Onchocerca Spp Research Articles

A footworm in the door: revising Onchocerca phylogeny with previously unknown cryptic species in wild North American ungulates

Onchocerca is an important genus of vector-borne filarial nematodes that infect both humans and animals worldwide. Many Onchocerca spp., most of medical and veterinary health relevance, are the focus of a variety of diagnostic and molecular research. However, despite the importance of these parasites, there is growing evidence of previously unexplored genetic diversity of these nematodes, particularly among wild ungulate hosts in North America. These understudied parasites prevent us from comprehending the evolutionary history of the genus Onchocerca, monitoring potential One Health threats, and improving our filarioid diagnostic capabilities. In order to fill these knowledge gaps, we identified five uncharacterized Onchocerca lineages and compared them with other well-known filarioid species using single and concatenated gene regions (i.e., nd5, cox1, 12S, 18S, 28S, hsp70, MyoHC, rbp1). Phylogenetic analyses revealed that the novel Onchocerca lineages of wild North American ungulates segregate into two clades. One clade comprised Onchocerca lineages II, IV, and V and other species found mainly in domestic animals and humans, and the second comprised Onchocerca lineages I and III and other species from a variety of hosts including cervids, bovids, and equids. The formation of two clearly separate clades supports the idea of at least two independent expansion events of ancestral Onchocerca spp. into the North American continent via the Bering land bridge. Cophylogenetic analysis shows evidence of ancestral Onchocerca spp. of Bovidae host-switching to wild Cervidae and giving rise to the novel Onchocerca spp. Lastly, pairwise analysis confirms informative molecular markers of diagnostic relevance in both mitochondrial and nuclear gene regions of filarioid nematodes. The overall information provides greater context to the genus Onchocerca and emphasizes the need to discover, characterize, and monitor neglected parasites, especially those of wildlife origin.

Morphological description and multilocus genotyping of Onchocerca spp. in red deer (Cervus elaphus) in Switzerland.

Onchocercosis is a parasitic disease caused by over 30 Onchocerca spp. (Nematoda: Filarioidea) and predominantly affecting ungulates. Four Onchocerca spp. have been described in the European red deer (Cervus elaphus). Onchocerca flexuosa and Onchocerca jakutensis form subcutaneous nodules in the back region. The other two species, Onchocerca skrjabini and the lesser-known Onchocerca garmsi, are found freely in the subcutaneous tissue of carpal and tarsal joints, and the sternal region, respectively. The presence of Onchocerca spp. in eight red deer shot in the hunting season during September 2020 in the Grisons region, Switzerland, was investigated by analysing nodules and free worms in the subcutaneous tissue. The obtained worms were morphologically and genetically identified as O. jakutensis, O. flexuosa and O. skrjabini. The latter two are first reports from Switzerland, and morphological redescriptions of these two species are presented. Onchocerca skrjabini and O. jakutensis are newly described from the sternal region of deer. One female of O. jakutensis was found free in the subcutaneous tissue of the sternal region, an atypical presentation for this species. Phylogenetic analyses were based on four mitochondrial and one nuclear loci, revealing that O. jakutensis belongs to a clade which so far only included non-cervid Onchocerca spp. Analysis of sequences from this study and GenBank entries revealed two distinct subpopulations of O. skrjabini: one from European red deer and another from Japanese serow and sika deer. Morphological identification can be challenging, also because worm location in the host is less strictly determined than previously described. Genetic identification is straightforward for O. flexuosa, O. jakutensis and O. skrjabini for which complete data of five loci are now available whereas genetic data of O. garmsi is still lacking.

The first report on internal transcribed spacer region-based characterization of microfilaria in Asian elephants (Elephas maximus) in Thailand.

Background and Aim:Filarial infections can significantly impact the health of both humans and animals. In elephants, filariasis has been associated with cutaneous dermatitis and skin nodules. However, molecular evidence for such infections is limited in Thailand. This study aimed to identify the morphological and molecular characteristics of microfilaria in captive Asian elephants in Thailand.Materials and Methods:Whole blood collected from the ear vein of 129 captive Asian elephants was hematologically analyzed, and the blood parasites were evaluated using three standard techniques: The microcapillary test, thin blood smears, and polymerase chain reaction (PCR).Results:Conventional PCR revealed that approximately 17% (22/129) of the sampled elephants were positive for microfilaria. Microscopy revealed that microfilariae are large, unsheathed, with extended nuclei, a short headspace, and a curved tail tapering at the end. Results of internal transcribed spacer region analysis show that the elephant microfilariae are closely related to Onchocerca spp. All of the elephants positive for microfilaria presented with neither skin lesion nor anemic signs. Microfilaria infection was not associated with age; however, microfilariae were more likely to be detected in male elephants due to differences in management systems.Conclusion:This is the first study to provide both morphological and molecular evidence of microfilaria in Thai elephants. There is an urgent need to investigate the long-term and large-scale effects of microfilaria on the health of elephants.

