Pyrosequencing analysis of the beta-tubulin gene in Spanish Teladorsagia circumcincta field isolates

Veterinary Parasitology 184 (2012) 371–376

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Pyrosequencing analysis of the beta-tubulin gene in Spanish Teladorsagia circumcincta field isolates María Martínez-Valladares a,∗ , Alison Donnan b , Peter Geldhof c , Frank Jackson b , Francisco-Antonio Rojo-Vázquez a , Philip Skuce b a b c

Instituto de Ganadería de Monta˜ na (CSIC-ULE), Finca de Marzanas, 24346 Grulleros, León, Spain Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Edinburgh EH26 OPZ, UK Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

a r t i c l e

i n f o

Article history: Received 15 April 2011 Received in revised form 28 August 2011 Accepted 5 September 2011 Keywords: Sheep Anthelmintic resistance Macrocyclic lactones Beta-tubulin Pyrosequencing

a b s t r a c t Benzimidazole (BZ) resistance in gastrointestinal nematodes has been associated with single nucleotide polymorphisms (SNPs) at codons 200, 167 and 198 in the beta-tubulin isotype 1 gene and, recently, these SNPs have also been found in macrocyclic lactone (ML) resistant strains of Haemonchus contortus. On this basis, we have studied the same putative SNPs in Spanish Teladorsagia circumcincta field isolates by pyrosequencing. Single L3 (infective 3rd stage larvae) from five sheep flocks were tested after confirming their BZ susceptibility and degree of ivermectin (IVM) resistance. According to the Faecal Egg Count Reduction Test (FECRT) one flock was classified as IVM susceptible, another one was resistant, and the rest had a suspicion of resistance to IVM. DNA extraction was carried out on 598 single L3 and 56% of these were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187. Results clearly indicate no resistance-associated SNPs were present at any codon, before or after treatment. Therefore, all T. cicumcincta L3 were designated as susceptible homozygous genotypes for all SNPs. The absence of the mutations in these populations would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in Spanish T. circumcincta. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Gastrointestinal nematodes (GIN) are the most common parasites infecting grazing sheep worldwide. Infections cause huge production losses relating to meat and milk yield and, therefore, their control is paramount. Treatment of infected animals with anthelmintic drugs is the most widely used method of control, however, inappropriate and over-use of them has led to the development of anthelmintic resistance (AR) (Wolstenholme et al., 2004).

∗ Corresponding author. Tel.: +34 987317064. E-mail address: [email protected] (M. Martínez-Valladares).

In the Northwest (NW) of Spain, the most prevalent GIN of ovines is Teladorsagia circumcincta, the predominant species present in temperate areas of the world. A study carried out previously during 1999–2003 in this same region (Alvarez-Sánchez et al., 2006), described several instances of AR against benzimidazoles (BZs), macrocyclic lactones (MLs) and imidazothiazoles in flocks infected with T. circumcincta. BZs are one of the most commonly used anthelmintic classes in sheep. Their nematocidal effect is due to the disruption of the tubulin-microtubule equilibrium, necessary for the maintenance of cellular homeostasis in cells. BZ molecules bind to the beta-tubulin preventing the polymerization of alpha and beta-subunits to produce heterodimers (Lacey, 1988). BZ resistance has

