Impact of gastrointestinal inoculation and benznidazole treatment on Trypanosoma cruzi II infection in mice

doi:10.21203/rs.3.rs-2239490/v1

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Impact of gastrointestinal inoculation and benznidazole treatment on Trypanosoma cruzi II infection in mice

https://doi.org/10.21203/rs.3.rs-2239490/v1

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The protozoan Trypanosoma cruzi causes Chagas disease and the most frequent form of transmission of the parasite is the oral route, associated with greater severity and worse response to benznidazole (BZ), the drug used in its treatment. This study aimed to evaluate the impact of gastrointestinal infection (GI) and BZ treatment on the histopathological alterations in mice inoculated with T. cruzi II. Swiss mice were inoculated by GI and intraperitoneal (IP) routes with 2x10⁶ culture-derived metacyclic trypomastigotes of the Y strain (TcII) of T. cruzi and were treated with BZ in the acute phase of the infection. Fresh blood examination, qPCR, histopathological and biochemical evaluations (enzymatic dosages and oxidative stress-OS) were performed. BZ treatment of uninfected animals caused changes in the liver, increased the activity of AST and ALT enzymes and OS, showing that the drug alone affects this organ. Inflammation and necrosis in the cardiac tissue were less intense and deaths occurred later in animals inoculated via the GI route than the animals inoculated via the IP route. BZ reduced the intensity of tissue lesions and avoided lethality in animals inoculated via the GI route, and decreased parasitemia and OS in those inoculated via both routes. Although BZ alone caused liver damage, it was less intense than that caused by both routes of inoculation. Infection with the Y strain of T. cruzi II via the GI route proved to be less virulent and pathogenic and responded better to treatment than the infection acquired via the IP route.

Trypanosoma cruzi

oral Chagas disease

benznidazole

qPCR

aminotransferases

oxidative stress

Trypanosoma cruzi is a hemoflagellate protozoan, belonging to the order Kinetoplastida and family Trypanosomatidae, which is the etiologic agent of Chagas disease (CD), also known as American trypanosomiasis [1]. Approximately 6 to 7 million people worldwide are currently infected with T. cruzi, particularly in Latin American countries [2]. The prevalence of CD is higher in countries where the vector transmission of T. cruzi occurs, whether in the classic form, after the blood meal of a triatomine bug, or by ingesting food contaminated with the crushed vector and/or its feces [3]. Oral transmission, although accidental, has emerged as the main form of transmission of CD in Brazil, accounting for 75% of new cases [4]. The majority of patients (95 to 98%) in the acute phase of CD are asymptomatic [5]. However, myocarditis and meningoencephalitis can occur, leading to death, especially in children. Other symptoms can include fever, lymphadenopathy, and hepatosplenomegaly. During the chronic phase, 50–70% of infected patients are asymptomatic (indeterminate form), 25–30% have the cardiac form, and 12 to 15% have digestive manifestations [6].

T. cruzi has widely heterogeneous populations, consisting of a variety of strains that circulate in the domestic and wild transmission cycles between insect vectors, humans, and animal reservoirs [7, 8]. The genetic lineages of the parasite are classified into six discrete typing units (DTUs), from TcI to TcVI [9]. Although a seventh DTU, TcBat, was recently described, after it was isolated from bats, which was subsequently found to be capable of infecting humans. These DTUs may have different geographic distributions and are associated with different clinical and epidemiological features. TcII, for example, is often isolated from the blood of chronic patients and is associated with the domestic cycle of transmission in the countries of the Southern Cone of South America, where cardiac manifestations of the disease predominate, but digestive manifestations are also found [10].

Oral transmission is probably the oldest strategy for the spread of T. cruzi among mammals. However, the intraperitoneal (IP) inoculation route is the most used in experimental in vivo infection, mainly in mice. Among the factors related to the parasite that can influence the evolution of the experimental infection are the origin of trypomastigotes (infective forms), in that they can either be isolated from blood or culture, the inoculation route, inoculum volume and concentration, and the parasite DTU [11].

T. cruzi strains may present differentiated tissue tropism, being classified as myotropic, preferentially parasitizing muscle tissue; or macrophagotropic, when they parasitize lymphoid organs. The latter is the case of the Y and Berenice 62 strains of T. cruzi, which, in addition to the myocardium, have been reported to cause intense tissue parasitism in the liver and spleen of Swiss mice inoculated via the IP route with 100,000 blood trypomastigotes (BT) [12]. On the other hand, it was observed that during the acute phase of the experimental infection of BALB/c mice with 1,000 BT of the Y strain (inoculation route not described), although amastigote forms were rarely found in the liver, the inflammation was commonly observed, indicating that this organ is also affected, even with a lower parasite inoculum [13].

The parasite is exposed to reactive oxygen species generated by its own aerobic metabolism, the host's immune response, and those released by drugs used in the treatment of CD [14]. Thus, T. cruzi infection can lead to tissue oxidative stress (OS), which, associated with toxic parasite secretions and a cytotoxic immune response, can contribute to tissue destruction [15]. In the blood of BALB/c mice orally inoculated with the Tulahuén (TcVI) strain, but not treated with BZ, an increase in AST and ALT enzymes was observed [3]. These authors also found that in infections via the oral or gastrointestinal (GI) routes, the stimulated immune response can lead to a more severe infection compared to the IP route, affecting the heart and, more intensely, the liver of the animal.

The drug used for the etiological treatment of CD is benznidazole (BZ), a nitroheterocyclic derivative that has low efficacy during the chronic phase and important adverse reactions, in addition to alterations in liver enzymes that can be indicative of tissue damage [16]. It has already been demonstrated, experimentally in vivo, that the response to treatment with BZ can vary according to the genotype of T. cruzi [17, 18], as well as its DTU [19, 20, 21]. In addition to altering aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, BZ treatment of mice that had been infected IP with an inoculum as low as 500 BT caused a further increase in OS [22].

