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Licensed Unlicensed Requires Authentication Published by De Gruyter March 25, 2015

Molecular diagnosis of natural fasciolosis by DNA detection in sheep faeces

Silvana Carnevale, María Laura Pantano, Laura Kamenetzky, Jorge Bruno Malandrini, Claudia Cecilia Soria and Jorge Néstor Velásquez
From the journal Acta Parasitologica

Abstract

Fasciolosis is an important parasitic zoonosis considered the most important helminth infection of ruminants in tropical countries. The aim of this study was to develop a PCR assay for the sensitive and specific detection of F. hepatica in formalin preserved sheep faeces. A 405-bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica was amplified from stool samples of infected sheep. The PCR assay showed a detection limit of 20 pg of F. hepatica DNA. No cross-reactions were observed with samples containing coccidian oocysts or gastrointestinal nematodes eggs. Our PCR technique showed to be effective for specific detection of F. hepatica infections in sheep.

References

Ai L., Dong S.J., Zhang W.Y., Elsheikha H.M., Mahmmod Y.S., Lin R.Q., Yuan Z.G., Shi Y.L., Huang W.Y., Zhu X.Q. 2010. Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. Annals of Tropical Medical Parasitology, 104, 65-72. DOI: 10.1179/136485910X12607012 373713Search in Google Scholar

Anderson N., Luong T.T., Vo N.G., Bui K.L., Smooker P.M., Spithill T.W. 1999. The sensitivity and specificity of two methods for detecting Fasciola infections in cattle. Veterinary Parasitology, 83,15-24Search in Google Scholar

Arias M.S., Pineiro P., Sanchez-Andrade R., Suarez J.L., Hillyer G.V., Diez-Banos P., Paz-Silva A., Morrondo P. 2013. Relationship between exposure to Fasciola hepatica in roe deer (Capreolus capreolus) and cattle extensively reared in an endemic area. Research in Veterinary Science, 95, 1031-1035. DOI: 10.1016/j.rvsc.2013.07.027Search in Google Scholar

Ashrafi K., Valero M.A., Panova M., Massoud J., Mas-Coma S. 2006. Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitology International, 55, 249-260Search in Google Scholar

Boch J., Supperer, R. 1988. Parasitología en Medicina Veterinaria. Editorial Hemisferio Sur, Buenos Aires, Argentina Boero J.J. 1976. Parasitosis Animales. EUDEBA, Buenos Aires, Argentina Bretagne S., Guillou J.P., Morand M., Houin R. 1993. Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification. Parasitology, 106, 193-199Search in Google Scholar

Carnevale S., Rodriguez M.I., Guarnera E.A., Carmona C., Tanos T., Angel S. 2001. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase. Diagnostic Microbiology and Infectious Diseases, 41, 43-49Search in Google Scholar

Carnevale S., Velasquez J.N., Labbe J.H., Chertcoff A., Cabrera M.G., Rodriguez M.I. 2000. Diagnosis of Enterocytozoon bieneusi by PCR in stool samples eluted from filter paper disks. Clinical and Diagnostic Laboratory Immunology, 7, 504-506Search in Google Scholar

Castro E., Freyre A., Hernandez Z. 2000. Serological responses of cattle after treatment and during natural re-infection with Fasciola hepatica, as measured with a dot-ELISA system. Veterinary Parasitology, 90, 201-208Search in Google Scholar

Chang Y.T., Loew G.H. 1994. Reaction mechanisms of formal dehyde with endocyclic imino groups of nucleic acid bases. Journal of the American Chemical Society, 11, 3548-3555Search in Google Scholar

Choubisa S.L., Jaroli V.J. 2013. Gastrointestinal parasitic infection in diverse species of domestic ruminants inhabiting tribal rural areas of southern Rajasthan, India. Journal of Parasitic Diseases, 37, 271-275. DOI: 10.1007/s12639-012-0178-0Search in Google Scholar

Cornelissen J.B.W.J., de Leeuw W.A., van der Heijden P.J. 1992. Comparison of an indirect haemagglutination assay and an ELISA for diagnosing Fasciola hepatica in experimentally and naturally infected sheep. Veterinary Quarterly, 14, 152-156Search in Google Scholar

Cucher M.A., Carnevale S., Prepelitchi L., Labbe J.H., Wisnivesky- Colli C. 2006. PCR diagnosis of Fasciola hepatica in fieldcollected Lymnaea columella and Lymnaea viatrix snails. Veterinary Parasitology, 137, 74-82Search in Google Scholar

