Alterations of the erythrocyte membrane by in vitro action of Trichinella spiralis muscle larvae.

The complex life cycle of Trichinella spiralis includes the migration of newborn larvae through the bloodstream to their encystment in muscle. The parasite establishes an intimate contact with the erythrocytes of the host both during the migration of the newborn larvae and when encysting, as this parasite causes intense vascularization in the muscle cell. The goal of this work was to study the effects of various concentrations of T. spiralis muscle larvae (ML) on erythrocyte membranes. The treatment was performed by incubating human erythrocytes with equal volume of different concentrations of ML for 30 minutes, with controlled agitation (37°C). The control erythrocytes (with no contact with the larvae) were incubated in the same way with an equal volume of physiological solution. To evaluate the alterations to the erythrocytes by the action of the larvae and in the respective controls, an Erythrocyte Rheometer and a Digital Image Analysis technique were used. The results indicated that when the larval concentration was higher, the aggregation and erythrocyte membrane alterations were also higher. Also, the erythrocyte deformability index and the erythrocyte elasticity increased. The values of isolated cell coefficient varied from 0.51 in the treatment with 100 larvae/ml to 0.91 in the incubation with 1000 larvae/ml. This experiment shows that T. spiralis muscle larvae affect significantly the red blood cell aggregation and the erythrocyte viscoelastic properties.

In vitro alterations of erythrocyte aggregation by action of Trichinella spiralis newborn larvae.

Possible changes in the erythrocyte membrane, by in vitro interaction with newborn larvae of T. spiralis (NL), were evaluated analyzing the alterations in erythrocyte aggregation by digital image analysis and laser transmission in a new optical chip aggregometer. NL were obtained from CBi mice infected with T. spiralis. RBCs samples from healthy donors where in vitro exposed to NL (concentration (3000 ± 500) larvae/mL) to assess its effect on RBC aggregation. Individual cell Coefficient (CCA) and aggregation parameter (S) were calculated by digitally processing RBC aggregate images, indicating the amount and size of the erythrocyte aggregates present. Also, size distribution of aggregate was analyzed. Kinetic aggregation parameters (Amp750 and t1/2) were calculated with a new optical chip aggregometer. Results show significant alterations in erythrocyte aggregability due the in vitro action of T. spiralis larvae increasing incubation time. These results are possibly related to the loss of surface sialic acid as it is captured by NL. Obtained results suggest that NL could produce hemorheological alterations in the host, which could be related to thrombosis and anemia reported in some patients with trichinosis.

Evaluation of three immunoserological techniques in the detection of porcine trichinellosis.

Three immunoserological tests (IST) used for the detection of porcine trichinellosis, immunofluorescence (IF), enzyme-inmunoanalysis (EIA), and Western blot (WB), were compared. Three groups of animals were analyzed: Group 1, animals naturally infected with parasite burdens (PB) of <1 muscle larvae (ML)/g (n=18); Group 2, animals naturally infected with PB of > or =2ML/g (n=23); Group 3, animals raised and home-slaughtered on farms in Argentina (n=59). Animals from Groups 1 and 2 were identified in outbreaks and were analyzed by individual artificial digestion (AD) of > or =30g of muscle. Animals in Group 3 were subjected to AD of 5g of muscle. The detection percentages in sera of swine with the lower PB were 100% for IF, 72% for EIA, and 50% for WB. Eighty-three percent of the animals were serologically positive by two or three techniques. In pigs with the higher PB, the detection percentage was similar for IF and EIA (100% vs. 91%, respectively), and was lower for the WB (61%). Ninety-six percent of the animals were serologically positive by two or three techniques. Group 3 animals had similar detection percentages for the three techniques (IF, 30%; EIA, 29%; WB, 42%). Twenty-five percent of the animals were serologically positive by two or three techniques. Two animals were positive by AD with PB of 0.33 and 2.4ML/g, and were positive for IF and WB, or IF, EIA, and WB. Results indicate that the sensitivity of each technique depends on the PB, and always ranked in sensitivity as IF>EIA>WB. For the lower PB, the decrease in the sensitivity is more pronounced for the EIA. Although the WB has a low sensitivity, the detection of the specific bands for Trichinella spiralis makes it a useful confirmatory tool. Considering that more than 83% of the parasitologically positive animals had 2 or 3 positive serological results using the techniques tested here, for the diagnosis of porcine trichinellosis, pigs positive by two of these serological techniques must be regarded as truly infected pigs.

Survey on porcine trichinellosis in Ecuador.

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.

Increased parasiticide activity against Trichinella spiralis newborn larvae during pregnancy.

