Molecular characterization of Mycobacterium avium subsp. paratuberculosis using multi-locus short sequence repeat (MLSSR) and mycobacterial interspersed repetitive units–variable number tandem repeat (MIRU–VNTR) typing methods

Veterinary Microbiology 149 (2011) 482–487

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Molecular characterization of Mycobacterium avium subsp. paratuberculosis using multi-locus short sequence repeat (MLSSR) and mycobacterial interspersed repetitive units–variable number tandem repeat (MIRU–VNTR) typing methods P.E. Douarre a, W. Cashman b, J. Buckley b, A. Coffey a, J. O’Mahony a,* a b

Cork Institute of Technology, Cork, Ireland Veterinary Department, Cork County Council, County Hall, Cork, Ireland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 June 2010 Received in revised form 30 November 2010 Accepted 2 December 2010

The aim of this study was to characterize 38 bovine strains of Mycobacterium avium subsp. paratuberculosis isolated in Ireland using 11 multi locus short sequence repeat (MLSSR) loci and 8 mycobacterial interspersed repetitive units–variable number of tandem repeat (MIRU– VNTR) loci. The discriminatory power of these two typing methods alone and combined was evaluated, as was the epidemiology of the isolates and the genotypes obtained. Using the MIRU–VNTR typing method (8 loci analysed), only 3 subtypes were detected with a discrimination index (DI) of 0.54, but one MIRU–VNTR type has not been identified in other studies. In contrast the MLSSR method (using 11 loci) differentiated the 38 MAP isolates into 18 types with DI of 0.92. Among these 18 types 6 have not been recorded previously. The combination of the 2 methods (MIRU–VNTR and MLSSR) produced 22 distinct genotypes giving a maximal DI of 0.94. According to the allelic diversity, some markers are more polymorphic than others and must be applied in priority for the differentiation of MAP bovine isolates. This is the first report of genotyping data for MAP on the island of Ireland and will be very useful for analysing its epidemiology, transmission and virulence. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Mycobacterium avium subsp. paratuberculosis Short sequence repeat (SSR) Mycobacterial interspersed repetitive units–variable number of tandem repeat (MIRU–VNTR) Ireland

1. Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne’s disease (or paratuberculosis), a chronic gastroenteritis of ruminants. Johne’s disease is prevalent in domestic livestock and is considered a serious animal pathogen. The disease occurs worldwide and causes considerable economic losses to the animal industry (Nielsen and Toft, 2009; Losinger, 2005). MAP is also considered a potential significant public health threat due to its possible association with Crohn’s disease in humans

* Corresponding author at: Cork Institute of Technology, Department of Biology, Rossa Avenue, Bishopstown, Cork, Ireland. Tel.: +353 21 4326833. E-mail address: [email protected] (J. O’Mahony). 0378-1135/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2010.12.001

(Hermon-Taylor, 2009). MAP has a wide host range and the evidence of interspecies has already been demonstrated (Stevenson et al., 2009). Control of paratuberculosis requires a better understanding of the genetic diversity of the pathogen. Strain differentiation by genotyping is useful for studying epidemiology, pathogenesis and interspecies transmission. The use of molecular typing methods has increased in the last 2 decades (Motiwala et al., 2006; Sohal et al., 2010), providing a better depiction of the genetic diversity of MAP. IS900 restriction fragment length polymorphism (RFLP) and pulse field gel electrophoresis (PFGE) are the most widely used (Thibault et al., 2007; Mo¨bius et al., 2008; Sevilla et al., 2007), but are only applicable to cultivable strains (large amount of high quality DNA needed) and require analysis of complex banding pattern. More recently, multilocus short sequence repeat (MLSSR), mycobacterial interspersed repetitive units (MIRU) and

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variable number tandem repeat (VNTR) have been used to differentiate subtypes based on the polymorphism of repetitive elements. The utility of the MLSSR and MIRU– VNTR typing approach for MAP strain differentiation has been evaluated in previous studies (Amonsin et al., 2004; Thibault et al., 2007) and has been shown to be rapid and highly discriminatory. In this study, 38 bovine MAP strains isolated in Ireland were typed using 11 SSR and 8 MIRU– VNTR. The aim of this study was to evaluate the discriminatory power of these 2 typing methods used alone or in combination and evaluate the epidemiology of the genotypes obtained. 2. Materials and methods 2.1. MAP strains A total of 38 M. avium subsp. paratuberculosis isolates were assembled in this study (Supplementary Table 1). The cultures were isolated on Herrold’s egg yolk medium by the centrifugation method according to the protocol of Ristow et al. (2006). One single colony of each individual MAP isolate was subcultured in Middlebrook 7H9 broth supplemented with OADC (Becton D), 2 mg/ml of Mycobactin J and 0.2% glycerol and incubated at 37 8C for 6–10 weeks.

