E. H. Sadun

The cellular and humoral immune response to Schistosoma mansoni infections in inbred rats. I. Mechanisms during initial exposure.

Fisher rats were infected with Schistosoma mansoni by skin exposure to penetrating cercariae. The subsequent development of immunity was ascertained through the rechallenge of animals with cercariae and the simultaneous quantitative assessment of the development of the worm burdens which resulted from each of the two infections. The basic nature of the immune response to rechallenge by S. mansoni was investigated through the adoptive transfer of cells or serum obtained from normal or preexposed animals. The results indicate that the rat develops a strong, immunologically mediated, augmented resistance to infection by S. mansoni which begins within 1 week of initial exposure. This response is reflected in both quantitative and qualitative changes in the development of the reinfecting population and has a general characteristic of an anamnestic or secondary response judged by a variety of immunologic criteria. The developing immunity apparently is both stimulated by, and directed exclusively against, the very early stages of infection. Although the mechanism of this immunity is not clear, it apparently involved the production of significant quantities of protective immunoglobulin.

Trypanosoma congolense. I. Clinical observations of experimentally infected cattle.

Abstract The course of disease was studied in 8 cattle infected with Trypanosoma congolense . Although the onset of patency was dependent on the numbers of infecting organisms, the duration of the infection was not. High fevers were present on the day of or the day after initial patency. Succeeding peaks of parasitemia, and a progressive weight loss of over 30% occurred. A decrease in packed cell volume (PCV) beginning the first week after infection was observed. Early in the course of the developing anemia, many polychromatophilic erythrocytes and occasional normoblasts were found in the blood. A leucopenia persisted for the duration of the disease. Total serum protein concentrations fell sharply during the first 5 weeks of infection, then gradually increased to low normal levels. Serum albumin levels followed a similar pattern for the first 5 weeks, and remained at a relatively low level. Although gamma globulin levels also declined during the first 5 weeks, their levels gradually surpassed those of preinfection samples. No marked changes in serum glucose were noted. A mild elevation of serum urea nitrogen values occurred early during infection, but subsided. The animals dying early after infection developed elevated total bilirubin levels.

Pathophysiology of Plasmodium berghei infection in mice

Abstract Significant biochemical changes were detected in the serum of mice infected with P. berghei . Marked increases in SGP-T and SGO-T transaminases were observed as early as 2 days after infection. Lower fasting glucose levels occurred in heavily infected mice 4 days after infection. A moderate reduction in alkaline phosphatase and albumin values was observed. Increases in BSP retention and positive cephalin flocculation reactions were also registered in the infected mice. Minimal or no changes in total protein, non-protein nitrogen, phosphorus, globulins, bilirubin, calcium, creatinine, carbon dioxide, chloride, sodium, and potassium resulted from infection.

Resistance produced in Mice by Exposure to Irradiated Schistosoma mansoni Cercariae.

Abstract An acquired resistance to Schistosoma mansoni was observed in mice following a previous exposure to irradiated cercariae. The resistance developed was sufficiently strong to eliminate or to prevent the development of a significant number of worms of the test dose, no matter whether the primary infection was as low as 200 or as high as 15,000 cercariae, or whether the immunizing infection was in a single dose or in three weekly doses. Resistance induced by a single exposure to irradiated cercariae was sufficiently powerful to protect mice from an otherwise lethal dose of normal cercariae. Mice stimulated with irradiated cercariae developed detectable antibodies to egg and larval antigens in spite of the fact that they received no egg stimulation.

Resistance produced in rats and mice by exposure to irradiated Plasmodium berghei.

