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Jennifer Keiser - One of the best experts on this subject based on the ideXlab platform.
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Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni
Parasites & Vectors, 2013Co-Authors: Gordana Panic, Katrin Ingram, Jennifer KeiserAbstract:Background Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Methods Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. Results The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. Conclusion NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.
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Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni
Parasites & Vectors, 2013Co-Authors: Gordana Panic, Katrin Ingram, Jennifer KeiserAbstract:Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.
Bernard Fried - One of the best experts on this subject based on the ideXlab platform.
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Helminth-Trematode: Echinostoma
Encyclopedia of Food Safety, 2020Co-Authors: R. Toledo, Bernard FriedAbstract:The term, echinostomes, includes those digeneans belonging to the family Echinostomatidae. Echinostomes are a rather heterogeneous group of cosmopolitan hermaphroditic digeneans that inhabit, as adults, the intestine of a great spectrum of vertebrate hosts, such as birds, mammals and, occasionally, reptiles and fishes. They can also parasitize humans causing the food-borne infection called Echinostomiasis. The definitive hosts become infected after ingestion of the second intermediate host harboring the encysted metacercariae. Clinical symptoms of Echinostomiasis include abdominal pain, violent watery diarrhea, and anorexia. The disease occurs focally and transmission is linked to fresh or brackish water habitats. Infections are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks, fish, crustaceans, and amphibians. Echinostomiasis is aggravated by socioeconomic factors such as poverty, and is mainly prevalent in poor, rural areas. In this article, the authors also review the main features of these trematodes with emphasis on their food-borne transmission and control.
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Postmetacercarial changes in Echinostoma caproni maintained in a defined medium plus calf serum.
Korean Journal of Parasitology, 2000Co-Authors: Bernard Fried, Aditya ReddyAbstract:Echinstoma caproni is a good model for various studies on the biology of Echinostoma and Echinostomiasis. The life cycle of this echinostome is easily maintained in the laboratory between Biomphalaria glabrata snails and Institute of Cancer Research (ICR) mice (see Fried and Huffman, 1996 for review). Although excysted metacercariae of this echinostome have been cultivated to ovigerous adults in chick embryos (Chien and Fried, 1992), surprisingly little work has been done on the in vitro maintenance or cultivation of the metacercarial stage of this echinostome. The encysted metacercariae of this species are easy to excyst in vitro in an alkaline trypsin-bile salts medium as described in Ursone and Fried (1995); therefore, excysted metacercariae are readily available to initiate in vitro cultivation or maintenance studies. The excysted metacercaria contains an abundant number of excretory concretions (calcareous corpuscles) in the protonephridial tubules as seen in Figure 15 in Fried and Huffman (1996). From observations of worms grown in chicks and mice, it is apparent that the excretory concretions are not present in the adults of E. caproni. Thus, they are probably voided between the metacercarial and adult stages. A purpose of the in vitro maintenance study reported herein was to note the disappearance of these concretions and also report other findings. Metacercariae of E. caproni were removed from experimentally infected B. glabrata snails and chemically excysted in an alkaline trypsin-bile salts medium at 41℃ as described in Ursone and Fried (1995). Excysted metacercariae were transferred through three changes of sterile Locke's solution containing 200 units penicillin/ml and 200 µg streptomycin/ml. The medium used to maintain the excysted metacercariae was the defined medium Mixture 199 + 20% calf serum (Mix 199 + 20 CS). The medium was fortified with penicillin/streptomycin as described for the Locke's solution. All chemicals were purchased from Sigma (St. Louis, MO, USA). Cultures were done in 15 ml plastic centrifuge tubes containing 10 ml of the medium. The cultures were capped and maintained upright in an incubator at 41℃ for up to 7 days. The gas phase was atmospheric air. Each culture was inoculated with 25 excysted metacercariae at the start of the experiment and one culture per day