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presence of the MAP bovine specific large sequence polymorphism LSPA20 (Semret et al., 2006). All the PCRs for the identification and the typing of MAP were carried out using the lightCycler 480 (Roche). 2.5. MLSSR typing Eleven short sequence repeat loci were analysed as described by Amonsin et al. (2004), with modifications. The reaction mixture consisted of 2X LightCycler1 480 SYBR Green I Master (Roche) (containing the FastStart Taq DNA Polymerase, reaction buffer, dNTP mix, SYBR Green I dye and MgCl2), 0.5 mM of each primer, 5% dimethyl sulfoxide (SIGMA) and PCR grade water. Sample tubes contained 50 ng of MAP DNA. Controls consisted of reaction mixture alone (negative control) and a positive control containing 1 ml of genomic DNA from MAP (strain 19698). Samples were run according to the following conditions: 1 cycle at 95 8C for 10 min and 35 cycles at 95 8C for 10 s, 60 8C for 10 s, and 72 8C for 10 s. The PCR amplicons were then sequenced by the DNA services at the University of Dundee, Scotland (http:// www.lifesci.dundee.ac.uk/sequencing). The sequence of each SSR locus for all 38 isolates was aligned and the number of short sequence repeat was identified using MegAlign program (DNASTAR Lasergene). MLSSR types were then allocated on the basis of the unique combination of alleles for each locus.

2.2. Epidemiological analysis 2.6. MIRU–VNTR typing All the strains were isolated in Ireland from bovine faecal samples between 2006 and 2008. MAP isolates were recovered from 18 distinct herds from 5 different locations in Ireland. 24 isolates were recovered from 4 herds (H1– H4) whereas the other 14 isolates were individual strains from 14 different herds. The number of isolates, herd identification and location of the herds are shown in Table 1 in Supplementary Material. 2.3. Preparation of mycobacterial DNA Ten ml of MAP culture, propagated as above, was centrifuged at 5000 rpm for 10 min and the pellet was resuspended with 750 ml of fresh GITC lysis buffer (50 mM Tris–HCl, 10 mM EDTA, 2%Triton X-100, 4 M GITC, 0.3 M sodium acetate) (O’Mahony and Hill, 2004) and transferred to a 2 ml screw capped tube containing 100 mg acid washed glass beads (150–212 mm, Sigma). The samples were sheared in a Ribolyser (MagNA Lyser, Roche) for 45 s at 6500 m s1 and then boiled for 5 min. The tubes were then spun at 12,000 rpm and the supernatant was transferred to a fresh tube. The DNA was extracted from the supernatant using a standard phenol–chloroform extraction procedure. The DNA was precipitated with 2 vol of 100% ethanol and 0.5 vol of ammonium acetate (7 M) at 20 8C overnight. After washing and drying, the DNA pellet was resuspended in 50 ml 10 mM TE buffer.

Eight specific MIRU–VNTR markers (292, X3, 25, 47, 3, 7, 10 and 32) were analysed using the same specific primers, annealing temperature and buffer that as previously described (Thibault et al., 2007). The reaction mixture consisted of 2X LightCycler1 480 SYBR Green I Master (Roche) (containing the FastStart Taq DNA Polymerase, reaction buffer, dNTP mix, SYBR Green I dye and MgCl2), 0.5 mM of each primer and PCR grade water. Samples and controls were set up as described above for MLSSR typing. Samples were run according to the following conditions: 1 cycle at 95 8C for 10 min and 35 cycles at 95 8C for 10 s [55– 64 8C]1 for 10 s, and 72 8C for 14 s. To detect differences in repeat numbers, PCR products were analysed by conventional gel analysis (1.5% agarose gel). 2.7. Calculation of discriminatory power The index of discrimination (HGDI) described by Hunter and Gaston (1988) was used as a numerical index for the discriminatory power of MLSSR and MIRU–VNTR typing methods. This index (DI) was calculated using the following equation:

