Differential expression of the estrogen-regulated proto-oncogenes c-fos, c-jun, and bcl-2 and of the tumor-suppressor p53 gene in the male mouse chronically infected with Taenia crassiceps cysticerci

Chronic infection with Taenia crassiceps cysticerci produces a 200-fold increase in serum estradiol levels in male mice. The aim of this study was to investigate the expression pattern of c-fos and c-jun, two estradiol-regulated genes, as well as that of p53 and bcl2 in the testes, spleen, and thymus of male mice infected with T. crassiceps cysticerci. In parasitized animals the c-fos mRNA content was significantly increased in all tissues studied, whereas the c-jun mRNA content was increased only in the thymus. The p53 mRNA content was markedly reduced in all tissues of the parasitized animals analyzed, whereas bcl-2 gene expression was abolished in the thymus. On the other hand, thymic cell analysis performed by flow cytometry showed a diminution in the content of CD3⁺, CD4⁺, and CD8⁺ subpopulations in the parasitized mice. Our results suggest that the increase in estradiol levels of the host should change the expression pattern of several genes that participate in apoptosis regulation in the thymus of male mice during chronic infection with T. crassiceps cysticerci. Received: 10 December 1997 / Accepted: 22 January 1998     

Development of Taenia saginata asiatica metacestodes in SCID mice and its infectivity in human and alternative definitive hosts

Development of Taenia saginata asiatica metacestodes in SCID mice, and its infectivity in humans, golden hamsters, and Mongolian gerbils as alternative definitive hosts, were investigated. Cysticerci were recovered from SCID mice that were subcutaneously injected with hatched oncospheres of T. s. asiatica. The morphological changes of metacestodes were observed. The recovered cysticerci were fed to gerbils, hamsters and humans, to check for their infectivity. Tapeworms were recovered from gerbils and hamsters fed with 20 to 45 week-old cysticerci, and proglottids excretions were observed in human volunteers fed with 45 week-old cysticerci. However, no tapeworms were recovered from gerbils fed with 10 week-old cysticerci. Our results suggest that T. s. asiatica oncospheres needed more than 20 weeks to develop to maturity in SCID mice to be infective to both their natural and alternative definitive hosts.     

MurineTaenia crassiceps cysticercosis: H-2 complex and sex influence on susceptibility

Several inbred strains of mice were infected by intraperitoneal injection of tenTaenia crassiceps cysticerci per mouse. Genes linked with the major histocompatibility complex (H-2) were found to influence parasite growth greatly, as demonstrated by the different parasite loads of H-2 congenic mice with BALB background: BALB/c (H-2d) mice were the most susceptible, whereas BALB/k (H-2k) and BALB/b (H-2b) animals were comparatively resistant. Non-H-2 genes had no significant effect on susceptibility in H-2d strains, as reflected by the similar parasite loads in BALB/c, DBA/2, and (BALB/cxDBA/2)F1 mice. Using the H-2b (BALB/b, C57BL/6J) and H-2k (C3H/HeJ, BALB/k, and C3HeB/FeJ) strains, we found that non-H-2 background genes caused a small but significant influence on parasite load. A recombinant mouse strain alleles (K^k, I^k, S^d, D^d) was also susceptible, indicating that S and/or D regions of the H-2d complex are probably involved in the control of resistance to murine cysticercosis. Females of all mouse strains were more susceptible than males. The same effects were observed for H-2 genes and sex, with two strains ofT. crassiceps differing in their rate of growth.     

Survival of destrobilated adults of Taenia crassiceps in T-cell-depleted Mongolian gerbils

In Mongolian gerbils (Meriones unguiculatus), prednisolone treatment induces the survival of strobilated Taenia crassiceps to sexual maturity followed by fecal release of gravid proglottides. The mechanism underlying the effects of prednisolone has not been elucidated in this taeniid/rodent model. Using a novel murine monoclonal antibody specific to a cell-surface determinant of gerbil T-cells (HUSM-M.g.15 of IgG_2b isotype) for in vivo depletion of the cells, we examined the T-cell dependence of the following two phenomena: (1) elimination of strobilated T. crassiceps from the intestine of naive gerbils, and (2) depressed egg formation by the cestode in prednisolone-treated gerbils. In T-cell-depleted gerbils, only destrobilated adults were recovered from the intestine, although the recovery rate was comparable with that observed in prednisolone-treated animals. Egg formation by the cestode in T-cell-depleted, prednisone-treated gerbils did not differ from that seen in gerbils treated with prednisolone alone. We conclude that one of main effects of prednisolone can be ascribed to the suppression of T-cell functions that work to eliminate strobilated T. crassiceps from gerbils. Received: 1 October 1999 / Accepted: 8 November 1999     

