Onchocerca sp. in an imported Zangersheide gelding causing suspensory ligament desmitis.

A 5-year-old imported Zangersheide gelding was evaluated for SC swellings over both forelimbs and lameness localized to the distal metacarpus. Ultrasound examination of the SC masses was compatible with verminous granulomas. Linear hyperechoic foci were present within the suspensory ligament branches of both forelimbs, suggestive of ligamentous parasitic infiltrates. A diagnosis of onchocerciasis was confirmed on biopsy of a SC mass. The gelding was treated with ivermectin and a tapering course of PO dexamethasone but was eventually euthanized. Necropsy confirmed the presence of SC eosinophilic granulomas and degenerative suspensory ligament desmitis, both with intralesional nematodes. Given the location and appearance of the nematode, a diagnosis of Onchocerca sp., most likely O. reticulata, was made. Onchocerciasis should be included as a differential diagnosis for multifocal suspensory ligament desmitis with these sonographic characteristics when paired with SC masses in imported European Warmbloods.

Onchocerciasis, an undiagnosed disease in Mozambique: identifying research opportunities.

BACKGROUND: The objective of this paper is to summarise and critically review the available data about onchocerciasis in Mozambique, in order to report epidemiological and clinical aspects related to the disease and identify gaps in knowledge. The paper is intended to raise awareness of the existence and importance of this disease and to define research priorities. METHODS: We examined the scarce epidemiological data at our disposal: two diagnostic studies in 1997 and 1998 (first reports on the existence of onchocerciasis in Mozambique), and two Rapid Epidemiological Mapping of Onchocerciasis (REMO) surveys in 2001 and 2007. We examined differences in study designs and methodologies as well as the differing geographical locations to explain the divergence in findings among the studies. RESULTS: Evidence indicates that onchocerciasis is hypoendemic in Mozambique (with national and imported cases), but still largely remains an undiagnosed illness. There is no awareness of the clinical aspects of the disease and nor of the differential diagnosis with lepromatous leprosy and dermatitis caused by Scabies spp. The use of skin biopsy and a symptom screening questionnaire, combined with nodule rate, in the first two studies may have captured even atypical or subacute presentations. Both REMO surveys relied solely on nodule detection and in the six years between the two studies, the prevalence of nodules detected more than doubled. CONCLUSIONS: The epidemiology and clinical aspects of the disease are unknown in Mozambique. Since the last REMO took place in 2007 and since the population is subject to large-scale movement and displacement, it is important to develop tools to identify and analyse populations that are at high risk for onchocerciasis. Cases of onchocerciasis may be misdiagnosed as leprosy or scabies that fail to improve despite being subjected to treatment against leprosy. Techniques to enable a differential diagnosis need to be established by training health professionals on the recognition of this undiagnosed disease. It is equally necessary to identify the blackfly vectors and where they breed.

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities.

Microscopic evaluation of skin biopsies is the monitoring and evaluation (M and E) method currently used by multiple onchocerciasis elimination programs in Africa. However, as repeated mass drug administration suppresses microfilarial loads, the sensitivity and programmatic utility of skin snip microscopy is expected to decrease. Using a pan-filarial real-time polymerase chain reaction with melt curve analysis (qPCR-MCA), we evaluated 1) the use of a single-step molecular assay for detecting and identifying Onchocerca volvulus microfilariae in residual skin snips and 2) the sensitivity of skin snip microscopy relative to qPCR-MCA. Skin snips were collected and examined with routine microscopy in hyperendemic regions of Uganda and Ethiopia (N= 500 each) and "residual" skin snips (tissue remaining after induced microfilarial emergence) were tested with qPCR-MCA. qPCR-MCA detected Onchocerca DNA in 223 residual snips: 139 of 147 microscopy(+) and 84 among microscopy(-) snips, suggesting overall sensitivity of microscopy was 62.3% (139/223) relative to qPCR-MCA (75.6% in Uganda and 28.6% in Ethiopia). These findings demonstrate the insufficient sensitivity of skin snip microscopy for reliable programmatic monitoring. Molecular tools such as qPCR-MCA can augment sensitivity and provide diagnostic confirmation of skin biopsies and will be useful for evaluation or validation of new onchocerciasis M and E tools.

