Granulomatous lymphadenitis in a dog caused by Mycobacterium intracellulare

Background: Mycobacteriosis is caused by bacteria belonging to the genus Mycobacterium, with considerable zoonoticpotential and risk to public health. Infection in dogs is rare and is usually associated with immunosuppression, resultingfrom eating meat or contact with contaminated soil or fomites. Dogs are also known as potential sources for the spread ofatypical tuberculosis in humans and other animals. This paper aims to describe the clinical, cytological, histopathological,and molecular findings of a male canine seen at University Veterinary Hospital of Cuiabá, Mato Grosso, with generalizedlymphadenomegaly associated Mycobacterium intracellulare infection.Case: A 2-year-old male Lhasa Apso dog was referred to the University Veterinary Hospital in Cuiabá city, located in theMidwest region of Brazil. The patient had a history of intermittent claudication of the left pelvic limb for approximately6 months and lymphadenomegaly with progression for approximately 2 months. The dog had wheezing and generalizedlymphadenopathy (submandibular, axillary, and popliteal lymph nodes); cryptorchidism was also observed. A completeblood count revealed nonspecific results, and in the serum biochemical profile, the values of urea, creatinine, albumin, andalanine aminotransferase were within the reference range. No changes were observed on the radiography of the femurotibiopatellar joints. Considering the generalised lymphadenopathy, fine needle aspiration cytology and histopathologicalexamination through biopsy of the lymph nodes was performed. On the cytology and histopathology, numerous negativeimages of moderately refringent bacillary structures distending the cytoplasm from the macrophages was found. Thesamples were also subjected to special Ziehl-Neelsen staining, which confirmed an accentuated and diffuse granulomatouslymphadenitis associated with alcohol...

Granulomatous lymphadenitis in a dog caused by Mycobacterium intracellulare

Background: Mycobacteriosis is caused by bacteria belonging to the genus Mycobacterium, with considerable zoonoticpotential and risk to public health. Infection in dogs is rare and is usually associated with immunosuppression, resultingfrom eating meat or contact with contaminated soil or fomites. Dogs are also known as potential sources for the spread ofatypical tuberculosis in humans and other animals. This paper aims to describe the clinical, cytological, histopathological,and molecular findings of a male canine seen at University Veterinary Hospital of Cuiabá, Mato Grosso, with generalizedlymphadenomegaly associated Mycobacterium intracellulare infection.Case: A 2-year-old male Lhasa Apso dog was referred to the University Veterinary Hospital in Cuiabá city, located in theMidwest region of Brazil. The patient had a history of intermittent claudication of the left pelvic limb for approximately6 months and lymphadenomegaly with progression for approximately 2 months. The dog had wheezing and generalizedlymphadenopathy (submandibular, axillary, and popliteal lymph nodes); cryptorchidism was also observed. A completeblood count revealed nonspecific results, and in the serum biochemical profile, the values of urea, creatinine, albumin, andalanine aminotransferase were within the reference range. No changes were observed on the radiography of the femurotibiopatellar joints. Considering the generalised lymphadenopathy, fine needle aspiration cytology and histopathologicalexamination through biopsy of the lymph nodes was performed. On the cytology and histopathology, numerous negativeimages of moderately refringent bacillary structures distending the cytoplasm from the macrophages was found. Thesamples were also subjected to special Ziehl-Neelsen staining, which confirmed an accentuated and diffuse granulomatouslymphadenitis associated with alcohol...(AU)

Diagnosis of goat tuberculosis using tuberculinization and molecular techniques

Tuberculosis is an infectious, chronic, and worldwide disease. It has been known since the beginning of humanity and still negatively influences public health and livestock, especially, in Brazil, in the northeast. Etiologic agents are the mycobacteria of the Mycobacterium tuberculosis complex, which is the most important in mammals' involvement. The state of Bahia has 68.7% of its territory located in the semiarid region and holds the largest goat herd in the country. Goat breeding is a social and economic activity that adds value to this region. Up to the present, data on goat tuberculosis is unknown in this state. Thus, this study seeks data on tuberculosis prevalence in goats in a semiarid region of Bahia by using the comparative tuberculin test and multiplex polymerase chain reaction (PCR). A total of 600 adult animals of both sexes were evaluated. A prevalence of 0.33% (2/600) and 33.33% (1/3) properties were found for positive animals. Each assessed property had a questionnaire to analyze the epidemiological data management and relevant aspects for the disease occurrence. To confirm the positive tuberculin test results, PCR was used to detect and identify the pathogenic mycobacteria involved in the infection. It is concluded that most of the properties performing goat breeding in the region show low technification levels and promote farming between different species. Low prevalence of the disease alerts preventive measures to avoid major proportion situations that could influence the goat breeding in the state.

The Rv2807 target gene: a determining factor to directly detect Mycobacterium bovis from suspected bovine tuberculosis lesions / Gene alvo Rv2807: um fator determinante para detectar Mycobacterium bovis diretamente de lesões suspeitas de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis, a species belonging to the Mycobacterium tuberculosis complex (MTC) group. Direct bTB diagnosis from suggestive lesions can be performed by nested q-PCR targeting the Rv2807 gene present in the MTC group, as well as the TbD1 gene, present in M. bovis. In this context, the aim of the present study was to assess the importance of considering positive MTC results for the Rv2807 target gene obtained through the nested real time polymerase chain reaction (nested q-PCR) applied to samples obtained directly from suspected bTB lesions. A total of 174 samples of suggestive bTB caseous lesions were obtained during cattle slaughter in slaughterhouses in the state of Mato Grosso, Brazil. DNA was extracted from the lesions and nested q-PCR was performed to detect both MTC and M. bovis. Both samples positive for the Rv2807 (41/174) and TbD1 (29/174) were submitted to bacterial culturing (23/41), and the DNA of the isolates (23) was extracted and submitted again to nested q-PCR. The Rv2807 gene (MTC) was previously amplified by nested q-PCR directly from the lesions, although the TbD1 gene specific for M. bovis was not amplified previously in four of the successfully isolated samples (4/23), only following isolation, and only the Rv2807 gene was amplified before and after isolation. In conclusion, the target gene Rv2807(MTC) exhibited higher positivity in the analyzed samples compared to the TbD1 gene (M. bovis).

