Showing papers in "Parasitology in 1989"

Detection of Trypanosoma congolense and Trypanosoma brucei subspecies by DNA amplification using the polymerase chain reaction.

Abstract: The nuclear DNA of Trypanosoma congolense contains a family of highly conserved 369 base pair (bp) repeats. The sequences of three cloned copies of these repeats were determined. An unrelated family of 177 bp repeats has previously been shown to occur in the nuclear DNA of Trypanosoma brucei brucei (Sloof et al. 1983a). Oligonucleotides were synthesized which prime the specific amplification of each of these repetitive DNAs by the polymerase chain reaction (PCR). Amplification of 10% of the DNA in a single parasite of T. congolense or T. brucei spp. produced sufficient amplified product to be visible as a band in an agarose gel stained with ethidium bromide. This level of detection, which does not depend on the use of radioactivity, is about 100 times more sensitive than previous detection methods based on radioactive DNA probes. The oligonucleotides did not prime the amplification of DNA sequences in other trypanosome species nor in Leishmania, mouse or human DNAs. Amplification of DNA from the blood of animals infected with T. congolense and/or T. brucei spp. permitted the identification of parasite levels far below that detectable by microscopic inspection. Since PCR amplification can be conducted on a large number of samples simultaneously, it is ideally suited for large-scale studies on the prevalence of African trypanosomes in both mammalian blood and insect vectors.

Infectivity of cultured Plasmodium falciparum gametocytes to mosquitoes

Abstract: Various factors that may influence routine and high levels of mosquito infection with cultured Plasmodium falciparum gametocytes are considered in this paper. One of the most important is the choice of an appropriate isolate, with facilities for cryopreservation and a good technique for initiation of cultures. The use of automated culture systems with strict adherence to detail and routine has eliminated much of the variability. The quality of the serum used for the culture of gametocytes and inclusion in the feed material for mosquitoes is of the highest importance. Blood collection for culture purposes must preferably involve alcohol as an antiseptic for cleaning donor skin or suitable receptacles. Mosquito blood meals should not include plasma with citrate phosphate dextrose or sera collected in microtainer tubes or from volunteers on proguanil-chloroquine prophylaxis. Sera of individuals on chloroquine alone do not influence transmission. Haematocrits of from 5 to 10% permit the culture of equally infective gametocytes. It was impossible to predict the outcome of an infection in mosquitoes based on the number of female gametocytes or gametes. Within any experiment, the oocyst load initially increased, followed by a decline with progressively lower numbers of gametocytes accompanied by a progressive increase in the efficiency of transmission. Some of the variability of mosquito infection within an experiment was due to individual differences in the speed of blood digestion of the mosquitoes. A new membrane feeder is described with three different sizes to accommodate a variety of goals.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-linear phenomena in host-parasite interactions.

Abstract: The paper examines non-linear dynamical phenomena in host-parasite interactions by reference to a series of different problems ranging from the impact on transmission of control measures based on vaccination and chemotherapy, to the effects of immunological responses targeted at different stages in a parasite's life-cycle. Throughout, simple mathematical models are employed to aid in interpretation. Analyses reveal that the influence of a defined control measure on the prevalence or intensity of infection, whether vaccination or drug treatment, is non-linearly related to the magnitude of control effort (as defined by the proportion of individuals vaccinated or treated with a drug). Consideration of the relative merits of gametocyte and sporozoite vaccines against malarial parasites suggests that very high leves of cohort immunization will be required to block transmission in endemic areas, with the former type of vaccine being more effective in reducing transmission for a defined level of coverage and the latter being better with respect to a reduction in morbidity. The inclusion of genetic elements in analyses of the transmission of helminth parasites reveals complex non-linear patterns of change in the abundance of different parasite genotypes under selection pressures imposed by either the host immunological defences or the application of chemotherapeutic agents. When resistance genes are present in parasite populations, the degree to which abundance can be suppressed by chemotherapy depends critically on the frequency and intensity of application, with intermediate values of the former being optimal. A more detailed consideration of the impact of immunological defences on parasite population growth within an individual host, by reference to the erythrocytic cycle of malaria, suggests that the effectiveness of a given immunological response is inversely related to the life-expectancy of the target stage in the parasite's developmental cycle.

High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections.

