Setaria cervi (Filarioidea, Onchocercidae) undressing in ungulates: altered morphology of developmental stages, their molecular detection and complete sequence cox1 gene.

This work introduces new morphological and molecular information on the filaroid nematode Setaria cervi (Rudolphi, 1819) obtained from 13 infected game ungulates out of 96 dissected. The hosts comprised the following: a single moose (Alces alces), ten red deer (Cervus elaphus) and two sika deer (Cervus nippon) originating from the western and northern regions of the Czech Republic. Based on the complete sequences of the gene encoding mitochondrial cytochrome c oxidase subunit 1 (cox1), all 20 females and four males belonged to the species S. cervi. We detected three developmental female stages (adult fertile females, juvenile L5 females and L4 female larvae) differing in size and some morphological traits as the subtle structure of peribuccal crown and shape and features of tail knob. Such differences were described in detail for the first time. The phylogenetic relationships within the family Onchocercidae have been evaluated using new information on the cox1 sequence of S. cervi (maximum likelihood method, GTR + I + G model). In accordance with the latest phylogenetic studies, the present analysis confirmed the ancient separation of the subclass Setariinae from the remaining two onchocercid lineages Dirofilariinae and Onchocerinae.

Setaria labiatopapillosa (Filarioidea, Nematoda) in Moroccan cattle: atypical localization and morphological characterization of females and microfilariae by light and scanning electron microscopy.

Filarioid nematodes are parasites of the tissues and tissue spaces of all vertebrates except fish. Females produce microfilariae that enter the host's blood circulation or skin and may cause ocular and neurological pathology, leading to important implications in veterinary and public health. The present work is the first investigation on Setaria labiatopapillosa conducted in Morocco to characterize the morphological features of both adult and microfilaria forms. Two adult female nematodes were found free in the thoracic cavity of a slaughtered 3.5-year-old (6 teeth) Moroccan enhanced cross-breed bull which was born and raised in Morocco. The worms were identified as S. labiatopapillosa by light microscope (LM) and scanning electron microscopy (SEM) on the basis of their characteristic features of the anterior and posterior parts of the worms. The two S. labiatopapillosa worms measured 90 mm and 105 mm in length and 0.55 and 0.64 mm in width, respectively. Microfilariae were detected in the fully developed eggs contained in the uterus of both nematodes. A detailed morphology of both the adult females and larvae of S. labiatopapillosa is described using LM and SEM. Although the origin of S. labiatopapillosa analyzed in the present study is unknown and there is currently no evidence that Setaria spp. have invaded Morocco, further surveillance is warranted to determine the incidence of setariasis, identify its vectors, and take appropriate measures to protect the livestock and cattle industry of the country.

Antifilarial effect of nanocomposite of silver nanoparticles with nitazoxanide against the microfilariae of Setaria cervi-infected albino rats.

The aim of the present study was to assess the effect of diethylcarbamazine (DEC), siver nanoparticles (AgNPs), nitazoxanide (NTZ), and a combination of nitazoxanide with silver nanoparticle (NTZ+AgNPs) against the microfilariae of Setaria cervi in experimentally infected albino rats. The NTZ+AgNPs was synthesized and subsequently characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV-visible absorption Spectra (UV-VIS), Fourier transforms infrared spectroscopy (FTIR), and energy dispersive X-ray (EDX) spectra. Twenty male albino rats were divided into 5 groups. Groups I, II, III, and IV were treated with DEC, AgNPs, NTZ, and NTZ+AgNPs, while group V was taken as untreated infected control. After the establishment of infection, microfilaraemic rats were treated with aforesaid drugs for 6 days at 100 mg/kg body weight. Efficacy of drugs was observed by counting the microfilariae in the blood of albino rats every 3rd day till microfilariae disappeared. Blood was taken at every 10 days interval till 40 days for biochemical studies to assess the level of antioxidant enzymes. NTZ+AgNPs proved to be the most effective drug which cleared the microfilariae within 18 days of infection when compared with DEC, AgNPs and NTZ where microfilariae persisted up to 24, 36, and 33 days, respectively. Oxidative stress is common inflammatory process associated with many diseases including filariasis. An enhanced antioxidant activity of NTZ+AgNPs was observed in the infected rats which was evident by quick disappearance of microfilariae due to increased oxidative stress. It clearly indicated positive contribution of the NTZ+AgNPs to the host together with harmful effect on the parasite. Hence, AgNPs improved the NTZ efficacy against S. cervi infection in albino rats and proved as a successful synergistic combination.

