A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance - PubMed (original) (raw)

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance

Nicola J Beesley et al. PLoS Pathog. 2023.

Abstract

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

Copyright: © 2023 Beesley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig 1. Experimental overview.

(A) Schematic of the in vivo work to produce an F2 cross from _Fh_LivS1 (a clonal population of susceptible parasites) and _Fh_LivR1 (a clonal population of resistant parasites). The parental parasites _Fh_LivS1 and _Fh_LivR1 were produced separately and used to co-infect sheep (n = 2). Some of these parental parasites would cross-fertilise to produce an F1 cross of _Fh_LivS1 and _Fh_LivR1. Eggs were collected from the adult parasites within these sheep. A single miracidium (obtained from these eggs) was used to infect snails (n = 28) and produce clonal F1 populations. The metacercariae were genotyped to ensure they were from an F1 cross and then combined together and used to infect sheep (n = 4). Some of these F1 parasites would cross-fertilise to produce an F2 recombinant population. Eggs were collected from the adult parasites within these sheep. Snails (n = 41 and n = 44 for Experiment 1 and 2, respectively) were exposed to multiple miracidia obtained from these eggs and combined to produce a common pool of F2 metacercariae. For each experiment, two groups of animals were infected with metacercariae from this common pool. Once the infection had reached patency, one group of animals in each experiment was treated with triclabendazole (TCBZ) at a dose of 10mg/kg. At post mortem, those animals which received no treatment had a mixture of triclabendazole susceptible (TCBZ-S) and triclabendazole resistant (TCBZ-R) parasites, whilst those animals that were treated had only TCBZ-R parasites remaining. These parasites were then used for pooled genotyping. (B) A haplotype schematic to show the genetic principle behind the in vivo F2 cross. The F1 cross consists of one haplotype from the susceptible parent: _Fh_LivS1 (_Fh_LivS1.Hap1 or _Fh_LivS1.Hap2) and one haplotype from the resistant parent: _Fh_LivR1 (_Fh_LivR1.Hap1 or _Fh_LivR1.Hap2). In the subsequent F2 generation, recombination events take place and the resistant haplotype becomes introgressed amongst the susceptible haplotype producing an F2 recombinant population for study. (C) Plot to show the reduction in the number of F2 parasites recovered from treated animals compared to untreated animals in Experiments 1 and 2. Boxplot indicates the median number of parasites, upper and lower quartiles, and outliers; overlaid points indicate the number of parasites in each animal. In both experiments a significant difference (Mann-Whitney W = 25 p < 0.05) is seen in the number of F2 parasites from untreated and treated animals.

Fig 2. Genome scan for regions associated with resistance to triclabendazole.

Data show the median likelihood ratio test (LRT) statistic from generalised linear models within moving windows of 1000 informative SNPs. Scaffolds are represented in alternating dark grey and light grey to allow visualisation. Scaffold order on the x-axis is arbitrary and does not imply physical proximity. (A) Results of the two replicate crossing experiments. Position of scaffolds under greatest selection (13, 157, 166, 324, 1853 and 2049) is indicated by arrows. Red crosses indicate where the median LRT appears in the top 1% quantile in both experiments. (B) Results from Field Isolate 1. Position of scaffold 157, under greatest selection, and scaffold 1853 are indicated by arrows. Red crosses indicate where the median LRT appears in the top 1% quantile.

Fig 3. Heat map (with no clustering or scaling) to show |D’| values between all pairs of loci in untreated F2 parasites.

Loci under selection are enclosed by the black horizontal and vertical lines, with neutral loci outside. Above the diagonal all |D’| values are shown and below the diagonal only |D’| values with significant _q_-values (q < 0.05 after false discovery rate correction) are shown. When comparing pairs of loci from scaffolds under selection, high |D’| values indicate that all six scaffolds are in linkage disequilibrium. The |D’| values for the majority of loci pairs containing neutral scaffolds are low or not-significant.

Fig 4. Schematic to demonstrate finer scale mapping of the genomic region under selection in recombinant F2s compared to parental haplotypes.

Parasites (treated and untreated) were individually genotyped at 36 loci across the six scaffolds under selection. PHASE 2.1.1 [30,31] was used to infer haplotypes from the SNP data and the parental haplotypes (_Fh_LivR1.Hap1; _Fh_LivR1.Hap2; _Fh_LivS1.Hap1; _Fh_LivS2.Hap2) were identified. The figure shows the individual genotypes for the loci on scaffolds 1853, 157 and 2049 (note that even though the sequences are consecutive in the diagram the individual loci are not physically next to each other; the nucleotide position of these loci across each scaffold can be found in S4 Table). Analysis of informative resistant recombinant haplotypes (Rec4/5 and Rec7; S5 Table) found within surviving F2 parasites (i.e. those from treated animals) allowed us to further localise the area needed for a parasite to be resistant. In these recombinants, recombination between SNPs delineates a single genomic locus from 1853_3 to 157_6 (~3.2Mbp; 0.3Mbp region of scaffold 1853 and a 2.9Mbp region of scaffold 157) that was consistently inherited in surviving F2 parasites (S5 Table).

