Multi-Method Evidence of Trypanosoma cruzi Infection in Wild European Rabbits in Chile: Implications for Reservoir Ecology and Surveillance

Simple Summary

Chagas disease is caused by a microscopic parasite that can infect many mammals and is mainly spread by blood-feeding “kissing bugs.” In central Chile, wild European rabbits are very abundant and live in the same habitats as these insects, but their role in the parasite’s natural cycle is still unclear. We examined eight wild rabbits using several complementary approaches: we tested their blood and many organs for parasite genetic material, we allowed laboratory-raised uninfected kissing bugs to feed on the rabbits and later checked whether the insects became infected (proving live parasites were present), and we examined tissues under the microscope to look for parasite forms. Most rabbits had parasite genetic material in their blood, every rabbit had it in at least one organ, and in half of the animals we could also see parasite forms in tissues. A smaller number of rabbits were able to infect the kissing bugs, providing evidence that some rabbits were carrying infecting parasites. These findings suggest that rabbits may contribute to the parasite’s wildlife cycle and could be useful as an early-warning species for Chagas surveillance.

Abstract

Chagas disease, caused by Trypanosoma cruzi, is maintained in nature by complex interactions among wild vertebrates and triatomine insect vectors, yet the role of many introduced hosts remains poorly resolved. Here, we assessed natural T. cruzi infection in wild European rabbits (Oryctolagus cuniculus) from central Chile, where introduced rabbits overlap ecologically with the sylvatic vector Mepraia spinolai. Eight free-ranging rabbits captured in Las Chinchillas National Reserve were evaluated using an integrative diagnostic approach combining xenodiagnosis with laboratory-reared, parasite-free M. spinolai nymphs, real-time polymerase chain reaction targeting T. cruzi satellite DNA in blood and 12–14 organs per animal, and histopathology with immunohistochemistry (anti-cruzipain) to identify tissue parasite forms. Blood molecular detection was positive in seven out of eight rabbits, while xenodiagnosis detected viable parasites in two out of seven evaluated individuals. Organ molecular screening detected T. cruzi DNA in at least one organ in all rabbits, with frequent positivity in the diaphragm, reproductive tissues, spleen, and kidney. Histopathology identified parasite forms in four out of eight animals, and immunohistochemistry confirmed hepatic amastigotes in one case. These findings provide multi-method evidence of natural infection in the sampled individuals, including evidence of parasite viability in some individuals, suggesting potential epidemiological relevance within this ecological context and possible utility for surveillance in Chilean sylvatic transmission settings.

1. Introduction

Chagas disease remains a major zoonotic parasitic disease in the Americas, sustained by complex transmission cycles involving triatomine vectors and a wide diversity of wild and domestic vertebrate hosts [1,2]. In sylvatic systems, reservoir competence varies substantially among host species and is influenced by ecological traits such as abundance, longevity, and vector contact [3]. However, the role of many invasive or introduced mammals in Trypanosoma cruzi (Chagas, 1909) (Kinetoplastida, Trypanosomatidae) transmission remains poorly characterized. Usually, vectorial transmission occurs while the triatomine is feeding, and the host disperses the contaminated feces through the skin by scratching, allowing the parasite to penetrate the skin at the bite site [4]. Throughout its development, the six hematophagous vector instars feed on humans and domestic and/or sylvatic animals [5,6,7,8].

The European rabbit (Oryctolagus cuniculus) is a successful invasive species in central Chile, where it reaches high densities in arid and Mediterranean ecosystems [9,10]. Its distribution overlaps extensively with Mepraia spinolai (Porter, 1934) (Hemiptera, Reduviidae), a sylvatic triatomine that feeds opportunistically on a broad range of vertebrates [11]. Blood-meal analyses have repeatedly confirmed the presence of rabbit blood in M. spinolai, suggesting regular contact and a potential for parasite exchange [5,12,13].

