To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Samples of serum, feces, and tissues from feral cats from St Kitts, West Indies were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test, and found in 71 of 96 (73·9%) of cats with titres of 1:10 in six, 1: 20 in six,1:40 in seven,1: 80 in three, 1: 160 in 10, 1:320 in 13, 1:640 in nine, and 1:1,280 or higher in 17. Tissues of 10 cats were bio-assayed in mice. Toxoplasma gondii was isolated from tissues of 7 cats; from hearts of 6, from tongue of 5, and brains of 3 cats. All 7 isolates were avirulent for mice. Toxoplasma gondii oocysts were not found in the feces of 51 cats. Genotyping of these 7 T. gondii isolates by 10 multi-locus PCR-RFLP markers, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico, revealed 4 genotypes, including clonal Type II, Type III and 2 unique genotypes. Five of the 7 cats had infection with 2 genotypes, indicating high frequency of mixed infection in the cat population on the St Kitts island.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0·27 μm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0·74 and 0·22 mm, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

Myxobolus cerebralis is the causative agent of whirling disease that has significant economical and ecological impacts on trout populations. Although intensive studies have been conducted to understand its effects on and interactions with its fish host, only limited information is available about how and to what extent M. cerebralis affects its oligochaete host, Tubifex tubifex. We investigated the effects of M. cerebralis on survival, growth, reproduction, and feeding activity of T. tubifex. Mature, immature and juvenile worms were exposed to myxospores and their infection prevalence, mortality, sexual development, reproduction and spore production were compared with unexposed worms. The parasite affected neither survival nor growth but inhibited clitellar development and reduced cocoon production by over 80%. Numbers of actinospores released from mature worms were nearly 9-fold higher than that of immature worms. When non-clitellated infected worms were kept at 30°C for 4 days, spore release ceased and they re-developed a clitellum. These results suggest parasite-induced castration. Comparative monitoring of defecation rate revealed that M. cerebralis reduced feeding activity of T. tubifex by approximately 40%. Low energy intake and impaired energetic allocation may be the underlying mechanism behind reduced fecundity of infected T. tubifex.

Proliferative kidney disease (PKD) of salmonids, caused by Tetracapsuloides bryosalmonae, can lead to high mortalities at elevated water temperature. We evaluated the hypothesis that this mortality is caused by increasing parasite intensity. T. bryosalmonae-infected rainbow trout (Oncorhynchus mykiss) were reared at different water temperatures and changes in parasite concentrations in the kidney were compared to cumulative mortalities. Results of parasite quantification by a newly developed real-time PCR agreed with the number of parasites detected by immunohistochemistry, except for very low or very high parasite loads because of heterogenous distribution of the parasites in the kidney. Two experiments were performed, where fish were exposed to temperatures of 12, 14, 16, 18 or 19°C after an initial exposure to an infectious environment at 12–16°C resulting in 100% prevalence of infected fish after 5 to 14 days of exposure. While mortalities differed significantly between all investigated water temperatures, significant differences in final parasite loads were only found between fish kept at 12°C and all other groups. Differences in parasite load between fish kept at 14°C to 19°C were not significant. These findings provide evidence that there is no direct link between parasite intensity and fish mortality.

Infection with a myotropic Trypanosoma cruzi clone induces maternal fertility failure. In the current work, we evaluated whether reduction of maternal parasitaemia before mating has beneficial effects on pregnancy outcome. Female mice were subjected to benznidazole (Bz) treatment after infection. On day 30 of therapy, mating was assessed and pregnancy outcome was determined on day 14 of gestation. Fetal resorptions diminished in T. cruzi-infected Bz-treated mice compared with T. cruzi-infected untreated mice. This was in agreement with the reduction in the blood/solid tissue parasite load and with the percentage of necrotic foci in placental samples from T. cruzi-infected Bz-treated females. To study eventual changes in the immune homeostasis of T. cruzi-infected Bz-treated mice, activation of the immune system was evaluated at the end of Bz therapy and before mating. We found specific IgG1 reduction resulting in a predominance of specific IgG2a, reduced numbers of CD69+ CD4+ cells and diminished frequency and numbers of CD44+ T cells. Concanavalin A-stimulated splenocytes from T. cruzi-infected Bz-treated mice produced higher amounts of IFN-γ than T. cruzi-infected untreated mice. These results indicate that reduction of maternal parasite load improves pregnancy outcome. These findings correlate with a favourable modulation of the immune response.

