Severe cryptosporidial infections were produced in gamma interferon (IFN-γ) knockout mice. Mean oocyst shedding increased from 332 to 30717 oocysts/100 μl of faecal suspension between day 4 and 9 after administration of 1×105 oocysts/mouse. No significant differences in oocyst shedding were observed in mice after being inoculated with 1×105, 1×104 or 1×103 oocysts/mouse (P>0·05). Infected mouse weights decreased an average 3–4 g before death or euthanization. Histological studies revealed heavy parasite colonization in small intestinal epithelium (approximately 250 organisms/high-power field at ×400). Mesenteric lymph nodes in infected mice were markedly enlarged compared to controls (P<0·05). Both CD4+ and CD8+ T cell populations increased in spleens of infected mice while the B cell population increased in mesenteric lymph nodes from infected mice. No significant proliferation was observed when pooled lymphocytes from infected mice were exposed to C. parvum antigens in vitro. Addition of recombinant mouse IFN-γ did not restore antigen responsiveness. While lymphoproliferative responses to specific antigen were not significant in the short period following infection, this mouse model provides unique features to study the characteristics of acute infection and the immune response against C. parvum.

A 298 bp region of the Cryptosporidium parvum 18S rDNA and a 390 bp region of the acetyl-CoA synthetase gene were sequenced for a range of human and animal isolates of Cryptosporidium from different geographical areas. A distinct genotype is common to isolates from cattle, sheep and goats and also an alpaca from Peru and is referred to here as the ‘calf’-derived Cryptosporidium genotype. Another genotype of ‘human’-derived isolates also appears to be conserved amongst human isolates although humans are also susceptible to infection with the ‘calf’ Cryptosporidium genotype. Mice and pigs carry genetically distinct genotypes of Cryptosporidium. Three snake isolates were also analysed, 2 of which exhibited C. muris genotypes and the third snake isolate carried a distinct ‘mouse’ genotype.

The relocation of several thousand members of the Kamba tribe from the Kyulu Hills to the Thange valley near Masongaleni in Kenya provides an excellent opportunity to study the development of the immune response to schistosomiasis mansoni in a population with little or no previous experience of the infection. An adjacent, well-established Kamba community with similar patterns of water contact provides a suitable endemic control population. The immigrants were, uniquely, examined shortly after their arrival in the endemic area, while the prevalence of infection was still low. At this time faecal egg counts peaked atypically around 30 years of age. Over the next 12–18 months infection increased rapidly, especially among teenagers, producing a pattern of infection more typical of endemic communities. This substantially narrows estimates of the time required to develop the important determinants of the age–intensity profile, supporting the notion that changes related to age per se, rather than duration of infection, dominate. Age-dependent factors might include behaviour or physiology, including immune response. This paper provides the background for continuing longitudinal studies on the development of immunological responses to this parasite.

The BALB/c strain of mice is comparatively more resistant to sporozoite infections of Plasmodium berghei than the C57BL6 strain. Infection with live sporozoites results in the formation of small hepatic forms in the BALB/c liver that persist for as long as 6 days. Upon infection with small numbers of sporozoites, some of the parasites are destroyed in the liver whereas the rest persist as blocked forms. When larger numbers of sporozoites are injected the same process occurs but, in addition, a fraction of the liver-stage parasites complete full development and give rise to blood forms. Although blocked liver forms persist until day 6 post-infection they actually develop to only 24 h of maturity. The nature of these persistent forms is similar to those obtained from irradiated sporozoite immunization. There is a stronger cell proliferation to liver-stage antigens by spleen lymphocytes of irradiated sporozoite-immunized BALB/c mice in comparison to that of immunized C57BL6 mice suggesting that a stronger priming to liver-stage antigens, probably due to the presence of blocked hepatic forms in the liver for a longer period of time (as compared to C57BL6), occurs in the BALB/c mice. This could be a reason for the long-lasting protective memory observed in BALB/c mice.

Mice infected with Toxoplasma gondii manifest a rapid decline in serum thyroxine (T4) levels. To locate the locus of the hypothyroxinaemia, the integrity of the pituitary–thyroid axis of infected mice was assessed by a thyrotropin-releasing hormone (TRH) assay. A rise in serum T4 after inoculation of TRH implies the release of thyrotropin (thyroid-stimulating hormone) from a functionally intact pituitary. Administration of a single, large-dose (1 μg) bolus of TRH to infected mice induced a positive, although subnormal, T4 response. In contrast, when infected mice were pre-treated with a series of low-dose (5 ng) pulses of TRH prior to the bolus challenge, the T4 response was markedly enhanced. We suggest that the multiple inoculations of low-dose pulses of TRH ‘primed’ the pituitary (and secondarily the thyroid) and led to replenishment of their readily available hormone reserves and the heightened response to stimulation. These observations indicate that the locus of thyroid dysfunction is in the hypothalamus, not the pituitary or thyroid, and apparently involves impairment of the pulsatile release of TRH.

