tyvelose and 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.

Topics: Animals; Antigens, Helminth; Gastrointestinal Tract; Hexoses; Larva; Molecular Structure; Muscle, Skeletal; Trichinella; Trichinellosis

A survey on porcine trichinellosis was organised in Ecuador between 2000 and 2003. Blood samples were taken in slaughterhouses (study 1, n=2000; study 2, n=331) and in a remote village where pigs are free roaming (study 3, n=646) and examined by ELISA using excretory/secretory (E/S) antigens. Seven samples (0.35%) in study 1 and none of the samples of study 2 were serologically positive. Thirty-seven (5.72%) village pigs tested positive by E/S ELISA in study 3. Sero-positive results by the E/S ELISA in study 1 were confirmed by ELISA using beta-tyvelose antigen, and by immunoblot. Muscle samples taken from pigs slaughtered in the abattoir (study 2) and from animals that showed a positive serology in study 3 were examined by trichinoscopy and artificial digestion. These techniques failed to demonstrate the presence of muscle larvae. The results of this survey need confirmation, but suggest that Trichinella is present in Ecuador; however, prevalence and parasite burdens are likely to be very low. The likelihood of detecting trichinellosis are higher in traditional settings than in pigs raised on improved farms.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Ecuador; Enzyme-Linked Immunosorbent Assay; Helminth Proteins; Hexoses; Immunoblotting; Muscles; Seroepidemiologic Studies; Swine; Swine Diseases; Trichinella; Trichinellosis

In Nepal, animal husbandry is a major source of income. Pig husbandry is practiced in rural, peri-urban, and urban communities. Free ranging "back yard" pigs and the practice of feeding offal is a very common management practice which potentially allows for the transmission of trichinellosis; however, this zoonosis has never been reported from this region. A total of 425 serum samples were collected from local pigs. These were initially screened by ELISA after which positive samples were examined by Western blot. This procedure identified two samples which had clear specific bands for Trichinella; however, muscle samples tested by HCL-pepsin digestion were found to be negative. If these highly specific serological analyses are confirmed, this would be the first report of trichinellosis in Nepal and a prevention program should be initiated to limit the access of pigs to open garbage dumps which exist both in towns and on farms.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Helminth Proteins; Hexoses; Meat; Muscles; Nepal; Seroepidemiologic Studies; Swine; Swine Diseases; Trichinella; Trichinellosis

For the surveillance of trichinellosis, the digestion method is reliable but also labour intensive. The serological methods for the detection of Trichinella-specific antibodies using ELISA offer a sensitive and relatively specific alternative. For serological studies, sera or plasma from blood samples are the most common source of antibodies, but although the concentration of antibodies is approximately 10-fold lower, muscle fluid can be a good alternative particularly for testing of wildlife samples. In the present study, an indirect ELISA technique was evaluated on both sera and muscle fluids from experimentally infected foxes, pigs, and wild boars using both excretory/secretory (E/S) antigens and a synthetic glycan antigen, beta-tyvelose. Although the synthetic antigen appears to be less sensitive than the E/S antigens, Trichinella-specific IgG antibodies were detected in both serum samples and muscle fluid samples from pigs, wild boars and foxes infected at levels which would be important for food safety or represent a significant reservoir for further transmission.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Enzyme-Linked Immunosorbent Assay; Foxes; Helminth Proteins; Hexoses; Muscle, Skeletal; Sus scrofa; Swine Diseases; Trichinella; Trichinellosis

