Development of Onchocerca volvulus microfilariae injected into Simulium species from Cameroon

Microfilariae (mff) of the savanna and forest strains of Onchocerca volvulus (Leuckart) were injected intrathoracically into adult females of Simulium damnosum Theobald sensu stricto, S.sirbanum Vajime & Dunbar, S.squamosum Enderlein and S.mengense Vajime & Dunbar. Nine days post infection (pi) 27-29% of the savanna mff and 31-38% of the forest strain had developed to third-stage larvae (L3), irrespective of the fly species, size or injection dose (5, 10 or 15 mff). Savanna flies supported the development of forest O.volvulus better than forest flies, in contrast to the results after per os infections. Therefore, in these four species of the S.damnosum complex from Cameroon, the peritrophic membrane is considered to be the main factor limiting the success rate of microfilarial development following the ingestion of blood infections, while the fly's haemolymph and intracellular environment play minor roles.     

Morphological differences between Venezuelan and African microfilariae of Onchocerca volvulus

Comparative morphological and biometric characteristics of microfilariae of Onchocerca gutturosa and O. volvulus from different geographical areas (Upper Orinoco, Venezuela; Togo; Liberia) were assessed. "Stepwise" discriminant analysis and Mahalanobis estimators were applied to measure distance between populations. The results indicate a strong similarity between the two strains from the Upper Orinoco (Venezuela) and the Togo strain, as well as a clear separation between these strains and that of O. gutturosa. The Liberian strain was easily distinguishable from microfilariae from Togo and Venezuela. Discriminant analysis showed the Liberian deme to be as different from the Venezuelan and Togo demes as these demes were from microfilariae of the reference species, O. gutturosa. Although it is necessary to confirm these data using formalin-fixed specimens obtained from the skin, the present findings suggest the existence of geographically-different strains of O. volvulus in America and Africa.     

Effect of diethylcarbamazine on the concentration of Onchocerca volvulus microfilariae in hydrocoele fluid and urine.

The effect of graded doses of diethylcarbamazine on the concentration of microfilariae in the hydrocoele fluid, urine and skin of patients suffering from onchocerciasis was studied. The results showed that a significant number of microfilariae migrated into hydrocoele fluid and urine immediately after the drug was given and thereafter returned to pre-treatment levels rather more quickly in the former than the latter. The increase was found to be roughly directly proportional to the intensity of infection in the skin. By contrast the skin concentration of microfilariae fell sharply after treatment and remained significantly lower than pre-treatment levels for over four weeks. The potential use to which these observations could be put with respect to clinical screening of drugs in onchocerciasis is discussed.     

Onchocerca volvulus: limited heterogeneity in the nuclear and mitochondrial genomes

West African populations of Onchocerca volvulus endemic to the rain forest and savanna bioclimes of West Africa differ in their ability to induce ocular disease in infected individuals. In recent years, both clinical- and animal-model-based studies have implicated particular parasite antigens in the development of ocular onchocerciasis. To test the hypothesis that the difference in pathogenic potential of blinding and nonblinding parasites might be reflected in qualitative differences in antigens that have been implicated in the development of ocular onchocerciasis, we compared the sequences of two parasite antigens implicated in the development of ocular disease in blinding- and nonblinding-strain parasites. The results demonstrated a high level of homogeneity between the parasite strains in these genes. The study was extended to include additional nuclear genes encoding antigens that are commonly recognized by individuals infected with O. volvulus and to the mitochondrial genome of the parasite. The results demonstrate a high degree of homogeneity in both the nuclear and the mitochondrial genomes among O. volvulus isolates collected from several different sites in Africa and in the Americas. This high degree of genetic homogeneity may reflect the passage of the parasite through a recent genetic bottleneck.     

Chromosomes of Onchocerca volvulus (Spirurida: Onchocercidae): a comparative study between Nigeria and Guatemala

Chromosomes of Nigerian Onchocerca volvulus were compared with those of Guatemalan O. volvulus. Both parasites had basically the same chromosomal construct (2n = 8, XY type). Autosomes consisted of a pair of large and two smaller pairs. Sex chromosomes were made up of medium sized X chromosome and very small Y chromosome. It was not possible to infer the position of the centromeres.     

A histocytochemical study of the macrophages present in tissue responses to adult Onchocerca volvulus

