Host immunity against newborn Trichinella spiralis larvae of different ages

The infectivity of newborn Trichinella spiralis larvae of different ages was studied in normal rats. Newborn larvae collected after incubation of adult worms in vitro for 2, 12, or 24 hr were injected intravenously (i.v.) into normal AO rats in 3 separate recipient groups. All recipient rats developed strikingly similar numbers of muscle larvae 20 days later. The susceptibility to immunity by newborn larvae of different ages was also studied. No difference was found when degree of protection was compared by assessing muscle larvae burden or peritoneal anti-newborn larvae effects after injection of newborn larvae of different ages either i.v. or intraperitoneally into immunized recipient rats. We conclude that newborn larvae of any age up to 24 hr have similar infectivity in normal rats and are equally susceptible to anti-newborn larvae immunity in vivo.     

Chemical composition of newborn larvae, muscle larvae and adult Trichinella spiralis

Chemical composition of newborn larvae, muscle larvae and adult Trichinella spiralis. International Journal for Parasitology16: 455–460. The chemical composition of newborn larvae (NBL), muscle larvae (ML) and 4-day-old and 7-day-old adult Trichinella spiralis were compared. Total protein constituted 44.1% of the dry wt of ML, 36.9% of NBL and 30.3% of 7-day-old adult worms. Glycogen content was 16.1% of worm dry wt in ML and 7.8% in NBL and 7.2% of worm dry wt in adults. Trehalose content was 5.2% in NBL, 8.3% in ML and 8.7% in 7-day-old adult worms. Significantly lower levels of trehalose and glycogen were found in 4-day-old worms than were present in 7-day-old worms. Free glucose amounted to 0.1% of dry wt in ML, 0.37% in NBL and 0.87% in 7-day-old adults. RNA accounted for 3.5% of the dry wt of ML, 3.2% of the dry wt of 7-day-old adults and 1.1% of the dry wt of NBL. The DNA content of NBL was 0.23% of worm dry wt, in ML 0.48% of worm dry wt and 0.51% of worm dry wt in 7-day-old adults. The three parasite stages examined agreed closely with regard to types of amino acids in the free pool. Exceptions were as follows: NBL lacked taurine which both adults and ML contained; adults lacked methionine which both ML and NBL contained; and, trace amounts of cysteine were present in ML but absent from the other two stages.     

Characterization of cellular and molecular immune effectors against Trichinella spiralis newborn larvae in vivo.

The cellular and molecular immune effectors that participated in host immunity against Trichinella spiralis newborn larvae were characterized in vivo using AO rats. Donor rats were immunized with 2,000 muscle larvae orally or 11,400 newborn larvae i.v. Immune serum and cells from spleen, peripheral lymph nodes, mesenteric lymph node, thoracic duct lymph and the peritoneal cavity were obtained from donor rats 10-21 days after infection and transferred into normal recipient rats. The control recipients received either no cells and serum or normal cells and normal serum obtained from normal donors. Newborn larvae (20,000-50,000) were injected either i.v. or ip into these recipients and immunity against newborn larvae was measured either by muscle larvae burden of the recipients three weeks later or by direct recovery of newborn larvae from the peritoneal cavity of the recipients. The experiments demonstrated that immune lymphocytes conferred no protection in the recipients but that immune serum and immune peritoneal cells were protective and these effects were synergistic. Cell adherence to the cuticle and killing of newborn larvae were observed in the peritoneal cavity of immune rats. Positive fluorescence was observed on newborn larvae incubated with fractionated IgM and IgG(E) antibody isotypes. Massive deposition of antibody molecules on newborn larvae was demonstrated by scanning electron microscopy. Studies using transmission electron microscopy revealed that the larval adherent cells were stimulated macrophages, neutrophils and eosinophils.     

Trichinella spiralis: vascular recirculation and organ retention of newborn larvae in rats

The recirculation of Trichinella spiralis newborn larvae was studied in inbred AO rats. Newborn larvae collected after in vitro incubation of adult T. spiralis worms for 2 or 24 hr were injected into rats through the tail vein or hepatic portal vein. Blood samples from the femoral vein, hepatic portal vein, and abdominal aorta were collected at intervals from 1 min to 24 hr after larval injection. Newborn larvae of both ages (24 hr or 2 hr old) persisted in femoral vein blood for less than or equal to 5 hr after injection, but they could be detected in portal vein blood by 24 hr after injection. The injection of larvae into a tail vein or the portal vein did not influence the pattern of larval circulation, although there was a 1-5 min delay in newborn larval appearance time after injection into the portal vein. Transcapillary migration through tissue and back to the circulation was evident in the appearance of newborn larvae in the thoracic duct lymph up to 24 (occasionally 48) hr after tail vein injection of newborn larvae. During the course of a natural primary infection, no evidence for trapping of larvae in the mesenteric lymph node could be found despite direct larval migration through this organ. Injected newborn larvae were retained in the lungs, and small numbers could be recovered 24 hr after intravenous injection. We conclude that a proportion of newborn larvae recirculates within the vasculature for several hours; a smaller population extravasates but can reenter the circulatory system via the lymphatics. Furthermore, some newborn larvae are found in organs rich in capillaries up to 24 hr after their entry into the blood.     

