A 30-h in vitro susceptibility test of Plasmodium falciparum wild isolates to artemisinin, chloroquine, sulfadoxine/pyrimethamine and mefloquine was performed in Kibaha, Tanzania. A sigmoid Emax model was fitted to all data for each isolate and drug combination. Artemisinin and mefloquine exhibited 100% growth inhibition against all isolates tested (n=69–74). The EC50 values for artemisinin and mefloquine were 44 and 146 nM respectively. Chloroquine and sulfadoxine/pyrimethamine resistance was 30% and 13% respectively. Susceptibility parameters (EC50, 90, 95 and 99 values and s) varied between compounds and isolates indicating the different sensitivity of P. falciparum isolates. No correlation between susceptibility parameters of artemisinin and the other compounds was found. The high in vitro activities of artemisinin and mefloquine indicate their potential role for the treatment of multidrug-resistant malaria in Africa.

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Strains of Entamoeba histolytica isolated in Brazil were characterized using the Low-Stringency Single Specific Primer PCR (LSSP–PCR), that detects single or multiple mutations in gene size DNA fragments. Using this technique, a 482-bp genomic DNA fragment from a structural gene in 8 strains and 2 clones of E. histolytica, isolated from symptomatic and asymptomatic patients in Brazil, including pathogenic and non-pathogenic zymodemes were studied. The results obtained indicate that LSSP–PCR is a valuable method for differentiating strains of E. histolytica. Moreover, the results are consistent with the concept that pathogenic and non-pathogenic strains of E. histolytica may represent distinct species or subspecies and are in accord with phenotypically characteristic isoenzyme patterns.

We have used a chromosome-specific approach to generate a 300 kb long ‘contig’ across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The ‘contig’ generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb ‘contig’ rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.

The physiological effects of synthetic replicates of the nematode FaRPs, AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), PF1 (SDPNFLRFamide), PF2 (SADPNFLRFamide), AF8/PF3 (KSAYMRFamide) and PF4 (KPNFIRFamide) were examined on muscle preparations of the liver fluke, Fasciola hepatica. Changes in contractility following the addition of the test compound were recorded using a photo-optic transducer system. Unlike the varied effects these peptides have on nematode somatic musculature, all were found to induce excitatory responses in the muscle activity of F. hepatica. While qualitative effects of the nematode peptides were similar in that they induced increases in both the amplitude and frequency of F. hepatica muscle contractions, they varied considerably in the potency of their excitatory effects. The threshold activity for each peptide was as follows: 10 μM, PF1 and PF2; 3 μM, AF1 and PF3; 1 μM, AF2; and 30 nM, PF4. The results demonstrate, for the first time, the cross-phyla activity of nematode neuropeptides on the neuromuscular activity of a trematode.

When Small-Ear-Miniature pigs subcutaneously injected once with frozen oncospheres of Taiwan Taenia, Korea Taenia, T. saginata or T. solium emulsified with Freund's complete adjuvant (1·6 × 104/0·4 ml) were challenged orally with 1·6 × 104 viable eggs of Taiwan Taenia 41 days later, they all showed strong resistance compared with pigs vaccinated with T. solium. Most pigs (5/8) of the former 3 groups harboured no cysticerci. The number of cysticerci was 5·5±9·1 (mean±S.D.), whereas pigs of the T. solium group and control group harboured 160±30·1 and 661±637·2 cysticerci, respectively. All cysticerci recovered from vaccinated pigs and most cysticerci in control pigs were degenerated or calcified at 36–55 days after oral egg challenge. These results strongly suggest that oncospheres of Taiwan Taenia and Korea Taenia are very similar to T. saginata in their immunogenicity in pigs.

The host–parasite relationship in taeniosis due to Taenia solium is practically unknown. Monoclonal antibodies were prepared against whole extracts of adult T. solium parasites and evaluated with tapeworms recovered from experimentally infected hamsters and with cysticerci from naturally infected pigs. With one antibody, mAb 4B3, it was possible to identify, purify and partially characterize a T. solium myosin. Some findings indicate that it corresponds to conventional myosin or myosin type II such as: purification with KCl, high molecular weight, size, structure (dimeric protein with globular and long tail portions), reaction with commercial anti-myosin antibodies, distribution in muscle fibres of parasites and cross-reactivity with antibodies against paramyosin from T. solium cysticerci. The reaction of the mAb was only with taeniids and not with other parasites. Also myosin was detected in faeces of infected animals and in supernatants of parasite cultures. Its presence in biological fluids may be useful for diagnosis of infected hosts.

The impact of diets varying in type and level of carbohydrates resistant to endogenous enzymes and lignin on the establishment and location of Ascaris suum and Oesophagostomum dentatum was investigated experimentally. Fifty worm-free pigs, from a specific pathogen-free farm were used. The animals were assigned randomly to 5 diets and infected with 600 infective A. suum eggs and 6000 infective larvae of O. dentatum per pig. The diets consisted of a traditional ground barley plus protein feed (diet A), commercial full-constituent pelleted feed (diet B), barley flour plus protein (diet C), barley flour, inulin (Raftiline® ST, ORAFTI, Tienen, Belgium), sugar beet fibre plus protein (diet D), and barley flour, wheat bran, and protein (diet E). The faecal egg excretion was followed and the pigs were slaughtered at 8 weeks p.i. and samples taken from the small and large intestine. Intestinal contents were analysed for worm burdens, worm location and female worm fecundity along with the concentration of insoluble (chromic oxide) and soluble (polyethylene glycol-4000) markers, lignin, non-starch polysaccharides (NSP) and organic acids. In all diet groups A. suum worm burdens were low and comparable, whereas the O. dentatum worm burdens were significantly higher in pigs fed the diets with high levels of NSP and lignin (diets A and E) than in pigs fed diets B, C, and D. The present study suggests that a diet rich in lignin and insoluble NSP's provides favourable conditions for the establishment of O. dentatum in the large intestine of pigs while it is unlikely that the concentration of short-chain fatty acids and pH plays any major role.

