A single gene determines rapid expulsion of Trichinella spiralis in mice.

In rats and some inbred mouse strains, one immune response, rapid expulsion, confers up to 95% protection against a challenge infection with Trichinella spiralis. Strain analysis in mice has shown that only three inbred strains, all originating from Swiss-line mice at the National Institutes of Health, Bethesda, Md., express rapid expulsion. Crosses between responder strain mice (NFR/N) and nonresponders (C3H/HeJ or B10 X BR) have indicated that rapid expulsion is dominant and autosomal (Bell et al., Exp. Parasitol. 53:301-314, 1982). In this study a segregation analysis of rapid expulsion in the F2 and backcross conformed to the Mendelian ratios expected of a single gene. This gene was not linked to the major histocompatibility complex (MHC) (chromosome 17) or the gene for albinism (c/c locus on chromosome 7). This locus has not previously been identified as conferring resistance to any infectious agent, and we have therefore designated the gene Ihe-1 (intestinal helminth expulsion 1).     

Delayed expulsion of adult Trichinella spiralis by mast cell-deficient W/Wv mice

Mast cell-deficient W/Wv mice and their mast cell-sufficient littermates were given infections of Trichinella spiralis. W/Wv mice were slower than their littermates to expel adult T. spiralis. Repair of the mast cell deficiency of W/Wv mice by bone marrow grafting was accompanied by accelerated expulsion of T. spiralis.     

Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis

Trichinellosis is a widespread zoonoses for which no effective drug treatment is available at this time. Though anthelmintics such as mebendazole and albendazole are commonly used to treat human trichinellosis, none of these drugs are fully effective against the encysted or new-born larvae of Trichinella spiralis. In recent years, there has been a growing interest in developing newer anthelminthics from medicinal plants, particularly the ones used in traditional medicines in many parts of the world, due to the increasing spread of anthelminthic resistance and/or decreasing activity against encapsulated larval stages of parasites. The aim of the present study was to investigate the efficacy of leaf extract of Lasia spinosa (Araceae) against different life cycle stages of T. spiralis, i.e. adult (days 3 and 4 post-infection), migrating larvae (days 8, 9 and 10 post-infection) and encysted muscle larvae (days 31–37 post-infection). The study showed that L. spinosa leaf extract is effective against all the three life cycle stages of parasite. Against the adult stage, an oral administration of plant extract at 800 mg/kg dose revealed a 75.30% reduction in the number of adult worms, as compared to untreated controls at day 10 post-infection. Whereas against migrating larvae, the same dose of plant extract given for 3 days, reduced the number of larvae recovered from musculature of treated animals by 72.23%. However, in comparison of preceding two stages, the extract showed comparatively less efficacy against the encysted larvae of parasite. In this case, the 800 mg/kg dose of extract given for 7 days (after 30 day of post-infection) revealed only 64.84% reduction in the number of encysted larvae, as was evident from larval count on day 49 post-infection. Therefore, the results of this study indicate that leaf extract of L. spinosa possesses significant anthelminthic efficacy against the adult stages and migrating larvae of T. spiralis. On the other hand, the encysted muscle larvae of parasite are comparatively less sensitive to L. spinosa leaf extract treatment.     

Acceleration of IgE responses by treatment with recombinant interleukin-3 prior to infection with Trichinella spiralis in mice.

Treatment of mice with recombinant interleukin-3 (rIL-3) accelerated an IL-4-dependent IgE production following infection with Trichinella spiralis. When mice were treated with a total of 1.5 x 10(4) units rIL-3 for 5 days before infection with 400 muscle larvae, the serum IgE level increased prominently on day 5. Acceleration of IgE responses was dependent on the dose of rIL-3 injected. Treatment of mice with a total of 10(3) units rIL-3 could accelerate IgE responses. IgE responses were detected by a sandwich enzyme-linked immunosorbent assay at least from day 3 in mice treated with rIL-3. Acceleration of IgE responses was inhibited by anti-IL-4 monoclonal antibody. In contrast to this, IgG1 and IgG2a responses were not suppressed by the anti-IL-4 treatment. IL-3 treatment could up-regulate IgE and IgG1 responses but not the IgG2a response. IL-3 treatment could also accelerate IgE responses in W/Wv mice infected with the parasites. These results suggest that IL-3 is involved in regulation of IgE responses in mice and that mast cells do not play an essential role in acceleration of IgE responses induced by rIL-3 treatment in this system.     

