An in vitro system for chemotherapeutic research using adult male Onchocerca gutturosa has been developed as a model for O. volvulus. Using a culture system consisting of medium MEM+10% heat inactivated foetal calf serum (IFCS)+LLCMK2 (monkey kidney) feeder cells in an atmosphere of 5% CO2 in air, we examined the effects of a range of antiparasitic drugs on worm motility. Ivermectin, levamisole, furapyrimidone, Mel W, chloroquine, metrifonate, flubendazole, amoscanate and the Ciba-Geigy compounds CGP 6140, CGP 20′376 and CGI 17658 either immobilized or significantly reduced motility levels at a concentration of 5x10−5M or less within a 7-day period. Worms were affected at very low concentrations by ivermectin (effective conc. to reduce motility levels to 50% of controls, 3.14x10−8M), levamisole (7.95x10−8M), CGP 6140 (8.87x10−9M) and CGP 20′376 (2.78x10−8M). Difficulties were experienced in accurately repeating the immotile endpoint for levamisole due to an inconsistent partial recovery of motility. Over a 7-day period diethylcarbamazine had little effect on motility levels, while suramin caused a slight increase in activity compared to controls at some timepoints. Subsequent experiments demonstrated some differences in drug efficacy depending on the presence or absence of serum and feeder cells in the culture system probably because of drug avidly binding to serum proteins. However, serum and cells were found to be essential ingredients of the culture system to maintain worms in good condition, indicating that new drugs should be evaluated both in the presence and absence of serum and cells. Comparisons were made between the responses of O. gutturosa and Brugia pahangi to certain drugs and these species were found to significantly differ in their sensitivities to ivermectin and a novel compound (Wellcome), indicating that Onchocerca parasites should be used wherever possible for compound identification and development intended for the treatment of onchocerciasis. The in vitro system described here, using male O. gutturosa, provides a basis for further research and a practical alternative to O. volvulus.

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5·7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into trichloracetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100 000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48 000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG anitbodies in positive serum. Especially the polypeptide with a molecular weight of 48 000 in ES products strongly reacted with IgG antibody in positive serum.

Antibodies directed against diagnostic Mr 31 000 polypeptide(s) of adult Schistosoma mansoni were already formed in mice during prepatency. In contrast, repeated immunization of mice with homogenates of adult schistosomes failed to elicit antibodies detectable in immunoblots in the Mr 31 000 region. Therefore, spleen cells of infected mice were used to produce hybridoma lines. The “miniblot technique” was developed in order to detect in hybridoma supernatants antibodies against schistosome Mr 31 000 components. Electrophoretically separated total S. mansoni proteins were transferred onto nitrocellulose, and the position of the Mr 31 000 components was determined with polyclonal antisera and immunoblotting. Pieces of about 3 square mm of nitrocellulose bearing the diagnostic proteins were incubated with about 100 μ1 of hybridoma supernatant in microtitre plates and subsequently probed with peroxidase-conjugated antibody to mouse IgG. This screening technique identified hybridomas secreting antibody to the relevant S. mansoni antigens. It is applicable to other defined parasit antigens, which are, however, not available in biochemically purified form. The monoclonal antibodies produced against the proteins with diagnostic potential reacted with antigens localized in the schistosome gut.

We have investigated the capacity of irradiated larvae of each of six different isolates of Schistosoma mansoni to protect mice against challenge infections from the homologous or a different isolate. The isolates differed in respect of their geographic origins, and in the lengths of time they had been maintained in the laboratory. All isolates induced significant protection against unattenuated challenge infections, and neither the degree of geographic disimilarity between vaccinating and challenge parasites, nor the numbers of laboratory cycles, appeared to influence the degree of protection induced by irradiation-attenuated larvae.

Mice presensitized with SEA and subsequently injected with S. japonicum eggs into their lungs or liver developed pathology similar to that found in infected mice and were consequently resistant to challenge (average 33% and 53%, respectively). However, soluble egg antigens were also capable of inducing low levels of resistance in mice (average 22·5%), but intact eggs alone injected into the lungs, liver or subcutance tissues were not. Thus, prior sensitization to ‘marginally’ protective soluble egg antigens is necessary for egg induced resistance.

Litomosoides carinii-infected Mastomys natalensis were treated 85 days post infection with cyclosporin A (CyA) or 8 derivatives with different immunosuppressive capacities. CyA (oral doses of 5x25 mg/kg. 5x50 mg/kg, 5x80 mg/kg on consecutive days) reduced parasitaemia levels in a dose dependent way, beginning 3 weeks after first drug administration. Using 5x50 and 5x80 mg/kg animals were free from circulating microfilariae on the day of necropsy (day 56). Derivatives were administered in 5 daily oral doses of 50 mg/kg. Compounds B–5–49 and G–7–53 had similar effects as CyA. Compounds A–4–16 and E–6–44 caused mean microfilaraemia reductions of about 80% until day 56. Compounds C–5–34, D–6–45, F–7–62 and H–7–94 were only marginally effective (10–40%). None of the drugs affected the number or the motility of adult worms. However, in the case of efficacious compounds the number of intrauterine microfilariae was considerably reduced and most of the intrauterine stages were pathologically altered. The efficacy of the various derivatives was independent of their immunosuppressive activity in vivo and in vitro, their anti-inflammatory activity and their activity against Plasmodium berghei. Effects on intrauterine stages were first detectable 7 days after treatment with 5x80 mg CyA/kg when the number of intrauterine microfilariae had decreased and the proportion of pathologically altered stages had increased. Alterations increased with time after treatment. Additionally, the uteri contained relatively large amounts of highly active microfilariae which were still included in an ovoid sheath. These stages seemed to try unsuccessfully to stretch their stiff sheath, and also degenerated by 35 days.

