Leucocytes and Trichinella spiralis: III. Importance of heat labile and heat stable substances in peritoneal exudate fluid for cell adherence reactions to infective larvae

The investigation in vitro of cell adherence reactions (CAR) to Trichinella spiralis infective larvae in peritoneal exudates obtained from rats and mice has shown that the adherence of leucocytes is almost entirely dependent upon factors present in the peritoneal exudate fluids (PEF). The heat-labile factor is primarily responsible for influencing adherences to worms in PEF obtained from non-infected animals. In the PEF obtained from infected animals, a heat stable factor is present, in addition. This heat stable factor can be adsorbed to surfaces of worms and was found up to at least 9 months after infection in rats. The heat stable factor can be absorbed from the PEF with infective larvae.     

Antigens of Toxoplasma gondii with a diagnostic and potential immunoprophylactic value: new strategies of identification

Toxoplasma gondii is an ubiquitous protozoan parasite which induces severe pathology in in utero infected children and in immunosuppressed patients (particularly in the case of AIDS). Previous work that focused on toxoplasma somatic antigens failed to demonstrate an efficient protection against highly virulent T. gondii strains. The authors therefore first studied the role of parasite excreted-secreted (ES) antigens in the immune response. They describe here the preparation of excreted-secreted antigens in cell-free medium from tachyzoites, the intracellular proliferative stage present during acute infection. Major ES antigens have Mr of 108 K, 97 K, 86 K, 57 K, 42 K, 39 K, 28.5 K, 27 K and 21 K. The protective role of ES antigens has been demonstrated using congenitally athymic (Nu/Nu) rats that are highly sensitive to T. gondii infection (+/+ Fischer rats are resistant). The humoral and cellular components of this protection have been studied by the passive transfer either of sera or of T lymphocytes from ES-immunized +/+ Fischer rats into Nu/Nu rats. Adoptive transfers were carried out 24 hours before infection with the highly virulent T. gondii RH strain. Based on the concept of concomitant immunity, the authors have characterized antigens from tachyzoites and bradyzoites (the encysted stage persisting during chronic infection) which share common epitopes. Four tachyzoite antigens, P63, GP43, P39 and GP 28.5 have been shown by immunoprecipitation to cross-react with bradyzoite antigens. Two monoclonal antibodies raised against ES antigens permitted to demonstrate the localization of the 28.5 K and 27 K antigens inside the dense granules of tachyzoites and bradyzoites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody responses in rats infected withAngiostrongylus cantonensis and the passive transfer of protective immunity with immune serum

Little haemagglutinating antibody was detected in the serum of rats with primaryAngiostrongylus cantonensis infections until the juvenile worms left the brain and lodged in the lungs about 35 days after infection. Antibody titres reached a peak 50 days after infection and were maintained for at least a further 95 days. Increasing the infective dose ofA. cantonensis larvae increased the peak antibody titres attained by the infected rats. Reinfection temporarily reduced the serum antibody titre, but later it increased and showed an anamnestic response. Reaginic antibody production was transient during primary infections; re-infection failed to stimulate further reaginic antibody production.Antiserum from immune donor rats protected recipients againstA. cantonensis. The transferred immune serum suppressed anti-A. cantonensis antibody production in recipient rats.     

Parthenogenesis in the genusSchistosoma: Electrophoretic evidence for this reproduction system inS. japonicum andS. mansoni

Mixed infections withSchistosoma japonicum andS. mansoni were carried out in mice.S. japonicum females paired withS. mansoni males developed normally and produced numerous viable eggs; very little sperm was found in the female genital tract. The eggs yielded many miracidia infective toOncomelania hupensis, the host ofS. japonicum. Cercariae arising from miracidia developed into male worms with an electrophoretic pattern of malate dehydrogenase (MDH) corresponding only to mae maternal speciesS. japonicum. S. mansoni females paired withS. japonicum produced few viable eggs; sperm was found in the female genital tract. Miracidia hatched from mime of these eggs were infective toBiomphalaria glabrata, the host ofS. mansoni. Cercariae arising from miracidia developed into female worms with an electrophoretic pattern of MDH typical of the maternal speciesS. mansoni. It was concluded thatS. japonicum females paired withS. mansoni males andS. mansoni females paired withS. japonicum males reproduce parthenogenetically. Parthenogenesis in schistosomes is discussed.     

