RESUMO

Hepatic injuries in COVID-19 are not yet fully understood and indirect pathways (without viral replication in the liver) have been associated with the activation of vascular mechanisms of liver injury in humans infected with SARS-CoV-2. Golden Syrian hamsters are an effective model for experimental reproduction of moderate and self-limiting lung disease during SARS-CoV-2 infection. As observed in humans, this experimental model reproduces lesions of bronchointerstitial pneumonia and pulmonary vascular lesions, including endotheliitis (attachment of lymphoid cells to the luminal surface of endothelium). Extrapulmonary vascular lesions are well documented in COVID-19, but such extrapulmonary vascular lesions have not yet been described in the Golden Syrian hamster model of SARS-CoV-2 infection. The study aimed to evaluate microscopic liver lesions in Golden Syrian hamsters experimentally infected with SARS-CoV-2. In total, 38 conventional Golden Syrian hamsters, divided into infected group (n=24) and mock-infected group (n=14), were euthanized at 2-, 3-, 4-, 5-, 7-, 14-, and 15-days post infection with SARS-CoV-2. Liver fragments were evaluated by histopathology and immunohistochemical detection of SARS-CoV-2 Spike S2 antigens. The frequencies of portal vein endotheliitis, lobular activity, hepatocellular degeneration, and lobular vascular changes were higher among SARS-CoV-2-infected animals. Spike S2 antigen was not detected in liver. The main results indicate that SARS-CoV-2 infection exacerbated vascular and inflammatory lesions in the liver of hamsters with pre-existing hepatitis of unknown origin. A potential application of this animal model in studies of the pathogenesis and evolution of liver lesions associated with SARS-CoV-2 infection still needs further evaluation.

Assuntos

RESUMO

ABSTRACT Hepatic injuries in COVID-19 are not yet fully understood and indirect pathways (without viral replication in the liver) have been associated with the activation of vascular mechanisms of liver injury in humans infected with SARS-CoV-2. Golden Syrian hamsters are an effective model for experimental reproduction of moderate and self-limiting lung disease during SARS-CoV-2 infection. As observed in humans, this experimental model reproduces lesions of bronchointerstitial pneumonia and pulmonary vascular lesions, including endotheliitis (attachment of lymphoid cells to the luminal surface of endothelium). Extrapulmonary vascular lesions are well documented in COVID-19, but such extrapulmonary vascular lesions have not yet been described in the Golden Syrian hamster model of SARS-CoV-2 infection. The study aimed to evaluate microscopic liver lesions in Golden Syrian hamsters experimentally infected with SARS-CoV-2. In total, 38 conventional Golden Syrian hamsters, divided into infected group (n=24) and mock-infected group (n=14), were euthanized at 2-, 3-, 4-, 5-, 7-, 14-, and 15-days post infection with SARS-CoV-2. Liver fragments were evaluated by histopathology and immunohistochemical detection of SARS-CoV-2 Spike S2 antigens. The frequencies of portal vein endotheliitis, lobular activity, hepatocellular degeneration, and lobular vascular changes were higher among SARS-CoV-2-infected animals. Spike S2 antigen was not detected in liver. The main results indicate that SARS-CoV-2 infection exacerbated vascular and inflammatory lesions in the liver of hamsters with pre-existing hepatitis of unknown origin. A potential application of this animal model in studies of the pathogenesis and evolution of liver lesions associated with SARS-CoV-2 infection still needs further evaluation.

