Detection of live Trypanosoma cruzi in tissues of infected mice by using histochemical stain for β-galactosidase

The pathogenesis of tissue damage in chronic Trypanosoma cruzi infection has been a subject of longstanding debate. Conventional staining methods reveal a paucity of parasites in tissues from chronically infected individuals, which has led to the theory that the pathologic findings may be primarily autoimmune in origin. Immunostaining for T. cruzi antigens or in situ PCR methods show evidence for parasite components in chronic tissues; however, these methods do not address whether the stained material represents parasite debris or live organisms. An improved method for detecting intact T. cruzi in tissues was developed by making a genetically engineered strain that expresses Escherichia coli P-galactosidase. The expression of this enzyme allows the detection of T. cruzi in tissues by using the histochemical stain 5-bromo-1-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). The technique was used to monitor tissue parasitism and its relation to pathologic findings in the mouse model of Chagas' disease. Parasites were easily visible as bright blue structures in skeletal muscle, heart, bladder, peripheral nerve, liver, spleen, adrenal gland, brain, and adipose tissue in acutely infected mice. The number of viable parasites diminished > 100-fold when tissues from 3-week-infected mice were compared with those from 10-month-infected mice. However, even at the lower level, parasites were clearly recognizable in sections of skeletal muscle and bladder at the 10-month time point. Inflammation remained robust in skeletal muscle, bladder, and sciatic nerve despite the near disappearance of parasites, suggesting three possibilities: exuberant host reactions to the few remaining parasites, autoimmune inflammation, or reactions to retained parasite antigens in the tissues.