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.

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 Mycobacterium avium subsp. paratuberculosis MAP1889c Protein Induces Maturation of Dendritic Cells and Drives Th2-Biased Immune Responses

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 944
Author(s):  
Hye-Soo Park ◽  
Yong Woo Back ◽  
Yeo-Jin Son ◽  
Hwa-Jung Kim
Keyword(s):  
Dendritic Cells ◽  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Inflammatory Cytokines ◽  
Mycobacterium Avium ◽  
Mycobacterium Avium Subsp ◽  
Effective Control ◽  
Th2 Response ◽  
Tnf Α ◽  
Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of chronic granulomatous bowel disease in animals and is associated with various autoimmune diseases in humans including Crohn’s disease. A good understanding of the host-protective immune response and antibacterial immunity controlled by MAP and its components may contribute to the development of effective control strategies. MAP1889c was identified as a seroreactive antigen in Crohn’s disease patients. In this study, we investigated the immunological function of MAP1889c in dendritic cells (DCs). MAP1889c stimulated DCs to increase expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) class molecules and to secret higher interleukin (IL)-10 and moderate IL-6, tumor necrosis factor (TNF)-α, and IL-12p70 levels through the Toll-like receptor (TLR) 4 pathway. MAP1889c-induced DC activation was mediated by mitogen-activated protein kinases (MAPKs), cAMPp-response element binding protein (CREB), and nuclear factor kappa B (NF-κB). In particular, the CREB signal was essential for MAP1889c-mediated IL-10 production but not TNF-α and IL-12p70. In addition, MAP1889c-matured DCs induced T cell proliferation and drove the Th2 response. Production of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokines and anti-inflammatory cytokines was suppressed and enhanced respectively by MAP1889c pretreatment in DCs and T cells. Furthermore, treatment of MAP1889c in M. avium-infected macrophages promoted intracellular bacterial growth and IL-10 production. These findings suggest that MAP1889c modulates the host antimycobacterial response and may be a potential virulence factor during MAP infection.

scholarly journals A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Sepideh Hosseiniporgham ◽  
Lucio Rebechesu ◽  
Pierangela Pintore ◽  
Stefano Lollai ◽  
Maria Dattena ◽  
...  
Keyword(s):  
Magnetic Separation ◽  
Mycobacterium Avium ◽  
Limit Of Detection ◽  
Goat Milk ◽  
Mycobacterium Avium Subsp ◽  
Milk Samples ◽  
Sheep Milk ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Higher Sensitivity

AbstractParatuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

scholarly journals Evaluation of Mycobacterium avium subsp. paratuberculosis faecal culture protocols and media

2006 ◽  
Vol 26 (1) ◽  
pp. 1-4 ◽  
Author(s):  
Paula Ristow ◽  
Marlei Gomes Silva ◽  
Leila de Souza Fonseca ◽  
Walter Lilenbaum
Keyword(s):  
Mycobacterium Avium ◽  
Egg Yolk ◽  
Mycobacterium Avium Subsp ◽  
Faecal Culture ◽  
Faecal Samples ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Centrifugation Protocol

Paratuberculosis is an important enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease is officially considered exotic in Brazil, but recent serological surveys and the isolation of the agent suggest it may occur in our herds. The aim of this study was to evaluate three different formulations of Herrold's egg yolk agar with mycobactin J (HEYM) and four faecal culture protocols considering their ability for Map growth as well as cost and ease of application. Three formulations of HEYM were inoculated with two suspensions of Map. Spiked faeces and naturally contaminated faecal samples were treated by the four faecal culture protocols. Centrifugation protocol and HEYM recommended by OIE showed the best results on the recovery of Map.

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