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.     

Leishmania in the chick embryo: IV. Effects of embryo age and hatching,and behavior of L. donovani in cultures of chick fibroblasts

On incubation Days 9, 11, 12, 14, or 15, chick embryos were injected intravenously with 4.0 × 10⁶L. donovani amastigotes. Embryos were incubated at 33 C immediately after infection. Numbers of amastigotes found in the liver 1 hr after injection increased as the age of embryo recipients increased. Most 14- or 15-day infected embryos hatched when allowed to do so, but many younger embryos were unable to survive at 33 C. Numbers of amastigotes in the liver of chicks, hatched after infection as embryos, decreased as the cloacal temperature of the chicks increased. Despite a 31 C incubation temperature, chicks exhibited a mean 38.3 C cloacal temperature 1 day after hatching.Chick fibroblast cultures were initiated as explants of embryo brain and infected with amastigotes from hamster spleen. Only amastigotes were seen in cultures kept at 37 C, but extracellular promastigotes and intracellular amastigotes were present in cultures at 33 C. Although promastigotes increased in number in the medium overlay at 33 C, amastigotes decreased in number at 33 C and 37 C. One intracellular amastigote was seen in a culture which had been incubated at 25 C after inoculation with promastigotes.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.     

Studies on pathogenesis of buffalo pox virus in rabbits: quantitative assay of virus in different organs

The buffalo pox virus was found to multiply in the skin, the primary site of inoculation with an eclipse phase of 12 hr. The virus was then detected in the skin after 15 hr followed by its appearance in regional lymph nodes 36 hr postinoculation. Primary viremia was detected 48 hr postinoculation, followed by detection of virus in the lungs, liver, and spleen. The virus multiplied in the lungs on day 4 and in the liver and spleen on day 5 postinoculation and its release led to secondary viremia. In a follow-up from day 7 to 14 postinoculation, the virus was detected in the kidneys, stomach, intestines, and gonads.     

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.     

Hypothermia: teratogenic and protective effects on the development of mouse embryos in vitro

Hypothermia often occurs in association with clinical conditions involving severe hypoglycemia, but its effect on embryonic development has not been well evaluated. Thus, the whole embryo culture method was used to expose day 9 (neurulating) and day 10 (early limb bud stage) mouse embryos to physiologic levels of hypothermia (35 degrees C and 32 degrees C) for 4 and 24 hr. Embryos were evaluated after 24 hours for growth and malformations and compared with controls grown at 37 degrees C. Lactate production was measured in embryos cultured for 4 hr at 32 degrees C and compared with those cultured at 37 degrees C. A 4-hr exposure to hypothermia produced little effect morphologically but reduced the rate of lactate production at both embryonic stages. A 24-hr exposure to hypothermia at 35 degrees C or 32 degrees C produced growth retardation and dysmorphogenesis in embryos undergoing neurulation. Early limb bud stage embryos were less sensitive to this treatment, with growth retardation produced only at the lower temperature. Since hypothermia is commonly associated with severe hypoglycemia in cases of diabetic insulin overdose, day 9 (neurulating) mouse embryos were exposed concurrently to short periods of hypothermia and hypoglycemia and compared with embryos cultured in hypoglycemic medium at normal temperature. The results demonstrate that hypothermia partially protects embryos against the dysmorphogenic effects of hypoglycemia. A balance of metabolic rate and available substrate is discussed as a possible mechanism for this protective effect.     

The influence of higher temperature on dengue-2 virus infected C6/36 mosquito cell line

Dengue-2 virus infection of C6/36 cells was studied at 28 and 37 degrees C. In infected cells maintained at 28 degrees C, syncytial development was seen on day 4 postinfection, whereas at 37 degrees C, extensive syncytial development was seen by 32 h. Extracellular virus titre was found to correlate with the cytopathic changes. Nine Dengue-2 virus specified proteins were observed in polyacrylamide analyses of cytoplasmic extracts of C6/36 infected cells. All the proteins were observed, although in varied intensities by 32 h postinoculation at 37 degrees C and only on day 4 postinoculation at 28 degrees C. The GP60 glycoprotein appeared at 32 h postinfection when the cells were maintained at 37 degrees C and became prominent only on day 5 at 28 degrees C. The results revealed that a higher temperature accelerated the onset of cytopathic effects, hastened the development of virus specified proteins, and also enhanced the titre of extracellular infectious virus. The importance of the accumulation of the envelope protein GP60 for the development of CPE was indicated.     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

The role of macrophages in the early resistance to mouse hepatitus virus infection in nude mice.

