Paul E. Thompson

Experimental amebic hepatitis in hamsters.

Summary Progressive and usually fatal amebic hepatitis can be induced readily in golden hamsters by the intrahepatic injection of a strain of Endamoeba histolytica and its bacterial associates from cultures. Amebae rather than bacteria appear to constitute the major etiologic agents in producing the hepatitis.

PAM 1392 [2,4-diamino-6-(3,4-dichlorobenzylamino) Quinazoline] as a chemotherapeutic agent: Plasmodium berghei, P. cynomolgi, P. knowlesi, and Trypanosoma cruzi

Abstract PAM 1392 was active orally against blood forms of Plasmodium berghei in mice, P. cynomolgi in rhesus monkeys, and P. knowlesi in the latter host. Against P. berghei , it (1) was 4 to 11 times as potent as quinine in mice, (2) acted rapidly, (3) suppressed the parasitemia for 6 days in an oral dose of 200 mg/kg, (4) acted synergistically with sulfadiazine without a commensurate increase in apparent toxicity for mice, and (5) was effective against four drug-resistant lines. The latter information suggests that it represents a new mode of action and may be useful in drug-resistant malaria. In monkeys, it acted somewhat slowly but when given appropriately cured infections of P. cynomolgi and knowlesi . The drug inhibited nuclear division of P. cynomolgi . Against Trypanosoma cruzi , PAM 1392 was active in culture against both extracellular and intracellular parasites at non-toxic levels for chick embryo cells. This activity was partially reversed by folinic acid but not by crithidia factor. PAM 1392 also was effective in protecting mice against the lethal effects of acute infections by T. cruzi but exhibited little capacity to effect radical cure of the infections. Based on gross examination, the compound is of relatively low toxicity for mice and monkeys on short-term administration.

Localization of acid phosphatase activity in Plasmodium berghei and P. gallinaceum: an electron microscopic observation.

The localization of acid phosphatase activity in the erythrocytic stages of Plasmodium gallinaceum and Plasmodium berghei was studied by electron microscope cytochemistry. Acid phosphatase activity was demonstrated in the food vacuoles and endoplasmic reticulum of both parasites. This study confirmed the food vacuoles as the site of digestion of host cell cytoplasm. Similarly, the enzyme activity was seen in clefts or slitlike structures of the infected host cells. This may indicate that these structures originate from the endoplasmic reticulum of the host cells. Acid phosphatase activity has been demonstrated in various protozoa such as Paramecium (Esteve, 1970), Tetrahymena (Elliott and Clemmons, 1966), Blepharisma (Demkitzer, 1968), and amebae, by means of electron microscopic cytochemistry. These studies have revealed that this enzyme often is associated with organelles related to the digestion of food as well as the endoplasmic reticulum and Golgi complex. There is, however, a lack of bioand cytochemical evaluation of this enzyme in malarial parasites. Therefore, we undertook this investigation to demonstrate the ultrastructural localization of acid phosphatase in the erythrocytic stages of Plasmodium gallinaceum and Plasmodium berghei. Studies such as this may lead to a better understanding of the digestive process of the host cell cytoplasm by the ma-

QUININE-RESISTANT PLASMODIUM BERGHEI IN MICE.

During induction of chloroquine resistance, Plasmodium berghei developed resistance to quinine administered in doses near the maximum amounts tolerated by mice. Resistant parasites did not form malarial pigment. Normal sensitivity to both quinine and chloroquine returned and pigment formation resumed during serial passage of the parasites through untreated mice.

Experimental studies on Trichuris muris in mice with an appraisal of its use for evaluating anthelmintics.

Attempts to infect white mice from five commercial sources with Trichuris muris yielded discouraging results. DBA-2 hybrid mice, particularly weanlings, were the most useful experimental hosts among nine types of rodents studied for susceptibility. In these mice, from 10 to 27 per cent of the administered eggs developed. Approximately 1 month was required for the worms to mature, and mature worms persisted for 2 to 3 months. However, the infections appeared to be of limited value in drug screening for trichuricidal activity, since they were refractory to treatment with the known trichuricides: dithiazanine iodide and pyrvinium chloride. Infection of laboratory rodents with Trichuris muris (Schrank, 1788) was first reported by Shikhobalova (1937). He experimentally infected white mice to study the developmental stages, with reference to the use of the worm for chemotherapeutic testing. Tissue reactions to the parasite in the cecum of white mice were described by Efremov and Shikhobalova (1939), and resistance of white mice to superinfection with T. muris was demonstrated by Shikhobalova (1940, 1941). Attempts to induce immunity in mice by oral or parenteral vaccination with extracts of T. muris were reported by Leikina (1944). Previously, T. vulpis in dogs had been the main experimental trichurid infection (Lammler, 1958). The limitations of work with this host, in contrast to a small rodent, are readily apparent. Fahmy (1954) induced T. muris infections in laboratory-reared albino mice with eggs from wild house mice. He described the main points of the life cycle, including the morphogenesis of the larval stages, but failed to disclose the strain of mice used, the proportion of exposed mice that became infected, or the relative susceptibility among strains. Each of these factors may influence decisively the maintenance of the infection on a routine basis in the

Effects of amodiaquine against Litomosoides carinii in gerbils and cotton rats.