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

BackgroundThe 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.MethodsThe 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.ResultsThe 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.ConclusionsThis 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.

Molecular Identification of Onchocerca spp. Larvae in Simulium damnosum sensu lato Collected in Northern Uganda.

Previous studies have demonstrated that the presence of larvae of other filarial species in Simulium damnosum sensu lato can distort estimates of transmission potential for Onchocerca volvulus in West Africa. However, studies conducted in foci of onchocerciasis in West Central Uganda indicated that larvae other than O. volvulus were not common in vectors collected there. Recent data collected in Northern Uganda revealed a striking discordance between estimates of the prevalence of flies carrying O. volvulus infective larvae obtained from molecular pool screening and dissection methods. To resolve this discrepancy, sequences from three mitochondrially encoded genes were analyzed from the larvae collected by dissection. All larvae analyzed were Onchocerca ochengi v. Siisa, a parasite of cattle, or Onchocerca ramachandrini, a parasite of warthogs. These results suggest that nonhuman parasite larvae are common in vectors in Northern Uganda, underscoring the necessity for molecular identification methods to accurately estimate O. volvulus transmission.

Molecular Identification of Onchocerca spp. Larvae in Simulium damnosum sensu lato Collected in Northern Uganda

Molecular Identification of Onchocerca spp. Larvae in Simulium damnosum sensu lato Collected in Northern Uganda

Whence river blindness? The domestication of mammals and host-parasite co-evolution in the nematode genus Onchocerca

The genus Onchocerca includes 34 described species and represents one of the largest genera of the filarial nematodes within the family Onchocercidae. Representative members of this genus are mainly parasites of ungulates, with some exceptions such as Onchocerca lupi and Onchocerca volvulus, infecting carnivores and/or humans. For a long time, the evolutionary relationships amongst onchocercids remained poorly studied, as the systematics of this genus was impaired by the high morphological variability of species included in the taxon. Although some molecular phylogenies were developed, these studies were mainly focused on bovine Onchocerca spp. and O. volvulus, including assessments of Wolbachia endosymbionts. In the present study, we analysed 13 Onchocerca spp. from a larger host spectrum using a panel of seven different genes. Analysis of the coxI marker supports its usefulness for the identification of species within the genus. The evolutionary history of the genus has been herein revised by multi-gene phylogenies, presenting three strongly supported clades of Onchocerca spp. Analyses of co-evolutionary scenarios between Onchocerca and their vertebrate hosts underline the effect of domestication on Onchocerca speciation. Our study indicates that a host switch event occurred between Bovidae, Canidae and humans. Cophylogenetic analyses between Onchocerca and the endosymbiotic bacterium Wolbachia indicate the strongest co-evolutionary pattern ever registered within the filarial nematodes. Finally, this dataset indicates that the clade composed by O. lupi, Onchocerca gutturosa, Onchocerca lienalis, Onchocerca ochengi and O. volvulus derived from recent speciation.

FILaRIoSIS hUMaNa eN La SeLVa PeRUaNa: RePoRTe De TReS CaSoS

We present three cases of male patients with filariasis from the Peruvian jungle (Junin, San Martin and Pucallpa). One case presented filariae in the eyeball and blood smears, which according to morphology, serology and molecular biology was established as a possible case of zoonotic filariasis by Onchocerca spp. The other two cases were caused by Dirofilaria spp. introduced a cheekbone in a lump sensation and mobility in the area and was diagnosed by serology and the last case was extracted filaria a thumb of the hand and was identified as such by morphology and molecular biology. These cases are the first reports in Peru by Dirofilaria spp. and Onchocerca spp.

