Helminth zoonoses remain a global problem to public health and the economy of many countries. Polymerase chain reaction-based techniques and sequencing have resolved many taxonomic issues and are now essential to understanding the epidemiology of helminth zoonotic infections and the ecology of the causative agents. This is clearly demonstrated from research on Echinococcus (echinococcosis) and Trichinella (trichinosis). Unfortunately, a variety of anthropogenic factors are worsening the problems caused by helminth zoonoses. These include cultural factors, urbanization and climate change. Wildlife plays an increasingly important role in the maintenance of many helminth zoonoses making surveillance and control increasingly difficult. The emergence or re-emergence of helminth zoonoses such as Ancylostoma ceylanicum, Toxocara, Dracunculus and Thelazia exacerbate an already discouraging scenario compounding the control of a group of long neglected diseases.

Trichinellosis is an important worldwide foodborne zoonosis. The gold standard test to detect Trichinella spp. larvae in muscle samples of animals intended for human consumption is the artificial digestion method. Handling and dispensing of conventional pepsin powder present significant safety risks for analysts. The use of pepsin powder that is resistant to aerosolization should alleviate these safety concerns. The aim of this study was to compare the efficacy of an aerosol-resistant pepsin powder to conventional pepsin powder in the artificial digestion method. Proficiency samples of pork diaphragm containing specific numbers of viable Trichinella spiralis larvae were tested in two laboratories. The results revealed that aerosol-resistant pepsin was simple, effective and convenient to use, and showed good solubility and larval recovery that met the requirements of the European Union regulation EU 2015/1375. Overall, the efficacy of the aerosol-resistant pepsin was comparable to the conventional pepsin and safer for analysts.

Horizontal gene transfer (HGT) has played an important role in the evolution of nematodes. Among candidate genes, cyanase, which is typically found only in plants, bacteria and fungi, is present in more than 35 members of the Phylum Nematoda, but absent from free-living and clade V organisms. Phylogenetic analyses showed that the cyanases of clade I organisms Trichinella spp., Trichuris spp. and Soboliphyme baturini (Subclass: Dorylaimia) represent a well-supported monophyletic clade with plant cyanases. In contrast, all cyanases found within the Subclass Chromadoria which encompasses filarioids, ascaridoids and strongyloids are homologous to those of bacteria. Western blots exhibited typical multimeric forms of the native molecule in protein extracts of Trichinella spiralis muscle larvae, where immunohistochemical staining localized the protein to the worm hypodermis and underlying muscle. Recombinant Trichinella cyanase was bioactive where gene transcription profiles support functional activity in vivo. Results suggest that: (1) independent HGT in parasitic nematodes originated from different Kingdoms; (2) cyanase acquired an active role in the biology of extant Trichinella; (3) acquisition occurred more than 400 million years ago (MYA), prior to the divergence of the Trichinellida and Dioctophymatida, and (4) early, free-living ancestors of the genus Trichinella had an association with terrestrial plants.

Genome assemblies can form the basis of comparative analyses fostering insight into the evolutionary genetics of a parasite's pathogenicity, host–pathogen interactions, environmental constraints and invasion biology; however, the length and complexity of many parasite genomes has hampered the development of well-resolved assemblies. In order to improve Trichinella genome assemblies, the genome of the sylvatic encapsulated species Trichinella murrelli was sequenced using third-generation, long-read technology and, using syntenic comparisons, scaffolded to a reference genome assembly of Trichinella spiralis, markedly improving both. A high-quality draft assembly for T. murrelli was achieved that totalled 63·2 Mbp, half of which was condensed into 26 contigs each longer than 571 000 bp. When compared with previous assemblies for parasites in the genus, ours required 10-fold fewer contigs, which were five times longer, on average. Better assembly across repetitive regions also enabled resolution of 8 Mbp of previously indeterminate sequence. Furthermore, syntenic comparisons identified widespread scaffold misassemblies in the T. spiralis reference genome. The two new assemblies, organized for the first time into three chromosomal scaffolds, will be valuable resources for future studies linking phenotypic traits within each species to their underlying genetic bases.

Comparison of antibody response and antigen recognition was made by ELISA and western-blot analysis in pig experimental infections by T-l and T-3 Spanish Trichinella isolates. Two groups of Iberian pigs were experimentally infected with 150 larvae/kg body weight of GM-1 and C-76 Spanish Trichinella isolates as representatives of T-1 and T-3 gene pools respectively. Antibody levels and antigen recognition were measured on days –14, 0, 6, 16, 20, 27, 34, 49, 63 and 82 after infection by ELISA and western-blotting assays. Antibody response against C-76 infection was significantly delayed and lower than against GM-1. The two Trichinella isolates were indistinguishable, however, by western blotting analysis, although recognition of larval antigens was quantitatively higher than adult ones. Interestingly, the principle larval antigenic components recognized by pigs were those recognized by the monoclonal antisera NIM-M1. Finally, there were no serological patterns indicative of the stage of infection (“antibody windows”) discriminating, for example between early versus late infections.

