Received 24 June 2008/Returned for modification 15 August 2008/Accepted 7 January 2009

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2009, p Vol. 47, No /09/$ doi: /jcm Copyright 2009, American Society for Microbiology. All Rights Reserved. Molecular Typing of Mycobacterium bovis Strains Isolated in Italy from 2000 to 2006 and Evaluation of Variable-Number Tandem Repeats for Geographically Optimized Genotyping M. Beatrice Boniotti, 1 * Maria Goria, 2 Daniela Loda, 1 Annalisa Garrone, 2 Alessandro Benedetto, 2 Alessandra Mondo, 3 Ernesto Tisato, 4 Mariagrazia Zanoni, 1 Simona Zoppi, 2 Alessandro Dondo, 2 Silvia Tagliabue, 1 Stefano Bonora, 3 Giorgio Zanardi, 1 and M. Lodovica Pacciarini 1 Centro Nazionale di Referenza per la Tubercolosi Bovina, Istituto Zooprofilattico Sperimentale Lombardia e Emilia-Romagna, Via Bianchi 9, Brescia, Italy 1 ; Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta, Via Bologna 148, Turin, Italy 2 ; Department of Infectious Diseases, University of Torino, Corso Svizzera 164, Turin, Italy 3 ; and Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell Università 10, Legnaro (PD), Italy 4 Received 24 June 2008/Returned for modification 15 August 2008/Accepted 7 January 2009 Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/etr analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/etr-a to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/etr methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains. Downloaded from Bovine tuberculosis (TB) is still an important disease in the cattle population in Italy. The first control measures started in 1964 on a voluntary basis. A compulsory national eradication program based on a single intradermal skin test (SIST), slaughter of positive animals, and postmortem meat inspection was introduced in Its application allowed for a gradual reduction of the national TB herd prevalence from 11% in 1965 to 1.1% in 2006, calculated as the proportion of outbreaks to herds under the control program in 1 year. As a result, the official TB-free status was achieved starting from 1970 in some Italian areas such as the Autonomous Region of Trentino Alto Adige, Friuli Venezia Giulia Region (northeast Italy), and some provinces of northern and central Italy. In the rest of the national territory, TB still persists with different prevalence rates: 6.6% in Sicilia, 0.77% in Puglia, and 0.59% in Lazio and Campania. In northern Italy, where about the 70% of the cattle population is reared, TB is still present in the Piedmont (0.4%), Lombardy (0.15%), Veneto (0.1%), and Emilia-Romagna (0.08%) regions. In order to accelerate the achievement of a TB-free status, the authorities in these areas are implementing specific TB control measures, taking into * Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia Romagna, Via Bianchi 9, Brescia, Italy. Phone: (39) Fax: (39) mariabeatrice.boniotti@bs.izs.it. Published ahead of print on 14 January account local situations and problems such as mean distance among herds, trade of animals from other territories, and common pasturing. In this respect, the origin of TB infection often remains undetermined despite its importance. Molecular typing of isolates has become a valuable tool in the study of M. bovis epidemiology, allowing investigators to better identify the sources of infection and achieve a wider knowledge of TB transmission routes. A genetic profile database collection of M. bovis isolates may help to confirm or reject hypotheses outlined by traditional epidemiological investigations. Genotyping of M. bovis probably lacks sufficiently informative methods. IS6110 restriction fragment length polymorphism (RFLP) typing has been considered a gold standard method for differentiation of M. tuberculosis strains for a long time; this has provided only limited discrimination among M. bovis populations where the majority of the isolates harbor only one or few IS copies (11). PCR-based spoligotyping (16) has been widely used to genotype M. bovis isolates (11); it is highly reproducible and rapid and represents the first universally recognized typing system for M. bovis populations. However, studies performed on M. bovis isolates in Northern Ireland (32, 33), France (12), Australia, Canada, the Republic of Ireland, and Iran (7) showed a limited discrimination power of this method. Over the last 10 years several PCR-based genotyping methods have become available for rapid molecular epidemiology on March 7, 2019 by guest 636

