The free-living amoeba Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM), a disease leading to death in the vast majority of cases. In patients suffering from PAM, and in corresponding animal models, the brain undergoes a massive inflammatory response, followed by haemorrhage and severe tissue necrosis. Both, in vivo and in vitro models are currently being used to study PAM infection. However, animal models may pose ethical issues, are dependent upon availability of specific infrastructural facilities, and are time-consuming and costly. Conversely, cell cultures lack the complex organ-specific morphology found in vivo, and thus, findings obtained in vitro do not necessarily reflect the situation in vivo. The present study reports infection of organotypic slice cultures from rat brain with N. fowleri and compares the findings in this culture system with in vivo infection in a rat model of PAM, that proved complementary to that of mice. We found that brain morphology, as present in vivo, is well retained in organotypic slice cultures, and that infection time-course including tissue damage parallels the observations in vivo in the rat. Therefore, organotypic slice cultures from rat brain offer a new in vitro approach to study N. fowleri infection in the context of PAM.

Two experiments (Exps 1 and 2) were carried out to study the effect of dietary carbohydrates on the establishment of Trichuris suis in pigs. Two experimental diets based on barley flour were used; Diet 1 was supplemented with non-fermentable carbohydrates from oat hull meal, while Diet 2 was supplemented with fermentable carbohydrates from sugar beet fibre and inulin. In Exp. 1, thirty-two pigs were allocated randomly into 4 groups. Two groups were fed Diet 1 and 2 groups were fed Diet 2. Pigs from one of each diet group were inoculated with 2000 infective T. suis eggs each and the other two groups were uninfected controls. All pigs were slaughtered 8 weeks post-inoculation (p.i.). In Exp. 2, twenty-four pigs were allocated randomly into 2 groups and fed Diet 1 or Diet 2, respectively. All the pigs were inoculated with 2000 infective T. suis eggs. Six pigs from each group were slaughtered 8 weeks p.i. and the remaining 6 pigs from each group were slaughtered 12 weeks p.i. Infections were followed by faecal egg counts and worm burdens were assessed at necropsy. Pigs fed Diet 2 had lower egg counts in both experiments; in Exp. 2 the difference was significant (P<0·05). No differences were found in worm burdens 8 weeks p.i. in both experiments, however, worms from pigs on Diet 2 were significantly shorter (P<0·0001). Pigs fed Diet 2 and slaughtered 12 weeks p.i. had significantly lower worm counts (P<0·01) compared to pigs fed Diet 1. The results indicate that fermentable carbohydrates do not affect the establishment of T. suis in naïve pigs, but result in earlier expulsion and reduced growth of the established worms. Thus, diets with highly fermentable carbohydrates may be used in the control of T. suis.

Parasites often induce behavioural changes in their host. However, it is not necessarily easy to determine whether these changes are representative of an adaptation of the parasite (parasite manipulation), an adaptive response of the host or a side-effect of infection. In a solitary parasitoid of Drosophila larvae (Leptopilina boulardi), viral particles (LbFV) modify the host acceptance behaviour of infected females by increasing their tendency to superparasitize. This behavioural alteration allows for the horizontal transmission of the virus within superparasitized Drosophila larvae. To add support for or against the ‘manipulation hypothesis’, we investigated whether other behavioural components of the parasitoid are affected by viral infection, and whether other forms of horizontal transmission exist. Neither the ability of females to locate host kairomones nor their daily rhythm of locomotor activity was affected by viral infection. However, infected females showed a lower rate of locomotor activity, suggesting a physiological cost of infection. The searching paths of females were also unaffected. Males from infected and uninfected lines showed the same ability to locate females'sexual pheromones. Moreover, alternative modes of horizontal transmission (through food consumption and/or contact with the same Drosophila larvae) did not lead to viral contamination of the parasitoid. The overall specificity of behavioural alteration and of viral horizontal transmission is consistent with the hypothesis that the virus manipulates the behaviour of the parasitoid.

