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Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called "slow-kill" effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The diversity and uniqueness of nematode heterotrimeric G-protein-coupled receptors (GPCRs) provides impetus for identifying ligands that can be used as therapeutics for treating diseases caused by parasitic nematode infections. In human medicine, GPCRs have represented the largest group of 'drugable' targets exploited in the market today. In the filarial nematode Dirofilaria immitis, which causes heartworm disease, the macrocyclic lactones (ML) have been used as the sole preventatives for more than 25 years and now there is confirmed ML resistance in this parasite. A novel anthelmintic emodepside, with antifilarial activity, can act on a GPCR. In view of the ML resistance, there is an urgent need to identify new drug targets and GPCRs of D. immitis may be promising receptors. Knowledge of polymorphism within the GPCR superfamily is of interest. A total of 127 GPCR genes have been identified, so far, in the genome of D. immitis. Whole genome sequencing data from four ML susceptible and four ML loss of efficacy populations was used to identify 393 polymorphic loci in 35 D. immitis GPCR genes. Out of 57 SNPs in exonic regions, 36 of them caused a change in an amino acid, out of which 2 changed the predicted secondary structure of the protein. Knowledge about GPCR genes and their polymorphism is valuable information for drug design processes. Further studies need to be carried out to more fully understand the implications of each of the SNPs identified by this study., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Filarial parasites are responsible for several serious human diseases with symptoms such as lymphoedema, elephantiasis, and blindness. An understanding of how these parasites pass through developmental checkpoints may suggest potential targets for intervention. A useful model system for the study of the human parasites is the closely related nematode, D. immitis, the causative agent of dog heartworm disease. Ecdysteroids have been identified in filarial nematodes and have been shown to have a biological affect both on molting and microfilarial production. The ecdysteroid, 20-hydroxyecdysone and its receptor, EcR, have a well-characterized developmental role in insects, where it is involved in the control of molting and metamorphosis. We have identified a D. immitis nuclear receptor, DiEcR that shows strong sequence similarity to the insect EcR and shares many of its biochemical properties, including ligand-dependent activation of transcription. However, unlike most insect EcRs, DiEcR requires a ligand-activated RXR partner to exhibit ligand-dependent transcriptional activation of a reporter gene in tissue culture., (Copyright 2010 Elsevier B.V. All rights reserved.)

Filarial parasites cause debilitating diseases in humans and domesticated animals. Brugia malayi and Dirofilaria immitis are transmitted by mosquitoes and infect humans and dogs, respectively. Their life cycle is punctuated by a series of cuticular molts as they move between different hosts and tissues. An understanding of the genetic basis for these developmental transitions may suggest potential targets for vaccines or chemotherapeutics. Nuclear receptor (NR) proteins have been implicated in molting in the free-living nematode Caenorhabditis elegans and have well characterized roles in molting during larval development of Drosophila melanogaster. For example, the D. melanogaster E75 (NR1D3) NR gene is required for molting and metamorphosis, as well as egg chamber development in adult females. We have identified Bm-nhr-11and Di-nhr-6, B. malayi and D. immitis orthologues of E75. Both genes encode canonical nuclear receptor proteins, are developmentally regulated, and are expressed in a sex-specific manner in adults.

Filarial parasites are responsible for several serious human diseases with symptoms such as lymphoedema, elephantiasis, and blindness. An understanding of how these parasites pass through developmental checkpoints may elucidate the general mechanisms of these illnesses and suggest potential targets for intervention. A useful model system for the study of human filariasis is the related nematode Dirofilaria immitis, the causative agent of dog heartworm disease. In D. immitis, molting from the third to the fourth larval stage can be induced in vitro by the insect hormone 20-OH ecdysone, suggesting that ecdysone, or some related hormone, may play a similar role in the development of D. immitis. Ecdysone has a well-characterized developmental role in insects, where it is involved in the control of molting and metamorphosis. We have identified a D. immitis orthologue of the Drosophila ecdysone response early gene E78, a member of the nuclear receptor (NR) superfamily. The D. immitis gene, Di-nhr-7 (NR1E1) encodes at least three isoforms, including two potential negative regulatory isoforms, and is expressed in a sex-specific manner. An MBP/Di-NHR-7 fusion protein is able to bind to DNA response elements that are recognized by the closely related mammalian NR Rev-erb(alpha).

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.