Nude mice which had received intraperitoneal injection of silica simultaneously with infection of mouse hepatitis virus, NuU strain, died of severe necrotic hepatitis within 2 weeks postinfection, whereas those having received no silica survived for 3 weeks or more after challenge. Silica given day 4 postinoculation had no effect. The virus titers of the liver and spleen at day 4 as well as serum interferon levels at day 2 were much higher in silica-treated mice than those without silica treatment. At day 2 or 3 postinoculation, silica-treated mice were found to have a considerable number of necrotic foci in the liver with some neutrophil and lymphocyte infiltration, and viral antigen was present in the cytoplasm of some hepatocytes around necrotic foci. In contrast, those without silica treatment showed only some necrotic foci with some lymphocyte infiltration. Viral antigen was detected only in a few littoral cells but not in hepatocytes. The role of macrophages in the resistance at early stage of inection in nude mice is discussed.     

Trypanosoma cruzi: morphogenesis in diffusion chambers in the mouse peritoneal cavity and attempted stimulation of host immunity

Sequential morphologic changes and antigen producing capacity of Trypanosoma cruzi in peritoneally implanted diffusion chambers were studied. Diffusion chambers were equipped with two Nuclepore filters (0.20 μm pore size) sandwiched between three Lucite rings. Epimastigotes or trypomastigotes and amastigotes were placed in diffusion chambers and surgically implanted into the peritoneal cavity of mice, or placed in in vitro cell culture, or in various types of culture media and incubated at 26 or 37 C.Epimastigotes maintained in diffusion chambers in mice changed into trypomastigotes as evidenced by the presence of numerous transitional stages and the concomitant decrease in the percentage of the former and increase in the percentage of the latter in chambers removed and examined at 16, 24, 36, 48, 72, and 84 hr after implantation. The maximum of 68% trypomastigotes was noted in chambers examined at 84 hr. Amastigotes subsequently appeared, apparently arising from trypomastigotes and reached the highest percentage (49%) obtained at 132 hr. The total number of parasites in chambers decreased slightly during the first 36 hr (20%). Little change in the total number of parasites was noted during the interval of 36–108 hr. A subsequent decrease in numbers of parasites was noted until by 280 hr after implantation, chambers contained less than 2% of the original number of organisms present in the chambers. No similar transformation of epimastigotes was noted in diffusion chambers maintained in cell culture at 37 C or in a cell culture growth medium or LIT medium at 37 or 26 C.No detectable morphological change was noted when trypomastigotes and amastigotes were implanted in diffusion chambers in the peritoneal cavity of mice. The total number of these parasites decreased notably (82%) after 24 hr.Mice receiving diffusion chambers containing epimastigotes implanted at two different intervals (21 days apart), developed only marginal protective immunity when challenged with virulent T. cruzi three weeks after the second implant of chambers, and no protection was afforded those mice implanted with chambers containing trypomastigotes and amastigotes. Sera collected from mice 6 wk after the second implantation of diffusion chambers containing parasites were observed to have antibody titers to T. cruzi as demonstrated by the fluorescent antibody technique and direct agglutination procedure.     

Leishmania mexicana: Serial cultivation of intracellular stages in a cell-free medium

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 × 10⁷/ml after 10 days of incubation at 33 C, and 5 × 10⁷/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 × 10⁷/ml after 12 days of incubation.     

First successful transfer of cryopreserved feline (Felis catus) embryos resulting in live offspring

Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.     

