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.

New method to disaggregate and analyze single isolated helminthes cells using flow cytometry: proof of concept.

In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

Guidelines for the detection of Trichinella larvae at the slaughterhouse in a quality assurance system.

SUMMARY: The European Community Regulation (EC) No. 2075/2005 lays down specific rules on official controls for the detection of Trichinella in fresh meat for human consumption, recommending the pooled-sample digestion method as the reference method. The aim of this document is to provide specific guidance to implement an appropriate Trichinella digestion method by a laboratory accredited according to the ISO/IEC 17025:2005 international standard, and performing microbiological testing following the EA-04/10:2002 international guideline. Technical requirements for the correct implementation of the method, such as the personnel competence, specific equipments and reagents, validation of the method, reference materials, sampling, quality assurance of results and quality control of performance are provided, pointing out the critical control points for the correct implementation of the digestion method.

Guidelines for the identification and development of sampling methods and design of suitable protocols for monitoring of Trichinella infection in indicator species.

SUMMARY: According to the European Community Regulation No. 2075/2005, a risk-based program should be implemented in those areas where wildlife and pig holdings applying for Trichinella-free status coexist, or for region where the risk of Trichinella in domestic swine is officially recognised as negligible. Trichinella nematode species are primarily parasites of wildlife which can transmit the infection to pigs intended for human consumption, acting as reservoir of the parasite. Since not all mammals play the same role for the different Trichinella species, the selection of the target animals for epidemiological surveillance is of utmost importance. This document provides indications on the most suitable wild animals to be targeted for epidemiological surveillance according to the four Trichinella species circulating in Europe, together with technical specifications on the muscles of choice and their amount to be analysed for each host species considered.

Comparative analysis of Trichinella spiralis and Trichinella nativa proteins by two-dimensional gel electrophoresis.

Trichinella spiralis and Trichinella nativa are both common wildlife parasites in Finland. However, they differ substantially in their resistance to below 0 degrees C temperatures in their natural hosts. T. nativa can live in frozen fox meat for years, whereas T. spiralis dies when frozen. In mouse muscle, the difference is not as evident; even T. nativa cannot maintain infectivity when kept at -20 degrees C for 1 week. Crude larval protein extracts of these two parasite species were analyzed by two-dimensional gel electrophoresis (2DE). The protein patterns showed clear differences, but matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) peptide mass fingerprint followed by database searches failed to identify these proteins, suggesting that they may still be uncharacterized. The patterns compared after freezing treatment at -20 degrees C revealed changes in the intensity of some protein spots. The antigenic differences of the species were analyzed with two-dimensional Western blots, which showed T. spiralis-specific proteins.

Endogenous morphine and parasitic helminthes.

The current paper summarizes the findings on endogenous morphine isolated by HPLC and characterized by mass spectroscopy in Schistosoma mansoni, Dracunculus medinensis and Ascaris suum. Morphine-6-glucuronide (M6G) has also been found by HPLC and confirmed by mass spectroscopy in Dracunculus medinensis and Ascaris suum. In addition, a morphine like substance has been isolated from Trichinella spiralis by HPLC and mice infected with Trichinella spiralis show a naloxone reversible analgesia. We discuss in greater detail the tissue distribution, course of secretion, and sex differences of morphine in Ascaris suum. Finally, we explore the function of morphine as both an internal signaling molecule and its use in immune evasion in Ascaris suum.

Trichinella infection in wildlife of the southwestern United States.

Several potential mammalian reservoirs of sylvatic species of Trichinella were examined from Texas, New Mexico, and Arizona. During 1998-99, tongues were collected from a black bear (Ursus americanus) in Arizona; from 9 black bears, a coyote (Canis latrans), and a mountain lion (Felis concolor) in New Mexico; and from 154 coyotes, 32 raccoons (Procyon lotor), 13 opossums (Didelphis marsupialis), 4 ocelots (Leopardus pardalis), 3 bobcats (Lynx rufus), and 5 feral hogs (Sus scrofa) in southern Texas. Larvae of Trichinella murrelli were identified by a multiple-polymerase chain reaction analysis in 1 black bear (11.1%) from New Mexico and in 7 coyotes (4.5%) of Texas, whereas Trichinella spiralis larvae were detected in the black bear of Arizona. This is the first report of Trichinella infection in wildlife of New Mexico and Texas and extends the distribution of T. murrelli into the southwestern United States near the border of Mexico.

DNA-binding activity in the excretory-secretory products of Trichinella pseudospiralis (Nematoda: Trichinelloidea).

