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Journal of Clinical Microbiology, March 2008, p. 972-981, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01801-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High Genetic Diversity among Mycobacterium avium subsp. paratuberculosis Strains from German Cattle Herds Shown by Combination of IS900 Restriction Fragment Length Polymorphism Analysis and Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem-Repeat Typing{triangledown}

Petra Möbius,1* Gabriele Luyven,2 Helmut Hotzel,3 and Heike Köhler1

Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany,1 Consortium Chemical and Veterinary Laboratories Agency (Rhine-Ruhr-Wupper), Deutscher Ring 100, 47798 Krefeld, Germany,2 Institute for Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany3

Received 10 September 2007/ Returned for modification 25 November 2007/ Accepted 23 December 2007


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ABSTRACT
 
Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease and is endemic to the national cattle herds of many countries. Because of the very low level of genetic heterogeneity of this organism, it is difficult to select a workable procedure for strain differentiation at a resolution sufficient to investigate epidemiological links between herds or different ruminant species and the suggested zoonotic potential of M. avium subsp. paratuberculosis for Crohn's disease. Analysis of restriction fragment length polymorphisms (RFLPs) based on the insertion element IS900 (IS900 RFLP) with four restriction enzymes and 10 markers of specific mycobacterial interspersed repetitive units (MIRUs) and variable-number tandem repeats (VNTRs) was applied to 71 bovine M. avium subsp. paratuberculosis isolates originating from 14 herds from different regions in Germany. Among these isolates, all of which belonged to the M. avium subsp. paratuberculosis type II group, 17 genotypes were detected by IS900 RFLP and consisted of a combination of seven BstEII, eight PstI, nine PvuII, and four BamHI restriction patterns. Novel RFLP types were found. The diversity of the M. avium subsp. paratuberculosis isolates inside the herds was different depending on the frequency of animal purchase. The results of typing by IS900 RFLP and MIRU-VNTR analyses were not associated. Fifteen MIRU-VNTR patterns were identified with a discriminatory index of 0.905. The most common BstEII-based IS900 RFLP type, type C1 (72%), was subdivided into 14 types by MIRU-VNTR analysis. A combination of fingerprinting and PCR-based techniques resulted in 24 M. avium subsp. paratuberculosis genotypes and achieved a discriminatory index of 0.997. By using only BstEII and PstI digestion together with typing by MIRU-VNTR analysis, a discriminatory index of 0.993 was achieved. This is high enough to support epidemiological studies on a national as well as a global scale.


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INTRODUCTION
 
Mycobacterium avium subsp. paratuberculosis is the etiologic agent of paratuberculosis (Johne's disease), a chronic infectious granulomatous enteritis principally affecting domestic and wild ruminants (20, 22). In Europe, cattle, sheep, goats, and farmed deer are predominantly affected (31, 34). Bovine paratuberculosis is endemic. The disease poses a significant economic problem primarily when it occurs in dairy herds (22) but also when it occurs in beef and sheep herds. The rapid spread of Johne's disease is caused by the trade in subclinically infected animals and infection in early calfhood (31). M. avium subsp. paratuberculosis is excreted in great quantities in the feces of the infected animals; it has a high tenacity, and it is therefore widely distributed in the environment. Furthermore, a role for M. avium subsp. paratuberculosis in the etiology of Crohn's disease in humans has long been discussed (7, 8, 16), but the zoonotic potential of the organism remains controversial.

So far, restriction fragment length polymorphism (RFLP) analysis based on the insertion element IS900 (IS900 RFLP) has been applied the most extensively to investigations of the genetic diversity among M. avium subsp. paratuberculosis strains for epidemiological studies of Johne's disease. Furthermore, macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) has been used. Based on the pattern groups obtained by these two techniques and on growth characteristics and pigmentation, M. avium subsp. paratuberculosis isolates are divided into three groups: the S (sheep) type, the C (cattle) type (3, 9), and the I (intermediate) type (13, 32). These three groups are also designated types I, II, and III, respectively (12, 37). These types show strong host preferences but no host specificity (3, 37). By using M. avium subsp. paratuberculosis isolates from different host species and different geographic regions of the world, a number of subtypes could be characterized, especially in the type II group of strains (9, 10, 25, 27, 32, 33, 46). Type II strains were mainly isolated from cattle; but they were also isolated from sheep, goats, different wildlife species, and humans.

