We describe a novel procedure for the immunofluorescent investigation of Plasmodium falciparum. This has allowed us to visualize clearly microtubular structures and their changing conformation through the erythrocytic cell-cycle, to the stage of cytodifferentiation leading to merozoite release. The images of spindle development we observed, together with an analysis of nuclear body numbers in large numbers of parasites, indicate that there is an apparent asynchrony in chromosomal multiplication within a single parasite. Using antibodies specific for post-translational modification of α- tubulin, we also demonstrate that the C-terminal tyrosine-containing epitope of P. falciparum α-tubulin I is similar to that of other organisms. Lysine-40 in the same molecule, a target for highly specific in vivo acetylation in some organisms, is unmodified in the blood stages we examined here. After in vitro acetylation of this residue, however, the epitope to which it contributes was recognized by antibody, showing that the conformation of this part of the molecule is also conserved, despite a lack of primary sequence homology immediately downstream of the target lysine residue.

Homologues of the mammalian multidrug resistance gene have been identified in isolates and clones of Plasmodium falciparum and designated pfmdr1 and pfmdr2. Mutations in pfmdr1 have been associated with chloroquine resistance but confirmation could not be obtained in a genetic cross. We have examined the copy number and expression of pfmdr1 and pfmdr2 in chloroquine-sensitive and -resistant P. falciparum and have found no relationship between the copy number of either gene and chloroquine resistance. However, a marked correlation was seen between levels of mRNA transcribed for each gene and chloroquine resistance. Two transcripts of pfmdr1 were detected, and in the asexual blood cycle an 8 kb transcript appeared first, followed by the appearance of a 7 kb species.

The diversity in a 925 bp portion of the Plasmodium vivax MSA1 gene in isolates from the Philippines, China, the Solomon Islands and Papua New Guinea was investigated. A total of 74 base pair changes was found in the amplified fragment from 18 isolates. Most of these changes were single or double base pair substitutions. In several regions, these point changes were tightly linked with one set always present or always absent in the different isolates. Seven such blocks were identified. These blocks were present in different combinations in the different isolates indicating that extensive intragenic recombination has occurred.

Oocyst prevalence and intensity have been recorded in 349 laboratory infections of Anopheles stephensi with Plasmodium berghei. Intensity and prevalence of infection are shown to be predictably related. The structure and heterogeneity in the infections has been analysed with the objective of describing the biological mechanisms by which the observed negative binomial oocyst distributions are generated. The analysis has revealed that the most likely processes lie within the population dynamic events of malaria within the mosquito, namely gametogenesis, fertilization and mortality. The distribution is similar in all Plasmodium – mosquito combinations examined so far, whether they are of laboratory (P. gallinaceum in Aedes aegypti) or field (P. vivax in An. albimanus and P. falciparum in An. gambiae s.l. and An. funestus) origin. Further we conclude that there is competition between parasites in the vector. Oocyst frequency distribution analysis shows that under natural conditions of transmission intensity, and even under the best laboratory conditions, significant numbers (> 10%) of fully susceptible mosquitoes will not be infected under conditions where the mean infection is as high as 250 oocysts. Failure to infect is not therefore an absolute indicator of refractoriness. In assessing transmission data it is shown that sample sizes should not be less than 50, and ideally 100 mosquitoes, if reliable data are to be obtained. In the field it is suggested that difficulties in determining the low natural intensity of oocyst infections indicate that prevalence estimates are a useful and accessible parameter to measure. In determining the impact of transmission blocking mechanisms we predict that under conditions where high oocyst intensities prevail, large reductions in intensity will be required before a reduction in prevalence can be expected i.e. here it will be necessary to measure intensity of infection. Conversely, under conditions where low oocyst intensities prevail, a rapid reduction in prevalence will occur with little concurrent reduction in intensity i.e. prevalence determination will be the more sensitive estimate.

The causal prophylactic activity of the novel hydroxynaphthoquinone, S66C80, was assessed against the exo-erythrocytic (EE) stages of Plasmodium berghei cultured in the human hepatoma cell line, HepG2. 566C80 was found to be highly active as an inhibitor of EE development and was more active than the established causal prophylactic pyrimethamine. A 566C80 concentration of 1·85 × 10−9 M, added 3 h after sporozoite invasion, reduced the numbers of EE forms visible at 48 h by 50%, while the equivalent concentration of pyrimethamine was 1·95 × 10−8 M.

