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 Effects of freezing on ability to detect Mycobacterium avium subsp. paratuberculosis from bovine tissues following culture

2018 ◽  
Vol 30 (5) ◽  
pp. 743-746
Author(s):  
Caroline S. Corbett ◽  
Jeroen De Buck ◽  
Herman W. Barkema
Keyword(s):  
Mycobacterium Avium ◽  
Gold Standard ◽  
Frozen Storage ◽  
Mycobacterium Avium Subsp ◽  
Freezing And Thawing ◽  
Tissue Samples ◽  
Milk Samples ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Autopsy Tissues ◽  
Numerical Change

Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium that causes Johne’s disease in cattle. Although infected cattle can be identified by examining fecal, blood, or milk samples, the gold standard is identification of MAP in tissue samples postmortem. Although tissue samples are commonly frozen, the ability to detect MAP in frozen–thawed tissue samples has apparently not been reported. We therefore determined the ability to detect MAP in tissue samples following freezing. Tissue samples were collected from calves that were either inoculated (IN) 3 mo prior, or contact-exposed (CE) for 3 mo. Following autopsy, tissues were immediately processed for culture, followed by DNA extraction and detection by qPCR. Samples were categorized as positive or negative based on the cycle threshold (Ct) value. The remaining unprocessed tissue samples were frozen at −80°C. After 18 mo, 50 tissue samples designated MAP-positive were thawed and processed for detection of MAP. Four (8%) samples were qPCR-negative, and Ct values of the remaining 46 samples were higher after freezing. Given the small numerical change in Ct values for MAP-positive samples after 18 mo of frozen storage, freezing and thawing may have had some deleterious effects on MAP detection in tissues. Although the decrease in ability to detect MAP-positive samples was minor for IN calves, there may be a greater effect for CE calves that should be considered when freezing tissue samples.

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 Mycobacterium avium subsp. paratuberculosis ELISA Responses in Milk Samples from Vaccinated and Nonvaccinated Dairy Goat Herds in The Netherlands

2019 ◽  
Vol 6 (2) ◽  
pp. 58 ◽  
Author(s):  
Saskia Luttikholt ◽  
Karianne Lievaart-Peterson ◽  
Maaike Gonggrijp ◽  
Marian Aalberts ◽  
Gerdien van Schaik ◽  
...  
Keyword(s):  
The Netherlands ◽  
Mycobacterium Avium ◽  
Relative Sensitivity ◽  
Herd Size ◽  
Mycobacterium Avium Subsp ◽  
Dairy Goat ◽  
Dairy Goats ◽  
Milk Samples ◽  
Goat Population ◽  
Mycobacterium Avium Subsp Paratuberculosis

The aims of our study were to calculate the most appropriate cut-off value for milk samples in a serum-validated Mycobacterium avium subsp. paratuberculosis (MAP) ELISA and to analyze MAP ELISA responses in milk samples from vaccinated and nonvaccinated dairy goats in the Netherlands. Analyzed herds were representative for location and herd size of dairy goat herds in the Netherlands. A significantly higher proportion of the analyzed 49 herds were organic as compared with the total Dutch dairy goat population. First, the MAP ELISA was optimized using 992 paired serum and milk samples. At a cut-off of 25 S/P%, the relative sensitivity (Se) was 58.4% (n = 992, 95% CI: 48.8%−67.6%) and relative specificity (Sp) was 98.5% (n = 992, 95% CI: 97.5%−99.2%), as compared to serum ELISA results. The percentage of positively tested herds was 78.2% (n = 49, 95% CI: 63.4%−88.1%). The percentage of positive milk samples per herd (n = 22) was on average 4.6% (median, min, and max of 4.7%, 0.0%, and 10.7%, respectively). Average age of ELISA-positive (3.2 years) and -negative goats (3.2 years) was not different. Significantly more vaccinated goats tested positive (6.7%) as compared with nonvaccinated goats (1.1%). This study shows that a high number of vaccinated and nonvaccinated commercial dairy goat herds in the Netherlands have MAP-ELISA-positive goats.

scholarly journals Identification of Mycobacterium avium subsp. paratuberculosis (MAP) in Sheep Milk, a Zoonotic Problem

2020 ◽  
Vol 8 (9) ◽  
pp. 1264
Author(s):  
Sepideh Hosseiniporgham ◽  
Tiziana Cubeddu ◽  
Stefano Rocca ◽  
Leonardo A. Sechi
Keyword(s):  
Mycobacterium Avium ◽  
Gold Standard ◽  
Dairy Product ◽  
Mycobacterium Avium Subsp ◽  
Individual Level ◽  
Reference Models ◽  
Sheep Milk ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
The Individual ◽  
Serum Elisa

