ABSTRACT
Trypanosoma cruzi is a vector-borne protozoan parasite that infects seven million individuals in Central and South America and is the etiologic agent of Chagas disease. There are increasing reports of endemic transmission within the southern US. Trypanosoma cruzi occurs in wild raccoons and dogs in Oklahoma, but its endemicity in the state is poorly studied. We suspected Mexican free-tailed bats (Tadarida brasiliensis) contributed to the endemicity of T. cruzi in Oklahoma due to their annual migration from Central America to their North American maternity roosts. During the summer of 2017, we sampled 361 Mexican free-tailed bats for T. cruzi at three maternity roosts in Oklahoma. We collected wing tissues, extracted T. cruzi DNA, amplified target DNA by PCR using the primers TCZ1/TCZ2, and observed amplification by gel electrophoresis. One juvenile Mexican free-tailed bat was positive for T. cruzi resulting in a 0.27% prevalence in the 361 sampled bats. Our finding of a wild bat naturally infected with T. cruzi in Oklahoma provided insight on the endemicity of T. cruzi in underrepresented endemic areas. The positive sample was sequenced, confirmed as T. cruzi, and uploaded to GenBank (no. MG869732). Future research will focus on monitoring T. cruzi prevalence in wild bats and insect vectors to better understand the enzootic emergence of this neglected tropical parasite.
Trypanosoma cruzi is a vector-borne protozoan parasite and the etiologic agent of American trypanosomiasis, commonly known as Chagas disease. Currently, seven million people worldwide are infected with T. cruzi and up to 40% will develop Chagas disease, which is a leading cause of cardiomyopathy in endemic regions (Bocchi et al. 2009). Trypanosoma cruzi infects over 400 mammalian species (Hoare 1972) and is transmitted by an infected triatomine (Hemiptera: Reduviidae: Triatominae) feeding on a host and defecating onto host skin or mucous membranes (Rassi et al. 2010). Triatomine vectors are found from South America to the central US, with two species documented statewide in Oklahoma (Lent and Wygodzinsky 1979).
Historically, the US lacked endemic T. cruzi; however, reports of autochthonous vectorial transmission suggest both endemic and enzootic transmission cycles occur in the southern US (Dorn et al. 2007). The prevalence of enzootic T. cruzi in Oklahoma is characterized by only three reported canine and raccoon infections (Fox et al. 1986; John and Hoppe 1986; Bradley et al. 2000). Given the presence of migratory Mexican free-tailed bat (Tadarida brasiliensis) maternity roosts in Oklahoma, we hypothesized that the Mexican free-tailed bat played a potential epidemiologic role in the endemicity of T. cruzi.
A migratory bat species, Mexican free-tailed bats are found from Argentina to Ohio (Wilkins 1989). During the spring and summer, Oklahoma is home to three million Mexican free-tailed bats who subsequently migrate south along the Sierra Madre Oriental into south-central Mexico for the winter (Fig. 1; Glass 1982). This species is gregarious and aggregates in maternity roosts (Wilkins 1989). Additionally, Mexican free-tailed bats are natural reservoirs for various Trypanosoma species, and Mexican free-tailed bats potentially support the bat-seeding hypothesis, which proposes that migratory bats have a unique epidemiologic role in the expansion of T. cruzi (Pinto et al. 2011; Hamilton et al. 2012; Hodo et al. 2016).
