Search

Large-scale mutagenesis screens have proved essential in the search for genes that are important to development in the fly, worm, and yeast. Here we present the power of large-scale screening in a vertebrate, the zebrafish Danio rerio, and propose the use of this genetic system to address fundamental questions of vertebrate developmental physiology. As an example, we focus on zebrafish mutations that reveal single genes essential for normal development of the cardiovascular system. These single gene mutations disrupt specific aspects of rate, rhythm, conduction, or contractility of the developing heart.

The liver fluke Platynosomum fastosum was identified upon necropsy of three ex-captive orangutans (Pongo pygmaeus) which had been part of a rehabilitation program for reintroduction to the wild. This trematode has not been reported in orangutans previously and is commonly found in cats in Southeast Asia. Cross infection from cats via intermediate hosts, to orangutans kept in captivity as pets, could explain their presence in the latter. Although P. fastosum caused intrahepatic and bile duct damage, death of the hosts could not be attributed solely to the presence of the liver fluke infection.

A serological survey of confiscated orangutans was conducted to determine the prevalence of specific viral infections cross reacting with human viruses. Antibodies specific for human hepatitis A (HAV) and B (HBV) viruses, herpes simplex viruses (HSV), and human T-lymphotropic virus (HTLV types I and II), as well as for the simian type D retroviruses (SRV types 1 to 3) and simian immunodeficiency virus (SIV) were tested in samples from 143 orangutans. Results revealed a high prevalence of potential pathogens. The most prevalent viral infection found was HBV (59.4% prevalence) of which 89.4% of infected individuals seroconverted to the non-infectious state and 10.6% remained as chronic carriers. Antibodies to HAV, HSV, HTLV-1, and SRV were also detected but at a lower prevalence. There was no evidence of lentiviral infections in this group of animals. The results confirm the importance of quarantine and the need for diagnostic differentiation of virus infections to determine if they are of human origin or unique orangutan viruses.

In a recent serological survey among 143 ex-captive orang-utans two individuals were found that reacted positive in an ELISA detecting antibodies which cross-react with human T-lymphotropic virus type I (HTLV-I) antigens. Infection of both animals with an HTLV-I or simian T-lymphotropic virus (STLV)-like virus was confirmed by Western blot analysis. A third wild-caught animal, which was not part of the original serological survey, was also found to be infected with an HTLV-related virus in a diagnostic PCR assay and Western blot assay. Nucleotide sequence analysis of the 709 bp PCR fragment from the tax/rex region of the HTLV/STLV genome confirmed infection of orang-utans with an STLV similar to but clearly distinct from other Asian STLVs.

At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role in vivo. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined slow mo (smo), a recessive mutation we discovered in the zebrafish Danio rerio. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from smo continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of Ih, a current described previously in the nervous system, pacemaker, and other cardiac tissue. smo cardiomyocytes have a reduction in Ih that appears to result from severe diminution of one kinetic component of the Ih current. This provides strong evidence that Ih is an important contributor to the pacemaking behavior of the intact heart.

The World Development Report 1993 announced that global life expectancy was then 65. Experience in the developed world suggests that the World Health Organization's dictum, 'health is a state of complete physical, mental and social well-being', is simply not attainable for the foreseeable future. As physical health has improved, mental problems have become more prominent and a sense of well-being has declined. Furthermore, as the population ages and medical technology improves, the cost of health care grows almost exponentially. Since the population of the developed world is continuing to age and aging is spreading rapidly throughout the developing world, knowledge is the principal way of dealing with this seemingly intractable problem: we must know, quantitatively, the age-specific causes of ill health, and we must know which means of prevention and treatment are effective. Finally, we must apply that knowledge rationally.

