Raccoons As an Important Reservoir for Trypanosoma cruzi : A Prevalence Study from Two Metropolitan Areas in Louisiana

Abstract

Raccoons are an important reservoir for Trypanosoma cruzi infection, having been reported to maintain a high and lengthy parasitemia. Although raccoon populations have historically been abundant in Louisiana, the prevalence rate of T. cruzi infection in raccoons in this state is unknown. Here, we tested raccoon tissues from two urban areas in Louisiana, namely Orleans Parish (OP) and East Baton Rouge Parish (EBRP), to investigate prevalence in these areas using direct detection through polymerase chain reaction. Overall, 33.6% of raccoons tested were positive. The prevalence in OP (42.9%) was significantly higher than the prevalence in EBRP (23.2%). There was no significant difference in prevalence between sexes or based on age, but there was a significant difference in infection prevalence based on season of trapping. These results suggest the importance of raccoons as a reservoir host, maintaining T. cruzi infection and potentially posing a risk to human health.

Introduction

Chagas disease, an anthropozoonosis caused by the protozoan parasite Trypanosoma cruzi, is transmitted mainly by the feces of insect vectors of the Triatomine subfamily, commonly known as kissing bugs. T. cruzi has been known to be enzootic in the southern United States for nearly a century (Roellig et al. 2009); however, much of the dynamics of its transmission in the sylvatic and peridomestic cycles is unknown. These cycles overlap with the domestic cycle, wherein humans are at high risk due to the potential intrusion of both vertebrate hosts and triatomine vectors into human dwellings and of humans into peridomestic and sylvatic habitats (Kribs-Zaleta 2010, Montgomery et al. 2014). In the United States, an estimated 300,000 humans are infected with the parasite, including a growing number of locally acquired cases (Garcia et al. 2015, 2017, Gunter et al. 2017). Autochthonous transmission is of growing concern, and analyses of triatomine blood sources in the southern United States, including Louisiana, suggest a significant rate of vector–human contact (Klotz et al. 2014a, 2014b, Waleckx et al. 2014, Gorchakov et al. 2016). Therefore, it is important to investigate mammalian reservoirs and their infection rates as part of assessing the risk to humans in local transmission cycles (Herrera et al. 2015, 2018, Pronovost et al. 2018).

Mammalian reservoirs are critical for the maintenance of T. cruzi in peridomestic and sylvatic cycles in the southern United States. At least 26 mammals have been recognized as natural sylvatic hosts for T. cruzi in this region, including woodrats (Neotoma spp.), Virginia opossums (Didelphis virginiana), raccoons (Procyon lotor), armadillos (Dasypus novemcinctus), skunks (Mephitis mephitis) (Bern et al. 2011, Herrera et al. 2015, Montgomery et al. 2016, Hodo and Hamer 2017), and mice (Peromyscus spp. and Mus musculus) (Herrera et al. 2015). Raccoons are a particularly important wild reservoir in southern states, with one of the highest estimates of infection prevalence at 36.4% (Hodo and Hamer 2017), followed by woodrats (Neotoma spp.) at 34.7% and opossums (Didelphis virginiana) at 22.9% (Hodo and Hamer 2017). Several studies have reported T. cruzi prevalence in wild reservoirs within the United States (Hodo and Hamer 2017). Studies conducted in different states of the United States have indicated that raccoons and Virginia opossums have the highest prevalence of T. cruzi compared with other mammals (Roellig et al. 2009) with an overall prevalence of 34.6% (Curtis-Robles et al. 2016, Hodo and Hamer 2017). However, to date, no study is specific to raccoons from Louisiana (Hodo and Hamer 2017), where local transmission from triatomines to humans has been documented (Dorn et al. 2007). Considering the close association of raccoons to humans, they likely play an epidemiological role helping link the sylvatic cycle of the parasite with the domestic cycle.

In Louisiana, Triatoma sanguisuga is the sole insect vector of T. cruzi. It has been found to be both infected with the parasite (Bern et al. 2011) and associated with autochthonous human infections (Dorn et al. 2007, Klotz et al. 2014a, 2014b, Beatty et al. 2018). Waleckx et al. (2014) demonstrated that in a sample of T. sanguisuga specimens from Louisiana positive for T. cruzi, 58% contained raccoon blood and 38.1% contained human blood, suggesting that local vectors are feeding on both raccoons and humans. Similarly, Gorchakov et al. (2016) reported the simultaneous presence of both raccoon and human blood in T. sanguisuga in Texas.