Abstract: Rates at which Trypanosoma brucei rhodesiense trypanosomes switch from expression of one variable antigen type (VAT) to that of another have been determined in cloned populations that have been recently tsetse-fly transmitted. Switching rates have been determined between several, specific pairs of VATs in each population. High rates of switching were observed in 2 cloned trypanosome lines, each derived from a separate cyclical transmission of the same parental stock and each expressing a different major VAT. Five estimates of the switching rate between one particular pair of VATs were consistently high (approximately 1 x 10(-3) switches/cell/generation). These high switching rates were similar both in bloodstream populations of mice and in populations confined to subcutaneously implanted growth chambers in mice, thus indicating that the interaction of the bloodstream population with other trypanosome populations in the lymphatics or extravascular sites in systemic infections did not influence the estimates of the rate of switching. Fourteen estimates were made of VAT-specific switching rates in bloodstream infections involving 8 combinations from among 6 VATs. Switching rate estimates were VAT-specific and showed considerable variation between different combinations of VATs--from 1.9 x 10(-6) to 6.9 x 10(-3) switches/cell/generation. The rates of switching to different metacyclic-VATs were, however, very similar. Summation of between 3 and 5 VAT-specific switching rate values in each of 4 experiments conducted in bloodstream infections has provided minimum estimates of the overall rate of antigenic variation: 2.0-9.3 x 10(-3) switches/cell/generation. These values are between 20 and 66,000-fold higher than previously published estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial and temporal heterogeneity in the population dynamics of Bulinus globosus and Biomphalaria pfeifferi and in the epidemiology of their infection with schistosomes.

Abstract: Populations of Bulinus globosus and Biomphalaria pfeifferi were studied in a river habitat in Zimbabwe over a period of 12 months. Data were obtained on the prevalences of infections of Schistosoma haematobium (also S. mattheei) and S. mansoni respectively. Population parameters showed the following patterns for both snail species. (1) A patchy distribution correlated with the distributions of aquatic plants. (2) Life-expectancies of only a few weeks. (3) Recruitment rates correlated with water temperature and showing a distinct seasonal peak. (4) Spatial variation in recruitment. (5) A redistribution of snails during the rainy season. Epidemiological parameters showed the following patterns. (1) Large seasonal variations in the prevalence of patent infections. (2) Evidence from size-prevalence curves that suggests a variable force-of-infection from man to snail, correlated with water temperature. (3) Prevalences of infection that were higher in the vicinity of (+/- 60 m from) major water contact sites. Local prevalences of infection for B. globosus sometimes exceeded 50% and may have approached 100% if pre-patent infections are included. Snail numbers may limit transmission at these locations. Attention is drawn to the need to make field observations at an appropriate spatial scale and also to the implications for the effectiveness of focal snail control as a means of reducing transmission.

The epidemiology of Ascaris lumbricoides and other soil-transmitted helminths in primary school children from Ile-Ife, Nigeria.

Abstract: An epidemiological survey of intestinal helminthiases was conducted on 766 primary school children aged 5-16 years from Ile-Ife, Nigeria. On the basis of stool examinations, the prevalence of Ascaris lumbricoides, Trichuris trichiura, hookworm and Strongyloides stercoralis was 88.5, 84.5, 33.1 and 3% respectively. Intensity of infection was measured indirectly by egg counts for each species of helminth and also by counting worms passed after chemotherapy in the case of A. lumbricoides. The influence of host age and sex on infection levels was assessed. Relationships between the intensities of A. lumbricoides, T. trichiura and hookworm in individual children were identified. After anthelmintic treatment with levamisole, the frequency distribution of A. lumbricoides per host and the relationship between parasite fecundity and worm burden were investigated. Reinfection patterns of A. lumbricoides were assessed at two 6-monthly intervals and even within the narrow age range described, differences were found. In addition, evidence was obtained for predisposition of individuals to heavy or light infection with A. lumbricoides.

Novel biochemical pathways in parasitic protozoa.

Abstract: Throughout evolution, enzymes and their metabolites have been highly conserved. Parasites are no exception to this and differ most markedly by the absence of metabolic pathways that are present in the mammalian host. In general, parasites are metabolically lazy and rely on the metabolism of the host both for a supply of prefabricated components such as purines, fatty acids, sterols and amino acids and for the removal of end-products. Nonetheless, parasites are metabolically highly sophisticated in that (1) they retain the genetic capacity to induce many pathways, when needed, and (2) they have developed complex mechanisms for their survival in the host. Certain unique features of the metabolism of trypanosomes, leishmania, malaria and anaerobic protozoa will be discussed. This will include (1) glycolysis and electron transport with reference to the unique organelles: the glycosome and the hydrogenosome, (2) purine salvage, pyrimidine biosynthesis and folic acid metabolism and (3) polyamine and thiol metabolism with special reference to the role of the unique metabolite of trypanosomes and leishmanias, trypanothione.

Molecular approaches to DNA diagnosis.

