Showing papers in "Journal of Parasitology in 1974"

Effect of host defenses on the feeding pattern of Culex nigripalpus when offered a choice of blood sources.

Abstract: Host defensive behavior was examined in several avian and mammalian species. Visual observations of host activity indicated that low mosquito feeding rates were caused by host defenses and not by a lack of attraction. Feeding rates were enhanced when this behavior was suppressed by immobilization. Several selection experiments were conducted in outdoor cages using paired combinations of different species which were situated in close horizontal proximity to each other (3 to 5 ft). Similar feeding rates were obtained when both hosts were immobilized. When only one host was restrained the feeding ratio always shifted in favor of the immobilized host. This shift was greatest when the free host was very defensive. If neither host was restrained, mosquitoes always fed to a lesser extent on the more defensive host. No evidence of any active diversion from defensive to tolerant hosts was observed in tests where hosts were vertically separated 8 ft apart. When mosquito densities were gradually increased, there was a corresponding increase in the proportion of the engorged feeding on the tolerant host. But at the same time there was a decrease in the overall engorgement rate. The engorgement rates remained relatively stable on the tolerant host but decreased on defensive hosts. In our previous studies of host defensive behavior different ciconiiform birds were exposed individually to predetermined numbers of mosquitoes in large outdoor cages. We demonstrated that the defensive behavior exhibited by different species has a marked effect on the feeding success and mortality of mosquitoes attempting to bite (Edman and Kale, 1971). Variation in feeding success was also influenced by the age of the bird, i.e., whether nestling or adult, and to a lesser extent, by certain individual characteristics (Kale et al., 1972). In addition, we found, as did Reeves (1972), that the density of biting mosquitoes directly affects the intensity of host defensive activity and thereby influences the interruption and success of feeding by the parasites as well (Edman et al., 1972). In the present study, hosts were tested in pairs or groups to determine if the behavior of different animals, when spaced or in close proximity, also influences the "selection of a host" by Culex nigripalpus. Numerous species of birds and mammals were first screened to establish their relative tolerance to mosquito attack. Then several species, manageable in captivity and yet representing a wide range of defensive behavior from intensely intolerant to tolerant, were selected for choice tests. Received for publication 9 August 1973. * Supported by National Institutes of Health Grants No. AI-06587 and AI-11201. t Florida Medical Entomology Laboratory, P.O. Box 520, Vero Beach, Florida 32960, U.S.A. MATERIALS AND METHODS Test mosquitoes were reared from eggs of wild females as previously described (Edman and Kale, 1971). Non-blood-fed unmated females 6 to 12 days old, the number varying with the experiment, were automatically released from electrically timed holding boxes (1 ft3) mounted inside the test cages containing hosts. All surviving mosquitoes were recovered from the cages the following morning with an aspirator, then counted and examined for blood. Partial or incomplete blood meals were also categorized as in past studies (Edman and Kale, 1971). In experiments with multiple hosts, engorged mosquitoes were individually extracted in buffered saline and precipitin tested (Edman, 1971) to determine which of the 2 or 3 host-bloods they contained. We employed the experimental cage apparatus previously described (Edman and Kale, 1971) as well as 2 modifications. Initial screening tests with individual birds were carried out in the original 4-cage (each 8 by 8 by 8 ft) system. In tests with paired hosts, cages 8 by 4 by 8 ft were used. Reducing the size allowed the utilization of 8 test cages per night and these were arranged in a circular pattern under the same rainproof canopy used previously. Two species of herons were tested using 2 of the original cages and 2 cages of the reduced size. Feeding success in the smaller cages was comparable to that in the larger cages. For the final test with 3 host species, a single large cage 20 by 20 by 16 ft was assembled under the elevated canopy. Unrestrained hosts were held in portable, wire cages varying in volume from about 1 ft3 for the sparrow and mouse to 22 ft3 for large birds. These cages allowed for movement of the animals within and mosquitoes could readily fly in and out. In tests with immobilized hosts, small birds were restrained in nylon stockings and mammals in tight-fitting hardware cloth tubes. Egrets were

Isospora felis: development in cultured cells with some cytological observations.

