EXPERtMENTALPARASITOLOGY73, 249-259 (1991)
Trypanosoma cruzi in the Opossum Didelphis marsupialis: Parasitological and Serological Follow-Up of the Acute Infection ANA M. JANSEN,* LEONOR LEON,? GEFUIA M. MAcHADo,t MARIA H. DA SILVA,$ SELMA M. SOUZA-LE,&o,* AND MARIA P. DEANE* *Department of Protozoology, Institute Oswald0 Cruz, 4365 Avenida Brash, Rio de Janeiro 21040, Rio de Janeiro, Brazil; fDepartment of Immunology, Institute Oswald0 Cruz, 4365 Avenida Brasil, Rio de Janeiro 21040, Rio de Janeiro, Brazil; and #Department of Immunology, Institute of Microbiology, Federal University of Rio de Janeiro, Ilha do Fundao 21941, Rio de Janeiro, Brazil
JANSEN, A. M., LEON, L., MACHADO, G. M., DA SILVA, M. H., SOUZA-LE.&O,S. M., DEANE, M. P. 1991. Trypanosoma cruzi in the opossum Didelphis marsupialus: Parasitological and serological follow-up of the acute infection. Experimental Parasitology 73, 249-259. The opossum Didelphis marsupialis is known to be among the most important wild reservoirs of Trypanosoma cruzi and one in which the trypanosome may go through both the usual vertebrate intracellular cycle in its tissues and an extracellular cycle in the lumen of its scent glands. The species is highly resistant to heavy inocula and, depending on the parasite strain, experimental infections may be permanent or self limited. Aiming to understand the mechanisms involved in this parasite-host interaction we made a study of the acute phase of infection with different T. cruzi strains. Strains F, G-49 and G-327 produced durable infections with relatively high parasitemia and invasion of the scent glands, while equivalent inocula of the Y strain resulted in scanty parasitemia of short duration, no invasion of the SG, and no evidence of persistent parasitism. A smaller inoculum of G-49 produced only subpatent though persistent parasitemia and no invasion of the scent glands. The humoral immune response was less marked in the Y group; among the other groups IgM and IgG antibodies increased to high levels, higher in the G-49 group. The increase in IgG coincided with a drop of parasitemia to subpatent levels. Two opossums inoculated directly in the scent glands with culture forms of the Y strain had a short-lived subpatent parasitemia, but the parasites remained in the glands and serum Ig antibodies reached high levels. Immunoblot analysis showed that the sera of the inoculated opossums recognized few T. cruzi antigens (more in the F strain) in comparison with those of mice. However, with the only exception of those subcutaneously inoculated with the Y strain and including two naturally infected specimens, all the opossum’s sera recognized a 90-kDa peptide in all T. cruzi strains. Our results confirm that opossums are able to selectively eliminate some strains of T. cruzj and indicate that the mechanism involved in this selection is probably not related to the humoral immune response. In infections by strains that are able to establish a permanent foothold in opossum tissues, there are indications that IgG antibodies participate in the control of the parasite population of the acute phase but are unable to prevent the chronic phase. It was once more demonstrated that the opossum infected scent glands function as diffusion chambers for parasite antigens but that, on the other hand, the parasites are here protected against the mechanisms developed by the host to control their population. 6 1991 Academic Press, Inc. INDEX DESCRIPTORSAND ABBREVIATIONS: Trypanosoma cruzi; Kinetoplastida; Trypanosomatidae; Opossum; Marsupialia; Didelphidae; Didelphis marsupialis; Wild reservoir; Natural resistance; Humoral immune response: Scent glands (SG); Indirect fluorescent antibody test (IFAT); Metacyclic trypomastigote (MT); McNeal, Novy, and Nicolle blood agar medium (NNN); Liver infusion tryptose (LIT); Sodium dodecyl sulphate (SDS); Phosphate-buffered saline (PBS); Glycoprotein (GP). AND
It is known that wild mammals may be completely refractory or highly resistant to
the African trypanosomes and that native breeds of cattle can survive infections that are fatal to breeds recently introduced into endemic areas (Murray et al. 1982). Spe249 0014-4894/91$3.00 Copyright 8 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
JANSEN ET AL.
