An extended proof of migration routes of immature parasites inside hosts: pathways of Nippostrongylus brasiliensis and Stronglyoides ratti in the rat are mutually exclusive N. R. Tindall and P. A. G. Wilson* Parasitology. April 1990, Vol. 100 pp. 281–288.

rRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

Mass-scale production of Plasmodium vivax sporozoites in Anopheles stephensi was achieved using the chimpanzee (Pan troglodytes) as a source of infective blood. Membrane feeding was as successful as feeding mosquitoes directly on the animal so long as the time between drawing the blood and feeding was restricted to 45 min. Longer delays such as 2–3 h resulted in loss of infectivity in terms of oocyst production. The selected strain of A. stephensi was highly susceptible to P. vivax (Chesson strain). A strain of A. stephensi relatively refractory to P. falciparum showed no cross-refractoriness to P. vivax. Mixed infections of P. falciparum and P. vivax did not interfere with each other in their development in A. stephensi. A second normal blood meal to mosquitoes infected with P. vivax increased the yield of salivary gland sporozoites.

Despite some years of enquiry, the mechanism that leads to intra-erythrocytic death of malarial parasites during the host's response to infection has not been elucidated. We report here that serum from mice undergoing a successful immune response to Plasmodium chabaudi does not inhibit Plasmodium falciparum unless the Pl. chabaudi is virulent enough to rise to at least 50% parasitaemia and to cause illness. This appears to be true of the 556 KA and DS strains of Pl chabaudi, and also other murine malaria parasites. In mice infected with Pl. chabaudi 556 KA inhibitory activity coincided with the presence of TNF in their serum. Exogenous TNF generated inhibitory activity in the serum of mice only if the animals were pretreated with Proprionobacterium acnes, implying a role for activated macrophages downstream from TNF in this process. The difference in inhibitory activity against Pl. falciparum in serum from mice infected with Pl. chabaudi of more or less virulence may be one of degree. Alternatively two distinct mechanisms may operate, the second coming into operation only in ill mice, with higher parasite burdens.

There is increasing support for the presence of viruses and virus-like particles inside protozoan cells. This study describes viral-like RNA molecules that have been detected in two species of Eimeria that infect the chicken. The RNA molecule identified in E. maxima has been characterized: subcellular fractionation studies have shown that the RNA is present in the cytoplasm, probably as an abundant ribonucleoprotein that is insensitive to RNAse A treatment. Electron microscopy has demonstrated that this RNA molecule is double stranded. In addition, all E. maxima strains examined so far contain this RNA molecule.

Intracellular differentiation of Leishmania promastigotes to amastigotes is a critical step in the establishment of infection. In this report three related features of mexicana subspecies amastigotes were used to follow the differentiation of the parasites within macrophages. Early after infection, (a) parasites did not contain ultrastructurally recognizable megasomes, (b) cysteine proteinase activity of parasite lysates was not detected in gelatin-containing acrylamide gels, and (c) parasites were essentially resistant to L-leucine-methyl ester (Leu-OMe). Typical megasomes were first identified on the 5th day, were more prevalent on day 7, and underwent swelling in macrophages exposed to Leu-OMe. Cysteine proteinase activity was first detected on day 3 and increased thereafter. Susceptibility to Leu-OMe of parasites studied in situ or isolated from infected macrophages increased with time of intracellular residence and by 7 days approached that of amastigotes isolated from mouse lesions. In contrast, parasites derived from either promastigotes or amastigotes were equally susceptible to another leishmanicidal compound, tryptophanamide (Trp-NH2). The results provide additional support for the involvement of megasomes and their cysteine proteinases in parasite killing by Leu-OMe, and highlight the slow pace of the intracellular differentiation of L. amazonensis promastigotes to amastigotes.

The haemolytic activity of live Trichomonas vaginalis organisms was investigated. Optimal haemolysis of human erythrocytes was observed at a parasite to erythrocyte ratio of 1:5 during a 2 h incubation period. No haemolytic activity was detected in concentrated culture supernatants after overnight growth of trichomonads or when parasites were separated from erythrocytes by a 3 μm filter, suggesting a contact-dependent mechanism for haemolysis. The haemolytic activity was temperature-dependent and maximal haemolysis occurred at 37 °C. Treatment of trichomonads with metronidazole reduced levels of haemolysis by > 50%. Maximal haemolysis occurred at the pH range of the vagina during trichomoniasis. N-μ-tosyl-L-lysyl-chloromethyl ketone and iodoacetamide, inhibitors of trichomonad cysteine proteinases, reduced the haemolytic activity of live parasites.