RESUMO

In Brazil, there have been few leptospires isolated from cattle, especially in the Amazon, implying that the epidemiology of the disease in this region is still largely unclear. In a previous study, 52 Leptospira isolates were obtained from urine of cattle raised in the Brazilian Amazon and, to achieve a greater understanding of Leptospira infection in cattle of this region, the present study aimed to serologically and molecularly characterizes all these isolates. The laboratory assays used were the microscopic agglutination test (MAT) adopting a panel of polyclonal antisera against Leptospira spp. for serogrouping the isolates, DNA sequencing (secY) and multiple locus variable number tandem repeat analysis (MLVA). The isolates belonged to five species: 20/52 were identified as L. borgpetersenii (38.5 %); 18/52 as L. kirschneri (34.6 %); 9/52 as L. santarosai (17.3 %); 3/52 as L. noguchii (5.8 %) and 2/52 as L. interrogans (3.8 %). With serogrouping, nine different serogroups were detected, with a high frequency of the Sejroe serogroup. MLVA showed that all L. borgpetersenii isolates had a profile compatible with serovar Hardjo; moreover, the other isolates demonstrated a diversity of patterns, and some of them may represent strains not yet characterized. In the Brazilian Amazon, the leptospires circulating in cattle revealed the unique aspects of infections in this area which, in addition to a variety of strains, were characterized by a high frequency of the Sejroe serogroup, highlighting the serovar Hardjo, which has not been reported in other regions of Brazil.

Assuntos

RESUMO

Leptospirosis is a zoonotic disease of worldwide distribution, affecting both humans and animals. The development of an effective vaccine against leptospirosis has long been pursued but without success. Humans are contaminated after direct contact with the urine of infected animals or indirectly by contaminated water or soil. The vaccines available consist of inactivated whole-bacterial cells, and the active immunoprotective antigen is the lipopolysaccharide moiety, which is also the basis for serovar classification. However, these vaccines are short-lasting, and protection is only against serovars contained in the preparation. The search for prevalent antigens, present in pathogenic species of Leptospira, represents the most cost-effective strategy for prevention of leptospirosis. Thus, the identification of these antigens is a priority. In this study, we examined the immunoprotective effect of eight leptospiral recombinant proteins using hamster as the challenge model. Animals received subcutaneously two doses of vaccine containing 50 μg of each recombinant protein adsorbed on alum adjuvant. Two weeks after the booster, animals were challenged with virulent leptospires and monitored for 21 days. All proteins were able to induce a specific immune response, although significant protective effects on survival rate were observed only for the proteins Lsa14, rLIC13259, and rLIC11711. Of these, only rLIC13259 and rLIC11711 were found to be highly prospective in promoting renal clearance. The sterilizing potential of both proteins will be further investigated to elucidate the immunoprotective mechanisms involved in leptospirosis control. These are the first proteins involved with human complement components with the capacity to protect against virulent challenge and to eliminate the bacteria from the host.

RESUMO

Leptospirosis is a disease of worldwide distribution that affects man and several animal species. Domestic and wild animals can behave as reservoirs of the agent. The aim of this study was to evaluate the frequency of anti-Leptospira spp. antibodies in dogs and wild small mammals from rural properties and conservation units of three municipalities of Rio Grande do Sul State, Brazil. Sera were collected from 192 domestic dogs and 132 small mammals, namely rodents of the Cricetidae and Caviidae families and marsupials of the Didelphidae family. The study used Microscopic Agglutination Test (MAT) against 23 serovars of Leptospira spp. Overall, 9.90% (19/192) dogs, and 1.51% (2/132) small mammals were seropositive. Leptospira interrogans serovar Australis was the most frequent serovar in dogs and antibodies against L. interrogans serovar Pomona were detected in a dog and a small mammal belonging to the same municipality. We conclude that in the regions studied the frequency of seropositivity in dogs is low, and leptospires do not seem to be circulating in small mammals, yet, further research is necessary to assess the real role that these animals may have for leptospirosis in the studied areas. This is the first serological survey in small mammals, including wild rodents, in the area that encompasses conservation units of great importance to Rio Grande do Sul state.

