• 1.

    Centers for Disease Control and Prevention, 2007. Health Information for International Travel 2008. Atlanta, GA: US Department of Health and Human Services, Public Health Service.

    • Search Google Scholar
    • Export Citation
  • 2.

    TDR, 2005. Chagas' Disease. Tropical Disease Research: Progress 2003–2004. Seventeenth Programme Report of the United Nations Childrens' Fund/United Nations Devlopment Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, 3133.

    • Search Google Scholar
    • Export Citation
  • 3.

    AABB, 2009. (Website Reference [101]) AABB: AABB Chagas' Biovigilance Network. Available at: www.aabb.org/Content/Programs_and_Services/Data_Center/Chagas/. Accessed October 25, 2009.

    • Search Google Scholar
    • Export Citation
  • 4.

    Kjos SA, Snowded KF, Craig TM, Lewis B, Ronald N, Olson JK, 2008. Distribution and characterization of canine Chagas disease in Texas. Vet Parasitol 152: 249256.

    • Search Google Scholar
    • Export Citation
  • 5.

    Williams JT, Dick EJ Jr, VandeBerg JL, Hubbard GB, 2009. Natural Chagas disease in four baboons. J Med Primatol 38: 107113.

  • 6.

    Barr SC, Brown CC, Dennis VA, Klei TR, 1991. The lesions and prevalence of Trypanosoma cruzi in opossums and armadillos from southern Louisiana. J Parasitol 77: 624627.

    • Search Google Scholar
    • Export Citation
  • 7.

    Brown EL, Roellig DM, Gomper ME, Monello RJ, Wenning KM, Gabriel MW, Yabsley MJ, 2009. Seroprevalence of Trypanosoma cruzi among twelve potential reservoir species from six states. Vector Borne Zoonotic Dis [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 8.

    Clark CG, Pung OJ, 1994. Host specificity of ribosomal DNA variation in sylvatic Trypanosoma cruzi from North America. Mol Biochem Parasitol 66: 175179.

    • Search Google Scholar
    • Export Citation
  • 9.

    Barnabé C, Yaeger R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99: 7379.

    • Search Google Scholar
    • Export Citation
  • 10.

    Roellig DM, Brown EL, Barnabé C, Tibayrenc M, Steurer FJ, Yabsley MJ, 2008. Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14: 11231125.

    • Search Google Scholar
    • Export Citation
  • 11.

    Bértoli M, Andó MH, de Ornelas Toledo MJ, de Araújo SM, Gomes ML, 2006. Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts. Parasitol Res 99: 713.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lisboa CV, Pinho AP, Monteiro RV, Jansen AM, 2007. Trypanosoma cruzi (kinetoplastida Trypanosomatidae): biological heterogeneity in the isolates derived from wild hosts. Exp Parasitol 116: 150155.

    • Search Google Scholar
    • Export Citation
  • 13.

    Wood SF, 1941. New localities for Trypanosoma cruzi Chagas in southwestern United States. Am J Trop Med Hyg 34: 113.

  • 14.

    Packchanian A, 1942. Reservoir hosts of Chagas' disease in the state of Texas: natural infection of nine-banded armadillo (Dasypus novemcinctus texanus), house mouse (Mus musculus), opossum (Didelphis virginiana), and wood rats (Neotoma micropus micropus), with Trypanosoma cruzi in the states of Texas. Am J Trop Med Hyg 22 (Suppl 1): 623631.

    • Search Google Scholar
    • Export Citation
  • 15.

    Walton BC, Bauman PM, Diamond LS, Herman CM, 1958. The isolation and identification of Trypanosoma cruzi from raccoons in Maryland. Am J Trop Med Hyg 7: 603610.

    • Search Google Scholar
    • Export Citation
  • 16.

    Olsen PF, Shoemaker JP, Turner HF, Hays KL, 1964. Incidence of Trypanosoma cruzi (Chagas) in wild vectors and reservoirs in east-central Alabama. J Parasitol 50: 599603.

    • Search Google Scholar
    • Export Citation
  • 17.