旋毛虫新生幼虫T668重组抗原对小鼠的保护性免疫

目的研究旋毛虫新生幼虫T668重组抗原的免疫原性,制备基因工程疫苗。方法以旋毛虫新生幼虫期特异性基因T668在大肠杆菌中的表达蛋白为抗原免疫小鼠,每间隔10d免疫1次,共免疫3次。末次免疫后10d,每只小鼠攻击感染200条旋毛虫感染性肌幼虫,感染后5周用消化法检查肌幼虫(ML)负荷。结果T668重组抗原免疫组肌幼虫减虫率明显高于佐剂组和对照组。结论T668重组抗原能诱导小鼠产生一定程度的保护性免疫,且可激发特异性体液免疫。     

Adaptive changes in muscle fibers infected with Trichinella spiralis.

Ultrastructural changes which occurred in infected skeletal muscle fibers after infection with larvae of Trichinella spiralis were followed on a daily basis utilizing synchronous infections. No changes were observed in muscle fiber architecture during the first 2 days of intracellular infection. However, on Day 3, a space containing various sarcoplasmic elements developed between the plasma membrane and myofilaments. Widening near the regions of triads was also observed at this time. On Day 4 the space at the outer edge had increased, as did the ones at the triads. In addition, the myofilaments throughout the infected fiber were in a state of partial disarray. Finally, the nuclei were enlarged and had migrated to the central portion of the infected cytoplasm. On Day 5 day, sarcomeres were highly disorganized, and an increase in sarcoplasmic reticulum (SR) was noted. By Day 8, only the extreme periphery of the infected fiber contained Z bands with actin filaments attached. Proliferation of the t tuble system was also evident. At Day 10, myofilaments were completely replaced with SR. Further, the plasma membrane became hyperinvoluted and was associated with a 36-fold increase in the thickness of the glycocalyx. No further enlargement of nuclei occurred after Day 10. Finally, a host-derived double membrane completely surrounded the larva.     

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.     

Genetic factors controlling the intestinal mast cell response in mice infected with Trichinella spiralis.

Inbred strains of mice showed marked variation in their mast cell (MC) response to infection with Trichinella spiralis. Variation was under genetic control, the ability to respond to infection being inherited as a dominant trait. MHC-linked genes may influence the absolute level of response, but overall response kinetics appear to be controlled by genes which are not linked to the MHC. An enhanced MC response was transferred adoptively with immune mesenteric lymph node cells (IMLNC), but reciprocal adoptive transfers between H-2 compatible rapid (NIH) and slow (B10.G) responder strains showed that the degree of enhancement was determined by the response phenotype of the recipient, not that of the donor. Similarly, in bone marrow (BM) chimaeras, produced by reconstituting lethally irradiated F1 (B10.G x NIH) mice with parental BM, the MC response to T. spiralis was determined by the response phenotype of the BM donor, whether or not rapid responder IMLNC were transferred. The data are discussed in terms of a T lymphocyte regulated, bone marrow stem cell origin of mucosal MC and interpreted as showing that genetic regulation of the MC response is expressed at the level of stem cell or precursor response to T cell derived mastopoietic factors.     

Rapid expulsion of Trichinella spiralis in suckling rats: mediation by monoclonal antibodies

Pups born to rats immunized with the excretory/secretory antigens (ESA) of Trichinella spiralis L1 larvae expressed rapid expulsion when challenged orally. Rat monoclonal antibodies specific for ESA were produced and tested for their specificity in Western blots, binding to intact larvae and protective capacity in suckling rats. Eight antibodies had apparently identical specificity in Western blots, each recognizing a polypeptide family that included three molecular weight species sized at 41,000-50,000 MW. These polypeptides formed a series of higher molecular weight aggregates that were also bound by the monoclonal antibodies. Four of eight antibodies were protective when serially transferred to suckling pups. Each protective antibody was able to bind to intact larvae. Antibodies of two subclasses, IgG1 and IgG2c, were strongly protective, delivering to pups the capacity to expel as much as 94% of the challenge dose.     

Cellular immunity in Peyer''s patches of rats infected with Trichinella spiralis.