RESUMO

In areas where human tuberculosis and bovine tuberculosis coexist, differentiation between M. bovis and M. tuberculosis is important for monitoring the spread of M. bovis among cattle and from cattle to humans. The objective of this study was to isolate and identify M. bovis in bovines with positive diagnosis identified on tuberculin test in the State of Paraíba, Northeastern Brazil. Thirty-two bovines that tested positive in the comparative tuberculin test were used, from which samples of any organ with lesions suggestive of tuberculosis were collected, as well as lymph nodes, when no gross lesions were observed. Samples were submitted to histopathological exam, mycobacterial culture, Ziehl-Neelsen staining and molecular diagnosis. Twenty-one (65.6%) animals presented lesions suggestive of tuberculosis. As to body region 77.7% of lesions were found in the thoracic cavity, 12.4% in the head and 9.9% in the abdominal cavity. Among 55 samples submitted to mycobacterial culture, mycobacteria were isolated in 31 (56.4%), being 13 (41.9%) identified as M. bovis and 18 (58.1%) as Mycobacterium spp. Conclusion is that isolation and identification of M. bovis and Mycobacterium spp. in cattle suggests that humans are exposed to the risk of infection. This reinforces the need for intensification and optimization of prevention and control measures foreseen in the Brazilian National Program for the Control and Eradication of Bovine Brucellosis and Tuberculosis. Mycobacteria isolation and identification surveys are, therefore, encouraged in other Northeastern states.(AU)

Em áreas onde a tuberculose humana e a tuberculose bovina coexistem, a diferenciação entre M. bovis e M. tuberculosis é importante para monitorar a disseminação de M. bovis entre bovinos e destes para os seres humanos. Objetivou-se neste estudo isolar e identificar M. bovis em bovinos com diagnóstico positivo pelo teste de tuberculinização no estado da Paraíba, nordeste do Brasil. Foram submetidos 32 bovinos positivos ao teste de tuberculinização comparativa, dos quais foram colhidas amostras de qualquer órgão com lesões sugestivas de tuberculose, e, nos casos em que não foram observadas lesões sugestivas, foram colhidas amostras de linfonodos. As amostras foram submetidas a exame histopatológico, cultivo micobacteriológico, coloração de Ziehl-Neelsen e diagnóstico molecular. Apresentaram lesões sugestivas de tuberculose 21 animais (65,6%). Com relação à distribuição das lesões de acordo com a região corporal, 77,7% localizavam-se na cavidade torácica, 12,4% na cabeça e 9,9% na cavidade abdominal. De 55 amostras submetidas ao cultivo de micobactérias, em 31 (56,4%) foram isoladas micobactérias, sendo que em 13 (41,9%) foi identificado M. bovis, e nas 18 restantes (58,1%) foi identificado Mycobacterium spp. Conclui-se que o isolamento e a identificação de M. bovis e Mycobacterium spp. em bovinos indicam que os seres humanos estão expostos ao risco de infecção. Isso reforça a necessidade de intensificação e otimização de medidas de prevenção e controle previstas no Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Bovina. Sugere-se a realização de estudos de isolamento e identificação de micobactérias em outros estados do Nordeste.(AU)

Assuntos

RESUMO

In areas where human tuberculosis and bovine tuberculosis coexist, differentiation between M. bovis and M. tuberculosis is important for monitoring the spread of M. bovis among cattle and from cattle to humans. The objective of this study was to isolate and identify M. bovis in bovines with positive diagnosis identified on tuberculin test in the State of Paraíba, Northeastern Brazil. Thirty-two bovines that tested positive in the comparative tuberculin test were used, from which samples of any organ with lesions suggestive of tuberculosis were collected, as well as lymph nodes, when no gross lesions were observed. Samples were submitted to histopathological exam, mycobacterial culture, Ziehl-Neelsen staining and molecular diagnosis. Twenty-one (65.6%) animals presented lesions suggestive of tuberculosis. As to body region 77.7% of lesions were found in the thoracic cavity, 12.4% in the head and 9.9% in the abdominal cavity. Among 55 samples submitted to mycobacterial culture, mycobacteria were isolated in 31 (56.4%), being 13 (41.9%) identified as M. bovis and 18 (58.1%) as Mycobacterium spp. Conclusion is that isolation and identification of M. bovis and Mycobacterium spp. in cattle suggests that humans are exposed to the risk of infection. This reinforces the need for intensification and optimization of prevention and control measures foreseen in the Brazilian National Program for the Control and Eradication of Bovine Brucellosis and Tuberculosis. Mycobacteria isolation and identification surveys are, therefore, encouraged in other Northeastern states.(AU)

