Ultrastructure of Trypanosoma cruzi in mouse myocardium. II. Crithidial and leishmanial forms

Albino mice infected with Trypanosoma cruzi were sacrificed 7, 14, 22, 35, 40, and 60 days after infection, and the myocardia were studied by means of the electron microscope. The intracellular leishmanial form consists of a cellular membrane under which is the striated periplast; a central ovoid nucleus; and a kinetonucleus situated on the side of the nucleus. In the concavity of the kinetonucleus are the flagellum basal corpuscles from which originate a short flagellum; mitochondria, vacuoles, lipid deposits and ribosomes are seen in the cytoplasm. The crithidial form is longer and has a longer flagellum which is joined to the parasite through an undulating membrane; rough endoplasmic reticulum and Golgi apparatus are seen in the crithidia cytoplasm. The intracellular nest is formed by binary division of the leishmanial form. The transformation of the crithidial form into the trypanosome is made through posterior migration of the kinetonucleus, which becomes transformed into the posterior vacuole. In the clear spaces between the myofibril are found isolated parasites with an elongated form and a central nucleus; their kinetonucleus is different than those of the leishmanial and crithidial forms, and they might be evolutionary stages in the transformation from trypanosome into leishmania after penetration into the myofiber. The observed destruction of the sarcomeres and alterations of the mitochondria within the parasitized myofiber characterize the acute experimental myocarditis in the white mouse.     

The Development and Survival of the Blood-Stream Forms of Trypanosoma conorhini in Culture

SYNOPSIS. Trypanosoma conorhini has been isolated from infected Triatoma rubrofasciata in Singapore. Parasites derived from the triatomid gut were inoculated into NNN medium and some cultures maintained at 25° to 27°C while others were kept at 37°. In cultures kept at lower temperatures crithidial forms predominated; however, at 37° blood-stream trypanosome forms developed and survived for 10 days or more.     

A Process of Reproduction of Trypanosoma conorhini Different from Binary or Multiple Fission

SYNOPSIS. In cultures of Trypanosoma conorhini the authors have detected cyst-like bodies (CLBs) that, in the initial phase, have the appearance of 2 or more overgrown crithidiae fused together; after disorganization of the internal structure of the parent flagellates and division of their organelles, new crithidiae are formed within the CLB. The wall of the CLB consists of an irregularly thick layer of cytoplasm surrounded by the periplast of the parent flagellates and a true cystic membrane is not evident. The daughter crithidiae eventually escape from the CLB leaving the periplast and other residues of the parent flagellates. It is suggested that this represents a method of reproduction entirely different from binary or multiple fission, that is probably common to other trypanosomes of the Section STERCORARIA, and that it may provide for genetic exchange even if it is not a true sexual process.     

Ultrastructure of Trypanosoma duttoni.

Trypanosoma duttoni exhibits the typical trypanosome morphology in that it is bounded by a unit membrane which included its anterior flagellum; it is surrounded by a framework of suppellicular fibrils, and it contains a nucleus and the posteriorly located kinetoplast-blepharoplast structures along with cytoplasmic organelles (mitochondria, Golgi, and enoplasmic reticulum). Very prominently displayed in T. duttoni is the contractile vacuole, which has not previously received wide-spread recognition. The kinetoplast is clearly continuous with mitochondria in some cases. Inclusion bodies are categorized into three distinct types, none of which can be conclusively designated as the very popular volutin granules, without cytochemical evidence. The existence of a third tubule among peripheral tubule doublets of the flagellum is observed.     

Life-Cycle of Trypanosoma conorhini. Influence of Temperature and Other Factors on Growth and Morphogenesis

SYNOPSIS. In continued observations on the in vitro growth and multiplication of the bloodstream trypanosome stage of Trypanosoma conorhini , a better medium was found for cultivating these forms at 37°C, but no subcultures could be obtained. The infectivity for mice of the blood type trypanosomes grown in vitro was comparable to that of the metacyclic trypanosomes. The only reproducing forms of T. conorhini found in the vertebrate were in the trypanosome stage.
It was also found that the in vitro reversion of the bloodstream trypanosome into crithidia, such as occurs in the invertebrate host and in the usual diphasic culture medium, is dependent on at least two factors: if incubated at 25–28° reversion did not occur in any of the liquid media tried (all containing blood serum and hematin or hemoglobin), unless total blood was part of the inoculum or washed red blood cells were added to the media; on the other hand, no reversion was seen, even in the presence of red blood cells if the cultures were incubated at 37°.     

