The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78–88%) 0–135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93–100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12–24h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24h. The data indicate that at 48h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT–PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms.Nucleotide sequence data reported in this paper are available in the EMBL, GenbankTM and DDJB databases under the acession numbers AF207904 and AF207905. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

The arbitrary genus Phytomonas includes a biologically diverse group of kinetoplastids that live in a wide variety of plant environments. To understand better the subdivisions within the phytomonads and the variability within groups, the exon, intron and non-transcribed spacer sequences of the spliced leader RNA gene were compared among isolates of the phloem-restricted members. A total of 29 isolates associated with disease in coconut, oil palm and red ginger (Alpinia purpurata, Zingibreaceae) were examined, all originating from plantations in South America and the Caribbean over a 12-year period. Analysis of non-transcribed spacer sequences revealed 2 main groups, I and II; group II could be further subdivided into 2 subgroups, IIa and IIb. Three classes of spliced leader (SL) RNA gene were seen, with SLI corresponding to group I, SLIIa to group IIa, and SLIIb to group IIb. Two isolates showed some characteristics of both major groups. Group-specific oligonucleotide probes for hybridization studies were tested, and a multiplex amplification scheme was devised to allow direct differentiation between the 2 major groups of phloem-restricted Phytomonas. These results provide tools for diagnostic and molecular epidemiology of plant trypanosomes that are pathogenic for commercially important flowers and palms.

The pig is a natural host of Schistosoma japonicum and a useful animal model of human disease. In the present study mRNA levels of Th1 (IFN-γ) and Th2 (IL-10 and IL-4) cytokines were assessed by RT-PCR within tissues from infected pigs. Twelve Danish crossbred pigs were infected by intramuscular injection or orally with 1000 cercariae. Six other pigs served as non-infected controls. Liver and intestinal tissues were collected 10 weeks post-infection, and analysed for their relative levels of cytokine mRNA. Infected pigs developed a Th2 response as characterized by the increased level of mRNA encoding for IL-4 and IL-10 in their large intestine (caecum and colon). In contrast, levels of IFN-γ did not differ between control and infected animals although variation between animals was observed. When comparing the immune response of orally and intramuscularly infected animals, we found that orally infected pigs produced higher IL-4 and IL-10 levels in their caecum and colon respectively. This stronger Th2 response correlated with a previously reported delay in maturation of infection following oral infection. The cytokine expression levels in tissue samples taken from lesion sites and in nearby areas, without obvious lesions, were then compared. Subsequent to an oral infection, the Th2 type cytokine production was higher in the lesion sites of the liver. In conclusion, this study is the first demonstration of IL-4 and IL-10 cytokine response in pig tissues during S. japonicum infection.

We investigated the involvement of nitric oxide in Schistosoma-induced liver injury. We found that inducible nitric oxide synthase mRNA became detectable in the liver at the onset of parasite egg laying and levels then increased as the eggs accumulated in the organ. Enzyme concentration and activity paralleled mRNA levels. The event was a direct effect of egg deposition, as it occurred in the liver after natural infection, or in the lungs after i.v. injection of the eggs. However, nitric oxide seems to have no direct effect on the eggs since in vitro assays showed that the nitric oxide donor SIN-1 did not alter the ability of the eggs to hatch. L-Arginine and L-NAME, a nitric oxide synthase inhibitor, were administered to infected mice in an attempt to increase or reduce nitric oxide production, respectively. Arginine had no effect on the disease, whereas the inhibitor led to a marked decrease of hepatic injury with, in particular, reduced fibrosis and decreased lipid peroxidation. In conclusion, not only is inducible nitric oxide synthase activity unlikely to exert an anti-microbicidal effect against the egg stage of S. mansoni but it might lead to deleterious effects in the liver and therefore contribute to the pathology.

Growth and development of adult schistosomes requires permanent communication processes of the parasites with their specific host environment and, additionally, between the two genders. Accumulating evidence suggests that, at the molecular level, the mandatory interactions are mediated by signal transduction processes. During recent years, a considerable interest has emerged in the identification of signalling molecules from this parasite and to elucidate their roles during development. In this organism, a number of different molecules have been identified which belong to diverse classes of evolutionary conserved signal transduction cascades. However, up to now no representative of the conserved family of cellular tyrosine kinases has been identified. In this study we present a suitable approach to identify this class of molecules and demonstrate the successful cloning and molecular characterization of one of the isolated genes, the tyrosine kinase 5 (TK5). An unexpected finding was that in the Liberian strain of Schistosoma mansoni the TK5 gene exhibits an allelic polymorphism.