A rat model of Trichinella spiralis gut infection was used to observe the sequence of developing cellular immunity in Peyer's patches and other lymphoid tissues. Whereas cellular reactivity (lymphocyte blastogenesis) for worm antigens was evident in mesenteric lymph nodes draining the gastrointestinal tract within 3 days after infection, Peyer's patch lymphocytes developed maximal reactivity 2 to 3 weeks later at the same time as the spleen and other lymphoid tissues. Furthermore, the immune reactivity found in Peyer's patches was only transient. Thus, in this parasitic gut infection, the Peyer's patch lymphoid tissue does not appear to be the first site of cellular responsiveness but rather to acquire cellular reactivity only when other lymphoid elements in the infected host have also acquired similar antigen-induced reactivity.     

Characterization of the immune mediator of rapid expulsion of Trichinella spiralis in suckling rats

In order to identify and characterize the mediator(s) of rapid expulsion, infant rats were passively immunized against oral challenge with 200 infectious Trichinella spiralis muscle larvae by oral or intraperitoneal (i.p.) administration of secretions or sera from infected rats. Milk whey from infected dams provided a modest level of protection when fed to pups. Immunoglobulins from T. spiralis-infected rat serum protected suckling pups when injected intravenously (i.v.) into lactating dams 2 days prior to pup challenge. Intraperitoneal injection of pups with serum immunoglobulins also enabled them to express rapid expulsion. The protective component of serum was precipitated with 40% (NH4)2SO4 and was not affected by heating to 56 degrees, although antibodies mediating passive cutaneous anaphylaxis were inactivated by both treatments. Oral transfer of biliary immunoglobulins collected from infected rats at various times during a primary infection provided no protection to pups. However, serum immunoglobulins from rats infected for 42 days or longer transferred rapid expulsion to pups. Absorption of protective serum immunoglobulins with subclass-specific reagents revealed that IgG1 played a significant role in protection.     

Release of leukotrienes during rapid expulsion of Trichinella spiralis from immune rats.

Rapid expulsion of the nematode Trichinella spiralis from immune rats is associated with an increase in volume of intestinal exudate and the presence of large numbers of tissue mucosal mast cells (MMC) and eosinophils. We have measured the concentrations of leukotrienes (LT) C4 (LTC4) and B4 (LTB4) in gut perfusates and mucosal homogenates at 30 min, 1, 3, 6 and 20 hr after challenge with larvae. Leukotrienes were identified by radioimmunoassay (RIA) combined with reverse-phase high-pressure liquid chromatography (RP-HPLC). There were significant elevations at 30 min and 1 hr in the concentrations of LTC4 in the perfusates from the gut of challenged immune rats compared to controls (infected unchallenged and uninfected naive rats). Similar increases in immunoreactive LTC4 and LTB4 were observed in mucosal homogenates from the gut of immune challenged animals. A second peak of LTB4 was also observed at 20 hr in both immune and naive challenged rats. There were also elevations in serum concentration of the MMC-associated specific serine protease, rat mast cell protease II (RMCPII). Since LTC4 causes smooth muscle contraction, increased vascular permeability and stimulation of mucus hypersecretion, and LTB4 recruits and activates inflammatory cells, leukotrienes may participate in the process of rapid expulsion of T. spiralis.     

Amelioration of influenza-induced pathology in mice by coinfection with Trichinella spiralis

Illness due to respiratory virus infection is often induced by excessive infiltration of cells into pulmonary tissues, leading to airway occlusion. We show here that infection with Trichinella spiralis results in lower levels of tumor necrosis factor in bronchoalveolar lavage fluid and inhibits cellular recruitment into the airways of mice coinfected with influenza A virus. Infiltration of neutrophils and CD4+ and CD8+ lymphocytes was reduced, resulting in animals gaining weight more rapidly following the initial phase of infection. Influenza resulted in a generalized increase in vascular permeability in pulmonary tissues, and this was suppressed by parasite infection, although the effects were restricted to the early phase of trichinosis. Moreover, the number of cells producing interleukin-10 (IL-10), and the local levels of this cytokine, were reduced, suggesting that amelioration of pulmonary pathology by parasite infection occurs independently of IL-10 production.     

