scholarly journals Search forMycobacterium aviumSubspeciesparatuberculosisAntigens for the Diagnosis of Paratuberculosis

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
María Laura Mon ◽  
Mariana Viale ◽  
Guido Baschetti ◽  
Fiorella Alvarado Pinedo ◽  
Andrea Gioffre ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Mycobacterium Avium ◽  
Bovine Tuberculosis ◽  
Cross Reactivity ◽  
Second Step ◽  
Initial Screening ◽  
Combined Application ◽  
Form Part

The aim of this study was to evaluate a wide panel of antigens ofMycobacterium aviumsubsp.paratuberculosis(MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n=25), healthy animals (n=10), and animals experimentally infected withM. bovis(n=8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected withM. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n=25), healthy animals (n=26), and animals experimentally infected withM. bovis(n=17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB.

scholarly journals Disparate Host Immunity to Mycobacterium avium subsp.paratuberculosisAntigens in Calves Inoculated with M. avium subsp.paratuberculosis, M. avium subsp.avium, M. kansasii, and M. bovis

2013 ◽  
Vol 20 (6) ◽  
pp. 848-857 ◽  
Author(s):  
J. R. Stabel ◽  
W. R. Waters ◽  
J. P. Bannantine ◽  
M. V. Palmer
Keyword(s):  
Immune Responses ◽  
Recombinant Proteins ◽  
Mycobacterium Avium ◽  
Mycobacterial Infection ◽  
Cross Reactivity ◽  
Antigenic Specificity ◽  
Cell Mediated Immunity ◽  
Content Type ◽  
Ifn Γ ◽  
Mycobacterial Antigens

ABSTRACTThe cross-reactivity of mycobacterial antigens in immune-based diagnostic assays has been a major concern and a criticism of the current tests that are used for the detection of paratuberculosis. In the present study,Mycobacterium aviumsubsp.paratuberculosisrecombinant proteins were evaluated for antigenic specificity compared to a whole-cell sonicate preparation (MPS). Measures of cell-mediated immunity toM. aviumsubsp.paratuberculosisantigens were compared in calves inoculated with liveM. aviumsubsp.paratuberculosis,M. aviumsubsp.avium(M. avium),Mycobacterium kansasii, orMycobacterium bovis. Gamma interferon (IFN-γ) responses to MPS were observed in all calves that were exposed to mycobacteria compared to control calves at 4 months postinfection. Pooled recombinantM. aviumsubsp.paratuberculosisproteins also elicited nonspecific IFN-γ responses in inoculated calves, with the exception of calves infected withM. bovis.M. aviumsubsp.paratuberculosisproteins failed to elicit antigen-specific responses for the majority of immune measures; however, the expression of CD25 and CD26 was upregulated on CD4, CD8, gamma/delta (γδ) T, and B cells for the calves that were inoculated with eitherM. aviumsubsp.paratuberculosisorM. aviumafter antigen stimulation of the cells. Stimulation with MPS also resulted in the increased expression of CD26 on CD45RO+CD25+T cells from calves inoculated withM. aviumsubsp.paratuberculosisandM. avium. Although recombinant proteins failed to elicit specific responses for the calves inoculated withM. aviumsubsp.paratuberculosis, the differences in immune responses toM. aviumsubsp.paratuberculosisantigens were dependent upon mycobacterial exposure. The results demonstrated a close alignment in immune responses between calves inoculated withM. aviumsubsp.paratuberculosisand those inoculated withM. aviumthat were somewhat disparate from the responses in calves infected withM. bovis, suggesting that the biology of mycobacterial infection plays an important role in diagnosis.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals Performance of recombinant proteins in diagnosis and differentiation of canine visceral leishmaniasis infected and vaccinated dogs

2020 ◽  
Vol 10 (3) ◽  
pp. 165-171
Author(s):  
Ingrid E. Pereira ◽  
Kyssia P. Silva ◽  
Laura M. Menegati ◽  
Aimara C. Pinheiro ◽  
Elaine A. O. Assunção ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Recombinant Proteins ◽  
Serological Test ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Reservoir Host ◽  
Superior Performance ◽  
Canine Visceral Leishmaniasis ◽  
Roc Curve Analysis

AbstractControl of canine visceral leishmaniasis (CVL), a major zoonotic disease in Brazil and many other tropical and subtropical countries, remains difficult as an accurate and reliable diagnosis is still missing. In endemic regions, infected dogs are the main parasitic reservoir host of human Visceral leishmaniasis (VL) infection. Vaccination of dogs against Leishmania infection constitutes an important strategy to prevent or to better control CVL, thus, a serological test that can discriminate between antibodies induced by immunization versus infection is highly desirable in order to improve and simplify diagnosis. Here, four recombinant proteins were evaluated for their ability to detect and differentiate between dogs that are infected with Leishmania or have been immunized with the anti-Leishmania vaccine Leish-Tec®. Receiver operating characteristic (ROC) curve analysis of the four Leishmania-specific IgG ELISA revealed superior performance of rK28, followed by rKLO8, rK39 and rLb6H. The rK28-based ELISA revealed not only the best accuracy against CVL, but also the lowest cross-reactivity with sera from Leish-Tec® immunized dogs. Our data show that the rK28-based ELISA is highly suitable for CVL screening as it shows high sensitivity with simultaneous low cross-reactivity. Further, the high specificity of the rKLO8 indicates its suitability for the confirmation of CVL diagnosis.

