Molecular identification of Sarcocystis spp. in foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) from Germany

MORÉ, G.; MAKSIMOV, A.; CONRATHS, F.J.; SCHARES, G.

Kusadasi

Encuentro; 2nd Apicowplexa meeting; 2013

Canids are definitive hosts (DH) of several Sarcocystis spp., which affect a wide range of intermediate hosts (IH) worldwide. The aim of the present study was to identify the Sarcocystis spp. present in the small intestine from foxes and raccoon dogs captured in Brandenburg, Germany, using molecular tools. A total of 50 or 38 mucosal scrapings from foxes (F) or raccoon dogs (RD), respectively, were collected as part of an Echinococcosis surveillance program, frozen at -80°C for at least one week and conserved at -20°C. The samples were analyzed by sugar flotation and when sporocysts/oocysts were detected, an overnight sedimentation was performed. DNA was extracted from aliquots of the sediment and the flotation concentrate using a commercial kit. A PCR was conducted using primers targeting a fragment of the 18S rRNA gene ( aprox. 850 bp). Amplicons were purified and sequenced. Samples with inconclusive sequencing results were cloned into plasmids and at least 3 plasmids from each amplicon were sequenced. Sarcocystis spp. infection was detected in 38% (19/50) and 52.6% (20/38), and mixed infection was recorded in 6 and 4 samples, of F and RD samples, respectively. Sequences from F samples showed ≥ 99% identity with S. capracanis (n=6), S. gracilis (n=5), S. capreolicanis (n=4), S. miescheriana (n=4), Sarcocystis spp. using birds as IH (n=3), S. grueneri (n=1), and Cystoisospora spp. (n=2). In addition, one sequence with 97% identity with S. cruzi was detected. Out of all RD samples, sequences with a ≥ 99% identity with S. miescheriana (n=7), S. gracilis (n=6), Sarcocystis spp. using birds as IH (n=5), S. capreolicanis (n=4), S. capracanis (n=1) and one sequence with 96% identity with S. cruzi were observed.The prevalence of Sarcocystis spp. infection observed in this study using mucosal scrapings was higher than related studies performed by analyzing stool samples. The present study represents the first molecular approach to identify Sarcocystis spp. infections from mucosal scrapings. It showed the utility of this approach to identify potential DH of these species.