Trypanosoma cruzi in wild raccoons and opossums in North Carolina.

Trypanosoma cruzi was isolated from 1 of 12 (8.3%) opossums and 3 of 20 (15%) raccoons from the piedmont area of North Carolina. Although T. cruzi has been isolated previously from wild mammals in the southern United States, the present study is the first published report of naturally occurring T. cruzi infection of wild mammals in North Carolina. All 4 isolates were maintained successfully in axenic culture and in murine fibroblasts. In addition, intraperitoneal injection of 1 x 10(6) culture forms of 1 of the opossum isolates into C3H mice resulted in low but detectable parasitemias as early as day 6 of infection. These mice resolved parasitemia and survived infection. Intraperitoneal injection of 1 x 10(6) culture forms of a raccoon isolate resulted in the death of 3 out of 4 mice. Surprisingly, parasitemias were never detected in the peripheral blood of these mice. Infection of murine fibroblasts in vitro resulted in the presence of intracellular amastigote stages characteristic of T. cruzi.     

Prevalence of antibodies to Trypanosoma cruzi, Toxoplasma gondii, Encephalitozoon cuniculi, Sarcocystis neurona, Besnoitia darlingi, and Neospora caninum in North American opossums, Didelphis virginiana, from southern Louisiana

We examined the prevalence of antibodies to zoonotic protozoan parasites ( Trypanosoma cruzi, Toxoplasma gondii, and Encephalitozoon cuniculi) and protozoans of veterinary importance ( Neospora caninum, Sarcocystis neurona, and Besnoitia darlingi) in a population of North American opossums ( Didelphis virginiana) from Louisiana. Samples from 30 opossums were collected as part of a survey for T. cruzi in Louisiana. Frozen sera from these 30 opossums were examined using an indirect immunofluorescent antibody test (IFAT) against in vitro-produced antigenic stages of these protozoans. Additionally, 24 of the 30 samples were examined using hemoculture, and all 30 were examined in the modified direct agglutination test (MAT) for antibodies to To. gondii. The prevalences of reactive IFAT samples were as follows: 60% for T. cruzi, 27% for To. gondii, 23% for E. cuniculi, 17% for S. neurona, 47% for B. darlingi, and 0% for N. caninum. Hemoculture revealed that 16 (67%) of 24 samples were positive for T. cruzi, compared to 18 of 30 (60%) by IFAT. The sensitivity and specificity for the IFAT compared to hemoculture was 100% for each. The modified direct agglutination test revealed that 9 (30%) of the 30 samples from opossums had antibodies to To. gondii , compared to 8 (27%) using the IFAT. The sensitivity and specificity of the IFAT compared to the MAT was 100% and 72%, respectively.     

Infections of inbred mice with three Trypanosoma cruzi isolates from Louisiana mammals

Trypanosoma cruzi isolates from a dog (Tc-D), opossum (Tc-O), and an armadillo (Tc-A) from southern Louisiana were inoculated into 6 inbred mouse strains. None of the isolates produced fatal infections in the mouse strains tested. Parasitemias were quantified over 34 days and found to be similar in mouse strains infected with Tc-O and Tc-A. Parasitemias in Tc-D-infected mice were detectable only by blood culture. Pseudocyst numbers, inflammatory changes, and weight changes were quantified in CF1 mice infected with the 3 isolates. Tc-O- and Tc-A-infected CF1 mice were shown to be myotropic and produced comparable weight increases, pseudocyst numbers, and inflammatory changes in similar tissues. Tissues found to contain pseudocysts were muscles of the bladder, abdominal wall, thigh, heart, and diaphragm. Clinical signs of infection, pseudocyst numbers, and inflammatory changes were minimal in Tc-D-infected CF1 mice. Tissue tropism of this isolate was not determined. The in vivo infectivity characteristics of these isolates suggests that Tc-O and Tc-A are similar and differ markedly from Tc-D.     

Origins of Chagas disease: Didelphis species are natural hosts of Trypanosoma cruzi I and armadillos hosts of Trypanosoma cruzi II, including hybrids

Trypanosoma cruzi, the causative agent of Chagas disease, has at least two principal intraspecific subdivisions, T. cruzi I (TCI) and T. cruzi II (TCII), the latter containing up to five subgroups (a-e). Whilst it is known that TCI predominates from the Amazon basin northwards and TCII to the South, where the disease is considered to be clinically more severe, the precise clinical and evolutionary significance of these divisions remains enigmatic. Here, we present compelling evidence of an association between TCI and opossums (Didelphis), and TCII and armadillos, on the basis of key new findings from the Paraguayan Chaco region, together with a comprehensive analysis of historical data. We suggest that the distinct arboreal and terrestrial ecologies, respectively, of these mammal hosts provide a persuasive explanation for the extant T. cruzi intraspecific diversity in South America, and for separate origins of Chagas disease in northern South America and in the southern cone countries.     