Onchocerca lupi infection in Turkey: a unique case of a rare human parasite.

Onchocerpa lupi was first isolated from a wolf in Russia. Since then, canine ocular onchocercosis has been increasingly reported, particularly in Europe and the United States. It is thought that blackflies and midges are the vectors of transmission, and it is possible that these vectors could transmit the parasite to humans. The first human case of O. lupi in Turkey was reported in 2011. In this report we present the third human case of O. lupi infection in Turkey. Our patient was a 28-year-old male who displayed a painless, immobile mass under the conjunctiva. The mass measured 10 × 12 mm in size. Pathological examination of the surgically excised tissue was suggestive of infection by a filarial nematode. Subsequently, the parasite was identified as O. lupi through molecular analysis. All of the previously reported cases of O. lupi in both humans and dogs were more symptomatic than in our patient, Onchocerca infection should not be ruled out during the differential diagnosis of the subconjunctival and orbital cystic mass in instances where there is little to no inflammation. It is important to consider biopsy and carry out molecular analysis to identify the parasite.

Redescription of Onchocerca lupi (Spirurida: Onchocercidae) with histopathological observations.

BACKGROUND: Onchocerca lupi is a dog parasite of increasing zoonotic concern, with new human cases diagnosed in Turkey, Tunisia, Iran, and the United States. Information about the morphology of this nematode is scant and a detailed re-description of this species is overdue. In addition, histopathological data of potential usefulness for the identification of O. lupi infections are provided. METHODS: Male and female nematodes, collected from the connective tissue of a dog, were examined using light microscopy and scanning electron microscopy (SEM), and an histological evaluation was performed on biopsy samples from periocular tissues. RESULTS: The morphological identification was confirmed by molecular amplification and partial sequencing of cytochrome oxidase subunit 1 gene. This study provides the first comprehensive morphological and morphometric description of O. lupi from a dog based on light microscopy, SEM, molecular characterization, and histological observations. CONCLUSIONS: Data herein presented contribute to a better understanding of this little known parasitic zoonosis, whose impact on human and animal health is still underestimated. The presence of granulomatous reactions only around the female adult suggests that the release of microfilariae from the uterus might be the cause of the inflammatory reaction observed.

Defining parasite biodiversity at high latitudes of North America: new host and geographic records for Onchocerca cervipedis (Nematoda: Onchocercidae) in moose and caribou.

BACKGROUND: Onchocerca cervipedis is a filarioid nematode of cervids reported from Central America to boreal regions of North America. It is found primarily in subcutaneous tissues of the legs, and is more commonly known as 'legworm'. Blackflies are intermediate hosts and transmit larvae to ungulates when they blood-feed. In this article we report the first records of O. cervipedis from high latitudes of North America and its occurrence in previously unrecognized host subspecies including the Yukon-Alaska moose (Alces americanus gigas) and the Grant's caribou (Rangifer tarandus granti). METHODS: We examined the subcutaneous connective tissues of the metacarpi and/or metatarsi of 34 moose and one caribou for parasitic lesions. Samples were collected from animals killed by subsistence hunters or animals found dead in the Northwest Territories (NT), Canada and Alaska (AK), USA from 2005 to 2012. Genomic DNA lysate was prepared from nematode fragments collected from two moose. The nd5 region of the mitochondrial DNA was amplified by PCR and sequenced. RESULTS: Subcutaneous nodules were found in 12 moose from the NT and AK, and one caribou from AK. Nematodes dissected from the lesions were identified as Onchocerca cervipedis based on morphology of female and male specimens. Histopathological findings in moose included cavitating lesions with multifocal granulomatous cellulitis containing intralesional microfilariae and adults, often necrotic and partially mineralized. Lesions in the caribou included periosteitis with chronic cellulitis, eosinophilic and lymphoplasmacytic infiltrate, and abundant granulation associated with intralesional adult nematodes and larvae. Sequences of the nd5 region (471bp), the first generated for this species, were deposited with Genbank (JN580791 and JN580792). Representative voucher specimens were deposited in the archives of the United States National Parasite Collection. CONCLUSIONS: The geographic range of O. cervipedis is broader than previously thought, and extends into subarctic regions of western North America, at least to latitude 66°N. The host range is now recognized to include two additional subspecies: the Yukon-Alaska moose and Grant's caribou. Accelerated climate change at high latitudes may affect vector dynamics, and consequently the abundance and distribution of O. cervipedis in moose and caribou. Disease outbreaks and mortality events associated with climatic perturbations have been reported for other filarioids, such as Setaria tundra in Fennoscandia, and may become an emerging issue for O. cervipedis in subarctic North America.

