Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus

Molecular and Biochemical Parasitology 110 (2000) 69 – 77 www.elsevier.com/locate/parasitology

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus  Myrthe Otsen a, Martijn E. Plas a, Johannes A. Lenstra a, Marleen H. Roos b, Ruurdtje Hoekstra b,* a

Faculty of Veterinary Medicine, Institute of Infectious Diseases and Immunology, Utrecht Uni6ersity, Utrecht, The Netherlands b Department of Molecular Recognition, Institute for Animal Science and Health (ID-Lelystad), Lelystad, The Netherlands Received 22 October 1999; received in revised form 29 March 2000; accepted 29 April 2000

Abstract The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2 – 10 tandems CA/GT repeats that are interrupted by sequences of 1–10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Nematode; Microsatellite; Drug resistance; HcREP1; Haemonchus contortus; Population

1. Introduction Abbre6iations: PCR, polymerase chain reaction; PIC, polymorphic information content; Tann, annealing temperature.  Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™, and DDJB databases under accession numbers AF178463-AF178492 * Corresponding author. Present address: Department of Experimental Surgery, Academic Medical Centre, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.: + 31-20-5665570; fax: +31-20-6976621. E-mail address: [email protected] (R. Hoekstra).

Infections by gastrointestinal parasitic nematodes cause considerable human and animal health problems [1–3]. In western countries problems mainly consist of widely spread infections in domestic livestock. Nematode infections are generally treated with drugs. However, this often leads to a selection of resistant genotypes from the genetically heterogeneous nematode populations [4–6]. Consequently, drug resistance has become

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a world-wide problem [7,8]. An understanding of the dynamics of population genetics as modulated by selective pressure is essential for developing effective treatments and monitoring their impact. Genetic markers with good discriminating power are required to study the genetic mechanisms of drug-resistance and to monitor or characterise populations with known susceptibility or acquired resistance to the different types of drugs. Microsatellite markers (or simple sequence repeats, SSR) are short, tandem repeats of di-, tri- or tetra-nucleotide motifs, which occur abundantly and randomly distributed over eukaryotic genomes [9 – 11]. Because microsatellites are polymorphic and easy to type by polymerase chain reaction (PCR), these repeats have within several years become the markers of choice for most mammalian genomes. Despite their popularity in higher eukaryotes, microsatellite markers have not been widely used in nematode parasites. So far, eight microsatellites were cloned and sequenced from Strongyloides ratti, but no amplification from single worms was achieved [12]. On the other hand, an imperfect compound microsatellite in the large subunit of the rDNA of Trichinella pseudospiralis was variable within and between different isolates [13]. Six polymorphic CA/GT microsatellites were characterised from the nematode Haemonchus contortus, a strongylid gastrointestinal parasitic nematode of sheep and goats [14]. Several polymorphic or monomorphic microsatellites were adjacent to the HcREP1 repeat, a tandem repeat of a 128 bp unit. These results show that microsatellites are genetic markers suitable for genotyping nematode species. For further genetic research on H. contortus, we characterised 30 new microsatellite markers and describe their application to the monitoring of experimental populations.

2. Materials and methods

2.1. Parasite populations, culti6ation and DNA isolation The benzimidazole resistant population RU, from Utrecht, the Netherlands, and the susceptible

field isolate SHS from Zimbabwe was used for the construction of genomic libraries [15,16]. Genotypings were carried out with the ISE, IRE, CAVR and RHS6 populations. ISE, susceptible to all drugs was inbred to 90% from the British susceptible population SE and IRE was inbred also to 90% from the benzimidazole resistant population RE4, which originates from population SE [15,17]. RHS6 is a levamisole resistant population from Zimbabwe [18] and CAVR is an ivermectin resistant population from Australia [19]. Nematode populations were maintained as described [15]. DNA was isolated from individual worms or from pooled L3 larvae as described previously [15].