cific and nonspecific mechanisms seem to be involved in the relative resistance, or trypanotolerance, such as the ability to recognize certain trypanosome antigens (Shapiro and Murray 1982), a more efficient phagocytic system (Rurangirwa et al. 1986), or the presence of cytotoxic factors in the hosts serum (Ferrante et al. 1982). About 200 species or subspecies of wild mammals are known to host Trypanosoma cruzi (Barrett0 and Ribeiro 1979). Their natural infection is usually subpatent but the parasite strains isolated from them show a wide spectrum of virulence for laboratory animals (Deane et al. 1963; Barretto 1965). However, very little has been done to clarify the mechanisms involved in the apparent resistance to infection among these reservoir hosts. The data available on resistance to T. cruzi come mostly from studies on experimentally infected inbred strains of mice. Levels of parasitemia and lethality are the parameters used to assess this resistance which seems to be related to the ability of the host to control the first intracellular cycles of the parasite (Trischmann et al. 1978; Wrightsman et al. 1982; Trischmann 1986). Results obtained with athymic (nude) mice, or mice pretreated with anti-CD4+ monoclonal antibody have clearly demonstrated that T lymphocytes play a decisive role in the control of parasite populations and inflammatory reactions in the acute phase of the infection (Goncalves da Costa et al. 1984; Russo et al. 1988). However, circulating Ig antibodies appear to participate in this control and the parasite strain has been considered to be the most important factor involved in the resistance of different mouse strains (Andrade et al. 1985). Studies are being performed in our laboratories on natural and experimental T. cruzi infections in didelphid opossums, which are the most common, ubiquitous, and, probably, most ancient reservoir hosts of the parasite. A series of new data on the biology of this association has been brought
to light, such as the cycle undertaken by the parasite in the lumen of the SG of the opossum (Deane et al. 1984a, 1986a). Among other findings it was seen that, since early age, opossums are resistant to inocula that kill adult mice in 10-12 days; they are able to eliminate infection with the more virulent (for laboratory animals) strains of T. cruzi while maintaining other strains indefinitely, with low or subpatent parasitemia and discrete pathology (Deane et al. 1984b; Carreira et al. 1986); and they are able to mount humoral and cellular immune responses to T. cruzi, Leishmania spp and other antigens (Jansen et al. 1985; SouzaLeao et al. 1987; Franc0 et al. 1987). In this paper we describe the humoral response (IgM and IgG levels and antigen recognition) in correlation with the parasitological follow-up, during the acute phase of infection of the opossum with various strains of T. cruzi. MATERIALSAND
The opossums used for inoculations were five litters (total 28 specimens) from our D. marsupialis colony, reared in captivity from birth, or taken as sucklings in the marsupium of wild caught females, weaned when 100days old, submitted to the routine tests to exclude previous T. cruzi infection (Deane and Jansen 1990) and subcutaneously inoculated when aged 120 days; and two adults captured as young, free from infection as ascertained by repeatedly negative tests, and inoculated directly in the lumen of their SG (Jansen et al. 1988). Two adults, equally free from infection, were kept as negative controls and the sera of two others that had a natural T. cruzi infection were used for comparison with those from experimentally infected specimens. All animals were individually caged and fed dog food, eggs, and fresh fruits. The parasite strains were: Y and F, of which the origin, maintenance procedures, and type of infection in mice are described elsewhere (Deane et al. 1984~); and G-49 and G-327, isolated from naturally infected opossums captured in localities of Rio de Janeiro State, in August 1982 and August 1987, respectively. The isolation of the opossum strains was through triatomid bugs, passaged in mice and into LIT and NNN cultures; parasitemia in mice was always subpatent. For the subcutaneous inoculations the inocula were MT shed with the urine of laboratory reared fourth and fifth instar nymphs of Rhodnius neglectus, infected
