Transcript Trypanosoma cruzi in Opossum

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.
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).
Figure 1. Worldwide distribution of Chagas Disease
RESULTS
Figure 2. Trypanosoma cruzi life cycle
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 PinebloomWest 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 4. Representative gel of
PCR products. Bottom Lane –
DNA ladder, Top Lane – Positive
Control
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 manufacturer’s 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.
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
30
25
20
15
2003
2004
10
5
0
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 > 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 PinebloomWest. 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.
REFERENCES
MCS35
MCS36
MCS35
MCS35
MCS36
MCS35
MCS36
330 bp
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Herwaldt, 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
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Karsten, V., C. Davis, and R. Kuhn. 1992. Trypanosoma crusi in wild raccoons and opossums in North Carolina.
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