We hypothesize that GI infection with the Y strain (TcII) of T. cruzi is more severe, in terms of virulence and pathogenicity for mice, and responds worse to treatment with BZ, than the infection via the IP route. Given the above, we aimed to investigate the impact of the parasite inoculation route (GI and IP), and treatment with BZ, in the experimental infection of Swiss mice with the Y strain (TcII, macrophagotropic) of T. cruzi during the acute phase of the infection, using parasitological (parasitemia and parasite load detected by qPCR), biochemical (dosage of liver enzymes and OS), and histopathological parameters in the evaluation of the animals.

• Infectiousness and survival

Mice inoculated with 2x10⁶ culture-derived metacyclic trypomastigotes (CMT) of the Y strain of T. cruzi (TcII) presented 100% of infectivity rate (%INF), when infected by either the GI or IP routes (data not shown). During the acute phase of the infection, the survival rate of infected animals, both GI and IP, and not treated with BZ, was 50%. However, as can be seen in the Kaplan-Meier graph, the deaths of animals inoculated by the GI route occurred later, indicating lower virulence by this inoculation route (p = 0.007) (Fig. 1). Treatment with BZ avoided lethality in animals inoculated by both routes, during the period of the experiments.

• Parasitological And Molecular Parameters

The mean parasitemia curves of animals inoculated by both routes (GI and IP) are shown in Fig. 2a. The untreated (NT) animals, inoculated via the GI route, had parasitemia levels three times lower than those inoculated via the IP route, in addition to a later day of maximum parasitemia peak (Dpmax, 13th day of infection), when compared to the IP-inoculated animals, whose maximum peak (Pmax) occurred on the 8th day of infection (d.i.) (Fig. 2a).

Regarding the other parasitological parameters derived from the parasitemia curve and obtained from the fresh blood examination (FBE) [pre-patent period (PPP), patent period (PP), Pmax, and the percentage of animals with a positive FBE (%+FBE)], and molecular parameters [percentage of mice with positive real-time polymerase chain reaction (%+qPCR) and the mean parasite load], in the comparison between NT animals inoculated via the GI and via the IP routes, significant differences were observed in three parameters (Table 1 and Fig. 2a). In animals inoculated via the GI route, only the PPP was higher than in the IP-inoculated animals, indicating lower virulence of the infection via the GI route, since the higher the PPP, the lower the virulence.

Table 1

Statistical comparisons (mean and standard deviation) of the parasitological and molecular parameters of mice inoculated via the gastrointestinal (GI) and intraperitoneal (IP) routes with 2x10⁶ culture-derived metacyclic trypomastigotes of the Y strain (TcII) of *Trypanosoma cruzi*, treated with benznidazole (BZ 100 mg/kg/day, 20X) from the 5th day of infection, and in untreated controls (NT).
	GI			IP
Parameter	NT	BZ	p	NT	BZ	p
PPP^A (days)	9.5 ± 1.4^*	AP^G	< 0.001	4.1 ± 0.6^*	3.6 ± 0.5	NS
PP^B (days)	11.6 ± 2.0^**	AP	< 0.001	14.3 ± 3.3^**	2.6 ± 0.5	< 0.001
Pmax^C (x10³)	75.7 ± 26.4^*	AP	< 0.001	250.9 ± 154.7^*	5.7 ± 1.3	< 0.001
Dpmax^D	13.2 ± 2.3	AP	< 0.001	10.3 ± 3.1	4.6 ± 0.5	< 0.001
%+FBE^E	100.0 (10/10)	AP	≤ 0.05	100.0 (10/10)	20.0 (2/10)	≤ 0.05
%+qPCR^F	100.0 (5/5)	100.0 (6/6)	NS^H	100.0 (3/3)	100.0 (5/5)	NS
Number of significant reductions			5/6			4/6
^A Pre-patent period; ^B Patent period; ^C Number of trypomastigotes / 0.1 mL of blood at parasitemia peak; ^D day of the maximum peak; ^E Percentage of animals with positive fresh blood examination; ^F Percentage of animals with positive qPCR; ^G Absent parasitemia; ^H Not significant. When comparing the inoculation routes of NT animals, values with * (p < 0.001) and ** (p ≤ 0.05) on the same line are significantly different. The nonparametric Mann-Whitney test was used.

The %+FBE and %+qPCR were 100% for animals infected by both routes and not treated (Table 1). In untreated animals, the parasite load was 0.6 equivalent parasites (par. eq.) per mL of blood for those inoculated via the GI route and 2.3 par. eq./mL for those inoculated via IP route, with no statistically significant difference between the groups (Fig. 2b).

• Impact Of Etiological Treatment On Parasitological And Molecular Parameters

The parasitemia of animals inoculated both by the GI and IP routes was suppressed by the treatment with BZ from the first day of treatment (Fig. 2a). In animals inoculated via the GI route, BZ treatment promoted a significant reduction in 5/6 parasitological parameters (PPP, PP, Pmax, Dpmax, and %+FBE), while in those inoculated via the IP route, the reduction occurred in 4/6 (PP, Pmax, Dpmax, and %+FBE) (Table 1).

The qPCR was positive in all animals inoculated via both the GI and IP routes, and in both the treated and untreated groups (Table 1), with the BZ treatment not significantly altering the parasite load of animals (Fig. 2b).

• Histopathological Parameters In Cardiac Tissue

Amastigote nests were not observed in the histological sections of cardiac tissue of infected animals in any of the groups, which were submitted to euthanasia on the 47th d.i. Except for the animals in the uninfected and untreated (NI) (Fig. 3a) group, inflammatory processes were observed in all other experimental groups, including the animals of the uninfected group that were treated with BZ (NI + BZ) (Fig. 3b, 3c, 3d, 3e and 3f).