Immunologic responses to experimental strongyloidiasis in rats

Humoral and cellular immune responses were evaluated in Lewis rats infected with high (3000) inocula ofStrongyloides ratti larvae (L3). The responses of peripheral and mesenteric node lymphocytes to crude larval antigen (by lymphocyte proliferation assay) sharply rose between days 12 and 16 of the primary infection and days 8 and 12 of the challenge, and decreased promptly to almost preinfection levels. Spleen lymphocytes failed to show any response to the primary infection, but did exhibit a modest response after challenge. The responses of peripheral lymphocytes to the mitogen phytohemagglutinin fell considerably on infection day 8 and then rose sharply to very high levels. Reinfection caused a small decrease in responsiveness, followed by another increase after 10 days. Inoculation of 3000 heat-killed larvae stimulated a response in peripheral lymphocytes similar to that elicited by the inoculation of live larvae. Spleen lymphocytes appeared to be stimulated more by the killed larvae inoculation, whereas only a limited response was found in mesenteric node lymphocytes in animals inoculated with dead larvae. IgG antibody titers againstS. ratti antigen were measured by the enzyme-linked immunoabsorbent assay (ELISA). A significant elevation was noted between days 12 and 20 with a return to pre-infection levels between days 80 and 150. Challenge with 3,000 L3 induced a rapid and significant elevation. These data indicate that in rats experimentally inoculated with large numbers ofS. ratti larvae a direct relationship exists between the development of cellular and humoral immune responses to larval antigens, the expulsion of the intestinal worms, and the acquisition of resistance to subsequent challenge.     

Chronic intestinal nematode infection exacerbates experimental Schistosoma mansoni infection

Mixed-parasite infections are common in many parts of the world, but little is known of the effects of concomitant parasite infections on the immune response or on disease progression. We have investigated the in vivo effects of a chronic gastrointestinal nematode infection on the infectivity and development of the immune response against the common trematode helminth Schistosoma mansoni. The data show that mice carrying an established chronic Trichuris muris infection and coinfected with S. mansoni, had significantly higher S. mansoni worm burdens than mice without coinfection. The increase in S. mansoni worm burden was accompanied by a higher egg burden in the liver. Kinetic analysis of S. mansoni establishment indicate reduced trapping of S. mansoni larvae during skin-to-lung migration, with T. muris-induced alterations in lung cytokine expression and inflammatory foci surrounding lung-stage schistosomula, suggesting that the immunomodulatory effects of chronic T. muris infection elicited at the gut mucosal surface extend to other organs and perhaps specifically to other mucosal surfaces. The data show that a preexisting chronic gastrointestinal nematode infection facilitates the survival and migration of S. mansoni schistosomula to the portal system, and as a result, increases the egg burden and associated pathology of S. mansoni infection.     

Action of diethylcarbamazine citrate on protective immunity in rats infected withNippostrongylus brasiliensis

Rats made immune toNippostrongylus brasiliensis and treated with diethylcarbamazine citrate (DEC) orally (250 mg/kg×6) exhibited significant suppression of functional immunity. Similarly, administration of compound 48/80 (100 g/rat i.p.) made the immune rats susceptible to challenge infection. Treatment of rats, with 22-day infection with compound 48/80, histamine (20 mg/rat, per os), orl-histidine (20 mg/rat, orally s.c.) did not accelerate worm expulsion. A massive complement-dependent adherence of peritoneal cells (1×10⁸), isolated from immune DEC-treated and untreated rats, to infective larvae (L₃) was observed. Likewise, heavy congregation of normal peritoneal cells to larvae was noticed when the cells were incubated with sera obtained from immune, DEC-treated or untreated rats. The rats receiving mesenteric lymph node cells (125×10⁶ i.v.) or sera (0.5 ml or 1 ml×3 i.p.), obtained from immune DEC-treated rats and challenged with infective larvae developed 50% more worms than those which received cells or serum from untreated immune donors. DEC appears to cause suppression of functional immunity and worm expulsion is not histamine mediated.     

Phospholipase B activity in congenitally athymic (nude) mice infected withTrichinella spiralis

The effects of an infection with 200Trichinella spiralis larvae on the intestinal phospholipase B activity and bone marrow eosinophilia of congenitally athymic (nude) mice (BALB/c; NU/NU) were studied. Nude mice were used since it had been shown that they do not undergo a typical worm expulsion and also they lack a thymus. The results showed that nude mice do not develop either an increased bone marrow eosinophilia or an elevation in intestinal phospholipase B activity. The findings thus support the hypothesis that phospholipase B is involved in the expulsion of parasitic worms and that elevated enzyme levels and expulsion are thymus cell dependent.     

