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J Clin Microbiol. Aug 2006; 44(8): 2970–2973.
PMCID: PMC1594651

Short-Sequence-Repeat Analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium Isolates Collected from Animals Throughout the United States Reveals Both Stability of Loci and Extensive Diversity

Abstract

We analyzed the multilocus short sequence repeats (SSRs) of 211 and 56 isolates of Mycobacterium avium subsp. paratuberculosis and M. avium subsp. avium, respectively. The M. avium subsp. paratuberculosis isolates could be differentiated into 61 genotypes. The M. avium subsp. avium isolates showed limited diversity. These SSRs are stable and suitable for studying the molecular epidemiology of M. avium subsp. paratuberculosis.

The Mycobacterium avium complex consists of a closely related group of microorganisms characterized by over 90% similarity at the nucleotide level; but its members differ widely in terms of their host tropisms, microbiological and disease phenotypes, and pathogenicities (8, 9, 11). In recent years, the M. avium complex has assumed greater importance in human medicine, largely because of often intractable infections in AIDS patients and because of the possible association of M. avium subsp. paratuberculosis with Crohn's disease (8, 11).

M. avium subsp. paratuberculosis is the causative agent of Johne's disease in cattle and other ruminants. This infection is characterized by chronic granulomatous enteritis, which is associated with a prolonged incubation period of months to years prior to the manifestation of clinical disease. Unambiguous differentiation of the infecting isolates is expected to provide useful epidemiologic information about the origin of an isolate to permit the rational design of more adequate intervention strategies. National Johne's disease control policies will gain from a thorough understanding of M. avium subsp. paratuberculosis transmission pathways.

The use of multilocus variable tandem repeats is well established for the genotyping of many pathogenic bacterial strains, such as Staphylococcus aureus (7), Escherichia coli (10), Salmonella species (16), Bacillus anthracis (18), and Mycobacterium tuberculosis (4). Within a population of bacterial species, variation in the number of copies of the repeat element at a specific locus is indicative of diversity. Due to the conserved nature of the M. avium subsp. paratuberculosis genome, most strain subtyping methods to date have provided limited information on the diversity of this organism. Recent sequencing of the M. avium subsp. paratuberculosis K-10 genome (12) has permitted the identification and application of multiple short sequence repeats (SSRs) to the study of the diversity, strain sharing, and host specialization among M. avium subsp. paratuberculosis isolates. However, only a limited number of studies have used this method with a restricted set of isolates (1, 3, 13, 14). Thus, the objective of this study was to analyze the in vitro stability of the most discriminatory SSR loci and provide information on the distribution and molecular diversity of M. avium subsp. paratuberculosis isolates from dairy herds throughout the United States. To understand the diversity and genotype distribution of specific SSR loci, we evaluated a set of four loci in both M. avium subsp. paratuberculosis and M. avium subsp. avium, using a collection of isolates derived from animals throughout the United States. We also performed studies of the stability of these loci in several isolates with different SSR genotypes. Isolates obtained from animal and environmental specimens were confirmed as M. avium subsp. paratuberculosis based on the presence of IS900 by PCR (2). The M. avium subsp. avium isolates analyzed in this study originated from diagnostic submissions to the National Veterinary Services Laboratories from 2003 through 2005.

To determine the most discriminatory and informative SSR loci, we first analyzed a subset of 68 bacterial isolates from different geographic locations using all 11 previously described SSR loci (1) Based on this analysis, locus 1, locus 2, locus 8, and locus 9 were identified as being associated with the highest Simpson's diversity indices and were selected and amplified for all isolates, as described previously (1). The PCR products were purified, sequenced by the use of standard dye terminator chemistry, and analyzed on an automated DNA sequencer (CEQ 8000; Beckman Coulter, Fullerton, CA). The number of tandem repeats for each locus was determined, and allele numbers were assigned to reflect the number of copies represented in the SSR sequence for each locus. Multilocus SSR (MLSSR) types were then assigned on the basis of the unique combination of alleles for each locus (Table (Table11).

TABLE 1.
Allele frequency among M. avium subsp. paratuberculosis isolates by geographic origin

To establish the stability of these four SSR loci, two experiments were performed. In the first experiment, the K-10 strain of M. avium subsp. paratuberculosis and two diagnostic submission isolates recovered from naturally infected cattle were passaged serially in vitro. Cultures were started from glycerol stocks in 15 ml of Middlebrook 7H9 broth supplemented with 0.05% Tween 20, oleic acid-albumin-dextrose-catalase, and mycobactin (Mycobactin J; 0.5 μg/ml) and grown to a turbidity approximately equal to a 1.0 McFarland standard. One hundred microliters of this culture was then used to inoculate a fresh 15 ml of culture medium, while the remaining bacterial growth was harvested for genomic DNA extraction. This procedure was repeated for a total of 10 serial passages. Each locus was then sequenced for each serial passage of all bacterial strains to determine the stability of the individual loci over time. In the second experiment, 10 colonies from a single isolation of each of these three isolates were used to harvest DNA for SSR analysis.

Both studies with three isolates with distinct SSR genotypes showed that there were no changes in the SSR genotype as a result of in vitro passages or culture, further adding to confidence in the utility of SSR analysis in molecular epidemiologic studies of M. avium subsp. paratuberculosis infection. These findings are also consistent with the 1-year-long serial passage and SSR stability analysis performed with strain K-10 by Amonsin et al. (1).