This study re-evaluates 13 out of 48 subjects involved in a trichinellosis outbreak that occurred in Central Italy (Umbria Region) in 1988 resulting from the consumption of raw boar meat harboring Trichinella britovi. During the outbreak, 28 of 48 serologically positive subjects were asymptomatic, whereas 20 subjects presented one or more clinical signs including but not limited to fever, myalgia, periorbital oedema and conjunctivitis. Several patients were hospitalized with severe clinical signs requiring treatment with mebendazole and corticosteroids. Upon re-evaluation of 13 patients, none presented clinical signs; however, three still had increased CPK or LDH serum levels with some signs of electromyographic changes. In this study, enzyme immunoassays (EIA) were used to test the 13 positive sera for reactivity with T. britovi antigens using both excretory/secretory (E/S) antigens and a synthetic antigen composed of beta-tyvelose conjugated to bovine serum albumin. Western blots (WB) were also carried out using a commercial kit. Studies using EIA with E/S antigen identified five positive sera; however, using beta-tyvelose as antigen, only one positive sample was identified. Nearly all sera reacted positively with one or more Trichinella antigens when analyzed by WB, in particular to the 45 k Da beta-tyvelose containing glycoprotein. Results indicate that T. britovi, though less pathogenic than other Trichinella species, is clearly capable of inducing sustainable sequelae.

Topics: Animals; Antigens, Helminth; Blotting, Western; Creatine Kinase; Disease Outbreaks; Electromyography; Female; Follow-Up Studies; Food Parasitology; Helminth Proteins; Hexoses; Host-Parasite Interactions; Humans; Immunoenzyme Techniques; Italy; L-Lactate Dehydrogenase; Male; Meat; Muscles; Swine; Trichinella; Trichinellosis

In the Balkan countries, where trichinellosis is a re-emerging zoonosis, it is of great importance to determine Trichinella infection prevalence among the major hosts, including horses. One method for monitoring prevalence is serological surveillance; however, the validity of serological methods in horses is not well understood. The dynamics of anti-Trichinella IgG production and circulating excretory/secretory (ES) antigens were investigated in three horses experimentally-infected with Trichinella spiralis. Horses were slaughtered at 32 week post infection (p.i.). Low worm burdens were found in all three animals. Anti-Trichinella IgG was detected up to 32 weeks p.i. by an indirect immunofluorescence assay (IFA) and by Western blot (Wb), but not by ELISA. The ELISA test detected antibodies for only a short period of time (up to 18 weeks p.i. using ES antigen or up to 20 weeks p.i. using tyvelose-BSA antigen). The presence of circulating muscle larvae ES antigen in sera of infected horses was observed by dot blot from the 4th week p.i. up to the 32nd week p.i.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Blotting, Western; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique, Indirect; Helminth Proteins; Hexoses; Horse Diseases; Horses; Tongue; Trichinella; Trichinellosis; Yugoslavia

Over the years, the opinions of clinicians on the existence of the so-called chronic trichinellosis or late sequelae of infection have differed. However, the persistence of a humoral immune response against Trichinella in these late-stage patients has been confirmed using specific tests such as the competitive inhibition assay (CIA). We evaluated sera from late-stage trichinellosis patients (2--8 years from acute infection), for their reactivity against Trichinella spiralis antigens. The following tests were carried out: (i) indirect immunofluorescence assay (IFA), performed on muscle sections from mice, 30 days following synchronous infection by intramuscular injection with T. spiralis newborn larvae (NBL); (ii) enzyme immunoassay, employing a synthetic beta-tyvelose antigen conjugated to bovine serum albumin (BSA-Ag); and (iii) western blot (WB) with both an "in house" kit and a commercial kit. The results of IFA obtained by confocal laser microscopy showed that sera reacted against both surface and internal structures of L(1) larvae but at varying levels. Employing the synthetic antigen, EIA showed that 50% of sera tested were positive for the presence of specific antibodies against beta-tyvelose. By WB, all sera were reactive with the 45 k Da glycoprotein (45 gp). These data suggest that reactivity against the beta-tyvelosylated 45 gp persists even in very late stages of human trichinellosis.

