Author:
- ABSTRACT.
- INTRODUCTION
- MATERIALS AND METHODS
- Study area.
- Snail sampling.
- Morphological identification of snails.
- Cercarial shedding.
- Morphological identification of trematode cercariae.
- DNA extraction from snails, cercariae, and adult worms of Amphimerus sp., Clonorchis sinensis, Paragonimus mexicanus, and Fasciola hepatica.
- Real time-polymerase chain reaction for detection of Amphimerus-specific DNA.
- DNA Sequencing and the phylogenetic tree analysis.
- Ethics.
- RESULTS
- DISCUSSION
- ACKNOWLEDGMENTS
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
WHO , 2017. Neglected Tropical Diseases Program. Available at: http://www.who.int/neglected_diseases/en/. Accessed July 18, 2020.
-
2.
Yamaguti S, 1958. Systema Helminthum. The Digenetic Trematodes of Vertebrates-Part 1, Vol. 1. New York, NY: Interscience Publishers Inc, 981–1575.
-
3.
Calvopiña M, Cevallos W, Kumazawa H, Eisenberg J, 2011. High prevalence of human liver infection by Amphimerus spp. flukes, Ecuador. Emerg Infect Dis 17: 2331–2334.
-
4.
Calvopiña M et al.2015. High prevalence of the liver fluke Amphimerus sp. in domestic cats and dogs in an area for human amphimeriasis in Ecuador. PLoS Negl Trop Dis 9: e0003526.
-
5.
Calvopiña M, Romero-Alvarez D, Diaz F, Cevallos W, Sugiyama H, 2018. A comparison of Kato-Katz technique to three other methods for diagnosis of Amphimerus spp. liver fluke infection and the prevalence of infection in Chachi Amerindians of Ecuador. PLoS One 13: e0203811.
-
6.
Romero-Alvarez D, Valverde-Muñoz G, Calvopina M, Rojas M, Cevallos W, Kumazawa H, Takagi H, Sugiyama H, 2020. Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador. PLoS Negl Trop Dis 14: e0008286.
-
7.
CDC , 2018. Parasites-Clonorchis. Atlanta, GA: Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/parasites/clonorchis/biology.html. Accessed May 20, 2021.
-
8.
Chai JY, Murrell KD, Lymbery AJ, 2005. Fish-borne parasitic zoonoses: status and issues. Int J Parasitol 35: 1233–1254.
-
9.
Font W, 1991. Life cycle of Amphimerus elongatus (Trematoda: Opisthorchiidae). J Helminthol Soc Wash 58: 24–30.
-
10.
Caron Y, Lasri S, Losson B, 2007. Fasciola hepatica: an assessment on the vectorial capacity of Radix labiata and R. balthica commonly found in Belgium. Vet Parasitol 149: 95–103.
-
11.
Pointier JP, Noya O, 2015a. Freshwater Molluscs of Venezuela and their Medical and Veterinary Importance. J. P. Pointier, ed. Harxheim, Germany: Conchbooks, 124–129.
-
12.
Pointier JP, Noya O, Alarcón de Noya B, 2015b. Freshwater molluscs of Venezuela and their medical and veterinary importance. J. P. Pointier, ed. Family Cochliopidae. Harxheim, Germany: Conchbooks, 94–101.
-
13.
Pointier JP, Noya O, Alarcón de Noya B, Théron A, 2015c. Freshwater molluscs of Venezuela and their medical and veterinary importance. JP Pointier, ed. Family PlanorbidaeHarxheim, Germany: Conchbooks, 129–169.
-
14.
Kiatsopit N, Sithithaworn P, Saijuntha W, Boonmars T, Tesana S, Sithithaworn J, Petney TN, Andrews RH, 2012. Exceptionally high prevalence of infection of Bithynia siamensis goniomphalos with Opisthorchis viverrini cercariae in different wetlands in Thailand and Lao PDR. Am J Trop Med Hyg 86: 464–469.
-
15.
Frandsen F, Christensen NØ, 1984. An introductory guide to the identification of cercariae from African freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. Acta Trop 41: 181–202.
-
16.
Ito J, 1962. Studies on cercariae from freshwater snails in Thailand. Jap J M Sc & Biol 15: 249–270.
