Animal models--Onchocerca ochengi and the development of chemotherapeutic and chemoprophylactic agents for onchocerciasis.
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Animals↗
Biomedical subjects
A Renz
Publications and source records attributed to A Renz.
Distribution of mast cells and their correlation with inflammatory cells around Onchocerca gutturosa, O. tarsicola, O. ochengi, and O. flexuosa.
In recent years, bovine Onchocerca species have been used as models for human onchocerciasis in drug screens. They have been suggested for immunology studies and evaluation of vaccine candidates. Therefore, mast cells and their association with other inflammatory cells were studied in five onchocercal species of cattle and deer using immunohistology. Intact mast cells occurred in large numbers in the capsule and septae of nodules, in fibrous tissue adjacent to nonnodular worms, and perivascularly. Inactive and, more frequency, activated and degranulating mast cells were observed within infiltrates in the nodule center or around nonnodular filariae. They were not detected in direct contact with the cuticle of adult worms or of microfilariae or among the macrophages, giant cells, and neutrophils forming the innermost layer around the worms. Eosinophils, but not mast cells, were obviously associated with microfilariae-producing females. The distribution, frequency, and activity of mast cells were similar for all five species and O. volvulus.
Animals↗
The entry of ivermectin and suramin into Onchocerca ochengi nodules.
No currently available drug, which is safe for mass treatment, effectively kills adults of Onchocerca volvulus, the causal agent of onchocerciasis in humans, or of O. ochengi, a cattle parasite used as a model of O. volvulus. Since adults of both of these filarial nematodes are found in well developed nodules, the lack of efficacy of these drugs may be a result of their poor penetration into the nodules. To check if this was the problem, the distributions of the microfilaricide, ivermectin, and the partial macrofilaricide, suramin, in plasma, skin, nodule capsules and nodule contents were determined in cattle naturally infected with O. ochengi in Cameroon. The cattle were treated with either a single, subcutaneous injection of 500 micrograms ivermectin/kg, or with intravenous injections of [14C]-labelled suramin, each of 10 mg/kg, given one a day for 6 days. Concentrations of ivermectin and suramin in various tissues were then assayed by high-pressure liquid chromatography and scintillation counting, respectively. On day 7 post-treatment (pt), suramin concentrations were consistently highest in the nodule, contents and capsule wall (11.0 and 8.9 nCi/g, respectively) and significantly less in skin and plasma (1.2 and 1.4 nCi/g, respectively; P < 0.05). The distribution of ivermectin on day 7 pt was similar, with the highest concentrations in the capsule wall, nodule contents and plasma (58.4 ng/g, 43 ng/g and 48.6 ng/ml, respectively; P > 0.05) and the concentration in the skin (6.4 ng/g) significantly lower than those in the capsule or plasma (P < 0.05). High intra-nodular concentrations of both drugs were maintained for 5-7 days at least and those of ivermectin would be expected to kill nematodes other than filariae. It is apparent that failure of ivermectin and suramin to kill adult Onchocerca spp. is not because the drugs penetrate nodules inadequately.
Animals↗
Purification and molecular cloning of the scaffold attachment factor B (SAF-B), a novel human nuclear protein that specifically binds to S/MAR-DNA.
We have purified to near homogeneity a novel nuclear protein from HeLa cells, that specifically binds to scaffold or matrix attachment region DNA elements (S/MAR DNA). The protein, designated SAF-B for scaffold attachment factor B, is an abundant component of chromatin, but not of the nuclear matrix and is expressed in all human tissues investigated. Antibodies against the purified protein were raised in rabbit and used to isolate the complete cDNA encoding SAF-B by immunoscreening. As predicted from the cDNA sequence, SAF-B contains 849 amino acids (96 696 Da), without significant homology to any known protein. SAF-B is rich in charged residues, leading to an aberrant migration on SDS gels, and has two putative bipartite nuclear localisation signals.
Animals↗
Evaluation of suramin, ivermectin and CGP 20376 in a new macrofilaricidal drug screen, Onchocerca ochengi in African cattle.
