Chagas disease, caused by the protozoan Trypanosoma cruzi, affects 6–7 million people worldwide, with an annual incidence of 28 thousand cases in the Americas (PAHO/WHO, 2022). Human infections occur via fecal feces released by contaminated insect vectors belonging to the Reduviidae family, blood transfusion, vertical transmission, organ transplantation, laboratory accidents, and oral infections (Schmunis and Yadon, 2010; Sánchez and Ramírez, 2013). As a result of various initiatives aimed at controlling vector transmission and eradicating household vectors, the incidence of Chagas disease has been reduced in recent years (Coura and Vĩas, 2010; Rassi et al., 2010; Coura, 2015). Similarly, the massive implementation of serological screening techniques in blood banks in endemic regions has significantly reduced post-transfusion transmission of Chagas disease (Rassi et al., 2010; de Arias et al., 2022). However, in recent decades, oral transmission as a result of the ingestion of contaminated food and beverages as a result of liquefaction of whole triatomines or contamination with feces from infected triatomines has begun to occupy a relevant place in the mechanisms of transmission of Chagas disease (Coura, 2006; Coura, 2015; de Noya and González, 2015). Currently, oral transmission of T. cruzi is the most significant transmission route in Brazil (70–80% of cases) (Coura, 2006). In addition, some outbreaks have been described in several South American countries, such as Venezuela, Colombia, Bolivia, Argentina, French Guyana and Ecuador (Coura et al., 1997; Benchimol Barbosa, 2006; Dias et al., 2008; De Noya et al., 2010; Shikanai-Yasuda and Carvalho, 2012; Ramírez et al., 2013; Sánchez and Ramírez, 2013). These outbreaks were associated with consuming contaminated food and beverages such as wild animal meat, vegetables, sugar cane extract, açaí pulp, guava juice, bacaba, babaçu, and palma wine (Ferreira et al., 2001; Pereira et al., 2009; Barbosa-Ferreira et al., 2010). Furthermore, from 1968 to 2000, 50% of acute cases in the Amazon region were attributed to oral transmission (Shikanai-Yasuda and Carvalho, 2012; Bruneto et al., 2021), and these numbers later reached 70% between 2000 and 2010 (Shikanai-Yasuda and Carvalho, 2012). Venezuela has also reported the most significant outbreak described to date, with two distinct occurrences affecting 103 and 88 people, respectively. These outbreaks affect adults and children from urban and rural schools (De Noya et al., 2010; Sánchez and Ramírez, 2013). The mortality rate of orally infected patients is higher (0–44%) than that of classical vectorial transmission through triatomine fecal contamination after biting (<5–10%) (López-García and Gilabert, 2023). Oral transmission is currently the most frequent form of transmission (Coura, 2015).

Many observations have suggested that carnivorism may play a central role in T. cruzi transmission, especially in wildlife, when predators devorate their prey (Coura et al., 2002; Coura, 2006). However, this hypothesis has not been experimentally demonstrated.

In this study, we demonstrate that consuming raw meat transmits the oral infection by T. cruzi, and both trypomastigote and amastigote stages are infective for mice when administered by a nasogastric tube. Additionally, cysteinyl proteinases (among them, cruzipain) and trans-sialidase (TS) appear to be involved in parasite adhesion or invasion of gastric cells since both molecules are associated with AGS stomach cells.

The ability of TCTs to traverse a mucin layer was evaluated in microtiter plates with transwell filters coated with gastric mucin, by counting the number of TCTs traslocated through the mucin layer after 30 minutes incubation. As shown in Figure 1A , when migration experiments in the presence or absence of mucin were performed, the migration of TCTs was reduced by approximately 30% in the presence of mucin ( Figure 1A ). An even more significant reduction in migration was observed in migration experiments in the presence of mucin and pepsin at pH 3.5. In these cases, only a small number of parasites (∼ 6%) had migrated ( Figure 1A ). To examine the susceptibility of TCTs to pepsin, they were incubated for 30 min in DMEM medium pH 3.5 containing pepsin (2 mg/mL^-1). TCTs were highly sensitive to pepsin lysis, and only 25.5% of C8C3hvir parasites survived ( Figure 1B ). To gain preliminary insights into the effect of pepsin on the surface proteins of TCTs, an SDS-PAGE gel was developed. As shown in Figure 1C , expression of at least four bands between 40 and 90 kDa was reduced after pepsin treatment, suggesting that they could probably be digested by pepsin. These differences were also visualized in the densitometric analysis, where statistically significant differences were observed in the intensity of the bands in the pepsin-treated parasite run with respect to the controls that did not receive this treatment ( Figure 1D ).

