Certain L-amino acid esters, such as L-leucine methyl ester (Leu-OMe), can kill intracellular and isolated Leishmania amazonensis amastigotes. Killing appears to involve ester trapping and hydrolysis within an acidified parasite compartment (M. Rabinovitch and S. C. Alfieri, 1987, Brazilian Journal of Medical and Biological Research 20, 665–74). We show here by acid phosphatase light microscopic cytochemistry and by ultrastructural morphometry that megasomes, lysosome-like amastigote organelles, are the putative parasite targets of Leu-OMe. This conclusion is supported by the following observations, (a) Control amastigotes displayed a string of electron-dense, acid phosphatase-positive megasomes mostly located in the cellular poles opposite the flagellar pockets. Incubation of the amastigotes with Leu-OMe resulted in concentration-dependent swelling and fusion of the organelles as well as decreased electron density of the internal contents. These changes, which preceded parasite disruption, were followed by the progressive loss of parasite viability and the release of acid phosphatase activity into the medium, (b) Incubation of the amastigotes with L-isoleucine methyl ester, a non-leishmanicidal compound, induced only moderate fusion of the megasomes. (c) Pre-incubation of the parasites with the proteinase inhibitors antipain and chymostatin, previously shown to confer protection from Leu-OMe toxicity, nearly completely prevented the morphological changes of megasomes. (d) Exposure of amastigotes to tryptophanamide (Trp-NH2), the leishmanicidal activity of which is not reduced by antipain and chymostatin, did not result in swelling and fusion of the megasomes. This last finding suggests that different mechanisms underlie the destruction of amastigotes by Trp-NH2 and Leu-OMe. Overall, the results are compatible with the hypothesis that Leu-OMe and other amino acid esters are trapped and hydrolysed within megasomes.

Acute toxoplasmosis was induced in mice, and interferon (IFN) production in serum and by spleen cells was evaluated during the infection period. Interferon was characterized by acid-treatment and anti-IFN-α/β neutralization. In order to verify the correlation between the unusual aspects of the IFN production and the induction of immunosuppression, splenocyte mitogen responsiveness was investigated concomitantly to IFN synthesis. The activity of Toxoplasma-induced serum IFN-α/β increased gradually throughout all post-infection days, but IFN-γ was not detected in the systemic circulation at any time during the infection. It was also observed that IFN-α/β production and the capacity to produce IFN-γ by spleen cells were closely and inversely correlated. As the infection progressed, more IFN-α/β was produced, and the ability of spleen cells to produce IFN-γ decreased. The observation that Toxoplasma-infected mice were concomitantly immunosuppressed (as documented by mitogen unresponsiveness and defective IFN-γ production) in direct correlation to IFN-α/β production, suggests that such IFN-α/β production is an important factor associated with acute toxoplasmosis-induced immunosuppression.

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.

Antibodies against the retrovirus envelope glycoprotien (gp70) of mouse xenotropic retrovirus, BALB virus 2 (Bv2) reacted with the adult worms of Schistosoma japonicum and S. mansoni. This reaction was completely inhibited after adsorption of the antibodies with virions of retrovirus. The reactive schistosome antigen was located in the subtegumental layer of the adult male fluke and in the vitelline gland of the adult female of S. japonicum and S. mansoni. Proteins extracted from both parasites were examined by immunoblot analysis. Anti-Bv2 gp70 antiserum reacted with those proteins from both schistosomes and the band patterns were different among sexes and species. Southern hybridization of the DNA extracted from adults of S. japonicum and S. mansoni demonstrated the presence of sequences homologous to the env gene of mouse ecotropic and xenotropic retroviruses. DNA sequences homologous to the gag and pol regions of the ecotropic murine leukaemia virus were also detected in the DNAs of schistosomes