Topics: Adult; Animals; Female; Fluorescent Antibody Technique, Indirect; Follow-Up Studies; Glycoproteins; Helminth Proteins; Hexoses; Host-Parasite Interactions; Humans; Immunoenzyme Techniques; Male; Mice; Microscopy, Confocal; Middle Aged; Muscles; Poland; Trichinella spiralis; Trichinellosis

The Inuit population of the Arctic has always been at risk of acquiring trichinellosis and severe outbreaks have been recorded in Alaska and Canada. In West Greenland, a number of large outbreaks took place during the 1940s and 1950s; they involved total 420 cases including 37 deaths. Since then only sporadic cases have been reported. Here, we describe an outbreak of infection with Trichinella spp. after consumption of infected meat presumably from walrus or polar bear caught in western Greenland. Six persons who had eaten of the walrus and polar bear meat were two males and four females, age range 6--47 years. Using ELISA and Western blot analysis (Trichinella-specific IgG antibodies against excreted/secreted antigen and synthetic tyvelose antigen, respectively) four of these persons were found to be sero-positive for Trichinella antibodies, with three of these having clinical symptoms compatible with trichinellosis. On re-test, 12--14 months later one of the two sero-negative persons had sero-converted, probably due to a new, unrelated infection. This study demonstrates that acquiring Trichinella from the consumption of marine mammals remains a possibility in Greenland, and that cases may go undetected. Trichinellosis in Greenland can be prevented by the implementation of public health measures.

Topics: Adult; Animals; Antibodies, Helminth; Antigens, Helminth; Blotting, Western; Child; Disease Outbreaks; Enzyme-Linked Immunosorbent Assay; Eosinophilia; Female; Food Parasitology; Greenland; Helminth Proteins; Hexoses; Humans; Male; Meat; Middle Aged; Trichinella; Trichinellosis; Walruses

Two enzyme-linked immunosorbent assay (ELISA) systems, one using natural excretory-secretory (ES) antigens and the other a synthetic glycan antigen (3,6-dideoxy-D-arabinohexose [tyvelose, TY]), were evaluated for the serological diagnosis of trichinellosis in swine. Sensitivity was estimated using samples (n = 113) collected 3-21 wk PI from 15 experimentally infected pigs, and specificity was estimated using samples (n = 397) from a population of Trichinella spp.-free pigs. Results were analyzed using 2 cutoff values recommended in international guidelines (Office Internationale des Epizooties [OIE]) and by the optimal cutoff level as determined by receiver-operator characteristic (ROC) analysis. The ROC-optimized TY-ELISA consistently performed better than all other combinations. None of the combinations of test and cut-off detected infected pigs sooner than 35 days; however, the ROC-optimized TY-ELISA identified 8 of 15 pigs earlier than the ES-ELISA and detected 2 pigs missed by all other tests. At 49 days PI the sensitivity and specificity of the ROC-optimized TY-ELISA were 94.3 and 96.7%, respectively, as compared with the ROC-optimized ES-ELISA at 84.9 and 96.0%, respectively. The ROC-optimized TY-ELISA was 100% specific at OIE-recommended cut-offs. This study indicates that the TY-ELISA is as good or better than the ES-ELISA for the detection of trichinellosis in swine.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Enzyme-Linked Immunosorbent Assay; Female; Hexoses; ROC Curve; Sensitivity and Specificity; Swine; Swine Diseases; Trichinella; Trichinellosis

The unusual sugar tyvelose is the immunodominant portion of the major larval glycoprotein antigens of Trichinella spiralis, which play an important role in generating immunity against the intestinal stages of infection. The possibility that the tyvelose component itself may have a host- or parasite-protective role in the intestine was tested by following the outcome of challenge infections in mice primed and boosted with tyvelose-BSA, or in mice primed with tyvelose-BSA before boosting with larval antigen. Although antibody responses were raised against tyvelose there was no evidence of protective immunity against the intestinal stages, as assessed by total adult worm recovery or by size and fecundity of female worms in immunized mice. Equally, priming with tyvelose-BSA before boosting with larval antigen had no effect on the expression of immunity against a challenge infection. The predominant antibody isotype recorded in all immunized mice was IgG1, suggesting the induction of type 2 T cell responses, and this was confirmed by cytokine analysis, mesenteric node lymphocytes of all mice showing production of IL-5 but not IFN-gamma. Clearly immunization with tyvelose had no significant effect on T cell polarization. The data show that, with the experimental design employed, there was no evidence for a functional role of tyvelose in either host- or parasite-protection during the intestinal phase of infection.

