Elvio H. Sadun

Studies on the host parasite relationships to Schistosoma japonicum IV. Resistance acquired by infection, by vaccination and by the injection of immune serum, in monkeys, rabbits and mice.

Although not always substantiated by experimental evidence, the suggestion has been made by various authors that an initial schistosome infection limits the severity of subsequent infections (Fisher, 1934; Pesigan et al, 1958). Numerous reports on the development of acquired immunity with various forms of human and animal schistosomes have been reviewed by Khalil (1931), Bouillon (1950), Fairley (1951), Newsome (1956) and Kagan (1958). With respect to the specific problem of acquired resistance to S. japonicum, Vogel and Minning (1953) reported that monkeys infected with male worms were still protected 1 to 3 years after treatment from otherwise lethal challenging doses. Lin and his coworkers (1954) also found that mice infected with male worms developed a moderate resistance to super infection. However, Hunter et al (1956) found no marked immunological response to reinfection in mice, hamsters and rabbits. They commented that these results are at variance with those obtained in the same laboratory 2 years earlier (Lin et al, 1954) and suggested that the selection of an experimental host for studies on immunological response to schistosomiasis may be a critical factor. Attempts by various investigators to induce resistance by means of artificial immunization have been met with but little success and have often given conflicting results. Ozawa (1930), Kawamura (1932) and Lin et al (1954) reported that experimental animals were protected to a small degree by previous injections of saline suspension of whole S. japonicum worms. However, Vogel and Minning (1953) were unsuccessful in immunizing monkeys against infection with S. japonicum by this method. Although diagnostic antibodies are present in the serum of infected animals, to demonstrate the presence of protective antibodies, the passive transfer of resistance should be successfully performed. Kawamura (1932) reported that he was able to protect dogs and rabbits by injecting them with serum from animals infected with S. japonicum. On the other hand, Vogel and Minning (1953) were unable to produce resistance in monkeys by injections with S. japonicum antiserum. These conflicting results, often based on limited experimentation in different hosts, suggested that further work was needed on this problem, with more attention paid to the type of experimental animals, the size of immunizing doses, and the time interval between immunizing and challenging exposures. The experiments re-

The use of purified antigens in the diagnosis of clonorchiasis sinensis by means of intradermal and complement fixation tests.

The limited research which has been conducted on the immunodiagnosis of clonorchiasis has yielded equivocal results. Chung and his coworkers used a saline extract of the adult worms in the intradermal and complement fixation tests. Two clonorchiasis and 18 paragonimiasis patients tested with this antigen showed a dermal sensitivity (Chung et al, 1955). The sera from 3 clonorchiasis and 25 paragonimiasis patients gave a positive complement fixation reaction (Chung et al, 1956). The intradermal test with a crude antigen was carried out in 136 infected individuals and 58 negative controls with unsatisfactory results (Hunter et al, 1958). Wykoff (1958) was able to detect antibody response in infected rabbits by the use of an ether extraction of the worms in the complement fixation test. However, even with this antigen, pre-infection rabbit sera reacted to some degree and antibody response had to be evaluated on a relative basis. These few reports based on limited experimentation suggested that further work on this problem was needed, with increased efforts toward the purification of the antigens. Therefore, the experiments reported in this paper were carried out.

Trypanosoma rhodesiense: variant specificity of immunity induced by irradiated parasites.

Abstract Rats immunized with irradiated Trypanosoma rhodesiense resisted infection with the homologous strain. When similarly immunized rats were challenged with parasites obtained from rhesus monkeys infected with the same strain, resistance depended on when parasites were obtained from the donor monkeys. Immunized rats challenged with trypanosomes obtained from a monkey during the first peak of parasitemia were solidly immune; immunized rats challenged with trypanosomes obtained from monkeys after their initial peak of parasitemia all succumbed to the challenging infection. These observations indicate that parasites of a variant antigenic specificity arose during the course of the monkey infections. Neutralization tests performed on the various isolates from rats and monkeys using antiserum obtained from immunized rats confirmed that the immunity produced by irradiated trypanosomes was variant specific.

Susceptibility of wild mammals to infection by Schistosoma mansoni.

