Typage génétique des souches de Mycobacterium avium subsp. de paratuberculosis isolées de 14 vaches excrétant des niveaux élevés de MAP dans les troupeaux des provinces de Québec et de l'Ontario
Citation
Byrne, A., O. Severine, T. Cochard, K. Tahlan, F. Biet, and N Bissonnette. 2022. Genetic typing of Mycobacterium avium subsp. paratuberculosis strains isolated from 14 cows shedding high levels of MAP in the Provinces of Quebec and Ontario Herds. 15th International Colloquium on Paratuberculosis, Dublin, Irland, June 12-16th 2022.
Résumé en langage clair
Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterium responsible for Johne's disease in ruminants. This disease is manifested by an inflammation of the intestine which is similar to Crohn's disease in humans. It is found especially in dairy herds. This incurable disease is chronic and sometimes progresses slowly (5-7 years). However, its progression is unpredictable. During this subclinical period (before showing symptoms), infected animals may excrete MAP in the faeces, allowing the bacteria to infect other animals. In Canada, we know of the existence of several foci of infection, also called strains of the MAP pathogen. This science that seeks to understand the origin and transmission of strains is called epidemiology. In order to trace the movement of strains, molecular epidemiology techniques based on PCR are used. This involves detecting portions of the bacteria, reading them by sequencing or another method of discrimination, to differentiate them. The aim is thus to establish the distinctive profile or a molecular signature of each outbreak of MAP infection. Widespread techniques allow the examination of targets distributed throughout the MAP genome. These techniques are known as MIRU-VNTR or SSR. Basically, the analysis of fragments of MAP that contain specific short sequence repeats makes it possible to differentiate the nature of the foci of infection.
An important consideration is the presence of multiple strain infections, i.e. from separate foci. The existence of multiple strains in animals with paratuberculosis is a phenomenon that has not been studied. Although reported for other infections, including tuberculosis in humans, it had not been studied in cows. In this study, we selected 14 cows with paratuberculosis in three farms, identified due to diagnostic blood tests (ELISA) and faecal PCR, both positive for the disease. Using bacterial culture, strains were identified from faecal analysis, as cows excreted MAP in their faeces. Similar to characterizing a single strain per cow, 10 strains were isolated per cow. A total of 139 strains were analyzed. The individual culture of the different strains made it possible to analyze their molecular signature. The results of the analysis by the MIRU-VNTR technique were identical for all. However, results from SSR fragment analysis showed up to 9 unique patterns in each animal suggesting that multiple foci of infection may be present.
The results of this analysis show that SSR can discriminate foci of infection with greater resolving power than the MIRU-VNTR technique. We do not know the extent of the phenomenon in the herds, but the presence of this phenomenon in all of the 14 cows analyzed suggests that the phenomenon is widespread. As always, this disease is unpredictable to see the appearance in some infected cows of symptoms of rapid progression of the disease. Knowing that several foci of infection are present, the question arises as to whether infection with an additional strain can influence the progression of the disease in certain animals.
Résumé
Mycobacterium avium subsp. paratuberculosis (MAP) is the causal bacteria for Johne’s Disease, a chronic granulomatous enteritis affecting both wild and domestic ruminants. During the subclinical stage of infection, infected animals may excrete MAP in feces, allowing disease to spread undetected throughout the herd. Epidemiological studies have used PCR-based techniques to examine loci found throughout the genome known as MIRU-VNTR. Additional studies have used DNA fragment analysis performed on specific short sequence repeats (SSRs), which also discriminate strains. An important consideration is the occurrence of mixed strain infections (MSIs), which may exhibit different phenotypes or antigens, complicating the immune response. Instances of MSIs are typically considered as rare events, with few studies successfully identifying MSIs within MAP host animals. This study examines strains of MAP isolated from 14 cows and evaluates molecular methods for rapid MSI identification, notably using MIRU-VNTR and SSR-based fragment analysis. Cows were selected from three farms and were categorized based on blood ELISA and fecal ISMAP02 qPCR results. Strains of MAP (10 per cow) were selected on solid cultures. For each strain, an axenic liquid culture was prepared, totalling 139 isolates. DNA extracted from each strain was examined using 8 MIRU-VNTR and 2 SSR loci. The MIRU-VNTR analysis results were identical across all isolates. However, the results from the SSR fragment analysis showed up to 9 unique patterns within each animal suggesting that MSIs may be present. As the rate of change of SSR could be independent of the rate of chromosomal evolution, further work will include whole-genome sequencing, which provides the greatest resolution for differentiation between strains. Results of this analysis show that SSR can discriminate strains with greater resolving ability than MIRU-VNTR. Our study suggests that MSI events may be overlooked within herds and should be considered in a phylogenetic context for affirming epidemiological connections.