The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78–88%) 0–135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93–100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs.

The role of interspecific interactions in the structure of gastrointestinal helminth communities has been at the core of most research in parasite community ecology, yet there is no consensus regarding their general importance. There have been two different approaches to the study of species interactions in helminths. The first one consists of measuring the responses of helminth species in concomitant infections, preferably in laboratory experiments. Any change in numbers of parasite individuals or in their use of niche space, compared with what is observed in single infections, provides solid evidence that the species are interacting. The second approach can only provide indirect, circumstantial evidence. It consists in contrasting observed patterns either in the distribution of species richness of infracommunities from wild hosts, in their species composition, or in pairwise associations between helminth species among infracommunities, with the random patterns predicted by appropriate null models. In many cases, observed patterns do not depart from predicted ones; when they do, alternative explanations are usually as plausible as invoking the effect of interactions among helminth species. The present evidence suggests that the role of species interactions in helminth community structure is often negligible, but that it must always be evaluated on a case-by-case basis.

Two studies were carried out to determine the effect of the rodent malaria Plasmodium yoelii nigeriensis on the blood feeding success of Anopheles stephensi. Initially, pairs of mice with similar packed cell volume (PCV) (measured by haematocrit) were selected. Following infection of one of the pair its PCV gradually fell. At various times post-infection, a comparison was made of the bloodmeal size (haemoglobin content) of mosquitoes feeding on these mice. The bloodmeal sizes increased with parasite-induced fall in PCV down to a haematocrit of 43–44%, which occurred approximately 48 h post-infection. Bloodmeals were significantly reduced, however, when mosquitoes fed on mice with higher parasitaemias and a haematocrit of 15–35%. Thus, at early stages of infection, mosquitoes ingested a bloodmeal significantly greater than did the mosquitoes feeding on the control mice. However, mosquitoes were not able to compensate for severe infection-associated anaemia. To compensate for variation due to innate differences in the mice, a second experiment was performed. Mosquitoes were fed on the same mice before (control) and after infection. Again, the bloodmeal size increased with decreasing PCV down to haematocrits of 42–45%, but declined thereafter. In this host–parasite–vector system, haematocrits that maximized erythrocyte intake were produced when gametocytes, capable of exflagellation, were present.

Fifty-two Leishmania strains, obtained from human patients and dogs in a visceral leishmaniasis focus in Sudan, were characterized by isoenzyme electrophoresis (15 enzymes). The phylogenetic analysis showed that the 7 Leishmania zymodemes obtained hold ancestral positions on the phylogenetic tree, supporting the hypothesis of an East African origin of visceral leishmaniasis.

Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem. This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation. As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT). The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13. All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60–70% is accounted for by coding sequence. All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance. These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle.

The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is describedNucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers TSU011991 and TSU011992.. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

A new mathematical model of Plasmodium falciparum asexual parasitaemia is formulated and fitted to 35 malaria therapy cases making a spontaneous recovery after primary inoculation. Observed and simulated case-histories are compared with respect to 9 descriptive statistics. The simulated courses of parasitaemia are more realistic than any previously published. The model uses a discrete time-step of 2 days. Its realistic behaviour was achieved by the following combination of features (i) intra-clonal antigenic variation, (ii) large variations of the variants' baseline growth rate, depending on both variant and case, (iii) innate autoregulation of the asexual parasite density, variable among cases, (iv) acquired variant-specific immunity and (v) acquired variant-transcending immunity, variable among cases. Aspects of the model's internal behaviour, concerning variant dynamics, as well as the respective contributions of the three control mechanisms (iii) – (v), are displayed. Some implications for pathogenesis and control are discussed.

A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. falciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (AGC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2.5×10−9 mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and anti-malarial drug therapy by demonstrating that even with low mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.

The establishment of Leishmania in mammals depends on the transformation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The appearance of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48h but probable precursors of these organelles were detected at 12h p.i. The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12–24h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24h. The data indicate that at 48h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The appearance of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host MHC molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

Using a combination of morphological, life-cycle and molecular data, we describe a new apicomplexan parasite Eimeria telekii n.sp. from a striped grass mouse Lemniscomys striatus captured in Kenya. Oocysts are oval to spherical or ellipsoidal, 20·4×15·7 (15·5–25·0×12·0–20·0)μm with a colourless, smooth and bilayered wall. Sporocysts are ellipsoidal, 11·2×7·8 (10·0–12·0×7·0–9·0)μm with a small Stieda body and granular sporocyst residuum and contain 2 elongated, banana-shaped sporozoites with a single refractile body. Life-cycle, pathogenicity and host specificity of this parasite were studied in laboratory-bred Lemniscomys barbarus and BALB/c mice. Two asexual stages and the sexual phase took place within the enterocytes of the caecum and colon of L. barbarus but not in inoculated BALB/c mice. An infectious dose of 5000 oocysts caused severe clinical illness and mortality in 2/2 (100%) L. barbarus. Phylogenetic analysis of the small subunit rRNA gene of E. telekii and members of the genera Eimeria, Cyclospora and Isospora placed E. telekii within the eimerian rodent clade.The nucleotide sequence was deposited in the GenBankTM under the accession number AF246717.