In searching for alternative antifilarial drugs, amodiaquine was found to be active against adult forms of Litomosoides carinii in Mongolian gerbils but not directly active against circulating microfilariae. Oral doses of 100, 50, or 25 mg/kg/day X 5 had significant activity. Female worms were more susceptible to the drug than males. In contrast, oral doses of 100 mg/kg/day X 5 had only feeble activity against the same strain of L. carinii in cotton rats. The generally unsatisfactory state of chemotherapy in filariasis encourages the development of alternative drugs (World Health Orgranization, 1967). Therefore, new types of antifilarial agents are being sought in these laboratories, mainly through the use of Litomosoides carinii in gerbils. The antimalarial drug amodiaquine (Camoquin?) was found to be active in this program. This report deals with its effects against adult and microfilarial stages of L. carinii in gerbils and cotton rats. MATERIALS AND METHODS Mongolian gerbils (Meriones unguiculatus, males) were purchased as weanlings from Tumblebrook Farms. Cotton rats (Sigmodon hispidus, juveniles of both sexes) were raised in the laboratory under conditions precluding natural infection. They were infected with Litomosoides carinii through a modification of the tank exposure method of Hawking and Sewell (1948) approximately 8 weeks before use. Briefly, this entailed allowing a large number of mites to feed on a gerbil or cotton rat with a patent L. carinii infection for 2 weeks and then allowing the mites to feed during an additional 2 weeks on the test animals. This schedule encompassed well the 2to 3-week incubation period in mites. The entire operation was carried out at 24 to 27.5 C. The animals used in an experiment were exposed concomitantly in the same tank. When placed on test, the gerbils weighed 70 to 100 g and the cotton rats weighed 140 to 200 g. Examination for microfilariae were made in Giemsa-stained thick films of blood drawn from the retro-ocular sinus. Films were prepared by spreading 20 mm3 of blood on a 25 by 15 mm area of a slide. Counts were made in 10 fields (100 X magnification). Multiplication of this count by 0.645 gave the approximate number of microfilariae/mm3 of blood. These examinations were started early in patency, 7 weeks after the 1st day of the 2-week exposure period, and were Received for publication 29 March 1968. continued at intervals of 1 to 4 days until the animals were examined for adult worms. Surviving animals were killed and examined for adult worms on day 15 by searching the pleural and peritoneal cavities. The thorax was excised intact, transferred to a petri dish containing heparinized physiologic saline, and dissected so as to permit a thorough search of the pleural cavity. The peritoneal cavity, which only occasionally contained worms, was carefully inspected. The numbers of live worms were scored or counted, as indicated. The numbers of dead worms were scored throughout since they could not be counted accurately owing to encapsulation and degeneration. Amodiaquine [ 4-[ 7-chloro-4-quinolylamino] -adiethylamino-o-cresol, dihydrochloride, dihydrate] was administered by gavage as solutions in aqueous 1% hydroxyethyl cellulose and 0.1% Tween 80 (volumes of 5 ml/kg). Doses were expressed in terms of the free base. The daily dose was given in two subdoses, approximately 6 hr apart. The controls were sham-dosed. The Mann-Whitney U test (Siegel, 1956) was used to test for significant differences (a = 0.05) between numbers of male or female worms in treated and control groups and between numbers of males and numbers of females within a group. Microfilarial counts were subjected to a 2-factor analysis of variance with repeated measures and, subsequently, the Neuman-Keuls procedure for testing differences between means (Winer, 1962).

Comparative Observations on Experimental Schistosoma mansoni Infections in African Green and Rhesus Monkeys.

