Real-Time PCR Detection of Mycobacterium bovis in Blood and Lymph Node Aspirates of Bovines Positive in Tuberculosis Screening Tests

2020 ◽  
Author(s):  
D. Narang ◽  
G. S. Sidhu ◽  
G. Filia ◽  
A. Singh ◽  
S. T. Singh ◽  
...  
Keyword(s):  
Lymph Node ◽  
Real Time ◽  
Mycobacterium Bovis ◽  
Real Time Pcr ◽  
Bovine Tuberculosis ◽  
Tuberculin Test ◽  
Screening Tests ◽  
Low Molecular Weight ◽  
Tuberculosis Screening ◽  
Three Samples

Bovine tuberculosis is a chronic infectious disease affecting broad range of mammalian hosts. ESAT-6 is a low molecular weight immunodominant protein coded by esxA gene located on RD1 region of genome and is responsible for virulence of Mycobacterium bovis. Out of 200 animals screened for bovine Tuberculosis (bTB), 38 animals (19%) were found positive for Comparative intradermal tuberculin test (CITT) (32 cattle, 6 buffaloes) and 41 animals were tested positive by IFN-g assay (29 cattle, 12 buffaloes). DNA extraction of blood (n=200) and lymph node aspirates (n=48) was done. Among 200 blood samples targeted for esxA (ESAT-6) gene, three samples (1.5%) whose CT was between 23-34 were considered positive by real-time PCR. Out of 48 animals (lymph node aspirates) that were positive either by CITT or IFN-g, one sample (2.08%) whose CT was between 23-34 were considered positive by real-time PCR. Remaining samples whose CT values were equal to or greater than 35 were considered negative. The sensitivity of esxA was 8 pg/ìl by real time PCR.

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

2017 ◽  
Vol 37 (6) ◽  
pp. 549-554
Author(s):  
Fabiana Q. Mayer ◽  
Emily M. dos Reis ◽  
André Vinícius A. Bezerra ◽  
Rogério O. Rodrigues ◽  
Thais Michel ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Bovine Tuberculosis ◽  
Tuberculin Test ◽  
Disease Diagnosis ◽  
Economic Losses ◽  
Nasal Swabs ◽  
In Vivo Diagnosis

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.

scholarly journals Duplex real-time PCR assay targeting insertion elements IS1081 and IS6110 for detection of Mycobacterium bovis in lymph nodes of cattle

2014 ◽  
Vol 30 (1) ◽  
pp. 45-59 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
W. Gaede
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Real Time ◽  
Mycobacterium Bovis ◽  
Real Time Pcr ◽  
Rapid Screening ◽  
Microscopical Examination ◽  
Pcr Assay ◽  
Specificity And Sensitivity ◽  
Insertion Elements

The development of a reliable and rapid screening test for detection of Mycobacterium bovis (M. bovis) helps in control of bovine tuberculosis. The aim of this study was evaluate a sensitive and specific assay for detecting M. bovis DNA in lymph nodes with lesions suggestive to tuberculosis taken from slaughtered cattle. A duplex real-time PCR assay was developed for the identification of M. bovis targeting insertion elements (IS) IS1081 and IS6110 in one internally controlled reaction. M. bovis DNA extraction protocols from tissue samples were evaluated. The specificity and sensitivity of the assay were evaluated for detecting serial dilutions of reference Mycobacteria strains as well as from spiked lymph node homogenate. Results revealed that microscopical examination of 600 lymph nodes with tuberculous-like lesion for detection of Acid-fast bacilli (AFB) showed a detection rate of 96.6%, compared to 98% M. bovis with duplex real-time PCR. The reproducible detection limit of the IS1081-PCR was 10 M. bovis cells/ml and the IS6110-PCR was 100 M. bovis cells/ml. Besides, both primer set of PCR protocol could detect 20 M. bovis cells/ml in spiked lymph node tissue. The assay was evaluated on 19 bacterial strains and was determined to be 100% specific for M. bovis. We suggest that the IS1081-PCR is a good candidate assay for routine screening of cattle lymph nodes and other tissue for M. bovis infection.

scholarly journals Validation of real-time PCR technique for detection of Mycobacterium bovis and Brucella abortus in bovine raw milk

