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Among the bacterial infections that impair the health status of marine mammals, those caused by Brucella spp. are the most reported worldwide. Brucella infections in marine mammals can result in acute or chronic disease and are associated with variable clinical outcomes, depending on the organ involved during the infectious process, infection route, host immunity, and strain pathogenicity. Asymptomatic infections may also occur. The current study expands the investigation of Brucella infection in northeast Brazil by analyzing 19 dead, stranded cetaceans and 52 Antillean manatees Trichechus manatus manatus. The manatees included 8 dead, captive manatees and 44 live specimens, of which 10 were analyzed only after reintroduction into the wild as part of a rehabilitation program, 9 were analyzed both while in captivity or semi-captivity and after reintroduction, 20 were sampled only in captivity or semi-captivity, and 5 were free-living manatees. Serological tests were used to screen for antibodies against smooth Brucella spp. Whole blood, swabs, and tissue samples were screened for Brucella spp. DNA by PCR. Samples with positive PCR results were cultured for Brucella spp. isolation. All manatees yielded negative results in serological and molecular tests. Brucella spp. DNA was detected in the kidney of one adult Guiana dolphin Sotalia guianensis exhibiting necrosis in the liver. No growth of Brucella spp. was observed via microbiological culturing. This study is the first report of Brucella spp. DNA detection in cetaceans in the state of Pernambuco, and it highlights the importance of conducting systematic monitoring for the presence of Brucella infection in marine mammals along the Brazilian coast, especially in the northeast region, where several cases have been reported.

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This work aimed at evaluating the antifungal susceptibility and production of virulence factors by Candida spp. isolated from sirenians in Brazil. The isolates (n = 105) were recovered from the natural cavities of Amazonian and West Indian manatees and were tested for the susceptibility to amphotericin B, itraconazole, and fluconazole and for the production of phospholipases, proteases, and biofilm. The minimum inhibitory concentrations (MICs) for amphotericin B ranged from 0.03 to 1 µg/mL, and no resistant isolates were detected. Itraconazole and fluconazole MICs ranged from 0.03 to 16 µg/mL and from 0.125 to 64 µg/mL, respectively, and 35.2% (37/105) of the isolates were resistant to at least one of these azole drugs. Concerning the production of virulence factors, phospholipase activity was observed in 67.6% (71/105) of the isolates, while protease activity and biofilm production were detected in 50.5% (53/105) and 32.4% (34/105) of the isolates, respectively. Since the natural cavities of manatees are colonized by resistant and virulent strains of Candida spp., these animals can act as sources of resistance and virulence genes for the environment, conspecifics and other animal species, demonstrating the potential environmental impacts associated with their release back into their natural habitat.

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The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogenhost interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.

O atual comércio internacional de carne tem aumentado a pressão para haver um melhor e mais rápido diagnóstico de tuberculose bovina. O tradicional cultivo continua a ser o método padrão ouro para confirmar a infecção por Mycobacterium bovis, apesar de ser excessivamente demorado e necessitar de micobactérias viáveis. Métodos moleculares representam a superação de todos os defeitos dos métodos bacteriológicos com detecção e identificação mais rápidas. Entretanto, características das micobactérias, como uma complexa parede celular e a interação patógeno-hospedeiro, torna-os um desafio. Três protocolos de extração de DNA (A, B e C) foram testados em tecidos bovinos para verificar qual técnica é mais adequada para diagnóstico de rotina, avaliando sua especificidade e sensibilidade. Trinta lesões granulomatosas positivas no cultivo e 30 lesões granulomatosas negativas no cultivo foram utilizadas no experimento. A partir de cada amostra, três homogeneizados com diferentes diluições (10-1, 10-2 e 10-3) foram preparadas e submetidas à extração de DNA. A PCR para o gene alvo IS6110 foi realizada empregando-se dois volumes de DNA: um com 5 µL para todas as três diluições e outro com 2,5 µL da diluição 10-1. O Protocolo A foi capaz de detectar membros do complexo M. tuberculosis na maior parte das amostras. À medida que a diluição dos homogeneizados aumentou, a sensibilidade diminuiu. Embora o Protocolo A tenha apresentado a maior sensibilidade, seguido por C e B, este revelou a menor especificidade, que pode ser devido à insuficiência de organismos viáveis ou tempo de incubação no primo isolamento. Apesar de os métodos bacteriológicos clássicos ainda serem recomendados pela OIE, através da avaliação da sensibilidade dos protocolos de extração de DNA e dos procedimentos de PCR, concluímos que a melhor estratégia para a detecção de M. bovis é usar o Protocolo A em homogeneizados mais concentrados.

