Development of a Polymerase Chain Reaction Test for Entamoeba invadens
American Association of Zoo Veterinarians Conference 2007

Carol M. Bradford, MS, DVM; Mary C. Denver, DVM; Michael Cranfield, DVM

The Maryland Zoo in Baltimore, Baltimore, MD, USA


Entamoeba invadens is a protozoal parasite of reptiles that causes colitis, abscessation of the liver and other organs, and sometimes, acute death.2 It is generally considered a commensal organism of chelonians, but has been implicated as a cause of disease in red-footed (Geochelone carbonaria) and leopard tortoises (Geochelone pardalis).3 Some of the apparent differences in species susceptibility may be due to the inability to distinguish nonpathogenic species of Entamoeba from E. invadens, rather than actual variation in severity of clinical signs among reptilian hosts. Diagnosis of E. invadens is currently by detection of trophozoites and/or cysts via direct fecal examination; however, definitive diagnosis of E. invadens has been difficult due to the almost identical morphology of nonpathogenic Entamoeba sp. such as E. ranarum, E. insolita, E. barreti, and E. terrapinae.1 Definitive speciation of Entamoeba sp. is important to avoid misdiagnosis or overtreatment for nonpathogenic protozoa and exposure of potentially susceptible species in mixed collections. In this study, polymerase chain reaction (PCR) primers were developed for E. invadens, E. ranarum, E. terrapinae, and E. insolita and PCR testing was conducted on purified DNA from cell cultures as well as purified DNA from reptile stool samples with E. invadens trophozoites added. This study has developed successful PCR primers for four species of Entamoeba and demonstrates that PCR is a promising diagnostic tool for the definitive identification of Entamoeba invadens.


The authors thank the individuals that have provided samples and technical assistance with this research including Daniel Eichinger, James Bradford, C. Graham Clark, Ellen Bronson, Christopher Bednarski, Jennifer Sohl, Steve Maltese, Nadia Bischoff, and many others.

Literature Cited

1.  Clark, C.G., F. Kaffashian, B. Tawari, J.J. Windsor, A. Twigg-Flesner, M.C.G. Davies Morel, J. Blessman, F. Ebert, B. Peschel, A.L. Van, C.J. Jackson, L. Macfarlane, and E. Tannich. 2006. New insights into the phylogeny of Entamoeba species provided by analysis of four new small-subunit rRNA genes. Int. J. Syst. Evol. Micro. 56:2235–2239.

2.  Jacobson, E., S. Clubb, and E. Greiner. 1983. Amebiasis in red-footed tortoises. J. Am. Vet. Med. Assoc. 183:1192–1194

3.  Greiner, E.C., and D.R. Mader. 2006. Parasitology. In: Mader, D.R. (ed.). Reptile Medicine and Surgery. 2nd ed. Elsevier, St. Louis, Missouri. Pp. 343–364.


Speaker Information
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Carol M. Bradford, MS, DVM
The Maryland Zoo in Baltimore
Baltimore, MD, USA

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