The American Journal of Tropical Medicine and Hygiene - Volume 37, Issue 1, 1987

Fatal cases of experimental Plasmodium falciparum (Indochina I) in Bolivian squirrel monkeys (Saimiri sciureus boliviensis) were examined by histologic and ultrastructural methods. Gross lesions were characterized by hepatosplenomegaly and interstitial pulmonary changes. Histologically, there was marked diffuse reticuloendothelial hyperplasia, pulmonary alveolar septal thickening, mesangioproliferative glomerulonephropathy, sequestration of parasitized erythrocytes in deep vascular beds, degenerative parenchymal changes in the liver and myocardium, and in one case retinal and cerebral hemorrhage. These data indicate that the Bolivian squirrel monkey is a good model for studying pathologic changes associated with human falciparum malaria.

Squirrel monkeys protected after vaccination with a particular protein fraction of Plasmodium falciparum elicit antibodies directed against two parasite proteins, Mr 90,000 and 72,000. We have used monoclonal antibodies and sera from protected monkeys to determine whether or not these polypeptides were polymorphic in 22 strains. In all the isolates studied, both polypeptides were conserved, as was the epitope defined by monoclonal antibody XIV/7 present on the Mr 90,000. Immunofluorescence of all strains showed the same pattern using 5 Mab produced against different fragments of the Mr 72,000 polypeptide from the FUP strain. All isolates examined were positive, indicating that this polypeptide was present in different strains and that the 5 epitopes were conserved. Peptide mapping of both the Mr 90,000 and 72,000 antigen purified from 3 different strains indicated that each antigen appeared to be conserved.

An enzyme-linked immunosorbent assay (ELISA) was developed to detect antibody in human sera to a synthetic peptide, Asn-Ala-Asn-Pro (NANP)3, derived from the repeating amino acid sequence found in the surface circumsporozoite protein of Plasmodium falciparum sporozoites. One hundred four sera from U.S. residents were used to determine a cut-off value for reactivity. Test sera were considered reactive when the absorbance was greater than that at the 95th percentile of the control sera. Sera from 112 Kenyans living in an area of holoendemic malaria transmission were tested. Of the total number of sera, 65% had detectable antibody to (NANP)3. The percentage of reactive sera increased from 41% in sera from children under 4 years of age to 85% in sera from adults 20 to 39 years of age. The high exposure to malaria parasites of the Kenyans was reflected in indirect fluorescent antibody assay titers to blood stage P. falciparum parasites. All of the Kenyan sera had antibody present at titers greater than 1:256.

Plasmodium falciparum polypeptide Pf155 is one of the main candidates for a vaccine against asexual blood stages of P. falciparum. Antibodies against Pf155 can be detected by a cell-ELISA technique with glutaraldehyde-fixed and air dried P. falciparum-infected erythrocytes as antigen. Using this assay, we measured the level of antibodies in sera from 230 subjects with various degrees of past exposure to P. falciparum. Significant levels of antibodies (OD492 > 0.5) were detected in 41/50 sera from Central African adults and in 34/50 sera from West African adults. All sera from 50 European adults suffering primary malarial attack and 28/30 sera from West African children (10 to 12 years old) were negative. Intermediate results were obtained with 50 sera from West African adults living in France for ≥2 years (12 positive). Mean OD values of the sera of these five groups of subjects varied in the same direction as the positivity rates. These preliminary results suggest that the level of anti-Pf155 antibodies as detected by cell-ELISA might provide an assessment of protective immunity against P. falciparum which could complement clinical or epidemiological criteria.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequences PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-µl samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3′ end of the 28S β coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.

In vitro tests for Plasmodium falciparum sensitivity to pyrimethamine, sulfadoxine, and both drugs in combination were performed in four kinds of culture medium, each differing in p-amino benzoic acid (PABA) and folic acid concentrations. Results of the tests using pyrimethamine-sensitive and pyrimethamine-resistant isolates indicated that drug activity was reduced proportionally to the concentrations of these two growth factors in the medium. The optimal concentrations of PABA and folic acid for parasite growth and drug susceptibility, as evaluated by microscopic examination and by the extent of incorporation of radioactive 14C-pyrimethamine and 14C-sulfadoxine, were 10 ng/ml and 2 ng/ml, respectively. Depletion of PABA and folic acid from the medium had no effect on drug-resistant parasites but multiplication of drug-sensitive isolates was markedly reduced. Medium containing 0.5 ng/ml PABA and 10 ng/ml folic acid was the best for parasite growth regardless of the degree of drug sensitivity. Results obtained by using this medium agreed most closely with results from in vivo observations.

Two leishmanial isolates from dogs from Alexandria, Egypt, were typed serologically and biochemically as Leishmania major. This is the second time that L. major has been shown to occur in dogs. The significance of these findings as a misleading phenomenon in relation to the relatively recent outbreak of infantile kala-azar in the area of Alexandria is discussed.

