococcus equi isolates

Rhodococcus equi é o agente etiológico da rodococose equina, importante doença respiratória de potros. Especialmente na última década, a emergência de cepas resistentes aos antimicrobianos empregados no tratamento da rodococose tem sido relatada. Nesse sentido, há a necessidade de estudos envolvendo terapias alternativas e novas tecnologias, incluindo o uso de plantas medicinais e nanotecnologia. Neste trabalho utilizou-se Melaleuca alternifolia nas seguintes formulações: óleo livre, nanocápsula, nanoemulsão e a combinação de óleo livre com nanocápsula e com nanoemulsão, além do seu composto majoritário, terpinen-4-ol, a fim de verificar a atividade antimicrobiana frente a isolados de R. equi de diferentes origens. Utilizou-se o método de microdiluição em caldo na determinação das concentrações inibitória mínima (CIM) e bactericida mínima (CBM) das diferentes formulações frente aos isolados (n=24). Verificou-se baixo potencial para atividade antibacteriana de M. alternifolia na formulação de óleo livre. Todavia, essa atividade foi potencializada quando se incorporou o óleo essencial às nanoformulações. O composto terpinen-4-ol demonstrou potencial atividade antibacteriana quando incorporado ao óleo essencial e quando utilizado isoladamente. Verificou-se que tanto M. alternifolia quanto terpinen-4-ol testados possuem atividade antimicrobiana contra isolados de R. equi, sugerindo seu emprego em estudos avaliando seu potencial para o tratamento da rodococose.(AU)

Rhodococcus equi causes rodococose in horses, characterized by bronchopneumonia in foals. Due to reports of antimicrobial resistance, it is important to develop studies involving alternative therapies and new technologies, including the use of medicinal plants and nanotechnology. In this work, the plant Melaleuca alternifolia in oil free formulations, nanocapsule, nanoemulsion and the combination of free and nanocapsule oil nanoemulsion, besides its major compound, terpinen-4-ol, were used in order to verify antimicrobial activity against isolates of R. equi. The broth microdilution method was employed to determine the minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations of different formulations against 24 isolates. There was low antibacterial activity of M. alternifolia in oil free formulation, however, it was observed that the activity was enhanced when incorporated as essential oil the nanoformulations. The major compound, terpinen-4-ol, showed bactericidal and bacteriostatic activity when used alone. It is suggested that M. alternifolia, in association with nanocarriers systems, as well as terpinen -4-ol, presents potential for future studies concerning the equine rodococosis therapy.(AU)

Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi.

Linfadenite suína por Rhodococcus equi: aspectos gerais da afeccção, virulência das linhagens em suínos e humanos / Swine lymphadenitis by Rhodococcus equi: general aspects of disease, virulence of the strains in swine and humans / Linfadenitis porcina por Rhodococcus equi: aspectos generales de afección, la virulencia de las cepas en cerdos y seres humanos

A linfadenite infecciosa (pio) granulomatosa é a principal manifestação clínica causada pelo Rhodococcus equi nos suínos gerando altos prejuízos com a condenação de carcaças em abatedouros e pelo potencial zoonótico dos micro-organismos. Recentemente tem-se investigado a virulência associada aos plasmídios (Vap) em linhagens de R. equi isolados de suínos. O perfil encontrado nos isolados de suínos é semelhante ao de humanos com rodococose (VapB), particularmente acometidos pela síndrome da imunodeficiência adquirida (Aids). O presente estudo revisou os principais aspectos da rodococose em suínos, com ênfase nas propriedades de virulência de R. equi, nos achados epidemiológicos e nos aspectos de Saúde Pública.

The (pyo)granulomatous infections lymphadenitis is major clinical manifestation caused by Rhodococcus equi in swine causing losses in the carcass condemnation in abattoirs and the zoonotic potential of agents. Recently, virulence associated to plasmids (Vap) of R. equi isolates has been investigated. Plasmid profile found in pigs is similar to observed in humans with rhodococcosis (VapB), particularly infected by acquired immunodeficiency syndrome (AIDS). The aim of present study was to review the major aspects of swine rhodococcosis, with emphasis to virulence mechanisms of bacterium, epidemiology and public health concern.

