Quantitative label-free proteomic analysis of excretory-secretory proteins in different developmental stages of Trichinella spiralis.

Trichinella spiralis (T. spiralis) is a zoonotic parasitic nematode with a unique life cycle, as all developmental stages are contained within a single host. Excretory-secretory (ES) proteins are the main targets of the interactions between T. spiralis and the host at different stages of development and are essential for parasite survival. However, the ES protein profiles of T. spiralis at different developmental stages have not been characterized. The proteomes of ES proteins from different developmental stages, namely, muscle larvae (ML), intestinal infective larvae (IIL), preadult (PA) 6 h, PA 30 h, adult (Ad) 3 days post-infection (dpi) and Ad 6 dpi, were characterized via label-free mass spectrometry analysis in combination with bioinformatics. A total of 1217 proteins were identified from 9341 unique peptides in all developmental stages, 590 of which were quantified and differentially expressed. GO classification and KEGG pathway analysis revealed that these proteins were important for the growth of the larvae and involved in energy metabolism. Moreover, the heat shock cognate 71 kDa protein was the centre of protein interactions at different developmental stages. The results of this study provide comprehensive proteomic data on ES proteins and reveal that these ES proteins were differentially expressed at different developmental stages. Differential proteins are associated with parasite survival and the host immune response and may be potential early diagnostic antigen or antiparasitic vaccine candidates.

Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella.

Genome assemblies provide a powerful basis of comparative multi-omics analyses that offer insight into parasite pathogenicity, host-parasite interactions, and invasion biology. As a unique intracellular nematode, Trichinella consists of two clades, encapsulated and non-encapsulated. Genomic correlation of the distinct differences between the two clades is still unclear. Here, we report an annotated draft reference genome of non-encapsulated Trichinella, T. pseudospiralis, and perform comparative multi-omics analyses with encapsulated T. spiralis. Genome and methylome analyses indicate that, during Trichinella evolution, the two clades of Trichinella exhibit differential expansion and methylation of parasitism-related multi-copy gene families, especially for the DNase II members of the phospholipase D superfamily and Glutathione S-transferases. Further, methylome and transcriptome analyses revealed divergent key excretory/secretory (E/S) genes between the two clades. Among these key E/S genes, TP12446 is significantly more expressed across three life stages in T. pseudospiralis. Overexpression of TP12446 in the mouse C2C12 skeletal muscle cell line could induce inhibition of myotube formation and differentiation, further indicating its key role in parasitism of T. pseudospiralis. This multi-omics study provides a foundation for further elucidation of the mechanism of nurse cell formation and immunoevasion, as well as the identification of pharmacological and diagnostic targets of trichinellosis.

Exploiting the potential of 2D DIGE and 2DE immunoblotting for comparative analysis of crude extract of Trichinella britovi and Trichinella spiralis muscle larvae proteomes.

The Trichinella genus poses an interesting puzzle for researchers, having diverged very early in the evolution of the nematodes. The Trichinella spiralis proteome is a cosmopolitan and well-studied model of Trichinella; however, Trichinella britovi also circulates in the sylvatic environment and both species infect humans, resulting in the development of trichinellosis. Few experiments have examined the proteins belonging to the T. britovi proteome. The aim of the present study was to compare the protein expression profiles of crude extracts of T. spiralis and T. britovi muscle larvae using a highly-sensitive two-dimensional differential in-gel electrophoresis (2D DIGE) technique coupled with 2DE immunoblotting. Selected immunoreactive protein spots were then identified by liquid chromatography coupled with mass spectrometry analysis (LC-MS/MS), and their function in Trichinella and the host-parasite interaction was determined by gene ontology analysis. Spots common to both T. spiralis and T. britovi, spots with different expressions between the two and spots specific to each species were labelled with different cyanine dyes. In total, 196 protein spots were found in both proteomes; of these 165 were common, 23 expressed exclusively in T. spiralis and 8 in T. britovi. A comparative analysis of volume ratio values with Melanie software showed that among the common spots, nine demonstrated higher expression in T. spiralis, and 17 in T. britovi. LC-MS/MS analysis of 11 selected spots identified 41 proteins with potential antigenic characteristics: 26 were specific for T. spiralis, six for T. britovi, and eight were found in both proteomes. Gene Ontology analysis showed that the identified T. spiralis proteins possess hydrolytic endopeptidase, endonuclease and transferase activities. Similarly, most of the T. britovi proteins possess catalytic activities, such as lyase, hydrolase, isomerase and peptidase activity. The applied 2D DIGE technique visualized Trichinella spp. protein spots with different molecular weights or isoelectric point values, as well as those with different expression levels. The identified immunoreactive proteins participate in multiple processes associated with host muscle cell invasion and larval adaptation to the host environment. Their reactivity with the host immune system makes them possible candidates for the development of a novel trichinellosis diagnostic test or vaccine against helminthiasis caused by T. spiralis or T. britovi.

