RESISTANCE OF T. GONDII TACHYZOITES AND BRADYZOITES TO ACID-PEPSIN DIGESTION

Until recently, acid-pepsin digestion was a generally accepted method to distinguish tachyzoites from bradyzoites and to recoverT. gondii from tissues. Biologically, bradyzoites are resistant to gastric digestion and thus remain orally infective whereas tachyzoites are often destroyed by gastric juice. This resistance of bradyzoites to digestion by gastric juice has been known for over 36 years. Jacobs et al. (93) found that bradyzoites can survive in acid-pepsin solution for 2 h or more whereas tachyzoites are killed within 1 h. These authors digested homogenates of liver, spleen, and lungs of mice, inoculated i.p. with RH strain tachyzoites, for 60 min in acid-pepsin solution and found that tachyzoites were rendered noninfective to mice. Direct microscopic examination of tachyzoites in acid-pepsin revealed that they were immediately damaged; they became more granular, less refractile, and ghostlike within 15 to 30 min (93). However, Jacobs et al. (93) tested the infectivity of tachyzoites for mice after digestion in acid-pepsin for only a 60-min period. Subsequently, most other researchers accepted that tachyzoites are immediately destroyed by acid-pepsin. Unlike acid-pepsin, tachyzoites survived in 1% trypsin for 3 h (93).

Sharma and Dubey (143) quantitatively studied survival of tachyzoites and bradyzoites in acid-pepsin and trypsin solutions. They reported that bradyzoites survived in acid-pepsin for 2 h but not for 3 h.

According to Pettersen (130), the destruction of tachyzoites in acid-pepsin was due to acid, not to pepsin, because no differences were found in survival rates when tachyzoites were incubated in acid-pepsin at room temperature or at 37°C (pepsin is active only at 37°C and at low pH). Pettersen (130) also reported that tachyzoites of two virulent strains (RH and 119) survived in acid-pepsin for 20 min but not 25 min. He thought that bradyzoites were present in the peritoneal exudate of mice inoculated with bradyzoites of two avirulent strains of T. gondii. The peritoneal exudate was obtained 4 or 6 days after i.p. inoculation with bradyzoites of strain DUE; the organisms in the peritoneal exudate survived 90 min in acid-pepsin at 37°C.

In a follow-up paper, Pettersen (131) proposed that bradyzoites can be excreted in the milk of mice 5 days after i.p. inoculation of lactating mice with 1,000 bradyzoites. The evidence for this was that milk treated with HCl for 60 min at room temperature produced T. gondii infection in bioassayed mice. This result disagrees with the conclusion reached by Dubey and Frenkel (48), who found that bradyzoites were not formed in any tissue of mice until 7 days after bradyzoite inoculation.

Popiel et al. (134) used the acid-pepsin digestion procedure to quantify the development of bradyzoites in cell cultures by using cell culture as a bioassay. Tachyzoites of the T-263 strain, obtained by a 2-day cultivation in cell culture, were killed after a 10 min digestion in acid-pepsin. These authors used the same concentration of acid as used by Jacobs et al. (93) but only 10% of the pepsin used by Jacobs et al. (93). Bradyzoites produced in cell culture survived acid-pepsin digestion for 30 to 60 min. Popiel et al. (134) concluded that organisms that resisted 30 min of acid-pepsin digestion were bradyzoites.

Lindsay et al. (113) compared the appearance of acid- pepsin-resistant organisms with the development of tissue cysts by TEM in cell cultures inoculated with the RH strain and a temperature-sensitive (ts4) mutant derived from it. Tissue cysts were not seen in cell cultures inoculated with these two strains, but the organisms survived acid-pepsin digestion.

To resolve whether the inoculum used to test the effect of pepsin digestion, the strain of T. gondii used, the method used to obtain tachyzoites, and the source of tachyzoites affected results, Dubey (43) conducted experiments with extracellular tachyzoites from the peritoneal exudate obtained 3 to 9 days after i.p. inoculation of mice with tachyzoites. The following conclusions were drawn from this study: (i) tachyzoites occasionally survived acid-pepsin digestion for 2 h, which was not due to protection within host cells; (ii) the strain of T. gondii did not affect the susceptibility of tachyzoites to acid-pepsin; and (iii) even extracellular tachyzoites were infective to mice orally, but the infectivity was dose dependent (the infective dose of tachyzoites by the oral route in mice was 1,000). Therefore, it was concluded that one cannot rely on oral infectivity in mice or digestion in acid-pepsin as the sole criterion to distinguish between tachyzoites and bradyzoites.

Date: 2016-01-03; view: 636