The pattern of egg excretion and the number of schistosomes are compared in five African green (Cercopithecus aethiops pygerythrus and C. aethiops sabaeus) and five rhesus monkeys. Egg excretion in the green monkey was lower during the acute phase and declined more slowly thereafter. The green monkeys maintained a significantly higher number of worms over a period of many months. The number of eggs passed by green monkeys during chronic infections was much less than would be expected from the number of live worms recovered at autopsy. Although primate hosts are valuable in schistosomiasis research, rhesus monkeys, which have been used mostly, rapidly develop strong immunity to schistosomes which reduces the worm burden and egg excretion (Meleney and Moore, 1954; Vogel, 1958; Naimark et al., 1960; Meisenhelder, Olszewski, and Thompson, 1960; Sadun et al., 1961; Thompson, Meisenhelder, and Najarian, 1962). Although improvisations permit the evaluation of drugs against Schistosoma mansoni infection in the rhesus monkey (Thompson et al., 1962), acquired immunity limits its value for this and many other purposes. The importance of searching for a primate host better than the rhesus monkey has been emphasized in a report to the World Health Organization (1959). Vogel (1958) stated, without elaboration, that the survival of S. mansoni and the duration of its egg excretion is much longer in African green monkeys than in the rhesus. These considerations stimulated the present observations dealing with the course of S. mansoni infection in African green monkeys relative to rhesus monkeys. MATERIALS AND METHODS The animals infected during this work included three Cercopithecus aethiops pygerythrus (the East African green monkey) and two C. aethiops sabaeus (the West African green monkey). Four of these were studied in parallel as pairs with a monkey of each subspecies in each pair, i.e., numbers 25 and 138, 38 and 39. Received for publication 9 January 1963. As detailed accounts appear elsewhere (Najarian and Thompson, 1958; Thompson et al., 1962), the materials and techniques will be described only briefly. The monkeys were anesthetized intravenously with sodium thiamylal (Surital ?, Parke, Davis and Co.) and exposed percutaneously for 45 min to S. mansoni cercariae (Puerto Rican strain) collected from 30 or more Australorbis glabratus. The course of infection was followed by semiquantitative stool examinations for eggs; acidether preparations of 1 ml of feces were examined twice weekly from the 5th week after exposure until autopsy. Egg counts were not adjusted for the water content of the feces. Infections were assessed terminally by the number of live worms found at autopsy; following perfusion, the mesenteric and portal veins, the ground liver, and the perfusate were examined for schistosomes.

The effects of reference anthelmintics against Nematospiroides dubius and oxyurids in mice relative to screening procedures for new drugs.

This paper deals with a continuing search for adequate screening procedures that would expedite the development of better anthelmintics, particularly for combatting intestinal nematodes. Reflection upon the general problem and a review of the literature attest so clearly to the inadequacy of available methods as to require no elaboration upon the need for better methods. Nematospiroides dubius Baylis, 1926, has been demonstrated to be infectious for laboratory mice (Baker, 1954). This contribution along with the oxyurids Aspiculuris tetraptera Schulz, 1924, and Syphacia obvelata Seurat, 1916, which occur as cosmopolitan natural infections, enables simultaneous study of drugs in mice against three intestinal nematodes that not only represent different taxonomic groups but also have predilections for different portions of the intestinal tract. N. dubius resides in the upper portion of the small intestine (unpublished observations) while S. obvelata and A. tetraptera have predilections, respectively, for the cecum and large intestine (Thompson and Reinertson, 1952). The effects of selected anthelmintics under various treatment regimens against these infections constitute the body of this report.

Experimental studies on Obeliscoides cuniculi, a trichostrongylid stomach worm of rabbits. II. Anthelmintic studies in the dutch rabbit.

