An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P. falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

This study focuses on the effect of acidic water and aqueous aluminium on the monogenean ectoparasite Gyrodactylus salaris, infecting Atlantic salmon (Salmo salar) parr. G. salaris-infected salmon were exposed to various combinations of acidity and aluminium concentrations. The most pronounced effect was the elimination of parasites after 4 days when 202 μg Al/l was added to the water. The effect of aluminium was concentration dependent, but was relatively independent of pH (5·2, 5·6 and 5·9). At the lowest pH of 5·0 the effect of aluminium was enhanced. Acidic aluminium-poor water had no or minor effects on the G. salaris infections except at pH 5·0 where all parasites were eliminated within 9 days. The G. salaris populations increased exponentially in untreated control water. The results show for the first time that aqueous aluminium can, to a limited extent, have a positive effect on fish health. This study emphasizes that basic knowledge about abiotic environmental factors is of importance in order to understand the population dynamics, range extension and dispersal of ectoparasites such as G. salaris. Finally, our results suggest that aluminium treatment could form an effective disinfection method against ectoparasites in hatcheries and laboratories, as well as complementing the controversial rotenone treatments used against natural populations of G. salaris.

Gyrodactylus arcuatus-like monogeneans collected from the gills of 3 goby species, Pomatoschistus minutus, P. lozanoi and P. pictus, from the Voordelta Area (SW Netherlands) were compared on a morphometric basis with G. arcuatus from its type host, the three-spined stickleback, Gasterosteus aculeatus. Univariate and multivariate analysis was carried out on measurements of 17 characters of the opisthaptoral hard parts of 268 specimens. The contribution of host-dependent and seasonal factors to the observed morphological variation are discussed. Univariate statistics (ANOVA) were used to detect features that were useful in separating the gyrodactylids from the different host groups. Subsequent factor analysis and discriminant analysis, combining all variables, led to the separation of 3 distinct forms (possibly species) dependent on the host species harbouring them. G. arcuatus (sensu stricto) was only present on G. aculeatus, while P. minutus and P. lozanoi carried a common species, Gyrodactylus sp. 1, and P. pictus hosted Gyrodactylus sp. 2. Seasonal variation in size of the sclerities was demonstrated on the Gyrodactylus species from P. minutus and P. lozanoi, and should be taken into consideration when specimens are compared. The findings are discussed in view of the possibility of phylogenetic host specificity of the gyrodactylids occurring on goby species that live sympatrically in the Voordelta area.

Infestations of post-smolt sea trout (Salmo trutta L.) by the salmon louse (Lepeophtheirus salmonis Krøyer) were characterized in 42 estuaries over a 5 year period in Ireland. Spatial variation in infestation was more significant than temporal trends and existed at 3 levels; between regions (regions >100 km of coastline), between bays within regions (bays <50 km in length) and between estuaries within bays (distance between estuaries <10 km). The observed spatial structure in infestations inferred that production of the infective larvae varied between regions and bays and that there was limited movement of fish and infective larvae between regions and bays. In addition the different levels of infestation recorded between estuaries in the same bay indicated short spatial scale variability in parasite transmission. Significantly higher infestations occurred in bays that contained lice-infested farmed salmon. Lice-infested wild spring salmon, which were present in estuaries of some systems, did not have a significant positive impact on infestations.

Humoral immune responses of the Indian leaf monkey (Presbytis entellus) experimentally infected with Brugia malayi and exhibiting disease manifestations were studied. Microfilaraemia, filaria-specific IgG and circulating immune complexes (CICs) were determined in the monkeys at different time-points after inoculation of B. malayi 3rd-stage larvae. Sera were analysed for recognition pattern of adult parasite antigen molecules by immunoblotting. More than 60% of the infected monkeys developed episodic or persistent limb oedema with or without fever and with low or no microfilaraemia. While both CIC and filaria specific IgG levels were comparable in animals showing no disease symptoms (asymptomatics) and some animals showing symptoms (symptomatics), IgG levels peaked during pre-patent stage in symptomatics and during latent stage in asymptomatic animals. However, some of the symptomatic animals showed a low level of filaria-specific IgG as compared to asymptomatic and other symptomatic animals. The immunoblot analysis showed non-reactivity of 17 and 55 kDa antigens with sera of symptomatic animals. The results thus suggest that humoral immune responses as measured in the present study do not precede the development of the manifestations. However, 2 non-reactive antigen molecules identified by symptomatic sera need further study to establish their possible involvement, if any, in the development of acute disease manifestations in this model.

