Dawn M. Roellig

Autochthonous Transmission of Trypanosoma cruzi, Louisiana

Autochthonous transmission of the Chagas disease parasite, Trypanosoma cruzi, was detected in a patient in rural New Orleans, Louisiana. The patient had positive test results from 2 serologic tests and hemoculture. Fifty-six percent of 18 Triatoma sanguisuga collected from the house of the patient were positive for T. cruzi by PCR.

Subtyping Cryptosporidium ubiquitum, a Zoonotic Pathogen Emerging in Humans

Cryptosporidium ubiquitum is an emerging zoonotic pathogen. In the past, it was not possible to identify an association between cases of human and animal infection. We conducted a genomic survey of the species, developed a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene, and identified 6 subtype families (XIIa-XIIf) of C. ubiquitum. Host adaptation was apparent at the gp60 locus; subtype XIIa was found in ruminants worldwide, subtype families XIIb-XIId were found in rodents in the United States, and XIIe and XIIf were found in rodents in the Slovak Republic. Humans in the United States were infected with isolates of subtypes XIIb-XIId, whereas those in other areas were infected primarily with subtype XIIa isolates. In addition, subtype families XIIb and XIId were detected in drinking source water in the United States. Contact with C. ubiquitum-infected sheep and drinking water contaminated by infected wildlife could be sources of human infections.

Seroprevalence of Trypanosoma cruzi Among Eleven Potential Reservoir Species from Six States Across the Southern United States

Trypanosoma cruzi, the causative agent of Chagas disease, is a substantial public health concern in Latin America. Although rare in humans and domestic animals in the United States, T. cruzi is commonly detected in some wildlife species, most commonly raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana). To increase our understanding of the reservoir host species range and geographic distribution, 11 species of mammals from six states spanning the known range of T. cruzi (Arizona, California, Florida, Georgia, Missouri, and Virginia) were tested for antibodies to T. cruzi using indirect immunofluorescent antibody testing. In addition, culture isolation attempts were conducted on a limited number of animals from Georgia and Florida. Evidence of T. cruzi was found in every state except California; however, low numbers of known reservoirs were tested in California. In general, the highest seroprevalence rates were found in raccoons (0-68%) and opossums (17-52%), but antibodies to T. cruzi were also detected in small numbers of striped skunks (Mephitis mephitis) from Arizona and Georgia, bobcats (Lynx rufus) from Georgia, two coyotes (Canis latrans) from Georgia and Virginia, and a ringtail (Bassariscus astutus) from Arizona. Culture-based prevalence rates for raccoons were significantly greater than those for opossums; however, seroprevalences of raccoons and opossums from several geographic locations in Georgia and Florida were not different, indicating that exposure rates of these two species are similar within these areas. For both raccoons and opossums, seroprevalence was significantly higher in females than in males. No difference was detected in seroprevalence between adults and juveniles and between animals caught in urban and rural locations. Our results indicate that T. cruzi prevalence varies by host species, host characteristics, and geographic region and provides data to guide future studies on the natural history of T. cruzi in the United States.

Oral Transmission of Trypanosoma cruzi with Opposing Evidence for the Theory of Carnivory

Abstract We present the first demonstration of oral transmission of Trypanosoma cruzi to raccoons (Procyon lotor), a natural reservoir host in the United States, by ingestion of trypomastigotes and infected bugs, but not infected tissue. To investigate an alternative, non-vector–based transmission method, we tested the hypothesis that raccoons scavenging on infected hosts results in patent infection. Macerated tissue from selected organs infected with amastigote stages of T. cruzi was orally administered to experimental groups of raccoons (n = 2/group) at 2, 12, or 24 hr after collection of the tissue samples. Additionally, raccoons (n = 1) in control groups were inoculated intravenously or per os with trypomastigotes. To further elucidate transmission routes of T. cruzi to raccoons, infected Rhodnius prolixus were fed to raccoons (n = 2). Raccoons did not become infected after ingestion of amastigote-infected tissues as evidenced by negative polymerase chain reaction results from blood and tissue, lack of seroconversion, and negative parasitemias. However, per os transmission can occur by ingestion of the infective trypomastigote stage or infected reduviid bugs. We conclude from these findings that oral transmission of T. cruzi may be a route of infection for wildlife in sylvatic cycles, but the scavenging behavior of animals is not likely a significant transmission route.

Genetic variation and exchange in Trypanosoma cruzi isolates from the United States.