Trypanosomes of the subgenus Megatrypanum from armadillos (Xenarthra:Dasypodidae)

A new species of trypanosome, Trypanosoma (Megatrypanum) peba, is described from the peripheral blood of the armadillo Euphractus sexcinctus setosus from Bahia State, Brazil. Ten out of 29 specimens of the armadillo Dasypus novemcinctus from Pará State were found to have trypanosomes, including epimastigote forms, in impression smears of subcutaneous lymph nodes. The trypanosomes from D. novemcinctus are illustrated and were identified as belonging to the subgenus Megatrypanum on the basis of their general appearance, although they failed to multiply in blood-agar culture medium and no bloodstream forms were seen. This is the first published record of trypanosomes of this subgenus from armadillos and the first demonstration of epimastigote trypanosomes in the mammalian host other than in the bloodstream, or in the anal glands of opossums.     

The opossum Didelphis virginiana as a synanthropic reservoir of Trypanosoma cruzi in Dzidzilché, Yucatán,México

In México, the role of mammals in the transmission cycle of Trypanosoma cruzi is poorly known. In the State of Yucatán, an endemic area of Chagas disease, both Didelphis virginiana and D. marsupialis occur sympatrically. However, until now, only the former species had been found infected with T. cruzi. To evaluate the role of D. virginiana in a peridomestic transmission, nine periods of capture-recapture were performed around the village of Dzidzilché, Yucatán. The sex, age, reproductive status, location, and presence of infection with T. cruzi were recorded for each opossum. The chromosome morphology was used to identify the opossum species. T. cruzi was identified by the presence of pseudocysts of amastigotes in cardiac muscle fibers of Balb/c mice inoculated with strains isolated from opossums. However, xenodiagnosis was the best diagnostic method. Triatoma dimidiata, the vector, were collected in and around the opossums' nests, and human dwellings; and were checked for T. cruzi. From 102 blood samples of D. virginiana examined 55 (53.9%) were positive to T. cruzi, the only two D. marsupialis captured were negative. Significant differences were found between infection, and both sex and reproductive condition. Eight out of 14 triatomines collected in peridomestic nests (57.1%), and 32 of 197 captured inside houses (16.3%) were found infected, suggesting a peridomestic transmission. The statistically high abundance of infected opossums and triatomines during the dry season (March to May) suggested the existence of a seasonality in the peridomestic transmission of T. cruzi in Dzidzilché.     

Trypanosoma cruzi infection in Didelphis marsupialis in Santa Catarina and Arvoredo Islands, southern Brazil

Between 1984 and 1993 the prevalence of the Trypanosoma cruzi infection in opossums (Didelphis marsupialis) was studied in Santa Catarina and Arvoredo Islands, State of Santa Catarina, Brazil. The association of the triatomine bug Panstrongylus megistus with opossums nests and the infection rate of these triatomines by T. cruzi was also studied. Thirteen different locations were studied in Santa Catarina Island (SCI), in which 137 D. marsupialis were collected. Sixty two opossums were collected at the Arvoredo Island (AI), located 12 miles north from SCI. All captured animals were submitted to parasitological examinations that revealed the presence of T. cruzi in 21.9% of the opossums captured in SCI and 45.2% among opossums captured in the AI. The presence of P. megistus was detected in most of the D. marsupialis nests collected in the SCI, however, in the non-inhabited AI only eight triatomines were collected during the whole study. The presence of T. cruzi-infected D. marsupialis associated with P. megistus in human dwellings in the SCI, and the high infection rate of D. marsupilais by T. cruzi in the absence of a high vector density are discussed.     

