The Leishmania donovani complex is considered to be composed of 3 species; L. donovani, L. infantum and L. chagasi, although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum/L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0·21 to 0·87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

Plasmodium ookinetes are elongate, motile and invasive while inside the mosquito gut but promptly metamorphose into spherical immobile oocysts upon coming in contact with the basement membrane surrounding the midgut. There they begin a prolonged growth period characterized by massive DNA synthesis for the production of sporozoites. Living Plasmodium gallinaceum ookinetes attached avidly to the murine extracellular matrix proteins, laminin and collagen type IV. In ELISA-type assays, the main ookinete surface protein, Pgs28 was implicated as a mediator of parasite attachment to these basement membrane constituents. Laminin and collagen IV adhered to ookinete and oocyst lysates spotted onto nitrocellulose membranes. Receptor–ligand blot assays demonstrated that Pgs28 and an oocyst-specific antigen recognized by the mAb 10D6 interact with murine collagen IV and laminin. 10D6 antigen was also recognized by monospecific antiserum against the human epidermal growth factor receptor. Mosquito-derived laminin was incorporated into oocyst capsules of P. gallinaceum growing in Aedes aegypti. We hypothesize that contact with the mosquito basement membrane triggers the transformation of ookinetes into oocysts. Coalescence of basement membrane proteins onto the capsules masks developing oocysts from the mosquito's immune system and facilitates their prolonged extracellular development in the mosquito body cavity.

Midgut proteases contribute to the success or failure of Plasmodium infection of the mosquito. This paper examines the reciprocal effect of Plasmodium yoelii nigeriensis on midgut trypsin, chymotrypsin, aminopeptidase and carboxypeptidase in the mosquito Anopheles stephensi. The total protein ingested and the rate of protein digestion were unaffected by the parasite, but more protein was ingested at the first than the second bloodmeal. All peptidases were unaffected by the presence of the parasite during the first gonotrophic cycle, when ookinetes were penetrating the midgut. In the second gonotrophic cycle, trypsin and chymotrypsin were unaffected by growing oocysts, but aminopeptidase activity was reduced in the midguts of infected mosquitoes. Chymotrypsin activity was depressed and aminopeptidase activity elevated during the second gonotrophic cycle. Plasmodium infection has a negligible effect on bloodmeal digestion and does not limit the availability of the protein for egg production. The significance of changes in aminopeptidase activity when oocysts are present is discussed.

An important element in most approaches to the subject of parasitism is the consideration of environment. Parasites are set apart within animal ecology because they experience two environments, one the ‘external’ conditions and the other created by the living body of the host. As in any ecological system, external environmental conditions have a major influence on life history parameters: these conditions may be experienced directly by ‘off-host’ stages of a parasite or, to a greater or lesser extent, indirectly through the body of the host. However, uniquely in parasitic associations, the internal (host) environment has a dual influence on the physiological conditions encountered by parasites: first, the host buffers the external conditions (by homeostatic mechanisms moderating environmental fluctuations, by behavioural responses selecting appropriate habitat conditions, etc.), but second, the host creates a suite of hostile factors associated with immune defence. This living, reactive environment has no parallels elsewhere in free-living animal ecology: it has the characteristic of reacting specifically to kill the organisms within its boundaries.

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P. falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Malaria-immune human sera were tested for their ability to affect the infectivity of Plasmodium falciparum gametocytes to Anopheles stephensi mosquitoes. Transmission-reducing effects of the sera were associated with the presence of antibodies to the gamete surface protein, Pfs230. Enhancement of transmission, manifest as elevated numbers of oocysts relative to controls, was observed for a number of sera, but was not found to be associated with antibodies against Pfs230. These results confirm that Pfs230 is a possible candidate for inclusion in a transmission-blocking malaria vaccine.

