COST Action 819: Entomopathogenic nematodes, supercedes Action 812: Cold active lines of insect parasitic nematodes in Agriculture and Biotechnology. It functions in the field of Agriculture and Biotechnology and began in July 1994 and will end in May, 1999. The main objective is to combine interrelated European expertise to increase the use of entomopathogenic nematodes (EPNs) in integrated pest management and to reduce the need for chemical control. Coordination of the Action is the responsibility of a management committee in accordance with a Memorandum of Understanding, which has been signed by representatives of 17 countries: Austria, Belgium, Czech Republic, Finland, France, Germany, Hungary, Ireland, Italy, The Netherlands, Norway, Poland, Portugal, Spain, Sweden, Switzerland and the UK. A research institute in Israel is also participating. Over 40 research institutions and 10 commercial companies participate in Action 819. The EU funding meets the costs related to coordination including the scientific secretariat, workshops, management committee and working group meetings, publications, short term scientific missions and evaluations.

A comparative study was undertaken between an enzymatic immunohistochemical technique (EIT) developed for the diagnosis of human trichinellosis and the indirect immunofluorescence test (IIF), analysing sera from outbreaks of human trichinellosis in Argentina. The HIT was developed using a biotinylated anti-human gammaglobulin and a preformed macromolecular complex of avidin and biotinylated peroxidase. In both tests, the antigen used consisted of infected rat tissue sections containing muscle larval cysts of Trichinella spiralis. Results showed that the HIT closely correlated with IIP and also allowed diagnosis at an early stage of infection, thus helping to provide effective treatment for the disease. When the test was performed on sera from healthy individuals and those with other parasitic infections, cross-reactions were observed only with sera from patients with toxocariasis (1/8), Chagas' disease (3/17) and four out of 100 healthy individuals. No cross-reactions were observed with sera from patients with toxoplasmosis (0/7) or hydatidosis (0/8). Assay sensitivity was 100% and its specificity 93%. Since it renders no false negative results, BIT is an effective screening tool for detecting infection and should prove to be an important diagnostic technique for trichinellosis in rural areas and for epidemiological surveys.

The Paragonimus ohirai group, named after P. ohirai Miyazaki, 1939, consists of three nominal species. Paragonimus iloktsuenensis Chen, 1940 and P. sadoensis Miyazaki et al., 1968, the other members of the group, were proposed primarily because of perceived differences in metacercarial morphology and/or host preferences. It has long been recognized that adults of the three were virtually indistinguishable. With the application of genetic techniques, it has become clear that the three forms can exchange genes freely, and that differences in metacercarial morphology constitute a polymorphism probably due to a single gene inherited in Mendelian fashion. Here, additional genetic data (DNA sequences from the second internal transcribed spacer of the nuclear ribosomal gene cluster and from the mitochondrial cytochrome c oxidase subunit I gene) are presented in support of the synonymy.

The larval development of Dicrocoelium dendriticum (Digenea: Dicrocoeliidae) in experimentally infected Cernuella (Xeromagna) cespitum arigonis (Schmidt, 1853), a species of mollusc important in the epidemiology of dicrocoeliosis in Spain, has been studied. A total of 948 specimens of this mollusc, distributed in five batches, were tested with individual doses of 50 to 150 parasite eggs, obtained from sheep, after 4 days without food. After infection these molluscs and control specimens were kept in an enviromental simulation chamber at 20°C, 50% relative humidity and 7h of light per day. To detect the parasite, a minimum of six molluscs were examined every 20 days from day 1 post-infection (p.i.). The eggs of D. dendriticum were eliminated in the molluscan faeces 48 h post infection. The percentages of molluscs harbouring the parasite ranged between 17.53% and 75%. Daughter sporocysts with undifferentiated germinal masses and occupying very reduced areas of the hepatopancreas were observed 50 days p.i. and in the period immediately following. After 110 days p.i. sporocysts with cercariae at different stages of development were found although slimeball emission was never observed.

Different organs and secretions/excretions of the swimbladder parasite, Anguillicola crassus (Nematoda), were tested for the presence of antigens to the humoral immune response previously detected in the European eel, Anguilla anguilla. Proteins from different fractions of Anguillicola crassus were separated using SDS–PAGE (sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis) under reducing conditions and electroblotted onto nitrocellulose membranes. Infected eels showed a specific antibody response to a 43 kDa antigen in the cuticle and towards two gonad antigens around 34 and 43 kDa. In protein released from the worms, two secretory/excretory antigens of approximately 28 kDa were found. The secretion/excretion rate of protein from the parasite to the surroundings was determined. Subsequently, an ELISA system was established applying these antigens as the first layer of coating. Furthermore, antigens from Anguillicola crassus were examined for the presence of glutathione-s-transferase (GST) using a specific antibody against GST. The antigens were found to be subunits of GST.

The interaction of DNA from filarial parasite Setaria cervi with polyamines was monitored by melting temperature (Tm) profile, condensation and B to Z DNA transition and compared with DNA of Escherichia coli, Micrococcus luteus and calf thymus having different GC content. Polyamines, viz. spermine and spermidine, stabilized the secondary structure of all DNAs as indicated by increase in Tm value. UV absorption studies indicated B to Z DNA transition in the presence of polyamines. The amount of polyamines required for B to Z transition was dependent upon base composition of DNA and charge of the polyamine. Filarial DNA (AT rich) required six times higher concentration of spermine as compared to GC rich DNA for B to Z DNA transition. Spermidine was not effective in causing transition of S. cervi DNA even at Spd:DNA-P ratio of 20. The antifilarial compound suramin significantly decreased melting temperature of filarial DNA as compared to GC rich DNAs of other parasites. Suramin adversely affected condensation and B to Z DNA transition of various DNAs but prior addition of polyamines protected the DNAs from the destabilizing effect of suramin.