Monthly data collected during 1993 in the Vaccarès lagoon (Rhône Delta, France) were used to investigate the influence of the trematode Aphalloïdes cœlomicola on the survival of the common goby Pomatoschistus microps, which acts both as second intermediate and definitive host. Prevalence of the parasite did not increase with fish size in either sex. Mean abundance and variance to mean abundance ratio gave evidence that the trematode could have an impact on host mortality. Gonad weight of parasitized females was reduced and the parasite seemed to provoke a gonadal regression above a certain parasitic biomass. Fecundity and egg diameters did not differ between parasitized and unparasitized females, we thus hypothesize that the energy content of eggs (composition in lipids and proteins) was probably modified as an adaptation to energy losses. Prevalence of infection in males compared to females and influence of the parasite on host survival and reproductive effort are discussed.

The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstrating the potential of the approach for the diagnosis of equine strongyloidosis. The establishment of this PCR assay also has implications for studying the biology and epidemiology of equine strongyles and anthelmintic resistance using faecal egg count reduction tests.

A gene encoding a protein of 646 amino acid residues with a molecular mass of 71·3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb). The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum. Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage). Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm. The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella.

The initial infection process of nematode-trapping fungi is based on an interaction between the trapping structure of the fungus and the surface of the nematode cuticle. A bioassay was designed to investigate the predatory response of several isolates of nematode-trapping fungi against 3 mutants of Caenorhabditis elegans (AT6, AT10 and CL261), which have been reported to differ in the reaction of their cuticle to antibodies and lectins. The bioassay was also applied to infective larvae of animal (Haemonchus contortus, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus axei) and plant (Meloidogyne spp.) parasitic nematodes. Differences in trapping ability were most marked in the first 24 h, and were density dependent. Although the isolate of Arthrobotrys responded very rapidly in the first 24 h, Duddingtonia flagrans was generally the most effective isolate and Monacrosporium responded relatively poorly throughout all experiments. All the fungi tested trapped the srf mutants of C. elegans more efficiently than the wild type, and there were differences between the different srf mutants of C. elegans. Differences in trapping ability were also observed between different isolates of D. flagrans; similarly, differences in trapping behaviour were observed not only amongst the different species of plant-parasitic nematodes, but also between the sheathed and exsheathed larvae of the animal-parasitic nematodes.

The efficacy of an isolate of the nematophagous fungus Duddingtonia flagrans against gastrointestinal nematodes of cattle was examined at 2 dose levels on 2 permanent pastures, with high and low stocking rates, respectively. Thirty calves, experimentally infected with Ostertagia ostertagi, were divided into 3 comparable groups and allocated to 3 similar paddocks in each of the 2 trials. Two of the 3 groups received fungal material once per day during the initial 2 months, either at high dose (106 fungal spores/kg body weight) or low dose (5×105 or 2·5×105 fungal spores/kg body weight). The third group remained as an untreated control group. Faecal, blood, and herbage samples were collected and animals were weighed every month from May to September. The pasture grazed at a high stocking rate had a large number of overwintering infective larvae, while the pasture grazed at a low stocking rate had a low overwintering herbage larval infectivity. The results showed that, at a high stocking rate, the recovery of infective larvae on pasture was diminished and calves were prevented from clinical ostertagiosis by using the D. flagrans Troll A-isolate. At low stocking rate, the parasite burden seemed not to be very heavy, and a conclusive effect of the fungi at the dose-level used could not be detected.