Giardia lamblia localize and multiply in the small intestine and may cause acute or chronic diarrhoea with malabsorption of fat, protein and other nutrients. Abnormal pancreatic function has been documented in giardiasis and trophozoites directly inhibit pancreatic lipase activity in vitro. The aim of this study was to examine the effect of Giardia trophozoites on pancreatic trypsin, chymotrypsin and amylase activity in vitro. Axenically cultured Giardia trophozoites (Portland-1 stock) were incubated with a range of concentrations of trypsin, chymotrypsin and amylase and enzyme activity assayed over time. Tryptic activity was decreased after incubation with Giardia trophozoites. This reduction was time dependent and linear over the incubation period of 2 h. At a trypsin concentration of 18 BAEE units/ml, there was a 35.5±4% reduction in enzyme activity after 2 h compared to controls. The total amount of activity lost was proportional to the initial trypsin concentration up to 185 BAEE units/ml. At this initial concentration, the activity was reduced by 46.5±3 units/ml after 2 h. Above this concentration, little further loss of enzyme activity was seen. To investigate the nature and specificity of this effect, similar experiments were conducted using killed trophozoites and with a related protozoan. Trichomonas vaginalis. No loss of enzyme activity was evident. Media previously incubated for 2 h with trophozoites did not diminish tryptic activity. Trophozoites had no effect on chymotrypsin or amylase activities over the range of concentrations tested. Giardia trophozoites directly diminish pancreatic tryptic activity in vitro, by an active process which appears specific and not mediated via a secretory product. This mechanism may enhance parasite survival in the small intestine by allowing trophozoites to escape destruction by luminal proteases and may also contribute to malabsorption.

We have investigated the effect of qinghaosu (QHS, artemisinin) and its derivatives on Leishmania major replication in vitro and on the disease development in mice infected with L. major. Artemisinin is effective against promastigotes in vitro, with an ED 50 (50% effective dose) at 7.5 × 10−7 m. Both artemisinin and artemether are leishmanicidal for amastigotes in infected murine macrophages in vitro, with ED50 at 3 × 10−5 m and 3 × 10−6 m respectively. These compounds have no effect on the viability of macrophages or on the phytohaemaglutinin-induced proliferation of normal spleen cells, even at 10−4 m. BALB/c mice infected in the footpad with L. major developed significantly smaller lesions and parasite loads when treated with the compounds. Intra-lesion injection of the compounds was the most effective route. The intramuscular and oral routes were also effective; however, intravenous injection with artesunate was not effective.

Ultra-violet irradiated larvae of Schistosoma mansoni stimulate high levels of resistance to challenge infection in experimental animals. In the experiments presented here, the binding patterns of antisera specific for the cercarial glycocalyx, and of various lectins, demonstrate that u.v. irradiation causes a pronounced modification of the carbohydrate antigens expressed at the surface of cercariae and newly transformed schistosomula.These alterations were dependent on the irradiation dose, and on the batch of cercariae used in each experiment. Our results strongly suggest that the changes in carbohydrate antigens consequent upon u.v. irradiation may be important in generating the enhanced immunogenicity of irradiated cercariae.

The polymerase chain reaction (PCR) has been used to amplify in vitro a semi-conserved region of a gene encoding an Mr 68–72000 surface antigen of Giardia intestinalis trophozoites. Using primers specific for conserved nucleotide sequences identified within the promoter-distal portion of two homologous genes (tsp11 and tsa417) cloned previously from the G. intestinalis isolates Ad-1 (from Australia) and WB (from Afghanistan), a single PCR-amplified DNA fragment of the expected size (0·52 kilobases) was obtained in high yield from either purified DNA or whole trophozoites of the Ad-1 isolate and from every 1 of 9 other axenic G. intestinalis isolates belonging to genetic groups I and II (defined previously on the basis of allozyme electrophoresis data—Andrews et al. 1989). Discernible product was recovered from as few as 2–4 trophozoites. In contrast, 6 G. intestinalis isolates that were assigned by allozymic analysis to genetic groups III/IV yielded small amounts of a 0·37-kilobase (kb) amplification product (with evidence in some samples of an additional 0·4 or 0·18 kb fragment) but no 0·52 kb product. Two animal-derived isolates of G. duodenalis (one from an Australian native rodent, Notomys alexis, the other from a domestic cat) also yielded a single 0·37 kb PCR-amplified fragment, whereas an isolate from another cat produced a 0·34 kb fragment. No product was recovered from G. muris, a morphologically distinct species of Giardia. The results demonstrate that different genotypes of G. duodenalis can be distinguished using this assay and that it is diagnostic for isolates belonging to two major clusters (groups I/II and III/IV) of G. intestinalis. The amplified DNA segment appears to be relatively conserved among group I and group II isolates of G. intestinalis. A related but clearly distinct sequence seems to be conserved among group III/IV isolates of G. intestinalis and some isolates of G. duodenalis.