Carbohydrate Transport in Trypanosoma gambiense*

SYNOPSIS. The uptake of ¹⁴C-labeled carbohydrates by Trypanosoma gambiense was studied. Glucose, mannose, glycerol, 2-deoxyglucose and fructose were rapidly absorbed by the parasite, and all had saturation kinetics. The glucose analog 3-O-methylglucose was not taken up by T. gambiense. Competitive inhibition experiments indicate that there are 2 transport loci for the tested substrates. It is suggested that there is a “glucose site” thru which glucose, mannose and glycerol, but not fructose, are transported and a “fructose site” thru which only fructose is transported. The specificity of the glucose-transporting mechanism appears to differ from those of other animals.     

Differentiation of Trypanosoma cruzi in Culture*

SYNOPSIS. A medium containing sodium and potassium chloride, phosphate buffer, glucose, calf serum, hemoglobin and lactalbumin hydrolysate permits some growth and differentiation of crithidiae into the metacyclic forms of Trypanosoma cruzi. The addition of ox liver infusion greatly enhances the growth promoting properties of this medium while making it very poor as far as differentiation is concerned. With dog heart infusion instead of ox liver, the medium is still good for growth but not much better for differentiation. If, however, the pH of the dog heart infusion medium is lowered from 7.2 to 6.7, it continues to be good for growth and becomes excellent for differentiation. Other factors were studied that stimulate or repress the rate of differentiation, such as preincubation at certain levels of temperature and the size and age of inocula.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

Development of Trypanosoma lewisi in Experimental Chimaeras*

SYNOPSIS. Development of Trypanosoma lewisi in chimaeric mice with and without rat serum supplements was studied in 5 protocols. Chimaeric mice were male and female C₃H/An Cum and 1C₃F₁, [(101/CUM × C₃H/An Cum) F₁] exposed to 950-1000 R of whole-body X-irradiation. Their hemopoietic systems were replaced with Sprague-Dawley rat bone marrow. Trypanosomes did not develop in any mice not supplemented with rat serum. Ih those chimaeric and normal mice given rat serum the trypanosomes grew and developed large populations, more so in chimaeric than in normal animals. The development of this trypanosome cell in heterologous mice is viewed as mediated principally thru nutritional factors supplied by rat serum with alteration of host-immune status being of lesser importance.     

Specificity of Sugar Transport in Trypanosoma gambiense*

SYNOPSIS Some carbohydrates inhibited glucose and fructose transport in Trypanosoma gambiense. Glucose transport was inhibited by glycerol, mannose, 2-deoxy-D-glucose, glucosamine and N-acetylglucosamine. Fructose transport was inhibited by glucose, glycerol, mannose, glucosamine and N-acetylglucosamine. Glucosamine transport appeared to be a mediated process and had a K_m of 1.20 mM and a V_max of 28.5 μM glucosamine/g dry wt/2 min. Glucosamine absorption was competitively inhibited by glucose, fructose and N-acetylglycosamine. N-Acetylglucosamine appeared to enter by passive diffusion. Reciprocal inhibition experiments suggested that glucosamine entered entirely via the “fructose site.” Specificity of sugar transport in T. gambiense differs from that of other organisms.     

Phosphate Metabolism in the Culture Form of Trypanosoma cruzi*

SYNOPSIS. The uptake, distribution and turnover of phosphorus have been studied for the culture form of Trypanosona cruzi. Following exchange reactions, phosphorus was accumulated at an approximate rate of 1.9 μg/10⁸ trypanosomes/hour in Krebs-Ringerphosphate. Of 3.8 mg P/g trypanosomes (wet weight), 60% occurred in acid-soluble, 13% in phospholipid, 22% in nucleic acid, and 5% in phosphoprotein fractions. Acid-soluble and phosphoprotein fractions incorporated P³² more rapidly than phospholipid and nucleic acid fractions, the phosphoprotein fraction having the highest specific activity by 8 hours of incubation.     