Discocotyle sagittata displays a clearly defined nocturnal egg-hatching rhythm. When eggs of D. sagittata were incubated in alternating 12 h periods of light and darkness at 13 °C, the majority of larvae hatched within the first 2 h of darkness. Larvae were rarely recovered after 4 h of darkness, and none hatched in the light. Reversal of the light/dark cycle resulted in immediate reversal of the hatching rhythm, so that larvae emerged at the beginning of the new dark period. This suggests that hatching is a direct response to exogenous cues and not endogenously driven. In continuous darkness hatching was arrhythmic; hatching occurred in continuous light although total numbers of larvae emerging were suppressed. Field experiments confirmed that eggs exposed to the natural day/night cycle throughout development also demonstrated the precise hatching rhythm seen under controlled conditions. Mechanical disturbance played no part as a hatching stimulus. However, larvae hatched in the presence of both host mucus and gill tissue. D. sagittata does not show an egg-laying rhythm. The hatching responses have adaptive advantage related to the host's activity pattern. Trout are generally visual predators, inactive in darkness, providing a stationary target for emerging larvae responding either to darkness or to host hatching factors.

The composition, richness and similarity of helminth communities in a tropical freshwater fish were determined in samples of Cichlasoma urophthalmus collected from 7 localities of broadly similar age and character situated along the northern coast of the Yucatan peninsula, Mexico. The component communities exhibited a unique combination of characteristics for a freshwater fish. They were dominated by digeneans, with all other helminth groups being numerically inferior. A common suite of species could be recognized, but many of its members were generalists and not cichlid specialists. Species richness and number of individuals per host were high, but diversity was low, reflecting high dominance by one species. Intra- and inter-locality similarity levels were low, and local variation high. In respect of species richness and digenean dominance, the communities resembled those in Australian tropical anguillids, but in respect of low diversity, similarity and high dominance they are more similar to helminth communities of northern temperate fish.

Infective juveniles (IJs) of Steinernema carpocapsae (All) are able to remain relatively highly infective even when they have almost exhausted their neutral lipid reserves. This is not seen in other steinernematid species so we proposed that carbohydrate may be important for infectivity in aging IJs of S. carpocapsae. The present study investigated glycogen utilization in IJs of 4 entomopathogenic nematodes, S. carpocapsae, S. riobravis (Biosys 355), S. feltiae (UK76) and S. glaseri (NC), stored in distilled water at 25 °C. The 4 species had appreciable amounts of glycogen; from ca. 8% dry weight in S. riobravis to ca. 18% in S. glaseri. Infective juveniles of S. carpocapsae and S. riobravis survived for 120–135 days and utilized ca. 90% of their glycogen reserve at an almost constant rate during a 112-day storage period. Steinernema feltiae and S. glaseri lived for much longer (>450 days) and during a 250-day storage period their glycogen content decreased by 27 and 40%, respectively. In contrast to the other 3 species, the rate of lipid decline preceded that of glycogen in S. carpocapsae. The rate of glycogen decline in S. carpocapsae IJs incubated with the glycolytic inhibitor, iodoacetamide (10−4 M) was significantly reduced (P<0·05) compared with untreated nematodes, and the infectivity of inhibitor-treated aged (>80 days) IJs was reduced compared with controls. Incubating aged (80-day) IJs of S. carpocapsae (mean neutral lipid content ca. 10% of initial level) with 10−4 M iodoacetamide for 24 h significantly reduced (P<0·05) their infectivity compared with freshly harvested inhibitor-treated IJs and untreated controls. Following an 11-day recovery period, the infectivity of inhibitor-treated aged IJs recovered significantly (P<0·05). The evidence suggests that glycogen is an important source of energy for maintaining infectivity in aged IJs of S. carpocapsae.

Factors affecting ‘invasion efficiency’ of steinernematid entomopathogenic nematodes into hosts were elucidated. The phenomenon that only part (10–40%) of the nematode population invades the target host has been recorded in many studies. It has been mainly ascribed to differences in the ability of individual nematodes to infect. In the present study the effect of an infected host, the wax moth Galleria mellonella, on subsequent infection of the entomopathogenic nematodes Steinernema carpocapsae Mexican, S. riobravus and S. feltiae was evaluated. The invasion rate of the 3 nematode species to a non-infected host was reduced by 40–60% after pre-exposure to infected hosts. These nematodes regained their full invasion potential after they were rinsed with water. Invasion into insects which were previously injected with nematodes was significantly reduced by 60–80% 6–9 h after injection. The reduction in subsequent invasion due to the initial infection was nematode species specific. This phenomenon was also observed with other lepidopteran pests (Helicoverpa armigera and Spodoptera littoralis). The data indicate that the initial infection by entomopathogenic nematodes induced the release of a substance which reduced the subsequent invasion. The chemical and biological characteristics of this substance are currently under investigation.

Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H. glycines. Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme. Both predicted proteins contain a short secretion signal sequence, a long pro-peptide and a mature protein of 219 amino acids. Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E. coli. HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-IΔD86. HGCP-II showed no activity against the synthetic substrates tested. The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.