Seroprevalence of Trypanosoma cruzi in raccoons from South Carolina and Georgia

Trypanosoma cruzi, the causative agent of American trypanosomiasis or Chagas' disease, is of both medical and veterinary importance as is evidenced by chronic phase myocarditis in humans and dogs. Further, T. cruzi has been reported from over 20 species of wildlife reservoir hosts in the USA, with raccoons (Procyon lotor) and opossums (Didelphis virginiana) being the most common. Whereas previous studies on T. cruzi in raccoons have included only culture and direct examination of blood, the indirect immunofluorescent antibody test (IFAT) was used in the current study to detect anti-T. cruzi antibodies in the serum of raccoons. Of 221 raccoons trapped at 13 sites representing the five physiographic regions of South Carolina plus five sites in the Piedmont region of Georgia (from April 1997 to February 2000), 104 (47%) were seropositive. A higher seroprevalence in raccoons was observed in the coastal regions, with seroprevalence in the Lower Coastal Plain South (61%) being significantly higher than that in the Foothills (37%), Piedmont (42%), and Upper Coastal Plain (40%) regions. However, at a seroprevalence of 52%, the Lower Coastal Plain North was not significantly different from any other region. Although more female raccoons were infected than males, no statistical difference in prevalence was observed between sexes. The high seroprevalence of T. cruzi in raccoons, together with a few reports of wildlife isolates being infective for other wildlife species and domestic/laboratory animals, suggests that risk of T. cruzi infection may be higher than previously suspected.     

The importance of the opossum (Didelphis albiventris) as a reservoir for Trypanosoma cruzi in Bambuí, Minas Gerais State.

In a survey realized on the sylvatic and peridomestic environments at Bambuí county, Minas Gerais State, 44 (37.9%) out of 116 opossums (Didelphis albiventris) captured were found to be naturally infected with Trypanosoma cruzi. One hundred and forty three parasite samples were obtained from 43 infected opossums using simultaneously hemoculture, xenodiagnosis (Triatoma infestans, Panstrongylus megistus and Rhodnius neglectus) and examination of anal glands contents. The parasite samples were characterized according to six isoenzyme patterns. All samples, independently of the method of isolation, presented an isoenzyme pattern similar to the standard T. cruzi Z1, showing that either xenodiagnosis or hemoculture can used without selecting parasite subpopulation from naturally infected opossums. Previous isoenzyme patterns reported for human T. cruzi isolates from the same region were completely different. This isoenzyme dissimilarity between sylvatic and domiciliar environments suggests the existence of two independent T. cruzi transmission cycles in Bambuí. The epidemiological implications of these results are discussed.     

Lack of Sarcocystis neurona antibody response in Virginia opossums (Didelphis virginiana) fed Sarcocystis neurona-infected muscle tissue

Serum was collected from laboratory-reared Virginia opossums (Didelphis virginiana) to determine whether experimentally infected opossums shedding Sarcocystis neurona sporocysts develop serum antibodies to S. neurona merozoite antigens. Three opossums were fed muscles from nine-banded armadillos (Dasypus novemcinctus), and 5 were fed muscles from striped skunks (Mephitis mephitis). Serum was also collected from 26 automobile-killed opossums to determine whether antibodies to S. neurona were present in these opossums. Serum was analyzed using the S. neurona direct agglutination test (SAT). The SAT was modified for use with a filter paper collection system. Antibodies to S. neurona were not detected in any of the serum samples from opossums, indicating that infection in the opossum is localized in the small intestine. Antibodies to S. neurona were detected in filter-paper-processed serum samples from 2 armadillos naturally infected with S. neurona.     

Trypanosoma cruzi: blood parasitism kinetics and their correlation with heart parasitism intensity during long-term infection of Beagle dogs

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.     

Comparison of serological methods and blood culture for detection of Trypanosoma cruzi infection in raccoons (Procyon lotor)

The indirect immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were compared with blood culture for the detection of Trypanosoma cruzi infection in 83 raccoons (Procyon lotor) trapped in 4 counties of southeast Georgia. Both IFAT and ELISA detected 24 of 25 culture-positive samples (96% sensitivity). Cultures from 25 raccoons (30%) were positive for epimastigotes, whereas a total of 50 raccoons (60%) was seropositive by either the IFAT or ELISA. Forty-five of 83 serum samples (54%) were positive for anti-T. cruzi antibodies with the ELISA, and 47 were IFAT positive (57%). Forty-two of the 50 seropositive raccoons (84%) were seropositive by both tests. Endpoint titers of IFAT-positive samples were determined by testing doubling dilutions from 1:40 to 1:1280. High titers of 640 and 320 were observed for 4 raccoons trapped in 1 county (St. Catherines Island, Liberty County) and titers of 160 for 1-2 raccoons from each of the 4 counties sampled. IFAT titers and ELISA optical density values were positively correlated. Both serological tests have a high sensitivity and should be excellent tools for studying the prevalence of T. cruzi in wildlife populations.     