2020 ◽  
Vol 51 (4) ◽  
pp. 2095-2100
Author(s):  
Débora R. Mascarenhas ◽  
David Germano G. Schwarz ◽  
Antônio A. Fonseca Júnior ◽  
Tatiana F.P. Oliveira ◽  
Maria A.S. Moreira
Keyword(s):  
Real Time ◽  
Mycobacterium Bovis ◽  
Brucella Abortus ◽  
Real Time Pcr ◽  
Raw Milk

scholarly journals Bovine tuberculosis in one cattle herd in Ibadan in Nigeria

2012 ◽  
Vol 49 (No. 11) ◽  
pp. 406-412 ◽  
Author(s):  
S. I B Cadmus ◽  
N. N Atsanda ◽  
S. O Oni ◽  
E. E U Akang
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Molecular Typing ◽  
Human Population ◽  
Tuberculin Test ◽  
Cattle Herd ◽  
Post Mortem ◽  
Risk Of Infection ◽  
Cultural Isolation ◽  
Male Animal

Bovine tuberculosis was investigated in one private herd with 171 cattle after five cases were suspected to be tuberculous following post mortem examination. Using the intradermal comparative cervical tuberculin test 18 (10.5%) animals (ages from 2 to 12 years) were positive: 11 animals of N’dama breed and seven animals of White Fulani (i.e. Bunaji) breed; 17 female and one male animal. In all 11 randomly selected positive reactors, a spectrum of tuberculous lesions affecting the lungs, spleen, heart, liver, and the lymph nodes were observed. All the smear samples obtained were positive for acid-fast bacilli; cultural isolation confirmed the growth of mycobacteria on pyruvate-enriched Loewenstein-Jensen medium, which were identified by molecular typing to be Mycobacterium bovis. This study demonstrates widespread infection in this cattle herd and potential risk of infection for the human population with M. bovis.

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR

2013 ◽  
Vol 173 (5) ◽  
pp. 117-117 ◽  
Author(s):  
W. Gan ◽  
X. Zhou ◽  
H. Yang ◽  
H. Chen ◽  
J. Qiao ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bovine Tuberculosis ◽  
Gamma Interferon

Diagnosis of Mycobacterium bovis infection in livestock using gamma interferon assay and single intradermal comparative tuberculin test in Assam and Meghalaya

2017 ◽  
Author(s):  
Acheenta G. Barua ◽  
Himangshu Raj ◽  
Ashok Kumar ◽  
Chandana C. Barua ◽  
Arundhati Purkayastha ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Screening Method ◽  
Tuberculin Test ◽  
Gamma Interferon ◽  
Tuberculosis Infection ◽  
Specific Detection ◽  
Biochemical Tests ◽  
Diagnostic Potential ◽  
Pcr Targeting

The present study was carried out to investigate the diagnostic potential of gamma interferon (IFN-ã) assay and single intradermal comparative tuberculin test (SICTT), including species specification of bovine tuberculosis infection in different livestock farms of Assam and Meghalaya. A total of 199 animals (cattle and buffalo) were examined for bovine tuberculosis symptoms and swab samples were cultured. Biochemical tests and PCR were used for species specification of bovine tuberculosis. Out of 199 cases examined, 33 (16.58%) showed positive for SICTT, 39 (19.59%) for IFN-ã and 35(17.59%) for PCR. Based on PCR targeting pncA region, the confirmation was done for M. Bovis. IFN-ã thus ensures a sensitive and specific detection of early bovine tuberculosis infection together with SICTT and hence may be considered as a screening method of choice.

Determination of microvessel density by real-time PCR in esophageal cancer: Correlation with histological methods, angiogenic growth factor expression and lymph node metastasis

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10053-10053
Author(s):  
S. Loges ◽  
H. Claussen ◽  
U. Reichelt ◽  
M. Bubenheim ◽  
A. Erbersdobler ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Lymph Node ◽  
Lymph Node Metastasis ◽  
Esophageal Carcinoma ◽  
Real Time ◽  
Microvessel Density ◽  
Real Time Pcr ◽  
Angiogenic Growth Factors ◽  
Node Metastasis