Leishmania turanica n.sp., found infecting the desert rodent Rhombomys opimus in the southern territories of the USSR and the Mongolian People's Republic, is described. This parasite exists sympatrically with L. major and L. gerbilli in R. opimus and is the predominant species. A total of 284 isolates of L. turanica from R. opimus, 3 from naturally infected Phlebotomus andrejevi and 1 from P. papatasi were characterized and found to be clearly distinguishable on isoenzyme and nuclear DNA characteristics from all other Old World taxa of Leishmania.

The cellular responses in regional lymphoid organs of C57B1/6 mice were examined, following protective immunization with 20 krad.-irradiated cercariae of S. mansoni. Marked changes in total cell number were observed, with peak increases of 13·5-fold in the skin-draining (axillary) lymph nodes and 6·9-fold in the lung-draining (mediastinal) lymph nodes. In contrast, cellular responses were small in the spleen and undetectable in the brachial lymph nodes. The temporal pattern of responses was coincident with the kinetics of parasite migration, events in the mediastinal lymph node being apparent only after day 7. Phenotypic analysis of the cellular changes revealed an increase both in T lymphocytes and, to a greater extent, in B lymphocytes. The changes in Thyl+ cells comprised an increase in both L3T4+ and Lyt2+ populations. A comparison of mice exposed to non-immunizing parasites (normal or 80 krad.-irradiated cercariae) with protected animals, revealed smaller and more transient cellular changes in the axillary lymph nodes of the former. We suggest that the successful immunization of mice with attenuated parasites depends upon the prolonged priming of lymphocytes within the lymph nodes draining the skin-exposure site and that the persistence of 20 krad.-irradiated parasites within these nodes provides the requisite stimulus.

Monoclonal IgM rheumatoid factor-like anti-globulins were produced by in vitro stimulation of naive BALB/c spleen cells with lipopolysaccharide, and by hyperimmunization of mice with merozoites of Plasmodium falciparum, followed by fusion of the spleen cells to mouse myelomas. In vitro, these anti-globulins augmented the inhibitory effects of P. falciparum-specific polyclonal mouse sera and monoclonal IgG1 and IgG2b antibodies by binding to Fc fragments of IgG molecules attached to blood-stage parasites. In some instances, the presence of anti-globulins correlated with an increase in the number of schizonts which failed to disperse merozoites. In other cases, parasitaemia remained low in the absence of the schizont inhibition phenomenon, suggesting that anti-globulins contribute to host cell protection not only by agglutinating merozoites, but also by increasing the density of the antibody coat surrounding the parasites, thus interfering with parasite receptor-erythrocyte ligand interactions. The anti-globulins were not inhibitory when added to parasite cultures containing IgG not specific for P. falciparum. These results may help explain the function of IgM anti-globulins found at elevated serum levels in some patients with malaria or other chronic infectious diseases.

Development and forward migration of Leishmania parasites in the sandfly gut is accompanied by morphological transformation to highly motile, non-dividing ‘metacyclic’ forms. Previous studies in vitro have demonstrated that this metacyclogenesis is associated with developmentally regulated changes in expression of two major surface glycoconjugates of Leishmania, the lipophosphoglycan (LPG) and the glycoprotein protease GP63. Studies presented here are the first to examine in situ the changes in expression of these two important surface molecules which occur during amastigote-initiated development of L. major in its natural vector Phlebotomus papatasi. Immunocytochemical analysis using a GP63-specific monoclonal (3.8). and others recognizing metacyclic-specific (3F12) and common (WIC79.3) epitopes of LPG on logarithmic and metacyclic promastigotes, demonstrates: (1) clear expression of LPG and GP63 from 2 and 7 days post-bloodfeeding, respectively; (2) developmental modification of the LPG molecule as parasites undergo forward migration and morphological changes associated with metacyclogenesis; and (3) striking deposition of large amounts of parasite-free excreted LPG on/in the epithelial cells of the gut wall.

When cercariae of Schistosoma mansoni and of S. japonicum were irradiated with various levels of u.v. light at 254 nm, their development to perfusable worms was reduced to below 1% at about 200 μW min cm−2. Cercariae attenuated with about 300 μW min cm−2 induced partial resistance against an homologous challenge infection in mice. No differences were observed between the two schistosome species when the same treatment was given to the cercariae. Thus the same u.v. dose can confer immunizing ability to cercariae of both S. mansoni and S. japonicum.