Topics: Animals; Antibodies, Helminth; Antigens, Helminth; Female; Hexoses; Immunization; Interleukin-5; Intestines; Larva; Mice; Serum Albumin, Bovine; Trichinella spiralis; Trichinellosis

Hosts infected with Trichinella produce antibodies specific for an epitope common to the TSL-1 family antigens. This epitope contained uncommon terminal 3, 6-dideoxy-D-arabinohexose (so called tyvelose) residues. The disaccharide moiety was synthesized and an immunodiagnostic assay was developed, which was specific and sensitive in swine trichinellosis. We aimed to verify the specificity and sensitivity of this immunodiagnostic test in human trichinellosis. 15 sera from normal subjects, 12 from patients with other parasitic diseases and 50 from trichinellosis patients were tested. Indirect enzyme linked immunosorbent assay (ELISA) for specific IgG and an amplified ELISA for specific IgE were performed using beta-tyvelose-GalNAc-bovine serum albumin (BSA) disaccharide conjugate or T. spiralis muscle larvae excretory/secretory (E/S) products, as antigens. Neither control sera nor other parasitic infection sera resulted positive both for IgG and IgE when synthetic or E/S antigens were used. In trichinellosis patient sera, specific IgG were present in 100% of cases, irrespective of the antigen used, but whereas specific IgE were detected in 78% using E/S antigens, a 100% positivity rate was obtained, using the beta-tyvelose-BSA conjugate.

Topics: Antibodies, Helminth; Antibody Formation; Antigens, Helminth; Disaccharides; Enzyme-Linked Immunosorbent Assay; Epitopes; Hexoses; Humans; Immunoglobulin E; Immunoglobulin G; Reference Values; Sensitivity and Specificity; Serologic Tests; Trichinellosis

Infection of mammalian skeletal muscle cells by Trichinella spiralis induces a series of changes that include: reentry of the terminally differentiated host cell into the cell cycle; suspension of infected cells in apparent G2/M; and transcriptional inactivation of the differentiated skeletal muscle gene program. Cell cycle repositioning and genetic reprogramming are chronic characteristics of host cells that can remain infected for years. Nuclear antigens (NA, 79, 86 and 97 kDa) that localize to host cell nuclei have been detected with antibodies against T. spiralis proteins. Since NA may play a role in regulating the infected cell phenotype, their origin, nuclear compartmentalization, and biochemical properties were investigated. We show that a monoclonal antibody to a defined epitope of T. spiralis glycans binds these NA, which indicates the parasite origin of these proteins. NA were not extracted under conditions that solubilized chromatin from infected cell nuclei. In contrast, NA were coextracted with B lamins (nuclear envelope) by 4 M urea. Urea extraction was pH dependent (8.0), suggesting ionic interaction of NA in protein complexes. Nevertheless, confocal microscopy demonstrated colocalization of NA with host chromatin, and not B lamins. Nuclear protein complexes containing NA were observed under non-reducing conditions, and NA were readily cross-linked in isolated nuclei by succinimidyl protein conjugating reagents. The results establish methods to extract NA from infected cell nuclei for further biochemical analysis, establish the existence of nuclear protein complexes containing NA and demonstrate colocalization of NA with host chromatin. Collectively, the results provide a foundation from which to investigate the role of NA in regulating the T. spiralis infected skeletal muscle cell phenotype.

Topics: Animals; Antigens, Helminth; Cell Nucleus; Chromatin; Epitopes; Gene Expression Regulation; Helminth Proteins; Hexoses; Hydrogen-Ion Concentration; Mice; Mice, Inbred BALB C; Muscle, Skeletal; Nuclear Envelope; Solubility; Trichinella spiralis; Trichinellosis; Urea