Summary Immunocytochemical and histochemical properties of macrophages present in the subcutaneous chronic inflammatory responses surrounding adultOnchocerca volvulus (nodules) in human tissues were examined. Macrophages with strong non-specific esterase (NSE) and acid phosphatase (AcPase) activities but weak adenosine triphosphatase (ATPase) activity and HLA-DR expression (NSE⁺⁺⁺, AcPase⁺⁺⁺, ATPase^–/+, HLA-DR^–/+) were present in the centre of nodules. Many of the cells adhering to the surface of worms were NSE⁺⁺⁺, AcPase⁺⁺⁺, ATPase^–, HLA-DR⁺⁺⁺. The inner zone of the fibrous capsule of nodules contained macrophages with the profile NSE⁺⁺⁺, AcPase^–, ATPase^–/+, HLA-DR^–/+. A fourth type, NSE⁺⁺⁺, AcPase^–/+, ATPase^–/+, HLA-DR⁺⁺⁺, was located in the outer zone of the capsule, frequently within perivascular accumulations of macrophages, lymphocytes and plasma cells. Active fibroblasts were identified at the inner edge of the fibrous capsule by alkaline phosphatase staining. A feature of all nodules examined was the presence of lipid-filled macrophages, demonstrated by Oil Red O stain; these cells were usually situated in zones adjacent to the centre of nodules, and were of the NSE⁺⁺, AcPase⁺⁺, ATPase^–/+, HLA-DR^–/+ type. Lipid accumulation was not found to be related to the clinical status of the patients studied. The origin and functional significance of this lipid is unknown.     

Onchocerca volvulus and epilepsy: A comprehensive review using the Bradford Hill criteria for causation

BackgroundThe possibility that onchocerciasis may cause epilepsy has been suggested for a long time, but thus far, an etiological link has not been universally accepted. The objective of this review is to critically appraise the relationship between Onchocerca volvulus and epilepsy and subsequently apply the Bradford Hill criteria to further evaluate the likelihood of a causal association.MethodsPubMed and gray literature published until September 15, 2020, were searched and findings from original research were synthesized. Adherence to the 9 Bradford Hill criteria in the context of onchocerciasis and epilepsy was determined to assess whether the criteria are met to strengthen the evidence base for a causal link between infection with O. volvulus and epilepsy, including the nodding syndrome.ResultsOnchocerciasis as a risk factor for epilepsy meets the following Bradford Hill criteria for causality: strength of the association, consistency, temporality, and biological gradient. There is weaker evidence supporting causality based on the specificity, plausibility, coherence, and analogy criteria. There is little experimental evidence. Considering the Bradford Hill criteria, available data suggest that under certain conditions (high microfilarial load, timing of infection, and perhaps genetic predisposition), onchocerciasis is likely to cause epilepsy including nodding and Nakalanga syndromes.ConclusionApplying the Bradford Hill criteria suggests consistent epidemiological evidence that O. volvulus infection is a trigger of epilepsy. However, the pathophysiological mechanisms responsible for seizure induction still need to be elucidated.     

Onchocerca volvulus: genetic diversity of parasite isolates from Sudan

Onchocerciasis in Sudan exists in three distinct foci which exhibit differing clinical presentations. Previous studies have demonstrated that a tandemly repeated Onchocerca sequence family with a unit repeat length of 150 bp (the O-150 family) is a useful marker for deducing relationships among different O. volvulus populations. In the current study, the O-150 repeat families of O. volvulus from Sudan were analyzed and compared to each other and to those of parasites from West Africa. Similar to West African and American O. volvulus, the O-150 families of the Sudanese parasites could be divided into clusters within which little or no intracluster variation was evident, suggesting that the O-150 family in these parasites was subject to the forces of concerted evolution. Statistical analysis of the O-150 families from the different Sudanese parasite isolates, employing a nested algorithm based on an analysis of variance, revealed that O. volvulus endemic to the northern focus at Abu Hamed were significantly different from all other O. volvulus populations examined to date. In contrast, parasites from the southern and eastern foci of Sudan were indistinguishable from those endemic to the West African savanna. The significance of these data are discussed in light of knowledge of the biogeography and biology of transmission of O. volvulus in Africa.     

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.     

A Four-Antigen Mixture for Rapid Assessment of Onchocerca volvulus Infection

Background

Onchocerciasis, an infection caused by the filarial nematode Onchocerca volvulus, is a major public health concern. Given the debilitating symptoms associated with onchocerciasis and concerns about recrudescence in areas of previous onchocerciasis control, more efficient tools are needed for diagnosis and monitoring of control measures. We investigated whether luciferase immunoprecipitation systems (LIPS) may be used as a more rapid, specific, and standardized diagnostic assay for Onchocerca volvulus infection.

Methods

Four recombinantly produced Onchocerca volvulus antigens (Ov-FAR-1, Ov-API-1, Ov-MSA-1 and Ov-CPI-1) were tested by LIPS on a large cohort of blinded sera comprised of both uninfected controls and patients with a proven parasitic infection including Onchocerca volvulus (Ov), Wuchereria bancrofti (Wb), Loa loa (Ll), Strongyloides stercoralis (Ss), and with other potentially cross-reactive infections. In addition to testing all four Ov antigens separately, a mixture that tested all four antigens simultaneously was evaluated in the standard 2-hour incubation format as well as in a 15-minute rapid LIPS format.