On the basis of partial amplification of a cloned fragment of kDNA of Leishmania infantum which is specific for this species, we developed a PCR–ELISA technique which avoids the problems associated with classical diagnostic techniques. This technique was tested on 33 L. infantum strains from 19 different zymodemes, which were recognized equally. It was also used on human and canine clinical samples. PCR–ELISA has a higher sensitivity than the other techniques used (IFAT, parasite cultures, optical microscopy of stained samples) and permits detection of a minimum of 0.1 promastigotes or 1 fg of genomic DNA. PCR–ELISA can be used to diagnose human cutaneous leishmaniasis using material obtained by scraping the lesion margin, and human visceral leishmaniasis in HIV(+) individuals and canine leishmaniasis with peripheral blood samples. The presence of L. infantum in dogs with low antibody titres with IFAT technique (20 and 40) was demonstrated indicating that seroprevalence data from epidemiological studies underestimate the true rates of infection.

Protozoan parasites of the Leishmania donovani complex (L. donovani, L. infantum/L. chagasi) are causative agents of visceral leishmaniasis. To understand phylogeny and taxonomy within this group better we have developed 2 new polymerase chain reaction-linked restriction fragment length polymorphism (PCR–RFLP) analyses of the major surface protease (msp or gp63) intergenic (ITG) regions. We have named this approach msp intergenic region RFLP typing (MIRT). One intergenic region lies between the constitutive msp (mspC) and stationary phase msp (mspS4) genes (ITG/CS) and the other between multicopy logarithmic phase msp (mspL) genes (ITG/L). The markers generated robust and congruent phylogenies, identifying 5 genetic clusters within L. donovani. One cluster was synonymous with L. infantum (L. chagasi); clusters strongly correlated with isoenzyme typing and some with geographical origin. These genetic groups may be important for epidemiological and clinical studies. The congruence of the groups identified indicates suitability of these genomic targets for taxonomic studies. Furthermore, subgroups of L. donovani were of equivalent phylogenetic status to L. infantum. No evidence was found to support the existence of L. archibaldi. It is likely to be necessary in future to re-evaluate the taxonomic status of L. donovani or of L. infantum, as discrete species.

The changes in prevalence and abundance of the three species of metacercariae in the humour of the eyes of perch Perca fluviatilis in Slapton Ley, Devon, have been monitored over a period of 29 years. Earlier studies had revealed that Diplostomum gasterostei was originally the sole occupant of this niche, but Tylodelphys clavata colonised in 1973 and T. podicipina in 1976. A decline in the number of perch with heavy infections of D. gasterostei was significantly negatively correlated with population abundance of T. clavata and a decline in recruitment rate of D. gasterostei coincided with the population increase in T. podicipina over the period 1976–1979. It was suggested that the decline in population size of D. gasterostei was due to inter-specific competition, but this hypothesis could not be tested experimentally. Subsequent investigations, reported here, confirmed the decline when the data set was extended to 1985. A severe decline in the perch population over the winter of 1984/1985 resulted in the disappearance of D. gasterostei and T. podicipina and this was followed by a slow recovery from 1990 onwards. This natural experiment provided an opportunity to test the hypothesis. Only T. clavata survived throughout the perch crash and the population continued at pre-crash levels up to 1999. Its congener T. podicipina did not re-appear until 1994 and was probably a re-introduction: it did not attain pre-crash levels until 1999. It is likely that D. gasterostei survived the crash as it re-appeared in 1991, but was confined to young of the year fish and barely approached pre-crash levels even in 1999. Its continual low levels cannot be explained by changes in the lake or in densities of snail intermediate or bird definitive hosts. New data revealed that the suspensory ligaments of the eye were the preferred site of all three species and that the eye was partitioned out between them. The data from the post-crash period do not refute but rather confirm earlier conclusions that inter-specific competition is responsible for the decline in D. gasterostei and this remains the preferred hypothesis.

Toxoplasma gondii has a unique enzyme, a NTPase, which has a wide specificity toward NTP. In the present study, we produced a monoclonal antibody (IgG1, 6C6) against the enzyme which could recognize NTPase isozymes among several strains of T. gondii. Three avirulent strains of T. gondii, ME49, Beverley and Nakayama, were found to have 1 NTPase (63kDa, pI 6·0), while a virulent strain RH and an avirulent strain Fukaya had 2 isozymes (63kDa) with different pIs (pIs 6·0 and 6·5 for the former, and pIs 6·2 and 6·4 for the latter, respectively), suggesting that this monoclonal antibody recognizes a common epitope of NTPase among T. gondii strains. Furthermore, 6C6 could inhibit NTPase activity in the presence of dithiothreitol in a dose-dependent manner, and immuno-EM study of NTPase revealed that this molecule is located on the surface membrane of T. gondii tachyzoites. When Vero cells were co-cultured with tachyzoites pre-treated with 6C6, the number of infected cells significantly decreased, suggesting that 6C6 inhibits invasion of the parasites to host cells. These data suggest that the molecule recognized by 6C6 might be considered a potential candidate antigen for vaccines against T. gondii tachyzoites.