Next, we evaluated the capacity of TCTs to invade AGS human stomach cells in the presence or absence of gastric mucin, in an attempt to mimic the in vivo situation in which trypomastigotes interact with and traverse the mucous layer before coming into contact with stomach epithelial cells. An invasion assay using two T. cruzi cell lines (high virulence and low virulence) showed that both T. cruzi, C8C3 hvir and C8C3 lvir could invade AGS cells. However, the C8C3 hvir cell line was at least three times more invasive than C8C3 lvir . Therefore, it was selected for experimental studies in mice. In the experiments in which mucin was present, a decrease of approximately 20% in the invasiveness of both cell lines was observed, as reflected by the lower number of intracellular parasites after 3 h of incubation compared to that observed in the absence of mucin ( Figure 2A ). As shown in Figure 2B , AGS cells were highly infective for TCTs of the virulent cell line (C8C3 hvir ), while the low virulence cell line (C8C3 lvir ) was poorly infective against these cells ( Figures 2B, C ).

To evaluate the possibility of T. cruzi transmission by carnivorism, we initially infected mice intraperitoneally, which were sacrificed in the acute phase at the peak of parasitemia ( Figure 3A ). The sacrificed animals were dissected, and raw meat was given as food to uninfected mice that had been starved for two days. Oral infection with T. cruzi in animals was monitored using parasitemia curve analysis. As shown in Figure 3B , three of the five mice (60%) were infected with T. cruzi and died due to the infection.

We determined whether the infection produced by raw meat from the infected mice resulted from trypomastigotes or intracellular amastigotes. Then, experimental infections were performed using a nasogastric probe and different parasite stages and inoculums. When a group of mice was experimentally infected with different amastigotes inocula using a nasogastric probe, all animals were infected ( Figure 4 ). One hundred % of mice were infected with an inoculum of 3 × 10⁵ cells ( Figure 4B ), whereas 80% were infected with an inoculum of 2 × 10⁵ cells ( Figure 4C ). Moreover, 60% of the mice were infected with the inoculum of 1x10⁵ ( Figure 4D ). Also, as shown in Figure 5A , trypomastigotes were infectives when inoculums of 4× 10⁵ cells were used, resulting in four of five infected mice (80%). When inoculums of 1×10⁵ were used, 4 of 5 mice inoculated (80%) were also infected ( Figure 5B ). It is well known t It is well known that the route of parasite administration is critical for the course of infection, with different courses of parasitemia and mortality. Thus, inoculation in the mouth (oral gavage) results in higher levels of parasitemia and mortality than intragastric or nasogastric infections (Barreto-de-Albuquerque et al., 2015). In oral infection experiments with TCTs or amastigotes, we preferred to use a nasogastric tube instead of oral infection. We chose this procedure for two reasons. First, since the objective was to evaluate the infectivity of TCTs and amastigotes, we decided to determine the infective capacity of the gastric mucosa, avoiding contact of the parasites with the oral mucosa or upper digestive tract. Second, we tried to avoid premature mortality in infected mice.

Next, we evaluated the virulence molecules that could be involved in parasite binding to mammalian stomach cells. To do this, we used trypomastigote or amastigote cell lysates of a recognized highly virulent T. cruzi cell line, C8C3 hvir , which expresses the main virulence factors described in T. cruzi. Thus, blots of human stomach AGS cells incubated with amastigote cell lysates were probed with different antibodies, and only antibodies raised against cruzipain and the ssp4 epitope, recognized by the monoclonal antibody 2C2, were detected ( Figure 6A ). In trials with trypomastigote lysates, we observed that only antibodies against cruzipain and transialidase were able to detect the proteins associated with AGS cells ( Figure 6B ). In contrast, CRP or gp85 were not detected by immunoblot assays (Data not shown).

