Trypanosoma cruzi from Opossums in Southwest Georgia and North Florida Jessica L. Gillis and J. Mitchell Lockhart Department of Biology Valdosta State University

Trypanosoma cruzi: Background Parasitic protozoan hemoflagellate Parasitic protozoan hemoflagellate Most species of Trypanosoma live in more than one host in their life cycle. Most species of Trypanosoma live in more than one host in their life cycle. Amastigotes of T. cruzi in muscle tissue

Trypanosoma cruzi: 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. cruzi changes morphotypes as it cycles from insects to humans.

The life cycle of T. cruzi WHO

Trypanosoma cruzi: Background The 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 population in many Latin American countries

Trypanosoma cruzi: Background 16–18 million people infected worldwide with Chagas disease. 16–18 million people infected worldwide with Chagas disease.  A significant percentage of Brazilians may eventually die as a result of T. cruzi infections. More than 50,000 deaths each year. More than 50,000 deaths each year.

Trypanosoma cruzi in the United States Five human cases have been reported from California, Tennessee, and Texas Five human cases have been reported from California, Tennessee, and Texas The T. cruzi strain that exists in the United States seems to be different than that found in South America. The T. cruzi strain that exists in the United States seems to be different than that found in South America. T. cruzi is thought to infect fifty to one hundred thousand immigrants living in the United States (Kirchhoff, 1993). T. cruzi is thought to infect fifty to one hundred thousand immigrants living in the United States (Kirchhoff, 1993).

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

Chagas’ Disease Prevention Treatment of home with residual insecticides. Treatment of home with residual insecticides. Blood screening to prevent transmission through transfusion. Blood screening to prevent transmission through transfusion. Housing improvements. Housing improvements.

T. cruzi Reservoirs Domestic as well as wild animals Domestic as well as wild animals  Infection of T. cruzi in domestic animals is thought to be under diagnosed.  Commonly infected wildlife hosts include: armadillos, deer, dogs, opossums, raccoons, rodents, bats, skunks, and gray fox.  T. cruzi is not limited to mammalian hosts but also affects reptiles and birds.

T. cruzi in the Virginia Opossum

T. cruzi in the Opossum Previous studies: 89 of 552 (16%) of opossums tested in Georgia and northwest Florida were positive for T.cruzi (McKeever et al., 1958). 89 of 552 (16%) of opossums tested in Georgia and northwest Florida were positive for T.cruzi (McKeever et al., 1958). In North Carolina, 1 of 12 (8.3%) of opossums were positive (Karsten et al., 1992). In North Carolina, 1 of 12 (8.3%) of opossums were positive (Karsten et al., 1992). In Georgia, 6 of 39 (15.4%) opossums tested were positive. (Pung, 1995). In Georgia, 6 of 39 (15.4%) opossums tested were positive. (Pung, 1995). In Louisiana, 37.5% of the opossums were positive (Barr, 1991). In Louisiana, 37.5% of the opossums were positive (Barr, 1991).

Current Study Mesomammalian predators were removed from three southwest Georgia/north Florida quail plantations (Pebble Hill, Tall Timbers, Pinebloom – 2 sites). Mesomammalian predators were removed from three southwest Georgia/north Florida quail plantations (Pebble Hill, Tall Timbers, Pinebloom – 2 sites). Included raccoons, opossums, foxes, armadillos, coyotes, feral dogs, feral cats and bobcats. Included raccoons, opossums, foxes, armadillos, coyotes, feral dogs, feral cats and bobcats. Animals were euthanized, frozen, transported to VSU and necropsied. Animals were euthanized, frozen, transported to VSU and necropsied. Various samples and data were collected. Various samples and data were collected.

Current Study Two-hundred and fourteen frozen heart tissue samples were examined for the presence of T. cruzi using polymerase chain reaction testing. Two-hundred and fourteen frozen heart tissue samples were examined for the presence of T. cruzi using polymerase chain reaction testing. Frozen samples were thawed and DNA isolations were performed using Qiagen DNeasy™ tissue kits following manufacturer’s protocol. Frozen samples were thawed and DNA isolations were performed using Qiagen DNeasy™ tissue kits following manufacturer’s protocol.

Current Study PCR utilizing T. cruzi-specific primers MCS-35 and MCS-36 was performed. PCR utilizing T. cruzi-specific primers MCS-35 and 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 of samples. Positive and negative controls were run for each set of samples. Precautions were taken and strict protocols were followed to prevent contamination. Precautions were taken and strict protocols were followed to prevent contamination.

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 MCS bp

Current Study /122 positive – 12.3% 15/122 positive – 12.3% /92 positive – 29.3% 27/92 positive – 29.3% Chi square = 8.62, df=1, p > Chi square = 8.62, df=1, p >

Current Study Females: 12/78 = 15.4% Females: 12/78 = 15.4% Males: 3/44 = 6.8% Males: 3/44 = 6.8% 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 NSD between sexes Chi sqaure = 1.57, df=1 NSD between sexes

Current Study

Conclusions Trypanosoma cruzi is present in opossums in southwest Georgia/north Florida. Trypanosoma cruzi is present in opossums in southwest Georgia/north Florida. No significant difference in infection level between sexes. No significant difference in infection level between sexes. Significant difference in infection level between years of collection. Significant difference in infection level between years of collection. Why? Why?

Future Studies 2 additional years of animal collections 2 additional years of animal collections Age data Age data Management practices on plantations Management practices on plantations Many other possibilities Many other possibilities

Trypanosoma cruzi in Georgia/Florida Acknowledgements Acknowledgements USDA- Wildlife Services USDA- Wildlife Services University of Georgia University of Georgia Auburn University Auburn University Tall Timbers Research Station Tall Timbers Research Station Valdosta State University Valdosta State University

Questions?