Binding of Plasmodium falciparum-infected erythrocytes (PE) to endothelial cells is mediated by the erythrocyte-membrane protein, band 3-related adhesin. To determine its role, the binding of infected cells treated with various chemical modifiers was investigated. Binding was inhibited by a lysine modifier (4,4′-diisothiocyanostilbene-2,2′-di-sulfonate (DIDS)) known to specifically bind to band 3, another lysine modifier (trinitrobenzene sulfonic acid), a tyrosine modifier (sodium iodide in conjunction with lactoperoxidase, hydrogen peroxide) and oxidants (diamide, sodium periodate and ADP-chelated ferric ion), but binding was unaffected by the histidine modifier (diethylpyrocarbonate) and the arginine modifier (phenyl glyoxyl monohydrate). To artificially expose the band 3-related adhesin, uninfected erythrocytes were treated with acridine orange or loaded with calcium. These cells bound to C32 amelanotic melanoma cells, were immunostained with a monoclonal antibody that specifically binds to the band 3-related adhesin on PE, and the binding was inhibited by this monoclonal antibody. The binding of acridine orange-treated and calcium-loaded uninfected erythrocytes, could also be blocked by DIDS. In the case of acridine orange-treated erythrocytes, the patterns of the effects of the chemical modification on binding were consistent with that of PE except for tyrosine modification. These results demonstrate that the band 3-related adhesin, even in the absence of parasite-encoded proteins, contributes to PE adhesion.

Schistosomiasis remains a serious world-wide public health problem with a still unfulfilled need for routine cost-effective methods of diagnosis. Such methods are required not only for people in endemic areas, but increasingly for tourists who may have become infected during visits to such places. This article reviews the wide range of immunoassays and antigenic preparations that have been shown to have potential for diagnosis of schistosomiasis by the indirect method of antibody detection. Antigens in native form derived from cercariae, adult worms and eggs are considered, as well as schistosome antigens produced by recombinant DNA technology and the schistosome cross-reactive antigen, keyhole limpet haemocyanin (KLH). Respective advantages and disadvantages of antibody detection, circulating antigen detection and parasitological methods of diagnosis are analysed. It is suggested that due to the relative insensitivity of both parasitology and antigen detection, antibody detection methods could find increasing use in situations of low infection intensity.

To identify the genes encoding novel immunodominant antigens of Theileria parva a λgt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host–sporozoite interaction.

Plasmodium falciparum merozoites have an array of 2–3 subpellicular microtubules, designated f-MAST. We have previously shown that colchicine inhibits merozoite invasion of erythrocytes, indicating a microtubular involvement in this process. Colchicine inhibition of invasion was reduced by the Taxol®-stabilization of merozoite microtubules prior to colchicine exposure. Immunofluorescence assays showed that the number and length of f-MASTs were reduced in colchicine-treated merozoites, confirming that microtubules were the target of colchicine inhibition. The dinitroaniline drugs, trifluralin and pendimethalin, were shown by immunofluorescence to depolymerize the f-MAST and both drugs were inhibitory in invasion assays. These results demonstrate that the integrity of the f-MAST is important for successful invasion. Fluorescence imaging demonstrated the alignment of mitochondria to f-MAST, suggesting that mitochondrial transport might be perturbed in merozoites with disorganized f-MAST. Depolymerizing mt in late-stage schizonts did not affect the allocation of mitochondria to merozoites.

The filarial nematodes Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus represent major public health problems in the Tropics. Effective diagnosis of infection with these parasites is required both for administration of drugs to infected individuals and for monitoring of control programs. However parasitological diagnosis is associated with a number of problems including frequently inadequate sensitivity, long pre-patency of infection and inconvenience for patients. For these reasons there has been considerable effort expended in developing other forms of diagnosis, in particular immunoassays for measuring antibody and circulating parasite antigen as well as molecular-biology-based assays for detecting parasite DNA. This article reviews the progress and achievements obtained to date. The latter include the development of ELISAs employing recombinant antigen for detection of antibody to O. volvulus which have both high sensitivity and specificity, the commercial availability of immunoassays to measure circulating antigen in W. bancrofti infection and the generation of specific DNA-based detection systems for all three parasites.