Em áreas onde a tuberculose humana e a tuberculose bovina coexistem, a diferenciação entre M. bovis e M. tuberculosis é importante para monitorar a disseminação de M. bovis entre bovinos e destes para os seres humanos. Objetivou-se neste estudo isolar e identificar M. bovis em bovinos com diagnóstico positivo pelo teste de tuberculinização no estado da Paraíba, nordeste do Brasil. Foram submetidos 32 bovinos positivos ao teste de tuberculinização comparativa, dos quais foram colhidas amostras de qualquer órgão com lesões sugestivas de tuberculose, e, nos casos em que não foram observadas lesões sugestivas, foram colhidas amostras de linfonodos. As amostras foram submetidas a exame histopatológico, cultivo micobacteriológico, coloração de Ziehl-Neelsen e diagnóstico molecular. Apresentaram lesões sugestivas de tuberculose 21 animais (65,6%). Com relação à distribuição das lesões de acordo com a região corporal, 77,7% localizavam-se na cavidade torácica, 12,4% na cabeça e 9,9% na cavidade abdominal. De 55 amostras submetidas ao cultivo de micobactérias, em 31 (56,4%) foram isoladas micobactérias, sendo que em 13 (41,9%) foi identificado M. bovis, e nas 18 restantes (58,1%) foi identificado Mycobacterium spp. Conclui-se que o isolamento e a identificação de M. bovis e Mycobacterium spp. em bovinos indicam que os seres humanos estão expostos ao risco de infecção. Isso reforça a necessidade de intensificação e otimização de medidas de prevenção e controle previstas no Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose Bovina. Sugere-se a realização de estudos de isolamento e identificação de micobactérias em outros estados do Nordeste.(AU)

Assuntos

RESUMO

The zoonotic potential of multidrug resistant (MDR) bacteria is a worldwide concern and companion animals have been implicated in the spread of resistant bacteria. Therefore, surveillance is important, as there are reports of transmission of these bacteria from dog to men, as well as from men to dog. A 5-year-old mixed-breed male dog was admitted with obstructive struvite urolithiasis relapsing for over 18 months, in Botucatu, in the state of São Paulo, Brazil. The strain, biochemically identified as Staphylococcus spp., was MDR and was treated off-label with vancomycin, which resulted in clinical cure. The strain was molecularly identified as Staphylococcus pseudintermedius and the mecA gene was identified. This is the main gene responsible for methicillin-resistant S. pseudintermedius (MRSP), which is often resistant to multiple antimicrobials. The hypotheses for this clinical case are the transmission from man to animal, since the tutor was an intensivist doctor, or the bacterium itself could be part of the animal's microbiota and due to other factors, such as stress or obstructive urinary disease, opened a doorway to infection by S. pseudintermedius. Further studies should elucidate the transmission of MDR bacteria between human and pets.(AU)

O potencial zoonótico de bactérias multirresistentes é uma preocupação global e os animais de companhia têm sido implicados na disseminação de bactérias resistentes; assim, é importante a vigilância, pois já existem relatos de transmissão destas bactérias do cão para o homem e vice-versa. Um cão, sem raça definida e de cinco anos de idade, foi atendido na cidade de Botucatu, São Paulo, Brasil, apresentando urolitíase obstrutiva de estruvita recorrente há um ano e meio. Na urocultura do animal foi isolada uma estirpe de Staphylococcus spp. multirresistente; o tratamento com vancomicina possibilitou acura clínica. A estirpe de Staphylococcus spp. isolada foi identificada molecularmente como S. pseudintermedius e nela foi identificada a presença do gene mecA, o principal responsável por S. pseuidintermedius resistente à meticilina (MRSP), e que é frequentemente resistente à múltiplos antimicrobianos. As hipóteses para este caso clínico são a transmissão do homem para o animal, pois o tutor era um médico intensivista, ou que a própria bactéria fazia parte da microbiota do animal e, devido a outros fatores como estresse e doença urinária obstrutiva, abriu-se uma porta de entrada para a infecção pelo S. pseudintermedius. Mais estudos são necessários para a elucidação da transmissão de bactérias multirresistentes entre animais de companhia e o ser humano.(AU)

