Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR.

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease") primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor α1 and α2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides.

Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR α subunits Cfα1, Cfα2, and Cfα3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) ß2 (Ggß2) subunit, these cat flea α subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cfα2/Ggß2 were resistant to α-bungarotoxin, while those of Cfα1/Ggß2 were sensitive to this snake toxin. The pharmacological profiles of Cfα1/Ggß2, Cfα2/Ggß2 and the chicken neuronal receptor Ggα4/Ggß2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cfα1/Ggß2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cfα2/Ggß2 or Ggα4/Ggß2: for the anti flea neonicotinoid market compound imidacloprid the respective EC50s were 0.02 µM, 1.31 µM and 10 µM. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dmα1 and Dmα2 subunits as hybrid receptors with Ggß2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Ggß2 hybrid receptor setting, insect α1 subunits confer higher sensitivity to neonicotinoids than α2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class.

Acetylcholinesterases of the cat flea Ctenocephalides felis: identification of two distinct genes and biochemical characterization of recombinant and in vivo enzyme activities.

Acetylcholinesterase (AChE, EC3.1.1.7.) is a prime target for insecticides and is the site of action of carbamate and organophosphate drugs used to combat the cat flea Ctenocephalides felis. In this paper we report the identification and cDNA cloning of two AChE-encoding genes from the cat flea, cface1 and cface2. Functional heterologous expression of the catalytic domains in Pichia pastoris shows that both genes encode functional enzymes, CfAChE1 and CfAChE2. Bioinformatical analysis of the predicted translation products and heterologous expression of the full length cDNAs in the human cell line HEK293 demonstrate, that CfAChE1 and CfAChE2 possess glycosylphosphatidylinositol membrane anchors and are transported to the cell surface. Recombinant CfAChE1 and CfAChE2 share high sensitivity towards the anti-flea carbamates propoxur and carbaryl, but can be distinguished by their specificity for different acylthiocholine AChE substrates and, particularly, by their differential sensitivity to the non-covalent inhibitor galanthamine. Comparison of substrate specificities and inhibitor sensitivities of both recombinant enzymes with those of AChE activities extracted from adult fleas suggest that CfAChE1, and not CfAChE2, is the dominant activity in C. felis imagoes. Three-dimensional structure models of CfAChE1 and CfAChE2 reveal similarities, but also differences, and compound docking experiments on these models provide potential rationales for the differential substrate and the inhibitor specificities observed experimentally.

Identification and characterization of two arginine kinases from the parasitic insect Ctenocephalides felis.

Arginine kinase (ATP:l-arginine omega-N-phosphotransferase, EC2.7.3.3.; AK) is an enzyme crucial for the energy metabolism of insects and other invertebrates, that has known allergenic potential in humans and that has been proposed as a pesticidal drug target. Here we report the identification, cDNA cloning, genomic gene structure and functional expression of AK genes from Ctenocephalides (C.) felis (cat flea). In contrast to other insect species investigated so far, C. felis possesses two AK genes, cfak1 and cfak2, encoding the functional enzymes CfAK1 and CfAK2 that can be distinguished by their guanidino substrate specificity and the kinetic parameters for their natural substrates. Molecular modelling on CfAK1 and CfAK2 based on the Limulus polyphemus AK X-ray structure (Zhou et al., 1998) and substrate docking studies provide a potential rational for the observed specificities. Evidence is provided that adult fleas express predominantly CfAK1 as an abundant soluble protein, and that in vivo in C. felis, the AK metabolites are present in concentration ranges relevant for this enzyme.

Succinic semialdehyde dehydrogenase from the parasitic cattle tick Rhipicephalus microplus: gene identification, biochemical characterization and comparison with the mouse ortholog.

The gamma-aminobutyric acid (GABA) degradation pathway consists of the enzymes GABA transaminase and succinic semialdehyde dehydrogenase (SSADH) and is essential for the development and functionality of the nervous system in mammals, while little is known on its role in invertebrates. In this study we report the gene identification, cDNA cloning and heterologous functional expression of a SSADH from the cattle tick Rhipicephalus (R.) microplus. In contrast to mammals and the insect model organism Drosophila melanogaster, which have one SSADH gene, R. microplus possesses several gene copies. One representative of these genes has been functionally expressed in Escherichia coli. This recombinant cattle tick protein has potent NAD(+)-dependent SSADH activity, but possesses also marked enzymatic activity on other aliphatic and aromatic aldehyde substrates. Comparison of R. microplus SSADH enzyme kinetic properties as well as substrate and inhibitor specificities with those of a recombinant mammalian SSADH reveals overall similarities, but also subtle differences, that may be exploited for the design of specific inhibitors with selective acaricidal activity.

Highly efficient transport of carboxyfluorescein diacetate succinimidyl ester into COS7 cells using human papillomavirus-like particles.

Human papillomavirus virus-like particles (VLPs) have recently been used to deliver genes into mammalian cells in vitro and in vivo. Here, we investigated whether VLPs may serve as an efficient carrier of low molecular weight compounds (e.g. hormones, vitamins, peptides etc.) into cells. COS7 cells were incubated with recombinant HPV-16L1/L2 VLPs labelled with the fluorescence dye carboxyfluorescein diacetate succinimidyl ester. Using flow cytometry, we demonstrate that labelled VLPs can specifically bind to the cell surface followed by their complete internalisation. Our results indicate that VLPs are promising vehicles for highly efficient delivery of low molecular weight compounds into cells.