Abstract Rats infected with Plasmodium berghei parasitized erythrocytes exposed to doses up to and including 16,000 r developed progressive parasitemias with obvious parasite multiplication and reinvasion of RBCs. Some rats receiving parasitized RBCs irradiated at levels of 17,000 and 18,000 r did not develop progressive infections. Infections were aborted by exposure to irradation at 19,000 r or higher. Inoculation with parasitized blood exposed to 20,000 r stimulated a resistance to a challenge infection with nonirradiated parasites. The peak parasitemias reached in the immunized animals were significantly lower than those of the nonimmunized controls and reduction in parasitemias began much earlier. The degree of acquired resistance induced by inoculation with irradiated parasites was influenced by the number of immunizing exposures before the challenging infection. The acquired resistance induced by irradiated infected RBCs could be detected also in a more susceptible animal such as the mouse. Twice-weekly inoculations of irradiated parasitized cells given three, five, and ten times extended the mean survival time after challenge considerably; a significant number of mice survived otherwise lethal infections. In both rats and mice the degree of acquired immunity was directly proportional to the number of immunizing doses. Similar results were obtained when immunizations occurred at weekly intervals or when given twice weekly. Likewise, no significant differences were detected when the challenge took place 1 or 2 weeks after the last immunizing dose.

Experimental production of bilharzial pipe-stem fibrosis in the chimpanzee

Abstract Clinical, pathologic, parasitologic, immunologic, and radiologic studies were conducted on five chimpanzees exposed to a single dose of 1000 or 2000 Schistosoma mansoni cercariae and on four chimpanzees exposed monthly for 2 years to 100 or 250 cercariae each. One chimpanzee developed the classical lesions of bilharzial pipestem fibrosis; another animal showed a precursor stage of this lesion and several others had variable lesser degrees of fibrosis predominately of smaller portal fields. The earliest identifiable precursor changes of pipe-stem fibrosis in large portal fields were seen at 7 months and the full-fledged picture developed within 2 years after exposure. Portal fibrosis was correlated with heavy egg deposition in the portal triads and intrahepatic portal radicles, accompanied by granulomatous as well as diffuse inflammation. The clinical evolution of pipe-stem fibrosis in the chimpanzee was similar to that in man. After an initial acute stage there was a prolonged relatively asymptomatic interval which evolved gradually into a compensated stage of pipe-stem fibrosis. As in man, this stage was characterized by extensive liver pathology, splenomegaly and portal collateral formation with relatively minor hepatocellular change. A sizeable submucosal esophageal varix was observed in one chimpanzee. This is believed to be a first finding of this kind in experimental liver disease induced without surgery.

A technique for the use of minute amounts of dried blood in the fluorescent antibody test for schistosomiasis.

Abstract A simple method for the collection, preservation, shipment, and testing of minute amounts of dried blood for the diagnosis of Schistosomiasis is described. A drop of blood obtained from a finger puncture and collected on filter paper was extracted in saline by use of flexible tube and carpenters vise. The extracted blood was tested by the indirect fluorescent antibody technique employing Schistosoma mansoni cercariae as antigen. The dried filter paper blood specimens were preserved at room temperature for as long as 90 days without detectable changes in antibody response. This technique is now being evaluated for field use in epidemiological surveys.

Serological cross reactivity between Plasmodium vivax and Plasmodium falciparum as determined by a modified fluorescent antibody test

Abstract The cross reactivity between P. vivax and P. falciparum was studied by an indirect fluorescent antibody (FA) technique in which homologous and heterologous antigen-antibody systems were used. Two modifications of the previously described test were employed: (1) the use of liquid nitrogen for the preservation of parasitized whole blood for use as the source of antigen, and (2) the use of Evans blue as a counterstain to decrease the intensity of nonspecific fluorescence. Six of 29 sera from natural P. vivax infections reactive with P. vivax antigen were also reactive with P. falciparum antigen. Eleven of 21 serologically positive sera from natural P. falciparum infections were reactive with P. vivax antigen, two of these with the P. vivax antigen only. Two groups of sera from human volunteers with either P. vivax or P. falciparum infections were titrated in parallel tests with homologous and heterologous antigen. In the first series the geometrical mean reciprocal titers (GMRTs) with P. falciparum, sera were 28.3 for the homologous antigen and 6.3 for the heterologous antigen. For the P. vivax sera the values were 17.2 and 9.3 for the homologous and heterologous cases, respectively. In the second series the values for the P. falciparum sera were 132 with the homologous antigen and 20.0 with the heterologous antigen; the P. vivax sera gave values of 30.0 and 11.9 with the homologous and heterologous antigens, respectively. A comparison of titers of blood collected on filter paper and matched serum samples from subjects with P. vivax or P. falciparum infections revealed GMRTs of 24.2 for the blood and 41.0 for the serum. Specificity tests of 246 sera from subjects presumptively free of malaria revealed 28 positive reactions. It is concluded that there is both a species-specific and a group-specific component in the reaction of these two organisms. The specificity of the reaction and the usefulness of the filter paper method of blood collection have been confirmed.