In this paper we investigate whether the assortative mating between individuals of the same genetic entity (i.e. coming from the same geographical area) of Echinostoma caproni, reported in a previous paper, may be explained by a post-zygotic isolating mechanism. The fecundity of the adults of 2 parental genetic entities and of their hybrids (i.e. F1, F2, F3) was quantified through 3 successive generations. Whereas the number of eggs released by F1 hybrids is similar to that of the mid-parent, that of recombinant hybrids (F2–3 hybrids) is significantly lower than that of F1 and that of the mid-parent. Since these results seem to demonstrate hybrid breakdown, 2 important factors maintaining reproductive isolation, i.e. pre-mating reproductive isolation and low hybrid fecundity, may influence the evolution of E. caproni.

A new and simple colorimetric method has been developed for determining activity in vitro against Giardia intestinalis. The microtitre plate assay is based upon the nucleoside hydrolase activity released from G. intestinalis by lysis. Action of the nucleoside hydrolase on the substrate analogue, 4-nitrophenyl β-d-ribofuranoside (NPR), gives rise to a coloured product which may be determined directly by the change in absorbance. A number of other such nucleoside analogues can be similarly used, but NPR is the preferred substrate, since it gives high enzymic activity at a relatively low substrate concentration. The IC50 values determined using this method for the known anti-giardials metronidazole, tinidazole and furazolidone were consistent with previously published values. The method is simple, does not involve radioisotopes or complex instrumentation, and thus provides a convenient method for screening potential anti-giardial agents.

Current theories assume that adaptive parasite evolution explains variation in the level of virulence and parasite success. In particular, mixed-genotype infections by parasites should generally be more virulent, and faster multiplying strains more successful, either because fixed strategies have evolved or because parasites facultatively alter virulence in response to co-infecting competitors. We compared several measures of parasite success and virulence between single-clone and mixed-clone infections of 2 strains of the trypanosome Crithidia bombi in its bumblebee host, Bombus terrestris. Contrary to expectation, we could not find differences between single-clone and mixed-clone infections in parasite prevalence, infection success, duration and clearance rate. However, a clearly significant effect of colony on infection intensity was present, and the colony effect emerged in virtually all other measures. We thus conclude that host environment as defined by the family (colony) genotype and thus host heterogeneity are more important in determining parasite virulence than the parasite characteristics. This does not invalidate modern theories of parasite evolution but suggests that variation in both hosts and parasites must be taken into account in more detail.

The intracellular development of Eimeria tenella sporozoites in in vitro cultured Madin–Darby Bovine Kidney (MDBK) cells was inhibited when parasite-infected MDBK cells were incubated with peripheral blood lymphocytes (PBL) from infected chickens. The inhibition mediated by PBL was quantified by [3H]uracil uptake and increased during the course of a series of oral infections of chickens with E. tenella. This was mirrored by the development of immunity in these birds, as assessed by counting the oocyst output following each re-infection. Similar levels of inhibition were observed using PBL from 3 inbred lines of chickens which differ in their relative susceptibility to infection with E. tenella, indicating that the genetic background of the host does not influence the production of this inhibitory activity. The inhibition could be transferred to freshly infected MDBK cells using supernatants prepared from parasite-infected monolayers incubated for 48 h with PBL from immune chickens. However, there was no inhibition using either supernatants from infected MDBK cells incubated with PBL from uninfected chickens, or supernatants from uninfected MDBK cells incubated with PBL from immune chickens. Experiments using Transwell plates showed that direct contact of PBL from immune birds with infected MDBK monolayers was not required to produce supernatants with inhibitory activity. Thus production of soluble inhibitory factor(s) by PBL from immune chickens can be specifically induced by soluble antigens present in the culture media of parasite-infected MDBK cells. These factors inhibit the intracellular development of sporozoites in in vitro culture.

