Trichinella spiralis: light microscope monoclonal antibody localization and immunochemical characterization of phosphorylcholine and other antigens in the muscle larva

A panel of monoclonal antibodies was used to examine the structure of the muscle larva of Trichinella spiralis under the light microscope. Immunofluorescence and, in some cases, immunoperoxidase staining were used. All four antibodies reacted with the cuticle of the organism, although differences in the staining pattern were observed for some of these. Interestingly, all the antibodies also reacted with the stichosome. One of the antibodies (Ts2Ab) is specific for the hapten, phosphorylcholine. In a binding assay, this antibody also reacted with extracts of Trichuris suis, Ascaris suum, and Fasciolopsis buski, but not with extracts derived from Cysticercus cellulosae, Candida albicans, Salmonella typhi, or Escherichia coli. This crossreactivity was confirmed microscopically in which the cuticle, oviduct and eggs of T. suis, the cuticle, muscle cells, and eggs of A. suum, and the cuticle and vitelline glands of F. buski were seen to be clearly stained by the antibody. In addition, Ts2Ab also reacted with the cuticle and stichosome of the adult T. spiralis worm. In Western blot analysis, Ts2Ab recognized a 43-kDa antigen from T. spiralis muscle larvae extracts, while a previously studied antibody (7C2C5Ab) identified four major antigens (48.5, 47, 43, and 39 kDa) in this preparation. Similar results were obtained when the 24-hr excretory-secretory (ES) antigens of T. spiralis were immunoblotted with the antibodies, although the reactivity shown by Ts2Ab was relatively weak. With the 72-hr ES material, on the other hand, major antigens of lower mol wt (44, 28, and 25 kDa) were revealed by 7C2C5Ab, and no reactivity was seen with Ts2Ab. However, this antigen preparation reacted well with both antibodies in an enzyme-linked immunoassay. Taken together, the findings suggest that the 72-hr ES antigens probably result from extensive degradation of material originally secreted or excreted by the worm. Similar binding studies on the 24-hr ES preparation indicated that this source may be relatively rich in 7C2C5Ab-reactive epitopes and relatively poor in the antigen identified by Ts2Ab. Other studies performed demonstrated that the antigens recognized by these two antibodies were distinct and physically unassociated.     

Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis.

Double-stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double-stranded breaks to the native DNA. The ES double-stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective-stage larvae of T. spiralis collected at 37 degrees C and was present in much smaller amounts in samples collected at 43 degrees C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated approximately 25, 30 and 58 kDa, respectively.     

Infectivity, persistence, and antibody response to domestic and sylvatic Trichinella spp. in experimentally infected pigs

Groups of pigs were inoculated with genotypes of Trichinella belonging to: Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (from Caucasus), T. pseudospiralis (from USA), Trichinella murrelli, Trichinella sp. (from North America), and Trichinella nelsoni. The pigs were sacrificed between 5 and 40weeks p.i., and the number of muscle larvae per gram (l.p.g.) of tissue was determined as an average of 18 muscles. All Trichinella genotypes were infective for pigs, but both their infectivity and persistence varied: 5weeks p.i., T. spiralis muscle larvae were present in high numbers (mean=427l.p.g.), while T. britovi, T. nelsoni, and T. pseudospiralis larvae were present in moderate numbers (means=24-52l.p.g.); larvae of the remaining genotypes were recovered only in low numbers (means=0.05-5. 00l.p.g.). The total larval burden (live weight of pigxl.p.g.) was constant over time for T. spiralis, T. britovi, and T. nelsoni, but declined significantly (P<0.05) for the other genotypes. Antibody responses could be detected 3-4weeks p.i. by seven different Trichinella ES antigens, but the antibody levels and dynamics differed significantly among the experimental groups. In pigs inoculated with T. spiralis, T. britovi, or T. nelsoni, the antibody level increased rapidly between weeks 3 and 5 p.i. and was stable or increased slightly throughout the experimental period. In pigs inoculated with T. nativa, T. murrelli, or Trichinella (T6) (from North America), a rapid increase was detected between weeks 3 and 5 p.i., but for these genotypes a reduction in the antibody levels was seen thereafter. In the pigs inoculated with T. pseudospiralis, the antibody level increased more gradually over a period from week 3 p. i. to weeks 15-20 p.i., and decreased thereafter. In general, all species of Trichinella were detected by any of the seven ES antigens, which points to the potential use of one common antigen for surveillance and epidemiological studies on both domestic and sylvatic Trichinella in pigs. Homologous ES antigens were slightly more sensitive in detecting antibodies to the corresponding Trichinella species.     