T. cruzi IN THE opossum 30-40 days before by feeding through a membrane (Garcia et al. 1975) on cultures of each T. cruzi strain. The urine was obtained according to N. Thomaz (unpublished): the infected nymphs, immediately after engorging through a membrane on sterile blood, were individually inserted uprightly in 1.5ml Eppendorf tubes; after about 2 hr the bugs were withdrawn and the clear, MT rich urine was collected with a Pasteur pipette from the bottom of the tubes. The parasites were counted in a Neubauer camera and their number adjusted with PBS so as to yield inocula of 200/g body wt for each animal of four litters (Sihtter), or SO/gbody wt in one litter comprising eight specimens. For the intraglandular inoculations cultures in LIT medium (Camargo 1964) were used, following a described technique (Jansen et al. 1988). During a follow-up period of at least 14 weeks (or about 100 days) the opossums were submitted to parasitemia evaluation by counting in a Neubauer camera every other day during the period of patency, hemoculture in NNN medium with a LIT overlay every 15th day when direct blood examinations became negative, weekly microscopical examinations of material obtained by manual expression of the SG, and weekly IFAT for IgM and IgG anti-T. cruzi antibodies. In some cases IFAT was done using secondary antibodies to the whole Ig molecule (Jansen et al. 1985). The titration of the antibody classes was made by using a fluorescein isothiocyanate conjugate of goat antiheavy chains p and y of opossum serum, according to the technique of Garvey et al. (1977). The antigen for all IFATS was prepared with the F strain epimastigotes from cultures, as described (Jansen et al. 1985). For immunoblotting parasite antigens of Y, F, and G-49 strains were obtained as described elsewhere (Leon et al. 1990). The soluble antigens were electrophoresed in a 10% polyacrylamide gel containing SDS under nonreducing conditions, following Laemmli’s (1970) technique. The gels were silver stained (Merrill
et al. 1981)or submitted to electroblotting of proteins. The proteins were transferred to nitrocegulose paper, following the method of Townbin et al. (1979). The nitrocellulose sheets were first incubated with opossum sera followed by incubation with rabbit antiopossum serum. The reaction was revealed with commercial peroxidase conjugated anti-rabbit IgG (Sigma). Except for one animal that had received the F strain and died at Day 90 ah the others survived through the period of observation and died or were killed at various intervals thereafter. However, some specimens were kept much longer, two of them for 3 years, chiefly to test the permanence of parasitism by hemoculture and antigenic recognition by immunoblotting.
Except for the groups infected with Y strain, inoculation of 200 MT/g body wt resulted in measurable levels of parasitemia in all opossums, the highest peak being reached in the group inoculated with the G-49 strains. After direct blood examinations became negative, repeated hemocultures were positive for these groups and in all these there was invasion of the SG by the parasite. In the Y strain group very small number of trypomastigotes appeared irregularly in the fresh blood preparations for a period after which hemocultures were negative, and there was no invasion of the SG. The smaller inoculum of 50 MT/g body wt of strain G-49 produced a subpatent infection revealed by hemocultures and the SG were negative (Table I).
TABLE I Parasitological Data on Opossums Didelphis marsupialis, Subcutaneously Inoculated with Triatomid-Derived Metacyclic Trypomastigotes of Trypanosoma cruzi Parasitemia
Inoculum MT/g body wt”
200 200 200 200 50
Number of opossum
Y F G-327 G-49 G-49
5 5 5 5 8
+ + + + -
Note. (?), irregular appearance in low numbers; (-),
Peakb no./ml ? 7.2 x 10s 1.2 x 16 1.7 x lo6
Percentage with parasite in SG
+ + + +
40 40 loo -
negative; (SG), scent glands. D MT/g body wt: Metacyclic trypomastigotes per gram of body weight. b Average count for the group in day of peak.
JANSEN ET AL.
The two opossums inoculated with cultures of the Y strain directly in the lumen of the SG had positive hemocultures, once in the second week, and twice in the first and third weeks, respectively; the glandular material was positive throughout the period of observation, and at necropsy, 5 and 11 months postinoculation. The serological follow-up revealed IgM antibodies in the second or third week after infection in all groups, but for those inoculated with strain G-49, independently of the size of the inoculum, the values were higher and the test was positive up to the 14th week, while in other groups it became negative between Weeks 9 and 11. (Figs. 14; data for G-49 50 MT/g body wt not shown). IgG immunoglobulins were detected on the fourth and fifth week and, with some variation, were maintained at their peak
2. T. cruzi strain G 327 in the opossum D. marsupialis. All the other data as in the legend to Fig. 1. FIG.
levels throughout the period of observation in all groups; the highest titers were recorded for the group inoculated SC with 200 MT/g body wt of strain G-49 and the lowest for the Y strain group with equivalent inocula (Figs. 1-l).
