Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado ​​para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados ​​para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients

Abstract Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

Resumo A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Detection of paratuberculosis in dairy cows from southern Brazil / Detecção da paratuberculose em vacas leiteiras no sul do Brasil

Bovine paratuberculosis causes chronic, incurable diarrhea and weight loss, resulting in decreased cattle production. The disease is caused by Mycobacterium avium subsp. paratuberculosis (MAP), an obligate intracellular mycobactin-dependent mycobacterium that replicates slowly in the host and has heightened environmental resistance. In countries where the disease is found and the damage has been quantified, direct and indirect economic losses are extremely high. Local epidemiological data is of paramount importance for the implementation of control programs. Our objective was to verify whether paratuberculosis is present in commercial dairy herds in different mesoregions of RS. Therefore, a prospective, cross-sectional and observational study was performed on dairy cattle from five mesoregions of the RS state, Brazil. Milk samples taken from individual cows on commercial farms were tested using indirect ELISA tests and classified as negative, suspicious, or positive. In herds containing at least one positive cow, we conducted convenience sampling of feces directly from the rectal ampulla to identify MAP through PCR. Of the 362 cows tested, 20 were seroreactive for paratuberculosis from two mesoregions. The PCR tests were all positive; cows with a negative ELISA and positive PCR results probably indicate that the MAP was ingested and eliminated without causing infection. We found that paratuberculosis is likely endemic in the northwest and northeast mesoregions.

A paratuberculose bovina causa diarreia crônica e incurável, perda de peso e resulta em diminuição da produção. A doença é causada pelo Mycobacterium avium subsp. paratuberculosis (MAP), micobactéria intracelular obrigatória, dependente de micobactina, que se replica lentamente no hospedeiro e possui elevada resistência ambiental. Nos países onde a doença é encontrada e os danos foram quantificados, as perdas econômicas diretas e indiretas são extremamente altas. Os dados epidemiológicos locais são de suma importância para a implementação de programas de controle. Nosso objetivo foi verificar se a paratuberculose está presente em rebanhos leiteiros comerciais em diferentes mesorregiões do RS. Para tanto, foi realizado um estudo prospectivo, transversal e observacional em bovinos leiteiros de cinco mesorregiões do estado do RS, Brasil. Amostras de leite individuais, provenientes de vacas leiteiras de fazendas comerciais foram testadas com ELISA indireto e classificadas como negativas, suspeitas ou positivas. Em rebanhos contendo pelo menos uma vaca positiva, realizamos amostragem por conveniência, em que foram coletadas fezes diretamente da ampola retal, para identificar MAP por meio da PCR. Das 362 vacas testadas, 20 foram sororreativas para paratuberculose, oriundas de duas mesorregiões. Os testes de PCR foram todos positivos. Vacas com resultado negativo no teste ELISA e PCR positivo provavelmente indicam que o MAP foi ingerido e eliminado sem causar infecção. Sugere-se que a paratuberculose é provavelmente endêmica nas mesorregiões noroeste e nordeste.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.

Comparison of different diagnostic modalities for isolation of Mycobacterium Tuberculosis among suspected tuberculous lymphadenitis patients / Comparação de diferentes modalidades de diagnóstico para isolamento de Mycobacterium tuberculosis entre pacientes com suspeita de linfadenite tuberculosa

Tuberculosis is a communicable disease with high morbidity and mortality rates in developing countries. The study's primary objective is to compare conventional methods such as acid-fast bacillus (AFB) culture and microscopy with rapid diagnostic methods. The secondary objective is to compare histopathological and microbiological findings in suspected patients with tubercular lymphadenitis. A total of 111 samples (August 2018 to September 2019) of lymph nodes were processed for AFB microscopy, AFB cultures, drug-susceptibility testing (DST), histopathology, and Xpert Mycobacterium Tuberculosis (MTB)/resistance to Rifampin (RIF) assays. Out of 111 lymph node samples, 6 (5.4%) were positive for AFB smear microscopy, 84 (75.6%) were positive for AFB culture, 80 (70.7%) were positive on Gene Xpert, and 102 (91.8%) were indicative of tuberculosis for histopathology studies. Mycobacteria growth indicator tube (MGIT) culture positivity was 84 (75.6%) higher than solid Lowenstein-Jensen (LJ) culture 74 (66.6%). Positive cultures underwent phenotypic DST. Two cases were Multidrug-resistant (MDR) on DST, while three cases were Rifampicin resistant on Gene Xpert. The sensitivity of Genexpert was (62%) against the conventional AFB culture method. The poor performance of conventional lymphadenitis diagnostic methods requires early and accurate diagnostic methodology. Xpert MTB/RIF test can help in the treatment of multidrug-resistant TB cases. Nonetheless, rapid and conventional methods should be used for complete isolation of Mycobacterium tuberculosis.(AU)

