Induction of protection in murine experimental models against Trichinella spiralis: an up-to-date review.

The parasitic nematode Trichinella spiralis, an aetiological agent of the disease known as trichinellosis, infects wild and domestic animals through contaminated pig meat, which is the major source for Trichinella transmission. Prevention of this disease by interrupting parasite transmission includes vaccine development for livestock; however, major challenges to this strategy are the complexity of the T. spiralis life cycle, diversity of stage-specific antigens, immune-evasion strategies and the modulatory effect of host responses. Different approaches have been taken to induce protective immune responses by T. spiralis immunogens. These include the use of whole extracts or excretory-secretory antigens, as well as recombinant proteins or synthesized epitopes, using murine experimental models for trichinellosis. Here these schemes are reviewed and discussed, and new proposals envisioned to block the zoonotic transmission of this parasite.

A prime-boost vaccination of mice with attenuated Salmonella expressing a 30-mer peptide from the Trichinella spiralis gp43 antigen.

Protection against Trichinella infections has been achieved using various parasite antigens and adjuvants. Recently, we reported that immunization of mice with an attenuated Salmonella strain displaying a 30-mer peptide (residues 210-239) from the Trichinella spiralis gp43 antigen using the ShdA autotransporter induced partial protection against T. spiralis infection. To improve the efficacy of vaccination, we used the MisL autotransporter system to display the Ts30mer peptide on the surface of Salmonella enterica ser. Typhimurium in combination with a prime-boost vaccination strategy. This vector and immunization regimen induced superior protection against T. spiralis when compared to our previously reported approach. Data presented herein showed a significant reduction in adult worm and muscle larvae burdens, high IgG titers, and increased production of intestinal mucus with entrapped adult worms. This prime-boost vaccination scheme is a suitable strategy to elicit enhanced protective immunity against T. spiralis.

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210-239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.

Encystation commitment in Giardia duodenalis: a long and winding road.

Cholesterol and bile salts are relevant modulators of Giardia encystation. Although several molecules within signaling cascades have been identified, and changes in their expression observed during giardial encystation, their underlying interactions leading to expression of cyst wall markers (CWPs and precursors of the GalNAc homopolymer) are not well defined. Recent experimental data and the completion of the Giardia Genome Project Database (GiardiaDB) allow us now to consider the role of bile salts as "natural stimuli" and the potential involvement of a Raf/MEK/ERK pathway mediating cholesterol-regulated expression of cyst-specific genes. These new findings may provide promising targets for diagnostics, drug design and prophylactic intervention against giardiasis.

Differential activation of mast cells by antigens from Trichinella spiralis muscle larvae, adults, and newborn larvae.

Mast cell (MC) hyperplasia and activation are prominent features in Trichinella spiralis infection. Indeed a temporal correlation has been shown between the kinetics of intestinal mastocytosis, release of inflammatory mediators from MC, and adult worm loss, which constitutes a major component of the defense against T. spiralis infection. It is well known that during the intestinal phase of trichinellosis, muscle larvae (ML) and adult worms (AD) enter into contact with the host; however, interaction with MC may also occur during migration of newborn larvae (NBL). Therefore, it is plausible that antigens from these developmental stages could activate MC. We have previously demonstrated by in vitro assays that T. spiralis muscle larval (TSL-1) antigens activate MC through an Ig-independent mechanism leading to the release of histamine, MC protease 5, IL-4 and TNF alpha. In this work we evaluated whether total antigens from AD or NBL could activate unsensitized MC and we compared this activation with the activation seen when MC are stimulated with TSL-1 antigens. MC activation was also tested with affinity chromatography purified antigens from NBL using the monoclonal antibody CE-4 that recognizes NBL surface components. The results obtained in this study showed that AD total extracts and TSL-1 antigens induced the release of histamine but not beta-hexosaminidase from unsensitized MC, suggesting a selective secretion of MC mediators. In contrast, NBL total extracts or purified NBL antigens did not induce the release of either histamine or beta-hexosaminidase from MC. Interestingly, AD and ML are the stages that interact with the host during the intestinal phase of infection. The mechanisms involved in TSL-1 and AD activation of unsensitized MC may function together with other mechanisms of MC activation in host protection against T. spiralis.