Dinkel A., Von Nickisch-Rosenegk M., Bilger B., Merli M., Lucius R., Roming T. 1998. Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. Journal of Clinical Microbiology, 36, 1871-1876Search in Google Scholar

Domke A.V., Chartier C., Gjerde B., Leine N., Vatn S., Stuen S. 2013. Prevalence of gastrointestinal helminths, lungworms and liver fluke in sheep and goats in Norway. Veterinary Parasitology, 194, 40-48. DOI: 10.1016/j.vetpar.2012.12.023Search in Google Scholar

Duenngai K., Boonmars T., Sithithaworn J., Sithithaworn P. 2013. Diagnosis of early infection and post chemotherapeutic treatment by copro-DNA detection in experimental opisthorchiasis. Parasitology Research, 112, 271-278. DOI: 10.1007/s00 436-012-3134-0Search in Google Scholar

Dumenigo B.E., Espino A.M., Finlay C.M., Mezo M. 2000. Kinetics of antibody-based antigen detection in serum and faeces of sheep experimentally infected with Fasciola hepatica. Veterinary Parasitology, 89, 153-161Search in Google Scholar

Dumenigo B.E., Mezo M. 1999. Monoclonal antibody sandwich immunoassay detection of coproantigen to evaluate the efficacy of treatment in natural ovine fasciolosis. Research in Veterinary Science, 66, 165-167Search in Google Scholar

El-Bahi M.M., Malone J.B., Todd W.J., Schnorr, K.L. 1992. Detection of stables diagnostic antigen from bile and feces of Fasciola hepatica infected cattle. Veterinary Parasitology, 45, 157-167Search in Google Scholar

Gonzalez-Lanza C., Manga-Gonzalez Y., Del-Pozo-Carnero P., Hidalgo-Arguello R. 1989. Dynamics of elimination of the eggs of Fasciola hepatica (Trematoda, Digenea) in faeces of cattle in the Porma Basin. Spain Veterinary Parasitology, 34, 35-43Search in Google Scholar

Gorman T., Valdes A., Fredes F., Ferreira A., Alcaino H. 1995. Reconocimiento antigenico de ovinos naturalmente infectados con Fasciola hepatica, monitoreado a traves de inmunoelectrotransferencia enzimatica (Westerm Blotting). Archivos de Medicina Veterinaria XXVII (2), 33-40Search in Google Scholar

Happich F.A., Boray J.C. 1969. Quantitative diagnosis of chronic fasciolosis. Australian Veterinary Journal, 45, 326-328Search in Google Scholar

Hillyer G.V., Soler de Galanes M. 1991. Initial feasibility studies of the FAST-ELISA for the immunodiagnosis of fascioliasis. Journal of Parasitology, 77, 362-365Search in Google Scholar

Kozłowska-Łoj J., Łoj-Maczulska A. 2013. The prevalence of Fasciola hepatica L. infection in cattle in the Lublin province in the years 2009-2012. Annals of Parasitology 59, 207-208Search in Google Scholar

Kuk S., Yazar S., Cetinkaya U. 2012. Stool sample storage conditions for the preservation of Giardia intestinalis DNA. Memorias do Instituto Oswaldo Cruz 107, 965-968.Search in Google Scholar

Langley R.J., Hillyer G.V. 1989. Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica. Journal of Parasitology, 75, 690-695Search in Google Scholar

Lelievre S., Verrez-Bagnis V., Jerome M., Vaz S. 2010. PCR-RFLP analyses of formalin-fixed fish eggs for the mapping of spawning areas in the Eastern Channel and Southern North Sea. Journal of Plankton Research, 32, 1527-1539. DOI: 10.1093/plankt/fbq067Search in Google Scholar

Locatelli A., Genchi C., Sartorelli P. 1984. Compendio di Parassitologia Veterinaria. Calderini, Bologna, Italia Mage C., Bourgne H., Toullieu J.M., Rondelaud D., Dreyfuss G. 2002. Fasciola hepatica and Paramphistomum daubneyi: changes in prevalences of natural infections in cattle and in Lymnaea truncatula from central France over the past 12 years. Veterinary Research, 33, 439-447Search in Google Scholar

Maniatis T., Fritsch E.F., Sambrook J. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA Martinez A., Martinez-Cruz M.S., Martinez F.J., Gutierrez P.N., Hernandez S. 1996. Detection of antibodies to Fasciola hepatica excretory-secretory antigens in experimentally infected goats by enzyme immunosorbent assay. Veterinary Parasitology, 62, 247-252Search in Google Scholar