Abstract: The DNA of a parasite is the ultimate blueprint of that parasite, the one characteristic which normally remains unchanged during every stage of the life-cycle. All the DNA sequence in the egg of a species of parasite are also in the larvae and adults of the same species. The same DNA is present in the parasite whether it is in a free-living stage, in an invertebrate vector or in a vertebrate host such as man. The molecular basis for DNA diagnosis is to allow labelled single-stranded species or strain-specific DNA sequences, selected from well-characterized reference species, to find and hybridize with homologous DNA from, or in, the unknown isolates of parasites. DNA probes are now available for most vector borne parasitic diseases. Parasitological identification problems are mostly concerned with distinguishing closely related strains or subspecies, for example detecting Taenia solium eggs as opposed to T. saginata eggs, or finding which of the 15 man-infecting subspecies of Leishmania is present in a single cutaneous lesion, the commonest clinical sign of the disease, or in a sandfly. For efficient hybridization by the present methods there has to be enough of a particular sequence present in a parasite's genome to make a feasible target. Therefore, DNA probes for parasites have been selected from repetitive, reiterated or multicopy DNA with intrinsic extensive sequence variation. DNA, which is free of coding restraint, can evolve rapidly to give differences between species, so that introns, ribosome gene spacers, variant genes, pseudo-genes and non-conserved DNA have all been used for DNA diagnosis. The major problems of sequence selection have been greatly aided by the use of recombinant DNA methods, which have the added advantage of economical production of DNA probes. The unique characteristics of kinetoplast mini-circle DNA in Leishmania has allowed the selection of a complex species, subspecies, strain and even isolate-specific DNA probes. These have been used successfully for Southern filter endonuclease fragment DNA identification, for dot-blot recognition of less than 200 parasites and non-radioactive detection of DNA sequence homology by ‘in situ’ hybridization and light microscopy in a single Leishmania cell. The adaptation of the forensic human genetic fingerprinting technique has allowed identification of L. braziliensis DNA in human biopsy material, even the presence of a vast excess of human DNA. Fingerprinting with ribosomal spacer-derived recombinant DNA probes has been used to discriminate Echinococcus species. The identification of Taenia species has been accomplished using probes from a genomic library of size-selected DNA fragments, and synthetic oligonucleotides are available for Onchocerca subspecies detection. The new technologies combining repeated genomic sequence probes with pulse field separation of the chromosomes of parasites, has opened up new avenues of research. Double probes for the simultaneous identification of the insect vector and the carried parasite have recently been reported. Lastly, the polymerase chain reaction technique has given us the opportunity of amplifying even single copy genes in one parasite to give sufficient DNA for positive identification. DNA diagnosis in parasitology is now on a par with the DNA diagnosis of viruses and human genetic disorders. The last ten years have seen many exciting developments in DNA diagnosis; the next years should see DNA diagnosis routinely used in epidemiological and clinical situations in countries where parasitic diseases are a major public health problem.

Experiences with vaccines against cutaneous leishmaniasis: of men and mice.

Abstract: The need for a vaccine(s) against cutaneous leishmaniasis and the populations at risk for whom such vaccines should be developed are briefly discussed. The current human vaccine studies are reviewed, as are some experimental mouse studies with emphasis on Leishmania major infection relevant to vaccine development. Based on the information available from the mouse model and those data which are being sought in human studies, the benign nature of the cutaneous disease, the ease with which L. major can be manipulated to yield the required material, and the ongoing practice of leishmanization which allows rapid evaluation of candidate vaccine(s), it is suggested that a vaccine, at least against L. major, is imminent in the not too distant future.

Analysis of a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei

Abstract: Two trypanosome clones, representing East and West African homozygotes at 2 isoenzyme loci (T. b. rhodesiense MHOM/ZM/74/58 [CLONE B] and T. b. brucei MSUS/CI/78/TSW 196 [CLONE A]), were cotransmitted through tsetse flies and the resulting trypanosome populations checked for the presence of non-parental karyotypes by pulsed-field gel electrophoresis. Ten clones isolated from these populations proved to have 5 different recombinant genotypes by analysis of nuclear and kinetoplast DNA (kDNA) polymorphisms. It is inferred that genetic exchange occurred between the 2 trypanosome clones in the fly, as previously reported for 2 other T. brucei spp. clones by Jenni and colleagues. For the most part, the hybrid clones shared many characteristics with both parents and their genotypes were consistent with segregation and reassortment of parental alleles. The least amount of genetic material exchanged was kDNA alone. Regarding the mechanism of genetic exchange, several hybrid clones had identical and unique nuclear DNA polymorphisms, but different kDNA type. Assuming that the same reassortment of nuclear markers is unlikely to occur by chance, these clones most probably arose from a predecessor carrying both types of kDNA.

