Trypanosoma cruzi in Opossums from South Georgia and North Florida

1
Trypanosoma cruzi in Opossums from South Georgia
and North Florida Jessica Gillis, Department of
Biology Faculty Sponsor Dr. J. Mitchell
Lockhart, Department of Biology
ABSTRACT Trypanosoma cruzi is a protozoan
parasite causing a disease that affects many in
Central and South America, but only few in the
United States. The disease is transmitted by
triatomine insects of the Reduviidae family. T.
cruzi is predicted to occupy numerous mammalian
hosts such as raccoons, coyotes, opossums, deer
and dogs. Over 1,000 opossums were acquired form
three southwest Georgia and north Florida
plantations as a result of an ongoing USDA
Wildlife Services bobwhite-quail predator
project. DNA isolations were performed on more
than 200 frozen opossum heart tissue samples and
were examined for the presence of T. cruzi via
polymerase chain reaction. Approximately 20 have
tested positive for T. cruzi as of submission
date. Season, host sex, and host age data will
also be analyzed. These results suggest that
there are significant levels of this parasite in
southwest Georgia opossum populations.
Figure 4. Representative gel of PCR products.
Bottom Lane DNA ladder, Top Lane
Positive Control
RESULTS From 2003 and 2004, two-hundred
and fourteen opossums samples were tested for
Trypanosoma cruzi and 42 (19.6) were positive
(see Figure 4 for representative gel). From
2003, 15/122 opossums (12.3) from Pebble Hill
and Pinebloom-East Plantations were positive for
T. cruzi. 27/92 (29.3) opossums from Tall
Timbers and Pinebloom-West plantations tested
positive for T. cruzi in 2004. 29/127 (22.8) of
female opossums and 13/87 (14.9) of male
opossums were positive.
Figure 1. Worldwide distribution of Chagas
Disease
Figure 2. Trypanosoma cruzi life cycle
Figure 5. Yearly prevalence of T. cruzi in
opossums. Prevalence is on y axis.
METHODS The United States Department of
Agriculture-Wildlife Services, Auburn University,
The University of Georgia, and Tall Timbers
Research Station in Florida began a project in
2001 to monitor the effects of mesomammalian
predator removal on bobwhite quail reproduction.
Valdosta State University became a research
collaborator on the project in 2003. Predators
were removed from Pebble Hill Plantation,
Pinebloom Plantation and Tall Timbers Research
Station by the USDA-WS. Animals were euthanized,
frozen and transported to VSU for study.
Predators included opossums, raccoons, foxes,
armadillos, coyotes, feral dogs, feral cats, and
bobcats. Animals were necropsied and tissue
samples were collected. Various natural history
parameters were recorded. Two-hundred and
fourteen frozen heart tissue samples were
examined for the presence of T. cruzi using
polymerase chain reaction. The frozen samples
were thawed and DNA isolations were performed
using Qiagen DNeasy tissue kits following the
manufacturers protocol. PCR, using 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 (Figure 3).
Products of PCR were electrophoresed in 2
agarose gels and visualized using ethidium
bromide and photographed for analysis (Figure 4).
Positive and negative controls were run for each
set of samples. Precautions were taken and
strict protocols were followed to prevent
contamination.
INTRODUCTION Trypanosoma cruzi, a
parasitic hemoflagellate and causative agent of
Chagas disease (American trypanosomiasis), has
devastated the human population in many Latin
American countries. T. cruzi infects 16-18
million people worldwide (World Health
Organization, 1993) (Fig.1), however, infections
in humans in the United States are rare.
Worldwide, T. cruzi is responsible from more than
50,000 deaths annually (Tanowitz, 1992).
Kissing bugs in the Reduviidae family are
responsible for the transmission of T. cruzi to
its mammalian hosts. Common hosts include
armadillos, deer, dogs, opossums, and raccoons.
The life cycle of T. cruzi is complex (Fig. 2).
Assassin bugs feed on the blood of an infected
host and the parasite reproduces asexually in the
gut of the bug. The assassin bug then feeds on a
new host by piercing the skin. Defecation occurs
during feeding on the skin of the new host.
Parasites then migrate from the feces into the
open wound of the host. Trypanosoma cruzi
has been detected from Maryland in the northeast
throughout the southern states and extending to
the west coast of California. Only five human
cases have been reported in the United States and
have come from California, Tennessee, and Texas
(reviewed by Herwakdt et. al., 2000). The T.
cruzi strain that exists in the United States
seems to be significantly less pathogenic than
that found in South America. The Virginia
opossum (Didelphis virginianus) is a frequent
carrier of Trypanosoma cruzi. In previous
studies, 89 of 552 (16) opossums tested in
Georgia and northwest Florida were positive for
T. cruzi (McKeever et al., 1958). In North
Carolina, 1 of 12 (8.3) opossums were carriers
of T. cruzi (Karsten et al., 1992). In
Louisiana, 37.5 of opossums were positive for T.
cruzi (Barr, 1991).
DISCUSSION From our data it is evident that
Trypanosoma cruzi exists in the opossum
population in the Red Hills region of southwest
Georgia and north Florida. There was no
significant difference between prevalence of
infection between opossum sexes, but there was a
statistically significant difference in
prevalence between year of collection (p gt
0.005). 2003 represented the third year of
collection from Pebble Hill and Pinebloom-East
and 2004 represented the first year of collection
from Tall Timbers and Pinebloom-West. We would
like to explore this further to delineate
potential host removal effects on prevalence of
T. cruzi.
FUTURE RESEARCH In the future, we would like to
incorporate specific age data into our analysis.
We would also like to evaluate data from the
final two years of the study. Other factors to
consider include habitat management strategies
and possibly to incorporate existing geographic
information systems data.
Figure 3. Schematic representation of a kDNA
minicircle of T. cruzi Arrows indicate the
relative hybridization locations of the MCS35
and MCS36 primers, which give an amplification
product of 330bp
MCS36
REFERENCES Barr, S.C., C.C. Brown, V.A. Dennis,
and T.R. Klei. 1991. The Lesions and Prevalence
of Trypanosoma cruzi in Opossums and
Armadillos from Southern Louisiana. Journal of
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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
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C. Davis, and R. Kuhn. 1992. Trypanosoma crusi in
wild raccoons and opossums in North Carolina.
Journal of Parasitology 78 547-549. McKeever,
S., G.W. Gorman, and L. Norman. 1958. Occurrence
of Trypanosoma cruzi-like organism in some
mammals from southwestern Georgia and
northwestern Florida. Journal of Parasitology 44
583-587. Tanowitz, H. B., L. V. Kirchhoff, D.
Simon, S. A. Morris, L. Weiss, and M. Wittner.
1992. Chagas disease. Clinical
Microbiology Review S 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 Program
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Diseases, Geneva, Switzerland, p. 67-75.
MCS35
MCS36
MCS36
MCS35
MCS35
MCS35
MCS36
330 bp