0304-4017/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.09.009

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been shown to be associated with single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype 1 gene. This has been reported in all nematode species studied to date (von Samson-Himmelstjerna et al., 2007) and, regarding T. circumcincta, the substitution of a phenylalanine (TTC) by a tyrosine (TAC) at codon 200 (F200Y) has been described in resistant populations (Silvestre and Humbert, 2002; Skuce et al., 2010). Silvestre and Cabaret (2002) also associated the substitution of a phenylalanine by a tyrosine at position 167 (F167Y) with resistant Haemonchus contortus and T. circumcincta populations from naturally infected goats and, more recently, a SNP at codon 198, characterized by a change of glutamate (GAA) to alanine (GCA) (E198A), was shown in BZ resistant H. contortus strains (Ghisi et al., 2007; Rufener et al., 2009). In respect of ivermectin (IVM) resistance, anthelmintic belonging to ML class, Freeman et al. (2003) reported an alteration of the microtubules in the amphid neurons in an IVM resistant strain of H. contortus with a shortening of the dendritic processes, when compared with a susceptible strain. This finding suggested a possible link between IVM resistance and beta-tubulin microtubules since amphidial neurons are formed by bundles of heterodimers of alpha and beta-tubulin subunits. On the other hand, Eng et al. (2006) showed, in a study on isotype 1 beta-tubulin sequence, that IVM appeared to be selecting for heterozygotes at codon 200 (TTC/TAC) in H. contortus, these represent a BZ susceptible phenotype since BZ resistance in trichostrongylid nematodes is thought to be recessive (Elard and Humbert, 1999). Subsequently, Mottier and Prichard (2008) confirmed the previous results after reporting that the frequency, not only of the SNP at position 200 but also at codon 167, was increased in the same IVM resistant and BZ susceptible isolates of H. contortus. They also analyzed the possible mutation at codon 198 in these isolates although without success. The SNPs at codons 167 and 200 after selection with IVM in H. contortus suggests some cross-talk between ML and BZ resistance mechanisms. This does not mean that IVM is directly involved in BZ resistance but may be linked to other changes in the isotype 1 beta-tubulin sequence (Prichard and Roulet, 2007). Taking into account all these findings in H. contortus, we studied the SNPs at codons 200, 167 and 198 of the beta-tubulin isotype 1 in T. circumcincta field isolates. With this objective, single L3 (infective 3rd stage larvae) were analyzed by pyrosequencing. According to von SamsonHimmelstjerna et al. (2009), this technique is quicker and easier to perform than Real-Time PCR and, furthermore, it is suitable for the testing of multiple nearby SNPs at the same time. Because of this, in the current study T. circumcincta L3 from five sheep flocks were tested after confirming their BZ susceptibility and degree of IVM resistance. 2. Materials and methods 2.1. T. circumcincta isolates With the aim of isolating different T. circumcincta field populations resistant to IVM, the FECRT (Faecal Egg Count Reduction Test) was carried out on five sheep flocks located

in the province of León (NW Spain). Previously, for the FECRT, faecal samples from 20 animals were taken to estimate the parasite infection level of each flock. According to the criteria established to conduct the test on a farm, the arithmetic mean of GIN eggs per gram (epg) in faeces should be higher than 150. After confirmation, 45 animals were selected and divided into three groups of 15 to investigate the efficacies of albendazole (ABZ) (Zodalben® ), oral ivermectin (IVM) (Oramec® ), and levamisole (LEV) (Endex® ), respectively, after administration at the manufacturers’ recommended dose rates. For nematode egg count determination, a modified McMaster technique, with a sensitivity of 15, was used and epg values were measured on the day of treatment (day 0) and 10–14 days post treatment (pt). The faecal egg count reduction, as well as the lower limit for a 95% confidence interval, was calculated according to the recommendations of WAAVP (World Association for the Advancement of Veterinary Parasitology) (Coles et al., 1992). When the FECRT value was less than 95% and the lower limit less than 90%, the flock would be considered resistant to the anthelmintic under study. If only one criterion was met, the flock would be classified as borderline but when none was shown, the flock would be deemed susceptible. The FECRT percentage was calculated according to the following formula:FECRT % = (Arithmetic mean epg day 0−Arithmetic mean epg days + 10−14) . Arithmetic mean epg day 0×100 Single L3 were isolated from coprocultures which were carried out on the treatment day, with a pool of faeces from all sheep, and the day 10–14 pt, with faeces from each treatment group. 2.2. DNA isolation and identification of T. circumcincta L3 Genomic DNA (gDNA) was extracted from single exsheathed L3 (exsheathed in 3.5% sodium hypochlorite) using a Qiagen extraction kit (Dneasy Blood and Tissue Kit® ), according to the manufacturer’s protocol. All samples were stored at −20 ◦ C until use. After the individual DNA extraction of L3 from the different isolates, T. circumcincta L3 were identified by the amplification of the ITS2 rRNA gene according to the protocol described by Wimmer et al. (2004). Between 66 and 88 individual L3 were analyzed per coproculture. 2.3. Sequencing of the beta-tubulin isotype 1 gene fragment around the BZ resistance SNPs Once T. circumcincta L3 were identified, a 847 bp fragment of beta-tubulin isotype 1 gDNA was amplified around the BZ resistance-related polymorphisms (Phe167; Glu198; Phe200) from six susceptible L3. Forward and reverse primers (5 -GGC AAA TAT GTC CCA CGT GC-3 /5 GAT CAG CAT TCA GCT GTC CA-3 ), were designed based on the mRNA sequence, GenBank accession number Z69258. The concentration of primers, dNTP and MgCl2 were 0.5 ␮M, 0.4 mM and 1.4 mM, respectively. The PCR conditions were an initial denaturation step of 94 ◦ C for 2 min, followed by 35 cycles of 94 ◦ C for 30 s, 57 ◦ C for 30 s and 72 ◦ C for 1 min and an additional final extension (72 ◦ C) for 10 min. Specific PCR products were run on a 1.5% agarose

