Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.

Molecular circumscription of the blackfly Simulium oyapockense from South America.

Problems related to the identity of Simuliidae species are impediments to effective disease control in Amazonia. Some of these species, such as Simulium oyapockense Floch & Abonnenc, 1946 (Diptera: Simuliidae), are vectors of the organisms that cause onchocerciasis and mansonellosis diseases. This blackfly species has a wide distribution in South America, and it is suspected of being a complex of cryptic species. The aim of this study is to characterize the nominal species S. oyapockense using partial COI gene sequences. Seven populations of S. oyapockense (morphologically identified) were analysed, including one from its type-locality. The other six populations were collected in Brazil and in Argentina. A taxon collected in Amazonas state, Brazil, with adults similar to S. oyapockense but with distinct pupae, was also included in the analysis (Simulium 'S'). The nominal species S. oyapockense is circumscribed, and its geographical distribution is restricted to areas north of the Amazon River. Populations of S. oyapockense s.l. collected south of the Amazon River comprise a species complex that needs to be evaluated using integrative taxonomy. Simulium 'S' represents a species with unique morphological and molecular characteristics. Distinguishing cryptic species is a prerequisite for reducing the taxonomic impediment, especially in medically important taxa.

Prospects for developing efficient targets for the xenomonitoring and control of Simulium damnosum s.l., the major vectors of onchocerciasis in Africa.

Despite the ethical issues concerning the continued use of Human Landing Catches (HLC) to monitor the Simulium damnosum complex for epidemiological monitoring of onchocericasis, few attempts to develop alternatives have been reported. In studies on a wild population of S. damnosum in Burkina Faso, we tested visual targets (different sizes and shapes) and olfactory stimuli (CO2 , and POCA and BG-lure® odour blends) for their ability to attract and collect host seeking blackflies. At each trap, blackflies were caught with appropriately sized electrocuting grids and results from Latin square design tests were compared. Throughout, HLCs captured more blackflies than the targets. Of the traps tested, small targets (0.0625 and 0.5 m2 ) were the most efficient visual lure in terms of the number of S. damnosum captured per unit area 1.7-5× more than larger targets. Overall, results suggested that sticky black targets of horizontal rectangular shape (0.125-0.5 m2 ) and baited with a POCA and/or CO2 mixture could provide a cheap practical field alternative to HLC for onchocerciasis xenomonitoring, subject to confirmation that the design has no inherent bias for certain members of the S. damnosum species complex.

Micro-CT imaging of Onchocerca infection of Simulium damnosum s.l. blackflies and comparison of the peritrophic membrane thickness of forest and savannah flies.

Onchocerciasis is a neglected tropical disease (NTD) caused by Onchocerca Diesing 1841 (Spirurida: Onchocercidae) nematodes transmitted by blackflies. It is associated with poverty and imposes a significant health, welfare and economic burden on many tropical countries. Current methods to visualize infections within the vectors rely on invasive methods. However, using micro-computed tomography techniques, without interference from physical tissue manipulation, we visualized in three dimensions for the first time an L1 larva of an Onchocerca species within the thoracic musculature of a blackfly, Simulium damnosum s.l. Theobald 1903 (Diptera: Simuliidae), naturally infected in Ghana. The possibility that thicker peritrophic membranes in savannah flies could account for their lower parasite loads was not supported, but there were limits to our analysis. While there were no statistically significant differences between the mean thicknesses of the peritrophic membranes, in the anterior, dorsal and ventral regions, of forest and savannah blackflies killed 34-48 min after a blood-meal, the thickness of the peritrophic membrane in the posterior region could not be measured. Micro-computed tomography has the potential to provide novel information on many other parasite/vector systems and impactful images for public engagement in health education.

Mosquito Magnet® traps as a potential means of monitoring blackflies of medical and veterinary importance.