A recombinant baculovirus-expressed hybrid protein containing epitopes for the C-terminal fragment of the Plasmodium falciparum precursor to the major merozoite surface antigens (PMMSA) and the tetrapeptide repeats of the circumsporozoite protein (CSP) was assessed for its immunogenicity. Murine MHC-II restriction of the antibody response to the CSP repeats was not overcome by the PMMSA component, the response to which showed no restriction. In an adjuvant trial the highest antibody titres in rabbits to both components of the hybrid were obtained using Freund's adjuvant. Lack of a boosting antibody response to the CSP repeats appeared to be linked to the conformation of the PMMSA component. Formulation of the hybrid protein into Iscoms gave antibody titres of only short duration to both components.

As animals we have been a remarkably successful species; but also as animals we are vulnerable to environmental, in particular climate change. Such change is accelerating as a result of human activity, and global warming may already be taking place. Although we can foresee the trends, we cannot yet be specific about the results. Change usually proceeds by steps rather than gradients. But warming would probably include new risks to human health and contribute to an increase in human displacement. Of course climate change is only one among other complex problems facing human society, but it is closely related to them all, including population increase, environmental degradation and loss of biodiversity. We cannot prevent global warming but we can anticipate and mitigate some of its worst effects. Peoples and governments still need persuading of the need for action and of the magnitude of the issue at stake.

A Neospora sp. was isolated from the brains of two aborted bovine foetuses and grown continuously in vitro in bovine cell cultures. A comparison of the antigenic reactivity of in vitro cultivated tachyzoites with polyclonal antisera to Neospora caninum, Hammondia hammondi or Toxoplasma gondii revealed that the bovine protozoal isolates were similar to N. caninum and antigenically distinct from T. gondii. Tachyzoites of both bovine isolates had similar ultrastructural features, including an apical polar ring, conoid, electron-dense rhoptries and micronemes. The orientation of the micronemes, presence of micropores and a large number of electron-dense granules in the posterior portion of the bovine isolate tachyzoites differed from previous descriptions of N. caninum in vivo. Tachyzoites of the bovine isolates were ultrastructurally more similar to in vitro cultivated N. caninum tachyzoites than to tachyzoites of T. gondii or H. hammondi. The antigenic and ultrastructural similarities between N. caninum and the protozoal parasites isolated from aborted bovine foetuses in this study support the proposition that these parasites belong to the genus Neospora.

Protocols for in vitro excystation of oocysts of Cryptosporidium parvum, including different chemical pre-incubation steps, were compared to examine some of the biochemical triggers involved in excystation and to define an in vitro excystation protocol of a reproducibly high efficiency. Pre-incubation steps which increased the permeability of the oocysts were found to enhance excystation dynamics and pre-treatment of oocysts with saliva was found to decrease the permeability and reduce excystation. Although excystation was maximal after incubation for 4 h, sporozoites tended to lyse over this period, and maximum sporozoite recovery occurred after 30 min. The results obtained are discussed in relation to excystation protocols adopted by different research groups and a number of recommendations are given for in vitro excystation of C. parvum oocysts.

An incomplete ring of electric nets was placed around uninfected cattle and cattle infected with Trypanosoma congolense. The numbers of fed and unfed Glossina pallidipes caught on the nets were used to estimate the attractiveness of infected and uninfected cattle to tsetse, and the feeding success of tsetse on the cattle. There was no difference in the attractiveness of infected and uninfected cattle to G. pallidipes. However, the feeding success of G. pallidipes on infected cattle was 75 % greater than on uninfected cattle. This suggests that certain effects of T. congolense on cattle behaviour or physiology act to increase the probability of transmission of the parasite by increasing the feeding success of the vector. The nature of the effects of T. congolense on cattle which caused this result is unknown, but several possibilities are discussed.