Several other typing methods that target different structures in the genome have been developed and are faster and easier to perform and require smaller quantities of bacteria than IS900 RFLP or PFGE. Among others, these include multiplex PCR of IS900 loci (MPIL) (5) and multilocus short-sequence-repeat (MLSSR) sequencing (1). Some methods (e.g., MPIL) were shown to have an insufficient power for the discrimination of M. avium subsp. paratuberculosis isolates, and others (e.g., MLSSR sequencing analysis) were successfully applied by using a set of isolates from different host species and different geographic locations of the world. Moreover, recent studies have identified loci containing variable-number tandem repeats (VNTRs) of specific mycobacterial interspersed repetitive units (MIRUs) in M. avium subsp. paratuberculosis isolates (4, 6, 27, 35, 41). Those investigations were based on the screening of parts of or the whole M. avium subsp. paratuberculosis K10 genome and tested the polymorphisms of a diverse panel of strains. A typing system that included at least eight loci was proposed by Biet et al. (4) and Thibault et al. (41).

However, M. avium subsp. paratuberculosis isolates show a very low level of genetic heterogeneity in comparison to that of Mycobacterium tuberculosis and other bacteria. The use of a combination of several typing methods might lead to an increase in the overall discriminatory power of DNA typing, an approach which has already been established for mycobacteria of the M. tuberculosis complex (11, 23). For M. avium subsp. paratuberculosis, Stevenson et al. (37) and Thibault et al. (41) applied a combination of typing methods by using M. avium subsp. paratuberculosis isolates originating from different hosts and diverse geographic regions of the world.

The aim of this study was to verify the possibilities provided by the use of a combination of independent typing techniques for the differentiation of bovine M. avium subsp. paratuberculosis field isolates of different origins in Germany. The use of such a combination of independent typing techniques would permit epidemiological investigations on a national level within temporal and geographic limits. The genetic diversity of M. avium subsp. paratuberculosis isolates was analyzed by typing the isolates by IS900 RFLP and MIRU-VNTR analysis of 10 genomic loci. For IS900 RFLP, four restriction enzymes were used in order to facilitate a comparison of the resulting restriction patterns with those published previously by other authors and with strains from different regions of the world. The results obtained by the different techniques were compared by calculation of the discriminatory power of the individual or the combined typing methods.


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MATERIALS AND METHODS
 
Collection and cultivation of bacteria. Field strains of M. avium subsp. paratuberculosis were isolated from the intestinal tissues (different lymph nodes, gut mucosa) or feces of dairy cattle between 2001 and 2004. The cattle originated from 14 herds (Table 1) from different geographic areas in Germany. Isolates originated from 1 to 34 single animals within the herds. The frequency of animal trade in these herds had differed in the past.


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  		TABLE 1. Origin of strains and results of typing by RFLP, MIRU, and VNTR analysis

Primary isolates were cultivated on Herrold's egg yolk medium containing mycobactin (BD Biosciences, Heidelberg, Germany). The presence of acid-fast organisms was proved by Ziehl-Neelsen staining. After the identification of M. avium subsp. paratuberculosis on the basis of mycobactin-dependent growth, incubation period, colony morphology, and PCR, mass cultivation was performed on modified Loewenstein-Jensen medium containing mycobactin (Bioservice Waldenburg, Germany). The identities of the M. avium subsp. paratuberculosis isolates were again confirmed. Isolates were stored as glycerol stocks at –80°C. M. avium subsp. paratuberculosis type strain ATCC 19698 and reference strain DSMZ 44135, both from the German Collection of Microorganisms and Cell Cultures (DSMZ), served as controls for all techniques.

Identification of bacteria by PCR. DNA was isolated before PCR. Briefly, the cells were heat inactivated at 80°C for 20 min and ultrasonicated for 10 min. After heat treatment (100°C, 10 min) and centrifugation at 12,000 x g twice for 5 min each time, the DNA-containing supernatant was used for PCR.

The isolates were identified as M. avium subsp. paratuberculosis by PCR amplification of IS900, which was carried out with the primers and the PCR conditions described by Englund et al. (15), and the presence of the M. avium subsp. paratuberculosis-specific sequence f57 was determined as described by Vansnick et al. (43). To ensure the purity of the DNA and the absence of other M. avium subspecies, a lack of IS901 and IS1245 was proven by PCR by the methods of Bartos et al. (2) and Guerrero et al. (19), respectively. A total of 71 isolates identified as M. avium subsp. paratuberculosis (with an IS900-positive, IS1245-negative, IS901-negative, and f57-positive genotype) was included in the study.

DNA extraction. Genomic DNA for typing by IS900 RFLP and MIRU-VNTR analysis was prepared by the cetyltrimethylammonium bromide method described by van Soolingen et al. (44). The concentration of DNA was measured with a spectrophotometer (Beckman Coulter).