2.2. Isolation and characterisation of microsatellite sequences Genomic DNA of pooled L3 larvae from H. contortus population RU was digested with TaqI or with the combinations HindIII with EcoRI or EcoRI with PstI, respectively. DNA from the SHS population was digested with Sau3AI [20]. Fragments were directly ligated into linearised pUC18 vector [21] and the constructs were transformed into Escherichia coli by electroporation. (CA)25, (CT)25 and (TA)25 oligonucleotides were synthesised by Pharmacia and labelled at the 5%-terminus by T4 polynucleotidekinase (Pharmacia) and [g32P]-ATP. Colony lifts were fixed to Hybond-N+ membrane (Amersham) and hybridised to the oligonucleotide probes at 48°C overnight in 6× SSPE (20× SSPE, 3 M NaCl; 0.2 M Na H2PO4; 0.02 M Na-EDTA), 5×Denhardt solution (100× Dendardt, 2% (w/v) bovine serum albumin; 2% (w/v) polyvinylpyrolidone; 2% (w/v) ficoll 400), 0.5% sodium dodecyl sulphate (SDS) and 0.1 mg ml − 1 denatured herring sperm DNA. Colony lifts were washed for 15 min in 5×SSPE, 0.1% SDS and 10 min in 2× SSPE, 0.1% SDS at 48°C. Colonies were further tested by growing overnight cultures, phenol/chloroform extraction, RNase treatment of 10 ml of extract, agarose gel electrophoresis, Southern blotting and hybridisation of the blots to the same probes. Inserts from positive colonies were sequenced with vector-specific primers on an ALF automatic sequence apparatus (Pharmacia) by following the instructions of the manufacturers. The

M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

microsatellites that are listed in Table 1 were found in clones originating from the following digests, EcoRI/HindIII (clones Hcms11, 15 – 17, 19 and 20); Sau3AI (Hcms18); TaqI (Hcms21 – 40); and EcoRI/PstI (Hcms41 – 43).

2.3. Microsatellite typing Primer pairs were designed from the complementary sequences flanking the dinucleotide repeats (Table 1). For six microsatellites one of the primers was a generic HcREP1 primer, complementary to nucleotides 5 – 27 of the HcREP1 consensus, 5%-ACAGGAGTTATGAATTTCCGG-3% [14]. One primer per set was endlabelled using T4 polynucleotidekinase (Pharmacia) and [g32P]-ATP. PCR was performed in a 10 ml volume containing about 5 ng genomic H. contortus DNA, 1.2 pmol of each primer, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 0.01% gelatine, 0.2 mM NTPs, and 0.2 U Taq polymerase (Promega) or Amplitaq Gold (Perkin Elmer). With the latter enzyme, the cycling was preceded by an incubation at 95°C for 10 min. After 40 cycles of 15 s at 94°C, 30 s at the indicated annealing temperature (Tann, Table 1), and 30 s at 72°C in a Perkin Elmer 9600 thermocycler, PCR products were separated on a 6% denaturing polyacrylamide gel [20]. Polymorphic information content (PIC) values were based on genotypings of eight individual worms per population and calculated as described [22]. Statistical significance of differences in number of alleles was tested by the Student’s t-test using the SPSS program package.

3. Results

3.1. Isolation sequences

of

H.

contortus

microsatellite

Four genomic libraries of H. contortus were hybridised with labelled (CA)25, (CT)25 and (TA)25 oligomers. No hybridisations to the (CT)25 and the (TA)25 probes could be detected. However, as found previously [14], the (CA)25

