Fig 1.
Egg hatch assay and isotype-1 β-tubulin 167Y(TAC) and 134H(CAT) mutation frequency data for 16 A. caninum samples from 25 greyhounds.
(A): Log-transformed Egg Hatch Assay (EHA) IC95 values (μM) for the 16 A. caninum fecal egg samples. The resistance threshold is indicated by the red dashed line; (B): 167Y(TAC) isotype-1 β-tubulin mutation frequencies; (C): 134H(CAT) isotype-1 β-tubulin mutation frequencies. Sample SF61-70 is a pool comprising A. caninum from 10 greyhounds. Barrow 1.0 is the first passage of an A. caninum benzimidazole-susceptible laboratory sample [10]. The remaining samples are from 25 greyhounds described in [12].
Fig 2.
In-silico protein structural model of the A. caninum isotype-1 β-tubulin bound to nocodazole.
Structural model for A. caninum isotype-1 β-tubulin made using AlphaFold2 (PMID: 34265844) (purple) aligned to Porcine β-tubulin (green) bound to nocodazole (white) (from PDB 5CA1). The inset shows a zoom into the binding site, with positions of residues at codons 134, 167, 198, and 200 indicated. These resistance mutation positions are all in direct van der Waals contact with the constant portion of benzimidazole drugs.
Fig 3.
Drug response assays for ben-1 Q134H(CAA>CAC) alleles introduced into C. elegans by CRISPR-Cas9 editing.
Regressed median optical density (median.EXT), a proxy measurement of animal length, from populations of nematodes after exposure to 30 μM albendazole is shown. ben-1(ean64) is a previously created deletion strain. ben-1(ean243) and ben-1(ean244) are two independent CRISPR-Cas9 edited Q134H(CAA>CAC) alleles. Each point represents the regressed phenotype of a well containing approximately 50 animals. Data are shown as Tukey box plots with the median as a solid horizontal line, and the top and bottom of the box representing the 75th and 25th quartiles, respectively. The top whisker is extended to the maximum point within the 1.5 interquartile range from the 75th quartile. The bottom whisker is extended to the minimum point within the 1.5 interquartile range from the 25th quartile. Statistical significance is shown as asterisk above each strain (p < 0.001 = ***, p < 0.0001 = ****, Tukey HSD).
Fig 4.
Prevalence and frequency of the F167Y(TTC>TAC) isotype-1 β-tubulin benzimidazole resistance mutation in A. caninum from pet dogs across the USA.
(A): Deep amplicon sequencing data of the 293 bp isotype-1 β-tubulin fragment of A. caninum from the 314/ 328 individual samples across the USA. The top panel is a bar chart showing the number of eggs used to make each genomic DNA preparation, in the middle is a bar chart showing the mapped read depth for each sample, and the lower chart shows the relative frequency of the F167Y(TTC>TAC) mutation. Red bars indicate the 167Y(TAC) resistance allele and the blue bars indicate the 167F(TTC) susceptible allele. (B): Geographical distribution of the individual samples and the frequency of the 167Y(TAC) resistance allele. The map of the USA was generated using the maps() package in R and is available from: https://rdrr.io/cran/maps/man/usa.html. The color of each circle indicates the frequency of the resistance mutation in each sample, ranging from 0–100%. (C): Violin Plot of the 167Y(TAC) resistance allele frequencies from samples in each of the four geographical regions. The mean frequency is indicated by a horizontal line and any statistically significant differences calculated using the pairwise Wilcoxon rank sum test (p<0.05) between the regions are indicated (p-value >0.05 not indicated).
Table 1.
Prevalence of the F167Y(TTC>TAC) mutation.
Fig 5.
Frequency and prevalence of the novel Q134H(CAA>CAT) benzimidazole resistance mutation in A. caninum from pet dogs across the USA.
(A): Deep amplicon sequencing data of the 293 bp isotype-1 β-tubulin fragment of A. caninum from the 314/ 328 individual samples across the USA. The top panel is a bar chart showing the number of eggs used to make each genomic DNA preparation, in the middle is a bar chart showing the mapped read depth for each sample, and the lower chart shows the relative frequency of the Q134H(CAA>CAT) mutation. Red bars indicate the 134H(CAT) resistance allele and the blue bars indicate the 134Q(CAA) susceptible allele. (B): Geographical distribution of the individual samples and the frequency of the 134H(CAT) resistance allele. The map of the USA was generated using the maps() package in R and is available from: https://rdrr.io/cran/maps/man/usa.html. The color of each circle indicates the frequency of the resistance mutation in each sample, ranging from 0–100%. (C): Violin Plot of the 134H(CAT) resistance allele frequencies from samples in each of the four geographical regions. The mean frequency is indicated by a horizontal line and any statistically significant differences calculated using the pairwise Wilcoxon rank sum test (p<0.05) between the regions are indicated (p-value >0.05 not indicated).
Table 2.
Prevalence of the Q134H(CAA>CAT) mutation.
Fig 6.
Frequency and prevalence of the novel Q134H(CAA>CAT) benzimidazole resistance mutation in A. caninum infecting greyhounds in the USA.
Deep amplicon sequencing data of the 293 bp isotype-1 β-tubulin fragment for A. caninum from greyhounds in adoption kennels in four locations in the USA [12]. The top panel is a bar chart showing the number of eggs used to make each genomic DNA preparation, in the middle is a bar chart showing the mapped read depth for each sample, and the lower chart shows the relative frequency of the Q134H(CAA>CAT) mutation. Red bars indicate the 134H(CAT) resistance allele and the blue bars indicate the 134Q(CAA) susceptible allele.