Showing papers in "Journal of Parasitology in 1971"

Cytological studies on the absorptive surfaces of cestodes. V. Incorporation of carbohydrate-containing macromolecules into tegument membranes.

Abstract: The origin of carbohydrate-containing macromolecules constituting the cestode tegument glycocalyx has been investigated by biochemical, cytochemical, and autoradiographic methods. Tapeworms incubated in vitro with 3H-galactose incorporate radioactivity into a protein fraction, much of which appears to be associated with membrane structure. Approximately 40% of the radioactivity which sediments at 50,000 g with a membrane-rich fraction from a diastase-digested homogenate can be solubilized by treating the pellet with sodium lauryl sulfate. Autoradiographs demonstrate the tegument perikarya as major sites of this radiogalactose incorporation. The labeled material is subsequently transported to more superficial regions of the tegument, much of the radioactivity becoming concentrated in the brush border. Autoradiographs of worms briefly "pulsed" with radiogalactose, then incubated with nonradioactive galactose for periods up to 24 hr, indicate continual replenishment of this tegument surface component, appreciable replacement occurring 6 to 8 hr following its synthesis and incorporation into the brush border. Carbohydrate-containing material was localized cytochemically within Golgi cisternae and vesicles in the perikarya and within vesicles concentrated beneath the brush border in the distal tegument cytoplasm. Ultrastructure suggestive of fusion between these vesicles and the tegument plasmalemma was frequently observed. Thus, carbohydrate constituents of the surface membrane appear to be added in quanta elaborated by the synthetic apparatus of the perikarya. Bennett (1963) coined the term glycocalyx for the polysaccharide coatings ubiquitously present on the external surfaces of prokaryotic and eukaryotic cells (Ito, 1969). These glycocalyces exhibit considerable structural diversity and in some cases can be visualized only by cytochemical staining or immunochemical methods (Revel and Ito, 1967). The body surface of many platyhelminths and acanthocephala is invested with a coating of fine filaments or more amorphous fuzz (Crompton and Lee, 1965; Nicholas and Mercer, 1965; Erasmus and Ohman, 1965; Burton, 1966; Lumsden, 1966a; Erasmus, 1967; Braten, 1968; Wright and Lumsden, 1968; Bogitsh, 1968; Smith et al., 1969; Bresciani and K0ie, 1970; Lyons, 1970; Lumsden et al., 1970a). This coat is otherwise demonstrable cytochemically by staining techniques for the vicinal hydroxy groups of carbohydrates and, in some cases, acidic functions (Morris and Threadgold, 1968; Bogitsh, 1968; Wright and Lumsden, 1968, 1970; Kemp, Received for publication 13 July 1971. * Supported by grants from the USPHS, NIH (AI 08673, 5 TI GM 669, AI 00002), and the NSF (GB 17992). The second author is the recipient of Career Development Award K04-AI 23449 from the USPHS, NIH, NIAID. 1970; Reissig, 1970; Rothman and Elder, 1970; Lumsden et al., 1970a). It would seem appropr ate, therefore, to designate the surface investment of these helminths a glycocalyx, though more specific terms such as external cuticle, epicuticle, pericercarial envelope, or Cercarienhiille have been used (Monne, 1959; Crompton, 1963; von Lichtenberg, 1967; Wright and Lumsden, 1968; Smith et al., 1969; Rothman and Elder, 1970). In cestodes and acanthocephala, the glycocalyx is intimately associated with the lipoprotein phase of the tegument plasmalemma and resistant to removal by physical means. Where visible, the glycocalyx in a given region of the tegument has consistent morphology among worms of the same species, but may vary considerably in structure among worms of different species. Moreover, larval tapeworms and acanthocephala not yet exposed to the mucins of the definitive host's digestive tract possess a surface coat structurally and cytochemically reminiscent of the adult glycocalyx. These characteristics (Wright and Lumsden, 1968; Lumsden et al., 1970a) favor the view that the glycocalyx of these helminths is not merely an adhering film of host mucins, but an integral component of the tegument membrane. It seems

Acanthocephalan infections of man, with two new records.