Phenothiazine, tetrachlorethylene, hexylresorcinol, CI-416 (a 2,4,5-trichlorophenolpiperazine salt), CI-474 (a cyanine compound), pyrvinium pamoate, and methyridine were effective against Obeliscoides cuniculi in single oral doses, as measured by reduction in worm burden and/or the percentage of rabbits cleared of worms. CI-474 and CI-416 showed the greatest potency of the ten compounds tested. The therapeutic failure of bephenium pamoate, thiabendazole, or Ruelene indicates an absence of direct correlation of effect of these drugs in rabbits with their action against trichostrongylid stomach worms of ruminants. The availability of nematode strains adapted to laboratory animals has assumed increasing importance as the search for new anthelmintics has intensified. Despite taxonomic dissimilarities between many laboratory host-parasite systems and the ultimate parasite problems of human or veterinary importance, the success of such laboratory screening is a matter of record. The development of bephenium as an anthelmintic for hookworms, Ascaris, and Nematodirus was based upon the initial discovery of its effect on Nematospiroides dubius and Nippostrongylus muris in rodents (Copp et al., 1958). Likewise, the oxyuricidal effect of pyrvinium pamoate was first detected against oxyurids in mice (Thompson et al., 1962). The present study was undertaken to evaluate the response of Obeliscoides cuniculi in the Dutch rabbit to a series of standard anthelmintics. A primary objective was to determine to what extent such rabbit bioassay data could be extrapolated to the chemotherapy of ruminant nematodiasis. MATERIALS AND METHODS The nematode strain and methods for its handling have been described (Worley, 1963). Dutch rabbits of either sex were infected with 40 to 150 larvae when 6 to 10 weeks old and were housed in groups of 3 or 4 in wire-bottomed cages. Dosing was deferred for 16 to 20 days postinoculation to permit the worms to mature. Composite fecal samples from each group were examined for worm eggs before treatment was started. Pelleted Rockland rabbit ration was fed ad libitum. Drugs were administered by gavage as a single dose suspended in 0.5% hydroxyethyl cellulose after an 18-hr fast. Dosages were expressed as the active component and were administered in a volume of 10 ml of vehicle per kg of body weight with a flexible plastic catheter. Drug effect was assessed by comparing the number of worms remaining in the stomach 3 to 5 days after treatment with the number in sham-dosed controls. Host tolerance of drugs was evaluated by post-treatment weight changes and by gross indications of intolerance such as diarrhea. The rabbits were fasted for a minimum of 32 hr before necropsy to permit the clearance of excessive ingesta from the stomach. This extended fasting period was necessary because the habitual reingestion of fecal pellets resulted in delayed emptying of the stomach. Post-mortem worm counts were made after removing the excised stomach of each animal to a petri dish containing 0.86% saline solution. The mucosa was scraped and examined for worms under a Dazor 3X magnifying lens, and any remaining ingesta were washed through a 20-mesh screen to remove finer debris. The mucosal scrapings and washed stomach contents were examined with a dissecting microscope, and the number, sex, and condition of the worms recorded. Statistical analysis of the percentage of rabbits cleared of worms was obtained with the fourfold contingency tables of Mainland, Herrera, and Sutcliffe (1956), and worm burden means were compared with Students t test. Anthelmintics tested, with key references to the newer ones, were: phenothiazine, CI-416 [bis (2,4,5-trichlorophenol)piperazine salt] (Short and Elslager, 1962; Thompson, Worley, and McClay, 1962), hexylresorcinol, tetrachlorethylene, pyrvinReceived for publication 20 August 1962. * Present address: Veterinary Research Laboratory, Montana State College, Bozeman.

Observations on Therapeutic and Prophylactic Effects by Homologous Immune Blood against Schistosoma mansoni in Rhesus Monkeys.

Although attempts to passively immunize mice against Schistosoma mansoni by the use of heterologous immune serum (from rats) have been unsuccessful (Stirewalt and Evans, 1953), it seems worthwhile to record the following brief efforts to induce immunity against mature and immature worms in rhesus monkeys by using homologous humoral substances. Parenthetically, it may be noted that conflicting results have been attained in attempts to confer passive immunity against S. japonicum. (For a review of this literature see Sadun and Lin, 1959.) Five monkeys were used in the present work. One served as the donor; the remaining four were used as two pairs of test subjects in which one member of the pair was treated and the other served as a parallel untreated control. The two pairs were used, respectively, for observations on the effects of passive immunization attempts against mature and immature worms. The pertinent features of the donor monkey, viz, the history of its infection and evidence that it had antischistosomal antibodies, are indicated by the following statements. The donor was a healthy male weighing 2.3 kg when infected percutaneously with 1000 S. mansoni cercariae; extensive experience in these laboratories has indicated such exposure induces infection with 200 to 250 mature worms. Egg production, as reflected by fecal examinations, reached a peak of 1250 eggs per ml of fecal material 2.5 months after exposure, gradually declined during the next 15 months, but continued at very low levels until the animal was sacrificed (final weight 11.2 kg) 32.5 months after exposure. Perfusion and search of the liver and portalmesenteric system yielded a single pair of living worms. Assays of circumoval precipitins (as performed by Oliver-Gonzalez et al, 1955) and of pericercarial membrane reactions (as performed by Kagan, 1955) were conducted on several serum samples between 4.5 and 30 months after exposure. All samples yielded highly positive reactions; precipitins formed around approximately 45 percent of the viable eggs and pericercarial membrane reactions occurred at serum dilutions of 1: 128. These findings are construed as evidence of a relatively high level of antibodies in the donor during the critical phases of the transfusion experiments. Heparinized whole blood was drawn from a peripheral vein of the donor and injected immediately intravenously into the recipients. The first few ml were given slowly so as to enable recognition of transfusion reactions; consistent with extensive transfusion work on monkeys in these laboratories, no reactiors were seen. It was then considered that transfusing whole blood would enable the most effective use of the available humoral substances with minimal manipulations. Table I depicts the experiment to see whether immune blood would exert an effect against mature worms sufficiently deleterious to influence egg counts (by a modification of the acid-ether procedures of Telemann, 1908), with due allowance