TrypanosomaTrypanosoma cruzicruzi from from Opossums in Southwest Opossums in Southwest

Georgia and North FloridaGeorgia and North Florida

Jessica L. Gillis and J. Mitchell LockhartJessica L. Gillis and J. Mitchell Lockhart

Department of BiologyDepartment of Biology

Valdosta State UniversityValdosta State University

Trypanosoma cruziTrypanosoma cruzi: Background: Background

Parasitic protozoan hemoflagellate Parasitic protozoan hemoflagellate Most species of Most species of Trypanosoma Trypanosoma livelive in more than one in more than one

host in their life cycle.host in their life cycle.

Amastigotes of T. cruzi in muscle tissue

Trypanosoma cruziTrypanosoma cruzi: Background: Background

Trypomastigotes:Pass from the insect's hindgut in the fecal

matter and burrow into the skin and penetrate

the bloodstream

Amastigotes:cluster into cysts and explode into

bloodstream where they move on to other tissue cells

Epimastigotes:travel to the insect's hindgut, where they

transform into trypomastigotes

T. cruziT. cruzi changes morphotypes as it cycles from insects to humans changes morphotypes as it cycles from insects to humans ..

The life cycle of The life cycle of T. cruziT. cruzi

WHO

Trypanosoma cruziTrypanosoma cruzi: Background: BackgroundThe VectorThe Vector

Order: Hemiptera (True Bugs)

Family: Reduviidae

Common name: “Kissing Bug”

Transmission of T. cruzi follows defecation by kissing bugs on the skin of bitten hosts

Chagas’ disease has devastated the human Chagas’ disease has devastated the human population in many Latin American countriespopulation in many Latin American countries

Trypanosoma cruziTrypanosoma cruzi: Background: Background

16–18 million people infected worldwide 16–18 million people infected worldwide with Chagas disease.with Chagas disease. A significant percentage of Brazilians may A significant percentage of Brazilians may

eventually die as a result of eventually die as a result of TT. . cruzicruzi infections. infections.

More than 50,000 deaths each year.More than 50,000 deaths each year.

Trypanosoma cruzi Trypanosoma cruzi in the United Statesin the United States

Five human cases have been reported from Five human cases have been reported from California, Tennessee, and TexasCalifornia, Tennessee, and Texas The The T. cruziT. cruzi strain that exists in the United States strain that exists in the United States

seems to be different than that found in South seems to be different than that found in South America. America.

T. cruziT. cruzi is thought to infect fifty to one hundred is thought to infect fifty to one hundred thousand immigrants living in the United States thousand immigrants living in the United States (Kirchhoff, 1993). (Kirchhoff, 1993).

TT. . cruzicruzi: Vector Biology: Vector Biology

Poor quality houses with thatched roofs, Poor quality houses with thatched roofs, cracked ceilings, and adobe walls are prime cracked ceilings, and adobe walls are prime dwellings for the kissing bug.dwellings for the kissing bug.

Chagas’ Disease PreventionChagas’ Disease Prevention

Treatment of home with residual insecticides.Treatment of home with residual insecticides. Blood screening to prevent transmission Blood screening to prevent transmission

through transfusion.through transfusion. Housing improvements.Housing improvements.

T. cruziT. cruzi Reservoirs Reservoirs

Domestic as well as wild animalsDomestic as well as wild animals Infection of Infection of T. cruziT. cruzi in domestic animals is thought in domestic animals is thought

to be under diagnosed. to be under diagnosed. Commonly infected wildlife hosts include: Commonly infected wildlife hosts include:

armadillos, deer, dogs, opossums, raccoons, armadillos, deer, dogs, opossums, raccoons, rodents, bats, skunks, and gray fox. rodents, bats, skunks, and gray fox.

T. cruzi T. cruzi is not limited to mammalian hosts but also is not limited to mammalian hosts but also affects reptiles and birds.affects reptiles and birds.

TT. . cruzicruzi in the Virginia Opossum in the Virginia Opossum

http://www.entm.purdue.edu/wildlife/opossum_pictures.htm

TT. . cruzicruzi in the Opossum in the Opossum

Previous studies:Previous studies: 89 of 552 (16%) of opossums tested in Georgia and northwest 89 of 552 (16%) of opossums tested in Georgia and northwest

Florida were positive for Florida were positive for T.cruziT.cruzi (McKeever et al., 1958). (McKeever et al., 1958). In North Carolina, 1 of 12 (8.3%) of opossums were positive In North Carolina, 1 of 12 (8.3%) of opossums were positive

(Karsten et al., 1992). (Karsten et al., 1992). In Georgia, 6 of 39 (15.4%) opossums tested were positive. In Georgia, 6 of 39 (15.4%) opossums tested were positive.

