Varroa jacobsoni is an ectoparasite of Apis mellifera which invades brood cells, on 8-day-old larvae several hours before cell capping. Reproduction of the parasite takes place in the capped brood cells during the nymphose of the bee. Cuticular hydrocarbons of unparasitized bees and of bees parasitized by Varroa jacobsoni were extracted and analysed by gas chromatography (GC) coupled with mass spectrometry (GC-MS). Three developmental stages of worker honey bees were studied: larvae, pupae and emergent adults. The comparison between unparasitized and parasitized hosts was performed with Principal Components Analysis coupled with a multivariate variance analysis. The cuticular hydrocarbon profiles of honey bees were qualitatively similar, for the 3 developmental stages and regardless of the presence of Varroa in the cells. Nevertheless, comparison of the relative proportions of hydrocarbons showed that the cuticular profiles of pupae and emergent adults parasitized by 1 mite and of larvae parasitized by 2 mites were significantly different from the corresponding unparasitized individuals. Such modifications could be regarded (i) as a cause of the multi-infestation in larvae during invasion of brood and (ii) as a consequence of stress and/or removal of proteins contained in the haemolymph of the host during its development.

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.

Multiple species infections with parasitic helminths, including nematodes, are common in wild rodent populations. In this paper we first define different types of associations and review experimental evidence for different categories of interactions. We conclude that whilst laboratory experiments have demonstrated unequivocally that both synergistic and antagonistic interactions involving nematodes exist, field work utilizing wild rodents has generally led to the conclusion that interactions between nematode species play no, or at most a minor, role in shaping helminth component communities. Nevertheless, we emphasize that analysis of interactions between parasites in laboratory systems has been fruitful, has made a fundamental contribution to our understanding of the mechanisms underlying host-protective intestinal immune responses, and has provided a rationale for studies on polyparasitism in human beings and domestic animals. Finally, we consider the practical implications for transmission of zoonotic diseases to human communities and to their domestic animals, and we identify the questions that merit research priority.

Saliva inoculated by sandfly females during feeding stimulated production of high levels of anti-saliva antibodies. To determine whether 3 species of the genus Phlebotomus have species-specific salivary antigens we performed dot-blots and immunoblots using sera from mice, hamsters and rabbits repeatedly bitten by sandflies. Important differences were found in the antigen components of the salivary gland lysates (SGL) of Phlebotomus papatasi, P. perniciosus and P. halepensis. In total 4–9 species-specific antigens were detected in each species by immunoblotting. Cross-reactivity was not detected between P. papatasi and the other species tested; in the SGL of P. papatasi sera from animals bitten by this species recognized 5–7 major antigens while sera from animals bitten by other species did not react. A weak cross-reaction was observed between P. perniciosus and P. halepensis; in SGL from P. perniciosus, the sera from rabbits and hamsters bitten by this species recognized about 8 intense bands while sera from animals bitten by P. halepensis reacted weakly with up to 4 saliva polypeptides.

Toxoplasma gondii is known to induce specific behavioural changes in its intermediate hosts. This is usually considered to be an evolutionary adaptation aimed to increase the probability of transmission of the parasite into its definitive host, the cat, by predation. In rodents an increase of reaction time as well as many other specific behavioural patterns have been observed. Here we report the results of our double blind study showing the significantly longer reaction times of 60 subjects with latent toxoplasmosis in comparison with those of 56 controls. Moreover, the existence of a positive correlation between length of infection and mean reaction time suggested that slow and cumulative effects of latent toxoplasmosis rather than a one-step (and possibly transient) effect of acute toxoplasmosis disease are responsible for the decrease of psychomotor performance of infected subjects. To our knowledge, this is the first study confirming the existence of such parasite-induced changes in human behaviour that could be considered in evolutionary history of the human species as adaptive from the point of view of parasite transmission.

