Two domestic guinea-pigs (Cavia porcellus), bought in Pinheiros, São Paulo State, Brazil, were taken by their owners a farm in the rural district of Capão Bonito, close to the Atlantic Forest, São Paulo, where they both developed tumour-like and ulcerating lesions on the ears. The causative agent was identified as Leishmania (L.) enriettii, based on biological characters and isoenzyme profiles. Sources of the parasite in wild mammals, and the possible sandfly vector species discussed.

Serum-free media were used to culture Plasmodium falciparum. A commercial preparation, Daigo's GF21 developed as a growth-promoting factor for many kinds of mammalian cells, and consisting of the 55–70% ammonium sulphate fraction of adult bovine serum, insulin, transferrin, ethanolamine and sodium selenite, was found to sustain growth of the parasite when Daigo's T was employed as a basal medium. The optimal Daigo's GF21 concentration for parasite growth was between 5 and 20% (v/v), with the best results at 10%. Differential counts indicated that Daigo's GF21 is essential for schizogony. Established serum-free medium, GIT, consisting of Daigo's T basal medium and Daigo's GF21, yielded good parasite growth without any supplementation. Growth-promoting factor derived from adult bovine serum in Daigo's GF21 was shown to be crucial to parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements for the parasite.

Plasmodium falciparum is an intracellular parasite of the red blood cell. During development it exports proteins which are transported to specific locations within the host erythrocyte. We have begun to identify and characterize exported membrane proteins of P.falciparum in order to obtain specific marker molecules for the study of the mechanisms involved in the distribution of parasite-derived proteins within the host cell. In this report we describe the characterization of a 35 kDa protein which is recognized by a monoclonal antibody. The protein is tightly associated with membranes isolated from infected erythrocytes; it is resistant to extraction with alkali and soluble after treatment with detergents. It is located at the membrane of the parasitophorous vacuole and in membrane-bound compartments which appear in the cytoplasm of the infected erythrocyte. The protein co-localizes with the previously described exported protein-1 (exp-1). Considering its localization and physical similarities to exp-1, we name the 35 kDa protein the exported protein-2 (exp-2).

Codon usage has been analysed in individual gene sequences, derived from a variety of parasitic protozoa in the class Sporozoa of the phylum Apicomplexa using metric multidimensional scaling. The two groups of codon usage patterns detected reflect the two main subgroups of organisms studied (the coccidia and the piroplasms), and it is the pattern usage of synonymous codons that has the largest influence on overall codon usage in the individual genes, rather than being the pattern of amino acid composition of the gene product. The magnitude of the codon usage bias in the sequences was determined using three commonly used indices – NC', GC3s and B. In general, although relatively low levels of codon usage bias were detected in these gene sequences, codon usage bias does explain at least some of the codon usage patterns observed. Codon usage bias was observed to be dependent on the overall base composition of the genes analysed, which in turn was reflected in the types of codons that were either over-or under-represented in the nucleotide sequences. keeping with observations on prokaryotic organisms, it is speculated that the codon usage patterns detected in these parasitic protozoa are the result of directional mutation pressure on the base composition of the genomic DNA.

A total of 736 outpatients diagnosed as having malaria using clinical criteria at a health centre in a highly endemic area of Papua New Guinea were investigated parasitologically. Plasmodium falciparum-attributable fractions were determined using a logistic regression model to compare parasite densities in cases with those of healthy individuals in community surveys. Thirty-seven percent of presumptive cases were found to have raised P. falciparum parasitaemia. This corresponds to an average reporting rate for the population of 0·53 attributable episodes per annum. Whilst the maximum prevalence of parasitaemia in the community was in children aged 5–9 years, the maximum age-specific incidence of attributable cases at the outpatient clinic was 2 cases per annum in the 2- to 4-year-old age group. The procedure for estimating attributable fractions makes it possible to compare morbidity rates between age groups, and to examine how the relationship between morbidity risk and parasite density changes with age, without diagnosing individual episodes. The average tolerance of parasites in an age group was measured by considering the level of parasitaemia associated with a given risk of malaria-attributable morbidity. In contrast to anti-parasite immunity, tolerance of parasites declines with age since at parasite isodensity the probability of being symptomatic increases with age.