A sandwich ELISA method using previously described E/S antigen-specific monoclonal antibodies has been developed to detect circulating immune complexes in patients infected with Toxocara canis. This technique could be used for the study of the dynamics of the parasite-host relationship, as we believe the detection of immune complexes and/or soluble antigen to be an improvement over detection of antibodies only. In this parasitosis, antibodies may be present in residual levels for prolonged periods after active infection.

The kinetics of Breinlia booliati infection in 3 inbred rat strains (Lewis, Wistar and Sprague Dawley) were investigated. One group of rats was infected as neonates (less than 24 hours of age) with third-stage larvae of B. booliati and the other group was infected as juveniles (4 weeks of age). The results showed that infection in the neonates were significantly different from the infection in the juveniles. The 60 rats infected as neonates, when necropsied between 8 to 10 months postinfection, yielded adult worms. The 2 neonatal infection groups of Lewis and Wistar strains showed highest susceptibility to the infections. The mean prepatent period was 85 days. Ninety to 95% of the infected rats were patent with microfilaraemia and a large percentage (33 to 47%) of them had high microfilaraemia counts exceeding 3000 mff/20 mm3 of blood and larger sizes (mean 157·11 mm for female adult worms and 61·88 mm for male adult worms. The adult worms were distributed equally in both the pleural (57%) and peritoneal cavity (43%). In most aspects, the neonatal infection group of the Sprague-Dawley strain was intermediate in susceptibility between the 2 neonatal infection groups of the Lewis and Wistar strains and the 3 juvenile infection groups. In contrast to neonatal infection groups, the 3 juvenile infection groups exhibited low infection rates (37%, 58% and 47% for the Lewis, Wistar and Sprague Dawley strains respectively), longer prepatent periods (mean 101 days), lower recovery rates (2 to 4%), lower adult worm loads (mean 0·4 to 0·8 female worms, and 0·2 to 0·8 male worms per rat), and smaller sizes (mean 141·24 mm for female adult worms and 53·75 mm for male adult worms). Forty-four to 57% of these infected rats harboured either single male or single female adult worms in the body cavity. Most (92%) of the adult worms recovered from the juvenile infection groups resided in the pleural cavity and the remaining 8% were recovered from the peritoneal cavity. Microfilaraemia could be detected in only 3/20 Lewis rats, 5/20 Wistar rats and 5/20 Sprague Dawley rats. The mean peak microfilaraemia of the 3 pooled juvenile infection groups was 632 mff/20 mm3 of blood, ranging from 7 mff/20 mm3 to 1856 mmf/20 mm3. Our results indicate that the susceptibility to B. booliati infection in white rats is both genetic and age-associated. The responses of the 2 distinct infection groups to B. booliati infections are discussed.

Onchocerca gutturosa and O. lienalis inflections in British cattle were studied by examination of cattle post-mortem originating from North Wales and Cheshire (north west England). In 463 adult animals, the microfilarial (mf) prevalence was 28·5%. In 95·3% of the inflected animals, gravid worms could not be found at either the ligamentum nuchae or the gastro-splenic omentum. Dermal mf at the head were identified as O. gutturosa on the basis of their highly significant assocation with the presence of gravid O. gutturosa at the ligamentum nuchae, which were found in only 3·2% of cattle. Mfs were isolated from different skin sites and from adult worms and a minimum of 10 mfs from each isolate were examined for width and acid phosphatase (AP) staining pattern. The width of O. gutturosa dermal mf was <4 μm (4 isolations), narrower than of putative O. lienalis mf isolated from umbilical skin of cattle without evidence of O. gutturosa, which were in 20/22 isolations >4 μm wide. The dermal mf were also distinguished on the basis of different AP staining patterns which, for each species, correlated closely with that of hatched intrauterine mf from their respective adult female worms. Based on the criteria of morphology and AP staining patterns the mf species prevalences in the survey population were estimated as O. lienalis 24·1% and O. gutturosa 2·2%, with a further 2·2% of cattle inflected with both species. The results indicate that the predilection site of adult O. lienalis is not the gastro-splenic omentum. In North Wales, the distribution of the two species was different; O. lienalis was widely distributed in all cattle rearing areas both lowland and upland, whereas O. gutturosa was largely restricted to valleys close to major rivers.