Abstract: Isopora felis undelwent development in monolayer cultures of human cells (embryonic intestine, esophageal epithelium, amnion, lung, and epithelioid carcinoma of the cervix) as well as cells from embryonic bovine trachea, canine kidney, and chick kidney. Pairs of daughter organisms developed by endodyogeny and possibly schizogony. Special stains were used to study the cytological changes that occurred during development. Disappearance of red-stained material from the refractile body in organisms stained with Whipf's polychrome stain suggested utilization or conversion of this material by the sporozoite or a change in its ionic nature; its reappearance in mature daughter organisms suggested replacement, reconversion, or return to the previous ionic composition for the next generation. Carbohydrate, identified as periodic acid-Schiff positive granules, also appeared to be utilized by the sporozoites and replaced in the mature daughter organisms. Nucleic acids were identified in the cytoplasm and the nucleus of sporozoites and daughter organisms by staining with gallocyaninchromalum after RNase or DNase digestion. The distribution of nucleic acids in these two stages was similar. Several species of Isospora have developed in cultured cells (Turner and Box, 1970; Fayer, 1972; Fayer and Kocan, 1972; Fayer and Mahrt, 1972). However, previous attempts to obtain development of I. felis have been unsuccessful. Sheffield and Melton (1970) inoculated monkey kidney cell cultures with a mixture of Toxoplasma gondii, I. felis, and I. rivolta and found that "a typical T. gondii infection resulted." They also inoculated similar cultures with a mixture of I. felis and I. rivolta sporozoites and found no intracellular organisms. Shibalova and Petrenko (1972) inoculated cultures of chick fibroblasts, quail fibroblasts, monkey kidney, human embryo, rabbit, sheep embryo, green marmoset, and human amniotic cells with sporozoites of I. bigemina, I. felis, and I. revolta. Each of the three species entered cells, but only I. bigemina appeared to have undergone development. The present study represents a further attempt to obtain development of I. felis by employing some of the same cell types used by previous investigators as well as several different cell types. This study also represents the first investigation of cytochemical changes of an isosporan in cell culture. Received for publication 5 July 1973. *Animal Parasitology Institute, Beltsville Agricultural Research Center, ARS, Beltsville, Maryland 20705. MATERIALS AND METHODS

Comparative biochemical studies of Litomosoides carinii, Dipetalonema viteae, and Brugia pahangi adults.

Abstract: The metabolisms and effects of several anthelmintics on 3 adult filarial nematodes, Litomo- soides carinii, Dipetalonema viteae, and Brugia pahangi, have been compared. The latter 2 differ from the obligate aerobe, L. carinii, in that they survive for extended periods of time anaerobically, indicating that they obtain their metabolic energy by utilizing anaerobic pathways. In accord with their apparent anaerobic nature, fermentation balance studies indicate that D. viteae and B. phangi are homolactate fermenters. L. carinii, on the other hand, forms appreciable quantities of acetate in addition to lactate and C02. Isotope studies indicate that the tricarboxylic acid cycle does not con- stitute a quantitatively significant energy-yielding pathway in L. carinii. Instead, findings suggest that the aerobic requirement of L. carinii may reside completely in one system, the oxidative decarboxyla- tion of pyruvate to acetate and CO2. Cytochrome c oxidase activity could not be demonstrated in any of the parasites as assayed by the oxidation of reduced cytochrome c. 1-Tetramisole at low concen- trations (4.2 X 10-7 M) completely stops motility of all 3 species within 1.5 min. d-Tetramisole also inhibits motility effectively, but it is considerably more readily reversible than the corresponding levo-isomer.

Development of Sarcocystis fusiformis in the small intestine of the dog.

Abstract: Coccidia-free dogs orally inoculated with Sarcocystis fusiformis sporocysts obtained from the feces of other dogs failed to pass sporocysts within 18 and 32 days after inoculation; these results suggest either that the sporocyst stage does not indicate infection leading to the production of sporocysts in this host or that the prepatent period after sporocyst inoculation is extremely long. Three groups of dogs fed raw bovine heart infected with S. fusiformis passed sporocysts beginning 13 to 22 days later. After passage of sporocysts stopped, dogs fed infected bovine heart a second time began passing sporocysts 9 and 11 days later. Freshly passed sporocysts contained four sporozoites and a residuum, but no micropyle or Stieda body. Twenty sporocysts averaged 15.7 by 9.9 /u. One dog passed unsporulated oocysts of Isospora bigemina (small form). Still other dogs fed raw bovine heart infected with S. fusiformis were killed 1 to 8, 10, 11, and 13 days later. In histological sections of the small intestine, parasites were observed from days 2 through 13 in the lamina propria of the villi. They were most abundant in the distal jejunum and proximal ileum. Macrogamonts were observed from days 2 through 13. Oocyst wall formation was first observed on day 7, and sporogony was first observed on day 8. Microgamonts were not positively identified, and no asexual stages were found. Dogs fed raw beef infected with Sarcocystis fusiformis have been found to pass in their feces coccidian sporocysts containing sporozoites (Heydorn and Rommel, 1972a; Mahrt, 1973; Fayer and Leek, 1973). The present report describes these sporocysts and the stages in the canine intestine that lead to their de-