Regression analysis of the relationship of serum circulating anodic and cathodic antigens (CAA and CCA), as a possible direct measure of worm burden, and fecal egg counts allows the study of phenomena like density-dependent fecundity in human Schistosoma mansoni infections. For a reliable analysis, variations in egg count measurements as well as in circulating antigen levels have to be taken into account, and an accurate estimation of these variations (represented by parameter λ in the so-called Deming regression) is of great importance. From a new, extensive data set of repeated measurements of fecal egg counts and CAA and CCA concentrations we determined the respective values for parameter λ, and (re)analysed the relationship between circulating antigens and egg counts in 3 data sets from Burundi, Senegal and Zaire by Deming regression. For comparison, ordinary linear regression was performed as well, which considerably biased the regression lines for CCA, but not for CAA. The analyses resulted in a clearly non-proportional relationship between egg counts and CAA, and, to a lesser extent, CCA. Assuming that egg counts and antigen measurements directly reflect egg production and worm burdens, respectively, our findings reinforce the indication of density-dependent fecundity in schistosomiasis mansoni, as suggested by others.

African trypanosomes of the subgenera Nannomonas and Pycnomonas have been recorded from both wild and domestic suids. However, complete descriptions of some of these trypanosomes with regard to host range, pathogenicity, transmission and distribution are still lacking. Neither the recently described Trypanosoma (Nannomonas) godfreyi nor Trypanosoma (Nannomonas) congolense Tsavo have been isolated from mammalian hosts, while Trypanosoma (Pycnomonas) suis remains the rarest of the Salivarian trypanosomes. The only isolate presumed to be of the latter species is maintained at the Kenya Trypanosomiasis Research Institute, Nairobi. We present here the results of characterization of this isolate by morphology, tsetse transmission, the use of species-specific DNA probes and DNA sequence analysis. Morphology in stained blood smears revealed a small trypanosome with a free flagellum. Experimental transmission through Glossina morsitans morsitans showed a developmental cycle typical of subgenus Nannomonas. A positive identification was obtained with species-specific PCR primers for T. congolense Tsavo; moreover, the sequence of the SSU rRNA gene was almost identical to that of T. congolense Tsavo on database. In phylogenetic analysis of the SSU rRNA genes of Salivarian trypanosomes, T. congolense Tsavo grouped with T. simiae rather than T. congolense, suggesting that the name T. simiae Tsavo is more appropriate.

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3·6–4·5; modest cleavage remained at pH 5·0, and no cleavage was detected above pH 5·0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1′ residues at the 2 CH2 cleavage sites were Phe254–Leu255 and Leu325–Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Heterologous immunity, or protection by one invading organism against another across phylogenetic divides, has been recognised for decades. It was initially thought to operate largely through enhancement of phagocytosis, but this explanation became untenable when it was realised it worked extremely well against intraerythrocytic protozoa and killed them while they were free in the circulation. Clearly a soluble mediator was called for. This review summarises the logic that arose from this observation, which led to a wider appreciation of the roles of pro-inflammatory cytokines, and then nitric oxide, in the host's response against invaders, as well as the ability of these mediators to harm the host itself if they are generated too enthusiastically. This has led to a discernable pattern across heterologous immunity as a whole, and its lessons influence a range of areas, including vaccine development.

Knowledge of the genetics underlying resistance to parasitic infection has important repercussions for our understanding of infection dynamics and the mechanisms of host–parasite co-evolution. The aim here was to determine for a Biomphalaria glabrata–Schistosoma mansoni system whether (1) resistance is dominant over susceptibility, (2) it is possible to crossbreed snails to be simultaneously resistant and/or susceptible to more than one parasite strain and (3) compatibility genotype affects reproductive strategy. Using replicate snail strains artificially selected for either resistance or susceptibility to single replicate parasite strains, individual snails from each line were paired with a selected partner of matched or non-matched compatibility status and cross-breeding was identified by RAPD–PCR. The resulting compatibility phenotype of all offspring was determined. Support for all 3 hypotheses were obtained. The results are discussed in terms of their applied and theoretical implications.