A tuberculose bovina (bTB) é uma zoonose causada pelo Mycobacterium bovis, uma espécie pertencente ao grupo do complexo Mycobacterium tuberculosis (MTC). O diagnóstico direto de bTB a partir de lesões sugestivas pode ser realizado por nested q-PCR visando o gene Rv2807 presente no grupo MTC, bem como o gene TbD1, presente em M. bovis. Nesse contexto, o objetivo do presente estudo foi avaliar a importância de considerar os resultados de MTC positivos para o gene alvo Rv2807 obtidos através da reação em cadeia da polimerase nested real time (nested q-PCR) aplicada a amostras obtidas diretamente de lesões suspeitas de bTB. Um total de 174 amostras de lesões caseosas sugestivas de bTB foram obtidas durante o abate de bovinos em frigoríficos do estado de Mato Grosso, Brasil. DNA foi extraído das lesões e nested q-PCR foi realizado para detectar tanto MTC quanto M. bovis. Ambas as amostras positivas para Rv2807 (41/174) e TbD1 (29/174) foram submetidas a cultura bacteriana (23/41), e o DNA dos isolados (23) foi extraído e submetido novamente à nested q-PCR. O gene Rv2807 (MTC) foi previamente amplificado por nested q-PCR diretamente das lesões, embora o gene TbD1 específico para M. bovis não tenha sido amplificado anteriormente em quatro das amostras isoladas com sucesso (4/23), apenas após o isolamento, e apenas o gene Rv2807 foi amplificado antes e após o isolamento. Em conclusão, o gene alvo Rv2807 (MTC) apresentou maior positividade nas amostras analisadas em relação ao gene TbD1 (M. bovis).

Detection of mycobacteria in coalho cheese sold in Northeastern Brazil / Detecção de micobactérias em queijo de coalho comercializado no Nordeste do Brasil

The aim of the present study was to detect and identify Mycobaterium spp. in 50 samples of coalho cheese sold in the Northeast region of Brazil. Of the 50 analyzed samples, 35 were produced by the artisanal process, using raw milk, and 15 originated from industrialized establishments that pasteurize milk. Conventional and real-time nested PCR for the rv2807 gene of the M. tuberculosis complex was performed directly from the 50 analyzed samples. Samples of coalho cheese were grown simultaneously in Stonebrink medium, and conventional PCR was performed from the bacterial isolates with primers that flank differentiation region 4 (DR4), specific to M. bovis, mb400F. Bacterial isolates negative by PCR for RD4 were subjected to PCR for hsp65 of Mycobacterium spp., with subsequent DNA sequencing. The cultures were negative for the M. tuberculosis complex, but two samples (4%) from the artisanal process (with raw milk) exhibited identity with hsp65 of Mycobacterium lehmanii (Sequence ID: KY933786.1, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, identities: 331/371 [89%]), showing to be indicative environmental contamination. Non-tuberculous mycobacteria are emergent and ubiquitous microorganisms; therefore, they deserve greater attention in the cheese production chain, both in terms of Good Agricultural Practices (GAP)

O presente estudo teve como objetivo detectar e identificar Mycobaterium spp. em 50 amostras de queijo de coalho comercializados na região Nordeste do Brasil. Das 50 amostras analisadas, 35 foram produzidas pelo processo artesanal, com leite cru e 15 provenientes de estabelecimentos industrializados que realizam a pasteurização do leite. Nested-PCR convencional e em tempo real para o gene rv2807 do Complexo M. tuberculosis foi realizada diretamente das cinquenta amostras analisadas. Paralelamente, as amostras de queijo de coalho foram cultivadas em meio de Stonebrink e dos isolados bacterianos foi realizada PCR convencional com iniciadores que flanqueiam a região de diferenciação 4 (RD4), específica de M. bovis, mb400F. Os isolados bacterianos negativos na PCR para RD4 foram submetidos à PCR para hsp65 de Mycobacterium spp., com posterior sequenciamento do DNA. As culturas mostraram-se negativas para o Complexo M. tuberculosis, porém duas amostras oriundas do processo artesanal (com leite cru) (4%) apresentaram identidade com hsp65 de Mycobacterium lehmanii (ID da sequência: KY933786.1, identidades: 312/363 [86%]); e Mycobacterium rutilum (ID da sequência: LT629971.1, identidades: 331/371 [89%]), apontando-se como indicativos de contaminação ambiental. As micobactérias-não-tuberculosas (MNT) são microrganismos emergentes e de natureza ubíqua. Devido a essas características merecem maior atenção na cadeia produtiva de queijos, tanto nas boas práticas agropecuárias (BPAs) quanto nas boas práticas de fabricação de alimentos (BPFs).