Findings

Antibody responses to the four different Ov antigens allowed for unequivocal differentiation between Ov-infected and uninfected control sera with 100% sensitivity and 100% specificity. Analysis of the antibody titers to each of these four antigens in individual Ov-infected sera revealed that they were markedly different and did not correlate (r_S = –0.11 to 0.58; P = 0.001 to 0.89) to each other. Compared to Ov-infected sera, patients infected with Wb, Ll, Ss, and other conditions had markedly lower geometric mean antibody titers to each of the Ov 4 antigens (P<0.0002 for each antigen). The simplified method of using a mixture of the 4 Ov antigens simultaneously in the standard format or a quick 15-minute format (QLIPS) showed 100% sensitivity and 100% specificity in distinguishing the Ov-infected sera from the uninfected control sera. Finally, the QLIPS format had the best performance with 100% sensitivity and specificity values of 76%, 84% and 93% for distinguishing Ov from Wb, Ll and Ss-infected sera.

Conclusions

The multi-antigen LIPS assay can be used as a rapid, high throughput, and specific tool to not only to diagnose individual Ov infections but also as a sensitive and potentially point-of-care method for early detection of recrudescent infections in areas under control and for mapping new areas of transmission of Ov infection.     

Structure of the major cytosolic glutathione S-transferase from the parasitic nematode Onchocerca volvulus

Onchocerciasis is a debilitating parasitic disease caused by the filarial worm Onchocerca volvulus. Similar to other helminth parasites, O. volvulus is capable of evading the host's immune responses by a variety of defense mechanisms, including the detoxification activities of the glutathione S-transferases (GSTs). Additionally, in response to drug treatment, helminth GSTs are highly up-regulated, making them tempting targets both for chemotherapy and for vaccine development. We analyzed the three-dimensional x-ray structure of the major cytosolic GST from O. volvulus (Ov-GST2) in complex with its natural substrate glutathione and its competitive inhibitor S-hexylglutathione at 1.5 and 1.8 angstrom resolution, respectively. From the perspective of the biochemical classification, the Ov-GST2 seems to be related to pi-class GSTs. However, in comparison to other pi-class GSTs, in particular to the host's counterpart, the Ov-GST2 reveals significant and unusual differences in the sequence and overall structure. Major differences can be found in helix alpha-2, an important region for substrate recognition. Moreover, the binding site for the electrophilic co-substrate is spatially increased and more solvent-accessible. These structural alterations are responsible for different substrate specificities and will form the basis of parasite-specific structure-based drug design investigations.     

Modelling the migration of Onchocerca volvulus in simuliids, using a simple compartmental process

A simple compartmental process, which is time-homogeneous and has differing transition rates between compartments, is discussed. When such a process is hierarchical, with all individuals released into the first compartment, the resulting distribution of individuals over the various compartments is multinomial. This process is applied to the migration of Onchocerca volvulus in simuliids and appears to represent successfully the early migration from the stomach through the abdomen to the thorax, provided that allowances are made for the engorgement period and the encapsulation of the blood meal by a peritrophic membrane.     

A preliminary analysis of the population genetics and molecular phylogenetics of Onchocerca volvulus (Nematoda: Filarioidea) using nuclear ribosomal second internal transcribed spacer sequences

Nuclear internal transcribed spacer 2 (ITS2) rDNA sequences were used for a molecular phylogenetics analysis of five Onchocerca species. The sister species of the human parasite O. volvulus was found to be the cattle parasite O. ochengi and not O. gibsoni, contrary to chromosomal evidence. The genetic differentiation of two African populations (representing the two African strains) and a Brazilian population of O. volvulus was also studied. Phylogenetic and network reconstruction did not show any clustering of ITS2 alleles on geographic or strain grounds. Furthermore, population genetics tests showed no indication of population differentiation but suggested gene flow among the three populations.     

Identification and characterization of onchoastacin, an astacin-like metalloproteinase from the filaria Onchocerca volvulus

The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections.     

Structure of the extracellular glutathione S-transferase OvGST1 from the human pathogenic parasite Onchocerca volvulus

Onchocerciasis or river blindness, caused by the filarial worm Onchocerca volvulus, is the world’s second leading infectious cause of blindness. In order to chronically infect the host, O. volvulus has evolved molecular strategies that influence and direct immune responses away from the modes most damaging to it. The O. volvulus GST1 (OvGST1) is a unique glutathione S-transferase (GST) in that it is a glycoprotein and possesses a signal peptide that is cleaved off in the process of maturation. The mature protein starts with a 25-amino-acid extension not present in other GSTs. In all life stages of the filarial worm, it is located directly at the parasite-host interface. Here, the OvGST1 functions as a highly specific glutathione-dependent prostaglandin D synthase (PGDS). The enzyme therefore has the potential to participate in the modulation of immune responses by contributing to the production of parasite-derived prostanoids and restraining the host’s effector responses, making it a tempting target for chemotherapy and vaccine development. Here, we report the crystal structure of the OvGST1 bound to its cofactor glutathione at 2.0 Å resolution. The structure reveals an overall structural homology to the haematopoietic PGDS from vertebrates but, surprisingly, also a large conformational change in the prostaglandin binding pocket. The observed differences reveal a different vicinity of the prostaglandin H₂ binding pocket that demands another prostaglandin H₂ binding mode to that proposed for the vertebrate PGDS. Finally, a putative substrate binding mode for prostaglandin H₂ is postulated based on the observed structural insights.