Lectin-blot analyses of Trichinella spiralis muscle larvae excretory-secretory components

The rapid recognition of invading pathogen polysaccharides by host lectins might have a significant role in the outcome of the infection. Oligosaccharide structures of the pathogens may provoke an antibody response and serve as a target for specific antibodies. It is well known that Trichinella spiralis antigens, either on the surface or excreted-secreted, are key modulators or targets of the host immune system. In our study of the role of lectins in host defense against T. spiralis infection, an investigation on sugar component of parasite glycoproteins was performed. Affino-blot analyses of T. spiralis muscle larvae excretory-secretory (ES) products by plant lectins revealed that these proteins possess: (1) N-glycans (ConA, PSA, PHA), and probably some O-linked structures (AAA), (2) oligosaccharide structures with mannose residues, especially of the oligomannose type (ConA) and the biantennary complex type with Fuc in the pentasaccharide core (PSA), (3) bisected oligosaccharides, probably some polyantennary glycophorms (PHA), (4) terminally positioned Gal (RCA I, AAA), (5) N-glycans containing oligomers of, or bisected GlcNAc (WGA), that lack alpha2,6 type of linkage (absence of SNA binding).     

Evaluation of Trichinella spiralis larva group 1 antigens for serodiagnosis of human trichinellosis

To identify Trichinella antigens suitable for high-specificity and high-sensitivity serodiagnosis of human trichinellosis, we evaluated assays using four antigens: (i) crude first-stage larval extract (CLE), (ii) O-deglycosylated CLE, (iii) tyvelose-bearing antigens (Trichinella spiralis larva group 1 [TSL-1] antigens) purified by US4 affinity chromatography and coupled directly to enzyme-linked immunosorbent assay (ELISA) plates (pTSL-1 antigens), and (iv) TSL-1 antigens immobilized on ELISA plates with the monoclonal antibody (MAb) US4 (cTSL-1 antigens). Assays using these antigens were compared by analysis of sera from healthy individuals (n = 224) (group 1), individuals with noninfectious intestinal pathologies (n = 114) (group 2), individuals with other parasitic infections (n = 107) (group 3), and individuals with confirmed trichinellosis (n = 42) (group 4). Our results indicate that capture ELISA using cTSL-1 antigens is the most effective method for serodiagnosis of human trichinellosis; this was the only method showing 100% specificity and 100% sensitivity at the patent stage of the infection, and it was also the most sensitive for sera obtained prior to patency in indirect immunofluorescence (IIF). Indirect ELISA with pTSL-1 antigens was also 100% specific but was slightly less sensitive, particularly with sera obtained before IIF patency. Inhibition ELISA with MAb US4 indicated (i) that in Trichinella-infected patients the immune response to TSL-1 antigens is directed mostly against tyvelose-containing epitopes (mean of 84.2% of total anti-TSL-1 immunoglobulin G1 [IgG1] antibody response [range, 51.3 to 97.6%]) and (ii) that in most individuals a large proportion of anti-CLE IgG1 antibodies (mean, 49.5%; range, 7.3 to 92.6%) are directed against tyvelose epitopes.     

Eosinophil adherence to infective larvae of Trichinella spiralis: quantification and modulation.

Eosinophils play an important role in host defence against helminthic infection. A quantitative system for studying the interactions of eosinophils and a large non-ingestible target was developed using infective muscle-stage larvae of Trichinella spiralis. At 37 degrees, this antibody-dependent system provided a reliable, reproducible index of eosinophil function. Adherence was equivalent under aerobic and anaerobic conditions. No significant differences were noted between peritoneal exudate eosinophils from Trichinella-infected and normal animals. Adherence was inhibited by leukotriene inhibitors [eicosaetraynoic acid (10(-3) M), nordihydroguaiaretic acid (10(-4) M), and BW755C (10(-3) M)] but not by prostaglandin inhibitors (aspirin and indomethacin), suggesting that leukotrienes mediate this eosinophil function. This assay provides another useful method for assessment of a crucial eosinophil function.     