Detection of mycobacteria in coalho cheese sold in Northeastern Brazil / Detecção de micobactérias em queijo de coalho comercializado no Nordeste do Brasil

The aim of the present study was to detect and identify Mycobaterium spp. in 50 samples of coalho cheese sold in the Northeast region of Brazil. Of the 50 analyzed samples, 35 were produced by the artisanal process, using raw milk, and 15 originated from industrialized establishments that pasteurize milk. Conventional and real-time nested PCR for the rv2807 gene of the M. tuberculosis complex was performed directly from the 50 analyzed samples. Samples of coalho cheese were grown simultaneously in Stonebrink medium, and conventional PCR was performed from the bacterial isolates with primers that flank differentiation region 4 (DR4), specific to M. bovis, mb400F. Bacterial isolates negative by PCR for RD4 were subjected to PCR for hsp65 of Mycobacterium spp., with subsequent DNA sequencing. The cultures were negative for the M. tuberculosis complex, but two samples (4%) from the artisanal process (with raw milk) exhibited identity with hsp65 of Mycobacterium lehmanii (Sequence ID: KY933786.1, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, identities: 331/371 [89%]), showing to be indicative environmental contamination. Non-tuberculous mycobacteria are emergent and ubiquitous microorganisms; therefore, they deserve greater attention in the cheese production chain, both in terms of Good Agricultural Practices (GAP)(AU)

O presente estudo teve como objetivo detectar e identificar Mycobaterium spp. em 50 amostras de queijo de coalho comercializados na região Nordeste do Brasil. Das 50 amostras analisadas, 35 foram produzidas pelo processo artesanal, com leite cru e 15 provenientes de estabelecimentos industrializados que realizam a pasteurização do leite. Nested-PCR convencional e em tempo real para o gene rv2807 do Complexo M. tuberculosis foi realizada diretamente das cinquenta amostras analisadas. Paralelamente, as amostras de queijo de coalho foram cultivadas em meio de Stonebrink e dos isolados bacterianos foi realizada PCR convencional com iniciadores que flanqueiam a região de diferenciação 4 (RD4), específica de M. bovis, mb400F. Os isolados bacterianos negativos na PCR para RD4 foram submetidos à PCR para hsp65 de Mycobacterium spp., com posterior sequenciamento do DNA. As culturas mostraram-se negativas para o Complexo M. tuberculosis, porém duas amostras oriundas do processo artesanal (com leite cru) (4%) apresentaram identidade com hsp65 de Mycobacterium lehmanii (ID da sequência: KY933786.1, identidades: 312/363 [86%]); e Mycobacterium rutilum (ID da sequência: LT629971.1, identidades: 331/371 [89%]), apontando-se como indicativos de contaminação ambiental. As micobactérias-não-tuberculosas (MNT) são microrganismos emergentes e de natureza ubíqua. Devido a essas características merecem maior atenção na cadeia produtiva de queijos, tanto nas boas práticas agropecuárias (BPAs) quanto nas boas práticas de fabricação de alimentos (BPFs).(AU)

Tuberculosis caused by Mycobacterium tuberculosis complex in a captive tapir (Tapirus terrestris)

Background: Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis complex has already been identified in a great number of wild species. Captive animals may have a potential source for zoonoses, because is related to factors such as cohabitation, direct contact with the public, presence of biological vectors, nutritional deficiencies, absence of sanitary barriers, precautionary hygienic measures and sanitary vigilance. In Brazil, there is little information on tuberculosis in captivity animals, and little attention is given to the risks of disease transmission from humans. The aim of this study was to report the first diagnosed case of tuberculosis Tapirus terrestris in Brazilian Amazon region.Case: One Tapirus terrestris was kept by local zoobotanic foundation in city of Marabá, Southeast of Pará state, Brazilian Amazon, and became ill. Physical examination revealed cough, sneezing, nasal outflow, dyspnea, hyperthermia and lethargy, leading to death. Necropsy demonstrated severe pulmonary alterations: thickening of the inter-alveolar septa, alveolar emphysema, and miliary nodules with dimensions up to 5 mm, which were yellowish-white, caseous, and sometimes calcified. Additionally, large areas of caseous compaction of the parenchyma, characteristic of caseous tuberculosis. Histopathological analysis revealed a process characteristic of mycobacterial infection, with alveoli filled with caseous exudate and thickened septa and fibrocytes, in addition to recently formed tubercles, some with caseous necrosis, calcifications and Langhans cells. In the Ziehl-Neelsen staining, alcohol-acid resistant bacilli were observed in the mesenteric lymph nodes. No mycobacterial growth was observed in Lowenstein-Jensen culture medium. A nested PCR followed by a sequencing assay targeting the hsp65 gene and M. tuberculosis complex member was detected. Water and M. tuberculosis H37Rv were used as negative and positive controls, respectively.[...](AU)

Tuberculosis caused by Mycobacterium tuberculosis complex in a captive tapir (Tapirus terrestris)

Background: Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis complex has already been identified in a great number of wild species. Captive animals may have a potential source for zoonoses, because is related to factors such as cohabitation, direct contact with the public, presence of biological vectors, nutritional deficiencies, absence of sanitary barriers, precautionary hygienic measures and sanitary vigilance. In Brazil, there is little information on tuberculosis in captivity animals, and little attention is given to the risks of disease transmission from humans. The aim of this study was to report the first diagnosed case of tuberculosis Tapirus terrestris in Brazilian Amazon region.Case: One Tapirus terrestris was kept by local zoobotanic foundation in city of Marabá, Southeast of Pará state, Brazilian Amazon, and became ill. Physical examination revealed cough, sneezing, nasal outflow, dyspnea, hyperthermia and lethargy, leading to death. Necropsy demonstrated severe pulmonary alterations: thickening of the inter-alveolar septa, alveolar emphysema, and miliary nodules with dimensions up to 5 mm, which were yellowish-white, caseous, and sometimes calcified. Additionally, large areas of caseous compaction of the parenchyma, characteristic of caseous tuberculosis. Histopathological analysis revealed a process characteristic of mycobacterial infection, with alveoli filled with caseous exudate and thickened septa and fibrocytes, in addition to recently formed tubercles, some with caseous necrosis, calcifications and Langhans cells. In the Ziehl-Neelsen staining, alcohol-acid resistant bacilli were observed in the mesenteric lymph nodes. No mycobacterial growth was observed in Lowenstein-Jensen culture medium. A nested PCR followed by a sequencing assay targeting the hsp65 gene and M. tuberculosis complex member was detected. Water and M. tuberculosis H37Rv were used as negative and positive controls, respectively.[...]