The miracidium-sporocyst transition in Schistosoma mansoni: surface changes in vitro with ultrastructural correlation.

Abstract: Miracidia of Schistosoma mansoni, hatched aseptically, readily shed their ciliated epidermal cells in a culture medium. Electron microscopy shows that within 200 min in culture a new sporocyst tegument has formed, presumably from material mobilized from the subepidermal region. Granules resembling the alpha and beta particles of glycogen are involved in this process, passing up into the new tegument where they appear to disintegrate. The surface at this time bears simple microvilli, which become abundant and branched by 20 hr incubation. Mother sporocysts cultured for 6, 8, or 10 days were injected into Biomphalaria glabrata snails in which some at each age continued development leading to production of cercariae. Within the first 24 to 48 hr after miracidial penetration, focal tissue responses in the snail, Biomphalaria glabrata, act to destroy certain individual sporocysts of Schistosoma mansoni while others proceed to normal development (Newton, 1954; Pan, 1963). Elicitation or absence of this host foreign body response is probably associated with surface features of very young mother sporocysts (Wright, 1971). Information about the nature and changes in the parasite surface during the critical period of transformation from miracidium to sporocyst is therefore basic to comprehension of specificity between host and parasite. It is difficult to follow these events in vivo because the precise moment of miracidial penetration usually cannot be determined, and the tiny organisms are hard to find within the snail tissue. Induction of the same changes in vitro, with viability of sporocysts demonstrated by subsequent normal development upon implantation into snails, offers a means whereby these phenomena may be readily observed. We present here a simple culture method together with a description of surface changes between miracidium and sporocyst of S. mansoni in vitro. MATERIALS AND METHODS Golden hamsters, Mesocricetus auratus, were infected with 150 to 200 cercariae of S. mansoni. After about 8 weeks an animal was killed by rapid cervical dislocation, rinsed with 82% alcohol, its liver removed aseptically and dropped into a Received for publication 8 July 1974. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. sterile, ice-cold stainless steel semi-micro blender container (Eberbach Corporation) along with 100 ml of sterile ice-cold distilled water containing 200 units of Penicillin G per ml. All subsequent procedures were done aseptically using sterile glassware and reagents. The liver was minced for about 10 sec at high speed and the container placed in the refrigerator for 20 to 30 min. The top 80 ml of fluid was then carefully decanted, and 100 ml of fresh water with antibiotics added. The container was again refrigerated for 20 to 30 min. Meanwhile 2 X 105 units of Penicillin G were added to each of 2 1-liter volumetric flasks which had earlier been filled to the base of the neck with filtered aquarium water, capped (aluminum 35-mm film cans were used), and autoclaved. After the second sedimentation of the comminuted liver, 10 ml of sediment was transferred by pipette to each volumetric flask. The body of each flask was covered with aluminum foil, sterile water added up to the liter mark, and the upper neck illuminated with a small desk lamp. When miracidia were abundant in the top few cm of the water column, 5-ml aliquots, usually containing several hundred organisms, were withdrawn at intervals and transferred to 15-ml screwcapped conical centrifuge tubes, which were then placed into shaved ice in an insulated bucket. After miracidia had concentrated at the bottom of the tubes, as much water as possible was removed with a drawn-out Pasteur pipette, while observing through a binocular dissecting microscope. Two to 3 ml of sporocyst culture medium (DiConza and Basch, 1974) was added and the tubes incubated at room temperature (25 ?+ 1 C). Medium was made up with horse or fetal calf serum (Grand Island Biological Co.) or human serum from our laboratory staff. All sera were inactivated (56 C, 30 min) before use. Some cultures were transferred to 10by 75-mm disposable glass culture tubes which were tightly sealed with 00 silicone stoppers. Medium usually was not changed during the culture period. Obser-

Schistosoma mansoni: adsorption of human blood group A and B antigens by schistosomula.