Abstract: Previous records of acanthocephalans in man are reviewed. Acanthocephalus bufonis (Shipley, 1903) Southwell et Macfie, 1925, is reported from man for the first time, recovered on autopsy of an Indonesian in Djakarta. Corynosoma strumosum (Rudolphi, 1802) Liihe, 1904, is reported from an Eskimo in Alaska, the first record of this parasite from man. Infections of man with acanthocephalan parasites are rare. Many species in this phylum are known to have low host specificity (Golvan, 1957) but for ecological reasons are not likely to be ingested by humans. The intermediate hosts of all species whose life cycles are known are either crustaceans, myriopods, or insects. However, several species are known to utilize fish, amphibians, or reptiles as paratenic hosts, thereby increasing the chances of accidental parasitism in persons eating such animals raw or undercooked. Lambl (1859) gave the first report of a thorny-headed worm in man when he recovered a Macracanthorhynchus hirudinaceus from a child in Prague. Lindemann (1865) stated the infection was common in Russia, where Schneider (1871) found that scarabaeid beetle grubs, the intermediate host, were commonly eaten raw. Gonzaga (1921) recovered eggs of M. hirudinaceus in two of 1,236 human stools in Brazil. A more recent case of proved chronicity was published by Pradatsundarasar and Pechranond (1965) in Thailand. Moniliformis moniliformis, a parasite of rats and cockroaches, has been reported from man in Italy, Sudan, and British Honduras (Faust and Russell, 1957), and Beck (1959) reported it from a patient in Florida. Grassi and Calandruccio (1888) reported on the symptoms displayed by the second author after voluntarily infecting himself with M. moniliformis. Kuntz (1960) reported acanthocephalan eggs, possibly those of this species, from stools of two children in Dacca, East Pakistan. Moore, Fry, and Englert (1969) made the interesting discovery of acanthocephalan eggs in human coprolites obtained in archeological excavations in Danger Cave, Utah. RadiocarReceived for publication 20 October 1970. bon dating placed the samples at 1869 BC ? 160 years, and AD 20 ? 240 years. The authors suggested that the parasite may have been Moniliformis clarki, which is common in the area. The only acanthocephalan outside the class Archiacanthocephala reported from man is A anthocephalus rauschi, recovered by Dr. Rob rt Rausch from the peritoneum of an Eskimo in Alaska, and described by Golvan (1969). The normal definitive host is probably a fish. The present paper reports the occurrence of two more palaeacanthocephalans in humans. Acanthocephalus bufonis (Shipley, 1903) Southwell et Macfie, 1925 (Syn. Acanthocephalus sinensis Van Cleave, 1937) In 1954, during a routine autopsy by Dr. Lie Kian Joe at the Medical School in Djakarta, Indonesia, four male acanthocephalans were recovered from the small intestine of an Indonesian man by screening the contents of the intestine with a metal sieve routinely used to collect hookworms. No wild animals were dissected in the autopsy room, and there was no chance that the specimens came from an outside source and had become mixed with material from human autopsies. Due to a lack of library facilities in Djakarta it was impossible to identify the worms to species; they were reported as Acanthocephalus sp. by Lie Kian Joe and Tan Kok Siang (1959). One specimen was lost prior to the present study. I now know them to be adult males of Acanthocephalus bufonis, a very common parasite of amphibians in Asia (Yuen and Fernando, 1967). Its life cycle is unknown but probably involves a terrestrial isopod crustacean as intermediate host. I can speculate that the man became infected in one of three ways: (1) by ingesting an infected arthropod; (2) by eating an am-

Preferential susceptibility of male jirds (Meriones unguiculatus) to infection with Brugia pahangi.

Abstract: In two trials, 17 litter-mate pairs of jirds (Meriones unguiculatus) were inoculated subcutaneously in the groin with infective-stage larvae of Brugia pahangi to determine if sex of the host was a factor in establishing infection. The mean prepatent period for 14 male jirds ranged from 67 to 76 days. Only 2 female jirds developed peripheral microfilaremia, one at 87 and the other at 94 days postinoculation. In 3 other females, microfilariae were found only in cardiac blood. More adult worms were recovered from male animals than from females; for males the percentage of inoculum recovered was 1 to 32%, in females it was 0 to 18%. In males, adult worms were found primarily in the testes and the heart and pulmonary arteries, whereas adult worms occurred almost exclusively in the heart and pulmonary arteries of female jirds. It appears that male animals, for unexplained reasons, are more susceptible to infection with lymphatic-dwelling filarial worms than are females. Earlier studies on the development of Brugia pahangi and subperiodic B. malayi in jirds indicated that male hosts appeared to be more readily infected than females (Ash and Riley, 1970a, b). This report summarizes observations made on two series of litter-mate pairs of jirds inoculated with B. pahangi to determine if one sex was more susceptible to infection than the other. MATERIALS AND METHODS The origin of the strain of B. pahangi used here was as reported earlier (Ash and Riley, 1970a). Armigeres subalbatus and Aedes aegypti (Liverpool strain) served as vector mosquitoes and were fed on an anesthetized cat that had a microfilarial density of 100 to 200 per 20 mm3 of blood. Maintenance of mosquitoes and the recovery of infective-stage larvae from them were as previously described (Ash and Riley, 1970a). Litter-mate pairs of Meriones unguiculatus, 4 to 6 weeks old, from a colony maintained in the UCLA Vivarium, were used. Male and female animals were inoculated alternately by pipetting a known number of larvae into a subcutaneous pocket close to the left testis in males and into the left groin in females. Animals were bled at weekly intervals beginning 56 days after inoculation; the prepatent period was computed as the mean day between the last bleeding in which no microfilariae were found and the day on which microfilariae were first found. The techniques of bleeding and Received for publication 12 January 1971. * This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the NIAID of the NIH, Department of Health, Education and Welfare, U. S. Public Health Service Research Grant AI-07770. necropsy of infected animals were as previously described (Ash and Riley, 1970a).