    Wood SF, 1975. Trypanosoma cruzi: new foci of enzootic Chagas' disease in California. Exp Parasitol 38: 153160.

  • 18.

    John DT, Hoppe KL, 1986. Trypanosoma cruzi from wild raccoons in Oklahoma. Am J Vet Res 47: 10561059.

  • 19.

    Barr SC, Brown CC, Dennis VA, Klei TR, 1990. Infections of inbred mice with three Trypanosoma cruzi isolates from Louisiana mammals. J Parasitol 76: 918921.

    • Search Google Scholar
    • Export Citation
  • 20.

    Pietrzak SM, Pung OJ, 1998. Trypanosomiasis in raccoons from Georgia. J Wildl Dis 34: 132136.

  • 21.

    Karsten V, Davis C, Kuhn R, 1992. Trypanosoma cruzi in wild raccoons and opossums in North Carolina. J Parasitol 78: 547549.

  • 22.

    Roellig DM, Ellis AE, Yabsley MJ, 2009. Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana). Int J Parasitol 39: 16031610.

    • Search Google Scholar
    • Export Citation
  • 23.

    Brisse S, Verhoef J, Tibayrenc M, 2001. Characterisation of large and small subunit rRNA and min-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31: 12181226.

    • Search Google Scholar
    • Export Citation
  • 24.

    Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B, 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83: 141152.

    • Search Google Scholar
    • Export Citation
  • 25.

    Castellani O, Ribeiro LV, Fernandes JF, 1967. Differentiation of Trypanosoma cruzi in culture. J Protozool 14: 447451.

  • 26.

    Yeo M, Acosta N, Llewellyn M, Sánchez H, Adamson S, Miles GAJ, López E, Gonzáles N, Patterson JS, Gaunt MW, de Arias AR, Miles MA, 2005. Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillo hosts of Trypanosoma cruzi II, including hybrids. Int J Parasitol 35: 225233.

    • Search Google Scholar
    • Export Citation
  • 27.

    Barnabé C, Yaegar R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99: 7379.

    • Search Google Scholar
    • Export Citation
  • 28.

    Wood SF, 1952. Mammal blood parasite records from Southwestern United States and Mexico. J Parasitol 38: 8586.

  • 29.

    Ritter DM, Rowland EC, 1984. Corpus Christi strain-induced protection to Trypanosoma cruzi infection in C3H(He) mice: effective dose, time, route, and number of vaccinations. J Parasitol 70: 755759.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Infectivity, Pathogenicity, and Virulence of Trypanosoma cruzi Isolates from Sylvatic Animals and Vectors, and Domestic Dogs from the United States in ICR Strain Mice and SD Strain Rats

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  • Department of Infectious Diseases, College of Veterinary Medicine, Southeastern Cooperative Wildlife Disease Study, Department of Population Health, and D. B. Warnell School of Forestry and Natural Resources, The University of Georgia, Athens, Georgia

Trypanosoma cruzi, the causative agent of Chagas disease, is widespread in the southern United States. In addition to detection in numerous wildlife host species, cases have been diagnosed in domestic dogs and humans. In the current investigation, groups of laboratory mice [Crl:CD1 (ICR)] were inoculated with one of 18 United States T. cruzi isolates obtained from a wide host range to elucidate their infectivity, pathogenicity, and virulence. In addition, laboratory rats (SD strain) were inoculated with four isolates. Mice and rats were susceptible to infection with all strains, but no morbidity or mortality was noted, which indicates that these T. cruzi isolates from the United States had low virulence for laboratory mice and rats.