Martinez-Perez J.M., Robles-Perez D., Rojo-Vazquez F.A., Martinez- Valladares M. 2012. Comparison of three different techniques to diagnose Fasciola hepatica infection in experimentally and naturally infected sheep. Veterinary Parasitology, 190, 80-86. DOI: 10.1016/j.vetpar.2012.06.002Search in Google Scholar

Martinez-Valladares M., Cordero-Perez C., Castanon-Ordonez L., Famularo M.R., Fernandez-Pato N., Rojo-Vazquez F.A. 2010. Efficacy of a moxidectin/triclabendazole oral formulation against mixed infections of Fasciola hepatica and gastrointestinal nematodes in sheep. Veterinary Parasitology, 174, 166-169Search in Google Scholar

Martinez-Valladares M., Robles-Perez D., Martinez-Perez J.M., Cordero-Perez C., Famularo Mdel R., Fernandez-Pato N., Gonzalez-Lanza C., Castanon-Ordonez L., Rojo-Vazquez F.A. 2013. Prevalence of gastrointestinal nematodes and Fasciola hepatica in sheep in the northwest of Spain: relation to climatic conditions and/or man-made environmental modifications. Parasites and Vectors, 6, 282. DOI: 10.1186/1756-3305-6-282Search in Google Scholar

Mas-Coma S., Bargues M.D., Valero M.A. 2005. Fascioliasis and other plant-borne trematode zoonoses. International Journal for Parasitology, 35, 1255-1278Search in Google Scholar

Mas-Coma S., Valero, M.A., Bargues M.D. 2009. Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Advances in Parasitology 69, 41-146. DOI: 10.1016/S0065-308X(09)69002-3Search in Google Scholar

Mathis A., Deplazes P. 2006. Copro-DNA tests for diagnosis of animal taeniid cestodes. Parasitology International 55, S87-S90Search in Google Scholar

Naidich A., McManus D.P., Canova S.G., Gutierrez A.M., Zhang W., Guarnera E.A., Rosenzvit M. C. 2006. Patent and pre-patent detection of Echinococcus granulosus genotypes in the definitive host. Molecular and Cellular Probes, 20, 5-10Search in Google Scholar

Ngategize P.K, Bekele T., Tilahun G. 1993. Financial losses caused by ovine fasciolosis in the Ethiopian highlands. Tropical Animal Health and Production, 25, 155-161.Search in Google Scholar

O’Neill S.M., Brady M.T., Callanan J.J., Mulcahy G., Joyce P., Mills K.H., Dalton J.P. 2000. Fasciola hepatica infection downregulates Th1 responses in mice. Parasite Immunology, 22, 147-155 Search in Google Scholar

Olaechea F.V. 1994. Epidemiologia y control de Fasciola hepatica en la Argentina. In (Eds. A. Nari and C. Fiel) Enfermedades Parasitarias de Importancia Económica en Bovinos. Editorial Agropecuaria, Hemisferio Sur SRL, Montevideo, Uruguay, 213-232Search in Google Scholar

Olaechea, F.V. 1995. Fasciola hepatica en bovinos. El tema elegido del mes: Fasciola hepatica. INTA, Sistema Regional de Salud Animal (SIRSA), Estacion Experimental Agropecuaria Bariloche, Unidad de Salud Animal, Bariloche, Argentina Search in Google Scholar

Pfister K., Daveau C., Ambroise-Thomas P. 1984. Partial purification of somatic and excretory-secretory products of adult Fasciola hepatica and their application for the serodiagnosis of experimental and natural fascioliasis using an ELISA. Research in Veterinary Science, 37, 39-43Search in Google Scholar

Rapsch C., Schweizer G., Grimm F., Kohler L., Bauer C., Deplazes P., Braun U., Torgerson P.R. 2006. Estimating the true prevalence of Fasciola hepatica in cattle slaughtered in Switzerland in the absence of an absolute diagnostic test. International Journal for Parasitology, 36, 1153-1158Search in Google Scholar

Reichel M.P. 2002. Performance characteristics of an enzyme-linked immunosorbent assay for the detection of liver fluke (Fasciola hepatica) infection in sheep and cattle. Veterinary Parasitology, 107, 65-72Search in Google Scholar

Richter B., Ladinig A., Nedorost N., Weissenbock H. 2010. A Taq- Man quantitative polymerase chain reaction assay for the detection of Lawsonia intracellularis in fecal and tissue samples from pigs. Journal of Veterinary Diagnostic Investigation, 22, 70-73. DOI: 10.1177/104063871002200112Search in Google Scholar