Vertebrate nonmuscle cells, such as human and rat fibroblasts, express multiple isoforms of tropomyosin, which are generated from four different genes and a combination of alternative promoter activities and alternative splicing. The amino acid variability among these isoforms is primarily restricted to three alternatively spliced exon regions; an amino-terminal region, an internal exon, and a carboxyl-terminal exon. Recent evidence reveals that these variable exon regions encode amino acid sequences that may dictate isoform-specific functions. The differential expression of tropomyosin isoforms found in cell transformation and cell differentiation, as well as the differential localization of tropomyosin isoforms in some types of culture cells and developing neurons suggest a differential isoform function in vivo. Tropomyosin in striated muscle works together with the troponin complex to regulate muscle contraction in a Ca(2+)-dependent fashion. Both in vitro and in vivo evidence suggest that multiple isoforms of tropomyosin in nonmuscle cells may be required for regulating actin filament stability, intracellular granule movement, cell shape determination, and cytokinesis. Tropomyosin-binding proteins such as caldesmon, tropomodulin, and other unidentified proteins may be required for some of these functions. Strong evidence for the distinct functions carried out by different tropomyosin isoforms has been generated from genetic analysis of yeast and Drosophila tropomyosin mutants.

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.

The World Development Report 1993 announced that global life expectancy was then 65. Experience in the developed world suggests that the World Health Organization's dictum, "health is a state of complete physical, mental and social well-being', is simply not attainable for the foreseeable future. As physical health has improved, mental problems have become more prominent and a sense of well-being has declined. Furthermore, as the population ages and medical technology improves, the cost of health care grows almost exponentially. Since the population of the developed world is continuing to age and aging is spreading rapidly throughout the developing world, knowledge is the principal way of dealing with this seemingly intractable problem: we must know, quantitatively, the age-specific causes of ill health, and we must know which means of prevention and treatment are effective. Finally, we must apply that knowledge rationally.

Previous studies have demonstrated that overexpression of the carboxyl-terminal fragment, CaD39, of human fibroblast caldesmon in Chinese hamster ovary cells protected endogenous tropomyosin from turnover and stabilized actin microfilament bundles [Warren et al., 1994: J. Cell Biol. 125:359-368]. To assess the consequences of having CaD39-stabilized microfilaments in living cell, we characterized the motile behaviors of stable CaD39-expressing lines. We here found that CaD39-expressing cells adhered faster to plastic, glass, fibronectin-coated glass, and collagen-coated glass than control cells. Moreover, the CaD39-expressing cells also exhibited enhanced spreading immediately after attachment. Despite these differences, overexpression of CaD39 had little effect on the velocity of intracellular granule movement, or the velocity and persistence of cellular translocation. However, CaD39-expressing cells were more elongate and encompassed less area than non-expressing cells during migration in a wound-healing assay. In interphase cells, the expressed CaD39 fragments were found associated with tropomyosin-enriched microfilaments. Like endogenous caldesmon, the CaD39 fragment was also modified at mitosis. Although a significant portion of CaD39 underwent only partial modification, the majority of the CaD39 was released from the microfilaments during mitosis. This is consistent with the finding that the CaD39-induced advantage for attachment and spreading was lost during mitosis. In CaD39-expressing cells, an incomplete release of the CaD39 from microfilaments at mitosis was found which may be responsible for the increase in the frequency of multinuclear cells in CaD39-expressing lines.

Libraries as presently constituted are essentially static paper-containing information systems often enclosed in massive specific spaces. The new electronic systems are by their very nature dynamic global systems that take up almost no space. Static information systems evolved from clay tablets to papyrus scrolls to parchment and finally to paper produced in vast quantities by the printing press. Medical and scientific journals have been growing exponentially since the seventeeth century as have the static paper-based libraries. This growth has been non-Darwinian, being outside of the laws of natural selection and survival of the fittest. Thus, the great majority of medical information is uncited and unread. Moreover, the vast investment in static libraries in the developed world has inhibited the development of modern electronic systems. In contrast, the developing world is largely bereft of such institutions, and has the unique opportunity to leap unencumbered by the static past into the far more efficient and dynamic electronic future.