Disseminated tuberculosis in a dog: a putative zooanthroponosis

Background: Zooantroponoses that affect pets are rare. Microorganisms involved in human tuberculosis has great importance to public health and has potentiality to produce reverse zoonoses. Infection by Mycobacterium tuberculosis is in top of the most important infectious diseases related to public health. In dogs, the infection occurs through prolonged exposure to aerosols and respiratory secretions from humans. Canine tuberculosis is little reported and known in Brazil, this work aims to describe the anatomopathological findings of the first case of canine tuberculosis in a 5-year-old dog in the Federal District and alert to the presence of this disease in this region.Case: The dog had history of close contact with its owner, who has been treated for pulmonary tuberculosis six months before. At necropsy scattered multifocal, firm, white to yellow-gray nodules, varying in size (3 mm to 10 mm) involving lungs, kidneys, heart, liver and spleen was observed. In frontal and occipital cerebral cortex, pale-gray circular lesions were detected with the same characteristics. The histological findings were, multifocal to coalescent granulomas, well organized with a central core of necrosis with foci of mineralization, surrounded by histiocytes, epithelioid macrophage, rare Langhans giant cells, some intact and degenerate plasma cells and neutrophils, and a rim of reactive fibroblasts producing fibrous connective tissue. Histological staining with ZN revealed free acid-fast bacilli and in the cytoplasm of macrophages and granulomas in all affected organs. The PCR allowed identification of DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues using the ReliaPrep™ FFPE gDNA Miniprep System®, according to the manufacturer’s instructions (Promega, EUA). The Speed-Oligo Mycobacteria assay was used to identify the presence of M. tuberculosis complex DNA in tissue samples.[...]

Disseminated tuberculosis in a dog: a putative zooanthroponosis

Background: Zooantroponoses that affect pets are rare. Microorganisms involved in human tuberculosis has great importance to public health and has potentiality to produce reverse zoonoses. Infection by Mycobacterium tuberculosis is in top of the most important infectious diseases related to public health. In dogs, the infection occurs through prolonged exposure to aerosols and respiratory secretions from humans. Canine tuberculosis is little reported and known in Brazil, this work aims to describe the anatomopathological findings of the first case of canine tuberculosis in a 5-year-old dog in the Federal District and alert to the presence of this disease in this region.Case: The dog had history of close contact with its owner, who has been treated for pulmonary tuberculosis six months before. At necropsy scattered multifocal, firm, white to yellow-gray nodules, varying in size (3 mm to 10 mm) involving lungs, kidneys, heart, liver and spleen was observed. In frontal and occipital cerebral cortex, pale-gray circular lesions were detected with the same characteristics. The histological findings were, multifocal to coalescent granulomas, well organized with a central core of necrosis with foci of mineralization, surrounded by histiocytes, epithelioid macrophage, rare Langhans giant cells, some intact and degenerate plasma cells and neutrophils, and a rim of reactive fibroblasts producing fibrous connective tissue. Histological staining with ZN revealed free acid-fast bacilli and in the cytoplasm of macrophages and granulomas in all affected organs. The PCR allowed identification of DNA was extracted from formalin fixed paraffin embedded (FFPE) tissues using the ReliaPrep™ FFPE gDNA Miniprep System®, according to the manufacturers instructions (Promega, EUA). The Speed-Oligo Mycobacteria assay was used to identify the presence of M. tuberculosis complex DNA in tissue samples.[...](AU)

Antimicrobial activity of essential oils from Lippia alba, Lippia sidoides, Cymbopogon citrates, Plectranthus amboinicus, and Cinnamomum zeylanicum against Mycobacterium tuberculosis / Atividade antimicrobiana de leos essenciais de Lippia alba, Lippia sidoides, Cymbopogon citrates, Plectranthus amboinicus, e Cinnamomum zeylanicum em Mycobacterium tuberculosis

The rise in cases of antibiotic-resistant Mycobacterium tuberculosis has become a major obstacle to the effective control of tuberculosis (TB) worldwide. Essential oils (EO) are complex mixtures that may contain between 20 and 60 components, with two or three major compounds at relatively high concentrations (20-70%) that are responsible for their pharmacological properties. The objective of this study was to assess the antimicrobial activity of the EOs, bushy lippia (Lippia alba), rosemary pepper (Lippia sidoides), lemon grass (Cymbopogon citratus), Mexican mint or Indian borage (Plectranthus amboinicus), and true cinnamon (Cinnamomum zeylanicum), against Mycobacterium tuberculosis H37Rv. Chemical characterization of the EOs was performed by gas chromatography coupled to mass spectrometry. The minimum inhibitory concentration (MIC) was determined by the microdilution-based resazurin microtiter assay. Four EOs were able to inhibit the growth of M. tuberculosis, with MICs of 286.5±130.2g/mL (C. zeylanicum), 299.5±117.2g/mL (L. sidoides), 351.6±39.06g/mL (P. amboinicus), and 1,250g/mL (C. citratus). Only the EO of L. alba showed no antimycobacterial activity at the tested concentrations, with an MIC greater than 1,250µg/mL. Results of this study suggested that C. zeylanicum, L. sidoides, and P. amboinicus could be important sources of bactericidal compounds against M. tuberculosis and require further investigation. The activity against M. tuberculosis of these three EOs has not been reported previously. The results show the high potential of the tested antimycobacterial EOs, making them a promising alternative for TB treatment. This data also confirms the importance of bioprospecting studies for active substances with antimycobacterial activity, which are still scarce.(AU)

