Beruflich Dokumente
Kultur Dokumente
Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 August 2015
Received in revised form
8 January 2016
Accepted 20 January 2016
Available online 28 January 2016
Oral transmission of the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease,
has been documented in Latin American countries. The reported cases of infection were due to the
ingestion of contaminated fresh fruit, juices, or sugar cane juice. There have been few studies on the
physiopathology of the disease in oral transmission cases. Gastritis is a common ailment that can be
caused by poor dietary habits, intake of alcohol or other gastric irritants, bacterial infection, or by the
widespread use of non-steroidal anti-inammatory drugs (NSAIDs). This study investigated in a mouse
model whether gastric mucosal injury, induced by aspirin, would affect the course of disease in animals
infected with T. cruzi by the oral route. The CL14 and G strains of T. cruzi, both of low infectivity, were
used. To this end, groups of BALB/c mice were treated during 5 days with aspirin (100 mg kg1) before
oral infection with T. cruzi metacyclic forms (4 105 or 5 107 parasites/mouse). Histological analysis
and determination of nitric oxide and TNF-a were performed in gastric samples obtained 5 days after
infection. Parasitemia was monitored from the thirteenth day after infection. The results indicate that
aspirin treatment of mice injured their gastric mucosa and facilitated invasion by both CL14 and G strains
of T. cruzi. Strain CL14 caused more severe infection compared to the G strain, as larger numbers of
amastigote nests were found in the stomach and parasitemia levels were higher. Our study is novel in
Keywords:
Chagas disease
Gastric mucosa
NSAIDs
Trypanosoma cruzi
Abbreviations: ASA, aspirin; COX, cyclooxygenase; MT, metacyclic trypomastigotes; NO, nitric oxide; p.i, post infection; PG, prostaglandins; PGE2, prostagandin
E2; iNOS, inducible nitric oxide synthase; NSAIDs, non-steroidal anti-inammatory
drugs.
^ncias Patolo
gicas, Centro de Cie
^n* Corresponding author. Departamento de Cie
gicas, Universidade Estadual de Londrina, CEP: 86051-970, Londrina,
cias Biolo
Paran
a, Brazil.
E-mail address: pingelho@uel.br (P. Pinge-Filho).
http://dx.doi.org/10.1016/j.exppara.2016.01.008
0014-4894/ 2016 Elsevier Inc. All rights reserved.
65
that it shows that gastric mucosal damage caused by aspirin, a commonly used NSAID, facilitates T. cruzi
infection by the oral route.
2016 Elsevier Inc. All rights reserved.
1. Introduction
Chagas disease is caused by the protozoan parasite Trypanosoma
cruzi. It is still a major public health problem in Latin America and a
potentially serious emerging threat in a number of countries
(Bonney, 2014; Carlier, 2011; Coura and Vinas, 2010). Transmission
occurs by blood-sucking triatomine bugs; metacyclic forms of the
parasite present in the triatomine feces contaminate the nearby
bite wound or mucous membranes and conjunctiva (ShikanaiYasuda and Carvalho, 2012). However, transmission by ingestion
of contaminated food or beverages has often been reported during
the last two decades and is presently considered an emergent
health problem (Benchimol-Barbosa, 2010).
Ingestion of fruit or fruit juices contaminated with infected
triatomines or their feces, which contain metacyclic trypomastigotes (MTs) of T. cruzi, is the most common cause of human
infection by the oral route (Yoshida et al., 2011). Gastric invasion
appears to be the main portal of entry for systemic T. cruzi infection
after oral challenge (Hoft et al., 1996). The parasites invade the
gastric epithelium where they initially replicate (de Meis et al.,
2013).
In addition, it was shown that infection by insect-derived MTs in
BALB/c mice was more severe when the parasites were administered via the mucosal route in comparison to the cutaneous route,
which emphasizes the hazard of oral contamination (Eickhoff et al.,
2013). However, mice inoculated with culture-derived MTs by
gavage or intraperitoneal routes developed comparable infections
(Dias et al., 2013). A recent study compared groups of mice infected
with T. cruzi by parasites inoculated in the oral cavity (oral infection) or by gavage (gastrointestinal infection); the orally infected
group developed more severe hepatic lesions and had higher
mortality rates than the latter, in which heart inammation was
predominant (Barreto-de-Albuquerque et al., 2015).
