Bruce Z. Lang

Host-parasite relationships of Fasciola hepatica in the white mouse. VIII. Successful vaccination with culture incubate antigens and antigens from sonic disruption of immature worms.

Mice were successfully vaccinated using culture incubate and sonicate antigens from 16-day-old flukes. Various injection schedules using the culture incubate antigen decreased challenge worm counts by 54 to 86%. The best results were achieved when the culture incubate was injected at 12 and 24 hr of incubation. Host mortality in the natural immunity controls ranged from 33 to 42%, and in the vaccinated animals from 12.5 to 25%. Functional antigens were present by 12 hr of incubation. A single immunizing injection with the sonicate antigen decreased challenge worm counts by 86%. Two immunizing injections with this antigen decreased challenge worm counts by 82%. However, the pathologic process in the liver was more severe than in animals that received a single injection.

Host-parasite relationships of Fasciola hepatica in the white mouse. VII. effects of anti-worm incubate sera on transferred worms and successful vaccination with a crude incubate antigen.

Mouse antisera against the 16-day-old worm incubate and sera from 25-day infections in mice debilitated migrating flukes in recipient animals as measured by worm recovery and host mortality. Mouse antisomatic and 100-day infection sera produced no such effects. Host mortality was significantly lower after challenge in mice given one ip immunizing injection of the worm incubate; however, worm recovery was not significantly reduced. Injections at 2, 7, 12, and 24 hr with the worm incubate elaborated over a 24-hr period protected 75% of the mice from infection after challenge, and reduced the worm burden by 83.3%.

Host-parasite relationships of Fasciola hepatica in the white mouse. V. Age of fluke responsible for the induction of acquired immunity.

Juvenile Fasciola hepatica 12, 14, 18, 20, and 24 days old were successfully transferred to normal recipient mice. Worms entered the common bile ducts when total worm age was 30 or 32 days. Liver migration time was controlled between 16 to 17 and 3 to 4 days. Flukes 12, 14, 16, 18, 20, and 24 days old were transferred to normal recipients in an attempt to relate the duration of liver migration and fluke age to the stimulation of acquired immunity. When transferred worms had a total age of 40 days immunized mice were given a challenge infection of 2 metacercariae per mouse. At 25 days after challenge these mice were killed and worms were recovered. Immunization with 12-, 14-, 16-, and 18-day-old worms produced a significant reduction in challenge worm burdens when compared to natural immunity controls. Immunization with 20and 24-day-old worms did not stimulate a significant acquired immunity. It is suggested that the duration of liver migration, at least 10 to 11 days, by young worms and not the specific age of the young worm is responsible for the stimulation of acquired immunity. Eightand 16-day-old Fasciola hepatica stimulated acquired immunity in recipient mice as measured by a significant reduction in worm burden following a two-worm challenge infection (Lang and Dronen, 1972). Transferred worms migrated in the liver for 21 to 22 days (8-day-old worms) or 13 to 14 days (16-dayold worms). Following an initial metacercarial infection this strain of F. hepatica did not migrate into the common bile duct until 32 days after infection (Lang, 1972). When total worm age was 32 days, regardless of the age of transfer, worms were located in the common bile ducts. Lang and Dronen (1972) did not determine if worms older than 16 days at transfer would complete development in mice and stimulate acquired immunity. The present study attempts to determine if immature worms older than 16 days can be successfully transferred and, if possible, to determine the maximum worm age for inducing acquired immunity. MATERIALS AND METHODS Mice utilized were an isologous strain used in previous studies with this parasite (Lang and Dronen, 1972). The strain of F. hepatica used in the present study behaves similarly to the one used in previous studies (Lang, 1972). Techniques for infection and necropsy of mice and infection of the snail host (Lymnaea bulimoides Received for publication 20 August 1973. * This work was supported by NSF grant GB31745. Lea) have been described (Lang, 1966, 1967, 1970, 1972). Procedures for the transfer of 16day-old and younger worms have been published (Lang and Dronen, 1972). Young worms, 12, 14, 16, 18, 20, and 24 days old, were transferred aseptically to recipient mice. EXPERIMENTAL PROCEDURES AND RESULTS

The life cycle of Cephalogonimus americanus Stafford, 1902 (Trematoda: Cephalogonimidae).

Mother and daughter sporocysts and xiphidiocercariae of Cephalogonimus americanus develop in the snails Helisoma antrosa and H. trivolvis. Following emergence, cercariae penetrate tadpoles in which they encyst. Cephalogonimus americanus develops in the anterior small intestine of Rana clamitans which acquires the parasite by eating infected tadpoles. It is suggested that young frogs may also become infected during metamorphosis. Stafford (1902) described adult Cephalogonimus americanus from the intestine of Rana clamitans Latreille. It has also been reported from R. pipiens Schreber by Brandt (1936) and Najarian (1955). Rai (1961) listed 20 species for the genus. To date no complete life history has been reported for any member of the genus. Cephalogonimus americanus is a common intestinal parasite of R. clamitans found in certain areas near Douglas Lake,

Host-parasite relationships of Fasciola hepatica in the white mouse. IV. Studies on worm transfer and the induction of acquired immunity by worms of different ages.

