Influence of the incubation conditions on culture media to optimize primary isolation of Mycobacterium bovis

The isolation of Mycobacterium bovis is critical to a surveillance system for bovine tuberculosis based on detection of lesions in abattoirs. Thus, four solid culture media and three incubation conditions were investigated to elucidate which combination overcomes the others by assessing growth, time to the first appearance of colonies and their number. Ninety-seven samples of granulomatous lesions were submitted to the decontamination procedure by 1-hexadecylpyridinium chloride at 0.75% w/v, and inoculated on two egg-based media, Stonebrink’s (ST) and Löwenstein-Jensen’s with sodium pyruvate (LJp), and two agar-based media, tuberculosis blood agar (B83) and Middlebrook 7H11 medium (7H11). Each medium was incubated at 37°C for 90 days in three incubation conditions: in air, in air containing 10% carbon dioxide (CO2), and in air in slopes closed with burned hydrophobic cotton and subsequently plugged with a cork to create a microaerophilic atmosphere. The colonies appeared faster and in higher number when incubated in air containing 10% CO2 (p < 0.01), independent of media. B83 showed a faster growth and detected more isolates at 30 days of incubation, when compared to ST (0.0178), LJp (p < 0.0001) and 7H11 (p < 0.0001), though there was no difference between B83, ST and LJp at 60 and 90 days of incubation. 7H11 presented the lowest number of isolates (p < 0.0001) and a longer period for the appearance of the first colony (p < 0.001). According to our findings, the concomitant use of ST and B83 media incubated in air containing 10% CO2 increases the isolation of M. bovis in a shorter period of time, which improves bovine tuberculosis diagnosis.

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Comparison of DNA extraction protocols to detect Mycobacterium bovis in bovine tissue by PCR

Semina Ciências Agrárias ◽

10.5433/1679-0359.2016v37n5supl2p3709 ◽

2016 ◽

Vol 37 (5Supl2) ◽

pp. 3709

Author(s):

Cássia Yumi Ikuta ◽

Daniella Carvalho Ribeiro Oliveira ◽

Gisele Oliveira de Souza ◽

Antonio Francisco de Souza Filho ◽

José Henrique de Hildebrand Grisi-Filho ◽

...

Keyword(s):

Dna Extraction ◽

Mycobacterium Bovis ◽

Gold Standard Method ◽

Primary Isolation ◽

Host Interaction ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Granulomatous Lesions ◽

Current Scenario ◽

Suitable Technique

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

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Identification and evaluation of new Mycobacterium bovis antigens in the in vitro interferon gamma release assay for bovine tuberculosis diagnosis

Tuberculosis ◽

10.1016/j.tube.2015.07.009 ◽

2015 ◽

Vol 95 (6) ◽

pp. 795-801 ◽

Cited By ~ 8

Author(s):

María E. Eirin ◽

Analia Macias ◽

Gabriel Magnano ◽

Claudia Morsella ◽

Laura Mendez ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Interferon Gamma ◽

Bovine Tuberculosis ◽

Interferon Gamma Release Assay ◽

Release Assay ◽

Tuberculosis Diagnosis

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Comparison between tests for tuberculosis diagnosis in slaughtered bovines

Arquivos do Instituto Biológico ◽

10.1590/1808-1657000652016 ◽

2018 ◽

Vol 85 (0) ◽

Cited By ~ 1

Author(s):

David Attuy Vey da Silva ◽

Márcio Junio Lima Siconelli ◽

Karina Paes Bürger ◽

Lara Borges Keid

Keyword(s):