Assuntos

RESUMO

The zoonotic potential of multidrug resistant (MDR) bacteria is a worldwide concern and companion animals have been implicated in the spread of resistant bacteria. Therefore, surveillance is important, as there are reports of transmission of these bacteria from dog to men, as well as from men to dog. A 5-year-old mixed-breed male dog was admitted with obstructive struvite urolithiasis relapsing for over 18 months, in Botucatu, in the state of São Paulo, Brazil. The strain, biochemically identified as Staphylococcus spp., was MDR and was treated off-label with vancomycin, which resulted in clinical cure. The strain was molecularly identified as Staphylococcus pseudintermedius and the mecA gene was identified. This is the main gene responsible for methicillin-resistant S. pseudintermedius (MRSP), which is often resistant to multiple antimicrobials. The hypotheses for this clinical case are the transmission from man to animal, since the tutor was an intensivist doctor, or the bacterium itself could be part of the animal's microbiota and due to other factors, such as stress or obstructive urinary disease, opened a doorway to infection by S. pseudintermedius. Further studies should elucidate the transmission of MDR bacteria between human and pets.(AU)

O potencial zoonótico de bactérias multirresistentes é uma preocupação global e os animais de companhia têm sido implicados na disseminação de bactérias resistentes; assim, é importante a vigilância, pois já existem relatos de transmissão destas bactérias do cão para o homem e vice-versa. Um cão, sem raça definida e de cinco anos de idade, foi atendido na cidade de Botucatu, São Paulo, Brasil, apresentando urolitíase obstrutiva de estruvita recorrente há um ano e meio. Na urocultura do animal foi isolada uma estirpe de Staphylococcus spp. multirresistente; o tratamento com vancomicina possibilitou acura clínica. A estirpe de Staphylococcus spp. isolada foi identificada molecularmente como S. pseudintermedius e nela foi identificada a presença do gene mecA, o principal responsável por S. pseuidintermedius resistente à meticilina (MRSP), e que é frequentemente resistente à múltiplos antimicrobianos. As hipóteses para este caso clínico são a transmissão do homem para o animal, pois o tutor era um médico intensivista, ou que a própria bactéria fazia parte da microbiota do animal e, devido a outros fatores como estresse e doença urinária obstrutiva, abriu-se uma porta de entrada para a infecção pelo S. pseudintermedius. Mais estudos são necessários para a elucidação da transmissão de bactérias multirresistentes entre animais de companhia e o ser humano.(AU)

Assuntos

RESUMO

Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20C freezer, directly in the -80C freezer, and at -196C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20C exhibited a lower recovery of M. bovis compared to storage at -80C (Dunns test, p=0.0018) and LN (Dunns test, p=0.1403), yet –80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedmans test, p=0.7765). Given the low loss proportion of 5% during storage at –20°C and the high cost equipment required for storage at –80°C and LN, we recommend storage at –20°C or –80°C, when this is available, for preservation of M. bovis field strains.(AU)