To aid the development of a macrofilaricidal agent for Onchocerca volvulus, the African bovine parasite, O. ochengi, was evaluated as a drug screen by testing three known filaricidal drugs. Groups of five Zebu cattle, naturally infected with more than 15 palpable O. ochengi nodules in the ventral skin, were treated with either suramin (10 mg/kg/day i.v. for 6 days), ivermectin (200 micrograms/kg, s.c.), CGP 20376 (20 mg/kg orally) or left untreated as controls and examined at intervals up to 137 days post-treatment (d.p.t.). After ivermectin treatment, microfilarial densities in the skin decreased within one week to virtually zero and remained at a very low level. A similar rapid and profound reduction was seen after CGP 20376 treatment, but by 137 d.p.t. microfilarial skin densities were approaching pre-treatment levels. With suramin, skin microfilarial densities fell to very low levels after 12 weeks but rose slightly by 137 d.p.t. Effects on the macrofilariae were assessed by sequential nodulectomies at -3 and 28, 84 and 137 d.p.t. By 137 d.p.t. embryogenesis was almost completely interrupted in the CGP 20376 and ivermectin treated animals, although not in the suramin treated group, but in all three groups the majority of remaining intrauterine microfilariae were pathologically altered. Degenerating intrauterine microfilariae accumulated in the ivermectin and in the CGP 20376, but not in the suramin treated worms. The motility of male and female worms was not reduced by any treatment except for female worms at 84 d.p.t. with CGP 20376. Viability of the worms as indicated by the MTT-formazan reduction assay was not reduced in any of the treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Africa↗
Nucleic-acid-binding properties of hnRNP-U/SAF-A, a nuclear-matrix protein which binds DNA and RNA in vivo and in vitro.
We show that SAF-A, a nuclear protein which specifically binds vertebrate scaffold-attachment-region (SAR) elements with high affinity is identical with hnRNP-U, assumed to be involved in packaging of hnRNA in ribonucleoprotein particles. Ultraviolet cross-linking experiments show that the protein, referred to as hnRNP-U/SAF-A, is bound to chromosomal DNA in vivo. In vitro, the isolated protein binds to double-stranded and single-stranded DNA and forms higher ordered nucleic-acid-protein complexes. Filter-binding experiments performed with different types of natural and synthetic nucleic acids as substrates show that the protein binds DNA and RNA with different affinities and most likely at different binding sites. We conclude that hnRNP-U/SAF-A thus may have functions in the organisation of chromosomal DNA in addition to its suggested role in hnRNA metabolism.
Amino Acid Sequence↗
Bovine onchocercosis in north Cameroon.
Ventral skin biopsies from 204 Gudali cattle of the Vina division in the Adamawa highlands revealed microfilariae of Onchocerca gutturosa, O. ochengi and O. dukei in 85%, 51% and 8% of the animals, respectively. In 60 Fulani cattle from the Tcholliré division in the Sudan savanna, the same microfilaria species were detected in 92%, 83% and 47% of the animals. Onchocerca armillata adult worms were found in 67% of the Gudalis and in 100% of the Fulanis. In areas of high transmission the prevalences declined in old animals, possibly indicating acquired resistance. For all species no significant difference in prevalence was found between male and female cattle. The microfilariae of O. ochengi and O. dukei were concentrated in the skin of the posterior and anterior belly, respectively. Onchocerca gutturosa microfilariae had highest densities on the hump and near the umbilicus, whereas those of O. armillata were distributed more evenly across the body surface. In infected hides the mean microfilarial densities of O. gutturosa, O. ochengi, O. dukei and O. armillata were respectively 3.1 microfilariae (mff) mg-1, 0.6 mff mg-1, 0.7 mff mg-1 and 0.092 mff mg-1 for the whole body surface and 9.3 mff mg-1, 3.8 mff mg-1 and 1.9 mff mg-1 for the sites of highest density (O. armillata had no predilection site). Ninety-five per cent of the microfilariae were located in the uppermost skin layer of 2 mm depth, 5% were in the corium and none were found in the subcutis. Two cattle had skin microfilariae of a hitherto unknown Onchocerca species.
Age Distribution↗
Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements.
We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF-A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF-A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF-A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T-stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures.
Animals↗
Age-related differences in parasitosis may indicate acquired immunity against microfilariae in cattle naturally infected with Onchocerca ochengi.