These findings, taken together, show a scenario of spillover risk (Hodo and Hamer 2017) in both the southern United States generally and Louisiana specifically. Transmission cycles vary locally, demonstrating a need for research regarding specific T. cruzi prevalence in raccoons to help inform estimates of local human health risk in Louisiana. Our study aimed to estimate the prevalence of T. cruzi in urban raccoon populations to better understand the impact of T. cruzi-infected raccoons in the ecoepidemiology of Chagas disease in two of the most populated areas in Louisiana, the metropolitan centers of Orleans Parish (OP) and East Baton Rouge Parish (EBRP).

Materials and Methods

Ethics statement

All animal procedures were approved by the Louisiana State University Health Science Center Institutional Animal Care and Use Committee (Protocol No. T3455) and the Tulane Institutional Animal Care and Use Committee (Protocol No. 4423). This study adhered to all Louisiana Department of Wildlife and Fisheries Immobilization and Euthanasia guidelines.

Study population

One hundred nineteen raccoons were sampled in the urban areas of OP and EBRP. One hundred seventeen paired hearts and colons were collected, along with one unpaired heart and one unpaired colon. Raccoons trapped in New Orleans were part of a study to estimate the prevalence of the raccoon roundworm Baylisascaris procyonis in a southern urban environment (Straif-Bourgeois et al. 2020). Overall, 65 raccoons were trapped between October 2014 and December 2017 by the New Orleans Mosquito, Rodent, and Termite Control team using Havahart^® (Woodstream Corp.; Lilitz, PA) traps in urban residential neighborhoods and parks in OP, totaling 32 trapping sites (Fig. 1). Trapped raccoons were brought to the Louisiana Society for the Prevention of Cruelty to Animals (LASPCA), where they were euthanized based on LASPCA's euthanasia protocol. Necropsies were performed at the New Orleans Mosquito, Termite and Rodent Control Board, at which point hearts and colons were harvested and kept at −20°C until processing. Necropsies yielded a total of 63 hearts and colons from raccoons in OP for further testing.

FIG. 1.

Maps of raccoon harvest sites demonstrating location of Louisiana in the southeastern United States and location of Orleans and Baton Rouge Parishes within the state. Black crosses indicate site where Trypanosoma cruzi-positive raccoons were trapped. Dots indicate sites where all raccoons tested negative for T. cruzi.

Between January 2015 and August 2018, 56 raccoons were trapped at 14 sites (Fig. 1) and removed from residential areas in EBRP by Nuisance Wildlife Control Officers. Upon euthanasia, hearts and colons were harvested and kept at −20°C until processing. Heart tissue was unavailable for one raccoon and colon tissue was unavailable for another, resulting in analysis of 55 hearts and 55 colons across 56 raccoons from EBRP. Hearts and colons were chosen for testing as cardiac and gastrointestinal pathologies are the most common morbidities of Chagas disease, and these tissues have been suggested as parasite reservoirs in mammalian hosts (Lewis and Kelly 2016). All trapping sites for both parishes were selected through a convenience sampling approach.

Molecular diagnostics

DNA extraction was performed for all raccoon tissue samples using Qiagen DNEasy Blood and Tissue Kit per manufacturer's instructions until the elution step, wherein only 70 μL of elution buffer was used for final elution in two separate spin steps of 50 and 20 μL. After extraction, total DNA content and quality were evaluated by nanodrop spectrophotometer. DNA was then diluted to 2.5 ng/μL in sterile water and used as template in a standard detection PCR assay, along with a negative control of sterile water and a positive control of DNA extracted from T. cruzi parasite culture of strain H1, using primers TcZ1 and TcZ2, targeting the repetitive satellite region, as previously described (Moser et al. 1989). Infection status was determined based on the electrophoretic results of PCR products on an ethidium bromine stained 2.0% agarose gel run for 50 min at 100 V, with the presence of a 188-bp band qualifying a sample as positive. To confirm T. cruzi infection, an additional PCR targeting the miniexon, either that described by Souto et al. (1996) (300–350 bp product) or a new assay recently described by our group (Majeau et al. 2019) (500 bp product), was performed. Each raccoon was considered to be overall positive for T. cruzi if either the heart or colon tested positive by PCR. For two raccoons from the New Orleans area, the initial PCR using the TcZ1 and TcZ2 primers presented ambiguous results, requiring the second PCR assay to confirm infection status, but for all other samples, PCR results were consistent for each of the assays targeting the satellite region and the miniexon.