La linfadenitis infecciosa (pio) granulomatosa es la principal manifestación clínica causada por Rhodococcus equi. En los cerdos causan elevadas pérdidas en la condena de las canales en los mataderos, y el potencial zoonótico de los microorganismos. Recientemente, se ha investigado la virulencia asociados con plásmideos (Vap) en cepas de R. equi aislados de cerdos. El perfil encontrado en aislamientos de cerdos es similar al de los seres humanos con rodococosis (VapB), especialmente afectado por el síndrome de inmunodeficiencia adquirido (SIDA). Este estudio examinó los aspectos principales de rodococosis en cerdos con énfasis en las propiedades de virulencia de R. equi, en los aspectos epidemiológicos y de salud pública.

Homeostasia do ferro em Rhodococcus equi: novas perspectivas a partir do perfil de expressão gênica de GAPDH e rhequichelin.

A rodococose equina manifesta-se principalmente através da ocorrência de pneumonia em potros, representando um grande desafio para a criação de equinos no Brasil e no mundo. Seu agente etiológico é uma bactéria intracelular facultativa, denominada Rhodococcus equi, cuja patogenicidade está associada à capacidade de replicar no interior de macrófagos. Os genes vapA e rhbC são necessários para sobrevivência intracelular de R. equi, uma vez que codificam a proteína associada à virulência A (VapA) e o sideróforo rhequichelin, respectivamente. A homeostasia do ferro (Fe) é regulada com precisão pelos micro-organismos, pois sua restrição limita a replicação destes, enquanto o excesso pode ser tóxico. R. equi é capaz de adquirir Fe através da transferrina (Tf), porém o mecanismo responsável por esta interação ainda não está elucidado.A aquisição de Fe ligado à Tf através da proteína Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) foi recentemente descrita em Mycobacterium tuberculosis, um patógeno intracelular similar ao R. equi. Em células eucarióticas, a GAPDH atua tanto na aquisição quando no efluxo de Fe. Nesse contexto, torna-se importante entender os mecanismos envolvidos na homeostasia do Fe por R. equi e buscar por alternativas terapêuticas e profiláticas com potencial para modular à imunidade nutricional. A presente dissertação descreve as alterações no crescimento bacteriano e na expressão dos genes GAPDH, rhbC e vapA em R. equi cultivado em diferentes concentrações de Fe, bem como na presença de apo-Tf (Tf insaturada) e holo-Tf (Tf saturada). Os cultivos bacterianos foram realizados em meio mínimo com lactato (controle) e suas variações (excesso de Fe, restrição de Fe e suplementação com apo-Tf e holo-Tf). O crescimento bacteriano foi avaliado através da contagem de unidades formadoras de colônias após 24 e 48 horas de incubação. Após o crescimento bacteriano foi realizada o isolamento do RNA e a síntese do cDNA, que posteriormente foi quantificado através da reação da cadeia polimerase em tempo real (PCR-RT). Em restrição de Fe observou-se a redução das taxas de crescimento de R. equi,onde em níveis extremos observou-se a sua morte. A taxa de transcrição dos genes GAPDH, vapA e rhbC elevaram-se em ambiente de restrição de Fe, quando comparadas ao controle. Todavia, para rhbC esta elevação é observada apenas em níveis críticos de restrição. Por outro lado, rhbC e vapA também aumentaram sua expressão em meio com excesso de Fe. Com base nestes resultados, acreditamos que além da função na aquisição de Fe, o sideróforo rhequichelin possivelmente desempenhe um papel antioxidante em R. equi, promovendo o sequestro deste micronutriente quando em excesso. Os resultados obtidos indicam que GAPDH participa da homeostasia de Fe em R. equi, possivelmente de forma similar a sua atuação em Mycobacterium tuberculosis. Futuros estudos são necessários a fim de elucidar os mecanismos pelos quais GAPDH e rhequichelin atuam na homeostasia do Fe e na sobrevivência intracelular de R. equi. É importante ressaltar que esses mecanismos podem representar potenciais alvos terapêuticos e de prevenção da rodococose em equinos jovens.

