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The Wuchereria bancrofti orthologue of Brugia malayi SXP1 and the diagnosis of bancroftian filariasis.

The gene encoding the Wuchereria bancrofti orthologue of the Brugia malayi-derived diagnostic antigen SXP1 was identified from a W. bancrofti L3 cDNA library and characterized. The Wb-sxp-1 cDNA encoded a basic protein with a calculated molecular mass of 20.8 kDa. Wb-SXP-1 was 85% identical to the SXP1 protein described from B. malayi (Bm-SXP-1). The Wb-SXP-1 sequence also showed significant identity with proteins described from B. pahangi, Onchocerca volvulus, Acanthochilonema vitea, Ascaris suum, Loa loa, Litomosoides sigmodontis and Caenorhabditis elegans. The presence of a number of invariant and conserved residues in all of these nematode-derived molecules suggests that Wb-SXP-1 is a member of a new protein family. A recombinant form of Wb-SXP-1 was produced and it was determined that the anti-Wb-SXP-1 antibody response in patients with W. bancrofti infections was restricted to the IgG4 subclass. An anti-Wb-SXP-1 IgG4 ELISA was developed and this assay was found to be 100% sensitive for patients with patent W. bancrofti infection. Sera from individuals experiencing chronic pathology, endemic normals or patients with non-filarial nematode infections had no detectable IgG4 against Wb-SXP-1. While patients with patent Onchocerca volvulus infections were uniformly negative in the Wb-SXP-1 assay, 40% of sera from patent Loa loa infections were positive. When Bm-SXP-1 was used as the antigen under identical conditions, the assay was 88% specific for patent W. bancrofti infections and the antigen was recognized by antibodies from both O. volvulus and L. loa infections. The results strongly suggested that, for certain diagnostic filarial antigens, the use of same-species molecules can enhance the specificity of diagnostic tests.

Amino Acid Sequence↗

Ecdysteroids and oocyte development in the black fly Simulium vittatum.

BACKGROUND: Oocyte development was studied in the autogenous black fly, Simulium vittatum (Diptera, Nematocera), a vector of Onchocerca volvulus, the causative agent of onchocerciasis. RESULTS: Oocyte growth was nearly linear between adult eclosion and was complete by 72 hours at 21 degrees C. The oocyte became opaque at 14 hours after eclosion indicating the initiation of protein yolk deposition. The accumulation of vitellogenin was measured using SDS-PAGE. The density of the yolk protein bands at about 200 and 65 kDa increased during the first and second days after eclosion. The amount of protein in the 200 kDa band of vitellogenin, determined using densitometry, rapidly increased between 12 and 25 hours after eclosion. Ecdysteroid levels were measured using a competitive ELISA. Ecdysteroid levels increased rapidly and subsequently declined during the first day after eclosion. CONCLUSION: These data show a correlation between the appearance of vitellogenin in the oocyte, and the rise in ecdysteroids. A possible relationship to molting of the nematode, Onchocerca volvulus, is discussed.

Animals↗

[Serodiagnosis of onchocerciasis using micro-ELISA. Study of 450 sera and comparison with indirect immunofluorescence].

The microtest ELISA has been used for human Onchocerciasis serological study. The antigens employed were adult Onchocerca volvulus extracts, collected from dissected nodules, delipidized and cleared from human proteins by affinity Chromatography. Under the circumstances, the positivity limit of the test seems excellent (maximum )D: 0,23) defined with 171 negative sera, 66 of them taken from Africans. Specificity controls were studied with 56 heterologous sera; cross-reactions occurred with hydatidosis and especially wit various nematode infections, in particular loasis. With reagents and technical conditions used, the specificity limit of the test corresponds to an OD of 0,4 (measured with a 3 mm optical course). The diagnosis value of the test was verified by studying sera from 90 individuals wit a positive skin biopsy and with sera from 233 adults living in endemic areas. For all the infected people, the global percentage of positivity with ELISA is not greater than that with indirect immunofluorescent antibody test (85%). On the other hand, the micro-test ELISA seems slightly more sensitive in detection of high serological positivities. We did not find any statistically relationship presence and quantity of microfilarial worms in skin biopsy and positivity with the microtest ELISA. Likewise, in some polyinfested patients (with Onchocerca volvulus and Dipetalonema perstans or Wuchereria bancrofti), we did not observe any correlation between the results given the microtest ELISA and the quantity of microfilariae in the blood stream.

