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.

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.

Alkaloids with Anti-Onchocercal Activity from Voacanga africana Stapf (Apocynaceae): Identification and Molecular Modeling.

A new iboga-vobasine-type isomeric bisindole alkaloid named voacamine A (1), along with eight known compounds-voacangine (2), voacristine (3), coronaridine (4), tabernanthine (5), iboxygaine (6), voacamine (7), voacorine (8) and conoduramine (9)-were isolated from the stem bark of Voacangaafricana. The structures of the compounds were determined by comprehensive spectroscopic analyses. Compounds 1, 2, 3, 4, 6, 7 and 8 were found to inhibit the motility of both the microfilariae (Mf) and adult male worms of Onchocerca ochengi, in a dose-dependent manner, but were only moderately active on the adult female worms upon biochemical assessment at 30 µM drug concentrations. The IC50 values of the isolates are 2.49-5.49 µM for microfilariae and 3.45-17.87 µM for adult males. Homology modeling was used to generate a 3D model of the O. ochengi thioredoxin reductase target and docking simulation, followed by molecular dynamics and binding free energy calculations attempted to offer an explanation of the anti-onchocercal structure-activity relationship (SAR) of the isolated compounds. These alkaloids are new potential leads for the development of antifilarial drugs. The results of this study validate the traditional use of V. africana in the treatment of human onchocerciasis.

Predictive Value of Ov16 Antibody Prevalence in Different Subpopulations for Elimination of African Onchocerciasis.

The World Health Organization currently recommends assessing elimination of onchocerciasis by testing whether Ov16 antibody prevalence in children aged 0-9 years is below 0.1%. However, the certainty of evidence for this recommendation is considered to be low. We used the established ONCHOSIM model to investigate the predictive value of different Ov16-antibody prevalence thresholds in various age groups for elimination of onchocerciasis in a variety of endemic settings and for various mass drug administration scenarios. According to our simulations, the predictive value of Ov16 antibody prevalence for elimination depends highly on the precontrol epidemiologic situation, history of mass drug administration, the age group that is sampled, and the chosen Ov16-antibody prevalence threshold. The Ov16 antibody prevalence in children aged 5-14 years performs best in predicting elimination. Appropriate threshold values for this age group start at 2.0% for very highly endemic areas; for lower-endemic areas, even higher threshold values are safe to use. Guidelines can be improved by sampling school-aged children, which also is operationally more feasible than targeting children under age 10 years. The use of higher threshold values allows sampling of substantially fewer children. Further improvement can be achieved by taking a differentiated sampling approach based on precontrol endemicity.

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.

Performance evaluation of 3 serodiagnostic peptide epitopes and the derived multi-epitope peptide OvNMP-48 for detection of Onchocerca volvulus infection.

Current diagnostic tools to determine infection with the helminth parasite Onchocerca volvulus have limited performance characteristics. In previous studies, a proteome-wide screen was conducted to identify linear epitopes in this parasite's proteome, resulting in the discovery of 1110 antigenic peptide fragments. Here, we investigated three of these peptides using peptide ELISA's and evaluated their sensitivity and specificity. Epitope mapping was performed, and peptides were constructed that contained only the minimal epitope, flanked by a linker. Investigation of the performance of these minimal epitope peptides demonstrated that all three of them have a specificity (as defined by lack of response in non-helminth-infected individuals) of 100%, low cross-reactivity (5.6%, 5.6%, and 9.3%, respectively), but low sensitivity (36.9%, 46.5%, and 41.2%, respectively). Some cross-reactivity was observed in samples from individuals infected with soil-transmitted helminths or Brugia malayi. Combining these three minimal epitopes in a single peptide, called OvNMP-48, resulted in a performance that exceeded the sum of the individual epitopes, with a sensitivity of 76.0%, a specificity of 97.4%, and a cross-reactivity of 11.1%. Cross-reactivity was observed in some STH and Brugia malayi-infected individuals. This work opens the opportunity to start exploring how these novel linear epitope markers might become part of the O. volvulus diagnostic toolbox.

