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J Clin Microbiol. Nov 2004; 42(11): 5022–5028.
PMCID: PMC525156

Use of Multilocus Variable-Number Tandem-Repeat Analysis for Typing Mycobacterium avium subsp. paratuberculosis

Abstract

The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900 restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity of M. avium subsp. paratuberculosis.

Paratuberculosis, also named Johne's disease in ruminants, is characterized by a chronic inflammation of the ileum and is caused by Mycobacterium avium subsp. paratuberculosis.

In humans, the symptoms of Crohn's disease partly resemble those of Johne's disease in ruminants (7). However, the etiology of Crohn's disease is much more complex and appears to be multifactorial (16, 27).

The finding that M. avium subsp. paratuberculosis has remarkable thermostability during pasteurization of milk (12) and the high prevalence of these bacteria both dead and alive in milk for consumers (13, 20) imply a regular exposure of humans to these bacteria. The role microbial agents, and especially M. avium subsp. paratuberculosis, may play in the induction of the immunological disorder characteristic of Crohn's disease has not yet been established (16, 27). In some studies, M. avium subsp. paratuberculosis was isolated from intestinal biopsies or feces of Crohn's disease patients (6, 10, 11, 19, 23). Furthermore, a recent study in the United Kingdom revealed a significant correlation between the presence of M. avium subsp. paratuberculosis in intestinal biopsies and active Crohn's disease (2). Others did not find evidence for a relation between Crohn's disease and the presence of M. avium subsp. paratuberculosis (4, 5). Unfortunately, M. avium subsp. paratuberculosis is very difficult to cultivate from human biopsies. Culture in liquid medium followed by PCR of presumed growth of M. avium subsp. paratuberculosis has increased the number of biopsies in which M. avium subsp. paratuberculosis is suspected (23). These cultures, however, seldom yield sufficient DNA for restriction fragment length polymorphism (RFLP) typing experiments, and many isolates fail to grow in sufficient quantities for RFLP typing.

Recently, pulsed-field gel electrophoresis of pigmented and nonpigmented M. avium subsp. paratuberculosis isolates resulted in a subdivision of a part of the IS900 RFLP types, providing more refined data for epidemiological interpretation (24). Unfortunately, the latter technique also requires large quantities of DNA.

Therefore, the question of whether these hardly cultivatable M. avium subsp. paratuberculosis strains are of the same genotype as animal M. avium subsp. paratuberculosis isolates remains unanswered. In view of this, a PCR-based typing technique for low quantities of bacterial DNA, as present in human intestinal biopsies, would be a valuable extension of typing methods for the molecular epidemiology of Crohn's disease.

Several PCR-based techniques for identification and typing of M. avium subsp. paratuberculosis strains have been described in the last few years. Whittington et al. (30, 31) and Marsh et al. (18) have described IS1311 PCR followed by restriction enzyme analysis to differentiate between cattle and sheep type strains and other M. avium complex isolates. However, these techniques have so far failed to distinguish between M. avium subsp. paratuberculosis isolates.

Another promising PCR-based method to study the epidemiology of tuberculosis is mycobacterial interspersed repetitive unit (MIRU) typing. This method is based on variation in the number of MIRUs of 40 to 100 bp in length. MIRUs are arranged mostly in tandem repeats and are dispersed in intergenic regions of M. tuberculosis complex bacteria (9, 25, 26). Not all genomic loci with MIRUs have been fully explored, but in several studies, variable-number tandem-repeat (VNTR) typing of M. tuberculosis complex isolates has revealed a higher resolution than that achieved with IS6110 RFLP typing (17).

Bull et al. (3) found four MIRU loci which can be used to discriminate between M. avium subspecies paratuberculosis, M. avium subspecies avium, and M. intracellulare strains. Furthermore, cattle and sheep strains were divided into four different groups on the basis of MIRU typing.

The aim of our study was to investigate the usefulness of multilocus VNTR typing analysis (MLVA) as a tool for typing M. avium subsp. paratuberculosis strains and to investigate whether a higher degree of discrimination can be reached among M. avium subsp. paratuberculosis isolates.

