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J Clin Microbiol. May 2005; 43(5): 2500–2502.
PMCID: PMC1153774

Distribution of IS1311 and IS1245 in Mycobacterium avium Subspecies Revisited

Abstract

We demonstrated that IS1245 is not present in Mycobacterium avium subsp. paratuberculosis by restriction fragment length polymorphism and that the designated three-banded bird pattern of IS1245 in M. avium subsp. avium consists of one copy of IS1245 and two copies of IS1311. Cross hybridization between the two elements can be avoided by using more specific probes.

The Mycobacterium avium complex (MAC) comprises a heterogeneous group of slow-growing, acid-fast bacilli that are divided into the two species Mycobacterium avium and Mycobacterium intracellulare (20). MAC organisms are ubiquitous in nature and can cause various diseases in animals and humans (8). M. avium subsp. avium can cause disease in different animal species such as poultry and swine and has been an increasingly important pathogen for humans because of the AIDS epidemic (6, 8). The bacteria can also cause pulmonary infections in patients without AIDS and cervical lymphadenitis in children (6). M. avium subsp. paratuberculosis causes paratuberculosis in ruminants and has been suggested as the etiologic agent of Crohn's disease (3, 10).

Different insertion sequences present in the MAC have been used for identification and strain differentiation. IS1245 has been used for restriction fragment length polymorphism (RFLP) of M. avium subsp. avium by the proposed standardized method of van Soolingen et al. (18). The 1,414-bp-long element was initially found to be present in M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum by PCR amplification of a 427-bp target sequence (7) and has also been found in some other mycobacteria (9). A closely related IS element is IS1311, which shows 85% sequence identity with IS1245 at the DNA level (9, 16, 17). The element is present in M. avium subsp. avium and M. avium subsp. paratuberculosis (21) and has been detected in strains of M. intracellulare, Mycobacterium malmoense, and Mycobacterium scrofulaceum (9). There is, however, considerably discrepancy in the literature about the presence and the copy number of IS1245 and IS1311 in the M. avium subspecies (4, 7, 16). The close relationship between the two elements makes cross hybridization a possible explanation for this dissention.

The objective of this study was to clarify some of the discrepancy in the literature by examining strains of M. avium subsp. avium and M. avium subsp. paratuberculosis for the presence of IS1245 and IS1311. A method for standardization of IS1245 RFLP has been proposed (18). However, the suggested 427-bp IS1245 probe (long IS1245 probe) has an identity of 82% (National Center for Biotechnology Information BLAST; http://www.ncbi.nlm.nih.gov/BLAST/BLAST.cgi) with IS1311 on the DNA level. We therefore designed probes from the 5′ end of each insertion element, where there is a lower homology of 75% between the two elements in order to reduce the possibility for cross hybridization. The primers with the location of the probes are described in Table Table11.

TABLE 1.
Primers and probes used in this study

The short IS1245 probe (175 bp) and the IS1311 probe (198 bp) were synthesized by PCR using AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) and the following conditions: 94°C for 3 min followed by 30 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s. Synthesis of the standardized long 427-bp IS1245 probe and the RFLP method were performed as described earlier (7, 18). Hybridizations with the short probes were performed at a low-stringency temperature of 45°C and at a high-stringency temperature of 60°C without any effect on the results. This shows that the use of the highly specific probes gave no cross hybridization even at low temperatures.

M. avium subsp. paratuberculosis.

Seven clinical isolates of M. avium subsp. paratuberculosis and the ATCC 19698 strain (Table (Table2)2) were cultured and DNA was prepared as described earlier (14). The strains showed an identical pattern of seven bands when RFLP was performed with the long IS1245 probe (Fig. (Fig.1a).1a). This is in accordance with the results described by some other authors (7, 16). However, the bands were faint, and we hypothesized that these bands were visualized due to hybridization with IS1311. RFLP was therefore performed with the short IS1245 probe, and no hybridization signals were seen in any of the strains (Fig. (Fig.1b).1b). RFLP on the M. avium subsp. paratuberculosis strains with the IS1311 probe revealed seven distinct bands with an identical pattern to what was observed with the long IS1245 probe (Fig. (Fig.1c).1c). Our results demonstrated that IS1245 was not present in the tested strains of M. avium subsp. paratuberculosis and that the bands that appeared when hybridizing with the long IS1245 probe were a result of cross-hybridization with IS1311. BLAST screening also confirmed that IS1311 was present in at least seven copies in the K10 strain of M. avium subsp. paratuberculosis, while IS1245 could not be found (National Center for Biotechnology Information BLAST).

