In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia.

INTRODUCTION: Over 170 municipalities in Colombia have been invaded by Lissachatina fulica, an African snail that can carry larvae of nematodes of interest in human and veterinary health. Nematodes enter the host snail as larvae L1 and then change to L2 and L3, the infectious form for vertebrates. OBJECTIVE: To standardize culture in vitro of L3 carried by L. fulica from Santa Fe de Antioquia. MATERIALS AND METHODS: Between July and November, 2014, 10 snails were collected, killed, and conserved with HCl 0.7%. Larvae were recovered using the Baermann technique and cultured for 36 days in Schneider, DMEM and RPMI media, with and without SFB 20% and distilled water with SFB 20%. Replacements were made every 36 hours; larvae were measured with an ocular micrometer on a microscope. Summary statistics were estimated; box and whisker plots were made; the t Student test was performed in SPSS 18™. A p-value below 0.05 was assumed as significant. RESULTS: Fifty per cent of the larvae survived. The highest survival and growth was 85% in supplemented DMEM. The final average length of larvae in supplemented media exceeded the initial one. There were significant differences between the average length of larvae cultured in supplemented media and the initial length. The initial width of larvae did not change. CONCLUSIONS: The best medium for the culture of L3 larvae was supplemented DMEM. The length provided more information than the width for the larval growth evaluation. The larvae studied did not correspond to Angiostrongylus cantonensis, A. costaricensis or Aelurostrongylus abstrusus.

Cultivo in vitro de larvas L3 de nematodos obtenidas del caracol gigante africano Lissachatina fulica (Mollusca: Gastropoda) en Santa Fe de Antioquia / In vitro culture of L3 larvae of nematodes obtained from the African giant snail Lissachatina fulica (Mollusca: Gastropoda) in Santa Fe de Antioquia

Introducción. Más de 170 municipios colombianos están invadidos por Lissachatina fulica, caracol africano que puede portar larvas de nematodos de interés en salud humana y veterinaria. Los parásitos entran al caracol huésped intermediario en el estadio de larva L1, y allí cambian a L2 y L3, formas estas capaces de infectar a vertebrados. Objetivo. Estandarizar el cultivo in vitro de las L3 portadas por especímenes de L. fulica recolectados en Santa Fe de Antioquia. Materiales y métodos. Entre julio y noviembre de 2014 se recolectaron 10 caracoles, se sacrificaron y se digirieron con ácido clorhídrico al 0,7 %. Las larvas se recuperaron mediante la técnica de Baermann; se cultivaron 36 días en los medios Schneider, mínimo esencial de Eagle modificado por Dulbecco (Dulbecco's Modified Eagles Minimal Essential Medium, DMEM), y Roswell Park Memorial Institute (RPMI), con suero fetal bovino (SFB) al 20 % y sin este, y agua destilada con SFB al 20 %. Los medios de cultivo se cambiaron cada 36 horas. Las larvas se midieron con el microscopio utilizando reglilla ocular, y se evaluaron la supervivencia, la longitud y el ancho. Se calcularon datos estadísticos de resumen y se hicieron gráficos de cajas y bigotes, así como la prueba t de Student. El nivel de significación (p) se estableció como menor de 0,05. Resultados. El 50 % de las larvas sobrevivió, 85 % en DMEM con SFB al 20 %, el 70 % con RPMI más SFB al 20 %, el 60 % en RPMI, el 50 % en Schneider más SFB al 20 %, el 45 % en Schneider y el 40 % en DMEM. El control sobrevivió diez días. Hubo diferencias significativas entre la longitud inicial promedio de las larvas y la longitud final promedio en los medios con suplementos: inicial, 645,83 µm; final en DMEM más SFB al 20 %, 732,65 µm (p<0,001); en RPMI más SFB al 20 %, 718,79 µm (p<0,001), y en Schneider más SFB al 20 %, 696,12 µm (p<0,01). No hubo diferencias significativas entre la anchura inicial promedio, de 24,99 µm, y la final. Conclusiones. El mejor medio para cultivar las L3 de L. fulica fue el DMEM más SFB al 20 %. En la evaluación del crecimiento larval, la longitud fue más informativa que la anchura. Las larvas estudiadas no correspondieron a Angiostrongylus cantonensis, A. costaricensis ni Aelurostrongylus abstrusus.

Introduction: Over 170 municipalities in Colombia have been invaded by Lissachatina fulica, an African snail that can carry larvae of nematodes of interest in human and veterinary health. Nematodes enter the host snail as larvae L1 and then change to L2 and L3, the infectious form for vertebrates. Objective: To standardize culture in vitro of L3 carried by L. fulica from Santa Fe de Antioquia. Materials and methods: Between July and November, 2014, 10 snails were collected, killed, and conserved with HCl 0.7%. Larvae were recovered using the Baermann technique and cultured for 36 days in Schneider, DMEM and RPMI media, with and without SFB 20% and distilled water with SFB 20%. Replacements were made every 36 hours; larvae were measured with an ocular micrometer on a microscope. Summary statistics were estimated; box and whisker plots were made; the t Student test was performed in SPSS 18™. A p-value below 0.05 was assumed as significant. Results: Fifty per cent of the larvae survived. The highest survival and growth was 85% in supplemented DMEM. The final average length of larvae in supplemented media exceeded the initial one. There were significant differences between the average length of larvae cultured in supplemented media and the initial length. The initial width of larvae did not change. Conclusions: The best medium for the culture of L3 larvae was supplemented DMEM. The length provided more information than the width for the larval growth evaluation. The larvae studied did not correspond to Angiostrongylus cantonensis, A. costaricensis or Aelurostrongylus abstrusus.

[Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum].

INTRODUCTION: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. OBJECTIVE: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. MATERIALS AND METHODS: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. RESULTS: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). CONCLUSIONS: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Estandarización de una prueba múltiple de reacción en cadena de la polimerasa en tiempo real para la identificación de Angiostrongylus cantonensis, A. costaricensis y A. vasorum / Standardization of a multiplex real-time PCR test for the identification of Angiostrongylus cantonensis, A. costaricensis and A. vasorum

Resumen Introducción. En el mundo, las angiostrongilosis de mayor impacto en salud humana y animal son ocasionadas por Angiostrongylus cantonensis, A. costaricensis y A. vasorum. En las personas, las formas clínicas son la meningitis eosinofílica y la angiostrongilosis abdominal, y, en los mamíferos cánidos, el daño cardiopulmonar. Se las consideran enfermedades emergentes debido a la propagación mundial del caracol africano Lissachatina fulica, un huésped intermediario de los parásitos. Los escasos métodos de identificación de Angiostrongylus spp. no son muy específicos ni sensibles y son costosos. Se necesita urgentemente una herramienta diagnóstica asequible, sensible y específica para el manejo de las angiostrongilosis humana y la animal. Objetivo. Desarrollar una prueba de PCR múltiple en tiempo real (qPCR) para identificar las tres especies patógenas de Angiostrongylus. Materiales y métodos. Mediante un análisis bioinformático se seleccionó una secuencia del genoma ITS-2 de Angiostrongylus para garantizar la especificidad del cebador y las sondas. El ADN de los parásitos adultos (control positivo) y de las larvas se extrajo con el estuche DNeasyBlood & Tissue®. Las reacciones de la PCR cuantitativa se ejecutaron en un termociclador Smartcycler Cepheid®, usando el estuche de mezcla maestra QuantiTect®. Como control negativo, se utilizó ADN humano, de otros parásitos y del caracol africano. Resultados. Los valores del ciclo umbral para los controles positivos de ADN fueron: 21 para Angiostrongylus cantonensis, 22 para A. costaricensis y 31 para A. vasorum. En los controles negativos, el ciclo umbral fue cero. La qPCR mostró una eficiencia de amplificación de 2 (100 %). Conclusiones. En el laboratorio se estandarizó una qPCR múltiple para tres especies clínicamente significativas de Angiostrongylus.

Abstract Introduction: Angiostrongyliasis is a disease caused by Angiostrongylus nematodes that is present worldwide. The infections with the highest impact on human and animal health are caused by A. cantonensis, A. costaricensis, and A. vasorum. Clinical forms of the disease in humans are eosinophilic meningitis and abdominal angiostrongyliasis, while the most common effect on dogs are cardiopulmonary damages. It is deemed as an emerging disease as the result of the global dissemination of the African snail Lissachatina fulica, an intermediary host of these parasites. The few diagnostic methods for Angiostrongylus spp. are unspecific, costly, and not very sensitive. It is urgent to develop a sensitive, specific and accessible diagnostic tool for the control of human and animal angiostrongyliasis. Objective: To develop a qPCR multiple test to identify the three pathogenic species of Angiostrongylus. Materials and methods: Through a bio-informatic analysis, we selected a sequence of the ITS-2 region of the Angiostrongylus genome to guarantee the specificity of primers and probes. We extracted DNA from adult parasites as positive control, and from larvae using the DNeasy Blood&Tissue® kit. Quantitative PCR reactions were conducted on a Smartcycler Cepheid® thermocycler using a master mix QuantiTect® kit. DNA from human beings, other parasites and the African snail was used as negative control. Results: The threshold cycle values for positive DNA controls were: 21 for Angiostrongylus cantonensis, 22 for A. costaricensis, and 31 for A. vasorum. In negative controls, the threshold cycle was zero. qPCR showed an amplification efficiency of 2 (100%). Conclusions: A multiple qPCR was standardized at the laboratory for three clinically significant species of Angiostrongylus.

Evaluación de la técnica modificada de Dennis para el diagnóstico de fasciolosis bovina / Evaluation of modified Dennis parasitological technique for diagnosis of bovine fascioliasis

Introducción. La fasciolosis bovina genera cuantiosas pérdidas económicas, que en Colombia se estiman en COP$12.483 millones anuales, y tiene una prevalencia de 25 % en vacunos lecheros. La enfermedad se diagnostica mediante pruebas parasitológicas. En el país se utiliza la técnica de Dennis modificada en el 2002, pero su sensibilidad, especificidad y validez se desconocen. Objetivo. Evaluar la validez y el desempeño de la técnica modificada de Dennis para el diagnóstico de fasciolosis bovina, usando como prueba de referencia la observación de parásitos en el hígado. Materiales y métodos. Se hizo un estudio de evaluación diagnóstica. Se seleccionaron por conveniencia bóvidos de descarte sacrificados entre marzo y junio de 2013 en Frigocolanta; de cada ejemplar se recolectaron 25 g de heces y se examinaron los conductos biliares y el hígado en busca de Fasciola hepatica . Se estimaron los valores de sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo e índice de validez, con intervalos de confianza de 95 %. Como prueba de referencia se usó la evaluación post mortem. Resultados. Se analizaron 180 bóvidos. La sensibilidad y la especificidad de la técnica modificada de Dennis fueron de 73,2 % (IC 95% 58,4-87,9) y 84,2 % (IC 95% 77,7-90,6), respectivamente. El valor predictivo positivo fue de 57,7 % (IC 95% 43,3-72,1) y el negativo fue de 91,4 % (IC 95% 86,2-96,6). La prevalencia de fasciolosis bovina fue de 22,8 % (IC 95% 16,4-29,2). Conclusión. La validez y el desempeño de la técnica modificada de Dennis fueron superiores a los de la técnica tradicional, por lo que constituye una buena heramienta de tamización para el diagnóstico de la fasciolosis en estudios poblacionales y de prevalencia, así como en jornadas de salud animal.