MATERIALS AND METHODS

Strains.

M. avium subsp. paratuberculosis strains were cultured on Herrold's egg yolk medium according to the method of Whipple et al. (29). The host species included cattle, sheep, red deer, fallow deer, roe deer, and humans.

Strains from the Czech Republic, Sweden, and the United States were obtained from the Veterinary Research Institute, Brno, Czech Republic. Human isolates of Dutch origin were obtained from the Institute for Animal Science and Health, Lelystad, The Netherlands. The isolates were identified as M. avium subsp. paratuberculosis by cultural and biochemical properties, and this identification was confirmed by DNA-DNA hybridization (14). The cattle isolates from The Netherlands were obtained from the Animal Health Service, Deventer, The Netherlands. All isolates from Argentina were obtained from the Instituto Nacional de Tecnologia Agropecuaria, Buenos Aires, Argentina, and the isolate from Venezuela was obtained from the Biolac United Nations University Institute, Baruta, Venezuala. DNA samples of the M. avium subsp. paratuberculosis strains SN 1 to SN 8, K05, K16, and K18 were kindly donated by the St. George Hospital Medical School Department of Surgery, London, United Kingdom.

IS900 RFLP typing.

IS900 RFLP typing is currently the standard method for typing M. avium subsp. paratuberculosis. This method has the highest discriminatory power and serves as the “gold standard” for MLVA analysis. IS900 RFLP type, geographic region, and host were used for the selection of strains to build a heterogeneous strain panel. IS900 RFLP typing was performed as described previously by van Soolingen et al. (28), with some modifications. Lysozyme incubation was performed overnight and proteinase K-sodium dodecyl sulfate treatment was done for 20 min at 65°C.

Digestion was performed with 3 μg of DNA and 7 U of BstEII (Roche) at 37°C for at least 4 h. Separation of the DNA restriction fragments was done on a 0.8% MP agarose (Roche) gel measuring 20 by 24 cm at 1.7 V/cm (40 V) overnight until the 1,150-bp fragment of Mw Marker IV (Roche) had reached a distance of 17 cm from the slots. Subsequently, the gel was blotted onto a Hybond N+ nylon (Amersham Biosciences) membrane followed by overnight incubation of the membrane at 42°C in the presence of an IS900 probe.

Preparation of the IS900 probe.

The IS900 DNA probe was prepared by PCR amplification of a 707-bp fragment of the IS900 insertion sequence specific for M. avium subsp. paratuberculosis.

Primers used were IS900A (5′ACG CCG CGG GTA GTT A) and IS900B (5′GGG GCG TTT GAG GTT TC).

PCRs were performed with 10 ng of chromosomal DNA of strain ATCC 19698 by using Ready to Go PCR beads (Amersham Biosciences). PCR conditions were as follows: 1 cycle of 5 min at 94°C; 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C; and 1 cycle of 4 min at 72°C. PCR products were purified on Qiaquick spin columns (QIAGEN) according to the manufacturer's instructions.

The probe was labeled with peroxidase (ECL DNA labeling kit; Amersham Biosciences) according to the manufacturer's instructions.

Analysis of the RFLP patterns.

Analysis of the RFLP patterns was performed by using Bionumerics version 2.0 software (Applied Maths, Sint-Martens-Latem, Belgium). Conserved bands in the IS900 RFLP pattern of 8.8, 5.2, 3.0, 2.4, 2.1, and 1.6 kb were used as the internal standard for the normalization of RFLP patterns.

Selection of VNTR loci.

In view of the close genetic relatedness of M. avium subsp. paratuberculosis and other M. avium complex strains, the recently published genomic sequence of M. avium strain 104 (GenBank accession number NC_002943 [http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi]) was used to identify VNTR sequences to be tested in MLVA typing. Tandem repeats were identified by using the Tandem Repeats Finder software (1) under the default settings of the program. Tandem repeats present in more than two copies and with a sequence match of 85% or higher were compared to the nucleotide sequence of M. avium subsp. paratuberculosis strain K10 (GenBank accession number NC_002944 [http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi]).