Enzyme-Linked Immunosorbent Assay↗

Onchocera volvulus: characterization of a highly immunogenic Gln-rich protein.

A pool of sera from individuals classified as putatively immune (PI) to Onchocerca volvulus infection was employed in the screening of a fourth-stage larval cDNA expression library. A highly immunogenic clone, encoding the Ov 53/80 protein, was identified. The full length cDNA of clone 4.21 contained 2527 nucleotides encoding 769 amino acids of which 100 are glutamine residues (13%). Antibodies raised against recombinant protein encoded by a partial cDNA sequence (clone 73-k) recognized a 53 and 80 kDa protein in O. volvulus larval and adult parasite extracts, respectively. The antibodies localized the native protein in the cuticle, hypodermis, secretory vesicles and in granules of the glandular esophagus of larvae and in the hypodermis and the cuticle of adult worms. The recombinant 73-k polypeptide (r73) was recognized by 90-100% of sera from PI and infected individuals from Liberia, but only by 67% of similar groups from Ecuador. r73 specific IgG2 and IgG3 levels in the PI from Liberia and Ecuador, respectively, were significantly lower than in the infected, whereas the r73 specific IgG1/IgG3 or IgG1/IgG2 in the PI and the infected individuals from Liberia or Ecuador, respectively, were similar. The IgG4 specific antibody response in the PI from Liberia and Ecuador were lower than in the infected. The T-cell proliferative responses to r73 in infected individuals from Cameroon were found to be inversely correlated with their levels of microfilariae.

Amino Acid Sequence↗

The identification of infective filarial larvae in Simuliidae.

Although it is recognized that the presence of animal filariae can lead to confusion in the interpretation of infection rates in mosquito vectors of filariasis, the filariae found in man-biting simuliids are usually assumed to be Onchocerca volvulus. The authors of this paper emphasize that it is unwise to calculate transmission indices from infection rates in man-biting simuliids unless there is confidence in the identification of the filarial larvae. In this respect they cite their observations on Mount Elgon in Uganda which show that the majority of the filarial larvae in Simulium neavei-the local vector of onchocerciasis-are of species that do not affect man.To assist in the correct interpretation of infection rates in the vectors the authors made a detailed study of the morphological character of O. volvulus infective larvae and established criteria for distinguishing O. volvulus from other filariae known to be transmitted by simuliids.

Animals↗

Characterization of two monoclonal antibodies raised in Btk(xid) mice that recognize phosphorylcholine-bearing antigens from Trichinella and other helminths.

This study investigated the binding properties of two monoclonal antibodies (mAbs US1 and US2) raised in (CBA/n x BALB/c)F1 (NBF1) Btk(xid) male mice. Both mAbs show unusual specificity for phosphorylcholine (PC)-containing TSL4 antigens of Trichinella. Specifically, and in contrast to mAbs raised in normal mice, US1 and US2 mAbs do not bind to artificial PC-protein conjugates and are not inhibited by either free PC or NPPC, although US2 was partially inhibited by NPPC at high concentration (10(-2) M). However, both mAbs completely abrogate the binding to Trichinella antigens of other anti-PC mAbs (e.g. BH8 and Mab-2). These results suggest that both US1 and US2 recognize complex PC-containing epitopes. The patterns of recognition of PC-bearing antigens from different helminths by US1, US2, Mab-2 and BH8 were broadly correlated with phylogenetic proximity. The closest similarities were observed between the members of the Trichinelloidea superfamily (Trichinella spiralis and Trichuris muris) and among the ascarids (Toxocara canis, Anisakis simplex, Hysterothylacium aduncum and Ascaris lumbricoides). However, US1 did not react with the filarial nematode Onchocerca volvulus and reacted only weakly with Onchocerca gibsoni, while US2 reacted only weakly with both species. Only BH8 recognized PC-bearing antigens from the trematode Fasciola hepatica and the cestode Bothriocephalus scorpii. These results suggest that PC is attached to identical or very similar structures on most different nematode species, although major differences exist with respect to helminth species from groups such as the trematodes and cestodes that are phylogenetically distant from the nematodes.

