Gray fox (Urocyon cinereoargenteus) identified as a new intermediate host for Sarcocystis neurona

The protozoan Sarcocystis neurona can cause severe disease in horses, marine mammals, and several other animal species in North America. The Virginia opossum (Didelphis virginiana) is its definitive host, and the raccoon (Procyon lotor) has been considered its primary intermediate host in the USA. Although canids have not previously been identified as important intermediate hosts for this parasite, we here report several natural cases in the gray fox (Urocyon cinereoargenteus). We identified muscular infections in 11 (23.9 %) of 46 gray foxes from Pennsylvania, USA. In hematoxylin and eosin (HE)-stained sections of tongue and limb muscles, only 13 sarcocysts were detected in 7 of 46 foxes, in limb muscle of 4 and in tongue of 4. In HE-stained sections, the sarcocyst wall was up to 2.7 μm-thick and contained finger-like villar protrusions. In unstained muscle squashes, 44 sarcocysts were detected; they were up to 1200 μm long and 69 μm wide. From each infected fox, only one morphologic type of sarcocyst was found. By transmission electron microscopy, these sarcocysts appeared identical to those in animals experimentally infected with S. neurona. Molecularly, sarcocysts were characterized using 18S rRNA, 28S rRNA, cox1, ITS1, rpoB, RON1, RON2, RON3, GAPDH1, ROP20, ROP21, ROP39, SnSRS21 and TUBA1; results confirmed the presence of S. neurona in the gray fox. This is the first report of muscular sarcocysts in the gray fox.