From the total of IS900 RFLP typed strains, a selection of 50 strains with variable features was made; different IS900 RFLP patterns (Fig. (Fig.1)1) isolated from various hosts in different countries (Table (Table1).1). Primers were designed to target flanking regions of the VNTRs and are listed in Table Table2.2. The details of the amplification of the various VNTRs are listed in Table Table3.3. PCR was performed with a Biometra T gradient thermocycler (Biometra, Göttingen, Germany) by using either Ready to Go PCR beads or Hotstar Taq (QIAGEN). VNTR 1067, 1605, 1658, 3527, 7661, and 9425 (Table (Table3)3) were amplified with Ready to Go PCR beads according to the instructions of the manufacturer. Due to poor results, VNTR 3249 was amplified with Hotstar Taq in standard PCR buffer and Q buffer (QIAGEN) for GC-rich templates according to the instructions of the manufacturer. All PCRs were performed by using 10 ng of purified DNA. To detect differences in numbers of repeats, the PCR products were analyzed on a 1% MP agarose gel in 1× Tris-borate-EDTA containing 0.5 μg of ethidium bromide (BDH)/ml.

FIG. 1.
IS900 RFLP types of the strains used for MLVA. The lanes show the patterns after normalization and analysis with Bionumerics version 2.0 (see Materials and Methods). R types are designated according to the nomenclature of the National Institute of Public ...
TABLE 1.
Strains used for MLVA and results
TABLE 2.
VNTR primer sequences
TABLE 3.
PCR protocols for MLVA

Sequence analysis.

DNA sequencing reactions were performed with purified PCR products by using an ABI Prism Big Dye Terminator kit as described above for multilocus sequence typing analysis. Products were analyzed with an ABI 3700 capillary sequencer (Applied Biosystems). Raw data were analyzed with DNASTAR software.

RESULTS

RFLP typing.

Over 250 isolates were subjected to IS900 RFLP typing. Thirty different RFLP types were found. The majority (37%) was of type R01, followed by R09 (26%), R10 (8%), and R17 (7%). The isolates were gifts of available strains at a certain moment from the donating institutes; therefore, the distribution of the different RFLP types may not reflect the natural distribution of these types.

In silico comparison of VNTR loci and flanking regions.

The whole genome sequence of M. avium strain 104 (GenBank accession number NC_2943 [available at http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi]) was analyzed for the presence of tandem repeats. In total, 376 different VNTR sequences with a length between 6 and 263 bp and with a mutual homology of 67 to 100% were found. The copy number of the repeats ranged from 2 to 56.

Sequences of VNTRs which met the following criteria were compared to the M. avium subsp. paratuberculosis K10 sequence: more than two copies, more than 18 nucleotides, and a mutual homology between 77 and 100%. A total of 47 VNTRs were selected. Other VNTRs were present in both strains but differed in the flanking regions.

Some of the VNTRs present in M. avium strain 104 were not present in the M. avium subsp. paratuberculosis K10 sequence. Finally, 27 VNTRs having two or more repeats of 20 to 70 bp in length and homology of 80 to 100% were used for a homology search of each VNTR and its flanking regions.

A comparison of 1-kb genomic regions containing each VNTR and its flanking region of M. avium strain 104 and M. avium subsp. paratuberculosis strain K10 revealed mutations in 20 loci. Thirteen loci had mutations in both the repeat and the flanking regions. Seven isolates had mutations only in the flanking regions.

The number of point mutations in the VNTR flanking regions varied from 2 to 13. The differences observed comprised inserts, gaps, and point mutations. The number of mutations found in repeat regions ranged from 0 to 4 nucleotides. Furthermore, deletions of whole repeats were observed.

Thirteen out of 20 VNTR loci in M. avium subsp. paratuberculosis strain K10 had fewer repeats than their corresponding locus in M. avium strain 104. Six loci had an equal number and one had an increased number of repeats.

Variation in repeat number among M. avium subsp. paratuberculosis isolates.

As a result of the in silico analysis, the 20 VNTR loci showing mutations compared to the M. avium strain 104 were tested. Polymorphism in these VNTR loci was determined in 49 M. avium subsp. paratuberculosis strains (Table (Table1),1), 22 of RFLP type R01, 7 of RFLP type R10, 19 of other RFLP types, and 1 of an unknown type.