2 VOL. 47, 2009 MYCOBACTERIUM BOVIS POPULATION IN ITALY 637 TABLE 1. Locus designations and PCR primer sequences used in this study VNTR locus Alias Repeat unit length (bp) PCR primer sequence (5 3 ) a Reference 2165 ETR-A 75 For, AAA TCG GTC CCA TCA CCT TCT TAT; Rev, CGA AGC CTG GGG TGC CCG CGA TTT ETR-B 57 For, GCG AAC ACC AGG ACA GCATCA TG; Rev, GGC ATG CCG GTG ATC GAG TGG ETR-C 58 For, GTG AGT CGC TGC AGA ACC TGC AG; Rev, GGC GTC TTG ACC TCC ACG AGT G MIRU For, TGG ACT TGC AGC AAT GGA CCA ACT; Rev, TAC TCG GAC GCC GGC TCA AAA T ETR-D 77 For, CAG GTC ACA ACG AGA GGA AGA GC; Rev, GCG GAT CGG CCA GCG ACT CCT C 10 MIRU 04 For, GCG CGA GAG CCC GAA CTG C; Rev, GCG CAG CAG AAA CGT CAG C MIRU For, GTT CTT GAC CAA CTG CAG TCG TCC; Rev, GCC ACC TTG GTG ATC AGC TAC CT MIRU For, TCG GAG AGA TGC CCT TCG AGT TAG; Rev, CCC GTC GTG CAG CCC TGG TAC MIRU For, TCG GAG AGA TGC CCT TCG AGT TAG; Rev, GGA GAC CGC GAC CAG GTA CTT GTA MIRU For, CTG TCG ATG GCC GCA ACA AAA CG; Rev, AGC TCA ACG GGT TCG CCC TTT TGT C MIRU For, CGA CCA AGA TGT GCA GGA ATA CAT; Rev, GGG CGA GTT GAG CTC ACA GAA MIRU For, TAG GTC TAC CGT CGA AAT CTG TGA C; Rev, CAT AGG CGA CCA GGC GAA TAG MIRU For, TCG AAA GCC TCT GCG TGC CAG TAA; Rev, GCG ATG TGA GCG TGC CAC TCA A 36 QUB5 For, TGA CCA ACG TCA GAT TCA; Rev, TGA CGG GGC ATC TTC GAT ETR-E 53 For, CTT CGG CGT CGA AGA GAG CCT C; Rev, CGG AAC GCT GGT CAC CAC CTA AG 10 MIRU 31 For, ACT GAT TGG CTT CAT ACG GCT TTA; Rev, GTG CCG ACG TGG TCT TGA T MIRU For, CGC ATC GAC AAA CTG GAG CCA AAC; Rev, CGG AAA CGT CTA CGC CCC ACA CAT MIRU For, GGG TTG CTG GAT GAC AAC GTG T; Rev, GGG TGA TCT CGG CGA AAT CAG ATA a QUB11a 69 For, CCC ATC CCG CTT AGC ACA TTC GTA; Rev, TTC AGG GGG GAT CCG GGA b QUB11b 69 For, CGT AAG GGG GAT GCG GGA AAT AGG; Rev, CGA AGT GAA TGG TGG CAT QUB15 54 For, TAC ATT CGC GGC CAA AGG; Rev, AGG GGT TCT CGG TCA CCC QUB18 78 For, GGC AAC TGA AAG CCG CTT; Rev, AAT ACC GGG GAT ATC GGT QUB23 21 For, GCT GCA CCG GTG CCC ATC; Rev, CAC CGG AGC CGG AAC GGC QUB For, AAC GCT CAG CTG TCG GAT; Rev, GGC CAG GTC CTT CCC GAT QUB For, GGT GCA CGG CCT CGG CTC C; Rev, AAG CCC CGC CGC CAA TCA A QUB /57 For, CAG ACC CGG CGT CAT CAA C; Rev, CCA AGG GCG GCA TTG TGT T QUB For, ATC CCC GCG GTA CCC ATC; Rev, GCC AGC GGT GTC GAC TAT CC 29 a For, forward; Rev, reverse. investigations. First, six variable-number tandem repeat (VNTR) loci described as exact tandem repeats A through F (ETR-A, -B, -C, -D, -E, and -F) (10) were reported to be more discriminative than spoligotyping. More recently, a novel class of genetic markers, collectively known as MIRU-VNTR, was described; this includes the ETRs, mycobacterial interspersed repetitive units (MIRUs) (36), and VNTRs (22, 29, 32). These markers were evaluated for genotyping M. bovis by using selected panels of field isolates. Different combinations of informative VNTR markers were described by Roring et al. (30), by Skuce et al. (32), by Hilty et al. (13), and by Allix et al. (1). All of these studies highlighted the use of few (ca. five to eight) informative loci to obtain a discrimination power greater than 90%. In this study, we characterized by spoligotyping and ETR typing 1,560 M. bovis-m. caprae isolates obtained in 747 different herd outbreaks in Italy; this work represents a contribution to a better understanding of the genetics of the M. bovis population present in Italy. In addition, we evaluated the stability, epidemiological significance, and discrimination capacity of 24 MIRU-VNTR loci on a selected panel of 100 strains characterized by spoligotype SB0120, found in more than 50% of the field isolates in our study. The final aim was to select one or more loci sets for discrimination of locally prevalent M. bovis population containing a different number of informative markers whose complexity depends on the epidemiological studies and investigation requirements. MATERIALS AND METHODS Mycobacterial strains. A total of 1,560 isolates were collected from 2000 to 2006 by Istituti Zooprofilattici (the Italian Network of Animal Health Agencies) throughout the whole Italian territory. Most isolates (n 1,259) were obtained from cattle herds of northern Italy (the Piedmont, Lombardy, Emilia Romagna, and Veneto regions), while 89 isolates originated from the Campania and 49 from the Umbria and Marche regions. The rest of the isolates (n 163) were obtained from animals of central or southern Italy and slaughtered in northern Italy. This nationwide collection of isolates was obtained from one or more animals of 747 different bovine TB outbreaks. All of the isolates were identified by microbiological methods (23) and by molecular methods described by Kulski et al. (21). Mycobacteria characterized by a spoligotype consistent with M. caprae identification were confirmed by a gyrb PCR/RFLP assay as reported by Kasai et al. (17) and Niemann et al. (25) and/or by the PCRs as described by Huard et al. (14). M. tuberculosis H37Rv, M. avium ATCC 25291, M. bovis ATCC 19210, M. fortuitum ATCC 6841, M. kansasii ATCC 12478, and M. tuberculosis ATCC were used as reference strains. Geographic information system. A total of 498 isolates from the Emilia Romagna, Lombardy, Piedmont, and Veneto Regions were associated with the geographical coordinates of the corresponding herds. Georeferencing was done by the GIS application ArcMap 8.2 (ESRI) format. DNA preparation. M. bovis strains frozen in tryptic soy broth with 10% glycerol were thawed and cultured on Lowenstein-Jensen or Stonebrink medium (prepared as solid slants in screw-cap tubes) for 3 to 4 weeks at 37 C. For each isolate, four to five colonies were transferred into 500 l of1 Tris-EDTA buffer. The suspended colonies were boiled for 15 min in a water bath, and the bacterial lysate was used directly in PCRs. Spoligotyping. Spoligotyping was performed as described by Kamerbeek et al. (16). The spacer sequences contained in the direct repeat locus were detected by hybridization onto a spoligotyping membrane (Isogen Bioscience BV, Maarssen, The Netherlands). MIRU-VNTR typing. Twenty-four genomic loci were amplified individually with the primers described in Table 1. PCRs were performed on 5 l of DNA sample in a final volume of 30 l. ETR-A to -E locus typing was performed according to the method of Frothingham and Meeker-O Connell (10) with the following modifications. The final MgCl 2 concentration was 1.5 mm, and 1.5 U of TaqGold DNA polymerase (Applied Biosystems) was used in each reaction. MIRU loci were amplified as described by Supply et al. (36). VNTR 2163a, 2163b, 1982, 1612, 4052, and 1895 loci were amplified by using the following conditions: 3 l of10 PCR buffer (Abgene, Epsom, Surrey, United Kingdom), 1.5 mm MgCl 2, 200 M concentrations of each of the four deoxynucleoside triphosphates, 0.4 M concentrations of each primer, and 0.62 U of Thermo-Start DNA polymerase (Abgene). The amplification program consisted of 15 min at 95 C, followed by 40 cycles of 30 s at 94 C, 60 s at 60 C, and 120 s at 72 C. The PCR conditions for the amplification of the VNTR 3232, 3336, and 3155 loci were: 3 l of10 PCR buffer (Qiagen, GmbH, Hilden, Germany), 1.5 mm MgCl 2, 200 M concentra-

3 638

4 VOL. 47, 2009 MYCOBACTERIUM BOVIS POPULATION IN ITALY 639 tions of each of the four deoxynucleoside triphosphates, 0.66 M concentrations of each primer, 6 lof5 Q-solution, and1uofhotstarttaq DNA polymerase (Qiagen). The amplification profile was as follows: 15 min at 95 C, followed by 40 cycles of 30 s at 94 C, 60 s at 55 C, and 120 s at 72 C. All of the PCRs were run using a GeneAmp9700 PCR system (Applied Biosystems). PCR products were electrophoretically separated on a 2% agarose gel (Bio-Rad), except VNTR 3232, 3336, and 2163a (1.5% Agarose) and VNTR 1612 (4% NuSieve GTG agarose gel) (BioWhittaker, Inc.), in 1 Tris-boric acid-edta buffer (containing 1 g of ethidium bromide/ml). DNA ladders of 100 and a 50 bp (Roche Diagnostics, GmbH, Mannheim, Germany) were used as size markers. Allele assignation. The reference strain M. tuberculosis H37Rv, M. bovis BCG 27290, and eight M. bovis field isolates from unrelated outbreaks were amplified for the 24 MIRU-VNTR loci, and the PCR fragments were sequenced by using an ABI Prism 3130 instrument (Applied Biosystems). An allele-calling table was designed correlating the amplicon size and the sequences obtained according to previous references (10, 29, 32, 36). Routinely, allele assignation of M. bovis and M. caprae strains was performed on the basis of PCR fragment size and comparison to PCR products from M. tuberculosis H37Rv. Allelic and genotypic diversity. The allelic diversity (h) of each VNTR was calculated by using the following equation: h 1 x i 2 [(n/n 1)], where n is the number of isolates and x i is the frequency of the ith allele at the locus (31). The discriminatory power of combined VNTR markers was evaluated by using the equation 1 x i 2 [(n/n 1)], where n is the total number of genotypes obtained and x i is the frequency of the ith genotype (15). Cluster analysis. Spoligotyping and MIRU-VNTR profiles were recorded as character data and analyzed using Bionumerics software (Applied Maths, St-Martin-Latem, Belgium). Dendrograms