Effect of various doses of infectiveToxocara canis andToxocara cati eggs on the humoral response and distribution of larvae in mice

The effect of 5–2,500 infectiveToxocara canis and 5–1,000T. cati eggs on the humoral immune response and on the distribution of larvae in the organism was studied in paratenic hosts — inbred C57BL6/J mice. With each dose ofT. canis eggs the maximal antibody level was recorded on day 56 post infection and was followed by a moderate decline that lasted until day 154 of the experiment. A correlation between the antibody level and the egg count was observed only with the infective dose of 5–50 eggs. A more rapid occurrence of antibodies was recorded in mice infected with a high dose of eggs. In those given 5 and 7T. cati eggs the antibody level exceeded the extinction threshold value only from day 21 to day 84. Low doses ofT. canis (n=5) andT. cati (n=7) eggs caused a comparable distribution of larvae in mice, and the larval recoveries on day 70 post infection ranged between 10.00% and 25.74%. Following a dose of 500T. cati eggs, 22.28% of the larvae were recovered, although only 1.08% were localized in the brain. A dose of 1,000T. canis eggs yielded, 36.37% of the larvae, with as much as 28.13% being found in the brain.     

Attempts to immunise rats and mice against infection withFasciola hepatica using antigens prepared fromTaenia hydatigena

Summary Attempts were made to immunise rats and mice against infection withF. hepatica by oral dosing withT. hydatigena eggs, or by vaccination with variousT. hydatigena antigen preparations. These antigens included extracts fromT. hydatigena cysticerci and cyst fluid, and antigens collected during short-term (48 h) and long-term (14 days) in vitro cultivation of larvae. Immunity was assessed by the numbers ofF. hepatica recovered from the challenge infection in rats, and the mortality rates of infected mice. None of the immunisation regimes withT. hydatigena antigens induced consistent, significant immunity. This was in contrast to the high level of immunity shown by rats dosed orally withF. hepatica metacercariae four weeks prior to challenge infection.     

Immunological recognition of larvalTaenia crassiceps glycolipids by sera from parasite-infected mice

The isolation and purification of a neutral glycolipid fraction fromTaenia crassiceps metacestodes (KBS strain), harvested from both male and female NMRI mice at 70–80 days following intraperitoneal infection, revealed 24 thin-layer chromatography-designated glycolipid bands. The glycolipids were defined as ceramide mono-(n=3), di-(n=3), tri-(n=4), tetra-(n=5), and >tetrasaccharides (n=9) according to their running properties as defined by thin-layer chromatography against standards of known structure. The defined glycolipids were tested for immunoreactivity with sera from noninfected andT. crassiceps-infected NMRI mice (intraperitoneal injection or implantation of 15 larvae/animal) using the enzyme-linked immunosorbent assay (ELISA) until day 33 p.i. (IgM and IgG reaction) and high-performance thin-layer chromatography (HPTLC) combined with immunostaining (IgG reaction) until day 7 p.i. ELISA-determined IgM and IgG titres were significantly elevated from day 5 p.i. Immunostaining revealed early reactivity for certain ceramide tetra- and >tetrasaccharides (n=6) on day 3 p.i. From day 5 p.i. onwards, nearly all glycolipids, including ceramide mono-and disaccharides, were recognized by the sera from metacestode-challenged mice. On day 7 p.i., a total of 22 bands were serologically active; of these, a considerable number (n=10) showed increased staining intensity. Remarkably, in many cases (10 of 20), 3 glycolipids (tetra-and >tetrasaccharides) were weakly recognized by mouse sera taken before infection.Dedicated to Prof. Dr. J. Eckert (Zürich) on the occasion of his 60th birthday     

The expulsion ofEchinostoma trivolvis (Trematoda) from ICR mice: scanning electron microscopy of the worms

Echinostoma trivolvis adults are rejected from ICR mice within 3 weeks postinfection (p.i.) but are retained in golden hamsters for >15 weeks. The present study used scanning electron microscopy (SEM) to examine worm topography in ICR mice, particularly that of the collar spines, and to correlate worm loss with tegumentary changes. The topography of the worm in ICR mice was similar to that observed in previous studies on this echinostome in domestic chick embryos, chickens, and golden hamsters. Observations were made on the pattern of collar spines in 115 worms from ICR mice at 3–14 days p.i. All worms examined at 3 days exhibited extended spines, whereas about 70% of the worms examined at 14 days displayed retracted or missing spines. Eight worms from golden hamsters examined at 14 days p.i. showed extended collar spines. The retraction or loss of collar spines may play a role in the expulsion ofE. trivolvis from ICR mice.     