Sex ratios of adult schistosomes in mice are almost invariably different from 1·0 and are biased towards males. The bias applies to wild rats infected with Schistosoma japonicum and trapped in an endemic area of the Philippines (male: female ratio = 1·7). It also applies to cercariae of snails collected in such areas and assessed by infection of laboratory mice using cercariae from individual snails (male: female ratio may approach 6·0). Experiments were designed to determine if duration of infection in the mammalian host was a factor that influenced the sex ratio of miracidia used for infecting snails and subsequently mice. BALB/c and C57BL/6 mice were infected with 100 cercariae of S. mansoni, and liver eggs harvested at early and late time points for infection of snails and production of cercariae. Two phenomena were demonstrated: firstly, a more pronounced male bias when eggs were harvested late compared with early in infection; secondly, a reduced apparent hatchability of eggs in BALB/c compared with C57BL/6 livers. The possibility is raised by the data that female miracidia within eggs of chronically infected individuals may be more prone to immune damage than male miracidia with important epidemiological consequences.

A non-polymorphic antigen associated with the rhoptry organelles of Plasmodium falciparum has been purified by immuno-affinity chromatography. The antigen, RAP-1 (rhoptry associated protein-1). which is defined by monoclonal antibodies which inhibit parasite growth in vitro, is a multi-component antigen consisting of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa. These proteins were electro-eluted from preparative sodium dodecyl sulphate polyacrylamide gels and protected Saimiri sciureus monkeys from a lethal blood-stage infection of P. falciparum malaria. Sera from the protected animals recognized only proteins of the RAP-1 antigen when used to probe a Western blot of total parasite protein extract, confirming that RAP-1 is responsible for eliciting the protective immune response.

The completed life-cycle of Cyclospora talpae, an extra-intestinal coccidian (Apicomplexa) of the liver of the mole (Talpa europaea). is reported. Oocysts sporulated within 12–14 days at room temperature (20–22 °C). Both macro- and micro-gametocytes developed independently within the nucleus of epithelial cells lining the bile duct as demonstrated by both light and electron microscopy. Merogony was seen in the mononuclear cells in the capillary sinusoids of the liver. Merozoites showed the typical ultrastructural features of the Apicomplexa. The life-cycle of C. talpae and the ultra-structure of the gametogonic and merogonic stages are reported; the ultrastructure of the merozoites is reported as the first description of this stage for the genus Cyclospora.

Evidence for the existence of 2 first intermediate host-specific sibling species of the 45 collar-spined echinostome Echinoparyphium recurvatum is presented. Experimental studies on their life-cycles were carried out under controlled laboratory conditions. The two entities were found to be morphologically indistinguishable in all major respects, yet they exhibited distinct biological characteristics. One of the sibling species utilizes the freshwater lymnaeid pulmonate snail Lymnaea peregra as first intermediate host, and the adults occur in the anterior small intestine of the wildfowl experimental definitive host Anas platyrhynchos. The other utilizes the freshwater mesogastropod prosobranch snail Valvata piscinalis as first intermediate host, and the adults occur in the posterior small intestine and rectum of A. platyrhynchos. The existence of the two sibling species in sympatry may be explained by the fact that the life-cycles of the two forms represent two distinct cycles of transmission serving to reduce excessive competition between them.

An in vitro bioassay was used to examine [14C]glucose incorporation into polysaccharides in albumen glands (AGs) of susceptible M-line Biomphalaria glabrata infected with the NMRI strain of Schistosoma mansoni. Polysaccharide and galactogen synthesis were unaffected by larval trematode infection in AGs of snails at days 14, 21, and 28 post-infection (p.i.) when compared to uninfected controls. Further experiments were conducted to determine if daughter sporocysts, hypothesized to be primary mediators of parasitic castration in this system, were able to exert direct effects on synthetic activity of uninfected AGs via haemolymph-borne molecules or in vitro culture-generated larval excretory–secretory (ES) products. When AGs were incubated in the presence of infected snail haemolymph, significant differences in quantities of polysaccharides and galactogen were detected only in test organs incubated in day 28 p.i. haemolymph. Daughter sporocyst ES products generated during the first 48 h of culture caused a significant reduction in polysaccharide and galactogen synthesis in test organs. When ES products from days 3 to 6 of in vitro culture were tested similarly, no significant differences in either polysaccharide or galactogen synthesis were observed between control and test organs. These data demonstrate that daughter sporocysts are able to modulate a specific aspect of the reproductive activity of the snail host through haemolymph-borne molecules of host or parasite origin, or directly through in vitro culture-generated ES products.

