Effects of Promastigote Growth Phase,Frequency of Subculture,and Host Age on Promastigote-initiated Infections with Leishmania donovani in the Golden Hamster*†

Promastigotes of Leishmania donovani, 3S strain, were cultured from homogenized infected hamster spleen incubated at 25 C in a particle-free modification of Tanabe's (1923) medium, and were subcultured in this medium from 1 to 4 times. Promastigotes were inoculated intracardially to golden hamsters (Mesocricetus auratus). Promastigotes that were subcultured frequently by transfer from log phase of growth retained their infectivity for hamsters, as assayed by numbers of amastigotes in the liver at 16 days post infection. Promastigotes that were subcultured infrequently by transfer from stationary phase declined in infectivity. The extent of the decline was roughly proportional to the length of the incubation periods of the primary culture plus 1st subculture. Promastigotes harvested from log phase of growth were significantly less infective for hamsters than those harvested from stationary phase of growth, in that numbers of amastigotes found in the liver after 16 days were lower, and times to death longer, when log phase organisms were used to infect hamsters. The age of the hamster at the time of inoculation was found to affect the apparent infectivity of promastigotes from a 1st or 2nd subculture. When weanling (age 4 weeks), juvenile (age 8 weeks) and adult (age 24 to 32 weeks) hamsters received the same numbers of promastigotes, the weanlings had the highest numbers of liver amastigotes at 16 days, and shortest times to death, of the 3 groups; juveniles were intermediate between weanlings and adults; and adults had the lowest numbers of parasites and longest times to death of the host. Differences were statistically significant only between weanlings and adults. Responses of weanling and adult hamsters to infection with promastigotes could be rendered indistinguishable if the promastigotes were inoculated on the basis of 10⁵ promastigotes per g of host body weight.     

Activities of Glucose-6-phosphate, 6-phosphogluconate,and Isocitrate Dehydrogenases from Leishmania donovani Cultivated at 25 and 37 C*

SYNOPSIS. The activities of glucose-6-phosphate dehydrogenase (G-6-PD) (EC No. 1.1.1.49), 6-phosphogluconate dehydrogenase (PGD) (EC No. 1.1.1.44), and isocitrate dehydrogenase (ICD) (EC No. 1.1.1.42) from promastigotes of Leishmania donovani strain 3S grown at 25 C in modified Tobie's (mT) medium and from promastigotes of the 37 C-adapted substrain of this strain cultivated in the mT at 37 C were assayed at 25 and 37 C. At 25 C ICD from both the strain and the substrain had the highest, and PGD, the lowest activity; the activity of G-6-PD was intermediate, but much closer to that of ICD. Irrespective of the temperature of the assay, the activities of G-6-PD and ICD from the 37 C substrain were significantly higher than those of these enzymes from the parental strain; however, the activity of PGD from the 25 C strain was slightly higher than that of this dehydrogenase from the 37 C-adapted stock. No significant activity losses of G-6-PD and ICD from either the strain or the substrain were noted after incubation of the extracts in the presence of 0.25 M sucrose at 37 C for 2 hr. PGD was unstable in such extracts, but it could be rendered stable by the addition of 4 mM 6-phosphogluconate. G-6-PD was the least and ICD the most dependent on Mg²⁺ ions. In the 15–25 C range, the Q₁₀ values of the enzymes from the 25 C strain were 2.83, 2.5, and 2.63 for G-6-PD, PGD, and ICD, respectively. These values for the respective enzymes in the 25–35 C range were 2.06, 1.67, and 1.62. The Q₁₀ values of the enzymes from the 37 C substrain in the 15–25 C range were 2.06 for G-6-PD, 3.25 for PGD, and 2.77 for ICD; in the 25–35 C range, the corresponding values were 1.67, 1.46, and 1.83. Cultivation of the 37 C substrain at 25 C was accompanied by a drop in G-6-PD and ICD activities.     

Leishmania in the Chick Embryo. II. Effects of Inoculum Size,Strain Origin,and Stage Injected and Infectivity of Embryo-Derived Parasites for Hamsters*

SYNOPSIS. Amastigotes of Leishmani donovani strains 2S, 3S, 3K, Hm, Gm, and Et were inoculated intravenously into 14-day chick embryos. The course of infection was followed by examinations of liver impression smears. With strain 33 at 33 C incubation, there was a 29-fold increase at 6 days postinfection when the inoculum contained ～4 × 10⁶ amastigotes, but only a ～6.3-fold increase when ～64 × 10⁶ parasites were injected. Infection courses of several geographic strains were compared at 30, 33, and 35 C incubation. Although the results were variable, Sudan strains 2S and 3S appeared to be separate isolations of a single strain. The Burma (Et), Kenya (3K), and Mediterranean (Hm, Gm) strains appeared to be distinct, but confirming evidence of their distinctness should be sought using serologic, epidemiologic, clinical, and biochemical criteria. Strains 2S and 3S multiplied best at 33 C or below, but the embryos usually failed to survive at 28 or 30 C. Multiplication was inhibited partially at 35 C and completely at 37 C. Inoculation of strain 3S promastigotes into chick embryos resulted in a loss of parasites in 1 hr to 2 days postinfection. Only amastigotes were seen in embryos incubated at 28 and 33 C for 4 days. Hamsters infected with parasites passaged once in chick embryos died at median postinoculation times that were closely comparable to those noted among hosts infected with amastigotes from hamster spleen.