Increase in Size of Eimeria separata Oocysts during Patency

Abstract: Examinations of 18 consecutive fecal samples collected at 6-hr intervals from infected rats showed that oocysts increased in length and width by approximately 40% from the beginning to the end of the patent period. Unsporulated oocysts were first observed in the 3rd fecal collection, 84 to 90 hr postinoculation (PI) and 100 of these measured 9.9 to 14.3 by 8.8 to 12.1 u (mean 11.7 by 10.1). Patency ended with the 15th fecal collection, 156 to 162 hr PI, and 100 unsporulated oocysts from this sample measured 14.3 to 17.6 by 13.2 to 15.4 ft (mean 16.3 by 14.2). Despite the size increase during patency, oocyst shape-index did not change. Sporulated oocysts did not differ significantly from unsporulated oocysts in length or width. The most significant increases in both length and width of the sporulated oocysts occurred 90 to 96 and 120 to 126 hr PI. During studies on interspecific interactions between coccidian species, I observed that oocysts of Eimeria separata seemed to become larger as the infection progressed. Increase in the size of coccidian oocysts, although apparently unusual, has been observed by some investigators (Becker et al., 1955, 1956; Cheissin, 1947; Cordero del Campillo, 1959; Fish, 1931; Jones, 1932). It was interesting to note that Becker et al. (1932) stated, "It is evident here also that the oocysts [of E. separata] exhibit no tendency toward a larger or smaller size as the infection progresses." Since my observations appeared to conflict with those of Becker et al., the following experiment was conducted in order to determine if there was an increase in the size of oocysts during the course of the infection. MATERIALS AND METHODS Experimental animals The hosts used in this study were 2 female, coccidia-free, SPF, Fischer 344 strain, inbred albino rats (National Laboratory Animal Company, Creve Coeur, Missouri). They were kept in a room with a relatively constant temperature (22 to 25 C) and were allowed food and water ad lib. Thus an attempt was made to standardize genetic, hormonal, age, and some environmental influences, all factors which may influence the course of an infection. The E. separata isolate was obtained by inoculating rats with fecal material from an infected wild Rattus norvegicus. This infected fecal material was Received for publication 23 February 1971. * Present address: Department of Biology, University of New Mexico, Albuquerque, New Mexico 87106. sent to me by Dr. J. V. Ernst, USDA Regional Parasite Research Laboratory, Auburn, Alabama. The specific status of this isolate was determined by measurement of 100 sporulated oocysts and comparison of observed characters with those from previously described species (Levine and Ivens, 1965). The species was further confirmed by observation of the living endogenous stages of the parasite in the cecum-colon using the technique of Marquardt (1966) and comparing these stages with those originally described by Roudabush (1937).

Immunity produced against Taenia ovis and T. taeniaeformis infection in lambs and rats following in vivo growth of their larvae in filtration membrane diffusion chambers.

Abstract: Activated embryos of Taenia taeniaeformis and T. ovis placed in filtration membrane diffusion chambers and implanted intraperitoneally in rats and lambs, respectively, for 3 weeks, developed at rates comparable with those reported in natural infections. The developing embryos of both cestodes induced a high degree of immunity to subsequent oral challenge in both rats and lambs. In the case of T. taeniaeformis, this immunity was greater after 3 weeks of in vivo growth than after either 1 or 2 weeks. T. ovis produced a solid immunity after only 1 week in vivo. Four main conclusions were drawn from the experiments. First, developing embryos released diffusible antigens capable of stimulating immunity in their host; direct contact between host cells and parasites was not necessary. Second, effective immunizing antigens are produced early in the development of cestode larvae. Third, sufficient antigen to immunize the host is produced by only small numbers of larvae. Fourth, the antigens are effective in immunizing the host via parenteral administration; an intestinal migration is not necessary to induce immunity to the establishment of an oral challenge infection. The relevance of these findings to the production of a vaccine or of improved antigens for diagnosis of cestode infections in man and animals is discussed. Immunity to taeniid cestode larvae has been induced experimentally in a variety of animals by means of infection with live organisms (Miller, 1931a; Sweatman, 1957; Froyd and Round, 1960), by the inoculation of live parasites into abnormal sites (Gemmell, 1962, 1964), and by the injection of extracts and suspensions of killed parasites (Miller, 1931b; Campbell, 1936). Each of these methods suffer from some disadvantages. In natural infections it is virtually impossible to control the period of exposure to antigen and it is often difficult to distinguish between parasites of the immunizing and test infections. It can be difficult also to distinguish between pathology associated with either of the two infections. When live organisms are injected into abnormal sites in the host, some may escape from these sites and develop in other parts of the body (Gemmell, 1964). "Killed" antigen preparations may be lacking in certain metabolic excretions or secretions (ES antigens) produced by live parasites; there is some evidence that these substances may play a part in stimulating immunity (Silverman, 1955). Filtration membrane diffusion chambers have Received for publication 8 September 1970. been used for in vivo cultivation of the nematode Trichinella spiralis in rats; partial development of this parasite occurred in intraperitoneally implanted diffusion chambers, and some degree of immunity developed in the host (Despommier and Wostmann, 1969a, b). Providing that the organisms survive, and preferably also develop, in diffusion chambers implanted into the host they provide a useful method for studying both growth of the parasite and the development of immunity in the host animal. Diffusion chambers allow strict control of the period of exposure to antigen, the host is exposed to a wide range of ES antigens, and there are no pathological changes in the host due to the immunizing infection. The present investigation was carried out to determine the suitability of diffusion chambers for studies on the growth and development of cestode larvae and the immune response of the intermediate host. MATERIALS AND METHODS

Essential amino acids in the culture of Leishmania tarentolae.