Cryopreservation of Trypanosoma cruzi Bloodstream Forms*

SYNOPSIS. Recovery rates of T. cruzi bloodstream forms subjected to several methods of cryopreservation in liquid nitrogen and at -73 C are reported. Inoculations of animals with cryopreserved and nonpreserved trypomastigotes revealed that prolonged storage at -196 C apparently did not change the biologic characteristics of different T. cruzi strains. The reproducibility and consistency of results suggest that “cryobanks'’or “reference centers'’may be established.     

Fine Structure of Trypanosoma cyclops in Noncellular Cultures*

The fine structure of the epimastigotes of Trypanosoma cyclops maintained in blood agar medium at 25 C is described. This organism was isolated from the Malaysian primates Macaca nemestrina and Macaca ira. A distinctive feature of T. cyclops is that it is pigmented when grown in the presence of hemoglobin. The pigment bodies apparently lack a substructure and are electron dense even in unstained sections. Most of the pigment is located posterior to the kinetoplast region but some is found adjacent and anterior to the kinetoplast. Cells from control cultures grown in medium lacking hemoglobin did not possess this type of pigment body. Similarly, pigment was not found in cells of an Indonesian trypanosome grown in medium containing hemoglobin. The cytoplasm of T. cyclops is bounded by a unit membrane which is specialized where it makes contact with the flagellum. A cytostome extends from the region of the flagellar pocket. The kinetoplast and nucleus are immediately posterior to the base of the flagellum. Transverse sections in the region of the flagellar pocket and flagellar base often reveal a group of 3 microtubules which are distinct from the pellicular microtubules.     

Cryoprotectants for Crithidia fasciculata Stored at -20 C, with Notes on Trypanosoma gambiense and T. conorhini

SYNOPSIS. Cryoprotectants were tested in both complex and semidefined media for the trypanosomatid Crithidia fasciculata. Near log-phase or end-of-log-phase cultures were frozen for 24–48 hr at ∼ -20 C, then warmed in air to room temperature. Immediate motility was correlated with viability. The best protectant of the 83 tested was glycerol at ∼ 10% (w/v). Survival without cryoprotectant was rare. Outstanding cryoprotectants (perhaps also useful solvents for drugs poorly soluble in water) were: ethylene glycol; 2,2'-dioxyethanol (diethylene glycol); 1,2,4-butanetriol; 1,4-cyclohexanediol; dimethylsulfoxide; propylene glycol; and N -acetylethanolamine. Several sugars were active, e.g., D-arabinose, sucrose, and sorbitol. Trypanosomes tolerated cryoprotectants much less; tolerance was better in growth media than in suspension media. Trypanosoma gambiense was grown in blood-enriched media + 2-2.5% glycerol, suspended in 20% (w/v) glycerol. then frozen; this permitted 3-week survival. T. conorhini survived 4 weeks after growth in media containing glycerol 2.5%+ ethylene glycol 4%+ rutin 1.0 mg per 100 ml.     

Specificity of Amino Acid Transport in Trypanosoma equiperdum*

Uptake of [¹⁴C] alanine, arginine, glutamic acid and phenylalanine by Trypanosoma equiperdum occurred by both a mediated mechanism and diffusion. Twenty amino acids were studied as inhibitors of absorption of the above amino acids. Results suggested that at least 4 distinct transport loci are involved in amino acid transport. These 4 loci have overlapping affinities for amino acids and seem to be involved, respectively, in the absorption of (a) arginine and phenylalanine; (b) arginine; (c) alanine, phenylalanine, and glutamic acid; (d) glutamic acid. The data also showed that multiple sites for substrate binding occur on each of 2 transport systems.     

Proteolytic Activities in Cell Extracts of Trypanosoma cruzi*

SYNOPSIS. Cell extracts of culture forms of Trypanosoma cruzi are capable of hydrolysing substances belonging to 4 different groups of protease substrates: (a) substrates for trypsin-like enzymes: benzoyl-arginine-p-nitroanilide and benzoylarginine-naphtylamide: (b) substrates for aminopeptidases: leucyl. lysyl and glutamyl-β-naphtylamide; (c) a substrate for chymotrypsin-like enzymes: carbobenzoxy-L-tyrosine-p-nitrophenylester, and (d) a nonspecific substrate for a broad range of proteases: azocasein. Some physico-chemical characteristics of each enzymic reaction were studied. They were found to be distinct enough to allow attributing each hydrolytic activity to a separate enzyme.