Translation products of mRNA from infective larvae of Trichinella spiralis include an antigenic polypeptide

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5.7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into the trichloroacetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100,000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48,000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG antibodies in positive serum. Especially the polypeptide with a molecular weight of 48,000 in ES products strongly reacted with IgG antibody in positive serum.     

Oxidative,heat and anthelminthic stress responses in four species of Trichinella: comparative study

The aim of this study was to compare levels of stress proteins in four Trichinella species when exposed to different stressors. Heat shock protein (HSP) 60, 70 and 90 responses were evaluated in infective larvae (L(1)) of four classic Trichinella species following exposure to oxidative, anthelminthic and thermal stress. Larvae of T. nativa, T nelsoni, T. pseudospiralis and T. spiralis were exposed to peroxide shock (0.2%, 1%, or 2% H(2)O(2)for 2h), high temperatures (40 degrees C or 45 degrees C for 2h), or 0.1 microg/ml of the benzimidazole anthelminthics: mebendazole (MBZ), albendazole (ALB) or thiabendazole (TBZ) for 4h. Following exposures, the L(1) were tested for induced morphological changes. Those observed were: (i) no change (in all species exposed to 40 degrees C) (ii) aberrant forms (in all species exposed to anthelminthics, in T. nativa, T. nelsoni and T. spiralis exposed to 45 degrees C, and in T. spiralis and T. nelsoni exposed to 0.2% H(2)O(2)) and (iii) severe degradation or death (in T. nativa and T. pseudospiralis exposed to 0.2% H(2)O(2), and in all species at 1% and 2% H(2)O(2)). In Western blot analyses, L(1) proteins were probed with monoclonal antibodies (mAbs) specific for the three HSPs. Greater changes in HSP levels occurred following H(2)O(2) exposure than with other stresses in all Trichinella species, while accumulation of a 50 kDa HSP was only observed in T. spiralis and T. pseudospiralis. Anthelminthic stress only caused decreased HSP levels in T. nativa. Thermal stress caused no significant changes in the HSP response of any species. It is suggested that other stress proteins (e.g., glucose-regulated proteins) may be involved in adaptation to thermal stress.     

Stimulated chemotactic response in neutrophils from Trichinella pseudospiralis-infected mice and the neutrophilotactic potential of Trichinella extracts.

During infection with Trichinella pseudospiralis a strong neutrophil response is evident in the peripheral circulation of the mouse. This study compared the chemotactic response of neutrophils from uninfected, T. pseudospiralis-infected and Trichinella spiralis-infected mice to extracts from adult worms, newborn larvae and muscle-stage larvae of both species of parasite. The chemotactic response of neutrophils from T. pseudospiralis-infected mice to Zymosan-activated mouse serum (ZAMS) was significantly greater than that seen with neutrophils from either uninfected or T. spiralis-infected mice. Unstimulated chemotactic response of neutrophils from these three groups of animals to medium alone was similar. The chemotactic response of neutrophils from the three groups of animals was unaffected by either the concentration or source of serum. The chemotactic response of neutrophils from T. pseudospiralis-infected mice was significantly greater than that observed with cells from uninfected or T. spiralis-infected mice. Among parasite extracts, those from newborn larvae displayed the strongest chemotactic potential for neutrophils. Extracts from muscle larvae of T. spiralis and T. pseudospiralis and extracts of T. spiralis adult worms showed the weakest attraction for neutrophils. Extracts from adult T. pseudospiralis and from newborn larvae of both species elevated the chemotactic response of uninfected mouse neutrophils to a significantly greater level than that seen with ZAMS alone, while a significant reduction in this response was evident only when ZAMS was presented to neutrophils with 500 micrograms of extract from muscle larvae of T. pseudospiralis or T. spiralis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mechanism for anti-asialo GM 1 antibody-induced anaphylactoid response in mice infected with Trichinella pseudospiralis