Low temperatures affect the rate of growth, development and metabolism of parasites and when temperatures fall below 0°C may expose the parasite to the potentially lethal risk of freezing. Some parasites have mechanisms, such as diapause, which synchronise their life cycle with favourable seasons and the availability of hosts. Parasites of endothermic hosts are protected from low temperatures by the thermoregulatory abilities of their host. Free-living and off-host stages, however, may be exposed to subzero temperatures and both freezing-tolerant and freeze-avoiding strategies of cold hardiness are found. Parasites of ectothermic hosts may be exposed to subzero temperatures within their hosts. They can rely on the cold tolerance adaptations of their host or they may develop their own mechanisms. Exposure to low temperatures may occur within the carcass of the host and this may be of epidemiological significance if the parasite can be transmitted via the consumption of the carcass.

Irreversible pathological lesions were noted in the organs of mice infected with 1 of 3 rodent malaria species: Plasmodium chabaudi chabaudi, P. vinckei petteri and P. yoelii nigeriensis at different times during the course of the primary parasitaemia and long after microscopical clearance of the parasites. Moreover, similar lesions were also obtained when parasite levels were kept below 1%by subcurative drug treatment. The frequency and severity of the lesions correlated with the duration of the infection. Accumulation of tissue damage during chronic low-grade malaria infections has implications for the design of control measures.

Antibodies against variable antigens expressed on the surface of Plasmodium falciparum-infected erythrocytes are believed to be important for protection against malaria. A target for these antibodies is the P. falciparum erythrocyte membrane protein 1, PfEMP1, which is encoded by around 50 var genes and undergoes clonal variation. Using agglutination and mixed agglutination tests and flow cytometry to analyse the recognition of variant antigens on parasitized erythrocytes by plasma antibodies from individuals living in Daraweesh in eastern Sudan, an area of seasonal and unstable malaria transmission, we show that these antibodies recognize different variant antigens expressed by parasites of different genotype. Comparing the levels and acquisition of antibody to variant antigens in pairs of parasite isolates expressing different variant types, there is a correlation between the acquisition of antibodies to some combinations of variant antigens but not to others. These results indicate that (1) a single infection will induce the production of antibodies recognizing several variants of surface-expressed antigens, (2) the repertoire of variable antigens expressed by different parasites is overlapping and the degree of overlap differs between isolates, and (3) the expression of at least some variant antigens is genetically linked.

In order to investigate the phenomenon of age-related immunity to visceral leishmaniasis, a 1 year prospective survey was carried out on 5671 people in a Leishmania infantum focus in north-west Iran. The average incidence rate of infection since 1985 was 2·8%/year with all ages equally at risk. One in 13 infections in children led to visceral leishmaniasis (VL), and this ratio decreased significantly with age. Seroprevalence also dropped rapidly with age, suggesting that the same process may affect both clinical outcome and the humoral immune responses. Cell-mediated immunity was associated with a reduction in the seroconversion rate and an increase in the serorecovery rate. Even amongst people with no detectable cell-mediated immunity to Leishmania, the seroconversion rate decreased and the serorecovery rate increased with age. All current VL patients had a negative leishmanin skin test response. Hence, adults may develop protection against L. infantum through 2 processes, 1 dependent and 1 independent of acquired cell-mediated immunity.

While anaemia has long been recognized as a consequence of acute infections with malaria, the relative contributions of direct erythrocyte destruction by parasites, destruction of uninfected erythrocytes and changes in erythropoiesis have been unclear. Fitting of parasitaemia and anaemia data from neurosyphilis patients undergoing malaria therapy to a mathematical model shows that in these patients, an average of 8·5 erythrocytes were destroyed in addition to each erythrocyte observed to become parasitized. The model also showed that dyserythropoiesis plays an insignificant role in the resulting anaemia. The anaemia occurs before a substantial antibody response to parasites or erythrocytes could be generated. We postulate that uninfected erythrocyte destruction occurs through phagocytosis of erythrocytes bound to merozoites killed as a result of the accompanying malaria paroxysms.