Onchocerca ochengi is a common parasite of cattle in savanna areas of West Africa and its adult stages inhabit intradermal nodules. We have exploited the accessibility of the macrofilariae to examine quantitative and qualitative differences in both the macro- and microfilariae (mff) in relation to age in naturally infected cattle. Autochthonous cattle at a site on the Adamawa plateau, North Cameroon, situated near a perennial Simulium damnosum s.l. breeding site, were examined in three age groups (1.5-2.5, n = 34; 3-5, n = 39 and greater than or equal to 8 years old, n = 21). Skin mff densities were assessed from ventral skin biopsies, total body counts of nodules were done by palpation and 1-4 nodules were excised from positive animals, collagenase digested and the numbers and condition of macrofilariae determined. Embryogenesis profiles (embryograms) were done on a representative number of female worms in each age group. The overall prevalence of infection in the study population was 71% for mff and 85% for nodules. Nodule prevalence increased significantly from the 1.5-2.5 to 3-5 years old groups, but no other inter-age prevalences were significantly different. While the mean number of nodules per animal increased with age, geometric means (gm) 3.7, 15.5 and 23.1, the skin mff density maximized in the middle age group and decreased significantly in the old animals (gm 15.8, 44.7 and 11.4 mff/g, difference between latter two parameters significant, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗
The development of Onchocerca dukei and O. ochengi microfilariae to infective-stage larvae in Simulium damnosum s.l. and in members of the S. medusaeforme group, following intra-thoracic injection.
Neonate Simulium females from an endemic area of human and bovine onchocerciasis in North-Cameroon were injected in one set of experiments with microfilariae (mff) of Onchocerca dukei and in another with mff of O. ochengi. Onchocerca dukei was found to develop to infective third-stage larvae in Simulium flies: 148 third-stage larvae (28.1% of injected mff) were recovered in S. hargreavesi, seven (10.4% of injected mff) in S. vorax, and 12 (1.3%) in S. damnosum s.l. Onchocerca ochengi mff developed in S. hargreavesi at a rate of 17.3% and in S. damnosum s.l. at 16.1%, yielding a total of 77 and 32 third-stage larvae respectively. Onchocerca dukei infective larvae had a mean length of 717.3 microns and a mean maximum diameter of 17.5 microns, and were slightly shorter and conspicuously thinner than those of O. ochengi (748.3 microns x 20.4 microns). All larval stages of O. dukei typically had an attenuated anterior end. The implications of these findings for the epidemiology of human onchocerciasis are discussed.
Animals↗
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.
Animals↗
Transmission of Onchocerca dukei by Simulium bovis in North-Cameroon.
"Wild" Simulium bovis females were collected after they had taken blood on a bait oxen with skin microfilariae (mf) of Onchocerca dukei and Onchocerca ochengi. Flies ingested O. dukei mf in high numbers (13.1 mf per blood fed fly) but rarely mf of O. ochengi (3 mf in 60 flies). In 32 flies dissected 14 to 17 hours after the blood meal, a total of 130 O. dukei mf but no of O. ochengi had migrated to the thorax. Six to 9 days after the blood meal, a total of 203 third stage larvae (L3) were found in 61 surviving flies. Invasive larvae from the head measured 738.0 x 17.8 microns and resembled those of O. dukei obtained by intrathoracic injection of mf in a previous study, but were shorter and thinner than O. ochengi L3. Thus, S. bovis can be seen as an efficient natural vector of O. dukei. Its role in the transmission of bovine and human onchocerciasis in North Cameroon is discussed.
Animals↗
A new PGM electromorph diagnostic for S. squamosum from Sierra Leone and Togo but not found in S. squamosum from Cameroun.
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Animals↗
Differential length of Onchocerca volvulus infective larvae from the Cameroon rain forest and savanna.
Infective larvae from the savanna strain of Onchocerca volvulus are significantly longer (662.6 +/- 66.8 microns; n = 99) than those from the forest strain (624.2 +/- 62.0 microns; n = 236). The length of the infective larvae is not influenced by the size, species or age post infectionem (pi) of the Simulium damnosum s. l. vectors nor by their localization in the fly's head, thorax or abdomen, the worm load per fly or the thoracic volume per larva. However, the lengths of infective larvae within one individual fly have a conspiciously low variance.
Analysis of Variance↗
An oligonucleotide probe specific for Onchocerca volvulus.