The retention and concentration of glycogen in Trichinella nativа in the winter-spring period.

The metabolism of Trichinella spp. is primarily anoxybiotic in nature. Their main energy source is glycogen, which is stored in the stichocites at the muscular stage of the larval development. When subject to tow temperatures the Trichinella larvae consume glycogen and neutral fats to provide for basal metabolism until the energy supplies reach the critical level. The present study establishes the glycogen concentration as well the invasive activity of T. nativа when affected by low temperatures in natural conditions. The carcasses of infected laboratory rats were placed in containers beneath the snow cover, in the natural conditions of a game husbandry in Central Russia. The viability, invasive capacity and the glycogen level were monitored in the Trichinella larvae monthly. The invasive capacity of Trichinella larvae was established based on the presence of the larvae in the muscular tissue of laboratory mice after the peroral administration of the helminth larvae. On the 45 day of the experiment, the mice were euthanized by cervical dislocation, and if the Trichinella larvae could be discovered in the muscular tissue with the help of the trichinelloscopic compression method, the invasive capacity of the Trichinella larvae was viewed as positive. To establish the quantitative value of glycogen content in Trichinella larvae a modified method was used. In order to measure the glycogen level in the T. nativa larvae isolated by fermentation larvae were counted in one drop of the suspended sedimentation in the Migacheva-Kotelnikov chamber. To establish the quantitative value of glycogen content in Trichinella larvae a method based on the treatment of glycogen with iodine, optical density measurement with a refractometer MКMФ-02 was used. For the purpose of measuring the concentration of glycogen in Trichinella larvae in the suspended sedimentation a calibration curve was used. The studies showed that the viability indicator of the Trichinella larvae which had been preserved in natural conditions in the four months of the winter-spring period, in the muscular tissue of laboratory rats remained high (over 90 %). The glycogen concentration in one helminth larva was 0.041 µg in January, 0.033 µg in February, 0.015 µg in April. The invasive capability of the preserved Trichinella larvae was considerably reduced to 33.3 %. In the winter period, under temperatures below 0 °C, a decrease in the glycogen concentration in the Trichinella larvae was observed.

Lipid profile of Trichinella papuae muscle-stage larvae.

Outbreaks of trichinellosis caused by Trichinella papuae have been reported in South-East Asia. Mebendazole and thiabendazole are the treatments of choice for trichinellosis; however, both drugs result in significant side effects and are less effective for muscle-stage larvae (L1). An alternative therapeutic agent is needed to improve treatment. Information on lipid composition and metabolic pathways may bridge gaps in our knowledge and lead to new antiparasitics. The T. papuae L1 lipidome was analysed using a mass spectrometry-based approach, and 403 lipid components were identified. Eight lipid classes were found and glycerophospholipids were dominant, corresponding to 63% of total lipids, of which the glycerolipid DG (20:1[11Z]/22:4[7Z,10Z,13Z,16Z]/0:0) (iso2) was the most abundant. Overall, 57% of T. papuae lipids were absent in humans; therefore, lipid metabolism may be dissimilar in the two species. Proteins involved T. papuae lipid metabolism were explored using bioinformatics. We found that 4-hydroxybutyrate coenzyme A transferase, uncharacterized protein (A0A0V1MCB5) and ML-domain-containing protein are not present in humans. T. papuae glycerophospholipid metabolic and phosphatidylinositol dephosphorylation processes contain several proteins that are dissimilar to those in humans. These findings provide insights into T. papuae lipid composition and metabolism, which may facilitate the development of novel trichinellosis treatments.

The Immunological Properties of Recombinant Multi-Cystatin-Like Domain Protein From Trichinella Britovi Produced in Yeast.