Induction of multiple chemokine and colony-stimulating factor genes in experimental Burkholderia pseudomallei infection

Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN-gamma in addition to the chemokines interferon-gamma-inducible protein 10 (IP-10) and monocyte interferon-gamma-inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G-CSF, M-CSF, GM-CSF, IP-10, Mig, RANTES, MCP-1, KC and MIP-2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high-level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) mRNA was demonstrated in spleen, while MIP-2, MCP-1, IP-10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.     

Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.     

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica)

The timing of development was examined in 496 embryos from female Monodelphis domestica, collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96–100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.© 1994 Wiley-Liss, Inc.     

Transfer of immunity to Babesia microti of human origin using T lymphocytes in mice

Lymph node and spleen cells from mice infected with Babesia microti of human origin developed the ability to transfer adoptive immunity to naive mice within 25 days after infection. This protective activity was greater in cells obtained at 32 days than in cells obtained at 25 days postinfection and remained stable up to 52 days postinfection. Recipients of lymph node cells and spleen cells displayed similar peak parasitemias although 2 days after peak parasitemia, immune spleen cell recipients had significantly lower parasitemias than immune lymph node cell recipients. Strong protective activity was demonstrated when cells were transferred 1 day postinfection, while equal numbers of cells, transferred 3 days postinfection did not confer significant protection over nonimmune cells. There was also a suggestion that the number of immune spleen cells necessary for significant protection was directly related to the number of parasites inoculated. The subpopulation of lymphocytes responsible for the transfer of adoptive immunity to B. microti of human origin was then studied in BALB/c mice depleted of T lymphocytes by thymectomy and lethal irradiation. One day after infection with B. microti, T-cell-depleted mice were given complement-treated immune spleen cells, anti-θ serum-treated immune spleen cells, nonimmune spleen cells, or no cells. Similar experiments were performed comparing the effects of anti-immunoglobulin serum-treated and unfractionated immune spleen cells on B. microti parasitemia. Treatment with anti-θ serum abrogated the protective activity of immune spleen cells while anti-immunoglobulin serum treatment had no effect. These results suggest that immunologic memory of B. microti in BALB/c mice is modulated by T rather than B lymphocytes.     

Trypanosoma congolense: calf erythrocyte survival.

Hereford calves infected with Trypanosoma congolense developed an anemia which was most severe 10 weeks after infection when packed cell volumes (PCV) averaged 21.1 ± 2.5% (±2 SE) as compared to 33.1 ± 2.1% for controls. At the termination of the study, at 28 weeks postinfection PCVs of infected animals had risen to 27.5 ± 1.0% as compared to 34.0 ± 1.7% for controls. In parallel with PCVs the apparent half-lives of ⁵¹Cr-labeled erythrocytes were reduced as early as the first 2 weeks postinfection. The greatest difference in erythrocyte half-lives between infected and control animals was found during the fourth to sixth weeks of infection when volumes for infected animals averaged 128 ± 46 hr as compared to 321 ± 30 hr for controls. During this period the parasitemia was at its highest level. At 28 weeks postinfection the average apparent half-life of infected animals was 243 ± 43 hr compared with 304 ± 11 hr for controls. No differences were observed in gastrointestinal loss of ⁵¹Cr between infected and control animals; however, urinary excretion of isotope was greatly increased in infected animals when compared to controls. No significant changes in total blood volumes were observed between infected and control animals but total plasma volumes increased and total erythrocyte volumes decreased significantly in infected animals.     

Leishmania mexicana: inhibition and stimulation of phagosome-lysosome fusion in infected macrophages

The polyanionic compound poly-d-glutamic acid was found to inhibit significantly the fusion of secondary lysosomes to phagosomes containing Leishmania mexicana mexicana amastigotes for at least 96 hr. This process was viewed both by dark-field vital fluorescence microscopy and transmission electron microscopy. In poly-d-glutamic acid-treated macrophages parasites multiplied at a significantly greater rate than in untreated macrophages. Conversely, the secondary amine chloroquine caused a marked reduction in parasite growth. When L. m. mexicana promastigotes were substituted for amastigotes these results were strikingly more pronounced.