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probe hybridised to several clones. Southern blotting of genomic DNA confirmed the abundance of CA/GT-rich relative to TA/AT and CT/GA-rich regions (results not shown). We obtained 118 positive clones, which were all unique. The lengths of the inserts were in the range 300–700 bp. Sequencing revealed that several contained a CA/GT-rich region rather than a CA/GT-repeat, while in other clones the repeats were too close to the ends of the insert for the design of PCR primers. Fifty-nine sequences remained for further analysis. Like the 12 CA/GT microsatellites of H. contortus reported earlier [14] all microsatellites were of the imperfect type [23], with one or more nucleotides interrupting the CA/GT repeats. The total number of repeated CA/GT elements in these microsatellites varied from eight up to over 100 (average 24914) and were distributed over 2–24 (average 5.69 2.9) tandem CA/GT repeats. These repeats were interrupted by non-repeated sequences of up to 43 bp. If a single mutation appeared to have interrupted a longer repeat, resulting in an imperfect repeat [23] as in TGTGTATGTGT, the 86% of the mutations was in a G/C bp, which was mostly replaced by A/T or T/A. A relative high mutation rate in G/C bp in the microsatellite was confirmed by the observation that 66% of the ends of the CA/GT repeats were T/A rather than G/C bp. In several cases, the sequence suggested duplication of segments as GTGTGTC or CACACATACT. The average length of perfect repeats was 4.09 1.1 CA/GT motifs, while the longest CA/GT stretch within a microsatellite varied from 4 to 90 bp with an average length of 16.4 bp. Although we did not isolate TA-positive clones, we found in some of the CA/GT regions TA/AT stretches of up to five dinucleotide motifs. Twenty-six (44%) of the 59 microsatellite sequences were associated with the 128 bp HcREP1 direct repeat [14]. Association with HcREP1 was not clearly correlated with the length of the imperfect microsatellite sequences or the perfect CA/GT stretches within.

M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

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Table 1 Data on 30 microsatellite loci Primer down (5%–3%)

Length (bp)a

Micro

HcREP1

Accession number

Primer up (5%–3%)

Hcms11

−

AF178463

Hcms15

−

AF178464

Hcms16

−

AF178465

Hcms17

+

AF178466

Hcms18

−

AF178467

Hcms19

−

AF178468

Hcms20

−

AF178469

Hcms21

+

AF178470

Hcms22

−

AF178471

Hcms23

−

AF178472

Hcms24

+

AF178473

Hcms25

+

AF178474

Hcms26

+

AF178475

Hcms27

−

AF178476

Hcms28

−

AF178477

Hcms29

+

AF178478

Hcms30

+

AF178479

Hcms31

+

AF178480

Hcms32

+

AF178481

Hcms33

−

AF178482

Hcms34

−

AF178483

Hcms35

+

AF178484

Hcms36

−

AF178485

Hcms37

−

AF178486

Hcms38

n.d.

AF178487

Hcms39

+

AF178488

CTTGTATCACAATCCT CGAAACATACTGAGCAAT 202 ACTACC GAGA GGGAGGTGTAAAGGC GCCGCCTGAAGCTCTTAC 285 TCAACTG AAAT CGAAAAGTACACAAAACATCATAATATGGCTGAG 255 TGACAA AAAG CTAGGGTAGGGAATG HcREP 228 GGAGAG CACACTGAGATACCAC GTGTGTCTCAGTGTGTGT 322 TACCGT CCCA TCTCTCGATACGTCAA GCTGATGCTACTTATCTA 238 GTTTGG TCCC GACTCTGGAAGTAAGCGCTTTCTAGCGGCACATA 232 GTGGGT CAAC CAAACAGACAGACAG CTGCATTCCTGCCAAATT 370 GTTGAGC AGAG CGGCTCCAATTCTGGC TTACGAGCGAGTGCGGCA 163 ATGT TC GTGATTGCCATATGTC ACACTTCGGCTCCAAGGA 173 TTCGG GAAA TAGAACCTTCATAGAG HcREP 178 TGTGC HcREP GCTTCAGTTTGAATTGCT 209 TCCC CACTAACAAGCTAGGA GTACTCCTACCTAGTTTG 162 TTATGG AACG ACATAAATCTAGGTAG ACAGAAGAACGATCAGA 346 GGTAGG ATCTC AGTGTGGAGATGAGA CCGACTAATCACTTCTTG 185 GAGAGCA TTTG GTAGCACAACCCACAC TAAGTGTTTCCATCCTGC 209 CCTAGT TCCC GGAAATAACGCCATCT TTTGCTACCTGTCATAGA 174 CATCCT TCGC AAACGCTCTAAGTCTA CTTAAAGGCACAGCTCAC 274 CGGGAA GATA CCTTGTAACCCAGTAG CCTGCCGTAACTGTTCCC 264 AGAAGC ATGT ATAGCGGTTCGGAGG CCCCGTCAAATAAAAGGC 225 GGTTTC TAGA CATAAGTGTGCGTATT TGTTGGGAACCCTTCTTC 270 TACGTG ATCA TTCGTACCAATCTTTG HcREP 194 TCCCTA GCATAGCGGCAAGGA CATGACGTACTCTGGTTG 151 CGTATGG TTCG CGAAACGTTGGCTGTA GACAAGAAGTTCCGGTTG 131 ATCGGT GATA ATTGAGGGTCCATCCC AGTCCTGCCGCCTATCGT 247 GGAGGT CACT GCGTAGCCCAGTATAA HcREP 188 CGAATG