Previous work in Chile demonstrated natural T. cruzi infection in wild rabbits, reporting a 38% prevalence and the presence of multiple discrete typing units (DTUs) [14]. These findings confirmed exposure and infection but did not address key aspects of reservoir competence under natural conditions.

Specifically, the extent of parasite distribution across organs in naturally infected rabbits has not been examined, nor has the detection of viable parasites through xenodiagnosis been assessed in this species. Additionally, histopathological characterization of tissue infection in free-ranging rabbits remains undocumented, despite extensive descriptions of experimental rabbit infections in laboratory settings [15,16,17]. Because parasite distribution within hosts is heterogeneous and parasitemia fluctuates over time, reliance on a single diagnostic method may underestimate infection or misrepresent epidemiological relevance [2].

Integrating complementary detection approaches can provide a more robust assessment of infection status and potential transmission capacity in wildlife hosts. Molecular detection allows sensitive identification of parasite DNA in blood and tissues [18,19,20], histopathology enables visualization of parasite forms and tissue involvement, and xenodiagnosis (XD) uniquely provides evidence of parasite viability and host-to-vector transmission potential [21,22]. XD has been successfully used in several mammalian species and a few lizard species [2,23,24,25] for detecting infection, even in specimens with low parasitic loads [22].

Here, we employed an integrative approach, including XD, real-time PCR of blood and organs, and histopathology with immunohistochemistry, to characterize natural T. cruzi infection in wild European rabbits captured in a protected semiarid reserve. Our aim was to establish infection status, unveil tissue parasitism, and clarify epidemiological relevance of this invasive lagomorph within the sylvatic transmission cycle.

2. Materials and Methods

2.1. Study Area and Animal Capture

Sampling was performed in Las Chinchillas National Reserve (31°28′ S, 71°03′ W), a semiarid shrubland where M. spinolai is abundant and T. cruzi circulation among small mammals is well-documented [26]. Trapping effort was performed using live baited traps. A total of 24 live traps, Havahart^® (Woodstream Corporation, Lancaster, PA, USA) and Tomahawk^® (Tomahawk Live Trap, Hazelhurst, WI, USA), were deployed each night over five consecutive nights within a 3 ha study area during each sampling period (autumn 2018 and summer 2019). Across both periods, eight rabbits were captured (six in autumn 2018 and two in summer 2019). Traps were placed within the same predefined study area in both sampling periods, with minor micro-location adjustments based on burrow activity and field conditions to maximize capture success. Sedation, handling, and euthanasia followed approved ethical protocols (CICUA 18136-FCS-UCH).

2.2. Xenodiagnosis

Five second-instar M. spinolai nymphs were individually placed in contact with each anesthetized rabbit and allowed to feed on the ear pinna for up to 20 min, or until voluntary detachment occurred. Nymphs were weighed immediately before and after feeding using an analytical balance (±0.1 mg) to confirm successful blood ingestion; these measurements were used as procedural quality control and are not presented as analytical data. After feeding, each nymph was individually housed in sterile plastic containers containing folded filter paper as refuge and maintained at 27 °C, 70–80% relative humidity, and a 14:10 light/dark cycle for 40 days to allow parasite multiplication in the vector gut. No refeeding occurred during incubation. After 40 days, nymphs were euthanized by freezing at −20 °C. Under sterile conditions, the abdomen was dissected, and the entire intestinal tract was removed using flame-sterilized forceps. Intestinal tissue was placed in 20 µL nuclease-free water and stored at −20 °C until DNA extraction. Second-instar nymphs were selected following a previously validated xenodiagnostic protocol in Chilean sylvatic systems [2]. Early instars were consistently available from synchronized laboratory cohorts and demonstrate high feeding success [23].