Contributions to the study of Trichinella spiralis TSL-1 antigens in host immunity.

The observation on different hosts infected with Trichinella spiralis that recognized similar muscle larvae (ML) antigens and the fact that different monoclonal antibodies (mAb) had a similar reactivity to ML components prompted a proposal to define a useful classification system for these antigens. For this purpose, an international workshop provided a platform for the classification of T. spiralis antigens. ML antigens were classified in eight groups -- Trichinella spiralis larvae groups, TSL-1 to TSL-8. TSL-1 antigens are highly immunogenic and a number of important studies have been performed to analyse the role of these antigens in the host-parasite interplay. In this context, we have focused on the analysis of the role of TSL-1 antigens in the induction of innate immune responses with particular emphasis on the activation of mast cells (MC) by an IgE-independent pathway. These studies provided evidence on the role of mediator release from TSL-1-activated MC in the development of Type 2 immune responses. The protective role of TSL-1 in T. spiralis-infected mice has been described. In addition, it has been demonstrated that the use of TSL-1 antigens allows for a more sensitive and specific diagnosis of human and animal trichinellosis.

A stage-specific open reading frame from three-day old adult worms of Trichinella spiralis encodes zinc-finger motifs.

The aim of the study was to isolate genes coding for stage-specific antigens of T. spiralis. Such antigens may then be associated with local and systemic immune responses against adult T. spiralis. Recombinant clones were obtained with an adult stage specific probe from a cDNA library of three-day old adult T. spiralis. Several cDNA clones encoding the same peptide were identified and their stage specificity was confirmed by northern blot analysis. Three independent clones were fully sequenced, and the resulting sequence found to code for a 487 amino acid peptide with a deduced molecular weight of approximately 55 kDa. Sequence analysis showed that the 55 kDa peptide contained putative DNA binding motifs, suggesting that this protein may be involved in transcriptional regulation during the early development of the parasite.

The stage-specificity of the IgA response to newborn larva and TSL-1 antigens of Trichinella spiralis in humans infected with the parasite.

Characterization of human IgA responses to newborn larva (NBL) and TSL-1 antigens was carried out by ELISA assays. Relevant and differential IgA antibody responses to these antigens were detected in humans infected with T. spiralis. The inhibition ELISA results showed that the IgA response to NBL antigens was inhibited significantly by both NBL and TSL-1 antigens and to a lesser extent when phosphorylcholine (PC) was used as inhibitor. In contrast, the IgA response to TSL-1 antigens was inhibited by the homologous antigen and to a lesser extent by the NBL and PC. Thus, the early IgA antibodies developed in trichinellosis patients contained a portion of IgA antibodies directed to PC which is present in TSL-1, A and NBL component. Another portion of antibodies to NBL are directed to other common non-defined epitopes present in TSL-1 and NBL antigens. All together these results suggest that the IgA response to common epitopes in antigens of both stages of the parasite may be useful for early diagnosis and epidemiological studies of human trichinellosis.

Cloning and analysis of a Trichinella britovi gene encoding a cytoplasmic heat shock protein of 72 kDa.

A gene encoding a protein of 646 amino acid residues with a molecular mass of 71.3 kDa showing homology to the cytoplasmic form of the 70 kDa heat shock protein was cloned and sequenced from the nematode parasite Trichinella britovi (Tb). The gene was expressed in vitro as a protein of 71 kDa that was immunoprecipitated by a Trichinella-infected rabbit serum. Monospecific polyclonal antibodies raised against the recombinant Tb Hsp70 expressed in Escherichia coli, recognized a protein of 70 kDa by Western blot analysis of Tb soluble antigen (muscular stage). Tb Hsp70 was located in the nuclei of the muscle larvae as determined by the indirect immunofluorescent pattern on cross-sections of the worm. The expression of this protein was not detected in adult worm nuclei suggesting a differential expression of Hsp70 between the 2 stages of Trichinella.

Phospholipid membranes form specific nonbilayer molecular arrangements that are antigenic.

Hexagonal phase (H(II))-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of H(II)-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.