To evaluate whether pregnancy has a synergetic effect on the host's immune response against Trichinella spiralis infection, immunological and parasitological parameters relating to the infection were assessed in pregnant rats and compared to those observed in virgin infected rats. The muscle parasite load was lower in pregnant infected rats but no differences were found in the intestinal worm burdens or the fecundity of female worms. The ability of sera to mediate death in newborn larvae (NBL) in an antibody-dependent cell cytotoxicity assay was higher for pregnant rats, even in the absence of specific anti-NBL antibodies. High levels of total and anti-NBL IgE were found in both groups, however, these levels were higher in the group of pregnant infected animals. No differences were found in anti-NBL IgGAM titers, nevertheless in some pregnant infected rats these antibodies were found earlier. No differences were found in peritoneal or blood eosinophil counts. Offspring born to infected dams were found to be infected. The results obtained in this model demonstrate that during pregnancy there is an enhanced helminthotoxic effect towards the NBL. Despite this immunoactivation, vertical transmission of the parasite is possible.

Detection of circulating and fecal Trichinella spiralis antigens during experimental infection using monoclonal antibodies against the new born larvae.

Different assays to detect antigens of Trichinella spiralis during current infection have been standardized, although sensitivity values have been the main limitation to use them as routine diagnostic test. We report the production and use of monoclonal antibodies (MAbs) against the new born larvae which recognize both somatic and metabolic antigens from adult and muscular larvae (ML). We used two IgG3 MAbs (4B1, 4B2) and two IgG2a (2D3, 2D4) to detect antigens during experimental infection. All MAbs detect fecal antigens starting the second to third week post infection (wpi), although it was less clear with MAbs 4B1 and 4B2, while circulating antigens were detected from third to fourth wpi. Thus, the recognition of shared antigens among T. spiralis developmental stages can be used for early diagnosis of trichinellosis.

Class specific antibody responses to newborn larva antigens during Trichinella spiralis human infection.

A follow-up study of the class antibody responses to newborn larva (NBL) antigens in individuals involved in an outbreak of human trichinellosis was carried out by ELISA assays. The data showed that similar kinetics of antibody responses of different magnitude developed in trichinellosis patients; it was low by week 3, a peak raised by week 5 and decreased from week 7 up to the end of the study. The IgA-ELISA assay was the most sensitive and specific while the IgM was the least sensitive and specific. IgA antibodies to NBL antigens were detected in 80% of patients while IgE, IgG and IgM responses were observed in 44, 31 and 19% of the patients by week 3, respectively. From weeks 5 to 7, IgA antibodies were found in 89 to 100% of the patients while lower percentages (0-82%) were found for the other isotypes. Reactivity of IgA, IgE, IgG and IgM to NBL antigens decreased from week 37 to 57 after infection (0-38%). These results suggest that detection of IgA antibodies may be useful for early diagnosis and epidemiological studies in human trichinellosis.

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.

A follow-up study of the human class and subclass antibody response developed against the adult stage of Trichinella spiralis.

We report the analysis by ELISA of class and subclass antibody response against a total soluble extract from T. spiralis adult stage (TSE-A) during a year after the infection in 17 symptomatic trichinellosis patients (SI) and five asymptomatic individual (AI) involved in an outbreak of trichinellosis occurred in the State of Mexico. Serum samples from 20 healthy individuals (HI) and 24 patients with other parasitosis were included as control. All SI showed a polyisotypic antibody response against the TSE-A, during the infection. Higher response of IgA, IgE, IgM were detected in SI during the acute phase of the infection, but only IgE remained at high levels all along the infection. None or a lower reactivity against TSE-A was observed in sera from AI and from HI. Some patients with trichuriosis and ascariosis showed a higher cross-reactivity, against TSE-A when IgG and their subclasses were analyzed.

Serological evaluation of thin-layer immunoassay-enzyme-linked immunosorbent assay for antibody detection in human trichinellosis.

A new immunoenzymatic test, named the thin-layer immunoassay-enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 microl each of antigen (7 microg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis.

Trichinella spiralis infection in pigs in eastern Bolivia.

Trichinellosis in pigs in Bolivia was first documented in 1993 following a small abattoir survey in a rural community in the Bolivian Altiplano. The present study investigated the presence of antibodies to Trichinella spiralis in pigs in the 2 largest departments in terms of pig production in Bolivia. Three geographically separate abattoir surveys were conducted to cover the major production areas in the Departments of Santa Cruz and Chuquisaca. Sera were tested using an enzyme-linked immunosorbent assay. Of the 1,327 sera analysed from the 3 areas, 13.4% overall tested positive. Results from the 3 individual surveys varied from 10.2% seropositivity to 17.1 per cent. However, within each of the 3 sample areas, highly significant variation in seropositivity was encountered, with those areas with the most extensive production systems having the highest percentage of positive sera. Such variation is probably due to differences in nutrition with foraging and household waste being important components of pig diets in extensive production systems. The results of this study were similar to those obtained from the previous survey in the Altiplano and indicate that trichinellosis is present throughout Bolivia and is a potentially important public health problem.