Abstract: Immediately following skin penetration, schistosomula of Schistosoma mansoni were found capable of passively adsorbing human A and B blood group antigens onto their surfaces. The antigens were adsorbed from intact erythrocytes, boiled human saliva, or alcohol extracts of erythrocyte membranes. Evidence is presented suggesting that a glycosphingolipid structure may be important in the binding of erythrocyte antigens to schistosomes. No uptake of various other blood group antigens could be demonstrated. Mouse erythrocyte antigens have been demonstrated on the surface of adult Schistosoma mansoni reared in mice (Smithers, Terry, and Hockley, 1969; Clegg, Smithers, and Terry, 1971a), and it has been shown that at least one such antigen is passively adsorbed by schistosomula (Dean, 1971; Sell and Dean, 1972; Dean and Sell, 1972). Clegg, Smithers, and Terry (1971b) reported that young schistosomes reared in a culture medium containing human erythrocytes were destroyed following surgical transfer into monkeys which had been immunized against human erythrocytes, indicating that human antigens are also acquired by schistosomes. In one experiment, the erythrocytes used to immunize recipient monkeys differed from the erythrocytes in the worm cultures with respect to five major blood group antigens. Fewer worms were killed upon transfer to monkeys so immunized than was the case with homologous transfers, and this led the authors to conclude that blood group antigens might be acquired by schistosomula. The present study was carried out to deReceived for publication 13 September 1973. * Supported by the Bureau of Medicine and Surgery Work Unit No. MR041.05.01.0023A6GI. The opinions or assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles outlined in the Animal Welfare Act (PL 89-544 as amended) and followed the guidelines prescribed in DHEW Publication No. (NIH) 72-23, formerly PHS Publication No. 1024, "Guide for Laboratory Animal Facilities and Care." termine which, if any, human blood group antigens are adsorbed onto the surfaces of schistosomula in vitro. MATERIALS AND METHODS Schistosomula were prepared from Puerto Ricanstrain cercariae of S. mansoni by allowing them to penetrate excised rat skins into Hanks' Balanced Salt Solution (HBSS), by methods described previously (Stirewalt and Uy, 1969; Clegg and Smithers, 1972). Alcohol extracts of erythrocyte membranes were prepared by the method of Koscielak (1963). Wet membranes prepared by hypotonic lysis (Dodge, Mitchell, and Hanahan, 1963) were mixed with ethanol to a final alcohol concentration of 85%, mixed on a shaker at room temperature for 3 days, centrifuged, and the superatant fluid stored at -15 C for 3 days. The resulting precipitate was collected by centrifugation and stored at -70 C. Koscielak showed that the blood group active molecules in these preparations are glycosphingolipids. Saliva of a known secretor of soluble blood group A substance was boiled and centrifuged, and the supernatant fluid employed in appropriate experiments. Neutr AB is a commercial preparation (Dade Reagents, Miami, Florida) containing both A and B blood group activities. The source of this preparation (equine gastric mucin) and the method of preparation (Dade Reagents, pers. comm.) indicate that the active molecules are probably glycoproteins rather than glycosphingolipids (Baer, Kabat, and Knaub, 1950; Kabat, 1956). Specific activities of alcohol-extracted antigens and Neutr AB were measured by means of the hemagglutination inhibition (HI) assay. Serial double dilutions of antigen were prepared in Vbottom wells of Microtiter plates (Cooke Engineering Company, Alexandria, Virginia). Dilutions were made in pH 7.4 phosphate-buffered saline (PBS) at a final volume of 0.05 ml per well. An equal volume of blood group typing antiserum

Immunity to Hymenolepis citelli by Peromyscus maniculatus: genetic control and ecological implications.