Trypanosoma cruzi, the causative agent of Chagas disease, infects approximately 10–12 million persons in the Americas; there are approximately 200,000 new cases annually.1,2 In the United States, only six autochthonously acquired human infections have been reported. However, > 1,000 seropositive persons have been detected during routine screening of blood donations in the United States since 2007.3 Although few autochthonous human cases in the United States have been reported, reports of domestic dog and captive exotic animal cases are increasing,4,5 and the prevalence of T. cruzi in wild mammal reservoir species can be as high as in South America.6,7 Trypanosoma cruzi is currently categorized into one of six discrete typing units (TcI, TcIIa, TcIIb, TcIIc, TcIId, TcIIe). To date, all isolates from humans, vectors, wild mammals, domestic animals, and non-human primates in the United States have been classified as TcI or TcIIa.810

Identifying the genotype of a T. cruzi strain is often important for characterizing biological differences among isolates, such as virulence, pathogenicity, tissue tropism, geographic locality, and host/reservoir capacity. Previous mouse infection studies using several sylvatic- and domestic-derived isolates from Brazil showed that those from marsupials were generally more infective and generated higher parasitemias than those from vectors or placental mammals.11,12 Patent infections were also more frequent in laboratory mice inoculated with TcII strains than TcI strains.12 In contrast, U.S. isolates rarely cause morbidity and mortality in laboratory rodents,1320 but in one study, a T. cruzi isolate from a raccoon caused hind limb paralysis in mice.21

Differences in infection outcome in these studies may be caused by different mouse strains, T. cruzi inoculum stage, inoculation route and/or dose, and source (host) species of the isolate. Additionally, many studies were conducted with genetically unclassified strains. The goal of the current study was to experimentally infect laboratory rodents with genetically classified T. cruzi isolates from the United States from a wide host range to determine infectivity, pathogenicity, and virulence. Based on previous studies on strains from the United States,1320 we hypothesized that sylvatic isolates would be infective but not virulent to mice.

A total of 18 T. cruzi isolates from seven mammalian host species and two vector species was used in the study (Tables 1 and 2). These isolates were chosen to represent both genotypes (TcI and TcIIa) present in the United States and a diverse geographic and host range. Two isolates from Brazil (Y and Brazil strains) were used as positive controls (kindly provided by Dr. Rick Tarleton, University of Georgia, Athens, GA). Parasites stored in liquid nitrogen (first passage for all but the two Brazil strains) were rapidly thawed and established in DH82 canine macrophage monolayers to yield the infective culture–derived trypomastigotes.22

Table 1

Detection of Trypanosoma cruzi in eight acutely-infected and one chronically-infected Crl:CD1 (ICR) mice*

IsolateHostOriginLineageNo. PCR-positive acute-stage mice (+, positive chronic mouse)No. hemoculture positive
BloodHeartQuadriceps muscle
FL Opo 18Didelphis virginianaWakulla County, FLI4 (+)171
FL Opo 3D. virginianaWakulla County, FLI526 (+)1
USA OpossumD. virginianaOrleans Parish, LAI6 (+)5 (+)7 (+)0
GA Opo 75D. virginianaClarke County, GAI67 (+)78
GA Opo 43D. virginianaChatham County, GAI2001
TxTg2Triatoma gerstackeriTXI53 (+)7 (+)0
Florida C16T. sanguisugaAlacua County, FLI506 (+)3
TX WR 22Neotoma micropusUvalde County, TXI5764
TX WR 30N. micropusUvalde County, TXIIa51 (+)6 (+)1
FL Rac 9Procyon lotorLiberty County, FLIIa2431
TX08 Rac 5P. lotorUvalde County, TXIIa52 (+)7 (+)2
FL Rac 13P. lotorLeon County, FLI/IIa52 (+)7 (+)2
OK DogCanis familiarisOsage and Washington Counties, OKIIa3571
Griffin DogC. familiarisCoffee County, TNI/IIa618 (+)4
Clarence RTLLemur cattaLiberty County, GAIIa2230
RTL MegL. cattaLiberty County, GAIIa303 (+)2
GA Sk 1Mephitis mephitisLong County, GAIIa4062
GA Arm 20Dasypus novemcinctusChatham County, GAIIa3140
BrazilHumanBrazilI888 (+)5
YHumanBrazilIIb6882

PCR = polymerase chain reaction.