Typing by IS900 RFLP. (i) DNA digestion and Southern blotting. Two micrograms of genomic DNA was digested separately with BstEII, BamHI, and PvuII (Roche Diagnostics) and PstI (New England Biolabs) under the conditions specified by the manufacturers. DNA fragments were separated by horizontal electrophoresis on a 240-mm 1% (wt/vol) agarose gel in 1x TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 65 V for 16 h. Bacteriophage lambda DNA cleaved with EcoRI and HindIII and labeled with digoxigenin-11-dUTP, with a size range of from 21.2 to 0.8 kbp (Roche Diagnostics), was run as an external molecular size marker in three lanes (both edges and the middle) of each gel. After incubation of the agarose gel with 0.25 N HCl for 15 min and washing with aqua bidest, the DNA was denaturized by incubation with 0.5 N NaOH for 30 min. The DNA fragments were vacuum transferred (model 785; Bio-Rad, United Kingdom) to a Hybond-N nylon membrane (Amersham Biosciences, United Kingdom) and fixed onto the membranes by treatment with heat for 2 h at 80°C.

(ii) DIG-labeled IS900 probe. The probe for IS900 RFLP was prepared by PCR amplification with the forward oligonucleotide 5'-TGG ACA ATG ACG GTT ACG GAG GTG G-3' and the reverse oligonucleotide 5'-GAT CGG AAC GTC GGC TGG TCA GGA T-3' (positions 209 to 233 bp and 662 to 638 bp, respectively, of IS900; GenBank/EMBL accession number X16293). These primers were previously described by Kunze et al. (24) and were recommended for use for probe amplification in the standardized protocol of Pavlik et al. (32). PCR was conducted with a digoxigenin (DIG)-labeled deoxynucleoside triphosphate (dNTP) mixture (DIG DNA labeling mix; Roche Diagnostics), according to the manufacturer's instructions. PCR mixtures of 20 µl contained 1x buffer, 1x DIG DNA labeling mixture, 0.5 µM (each) primer, 2.5 U Taq polymerase (Roche Diagnostics), and 50 ng of purified genomic DNA. The M. avium subsp. paratuberculosis reference strain (ATCC 19698) was used as the matrix for probe amplification. PCR was performed with the following cycling conditions: 94°C for 30 s for initial denaturation; 29 cycles consisting of 94°C for 30 s, 68°C for 30 s, and 72°C for 2 min 30 s; and then 1 cycle consisting of 94°C for 30 s, 68°C for 2 min, and 72°C for 3 min. The amplification product was visualized by ethidium bromide staining after electrophoresis in a 1% (wt/vol) agarose gel and was purified by use of a QIAquick gel extraction kit (Qiagen). Sequence analysis confirmed the probe sequence (GenBank/EMBL accession number X16293).

(iii) Hybridization and detection. Hybridization was performed at 68°C overnight with the DIG-labeled IS900-specific probe. The membrane was subsequently washed under high-stringency conditions (twice with 2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate] and 0.1% sodium dodecyl sulfate at room temperature for 5 min; twice with 0.1x SSC and 0.1% sodium dodecyl sulfate at 68°C for 15 min, and once with wash buffer at room temperature). The targets were submitted to immunological detection with anti-DIG antibody conjugated to alkaline phosphatase (DIG luminescent detection kit; Roche Diagnostics). The CSPD detection system was used according to the manufacturer's protocol to visualize the hybridized probe (Roche Diagnostics). The membrane was exposed to Hyperfilm ECL (GE Healthcare, Freiburg, Germany) and was developed for 1 to 30 min for optimal exposure.

(iv) Analysis of IS900 RFLP results. The DNA fingerprints obtained by IS900 RFLP were scanned and analyzed by using Gel Compare software (version 4.011; Applied Maths, Kortrijk, Belgium). All patterns were confirmed by retesting. The types obtained by IS900 RFLP were analyzed by geographic location, by farm, and by individual animal.

(v) Nomenclature for IS900 RFLP types. When the IS900 RFLP profiles matched the patterns described by other authors, the designations of the other authors were used. Unknown new patterns were given a new numeric code. The IS900 RFLP type designation after BstEII digestion was that described by Pavlik et al. (32); the IS900 RFLP type designation after BamHI digestion was that described by Cousins et al. (10). Comparable RFLP patterns based on a specific RFLP probe on the left side of IS900 have been described previously for patterns based on PstI digestion by Whipple et al. (46), Saunders et al. (36), and Palmer et al. (28) and for patterns based on PvuII digestion by Whipple et al. (46), Cousins et al. (10), Stevenson et al. (37), and de Juan et al. (12).