Immunocytochemical demonstration of neuropeptides in the nervous-system of the liver fluke, fasciola-hepatica (trematoda, digenea)

Abstract: The localization and distribution of neuropeptides in the nervous system of the liver fluke, Fasciola hepatica at different stages in the development of the adult fluke have been determined by an indirect immunofluorescence technique, using antisera to 19 vertebrate peptides and the invertebrate neuropeptide, FMRFamide. Positive immunoreactivity was obtained with antisera to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), substance P (SP) and FMRFamide. Cell bodies and nerve fibres immunoreactive to the 4 peptides are present in the anterior ganglia and the 3 pairs of longitudinal nerve cords and their commissures in the central nervous system. In the peripheral nervous system, immunoreactivity occurs in the nerve plexuses supplying the subtegumental musculature, the oral and ventral suckers, and the muscular lining of the male and female reproductive ducts, including the ootype, uterus, cirrus pouch and gonopore. Cells displaying immunoreactivity to PYY and FMRFamide lie amongst the Mehlis' gland cells that surround the ootype. Processes from these cells extend into the wall of the ootype. One group of PP-immunoreactive cells occurs at the junction of the vitelline and ovovitelline ducts, whilst another group is situated at the entrance to the uterus from the ootype. The results are discussed in relation to the possible roles of the peptides in the neurophysiology and egg production of the fluke.

Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae.

Abstract: The excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [35S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS-PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified 125I-labelled material was measured in naive and infected jird hosts. It was reduced from 2-7 h in naive animals to less than 30 min in 4-10 week infected rodents, a finding which correlated with clearance of antigen by antibody in the infected group. In animals infected for longer time periods the serum half-life returned to the values observed in naive jirds. The idea that this change in half-life may reflect differences in the nature of 62 kDa antigen containing circulating immune complexes as infection progresses is discussed. The 125I-labelled antigen is predominantly removed from the circulation via the liver and ultimately excreted in the urine in a non-antigenic form. This work provides the first description of the origin, kinetics of circulation and fate of a defined filarial E-S product and may aid in determining the function and assessing the diagnostic utility of PC-bearing E-S components.

Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes.

Abstract: A segment of the ribosomal RNA gene of Schistosoma mansoni and a DNA fragment specific to Echinococcus granulosus, cloned in plasmids, have been used as DNA probes to assess the extent of genetic variability within E. granulosus and some distinct strains have been identified. The DNA analysis, involving restriction endonuclease digestion and Southern blot hybridization with the probes, did not demonstrate any significant genetic variation within the U.K. horse/dog or sheep/dog strains but confirmed the distinctiveness of the two strains shown in previous studies. The sheep/dog strain was shown to be cosmopolitan in its distribution and fertile bovine material originating from the United Kingdom, Kenya, Spain and India conformed to this strain by DNA hybridization. In contrast, cattle isolates from Holland produced markedly different DNA hybridization banding profiles indicating that cattle can harbour more than one strain of E. granulosus. Similarly, it was shown that goats can harbour two different strains of E. granulosus, the sheep/dog strain and a form which infects camels. The strain of E. granulosus infecting equines in Spain and Ireland is genetically identical to that infecting horses in the United Kingdom. There is also a different strain infecting pigs in Poland and Yugoslavia. This pig/dog strain appears to be very similar genetically to the forms of E. granulosus which use camels and goats as intermediate hosts and is similar, though not identical, to the variant infecting Dutch cattle. It has been shown that E. granulosus material, fixed for a prolonged period in ethanol, or lyophilized, is amenable to DNA analysis and that it is possible to characterize the DNA of a single adult worm.

Ancylostoma ceylanicum in the hamster: observations on the host-parasite relationship during primary infection.

Abstract: The course of primary infection with a hamster-adapted strain of Ancylostoma ceylanicum was studied in inbred DSN and randomly bred WO/GD and WO/CR hamsters. Infective larvae were administered orally and began to develop in the small intestine without embarking on a tissue migration. Only the occasional larva was detected in other organ sites. It was concluded that the developing larvae moulted on days 3-4 and again to the pre-adult stage about 9-11 days post-infection. Worm burdens in infected hamsters were stable for at least 11 weeks after infection. There was no sudden expulsive phase and some adult worms survived for over 200 days. Overall the sex ratio of worms in groups of hamsters killed concurrently was about 50% although occasionally the ratio was biased in favour of one sex in individual animals. The blood packed cell volume (PCV) was significantly depressed 2 weeks following infection and continued to decline until a point of stability was achieved 4-5 weeks post-infection. The PCV subsequently remained depressed throughout the period of observation. Infected hamsters lost weight if kept in groups, but not when housed in separate cages. Groups of animals which lost weight did not recover to normal values within 11 weeks of infection. It is suggested that this model of hookworm infection has scope for exploring aspects of the host-parasite relationship which the canine models cannot fulfill adequately.

