Protein malnutrition impairs the immune control of Trichinella spiralis infection.

OBJECTIVES: We aimed to analyze the effect of a protein-deficient diet on mucosal and systemic immunity during a Trichinella spiralis infection. METHODS: Two groups of weaning Wistar rats received a protein-deficient diet (6.5% casein) and the other two groups received a control diet (20% casein). After 10 d, one group of each diet was infected (PDI and CI) with muscle larvae (infecting stage). Food intake and body weight were assessed over time. Blood eosinophils counts, antibodies in serum, and tissue extracts were assessed at different days postinfection. Histologic studies were done in the lungs and intestines, and adult worm (AW) fecundity index score and muscle parasite burden were determined. RESULTS: Food and protein intake were lower in PDI than in CI. Body weight was lower in PDI than in a non-infected protein-deficient diet. Eosinophils counts were lower in PDI than in CI. Total and specific antibodies were lower in PDI than CI. PDI had a reduced number of mast and goblet cells in the lungs and intestines compared with CI. The persistence of AW in the intestines and migrant larvae at the lungs was longer in PDI than in CI.. The AW fecundity index score was higher in PDI than in CI. Finally, PDI evidenced a higher muscular parasite burden than CI. CONCLUSIONS: Protein deficiency affects the mucosal and systemic immune response to Trichinella spiralis and delays the expulsion and increases the fecundity index score of AW, which leads to a higher parasite burden in the muscles.

Serum IgE and IgG4 against muscle larva excretory-secretory products during the early and late phases of human trichinellosis.

In human trichinellosis, the relevance of the presence and persistence of specific serum IgE and IgG4 during the early and late phases of infection is still controversial.The aim of this work was to determine the percentage of human sera presenting IgE and IgG4 against Trichinella spiralis muscle larvae excretory-secretory products as well as their levels during the early and late phases of the infection. The antigen recognition pattern by serum total immunoglobulins (IgGAM), IgE, and IgG4 was assessed over time. Serum samples during early and late phases were analyzed by ELISA and immunoelectrotransfer blot (IETB).Results showed that (a)-IgE and IgG4 are present at constant levels in both phases; (b)-IgE recognized the glycoproteins of ~ 45 and ~ 55 kDa and IgG4 only the ~ 45 kDa;

Trichinella spiralis infection and transplacental passage in human pregnancy.

Infection by Trichinella spp. during pregnancy still arises many questions. Worldwide an important number of trichinellosis outbreaks have occurred over time where pregnant women were or might have been involved. Parasitological and immunoserological parameters, clinical background and transplacental passage of Trichinella spiralis of six pregnant women, from different outbreaks, were studied. Serum samples were collected from patients and their children: at pregnancy, at delivery, newborn and/or infant (≥1 year-old). Total immunoglobulins (Igs) and isotypes against excretion-secretion products from muscle larvae (ESP-ML) and newborn larvae (NBL) of T. spiralis were detected by immunoserological techniques. Sera helminthocytotoxic activity against NBL was evaluated by antibodies dependent cell cytotoxicity assay. Patients presented similar clinical symptoms. Mother's sera showed differences in the presence and titres of Igs, IgE, IgG1 and IgG4 against NBL surface. All of them showed helminthocytotoxic activity against NBL, four in progesterone dependent manner and the other two by antibodies. Igs anti ESP-ML were present in mother's sera, with differences in the isotypes detected. Anti-ESP-ML IgE, IgA and/or IgM in newborn sera were observed. Three out of four infants showed specific Igs; one even at 8 years-old. The distinct immune response between mothers at pregnancy evidenced by sera helminthocytotoxic activity and immunoserological parameters may be associated with a different parasite burden exposition. Specific Igs observed in newborns would be a suggestion of transplacental passage of this Igs and/or their production by the fetus. Specific Igs found in infants sera suggests that newborn larvae transplacental passage is possible.

Regulatory parameters of the lung immune response during the early phase of experimental trichinellosis.