Pesquisa, desenvolvimento de novos métodos biotecnológicos, testes diagnósticos, confirmação de resultados e reinvestigações são possíveis por causa da disponibilidade de organismos vivos bem preservados e mantidos inalterados. A criopreservação tem mostrado ser um método de manutenção de cultura mais simples, mais confiável e estável em longo prazo. Temperatura de armazenamento e composição do veículo de suspensão são fatores que afetam a viabilidade de cepas micobacterianas. Três veículos e três temperaturas de armazenamento foram avaliados para definir um meio crioprotetor adequado para preservação de estirpes de Mycobacterium bovis. Colônias de 16 isolados de M. bovis foram usadas na preparação de suspensões, as quais foram adicionadas a três veículos: solução salina a 0,85% estéril, caldo Middlebrook 7H9 (7H9) e caldo Middlebrook 7H9 com piruvato de sódio (7H9p) substituindo o glicerol. Alíquotas dessas suspensões foram congeladas por três métodos diferentes, direto em freezer a -20C, direto em freezer a -80C e a -196C por imersão em nitrogênio líquido (LN). As amostras foram descongeladas a temperatura ambiente após 45, 90 e 120 dias. A viabilidade das micobactérias foi avaliada pela contagem de células vivas em placas com meio Stonebrink, antes e depois dos processos de congelamento. Armazenamento a -20C exibiu menor recuperação de M. bovis comparado a -80C (Teste de Dunn, p=0.0018) e LN (Teste de Dunn, p=0.0352). Não houve diferença entre armazenamento a –80°C e LN (Teste de Dunn, p=0.1403), mas –80°C apresentou melhores resultados do que LN. Os três veículos de suspensão não apresentaram diferença estatística (Teste de Friedman, p=0.7765). Dada à baixa proporção de perda (5%) durante o armazenamento a –20°C e ao alto custo do equipamento necessário para o armazenamento a –80°C, nós recomendamos o armazenamento a –20°C ou –80°C, quando disponível, para a preservação das estirpes de campo de M. bovis.(AU)

Assuntos

RESUMO

Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20C freezer, directly in the -80C freezer, and at -196C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20C exhibited a lower recovery of M. bovis compared to storage at -80C (Dunns test, p=0.0018) and LN (Dunn’s test, p=0.1403), yet –80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at –20°C and the high cost equipment required for storage at –80°C and LN, we recommend storage at –20°C or –80°C, when this is available, for preservation of M. bovis field strains.

Pesquisa, desenvolvimento de novos métodos biotecnológicos, testes diagnósticos, confirmação de resultados e reinvestigações são possíveis por causa da disponibilidade de organismos vivos bem preservados e mantidos inalterados. A criopreservação tem mostrado ser um método de manutenção de cultura mais simples, mais confiável e estável em longo prazo. Temperatura de armazenamento e composição do veículo de suspensão são fatores que afetam a viabilidade de cepas micobacterianas. Três veículos e três temperaturas de armazenamento foram avaliados para definir um meio crioprotetor adequado para preservação de estirpes de Mycobacterium bovis. Colônias de 16 isolados de M. bovis foram usadas na preparação de suspensões, as quais foram adicionadas a três veículos: solução salina a 0,85% estéril, caldo Middlebrook 7H9 (7H9) e caldo Middlebrook 7H9 com piruvato de sódio (7H9p) substituindo o glicerol. Alíquotas dessas suspensões foram congeladas por três métodos diferentes, direto em freezer a -20C, direto em freezer a -80C e a -196C por imersão em nitrogênio líquido (LN). As amostras foram descongeladas a temperatura ambiente após 45, 90 e 120 dias. A viabilidade das micobactérias foi avaliada pela contagem de células vivas em placas com meio Stonebrink, antes e depois dos processos de congelamento. Armazenamento a -20C exibiu menor recuperação de M. bovis comparado a -80C (Teste de Dunn, p=0.0018) e LN (Teste de Dunn, p=0.0352). Não houve diferença entre armazenamento a –80°C e LN (Teste de Dunn, p=0.1403), mas –80°C apresentou melhores resultados do que LN. Os três veículos de suspensão não apresentaram diferença estatística (Teste de Friedman, p=0.7765). Dada à baixa proporção de perda (5%) durante o armazenamento a –20°C e ao alto custo do equipamento necessário para o armazenamento a –80°C, nós recomendamos o armazenamento a –20°C ou –80°C, quando disponível, para a preservação das estirpes de campo de M. bovis.