The effectors responsible for gastrointestinal nematode parasites, Trichinella spiralis, expulsion in rats

Helper T (Th2) cells type 2 have a central role in host protective responses to gastrointestinal nematode parasite, Trichinella spiralis infection, but the actual effector mechanisms involved in parasite expulsion are still uncertain. Recent evidences suggest that mast cell recruitment and activation may associate with parasite elimination from host intestines in mice. On the other hand, IgE production may induce defensive responses to primary infection with the helminth in rats. The differences of host effector mechanisms to the same experimental infections might disturb our understanding of the host protective mechanisms to gastrointestinal nematode parasite infection. In order to redefine these differences, we investigated in detail the relationship between intestinal immune responses and worm expulsion following T. spiralis infection among several rat strains including mutants. As a result, there were significant correlations of parasite expulsion with mast cell hyperplasia in addition to serum IgE level. Moreover, mast cell-deficient and dysfunction rats showed delayed worm elimination from their gut. Therefore, the present study suggests that mast cells should also be one of the prominent effector cells involved in T. spiralis parasite expulsion in rats as well as mice.     

The role of mucus in antibody-mediated rapid expulsion of Trichinella spiralis in suckling rats.

Rat pups suckling dams parasitized by Trichinella spiralis express rapid expulsion, a protective response that is associated with the entrapment of infectious muscle larvae in intestinal mucus. Immunofluorescent studies revealed that antibodies were bound to the surfaces of the entrapped larvae. Mucus binding and rapid expulsion occurred in normal pups dosed with larvae coated with antibodies prepared from infected rat serum. Subsequent experiments revealed that entrapped larvae escaped from mucus after 2 hr in vitro incubation in saline. Escape correlated with the loss of the surface-bound antibodies, suggesting that mucus entrapment was reversible and dependent on antibody coating. Finally, when protective antibodies were injected 1, 2 or 6 hr after larvae were administered to pups, the parasites were forced to leave their epithelial niche and became enveloped in mucus. The above findings suggest that mucus trapping of T. spiralis larvae is dependent upon the coating of larvae by antibody, but that trapping is reversible, and is not in itself the pivotal event in rapid expulsion. The primary mechanism of rapid expulsion appears to be antibody-mediated inhibition of processes required for the parasite to maintain itself in the epithelium.     

Identification and partial characterization of a T-cell-derived antigen-binding factor from mice infected with the intestinal helminth Trichinella spiralis

Immunochemical and biological characterization was performed of an antigen-binding factor derived from culture supernatants of T cells from mice infected 4 days previously with the intestinal helminth Trichinella spiralis. Affinity chromatography with T. spiralis antigen resulted in the purification of a protein, provisionally designated Trichinella factor (Tric-F), that shared antigenic and other properties with a known T-cell-derived antigen-binding factor of different antigenic specificity, picryl chloride factor, which mediates an early 2-hour component of contact sensitivity. Tric-F lacked determinants of immunoglobulins and possessed determinants shared by other antigen-specific T cell factors, as determined by ELISA and antibody affinity chromatography. Biological activity of Tric-F was assayed in vivo and in vitro. Mice injected intravenously with Tric-F developed an antigen-specific early 2-hour ear swelling response following local challenge with T. spiralis antigen. These results corresponded to delayed-type hypersensitivity responses in the ears of T. spiralis-infected mice that comprised early 2-hour and late classical 24-hour responses. In vitro, Tric-F induced serotonin release by mast cells in the presence of T. spiralis antigen. Mast cells sensitized with Tric-F formed rosettes with antigen-coated sheep erythrocytes. It is suggested that Tric-F, an antigen-binding molecule that is T-cell-derived, mediates the early 2-hour component of delayed-type hypersensitivity and is involved in the initiation and regulation of T-cell-mediated intestinal inflammation during a T. spiralis infection in mice.     

The detection and occurrence of circulating antigens of Trichinella spiralis during worm development

