Mycobacterium avium subsp. paratuberculosis and Crohn's disease: characterization of the interaction with different aspects of the disease

Abstract

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease with no fully understood etiology and cure. Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of paratuberculosis, is also isolated from samples from human patients with CD. Paratuberculosis is characterized by persistent diarrhea and progressive weight loss and primarily affects ruminants, which eliminate the agent via feces and milk. The involvement of MAP in the pathogenesis of CD and other intestinal diseases is unclear. Thus, the present study aimed to analyze immunological, socioepidemiological, biochemical, and therapeutic variables that may be related to the occurrence of MAP in blood samples and CD patients. The sampling was random, and the population of origin was the patients from the Bowel Outpatient Clinic of the Alpha Institute of Gastroenterology (IAG), Hospital das Clínicas, Universidade Federal de Minas Gerais (HC-UFMG). Blood samples were collected from 20 patients with CD, eight with ulcerative rectocolitis (UCR), and 10 control patients without inflammatory bowel diseases. Samples were subjected to real-time PCR for detection of MAP DNA, oxidative stress analyses, and socioepidemiological variables. MAP was detected in 10 (26.3%) of the patients, seven (70%) were CD patients, 2 (20%) were URC patients, and one (10%) was a non-IBD patient. MAP was found more frequently among CD patients, but not restricted to CD patients. The presence of MAP in the blood of these patients occurred simultaneously with an inflammatory response with an increase in neutrophils and significant alterations in the production of antioxidant enzymes such as catalase and GST.

Keywords: Inflammation; Inflammatory bowel disease; Oxidative stress; Paratuberculosis.

Fig. 1

qt-PCR of the 900 positive samples, confirmed with F57. All reactions were performed in triplicate in 96-well plates (Bio-Rad) and sealed with Microseal “B” film (Bio-Rad). Real-time PCR was performed on a CFX96 real-time PCR detection system (Bio-Rad). The reaction conditions were: 98 °C for 2 min, then 40 cycles at 98 °C for 5 s, and pairing/extension at 55.0 °C for 10 s. Then, the PCR products were heated to 95.0 for 1 min and then cooled to 70.0 for 1 min. Finally, the melting curve was generated by measuring the fluorescence during a temperature increase from 70 to 95 °C (with 0.2 °C/10 s increments). Only samples that had a CT (“cycle threshold”) less than 38 in at least two of the three positive repeats for both target sequences (IS900 and F57) were considered positive. The results were analyzed with Bio-Rad CFX Manager v.1 software and then with Bio-Rad Precision Melt Analysis software

Fig. 2

Percentage of segmented leukocytes in the blood of sampled individuals (total population = 38), categorized according to the presence of Mycobacterium avium subspecies paratuberculosis in blood detected by the real-time PCR technique, compared by the t-test, considering p = 0.05

Fig. 3

Percentage of segmented leukocytes in the blood of individuals with Crohn’s disease sampled, categorized according to the presence of Mycobacterium avium subspecies paratuberculosis in blood detected by the real-time PCR technique, compared by the t-test, considering p = 0.05

Fig. 4

Oxidative stress analysis of patients with Crohn’s disease. A Comparison between serum levels of catalase from Mycobacterium avium subspecies paratuberculosis–positive and Mycobacterium avium subspecies paratuberculosis–negative individuals using the t-test, considering p = 0.05. B Comparison between serum levels of FRAP (“ferric reducing antioxidant power”) of positive and negative patients for Mycobacterium avium subspecies paratuberculosis using the t-test, considering p = 0.05

Fig. 5

Oxidative stress analysis of patients with Crohn’s disease. A Comparison between serum levels of superoxide dismutase (SOD) of positive and negative individuals using the t-test, considering p = 0.05. B Comparison between serum levels of glutathione-S-transferase (GST) of Mycobacterium avium subspecies paratuberculosis–positive and Mycobacterium avium subspecies paratuberculosis–negative patients using the t-test, considering p = 0.05

Fig. 6

Oxidative stress analysis of patients with Crohn’s disease. A Comparison between serum levels of hydrogen peroxide (H₂O₂) of positive and negative individuals for Mycobacterium avium subspecies paratuberculosis using the t-test, considering p = 0.05. B Comparison between serum nitric oxide levels of positive and negative patients for Mycobacterium avium subspecies paratuberculosis using the t-test, considering p = 0.05