Bovine Tuberculosis ◽

Dna Detection ◽

Bacterial Isolation ◽

Solid Culture ◽

Chain Reaction ◽

Bacterial Dna ◽

Tuberculosis Diagnosis ◽

Polymerase Chain ◽

Low Sensitivity ◽

Different Sources

ABSTRACT: Our goal for this article is to compare several different diagnosis tests for bovine tuberculosis identification. We have performed bacterial isolation, histopathological characterization, acid-fast bacilli (AFB) identification and M. bovis DNA detection. Lesions suggestive of Tuberculosis were sampled from bovine lymph nodes during slaughtering of bovines at an abattoir that operates under federal inspection. The bacterial isolation was performed in solid culture mediums, the histopathological characterization was made by Hematoxylin-eosinstaining, and AFB identification by Ziehl-Neelsen staining. Bacterial DNA detection was performed by Polymerase Chain Reaction (PCR) using DNA from two different sources, directly collected from the tuberculosis-like lesions (PCR followed by nested PCR) and from isolated bacteria. We have concluded that the multi-step approach, including histopathological characterization, bacterial isolation and AFB identification, is strongly recommended to diagnose tuberculosis in bovines. Furthermore, PCR assays using specimens of lesions suggestive of tuberculosis are a faster and more promising way to diagnose the disease. However, it should not be used alone due to the low sensitivity shown in this study.

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Bovine Tuberculosis in Colombia; Findings from Histopathological, Microbiological and Molecular Tests

10.21203/rs.3.rs-862971/v1 ◽

2021 ◽

Author(s):

Paula Palomino Cadavid ◽

Dubel Ignacio Balvin ◽

Jhon Ruiz Buitrago ◽

Rafael Villarreal Julio ◽

Enderson Murillo Ramos ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Bovine Tuberculosis ◽

Diagnostic Tests ◽

Bubalus Bubalis ◽

Comparative Test ◽

Tuberculosis Complex ◽

Water Buffaloes ◽

Molecular Tests ◽

Wild Fauna ◽

Granulomatous Lesions

Abstract Tuberculosis is a zoonotic infectious disease, caused by bacteria of the tuberculosis complex. Mycobacterium bovis causes tuberculosis in water buffaloes (Bubalus bubalis), and it can also infect other domestic animals, wild fauna, and humans. The diagnosis of bovine tuberculosis (bTB) through intradermal injections is challenging, and to understand the behaviour of other diagnostic tests is crucial. The aim of this research is to analyse three diagnostic tests against bTB in water buffaloes with positive test DPP. Different diagnostic tests were tested in 50 buffaloes diagnosed with bTB Cervical Comparative Test, from the Colombian lower tropic. Lesions compatible with bTB in 26 buffaloes with a positive DPP ; Four out of 2 samples of Mycobacterium bovis in DPP-positive buffaloes were isolated and confirmed positively in tissues using PCR-HRM, three buffaloes showed granulomatous lesions in histological analyses with positive microbiological isolation; 17 DPP-positive buffaloes had a positive PCR-HRM test and nine of these buffaloes showed no histological findings compatible with bTB, leading to purely molecular diagnosis. Evidence in histological, microbiological, and molecular findings in DPP is positive for the water buffaloes. None of the complementary tests showed 100% concordance with the intradermal results obtained with the Cervical Comparative Test for bTB.

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Recording the Presence of Peanibacillus larvae larvae Colonies on MYPGP Substrates Using a Multi-Sensor Array Based on Solid-State Gas Sensors

Sensors ◽

10.3390/s21144917 ◽

2021 ◽

Vol 21 (14) ◽

pp. 4917

Author(s):

Beata Bąk ◽

Jakub Wilk ◽

Piotr Artiemjew ◽

Jerzy Wilde

Keyword(s):