Tann (°C)b

45* 58* 55 55 50 50* 58* 45* 60 55 60 60 55 60 55 58* 53* 55* 50* 60* 60* 67 60 67 58* 60

M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

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Table 1 (Continued) HcREP1

Accession number

Primer up (5%–3%)

Hcms40

−

AF178489

Hcms41

−

AF178490

Hcms42

n.d.

AF178491

Hcms43

+

AF178492

TCCCATCCAGCCATCT TGAGTGAAGGCGAGCCG 192 ATCTCT TATGA ACGCCCAAGGTTCAAG GACAGCCGTTTATGCCAC 234 AGAACA AACT GGAAGGATGGTTTGA AAACTCCAGGTCCATGAG 356 AGCTGGT AACA ATCAACGGGACGACA AACGCTTTGGGTCCACTG 164 GGTGAGA AAAC

a b

Primer down (5%–3%)

Length (bp)a

Micro

Tann (°C)b

58* 60* 60 55*

The length of the PCR product with as template the cloned DNA. A Tann with asterisk indicates that Amplitaq Gold (Perkin Elmer) was required.

3.2. Microsatellites as genetic markers

Fig. 1. Details of the H. contortus microsatellites. The total number of CA/GT motifs of a microsatellite, the average length of the CA/GT stretch in a microsatellite and the average length of the longest perfect CA/GT stretch in a microsatellite for highly polymorphic markers (PIC \ 0.5) and moderately polymorphic markers (PIC B 0.5).

Fig. 2. Number of microsatellite alleles for the four populations, averaged over 30 microsatellites and eight individuals.

PCR primers were designed on the basis of flanking sequences of the 59 microsatellites and were tested on samples from the ISE, IRE, RHS6 and CAVR populations. Six microsatellites that are associated with the HcREP1 repeat were amplified by using a generic HcREP1 primer (Table 1). This primer was designed on the basis of an alignment of HcREP1 sequences, which revealed that the conservation of consecutive HcREP1 elements decreases with the distance from the CA/ GT [14]. Thus, under the PCR conditions used the shortest possible product was expected to be predominantly amplified. For thirty microsatellites a product with the expected size was obtained in at least one of the populations (Table 2). Six microsatellites gave null-alleles that were specific for one, two or three of the tested populations. Twenty-seven microsatellites were polymorphic and provided genetic markers with up to seven alleles, including null-alleles, and an average polymorphic information content of 0.469 0.21. As shown in Fig. 1, there was no clear relation between the total number of CA/GT elements, the average length of the perfect stretches within the imperfect microsatellite, or the longest perfect CA/ GT stretch within the imperfect microsatellite and the degree of polymorphism (PIC values) of these microsatellites. However, HcREP1-associated microsatellites had a significantly (PB 0.05) lower number of alleles per marker (2.89 0.9) than the

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Table 2 Characterisation of 30 microsatellite loci in four H. contortus populations Micro

a

− − − + − − − + − − + + + − − + + + + − − y + − n.d. + − − n.d. +

Number of allelesa

5 3 1 5 6 6 5 3 5 4 2 3 3 4 7 3 3 3 2 3 2 3 4 3 5 3 3 2 1 1

PICgenerala

0,6936 0,5645 0 0,5476 0,7388 0,6558 0,6294 0,648 0,4206 0,4136 0,0308 0,5657 0,4904 0,3374 0,5129 0,5859 0,48 0,4896 0,2604 0,5137 0,375 0,5645 0,532 0,5404 0,6987 0,5717 0,5676 0,375 0 0