[International Workshop on Molecular Epidemiology. Proposals of the Interdisciplinary Group on American Trypanosomiasis and Leishmaniasis]. / Taller Internacional sobre Epidemiología Molecular. Propuestas del Grupo Interdisciplinario sobre Trypanosomosis Americana y Leishmaniosis.

Parasite diseases such as Leishmaniases and American Trypanosomiases have been increasingly important in Mexico and other countries of the American Region. In known areas, these diseases are highly endemic, and in recently opened developing areas became a new threat to public health. Some social groups working in natural resources exploitation, agriculture, animal stock and public labor are particularly affected. The molecular epidemiology approach to these diseases is linking valuable capabilities and resources within the academic and operational institutions actual working in genetic polymorphism, strain characterization and PRC identification of Trypanosoma and Leishmania parasites. Clinical and epidemiological aspects of American trypanosomiasis infections and Chagas's disease and of cutaneous, mucocutaneous, disseminated and visceral leishmaniases, as well as genetic susceptibility studies have been initiated by Mexican scientists. In order to organize and coordinate the molecular epidemiology activities and support effective prevention and control programs against these diseases, political decision from the health, and academic authorities is urgently needed to adopt and support the research strategy for typing Trypanosoma and Leishmania species through exposition and biological markers (analysis of chromosomal DNA, ribosomal genes restriction patterns, DNA sequence, and DNA plasmids); the study of the membrane proteins and isoenzymes and monoclonal antibodies; detecting antigens and nucleic acids; defining susceptibility to infection with genetic markers, and searching for species, variants and mutant strains responsible for high virulence. The support for the establishment of a Reference Center for identification, cryopreservation and registration of parasites, vectors and reservoires is of paramount importance.

Serologic survey of trichinellosis in wild mammals kept in a Mexico City Zoo.

A serologic survey of Trichinella infection was carried out to determine the prevalence of this parasitosis among wild mammals kept in captivity at the Chapultepec Zoo. This was prompted by the necropsy finding of a heavy Trichinella infection in a Canadian polar bear (Ursus maritimus) that had been kept at the Zoo for more than 11 years. The parasites recovered were identified as T. nativa (T2). A serologic study based on ELISA and Western blot analysis was performed in serum samples from two polar bears (U. maritimus), six wolves (Canis lupus); nine foxes (Urocyon cinereoargenteus); seven coyotes (Canis latrans); nine jaguars (Panthera onca); ten lions (Panthera leo); 11 tigers (Panthera tigris); six panthers (Panthera pardus); eight leopards (Panthera pardus); two lynxes (Lynx rufus); five pumas (Felis concolor); one yagouaroundi (Felis yagouaroundi); and one ocelot (Felis pardalis). In these assays, 25% and 27% of the samples studied were positive using total muscle larva extract from T. nativa (T2) or T. spiralis (T1), respectively. When T. spiralis (T1) excretory/secretory products or surface/stichosomal antigens were used, 15 and 13% positivity was obtained respectively. The reactivity rates obtained among the different groups varied from 11 to 83%, wolves having the highest infection rate. Western blot analysis of positive ELISA sera showed an antigenic recognition pattern characteristic of animals infected with Trichinella.

Detection of circulating Trichinella spiralis muscle larva antigens in serum samples of experimentally and naturally infected swine.

A follow up study was carried out to determine the kinetics of appearance of surface/stichosomal (S/S) components, recently included in the TSL-1 group of Trichinella spiralis muscle larva (ML), in serum samples from 13 experimentally infected pigs. Detection of circulating antigens in these animals was done by a sandwich enzyme-linked immunosorbent assay (ELISA) using T. spiralis specific rabbit polyclonal immunoglobulins to capture free antigens and monoclonal antibody NIM-M1 to recognize S/S antigens. The assay developed was able to detect as little as 35 ng ml-1 of S/S components added to normal pig serum. Antigenemia was observed in 54% of the experimentally infected swine with two peaks of appearance, one early at 1-4 weeks post-infection (pi) and one late at 10-14 weeks pi. Specific antibodies against S/S components were demonstrated in serum samples from all experimentally infected pigs starting at 3-4 weeks pi. Free antigen was also detected in serum samples from naturally infected backyard pigs with a sensitivity of 56% compared with 94% when antibody production was determined using purified S/S components in an ELISA.