Abstract: The following amino acids may be singly deleted from Trager's Defined Medium C without inhibiting the continuous culture of Leishmania tarentolae promastigotes: L-glycine, L-alanine, L-aspartic acid, L-methionine, L-proline, L-glutamic acid, and L-isoleucine. Deletion of L-proline prevents continuous culture of L. tarentolae when either L-methionine, L-alanine, L-glutamic acid, L-aspartic acid, or L-isoleucine is removed as well. Lack of glucose in Medium C is detrimental when either L-glutamic acid or L-proline is missing. The following amino acids are considered essential for the continuous culture of L. tarentolae in Medium C: L-arginine, L-histidine, L-tryptophan, DL-phenylalanine, DL-serine, L-tyrosine, L-threonine, L-valine, L-leucine, and L-lysine. L-proline may also be considered an essential amino acid because continuous culture in its absence is impossible if any other amino acid, except Lglycine, is deleted from Medium C as well. Cultivation of parasitic organisms in chemically defined media is an important prerequisite for determining their nutritional requirements, the understanding of which is of value in the development of rational methods of chemotherapy. In 1957 Trager devised one of the first chemically defined media for a parasitic protozoan, Defined Medium C, which was used to culture promastigotes (leptomonads) of the hemoflagellate Leishmania tarentolae. Trager's Medium C has been used in studies involving L. tarentolae metabolism (see review by Trager, 1968), transformation of L. donovani (Simpson, 1968), and culture of parasitic nematodes (Jackson, 1962). Since the amino acid requirements of L. tarentolae are complex (Trager, 1968), a study was undertaken to determine which ones were essential for continuous culture by singly deleting each of the amino acids in Medium C. This paper reports the results of deletion tests on all of the amino acids in Medium C. The effect of additional deletion of the energy sources D-glucose and L-proline (Krassner, 1969a) on growth was also followed. Preliminary accounts of portions of this study have appeared in earlier communications (Krassner, 1969a, b). MATERIALS AND METHODS Stock cultures of L. tarentolae, strain TAR II (obtained from Dr. L. Parrot of the Institut Pasteur d'Algerie) were maintained at 26 C in 25-ml Erlenmeyer flasks containing 3.0 ml of Trager's Received for publication 5 January 1971. * This work was supported by U. S. Public Health Service Grant AI06827. Defined Medium C (Trager, 1957). The concentration and sources of amino acids used in this study are listed in Table I. Promastigotes cultured in complete Medium C, in the log phase of growth, were used to inoculate flasks containing 3.0 ml of Medium C lacking: (1) one of the amino acids or (2) an amino acid and either L-proline or D-glucose. Inocula contained 0.2 ml (. 4 X 106 cells) of unwashed organisms so that a small residual amount of the deleted material was carried over into the first culture. Successful cultures were transferred every 5 to 7 days into fresh identical media and growth was considered continuous if cells grew for at least 6 successive subcultures. Culture studies were carried out in triplicate and each experiment was repeated at least 3 times. Control cultures containing complete media were run in parallel with test cultures. Each amino acid was tested for purity and homogeneity on Cellulose MN300 (Brinkman Instruments, Inc., Westbury, New York) thin-layer chromatogram plates according to the method devised by Jones and Heathcote (1966). In most cases the L-form of the amino acid was used. Promastigotes from each subculture were studied under phase contrast microscopy to determine if there were any gross morphological changes.

Penetration of Eimeria larimerensis sporozoites into cultured cells as observed with the light and electron microscopes.

Abstract: Cell cultures of Madin-Darby bovine kidney (MDBK) were used to study with the light and electron microscopes the process of penetration of cells by Eimeria larimerensis sporozoites. The fine structure of these sporozoites was also studied. Monolayers from Leighton tubes were covered with a concentrated suspension of sporozoites and immediately observed in double-coverslip preparations with phase-contrast microscopy. For electron microscope study, sporozoites were added to and mixed well with a suspension of MDBK cells, centrifuged for 4 min, fixed immediately, and prepared for study with the electron microscope. Frequently, the Golgi complex of the sporozoites was located in an indentation of the nucleus and partially surrounded by a fold of the nuclear envelope. The inner layer of the pellicle consisted of 2 unit membranes. Wavelike elevations of the pellicle were seen; these may be involved in locomotion. An intranuclear inclusion, consisting of microtubulelike fibrils, was observed. Vacuoles with particulate matter similar to that of the central vacuoles of the Golgi complex and with extensions running anteriorly into the conoid area were seen in extracellular sporozoites and in sporozoites entering cells. The bodies of sporozoites were usually constricted as they entered and left host cells and host cell nuclei; this was also observed in sporozoites moving through the cytoplasm of the host cell. During penetration, the host cell membrane was either interrupted at the initial site of entry or was interrupted after becoming invaginated for a short distance. Escape of host cell cytoplasm occurred frequently after sporozoites left host cells, but only seldom after entrance. Some intracellular sporozoites fixed 4 min or less after inoculation were surrounded by a host cell membrane; others were not. Some sporozoites which were fixed in the process of leaving host cells had a thin layer of host cell cytoplasm covering the portion of the body which was outside of the host cell, and some extracellular sporozoites with such a covering were seen. The process of host cell penetration by sporozoites of several Eimeria species has been studied in vitro with the light microscope. E. larimerensis is especially favorable for such a study because of the relatively large size of its sporozoites and because they enter cultured cells readily (Speer and Hammond, 1970). A detailed study with the light and electron microscopes of sporozoite penetration into cultured cells, as well as the fine structure of extraand intracellular sporozoites of E. larimerensis, is reported herein. MATERIALS AND METHODS Oocysts of Eimeria larimerensis were collected from experimentally infected ground squirrels (Spermophilus armatus), cleaned, sporulated, and sterilized as described previously (Speer, Hammond, and Anderson, 1970). They were then Received for publication 15 September 1970. * Supported in part by research grant AI-07488 from the NIAID, U. S. Public Health Service, and by Public Health Service Fellowship 1-F01GM44456-01 from the Institute of General Medical Sciences. Published as Journal Paper No. 1080, Utah Agricultural Experiment Station. incubated for 15 min in an excysting medium consisting of 0.25%o trypsin and 0.75% sodium taurocholate in saline A. The free sporozoites were washed and resuspended in minimum essential medium (MEM). For studying penetration, 2day-old cultures of Madin-Darby bovine kidney (MDBK) cells in Leighton tubes or 8-oz Brockway culture flasks were used. In the light microscope study, cover slips with monolayers were removed from Leighton tubes, covered with a few drops of suspended sporozoites (0.8 to 1 million sporozoites/ml), and immediately observed in doublecoverslip preparations (Parker, 1962). In the electron microscope study, the cell monolayer in an 8-oz Brockway culture flask was harvested with a trypsin-versene solution. The suspension was centrifuged and the pellet was resuspended in 3 ml of MEM. Five million sporozoites in 3 ml of MEM we added to the tube containing suspended MDBK cells. These were mixed well and immedi tely centrifuged at 750 g for 4 min. The fixative was added immediately after removing the supernatant. Some pellets were fixed according to the method described by Karnovsky (1965), using cacodylate buffer. Other pellets were fixed with 2.67% glutaraldehyde in cacodylate buffer for 1 hr, washed with buffer for 1/2 hr, and postfixed in 2.5% osmium tetroxide in cacodylate buffer for 1 hr. The fixed cells were dehydrated in 35 and 50% ethanol for 10 min each and stained with 1% uranyl acetate and 1% phosphotungstic

