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An evaluation of the bovine -- Onchocerca gibsoni, Onchocerca gutturosa model as a tertiary screen for drugs against Onchocerca volvulus in man.

In this trial suramin, diethylcarbamazine, trichlorphon, levamisole, mebendazole, melarsonyl potassiu, Hoechst 33258 and tinidazole were administered to cattle infected with O. gibsoni and O. gutturosa to determine the usefulness of this screen in predicting the effect of drugs in man against. O. volvulus except for melarsonyl potassium which was macrofilarticidal against O. gutturosa but not O. gibsoni when cattle were slaughtered 6 weeks after treatment. It was concluded that cattle infected with O. gibsoni are a satisfactory substitute for chimpanzees infected with O. volvulus, as a tertiary screen for drugs against O. volvulus, but that their use would be restricted to centres in O. gibsoni endemic areas where the necessary facilities and specialised knowledge required to use cattle as experimental animals exist.

Animals

Identification of antigens of Onchocerca volvulus and Onchocerca gibsoni for diagnostic use.

Adults of Onchocerca volvulus and Onchocerca gibsoni were identically fractionated into a surface-enriched fraction, a phosphate buffered saline (PBS) extract and a PBS insoluble-detergent soluble fraction. Glycoproteins were prepared from these extracts and all fractions were examined by the Western blot technique using sera from individuals infected with a variety of filarial and non-filarial nematode worms. Using antisera to O. volvulus, a number of antigens were demonstrated in all of the extracts, with some antigens of each extract being unique. Many antigens were glycoproteins, and a high cross-reactivity was observed between O. volvulus and O. gibsoni. The different fractions of both species were also analysed using a panel of different sera in order to identify Onchocerca-specific antigens. The studies revealed that the lower molecular weight antigens showed greater Onchocerca specificity in all of the extracts examined. The surface-enriched fraction, however, clearly contained less widely cross-reacting components than the somatic and glycoprotein fractions. Finally, using surface labelling and coprecipitation techniques, O. gibsoni was shown to possess a 20 kDa Onchocerca-specific antigen, previously described for O. volvulus. The findings indicate a number of Onchocerca-specific antigens which may have potential in diagnosis of human onchocerciasis. They also show that the related bovine parasite O. gibsoni, may be an alternative source of material.

Animals

Resistance to Onchocerca lienalis microfilariae in mice conferred by egg antigens of homologous and heterologous Onchocerca species.

Embryonic stages of various Onchocerca species have been used to stimulate resistance in CBA mice to challenge injections with the microfilariae of Onchocerca lienalis. Comparable levels of resistance to challenge (29-37% reductions) were conferred by living, freeze-killed, or sonicated organisms administered with Freunds' Complete Adjuvant (FCA). Antigens extracted in saline, or with the detergent sodium deoxycholate, were also protective. Adjuvants enhanced the protective effect, particularly FCA (78% reduction), Freunds' Incomplete Adjuvant (74% reduction), aluminum hydroxide (70% reduction) and Bordetella pertussis (70% reduction). Detergent extracts prepared from intact embryos with n-octyl glucoside also stimulated significant levels of protection against microfilarial challenge when given with FCA (37-45% reductions). Levels of resistance induced by immunizations with intact organisms were greatest following subcutaneous (s.c.) injection over the neck or by intramuscular inoculation. Soluble extracts were also particially effective given by s.c. inguinal or intraperitoneal injection. A time-interval of greater than 3 weeks between the completion of immunization and challenge was required for the expression of immunity. Cross-protection against challenge with O. lienalis microfilariae was also afforded to mice by immunization with intact embryos or detergent extracts of Onchocerca gutturosa (45 and 34% reductions), Onchocerca gibsoni (66 and 47% reductions) or Onchocerca volvulus (58 and 41% reductions). It is concluded that the embryonic stages of both human and animal parasites provide a source of cross-protective antigens of value in studies on resistance to Onchocerca microfilariae in experimental hosts.

Animals

Onchocerca armillata Railliet and Henry, 1909 and Onchocerca gutturosa (Neumann, 1910) in camels (Camelus dromedarius L.) in the Sudan.