MIRU typing (MIRU PCR). (i) MIRU PCR. The amplification of specific MIRUs at four specific loci within the M. avium subsp. paratuberculosis genome, described as MIRU PCR by Bull et al. (6), was applied. The same DNA templates used for typing by IS900 RFLP were used for the MIRU PCR. The primer sequences specific for loci 2 to 4 were those of Bull et al. (6). Reverse primer M1.rr (5'-CCG TTG TCC AGG TGG AGT-3') was used for locus 1. M. avium subsp. paratuberculosis-specific DNA (approximately 50 ng) was amplified in a 50-µl reaction volume containing a 2 µM concentration of each primer, 1x reaction buffer containing 1.5 mM MgCl2, 10% dimethyl sulfoxide, 100 µM dNTPs, and 1 U of HotStarTaq DNA polymerase (Qiagen). The PCR started with an initial denaturation step at 94°C for 15 min, followed by 35 cycles of 94°C for 30 s, 58°C (for the MIRU PCRs for loci 1 and 2) for 1 min, and 72°C for 30 s, with a final extension at 72°C for 1 min. The annealing temperature for the MIRU PCR for locus 3 was 52°C, and that for the MIRU PCR for locus 4 was 53°C. The MIRU PCR primers specific for locus 3 were used at a concentration of 6 µM. The PCR-amplified VNTRs were analyzed for their amplicon sizes on a 1.5% agarose gel. The results were proven by sequencing of the amplification products of isolates with different numbers of repetitive units at the four loci.

(ii) Nomenclature for MIRU types. The MIRU types were designated I to VII and represented the PCR results at the different MIRU loci (Table 2). Types I, II, and VII correspond to the MIRU patterns described by Bull et al. (6).


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  		TABLE 2. MIRU profiles and their frequencies

Typing by VNTR and MIRU analysis. Additionally, six other PCR-based typing markers that were identified by Thibault et al. (41) and that consisted of VNTRs at five specific genome loci (designated tandem repeats [TRs] 25, 47, 3, 7, and 32) and one TR with an MIRU structure (TR 292) were included in this study. The specific primer sequences and annealing temperatures described by Thibault et al. (41) were employed. DNA (approximately 50 ng) was amplified in a 20-µl reaction volume containing a 2 µM concentration of each primer, 1x reaction buffer containing 1.5 mM MgCl2, 10% dimethyl sulfoxide, 100 µM dNTPs, and 0.5 U HotStarTaq DNA polymerase (Qiagen). The PCR started with an initial denaturation step at 95°C for 15 min, followed by 35 cycles at 95°C for 1 min, 57°C (TR 32) for 1 min, and 72°C for 1 min 30 s, with a final extension at 72°C for 15 min. The annealing temperature for TR 25 and TR 292 was 58°C, that for loci 3 and 7 was 60°C, and that for TR 47 was 64°C. The PCR products were analyzed for their amplicon sizes on a 1.5% agarose gel to detect differences in repeat numbers. The results were proven by sequencing of the amplification products with different sizes at the different TR loci. The VNTR-MIRU profiles were designated by the number of complete TRs in the following order: TR 292-25-47-3-7-32.

Calculations. The discriminatory index of Hunter and Gaston (21) was calculated as a numerical index of the discriminatory powers of each typing technique and different combinations of these methods. The following formula was used to calculate the discriminatory index (DI):

Formula
where N is the total number of epidemiologically unrelated strains in the respective typing scheme. Therefore, only isolates with different genotypes from a distinct herd detected by the method of interest were included in the particular calculation. S is the total number of distinct patterns or profiles discriminated by each typing method or by the combination of individual methods. Nj is the number of epidemiologically unrelated strains belonging to the jth pattern.

Calculation of the allelic diversity at the different MIRU loci was done as described by Supply et al. (38).

Definitions. Epidemiologically unrelated strains were defined as strains that were not derived from a common source; that is, they originated from different areas. A strain was defined as described by Tenover et al. (40) as an isolate or a group of isolates that can be distinguished from other isolates of the same genus and species by genotypic characteristics. In contrast, an isolate was defined as an isolate for which no information apart from its genus and species was available.


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RESULTS
 
Characterization of strain types by growth characteristics and IS900 RFLP. All 71 bovine M. avium subsp. paratuberculosis isolates from Germany analyzed had the following primary isolation characteristics: 6 to 12 weeks of growth for the appearance of cream-colored colonies, a rough colony morphology, and mycobactin dependence. During subcultivation they were moderately slowly growing. All isolates, including type strain ATCC 19698 and reference strain DSMZ 44135, showed the IS900 cattle type after digestion with BstEII by IS900 RFLP (Table 1). Therefore, they all belonged to the cattle group designated type II (12).