A tuberculose é uma doença transmissível com altas taxas de morbimortalidade nos países em desenvolvimento. O objetivo principal do estudo é comparar métodos convencionais, como cultura de bacilo álcool-ácido resistente (BAAR) e microscopia, com métodos de diagnóstico rápido. O objetivo secundário é comparar os achados histopatológicos e microbiológicos em pacientes com suspeita de linfadenite tubercular. Um total de 111 amostras (agosto de 2018 a setembro de 2019) de gânglios linfáticos foi processado para microscopia de AFB, culturas de AFB, teste de susceptibilidade a drogas (DST), histopatologia e Xpert Mycobacterium tuberculosis (MTB)/ensaios de resistência à rifampicina (RIF). Das 111 amostras de linfonodos, 6 (5,4%) foram positivas para baciloscopia de AFB, 84 (75,6%) foram positivas para cultura de AFB, 80 (70,7%) foram positivas para o GeneXpert e 102 (91,8%) foram indicativas de tuberculose para estudos histopatológicos. A positividade da cultura do tubo indicador de crescimento de micobactérias (MGIT) foi 84 (75,6%), maior que a cultura sólida de Lowenstein-Jensen (LJ), 74 (66,6%). As culturas positivas foram submetidas a DST fenotípico. Dois casos eram multirresistentes (MDR) ao DST, enquanto três casos eram resistentes à rifampicina no GeneXpert. A sensibilidade do GeneXpert foi 62% contra o método convencional de cultura AFB. O fraco desempenho dos métodos convencionais de diagnóstico de linfadenite requer metodologia de diagnóstico precoce e precisa. O teste Xpert MTB/RIF pode ajudar no tratamento de casos de tuberculose multirresistente. No entanto, métodos rápidos e convencionais devem ser usados para o isolamento completo do Mycobacterium tuberculosis.(AU)

Avian mycobacteriosis in a naturally infected captive nocturnal curassow (Nothocrax urumutum)

A senile female nocturnal curassow (Nothocrax urumutum) kept under human care was found dead. Grossly, the lungs were congested and had a nodule with 2.0 cm in diameter adhered to the left lung. Histologically, there were multifocal to coalescent areas of necrosis characterized by deposition of amorphous and eosinophilic material with large amount of cellular debris, surrounded by numerous epithelioid macrophages, multinucleated Langhans giant cells, occasional lymphocytes and plasma cells. Ziehl-Neelsen stained sections demonstrated myriad of intralesional alcohol-acid resistant bacilli (BAAR). DNA extracted from lung samples were evaluated by nested polymerase chain reaction resulting in mycobacterial DNA identification. This is the first reported case of mycobacteriosis in a nocturnal curassow.(AU)

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)

Mastitis in cattle due to Mycobacteroides abscessus - case report / [Mastite bovina por Mycobacteroides abscessus - relato de caso]

Mastitis is considered the main disease that affects dairy cattle worldwide, and it is caused mainly by Staphylococcus aureus and environmental Streptococcus spp. Eventually, nonconventional pathogens, as rapidly growing mycobacteria (RGM), may also cause chronic mastitis, which will not be responsive to antibiotic treatments. Diagnosis of mastitis caused by RGM is a difficult task, and most of time this agent may be misdiagnosed. Here we describe a case of clinical mastitis caused by the RGM Mycobacteroides abscessus in a cow from Southern Brazil, confirmed by microbiological and molecular characterization. Our results reinforce the necessity of a detailed laboratorial identification of the agent and to include this agent in differential diagnosis of chronical clinical mastitis nonresponsive to treatment.