A study undertaken to determine the Onchocerca species infecting camels that live in the same localities as cattle in the Dinder Region, Blue Nile Province, Sudan revealed concurrent infections with the bovine parasites Onchocerca armillata in the thoracic aorta, brachiocephalic trunks and brachial arteries and Onchocerca gutturosa in the ventral side of the lamellar parts of the ligamentum nuchae. The microfilariae of both species had the same predilection sites in the skin of the ears, head and neck regions. Those of O. gutturosa outnumber those of O. armillata but both are smaller than the respective uterine microfilariae. Males and microfilariae of both species are smaller than those of cattle origin.

Animals

Distribution of microfilariae of Onchocerca lienalis and Onchocerca gutturosa in the skin of cattle in Germany and their development in Simulium ornatum and Culicoides nubeculosus following artificial infestation.

Onchocerca microfilariae were isolated form the umbilicus and neck of 438 cow hides at the abattoir in Tübingen, F.R.G. The overall Onchocerca infection rate was 40.4%. The presence of Onchocerca lienalis and O. gutturosa microfilariae, which are difficult to distinguish by morphological criteria, was retrospectively demonstrated after artifically infesting Simulium ornatum and Culicoides nubeculosus and identifying the infective larvae recovered. Nine of 16 samples of umbilical microfilariae fed to C. nubeculosus through a latex membrane developed to O. gutturosa third stage larvae (L3). Six of seven umbilical samples injected into the thorax of S. ornatum yielded O. lienalis L3. In six infestation trials in which microfilariae were introduced both into S. ornatum and C. nubeculosus, O. lienalis L3 were recovered exclusively from simuliids, while O. gutturosa L3 developed only in midges. Of six umbilical skins tested by cross-infestation, one contained exclusively O. gutturosa microfilariae, four only O. lienalis microfilariae and one was infected with both species. Developmental success of O. lienalis microfilariae to L3 in S. ornatum following intrathoracic injection was 22% of the mean inoculum. O. gutturosa microfilariae, ingested by C. nubeculosus through a latex membrane, developed to L3 at a rate of 2.3% of the mean microfilarial uptake.

Animals

An evaluation of implanted male Onchocerca gibsoni in mice as a screen for macrofilaricides against Onchocerca volvulus.

An in vivo drug screen for identifying new compounds with activity against Onchocerca macrofilariae was developed using male Onchocerca gibsoni implanted subcutaneously in outbred mice. There were several similarities (Mel W, CGP 20376, CGP 6140, levamisole) and two differences (suramin, furapyrimidone) between levels of drug efficacy in this model and activity against natural infections of O. gibsoni and O. volvulus. There was considerable variation in the mouse reaction. This mouse model is a potentially useful primary screen for macrofilaricidal drugs against Onchocerca.

Animals

Chitin in egg shells of Onchocerca gibsoni and Onchocerca volvulus.

Chemical analysis of adult females of Onchocerca gibsoni gave estimated chitin contents of 200-500 micrograms (g dry weight)-1. Egg shells from both O. gibsoni and Onchocerca volvulus stained with Calcofluor white and with fluorescent wheat germ agglutinin as shown by fluorescent light microscopy, and bound gold-labelled wheat germ agglutinin as shown by electron microscopy, under conditions specific for chitin. The egg shells appeared as single electron dense layers from 50 to 85 nm in thickness. Purified chitinase digested these egg shells, leaving coiled microfilariae unattacked. We conclude that chitin is a major component of the egg shells.

Animals

Tissue pathology and comparative microanatomy of Onchocerca from a resident of Ontario and other enzootic Onchocerca species from Canada and the U.S.A.

The microanatomy of one male and one female specimen of a zoonotic Onchocera sp in a fibrous nodule excised from the wrist of an Ontario resident, two bovine Onchocerca from Saskatchewan and Manitoba, Canada, and O. cervicalis from the ligamentum nuchae of a horse in Georgia, U.S.A., were compared. In dimensions and cuticular morphology, the three Canadian specimens of Onchocera shared certain features and closely resembled both O. cervicalis and O. gutturosa. The number of cuticular striae in some species of Onchocerca, particularly in male worms, is briefly discussed and its taxonomic value questioned. The Canadian patient visited Radlett, Hertfordshire, England, and then her summer cottage in the Muskoka region, Ontario, prior to the appearance of her symptoms, so that it is uncertain where she acquired the infection. Zoonotic onchocerciasis has yet to be described from Ontario. All nine cases of bovine onchocerciasis (the two described here and seven others) have occurred in Manitoba and Saskatchewan.