Changes in worm burden,haematological and serological response in rats after single and multipleAngiostrongylus cantonensis infections

Summary Thirteen groups of rats were first sensitized with single or double doses of 5–30 third-stage larvae ofAngiostrongylus cantonensis, followed by a challenge infection with 100 larvae at various periods after the primary infection. Seven other groups of rats receiving only the sensitizing infection served as the controls. In all the sensitized rats, a significantly (p<0.05) smaller mean number of adult worms was found established in the challenge infection as compared to the control. The frequency of the sensitizing dose and timing of the challenge infection appeared to influence the intensity of the host's response. There was no conclusive evidence to indicate that the immune response could retard the growth, development, or sex ratios of the worms established in subsequent infections. A positive haemagglutinating antibody response was first observed in some rats as early as four weeks post-infection with 100 larvae when the worms began migrating from the brain to the lungs. The antibody response and eosinophilia were most pronounced during the oviposition of the female worms and hatching of firststage larvae. Changes in white blood cell, lymphocyte, and neutrophil counts were also followed in some groups.     

Unterdrückung der Parasitämie bei der Nagetierfilariose (Litomosoides carinii) durch Immunisierung mit BCG und Mikrofilarien

Zusammenfassung Bei intrakutaner Inokulation von BCG und einer 5–11 Wochen später erfolgenden subkutanen Infektion mit metazyklischen Larven vonLitomosoides carinii kommt es bei Baumwollratten zu einer Infektion, deren Parasitämie signifikant reduziert ist, wenn BCG-Inokulation und Larveninjektion im Einzugsgebiet der gleichen poplitealen und iliacalen Lymphknoten erfolgen.Werden die metazyklischen Larven jedoch über andere regionäre Lymphknoten (cubitale und axilläre) geleitet, so bleibt die Depression der Mikrofilarämie in der Patenz der Belastungsinfektion aus. Auf die Wurmlast, d.h. die Rückfindungsrate, sowie den Mikrofilarienausstoß durch die adulten Würmer hat die BCG-Inokulation keinen Einfluß. Offenbar wird das lymphatische Gewebe durch die BCG-Inokulation unspezifisch stimuliert, die metazyklischen Larven bilden den ersten spezifischen Antigenreiz; die bei Einsetzen der Patenz ausgestoßenen Mikrofilarien führen dann zu einer Boosterung.Kombiniert man die intrakutane BCG-Inokulation mit dem spezifischen Antigen in Form einer gleichzeitigen subkutanen Injektion von Blutmikrofilarien an der gleichen Hinterextremität, so kommt es zu einer Steigerung der gegen die Mikrofilarien gerichteten Immunität; die Parasitämie der späteren Belastungsinfektion bleibt dann ganz aus oder ist extrem niedrig.

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Suppression of parasitaemia fromLitomosoides carinii by immunisation with BCG and microfilariaeI. Intracutaneous inocultion of BCG

After intracutaneous inoculation of BCG and challenge by subcutaneous injection of infective larvae ofLitomosoides carinii, the parasitaemia of the filarial infection in cotton rats remains significantly lower when BCG and larvae are applied in the region of the same popliteal and ileal lymph nodes. However, when the infective larvae are directed to other regional lymph nodes (Ln cubitales and axillares), the depression of microfilaraemia is missed. The worm load (recovery rate) and the expulsion of microfilariae by the adult worms are not influenced by the BCG inoculation. Obviously BCG stimulates the lymphatic tissue unspecifically, and the infective larvae produce the first antigen contact, which is boostered by the microfilariae at the onset of patency. When the intracutaneous BCG inoculation is combined with specific antigen stimulation by simultaneous injection of blood microfilariae in the region of the same lymph nodes, the microfilaraemia of the challenge infection disappears completely or remains extremely low.

     

Nonspecific suppressor cell activity and specific cellular unresponsiveness in rat schistosomiasis.

Spleen cells from inbred Fischer rats infected with Schistosoma mansoni were cultured with S. mansoni antigen, phytohemagglutinin (PHA) or concanavalin A (Con A). During the first 3 weeks of infection, both a significant proliferative response and a delayed cutaneous hypersensitivity to S. mansoni antigen were observed. In contrast, during the fourth to the seventh week both responses were reduced to a nonsignificant level, and the proliferative response to PHA or Con A also decreased during this period. At the time of minimal reactivity to PHA or Con A, the depletion of cells adherent to nylon wool (but not depletion of mononuclear phagocytic cells by plating on plastic) restored a significant response to Con A. At this time, infected rat lymphocytes reduced the proliferative response of noninfected syngeneic cells induced by Con A. This inhibition was not observed when infected cells were previously passed through a nylon wool column. Inversely, nylon wool depletion of day-28 or 35-infected spleen cells did not restore lymphocyte response to S. mansoni antigen. Serum (or the dialyzable fraction thereof) from day-29-infected rats inhibited thymidine incorporation of PHA-stimulated normal lymphocytes and the response to S. mansoni of spleen cells from infected rats. This information suggests that at the end of the first month of infection in the rat, lymphocyte unresponsiveness to S. mansoni antigen could not be related to a demonstrable suppressor cell activity. Low molecular weight inhibitory factor(s) released by the parasite and acting directly on lymphocytes could partly account for this unresponsiveness in rat schistosomiasis.     