Unless noted, lineage was determined according to methods of Roellig and others.10

Lineage was determined according to methods of Brisse and others.23

Table 2

Detection of Trypanosoma cruzi in three acutely infected and one chronically infected white Crl:CD (SD) laboratory rats*

IsolateHostOriginLineageNo. PCR-positive acute stage rats (+, positive chronic rat)No. hemoculture positive
BloodHeartQuadriceps muscle
USA OpossumD. virginianaOrleans Parish, LAI3 (+)322
Florida C16T. sanguisugaAlacua County, FLI32 (+)3 (+)2
TX WR 30N. micropusUvalde County, TXIIa0001
GA Sk 1Mephitis mephitisLong County, GAIIa0021

PCR = polymerase chain reaction.

Unless noted, lineage was determined according to methods of Roellig and others.10

Lineage was determined according to methods of Brisse and others.23

One hundred eighty-two outbred, eight-week-old male Crl:CD1 (ICR) mice and 16 white Crl:CD (SD) laboratory rats (Charles River Laboratory International, Inc, Wilmington, MA) were housed in microisolator cages in climate-controlled animal facilities at the College of Veterinary Medicine, University of Georgia (Athens, GA). All methods were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Georgia. Mice were weighed and randomly separated into one of 21 groups (18 United States isolate groups, two positive control groups, and one negative control group). Rats were weighed and separated into one of five groups (four United States isolates groups and one negative control). Individuals animals (nine mice and four rats) from each experimental and positive control group were inoculated intraperitoneally with 1 × 106 culture-derived trypomastigotes of one of the representative isolates (Tables 1 and 2). Two negative controls were similarly inoculated with an equivalent volume of culture medium. Any physical or behavioral changes indicative of Chagas clinical signs, such as lethargy, hind limb paralysis, weight loss, or ruffed coat, were noted daily.

At days 3, 7, 10, 14, 17, 21, 24, 28, and 112 post-inoculation (DPI), one mouse from each experimental and positive control group was humanely killed. At days 3, 7, 28, and 112 DPI, one rat from each experimental group was humanely killed. Acutely infected animals were those killed between 3 and 28 DPI; chronically infected animals were killed at 112 DPI. After killing, approximately 1–1.5 mL of whole blood from mice and 3 mL of whole blood from rats were collected by cardiocentesis into tubes containing EDTA.

Infection was determined by detecting T. cruzi DNA by polymerase chain reaction (PCR) or culture of blood. For PCR, DNA was extracted from 100 μL of whole blood, and sections of heart and quadriceps muscle were obtained at necropsy. Amplification of the 24Sα ribosomal DNA gene of T. cruzi using a modified nested reaction was performed as described.2224 For culture, remaining whole blood was centrifuged at 1,620 × g for 15 minutes. Plasma was removed and approximately 3 mL of liver infusion tryptose medium was added to the remaining buffy coat and erythrocytes.25 Cultures were evaluated 2–4 months later.

A t-test was performed to compare mean number of positive animals between genotype groups for each observed variable (P < 0.05 was considered statistically significant), excluding positive controls and results from the FL Rac 13 and Griffin Dog. A t-test was also performed to detect mean differences between heart and quadriceps muscle PCR results to determine whether tissue predilection exists (P < 0.05).

Based on PCR and culture results, all 18 isolates from the United States caused patent infections in mice (Table 1). Results from the first eight bleeding periods (3–28 DPI) were combined as a measure of infection status during the acute stage. In the groups of isolates from the United States, at least one sample from at least one animal was acutely positive either by hemoculture or PCR. Mice infected with the strain from Brazil caused infections in each mouse at all time points. Blood samples taken from acute-stage mice infected with TcI from the United States were PCR positive at a significantly higher rate than those infected with TcII isolates (F = 4.9532, P = 0.043). Additionally, significant differences in detecting T. cruzi DNA present in tissue (heart and/or quadriceps) between genotype were noted. More mice infected with TcI isolates from the United States were T. cruzi positive in all tissues compared with TcIIa-inoculated mice (F = 5.1317, P = 0.0399). However, no difference was noted in tissue predilection (heart versus quadriceps muscle) (F = 1.5217, P = 0.2377).