Mapping and statistical analysis

Maps of trapping sites were constructed using QGIS 2.18.11 with map data from ESRI, the Louisiana Department of Transportation and Development (Louisiana Department of Transportation and Development, February 5, 2007, Louisiana Parish Boundaries, Geographic NAD83, LDOTD (2007) [Parishes_LDOTD_2007]). Sites were plotted by GPS coordinates associated with the trapping site addresses and coded as positive if any raccoon trapped at the site tested positive by PCR, as described above, or negative if no raccoon trapped at the site tested positive by PCR. All statistical analyses were performed using RStudio. Fisher's exact test was used to investigate differences in infection based on parish, age of raccoon, or season trapped. McNemar's test was used to determine if there was a significant difference in infection prevalence between the two tissue types in each parish individually and McNemar's test with an adjustment for clustered data was used to determine if there was a significant difference in tissue-specific infection prevalence overall.

Results

In both OP and EBRP, 40/119 (33.6%) raccoons tested positive for T. cruzi infection by PCR (Fig. 1).

Twenty-two of 32 OP trapping sites and 5 of 14 EBRP sites resulted in raccoons positive for T. cruzi (Fig. 2). Of note, some trapping sites resulted in more than one individual. However, infection status was not always consistent between individuals trapped at the same site. Therefore, any site from which any positive raccoon was trapped was considered to be a “positive site,” though this does not mean every animal trapped at this site tested positive.

FIG. 2.

Sample gel electrophoresis of detection PCR. Lane 1: 1-kb Ladder Lanes 2–16: PCR product from raccoon tissue template DNA with lanes 8, 14, and 15 demonstrating 188-bp positive band, indicated by arrow heads. Lane 17: T. cruzi-positive control (Strain H1); Lane 18: no template control.

Raccoons from OP were significantly more likely to be infected (p = 0.0322) than raccoons from EBRP, with respective prevalence of 42.9% (27/63) and 23.2% (13/56) (Table 1). There was no significant difference in infection status based on sex or age overall, though a significant difference in infection between juveniles and adults was observed in OP (p = 0.0426). There was a highly significant difference in infection based on season trapped overall (p = 0.0034) and in EBRP alone, but this difference was not significant in OP (Table 1). As several studies have demonstrated the tropism of certain strains of T. cruzi to different tissues (Herrera et al. 2015, Zingales 2018), we were interested to see if there was a significant difference in infection status between the two tissue types. In both parishes, a greater proportion of hearts were infected than colons, but this was only significant in EBRP (p = 0.01586) and was not significant in OP nor overall. Nearly 30% (8/27) of the positive raccoons from OP and 15% (2/13) of the positive raccoons from EBRP were positive for the parasite in both tissues tested, suggesting that while strains circulating in both areas have tropism for both heart and colon tissues, there may be a more pronounced tropism for heart tissue compared with colon tissue in strains circulating in EBRP.

Table 1.

Prevalence of Trypanosoma cruzi Infection in Urban Louisiana Raccoons from Two Parishes

	Positive	Negative	p-Value
Parishes combined (n = 119)
Total	40 (33.6%)	79
Parish			0.0322^*
OP	27 (42.9%)	36
EBRP	13 (23.2%)	43
Sex			0.3323
Male	16 (28.6%)	40
Female	24 (38.1%)	39
Age			0.2990
Juvenile	10 (25.0%)	30
Adult	28 (35.9%)	50
Season			0.0034^*
Winter	9 (50.0%)	50
Spring	5 (25.0%)	15
Summer	5 (12.2%)	36
Fall	15 (44.1%)	19
Tissue^a			0.2214
Heart	31 (26.3%)	87
Colon	18 (15.3%)	100
OP (n = 63)
Total	27 (42.9%)	36
Sex			0.2074
Male	11 (34.4%)	21
Female	16 (51.6%)	15
Age			0.0426^*
Juvenile	3 (18.8%)	13
Adult	23 (48.9%)	24
Season			0.6826
Winter	7 (46.7%)	8
Spring	5 (27.7%)	13
Summer	4 (44.4%)	5
Fall	5 (35.7%)	9
Tissue^b			0.4795
Heart	19 (30.2%)	44
Colon	15 (23.8%)	48
EBRP (n = 56)
Total	13 (23.2%)	43
Sex			0.7603
Male	5 (20.8%)	19
Female	8 (25.0%)	24
Age			0.5237
Juvenile	7 (29.2%)	17
Adult	6 (18.8%)	26
Season			0.0000^*
Winter	2 (66.7%)	1
Spring	0 (0.0%)	1
Summer	1 (3.1%)	31
Fall	10 (50%)	10
Tissue^b			0.0159^*
Heart	12 (21.8%)	43
Colon	3 (5.5%)	52

McNemar's test with adjustment for clustering.

McNemar's test; p < 0.05.

Fisher's exact test; ^* p < 0.05.

OP, Orleans Parish; EBRP, East Baton Rouge Parish.