Effects of estradiol on worm burden and peripheral leukocytes inParastrongylus malaysiensis-infected rats

Gonadectomized male laboratory rats were given 0.06 mg/kg estradiol benzoate daily for 14 days before being inoculated with 50 third-stage larvae ofParastrongylus malaysiensis. Hormone treatment was continued until the rats were killed. The numbers of larvae in the brain and of adult worms in the pulmonary area of the rats were determined every 7 days after the inoculation. It was found that the rats treated daily with estradiol benzoate had significantly and consistently higher numbers of larvae and adult worms as compared with the controls. The number of total leukocytes increased significantly after the rats were infected. The results show that estradiol-treated rats become susceptible toP. malaysiensis infection, which may indicate that the immunosuppressive effects of testosterone observed in earlier studies may partly be caused by estradiol that was peripherally aromatized from testosterone.     

The cellular transfer of immunity to Nippostrongylus brasiliensis in inbred rats (Lewis strain)

Mesenteric lymph node cells obtained from highly inbred donor rats (Lewis strain), resistant to Nippostrongylus brasiliensis infection, were syngeneically transferred by intravenous injection into previously uninfected recipients. The adoptively immunized recipients were then challenged with either 1500 or 3000 third stage N. brasiliensis larvae on the day of cell transfer. The degree of resistance transferred was assessed by monitoring daily faecal egg output, differential worm burdens on days 6 and 10 of infection and the number of eggs per uterus in gravid worms.

The syngeneic transfer of 100 × 10⁶ immune mesenteric lymph node cells invariably resulted in suppression of egg production, a two- to four-fold reduction in the number of eggs per uterus in gravid females and rejection of at least 75 per cent of adult worms by days 6 and 10 of infection.

It was also noted that mesenteric lymph node cells obtained from donors on day 15 of a primary infection were more effective than those obtained from donors immunized by multiple infections.

Immune cells transferred from donors on day 4 of infection were equally effective with those transferred on day 0. However, immune cells transferred on or after day 10 of infection had little or no effect and this shows that the parasite is less susceptible to an attack mounted by the transferred cells during the later stages of infection.

     

Observations on the transmission and development ofToxocara pteropodis (Ascaridoidea: Nematoda) in the Australian Grey-Headed Flying-Fox,Pteropus poliocephalus (Pteropodidae: Megachiroptera)

Findings in the Australian Grey-Headed Flying-Fox,Pteropus poliocephalus, have elucidated the life-cycle ofToxocara pteropodis. In adult bats, other than parturient females, larvae were found only in the livers. Following parturition, larvae were recovered only from mammary glands up to 2 weeks post-partum. Developing larvae were found only in the intestine of young bats from the age of two days onwards; there was no evidence of pulmonary migration. The evidence indicates that juvenile bats commence passingToxocara eggs in their faeces at about 2 months of age and expel the worms spontaneously following weaning at about 5 months. The eggs passed in the faeces of the young bat and its mother are disseminated through-out their environment and embryonate rapidly, being infective to mice after 10 days. Under natural conditions the eggs remain viable for 6 weeks or less and are infective to bats by the oral route.     

Biochemical characterization and role of the proteasome in the oxidative stress response of adult Schistosoma mansoni worms

The trematode Schistosoma mansoni, an important parasite of humans, is the principle agent of the disease schistosomiasis. In the human host, one of the most important stress factors of this parasite is the oxidative stress generated by both the metabolism of the worm and the immune system of the host. The proteasomal system is responsible for protein homeostasis during oxidative stress. The 26S proteasome is a multicatalytic protease formed by two compartments, a 20S core and regulatory particle 19S, and controls the degradation of intracellular proteins, hence regulating many cellular processes. In the present report, we describe the biochemical characterization and role of the 20S proteasome in the response of adult S. mansoni worms exposed to hydrogen peroxide. Characterization of the response to the oxidative stress included the evaluation of viability, egg production, mortality, tegument integrity, and both expression and activity of proteasome. We observed decreases in viability, egg production as well as 100 % mortality at the higher concentrations of hydrogen peroxide tested. The main changes observed in the tegument of adult worms were peeling as well as the appearance of bubbles and a decrease of spines on the tubercles. Furthermore, there were increases in 26S activity to the same extent as 20S proteasome activity, although there was increase of 20S proteasome content, suggesting that degradation of protein oxidized in adult worms is due to the 20S proteasome. It was demonstrated that adult S. mansoni worms are sensitive to oxidative stress, and that a variety of processes in this parasite are altered under this condition. The work contributes to a better understanding of the mechanisms employed by S. mansoni to survive under oxidative stress.     