No animal in the NI group had cardiac tissue damage (Fig. 3a and 3g). A moderate inflammatory process was observed in 83.33% (5/6) of the animals in the infected via GI route and untreated (GINT) group (Fig. 3g), with focal epicarditis and myocarditis (Fig. 3b), while an intense inflammatory process was observed in 100% (2/2) of the surviving animals in the infected via IP route and untreated (IPNT) group (Fig. 3g), with diffuse myocarditis, necrotic cardiac fibers, areas of myocardial fibrosis, and connective tissue deposition as the initial form of the healing process (Fig. 3c). The animals in the GINT group presented mild (50%; 3/6) to moderate necrosis (33.3%; 2/6), while the two survivors (100%) in the IPNT group presented intense necrotic tissue. Five (83.3%) animals in the GINT group had mild fibrotic tissue, while the other (16.6%) had moderate fibrotic tissue, whereas the two surviving animals in the IPNT group had intense fibrotic tissue (p < 0.05) (Fig. 3g). Although these changes were observed in animals infected by both routes and not treated, they were more intense (+++) in animals from the surviving IPNT group (Fig. 3g).

• Histopathological Parameters Of Liver Tissue

Histological analysis of the liver of NI animals showed normal morphology with hepatocytes organized in well-defined cell strands, as expected (Fig. 4a).

In the liver of animals infected both by the GI and IP routes and not treated, amastigote forms were also not detected. However, focal inflammatory processes were observed in the liver parenchyma and the periphery of the portal triad region (Fig. 4b) and, in some cases, of the central vein (Fig. 4c). Animals of the GINT group displayed a focus of hepatitis with intensity ranging from mild (4/6; 66.6%) to moderate (2/6; 33.3%), while in those of the IPNT group, focal hepatitis was classified as intense in the two surviving animals (100%) (Fig. 4g). Although no statistical differences were observed between untreated animals inoculated via the GI and IP routes, in the animals of the GINT group, necrotic tissue was not observed, and 100% (6/6) of them had mild fibrotic tissue. On the other hand, the two surviving animals in the IPNT group had moderate necrotic tissue (100%) and fibrotic tissue ranging from moderate (1/2; 50%) to intense (1/2; 50%).

• Impact Of Etiological Treatment On Histopathological Lesions

The animals in the uninfected but BZ-treated group had a mild inflammatory process in the heart (Fig. 3d), with a significant difference (p < 0.05) compared to the animals in the NI group (Fig. 3g).

When comparing the hearts of animals in the infected and BZ-treated groups with their respective untreated controls, for each inoculation route, significant differences were observed only between the GI-inoculated animals, regarding inflammatory process and necrotic tissue (Fig. 3g). Treatment with BZ promoted a significant reduction (p < 0.05) in the intensity of the inflammatory process with 6/6 (100%) of the infected via GI route and treated (GI + BZ) animals presenting a mild inflammatory process (Fig. 3e), against 5/6 (83.3%) and 1/6 (16.6%) of the GINT group that presented a moderate or an intense inflammatory process, respectively (Fig. 3g). The treatment of GI-inoculated animals also promoted a significant reduction (p < 0.05) in the number of animals with necrotic tissue and the intensity of these lesions, with no animal in the GI + BZ group presenting necrotic tissue against 3/6 (50.0%) with moderate and 2/6 (33.3%) with mild necrotic tissue in the untreated group (GINT) (Fig. 3g). Therefore, treatment with BZ promoted benefits in animals that had been infected via the GI route. On the other hand, due to the high mortality caused by IP infection route, the number of animals remaining for evaluation in both IP groups was reduced, infected via IP route and treated (IP + BZ) (n = 4) and IPNT (n = 2). Thus, no significant differences were observed regarding heart lesions, between treated and untreated animals, inoculated by the IP route (Fig. 3g).

The liver tissue of uninfected animals that had been submitted to treatment with BZ presented a mild inflammatory process (Fig. 4d- red arrow) in 3/5 (60%) animals and moderate inflammatory process in 1/5 (20%) of the animals evaluated, with a significant difference (p < 0.05) in relation to the NI group, indicating that there was tissue damage in the liver caused by BZ (Fig. 4g), with greater intensity than that observed in the cardiac tissue.

Regarding the histopathological lesions of the liver, there were no significant differences between the animals infected by both routes and treated with BZ (Fig. 4e and 4f) and their respective untreated controls (Fig. 4b and 4c). Although without a statistical difference, the BZ treatment of the animals inoculated via the GI route apparently promoted an increase in the intensity of the lesions compared to the untreated animals (Fig. 4e). The opposite occurred with the treatment of animals inoculated by the IP route (Fig. 4f). Since only two animals in the IPNT group survived, suggesting greater lethality of the CMT forms of the Y strain (TcII) when inoculated by the IP route, the small number (n) of animals in this group interfered in the result of the statistical analyses. In animals infected with the macrophagotropic Y strain, these data suggest that BZ may increase the intensity of lesions in the liver (Fig. 4g), the organ where it is metabolized, promoting fewer benefits for the GI infection compared to IP infection, and differently from what was observed in the heart (Fig. 3g).

• Biochemical Parameters

The results of the measurements of plasma liver enzymes and hepatic OS from the different experimental groups are shown in Table 2. In NT animals infected via the GI route, the enzymes AST and ALT, but not alkaline phosphatase (ALP), presented higher values (p ≤ 0.05) than the NI animals. In NT animals infected via the IP route, only the ALT enzyme showed a significant change (p ≤ 0.05) compared to NI animals (Table 2). These results show that infection, mainly via the GI route, promotes an increase in liver enzymes.