Abstract: Acquired resistance to Hymenolepis citelli in Peromyscus maniculatus, the white-footed deer mouse, is shown to be controlled by a single autosomal dominant gene R (recessive gene designated r). This resistance could be transferred to uninfected hosts with "immune" lymphoid cells but not serum from infected resistant animals. Treatment with heterologous antilymphocyte serum depressed the ability of competent hosts to resist infections. A comparison of the response elicited against 2 strains of H. citelli (Utah and California) illustrated that chance-bred P. m. sonoriensis respond more rapidly and in greater incidence to the California strain than to the Utah strain. Homozygous recessive (r/r) hosts, susceptible to H. citelli (Utah), respond in significant numbers to the California strain of the tapeworm. Natural infections of H. citelli in Peromyscus do not occur uniformly throughout the environment but occur in small foci favoring existence of both intermediate and definitive hosts. Infections rarely occur outside such foci. We postulate that H. citelli is propagated in such foci by genetically nonimmune (r/r) hosts comprising about 25% of the host population. A previous study (Wassom et al., 1973), utilizing the tapeworm Hymenolepis citelli and its natural host Peromyscus maniculatus, illustrated that a tapeworm living exclusively in the gut lumen could elicit a protective resistance in some individuals. Such hosts, after initial acceptance of infection, responded by eliminating the worms and resisting reinfection. Other individuals were incapable of developing resistance. Primary infections were maintained and secondary infections could be imposed upon initial ones. The present study was conducted to evaluate the genetic aspects of this host resistance, and to determine whether the response is mediated by specific immune mechanisms or by nonspecific factors. MATERIALS AND METHODS All experimental animals were laboratory-reared Peromyscus maniculatus sonoriensis fed "Purina Laboratory Chow." The isolation of H. citelli from wild-caught P. maniculatus and its maintenance in the laboratory as well as procedures for infection have been previously reported (Wassom et al., 1973). "Immune" serum was pooled from 2 groups of chance-bred P. maniculatus infected on day 0 with 10 cysticercoids of H. citelli, challenged with reinfection on day 14 or 21, and bled 7 days later. Received for publication 16 July 1973. * Department of Biology, University of Utah, Salt Lake City, Utah 84112. t Department of Pathology, University of Utah, Salt Lake City, Utah 84112. 47 Lymphoid-cell suspensions from the thymus, spleen, and lymph nodes of infected animals were prepared by mincing the tissues in Minimal Essential Medium for Suspension-Earle's BSS (MEMMicrobiological Associates), filtering through nylon gauze, and centrifuging at 60 g for 10 min. Red blood cells were lysed with Tris-buffered hypotonic ammonium chloride (Boyle, 1968), and the cells washed 4 times in MEM. Viability of cells was determined by trypan blue exclusion (Boyse et al., 1962). Cell concentrations were determined with standard microscopic counting chambers. Antilymphocyte serum (ALS) was prepared in rabbits by injection of Mus musculus lymphoid cell membranes in Freund's complete adjuvant. Injection of 0.5 ml of this ALS intraperitoneally on days 0, 2, and 4 was shown to delay median rejection times of Mus musculus allogeneic skin grafts (H2' to H2k) from 10.5 to 13.7 days. Skin grafts were not attempted in P. maniculatus. However, injection of 0.4 ml ALS intraperitoneally to P. maniculatus on days 0, 2, and 4 was shown to reduce hemagglutinin titers to SRBC by 4 doubling dilutions. This ALS is thus an effective immunosuppressive agent in P. maniculatus.

Anaerobic excretion of fermentation acids by Hymenolepis diminuta during development in the definitive host.