2.3. Tissue Collection

Blood was collected from the ear marginal vein and preserved in Guanidine-HCl 6 M (Thermo Scientific™, Waltham, MA, USA)-EDTA 0.2 M (Thermo Scientific™, Waltham, MA, USA) buffer. Fourteen organs/tissues were sampled during necropsy. For each organ, approximately 50 mg of tissue was excised from the central region using sterile instruments. Separate sterile instruments were used for each organ to prevent cross-contamination. Instruments were cleaned with 70% ethanol and flame-sterilized between samples. Each organ sample was bisected: one half was fixed in formalin (Winkler, Santiago, Chile) for histology/immunohistochemistry, and one half was preserved in ethanol for DNA extraction. Tissue fragments were mechanically homogenized prior to DNA extraction to increase representativeness.

2.4. Molecular Detection

DNA isolation: DNA from XD intestines, blood, and organ tissues was extracted using InnuPREP kits (IST Innuscreen GmbH, Berlin, Germany), including an internal amplification control DNA (Arabidopsis-derived, IAC), extraction blanks, negative template controls, and positive T. cruzi controls [25].

Real-time PCR: The T. cruzi satDNA target was amplified using the Cruzi 1/2/3 primer-probe system [27,28]. Each real-time PCR reaction was performed in a final volume of 20 µL, containing 0.45 µM of each primer, 0.15 µM of probe, 10 µL of Luna^® Universal Probe qPCR Master Mix (New England Biolabs^®, Ipswich, MA, USA), and 2 µL of extracted DNA template. All samples were run in duplicate. A sample was considered positive when both replicates yielded a Ct < 38 with appropriate amplification curves and valid IAC signal. Samples with Ct ≥ 38 were re-extracted and re-tested by real-time PCR once in duplicate. Negative extraction controls and no-template controls were included in each run to monitor contamination.

2.5. Histopathology and Immunohistochemistry (IHC)

Formalin-fixed tissues were paraffin-embedded, sectioned (5 µm for hematoxylin–eosin (HE); 3 µm for IHC), stained with HE, and screened for amastigotes, trypomastigotes, and inflammatory lesions. IHC used anti-cruzipain primary antibody and HRP-conjugated secondary antibody, with DAB chromogen detection. Antigen retrieval was performed in a pressure-based heating system at 95–98 °C for 20 min using the Universal HIER reagent (Abcam, Cambridge, UK). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 30 min at room temperature. Primary antibody incubation was conducted overnight at 4 °C in a humidified chamber. DAB chromogen development was monitored under microscopy and stopped after 2–5 min to avoid overstaining.

2.6. Statistical Analysis

Fisher’s exact tests were used to compare detection frequencies among diagnostic methods (α = 0.05). Analyses were performed using R 4.4.2. Given the limited sample size, inferential statistics were used conservatively and are presented primarily to illustrate trends in detection frequency.

3. Results

3.1. Non-Lethal Detection

All XD nymphs fed successfully and survived the incubation period. Blood PCR detected more positive individuals than XD (7/8 vs. 2/7). Although Fisher’s exact test yielded p = 0.04, this comparison should be interpreted cautiously given the small sample size.

3.2. Organ-Level Detection

Organ PCR identified T. cruzi DNA in at least one organ of every rabbit (100%). The most frequently positive tissues were the diaphragm (50%), reproductive organs (37.5%), spleen (37.5%), and kidney (37.5%). Individual rabbits showed one to five positive organs (

Table 1. Trypanosoma cruzi infection status by histological (HIS) and molecular analysis (rtPCR) of rabbit tissues. Positive HIS organs are indicated with the presence of amastigotes (A), trypomastigotes (T), or intracellular form (IC).