Table 2

Plasma markers and oxidative stress parameters of liver injury in mice inoculated via the gastrointestinal (GI) and intraperitoneal (IP) routes with 2x10⁶ culture-derived metacyclic trypomastigotes/animal of the Y strain (TcII) of *Trypanosoma cruzi*, treated with benznidazole (BZ) (100 mg/kg/day, 20X), and untreated controls. Data are the mean ± standard error of 3–6 animals.
Experimental groups
Parameters	NI	NI + BZ	GINT	GI + BZ	IPNT	IP + BZ
Plasma parameters of liver damage (U·L^− 1)
AST	70.9 ± 5.0	97.5 ± 7.6^*	140.0 ± 9.8^*	91.7 ± 8.3^**	93.7 ± 12.8	82.9 ± 4.7
ALT	51.1 ± 4.4	94.7 ± 6.3^*	81.6 ± 5.3^*	61.1 ± 4.3^**	76.0 ± 9.1^*	43.2 ± 3.9^**
ALP	143.7 ± 12.0	132.7 ± 12.5	124.4 ± 13.3	157.5 ± 13.4	212.8 ± 31.6	210.6 ± 27.8
Hepatic oxidative stress parameters
GSH (nmol·mg^− 1)	8.24 ± 0.11^*	6.02 ± 0.65^*	3.47 ± 0.35^**	6.19 ± 0.72^*	6.12 ± 0.21^*	7.80 ± 0.63^*
Catalase (mmol·min^− 1·mg^− 1)	0.26 ± 0.03	0.43 ± 0.04	0.39 ± 0.04	0.34 ± 0.05	0.33 ± 0.03	0.29 ± 0.05
Carbonyl groups (nmol·mg^− 1)	2.60 ± 0.14^*	3.87 ± 0.20^**	3.46 ± 0.28^**	3.95 ± 0.1^**	3.98 ± 0.12^**	3.21 ± 0.09^,*
NI, uninfected; NI + BZ, uninfected and treated; GINT, inoculated via the gastrointestinal (GI) route and untreated; GI + BZ, inoculated via the GI route and treated; IPNT, inoculated via the intraperitoneal (IP) route and untreated; IP + BZ, inoculated via the IP route and treated. GSH, Reduced glutathione.
For plasma parameters of liver damage: values with * are different from NI; and ** are different from NT, within the same route (p ≤ 0.05).
For hepatic OS parameters, values with different symbols (* and *), on the same row, are different (p ≤ 0.05*).

In the liver OS parameters of NT animals, only infection via the GI route resulted in decreased levels of reduced glutathione (GSH), which means an increase in tissue OS in this condition. Infection both by the GI and IP routes caused an increase in protein carbonyl groups, suggesting an increase in tissue injury (Table 2).

• Impact Of Etiological Treatment On Biochemical Parameters

In the plasma parameters of liver damage, treatment with BZ in uninfected animals (NI + BZ group) caused a significant increase (p ≤ 0.05) in the levels of AST and ALT enzymes, showing that the drug, by itself, affects the liver (Table 2). Treatment with BZ, however, reduced the AST enzyme level in animals infected via the GI route, and ALT in animals infected by both routes, showing that the treatment reversed the effects of infection and promoted some benefits (Table 2). For the ALP enzyme, there was no significant alteration caused either by the infection or the drug.

In the analysis of the OS parameters, it was observed that BZ caused a significant increase only in the carbonyl groups of proteins for the animals in the NI + BZ group (Table 2). For animals infected via the GI route, a significant increase in GSH was observed in treated animals compared to GINT (Table 2). In the animals infected via the IP route, although BZ promoted a significant reduction in the carbonyl groups, the values of this parameter did not return to the level of NI animals.

Despite the wide diversity in T. cruzi strains and DTUs, the Y strain of T. cruzi, belonging to the DTU TcII, is one of the most used in experimental studies around the world. Initially, in the treatment with BZ and nifurtimox of Swiss mice inoculated via the IP route with BT forms, the Y strain was considered partially resistant to drugs in vivo, since around 50% of treated animals were not considered cured [23, 24]. However, in these studies, the authors did not use cyclophosphamide (Cy) immunosuppression or the qPCR technique to monitor the cure of animals undergoing etiological treatment. In the current study, in which stricter cure criteria was used, we obtained a cure rate of 0% in Swiss animals inoculated with the Y strain by both the GI and IP routes with the same inoculum and treated with the same BZ regime. This T. cruzi strain should therefore be considered resistant to BZ, since no animal treated was cured, which is corroborated by the literature when similar cure criteria was used [25].

In the present study, the infection of mice with CMT of the Y strain (TcII) by both inoculation routes (GI and IP) caused 50% mortality in these animals within the experimental period. However, deaths occurred later in the animals inoculated via the GI route, suggesting a lower severity of this inoculation route for this T. cruzi strain. Nonetheless, this needs to be verified with other strains of the same DTU. Dias et al. [11], using trypomastigotes of the same origin and strain, and in the same inoculum, in the infection of animals, also found no differences in the mortality rate between the two inoculation routes; however, they observed mortality rates higher than those in the present study: 90% and 100%, respectively, for the GI and IP routes. Differences in the maintenance time of the strain in the culture medium, before inoculation of the animals, can lead to differences in its infectivity and lethality for mice. However, in the current study, BZ prevented lethality in the infection by both routes, even though the mice were not parasitologically cured by the etiological treatment, which is similar to that which was observed in the study of Zanusso Jr et al. [25].