Maintenance of the life cycle of Echinostoma trivolvis (Trematoda) in dexamethasone-treated ICR mice and laboratory-raised Helisoma trivolvis (Gastropoda)

 Echinostoma trivolvis is a ubiquitous 37-collar-spined echinostome found in aquatic birds and mammals and in the planorbid snail Helisoma trivolvis. This echinostome has not been cycled continously in the laboratory. The present report provides details on the continuous life cycle of E. trivolvis in dexamethasone-treated ICR mice and laboratory-raised H. trivolvis snails. Previous attempts to obtain patent adult of E. trivolvis in mice hosts failed because of worm rejection within 2 weeks of infection. ICR mice infected with encysted metacercariae and injected with 2 mg/kg dexamethasone daily for 28 days yielded gravid worms that produced 250–500 eggs/worm at 21 and 28 days postinfection (p.i.). Miracidia derived from these eggs or eggs containing fully developed miracidia were capable of infecting 3- to 5-mm shell-diameter, laboratory-reared H. trivolvis snails. These snails released cercariae by 35 days p.i. Cercariae encysted in the kidney-pericardium of the snails. Encysted metacercariae could be excysted in vitro in an alkaline trypsin-bile salts medium or in vivo when fed to domestic chicks. Received: 13 May 1996 / Accepted: 25 June 1996     

An enzyme-linked immunosorbent assay for detecting anti-Echinostoma trivolvis (Trematoda) IgG in experimentally infected ICR mice

An enzyme-linked immunosorbent assay (ELISA) utilizing surface glycocalyx-membrane crude antigen of adultEchinostoma trivolvis was developed for the detection of circulating anti-E. trivolvis IgG in experimentally infected ICR mice. An antigen concentration of 10.0 g/ml was used, and it was possible to detect anti-E. trivolvis IgG at a dilution of 1/3,200. On day 10 postinfection (p.i.), all infected mice had anti-E. trivolvis IgG reactive with the surface glycocalyx antigen. The IgG level varied over a 40-day period, showing a timedependent pattern with a peak on day 16 p.i. The results concerning reciprocal cross-reactivity indicate that adultE. trivolvis andE. caproni share at least some of the surface antigens and express species-specific antigenic determinants in ICR mice at similar, nonsignificantly (P=0.76) different levels.     

Oral transmission of trypanosomes of the subgenusHerpetosoma from small mammals

The rodentsMicrotus agrestis, Clethrionomys glareolus, Apodemus sylvaticus and white BK rats were given either a single intraperitoneal (i.p.) injection, an intragastric (i.g.) inoculation or an oral (p.o.) inoculation of the culture forms, including metacyclic trypomastigotes, ofTrypanosoma microti, T. evotomys, T. grosi andT. lewisi, respectively. Similar levels of parasitaemia were produced by each of the three routes of infection, although the prepatent period was 3–5 days shorter in the case of the i.p.-injected animals. The oral inoculation of blood from mice infected withT. musculi into uninfected mice (outbred) and from rats infected withT. lewisi into uninfected BK rats produced parasitaemia after 6–8 days. This is the first report of the oral and i.g. transmission ofT. microti, T. evotomys andT. grosi into their specific homologous hosts.     

The common vole,Microtus arvalis pall. as intermediate host ofMesocestoides (Cestoda) in Germany

Tetrathyridia ofMesocestoides leptothylacus Loos-Frank, 1980 were found in 1.4% of 513 common voles (Microtus arvalis) in a district of Southwest Germany where foxes (Vulpes vulpes) are frequently infected with this tapeworm. The tetrathyridia measured 1 to 1.5 mm in length and 0.5 to 1 mm in width. When injected intraperitoneally into white mice, jirds, or common voles, the tetrathyridia did not multiply. Cats fed with the larvae shed proglottids from the 21^st day onwards. In one experimentally infected silver-fox proglottids were passed from day 12 onwards. One human subject infected twice with tetrathyridia ofM. leptothylacus, did not develop patent infections. One common vole from another district contained tetrathyridia of aMesocestoides species, which is rarely found in indigenous foxes and which is characterized by a broad-oval cirrus pouch with a much convoluted cirrus.Dedicated to the 70th birthday of Professor Piekarski     