Newly transformed schistosomula and day 8 lung parasites, derived from optimally irradiated cercariae, were used to immunize groups of C57B1/6 mice via 4 different injection routes. Schistosomula administered intradermally induced high levels of protection, comparable with that achieved by percutaneous vaccination. Intermediate levels were elicited by delivery of parasites via intraperitoneal or intratracheal routes. In contrast, intravenous injection of schistosomula to the lungs resulted in little or no resistance. Attenuated day 8 schistosomula administered intradermally were at least as immunogenic as irradiated cercariae. The fate of radio-isotope labelled attenuated lung schistosomula, injected via the various routes, was examined by compressed organ autoradiography. After intradermal vaccination, a proportion of parasites migrated from the site of injection to the draining lymph node and lungs. Conversely, schistosomula administered via the other 3 routes persisted to varying degrees at the injection site, but little onward migration was observed. We suggest that successful vaccination requires that some attenuated parasites migrate to, and sequester in, lymph nodes draining the vaccination site; persistence at the site of administration alone is not an adequate stimulus.

The prevalence of schistosome infections in intermediate host snails varies with snail age. The relationship between age and prevalence, the age—prevalence curve, is complex and may vary in space and time, and among parasite—host species. Field studies show that the shape of the age—prevalence curve may be seasonally variable, and that at some times there may be a decline in prevalence among older snails. This paper attempts to explain these observations in terms of the underlying epidemiological processes. A discrete-time version of Muench's catalytic model for age-dependent infection is developed. Model simulations were carried out using life-history and epidemiological parameters derived from studies of Schistosoma haematobium—Bulinus globusus in Zimbabwe. Analysis of model behaviour identifies aspects of the schistosome— snail interaction that affect the shape of the age—prevalence curve. The following features can result in a decline in prevalence among older snails. (1) A decrease in the survival rate of patent infected snails with age. (2) A decrease in the force of infection with age. (3) A high rate of loss of infection. (4) A heterogeneity in the snail population such that the probability of infection is correlated with snail fecundity. (This would occur if there existed a spatial correlation between force of infection and fecundity, or if there were a correlation between fecundity and susceptibility.) The evidence for the occurrence of these features in the field is assessed. Survival rate is related more closely to the duration of patent infection than to age per se. The evidence for age-dependent force of infection is equivocal. Significant loss-of-infection rates have yet to be demonstrated. Heterogeneities in force of infection and fecundity have been reported and, for the Zimbabwe data, this mechanism can explain seasonally in the age—prevalence curve as a function of known seasonal variation in the force of infection and snail fecundity.

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.

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 × 103 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 × 10−6 to 6.9 × 10−3 switches/cel/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 × 10−3 switches/cell/generation. These values are between 20 and 66000-fold higher than previously published estimates. It is likely that at least 1 in every 100 trypanosomes switches its VAT expression every generation.

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.

A comparison was made of the development, survival and reproduction of 5 isolates of Trichinella spiralis in inbred mice. Low responder C57BL/10 mice allowed worms of all isolates to survive longer and reproduce more successfully than did high responder NIH mice, suggesting that host immunity exerted a dominant influence upon infectivity. One isolate (Is-5 (W) — an arctic isolate) had a markedly lower infectivity than all other isolates, and was selected for more detailed study, together with isolate Is-1 (S) (a temperate isolate) which showed high infectivity. The lower infectivity of Is-5 (W) reflected a more rapid onset of immunity in mice infected with this parasite, immunity reducing the reproductive potential of female worms and causing an early expulsion from the intestine. No evidence of a dose-dependent suppression of immunity was found to explain the higher infectivity of Is-1 (S). Is-5 (W) provided a very rapid mucosal mastocytosis in infected mice, whereas Is-1 (S) caused no measurable response. In contrast, Is-1 (S) elicited higher levels of circulating parasite-specific antibodies than did Is-5 (W). These results are discussed in relation to the interplay of parasite immunogenicity and host immune responsiveness in determining infectivity, and point to the importance of identifying those immunodominant parasite molecules which control the balance of the host—parasite relationship.