The prevalence and abundance of infections with haemoparasites were studied over a 3 year period in Clethrionomys glareolus (bank vole, n=420) sampled from forests in the NE of Poland. Total species richness was 5 (Prevalence=Haemobartonella sp. 63·1%, Bartonella grahamii 27·4%, Hepatozoon erhardovae 31·4%, Trypanosoma evotomys 15% and Babesia microti 1·0%) with 81·9% of the voles carrying at least 1 species and a mean infracommunity species richness of 1·4. Variation in species richness was determined primarily by season and year, and the interaction of these factors. The observed frequency distribution of infracommunity species richness did not differ from that predicted by a null model, suggesting that there were no marked associations between the species. Analyses of prevalence and abundance of infection with each species in turn, revealed that overall the principal causes of variation were temporal and seasonal and their interaction, intrinsic factors such as age and sex playing only a minor role. However, the relative importance of specific extrinsic, and rarely intrinsic, factors varied and was distinct for each of the species in the study. Prevalence data revealed 4 sets of 2-way associations between species, mostly varyingly dependent on combinations of intrinsic and extrinsic factors. Analysis of quantitative associations suggested 4 sets of positive 2-way interactions, 3 of which remained after controlling for the effect of extrinsic and intrinsic factors on the abundance of each species, but only one could be unequivocally accepted (Haemobartonella sp.+B. grahamii) after correction for multiple comparisons. These data are discussed in the context of the changing ecological profiles in this region of Eastern Europe and, in a wider context, in relation to current understanding of the factors which shape component community structures of haemoparasites in wild rodents.

Infective metacyclic promastigote forms of Leishmania mexicana are introduced by the bite of sandfly vectors into their human hosts where they transform into the amastigote form. The kinetics of this process was examined in vitro in response to different combinations of temperature (26 °C or 32 °C), pH (7.2 or 5.5), and exposure to human serum. Little transformation occurred at 26 °C/pH 7.2, intermediate levels at 26 °C/pH 5.5 and 32 °C/ pH 7.2, and the greatest response at 32 °C/pH 5.5. Transformation was stimulated by exposure to normal human serum, but was markedly reduced when serum previously incubated at 56 °C for 1 h was used (complement heat-inactivated). This stimulatory effect was reproduced by exposure to a single purified component of human serum, C-reactive protein (CRP). Binding of CRP to the whole surface of L. mexicana metacyclic promastigotes, including the flagella, was demonstrated by an indirect fluorescent antibody test. The effect of purified CRP was dose dependent and occurred using normal serum concentrations. The stimulatory effect of whole serum was oblated by CRP depletion and restored by addition of purified CRP. The effects of cAMP analogues indicated that transformation could be mediated via an adenylate cyclase cascade.

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.

It is generally accepted that free-living stages of parasitic helminths are dependent on aerobic degradation of endogenous energy sources for their energy generation. This concept, however, is not the result of extensive experimental evidence, but originated mainly intuitively as oxygen is widely available in their habitat and these stages generally have a small size. Schistosoma mansoni, the sole parasitic helminth whose energy metabolism has been studied throughout its life-cycle indeed has aerobically functioning free-living stages. However, large differences exist in energy metabolism between adult stages of distinct parasitic helminths, and caution should be taken in predicting that all free-living stages of all parasitic helminths have the same, aerobic energy metabolism. Hence, this report studied the energy metabolism of Fasciola hepatica miracidia and demonstrated that F. hepatica miracidia are also dependent on aerobic degradation of their endogenous glycogen stores by glycolysis and on Krebs cycle activity for energy generation. However, in contrast to S. mansoni, F. hepatica miracidia cannot function anaerobically, as inhibition of the respiratory chain blocked motility and carbohydrate degradation, and finally resulted in death of the miracidia. Therefore, this report demonstrated that differences exist between miracidia of distinct species, in pre-adaptation of their energy metabolism to the occasional hypoxic conditions within their next host.

To dissociate the influence of host age from length of exposure on acquisition of filarial infection, we examined the development of microfilaraemia and anti-filarial IgG4 in all ages of a naive population that became suddenly exposed to Brugia malayi as a result of transmigration. Responses in 247 transmigrants, who had settled for periods of several months up to 6 years in their new homesteads, were compared with those of 133 life-long residents. As shown in earlier studies, anti-filarial IgG4 increased with age in the indigenous population, whose age is equivalent to length of exposure. However, by examining transmigrants, it became clear that development of specific IgG4 was influenced by age, since levels of this antibody were consistently higher in transmigrant adults than in transmigrant children, despite an equal length of exposure to filarial infection. Examining microfilaraemia, it was confirmed that infection establishes more rapidly in adults than in children.