Trypanosoma cruzi, the causative agent of Chagas disease, is a multiclonal parasite with high levels of genetic diversity and broad host and geographic ranges. Molecular characterization of South American isolates of T. cruzi has demonstrated homologous recombination and nuclear hybridization, as well as the presence of 6 main genetic clusters or “discrete typing units” (DTUs). Few studies have extensively investigated such exchange events and genetic diversity in North American isolates. In the current study, we genetically characterized over 50 US isolates from wildlife reservoirs (e.g., raccoons, opossums, armadillos, skunks), domestic dogs, humans, nonhuman primates, and reduviid vectors from nine states (TX, CA, OK, SC, FL, GA, MD, LA, TN) using a multilocus sequencing method. Single nucleotide polymorphisms were identified in sequences of the mismatch-repair class 2 (MSH2) and Tc52 genes. Typing based on the two genes often paralleled genotyping by classic methodologies using mini-exon and 18S and 24Sα rRNA genes. Evidence for genetic exchange was obtained by comparing sequence phylogenies of nuclear and mitochondrial gene targets, dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the cytochrome oxidase subunit II- NADH dehydrogenase subunit I region (COII-ND1), respectively. We observed genetic exchange in several US isolates as demonstrated by incongruent mitochondrial and nuclear genes phylogenies, which confirms a previous finding of a single genetic exchange event in a Florida isolate. The presence of SNPs and evidence of genetic exchange illustrates that strains from the US are genetically diverse, even though only two phylogenetic lineages have been identified in this region.

Genetically different isolates of Trypanosoma cruzi elicit different infection dynamics in raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana).

Trypanosoma cruzi is a genetically and biologically diverse species. In the current study we determined T. cruzi infection dynamics in two common North American reservoirs, Virginia opossums (Didelphis virginiana) and raccoons (Procyon lotor). Based on previous molecular and culture data from naturally-exposed animals, we hypothesised that raccoons would have a longer patent period than opossums, and raccoons would be competent reservoirs for both genotypes T. cruzi I (TcI) and TcIIa, while opossums would only serve as hosts for TcI. Individuals (n=2 or 3) of each species were inoculated with 1x10(6) culture-derived T. cruzi trypomastigotes of TcIIa (North American (NA) - raccoon), TcI (NA - opossum), TcIIb (South American - human), or both TcI and TcIIa. Parasitemias in opossums gradually increased and declined rapidly, whereas parasitemias peaked sooner in raccoons and they maintained relatively high parasitemia for 5weeks. Raccoons became infected with all three T. cruzi strains, while opossums only became infected with TcI and TcIIb. Although opossums were susceptible to TcIIb, infection dynamics were dramatically different compared with TcI. Opossums inoculated with TcIIb seroconverted, but parasitemia duration was short and only detectable by PCR. In addition, raccoons seroconverted sooner (3-7days post inoculation) than opossums (10days post inoculation). These data suggest that infection dynamics of various T. cruzi strains can differ considerably in different wildlife hosts.

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

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.

Experimental infection of two South American reservoirs with four distinct strains of Trypanosoma cruzi.

Trypanosoma cruzi (Tc), the causative agent of Chagas disease, is a diverse species with 2 primary genotypes, TcI and TcII, with TcII further subdivided into 5 subtypes (IIa-e). This study evaluated infection dynamics of 4 genetically and geographically diverse T. cruzi strains in 2 South American reservoirs, degus (Octodon degus) and grey short-tailed opossums (Monodelphis domestica). Based on prior suggestions of a genotype-host association, we hypothesized that degus (placental) would more readily become infected with TcII strains while short-tailed opossums (marsupial) would be a more competent reservoir for a TcI strain. Individuals (n=3) of each species were intraperitoneally inoculated with T. cruzi trypomastigotes of TcIIa [North America (NA)-raccoon (Procyon lotor) origin], TcI [NA-Virginia opossum (Didelphis virginiana)], TcIIb [South America (SA)-human], TcIIe (SA-Triatoma infestans), or both TcI and TcIIa. Parasitaemias in experimentally infected degus peaked earlier (7-14 days post-inoculation (p.i.)) compared with short-tailed opossums (21-84 days p.i.). Additionally, peak parasitaemias were higher in degus; however, the duration of detectable parasitaemias for all strains, except TcIIa, was greater in short-tailed opossums. Infections established in both host species with all genotypes, except for TcIIa, which did not establish a detectable infection in short-tailed opossums. These results indicate that both South American reservoirs support infections with these isolates from North and South America; however, infection dynamics differed with host and parasite strain.

Multilocus Sequence Typing Tool for Cyclospora cayetanensis

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

Detection of Anaplasma phagocytophilum in Ixodid Ticks from Equine-Inhabited Sites in the Southeastern United States

Anaplasma phagocytophilum is a vector-borne, obligate intracellular bacterium that invades the neutrophils and eosinophils of infected individuals, causing granulocytic anaplasmosis. Equine cases have previously been reported in the United States from California, Florida, and Connecticut, but limited surveillance studies in the Southeast have been conducted. The objective of this study was to determine A. phagocytophilum prevalence in Ixodes scapularis ticks at southeastern U.S. horse-inhabited sites to evaluate the potential risk for equine exposure to A. phagocytophilum-infected ticks in these areas. Samples of I. scapularis were collected from selected barrier islands and Georgia mainland sites where feral and domestic equine populations are present, respectively. Ticks were individually tested for infection by amplification of the A. phagocytophilum ankA gene. The collective prevalence of A. phagocytophilum in I. scapularis ticks was 20% (n=808).