The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are describedThe sequence data reported herein have been deposited in EMBL under Accession No. AJ422214. . In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

Pulsed-field gel electrophoresis of intact chromosomes from 2 isolates of each of the 2 most pathogenic species of large Babesia parasites that infect dogs, i.e. Babesia canis (European species) and B. rossi (South African species), revealed 5 chromosomes in their haploid genome. The size of chromosomes 1–5 was found to be different in the 2 species, ranging from 0.8 to 6.0 Mbp. The genome size was estimated to be approximately 14.5 Mbp for B. canis and 16 Mbp for B. rossi, respectively. Within each species, the size of chromosomes 1–3 of B. canis and 1–2 of B. rossi was conserved between the 2 isolates, whereas the size of chromosomes 4–5 of B. canis and 3–5 of B. rossi was variable. Chromosomes 1–5 hybridized with a 28-mer telomeric oligonucleotide probe derived from Plasmodium berghei. When NotI-digested chromosomes of the 4 isolates were hybridized with the telomeric probe a maximum of 10 fragments was revealed. Moreover, hybridization of this telomeric probe to a Southern blot of genomic DNA from the 4 isolates, digested with a series of restriction enzymes, revealed a species-specific restriction map. Hybridization of intact or NotI-digested chromosomes of both species with 2 sets of 3 cDNA-antigen probes derived from each species, revealed no cross-hybridization between these B. canis and B. rossi genes.

Perkinsus marinus is a protozoan responsible for dramatic mortality in the Eastern oyster, Crassostrea virginica, but not in the Pacific oyster, C. gigas. To understand the host–parasite relationship, we inoculated P. marinus trophozoïtes into the shell cavity of C. gigas and measured, over 2 months, (i) intensity of infection, (ii) protease inhibitory activities against P. marinus proteases and against bovine α-chymotrypsin, (iii) plasma haemagglutinin titre, (iv) plasma protein concentration, (v) plasma lysozyme activity and (vi) total haemocyte count. We observed that the highest protease inhibitory activities and haemagglutinin titres (3–10 days post-challenge) preceded parasite elimination (initiated 7 days post-challenge). In contrast, plasma protein concentration, lysozyme activity and total haemocyte count showed no significant modification following the challenge. It is hypothesized that the capacity of C. gigas to increase its protease inhibitors represents the key event in resistance to parasite infection by neutralizing the proteases secreted by P. marinus, thus preserving the oyster haemagglutinins from degradation. Such haemagglutinins will be ready to act as opsonins stimulating phagocytosis of parasites.

The genetic polymorphism among 271 Danish Cryptosporidium isolates of human and animal origin was studied by partial amplification and sequencing of the Cryptosporidium oocyst wall protein (COWP) gene, the 18S rDNA, and a microsatellite locus.The nucleotide sequence data reported in this paper are available in the GenBank database under the accession number AF469174. Furthermore, the microsatellite locus was studied directly using fragment analysis. A comparative analysis of DNA sequences showed the presence of 3 different subgenotypes (C1, C2 and C3) in C. parvum isolates from Danish cattle, with prevalences of 16.7, 17.2 and 73.1% including 13 (7.0%) mixed infections. Subgenotype C1 was significantly more prevalent (P<0.001) in the southern part of Denmark. In Cryptosporidium isolates of human origin the anthroponotic subgenotype H1 was identified, in addition to the zoonotic subgenotypes C1, C2, and C3. Of 44 human samples, 56.8% were anthroponotic, whereas 40.9% were zoonotic genotypes. One human isolate was characterized as C. meleagridis. The porcine Cryptosporidium isolates (N = 4) revealed a pattern which was genetically distinct from human and bovine isolates. Cryptosporidium in a hedgehog (Erinaceus europaeus L.) was identified for the first time. By microsatellite sequencing the hedgehog isolate showed a subgenotype distinct from the previously detected types. The assignment to subgenotype by microsatellite sequencing and fragment typing was 100% identical in samples where results were achieved by both methods. In addition, the fragment analysis proved more sensitive, easier, faster, and less expensive compared to sequencing.

Phlebotomus papatasi sand flies, the vectors of Leishmania major, differ genetically in their susceptibility to the pathogens. The costs of infection appear to be so great that selection against Leishmania-susceptible flies could presumably occur, unless susceptibility is compensated for by some advantage. Foci of P. papatasi-transmitted L. major are mainly found in arid habitats where seasonal stress of dehydration and heat reduces the quantity of sugar in plant leaves. The sand flies feed on these leaves and with the lack of essential sugar only a few survive long enough to deposit eggs and transmit Leishmania. This association suggested that susceptibility to L. major infection may be linked with advantageous tolerance of sugar deprivation. Here we show that desert sand flies, provided with excess sugar, became progressively resistant to infection. Selection for survival under sugar-poor conditions increased the susceptibility of parent and first-generation offspring by more than 2-fold. The leaves of plants, on which flies naturally feed, contain more sugar in irrigated than in arid habitats. About 85% of first-generation flies colonized from a desert habitat retain experimental Leishmania infection compared to 25% of offspring of flies from irrigated sites.