Functional characterization and immune recognition of the extracellular superoxide dismutase from the human pathogenic parasite Onchocerca volvulus (OvEC-SOD).

Onchocerca volvulus is a human pathogenic filarial nematode causing chronic onchocerciasis, a disease characterized by chronic skin and eye lesions. Despite attempts to control this infection from many perspectives, it still remains a threat to public health because of adverse effects of available drugs and recent reports of drug resistance. Under control of an intact immune system, O. volvulus survives for a long time in the host by employing a variety of strategies including the utility of antioxidant enzymes. In the present study, we focus on the extracellular superoxide dismutase from O. volvulus (OvEC-SOD) found in the excretory/secretory products of adult worms. Contrary to previous studies, the OvEC-SOD was found to have a 19 amino acid long signal peptide that is cleaved off during the process of maturation. To validate this result, we designed a novel method based on Caenorhabditis elegans cup5(ar465) mutants to specifically evaluate signal peptide-mediated secretion of nematodal proteins. Following purification, the recombinant OvEC-SOD was active as a dimer. Site-directed mutagenesis of the three cysteines present in the OvEC-SOD shows that enzyme activity is markedly reduced in the Cys-192 mutant. A homology model of the OvEC-SOD underlines the importance of Cys-192 for the stabilization of the adjacent active site channel. The generation of a humoral immune response to secretory OvEC-SOD was indicated by demonstrating IgG reactivity in sera from patients infected with O. volvulus while the cross-reactivity of IgG in plasma samples from cows, infected with the most closely related parasite Onchocerca ochengi, occurred only marginally. High IgG1 and IgM titres were recorded in sera from mice infected with the filaria Litomosoides sigmodontis, however, low or no cellular proliferative responses were observed. Thus, the present data suggest that secretory OvEC-SOD is a target of the humoral immune response in human onchocerciasis and induced strongest IgG responses in hyperreactive onchocerciasis. Furthermore, humoral response during murine infection induced SOD-specific IgG that cross-reacted with OvEC-SOD.

Immunohistology of ectopic secondary lymph follicles in subcutaneous nodules from patients with hyperreactive onchocerciasis (sowda).