Parasitic infection caused by Trichinella spiralis provokes an early stimulation of the mucosal immune system which causes an allergic inflammatory response in the lungs. The present work was intended to characterize the kinetics of emergence of regulatory parameters in Wistar rat lungs during this early inflammatory response, between days 0 and 13p.i. The presence of regulatory cells such as regulatory T cells (Tregs) and alternatively activated macrophages (AAM) was analyzed in lung cell suspensions. Moreover, a regulatory cytokine (TGF-ß) was studied in lung tissue extracts. Considering that newborn larvae (NBL) travel along the pulmonary microvasculature, the ability of this parasite stage to modulate the activation of lung macrophages was evaluated. For this purpose, lung macrophages from non-infected or infected rats (day 6p.i.) were cultured with live or dead NBL. Arginase activity (characteristic of AAM) and nitric oxide (NO produced by iNOS, characteristic of classical activated macrophages) were measured after 48h. Our results revealed a significant increase in the percentage of Tregs on days 6 and 13p.i., arginase activity on day 13p.i. and TGF-ß levels on days 6 and 13p.i. Lung macrophages from non-infected rats cultured with live NBL showed a significant increase in arginase activity and NO levels. Live and dead NBL induced a significant increase in arginase activity in lung macrophages from infected rats. Only live NBL significantly increased NO levels in these macrophages. The present work demonstrates for the first time, the emergence of regulatory parameters in the early lung immune response during T. spiralis infection. The immumodulatory properties exerted by NBL during its passage through this organ could be the cause of such regulation. Moreover, we have shown the ability of NBL to activate macrophages from the lung parenchyma by the classical and alternative pathways.

Trichinella spiralis: killing of newborn larvae by lung cells.

The migratory stage of Trichinella spiralis, the newborn larva (NBL), travels along the pulmonary microvascular system on its way to the skeletal muscle cells. The present work studies the capability of lung cells to kill NBL. For this purpose, in vitro cytotoxicity assays were performed using NBL, lung cell suspensions from Wistar rats, rat anti-NBL surface sera, and fresh serum as complement source. The cytotoxic activity of lung cells from rats infected on day 6 p.i. was compared with that from noninfected rats. Two and 20 h-old NBL (NBL2 and NBL20) were used as they had shown to exhibit different surface antigens altering their biological activity. Sera antibodies were analyzed by indirect immunofluorescence assay, and cell populations used in each assay were characterized by histological staining. The role of IgE in the cytotoxic attack against NBL was analyzed using heated serum. The FcεRI expression on cell suspensions was examined by flow cytometry. Results showed that lung cells were capable of killing NBL by antibody-dependent cell-mediated cytotoxicity (ADCC). Lung cells from infected animals yielded the highest mortality percentages of NBL, with NBL20 being the most susceptible to such attack. IgE yielded a critical role in the cytotoxic attack. Regarding the analysis of cell suspensions, cells from infected rats showed an increase in the percentage of eosinophils, neutrophils, and the number of cells expressing the FcεRI receptor. We conclude that lung cells are capable of killing NBL in the presence of specific antibodies, supporting the idea that the lung is one of the sites where the NBL death occurs due to ADCC.

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 lung's 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 pathogen's migration stage.

Trichinella infection in wild boars (Sus scrofa) from a protected area of Argentina and its relationship with the presence of humans.

In Argentina, Trichinella infection has been documented in humans and animals of several provinces since 1930. This zoonotic parasite infection has been recently detected in humans and pigs of a region historically considered as Trichinella-free, suggesting the spread of these pathogens. The aim of the present work was to investigate the presence of Trichinella infection in wild boars (Sus scrofa) and in the human population living in a protected area. Trichinella infection has been investigated by serology (in humans and wild boars) and by artificial digestion of wild boar muscles. The isolated Trichinella larvae have been identified at the species level by multiplex PCR. A geographical information system has been used to collect environmental data. The results showed the circulation of Trichinella spiralis in wild boars with a low parasite burden, and suggest the influence of human behavior on the transmission. The transplacental passage of parasite is postulated. It follows that the declaration of region as Trichinella-free should be carefully established by means of extensive monitoring programs, not only in humans and domestic animals but also in wildlife.

[Trichinellosis serology in blood-donors from a non-endemic area of Argentina]. / Serología para triquinosis en dadores de sangre en área no endémica de la Argentina.