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The current scenario of international beef trading has increased the pressure for better and faster diagnosis of bovine tuberculosis. Although traditional culture remains the gold standard method to confirm Mycobacterium bovis infection, it is exceedingly time consuming, and demands viable mycobacteria. Molecular methods overcome the flaws of the bacteriological methods with faster detection and identification. However, mycobacterial features like a complex cell wall and pathogenhost interaction make the molecular detection a challenge. Three protocols for DNA extraction (A, B and C) from bovine tissues were tested to verify the most suitable technique for routine diagnostic assessment of their specificity and sensitivity. Thirty culture-positive and thirty culture-negative granulomatous lesions were included in the trial. From each sample, three tissue suspensions at different dilutions (10-1, 10-2 and 10-3) were prepared and submitted to DNA extraction. PCR procedures targeting IS6110 were performed, employing two volumes of DNA: 5 µL of all three dilutions, and 2.5 µL of the 10-1 dilution. Protocol A was able to detect members of the M. tuberculosis complex in most samples. The sensitivity of the test decreased with increase in tissue-suspension dilution. Although Protocol A presented the highest sensitivity followed by C and B, it showed the lowest specificity, which can be due to a failure in primary isolation caused by the lack of viable organisms or incubation time. Regardless classical bacteriological methods are still recommended by OIE, after evaluating the sensitivity of DNA extraction protocols and PCR procedures, we conclude that the best strategy for M. bovis detection is to follow Protocol A on concentrated tissue suspensions.(AU)

O atual comércio internacional de carne tem aumentado a pressão para haver um melhor e mais rápido diagnóstico de tuberculose bovina. O tradicional cultivo continua a ser o método padrão ouro para confirmar a infecção por Mycobacterium bovis, apesar de ser excessivamente demorado e necessitar de micobactérias viáveis. Métodos moleculares representam a superação de todos os defeitos dos métodos bacteriológicos com detecção e identificação mais rápidas. Entretanto, características das micobactérias, como uma complexa parede celular e a interação patógeno-hospedeiro, torna-os um desafio. Três protocolos de extração de DNA (A, B e C) foram testados em tecidos bovinos para verificar qual técnica é mais adequada para diagnóstico de rotina, avaliando sua especificidade e sensibilidade. Trinta lesões granulomatosas positivas no cultivo e 30 lesões granulomatosas negativas no cultivo foram utilizadas no experimento. A partir de cada amostra, três homogeneizados com diferentes diluições (10-1, 10-2 e 10-3) foram preparadas e submetidas à extração de DNA. A PCR para o gene alvo IS6110 foi realizada empregando-se dois volumes de DNA: um com 5 µL para todas as três diluições e outro com 2,5 µL da diluição 10-1. O Protocolo A foi capaz de detectar membros do complexo M. tuberculosis na maior parte das amostras. À medida que a diluição dos homogeneizados aumentou, a sensibilidade diminuiu. Embora o Protocolo A tenha apresentado a maior sensibilidade, seguido por C e B, este revelou a menor especificidade, que pode ser devido à insuficiência de organismos viáveis ou tempo de incubação no primo isolamento. Apesar de os métodos bacteriológicos clássicos ainda serem recomendados pela OIE, através da avaliação da sensibilidade dos protocolos de extração de DNA e dos procedimentos de PCR, concluímos que a melhor estratégia para a detecção de M. bovis é usar o Protocolo A em homogeneizados mais concentrados.(AU)

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The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

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