The range of the migratory Mexican free-tailed bat (Tadarida brasiliensis; IUCN 2018). The range spans multiple Trypanosoma cruzi endemic foci. During the fall, the sampled population in Oklahoma, USA migrate south through Texas, USA and into Mexico along the Sierra Madre Oriental before returning in the spring to give birth at maternity roosts (Glass 1982). The sequence obtained from the positive sample in our study, Woodward 1, matched closely to multiple GenBank sequences that were collected from rodents in the Las Palomas Wildlife Management Area (Aleman et al. 2017), which is along the migratory pathway of the bats. The representative T. cruzi lineages we used in phylogenetic analysis are illustrated. Sampling occurred at three maternity roosts in Oklahoma during the summer of 2017
The range of the migratory Mexican free-tailed bat (Tadarida brasiliensis; IUCN 2018). The range spans multiple Trypanosoma cruzi endemic foci. During the fall, the sampled population in Oklahoma, USA migrate south through Texas, USA and into Mexico along the Sierra Madre Oriental before returning in the spring to give birth at maternity roosts (Glass 1982). The sequence obtained from the positive sample in our study, Woodward 1, matched closely to multiple GenBank sequences that were collected from rodents in the Las Palomas Wildlife Management Area (Aleman et al. 2017), which is along the migratory pathway of the bats. The representative T. cruzi lineages we used in phylogenetic analysis are illustrated. Sampling occurred at three maternity roosts in Oklahoma during the summer of 2017
In the summer of 2017, we sampled 361 Mexican free-tailed bats from three maternity caves in Woodward, Woods, and Major counties of Oklahoma. While collecting samples, we followed mandatory white-nose syndrome decontamination protocols (US Fish and Wildlife Service 2016) to reduce the spread of Pseudogymnoascus destructans, and all study methods were approved by the University of Central Oklahoma Institutional Animal Care and Use Committee (IACUC 17004). We used insect sweep nets to catch emerging Mexican free-tailed bats at cave mouths and collected tissues from the uropatagium and plagiopatagium using sterile, 3- mm biopsy punches. We collected tissues in the vascular patagia to sample both intracellular amastigotes and circulating trypomastigotes, which are found in multiple tissues throughout infected hosts (Rassi et al. 2010). Bats were released on site following sample collection. Following the DNeasy Blood and Tissue Kit (Qiagen, Germantown, Maryland, USA) protocol, we placed biopsy punches in labeled, 1.5-mL microcentrifuge tubes containing 180 µL ATL tissue lysis buffer and 20 µL proteinase K and stored the tubes at room temperature.
To increase sample diversity, we divided collection into monthly trips. We expected T. cruzi to be in both adults and juveniles because T. cruzi can be transmitted congenitally (Añez et al. 2009). In May, we sampled 91 pregnant females. In June and July, we sampled 198 lactating females and 8 adult males. In August, we sampled 29 female juveniles and 35 male juveniles. Females constituted 86.5% of the sample size because females congregated at maternity roosts and males roosted separately. The sharp increase of sampled males during August was attributed to the 1:1 ratio of pup gender (Wilkins 1989). We determined age by transillumination of the wing using a headlight to visualize the epiphyseal fusion of the metacarpalphalangeal joint.
Trypanosoma cruzi strain Sylvio X10 (American Type Culture Collection, Manassas, Virginia, USA) target DNA was amplified and cloned into a plasmid using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, California, USA) and transformed into Escherichia coli strain Mach1-T1. We purified plasmid constructs from the transformants using the PureLink HiPure Plasmid Miniprep Kit (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) to serve as a positive control DNA for PCR. We extracted DNA from wing punches using the DNeasy Blood and Tissue Kit (Qiagen) following manufacturer protocols.
We amplified a 195-base pair satellite repeat from T. cruzi nuclear DNA via PCR using the primers TCZ1/TCZ2 (Virreira et al. 2003). The TCZ1/TCZ2 primers amplify nDNA from all T. cruzi lineages and subspecies (Virreira et al. 2003) but do not amplify other Trypanosoma species, including nonpathogenic Trypanosoma rangeli. We followed the protocol of Virreira et al. (2003) for PCR. The PCR amplicons were electrophoresed on a 1% agarose gel with Trisacetate- EDTA buffer in the presence of 0.5 µL/mL of ethidium bromide. Additionally, we ran negative controls for each PCR.
We detected T. cruzi DNA in one juvenile Mexican free-tailed bat, resulting in a 0.27% prevalence in the sampled bats (n=361). The positive sample and control were Sangarsequenced at Eton Biosciences (San Diego, California, USA), and we aligned the forward and reverse sequences using Sequencher 5.4.6 (GeneCodes, Ann Arbor, Michigan, USA). We did not determine the discrete typing unit of the positive sample due to sequencing equipment restraints. We entered the aligned sequences into BLAST (National Center for Biotechnology Information 2018) and confirmed the organismal DNA as T. cruzi. We compared closely related sequences (Fig. 2) and uploaded the sample to GenBank (no. MG869732). Multiple, closely related sequences were collected from rodents in the Las Palomas Wildlife Management Area in southern Texas (Aleman et al. 2017), which is along the Mexican free-tailed bat migratory pathway. We suspect a female Mexican freetailed bat had acquired T. cruzi while migrating through this area, as all published Las Palomas sequences and the Sylvio X10 strain belong to the TcI lineage (Hodo et al. 2016).