The variation in the prevalence and severity of contamination
among persons exposed to a common source of T. cruzi-infected
food or beverage indicates a wide range of individual susceptibility
(Yoshida, 2008). The numbers of ingested parasites as well as the
genetic background of each person certainly are major determinants of each person's susceptibility. Nevertheless, given the
prevalence of gastritis of different etiologies in the population, it is
conceivable that gastric mucosal injury might favor invasion by
ingested T. cruzi parasites.
In order to investigate this question we chose to verify the effect
of aspirin-induced gastric injury on the outcome of oral infection by
T. cruzi metacyclic forms. Gastric mucosal damage is a common side
effect of nonsteroidal anti-inammatory drugs (NSAIDs) (Katzka
et al., 1987).
Aspirin and other NSAIDs, such as celecoxib and indomethacin,
act on cyclooxygenases and inhibit the synthesis of prostaglandins
(PGs). Mice infected by the intraperitoneal route with T. cruzi blood
forms and treated with NSAIDs showed a worsening of the infection, regardless of the mouse or parasite strain under investigation
(Celentano et al., 1995; Hideko Tatakihara et al., 2008, Mukherjee
et al., 2011; Pinge-Filho et al., 1999; Sterin-Borda et al., 1996). The
increased synthesis of arachidonic acid-derived lipids and of nitric
oxide (NO) seen in the early phase of T. cruzi infection of mice was
correlate with resistance; however, at later stages of infection these
66
Fig. 1. Experimental design. BALB/c mice were treated with aspirin (ASA, 100 mg kg1) for ve days before T. cruzi infection. Control mice received vehicle (DMSO PBS) alone. The
mice were infected by gavage with 5 107 or 4 105 living forms of T. cruzi, strains G or CL14. Four days after infection the stomach of each animal was collected and processed for
histological analysis and quantication of nitric oxide and TNF-a. Parasitemia was monitored in alternate days starting from day 13 until day 31 after infection.
67
Fig. 2. Effect of ASA treatment on the gastric mucosa. ASA (100 mg kg1) treatment during 5 days caused: elevated gastric mucosal damage scores (A), and reduced nitrite (B) and
increased TNF-a levels (C) in the gastric mucosa in comparison to samples obtained from vehicle treated mice. The means SEM from 3 mice/group are presented (two different
sections from each mouse were evaluated). *P < 0.05, compared to the control mice that received vehicle.
(Fig. 3A). Prior treatment by gavage with ASA of mice infected with
CL14 caused a four-fold increased in the number of amastigote
nests in the stomach in comparison with control mice given only
vehicle solution. In contrast, gastric parasitism of mice infected
with G strain was not changed by similar ASA treatment (Fig. 3A).
These numerical results are illustrated by photomicrographs in
Fig. 3B and C showing T. cruzi gastric parasitism by strains G and
CL14 respectively. To examine the effect of ASA in the intracellular
parasite development in vivo, BALB/c mice treated with ASA
(100 mg kg1) were inoculated orally with 5 107 MT forms of CL
14 and G (both lowly infective). It was observed that the number of
CL14 strain-amastigote nests was four times higher as compared to
G strain-ones (Fig. 3A). No difference was observed in the number
of amastigote nests in the stomach of ASA-treated mice inoculated
with G strain (Fig. 3A). The distribution of parasite nests of G strain
was sparser (Fig. 3B) than that for the CL14 strain (Fig. 3C).
3.3. Treatment with aspirin augmented the frequency of mice
developing patent and elevated parasitemia levels
The frequency of mice that developed patent parasitemia when
infected with strains G or CL14 was increased in the groups given
ASA prior to oral inoculation (Table 1). As expected, no mortality
was observed in either group of animals (Table 1). Parasites became
detectable by the 13th day and the peak of parasitemia occurred on
the 17th day after infection for both T. cruzi strains (Fig. 4A and B).
Peak parasitemia counts in CL14-infected mice were approximately
twice those observed in G-infected mice. By day 31 parasite counts
in the blood fell below the detection threshold of the method
(Fig. 4A and B).
4. Discussion
Invasion of the vertebrate host by T. cruzi depends primarily on
events at the point of entry. In the case of oral ingestion, the gastric
mucosa appears to be the major entry portal. We chose to verify
infectivity by the oral route of the laboratory-adapted strains CL14
and G because they are representative of the low-infectivity T. cruzi
strains that predominate in endemic areas (Atayde et al., 2004;
Yoshida, 2006). As expected, parasites in the blood were detected
only in a limited percentage of mice when the infective inoculum
was deposited inside normal stomachs.