Molecular genetic comparison of Onchocerca sp. infecting dogs in Europe with other spirurid nematodes including Onchocerca lienalis

In the past 15 years, subconjunctival onchocercosis has been reported from 63 dogs in south-western United States (Arizona, California, Utah) and Southern and Central Europe (Germany, Greece, Hungary, Portugal, Switzerland). To reveal the taxonomic status of the parasite responsible for these infections, fragments of the mitochondrial cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) genes of three European strains of canine Onchocerca sp. and the 16S ribosomal RNA (16S rRNA) gene of their Wolbachia endosymbionts were sequenced and compared to the homologous sequences of other spirurid nematodes. The evolutionary divergence between COI and ND5 gene sequences of Greek, Hungarian and Portuguese strains of canine Onchocerca sp. were similar in magnitude to that seen within Thelazia callipaeda or Onchocerca lienalis. The evolutionary divergence between the sequences of canine Onchocerca sp. and other Onchocerca spp. including O. lienalis were similar or higher in magnitude to that seen between other Onchocerca spp. The results of the current and earlier phylogenetic analyses indicate that canine Onchocerca sp. separated from other Onchocerca spp. early in the evolution. Based on the similar clinical pictures, the identical morphology of nematodes and the sequence analyses of COI and ND5 genes of the worms and 16S rRNA gene of their wolbachiae, the Onchocerca worms isolated from European dogs appear to belong to the same species. The results support the earlier biological and morphological arguments that a distinct species, most likely O. lupi originally described from the subconjunctival tissues of a Caucasian wolf is responsible for canine ocular onchocercosis in Europe.

Nodular onchocercosis of red deer in central Spain

The presence of nodular onchocercosis was investigated post- mortem in 142 red deer (11 calves, 35 yearlings and 96 adults) shot from February 1998 to January 1999, and July–November 1999 in “Quintos de Mora” (Toledo, central Spain), a game property belonging to the National Wildlife Reserves. Between 6 and 13 animals were analysed monthly by inspection for subcutaneous nodules of Onchocerca spp. Subcutaneous nodules of Onchocerca sp. were detected in 24% of the animals. Anatomical locations for nodules were the back and flanks. Infection ranged from 1 to 29 nodules per animal. Adult worms collected from nodules were identified as O. flexuosa. No apparent seasonal trend was observed either in prevalence or in mean intensity of infection, which fluctuated between 48% (5±8 nodules) in winter 1998 and 5% (1±0 nodules) in summer 1999. Prevalence of infection was significantly higher in adult (30%) than in young animals (9% in calves, 11% in yearlings), although no age effect on intensity was observed. The size of the nodules was measured to evaluate the age of infection. Small (recent) nodules (5–6 mm of diameter) were collected in late spring, summer and fall; medium-sized nodules (12–15 mm) were in second half of summer, fall and winter, and large (mature) nodules (20–25 mm) in fall, winter and part of spring. Significant differences were found among host age groups.

Orientation of Onchocerca lienalis stiles (Filarioidea: Onchocercidae) microfilariae to black fly saliva.

Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host-skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor [s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host's skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.

Onchocerca spp: A "Family" of Secreted Acidic Proteins Expressed by Infective Larvae in Blackflies

Biosynthetic labeling of developing larvae of Onchocerca in blackflies has been used to characterize a group of stage-specific, secreted proteins produced by vector-stage parasites. These are highly acidic molecules (pI 4.4-5.1) present in at least three members of the genus (O. volvulus, O. lienalis, O. ochengi) that exhibit minor heterogeneity among species in apparent molecular mass (between 18 and 23 kDa). In 0. volvulus, there are two polypeptides that run as a doubler of 18 and 20 kDa. In O. lienalis and O. ochengi, single polypeptides of 23 and 20 kDa were detected. The processes of synthesis and secretion appear to be temperature-sensitive and dissociated events. Experiments with O. volvulus in Simulium damnosum sl revealed that synthesis is initiated in second stage larvae and increases in infective-stage parasites: Secretion occurs when larvae leave the vector and enter the phase of development associated with the vertebrate host. Third-stage larvae of O. lienalis were shown to continue to express and accumulate the 23-kDa protein with age. The primary organ of secretion, as indicated by dissection, was the glandular esophagus. These data point to an important biological role for this group of molecules and suggest that they may belong to a family of related products. Because they have the distinctive characteristics of being secreted larval acidic proteins, we propose the acronym SLAP pending further insights into their functional properties.

Identification and characterisation of a novel repetitive antigen from Onchocerca spp.