Four enzyme-based immunoassays were compared in detecting circulating antigens (CA) of Trichinella spiralis, i.e. microfluorescence, dissociated enhanced lanthanide fluoroimmunoassay (DELFIA), enhanced chemiluminescence and enzyme-linked immunosorbent assay (ELISA). Parameters which could affect the sensitivity and specificity of the assays were evaluated. Different combinations of polyclonal antibody (PA) and five monoclonal antibodies (mAbs) against the excretory/secretory antigens of the nematode were tested to produce an optimal antigen-detecting system. DELFIA and a “sandwich” consisting of PA as capturing antibody and mAb 7C₂C₅ as detecting antibody, yielded the most stable and sensitive results; and 1 ng CA/ml could be detected. The assay did not cross-react with heterologous antigens of Angiostrongylus cantonensis, Ascaris suum, Cysticercus cellulosae, Fasciolopsis buski, Gnathostoma hispidum and Trichuris suis. Fluctuating levels of CA were observed in the serum of experimentally infected mice at various periods post-infection. As early as days 4 and 6, a significant amount of CA was detected. The level reached a peak at day 10, then declined and another peak was observed at day 18. The monitoring of the corresponding antibody response by ELISA showed that IgM was first detected at day 10, reaching a peak at day 16. A marked increase in IgG₁ was noted from day 16 and its level was significantly higher than that of IgG₂. Received: 23 June 2000 / Accepted: 5 September 2000     

An antigenic recombinant serine protease from Trichinella spiralis induces protective immunity in BALB/c mice

In this study, we report the cloning and characterization of a cDNA encoding a Trichinella serine protease gene (TspSP-1.3) from GenBank. The recombinant TspSP-1.3 protein (rTspSP-1.3) was expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR analysis revealed that TspSP-1.3 was expressed at significantly higher levels in muscle larvae and adult worms than in newborn larvae. TspSP-1.3 was detected in excretory–secretory proteins of Trichinella spiralis with western blotting. Immunization with the rTspSP-1.3 antigen induced humoral immune responses, which manifested as elevated specific anti-rTspSP-1.3 IgG and IgE antibodies and a mixed Th1/Th2 response. To determine whether purified rTspSP-1.3 had good antigenicity and could be a vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with rTspSP-1.3 and subsequently challenged the mice with T. spiralis larvae. The results showed that mice vaccinated with rTspSP-1.3 exhibited an average reduction in the muscle larvae burden of 39 % relative to the control group. These results suggest that TspSP-1.3 could be a novel vaccine candidate for controlling Trichinella infection.     

The immunological response of mice infected with Trichinella spiralis: Biological and physico-chemical distinction of two homocytotropic antibodies

(1) Infection of mice with Trichinella spiralis induced the appearance in serum of two homocytotropic antibodies that could be distinguished by their biological and chromatographic behaviour.

(2) Biologically, the antibodies could be distinguished by their ability to persist in homologous skin after passive transfer. One antibody was able to induce PCA only when a short latent period was used, whereas the other was able to induce PCA even after 72 hours.

(3) They could also be separated when antiserum was passed through a DEAE-cellulose column. Antibody present in the first eluates was able to induce PCA only if a short latent period was used whereas antibody present in the subsequent eluates was able to induce PCA 72 hours after sensitization.

(4) Both antibodies appeared in the circulation 5 weeks after infection and reached their highest levels around the 9th week. Later, the 72-hour PCA antibody disappeared from the serum in some animals, whereas the 4-hour PCA antibody remained.

(5) Re-infection resulted in an increase in the levels of both antibodies.

(6) In animals subjected to repeated reinfections the reagin-like antibody either decreased or disappeared from the serum. On the other hand, the 4-hour PCA antibody increased.

(7) Immunization with `dead' T. spiralis antigen led to the appearance of both antibodies in the serum. A second dose of antigen resulted in increases in the levels of both antibodies, but further injections resulted in a high level of 4-hour PCA antibody and in the disappearance of the reagin-like antibody.

     

Increase of mucosal mast cells in the jejunum of patients infected with Trichinella spiralis

The mast cell response in the mucosa and connective tissue of 36 jejunal biopsies of patients with clinically diagnosed trichinellosis, teniasis and lambliasis has been studied. Biopsy material was fixed in standard formalin or Carnoy's fixative, enabling differentiation between mucosal mast cells (MMC) and connective tissue mast cells (CTMC). With both fixatives CTMC could equally well be recognized. With Carnoy's fixative an additional population of mast cells (MMC) could be visualized both in the mucosa and the connective tissue. In the mucosa small mucosal mast cells were observed as well. Compared to the numbers of mast cells in the mucosa and the connective tissue of teniasis and lambliasis patients, the number of mast cells in trichinellosis patients only visualized using Carnoy's fixative was markedly higher. It was concluded that also in man trichinellosis is accompanied by an increase of cells with MMC characteristics. Further studies are needed to clarify the morphological and histochemical features of these cells and their possible role in this parasitic infection.