was examined for 7 days. All metacercariae examined were alive until 4 days post-inoculation. Many of the metacercariae were attached to each other in a clump at the bottom of the tubes, but some metacercariae were solitary. Metacercariae began to void their excretory concretions by day 1 so that by day 2, most metacercariae contained 4 to 8 concretions. By 2 days the collar and tegumentary spines were flared out, making them more visible than spines seen in the excysted metacercariae. By 4 days in culture, most excysted metacercariae were completely devoid of excretory concretions (Fig. 1) and some contained bumps or protuberances (Fig. 2); others contained a vacuolar bleb in the posterior aspect of the organism (Fig. 3). Some mortality was seen by day 5 with 5 of the 25 metacerariae being dead. Mortality increased to 10 of 25 dead organisms by 7 days of culture at which time the cultures were terminated. The surviving organisms looked the same at 7 days as they did at 4 days with some also showing bumps, protuberances, and blebs and all being devoid of the excretory concretions. Figs. 1-3 Light micrographs of excysted metacercaria of Echinostoma caproni maintained for 4 days in Mixture 199 plus 20% calf serum. Fig. 1. Intact organism showing the absence of excretory concretions. Fig. 2. Organism showing the flaring of the oral collar and ... There has been a decline in studies on the in vitro cultivation or maintenance of digeneans. Reasons for the decline have been discussed by Irwin (1997). In brief, these studies are labor intensive and often give negative results which are not publishable. The present study used a defined medium supplemented with calf serum to examine some postmetacercarial changes in E. caproni, i.e., voidance of the excretory concretions and development of body protuberances in vitro. Future in vitro studies on this and other echinostomes may provide interesting information on the many transformational events that must occur in the development from the metacercarial to the adult stage. In conclusion, no sexual organs appeared in 6- or 7-day-old worms. Therefore, although some somatic changes were observed, there were no indications of germinal changes in worms maintained under our in vitro conditions. Cultures were terminated at day 7 post-incubation because the culture conditions were becoming unfavorable, and many worms were dead. The appearance of the blebs, protuberance, and vesicles in the worms may be an indication of worm degeneration in an inhospitable environment. Although some digeneans have been maintained in the serum-free cultures, attempts to cultivate the excysted metacercariae of E. caproni in serum-free media were not made in our study.
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Echinostomiasis a common but forgotten food borne disease
American Journal of Tropical Medicine and Hygiene, 1998Co-Authors: Thaddeus K Graczyk, Bernard FriedAbstract:Human Echinostomiasis, endemic to southeast Asia and the Far East, is a food-borne, intestinal, zoonotic parasitosis attributed to at least 16 species of digenean trematodes transmitted by snails. Two separate life cycles of echinostomes, human and sylvatic, efficiently operate in endemic areas. Clinical symptoms of Echinostomiasis include abdominal pain, violent watery diarrhea, and anorexia. The disease occurs focally and transmission is linked to fresh or brackish water habitats. Infections are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks, fish, crustaceans, and amphibians, promiscuous defecation, and the use of night soil (human excrement collected from latrines) for fertilization of fish ponds. The prevalence of infection ranges from 44% in the Philippines to 5% in mainland China, and from 50% in northern Thailand to 9% in Korea. Although the patterns of other food-borne trematodiases have changed in Asia following changes in habits, cultural practices, health education, industrialization, and environmental alteration, human Echinostomiasis remains a health problem. The disease is most prevalent in remote rural places among low-wage earners and in women of child bearing age. Echinostomiasis is aggravated by socioeconomic factors such as poverty, malnutrition, an explosively growing free-food market, a lack of supervised food inspection, poor or insufficient sanitation, other helminthiases, and declining economic conditions. Furthermore, World Health Organization control programs implemented for other food-borne helminthiases and sustained in endemic areas are not fully successful for Echinostomiasis because these parasites display extremely broad specificity for the second intermediate host and are capable of completing the life cycle without involvement of the human host.