Mosquito Magnet® traps, deployed in widespread parts of England as part of nationwide mosquito surveillance projects, also caught blackflies. As many as 1242 blackflies were caught in a trapping session lasting 4 days. Principal among the species caught were Simulium equinum, Simulium lineatum and Simulium ornatum s.l. As S. ornatum s.l. is a vector that transmits Onchocerca linealis to cattle and S. equinum is responsible for dermatitis ('sweet itch') in cattle and horses, it is suggested that Mosquito Magnet® traps could be used to monitor and partially control these pests, as well as nuisance anthropophilic blackflies such as Simulium posticatum that can cause simuliidosis in southern England.

Onchocerca ochengi male worms implanted in SCID mice and Gerbil: Relationship between microfilaridermia status of cows, nodular worm viability and fertility and worm survival in the rodents.

BACKGROUND: Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. METHODS: Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. RESULTS: Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf-. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf- cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf- cows, respectively. Female worms from nodules of mf- cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. CONCLUSION: Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents.

Coyotes as Reservoirs for Onchocerca lupi, United States, 2015-2018.

The Onchocerca lupi nematode infects dogs, cats, and humans, but whether it can be spread by coyotes has been unknown. We conducted surveillance for O. lupi nematode infection in coyotes in the southwestern United States. We identified multiple coyote populations in Arizona and New Mexico as probable reservoirs for this species.

Survey of infection and determination of the transmission vector of Onchocerca fasciata in camels (Camelus bactrianus) in Inner Mongolia, China.

Onchocerciasis in camels is caused by adult Onchocerca spp. and results in great economic losses to the camel industry. However, only a few studies on Onchocerca have been conducted, especially regarding the intermediate host and vector(s). In the present study, 192 camels were examined from December and January during 2013 and 2016, and the filarial larvae suspected to be Onchocerca spp. were further identified. Furthermore, aquatic dipteran insects in the living environment of camels were collected from May to September between 2013 and 2017 and dissected. Eventually, onchocercal lesions were observed in 95 of 192 (49%) camels and the captured insects were classified into 49 species from 42 genera in 21 families, among which 18 species were newly recorded in Inner Mongolia and 14 were haematophagous species. The filarial larvae were found in Culicoides puncticollis and identified as Onchocerca fasciata, indicating that C. puncticollis is the vector of O. fasciata in Inner Mongolia. These findings provide an estimate of onchocerciasis infection in camels and an alternative method of identifying insects and screening vectors using molecular methods. Important data are also provided for the diagnosis and control of onchocerciasis, thereby further filling the gap in knowledge regarding transmission vectors in China.

First molecular detection of Onchocerca flexuosa (Wedl, 1856) in red deer in Slovakia.

The present paper deals with the post-mortem diagnostics of onchocerciasis and the molecular detection of causative agents of this disease in wild ruminant ungulates (Cervus elaphus, Dama dama and Capreolus capreolus). The animals were shot in hunting seasons 2017 and 2018, in two regions of the Eastern Slovakia. The total number of examined skins was fifty-eight. The presence of subcutaneous nodules was confirmed in 27.59% (95% CI 16-39) of animals. All positive skins belonged to red deer individuals (47.06%; 95% CI 30-64). The nodules were present mainly in the back area and in the lumbar area, and their sizes ranged from 2.9 to 24.1 mm, with the average count of 10 nodules per animal. Thirteen worms, isolated from the nodules collected from 13 animals, were subjected to molecular identification. Applying the standard PCR method, targeting the mitochondrial 12S rRNA, 16S rRNA and NADH-dehydrogenase gene, and subsequent sequencing, all the worms were identified as Onchocerca flexuosa Wedl, 1856. The sequences were submitted to GenBank under specific accession numbers. Two samples were identified as Onchocerca flexuosa haplotype B, in which T176A and A177T were present. Despite the presence of mutations in the 12S rRNA of the Onchocerca flexuosa, the standardized PCR remains to be a very specific and sensitive method that uses this fragment as a selectable marker for the detection of the studied parasite.

Characterization of a novel glycosylated glutathione transferase of Onchocerca ochengi, closest relative of the human river blindness parasite.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

Onchocerca - infected cattle produce strong antibody responses to excretory-secretory proteins released from adult male Onchocerca ochengi worms.

