Development of cell mediated immunity to flagellar antigens and acquired resistance to infection by Trypanosoma cruzi in mice

Modulation by BCG and/or cyclophosphamide of sensitization of mice with flagellar fraction (a tubulin-enriched fraction) prevented death of mice challenged with T. cruzi CL strain trypomastigotes recovered from Vero cells. A methodology was ceveloped to assay specific antigens and to determine optimal doses for sensitization and elicitation of DTH in mice. CL strain is predominantly myotropic strain which does not produce important parasitism of mononuclear phagocyte cells; these cells appear to control infection when activated in vivo. Maximum protection was seen in this study when BCG and cyclophosphamide were associated, but protection was observed also when cyclophosphamide, that prevents supressor T cells, was applied 2 days before flagellar fraction sensitization in normal mice. These experiments suggested that the macrophage may have an important role in the early phases of infection particularly when nonspecific stimulation is associated with specific sensitization. A correlation betwen delayed hypersensitivity to parasite antigens and protection was observed.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease. 1 - Comparative xenodiagnosis with nine triatomine species of animals with acute infections by Trypanosoma cruzi

In search of a suitable vector species for xenodiagnosis of humans and animals with chronic Chagas' disease we first investigated the reactions of different vector species to acute infection with Trypanosoma cruzi. Vector species utilized in this study were: Triatoma infestans, Rhodnius prolixus and Triatoma dimidiata, all well adapted to human habitats; Triatoma rubrovaria and Rhodnius neglectus both considered totally wild species; Panstrongylus megistus, Triatoma sordida, Triatoma pseudomaculata and Triatoma brasiliensis, all essentially sylvatic but some with domiciliary tendencies and others restricted to peridomestic biotopes with incipient colonization of human houses after successful eradication of T. infestans. Results summarized in Table IV suggest the following order of infectivity among the 9 studied vector species: P. megistus with 97.8% of infected bugs, T. rubrovaria with 95% of positive bugs a close second followed by T. Pseudomaculata with 94.3% and R. neglectus with 93.8% of infected bugs, almost identical thirds. R. prolixus, T. infestans and T. dimidiata exhibited low infection rates of 53.1%, 51.6% and 38.2% respectively, coupled with sharp decreases occuring with aging of infection (Fig. 1). The situation was intermediate in T. brasiliensis and T. sordida infection rates being 76.9% and 80% respectively. Results also point to the existence of a close correlation between prevalence and intensity of infection in that, species with high infection rates ranging from 93.8% to 97.8% exhibited relatively large proportions of insects (27.3% - 33.5%) harbouring very dense populations of T. cruzi. In species with low infection rates ranging from 38.2% to 53.1% the proportion of bugs demonstrating comparable parasite densities was at most 6%. No differences attributable to blood-meal size or to greater susceptibility of indigenous vector species to parasites of their own geographical area, as suggested in earlier...

Acquired resistance of mice against S. mansoni and lung granulomatous reaction induced by BCG

Studies carried out in Sw outbred mice showed that there is no correlation between the degree of lung granulomatous reaction and the level of acquired resistance against S. mansoni infection induced by BCG.

Efficacy of ivermectin against the bloodsucking insect, Rhodnius prolixus (Hemiptera, Triatominae)

Ivermectin (0.2 mg/kg body weight) caused a high mortality in nymphs and adults of Rhodnius prolixus following a single meal in mice sub-cutaneously injected with the drug. This effect was more evident in nymphs of 1st-and 2nd-instar than in older nymphs and adults. Third-instar nymphs presented a high mortality when fed on mice treated with ivermectin 24 and 48 hours previously, while mortality was significantly reduced in nymphs fed on mice treated 72 hours before. Surviving 3rd-instar nymphs did not molt. When adult females were fed once on mice treated for 24 hours with ivermectin there was a considerable reduction in egg production. This inhibition was not reversed by a second feeding on normal mice. We concluded that sub-lethal doses of ivermectin caused toxic effects interfering in the neuro-endocrine control of development and reproduction of this bloodsucking insect.

Studies in search of a suitable experimental insect model for xenodiagnosis of hosts with Chagas' disease: 2 - Attempts to upgrade the reliability and the efficacy of xenodiagnosis in chronic Chagas' disease