Phylogenetic analysis of the positive sample, Woodward 1, from migratory Mexican free-tailed bats (Tadarida brasiliensis) that we sampled in Oklahoma, USA and closely related sequences. Woodward 1 is closely related to multiple Las Palomas sequences, which were collected from rodents in the Las Palomas Wildlife Management Area in southern Texas, USA (Aleman et al. 2017). All six Trypanosoma cruzi discrete typing units are represented. Leishmania donovani is a sympatric trypanosomatid and is included as the outgroup. All sequences were acquired from GenBank. The evolutionary history was inferred by using the maximum likelihood method based on the Jukes-Cantor model with 1,000 bootstrap replicates. Evolutionary analyses were constructed using MEGA7 (Kumar et al. 2016)
Phylogenetic analysis of the positive sample, Woodward 1, from migratory Mexican free-tailed bats (Tadarida brasiliensis) that we sampled in Oklahoma, USA and closely related sequences. Woodward 1 is closely related to multiple Las Palomas sequences, which were collected from rodents in the Las Palomas Wildlife Management Area in southern Texas, USA (Aleman et al. 2017). All six Trypanosoma cruzi discrete typing units are represented. Leishmania donovani is a sympatric trypanosomatid and is included as the outgroup. All sequences were acquired from GenBank. The evolutionary history was inferred by using the maximum likelihood method based on the Jukes-Cantor model with 1,000 bootstrap replicates. Evolutionary analyses were constructed using MEGA7 (Kumar et al. 2016)
Low prevalence in the sampled population is likely due to roost location on the northern boundary of the historic triatomine range and low endemic triatomine populations near these sites (Lent and Wygodzinsky 1979). We were unable to trap and confirm vector prevalence and infectivity due to sampling restrictions imposed by the risk of anthropogenic P. destructans contamination.
We present a report of a wild bat naturally infected with T. cruzi in Oklahoma, the second report of a bat naturally infected in the US, and the fourth reported animal infection in the state (Hodo et al. 2016). We report that the detection of T. cruzi in bat patagia is a convenient and sensitive method for T. cruzi disease surveillance and is applicable to a variety of wild mammals in underrepresented and endemic areas.
We suggest that Mexican free-tailed bats potentially contribute to the endemicity of T. cruzi in Oklahoma and may contribute to future enzootic expansion. Despite a low prevalence in our samples, Mexican freetailed bats may play a unique role in the epidemiology of T. cruzi through their annual migration from classical endemic foci to other areas. Although the distribution of triatomines in Oklahoma is poorly studied, Triatoma lecticularia and Triatoma sanguisuga have been identified statewide, including in Oklahoma City (Griffith 1947; Drew and Schaefer 1962). In Texas, up to 63% of sampled triatomines statewide are positive for T. cruzi (Curtis-Robles et al. 2015), and there are reports of sylvatic transmission cycles in Texas mammals along the Mexican free-tailed bat migratory pathway (Kjos et al. 2009). Future research should focus on assessing T. cruzi prevalence in wild and domestic Oklahoma mammalian reservoirs, identifying foci of sylvatic and peridomestic transmission, increasing surveillance of classic and potentially novel arthropod vectors, and assessing the impending impact of climate change on vector, Mexican free-tailed bat, and T. cruzi biogeography in Oklahoma.
We thank the research volunteers who assisted in sample collection, the Oklahoma Department of Wildlife Conservation, and the landowners of the maternity roosts. We thank William Caire for the project structure and mammalogy training and James Creecy for verifying our work with Sequencher, providing molecular analyses advice, and manuscript review. We thank Sarah Vrla for her editorial comments. Support for this research was provided by the University of Central Oklahoma, Student Transformative Learning Record; Research, Creative, and Scholarly Activities; the University of Central Oklahoma Center for Wildlife Forensic Science and Conservation Studies; the W. Roger Webb Forensic Science Institute; and the College of Mathematics and Sciences.