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): dose-dependent expulsion of adult worms.

Abstract: The survival of Heligmosomoides polygyrus was monitored during primary infections in female C57Bl10, NIH and BALB/c mice at low and high intensities of infection. Survivorship curves were fitted for each data set and analysed. C57Bl10 mice, given either low or high intensities of infection, harboured parasites for 28-37 weeks, heavier infections surviving marginally but significantly longer. Essentially the survivorship curves of H. polygyrus in C57Bl10 mice could be accounted for by senility, the increased probability of worms with a longer life-span occurring at high infection intensities and, possibly, by a contribution from host-protective immune mechanisms in the terminal stages of infection. The pattern of survivorship was different in NIH and BALB/c mice. NIH mice showed weak but significant density-dependent suppression of parasite loss and infections in this strain did not exceed 27.5 weeks in duration. Primary infections in BALB/c mice were briefer still and showed marked dependence on parasite density. Thus low-level infections lasted 10-15 weeks whereas heavier infections survived for 21-34 weeks. The data suggested that both strains developed host-protective responses to adult H. polygyrus and that parasite survival was curtailed earlier than would be expected if senility alone was involved. The hybrid strains (C57Bl10 x NIH)F1 and (B10G x NIH)F1 both expelled H. polygyrus in a dose-dependent manner, worm loss commencing within 10 weeks of infection. In some experiments worm loss was clearly evident by weeks 4 and 6. These hybrid strains showed gene complementation in that adult worms were cleared considerably earlier than in parental strains.

The influence of individual, social group and household factors on the distribution of Ascaris lumbricoides within a community and implications for control strategies.

Abstract: The distribution of Ascaris lumbricoides within a community was examined at an initial mass anthelmintic treatment programme (January 1984) and following an 11-month period of reinfection (November 1984). Similar patterns of the negative binomial parameter k (an inverse measure of parasite aggregation) and the proportion of parasites within the most heavily infected quartile of the community were recorded at the two dates. The pattern of parasite aggregation within individuals, measured by parameter k, appears to be a stable characteristic of this host-parasite relationship. Significant variation in the intensity of infection was observed between households in the community. The number of family members living in the house strongly influenced the mean Ascaris burden and proportion of relatively heavy infections within adults and children. This finding suggests that the density of people in a house positively influences the frequency of exposure to infective stages of Ascaris, which in turn plays a major role in determining which individuals will harbour heavy infections. Positive correlations were recorded between the initial and reinfection burdens of individuals, relative to others in the community. The correlations were strongest in the youngest and oldest age groups and were more frequently significant among age-stratified groups of females, compared to males. A comparative examination of hypothetical treatment strategies suggests that for Ascaris infections in this community, targetting age groups with anthelmintic treatment would probably be more cost-effective in the long term in reducing the abundance of this parasite than selective treatment of individually identified heavy infections.

Schistosome vaccines: current progress and future prospects.

Abstract: Vaccination against human schistosomes in laboratory hosts is now a reality. A number of different parasite molecules have been shown to confer partial protective immunity against challenge infection with Schistosoma mansoni or Schistosoma japonicum in rodent or primate hosts. These antigens are unusually diverse in their structure and stage specificity. Interestingly, although all of the vaccine molecules characterized are situated in the tegument, their exposure on the parasite surface, in most instances, is transient and/or non-essential. The properties of four of these immunogens, glutathione-S-transferase (P26,28), paramyosin (Sm97), GP38, and GP18 are discussed. Despite the identification and recombinant synthesis of several promising protective antigens, vaccination of humans against schistosomiasis remains in the realm of fantasy. At the technical level, a major problem is the failure of any of the current vaccine immunogens and immunization protocols to induce levels of resistance sufficient for significant reduction of human infection or disease. Once this important hurdle is passed, human immunization trials should be attempted as the potential beneficial impact of a vaccine against schistosomiasis remains enormous.

