scholarly journals Evaluation of real time PCR for the detection of Mycobacterium avium subsp. paratuberculosis in faecal samples of cattle

2021 ◽  
Vol 52 (4) ◽  
Author(s):  
Chaitanya R.K. ◽  
Priyanka G. ◽  
Sreedevi B.
Keyword(s):  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Previous History ◽  
Mycobacterium Avium Subsp ◽  
Standard Curve ◽  
Government Sector ◽  
Faecal Samples ◽  
History Of ◽  
Mycobacterium Avium Subsp Paratuberculosis

The efficiency and suitability of a MAP F57 based SYBR Green qPCR assay for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) using a set of known MAP positive (12) and MAP negative (23) DNA samples that were previously identified by conventional IS 900 PCR were assessed. These DNA samples were isolated in our previous study from faecal samples collected from cattle in the livestock farms under government sector with a previous history of Johne’s disease. The MAP F57 qPCR was able to identify all the positive samples accurately and rapidly with Cq values ranging from 20-29. The efficiency of qPCR using recombinant plasmid for standard curve was 0.991 and limit of detection was 10 MAP organisms per microlitre of DNA sample.

Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk

2005 ◽  
Vol 101 (1) ◽  
pp. 93-104 ◽  
Author(s):  
David Rodríguez-Lázaro ◽  
Martin D'Agostino ◽  
Arnold Herrewegh ◽  
Maria Pla ◽  
Nigel Cook ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Mycobacterium Avium Subsp ◽  
Mycobacterium Avium Subsp Paratuberculosis

Detection of Mycobacterium avium subsp. paratuberculosis (MAP) from Subclinical Caprine Paratuberculosis Cases of Odisha

2020 ◽  
Author(s):  
Sangram Biswal ◽  
Adya Prakash Rath ◽  
Shoor Vir Singh ◽  
Niranjana Sahoo ◽  
Saurabh Gupta ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Animal Health ◽  
Restriction Endonuclease Analysis ◽  
Mycobacterium Avium Subsp ◽  
Serum Samples ◽  
Blood Samples ◽  
Blind Review ◽  
Faecal Samples ◽  
Goat Population ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP) and is a chronic, intestinal tract infection in the ruminant sector globally. A total of 122 EDTA mixed blood samples, 121 serum and 16 pooled faecal samples were collected from farms of 4 different districts i.e. Nayagarh, Cuttack, Khordha and Angul and a blind review was conducted at the Animal Health Division, CIRG, Mathura. Microscopic examination of 16 pooled faecal samples revealed +2 reactivity to Acid-Fast Bacilli. All the serum samples were subjected to indirect ELISA. Out of them, 23 (19.01%), 85 (70.25%), shows strongly positive, positive, antibody titre respectively. EDTA blood samples of 23 ELISA-strongly positive were subjected to 413 bp IS900 PCR and 11 (9%) of them were found positive for Mycobacterium avium subsp. Paratuberculosis (MAP). MAP isolates were further subjected to genotyping using 608 bpIS1311 PCR and restriction endonuclease analysis (IS1311 PCR-REA) and 2 (1.64%) of them matched with “Indian Bison Type”. Genotyping of the isolates using IS1311 PCR-REA revealed that goat population of Odisha are primarily infected with “Indian Bison Type” strains.

scholarly journals Faecal bacterial composition in dairy cows shedding Mycobacterium avium subsp. paratuberculosis in faeces in comparison with nonshedding cows

2016 ◽  
Vol 62 (6) ◽  
pp. 538-541 ◽  
Author(s):  
Marija Kaevska ◽  
Petra Videnska ◽  
Karel Sedlar ◽  
Iva Bartejsova ◽  
Alena Kralova ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subsp ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Bacterial Composition ◽  
Bacterial Populations ◽  
Faecal Samples ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
The 16S Rrna Gene

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne’s disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

scholarly journals Detection of Mycobacterium avium subsp. paratuberculosis by several diagnostics techniques in clinical suspected sheep

