Donald V. Moore

A technique for the perfusion of laboratory animals for the recovery of schistosomes.

The search for and the recovery of schistosomes from laboratory animals presents a problem if the total number of worms in the animal must be ascertained, or if comparative figures must be compiled on the number of worms present in the blood vessels of various organs and regions. The most important area involved is the mesenteric-portal system, but worms may also be present, especially during their migration, in the pulmonary and systemic circulations. Inspection of the mesenteric veins and main portal vein, and manual dissection or comminution of the liver are not likely to reveal all of the worms, especially the small ones, and the time consumed in removing them individually is considerable. A search for worms early in the infection would be impossible by this technique. A desirable method is one which will standardize the individual differences of the operators, effect a reliable removal of worms, and be applicable to early infections. A modification of the perfusion technique of Faust and Meleney (1924) serves these purposes. The principle of this technique is to isolate the organs and regions to be examined and to perfuse the vessels in which the worms may be present from their smallest to their largest branches. They accomplished this in the case of the mesenteric system and the extremities by perfusing from artery to vein, in the case of the liver by perfusing from hepatic vein to the portal vein, and in the case of the lungs by perfusing from artery to vein. Improvements of the technique, made in this laboratory, include supplying a continuous flow of the perfusing fluid, arranging the apparatus in such a way that several animals may be perfused simultaneously, and adding details to obtain more complete collection of worms.

The relationship of male worms to the sexual development of female Schistosoma mansoni.

Of course, the failure of the dog to pass cysts under most circumstances prevents a fair comparison between concentration and the other techniques in the present case; however, the efficiency of the direct examination of feces in saline and the examination of the PVA preserved specimens, and the efficiency of the culture method in the detection of small numbers of trophozoites are directly comparable.

Post-cercarial development of Schistosoma mansoni in the rabbit and hamster after intraperitoneal and percutaneous infection.

It is agreed by most authorities (Miyagawa and Takemoto 1921, Faust and Meleney 1924, Faust, Jones and Hoffman 1934) that the normal route of migration of the schistosomes of man which gain access to the mammalian host by penetration of the skin is as follows. They enter capillaries or venules of the peripheral circulation, are carried in the venous blood stream through the heart to the lungs, work their way through the lung capillaries, are carried in the arterial blood stream to the mesenteric capillaries from which they enter the portal circulation, are carried to the liver and finally, as they mature, return by active movement against the portal blood stream into the mesenteric veins.

Observations on immunity to superinfection with Schistosoma mansoni and S. haematobium in monkeys

Abstract 1. 1. One monkey infected with Schistosoma mansoni ceased to pass eggs after several months, and thereafter failed to pass eggs following four re-exposures to cercariae of S. mansoni. At autopsy 71 2 years after initial infection there was marked fibrosis of the liver, but no evidence of eggs or worms in the viscera. 2. 2. One monkey infected with S. haematobium, ceased to pass eggs after several months, and failed to pass eggs after one re-exposure to cercariae of S. haematobium. Subsequent exposure to cercariae of S. mansoni produced an infection of normal prepatent duration, and egg passage which ceased after several months. At autopsy one year after S. mansoni infection minimal egg lesions were present in liver and intestines. Many S. mansoni worms, but no S. haematobium worms, were recovered from the mesenteric-portal veins. The average length of these worms was abnormally short, and some of the females showed atrophy of the reproductive organs. 3. 3. Two monkeys, one first infected with male worms of S. mansoni, the other with female worms, and each subsequently infected with worms of the other sex, developed egg passage after the normal prepatent interval. At autopsy active egg lesions were present in the liver and intestines, and normal adult worms were recovered from the mesenteric-portal veins. In one of these monkeys, which had a large preponderance of female worms, many of these were sexually immature. 4. 4. These observations corroborate those of other workers that bisexual infection of monkeys with a species of schistosome produces immunity against reinfection with the same species. They also indicate that such infection may product slight evidence of immunity to another species. No evidence was observed, however, that one sex of S. mansoni produced immunity against normal development of the other sex.

Morphology, life history and development of the acanthocephalan Mediorhynchus grandis Van Cleave, 1916.