Establishment and survival of the strobilar stage ofTaenia crassiceps in hamsters,gerbils, and mice,with reference to different helminth isolates

Following the oral administration of metacestodes of two isolates ofTaenia crassiceps, the enteral establishment and survival of the strobilar stage were examined in golden hamsters (Mesocricetus auratus), Mongolian gerbils (Meriones unguiculatus) and laboratory mice. The origin of the isolates wasMicrotus montebelli caught in Japan in 1985 orClethrionomys rutilus captured on St. Lawrence Island, Bering Sea, in 1988 (abbreviated as JPN and SLI isolates), respectively. The enteral establishment of the SLI isolate was distinctly higher than that of the JPN isolate in golden hamsters and mice, whereas the difference was marginal in Mongolian gerbils. All initially-established parasites survived to become gravid adults in prednisolone-treated golden hamsters and Mongolian gerbils; the average recovery of cestodes of the SLI and JPN isolates were 55.8%–76.7% vs 11.7%–35.0% in the former and 28.0%–52.7% vs 25.8%–32.2% in the latter. The distinctly higher level of enteral establishment of the SLI isolate in golden hamsters makes available a model for quantitative studies on parasite-host relationships in experimental taeniasis.This study was supported by grants 01790490, 02044083 and 03044016 from the Ministry of Education, Science and Culture, Japan     

Animal models for Opisthorchis viverrini infection

We investigated the utility of various animal models for the study of opisthorchiasis in humans and its common sequel of cholangiocarcinoma (CCA). Rats, mice, gerbils, and hamsters were infected with Opisthorchis viverrini metacercariae. Worms from the infected animal hosts were recovered from livers and counts made of eggs per gram of feces. Worms were observed in and recovered from hamsters and gerbils but not rats and mice. The recovered worms from the infected gerbils were larger and more physiologically developed than those from the infected hamsters. The results suggest that gerbils are more susceptible to infection by Opisthorchis viverrini and thus more suitable for modeling opisthorchiasis and its connection to CCA.     

Homologous and heterologous immunization against the metacestodes ofTaenia saginata andTaenia taeniaeformis in cattle and mice

Summary Cattle and mice were immunized against infection withTaenia saginata andTaenia taeniaeformis, respectively, using antigens obtained from both homologous and heterologous species of cestodes. Mice were protected against infection withT. taeniaeformis when they were immunized intramuscularly or orally with either a somatic antigen extracted from the metacestodes or an excretory/secretory (E/S) antigen collected during the in vitro culture of oncospheres ofT. taeniaeformis. Also, the intramuscular or oral immunization of mice with the E/S antigens from the oncospheres ofT. saginata was highly effective in inducing protection against infection withT. taeniaeformis, as was intramuscular immunization with a somatic antigen extracted from the metacestodes ofTaenia crassiceps and prior infection with viable metacestodes ofT. crassiceps. Furthermore, three-month-old calves developed a protective immunity against infection withT. saginata when they were immunized intramuscularly with the E/S products of oncospheres of the homologous parasite or a heterologous parasite,T. taeniaeformis. In addition, the E/S products of the heterologous parasite,T. taeniaeformis, as well as the homologous parasite,T. saginata, were highly effective when used to immunize pregnant heifers, either intramuscularly or via the intramammary route, resulting in a passive transfer of immunity againstT. saginata to newborn calves.     

Decrease of peritoneal inflammatory CD4+, CD8+, CD19+ lymphocytes and apoptosis of eosinophils in a murine Taenia crassiceps infection

After an intraperitoneal infection of mice with Taenia crassiceps metacestodes, peritoneal inflammatory cells labeled with fluoresceinated MoAb anti-mouse were analyzed by flow cytometry. Apoptosis was studied by annexin A/PI, TUNEL assays, DNA laddering, caspase-3 activity, and electron microscopy. An important continuous decrease of CD4+, CD8+ and CD19+ lymphocytes, and an increase of eosinophils and macrophages throughout the observation time were found. Apoptosis of eosinophils was quantified during the observation period with a peak at 6 days post-infection (67.27%). In an additional experiment at 12 days post-infection using TUNEL staining, a high level of apoptosis of eosinophil (92.3%) and a significant decrease of CD4+, CD8+, and CD19+ lymphocytes were confirmed. Caspase-3 activity in peritoneal fluid, peritoneal cells' DNA fragmentation, and apoptosis of eosinophils and monocytes were found. The dramatic decrease of peritoneal inflammatory T and B cells and the high level of apoptosis of inflammatory eosinophils induced in mice by infection with T. crassiceps cysticerci may be important factors of the immunosuppression observed in cysticercosis.     