Introduction: Bovine fascioliasis causes important economic losses, estimated at COP$ 12,483 billion per year; its prevalence is 25% in dairy cattle. Parasitological techniques are required for it diagnosis. The Dennis technique, modified in 2002, is the one used in Colombia, but its sensitivity, specificity and validity are not known. Objective: To evaluate the validity and performance of the modified Dennis technique for diagnosis of bovine fascioliasis using as reference test the observation of parasites in the liver. Materials and methods: We conducted a diagnostic evaluation study. We selected a convenience sample of discarded bovines sacrificed between March and June, 2013, in Frigocolanta for the study. We collected 25 g of feces from each animal and their liver and bile ducts were examined for Fasciola hepatica . The sensitivity, specificity, predictive positive value, predictive negative value, and validity index were calculated with 95% confidence intervals. The post-mortem evaluation was used as the gold standard. Results: We analyzed 180 bovines. The sensitivity and specificity of the modified Dennis technique were 73.2% (95% CI=58.4% - 87.9%) and 84.2% (95% CI= 77.7% - 90.6%), respectively. The positive predictive value was 57.7% (95% CI= 43.3% - 72.1%) and the negative one 91.4% (95% CI= 86.2% - 96.6%). The prevalence of bovine fascioliasis was 22.8% (95% CI= 16.4% - 29.2%). Conclusion: The validity and the performance of the modified Dennis technique were higher than those of the traditional one, which makes it a good screening test for diagnosing fascioliasis for population and prevalence studies and during animal health campaigns.

Evaluation of modified Dennis parasitological technique for diagnosis of bovine fascioliasis.

INTRODUCTION: Bovine fascioliasis causes important economic losses, estimated at COP$ 12,483 billion per year; its prevalence is 25% in dairy cattle. Parasitological techniques are required for it diagnosis. The Dennis technique, modified in 2002, is the one used in Colombia, but its sensitivity, specificity and validity are not known.  OBJECTIVE: To evaluate the validity and performance of the modified Dennis technique for diagnosis of bovine fascioliasis using as reference test the observation of parasites in the liver.  MATERIALS AND METHODS: We conducted a diagnostic evaluation study. We selected a convenience sample of discarded bovines sacrificed between March and June, 2013, in Frigocolanta for the study. We collected 25 g of feces from each animal and their liver and bile ducts were examined for Fasciola hepatica. The sensitivity, specificity, predictive positive value, predictive negative value, and validity index were calculated with 95% confidence intervals. The post-mortem evaluation was used as the gold standard.  RESULTS: We analyzed 180 bovines. The sensitivity and specificity of the modified Dennis technique were 73.2% (95% CI=58.4% - 87.9%) and 84.2% (95% CI= 77.7% - 90.6%), respectively. The positive predictive value was 57.7% (95% CI= 43.3% - 72.1%) and the negative one 91.4% (95% CI= 86.2% - 96.6%). The prevalence of bovine fascioliasis was 22.8% (95% CI= 16.4% - 29.2%).  CONCLUSION: The validity and the performance of the modified Dennis technique were higher than those of the traditional one, which makes it a good screening test for diagnosing fascioliasis for population and prevalence studies and during animal health campaigns.

[Lymnaea cousini , intermediate host of Fasciola hepatica in the Colombian high tropical Andes, and its new haplotypes confirmed with the mitochondrial marker cytochrome oxidase I]. / Lymnaea cousini , huésped de Fasciola hepatica en el trópico alto andino de Colombia, y sus nuevos haplotipos, confirmados con el marcador mitocondrial del gen de la citocromo oxidasa I.

INTRODUCTION: Fasciolosis is the disease transmitted by vectors with the highest latitudinal, longitudinal, and altitudinal distribution due to the colonizing capacity of the parasite Fasciola hepatica and its intermediate hosts, Lymnaeidae mollusks. These snails are under research due to their epidemiological importance, but their taxonomic identification is difficult given their interspecific phenotypical similarity. For this reason, there is uncertainty regarding Lymnaea cousini -a host of F. hepatica in Colombia- due to the morphological similarity it has with Lymnaea meridensis , recently described for Venezuela. OBJECTIVE: To confirm with the COI marker (ADNmt) the taxonomic status of individuals morphologically identified as L. cousini from Nariño, Norte de Santander, and Santander (Colombia), deposited in the Vector Mollusks Collection VHET No. 37 of Universidad de Antioquia. MATERIALS AND METHODS: The amplification of the mitochondrial COI required total DNA extraction of each individual´s foot using the DNeasy Blood and Tissue Kit (Qiagen®). Products amplified were sent for sequencing to Macrogen Inc., Korea. Twenty seven sequences generated in this research were compared to sequences published in the GenBank, including sequences of the type locality of L. cousini . RESULTS: Two new haplotypes of L. cousini were obtained for Colombia. Specimens from Nariño correspond to haplotype A, referenced for Ecuador, and specimens from Santander and Norte de Santander belong to a new haplotype we called haplotype D. CONCLUSION: By using the mitochondrial COI marker, we confirmed that the species under study did correspond to L. cousini . The number of known haplotypes of the species for Colombia has been duplicated and its geographical distribution has been extended to the southwest and northeast of the Colombian high Andean region.