Two TcI isolates from Virginia opossums (GA Opo 75 and GA Opo 43) yielded dramatically different results; many more mice had detectable infections with the GA Opo 75 strain than with the GA Opo 43 strain (blood: t = 2.16, P = 0.0486; heart and quadriceps: t = 4.24, P = 0.0008). Mortality and weight loss were not observed in mice inoculated with any isolates of T. cruzi from the United States. However, one Y-strain-inoculated mouse had marked weight loss and lethargy and was humanely killed at 28 DPI. Parasites were detected in the blood and tissues of this animal by PCR.

Similarly, all four isolates caused patent infections in laboratory rats (Table 2). Trypanosoma cruzi DNA was detected in the blood and tissues of rats infected with both TcI isolates from the United States at multiple time points, and one rat was positive at 112 DPI (USA Opossum). No rats inoculated with TcIIa isolates had detectable parasitemias by PCR, but an animal from each TcIIa group was culture positive on 3 DPI. Although infection differences were noted between genotypes, small sample sizes and few isolates precluded statistical inferences.

In this study, the two T. cruzi genotypes caused differential infection dynamics in mice and rats as determined by PCR detection of T. cruzi DNA in the blood and tissues. In general, significantly more TcI-inoculated mice had detectable infections than TcIIa-inoculated mice. Additionally, mice and rats inoculated with two TcI isolates (FL Opo 18 and/or USA Opossum) had positive PCR results for blood well into the chronic stage (112 DPI). No TcIIa-inoculated mice or rats were positive beyond the acute stage (28 DPI). These findings suggest that sylvatic TcI isolates from the United States in this study had greater infectivity to laboratory rodents than TcIIa isolates from the United States. Previous studies of T. cruzi isolates from the United States have not investigated infectivity differences based on genotype. However, a study of T. cruzi isolates from Brazil found TcII strains more infective and virulent than TcI strains to Swiss mice.12 The population of T. cruzi from countries in South American is more diverse than T. cruzi from the United States, particularly TcII strains, because all TcII subtypes are found in South America but only TcIIa has been detected in the United States26 (Roellig DM, unpublished data). Additionally, TcIIa strains from the United States are genetically distinct from TCIIa strains from South American at numerous loci27 (Roellig DM, unpublished data). Molecular differences between T. cruzi from South America and the United States may account for biological differences, including infectivity to mice and rats.

Interestingly, T. cruzi isolates from a wildlife rodent reservoir (Neotoma micropus) did not readily infect the laboratory mice or rats. Vectors live within woodrat nests, presumably leading to a continuous transmission cycle where bugs and animals are infected and often reinfected. Therefore, patency would be extended, and T. cruzi infections are easily detected in field samples as in previous surveillance studies.14,28 Additionally, the isolates may be adapted to woodrats and not infective to all rodent species; host adaptation has been suggested.8,10,22,26

We also noted in this study that none of the laboratory rodents inoculated with strains from the United States resulted in observable clinical signs or mortality. These data support previous studies, which reported that sylvatic isolates from the United States were largely avirulent and did not cause morbidity or mortality in rodent models.1320 In contrast, T. cruzi isolates from South America readily infect a wide variety of laboratory mice strains and many cause significant morbidity and mortality.11,12 In the current study, one control mouse inoculated with Y strain displayed lethargy and marked weight loss. Although no clinical signs were observed in mice inoculated with the strain from Brazil in this study, this strain has previously been shown to cause disease and mortality.29 A previous study reported mortality in three of four C3H mice inoculated with a T. cruzi isolate from a raccoon in North Carolina; these mice were not parasitemic but amastigotes were observed in muscle tissue.21 Furthermore, natural infections of captive baboons in Texas have resulted in mortality, indicating that some strains of T. cruzi from the United States can cause disease and death.5 These data suggest that the biological characteristics of T. cruzi isolates from the United States may vary considerably.

Acknowledgments:

We thank B. Shock, E. L. Blizzard, and E. Gleim for laboratory assistance and the Animal Resource staff at The University of Georgia College of Veterinary Medicine for assistance with mouse care.

  • 1.