O aumento no número de casos de Mycobacterium tuberculosis resistentes tem se tornado um grande obstáculo no controle efetivo da tuberculose (TB) mundialmente. Os óleos essenciais (OE), que são misturas complexas que podem conter entre 20 a 60 componentes, apresentam dois ou três compostos principais, em concentrações relativamente elevadas, 20 a 70%, que são responsáveis pelas suas propriedades farmacológicas. O objetivo deste estudo foi avaliar a atividade antimicobacteriana dos seguintes óleos essenciais (OEs) em Mycobacterium tuberculosis H37Rv: erva-cidreira (Lippia alba), alecrim-pimenta (Lippia sidoides), capim-limão (Cymbopogon citratus), orégano (Plectranthus amboinicus) e canela (Cinnamomum zeylanicum). A caracterização química dos OEs foi realizada por cromatografia gasosa acoplada a espectrometria de massa. A Concentração Inibitória Mínima (CIM) foi determinada pela técnica de microdiluição da resazurina. Quatro OEs foram capazes de inibir o crescimento de M. tuberculosis, com CIM de 286,5±130,2g/mL (C. zeylanicum), 299,5±117,2g/mL (L. sidoides), 351,6±39,06g/mL (P. amboinicus) e 1250g/mL (C. citratus). Somente o OE de L. alba não mostrou atividade antimicobacteriana nas conscentrações testadas, considerando CIM maiores que 1250µg/mL. Os resultados deste estudo sugerem que L. sidoides, C. zeylanicum e P. amboinicus podem ser fontes importantes de compostos bactericidas contra M. tuberculosis e prováveis candidatos a serem investigados. A atividade contra M. tuberculosis desses três OEs não foi relatada em estudos anteriores. Os resultados mostram o elevado potencial antimicobacteriano dos OEs analisados, fazendo deles uma alternativa promissora para o tratamento da TB. Os resultados obtidos demonstraram a importância de pesquisas para bioprospecção de substâncias ativas com ação antimicobacteriana, que ainda são escassas.(AU)

Isolation and identification of Mycobacterium bovis in cattle slaughtered from an abattoir in Garanhuns, Pernambuco / Isolamento e identificação de Mycobacterium bovis em bovinos abatidos em matadouro público de Garanhuns, Pernambunco

Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (CMT), members including M. bovis. The sanitary inspection in slaughterhouses has a great importance on public health for the removal of contaminated meat with pathological lesions. Cattle slaughtered in the abattoir of Garanhuns were subjected to macroscopic, histological, bacteriological, and molecular analyses. Lung inspection revealed gross lesions suggestive of tuberculosis. The characterization of tuberculous granulomas was performed by histopathology and hematoxylin and eosin (HE) staining. The identification of acid-fast bacilli (AFB) was performed on smears with fresh material and histological staining using Ziehl-Neelsen stain. Bacteriological diagnosis was carried out using Stonebrink and Lowenstein-Jensen media, and polymerase chain reaction (PCR) was performed from the DNA extracted from colonies for the identification of Mycobacterium spp. We found that 1% (32/3,.180) of the collected lungs showed lesions suggestive of tuberculosis. Histopathological analysis revealed that 65.62% (21/32) samples had granulomatous pneumonia. AFB was detected in 46.88% (15/32) smears and 4.16% (1/24) histological sections, as analyzed by Ziehl-Neelsen staining. Moreover, 68.75% (22/32) samples showed bacterial growth and molecular identification 90% (18/20) of the samples were positive...

A tuberculose é uma doença infecciosa crônica causada por membros do complexo Mycobacterium tuberculosis (CMT), incluindo M. bovis. A inspeção sanitária em matadouros frigoríficos possui grande importância para a saúde pública pela remoção de carne contaminada com lesões patológicas. Os bovinos abatidos no matadouro de Garanhuns-PE foram submetidos a análises macroscópicas, histológicas, bacteriológicas e moleculares. A inspeção pulmonar revelou lesões sugestivas de tuberculose.A caracterização de granulomas tuberculoides foi realizada através da histopatologia e coloração de hematoxilina e eosina (HE). A identificação de bacilo álcool - ácido resistente (BAAR) foi realizado em esfregaços com material fresco e em cortes histológicos sob a coloração de Zihel-Neelsen. O diagnóstico bacteriológico foi realizado em meio de cultivo Stonebrink e Lowenstein-Jensen e a reação em cadeia da polimerase (PCR) sucedeu a partir de DNA extraído de colônias para identificação do Mycobacterium sp... Verificou-se que1% (32/3.180) dos pulmões coletados apresentaram lesões sugestivas de tuberculose. Na análise histopatológica 65.62% (21/32) amostras tinham pneumonia granulomatosa. BAAR foram detectados em 46,88% (15/32) esfregaços e 4,16% (1/24) em corte histológico conforme analisado pela coloração de Ziehl-Neelsen. Além disso, em 68,75% (22/32) das amostras houve crescimento bacteriano e na...