ISEb

IREb

RHS6b

CAVRb

Alleles

PIC

Alleles

PIC

Alleles

PIC

Alleles

PIC

A A, B A E D C, F A, C A D B, C A C A B C B A, B A A A A A, C D A, B B, C A A, B A A A

0 0,5 0 0 0 0,67 0,43 0 0 0,38 0 0 0 0 0 0 0,43 0 0 0 0 0,5 0 0,24 0,12 0 0,47 0 0 0

A, B, E A, B A A, E E D A 0 D B, C A B B B C A B, C A A C 0 A, B, C B, D A, C 0 A, B B 0 A A

0,32 0,22 0 0,28 0 0 0 0 0 0,3 0 0 0 0 0 0 0,3 0 0 0 0 0,59 0,46 0,5 0 0,49 0 0 0 0

B, C, D A, B, C A B, C, D, E A, C, D, E B, C, E, F B, C, D, E B A, B, C, D A, B, C A, B A, B A, B, C A, B, D A, B, D, E, F, G B, C B, C 0 B A, B, C A A, B A, B A, B, C A, B, C, D A, B, C B, 0 0 A A

0,61 0,59 0 0,7 0,7 0,63 0,74 0 0,68 0,4 0,12 0,22 0,57 0,53 0,81 0,58 0,3 0 0 0,64 0 0,22 0,44 0,56 0,56 0,6 0,12 0 0 0

B, C, E A, C A E B, F A, D A, C 0 D, E B, D A C B C, D B, C, D B, C B A, B A C A A, B B, C, D A, B D A, C 0 0 A A

0,61 0,5 0 0 0,5 0,22 0,3 0 0,38 0,43 0 0 0 0,28 0,57 0,43 0 0,46 0 0 0 0,5 0,64 0,47 0 0,46 0 0 0 0

The number of alleles and PICgeneral indicate the polymorphism of the marker with respect to all four populations. The alleles found for the different populations are indicated in alphabetical letters. \A, indicates the largest allele; B, the second largest; etc. 0, indicates the absence of amplification. The most frequent allele is indicated in bold. b

M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

Hcms11 Hcms15 Hcms16 Hcms17 Hcms18 Hcms19 Hcms20 Hcms21 Hcms22 Hcms23 Hcms24 Hcms25 Hcms26 Hcms27 Hcms28 Hcms29 Hcms30 Hcms31 Hcms32 Hcms33 Hcms34 Hcms35 Hcms36 Hcms37 Hcms38 Hcms39 Hcms40 Hcms41 Hcms42 Hcms43

HcREP1

M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

other microsatellites (3.99 1.6), but the overall PIC values did not differ significantly. PIC values predict the chance that an allele of an individual can be assigned to one of the parents and range from 0 to 1 [22].

3.3. Genotyping of H. contortus populations The four H. contortus populations were characterised with the 30 microsatellites (Table 2). The average numbers of alleles and PIC values are indicated in Fig. 2. Populations ISE and IRE showed little polymorphism within the population. The CAVR population was only slightly more variable than the two inbred populations, but RHS6 appeared to have significantly (P B 0.05) more alleles and higher PIC values than the other three populations. Eighteen of the 30 microsatellites could be used to discriminate completely between two or more populations. The less variable populations ISE, IRE and CAVR could easily be discriminated, since several microsatellite alleles are fixed within these populations. This enabled us to check the genetic purity of an ISE isolate. This inbred line is completely or to a large extent monomorphic for microsatellite Hcms25 (Table 2). After propagation of this inbred line, new alleles appeared in offspring, indicating genetic contamination. By testing two earlier, successive generations (Fig. 3), it became clear when the contamination had taken place and possible sources of contamination could be identified.