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

ATP-dependent Ca2+ uptake was studied in a subcellular fraction from Herpetomonas sp. prepared by mechanical disruption and using 45Ca2+ as a tracer. The uptake was stimulated by Ca2+ with a K0·5 of 0·1 μm and a Hill number (nH)=2·8±0·4. The Ca2+-dependent ATP hydrolysis was optimal at pH 7·0 and had a Ca2+ dependence identical to uptake. The uptake was highly stimulated by oxalate whereas calmodulin had no activating effect. ATP stimulated Ca2+ uptake with a biphasic pattern that resembled the curves described for the purified preparations of rabbit sarcoplasmic reticulum. The ATP stimulation is described as the sum of two Michaelis-Menten curves with Km1=0·25±0·19 μm and Km2=29·6±6·8 μm. GTP or UTP could also promote Ca2+ uptake, but with less efficiency than ATP. Vanadate inhibited the uptake with low apparent affinity. Thapsigargin and cyclopiazonic acid were almost ineffective. The Ca2+ uptake was insensitive to H+ ionophores and to bafilomycin suggesting no participation of acidocalcisomes. The results are comparable to those obtained using cells permeabilized with digitonin and using arsenaze III as Ca2+ indicator. The Ca2+ uptake activity described here seems to belong to the endoplasmic reticulum of Herpetomonas sp. and is suitable for further studies on the mechanisms of calcium homeostasis in parasites.

Sheep infected with the triclabendazole-susceptible, Cullompton isolate of Fasciola hepatica were dosed with 15 mg/kg of compound alpha at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24, 48 and 72 h post-treatment (p.t.). Ultrastructural changes to the flukes were assessed using transmission electron microscopy (TEM), with a view to gathering information on the mechanism(s) of action for compound alpha and on the possible route of its entry into F. hepatica. The tegumental syncytium was more severely affected than the gut at all time-points p.t. with compound alpha, suggesting a predominantly trans-tegumental route of uptake. Disruption to the tegumental system became increasingly severe over time. A stress response was observed at 24 h p.t. and took the form of blebbing and increases in the production and transport of secretory bodies. By 72 h p.t., extensive tegumental loss and degeneration of the tegumental cell bodies had occurred. Degeneration of subtegumental tissues and internal flooding were also observed. Changes in the gastrodermal cells were slow to develop: reduced secretory activity was evident at 72 h p.t.. There was progressive disruption to the somatic muscle layers, with disorganization of the muscle blocks and loss of muscle fibres.

Seroprevalence of Toxocara and Taenia solium and risk factors for infection with these parasites were explored in a long-term rural settlement in São Paulo state, Brazil. An ELISA for the detection of anti-Toxocara IgG and IgE and anti-T. solium cysticerci was standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis and by vesicular fluid antigen from Taenia crassiceps cysticerci (VF). For cysticercosis, the reactive ELISA samples were assayed by Western blot using 18 kDa and 14 kDa proteins purified from VF. Out of 182 subjects, 25 (13·7%) presented anti-Toxocara IgG and a positive correlation between total IgE and the reactive index of specific anti-TES IgE (P=0·0265) was found amongst the subjects found seropositive for anti-Toxocara IgG. In these individuals 38·0% showed ocular manifestations. The frequency of anti-T. solium cysticerci confirmed by Western blot was 0·6%. Seropositivity for Toxocara was correlated with low educational levels and the owning of dogs. Embryonated eggs of Toxocara spp. were found in 43·3% of the analysed areas.

The population dynamics of Trichuris suis in pigs was studied during long-term experimental infections. Twenty-three 10-week-old pigs were inoculated with 5 T. suis eggs/kg/day. Seven, 8, and 8 pigs were necropsied at weeks 4, 8, and 14 post-start of infection (p.i.), respectively. The median numbers of worms in the colon were 538 (min–max: 277–618), 332 (14–1140) and 0 (0–4) at 4, 8, and 14 weeks p.i. respectively, suggesting an increased aggregation of the worms with time and acquisition of nearly sterile immunity. The serum levels of T. suis specific antibodies (IgG1, IgG2 and IgA) peaked at week 8 p.i. By week 14 p.i. the IgG2 and IgA antibody levels remained significantly elevated above the level of week 0. The population dynamics of T. suis trickle infections in pigs is discussed with focus on interpretation of diagnostic and epidemiological data of pigs, the use of pigs as a model for human Trichuris trichiura infections and the novel approach of using T. suis eggs in the treatment of patients with inflammatory bowel disease.