Cardiac troponin T (cTnT), a tropomyosin (TM)-binding subunit of the troponin complex, undergoes a developmentally regulated isoform switch from embryonic form to adult form in the rat heart. To investigate the in vivo assembly of cTnT isoforms, we transiently transfected cDNA clones of either rat cTnT isoform into nonmuscle CHO cells and chick embryo myogenic (CEM) cells. As determined by Western blotting, both isoforms can be expressed in CHO and CEM cells. The expressed proteins had the same mobility as native rat cTnT proteins on SDS polyacrylamide gels and were recognized by anti-TnT antibodies. Conventional and confocal microscopy of transfected cells, double-labelled with antibodies against cTnT and against TM, revealed that neither isoform appears to associate with the nonmuscle TM in CHO cells, although both are able to colocalize with muscle TM-containing microfilament bundles in the myogenic CEM cells. There was no appreciable cTnT isoform-related difference in association with TM, suggesting that the functional significance of isoform variability in rat cTnT does not correspond to an assembly advantage for the maturing cardiac thin filament. To help determine whether cTnT nonassembly in CHO environment is primarily due to the nonmuscle nature of the endogenous TM, or if it involves the absence of other factors specific to muscle, we have isolated several stably-transfected clones of skeletal beta TM-expressing CHO cells which incorporate this muscle TM onto stress fibres. When either isoform of cTnT was transiently expressed in these beta TM-CHO cells, the strictly filamentous beta TM staining pattern was no longer observed. Instead, beta TM codistributed with cTnT in brightly staining aggregates not associated with the intact stress fibres. This suggests that both isoforms of cTnT are interacting with the beta TM in the nonmuscle environment and that other muscle-specific proteins may indeed be required for stable assembly of cTnT onto microfilaments. It also suggests that the interaction between cTnT and muscle TM is stronger than that between muscle TM and nonmuscle microfilaments.

In the latter half of this century there has been a dramatic change in the treatment of helminthic infections. Two factors have contributed to this: an understanding of the major differences between helminths and other infectious agents, and the development of effective low-cost, single-dose, broad spectrum drugs. A program of administration of these drugs to school-aged children in developing countries is now being implemented and will result in a significant improvement in their health at a crucial time in their growth and development.

Tropical medicine as a specialty began during colonialization of the tropics. From the outset, controversy focused on scientific research (tropical medicine) vs. public health (tropical health). The former became associated with parasitology to the relative exclusion of microbiology. Remarkable discoveries made before 1912 were followed by sixty years of slow progress. In the last decade, however, not only has parasitic disease research flourished, but the major importance of bacterial and viral diarrheas and respiratory infections has also been revealed. Tropical health did not evolve as a major strategy in the colonial era. Later, a global eradication policy developed, first for hookworm infection, then for yellow fever and malaria, but failure led to disillusionment with technology and development of an undifferentiated approach to primary health care. In the last decade a selective approach has focused on diseases for which cost-effective control measures exist. Moreover, several developing countries have achieved good health at low cost by equitable distribution of health care, education, and food. Today, the conflict between tropical medicine and tropical health is being resolved with the realization that they are truly complementary disciplines.

Disease due to virtually all of the human helminth parasites can be prevented simultaneously by a simple system based on the following premises: 1) helminths do not multiply within their definitive hosts, and their distribution in host populations is overdispersed; 2) disease manifestations occur largely in the small proportion of hosts, often school children, with heavy worm burdens; and 3) a few single-dose, broad-spectrum anthelmintics given in low doses at prolonged intervals can maintain worm burdens below pathogenic levels for almost all of the major human helminth parasites. The three drugs might be albendazole or one of the other benzimidazoles for hookworm, ascaris and trichuris; ivermectin for the filaria, including onchocerca and many other nematodes such as strongyloides; and praziquantel for virtually all trematodes and cestodes. The target group would be school children treated at intervals set to maintain worm burdens below the disease-inducing threshold. It is possible that all three drugs could be administered in low doses simultaneously at yearly intervals. Studies will be necessary to examine drug interactions, development of drug resistance, optimal dosages and timing, and effects on morbidity.