A qualitative and quantitative analysis was made of the release of surface-associated molecules from developing Dirofilaria immitis infective-stage larvae (L3). D. immitis L3s were labelled with 125I using an Iodogen catalysed reaction and either maintained in culture or placed in chambers that were implanted into Lewis rats. The larvae released 10–20% of the labelled material each day during the first 4 days of in vitro and in vivo development. The loss of surface-labelled peptides from developing larvae corresponded with an increase in the amount of trichloroacetic acid-precipitable radioactivity found in the culture medium. SDS—PAGE analysis of the labelled material showed that the same 35 and 6 kDa components found in larval extracts were shed into culture medium by the developing parasites. Metabolic labelling studies and experiments in which larvae were labelled after different times in culture indicated that, once released, the surfaceassociated molecules were not replaced, and that this net loss of surface peptides resulted in a reduction in the antigenic potential of the cuticular surface. Antibodies from both immunized rabbits and naturally infected dogs immunoprecipitated the 35 kDa component. In contrast, the 6 kDa molecule was not recognized by the antibodies in any of the sera tested. Shedding of surface peptides and reducing surface antigenicity may represent mechanisms by which D. immitis infective-stage larvae evade immune attack.

Phase-interference microscopic examination of the infective, post-infective 3rd-stage and 4th-stage larvae of Dirofilaria immitis has identified a single cell body in each of the paired lateral amphidial nerves which undergoes characteristic morphological change during the development from 3rd- to 4th-stage larvae. Acetaldehyde—fuchsin staining of worm sections revealed fuchsinophilic material in the precise location of the amphidial nerve-cell bodies observed by phaseinterference microscopy. This material was found in 70% of infective larvae recovered from mosquitoes and in 100% of larvae recovered from micropore chambers 24 h after implantation into BALB/C mice. In pre-moult larvae recovered at 42 h (Experiment 1) and at 48 h (Experiment 2) fuchsinophilic material was demonstrable, but no staining was observed in those larvae in which separation of the 3rd- and 4th-stage cuticles had occurred. No such material was observed in 4thstage larvae recovered from chambers after 74 h, and in these larvae the amphidial nerve-cell bodies were not discernible. These cytological observations are consistent with a cycle of elaboration and release of neurosecretion associated with moulting in D. immitis.

In the laboratory, yields of infective 3rd-stage larvae of Trichostrongylus tenuis were determined by temperature when moisture was adequate. On the moor, eggs in caecal faeces did not develop to infective larvae during the winter. In summer, development did occur and yields of infective larvae were related to temperature: greatest yields were obtained when mean monthly soil surface temperatures ranged between 76 and 103°C. Rainfall was unimportant, as was desiccation. During summer the mean monthly mortality of infective larvae was 44% but over winter survival was negligible.

The growth of individual proglottides of Hymenolepis nana has been studied by measuring the width and length of the proglottides between days 3 and 8 post-infection in mice. Two sets of measurements were obtained. The first involved proglottides immediately posterior to the point of proglottis formation and proglottides at particular stages of development. For the second set, measurements were made of proglottides at certain positions in the worm, specifically the 20th, 100th and 200th proglottides, as counted from the posterior end of the worm. For proglottides at different stages in development, the width of a particular stage was greater in the later days of infection, while the length was almost constant. The length of newly-formed proglottides varied only slightly with time despite differences in the width and in the 3-fold increase in proglottis production that occurs between days 3 and 8 post-infection. The width of the 20th, 100th and 200th proglottides followed parallel growth curves that coincided with each other after an appropriate shift along the time axis. The more anterior the position of the proglottis, the greater was the width at the time of proglottis formation, and so its growth began later on the common growth curve. Growth in length of the 20th proglottis was at first faster than the 100th and 200th proglottides, but later slowed down to a level comparable with them, and the growth curves for the length of the three proglottides were very similar to each other. The rates of growth in the volume of the three proglottides were also estimated. The results are also discussed in relation to other approaches to the study of tapeworm growth and to factors that may be responsible for the growth patterns observed.