were generated by using the categorical character option and the UPGMA (for unweighted pair-group method with arithmetic averages) clustering method. RESULTS Spoligotyping and ETR-A to -E typing. The molecular typing techniques used first were spoligotyping and ETR-A, -B, -C, -D, and -E analysis. Before their application, we verified the organization of direct repeat and ETR-A to -E loci of Italian strains by sequencing four and eight M. bovis strains, respectively, isolated in unrelated outbreaks. The results confirmed the genetic organization previously described in other countries (data not shown). A total of 1,560 isolates, collected in 747 outbreaks from 2000 to 2006, were analyzed by these typing techniques. Spoligotyping produced 81 different profiles, which are schematically represented in Fig. 1; they were differentiated in 44 clusters containing 2 to 408 isolates and 37 unique isolates. The genetic diversity provided by this genotyping method was 0.7. The predominant spoligotype was SB0120, named BCG-like by Haddad et al. (12), accounting for 54.6% of the Italian infected herds under study, followed by two spoligotypes at a much lower percentage: SB0134 (5.7%) and SB0841 (4.8%), characterized, respectively, by the lack of spacers 4 to 5 and spacers 6 to 7 in addition to the ones already absent in SB0120. Fortyfive isolates (representing 6.0% of the herds) showed spoligotypes consistent with a M. caprae identification: the seven profiles found in this study were characterized by the common lack of spacers 2 to 13, 15, and 28. However, isolates with similar patterns and included in the same cluster (Fig. 1) Locus TABLE 2. Allelic diversity of individual ETR loci among 747 isolates No. of isolates with ETR allele: Allelic diversity (h) ETR-A ETR-B ETR-C ETR-D ETR-E were identified as M. bovis by molecular assays (see Materials and Methods). ETR analysis identified 113 different genotypes; the most common ones were 45533, 54534, 55533, and 54533, found in 110, 82, 78, and 47 isolates, respectively. A total of 51 were unique, while the 680 left were clustered into 62 groups containing from 2 to 110 isolates. The most polymorphic ETR loci were ETR-A and ETR-B with allelic diversity scores of 0.57 and 0.68, respectively, while ETR-C had the lowest value (0.21) (Table 2). The overall genetic diversity of ETR-A to -E was A combination of the two typing techniques increased the number of genotypes to 228 (141 unique and 87 clustered); in particular, the prevailing SB0120 spoligotype was split into 64 different patterns, while the seven M. caprae spoligotypes were subdivided into 17 genotypes. The discrimination provided by combination of spoligotyping and ETR methods allowed for a genotypic diversity of Geographical distribution of spoligotypes. Figure 2 shows the distribution of the most common M. bovis spoligotypes (SB0120, SB0134, and SB0841) and the presence of M. caprae in the Piedmont, Lombardy, Veneto, and Emilia-Romagna regions. SB0120 is widely distributed with higher prevalence in Piedmont (63% of the outbreaks). With the exception of one cow imported from Germany and directly delivered to a slaughterhouse in Piedmont, M. caprae was found in the Lombardy and Emilia-Romagna regions, mainly clustered in two different genotypes, one involved in eight outbreaks in the Po Valley and the other one in eleven epidemiologically linked outbreaks in the mountain area of Valcamonica (28). A preliminary evaluation of M. bovis genotypes in central and southern Italy shows that spoligotype SB0120 is widespread also throughout the rest of Italy and that M. caprae is present as 11 additional genotypes (spoligotype/etrs). The geographical distribution of ETR types A to E did not highlight any significant profile clusterization (data not shown). Performance of MIRU-VNTR genotyping on a panel of 100 SB0120 strains. Genotypes characterized by SB0120 and the most frequent ETRs (45533, 54534, 55533, and 54533) were still too prevalent in the bacterial population (10.2, 6.7, 4.8, and FIG. 1. Dendrogram and schematic representation of 81 different spoligotypes obtained from the analysis of 747 isolates. Most spoligotypes are clustered in one major BCG-like group of similarity. A divergent group includes M. caprae patterns and similar M. bovis spoligotypes (indicated by asterisks). The dendrogram shows genetic distances and was generated by using the categorical character option and UPGMA clustering method (Bionumerics software). When available, the spligotype code is given.

