Insights into the Giardia intestinalis enolase and human plasminogen interaction.

Giardia intestinalis is an intestinal parasite that causes diarrhea in humans and animals worldwide. The enolase of G. intestinalis (GiENO) participates in its glycolysis pathway and is abundantly expressed in the parasite cytosol; however, its localization on the surface of trophozoites and cysts has been demonstrated. Enolases from bacteria and parasites can have different functions and are considered moonlighting proteins, for example, as a cell surface plasminogen receptor. In relation to GiENO, no studies have been performed about its possible participation as a plasminogen receptor. In this work, we employed molecular docking and multiscale molecular dynamics (MD) simulations to explore the possible interactions of human plasminogen (HsPLG) with the open and closed GiENO conformations. Our proposed GiENO plasminogen binding site (PLGBs) was identified at Lys266 based on the sequence comparison with bacterial enolase known to act as a plasminogen receptor. Our docking results performed with multiple MD snapshots of the closed GiENO conformation showed that Lys266 preferentially binds to the K5 domain of HsPLG. On the other hand, open GiENO conformations from all-atom and coarse-grained simulations indicated a high preference of the HsPLG K4 domain for lysine residues 186 and 188. Furthermore, we identified a potential N-glycosylation site of GiENO which suggests a possible explanation for the parasite cell surface localization or host mucin oligosaccharide adhesion mechanism. Our study constitutes the first multiscale computational study to explore the plasminogen receptor function of GiENO for its further consideration as a potential therapeutic target for giardiasis treatment.

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.

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.

Interleukin mRNA changes in mast cells stimulated by TSL-1 antigens.

In this work we analyzed by RT-PCR, the mRNA changes for IL-4, IL-10, TNF and IFN (induced by TSL-1 antigens in a rat mast cell line (HRMC) with mucosal characteristics. The data obtained showed an increase of 65 and 52% in mRNA expression for IL-4 and TNF respectively and a decrease of 59 and 55% in mRNAs for IFN gamma and IL-10. Our results suggest that TSL-1 antigens induce the release from MC of regulatory molecules, such as IL-4 by an IgE independent mechanism. Our data also provides important information related to the ability of MC to participate not only in the effector phase against the infectious agents, but also in the orchestration of the immune response by the host against parasites.

Synthesis and hydrolytic stability studies of albendazole carrier prodrugs.

Three N-acyl (2, 3, and 4), two N-alkoxycarbonyl (5 and 6), and one N-acyloxymethyl (7) derivatives of albendazole (1) have been prepared and assessed as potential prodrugs. The determination of the aqueous solubility and partition coefficient, as well as the conversion of these derivatives to 1 in buffer solution, human plasma, and pig liver esterase were determined.

Epidemiology of trichinellosis in Mexico, Central and South America.

Trichinella species are widely distributed throughout the world and are found in a large number of carnivorous animals, humans and incidental hosts. The data presented in this review show that Trichinella infection has been reported in both humans and animals in Mexico, Argentina and Chile since the end of the 19th century, and more recently in Bolivia. This parasitic infection is still a public health problem in countries such as Argentina and Chile. Although efforts have focused on the control and prevention of trichinellosis in these countries, there were still human cases and outbreaks of trichinellosis reported. Diagnosis of infection in animals such as pigs still includes, in many endemic areas, the use of trichinoscopy. In Argentina, however, artificial digestion has been recently introduced in slaughterhouses to detect Trichinella infection in pigs. In some endemic areas in Mexico, the use of serological assays for human trichinellosis and pig infections have resulted in improved detection. Most of the outbreaks of human trichinellosis in Mexico, Argentina and Chile have occurred as a result of the consumption of undercooked pork or pork products from animals raised under poor hygienic conditions and which are clandestinely slaughtered. In several studies, rats, dogs and cats have been found to be infected with Trichinella and may be considered a risk for transmission of the infection to pigs or other animals intended for human consumption. Another potential source of transmission of Trichinella to humans is horsemeat; however, information related to horse trichinellosis in Latin-American countries is scarce. In most cases the etiological agent of human trichinellosis in Central and South America has been reported to be Trichinella spiralis; however, only in a few cases has the parasite species been properly identified. Based on the reports available, it is clear that there is a need to carry out better controlled epidemiological studies to determine the true prevalence of the infection in this region of the world. Also, more sensitive methods of diagnosis and improvements in conditions for pig production as well as better sanitary inspection systems, are needed for controlling and preventing transmission of trichinellosis in these countries.

Evaluation of albendazole prodrugs in experimental trichinellosis.