Trichinella spiralis: nonspecific resistance and immunity to newborn larvae in inbred mice

The implantation and development of intravenously injected Trichinella spiralis newborn larvae were examined in different strains of inbred mice by determining muscle larvae burden. This was compared to the numbers of muscle larvae that established after a natural infection during which a quantitative assessment of intestinal newborn larvae production was made. In most inbred strains of mice, newborn larvae do not all successfully implant in muscle. Mice of the DBA/1 strain are the most resistant to successful implantation, and C3H mice are the most permissive. This pattern is evident in the strains studied whether newborn larvae are injected intravenously or are produced by intestinal adults. Thus, after a natural infection, 100% of intestinally produced newborn larvae implanted in C3H mice, whereas in NFR 68% and DBA/1 mice 62% successfully matured in muscle. Immunity to newborn larvae could be demonstrated as early as 10 days after exposure to this stage of the life cycle. This immunity was protective against a complete challenge infection given 9 days after newborn larvae had been injected intravenously. Protection against newborn larvae was identical in male and female mice or in mice from 1 to 9 months of age. We conclude that there are two mechanisms by which mice impair newborn larvae establishment or development in muscle. The first appears to be nonimmunological (non-specific resistance), and the second is immunological. Genetically determined variation in strain-specific expression is apparent with both mechanisms. In strains displaying high intrinsic "resistance" (DBA/1), this process is likely to account for most of the 38% reduction in newborn larvae establishment in a primary infection. However, immunity against newborn larvae develops quickly enough to have a significant effect on migratory larvae in primary infections where adults persist in the intestine (e.g., the B10 congenic mice), or when high adult worm burdens delay adult worm rejection. Muscle larvae burden, therefore, reflects systemic nonspecific resistance to newborn larvae as well as immunological processes that occur in the intestine and systemically.     

Trichinella spiralis: activation of complement by infective larvae, adults, and newborn larvae.

The ability of Trichinella spiralis to activate complement (C) has been addressed by several investigators. However, these investigators employed methods in which either detection of C fragments on the parasite surface or the adherence of leukocytes to the parasite was considered an indication of C activation. The present studies were undertaken to examine: (a) whether activation of C occurs via the classical and/or alternative pathway, (b) at which stage(s) of the parasite C activating capacity is acquired, and (c) what molecular entities of the epicuticle and/or cuticle are responsible for initiating C activation. Our studies indicate that T. spiralis activates C primarily via the alternative pathway (and weakly via the classical pathway) since incubation of parasites obtained from infected mice with either normal human serum (NHS) or Mg.EGTA-NHS, followed by incubation (1 hr, 37 degrees C) with antibody-sensitized sheep erythrocytes or rabbit erythrocytes, respectively, showed a time-and parasite number-dependent depletion of C. Although the three stages of T. spiralis, i.e., infective larvae, adults and newborn larvae, are capable of activating C, the newborn appears to be the most potent activator, especially when parasite number and size are taken into consideration. Further evidence of C activation is obtained from SDS-PAGE and Western blot analysis in which homogenates of parasites preincubated with NHS showed the presence of C3, C9, and C1q, whereas controls without serum were negative. Since isolated C1q was also capable of directly binding to the surface of adults and infective larvae, it is postulated that their cuticle and/or epicuticle may possess surface structures which serve as binding sites for C1q.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis: macrophage activity and antibody response in chronic murine infection

The role of macrophages, their products, and the specific antibody response were examined during chronic Trichinella spiralis infection in BALB/c mice. Adult T. spiralis in intestines were detected from 5 to 20 dpi. Muscle larvae numbers peaked at 45 dpi and thereafter a reduction was noted. The highest numbers of macrophages in the peritoneal cavity of infected mice were obtained up to 30 dpi. The production of NO by macrophages in infected mice was suppressed at 5 dpi, and then NO release increased until 45 dpi. The levels of NO in plasma and urine were lower in infected mice during the entire experiment in comparison to control. The production of O(2)(-) in peritoneal macrophages was inhibited during the first two weeks after infection and then increased until 90 dpi. Circulating T. spiralis antigens in plasma and urine were detected from 5 to 30 dpi. Specific IgM and IgA in serum increased until 20 dpi. IgG, IgG(1), and IgG(2) levels in serum increased until 60 dpi.     

