Detection of Mycobacterium avium Subspecies paratuberculosis (MAP) Microorganisms Using Antigenic MAP Cell Envelope
Shanmugasundaram Karuppusamy1, Lucy Mutharia2, David Kelton3
1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Abstract:
Cell envelope proteins from Mycobacterium avium subspecies paratuberculosis (MAP) that are antigenically distinct from closely related mycobacterial species are potentially useful for Johne's Disease (JD) diagnosis. We evaluated the potential of ELISAs, based on six antigenically distinct recombinant MAP cell envelope proteins (SdhA, FadE25_2, FadE3_2, Mkl, DesA2, and hypothetical protein MAP1233) as well as an extract of MAP total cell envelope proteins, to detect antibodies against MAP in the sera of infected cattle. The sensitivity (Se) and specificity (Sp) of an ELISA based on MAP total cell envelope proteins, when analyzing 153 bovine serum samples, was 75 and 96%, respectively. Analysis of the same samples, using a commercial serum ELISA resulted in a Se of 56% and Sp of 99%. Results of ELISA analysis using plates coated with recombinant cell envelope proteins ranged from a highest Se of 94% and a lowest Sp of 79% for Sdh A to a lowest Se of 67% and a highest Sp of 95% for hypothetical protein MAP1233. Using polyclonal antibodies to MAP total cell envelope proteins, immunohistochemical analysis of intestinal and lymph node tissues from JD-positive cattle detected MAP organisms whereas antibodies to recombinant proteins did not. Finally, polyclonal antibodies to MAP total cell envelope protein and to recombinant SdhA, FadE25_2, and DesA2 proteins immunomagnetically separated MAP microorganisms spiked in PBS. These results suggest that antigenically distinct MAP cell envelope proteins and antibodies to these proteins may have potential to detect MAP infection in dairy cattle.
Insights
Researchers explored new diagnostic tools for Johne
Area of Science:
- Veterinary Immunology
- Bacteriology
- Diagnostic Microbiology
Background:
- Johne's Disease (JD) is caused by Mycobacterium avium subspecies paratuberculosis (MAP).
- Accurate diagnosis of JD in cattle is crucial for disease control.
- Identifying antigenically distinct MAP cell envelope proteins is key for developing specific diagnostic assays.
Purpose of the Study:
- To evaluate the diagnostic potential of recombinant MAP cell envelope proteins and total MAP cell envelope proteins in ELISAs for detecting antibodies against MAP in cattle sera.
- To assess the sensitivity and specificity of these novel ELISAs compared to a commercial assay.
Main Methods:
- ELISAs were developed using six recombinant MAP cell envelope proteins and total MAP cell envelope proteins.
- 153 bovine serum samples from infected and control cattle were analyzed.
- Immunohistochemistry and immunomagnetic separation were used to validate findings.
Main Results:
- ELISA with total MAP cell envelope proteins showed 75% sensitivity and 96% specificity.
- Recombinant SdhA achieved 94% sensitivity and 79% specificity.
- Commercial ELISA had 56% sensitivity and 99% specificity.
- Immunohistochemistry detected MAP organisms using antibodies to total cell envelope proteins.
Conclusions:
- Antigenically distinct MAP cell envelope proteins show promise for JD diagnosis.
- Further development of these proteins into diagnostic tools could improve detection of MAP infection in dairy cattle.
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