Growth rate and histology of splenic autotransplants in subcutaneous pockets were compared with those of autotransplants in the extrahepatic portal bed in splenectomized mice infected with cercariae of Schistosoma mansoni and in splenectomized uninfected controls. By the fifteenth week after transplantation (and tenth week after injection of cercariae) subcutaneous transplants gained 6.5 times and omental transplants 8.2 times more weight in infected animals than corresponding transplants in uninfected animals. Portal pressures averaged 11 to 13 cm of water in infected animals with transplants and 17 cm in those with intact spleens (compared to that of 6 to 7 cm in controls). Hyperplasia of white pulp with increase in germinal center activity characterized transplants as well as intact spleens of infected animals. The results suggest that a) During the first 10 weeks of experimentally induced infection, portal congestion is not the predominant mechanism regulating increased spleen growth; and b) An intact enlarged spleen appears to contribute to elevated portal pressure.

Although Schistosoma japonicum egg granulomas are generally considered to be similar to those of S. mansoni (which are largely immunologic reactions of the delayed hypersensitivity type) there are suggestions that the histopathology and perhaps the etiology of the lesions are different. In mice with light S. japonicum infections, at 5 weeks after infection (2 weeks after egg production began), the livers contained 36,000 eggs each, but there was no reaction to the eggs, nor any evidence of hepatosplenic disease. By 6 weeks, large abscesses replete with cosinophils occurred around some of the eggs, and there was periportal inflammation consisting predominantly of plasma cells. From this time on, major lesions occurred mainly around large aggregates of eggs, and there was hepatosplenomegaly and portal hypertension. Living S. japonicum eggs injected into the pulmonary microvasculature of mice did not evoke significant granulomatous reactions on either primary or secondary exposure. Even when the eggs were injected into the lungs of infected animals, which had large granulomas around egg aggregates in the liver, little or no inflammatory reaction was seen around the eggs distributed singly throughout the pulmonary vessels. When the priming dose of eggs or soluble egg antigens was injected subcutaneously with or without complete Freund's adjuvant, significant granuloma formation occurred around eggs subsequently injected into the lungs. On the basis, therefore, of differences in the parasite factor (eggs) and host factors (histopathology and responses to routes of injection) it is suggested that the immunologic factors responsible for granuloma formation around S. mansoni and S. japonicum eggs may differ significantly.

Schistosoma mansoni egg antigens that elicit delayed hypersensitivity in appropriately sensitized guinea pigs were partially characterized by using ion exchange chromatography and preparative electrophoresis. At least three skin-reactive antigens were found, one of which was purified to homogeneity, as analyzed by polyacrylamide gel electrophoresis (PAGE). This antigen was not adsorbed to CM cellulose, migrated cathodal to guinea pig albumin on electrophoresis, and was adsorbed to DEAE cellulose. A second pool of antigenic activity was obtained by adsorption to CM cellulose and subsequent elution. DEAE cellulose chromatography and preparative electrophoresis of this pool indicated the presence of more than one antigen.

A test for the lymphokine eosinophil stimulation promoter (ESP) has been adapted to the study of Trichinella spiralis infections in mice and has been used as an aid in the diagnosis of trichinosis in a patient. With use of soluble antigen extracted from T. spiralis larvae and peritoneal exudate cells rich in eosinophils obtained from mice with trichinosis of four weeks' duration, a dose-response curve was constructed and analyzed. The soluble larval antigens greatly enhanced the migration of eosinophils from the mice with trichinosis but had no effect on the eosinophils from mice with schistosomiasis, nor did soluble schistosome egg antigens have any effect on the cells from mice with trichinosis. On two occasions peripheral white blood cells from a patient with presumed T. spiralis infection gave strongly positive results in ESP tests on stimulation with soluble larval antigen, but the cells did not respond significantly to soluble schistosome egg antigen; the result in the ESP test was positive before the patient's serum produced a weakly positive agglutination response.