A novel DNA-binding peptide of Mr approximately 30 kDa was documented for the first time in the excretory-secretory (E-S) products of the infective-stage larvae of Trichinella pseudospiralis. Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E-S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of the Mr of proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E-S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E-S product.

Possible presence of common tyvelose-containing glycans in Trichinella L1 larvae and embryonated eggs of several nematodes.

A monoclonal antibody (mAb US4) recognising an epitope containing tyvelose within the T. spiralis L-1 muscle larvae (TSL-1) antigens was tested in western-blot against various antigenic preparations from different stages of the following nematodes: T. spiralis (L1, adult), T. muris (egg, L1, L3, adult), Ascaris suum (egg, adult), Toxocara canis (egg, adult), Anisakis simplex (L3) and Haemochus contortus (egg). Positive reaction was present in antigen preparations from L1 larvae of T. spiralis and T. muris and from embryonated eggs of T. muris, A. seum, T. canis and H. contortus.

Immunofluorescent localization of intermediate filaments (IFs) in helminths using anti-mammalian IFs monoclonal antibody.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

The genetic analysis of F1 hybrid larvae between female Trichinella spiralis and male Trichinella britovi.

Hybrids between female Trichinella spiralis and male Trichinella britovi were constructed. Then, hybrid genotype was characterized by DNA markers including mitochondrial cytochrome c oxidase subunit I (CO I) gene, the gene encoding the 43-kDa excretory-secretory (ES) protein, and genomic DNA fragments specific for T. spiralis and T. britovi identified from random amplified polymorphism DNA (RAPD). PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial CO I gene revealed that all hybrids carried a T. spiralis pattern. The same analysis of the gene encoding the 43-kDa ES protein showed that each hybrid carried both T. spiralis and T. britovi gene type simultaneously. In the analysis of genomic DNA using RAPD-derived PCR primers, some hybrids carried T. spiralis and T. britovi-specific RAPD markers, while others carried the RAPD marker of T. spiralis only.

Anaphylactic response to parasite antigens: IgE and IgG1 independently induce death in Trichinella-infected mice.

The response of animals infected with different Trichinella species (T. spiralis, T. britovi, T. pseudospiralis) to antigens of different Trichinella species was evaluated in outbred mice (CD1) and inbred mice (BALB/c, C3H, SJL, C57BL/6). In mice deficient for IgE or IgG1, T. spiralis only was used. In homologous conditions (i. e., same Trichinella species for antigens and infectious agent) mortality was very high (up to 100% in SJL and C57BL/6). The anaphylactic response in heterologous conditions was much lower and death was observed only in C57BL/6 and SJL mice. Experiments with deficient mice have shown that both specific IgE and IgG1 independently induce a fatal anaphylactic response.

Trichinella pseudospiralis: X-ray analysis of the infected muscle cells and the muscle larva compartments after exponential growth of the larva in situ.

The distribution of nutrients such as Na, Cl, K, P, S, and Ca was investigated by energy-dispersive X-ray microanalysis (EDXA) on bulk-frozen mouse muscles infected with Trichinella pseudospiralis. In an attempt to gain information as to whether muscle larvae would modify the element status within muscles of their host. Significant increases in phosphorus concentrations within nurse cells and internal cells of the larva, e.g., the somatic muscle cells and the stichocytes, were shown. This could reflect changes in the energy metabolism and/or in the turnover of nucleic acids of the nurse cell. High phosphorus concentrations within the internal larva cells corresponded to high levels of expression of thymidylate synthase shown elsewhere in both T. pseudospiralis and T. spiralis muscle larvae, most likely reflecting more intensive DNA replication. Furthermore, the Na+-concentration gradient between the nurse cell and neighboring host muscle cells indicated the plasma membrane as a diffusion barrier for Na+. Moreover, the cuticle seemed to maintain the Cl--concentration gradient between the nurse cell and the internal cells of the larva. Nevertheless, the mechanisms involved in the transport of both ions, the collagen cuticle appeared, strong permeability that serves the transcuticular transport maintaining the Cl--concentration gradient.

Differences and similarities between Trichinella spiralis and T. pseudospiralis in morphology of stichocyte granules, peptide maps of excretory and secretory (E-S) products and messenger RNA of stichosomal glycoproteins.

