scholarly journals Maximizing Capture Efficiency and Specificity of Magnetic Separation for Mycobacterium avium subsp. paratuberculosis Cells

2010 ◽  
Vol 76 (22) ◽  
pp. 7550-7558 ◽  
Author(s):  
Antonio Foddai ◽  
Christopher T. Elliott ◽  
Irene R. Grant
Keyword(s):  
Magnetic Separation ◽  
Rapid Method ◽  
Mycobacterium Avium ◽  
Limit Of Detection ◽  
Skim Milk ◽  
Capture Efficiency ◽  
Nonspecific Binding ◽  
Amplification Assay ◽  
The Mean ◽  
Mycobacterium Avium Subsp Paratuberculosis

ABSTRACT In order to introduce specificity for Mycobacterium avium subsp. paratuberculosis prior to a phage amplification assay, various magnetic-separation approaches, involving either antibodies or peptides, were evaluated in terms of the efficiency of capture (expressed as a percentage) of M. avium subsp. paratuberculosis cells and the percentage of nonspecific binding by other Mycobacterium spp. A 50:50 mixture of MyOne Tosylactivated Dynabeads coated with the chemically synthesized M. avium subsp. paratuberculosis-specific peptides biotinylated aMp3 and biotinylated aMptD (i.e., peptide-mediated magnetic separation [PMS]) proved to be the best magnetic-separation approach for achieving 85 to 100% capture of M. avium subsp. paratuberculosis and minimal (<1%) nonspecific recovery of other Mycobacterium spp. (particularly if beads were blocked with 1% skim milk before use) from broth samples containing 103 to 104 CFU/ml. When PMS was coupled with a recently optimized phage amplification assay and used to detect M. avium subsp. paratuberculosis in 50-ml volumes of spiked milk, the mean 50% limit of detection (LOD50) was 14.4 PFU/50 ml of milk (equivalent to 0.3 PFU/ml). This PMS-phage assay represents a novel, rapid method for the detection and enumeration of viable M. avium subsp. paratuberculosis organisms in milk, and potentially other sample matrices, with results available within 48 h.

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 Optimization of a Phage Amplification Assay To Permit Accurate Enumeration of Viable Mycobacterium avium subsp. paratuberculosis Cells

2009 ◽  
Vol 75 (12) ◽  
pp. 3896-3902 ◽  
Author(s):  
Antonio Foddai ◽  
Christopher T. Elliott ◽  
Irene R. Grant
Keyword(s):  
Mycobacterium Avium ◽  
Incubation Time ◽  
Test Parameters ◽  
Amplification Assay ◽  
The Mean ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Optimal Incubation ◽  
Burst Time ◽  
Assay Conditions ◽  
Accurate Quantification

ABSTRACT A commercially available phage amplification assay, FASTPlaqueTB (Biotec Laboratories, Ipswich, United Kingdom), when used according to the manufacturer's instructions, does not permit accurate enumeration of Mycobacterium avium subsp. paratuberculosis. The aim of this study was to optimize the phage amplification assay conditions to permit accurate quantification of viable M. avium subsp. paratuberculosis cells. The burst time for M. avium subsp. paratuberculosis was initially determined to inform decisions about optimal incubation time before plating, and then other test parameters were altered to evaluate how the correlation between plaque and colony counts was affected. The D29 mycobacteriophage replicates more slowly in M. avium subsp. paratuberculosis than in Mycobacterium smegmatis (used to optimize the commercial test originally), and the mean burst time for four M. avium subsp. paratuberulosis strains was 210 ± 36.8 min at 37°C compared to 63 ± 17.5 min for M. smegmatis mc2 155. To achieve 100% correlation between plaque and colony counts, the optimized phage assay includes the following: (i) resuspension of the samples to be tested in Middlebrook 7H9 broth containing 10% oleic acid-albumin-dextrose-catalase and 2 mM calcium chloride, followed by overnight incubation at 37°C before performance of the phage assay; (ii) a 2-h incubation of the sample with D29 mycobacteriophage before viricide treatment; and (iii) a further 90-min incubation after viricide treatment and neutralization up to the burst time (total incubation time, 210 min) before plating with M. smegmatis mc2 155 in 7H9 agar. The optimized phage amplification assay was able to detect 1 to 10 CFU/ml of M. avium subsp. paratuberculosis in spiked milk or broth within 48 h, as demonstrated by the results of several blind trials.

Mycobacterial infection influences bone biomarker levels in patients with Crohn’s disease

2018 ◽  
Vol 96 (7) ◽  
pp. 662-667 ◽  
Author(s):  
Amna Naser ◽  
Ahmad Qasem ◽  
Saleh A. Naser
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Mycobacterium Avium ◽  
Disease Model ◽  
Mycobacterial Infection ◽  
Undercarboxylated Osteocalcin ◽  
Healthy Cattle ◽  
The Mean ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Active Inflammation