Filarial infections of humans are chronic diseases. Despite an ongoing immune response, adult filariae continuously produce their offspring, the microfilariae (Mf), which are able to persist in sufficient numbers to ensure transmission. In this study, host- and parasite-derived factors, which contribute to persistence of Mf, were investigated using the filariasis model of Litomosoides sigmodontis in mice. Different strains of mice were found to differ widely in their capability to eliminate circulating Mf. Studies of congenic mouse strains showed that early and rapid clearance of Mf was mediated by activation pathways relevant to innate immunity, whereas late or delayed clearance of Mf was pre-determined by MHC-related factors. Genetic knock-out of genes for the MHC class-II molecules totally abrogated resistance. Most interestingly, the presence of only 1 adult female, but not male worms, renders all mice susceptible, irrespective of the genetic background, enabling Mf to circulate for extended periods of time. Such prolonged microfilaraemia was also observed in L. sigmodontis-infected animals challenged with heterologous Mf of Acanthocheilonema viteae. The use of cytokine gene knock-out mice showed that persistence of L. sigmodontis Mf was facilitated by IL-10, but not by IL-4 or IFN-γ. In conclusion, irrespective of a resistant or susceptible host genetic background, survival of Mf of L. sigmodontis in mice is decisively regulated by the presence of adult female L. sigmodontis which will skew and exploit immune responses to facilitate the survival and persistence of their offspring in the infected host.

Injection trials with compatible and non-compatible Onchocerca species into S. damnosum s.l., the vector of human and bovine onchocerciasis, demonstrated that the rapid killing of microfilariae within the blackfly's haemocoel is species specific. In the presence of the peptide RGDS as a blocking agent for integrin-like receptors of haemocytes, the survival of O. ochengi microfilariae in its natural intermediate host was significantly increased. This increased survival 24 h p.i. correlated with a significant decrease of apoptosis levels in the microfilariae following a 2 h exposure to the haemolymph in vivo. These findings suggest that haemocytes are directly involved in the killing of Onchocerca microfilariae in the blackfly.

Opisthorchis viverrini infection is an endemic disease that causes a serious public health problem in southeast Asia, especially in northeast Thailand. We have developed a PCR method using a pair of primers named OV-6F/OV-6R for detecting O. viverrini eggs in stool samples and compared it with Stoll's egg-count method. The primers were designed based on the pOV-A6 specific DNA probe sequence which gave a 330 base pair product. The PCR method can detect a single egg in artificially inoculated faeces or as little as 2×10-17ng of O. viverrini genomic DNA. The method gave 100% sensitivity in all hamster groups except in animals exposed to the lowest intensity of infection (1 metacercaria/hamster). In the first month of infection, the PCR method was more sensitive than using the egg-count method in all infected groups especially in the light infections. The PCR method was also successfully used in monitoring a therapeutic study. Since the PCR method showed no cross-reaction with Heterophyid flukes, it can be useful for specific identification of O. viverrini eggs in stool samples without the risk of false positives. It also has great potential for application in clinical epidemiological studies.

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.

Six myxosporidian species were found in chub (Leuciscus cephalus) originating from Lower Austrian rivers. The frequency of the parasites and their localization was recorded. In all chub, independent of size and origin, Myxobolus cyprini occurred predominantly in the macrophage centres (MCs) of the haematopoietic organs, spleen and kidney. Exclusively in the head kidney of young fish not yet described vermicular plasmodia containing spores of M. cyprini were found. In muscle tissue the prevalence of M. cyprini was comparatively low. Other species of Myxobolus characterized by plasmodial cysts frequently occurred in gills and swimbladder but were rarely detected, and only in small numbers, in the haematopoietic organs. The number of M. cyprini spores and the relative volume of MCs in the haematopoietic organs were estimated in order to examine possible correlations. Significant interrelated changes were found only in juvenile fish up to a size of 15cm. In bigger fish, the number and size of macrophage aggregates were highly variable and independent of infection intensity and fish size, but the number of spores never exceeded that of the aggregated macrophages. The data suggest that due to an early date of infection M. cyprini is the only species which is closely associated with macrophage aggregation.