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20 000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6·8±1·2 million (mean±s.e., n = 10) or 5·8±1·2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0·9±0·4 million (n = 5) E. maxima oocysts or 2·2±0·4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella. Thus, maternally-derived, IgG-mediated cross- resistance to different species of Eimeria occurs in the chicken, most likely as a result of cross-recognition of conserved epitopes or proteins.

The transmission-blocking monoclonal antibody 13.1, which recognizes the ookinete surface antigen Pbs21 of Plasmodium berghei, and an IgG2a isotype control antibody 26.37 were purified by caprylic acid and ammonium sulphate precipitation. Fab fragments were prepared by papain digestion. IgG but not Fab from antibody 13.1 reduced ookinete formation by P. berghei in culture by as much as 94% at a concentration of 100 μg/ml. There was little difference in antibody efficacy in the range 6·25–400 μg/ml in this assay. The parasite was most sensitive to antibody activity in the first 6–9 h of culture, i.e. the gamete/zygote and early retort stages. Peripheral blood leucocytes (PBL) were essential to achieve maximal inhibition by mAb 13.1 (activity was abrogated totally if PBL were removed). Together the data suggest that one of the mechanisms of action of this antibody is antibody-mediated PBL killing. Phagocytosis of parasites was noted in these experiments in all cultures. We have not attempted in this study to distinguish between Fc-mediated opsonization, as opposed to antibody-dependent cellular cytotoxicity.

The paper describes a longitudinal field study of canine leishmaniasis in sympatric domestic dog and crab-eating fox (Cerdocyon thous) populations. Dogs were studied in house-to-house surveys, and foxes were studied by live-trapping and radio-telemetry. Because serological tests (IFAT in this case) for leishmaniasis are often of uncertain sensitivity and specificity, we draw conclusions comparatively. Both cross-sectional (age–prevalence) and longitudinal analyses indicate that incidence in dogs was highest in the dry season. Seasonal changes in the age–prevalence relationship for dogs suggest that serological conversion and recovery rates decline with prior exposure to infection, where ‘recovery’ may be due to loss of a positive antibody titre or death from leishmaniasis. The mean incidence in dogs was higher in the rural than in the urban population and higher in hunting dogs than pet dogs. Cross-sectional and longitudinal analyses of fox serological data suggest that foxes remain positive for longer than dogs on average, either because detectable antibody is more persistent or because they experience a lower mortality rate due to leishmaniasis.

Receptors interacting with terminal sugars as ligands are involved in the binding of Leishmania donovani promastigotes to the macrophage surface and their subsequent internalization. Mannose and glucose are specifically involved in the binding process. Decreased binding occurs to macrophages already infected with L. donovani either in vivo or in vitro. When mannose- or glucose-bearing liposomes are used as ligands the binding shows similar trends and the percentage inhibition of binding with mannose-bearing liposomes increases when compared to that for the glucose-bearing ones. The decreased binding of the ligand seems to be due to a decrease in the number of receptors after infection. The affinity of the ligands for the binding sites either on the normal macrophages or on the infected macrophages apparently remains the same. The results based on the incorporation of [3H]phenyl alanine and supported by the binding of glycosylated liposomes to both infected and non-infected macrophages suggest that protein synthesis, in general, is suppressed in L. donovani infected macrophages thus affecting also mannose/glucose receptor protein synthesis, resulting in fewer receptors on the macrophage surface.