The ultrastructure of muscle larvae of Trichinella pseudospiralis was studied by electron microscopy. The overall structure of muscle larvae of T. pseudospiralis resembled that of T. spiralis except for the stichocyte granules. T. pseudospiralis had at least 3 kinds of stichocyte granules distinguishable from each other by their shape, size and inclusions. The granules had some resemblance to alpha granules or beta granules of T. spiralis, but no resemblance to gamma granules. In favour of these morphological differences and similarities among T. spiralis and T. pseudospiralis, excretory and secretory (E-S) products (originating from stichocyte granules) of the 2 species differed to some degree. In an analysis by 2-dimensional electrophoresis, some peptide spots migrating at 45 kDa were shared by the 2 species but the other spots were unique to each of the 2 species. Messenger RNA encoding the 43 kDa glycoprotein of stichocyte granules was detected in the muscle larvae of both species but mRNA encoding the 53 kDa glycoprotein was detected only in muscle larvae of T. spiralis.

Trichinella pseudospiralis secretes a protein related to the Trichinella spiralis 43-kDa glycoprotein.

A 43-kDa secreted glycoprotein from the intracellular parasitic nematode Trichinella spiralis has been considered as a factor involved in the formation of the Nurse cell in infected muscle. The closely related intracellular parasitic nematode Trichinella pseudospiralis that also infects muscle cells, does not form Nurse cells and was thought not to secrete the 43-kDa glycoprotein. This implied a unique role for the 43-kDa glycoprotein in T. spiralis infection and supported the hypothesis of involvement of the 43-kDa glycoprotein in Nurse cell formation. Following cloning of a full length cDNA encoding the 43-kDa protein, antibodies were raised against several domains of the 43-kDa glycoprotein. Here we show that a protein related to the 43-kDa glycoprotein exists in T. pseudospiralis. Immunohistochemical studies reveal important similarities in the distribution of the 43-kDa glycoprotein and the related protein from T. pseudospiralis in muscle infections with either of the two parasites. The 43-kDa glycoprotein may therefore play a common role in the life cycles of these two parasites and probably is not involved in Nurse cell formation.

Ultrastructure, antigenicity, and histochemistry of stichocyte granules of adult Trichinella spiralis.

The stichosome of adult Trichinella spiralis was studied to determine its ultrastructural, antigenic, and histochemical characteristics. Stichocytes of adult worms had 2 types of granules, type I and type II, the ultrastructure of which was different from those of muscle larvae. Both types of granules consisted of a membrane surrounding a homogeneous matrix, and type I granules were rounder than type II granules. Sera from C3H mice immunized against excretory-secretory products of muscle larvae produced positive immunostaining of type I but not type II granules. Differences in antigenicity were observed between larval and adult stichocyte granules; monoclonal antibodies against alpha-granules of muscle larvae failed to label the adult granules. Azan staining revealed a histochemical difference between larval and adult stichocytes; adult stichocytes stained yellow, whereas larval stichocytes are known to stain red or blue. Thus, the present contribution revealed the existence of 2 distinct types of stichocyte granules in adult T. spiralis and showed them to differ profoundly from those characterized previously in muscle larvae.

Comparative study of soluble protein profiles of different Trichinella isolates.

The comparison and characterization of different populations of Trichinella has become possible through the study of their complex polypeptide profiles. Techniques such as polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) provide a high capacity of resolution. We used both techniques to compare proteinograms of different Trichinella isolates, in order to characterize an isolate detected in a wild boar captured in Güejar-Sierra (Province of Granada, Spain).

[The fatty acids of trichinellae]. / Zhirnye kisloty trikhinell.

The composition of fatty acids of lipids in the muscles of rats and larvae of Trichinella spiralis and T. nativa developing in them were studied. Both species are characterized by practically the same composition of fatty acids, only in the frost-resistant species T. nativa there was a sufficient amount (up to 3.5%) of docosapenta- and docosahexaenic acids (22:5 and 22:6). The comparison of the content of individual fatty acids in larvae and in muscles of the host by means of statistical correlation analysis suggests that larvae obtain a considerable portion of palmitic acid from the host and transform in into necessary long-chain saturated and unsaturated fatty acids by means of elongating and desaturating enzymes. Changes in the contents of fatty acids in larvae extracted from dead rats, which during some days were undergone freezing at negative temperatures (-8-10 degrees), are the same in quality for both species. These changes can be explained if we assume that the activity of elongases and desaturases of Trichinella decreases with cooling to a greater extent than the supply of palmitic acid from the host's tissues. A higher frost-resistance of T. nativa may be associated as with a greater protection of enzymes in the membranes by long-chain polyene acyls so with a higher thermal stability of proteins themselves.