It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT–PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms.Nucleotide sequence data reported in this paper are available in the EMBL, GenbankTM and DDJB databases under the acession numbers AF207904 and AF207905. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

The gp63 encoding genes were characterized by PCR–RFLP in 35 isolates representative of the Leishmania donovani complex (L. infantum, L. donovani, L. archibaldi and L. chagasi), with special attention to Mediterranean L. infantum from different geographical origins, and in separate groups from Old World Leishmania (L. major, L. tropica and L. aethiopica). The aim was to evaluate how the possible selective pressure by the host on these important surface proteins would influence structuring of our sample. Comparison was carried out with the structure obtained (i) from reported isoenzyme data, characters supposed to vary neutrally, and (ii) from PCR–RFLP analysis of gp63 inter-genic regions, containing non-translated spacers and regulatory genes. Polymorphism within the gp63-encoding region, was much higher than in gp63 inter-genic regions. In the gp63 intra-genic dendrogram, the 4 species of L. donovani complex were discriminated and quite distinct from outgroups. Within L. infantum, geographical structuring was observed and did not overlap with the structure built-up from isoenzymes and inter-genic data. These results support the idea of a strong host-selection on gp63, at vector level but most of all at vertebrate (human or dog) immunological level. Furthermore, they illustrate how the nature of genetic characters may influence the perception of population structuring.

Intrapredatorus barri, a microsporidian parasite of the predator mosquito Culex fuscanus Wiedemann has been shown to be heterosporous. In many features this species has been reported to be similar to Amblyospora trinus, another microsporidian parasite of a different predator mosquito which was thus proposed for reassignation to the genus Intrapredatorus. In this report small subunit rDNA has been sequenced from I. barri and phylogenetic analysis of 42 microsporidia sequences has been performed.Nucleotide sequence data reported in this paper are available in the Gen Bank™ database under the accession number AY013359. The molecular data show that I. barri can not be a member of the genus Amblyospora. Furthermore, the phylogenetic analysis shows, with high confidence, that the Amblyospora spp. sequences in the present study are not monophyletic.

In a 3-year study, we examined landscape features (aspect, slope, sun exposure, canopy cover, type of ground cover, and nearest water source) that were potentially related to prevalence of infection with Plasmodium mexicanum in fence lizards (Sceloporus occidentalis) within a 4.5 ha study area in northern California, USA. Logistic regression analysis showed that ground cover type was the primary mediator of the probability of P. mexicanum infection. Infected lizards were captured more often in rock and/or leaf litter locations than in grassy ones. In another experiment, the study area was divided into 9 sites (0.07–0.33 ha), and infection prevalence was calculated for each. Three sites with high (>30%) infection prevalence had significantly more rocky outcrops and leaf litter than those with low (<20%) or moderate (20–30%) infection prevalence (N = 3 sites each). We conclude that lizard site selection may influence the probability of exposure to infected vectors and thus the likelihood of P. mexicanum infection. We also demonstrate that studies at different spatial scales may be required to understand fully the relationship between landscape features and parasite distribution.

As shown by RFLP analysis, there is a high variability in the β-tubulin gene region of Leishmania sp. Such variability has been used in the identification of these parasites, establishing differences between subgenera of New World Leishmania. We have found a region of 500 bp (β500) upstream of the coding region of the β-tubulin gene that is present in all strains tested belonging to the L. (Viannia) subgenus. This region apparently is a repetitive sequence and we have shown that it is specific to the Leishmania (Viannia) subgenus. This sequence has no homology with the genomic DNA isolated from either the species belonging to the L. (Leishmania) subgenus or other Kinetoplastida, such as Trypanosoma cruzi, T. brucei, Leptomonas samueli, or Crithidia fasciculata. The β500 sequence showed sufficient variation to be used as a molecular marker in the identification of parasites. We established inter- and intrasubgenus differentiation and were able to discriminate at the species level in the Vianna subgenus. A PCR assay confirmed the specificity of the β500 sequence.

Concomitant infections are common in nature and often involve parasites. A number of examples of the interactions between protozoa and viruses, protozoa and bacteria, protozoa and other protozoa, protozoa and helminths, helminths and viruses, helminths and bacteria, and helminths and other helminths are described. In mixed infections the burden of one or both the infectious agents may be increased, one or both may be suppressed or one may be increased and the other suppressed. It is now possible to explain many of these interactions in terms of the effects parasites have on the immune system, particularly parasite-induced immunodepression, and the effects of cytokines controlling polarization to the Th1 or Th2 arms of the immune response. In addition, parasites may be affected, directly or indirectly, by cytokines and other immune effector molecules and parasites may themselves produce factors that affect the cells of the immune system. Parasites are, therefore, affected when they themselves, or other organisms, interact with the immune response and, in particular, the cytokine network. The importance of such interactions is discussed in relation to clinical disease and the development and use of vaccines.