BACKGROUND: Two albendazole (ABZ) prodrugs, N-methoxycarbonyl-N'-[(2-nitro-4-propylthio) phenyl] thiourea (compound 2), and N-methoxycarbonyl-N'-[(2-nitro-5-propylthio) phenyl] thiourea (compound 3) have recently been synthesized. These compounds showed greater solubility than ABZ itself. METHODS: In order to evaluate the biotransformation of compounds 2 and 3 to ABZ and/or ABZ-sulphoxide (ABZ-SO), plasma samples taken from mice treated with the prodrugs were analyzed by HPLC. Also, the anthelmintic activity of compounds 2 and 3 against Trichinella spiralis was evaluated in the mice experimentally infected with the parasite. RESULTS: The presence of ABZ and/or ABZ-SO was demonstrated in plasma samples taken at different time intervals after prodrug administration, although their levels were low compared to those reached in mice treated with ABZ. Additionally, prodrugs 2 and 3 were also detected in these samples. In regard to the anthelmintic activity of ABZ prodrugs, it was shown that compound 3 was more active than compound 2. Additionally, it was as effective as ABZ against T. spiralis pre-adult, adult, and female fecundity. However, compound 3 was not as active as ABZ against the muscle stage of the parasite. CONCLUSIONS: Compound 3 had better anthelmintic activity against T. spiralis than compound 2. The bioconversion of compounds 2 and 3 to ABZ and/or ABZ-SO was demonstrated by HPLC, but they did not reach equivalent concentrations to that of ABZ. Prodrugs 2 and 3 were also present in plasma samples, suggesting that prodrugs were not efficiently reduced in the intestine of mice.

Detection of Trichinella infection in slaughter horses by ELISA and western blot analysis.

In order to determine the presence of Trichinella infections in horses slaughtered at an abattoir in Mexico, 147 serum samples were examined by two immunoenzymatic methods. Specific antibodies were detected by ELISA in 7% of the serum samples at a dilution 1:400 and in 10% at lower dilutions (1:20, 1:40) using Trichinella spiralis muscle larvae (ML) excretory/secretory (E/S) products. Serum samples from four naturally infected horses (confirmed by direct methods) gave negative O.D. values in an ELISA at a 1:400 dilution and only two of them were positive at a 1:20 and 1:40 dilutions. Serum samples from experimentally infected horses reacted by Western blotting with ML components with molecular weights of 47, 52, 59, 67, 72 and 105 kDa which correspond to the TSL-1 antigens. Serum samples from the four naturally infected horses and from the abattoir horses that were positive in ELISA using E/S antigens recognized several ML components, some of them reacted with all the TSL-1 antigens mentioned above and others recognized preferentially two or three of these molecules. Since the serologic assays may not offer the sensitivity required in the diagnosis of horses trichinellosis and the direct methods had not always been useful in the detection of larvae in horsemeat related to trichinellosis outbreaks in Europe, it is proposed that additional assays are performed to determine Trichinella infection in horses. These can include detection of parasite antigens by ELISA and Dot ELISA or PCR, which in turn may also help to determine the presence of the parasite in early and late infections of horses.

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.

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite.

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.

[Updates on equine trichinellosis]. / Actualidades sobre la triquinelosis equina.

Trichinellosis is a zoonosis caused by parasites of the genus Trichinella. Transmission of trichinellosis to humans has been shown to occur mainly by the ingestion of meat from pigs, bears of foxes parasitized with muscle larvae of this parasite. However, in Europe, the major human outbreaks of the disease have occurred due to the ingestion of parasitized horse meat. Although the larvae were not isolated from the horse meat, the identification of larvae as T. nativa, T. britovi and T. spiralis was done in biopsy samples obtained from infected individuals. More recently T. spiralis muscle larvae have been isolated and identified, for the first time, in muscle tissue of horses slaughtered at an abattoir in the State of Mexico. Furthermore, in ELISA assays using total extracts or TSL-1 antigens, circulating antibodies against Trichinella have been detected in horses slaughtered at abattoirs from various countries in Europe and Mexico. On the other hand, the experimental infection of horses with parasites of the genes Trichinella has been achieved by several research groups and data obtained regarding the kinetics of antibody production in these animals are important in the development of sensitive and specific diagnostic assays for horse trichinellosis. This will allow to determine the frequency of this infection in horses which are used for animal and human feeding. These assays will also be very helpful for designing strategies to control transmission on the disease by horse meat.

Detection of Trichinella spiralis muscle larvae in naturally infected horses.

Human trichinellosis outbreaks related to horsemeat consumption have been reported in France and Italy in recent years. In order to determine if Trichinella is present in horses slaughtered at an abattoir in the State of Mexico, diaphragm muscle tissue samples (22-37 g) from 80 horses were examined by artificial digestion. Four of these samples had larvae that were characterized as Trichinella sp. by morphological criteria and as Trichinella spiralis by the polymerase chain reaction.

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.

[Current aspects of trichinosis diagnosis]. / Aspectos actuales sobre el diagnóstico de la triquinelosis.

An important approach to the study of trichinellosis is the development of sensitive diagnostic methods that allow the detection of Trichinella spiralis. Recently, ELISA assays that use surface/stichosome and/or secretion/excretion antigens from muscle larvae have been proved to be highly sensitive and specific in the diagnosis of the infection. Furthermore, the high immunodominance of carbohydrate residues on these molecules in a broad host range make them useful diagnostic markers for trichinellosis.

Improved methodology for the production of monoclonal antibodies against parasites.

A modification of the standard fusion methodology is described which results in greatly increased yields of monoclonal antibodies against certain organ-specific parasites. Several fusions were carried out using mice infected with Schistosoma mansoni or Nematospiroides dubius, using B lymphocytes harvested from either the spleen or the mesenteric lymph nodes. Results indicated a greatly improved yield of positive clones using the lymph nodes as a source of B cells for fusion. A 7-fold increase in the number of positive clones was seen with N. dubius injections, while S. mansoni fusions showed a 2-fold increase.