5 640 BONIOTTI ET AL. J. CLIN. MICROBIOL. FIG. 2. Geographical distribution of isolates from the Piedmont, Lombardy, Emilia-Romagna, and Veneto regions of Italy. The most predominant spoligotypes are SB120 (55.4%), SB0134 (5.7%), and SB0841 (4.1%). Thirty strains showed spoligotypes consistent with M. caprae identification. 4.5%, respectively). Therefore, the next step was the selection of new markers to improve the discrimination capacity of our typing procedure. Twenty-four MIRU-VNTR markers (Table 1) were thus evaluated on 100 isolates characterized by spoligotype SB0120 and seven ETRA-E patterns: 45533, 55533, 54533, 53533, 54534, 44533, and (26, 17, 16, 15, 13, 7, and 6 isolates, respectively). The panel represents 60% of the predominant SB0120 spoligotype population. MIRU-VNTR markers further differentiate this panel into 89 profiles with only nine clusters of two to three isolates each, which corresponds to a genotypic diversity of (Fig. 3). The isolates included in this study were assumed to be without epidemiological links, but an investigation conducted after the acquisition of the MIRU-VNTR genotype data demonstrated that two of these groups were correlated (Fig. 3). These clusters displayed a fully identical profile by the 24-locus MIRU-VNTR genotyping. The allelic diversity of individual loci greatly differed, with values ranging from 0.00 for MIRU 2 to 0.64 for ETR-B (Table 3). As for loci ETR-C and -D, MIRU 10 and MIRU 39 displayed no allelic diversity within this panel. Loci with high allelic diversity included ETR-B, VNTR 3336, 3232, and ETR-A, while loci with intermediate values were VNTR 11a, MIRU 26, VNTR 26, VNTR 11b, VNTR 15, VNTR 1895, and ETR-E. By testing multiple combinations of markers within the set of 24 loci, we found that the maximal resolution of 89 types in this collection could already be achieved using a minimal group of 13 markers: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336 (Table 4), whereby all of the loci with a low h value were excluded. By decreasing the number of typing loci, we observed a slight decrease of resolution with ten loci and a more drastic reduction with nine loci. Similar results were obtained in other studies (1, 30). Stability of MIRU-VNTR loci. The clonal stability of the 24 MIRU-VNTR loci was evaluated using two panels for a total of 91 isolates. The first panel included 26 series of two to seven isolates collected from different animals coming from a single herd. The rationale behind this test is that a farm outbreak is most often caused by a single clonal source in low-prevalence countries such as Italy. The second panel included 19 isolates belonging to three groups obtained from different herds where epidemiological links were established. Spoligotyping patterns were identical within each group of two panels. Among the 24 loci examined, a total of eight single-locus variants (SLVs) were detected. Four SLVs (VNTR 2163b, VNTR 4052, VNTR 3232, and VNTR 3336) were found in 4 groups of 26 in the first panel, whereas two SLVs were found in two clusters of the second panel: ETR-A and VNTR 2163a in two independent isolates of the first cluster and VNTR 2163b and VNTR 3336 in two different isolates of the second cluster. DISCUSSION Strain genotyping is considered a valuable tool for epidemiological tracing of TB to identify infection sources and routes of transmission. In addition, a combination with spatial data highlights the geographical distribution of the M. bovis strains under study and fosters the study of population genetics. In particular, spoligotyping data have been used to study the structure and diversity of M. bovis populations in France (12) and in the British Isles (34), leading to a better knowledge of the history and distribution of TB in these countries. The Italian situation can be considered intermediate between the British Isles and France, with a predominant spoligotype combined with a considerable number of spoligotype patterns. SB0120 represents 54% of the Italian isolates, similar to the percentage of the predominant spoligotype (SB0140) in Ireland (52%) and Northern Ireland (66%) (34). SB0120 is also the most common spoligotype in France and the second most common one in Spain (6) and is not present United Kingdom or Ireland (34). Similarly, SB0140 is present at a very low level in France and Italy. Although SB0120, together with three other spoligotypes, represents more than 70% of all Italian isolates, we found 81 further spoligotype patterns, 45% of them being found in one isolate only. Moreover, cluster analysis showed that most spoligotypes were included in one major group of similarity (Fig. 1) and that 24 profiles derived from

6 FIG. 3. Dendrogram and schematic representation of 100 SB0120 M. bovis strains typed for 24 MIRU-VNTR loci. The panel was differentiated into 89 different profiles with 9 clusters only. The dendrogram shows genetic distances and was generated as described in Materials and Methods. *, epidemiologically linked isolates. 641