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gel stained by gel red and fragments were purified with the Geneclean® kit. The purified PCR products were ligated into a p-GEMT easy vector (Promega) and cloned into DH5␣ cells. Afterward, five clones were sequenced using universal primers SP6 and T7 (96-capillary ABI-3730xl sequencer) (Applied Biosystems). Alignment and analysis of the partial sequences were carried out with the DNASTAR software program (DNAstar INC). 2.4. Pyrosequencing analysis The number of L3 larvae tested in the pyrosequencing assay was 187 for the Phe167Tyr polymorphism and 255 for both the Glu198Ala and Phe200Tyr polymorphism. For codon 167, we used a forward primer (F1), a biotinylated reverse primer (R1-Bio) spanning a fragment of 207 bp, and a sequencing primer (Seq1). For codon 198 and 200 we used a shared biotinylated forward primer (F2-Bio) and a reverse primer (R2) spanning a fragment of 97 bp. Subsequently, the sequencing primer (Seq2) was used to examine the SNPs 198 and 200 jointly. All primers are shown in Table 1. All PCR amplifications were carried out in a 50 ␮l reaction volume using 5 ␮l of gDNA from a single L3 as template. The PCR conditions were an initial denaturation step of 95 ◦ C for 4 min, followed by 40 cycles of 95 ◦ C for 30 s, 60 ◦ C for 30 s and 72 ◦ C for 1 min and an additional final extension (72 ◦ C) for 10 min. Prior to pyrosequencing, a 7 ␮l sample of each PCR product was tested by agarose gel electrophoresis to confirm the size and specificity of the products. The pyrosequencing assay was carried out with a PyroMark ID Pyrosequencer (Biotage) according to the manufacturer’s recommendations. After PCR amplification, 40 ␮l of PCR product was added to 37 ␮l 2× Binding buffer (Biotage), 3 ␮l streptavidin sepharose beads (Roche) in a 96 well plate and then agitated for 5 min at room temperature to allow binding of biotin-labelled DNA to the beads. The beads were processed using the sample preparation tool and reagents (Biotage) dispensed into the assay plate with 40 ␮l of 0.4 ␮M sequencing primer per well. Positive controls representing gDNA extracted from individual adults from a triple resistant T. circumcincta UK isolate (MTci5, Skuce et al., 2010) was included in each assay. 3. Results The results of the FECRT showing the IVM resistance status in T. circumcincta isolates are described in Table 2. Flocks 1 and 5 were classified as IVM susceptible and resistant, respectively, and the rest of the flocks were deemed borderline between susceptibility and resistance with reductions between 92% and 94%. All flocks were susceptible to ABZ and LEV with the exception of flock 5 which was resistant to LEV (data not shown). Regarding the genomic organisation of the beta-tubulin isotype 1 gene in T. circumcincta, the gDNA analysis was based on a consensus sequence from six T. circumcincta L3. A 847 bp fragment was amplified around the BZ resistance-related polymorphisms (Phe167; Glu198; Phe200). Throughout this fragment, three introns were described with 64, 126 and 77 bp respectively (Fig. 1)