Synonyms: Heteroplus grandis (Van Cleave, 1916) of Van Cleave, 1918. Heteroplus grandis (Van Cleave, 1916) of Baer, 1925. Empodius grandis (Van Cleave, 1916) of Travassos, 1924. Mature females 27 to 51 mm in length by 0.8 to 1.4 mm in width. Mature males 7 to 12 mm in length by 0.6 to 0.8 mm in width. Anterior and posterior ends of body flexed ventrally. Body slightly expanded just posterior to proboscis. Fully extended proboscis, cone-shaped, 0.57 to 0.86 mm in length, divided into anterior and posterior regions by insertion of proboscis receptacle. Anterior region 0.28 to 0.42 mm long, posterior region 0.29 to 0.44 long. Diameter of proboscis near apex (level of most anterior hooks) 0.14 to 0.21 mm; at level of insertion of proboscis receptacle 0.28 to 0.43 mm; at base 0.35 to 0.79 mm. Anterior region of proboscis possesses 18 more or less longitudinal rows of hooks with 4 to 5 hooks per row, each hook provided with posteriorly directed root. Average total number of hooks

Experimental infection of Bulinus truncatus with Schistosoma haematobium .

establishment of the complete life cycle of the parasite under laboratory conditions. An active breeding colony of the suitable snail host must be established to provide a reliable source of snails for exposure to miracidia at regular intervals so as to insure a steady supply of cercariae for animal infections. The study of Schistosoma haematobium infections under laboratory conditions has been seriously hampered by the inability to infect, routinely, Bulinus truncatus or other suitable molluscan hosts with this parasite. REVIEW OF LITERATURE Cawston (1922 a and b), working in South Africa, was the first to successfully infect laboratory reared snails (Physopsis africana) with S. haematobium. He observed the discharge of cercariae from the snails 35 days after exposure. Brumpt (1928) exposed Bulinus contortus to miracidia obtained from the crushed liver of an infected mouse. After 9 weeks he found 7 snails shedding cercariae out of 35 snails surviving. In another study, Brumpt and Werblunsky (1928), working in Corsica, exposed 35 snails; 28 of these shed cercariae. In this study, Brumpt and Werblunsky emphasized the importance of temperature and recommended 2425? C. as the optimum temperature for snails exposed to infection. Archibald (1933) studying schistosomiasis in the Sudan reported that young Bulinus snails, 3-4 weeks old, were more susceptible to infection than adult snails. He reported that the interval between exposure of the snails and shedding of cercariae was 6-8 weeks. He observed a maximum shedding period of 75 days. Gordon et al (1934), working in Sierra Leone, exposed a total of 262 laboratory reared Physopsis globosa to varying numbers of S. haemtatobium miracidia. Seventy-five snails died during the course of the experiment. They examined 48 snails by dissection and found 23 (47.9%) harboring sporocysts. They allowed 114 snails to show evidence of infection by discharging cercariae and found 95 (83.3%) infected snails. They also performed several experiments to determine the effect of temperature on the development of the infection in the snails. They found that at 26-28? C., which was considered normal, the snails shed cercariae from the 36th day on. When the temperature was raised (27-32? C.), the incubation period was shortened to 23 days but fewer snails survived the incubation period. Lower temperatures retarded the development of the infection, and at 14? C. little or no development occurred. Stunkard (1946) reported the successful laboratory infection of Bulinus truncatus

Comparative susceptibility of common laboratory animals to experimental infection with Schistosoma haematobium.

Brumpt (1928) listed the animals which at that time had been reported by various investigators to have been successfully infected with Schistosoma haematobium. This list included the following: various species of monkeys, guinea pigs, white mice, white rats and hedgehogs. Stunkard (1946), in a search for a suitable molluscan host for S. haematobium among snails native to the United States, used jerbils and baboons, which had been infected in Egypt, as a source of eggs. Watson, AbdelAzim and Halawani (1948) used mice as experimental hosts for S. haematobium in carrying out chemotherapeutic studies. Standen (1949) reported the successful infection of the golden hamster with S. haematobium, but lacked sufficient material to make any observations on the course of the infection in the hamster. In the above reports, no mention is made of the number of cercariae used for exposure or of the resulting worm yield or other pertinent information which is of value in experimental schistosome infections.

Studies on schistosomiasis. XX. Further studies on the immunogenic significance of Schistosoma mansoni eggs in albino mice when subjected to homologous challenge.