Whilst most animals invest equally in males and females when they reproduce, a variety of vertically transmitted parasites has evolved the ability to distort the offspring sex ratios of their hosts. One such group of parasites are male-killing bacteria. Here we report the discovery of females of the ladybird Anisosticta novemdecimpunctata that produced highly female-biased offspring sex ratios associated with a 50% reduction in egg hatch rate. This trait was maternally transmitted with high efficiency, was antibiotic sensitive and was infectious following experimental haemolymph injection. We identified the cause as a male-killing Spiroplasma bacterium and phylogenetic analysis of rDNA revealed that it belongs to the Spiroplasma ixodetis clade in which other sex ratio distorters lie. We tested the potential for interspecific horizontal transfer by injection from an infected A. novemdecimpunctata line into uninfected individuals of the two-spot ladybird Adalia bipunctata. In this novel host, the bacterium was able to establish infection, transmit vertically and kill male embryos.

Genetic variation in susceptibility to pathogens is a central concern both to evolutionary and medical biologists, and for the implementation of biological control programmes. We have investigated the extent of such variation in Drosophila melanogaster, a major model organism for immunological research. We found that within populations, different Drosophila genotypes show wide-ranging variation in their ability to survive infection with the entomopathogenic fungus Beauveria bassiana. Furthermore, striking divergence in susceptibility has occurred between genotypes from temperate and tropical African locations. We hypothesize that this may have been driven by adaptation to local differences in pathogen exposure or host ecology. Genetic variation within populations may be maintained by temporal or spatial variation in the costs and benefits of pathogen defence. Insect pathogens are employed widely as biological control agents and entomopathogenic fungi are currently being developed for reducing malaria transmission by mosquitoes. Our data highlight the need for concern about resistance evolution to these novel biopesticides in vector populations.

Staurosporine, Ro-31-7549, and KN-93, which are inhibitors of serine/threonine protein kinase, protein kinase C, and calcium-modulin kinase, respectively, were tested for their effects on the in vitro growth of Babesia bovis. Staurosporine was the most effective inhibitor, completely clearing the parasitaemia as early as the first day of exposure at a concentration of 100 μM. Moreover, staurosporine caused a significant increase in the percentage of extracellular merozoites, most likely due to the inhibition of erythrocyte invasion by the parasite. Although 5 mM Ro-31-7549 and KN-93 had a suppressive action, this was not enough to destroy the parasite. Interestingly, concentrations of 0·5 to 5 mM KN-93 influenced the parasitic development within the infected erythrocytes. The present study suggests that B. bovis requires, to a certain extent, the phosphorylations mediated by parasite- or host erythrocyte-protein kinases, in particular, for the processes of successful invasion of erythrocytes and intraerythrocytic development.

Species of the phylum Myxozoa are common parasites of fish and can cause severe losses in cultured species. Although a number of myxozoan life-cycles have now been elucidated, little is known about the biology of these organisms in the fish host. Monoclonal antibody B4 raised to the myxozoan Tetracapsuloides bryosalmonae has been previously noted to react with a number of species infecting fish kidney. We present the results of a survey of 55 myxosporean species that determined that this antibody detects an antigen on the spore surface of 33 of these species in the genera Myxobolus, Sphaerospora and Thelohanellus. However, there appears to be no clear relationship between those spores that contain the MAb B4 reactive antigen and the host or organ in which they are detected. The antigen appears to be synthesized in the plasmodial cytoplasm and is intimately associated with the surface of the spore capsules and, where present, the mucus envelope. The nature of this envelope is further discussed in relation to its formation and distinctive properties.

Toxocara canis, the parasitic roundworm of dogs, can infect a number of paratenic hosts, such as mice and humans, due to the widespread dissemination of its ova in the environment. In these paratenic hosts, larvae have been shown to exhibit a predilection for the central nervous system, resulting in an increasing number of parasites migrating to the brain as infection progresses. In an initial experiment, we investigated the differential brain involvement of T. canis in 7 strains of inbred mice, and chose 2 strains, susceptible (BALB/c) and resistant (NIH) to cerebral infection. In a second experiment, both strains were investigated in terms of course of migration, larval accumulation, and behavioural response to T. canis infection. Results revealed that infected BALB/c mice took significantly longer to drink from a water source (following a period of deprivation), compared with control mice, indicating some degree of memory impairment. Cerebral larval recoveries from both strains of mice demonstrated variation between the two experiments, suggesting that larval burdens may not be a reliable indicator of susceptibility or resistance to T. canis infection. The percentage of total recovered larvae in each organ may be a better representation of larval distribution. Our model system may provide insights into the impact of chronic geohelminth infection on cognitive development.