Trichinella spiralis : proteinases in the larvae

Under in vitro conditions, muscle larvae of Trichinella spiralis secreted minute amounts of a cysteine proteinase into the outer environment from the stichosome. The proteinase hydrolyzed azocoll at pH 5.0 but not a number of synthetic N-blocked and N-unsubstituted proteinase substrates at this pH. The reducing compound dithioerythritol enhanced the enzyme activity, but the thiol-blocking reagent sodium-p-hydroxymercuribenzoate (0.1 mM) was without effect. Phenylmethylsulfonyl fluoride (PMSF) (2 mM) and leupeptin (100 mM) produced partial and complete inhibition, respectively, whereas soybean trypsin inhibitor, pepstatin A, and 1,10-phenanthroline were non-inhibitory. Calcium (1 mM) produced a slight decrease in the activity that was reversed by 1 mM EGTA. Although multiple proteinase activities were detected histochemically in the somatic muscles, stichosome, midgut, and genital primordium of the muscle larvae, none of these enzymes appeared to be the one secreted. Several histochemically demonstrable proteinases were also found in the cells of 48- to 72-h-old juveniles of the parasite. One was localized in the esophageal lumen and at or around the anterior esophagus of the larvae, where developing stichocytes are believed to occur. The proteinase hydrolyzed N-acetyl-l-methionine-1-naphthyl ester and was sensitive to the metal cation-complexing compound EGTA as well as to PMSF, an inhibitor of serine proteinases. Received: 28 March 1998 / Accepted: 29 June 1998     

两种方法制备的旋毛虫ES抗原对小鼠免疫保护作用研究

旋毛虫病流行于世界各地,是一种危害严重的人兽共患病.旋毛虫的抗原可分为虫体抗原、表面抗原、杆细胞颗粒相关抗原以及排泄分泌抗原(excretory-secretory antigen,简称ES抗原).ES抗原是旋毛虫的代谢分泌产物,与宿主免疫系统直接接触,具有较强的免疫原性[1].ES抗原也可用于人旋毛虫病的诊断[2].     

Helminthotoxic responses of intestinal eosinophils to Trichinella spiralis newborn larvae.

Because the gastrointestinal lamina propria is the first line of defense against invasion with Trichinella spiralis newborn larvae, we investigated the helminthotoxic characteristics of isolated lamina propria eosinophils. Eosinophils were isolated from the intestinal lamina propria of rats and purified to nearly 90% purity by a combination of velocity sedimentation through Percoll and unit gravity sedimentation through a continuous gradient of bovine serum albumin. Isolated eosinophils were of high viability and responded to surface receptor stimulation. Freshly isolated intestinal eosinophils lacked cytotoxic capacity when incubated with newborn larvae in the presence of specific antiserum. Peritoneal eosinophils from the same rats exhibited 100% helminthotoxicity after 24 h. Cytotoxicity could be stimulated in the intestinal eosinophils by the addition of recombinant murine interleukin-5.     

Preotection againstTrichinella spiralis induced by purified stage-specific surface antigens of infective larvae

The stage-specific surface antigens of the infective larvae ofTrichinella spiralis, isolated using an affinity column of monoclonal antibody (Mab) NIM-M1, consisted of four components with molecular weights of 72, 65, 52, and 47 kDa, respectively. These four components may have unique as well as shared structural features and appear to be products of the stichosome. When injected i.p. as an emulsion in complete Freund's adjuvant, the purified antigens induced protection against infection of BALB/c mice withT. spiralis, as assessed by reductions in both the muscle larvae load and the number of adult intestinal worms.     