We have investigated the development of the schistosome egg and its secretions in order to understand how it migrates through gut tissues and also initiates pathology in the liver. We show by electron microscopy that the subshell envelope is absent in the newly deposited egg, but appears very early and differentiates as development progresses. In the mature egg, this nucleated envelope contains extensive endoplasmic reticulum, suggestive of a protein synthetic capacity. Furthermore, Reynolds' layer only appears between the envelope and the egg-shell in the mature egg and may represent its accumulated secretions. We have biosynthetically labelled and collected the secretions (ESP) released by mature but not immature eggs during culture. Their fractionation by SDS–PAGE reveals a simple pattern of 6 bands, differing markedly in composition from soluble egg antigen preparations. Electrophoresis in casein substrate gels demonstrates the presence of 2 distinct proteases in the egg secretions. By immunocytochemistry, ESP localized predominantly to the envelope of the mature egg, suggesting that this layer rather than the miracidium is the source of egg secretions.

Immunological and structural changes in the thymus and pronephros of Cyprinus carpio infected with the blood fluke, Sanguinicola inermis for 30 days, and exposed to 0·5mg NH+4/l for 48 and 168h were investigated. Ultrastructural observations revealed cell disruption and highly vacuolated cytoplasm in the thymus. Of the cells that remained intact there was a significant increase in thrombocytes after 48h exposure to the pollutant. In addition, there was a decrease in lymphocytes following exposure to ammonia at both time-periods studied. In contrast the pronephros of fish exposed to the pollutant underwent relatively mild changes in cellular architecture although ammonia and time of exposure had significant effects on the proportions of several leucocyte types. A significant decrease in neutrophils, thrombocytes and lymphocytes occurred in fish exposed to the pollutant for 168h. Pronephric lymphocyte stimulation (cpm) by Con A and PWM increased in vitro, whereas the stimulation index was reduced in infected fish exposed to ammonia. Changes in the immune organs of S. inermis-infected carp treated with pollutant were both organ- and time-specific. The possible reasons for this are discussed and significance in relationship to parasitization assessed.

Electrophysiological techniques were used to obtain recordings of extracellular electrical activity from the anterior end of live adult females of Brugia pahangi. Stimulation with 100mM, 10mM and 1mM acetylcholine resulted in an increase in spike activity which was concentration dependent, whereas stimulation with phosphate-buffered saline and 0·1mM acetylcholine gave no increase in activity. The delay in response was not concentration dependent. The action of possible host cues was investigated. Stimulation with heat-inactivated foetal calf serum (IFCS) and 10mM glutathione gave an increase in spike activity but exposure to 5mg/dl haemoglobin elicited no response. The response to IFCS was found to be suppressed completely by pre-incubation for 30min in ivermectin.

The glycolipids of Ascaris suum represent either neutral, zwitterionic or acidic structures. The acidic fraction comprises a sulphatide and an unusual phosphoinositolglycosphingolipid (Lochnit et al. 1998b). The sulphatide was previously localized to the hypodermis, contractile zone of somatic muscle cells and the external musculature of the female uterus, whereas the presence of the phosphoinositolglycosphingolipid species was restricted to the intestine. The neutral and zwitterionic components belong to the arthro-carbohydrate series, which are substituted in their zwitterionic structures by phosphocholine (PC) and in one glycolipid by an additional phosphoethanolamine residue. In previous immunohistochemical localization studies, however, the chemical nature of the PC-substituted biomolecules has not been investigated in detail. Here, we report on the immunohistochemical localization and differentiation of phosphocholine-containing structures into lipid- and protein-bound species in adult A. suum. The patterns of immunostaining, obtained with a PC-specific monoclonal antibody and anti-zwitterionic glycolipid hyperimmune serum in the female worm, indicated a parallel organ distribution for glycolipid- and protein-bound PC-epitopes. Immunoreactivity was localized to specific tissues of the body wall, intestine and reproductive tract. This is the first report of surface-located PC-epitopes for ascarids. The patterns of immunolabelling obtained with antibodies directed against the unsubstituted arthro-carbohydrate series backbone suggested that the glycolipid-bound epitope was restricted to the hypodermis, whilst the protein-bound antigenic determinant resembled that for PC.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatinNucleotide and amino acid sequence data reported in this paper are available in the GenBankTM database under the accession number AF035945. . Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.