Animals↗

Anthropophilic blackflies (Diptera: Simuliidae) and onchocerciasis transmission in southwest Ethiopia.

From May 1984 to May 1985, the transmission of onchocerciasis by blackflies (Dipteria: Simuliidae) was studied around Gilgel Ghibe and Gojeb rivers in southwest Ethiopia. The predominant anthropophilic species of Simulium at both localities were S. (Edwardsellum) damnosum sensu lato and S. (Lewisellum) ethiopiense. Some S. (Anasolen) dentulosum also landed on human bait at Gilgel Ghibe river. Dissections of S. damnosum from Gilgel Ghibe and Gojeb revealed mean parous rates of 92% (n = 18,291) and 84% (n = 9530), respectively. S. ethiopiense from Gilgel Ghibe and Gojeb showed mean parous rates of 53% (n = 322) and 93% (n = 14), respectively. Of the parous S. damnosum, 1.3% at Gilgel Ghibe and 0.5% at Gojeb harboured infective third-stage larvae (L3) of Onchocerca volvulus (or morphologically indistinguishable from it). Unknown filariae of animal origin, indicative of zoophily, were found in 0.3% and 0.7% of Simulium damnosum complex females from Gilgel Ghibe and Gojeb, respectively. S. ethiopiense harboured developing (L1) larvae only, with 7.7% infection rate in both localities. In contrast, S. dentulosum did not harbour any filaria larvae. The annual infective biting rate (AIBR) and transmission potential (ATP) of the S. damnosum complex at Gilgel Ghibe river were 858 and 5478, respectively. The AIBR and ATP of S. damnosum s.l. at Gojeb river were 519.5 and 1963, respectively. These results emphasize the predominant role of the S. damnosum complex in the transmission of O. volvulus in southwest Ethiopia.

Animals↗

Diagnosis of onchocerciasis using highly specific and sensitive native proteins.

We describe a highly immunogenic low molecular weight fraction of proteins obtained from Onchocerca volvulus. It is comprised of a mixture of native proteins ranging in molecular weight from 10 to 40 kDa and is collectively designated PakF. Unlike other Onchocerca (and other filarial) antigens previously described, IgG4 antibody responses to this protein mixture were absent or low. In a simple, IgG-based dot blot assay, this mixture of native proteins showed high sensitivity, comparable to or better than that of other previously described recombinant proteins, when used to detect infected individuals in endemic areas of Ghana and Guatemala. The specificity was also high when evaluated using sera from patients with other filarial infections. Arguments are put forward to suggest that such a native fraction could present an interesting alternative to the use of well-defined molecules, which are expensive, labile or prone to denaturation, in endemic areas.

Animals↗

Design of Onchocerca DNA probes based upon analysis of a repeated sequence family.

Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.

Animals↗

The prevalence of epilepsy follows the distribution of onchocerciasis in a west Ugandan focus.

Epidemiological surveys indicate that the prevalence of epilepsy is higher in developing countries than in industrialized countries. Except for neurocystocercosis due to Taenia solium, little is known about possible underlying causes. This article reports the relationship between epilepsy and onchocerciasis in an Onchocerca volvulus endemic area in West Uganda. Individuals complaining of seizures were identified by means of a population census in 12 villages. Active epilepsy was confirmed in 61 of 4743 inhabitants (crude prevalence rate = 1.3%; age-standardized rate = 1.1%). Distribution of epilepsy in the study area was clustered, ranging from a prevalence of 0.2% to 3.4% in different villages. Age-specific prevalence was highest between 10 and 19 years, with a rate of 3.6% for the study are as a whole, and up to 10.0% in villages of high epilepsy prevalence. The prevalence of onchocerciasis in the 10-19-year-old age group was assessed by skin-snip biopsy and ranged from 15% to 85% in different villages. Epilepsy was significantly more frequent in the three villages with the highest levels of O. volvulus endemicity than in other villages (P < 0.0001). Serological testing for T. solium infection was positive in one and borderline in three of 53 epilepsy patients tested. The significant correlation between epilepsy and onchocerciasis did not change when these four patients were excluded from the analysis. These findings suggest a strong association between epilepsy and onchocerciasis in this area. This could have significant implications for the concept of morbidity due to O. volvulus.