2

3 i X 1 DI ¼ 1  4 n ðn  1Þ5 NðN  1Þ j¼1 j j

2.4. Molecular identification of MAP All 38 cultured MAP isolates were tested by PCR for the presence of the IS900 element (Bull et al., 2003) and the

1 Primers for each individual tandem repeat had specific annealing temperature comprised between 55 and 64 8C (Thibault et al., 2007).

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Only one isolate contained 2 TR at the locus 25 giving a unique type (MIRU–VNTR type 3, 42232228). Due to the rarity of this allele (at locus 25), the PCR product was sequenced to confirm the validity of this result. The allelic diversity was 0.49 for the locus 292 and 0.02 for the locus 25 with an overall DI of 0.54 (Table 2).

where N is the total number of isolates in this study, s is the total number of distinct types, and nj is the number of isolates belonging to the jth type. 3. Results All 38 MAP isolates were positive for the presence of the IS900 element and were characterized as ‘‘Cattle Type’’ after amplification of the MAP bovine specific LSPA20.

3.3. Combination of MLSSR and VNTR typing The combination of the two methods based on the most useful 8 markers (6 SSR and 2 TR) created 22 distinct subtypes. Upon analysis, 6 MLSSR–VNTR types appeared more than once (Supplementary Table 2). Of these, the most prevalent type, MLSSR–MIRU–VNTR 20 was detected in 8 isolates (21%). The addition of 2 polymorphic TR created an extra 4 types (18 MLSSR types and 22 MLSSR + MIRU–VNTR). An overall DI of 0.94 was obtained by using SSR and MIRU–VNTR typing in combination (Table 2). Interestingly, one sample generated a unique MIRU–VNTR type 3 which also generated a unique MLSSR type 7 which thereby validated the consistency of both methods (Table 4).

3.1. MLSSR typing The 11 SSR loci were characterized for all 38 MAP isolates. The analysis identified 18 types among the 38 MAP isolates recovered from the bovine faecal samples (Table 1). Out of the 18 MLSSR types detected, 10 were unique for one isolate (2.6%). The other 8 genotypes where shared among the remaining 28 isolates. The most prevalent type (MLSSR type 4) was identified for 9 isolates which represented 24% of the strains characterized. The loci 1 and 2 were highly polymorphic with four and five different alleles respectively (Supplementary Fig. 1) whereas loci 3, 4, 5, 10 and 11 did not show any polymorphism. The allelic diversity ranged from 0.12 (locus 7) to 0.70 (locus 2) and the overall index of discrimination (DI) of the MLSSR typing was 0.92 (Table 2). Six of the MLSSR types detected in this study (6, 7, 8, 9, 12 and 13) have not been recorded elsewhere to the best of our knowledge.

3.4. Genotyping and epidemiology Two main MIRU–VNTR types were detected across the 18 herds (MIRU–VNTR 1 and 2) accounting for 97.4% of all the isolates. In contrast, 18 MLSSR types were recorded. In some herds a predominant MLSSR type emerged. For example in herd H1 73% were recorded as type 4, whereas in others (H2 and H3) no single dominant type was detected. As the discriminatory power of MIRU–VNTR is less informative, limited epidemiological information was obtained from these results. MLSSR typing on the other hand proved far more useful. Some types such as 2, 4, and 5 were each detected in three unrelated herds. Other types (e.g. 6, 7, 11, 12 and 15) were unique across this study and were only detected once. Collectively, the largest cohort in this study was 13 isolates recovered from 4 herds in the

3.2. MIRU–VNTR typing The analysis of the 8 TR loci identified 3 subtypes among the 38 MAP strains isolated from different locations in Ireland (Table 3). Respectively, MIRU–VNTR type 1 and type 2 were present in 47% and 50% of the MAP isolates whereas MIRU–VNTR type 3 was only characterized for 1 strain (2.6%). Only 2 loci (MIRU 292 and VNTR 25) were polymorphic with two variable alleles (3–4 and 3–2) but the allelic distribution for these markers was not equal. Table 1 MLSSR profiles of 38 MAP bovine strains isolated in Ireland. MLSSR types

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 a

No. of isolates

2 4 2 9 3 1 1 1 3 3 1 1 1 1 1 1 2 1

SSR identified by Amonsin et al., 2004.