Abstract: Succinic, acetic, and lactic acids are the major anaerobic fermentation acids excreted in vitro by Hymenolepis diminuta. In parasites recovered from immature (6-day) infections in white rats that were fed ad lib. in the dark for 12 hr, approximately equal amounts of succinic and lactic acids, and less acetic acid, were excreted. In the succeeding 12-hr period of fasting in the light, production of succinic and acetic acids from endogenous glycogen reserves was unaffected, whereas that of lactic acid decreased markedly. The pattern was different in parasites recovered from 10and 14-day infections, in that quantitative differences in acid excretion during feeding and fasting periods were not observed, lactic acid production was relatively small, and succinic acid constituted about half of the total excreted acids. An active pyruvate decarboxylase enzyme complex presumably accounted for the formation of acetate from pyruvate. The existence of lactate dehydrogenase, and a phosphoenolpyruvate carboxykinase leading to succinate production were already known. A calculation of the carbon content of excreted acids in terms of ,moles phosphoenolpyruvate carbon/mg protein showed that the rates of excretion of fermentation acids in 6and 14-day infections did not differ. Succinic, acetic, and lactic acids comprise most of the total fermentation acids excreted in vitro by Hymenolepis diminuta recovered from patent (15 day) infections in rats (Fairbairn et al., 1961). It is known that phosphoenolpyruvate is a key intermediate in the formation of these acids since it reacts with carbon dioxide in the presence of phosphoenolpyruvate carboxykinase and GDP or IDP to form oxaloacetate and thence succinic acid, and is also converted to pyruvate by pyruvate kinase (Bueding and Saz, 1968) and hence to L(+) lactate by lactate dehydrogenase (Walkey and Fairbairn, 1973). Acetic acid is presumably formed by oxidative decarboxylation of pyruvate, although the existence of this enzyme complex has not been studied. Different isoenzymes of L(+) -lactate dehydrogenase occur in the anterior regions of the mature tapeworm strobila or in immature (6 day) parasites, and in infective eggs obtained from mature proglottids (Walkey and Fairbairn, 1973). Similar observations have been made on pyruvate kinase (Carter and Fairbairn, unpublished). There is reason to believe, therefore, that quantitative differences in the forReceived for publication 11 February 1974. * This work was supported by Grants AI-08491 and 5 TOI AI-226 from the NIH, Bethesda, Maryland 20014. t Present address: The Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, England. mation and excretion of succinic, acetic, and lactic acids may exist in different regions of the strobila, or in parasites at different stages of development in the definitve host. This is not surprising, as the strobila of H. diminuta contains hundreds of proglottids representing all stages of continuous growth and differentiation. In the present investigation we have compared the in vitro anaerobic excretion of succinic, acetic, and lactic acids by H. diminuta recovered from infected rats after 6 and 14 days, respectively, and have demonstrated the decarboxylation of pyruvate by a pyruvate decarboxylase enzyme complex. MATERIALS AND METHODS In order to make possible a comparison of the present experiments with other recently completed experiments (Carter and Fairbairn, unpublished), white, male Sprague-Dawley rats (Holtzman strain) weighing about 150 g each were infected with 30 cysticercoids of H. diminuta injected into the stomach through a catheter. After ensuring that all cysticercoids had been delivered from the catheter, the rats were caged in pairs and maintained on a 12 hr light-12 hr dark daily photoperiod. Food (commercial pellets) was provided only during the dark half of the cycle, from 8:00 PM to 8:00 AM. Rats harboring parasites to be recovered on the 10th or 14th day after infection were maintained in this manner. When parasites were to be recovered after infection for 6 days the procedure was the same except that greater numbers of rats were infected, each with 300 to 500 cysticercoids, in order that sufficient weight of tissue would be available for

Host-parasite relationship in echinococcosis. I. Parasite biomass and antibody response in three strains of inbred mice against graded doses of Echinococcus multilocularis cysts.

Abstract: Susceptibility studies in C57L, DBA, and C57B1 mice, infected intraperitoneally with 1, 5, 20, and 100 cysts of Echinococcus multilocularis, showed a significant difference in the cyst weights, after a 12-week period. With all 4 inocula C57L mice supported a greater larval load than DBA and C57B1 mice. Sera obtained at 2, 4, 8, and 12 weeks postinfection (p.i.) from these mice showed hemagglutinating antibodies (HAA) only at 12 weeks p.i. in the 1and 5-cyst groups of mice but much earlier in the 20and 100-cyst groups. C57L and DBA mice showed similar HAA titers and had 1 to 4 precipitin bands depending on the stage of infection. These results are discussed with regard to a possible nonprotective role of antibodies in echinococcosis. No specific data are available on the functional relationship between the immune mechanisms of the host in either unilocular (Echinococcus granulosus) or the alveolar (E. multilocularis) types of hydatid disease. The former species has been of continuing interest because of its worldwide distribution and consequent higher incidence of infection in man and animals (Kagan, 1968; Gore et al., 1970; Williams et al., 1971). In recent years screening of experimental animals for laboratory maintenance and studies on the histogenesis of E. multilocularis have been described (Rausch, 1954; Yamashita et al., 1958; Lubinsky, 1964; Webster and Cameron, 1961), but quantitative data on parasite burden, antibody response, and ensuing immunopathological changes in response to various infective inocula in inbred strains of laboratory animals are lacking. The development of an experimental model using an inbred strain of mice and establishment of measurable parameters in order to study the host response or, conversely, the apparent inability of suitable strains of mice to contain rapid proliferation and elimination of alveolar cysts, appear to be in order. This paper reports the parasite biomass and antibody response in three strains of mice against variable inocula of E. multilocularis

Dipetalonema viteae in the experimentally infected jird, Meriones unguiculatus. I. Insemination, development from egg to microfilaria, reinsemination, and longevity of mated and unmated worms.