Patients with Crohn’s disease (CD) have higher risk for osteoporosis following decreased level of osteocalcin. We hypothesize that active inflammation following Mycobacterium avium subsp. paratuberculosis (MAP) infection results in elevation of undercarboxylated osteocalcin (ucOC) and downregulation of active osteocalcin in CD patients and cow-disease model (Johne’s disease). In this study, we measured ucOC, active osteocalcin, and calcium levels in sera from 42 cattle (21 infected with MAP and 21 healthy cattle), 18 CD patients, and 20 controls. The level of ucOC in MAP+ bovine samples was higher than that in MAP− controls (318 ± 57.2 nmol/mL vs. 289 ± 95.8 nmol/mL, P > 0.05). Consequently, mean calcium level in bovine MAP+ was significantly higher than that in bovine-MAP− samples (9.98 ± 0.998 mg/dL vs. 7.65 ± 2.12 mg/dL, P < 0.05). Also, the level of ucOC was higher in CD-MAP+ than in CD-MAP− (561 ± 23.7 nmol/mL vs. 285 ± 19.6 nmol/mL, P < 0.05). Interestingly, the mean osteocalcin level in MAP+ bovine was lower than that in MAP− bovine (797 ± 162 pg/mL vs. 1190 ± 43 pg/mL) and it was lower in CD-MAP+ than in CD-MAP− infection (1.89 ± 0.184 ng/mL vs. 2.19 ± 0.763 ng/mL) (P < 0.05). The correlation between MAP infection and elevation of sera ucOC, reduction of active osteocalcin and increased calcium supports MAP infection role in CD and complications with osteoporosis.

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 Rapid Assessment of the Viability of Mycobacterium avium subsp. paratuberculosis Cells after Heat Treatment, Using an Optimized Phage Amplification Assay

2010 ◽  
Vol 76 (6) ◽  
pp. 1777-1782 ◽  
Author(s):  
Antonio Foddai ◽  
Christopher T. Elliott ◽  
Irene R. Grant
Keyword(s):  
Mycobacterium Avium ◽  
Thermal Inactivation ◽  
Rapid Assessment ◽  
Egg Yolk ◽  
Inactivation Kinetics ◽  
Complete Inactivation ◽  
Conventional Culture ◽  
Amplification Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Thermal Cycler

ABSTRACT Thermal inactivation experiments were carried out to assess the utility of a recently optimized phage amplification assay to accurately enumerate viable Mycobacterium avium subsp. paratuberculosis cells in milk. Ultra-heat-treated (UHT) whole milk was spiked with large numbers of M. avium subsp. paratuberculosis organisms (106 to 107 CFU/ml) and dispensed in 100-μl aliquots in thin-walled 200-μl PCR tubes. A Primus 96 advanced thermal cycler (Peqlab, Erlangen, Germany) was used to achieve the following time and temperature treatments: (i) 63°C for 3, 6, and 9 min; (ii) 68°C for 20, 40, and 60 s; and (iii) 72°C for 5, 10, 15, and 25 s. After thermal stress, the number of surviving M. avium subsp. paratuberculosis cells was assessed by both phage amplification assay and culture on Herrold's egg yolk medium (HEYM). A high correlation between PFU/ml and CFU/ml counts was observed for both unheated (r 2 = 0.943) and heated (r 2 = 0.971) M. avium subsp. paratuberculosis cells. D and z values obtained using the two types of counts were not significantly different (P > 0.05). The D 68°C, mean D 63°C, and D 72°C for four M. avium subsp. paratuberculosis strains were 81.8, 9.8, and 4.2 s, respectively, yielding a mean z value of 6.9°C. Complete inactivation of 106 to 107 CFU of M. avium subsp. paratuberculosis/ml milk was not observed for any of the time-temperature combinations studied; 5.2- to 6.6-log10 reductions in numbers were achieved depending on the temperature and time. Nonlinear thermal inactivation kinetics were consistently observed for this bacterium. This study confirms that the optimized phage assay can be employed in place of conventional culture on HEYM to speed up the acquisition of results (48 h instead of a minimum of 6 weeks) for inactivation experiments involving M. avium subsp. paratuberculosis-spiked samples.

scholarly journals Mycobacterium avium Subsp. paratuberculosis Induces Specific IgE Production in Japanese People with Allergies

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
D. Cossu ◽  
S. Otsubo ◽  
Y. Otsubo ◽  
S. Eda ◽  
T. Suzuki ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Allergic Inflammation ◽  
Specific Ige ◽  
Total Serum ◽  
Japanese People ◽  
Th2 Response ◽  
Dairy Foods ◽  
Patient Groups ◽  
The Mean ◽  
Mycobacterium Avium Subsp Paratuberculosis

Background. The prevalence of allergies is steadily increasing worldwide; however, the pathogenesis is still unclear. We hypothesized that Mycobacterium avium subsp. paratuberculosis (MAP) may contribute to allergy development. This organism can be present in dairy foods, it can elicit an immunomodulatory switch from a Th1 to a Th2 response, and it has been speculated that it is linked to several human autoimmune diseases. To determine the contribution, sera from 99 individuals with various atopic disorders and 45 healthy nonallergic controls were assessed for total IgE levels and successively for MAP-specific IgE by ELISA. Results. The mean total serum IgE level in allergic patients was 256±235 IU/mL, and in the healthy controls it was 62±44 IU/mL (AUC = 0.88; p<0.0001). Among the patient groups, 50 of the 99 subjects had increased IgE total level ≥ 150 IU/mL, while 49 subjects had IgE ≤ 150 IU/mL (mean level: 407±256 IU/mL versus 106±16 IU/mL; p<0.0001). Additionally, 6 out of 50 subjects (12%) with IgE ≥ 150 IU/mL and none (0%) with IgE ≤ 150 IU/mL were positive for specific MAP IgE (AUC = 0.63; p=0.03). Conclusion. The present study revealed that MAP has the ability to induce specific IgE and might contribute to the induction of allergic inflammation in genetically predisposed individuals.

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