Ectopic secondary lymph follicles emerge in patients with autoimmune or infectious diseases, e.g. in the synovium in rheumatoid arthritis or the skin in Borrelia burgdorferi infection, but ectopic localisations in the skin are rarely described for helminth infections. We investigated the cellular composition of secondary lymph follicles in subcutaneous nodules from eight patients with hyperreactive onchocerciasis (synonymous "localised" form or sowda) using immunohistology. CD3- and CD45RO-positive T cells and CD20-positive B cells were present in the mantle zone. The germinal centre was characterised by many B cells and CD35-positive follicular dendritic cells, which formed a network of attached IgE- and CD23-positive cells with the low-affinity IgE (epsilon) receptor. Few of the B cells were labelled for IgG1, IgG2 and IgG4, whereas in other zones of the nodule IgG1 was expressed by plasma cells and IgG1-coated dead microfilariae. B cells and few macrophages expressed the MHC class II molecule HLA-DR. Mature CD68-positive tingible body macrophages with phagocytosed leukocytes and CD57-positive lymphocytes occurred in the germinal centre. Macrophages in the germinal centre only weakly expressed alpha1-antichymotrypsin in contrast to macrophages in other zones of the onchocercoma. Furthermore, the multifunctional cytokine TGF-beta was only weakly expressed by macrophages and lymphocytes in the secondary follicles. Only few tryptase-positive mast cells, calprotectin-positive young macrophages, eosinophils and neutrophils occurred in the secondary follicles, although these cells were abundant in the onchocercomas. In conclusion, the ectopic secondary lymph follicles in onchocercomas and lymph nodes from hyperreactive onchocerciasis patients are equally composed.

Immunohistological studies on neoplasms of female and male Onchocerca volvulus: filarial origin and absence of Wolbachia from tumor cells.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E(2). These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

Hyperendemicity of onchocerciasis in Ovia Northeast Local Government Area, Edo State, Nigeria.

OBJECTIVE: The present study investigates the prevalence of onchocerciasis in 40 communities of the LGA and provides data, which could contribute towards the control programme of the disease in the local government Area, Edo state, Nigeria. METHODS: A physician performed the skin examination and thereafter a skin biopsy taken and immersed in normal saline solution in the wells of micro titration plates and allowed to stand for 24 hours to allow microfilaria to emerge and then examined microscopically. RESULTS: The prevalence of onchocerciasis was investigated amongst 40 communities in Ovia North-East LGA of Edo state, Nigeria. Of the 2020 subjects examined between March 2007 to April, 2008, 1674 (83%) of them harboured microfilaria in their skin tissues.Females had significantly (P < 0.001) higher prevalence than their male counterparts (93.1% vs 74.5%). CONCLUSION: The hyperendemicity of the disease may however be due to poor distribution of ivermectin and non compliance on the part of the subjects taking the drug as some often complained about the side effects of ivermectin. The findings demonstrated hyperendemicity of infection and require urgent attention with ivermectin treatment and other control measures.

Lymphatic vascularisation and involvement of Lyve-1+ macrophages in the human onchocerca nodule.

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e.g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.

Identification of the lipid mediator prostaglandin E2 in tissue immune cells of humans infected with the filaria Onchocerca volvulus.

Prostaglandins generated by multiple tissue and immune cells exhibit regulatory effects on the vascular and immune systems. Prostaglandin E(2) (PGE(2)), in particular, affects innate as well as adaptive immune mechanisms. We identified PGE(2) in host immune cells adjacent to Onchocerca volvulus in subcutaneous onchocercomas and the affected skin. Using immunohistology, PGE(2) was predominantly detected in infiltrating macrophages but also in plasma cells. Consecutive sections revealed concomitant presence of PGE(2) and transforming growth factor-beta (TGF-beta), representing a second immunoregulative mediator in macrophages and plasma cells. TGF-beta was preferentially observed in the infiltrating macrophages in patients with a generalized hyporeactive onchocerciasis and less in patients with the hyperreactive form. The presence of PGE(2) and TGF-beta in adjoining host cells infiltrating in the onchocercoma and dermis may indicate containment of inflammatory responses that could favour survival of the filarial parasite.

Onchocercosis mansonella co-infection presenting as a subcutaneous nodule in a child.

A 11-year-old girl presented with a subcutaneous lesion on the forehead, thought to be an implantation dermoid cyst. Microscopic examination revealed an onchocercoma. Microfilariae were found in the blood indicating a co-infection with Mansonella perstans. This case demonstrates the diagnostic difficulties of a parasitic co-infection uncommonly encountered in Europe.

Hanging groin and persistent pruritus in a patient from Burkina Faso.

