title: A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium.
abstract: DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to -80 °C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs.
authors: Departamento de Sanidad Animal, Instituto de Ganadería de Montaña, Consejo Superior de Investigaciones Científicas (CSIC)- Universidad de León (ULE), Grulleros, 24346, León, Spain. mhsanh@unileon.es
authors: H
authors: H
authors: Sandoval
authors: M Yolanda
authors: MY
authors: Manga-González
authors: José M
authors: JM
authors: Castro
mesh_terms: D000818:Animals; D017931:DNA Primers / Q000235:genetics; D018509:DNA, Helminth / Q000235:genetics / Q000302:isolation & purification; D004011:Dicrocoeliasis / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D004013:Dicrocoelium / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D005243:Feces / Q000469:parasitology*; D005615:Freezing; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000469:parasitology; D013048:Specimen Handling / Q000379:methods; D014730:Veterinary Medicine / Q000379:methods
chemical_list: D017931:DNA Primers; D018509:DNA, Helminth; D012337:RNA, Ribosomal, 18S
references: Parasitol Res. 1998 Jun;84(6):505-8
references: 9660142
references: Prev Vet Med. 2003 Feb 15;57(1-2):1-5
references: 12547170
references: Parasitol Res. 2007 Aug;101(3):547-55
references: 17393185
references: Parasitol Res. 2000 Jun;86(6):472-9
references: 10894473
references: J Environ Sci Health B. 1991 Apr;26(2):241-57
references: 1906907
references: Vet Parasitol. 1996 Dec 31;67(3-4):185-95
references: 9017867
references: J Parasitol. 1976 Oct;62(5):715-20
references: 988153
references: Parasitology. 2011 Dec;138(14):1916-23
references: 21861946
references: Vet Parasitol. 1996 Jan;61(1-2):151-6
references: 8750692
references: Parasitology. 1993 Feb;106 ( Pt 2):193-9
references: 8446472
references: Vet Parasitol. 1983 Oct;13(3):245-9
references: 6686380
references: J Helminthol. 2005 Dec;79(4):291-302
references: 16336711
references: J Egypt Soc Parasitol. 1991 Aug;21(2):439-44
references: 1875073
references: J Parasitol. 1986 Aug;72(4):507-11
references: 3783344
references: Parasitol Res. 2008 Apr;102(5):927-9
references: 18214542
references: Z Parasitenkd. 1975 Aug 21;47(1):35-43
references: 242135
references: Parasitol Int. 1999 Mar;48(1):21-6
references: 11269322
references: Mol Biochem Parasitol. 1993 Jul;60(1):71-9
references: 8366896
references: Vet Q. 1991 Apr;13(2):81-7
references: 1909064
references: Parasitology. 1993 Aug;107 ( Pt 2):147-56
references: 8414669
references: Genetika. 2002 Aug;38(8):1155-62
references: 12244701
references: J Helminthol. 1998 Jun;72(2):109-16
references: 9687590
references: Vet Parasitol. 2000 Jan;87(2-3):139-49
references: 10622605
references: Parasitol Res. 2007 Oct;101(5):1251-5
references: 17605007
references: Vet Parasitol. 2012 Nov 23;190(1-2):80-6
references: 22749330
references: Parasitol Res. 2004 Aug;93(5):344-55
references: 15197580
references: Acta Trop. 2007 Nov-Dec;104(2-3):91-8
references: 17803950
references: Parasitology. 2001;123 Suppl:S91-114
references: 11769295
references: Exp Parasitol. 1968 Aug;23(1):67-78
references: 5673258
references: Z Parasitenkd. 1975 Dec 11;48(1):35-45
references: 3901