The recommended doses of the drugs now used to cure schistosomiasis mansoni may be associated with toxic side-effects. Since Schistosoma mansoni does not multiply in the human host and the disease seems to be closely associated with the intensity of infection, it may not be necessary to use 100% lethal antischistosomal doses, particularly in endemic areas. A dose-response to the antischistosomal drug, hycanthone was established for three different doses in 169 patients with heavy S. mansoni infections in the Machakos district of Kenya. The highest dose used (1.5 mg/kg or half the recommended package-insert dose) resulted in a 96% decrease in egg output (equivalent ot death of the worms); 0.75 mg/kg in an 85% decrease; and 0.375 mg/kg in an 11% decrease one month after treatment. In contrast to the vomiting common with the package-insert dose (3.0 mg/kg), there were no side-effects with any of the lower doses.

Niridazole, a nitroheterocyclic drug used to treat helminth infections is reported for the first time to suppress cell-mediated immunity, as ascertained by multiple in vivo assays. Skin homograft survival time was significantly prolonged in mice, without loss of animals due to drug toxicity.

In vivo microscopy of the liver was undertaken to determine the extent of involvement of the hepatic microvascular system in mice infected with viral hepatitis (MHV-3). It was found that the major effects of the disease were produced in localized areas where parenchyma, sinusoids and blood were obliterated. However, immediately peripheral to these lesions blood flow and vessels were unaffected other than by localized hypertrophy of Küpffer cells. While each lesion produced a sinusoidal block to blood flow, portal hypertension did not occur even in the presence of extensive focal and confluent necrosis.

Niridazole, an anthelminthic drug, has been shown to be a potent long-acting suppressant of cell-mediated immune responses both in man and in experimental animals. In the present study the effect of niridazole on the magnitude and kinetics of the primary and secondary antibody responses of mice to sheep erythrocytes, human serum albumin, and keyhold limpet hemocyanin was investigated. The therapeutic dose of niridazole for murine schistosomiasis moderately and transiently inhibited the primary antibody response to these antigens, but had no effect on ongoing antibody production against keyhole limpet hemocyanin or on secondary responses to any of the antigens. Single doses of niridazole (which suppress cell-mediated immune responses) had no effect whatsoever on the most strongly inhibited of the primary antibody responses, that to alum-precipitated polymerized human serum albumin.

Three highly purified Schistosoma mansoni egg antigens reactivity with sera from mice with chronic schistosomiasis mansoni (major serologic antigens-MSA1, MSA2, and MSA3) were utilized in radioimmunoassays for determining their stage and species specificity. Inhibition studies revealed that MSA1 was completely stage specific, since its binding to S. mansoni chronic infection serum (CIS) could not be inhibited either with cercarial or adult worm antigen. MAS2 and MSA3 were specific with regard to adult worm antigen but their binding to CIS was slightly inhibited by cercarial antigen. Immature eggs in comparison with mature eggs contained only negligible amounts of MSA1 whereas MSA2 and MSA3 were present in large quantities. Fluid from eggs hatched in spring water (hatch fluid-HF) contained considerable amounts of MSA1 as well as MSA2 and MSA3. With regard to species specificity, MSA1 essentially did not cross-react with crude soluble egg antigen (SEA) from Schistosoma japonicum and Schistosoma haematobium whereas MSA2 and MSA3 showed partial cross-reactivity. Antibody dilution studies with sera from animals and humans infected with heterologous schistosome species (i.e., S. japonicum and S. haematobium) indirectly confirmed the high degree of species specificity of MSA1. The degree of serologic specificity of MSA1 as demonstrated in this study parallels that of the sensitizing activity of S. mansoni eggs and SEA for granuloma formation, as demonstrated in previous investigations. This study, therefore, indicates that MSA1 may be a major immunopathologically active egg antigen. In addition, the high degree of species specificity of MSA1 suggests its use in the serodiagnosis of schistosomiasis mansoni.

