title: Specific immunoassays confirm association of Mycobacterium avium Subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus.
abstract: BACKGROUND
  Mycobacterium avium subspecies paratuberculosis (MAP) is a versatile pathogen with a broad host range. Its association with type-1 diabetes mellitus (T1DM) has been recently proposed. Rapid identification of infectious agents such as MAP in diabetic patients at the level of clinics might be helpful in deciphering the role of chronic bacterial infection in the development of autoimmune diseases such as T1DM.
  
  
  RESULTS
  We describe use of an ELISA method to identify live circulating MAP through the detection of a cell envelope protein, MptD by a specific M13 phage--fMptD. We also used another ELISA format to detect immune response to MptD peptide. Both the methods were tested with blood plasma obtained from T1DM, type-2 diabetes (T2DM) patients and non-diabetic controls. Our results demonstrate MptD and fMptD ELISA assays to be accurate and sensitive to detect MAP bacilli in a large fraction (47.3%) of T1DM patients as compared to non-diabetic controls (12.6%) and those with confirmed T2DM (7.7%). Comparative analysis of ELISA assays performed here with 3 other MAP antigen preparations, namely HbHA, Gsd and whole cell MAP lysates confirmed comparable sensitivity of the MptD peptide and the fMptD based ELISA assays. Moreover, we were successful in demonstrating positive bacterial culture in two of the clinical specimen derived from T1DM patients.
  
  
  CONCLUSIONS
  The MptD peptide/fMptD based ELISA or similar tests could be suggested as rapid and specific field level diagnostic tests for the identification of MAP in diabetic patients and for finding the explanations towards the occurrence of type-1 or type-2 diabetes in the light of an active infectious trigger.
authors.affiliation: Dipartimento di Scienze Biomediche, Sezione di Microbiologia, Università degli studi di Sassari, Sassari, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Rosu
authors.forename: Niyaz
authors.initials: N
authors.lastname: Ahmed
authors.forename: Daniela
authors.initials: D
authors.lastname: Paccagnini
authors.forename: Gerald
authors.initials: G
authors.lastname: Gerlach
authors.forename: Giovanni
authors.initials: G
authors.lastname: Fadda
authors.forename: Seyed E
authors.initials: SE
authors.lastname: Hasnain
authors.forename: Stefania
authors.initials: S
authors.lastname: Zanetti
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Sechi
mesh_terms: D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D016022:Case-Control Studies; D002457:Cell Extracts / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000276:immunology / Q000382:microbiology*; D003924:Diabetes Mellitus, Type 2 / Q000276:immunology / Q000382:microbiology*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000276:immunology* / Q000302:isolation & purification; D010455:Peptides / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D002457:Cell Extracts; D010455:Peptides; D011994:Recombinant Proteins