	C01		C02		C03		C04		C05		C06		C07		C08
Organ/Tissue	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR	HIS	rtPCR
Skin	−	+	−	−	−	−	−	−	−	−	−	−	−	ns	−	+
Muscle	−	−	−	+	−	−	−	+	−	−	−	−	−	−	−	−
Small intestine	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−
Large intestine	−	−	−	−	−	+	−	−	−	−	IC	−	−	−	−	−
Stomach	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	+
Pancreas	−	−	A	−	−	−	−	−	−	−	−	−	−	ns	−	−
Kidney	−	+	−	−	−	−	−	−	−	+	−	−	−	−	−	+
Liver	−	−	−	−	A	+	−	−	−	−	−	−	−	−	−	−
Ovaries-Uterus	−	−	−	−	ns	ns	−	−	−	−	−	−	ns	+	ns	+
Testicles–Penis	ns	ns	ns	ns	−	+	ns	ns	ns	ns	ns	ns	ns	ns	ns	ns
Bladder	−	+	−	+	−	−		−	−	−	−	−	IC	−	−	−
Diaphragm	−	−	−	−	−	+	−	+	−	+	−	−	−	−	−	+
Heart	−	−	−	−	−	−	−	−	−	−	T	−	T	−	−	−
Lungs	−	−	−	+	−	−	−	−	−	−	−	+	−	−	−	−
Spleen	−	−	−	+	−	−	−	−	−	+	−	−	−	+	−	−

+: PCR positive, −: negative to PCR or histopathology, ns: no sample.

).

3.3. Histopathology and IHC

Histopathological screening detected parasite forms in four out of eight animals (50%). Findings included trypomastigotes in the heart tissue of two individuals, intracellular forms in the bladder and cecum, and amastigotes in the pancreas and liver (Table 1). IHC confirmed hepatic amastigotes in one rabbit, validating the presence of T. cruzi antigen where HE morphology suggested infection (Figure 1).

3.4. Concordance Among Methods

Agreement across methods was limited; some histology-positive tissues were PCR-negative and vice versa. The combined use of all methods yielded consistent evidence of systemic infection across the sample set (

Table 2. Lethal and non-lethal Trypanosoma cruzi detection techniques’ outcomes in naturally infected rabbits.

	Non-Lethal Detection		Lethal Detection
Rabbit ID	XD Result	Blood rtPCR	Organ rtPCR	Organ Histopathology
C01	−	−	+ (3/14)	−
C02	−	+	+ (4/14)	+ (1/14)
C03	+	+	+ (4/14)	+ (1/14)
C04	−	+	+ (2/14)	−
C05	+	+	+ (5/14)	−
C06	ns	+	+ (1/14)	+ (2/14)
C07	−	+	+ (2/12)	+ (2/14)
C08	−	+	+ (5/14)	−

+: positive, −: negative, ns: no sample. XD: xenodiagnoses. Numbers between parenthesis show positive sample proportion in lethal techniques.

).

4. Discussion

All rabbits exhibited evidence of T. cruzi infection with at least one diagnostic method. The 87.5% positivity of blood PCR is higher than previously reported in Chile [14], potentially reflecting either local vector–host interaction dynamics, repeated exposure, or differences in the detection method, as this work used real-time PCR instead of conventional PCR. Real-time PCR targeting satellite DNA increases analytical sensitivity, particularly in hosts with fluctuating or low parasitemia, which may partially explain the discrepancy with earlier prevalence estimates.

Organ PCR provided the broadest detection, aligning with the known heterogeneous tissue tropism of T. cruzi [29,30]. Frequent detection in the diaphragm, spleen, and reproductive organs shows systemic dissemination in naturally infected lagomorphs. Experimental and molecular studies have demonstrated that circulating parasite populations can differ from those established in specific tissues and that tissue-level parasite burden may vary substantially even within the same host [2]. Such heterogeneity has been associated with strain-dependent tropism, parasite load dynamics, and host immune modulation [31,32,33]. Therefore, the organ-level variability observed here is biologically plausible and consistent with the current understanding of T. cruzi within-host ecology.

Xenodiagnosis, the only direct measure of parasite viability, yielded a modest positivity (28.6%). While lower than blood PCR, XD positivity indicates that at least some individuals can infect vectors under natural conditions. Although later instars ingest larger blood volumes, parasite replication during the 40-day incubation period allows detection even when smaller blood meals are taken. Thus, the modest xenodiagnosis positivity observed here likely reflects naturally low parasitemia rather than methodological limitation alone [34,35].