Lymnaea cousini , huésped de Fasciola hepatica en el trópico alto andino de Colombia, y sus nuevos haplotipos, confirmados con el marcador mitocondrial del gen de la citocromo oxidasa I / Lymnaea cousini , intermediate host of Fasciola hepatica in the Colombian high tropical Andes, and its new haplotypes confirmed with the mitochondrial marker cytochrome oxidase I

Institución donde se ejecutó el trabajo: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Unidad de Malacología Médica y Trematodos (UMMT), Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia Introducción. La fasciolosis es la enfermedad transmitida por vectores con mayor distribución latitudinal, longitudinal y altitudinal, debido a la capacidad colonizadora del parásito Fasciola hepatica y de sus huéspedes intermediarios, los moluscos limneidos. Estos caracoles se investigan por su importancia epidemiológica, pero su identificación taxonómica es difícil por la similitud fenotípica entre especies. En este sentido, con respecto a Lymnaea cousini , un huésped de F. hepatica en Colombia, existe incertidumbre en razón de su similitud morfológica con L. meridensis , descrita recientemente en Venezuela. Objetivo. Confirmar con el marcador del gen de la citocromo oxidasa I en el ADN mitocondrial COI (ADNmt), el estatus taxonómico de ejemplares morfológicamente caracterizados como L. cousini provenientes de Nariño, Norte de Santander y Santander (Colombia), depositados en la Colección de Moluscos Vectores de la Universidad de Antioquia, VHET N° 37. Materiales y métodos. Para la amplificación del COI mitocondrial, se extrajo ADN total del pie de cada ejemplar con el estuche DNeasy Blood and Tissue (Qiagen ® ). Los productos amplificados se enviaron a secuenciar a Macrogen Inc., Corea. Las 27 secuencias generadas en esta investigación se compararon con secuencias publicadas en el GenBank, incluidas las secuencias de la localidad tipo de L. cousini. Resultados. Se encontraron dos nuevos haplotipos de L. cousini para Colombia. Los especímenes de Nariño correspondían al haplotipo A, referenciado en Ecuador, y los especímenes de Santander y Norte de Santander, a un nuevo haplotipo al que se denominó D. Conclusión. Mediante el marcador mitocondrial del COI , se confirmó que los especímenes pertenecían a la especie L. cousini . Con el hallazgo se duplicó el número de haplotipos conocidos de la especie en Colombia y se amplió su distribución geográfica al suroeste y nordeste de la región altoandina colombiana.

Introduction: Fasciolosis is the disease transmitted by vectors with the highest latitudinal, longitudinal, and altitudinal distribution due to the colonizing capacity of the parasite Fasciola hepatica and its intermediate hosts, Lymnaeidae mollusks. These snails are under research due to their epidemiological importance, but their taxonomic identification is difficult given their interspecific phenotypical similarity. For this reason, there is uncertainty regarding Lymnaea cousini -a host of F. hepatica in Colombia- due to the morphological similarity it has with Lymnaea meridensis , recently described for Venezuela. Objective: To confirm with the COI marker (ADNmt) the taxonomic status of individuals morphologically identified as L. cousini from Nariño, Norte de Santander, and Santander (Colombia), deposited in the Vector Mollusks Collection VHET No. 37 of Universidad de Antioquia. Materials and methods: The amplification of the mitochondrial COI required total DNA extraction of each individual´s foot using the DNeasy Blood and Tissue Kit (Qiagen®). Products amplified were sent for sequencing to Macrogen Inc., Korea. Twenty seven sequences generated in this research were compared to sequences published in the GenBank, including sequences of the type locality of L. cousini . Results: Two new haplotypes of L. cousini were obtained for Colombia. Specimens from Nariño correspond to haplotype A, referenced for Ecuador, and specimens from Santander and Norte de Santander belong to a new haplotype we called haplotype D. Conclusion : By using the mitochondrial COI marker, we confirmed that the species under study did correspond to L. cousini . The number of known haplotypes of the species for Colombia has been duplicated and its geographical distribution has been extended to the southwest and northeast of the Colombian high Andean region.

The disease is another: therapeutic itinerary of bovine fascioliosis inRionegro (Antioquia) / La enfermedad es otra: itinerario terapéutico de la fasciolosis bovina enRionegro (Antioquia) / A doença é outra: itinerário terapêutico de fasciolose bovina em Rionegro(Antioquia)

This article presents results of an anthropological research project that was carried out in the municipality ofRionegro in the department of Antioquia, Colombia. The disease caused by hepatic fasciola, a parasite native toEurope, and is now present on every continent and is considered to be the pathogenic disease with the greatestlatitudinal, longitudinal, and altitudinal distribution. There are no specific observable symptoms in infected animals that allow a diagnosis, which must be done in veterinary laboratories, which is scarcely ever the case given that thedisease rarely causes death. Through the use of ethnographic methods the researchers accessed various interlocutors linked to the pecuary field, which relayed their interpretations about bovine fasciolosis and about the common practices dealing with it ranging from lay knowledge to dialogues with expert and professional understanding. Thetext illustrates the therapeutic itinerary that is undertaken when certain changes in behavior and appearance areperceived, it describes the course of action that is followed in order to restore the animals’ health, and it considers the decisions that livestock farmers take when cures fail, all of which are the result of the fact that it is not mandatory to declare bovine fasciolosis. Academics in the field of national veterinary medicine who research the basic aspects related to the parasite’s life cycle are not familiar with the lay knowledge produced about this disease. An analysis of this knoweldge could contribute to the design of control strategies given that it presents in detail the doubts anduncertainties that have historically surrounded fasciolosis. The tendency to conceive it as a “new” disease, and the scarce attention that it has received in comparison with other livestock diseases that are well-defined in sanitation plans work against the control and prevention of fasciolosis in social practices of bovine livestock management inthe area under study.