Apoptosis patterns in experimental Taenia solium and Taenia crassiceps strobilae from golden hamsters

Apoptosis or programmed cell death (PCD) patterns of two taeniid species, Taenia solium and Taenia crassiceps, were explored in adult tapeworms grown in golden hamsters. Animals were fed either ten viable T. solium cysticerci from naturally infected pigs or from T. crassiceps WFU strain maintained in Balb/c mice. Adult strobilae were recovered from the intestine at different times after infection and either frozen at –70°C or fixed in paraformaldehyde–glutaraldehyde. Frozen sections were processed using the DNA fragmentation fluorescent TUNEL reagents and examined in an epifluorescent microscope. Fixed tissues were processed for light and electron microscopy. Typical apoptotic cells were found in the central core of scolex and strobilar tissues, mainly in the germinal tissue and subtegumentary areas. By the TUNEL technique, cells exhibited the characteristic fluorescent images of condensed nuclear chromatin. By light microscopy of thick sections stained with toluidine blue, we found a number of small rounded cells which had lost their cytoplasmic bridges and had shrunken nuclei with aggregated chromatin, cells which were found interspersed with normal syncytial cells. Similar cell morphology was confirmed by electron microscopy. Stunted viable worms, recovered with longer mature specimens, had very short strobilae and exhibited a large number of apoptotic cells in the germinal neck tissues. The results are consistent with the syncytial nature of these parasites, and strongly suggest that cell proliferation and PCD in these adult cestodes are continuous processes of the germinal tissue and tegumentary cytons.     

Modified expression of steroid 5α-reductase as well as aromatase, but not cholesterol side-chain cleavage enzyme, in the reproductive system of male mice during (Taenia crassiceps) cysticercosis

Infection with Taenia crassiceps cysticerci in male mice produces an increase in serum estradiol levels, whereas serum testosterone is abolished. Concomitantly, complete atrophy of the reproductive tract of infected male mice is observed. The present study was undertaken to determine the expression pattern of three key steroidogenic enzymes in the reproductive tissues of normal and infected male mice. In infected mice, serum estradiol levels were increased 97 times as compared with control mice of the same age. Testosterone and dihydrotestosterone levels were completely inhibited. The expression of 5α-reductase in the reproductive tract was markedly reduced, whereas aromatase mRNA levels were highly elevated in the testes of parasitized mice. No change in the mRNA content for cholesterol side-chain cleavage enzyme was evident. The overall results suggest that the change in the normal production of sex steroids in infected male mice is produced concomitantly by the inhibition of expression of the 5α-reductase enzyme and the activation of aromatase gene expression. This induces a preferential metabolism from testosterone to estradiol instead of the normal metabolism from testosterone to dihydrotestosterone. Received: 29 July 1998 / Accepted: 4 November 1998     

Circulating antigens in infections of mice by tetrathyridia ofMesocestoides corti Hoeppli, 1925

Two circulating antigens were detected in the serum of ICR/Timco female mice infected intraperitonealy with tetrathyridia of the cestodeMesocestoides corti Hoeppli, 1925. One circulating antigen appeared by day 2 postinfection (p.i.) and remained in all mice until at least 90 days p.i. A second antigen appeared in the serum on day 14 p.i. and disappeared from all mice by day 28 p.i. Infected mouse serum also contained antibodies against one secretory/excretory antigen and two antigens in crude homogenate, as judged by double diffusion in two dimensions (Ouchterlony). Immune deposits were observed in the kidney tissue of Rockland mice by transmission electron microscopy, and their identity as products of tetrathyridia was confirmed by immunofluorescence. Further studies showed that the main antibody subclass associated with the mesangial immune deposits was 7Sl, and that other subclasses of IgG and IgM were not involved. Antigen was found in the proximal renal tubules of infected mice, as demonstrated by fluorescein-labeled IgG fraction of rabbit antitetrathyridia secretory/excretory antigen antisera. The presence of tetrathyridia antigen in the urine of infected mice was confirmed using the Ouchterlony technique.