Fig 5. Median likelihood ratio test (LRT) statistic from generalised linear models within moving windows of 1000 informative SNPs for in vivo Experiment 1 and Experiment 2 and Field Isolate 1 are plotted against the position within the 3.2Mbp locus (0.3Mbp region of scaffold 1853 and a 2.9Mbp region of scaffold 157).

Positions of the 30 genes are indicated across the locus and are represented in alternating green and blue colours to allow visualisation. Gene numbering corresponds with Table 5: 1: maker-scaffold10x_1853_pilon-snap-gene-0.15 (26S proteasome non-ATPase regulatory subunit 14; *gene crosses locus boundary); 2: maker-scaffold10x_1853_pilon-snap-gene-0.14 (26S proteasome non-ATPase regulatory subunit 14); 3: maker-scaffold10x_1853_pilon-snap-gene-0.13 (Uncharacterised protein); 4: maker-scaffold10x_157_pilon-snap-gene-0.196 (EGF-like protein); 5: maker-scaffold10x_157_pilon-snap-gene-0.179 (Putative multidrug resistance protein 1, 2, 3 (P glycoprotein 1, 2, 3); ATP binding cassette subfamily B MDR TAP); 6: maker-scaffold10x_157_pilon-snap-gene-0.180 (SANT/Myb-like DNA-binding domain protein); 7: maker-scaffold10x_157_pilon-snap-gene-0.197 (ADP-ribosylation factor 2); 8: maker-scaffold10x_157_pilon-snap-gene-0.181 (RNA-binding protein sym-2/ Heterogeneous nuclear ribonucleoprotein); 9: maker-scaffold10x_157_pilon-snap-gene-0.198 (DNA directed RNA Polymerase I and III (A/C) shared subunit); 10: maker-scaffold10x_157_pilon-snap-gene-0.182 (Ras-related protein Rap-1); 11: maker-scaffold10x_157_pilon-snap-gene-0.183 (Receptor protein serine/threonine kinase); 12: maker-scaffold10x_157_pilon-augustus-gene-0.97 (D-amino-acid oxidase/ D-aspartate oxidase); 13: maker-scaffold10x_157_pilon-snap-gene-0.184 (Max-like protein X); 14: maker-scaffold10x_157_pilon-snap-gene-0.185 (EGF-like protein); 15: maker-scaffold10x_157_pilon-snap-gene-0.186 (Surfeit locus protein 4); 16: augustus_masked-scaffold10x_157_pilon-processed-gene-0.14 (TFIIH basal transcription factor complex helicase XPD subunit); 17: maker-scaffold10x_157_pilon-snap-gene-0.187 (Fatty acid binding protein V); 18: maker-scaffold10x_157_pilon-snap-gene-0.200 (Stomatin-2 / SPFH Domain / Band 7 family protein); 19: maker-scaffold10x_157_pilon-snap-gene-0.201 (Glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase 1); 20: maker-scaffold10x_157_pilon-pred_gff_StringTie-gene-0.138 (Sugar phosphate exchanger 3); 21: maker-scaffold10x_157_pilon-snap-gene-0.203 (Ribonuclease 3); 22: maker-scaffold10x_157_pilon-snap-gene-0.188 (Putative serine-rich repeat protein); 23: maker-scaffold10x_157_pilon-snap-gene-0.204 (Putative transferase CAF17, mitochondrial); 24: maker-scaffold10x_157_pilon-snap-gene-0.205 (Lamin-1/ Neurofilament protein); 25: maker-scaffold10x_157_pilon-snap-gene-0.189 (Gyf domain protein); 26: snap_masked-scaffold10x_157_pilon-processed-gene-0.72 (Prominin); 27: maker-scaffold10x_157_pilon-snap-gene-0.206 (Phospholipid transport protein / CRAL-TRIO / SEC14-like); 28: maker-scaffold10x_157_pilon-snap-gene-0.190 (Ubiquitin carboxyl-terminal hydrolase); 29: maker-scaffold10x_157_pilon-snap-gene-0.207 (Ubiquitin carboxyl-terminal hydrolase); 30: maker-scaffold10x_157_pilon-augustus-gene-0.89 (Ubiquitin carboxyl-terminal hydrolase).

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A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance - PubMed (original) (raw)