,p\ ) , s,/ ,
8’ ; ,:
,, 7 AFI-ERS
FIG. 1. T. cruzi strain F in the opossum D. marsupialis. Levels of IgM and IgG antibodies and of patent
parasitemia in five opossums of a litter subcutaneously inoculated with 200 triatomid-derived metacyclic trypomastigotes per gram of body weight. Each point represents mean values for the litter. The immunoglobulins were detected by an indirect immunofluorescent antibody test and the curves were plotted as log 2 of the dilution titers. The arrow indicates the first positive examination of the scent glands.
3. T. cruzi strain G 49 in the opossum D. marsupialis. All the other data as in the legend to Fig. 1. FIG.
T. cruzi IN THE OPOSSUM
FIG. 4. T. cruzi strain Y in the opossum D. marsupi&s. Levels of IgM and 1gG antibodies. Patent parasitemia was very light, irregular, and transient and there was no invasion of the scent glands. Other data as in the legend to Fig. 1.
For the two opossums inoculated in the SG with cultures of the Y strain, IFAT was done to determine total Ig only; it was positive from the second week reaching a titer of 1:640 that was maintained, with little variation, though the follow-up period. Most of the infected opossum sera recognized about 15 T. cruzi antigens in the range of 2&180 kDa, but there were marked differences depending on the parasite strain, type, and time of infection. Strain F had the most, and Y the least, antigens recognized by homologous and heterologous sera (Figs. 5-7). A polypeptide of 90 kDa was recognized in all strains, chiefly in the F, by sera of opossums infected with any of the strains (Figs. 5-7). For the specimens inoculated subcutaneously with 200 MT/g body wt of strains F, G-327, and G-49 that had the SG infected during the follow-up period (Table I), recognition of the 90-kDa antigen started on the sixth week and the corresponding band became progressively more intense;
FIG. 5. An immunoblot showing T. cruzi antigens recognized by opossum sera. Lanes l-3: opossum naturally infected with Trypanosoma (Megattypanum) freitasi. Lanes 4-12: opossums infected with T. cruzi strain G-49, subcutaneous inoculation, 3-year infection (lanes 4-6); strain Y, inoculation in the scent glands (lanes 7-9); late natural infection (lanes 10-12). Sources of antigens were: F strain in lanes 2, 5, 8, 11; G-49 strain in lanes 3, 6, 9, 12; Y in lanes 1, 4, 7, 10.
for those in the same groups in which the SG were negative in the course of the above period, recognition started much later, in the eighth month and the corresponding
I 2 3 4 5 6 7 8 9 10II 121314 15 18u 18 FIG. 6. An immunoblot showing T. cruzi antigens recognized by the serum of an opossum D. marsupialis subcutaneously inoculated with strain G 49, 200 triatomid derived metacyclic trypomastigotes per gram of body weight. A follow-up study at different intervals postinoculations: 15 days, lanes l-3; 45 days, lanes 4-6; 60 days, lanes 7-9; 90 days, lanes l&12; 100 days, lanes 13-15; 120 days, lanes 16-18. Strains used as sources of antigen were: Y, in lanes 1, 4, 7, 10, 13, and 16; F, in lanes 2,5,8, 11, 14, and 17; G 49, in lanes 3, 6, 9, 12, 15, and 18.
nized by sera of opossums infected with T. (Fig. 5) or Leishmania spp.