Adolescent↗

[Molecular approaches to the control of onchocerciasis in Mexico].

Onchocerciasis is one of the major causes of blindness in the World, with about 17.7 million infected, particularly in West Africa. In Mexico, onchocerciasis is also present and has been subjected to control since 1923. The standard diagnosis of onchocerciasis is by the detection of microfilariae by skin biopsy and transmission is evaluated by detection of Onchocerca volvulus larvae in the vector. Classically, this was carried out by manual dissection of Simuliumn ochraceun s.l. However, with the use of ivermectin, a drug that kills microfilariae but not the adult worms, the skin biopsy is becoming no longer useful for detecting microfilariae levels and due to the reduced transmission, fly dissection is no longer viable. The subject of this paper is to present the immunological and molecular techniques developed to supersede the skin biopsy and fly dissection, and their diagnostic ability to assess the impact of multiple bi-annual mass ivermectin treatments on O. volvulus transmission in Mexico.

Africa, Western↗

Host-parasite interaction in human onchocerciasis: identification and sequence analysis of a novel human calgranulin.

A novel S100 ca2+-binding protein, termed calgranulin-related protein (CGRP), was purified to homogeneity from extracts of adult Onchocerca volvulus, a human tissue-dwelling parasite. Its complete amino acid sequence was determined using microanalytical techniques. The primary structure of CGRP consists of 91 residues and displays identity with the recently reported partial sequence of an S100 protein present in human neutrophils. The human origin of CGRP is supported by the occurrence in O. volvulus extracts of additional human neutrophil proteins, including migration inhibitory factor-related protein 8 and defensins. The results suggest that these proteins interact with the worm surface following their release by activated neutrophils in the course of inflammatory reactions caused by O. volvulus infection.

Amino Acid Sequence↗

Future directions for vaccine-related onchocerciasis research.

Although the general perception has been that Onchocerca volvulus infection is well on its way towards extinction, it is becoming increasingly clear that without an additional therapeutic or preventative modality (separate from ivermectin distribution or vector control), elimination of onchocercal infection will remain an unfulfilled goal. Vaccination aimed at preventing infection (directed toward infective-stage or adult worms), transmission and/or pathology (directed toward skin microfilariae) could provide the necessary 'final punch' to knockout human infection with O. volvulus altogether. Thus, efforts toward the development of a useful vaccine need to be redoubled rather than shelved, and, to this end, major gaps in our knowledge need to be filled.

Animals↗

DNA probe-based classification of Simulium damnosum s. l.-borne and human-derived filarial parasites in the onchocerciasis control program area.

The development of polymerase chain reaction-based methods using strain- and species-specific DNA probes for Onchocerca volvulus has permitted classification of individual parasites from every stage of the parasite's life cycle. This technology has been applied on a large scale basis by Onchocerciasis Control Program (OCP) in West Africa. The primary objective of the OCP in using the DNA probes was to obtain accurate estimates of the annual transmission potential of the blinding strain of O. volvulus. The DNA probe classification of larvae collected throughout the OCP area demonstrated that larvae of less pathogenic strains of O. volvulus and other filarial parasites carried by Simulium damnosum s.l. have resulted in a significant overestimation of the annual transmission potential for blinding onchocerciasis. This effect is particularly pronounced along the southern border of the OCP, where the blinding and less pathogenic strains of O. volvulus coexist, and in the north of the control area, where animal parasites, particularly O. ochengi, may even predominate. A second objective of the OCP in applying the DNA probe technology was to determine the distribution of blinding and less pathogenic O. volvulus in infected individuals along the southern border of the control area. Results obtained from these studies have generally confirmed the distribution pattern established by previous epidemiologic studies. In addition, DNA probe classifications have demonstrated that in areas where the blinding and less pathogenic strains of O. volvulus coexist, a single individual may simultaneously be infected with both strains of the parasite.

Africa, Western↗

Down-regulated lymphoproliferation coincides with parasite maturation and with the collapse of both gamma interferon and interleukin-4 responses in a bovine model of onchocerciasis.

