Dirofilaria immitis JYD-34 isolate: whole genome analysis

Abstract

Background: Macrocyclic lactone (ML) anthelmintics are used for chemoprophylaxis for heartworm infection in dogs and cats. Cases of dogs becoming infected with heartworms, despite apparent compliance to recommended chemoprophylaxis with approved preventives, has led to such cases being considered as suspected lack of efficacy (LOE). Recently, microfilariae collected from a small number of LOE isolates were used as a source of infection of new host dogs and confirmed to have reduced susceptibility to ML in controlled efficacy studies using L3 challenge in dogs. A specific Dirofilaria immitis laboratory isolate named JYD-34 has also been confirmed to have less than 100% susceptibility to ML-based preventives. For preventive claims against heartworm disease, evidence of 100% efficacy is required by FDA-CVM. It was therefore of interest to determine whether JYD-34 has a genetic profile similar to other documented LOE and confirmed reduced susceptibility isolates or has a genetic profile similar to known ML-susceptible isolates.

Methods: In this study, the 90Mbp whole genome of the JYD-34 strain was sequenced. This genome was compared using bioinformatics tools to pooled whole genomes of four well-characterized susceptible D. immitis populations, one susceptible Missouri laboratory isolate, as well as the pooled whole genomes of four LOE D. immitis populations. Fixation indexes (F_ST), which allow the genetic structure of each population (isolate) to be compared at the level of single nucleotide polymorphisms (SNP) across the genome, have been calculated. Forty-one previously reported SNP, that appeared to differentiate between susceptible and LOE and confirmed reduced susceptibility isolates, were also investigated in the JYD-34 isolate.

Results: The F_ST analysis, and the analysis of the 41 SNP that appeared to differentiate reduced susceptibility from fully susceptible isolates, confirmed that the JYD-34 isolate has a genome similar to previously investigated LOE isolates, and isolates confirmed to have reduced susceptibility, and to be dissimilar to the susceptible isolates.

Conclusions: These results provide additional evidence for the link between genotype and the reduced susceptibility phenotype observed in such isolates as JYD-34. Further work on other isolates showing reduced susceptibility to ML is required to demonstrate the value of genetic analysis in predicting the response to ML chemoprophylaxis. The authors suggest that genetic analysis may be useful in helping to interpret the results of in vivo efficacy testing of ML heartworm preventives against D. immitis isolates.

Keywords: Dirofilaria immitis; Heartworm preventives; JYD isolate; Macrocyclic lactone resistance; Whole genome analysis.

Fig. 1

Means F_ST calculated from 1,602,214 SNPs over the whole D. immitis nDi.2.2 genome, for comparisons between isolates: Susceptible versus JYD-34, Susceptible versus LOE, Missouri versus JYD-34, Missouri versus LOE, and JYD-34 versus LOE. F_ST = 0 means no divergence between two populations and F_ST = 1 means complete divergence

Fig. 2

Dendrogram visualization based on mean F_ST and different level of F_ST threshold (0, 0.1, 0.5 and 0.9 (panels a, b, c and d, respectively). The dendrograms illustrate the divergence that exists between JYD-34 isolate and, respectively, Missouri (susceptible laboratory isolate), pooled susceptible and pooled LOE isolates; where F_ST = 0 means no divergence between two populations, and F_ST = 1 means complete divergence. Additional dendrograms and information are presented in Additional file 1

Fig. 3

Comparison of the genetic profile of JYD-34 isolate with SUS and (LOE + RES) isolates based on 41 SNP previously reported. Difference in the percentage of the alternative nucleotide frequencies of the 41 alternative nucleotides between the (LOE + RES) isolates and the SUS isolates (blue square shape and line) and between JYD-34 isolate and the SUS isolates (black triangle shape and line) are illustrated. Green round shape and line represent the percentage of the alternative nucleotide frequencies in the SUS isolates. The information of the SNP on the x axis is provided in Additional file 2

Fig. 4

Summary of locations where confirmed resistant isolates to heartworm preventives were reported