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.

Effect of the somatic proteins from Trichinella spiralis muscle larvae on apoptosis of H446 small cell lung cancer cells / 中国人兽共患病学报

To probe the effects of Trichinella spiralis muscle larval somatic proteins on small cell lung cancer H446 cells and the possible mechanism of anti-tumor,H446 cells were culture with 0.2 mg/mL,0.4 mg/mL,0.6 mg/mL,0.8 mg/mL,1.0 mg/mL,and 1.2 mg/mL somatic proteins respectively.The experimental group was set and no dosing as control group.MTT colorimetric assay was used to test the effects of T.spiralis muscle larval somatic proteins on the proliferative activity of H446 cells.We used flow cytometry (FCM) to test the influence of T.spiralis muscle larval somatic proteins induced H446 cells apoptosis.The real-time PCR and Western blot methods were used to detect the expression of Cyt-C and apoptotic protease activating factor 1(Apaf-1) mRNA and protein.The MTT colorimetric assay showed that T.spiralis muscle larval somatic proteins could inhibit the proliferation of H446 cells;the flow cytometry showed that polypide proteins acted on H446 cells after 24 h appeared an obvious effect on promoting apoptosis.Results of real-time PCR and Western blot analysis indicated that compared with the control group,Cyt-C and Apaf-1 showed up-regulated expression.T.spiralis muscle larval somatic proteins could inhibit proliferation activity and induce the apoptosis of H446 cells,and its effects may be related to up-regulated expression of Cyt-C and Apaf-1.

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.

Captación de ácido siálico por larvas musculares de Trichinella spiralis durante incubación in vitro / Sialic acid capture by Trichinella spiralis muscle larvae during incubation in vitro / Captação de ácido siálico por larvas musculares de Trichinella spiralis durante a incubação in vitro

Se ha comunicado que los eritrocitos (GR) incubados in vitro con larvas infectantes (LM) de T. spiralis presentan mayor agregación que los GR Controles incubados con solución salina, lo que indica que el parásito capta el ácido siálico globular. El objetivo del trabajo fue estudiar la captación de ácido siálico por LM durante incubación in vitro. Se incubaron 30, 60, 90 y 180 larvas con 30 mL del sedimento de GR en 1 mL de solución salina durante 24 y 48 h (37 °C). Se aplicó el Método de Agregación por Polibrene y se calculó Tïtulo, Score Total y CexpCASP. Los resultados mostraron una disminución significativa de estos 3 valores en relación al Control, excepto en el cultivo con 30 LM donde solo se observó un descenso moderado del Score Total y CexpCASP. La captación fue máxima a las 24 h, sin diferencias con los valores obtenidos a las 48 h. Posteriormente se incubaron 60 LM durante 1, 2 y 3 h. Se observó que no hubo captación de ácido siálico en la primera hora de incubación y que fue moderada a las 3 h. La experiencia demostró la captación de ácido siálico globular por las LM in vitro, lo que sugiere que podrían secuestrarlo del miocito con el objeto de evadir y/o interferir la respuesta inmune a los fines de asegurar su permanencia en el hospedador.

It has been reported that erythrocytes (RBC) incubated with infective larvae of T. spiralis (ML) exhibit higher aggregation than Control RBC incubated with saline solution, indicating that the parasite captures erythrocyte sialic acid. The objective of this work was to study sialic acid capture by ML during in vitro incubation. A total of 30, 60, 90 and 180 larvae were incubated with 30 mL of GR sediment in 1 mL of saline solution for 24 to 48 hours (37 °C). Aggregation by Polybrene Method was used, and Titre, Total Score and CexpCASP were calculated. The results showed a significant decrease of these three values compared to Control except in the culture with 30 ML where only a moderate decrease of Total Score and CexpCASP were observed. The capture was maximal at 24 hours, with no difference with the values obtained at 48 hours. Then, 60 ML were incubated for 1, 2 and 3 hours. It was noted that ML did no capture sialic acid in the first hour of incubation and the capture was moderate at 3 hours. The experience showed globular sialic acid capture by ML in vitro, suggesting that they could sequester it from the myocyte in order to evade and/or interfere with the immune response for the purposes of assuring their permanence in the host.

Foi informado que os eritrócitos (GV) incubados in vitro com larvas infetantes (LM) da T. spiralis apresentam maior agregação que os GV Controles incubados com solução salina, indicando que o parasita capta o ácido siálico globular. O objetivo do trabalho foi estudar a captação de ácido siálico por LM durante a incubação in vitro. Foram incubadas 30 , 60 , 90 e 180 larvas com 30 μL do sedimento de GV em 1 mL de solução salina durante 24 a 48 horas (37 °C). Foi aplicado o Método de Agregação por Polibrene e se calculou o Título, Pontuação Total e CexpCASP. Os resultados mostraram uma diminuição significativa desses três valores em relação ao Controle, exceto na cultura com 30 LM, onde foi observada apenas uma redução moderada da Pontuação Total e CexpCASP. A captação foi máxima às 24 horas, sem diferença com os valores obtidos às 48 horas. Posteriormente 60 LM foram incubadas durante 1, 2 e 3 horas. Observou-se que não houve captação alguma de ácido siálico na primeira hora de incubação e que foi moderada às 3 horas. A experiência mostrou captação de ácido siálico globular pelas LM in vitro, sugerindo que poderiam sequestrá-lo do miócito visando a evadir e/ou interferir a resposta imune, para garantir sua permanência no hospedeiro.