Typing by IS900 RFLP with four different restriction enzymes. Hybridization of total DNA, digested separately with the enzymes BstEII, PstI, PvuII, and BamHI, resulted in at least 9 to 18 bands that ranged in size from less than 1 to 17 kb (Fig. 1 and 2). The use of DIG labeling for IS900 RFLP resulted in band patterns with very high resolving powers. New IS900 RFLP patterns could be identified. Depending on the restriction enzyme used, the number of distinct IS900 RFLP profiles was different (Table 1). Seven BstEII-based IS900 RFLP profiles, six of which (profiles C1, C17, C5, C16, C18, and C22) were described by Pavlik et al. (32) and one of which was a new profile (Fig. 1a), were detected at different frequencies (Table 1). Restriction analysis by PstI resulted in eight profiles; two of them (types 1 and 3) were previously described by Whipple et al. (46). The other six profiles (types 7 to 12) have not been published before (Fig. 2a). Nine IS900 RFLP patterns were identified by PvuII restriction digestion (Fig. 2b). The clear differentiation of single bands, also within a range from 0.8 to 1.5 kbp, was remarkable. Three of these patterns (types 1, 2, and 3) were previously described by Whipple et al. (46), and type 1 and type 2C were also described by Cousins et al. (10). Five new IS900 RFLP profiles were obtained. After BamHI digestion, four profiles were identified: two (types 1 and 11) were previously described by Cousins et al. (10), and two profiles were new (Fig. 1b).


Figure 1
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  		FIG. 1. IS900 RFLP profiles of German M. avium subsp. paratuberculosis isolates from cattle. The numbers above the lanes are the IS900 RFLP type designations. The numbers on the left or right side of the patterns indicate the sizes of the reference bands. The arrowheads in the schematic diagrams show the differences between type 1 and the other RFLP profiles. (a) IS900 RFLP profiles after digestion with restriction endonuclease BstEII, designated according to the nomenclature of Pavlik et al. (32). One novel profile was detected. (b) IS900 RFLP profiles after digestion with restriction endonuclease BamHI, designated according to the nomenclature of Cousins et al. (10). Types 14 and 15 have not been described previously. Lanes M, molecular weight (Mw) markers.


Figure 2
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  		FIG. 2. IS900 RFLP profiles of German M. avium subsp. paratuberculosis isolates from cattle. The numbers above the lanes are the IS900 RFLP type designations. The numbers on the left or right side of the patterns indicate the sizes of the reference bands. The arrowheads in the schematic diagrams show the differences between type 1 and the other RFLP profiles. (a) RFLP profiles after digestion with PstI. Types 1 and 3 were designated according to Whipple et al. (46). Types 7 to 12 new profiles not previously published. (b) RFLP profiles after digestion with PvuII. Types 1 to 3 were designated according to the nomenclature of Whipple et al. (46), and type C2 is designated according to the nomenclature of Cousins et al. (10). Profiles N13 to N17 are novel profiles not described previously. Lanes M, molecular weight (Mw) markers.

As shown in Table 1, BstEII-based IS900 RFLP type C1 was the most common type (identified in 11 herds [79%] and in 43% of the isolates) but could be subdivided into 10 different genotypes by use of the other three digestion enzymes. Likewise, BstEII-based IS900 RFLP type C17, found in a total of 34 isolates (46%) from herds 1 and 4, was divided into two genotypes. Types C5, C16, C18, C22, and Cnew were found in individual isolates.

Combination of the results obtained with all enzymes yielded 17 different restriction pattern profiles. Without BamHI digestion, 16 profiles could be found. By using the results of only BstEII and PstI digestion or only BstEII and PvuII digestion, 11 profiles were detected. Frequently observed IS900 RFLP types (after BstEII, PstI, PvuII, and BamHI digestion) consisted of profiles C1-1-1-1 (herds 1, 2, and 5; 13 isolates), C1-7-1-1 (herds 7 to 9; 3 isolates), and C1-3-1-11 (herds 4 and 6; 6 isolates). Thirty isolates had the IS900 RFLP pattern combination C17-9-3-11, but they originated from only one herd in Thuringia.

Typing by MIRU and VNTR analysis. As a result of MIRU PCR, three of the four MIRU loci studied showed polymorphisms in the number of repeat motifs (Table 2). Therefore, the results for MIRU locus 4, which did not contain polymorphisms, were not included in the subsequent analysis (Table 3 to 6). Altogether, seven different MIRU pattern profiles were identified (Table 1 and 2). MIRU profiles I to IV were each detected in several isolates, whereas MIRU profiles V to VII were found in individual isolates. M. avium subsp. paratuberculosis strains exhibiting MIRU profile I or II were designated M. avium subsp. paratuberculosis groups 1 and 2, respectively, by Bull et al. (6). Type strain ATCC 19698 and reference strain DSMZ 44135, which had MIRU profile I, belong to one of these large M. avium subsp. paratuberculosis groups. For one strain from Baden-Wurttemberg (herd 10), MIRU profile VII combined with BstEII-based IS900 RFLP type C1 was detected. This pattern was recently shown for M. avium subsp. paratuberculosis vaccine strain 316F by Bull et al. (6).