BACKGROUND: The front line molecules from filarial worms and other nematodes or helminthes are their Excretory-Secretory (ES) products. Their interaction with the host cells, proteins and immune system accounts for the skin and eye pathology or hyposensitivity observed in human onchocerciasis. ES products and adult worms' crude extracts from Onchocerca ochengi, a filarial nematode that infects the African zebu cattle, were utilized in the present study as a model for studying Onchocerca volvulus that causes river blindness in man. METHODS: The ES products were generated from adult male and female worms in vitro and analyzed with poly acrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) using sera from Onchocerca-infected cattle and humans. The cattle sera were collected from a herd that had been exposed for six years to natural transmission of Onchocerca spp. The expressed reactivity was evaluated and differences analyzed statistically using Kruskal-Wallis rank and Chi-square tests. RESULTS: The gel electrophoretic analyses of 156 ES products from O. ochengi female and male worms and of two somatic extracts from three females and 25 males revealed differences in the protein pattern showing pronounced bands at 15, 30-50 and 75 kDa for male ES proteins and 15, 25 and 40-75 kDa for somatic extracts, respectively and less than 100 kDa for female worms. Proteins in the ES products and somatic extracts from female and male Onchocerca ochengi worms were recognized by IgG in sera from both Onchocerca-exposed cattle and humans. Bovine serum antibodies reacted more strongly with proteins in the somatic extracts than with those in the ES products. Interestingly, the reaction was higher with male ES products than with ES products from female worms, suggesting that the males which migrate from one nodule to another are more exposed to the host immune system than the females which remain encapsulated in intradermal nodules. CONCLUSIONS: This study demonstrates that O. ochengi ES products and, in particular, extracts from male filariae may represent a good source of immunogenic proteins and potential vaccine candidates.

Stage-specific Proteomes from Onchocerca ochengi, Sister Species of the Human River Blindness Parasite, Uncover Adaptations to a Nodular Lifestyle.

Despite 40 years of control efforts, onchocerciasis (river blindness) remains one of the most important neglected tropical diseases, with 17 million people affected. The etiological agent, Onchocerca volvulus, is a filarial nematode with a complex lifecycle involving several distinct stages in the definitive host and blackfly vector. The challenges of obtaining sufficient material have prevented high-throughput studies and the development of novel strategies for disease control and diagnosis. Here, we utilize the closest relative of O. volvulus, the bovine parasite Onchocerca ochengi, to compare stage-specific proteomes and host-parasite interactions within the secretome. We identified a total of 4260 unique O. ochengi proteins from adult males and females, infective larvae, intrauterine microfilariae, and fluid from intradermal nodules. In addition, 135 proteins were detected from the obligate Wolbachia symbiont. Observed protein families that were enriched in all whole body extracts relative to the complete search database included immunoglobulin-domain proteins, whereas redox and detoxification enzymes and proteins involved in intracellular transport displayed stage-specific overrepresentation. Unexpectedly, the larval stages exhibited enrichment for several mitochondrial-related protein families, including members of peptidase family M16 and proteins which mediate mitochondrial fission and fusion. Quantification of proteins across the lifecycle using the Hi-3 approach supported these qualitative analyses. In nodule fluid, we identified 94 O. ochengi secreted proteins, including homologs of transforming growth factor-ß and a second member of a novel 6-ShK toxin domain family, which was originally described from a model filarial nematode (Litomosoides sigmodontis). Strikingly, the 498 bovine proteins identified in nodule fluid were strongly dominated by antimicrobial proteins, especially cathelicidins. This first high-throughput analysis of an Onchocerca spp. proteome across the lifecycle highlights its profound complexity and emphasizes the extremely close relationship between O. ochengi and O. volvulus The insights presented here provide new candidates for vaccine development, drug targeting and diagnostic biomarkers.

In vivo confocal microscopy for detection of subconjunctival Onchocerca lupi infection in a dog.