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.

Full mitochondrial and nuclear genome comparison confirms that Onchocerca sp. "Siisa" is Onchocerca ochengi.

Onchocerca ochengi is a nodule-forming filarial nematode parasite of cattle. It is the closest known relative of the human parasite Onchocerca volvulus, with which it shares the black fly vector Simulium damnosum. Onchocerca sp. "Siisa" was described in black flies and in cattle and, based on limited mitochondrial sequence information, appeared to be about equally phylogenetically distant from O. ochengi and O. volvulus. Based on molecular genetic markers and apparent interbreeding, we later proposed that O. sp. "Siisa" belongs to the species O. ochengi. However, we did not demonstrate directly that the hybrids were fertile, and we were still unable to resolve the phylogenetic relationship of O. ochengi, O. sp. "Siisa," and O. volvulus, leaving some concerns with the conclusion mentioned above. Here, we present fully assembled, manually curated mitochondrial genomes of O. ochengi and O. sp. "Siisa," and we compare multiple individuals of these two taxa with respect to their whole mitochondrial and nuclear genomes. Based on the mitochondrial genomes, O. ochengi and O. sp. "Siisa" are phylogenetically much closer to each other than to O. volvulus. The differences between them are well within the range of what is expected for within-species variation. The nuclear genome comparison provided no indication of genetic separation of O. ochengi and O. sp. "Siisa." From this, in combination with the earlier literature, we conclude that O. ochengi and O. sp. "Siisa" should be considered one species.

Review on medicinal plants and natural compounds as anti-Onchocerca agents.

Onchocerciasis is a filarial vector borne disease which affects several million people mostly in Africa. The therapeutic approach of its control was based on a succession of drugs which always showed limits. The last one: ivermectin is not the least. It was shown to be only microfilaricidal and induced resistance to the human parasite Onchocerca volvulus. The approach using medicinal plants used in traditional medicine is a possible alternative method to cure onchocerciasis. Onchocerca ochengi and Onchocerca gutturosa are the parasite models used to assess anthelmintic activity of potentially anthelmintic plants. Numerous studies assessed the in vitro and/or in vivo anthelmintic activity of medicinal plants. Online electronic databases were consulted to gather publications on in vitro and in vivo studies of anti-Onchocerca activity of plants from 1990 to 2017. Globally, 13 plant families were investigated for anti-Onchocerca activity in 13 studies. The most active species were Anacardium occidentale, Euphorbia hirta and Acacia nilotica each with an LC50 value of 2.76, 6.25 and 1.2 µg/mL, respectively. Polycarpol, voacamine, voacangine, ellagic acid, gallic acid, gentisic acid, 3-O-acetyl aleuritolic acid and (-)-epigallocatechin 3-O-gallate were the isolated plant compounds with anti-Onchocerca activity. Most of the assessed extract/compounds showed a good safety after in vivo acute toxicity assays and/or in vitro cytotoxicity test. The exception was the ethanol extract of Trichilia emetica, which killed completely and drastically mice at a dose of 3000 mg/kg. Several plant groups of compounds were shown active against Onchocerca sp. such as tannins, alkaloids, triterpenoids and essential oils. Nevertheless, none of the active compounds was subjected to clinical trial, to assessment of its diffusibility through nodular wall or its capability to induce genetic resistance of Onchocerca sp.

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.

Inverse density dependence of parity rates in the onchocerciasis vector Simulium damnosum s.l.

A correlation between parity rates and an index of adult numbers of Simulium damnosum s.l. (Diptera: Simuliidae) indicates an association, but does not prove causality or show the direction of any causal relationship. The question of whether adult numbers affect parity rates or vice versa is reminiscent of the age-old query of which of the chicken and the egg came first. A method for resolving such issues based on analyses of pairs of time series was proposed by Granger in 1969. When Granger's method was applied to monthly numbers of adult female S. damnosum s.l. caught attempting to bite humans at Asubende, Ghana, and their parity rates, a significant relationship (P = 0.005) emerged, clearly showing that parity rates were dependent on adult numbers. Implications of this inverse density dependence and the results of analyses of other similar time series are presented and discussed.

