Trypanosoma cruzi specific sequences were amplified by the polymerase chain reaction from total blood of human chagasic patients and normal individuals. A 330 bp fragment originating from kinetoplast DNA was specifically detected in most chagasic individuals. We tested the sensitivity and specificity of this method in normal and affected individuals attending the Evandro Chagas Hospital, Rio de Janeiro. The results of these tests were compared with serological diagnosis performed using standard techniques, and in some cases with xenodiagnosis. We found that none of the serologically negative individuals gave any specific amplification product, whereas 55 out of 61 patients previously serodiagnosed as chagasic were positive using the PCR method (sensitivity: 90%). Xenodiagnosis, which is currently considered to be the most sensitive parasitological technique for Chagas' disease diagnosis, detected only 12 out of 28 serologically positive patients (sensitivity: 43%). The usefulness of the PCR method was further investigated with chagasic patients who had received anti-parasite treatment with benznidazole. It has always been difficult to evaluate the incidence of cure in such cases by serology, since a humoral response against T. cruzi antigens may remain for years even in the absence of the parasite. We observed a positive amplification result in only 9 out of 32 treated patients who remained reactive when tested using classical serology. These observations suggest that PCR is the most sensitive technique available for direct detection of T. cruzi in chagasic patients and that it can be a very useful instrument for the follow-up of patients after specific treatment.

Trypanosome infection of mammalian hosts leads, within days, to a strong early response against a small, distinct number of parasite proteins. One of these proteins is the variable surface glycoprotein (VSG). Most of the others are apparently non-variable, intracellular trypanosome proteins. Two of these antigens I2 and I17 are now characterized at the molecular level. Both exhibit a highly repetitive amino acid sequence organization, but they show no sequence similarity either to each other or to any other proteins known to date. Preliminary serological analyses indicate that both allow the early, sensitive and specific detection of infections with different species of trypanosomatids, making them interesting candidates for the development of diagnostic tools for trypanosomiasis detection.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immuno-fluorescence antibody test monoclonal antibody (MAb) IB5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

A polymerase chain reaction (PCR) based on the detection of the Lmet2 repeat sequence specific to members of the Leishmania donovani–complex is described. To improve PCR specificity, a post-PCR hybridization step is often performed but this usually involves an entirely new procedure with additional manipulations, expense and time. We have simplified this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed automatically as part of the PCR programme. The hybrids are afterwards detected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-linked immunoassay (PCR–SHELA), is rapid, able to detect less than 5 cultured parasites and is specific for parasites of the Leishmania donovani–complex. We also describe the application of PCR–SHELA to the detection of amastigotes in various tissues of infected laboratory animals.

Th relationships between the Encephalitozoon-like Septata intestinalis and other microsporidia that occur in humans, notably Encephalitozoon cuniculi and Encephalitozoon hellem, is insufficiently documented using morphological descriptions alone. To assess mutual relationships, we have examined other phenotypic as well as genetic aspects of S. intestinalis, obtained both from tissue culture and clinical specimens, in comparison with a number of other microsporidia. Phenotypic characterization was performed by analysis of the protein composition and antigenic structure of various microsporidian spores by SDS-PAGE and Western blotting. The genetic characterization consisted of the determination of the sequence of the S. intestinalis rrs gene encoding the small subunit ribosomal RNA (srRNA), restriction fragment length polymorphism (RFLP) analysis of amplified rrs genes and establishment of the degree of sequence identity between rrs genes of various microsporidian species. The unique sequence of rrs of S. intestinalis as well as the distinct RFLP and SDS-PAGE profiles indicate that S. intestinalis is clearly different from other human microsporidian species. However, its rrs gene shared about 90% sequence identity with rrs of both Encephalitozoon spp., E. cuniculi and E. hellem. This is remarkably higher than the about 70% identity observed between rrs of microsporidian species which belong to different genera and thus suggests that S. intestinalis should be regarded as a species of the genus Encephalitozoon. Western blots revealed a marked cross-reactivity between S. intestinalis and both species of Encephalitozoon which also stresses the close relationship between these organisms. It is concluded that S. intestinalis is so closely related to E. cuniculi, the type species of Encephalitozoon, that it should be reclassified as Encephalitozoon intestinalis.