Se presentan resultados de una investigación antropológica realizada en el municipio de Rionegro, departamentode Antioquia, Colombia. La enfermedad ocasionada por Fasciola hepatica, un parásito originario de Europa, seencuentra en la actualidad en todos los continentes y es considerada la enfermedad patogénica transmitida porvectores de mayor distribución latitudinal, longitudinal y altitudinal. En los animales infectados no se observansíntomas específicos que faciliten el diagnóstico, este debe hacerse en un laboratorio veterinario, al que en muypocas ocasiones se recurre, debido a que la dolencia rara vez causa la muerte. Mediante el método etnográfico los investigadores accedieron a diferentes interlocutores ligados al ámbito pecuario que expresan sus interpretaciones acerca de la fasciolosis bovina y de las prácticas corrientes para enfrentarla desde el conocimiento lego, en diálogo con el conocimiento profesional o experto. El texto ilustra el itinerario terapéutico iniciado al percibir ciertos cambios en el comportamiento y en el aspecto de los animales, describe el curso de acción para restituír la salud de éstos y se detiene en las decisiones que toman los ganaderos ante el fracaso de la cura, todo ello a expensas de que la fasciolosis bovina no constituye una enfermedad de declaración obligatoria.

No presente artigo apresentam-se resultados de uma pesquisa antropológica no município de Rionegro, departamentoou província de Antioquia, Colômbia. A doença produzida pela fasciola hepatica, um parasita originário da Europa,encontra-se atualmente em todos os continentes, sendo considerada uma doença patogênica transmitida por vetores de maior distribuição latitudinal, longitudinal e altitudinal. Nos animais infetados não se observam sintomas específicosque facilitem o diagnóstico, sendo que este dever ser realizado em um laboratório veterinário. Contudo, o diagnóstico é feito com pouca frequência devido a que a doença raramente causa a morte. Mediante o método etnográfico, ospesquisadores entraram em contato com diferentes interlocutores ligados ao âmbito pecuário que expressam suasinterpretações acerca da fasciolose bovina e das práticas mais comuns para enfrentá-la a partir de um conhecimentoleigo que dialoga com o conhecimento profissional ou especializado. Isto tudo à custa do fato de a fasciolose bovina não constituir uma doença que deva ser declarada. Os acadêmicos, no âmbito da medicina veterinária na Colômbia,conhecem os aspectos básicos relacionados com o ciclo de vida do parasita, mas não estão familiarizados como o conhecimento leigo da doença. Um olhar a partir deste aspecto poderia contribuir para a criação de estratégias de controle visto que apresenta, para um público mais amplo, um mundo de dúvidas e incertezas que foi construídohistoricamente em torno da fasciolose. A tendência a concebê-la como uma doença “nova” e a pouca atenção dada,quando comparada com outras doenças do gado definidas nos programas e planos da saúde pública, desfavorecemo controle a prevenção da fasciolose nas práticas sociais de manejo do gado bovino observadas na área de estudo.

Biodiversidad y endemismo de los caracoles terrestres Megalobulimusy Systrophia en la Amazonia occidental / Biodiversity and endemism of the western Amazonia land snails Megalobulimus and Systrophia

En este trabajo realizamos un estudio biogeográfico de dos géneros de caracoles terrestres amazónicos, Megalobulimus (Strophocheilidae) y Systrophia (Scolodontidae). Se utilizaron individuos colectados en diversas localidades de la Amazonia peruana así como información bibliográfica. Se utilizaron los marcadores moleculares 5.8S-ITS2-28S rRNA y 16S rRNA para reconstruir filogenias y obtener hipótesis sobre las relaciones evolutivas entre los géneros amazónicos y otras especies de distribución global. La filogenia nuclear permitió determinar la posición evolutiva de ambos géneros y la filogenia mitocondrial permitió la diferenciación de las especies a nivel intragenérico. Megalobulimus formó parte del clado no-achatinoideo en la filogenia de los gastrópodos Stylommatophora, como lo esperado, pero no pudo ser demostrada su cercanía a la familia Acavidae, mientras que Systrophia quedó fuera de los dos clados establecidos, formando uno basal dentro de los Stylommatophora. El gen mitocondrial 16S rRNA permitió diferenciar a las especies de Megalobulimus, actuando como código de barras de ADN de estos caracoles comestibles. El análisis de distribución geográfica reveló varios endemismos para la Amazonia peruana para especies de ambos géneros, resaltando las unidades biogeográficas de Chanchamayo e Inambari.

In this work we performed a biogeographic study of two genera of Amazonian land snails, Megalobulimus (Strophocheilidae) and Systrophia (Scolodontidae). We used samples from different regions of the Peruvian Amazon, as well as bibliographic information. We analyzed both nuclear (5.8S-ITS2-28S rRNA) and mitochondrial (16S rRNA) genes to reconstruct phylogenies and obtain hypotheses concerning the evolutionary relationships among Amazonian genera and other species with global distribution. The nuclear phylogeny allowed us to determine the evolutionary position of both genera, and the mitochondrial phylogeny permitted the differentiation of species at the intrageneric level. We found that Megalobulimus clustered with the non-achatinoid clade within Stylommatophora, as expected, but its relationship to family Acavidae could not be demonstrated. Systrophiadid not cluster with any of the two established clades, but formed a basal one within Stylommatophora. The mitochondrial gene 16S rRNA allowed us to differentiate Megalobulimus species, and performed well for DNA barcoding of these edible snails. Biogeographical analysis revealed several endemic species in the Peruvian Amazon within both genera, highlighting the Chanchamayo and Inambari biogeographic units.