Excretory/secretory (ES) products obtained by in vitro culture of infective-stage larvae of Trichinella spiralis and T. pseudospiralis were injected intramuscularly at various intervals into mice. Mini-osmotic pumps containing T. spiralis ES products were also implanted subcutaneously and intraperitoneally into rats. The introduction of ES materials into muscles elicited extensive lesions which included dissolution of myofibres, mobilization of mononuclear and polymorphonuclear leucocytes, angiogenesis, hypertrophy of myonuclei, myotube formation, mitosis, muscle bundles becoming rounded and separated from each other, disappearance of Z, I and A bands of sarcomeres, increase in endoplasmic reticulum and Golgi complexes, decrease in glycogen and relocation of mitochondria. These are considered as degenerative/regenerative changes of muscles to injury. Immunodominant epitopes of specific 45–53 kDa glycoproteins in ES antigens of T. spiralis could not be detected in hypertrophic nuclei of injected muscles by using polyclonal and monoclonal antibodies and immunocytochemical methods. ES products of T. spiralis failed to stimulate unsensitized lymphocytes in the lymphocyte transformation test. Infective-stage larvae of T. spiralis released from muscles were found capable of forming nurse cells after injection subcutaneously into rats. It is postulated that the invasion of muscles by trichinellids elicits two independent events, i.e. a general degenerative/regenerative response of muscles and a specific change in genomic expression of myonuclei. The two events are probably mediated by different effector molecules.

Monoclonal antibodies raised against antigens present in the excretions/secretions (E/S) of larval Trichinella spiralis, polyclonal antibodies raised against E/S and antisera from rabbits and pigs infected with T. spiralis were used in conjunction with immunocytochemical techniques to detect antigens in sections of muscle from mice that had been infected with T. spiralis for 15 or 30 days. The antibodies recognized epitopes in the stichocytes, on the surface of the cuticle, in the lumen of the oesophagus and in the lumen of the intestine of encysted larvae. Monoclonal antibodies 7C2C5 and 1H7 and the polyclonal antibodies recognized epitopes in the cavity occupied by the larva, in the cytoplasm of the nurse cell, and in the hypertrophic nuclei of the nurse cell, but did not recognize material in the smaller nuclei of the nurse cell, in the cyst wall or in the surrounding muscle. Monoclonals 3B2E6 and 1D11G8B2, which recognized epitopes in the stichocytes and on the surface of the cuticle of the larvae, gave negative results with the cytoplasm and nuclei of the nurse cell. A polyclonal antibody raised against Trichuris suis recognized epitopes in the muscle and hypodermis of encysted T. spiralis but gave negative results with material in the nurse cell and nurse cell nuclei. The possibility that the antigen detected in the cytoplasm and nuclei of the nurse cell is produced by the stichocytes of the nematode and that it is controlling genes of the altered muscle fibre, either directly or indirectly, is discussed.

Eight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

To assess the potential role of IgA antibody in expulsion of the nematode of the genus Trichinella from the intestine, a panel of IgA monoclonal antibodies (mAbs) were produced from the mesenteric lymph node cells from BALB/c mice orally vaccinated with irradiated muscle larvae of Trichinella britovi. One IgA mAb, HUSM-Tb1, formed immunoprecipitates on the surface of live muscle larvae, and by immunohistochemistry reacted with their stichocytes and cuticular surface, but not with those tissues of the adult stage or newborn larvae. Intraperitoneal injection of BALB/c mice with this mAb 5 h before challenge conferred a high level of protection (more than 95%) against T. britovi infection, when 2·0 mg of specific IgA/20 g body weight was given to a mouse. The same treatment produced a similar effect in SCID mice lacking functional T- and B-cells, indicating no requirement of synergistc T-cell factors for the effect. Passive transfer of the mAb at the time of challenge or later showed less or no effect upon worm expulsion. It is concluded that the mucosal IgA response, when adequately induced, can impede the establishment of infective Trichinella parasites in the mouse intestine.

Genetically determined variation in host capacity to express resistance to a given parasite plays a major role in determining the outcome of infection. It can be assumed that the same is true of variation in parasites, but very much less is known of its influence on the host–parasite relationship. Phenotypic and genotypic variation within species of intestinal worms is now well documented, detailed studies having been made of parasites such as Ascaris in humans and trichostrongyles in domestic animals. However, the extent to which this variation affects the course of infection or the host immune response in these hosts is limited. Of the nematodes used as experimental models in laboratory rodents, detailed data on phenotypic or genotypic variation are limited to Strongyloides and Trichinella. Parasite variation is known to be subject to host-mediated selection, the emergence of anthelmintic resistance being a good example. Repeated passage has been used to select lines of parasite that survive in abnormal hosts or which show adaptation to host immunity. Experimental studies with Trichinella genotypes in mice have demonstrated the extent to which parasite variation influences the nature and degree of the host's immune and inflammatory responses, the complex interplay between immunogenicity and pathogenicity influencing both partners in the relationship. Recent studies with isolates of Trichuris muris have shown how parasite variation influences the capacity of mice to express the T helper cell responses necessary for resistance. Molecular differences between T. muris isolates have been shown in their excreted/secreted products as well as at the level of their DNA. Knowledge of the functional consequences of parasite variation will add to our understanding of host-parasite evolution as well as providing a rational basis for predicting the outcome of controls strategies that rest on the improvement of host resistance through vaccination or selective breeding.