Intravenous injection of anti-asialo GM 1 antibody into mice infected with Trichinella pseudospiralis resulted in rapid acute illness or death accompanied by a dramatic rise in hematocrit values in these animals. The described antibody-induced changes were reversible by intravenous infusion of Hanks' balanced salt solution (HBSS). These effects were not seen in uninfected mice or in Trichinella spiralis-infected mice injected with anti-asialo GM 1 antibody. Viability of T. spiralis or T. pseudospiralis infective L1 larvae, both isolated worms and those housed in muscle, was unaffected by exposure to anti-asialo GM 1 antibody and complement. Infectivity of larvae of T. pseudospiralis decreased significantly following exposure to anti-asialo GM 1 antibody. Release of protein by T. pseudospiralis infective L1 larvae during incubation in the presence of anti-asialo GM 1 antibody was significantly greater than that released by worms incubated in normal rabbit serum or HBSS. Protein released by infective L1 larvae of T. pseudospiralis was identified as Trichinella excretory/secretory antigens by immunoblot. Intravenous injection of T. pseudospiralis excretory/secretory products resulted in anaphylaxis in T. pseudospiralis-infected mice but not in uninfected or T. spiralis-infected mice. Excretory/secretory product-induced anaphylactoid response also was reversible by the intravenous injection of HBSS or by injection of an antihistamine. Significantly higher levels of total IgE were observed in sera from mice infected with T. pseudospiralis compared to uninfected or T. spiralis-infected mice. Binding of anti-asialo GM 1 antibody to the surface of T. pseudospiralis muscle larvae induced release of excretory/secretory antigen by the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichinella spiralis: recognition of muscle larva antigens during experimental infection of swine and its potential use in diagnosis

Longitudinal studies with Trichinella spiralis experimentally infected pigs were carried out to identify muscle larva antigens recognized during infection. This was approached using Western blot analysis and ELISA assays. Immunoblots of sera from experimentally infected pigs using total parasite extracts revealed five principal parasite antigens throughout infection. A similar pattern of antigen recognition was given by sera from backyard pigs in areas of Mexico, some of them endemic for Trichinella. Four of the five antigens recognized (MW 47, 52, 67, and 72 kDa) corresponded to surface/stichosomal antigens purified by monoclonal antibody NIM-M1. In addition, Western blots of excretions-secretions of muscle larva contained three (MW 52, 67, and 72 kDa) of the four surface/stichosomal components recognized by NIM-M1. Affinity-purified surface/stichosomal components, total soluble extracts, and excretory-secretory antigens of muscle larva were then evaluated in ELISA for detection of T. spiralis infections in experimentally infected, noninfected control, and 295 backyard pigs. These assays showed that purified surface/stichosomal components and excretory-secretory antigens increased the specificity of ELISA. These results suggest that muscle larva components purified by monoclonal antibody NIM-M1 are the major antigens recognized during infection of pigs with T. spiralis and therefore potentially useful for diagnosis of swine trichinellosis.     

Sylvatic and domestic Trichinella spp. in wild boars; infectivity, muscle larvae distribution, and antibody response

Thirty-six wild boars were inoculated with Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella pseudospiralis (USSR), T. pseudospiralis (USA), T. pseudospiralis (AUST), Trichinella murrelli, Trichinella T6, and Trichinella nelsoni. The wild boars were killed at 5 and 10 wk postinoculation (PI), and the number of muscle larvae per g (lpg) of tissue was determined for 18 muscles or muscle groups. Five weeks PI, all Trichinella genotypes had established as muscle larvae, but their infectivity varied widely: T. spiralis established in high numbers (mean = 296 lpg), T. britovi, T. nelsoni, and 1 of the T. pseudospiralis genotypes (AUST) in moderate numbers (mean = 53-74 lpg), whereas the remaining genotypes were poorly infective (mean 2-16 lpg). Because of considerable weight gain of the wild boars, an estimated total larval burden (live weight x lpg) was calculated for each animal. The total larval burden did not change significantly over time for T. spiralis, T. murrelli, T. britovi, T. nelsoni, and T. pseudospiralis (USA and USSR), whereas a significant reduction could be demonstrated for T. nativa, Trichinella T6, and T. pseudospiralis (AUST). Diaphragm and tongue were predilection sites in wild boars, independent of Trichinella genotype and infection level. At low infection levels, a greater percentage of larvae were found in diaphragm and tongue at 10 wk than 5 wk PI. Antibody responses increased rapidly between weeks 3 and 5 PI. For T. spiralis and T. nelsoni, the high antibody level persisted throughout the experimental period, but for T. nativa, T. britovi, T. murrelli, or Trichinella T6, the levels declined. For T. pseudospiralis, the antibody response increased more gradually between weeks 3 to 10 PI. Infection with all genotypes of Trichinella were detected using any of 7 excretory-secretory antigens, which points to the potential use of 1 common antigen for epidemiological studies on Trichinella in wild boars. In conclusion, T. spiralis is highly infective to wild boars, T. britovi, T. nelsoni, T. pseudospiralis (USA), and T. pseudospiralis (USSR) are moderately infective, and T. nativa, T. murrelli, T. pseudospiralis (AUST), and Trichinella T6 are poorly adapted to this host species.     