In the past it was believed that mlan was the only epidemiologically important malnmalian host of Schistosoma mansoni. Recent studies, however, have shown that a few other hosts may play a role in the dissemlination of this parasite (Barbosa et al 1958; Kuntz and Malakatis, 1955; Price, 1953; AMartins, 1958). The present investigation was undertaken to determine if wild lmanInals trapped in the Washington, D. C.-Baltimore, Maryland, area were susceptible to S. mansoni. It was regarded as unlikely that any of these aniimals were naturally infected because no recognized snail host of S. mansoni is present in this area. The degree of susceptibility of the various imammals tested was deteriiined by the percentage of parasites developing in the host; the growth and structural development of the worms; the ability of worims to produce eggs; the viability of eggs recovered from the feces, intestine and liver; the infectivity of miracidia for suitable snails; the sex ratio and location of the worms in the host; the length of the prepatent period; and the pathological manifestations of infection.

The effect of maklua (Diospyros mollis) in the treatment of human hookworm.

AND 1951 Efficacy of 0.0055 per cent nitrofurazone fed continuously for the control of avian coccidiosis under conditions of natural infection. J. Parasit. 37 (suppl.) : 13. AND 1953 A search for drug-fast strains of Eimeria tenella. J. Parasit. 39: 268-271. HORTON-SMITH, C. AND LONG, P. L. 1952 Nitrofurazone in the treatment of caecal coccidiosis in chickens. Brit. Vet. J. 108: 47-57. PETERSON, E. H. AND HYMAS, T. A. 1950 Sulfaquinoxaline, nitrofurazone, and nitrophenide in the prophylaxis of experimental Eimeria necatrix infection. Am. J. Vet. Res. 11: 278-283. SWALES, W. E. 1950 On the chemotherapy of caecal coccidiosis (Eimeria tenella) of chickens. VII. The use of a standardized test to determine coccidiostatic properties of drugs. Canad. J. Comp. Med. 14: 269-274.

CHOLESTEROL-LECITHIN SLIDE (TSSF) AND CHARCOAL CARD (TSCC) FLOCCULATION TESTS USING AN ACID SOLUBLE FRACTION OF TRICHINELLA SPIRALIS LARVAE.

Two simple sensitive flocculation tests were developed employing acid soluble T. spiralis larvae antigen absorbed onto cholesterol-lecithin crystals. Washing of the emulsion greatly increased the sensitivity of the antigen in the slide flocculation test. The addition of charcoal to the antigen emulsion permitted its use in a simple card test using serum or minute amounts of plasma collected from finger puncture. Absorption of reactive sera with a single dose of washed, packed, and essentially dry antigen-cholesterol-lecithin complex resulted in a complete removal of serologically detectable antibody indicating that the tests were based upon a true antigen-antibody system. Sensitivity of both tests was excellent. In the slide flocculation test, 64 sera from 68 patients were reactive, 2 were weakly reactive and 2 were nonreactive. All 21 sera from trichinosis patients were reactive in the charcoal card test. The tests also appear to be relatively specific. Only 3 sera from a total of 96 normal persons and patients with other diseases were reactive in the slide flocculation test; 13 sera showed weak reactions. With the charcoal card test 3 reactions were obtained with sera from a total of 64 normal individuals and patients with other diseases. The tests are especially well suited to small laboratory and field conditions because they are simple to perform and utilize antigenic emulsions which may be kept for prolonged periods without apparent loss of reactivity. Slide flocculation tests are among the simplest procedures for the serodiagnosis of infections. These techniques have not yet found wide application in the field of parasitology primarily because of the difficulty in many instances of combining the parasitic antigens with the carrier (cholesterol, latex, or bentonite). Suessenguth and Kline (1944) found that an aqueous extract of Trichinella larvae coated onto cholesterol crystals could be used as antigen in a simple and rapid flocculation slide test for trichinosis. They reported that this test was more sensitive than the complement fixation test (Suessenguth et al., 1957). However, other investigators have presented evidence indicating that this test lacks the specificity of other serological tests for trichinosis (Bozicevich et al., 1951; Sadun and Norman, 1955a; Greene and Brazeale, 1951). Recently Anderson (1960) developed a slide Received for publication 26 April 1963. flocculation test for schistosomiasis using antigen prepared by coating a buffered saline extract of cercariae onto cholesterol-lecithin particles and subsequently washing this complex to remove the excess antigen. This method has been shown to be one of the most sensitive and specific serodiagnostic procedures for schistosomiasis (Anderson, 1960; Anderson and Naimark, 1960; Jachowski and Anderson, 1961). Following the development of the rapid plasma reagin card test (Portnoy et al., 1962) for syphilis, the schistosome slide flocculation te t antigen was adapted to the card procedure and evaluated under laboratory and field conditions (Sadun et al., 1963). The card test in schistosomiasis gave results comparable to those obtained with the standard slide flocculation procedure and permitted the test to be completed within a few minutes on plasma obtained by finger puncture. The purpose of the current study was to determine whether similar procedures would provide a slide flocculation test for trichinosis