373

after alignment with the mRNA sequence (Z69258). Primer design for pyrosequencing analysis was based on the obtained consensus sequence. The DNA extraction was carried out on 598 single L3 and 336 (56%) were identified as T. circumcincta after the amplification of a species-specific ITS2 fragment. The number of L3 analyzed for the SNPs 198/200 was 255 and for the SNP 167 was 187 (Table 2). After carrying out the pyrosequencing, no resistant SNPs were found at any codon, before or after treatment (Table 3). Therefore, all T. cicumcincta L3 were designated as susceptible homozygous for all SNPs. It should be noted that the assays were capable of detecting BZ resistance-associated SNPs because, with gDNA from a triple resistant adult sample included as a positive control (MTci5, Skuce et al., 2010), results were as expected, with 14% heterozygous and 86% homozygous to result in a frequency for the resistant allele of 93% at codon 200 (Table 3). 4. Discussion Conventional in vivo and in vitro methods such as the FECRT and Egg Hatch Test (EHT) have been carried out to determine the therapeutic efficacy of different anthelmintics with the aim of detecting emerging resistance. However, the low sensitivity of these tests necessitates the development of new sensitive tools based on molecular techniques such as pyrosequencing. In the current study, we have analyzed a fragment of the betatubulin isotype 1 sequence with the objective to search for SNPs related with AR. Previous reports have related SNPs at codons 200, 198 and 167 of this gene with the AR against BZs in trichostrongylid nematodes (reviewed by von Samson-Himmelstjerna et al., 2007). In addition, new findings have also associated these same mutations with the selection, after repeated administrations, with IVM (Prichard and Roulet, 2007). We have analyzed a total of 336 T. circumcincta individual L3 from five sheep flocks with different degrees of resistance against IVM, but all of them susceptible to ABZ. After the analysis of the codons 200 and 198 in 255 L3 and the analysis of the codon 167 in 187 L3, we did not detect any mutation at these sites in any L3. Taking into account the hypothesis that IVM treatment could also select for the beta-tubulin polymorphisms, these results would be indicative of a susceptible phenotype while, according to the FECRT, four out of the five flocks were suspected IVM resistant or even resistant. Although the degree of resistance of these field isolates is low, it is important to note that the FECRT only permits the detection of resistance when at least 25% of the population is resistant (Martin et al., 1989). Therefore, at least the L3 collected after treatment (from flocks 2, 3 and 4), which would be anticipated to be IVM resistant, might have been expected to show polymorphism in their beta-tubulin sequence bearing in mind Mottier and Prichard (2008) findings in H. contortus. According to the present study, the mutations at 167, 198 and 200 SNPs are absent in T. circumcincta field isolates, either susceptible or with low level of resistance after treatment with IVM. This fact suggests that the frequency of the resistant genotypes in T. circumcincta seems to be

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Table 1 Primers used in the pyrosequencing for codons 167, 198 and 200. Codons

167

198/200

Primers

Sequence

F1 R1-Bio Seq1 F2-Bio R2 Seq2

5 -CCG GAT AGA ATC ATG GCT TCA-3 5 Biotin-AGT GGC ATT GTA AGG TTC CAC AAC-3 5 -CAT GGC TTC ATT CTC A/C GT-3 5 Biotin-ACC TTA CAA TGC CAC TCT TTC TG-3 5 -GCG GAA GCA GAT ATC GTA CAG-3 5 -A/GGA GCC/T TCA TTA TCG ATA/G-3

Table 2 Flock identification and number of single T. circumcincta L3 analyzed. Flock identification

FECRT

Coproculture

Number of L3 analyzed

98%

Pre-treatment

88

67

56

64

2

94%

Pre-treatment Post-treatment

72 72

57 6

17 6

16 3

3

93%

Pre-treatment Post-treatment

80 66

38 2

27 1

6 1

4

92%

Pre-treatment Post-treatment

80 74

52 63

52 56

36 40

90%

Pre-treatment Total

66 598

51 336

40 255

21 187

DNA extraction 1

5

very low in field conditions. In other nematode species, such as H. contortus, even when the isolates are shown to be BZ and/or IVM susceptible, the homozygous resistant genotype at codon 200 is clearly demonstrable as it is described in the following studies. This was the case in the study of Cudeková et al. (2010), who reported the presence of the resistant genotype at codon 200 in four H. contortus isolates designated susceptible to BZ, although the allele frequency values were only between 3% and 6%. More significant is the study carried out by Höglund et al. (2009), who analyzed the beta-tubulin gene of H. contortus in Swedish sheep farms following treatment with oral IVM and ABZ. The presence of H. contortus was confirmed by PCR in pre-treatment coprocultures. After analysis of the codon 200 by pyrosequencing, the resistant allele frequency in flocks treated with oral IVM was 66% (with a range of 0–100%), although all flocks were clinically susceptible to IVM, with a reduction in the epg > 95% by FECRT. When the same flocks were treated with ABZ, the allele frequency was the same but, in this case, two flocks were resistant to the drug (81% and 94%) and another was suspected of resistance (97%). The allele frequency for these last three flocks was between 95% and 100%. What is worth noting is that the resistant allele frequency was 100% in 3 flocks deemed susceptible to orally administered IVM which, at the same time, were the 3 farms resistant and