A novel repetitive antigen from the cattle parasite Onchocerca gibsoni was shown to be recognised by sera from humans infected with Onchocerca volvulus, Wuchereria bancroftii or Brugia malayi. The O. gibsoni protein was produced in a recombinant form, and antibodies raised to this protein used to screen cDNA libraries for O. volvulus. A series of clones were isolated which encoded repetitive regions very similar to those in O. gibsoni, but interspersed between these were longer repeating units which we have not so far found in O. gibsoni. The repetitive antigen was shown to be of high molecular weight and present only in the insoluble (membrane) fraction of O. gibsoni microfilariae. Immunofluoresence techniques demonstrated that the antigen was associated both with muscle and with specific membrane layers, including a peripheral layer which corresponds to either the outer hypodermis or an inner region of the cuticule in adult female O. gibsoni. In many respects, the proteins encoded by the O. gibsoni and O. volvulus cDNA clones resembled repetitive antigens from several distantly related eukaryotic parasites, and a possible common role in immune evasion is discussed.

Morphometric differentiation of Onchocerca volvulus and O. ochengi infective larvae

By microinjection of cryopreserved microfilariae (mf) into nulliparous flies, a comparison of the lengths of the infective larvae (L 3) of Onchocerca volvulus and O. ochengi from the head of Simulium damnosum s.l. (presumed S. sirbanum) has been made. The suitability of S. sirbanum as a host was similar for both Onchocerca spp. The mean length ± standard deviation of O. ochengi infective larvae measured in aqueous medium after storage of infected flies in liquid nitrogen was 762 ± 63 μm ( n = 39), significantly longer ( P ⪡ 0·0001) than those of a savanna isolate of O. volvulus (676 ± 56 μm, n = 26). Although the frequency distributions of the lengths of larvae of the 2 species overlapped, a critical value for discrimination of 719 μm applied to normally distributed populations with means and standard deviations of these samples would result in correct classification of 78% of true O. volvulus and 75% of true O. ochengi. A discriminant function analysis incorporating width measurements did not usefully improve the level of accuracy of discrimination. Larvae from flies stored in 70% ethanol and stained with acid haemalum were about 10% shorter, but O. ochengi infective larvae were still proportionately longer than those of O. volvulus (693 ± 40 μm, n = 45 compared to 580 ± 38 μm, n = 6, respectively). These data show that the infective L 3 of O. volvulus and O. ochengi differ morphologically. Although the population length distributions overlap, by classifying larvae >719 μm long as O. ochengi and those <719 μm long as O. volvulus a more accurate estimation of true O. volvulus infection rates in S. damnosum s.l. can be derived than is currently possible.

Perspectives on the in vitro culture of filariae

A primary constraint in the culture of human filariae is in obtaining starting material--either microfilariae (mfs), which infect invertebrates, or third stage larvae (L3s), which are infective to humans. Cryopreservation methods which partially overcome this difficulty have been developed for both mfs and L3s. Complete development of mfs to L3s outside an intact host was obtained recently when mosquito thoraces infected by Brugia malayi (24 h after the bloodmeal) were maintained in vitro. In another recent study in which no host tissues were present, a semidefined culture medium was used to investigate the properties of reduced glutathione (GSH) that stimulate early development of Onchocerca lienalis mfs. An extended cysteinyl backbone and a free sulfhydryl were identified as the key structural elements provided by GSH. Stimulation also required the presence of low and high molecular weight components of serum as well as oxygen. Molting of Onchocerca spp. L3s to the fourth stage at the rate of 50 to 70% has been reported by several researchers. Key factors identified in those successes have been temperature and serum lot. Improved long-term viability occurred with cellular co-culture. Beneficial effects of co-culture were shown to be due both to cellular conditioning of the medium as well as to lowered dissolved oxygen levels as a result of cellular metabolism. With the use of cell-conditioned medium and decreased incubator oxygen levels, long-term viability of Onchocerca larvae in culture exceeded that previously reported. Recently, Brugia malayi adults of both sexes were cultured from L3s using a semidefined medium supplemented with human serum. Many of these sexually matured adults mated and produced viable microfilariae.