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4. Discussion In this study, we present the isolation and characterisation of new microsatellite loci for H. contortus. The screening generated only CA/GT microsatellites, which are also by far the most common type of dinucleotide repeats in mammalian genomes [24]. The sequencing of 59 microsatellites allows a generalisation of the earlier finding that all isolated H. contortus CA/GT microsatellites are complex combinations of CA/GT motifs of varying lengths alternated by other sequences [14]. Since CA/GT repeats were never found in combination with complementary GT repeats on the same DNA strand, the neighbouring CA/GT stretches are likely to have a common origin and may originate from mutations in a longer CA/GT repeat or from sequence duplications. The unusual high proportion of imperfect microsatellites in the H. contortus is not a universal feature of the genomes of nematodes, since a number of perfect microsatellites have been found for Strongyloides ratti [12]. This difference in genetic variability between nematode species has also been observed in mitochondrial DNA and is related with differences in mutation rate, both for mitochondrial and genomic DNA [6]. The substitutional bias towards A/T nucleotides in the CA/GT microsatellites we have detected is also found in mitochondrial DNA sequences of H. contortus [25]. About 44% of the microsatellite loci were associated with the direct repeat HcREP1. We found no correlation between the length of the imperfect CA/GT repeat and HcREP1-association. A conve-

Fig. 3. Alleles of Hcms25 for two subsequent generations of the inbred strain ISE. As shown for four of the 48 tested individuals, the previous ISE generation has only allele C. The next ISE generation has alleles B, C, D and E (PIC=0.70).

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M. Otsen et al. / Molecular and Biochemical Parasitology 110 (2000) 69–77

nient universal HcREP1 primer proved successful in amplifying the HcREP1-associated microsatellites. This facilitates the future typing of HcREP1associated microsatellites significantly, since the number of labelled primers will be reduced to only the HcREP1 primer. The variability of the microsatellites was high, since 27 of the 30 microsatellites, which could be amplified, were polymorphic within and/or between the tested populations. Generally, the information content of mammalian microsatellites is positively correlated with the length of the perfect repeat [26]. However, we did not establish a significant correlation of the PIC value with the length of the microsatellite, the average length of the perfect CA/GT stretches within a microsatellite, or with the length of the longest repeat of a microsatellite. The occurrence of null-alleles may be explained by the variability of the H. contortus genome [6,14,27], which may change the sequence of the primer binding site. We characterised four frequently used populations in research on drug resistance with the 30 microsatellites. ISE and IRE are inbred strains [17] and have the lowest average PIC values. RHS6 was obtained after six consecutive rounds of in vivo selection of worms of the SHS isolate from Zimbabwe [16] for levamisole resistance. The genetic variation of the SHS isolate was higher than the variation of other isolates and [14] was hardly decreased by the selection for levamisole resistance [28]. This agrees with our observation that also RHS6 has significantly more allelic variation than the populations ISE, IRE and CAVR. The high level of differentiation found among the different isolates can be explained by several factors. First, by geographical isolation, which has already been suggested to contribute to the genetic variation between four susceptible outbred populations of H. contortus [14]. Furthermore, selection for resistance to different types of drugs may have caused genetic drift and also selection for specific alleles of loci associated with the different types of resistance. Finally, inbreeding has reduced the heterogeneity markedly and often led to the fixation of specific microsatellite alleles.

Inbred lines or well-defined and selected populations have developed as tools for studying genetic drug resistance. However, propagation of these populations in sheep carries the risk of genetic contamination, which may not be apparent from any change in phenotype. Since such contamination would drastically affect the outcome of an experiment, genetic monitoring of new generations is essential. As shown in Fig. 3, we detected contamination in the inbred line ISE after propagation in sheep. This contamination would have remained undetected without appropriate genetic markers. In addition, these markers will be essential in linkage studies with hybrid offspring of isolates with different drug resistance phenotypes.

Acknowledgements The work was supported by the Netherlands Organisation for the Advancement of Research SLW-STW 790.43.805. We thank J. Tibben for expert technical assistance.

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