Assuntos

RESUMO

Mycobacterium bovis is the agent of bovine tuberculosis, a disease endemic to all Brazilian states. Molecular typing techniques help to stratify and refine data, providing information that facilitates epidemiological research. In this study, MIRU-VNTR, targeting 24 loci, was employed to identify and characterize the genetic groups of M. bovis isolates obtained from an outbreak of bovine tuberculosis. Eighteen acid-fast bacilli isolates, obtained from bovine tissue samples, and reactive to the comparative cervical tuberculin test, were identified as species of the M. tuberculosis complex, and were genotyped by MIRU-VNTR with 24 primer pairs. Genotyping revealed three genetic profiles comprising one with 15 isolates (83.3%), one with two isolates (11.1%), and one profile with one unique isolate (5.6%). This distinction was achieved with the MIRU 31 primer, which resulted in clustering of two isolates into the same profile, and ETR A, B, and C, which discriminated the isolate with a unique profile. The occurrence of clustered isolates is indicative of recent transmission, whereas isolates with a unique profile suggest reactivation of latent infection. The presence of different M. bovis genotypes in the same herd suggests movement of infected animals or different sources of intra-herd infection. Use of the MIRU-VNTR molecular epidemiology technique in M. bovis isolates obtained from an outbreak of bovine tuberculosis in Rio Grande do Sul state demonstrated the genetic diversity of circulating strains, despite the presence of a predominant group.

Mycobacterium bovis é o agente da tuberculose bovina, enfermidade endêmica diagnosticada em todos os estados brasileiros. As técnicas de tipagem molecular auxiliam a estratificar e refinar dados, fornecendo informações que facilitam as atividades epidemiológicas. Neste estudo aplicou-se a técnica MIRU-VNTR com 24 loci para caracterizar e identificar os agrupamentos genéticos de isolados de M. bovis oriundos de um foco de tuberculose bovina. Dezoito isolados de bacilos álcool-ácido resistentes, obtidos de amostras de tecido de bovinos reagentes ao teste cervical comparativo e identificados como espécies do complexo M. tuberculosis, foram genotipados por meio de MIRU-VNTR com 24 pares de iniciadores. A genotipagem revelou três perfis genéticos distintos: um para 15 isolados (83,3%); um para dois isolados (11,1%) e, por fim, um isolado com perfil único (5,6%). Os iniciadores responsáveis por essa distinção foram MIRU 31, que agrupou dois isolados em um perfil, e ETR A, B e C, que discriminaram o isolado de perfil único. A ocorrência de agrupamentos de isolados é indicativa de transmissão recente, ao passo que isolados de perfil único sugerem reativação de infecção latente. A presença de diferentes genótipos de M. bovis no mesmo rebanho sugere haver circulação de animais doentes ou existirem diferentes fontes de infecção intrarrebanho. A aplicação da técnica de epidemiologia molecular MIRU-VNTR a isolados de M. bovis obtidos a partir de um foco de tuberculose bovina no Rio Grande do Sul demonstrou haver diversidade genética entre as estirpes em circulação, embora exista um agrupamento predominante.

Assuntos

RESUMO

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.

Assuntos

RESUMO

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)

Assuntos

RESUMO

Mycobacterium bovis is the agent of bovine tuberculosis, a disease endemic to all Brazilian states. Molecular typing techniques help to stratify and refine data, providing information that facilitates epidemiological research. In this study, MIRU-VNTR, targeting 24 loci, was employed to identify and characterize the genetic groups of M. bovis isolates obtained from an outbreak of bovine tuberculosis. Eighteen acid-fast bacilli isolates, obtained from bovine tissue samples, and reactive to the comparative cervical tuberculin test, were identified as species of the M. tuberculosis complex, and were genotyped by MIRU-VNTR with 24 primer pairs. Genotyping revealed three genetic profiles comprising one with 15 isolates (83.3%), one with two isolates (11.1%), and one profile with one unique isolate (5.6%). This distinction was achieved with the MIRU 31 primer, which resulted in clustering of two isolates into the same profile, and ETR A, B, and C, which discriminated the isolate with a unique profile. The occurrence of clustered isolates is indicative of recent transmission, whereas isolates with a unique profile suggest reactivation of latent infection. The presence of different M. bovis genotypes in the same herd suggests movement of infected animals or different sources of intra-herd infection. Use of the MIRU-VNTR molecular epidemiology technique in M. bovis isolates obtained from an outbreak of bovine tuberculosis in Rio Grande do Sul state demonstrated the genetic diversity of circulating strains, despite the presence of a predominant group.(AU)

