Paramphistomum leydeni: experimental infections in lambs and characterization of the rDNA ITS markers from flukes and snails

SANABRIA, R.E.F.; MORÉ, G.; ROMERO, J. R.

Buenos Aires

Congreso; The 23rd International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP 2011); 2011

WAAVP

Two intermediate hosts (IH) of Paramphistomum leydeni have been found in Argentina: Drepanotrema depressissiumum (Dd) and Lymnaea viatrix (Lv). Two lambs were experimentally infected with 50 metacercariae each, obtained from naturally infected Dd, and another four lambs were inoculated with metacercariae (250 each) recovered from experimentally infected Lv. Only those lambs that received metacercariae from Dd resulted infected and adult flukes were recovered. DNA was extracted from these adult flukes, from rediae and cercariae obtained from Lv and from P. leydeni eggs collected from adult flukes in order to compare with P.leydeni adult flukes from natural infections. For this purpose a PCR-RFLP assay was performed using the ITS2+ marker and endonucleases HhaI and HinfI. Simultaneously, the same procedure using the ITS1+ segment as target restricted by DdeI enzyme, was applied to characterize the infected snails: two Lv strains (U and SP) and one of Dd. All the paramphistome samples given ~500 bp bands, and all restriction products resulted in 220 and 280 bp fragments for HhaI, and 130, 170 y 200 bp fragments for HinfI. Regarding snails, the PCR for ITS1+ segments results in fragments of ~600 bp for Lv strain SP, and ~650 for Lv strain U and Dd. Restriction products were also different, being 120, 130 and 400 bp for LvU; 80, 130 and 390 for LvSP; and 280 and 370 for Dd. Differences between paramphistomids using ITS2+ marker were not detected, but, differential infection patterns were recorded, depending on the infection source (Dd or Lv). Differences  between both Lv strains may suggest that more than one Lymnaeids species or subspecies could be implicated in the life cycle development. Despite of this the specific influence of Lv in metacercarie infectivity remains unknown. P. leydeni has emerged over the last 10 years, for the east-central region of Argentina, may be due to a specific adaptation to the IH like Drepanotrema depressissiumum. Additional molecular markers and sequences analysis could be helpful to improve characterization of amphistomes and snails species and allows a better knowledge of the biology of these parasites.