The American Journal of Tropical Medicine and Hygiene - Volume 29, Issue 3, 1980

Two years ago, when I had the privilege of giving the Craig lecture before this Society, my subject was the cultivation of parasites. With the very similar title now, I cannot blame you if you begin to think I have gotten into a rut and can't get out. That's so likely to happen with advancing age. Actually, however, this time I will be telling more about people and places than about parasites. The parasites served to bring me to these people and places.

It was 3 years ago that it became clear that we had efficient, reproducible methods for continuous culture of the human malaria parasite Plasmodium falciparum. The Special Programme for Research and Training in Tropical Diseases of the World Health Organization then laid plans to spread this new technique to laboratories in countries where malaria is a major problem.

Presentations like this have several objectives. The main one is, of course, to honor the person whose name is given to the lecture; that is, to make a public and distinct recognition of his activities. The occasion could be used also to remember his qualities, to point out aspects of his experiences which might be useful for the understanding or solution of current problems, and to point out examples which deserve to be followed. In summary, to find inspiration in his life. Another objective is to discuss some topics which might be related to the ideals of the person honored and to perpetuate his memory in this way.

Having been invited to deliver the second Fred L. Soper Lecture, I appreciate the undeserved honor which has been bestowed upon me. There are indeed many persons with higher qualifications than myself to accomplish this important task.

Three recently isolated African strains of Plasmodium falciparum were tested in vitro for their response to chloroquine. Both the 48-hour method described earlier and a modified 48-hour test were used, yielding comparable results. Strain FCR-7/Kenya, isolated from a clinically chloroquine-resistant case, was more resistant to the drug in vitro than the two other strains (FCR-8/West Africa and FCN-1/Nigeria, both isolated from chloroquine-sensitive cases). Complete inhibition of parasite growth occurred for strain FCR-7/Kenya in a drug concentration range ten times higher than for strains FCR-8/West Africa and FCN-1/Nigeria. In the modified 48-hour test, a lower erythrocyte suspension (2%) allows continuous growth of the parasites over a 48-hour cycle without necessitating change in medium. It thus offers distinct advantages for routine laboratory work as well as for potential field trials.

The preparation and storage of Plasmodium berghei sporozoites for immunization purposes is described. The sporozoites were harvested from the salivary glands of infected mosquitoes, and maintained in cold tissue culture medium M199 with or without mouse serum. They were irradiated and frozen either at -75°C or in liquid nitrogen. After various periods sporozoites were thawed and injected into A/J mice. At the end of the immunization period the animals were challenged with infective sporozoites of P. berghei and monitored for parasitemia. It was found that the storage did not appreciably alter the ability of the irradiated sporozoites to induce protective immunity in the recipient animals. The highest protection (80–100%) was induced with sporozoites maintained in 10% serum and stored at -75°C.

Five strains of inbred rats (ACI, BUF, F344, WF and WL) were infected with Trypanosoma rhodesiense and killed at 28 days for renal pathology and serological studies. The BUF strain demonstrated the most consistent and severe glomerulonephritis, accompanied by high titer IgM antibodies to trypanosomes and high total IgM levels. The WL strain demonstrated mild glomerulonephritis but prominent PAS (+) intracytoplasmic inclusions in proximal convoluted tubules, accompanied by low titer IgM antibodies and slightly elevated total IgM levels. All strains had low to medium titer IgG1 and IgGa antibodies to trypanosomes. Serial serologic studies in BUF rats showed peak titers of IgM, IgGa and IgG1 antibodies to trypanosomal antigens at 10 days. Immune complexes as measured by a C1q binding assay were elevated at 10 days, 3 days following the first detectable antibody responses, and remained so through the 30th day.

Sera from nine populations in Surinam, South America, were screened for antibodies against Entamoeba histolytica by the enzyme-linked immunosorbent assay (ELISA) technique and a precipitin test (gel diffusion or counterimmunoelectrophoresis). In two small surveys in 1974 and 1978, a high rate of clinically significant ELISA titers was found in a rather isolated group of Amerindians living near the Brazilian border, predominately in the youngest age groups. Amerindians living near the coast showed a low level of seropositivity. Low levels were also found in Bush Negro villages in the interior of Surinam and in Hindustani school children from the savannah area. A high frequency of ELISA titers ⩾320, indicating a high rate of invasive amebiasis, was detected in persons in a mental institution in Paramaribo. It is concluded that the ELISA, which is much more sensitive than the precipitin tests, is a useful screening test for assessing the prevalence of invasive amebiasis on an epidemiological scale.

Labrador, a previously unsurveyed area of Canada, has been sampled for human intestinal parasites. Four hundred and one asymptomatic volunteers between 1 and 72 years of age, including Inuit, Naskapi and whites, were examined during the summer of 1977. They harboured: Entamoeba coli, E. histolytica, E. hartmanni, Giardia lamblia and Diphyllobothrium sp. The infection rates are considerably lower than those found in other studies of Northern Canadian communities.