Isolation and axenization of Giardia lamblia isolates from symptomatic and asymptomatic patients in Mexico.

Infection of the small intestine of humans with the parasitic protozoan Giardia lamblia may have an asymptomatic course, or else, may produce acute or chronic diarrhea. In order to establish if the different clinical outcome of giardiasis in children could be due, at least partially, to strain differences, isolates from asymptomatic and symptomatic cases studied in Mexico City during 1986 and 1987 were cultured under axenic conditions. With modifications of available methods for the isolation of G. lamblia from cysts in stools, we obtained 19 axenic isolates: 5 from symptomatic patients and 14 from asymptomatic cyst carriers. The isolation procedure involved: (1) concentration and cleaning of cysts through centrifugation in sucrose gradients; (2) excystment induction in acid solution; (3) culture in modified TYI-S-33 medium, and (4) axenization of isolates using ceftriaxone and Amphotericin B. Results indicate that isolates from carriers and from symptomatic cases of giardiasis are equally amenable to isolation and axenization. The Giardia isolates obtained are being studied to analyze differences in isoenzyme pattern, antigenicity, and molecular markers.

Characterization of two DNA populations of Giardia lamblia.

Total DNA was isolated from the parasitic protozoan Giardia lamblia and separated into two distinct populations of different densities by centrifugation through CsCl gradients containing Hoechst dye 33258. The two populations obtained were characterized by restriction enzyme analysis and nucleic acid hybridization. The less-dense population contains non-repetitive DNA and may encode mainly structural genes, such as those for alpha- and beta-tubulin. Digestion of the DNA with several restriction endonucleases showed that the denser band was composed of a 5.5 kb unit which contains the G. lamblia ribosomal RNA cistron in tandem repeated organization.

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.

Trichinella spiralis: ultrastructure of the body wall of newborn and muscle larvae.

The body wall structure of muscle and newborn larvae of Trichinella spiralis was studied using transmission and scanning electron microscopy. Differences were found in the structure of the cuticle of the two developmental stages. In the case of the cuticle of the muscle larvae only transverse striae were present whereas the newborn larvae cuticle showed both transverse and longitudinal striations. In the two parasite stages the outer surface of the cuticle appears as a three-layered structure. The hypodermis presents well defined cellular components similar in fine structure in both stages. The plasma membrane of the hypodermal cells in the muscle larvae shows abundant short finger-like projections, that are not present in newborn larvae.

Giardia lamblia: surface charge of human isolates in culture.

The surface charge of Giardia lamblia trophozoites from axenic cultures of strains recently isolated in Mexico from human cases of symptomatic and asymptomatic giardiasis was studied by means of cellular microelectrophoresis and ultrastructural cytochemistry. It is concluded that ionogenic surface groups confer a negative surface charge on trophozoites of G. lamblia and that no significant differences exist between the surface charge of trophozoites of symptomatic and asymptomatic origin.

Giardia lamblia: isoenzyme analysis of 19 axenic strains isolated from symptomatic and asymptomatic patients in Mexico.

Infection of the small intestine of humans with the parasitic protozoon Giardia lamblia may have an asymptomatic course, or it may produce acute or chronic diarrhoea. In order to establish if the different clinical outcome of giardiasis in children could be due, at least partially, to strain differences, 19 isolates from asymptomatic and symptomatic cases studied in Mexico City were cultured under axenic conditions and the isoenzyme electrophoretic patterns of 10 different enzymes were compared. Strains from carriers and from symptomatic cases of giardiasis were equally amenable to isolation and axenization. Isoenzyme electrophoresis demonstrated remarkable homogeneity in 7 enzyme patterns for all 19 isolates, except for phosphoglucomutase, for which 3 different zymodemes were found. Therefore, these isolates of G. lamblia, obtained from a single geographical location, tended to be genetically homogeneous. In addition, there were no consistent zymodeme differences between isolates from symptomatic and asymptomatic human infections.