Trichinellosis is a globally-distributed zoonotic parasitic disease caused by nematode worms of the genus Trichinella. One of the most common species of Trichinella known to affect human health is T. britovi; however, it is relatively poorly investigated. A thorough knowledge of the proteins expressed by Trichinella is important when developing immunological detection methods and vaccines and studying its interactions with the host. The present study uses the Pichia pastoris expression system to produce a soluble TbCLP antigen which induces strong antibody responses in the host during natural infection. Our results demonstrate the feasibility of TbCLP antigen production in yeasts, which are able to carry out post-translational modifications such as glycosylation and disulfide bond formation; they also indicate that the glycosylated TbCLP antigen had immunogenic effects in the tested mice and induced a mixed Th1/Th2 response, and was associated with a reduced larval burden after challenge with T. britovi. Subsequent in vitro stimulation of mice splenocytes revealed that TbCLP most likely possesses immunomodulatory properties and may play a significant role in the early phase of infection, affecting host immunological responses.

Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae.

BACKGROUND: The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES) proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. RESULTS: In this study, we used two-dimensional gel electrophoresis (2-DE)-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS) to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10, poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially deoxyribonuclease II (DNase II) activity and serine-type endopeptidase activity. CONCLUSIONS: Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis infection and suppress the host immune response. Further, they are the most likely antigen for early diagnosis and the development of a vaccine against the parasite.

Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues.

Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

Wnt signaling in regulation of biological functions of the nurse cell harboring Trichinella spp.

BACKGROUND: The nurse cell (NC) constitutes in mammalian skeletal muscles a confined intracellular niche to support the metabolic needs of muscle larvae of Trichinella spp. encapsulating species. The main biological functions of NC were identified as hypermitogenic growth arrest and pro-inflammatory phenotype, both inferred to depend on AP-1 (activator protein 1) transcription factor. Since those functions, as well as AP-1 activity, are known to be regulated among other pathways, also by Wnt (Wingless-Type of Mouse Mammary Tumor Virus Integration Site) signaling, transcription profiling of molecules participating in Wnt signaling cascades in NC, was performed. METHODS: Wnt signaling-involved gene expression level was measured by quantitative RT-PCR approach with the use of Qiagen RT(2) Profiler PCR Arrays and complemented by that obtained by searching microarray data sets characterizing NC transcriptome. RESULTS: The genes involved in inhibition of canonical Wnt/ß-catenin signaling cascade as well as leading to ß-catenin degradation were found expressed in NC at high level, indicating inhibition of this cascade activity. High expression in NC of genes transmitting the signal of Wnt non-canonical signaling cascades leading to activation of AP-1 transcription factor, points to predominant role of non-canonical Wnt signaling in a long term maintenance of NC biological functions. CONCLUSIONS: Canonical Wnt/ß-catenin signaling cascade is postulated to play a role at the early stages of NC formation when muscle regeneration process is triggered. Following mis-differentiation of infected myofiber and setting of NC functional specificity, are inferred to be controlled among other pathways, by Wnt non-canonical signaling cascades.

First report of Trichinella pseudospiralis in Poland, in red foxes (Vulpes vulpes).

Nematode worms of the genus Trichinella are one of the most widespread zoonotic pathogens. Natural transmission between hosts can only occur through the ingestion of infected meat. To date, two Trichinella species are known to be etiological agents of disease among domestic animals and wildlife in Poland: T. spiralis and T. britovi. In the last decades, since the administration of an oral vaccination against rabies, the red fox population in Poland has increased exponentially. The study area covers the Nowy Targ region: a mountainous area (585-1138 m above the sea) in southern Poland. Of 24 red foxes examined in the study, four were infected with Trichinella isolates: three were identified as T. britovi and one as T. pseudospiralis. The muscle of red foxes infected with T. britovi harboured 2.75, 3.11, 4.4 LPG and with T. pseudospiralis 0.36 LPG. Trichinella larvae were identified at species level by genomic and mitochondrial multiplex PCR, the products of which were sequenced for comparison with other sequences available in GenBank. The sequences obtained from the Polish T. pseudospiralis isolate, deposited in GenBank under the accession numbers JQ809660.1 and JQ809661.1, matched sequences already published in GenBank. Sequence comparison showed a 100% match with the large subunit ribosomal RNA gene of T. pseudospiralis isolate ISS 013, and a 96-95% match with those of T. pseudospiralis isolates ISS 141 and ISS 470. This is the first report of the identification of T. pseudospiralis larvae from red fox in Poland.

Discernible but limited introgression has occurred where Trichinella nativa and the T6 genotype occur in sympatry.