Mycobacterium bovis é o agente da tuberculose bovina, enfermidade endêmica diagnosticada em todos os estados brasileiros. As técnicas de tipagem molecular auxiliam a estratificar e refinar dados, fornecendo informações que facilitam as atividades epidemiológicas. Neste estudo aplicou-se a técnica MIRU-VNTR com 24 loci para caracterizar e identificar os agrupamentos genéticos de isolados de M. bovis oriundos de um foco de tuberculose bovina. Dezoito isolados de bacilos álcool-ácido resistentes, obtidos de amostras de tecido de bovinos reagentes ao teste cervical comparativo e identificados como espécies do complexo M. tuberculosis, foram genotipados por meio de MIRU-VNTR com 24 pares de iniciadores. A genotipagem revelou três perfis genéticos distintos: um para 15 isolados (83,3%); um para dois isolados (11,1%) e, por fim, um isolado com perfil único (5,6%). Os iniciadores responsáveis por essa distinção foram MIRU 31, que agrupou dois isolados em um perfil, e ETR A, B e C, que discriminaram o isolado de perfil único. A ocorrência de agrupamentos de isolados é indicativa de transmissão recente, ao passo que isolados de perfil único sugerem reativação de infecção latente. A presença de diferentes genótipos de M. bovis no mesmo rebanho sugere haver circulação de animais doentes ou existirem diferentes fontes de infecção intrarrebanho. A aplicação da técnica de epidemiologia molecular MIRU-VNTR a isolados de M. bovis obtidos a partir de um foco de tuberculose bovina no Rio Grande do Sul demonstrou haver diversidade genética entre as estirpes em circulação, embora exista um agrupamento predominante.(AU)

Assuntos

RESUMO

Milk samples from 16 cows that tested positive on the tuberculin test in the state of Paraíba, northeastern Brazil, were used for mycobacteria isolation and identification. Mycobacteria were isolated from five (31.25%) of the 16 milk samples; three samples were classified as M. bovis, and two as belonging to the Mycobacterium genus. This is probably the first study of isolation and identification of M. bovis in milk from cows in Northeastern Brazil, which suggests that humans are at risk of contamination by ingestion.(AU)

Amostras de leite de 16 vacas positivas no teste da tuberculinização no Estado da Paraíba, Nordeste do Brasil, foram utilizadas para isolamento e identificação de micobactérias. Foram isoladas micobactérias em cinco (31,25%) das 16 amostras de leite: três amostras foram classificadas como M. bovis e duas como pertencentes ao gênero Mycobacterium. De acordo com nosso conhecimento, este é o primeiro estudo de isolamento e identificação de M. bovis em leite de vacas no Nordeste do Brasil, o que sugere que os seres humanos estão em risco de contaminação por ingestão.(AU)

Assuntos

RESUMO

Milk samples from 16 cows that tested positive on the tuberculin test in the state of Paraíba, northeastern Brazil, were used for mycobacteria isolation and identification. Mycobacteria were isolated from five (31.25%) of the 16 milk samples; three samples were classified as M. bovis, and two as belonging to the Mycobacterium genus. This is probably the first study of isolation and identification of M. bovis in milk from cows in Northeastern Brazil, which suggests that humans are at risk of contamination by ingestion.

Amostras de leite de 16 vacas positivas no teste da tuberculinização no Estado da Paraíba, Nordeste do Brasil, foram utilizadas para isolamento e identificação de micobactérias. Foram isoladas micobactérias em cinco (31,25%) das 16 amostras de leite: três amostras foram classificadas como M. bovis e duas como pertencentes ao gênero Mycobacterium. De acordo com nosso conhecimento, este é o primeiro estudo de isolamento e identificação de M. bovis em leite de vacas no Nordeste do Brasil, o que sugere que os seres humanos estão em risco de contaminação por ingestão.

Assuntos