To deepen our knowledge of the interaction of TCTs with human stomach cells, the experimental shaving approach and subsequent protein identification by LC/MS-MS showed 33 proteins that would eventually interact during the T. cruzi adhesion/invasion process. Considering that the AGS cell cultures were incubated with the TCTs lysate, it is possible that the proteins located in the cytoplasm and organelles are not likely to participate in the invasive processes. Therefore, considering only parasite membrane proteins, it was possible to identify trans-sialidases, cysteinyl proteinases, and Gp63 ( Table 1 ).

Based on protein LC-MS/MS identification and in silico protein-protein interaction (PPI) analysis, we mapped the interaction network of T. cruzi membrane proteins and human proteins. Nine proteins were mapped, interacting with 34 human proteins ( Figure 7 ). Among the T. cruzi proteins are cruzipain, cysteine peptidase, GP63, trans-sialidases, and mucin, among others. Human proteins involved in PPIs such Apolipoprotein L1, Cathepsin D heavy chain, Cathepsin B heavy chain, Catenin beta-1, Serum albumin, T-cell surface glycoprotein CD4 shown a high level of stomach expression level ( Table 2 ).

We then analyzed PPIs between human mucins and T. cruzi proteins ( Figure 8 ). We found six mucin proteins that interacted with 33 T. cruzi proteins. All mucins shown a high level of expression in stomach tissue, except for Mucin-15 ( Table 3 ).

Our search for cysteine protease cleavage sites in human mucins using in silico prediction returned tens to thousands of cleavage sites in mucins ( Supplementary Table 2 ). We mapped the ten best-ranked sites based on their prediction scores and found that all mucins had this score ( Figure 9A ). Our analysis allowed us to extrapolate the cleavage sites for calpain and cruzipain proteases based on the amino acid motifs present in the P1 and P2 sites of the substrates, in this case in mucins ( Figure 9B ).

Finally, to corroborate in vivo the possible role of cysteine proteinases like cruzipain, the main T. cruzi cysteine proteinase seems to play an important role in parasite binding to the stomach cells. We performed oral infection in mice using a nasopharyngeal probe, in which trypomastigotes or amastigotes were treated with or without E 64d ( Figure 10 ). Thus, as shown in Figure 10A , the infectivity of trypomastigotes previously treated with E 64d was dramatically reduced, while the three mice infected with untreated trypomastigotes developed high parasitemia ( Figure 10A ). In the same way, when amastigotes were pretreated with E64d, parasitemia was significantly decreased ( Figure 10B ).

T. cruzi infection is still highly prevalent in Latin America, where active transmission still occurs, especially in Venezuela, Colombia, and Bolivia, where vectors have reappeared (Lidani et al., 2019). However, 70% of the 1,000 cases of acute Chagas disease described in the first 10 years of this century have resulted from oral infections (Shikanai-Yasuda and Carvalho, 2012; Bruneto et al., 2021).

Initially, Chagas disease was endemoenzootic, where the T.cruzi cycle in wild environments was maintained in great part by oral infections, when different animals ingested infected vectors or through carnivorism in the prey-predator relationship (Coura et al., 2002; Coura, 2006). However, the latter hypothesis states that it naturally occurs and still occurs in the wild environments of the continent has not been confirmed, and there is no experimental evidence that meat consumption from animals infected with T. cruzi can transmit infection. Therefore, this study aimed to demonstrate the role of meat intake from infected animals in the oral transmission of T. cruzi.

To approach this question, we fed mice meat from mice in the acute phase of T. cruzi infection at the parasitemia peak ( Figure 3A ). Three of the five mice (60%) were infected with T. cruzi and died from infection ( Figure 3B ). This observation demonstrated that the intake of infected animal meat could play a role in the oral transmission of T. cruzi infection. To our knowledge, this is the first experimental evidence demonstrating the role of carnivorism in Chagas disease transmission. However, Aufderheide et al. (2004) studied 283 mummified bodies from sites in southern Peru and northern Chile and demonstrated a Chagas’ disease prevalence rate of approximately 41% over the past 9,000 years. They suggested that oral transmission might have occurred by ingesting infected, uncooked meat, such as guinea pigs (Cavia porcellus). If the raw meat from T.cruzi infected mice was used to infect the mice that were consumed, trypomastigotes or intracellular amastigotes could be responsible for the infection. Experimental infections were performed using different inoculums at these two parasite stages to investigate this.