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  		TABLE 3. TR copy numbers at the nine MIRU-VNTR loci


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  		TABLE 6. Numerical index of discriminatory power calculated by separate and combined typing methods for the current strain panel

Typing by VNTR-MIRU analysis with six of eight TR loci, as described by Thibault et al. (41), subdivided the M. avium subsp. paratuberculosis strains into strains with nine different profiles, as shown in Table 1. The most frequently identified profiles were 333228 (six herds), 433218 (four herds), and 433228 (three herds). By combining the results obtained for MIRU loci 1 to 3 and the six TR loci mentioned above, a total of 15 distinct MIRU-VNTR profiles (Tables 1, 3, and 4) were obtained. The allelic diversities of these nine TR loci calculated for the current strain panel were different (Table 5).


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  		TABLE 4. Results of typing by MIRU-VNTR analysis in combination with IS900 RFLP


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  		TABLE 5. Allelic distribution among epidemiologically unrelated M. avium subsp. paratuberculosis isolates determined by MIRU-VNTR analysisc

Comparison and combination of typing results obtained by IS900 RFLP and MIRU-VNTR analysis. Altogether, IS900 RFLP with the four digestion enzymes in combination with the results of typing by MIRU-VNTR analysis subdivided the 71 isolates originating from 14 farms from different geographic locations into 24 genetically distinguishable strains. The predominant BstEII-based IS900 RFLP type, type C1, could be split into 14 different types by MIRU-VNTR analysis. The results of typing by IS900 RFLP and MIRU-VNTR analysis were not associated (Tables 1 and 4). The discriminatory indices of the different typing techniques, calculated for each technique separately or in combination, are shown in Table 6. A combination of fingerprinting and PCR-based techniques resulted in a maximal discriminatory index (21) of 0.997. The individual methods had discriminatory indices if less than 0.9. For strain differentiation by IS900 RFLP with BstEII digestion only together with typing by MIRU-VNTR analysis (nine loci), a discriminatory index of 0.986 was estimated.

Epidemiological results. The combination of results of all typing methods used enabled us to find isolates with different genotypes within distinct regions of origin and individual dairy cattle herds in Germany. Six to seven strains were found in three geographic regions. Up to seven different genotypes were found among the isolates from cattle on one farm (herd 4). The diversity of the M. avium subsp. paratuberculosis strains within herds was different. Among the isolates in herd 1 (n = 34) (Table 1), 91% of the isolates were genetically indistinguishable and the isolates thus corresponded to one strain. The other three profiles detected for the isolates in herd 1 appeared only once or twice. In contrast, the seven profile combinations detected among the isolates in herd 4 (n = 15) were more or less evenly distributed. In the past, cattle in this herd had frequently been purchased from other sources and had unknown infection states. The six isolates from the cattle in herd 5 showed the same genotype and thus corresponded to a single strain. The diversities of the isolates detected within these herds reflect the differences in the frequency of animal movements among these farms.

Profile C1-1-1-1/2 (IS900 RFLP/MIRU-VNTR analysis; Table 1) was detected in herds 1 and 2 from Thuringia. The isolates are likely epidemiologically linked. The trade of animals between these herds in the past could not be excluded.


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DISCUSSION
 
This is the first report of a high number of genetically distinguishable M. avium subsp. paratuberculosis isolates in cattle on a national scale. A combination of independent typing techniques was applied to increase the discriminatory power of typing and to differentiate between M. avium subsp. paratuberculosis isolates.

IS900 RFLP has been the technique used in most cases for the comparison of isolates in national and international studies. Standardization of this fingerprinting technique was proposed for the first time in 1999 (32). In preceding studies, a variety of restriction enzymes, labeling methods, and hybridization probes was used. In the present study, the IS900 RFLP patterns resulting from restriction with four different enzymes were compared with the available reference patterns. However, by examination of the reference patterns obtained by IS900 RFLP with PvuII or PstI digestion, the pattern types diverged from each other. The reason is that 1,451-bp insertion element IS900 contains a restriction site for PstI as well as one for PvuII at position 891 bp and position 707 bp, respectively (based on the sequence with GenBank/EMBL accession number X16293). Therefore, the IS900 RFLP profiles differ depending on whether the hybridization site of the probe is to the 5' or 3' site of the digestion site of the restriction enzyme or covers the digestion site. In the latter case, the probe recognizes IS900 on both sides of the PstI site (25) and IS900 RFLP results in a higher number of bands. Such patterns obtained by PvuII digestion have been published by Kunze et al. (24); and such patterns obtained by PstI digestion have been published by Thoresen and Olsaker (42), Pavlik et al. (29, 32), Moreira et al. (25), and Djonne et al. (14). The band pattern types obtained in the current study are in contrast to those results. The probe used here, originally recommended for use in the standardized protocol for IS900 RFLP (32), was located on the left arm of IS900 and did not cover a digestion site for PstI and PvuII. Comparable probes were applied by Whipple et al. (46), Francois et al. (17), Moreira et al. (25), Cousins et al. (10), Stevenson et al. (37), Saunders et al. (36), and de Juan et al. (12); and corresponding patterns were shown after digestion with one or both of these enzymes. In contrast, IS900 contains no restriction site for BstEII or BamHI. Therefore, the BstEII- and BamHI-based IS900 RFLP patterns are independent of the hybridization site of the probe. The numerous BstEII patterns published worldwide are quite comparable to those obtained in the current study.