7 642 BONIOTTI ET AL. J. CLIN. MICROBIOL. TABLE 3. Allelic diversity at each locus among the 100 SB0120 panel Locus SB0120 by the loss of one spacer only. In conclusion, we might postulate that SB0120 was the founder strain in Italy and that diversification of the other spoligotype patterns occurred later. An important contribution to variability might be the introduction of foreign strains through the considerable import of dairy and fattening cattle, mainly from Central Europe. Alternatively, SB0120 could theoretically define a specific strain lineage with increased ability to escape from skin tests (and the accompanying slaughter protocol), a hypothesis that can now be investigated with new advanced genomic techniques. Moreover, in the United Kingdom and Ireland M. bovis No. of isolates with MIRU-VNTR allele: MIRU 4/ETR-D MIRU MIRU ETR-C MIRU MIRU MIRU MIRU QUB18/VNTR QUB QUB5/MIRU MIRU MIRU MIRU 31/ETR-E QUB1895/VNTR QUB QUB11b QUB MIRU QUB11a ETR-A VNTR VNTR ETR-B TABLE 4. Number of different profiles and genotypic diversity among the 100 SB0120 panel generated by the 24 MIRU-VNTR or different subsets of MIRU-VNTR Typing loci No. of loci No. of genotypes Genotypic diversity MIRU-VNTR loci ETR-A, ETR-B, ETR-E, QUB11a, QUB11b, QUB15, QUB23, QUB26, MIRU 26, MIRU 40, VNTR 1895, VNTR 3232, VNTR 3336 ETR-A, ETR-B, ETR-E, QUB11a, QUB11b, QUB15, QUB23, QUB26, MIRU 26, VNTR 1895, VNTR 3232, VNTR 3336 ETR-A, ETR-B, ETR-E, QUB11a, QUB11b, QUB15, QUB26, MIRU 26, VNTR 1895, VNTR 3232, VNTR 3336 ETR-A, ETR-B, QUB11a, QUB11b, QUB15, QUB26, MIRU26, VNTR 3232, VNTR Allelic diversity (h) strains appear to cluster in defined geographical areas. This suggests that sources of infection are stable and local, while in Italy we could not find any evident territorial clustering, based on spoligotypes or ETR-A to -E types (data not shown), the only exception being the M. caprae strains, mostly present in northeast Italy. This geographical localization is consistent with the frequent trade of animals from countries such as Austria and Germany, where this strain is widespread (28). In Italy M. caprae is present in 17 genotypes (spoligotype/etrs), some of them never reported in central Europe (28) or Spain (5, 6). Few M. caprae strains are present in central and southern Italy, in some cases with unique genetic patterns. Unfortunately, the lack of data coming from southern Italy does not allow us to grasp the real distribution of M. caprae isolates in Italy. Interestingly, five spoligotypes were characterized by the presence of spacer 14, which is rarely found in the M. caprae strains isolated in central Europe, France, and Spain (5, 6, 9, 12, 28). In some strains, the absence of spacer 14 could be due to technical problems of the spoligotype filter, as demonstrated by the different results obtained in two independent laboratories (W. M. Prodinger, unpublished data). The routine application of spoligotyping and ETR-A to -E typing generated a genotype data collection that is currently used successfully to trace back the infection chains, contiguous herd contacts, or reinfection within the same herd. However, when the M. bovis specific pattern is characterized by the combination of SB0120 with one of the most frequent ETR-A to -E type, more markers are needed. Among the 24 MIRU-VNTR loci tested on 100 SB0120 isolates, the maximal resolution was achieved using a subset of 13 loci (ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336). For local application, not all of the 13 loci are always re-

8 VOL. 47, 2009 MYCOBACTERIUM BOVIS POPULATION IN ITALY 643 quired to genotype all of the isolates in any given situation. A restricted set of loci among the 13 could be applied, along with spoligotyping as a first characterization step of all of the isolates and, subsequently, a second locus group could be used, depending on the genotype, as well as on the need to improve epidemiological investigations. The 100 SB0120 panel is not representative of the genetic complexity of the Italian M. bovis population and the allelic diversity index of individual MIRU-VNTR loci obtained with this panel can be different from that of a population-based evaluation. For example, the allelic diversity of ETR-E varies greatly depending on the population considered, i.e., whole population (0.51), the 400 SB0120 strains isolated in Italy (0.47) or the 100 SB0120 panel (0.25). Therefore, the selection of the studied panel is not representative of the ETR-E allele distribution in the Italian population. Allelic diversity for ETR-B is less variable: values are high in the whole population (0.68), in the 400 SB0120 strains (0.64), and even in the 100 SB0120 selected panel (0.64). Moreover, a locus could have a linkage disequilibrium with SB0120, but it could be very useful in the case of other spoligotypes. On the contrary, the VNTR 3336 showed high allelic diversity with this panel but displays no allelic diversity with M. caprae strains, which always showed two repeats in this locus. Despite these limitations, it is remarkable that some loci systematically emerge among the most discriminatory markers across distinct bacterial populations. Comparison of our results to those of other studies on optimization of genotyping on isolates from Irish, Chadian, and Belgian cattle (1, 13, 30) shows that ETR-A, ETR-B, MIRU 26, and VNTR 2163a, 2163b, 4052, 3232, and 3336 are also the most discriminating MIRU-VNTR loci selected in these studies. However, a global marker evaluation should also consider the reproducibility of the amplification product and the stability of the locus itself. Although reproducibility can be assessed only by a ring trial study, stability could be tested on 26 groups of isolates from different animals of the same herd and on three groups of epidemiologically linked outbreaks. In the case of the first set of isolates, we