When mice were infected with a nasogastric probe with different amastigotes inoculums, all animals were infected ( Figure 4 ). One hundred percent of the mice were infected with an inoculum of 3×10⁵ ( Figure 4B ), whereas 80% were infected with an inoculum of 2×10⁵ ( Figure 4C ). Moreover, 60% of the mice were infected with the inoculum of 1x10⁵ ( Figure 4D ). Similarly, trypomastigotes were infective when inoculums of 4 × 10⁵ were used, resulting in four of the five infected mice (80%) ( Figure 5A ). When inoculums of 1 × 10⁵ were used, 4 of 5 mice inoculated (80%) were also infected ( Figure 5B ). This evidence shows that both, TCTs and amastigotes can invade the gastric mucosa and cause oral infections. To our knowledge, the role of amastigotes in oral transmission of T. cruzi has not been evaluated. However, unlike metacyclics trypomastigotes (MT), it has been proposed that blood trypomastigotes are inefficient in infecting mice via the oral route (Hoft, 1996). The reason for these differences in infectivity between these two stages is not well known but could be due to the migration capacity of the two infectious forms through the gastric mucin layer (Cortez et al., 2012). When tested for the possible use of TCTs, it was found that the parasite traversed the mucin layer at a two-fold lower number than metacyclic trypomastigote (MT). The possibility of blood trypomastigotes being more susceptible to pepsin digestion at an acidic pH has also been proposed (Cortez et al., 2006). In our study, we observed that TCTs from the C8C3 hvir cell line were highly susceptible to pepsin lysis. This susceptibility and reduced ability to pass through mucin may explain why not all mice were infected. Similarly, differences in parasitemia peaks with respect to MT infections were observed. How can this TCTs delay the peak of parasitemia be explained? One of the most likely explanations could be based on the fact that MTs are highly resistant to degradation by pepsin (Cortez et al., 2012)). Therefore, the total inoculum administered can be used to infect stomach host cells. However, our experiments on migration through mucin in the presence or absence of pepsin at acidic pH and the in vitro infection of AGS cells in the presence of mucin showed that the situation with TCTs is completely different from that with MTs. Only a small number of TCTs cross mucin and can resist pepsin and acidic pH. Therefore, if we extrapolate this condition to what could occur in vivo, it is possible to hypothesize that only a very small portion of the TCTs inoculum would survive and invade the stomach cells. Since there is a small amount of infecting inoculum, the development of the infectious process would be delayed; therefore, the parasitemia peak would also be delayed.

However, we demonstrated that TCTs from the virulent cell line C8C3hvir and amastigotes were highly infectious in mice. Nevertheless, the origin of the observed differences in the infectivity of blood trypomastigotes and TCTs may also be due to their genetic background. Little information is available regarding the genotypes associated with oral T. cruzi infections. However, DTU 1 seems to be the most frequently encountered, where haplotipes TcId has been identified in T cruzi isolates from rats, triatomines, and patients (Marcili et al., 2009; Andrade et al., 2011; Ramírez et al., 2013; Díaz-Bello et al., 2014). As the C8C3 hvir cell line corresponds to DTU 1, this may partly explain its ability to cause oral infections. Nevertheless, studies of metacyclic forms of different strains have suggested that they are poorly infective (Maeda et al., 2016). However, our evidence using TCTs suggests otherwise. Thus, it is possible to theorize that DTU 1 could be poorly infective if transmitted by contamination with triatomine feces containing metacyclic forms of the parasite but may be highly infective when consumed in meat from infected animals having blood trypomastigotes or amastigotes.