A microsporidium was isolated from larvae of Spodoptera litura (Fabricius) collected from Tamil Nadu, India. This microsporidian species is monomorphic, disporous and develops in direct contact with the cytoplasm of the host cell. The nuclear configuration of merogonic and sporogonic stages was diplokaryotic. The merogonic proliferative stage was unusual that normal development with 1, 2 and 4 binucleated forms were common, while large multinucleate meronts containing 8 and 12 small compact horseshoe-like diplokaryotic nuclei were also observed. The fresh spores were typically ovocylindrical in shape, with a mean size of 3·91×1·91 μm and the polar filament length was ~90 μm. Infection was systemic with mature spores produced in the midgut, nervous tissue, muscles, labial glands, gonads, tracheae, epidermis, Malpighian tubules and, most extensively, fat body tissues. The new isolate was highly pathogenic to S. litura larvae. Host specificity tests performed on 37 non-target hosts of 5 different insect orders revealed that the new isolate is pathogenic only to lepidopteran insects. We sequenced the 16S small subunit rRNA (SSU rRNA) gene of the isolate and compared it with 72 non-redundant microsporidian sequences from the GenBank. Based on the light microscopic studies and phylogenetic analyses, the new isolate is assigned to the genus Nosema. Significant differences in the SSU rRNA sequence were identified when compared with the type species Nosema bombycis and other closely related species viz., Nosema spodopterae. Structural differences were also observed in the 16S SSU rRNA between the new isolate and the two above-mentioned microsporidian pathogens. We conclude that the microsporidian isolate reported here is distinctly different from the other known species and is likely to be a new species.

The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that develops asynchronously inside parasitophorous vacuoles. Spore differentiation involves the construction of a cell wall commonly divided into an outer layer (exospore) and a thicker, chitin-rich inner layer (endospore). The developmental patterns of protein deposition and mRNA expression for 2 different spore wall proteins were studied using immunocytochemical and in situ hybridization procedures with ultrathin frozen sections. The onset of deposition of an exospore-destined protein (SWP1) correlated with the formation of lamellar protuberances during meront-to-sporont conversion. No evidence for a release of SWP1 towards the parasitophorous vacuole lumen was obtained. An endospore-destined protein (EnP1) was detected early on the plasma membrane of meronts prior to extensive accumulation within the chitin-rich layer of sporoblasts. swp1 mRNA was preferentially synthesized in early sporogony while enp1 mRNA was transcribed during merogony and a large part of sporogony. The level of both mRNAs was reduced in mature spores. Considering the availability of the E. cuniculi genome sequence, the application of nucleic and/or protein probes to cryosections should facilitate the screening of various genes for stage-specific expression during microsporidian development.

Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica on the East Coast of the United States. Transmission dynamics of this parasite were investigated in situ for 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity. Perkinsus marinus weighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1200 cells/l) and was significantly related to P. marinus weighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Ivermectin treatment may induce severe adverse reactions in some individuals heavily infected with Loa loa. This hampers the implementation of mass ivermectin treatment against onchocerciasis in areas where Onchocerca volvulus and L. loa are co-endemic. In order to identify factors, including co-infections, which may explain the presence of high L. loa microfilaraemia in some individuals, we analysed data collected in 19 villages of central Cameroon. Two standardized skin snips and 30 μl of blood were obtained from each of 3190 participants and the microfilarial (mf) loads of both O. volvulus and L. loa were quantified. The data were analysed using multivariate hierarchical models. Individual-level variables were: age, sex, mf presence, and mf load; village-related variables included the endemicity levels for each infection. The two species show a certain degree of ecological separation in the study area. However, for a given individual host, the presence of microfilariae of one species was positively associated with the presence of microfilariae of the other (OR=1·79, 95% CI [1·43–2·24]). Among individuals harbouring Loa microfilariae, there was a slight positive relationship between the L. loa and O. volvulus mf loads which corresponded to an 11% increase in L. loa mf load per 100 O. volvulus microfilariae. Co-infection with O. volvulus is not sufficient to explain the very high L. loa mf loads harboured by some individuals.