Partial characterization of two antigens secreted by L1 larvae of Trichinella spiralis

Two protein antigens were isolated from excretory-secretory products of Trichinella spiralis by biochemical methods and characterized with respect to their chemical and immunological properties. One antigen, of apparent Mr 43,000, is an abundant secreted protein of infective L1 larvae, while the other, of 45-50 kDa, is present in smaller amounts. Yields, extinction coefficients, isoelectric points, amino acid compositions, and partial N-terminal amino acid sequences for each are reported. Partial amino acid sequences of peptides derived from the 43-kDa protein by cyanogen bromide cleavage have been determined. Treating a reduced-pyridylethylated derivative of the 43-kDa protein with glycopeptidase F (N-glycanase) resulted in formation of a transient product of 37 kDa followed by a stable polypeptide of 32 kDa (by SDS-PAGE), suggesting the presence of two N-linked carbohydrate groups. A similar result was obtained with the 45-50-kDa protein, which gave a transient doublet of 38 and 40 kDa and a final, stable product of 33 kDa, with a minor component of 35 kDa. Two glycosylation sites of the 43-kDa protein and one site of the 45-50-kDa protein can be identified in the amino acid sequences. Polyclonal antibodies prepared against the two proteins cross-reacted extensively, but failed to react with the doubly deglycosylated polypeptides in Western blots. The dominant epitopes present in the reduced-pyridylethylated polypeptides are, therefore, N-linked carbohydrate, although the presence of peptide epitopes in the native proteins cannot be excluded.     

A putative serine protease among the excretory-secretory glycoproteins of L1 Trichinella spiralis

Trichinella spiralis first-stage larvae infect susceptible hosts by invading epithelial cells that line the small intestine. During this process the larva disgorges several glycoproteins that bear an unusual, highly antigenic sugar moiety, tyvelose (3,6-dideoxy arabinohexose). Monoclonal antibodies specific for tyvelose protect the intestine against infection, implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment in the intestinal epithelium. In order to investigate these glycoproteins at the molecular level, we first prepared monoclonal anti-peptide antibodies. The antibodies bind a family of glycoproteins that are present in excretory-secretory products of first-stage larvae and are delivered to epithelial cells during invasion by T. spiralis. The major species present in an affinity purified fraction of crude T. spiralis antigens were subjected to tryptic peptide digestion. De novo amino acid sequencing of the peptides using Q-TOF tandem mass spectrometry, in combination with database searches and antibody screening of an L1 cDNA library, showed that the glycoproteins are variably glycosylated homologues of the serine protease family.     

Cryopreservation of Dictyocaulus viviparus third-stage larvae and Trichinella spiralis muscle larvae

In cryopreservation studies with third-stage larvae of Dictyocaulus viviparus, best results were achieved by incubating larvae in 0.05% NaOCl at 37 C to remove the sheath, followed by cooling at a rate of 1 C min per min down to about 0 C. After an equilibration time of 10 min at +4 C with or without 4% polyethylene glycol-400 as cryoprotectant, samples were frozen at the same cooling rate to an intermediate temperature of –20 C, maintained at this temperature for 10 min and finally plunged into liquid nitrogen for storage. Three groups of 3 calves were infected with the following batches of third-stage larvae: (a) fresh, sheated; (b) fresh, exsheathed; (c) exsheathed, cryopreserved for 13 weeks in liquid nitrogen and subsequently thawed. Although 62% of group (c) were regarded as viable in vitro, their infectivity to calves was low and only an average of 0.08% of the inoculated larvae (3000 per animal) developed into adult lungworms (=infectivity rate). Average infectivity rates of fresh, sheathed (a) and fresh, exsheathed (b) larvae were much higher (38.3% and 29.7%) and not significantly different from each other. Two of the calves inoculated with previously frozen larvae and all of the calves infected with fresh larvae excreted first-stage larvae in their faeces, but the latter groups in higher quantities. The results show that cryopreservation of exsheathed third-stage larvae of D. viviparus is possible, but for strain maintenance infection doses greater than 3000 larvae should be used for inoculation of calves. Thawed Trichinella spiralis larvae, which had been cryopreserved in tissue blocks (approx. 2 g) with addition of undiluted ethylene glycol (0.2 ml), revealed in vitro viability rates of 60% after storage at –10 C for 2 and 13 weeks and were still infective to rats.Part of the Dissertation of I. Andermatt-Mettler under supervision of J. Eckert at the Faculty of Veterinary Medicine, University of Zürich     