A mastite é considerada uma das principais doenças que afetam rebanhos leiteiros ao redor do mundo e é causada principalmente por Staphylococcus aureus e Streptococcus spp. ambientais. Eventualmente, patógenos não convencionais, como Mycobacterium de crescimento rápido (RGM), podem também causar mastite crônica, a qual não respondera ao tratamento com antimicrobianos. O diagnóstico de mastite causada por RGM é uma tarefa difícil, e a maioria das vezes esse agente pode ser subdiagnosticado. Neste relato, descreveu-se um caso de mastite clínica em uma vaca, no sul do Brasil, causada por RGM Mycobacteroides abscessus, confirmado por caracterização microbiológica e molecular. Os resultados reforçam a necessidade de um diagnóstico laboratorial detalhado para a identificação do agente e a inclusão deste como diagnóstico diferencial no reconhecimento de mastite crônica não responsiva ao tratamento.

Mycobacteriosis in an appearantly healty atlantic mackerel (Scomber Scombrus, L) and zoonotic potential / Micobacteriose em uma cavala atlântica aparentemente curada (Scomber Scombrus, L) e potencial zoonótico

Mycobacteriosis was detected in seven out of one Atlantic mackerel (Scomber scombrus) that was purchased for human consumption from a fish market. The fish was apparently healthy but during cleaning, several granulomatous foci were noticed in the visceral organs. Histopathological examination of the lesions revealed numerous foci characterized by caseous necrosis in the center of the lesion surrounded by epithelioid giant cells. Ziehl-Neelsen staining revealed the presence of rod-shaped, acid-fast bacteria. Furthermore, immunohistochemical examination revealed the presence of a protein of mycobacterial origin in giant cells and macrophages. Based on gross and microscopic findings, mycobacteriosis was diagnosed. This report showed that due to its zoonotic potential, mycobacteriosis should be considered even in healthy-appearing fishes for human consumption.

A micobacteriose foi detectada em uma de sete cavalas do Atlântico (Scomber scombrus) que foi comprada para consumo humano em um mercado de peixe. O peixe era aparentemente saudável, mas durante a limpeza, vários focos granulomatosos foram notados nos órgãos viscerais. O exame histopatológico das lesões revelou numerosos focos caracterizados por necrose caseosa no centro da lesão circundada por células gigantes epitelioides. A coloração de Ziehl-Neelsen revelou a presença de bactérias em forma de bastonete, com rápida acidez. Além disso, o exame imunohistoquímico revelou a presença de uma proteína de origem micobacteriana em células gigantes e macrófagos. Com base em achados grosseiros e microscópicos, a micobacteriose foi diagnosticada. Este relatório mostrou que devido a seu potencial zoonótico, a micobacteriose deve ser considerada mesmo em peixes saudáveis para consumo humano.

Mycobacterium avium subsp. paratuberculosis (MAP) in goat milk in the semiarid region of the Brazilian Northeast - molecular characterization

Background: Goat farming has been on the rise in Brazil in recent years. Overall, 93% of the national herd is concentrated in the Northeast, with the state of Paraíba being the largest goat milk producer in the country. Considering Mycobacterium avium subsp. paratuberculosis (MAP) as a sanitary issue for the development of animal farming with risks for human health and that is a notifiable disease, this research was structured with the objective of confirming the presence and performing a molecular characterization of MAP in goat milk destined for processing plants in the semiarid region of the Brazilian Northeast. Materials, Methods & Results: Samples from 179 production units and 5 collective bulk tanks and 4 samples of pasteurized goat milk were analyzed through Real-time polymerase chain reaction (qPCR). Genetic material (DNA) for MAP was found in the goat milk sample from 1 production unit (1/179). From this positive sample, 9 lactating goats were identified in the original property, 7 of which showed MAP DNA in milk samples (77.77%). The characterization of the nucleotide sequence detected in the positive sample has 99% identity with KJ173784. Discussion: One sample (1/179), from the production units, had MAP genetic material (DNA) detected using the molecular test. Samples from these production units represent the milk from all lactating goats from each producer. Therefore, it was possible to identify from which farm the samples originated, allowing individual animals to then be tested, with milk samples collected from 9 goats and MAP DNA detected in 7 of them (77.77%) via PCR. Control and/or prevention programs need this type of surveillance in reason that it allows the tracking of possible foci from milk samples collected from dairy products or cooling stations. The use of PCR to detect MAP foci via goat milk is thus advantageous because samples are obtained in a non-invasive manner, with faster results when compared to the culture technique. The low detection via PCR in goat milk may be related to factors such as the small amount of MAP eliminated and the intermittent excretion in asymptomatic animals, as also false-positive samples. Samples from the collective bulk tanks was negative. It is possible that the combination of milk from all the properties diluted the amount of MAP. This suggests that the sensitivity of the PCR can be improved if the samples are obtained from the pooled milk from the same property. In some regions of Brazil, for example, showed the frequency of Zona da Mata region of the state of Minas Gerais, Brazil, found 1.94% of positive samples (9/464) and 9.76% (4/41) of properties with at least 1 positive sample for MAP. Different results to what were found in the semiarid region of Paraíba, where climate and production characteristics are different. Goats are susceptible to 3 strains: type "S" (Sheep), "Bison type" and type "C" (Cattle). Previous contact with this species may explain the similarity between the strain found in goat milk and those detected from bovine samples. This must also be taken into consideration during diagnosis and upon implementation of control measures for paratuberculosis in goats. Mycobacterium avium subsp. paratuberculosis was recorded for the first time in goat milk in the semiarid region, which may reveal a potential biological risk to humans and suggests the need for active surveillance of the agent.