When comparing the parasitological parameters between the routes (GI x IP), the levels of parasitemia were about three times lower following inoculation by the GI route compared to the IP route, suggesting lower virulence of the Y strain via the GI route. These data corroborate the histopathological findings observed in the cardiac tissue of these animals, in which an inflammatory process (epicarditis and myocarditis), necrotic cardiac fibers, and a healing process (tissue fibrosis) of lower intensity were observed in animals inoculated via the GI route when comparing with the animals inoculated via the IP route.

Animals infected via the GI route also had higher PPP and lower parasite compared to those inoculated by the IP route, although the difference in parasite load was not significant, due to the low number of animals (3/6) surviving in the group infected via the IP route (Fig. 2b). Blood samples used to determine parasite load by qPCR were collected on the 27th d.i., which corresponds to the beginning of the chronic phase in the experimental infection. Dias et al. [11] also observed lower virulence for mice infected with CMT of the Y strain via the GI route compared to the IP route, when considering the parameters PPP, PP, and maximum peak of parasitemia.

When evaluating the impact of BZ treatment on parasitological parameters derived from the parasitemia curve (PPP, PP, Pmax, and Dpmax) and %+FBE, as well as %+qPCR, a significant reduction was observed in 4/6 parameters, in animals infected by both the GI and IP routes. In addition, BZ suppressed the parasitemia in animals infected by both inoculation routes evaluated (GI and IP) during the entire course of treatment, which is in agreement with the literature, where a cure rate of 50% has been reported for IP-inoculated animals [23, 24], and 0 to 12.5% for GI-inoculated animals [25] for the Y strain infection. In other words, parasitemia is suppressed by treatment with BZ, even in mice infected via the GI route despite lower rates of cure. However, in the present study no significant differences were observed in the parasite load between the inoculation routes or between treated animals and untreated controls.

Important adverse reactions caused by the antiparasitic drugs BZ and nifurtimox (NX), used in the treatment of CD, have already been reported in the literature, including areas of necrosis, periportal inflammatory infiltrates, and vascular congestion in the liver [26, 27]. On the other hand, in the Brazilian Amazon region, familial microepidemics of acute CD, acquired by oral transmission, through the ingestion of food contaminated by the parasite, mainly palm fruits such as açaí, have also been observed. These cases present more severe conditions than those acquired by the classical vector route (contamination by triatomine excreta) and may present greater lethality [19, 28]. Medicines administered orally, as well as pathogens acquired by this route, after being absorbed by the intestinal gastric mucosa, are transported to the hepatic portal system [3]. In addition, the liver is affected in the acute T. cruzi infection and has the function of clearing blood trypomastigotes [13, 29].

In the cardiac tissue of the groups infected and treated with BZ, a decrease in tissue damage was observed only in the animals inoculated via the GI route. The intensities of the lesions went from moderate to mild, which did not happen with the animals inoculated by the IP route, in which the lesions remained intense, even after the etiological treatment. That is, in infection with the Y strain (TcII), in addition to the GI route causing less intense histopathological changes in the heart, the animals infected via this route responded better to treatment with BZ than those infected via the IP route in terms of reducing cardiac lesions. In a study with Swiss mice inoculated by the IP route with two strains of TcII (PR1219 and PR2259), an increase in the number of tissue lesions in different organs (including the heart) of animals treated in the acute phase with BZ, was observed during the chronic phase of infection. However, this number was still lower than in infected animals that were left untreated [21]. As the parasite load becomes controlled, inflammatory and oxidative processes decrease in skeletal muscle, colon, and stomach tissues; however, mitochondrial function deterioration, which is responsible for the production of free radicals, persists in the heart muscle, contributing to the lesions found in CD [27, 28].

In the present study, the analysis of plasma enzymes showed an increase in AST and ALT levels only in the infection via the GI route, and there was no significant change in ALP by either parasite inoculation route. These data suggest damage at the hepatocellular level in GI-inoculated animals, leading to the release of these enzymes into the bloodstream and an increase in their serum levels [30]. Increased AST levels indicate hepatic necrosis and may occur in parasitic infections. The ALT enzyme is found in high concentrations in the liver and its origin is predominantly cytoplasmic, being a sensitive marker of liver damage. These data suggest that the Y strain (TcII, macrophagotropic), when inoculated via the GI route, unlike what was observed in the heart, causes more changes in the liver than when inoculated via the IP route. These data corroborate Barreto-de-Albuquerque et al. [3], who found that BALB/c mice orally inoculated with the Tulahuén (TcVI) strain had liver damage, given the increased serum levels of AST and ALT, which was further confirmed by histology.

However, our histopathological findings showed more intense lesions in the liver tissue of animals inoculated via the IP route, even though they were not significant due to the low number of surviving animals; there was an increase in hepatic sinusoids due to obstruction of blood vessels, caused by fibrosis, and also by a greater blood supply due to heart failure caused by the intense myocardial lesions found in this group. The group of IPNT animals showed increased serum levels only for the ALT enzyme. For AST, even with a tendency to increase, it was not significant, due to the greater virulence of the IP route, with a consequent higher mortality, making these analyses difficult. Despite these limitations, these findings confirm the study by Novaes et al. [31], carried out with male C57BL/6 mice infected via the IP route with the Y strain (5,000 BT forms), which presented high serum levels of AST and ALT enzymes. However, this same study also recorded an increase in these enzymes in animals that received BZ only.

In the present study, BZ administered to uninfected mice caused a significant increase in AST and ALT enzymes, confirming that this drug causes liver damage in uninfected animals [31, 32, 33]. This was also confirmed by the increase in liver OS in NI + BZ animals, demonstrated by the increase in the carbonyl groups of proteins and by histopathological analyses that showed a mild inflammatory process in the organ. On the other hand, the etiological treatment promoted a reduction in the dosage of the two enzymes in animals infected via the GI route, and only in the ALT of those infected via the IP route, showing a reversal of the condition caused by the drug. This result suggests a greater benefit of etiological treatment in the infection via the GI route.