First report of the isolation and phylogenetic characterization of equine Setaria digitata from India based on mitochondrial COI, 12S rDNA, and nuclear ITS2 sequence data.

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.

First report of equine Setaria digitata (von Linstow 1906) infestation in Malaysia.

The occurrence of Setaria digitata in a horse is reported for the first time in Malaysia. An 8-year-old Thoroughbred cross mare was referred to the University Veterinary Clinic with the primary complaint of corneal opacity and excessive eye discharge. After initial treatment with Terramycin eye ointment, corneal opacity cleared partially to reveal a moving thread-like cylindrical worm in the anterior chamber of the eye. The parasite was successfully removed surgically, and examination under the light microscope revealed that the isolated worm (length = 45 mm) was a 5th stage larva of S. digitata based on morphological criteria. Confirmation of the species of the worm was through molecular methods. The 12S rRNA gene was PCR-amplified, and the purified amplicon was directly sequenced. Phylogenetic analyses revealed that the isolated roundworm showed 100% sequence similarity with that of S. digitata in NCBI GenBank database (Accession no.: KY284626.1). This report is the first confirmed case of equine ocular setariasis by S. digitata in Malaysia. The current study provides evidence that S. digitata is an etiological agent of ocular infection and its presence in Malaysia.

Screening of mosquitoes for filarioid helminths in urban areas in south western Poland-common patterns in European Setaria tundra xenomonitoring studies.

In recent years, numerous studies screening mosquitoes for filarioid helminths (xenomonitoring) have been performed in Europe. The entomological monitoring of filarial nematode infections in mosquitoes by molecular xenomonitoring might serve as the measure of the rate at which humans and animals expose mosquitoes to microfilariae and the rate at which animals and humans are exposed to the bites of the infected mosquitoes. We hypothesized that combining the data obtained from molecular xenomonitoring and phenological studies of mosquitoes in the urban environment would provide insights into the transmission risk of filarial diseases. In our search for Dirofilaria spp.-infected mosquitoes, we have found Setaria tundra-infected ones instead, as in many other European studies. We have observed that cross-reactivity in PCR assays for Dirofilaria repens, Dirofilaria immitis, and S. tundra COI gene detection was the rule rather than the exception. S. tundra infections were mainly found in Aedes mosquitoes. The differences in the diurnal rhythm of Aedes and Culex mosquitoes did not seem a likely explanation for the lack of S. tundra infections in Culex mosquitoes. The similarity of S. tundra COI gene sequences found in Aedes vexans and Aedes caspius mosquitoes and in roe deer in many European studies, supported by data on Ae. vexans biology, suggested host preference as the most likely cause of the mosquito genus-biased infections. High diversity of the COI gene sequences isolated in the city of Wroclaw in south western Poland and the presence of identical or almost identical sequences in mosquitoes and roe deer across Europe suggests that S. tundra has been established in most of Europe for a very long time.

Exploring the homolog of a novel proinflammatory microfilarial sheath protein (MfP) of Wuchereria bancrofti in the adult-stage bovine filarial parasite Setaria cervi.

A novel microfilarial sheath protein (MfP) of the human filarial parasite Wuchereria bancrofti and its proinflammatory activity on host macrophages were identified recently. MfP is a homolog of the nematode bestrophin-9 superfamily that acts as a ligand of macrophage Toll-like receptor 4 (TLR4) to induce inflammation through NF-κB activation. Therefore, the presence and functional implication of this novel protein in adult-stage parasites were open questions to answer. In this study, the bovine filarial parasite Setaria cervi was used to simulate adult W. bancrofti. We detected the presence of MfP in adult-stage S. cervi through clear immunological cross-reactivity and immunolocalization employing an anti-MfP antibody developed in mice. Therefore, our findings put forward S. cervi as a cost-effective source of immunodominant filarial antigen MfP to simulate its future utilization in the immunotherapeutic intervention of lymphatic filariasis.

The annual, temporal and spatial pattern of Setaria tundra outbreaks in Finnish reindeer: a mechanistic transmission model approach.