Infection of an intracellular parasitic nematode, Trichinella spiralis, resulted in severe damage in muscle cells which was followed by activation and proliferation of satellite cells. The repairing process, shortly after the damage, histopathologically resembled those seen after mechanical injury. Resemblance was also true for kinetics of expression of myogenic regulatory factors (MyoD, myogenin and MRF4). The difference resided in the next step where the muscle cell infected with T. spiralis transformed to a unique cell which is parasitologically known as the nurse cell, and the proliferated satellite cells did not differentiate to the muscle cell but to the nurse cell (misdifferentiation). Thus the nurse cell was a fusion of the transformed infected muscle cell and misdifferentiated satellite cells. Infection with another species of Trichinella, T. pseudospiralis, also caused cell damage, but more extensively involving the entire length of the infected muscle cells because no septum was formed to minimize the affected area. Therefore, a large number of satellite cells were activated and proliferated. The myogenic regulatory factors such as MyoD and myogenin were activated for a longer period than in the case with T. spiralis infection. The infected muscle cell transformed to the nurse cell, whose cytoplasm was characterized by extensive smooth endoplasmic reticulum. Satellite cells misdifferentiated to the nurse cell, whose cytoplasm was amorphous, void of distinct cell organelles. The two kinds of cytoplasm did not fuse as examined thus far. Thus infection with T. spiralis and T. pseudospiralis caused misdifferentiation of satellite cells, but in a different way.

Diagnostic PCR primers for Trichinella were constructed. Twelve pairs of primers were designed based on the sequences of random amplified polymorphic DNA, and 4 pairs of primers were designed based on the reported sequences of complementary DNA encoding excretory–secretory glycoproteins. With these primers, 31 samples of DNA from different strains of Trichinella including 5 species (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella nelsoni and Trichinella pseudospiralis) and 3 phenotypes of uncertain taxonomic level (Trichinella T5, T6 and T8) were tested with PCR. Genus Trichinella can be identified by 4 different primer pairs (SB147D, SB372A, SB153, or Ts43). Trichinella spiralis can be identified by the presence of a 673 bp amplicon in PCR with the primer pair SB147B. Trichinella nelsoni can be identified using primer pair SB147F or by the presence of 673 bp and ca. 380 bp amplicon in PCR with the primer pair SB147B. Trichinella pseudospiralis can be identified by 2 primer pairs (SB147E or SB372B). Trichinella T5 can be identified by the primer pair SB147G. Trichinella T8 can be identified by its positivity by the primer pair SB147C and its negativity by the primer pair SB372C. A group of Trichinella species (T. britovi, T. nativa and Trichinella T6) can be identified by the primer pair SB372C.

Several monoclonal antibodies (Mabs) were raised against the L1 muscle stage (L1M) of Trichinella spiralis (Ts) and Trichinella pseudospiralis (Tp). Western blot analysis of various antigenic preparations established that Mabs described by different authors recognized 8 antigenic fractions (TSL1–TSL8) in crude extracts of infective larvae. The TSL1 fraction was immunodominant and present on the cuticle of different parasite stages. Mabs against Trichinella T5 (T5) and Ts were selected in order to extend the previous studies to another Trichinella phenotype. Only 35% of the selected Mabs recognized linear epitopes and 71% reacted with soluble or excretory–secretory antigens in a dot blot procedure and ELISA test. The targets of the Mabs were identified by immunoprecipitation with [35S]methionine-labelled L1M worm lysate. Mabs prepared from mice immunized with the whole parasite (T5) recognized a wider panel of antigens in different parasitic organs. Seven antigenic structures were distinguished on the cuticle and several epitopes were identified in the gut, haemolymph and stichocytes. Eleven antigenic groups were established according to their indirect immunofluorescence pattern on cross-sections of the worm. Monoclonal antibodies raised against Ts soluble antigen mainly recognized epitopes in stichocytes and on the cuticle surface. All the selected Mabs recognized T5 and Trichinella britovi (Tb) strengthening the link between these 2 species. Four Mabs were used to differentiate antigenic structures among 6 Trichinella phenotypes and to develop a new tool to follow gene flow within the Trichinella genus.