If trypomastigotes and amastigotes are infectives by the oral route, we raise the question of which molecules could be involved in parasite binding to mammalian stomach cells. When the immunoblots of AGS cells incubated with amastigote cell lysates were incubated with different antibodies, only antibodies raised against cruzipain and the Ssp-4 epitope, recognized by the monoclonal antibody 2C2, were detected ( Figure 6A ). In trials with trypomastigote lysates, we observed that only antibodies against cruzipain and transialidase were able to detect the proteins associated with AGS cells ( Figure 6B ), whereas immunoblots in which antibodies against CRP or gp85 did not detect reactivity.

A shaving experiment was performed to confirm these observations, and proteins associated with the surface of human stomach AGS cells were identified by LC-MS/MS ( Table 1 ). Among the T. cruzi TCT membrane proteins identified, several trans-sialidases were observed in addition to cysteinyl proteinases and Gp63. The presence of trans-sialidase and cysteine proteinases was consistent with our observations derived from the western blot assays. Gp63 was not studied by western blotting. However, the role of Gp63 in T. cruzi infectivity has been suggested by Cuevas et al. (2003). On the other hand, trans-sialidase is a unique T. cruzi enzyme involved in transferring sialic acid from host glycoconjugates to mucins (Eugenia Giorgi and De Lederkremer, 2011). TS is involved in several virulence mechanisms, including invasion and host cell recognition (Magdesian et al., 2007; Dc-Rubin and Schenkman, 2012; Freire-de-Lima et al., 2012). The presence of sialylated epitopes on the surface of infective forms of T. cruzi increases infectivity (Piras et al., 1987; Schenkman et al., 1991). Similarly, sialylation and desialylation of the host cell surface can modulate parasite adherence and penetration (Freire-de-Lima et al., 2012). Therefore, it is possible to hypothesize that stomach mucus could be a source of sialic acid that positively regulates the invasion of the stomach epithelium through parasite TS activity. Considering that among the molecules detected both in the blots and in the shaving experiment associated with LC-MS/MS, we identified cruzipain and cysteine proteases, we decided to deepen our knowledge regarding the potential role of this protease in oral T. cruzi infection.

We then performed oral infection in mice, in which trypomastigotes or amastigotes treated with or without E64d were used to infect the mice ( Figure 10 ). As shown in Figure 10A , the infectivity of trypomastigotes previously treated with E64d was dramatically reduced, whereas three mice infected with untreated trypomastigotes developed high parasitemia ( Figure 10A ). In contrast, parasitemia significantly decreased when amastigotes were pretreated with E64d ( Figure 10B ). These results suggest that cruzipain, the principal T. cruzi cysteinyl proteinases (and other parasite cysteine proteinases), plays an important role in parasite association with stomach cells. According to electron microscopy studies, Czp is located in the flagellar pocket of the TCTs being the main secreted lysosomal peptidase in T. cruzi (Souto-Padrón et al., 1990). Several studies using synthetic irreversible cysteine peptidase inhibitors have demonstrated that T. cruzi infection, host immune evasion, and intracellular growth depend on Czp activity (Meirelles et al., 1992; Tomas et al., 1997; Scharfstein et al., 2000; McKerrow et al., 2008; Doyle et al., 2011). Pretreatment of TCTs of the C8C3 hvir cell line with E64d has been shown to significantly inhibit their infectivity both in vivo and in vitro (San Francisco et al., 2017). In the stomach, the gastric mucosa is the first natural barrier responsible for protecting the host against internal insults, such as the acidic pH of gastric juice, and external insults, such as microorganisms (Deng et al., 2021). Thus, cruzipain and other cysteinyl proteinases may play a critical role in the invasion of TCTs into the gastric mucosa. Although the molecular mechanisms involved in this process are unknown, it is possible to speculate that these proteases could participate in mucus degradation, an event before parasite contact with stomach epithelial cells. The participation of cysteinyl proteinases in the degradation of gastric mucus before parasite contact with the intestinal epithelium has been described in Entamoeba histolytica (Lidell et al., 2006).