旋毛虫丝氨酸蛋白酶基因的结构与功能分析

目的：运用生物信息学原理,对由旋毛虫新生幼虫（NBL）差减文库调取的新生幼虫期特异性基因进行分析。方法：通过对BLASTn核酸数据库及BLASTp蛋白数据库的相似性检索,登陆PROSITE数据库进行蛋白位点和序列模式分析,并采用AN-THEPROT4.3软件包对其理化特性进行分析。结果：相似性检索及序列模式分析显示,该基因为胰蛋白酶家族丝氨酸蛋白酶的编码基因,并对其编码蛋白的理化特性,疏水性,抗原性,信号肽及其二级结构进行了预测,登陆SWISS-MODEL自动蛋白质同源建模服务器,搜索,优化后预测了其3D结构模型。结论：该期特异性基因为旋毛虫新生幼虫胰蛋白酶家族丝氨酸蛋白酶的编码基因。     

Trichinella spiralis: migration of larvae in the rat

Counts were made of Trichinella spiralis “migratory” larvae recovered from blood, abdominal cavity, lungs, liver, kidneys, and thoracic duct lymph of male albino rats from 4–15 days postinoculation. From these data, the pathways the larvae utilized to travel from the small intestine to skeletal muscle were determined. Approximately 70% of the encysted muscle larvae were accounted for by the lymph-blood circulatory system pathway. The data indicate that the other 30% probably migrated by way of both (a) the hepatic portal circulatory system to the heart and then to the general circulation and skeletal muscle; and (b) the abdominal cavity and/or abdominal fluids to skeletal muscle.     

Effects of rat and human intestinal lamina propria cells on viability and muscle establishment of Trichinella spiralis newborn larvae

Although eosinophils and other inflammatory cells from the circulation and peritoneal cavity can damage Trichinella spiralis newborn larvae (NBL) in vitro, the cytotoxic potential of cells from the intestinal lamina propria, a site that may be the first line of defense against NBL migration, is unknown. Accordingly, we examined the interaction between NBL and isolated intestinal lamina propria cells (ILPC), including an enriched eosinophil population, from rats and humans. Rat ILPC killed NBL in vitro only after a prolonged incubation of 6 days. However they strongly adhered to NBL after only 4 hr incubation and prevented muscle establishment of NBL injected intravenously. Human ILPC showed similar adherence as rat ILPC but no killing was seen at the incubation time tested (36 hr).     

Trichinella spiralis: antifecundity and antinewborn larvae immunity in swine

The development of antifecundity and antinewborn larvae immunity in swine infected with Trichinella spiralis was investigated. In primary infections, adult female worm fecundity dropped sharply after 3 weeks, although adults could be recovered from the small intestine for at least 7 weeks after infection. In challenge infections of pigs infected previously, adult female worm fecundity was depressed up to 51% and the adults were expelled within 3 weeks. Since immune pigs are almost completely resistant to the secondary establishment of muscle larvae, this suggested the existence of immune effector mechanisms also acting on the newborn larvae. This was supported by observations, using an indirect fluorescent antibody assay, that pig antibody bound to the surface of the newborn larvae. Passive transfer of immune pig serum resulted in a large reduction in muscle larvae burden in both infected pig and rat recipients. Adult female worm fecundity in such immune serum recipients was reduced only by 20% and worm survival in the intestine was unaffected. These results indicate that immunity to the newborn larvae, in addition to antifecundity effects, are responsible for the high levels of acquired resistance to T. spiralis in swine.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

Comparison of cholinesterase activities in the excretion-secretion products of Trichinella pseudospiralis and Trichinella spiralis muscle larvae

The presence of cholinesterases (ChE) is reported in T. pseudospiralis excretion-secretion products (ESP) by spectrophotometric method, using acetylthiocholine (ATCI) and butyrilthiocholine (BTCI) as substrates. By inhibition assays, we found that T. pseudospiralis release both acetyl- and butiryl-cholinesterases (AchE and BchE, respectively). The sedimentation coefficientes of these enzymes were determined by sucrose density gradient. We studied the in vivo ChE secretion by immunoblot assays using AchE from Electrophorus (electric eel) and sera from normal or infected mice with T. pseudospiralis or T. spiralis. The presence of anti-AchE antibodies was only demonstrated in the sera from T. pseudospiralis infected mice. Moreover the in vivo secretion was corroborated by the high difference determinate between the ChE activity of the immuno complexes from T. pseudospiralis infected sera and the immunocomplexes from T. spiralis infected sera as well as normal sera. Finally, we analyzed the effect of the organophosphate Neguvón (metrifonate) on the ChE activity from the T. pseudospiralis ESP. The drug inhibits in part this activity. Moreover Neguvón (metrifonate) showed a high activity against the T. pseudospiralis viability.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.