The Fine Structure of Ciliated Nerve Endings in the Cercaria of Schistosoma mansoni

Abstract: The fine structure of ciliated nerve endings located in the tegument of Schistosoma mansoni cercariae is described. These include a previously undescribed multiciliate type as well as relatively simpler, uniciliate endings similar to those found in the adult. Comparison is made with similar structures seen in other animal groups. Specialized, uniciliate nerve endings in the adult of Schistosoma mansoni have been described by Morris and Threadgold (1967), Smith et al. (1969), and Silk and Spence (1969). These are generally assumed to have a sensory function. Little is known, however, about the nervous system of the cercaria of this species. Wagner (1961) reported large numbers of "papillae" distributed over the body and tail; Morris and Threadgold (1967) referred to the presence of a cercarial process similar to that found in the adult. The present study describes three distinct types of ciliated nerve endings in the cercaria, and their possible role as sensory receptors is discussed. MATERIALS AND METHODS Cercariae were obtained from infected snails maintained at this laboratory and fixed within 2 hr of emergence. Initial fixation was carried out at 4 C for 1 hr in cacodylate-buffered 2% glutaraldehyde, and postfixation was in Zetterquist's buffered osmium tetroxide (Glauert, 1965). Cercariae were dehydrated in a series of alcohols from 30 to 100% and finally embedded in an araldite-epon mixture. Sections were cut on an LKB 4800A ultratome, mounted on copper grids coated with formvar and carbon, and stained with uranyl acetate and lead citrate. Material was examined in a Hitachi HS8 electron microscope operating at 50 kv.

Effect of the "salmon poisoning" trematode, Nanophyetus salmincola, on the swimming ability of juvenile salmonid fishes.

Abstract: Coho salmon, Oncorhynchus kisutch, and steelhead trout, Salmo gairdneri, 57 to 60 mll in total length, were exposed either once for 1 hr to 1,500 cercariae of Nanophyetus salmincola, or daily for 1 hr to 100 of these parasites for 15 days. The effect of the parasites on the swimming ability of fish exposed only once was determined immediately after exposure, or after 6, 12, 24, or 96 hr, or after 15 days; fish exposed daily were tested immediately after the last exposure. Groups of control and infected fish were tested together in a swimming tube. In some tests, the water velocity in the tube was increased gradually at 10-min intervals, and the velocity at which each fish became fatigued was recorded. In other tests, the water velocity was suddenly increased to a high, constant level, and the duration of swimming of each fish at that velocity was recorded. In both types of tests, there was a marked effect of the parasite on the swimming ability of fish exposed once and tested 0 to 96 hr later and on those exposed repeatedly and tested after their last exposure. The mean swimming speeds or times of groups of steelhead trout were reduced by 10 to 58%, as compared with those of controls, and corresponding per cent reduction values for coho salmon ranged from 4 to 95%. This impairment of the swimming ability of infected fish is attributed to tissue injury caused by migrating parasites. The swimming ability of fish that were exposed once and tested 15 days later, when the parasites were encysted, was little affected. However, it is likely that the effect would have been greater had the infections been heavier. The "salmon poisoning" trematode, Nanophyetus salmincola, can kill experimentally infected fish, and the same may be true for fish in nature (Millemann and Knapp, 1970). But we have no information on the sublethal effects of the parasite on fish, such as impairment of swimming ability and retardation of growth. This paper reports the results of a study, carried out from September 1968 to August 1970, on the effects of N. salniincola on the swimming ability of juvenile coho salmon, Oncorhlynchus kisutch, and summer-run steelhead trout (anadromous rainbow trout), Salito gairdneri. Effects of the parasite on the growth of these fish species will be reported in a subsequent paper. MATERIALS AND METHODS Experimental animals Coho salmon or fertilized salmon eggs were obtained from the Alsea River Salmon Hatchery of the Fish Commiission of Oregon, and steelhead Received for publication 16 February 1971. * This investigation was supported in part by Public Health Service Research Grant 5 RO1 AI06599 from the NIAID, and in part by the National Science Foundation Institutional Sea Grant GH 97, and constitutes part of doctoral thesis research done by the senior author at Oregon State University. Technical Paper No. 3026, Oregon Agricultural Experiment Station. trout from the Oregon State Game Commission's Oak Springs Fish Hatchery. The former hatchery is in the enzootic area of the trematode, but the salmon used in the experiments were obtained as fry in late winter, when N. salmincola cercariae are not present in the hatchery water supply, or were hatched and reared in parasite-free well water. The hatchery at which the steelhead trout had been reared was outside the enzootic area, but the fish were hatched there from eggs obtained from adult fish taken in the Siletz River (Lincoln County, Oregon), which is within the enzootic area. Thus, all experimental fish were initially uninfected but originated from stocks that have had long evolutionary association with the parasite. The fish were maintained in outdoor cement tanks provided with flowing dechlorinated tap water and were fed commercial fish pellets several times a week. When the fish were used in experiments, their total lengths ranged from 57 to 60 mm and their weights from 1.4 to 1.7 g, but the fish used in any one experiment never differed from each other in weight by more than 0.2 g. Oxyjtrema silicula snails were collected from the Big Elk River and Beaver Creek in Lincoln County, Oregon. Those found to be infected with N. salmincola were maintained thereafter in the laboratory in plastic trays provided with flowing chlorine-free tap water at 15 to 16 C. Clam shells in the trays served as a calciium source. Snails were fed either lettuce or alder leaves. When cercariae were needed for an experiment, approximately 25 snails were placed in 1.0 liter of dechlorinated, aerated tap water at room temperature (near 21 C) and left there overnight, because exposure to the elevated temperature for 8

Developmental physiology of cestodes. IX. Cytological characteristics of the germinative region of Hymenolepis diminuta.

