Parasitol Res (1995) 81:6-12 9 Springer Verlag 1995 Morgana T. Lima- Henrique L. Lenzi 9 Cerli R. Gattass Negative tissue parasitism in mice injected with a noninfective clone of Trypanosoma cruzi Received: 18 February 1994/Accepted: 1 July 1994 Abstract Trypanosoma cruzi is a heterogeneous popula- tion of parasites as shown by differences between strains and cloned stock from the same strain. Herein we present evidence of the noninfectivity of CL-14, a clone derived from the CL strain of T. cruzi. In a previous paper we re- ported the absence of parasitemia and mortality in mice injected with metacyclic trypomastigotes of this clone. To investigate further this lack of infectivity we did and extensive histopathological analysis in mice at different intervals after i.p. (5 and 15 days as well as 1, 4, and 12 months) or i.v. (5 and 30 days) injection of trypomas- tigotes. In spite of a systematic search in all tissues and organs of the animals, no parasite or significant patho- logical change was detected in any of the tissue sections. These data suggest the inability of this clone to mediate infection and/or cause pathological alterations in vivo. Introduction The early phase of Trypanosoma cruzi infection in mam- malian hosts is characterized by extensive tissue and blood parasitism and depression of the cellular immune response. As the infection progresses, parasitemia is con- trolled and pathological alterations can be observed. However, effective immunity is never attained and the animal dies infected. In contrast to this pattern, BALB/c mice injected with culture- or insect-derived try- pomastigotes of CL-14, a clone isolated from the CL strain of T. cruzi (Chiari 1981), show negative parasite- mia when evaluated by direct blood examination, hemo- culture, or xenodiagnostics (Lima et al. 1991). The ab- sence of circulating parasites was also observed in C3H M. T. Lima. C.R. Gattass ( ~ ) Institute of Biophysics Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Ilha do Fund~o, 21949-900, Rio de Janeiro, RJ, Brasil H. L. Lenzi Department of Pathology, Institute Oswaldo Cruz, FIOCRUZ, Manguinhos, 21045-900 - Rio de Janeiro, RJ, Brasil mice or newborn (1- to 2-day-old) BALB/c mice injected with metacyclic trypomastigotes of this clone (Lima et al. 1991). The lack of parasitemia and the inability to in- fect susceptible hosts indicate that this clone is a nonin- fective parasite. However, in chagasic infection there is no direct correlation between acute-phase parasitemia and pathology. Indeed, the existence of clones showing negative blood parasitemia and extensive tissue parasit- ism has been reported (Postan et al. 1983). To check the possibility that clone 14 might display similar behavior, we carried out an extensive histopatho- logical analysis to investigate whether injection of CL-14 would induce either tissue parasitism or pathological le- sions in mice. For this purpose, BALB/c mice were euthanized at different times after injection of the clone and all tissues and organs were processed. The negative results obtained indicate that in addition to being nonin- fective, this clone is also nonpathogenic. Materials and methods Clone 14 was isolated from the CL strain by Chiari (1981) and was maintained as epimastigotes in LIT medium at 29 ~ C. For in- duction of metacyclogenesis, epimastigotes in exponential growth were harvested and resuspended in M-16 medium to a final con- centration of 2• parasites/ml. After 5 days, the proportion of metacyclic trypomastigotes in the culture was 70%-95% (Chiari 1981; Rondinelli et al. 1988). Metacyclic trypomastigotes were purified by passage through a diethylaminoethyl cellulose (DE52) column, resuspended in phosphate-buffered saline (PBS), and ad- justed to the desired concentration. Mice were injected i.p. or i.v. Fig. 1A-H Histopathology of muscular tissues from mice injected with the CL-14 clone (A, C, E, G) or the CL strain (B, D, F, H). Intensive tissue parasitism and inflammatory infiltrates are visible in cardiac ventricular (B, x200) and auricular muscles (D, x320) as well as in striated (F, x200) and intestinal smooth muscles (H, x500). The inflammatory infiltrate is mainly composed of lympho- cytes and macrophages with neutrophilic foci and apoptotic resi- dues. No histopathological alteration is visible in ventricular (A, x256), auricular (C, x320), striated (E, x400), or intestinal smooth muscles (G, x500) from mice injected with the CL-14 clone (time of infection: 15 days; H&E stain)