Isolation and identification of Mycobacterium bovis in cattle slaughtered from an abattoir in Garanhuns, Pernambuco / Isolamento e identificação de Mycobacterium bovis em bovinos abatidos em matadouro público de Garanhuns, Pernambunco

Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (CMT), members including M. bovis. The sanitary inspection in slaughterhouses has a great importance on public health for the removal of contaminated meat with pathological lesions. Cattle slaughtered in the abattoir of Garanhuns were subjected to macroscopic, histological, bacteriological, and molecular analyses. Lung inspection revealed gross lesions suggestive of tuberculosis. The characterization of tuberculous granulomas was performed by histopathology and hematoxylin and eosin (HE) staining. The identification of acid-fast bacilli (AFB) was performed on smears with fresh material and histological staining using Ziehl-Neelsen stain. Bacteriological diagnosis was carried out using Stonebrink and Lowenstein-Jensen media, and polymerase chain reaction (PCR) was performed from the DNA extracted from colonies for the identification of Mycobacterium spp. We found that 1% (32/3,.180) of the collected lungs showed lesions suggestive of tuberculosis. Histopathological analysis revealed that 65.62% (21/32) samples had granulomatous pneumonia. AFB was detected in 46.88% (15/32) smears and 4.16% (1/24) histological sections, as analyzed by Ziehl-Neelsen staining. Moreover, 68.75% (22/32) samples showed bacterial growth and molecular identification 90% (18/20) of the samples were positive...(AU)

A tuberculose é uma doença infecciosa crônica causada por membros do complexo Mycobacterium tuberculosis (CMT), incluindo M. bovis. A inspeção sanitária em matadouros frigoríficos possui grande importância para a saúde pública pela remoção de carne contaminada com lesões patológicas. Os bovinos abatidos no matadouro de Garanhuns-PE foram submetidos a análises macroscópicas, histológicas, bacteriológicas e moleculares. A inspeção pulmonar revelou lesões sugestivas de tuberculose.A caracterização de granulomas tuberculoides foi realizada através da histopatologia e coloração de hematoxilina e eosina (HE). A identificação de bacilo álcool - ácido resistente (BAAR) foi realizado em esfregaços com material fresco e em cortes histológicos sob a coloração de Zihel-Neelsen. O diagnóstico bacteriológico foi realizado em meio de cultivo Stonebrink e Lowenstein-Jensen e a reação em cadeia da polimerase (PCR) sucedeu a partir de DNA extraído de colônias para identificação do Mycobacterium sp... Verificou-se que1% (32/3.180) dos pulmões coletados apresentaram lesões sugestivas de tuberculose. Na análise histopatológica 65.62% (21/32) amostras tinham pneumonia granulomatosa. BAAR foram detectados em 46,88% (15/32) esfregaços e 4,16% (1/24) em corte histológico conforme analisado pela coloração de Ziehl-Neelsen. Além disso, em 68,75% (22/32) das amostras houve crescimento bacteriano e na...(AU)

Combination of commercially available molecular assays and culture based methods in diagnosis of tuberculosis and drug resistant tuberculosis

ABSTRACT Early diagnosis of tuberculosis is of major clinical importance. Among 4733 clinical specimens collected from 3363 patients and subjected to Ziehl-Neelsen microscopy, 4109 were inoculated onto Lõwenstein-Jensen slants and 3139 in Bactec/9000MB. Polymerase Chain Reaction (PCR) was performed in 3139 specimens, whereas, a genotypic assay was directly applied in 93 Mycobacterium tuberculosis complex PCR-positive for isoniazid and rifampicin resistance detection specimens (GenoType MTBDRplus). Recovered M. tuberculosis isolates (64) as well as, 21 more sent from Regional Hospitals were tested for antimycobacterial resistance with a phenotypic (manual MGIT-SIRE) and a genotypic assay (GenoType MTBDRplus). PCR in the clinical specimens showed excellent specificity (97.4%) and accuracy (96.8%), good sensitivity (70.4%), but low positive predictive value (40.3%). MGIT-SIRE performed to M. tuberculosis did not confer a reliable result in 16 isolates. Of the remaining 69 isolates, 15 were resistant to streptomycin, seven to isoniazid, seven to ethambutol and five to rifampicin. GenoType MTBDRplus correctly detected isoniazid (seven) and rifampicin-resistant M. tuberculosis strains (five), showing an excellent performance overall (100%). Susceptibility results by the molecular assay applied directly to clinical specimens were identical to those obtained from recovered isolates of the corresponding patients. Combining molecular and conventional methods greatly contribute to early diagnosis and accurate susceptibility testing of tuberculosis.(AU)

Diagnóstico histopatológico e molecular de lesões sugestivas de tuberculose em búfalos abatidos nos municípios de Macapá e Santana, estado do Amapá / Histopathological and molecular diagnosis of lesions suggestive of tuberculosis in buffaloes slaughtered in the municipalities of Macapá and Santana, Amapá state, Brazil

Este estudo teve como objetivo avaliar lesões sugestivas de tuberculose em búfalos abatidos em matadouros oficiais no Estado do Amapá, Brasil, a fim de confirmar o diagnóstico de tuberculose por avaliação histopatológica e molecular. As amostras de tecido de 20 búfalos que apresentavam lesões sugestivas de tuberculose, dos municípios de Macapá e Santana, foram coletadas. As amostras foram divididas em duas partes: uma delas foi fixada em formalina a 10% tamponada e rotineiramente processadas para avaliação histopatológica, coradas pela hematoxilina-eosina e Ziehl-Neelsen; e o outra parte foi usado para Nested-PCR para o complexo de Mycobacterium tuberculosis (CMT) e para Mycobacterium bovis. As lesões macroscópicas sugestivas de tuberculose foram observadas nos pulmões, linfonodos brônquicos, mediastínicos, retrofaríngeos e submandibulares, fígado e pleura. Histopatologicamente, todas as amostras apresentaram lesões sugestivas de tuberculose, caracterizadas por granulomas compostos por grande quantidade de infiltração de células epitelióides, células de Langerhans e linfócitos, margeando um centro necrótico, calcificado ou não, rodeado por cápsula de tecido conjuntivo fibroso. Bacilos álcool-ácido resistentes foram observados nos tecidos de 3/20 (15%) búfalos. Com relação à detecção molecular, 13/20 (65%) bubalinos apresentaram amostras de tecidos positivos: 6 foram positivos nas Nested-PCRs para CMT e M. bovis, um foi positivo apenas na Nested-PCR para CMT, e 6 foram positivos apenas na Nested-PCR para M. bovis. Os resultados deste estudo demonstram a importância de diagnosticar a tuberculose em búfalos na região e apontam para a necessidade de implementar medidas eficazes para controlar e erradicar a enfermidade.(AU)