Rickard L.G. 1995. Development and application of a dot-ELISA test for the detection of serum antibodies to Fasciola hepatica antigens in llamas. Veterinary Parasitology, 58, 9-15Search in Google Scholar

Robles-Perez D., Martinez-Perez J.M., Rojo-Vazquez F.A., Martinez- Valladares M. 2013. The diagnosis of fasciolosis in feces of sheep by means of a PCR and its application in the detection of anthelmintic resistance in sheep flocks naturally infected. Veterinary Parasitology, 197, 277-282. DOI: 10. 1016/j.vetpar.2013.05.006Search in Google Scholar

Rodriguez-Perez J., Hillyer G.V. 1995. Detection of excretorysecretory circulating antigens in sheep infected with Fasciola hepatica and with Schistosoma mansoni and Fasciola hepatica. Veterinary Parasitology, 56, 57-66 Search in Google Scholar

Rojo-Vazquez F.A., Meana A., Valcarcel F., Martinez-Valladares M. 2012. Update on trematode infections in sheep. Veterinary Parasitology, 189, 15-38Search in Google Scholar

Santiago N., Hillyer G.V. 1988. Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica. Journal of Parasitology, 74, 810-818Search in Google Scholar

SENASA. 1998. Manual de Procedimientos para la Inspección Veterinaria, MP3. SENASA, Argentina Serra-Freire N.M., Bordin E.L., Lessa C.S.S., Scherer P.O., Farias M.T., Malacco M.A., Correa T.C., Tschumi J.A. 1995. Reinvestigacao sobre a distribuicao da Fasciola hepatica no Brasil. A Hora Veterinária, 1, 19-21Search in Google Scholar

Skage M., Schander C. 2007. DNA from formalin-fixed tissue: extraction or repair? That is the question. Marine Biology Research, 3, 289-295. DOI: 10.1080/17451000701473942Search in Google Scholar

Spithill T.W., Dalton J.P. 1998. Progress in development of liver fluke vaccines. Parasitology Today, 14, 224-228Search in Google Scholar

Stoll N.R., Hausheer W.C. 1926. Concerning two options in dilution egg counting: small drop and displacement. American Journal of Hygiene, 6, 134-145Search in Google Scholar

Taylor S.M. 1989. Control of fasciolosis in the British Isles. Magyar Allartorv Lapja, 44, 651-655Search in Google Scholar

Telemann W. 1908. Eine methode zur erleichterung der auffindung von parasiteneiern in den faeces. Deutsche Medizinische Wochenschrift, 35, 1510-1511Search in Google Scholar

Torgerson P., Claxton J. 1999. Epidemiology and control. In: (Ed. J. P. Dalton) Fasciolosis. CAB International Publishing, Wallingford, Oxon, UK, 113-149Search in Google Scholar

Tum S., Puotinen M.L., Skerratt L.F., Chan B., Sothoeun S. 2007. Validation of a geographic information system model for mapping the risk of fasciolosis in cattle and buffaloes in Cambodia. Veterinary Parasitology, 143, 364-367Search in Google Scholar

Velasquez J.N., Osvaldo G.A., di Risio C., Etchart C., Chertcoff A.V., Perisse G.E., Carnevale S. 2011. Molecular characterization of Cystoisospora belli and unizoite tissue cyst in patients with Acquired Immunodeficiency Syndrome. Parasitology, 138, 279-286. DOI: 10.1017/S0031182010001253Search in Google Scholar

Velasquez J.N., Carnevale S., Guarnera E.A., Labbe J.H., Chertcoff A., Cabrera M.G., Rodriguez M.I. 1996. Detection of the microsporidian parasite Enterocytozoon bieneusi in specimens from patients with AIDS by PCR. Journal of Clinical Microbiology, 34, 3230-3232Search in Google Scholar

Velasquez J.N., Carnevale S., Oelemann W.M.R., Etchart C., Peralta J.M. 2005. Diagnosis of Enterocytozoon bieneusi by the polymerase chain reaction in archival fixed duodenal tissue.Enfermedades Infecciosas y Microbiología Clínica, 23, 218-220Search in Google Scholar

Zimmerman G.L., Jen L.W., Cerro J.E., Farnsworth K.L., Wescott R.G. 1982. Diagnosis of Fasciola hepatica infections in sheep by an enzyme linked immunosorbent assay. American Journal of Veterinary Research, 43, 2097-2100 Search in Google Scholar

Received: 2014-5-28
Revised: 2014-10-26
Accepted: 2014-11-28
Published Online: 2015-3-25
Published in Print: 2015-6-1

© 2015 W. Stefański Institute of Parasitology, PAS