A genomic DNA library of a Liberian strain of Onchocerca volvulus was prepared in the vector bacteriophage lambda gt10. The library was differentially screened by hybridisation with radiolabelled total DNA from the homologous parasite, two heterologous Onchocerca 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.
Animals↗
Construction of Onchocerca volvulus cDNA libraries and partial characterization of the cDNA for a major antigen.
Adult Onchocerca volvulus were isolated from nodules removed from onchocerciasis patients at four locations--two in the West African Sudan-savanna region (near Bamako, Mali, and Touboro, Cameroon), one in a West African forest region (Kumba, Cameroon) and one near Guatemala City, Guatemala. Four different cDNA expression libraries were constructed in bacteriophage lambda gt11 using poly(A)+ RNA from the adult female worms. Individual cDNA clones of single copy genes were used to compare the genomes of parasites from the different locales and to show that the haploid genome of O. volvulus is 1.5 x 10(8) base pairs. About 1 in 700 recombinant clones in each of the four amplified cDNA libraries produces a fusion protein recognized by pooled human anti-O. volvulus antisera. Partial sequence determination of a 2.0 kb cDNA clone for an O. volvulus protein that induces an immunodominant response in rabbits revealed that this antigen has sequence similarities with Caenorhabditis elegans myosin and with schistosome paramyosin (which confers partial protection against schistosome infection). The four cDNA libraries have been deposited with American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20852, U.S.A., for general distribution under ATCC Number 37509.
Animals↗
Studies on the dynamics of transmission of onchocerciasis in a Sudan-savanna area of North Cameroon I. Prevailing Simulium vectors, their biting rates and age-composition at different distances from their breeding sites.
Along the main water courses in the sparsely populated areas of the Sous-Préfecture of Tcholliré, the vectors of onchocerciasis were mainly Simulium damnosum s. str. and S. sirbanum, together with a small proportion of S. squamosum. Over a period of one to three years, vector biting rates were measured at 23 fly-catching sites in the vicinities of nine villages with different endemicities of onchocerciasis. Annual Biting Rates (ABR) on man were estimated as 26,100-83,800 fly-bites per man per year along the rivers Mayo Rey and Vina du Nord, and 11,000-37,400 at rainy season tributaries. Biting rates decreased rapidly at increasing distances from the river, and were between 10,700 and 2400 at 2-10 km cross-country from the breeding site. Lowest biting rates (50-6000) were measured at the village centres. The ABR varied from year to year in relation to the water-discharge of the main rivers, the coefficient of variation of the mean being 34-49%. The parous rates were 64-73% at the perennial breeding sites and only 17-44% away from the breeding sites, indicating dispersal mainly of young nulliparous flies and a reduced flight-range after oviposition.
Age Factors↗
Studies on the dynamics of transmission of onchocerciasis in a Sudan-savanna area of North Cameroon II. Seasonal and diurnal changes in the biting densities and in the age-composition of the vector population.
Near the perennial breeding sites in the main water courses the highest monthly biting rates were recorded from June to August (early rainy season) and again from December to February (dry season), but near the rainy season breeding sites in the affluents, or at fly-catching sites, more than 2 km inland from the nearest main river breeding site, the highest rates were recorded between August and December (mid-rainy to early dry season). The variation of the biting rates over the year depended on the waterflow of the main rivers and on the differential dispersal of the flies from the breeding sites during the dry and rainy seasons. At the beginning of the rainy season, when the rivers started flowing, an immigration of non-local flies was observed at the river Mayo Rey near Tcholliré, presumably coming from Simulium breeding sites in the Adamaoua mountains to the South. During three years 77,374 flies were caught by daily catches between 06.00 and 18.30 hours. During the dry season the biting activity of nulliparous and parous flies was highest between 09.00 and 10.00 hours and 16.00 and 17.00 hours, 9% and 22% of the total day-catches respectively. In the rainy season the peak biting rates occurred between 06.00 and 07.00 hours (9%) and again from 16.00 to 17.00 hours (14%). The lowest biting rates were observed between 11.00 and 12.00 hours in both dry and rainy seasons (4% and 3% of the total catch). Nulliparous flies showed higher variations in biting activity than did parous flies, leading to the highest proporation of parous flies during midday.(ABSTRACT TRUNCATED AT 250 WORDS)
Age Factors↗