Characterization of the second internal transcribed spacer of ribosomal DNA of Calicophoron daubneyi from various hosts and locations in southern Italy.
Isolates of the rumen fluke Calicophoron daubneyi (Digenea: Paramphistomidae) from various hosts and three locations in southern Italy were characterized genetically. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified from individual rumen flukes by PCR. PCR-linked restriction fragment length polymorphism (PCR-RFLP) analysis was performed using four different restriction endonucleases, and PCR products were sequenced. The PCR analyses from all the C. daubneyi specimens produced identical fragments, and the PCR-RFLP analyses did not show, with respect to any of the four restriction endonucleases, any differences between the C. daubneyi specimens. The sequence analyses of the ITS-2+ from each of the C. daubneyi specimens showed them all to be 428 bp, and composed of the entire ITS-2 sequence (282 bp) plus the two partial flanking conserved sequences, 5.8S (99 bp) and 28S (47 bp). No intra-specific variation was observed in the nucleotide composition of the ITS-2+ (homology=100%). There was, however, an observable inter-specific variation between the ITS-2+ of C. daubneyi and the ITS-2+ of both Calicophoron calicophorum (homology=97.2 %) and Calicophoronmicrobothrioides (homology=97.4 %), both previously deposited in the GenBank. The finding of the present study shows that, as has already demonstrated for other parasitic helminths, ITS-2 can serve as an effective genetic marker for the molecular identification of paramphistomes, and as a useful tool for developing molecular epidemiological techniques for the study of C. daubneyi transmission patterns and prevalence in definitive and intermediate hosts.
Rinaldi
L
L
Dipartimento di Patologia e Sanità Animale, Università degli Studi di Napoli "Federico II" - CREMOPAR Regione Campania, Napoli, Italy.
Perugini
A G
AG
Capuano
F
F
Fenizia
D
D
Musella
V
V
Veneziano
V
V
Cringoli
G
G
D000818:Animals; D001483:Base Sequence; D002020:Buffaloes / Q000469:parasitology*; D002417:Cattle; D002418:Cattle Diseases / Q000469:parasitology*; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D007411:Intestinal Diseases, Parasitic / Q000469:parasitology / Q000662:veterinary*; D007558:Italy; D008969:Molecular Sequence Data; D010250:Paramphistomatidae / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012339:RNA, Ribosomal, 28S / Q000737:chemistry / Q000235:genetics; D012340:RNA, Ribosomal, 5.8S / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012756:Sheep; D012757:Sheep Diseases / Q000469:parasitology*; D014201:Trematode Infections / Q000469:parasitology / Q000662:veterinary*
D018509:DNA, Helminth; D021903:DNA, Ribosomal Spacer; D012339:RNA, Ribosomal, 28S; D012340:RNA, Ribosomal, 5.8S