68
Fig. 3. Effect of ASA treatment on the invasion capacity of strains G and CL14 of T. cruzi in the gastric mucosa. Stomach tissue sections of mice (n 3/group) were performed 5 days
after oral infection with metacyclic trypomastigotes (5 107). Number of amastigotes nests and distribution of amastigote-containing nests of G (B) and CL14 (C) strains. Note the
multiple parasite nests of strain CL14 and a single nest of strain G, in equivalent sections of the stomach stained by were stained by Hematoxylin-Eosin (H.E). Bar: 25 mm. Inserts
show amastigote nests.*P < 0.05, compared to the untreated mice.
Table 1
Parasitological parameters of the infection with T. cruzi (CL14 and G strains) in ASA treated and untreated mice.
Strain
Treatment
(For 5 days)a
CL14
Vehicle
ASA
Vehicle
ASA
4
4
4
4
G
a
b
105
105
105
105
% of survival
30 days post infection
3/12
9/12
1/8
5/8
100
100
100
100
Mice were fasted for 16 h before administration of one of the following oral suspensions: ASA (100 mg kg1) or vehicle (PBS DMSO 0.05%) by gavage.
Suspension of metacyclic trypomastigotes (MT) was used as the inoculum.
69
70
Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14. Int. J. Parasitol. 34, 851e860.
Barreto-de-Albuquerque, J., Silva-dos-Santos, D., Perez, A.R., Berbert, L.R., de Santana-van-Vliet, E., Farias-de-Oliveira, D.A., Moreira, O.C., Roggero, E., de Carvalho-Pinto, C.E., Jurberg, J., Cotta-de-Almeida, V., Bottasso, O., Savino, W., de
Meis, J., 2015. Trypanosoma cruzi infection through the oral route promotes a
severe infection in mice: new disease form from an old infection? PLoS Negl.
Trop. Dis. 9, e0003849.
Benchimol-Barbosa, P.R., 2010. Further comments on oral transmission of Chagas'
disease in Brazil: epidemiology, geographical distribution and viability of the
infective parasite. Int. J. Cardiol. 141, 203e204.
Bonney, K.M., 2014. Chagas disease in the 21st century: a public health success or an
emerging threat? Parasite 21, 11.
Brener, Z., 1962. Therapeutic activity and criterion of cure on mice experimentally
infected with Trypanosoma cruzi. Rev. Inst. Med. Trop. Sao Paulo 4, 389e396.
Cardoni, R.L., Antunez, M.I., 2004. Circulating levels of cyclooxygenase metabolites
in experimental Trypanosoma cruzi infections. Mediat. Inamm. 13, 235e240.
Carlier, Y., 2011. Globalization of Chagas disease (American trypanosomiasis): the
situation in Europe and Belgium. Bull. Mem. Acad. R. Med. Belg. 166, 347e355
discussion 356e347.
Celentano, A.M., Gorelik, G., Solana, M.E., Sterin-Borda, L., Borda, E., Gonzalez
Cappa, S.M., 1995. PGE2 involvement in experimental infection with Trypanosoma cruzi subpopulations. Prostaglandins 49, 141e153.
Cortez, M., Silva, M.R., Neira, I., Ferreira, D., Sasso, G.R., Luquetti, A.O., Rassi, A.,
Yoshida, N., 2006. Trypanosoma cruzi surface molecule gp90 downregulates
invasion of gastric mucosal epithelium in orally infected mice. Microbes Infect.
8, 36e44.
Coura, J.R., Vinas, P.A., 2010. Chagas disease: a new worldwide challenge. Nature
465, S6eS7.
de Meis, J., Barreto de Albuquerque, J., Silva Dos Santos, D., Farias-de-Oliveira, D.A.,
Berbert, L.R., Cotta-de-Almeida, V., Savino, W., 2013. Trypanosoma cruzi
entrance through systemic or mucosal infection sites differentially modulates
regional immune response following acute infection in mice. Front. Immunol. 4,
216.