Blood digestion was studied in strains of Anopheles stephensi which had been genetically selected for either refractoriness or susceptibility to infection by Plasmodium falciparum. Females of the refractory Pb3—9a strain ingested more blood than selected (Sda-500) and unselected (Punjab) susceptible females and began to degrade the haemoglobin soon after feeding. In susceptible females, haemoglobin degradation started only after a significant post-feeding lag period. Total protein content of the midgut after the bloodmeal was correspondingly higher for refractory than for susceptible females, but absolute and relative rates of protein degradation were not significantly different between the different mosquito strains. Bloodmeal induction of midgut trypsin activity and the maximal trypsin activity were the same for the different strains. The residual aminopeptidase activity and its relative post-feeding activity (enzyme units per midgut) were significantly higher in refractory females. However, when converting to specific aminopeptidase activity, no differences between strains were evident. The results indicate that both the early initiation of haemoglobin degradation and higher aminopeptidase activity in the Pb3—9a refractory strain are important in the limitation of parasite development within the mosquito midgut, whereas trypsin plays no role in this process.

Transmission of cercariae of the echinostome digenean Echinoparyphium recurvatum was observed in experimental populations of the second intermediate host snail Lymnaea peregra. Populations of 100 snails were distributed among plastic mesh cages in an infection arena to produce different patterns of host dispersion. Cercarial transmission was investigated in host populations showing three separate patterns of host dispersion; Ideal Regular, Random and Contagious (‘Clumped’). Levels of cercarial transmission in populations showing each type of dispersion pattern were found to be significantly different. The highest rate of cercarial transmission occurred in the contagiously dispersed host population, and the lowest in the randomly dispersed population. Analysis of results from the randomly dispersed population also showed that both the mean percentage prevalence of infection, and also the mean number of cysts recovered per snail, increased significantly with the number of snails per cage. The positive relationship between the mean number of cysts per snail and the number of snails per cage was found to be best described by an exponential function. Chemosensory location of L. peregra snail hosts by E. recurvatum cercariae may provide an explanation for the patterns of cercarial transmission observed.

Two natural human interferon alpha preparations, (nHuIFN-μ [Cantell]) and (Nhuifn-μ [ISI]), were used for the oral treatment of cattle experimentally infected with Theileria parva parva. In the first experiment, 8 Friesian bulls were inoculated with a 1 in 10 dilution of a sporozoite stabilate of T. p. parva (Marikebuni) stock. Four of the cattle were treated daily with 1 international unit/kg body weight (i.u./kg bwt) of nHuIFN-μ (Cantell) from day –2 to day 8 p.i. None of the 4 calves given IFN developed clinical theileriosis, but 3 of the 4 control calves died of theileriosis while the fourth had a mild infection. Three of 4 treated calves and the 1 surviving control calf developed a detectable antibody response to T. p. parva schizont antigen but, on challenged with a 10-fold higher dose of stabilate, the surviving control animal and only 1 of the 4 treated calves proved to be immune. In a second experiment, 4 groups of 4 calves were inoculated with the same stabilate dilution. Three treatment groups were given either 1 i.u. nHuIFN-μ (Cantell), 1 i.u. nHuIFN-μ (ISI), or 10 i.u. nHuIFN-μ (ISI)/kg bwt from day –2 to day 8 p.i. once daily and the fourth group were controls. Clinical theileriosis occurred in 2 controls, 2 calves given 10 i.u. nHuINF-μ (ISI), 1 calf given 1 i.u. nHuIFN-μ (ISI) and no calves given 1 i.u. nHuIFN-μ (Cantell)/kg bwt. Of these, 2, 1, 0 and 0 cattle died in the respective groups. All the surviving cattle proved to be immune on homologous challenge with 10-fold higher dose of stabilate except the 2 cattle which did not develop high antibody responses. A third experiment using an undiluted challenge of T. p. parva (Muguga) sporozoite stabilate (10α) on 8 steers. Four steers were treated with 1 i.u. nHuIFN-α (Cantell)/kg bwt and 4 were controls. All calves developed acute theileriosis and the experiment was terminated. Cells of the C2 lymphoblastoid cell line, infected with T. p. parva (Muguga) schizonts, were cultured in vitro with various concentrations (0-01-100 i.u./ml) of nHuIFN-a (Cantell). The IFN appeared to have no effect on host cell or parasite developmental variables when compared to untreated control cultures.