The genetic diversity within and among parasite populations provides clues to their evolutionary history. Here, we sought to determine whether mitochondrial and microsatellite DNA variation could be used to evaluate the extent of differentiation, gene flow and historical reproductive isolation among the freeze resistant parasites Trichinella nativa and the Trichinella T6 genotype infecting wolverines (Gulo gulo) in Nunavut, Canada. To this end, we genotyped Trichinella isolates derived from the diaphragms of 39 wolverines from this locale to reference strains of T. nativa and the Trichinella T6 genotype. Results showed that among a subset of 13 isolates examined, individuals resembled T. nativa in their mitochondrial DNA, but resembled the Trichinella T6 genotype when assayed at expansion segment V and the internal transcribed spacer of the nuclear rDNA. To adjudicate among these conflicting diagnoses, we further characterized each isolate at several nuclear microsatellite loci and again compared these to data from reference strains. Statistical assignment established that the nuclear genomes of most Nunavut isolates corresponded to those of the Trichinella T6 genotype; however, two isolates corresponded to T. nativa, and one isolate exhibited equal similarity to both reference strains. Taken as a whole, the evidence suggests that these isolates derive from the T. nativa matrilineage, but that their nuclear genomes resemble individuals previously designated as Trichinella T6. Assuming distinct lineages, this argues for cross-hybridization among these genotypes. Although introgression has occurred, recognizable genetic distinctions persist. One possibility is that selection disfavors the survival of hybrid offspring in most instances. Alternatively, the recent disappearance of glacial barriers might have increased contact, and therefore introgression. Broader geographic sampling will be required to determine the extent to which hybridization occurs beyond this particular geographic focus.

Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp.

A cDNA library was constructed from Trichinella pseudospiralis muscle larvae. One cDNA clone, designated Tp4, contained a cDNA transcript of 783 bp in length, with a single open reading frame that encoded 153 amino acids (16,793 Da as the estimated molecular mass). The predicted amino acid sequence of Tp4 showed that the clone had a calponin homology domain and was approximately 50% identical to the transgelin-like proteins (calponin-family members) present in Bombyx mori or Tribolium castaneum. A homologue of the Tp4 clone was also present in cDNA from Trichinella spiralis, and this clone was designated Ts4. A comparison of the amino acid sequence of the transgelin-like proteins from T. spiralis (Ts4 protein) with the Tp4 protein indicated that the two proteins are very similar (about 94% homology). Real time quantitative polymerase chain reaction results showed that the transcription level of the Tp4 and Ts4 genes was highest in newborn larvae. On Western blot, the recombinant Tp4 and Ts4 proteins migrated at 20 kDa when reacted to an antibody against the recombinant Tp4 and Ts4 proteins, respectively. An antibody against the recombinant Tp4 and Ts4 proteins strongly stained two bands migrating at approximately 9 and 8 kDa in the crude extracts from adult worms and newborn larvae, but only weakly stained proteins in muscle larvae. However, an immunocytochemical study showed that the Tp4 protein was present within the muscle of the muscle larvae of T. pseudospiralis. The antibody level against the recombinant Tp4 antigens in infected mice began to increase from 8 days post-infection, was highest in 13 days post-infection, and then slowly decreased.

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells.

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.

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.

Molecular cloning and characterization of an Rcd1-like protein in excretory-secretory products of Trichinella pseudospiralis.

A cDNA library was constructed from muscle larvae of Trichinella pseudospiralis. A cDNA clone, designated as Tp8 contained a cDNA transcript of 1326 bp length with a single open reading frame, which encoded 303 amino acid residues (34,187 Da, estimated molecular mass). The predicted amino acid sequence of the clone had an identity of approximately 60% to the Rcd1 (Required cell differentiation 1) -like proteins among a wide range of organisms. Real-time quantitative polymerase chain reaction results showed that the transcription level of Tp8 gene reached the highest value in adult worms, and that the transcription level in muscle larvae before stichosome formation was higher than in muscle larvae after stichosome formation. The recombinant Tp8 protein migrated at 37 kDa and reacted to antibody against T. pseudospiralis excretory-secretory (E-S) products and sera from mice infected with T. pseudospiralis. An antibody against the Tp8 recombinant protein could stain proteins migrating at approximately 34 kDa (which is the expected size from the sequence) on Western blotting of E-S products from muscle larvae. An immunocytochemical study showed that the Tp8 protein was present within the stichocyte of muscle larvae and adults worms.

TSL-1 antigens of Trichinella: an overview of their potential role in parasite invasion, survival and serodiagnosis of trichinellosis.