This study aimed to evaluate suggestive lesions of tuberculosis in buffaloes slaughtered in official slaughterhouses in the State of Amapá, Brazil, in order to confirm the diagnosis of tuberculosis by histopathological and molecular evaluation. Tissue samples of 20 buffaloes showing lesions suggestive of tuberculosis, from the municipalities of Macapá and Santana, were collected. The samples were divided into two parts: one was fixed in 10% buffered formalin and routinely processed for histopathological evaluation, stained by hematoxylin-eosin and Ziehl-Neelsen; and the other was used for Nested-PCRs for Mycobacterium tuberculosis complex (MTC) and for Mycobacterium bovis. Gross lesions suggestive of tuberculosis were observed in the lungs, bronchial, mediastinic, retropharyngeal and submandibular lymph nodes, liver and pleura. Histopathologically, all samples showed lesions suggestive of tuberculosis, characterized by granulomas composed of large amount of infiltration of epithelioid cells, Langhans cells and lymphocytes, bordering a necrotic core, calcified or not, surrounded by a fibrous connective tissue capsule. Acid-fast bacilli were observed in the tissues of 3/20 (15%) buffaloes. With regards to the molecular detection, 13/20 (65%) buffaloes showed positive tissue samples: 6 were positive both in the MTC and M. bovis Nested-PCRs, one was positive only in the MTC Nested-PCR, and 6 were positive only in the M. bovis Nested-PCR. The results of this study demonstrate the importance of diagnosing TB in buffaloes in the region and point to the requirement to implement effective measures to control and eradicate the disease.(AU)

Diagnóstico histopatológico e molecular de lesões sugestivas de tuberculose em búfalos abatidos nos municípios de Macapá e Santana, estado do Amapá / Histopathological and molecular diagnosis of lesions suggestive of tuberculosis in buffaloes slaughtered in the municipalities of Macapá and Santana, Amapá state, Brazil

Este estudo teve como objetivo avaliar lesões sugestivas de tuberculose em búfalos abatidos em matadouros oficiais no Estado do Amapá, Brasil, a fim de confirmar o diagnóstico de tuberculose por avaliação histopatológica e molecular. As amostras de tecido de 20 búfalos que apresentavam lesões sugestivas de tuberculose, dos municípios de Macapá e Santana, foram coletadas. As amostras foram divididas em duas partes: uma delas foi fixada em formalina a 10% tamponada e rotineiramente processadas para avaliação histopatológica, coradas pela hematoxilina-eosina e Ziehl-Neelsen; e o outra parte foi usado para Nested-PCR para o complexo de Mycobacterium tuberculosis (CMT) e para Mycobacterium bovis. As lesões macroscópicas sugestivas de tuberculose foram observadas nos pulmões, linfonodos brônquicos, mediastínicos, retrofaríngeos e submandibulares, fígado e pleura. Histopatologicamente, todas as amostras apresentaram lesões sugestivas de tuberculose, caracterizadas por granulomas compostos por grande quantidade de infiltração de células epitelióides, células de Langerhans e linfócitos, margeando um centro necrótico, calcificado ou não, rodeado por cápsula de tecido conjuntivo fibroso. Bacilos álcool-ácido resistentes foram observados nos tecidos de 3/20 (15%) búfalos. Com relação à detecção molecular, 13/20 (65%) bubalinos apresentaram amostras de tecidos positivos: 6 foram positivos nas Nested-PCRs para CMT e M. bovis, um foi positivo apenas na Nested-PCR para CMT, e 6 foram positivos apenas na Nested-PCR para M. bovis. Os resultados deste estudo demonstram a importância de diagnosticar a tuberculose em búfalos na região e apontam para a necessidade de implementar medidas eficazes para controlar e erradicar a enfermidade.(AU)

This study aimed to evaluate suggestive lesions of tuberculosis in buffaloes slaughtered in official slaughterhouses in the State of Amapá, Brazil, in order to confirm the diagnosis of tuberculosis by histopathological and molecular evaluation. Tissue samples of 20 buffaloes showing lesions suggestive of tuberculosis, from the municipalities of Macapá and Santana, were collected. The samples were divided into two parts: one was fixed in 10% buffered formalin and routinely processed for histopathological evaluation, stained by hematoxylin-eosin and Ziehl-Neelsen; and the other was used for Nested-PCRs for Mycobacterium tuberculosis complex (MTC) and for Mycobacterium bovis. Gross lesions suggestive of tuberculosis were observed in the lungs, bronchial, mediastinic, retropharyngeal and submandibular lymph nodes, liver and pleura. Histopathologically, all samples showed lesions suggestive of tuberculosis, characterized by granulomas composed of large amount of infiltration of epithelioid cells, Langhans cells and lymphocytes, bordering a necrotic core, calcified or not, surrounded by a fibrous connective tissue capsule. Acid-fast bacilli were observed in the tissues of 3/20 (15%) buffaloes. With regards to the molecular detection, 13/20 (65%) buffaloes showed positive tissue samples: 6 were positive both in the MTC and M. bovis Nested-PCRs, one was positive only in the MTC Nested-PCR, and 6 were positive only in the M. bovis Nested-PCR. The results of this study demonstrate the importance of diagnosing TB in buffaloes in the region and point to the requirement to implement effective measures to control and eradicate the disease.(AU)