An oligonucleotide probe specific for Onchocerca volvulus

A genomic DNA library of a Liberian strain of Onchocerca volvulus was prepared in the vector bacteriophage λgt10. The library was differentially screened by hubridisation with radiolabelled total DNA from the homologous parasite, two heterologous Onchocerco parasites ( Onchocerca gibsoni and Onchocerca gutturosa) and human liver cells. A clone (C1A1) was isolated whose binding to O. volvulus DNA was at least 50 times stronger than to the other parasite DNA samples. No binding was observed with human DNA. The insert of C1A1 was subcloned into the filamentous phage vector M13 mp18 and sequenced. Two oligonucleotides, each corresponding to a unique region of 60 nucleotides (out of a total of 154) were synthesised and examined for hybridisation with three different geographical isolates of O. volvulus (including forest and savannah strains) and six other Onchocerca spp. One of the oligonucleotides (C1A1-2) was found to hybridise to the three O. volvulus isolates with an intensity in the region of 300 times greater than to any other Onchocerca spp. Since the other species include the two which may be most closely related to O. volvulus, i.e., O. gibsoni and Onchocerca ochengi, it is concluded that C1A1-2 is likely to represent a truly species-specific probe.

Improved development of Brugia microfilariae following cryopreservation in liquid nitrogen using a technique suitable for field conditions

A technique for improved cryopreservation at −196 °C of Brugia spp. microfilariae has been developed by modifications of a procedure previously used with Onchocerca spp. A double incubation in ethanediol (ED) solutions, firstly at 37 °C in 10% ( v v ) ED for 15 min and secondly at 0 °C in 40% ( v v ) ED for 45 sec followed by plunging into liquid nitrogen, resulted in over 90% of the microfilariae of B. malayi exhibiting normal motility. When used with B. pahangi microfilariae, followed by blood-feeding to Aedes aegypti (Bels New strain) a third-stage larvae yield of 4·04 per fly was obtained, compared with 5·16 in the unfrozen group. This represents an infectivity level of 78% compared with unfrozen controls. The technique also has the benefits of being suitable for field conditions, requiring no sophisticated equipment, merely some ice, ethanediol and liquid nitrogen.

Diagnostic features of zoonotic filariae in tissue sections

The diagnostic features in tissue sections from patients with zoonotic filarial infections are reviewed. In general, two types of infections are recognized: 1) those presenting radiologically in healthy patients as coin lesions, which are usually removed because a clinical diagnosis of carcinoma of the lung is made and infarcts containing Dirofilaria immitis parasites in pulmonary arteries are found; and 2) those presenting as subcutaneous nodules, in which D. tenuis, a parasite from raccoons, D. repens, from dogs and cats, D. ursi , from bears, and Onchocerca spp, from horses or cattle, require delineation. Moreover, nodules that on microscopic examination are seen to be lymph nodes may harbor Brugia spp. The geographic distribution of these infections, with emphasis on the United States, is discussed.

The fecundity of Simulium damnosum s.l. in northern Togo and infections with Onchocerca spp.

The numbers of maturing oocytes in gravid Simulium damnosum s.l. were related to the sizes of the flies and when this was taken into account the numbers fell into two groups. It was concluded that the group with the highest oocyte counts were nulliparous and that the remainder were parous flies. No evidence was found to suggest that flies in their third or subsequent gonotrophic cycles matured fewer oocytes than those in their second cycles. However, the results did suggest reduced fecundity of those flies infected with Onchocerca spp. The frequency distributions of the Onchocerca spp. infections for each of three different stages can be fitted to negative binomial distributions with similar values (0·04–0·07) for the parameter k.

Aedes aegypti as a surrogate host for Onchocerca spp.

Experimental infections of Onchocerca gutturosa, O. cervicalis, and O. volvulus were induced in males and females of a strain of Aedes aegypti originally selected for susceptibility to Brugia malayi. Mosquitoes were inoculated intrathoracically with microfilariae from the skins of animals infected with the three parasites. Percentages of female Ae. aegypti harboring third-stage larvae 9 or more days after inoculation were 22.5% for O. gutturosa, 11.1% for O. cervicalis and 14.4% for O. volvulus. Infection rates among male mosquitoes were 39.5% for O. gutturosa and 16.15% for O. cervicalis. Although O. volvulus did develop to the third larval stage in males, high mosquito mortality prevented estimation of the rate of susceptibility. Infection rates among mosquitoes inoculated with cryopreserved microfilariae of O. gutturosa were similar to those in mosquitoes inoculated with microfilariae from fresh skin snips. Of the three species of filariae studied, only O. volvulus had a pronounced pathological effect on the inoculated mosquitoes. While most larvae recovered from Ae. aegypti were normal, pathologies similar to those exhibited by other filariae developing in abnormal hosts were noted in some individuals.