    Centers for Disease Control and Prevention, 2007. Health Information for International Travel 2008. Atlanta, GA: US Department of Health and Human Services, Public Health Service.

    • Search Google Scholar
    • Export Citation
  • 2.

    TDR, 2005. Chagas' Disease. Tropical Disease Research: Progress 2003–2004. Seventeenth Programme Report of the United Nations Childrens' Fund/United Nations Devlopment Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases, 3133.

    • Search Google Scholar
    • Export Citation
  • 3.

    AABB, 2009. (Website Reference [101]) AABB: AABB Chagas' Biovigilance Network. Available at: www.aabb.org/Content/Programs_and_Services/Data_Center/Chagas/. Accessed October 25, 2009.

    • Search Google Scholar
    • Export Citation
  • 4.

    Kjos SA, Snowded KF, Craig TM, Lewis B, Ronald N, Olson JK, 2008. Distribution and characterization of canine Chagas disease in Texas. Vet Parasitol 152: 249256.

    • Search Google Scholar
    • Export Citation
  • 5.

    Williams JT, Dick EJ Jr, VandeBerg JL, Hubbard GB, 2009. Natural Chagas disease in four baboons. J Med Primatol 38: 107113.

  • 6.

    Barr SC, Brown CC, Dennis VA, Klei TR, 1991. The lesions and prevalence of Trypanosoma cruzi in opossums and armadillos from southern Louisiana. J Parasitol 77: 624627.

    • Search Google Scholar
    • Export Citation
  • 7.

    Brown EL, Roellig DM, Gomper ME, Monello RJ, Wenning KM, Gabriel MW, Yabsley MJ, 2009. Seroprevalence of Trypanosoma cruzi among twelve potential reservoir species from six states. Vector Borne Zoonotic Dis [Epub ahead of print].

    • Search Google Scholar
    • Export Citation
  • 8.

    Clark CG, Pung OJ, 1994. Host specificity of ribosomal DNA variation in sylvatic Trypanosoma cruzi from North America. Mol Biochem Parasitol 66: 175179.

    • Search Google Scholar
    • Export Citation
  • 9.

    Barnabé C, Yaeger R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99: 7379.

    • Search Google Scholar
    • Export Citation
  • 10.

    Roellig DM, Brown EL, Barnabé C, Tibayrenc M, Steurer FJ, Yabsley MJ, 2008. Molecular typing of Trypanosoma cruzi isolates, United States. Emerg Infect Dis 14: 11231125.

    • Search Google Scholar
    • Export Citation
  • 11.

    Bértoli M, Andó MH, de Ornelas Toledo MJ, de Araújo SM, Gomes ML, 2006. Infectivity for mice of Trypanosoma cruzi I and II strains isolated from different hosts. Parasitol Res 99: 713.

    • Search Google Scholar
    • Export Citation
  • 12.

    Lisboa CV, Pinho AP, Monteiro RV, Jansen AM, 2007. Trypanosoma cruzi (kinetoplastida Trypanosomatidae): biological heterogeneity in the isolates derived from wild hosts. Exp Parasitol 116: 150155.

    • Search Google Scholar
    • Export Citation
  • 13.

    Wood SF, 1941. New localities for Trypanosoma cruzi Chagas in southwestern United States. Am J Trop Med Hyg 34: 113.

  • 14.

    Packchanian A, 1942. Reservoir hosts of Chagas' disease in the state of Texas: natural infection of nine-banded armadillo (Dasypus novemcinctus texanus), house mouse (Mus musculus), opossum (Didelphis virginiana), and wood rats (Neotoma micropus micropus), with Trypanosoma cruzi in the states of Texas. Am J Trop Med Hyg 22 (Suppl 1): 623631.

    • Search Google Scholar
    • Export Citation
  • 15.

    Walton BC, Bauman PM, Diamond LS, Herman CM, 1958. The isolation and identification of Trypanosoma cruzi from raccoons in Maryland. Am J Trop Med Hyg 7: 603610.

    • Search Google Scholar
    • Export Citation
  • 16.