A 21-mm filarial worm appeared suddenly in the anterior chamber of the right eye of a 32-year-old man in western Oregon. By a simultaneous irrigation-aspiration procedure, it was removed alive and only slightly damaged and was identified as a female Dipetalonema in the fourth stage of development. It was the third such case to be reported from western Oregon. In this and one other case the worms were morphologically similar to adult worms identified as Dipetalonema arbuta Highby 1943 from the body cavity of the porcupine (Erethizon dorsatum) and a similar species, Dipetalonema sprenti Anderson 1953, from the body cavity of the beaver (Castor canadensis).

Mansonella ozzardi infections were found in 80/139 (58%) of Indians living in the forest of the upper Caura River Basin located in the southwestern part of Bolivar State, Venezuela. In four (3%) of the blood samples a second type of microfilaria which could not be identified was also found.

Simulium exiguum were collected in the San Antonio area on the Rio Micay as they blood-fed on a volunteer naturally infected with Onchocerca volvulus. The daily dissection of specimens revealed that delayed larval development was common and 23% of the larvae were abnormally formed. However, third-stage larvae were recovered by late day 5 (140 hours) following engorgement. It is concluded that S. exiguum is the vector of O. volvulus at this Colombian endemic focus even though its host efficiency is somewhat limited. S. metallicum collected near Cali from a naturally infected San Antonio volunteer was shown to be a relatively poor experimental host for O. volvulus.

Experimental infections of Onchocerca gutturosa, O. cervicalis, and O. volvulus were induced in males and females of a strain of Aedes aegypti originally selected for susceptibility to Brugia malayi. Mosquitoes were inoculated intrathoracically with microfilariae from the skins of animals infected with the three parasites. Percentages of female Ae. aegypti harboring third-stage larvae 9 or more days after inoculation were 22.5% for O. gutturosa, 11.1% for O. cervicalis and 14.4% for O. volvulus. Infection rates among male mosquitoes were 39.5% for O. gutturosa and 16.15% for O. cervicalis. Although O. volvulus did develop to the third larval stage in males, high mosquito mortality prevented estimation of the rate of susceptibility. Infection rates among mosquitoes inoculated with cryopreserved microfilariae of O. gutturosa were similar to those in mosquitoes inoculated with microfilariae from fresh skin snips. Of the three species of filariae studied, only O. volvulus had a pronounced pathological effect on the inoculated mosquitoes. While most larvae recovered from Ae. aegypti were normal, pathologies similar to those exhibited by other filariae developing in abnormal hosts were noted in some individuals.

Large scale studies were performed to determine the prevalence of various intestinal helminth infections in urban and rural areas of Khuzestan Province, southwest Iran. A total of 16,361 stool samples from people in 105 villages and 14 small towns were examined by the formalin-ether concentration method. Ascaris, hookworms, Trichostrongylus, Trichuris, and Hymenolepis nana showed the highest prevalence. Hookworms and Trichostrongylus were twice as prevalent in rural than in urban areas; in contrast, H. nana was more prevalent in urban areas while the level of Ascaris infections was not significantly different. The intensity of Ascaris and hookworm infections, as estimated by the number of eggs/g of feces, was higher in rural than in urban areas.

Fecal pellets of mice infected with Trichinella spiralis, Hymenolepis nana, and Schistosoma mansoni have been found to contain high levels of phospholipase B activity. The rise, time course and decline of the enzymatic content of the pellets correlate with the known patterns of intestinal injury and reaction due to the parasites or their eggs. Treatment with drugs (thiabendazole, niclosamide, niridazole) which are effective in suppressing the infection also prevents the rise, or causes an early decline, in the titers of phospholipase B appearing in the excreta. These findings complement the previous reports of a close correlation between accumulation of this enzyme in the intestine and infection of mice with T. spiralis and H. nana. Determination of fecal phospholipase B activity constitutes a relatively simple, quantitative, and blood-free method of following the course of infection and its response to treatment, which might be of particular advantage in long term studies and preliminary therapeutic screening.

The applicability of seven different antigen preparations for the detection of antibodies against Schistosoma mansoni was tested in the enzyme-linked immunosorbent assay. For this purpose, sera from children and adults from Surinam infected with S. mansoni were screened for the presence of specific antibodies against the various antigens. With all antigens, generally better results were obtained with the sera from children than with those from adults. The best results were obtained when the trichloroacetic acid-soluble fraction of adult worm antigen (containing the proteoglycan circulating anodic antigen) was used.