Number of Tc

SNPs 200/198

SNP 167

suspected of resistance against ABZ. These results indicate that the species most likely to develop resistance to ABZ and IVM was H. contortus, although if the proportion of eggs is low, resistance may be overlooked by the FECRT. Regarding this last study, it seems that in H. contortus the selection pressure to have a resistant genotype at codon 200 is higher after the treatment with ABZ rather than with oral IVM. After the selection with both drugs, the resistant allele frequency was the same in both groups (66%), however, all flocks were IVM susceptible and some flocks were ABZ resistant. If this was also true for T. circumcincta, the results presented in this paper would be expected since all strains were susceptible to ABZ but had low level resistance against IVM. Mottier and Prichard (2008) also compared the effect of both treatments on H. contortus populations. Three BZ susceptible and ML resistant H. contortus isolates were genotyped, showing frequencies of the resistant allele in the SNPs 200, 167 and 198 of 0–14.7%, 0–33% and 0%, respectively. However, when the isolates were ABZ resistant and IVM susceptible, the frequencies were higher, between 40.6% and 100%, 0% and between 0% and 59.4% for the same SNPs. In relation to T. circumcincta, Skuce et al. (2010) carried out a study in which single T. circumcincta L3 were genotyped by pyrosequencing from different isolates with different resistance degrees to BZ and IVM. The homozygous

Table 3 Frequencies of SNPs at codons 200, 198 and 167 in all T. circumcincta samples analyzed. Codons

Frequencies of SNPs

Phenotype

L3

200 198 167

Positive sample

200

100% T/T 100% C/C 100% T/T 86% A/A 14% A/T

Susceptible Susceptible Susceptible Resistant Susceptible

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Fig. 1. Alignment of T. circumcincta partial beta-tubulin isotype 1 gene (cDNA and gDNA). The codons 167,198 and 200 are indicated in the black boxes.

resistant genotype at codon 200 was not found either in a BZ susceptible population or in a BZ suspect resistance population although the heterozygous susceptible genotype was described in this last one with a frequency around 15%. Mutations at codons 167 and 198 were not observed in any L3, including those resistant to both BZs and IVM. This result is in agreement with the findings of the present study, which also did not find any resistance-associated SNPs at the codons 167 and 198. Therefore, the mutations F167Y and E198A in T. circumcincta field isolates may be less common than in H. contortus. Comparing all these previous results with the current study in T. circumcincta, it seems that the selection after the administration of IVM for the resistant allele at codon 200 takes place more readily in H. contortus. Moreover, the absence of mutations in the IVM resistant populations of this study suggests that mechanisms other than beta-tubulin could be implicated in the development of resistance against IVM in T. circumcincta. It is interesting to note that as the fecundity of H. contortus is higher than T. circumcincta, one resistant worm can be produced in the population of a flock every 20 days for H. contortus and

every 200 days for T. circumcincta (Silvestre and Humbert, 2002). In conclusion, the absence of the mutations in the T. circumcincta populations described in the current study would agree with the findings of the recent UK study (Skuce et al., 2010), and would argue against resistance haplotypes being present in the parasite population prior to drug treatment, at least in T. circumcincta. Acknowledgements This study has been funded by the National Project INIA-MEC, RTA2006-00183-C03-02. The work of MartínezValladares M. has been supported by a postdoctoral Jae-Doc contract from Consejo Superior de Investigaciones Científicas (CSIC). References Alvarez-Sánchez, M.A., Pérez-García, J., Cruz-Rojo, M.A., Rojo-Vázquez, F.A., 2006. Anthelmintic resistance in trichostrongylid nematodes of sheep farms in Northwest Spain. Parasitol. Res. 99, 78–83. Coles, G.C., Bauer, C., Borgsteede, F.H., Geerts, S., Klei, T.R., Taylor, M.A., Waller, P.J., 1992. World Association for the Advancement of

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