旋毛虫成虫抗原和肌幼虫抗原保护性免疫的比较研究

目的:比较旋毛虫成虫抗原和肌幼虫抗原诱导小鼠产生保护性免疫效果的异同.方法:制备旋毛虫成虫抗原和肌幼虫抗原,分组免疫小鼠,并于末次免疫后10 d用旋毛虫感染性幼虫对各组小鼠实施攻击感染,检测攻击感染后各组小鼠肠道成虫和肌肉幼虫数并计算减虫率,同时采用ELISA法检测各组小鼠血清IgG抗体水平.结果:成虫抗原免疫组小鼠和肌幼虫抗原免疫组小鼠检获的肠道成虫数和肌幼虫数均显著少于对照组(P＜0.05),其中以成虫抗原免疫组小鼠减虫率最高(P＜0.01).成虫抗原免疫组和肌幼虫抗原免疫组小鼠于实施旋毛虫感染性幼虫进行攻击感染的当日所检测的血清IgG抗体水平与其免疫前相比均升高(P＜0.01,P＜0.05),与对照组相比亦升高(P＜0.01,P＜0.05),其中也以成虫抗原免疫组小鼠血清IgG抗体升高最为显著(P＜0.01).结论:旋毛虫成虫抗原和肌幼虫抗原均能激发小鼠产生保护性免疫,其中成虫抗原免疫组小鼠显示出更好的抗感染保护性.     

Identification of diagnostic antigens fromTrichinella spiralis

The Western blotting technique was used to determine the antigens ofTrichinella spiralis muscle larvae that were recognized by antibodies in sera from humans and pigs displayingT. spiralis infections. This resulted in the identification of several antigens that were recognized by all sera. Some of these antigens, notably those that were recognized during the early stage of infection, cross-reacted with antibodies to other parasites. This cross-reactivity was caused by the presence of phosphorylcholine on these antigens. A large portion of the antigens that were recognized by antibodies from infected humans and pigs were found to share a singleTrichinella-specific determinant. TheTrichinella-specific antigen population could be isolated from phosphorylcholine-containing antigens by a simple two-step affinity chromatography procedure using monoclonal antibodies to both determinants. The resulting preparation consisted primarily of a single antigen showing an apparent molecular weight of 45 kDa that corresponded to a mamor constituent of excretory-secretory (ES) products of muscle larvae. When tested in an enzyme-linked immunosorbent assay (ELISA), this antigen displayed diagnostic specificity that was comparable with the ES fraction and diagnostic sensitivity comparable with the crude muscle-larvae extract.     

Stage specific secreted and somatic antigens of Trichinella spiralis

Infective larvae, adult males and newborn larvae of Trichinella spiralis were cultured with [35S]methionine in vitro. Total secreted and total somatic (sodium deoxycholate-soluble) proteins were analyzed by electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Secreted proteins were relatively few in number and were different for each stage, whereas somatic proteins gave an unresolved smear in all cases. Immune precipitation with serum from infected mice revealed only one major antigen in secretions of all stages. In a similar investigation of the solubilised somatic antigens, the complexity of antigens ranged from none in infective larvae, through few in the adult, to many in the newborn larvae. The total concanavalin A-binding glycoproteins of each stage exhibited considerable individuality, and hence stage specificity, when resolved by two dimensional gel analysis. These results extend our knowledge of stage specific components of T. spiralis, and allow a rational approach towards the construction of diagnostic procedures.     

Detection of circulating Trichinella spiralis larvae by polymerase chain reaction

Several investigators have successfully applied the polymerase chain reaction to the amplification of DNA from Trichinella spiralis muscle-stage larvae. We show herein that specific DNA can be amplified from T. spiralis migratory larvae in the blood of experimentally infected mice. The polymerase chain reaction detected the presence of migratory larvae in mouse blood from day 5 to day 14 of infection. The technique may be applied to human trichinosis, but its diagnostic value will depend on the severity and stage of the infection.     