Adult

Characterization of an Onchocerca-specific DNA clone from Onchocerca volvulus.

A genomic library of a savanna isolate of Onchocerca volvulus was screened to detect recombinant plasmids containing highly repeated DNA sequences of this parasite. Four recombinant plasmids were identified which hybridized specifically to Onchocerca DNA, but not to DNA from humans, black flies, Brugia malayi, B. pahangi, or Wuchereria bancrofti. The recombinant plasmids had a low level of homology to Dirofilaria immitis. All recombinant plasmids contain related DNA sequences based on Southern hybridization analysis. Sequences related to these recombinant plasmids are present in different geographic isolates of O. volvulus and O. ochengi, an animal parasite. Two of the recombinant plasmids contain sequences also found in O. lienalis. One recombinant plasmid, puOvs3, has been characterized in detail, including DNA sequence determination. Radiolabeled puOvs3 is able to detect 100 pg of genomic DNA isolated from O. volvulus worms from both savanna and forest regions. It can differentiate O. volvulus from O. ochengi by Southern blot analysis.

Animals

Onchocerca gutturosa and Onchocerca lienalis in cattle: effect of age, sex, and origin on prevalence of onchocerciasis in subtropical and temperate regions of Florida and Georgia.

Evaluation of 845 cattle from Florida and Georgia for onchocerciasis indicated a significantly higher prevalence of infection with Onchocerca gutturosa and a significantly lower prevalence of infection with Onchocerca lienalis in the southern portion of Florida (subtropical region) than in the northern portion of Florida or in Georgia (temperature region). In all regions, prevalence of infection with O gutturosa was significantly higher than infection with O lienalis. In the temperate region, bulls were infected more often with O lienalis than were cows. Other sex-related differences in infection with either filariid was not found. Prevalence of infection with O lienalis was similar between age groups in all regions. Rates of infection with O gutturosa differed between age groups within and between subtropical and temperate regions. Examination of cervical and umbilical skin specimens for microfilariae detected an additional 6.6% of infected animals that had not been found by examination for adult worms. However, microfilariae were not found in 21.4% of animals that were positive for adult worms.

Age Factors

Studies on Onchocerca cervicalis Railliet and Henry 1910: V. The development of Onchocerca cervicalis larvae in the vectors.

Colonies of Culicoides nubeculosus and C; variipennis were established at Winches Farm Field Station, St. Abans. Both species of midge were found to be susceptible to infection with Onchocerca cervicalis microfilariae. Infection was achieved either by engorging through a chick skin membrane on a suspension of microfilariae in blood, or by feeding upon a horse naturally infected with O. cervicalis microfilariae. A large proportion of the microfilariae ingested by the midges penetrated into the haemocoel and migrated first to the thoracic flight muscles and then to the head. Larval development occurred during migration and 3rd stage (infective) larvae of O. cervicalis reached the head of infected midges (kept at 23 degrees C.) 14-15 days after engorgement.

Animals

Onchocerca gutturosa and Onchocerca lienalis in cattle: variation in length of microfilariae by site of recovery.

Dermal microfilariae recovered from specimens obtained from umbilical and cervical sites of cattle infected with adult Onchocerca gutturosa alone or with adults of O gutturosa and O lienalis were measured and compared with uterine microfilariae obtained directly from gravid female worms of each species. Uterine microfilariae of O gutturosa were longer than dermal microfilariae obtained from cattle harboring only adults of O gutturosa. Dermal microfilariae were recovered from umbilical and cervical sites in these cattle. Those found at the cervical site had lengths equal to or greater than lengths of microfilariae recovered from the umbilical site. There was a significant (P less than 0.0001) shift in length across populations of microfilariae of O gutturosa from various sites in its bovine host, with a progressive decrease in length between microfilariae recovered from the worm's uterus, microfilariae from the cervical dermis, and microfilariae from the umbilical dermis, respectively. A similar direct comparison was not possible for microfilariae of O lienalis, because none of the cattle was infected with only adult worms of this species. In an indirect comparison, microfilariae of O lienalis were identified at the umbilicus, but their presence in the cervical region could not be determined unequivocally because of confounding of microfilariae length by concurrent infection with O gutturosa. Uterine microfilariae from O lienalis were longer than uterine microfilariae of O gutturosa, although a degree of overlap in the range of measurements existed between species.

Algorithms

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