A seven-year-old male castrated mixed-breed dog was diagnosed with bilateral subconjunctival masses. In vivo confocal microscopy facilitated visualization of Onchocerca lupi adult nematodes and their characteristic cuticular morphology. Long, thin, white nematodes were extracted during excisional biopsy. Histopathologic and parasitologic evaluation confirmed the diagnosis of O. lupi. In addition to surgical debulking of the parasitic granulomas, the dog received systemic doxycycline, prednisone, and ivermectin therapy. In vivo confocal microscopy was repeated one year after initial diagnosis, and no remaining nematodes were visible. To the authors' knowledge, this is the first report of use of in vivo confocal microscopy as a noninvasive diagnostic and monitoring tool for canine onchocerciasis.

Filarial infection caused by Onchocerca boehmi (Supperer, 1953) in a horse from Italy.

Equids can be infected by a range of skin-dwelling filarial nematodes, including four species of the genus Onchocerca. Current literature on equine onchocercosis is fragmentary and often limited to isolated case reports. The present study aimed to describe a clinical case of equine onchocercosis caused by Onchocerca boehmi (Supperer, 1953) (syn. Elaeophora boehmi) in an 8-year-old gelding Belgian show jumper from northern Italy. The horse was presented with a firm and painless mass on the proximal third of the right metacarpal region. Ultrasound examination showed a peritendinous enlargement around the palmaro-lateral area of the tendons, characterized by an elongated hypoechoic and well-defined structure, embedding a coiled hyperechoic line. The metacarpal nodule was resected and histologically examined. Fragments of a parasitic nematode were detected, isolated and examined. The morphological analysis allowed identifying the nematode as O. boehmi. In addition, total genomic DNA was extracted from individual fragments using a commercial kit for the nematode identification and a comparative sequence analysis of the nematode cytochrome oxidase subunit 1 (cox1) sequence with data available in the GenBankTM database revealed the closest identity (i.e. 91 %) with that of Onchocerca lupi. Thus far, O. boehmi has only been reported in Austria and Iran, and information about its life-cycle and vectors is lacking. The systematic position of this species within the genus Onchocerca, not in Elaeophora where it was originally described, is in concordance with the morphological and molecular analysis. In this article, we describe the first autochthonous case of equine onchocercosis in Italy caused by O. boehmi and discuss novel parasitological, clinical, and pathological data on these pathogens of horses.

Phytochemical analysis and in vitro anthelmintic activity of Lophira lanceolata (Ochnaceae) on the bovine parasite Onchocerca ochengi and on drug resistant strains of the free-living nematode Caenorhabditis elegans.

BACKGROUND: Onchocerciasis is one of the tropical neglected diseases (NTDs) caused by the nematode Onchocerca volvulus. Control strategies currently in use rely on mass administration of ivermectin, which has marked activity against microfilariae. Furthermore, the development of resistance to ivermectin was observed. Since vaccine and safe macrofilaricidal treatment against onchocerciasis are still lacking, there is an urgent need to discover novel drugs. This study was undertaken to investigate the anthelmintic activity of Lophira lanceolata on the cattle parasite Onchocerca ochengi and the anthelmintic drug resistant strains of the free living nematode Caenorhabditis elegans and to determine the phytochemical profiles of the extracts and fractions of the plants. METHODS: Plant was extracted in ethanol or methanol-methylene chloride. O. ochengi, C. elegans wild-type and C. elegans drug resistant strains were cultured in RPMI-1640 and NGM-agar respectively. Drugs diluted in dimethylsulphoxide/RPMI or M9-Buffer were added in assays and monitored at 48 h and 72 h. Worm viability was determined by using the MTT/formazan colorimetric method. Polyphenol, tannin and flavonoid contents were determined by dosage of gallic acid and rutin. Acute oral toxicity was evaluated using Swiss albino mice. RESULTS: Ethanolic and methanolic-methylene chloride extracts killed O. ochengi with LC50 values of 9.76, 8.05, 6.39 µg/mL and 9.45, 7.95, 6.39 µg/mL respectively for leaves, trunk bark and root bark after 72 h. The lowest concentrations required to kill 50% of the wild-type of C. elegans were 1200 and 1890 µg/mL with ethanolic crude extract, 1000 and 2030 µg/mL with MeOH-CH2Cl2 for root bark and trunk bark of L. lanceolata, respectively after 72 h. Leave extracts of L. lanceolata are lethal to albendazole and ivermectin resistant strains of C. elegans after 72 h. Methanol/methylene chloride extracted more metabolites. Additionally, extracts could be considered relatively safe. CONCLUSION: Ethanolic and methanolic-methylene chloride crude extracts and fractions of L. lanceolata showed in vitro anthelmintic activity. The extracts and fractions contained polyphenols, tannins, flavonoids and saponins. The mechanism of action of this plant could be different from that of albendazole and ivermectin. These results confirm the use of L. lanceolata by traditional healers for the treatment of worm infections.