An enzyme-linked immunosorbent assay done with microtiter plates and capable of testing hundreds of sera per day has been developed and evaluated for the detection of antibodies to DNA. An increase of antibody levels with age in New Zealand White mice was demonstrated. We demonstrated for the first time that New Zealand White mice infected with Schistosoma mansoni for 10 weeks have higher anti-DNA antibody levels than controls. We demonstrated that NIH outbred albino mice infected with S. mansoni for 18 weeks developed anti-DNA antibodies by 6 weeks of infection, and found a suggestion that immune complexes are present in the circulation by 9 and 11 weeks of infection. A reasonable correlation between visual end-point titration readings and spectrophotometric readings was found. However, spectrophotometric measurements revealed more subtle changes in antibody concentrations that did visual readings. These results demonstrate the usefulness of the micro-ELISA technique for the detection of anti-DNA antibodies in experimental animals, and warrant further evaluation of this test with the serum of humans with autoimmune diseases such as systemic lupus erythematosus.

We report details of a population-based clinical morbidity study of 77 subjects from the Qalyub Governorate, Egypt, with high intensity schistosomal infections. Male Egyptians, mostly farmers, infected with either Schistosoma mansoni, S. haematobium, or both were hospitalized for a routine battery of screening tests, including a standardized history and physical examination, quantitative schistosomal egg counts from serial urine and stool samples, examination of stools for parasites, urinalysis, urine and blood cultures, hepatitis-B surface antigen, complete blood count, liver and kidney function tests, blood chemistries, electrocardiogram, proctoscopy, chest and abdominal X-rays, and intravenous pyelography in those with high intensity S. haematobium infections. Study participants were selected on the basis of schistosomal egg count and age (not on the basis of symptoms of physical findings), agreed to participate, and had not received antischistosomal therapy during the previous 3 months. Schistosomal egg counts ranged from 10 to 3,580 eggs per gram of stool for S. mansoni, and 0.26 to 2,432.8 per 10 cc of urine for S. haematobium. Despite the high intensity of S. mansoni infections the degree of morbidity was not striking, with the possible exception of rectocolonic polyposis noted in 12% of our sample. Infections with S. haematobium were associated with considerable damage to the urinary tract, although significant urinary tract infections and the nephrotic syndrome were notably absent. The findings are interpreted in light of recent changes in the pattern of schistosome transmission in the Nile Delta.

Metrifonate (0,0-dimethyl-[1-hydroxy-2,2,2-trichloroethyl]-phosphonate), and its rearrangment product dichlorvos (2,2-dichlorovinyl dimethyl phosphate) (DDVP), were studied in plasma from two patients with schistosomiasis who were treated with Bilarcil®. A mass fragmentographic technique was used. Isotopic variants of the substances were used as internal standards and to compensate for DDVP formed during the workup procedure. The results were related to erythrocyte and plasmacholinesterase determinations. The method described makes it possible to study pharmacokinetics in man and to relate this information to therapeutic effects. It is proposed that metrifonate acts as a slow release formulation for DDVP.

Morphologically distinct circumoval precipitin reactions, not having the usual “segmented” appearance, are characteristic of recently acquired infection of man with Schistosoma japonicum. In contrast to the precipitins seen in plasma of those with chronic infections, reactions of recent infection (RRI) are commonly, large, vacuolated blebs or, if elongate, are smooth and vacuolated in structure. Recognition of these RRI provides a practical, simple method to study incidence of infection and the techniques used are readily accepted by children in primary school grades.

A 2-year epizootiological study was carried out on Schistosoma bovis in cattle in an enzootic area of the Sudan. The prevalence of infection, as judged by the Pitchford fecal egg counting technique, was very high, approaching 90% in 1.5-year-old animals. There was, however, a lower prevalence in older cattle, and this trend was also seen with the fecal egg counts. This suggests that the cattle gradually acquire resistance to reinfection, particularly since there seems to be little age-related variation in the amount of water contact. Monthly incidence rates, estimated from fecal examinations of initially uninfected “tracer” calves showed a marked seasonal pattern, being much higher in the hot summer months, and snail infection rates showed a similar seasonnal pattern. In addition, both the monthly incidence measurements and the snail infection rates showed that transmision was much heavier in 1976 than in 1977. Epizootics such as those recorded previously in this area presumably arise from exceptionally intense transmission years.

Epizootiological observations on Schistosoma bovis in cattle at Kosti, Sudan, showed a significant fall in age-specific prevalence and intensity with age, based on fecal egg count. To test the possibility that this is due to acquired resistance, Kosti cattle and a control group of cattle of similar breed and age from a nonenzootic area were experimentally challenged with 70,000 S. bovis cercariae. Clinical observations showed very clearly that the Kosti cattle were able to withstand almost completely the effects of the challenge, whereas the controls developed lethal infections. Resistance was further demonstrated by clear differences between the two groups in terms of their body weights, hematological measurements, histopathological and pathophysiological responses, and worm and egg counts. The data suggested that the main basis of the resistance was a suppression of egg production by the worms from the challenge, rather than absolute prevention of their maturation. There was also evidence of a suppression of the fecundity of worms in the naturally infected Kosti cattle.