The impact of immune sera, and peripheral blood cells (PBC) from mice immunized with Plasmodium berghei ookinetes; and of purified immunoglobulin or Fab fragments from anti-Pbs21 monoclonal antibody 13.1, upon establishment of oocyst infections in the mosquito was studied. Infections were initiated either from gametocyte-infected mice, or membrane feeders which contained either gametocytes or mature ookinetes. PBC from ookinete-immunized mice presented with non-immune serum failed to show any transmission-blocking activity. Anti-ookinete serum, intact anti-Pbs21 monoclonal antibody 13.1 or its Fab fragments, all inhibited oocyst formation significantly. When gametocyte-infected mice or gametocytes in membrane feeds were used, inhibition did not directly correlate with antibody concentration. In membrane feeders that contained ookinetes and antibody, concentration-dependent inhibition usually occurred. The efficacy of purified 13.1 IgG was dependent upon the ookinete concentration. The ookinete plasmalemma and cytoplasm were significantly disturbed after 12h in bloodmeals that contained antibody 13.1, but not in the isotype controls. These changes may have caused the observed failure of the ookinete to migrate as rapidly as the controls from the destructive environment of the bloodmeal.

The polymerase chain reaction (PCR) was used to produce a 556 bp nucleotide stretch, employing primers based on the published sequence of the 18S rRNA genes in Cryptosporidium parvum and C. muris. This sequence was found to contain 3 Mae I endonuclease restriction sites, 1 of which was present only in C. parvum. Mae I restriction of PCR products from 2 C. parvum isolates (one of human origin and the other of bovine origin), 1 C. muris isolate, and 1 C. baileyi isolate, showed a specific and reproducible profile for C. parvum that was different from the one obtained for both C. muris and C. baileyi. From these data, new Mae I restriction maps were proposed for the three species. The system was then used to screen 6 C. parvum isolates (from human and bovine hosts), and the C. parvum-specific profile was obtained for all isolates examined. It should be possible to adapt this protocol to detect small numbers of C. parvum oocysts in environmental samples (e.g. in water supplies).

Two species of microsporidia, Enterocytozoon bieneusi and Septata intestinalis have been reported as intestinal parasites of AIDS patients. In attempts to establish E. bieneusi in vitro, spores were concentrated from stool samples from 4 AIDS patients with biopsy-proven E. bieneusi infections. After sterilization of the concentrate in antibiotic solution, the spores were added to monolayers of RK13 cells grown on the membranes of Transwells. Cultures were established from 7 stool samples from the 4 patients but in every case the species established was S. intestinalis not E. bieneusi. On retrospective examination of the stools, a very small number of spores of a size comparable to that of S. intestinalis was found but this species was not detected in biopsies. Typical septate vacuoles containing Type I tubules were observed in vitro but in contrast to the original description, meronts were intravacuolar and sporogony was mainly disporoblastic. The cultivation system, used for the first time for microsporidia, revealed the presence of unsuspected S. intestinalis infections and indicates that this species may be much more common than hitherto suspected. S. intestinalis has not previously been cultured.

The genetic structure of a population of the malaria parasite Plasmodium falciparum has been examined in a village in Tanzania. Seventeen alleles of the merozoite surface protein MSP-1 and 23 of MSP-2 were detected by the polymerase chain reaction (PCR) among the blood parasites of the inhabitants. Most infections contained mixtures of genetically distinct parasite clones. PCR was then used to examine individual P. falciparum oocysts, the products of fertilization events, in wild-caught mosquitoes. Forty-five out of 71 oocysts were heterozygous for one or both genes, showing that crossing between clones was taking place frequently, following uptake of mixtures of gametocytes by the mosquitoes. The frequency of heterozygous forms showed that random mating events probably occurred within mosquito bloodmeals between gametes belonging to different parasite clones.

Cercariae, like miracidia, are non-parasitic larval stages implicated in the life cycle of all trematodes for the host-to-host parasite transmission. Almost all cercariae are free-living in the external environment. With a few exceptions (cercariae of Halipegus occidualis (Halipegidae) can live several months, Shostak & Esch, 1990a), cercariae have a short active life during which they do not feed, living on accumulated reserves. Most cercariae encyst as metacercariae in second intermediate hosts which are prey of the definitive host; in certain species, the interruption of the active life is achieved by an encystment in the external environment (or a simple immobile waiting strategy in a few species). In some two-host life cycles, the cercariae develop into adults after penetration (this is the case for various species causing human schistosomiasis). Some cercariae do not leave the mollusc which must then be ingested by the definitive host.