Density dependence in establishment, growth and worm fecundity in intestinal helminthiasis: the population biology of Trichuris muris (Nematoda) infection in CBA/Ca mice

Abstract: The results are presented of an experimental study of the population biology of chronic Trichuris muris (Nematoda) infection in cortisone-treated CBA/Ca mice. Attention is focused upon both the validity of the common use of faecal egg counts to demonstrate density dependence in helminth fecundity, and the identification of other possible density-dependent mechanisms that may regulate worm numbers in chronic trichuriasis. The results show that faecal egg counts, although demonstrating high daily variation, are not an artefact of host faecal output but a significant density-dependent function of worm burden. This finding contrasts with the observations on Heligmosomoides polygyrus infection in outbred MFI mice, but accords with similar studies in a wide variety of host - helminth systems. Worm establishment in the murine host is found to be a density related function of infection dose. This is attributed to the probable existence of a physical gut-carrying capacity in the murine host for T. muris. Worm distribution in the gut is also shown to the density dependent, with worms being displaced from the caecum to the colon at increasing intensities of infection. The sex ratio of the adult parasites, however, is found to be both unitary and independent of worm burden. Evidence for a significant density-dependent decline in female T. muris growth or size is presented. The results also show a significant positive association between female T. muris weight and per capita fecundity. These findings indicate that the stunted growth of individual worms at high parasite densities may be a potential mechanism underlying density dependence in helminth fecundity.

Characterization of buffalo-derived theilerial parasites with monoclonal antibodies and DNA probes.

Abstract: The characteristics of intra-lymphocytic Theileria isolated from African buffalo and from cattle that were infected with buffalo-derived parasites were evaluated using anti-schizont monoclonal antibodies (mAbs) and DNA probes. Antigenic differences were revealed by the reactivities of 27 mAbs with the buffalo-derived parasites isolated from different animals. Antigenic diversity was also seen with Theileria-infected lymphoblastoid cell isolates taken from the lymph nodes and lambda gt11, showed specific hybridization to parasite DNA in Southern blots of restriction enzyme-digested, lymphoblastoid cells infected with buffalo-derived theilerial parasites. Genotypic differences between the buffalo-derived parasites were revealed by the restriction fragment length polymorphisms seen with hybridization of those probes to DNA from cloned and uncloned Theileria-infected cell lines. The evaluation of theilerial parasites derived from buffalo and from cattle which underwent typical T. p. lawrencei reactions, after being infected with buffalo-derived theilerial parasites, did not show any specific phenotypic or genotypic characteristics of these parasites that would distinguish them from T. p. parva and T. p. bovis parasites. The validity of these subspecies distinctions is discussed.

A comparison of fluorescein diacetate and propidium iodide staining and in vitro excystation for determining Giardia intestinalis cyst viability.

Abstract: The viability of 4 human isolates of Giardia intestinalis cysts using either the fluorogenic vital dyes fluorescein diacetate (FDA) and propidium iodide (PI) or in vitro excystation was assessed. Whereas viable cysts, as defined by in vitro excystation were present in each of the 4 isolates, cysts from only 3 of the 4 isolates took up the vital dyes. FDA consistently over-estimated cyst viability whilst PI under-estimated non-viable cysts when compared with in vitro excystation. Following in vitro excystation, both FDA and PI stained a proportion of unexcysted cysts indicating that FDA stained cysts which were incapable of excystation, whereas PI did not stain all cysts which were incapable of excystation. One human cyst isolate, which underwent in vitro excystation, could not be stained with either FDA or PI. In the absence of currently more specific fluorescent indicators of viability, PI alone could be used to determine the lower limit of nonviability in positive water-related samples, where small numbers of cysts are to be expected.

Effects of infection with Echinostoma paraensei on the circulating haemocyte population of the host snail Biomphalaria glabrata.

Abstract: Circulating haemocytes from Echinostoma paraensei-infected M line Biomphalaria glabrata snails, or from age- and size-matched control snails, were studied on plastic slides with phase-contrast optics. Granulocytes, hyalinocytes, and round cells were consistently present; granulocytes were further categorized as 'fully spread' (FS) or 'partially spread' (PS). Among control snails, the relative percentage and estimated number/mm3 of round cells declined significantly with increased snail size, and the corresponding values for both categories of granulocytes increased. At 1 day post-infection (p.i.) with E. paraensei, overall composition of the haemocyte population was relatively unaffected, but by 8 days p.i. infected snails had significantly higher relative percentages of round cells and PS granulocytes than controls. Because a marked increase in the number of circulating haemocytes is also evident by 8 days p.i., infected snails had approximately 12 times more round cells and PS granulocytes/mm3 of haemolymph than did controls. At 30 days p.i. the relative and absolute abundance of PS granulocytes was still significantly elevated, but otherwise haemocyte populations did not differ from control snails. Alterations in granulocyte size in infected snails were also noted. Infection with E. paraensei has a striking impact of circulating haemocyte populations and also increases the relative concentration of haemocytes with less ability to adhere to a foreign surface.