Artemisinin (QHS) and its derivatives are new antimalarials which are effective against Plasmodium falciparum parasites resistant to chloroquine (CQ). As these drugs are introduced it is imperative that resistance is monitored. In this paper we demonstrate that the inoculum size used in in vitro testing influences the measured in vitro susceptibility to QHS and its derivative dihydroartemisinin (DHA) and to mefloquine (MEF) and CQ over the range of parasitaemias routinely used in testing with the WHO in vitro microtest. An increase in parasitaemia and/or haematocrit was accompanied by a decrease in the measured sensitivity of 2 laboratory lines. In the context of a field study testing in vitro susceptibility of parasite isolates from patients with uncomplicated malaria in Fajara, The Gambia we demonstrate that failure to control for inoculum size significantly overestimates the level of resistance to QHS and DHA as well as MEF, halofantrine (HAL) and quinine (QUIN). When controlling for the inoculum effect, cross-resistance was observed between QHS, MEF and HAL suggesting the presence of a multidrug resistance-like mechanism. These studies underline the importance of inoculum size in in vitro susceptibility testing.

A cross-sectional sero-epidemiological study was performed in Magoda, Tanzania, an area where malaria is holoendemic. Blood samples were collected from children (1–4 years) and tested for IgG antibody reactivity against 2 recombinant protein fragments of Plasmodium falciparum Rhoptry-Associated Protein-1 (rRAP-1). The data were related to the prevalence of malarial disease and single P. falciparum or mixed Plasmodium infections. Fever ([ges ]37·5 °C) in combination with parasite densities >5000/μl were used to distinguish between children with asymptomatic malaria infections and those with acute clinical disease. Furthermore, C-reactive protein (CRP) was applied as a surrogate marker of malaria morbidity. The prevalence of Plasmodium infections was 96·0%. Eleven children were defined as clinical malaria cases, all with single P. falciparum infections. The density of P. falciparum was significantly lower in children with mixed Plasmodium infections compared to those with single P. falciparum infections. Children with asymptomatic P. falciparum infections had higher IgG reactivities to rRAP-1, compared to IgG reactivities of children with malarial disease. Children with mixed Plasmodium infections generally showed elevated IgG reactivity to rRAP-1, when compared to children with single P. falciparum infections. The possible relationship between mixed species infections, clinical outcome of the disease and antibody responses to RAP-1 is discussed.

Proliferative kidney disease (PKD), a condition associated with high mortality in salmonid fish, represents an abnormal immune response to the presence of an enigmatic myxozoan, which has been designated simply as PKX organism because its generic and specific status are obscure. Phylogenetic analyses of partial sequences of the 18S rDNA of PKX and of myxozoan parasites infecting the bryozoans Cristatella mucedo, Pectinatella magnifica and Plumatella rugosa, including the previously named Tetracapsula bryozoides from C. mucedo, showed that these taxa represent a distinct clade that diverged early in the evolution of the Myxozoa before the radiation of the other known myxozoan genera. A common feature of the myxozoans in this clade may be the electron-dense sporoplasmosomes with a lucent bar-like structure, which occur in T. bryozoides and PKX but not in the myxozoans belonging to the established orders Bivalvulida and Multivalvulida. Variation of 0·5–1·1% was found among the PKX 18S rDNA sequences obtained from fish from North America and Europe. The 18S rDNA sequence for T. bryozoides showed that it is a distinct taxon, not closely related to PKX but some sequences from myxozoans infecting 2 of the bryozoan species were so similar to those of PKX as to be indistinguishable. Other sequences from the new myxozoans in bryozoans at first appeared distinct from PKX in a maximum likelihood tree but, when analysed further, were also found to be phylogenetically indistinguishable from PKX. We propose that at least some variants of these new myxozoans from bryozoans are able to infect and multiply in salmonid fish, in which they stimulate the immune reaction and cause PKD but are unable to form mature spores to complete their development.