Estudio de la desialización producida por larvas musculares de Trichinella spiralis / Desialylation study produced by muscle larvae of Trichinella spiralis

Trichinella spiralis es agente causal de una zoonosis endémica en Argentina. El objetivo fue estudiar la desialización eritrocitaria producida por larvas musculares (LM) de T. spiralis. Se trabajó con concentrados de LM, incubados en partes iguales con glóbulos rojos (GR) Grupo O (37 °C) durante 3 horas (con y sin agitación controlada) durante 150 minutos, a intervalos de 30 minutos, para estudiar el curso de la desialización en el tiempo. Los GR controles fueron incubados de la misma manera, con igual volumen de solución fisiológica. Se aplicó el método de titulación de la agregación, calculando título y coeficiente de puntuación total (CexpST). Se encontró que los eritrocitos incubados con LM presentaron mayor agregación que los controles. El valor medio de CexpST con agitación (0,43) fue significativamente menor que cuando los GR no se agitaron (0,72). El estudio de la desialización eritrocitaria en el tiempo mostró que el título de los GR control disminuyó significativamente a los 90 minutos en 5/10 repeticiones y a los 150 minutos en 9/10. El aumento del tiempo de incubación produjo el incremento de la desialización excepto a los 120 y 150 minutos donde no existieron diferencias significativas en el valor de CexpST. La experiencia realizada in vitro, sugeriría que en la infección in vivo, las LM podrían captar el ácido siálico a partir de los residuos sializados presentes en la célula muscular.

Trichinella spiralis is the cause of an endemic zoonosis in Argentina. The objective was to study the erythrocyte desialylation by T. spiralis muscle larvae (ML) applying an aggregation titulation method. We worked with ML concentrates, which were incubated with an equal volume of O Group erythrocytes (RBCs) at 37° C for 3 hours, (with and without controlled agitation) and for 150 minutes, taking samples at 30 minutes intervals to study the course in time of the desialylation. RBCs control were incubated with an equal volume of physiological saline solution. Aggregation titulation method was applied and the title and total score coefficient (TSexpC) were calculated. The results showed that erythrocytes incubated with ML showed more aggregation than controls. The average TSexpC with agitation (0.43) was significantly lower than when erythrocytes were not stirred (0.72). The course in time of the erythrocyte desialylation showed that the RBCs contol title decreased significantly at 90 minutes in 5/10 repetitions and at 150 minutes in 9/10. Increasing the incubation time produced an increase in erythrocyte desialylation, except at the 120 and 150 minutes interval where no significant differences in TSexpC values were found. The in vitro experience would suggest that in cases of in vivo infection, ML could capture sialic acid from sialylate residues present in the muscle cell.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.

Application of Giemsa stain for easy detection of Trichinella spiralis muscle larvae

The application of Giemsa technique to stain compressed diaphragm samples obtained from rodents experimentally infected with Trichinella spiralis is described. Diaphragm samples from rats heavily infected with 20 muscle larvae per gram of body weight (20 ML/gbw) were cut into several pieces and stained with Giemsa; on the other hand, whole diaphragms from slightly infected mice (1 ML/gbw) were also stained with Giemsa. Besides, muscle samples were also stained with Giemsa. Observation at 10 x magnification revealed that both ML and nurse cells (NC) look as bluish structures clearly contrasting with the pinkish color of the non-infected muscle fibers. NC in the diaphragms of mice could be easily observed at naked eye as blue points contrasting with the pink surrounding areas formed by the non-infected muscle fibers. Among NC observed in the diaphragms of rats infected with 20 ML/gbw, 4.4% was multiple infection. These findings were confirmed in sectioned and hematoxylin-eosin stained specimens. This data could be usefulness for a rapid diagnosis of trichinellosis in post-mortem mammals without magnification procedures.

IDENTIFICATION OF SPECIFIC ANTIGEN OF TRICHINELLA SPIRALIS MUSCLE LARVAE / 中国血吸虫病防治杂志

Objective To find out the specific antigens for immunodiagnosis of trichinellosis. Methods The soluble antigens of Trichinella spiralis muscle larvae were analyzed by SDS-PAGE and Western blot. Results SDS-PAGE revealed that the soluble antigens of T. spiralis muscle larvae had 29 protein bands with molecular weight (MW) from 112 kDa to 12 kDa, among them the protein bands with MW 65,43,42,31,30,20,17,16 kDa were the major bands. Western blot results showed that the protein bands with 112,110,108, 102,97,95,65,63,58,55,53,49,45,43,42 kDa in T.spiralis muscle larval soluble antigens were cross-reacted with sera from rats and patients with paragonimiasis, sera from patients with clonorchiasis, schistosomiasis, and cys-ticercosis. The protein components with 24 - 20 kDa were only reacted with sera from rats, mice infected with T.spiralis and patients with trichinellosis, and not reacted with sera from animals and patients infected with other parasites,and sera from normal rats, mice and healthy persons. Conclusion The protein components with 24-20 kDa in T.spiralis muscle larval soluble antigens are the specific antigen for T.spiralis muscle larvae, it could be applied to the immunodiagnosis and seroepidemiological investigation on trichinellosis.

Distribution of Trichinella spiralis larvae in muscles of infected mice / 中国血吸虫病防治杂志

Objective To study the distribution of larvae of Trichinella spiralis in muscles of infected mice.Methods The muscles of mice infected with Trichinella spiralis were examined with microscopy.These muscles included lingua,masseter,pectoral muscle,abdominal muscle,forelimb muscle,crus muscle,diaphragma and dorsal muscle.Results The densities of the larvae were highest in diaphragma and lowest in abdominal muscle.Conclusion The larvae infection rate of diaphragma was higher than that of other muscles of mice with Trichinella spiralis infection.