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Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.
- Source :
-
PloS one [PLoS One] 2024 Jul 17; Vol. 19 (7), pp. e0307261. Date of Electronic Publication: 2024 Jul 17 (Print Publication: 2024). - Publication Year :
- 2024
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Abstract
- Background: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood.<br />Methods: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families.<br />Results: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes.<br />Conclusions: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.<br />Competing Interests: The authors have declared that no competing interests exist.<br /> (Copyright: © 2024 Hampton 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.)
- Subjects :
- Animals
Dogs
Dog Diseases parasitology
Dog Diseases blood
Ivermectin pharmacology
Doxycycline pharmacology
Drug Resistance genetics
ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism
ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics
Dirofilaria immitis genetics
Dirofilaria immitis metabolism
Microfilariae genetics
Dirofilariasis parasitology
Dirofilariasis blood
Dirofilariasis diagnosis
Subjects
Details
- Language :
- English
- ISSN :
- 1932-6203
- Volume :
- 19
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- PloS one
- Publication Type :
- Academic Journal
- Accession number :
- 39018313
- Full Text :
- https://doi.org/10.1371/journal.pone.0307261