Individually, infection with T. cruzi and the treatment with BZ caused an increase in OS and tissue alterations in the liver, corroborating the literature [31, 34]. Although the etiological treatment reduced the serum levels of the plasma enzymes (AST and ALT) in infected and treated animals, the analysis of OS markers in the liver indicated an increase in the production of carbonyl groups in the treated animals inoculated by both routes. The production of carbonyl groups is used as a marker of free radical-mediated protein oxidation, which is indicative of tissue damage [35], corroborating our histopathological findings in the animals infected and treated with BZ.

Although exposure to BZ was harmful to liver tissue [31], treatment attenuated the inflammatory processes caused by both infection routes, mainly in the heart. Gruendling et al. [21] also observed a significant reduction in the number of organs undergoing an inflammatory process in Swiss mice infected with strains belonging to three DTUs (TcI, TcII, and TcIV) and submitted to treatment with BZ. Our results also showed positive changes in OS parameters in the liver after treatment. After the reduction of GSH values in animals infected via the GI route, the BZ treatment promoted a return to its normal levels, suggesting a reduction in OS and another benefit of the treatment for infection caused by the GI route. In the IP infection, BZ also promoted benefits, demonstrated by the reduction of carbonyl groups; however, a study by Novaes et al. [31], who also used the Y strain, but with an inoculum of 5,000 BT, contradict our findings, associating the BZ treatment of infected animals with a more severe OS.

The lower severity of infection with Y strain (TcII) in mice when inoculated via the GI route, compared to IP route infection, observed in the present study, contrasts with the greater severity and worse response to treatment observed in mice infected via the GI route with TcIV strains [36, 37]. It is possible that different strains/DTUs of T. cruzi, whose composition of surface glycoproteins of their developmental forms may differ [37], can stimulate different reactions in combination with treatment and infection routes, given that the genetic diversity of the species may have an important impact on the biological and medical properties of the parasite such as virulence, pathogenicity, and drug resistance [17, 19, 21, 38].

The Y strain used in this study was maintained through alternating passages in mice and liver infusion tryptose (LIT) acellular culture medium in vitro. It is known that the Y strain, isolated from a patient in 1950, becomes more virulent after successive passages in mice [39]. The conditions used in the maintenance of T. cruzi strains may result in a artificial selection for some subpopulations, and a strain may be the result of the interaction of these subpopulations that are selected after a few years of maintenance in the laboratory [40]. In addition to biological and genetic diversity, which can influence the behavior of the T. cruzi strain, differences in the time of maintenance of the strain in the culture medium before inoculation, and inoculum volume and concentration, may also contribute to differences in its infectivity, pathogenicity, and lethality in mice. The results obtained in this study, in general, allow us to reject our working hypothesis, since the infection with CMT of the Y strain (TcII) by the GI route was less severe and responded better to BZ treatment than the infection via the IP route, contradicting the findings obtained in BALB/c mice with the Tulauhen (TcVI) strain [3], which found greater severity of infection via the oral and GI routes compared to the IP route. However, the strain influences the resistance of the host, some can be extremely virulent, such as the Y strain [39], while others not, such as Tulahuen [41]. They also contradict the findings obtained in Swiss mice inoculated with TcIV strains from Amazonas state (Brazilian Amazon region), which observed, in addition to greater virulence by the GI route [42], a worse response to treatment with BZ [36]. Therefore, the results of the present study suggest that both the severity and the response to treatment of oral/GI infection by T. cruzi depend on the strain or DTU of the parasite. We must also consider the conditions of origin, maintenance of these strains, and experimental models used, all of which may influence the course of T. cruzi infection.

Swiss mice infected with the Y strain (TcII) via the GI route displayed lower levels of parasitemia, with a later peak, and less intense lesions in the heart and liver than mice infected via the IP route, suggesting a lower severity of infection for this strain of T. cruzi when inoculated by the GI route. Although animals infected by both the GI and IP routes had a mortality rate of 50%, deaths occurred later in those inoculated via the GI route, also suggesting a lower lethality rate of this route. Furthermore, these data suggest that the severity of the infection route depends on the strain or DTU of T. cruzi.

BZ administered to uninfected mice caused a focal inflammatory process in the heart and liver of animals, although mild, and increased plasmatic parameters of liver damage and OS, confirming that the drug alone causes liver damage in these animals.

Although liver damage and OS parameters suggested greater severity of the GI route, with increased levels of AST and ALT enzymes and reduced levels of GSH, compared to an increase only in ALT in IP-inoculated animals, both BZ and T. cruzi infection alone caused tissue alterations and increased OS in the liver of mice. However, the BZ treatment of the infected animals reverted this situation with the return to normal enzymatic levels, suggesting reversion of liver damage and OS, another benefit of the treatment in addition to parasite clearance.

• Ethical aspects

The use, maintenance, and care of the mice followed the guidelines of the National Council for the Control of Animal Experimentation (CONCEA), and we complied with the ARRIVE guidelines. The research was approved by the Ethics Committee on the Use of Animals of the State University of Maringá (UEM) (registration number 9659251017/2017). The animals were kept in polyethylene cages (dimensions 20x32x21 cm) on microenvironmental shelves (AL20 - Alesco®) with water and food ad libitum and light/dark cycle (12/12 h). For organ collection (heart and liver), animals were euthanized by deepening anesthetic with administration of the association of ketamine (50 mg/kg) and xylazine (10 mg/kg) intraperitoneally.

