Phylogenetic characterization of Setaria equina and its association with other filarids.

Molecular characterization studies on Setaria equina are limited. The present study aimed to characterize S. equina at the cytochrome c oxidase gene and to examine its phylogenetic relationships with other filarid species. Sequence analysis showed 100% nucleotide homology with an S. equina sequence from Italy (AJ544873). However, both sequences exhibited 7 nucleotide substitutions from a S. equina donkey isolate from Egypt (MK541847). Overall, S. equina formed a monophyletic sister group to Setaria tundra. All Setaria spp. examined formed a separate group on the phylogenetic tree that was related to corresponding Onchocerca spp. and Dirofilaria spp. clades. Human filarid worms-Brugia spp. and Wuchereria spp. grouped in a separate clade alongside Theilezia spp. Dipetalonema spp.-formed a separate group at the top of the tree.

Identification of the complement 9-binding protein in Setaria equina excretory-secretory products.

The current study aimed to detect the complement-binding proteins in the excretory-secretory (ES) products of adult filarial parasite Setaria equina (SeqES). Tests for complement activation pathways (CH50 and APH50 ) in normal human serum (NHS) after incubation with SeqES were performed. Quantitative detection of complement activation products like C3d and sC5b-9 by ELISA in inulin-activated NHS before and after addition of SeqES was estimated. Immunoblotting for 1D and 2D electrophoresed SeqES were performed for detection of C9-binding protein. MALDI mass sequencing and multiple sequence alignment were performed for identification of the protein. The results showed an inhibitory effect of SeqES for complement activation pathways. This was confirmed by an obvious reduction in C3d and sC5b-9 in inulin-activated NHS. Immunoblotting showed the reaction of a protein at 21 kDa with human C9. The latter protein was identified as OV-16 based on MALDI mass sequencing and multiple sequence alignment. In conclusion, S equina OV-16 is the complement regulatory protein by its ability to bind C9 and inhibit the classical and alternative pathways of complement activation. This protein can be used as a target for therapeutic treatment or as an anti-inflammatory agent in human diseases.

Subconjunctival nodule due to Setaria equina erratic migration in a horse: First case report.

An 18-month-old Arabian-English filly resident in southwest France was referred for evaluation of a conjunctival mass in the right eye (OD). A pink, solid, and mobile nodular formation, measuring approximately 1.2 × 0.8 cm was found under the superior nasal bulbar conjunctiva during an ophthalmic examination that was otherwise normal. The mass was surgically removed using a standing procedure. Cytological examination of fine-needle aspirates from the mass revealed a mixed eosinophilic-lymphocytic inflammation. Histological examination confirmed the dense and diffuse eosinophilic-lymphocytic infiltrate of the mass, and it revealed several cross sections of a parasitic nematode. The morphometric diagnosis identified an immature form of a filarial worm, and molecular analysis of the mitochondrial cytochrome c oxydase subunit 1 (cox1) and 12S rRNA gene sequences led to further identification of the specimen as Setaria equina. Microfilaremia was not observed on fresh blood smears. There have been no signs of local recurrence after 18 months, nor any evidence of intraocular involvement. To the authors' knowledge, this is the first documented case of subconjunctival setariasis due to S equina in a horse.

Heat shock protein 70 of filarial parasite Setaria equina: Cloning, expression, and analysis of binding with diethylcarbamazine citrate.

Setaria equina heat shock protein (SeqHSP) 70 gene was characterized, cloned and expressed to recombinant protein (rSeqHSP70). The protein was tested for binding with an anti-filarial drug "diethylcarbamazine citrate (DEC)" by equilibrium dialysis method. Molecular docking was also used to determine the binding sites and residues of binding with DEC. The mice were immunized with the protein alone or bound to DEC. Serum IFN-γ levels in the immunized group with protein-drug complex were significantly higher (P < 0.05) than the protein-immunized group. Mouse anti-SeqHSP70 polyclonal IgG recognized 2 bands at 70 and 75 kDa in S. equina adult worm and human cancer cell lines (HepG2 and MCF-7) extracts. The proliferation assay for mice splenocytes revealed a potentiation and down-regulating effects in non-immunized and immunized groups, respectively with the drug-protein complex. The proliferation and IFN-γ assays for purified human NK cells indicated a potentiating effect of the drug-protein complex (DEC concentration is 50 µM) comparable to the protein. DEC at lower concentration (25 mM) could also show a significant increase (P < 0.05) in IFN-γ. From the results, DEC was postulated to induce conformational changes in the protein exposing more epitopes for NK cell binding and activation.

Filarial worm circulation by mosquitoes along an urbanization gradient in southern Spain.

Mosquitoes are the main vectors of pathogens affecting wild animals, livestock and humans. Here, we used molecular tools to assess the local circulation of filarial parasites in mosquitoes collected during 2013 from natural, rural and urban habitats from southern Spain. We screened parasites in 22,791 female mosquitoes of the genera Aedes, Culex and Culiseta. Filarial worms were only detected in two mosquito pools. An Ae. caspius pool was positive for Setaria equina and an unidentified worm related to Onchocerca was detected in a Cx. pipiens pool. None of the mosquito pools were positive for Dirofilaria. These results underlay the role of Ae. caspius in the transmission of Setaria parasites among livestock and/or wildlife to humans in southern Spain.

Subconjunctival setariasis due to Setaria equina infection; a case report and a literature review.

A rare case of human subconjunctival setariasis due to Setaria equina infection is reported herein. A 15-years old girl was referred with a 24h history of edema and redness in her left eye. On slit lamp examination, a thread-like cylindrical worm was moving in the subconjunctival area. The worm was extracted, stained and measured 110mm in length 510µm in width. The isolated worm was identified as adult female S. equina based on morphometric criteria. Identification of the species of the worm was confirmed using molecular methods. For this purpose, the 12S rRNA gene was PCR-amplified and the purified amplicon was directly sequenced. After alignment, phylogenetic analysis revealed that the 12S rRNA sequence of this worm (Accession no.: KU291446) showed 100% identity with that of S. equina. This is the first case in Iran and provides evidence that S. equina can be an etiological agent of subconjunctival infection was isolated and diagnosed as where it located Middle East.