Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis : Evidence for Limited Strain Diversity, Strain Sharing, and Identification of Unique Targets for Diagnosis

Abstract

The objectives of this study were to understand the molecular diversity of animal and human strains of Mycobacterium avium subsp. paratuberculosis isolated in the United States and to identify M. avium subsp. paratuberculosis-specific diagnostic molecular markers to aid in disease detection, prevention, and control. Multiplex PCR of IS900 integration loci (MPIL) and amplified fragment length polymorphism (AFLP) analyses were used to fingerprint M. avium subsp. paratuberculosis isolates recovered from animals (n ‫؍‬ 203) and patients with Crohn's disease (n ‫؍‬ 7) from diverse geographic localities. Six hundred bacterial cultures, including M. avium subsp. paratuberculosis (n ‫؍‬ 303), non-M. avium subsp. paratuberculosis mycobacteria (n ‫؍‬ 129), and other nonmycobacterial species (n ‫؍‬ 168), were analyzed to evaluate the specificity of two IS900 integration loci and a newly described M. avium subsp. paratuberculosis-specific sequence (locus 251) as potential targets for the diagnosis of M. avium subsp. paratuberculosis. MPIL fingerprint analysis revealed that 78% of bovine origin M. avium subsp. paratuberculosis isolates clustered together into a major node, whereas isolates from human and ovine sources showed greater genetic diversity. MPIL analysis also showed that the M. avium subsp. paratuberculosis isolates from ovine and bovine sources from the same state were more closely associated than were isolates from different geographic regions, suggesting that some of the strains are shared between these ruminant species. AFLP fingerprinting revealed a similar pattern, with most isolates from bovine sources clustering into two major nodes, while those recovered from sheep or humans were clustered on distinct branches. Overall, this study identified a high degree of genetic similarity between M. avium subsp. paratuberculosis strains recovered from cows regardless of geographic origin. Further, the results of our analyses reveal a relatively higher degree of genetic heterogeneity among M. avium subsp. paratuberculosis isolates recovered from human and ovine sources.Paratuberculosis or Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (48). It is estimated that 35% of the U.S. herds are infected with M. avium subsp. paratuberculosis, resulting in annual losses of $200 million (7). Crohn's disease (CD) is also a chronic inflammation of distal intestines exhibiting a pathology similar to that of JD in ruminants. The prevalence of CD is estimated to be 0.15% among the U.S. population resulting in substantial morbidity and medical costs (1). M. avium subsp. paratuberculosis has been implicated as a cause of CD in humans (13,21,36). Currently, the evidence for a link remains inconclusive since strain sharing or a causal role of M. avium subsp. paratuberculosis has not been demonstrated.Comprehensive analysis of the molecular diversity and comparative molecular pathology of M. avium subsp. paratuberculosis will help establish the degree of heterog...

Experimental challenge models for Johne's disease: A review and proposed international guidelines

“…Until 2000, the majority of experimental studies in sheep did not use characterized ovine MAP strains, in contrast to later studies which have used tissue homogenates or low passage ovine MAP strains grown in vitro (Gwozdz et al, 2000a,b;Stewart et al, 2004;. The most common MAP isolates from clinical JD in sheep belong to the ovine genotype (Motiwala et al, 2004;Sevilla et al, 2005;Sevilla et al, 2007). Marked genetic diversity was detected among ovine isolates, as well as between ovine and both bovine and goat isolates (Motiwala et al, 2004).…”

“…Twenty-six to 50% of isolates from clinical cases in goats have been of bovine genotypes (Motiwala et al, 2004;Sevilla et al, 2005;Sevilla et al, 2007). Motiwala et al (2004) detected little genetic diversity between and within bovine and goat isolates, but Sevilla et al (2007) showed that goat isolates had a high degree of genetic heterogeneity.…”

“…The most common MAP isolates from clinical JD in sheep belong to the ovine genotype (Motiwala et al, 2004;Sevilla et al, 2005;Sevilla et al, 2007). Marked genetic diversity was detected among ovine isolates, as well as between ovine and both bovine and goat isolates (Motiwala et al, 2004). Therefore, to most closely reproduce natural ovine infections, any confirmed-virulent, ovine clinical isolate that can be cultivated in vitro is considered acceptable.…”

Mycobacterium avium ssp . paratuberculosis detection in animals, food, water and other sources or vehicles of human exposure: A scoping review of the existing evidence

“…Most of these studies were based on existing isolate collections, and showed a fair amount of homogeneity within M. paratuberculosis strains and revealed that M. paratuberculosis strains from human infections were less diverse compared to cattle (Wynne et al, 2011). Results were derived from several methods making it difficult to make direct comparisons, including IS1311 restriction fragment length polymorphism (RFLP) analyses (Whittington et al, 2000;Motiwala et al, 2003;Singh et al, 2010b;Okuni et al, 2012;Liapi et al, 2015), (Stevenson et al, 2009;Fernandez-Silva et al, 2012) randomly amplified polymorphic DNA (RAPD) (Pillai et al, 2001), multi-locus short sequence repeat sequencing (MLSSR) (Fernandez-Silva et al, 2012), Multiplex PCR of IS900 integration loci (MPIL) (Motiwala et al, 2004), short sequence repeats (SSR) (Ghadiali et al, 2004), and single nucleotide polymorphisms (SNPs) (Wynne et al, 2011). A tally of the type of M. paratuberculosis (e.g.…”

Distribution of Mycobacterium avium subsp.paratuberculosis in tissues of naturally infected cattle as affected by age

“…Methods for differentiation or subtyping of MAP, such as IS900 multiplex PCR (Bull et al, 2000), RFLP -random fragment length polymorphism (Pavlik et al, 1999), AFLP -amplified fragment length polymorphism (Motiwala et al, 2003) or MLSSR -multilocus short sequence repeat (Amonsin et al, 2004) have been previously developed. RFLP analysis of the examined tissue isolates identified RFLP type B-C1 in all cases.…”