• Parasites

The Y strain of T. cruzi genotyped as TcII was used [9]. This strain presents tropism for lymphoid organs, parasitizing preferentially the liver and spleen, in addition to the heart [12]. Furthermore, it is highly virulent to albino mice and was found to be partially resistant to BZ and NX in animals inoculated via the IP route with BT forms [23, 24], and resistant to BZ in mice inoculated via the GI route with 2x10⁶ CMT/animal [25].

The parasites were maintained through alternating passages between Swiss mice and LIT acellular culture medium supplemented with 10% heat-inactivated PBS, pH 7.4. To obtain the metacyclic forms, the blood with the trypomastigote forms of the Y strain was incubated in LIT culture medium in a Biochemical Oxygen Demand (B.O.D.) to obtain the epimastigote forms. Cultures were maintained axenic at 28°C in a stationary growth phase for at least 15 days before inoculation of experimental animals. The parasites present in the culture were counted in a Neubauer chamber and the inoculum was adjusted to 2x10⁶ CMT/ 1.0 mL.

• Inoculation Of Animals

We used 60 Swiss male mice, 21 to 28 days old, from the Central Animal Facility of UEM. Twenty animals were inoculated via the GI route and 20 via the IP route, with 2x10⁶ CMT in 1.0 mL of LIT medium [11]. GI inoculation was performed by gavage with a special cannula (Biomedical Needles, Popper & Sons, Inc., New York, USA) and a volume of 1.0 mL of the inoculum was injected into the stomach, as used for IP inoculation. The animals inoculated via the GI route have previously fasted for approximately 12 h. The remaining 20 animals were not inoculated and served as uninfected controls.

• Experimental Groups

The animals were divided into six experimental groups with 10 animals each, as follows: 1) Uninfected and untreated (NI); 2) Uninfected and treated (NI + BZ); 3) Infected via GI route and untreated (GINT); 4) Infected via GI route and treated (GI + BZ); 5) Infected via IP route and untreated (IPNT); 6) Infected via IP route and treated (IP + BZ).

• Etiological Treatment

The etiological treatment consisted of administering a dose of 100 mg/kg/day of BZ (LAFEPE®, Pernambuco, Brazil) by gavage, for 20 consecutive days, starting on the 5th d.i. All infected groups, ~ 48 h after the end of treatment with BZ, underwent immunosuppression with cyclophosphamide (Cy, Genuxal, Baxter, Brazil), at a dose of 50 mg/kg/day, for four consecutive days in the first week and three alternate days in the following two weeks [43].

• Parasitological Assessments

Fresh blood examination (FBE)

This test was performed on all inoculated animals before, during, and after treatment with BZ to confirm the infection and plot the parasitemia curve. The parasitemia was evaluated daily from the 3rd d.i. until negative results for three consecutive days [44]. Parasitemia was also evaluated, on alternate days, during and after the administration of the immunosuppressive agent (Cy), to verify the reactivation of the infection, characterized by the return of patent parasitemia and increased mortality. From the mean parasitemia curve, the following parameters were obtained: PPP, first day on which positive FBE was detected; PP, mean of the periods in which each animal had the parasitemia detected by the FBE; Pmax, mean obtained from the detected parasitemia peak for each animal; and Dpmax, mean of the days on which each animal had the peak of parasitemia detected. With the results of this test, the %+FBE was obtained.

Real-time polymerase chain reaction (qPCR)

The qPCR test was performed to detect T. cruzi DNA and quantify the blood parasite load. Blood samples for qPCR analysis were collected on the 47th d.i., after the Cy immunosuppression period. DNA was extracted by the phenol/chloroform method, as described by Caldas et al [45], and modified by Gruendling et al. [21]. The DNA obtained was analyzed using the QuantiNova SYBR Green PCR kit (Qiagen) with 100 ng of total genomic DNA, using the primers TCZ-F (5' –GCTCTTGCCCACAMGGGTGC– 3') and TCZ-R (5' –CCAAGCAGCGGATAGTTCAGG– 3') [28]. Samples were amplified in LightCycler® 480 by denaturation at 95°C for 2 minutes, 35 cycles of amplification at 95°C for 15 seconds, and 60°C for 10 seconds. To monitor primer-dimers or the formation of non-specific products, an analysis of the melting curve from 65°C to 97°C was performed at the end of each assay.

A standard curve was established using purified T. cruzi DNA; serial dilutions ranging from 100 to 0.001 ng of DNA were added to the reaction plate wells in triplicate. The standard curve was generated by the LightCycler® 96 software and was used to calculate the equivalent parasite ratio in each sample. The equivalent parasites (par. eq.) per mL of blood were based on the amount of DNA per epimastigote cell, in which 200 fg/parasite were considered [46]. With these results, the percentage of mice with positive qPCR (%+qPCR) and the mean parasite load for each experimental group were obtained.

Infectiousness and survival rate

Animals with positive FBE and/or qPCR were considered infected. The %INF was obtained by the ratio between the number of infected animals and the number of inoculated animals X 100. Deaths were recorded throughout the experiments and the survival rates of the different groups were compared using the Kaplan-Meyer plot.

• Histopathological Evaluations

We evaluated 2–6 animals per experimental group. The animals were euthanized and had their heart and liver collected 48 h after the end of immunosuppression, on the 47th d.i.

Fragments of these organs were first fixed in 10% formalin and were preserved in 70% alcohol after 24 h. After dehydration and diaphanization, the tissues were embedded in paraffin. Subsequently, they were cut into 5 µm thick sections, separated by 25 µm intervals, placed on microscope slides (four sections per slide), and then were stained with hematoxylin-eosin. The sections were examined under an optical microscope in a 40X objective, and the photomicrographs were obtained using the Optical Microscopy OPTHD software Copyright© 2003–2017.