-
17.
Yamaguti S, 1975. A Synoptical Review of Life Histories of Digenetic Trematodes of Vertebrates. Tokyo, Japan: Keigaku Publishing., 590.
-
18.
Ditrich O, Scholz T, Aguirre-Macedo L, Vargas-Vasquez J, 1997. Larval stages of trematodes from freshwater molluscs of the Yucatan Peninsula, Mexico. Folia Parasitol (Praha) 44: 109–127.
-
19.
Suenaga E, Nakamura H, 2005. Evaluation of three methods for effective extraction of DNA from human hair. J Chromatogr B Analyt Technol Biomed Life Sci 820: 137–141.
-
20.
Herschler R, Thomson FG, 1992. A review of the aquatic gastropod subfamily Cochliopinae (Prosopbranchia: Hydrobiidae). Malacol Rev 5: 140p.
-
21.
Ponder WF, Walker KF, 2003. From mound springs to mighty rivers: the conservation status of freshwater molluscs in Australia. Aquat Ecosyst Health Manage 6: 19–28.
-
22.
de Moraes Neto AH, Thatcher VE, Lanfredi RM, 1998. Amphimerus bragai n. sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nectomys squamipes (Cricetidae) from Minas Gerais, Brazil. Mem Inst Oswaldo Cruz 93: 181–186.
-
23.
Thatcher VE, 1970. The genus Amphimerus Barker, 1911. (Trematoda: Opisthorchiidae) in Colombia with the description of a new species. Proc Helminthol Soc Wash 37: 207–211.
-
24.
Miyazaki I, Kifune T, Habe S, Uyema N, 1978. Reports of Fukuoka University scientific expedition to Peru, 1976. Med. Bull. Fukuoka University 1: 1–28.
-
25.
WHO , 1995. Control of foodborne trematode infections. Report of a WHO study Group. Technical Report Series 849. Geneva, Switzerland: World Health Organization.
-
26.
Amunárriz M, 1991. Intermediate hosts of Paragonimus in the eastern Amazonic region of Ecuador. Trop Med Parasitol 42: 160–164.
-
27.
Calvopiña M, Romero D, Castañeda B, Hashiguchi Y, Sugiyama H, 2014. Current status of Paragonimus and paragonimiasis in Ecuador. Mem Inst Oswaldo Cruz, Rio de Janeiro 109: 849–855.
-
28.
Calvopina M et al.2018. Asymptomatic Fasciola hepatica infection presenting with hypereosinophilia. Arch Clin Microbiol 9: 73.
-
29.
Scholz T, Aguirre-Macedo ML, de León SFD, Ditrich O, 2000. Larval stages of trematodes in Mexican freshwater mollusc: a review of present state and methodology for future research. Salgado-Maldonato GA, Vidal-Matínez VM, eds. Metazoan Parasites in the Neotropics: A Systematic and Evolutionary Perspective. Mexico D.F.: Instituto de Biología, UNAM, 62–89.
-
30.
Vergara D, Velásquez LE, 2009. Larval stages of digenea from Melanoides tuberculata (Gastropoda: Thiaridae) in Medellín, Colombia. Acta Biol Colomb 14: 135–142.
-
31.
Pinto HA, de Melo AL, 2011. A checklist of trematodes (Platyhelminthes) transmitted by Melanoides tuberculata (Mollusca: Thiaridae). Zootaxa 2799: 15–28.
-
32.
Pulido-Murillo EA, Furtado LFV, Melo AL, Rabelo ÉML, Pinto HA, 2018. Fishborne zoonotic trematodes transmitted by Melanoides tuberculata snails, Peru. Emerg Infect Dis 24: 606–608.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1233 | 529 | 108 |
| Full Text Views | 79 | 46 | 0 |
| PDF Downloads | 79 | 37 | 0 |
Molecular Identification of the Human Pathogen Amphimerus sp. in the Freshwater Snail Aroapyrgus sp. in Ecuador
Manuel Calvopiña
Manuel Calvopiña
One Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador;
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Carlos Bastidas-Caldes
Carlos Bastidas-Caldes
One Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador;
Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador;
Programa de Doctorado en Salud Pública y Animal, Universidad de Extremadura, Extremadura, Caceres, Spain;
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Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador;
Programa de Doctorado en Salud Pública y Animal, Universidad de Extremadura, Extremadura, Caceres, Spain;
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Francisco Romero
Francisco Romero
Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador;
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Irina Villacrés-Granda
Irina Villacrés-Granda
Programa de Doctorado Interuniversitario en Ciencias de La Salud, Universidad de Sevilla, Sevilla, Spain;
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Jean-Pierre Pointier
Jean-Pierre Pointier
PSL Research University, USR 3278 CNRS–EPHE, CRIOBE Université de Perpignan, Perpignan, France;
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Hidekazu Takagi
Hidekazu Takagi
Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi, Japan;
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Hiromu Sugiyama
Hiromu Sugiyama
Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan
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ABSTRACT.