[Paragonimus sp. in crabs and awareness of the educational community to aquatic ecosystems in La Miel and La Clara, Caldas, Antioquia]. / Paragonimus sp. en cangrejos y sensibilización de la comunidad educativa hacia los ecosistemas acuáticos de La Miel y La Clara, Caldas, Antioquia.

INTRODUCTION: Previous research has demonstrated the presence of Paragonimus spp. in crabs of three municipalities located in the Medellín river basin. To design a plan for the control and prevention of paragonimosis, parasite presence must be demonstrated in the upper area of the river, and an awareness campaign of its risks must involve the participation of the educational community in focusing attention on the local aquatic resources. OBJECTIVE: The presence of Paragonimus spp. metarcercariae was established in freshwater crabs from La Miel and La Clara streams, and an awareness campaign created public awareness to management of the aquatic ecosystems for risk reduction. MATERIALS AND METHODS: Nine crabs were collected from streams of La Miel and La Clara villages in 2007 and 2008. The crabs were indentified, sacrificed in the laboratory and examined for presence of Paragonimus spp. metacercariae. Five workshops were conducted with students of educational institutions of the two villages to improve the student perception of paragonimosis risks associated with the local streams. RESULTS: The crabs were identified as Hypolobocera bouvieri. One crab was found with Paragonimus spp. in La Miel. A mollusk, Aroapyrgus colombiensis, another host of the parasite was found in La Clara. That aquatic ecosystems are a great attraction for the local was well established. CONCLUSIONS: Foci of Paragonimus spp. flatworms are present in the upper area of Medellín river which includes Caldas. However, local school children were uninformed about the risk of paragonimosis associated with the local aquatic ecosystem. Therefore this province must be included in programs for prevention and control of paragonimosis. The prevalence of this disease recommends educational programs that promote awareness of local aquatic ecosystems and their parasitic inhabitants.

Paragonimus sp. en cangrejos y sensibilización de la comunidad educativa hacia los ecosistemas acuáticos de La Miel y La Clara, Caldas, Antioquia / Paragonimus sp. in crabs and awareness of the educational community to aquatic ecosystems in La Miel and La Clara, Caldas, Antioquia

Introducción. Estudios previos demostraron la presencia de Paragonimus sp. en cangrejos de tres municipios en la cuenca del río Medellín. Para diseñar un plan para el control y la prevención de la paragonimosis, es necesario determinar la presencia del parásito en la parte alta del río, con la participación de la comunidad educativa, para dirigir su atención hacia el recurso hídrico. Objetivo. Establecer la presencia de metacercarias de Paragonimus sp. en cangrejos de las quebradas La Miel y La Clara, y sensibilizar a la comunidad educativa hacia el manejo adecuado de sus ecosistemas acuáticos. Materiales y métodos. En 2007 y 2008 se recolectaron cangrejos de quebradas de las veredas La Miel y La Clara. En el laboratorio se relajaron y sacrificaron para buscar metacercarias de Paragonimus sp. y determinar la especie. Con estudiantes de instituciones educativas de ambas veredas, se llevaron a cabo cinco talleres lúdicos, para mejorar la percepción de su relación con sus quebradas. Resultados. Se capturaron nueve cangrejos Hypolobocera bouvieri. En La Miel se halló uno con Paragonimus sp. En La Clara se encontró, además, Aroapyrgus colombiensis, molusco huésped del parásito. Con los estudiantes se estableció que los ecosistemas acuáticos ejercen una gran atracción sobre ellos; sin embargo, desconocen los riesgos y no sostienen una relación responsable. Conclusiones. Se señala la presencia de focos de Paragonimus sp. en la parte alta del río Medellín; por lo tanto, el municipio de Caldas debe incluirse en los programas de prevención y control. La población estudiantil requiere nuevos programas educativos para mejorar la interacción con los ecosistemas acuáticos y disminuir el deterioro de su salud.

Introduction. Previous research has demonstrated the presence of Paragonimus spp. in crabs of three municipalities located in the Medellín river basin. To design a plan for the control and prevention of paragonimosis, parasite presence must be demonstrated in the upper area of the river, and an awareness campaign of its risks must involve the participation of the educational community in focusing attention on the local aquatic resources. Objective. The presence of Paragonimus spp. metarcercariae was established in freshwater crabs from La Miel and La Clara streams, and an awareness campaign created public awareness to management of the aquatic ecosystems for risk reduction. Materials and methods. Nine crabs were collected from streams of La Miel and La Clara villages in 2007 and 2008. The crabs were indentified, sacrificed in the laboratory and examined for presence of Paragonimus spp. metacercariae. Five workshops were conducted with students of educational institutions of the two villages to improve the student perception of paragonimosis risks associated with the local streams. Results. The crabs were identified as Hypolobocera bouvieri. One crab was found with Paragonimus spp. in La Miel. A mollusk, Aroapyrgus colombiensis, another host of the parasite was found in La Clara. That aquatic ecosystems are a great attraction for the local was well established. Conclusions. Foci of Paragonimus spp. flatworms are present in the upper area of Medellín river which includes Caldas. However, local school children were uninformed about the risk of paragonimosis associated with the local aquatic ecosystem. Therefore this province must be included in programs for prevention and control of paragonimosis. The prevalence of this disease recommends educational programs that promote awareness of local aquatic ecosystems and their parasitic inhabitants.