Onchocerciasis is a filarial disease caused by Onchocerca volvuvus which can lead to skin, bladder or ocular signs. The authors report a 6-month history of generalized pruritus and two bilateral and symmetric masses in inguinal areas in a 57-year-old black woman. Skin snips and a biopsy of a subcutaneous nodule were diagnosed as onchocerciasis with hanging groin and the patient was treated with ivermectin with a dramatic improvement. If pruritus is the most constant symptom of onchocerciasis, hanging groin is usually observed only in hyperendemic areas in association with high microfilarial density. As no protective immune response is acquired after onchocerciasis, it is recommended that observation is maintained in communities where blindness is less common but where skin manifestations remain prevalent. There is a risk of misdiagnosing onchocerciasis owing to its long incubation period and its unfamiliarity to physicians in nonendemic areas.

Onchocercosis of an intervertebral joint capsule causing cervical vertebral stenotic myelopathy in a horse.

A novel case where onchocercosis was identified as a cause of cervical myelopathy in the horse is described. A 15-year-old Connemara mare was euthanized due to progressive locomotion disturbance. Postmortem examination revealed soft-tissue swelling in the intervertebral joint capsule of C6-7 with narrowing of the vertebral canal. On light microscopy, axonopathy was pronounced in the corresponding segment of the spinal cord. Fibrous tissue and eosinophilic granulomas were found in the joint capsule, together with parasites identified histologically as Onchocerca sp.

Onchocerciasis in Imo state, Nigeria (2): the prevalence, intensity and distribution in the upper Imo river basin.

Human infection with Onchocerca volvulus was investigated in 13 rural communities in the Upper Imo River basin, Imo State, Nigeria between March 1997 and December 2000 using the skin snip method. Of the 3,311 persons examined, 889 (26.8%) had microfilariae. There were significant differences between communities in microfilariae prevalence and intensity of infection (P<0.05). Sex infection was similar in males (26.8%) and females (26.9%); whereas farmers (46.6%) and fishermen (28.4%) were more affected than people in other occupations. The proportion of infected persons increased with age coinciding with the period when most clinical signs manifest. A significant proportion of the 889 persons with positive microfilariae skin biopsy had musculoskeletal pain (57.1%). Thirty-four (47.2%) of the 72 epileptics identified had positive skin biopsy in addition to other manifestations. The present findings show that onchocerciasis is a public health problem in the area. The use of ivermectin has reduced the incidence of the disease when compared with data from the pre-ivermectin era. This shows that Community Directed Treatment with Ivermectin (CDTI) campaign is successful and should be sustained until onchocerciasis is eliminated as a public health problem in the area.

Isolation and characterization of the regulatory subunit of cAMP-dependent protein kinase from the filarial parasite Onchocerca volvulus.

Protein kinases exert major regulatory effects in eukaryotic signaling events. As these proteins play central regulatory and sensory functions they are interesting targets for antiparasitic drug development and serve as vaccine candidates. A cDNA with an open reading frame of 1122 bp coding for the regulatory subunit of the cAMP-dependent protein kinase (Ov-pka-r) of the pathogenic human nematode Onchocerca volvulus has been isolated. The predicted protein displays 84% homology to the corresponding protein of Caenorhabditis elegans and 71% to the human homologue. The O. volvulus protein has unique features, it includes six cysteine residues, as compared to four residues in mammals. Ov-PKA-r was recombinantly expressed as His-tagged protein and under reducing conditions showed a molecular mass of 52 kDa. In sera from O. volvulus patients IgG antibodies were found that strongly reacted with the recombinant Ov-PKA-r. Using rabbit antisera raised against the recombinant protein for immunohistology allowed the localization of the native Ov-PKA-r within the nervous system and sensory organs of adult O. volvulus worms and of microfilariae. The predominant expression in the nervous system and sensory organs as well as the unique structural features identify this signaling molecule of O. volvulus as a new and interesting target for drug or vaccine development.