Discussion

Characterizing T. cruzi prevalence in mammalian hosts is a crucial first step toward understanding the role of these hosts in the transmission networks as well as the risk of human infection in the US. Previous studies in different mammal species have demonstrated a high T. cruzi parasitemia in the naturally infected host (Hodo and Hamer 2017). The need to investigate peridomestic mammals in Louisiana to asses risk of human infection is further highlighted by a recent study of T. cruzi infection in shelter dogs in Louisiana that observed average seropositivity of ∼6.9% (Elmayan et al. 2019). While this suggests that dogs may represent potentially significant peridomestic hosts, raccoons are likely a more important reservoir to investigate for T. cruzi infection in the United States. They demonstrate one of the highest T. cruzi prevalence of mammalian hosts in the eastern United States, with some statewide prevalence estimates reaching as high as 68% (Bern et al. 2011). In addition, raccoons maintain a high parasitemia for up to 5 weeks postinfection and are subject to infection by most, if not all, discrete typing units of the parasite (Roellig et al. 2009). Although the parasite and raccoon host exist in an enzootic cycle, there is additional human health risk due to the peridomestic nature of both raccoons and some triatomines, increasing the potential interactions of reservoir host, vector, human host, and pathogen (Prange et al. 2004, Kribs-Zaleta 2010, Klotz et al. 2014a, 2014b).

Raccoons' home range in urban areas is ∼5–79 ha as a result of artificial resource abundance, though range may increase seasonally or when resources are limited (Prange et al. 2004). Raccoons in urban areas also demonstrate an aggregated spatial distribution, increasing their likelihood to be reservoirs of disease (Prange et al. 2004). In this study, raccoons were trapped in residential backyards, near playgrounds, urban parks, schools, and abandoned houses, further highlighting the risk for the aforementioned interactions. Currently, a precise estimate of raccoon population size in the urban areas studied is unavailable, but historically, populations in Louisiana have reached in the hundreds of thousands and raccoons are known to abundantly occur in every parish (Lowery 1974). While investigating the parasite prevalence in raccoons is not sufficient to fully elucidate local spillover risk to humans, it is an important piece of the puzzle that must be considered.

Both OP and EBRP, though urban, present variability in their landscapes. Some areas within each parish and city are well-developed urban swaths, while others are large stretches of green space or forest and still others may be largely abandoned or poorly developed urban areas. Therefore, while the overall classification of both of these areas is “urban,” the nuanced ecologies that exist within these urban areas should still be considered. Given the difference in prevalence between the two regions, a more robust analysis of land-use differences may be interesting for further research, though this may simply be suggestive of highly localized transmission patterns.

Based on our sampling, there does not appear to be evidence for any difference in T. cruzi infection based on sex in raccoons. Although there seems to be a stronger parasite tropism for heart tissue versus colon tissue, this was only found to be significant in EBRP. This may be attributable to differences in locally circulating strains, as noted above, or may suggest a difference in transmission routes between the two locations. Eating insect vectors is considered an important part of the zoonotic transmission cycle and, experimentally, there has not been evidence of a large difference in parasite localization to either heart or colon tissue in small mammals based on oral parasite introduction (Silva-Dos-Santos et al. 2017). Thus, it is possible that raccoons in OP are infected more commonly through oral parasite introduction, while raccoons in EBRP are infected more commonly through stercorarian transmission.

Conclusion

Given the potential for a T. cruzi reservoir pertinent to human health being quite literally in our own backyards, it is crucial to elucidate the infection prevalence. Raccoon prevalence determined in this study is rather close to those reported from other mammals in Louisiana, namely opossums (33%) and armadillos (29–38%) (Bern et al. 2011). Given the prevalence of raccoons and T. cruzi parasite infection in two of the most populated areas of Louisiana, raccoons represent an important reservoir in the T. cruzi transmission cycle.

Footnotes

Authors' Contributions

C.H. and S.C.S.-B. conceived the study and designed the study protocol. S.C.S.-B., E.C., A.N.A., and G.B. performed necropsies and provided heart and colon samples. A.M., H.P., and A.S. carried out the molecular analysis and interpretation of the data. All authors drafted the article and critically revised the article for intellectual content. All authors read and approved the final article. C.H. and S.C.S.-B. are guarantors of the article.

Acknowledgments

We thank the Louisiana Society for the Prevention of Cruelty to Animals (LASPCA) for their support with this study and David Perault for animal capture in East Baton Rouge Parish. We also thank Logan Stuck and Brendan Carter from the department of Tropical Medicine-Tulane University, for their assistance with statistical analyses and mapping tools.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

This work was funded in part by the Tulane ByWater Institute-Faculty Fellowships in Interdisciplinary Collaboration, the Carol Lavin Bernick Faculty Grant Program-Tulane University to C.H., and Louisiana State University School of the Public Health, Department of Epidemiology.

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