LARVAS DE DIGENEA EN Melanoides tuberculata (GASTROPODA: THIARIDAE) EN MEDELLÍN, COLOMBIA / Larval stages of digenea from Melanoides tuberculata (Gastropoda: Thiaridae) in Medellín, Colombia

Se describen las larvas de digeneos que se obtuvieron en Melanoides tuberculata (Gastropoda: Thiaridae), molusco dulceacuícola del que se colectaron 125 especímenes en el lago del Jardín Botánico Joaquín Antonio Uribe de Medellín. En el laboratorio se individualizaron y se estimuló la emisión cercariana con una fuente luminosa. El 9,6 % de los caracoles emitió cercarias. Los moluscos emisores se sacrificaron para obtener los demás estadios larvarios. Las larvas se montaron al microscopio, se midieron y luego se dibujaron. Los caracteres morfológicos permitieron establecer la presencia de Centrocestus formosanus (Heterophyidae) y de dos Philophthalmidae. Uno de estos es primer registro para Colombia. Se confirma la sensibilidad de M. tuberculata a infecciones por digeneos, así como la especificidad de los filoftálmidos por moluscos hospedadores de la superfamilia Cerithioidea.

The larvae of two trematodes found in the freshwater mollusk Melanoides tuberculata (Gastropoda: Thiaridae) are described. 125 snails from the Medellin’s Botanical Garden Joaquín Antonio Uribe were collected. In the laboratory, each individual was placed alone and stimulated with light in order to have cercaria emission. 9.6% of the snails released cercariae. These were separated in three morphologies. From each one, 15 individuals were measured and drawn. Photographs were taken. Based on their morphological characteritics the presence of Centrocestus formosanus (Heterophyidae) was determined. The other two morphologies were classified in the family Philophthalmidae. One of them constitutes the first register for Colombia. The sensibility of M. tuberculata to infections by digeneans, and the specificity of the family Philophthalmidae to mollusk hosts of the superfamily Cerithioidea are confirmed.

Distribución parcial de Paragonimus (Digenea: Troglotrematidae) en Antioquia, por presencia de metacercarias en cangrejos dulciacuícolas / Distribution of Paragonimus (Digenea: Troglotrematidae) in Antioquia Province, Colombia, based on metacercariae counts in freshwater crabs

Introducción. La paragonimosis, o distomatosis pulmonar, es una enfermedad con sintomatología similar a la observada en la tuberculosis. Es causada por parásitos del género Paragonimus (Digenea: Troglotrematidae). Las personas se infectan al consumir cangrejos crudos o mal cocidos, con metacercarias del parásito. El primer foco de paragonimosis humana en Colombia se registró durante 1995 en Urrao, Antioquia, donde se hallaron dos especies de cangrejos que hospedaban el parásito. En el 2005 se capturaron cangrejos con metacercarias de Paragonimus en Medellín, lo que motivó la búsqueda del parásito en otras localidades, mediante su presencia en estos crustáceos. Objetivo. Establecer la distribución de Paragonimus en Antioquia, evaluando la presencia demetacercarias en macrocrustáceos braquiuros, dulciacuícolas. Materiales y métodos. Desde 2005 hasta 2007 se capturaron cangrejos en 13 municipios antioqueños. Se relajaron y sacrificaron para la búsqueda del digeneo y la identificación taxonómica. Resultados. En nueve municipios se capturaron 52 cangrejos, 42 (80,76 por ciento) con metacercarias de Paragonimus. Todos los crustáceos se determinaron como Pseudothelphusidae, de los géneros Hypolobocera y Strengeriana, y se asignaron a cuatro especies. Tres se registran por primera vez como huéspedes del parásito. Conclusión. Se inicia la construcción de un mapa con la distribución de Paragonimus en Antioquia que incluye por primera vez zonas urbanizadas. Se ratifican el consumo y la manipulación de los cangrejos crudos y mal cocidos como factores de riesgo para la infección humana. Se propone a los cangrejos como agentes focalizadores de paragonimosis por ser asequibles y de fácil diagnóstico.

[Paragonimosis in the peri-urban zone of Medellín, Antioquia]. / Estudio de foco de paragonimosis en Fuente Clara, Robledo, área periurbana de Medellín, Antioquia.

INTRODUCTION: Human paragonimosis in Colombia was assumed to be restricted to the sylvatic areas. However, in 2005, crabs infected with Paragonimus were found in Fuente Clara, an urban sector in Medellín. OBJECTIVE: A study was designed to understand the ecoepidemiology of paragonimosis. Programs were initiated to educate the community in the suitable use of wetland ecosystems. MATERIALS AND METHODS: Infection rates of Paragonimus in human and wild hosts was documented in the Fuente Clara sector. The presence of larvae and digenic adult worms was detected in mollusks (1,312), crabs (27) and mammals (4). Sputum diagnosis was performed on samples from 18 volunteer individuals. The following determinants of water quality were measured in the stream La Puerta: total fecal/coliform, pH, conductivity and dissolved oxygen were measured. Recreational workshops were conducted with children and teenagers for educational purposes. RESULTS: The percentage of infections found in hosts was as follows: snails, 0.07%; crabs, 55.5%; (wild) mammals, 25%; humans, 0%. During the workshops, children and teenagers identified the Paragonimus hosts and the risk factors for acquiring the disease. The water of the stream was found to be unsuitable for consumption and recreation (the most probable number of total coliforms/100 ml was in a scale of 104). CONCLUSIONS: Fuente Clara is the first urban locality in Colombia where a focus of Paragonimus was found. Exposure to and consumption of crabs may constitute a risk for human infection.