Four sections per animal were counted and individual data were determined as the average of the results of the four sections. The histopathological parameters analyzed included of tissue parasitism, inflammatory process, tissue necrosis, and tissue fibrosis.

For the analysis of the inflammatory infiltrate, the presence of 10 or more inflammatory cells per field was considered as an inflammatory process and was classified as follows: absent (–) (without the presence of minimal inflammatory cells), mild (+) (10–25 cells), moderate (++) (26–50 cells) and intense (+++) (> 50 cells) [21].

Tissues with nuclear pyknosis (nucleus with basophil staining and reduced size), karyolysis (absence of nucleus) and dispersion of the nucleus in the cytoplasm were considered as necrosis. Tissue fibrosis was characterized by the presence of fibroblasts, accumulation of connective tissue and excessive deposition of collagen fibers occupying the space of lost cardiac cells [30]. Tissue necrosis and fibrosis alterations were classified according to lesion severity and distribution, (-) normal histological appearance; (+) mild and focal lesion; (++) moderate multifocal or diffuse lesion; (+++) intense multifocal or diffuse lesion.

• Biochemical Assessments

The following plasma parameters of liver damage were evaluated: dosages of the enzymes AST, ALT, and ALP; and OS parameters: dosages of GSH, catalase and protein carbonyl groups.

Dosage of plasma liver enzymes

The enzymes AST, ALT, and ALP were measured in blood plasma by spectrophotometry (Hitachi U-3000 spectrophotometer) with commercial kits from Gold Analyze Diagnostica Ltda. (Belo Horizonte, MG, Brazil). The blood of the animals was collected by cardiac puncture, 48 h after the end of treatment with BZ and 48 h after the end of immunosuppression, using sodium heparin as an anticoagulant, and was centrifuged at 252 g for 20 minutes to separate the plasma. Subsequently, the plasma samples were transferred to Eppendorf microtubes and were frozen at -4 ºC, until use.

Oxidative stress (OS) in the liver

After euthanasia, the liver was clamped and kept in liquid nitrogen. For the preparation of the organ homogenate, the Van Potter-Elvehjem homogenizer was used with 10 volumes of 0.1 M potassium phosphate buffer (pH 7.4), in an ice bath, and an aliquot was separated for use as the total homogenate. The remaining homogenate was centrifuged at 11,000 g for 15 minutes and the supernatant was separated as a soluble fraction of the homogenate. GSH was measured spectrofluorimetrically (excitation at 350 nm and emission at 420 nm) using the o-phthaloaldehyde assay as described previously [48]. Catalase activity was estimated by measuring the change in absorbance at 240 nm using H₂O₂ as substrate and the results were calculated using the molar extinction coefficient (ε) of 9.6x10-3 M-1 • cm-1. Protein carbonyl groups were measured spectrophotometrically using 2,4-dinitrophenylhydrazine [49] and their levels were calculated using the molar extinction coefficient (ε) of 2.20x10-4 M-1 • cm -1.

• Statistical analysis

Statistical analysis of parasitological parameters was performed as follows: PPP, PP, Pmax, Dpmax, and parasite load were compared with the non-parametric tests of Mann-Whitney or Krust-Wallis, using the Biostat software, version 5.3 (Belém, PA, Brazil). The parameters %+FBE, %+qPCR, and %INF were compared by the chi-square test (χ²). In the analysis of biochemical parameters, the mean and standard error were used. The Kaplan-Meier graph of the GraphPad Prism 5.0 software was used to compare the survival rate. Fisher’s exact test was used to compare the intensity of histopathological lesions and the proportion of organs with alterations, using the Biostat software, version 5.3.

Statistical comparisons were made between uninfected and infected animals, between inoculation routes (GI x IP) and between BZ-treated and untreated animals (GI + BZ x GINT, IP + BZ x IPNT). Differences with a p-value ≤ 0.05 were considered significant.

Additional information

Competing interests

The authors declare no competing interests.

Acknowledgments

To the Coordination for the Improvement of Higher Education Personnel (CAPES), Brazil, Financing Code 001.

Funding

This work was supported by grants from the Araucaria Foundation for Scientific and Technological Development through the Basic and Applied Research Support program (251/2014, number 10943812) and from the National Council for Scientific and Technological Development (CNPq) through the Research Productivity program (PQ – 2014, number 305853/2014–7) to MJOT. The funders did not have any role in study design, data collection, management, analysis, data interpretation, manuscript writing, and the decision to submit the manuscript for publication.

Ethics approval

All methods were performed in accordance with the relevant guidelines and regulations of the National Council for the Control of Animal Experimentation (CONCEA), and ARRIVE guidelines. The research was approved by the Ethics Committee for the Use of Animals of Maringá State University (UEM) (registration number 9659251017/2017).

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on request.

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Impact of gastrointestinal inoculation and benznidazole treatment on Trypanosoma cruzi II infection in mice

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Abstract

Figures

Introduction

Results

• Infectiousness and survival

• Parasitological And Molecular Parameters

• Impact Of Etiological Treatment On Parasitological And Molecular Parameters

• Histopathological Parameters In Cardiac Tissue

• Histopathological Parameters Of Liver Tissue

• Impact Of Etiological Treatment On Histopathological Lesions

• Biochemical Parameters

• Impact Of Etiological Treatment On Biochemical Parameters

Discussion

Conclusion

Methods

• Ethical aspects

• Parasites

• Inoculation Of Animals

• Experimental Groups

• Etiological Treatment

• Parasitological Assessments

Fresh blood examination (FBE)

Real-time polymerase chain reaction (qPCR)

Infectiousness and survival rate

• Histopathological Evaluations

• Biochemical Assessments

Dosage of plasma liver enzymes

Oxidative stress (OS) in the liver

• Statistical analysis

Declarations

References

Additional Declarations

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