Pneumonia in foals is the main clinical manifestation of equine rhodococosis, and represents an important challenge in equine production in Brazil and world-wide. The etiologic agent is an intracellular facultative bacterium, named Rhodococcus equi. Its pathogenicity is associated to its ability to replicate inside macrophages. The genes vapA e rhbC, which encode virulence associated protein A (VapA) and the siderophore rhequichelin, respectively, are required for intracellular survival of R. equi. Iron (Fe) homeostasis is precisely regulated by microorganisms: its restriction limits cell replication, and its excess might be toxic. R. equi acquires Fe from holo-tranferrin, however, the mechanisms involved are unknown. The acquisition of Fe bound to Tf by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was described on Mycobacterium tuberculosis, a pathogen similar to R. equi. In eukaryotic cells, GAPDH has a role in both up-take and efflux of Fe. It is important to understand the mechanisms involved in R. equi Fe homeostasis in order to develop therapeutic and prophylactic alternatives with potential to modulate nutritional immunity. The present dissertation describes the changes in bacterial growth rates and in gene expression of GAPDH, rhbC and vapA in R. equi cultures under different concentration of Fe as well as presence or absence of apo-Tf (unsaturated-Tf) and holo-Tf (saturated-Tf). Quantitative culture of R. equi was performed in lactate minimal media (control) e its variations (Fe excess, Fe deprivation, and apo-Tf and holo-Tf supplementation). Bacterial growth rates were measured at 24 and 48 hours postincubation, by counting the number of colony-forming units. The RNA was isolated from the bacterial culture and the cDNA synthesized for latter quantification by real-time polymerase chain reaction (PCR-RT). Under Fe deprivation, there were a decrease in R. equi growth rates and in the critical level bacterial death were observed. It was found that GAPDH, rhbC and vapA transcription increase in a Fe deprived environment, however, the rhbC gene expression increase was observed only in critical levels of Fe starvation. Conversely, rhbC and vapA were also up regulated in media with Fe excess. Based on these results, we believe that in addition to the role in the Fe acquisition, rhequichelin might have an antioxidant role in R. equi by sequestration of this micronutrient in excessive concentration. The results indicate that GAPDH participate on Fe homeostasis in R. equi, possibly with similar function as observed in Mycobacterium tuberculosis in which the GAPDH expression increased in Fedepleted environment, indicating a possible role R. equi Fe acquisition. Further studies are needed to elucidate the mechanisms involved in GAPDH and rhequichelin functions in Fe homeostasis and in intracellular survival of R. equi. It is important to highlight that these mechanisms might be potential targets for rhodococcosis prevention and therapy in young foals.

Perfil de suscetibilidade antimicrobiana e presença do gene vapA em Rhodococcus equi de origem humana, ambiental e equina / Susceptibility profile of Brazilian Rhodococcus equi isolates to different antimicrobial classes and the presence of vapA gene

Rhodococcus equi é um micro-organismo intracelular facultativo, agente etiológico da rodococose, uma importante enfermidade que acomete principalmente potros com menos de seis meses de idade, causando a morte geralmente em decorrência de lesões pulmonares. Este agente também tem potencial zoonótico e emergiu como um patógeno oportunista no mundo, acometendo humanos imunocomprometidos, especialmente os transplantados e infectados pelo vírus da imunodeficiência humana (HIV). Entretanto, infecções por R. equi em hospedeiros hígidos tem sido relatadas, principalmente em crianças e idosos. Estudos tem mostrado um nível crescente na resistência de isolados de R. equi em relação aos antimicrobianos comumente utilizados no tratamento de animais e seres humanos infectados por este agente. A virulência deste pode estar associada a fatores como a cápsula de polissacarídeo, fosfolipase C e à enzima colesterol oxidase (fator equi). No entanto, uma proteína localizada em um plasmídeo, designada vapA, é essencial para a sobrevivência e replicação do agente em macrófagos. Com isso, os objetivos deste estudo foram avaliar o perfil de suscetibilidade de isolados de R. equi de diferentes fontes em relação aos antimicrobianos mais comumente utilizados na terapêutica animal e humana, bem como verificar a associação entre a presença do gene vapA e o índice de resistência múltipla aos antimicrobianos (IRMA). Neste estudo, 67 isolados brasileiros de R. qui de diferentes fontes foram analisados: 30 provenientes de amostras clínicas de equinos, sete de humanos e 30 ambientais (seis do solo e 24 de fezes de equinos). Para avaliar o perfil de suscetibilidade dos isolados utilizou-se o método de disco difusão, sendo testadas 16 drogas de diferentes classes de antimicrobianos. As amostras clínicas de equinos apresentaram as maiores taxas de resistência à penicilina (86,7%) e lincomicina (30%). Além disso, foram também resistentes a macrolídeos (azitromicina a 6,7%, eritromicina a 6% e ... (AU)