Administration of the antischistosomal compound niridazole to mice, guinea pigs, and humans results in the suppression of several manifestations of cell-mediated immunity. Sera from animals treated with niridazole blocked the in vitro production of migration inhibitory factor (MIF) while niridazole itself was inactive, suggesting that these effects are caused by water soluble mediators. We now report that crude extracts prepared from the urine of rats and a patient receiving nirdazole, but not from pretreatment control urine, similarly suppress antigen-induced inhibition of migration of peritoneal exudate cells from sensitized guinea pigs. With immunosuppressive activity monitored by the direct MIF assay, combined solvent extraction and chromatographic techniques were used to fractionate immunosuppressive activity from the urine of niridazole-treated rats and the patient; the most active fractions, purified about 100-to 1000-fold as compared to methanol-water extracts of dried voided urine, inhibited MIF production at 0.1 to 0.01 ng/ml of assay mixture. These purified fractions also showed immunosuppressive activity by an in vivo assay wherein doses as low as 1 mug/kg injected intravenously (i.v.) into mice suppressed cell-mediated granuloma formation around Schistosoma manisoni eggs. Identically purified fractions prepared from urine of rats and the patient before they received niridazole showed no immunosuppressive activity either in the MIF or in the granuloma assay systems.

Although the general pathology of schistosomiasis has been well understood for more than 70 years, it is only recently that it has been possible to analyse the disease at the molecular level and to understand the relationship between the number of parasites in an infected individual and the appearance of overt disease.

Investigations of schistosomiasis mansoni in 218 Yemeni agricultural workers in the San Joaquin Valley of California revealed a prevalence of 56%. In those infected, quantitative egg counts performed by the Kato thick smear method revealed that 57% had light infections (1-100 eggs/g), 27% moderate infections (101-400 eggs/g), and 16% heavy infections (greater than 400 eggs/g; mean--918 eggs/g). The Yemeni had been migrating to the USA for the past 20 years, a period in which the prevalence of schistosomiasis had remained constant in the Yemen. The prevalence of schistosomiasis in those who had been away from Yemen for less than 5 years was 59% with a mean egg output of 236 eggs/g, but in those away for more than 5 years (up to 20 years) it was 32% with a mean egg output of 75% eggs/g. This is in spite of the fact that 75% of the latter had returned to Yemen for short visits. Statistical analysis by the Fisher's exact probability test revealed a significantly lower egg output in those away from Yemen more than 5 years. On the basis of these findings the mean life span of the Yemen strain of Schistosoma mansoni in man was estimated to be between 5 and 10 years. The presence of disease was assessed in this population by traditional medical means without prior knowledge of the status of the infection in the individuals examined. Under these circumstances, there were no differences in symptoms such as weakness, diarrhea and abdominal pain among the uninfected proportion of the population, total infected group and a small subgroup of those most heavily infected. None of the patients had hepatomegaly and only two had splenomegaly--one lightly and the other heavily infected. Because of the toxicity of antischistosomal drugs and the lack of treatment facilities only the 22 most heavily infected (greater than 200 eggs/g) of the 122 individuals with schistosomiasis mansoni were treated with niridazole. In addition the two individuals with splenomegaly were treated with antimony dimercaptosuccinate. Side effects, though common, were not severe. Although follow-up in this migrant population was poor the nine patients examined 3 to 7 months after treatment showed a mean decrease in egg output of 97%.

Egyptian school children infected with Schistosoma haematobium and treated with a single dose of metrifonate, 10 mg/kg body weight, had a marked reduction in urine egg counts reaching 90% during a 30-week follow-up. While cure rate was higher among lightly infected persons, percentage reduction in egg counts was greater among the heavily infected. After a summer period of probable high risk exposure reinfection rate in those children who were treated and cured was 4.7% as compared to 6% in previously uninfected children.