This study focuses on the effect of acidic water and aqueous aluminium on the monogenean ectoparasite Gyrodactylus salaris, infecting Atlantic salmon (Salmo salar) parr. G. salaris-infected salmon were exposed to various combinations of acidity and aluminium concentrations. The most pronounced effect was the elimination of parasites after 4 days when 202 μg Al/l was added to the water. The effect of aluminium was concentration dependent, but was relatively independent of pH (5·2, 5·6 and 5·9). At the lowest pH of 5·0 the effect of aluminium was enhanced. Acidic aluminium-poor water had no or minor effects on the G. salaris infections except at pH 5·0 where all parasites were eliminated within 9 days. The G. salaris populations increased exponentially in untreated control water. The results show for the first time that aqueous aluminium can, to a limited extent, have a positive effect on fish health. This study emphasizes that basic knowledge about abiotic environmental factors is of importance in order to understand the population dynamics, range extension and dispersal of ectoparasites such as G. salaris. Finally, our results suggest that aluminium treatment could form an effective disinfection method against ectoparasites in hatcheries and laboratories, as well as complementing the controversial rotenone treatments used against natural populations of G. salaris.

This study examines polymorphisms in 2 genes (pfmdr1 and cg2), which have been associated with resistance to chloroquine in Plasmodium falciparum, to determine their value as predictors of resistance status. Among field samples from children in Zaria, northern Nigeria, the Tyr-86 polymorphism in pfmdr1 and Ala-281 and the Dd2κ repeat of cg2, were significantly associated. In 8 samples classified resistant by the micro-in vitro test, or, where this failed, by in vivo trial, 7 showed the cg2 Dd2 type κ repeat, and 6 of these had both the Ala-281 allele and the pfmdr1 Tyr-86 allele. In 26 chloroquine-sensitive samples, none had this combination of 3 polymorphisms (P = 0·00002). This indicates 75% sensitivity and 100% specificity in detection of resistance and shows a positive predictive value for resistant infections of 100%. The negative predictive value, because of sensitivity less than 100%, would depend on the prevalence of resistance. Where prevalence of resistance is approx. 21% as in Zaria, the negative predictive value would be 94%, while in Gabon, with a prevalence of ca 73% it would be 60%. The use of (cg2: Ala-281, Dd2κ. pfmdr1: Tyr-86) genotype detection as a predictive epidemiological tool to examine the distribution of chloroquine-resistance in parts of Africa is therefore possible. The sensitivity of detection of resistant strains still requires improvement.

The ability of certain species of parasitic nematodes to survive desiccation for considerable periods is a fascinating example of adaptation to the demands of fluctuating environments that occasionally can become extreme and life threatening. Behavioural and morphological adaptations associated with desiccation survival serve primarily to reduce the rate of drying, either to prolong the time taken for the nematode's water content to reach lethal low levels or, in true anhydrobiotes, to enable the structural and biochemical changes required for long-term survival to take place. Examples of these adaptations are reviewed, together with information on the factors involved in rehydration that ensure successful exit from the dormant state. Information on desiccation survival is central to effective management and control options for parasitic nematodes. It is also required to assess the feasibility of enhancing the longevity of commercial formulations of entomopathogenic nematodes, both before and after application; current research and future prospects for enhancing survival of these bio-insecticides are discussed.

The complete sequence of the 18S small subunit (SSU) ribosomal DNA of Hammondia hammondi and Sarcocystis mucosa was obtained and compared to SSU rDNA sequences of Neospora caninum, Toxoplasma gondii, Besnoitia besnoiti, 2 species of Frenkelia, 3 species of Isospora, and 13 species of Sarcocystis. Analyses showed that H. hammondi and T. gondii are monophyletic and that these taxa shared a common ancestor with N. caninum and B. besnoiti. The weight of evidence shows that S. mucosa, S. neurona, and Frenkelia species form a clade thereby supporting the conclusion that Sarcocystis is paraphyletic.

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.