assume that an outbreak in a farm is most often caused by a single source in low-prevalence countries such as Italy. In fact, MIRU-VNTR genotypes were fully conserved within most groups. Four groups of this panel showed SLVs (loci VNTR 2163b, VNTR 4052, VNTR 3232, and VNTR 3336), suggesting the presence of clonal variants. Two clusters of related outbreaks showed two SLVs, each involving ETR-A, VNTR 2163a in the first cluster and VNTR 2163b and VNTR 3336 in the second one. The two variant strains of the first cluster were consistent with an independent TB introduction. On the contrary, in the second cluster all of the epidemiological information pointed to a single source of infection. Therefore, the presence of genotypic heterogeneity in the studied panels suggests a more rapid evolution of markers VNTR 2163b, VNTR 3336, VNTR 3232, and VNTR Moreover, a source of variation might be the reliability of locus amplification. In our hands, VNTR 3336, VNTR 3232 and, to a lesser extent, VNTR 1895 and VNTR 4056 needed repeated PCR assays in order to obtain a clear interpretable amplification product. In particular, VNTR 3336 is already reported as a hypervariable and inconsistent marker (1, 19, 20). In conclusion, the most discriminative and stable loci identified in this study and in previous studies (1, 13, 30) are ETR-A, ETA-B, MIRU 26, and VNTR 2163a. This group can represent the first core set for M. bovis genotyping in different countries. Further studies are needed for a better evaluation of other eligible loci such as VNTR 3232, 2163b, and Many efforts have been made to establish a reference method for a high-resolution genotyping of Mycobacterium tuberculosis strains (2, 3, 26, 35) that could be used for epidemiological and phylogenetic studies and, recently, a standardized MIRU-VNTR set of 15 or 24 loci optionally combined with spoligotyping has been proposed (2). Fewer attempts have been made to find an optimized genotyping method for M. bovis and M. caprae strains, and probably the M. bovis populations in the different countries are too diverse to achieve a consensus set of markers useful in any situation. Recent publications show that historical and economical differences, the application of eradication programs, the presence of wildlife reservoirs, and control measures for other infection diseases can influence the evolution of M. bovis populations and lead to different outcomes (4, 8, 18, 24, 27). However, in a not too distant future genotyping data should be available and comparable at an international level in order to share information and globally understand the spatiotemporal distribution of M. bovis strains. For the comparison of the typed strains, a possible scenario might be a two-level genotyping analysis including a first consensus set of VNTR markers applied universally and a second set of markers that needs to be empirically determined, depending on the prevalent strains in a given country. ACKNOWLEDGMENTS We thank A. Mangeli, L. Tonoli, A. Moneta, and A. Scalvenzi, for excellent technical assistance and D. Avisani, V. Bonazza, and C. Nassuato for epidemiological inquires. We are also grateful to M. Amadori for critical reading of the manuscript. We thank A. Di Sarno, M. Cagiola, and P. Mazzone for providing M. bovis strains coming from the Campania and Umbria-Marche regions, respectively. This study was supported by Italian Ministry of Health (projects PRC and PRC ). REFERENCES 1. Allix, C., K. Walravens, C. Saegerman, J. Godfroid, P. Supply, and M. Fauville-Dufaux Evaluation of the epidemiological relevance of variable-number-tandem repeat genotyping of Mycobacterium bovis and comparison of the method with IS6110 restriction fragment length polymorphism analysis and spoligotyping. J. Clin. Microbiol. 44: Allix-Béguec, C., M. Fauville-Dufaux, and P. Supply Three-year population-based evaluation of standardized mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 46: Alonso-Rodriguez, N., M. Martinez-Lirola, M. Herranz, M. Sanchez-Benitez, P. Barroso, E. Bouza, D. Garcia de Viedma, et al Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies. BMC Microbiol. 8: Ameni, G., A. Aseffa, A. Sirak, H. Engers, D. B. Young, G. R. Hewinson, M. H. Vordermeier, and S. V. Gordon Effect of skin testing and segregation on the incidence of bovine tuberculosis, and molecular typing of Mycobacterium bovis in Ethiopia. Vet. Rec. 161: Aranaz, A., L. de Juan, N. Montero, C. Sanchez, M. Galka, C. Delso, J. Alvarez, B. Romero, J. Bezos, A. I. Vela, V. Briones, A. Mateos, and L. Dominguez Bovine tuberculosis Mycobacterium bovis in wildlife in Spain. J. Clin. Microbiol. 42: Aranaz, A., E. Liebana, A. Mateos, L. Dominguez, D. Vidal, M. Domingo, O. Gonzolez, E. F. Rodriguez-Ferri, A. E. Bunschoten, J. D. Van Embden, and D. Cousins Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis. J. Clin. Microbiol. 34: Cousins, D., S. Williams, E. Liebana, A. Aranaz, A. Bunschoten, J. Van