2018 ◽  
Vol 68 (2) ◽  
pp. 167
Author(s):  
A. C. COELHO ◽  
A. M. COELHO ◽  
J. GARCÍA-DIEZ ◽  
M. A. PIRES ◽  
M. L. PINTO
Keyword(s):  
Mycobacterium Avium ◽  
Bacterial Culture ◽  
Histopathological Examination ◽  
Diagnostic Techniques ◽  
Clinical Findings ◽  
Mycobacterium Avium Subsp ◽  
Clinical Symptomatology ◽  
Faecal Samples ◽  
Polymerase Chain ◽  
Mycobacterium Avium Subsp Paratuberculosis

A total of thirty sheep with clinical symptomatology of paratuberculosis (Johne’s disease) were subjected to four diagnostic techniques: histopathological examination, bacteriological culture (in faeces and tissues), polymerase chain reaction (PCR) (in blood, tissue and faecal samples) and antibody responses (ELISA). Twenty-one (70.0%) animals showed histological lesions. Bacterial culture of both faeces and tissue revealed that 2 (6.7%) and 6 (20.0%) of the 30 sheep were infected, respectively. Mycobacterium avium subsp. 24 paratuberculosis (Map) was identified in 4 animals via PCR of faeces (13.3%), and in 19 (63.3%) by PCR in tissues. PCR in blood revealed 7 (23.3%) infected animals. Three (10.0%) animals showed antibodies against Map. A greater number of positive animals were detected by histopathological examination and PCR in tissues than by culture or ELISA. This study confirmed the clinical findings and results suggest that histopathology, PCR in tissues and in blood can improve the detection of Map in physically suspected animals and should be considered useful tools in the diagnosis of Map in suspected sheep.

scholarly journals Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces

Materials ◽  
2020 ◽  
Vol 13 (22) ◽  
pp. 5112
Author(s):  
Marketa Husakova ◽  
Petr Kralik ◽  
Vladimir Babak ◽  
Iva Slana
Keyword(s):  
Magnetic Separation ◽  
Mycobacterium Avium ◽  
Magnetic Beads ◽  
Dna Isolation ◽  
Mycobacterium Avium Subsp ◽  
Effective Control ◽  
Silica Column ◽  
Faecal Samples ◽  
Isolation Methods ◽  
Mycobacterium Avium Subsp Paratuberculosis

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

scholarly journals Validation of an in-house real-time PCR fecal assay and comparison with two commercial assays for the antemortem detection of Mycobacterium avium subsp. paratuberculosis infection in culled sheep

2018 ◽  
Vol 31 (1) ◽  
pp. 58-68 ◽  
Author(s):  
Julie Arsenault ◽  
Jagdip Singh Sohal ◽  
Anne Leboeuf ◽  
Pierre Hélie ◽  
Gilles Fecteau ◽  
...  
Keyword(s):  
Real Time ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Positive Culture ◽  
Cost Effective ◽  
Mycobacterium Avium Subsp ◽  
Fecal Culture ◽  
Convenience Sample ◽  
Promising Tool ◽  
Mycobacterium Avium Subsp Paratuberculosis

Paratuberculosis is a chronic infectious enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). In sheep, the antemortem detection of the infection is challenging given the slow progression of the disease and the lack of sensitive, specific, and cost-effective validated tests. We adapted an in-house real-time PCR (rtPCR) assay targeting the multi-copy IS 900 element of MAP. The sensitivity and specificity of this essay for the detection of MAP infection were estimated in a convenience sample of culled ewes from 7 infected flocks and compared to a commercial fecal rtPCR, a commercial ELISA, and fecal culture. An infected ewe was defined as a ewe with a positive culture of the ileum and/or mesenteric lymph node. A non-infected ewe was defined as a ewe negative in intestinal tissue culture, negative in fecal culture, and with no lesions consistent with paratuberculosis. The in-house rtPCR had a sensitivity estimate of 84% (95% confidence interval [CI]: 59%, 97%) among the 44 infected ewes, which was significantly higher ( p ⩽ 0.05) than the sensitivity of a commercial fecal rtPCR (52%, 95% CI: 27%, 76%; or 63%, 95% CI: 35%, 87% depending on the cutoff used), an ELISA (14%, 95% CI:2.0%, 41%), and fecal culture (21%, 95% CI: 2.7%, 59%). No statistical difference in assay specificities was observed for the 30 non-infected ewes. The in-house rtPCR is a promising tool that could be used advantageously for the antemortem detection of MAP infection in sheep.

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