scholarly journals Uterus Transplantation with Live Donors: Screening Candidates in One French Center

2020 ◽  
Vol 9 (6) ◽  
pp. 2001 ◽  
Author(s):  
Marie Carbonnel ◽  
Aurelie Revaux ◽  
Elena Menzhulina ◽  
Lea Karpel ◽  
Renaud Snanoudj ◽  
...  
Keyword(s):  
International Society ◽  
Limiting Factors ◽  
Second Step ◽  
Initial Screening ◽  
Inclusion Criteria ◽  
Screening Process ◽  
Uterus Transplantation ◽  
Individual Interviews ◽  
Live Donors ◽  
The Future

We report our experience regarding the profile and screening process of potential recipients (R) and their live donors (D) in our Uterus transplantation (UTx) trial from 2014 to 2020. The initial screening was performed using medical questionnaires and consultations. The second step of the screening consisted of two individual interviews with an independent multidisciplinary committee. Then, a complete medical, biological and imaging assessment of the directed living D, the R, and her partner was performed over a two-day hospitalization. A total of 239 women contacted our department: 165 potentials R and 74 potentials D. During the first step of screening, 141 R and 45 D were excluded. Only 12 R/D pairs were pursued. During inclusion, 10 R/D pairs were excluded. One R/D pair is still under evaluation. Finally, only 1 R/D pair was definitively included (0.6%), which led us to perform the first French UTx in March 2019 with a successful graft. The primary limiting factors of inclusion were due to very strict criteria and difficulty of having a suitable directed living D. The International Society of UTx (ISUTx) guidelines based on worldwide results of trials can help ease our inclusion criteria in the future while remaining safe for patients.

scholarly journals Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Maor Sheva ◽  
Uri Hanania ◽  
Tami Ariel ◽  
Albina Turbovski ◽  
Vishal Kumar Rameshchandra Rathod ◽  
...  
Keyword(s):  
Host Cell ◽  
Genome Editing ◽  
Cell Lines ◽  
Recombinant Proteins ◽  
Plant Cells ◽  
Second Step ◽  
Selectable Markers ◽  
Additional Gene ◽  
Bright Yellow ◽  
Foreign Dna

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

scholarly journals Paratuberculosis Vaccination Causes Only Limited Cross-Reactivity in the Skin Test for Diagnosis of Bovine Tuberculosis

PLoS ONE ◽  
2013 ◽  
Vol 8 (11) ◽  
pp. e80985 ◽  
Author(s):  
Joseba M. Garrido ◽  
Patricia Vazquez ◽  
Elena Molina ◽  
Jose M. Plazaola ◽  
Iker A. Sevilla ◽  
...  
Keyword(s):  
Skin Test ◽  
Bovine Tuberculosis ◽  
Cross Reactivity

scholarly journals Development and Validation of a Novel ELISA for the Specific Detection of Antibodies against Mycobacterium avium Subspecies paratuberculosis Based on a Chimeric Polyprotein

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Roberto Damián Moyano ◽  
Magali Andrea Romero ◽  
María Alejandra Colombatti Olivieri ◽  
María Fiorella Alvarado Pinedo ◽  
Gabriel Eduardo Traveria ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Roc Curve ◽  
Bovine Serum ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Cross Reactivity ◽  
Specific Detection ◽  
Serum Samples ◽  
Specific Antigens ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Development And Validation

Bovine paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The optimization of detection tests specific for MAP is crucial to improve PTB control. In this work, we aimed to develop and validate a diagnostic tool based on an ELISA to specifically detect anti-MAP antibodies from bovine serum samples. For that purpose, we designed a recombinant polyprotein containing four specific antigens from MAP and optimized the ELISA. The validation consisted of the assessment of 10 sera from PTB-infected and healthy bovines with different OD values. The diagnostic performance of the polyprotein-ELISA was evaluated by testing 130 bovine serum samples (47 healthy, 48 MAP-infected, and 35 M. bovis-infected bovines). The ELISA using the polyprotein yielded an area under the ROC curve (AUC) of 0.9912 (95% CI, 0.9758–1.007; P  < 0.0001). Moreover, for this ELISA, the cut-off selected from the ROC curve based on the point with a sensitivity of 95.56% (95% CI, 0.8485–0.9946) and specificity of 97.92 (95% CI, 0.8893–0.9995) was 0.3328. Similar results were obtained with an ELISA using the commercial Paratuberculosis Protoplasmatic Antigen (PPA). However, the ELISA with the polyprotein antigen showed a better performance against sera from animals infected with Mycobacterium bovis compared to the ELISA with PPA: lower cross-reactivity (2.85% versus 25.71%). These results demonstrate a very low cross-reactivity of the polyprotein with antibodies present in serum samples from animals infected with M. bovis. The designed polyprotein and the validated ELISA could be very useful for the specific identification of MAP-infected animals in herds.

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