Dias, G.B., Gruendling, A.P., Araujo, S.M., Gomes, M.L., Toledo, M.J., 2013. Evolution of
infection in mice inoculated by the oral route with different developmental
forms of Trypanosoma cruzi I and II. Exp. Parasitol. 135, 511e517.
Eickhoff, C.S., Dunn, B.A., Sullivan, N.L., Hoft, D.F., 2013. Comparison of the infectivity of Trypanosoma cruzi insect-derived metacyclic trypomastigotes after
mucosal and cutaneous contaminative challenges. Mem. Inst. Oswaldo Cruz
108, 508e511.
Fichera, L.E., Albareda, M.C., Laucella, S.A., Postan, M., 2004. Intracellular growth of
Trypanosoma cruzi in cardiac myocytes is inhibited by cytokine-induced nitric
oxide release. Infect. Immun. 72, 359e363.
Fiorucci, S., 2009. Prevention of nonsteroidal anti-inammatory drug-induced ulcer: looking to the future. Gastroenterol. Clin. North Am. 38, 315e332.
Fiorucci, S., Santucci, L., Federici, B., Antonelli, E., Distrutti, E., Morelli, O.,
Renzo, G.D., Coata, G., Cirino, G., Soldato, P.D., Morelli, A., 1999. Nitric oxidereleasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine
proteases and protect endothelial cells from apoptosis induced by TNFalpha.
Aliment. Pharmacol. Ther. 13, 421e435.
Fiorucci, S., Mencarelli, A., Cipriani, S., Renga, B., Palladino, G., Santucci, L.,
Distrutti, E., 2011. Activation of the farnesoid-X receptor protects against
gastrointestinal injury caused by non-steroidal anti-inammatory drugs in
mice. Br. J. Pharmacol. 164, 1929e1938.
Freire-de-Lima, C.G., Nascimento, D.O., Soares, M.B., Bozza, P.T., Castro-FariaNeto, H.C., de Mello, F.G., DosReis, G.A., Lopes, M.F., 2000. Uptake of apoptotic
cells drives the growth of a pathogenic trypanosome in macrophages. Nature
403, 199e203.
Hernadez, C., Barrachina, M.D., Vallecillo-Hernandez, J., Alvarez, A., Ortiz-Masia, D.,
Cosin-Roger, J., Esplugues, J.V., Calatayud, S., 2015. Aspirin-induced gastrointestinal damage is associated with an inhibition of epithelial cell autophagy.
J. Gastroenterol. http://dx.doi.org/10.1007/s00535-015-1137-1.
Hoft, D.F., Farrar, P.L., Kratz-Owens, K., Shaffer, D., 1996. Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses. Infect.
Immun. 64, 3800e3810.
Hunt, R.H., Yuan, Y., 2011. Acid-NSAID/aspirin interaction in peptic ulcer disease.
Dig. Dis. 29, 465e468.
Katzka, D.A., Sunshine, A.G., Cohen, S., 1987. The effect of nonsteroidal antiinammatory drugs on upper gastrointestinal tract symptoms and mucosal
integrity. J. Clin. Gastroenterol. 9, 142e148.
Lichtenberger, L.M., 2001. Where is the evidence that cyclooxygenase inhibition is
the primary cause of nonsteroidal anti-inammatory drug (NSAID)-induced
gastrointestinal injury? topical injury revisited. Biochem. Pharmacol. 61,
631e637.
Lima, M.T., Lenzi, H.L., Gattass, C.R., 1995. Negative tissue parasitism in mice injected
with a noninfective clone of Trypanosoma cruzi. Parasitol. Res. 81, 6e12.
Machado, F.S., Mukherjee, S., Weiss, L.M., Tanowitz, H.B., Ashton, A.W., 2011.
Bioactive lipids in Trypanosoma cruzi infection. Adv. Parasitol. 76, 1e31.
MacNaughton, W.K., Cirino, G., Wallace, J.L., 1989. Endothelium-derived relaxing
factor (nitric oxide) has protective actions in the stomach. Life Sci. 45,
1869e1876.
Michelin, M.A., Silva, J.S., Cunha, F.Q., 2005. Inducible cyclooxygenase released
prostaglandin mediates immunosuppression in acute phase of experimental
Trypanosoma cruzi infection. Exp. Parasitol. 111, 71e79.