The estimation of two parameters in the transmission of Babesia microti by the tick Ixodes trianguliceps amongst small mammals, (1) the duration of infectivity in natural hosts and (2) the probability of transmission from an infected to a susceptible vole, is described. When B. microti was maintained by direct tick transmission, the probability of a complete cycle of transmission via the larval-nymphal and nymphal-adult transstadial routes was 1·0 and 0·71 respectively, but only if the larvae or nymphs had engorged, as distinct from feeding slowly, while the source parasitaemia exceeded 2 or 0·2% respectively, but had not yet passed the peak level. The duration of this condition for infectivity in voles infected by nymphal bites was only 1–4 days, whilst infections delivered by adult ticks barely reached the threshold level necessary for successful transmission. When syringe passage was introduced into the parasite maintenance schedule (a) the probability of transmission declined markedly and (b) the time-course of the parasitaemia was altered. If these parameter values are put into a simple model, together with field data on tick and host survival rates, it becomes apparent that additional factors, such as the highly aggregated distribution of ticks on their hosts, must account for the maintenance of B. microti at the levels seen in wild small mammal populations.

An incomplete ring of electric nets was placed around oxen which were either uninfected, infected with Trypanosoma vivax, or infected with T. congolense. The numbers of fed and unfed Glossina pallidipes caught on the nets were used to estimate the attractiveness of the oxen to tsetse, and the feeding success of the tsetse on the oxen. Oxen infected with T. congolense attracted more G. pallidipes than the other groups of oxen. Taking into consideration daily variation in the abundance or activity of the flies, oxen infected with T. congolense were about 70% more attractive to G. pallidipes than were uninfected oxen or oxen infected with T. vivax. The latter two groups mostly attracted high numbers of G. pallidipes on days when the flies were especially abundant or active. The feeding success of G. pallidipes declined with increase in the rate at which oxen made anti-fly movements. Taking this movement rate into consideration, the feeding success of G. pallidipes on oxen infected with T. congolense was approximately 60% greater than on uninfected oxen or oxen infected with T. vivax. It is suggested that vasodilation induced by T. congolense may account for the difference in feeding success. The level of parasitaemia of T. congolense or T. vivax was not found to affect either the attractiveness of oxen or the feeding success on oxen. There was significant daily variation in the mean fat content of male G. pallidipes caught around the oxen but no effect of mean daily fat content on the proportion of males that fed. The mean haematin content of fed male G. pallidipes was positively correlated with the packed cell volume of the ox on which they fed.

Dynamic models which predict changes in the intensity of schistosome infection with host age are fitted to pre-intervention Schistosoma mansoni data from Kenya. Age-specific post-treatment-reinfection data are used to estimate the force of infection, thus enabling investigation of the rate of worm death. An empirical and statistical approach is taken to the model fitting: where possible, distributional properties and function relationships are obtained from the data rather than assumed from theory. Attempts are made to remove known sources of bias. Maximum likelihood techniques, employed to allow for error in both the pre-intervention and reinfection data, yield confidence intervals for the worm life-span (CI95% = 5·7–10·5 years) and demonstrate that the worm death rate is unlikely to vary with host age. The possibilities and limitations of fitting dynamic models to data are discussed. We conclude that a detailed, quantitative approach will be necessary if progress is to be made with the interpretation of epidemiological data and the models intended to describe them.

Three nucleotide data sets, two mitochondrial (COI and ND1) and one nuclear (ribosomal ITS1), have been investigated in order to resolve relationships among species and strains of the genus Echinococcus. The data have some unusual properties in that mitochondrial heteroplasmy was detected in one strain of E. granulosus, and more than one class of ITS1 sequence variant can occur in a single isolate. The data failed to support the hypothesis that E. granulosus, as it is currently viewed, is a single valid species. Rather, the strains of E. granulosus seem to comprise at least three evolutionarily diverse groups, the sheep strain group, bovine strain group and horse strain group. Molecular distances between them are comparable to, or greater than, molecular evolutionary distances observed between recognized species. The affinities of the cervid strain of E. granulosus are unclear because of ambiguous data, but this strain does not appear to be ancestral to others. E. multilocularis may not be distinct from E. granulosus. However, the remaining two species, E. vogeli and E. oligarthrus appear distinct and rather distant from the first two. Based on the results presented here, taxonomic revision of the genus is clearly warranted.

The behavioural responses of Onchocerca lienalis microfilariae (mf) to tissue factors of the surrogate black fly host, Simulium vittatum, were studied using a novel in vitro bioassay. Mf accumulated towards thoracic tissues to a density 4 times higher than towards abdominal tissues, despite the larger surface area and volume of abdominal tissues. Mf migrated toward thoracic tissues regardless of whether or not contact with thoracic tissues was possible. Therefore, mf directed themselves toward the thorax rather than arresting their movement after they make contact with thoracic tissues. Chemical cue(s) provided a principal guidance for mf to locate thoracic tissues. Mf lost their ability to differentiate thoracic and abdominal tissues following addition of thoracic attractant(s) to excised abdomens and reversed their differential response when excised thoraces were depleted of chemical cue(s). Mf did not respond to salivary gland product, but to other thoracic factor(s). Intact thorax that was connected to 1–2 abdominal segments attracted considerably less mf than excised thorax. However, intact thorax attracted more mf than excised abdomen. Abdominal tissue size did not affect thorax attractiveness. Involvement of a large molecular weight protein(s) was suggested by the high attractiveness of the 100 kDa Centricon concentrator retentate and complete precipitation of attractant(s) by ethanol.