Plasmodium falciparum growth inhibition by human platelets in vitro

Abstract: Platelets take an active part in immunological processes as well as in haemostasis, especially in the host-parasite relationship. Our aim is to assess the growth of Plasmodium falciparum, cultured in human erythrocytes in the presence of fresh washed human platelets, since thrombocytopaenia is frequently observed during malarial infections. Our results show that platelets induce a dose-related growth inhibition of P. falciparum. Both proliferation and maturation of intraerythrocytic stages of the parasite are inhibited. This growth inhibition is triggered by the parasite itself as neither specific antibodies nor any other components are needed to activate platelets. Activated platelets are directly toxic since complement is not involved. Furthermore, inhibition is not mediated by erythrocyte lysis or by toxic oxygen metabolites. Platelets induce an inhibition of P. falciparum growth, at least in vitro, although the importance of their role played in vivo in malarial immunity has yet to be evaluated.

Hybrids between Schistosoma mansoni and S. rodhaini : characterization by cercarial emergence rhythms

Abstract: Hybridization between Schistosoma mansoni, with a diurnal cercarial emergence rhythm and S. rodhaini, with a nocturnal cercarial shedding pattern leads to F1 and F2 generations, hybrid schistosomes whose chronobiological phenotype of cercariae is characterized by two unequal emergence peaks, one diurnal and the other nocturnal. The relative importance of diurnal and nocturnal peaks depends upon which S. mansoni strain (early or late) is used for the hybridization with S. rodhaini. The results are compared and discussed with those resulting from crosses between intraspecific sympatric and allopatric chronobiological variants (early and late) of S. mansoni. The genetic determinism of the cercarial emergence of schistosomes and the significant differences observed between cercarial shedding patterns of parental species and their hybrids allow the use of this behavioural marker in biological and genetical studies in schistosome populations.

The sex ratio of Plasmodium gametocytes.

Abstract: Sex ratio theory usually predicts an equilibrium sex ratio and equal proportions of males and females in a population, including the progenitors of the reproductive cells of protozoans. This proposal was tested with three species of malarial parasites of lizards, Plasmodium mexicanum of the western fence lizard, and P. agamae and P. giganteum of the African rainbow lizard, using single samples from naturally infected lizards, repeated samples from free-ranging lizards (P. mexicanum only), and repeated samples from laboratory maintained animals. Macrogametocytes were usually more abundant than microgametocytes, and were slightly larger, revealing a typically greater investment of resources by the progenitors of female reproductive cells. However, the proportion of microgametocytes varied among the three species and among infections within each species of Plasmodium. The sex ratio of gametocytes often remained constant within infections followed over time even if the absolute number of gametocytes was changing. However, the equilibrium sex ratio of gametocytes varied among those infections that had an unchanging microgametocyte proportion. Thus, although an equilibrium sex ratio apparently occurs for most infections, there appears to be no characteristic proportion of microgametocytes for any of the species. Potential explanations for this conflict with theory are presented.

Biochemical characteristics of the metacyclic forms of Leishmania major and L. mexicana mexicana.

Abstract: Metacyclic forms of Leishmania major and putative metacyclics of L. mexicana mexicana were found to occur in abundance in stationary phase cultures. These forms have been compared in several ways with promastigotes from mid-log phase cultures and, in the case of L. m. mexicana, amastigotes. Metacyclics are smaller, contain less protein and appear more active than other promastigotes. Both forms of promastigote respire at a high rate in the absence of exogenous substrate. The free amino-acid contents of the various forms of the two species have been analysed. They differ in detail but alanine was the major amino acid in all cases. The isoenzyme content of the different forms differed significantly. That of the putative metacyclics of L. m. mexicana was in several respects more similar to amastigotes than promastigotes, suggesting that the form is pre-adapted for life in a mammal. Metacyclics of L. major apparently did not divide in culture but transformed back over a period of 48 h to mid-log phase cells. The results provide further detail of the molecular differences between mid-log phase and metacyclic promastigotes and confirm that metacyclics are a distinct form in the life-cycle.

Infective juvenile formation in the insect parasitic nematode Steinernema feltiae

Abstract: Steinernematid nematodes form a developmentally arrested infective juvenile (IJ) stage at the second moult, when conditions inside the insect host are no longer suitable for further reproduction. In a liquid culture micro-assay two environmental cues were shown to influence the formation of Steinernema feltiae IJs. High nematode population density induced IJ formation, suggesting the presence of a nematode pheromone. Bacterial food and soluble nutrients acted competitively to reduce the frequency of IJ formation. Frequency of IJ formation was greatest when lst-stage juveniles were subjected to IJ-inducing conditions. The optimum temperature range for the IJ induction response was 25–30°C. These findings suggest that maximal IJ production in large-scale liquid culture will depend on the correct balance of nematode population density and nutrient availability at the peak of egg hatching.