Fluorescent Antibody Test for the Serodiagnosis of African and American Trypanosomiasis in Man.

A fluorescent antibody technique for the serodiagnosis of African and American trypanosomiasis in man is described. The test can be performed simply and rapidly through the use of trypanosomes in blood smears as antigen. There were extensive cross-reactions with sera from patients with other species of trypanosomes. Tests of sera from healthy controls and from patients with nontrypanosomal diseases and disorders revealed relatively few cross-reactions, indicating a high degree of specificity. The finding that dried blood on absorbent paper could be tested successfully with the FA technique may indicate its usefulness for studies in endemic areas. Human infections with African and American trypanosomiasis constitute important clinical and public health problems in large regions of the world. An unequivocal diagnosis of trypanosomiasis is often difficult to obtain since the clinical picture is not always well defined and the organisms frequently cannot be recovered by blood examinations. Consequently, there is a need for reliable, rapid, and inexpensive procedures which could provide the basis for an adequate diagnosis of these infections, especially during the latent and chronic phases of the disease. The fluorescent antibody technique (Coons et al., 1941) is rapidly developing into a practical, sensitive, and specific diagnostic method for several parasitic infections. Fife and Muschel (1959) described a fluorescent antibody technique (FA) for the serodiagnosis of Trypanosoma cruzi infections. T. cruzi cultured on a diphasic blood agar medium was utilized as a source of antigen. This technique, which required all the reactions to be carried out in test tubes to prevent drying of organisms, appeared to be less specific than the complementfixation test using purified T. cruzi antigen. Studies with T. rhodesiense and T. gambiense in experimental animals (Williams et al., 1963) have resulted in a rapid and practical FA technique which can be run on slides using as Received for publication 21 December 1962. antigen thin blood smears from infected rats. A technique for the use of minute amounts of dried blood in the fluorescent antibody test was developed recently (Anderson et al., 1961). This technique, which permitted mailing of specimens under adverse conditions, was found to be particularly useful in epidemiological surveys of schistosomiasis and trichinosis (Sadun et al., 1961, 1962). The current report summarizes results of studies in which the FA technique was used to stain fixed blood forms of T. rhodesiense, T. gambiense, and T. cruzi toward the development of a reliable and practical test for the laboratory diagnosis of African and American trypanosomiasis. The procedures were evaluated with sera obtained from humans in endemic areas. Furthermore, in the present work attempts were made to determine the degree of cross-reactivity of different trypanosome species and to determine whether blood smears dried on absorbent paper could be used in the serological diagnosis of trypanosomiasis. MATERIALS AND METHODS Sera: Human sera were obtained from individuals with well-documented trypanosomiasis infections. The diagnosis of trypanosomiasis in the patients was established by the recovery and identification of organisms from the blood or spinal fluid. To determine the specificity of the FA test, control sera from individuals with viral, bacterial, and parasitic infections other than trypanosomiasis were used. To test whether the presence of auto-

Fluorescent antibody test for the serological diagnosis of trichinosis.

Abstract A fluorescent antibody test for trichinosis, employing T. spiralis larvae as antigen and possessing a high degree of sensitivity and specificity, is described. Reliable qualitative and quantitative results were obtained either with fresh sera or dried blood specimens on absorbent paper.