The Emergence of Zoonotic Onchocerca lupi Infection in the United States--A Case-Series.

This case-series describes the 6 human infections with Onchocerca lupi, a parasite known to infect cats and dogs, that have been identified in the United States since 2013. Unlike cases reported outside the country, the American patients have not had subconjunctival nodules but have manifested more invasive disease (eg, spinal, orbital, and subdermal nodules). Diagnosis remains challenging in the absence of a serologic test. Treatment should be guided by what is done for Onchocerca volvulus as there are no data for O. lupi. Available evidence suggests that there may be transmission in southwestern United States, but the risk of transmission to humans is not known. Research is needed to better define the burden of disease in the United States and develop appropriately-targeted prevention strategies.

Onchocerca lupi Nematodes in Dogs Exported from the United States into Canada.

The Onchocerca lupi nematode is an emerging helminth capable of infecting pets and humans. We detected this parasite in 2 dogs that were imported into Canada from the southwestern United States, a region to which this nematode is endemic. We discuss risk for establishment of O. lupi in Canada.

Isolation of Onchocerca lupi in Dogs and Black Flies, California, USA.

In southern California, ocular infections caused by Onchocerca lupi were diagnosed in 3 dogs (1 in 2006, 2 in 2012). The infectious agent was confirmed through morphologic analysis of fixed parasites in tissues and by PCR and sequencing of amplicons derived from 2 mitochondrially encoded genes and 1 nuclear-encoded gene. A nested PCR based on the sequence of the cytochrome oxidase subunit 1 gene of the parasite was developed and used to screen Simulium black flies collected from southern California for O. lupi DNA. Six (2.8%; 95% CI 0.6%-5.0%) of 213 black flies contained O. lupi DNA. Partial mitochondrial16S rRNA gene sequences from the infected flies matched sequences derived from black fly larvae cytotaxonomically identified as Simulium tribulatum. These data implicate S. tribulatum flies as a putative vector for O. lupi in southern California.

Canine Infections with Onchocerca lupi Nematodes, United States, 2011-2014.

Infections with Onchocerca lupi nematodes are diagnosed sporadically in the United States. We report 8 cases of canine onchocercosis in Minnesota, New Mexico, Colorado, and Florida. Identification of 1 cytochrome c oxidase subunit 1 gene haplotype identical to 1 of 5 from Europe suggests recent introduction of this nematode into the United States.

Litomosoides sigmodontis: a jird urine metabolome study.

The neglected tropical disease onchocerciasis affects more than 35 million people worldwide with over 95% in Africa. Disease infection initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious L3 larvae. New treatments and diagnostics are required to eradicate this parasitic disease. Herein, we describe that a previously discovered biomarker for onchocerciasis, N-acetyltyramine-O-glucuronide (NATOG) is also present in urine samples of jirds infected with the onchocerciasis model nematode Litomosoides sigmodontis. Increased NATOG values paralleled a progressing infection and demonstrated that quantification of NATOG in this rodent model can be utilized to track its infectivity. Moreover, our findings suggest how NATOG monitoring may be used for evaluating potential drug candidates.