Persistence of Trichinella spiralis muscle larvae in natural decaying mice

The influence of natural weather conditions on the viability and reproductive capability of Trichinella spiralis muscle larvae in mouse corpses exposed to summer and winter conditions in the Buenos Aires Province, Argentina, was studied. For this purpose, a total of 49 mouse corpses harbouring muscle larvae of T. spiralis were exposed for a period of 1, 2, 4 and 6 weeks in each of the seasons. Control corpses maintained at 8°C were also included. In summer, T. spiralis muscle larvae were recovered from corpses exposed up to 1 week only. The viability of these larvae was 54.2%, and the reproductive capability index in mice (RCI) was 13.1 and significantly lower than the control (p<0.0005). Morphologic deterioration and reduction in the glycogen content of cysts and larvae were observed at the second week of exposition. By week 4, larval stages of Dermestes maculatus were observed inside corpses, and 22 live muscle larvae of T. spiralis were obtained by artificial digestion of their bodies. In winter, T. spiralis muscle larvae were always recovered, the viability being almost 100% except for a significant reduction by week 6 of exposition (p<0.0001). For this season, the RCI were 50.5, 46.9, 59.7 and 45.2 for the periods of 1, 2, 4 and 6 weeks of exposition, respectively. The morphology of cysts and larvae did not show alterations, and no variations were observed as well in glycogen reserves during the 6-week period of exposition. RCI of non-exposed muscle larvae were always significantly higher that any of those recorded from muscle larvae that belonged to exposed corpses (p=0.0005). The present results demonstrate that muscle larvae of T. spiralis are able to survive in nature and keep infective for a 1-week period in summer and at least for 6 weeks in winter, becoming an important source of infection for scavengers. In summer, larvae stages of D. maculatus, and probably other insects, may play an important role in the survival and transmission of T. spiralis in the sylvatic cycle.     

Taurine drinking attenuates the burden of intestinal adult worms and muscle larvae in mice with Trichinella spiralis infection

The parasitic nematode Trichinella spiralis can cause trichinellosis, which leads to pathological processes in the intestine and muscle. The intestinal invasion determines the development, subsequent course, and consequences of the disease. Gastrointestinal nematode infection, including with T. spiralis, is accompanied by a rapid and reversible expansion of mucosal mast cell and goblet cell in the intestinal epithelium, which play important roles in the host immune response to parasite and worm expulsion from the intestine. Taurine and its derivatives have anti-infection and anti-inflammatory properties. We investigated whether taurine supplementation in mice could influence the development and pathological processes of infection with T. spiralis. Supplementing 1 % taurine in drinking water in mice infected with T. spiralis could alleviate the burden of intestinal adult worms on days 7 and 10 postinfection (all p?<?0.01) and the formation of infective muscle larvae in striated muscle during T. spiralis infection (p?<?0.01). As compared with T. spiralis infection alone, taurine treatment increased the number of goblet cells on days 7, 10, and 15 (p?<?0.01 and p?<?0.05) and alleviated intestinal mucosal mast cell hyperplasia on days 10 and 15 (all p?<?0.01). So taurine supplementation in drinking water increased infection-induced intestinal goblet cell hyperplasia and ameliorated mucosal mastocytosis. Thus, taurine can ameliorate the pathological processes of trichinellosis and may be of great value for the treatment and prevention of infection with T. spiralis and other gastrointestinal nematodes.     

Trichinella spiralis: the infectivity of synchronous newborn larvae of different ages inoculated intraocularly

Trichinella spiralis infection results in the transformation of muscle cells into a new, non-muscular cell called the nurse cell, and the nurse cell-muscle larva complex is finally created. To investigate whether T. spiralis infectivity is NBL age-dependent, five groups of synchronous newborn larvae (sNBL) were obtained at 1, 9, 24, 48, and 72 h of age and were inoculated into mice by intravenous injection into the retro-orbital venous plexus. When both “young” groups of sNBL (1 and 9 h old) were injected, the highest number of larvae were capable of infecting the muscle cells. The highest infectivity of 80.0% was observed for 9-h-old sNBL. In older sNBL the infectivity gradually decreased; thus, for 72-h-old sNBL the lowest level – 0.1% – was detected. Therefore, an “age limit” for NBL infectivity in the present study was precisely determined. Received: 14 September 1998 / Accepted: 15 October 1998