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).

Isolation and molecular identification of Mycobacterium bovis in tissue lesions of cattle slaughtered in slaughterhouses located in the State of Ceará / [Isolamento e identificação molecular de Mycobacterium bovis em lesões teciduais de bovinos abatidos em matadouros-frigoríficos localizados no estado do Ceará]

The objective of this research was to identify Mycobacterium bovis in lesions suggestive of tuberculosis in bovine carcasses in the State of Ceará, by means of bacteriological and molecular diagnostic tests. Between August 2017 and January 2019, the State inspection service (SIE) inspected 59,512 cattle, of which 7.4% (44 / 59,512) presented suggestive lesions. Of these animals, 68 samples were sent, of which 4.5% (31/68) located in the lung, 2.9% (20/68) in lymph nodes, 2.0% (14/68) in the liver, and 0.4% in the carcass (3/68). When performing bacteriological isolation, 15.9% (7/44) of bovines showed colony growth in the samples. The smears of the isolates were submitted to Zielh-Neelsen staining and all confirmed acid-fast bacilli. The polymerase chain reaction identified all isolates 100% (7/7) as M. bovis. The association of diagnostic techniques allowed to identify the presence of the agent in the State and the molecular analysis proved to be a beneficial technique in the monitoring of bovine tuberculosis and can be used as an auxiliary method in the bovine tuberculosis control and eradication program in the State of Ceará.

O objetivo do trabalho foi pesquisar Mycobacterium bovis em lesões sugestivas de tuberculose nas carcaças de bovinos no estado do Ceará, por meio dos testes de diagnóstico bacteriológico e molecular. Entre agosto de 2017 e janeiro de 2019, o Serviço de Inspeção Estadual (SIE) inspecionou 59.512 bovinos; destes, 7,4% (44/59.512) apesentaram lesões sugestivas. Desses animais foram enviadas 68 amostras, das quais 4,5% (31/68) estavam localizadas no pulmão, 2,9% (20/68) nos linfonodos, 2,0% (14/68) no fígado e 0,4% (3/68) na carcaça. Ao realizar o isolamento bacteriológico, 15,9% (7/44) dos bovinos evidenciaram crescimento de colônias nas amostras. Os esfregaços dos isolados foram submetidos à coloração de Zielh-Neelsen e todos eles confirmaram bacilo álcool-ácido resistente. A reação em cadeia da polimerase identificou todos os isolados, 100% (7/7), como M. bovis. A associação das técnicas de diagnóstico permitiu identificar a presença do agente no estado, e a análise molecular demonstrou ser uma técnica benéfica no monitoramento da tuberculose bovina, podendo ser utilizada como um método auxiliar no programa de controle e erradicação da tuberculose bovina no estado do Ceará.

Indirect ELISA as a complementary diagnostic method of bovine tuberculosis / ELISA indreto como método complementar de diagnóstico da tuberculose bovina