Gas Sensors ◽

Culture Media ◽

Laboratory Diagnosis ◽

Baseline Correction ◽

American Foulbrood ◽

Sensor Signal ◽

Sodium Pyruvate ◽

Mueller Hinton ◽

Regeneration Phase ◽

Mueller Hinton Broth

American foulbrood is a dangerous disease of bee broods found worldwide, caused by the Paenibacillus larvae larvae L. bacterium. In an experiment, the possibility of detecting colonies of this bacterium on MYPGP substrates (which contains yeast extract, Mueller-Hinton broth, glucose, K2HPO4, sodium pyruvate, and agar) was tested using a prototype of a multi-sensor recorder of the MCA-8 sensor signal with a matrix of six semiconductors: TGS 823, TGS 826, TGS 832, TGS 2600, TGS 2602, and TGS 2603 from Figaro. Two twin prototypes of the MCA-8 measurement device, M1 and M2, were used in the study. Each prototype was attached to two laboratory test chambers: a wooden one and a polystyrene one. For the experiment, the strain used was P. l. larvae ATCC 9545, ERIC I. On MYPGP medium, often used for laboratory diagnosis of American foulbrood, this bacterium produces small, transparent, smooth, and shiny colonies. Gas samples from over culture media of one- and two-day-old foulbrood P. l. larvae (with no colonies visible to the naked eye) and from over culture media older than 2 days (with visible bacterial colonies) were examined. In addition, the air from empty chambers was tested. The measurement time was 20 min, including a 10-min testing exposure phase and a 10-min sensor regeneration phase. The results were analyzed in two variants: without baseline correction and with baseline correction. We tested 14 classifiers and found that a prototype of a multi-sensor recorder of the MCA-8 sensor signal was capable of detecting colonies of P. l. larvae on MYPGP substrate with a 97% efficiency and could distinguish between MYPGP substrates with 1–2 days of culture, and substrates with older cultures. The efficacy of copies of the prototypes M1 and M2 was shown to differ slightly. The weighted method with Canberra metrics (Canberra.811) and kNN with Canberra and Manhattan metrics (Canberra. 1nn and manhattan.1nn) proved to be the most effective classifiers.

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Characterization of Drug-Resistant Lipid-Dependent Differentially Detectable Mycobacterium tuberculosis

Journal of Clinical Medicine ◽

10.3390/jcm10153249 ◽

2021 ◽

Vol 10 (15) ◽

pp. 3249

Author(s):

Annelies W. Mesman ◽

Seung-Hun Baek ◽

Chuan-Chin Huang ◽

Young-Mi Kim ◽

Sang-Nae Cho ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture Media ◽

Multidrug Resistant ◽

Culture Conditions ◽

Sputum Smear ◽

Smear Microscopy ◽

Solid Culture ◽

Lowenstein Jensen ◽

Genetic Mechanisms ◽

Lipid Resuscitation

An estimated 15–20% of patients who are treated for pulmonary tuberculosis (TB) are culture-negative at the time of diagnosis. Recent work has focused on the existence of differentially detectable Mycobacterium tuberculosis (Mtb) bacilli that do not grow under routine solid culture conditions without the addition of supplementary stimuli. We identified a cohort of TB patients in Lima, Peru, in whom acid-fast bacilli could be detected by sputum smear microscopy, but from whom Mtb could not be grown in standard solid culture media. When we attempted to re-grow Mtb from the frozen sputum samples of these patients, we found that 10 out of 15 could be grown in a glycerol-poor/lipid-rich medium. These fell into the following two groups: a subset that could be regrown in glycerol after “lipid-resuscitation”, and a group that displayed a heritable glycerol-sensitive phenotype that were unable to grow in the presence of this carbon source. Notably, all of the glycerol-sensitive strains were found to be multidrug resistant. Although whole-genome sequencing of the lipid-resuscitated strains identified 20 unique mutations compared to closely related strains, no single genetic lesion could be associated with this phenotype. In summary, we found that lipid-based media effectively fostered the growth of Mtb from a series of sputum smear-positive samples that were not culturable in glycerol-based Lowenstein–Jensen or 7H9 media, which is consistent with Mtb’s known preference for non-glycolytic sources during infection. Analysis of the recovered strains demonstrated that both genetic and non-genetic mechanisms contribute to the observed differential capturability, and suggested that this phenotype may be associated with drug resistance.

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Immune Responses to Defined Antigens of Mycobacterium bovis in Cattle Experimentally Infected with Mycobacterium kansasii

Clinical and Vaccine Immunology ◽

10.1128/cvi.00054-06 ◽

2006 ◽

Vol 13 (6) ◽

pp. 611-619 ◽

Cited By ~ 45

Author(s):

W. R. Waters ◽

M. V. Palmer ◽

T. C. Thacker ◽

J. B. Payeur ◽

N. B. Harris ◽

...

Keyword(s):

Mycobacterium Bovis ◽

Bovine Tuberculosis ◽

Nontuberculous Mycobacteria ◽

Gamma Interferon ◽

Delayed Type Hypersensitivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Antibodies ◽

Specific Serum ◽

Specific Proteins

ABSTRACT Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. kansasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. kansasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests.

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