Rhodococcus equi is a facultative intracellular bacterium and etiological agent of rodococosis, an important disease that affects specially foals under six months old and leading the death generally due to pulmonary lesions. R. equi also has zoonotic potential, and it has emerged as an opportunistic pathogen in the world, specially infecting solid organ transplant recipients and immunocompromised human patients, mainly those infected by human immunodeficiency virus (HIV). Additionally, R. equi infections by healthy hosts have been reported principally in children and elderly individuals. Studies have shown an increasing level of resistance of isolates of R. equi against antibiotics commonly used to treat affected animals and humans. The virulence of this agent is associated with a protein located on a plasmid, designated vapA, it is essential for survival and replication of R. equi within macrophages. The present study evaluated the susceptibility profile of R.equi isolates from different sources against the antimicrobials most commonly used to treat animals and humans, as well as the occurrence and association of vapA gene and the antimicrobial multiple resistance index (AMRI). Sixty-seven Brazilian isolates of R. equi from different sources were analyzed: 30 clinical samples of horses, seven of human and 30 of environmental (six from soil and 24 from horse feces). To evaluate the susceptibility profile of R. equi isolates, the Kirby Bauer method was performed by using 16 drugs of 11 distinct antimicrobials classes. Additionally, those samples were also resistant to macrolides (azithromycin 6.7%, erythromycin 6% and clarithromycin 3.3%) as well as rifamycin (13%). All human and environmental samples were sensitive to macrolides and rifamycin. However, environmental isolates demonstrated high levels of resistance to penicillin and chloramphenicol. Similarly, human isolates had high level of resistance to ceftiofur, lincomycin and sulfazotrim. AMRI...(AU)

Infecção Natural por Theileria equi em Tapirus terrestris (Perissodactyla: Tapiridae) na Amazônia Brasileira.

As antas são consideradas um dos maiores mamíferos terrestres e são grandes dispersores de sementes, tendo um papel importante na preservação e reconstrução de habitats. Esses mamíferos sofrem grande pressão antrópica como a caça furtiva, a fragmentação e a perda de habitats, acarretando o declínio de suas populações. A redução do habitat natural além de ameaçar a vida desses mamíferos, pode favorecer a transmissão de agentes patogênicos, pois aumenta o contato entre fauna doméstica e a fauna silvestre, representando riscos para a saúde desses animais. O presente estudo teve como objetivo detectar a infecção natural por Theileria equi em antas (Tapirus terrestris) na Amazônia brasileira. Foram coletadas amostras de 19 antas cativas em parques zoobotânicos e mantenedores conservacionistas nos estados do Pará (n=18) e Amazonas (n=1). Amostras de sangue total foram coletadas e armazenadas em tubos contendo EDTA para posterior análise molecular. Esfregaços sanguíneos foram confeccionados para pesquisa de trofozoítas de T. equi. A extração de DNA genômico das amostras de sangue foi realizada com um kit comercial e para a pesquisa de DNA de T. equi foi realizada uma PCR com os iniciadores BEC-UF2 e EQUI-R, que amplificam um produto de aproximadamente 392 pares de base. As análises microscópicas dos esfregaços sanguíneos revelaram formas sugestivas de trofozoítas de T. equi em 36,8% (7/19) dos animais examinados. DNA de T. equi foi detectado em 57,9% (11/19) das amostras sanguíneas analisadas. O sequenciamento confirmou os resultados obtidos pela PCR e a análise filogenética revelou a homologia dos isolados de T. equi com os isolados disponíveis no GenBank. As antas mantidas em cativeiro estão infectadas por T. equi nas áreas estudadas e podem atuar como reservatórios e fonte de infecção para os carrapatos vetores.

The tapirs are considered one of the largest terrestrial mammals and are large seed dispersers, having an important role in the preservation and reconstruction of habitats. These mammals suffer from great anthropogenic pressure such as poaching, fragmentation and loss of habitats, leading to declining populations. The reduction of the natural habitat, besides threatening the life of these mammals, can favor the transmission of pathogens, since it increases the contact between domestic fauna and the wild fauna, representing risks for the health of these animals. The present study aimed to detect the natural infection by Theileria equi in tapirs (Tapirus terrestris) from the Brazilian Amazon. Samples of 19 captive tapirs were collected in zoobotanical parks and conservationists in the states of Pará (n=18) and Amazonas (n=1). Whole blood samples were collected and stored in tubes containing EDTA for further molecular analysis. Blood smears were made for T. equi trophozoite research. Extraction of genomic DNA from blood samples was performed with a commercial kit and for searching of T. equi DNA a PCR was performed with primers BEC-UF2 and EQUI-R, which amplify a product of approximately 392 base pairs. Microscopic analyzes of the blood smears revealed suggestive forms of T. equi trophozoites in 36.8% (7/19) of the animals examined. T. equi DNA was detected in 57.9% (11/19) of the blood samples analyzed. Sequencing confirmed the results obtained by PCR and the phylogenetic analysis revealed the homology of the T. equi isolates with the available GenBank isolates. The tapirs kept in captivity are infected by T. equi in the studied areas and can act as reservoirs and source of infection for the vector ticks.