In order to upgrade the reliability of xenodiagnosis, attention has been directed towards population dynamics of the parasite, with particular interest for the following factors: 1. Parasite density which by itself is not a research objective, but by giving an accurate portrayal of parasite development and multiplication, has been incorporated in screening of bugs for xenodiagnosis. 2. On the assumption that food availability might increase parasite density, bugs from xenodiagnosis have been refed at biweekly intervals on chicken blood. 3. Infectivity rates and positives harbouring large parasite yields were based on gut infections, in which the parasite population comprised of all developmental forms was more abundant and easier to detect than in fecal infections, thus minimizing the probability of recording false negatives. 4. Since parasite density, low in the first 15 days of infection, increases rapidly in the following 30 days, the interval of 45 days has been adopted for routine examination of bugs from xenodiagnosis. By following the enumerated measures, all aiming to reduce false negative cases, we are getting closer to a reliable xenodiagnostic procedure. Upgrading the efficacy of xenodiagnosis is also dependent on the xenodiagnostic agent. Of 9 investigated vector species, Panstrongylus megistus deserves top priority as a xenodiagnostic agent. Its extraordinary capability to support fast development and vigorous multiplication of the few parasites, ingested from the host with chronic Chagas' disease, has been revealed by the strikingly close infectivity rates of 91.2% vs. 96.4% among bugs engorged from the same host in the chronic and acute phase of the disease respectively (Table V), the latter comporting an estimated number of 12.3 x 10[raised to the power of 3] parasites in the circulation at the time of xenodiagnosis, as reported previously by the authors (1982).

The study of insect blood-feeding behaviour: 1. Feeding equipment, physical and endogenous factors, dose effect analysis, and diet destination

Experimental techniques that we have found useful during our studies of insect blood-feeding behaviour are reviewed. Some of the principal findings resulting from these techniques are discussed. Where directly applicable, the work of others is included, but no complete review of the subject has been attempted.

Evolution of holometabola insect digestive systems: physiological and biochemical aspects

This review takes into account primarily the work done in our laboratory with insects from the major Holometabola orders. Only the most significant data for each insect will be presented and a proposal on the evolution of Holometabola insect digstive systems will be advanced.

The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.

The Azadirachtins: potent insect growth inhibitors

In the course of their coevolution with insects, plants have learnt to protect themselves by chemical means. Semiochemical act as antifeedants or deterrents, others by disrupting growth and development. By use of the Epilachna varivestis bioassay we isolated from Azadirachta indica seed a group of triterpenoids which interfee with larval growth and development in ppm range. Main components are the azadirachtins A and B with identical biological activity. Various other azadirachtins were obtained, either as minor seed components or by chemical modification of the naturally occuring compounds. Structure vs. activity relation studies enabled us to postulate a basic structural element that should still be biologically active and with much simpler chemical structure than natural compounds. What underlies the biological activity of these insect growth inhibitors? Their interference with the hormonal regulation of development and reproduction has been studied in Locusta migratoria and Rhodnius prolixus. In addition, tritiated dihydroazadirachtin A was used. With this approach, a precise correlation between administered dose, resulting effects, and retention of the compound was established. The azadirachtins either interrupt, delay, or deviate whole developmental programs. Results from these studies provide another chemical probe for studies in insect endocrinology and physiology.

Prospects for a nonliving vaccine against Schistosomiasis based on cell-mediated immune resistance mechanisms

We have designed a vaccine model based on induction of cell-mediated immunity and shown that it protects mice against Schistosoma mansoni infection. Mice are immunized by intradermal injection with schistosome antigens plus BCG. Resistance is dependent on the route of antigen presentation and the adjuvant chosen. The pattern of resistance correlates with sensitization of T lymphocytes for production of gamma interferon, a macrophage activating lymphokine that stimulates the cellular effector mechanism of protection. Purified schistosome paramyosin, a muscle cell component present in soluble parasite antigenic preparations, is immunogenic for T lymphocytes and induces resistance when given intradermally with BCG. It is likely that this protein, and possibly other soluble molecules that are released by the parasites of a challenge infection, induce a cellular inflammatory response resulting in larval trapping and/or killing by activated macrophages. These results verify the feasibility of a vaccine against schistosomiasis based on induction of cell-mediated immune resistance mechanisms.

Drug resistance in Schistosomiasis: a review

Drug resistance associated with the treatment of human schistosomiasis appears to be an emerging problem requiring more attention from the scientific community than the subject currently receives. Drug-resistant strains of Schistosoma mansoni have been isolated by various investigators as a result of laboratory experimentation or from a combination of field and laboratory studies. Review of this data appears to indicate that the lack of susceptibility observed for some of the isolated strains cannot be ascribed solely to previous administration of antischistosome drugs and thus further studies are required to elucidate this phenomena. Strains of S. mansoni have now been identified from Brazil which are resistant to oxamniquine, hycanthone and niridazole; from Puerto Rico which are resistant to hycanthone and oxamniquine; and from Kenya which are resistant to niridazole and probably oxamniquine. Strains derived by in vitro selection and resistant to oxamniquine and possibly to oltipraz are also available. All of these strains are currently maintained in the laboratory in snails and mice, thus providing for the first time an opportunity for indepth comparative studies. Preliminary data indicates that S. haematobium strains resistant to metrifonate may be occurring in Kenya. This problem could poise great difficulty in the eventual development of antischistosomal agents. Biomphalaria glabrata from Puerto Rico and Brazil were found to be susceptible to drug-resistant S. mansoni from each country.