Surveillance for naturally acquired leprosy in a nine-banded armadillo population

Samples from 77 nine-banded armadillos (Dasypus novemcinctus) inhabiting a 16.7 km portion of the East Atchafalaya River Levee, Point Coupee Parish, Louisiana, were serologically tested and/or histopathologically examined for evidence of naturally acquired leprosy. Five of 67 (7.5%) armadillos tested sero-positive with ELISA test for IGM class antibodies to the phenolic-glycolipid-1 antigen of Mycobacterium leprae. One of 74 (1.3%) was histopathologically positive as determined by presence of acid-fast bacteria in nerves.     

Helminth fauna of the nine-banded armadillo (Dasypus novemcinctus) in north-central Florida

The gastrointestinal tracts of 32 free-ranging, 9-banded armadillos (Dasypus novemcinctus) from north-central Florida were examined for the presence of helminths during July 1991 to November 1993. Aspidodera sp. (Nematoda: Aspidoderidae), most closely resembling Aspidodera sogandaresi, were recovered from 20 of 32 armadillos examined. Total numbers of A. sogandaresi ranged from 1 to 1,021 per infected animal, and followed an inverse correlation to body condition index for those animals. The cystacanth stage of 1 acanthocephalan, Macracanthorhynchus ingens, was present in 1 armadillo, and is the first report of M. ingens in the 9-banded armadillo. The present study contributes to the known natural history of the 9-banded armadillo, an important animal research model.     

Sylvatic focus of American Trypanosomiasis in the State of Morelos, Mexico

Wild vectors and reservoir hosts of Trypanosoma cruzi were surveyed from February 1993 to June 1994 in Ticumán (18 degrees 46'N, 99 degrees 07'W), Mexico (Deciduous Tropical Forest). Direct faeces examination showed that 87% of Triatoma pallidipennis hosted the parasite; T. cruzi forms were present in cultures inoculated with faeces of fifty 67% triatomine bugs and thirty CD-1 strain mice (10 d old) inoculated (peritoneum) with faeces of positive insects T. cruzi amastigotes were found in heart 67%, kidneys 47%, liver 80%, lungs 50%, oesophagus 60%, skin 23%, spleen 73% and stomach 60%. T. cruzi was isolated by direct blood examination from seven 21% chiropterans and five 38% rodents and T. cruzi forms were present in cultures inoculated with blood of twenty-three 68% chiropterans and seven 54% rodents and T. cruzi amastigotes were seen in the kidneys of one 3% chiropterans and four 31% rodents and only in one Pteronotus parnellii mexicanus, organisms were seen in skin 2%. There was no association between organs and T. cruzi infection (p > 0.05).     

Earthworms near leprosy patients' homes are negative for acid-fast bacilli by fite stain,providing no link between leprous armadillos (Dasypus novemcinctus) and human leprosy

Enzootic leprosy has been recognized in armadillos in Louisiana since 1975. Contact with armadillos is being assessed as a risk factor for leprosy in three white women, lifelong residents of separate rural areas in northern Louisiana, which is a region without endemic leprosy. None has had any known exposure to human leprosy. Each was aware of armadillos (Dasypus novemcinctus) near or under her home for decades. In considering Possible environmental sources forMycobacterium leprae, we observed that all three had earthworm growth areas for fishing bait where soil was kept moist near their homes. The worms attracted armadillos. Since armadillos subsist on worms, grubs, and insects and because of the common feature of a worm farm near each home, we reasoned that earthworms might containM. leprae and be part of a cycle involving the armadillo and human beings. Worms from each home worm farm were studied. One site was sampled twice at patient 1's home, five sites were sampled once at patient 2's home, and three sites were sampled once at patient 3's home. A sample consisted of 3–4 worms, which were washed, purged, fixed live in 10% formalin, embedded in paraffin, sectioned, and stained with the Fite stain. Each was sagittally sectioned and examined by three independent observers. No acid-fast bacilli or other acid-fast structures were identified. We conclude that it is unlikely that earthworms are an environmental source or reservoir ofM. leprae.     