Pneumonia causada por Rhodococcus equi em um potro da raça Crioula / Pneumonia by Rhodococcus equi in a Criollo Foal

Background: Rhodococcus equi is a facultative intracellular bacteria, gram positive and telluric. The airway is the main form of transmission. Clinical manifestations include enteritis, lymphadenitis and pneumonia in foals. Foals with pneumonia are infected during the first days of life, although clinical symptoms manifest mainly between 1 and 6 months of age. Susceptibility to infection is related to: amount of microorganism inhaled or ingested; decline of humoral immunity; immune system immaturity and failure in cellular response. The objective of this study is to report the case of a Criollo foal with pneumonia by Rhodococcus equi, with the description of diagnosis and therapeutic methods. Case: In the Hospital of Veterinary Clinics of UFPel, it was attended a Criollo foal, 3 months old, with tachycardia, dyspnea, hyperthermia, mild bilateral nasal mucopurulent secretion and bilateral pulmonary rattling sounds. In CBC it was found hyperfibrinogenemia, leukocytosis with neutrophilia and normocytic normochromic anemia. In the thorax radiographic examination it was evidenced areas with increased radiopacity and irregular edges in dorsal and caudal lung field. In the transtracheal fluid it was isolated Rhodococcus equi bacteria and submitted to antibiogram test. Treatment was based on azithromycin (10 mg/kg), rifampicin (2.5 mg/kg) orally (PO) for 60 days, flunixin meglumine (1.1 mg/kg) intravenously (IV), omeprazole (4 mg/kg) PO for 45 days and fluid was administered from daily clinical evaluation. The foal accompanied by his mother was transferred to a field with improved native pasture, shaded, with water ad libitum and feed 3x a day. After 30 days of hospitalization, the animal presented anorexia, lethargy, fever associated with leukocytosis. At this time it was added to treatment metronidazole (30 mg/kg) PO every 12 h for 10 days. After 35 days, the foal presented severe diarrhea, which ceased in 5 days, and in the first 3 days it was administered activated charcoal PO. The CBC results indicated a reduction in the number of leukocytes and fibrinogen during the hospitalization period. In the same period, the ultrasound evidenced a reduction in lesions diameter observed previously in pulmonary area. After 75 days the foal showed an improvement in overall clinical frame and he was discharged. Discussion: Early diagnosis of R. equi infection allied with the results of laboratory tests, allowed specific and immediate treatment improving the foal prognosis of living. The description of the occurrence of pneumonia R. equi in Criollo foals is unusual. In this case, the animal developed infection during the breeding season (February), when it was housed in a reproduction centre. In this centre, the foal remained part of the day in a sand hose, high ambient temperature, overcrowded paddocks and large turnover of equines from various age groups. These factors predispose to installation and permanence of the disease. Due to Criollo breed appreciation and increased use of assisted reproductive in this breed, the raisin system implies in gathering of factors risk that predispose the occurrence of R. equi in foals. The clinical evaluation associated with laboratory tests was imperative in order to obtain definitive diagnosis of R. equi infection and foals monitoration. The treatment based on the antibiotics azithromycin and rifampin associated with supportive therapy was effective in resolving a clinical case of Rhodococcus equi pneumonia in a Criollo foal 3 months old.

Identification of pathogens and virulence profile of Rhodococcus equi and Escherichia coli strains obtained from sand of parks

The identification of pathogens of viral (Rotavirus, Coronavirus), parasitic (Toxocara spp.) and bacterial (Escherichia coli, Salmonella spp., Rhodococcus equi) origin shed in feces, and the virulence profile of R. equi and E. coli isolates were investigated in 200 samples of sand obtained from 40 parks, located in central region of state of Sao Paulo, Brazil, using different diagnostic methods. From 200 samples analyzed, 23 (11.5%) strains of R. equi were isolated. None of the R. equi isolates showed a virulent (vapA gene) or intermediately virulent (vapB gene) profiles. Sixty-three (31.5%) strains of E. coli were identified. The following genes encoding virulence factors were identified in E. coli: eae, bfp, saa, iucD, papGI, sfa and hly. Phylogenetic classification showed that 63 E. coli isolates belonged to groups B1 (52.4%), A (25.4%) and B2 (22.2%). No E. coli serotype O157:H7 was identified. Eggs of Toxocara sp. were found in three parks and genetic material of bovine Coronavirus was identified in one sample of one park. No Salmonella spp. and Rotavirus isolates were identified in the samples of sand. The presence of R. equi, Toxocara sp, bovine Coronavirus and virulent E. coli isolates in the environment of parks indicates that the sanitary conditions of the sand should be improved in order to reduce the risks of fecal transmission of pathogens of zoonotic potential to humans in these places.(AU)