The population biology of the schistosome–vector snail Bulinus truncatus was studied in an irrigation system near Marrakech, Morocco using both genetic and demographic approaches. The population genetic survey was conducted in 4 sites, 2 sites being sampled on 2 separate occasions. Individuals were genotyped at 6 microsatellite loci. No variability was found at 4 loci, and the 2 other loci had less than 4 alleles. The differentiation, both spatial and temporal, among populations was extremely weak. The demographic survey was conducted using 2 capture-mark-recapture analyses in 2 separate sites, the first in 1999 and the second in 2000. The second analysis permitted the estimation of parameters based on recent methodological developments (multisite models). Although these studies provided information on several traits, we report here on dispersal only. Both analyses showed that individual dispersal is of the order of a few hundreds of metres per reproductive life, that is the scale of the whole irrigation area. Both the genetic and demographic studies indicated that this area harbours a single – or no more than a few – populations of B. truncatus. This has implications for our understanding of the coevolutionary process between snails and flukes.

The time-course of the humoral immune response of female blackflies after a challenge with bacteria, different Onchocerca microfilariae species, bacterial endotoxin and microfilarial extract was investigated. Strong bacteriolytic and growth inhibition activities against the Gram-positive bacterium Micrococcus luteus were induced by all agents. Specific differences were found in activity levels and time-course. Notably the endotoxin lipopolysaccharide (LPS) induced a very early, profound bacteriolytic and antibacterial response, which declined within a day after injection. In contrast, the bacteriolytic activities after Escherichia coli D31 and Onchocerca microfilariae infections were lower, but remained elevated over the observation period of 4 days. The bacteriolytic activity was correlated to a haemolymph protein with a molecular weight of around 14 kDa. Anti-Gram-positive activity in the E. coli infected group appeared within the first 6 h. However, it took 4 days in the microfilarial infected blackflies to reach significant levels. The active agent was identified to be a peptide with a molecular weight of around 4–4.5 kDa. Activity against the Gram-negative bacteria E. coli was detected in blackflies injected with E. coli D31, O. dukei microfilariae and microfilarial extract on days 1 and 4 after injection. The immune response in S. damnosum s.l. naturally infected via a bloodmeal on cattle supported the findings of the experimental infections. Similarities of the immune response kinetics between bacterial and filarial infections suggested that intracellular Wolbachia bacteria, released from microfilariae, could be responsible for the antibacterial response. This is supported by the observation that the induction of an immune response in the Drosophila melanogaster mbn-2 cell line by the filarial extract is blocked by polymyxin B, which forms inactive complexes with bacterial LPS.

Iran is an important endemic focus of cystic hydatid disease (CHD) where several species of intermediate host are commonly infected with Echinococcus granulosus. Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Iran and characterized using both DNA (PCR-RFLP of ITS1) and morphological criteria (metacestode rostellar hook dimensions). The sheep and camel strains/genotypes were shown to occur in Iran. The sheep strain was shown to be the most common genotype of E. granulosus affecting sheep, cattle, goats and occasionally camels. The majority of camels were infected with the camel genotype as were 3 of 33 human cases. This is the first time that cases of CHD in humans have been identified in an area where a transmission cycle for the camel genotype exists. In addition, the camel genotype was found to cause infection in both sheep and cattle. Results also demonstrated that both sheep and camel strains can be readily differentiated on the basis of hook morphology alone.

Nine groups of lambs were challenged with 500 drug-resistant (R) Ostertagia circumcincta infective-stage larvae (L3) twice-weekly for 6 weeks; 3 of the groups were also given 500 susceptible (S) L3 and another 3 given 3000 L3 twice-weekly. One week prior to parasite challenge, 1 group of each dose rate was administered ivermectin-capsules (IVM-CRCs). Following challenge, a second group from each dose rate was administered oxfendazole to remove susceptible parasites. The remaining groups received no drug treatment throughout the trial. No significant difference in faecal egg count was observed between the groups. The groups left untreated did not differ significantly in resulting worm burden. However, the percentage of the total L3 challenge recovered as 4th-stage larvae or adult worms demonstrated a density-dependent relationship and declined with increasing challenge. Those groups treated with anthelmintic to remove S worms prior to necropsy also showed density dependency as worm burdens were lower at the highest dose rate. In the groups administered IVM-CRCs, no dose-dependent effect on worm burden was observed. These differences resulted in more worms being present at the highest dose rate in the IVM-CRC-treated animals than in those animals treated orally with anthelmintic. Adult female worms from the IVM-CRC high challenge group were significantly longer and contained more eggs in utero than those in the other high challenge groups. These results demonstrate that where resistance is present, IVM-CRCs can modify the density-dependent regulation of the parasite population, resulting in the acquisition of larger resistant worm burdens than might otherwise be expected. This could accelerate the build-up of resistant worm populations and decrease the time until treatment failure.

The identification of protective helminth antigens remains the most important challenge in the development of parasitic vaccines. To identify protective antigens of Ostertagia ostertagi, an important abomasal parasite of cattle, parasite-specific local antibodies from the abomasal mucus and from the draining lymph nodes were collected from calves immunized with multiple infections and from ‘primary infected’ animals. With these probes, Western blots of extracts and excretion/secretion (E/S) material from L3, L4 and adult life-stages as well as cDNA expression libraries were screened to identify antigens that were exclusively recognized by antibodies from ‘immunized’ calves. In the adult stage, a protein of 32 kDa was specifically detected on Western blot by mucus antibodies from ‘immunized’ animals. In the L3 and L4 larval stages, proteins situated in the regions of 28–29 kDa were recognized by mucus antibodies and a 59 kDa antigen was specifically recognized by lymph node antibodies from ‘immunized’ animals. Screening E/S material revealed no specific difference in recognition pattern between ‘immunized’ and ‘primary infected’ animals. Screening of the cDNA libraries revealed 26 relevant clones, coding for 15 proteins, among these several with potential protective capacity, immunodominant properties or functional and physiological importance e.g. metalloproteases, an aspartyl protease inhibitor and collagen.