Abstract: Dipetalonema viteae in Meriones unguiculatus usually completed the 3rd molt at 7 days and the 4th molt at 23 days of infection. Insemination, first recorded at 23 days of infection, occurred most frequently at 25 to 28 days. Microfilariae were first seen in the uterus at 42 days and in the peripheral blood at 47 days. The minimal time between insemination and detection of microfilariae in the blood was 22 days, and between implantation of mature uninseminated females together with mature males and the detection of microfilaremia, 26 days. In inseminated females isolated soon after the first mating, sperm was depleted as early as 14 days and as late at 85 days, indicating that for continuous fertility when few worms are present frequent mating is required. Mated and unmated worms of both sexes lived for periods cf up to about 2 years. Studies on the development of filariae in the vertebrate host have emphasized mainly the gross morphological changes and molting of the worms, and the duration of prepatency and patency of the infection. More detailed observations on the development of the organ systems were reported for Dirofilaria immitis by Orihel (1961) and for Brugia pahangi by Schacher (1962). Development of the microfilaria of different species was observed by Kotcher (1941) and McFadzean and Smiles (1956), and spermiogenesis was described by Taylor (1960). Observations on the filarial reproductive system have included studies by Webber (1954a, b), in vitro examinations by Weinstein and Sawyer (1961) and by Raghavan et al. (1956). Hawking (1954) studied the reproductive capabilities of Litomosoides carinii both in vitro and in vivo. Received for publication 18 September 1973. * Conducted under the sponsorship of the Commission on Parasitic Diseases, Armed Forces Epidemiological Board, and supported by the Office of the Surgeon General, Department of the Army, and by NIH research grant AI-04919 from the NIAID. Address reprint requests to: Dr. Paul C. Beaver, Department of Tropical Medicine and Parasitology, Tulane Medical Center, 1430 Tulane Avenue, New Orleans, La. 70112. t Present address: Department of Biology, University of Southern Mississippi, Southern Station, Box 18, Hattiesburg, Mississippi 39401. Dipetalonema viteae (Krepkogorskaja, 1933), a natural parasite of some members of the Gerbillidae (Meriones spp. and Rhombomys opimus), was studied by Chabaud (1954), Worms et al. (1961), and Bain (1967). Weiss (1970) compared the rate of development and persistence of infection in various laboratory hosts, mainly Meriones libycus, Gerbillus hirtipes, and the golden hamster, Mesocricetus

Proline in fascioliasis: I. Comparative activities of ornithine-delta-transaminase and proline oxidase in Fasciola and in mammalian livers.

Abstract: The source was sought for the high levels of proline observed in the bile of animals infected with Fasciola hepatica. Toward this end the activities of 2 key enzymes in the metabolism of proline were measured. Ornithine-4-transaminase was compared in homogenates of Fasciola, rabbit liver, and rat liver. The specific activity of the Fasciola enzyme is more than 10 times greater than that of rabbit liver and more than 7 times greater than that of rat liver. Furthermore, proline oxidase which breaks down proline in mammalian liver was absent or very low in homogenates of Fasciola. These observations strongly suggest that the worm is the source of the excessive free proline. Recent studies by Kurelec and Rijavec (1966) and Isseroff et al. (1972) have shown that free proline makes up 25 to 30% of the total free amino acid nitrogen in the trematode, Fasciola hepatica. In the latter study an increase in the total free amino acid concentration was observed in the bile of infected animals. While most free amino acids increased by approximately 40 times normal concentration, proline exhibited the greatest change in concentration with increases up to 10,000-fold. Because an increase of this magnitude in a single amino acid was likely to be of significance in the host-parasite relationship, it was of interest to determine the origin of the excessive proline. From data in the study cited above (Isseroff et al., ibid.) it was known that the concentration of proline in the worm itself was always greater than in the bile, while analysis of host tissues revealed proline in concentrations as low as those in uninfected controls. Yet it was not likely that the worms were concentrating proline from the bile. Studies by Isseroff and Read (1969) had shown that proline uptake by the worm in 2min incubations is a linear function of increasing concentration, indicating uptake by simple diffusion. Furthermore, Moss (1970) observed that proline was one of the major free amino acids excreted by Fasciola in in-vitro