Antimicrobial activity of copper (ii) complex with 1, 2-bis [(1, 3-diphenylpyrazol-4-yl)methyl] diaminoethane

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to find new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for today's research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supplement from the Microsoft Excel program. Cytotoxicity (growth inhibition) was evaluated by tetrazolium colorimetric MTT assay, after exposure of cells to the tested compound for 48 h. Inhibition of growth was expressed as a percentage of cytotoxicity and calculated according to the following equation: (1-A test/A control) x 100. MBC99.9 and MIC99 of the test substance were lowest for Arcanobacterium haemolyticum being 0.2 mg/L and 0.0054 mg/L, respectively. The highest values were obtained for Arcanobacterium pyogenes and methicillin-resistant Staphylococcus aureus (MRSA) 488.002 mg/L and 20.2 mg/L. MIC80 for all four strains ranged from 0.00002 to 0.0023 mg/L. Measured values for MIC99 are 0.00545 mg/L for A. haemolyticum, 0.0443199 mg/L for R. equi, 0.0520712 mg/L for S. aureus and 2.36378 mg/L for A. pyogenes. Values for MIC99.9 ranged from 0.236134 to 488,002 mg/L. Most of the MIC values obtained in this study are significantly lower than those reported by other researchers. The values we obtained were lower as compared to MIC values for standard antibiotics, which were considered acceptable by the relevant institutions. This speaks in favor of a stronger antibacterial effect of our tested substances. In regards to cytotoxicity, the obtained MIC80 doses were lower than toxic, whereas MIC90 could be classified as low-toxic (less than 0.0625 µM), except of Arcanobacterium pyogenes only. According to the IC50 values, the compound Cu (L) Br2·MeOH was 6.4-fold and 4.8-fold more potent against HCT116 cells compared to normal lung fi broblasts and SW620 cells, respectively. Discussion: Copper (II) complex with an arylpyrazole ligand exhibits strong antibacterial properties, and it shows bacteriostatic effect at concentrations where there is no cytotoxic effect in normal human cells. The emergence of multi-resistant strains of pathogenic bacteria is a growing problem worldwide. Therefore, each new compound with potential antimicrobial activity, especially if it is not cytotoxic in effective dosage, deserves the attention of the scientific community. In this paper, we presented a newly synthesized substance with such properties.

OCORRÊNCIA E DETECÇÃO MOLECULAR DE ESPÉCIES DE MYCOBACTERIUM, E DOS GENES DE VIRULÊNCIA VAPA, VAPB E VAPN EM LINHAGENS DE RHODOCOCCUS EQUI, ISOLADAS DE LINFONODOS DE TAIASSUÍDEOS DE CATIVEIRO

Foram investigadas, neste estudo, proteínas associadas à virulência (genes vapA, vapB e vapN) das espécies de Rhodococcus equi e Micobactérias isoladas de 330 linfonodos de catetos (Tayassu tajacu) e queixadas (Tayassu pecari) destinados ao consumo humano. Trinta e seis (10,9%) linhagens de R. equi foram isoladas, 3,3% (11/330) dos linfonodos de queixadas e 7,6% (25/330) dos catetos. Entre os 11 isolados de R. equi das queixadas, 90,9% (n=10/11) foram obtidos de linfonodos mesentéricos e apenas 9,1% (n=1/10) de linfonodo mediastínico. Nos 25 isolados de R. equi dos catetos, 40,0% (10/25) foram obtidos de linfonodos mesentéricos, 36,0% (9/25) de submandibulares e 24,0% (6/25) de mediastínicos. Não foram identificados genes vapA, vapB e vapN entre os isolados de R. equi. Foi isolado Mycobacterium sp. de 3,03% (10/330) do total de linfonodos. Entre os 10 isolados de micobactérias, 60% (n=6/10) dos linfonodos eram de queixadas e 40% (4/10) de catetos. Dez espécies de Mycobacterium foram detectadas por PCR-PRA, com predominância de M. avium tipo 1. O sequenciamento dos genes hsp65 e rpob revelaram micobactérias saprófitas (M. sinense, M. kumamotonense) e potencialmente patogênicas (M. colombiense, M. intracellulare) para humanos e animais. Esta é a primeira descrição de R. equi e / ou espécies de micobactérias identificadas nos linfonodos de espécimes de Tayassuideos. Apesar da ausência de genes Vap, a identificação de R. equi, bem como de micobactérias não tuberculosas e saprófitas, destacam o risco de transmissão desses patógenos das espécies de Tayassuideos para humanos por meio de carne ou produtos à base de carne contaminados com conteúdo de linfonodos, uma vez que R. equi sem plasmídeo de virulência e as espécies de micbactérias descritas aqui já foram relatadas como causas de infecções pulmonares e extrapulmonares em seres humanos imunocompetentes e imunocomprometidos.