%

5.2 10.5 5.2 23.6 7.8 2.6 2.6 2.6 7.8 2.6 2.6 2.6 2.6 2.6 2.6 2.6 5.2 2.6

No. of copies of SSRa 1

2

3

4

5

6

7

8

9

10

11

7 7 7 7 7 8 8 8 7 7 7 7 7 7 7 11 7 10

10 9 11 10 11 >11 11 9 11 >11 >11 10 >11 >11 11 11 11 11

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

4 4 4 4 4 4 4 5 4 4 4 4 5 4 4 5 5 4

5 5 5 5 5 5 6 5 5 5 5 5 5 6 6 5 5 5

4 4 4 4 4 4 4 5 5 4 5 5 5 4 4 5 5 4

4 4 4 5 5 5 5 5 4 5 4 4 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

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Table 2 MLSSR and MIRU–VNTR allelic distribution among MAP isolates and discriminatory power of the two typing methods Locus. No. of isolates with the specified allele 1 MLSSRa 1 2 6 7 8 9 MIRU–VNTRb 292 25 a b c d

2

3

4

33 27 14

1

18 37

5

6

5 35 11 24

7

8

33

3

9

10

11

>11

5

1 12

1 14

7

3

20

Allelic diversityc

Discrimination index (DI)d

0.22 0.70 0.21 0.12 0.40 0.45

0.92

0.49 0.02

0.54

0.94

SSR loci 1, 2, 6, 7, 8 and 9 according to Amonsin et al. (2004). MIRU–VNTR loci 292 and 25 according to Thibault et al. (2007). Allelic distribution was calculated as described by Mazars et al. (2001). DI was calculated using the equation described by Hunter and Gaston (1988).

county of Region 2 (which comprises 11% of the Irish state). When this group was analysed using MIRU–VNTR results the majority (77%) belonged to MIRU–VNTR 1. When the same cohort was analysed using MLSSR typing, 8 MLSSR genotypes were recorded, the most predominant of which was MLSSR type 2 (31%) occurring in three of the herds (Table 4). 4. Discussion MIRU–VNTR types 1 and 2 accounted for 97.4% of all isolates characterized. These 2 types were designated INMV2 and INMV1 by Thibault et al. (2007) and were also the most represented in their study (70% of all the isolates tested). MIRU–VNTR type 3 (which was found in a single isolate here) has not been detected in previous studies. In our collection, only MIRU 292 and VNTR 25 were polymorphic and only one isolate contains 2 tandem repeats for the VNTR 25, which was confirmed by sequencing. This particular allele was also identified for one cattle strain isolated in Germany (Mo¨bius et al., 2008). In this study 18 MLSSR subtypes were identified among the 38 MAP isolates. As seen in previous studies (Thibault et al., 2008; Amonsin et al., 2004) the SSR loci 3, 4, 5, 10 and 11 were not variable for MAP cattle strains. In relation to the allelic diversity, the best SSR markers for this strain collection were 2, 9, 8, 1, 6 and 7 in decreasing order. By using the two most discriminative SSR markers in this study (SSR markers 2 and 9), nine sub-types were detected with a DI of 0.86 which highlights the importance of selecting multiple loci. A potential disadvantage of MLSSR

