Molecular identification of Sarcocystis species in imported frozen beef in Egypt

Abstract

Key words:Sarcocystis, imported frozen beef, PCR, Histopathology, SequencingIn the present study, we set out to implement and compare the different detection methods that include the peptide digestion, histopathological examination and PCR, to identify Sarcocystis species that can be found in the imported frozen beef. Comparing results from the three identification methods, along with their compatibility and accuracy in diagnosing Sarcocystis spp. are also presented. Finally, a special focus is given to the use of PCR followed by sequencing and the bioinformatics analysis in detecting and comparing Sarcocystis found in the imported frozen beef. A total number of 384 samples of imported frozen beef (280 Indian buffalo and 104 Brazilian cattle meat) were collected from different retail meat shops in Alexandria province, Egypt. Visual meat inspection revealed a low prevalence of macroscopic Sarcocystis spp. in buffalo beef (3.9%) and no macrocysts were detected in cattle beef. Fifty samples were taken from buffalo beef and subjected to peptic digestion, histopathology and PCR resulting in 24%, 38% and 34% positive samples respectively. While the same number of samples were taken from cattle beef resulting in lower infection rates (6%, 2% and 2%). There were no significance difference found between the infection rate according to diagnostic method or type of meat cut. For molecular characterization newly designed primer sets were used to specifically amplify the 18S DNA sequences of the four Sarcocystis spp. in both buffalo and cattle (S.fusiformis, S.buffalonis, S.hirsuta and S.hominis) using the primer BLAST tool on the NCBI of the Genbank. Aligning the 18S rDNA sequences of three positive PCR products revealed high identity percents with three Sarcocystis spp.; S. fusiformis, S. buffalonis and S. hominis. (85%, 92% and 100% respectively).