Virulence-associated proteins (vapA, vapB and vapN genes) of Rhodococcus equi and Mycobacterium species isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated, 3.3% (11/330) from white-lipped peccary and 7.6% (25/330) of collared peccary lymph nodes. Among the 11 isolates of R. equi of the white-lipped peccaries, 90.9% (n = 10/11) were obtained from mesenteric lymph nodes and only 9.1% (n = 1/10) of the mediastinal lymph node. In the 25 isolates of R. equi obtained from collared peccaries, 40.0% (10/25) were recovered from mesenteric lymph nodes, 36% (9/25) from submandibular, and 24.0% (6/25) from mediastinal ones. No vapA, vapB, and vapN genes (plasmidless type) were identified among R. equi isolates. Mycobacterium sp. was isolated in 3.03% (10/330) of all lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from white-lipped peccary, and 40% (4/10) from collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA, with predominance of M. avium type 1. Sequencing of hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense, M. kumamotonense) and potentially pathogenic (M. colombiense, M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacteria species identified in the lymph nodes of Tayassuid specimens. Despite the absence of Vap genes, the identification of R. equi, as well as nontuberculous and saprophytic mycobacteria highlight the risk of transmission of these pathogens from Tayassuidae species to humans by means of meat or meat products contaminated with lymph node contents, since R. equi plasmidless type and mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections both in immunocompetent and immunocompromised humans.

Agentes da Piroplasmose equina: variabilidade genética de Theileria equi e Babesia caballi em equinos naturalmente infectados do estado do Rio de Janeiro e a dinâmica da infecção de Theileria equi em Rhipicephalus (Boophilus) microplus

Os objetivos deste trabalho foram caracterizar molecularmente Theileria equi e Babesia caballi de amostras de equinos do Rio de Janeiro, bem como realizar um estudo da dinâmica da infecção de T. equi em Rhipicephalus (Boophilus) microplus. No primeiro capítulo realizou-se a caracterização molecular de um fragmento do gene rap-1 de B. caballi e a análise do grau de heterogeneidade do gene 18S rDNA em T. equi em amostras de equinos naturalmente infectados, do estado do Rio de janeiro. Foram obtidas vinte sequências do gene 18S rDNA de T. equi. As análises filogenéticas foram realizadas baseadas no ARB-Silva. Para a caracterização molecular de um fragmento do gene rap-1 de B. caballi foram utilizadas amostras positivas por cELISA e qPCR. Uma nested PCR foi desenvolvida para amplificar um fragmento do gene rap-1, e uma PCR convencional, para o gene completo. As sequências de aminoácidos e nucleotídeos sequenciados foram submetidas ao método Maximum- Likelihood. Na análise das sequências do 18S rDNA de T. equi, doze diferentes isolados foram observados em equinos no estado do Rio de Janeiro, sendo agrupados em dois dos três clados formados (I, II). As sequências de T. equi se agrupam dentro de um clado diferente dos demais Theilerídeos. Uma das 23 sequências de aminoácidos do gene rap-1 apresentou doze pontos de mutação. Na análise de agrupamentos, as amostras brasileiras agruparam no clado A, sendo estas analisadas como aminoácidos (152 aas) ou nucleotídeos (1244 pb). No segundo capítulo foram avaliados aspectos da dinâmica da infecção de T. equi em R. (B.) microplus alimentados em equino cronicamente infectado. Larvas de uma colônia livre de hemoparasitos foram utilizadas. Carrapatos e sangue do equino foram coletados de dois em dois dias. Os carrapatos foram dissecados e armazenados inteiros em pools de acordo com o dia pós-infestação, estágio evolutivo e gênero. Amostras de glândula salivar e ovário foram armazenados para Microscopia Eletrônica de Transmissão. As amostras foram testadas pela qPCR. Com relação aos carrapatos inteiros, mais de 90% das amostras foram positivas para T. equi. A carga parasitária média das amostras de carrapatos inteiros e glândulas salivares apresentaram aumento gradual com o avançar do período de alimentação. Das 45 amostras de glândula salivar, 77,78% foram positivas para T. equi e tiveram maior taxa de infecção no final do período experimental. Foi possível observar positividade em carrapatos recém emergidos, ninfas e adultos, indicando a ocorrência de transmissão transestadial. A positividade de amostras de ovários foi de 84,38%. A maior permanência de machos sobre o equino, associado à alta positividade deste tipo de amostra indicam que o macho pode ter participação significativa na transmissão. Visualizaram-se estruturas morfologicamente compatíveis com um piroplasma através de micrografia de transmissão em ovários e glândulas salivares de em R. (B.) microplus. A análise do gene 18S rDNA de T. equi e do gene rap-1 de B. caballi de amostras brasileiras demonstrou que diferentes isolados coexistem na mesma área. Experimentalmente, observou-se que R. (B.) microplus é capaz de se infectar com T. equi e que as formas multiplicam-se nas glândulas salivares e, possivelmente, em ovários.