In MT, it has been described that the surface glycoprotein gp82 plays a central role in establishing T. cruzi infection in mice via the oral route (Yoshida, 2008). gp82 is critical for oral T. cruzi infection because it binds gastric mucin in the mucus layer, which protects the stomach mucosa (Staquicini et al., 2010). It has been proposed that upon binding to gastric mucin, MT migrates through the mucus layer and reaches the underlying epithelial cells, invading in a gp82-mediated manner (Neira et al., 2003; Cortez et al., 2006; Covarrubias et al., 2007). However, no trypomastigote molecules have been reported to play a role in bloodstream trypmomastigotes or their counterparts in TCTs.

Here, we observed that cruzipain and transialidase were involved in parasite binding to stomach cells ( Figure 6 ). In contrast, cruzipain digests fibronectin (FN). The interaction between fibronectin and T. cruzi has also been previously described. Experiments with TCT revealed the involvement of fibronectin in target cell adhesion/invasion (Ouaissi et al., 1985). Using T. cruzi CL strain metacyclic forms, it was reported that fibronectin present in the cell culture medium bound to metacyclic forms is digested by cruzipain. Treating HeLa cells with purified recombinant cruzipain increased parasite internalization, whereas treatment with cysteinyl proteinase inhibitors had the opposite effect. This evidence confirms that the cysteine proteinase of T. cruzi metacyclic forms, through its fibronectin-degrading activity, is involved in host cell invasion (Maeda et al., 2014). We speculated that cruzipain acts on stomach cells to degrade fibronectin and other glycoproteins in the extracellular matrix and promotes parasite invasion.

Several pieces of evidence strongly suggest that C8C3 hvir virulence is related to the differential expression of well-known virulence factors, including Czp and TS (San Francisco et al., 2017). In addition, parasite treatment with E64d strongly reduced parasite oral infection capacity and parasitemia levels in BALB/c mice. Thus, the concerted action of cysteinyl proteinases and TS could contribute to the success of TCTs in the invasion of the gastric mucosa. Although the molecule that binds gastric mucins is unknown, trans-sialidase could act on the O-linked mucins present in the mucous layer of the stomach, removing sialic acid and sialydating the parasite membrane, making it more invasive. On the other hand, cysteinyl proteinases could degrade the gastric mucins, favoring the progression of TCTs through the mucous layer to finally access the stomach epithelium, where the classical mechanisms of invasion could operate.

Our in silico analysis of PPIs allowed us to identify important T. cruzi proteins involved in interactions with host proteins, including trans-sialidases and cruzipain, as well as proteins identified by LC-MS/MS in our proteomic dataset. Furthermore, these proteins interact with human proteins with high expression in stomach tissue, as seen in the gene-tissue association analysis.

Our interaction analysis involving host mucins, despite having demonstrated interaction with gp85 or gp82, nevertheless demonstrated that human mucins interact with T. cruzi protein complexes that are related to the transfer of sialic acid, such as the CMP-sialic acid transporter, putative, and trans-sialidase itself.

Since the activity of cysteine proteases is important in host cell interaction and invasion, we predicted substrate cleavage sites for cysteine proteases in mucin proteins, specifically for calpains, an important family of Ca⁺²-dependent cysteine proteases that contain a nucleophilic cysteine in the catalytically active site. The expression and cellular localization of T. cruzi calpains revealed that are distributed in different cellular components in all life cycle forms of the parasite, including plasma membrane (Ennes-Vidal et al., 2020) and thus, if calpains cleave mucins, they can regulate the interaction of the parasite in stomach cells. Thus, we predicted the calpain cleavage sites in mucins that were mapped in the interaction network with T. cruzi proteins. Site prediction showed that mucins have several cleavage sites for the cysteine protease calpains ( Figure 9A ). Another important family of cysteine proteases are cathepsins, which include cruzipain. The studies on substrate profiling of cysteine proteases showed that cruzipain has a preference for hydrophobic residues (such as Leucine and Glycine) in the P2 position of the substrates (Choe et al., 2006; Dos Reis et al., 2006; Ren et al., 2009; Wang et al., 2023). Our k-mer probability logo analysis of sequence window upstream predicted sites ( Figure 9B ) corroborates these previous results for cruzipain and reinforces the potential relationship of T. cruzi cysteine proteases, both calpain and cruzipains, with the invasion of stomach cells.