As expected and in line with current knowledge, all isolates of the current study, which originated from cattle, belonged to the C type (type II). BstEII-based IS900 RFLP type C1 was the predominant pattern found among the isolates in the panel of strains examined, in agreement with previous data for cattle herds from different countries in Europe (30, 31, 33). By using a second restriction enzyme, PstI, B-C1 was the most frequent of three IS900 RFLP types (B-C1, E-C1, and L-C13) found among strains from cattle in Germany, as well as the most common type in Europe; this type has also been detected in the United States and Australia (33). The results obtained with the current panel of strains comprising 71 isolates from 14 German herds supplements these data with BstEII-based IS900 RFLP types C17, C5, C16, C18, C22, and Cnew, which are otherwise rare. Besides Europe, IS900 RFLP type C1 is highly prevalent in the United States (33, 46) and New Zealand (9), whereas type C3 predominates in Australia (10, 36, 47) and type C17 predominates in specific regions of Argentina (25). As documented with our results for Germany, type C17 is rare in Europe and up to now has only occasionally been detected in isolates from Scotland (12, 18).

Using PvuII-based IS900 RFLP, Bauerfeind et al. (3) detected only two different IS900 RFLP patterns among nine German isolates, each of which was from an individual herd. The types were not comparable to the patterns obtained in the current study because a different hybridization probe was applied. However, the large panel of isolates in the present study yielded a higher diversity, as nine profiles were found only by PvuII digestion.

To increase the discriminatory power of M. avium subsp. paratuberculosis typing and to subdivide predominant BstEII-based IS900 RFLP type C1, a combination of different methods was used by various authors. Whipple et al. (46) and Cousins et al. (10) applied four digestion enzymes for IS900 RFLP to differentiate isolates from different regions in the United States and Australia. They detected 8 and 11 subtypes, respectively, among the cattle type II strains, whereas 17 IS900 RFLP subtypes were detected in the present study. Depending on the distinct panel of strains used, different digestion enzymes showed the highest potential for differentiation. Whipple et al. (46) and Pavlik et al. (32) were able to subdivide isolates with BstEII-based IS900 RFLP profile C1 into three subtypes by further digestion with another restriction enzyme. de Juan et al. (12) applied multiplex PFGE and found eight subtypes among 19 caprine isolates exhibiting IS900 RFLP profile C1. Depending on the number of different isolates, multiplex PFGE subdivided BstEII-based profile C1 of cattle isolates into two types, BstEII-based profile C5 into three types, and BstEII-based profile C17 into six types (37).

By using PCR-based techniques but different markers and strain panels, IS900 RFLP type C1 was divided into 4 (27), 11 (4), and 15 (41) different subtypes by MIRU-VNTR analysis. In the current study, 14 subtypes of the IS900 RFLP C1 type were identified by typing by MIRU-VNTR analysis and 17 subtypes were identified by using a combination of IS900 RFLP and this PCR-based technique. In contrast to the previous studies, however, our isolates originated from the cattle herds in only one country.

The overall hierarchy of the polymorphisms of the MIRU-VNTR loci used was similar to that observed in previous studies (6, 41). The six loci, loci 2, 3, 292, 7, 25, and 47 (Table 5), largely showed comparable variabilities in the current population. This suggests that the relative degrees of genetic information carried by the different loci are globally conserved among distinct epidemiological settings and bacterial populations, as described by Supply et al. (38) for some MIRU-VNTR loci of M. tuberculosis.

Motiwala et al. (26) compared the discriminatory powers of various M. avium subsp. paratuberculosis genotyping techniques by analysis of the data from former studies by various authors: IS900 and IS1311 RFLP with different digestion enzymes, MPIL, MIRU PCR, PFGE, and other methods. Except for the MLSSR sequencing approach (1), a discriminatory index of less than 0.9, which is comparable to that achieved in the current study, was achieved for all methods. This is not sufficient for epidemiological studies (21). It must be taken into account that only epidemiologically unrelated strains can be included in the calculation of discriminatory indices. Isolates from one herd with the same profile can be counted only once. However, although the current panel of strains exclusively originated from cattle herds in Germany, the discriminatory power was markedly increased by use of a combination of different typing methods. The discriminatory indices achieved (>0.98) were higher than the discriminatory index of 0.96 published by Amonsin et al. (1) for the typing of isolates originating from distinct geographic regions of the world and different host species by MLSSR sequencing analysis.