PCR on four loci yielded either no product or multiple nonspecific products. These loci were excluded from further study. Eleven loci gave no polymorphism in the number of repeats. Four loci showed two different VNTR types, and one locus revealed three VNTR types. Based on analysis of the five VNTR loci in 49 M. avium subsp. paratuberculosis isolates, six different MLVA types were found (Table (Table11).

DNA sequence analysis of variable VNTR loci in M. avium subsp. paratuberculosis strains.

All VNTR loci with variation in the number of repeats were subjected to DNA sequence analysis. In addition, three VNTR loci (2495, 7661, and 9425) without variation in the number of repeats were analyzed to detect possible nucleotide polymorphisms.

In the loci 1605 and 1658, point mutations between M. avium subsp. paratuberculosis strains were observed in the genomic regions flanking the VNTRs. In three strains, a C-T mutation at position 119 was observed in VNTR 1605. The location of this mutation is outside the repeat sequence. In two strains, a C-T mutation was observed at position 74 of VNTR 1658, and this mutation was positioned inside the second repeat. The DNA sequence of the three loci 2495, 7661, and 9425 showed no variation in the number of tandem repeats analyzed nor did they reveal any mutations at the single-nucleotide level.

In conclusion, MVLA typing of 49 M. avium subsp. paratuberculosis isolates yielded six different genotypes, and DNA sequence analysis of the VNTR loci resulted in two additional alleles.

Comparison of IS900 RFLP typing and MLVA-associated variation.

The 22 strains of RFLP type R01 could be subdivided into six different combined VNTR types, providing a subdivision of the largest group of IS900 RFLP types. Seven strains of RFLP type R10 have an MLVA type identical to 17 other different RFLP types. No association was found between the MLVA types of the M. avium subsp. paratuberculosis strains and the animal source or the country of isolation. All human isolates revealed the same MLVA type, 22222, as found for the majority of the cattle strains. This finding indicates that these isolates are genetically closely related to the most common and widespread cattle isolates.

Diversity index.

Diversity indices of RFLP and MLVA typing were calculated according to the method of Hunter and Gaston (15).The index ranges between 0 and 1. The closer the diversity index is to 1, the higher the discriminatory power of the method. The RFLP index is based on 49 strains of known IS900 RFLP type (Table (Table1).1). The MLVA index is based on 50 strains of known MLVA type. The indices are 0.448 and 0.316, respectively.

DISCUSSION

The detection of four different VNTR types and two point mutations in the VNTR flanking regions among 24 M. avium subsp. paratuberculosis isolates of the R01 RFLP type analyzed is promising for VNTR typing as a tool to study the epidemiology of M. avium subsp. paratuberculosis. Further analysis of other VNTRs in the genome of M. avium subsp. paratuberculosis may yield an epidemiological tool with an even higher resolution, enabling a clear interpretation of comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from animal and human sources.

The C-T mutation in VNTR 1605 causes the loss of an HaeII restriction site, which can be used for the detection of this mutation.

The search for VNTRs in M. avium subsp. paratuberculosis was based on the genome sequence of M. avium strain 104. Since the complete nucleotide sequence of M. avium subsp. paratuberculosis strain K10 has recently become available for BLAST searches, it is possible to compare VNTRs present in M. avium strain 104 with the genome of M. avium subsp. paratuberculosis. It is conceivable that other VNTRs will be disclosed which also show polymorphism among M. avium subsp. paratuberculosis strains and can be used in MLVA typing.

Evaluation of RFLP versus MLVA shows that on the one hand, the group of R01 could be successfully divided in subgroups, but on the other hand, 17 different RFLP types have the same MLVA type. More strains of each RFLP type need to be tested by MLVA to establish the absolute value of MLVA typing compared to the different RFLP types. Evaluation of RFLP versus MLVA typing by calculating the diversity index gives an idea about the discriminative value of these methods. The results suggest that RFLP is more discriminative than MLVA (0.448 and 0.316, respectively), but one has to keep in mind that the strain panel was based on RFLP types and as a result of that favors RFLP over MLVA in the number of different types assigned to it. Once the calculation was based upon the group R01 RFLP type isolates, among which six different MLVA types were found, the MLVA method is much more discriminative. The index is more reliable for large representative collections of strains (15). In this study, there is neither a representative collection nor a large number of strains, so limiting conditions are not applicable. Therefore, the reliability of the indices is low.