9 644 BONIOTTI ET AL. J. CLIN. MICROBIOL. Embden, and T. Ellis Evaluation of four DNA typing techniques in epidemiological investigations of bovine tuberculosis. J. Clin. Microbiol. 36: Duarte, E. L., M. Domingos, A. Amado, and A. Botelho Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates. Vet. Microbiol. 25: Erler, W., G. Martin, K. Sachse, L. Naumann, D. Kahlau, J. Beer, M. Bartos, G. Nagy, Z. Cvetnic, M. Zolnir-Dovc, and I. Pavlik Molecular fingerprinting of Mycobacterium bovis subsp. caprae isolates from central Europe. J. Clin. Microbiol. 42: Frothingham, R., and W. A. Meeker-O Connell Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem repeats. Microbiology 144: Haddad, N., M. Masselot, and B. Durant Molecular differentiation of Mycobacterium bovis isolates: review of techniques and applications. Res. Vet. Sci. 76: Haddad, N., A. Ostyn, C. Karoui, M. Masselot, M. F. Threl, S. L. Hughes, J. Inwald, R. G. Hewinson, and B. Durand Spoligotype diversity of Mycobacterium bovis strains isolated in France from 1979 to J. Clin. Microbiol. 39: Hilty, M., C. Digimbaye, E. Schelling, F. Baggi, M. Tanner, and J. Zinsstag Evaluation of the discriminatory power of variable number of tandem repeat (VNTR) typing of Mycobacterium bovis strains. Vet. Microbiol. 109: Huard, R. C., L. C. de Oliveira Lazzarini, W. Ray Butler, D. van Soolingen, and J. L. Ho PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions. J. Clin. Microbiol. 41: Hunter, P. R., and M. A. Gaston Numerical index of the discriminatory ability of typing systems: an application of Simpson s index of diversity. J. Clin. Microbiol. 26: Kamerbeek, J., L. Schouls, A. Kolk, M. van Agterveld, D. van Soolingen, S. Kuijper, A. Bunschoten, H. Molhuizen, R. Shaw, M. Goyal, and J. van Embden Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35: Kasai, H., T. Ezaki, and S. Harayama Differentiation of phylogenetically related slowly growing Mycobacteria by their gyrb sequences. J. Clin. Microbiol. 38: Kazwala, R. R., L. J. M. Kusiluka, K. Sinclair, J. M. Sharp, and C. J. Daborn The molecular epidemiology of Mycobacterium bovis infections in Tanzania. Vet. Microbiol. 112: Kremer, K., C. Arnold, A. Cataldi, M. C. Gutierrez, W. H. Haas, S. Panaiotov, R. A. Skuce, P. Supply, A. G. M. van der Zanden, and D. van Soolingen Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains. J. Clin. Microbiol. 43: Kremer, K., D. van Soolingen, R. Frothingham, W. H. Haas, P. W. M. Hermans, C. Martin, P. Palittapongarnpim, B. B. Plykaytis, L. W. Riley, M. A. Yakrus, J. M. Musser, and J. D. A. van Embden Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J. Clin. Microbiol. 37: Kulski, J. K., C. Khinsoe, T. Pryce, and K. Christiansen use of a multiplex PCR to detect and identify Mycobacterium avium and M. intracellulare in blood culture fluids of AIDS patients. J. Clin. Microbiol. 33: LeFleche, P., M. Fabre, F. Denoend, J. L. Koeck, and G. Vergnaud High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol. 2: Metchock, B. G., F. S. Nolte, and R. J. Wallace Mycobacterium, p In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. ASM Press, Washington, DC. 24. Michel, A. L., T. M. Hlokwe, M. L. Coetzee, L. Marè, L. Connoway, V. P. M. G. Rutten, and K. Kremer High Mycobacterium bovis genetic diversity in a low prevalence setting. Vet. Microbiol. 126: Niemann, S., D. Harmsen, S. Rüsch-Gerdes, and E. Richter Differentiation of Mycobacterium tuberculosis complex isolates by gyrb DNA sequence polymorphism analysis. J. Clin. Microbiol. 38: Oelemann, M. C., R. Diel, V. Vatin, W. Haas., S. Rüsch-Gerdes, C. Locht, S. Niemann, and P. Supply Assessment of an optimized mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis. J. Clin. Microbiol. 45: Oloya, J., R. Kazwala, A. Lund, J. Opuda-Asibo, B. Demelash, E. Skjerve, T. B. Johansen, and B. Djønne Characterisation of mycobacteria isolated from slaughter cattle in pastoral regions of Uganda. BMC Microbiol. 7: Prodinger, W. M., A. Brandstatter, L. Naumann, M. Pacciarini, T. Kubica, M. L. Boschiroli, A. Aranaz, G. Nagy, Z. Cvetnic, M. Ocepek, A. Skrypnyk, W. Erler, S. Niemann, I. Pavlik, and I. Moser Characterization of Mycobacterium caprae isolates from Europe by mycobacterial interspersed repetitive unit genotyping. J. Clin. Microbiol. 43: Roring, S., A. Scott, D. Brittain, I. Walker, G. Hewinson, S. Neill, and R. Skuce Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping. J. Clin. Microbiol. 40: Roring, S., A. N. Scott, R. Glyn Hewinson, S. D. Neill, and R. A. Skuce Evaluation of variable number tandem repeat (VNTR) loci in molecular of Mycobacterium bovis isolates from Ireland. Vet. Microbiol. 101: Selander, R. K., D. A. Caugant, H. Ochman, J. M. Musser, M. N. Gilmour, and T. S. Whittan Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 51: Skuce, R. A., T. P. McCorry, J. F. McCarroll, S. M. Roring, A. N. Scott, D. Brittain, S. L. Hughes, R. G. Hewinson, and S. D. Neill Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology 148: Skuce, R. A., S. W. McDowell, T. R. Mallon, B. Luke, E. L. Breadon, P. L. Lagan, C. M. McCormick, S. H. McBride, and J. M. Pollock Discrimination of isolates of Mycobacterium bovis in Northern Ireland on the basis of variable numbers of tandem repeats (VNTRs). Vet. Rec. 157: Smith, N. H., S. V. Gordon, R. de la Rua-Domenech, R. S. Clifton-Hadley, and R. G. Hewinson Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis. Nat. Rev. Microbiol. 4: Supply, P., C. Allix, S. Lesjean, M. Cardoso-Oelemann, S. Rüsch-Gerdes, E. Willery, E. Savine, P. de Haas, H. van Deutekom, S. Roring, P. Bifani, N. Kurepina, B. Kreiswirth, C. Sola, N. Rastogi, V. Vatin, M. C. Gutierrez, M. Fauville, S. Niemann, R. Skuce, K. Kremer, C. Locht, and D. van Soolingen Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis. J. Clin. Microbiol. 44: Supply, P., E. Mazars, S. Lesjean, V. Vincent, B. Gicquel, and C. Locht Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36: