Trichinella spiralis excretory-secretory antigens selectively inhibit the release of extracellular traps from neutrophils without affecting their additional antimicrobial functions.

Neutrophil extracellular traps (NETs) are fiber structures composed of chromatin and granular proteins that capture and eliminate microorganisms. The NETs formation is induced in response to pathogens and physiological stimuli; however, some pathogens have developed strategies to evade NETs activity. Trichinella spiralis excretory-secretory (ES) antigens are proteins that allow the establishment of the parasite in the host, facilitating penetration, migration, nutrition, and survival. In this paper we described that ES antigens inhibit NETs release, since neutrophils incubated with these antigens maintains a delobulated nucleus, without the release fibers structures indicative of NETs. We also found that other antimicrobial functions of neutrophils, such as phagocytic activity, degranulation, and ROS production, remain unchanged after incubation with ES antigens. This is relevant since it could constitute a novel strategy for the treatment of autoimmune pathologies in which the formation of NETs performs an important role.

Kinetics of Eosinophils during Development of the Cellular Infiltrate Surrounding the Nurse Cell of Trichinella spiralis in Experimentally Infected Mice.

We study the kinetics of eosinophils during the development of the cellular infiltrate surrounding the nurse cell of Trichinella spiralis (T. spiralis) in experimentally infected mice. Male CD1 mice were experimentally infected with 50 viable muscle larvae of the MSUS/MEX/91/CM-91 T. spiralis strain. Tongues and diaphragms were obtained daily from days 13 to 39 post infection. Diaphragms were compressed and subjected to Giemsa stain. Tongues were histologically sectioned and stained with erythrosine B or hematoxylin and eosin. The cellular infiltrate and the nurse cell-larva complex were detected by optical microscopy since day 16 post infection. The size of the larva increased exponentially during the course of the infection. The kinetics of eosinophils showed a multimodal trend, with a bimodal predominance. The maximum peaks were reached on days 21 and 27 post infection. The results of this study demonstrate that eosinophils occur abundantly in two transcendent moments of the T. spiralis life cycle: first, when the stage 1 larva invades the myocyte and second when the nurse cell-larva complex has been fully developed. These results help one to understand the immunobiology of T. spiralis, highlighting the importance of eosinophils in the survival of the larva in skeletal muscle. Further studies are needed to characterize the cell populations that comprise the cellular infiltrate during the development of the mother cell.

Protein deficiency during Trichinella spiralis infection impairs lung immunity against newborn larvae.

The goal of this study was to analyse the effects of a protein-deficient (PD) diet on antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro against newborn larvae (NBL) of Trichinella spiralis in the lungs of infected rats. Two groups of weaning Wistar rats received a PD diet (6.5% casein) and other two received a control diet (C, 20% casein). After ten days, one group of each diet was infected (PDI and CI ) with muscle larvae. Lung tissue extracts (LTE) and lung cell suspension (LCS) were obtained. PDI had lower titres of anti-NBL antibodies in LTE than CI . In ADCC assays using control cells, NBL mortality percentage was lower with LTE from PDI than LTE from CI (P < .01). In assays using control cytotoxic sera, ADCC was exerted by LCS from CI at all days post-infection (p.i.), but only by LCS from 13 days p.i. from PDI . ADCC assays combining LTE and LCS from the same group showed a lower response for PDI than for CI (P < .0001). LCS from PDI contained lower numbers of neutrophils, eosinophils and FcεRI+ cells than CI . PD may diminish ADCC activity against T spiralis NBL in lungs through alterations in specific antibodies and effector cells.

Effect of sex and genotype of the host on the anthelmintic efficacy of albendazole microcrystals, in the CBi-IGE Trichinella infection murine model.

Albendazole (ABZ) is an anthelmintic pharmaceutical commonly used in the treatment of nematode infections. It is a Class II drug poorly water-soluble, with very low bioavailability, a feature particularly limiting to treat the trichinellosis chronic phase. Microcrystals obtained by controlled precipitation using hydroxyethyl cellulose and chitosan have previously been shown to improve ABZ biopharmaceutical properties. This investigation aimed to test the systems' in vivo efficacy in the CBi-IGE murine model of Trichinella spiralis infection in the infection's different phases and parasite' stages. Treatment in the enteral phase led to a 90% decrease in the larval muscle load, probably due to its effect on T. spiralis female fecundity. Both microcrystal systems given in the migratory phase halved muscle load in males, a response not observed in females. The chitosan-based microcrystals proved to be the best when administered in the chronic phase of the infection ­ an increased proportion of L1 dead larvae was found compared to controls, except in CBi+-treated females. Males and females from the highly susceptible CBi+ line presented a significantly different treatment response in this phase. In vivo efficacy depended on the host genotype and sex and was related to the parasite cycle stage in which the formulations were administered.

Cellular and molecular changes and immune response in the intestinal mucosa during Trichinella spiralis early infection in rats.

BACKGROUND: The main targets of the host's immune system in Trichinella spiralis infection are the adult worms (AW), at the gut level, and the migrant or newborn larvae (NBL), at systemic and pulmonary levels. Most of the studies carried out in the gut mucosa have been performed on the Payer's patches and/or the mesenteric lymph nodes but not on the lamina propria, therefore, knowledge on the gut immune response against T. spiralis remains incomplete. METHODS: This study aimed at characterizing the early mucosal immune response against T. spiralis, particularly, the events taking place between 1 and 13 dpi. For this purpose, Wistar rats were orally infected with muscle larvae of T. spiralis and the humoral and cellular parameters of the gut immunity were analysed, including the evaluation of the ADCC mechanism exerted by lamina propria cells. RESULTS: A marked inflammation and structural alteration of the mucosa was found. The changes involved an increase in goblet cells, eosinophils and mast cells, and B and T lymphocytes, initially displaying a Th1 profile, characterised by the secretion of IFN-γ and IL-12, followed by a polarization towards a Th2 profile, with a marked increase in IgE, IgG1, IL-4, IL-5 and IL-13 levels, which occurred once the infection was established. In addition, the helminthotoxic activity of lamina propria cells demonstrated the role of the intestine as a place of migrant larvae destruction, indicating that not all the NBLs released in the gut will be able to reach the muscles. CONCLUSIONS: The characterization of the immune response triggered in the gut mucosa during T. spiralis infection showed that not only an effector mechanism is directed toward the AW but also towards the NBL as a cytotoxic activity was observed against NBL exerted by lamina propria cells.

Trichinella spiralis y Trichinella patagoniensis: estudio de la carga globular aplicando el método de Azul Alcian / Trichinella spiralis and Trichinella patagoniensis: study of globular charge applying Alcian Blue method / Trichinella spiralis e Trichinella patagoniensis: estudo da carga globular pelo método de Azul de Alcian

Resumen Los residuos de ácido siálico de las glicoproteínas de superficie son los principales responsables de la carga negativa eritrocitaria. El objetivo de este trabajo fue estudiar alteraciones de carga globular producidas por Trichinella spiralis y Trichinella patagoniensis. Se trabajó con concentrados de larvas musculares de ambas especies y con eritrocitos frescos. Se incubó el sedimento globular con igual volumen de concentrado larval (1 y 2 horas). Los controles fueron incubados con solución salina. Se aplicó el método de Azul Alcian y se determinó el coeficiente experimental de carga aniónica eritrocitaria (CexpCAE). Los resultados mostraron que la carga disminuyó con el aumento del tiempo de tratamiento para ambas especies. Los valores de CexpCAE de las suspensiones incubadas con T. spiralis fueron menores que con T. patagoniensis, indicando que T. spiralis produjo mayor disminución de carga que T. patagoniensis. Se concluye que la desialización producida por ambas especies no es la misma, lo que sugiere que la relación hospedador-parásito que se establecería in vivo sería distinta.

Abstract The sialic acid residues of the surface glycoproteins are mainly responsible for the erythrocyte negative charge. The objective of this work was to study alterations of globular charge produced by Trichinella spiralis and Trichinella patagoniensis. Work was carried out on muscle larvae concentrates of both species and fresh erythrocytes. The treatment was performed by incubating the globular pellet with equal volume of larval concentrate (1 and 2 hours). Controls were incubated with saline solution. The Alcian Blue method was applied and the experimental coefficient of erythrocyte anion charge (expCEAC) was determined. The results showed that the globular charge decreased with increasing treatment time for both species. The expCEAC values of the suspensions incubated with T. spiralis were lower than with T. patagoniensis, indicating that T. spiralis produced a greater decrease in charge than T. patagoniensis. It is concluded that the desialization produced by both species is not the same, suggesting that the host-parasite relationship that would be established in vivo would be different.

Resumo Os resíduos de ácido siálico das glicoproteínas de superfície são os principais responsáveis pela carga negativa dos eritrócitos. O objetivo desse trabalho foi estudar as alterações da carga globular produzidas por Trichinella spiralis e Trichinella patagoniensis. Trabalhamos com concentrados de larvas musculares de ambas as espécies e com eritrócitos frescos. O tratamento foi realizado incubando o sedimento globular com igual volume de concentrado larval (1 e 2 horas), Os controles foram incubados com solução salina. Foi aplicado o método de Azul de Alcian e se determinou o coeficiente experimental de carga aniônica de eritrócitos (CexpCAE). Os resultados mostraram que a carga diminuiu com o aumento do tempo de tratamento para ambas as espécies. Os valores de CexpCAE das suspensões incubadas com T. spiralis foram menores que com T. patagoniensis, indicando que T. spiralis produziu uma diminuição maior na carga que T. patagoniensis. Conclui-se que a dessalinização produzida por ambas as espécies não é a mesma, sugerindo que a relação hospedeiro parasita que seria estabelecida in vivo é diferente.

Trichinella spiralis y Trichinella patagoniensis: cinética de desialización eritrocitaria / Trichinella spiralis and Trichinella patagoniensis: kinetics of erythrocyte desialization / Trichinella spiralis e Trichinella patagoniensis: cinética de dessialização eritrocitária

El objetivo del trabajo fue estudiar la cinética de desialización eritrocitaria producida por larvas infectantes de Trichinella spiralis y Trichinella patagoniensis. Se trabajó con 7 suspensiones eritrocitarias incubadas con 1.000±200 larvas musculares/mL, durante 2 horas, tomando muestra al tiempo inicial y cada 15 minutos. Los respectivos eritrocitos controles se incubaron de la misma manera con solución salina. Se aplicaron el método de titulación por Polibrene calculando el CexpST y un análisis de varianza (ANOVA) con las comparaciones múltiples según Tukey. Los resultados mostraron que el valor promedio de CexpST disminuyó con el aumento del tiempo de incubación, para ambas especies. En el tratamiento con T. spiralis no hubo diferencias significativas entre el valor medio del coeficiente a tiempo 60 y 75 minutos, mientras que con T. patagoniensis, a 45 y 60 minutos. Todas las restantes diferencias fueron significativas. La comparación entre los tratamientos, para cada uno de los tiempos, mostró que al tiempo inicial el coeficiente promedio no difirió entre las especies, pero que a todos los otros tiempos fue significativamente menor en la incubación de los eritrocitos con T. spiralis. Se concluye que la relación hospedador-parásito que se establece en ambos casos es distinta y probablemente también la capacidad de adaptación y de daño al hombre.

The objective of this work was to study the kinetics of erythrocyte desialization produced by infective larvae of Trichinella spiralis and Trichinella patagoniensis. It was performed on 7 erythrocyte suspensions incubated with 1,000±200 muscle larvae/ mL for 120 minutes, taking samples at the initial time and every 15 minutes. The respective control erythrocytes were incubated in the same way with saline solution. The Polybrene Titration method calculating the CexpST and variance analysis (ANOVA) with the multiple comparisons according to Tukey were applied. The results showed that the average value of CexpST decreased with the increase in incubation time, for both species. There were no significant differences between the mean value of the coefficient at 60 and 75 minutes in the treatment with T. spiralis, while neither were there any differences between 45 and 60 minutes in the incubation with T. patagoniensis. All other differences were significant. The comparison between the two treatments, for each of the times, showed that at the initial time the average coefficient did not differ between the species, but at all other times it was significantly lower in the incubation of the erythrocytes with T. spiralis. It is concluded that the parasite host relationship that is established in both cases is different and probably also is the ability to adapt and cause harm to man.

O objetivo do trabalho foi estudar a cinética de dessialização eritrocitária. produzida por larvas infectantes de Trichinella spiralis e Trichinella patagoniensis. O trabalho foi feito com 7 suspensões eritrocitárias incubadas com 1.000±200 larvas musculares/mL por 2 horas, colhendo amostras no tempo inicial e a cada 15 minutos. Os respectivos eritrócitos-controle foram incubados da mesma forma com solução salina. Foi aplicado o método de titulação por Polibreno calculando o CexpST e também uma análise da variância (ANOVA) com as comparações múltiplas de acordo com Tukey. Os resultados mostraram que o valor médio de CexpST diminuiu com o aumento do tempo de incubação para ambas as espécies. No tratamento com T. spiralis não houve diferenças significativas entre o valor médio do coeficiente no tempo 60 e 75 minutos, ao passo que com T. patagoniensis, aos 45 e 60 minutos. Todas as diferenças restantes foram significativas. A comparação entre os tratamentos, para cada um dos tempos, mostrou que no tempo inicial o coeficiente médio não diferiu entre as espécies, mas que em todos os outros tempos foi significativamente menor na incubação dos eritrócitos com T. spiralis. A conclusão é que a relação hospedeiro-parasita, estabelecida em ambos os casos, é diferente e provavelmente também a capacidade de adaptação e dano ao homem.

Compromiso neurológicoy cardiovascularen pacientes con triquinosis / Neurological and cardiovascular involventin patients with trichinosis

Introducción: La triquinosis es una infección parasitaria causada por nematodos del género Trichinella. El compromiso cardíaco no es habitual, pero representa la causa más frecuente de muerte por triquinosis, mientras que la afectación neurológica ocurre principalmente en pacientes severamente enfermos. Se sabe que a mayor cantidad de larvas ingeridas menor es el tiempo de incubación y mayor es la severidad de la enfermedad. Se presentan dos pacientes con compromiso del sistema nervioso central, uno de ellos cardiovascular, ambos pertenecientes a un brote de triquinosis ocurrido en la ciudad de Bahía Blanca.

Background: Trichinosis is a parasitic infection caused by nematodes of the genus Trichinella. Cardiac involvement is not usual but represents the most frequent cause of death by trichinosis, while neurological affectation occurs mainly in severely ill patients. A greater number of larvae ingested, the shorter the incubation time and the greater the severity of the disease. We present two patients with compromise of the central nervous system and one of them cardiovascular, both belonging to an outbreak of trichinosis in the city of Bahía Blanca

Larvas recién nacidas de Trichinella spiralis: cinética de agregación eritrocitaria / Newborn larvae of Trichinella spiralis: erythrocyte aggregation kinetics

El objetivo de este trabajo fue estudiar la cinética de agregación eritrocitaria producida por dos concentraciones de larvas recién nacidas (LRN) de Trichinella spiralis. Se trabajó con 5 suspensiones eritrocitarias incubadas con 500 y 1.000±100 LRN/mL durante 120 minutos, con tomas de muestra al tiempo inicial y cada 15 minutos. Los respectivos controles se incubaron de la misma manera con solución salina. Se aplicaron el método de titulación por Polibrene calculando el CexpST y la técnica de análisis de la variancia con las comparaciones múltiples según Tukey. Los resultados mostraron para ambas concentraciones de LRN, que el coeficiente promedio disminuyó con el aumento del tiempo de incubación. En el tratamiento con 1.000 LRN/mL, el coeficiente promedio no presentó diferencias significativas a tiempo 0 y 15 minutos, ni entre 60 y 75 minutos, mientras que con 500 LRN/mL no hubo diferencias entre los tiempos 0, 15 y 30 minutos. Todas las restantes diferencias fueron significativas para ambas concentraciones larvales. El valor promedio de CexpST no difirió significativamente entre los dos tratamientos a tiempo 0 y 15 minutos, pero a todos los otros tiempos fue menor a mayor concentración de larvas. La experiencia realizada indicaría que in vivo, la cantidad de LRN y el tiempo que permanecen en circulación determinan el grado de desializacion eritrocitaria, y por lo tanto el riesgo de activación T y de alteraciones hemorreológicas en el hospedador.

The aim of this work was to study the kinetics of erythrocyte aggregation produced by two concentrations of Trichinella spiralis newborn larvae (NL). Work was performed with 5 erythrocyte suspensions incubated with 500 and 1000 ± 100 NL/mL for 120 minutes, taking samples at the initial time and every 15 minutes. The respective controls were incubated in the same way with saline solution. The Polybrene titration method calculating the CexpST and the variance analysis technique with the multiple comparisons according to Tukey were applied. The results showed that the average coefficient decreased with the rise in incubation time for both NL concentrations. The average coefficient did not present significant differences between the initial time and 15 minutes, nor between 60 and 75 minutes in the treatment with 1000 NL/mL, while there were no differences between 0,15 and 30 minutes in the treatment with 500 NL/mL. All other differences were significant for both larval concentrations. The average value of CexpST did not differ significantly between the two time treatments at zero time and 15 minutes, but at all other times it was less at a higher concentration of larvae. The experience carried out would indicate that in vivo, the amount of NL and the time that they remain in circulation determines the degree of erythrocyte desialylation, and therefore, the risk of T activation and hemorrheological alterations in the host.

O objetivo desse trabalho foi estudar a cinética de agregação eritrocitária produzida por duas concentrações de larvas recém nascidas (LRN) de Trichinella spiralis. O trabalho foi feito com 5 suspensões eritrocitárias incubadas com 500 e 1.000 ± 100 LRN/mL por 120 minutos, colhendo amostras no tempo inicial e a cada 15 minutos. Os respectivos controles foram incubados da mesma forma com solução salina. Foi aplicado o método de titulação por Polibreno e se calculou CexpST. e a técnica de análise da variância com as comparações múltiplas de acordo com Tukey. Os resultados mostraram, para ambas as concentrações de LRN, que o o coeficiente médio diminuiu com o aumento do tempo de incubação. No tratamento com 1.000 LRN/mL o coeficiente médio não mostrou diferenças significativas no tempo 0 e 15 minutos ou entre 60 e 75 minutos, ao passo que não houve diferenças com 500 LRN/mL entre tempos 0, 15 e 30 minutos. Todas as restantes diferenças foram significativas para ambas as concentrações de larvas. O valor médio de CexpST não diferiu significativamente entre os dois tratamentos no tempo de 0 e 15 minutos, mas em todos os outros tempos foi menor em maior concentração de larvas. A experiência realizada indicaria que in vivo a quantidade de LRN e o tempo que permanecem em circulação determina o grau de dessialização dos eritrócitos e, portanto, o risco de ativação T e de alterações hemorreológicas no hospedeiro.

Efecto de Trichinella spiralis y de Trichinella patagoniensis n.sp. sobre la desialización / Effect of Trichinella spiralis and Trichinella patagoniensis n.sp. on desialization

El ácido siálico tiene importantes funciones biológicas, muchas de las cuales determinan su participación en la respuesta inmune. El objetivo del trabajo fue comparar el efecto de Trichinella spiralis y Trichinella patagoniensis n.sp. sobre la desialización eritrocitaria. Se trabajó con 10 concentrados de larvas musculares de T. spiralis y 10 de T. patagoniensis de la misma concentración larval. Se realizó el tratamiento incubando el sedimento de eritrocitos frescos con igual volumen de concentrado larval (37 ºC), tomando muestra a los 30, 60 y 90 minutos. Los controles fueron incubados de la misma forma con solución salina. Se aplicó el método de Titulación de la Agregación por Polibrene y se determinó el CexpST. Los resultados mostraron que el valor medio del CexpST en los eritrocitos tratados con T. spiralis fue significativamente menor que en los glóbulos tratados con T. patagoniensis, para todos los tiempos estudiados. El aumento del tiempo de tratamiento también disminuyó significativamente el valor medio del CexpST para las dos especies. Éste fue significativamente menor a los 90 minutos de incubación que a los 60 minutos y éstos a su vez menores que a los 30 minutos. Se concluye que T. spiralis provocó mayor desialización eritrocitaria que T. patagoniensis en las condiciones experimentales estudiadas.

Sialic acid has important biological functions, many of which determine its participation in the immune response. The objective of this paper was to compare the effect of Trichinella spiralis and Trichinella patagoniensis n.sp. on erythrocyte desialization. Work was performed on 10 larval concentrates of muscle larvae of T. spiralis and 10 of T. patagoniensis of the same larval concentration. The treatment was carried out incubating the sediment of fresh erythrocytes with an equal volume of larval concentrate (37 °C), taking samples at 30, 60 and 90 minutes. The controls were incubated in the same way treated with saline solution. Titration of aggregation by Polybrene Method was applied and the CexpST was determined. The results showed that the mean value of CexpST in erythrocytes with T. spiralis was significantly lower than in the globules treated with T. patagoniensis, for all the studied times. The increase in treatment time also significantly decreased the mean value of CexpST for the two species, being significantly lower at 90 minutes of incubation than at 60 minutes and these in turn lower than at 30 minutes. It is concluded that T. spiralis caused greater erythrocyte desialization than T. patagoniensis in the experimental conditions studied.

O ácido siálico tem importantes funções biológicas, muitas das quais determinam sua participação na resposta imune. O objetivo foi comparar o efeito de Trichinella spiralis e Trichinella patagoniensis n.sp. sobre a dessialização eritrocitária. Trabalhou-se com 10 concentrados de larvas musculares de T. spiralis e 10 de T. patagoniensis da mesma concentração larval. Realizou-se o tratamento incubando o sedimento de eritrócitos frescos com igual volume de concentrado larval (37 ºC), tomando amostra aos 30, 60 e 90 minutos. Os controles foram incubados da mesma forma com solução salina. Foi aplicado o método de Titulação da Agregação por Polibrene e se determinouo CexpST. Os resultados mostraram que o valor médio do CexpST nos eritrócitos Tratados com T. spiralis foi significativamente menor que nos glóbulos tratados com T. patagoniensis, para todos os tempos estudados. O aumento do tempo de tratamento também diminuiu significativamente o valor médio do CexpST para as duas espécies, sendo significativamente menor aos 90 minutos de incubação que aos 60 minutos e eles por sua vez menores que aos 30 minutos. Conclui-se que T. spiralis provocou maior dessialização eritrocitária que T. patagoniensis nas condições experimentais estudadas.

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.

Alteración del ácido siálico eritrocitario por efecto de Trichinella spiralis aplicando el método de Azul Alcian / Alteration of erythrocyte sialic acid by the effect of Trichinella spiralis applying the Alcian Blue method / Alteração do ácido siálico eritrocitário por efeito da Trichinella spiralis aplicando o método de Alcian Blue

Actualmente se reconoce que los ácidos siálicos están involucrados en múltiples funciones biológicas y que tienen un papel importante en la interacción parásito-hospedador. El objetivo del trabajo fue estudiar la alteración del ácido siálico eritrocitario por efecto de T. spiralis aplicando el método de Azul Alcian. Se trabajó con 11 concentrados de larvas musculares de T. spiralis (LM) y 9 de larvas recién nacidas de T. Spiralis (LRN) (total: 20) y con suspensiones de eritrocitos frescos. Se realizó el tratamiento incubando el sedimento globular con igual volumen de concentrado larval (37 ºC durante 1 hora). Los controles fueron incubados de la misma forma con solución salina. Se aplicó el método de Azul Alcian y se determinó CAE% en el control y en los glóbulos tratados. Se calculó CexpCAE. Los resultados mostraron que 5/9 de los concentrados de LRN y 9/11 de LM modificaron la carga globular. La media y la desviación estándar de los CexpCAE de las suspensiones en que la carga disminuyó por contacto con LRN y LM, fueron 0,614±0,1946 y 0,656±0,1865 respectivamente, mientras que en las que no se modificó resultó 0,955±0,0289 en el tratamiento con LRN y 0,93±0,0141 con LM. Se concluye que el Método de Azul Alcian es útil para estudiar las variaciones en la carga eritrocitaria por efecto de LRN y LM de T. spiralis.

It is now recognized that sialic acids are involved in multiple biological functions and that they have an important role in the parasite-host interaction. The objective of this work was to study the alteration of erythrocyte sialic acid by the effect of T. spiralis applying the Alcian Blue method. Work was carried out with 11 larval concentrates of ML and 9 of NL (total 20) and with fresh erythrocyte suspensions. The treatment was performed incubating the globular sediment with equal volume of larval concentrate (37 °C for 1 hour). The controls were incubated in the same way with saline solution. The Alcian Blue method was applied and CAE% was determined in the control and in the treated globules. CexpCAE was calculated. The results showed that 5/9 of the NL concentrates and 9/11 of ML modified the globular charge. The mean and standard deviation of the CexpCAE of the suspensions in which the charge decreased by contact with NL and ML were 0.614±0.1946 and 0.656±0.1865 respectively, whereas in those that did not change, it was 0.955±0.0289 in the NL treatment and 0.93±0.0141 in the ML. It is concluded that the Alcian Blue Method is useful to study the variations in erythrocyte charge due to NL and ML of T. spiralis.

Atualmente se reconhece que os ácidos siálicos estão envolvidos em múltiplas funções biológicas e que têm um papel importante na interação parasita-hospedeiro. O objetivo do trabalho foi estudar a alteração do ácido siálico eritrocitário por efeito de T. spiralis aplicando o método do Alcian Blue. Trabalhou-se com 11 concentrados de larvas musculares de T. spiralis (LM) e 9 de larvas recém-nascidas de T. Spiralis (LRN) (total: 20) e com suspensões de eritrócitos frescos. Realizou-se o tratamento incubando o sedimento globular com igual volume de concentrado larval (37 ºC durante 1 hora). Os controles foram incubados da mesma forma com solução salina. Aplicou-se o método de Azul Alcian e determinou-se CAE% no controle e nos glóbulos tratados. Calculou-se CexpCAE. Os resultados mostraram que 5/9 dos concentrados de LRN e 9/11 de LM modificaram o carga globular. A média e o desvio padrão dos CexpCAE das suspensões em que a carga diminuiu por contato com LRN e LM, foram 0,614±0,1946 e 0,656±0,1865 respectivamente, enquanto que naquelas onde não se modificou, resultou 0,955±0,0289 no tratamento com LRN e 0,93±0,0141 com LM. Conclui-se que o método de Alcian Blue é útil para estudar as variações na carga eritrocitária por LRN e LM de T. spiralis.

Prevalencia de Trichinella Spiralis en carnes de cerdo comercializadas en el mercado de la ciudad de Posadas y caracterización de su producción primaria / Prevalence of Trichinella Spiralis in pork meat sold in the market of the city of Posadas and characterization of its primary production

INTRODUCCIÓN En los últimos años, el consumo de carne de cerdo en Argentina denota un incremento sostenido. En Misiones, gran parte de la producción proviene de unidades familiares rurales que abastecen al mercado interno. La triquinosis es una zoonosis cuyo agente es Trichinella spiralis, y está ligada con la producción porcina. Su contagio humano se produce principalmente por ingestión de carne cruda o mal cocida de cerdo o sus derivados. OBJETIVOS Determinar la prevalencia de Trichinella spiralis en carnes de cerdo destinadas a la comercialización en mercados de la ciudad de Posadas y caracterizar su producción primaria. MÉTODOS Se recolectaron muestras de carne de cerdo de cuatro mercados de Posadas, que provenían de granjas productoras de diferentes localidades, y se procesaron en busca del parásito por la técnica de digestión de muestras agrupadas con utilización de un agitador magnético, remitiendo resultados previamente a su comercialización. Se realizaron encuestas estandarizadas en dichas granjas para la caracterización productiva. Los datos se procesaron con planillas de cálculo de uso libre. RESULTADOS Tras el análisis de 1235 muestras, la prevalencia obtenida fue de 0%. Las muestras provenían de 8 establecimientos productores de 5 municipios. La caracterización productiva arrojó los siguientes resultados escaso aislamiento sanitario (25%, n=8), desconocimiento de las enfermedades animales (100%, n=8), presencia de fauna sinantrópica y silvestre en las proximidades con abundante vegetación que pueda servir de refugio (75%, n=8), adecuada asistencia profesional (100%, n=8) y realización de tratamientos para el control de roedores (100%, n=8). DISCUSIÓN Los resultados contribuyen a mejorar el conocimiento epidemiológico regional y la planificación estratégica de acciones preventivas. La replicación de este modelo de control sanitario previo a la comercialización se constituye como una herramienta de vigilancia sanitaria eficaz frente a esta zoonosis.

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.

Trichinella spiralis in a South American sea lion (Otaria flavescens) from Patagonia, Argentina.

Trichinella spp. from a sylvatic cycle has been found in several animal species such as pumas (Puma concolor), armadillos (Chaetophractus villosus), rats (Rattus norvegicus), and wild boars (Sus scrofa) in Argentina. Moreover, Trichinella infection has been detected in a wide range of marine mammals around the world, including polar bears (Ursus maritimus) and walruses (Odobenus rosmarus). Until the present time, Trichinella spp. infection has not been detected in marine mammals of South America. Samples from four South American sea lions (Otaria flavescens) found dead in Rio Negro, Argentina, were analyzed by artificial digestion, and in the case of one animal, Trichinella larvae were identified at the species level by nested multiplex PCR as Trichinella spiralis. This is the first report of a Trichinella species infecting marine mammals from South America.

Desialización eritrocitaria con y sin exposición del antígeno T producida por Trichinella spiralis / Erythrocyte desialylation with and without T antigen exposure produced by Trichinella spiralis / Desialylation eritrocitaria com e sem exposição antigénio T produzida por Trichinella spiralis

El objetivo del trabajo fue estudiar la desialización de eritrocitos que desenmascaran el antígeno T y de los glóbulos que no lo exponen, por contacto con larvas recién nacidas (LRN) de T. spiralis. Se trabajó con 15 suspensiones eritrocitarias en medio enzimático, incubadas con LRN. Los GR control se incubaron con solución salina. Para analizar el efecto del parásito sobre la exposición del antígeno T, se aplicó el Método de Aglutinación anti-T con antígeno T y se estudió la agregación por los métodos de Polibrene y de Análisis Digital de Imágenes, determinando CexpST, CCA y Distribución de los agregados eritrocitarios (DAE). La media de los coeficientes en los eritrocitos que expusieron el antígeno T fueron CexpST=0,22±0,179 y coeficiente de células aisladas (CCA)=0,73±0,108 y en los que no 0,86±0,125 y 0,205±0,163 respectivamente. La DAE mostró que los GR que lo expusieron, presentaron marcada disminución de células aisladas y pronunciado aumento de grandes agregados en relación a los controles, mientras que los GR en los que no hubo exposición, la disminución de células aisladas con respecto a los controles fue menor y el aumento de agregados estuvo homogéneamente distribuido entre todas las categorías. La experiencia realizada concluye que los valores obtenidos de los coeficientes difieren significativamente en los GR que exponen el antígeno T y en los que no, por lo que estas técnicas podrían ser predictivas para detectar desenmascaramiento T.

The aim of this work was to study the desialylation of erythrocytes that expose the T antigen by contact with T. spiralis newborn larvae (NL) and of those that do not. Work was carried out with 15 red cell suspensions in enzymatic medium, which were incubated with NL. The respective Control RBC were incubated with saline solution. To analyze the effect of the parasite on the exposure of the T antigen, the Anti T- antigen T Agglutination Method was applied and the aggregation was studied by Polybrene and Digital Image Analysis Methods determining CexpTS, ICC and Distribution of Erythrocyte Aggregates (DEA). The mean of coefficients in the erythrocytes that exposed the T antigen were CexpTS= 0.22 ± 0.179 and ICC= 0.73 ± 0.108 and of those that did not it was 0.86±0.125 y 0.205±0,163 respectively. DEA showed that the RBCs that exposed it showed a marked decrease of isolated cells and a pronounced increase of large aggregates in relation to the Controls, while the RBCs in which there was no exposure, the decrease of isolated cells with respect to Controls was lower and the increase of aggregates was homogeneously distributed among all categories. The experience concludes that the obtained values of the coefficients differ significantly in the RBCs that expose the T antigen from those that do not, so these techniques could be predictive to detect T unmasking.

O objetivo do trabalho foi estudar a desialização de eritrócitos que desmarcaram o antígeno T e dos glóbulos que não o expõem, por contato com LRN de T. spiralis. Trabalhou-se com 15 suspensões de eritrócitos em meio enzimático, incubadas com LRN. Os GV controle foram incubados com solução salina. Para analisar o efeito do parasita sobre a exposição do antígeno T, foi aplicado o Método de Aglutinação anti T com antígeno T e se estudou a agregação pelos Métodos de Polibrene e de Análise Digital de Imagens, determinando CexpST, CCA e Distribuição dos agregados eritrocitários (DAE). A média dos coeficientes nos eritrócitos que expuseram o antígeno T foram CexpST= 0.22±0.179 e CCA= 0.73±0.108 e naqueles que não o expuseram 0.86±0.125 y 0.205±0,163 respectivamente. Foi demonstrado que os GV que o expuseram, apresentaram marcada diminuição de células isoladas e pronunciado aumento de grandes agregados em relação aos controles, enquanto que os GV nos quais não houve exposição, a diminuição de células isoladas com respeito aos controles foi menor e o aumento de agregados esteve homogeneamente distribuído entre todas as categorias. A experiência realizada conclui que os valores obtidos dos coeficientes diferem significativamente nos GV que expõem o antígeno T e nos que não, portanto estas técnicas poderiam ser preditivas para detectar desmascaramento T.

Trichinella spiralis muscle larvae excretory-secretory products induce changes in cytoskeletal and myogenic transcription factors in primary myoblast cultures.

Trichinella spiralis infection in skeletal muscle culminates with nurse cell formation. The participation of excretory-secretory products of the muscle larvae has been implicated in this process through different studies performed in infected muscle and the muscle cell line C2C12. In this work, we developed primary myoblast cultures to analyse the changes induced by excretory-secretory products of the muscle larvae in muscle cells. Microarray analyses revealed expression changes in muscle cell differentiation, proliferation, cytoskeleton organisation, cell motion, transcription, cell cycle, apoptosis and signalling pathways such as MAPK, Jak-STAT, Wnt and PI3K-Akt. Some of these changes were further evaluated by other methodologies such as quantitative real-time PCR (qRT-PCR) and western blot, confirming that excretory-secretory products of the muscle larvae treated primary mouse myoblasts undergo increased proliferation, decreased expression of MHC and up-regulation of α-actin. In addition, changes in relevant muscle transcription factors (Pax7, Myf5 and Mef2c) were observed. Taken together, these results provide new information about how T. spiralis could alter the normal process of skeletal muscle repair after ML invasion to accomplish nurse cell formation.

Exposición de criptoantígeno eritrocitario por larvas recién nacidas de Trichinella spiralis / Exposure of erythrocyte cryptic antigen by Trichinella spiralis newborn larvae / Exposição do cripto antígeno eritrocitário por larvas recém-nascidas de Trichinella spiralis

La desialización del eritrocito puede exponer determinantes antigénicas crípticas de la membrana, entre las cuales se encuentra el antígeno T. Se ha comunicado que las larvas recién nacidas de Trichinella spiralis (LRN), las cuales circulan por el torrente sanguíneo del hospedador, captan el ácido siálico eritrocitario. El objetivo de este trabajo fue estudiar in vitro la exposición del criptoantígeno T por la desialización producida por distintas concentraciones de LRN. Se trabajó con 30 suspensiones eritrocitarias en medio enzimático, que fueron incubadas en partes iguales con concentrados de LRN (GR Tratados) durante 2 horas a 37 ºC con agitación continua. Los respectivos GR Controles se pusieron en contacto con solución salina. El tratamiento de 10/30 suspensiones globulares se realizó con 500 LRN/mL, 10 con 300 LRN/mL y las últimas 10 con 150 LRN/mL. Se aplicó el Método de Aglutinación anti-T con antígeno T (en Placa y Tubo) enfrentando las suspensiones a suero de adulto y suero de cordón. El tratamiento de 9/10 de las suspensiones con 500 LRN/mL, el de 7/10 con 300 LRN/mL y el de 2/10 con 150 LRN/mL las expuso al criptoantígeno. Se concluye que en pacientes diabéticos, hipertensos o con otra patología que produzca disminución de ácido siálico eritrocitario y que cursen simultáneamente una infección por T. spiralis, en la etapa de circulación de larvas por el torrente sanguíneo podría producirse la activación T con la consecuente hemólisis, trombocitopenia y trombosis.

Erythrocyte desialylation can expose cryptic antigenic determinants of the membrane, among which is the T antigen. It has been reported that newborn larvae (NL), which circulate in the bloodstream of the host, capture erythrocyte sialic acid. The aim of this study was to study in vitro exposure of cryptic T antigen by the desialylation produced by different concentrations of NL. Work was carried out on 30 red cell suspensions in enzymatic medium, which were incubated in equal parts with NL concentrates (Treated RBC) for 2 hours at 37 ºC with continued agitation. The respective Control RBC was incubated with saline solution. Treatment of 10/30 globular suspensions was performed with 500 NL/mL, 10 with 300 NL/mL and the last 10 with 150 NL/mL. Anti T- antigen T Agglutination Tests (Plate and Tube) were made, facing the globular suspensions against adult and cord human sera. Treatment of 9/10 suspensions with 500 NL/mL, 7/10 with 300 NL/mL and 2/10 with 150 NL/mL exposed T antigen. It is concluded that in patients with diabetes, hypertension or other diseases with lower content of erythrocyte sialic acid and who simultaneously have a T. spiralis infection, T activation may occur in the stage of larvae circulating through the bloodstream, with consequent haemolysis, thrombocytopenia and thrombosis.

A dessialização do eritrócito pode expor determinantes antigênicos crípticos da membrana, entre os quais se encontra o antígeno T. Foi comunicado que as larvas recém-nascidas de Trichinella spiralis (LRN), as quais circulam na corrente sanguínea do hospedeiro, capturam o ácido siálico eritrocitário. O objetivo foi estudar in vitro a exposição do cripto antígeno T pela dessialização produzida por diferentes concentrações de LRN. Trabalhou-se com 30 suspensões eritrocitárias em meio enzimático, incubadas em partes iguais com concentrados de LRN (GV Tratados) durante 2 horas a 37 °C com agitação contínua. Os respectivos GV Controles entraram em contato com solução salina. Tratamento de 10/30 suspensões globulares foi realizado com 500 LRN/mL, 10 com 300 LRN/mL e as últimas 10 com 150 LRN/mL. Foi aplicado o Método de aglutinação anti T-antígeno T (em Placa e Tubo) enfrentando as suspensões a soro de adulto e soro de cordão. O tratamento de 9/10 suspensões com 500 NRL / mL, o de 7/10 com 300 LRN/mL e o de 2/10 com 150 LRN/mL expuseram o cripto antígeno. Conclui-se que em pacientes diabéticos, hipertensos ou com outra patologia que produza diminuição do ácido siálico eritrocitário, e que padeçam simultaneamente uma infecção por T. spiralis, na fase de circulação de larvas pela corrente sanguínea, poderia ocorrer a ativação T com a consequente hemólise, trombocitopenia e trombose.

Análisis proteómico de los productos de excreción-secreción de cuatro aislados de Trichinella spiralis obtenidos de hospederos accidentales / Proteomic analysis of the excretion-secretion products of four Trichinella spiralis isolates obtained from accidental hosts

Resumen: Introducción: Trichinella spiralis es un nemátodo tisular que se aloja en el músculo esquelético de humanos y otros mamíferos y causa una serie de alteraciones fisiológicas. Las proteínas de los productos de excreción-secreción de T. spiralis juegan un papel importante en la aparición y regulación de estas alteraciones. Sin embargo, aún no se conoce el efecto de estos productos en la infección e invasión del parásito al hospedero. Métodos: Mediante un análisis electroforético en una dimensión, Western blot y espectrometría de masas, se evaluaron las diferencias y similitudes entre proteínas antigénicas y de superficie de cuatro aislados de T. spiralis obtenidos de hospederos accidentales (perros) y la cepa de referencia aislada de cerdos (MSUS/MEX/91/CM). Resultados: Utilizando ontología de genes, se encontraron cinco proteínas exclusivas de los hospederos accidentales. Después del análisis, se encontró que estas proteínas forman parte de la matriz extracelular del parásito, cuentan con actividad catalítica y están implicadas en la adhesión a las células del hospedero. La actividad antigénica de las cuatro cepas aisladas de hospederos accidentales es idéntica a la reportada para T. spiralis, visualizándose el triplete antigénico característico de 43, 45 y 47 kDa. Conclusiones: Las proteínas exclusivas de los hospederos accidentales proveen información para entender el mecanismo de acción de este parásito para penetrar el músculo y evadir la respuesta inmune en el hospedero.

Abstract: Background: Trichinella spiralis is an intestinal and tissue nematode specific for mammalian skeletal muscle, causing a series of physiological alterations. T. spiralis excretory-secretion products play an important role in the appearance and regulation of these alterations. However, the effect of these products on the infection and invasion of the parasite to the host is unknown. Methods: Differences and similarities between antigenic proteins and surface proteins of four accidental hosts isolates (dogs) of T. spiralis and the reference strain isolated from pigs (MSUS/MEX/91/CM) were assessed by electrophoresis, western blot and mass spectrometry. Results: Using gene ontology, five proteins exclusive to the accidental hosts were analyzed. The results showed that these proteins are part of the extracellular matrix of the parasite, present catalytic activity, and bind to host cells. The antigenic activity the four strains showed the antigenic triplet characteristic of T. spiralis of 43, 45 and 47 kDa. Conclusions: Five proteins exclusive to dog isolates provided information to understand the mechanism of action of this parasite to penetrate the muscle and evade the immune response in the host.

Cinética de desialización eritrocitaria producida por larvas musculares vivas de Trichinella spiralis / Erythrocyte desialylation kinetics produced by living Trichinella spiralis muscle larvae / Cinética de dessialização eritrocitária causada por larvas musculares vivas da Trichinella spiralis

Las larvas musculares (LM) de T. spiralis alteran la agregación eritrocitaria. El objetivo del trabajo fue estudiar la cinética de desialización eritrocitaria producida por LM vivas de T. spiralis. Se realizaron 3 experiencias en las que se incubaron 60 larvas con 30 μL de eritrocitos en 1 mL de solución salina durante 1, 2, 3, 4, 5, 6, 7, 10, 15, 18, 20, 22 y 24 horas. Se aplicó el Método de Titulación de la Agregación por Polibrene y se calculó Título, Score Total y CexpCASP en los eritrocitos Control e incubados con LM. Los resultados mostraron que en la primera hora no hubo captación de ácido siálico. A las 2 horas el coeficiente comenzó a decrecer y a las 3 horas el Título disminuyó en una dilución y el coeficiente fue 0,62±0,021. En los siguientes tiempos el Título se mantuvo y el valor del coeficiente presentó pequeñas disminuciones, hasta alcanzar el valor de 0,45±0,010 a las 22 horas, tiempo en que se produjo la disminución significativa del Título. A las 24 horas hubo una nueva disminución del Título del Control y CexpCASP fue 0,13±0,093. Se concluye que las LM vivas durante incubación in vitro con eritrocitos comenzarían a captar ácido siálico a partir de las 2 horas de contacto logrando la desialización casi completa del eritrocito a las 24 horas.

T. spiralis muscle larvae (ML) alter erythrocyte aggregation. The objective of this work was to study erythrocyte desialylation kinetics produced by living T. spiralis ML. Three experiments were conducted in which 60 larvae were incubated with 30 μL of erythrocytes in 1 mL of saline solution for 1, 2, 3, 4, 5, 6, 7, 10, 15, 18, 20, 22 and 24 hours. Titration of Aggregation by Polybrene Method was used and Title, Total Score and CexpCASP were calculated in Control erythrocytes and erythrocytes incubated with ML. The results showed that in the first hour there was no capture of sialic acid. The coefficient began to decrease at 2 hours, and at 3 hours the Title decreased in one dilution and the coefficient was 0.62±0.021. The title was maintained at the following times and the coefficient value presented small decreases, until reaching 0.45±0.010 value at 22 hours. It was then that, a significant decrease in Title occurred. Within 24 hours, there was a further decrease of Control Title, and CexpCASP was 0.13±0.093. It can be concluded that living ML during in vitro incubation with erythrocytes began to capture sialic acid after 2 hours of contact, getting almost complete desialylation of erythrocytes at 24 hours.

As larvas musculares (LM) de T. spiralis alteram a agregação eritrocitaria. O objetivo do trabalho foi estudar a cinética de dessialização eritrocitária produzida por LM vivas de T. spiralis. Realizaram-se 3 experiências nas quais foram incubadas 60 larvas com 30 μL de eritrócitos em 1 μL de solução salina durante 1, 2, 3, 4, 5, 6, 7, 10, 15, 18, 20, 22 y 24 horas. Foi aplicado o Método de Titulação da Agregação por Polibreno e se calculou Título, Pontuação Total (ST) e CexpCASP. Os resultados mostraram que na primeira hora não houve captura de acido siálico. Às 2 horas o coeficiente começou a decrescer e às 3 horas o Título diminuiu numa diluição e o coeficiente foi 0,62±0,021. Nos tempos seguintes o Título se manteve e o valor do coeficiente apresentou pequenas diminuições, até atingir o valor de 0,45±0,010 às 22 horas, tempo em que se produziu a diminuição significativa do Título. Às 24 horas houve uma nova diminuição do Título do Controle e o CexpCASP foi 0,13±0,093. Conclui-se que as LM vivas durante incubação in vitro com eritrócitos, começariam a captar ácido siálico a partir das 2 horas de contato conseguindo a dessialização quase completa do eritrócito às 24 horas.