freitasi 90 kd-
Results of the parasitological follow-up were as expected from previous work demonstrating that the susceptibility to T. cruzi varies with the strain of the parasite (Deane et al. 1984b). The low infectivity of the Y strain for the opossum was confirmed by the fact that the group inoculated subcutaI 2 3 4 5 6 7 8 9 IUII 12 neously had only a light and transient infection, while equivalent inocula of strains GFIG. 7. An immunoblot showing T. cruzi antigens recognized by the serum of an opossum D. marsupialis 49, G-327, and F produced infections of inoculated in the scent glands with axenic culture of long duration revealed by patent parasitthe Y strain. A follow-up study at different intervals emia and/or hemoculture. G-49 was the postinoculation: 1 month, lanes 1-3; 3 months, lanes 4-6; 4 months, lanes 7-9; 5 months, lanes 10-12. most infective and was able to produce a lasting (although subpatent) parasitemia Strains of T. cruzi used as sources of antigen were: Y, in lanes 1,4,7, and 10; F, in lanes 2,5, 8, and 11; G 49, even when used in a relatively low inocuin lanes 3, 6, 9, and 12. lum. The data on the positivity of the SG suggest a certain correlation between the level band became progressively more marked of the parasitemia and the presence of the and persisted in two specimens followed for parasite in these glands. However, the facas long as three years (Fig. 5), the maxi- tors responsible for this occurrence are far mum period of observation. All these spec- from clear. T. cruzi invasion of the opossum SG unimens also had a persistent, although subpatent, parasitemia revealed by hemocul- der natural conditions has been reported by various workers (Naiff et al. personal comture. The group inoculated with 50 MT/g body munication; Femandes et al. 1987, 1989; wt of strain G-49 recognized antigens in the Steindel et al. 1987, 1988) from widely dissame range of 20 and 180 kDa (data not tant areas in Brazil (States of Amazonas, shown), while those infected with 200 MT/g Minas Gerais, and Santa Catarina), but it body wt of the Y strain recognized only seems to be rare in some places, despite a four bands (data not shown). The 90-kDa high incidence of infection detected by xeantigen was not recognized by any serum in nodiagnosis and hemoculture. Yet, isolates these two groups that, as mentioned (Table from opossums captured in these places I), were always negative in the SG. The two have been found colonizing the glands opossums inoculated directly in the SG when inoculated subcutaneously in laborawith Y strain cultures recognized the pep- tory reared specimens. tide, although much later (12 weeks) than It was suggested that the opossum SG (or the groups infected subcutaneously (Figs. similar glands in other mammals) might 6,7). On the other hand, the two specimens have been a primitive habitat for monogewith a natural T. cruzi infection recognized netic trypanosomatids that later invaded the 90-kDa antigen, but had no parasites in the vertebrate host tissues and adopted ditheir SG at the occasion of their capture or genetic life cycles. Indeed it was shown thereafter (Fig. 5). that several monogenetic species can mulThe 90-kDa polypeptide was not recog- tiply in these glands for prolonged periods.
Once a species is fully adapted to a digenetic lifecycle, dependence on the SG cycle would cease and the primitive habitat would remain as an occasional refuge or an evolutionary reliquary (Deane and Jansen 1988). T. cruzi is, doubtless, a successful digenetic species that, at least under certain conditions, can go through a double cycle in the opossum (Deane et al. 1984a). According to Jansen (1988), SG invasion happens when a bloodstream population reaches some magnitude, and this would favour the opposite hypothesis that, in the case of T. cruzi, SG adaptation would have been secondary to multiplication in the host tissues. On the other hand, some T. cruzi populations, such as Y strain, produce only poor and fleeting infections in the opossum tissues when injected subcutaneously but, as demonstrated in the present work, can build a thriving extracellular colony when injected directly in the SG. It was previously reported that the opossum is able to mount humoral responses to T. cruzi antigens and that the level of serum Ig antibodies is dependent on the strain of the parasite (Jansen et al. 1985). The analysis of humoral response by Ig classes showed that among the subcutaneously inoculated opossums the highest titers for both IgM and IgG were for strain G-49 and the lowest for Y strain. In all cases serum conversion IgM-IgG was slower than that observed in mice (Araujo et al. 1984), Cebus apella (Rosner et al. 1988), and in a human accidental infection (Israelsky et al. 1988). The difference seen in the immunoglobulin levels between strains G-49 and G-327, both of sylvatic origin and both isolated from naturally infected opossums, illustrates the great diversity of T. cruzi, even when it comes from apparently homogeneous populations and even in its antigenic repertoire (Deane et al. 1984b; Dvorak et al. 1988; Bongertz and Dvorak 1983).
