Parasitol Res (2007) 100:1395–1399 DOI 10.1007/s00436-006-0403-9
Cornea as a tissue reservoir of Trypanosoma cruzi Leidi Herrera & Clara Martínez & Hernán Carrasco & Ana Maria Jansen & Servio Urdaneta-Morales
Received: 12 September 2006 / Accepted: 21 November 2006 / Published online: 20 December 2006 # Springer-Verlag 2006
Abstract Trypanosoma cruzi causal agent of Chagas’ disease is a paninfective parasite of mammals transmitted through skin fecal contamination by Triatominae vectors. Studies of alternative routes for infection are scarce; therefore, eye infection should be important, because of the eye’s high blood irrigation and brain proximity, as port of entry of the parasite. Trypanosoma cruzi parasites and/or their genetic material in ocular and adjacent muscle tissues were studied in batches of six NMRI mice (15 g) and Trichomys apereoides, an ancient caviomorph (250 g) inoculated with T. cruzi metacyclics from Brazilian (2) and Venezuelan (3) isolates genetically typified as T. cruzi I and II. Two animals/batch in the acute or chronic phase were killed and necropsies of cardiac and skeletal muscles, eyeball, and surrounding ocular muscle were processed for hematoxylin–eosine staining. Tissue parasitism was determined. DNA of the digested sections of the eyeball (5– 10 μm) was extracted for T. cruzi k-DNA amplification by PCR, with S35 and S36 primers. The PCR products were analyzed. The average of maximum values of parasitemia of all infected animals was of 105 trypomastigotes/ml Declaration: The experiments conducted in this study comply with the current laws of Venezuela. L. Herrera (*) : S. Urdaneta-Morales Instituto de Zoología Tropical, Universidad Central de Venezuela, Apartado 47058, Los Chaguaramos, Caracas, Venezuela e-mail: [email protected]
C. Martínez : H. Carrasco Instituto de Medicina Tropical, Universidad Central de Venezuela, Caracas, Venezuela A. M. Jansen Instituto Oswaldo Cruz, Rio de Janeiro, Brazil
blood. Skeletal muscle and heart were colonized in patent infection for all isolates. Amastigote nests were found in corneal tissue of 2/3 of the used isolates and adjacent ocular muscle and connective tissue were parasitized. Trypanosoma cruzi k-DNA (330-bp band) was observed in ocular tissue of 4/6 of the isolates studied in both animal models. Investigations concerning infection of the eye globe tissues by T. cruzi are extremely scarce. The presence of stages of T. cruzi and/or its genetic products in ocular tissues indicate a broad colonization from a systemic infection. The results show the ocular environment as a possible appropriate microniche for T. cruzi and emphasize the risk of transmitting T. cruzi by ocular fluids and by parasitized cornea through transplants.
Introduction Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae), the causal agent of Chagas’ disease, is an intracellular hemoflagellate with different biological and genetic properties inducing considerable diversity in infectivity, virulence, pathology, and tissue parasitism in a broad range of mammalian hosts (Andrade 2000). The natural parasite transmission is produced through fecal contamination with metatrypomastigotes, deposited by vectors (Hemiptera, Reduviidae, Triatominae), in the mammalian host skin or mucous membranes, especially the oral and ocular membranes, differentiating into intracellular amastigote and trypomastigote stages, which are disseminated by hematogenous and lymphatic routes in organs and tissues (Schuster and Schaub 2000). Eyeball infection should be important because of its role as a port of parasite entry (Romaña sign), high blood irrigation, and proximity to the brain (Dias 2000). Therefore, we studied the presence of
1396 Table 1 Strain and isolates of Trypanosoma cruzi used
H hemoculture isolation (NNN medium), X xenodiagnosis isolation (R. prolixus nymphs), G anal gland metacyclic culture a Codified according to Anonymous (1999)
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MTRI/BR/1999/R4 (T. cruzi II) MDID/BR/1999/M1 (T. cruzi I) MHOM/VE/1970/EP (T. cruzi I) MRAT/VE/1996/CO22 (T. cruzi I) MDID/VE/1995/CO79 (T. cruzi I) MHOM/BR/2000/Y (T. cruzi II)
Trichomys apereoides Didelphis albiventris Human Rattus rattus Didelphis marsupialis Human
H H H X G H
Piauí, Northeast Brazil Piauí, Northeast Brazil Guárico State, Venezuela Caracas, Venezuela Caracas, Venezuela Laboratory reference strain
stages of T. cruzi and/or its genetic material in the eyeball, adjacent ocular muscle tissue, and heart from the ancient caviomorph Trichomys apereoides (Rodentia, Echimyidae), a typical and abundant T. cruzi host in some areas of Brazil and Paraguay (Herrera et al. 2005; Roque et al. 2005), and also from albino mice (NMRI outbreed strain).