Estudio de foco de paragonimosis en Fuente Clara, Robledo, área periurbana de Medellín, Antioquia / Paragonimosis in the peri-urban zone of Medellín, Antioquia

Introducción. La información sobre paragonimosis humana en Colombia llevó a suponer que los focos de la enfermedad estaban localizados en sectores selváticos; sin embargo, durante 2005 se hallaron cangrejos infectados con Paragonimus sp. en Fuente Clara, zona urbana de Medellín. Esto motivó la investigación. Objetivo. Realizar un estudio ecoepidemiológico de la paragonimosis con la participación de la comunidad para promover un manejo adecuado de los ecosistemas acuáticos. Materiales y métodos. Se realizó búsqueda de huéspedes silvestres y humanos de Paragonimus sp. en Fuente Clara. La presencia de formas larvarias y gusanos adultos del digéneo se evaluó en moluscos, crustáceos y mamíferos. En las personas voluntarias se hizo diagnóstico en esputo. Al agua de la quebrada La Puerta se le midieron: coliformes totales/fecales, pH, conductividad y oxígeno disuelto. Con niños y adolescentes se realizaron talleres educativos utilizando técnicas lúdicas. Resultados. Los porcentajes de infección encontrados fueron: caracoles, 0,07 por ciento; cangrejos, 55,5 por ciento; mamíferos, 25 por ciento, y personas, 0 por ciento. Durante los talleres educativos se manifestó la importancia de los recursos naturales del barrio. Los niños identificaron los huéspedes de Paragonimus sp. y detectaron los factores de riesgo para adquirir la enfermedad. Se determinó que el agua de la quebrada La Puerta no es apta para el consumo y la recreación de las personas. Conclusiones. Se señala a Fuente Clara como el primer foco de paragonimosis en zona urbana de Colombia, donde la manipulación y el consumo de cangrejos ponen en riesgo de adquirir la infección a sus habitantes; se sugiere realizar vigilancia de la enfermedad en el sector.

[Distribution of Paragonimus (Digenea: Troglotrematidae) in Antioquia Province, Colombia, based on metacercariae counts in freshwater crabs]. / Distribución parcial de Paragonimus (Digenea: Troglotrematidae) en Antioquia, por presencia de metacercarias en cangrejos dulciacuícolas.

INTRODUCTION: Paragonimosis or lung fluke disease courses with signs similar to those seen in tuberculosis. The causative agent is a parasite of the genus Paragonimus (Digenea: Troglotrematidae). People become infected by ingesting raw or partially cooked crabs containing metacercariae. The first focus of human paragonimosis in Colombia was recorded in the county of Urrao, where two species of crabs infected with Paragonimus were found. In 2005, crabs with Paragonimus' metacercariae were captured near Medellín, western Colombia. This prompted a search for the parasite in other locations through its presence in the crabs. OBJECTIVE: To establish the distribution of Paragonimus in Antioquia, we evaluated the presence of metacercaria in freshwater brachyuran crabs. MATERIALS AND METHODS: From 2005 to 2007, crabs were captured in 13 counties of Antioquia. The crabs were relaxed and dissected to determine presence of trematodes and then to make the taxonomic identifications. RESULTS: From 52 crabs captured in 9 counties, 42 (80.8%) were found with Paragonimus metacercariae. The crabs were identified as Pseudothelphusidae in 2 genera--Hypolobocera and Strengeriana--and were assigned to four species. Three of the species were recorded for the first time as hosts of Paragonimus. CONCLUSIONS: A Paragonimus' distribution map was constructed for Antioquia; for the first time urban zones were included. Because of the high rate of infection, the handling and consumption of raw and poorly cooked crabs pose risk factors for human infection. Because crabs are affordable and provide means of easy diagnosis, crabs are targeted as primary agents of and diagnostic tools for paragonmosis.

Synonymy between Lymnaea bogotensis Pilsbry, 1935 and Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae).

The lymneid snail Lymnaea bogotensis Pilsbry 1935 is synonymized with L. cousini Jousseaume 1887, based on morphological comparisons of the reproductive systems. The shell, renal tube, and reproductive system are described and illustrated from specimens collected in the type locality and the municipality of Paipa, Colombia. Bibliographical records reveal L. columella to be the commonest lymneid in the country. The latter two species can be differentiated based on the morphology of the penial complex.

Synonymy between Lymnaea bogotensis Pilsbry, 1935 and Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae)

The lymneid snail Lymnaea bogotensis Pilsbry 1935 is synonymized with L. cousini Jousseaume 1887, based on morphological comparisons of the reproductive systems. The shell, renal tube, and reproductive system are described and illustrated from specimens collected in the type locality and the municipality of Paipa, Colombia. Bibliographical records reveal L. columella to be the commonest lymneid in the country. The latter two species can be differentiated based on the morphology of the penial complex.

Purificación y caracterización parcial de una toxina (Hm3) del veneno de Hadruroides mauryi (Francke y Soleglad, 1980) (Scorpiones, luridae) / Purification and partial characterization of a toxin (Hm3) from Hadruroides mauryi (Francke y Soleglad, 1980) (Scorpiones, luridae) venom

Se ha purificado una toxina (Hm3) del veneno del escorpión Hadruroides mauryi, por cromatografía de intercambio iónico en CM-Sephadex C-25 con buffer acetato de amonio 0,05M, pH 7. La toxina produce contracción y parálisis en la extremidad inoculada de ratones albinos y se caracteriza por ser de naturaleza básica y tener un peso molecular de 4,5 kDa. Después de 45 minutos de ser inoculada en el músculo gastrocnemius de ratones albinos, Hm3 (30,4 µg) aumenta los niveles plasmáticos de creatina kinasa desde 252,6 UI/L hasta 3779,3 UI/L y de lactato deshidrogenasa, desde 142,7 UI/L hasta 248,2 UI/L; igualmente incrementa los niveles de calcio intramuscular desde 34,1 nmoles hasta 69,3 nmoles. Esta toxina carece de actividad proteolítica y fosfolipásica.