Abstract: The germinative region of developing Hymenolepis diminuta is defined based on cytological characteristics and DNA synthetic activity as beginning 200 ,u posterior to the apex of the scolex, and extending posteriorly to the point of germinal primordia formation. The structure of the germinative cell type in this region is described. The kinetics of cell division in the germinative region are studied, and estimates of the duration of G1, S, and G2 of prophase are 3.0, 2.3, and 3.2 hr, respectively. The time for a complete mitotic division is estimated to be 8.5 + hr. Although proglottisation in most cyclophyllidean cestodes has been attributed to budding from the unsegmented region immediately posterior to the scolex (Wardle and McLeod, 1952; Roberts, 1961), no cytological definition or exact location has been given to the so-called germinative area. Roberts (1961) indirectly defined the germinative area of Hymenolepis diminuta as the area behind the scolex in which genital primordia are not observed. Sandeman (1959) defined the germinative area of Capsulata edenesis, a parasite of Limosa lapponica, as a posteriorly located diffuse area of the strobila from which the rest of the strobila originates. If the germinative region is indeed responsible for proglottid production, the cellular activity in this region should be significantly different from the rest of the strobila, and cell division indices and an index of cells involved in DNA synthesis could be used as criteria of definition. This region should also contain a cytologically identifiable cell type which could act as a "stem cell" for production of proglottids. Bell and Smyth (1958) and Wikgren (1964) have used the criterion of a mitotic index to estimate growth in trematodes and cestodes. The use of the incorporation of tritiated thymidine into DNA as an index of DNA synthesis and, therefore, as an index of cell replication was discussed by Clever (1967). Received for publication 30 July 1970. * This investigation was supported by Research Grant AI-06153 and Training Grant 5 TOI AI226 from the NIH, U. S. Public Health Service, Bethesda, Maryland 20014. tPresent address: Department of Biology, Notre Dame University, Notre Dame, Indiana 46556. In the present study we have investigated the cytological characteristics of the germinative region of H. diminuta and a possible germinative cell type within this region. The duration of the cell cycle of the proposed germinative cell has been estimated. MATERIALS AND METHODS Recovery and incubation of H. diminuta Male Holtzman rats (120 to 180 g) were inoculated with either 100 or 10 cysticercoids and worms were recovered 2, 4, and 6 to 14 days postinfection, respectively. Worms were recovered 2 and 4 days postinfection from the hosts' small intestine by scraping the intestinal mucosa with a glass microscope slide into phosphate-buffered (pH 7.4) balanced salt solution (BSS) (Roberts and Fairbairn, 1965) or into tris-maleate-buffered Ringer's solution (Read et al., 1963) with 0.1% added glucose (TMR + G; pH 7.4). The gut scrapings were examined under a dissecting microscope, and the worms were separated from the debris with a Pasteur pipette. Older worms, 6 to 14 days postinfection, were flushed from the rat small intestine with a small amount of salt solution. The worms were washed, incubated 15 min in BSS or TMR + G containing 0.4 mg streptomycin and 500 units potassium penicillin-G per ml, and then incubated in the salt solution containing an experimental substrate. All incubations were done in 25-ml Erlenmeyer flasks with a tissue-to-medium ratio of 60 mg worm fresh weight/ml incubation fluid. Incubations were at 37 C in an atmosphere of N2:CO2 (95:5). Following the incubation, the worms were washed with salt solution and fixed for cytological studies. Preparation for determination of mitotic index Worms were incubated in a concentration of colchicine for a length of time necessary to obtain the maximum number of arrested metaphase figures. Entire 2and 4-day-old worms and the ant rior 2 cm of 6to 14-day-old worms were fixed immediately in 3:1 (v:v) 70% ethanol:neutral

A comparison of genome sizes and thermal-denaturation-derived base composition of DNAs from several members of Entamoeba (histolytica group).