Since pigs are important in the zoonotic transmission of schistosomiasis japonica in China, a veterinary vaccine might contribute to the control of the disease in humans. Pigs were immunized with three doses each of 10000 cercariae of Schistosoma japonicum attenuated with ultraviolet light (400 μWatt·min/cm2). The experiment was performed with portable irradiation equipment in a rural area of the Hubei Province (P.R. China). A challenge infection of 1000 untreated cercariae was given 2·5 or 6 months after the last immunization, and age-matched naive pigs were challenged as a control. Immunized pigs developed about 90% resistance against the challenge. The liver egg load of these animals was reduced by over 90%. Less than 0·01 % of the immunizing cercariae developed to adult parasites and the vaccination had no apparent adverse influence on the pigs' health.

The snail Bulinus nasutus 1214 possesses a potent haemagglutinin (end-point titre with human erythrocytes, 2−18) in its cell-free haemolymph which also binds to the miracidia (but not other larvae) of the incompatible parasite Schistosoma margrebowiei. We have purified a protein possessing this haemagglutinating property from the plasma of this snail. The native Mr of this protein was estimated by SDS polyacrylamide gel electrophoresis to be 210 kDa; under denaturing conditions in a 7.5% PAGE gel it ran as a major band of 135 kDa. Proteins of similar Mr were also found in the haemolymph of 16 other Bulinus spp. (the major intermediate hosts of human and veterinary schistosomiasis in Africa) although the plasma of none of these agglutinated human erythrocytes. Nonetheless, Cleveland mapping of the Mr 135 kDa bands from these different Bulinus spp. revealed 4 identical major peptide fragments (30, 28, 19 and 16 kDa) in each, thus demonstrating a similarity in the primary structure of these plasma proteins. Antisera from Balb/C mice immunized with the 135 kDa polypeptide from Bulinus truncatus 1521 cross-reacted in Western blots with the 135 kDa band of other members of the same truncatus/tropicus species complex but not with species from the africanus or forskalii species groups.

The atmosphere plays a key role in plant disease, but only recently has it become understood that atmospheric pollutants can influence the response of plants to attack by pests and pathogens. This paper reviews the evidence for this phenomenon, considering impacts of sulphur dioxide, nitrogen dioxide and ozone, mainly on fungal pathogens and aphid pests. Field observations in polluted areas have indicated changes in abundance of pests and pathogens and in some cases a causal link has been demonstrated in controlled experiments. A major study is described in which consistent marked positive impacts of SO2 and NO2 have been shown on a range of British agricultural aphid pests, using four different approaches: fumigations, nitration studies, exposure along air pollution gradients and a nation-wide field survey. Ozone, in contrast, produces a more complex range of responses. These effects are apparently mediated via chemical changes in the plant. Fungal pathogens show both positive and negative responses to air pollutants. A study is described in which these opposite responses in two different fungal species were observed in a field SO2–fumigation system and confirmed in controlled laboratory fumigations. Models are presented to describe the complex pathways by which air pollutants could influence host plant performance via impacts on pests and pathogens.

In order to estimate the value of population genetics for both the taxonomy of trypanosomes belonging to the species Trypanosoma brucei and a better understanding of Human African Trypanosomiasis (HAT), we undertook a cellulose acetate electrophoresis isoenzyme study involving 55 stocks isolated from man and animals in Congo, Zaire and Cameroun. Out of the 24 loci surveyed, 15 exhibited variability, which made it possible to delimit 23 zymodemes, divided into 2 groups. The first group equated to the classical subspecies Trypanosoma brucei gambiense, while the second corresponded to the classical subspecies Trypanosoma brucei brucei. These results broadly agree with the current taxonomy, and are corroborated by RFLP analysis of kDNA. Statistical analysis indicates a basically clonal reproduction system of the trypanosomes in the area studied; the zymodemes are equivalent to natural clones (or a family of closely related clones), stable in space and time. Epidemiological hypotheses are proposed according to the geographic distribution of the clones in this area.