Eightand 16-day-old worms were transferred to normal recipient mice via peritoneal injection, contacted the liver 72 hr after transfer, and migrated into the common bile duct when total worm age was 32 days. Eight-day-old transferred worms were present in the liver parenchyma of recipient mice for 21 to 22 days, while 16-day-old transferred worms were in the liver parenchyma for 13 to 14 days. Transferred worms (8and 16-day-old) were utilized as immunizing infections in 2 groups of normal recipient mice to control the duration of liver migration. When transferred worms had a total age of 40 days, immunized mice were given a challenge infection of 2 metacercariae per mouse. At 25 days after challenge, these mice were killed and worms were recovered. Immunization with 8and 16-day-old worms produced a significant reduction in challenge worm burden when compared to natural immunity controls. Apparently, young flukes are capable of inducing acquired immunity during the entire liver migration period. Studies on the immunology of Fasciola hepatica L. in mice have shown that male mice develop an acquired immunity against a challenge infection after a single two-worm immunizing infection (Lang, 1967; 1968a). Young worms 8 to 17 days old may be responsible for inducing functional acquired immunity in this system (Lang, 1968a). Acquired immunity in this system can be transferred to normal recipients via peritoneal exudate cells from immunized donors (Lang et al., 1967), and it is felt that the lymphocytes seen in the liver by 16 days in a first infection and in immune mice at the time of challenge are effector cells of a specific cellular immunity. As these cells respond almost immediately to the challenge worms after they enter the liver, these 8to 10-day-old worms are no doubt producing the specific antigens. No such response is seen prior to this period in the intestine or body cavity. Young worms removed from livers of normal mice 17 days after infection can be transferred to normal recipients and the worms migrate to and mature in the common bile duct; however, worms of the same Received for publication 15 June 1971. * This work was supported by NSF grant GB8713. t Present address: Department of Biology, New Mexico State University, Las Cruces, New Mexico 88001. age when removed from the livers of immune mice do not complete migration to the common bile duct in normal recipients. Apparently, as these worms were unable to establish, the immune response is against young worms during the liver migratory phase (Lang, 1968b). The present study attempts to determine the age of worm responsible for inducing acquired immunity in the mouse-Fasciola system using the technique of worm transfer. For this work on acquired immunity, a different population of Fasciola (eastern Washi gton) was used; however, it has been shown that it produces nearly the same host responses, with some differences in parasite behavior, in the strain of mouse used for this and previous work. MATERIALS AND METHODS Mice utilized were an isologous strain maintained in the Department of Parasitology, University of North Carolina. Techniques for infection and necropsy of mice, handling of recovered worms, determination of host responses, and infection of snail hosts have been described (Lang, 1966, 1970). In this study, Lymnaea bulimoides Lea was utilized as the snail host. For worm transfer, donor mice were inoculated with 15 to 22 metacercariae and necropsied 8 or 16 days later. Young worms, recovered from the livers and washed in sterile saline at 37 C, were placed in fresh saline at 13 C for 7 min stimulating them to ball up. Two worms were then injected into the body cavity of each recipient mouse using a 16-gauge needle.

Host-parasite relationship of Fasciola hepatica in the white mouse. VI. Studies on the effects of immune and normal sera on the viability of young worms transferred to normal recipients.

ABSThACT: Incubation of 12-, 16-, 18-, 20-, and 24-day-old worms for 4 hr in immune sera, followed by intraperitoneal transfer to normal mice, resulted in a significant reduction in worm burden compared to treatment with heat-inactivated immune sera, normal sera, Medium 199, or buffered saline. Significant differences were not observed between control groups. The effect of immune sera was decreased by heatinactivation. Mice were necropsied when total worm age was 34 days and at this time all transferred flukes were located in the common bile ducts of recipient mice.

SCANNING ELECTRON MICROSCOPY OF IN VITRO SERUM-MEDIATED DESTRUCTION OF JUVENILE FASCIOLA HEPATICA

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.

The Life Cycle of Cephalogonimus salamandrus sp. n. (Digenea: Cephalogonimidae) from Ambystoma tigrinum (Green) from Eastern Washington