Johne’s disease (JD) is a life-threatening gastrointestinal disease affecting ruminants, which causes crucial economical losses globally. This ailment is caused by Mycobacterium avium subsp. paratuberculosis (MAP), a fastidious intracellular pathogen that belongs to the Mycobacteriaceae family. This acid-fast, hard-to-detect bacterium can resist milk pasteurization and be conveyed to dairy product consumers. Many studies have emphasized the zoonotic nature of MAP, suggesting an association between MAP and some gastroenteric conditions such as Crohn’s disease in humans. This underlines the importance of utilizing efficient pasteurization alongside a state-of-the-art diagnostic system in order to minimize the possible ways this pathogen can be conveyed to humans. Until now, no confirmatory MAP screening technique has been developed that can reveal the stages of JD in infected animals. This is partially due to the lack of an efficient gold-standard reference method that can properly evaluate the performance of diagnostic assays. Therefore, the following research aimed to compare the merits of qPCR and ELISA assessments of milk for the detection of MAP in a total of 201 Sardinian unpasteurized sheep milk samples including 73 bulk tank milk (BTM) and 128 individual samples from a MAP-infected flock (MIF) applying various reference models. Accordingly, milk qPCR and ELISA assessments, together and individually, were used as reference models in the herd-level study, while serum ELISA and fecal PCR were similarly (together and in isolation) considered as the gold standards in the individual-level diagnosis. This study showed that the type of gold-standard test affects the sensitivity and specificity of milk qPCR and ELISA significantly. At the individual level in the MAP-infected flock, serum ELISA in isolation and together with fecal PCR were recognized as the best references; however, the best correlation was seen between milk and serum ELISA (p < 0.0001). Regarding the detection of MAP in BTM, qPCR IS900 was recognized as the most sensitive and specific diagnostic test (p < 0.0001) for monitoring the MAP shedders and animals with clinically developed symptoms within herds, under the condition that both milk qPCR and milk ELISA tests formed a binary reference model. The BTM analyses (qPCR and ELISA) revealed that MAP positivity has a seasonal pattern. This hypothesis was proven through a longitudinal study on 14 sheep herds.

scholarly journals A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Vaccines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 997
Author(s):  
Sepideh Hosseiniporgham ◽  
Franck Biet ◽  
Christelle Ganneau ◽  
John P. Bannantine ◽  
Sylvie Bay ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Elisa Test ◽  
Cross Reactivity ◽  
Mycobacterium Avium Subsp ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Individual Level ◽  
Milk Samples ◽  
Milk Elisa ◽  
Mycobacterium Avium Subsp Paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.

Modeling the Occurrence of Mycobacterium avium subsp. paratuberculosis in Bulk Raw Milk and the Impact of Management Options for Exposure Mitigation

2011 ◽  
Vol 74 (7) ◽  
pp. 1126-1136 ◽  
Author(s):  
CHRISTOPHE BOULAIS ◽  
RON WACKER ◽  
JEAN-CHRISTOPHE AUGUSTIN ◽  
MOHAMED HEDI BEN CHEIKH ◽  
FABRICE PELADAN
Keyword(s):  
Mycobacterium Avium ◽  
Raw Milk ◽  
Mycobacterium Avium Subsp ◽  
Dairy Herds ◽  
Management Options ◽  
Scientific Debate ◽  
Milk Samples ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Exposure Mitigation ◽  
The Impact

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis (Johne's disease) in cattle and other farm ruminants. The potential role of MAP in Crohn's disease in humans and the contribution of dairy products to human exposure to MAP continue to be the subject of scientific debate. The occurrence of MAP in bulk raw milk from dairy herds was assessed using a stochastic modeling approach. Raw milk samples were collected from bulk tanks in dairy plants and tested for the presence of MAP. Results from this analytical screening were used in a Bayesian network to update the model prediction. Of the 83 raw milk samples tested, 4 were positive for MAP by culture and PCR. We estimated that the level of MAP in bulk tanks ranged from 0 CFU/ml for the 2.5th percentile to 65 CFU/ml for the 97.5th percentile, with 95% credibility intervals of [0, 0] and [16, 326], respectively. The model was used to evaluate the effect of measures aimed at reducing the occurrence of MAP in raw milk. Reducing the prevalence of paratuberculosis has less of an effect on the occurrence of MAP in bulk raw milk than does managing clinically infected animals through good farming practices.

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 Application of the Actiphage® Assay to Detect Viable Mycobacterium avium subsp. paratuberculosis Cells in Fresh Sheep and Goat Milk and Previously Frozen Milk and In-Line Milk Filters

2021 ◽  
Vol 8 ◽  
Author(s):  
Monika Beinhauerova ◽  
Iva Slana
Keyword(s):  
Mycobacterium Avium ◽  
Limit Of Detection ◽  
Goat Milk ◽  
Time Lapse ◽  
Cow Milk ◽  
Economic Losses ◽  
Diagnostic Tools ◽  
Lytic Bacteriophage ◽  
Milk Samples ◽  
Detection And Identification

Mycobacterium avium subsp. paratuberculosis (MAP) is a well-known causative agent of paratuberculosis, a chronic infectious granulomatous enteritis of ruminants contributing to significant economic losses worldwide. Current conventional diagnostic tools are far from being sufficient to manage and control this disease. Therefore, increased attention has been paid to alternative approaches including phage-based assays employing lytic bacteriophage D29 to detect MAP cells. The aim of the present study was to assess the applicability and efficiency of the recently developed phage-based kit termed Actiphage® combined with IS900 real-time PCR (qPCR) for rapid detection and quantification of viable MAP in milk samples. We demonstrated that Actiphage® in combination with IS900 qPCR allows for rapid and sensitive detection and identification of viable MAP in milk samples with a limit of detection of 1 MAP per 50 ml milk. Using this method, the presence of viable MAP cells was successfully determined in 30.77% of fresh goat, sheep and cow milk samples originating from paratuberculosis-affected herds. We further used Actiphage assay to define the time-lapse aspect of testing naturally contaminated milk and milk filters frozen for various lengths of time by phage-based techniques. Viable MAP was detected in 13.04% of frozen milk samples and 28.57% of frozen milk filters using Actiphage-qPCR. The results suggest the ability to detect viable MAP in these samples following freezing for more than 1 year. The obtained results support the views of the beneficial role of this technology in the control or monitoring of paratuberculosis.

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