Experimental inoculation of North American opossums (Didelphis virginiana) with Mycobacterium bovis

Eight North American opossums (Didelphis virginiana) were inoculated with 1 x 10(5) colony forming units of Mycobacterium bovis to investigate their potential as reservoir hosts for bovine tuberculosis in Michigan. Four animals received this dose orally and four were inoculated intramuscularly (i.m.). In each group, two animals were euthanized 1 mo postinoculation (PI) and two at 2 mo PI. Four control animals were housed separately and sacrificed in the same manner as those inoculated. One of four orally inoculated opossums and three of four i.m.-inoculated opossums were positive for M. bovis by culture of tissues obtained at necropsy. The oral recipient had positive cultures from intestine and pooled lymphoid samples. Pooled lymphoid samples were positive in three i.m.-inoculated animals and two of these also had positive liver and lung cultures. One animal with gross and histologic lesions compatible with tuberculosis had negative tissue cultures. The findings suggest that opossums are susceptible to M. bovis infection by multiple routes, although their relative susceptibility compared to true reservoir hosts appears to be low.     

Trypanosoma cruzi in the opossum Didelphis marsupialis: parasitological and serological follow-up of the acute infection

The opossum Didelphis marsupialis is known to be among the most important wild reservoirs of Trypanosoma cruzi and one in which the trypanosome may go through both the usual vertebrate intracellular cycle in its tissues and an extracellular cycle in the lumen of its scent glands. The species is highly resistant to heavy inocula and, depending on the parasite strain, experimental infections may be permanent or self limited. Aiming to understand the mechanisms involved in this parasite-host interaction we made a study of the acute phase of infection with different T. cruzi strains. Strains F, G-49 and G-327 produced durable infections with relatively high parasitemia and invasion of the scent glands, while equivalent inocula of the Y strain resulted in scanty parasitemia of short duration, no invasion of the SG, and no evidence of persistent parasitism. A smaller inoculum of G-49 produced only subpatent though persistent parasitemia and no invasion of the scent glands. The humoral immune response was less marked in the Y group; among the other groups IgM and IgG antibodies increased to high levels, higher in the G-49 group. The increase in IgG coincided with a drop of parasitemia to subpatent levels. Two opossums inoculated directly in the scent glands with culture forms of the Y strain had a short-lived subpatent parasitemia, but the parasites remained in the glands and serum Ig antibodies reached high levels. Immunoblot analysis showed that the sera of the inoculated opossums recognized few T. cruzi antigens (more in the F strain) in comparison with those of mice. However, with the only exception of those subcutaneously inoculated with the Y strain and including two naturally infected specimens, all the opossum's sera recognized a 90-kDa peptide in all T. cruzi strains. Our results confirm that opossums are able to selectively eliminate some strains of T. cruzi and indicate that the mechanism involved in this selection is probably not related to the humoral immune response. In infections by strains that are able to establish a permanent foothold in opossum tissues, there are indications that IgG antibodies participate in the control of the parasite population of the acute phase but are unable to prevent the chronic phase. It was once more demonstrated that the opossum infected scent glands function as diffusion chambers for parasite antigens but that, on the other hand, the parasites are here protected against the mechanisms developed by the host to control their population.     

The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona

Sarcocysts were dissected from the tongue of a nine-banded armadillo (Dasypus novemcinctus). DNA was extracted and characterised by PCR amplification followed by restriction fragment length polymorphism analysis and nucleotide sequencing. A total of 1879 nucleotides were compared; the sarcocyst DNA sequence was identical to that reported for Sarcocystis neurona. DNA was extracted from the sarcocysts of five more nine-banded armadillos. A 254-nucleotide sequence was determined for each and found to be identical to S. neurona. Western blot techniques for detection of anti-S. neurona antibody were developed for use with armadillo plasma and samples from 19 wild-caught and 17 captive-raised armadillos were examined. Whereas all of the 19 wild-caught armadillos had antibodies to S. neurona, only one of 17 captive-raised armadillos did. These results suggest that the nine-banded armadillo are naturally infected with S. neurona.     

Natural infection of the anal glands of the opossum (Didelphis albiventris) by Trypanosoma cruzi in the municipality of Bambuí--MG, Brazil

Out of 87 opossums, Didelphis albiventris, captured in the Bambuí area (Minas Gerais State), 32 (36.7%) were found infected by Trypanosoma cruzi; the rates varied according to whether the specimens originated from sylvan, rural peridomiciliar or urban surroundings, being 34.9, 81.8 and 7.7 respectively. From 20 of the infected opossums the anal glands were repeatedly examined and found positive in only one (5%) specimen (GA 9), with 7 positive examinations out of 17 performed through an 18-months periods. Material from these glands produced patent parasitemia in opossums and sub-patent infections in mice. Isolates from the opossum GA 9, obtained through xenodiagnoses and hemocultures and from cultures of the infected anal glands fitted into zymodeme Z1.