The course ofAngiostrongylus cantonensis infection in athymic nude and neonatally thymectomized mice

BALB/c athymic nude and thymus-reconstituted nude mice and neonatally thymectomized BALB/c mice were infected with stage 3 larvae ofAngiostrongylus cantonensis and the worm burdens of the mice were determined at various times after infection. When the nude and thymectomized mice were exposed to the parasite, some worms were found to migrate from the brain to lungs but died there without reaching maturity. This pulmonary arterial migration of the worms in the nude mice did not occur following thymic reconstitution. These data suggest that the inability of murine intracranial worms to migrate to the lungs is at least in part due to thymus-dependent mechanisms, and also that the failure of worm maturation in mouse lungs might be due to thymus-independent immune mechanisms and/or nonimmunological mechanisms.     

Intestinal mucus trapping in the rapid expulsion of Trichinella spiralis by rats: induction and expression analyzed by quantitative worm recovery

Rats were immunized with a Trichinella spiralis infection restricted by chemotherapy to the intestine (the T/M regime) or with a complete infection that resulted in the deposition of muscle larvae. After an oral challenge infection, rapid expulsion could be demonstrated in both groups within 20 min and with 100% recovery of the infectious dose from the stomachs and intestines of infected animals. Immune and nonimmune groups were distinguished by the large numbers of worms in the intestinal lumens of immune rats and large numbers of worms in the intestinal walls in nonimmune rats. Infectious larvae persisted in the stomach lumens for longer in the immune rats. There was no quantitative difference in worm distribution in the intestine during rapid expulsion in rats immunized with the T/M regime or those given a complete infection. However, in the complete infection group 69% of the luminal worms were trapped in mucus; this did not occur during rapid expulsion in rats immunized with the T/M regime. Mucus trapping was observed only when muscle larvae had matured to the infectious stage in muscle (28 days after the primary infection). Complete infection rats challenged at 14 or 21 days did not display significant mucus trapping of larvae in the intestinal lumen. We conclude that (i) mucus trapping is not essential for rapid expulsion and (ii) mucus trapping is produced by systemic exposure to target antigens of the infectious larvae.     

Trichinella spiralis infection in congenitally athymic (nude) mice. Parasitological, serological and haematological studies with observations on intestinal pathology.

In six experiments the course of a Trichinella spiralis infection in congenitally athymic (nu/nu) mice and their heterozygous thymus-bearing littermates (+/nu) was followed. In the +/nu mice worms were expelled at day 10 post infection. In nu/nu mice worms remained in the intestine until the end of the observation period (83 days post infection). In testing the yield of muscle larvae in +/nu and nu/nu mice 4--5 times more muscle larvae were isolated from nu/nu mice than from infected +/nu mice. The following phenomena were observed in +/nu mice only: anti-T. spiralis antibodies detected by immunofluorescence, intestinal plasma-cell production and intestinal eosinophilia. In nu/nu mice no blood eosinophilia was observed in contrast to the induction of eosinophilia both in infected +/nu and infected nu/nu mice reconstituted with thymuses from heterozygous littermates. Intra-epithelial lymphocytes, more numerous in +/nu than in nu/nu mice, were not attracted by Trichinella antigen. The data supported the hypothesis that worm expulsion is a T cell-dependent phenomenon. Plasma cell and antibody production as well as tissue and blood eosinophilia were shown to be thymus-dependent in a T. spiralis infection.     

Enzymatic basis for the lack of oxamniquine activity in Schistosoma haematobium infections

The notion that oxamniquine is active against Schistosoma mansoni but inactive against S. haematobium was confirmed using in vitro cultures of adult worms. Since oxamniquine and hycanthone have been shown to become effective upon activation by a schistosome enzyme, enzymatic tests were carried out to detect possible differences between the enzyme of S. mansoni and that of S. haematobium . It was found that the S. mansoni enzyme could activate hycanthone and, to a lesser extent, oxamniquine. The S. haematobium enzyme, on the other hand, was capable of activating hycanthone but virtually incapable of activating oxamniquine. It is concluded that the different activity of oxamniquine in the two species is due to differences in the drug-activating enzyme.