Materials and methods Trichomys apereoides (250 g mean weight; furnished by Dr. P. D’Andrea, Instituto Oswaldo Cruz, Brazil) and mice (15 g mean weight) were distributed in batches of six animals each and inoculated subcutaneously with 200 T. cruzi metacyclics/gram body weight from genetically typified Brazilian and Venezuelan isolates (Herrera et al. 2003, 2005) and reference strains indicated in Table 1. Control animals were also used. The prepatent period and parasitemia in the inoculated animals were microscopically determined at 400× every other day using a Neubauer chamber; mortality was recorded daily. Persistence of parasitism was estimated in survivor animals by xenodiagnosis with 12 nymphs of Rhodnius prolixus and/or hemoculture in liver infusion tryptose medium. Two animals per batch in the acute or chronic infection phase were killed by anesthetic overdose with Ketaset (Ketamine HCl 100 mg/ml, Fort Dodge, IA, USA). Tissue necropsies of heart, muscle of the thigh, ocular globe, and surrounding
Table 2 Parasitological and molecular results obtained in the experimental infections with the strain and isolates used
(1) Infection in T. apereoides model, (2) infection in NMRI mice model
skeletal muscle were routinely fixed and processed for paraplast embedding, sectioning (3 μm), and hematoxylin– eosine staining. Tissue parasitism was determined at 1,000× as trypomastigote or amastigote nests. Sections cut through the middle of the embedded eyeball (5–10 μm) were treated and digested as described by Wright and Manos (1990). DNA of the digested tissues was extracted with the Wizard Genomic DNA Purification System (Promega, Madison, WI, USA), and its integrity was confirmed by electrophoretic loading. Trypanosoma cruzi k-DNA amplification was carried out using the polymerase chain reaction (PCR), with specific primers for the conserved region of the kinetoplast minicircles: S35 (5′-GGTTCGATTGGGTTGGTGTAA TATA-3′) and S36 (5′-AAATAATGTACGGGKGAGATG CATGA-3′) (modified from Lane et al. 1997). The β-actin protein for mice was simultaneously amplified with the primers BACTH1 (5′ GCTGTGCTATGTTGCCCTA GAATTCGAGC-3′) and BACTH2 (5′-CGTACTCCTG CTTGCTGATCCACATGTGC-3′), determining the integrity of the constitutive DNA. The PCR products were analyzed by electrophoresis on a 2.5% agarose gel (Ultra Pure, Gibco Life Tech, Gaithersburg, MD, USA) and visualized after ethidium bromide staining in a FBTIV Fisher Biotech Transiluminator (Fisher Scientific, Pittsburgh, PA, USA). The size of the bands obtained was contrasted with a 1-Kb DNA Ladder standard (Gibco Life Tech) and the gel registered with a computerized photographic system, KODAK 1D.
Colonization in ocular tissue and annexes
PCR in ocular tissue
MTRI/BR/1999/R4 MDID/BR/1999/M1 MHOM/BR/2000/Y MHOM/VE/1970/EP
Acute (1) Chronic (1) Chronic (1) Acute (2)
Negative Positive Positive Positive
Acute (2) Acute (2)
Negative Negative Negative Amastigotes in conjunctiva, corneal stroma, adjacent ocular muscle and interstitial macrophages Negative Amastigotes in fibroblasts of corneal stroma and adjacent muscle
Parasitol Res (2007) 100:1395–1399
Results and discussion Trichomys apereoides and mice were susceptible to infection by T. cruzi I and T. cruzi II isolates, displaying mean parasitemia of 105 flagellates/ml blood (data not shown). All infected mice died, while 50% of T. apereoides survived the infection by the MTRI/BR/1999/R4 isolate; no mortality was produced by the MDID/BR/1999/M1 isolate and MHOM/BR/2000/Y reference strain. Spontaneous urination and scarce paralysis were observed in mice; in contrast, T. apereoides displayed asymptomatic infection by all isolates. The parasitological and molecular results are recorded in Table 2 and Fig. 1. Trypanosoma cruzi k-DNA was observed in ocular tissues of 67% (4/6) of the isolates studied in both animal models.