In summary, it was concluded that, under our experimental conditions, consuming meat from infected animals in the acute phase allows the transmission of T. cruzi infection. However, the possibility that oral transmission may be feasible when animals are in the indeterminate or chronic phase cannot be ruled out. At these stages of T. cruzi infection, the success of oral transmission may depend on the organ and tissue parasite load and the genetic haplotype of T. cruzi, which could determine its ability to resist acidic pH, cross mucins, and infect gastric cells. Thus, it is possible to postulate that the DTUs involved may be critical to the success of oral infection. Indeed, Hoft et al. (1996) reported poor infectivity of blood trypomastigotes in Tulahuen strain (TC VI). On the other hand, Cortez et al. 2012, also suggested that trypomastigotes were poorly infective but used Y strain (Tc II). In addition, the intrinsic virulence of each strain or isolate, indicated by a higher expression of virulence factors such as Cz, TS, CRP, and gp63, among others, should be considered (Osorio et al., 2012). Furthermore, upregulation of bioenergetic and biosynthetic pathways has been proposed as a new aspect to be considered in T. cruzi virulence (San Francisco et al. 2022). In addition, the genetic background of the host should also be considered, and a strain or isolate of a given DTU that is infective for one species might not be infective for another.

Transmission via the oral route is an emerging scenario in Chagas disease. This transmission mechanism displays a habitual character in the primitive and endemic cycle of the parasite, which occurs through consuming contaminated food and/or beverages by dejection of triatomine feces (PAHO/WHO, 2022). Here, we experimentally demonstrate that carnivorism is a mechanism of T. cruzi transmission. Although this is perhaps the major mechanism by which parasite infection is transmitted in the wild, the possibility of human transmission cannot be ruled out. In endemic areas where peridomestic and wild vectors are present concomitantly with rabbits, guinea pigs, lambs, and others, the meat of infected animals could be a source of human infection. Therefore, sanitary education of the population is necessary to prevent the ingestion of raw meat and to encourage the use of previously frozen or cooked meat. Therefore, if Chagas disease is a food-borne infection, implementing prophylactic measures, such as sanitization, freezing, or using heat, accompanied by qPCR detection methods, should be considered (de Oliveira et al., 2019). Furthermore, implementing health education programs for populations living in endemic areas of potential risk from the perspective of oral transmission of Chagas disease and the implementation of traveler advisories for tourists visiting these areas is an absolute necessity. These traveler advisories should emphasize avoiding the consumption of tropical fruit juices, pulps, and exotic meats (Franco-Paredes et al., 2020; López-García and Gilabert, 2023). Thus, considering that oral transmission of Chagas disease involves animals, the environment, and humans, only a concerted strategy under the One Health concept would succeed in controlling this form of transmission of this parasitic infection (López-García and Gilabert, 2023).

Finally, experimental studies will be necessary to define the differences observed in the infectivity of blood trypomastigotes or their equivalents, the TCTs. These should be oriented to define whether their infectivity depends on the genetic background of the parasite, on the infected species and its genetic background, or both.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

The animal study was approved by Comité de Ética de la Investigación Científica de la Universidad de Antofagasta. The study was conducted in accordance with the local legislation and institutional requirements.

VT: Conceptualization, Investigation, Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Methodology, Validation. VC: Investigation, Data curation, Formal analysis. BG: Data curation, Investigation, Methodology, Writing – review & editing, Software, Supervision. JSF: Investigation, Methodology, Data curation, Software, Supervision, Writing – review & editing. AC: Data curation, Investigation, Software, Formal analysis, Resources, Writing – review & editing. JLV: Data curation, Investigation, Methodology, Formal analysis, Funding acquisition, Resources, Writing – review & editing. KM: Data curation, Investigation, Resources, Software, Methodology, Writing – original draft. LJF: Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Writing – review & editing, Conceptualization, Software. RdA: Bioinformatic analysis, Writing-review and edition. AK: Funding acquisition, Writing – review & editing. JG: Conceptualization, Funding acquisition, Resources, Writing – review & editing, Investigation, Supervision, Visualization, Writing – original draft.