The cercariae of Schistosoma haematobium showed a diurnal periodicity of emergence from Bulinus globosus in a twelve hour light/dark cycle. Peak emission occurred at 11.00 hrs with a smaller peak at 20.00 hrs, following the start of the period of darkness. In continuous illumination this second peak was not seen, indicating that only the morning peak is circadian in origin. The evening peak occurs in response to dark treatment and can be produced by periods of darkness ranging from eight seconds to one hour. The longer the period of dark treatment the longer the rise in output is maintained on return to light conditions. Subjection of snails to periods of dark treatment during the normal light period caused a reduction in the evening peak with the largest effect seen following the longest period of darkness. An increased output of cercariae was seen following fifteen minutes exposure to a range of light intensities, the largest increase occurring at 10 000 and 7000 lux and complete darkness. The rapidity of this reaction to variations in light intensity suggests that the cercariae of S. haematobium are showing emergence in response to shadows.

The implications of the confused taxonomy of the genus Mesocestoides and the misuse of the name M. lineatus are described. In Southwest Germany rodents are intermediate hosts and red foxes are definitive hosts of M. leptothylacus. The shedding patterns of experimentally infected foxes showed that destrobilation occurs frequently and that there are long periods during which no gravid proglottids are shed at all. Lengths of worms can be taken as a measure of a possible crowding effect only when worms with gravid segments are present, i.e. at the end of the prepatent period (11 to 13 days) or at the beginning of a shedding period.

Mechanisms of protective immunity to larvae of Brugia pahangi were studied in congenitally athymic nude C3H/HeN mice and their syngeneic heterozygous littermates. An average 11% if subcutaneous larval inocula was recovered from control nudes 28 days after inoculation. No worms were recovered from nude recipients of viable splenic Thy 1·2+ T lymphocytes from heterozygotes which had killed a priming does of B. pahangi larvae. Primed T lymphocytes, depleted of either Lyt 1·1+of Lyt 2·1+cells or incubated with anti-Thy 1.2 monoclonal antibody and complement, failed to protect nude mic against a larval challenge. Nor were primed B lymphocytes depleted by Thy 1·2+T cell contaminants protective. Treatment with cyclosporin A (CsA) did not increase the numbers of worms recovered from heterozygotes nor did CsA treatment of heterozygous cell donor abolish the ability of primed Thy 1·2+T lymphocytes to transfer protection to nude mice. IgG but not IgM antibody titres to B. pahangi antigens were depressed in all CsA-treated mice. CsA treatment of nude mice had no direct effect upon development of B. pahagi larvae. These results show that ptotective immunity to larvae of B. pahangi in mice depends upon small numbers of Thy 1·2+T cells which are CsA-resistant.

In 6 week-old Paramphistomum cervi, vitelline cells appear in the form of a few scattered cells, present below the body wall which divide mitotically to form vitelline follicles in 8 week-old worms. Small shell globules appear first in 10 week-old worms, the number and size of which continue to increase till the worm becomes adult. In the latter the vitelline follicles contain stem cells, immature, maturing and mature vitelline cells, which differ due to the distribution of shell globules as well as their histochemical nature. The shell globules are keratin in nature. The vitelline cells showed activities of various phosphatases but did not show the presence of any of the dehydrogenases studied.

Shelled acanthors (=eggs) of two isolates of Moniliformis moniliformis (Acanthocephala) were tested for their capacity to respond to a hatching stimulus in vitro and for their retention of infectivity to a natural intermediate host (Periplaneta americana). The shelled acanthors were stored for more than 120 weeks (Australian isolate) and for 104 weeks (Texan isolate) together with rat faeces in an incubator maintained at 22·2±0·1°C. In both cases, infectivity to P. americana was shown to have been retained. In vitro tests of the hatching response were carried out on many occasions during this period of faecal storage. Shelled acanthors continued to respond and no differences were detected either between isolates or within an isolate through time.

Employing papain as the enzyme and agarose bound Ricinus communis agglutinin as the affinity gel, a glycoprotein has been isolated and purified from the surface of Ascaridia galli. The glycoprotein shows an apparent molecular weight of 68 kilo daltons and contains fucose, galactose, rhamnose and glucosamine as sugar moieties. Only 2% of its entire molecule has been found to possess α-helical configuration.

A series of experiments was carried out using adult outbred Polish race rabbits of both sexes infected, during spring or autumn. with 10 000 larvae of Obeliscoides cuniculi, either fresh or stored at 4°C. Extracts of mucosal proteins and bile were collected at postmortem 6 or 12 weeks after infection. Antibody levels were determined in antisera. bile and stomach mucosa by haemagglutination and precipitation tests. Local antibody respones were demonstrated in the stomach and bile, and reactions were obtained with the tissue fluids by haemagglutination and precipitation tests with worm antigens and ES products. Additionally, some specific immunological response was observed in the circulation during the primary infection. These results suggest a clear-cut relationship between increased levels of these antibodies and either larval inhibition or worm expulsion during O. cuniculi infections.

Heterophyes heterophyes is reported for the first time from the snail host Pirenella conica in Greece. H. aequalis and H. dispar are reported for the first time from Greece and from Europe. The results presented, together with the available data on necropsies of dogs and cats, human infections and the biotope preference of the snail, suggest that Greece is an important focus of infection in Europe.