Fig. 1 Histological and molecular parasitism in NMRI mice experimentally infected with different isolates and strains of T. cruzi (a, a1, a2) Sequence of microphotographs with amplification of a nest of amastigotes in a fibroblast (F, arrows, 40×, 400×, and 1,000×, respectively; MDID/VE/1995/CO79 isolate) of corneal stroma (S). b Trypomastigote nest in thigh skeletal muscle (arrow; MHOM/VE/ 1970/EP isolate). c Amastigote nest in heart muscle (arrow; MDID/VE/1995/CO79 isolate) (HE; scale bar 15 μm). d PCR amplification of the 330-bp fragment from the conserved regions of kDNA (short arrow) extracted from ocular tissues of NMRI experimentally infected mice in 2.5% agarose gel electrophoresis (ethidium bromide stain): Lane 1 1-Kb Ladder molecular marker (Gibco BRL Life Technologies), lane 2 nude T. cruzi DNA, lane 3 negative PCR control, lane 4 MRAT/VE/ 1996/CO22 isolate, lane 5 MHOM/BR/1950/Y strain, lane 6 MHOM/VE/1970/EP isolate, lane 7 MDID/BR/1999/M1 isolate, lane 8 MDID/VE/1995/ CO79 isolate
The presence of k-DNA in ocular tissue could explain the potential broad colonization of T. cruzi from a systemic infection. Trypanosoma cruzi paninfectivity is a property related to parasite–host interactions and capacity of the tissue microenvironment to control parasite proliferation (Lenzi et al. 1996). However, the strict classification of this parasite in relation to tissue stenotropism should be reconsidered, based on the results of new molecular approaches. The variability in T. cruzi parasites and their genetic products in eye globe tissues observed in experimentally infected rodents could be a consequence of T. cruzi genetic heterogeneity (Macedo and Penna 1998). Numerous species of microorganisms can infect the ocular globe (Klotz et al. 2000; Hiroshige et al. 2002). Viruses, frequently implicated as pathogens using the
cornea as a microhabitat (Biswas et al. 2005), are able to pass to the tears, which become infectious fluids (Yamamoto et al. 1994; Kouichi et al. 2002). Among protozoa, Kinetoplastida (Trypanosomatidae) such as Trypanosoma evansi, Trypanosoma equinum, Trypanosoma vivax, Trypanosoma brucei, Trypanosoma congolense (references in García et al. 1983; Whitelaw et al. 1988), and Leishmania spp. (Gontijo et al. 2002; Leiva et al. 2005) infect most of the components of the eyeball. On the other hand, 15% of the cases of mucocutaneous leishmaniasis present initial nasal lesions and can affect, by contiguity, eyelids, ocular conjunctiva, and the nasolachrymal conduit (Hoyama et al. 2001) with the presence of parasite blood stages in the nasal secretions (Mebrahtu et al. 1993) and other fluids such as semen (Diniz et al. 2005) and saliva and urine (Marsden and Hagstrom 1968). We believe it necessary to emphasize the importance of determining the possibility that the behavior of T. cruzi could be similar in ocular infections. To the best of our knowledge, studies on the determination of parasitic stages as well on the ocular target tissues recognized by T. cruzi for its growth and morphological differentiation are extremely scarce (Marsden and Hagstrom op. cit.; Lenzi et al. op. cit.). In spite of the frequent tropism for the eye by these Trypanosomatidae, investigations on the parasitism caused by T. cruzi have been restricted to demonstrate, comparatively, the infectivity of metacyclics and blood trypomastigotes on the ocular globe (Zeledón et al. 1977), as well as the clinical manifestations in the course of the infection (Idiaquez 1992; Prata et al. 1996). Chronic degenerative diseases or traumatisms causing the failure of solid organs, tissues, or cells are treated by transplantation. Allotransplantation includes organs (heart, kidneys, lungs, liver, and pancreas) and tissues (bone, bone marrow, eye tissues, and skin) among animals within the same species. Xenotransplantation involves the passage of live cells, tissues, or organs from one species to another, such as from non-human animals (chimpanzees, baboons, and pigs) to humans. In both cases, the risks exist of transmitting infections (xenozoonoses), such as Chagas’ disease, from the donor cornea, as well as the complications resulting from achieving sufficient immunosuppression to ensure lack of rejection in xenotransplantation (Boneva et al. 2001). Viruses, bacteria, fungi, protozoa, and helminthes have been transmitted via allotransplantation of those organs and tissues (Eastlund 1995). Transplantation and immunosuppression could enhance natural T. cruzi intracellular multiplication; discarding T. cruzi cornea infection should be mandatory in cornea banks. Acknowledgements The authors thank Marlene Rodríguez and Estefanía Flores for technical assistance. We are most grateful to Dr. Marian Ulrich for her aid in preparing the manuscript. This work was supported by the Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (Grant No. PG 03-00-5609-2004)
Parasitol Res (2007) 100:1395–1399 and by the Fondo Nacional de Ciencia y Tecnología de Venezuela (Grant No. G-2005000406 FONACIT).
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