Fresh normal human serum was observed to have a lethal effect on Leishmania mexicana amazonensis promastigotes obtained from laboratory-bred Lutzomyia longipalpis or on promastigotes grown in liquid culture medium, inoculated with the same isolates. Heat inactivation abolished the Leishmania lytic activity from the sera.

Resistance of culture promastigotes to lysis by normal human serum was investigated in three isolates of L. m. amazonensis. Development of resistance (up to 7%) was found in only one isolate, obtained from the bone marrow in a human case of visceral leishmaniasis.

Domestically bred South American guinea pigs received 3 to 5 immunizing intradermal inocula of 28 × 106 live attenuated Trypanosoma cruzi epimastigotes (TCC strain) per kg. These inocula were unable to produce patent infections or to propagate through vectors. Groups of experimental and control guinea pigs were exposed to natural T. cruzi infection in a field yard for periods of up to 551 days. Xenodiagnoses were applied periodically to all animals. This showed that the incidence of natural T. cruzi infection was significantly lowered at various periods post-exposure. The final proportion of infected animals was 39% (20/51) among vaccinees vs. 63% (32/51) among controls (P < 0.02). The protective effect was exerted particularly upon males and lasted for over a year in one experimental series (infection in 1/7 vaccinees vs. 6/7 controls, P = 0.014). Vaccination reduced vector transmission rates from 38% to 18% (P < 0.001). These results agree with previous laboratory experiments in showing a partial resistance which does not eliminate residual T. cruzi infection. However, the field work indicates that even this kind of resistance may have epidemiological impact, reducing both the number of reservoirs spreading the disease and the rate of vector transmission.

Antigenic differences between extracts prepared from insect- and axenic culture-derived T. cruzi were demonstrated by a protein A-binding immunoradiometric assay.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Upon exposure to Entamoeba histolytica antigen, lymphocytes from patients treated for amebic liver abscess produce lymphokines which activate monocyte-derived macrophages to kill E. histolytica trophozoites. We now demonstrate that gamma interferon (IFN-γ) is produced by these stimulated lymphocytes and is sufficient but not exclusively necessary to activate monocyte-derived macrophage amebicidal activity. Supernatants from mononuclear cells of 7 patients when stimulated with amebic antigen contained more IFN-γ than comparable supernatants derived from control cells (1,862 U/ml vs. 174 U/ml geometric means, P < 0.01); IFN-γ levels were similar in patient and control supernatants following concanavalin A stimulation. Macrophages activated solely by partially purified IFN-γ or recombinant human IFN-γ (300 U/ml) killed 47% of virulent amebae by 6 hr at 37°C. Monocyte-derived macrophages stimulated with lymphokines elicited by amebic antigen or concanavalin A killed 48% and 57% of axenic E. histolytica trophozoites, respectively, over 6 hr at 37°C (P < 0.001 for each compared to control). Macrophages incubated with the identical lymphokines, but in the presence of monoclonal antibody to IFN-γ, were only able to kill 18% and 27% of amebae, respectively, at 6 hr (P < 0.05 to control or when antibody to IFN-γ was not present). If antibody to IFN-γ was added to the stimulating lymphokine, more macrophages died during interaction with amebae (P < 0.05). In summary, IFN-γ has a major but not exclusive role in activating human monocyte-derived macrophages in vitro to kill virulent E. histolytica trophozoites.

Conflicting interpretations regarding the fecundity of schistosomes infecting human beings have arisen and are, in part, due to the inability to directly measure the parameters. The inability to experimentally manipulate human beings necessitates the use of surrogate variables, i.e., number of eggs per gram of feces, as an indicator of worm burden. This study reanalyzes data from quantitative autopsies performed in Egypt by Cheever and colleagues on individuals with active Schistosoma mansoni infections. Exploratory regression analysis of the relationship of female worms recovered to eggs/g of feces and of female worms to eggs retained in host tissues suggests a linear relationship in both cases. Over the observed range of female worms recovered from an individual human being, the estimated worm fecundity, as measured by the number of eggs either in feces or retained in tissues per female worm, is not significantly different from a constant value. Hence, density-dependent fecundity of S. mansoni in the human host, as suggested by others, is not demonstrated in these data.