    Olsen PF, Shoemaker JP, Turner HF, Hays KL, 1964. Incidence of Trypanosoma cruzi (Chagas) in wild vectors and reservoirs in east-central Alabama. J Parasitol 50: 599603.

    • Search Google Scholar
    • Export Citation
  • 17.

    Wood SF, 1975. Trypanosoma cruzi: new foci of enzootic Chagas' disease in California. Exp Parasitol 38: 153160.

  • 18.

    John DT, Hoppe KL, 1986. Trypanosoma cruzi from wild raccoons in Oklahoma. Am J Vet Res 47: 10561059.

  • 19.

    Barr SC, Brown CC, Dennis VA, Klei TR, 1990. Infections of inbred mice with three Trypanosoma cruzi isolates from Louisiana mammals. J Parasitol 76: 918921.

    • Search Google Scholar
    • Export Citation
  • 20.

    Pietrzak SM, Pung OJ, 1998. Trypanosomiasis in raccoons from Georgia. J Wildl Dis 34: 132136.

  • 21.

    Karsten V, Davis C, Kuhn R, 1992. Trypanosoma cruzi in wild raccoons and opossums in North Carolina. J Parasitol 78: 547549.

  • 22.

    Roellig DM, Ellis AE, Yabsley MJ, 2009. Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana). Int J Parasitol 39: 16031610.

    • Search Google Scholar
    • Export Citation
  • 23.

    Brisse S, Verhoef J, Tibayrenc M, 2001. Characterisation of large and small subunit rRNA and min-exon genes further supports the distinction of six Trypanosoma cruzi lineages. Int J Parasitol 31: 12181226.

    • Search Google Scholar
    • Export Citation
  • 24.

    Souto RP, Fernandes O, Macedo AM, Campbell DA, Zingales B, 1996. DNA markers define two major phylogenetic lineages of Trypanosoma cruzi. Mol Biochem Parasitol 83: 141152.

    • Search Google Scholar
    • Export Citation
  • 25.

    Castellani O, Ribeiro LV, Fernandes JF, 1967. Differentiation of Trypanosoma cruzi in culture. J Protozool 14: 447451.

  • 26.

    Yeo M, Acosta N, Llewellyn M, Sánchez H, Adamson S, Miles GAJ, López E, Gonzáles N, Patterson JS, Gaunt MW, de Arias AR, Miles MA, 2005. Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillo hosts of Trypanosoma cruzi II, including hybrids. Int J Parasitol 35: 225233.

    • Search Google Scholar
    • Export Citation
  • 27.

    Barnabé C, Yaegar R, Pung O, Tibayrenc M, 2001. Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States. Exp Parasitol 99: 7379.

    • Search Google Scholar
    • Export Citation
  • 28.

    Wood SF, 1952. Mammal blood parasite records from Southwestern United States and Mexico. J Parasitol 38: 8586.

  • 29.

    Ritter DM, Rowland EC, 1984. Corpus Christi strain-induced protection to Trypanosoma cruzi infection in C3H(He) mice: effective dose, time, route, and number of vaccinations. J Parasitol 70: 755759.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Dawn M. Roellig, College of Veterinary Medicine, The University of Georgia, 589 D.W. Brooks Drive, Wildlife Health Building, Athens, GA 30602. E-mail: droellig@uga.edu

Financial support: This study was primarily supported by the National Institutes of Health grant R15 AI067304. Additional support was through funding provided to the Southeastern Cooperative Wildlife Disease Study by the Federal Aid to Wildlife Restoration Act (50 Stat. 917) and through sponsorship of the fish and wildlife agencies of Alabama, Arkansas, Florida, Georgia, Kansas, Kentucky, Louisiana, Maryland, Mississippi, Missouri, North Caroling, Oklahoma, Puerto Rico, South Carolina, Tennessee, Virginia, and West Virginia.

Authors' addresses: Dawn M. Roellig and Michael J. Yabsley, College of Veterinary Medicine, The University of Georgia, Wildlife Health Building, Athens, GA, E-mails: droellig@uga.edu and myabsley@uga.edu.

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