Guillermo G. Nuñez

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis.

An immunoelectrotransfer blot assay (IETB) using excretory–secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin–biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at ∼55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at ∼55 kDa was present in all trichinellosis sera, whereas the ∼14-kDa band was present in only a small percentage of sera. The development of ∼36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.

Increased parasiticide activity against Trichinella spiralis newborn larvae during pregnancy

Abstract. To evaluate whether pregnancy has a synergetic effect on the hosts 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.

Trichinella spiralis infection rapidly induces lung inflammatory response: the lung as the site of helminthocytotoxic activity.

In the present work, we studied the kinetics of the appearance of different immunological parameters in the lungs during the intestinal phase of infection with Trichinella spiralis. We also evaluated the lungs role in the retention and death of this helminth in its migratory stage. To study these parameters, we used lung extracts, lung cell suspensions and rat lung tissue sections. During the intestinal phase of infection (days 0-13 post-infection, p.i.), an inflammatory response is elicited in the lungs, which reflects humoral, cellular and functional changes. These changes included an increased number of mast cells and eosinophils and the local production of IL-4, IL-5, IL-10, TNFα, IFNγ, IL-13, CCL11 and CCL28. We found hyperplasia of the bronchus-associated lymphoid tissue (BALT). Total and specific IgA, IgE, IgG1 and IgG2a were detected locally. The retention of the migratory larvae in the lung, together with the ex vivo cytotoxic capacity of the lung cells and antibodies present in the lung extracts, suggested that the lung was one of the immune defense organs against the pathogens migration stage.

Diagnosis of porcine trichinellosis: parasitological and immunoserological tests in pigs from endemic areas of Argentina

In order to compare the reliability of serological and parasitological techniques for the diagnosis of porcine trichinellosis from endemic areas in Argentina, 116 pigs were studied: 61 animals from two separate outbreaks and 55 from a small abattoir. Direct diagnostic techniques included trichinoscopy and the artificial digestion method. Indirect diagnostic tests used in this study were the enzyme-linked immunosorbent assay (ELISA), employing the excretory-secretory products of muscle larvae (ML) as antigen, and the indirect immunofluorescence assay using as antigen ML in suspension (IIF-susp), cryostat sections of infected rat muscle or of free ML (IIF-slide). The percentage of parasitologically positive pigs was invariably lower than that of serologically positive animals (IIF-slide), even when digestion studies were carried out individually with a greater amount of muscle sample than required by current regulations. Close correlation was found between IIF using as antigen tissue sections and IIF using free ML sections, while IIF-susp proved unsuitable for diagnosis since this assay presented a high percentage of false negative results (20%). The IIF-slide technique proved positive in all parasitologically positive animals. ELISA rendered a lower percentage of positive reactions than IIF-slide, especially when worm burden was low. Since most parasitologically positive animals rendered at least two positive serological tests (two variations of IIF or IIF plus ELISA), those negative by digestion and positive by two serological methods were strongly suspected of having trichinellosis. Upon studying swine from a abattoir it was found that 9% of the pigs were positive when assayed by two serological techniques, but Trichinella spiralis infection could not be parasitologically confirmed. To sum up, serological methods may be used for screening all pigs and positive findings should be tested by the digestion method by analysing a greater quantity of pork than that required by current regulations, above all in areas with reported clinical trichinellosis in humans, to ensure that the pork is safe for human consumption.

In vitro and in vivo effects of progesterone on Trichinella spiralis newborn larvae

We have previously demonstrated that during pregnancy there exists an increased parasiticide activity against Trichinella spiralis newborn larvae (NBL) in infected rats. In this work we analysed the contribution of peritoneal cells from noninfected pregnant rats to the mortality of the NBL in cytotoxicity assays, and evaluated the role of progesterone in this effector mechanism. Our findings suggest that progesterone can induce activation of effector peritoneal cells to destroy the NBL in a rapid and antibody-independent manner. The administration of progesterone to ovariectomized rats also led to a significant decrease in the parasite load of the animals, thus demonstrating that progesterone induces the increase of the parasiticide activity of the leukocytes involved in the mechanisms of NBL death.

Cytotoxicity-blocking antibodies in human chronic trichinellosis.

Abstract Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.

Detection of coproantibodies and faecal immune complexes in human trichinellosis

Trichinella spiralis is the nematode causative agent of trichinellosis, an intestinal and tissular parasitosis. Even though an early diagnosis during the intestinal phase is essential to limit the infection in humans, to date, there are no available tests to achieve this goal. Based on the immune response generated by the hosts intestinal mucosa, the aim of this work was to develop ELISAs to assess the presence of coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes in stool samples of 18 individuals belonging to different outbreaks that have arisen in Argentina. By the methodologies developed in this work it was found that anti-muscle larva excretory-secretory products (ML-ESP) CAbs were detected in 89% of the samples analysed regardless of the time p.i. Anti-ML-ESP IgA, IgG, IgE and IgM were detected in 56%, 56%, 28% and 22% of the individuals respectively. Those samples negative for anti-ML-ESP total immunoglobulins proved positive for anti-adult worm-ESP CAbs. No CAgs were detected in any of the samples. The results obtained in this work indicate that the intestinal immune response in human trichinellosis is featured by all the isotypes of specific immunoglobulins. Furthermore, the detection of antibodies in stool samples, in either the free or complexed form, could be applied to confirm early human trichinellosis.

Immunoparasitological parameters of the intestinal phase of trichinellosis in rats.

Enzyme-linked immunosorbent assays (ELISAs) were developed in order to detect coproantigens (CAgs), coproantibodies (CAbs) and faecal immune complexes (FIC) in rats experimentally infected with Trichinella spiralis. The usefulness of these assays was compared to that of a conventional ELISA for detection of serum antibodies (Abs) to muscle larvae excretory-secretory products (ML-ESP). The ELISA for CAgs was the first parameter to give a positive result but the detection was limited only to day 2 p.i. CAbs against ML-ESP and adult worm excretory-secretory products (AW-ESP) was first positive on day 4 p.i. Anti-ML-ESP remained positive until day 12 p.i. while CAbs against AW-ESP remained positive throughout the study period. Specific IgE and IgA were found. FIC were detected between days 2 and 8 p.i. Serum Abs began to appear on day 10 p.i. Therefore, the ELISA for CAbs was a suitable assay for the detection of the enteral and early phases of the infection.

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.