The in vitro activity of artemether against 63 African isolates of Plasmodium falciparum from Libreville, Gabon was evaluated using an isotopic drug susceptibility semi-microtest. The 50% inhibitory concentration (IC50) values for artemether were in a narrow range from 0·8 to 34·8 nm (mean IC50 5·0 nm) and the 95% confidence interval (CI95%) was 3·6–6·3 nm. In vitro decreased susceptibility or resistance were observed with artemether (14%), to chloroquine (90%), to quinine (32%). Isolate susceptibility to amodiaquine and halofantrine was high i.e. 100% and 98%, respectively. There was a significant positive correlation between responses to artemether and amodiaquine (r2=0·45, P<0·001), artemether and chloroquine (r2=0·36, P<0·001), artemether and quinine (r2=0·31, P<0·001), and artemether and halofantrine r2=0·19, P<0·01). Positive correlation between these drugs suggests in vitro cross-resistance or at least common features in drug uptake and/or mode of action or resistance.

The sequences for the entire intergenic spacer (IGS) and 3′ end region of the large subunit (28S) ribosomal RNA gene are presented for Schistosoma intercalatum, S. haematobium and S. mansoni. The IGS was found to have at least 2 size formats for S. intercalatum and S. haematobium and the region for all 3 species contains numerous repeats and evidence of recombination. An A+T rich sequence found in S. intercalatum and S. haematobium is described and its possible function and origins are discussed. The amplification of this region by means of the polymerase chain reaction can be used to discriminate clearly between the 3 species involved in this study. The putative end of the 28S gene is identified.

The development of molecular diagnostic methods, particularly those utilizing PCR for the detection of parasitic protozoa will contribute greatly to the identification and control of these pathogens, by increasing speed of diagnosis, specificity and sensitivity, reproducibility and ease of interpretation. PCR methods are not without their problems however, and there is a need for laboratory procedures to be refined before PCR-based assays are accepted as the tools of choice for the routine detection of protozoan parasites. The application of PCR detection to various parasites is discussed.

Five chromosomes and 17 isoenzyme loci were analysed in 4 allopatric populations of Leishmania (Viannia) peruviana, and molecular distances calculated with 2 estimators, Chromosomal Size Difference Index and Jaccard Distance. Chromosome and isoenzyme data were in overall concordance: 13/30 isolates clustered similarly on the dendrograms constructed from the different estimators, and a significant correlation (P<0·001) was observed between the molecular distances calculated from the two sets of characters. This indicates an evolutionary association between chromosomal size polymorphism and isoenzymes. Chromosomes have a faster molecular clock than isoenzymes; twice as many genotypes were identified by chromosome analysis and significant size differences (for a total of up to 500 kb for 5 chromosomes together) were observed within a given zymodeme. Chromosomes most likely represent better indicators of genetic drift than isoenzymes, as suggested by the higher correlation between both estimators of chromosomal size-polymorphism and eco-geography. Some chromosomes might present an adaptive response to environmental variation.

Entomopathogenic nematodes (EPN) are useful biological control agents of insect pests. However, the infective juvenile (IJ) stage which is the only stage to occur outside the host is susceptible to environmental extremes such as desiccation. We have isolated desiccation-tolerant strains of the EPN Heterorhabditis megidis. In this paper we describe the surface properties of these desiccation-tolerant mutants. Heterorhabditid IJs retain the sheath of the previous larval stage. The mutant lines possess alterations in the surface properties of the sheath. Differences were observed in fluorescent lipid analogue insertion into the surface of the sheath. Furthermore, cationized ferritin-binding studies demonstrated that the mutant lines possessed an increase in net negative surface charge. Removal of the surface layer of the sheath resulted in the loss of the mutant phenotype and in a reduction in the desiccation tolerance of the parental strain. Therefore, the negatively charged ‘surface coat’ appears to play an important role in the desiccation tolerance of Heterorhabditis species.

A mathematical model of the transmission dynamics of Cowdria ruminantium by the ixodid tick Amblyomma hebraeum in the bovine host is developed and used to investigate the epidemiology of heartwater across a range of vector challenge. The processes described are supported by empirical data. The pattern of outcome measures (incidence, case-fatality and proportion of infected hosts) predicted agrees with those described anecdotally from field experience and empirical observation, and demonstrates the concept of endemic stability. The underlying theory is explored and it is shown that endemic stability may be due principally to the protection of calves against disease by either innate or maternally derived factors. The role of vertical infection in the establishment and maintenance of endemic stability is also investigated. Although increasing the vertical infection proportion results in endemic stability occurring at progressively lower levels of tick challenge, the concomitant reduction in incidence and case-fatality predictions across the range of tick challenge means the endemically stable state simultaneously becomes less discernible. Model limitations and future developments are discussed. The essential role of a transmission dynamics model in assessing the impact of new vaccines in conjunction with vector control programmes is highlighted.