Tc13 is a trans-sialidase family protein of Trypanosoma cruzi, the aetiological agent of Chagas' disease. Recently, in vitro studies had suggested that Tc13 might participate in the pathogenesis of the disease. In order to study the role of Tc13 antigens in an in vivo model, we administered plasmid DNA encoding a Tc13 antigen from the Tulahuén strain (Tc13 Tul) to BALB/c mice and evaluated the immunological and pathological manifestations as well as the capacity of this antigen to confer protection against T. cruzi infection. Tc13 Tul immunization did not elicit a detectable humoral immune response but induced specific memory T-cells with no capacity to produce IFN-γ. Five months after DNA-immunization with Tc13 Tul, signs of hepatotoxicity and reactive changes in the heart, liver and spleen were observed in 40–80% of mice. When Tc13 Tul DNA-immunized animals were challenged with trypomastigotes, a significant decrease in parasitaemia in early and late acute phase was observed without modification in the survival rate. Surprisingly, Tc13 Tul-immunized mice chronically infected with T. cruzi showed a decrease in the severity of heart damage. We conclude that, in BALB/c mice, genetic immunization with Tc13 Tul mainly induces immune responses associated with pathology.

The Trypanosoma cruzi cyclophilin gene family comprises 15 paralogues whose nominal masses vary from 19 to 110 kDa, namely TcCyP19, TcCyP20, TcCyP21, TcCyP22, TcCyP24, TcCyP25, TcCyP26, TcCyP28, TcCyP29, TcCyP30, TcCyP34, TcCyP35, TcCyP40, TcCyP42 and TcCyP110. Under the conditions used, only some of the T. cruzi cyclophilin paralogue products could be isolated by affinity chromatography. The 15 paralogues were aligned with 495 cyclophilins from diverse organisms. Analyses of clusters formed by the T. cruzi cyclophilins with others encoded in various genomes revealed that 8 of them (TcCyP19, TcCyP21, TcCyP22, TcCyP24, TcCyP35, TcCyP40, TcCyP42 and TcCyP110) have orthologues in many different genomes whereas the other 7 display less-defined patterns of their sequence attributes and their classification to a specific group of cyclophilin's orthologues remains uncertain. Seven epimastigote cDNA clones encoding cyclophilin isoforms were further studied. These genes were found dispersed throughout the genome of the parasite. Amastigote and trypomastigote mRNAs encoding these 7 genes were also detected. We isolated 4 cyclosporin A-binding proteins in T. cruzi epimastigote extracts, which were identified by mass spectrometry as TcCyP19, TcCyP22, TcCyP28 and TcCyP40. Cyclosporin A-binding to these cyclophilins might be of importance to the mechanism of action of Cyclosporin A and its non-immunosuppressive analogues, whose trypanocidal effects were previously reported, and therefore, of potential interest in the chemotherapy of Chagas' disease.

We have identified and partially sequenced 8 ABC transporters from an EST dataset of Sarcoptes scabiei var. hominis, the causative agent of scabies. Analysis confirmed that most of the known ABC subfamilies are represented in the EST dataset including several members of the multidrug resistance protein subfamily (ABC-C). Although P-glycoprotein (ABC-B) sequences were not found in the EST dataset, a partial P-glycoprotein sequence was subsequently obtained using a degenerate PCR strategy and library screening. Thus a total of 9 potential S. scabiei ABC transporters representing the subfamilies A, B, C, E, F and H have been identified. Ivermectin is currently used in the treatment of hyper-infested (crusted) scabies, and has also been identified as a potentially effective acaricide for mass treatment programmes in scabies-endemic communities. The observation of clinical and in vitro ivermectin resistance in 2 crusted scabies patients who received multiple treatments has raised serious concerns regarding the sustainability of such programmes. One possible mechanism for ivermectin resistance is through ABC transporters such as P-glycoprotein. This work forms an important foundation for further studies to elucidate the potential role of ABC transporters in ivermectin resistance of S. scabiei.

The ability to increase the chances that infectious prey are taken by predators is an observed feature of many parasites that rely on one or more predator-prey relationships to complete their life-cycle. In the sylvatic life-cycle of Echinococcus multilocularis – the causative agent of human alveolar echinococcosis – foxes are the final host, with voles acting as intermediate hosts. Here we review the evidence that E. multilocularis causes increased susceptibility to predation and present a general mathematical model for the sylvatic life-cycle. The ability to increase susceptibility to predation in the intermediate host reduces the sensitivity of the parasite population to adverse conditions. For example, there is no critical density of foxes below which the parasite is expected to die out, even if the effect of the parasite on infected prey is very small. We suggest that increased susceptibility to predation is a plausible explanation for the observed resilience of E. multilocularis during and following field trials of praziquantel baiting.