BACKGROUND: In northern Finland (Lapland), reindeer are reared as semi-domesticated animals. The region has a short summer season of 2-3 months, yet reindeer are infected with the mosquito-borne filarioid parasite Setaria tundra. The infection causes peritonitis and perihepatitis, which cause significant economic losses due to reduced body weight of infected animals. The objective of this study was to: (i) describe the spatial and temporal pattern of outbreaks in three different areas across Finnish Lapland; and (ii) construct a temperature-driven mechanistic transmission model to quantify the potential role of temperature on intensity of S. tundra transmission in reindeer. METHODS: We developed a temperature-driven transmission model able to predict the number of S. tundra potentially transmitted from an infectious reindeer. We applied the model to the years 2004-2015, and compared the predictions to the proportion of reindeer whose livers were condemned due to S. tundra infection at the time of slaughter. RESULTS: The mean proportion of liver condemnation increased in reindeer slaughtered in late autumn/winter compared to earlier dates. The outbreaks were geographically clustered each year but there were no fixed foci where outbreaks occurred. Larger outbreaks were recorded in the southern regions of reindeer-herding areas compared to the central or northern parts of Lapland. Our model showed that temperatures never allowed for transmission of more than a single generation of S. tundra each season. In southern (Kuusamo) and central (Sodankylä) Lapland, our model predicted an increasing trend from 1979 to 2015 for both the duration of the effective transmission period of S. tundra (P < 0.001) and for the potential number of L3 S. tundra larvae being transmitted from an infectious reindeer (P < 0.001). CONCLUSIONS: The effective transmission period for S. tundra in reindeer is very short in Lapland, but it increased over the period studied. Only one generation of S. tundra can be transmitted in one season among reindeer in Lapland. Increasing temperatures may facilitate a range expansion and increasing duration of effective transmission period for S. tundra.

Oleanolic acid from antifilarial triterpene saponins of Dipterocarpus zeylanicus induces oxidative stress and apoptosis in filarial parasite Setaria digitata in vitro.

Absence of a drug that kills adult filarial parasites remains the major challenge in eliminating human lymphatic filariasis (LF); the second leading cause of long-term and permanent disability. Thus, the discovery of novel antifilarial natural products with potent adulticidal activity is an urgent need. In the present study, methanol extracts of leaves, bark and winged seeds of Dipterocarpus zeylanicus (Dipterocarpaceae) were investigated for macro and microfilaricidal activity. Two antifilarial triterpene saponins were isolated from winged seed extracts by bioactivity guided chromatographic separation and identified using Nuclear Magnetic Resonance and mass spectroscopic analysis as oleanolic acid 3-O-ß-D- glucopyranoside (1) (IC50 = 20.54 µM for adult worms, 19.71 µM for microfilariae ) and oleanolic acid 3-O-α-L-arabinopyranoside (2) (IC50 = 29.02 µM for adult worms, 25.99 µM for microfilariae). Acid hydrolysis of both compounds yielded oleanolic acid (3) which was non or least toxic to human peripheral blood mono nuclear cells (Selectivity index = >10) while retaining similar macrofilaricidal (IC50 = 38.4 µM) and microfilaricidal (IC50 = 35.6 µM) activities. In adult female worms treated with 50 and 100 µM doses of oleanolic acid, condensation of nuclear DNA, apoptotic body formation and tissue damage was observed by using Hoechst 33342 staining, TUNEL assay and Hematoxylin and Eosin staining respectively. A dose dependent increase in caspase 3/CED3 activity and decrease in total protein content were also observed in these parasites. A dose dependant DNA fragmentation was observed in adult parasites and microfilariae. Decreased levels of reduced glutathione (GSH) and elevated levels of glutathione S transferase (GST), superoxide dismutase (SOD) and reactive oxygen species (ROS) were also observed in parasites treated with oleanolic acid indicating an oxidative stress mediated apoptotic event. Compound 3/oleanolic acid was thus identified as a potent and safe antifilarial compound in vitro.

Characterization of the complete mitochondrial genome of Setaria digitata (Nematoda: Setariidae) from China.

Setaria digitata is a filarial parasite that causes fatal cerebrospinal nematodiasis in goats, horses and sheep, resulting in substantial economic losses to livestock farmers. In the present study, the complete mitochondrial (mt) genome of S. digitata from China was determined, characterized and compared with that of S. digitata from Sri Lanka. The identity of the mt genomes was 98.3% between S. digitata from China and Sri Lanka, and the complete mt genome sequence of S. digitata from China was slightly shorter (25 bp) than that from Sri Lanka. For the 12 protein genes, this comparison revealed sequence differences at both the nucleotide (1.4%) and amino acid (2.2%) levels. The present study determined the complete mt genome sequence of S. digitata from China, providing novel genetic markers for the study of the population genetics and molecular epidemiology of S. digitata in animals.

Congenital Filariasis Caused by Setaria bidentata (Nematoda: Filarioidea) in the Red Brocket Deer (Mazama americana).