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In vitro excystation of metacercarial cysts of Echinostoma trivolvis from Rana species tadpoles
Korean Journal of Parasitology, 1997Co-Authors: Bernard Fried, J. David BradfordAbstract:In uitro excystation studies were done on the metacercarial cysts of Echinostomn triuolvis obtained from the kidneys of naturally infected Rnna species tadpoles. Cysts were excysted in an alkaline trypsin-bile salts medium and the percentage of excystation was compared with that from previous studies done on cysts obtained from the kidneys of snails. The percentage of excystation of E. triuoluis metacercariae from tadpole kidneys was similar to that reported for previous studies on cysts obtained from experimentally infected gastropod hosts. The possible role of tadpoles as an agent for the transmission of Echinostomn and Echinostomiasis to humans is discussed. Key words: Rana species, tadpoles, trematoda, Uchinostoma triwzuis, metacercarial cysts, in vitro excystation
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echinostoma and Echinostomiasis
Advances in Parasitology, 1990Co-Authors: Jane E Huffman, Bernard FriedAbstract:Publisher Summary This chapter discusses the studies on the biology, life history, infectivity, immunology, pathology, epidemiology, physiology, and biochemistry of Echinostoma . Some studies on other genera of Echinostomatidae are considered as they relate to Echinostoma. The systematics of the 37-collar-spined Echinostoma in the E. revoluturn group is discussed and is based on morphological, biometrical, isoenzymatical, and biological characteristics. The chapter focuses on E. trivolvis, E. caproni, and E. revolutum. Echinostome adults are cosmopolitan, hermaphroditic digeneans that live in the intestines and bile ducts of numerous vertebrate hosts, particularly, aquatic or semi-aquatic birds and mammals. Results derived from the experimental infection of laboratory rodents have provided insight into the pathogenesis of echinostomes. The diagnosis of Echinostomiasis is made by recovering the eggs in the feces. The time of deposition of eggs in the feces will vary between species. Species diagnosis is based mainly on the morphological study of the adult worm, which can be obtained after anthelmintic treatment. The transplantation studies and cultivation of Echinostoma in vivo and in vitro are also discussed. The use of echinostomes in physiological, biochemical, and behavioral studies is an untapped resource. Many of the studies done on E. trivolvis need to be performed on E. caproni , E. revohturn , and other echinostomes for comparative purposes. The gross and histopathological effects of echinostome infections are well documented in experimental infections in mice and hamsters.
Sagar Poudyal - One of the best experts on this subject based on the ideXlab platform.
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Human Echinostomiasis: a case report
BMC Research Notes, 2018Co-Authors: Shusila Khadka, Rabin Hamal, Sagar PoudyalAbstract:Background Echinostomiasis is a food-borne infection caused by an intestinal trematodes belonging to the family Echinostomatidae. They infect the gastrointestinal tract of humans. Patients are usually asymptomatic. However, with heavy infections, the worms can produce catarrhal inflammation with mild ulceration and the patient may experience abdominal pain, anorexia, nausea, vomiting, diarrhea and weight loss. Infection are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks and fish. Case presentation We report a first case of Echinostomiasis from Nepal in a 62 years old, hindu male who presented to Tribhuvan University Teaching Hospital, Kathmandu with a complaint of abdominal pain and distension with vomiting on and off for 3–4 months. He had history of consumption of insufficiently cooked fish and snail with alcohol. During endoscopy, an adult flat worm was seen with mild portal hypertensive gastropathy (McCormack’s classification) and erosive duodenopathy. The adult worm was identified as Echinostoma species based on its morphology and characteristic ova found on stool routine microscopic examination of the patient. Patient was treated with praziquantel 40 mg/kg (single dose) which is the drug of choice for Echinostoma species infection by which he got improved and on follow up stool examination after 2 weeks revealed no ova of Echinostoma species. Conclusions The patients having history of consumption of insufficiently cooked snail and fish with suggestive clinical features of Echinostomiasis should be suspected by physicians and ova of Echinostoma species should be searched by trained microscopists. An epidemiological survey is required to know the exact burden of Echinostoma species infection in the place where people have habit of eating insufficiently cooked fish and snails, as it can be endemic in that community or geographical area.
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human Echinostomiasis a case report
BMC Research Notes, 2018Co-Authors: Shusila Khadka, Rabin Hamal, Sagar PoudyalAbstract:Background Echinostomiasis is a food-borne infection caused by an intestinal trematodes belonging to the family Echinostomatidae. They infect the gastrointestinal tract of humans. Patients are usually asymptomatic. However, with heavy infections, the worms can produce catarrhal inflammation with mild ulceration and the patient may experience abdominal pain, anorexia, nausea, vomiting, diarrhea and weight loss. Infection are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks and fish.
Gordana Panic - One of the best experts on this subject based on the ideXlab platform.
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Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni
Parasites & Vectors, 2013Co-Authors: Gordana Panic, Katrin Ingram, Jennifer KeiserAbstract:Background Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Methods Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. Results The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. Conclusion NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.
