CONTINUING STUDIES OF TRYPANOSOMA CRUZI IN GEORGIA RACCOONS Kevin Tyler Ward, Department of Biology

Faculty Sponsor: Dr. J. Mitchell Lockhart, Department of Biology

ABSTRACT Trypanosoma cruzi, a parasite known to cause Chagas’ disease occupies several mammalian hosts including canids, opossums, and raccoons. Although most cases of Chagas’ disease are found in Central and South America, the parasite is present in wildlife populations in North America and warrant further investigation. More than 200 raccoons were obtained in 2005 from south Georgia and north Florida as part of a USDA-Wildlife Services bobwhite quail predator removal study and were evaluated for the presence of T. cruzi by PCR. For 2005, roughly 25.0% of 100 samples tested so far have been positive. This data will be compared with data collected in 2003 and 2004 and will be analyzed using various natural history variables. Collectively, data from these 3 years suggests that there are considerable levels of T. cruzi in wildlife populations in these locations.

INTRODUCTION An increasingly persistent parasite, Trypanosoma cruzi, is the source of

Chagas’ disease, a condition accountable for over 50,000 deaths per year (Tanowitz et. al. 1992). The principal location of these deaths are several Latin American countries. Although T. cruzi is more established in these countries, it infects 16-18 million people worldwide every year (World Health Organization, 1993). There have been five reported cases in the United States all of which occurred in Tennessee, Texas, or California (reviewed by Herwaldt et. al., 2000). A probable explanation for the extreme disparity between cases in the United States and Latin American countries involves living conditions. It is now believed that there may be up to one hundred thousand immigrants living within the United States that are infected with T. cruzi (Kirchhoff, 1993).

Upon reaching maturity and with the aid of a blood sucking invertebrate host, T. cruzi can be transmitted to various mammals including deer, armadillos, opossums, raccoons, and also reptiles and birds. T. cruzi inhabits the intestines of the invertebrate host and is transmitted after the host bites a mammal, where it then resides in the blood and/or tissues (Figure 1).

Raccoons and opossums are thought to be frequent carriers of T. cruzi in the United States. Recent research demonstrates that 15% of raccoons tested in North Carolina (Karsten et. al., 1992) and 22.2% in Georgia (Pung, 1995) were infected with T. cruzi, whereas 16% of opossums tested in Georgia and northwest Florida were positive for T.cruzi (McKeever et al., 1958).

Figure 2. Representative gelfrom T. cruzi PCR. Note ladderon bottom and positive controlon top.

Figure 1. Life cycle of Trypanosoma cruzi.

METHODS Animals were collected from Tall Timbers Research Station and the Eastern and Western section of Pinbebloom Plantation. The locations are found in the Red Hills region of south Georgia and north Florida. Animals were euthanized, frozen, and transported to Valdosta State University. Animals were necropsied and various tissue samples and morphometric data was collected. From 2003-2005, 423 raccoons have been evaluated for the presence of T. cruzi using polymerase chain reaction. Frozen tissue samples were thawed and DNA isolations were performed using Qiagen DNeasyTM tissue kits following manufacturer’s protocol. PCR, utilizing T. cruzi specific primers MCS-35 and MCS-36 found in a 330 base pair region of the kinetoplast DNA minicircle of T. cruzi, was performed. PCR products were electrophoresed in 2% agarose gels and visualized using ethidium bromide and photographed. Positive and negative controls were run for each set of samples and strict protocols were followed to avoid contamination.

Prevalence of Trypanosoma cruzi in Raccoons

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RESULTS and DISCUSSION From 2003-2005, a total of 423 raccoons were evaluated for the presence of T. cruzi. In 2003, 36/150 (24.0%) raccoons tested positive; in 2004, 50/202 (24.7%) raccoons tested positive; and in 2005 15/66 (22.7%) raccoons have tested positive for the presence of T. cruzi (Figure 2). No significant trends were noted with regard to seasonal prevalence (Figure 3). 58/284 male raccoons (20.4%) were positive for T. cruzi versus 43/133 (32.3%) of females raccoons (Figure 5). This difference was statistically significant (p > 0.005). We theorize that reproductive status may influence prevalence of T. cruzi, but we would like to investigate this hypothesis further. This study represents the first three years of a four year study. We will analyze the final years data and incorporate specific age data in the future.

Figure 3. Seasonal prevalence of T. cruzi in Raccoons

Acknowledgements We would like to thank the USDA-WS for trapping animals, all other principals associated with the Predator Removal Study, and the Valdosta State University Faculty Research Fund for providing funds to complete this project.

REFERENCESHerwaldt, B.L., M. J. Grijalva, A. L. Newsome, C. R. McGhee, M. R. Powell, D. G. Nemec, F. J. Steurer, and M. L. Eberhard. 2000. Use of polymerase chain reaction to diagnose the Fifth reported U. S. case of autochthonous transmission of Trypanosoma cruzi, in Tennessee, 1998. Journal of Infectious Diseases 181: 395-399.John, D. T., and K. L. Hoppe. 1986. Trypanosoma cruzi from wild raccoons in Oklahoma. American Journal of Veterinary Research 47: 1056-1059.Kirchhoff, L. V. 1993. American Trypanosomiasis (Chagas’ disease)- A Tropical disease now I the United States. The New England Journal of Medicine 329: 639-644.McKeever, S., G. W. Gorman, and L. Norman. 1958. Occurrence of a Trypaosoma cruzi-like organism in some mammals from southwestern Georgia and northwestern Florida. Journal of Parasitology 44: 583- 587.Pung, O. J., C. W. Banks, D. N. Jones, and M. W. Krissinger. 1995. Trypanosoma cruzi in wild raccoons, opossums, and triatomine bugs in southeast Georgia, U.S.A. Journal of Parasitology 81: 324-326Tanowitz, H. B., L. V. Kirchhoff, D. Simon, S. A. Morris, L. Weiss, and M. Wittner. 1992. Chagas’ disease. Clinical Microbiology Review 5: 400-419.World Health Organization. 1993. Chagas’ disease. In Tropical diseases. Tropical disease research. Progress 1991-1992. Eleventh Programme Report of the UNDP/World Bank/ WHO Special Programme for Research and Training in Tropical Diseases, Geneva, Switzerland, p. 67-75.