The development and mortality of the eggs of Ancylostoma duodenale and Necator americanus in distilled water were monitored over a range of temperatures between 15 and 35°C. Egg demography was examined within the context of a fourparameter mathematical model of development and mortality. Over the range of temperatures studied, egg mortality (μ) was an increasing exponential function of temperature (T) measured in degrees Celsius. A single model adequately described the mortality of both species (ln [μ] = 0·041*T − 6·87). The minimum time (τ) to hatching was consistently less for A. duodenale (ln [1/T] = −(0·79+53·05*[l/T]) than N. americanus (ln [1/T] = −(0·99 + 53·05*[l/T])). The hatching rate [σ when t > τ) was an increasing function of time and temperature in both cases, but the precise functional relationship was species specific.

A method is described for the isolation of high molecular weight DNA from females of potato cyst nematodes. Restriction enzyme analyses of repetitive DNA revealed 30 bands specific for Globodera rostochiensis and 8 bands specific for G. pallida. None of the repetitive DNA bands resolved was common to both species. Hybridization patterns, obtained with a DrosophilaβJ-tubulin probe, revealed 4 common bands, 3 bands specific for G. rostochiensis and 12 bands specific for G. pallida.

Groups of cattle were immunized with 10−2 dilutions of sporozoite stabilates of Theileria parva lawrencei derived from African buffaloes either alone or in combination with Theileria parva parva derived from cattle and concomitant treatment with either long or short-acting formulations of oxytetracyline. At 90 or 120 days after infection, uninfected Rhipicephalus appendiculatus nymphal ticks were applied to individual immunized cattle and the resultant adults ticks were applied to individual susceptible cattle. Theilerial infection developed from ticks fed on 6 out of 11 animals investigated for evidence of a carrier state. Two additional animals were shown by cell-culture isolation to have persistent theilerial infections. Nine cattle infected with the parasites from carrier animals were treated with parvaquone and 7 recovered. These recovered cattle were then challenged with the original immunizing stabilates at 10° dilution together with the original immunized and carrier cattle. Six out of 7 cattle which had recovered from carrier-derived infection succumbed to this challenge and died but none of the original immunized cattle showed theilerial reactions. When a carrier-derived sporozoite stabilate was used to challenge cattle immune to the original immunizing parasite, they proved to be immune. Cattle immune to the carrier-derived parasites were all immune to challenge with the original parasite. A monoclonal antibody profile aginst T. parva schizonts isolated by cell culture from samples of the experimental animals did not appear to be sensitive enough to determine the antigenic differences between the carrier-derived parasite and the original immunizing parasite. Indications are that the carrier state is not likely to produce new antigenic strains which would be dangerous to immunized cattle.

A comparison of feeding and breeding performance was made between a laboratory strain (LS) and two freshly collected field strains (FS1, FS2) of Rhipicephalus appendiculatus. The LS had been cultured for over 30 years on tick-susceptible rabbits. When fed on susceptible rabbits LS produced smaller eggs and unfed larvae, nymphs and adults than those of FS. Eggs and larvae from females engorged on cattle were larger than those from females engorged on rabbits. LS and FS females engorged on susceptible hosts were of similar weight, but the FS females were twice as heavy as LS females when tick-resistant hosts were used. A higher proportion of LS ticks than of FS ticks engorged on susceptible hosts, but a lower proportion on resistant hosts. Thus on susceptible hosts LS showed a higher reproduction efficiency than FS, but a lower one than FS on resistant hosts. The proportional reductions of engorged weights and numbers feeding successfully on resistant hosts were significantly lower for LS than FS ticks. Cross-resistance was low between LS and FS. Although all strains were affected by host resistance, it was concluded that the field strains were better adapted to withstand resistance than the laboratory strain.