So far, only a limited number of M. avium subsp. paratuberculosis isolates from humans is available for typing. In this study, 10 M. avium subsp. paratuberculosis isolates from humans have been subjected to IS900 RFLP typing. This typing revealed only cattle types: three isolates belong to type R01 and seven belong to type R10. However, in The Netherlands, only 10% of the cattle isolates are of the R10 (C5) type (P. Overduin and D. van Soolingen, unpublished data), and in an international study, only 0.5% of the cattle isolates were assigned to the R10 type (22). In view of this finding, it is remarkable that six out of seven M. avium subsp. paratuberculosis isolates originating from the United States have RFLP type R10. One could hypothesize that the high percentage of R10 (C5) strains found in humans indicates that these M. avium subsp. paratuberculosis strains are more capable of infecting humans or that there is a (unknown) reservoir of M. avium subsp. paratuberculosis different from cattle. More isolates are needed for further study of this question.

All isolates from humans were of the most common MLVA type (22222). In addition to IS900 RFLP typing, this provides evidence that the human M. avium subsp. paratuberculosis strains are identical to a subpopulation of the strains found in cattle. This finding suggests that humans can be infected with M. avium subsp. paratuberculosis strains from cattle. Unfortunately, cultivation of M. avium subsp. paratuberculosis from human tissue is still very difficult. Although modern liquid cultures produce higher yields than solid media (23), the number of human isolates available for typing is still very limited (2). Therefore, until more suitable cultivation methods have become available and the nature of noncultivatable M. avium subsp. paratuberculosis bacteria has been disclosed, large epidemiological studies on human isolates are future prospects.

Spoligotyping is frequently used as an alternative for, or in addition to, RFLP typing of M. tuberculosis. We have explored the use of spoligotyping for discrimination of M. avium subsp. paratuberculosis strains (P. Overduin, A. Herrewegh, and D. van Soolingen, unpublished data). As a model, we have used the genome sequence of M. avium strain 104 which has a direct repeat region containing a 27-bp repeat and 12 spacers. Detailed investigation of other M. avium strains with a DNA probe covering a large part of the direct repeat region of M. avium strain 104 revealed that only 2 out of 20 M. avium strains had an M. avium strain 104 homologous direct repeat region. Furthermore, study of the complete genome sequence of M. avium subsp. paratuberculosis strain K10 revealed that neither this direct repeat region nor the spacers found in M. avium strain 104 are present in M. avium subsp. paratuberculosis strain K10. These results indicate that the direct repeat region found in M. avium strain 104 is not common in other M. avium complex strains. It remains unclear whether there is a direct repeat region present in M. avium subsp. paratuberculosis at all.

In this study, we tested a number of VNTRs for the presence of polymorphisms in the conserved mycobacterial subspecies M. avium subsp. paratuberculosis. Five out of eight studied VNTRs showed polymorphism in the number of repeats. Sequencing revealed point mutations in two VNTRs. Both polymorphisms divided the major R01 RFLP group into several subgroups, demonstrating that VNTR typing could be a useful (additional) tool for typing M. avium subsp. paratuberculosis.

Acknowledgments

We thank Ivo Pavlik, Douwe Bakker, Cees Kalis, Mariso Romano, Jacobus de Waard, and John Hermon-Taylor for kindly donating M. avium subsp. paratuberculosis isolates and DNA.

M. avium strain 104 was sequenced by The Institute for Genomic Research, Rockville, Md. M. avium subsp. paratuberculosis strain K10 was sequenced at the Department of Microbiology and Biomedical Genomics Center, University of Minnesota, St. Paul, Minn., by L. Li, J. Bannantine, Q. Zhang, A. Amonsin, D. Alt, and V. Kapur.

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