Three proteins isolated from the excretory-secretory products (ES) of larval Dirofilaria immitis have been previously characterized and termed the 20, 22L and 22U kDa proteins. Two of the proteins (20 and 22L) were produced and released around the time of the third molt and were specifically recognized by immune dog sera. An amino acid sequence common to both proteins was used to synthesize a DNA probe to molecularly clone these molecules from a 48-h third stage larval cDNA library. The DNA sequence of the isolated clones encoded a 17.5 kDa protein with a 21 amino acid hydrophobic leader sequence that when removed yielded a 15.3 kDa protein starting with the N-terminal sequence obtained from the 20 kDa protein and containing all sequences obtained from tryptic peptides of the 20 and 22L kDa proteins. It was hypothesized that the 20 and 22L kDa proteins were the same, differing only by a 21 amino acid hydrophobic leader sequence which was later cleaved. The calculated molecular masses were consistent with those determined by reducing Tris-tricine SDS-PAGE. Expression of the protein without the leader sequence was accomplished in Escherichia coli. Antiserum raised against the expressed protein demonstrated the presence of the protein in L3 and L4, but not in adults or microfilariae. Expression of the protein with the leader sequence using a baculovirus system demonstrated processing of the signal sequence at the same time as found in larval D. immitis ES. Sera from dogs immune to infection were reactive with the D. immitis proteins expressed in either E. coli or insect cells.

Dirofilaria immitis, a filarial nematode, is the causative agent of canine and feline heartworm disease. Previous research has demonstrated that immunity to D. immitis can be induced in dogs by repeated chemical abbreviation of infections while the parasite is a fourth-stage larva. Sera obtained from dogs immunized in this manner has been effective in passively transferring larval killing and stunting. These immune sera, by comparison to nonimmune sera from infected cohorts, recognize a number of unique D. immitis antigens, some of which are larval specific. In this study immune dog sera were used to screen a D. immitis larval cDNA expression library. Three overlapping cDNA clones, Di22, Di18 and Di16, were obtained that encode a portion of a large molecule, greater than 150 kDa, that is composed of multiples of a 399-bp repeat. This protein when immunoblotted with antibody against a recombinant expressed Di22 fusion protein is found in larval as well as adult extracts and excretory-secretory products, and is seen as a series of ascending subunits, each approximately 15 kDa larger than the previous one. This antigen is highly immunogenic, as evidenced by the strong reactivity of the recombinant expressed Di22 fusion protein with sera from immune dogs, microfilaremic dogs and infected amicrofilaremic dogs. While the function of this antigen is unknown it has significant sequence similarity with an allergen found in Ascaris.

The nucleotide sequence of a cDNA copy of the Dirofilaria immitis paramyosin gene was determined. The sequence was 2545 nucleotides in length, consisting of a single open reading frame of 848 amino acids capable of encoding a protein with a calculated molecular weight of 98,000. The cDNA clone was not complete, but probably includes over 97% of the coding region of the gene. We have previously observed that the cloned D. immitis paramyosin is recognized by sera from humans infected with Onchocerca volvulus. To determine the extent of homology at the protein level, we screened a cDNA library of O. volvulus with an antiserum made against D. immitis paramyosin. Ten recombinant clones were partially sequenced, comprising a total of 1186 nucleotides or 389 amino acids. The amino acid sequence of D. immitis paramyosin was 99% identical to the O. volvulus paramyosin. We also compared the amino acid sequence to other cloned paramyosins, and noted that 92% of the amino acids were identical to those of Caenorhabditis elegans, and 34% identical to those of Schistosoma mansoni. Comparison of the paramyosin sequence between different species revealed a hierarchy of similarities: (1) a 7-amino-acid repeat with apolar residues in the a and d position as the most conserved, followed by (2) the amino acid sequence and (3) the DNA sequence.

This report describes a specific and sensitive DNA probe for the identification of Brugia malayi. A genomic DNA library produced from subperiodic B. malayi microfilariae was screened to detect clones containing DNA sequences which are highly repeated within the parasite genome. Several clones were further analyzed to identify those which hybridize specifically with B. malayi DNA but not with DNA from B. pahangi and Dirofilaria immitis. From these, clone pBm15 was selected because it hybridized with high sensitivity to B. malayi DNA as detected by autoradiography. Clone pBm15 was sensitive enough to detect two infective larvae or five microfilariae or 300 pg of purified B. malayi microfilarial DNA. This study forms the basis for the development of a specific and sensitive DNA probe for the identification of B. malayi in field specimens.

The structure of ribosomal DNA (rDNA) clone pBmr7 from microfilariae of the human parasite Brugia malayi has been examined in detail by Southern blot analysis and S-1 mapping techniques. The results demonstrate that this clone contains regions homologous to 28S, 18S and 5.8S rDNAs. A noncoding or 'spacer' region lies between the 3' end of 28S rDNA and the 5' end of 18S rDNA. An AccI-Sau3AI fragment of approximately 900 bp from this spacer region cross-hybridizes to genomic DNA fragments of different sizes from Brugia pahangi and Dirofilaria immitis. The differences observed in hybridization suggest that this rDNA fragment can be used to differentiate between various filariid species.