Abstract: The DNA base composition and genome size of several strains of Entamoeba that form mature 4-nucleated cysts (histolytica group) was compared. These 2 measurements of genetic potential were used to differentiate species. Significant differences in genome size were observed for "classical" E. histolytica, E. histolytica-like amebae, and E. moshkovskii. Significant differences in DNA base composition were found in strains of amebae thought to be of the same species. These data clearly indicate the existence of more than one genospecies for classical E. histolytica, E. histolytica-like amebae, and E. invadens. Members of the genus Entamoeba can be readily divided into four major morphological groups on the basis of the existence or absence of a cyst stage and by the number of nuclei in the mature cyst. One group is characterized by having uninucleated cysts, another has 4-nucleated cysts, a third has 8-nucleated cysts, and one is without any known cyst stage. Levine (1961) conveniently designates these groups as bovis, histolytica, coli, and gingivalis, respectively. Within each of these groups separation into species is especially difficult. Currently, the classification is based primarily on morphology, host specificity, and pathogenicity. This system is inadequate. There are few consistent morphological differences among members of a group other than size. Host specificity is not a particularly reliable character, and pathogenicity is a highly variable property. Additional criteria for classification must be sought. In recent years, advances in our knowledge of the biology of the Entamoeba, gained primarily through study of monoxenically and axenically cultivated organisms, gives promise that the antigenic, biochemical, metabolic, and physiological properties of the amebae can be used to develop more meaningful systems of classification as has been done for bacteria. We have been characterizing entamoebal DNA and using specific differences as a means of distinguishing members of the histolytica group, or those Entamoeba forming 4-nucleated Received for publication 1 December 1970. cysts. In our previous study (Gelderman et al., 1971) we demonstrated that three members of the group, classical Entamloeba histolytica, E. histolytica-like ameba, and E. moshkovskii were sufficiently different with respect to genome size, DNA reassociation kinetics, mole per cent guanine plus cytosine (%GC), and DNA to DNA homologies to be considered distinct species. In that work only one strain of each type was studied. In this study, the results of two parameters of genetic potential (genome size and %GC) are reported for several strains of the histolytica group. GENOSPECIES AND TAXONOMY The ideal taxonomic classification must consider the widest possible range of relatedness such as morphology, host specificity, metabolism, growth requirements, immunological characteristics, specific isozymes, and DNA composition. Many of the parameters change with differing environmental conditions, thus requiring a different part of the DNA to be expressed. However, genome size and %GC of a cell is a biological constant and any significant change in the nucleotide composition of the DNA of an organism with 107 to 108 nucleotide pairs would be lethal to the cell. The DNA base ratio has been extensively studied in bacteria and used as a taxonomic tool (Mandel, 1969). The %GC of the DNA from all bacteria studied varied from 27 to 72%(Shapiro, 1968). The range of the DNA base ratios from the whole family of Enterobacteriaceae varied by only 20% (Shapiro, 1968). The

The molting pattern in Trichinella spiralis. I. A light microscope study.

Abstract: Development of Trichinella spiralis in rats was studied in detail to determine where and how many times the larvae molt. Four molts were observed during the intestinal phase of development, none earlier. In synchronously developing populations obtained by injecting excysted larvae directly into the duodenum, male worms molted at approximately 9, 13, 18, and 25 hr, females at approximately 10, 15, 21, and 28 hr, and inseminated females were first observed at 30 hr after inoculation. Each successive larval stage could be recognized by the thickness of the cast cuticle and the presence or absence of copulatory appendages, tuberculate papillae, and sperm in the male, and by the state of development of the vagina and uterus and the thickness and cross striations of the cuticle in the female. The development of T. spiralis can be divided into two periods, one in the tissues where, although there is no molt, the level of sexual differentiation reaches that of the fourth or early fifth-stage larva of phasmid nematodes, the other in the intestine where there are 5 stages, separated by 4 successive molts. The high level of sexual differentiation reached in the tissue phase of development together with the rapid succession of molts occurring in the intestine renders the Trichinella life cycle atypical among the trichuroids. Although more than a century has elapsed since the general life cycle of Trichinella spiralis (Owen, 1835) Railliet, 1895, was described by Leuckart and Virchow (Gould, 1945), its molting pattern is still disputed. It has been claimed that the first molt occurs within the egg in the uterus of the female, that there are two subsequent molts during the intramuscular phase, and one during the intestinal phase of development (Berntzen, 1965). It has also been reported that only two molts occur, both in the intestine (Podhajecky, 1964; Thomas, 1965; Shanta and Meerovitch, 1967a). One report (Kreis, 1937) suggested that males molt three times, females four times, but some observers have found that both males and females molt four times (Villella, 1958; Ali Khan, 1966). In vitro studies on the development of T. spiralis by Weller (1943) and by Tarakanov (1964) also suggested that the larvae of both Received for publication 2 March 1971. * Portion of a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This study was supported by grants AI-04919 and AI-00002 from the NIH, U. S. Public Health Service. Presented at the 44th Annual Meeting of American Society of Parasitologists, 6 November 1969, Washington, D.C. t Present address: Department of Microbiology, University of Chicago, 939 East 57th Street, Chicago, Illinois 60637. xes molt four times. Kim (1961, 1962) likew se proposed that four, or more, molts occurred in his in vitro system, but Meerovitch (1965) reported only two molts, and Berntz (1965) only one in their respective culture systems. Microand ultrastructural studies were conducted on stages of T. spiralis larvae from the female worms, and from the muscles and intestine of the host, in an attempt to determine how many molts occur during the development of this trichuroid, during which phases of the life cycle they occur, and what morphological characteristics can be used to differentiate the various larval stages. The present report is limited to the results obtained from the light microscopic studies. The ultrastruct-ural studies are presented separately (Kozek, 1971). MATERIALS AND METHODS Larvae were obtained from rats whose infections were 3 to 6 months old. Following a brief mincing in a Waring Blendor, the muscles were digested in 1% pepsin solution containing 1% HC1, for 2 to 6 hr at temperatures ranging between 37 and 40 C. Larvae were subsequently separated from tissue debris by sieving through a double layer of cheesecloth followed by sedimentation. The inoculating dose was administered to experimental[ animals in a 0.4or 0.5-ml volume of pepsin-muscle-digest fluid. The larvae and adult worms were observed alive, or after fixation with either Bles' fluid, AFA, 10% f rmalin, or 3% glutaraldehyde solution. The fixed worms were cleared in 70% ethanol + 5% glycerin mixture and mounted in glycerin.

Leishmania hertigi sp. n., from the tropical porcupine, Coendou rothschildi Thomas.