10053 Background: Neoangiogenesis and lymphangiogenesis represent prognostic factors in human malignancies. Determination of microvessel density (MVD) by immunohistology is labor-intensive and subject to interobserver variability. We evaluated real time PCR to quantify MVD in primary tumor samples from patients with esophageal cancer. Methods: We performed real-time PCR analyzes of endothel-specific (VE-Cadherin, P1H12, VEGFR-2, tie-2), lymphendothel-specific (Prox, LYVE, VEGFR-3) antigens and of angiogenic growth factors (VEGF-A, VEGF-C, VEGF-D, Ang-1, Ang-2) in primary esophageal carcinoma tissue of 54 patients. These results were compared to MVD determined immunohistochemically by CD31 staining. Results: For validation, MNC samples spiked with HUVECs were analyzed by qPCR for VE-CAD and P1H12 yielding a linear correlation (r=0.99 and 0.96 respectively). Expression of endothelial markers was highly correlated in tumor samples, e.g. CD144 with CD146 τb=0.451, CD144 with VEGFR-2 τb =0.744 and CD144 with tie-2 τb=0.684 (p for all comparisons < 0.0001). QPCR results were compared to MVD determination by CD31 staining in a subgroup of 33 patients. The highest association between both methods was found for CD144 (τb=0.258, p=0.0379) and VEGFR-2 (τb=0.222, p=0.0745) indicating that immunohistology and qPCR yield comparable results. MVD was significantly linked to the expression of VEGF-A, -C,-D and Ang1 and Ang2 (p for all comparisons <0.0001). We analyzed expression of lymphendothelial cell antigens Prox, LYVE and VEGFR-3 for quantification of lymphatic vessels. A close association between the expression of different lymphendothelial factors was seen (LYVE with Prox τb=0.334, p=0.0021, LYVE with VEGFR-3 τb=0.450, p=0.0150). MVD and lymphvessel density was not linked. Lymph node metastases detected on surgical specimen were associated with MVD determined immunohistologically (p=0.003)or by qPCR (p=0.048) and to VEGF-C expression (p=0.04). Conclusions: QPCR analysis of CD144 and VEGFR2 represents a novel tool for quantification of MVD in tumor samples. Expression of VEGFR2 and VEGF-C is associated with lymph node metastasis in patients with esophageal carcinoma. No significant financial relationships to disclose.

scholarly journals Detection of Shiga Toxin-ProducingEscherichia coliSerotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in Raw-Milk Cheeses by Using Multiplex Real-Time PCR

2011 ◽  
Vol 77 (6) ◽  
pp. 2035-2041 ◽  
Author(s):  
Jordan Madic ◽  
Noémie Vingadassalon ◽  
Carine Peytavin de Garam ◽  
Muriel Marault ◽  
Flemming Scheutz ◽  
...  
Keyword(s):  
Real Time ◽  
Genetic Markers ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Raw Milk ◽  
Immunomagnetic Separation ◽  
Genetic Profile ◽  
High Genetic Diversity ◽  
E Coli ◽  
Three Samples

ABSTRACTShiga toxin (Stx)-producingEscherichia coli(STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive forstx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28fliCalleles were highly prevalent and could not be used as reliable targets for screening. Combinations ofstx,eaevariants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and includedstx-wzxO26-eae-β1(4.8%; 19 samples),stx-wzxO103-eae-ε (1.3%; five samples),stx-ihp1O145-eae-γ1(0.8%; three samples), andstx-rfbEO157-eae-γ1(0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seveneaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Threestx-negative andeaeβ1-positiveE. coliO26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC fromstx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagicE. coli(EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA,katP, andespP, as well as genomic O islands 71 and 122. Except for one strain, they all contained thestx1variant only, which was reported to be less frequently associated with human cases thanstx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

scholarly journals Environmental Monitoring of Mycobacterium bovis in Badger Feces and Badger Sett Soil by Real-Time PCR, as Confirmed by Immunofluorescence, Immunocapture, and Cultivation

2007 ◽  
Vol 73 (22) ◽  
pp. 7471-7473 ◽  
Author(s):  
F. P. Sweeney ◽  
O. Courtenay ◽  
V. Hibberd ◽  
R. G. Hewinson ◽  
L. A. Reilly ◽  
...  
Keyword(s):  
Environmental Monitoring ◽  
Cell Viability ◽  
Real Time ◽  
Species Identification ◽  
Mycobacterium Bovis ◽  
Real Time Pcr ◽  
Immunomagnetic Capture

ABSTRACT Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

scholarly journals Genetic profiles of Mycobacterium bovis from a cattle herd in southernmost Brazil

2016 ◽  
Vol 37 (5Supl2) ◽  
pp. 3719
Author(s):  
Antonio Francisco de Souza Filho ◽  
Ana Luiza Alves Rosa Osório ◽  
Klaudia Dos Santos Gonçalves Jorge ◽  
Flábio Ribeiro Araújo ◽  
Carlos Eugênio Soto Vidal ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Bovine Tuberculosis ◽  
Tuberculin Test ◽  
Tissue Samples ◽  
Genetic Groups ◽  
As Species ◽  
Recent Transmission ◽  
Bovine Tissue ◽  
Genetic Profiles ◽  
Different Sources

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

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