Molecular and microscopic techniques for detection of Sarcocystis neurona sporocysts in fecal samples

Diagnosis of Sarcocystis sp. in the definitive host is generally by microscopic detection of the sporocysts in feces. This method is insensitive and cannot differentiate between species because sporocysts lack specific staining criteria. The hypothesis suggested that molecular techniques provide better alternatives to classical detection of Sarcocystis sporocysts. The sensitivity of two PCR assays was compared to one another and to microscopic examination by conventional fecal flotation and Diamant-Fuchsin staining procedures for detection of sporocysts spiked into mice feces. PCR1 assay using LSM1 and LSM2 primers that amplified 496 bp of the ssurRNA gene was more sensitive than the PCR2 method using JNB25 and JD396 primers that amplified 334 bp of a RAPD-derived marker. PCR1 gave positive results with 200 micro1 of fecal suspension spiked with as little as 5 sporocysts compared to 75 sporocysts detected by JNB25 and JD396 primers. PCR1 was more sensitive than conventional microscopy. PCR1 or PCR2 followed by sequencing or RFLP analysis not only detected Sarcocystis sporocysts in feces but also enabled to ascertain the genotype of the species as S. neurona

Sarcocystosis of sarcocystis felis in cats

The features of S. felis sarcocystosis in muscles of the domestic cats [Felis domesticus] were studied. A complete clinical history, post mortem, and histopathologic examinations were done for each cat. Multiple protozoan elliptical cysts were in the skeletal muscles, heart, and diaphragm muscles of 3/17 [17.6%] adult cats. Ultrastructural characteristics of the brady-zoites and cyst wall were consistent with those descried for S. felis in bobcat and domestic cat. Clinicopathological study in 3 cats showed hypertrophy cardiomyopathy and lymphosarcoma associated with S. felis. Tissue samples showed a spectrum of pathological changes such as multi-focal subacute myocarditis and multi-focal subarachnoid lymphocytic infiltration. DNA extracted from muscles diaphragm with cysts was tested by PCR and sequence analyses of ssurRNA gene. The phylogenetic reconstructions using neighbor-joining method showed that S. felis is closely related to S. neurona. The results were illustrated and photographed and peer discussed.