Here, we report for the first time the snail intermediate host for the Amphimerus liver fluke, a foodborne trematodiasis. In Ecuador, Amphimerus of the Opisthorchiidae family, infects humans, cats, and dogs, in the tropical Pacific-coast region. Opisthorchiidae comprising also Clonorchis sinensis, Opisthorchis sp., and Metorchis sp., have complex life cycles involving a definitive and two intermediate hosts. We identified morphologically and investigated the presence and prevalence of Amphimerus cercaria and DNA in freshwater snails collected in a human-amphimeriasis endemic region in Ecuador, extracted DNA from snail tissue and emerged cercariae, performed real-time polymerase chain reaction (PCR) with the newly developed primers and probe amplifying the Amphimerus ribosomal internal transcribed spacer 2 (ITS2) region, and sequenced the amplified DNA fragment. We collected 2,800 snails, characterized four species Aroapyrgus sp., Melanoides tuberculata, Biomphalaria cousini, and Aplexa marmorata, isolated three cercariae morphotypes. Of the 640 snails analyzed by qPCR, only Aroapyrgus and one of the three cercariae resulted positive, at a 15% infection prevalence. Polymerase chain reaction revealed that the Aroapyrgus snail and cercaria-morphotype-3 corresponded to Amphimerus, but not to C. sinensis, Fasciola hepatica, or Paragonimus mexicanus. The sequence of amplified DNA product matched that of human-isolated Amphimerus. This finding constitutes the first documentation that Aroapyrgus sp. is the first intermediate host for the Amphimerus sp. that infect humans in Ecuador. The ITS2–gene PCR and sequencing analysis demonstrated a high prevalence of snail infection and proved useful for detecting the infection in snails, which findings can help the establishment of suitable control programs against transmission in any endemic region of interest.
- Supplemental Materials (PDF 499 KB)
Author Notes
Financial support: This research was supported by a grant from the Direccion General de Investigacion y Vinculacion (DGIV) de la Universidad de las Américas (UDLA), Quito-Ecuador (BIO.CBC.20.02) and by the Japan Agency for Medical Research and Development (AMED 21fk0108136j0102) to HS.
Authors’ addresses: Manuel Calvopiña, One Health Research Group, Facultad de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador, E-mail: manuelcalvopina@gmail.com. Carlos Bastidas-Caldes, One Health Research Group, Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de las Américas (UDLA), Quito, Ecuador, Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador, and Programa de Doctorado en Salud Pública y Animal, Universidad de Extremadura, Extremadura, Caceres, Spain, E-mail: cabastidasc@gmail.com. Francisco Romero, Ingeniería en Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas (FICA), Universidad de las Américas (UDLA), Quito, Ecuador, E-mail: francisco.romero@udla.edu.ec. Irina Villacrés-Granda, Programa de Doctorado Interuniversitario en Ciencias de La Salud, Universidad de Sevilla, Sevilla, Spain, E-mail: irinamaribel@gmail.com. Jean-Pierre Pointier, PSL Research University, USR 3278 CNRS–EPHE, CRIOBE Université de Perpignan, Perpignan, France, E-mail: pointier@univ-perp.fr. Hidekazu Takagi, Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Aichi, Japan, E-mail: htakagi@aichi-med-u.ac.jp. Hiromu Sugiyama, Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan, E-mail: hsugi@nih.go.jp.
- ABSTRACT.
- INTRODUCTION
- MATERIALS AND METHODS
- Study area.
- Snail sampling.