A total of 61 distinct genotypes were identified among the 211 M. avium subsp. paratuberculosis isolates analyzed. The Simpson's diversity indices were 0.72, 0.8, 0.32, and 0.33 for loci 1, 2, 8, and 9, respectively (Table (Table2).2). Five genotypes were clustered in 35.5% (75/211) of all isolates analyzed (Table (Table3).3). Several distinct SSR genotypes were identified within both the herds and the individual animals studied. These findings are in agreement with those of other recent studies in which several SSR genotypes were demonstrated within some herds in Ohio (14). A similar polyclonal infection within a herd has also been described by the use of IS900-based restriction fragment length polymorphism analyses (15). These studies may reflect multiple introductions of infected animals into herds, the presence of animals infected with different strains of M. avium subsp. paratuberculosis, or possibly, host factors that may be serving as a genetic bottleneck. However, the evolutionary history of mycobacteria suggests that these bottlenecks occur infrequently. Thus, this hypothesis is unlikely because of the short time span of this study. While it is reasonable to speculate that coinfection with multiple genotypes is possible, it is also recognized that this may be a rare event. The presence of a major shared genotype among M. avium subsp. paratuberculosis isolates within herds and between geographically distinct sites suggests that SSRs can be applied in molecular epidemiologic studies to track transmission pathways in Johne's disease. While some cross-sectional studies have used these loci (1, 3, 14) to epidemiologically track infection, further research by the use of well-designed longitudinal studies in multiple states will be necessary to apply these loci in understanding the epidemiology of Johne's disease.

TABLE 2.
Characterization of the SSR loci in M. avium subsp. paratuberculosis and M. avium subsp. avium strains
TABLE 3.
Most predominant strains of M. avium subsp. paratuberculosis present in a geographical survey of the United States

The existence of diversity and the stability of these loci through in vitro passages suggest that the SSR analysis could also be applied to track laboratory contamination.

Sixteen herds from which M. avium subsp. paratuberculosis isolates from both cattle and environmental sources were analyzed. Eight herds showed at least one isolate from each source that carried a shared genotype. The recovery of identical genotypes from both environmental samples and animal feces on the same premises suggests that passive transfer of infected manure to other animals through farm equipment or unsanitary management practices may be contributing to the spread of the disease. Animals may also transiently contaminate the environment by intermittent shedding, and thus, the environment may serve as a proxy for the SSR genotypes within a herd. While the SSR genotypes across geographic localities did not show a nonrandom distribution pattern of specific SSR types, the overall distribution was suggestive of a clonal structure with clustering of isolates into major subcategories (Tables (Tables11 and and3)3) across several geographic locations. Similar observations have recently been reported for M. tuberculosis complex organisms (5, 6).

Analysis of the multiple SSR loci for 56 M. avium subsp. avium isolates from a wide range of animal sources identified restricted variation. For example, all isolates had the same allele (GGCGGGG) for locus 1 and three GGT repeats for locus 8 (Table (Table2).2). These were highly polymorphic in the M. avium subsp. paratuberculosis isolates studied. The major polymorphic site in the M. avium subsp. avium isolates was locus 2, with five alleles. Also, the most predominant subtypes observed in M. avium subsp. avium for loci 2, 8, and 9 were entirely absent from the M. avium subsp. paratuberculosis strains in this study. The idea that these loci are highly conserved in the relatively polymorphic M. avium subsp. avium strains is suggestive of the possibility that the polymorphisms have occurred more recently in M. avium subsp. paratuberculosis and provides meaningful information on the diversity within this subspecies. Although one of the principal findings from this study was that we could apply these SSR loci as a genotyping tool to track M. avium subsp. paratuberculosis, we also demonstrated that more diverse M. avium subsp. avium isolates from animal sources were conserved at these loci. This finding is significant, because it not only adds to the fact that SSRs are conserved in a closely related, more rapidly growing subspecies but also shows the utility of MLSSR analysis as a potential typing tool for the differentiation of M. avium subsp. avium isolates from M. avium subsp. paratuberculosis isolates.

Observations from several epidemiologic studies suggest that most transmission of Johne's disease occurs very early in the life of infected cattle. This has placed the focus of current control programs on neonatal calf management, including management strategies which aim to interrupt the transmission of the pathogen to susceptible cattle (2, 19). The current belief is that M. avium subsp. paratuberculosis is usually introduced into dairy herds through the purchase of infected but clinically healthy cattle as herd replacements, which later serve as a source of infection to other cattle (19). In reality, our understanding of the transmission of M. avium subsp. paratuberculosis is incomplete, based on a limited number of studies, some of which are quite dated and do not reflect the use of the herd management systems used today. As indicated in a recent National Research Council report (17), “significant gaps exist in our understanding of the epidemiology of Johne's disease, which could affect the success of control programs being proposed.” With limited cattle producer participation in control programs to date and limitations in the available diagnostic methods, the risk of continuing unchecked transmission of Johne's disease in the United States is an important concern.

Until relatively recently it had not been possible to trace the pathways of M. avium subsp. paratuberculosis transmission within populations. Although phenotypic differences among clinical isolates have long been recognized, it was not previously possible to determine whether they were stably associated with specific lineages of M. avium subsp. paratuberculosis circulating in the population. Most of the isolates identified have been associated with large clusters that are widely dispersed both geographically and temporally, raising the possibility that they are either more transmissible or more likely to cause disease, once they are transmitted, than other isolates. The advent of molecular typing of M. avium subsp. paratuberculosis, specifically, the described MLSSR method described here, shows promise (in terms of both the discriminatory index and in vitro stability) and may allow researchers to describe the pathways of transmission of Johne's disease in well-designed longitudinal studies and strain-specific variations in clinical phenotypes, such as virulence, growth characteristics, immunogenicity, and transmissibility.

Acknowledgments

This study was supported in part by a USDA-NRICAP (Johne's Disease Integrated Program) grant awarded to S.S.

We acknowledge Tod Stuber for his excellent technical assistance in this project.

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