Hemoglobin absorption in the gut of a monogenetic trematode, diclidophora merlangi

Abstract: The absorption of hemoglobin in the gut of D. merlangi takes place in pigmented hematin cells. The process is an endocytotic one involving incorporation of the protein in bristle-coated in- vaginations of the cell surface which are pinched off to form small coated vesicles. Vesicle counts on worms in different nutritional states show there is a direct correlation between the number of coated vesicles in a hematin cell and the presence of host blood in the adjacent gut lumen. The vesicles transport the hemoglobin to an extensive series of channels of irregular dimension and bulbous ex- panded portions forming a reticulum. The reticular system forms a semipermanent feature of each hematin cell and represents the site of intracellular digestion of blood and formation of the pigment hematin.

Sparganum proliferum, a sparganum infected with a virus?

Abstract: Electron micrographs of material from the case of proliferating sparganosis reported by Stiles, 1908, from Manatee County, Florida, show what appear to be type-c virus particles abundantly budding from the walls of internal cavities, presumably excretory ducts. The particles are spherical, approximately 85 mu in diameter. No similar structures were seen in corresponding thin sections of normal spargana. It is suggested that Sparganum proliferum is a worm whose genetic blueprint and organization have been profoundly altered by infection with a virus which simultaneously stimulates exaggerated growth and loss of morphogenetic control, as well as endowing the worm with a greatly extended life span. Sparganum proliferum is a rare aberrant tapeworm larva known only from man. It is characterized by continuous branching and budding, the resulting progeny invading the entire limb or region involved, and appearing under the skin as acneform pustular nodules from which living worms may be expressed. All tissues are invaded except bone. The buds have the histological components of ordinary spargana, but their organization and symmetry are profoundly disturbed, and for want of a scolex they do not mature when fed to presumptive definitive hosts such as the dog and cat (Mueller, 1938). In 1966 the senior author suggested that this peculiar form might be explained on the basis of "hyperparasitism of a sparganum by an agent which simultaneously causes exaggerated growth stimulation and breakdown of morphogenetic control, as in the case of certain tumor-producing viruses." The present paper tends to validate this conjecture. MATERIALS AND METHODS When the senior author first began work on sparganosis in the 1930's, the late Maurice C. Hall, of the NIH, presented him with a vial of material from Gates' case, in Manatee County, Florida, reported by Stiles (1908). Recently the Armed Forces Institute of Pathology (AFIP) requested confirmation of a suspected case of sparganosis in a man who had died of Hodgkins' disease. Since only tissue sections were available a specific diagnosis could not be made, although the organism appeared to be a sparganum. In the course of a telephone conversation the above-mentioned viral Received for publication 10 November 1973. * This research has been supported by a continuing Grant AI-01876 from the NIH. t State University of New York. Upstate Medical Center, Syracuse, New York 13210. t Armed Forces Institute of Pathology, Washington, D. C. 20305. hypothesis was alluded to, along with the fact that material of Sparganum proliferum from the Florida case was available, although probably useless because it had been fixed and preserved since 1907 (Fig. 1). At this point the junior author stated that virus particles, if present, could be detected in fixed material even after a lapse of many years. Thus the present collaboration was born. Several pieces of Stiles' material were sent to the AFIP, and thin sections were examined by electron microscopy. Typical specimens of Spirometra mansonoides spargana were processed at the same time and served as controls for compari-

A reexamination of neochordodes occidentalis (montg.) comb. n. (chordodidae: gordioidea): larval penetration and defense reaction in culex pipiens l.

Abstract: The eggs, preparasitic larvae, and certain adult characters of Neochordodes occidentalis (Montg.) (Chordodidae: Gordioidea) are newly described. The method of penetration of preparasitic hairworms into larvae of Culex pipiens L. was examined. The parasites bored directly through the peritrophic membrane and epithelial gut cells to reach the host's body cavity. The hairworms encysted normally in most 2nd- and 3rd-stage mosquito larvae but elicited a rapid and often lethal defense reaction in 4th-stage hosts. This reaction consisted of the deposition of melanin on the surface of the parasite. Cyst material originated from a bilobed intestinal gland in the preparasitic larva and was not considered of host origin. Findings here indicate that characters found in the preparasitic larvae could be used for distinguishing between hairworm species.