Immune cytotoxic activity of human eosinophils against Trichinella spiralis newborn larvae.

The ability of human eosinophils to kill the newborn larvae (NBL) of Trichinella spiralis of different maturation status, in the presence of antibody, was studied. A cytotoxic in vitro test was performed using NBL less than 2 h of age (NBL2) or NBL maintained in culture at 37 degrees C for 20 h (NBL20), peripheral blood eosinophils, anti-Trichinella serum and human fresh serum as source of complement. Under these experimental conditions eosinophils from normal individuals attached to NBL2 as well as to NBL20 but only the latter were killed. On the other hand, eosinophils from volunteers with eosinophilia killed NBL regardless of larval age. Neither adherence nor significant mortality was observed in the absence of immune serum. These results indicate that NBL maturation and eosinophil activation status are crucial for antibody-dependent cellular cytotoxic reaction (ADCC).

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.

Recognition of Trichinella spiralis muscle larvae antigens by sera from human infected with this parasite and its potential use in diagnosis.

Human antibody response to total soluble extract of Trichinella spiralis muscle larvae (TSE) was analyzed by Western blot. The most frequently recognized antigens had molecular weights of 96, 67, 63, 60, 55 and 47 kDa. An antigenic fraction containing two peptides with M.W. of 43, 47 kDa from the parasite (p43, 47 Ts L1) was isolated by elution from polyacrylamide gel slabs. It was used as antigen in an ELISA test and compared to that of TSE. Serum samples from 51 symptomatic trichinellosis patients--43 with high antibody levels to TSE, 5 of them with positive biopsy and 8 with low levels of these antibodies--as well as 38 from asymptomatic individuals from the area where the trichinellosis outbreaks had occurred and 43 from apparently healthy individuals from a non-endemic area, 37 from patients with intestinal parasitic infections caused by helminth and protozoan parasites--11 from recurrent and 26 from non-recurrent disease--were analyzed by ELISA using both antigens. The ELISA using p43, 47 Ts L1 detected all trichinellosis patients with high antibody levels as well as 6 out of 8 of those with low antibody levels. All control groups were negative. Therefore, this purified fraction allowed the ELISA to be more specific and sensitive for human trichinellosis diagnosis.

[The indirect hemagglutination reaction in the diagnosis of trichinosis]. / La reacción de hemaglutinación indirecta en el diagnóstico de la triquinosis.

An indirect hemagglutination test (IHAT) for trichinosis using a Melcher's antigen was evaluated and compared with a precipitin test (PT) and a bentonite flocculation test (BFT). One hundred and forty eight serum samples from patients from the whole country confirmed or suspected to have trichinosis by clinical or epidemiological evidences were studied: 117 (79.1%) samples resulted positive by IHAT, 138 (93.2%) by PT and 65 (43.9%) by BFT. Sixty three serum samples from patients with strong clinical suspect of trichinosis presented the PT and the BFT positive and were compared with the IHAT for sensitivity study. IHAT was positive in 60 (95.2%) serum samples. In order to determine the specificity of IHAT 25 serum samples from healthy volunteers and 124 serum samples from individuals with other parasitoses, such as cysticercosis (48), hydatidosis (45) and fascioliasis (31) were studied. The specificity, using a titre > or = 1:16 as a possible diagnostic value was 96%. The use of IHAT with RP and BFT in the diagnosis of human trichinosis is discussed.

Basophils and eosinophils in three strains of rats and in athymic (nude) rats following infection with the nematodes Nippostrongylus brasiliensis or Trichinella spiralis.

A previous report showed that infection with the nematode Nippostrongylus brasiliensis stimulates a basophilia as well as an eosinophilia in the blood of August rats. The present study shows that blood levels of basophils and eosinophils were increased in two other rat strains, one inbred and one outbred, after infection with N. brasiliensis, and infection of two inbred rat strains with Trichinella spiralis also stimulated a basophilia as well as an eosinophilia. No increase occurred in basophils or eosinophils in athymic (nude) rats infected with N. brasiliensis, although both these cell types were found in the blood of control, specific pathogen free, nude rats in numbers comparable to those in specific pathogen free, heterozygote controls of the same strain. Rat basophils usually have few granules and in blood smears often appear as if they were partly degranulated. Basophils from uninfected nude rats contained more negative than positive staining granules compared with basophils from parasitized heterozygotes. The possession of small numbers of granules which vary in their reaction to stains of the Romanowski type is a normal feature of rat basophils in blood smears. Consequently rat basophils differ in these respects from those of other species.