Fasciola hepatica secretes proteolytic enzymes to aid it to penetrate and migrate through the host tissues. Two of these proteinases have been purified and shown to be cathepsin L-like, and are termed, CL1 (27.5 kD) and CL2 (29 kD). The immunogenicity of these proteinases was investigated over the course of an experimental infection and following drug treatment. Four groups of rabbits were studied: group 1: orally infected with 50 metacercariae; group 2: infected and treated 8 weeks after infection; group 3: infected, treated at week 8 and reinfected at week 13 and group 4: non-infected control group. Sera were collected weekly from each group until week 20 postinfection. CL1 and CL2 were incubated with the different sera and then analysed by gelatin substrate polyacrylamide gel electrophoresis (GS-PAGE). Analysis of groups 1, 2 and 3 showed that CL1 and CL2 neutralizing antibodies appear at week 5 post-infection. In group 1, these remained throughout the 20 weeks of infection. In group 2, neutralizing antibodies disappeared at week 13, that is, 5 weeks after anti-Fasciola treatment. In group 3, CL1 and CL2 neutralizing antibodies disappeared at week 13 but reappeared by week 15, that is 2 weeks after reinfection. Pooled sera from group 4, showed no inhibitory capacity. ELISA results using CL1 and CL2 as antigen, correlate very well with the inhibitiory time course observed by GS-PAGE. These results suggest that purified cathepsin Ls are antigenic molecules recognized early in the infective process and capable of inducing a specific humoral response, strong enough to neutralize, at least partially, their enzymatic activity.

The subchronic effect of copper and mercury on selected immunological parameters in guinea pigs with experimental ascariasis was studied. Cupric sulphate and mercuric chloride administered for 28 days did not suppress the levels of specific circulating antibody in a subsequent Ascaris suum infection. Intoxication of animals significantly inhibited the phagocytic activity of peritoneal macrophages throughout the experiment. Ascaris suum infection elevated macrophage phagocytic activity but values remained significantly lower in comparison with infected non-intoxicated animals. Both the concurrently administered metal compounds changed the phagocytic ability of polymorphonuclear leucocytes in the blood.

The intensity of infection assessed by the number of Ascaris larvae migrating in the lungs of animals currently intoxicated with both the metal compounds increased by 28.6%, compared with controls.

The surface morphology of metacercariae isolated from Puntius spp., adult worms from infected hamsters and eggs of Centrocestus formosanus (Digenea: Heterophyidae) were studied using scanning electron microscopy. It was found that the surfaces of the metacercariae and adult worms were closely similar in appearance. The oral sucker was surrounded by a circumoral expansion with two rows of 32 spines (16 spines each). The ventral sucker, with six large nonciliated papillae on the lip, and the genital opening were located midventrally. The excretory pore was terminal. The body surface was covered with pectinate scale-like spines of varying sizes. The scales in the middle area were larger than those in the anterior and posterior parts of the body. Sensory papillae, mostly uniciliated, were present in greater abundance at the anterior region of both stages. However, morphological variations were observed among the adults depending on the age of the worms. At 4 weeks, the oral sucker and circumoral expansion were cobblestone-like in structure and the grooves of spines split compared with their smooth surfaces at 10 days. In addition, the ventral sucker became depressed and its large papillae on the lip disappeared at 10 weeks post infection. Multiciliated papillae were also present in 10-week old worms. Each egg of this parasite possessed a prominent operculum and a latticed design on the egg shell.

The effects of a high protein diet on the host-parasite relationship of Echinostoma caproni in ICR mice were studied. The customized high protein diet (CHPD) contained 64% casein as a source of protein. The control diet consisted of a standard laboratory diet containing 23% casein as a source of protein. Mice were each fed 25 cysts of E. caproni by stomach tube and necropsied 2, 3, 4 and 5 weeks postinfection. The weight of mice on the CHPD did not differ significantly from mice on the control diet. Worm recoveries were also unaffected by the high protein diet. There was a significant decline in worm dry weight, body area and uterine egg counts in worms from mice on the CHPD compared with those on the control diet. Worms from hosts on the CHPD were located more posteriad in the gut than those recovered from mice on the control diet. Changes in the mouse diet adversely affected E. caproni maturation and growth, possibly by altering the immediate host mucosal environment and making it less conducive to worm development.

In order to characterize GATA transcription factors in Echinococcus granulosus, a PCR-based cloning strategy was developed. Degenerate oligonucleotides were designed for the most conserved sequence in GATA proteins that include 20 amino acids of the zinc domain. A 60 bp fragment was isolated that had high homology among this sequence and those reported in other species. An analogous sequence was obtained by performing the same procedure with DNA from the free living platyhelminth Dugesia tigrina. High stringency Southern blotting experiments confirmed the presence of this sequence in the parasite genome.

The occurrence of specimens in a laboratory strain of Trichostrongylus vitrinus exhibiting unusual spicules resembling those of T. minor and T. angistris is reported and the abnormal spicules described. The specimens include individuals with one spicule typical of T. vitrinus and the other spicule of the abnormal type. Possible reasons for the occurrence of such abnormalities are discussed.