In an immunocytochemical study, using an antiserum and a monoclonal antibody specific for the amino acid, γ-aminobutyric acid (GABA), GABA-like immunoreactivity (GLIR) has been demonstrated for the first time in parasitic flatworms. In Moniezia expansa (Cestoda), GLIR was seen in nerve nets which were closely associated with the body wall musculature and in the longitudinal nerve cords. In the liver fluke Fasciola hepatica (Trematoda), the GLIR occurred in the longitudinal nerve cords and lateral nerves in the posterior half of the worm. GLIR was also detected in subtegumental fibres in F. hepatica. The presence of GABA was verified, using high-pressure liquid chromatography coupled with fluorescence detection. The concentration of GABA (mean ± S.D.) in M. expansa anterior region was 124·8 ± 15·3 picomole/mg wet weight, while in F. hepatica it was 16·8 ± 4·9 picomole/mg. Since several insecticides and anti-nematodal drugs are thought to interfere with GABA-receptors, the findings indicate that GABAergic neurotransmission may be a potential target for chemotherapy in flatworms too.

The relationship between varying intensities of Trichuris trichiura infection and iron status was examined in Jamaican schoolchildren, aged 7 to 11 years. A total of 409 children was identified with T. trichiura (epg > 1200). A control group comprised 207 uninfected children who were matched by school and class to every pair of infected subjects. Blood samples were obtained from 421 children: 264 infected and 157 controls. Compared to the rest of the children, those with heavy infections (epg > 10 000) had significantly lower (P < 0·05) Hb (11·5 ± 1·3 vs. 12·1 ± 1·1 g/dl), MCV (78·6 ± 6·3 vs. 81·2 ± 5·5 fl), MCH (26·2 ± 2·9 vs. 27·5 ± 2·5 pg) and MCHC (33·2 ± 1·5 vs. 33·9 ± 1·4 g/dl). Similarly, the prevalence of anaemia (Hb < 11·0 g/dl) amongst heavily infected children (33%) was significantly higher (P < 0·05) than the rest of the sample (11%). These differences remained significant after controlling for confounding variables including socio-economic status, age, gender, area of residence and the presence of Ascaris infections. Differences in red cell count, ferritin, and free erythrocyte protoporphyrin were not statistically significant and showed no association with the infectious load. These results suggest that in the Jamaican children studied, iron deficiency anaemia is associated with Trichuris infections over 10 000 epg, but not with less intense infections.

AF2 is an endogenous RFamide-like peptide from the parasitic nematode Ascaris suum. The potent stimulatory effects of this peptide on the somatic musculature of Ascaris strongly suggest that it may have an important role in the motornervous system. Here we have investigated the possibility that AF2 may elicit a stimulatory action on Ascaris muscle by potentiating the actions of the excitatory cholinergic motornervous system either pre-synaptically, post-synaptically or both. In in vitro pharmacological experiments AF2 produced a dose-dependent increase in the frequency and amplitude of spontaneous contractions of Ascaris muscle strip which lasted for more than 1 h after a 3 min application of AF2 (10 nM–10µM; N = 7). In addition, AF2 (100 nM) potentiated the contraction elicited by ACh by 43 ± 9% (P < 0·01; N = 8). In electro-physiological recordings from muscle cells, AF2 (10–100 nM; N = 10) potentiated the amplitude of EJPs (excitatory junction potentials). For 100 nM AF2, the potentiation of the EJP was 218 ± 48% (N = 7; P < 0·01). This effect reversed after a wash of 10 min. AF2 did not potentiate the depolarization of the muscle cell elicited by bath applied ACh. These latter two observations are consistent with a presynaptic action of AF2. AF2 (10–100 nM) generated spontaneous muscle cell action potentials in previously quiescent cells. This effect took more than 1 h to wash out. These observations are discussed in terms of the paralysis of Ascaris that is elicited by AF2.

Comparison of the proteins in salivary glands, saliva and haemolymph of Rhipicephalus appendiculatus female ticks during feeding

Parasitology (1994), 109, 517–523

Fig. 2. Delete 24 kDa marker between 36 kDa and 29 kDa markers.

Fig. 6. Replace 24 to 17·6 kDa with 34, 33·5, 32·5, 31·5, 30, 29 kDa.

Fig. 7. Replace 33·2 kDa with 33·5 kDa.

Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands

Parasitology (1994), 109, 525–530

Fig. 4. Replace 19·2 kDa with 14·2 kDa.