- Morphological identification of snails.
- Cercarial shedding.
- Morphological identification of trematode cercariae.
- DNA extraction from snails, cercariae, and adult worms of Amphimerus sp., Clonorchis sinensis, Paragonimus mexicanus, and Fasciola hepatica.
- Real time-polymerase chain reaction for detection of Amphimerus-specific DNA.
- DNA Sequencing and the phylogenetic tree analysis.
- Ethics.
- RESULTS
- DISCUSSION
- ACKNOWLEDGMENTS
- REFERENCES
ProCite
RefWorks
Reference Manager
BibTeX
Zotero
EndNote
-
1.
WHO , 2017. Neglected Tropical Diseases Program. Available at: http://www.who.int/neglected_diseases/en/. Accessed July 18, 2020.
-
2.
Yamaguti S, 1958. Systema Helminthum. The Digenetic Trematodes of Vertebrates-Part 1, Vol. 1. New York, NY: Interscience Publishers Inc, 981–1575.
-
3.
Calvopiña M, Cevallos W, Kumazawa H, Eisenberg J, 2011. High prevalence of human liver infection by Amphimerus spp. flukes, Ecuador. Emerg Infect Dis 17: 2331–2334.
-
4.
Calvopiña M et al.2015. High prevalence of the liver fluke Amphimerus sp. in domestic cats and dogs in an area for human amphimeriasis in Ecuador. PLoS Negl Trop Dis 9: e0003526.
-
5.
Calvopiña M, Romero-Alvarez D, Diaz F, Cevallos W, Sugiyama H, 2018. A comparison of Kato-Katz technique to three other methods for diagnosis of Amphimerus spp. liver fluke infection and the prevalence of infection in Chachi Amerindians of Ecuador. PLoS One 13: e0203811.
-
6.
Romero-Alvarez D, Valverde-Muñoz G, Calvopina M, Rojas M, Cevallos W, Kumazawa H, Takagi H, Sugiyama H, 2020. Liver fluke infections by Amphimerus sp. (Digenea: Opisthorchiidae) in definitive and fish intermediate hosts in Manabí province, Ecuador. PLoS Negl Trop Dis 14: e0008286.
-
7.
CDC , 2018. Parasites-Clonorchis. Atlanta, GA: Centers for Disease Control and Prevention. Available at: https://www.cdc.gov/parasites/clonorchis/biology.html. Accessed May 20, 2021.
-
8.
Chai JY, Murrell KD, Lymbery AJ, 2005. Fish-borne parasitic zoonoses: status and issues. Int J Parasitol 35: 1233–1254.
-
9.
Font W, 1991. Life cycle of Amphimerus elongatus (Trematoda: Opisthorchiidae). J Helminthol Soc Wash 58: 24–30.
-
10.
Caron Y, Lasri S, Losson B, 2007. Fasciola hepatica: an assessment on the vectorial capacity of Radix labiata and R. balthica commonly found in Belgium. Vet Parasitol 149: 95–103.
-
11.
Pointier JP, Noya O, 2015a. Freshwater Molluscs of Venezuela and their Medical and Veterinary Importance. J. P. Pointier, ed. Harxheim, Germany: Conchbooks, 124–129.
-
12.
Pointier JP, Noya O, Alarcón de Noya B, 2015b. Freshwater molluscs of Venezuela and their medical and veterinary importance. J. P. Pointier, ed. Family Cochliopidae. Harxheim, Germany: Conchbooks, 94–101.
-
13.
Pointier JP, Noya O, Alarcón de Noya B, Théron A, 2015c. Freshwater molluscs of Venezuela and their medical and veterinary importance. JP Pointier, ed. Family PlanorbidaeHarxheim, Germany: Conchbooks, 129–169.
-
14.
Kiatsopit N, Sithithaworn P, Saijuntha W, Boonmars T, Tesana S, Sithithaworn J, Petney TN, Andrews RH, 2012. Exceptionally high prevalence of infection of Bithynia siamensis goniomphalos with Opisthorchis viverrini cercariae in different wetlands in Thailand and Lao PDR. Am J Trop Med Hyg 86: 464–469.
-
15.