This study aimed to perform the molecular characterization of the B. caballi rap-1 gene and to analyze the degree of heterogeneity of the T. equi 18S rDNA gene of naturally infected horses in the state of Rio de Janeiro. Complete sequences of the T. equi 18S rDNA gene (n = 20) were obtained. The PCR products were cloned and sequenced. Phylogenetic analyzes were performed on ARB-Silva basis. For the molecular characterization of a fragment of the B. caballi rap-1 gene, 32 cELISA and qPCR positive equine samples were used. A nested PCR was developed to amplify a fragment of the rap-1 gene and a conventional PCR, aiming the amplification of the complete gene. The products were sequenced. Sequences were submitted to the Maximum-Likelihood reconstruction method with Poisson model and 1000 replicates. In the analysis of T. equi, twelve different isolates were observed in equines from the state of Rio de Janeiro and were grouped into two of the three clades formed (I, II), most of them grouped in clade I (70%, n = 14/20). The sequences of T. equi are grouped within a clade different from other Theilerids. It was possible to amplify a fragment of the B. caballi rap-1 gene from 23 sequences using nested PCR, identified as single sequences. One of the sequences showed twelve mutation points. In cluster analysis, the Brazilian samples belonged to clade A, being analyzed as amino acids (152a) or nucleotides (1244 bp). The aim of the second chapter was to evaluate the dinamic of infection of T. equi in R. (B.) microplus fed in chronically infected horses. The larvae obtained from a hemoparasite-free colony were used in the infestation of an equine chronically infected by T. equi. Ticks and the horse blood were collected every two days. Ticks were dissected and stored in pools according to the pos- infestation day, evolutionary stage and gender. Some of these specimens were stored entire. Salivary gland and ovary samples were stored properly for Transmission Electron Microscopy. Some stages, prior to ecdysis, were stored in B.O.D. to molt. Samples were tested by qPCR. Regarding to entire tick samples, more than 90% of the samples were positive for T. equi. The mean of parasite load in entire ticks and salivary glands presented gradual increasing with the progress of the feeding period. Of the 45 salivary gland samples, 77.78% were positive for T. equi and had a higher infection rate at the end of the experimental period. It was found freshly molted ticks positives for T. equi by qPCR, indicating transestadial transmission. 84,38% of R. (B.) microplus ovary samples were considered positives for T. equi by molecular tools. Males of R. (B.) microplus were found parasiting the equine for 60 days. This fact associated with the high positivity for T. equi indicates males as an epidemiologically important gender. Transmission micrographs revealed morphologic structures compatible with a Piroplasmid in the acini of salivary glands and ovaries. The phylogenetic analysis of the T. equi 18S rDNA gene and of B. caballi rap-1 gene showed that different isolates co-exist in the same area in Rio de Janeiro, Brazil. Experimently, it was possible to observe that R. (B.) microplus could be infected with T. equi and that the forms of development of this parasite are capable of multiply inside salivary glands and, possibly, ovaries.