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Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

ABSTRACT: Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

SUBMITTER: Jesudoss Chelladurai JRJ

PROVIDER: S-EPMC7410292 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Jesudoss Chelladurai Jeba R J JRJ Martin Katy A KA Chinchilla-Vargas Krystal K Jimenez Castro Pablo D PD Kaplan Ray M RM Brewer Matthew T MT

PloS one 20200806 8

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeabil ...[more]

PMID: 32760111

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Project description:BackgroundCurrent measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation.MethodsDirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR).ResultsIn vitro conditions that best promote early development of D. immitis microfilariae to the "late sausage stage" have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro.ConclusionsThe arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.

Project description:BackgroundThe ability of blood-feeding arthropods to successfully acquire and transmit pathogens of medical and veterinary importance has been shown to be interfered with, or enhanced by, the arthropod's native microbiome. Mosquitoes transmit viruses, protozoan and filarial nematodes, the majority of which contribute to the 17% of infectious disease cases worldwide. Dirofilaria immitis, a mosquito-transmitted filarial nematodes of dogs and cats, is vectored by several mosquito species including Aedes aegypti.MethodsIn this study, we investigated the impact of D. immitis colonization on the microbiome of laboratory reared female Ae. aegypti. Metagenomic analysis of the V3-V4 variable region of the microbial 16S RNA gene was used for identification of the microbial differences down to species level.ResultsWe generated a total of 1068 OTUs representing 16 phyla, 181 genera and 271 bacterial species. Overall, in order of abundance, Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the most represented phylum with D. immitis-infected mosquitoes having more of Proteobacteria (71%) than uninfected mosquitoes (56.9%). An interesting finding in this study is the detection of Klebsiella oxytoca in relatively similar abundance in infected and uninfected mosquitoes, suggesting a possible endosymbiotic relationship, and has been previously shown to indirectly compete for nutrients with fungi on domestic housefly eggs and larvae. While D. immitis colonization has no effect on the overall species richness, we identified significant differences in the composition of selected bacterial genera and phyla between the two groups. We also reported distinct compositional and phylogenetic differences in the individual bacterial species when commonly identified bacteria were compared.ConclusionsTo the best of our knowledge, this is the first study to understand the impact of a filarial infection on the microbiome of its mosquito vector. Further studies are required to identify bacteria species that could play an important role in the mosquito biology. While the microbiome composition of Ae. aegypti mosquito have been previously reported, our study shows that in an effort to establish itself, a filarial nematode modifies and alters the overall microbial diversity within its mosquito host.

Dataset Information

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Publications

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

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