Infection by Mycobacterium bovis in a dog from Brazil

Tuberculosis (TB) is a chronic disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MtbC). This disease rarely affects dogs. Canine infections are usually caused by M. tuberculosis. Mycobacterium bovis infections are rare in dogs and associated with consumption of raw milk or contaminated products. Here, we report a Boxer dog who had a M. bovis infection and was admitted to a Brazilian veterinary hospital with a presumptive diagnosis of chronic ehrlichiosis. Despite receiving treatment for chronic ehrlichiosis, it progressed to death. TB was diagnosed during post-mortem examinations using histopathological analysis. Ziehl-Neelsen staining revealed acid-fast bacilli in the kidneys, liver, mesentery, and a mass adhered to the liver. Further, PCR-restriction analysis was performed to identify mycobacteria in the samples. A restriction profile compatible with MtbC was found in the lungs. In addition, PCR-based MtbC typing deletions at different loci of chromosome 9 enabled the identification of M. bovis in the lungs. Therefore, it is very essential to perform differential diagnosis of TB in dogs with non-specific clinical signs and who do not respond to treatment, particularly those who had been in contact with TB-infected cattle or owners. Further, we highlight the use of molecular methods for the identification of bacilli, improving the diagnosis and aiding epidemiological studies.(AU)

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis

ABSTRACT: Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.

RESUMO: A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)

Nasal swab real-time PCR is not suitable for in vivo diagnosis of bovine tuberculosis / A PCR em tempo real de swab nasal não é adequada para o diagnóstico in vivo de tuberculose bovina

Bovine tuberculosis (bTB) is a zoonosis causing economic losses and public health risks in many countries. The disease diagnosis in live animals is performed by intradermal tuberculin test, which is based on delayed hypersensitivity reactions. As tuberculosis has complex immune response, this test has limitations in sensitivity and specificity. This study sought to test an alternative approach for in vivo diagnosis of bovine tuberculosis, based on real-time polymerase chain reaction (PCR). DNA samples, extracted from nasal swabs of live cows, were used for SYBR® Green real-time PCR, which is able to differentiate between Mycobacterium tuberculosis and Mycobacterium avium complexes. Statistical analysis was performed to compare the results of tuberculin test, the in vivo gold standard bTB diagnosis method, with real-time PCR, thereby determining the specificity and sensitivity of molecular method. Cervical comparative test (CCT) was performed in 238 animals, of which 193 had suitable DNA from nasal swabs for molecular analysis, as indicated by amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and were included in the study. In total, 25 (10.5%) of the animals were CCT reactive, of which none was positive in the molecular test. Of the 168 CCT negative animals, four were positive for M. tuberculosis complex at real time PCR from nasal swabs. The comparison of these results generated values of sensitivity and specificity of 0% and 97.6%, respectively; moreover, low coefficients of agreement and correlation (-0.029 and -0.049, respectively) between the results obtained with both tests were also observed. This study showed that real-time PCR from nasal swabs is not suitable for in vivo diagnosis of bovine tuberculosis; thus tuberculin skin test is still the best option for this purpose.(AU)

A tuberculose bovina (bTB) é uma zoonose que causa perdas econômicas e riscos à saúde pública em muitos países. O diagnóstico da doença em animais vivos é realizado pelo teste intradérmico da tuberculina, que é baseado em reações de hipersensibilidade tardia. Como a tuberculose tem resposta imunológica complexa, este teste tem limitações em termos de sensibilidade e especificidade. Este estudo procurou desenvolver uma abordagem alternativa para o diagnóstico in vivo da tuberculose bovina, com base na reação em cadeia da polimerase (PCR) em tempo real. As amostras de DNA, extraídas de suabes nasais de vacas vivas, foram usadas para PCR em tempo real com SYBR® Green, capaz de diferenciar os complexos Mycobacterium tuberculosis e Mycobacterium avium. A análise estatística foi realizada para comparar os resultados de teste de tuberculina, padrão ouro para o diagnóstico in vivo da bTB, com PCR em tempo real, determinando-se assim a especificidade e sensibilidade do método molecular. O teste cervical comparativo (TCC) foi realizado em 238 animais, dos quais 193 tiveram DNA dos suabes nasais adequados para análise molecular, como indicado pela amplificação do gene gliceraldeído-3-fosfato-desidrogenase (GAPDH), e foram incluídos no estudo. No total, 25 (10,5%) animais foram reativos no TCC, dos quais nenhum foi positivo no teste molecular. Dos 168 animais negativos no TCC, quatro foram positivos para o complexo M. tuberculosis na PCR em tempo real a partir dos suabes nasais. A comparação destes resultados gerou valores de sensibilidade e especificidade de 0% e 97,6%, respectivamente; além disso, baixos coeficientes de concordância e correlação (-0,029 e -0,049, respectivamente) entre os resultados obtidos com ambos os testes também foram observados. Este estudo mostrou que a PCR em tempo real a partir de suabes nasais não é adequada para o diagnóstico in vivo da tuberculose bovina; portanto, o teste da tuberculina ainda é a melhor opção para este fim.(AU)