Comparative analysis of the excretory-secretory proteome of the muscle larva of Trichinella pseudospiralis and Trichinella spiralis

The nematodes Trichinella spiralis and Trichinella pseudospiralis are both intracellular parasites of skeletal muscle cells and induce profound alterations in the host cell resulting in a re-alignment of muscle-specific gene expression. While T. spiralis induces the production of a collagen capsule surrounding the host-parasite complex, T. pseudospiralis exists in a non-encapsulated form and is also characterised by suppression of the host inflammatory response in the muscle. These observed differences between the two species are thought to be due to variation in the proteins excreted or secreted (ES proteins) by the muscle larva. In this study, we use a global proteomics approach to compare the ES protein profiles from both species and to identify individual T. pseudospiralis proteins that complement earlier studies with T. spiralis. Following two-dimensional gel electrophoresis, tandem mass spectrometry was used to identify the peptide spots. In many cases identification was aided by the determination of partial peptide sequence from selected mass ions. The T. pseudospiralis spots identified included the major secreted glycoproteins and the secreted 5'-nucleotidase. Furthermore, two major groups of T. spiralis-specific proteins and several T. pseudospiralis-specific proteins were identified. Our results demonstrate the value of proteomics as a tool for the identification of ES proteins that are differentially expressed between Trichinella species and as an aid to identifying key parasite proteins that are involved in the host-parasite interaction. The value of this approach will be further enhanced by data arising out the current T. spiralis genome sequencing project.     

Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards

Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus.     

Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC-MS/MS approach

Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24 kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24 kDa) were studied employing shotgun LC-MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10-70 kDa, and 26 proteins (40.6%) were in the range of pI 5-6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis.     

Alterations in the longevity and fecundity of adult Trichinella pseudospiralis related to method of isolation of infective larvae

The infectivity of Trichinella pseudospiralis infective larvae was reduced significantly following exposure to low pH or a combination of 1% pepsin at low pH compared to that for larvae isolated in phosphate-buffered saline (PBS) at pH 7.0. Reduction of host gastric pH by administration to mice of sodium bicarbonate solution in PBS was accompanied by an increase in the infectivity of larvae isolated in 1% pepsin/HCl (P/HCl) compared to that for worms inoculated into hosts given PBS alone. Fewer adult worms developing from larvae isolated in P/HCl became established in the host small bowel than was seen with larvae isolated in PBS; moreover, the fecundity in vitro of adult worms developing from P/HCl-isolated larvae was reduced below that for adults developing from larvae isolated from host muscle in PBS. More adult worms were recovered following infection of immune hosts with PBS-isolated larvae than were recovered from immune mice challenged with larvae isolated in P/HCl. Similar findings were observed in mice immunized by infection with Trichinella spiralis and challenged with T. pseudospiralis larvae isolated in either P/HCl or PBS. Immunization of mice with T. pseudospiralis larvae isolated by either method and challenged with larvae of T. spiralis resulted in recovery of similar percentages of the challenge inoculum.     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。     

Trichinella spiralis: influence of an immunodominant, carbohydrate-associated determinant on the host antibody response repertoire.

Host antibody responses to the G2.1 epitope, a carbohydrate-associated determinant shared by several Trichinella spiralis glycoproteins, were examined by competitive inhibition enzyme-linked immunosorbent assay (ELISA). The G2.1 epitope dominated the AKR/J mouse antibody response whether the antigens were injected or introduced through infection, as determined by the serum blocking ability of a G2.1 epitope-specific monoclonal antibody (mAb). Serum T. spiralis-binding activity from several other infected mouse strains was blocked 22 to 86% by the G2.1 epitope-specific mAb. In addition to mice, the G2.1 epitope evoked powerful antibody responses in four other species. The binding activity of Trichinella-reactive antibodies from infected rats and pigs was inhibited 56 and 34%, respectively, by the mAb. Greater than 48% of the T. spiralis serum-binding activity from 4/5 infected humans was G2.1-specific. Most of the rabbit antibody response induced by injection of a previously characterized 43-kDa antigen was also directed to the G2.1 determinant. The specificities of 10 T. spiralis-reactive mAb were examined, and 7 reacted with the immunodominant epitope. Finally, of nine helminth species examined, only T. spiralis and T. pseudospiralis extracts efficiently blocked G2.1-specific antibody binding to solid-phase antigens. These results suggest that the responses to G2.1 epitope may play an important role during infection.