Changes in nematode antigens recognized by monoclonal antibodies during early infections of soya beans with the cyst nematode Heterodera glycines

Abstract: The invasion and establishment of Heterodera glycines has been studied for 0–120 h after addition to host roots using a synchronized infection system. A high degree of synchrony in development was noted, with juvenile nematodes reaching their feeding sites adjacent to the root endodermis and discharging their subventral pharyngeal glands by 24 h post-invasion (p.i.). The moult to the 3rd-stage juvenile occurred approximately 89 h after entry to the root. Using an enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) whose tissue specificities had already been defined by immuno-cytochemistry, it was found that MAbs recognizing intestinal lipid droplets and granules showed a major increase in reactivity 18–24 h p.i. before declining by 39 h p.i. In contrast, the reactivity of two MAbs recognizing adult cuticle increased significantly only after 89 h p.i., following the second moult. MAbs to the two subventral pharyngeal gland cells showed a variety of patterns of changing reactivity during the experiment. One increased within 24 h of invasion before declining whereas a second fell without any initial rise. Two further MAbs specific for the subventral glands initially declined but showed a secondary increase in reactivity after 72 h and could be detected in adult females. This pattern was also seen in a MAb specific for the dorsal pharyngeal gland cell. These changes are discussed in the context of events taking place following invasion, with particular reference to the initiation and maintenance of a feeding syncytium by the developing nematode.

Spatial and temporal variation in the infracommunity structure of helminths of Apodemus sylvaticus (Rodentia: Muridae)

Abstract: Mean species richness and diversity of the helminth infracommunity of Apodemus sylvaticus in woodland areas of Co. Down, Northern Ireland, varied in time and space. Variation in infracommunity structure among individual hosts, however, always accounted for more than 60% of the variation in the data from different places or different times. Helminth species richness increased with increasing population density, the percentage of the host population 16 weeks old or older, and the proportion of the host population with animal material in their stomachs, at two sites monitored over 33 months. The basis for spatial variation in infracommunity structure is less certain but host dynamics and differences in diet are likely to play some role. It is concluded that analysis at the infracommunity level focuses closely on the potential for species interactions and overlap in resource utilization. Infracommunity structure, at least in the case of A. sylvaticus, varies markedly in time and space and between individual hosts. Such variations should not be ignored in comparative studies.

The role of parasite fecundity and longevity in the success of Trichostrongylus tenuis in low density red grouse populations.

Abstract: The prevalence of the caecal threadworm Trichostrongylus tenuis in red grouse in the north of Scotland was high despite low grouse densities. Prevalence, intensity and aggregation of threadworms was higher in old than in young grouse. Infections were long-lasting: populations of adult worms could survive for over 2 years in grouse, with little mortality. Parasite egg output decreased with the age of a worm population, largely as a result of a decrease in the fecundity of ageing female worms. Seasonal variations in worm fecundity were also evident. However, there was no evidence of an intensity-dependent decrease of worm fecundity with increasing worm numbers in either captive or wild grouse. The long life and high reproductive capacity of T. tenuis probably contribute to its effective transmission and high prevalence.

Host predisposition to trichuriasis: the mouse--T. muris model.

Abstract: Predisposition to trichuriasis in mice is reflected in the inability of certain strains, or certain individuals within strains, to express protective immunity. Poor responders fail to expel worms and harbour chronic patent infections. The mechanisms underlying this phenomenon were studied in poor responder mice challenged after abbreviated or prolonged primary infections. Mice exposed to a complete primary infection were fully susceptible when challenged after the removal of the primary infection by anthelmintic. Failure to expel either infection suggests (a) that non-responsiveness to a primary infection does not reflect an inability to expel worms of a certain size, i.e. is not a consequence of the speed of the immune response in relation to parasite growth and (b) that non-responsiveness is long-lasting. Challenge after abbreviation of primary infections at different stages of worm development showed that persistence of larvae beyond day 21 was critical in determining poor response to reinfection. By inference the same conclusion can be drawn about the inability of such mice to expel primary infections. Serological analysis suggested a relationship between low antibody titres, restricted antigen recognition profiles and resistance to infection. It is suggested that the later stages of parasite development are immunosuppressive; the implications for human trichuriasis are discussed.