Cultivation of Endamoeba histolytica in the chick embryo

Abstract After chorio-allantoic inoculation of chick embryos with Endamoeba histolytica , two-thirds of the embryos were dead when examined after 2–6 days, and no amebae were found, although survival up to 12 hours was demonstrated. After allantoic inoculation 10 of 16 embryos died but 2 of the dead ones were positive for amebae. Yolk sac inoculations were negative. After amniotic inoculations, a considerable number were positive for amebae up to 72 hours, although a large proportion of these embryos were dead. The amebae were in the amniotic fluid as well as on and in the membrane. No proof of multiplication was obtained. Death of the embryos appeared to be due mainly to accompanying bacteria which were insufficiently inhibited by the antibiotics used.

The Comparative Growth Cycle of different Strains of Endamoeba histolytica in Transparent Medium and of the same Strain in different Media.

Many investigations have been undertaken in the attetnpt to provide a better understanding of the factors responsible for the growth of Endalmoeba histolytica in vitro. In order to evaluate the culturability of different strains of this ameba in the same medium, Faust and his co-workers (1946) cultured stools from 18 infected persons. Although no numerical determinations were carried out, these workers reported marked differences in population growth among the different strains. Since the bacterial flora present in these amebic cultures varied considerably, it was impossible to determine whether the differences in population growth were due to intrinsic properties of the amebae or to their accompanying bacterial flora. The cultivation of E. histolytica in a transparent medium without demonstrable bacterial growth has been reported by Shaffer and Frye (1948). This medium appears to be ideally suited for the study of factors influencing the growth of E. histolytica, since very little, if any, multiplication of bacteria appears to take place in it. The present study was carried out in an attempt to trace the growth of different strains of E. histolytica in this medium with an inhibited monobacterial flora and to compare it to that that in standard media with a multibacterial flora.

Flocculation test in sera from hogs experimentally and naturally infected with Trichinella spiralis.

Many investigators have reported observations on the use of serological tests in the diagnosis of infections with Trichinella spiralis, but most of the work has been with infections in man or small laboratory animals. The relatively smaller amount of work that has been done using the hog as a test animal has given conflicting results (Augustine and Theiler, 1932; Bachman and Rodriguez-Molina, 1933; Spindler, Cross, and Avery, 1941; Gaase, 1949; Suessenguth, 1947; and Wagner, 1949), and the value of the serological tests for trichinosis in swine is still debatable. Bozicevich, Tobie, Thomas, Hayem, and Ward (1951) described a simple rapid flocculation test for the diagnosis of trichinosis employing as antigen a saline extract of trichina larvae adsorbed on uniform-sized bentonite particles. In view of the great practical importance of being able to detect infections in pigs in the abattoir before the meat reaches the population for general consumption, it was felt that further work with this test with attention to the time of infection and quantitative relationships was needed. Therefore, three experiments were undertaken to study the possibility of detecting serologically infections of various duration in hogs infected with graded doses of larvae. A fourth experiment was designed to test the sensitivity of the flocculation test in detecting normally occurring infections in pigs at the time they are slaughtered.

Effect of Single Inocula of Endamoeba histolytica Trophozoites on Guinea Pigs.

Summary Guinea pigs were inoculated intracecally with graded single doses of from 100 to 5,000,000 amebae. None of the animals that were given less than 1,000 amebae developed infection. The 50% infective dose under the experimental conditions was 10,000 amebae. The mean weight of the guinea pigs that later became infected was significantly smaller than that of those which resisted infection. Thirty-one out of 50 guinea pigs that received single inocula of from 5,000 to 5,000,000 amebae died as a result of the infection. The LD50 under the experimental conditions was 63,000 amebae. Death occurred in all cases between 5 and 17 days after inoculation, on the average 9.3 days after inoculation, and the greatest number of guinea pigs died on the eighth day. In general, the size of the inoculum was found to have an inverse relationship to the average time between inoculation and death. Histopathologic studies confirmed necropsy examination. All animals found to be positive by gross examination and by direct smears of the cecal content were found to have typical amebic lesions, while in none of the controls and of those reported as negative were ulcers of any kind observed by histologic sections.