The filarial nematode Setaria bidentata was found in 10 of 31 fetuses of the red brocket deer ( Mazama americana ) from the Loreto region of the Peruvian Amazon. A total of 25 specimens were collected and morphologically identified as S. bidentata. Filarial nematodes were found in the peritoneal cavity of 9 deer fetuses and the thoracic cavity of 1 fetus. Most specimens were adult stage. In this report, we provide morphometric data for these filarial specimens. This is the first study to demonstrate prenatal S. bidentata infection in cervid fetuses. Also, the finding of S. bidentata in Peru expands the geographic range of this parasite.

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.

First Blindness Cases of Horses Infected with Setaria Digitata (Nematoda: Filarioidea) in the Republic of Korea.

Ocular setariases of cattle were reported but those of equine hosts have never been reported in the Republic of Korea (Korea). We found motile worms in the aqueous humor of 15 horses (Equus spp.) from 12 localities in southern parts of Korea between January 2004 and November 2017. After the affected animals were properly restrained under sedation and local anesthesia, 10 ml disposable syringe with a 16-gauge needle was inserted into the anterior chamber of the affected eye to successfully remove the parasites. The male worm that was found in 7 of the cases showed a pair of lateral appendages near the posterior terminal end of the body. The papillar arrangement was 3 pairs of precloacal, a pair of adcloacal, and 3 pairs of postcloacal papillae, plus a central papilla just in front of the cloaca. The female worms found in the eyes of 8 horses were characterized by the tapering posterior terminal end of the body with a smooth knob. Worms were all identified as Setaria digitata (von Linstow, 1906) by the morphologic characteristics using light and electron microscopic observations. This is the first blindness cases of 15 horses infected with S. digitata (Nematoda: Filarioidea) in Korea.

Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis.

Evidence of reactive oxygen species (ROS) mediated apoptosis in Setaria cervi induced by green silver nanoparticles from Acacia auriculiformis at a very low dose.

Green synthesis of silver nanomaterial plays a pivotal role in the growing field of nanotechnology. Development of anti-parasitic drugs from plant metabolites has been in regular practice from the ancient period but most of them were discarded due to their inefficiency to control diseases effectively. At present, nanoparticles are used for developing anti-parasitic therapy for their unique properties such as smallest in size, bio-ability, bio-compatibility and penetration capacity into a cell. The present study aims at synthesis of silver nanoparticles (AgNPs) by using funicles extract of Acacia auriculiformis and tests its efficacy as antifilarial. Experimental evidence show that AgNPs are effective at a very low concentration compared to crude plant extracts. Synthesis of these nanoparticles is a single-step, biogenic, cost effective and eco-friendly process. Synthesized nanoparticles were characterized by UV-Vis spectroscopy, TEM, SAED, FTIR, EDX, FESEM and Z-potential. The antifilarial efficacy of AgNPs was tested against different life cycle stages of bovine filarial parasite Setaria cervi by morphological study, motility assessment and viability assay. These nanoparticles are found to have antifilarial activity with LC50 of 5.61 µg/mL and LC90 of 15.54 µg/mL against microfilaria of S. cervi. The microscopic findings and the detailed molecular studies confirmed that green synthesized AgNPs were effective enough to induce apoptosis through up regulation of ROS (reactive oxygen species).

Setaria digitata in advancing our knowledge of human lymphatic filariasis.

Setaria digitata is a filarial parasite that causes fatal cerebrospinal nematodiasis in goats, sheep and horses, resulting in substantial economic losses in animal husbandry in the tropics. Due to its close resemblance to Wuchereria bancrofti, this nematode is also frequently used as a model organism to study human lymphatic filariasis. This review highlights numerous insights into the morphological, histological, biochemical, immunological and genetic aspects of S. digitata that have broadened our understanding towards the control and eradication of filarial diseases.

Molecular mass screening of mosquitoes for filarial parasites in Germany--re-interpretation of PCR xenomonitoring results would be required.

Comments concerning interpretation of the PCR xenomonitoring results in the article "Molecular detection of Setaria tundra (Nematoda: Filarioidea) and an unidentified filarial species in mosquitoes in Germany" Parasites & Vectors 2012, 5:14.

Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis.

Molecular characterization of Setaria digitata from Mithun (Bos frontalis).

Mithun is a strongly built hill animal of Southeast Asia and plays an important role in the socio-economic and cultural life of the tribal population. Setaria digitata isolated from peritoneal cavity of mithun both from Arunachal Pradesh and Nagaland were characterized based on conserved region of 12SrDNA, 28SrDNA and ITS-2 and mitochondrial gene CoxI. Based on sequence analysis, it was found to be 99% similarity with Srilankan isolate of S. digitata.

Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 µM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 µM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.