Frandsen F, Christensen NØ, 1984. An introductory guide to the identification of cercariae from African freshwater snails with special reference to cercariae of trematode species of medical and veterinary importance. Acta Trop 41: 181–202.
-
16.
Ito J, 1962. Studies on cercariae from freshwater snails in Thailand. Jap J M Sc & Biol 15: 249–270.
-
17.
Yamaguti S, 1975. A Synoptical Review of Life Histories of Digenetic Trematodes of Vertebrates. Tokyo, Japan: Keigaku Publishing., 590.
-
18.
Ditrich O, Scholz T, Aguirre-Macedo L, Vargas-Vasquez J, 1997. Larval stages of trematodes from freshwater molluscs of the Yucatan Peninsula, Mexico. Folia Parasitol (Praha) 44: 109–127.
-
19.
Suenaga E, Nakamura H, 2005. Evaluation of three methods for effective extraction of DNA from human hair. J Chromatogr B Analyt Technol Biomed Life Sci 820: 137–141.
-
20.
Herschler R, Thomson FG, 1992. A review of the aquatic gastropod subfamily Cochliopinae (Prosopbranchia: Hydrobiidae). Malacol Rev 5: 140p.
-
21.
Ponder WF, Walker KF, 2003. From mound springs to mighty rivers: the conservation status of freshwater molluscs in Australia. Aquat Ecosyst Health Manage 6: 19–28.
-
22.
de Moraes Neto AH, Thatcher VE, Lanfredi RM, 1998. Amphimerus bragai n. sp. (Digenea: Opisthorchiidae), a parasite of the rodent Nectomys squamipes (Cricetidae) from Minas Gerais, Brazil. Mem Inst Oswaldo Cruz 93: 181–186.
-
23.
Thatcher VE, 1970. The genus Amphimerus Barker, 1911. (Trematoda: Opisthorchiidae) in Colombia with the description of a new species. Proc Helminthol Soc Wash 37: 207–211.
-
24.
Miyazaki I, Kifune T, Habe S, Uyema N, 1978. Reports of Fukuoka University scientific expedition to Peru, 1976. Med. Bull. Fukuoka University 1: 1–28.
-
25.
WHO , 1995. Control of foodborne trematode infections. Report of a WHO study Group. Technical Report Series 849. Geneva, Switzerland: World Health Organization.
-
26.
Amunárriz M, 1991. Intermediate hosts of Paragonimus in the eastern Amazonic region of Ecuador. Trop Med Parasitol 42: 160–164.
-
27.
Calvopiña M, Romero D, Castañeda B, Hashiguchi Y, Sugiyama H, 2014. Current status of Paragonimus and paragonimiasis in Ecuador. Mem Inst Oswaldo Cruz, Rio de Janeiro 109: 849–855.
-
28.
Calvopina M et al.2018. Asymptomatic Fasciola hepatica infection presenting with hypereosinophilia. Arch Clin Microbiol 9: 73.
-
29.
Scholz T, Aguirre-Macedo ML, de León SFD, Ditrich O, 2000. Larval stages of trematodes in Mexican freshwater mollusc: a review of present state and methodology for future research. Salgado-Maldonato GA, Vidal-Matínez VM, eds. Metazoan Parasites in the Neotropics: A Systematic and Evolutionary Perspective. Mexico D.F.: Instituto de Biología, UNAM, 62–89.
-
30.
Vergara D, Velásquez LE, 2009. Larval stages of digenea from Melanoides tuberculata (Gastropoda: Thiaridae) in Medellín, Colombia. Acta Biol Colomb 14: 135–142.
-
31.
Pinto HA, de Melo AL, 2011. A checklist of trematodes (Platyhelminthes) transmitted by Melanoides tuberculata (Mollusca: Thiaridae). Zootaxa 2799: 15–28.
-
32.
Pulido-Murillo EA, Furtado LFV, Melo AL, Rabelo ÉML, Pinto HA, 2018. Fishborne zoonotic trematodes transmitted by Melanoides tuberculata snails, Peru. Emerg Infect Dis 24: 606–608.
| Past two years | Past Year | Past 30 Days | |
|---|---|---|---|
| Abstract Views | 1233 | 529 | 108 |
| Full Text Views | 79 | 46 | 0 |
| PDF Downloads | 79 | 37 | 0 |