Nodding syndrome since 2012: recent progress, challenges and recommendations for future research.

We aim to review the current epidemiology of nodding syndrome (NS) and discuss relevant gaps in research. NS and convulsive epilepsy of unknown aetiology are clustered within the same villages and families in onchocerciasis-endemic areas. They are therefore potentially different clinical expressions of the same disease. It has been difficult to perform full autopsies on NS patients who die in remote villages. Adequate fixation of tissue immediately after death is critical for the examination of brain tissue. Therefore, post-mortem transsphenoidal brain biopsies, performed immediately after death by trained nurses, will provide the best option for obtaining tissue for analysis. We suspect that certain blackflies in onchocerciasis-endemic areas may transmit a novel pathogen that could cause NS and epilepsy. This is supported by a recent drop in the number of new NS cases coinciding with vector control activities aimed at reducing blackfly populations in northern Uganda. We propose that metagenomic studies of human samples, blackflies and microfilariae are conducted to screen for pathogens, and that a clinical trial is planned to evaluate the impact of larviciding against NS and epilepsy epidemics.

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.

Onchocerciasis caused by Onchocerca lupi: an emerging zoonotic infection. Systematic review.

Globalization has increased circulation of people, their food, livestock and pets in the world, and changes in the environment, climate and human behaviour have led to the rapid expansion of emerging infections throughout the world. One of the reasons of a new pathogen affecting humans is the passage from an animal to a human being. Onchocerca (O.) lupi, a filarial worm first described in a wolf in 1967, is an emerging pathogen which has been incriminated as the etiological agent for 205 canine, 2 feline and 18 human infections in Europe, Tunisia, Turkey, Iran and the USA. Most frequent findings in animals and humans are monolateral or asymmetrical variably painful subconjunctival swellings and nodules containing immature or mature worms affecting the eye and/or adjacent tissues accompanied by conjunctival hyperemia. Occasionally, subcutaneous nodules and masses affecting the spinal cord have been observed in humans. Diagnosis of O. lupi is achieved by microscopy of excised adult female worms which exhibit a particular cuticular structure and molecular analysis. Treatment consists in worm removal accompanied by antihelminthic, antibiotic and anti-inflammatory therapy.

Levels of infection, pathology and nodule size of Onchocerca flexuosa (Nematoda: Onchocercidae) in red deer (Cervus elaphus) from northern Spain.

Between 2005 and 2007, the presence of Onchocerca flexuosa (Wedl, 1856) was discovered and investigated in 110 red deer (Cervus elaphus) shot in the Riaño Regional Hunting Reserve, in the province of León (north-western Spain). Nodules containing O. flexuosa were located in the dorsal region and flanks of the deer. These were collected and measured, and some adult parasites were extracted from the nodules and identified by morphology and by obtaining mitochondrial 12S rDNA sequences, which were identical to those of previously published sequences for O. flexuosa. Some nodules were prepared for histology, embedded in paraffin, sectioned and stained with haematoxylin-eosin. Histologically, the worms were found in several compartments separated by an infiltrated fibrous tissue. These compartments were inhabited by several females and males, surrounded by a fibrous capsule. A total of 85.45% (95% confidence interval (CI): 78.86-92.04%) of red deer were parasitized, with a mean intensity of 9.53 ± 12.27 nodules/host, ranging between 1 and 74 nodules/deer. Significant differences in prevalence and intensity of infection were found between young and adult red deer, and also between seasons. However, no significant differences between males and females were observed. Five hundred and ninety-seven nodules were measured (15.81 ± 3.94 mm) and classified by sizes into small ( < 10 mm), medium (10-20 mm) and large (>20 mm). No relation was found between the size of the nodules and the time of infection. The high values found in the studied parameters show that northern Spain is an area of high-intensity infection for deer.