The aim of the present study was to determine the presence of human trichinellosis in an area of Argentina historically regarded as free of this parasitic disease. This parasitosis was assessed on 150 blood donors by means of immunoserological tests, and their relation with risk alimentary habits by a questionnaire. The questionnaires showed that 98.0% (n=147) of the individuals consumed pork and pork products at a high frequency. The main pork products incorporated to the habitual diet were the stuffed ones. A 80.3% (n=118) of the individuals had acquired such products at butchers, a 38.1% (n=56) at home slaughters and a 34.0% (n=50) at supermarkets. The immunoserological techniques employed were: enzyme immunoanalysis, indirect immunofluorescence and/or immunoelectrotransfer blot assay. Anti-Trichinella antibodies were detected in 8.0% (n=12) of the serum samples by more than one of the methodologies employed, considering these results as confirmatory of trichinellosis. The studies carried out herein demonstrate the existence of human trichinellosis in an area historically regarded as free of this parasitic disease and suggest its relationship with the ingestion of pork or pork products as a risk factor. These results indicate that the lack of reports should not be the only criterion for an area to be considered as Trichinella-free. The awareness of the existence of the parasite in a region will favor the establishment of control and prevention strategies which is of fundamental importance to avoid the spread of the disease.

Serología para triquinosis en dadores de sangre en área no endémica de la Argentina / Trichinellosis serology in blood-donors from a non-endemic area of Argentina

El presente estudio tuvo como objetivo determinar la presencia de triquinosis humana en un área considerada históricamente libre de esta parasitosis en la Argentina. Se evaluó la parasitosis mediante técnicas inmunoserológicas y su relación con los hábitos alimenticios de riesgo mediante un cuestionario realizado a 150 donantes de sangre. Las encuestas revelaron que el 98.0% (n=147) de los individuos consumía carne de cerdo y/o derivados con elevada frecuencia. Los principales productos porcinos incorporados a la alimentación habitual fueron los chacinados. El 80.3% (n=118) de los individuos adquirían los productos en carnicerías, el 38.1% (n=56) en faenas domiciliarias y el 34.0% (n=50) en supermercados. Las técnicas inmunoserológicas utilizadas para el diagnóstico de triquinosis fueron: enzimoinmunoanálisis, inmunofluorescencia indirecta y/o inmunoelectrotransferencia. En el 8.0% (n=12) de los sueros de los individuos se detectaron anticuerpos anti-Trichinella por más de una de las técnicas realizadas, considerándose este resultado diagnóstico confirmatorio de triquinosis. Los estudios realizados demuestran la existencia de triquinosis humana en un área considerada libre de esta parasitosis y sugieren su relación con la ingesta de carne porcina y/o sus subproductos, hábito alimenticio de riesgo. Estos resultados indican que la falta de denuncia de casos de la enfermedad no debe ser el único criterio a tener en cuenta para considerar a una región área libre de triquinosis. El conocimiento de la presencia del parásito en un área determinada favorece la instauración de medidas de control y prevención evitando la diseminación de la parasitosis.

The aim of the present study was to determine the presence of human trichinellosis in an area of Argentina historically regarded as free of this parasitic disease. This parasitosis was assessed on 150 blood donors by means of immunoserological tests, and their relation with risk alimentary habits by a questionnaire. The questionnaires showed that 98.0% (n=147) of the individuals consumed pork and pork products at a high frequency. The main pork products incorporated to the habitual diet were the stuffed ones. A 80.3% (n=118) of the individuals had acquired such products at butchers, a 38.1% (n=56) at home slaughters and a 34.0% (n=50) at supermarkets. The immunoserological techniques employed were: enzyme immunoanalysis, indirect immunofluorescence and/or immunoelectrotransfer blot assay. Anti-Trichinella antibodies were detected in 8.0% (n=12) of the serum samples by more than one of the methodologies employed, considering these results as confirmatory of trichinellosis. The studies carried out herein demonstrate the existence of human trichinellosis in an area historically regarded as free of this parasitic disease and suggest its relationship with the ingestion of pork or pork products as a risk factor. These results indicate that the lack of reports should not be the only criterion for an area to be considered as Trichinella-free. The awareness of the existence of the parasite in a region will favor the establishment of control and prevention strategies which is of fundamental importance to avoid the spread of the disease.

Detection of trichinellosis in a historically Trichinella-free area of Argentina.

The aim of the present work was to determine the presence of human and porcine trichinellosis in an area of Argentina historically regarded as Trichinella-free. Human blood donors (n=216) and swine destined for consumption (n=57) were evaluated by serological techniques (ELISA, immunofluorescence, and/or Western Blot). Muscle tissues from 26 of the pigs were evaluated for the presence of Trichinella larvae by the artificial digestion method. A questionnaire was used to collect and evaluate data on eating habits of the human population under study and on swine-raising conditions. The survey showed that 98.1% of the individuals (n=212) were regular consumers of pork in the form of stuffed products such as sausages produced by local butchers. The seroprevalence (positive sera by at least two of the three methods) was 8.3% (n=18) for human trichinellosis and 24.5% (n=14) for porcine trichinellosis. Trichinellaspiralis larvae were found in 2 of the 26 pigs (7.7%) with parasite loads of 0.33 and 2.4 muscle larvae per gram. Twelve swine found positive by serological and/or parasitological tests were raised under poor sanitary conditions (presence of rubbish in the surroundings, with cannibalism and scavenging behaviors, presence of rodents, etc.). Our study confirms the existence of porcine trichinellosis in an area regarded as Trichinella-free, provides supporting serological evidence of human infection in this area, and indicates that failure to report cases of trichinellosis based on inadequate surveillance can result in incorrect prevalence classification of an area.

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.

Immunoparasitological evaluation of Trichinella spiralis infection during human pregnancy: a small case series.

The clinical background, vertical transmission of Trichinella spiralis and helminthotoxic activity of sera were evaluated in four pregnant women infected at different gestation times. Parasitological and immunological parameters were analyzed. Patients presented clinical symptoms and a seroconversion pattern typical of Trichinella infection, independently of the gestation trimester. All patients, including one patient treated with mebendazole, gave birth to healthy infants at full term. Studies performed in placentas and umbilical cords by artificial digestion and/or immunofluorescence did not reveal the presence of parasites or parasite antigens. Specific antibodies were found in placental extraction and maternal sera by immunofluorescence, ELISA and/or immunoelectrotransfer blot assay. Specific IgG, IgE and IgA were found in the umbilical cord sera. One umbilical cord demonstrated immune-complexed specific IgM and circulating parasite antigens were found in the corresponding infant up to 10 months after birth. Patients' sera were able to kill newborn larvae in cytotoxicity assays, even in the absence of specific antibodies; this effect was abrogated by mifepristone. The results suggest that in human trichinellosis during pregnancy there is an enhanced helminthotoxicity to newborn larvae, dependent in part on progesterone, leading to a mild or moderate course of the infection. The results also indicate that the transplacental passage of migrating larvae is possible.

[Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina. The acute and chronic phases of the infection]. / Aspectos clínicos, serológicos y parasitológicos de un brote de triquinelosis humana en Villa Mercedes, San Luis, Argentina. Las fases aguda y crónica de la infección.

Clinical and serological features of individuals undergoing the acute (n: 54) and chronic (n: 32) phases of trichinellosis belonging to an outbreak originated by the consumption of pork products of a commercial source, arisen in Argentina, are described. Epidemiological data, signs, symptoms, and laboratory studies were assessed. Parasitological studies were performed in patients and pork products. Results showed that: a) the parasite burden of pork products was 200.0 +/- 18.3 larvae/g; b) muscle larvae were demonstrated in 10 out of the 11 patients studied; c) during the acute phase, fever (94%), eosinophilia (90%), myalgia (85%), headache (81%), facial edema (54%), diarrhoea (52%) and anti-Trichinella antibodies (64%) were observed; d) 15% of the patients had to be hospitalized, 7% having complications; e) during the chronic phase, myalgia (72%), sight abnormalities (22%), gastrointestinal disorders (31%) and the persistence of antibodies (77%) were observed. This study highlights the socioeconomical impact of trichinellosis due to the clinical characteristics of the acute phase and the presence of symptoms in the chronic phase.