The reason for this high index is the lack of congruence of the typing results obtained by multiplex IS900 RFLP and MIRU-VNTR analysis, as they are two fully independent genotyping methods. The two typing tools are apparently directed toward different targets of the M. avium subsp. paratuberculosis genome and provide complementary information (35). In contrast to typing by MIRU-VNTR analysis, IS900 RFLP as well as PFGE provides information about alterations in the whole genomes of strains. Although RFLP is a cumbersome technique that requires the preparation of high-quality DNA, at present only a combination of such independent typing techniques allows the identification of sufficient heterogeneity among isolates in the M. avium subsp. paratuberculosis population. For phylogenetic studies, it should be considered that the modes of the evolutionary processes of the different independent target regions will differ and that the target regions will exhibit different evolutionary rates. According to Vergnaud and Pourcel (45), multiple-locus VNTR analysis does not provide a molecular clock. The mutation of TRs directly influences the phenotypes of the corresponding strains, so that these mutations are not neutral and probably contribute to the adaptation of the species to its environment in a reversible way. A simple replacement of RFLP by the use of additional TR markers to reduce the duration and the special technical demands is not recommended.

By applying discrimination criteria based on M. tuberculosis (39) to M. avium subsp. paratuberculosis, the high number of strains with IS900 RFLP type C1 in frequent association with MIRU profiles I and II could characterize closely related, possibly clonally related strains. In contrast, typing by VNTR analysis resulted in diverse genotypes within these groups; therefore, their relations could not be confirmed. While the current multiplex IS900 RFLP patterns suggest an association between the genotypes and the origins of many isolates, isolates with identical or similar MIRU-VNTR patterns were distributed rather randomly. As described in several studies that used microsatellites (2-bp to 8-bp repeat units) for multilocus VNTR analysis (45), highly polymorphic VNTR markers, which usually result from a higher rate of mutation events, usually have high homoplasy levels. To which of the currently used M. avium subsp. paratuberculosis markers this can be applied should be studied in further investigations. Evolutionary processes are certainly influenced by the extremely long generation time of M. avium subsp. paratuberculosis and the incubation time of paratuberculosis as a chronic disease. However, MIRU-VNTR typing must be combined with complementary methods not only for evolutionary studies but also for epidemiological studies.

In conclusion, despite the high level of homogeneity of the M. avium subsp. paratuberculosis isolates, typing by use of the combination of IS900 RFLP and VNTR-MIRU analysis described here has shown a sensitivity sufficient to distinguish strains of type II, even within the bovine population, on a national scale. It offers the possibility of tracing the transmission of M. avium subsp. paratuberculosis among individual animals in one herd, between herds, and between different host species. For IS900 RFLP we propose the selection of BstEII because it is the restriction enzyme most commonly used worldwide, and the resulting fingerprint patterns are quite comparable to those of previous epidemiological studies. When PstI or PvuII is also applied, a specific probe for hybridization situated on the left site of the IS900 element without a restriction site for these enzymes should be used in further IS900 RFLP studies. The standardized protocol should be changed accordingly. Use of the restriction enzyme PstI would be preferred and is recommended. Additional M. avium subsp. paratuberculosis-specific markers can further be applied and verified for use in a still more complex typing technique. Combined analysis of the types determined on the basis of independent genomic characteristics constitutes an approach to studying the population structure, the global epidemiology, and the evolution of M. avium subsp. paratuberculosis.


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ACKNOWLEDGMENTS
 
This work was supported by the Federal Ministry of Nutrition, Agriculture and Consumer Protection of Germany.

Strains were kindly provided by Sabine Bracknies, Aulendorf, Germany; Hartmut Helbing, Bad Langensalza, Germany; Martina Schulze and Dieter Klarmann, Oldenburg, Germany; Annette Schliephake, Stendal, Germany; and Peter Kutzer, Frankfurt (Oder), Germany. We thank Jutta König, Kerstin Steger, Angela Dramburg, and Sandra Müller from the Friedrich-Loeffler-Institut for their excellent technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany. Phone: (49) 3641 804280. Fax: (49) 3641 804228. E-mail: petra.moebius{at}fli.bund.de Back

{triangledown} Published ahead of print on 3 January 2008. Back


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Journal of Clinical Microbiology, March 2008, p. 972-981, Vol. 46, No. 3
0095-1137/08/$08.00+0     doi:10.1128/JCM.01801-07
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