The majority of studies on the immunobiology of Trichinella species have centred on the larval muscular phase (L1) with a view to identifying immunodominant antigens located on the surface of the cuticle and in the larval secretions; the nucleus of the parasite-host interaction. These antigens have been classified as eight groups (TSL-1-TSL-8), of which those belonging to the group TSL-1 have been most intensely studied. The principal constituents are glycoproteins, glycan carriers that contain a unusual sugar, the tyvelose (3,6-dideoxy-d-arabinohexose). Studies aimed at improving serodiagnostic techniques to detect trichinellosis indicate that these antigens are ideal candidates. They are capable of inducing a strong humoral response involving the generation of specific antibodies against beta-tyvelose, a sugar that seems to be exclusive to the Trichuroidea. Furthermore, these glycoproteins appear to fulfil an important function in the development and maintenance of the parasite in the muscular niche, and they appear to be fundamental for the invasion of the intestinal epithelium. It has also been demonstrated that specific monoclonal antibodies against tyvelose can mediate a degree of immunoprotection in the rat through the phenomenon known as rapid expulsion.

Effects of raw biles and their non-protein fractions from fox, pig, sheep and chicken on the survival of Trichinella spp. in vitro.

The in vitro differential effect of fox, pig, sheep and chicken bile and corresponding non-protein fractions at various concentrations on the motility of released muscle larvae of Trichinella spiralis, Trichinella nativa and Trichinella nelsoni was examined. In many cases, the percentages of motile (live) larvae of the three Trichinella species cultured in the presence of the non-protein fractions of bile from the study animals were significantly higher (p<0.001) compared to their respective control cultures. In addition, the percentages of motile (live) larvae of all Trichinella species cultured in the presence of the non-protein fraction of bile at every concentration from all study animals were significantly higher (p<0.001) compared to their respective cultures in the presence of raw bile. Not only did results with non-protein fractions of bile differ from those with raw bile, but also the non-protein fraction, with increased dilution, showed a decrease in the percentages of motile larvae while the opposite was true with the raw bile (p<0.001). These observations indicate that the non-protein fraction of bile prolongs the in vitro survival of larvae.

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock.

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.

Expression of excretory and secretory protein genes of Trichinella at muscle stage differs before and after cyst formation.

By adapting a semi-quantitative reverse transcriptase-PCR (RT-PCR) method, we investigated kinetics of gene expression at different developmental stages of Trichinella spiralis and T. pseudospiralis. The analyzed genes included four kinds of excretory and secretory (ES) proteins, a heat shock protein (HSP) and a DNA binding protein and showed that T. spiralis and T. pseudospiralis expressed ES proteins in a stage-specific manner. The gene encoding a 43 kDa ES protein was expressed by muscle larvae, either pre-cyst or post-cyst larvae. The genes encoding: the 53 kDa ES protein of T. spiralis; 53 kDa ES protein of T. pseudospiralis; and 19.6 kDa ES protein of T. spiralis were expressed by post-cyst larvae and adult worms, but not expressed by pre-cyst larvae or newborn larvae. The results showed that pre-cyst larvae and post-cyst larvae are similar but different in the expression of 53 and 19.6 kDa ES proteins. On the other hand, genes of housekeeping proteins, such as HSP and the DNA binding protein, were expressed at all stages although there were some differences in the expression level.

Molecular cloning and characterization of a novel protein of Trichinella pseudospiralis excretory-secretory products.

A novel excretory-secretory (ES) protein of Trichinella pseudospiralis was produced. A cDNA library was constructed from mRNA of muscle larvae at 30 days post infection (p.i.) and immunoscreened with the antibody against ES products. A clone, designated Tp22-3, contained a cDNA transcript of 815 bp in length with a single open reading frame which encoded 244-amino acids (28407 Da in the estimated molecular mass). A database search revealed that no sequences had a homology to this predicted protein. The recombinant protein was produced in an Escherichia coli expression system. Stage specific expression of this protein was suggested from the following experiments. An antibody against the recombinant protein could stain proteins migrating at about 28 kDa (which is the expected size from the sequence) on Western blotting of crude extracts or ES products from 30 days p.i. muscle larvae, but failed to stain any proteins in crude extracts from newborn larvae or 15 days p.i. muscle larvae. The antibody reacted to the stichocytes of larvae at 30 days p.i., but did not react to 15 days p.i. muscle larvae. The production of an mRNA transcript for Tp22-3 gene was restricted largely to the 30 days p.i. muscle larvae and adult worms.