(Pung, 1995).(Pung, 1995). In Louisiana, 37.5% of the opossums were positive (Barr, In Louisiana, 37.5% of the opossums were positive (Barr,

1991).1991).

Current StudyCurrent Study Mesomammalian predators were removed Mesomammalian predators were removed

from three southwest Georgia/north Florida from three southwest Georgia/north Florida quail plantations (Pebble Hill, Tall Timbers, quail plantations (Pebble Hill, Tall Timbers, Pinebloom – 2 sites).Pinebloom – 2 sites). Included raccoons, opossums, foxes, Included raccoons, opossums, foxes,

armadillos, coyotes, feral dogs, feral cats armadillos, coyotes, feral dogs, feral cats and bobcats.and bobcats.

Animals were euthanized, frozen, transported Animals were euthanized, frozen, transported to VSU and necropsied.to VSU and necropsied. Various samples and data were collectedVarious samples and data were collected ..

Current StudyCurrent Study

Two-hundred and fourteen frozen heart tissue Two-hundred and fourteen frozen heart tissue samples were examined for the presence of samples were examined for the presence of T. cruziT. cruzi using polymerase chain reaction testing. using polymerase chain reaction testing.

Frozen samples were thawed and DNA isolations Frozen samples were thawed and DNA isolations were performed using Qiagen DNeasy™ tissue kits were performed using Qiagen DNeasy™ tissue kits following manufacturer’s protocol.following manufacturer’s protocol.

Current StudyCurrent Study

PCR utilizing PCR utilizing TT. . cruzicruzi-specific primers MCS-35 and -specific primers MCS-35 and MCS-36 was performed.MCS-36 was performed.

MCS-35 5’- AAATAATGTACGGG(T/G)GAGATGCATGA - 3’MCS-35 5’- AAATAATGTACGGG(T/G)GAGATGCATGA - 3’ MCS-36 5’- GGGTTCGATTGGGGTTGGTGT- 3’MCS-36 5’- GGGTTCGATTGGGGTTGGTGT- 3’

Ethidium bromide/gel visualization.Ethidium bromide/gel visualization. Positive and negative controls were run for each set Positive and negative controls were run for each set

of samples. of samples. Precautions were taken and strict protocols were Precautions were taken and strict protocols were

followed to prevent contamination. followed to prevent contamination.

Schematic representation of a kDNA minicircle of T. cruziArrows indicate the relative hybridization locations of the

MCS35 and MCS36 primers, which give an amplification product of 330bp

MCS36

MCS36

MCS36

MCS36

MCS35

MCS35

MCS35

MCS35

330 bp

Current StudyCurrent Study

20032003 15/122 positive – 12.3%15/122 positive – 12.3%

20042004 27/92 positive – 29.3%27/92 positive – 29.3%

Chi square = 8.62, df=1, Chi square = 8.62, df=1, p > 0.005 p > 0.005

0

5

10

15

20

25

30

2003 2004

29.3

12.3

Current StudyCurrent Study

20032003 Females: 12/78 = 15.4%Females: 12/78 = 15.4% Males: 3/44 = 6.8%Males: 3/44 = 6.8%

20042004 Females: 17/49 = 34.7%Females: 17/49 = 34.7% Males: 10/43 = 23.3%Males: 10/43 = 23.3%

Chi sqaure = 1.57, df=1 Chi sqaure = 1.57, df=1 NSD between sexesNSD between sexes

Current StudyCurrent Study

Monthly Prevalence of T. cruzi

0

10

20

30

40

50

60

Feb Mar Apr May Jun Jul Aug Sep

Month

Pre

vale

nce

ConclusionsConclusions

TrypanosomaTrypanosoma cruzicruzi is present in opossums in is present in opossums in southwest Georgia/north Florida.southwest Georgia/north Florida.

No significant difference in infection level No significant difference in infection level between sexes.between sexes.

Significant difference in infection level Significant difference in infection level between years of collection.between years of collection. Why?Why?

Future StudiesFuture Studies

2 additional years of animal collections2 additional years of animal collections Age dataAge data Management practices on plantationsManagement practices on plantations Many other possibilitiesMany other possibilities

Trypanosoma cruziTrypanosoma cruzi in Georgia/Florida in Georgia/Florida

AcknowledgementsAcknowledgements USDA- Wildlife ServicesUSDA- Wildlife Services University of GeorgiaUniversity of Georgia Auburn UniversityAuburn University Tall Timbers Research StationTall Timbers Research Station Valdosta State UniversityValdosta State University

Questions?Questions?