Bovine tuberculosis is an economic and health problem, requiring precise diagnostic methods for its control and eradication. The aim of this study was to evaluate the performance of a commercial enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of bovine tuberculosis. A total of 1,644 cattle from eight dairy herds were evaluated using the comparative cervical tuberculin test (CCTT). Three of the herds had no recent tuberculosis infection, and the other five had shown positive results in a previous tuberculin test. For the serological diagnosis of tuberculosis, a commercial ELISA antibody test kit for Mycobacterium bovis was used. Serum samples from 846 cattle from the eight herds were evaluated using ELISA for M. bovis. Animals that were positive based on either CCTT or ELISA for M. bovis or both were sent to slaughter. Samples of their lungs, livers, and lymph nodes were collected and stored under refrigeration for microbiological culture and subsequent confirmation by polymerase chain reaction. Samples from the same tissues were also fixed with 10% formaldehyde in bottles for histopathological examination and stained with hematoxylin and eosin (HE). Of the 1,644 cattle, 61 were considered positive and 65 inconclusive based on CCTT. Retesting of the inconclusive samples identified an additional 19 positive cases, totaling 80 (4.8%) CCTT-positive animals from five herds. ELISA for M. bovis identified 4.2% (36/846) positive cattle, of which 35 were considered negative and one inconclusive based on CCTT. Of the 36 positive cases identified by ELISA for M. bovis, 27 were euthanized, 11% (3/27) showed suggestive lesions of tuberculosis on macroscopic examination, and two were confirmed by histological, microbiological, and PCR methods. The weak association of ELISA for M. bovis with the results obtained by macroscopic, histological, and microbiological isolation indicates the fragility of ELISA performance in field conditions. Therefore, it is suggested that its use as a complementary method for herd sanitation be based on the local epidemiological situation.

A tuberculose bovina é um problema econômico mundial e de saúde, que requer métodos de diagnóstico precisos para controle e erradicação. Objetivou-se com este estudo avaliar o desempenho de um teste comercial de ELISA no diagnóstico da tuberculose bovina. Foram avaliados pelo teste cervical comparativo (TCC) 1644 bovinos, provenientes de oito rebanhos de exploração leiteira, sendo três deles sem histórico recente da doença, e outros cinco com resultados positivos no último exame de tuberculinização. Para o diagnóstico sorológico da tuberculose utilizou-se um kit comercial de ELISA Mycobacterium bovis antibody test (ELISA M. bovis). Amostras de soro sanguíneo de 846 bovinos provenientes dos mesmos rebanhos foram também avaliadas no ELISA M. bovis. Os bovinos positivos no TCC e/ou ELISA M. bovis foram encaminhados para o abate sanitário. Dos positivos no ELISA para M. bovis foi realizado o exame macroscópico das carcaças e classificação das mesmas em: com presença ou ausência de lesões. Amostras de pulmão, fígado, e linfonodos, foram colhidas em duplicata para realização dos exames de cultivo microbiológico com posterior confirmação por PCR, e exame histopatológico com coloração de hematoxilina e eosina (HE). Dos 1,644 bovinos, 61 foram considerados positivos e 65 inconclusivos no TCC. O reteste dos inconclusivos identificou mais 19 positivos. No total, 80 (4,8%) bovinos positivos no TCC, provenientes de 5 rebanhos foram encaminhados para abate. O ELISA para M. bovis identificou 4,2% (36/846) de bovinos positivos, sendo 36 considerados negativos e um inconclusivo no exame de tuberculinização. Dos positivos no ELISA para M. bovis, 27 foram eutanasiados, e no exame macroscópico das carcaças 11% (3/27) dos animais apresentaram lesões sugestivas de tuberculose, e em apenas dois houve confirmação da doença pelos métodos histológico, microbiológico e PCR. A baixa associação dos resultados obtidos no ELISA para M. bovis com os exames macroscópico, histológico e isolamento microbiológico apontam para a fragilidade do desempenho do ELISA para o diagnóstico de tuberculose. Sugere-se assim que seu uso como método complementar para saneamento de rebanhos, seja adotado com cautela e considere a situação epidemiológica local.

Frequência de lesões sugestivas de tuberculose em bovinos abatidos no estado do Espírito Santo e perdas econômicas aos produtores / Frequency of suggestive lesions of tuberculosis in bovine slaughtered in the state of Espírito Santo and economic losses for farmers

A bovinocultura de corte tem papel fundamental na economia do Brasil, que atualmente é um dos maiores produtores, consumidores e exportadores de carne bovina, e consequentemente se faz necessária a inspeção sanitária desses animais abatidos no país. A legislação vigente determina que quando as carcaças possuem indicativos de doenças, como a tuberculose bovina, podem ser condenadas ou aproveitadas condicionalmente. A tuberculose bovina é uma zoonose crônica causada pelo bacilo Mycobacterium bovis que carece de estudos para estabelecer a sua frequência em frigoríficos no país, bem como as consequentes perdas econômicas aos pecuaristas decorrentes da condenação de carcaças e vísceras. Assim, esse trabalho objetivou analisar a frequência de condenação de carcaças de bovinos do Estado do Espírito Santo, em um frigorífico que condena todos os casos de tuberculose, durante os anos de 2019 a 2021, e estimar as perdas econômicas aos produtores. Verificou-se que entre os 156.800 animais abatidos, 120 (0,076%) apresentaram lesões sugestivas de tuberculose, o que ocasionou uma perda econômica de R$ 461.160,00 (U$ 85.338,40). Conclui-se que a tuberculose é endêmica e responsável por importantes perdas econômicas para a cadeia produtiva da carne nesse estado. Também se destacou a importância da inspeção sanitária em abatedouros-frigoríficos de bovinos para reduzir os riscos à saúde pública.