Mother and daughter sporocysts and xiphidiocercariae develop in Helisoma trivolvis. Cercariae penetrate tadpoles of Rana pretiosa and small A. tigrinutm larvae in which they encyst. The adult develops in the anterior small intestine of A. tigrilnum after ingestion of an infected 2nd intermediate host. The life cycle of C. salamandrus is compared with C. americanus from Rana clamitans. C. salamandrus is described as a new species on the basis of cercarial and adult morphology, and the failure to achieve cross-infections in the respective definitive hosts for C. americanus and C. salamandrus. Cephalogonimus Poirier, 1886, is the only genus of the family Cephalogonimidae in amphibians. Rai (1961) listed Cephalogonimus brevicirrus (Ingles, 1932), C. amphiumae (Chandler, 1923), C. letusus Dujardin, 1845, and C. am?ericanus (Stafford, 1902) from amphibians of North America. Premvati (1969) described C. sireni from Florida mud eels. The only life cycle reported is for C. americanus from Rana clamitans Latreille (Lang, 1968). This parasite has also been reported from R. catesbeiana Shaw (Rankin, 1945), and R. pipiens Schreber (Brandt, 1936; Najarian, 1955). An undescribed cephalogonimid is a common intestinal parasite of neotenic and adult Ambystoma tigrinum (Green) of eastern Washington. Of the other species of Cephalogonimus, this parasite most closely resembles C. americanus. Comparison of the life cycle stages of this parasite with those of C. americanus and the demonstrated specificity of these two parasites to their respective definitive hosts establishes the validity of this new species to which the name Cephalogonimus salamandrus is assigned. MATERIALS AND METHODS Eggs were teased from adult flukes and stored in filtered spring water at 5 C until used. Fully embryonated eggs were fed to young laboratoryreared Helisoma trivolvis (Say). Exposed snails were crushed and examined at intervals after Received for publication 9 April 1973. *A portion of this study was conducted at the University of Michigan Biological Station. tPresent address: Department of Biology, New Mexico State University, Las Cruces, New Mexico 88003. exposure. Naturally infected H. trivolvis were collected for cercarial study and laboratory infection of the second intermediate host. Cercariae were studied alive with and without vital stains. Cercariae were placed wvith various aquatic invertebrates, small salamander larvae, tadpoles, and small frogs. Penetration and encystment occurred only in amphibian skin. Metacercariae were studied at 2 hr, 1 day, 5 days, 10 days, 12 days, and 21 days after infection. Tadpoles and salamander larvae used as intermediate hosts were laboratory-reared. Large neotenic A. tigrinum for use in laboratory infections were collected from a lake where no trematode or cestode parasites were found in 150 A. tigrinum and no Helisoma spp. have been found in over 6 years of study. Salamanders were maintained in filtered spring wrater on a diet of earthworms. Before exposure 13 neotenic salamanders (20 to 30 cm in length) were starved for 2 weeks. Feeding was resumed the day after exposure. Each of 11 salamanders was permitted to ingest two infected tadpoles, each containing 20 to 30 metacercariae which were 12 days old at exposure. Salamanders were necropsied at 52 hr, 5 days, 10 days, 15 days, 20 days, and 48 days after exposure. Also each of two salamanders was permitted to ingest two infected salamander larvae, each containing 20 to 30 metacercariae which were 12 days old at exposure. These were necropsied at 5 days. Recovered flukes were studied alive, measured, heat-killed, stored in AFA, and stained in Semichons Carmine. To determine if A. tigrinum and R. pretiosa of eastern Washington could be infected with C. americanus, hosts were transported to the University of Michigan Biological Station. Methods for handling C. americanus and collection of Cephalogonimus-free R. clamitans have been reported (Lang, 1968). Two salamanders were exposed to 10-day-old metacercariae of C. americanus. Each salamander received 50 to 70 metacercariae and was necropsied 10 or 30 days after exposure. During this experiment two A. tigrinum

Prevalence of the Ectoparasitic Copepod Lernaea cyprinacea L. on Four Species of Fish in Medical Lake, Spokane County, Washington

musculature at necropsy but none of the inoculated, or inoculated and dexamethasone treated voles or gerbils had sarcocysts. Results of the in vitro excystation experiments are shown in Figure 1. Results with mouse and vole or gerbil bile were significantly different (P < 0.05) through the 1:16 dilution. The results of these experiments indicated that S. muris cannot infect voles or gerbils as intermediate hosts. Furthermore, this specificity was not affected by the application of dexamethasone to the hosts. The dexamethasone treatment used in this study can increase both the prevalence and intensity of S. muris infections in mice (Rommell et al., 1981, loc. cit.). Our report shows that the source of bile can have an effect on the in vitro excystation of Sarusculature at necropsy but none of the inocucocystis sporocysts. These data are consistent with observations on the in vitro excystation of a bovine Sarcocystis species (Fayer and Leek, 1973, Proc. Helm. Soc. Wash. 40: 294-296) and observations of in vivo excystation of S. cernae (Cema et al., 1978, Folia Parasitol. 25:201-207). Although it is commonly held that differences in excystation do not play a role in the host specificity of coccidians (Kartchner and Becker, 1930, J. Parasit. 17: 90-94; Lotze and Leek, 1963, J. Parasit. 49(Suppl.): 32; Doran, 1980, Proc. Helm. Soc. Wash. 47: 114-117), the correlation between infectivity and excystation rate observed in our study suggests that these differences should not be overlooked in the search for the physiological basis of host specificity in S. muris. siste t it a i o a o-