typing was the need to take account of errors due to strand slippage during either PCR or sequence reactions that may result in an erroneous assignment of genotype. This limitation is particularly directed at the 2 loci (1 and 2) for which there are a high number of G repeats. It is therefore important to sequence the locus at both direction and repeat the sequencing. In this study, in order to avoid ambiguities, we assign all alleles >11 as the same type, as previously described by Thibault et al. (2007). However, two recent studies reported that the four most discriminatory loci in the USA (1, 2, 8 and 9) were stable in 3 MAP strains tested over ten subcultures (Harris et al., 2006) and the two most commonly used SSR loci were invariant in 98 isolates of the bison type from different parts of India (Singh et al., 2009). In general the MIRU–VNTR typing method described here was found to be useful for producing broad epidemiological data especially in terms of establishing the inter-relatedness of strains. For example, within the herd H1 MIRU–VNTR type 2 was found to be predominant (10 isolates out of 11). Furthermore, all isolates from H3 (MIRU–VNTR1) and H4 (MIRU–VNTR 2) shared the same MIRU–VNTR type (n = 5 and n = 2, respectively). When examined for geographic trends this technique proved that within the largest cohort of samples collected (Region 2, n = 13), MIRU–VNTR type 1 (n = 10) was more prevalent than MIRU–VNTR type 2 (n = 3) (Table 4). MLSSR typing proved to be much more extensive and informative epidemiologically, particularly for producing unique genetic fingerprints. For example in herd H3, all 5 isolates were found to be MIRU–VNTR type 1 which suggests that the isolates were genetically identical. When examined using MLSSR

Table 3 MIRU–VNTR profiles of 38 MAP bovine strains isolated in Ireland. MIRU–VNTR types

1 2 3 a b c

No. of isolates

18 19 1

%

47.4 50 2.6

No. of copies of MIRU–VNTRa

INMV profileb

292

X3

25

47

3

7

10

32

3 4 4

2 2 2

3 3 2

3 3 3

2 2 2

2 2 2

2 2 2

8 8 8

MIRU–VNTR described by Thibault et al. (2007). INMV: INRA Nouzilly MIRU–VNTR, Thibault et al. (2007). New INMV profile.

INMV2 INMV1 c

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Table 4 Geographic distribution of MLSSR and MIRU–VNTR types identified for 38 Irish MAP isolates. Location

Herd ID

MLSSR type

MIRU–VNTR type

N

Region 1

HA HD HE HN

Type Type Type Type

3 3 5 17

Type Type Type Type

1 2 2 2

1 1 1 1

Region 2

HB HC H2 H2 H2 H2 H3 H3 H3 H3

Type Type Type Type Type Type Type Type Type Type

1 2 2 10 5 4 2 12 9 13

Type Type Type Type Type Type Type Type Type Type

1 1 1 2 2 1 1 1 1 1

1 1 2 2 1 1 1 1 2 1

Region 3

HF HI HJ HK

Type Type Type Type

6 5 16 9

Type Type Type Type

2 1 1 1

1 1 1 1

Region 4

HM H1 H1 H1 H1

Type Type Type Type Type

1 4 10 12 18

Type Type Type Type Type

1 2 2 1 2

1 8 1 1 1

Region 5

H4 H4

Type 14 Type 15

Type 2 Type 2

1 1

Unknown

HG HH HL

Type 7 Type 8 Type 17

Type 3 Type 1 Type 1

1 1 1

N, number of isolates.

analysis these 5 isolates were classified under 4 distinct MLSSR types (Table 4). Furthermore, upon initial investigation of the Region 5 isolates, both strains were typed as MIRU–VNTR 2. This implies a close link with the other 17 type 2 isolates recovered throughout the country. MLSSR analysis was more discriminative and generated 2 distinct types (14, 15) which were not recorded elsewhere in this study (Table 4) indicating that although these 2 isolates are related to other VNTR type 2 isolates they are not identical (Table 4). Furthermore, although a predominant MLSSR type emerged in herd H1 (type 4), the presence of 3 other MLSSR types within the same herd may reveal the introduction of many infected animals with different genotypes (MLSSR types 10, 11 and 18) within the same farm (Table 4). This highly discriminative epidemiological data could not be picked up by MIRU–VNTR typing alone in this study. This is the first molecular epidemiology study for MAP in Ireland. Despite the limited number of samples available, combined SSR and MIRU–VNTR typing is highly discriminative for Irish MAP isolates. Genotyping data are invaluable when attempting to track the transmission and persistence of a particular genotype nationally and internationally. Both methods produce numerical genotypes and are therefore convenient for online comparisons and phylogenetic analysis which are greatly required in epidemiology studies. The introduction of an internationally standardised genotyping system for MAP is urgently needed.

Acknowledgments This work was financed by a grant from the Department of Agriculture from the Food Institutional Research Measure (06RDCIT414).

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