Genome-wide analysis of the response to ivermectin treatment by a Swedish field population of Haemonchus contortus

Abstract

Haemonchus contortus is a pathogenic gastrointestinal nematode of small ruminants and, in part due to its capacity to develop resistance to drugs, contributes to significant losses in the animal production sector worldwide. Despite decades of research, comparatively little is known about the specific mechanism(s) driving resistance to drugs such as ivermectin in this species. Here we describe a genome-wide approach to detect evidence of selection by ivermectin treatment in a field population of H. contortus from Sweden, using parasites sampled from the same animals before and seven days after ivermectin exposure followed by whole-genome sequencing. Despite an 89% reduction in parasites recovered after treatment measured by the fecal egg count reduction test, the surviving population was highly genetically similar to the population before treatment, suggesting that resistance has likely evolved over time and that resistance alleles are present on diverse haplotypes. Pairwise gene and SNP frequency comparisons indicated the highest degree of differentiation was found at the terminal end of chromosome 4, whereas the most striking difference in nucleotide diversity was observed in a region on chromosome 5 previously reported to harbor a major quantitative trait locus involved in ivermectin resistance. These data provide novel insight into the genome-wide effect of ivermectin selection in a field population as well as confirm the importance of the previously established quantitative trait locus in the development of resistance to ivermectin.

Keywords: Anthelmintic resistance; Haemonchus contortus; Ivermectin; Pool-seq; Whole-genome sequencing.

Graphical abstract

Fig. 1

Genetic diversity within pre- and post-treatment groups. Within treatment group nucleotide diversity (a and b) and Tajima's D (c and d) comparisons per 100 kbp genomic windows per chromosome based on pooled sequencing of H. contortus L3, recovered before and seven days after IVM treatment from the same flock of sheep. (e and f) Tajima's D estimates per 100 kbp genomic window were evaluated specifically for chromosome 5. The blue line represents a LOESS function drawn through the data points. The dashed line in (c and d) and (e and f) is drawn through value 0 which indicates neutrality and otherwise serves to separate windows wherein Tajima's D is either positive or negative. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Genetic differentiation between pre- and post-treatment groups. Pairwise genetic differentiation between the two treatment groups was calculated as F_ST values per (a) 10 kbp windows throughout the genome or (b) for entire genes. In both (a) and (b), the level of significance is indicated by dashed black line (mean F_ST + 3 SDs) and dotted black line (mean F_ST + 5 SDs). (a) Three consecutive 10kbp windows above the mean F_ST + 5 SDs present in chromosome 5 are displayed in red. (b) F_ST values (left to right; in a decreasing manner) for genes lgc-37, haf-6, osm-3, osm-5, lgc-55, pgp-9.1, pgp-9, avr-15, glc-1, avr-14, che-11, dyf-11, pgp-1, che-2, osm-1, lgc-36, mrp-1, che-3, pgp-12, che-12, glc-3, glc-2, che-13, glc-5, ggr-3, osm-6, pgp-3, unc-9, unc-38, dyf-7 are shown as black dots (standalone figure with gene names is shown as Supplementary Fig. 3), whereas the points in red represents the three top most F_ST value having genes (located in chromosomes 5 - HCON_00141660 and 4 - HCON_00128970 and HCON_00115660). Colours in (a) represent different chromosomes as indicated in the legend of Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Ratios of nucleotide diversity and Tajima's D highlight outlier variation in response to treatment in chromosome 5. Genome-wide nucleotide diversity (a) and chromosome 5 Tajima's D (b) ratios were analyzed per every genomic 100 kbp window to identify subtle signs of selection in the post-treatment group. In both (a) and (b), the level of significance is indicated by dashed black line (mean ± 3 SDs) and dotted black line (mean ± 5 SDs). Colours represent different chromosomes as indicated in the legend of Fig. 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)