Mukherjee, S., Machado, F.S., Huang, H., Oz, H.S., Jelicks, L.A., Prado, C.M., Koba, W.,
Fine, E.J., Zhao, D., Factor, S.M., Collado, J.E., Weiss, L.M., Tanowitz, H.B.,
Ashton, A.W., 2011. Aspirin treatment of mice infected with Trypanosoma cruzi
and implications for the pathogenesis of Chagas disease. PLoS One 6, e16959.
Navarro-Gonzalvez, J.A., Garcia-Benayas, C., Arenas, J., 1998. Semiautomated measurement of nitrate in biological uids. Clin. Chem. 44, 679e681.
Nishino, M., Sugimoto, M., Kodaira, C., Yamade, M., Shirai, N., Ikuma, M., Tanaka, T.,
Sugimura, H., Hishida, A., Furuta, T., 2010. Relationship between low-dose
aspirin-induced gastric mucosal injury and intragastric pH in healthy volunteers. Dig. Dis. Sci. 55, 1627e1636.
Paiva, C.N., Arras, R.H., Lessa, L.P., Gibaldi, D., Alves, L., Metz, C.N., Gazzinelli, R.,
Pyrrho, A.S., Lannes-Vieira, J., Bozza, M.T., 2007. Unraveling the lethal synergism
between Trypanosoma cruzi infection and LPS: a role for increased macrophage
reactivity. Eur. J. Immunol. 37, 1355e1364.
Pinge-Filho, P., Tadokoro, C.E., Abrahamsohn, I.A., 1999. Prostaglandins mediate
suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection. Cell Immunol. 193, 90e98.
Shikanai-Yasuda, M.A., Carvalho, N.B., 2012. Oral transmission of Chagas disease.
Clin. Infect. Dis. 54, 845e852.
Shimamoto, C., Fujiwara, S., Kato, M., Ito, S., Katsu, K., Mori, H., Nakahari, T., 2005.
Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous
cells: autocrine regulation of mucin secretion by PGE2. Am. J. Physiol. Gastrointest. Liver Physiol. 288, G39eG47.
Sterin-Borda, L., Gorelik, G., Goren, N., Cappa, S.G., Celentano, A.M., Borda, E., 1996.
Lymphocyte muscarinic cholinergic activity and PGE2 involvement in experimental Trypanosoma cruzi infection. Clin. Immunol. Immunopathol. 81,
122e128.
Takao, Y., Shimamoto, C., Hirata, I., Ohshiba, S., 1993. The effect of acid secretagogues
on mucin synthesis using primary monolayer culture of the guinea pig gastric
mucous cells. Gastroenterol. Jpn. 28, 638e646.
Hideko Tatakihara, V.L., Cecchini, R., Borges, C.L., Malvezi, A.D., Graca-de Souza, V.K.,
Yamada-Ogatta, S.F., Rizzo, L.V., Pinge-Filho, P., 2008. Effects of cyclooxygenase
inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection. FEMS Immunol. Med. Microbiol. 52, 47e58.
Tatakihara, V.L., Malvezi, A.D., Panis, C., Cecchini, R., Zanluqui, N.G., Yamauchi, L.M.,
Martins, M.I., da Silva, R.V., Yamada-Ogatta, S.F., Rizzo, L.V., Martins-Pinge, M.C.,
Pinge-Filho, P., 2015. Nitric oxide-releasing indomethacin enhances susceptibility to Trypanosoma cruzi infection acting in the cell invasion and oxidative
stress associated with anemia. Chem. Biol. Interact. 227, 104e111.
Teixeira, M.M., Yoshida, N., 1986. Stage-specic surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identied by monoclonal antibodies. Mol.
Biochem. Parasitol. 18, 271e282.
Yoshida, N., 2006. Molecular basis of mammalian cell invasion by Trypanosoma
cruzi. An. Acad. Bras. Cienc. 78, 87e111.
Yoshida, N., 2008. Trypanosoma cruzi infection by oral route: how the interplay
between parasite and host components modulates infectivity. Parasitol. Int. 57,
105e109.
Yoshida, N., 2009. Molecular mechanisms of Trypanosoma cruzi infection by oral
route. Mem. Inst. Oswaldo Cruz 104 (Suppl. 1), 101e107.
Yoshida, N., Tyler, K.M., Llewellyn, M.S., 2011. Invasion mechanisms among
emerging food-borne protozoan parasites. Trends Parasitol. 27, 459e466.