Rabbits meet several ecological criteria of reservoir competence, such as abundance, long-term infections, and vector contact [3,36,37]. Reservoir competence encompasses multiple biological components: (i) susceptibility to infection, (ii) ability to sustain parasite persistence within tissues, and (iii) capacity to transmit viable parasites to feeding vectors [3,38]. Detection of parasite DNA alone does not necessarily imply epidemiological relevance [38], as it does not confirm viability or transmissibility. In this study, molecular detection shows infection, multi-organ positivity supports systemic persistence, and XD positivity provides evidence of viable parasites and potential host-to-vector transmission in a subset of individuals [39]. Therefore, our findings support the potential for reservoir competence under natural conditions, while acknowledging that population-level transmission efficiency cannot be inferred from the present sample size [40]. Feeding studies of M. spinolai have confirmed frequent interaction with rabbits, and experimental observations indicate efficient blood ingestion and defecation behavior on lagomorph hosts [41]. However, our findings should be interpreted as evidence of a potential reservoir role rather than definitive reservoir status. Our results complement previous findings in wild rabbits from Chile [14], extending knowledge beyond prevalence and adding multi-organ tissue distribution, histopathological confirmation of parasite presence, and detection of viable parasites via XD.

Experimental rabbit infections described marked myocarditis and systemic lesions [15,16,17]; in contrast, naturally infected individuals exhibited parasite presence with minimal inflammation, consistent with chronic or repeatedly exposed wildlife hosts. No single diagnostic method captured all infections. Histopathology was specific but insensitive; PCR was sensitive but could not confirm viability; XD provides viability but requires detectable parasitemia. Our study supports the use of integrative approaches in wildlife disease surveillance, particularly for hosts with uneven parasite loads.

Our limitations include a small sample size inherent to field studies and thus may underestimate variability. The small sample size (n = 8) limits statistical power and precludes population-level inference regarding prevalence, transmission intensity, or broader ecological patterns. The findings presented here should therefore be interpreted as descriptive and exploratory within the specific study area and sampling period. Larger-scale studies incorporating greater numbers of individuals across multiple sites and seasons will be necessary to confirm the epidemiological relevance of European rabbits in Chilean sylvatic transmission cycles. Discrete typing unit (DTU) identification was not performed in this study. The real-time PCR assay targeted the satellite DNA region, which generates short amplicons optimized for high sensitivity but unsuitable for genotyping. Furthermore, the generally low parasite loads detected in the organ tissues limit the feasibility of multilocus typing approaches from field-preserved samples. In central Chile, previous studies have documented circulation of TcII, TcV, and TcVI in wild and domestic hosts, with occasional reports of TcI [2,42]. Experimental infections have shown that different DTUs can exhibit distinct tissue tropisms and pathogenic profiles [29,43,44]. Therefore, the heterogeneous organ distribution observed in our study could potentially reflect DTU-dependent tropism, mixed infections, or repeated exposure events. Future investigations incorporating DTU typing would help clarify these patterns in naturally infected lagomorphs. In paraffin tissue sections, only one section per organ was analyzed; since T. cruzi aggregates may be missed in small fragments, it is possible that we reported false negative organs; also, brain tissues were not obtained. Despite these limitations, rigorous contamination controls, multi-method agreement, and histological confirmation strengthen confidence in these findings.

5. Conclusions

European rabbits may serve as a practical sentinel species to detect T. cruzi circulation in semiarid ecosystems, given their high abundance, invasive status, and greater bioethical accessibility for sampling than native fauna. Sentinel implementation would require structured sampling strategies, such as periodic molecular screening of rabbits in areas with a known vector presence, integration with entomological surveillance of M. spinolai, and longitudinal monitoring to detect temporal changes in infection dynamics. Because rabbits are already subject to population control and hunting, surveillance could be integrated into existing management programs without additional ecological impact. However, the applicability of this sentinel model is likely context-specific to ecosystems where rabbits and M. spinolai overlap and should not be generalized beyond comparable ecological settings in central Chile.