Abstract: A skin-inhabiting species of Leishmania found in Panama in the tropical porcupine, Coendou rothschildi, is considered new and described as Leishmania hertigi sp. n. L. hertigi is differentiated from other species of Leishmania found in mammals mainly by the nature and course of the infection in its natural host. It is apparently harmless to its host, in which it produces a long-lasting infection, showing a well-established host-parasite relationship. There is a complete absence of any gross skin alteration due to the infection, although the parasite frequently is found in skin over the whole body. L. hertigi seems to be host-specific. In the search for reservoir hosts of human cutaneous leishmaniasis in Panama a new approach was initiated at the Gorgas Memorial Laboratory in March 1965. Starting from the hypothesis that natural leishmanial infections among animals might occur with no gross skin alterations, wild-caught mammals were investigated using skin smears and the recently described biopsy-skin-culture technique (Herrer et al., 1966). The latter technique greatly simplified the detection of parasites in the skin and proved to be very useful in these studies. Early in the investigation, a tropical porcupine, Coendou rothschildi Thomas, was found lightly infected. Subsequent studies showed a high prevalence rate. Morphological and other studies indicate that this is a new species which is described in this paper and named Leishmania hertigi. MATERIALS AND METHODS Porcupines (C. rothschildi) were obtained alive from different localities throughout the central part of the Republic of Panama. Skin from several parts of the body was cultured by the biopsy-skin-culture technique. Skin cultures were made periodically from porcupines maintained in captivity to study the course of the infection. Cultures were also made from skin, liver, and spleen at autopsy. Skin smears were prepared only in certain cases, and skin samples were preserved for sections from animals with positive skin smears. Senekjie's culture medium was used throughout this study, and all cultures were incubated at 19 to 22 C. The parasite could be maintained for about Received for publication 8 September 1970. * The work reported here was supported in part by the research grant AI-01251 from the NIAID, NIH, USPHS. a month before transfer to a new culture. Promastigote (leptomonad) flagellates used for the morphological description were from 2 strains maintained for several months after isolation and were taken from the culture slant 6 to 8 days after a transfer. Drawings were made with the aid of a camera lucida and 80 flagellates were measured for the description. Leishmania hertigi sp. n. (Figs. 1-18) Morphological description (all measurements in microns)

Increasing the yield of Eimeria tenella oocysts in cell culture.

Abstract: Eimeria tenella sporozoites were inoculated into partially confluent primary chick kidney cell cultures. Counts were made of the number of intracellular sporozoites at 4 and 24 hr, and oocysts at 6 and 7 days in cultures inoculated and maintained under a variety of conditions. By using ( 1 ) a single medium composed of 90% Hanks' balanced salt solution, 5% lactalbumin hydrolysate (2.5% solution in Hanks' BSS), and 5% fetal calf serum for growth of cells, inoculation of sporozoites, and maintenance of cells, (2) sporozoite suspensions free of debris, and (3) 10 rather than 2 ml of media for maintaining the cultures, an OI (oocyst index, or relationship between intracellular oocysts at 6 or 7 days and sporozoites at 4 hr) was obtained that was nearly 7 times greater than that obtained using the procedure previously described (Doran, 1970c). In a previous report (Doran, 1970c), it was shown that Eimeria tenella undergoes its complete life cycle from excysted sporozoites to viable oocysts in primaly cell cultures of embryonic and nonembiyonic chick kidney. This report pertains to the effect of media, size of the sporozoite inoculum, and sporozoite inocula freed of debris (oocyst and sporocyst walls), on infection of primary chick kidney cells and subsequent production of oocysts. It shows how a greater yield of oocysts is obtained by altering the growth conditions for cells and eliminating certain of the procedures previously used. MATERIALS AND METHODS

Characterization of the deoxyribonucleic acid of representative strains of Entamoeba histolytica, E. histoytica-like amebae, and E. moshkovskii.

Abstract: DNAs isolated from Entamoeba histolytica (HK-9 strain), an E. histolytica-like ameba (Laredo strain), and E. moshkovskii (FIC strain) were quantitatively and qualitatively compared. Quantitative determinations included DNA content per cell derived from the measurement of the extent of reaction with diphenylamine, and the degree of DNA sequence repetition and genome size derived from measurements of the reassociation kinetics. Qualitative determinations included guanine and cytosine content derived from DNA thermal denaturation data, and sequence relatedness of Laredo and HK-9 DNA derived from hybrid formation data. Two DNA isolation techniques (CsCl buoyant density and hydroxyapatite fractionation) yielded preparations that had identical thermal denaturation profiles. DNA from each ameba was quantitatively and qualitatively different from the others. Although the amebae are morphologically indistinguishable, differences in their DNA are sufficient to constitute clearly separate species. The species Entamoeba histolytica currently includes at least two groups of amebae, the "classical" E. histolytica and the E. histolyticalike amebae designated Laredo-type amebae by Goldman (1969) after the first of these strains to be recognized (Dreyer, 1961). The two, though morphologically indistinguishable, are clearly distinct with respect to many other parameters. Xenic cultures of classical E. histolytica have an optimum growth temperature of 35 to 37 C with a range of 29 to 42.5 C and the amebae are osmotically fragile (Richards, Goldman, and Cannon, 1966). Xenic cultures of the Laredo-type ameba have an optimum growth temperature of 25 to 30 C with a range of 10 to 35 C and will multiply in hypotonic medium (Richards et al., 1966). In addition to the parameters mentioned above there are dissimilarities in pathogenicity, drug sensitivity, antigenicity, and biochemical composition (Goldman, 1969). Entamoeba moshkovskii, an Entamoeba known only from free-living environments at present, is similar to the Laredo-type ameba with respect to the above parameters. Goldman (1969) has suggested that this species might actually be a parasitic form incidentally found in free-living environments and able to survive because of a wide temperature and tonicity tol-