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  • Acknowledgement This work was supported by


    Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (No.81802749), Shenzhen Healthcare Research Project (No.SZBC2017029), the Science and Technology Foundation of Shenzhen (JCYJ20170412155231633, JSGG20170414104216477), National Key Research and Development Program of China (2018YFC1002801), and the Shenzhen Public Service Platform on Tumor Precision Medicine and Molecular Diagnosis.
    INTRODUCTION Trypanosomiasis is generally considered an exotic tropical disease. American trypanosomiasis (Chagas’ disease), which is caused by Trypanosoma cruzi, affects dogs, cats, armadillos, monkeys, and small wild animals.1, 2, 3, 4, 5 Wildlife, people, livestock, and pets may be affected and increased surveillance is necessary to monitor potential zoonotic outbreaks. T. cruzi, unlike its relatives, multiplies within the cytoplasm of the mammalian host's cells. It has a predilection for cardiac and skeletal muscle, where transformation to amastigotes, a form closely resembling Leishmania, occurs within the potassium hydrochloride and the collections form pseudocysts. As an example of its northward movement, Trypanosoma cruzi is known to be common in the triatomid bugs of south-central Texas and has caused chronic heart disease in dogs. In Texas, it is maintained by a sylvatic cycle in domestic and wild animals.4, 5, 6 Marsupials in South America have been intensively studied in relationship to triatomid/Trypanosoma life cycles. Most New World species of small mammals appear to be highly resistant to pathological disease, and function primarily as reservoirs of trypanosomes.7, 8 Although T. cruzi has been detected in captive chimpanzees and cynomolgus monkeys as well as experimentally infected Australian phalanger marsupials (Trichosurus vulpecula), this is the first time this organism has been detected in naturally infected pet sugar gliders (Petaurus breviceps) and African hedgehogs (Atelerix albiventris).9, 10, 11 This is the first report of natural infection of Chagas’ disease in Old World marsupials and African insectivores. A polymerase chain reaction (PCR)-based method was used to detect T. cruzi organisms in formalin fixed paraffin embedded blocks from 3 sugar gliders and hedgehogs, which were previously diagnosed as T. cruzi-positive using the histopathological method.
    MATERIALS AND METHODS Tissue samples were referred to the Zoo/Exotic Pathology Service (West Sacramento, CA, USA). Cardiac tissue samples were harvested, fixed in formalin, and wax blocks made using conventional methods. The most significant histologic lesion was a diffuse lymphoplasmacytic myocarditis with intralesional protozoa. Immunohistochemistry was negative for Toxoplasma gondii, Neospora sp., and Sarcocystis neurona. On thin sectioning, the protozoa were determined to have a distinct kinetoplast. Electron microscopic examination demonstrated the salient features of flagella (some in and some out of cytoplasm) and kinetoplasts supporting an identification of Trypanosoma sp. PCR assay samples were obtained from the paraffin wax blocks using a 3-mm tissue punch (Acuderm. Inc, Fort Lauderdale, FL, USA) and deparaffinized overnight in xylene. DNA was extracted using the DNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's specifications. PCR was carried out using primers TCZ1 and TCZ2 and platinum Taq polymerase (Invitrogen) under touchdown conditions.2, 12 The PCR products were stained with ethidium bromide, visualized under UV illumination and photo-documented.
    RESULTS This is the first report of trypanosomiasis detected in naturally infected “pet” sugar gliders and African hedgehogs. A PCR-based method was used to detect T. cruzi organisms in formalin fixed paraffin embedded blocks, which were previously diagnosed as T. cruzi-positive using the histopathological method and in several samples that were not detected by histopathological techniques alone. Formalin-fixed histopathological samples combined with subsequent PCR testing were useful for diagnosis in all samples.