FIG. 1.
RFLP patterns of four clinical isolates and one reference strain of M. avium subsp. paratuberculosis. Hybridization with the IS1245 long probe (a), IS1245 short probe (b), and IS1311 probe (c). Lanes 1 to 3 are bovine strains, lane 4 is a caprine strain, ...
TABLE 2.
Mycobacterial strains examined in this study

M. avium subsp. avium.

Four clinical isolates of M. avium subsp. avium and three reference strains (Table (Table2)2) were cultured, and DNA was prepared as described earlier (14). Strains of M. avium subsp. avium isolated from wild birds and poultry have been described in the literature as highly conserved. It has been proposed to reserve the term M. avium subsp. avium for these strains and to designate the heterogeneous human and porcine isolates M. avium subsp. hominissuis (11). The bird strains belong to the serovar 1, 2, or 3 and contain IS901 (2, 16). They also have an identical three-banded pattern with the standardized IS1245 RFLP, often referred to as the “bird-type” profile (1, 11, 13, 16). The bird strains in the present study showed the same three-banded pattern with the standardized long IS1245 probe (Fig. (Fig.2a).2a). The upper and the lower bands were, however, faint and inconsistent, while the middle band was distinct. Similar results can be seen by observing the figures in several other studies (1, 7, 11, 16). Performing RFLP with the short IS1245 probe gave only one band in the same position as the middle of the three bands (Fig. (Fig.2b),2b), while IS1311 RFLP revealed the other two bands (Fig. (Fig.2c).2c). Collins et al. obtained similar results, and they also argued that the contradicting results in other studies were due to cross hybridization with IS1311 (4).

FIG. 2.
RFLP patterns of four clinical isolates and three reference strains of M. avium subsp. avium. Hybridization with the IS1245 long probe (a), IS1245 short probe (b), and IS1311 probe (c). Lane 1, ATCC 25291; lane 2, R13; lane 3, 1794; lane 4, IWGMT49; lane ...

The strains of M. avium subsp. avium isolated from pigs and the human strain showed multibanded complex patterns when the standardized IS1245 RFLP was performed (Fig. (Fig.2a).2a). A problem with this method has been many weak and inconsistent bands, making interpretation of the results difficult (15-17). The use of the shorter IS1245 probe helped to resolve some of these problems. The faint smaller fragments from hybridization with the long IS1245 probe were not visible when hybridizing with the short IS1245 probe (Fig. (Fig.2b)2b) and appeared distinct with the IS1311 probe (Fig. (Fig.2c).2c). RFLP with the IS1311 probe gave very distinct and easily interpretable results compared to the IS1245 probes. IS1311 has been used for RFLP by several authors, and the discriminatory power has been judged to be almost equal to that of IS1245 (4, 5, 12, 17). Standardization of the method and further studies are necessary for testing the potential of using the IS1311 element in RFLP.

We conclude that IS1245 is not present in M. avium subsp. paratuberculosis and that the designated three-banded bird pattern of IS1245 consists of one copy of IS1245 and two copies of IS1311. Cross hybridization between the two insertion elements can be avoided by using shorter and more specific probes. Combining the two probes in RFLP may also give a more detailed typing result.

Acknowledgments

We would like to thank Dick van Soolingen (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) for providing the plasmid pMA12 and the reference strains IWGMT49 and R13 and Ulf Dahle (The Norwegian Institute of Public Health) for providing the human strain H6 and for valuable support. We would also like to thank Nina Fundingsrud and Vivi Myrann for excellent technical support.

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