A chloroform extract of molluscicidal plant secondary compounds from the seeds of a West African legume Millettia thonningii was used to attenuate cercariae of Schistosoma mansoni prior to infection of NMRI female mice. Exposure of cercariae to 0·3, 0·6, 1·3, 2·5, 10 or 20 mg/l concentrations of extract for 30 mm, immediately before standardized infection, was associated with a concentration-dependent decline in worm establishment at 55 days post-infection. The mean numbers of adult worms established declined from about 17 worms/mouse with control cercariae and those exposed to 0·3 mg/l Millettia to 0·1 worms/mouse after 10 mg/l pre-treatment. Mice exposed to cercariae after 20 mg/l pre-treatment had no adult worms at 55 days post-infection. The activities of cercariae 30 mm after exposure to Millettia extract at concentrations up to 2·5 mg/l were similar to those of control larvae: none was immobile. Exposure to higher concentrations of Millettia progressively reduced swimming activity and increased the proportion of immobile cercariae. After pretreatment with 20 mg/l Millettia the majority of larvae were immobile. Levels of anti-parasite antibodies (estimated by ELISA assay using an adult worm-based antigen preparation) rose between 21 and 55 days p.i. with control cercariae and those pre-treated with 2·5 mg/l Millettia. After 5 mg/l pre-treatment, cercariae induced a reduced antibody rise, while mice exposed to cercariae after 10 mg/l pre-treatment showed no rise in anti-parasite antibody levels. These results are discussed in the context of protocols which could possibly use Millettia-attenuated cercariae to induce useful levels of protection in mice towards further cercarial challenge.

The severity of the trypanosomiasis problem in a particular location is traditionally assessed in terms of a challenge index – the product of some measure of tsetse abundance and infection rate – which is assumed to be proportional to the force of infection. However, this index masks variation in the force of infection between herds and among individuals within herds. It is also not comparable between sites since the relative abundance of tsetse to hosts may vary. We have studied spatial distribution of herds of cattle in relation to tsetse in The Gambia and calculated an index of challenge based on the ratio of vectors to hosts over the livestock ranging area. This index is strongly correlated with estimates of the force of infection calculated from the incidence of infection in susceptible zebu; and it provides information on heterogeneity in exposure of different herds to tsetse.

Haemoglobin or blood in the growth medium of Leishmania major inhibited the formation of infective promastigotes and the secretion of chitinases. Inoculation of mice with stationary-phase parasites from control medium caused infections in 20/29 mice, compared to 3/20 mice injected with parasites grown with 10% rabbit blood, or 1/30 mice that received parasites grown with rabbit haemoglobin. The concentration of peanut lectin (PNA) required to agglutinate promastigotes was used as an index of their infectivity, ranging from a high concentration for infective populations to a low concentration for relatively non-infective populations. Agglutination of 50% of the parasites from control medium or from medium containing rabbit haemoglobin required 4·1 μg PNA/ml and 0·1 μg PNA/ml, respectively. Chitinase activities/107 parasites decreased from 4·8 units chitinase and 12·5 units N-acetylglucosaminidase (NAGase) in the control to 2·0 units chitinase and 8·5 units NAGase in cultures containing rabbit haemoglobin. Rabbit, human, bovine and pigeon haemoglobins had various inhibitory effects on the activity of chitinases and not on the virulence, as expressed by PNA agglutination. The relevance of the results to the cycle of Leishmania is discussed.

Following cloning of Leishmania (L.) amazonensis kinetoplast DNA two recombinant clones were identified: one specific for L. (L.) amazonensis and the other specific for L. (L.) amazonensis and closely related isolates. DNA sequences from these clones were compared with those of other kinetoplastids and oligonucleotide primers were designed to be used in the polymerase chain reaction. A pair of these primers has been shown not only to be highly specific for L. mexicana complex isolates but can also be used to distinguish between L. (L.) mexicana and L. (L.) amazonensis isolates. These primers have been tested with water-lysed cultures, crude DNA extracts from human patients, potential host reservoirs, sandfly vectors and with cell pellets after isoenzyme characterization. The results of these tests indicate that the primers can be used specifically in the presence of excess host DNA originating from the majority of South American countries.