To investigate the relation between the size of circumoval granulomas and hepatic fibrosis, a variety of mouse strains infected with Schistosoma mansoni were examined and the number of eggs in the tissues, the fibrotic responses to the eggs, and the volume of the granulomas were determined. Marked differences in granuloma volume and in hepatic fibrosis were found between mouse strains, and those strains with the largest granulomas also showed the most hepatic fibrosis. On the other hand no significant correlation between granuloma size and hepatic fibrosis was found in the progeny of the F2 generation and backcrosses between F1 mice and the parental strains when crosses were made between Nmri mice (high granuloma volume and high fibrosis) and C57BL/6 mice (low granuloma volume and low fibrosis). Hepatic fibrosis per egg decreased with increasing infection intensity while granuloma volume was unaffected, indicating that fibrosis and granuloma size are at least in part modulated by different factors. The number of eggs found in the tissues per worm pair and the proportion of eggs in the liver also decreased as infection intensity increased. Some influence of the major histocompatibility complex on both granuloma size and fibrosis was found. Congenic mice on the C57BL/10 and C3H/HeSn backgrounds showed larger granulomas in H-2b than in H-2k mice, but no such correlation was found in comparing C57BL/6 mice with B6.H-2k mice. Less hepatic fibrosis was found in B10.M (H-2f), B10.SM (H-2v), and B10.RIII (H-2r) animals than in C57BL/10 mice. The regulation of granuloma size and of hepatic fibrosis is clearly complex and involves genes both outside of and within the major histocompatibility complex.

Experiments were performed to determine the level of protection in immunized mice to multiple small (trickle) Schistosoma mansoni cercarial challenges. C57BL/6 mice were immunized with either 50 Krad-irradiated cercariae or a soluble worm antigenic extract injected intradermally in conjunction with the adjuvant BCG. A series of cercarial challenges (40 cercariae per exposure), beginning approximately one month after immunization, was given at 4-week intervals. To prevent any contribution of egg-related pathology, the mice were exposed to male cercariae only. The results showed that highly significant levels of protection developed at all times after cercarial challenge. Regardless of the type of immunization or the number of cercarial challenges, the levels of protection which appeared after one, two, three, or four challenge exposures were nearly identical, and equalled those in mice exposed at a single time to a cumulative total of the several exposures. As expected, titers of anti-schistosome antibodies were higher in the immunized and challenged groups than in those challenged alone, and titers increased anamnestically after challenge infection. However, increased antibody titers were not associated with increased resistance in trickle challenged mice. Overall, we showed that the level of vaccine-induced immunity does not change, under these conditions, in the face of repeated cercarial challenges. Also, these results show that single mass cercarial challenges may legitimately be used in experimental situations to assess the effectiveness of vaccines which might be used in subjects repeatedly exposed to cercariae in field situations.

Eosinophils generate hypochlorous acid when stimulated with opsonized particles. The hypochlorous acid can react with β-amino acids such as taurine to produce chloramines with a long lifetime. In the presence of bromide, eosinophils generate hypobromous acid which can react with taurine to generate taurine bromamine. As taurine is abundantly present in leukocytes, eosinophils have the potential to generate taurine chloramine or taurine bromamine. With regard to the role of eosinophils in killing schistosomula, we have now shown that both taurine chloramine and taurine bromamine in physiological concentrations are able to kill the schistosomula of Schistosoma mansoni.

I suggest the hypothesis that bancroftian filariasis, endemic since the early days of slavery in Charleston, South Carolina, disappeared around 1930 by virtue of the long-term effects of a municipal sewerage-water system begun in the 1890s or thereabouts. These public works, originally intended to abate typhoid and related diseases, helped to eliminate filariasis by reducing the availability of polluted domestic waters which are the preferred breeding sites for the urban vector, Culex quinquefasciatus (=fatigans). Cause and effect having been separated by some three decades, the epidemiologic connections between these events in Charleston seem not to have been appreciated hitherto.

A campaign to eradicate dracunculiasis has been underway from the beginning of the International Drinking Water Supply and Sanitation Decade (1981–1990), since providing safe drinking water is the most effective means to prevent that disease. About 120 million persons are estimated to be at risk of the infection in Africa, and 20 million more in India and Pakistan. Both major endemic countries in Asia have begun efforts to eliminate the disease, and by the end of 1986, national anti-dracunculiasis programs were underway or planned in 8 of the 19 affected African countries. In May 1986, the World Health Assembly adopted a resolution on the elimination of dracunculiasis—the first such resolution since the successful Smallpox Eradication Program. India, which began its Guinea Worm Eradication Program in 1980, has already eliminated the disease from one of seven endemic states, and reduced the total number of cases found through active surveillance by 35% between 1983 and 1985. In Côte d'Ivoire (Ivory Coast), the only African country to conduct active surveillance for dracunculiasis so far, an aggressive combined program of rural water supply, health education, and active surveillance has reduced the disease from 4,971 cases in 1976 to 592 cases in 1985.

This paper presents a preliminary assessment of the geographic extent and estimates of the incidence and the population at risk of dracunculiasis in Africa. Nineteen countries are known to be affected, in a belt extending right across the northern part of the continent south of 18°N, and in east Africa extending almost to the equator. Annual incidence is estimated to be 3.32 million, and the at-risk population is approximately 120 million. These data provide an initial baseline on which the success of control measures now being initiated in Africa can be assessed.