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Development of an in vitro drug sensitivity assay based on newly excysted larvae of Echinostoma caproni
Parasites & Vectors, 2013Co-Authors: Gordana Panic, Katrin Ingram, Jennifer KeiserAbstract:Echinostomiasis is one of the major food-borne trematodiases and the species Echinostoma caproni serves as a useful model for trematocidal drug discovery. The current in vitro drug sensitivity assay uses adult E. caproni worms that are incubated with candidate drugs and scored microscopically for viability at 72 hrs. The aim of this study was to investigate the use of newly excysted larvae (NEL) of E. caproni for in vitro drug testing, which would be faster, more cost effective and more ethical compared to adult worm assays. Larvae were obtained by collecting metacercariae from snails and triggering their excystation using the trypsin-bile salt excystation method. Studies concerning various parameters of this chemical transformation process as well as appropriate NEL culturing conditions were carried out and findings evaluated. NEL and adult worms were incubated with praziquantel, tribendimidine, albendazole and quinine and evaluated microscopically 72 hrs post-incubation. In addition, the colorimetric markers resazurin, CellTiter-Glo® and Vybrant® were tested as an alternative assay read-out method. The chemical excystation method successfully induced E. caproni metacercariae to excyst at a rate of about 20-60%. NEL remained viable in culture medium for 5–7 days. The results of an in vitro drug assay using NEL mirrored the results of an assay using adult worms incubated with the same drugs. None of the markers could reliably produce signals proportional to NEL viability or cytotoxicity without significant complications. NEL are adequate for in vitro drug testing. Challenges remain in further improving the excystation yield and the practicability of the assay setup. Resolving these issues could also improve read-outs using colorimetric markers. Using NEL is in alignment with the 3 R rules of the ethical use of laboratory animals and can greatly increase the rate and affordability with which drugs are screened in vitro against this intestinal trematode.
Shusila Khadka - One of the best experts on this subject based on the ideXlab platform.
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Human Echinostomiasis: a case report
BMC Research Notes, 2018Co-Authors: Shusila Khadka, Rabin Hamal, Sagar PoudyalAbstract:Background Echinostomiasis is a food-borne infection caused by an intestinal trematodes belonging to the family Echinostomatidae. They infect the gastrointestinal tract of humans. Patients are usually asymptomatic. However, with heavy infections, the worms can produce catarrhal inflammation with mild ulceration and the patient may experience abdominal pain, anorexia, nausea, vomiting, diarrhea and weight loss. Infection are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks and fish. Case presentation We report a first case of Echinostomiasis from Nepal in a 62 years old, hindu male who presented to Tribhuvan University Teaching Hospital, Kathmandu with a complaint of abdominal pain and distension with vomiting on and off for 3–4 months. He had history of consumption of insufficiently cooked fish and snail with alcohol. During endoscopy, an adult flat worm was seen with mild portal hypertensive gastropathy (McCormack’s classification) and erosive duodenopathy. The adult worm was identified as Echinostoma species based on its morphology and characteristic ova found on stool routine microscopic examination of the patient. Patient was treated with praziquantel 40 mg/kg (single dose) which is the drug of choice for Echinostoma species infection by which he got improved and on follow up stool examination after 2 weeks revealed no ova of Echinostoma species. Conclusions The patients having history of consumption of insufficiently cooked snail and fish with suggestive clinical features of Echinostomiasis should be suspected by physicians and ova of Echinostoma species should be searched by trained microscopists. An epidemiological survey is required to know the exact burden of Echinostoma species infection in the place where people have habit of eating insufficiently cooked fish and snails, as it can be endemic in that community or geographical area.
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human Echinostomiasis a case report
BMC Research Notes, 2018Co-Authors: Shusila Khadka, Rabin Hamal, Sagar PoudyalAbstract:Background Echinostomiasis is a food-borne infection caused by an intestinal trematodes belonging to the family Echinostomatidae. They infect the gastrointestinal tract of humans. Patients are usually asymptomatic. However, with heavy infections, the worms can produce catarrhal inflammation with mild ulceration and the patient may experience abdominal pain, anorexia, nausea, vomiting, diarrhea and weight loss. Infection are associated with common sociocultural practices of eating raw or insufficiently cooked mollusks and fish.