Aspectos clínicos, serológicos y parasitológicos de un brote de triquinelosis humana en Villa Mercedes, San Luis, Argentina. Las fases aguda y crónica de la infección. / [Clinical, serological and parasitological aspects of an outbreak of human trichinellosis in Villa Mercedes, San Luis, Argentina. The acute and chronic phases of the infection]

Clinical and serological features of individuals undergoing the acute (n: 54) and chronic (n: 32) phases of trichinellosis belonging to an outbreak originated by the consumption of pork products of a commercial source, arisen in Argentina, are described. Epidemiological data, signs, symptoms, and laboratory studies were assessed. Parasitological studies were performed in patients and pork products. Results showed that: a) the parasite burden of pork products was 200.0 +/- 18.3 larvae/g; b) muscle larvae were demonstrated in 10 out of the 11 patients studied; c) during the acute phase, fever (94

), eosinophilia (90

), myalgia (85

), headache (81

), facial edema (54

), diarrhoea (52

) and anti-Trichinella antibodies (64

) were observed; d) 15

of the patients had to be hospitalized, 7

having complications; e) during the chronic phase, myalgia (72

), sight abnormalities (22

), gastrointestinal disorders (31

) and the persistence of antibodies (77

) were observed. This study highlights the socioeconomical impact of trichinellosis due to the clinical characteristics of the acute phase and the presence of symptoms in the chronic phase.

Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires / Serological, clinical and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires

Se estudió un brote de triquinosis ocurrido en el sur de la provincia de Buenos Aires, zona endémica para esta zoonosis. Dieciocho personas fueron afectadas por la ingesta de productos elaborados con carne de cerdo (chacinados sin el correspondiente control veterinario. Se describen síntomas y signos de los apcientes, como así también estudios serológicos relacionados con la formación de anticuerpos y la capacidad citotóxica de los sueros. Se analizaron los producto elaborados (chacinados), así como ocho cerdos interdictos de la granja donde se originó el brote. Con el fin de estudiar la cadena de transmisión se capturaron y analizaron ratas de los alrededores del hábitar de los porcinos. Este trabajo demuestra que los principales síntomas y signos fueron: mialgia, edema palpebral, fiebre y cefalea, que el 61// de los pacientes presentó anticuerpos contra el estadio de larva muscular entre los 15-30 días posteriores a la ingesta, que sólo presentaron anticuerpos contra la larva recien nacida a los 40-60 días postinfección cinco de los dieciocho pacientes, que la carga parasitaria en los chacinados fue de 5,3 larvas/g, que dos de los ocho cerdos se diagnosticaron por técnicas serológicas, mientras que por técnicas parasitológicas se detectó sólo uno y que la rata es uno de los principales reservorios en la cadena epidemiológica

Aspectos serológicos, clínicos y epidemiológicos de un brote de triquinosis en Azul, Provincia de Buenos Aires / Serological, clinical and epidemiological aspects of an outbreak of trichinosis in Azul, province of Buenos Aires

Se estudió un brote de triquinosis ocurrido en el sur de la provincia de Buenos Aires, zona endémica para esta zoonosis. Dieciocho personas fueron afectadas por la ingesta de productos elaborados con carne de cerdo (chacinados sin el correspondiente control veterinario. Se describen síntomas y signos de los apcientes, como así también estudios serológicos relacionados con la formación de anticuerpos y la capacidad citotóxica de los sueros. Se analizaron los producto elaborados (chacinados), así como ocho cerdos interdictos de la granja donde se originó el brote. Con el fin de estudiar la cadena de transmisión se capturaron y analizaron ratas de los alrededores del hábitar de los porcinos. Este trabajo demuestra que los principales síntomas y signos fueron: mialgia, edema palpebral, fiebre y cefalea, que el 61// de los pacientes presentó anticuerpos contra el estadio de larva muscular entre los 15-30 días posteriores a la ingesta, que sólo presentaron anticuerpos contra la larva recien nacida a los 40-60 días postinfección cinco de los dieciocho pacientes, que la carga parasitaria en los chacinados fue de 5,3 larvas/g, que dos de los ocho cerdos se diagnosticaron por técnicas serológicas, mientras que por técnicas parasitológicas se detectó sólo uno y que la rata es uno de los principales reservorios en la cadena epidemiológica (AU)