Among specimens with patent parasitemia a decrease in the parasitemic curve coincided with an increase in the IgG level. This coincidence is an indication of the probable role of specific antibodies in the control of the parasite population. Indeed, experiments in mice demonstrated that the transference of immune serum afforded protection and that the efficiency of this passive protection was related to the level of IgG antibodies in the serum and coincided with the onset of the descent of the parasitemic curve in the donor mice (Krettli and Brener 1976; McHardy 1977; Takehara et al. 1981; Hanson 1976; Brodskyn et al. 1988). The control of the parasite population by circulating antibodies was only partial, since parasitemia persisted, although at subpatent levels, in the subcutaneously infected animals (with the exception of those inoculated with the Y strain), despite the persistence of high levels of serum IgG through the whole period of observation. As in other mammals, the control of the parasite population during the acute phase in the opossums does not prevent the chronic phase of the infection. Follow-up studies of natural and experimental T. cruzi infections in opossums revealed the presence of both subpatent parasitemia and circulating specific immunoglobulins for several years (Jansen et al. 1985). The low levels of serum antibodies among specimens inoculated subcutaneously with the Y strain were probably due to early restraint of the parasite population by the opossum and not to an intrinsic low antigenicity of the strain, since SG inoculations resulted in the production of high levels of specific Ig antibodies. It has been suggested (Trischmann 1983, 1986) that resistance of C-57/B/10 mice against T. cruzi is due to precocious control of the parasite populations by a thymus-dependent mechanism. It is possible that similar mechanism operates in opossums. On the other hand, the serum of experi-
mentally infected opossums was shown to display the complement-mediated lytic activity against living trypomastigotes (Thomaz and Deane, unpublished) that was described in mouse and human sera and associated with the extremely low levels of parasitism which characterize the chronic stage of T. cruzi infections (Krettli and Brener 1982; Krettli et al. 1982). The passive protection following transference of IgG antibodies through lactation from infected mothers to sucklings (Jansen et al. 1989) is one more evidence of the capacity of the opossum D. marsupialis to mount humoral responses to T. cruzi infection and of the protective role of the antibodies produced. Antigenic recognition by the sera of inoculated opossums started later and included less antigens than the sera of susceptible or resistant mice (Grogi and Kuhn 1983; Zweerink et al. 1985) and of an accidentally infected human patient (Israelsky et al. 1988). Except that fewer antigens were recognized by the specimens inoculated subcutaneously with the Y strain and despite some other minor differences, the recognition pattern had the same amplitude and was quite similar for all strains. Most striking was the constancy of the band corresponding to a 90-kDa polypeptide which appeared earlier in the sera of the specimens that had infected the SG during the acute phase and later in those in the chronic phase. The kinetics of the appearance and the progressive intensity of this band indicate a quantitative relationship with parasite antigens that are either massively filtered from the infected SG, or gradually accumulate during the course of a prolonged infection. This assumption is corroborated by the fact that the polypeptide was not detected by the sera of the opossums that had only a light and transient infection with the Y strain, but was demonstrated when the sera came from opossums with growing Y populations in their SG. However, the two specimens captured as
naturally infected adults that recognized the 90-kDa peptide had no parasites in the SG; of course we do not know for how long they had been infected or if the parasite had ever sojourned in their glands. That parasite antigens actually filter from infected SG was proved by the presence of specific Ig antibodies in the sera of opossums inoculated in the glands with several species of monogenetic trypanosomatids (Jansen ef al. 1988). A 90-kDa antigen, a surface glycoprotein characterized as the major T. cruzi antigen, is recognized by the sera of various strains of mice and by humans in acute and chronic stages of the infection with various parasite strains of different origins and all zymodemes (Snary and Hudson 1979; Snary 1983, 1985; Zingales et al. 1984; Grogi and Kuhn 1983; Zweerink et al. 1985; Israelsky et al. 1988). Antibodies to this 90-kDa antigen afford some protection against infection by bloodstream and metacyclic trypomastigotes (Snary 1985). The GP 90 has also been implicated in the process of interiorization of the trypomastigote in mammalian cells (Zingales et al. 1982), in the production of lytic antibodies, and in possibly stimulating cell mediated immunity (Mortara et al. 1988). There is some controversy about the stages at which the parasite expresses the 90-kDa protein (Nogueira et al. 1981, 1982; Snary 1983; Araujo et al. 1984; Zingales et al. 1984). Since in the opossum T. cruzi is able to go through a double cycle, vertebrate and invertebrate, the latter including epimastigotes and metatrypomastigotes, it is impossible to know which stages express the antigen. However it may be significant that the specimens in which the Y strain was practically restrained to the invertebrate cycle in the SG produced the 90-kDa specific antibody. In conclusion, we think that antibodies have a significant role in controlling T. cruzi infection in the opossum; however, other
T. cruzi IN THE OPOSSUM
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