Cattle farming has a fundamental role in the economy of Brazil, which is currently one of the largest producers, consumers, and exporters of beef, and consequently it is necessary to inspect meat from cattle slaughtered in the country. The current legislation establishes that when carcasses have signs of diseases, such as bovine tuberculosis, should be condemned or conditionally used. Bovine tuberculosis is a chronic zoonosis caused by Mycobacterium bovis that requires epidemiological studies to establish its frequency in abattoirs and the economic losses due condemnation of viscera and carcasses for beef farmers. Thus, this study aimed to analyze the frequency of condemnation of bovine carcasses slaughtered in the state of Espírito Santo, in an abattoir that condemned all cases of tuberculosis, during the years 2019 to 2021, and to estimate the economic losses to farmers. Among the 156,800 animals slaughtered, 120 (0.076%) had lesions suggestive of tuberculosis, which caused an economic loss of U$ 85,338.40 (R$ 461,160.00). Bovine tuberculosis is endemic and responsible for important economic losses to the meat production chain in this state. Additionally, the importance of inspecting beef in abattoirs to reduce the risks for public health was highlighted.

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.

Reverse vaccinology and subtractive genomics approaches for identifying common therapeutics against Mycobacterium leprae and Mycobacterium lepromatosis

Mycobacterium leprae and Mycobacterium lepromatosis are gram-positive bacterial pathogens and the causative agents of leprosy in humans across the world. The elimination of leprosy cannot be achieved by multidrug therapy alone, and highlights the need for new tools and drugs to prevent the emergence of new resistant strains. Methods In this study, our contribution includes the prediction of vaccine targets and new putative drugs against leprosy, using reverse vaccinology and subtractive genomics. Six strains of Mycobacterium leprae and Mycobacterium lepromatosis (4 and 2 strains, respectively) were used for comparison taking Mycobacterium leprae strain TN as the reference genome. Briefly, we used a combined reverse vaccinology and subtractive genomics approach. Results As a result, we identified 12 common putative antigenic proteins as vaccine targets and three common drug targets against Mycobacterium leprae and Mycobacterium lepromatosis. Furthermore, the docking analysis using 28 natural compounds with three drug targets was done. Conclusions The bis-naphthoquinone compound Diospyrin (CID 308140) obtained from indigenous plant Diospyros spp. showed the most favored binding affinity against predicted drug targets, which can be a candidate therapeutic target in the future against leprosy.(AU)

Reverse vaccinology and subtractive genomics approaches for identifying common therapeutics against Mycobacterium leprae and Mycobacterium lepromatosis

Mycobacterium leprae and Mycobacterium lepromatosis are gram-positive bacterial pathogens and the causative agents of leprosy in humans across the world. The elimination of leprosy cannot be achieved by multidrug therapy alone, and highlights the need for new tools and drugs to prevent the emergence of new resistant strains. Methods In this study, our contribution includes the prediction of vaccine targets and new putative drugs against leprosy, using reverse vaccinology and subtractive genomics. Six strains of Mycobacterium leprae and Mycobacterium lepromatosis (4 and 2 strains, respectively) were used for comparison taking Mycobacterium leprae strain TN as the reference genome. Briefly, we used a combined reverse vaccinology and subtractive genomics approach. Results As a result, we identified 12 common putative antigenic proteins as vaccine targets and three common drug targets against Mycobacterium leprae and Mycobacterium lepromatosis. Furthermore, the docking analysis using 28 natural compounds with three drug targets was done. Conclusions The bis-naphthoquinone compound Diospyrin (CID 308140) obtained from indigenous plant Diospyros spp. showed the most favored binding affinity against predicted drug targets, which can be a candidate therapeutic target in the future against leprosy.(AU)

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)