Onchocerciasis is a debilitating parasitic infection caused by the filarial nematode Onchocerca volvulus. Infections are chronic, and persistence of the parasites for several years argues for highly adapted mechanisms of immune evasion. Due to the restricted host repertoire of O. volvulus, we have used the cattle parasite Onchocerca ochengi to investigate the nature of immunomodulation underpinning these long-term infections. Cattle were infected with a single inoculation of 350 infective-stage larvae under laboratory conditions (n = 6). Intradermal nodules containing immature adult worms were detected from 110 days postinfection, and microfilariae in skin were detected from day 280 postinfection. Parasite-specific responses during early infection were nonpolarized with respect to the major Th cytokines (interleukin-4 [IL-4], IL-2, and gamma interferon [IFN-gamma]) produced by antigen-stimulated peripheral blood mononuclear cells (PBMC) or serum antibody isotypes. Antigen-induced proliferation of PBMC peaked shortly after exposure and remained high during the prepatent infection. As the parasites matured and animals developed patent infections, there was a profound down-regulation of lymphoproliferation, accompanied by sharp falls in the expression of both IL-4 and IFN-gamma and a gradual decline in IL-2. Levels of immunoglobulin G2 (IgG2) fell, while those of IgG1 remained high. We conclude that neither a classical Th2 response nor a simple Th1-to-Th2 switch is sufficient to explain the immunomodulation associated with patent Onchocerca infections. Instead, there is an initial Th0 response, which matures into a response with some, but not all of the features of a Th2 response. The natural host-parasite relationship of O. ochengi in cattle may be useful as both a descriptive and predictive tool to test more refined models of immunomodulation in onchocerciasis.

Animals↗

Adverse systemic reactions to treatment of onchocerciasis with ivermectin at normal and high doses given annually or three-monthly.

In Cameroon, a 3-year randomized, double-blind controlled trial was conducted to determine if ivermectin, given at 3-monthly intervals and/or at high doses (800 microg/kg), had a greater effect on adult Onchocerca volvulus than standard annual doses of 150 microg/kg. Adverse reactions were recorded and analysed in a logistic regression model with random effects to assess the influence of the dose and rhythm of treatment on their occurrence. After the first dose, 3-monthly treatment was associated with a clearly reduced risk of reactions, especially oedematous swellings, pruritus and back-pain. Oedematous swellings and subjective ocular troubles were found to be associated with high doses of ivermectin. These results reinforce former parasitological conclusions that it would be desirable to evaluate the feasibility and effects on transmission of large-scale 3-monthly treatments with standard doses of ivermectin for onchocerciasis control. Owing to the unexpected ocular reactions, the use of high doses to counteract any future resistance of O. volvulus to ivermectin should be considered with caution.

Adolescent↗

Filariasis in the Kaiyamba Chiefdom, Moyamba District Sierra Leone: an epidemiological and clinical study.

In a cross-sectional epidemiological and clinical study of human filariasis, 630 individuals were examined for Onchocerca volvulus, Wuchereria bancrofti and Mansonella perstans infections in five communities in the Kaiyamba Chiefdom, Moyamba District, Sierra Leone. The overall prevalence of O. volvulus infection in males 144(39.1%) and females 94(35.9%) was not significantly different and the sex prevalence rate between communities was also not significant (G = 3, d.f. = 4, P > 0.05). Prevalence of O. volvulus was significantly lower (G = 42.331, d.f. = 5, P < 0.001) in the 5-9 age group (13.3%) compared to the 40-49 age group (61.9%). Sixty-four (10.2%) and 38(6.0%) of individuals examined were positive for W. bancrofti and M. perstans infections respectively and prevalence of both infections in the five communities was not significant. Mixed infections with the all three filaria parasites were recorded in 10(3.2%) of the individuals. One hundred and sixty-four (71.3%) clinical cases due to W. bancrofti were inflammatory in nature; 36.5% were chronic, of which, 26.6% were hydroceles and 9.4% involved elephantiasis of both the scrotum and the lower legs. All 19(3.0%) of M. perstans-related clinical cases were inflammatory. Ninety-three(63.3%) of O. volvulus positive individuals that presented symptoms were inflammatory in nature, 14(9.5%) had ocular symptoms and 57(38.8%) had subcutaneous nodules. These data indicate that infections due to O. volvulus, W. bancrofti and M. perstans may be of public health importance in Sierra Leone.

Adolescent↗