A group of experimental mice was given ten subcutaneous injections of living S. mansoni eggs at 4to 7-day intervals. Each mouse received an approximate total of 5,500 eggs. One group of control mice received a like number of injections of the supernate from the egg suspensions; another control group remained untreated. All groups were challenged by percutaneous exposure to 100 S. mansoni cercariae at intervals ranging from 2 to 42 days after the last egg injection. Antibody response due to the egg injections was demonstrated by the miracidial immobilization test, but no increased resistance to S. mansoni challenge infection attributable to the egg injections could be demonstrated. The immunogenic significance of schistosome eggs deposited in the tissues of the mammalian host remains uncertain. Antibody reactive with eggs has been demonstrated by Oliver-Gonzalez (1954) and Oliver-Gonzalez, Bauman, and Benenson (1955) who showed that the anti-egg precipitins were species specific. Circumstantial evidence that eggs in tissues are necessary for the development of acquired resistance to Schistosomatium douthitti and Schistosoma mansoni in mice has been presented (Kagan and Lee, 1953; Crandall and Hunter, 1961; Hunter et al., 1962a). Hunter and Crandall (1962b) in preliminary experiments produced some resistance to homologous challenge infection in mice by the injection of living schistosome eggs, but the results were not entirely consistent. Recently Hsui and Hsii (1961) and Sadun, Yamaki, and Burke (1961) using the zoophilic Formosan strain of S. japonicum as an immunizing infection and the human Japanese strain as a challenge infection have suggested that eggs in the tissues are not necessary to demonstrate some degree of resistance in experimental animals. Hsui, Hsii, and Osborne (1962) using X-irradiated cercariae of S. japonicum for immunization of monkeys produced evidence of partial resistance to homologous challenge as manifested by reduced egg production in immunized animals. Villella, Gomberg, and Gould (1961, 1962) working with S. mansoni in mice have shown that irradiation of cercariae with cobalt 60 results in sterile worms, and that mice infected with irradiated cercariae exhibit some degree of resistance to challenge infections. Smithers (1962) using X-irradiated S. mansoni cercariae and monkeys as the test animal generally confirmed the work of Villella et al. and Hsui et al. in that a partial resistance to reinfection was produced by exposure to irradiated cercariae. Smithers states that even large numbers of irradiated cercariae produce only an incomplete protection against S. mansoni in rhesus monkeys. In some of the studies on irradiation complete sterilization of the worms was not always attained. Ritchie, Garson, and Erickson (1962) failed to demonstrate resistance to S. mansoni in mice after the injection of homogenates of lyophilized cercariae, adult worms, and eggs. These recent findings do not exclude the possibility that eggs, when present, contribute significantly to immunity in schistosome infections. Because previous experiments on inoculation Received for publication 27 August 19Q2. * With the aid of Grants E-1893 and E-2500 from the Division of Allergy and Infectious Diseases of the National Institutes of Health, U. S. Public Health Service. t Department of Microbiology, The University of Texas Southwestern Medical School, Dallas. + Department of Microbiology, College of Medicine, University of Florida, Gainesville. ? Resident Coordinator, L.S.U.-International Center for Medical Research and Training, San Jose, Costa Rica on leave, Department of Microbiology, College of Medicine, University of Florida, Gainesville.

Adaptability of Schistosoma mansoni of Human Origin to Mice and Hamsters.

Abstract Cercariae derived from A. glahratus exposed to miracidia hatched from stools of Puerto Rican patients infected with S. mansoni were used for exposure of mice and hamsters. In every instance viable eggs of S. mansoni were demonstrated in the stools of the infected animals 6 to 7 1 2 weeks after exposure. In view of the present findings the gradual adaptation of human strains of S. mansoni to mice and hamsters would appear to be unnecessary.

Laboratory studies of combinations of piperonyl cyclonene, piperonyl butoxide, pyrethrins, and rotenone for thé control of ticks on dogs.

It has been reported by Wachs (1947) that synergism could be demonstrated when piperonyl butoxide or piperonyl cyclonene was used in combination with pyrethrins for the control of houseflies. Brannon (1947) likewise demonstrated the principle of synergism when either piperonyl butoxide or piperonyl cyclonene was used in combination with rotenone for the control of the Mexican bean beetle. When Dove (1947) reported on the relative safety of piperonyl butoxide to warm-blooded animals, it was felt that investigations should be made on similar combinations to determine the relative effectiveness against dog ticks. The present paper summarizes the results of several laboratory tests with these combinations for the control of the American dog tick, Dermacentor variabilis Say and the brown dog tick, Rhipicephalus sanguineus Latr.