Single Nucleotide Polymorphisms in the IS900 Sequence of Mycobacterium avium subsp. paratuberculosis Are Strain Type Specific

ABSTRACT Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.
The insertion sequence IS900 (13) is one of the 20 members of the IS110 family and is considered to be unique to Mycobacterium avium subsp. paratuberculosis, although IS900-like sequences have been found rarely in other environmental mycobacteria (9,12). IS900 is present in multiple copies (14)(15)(16)(17)(18) within the M. avium subsp. paratuberculosis genome (13) and therefore is an ideal target for identification (16,31). The heterogeneity of M. avium subsp. paratuberculosis isolates based on the number of IS900 copies and their positions within the genome has been exploited for epidemiological purposes. The IS900 sequence has been used in different molecular techniques such as multiplex PCR and restriction fragment length polymorphism analysis with hybridization to IS900, leading to the identification of individual strains and the classification of M. avium subsp. paratuberculosis into strain types (3,17,19,30).
M. avium subsp. paratuberculosis strains have been divided into three clusters named type I (also called sheep [S] strains), type II (also called cattle [C] strains), and type III (also called intermediate strains), although some of the studies include types I and III in the S type (8,35). This classification into three clusters is based on results from molecular characterization techniques such as restriction fragment length polymorphism analysis with hybridization to IS900 (19), pulsed-field gel electrophoresis (PFGE) (11,29), PCR-restriction enzyme analysis (PCR-REA) of gyrB (5), PCR-REA of inhA (6), PCR and denaturing gradient gel electrophoresis analyses of MAP1506 (14), PCR sequencing of recF (31), and comparative genomic hybridization analyses (4). The genotypic and phenotypic dissimilar-ities among types of M. avium subsp. paratuberculosis may be reflected in the differences detected in the progression of paratuberculosis or Johne's disease among infected herds (33,34).
Of special interest are the results reported by Semret et al. (24), who observed major variance in the IS900 sequences in M. avium subsp. paratuberculosis S strains (classification into type I or III was not indicated in the study) compared to the IS900 sequences in C strains.
The aim of this research was to follow up these findings and to analyze the IS900 sequences from a panel of type I, II, and III M. avium subsp. paratuberculosis isolates to establish the degree of nucleotide identity among them and the relationship between M. avium subsp. paratuberculosis types and IS900 sequence profiles.
A fragment of 662 bp of the IS900 sequence from each of the isolates was amplified by PCR directed to the 5Ј end of the insertion sequence, considered to be specific to M. avium subsp. paratuberculosis (9,18) and found previously to be poly-morphic (24). Primers IS900-F (5Ј CCTTTCTTGAAGGGTG TTCG 3Ј [24]) and IS900-R (5Ј CCACCAGATCGGAA CGTC 3Ј) were used in the amplification reaction, and then amplicons were purified with a QIAquick PCR purification kit   (24). The analysis of the chromatograms showed IS900 SNPs that were homogeneous, conserved, and dependent on the M. avium subsp. paratuberculosis type (Table 3). Every type I ovine isolate showed an SNP at position 216, with a G instead of an A, and no other sequence modifications. This SNP was observed previously in a sample extracted directly from river water (20,21), in an isolate from a sheep in Australia (35), and in some ovine isolates analyzed by Semret et al. (24). The eight isolates of type II subjected to analysis displayed complete homology to M. avium subsp. paratuberculosis K-10. However, for the type III isolates tested, double peaks of the same size at bp 169 (T/C) and bp 216 (G/A) or a single T peak and a G were noticed. For four of these isolates with ambiguities, another DNA extraction from a single colony was per-formed with an inoculating needle, but the results were corroborated, ruling out the possibility of contamination by two different strain types. The results of this analysis match previous observations of these SNPs in some of the S isolates tested by Semret et al. (24). This pattern is probably indicative of the presence of a point mutation in some of the copies of IS900 (Table 3) and also confirms the presence of type III isolates outside Spain (10). To our knowledge, this is the first report to establish a correlation between IS900 polymorphisms and strain types I, II, and III. Interestingly, in two previous studies, an irresolvable A/G polymorphism at position 216 (reported initially to be at bp 214) was observed in two samples obtained from a river flow (20,21), due maybe to the presence of both types I and II in the samples collected. On the other hand, none of our isolates revealed any other SNPs described previously in the literature (2,23,27,28,36). The type III strains exhibited polymorphisms identical to those of type I strains, as determined by PCR analysis of DMC, IS1311 REA, and LSP A 20 and hsp65 sequencing (Table 3), confirming previous observations (1) and showing the genetic C/T G a PCR analysis of DMC was performed as described by Collins et al. (8). Isolates were identified as S or C strains. b PCR-REA was performed as described by Marsh et al. (15). Isolates were identified as S or C strains. c The PCR method used was developed by Semret et al. (25). d hsp65 sequevar. e SNPs found in the IS900 sequences. f IS1311 data were also confirmed in previous work (29). g The DNA template was insufficient and did not yield a visible product in the PCR. similarity of these two strain types. However, this report provides further evidence of additional polymorphic loci that can be used to distinguish between these two strain types. Also, it supports the theory that less genomic divergence exists among type II (bovine) isolates than among type I and III (S) isolates (4,26,31). The stability and conservation of the IS900 sequence drift is reflected at positions 169 and 216 in some copies of IS900 in type I and III strains from herds in different locations. These results are consistent with suggestions in previous studies that M. avium subsp. paratuberculosis strains tend to be clonal (34). The IS900 sequence analysis could be used as a complementary diagnostic tool for epidemiological purposes to study the geographical distribution patterns of the three clusters within the M. avium subsp. paratuberculosis group. On the other hand, from this evidence, it is still not possible to obtain a correlation between pathogen adaptation to environmental factors or virulence pathways and the divergences in IS900 sequences.
Notwithstanding these results, M. avium subsp. paratuberculosis nomenclature and the subdivision of strains into groups are still controversial. Current classifications have been based on the data gathered by several research groups. It would be necessary to perform whole-genome sequencing of M. avium subsp. paratuberculosis types I and III to further clarify the taxonomy of the lineages.
Nucleotide sequence accession numbers. The GenBank accession numbers for IS900 partial sequences from M. avium subsp. paratuberculosis type I and type III are FJ775181 and FJ775182, respectively.