Infectivity of Trypanosoma rhodesiense Cultivated at 28°C with Various Tsetse Fly Tissues1

ABSTRACT. Metacyclic trypanosomes developed in populations of procyclic forms of four stocks of Trypanosoma brucei rhodesiense cultivated at 28°C in a liquid medium containing explants of tsetse fly head-salivary glands, alimentary tract, abdominal body wall, or thoracic muscle. The cultures became infective for mice 7–16 days after they were prepared, and infective trypanosomes were present for prolonged periods. In the culture series of stock TRUM 545, infectivity persisted for 138 days when the cultures were terminated. Only one explant of thoracic muscle tissue was required for the production of metacyclic stages in stock TRUM 497 cultures. Infectivity titrations on trypanosome suspensions from cultures of stocks TRUM 497, TRUM 545, and TRUM 567 revealed that only a small proportion of the culture population was infective. Using stock TRUM 530, mice were infected consistently from inoculations of trypanosomes grown in the presence of explants; infectivity of the trypanosomes ceased when the explants were removed from the flasks, but reappeared when they were returned to the cultures. Parasites grown in medium “conditioned” by explants produced sporadic infections in mice. The control cultures of trypanosomes grown in medium alone were generally not infective, but two of the stocks produced occasional parasitemias. Stained samples of infective inocula contained a few epimastigote-like and metacyclic-like trypanosomes.     

Antigenic Analysis By Immunofluorescence of In Vitro-Produced Metacyclics of Trypanosoma Brucei and Their Infections In Mice*

SYNOPSIS. the antigenic types in populations of metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms were heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

In Vitro Binding of Trypanosoma congolense to Erythrocytes*

Synopsis. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin. Trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

The Effects of Bacterial Endotoxin on the Infection of Mice with Trypanosoma cruzi*

Mice (Rockland strain) infected with Trypanosoma cruzi strain Tulahuén were treated with Escherichia coli endotoxin before, simultaneously with, and after inoculation of the parasites. The peak parasitemias of endotoxin-treated mice were higher than those of nontreated infected animals, regardless of the time of endotoxin administration. Peak parasitemias occurred at the same time in infected nontreated mice as in animals given endotoxin before or simultaneously with the trypanosomes. If endotoxin was administered 24 hr after the infection, a delay in the peak parasitemia was noted. Changes in the survival time were not observed unless endotoxin was given 24 hr postinfection. Infected mice had an increasing susceptibility to the lethal effect of endotoxin. The LD₅₀ of endotoxin decreased from 675 μg for normal mice to 230, 92, and 18 μg for infected animals 1, 3, and 8 days after the infection, respectively. In the infected mice, the endotoxin-detoxifying ability of the spleen was found to be impaired.     

Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics

Okanla E. O., Stumpf J. L. &; Dusanic D. G. 1982. Resistance of mice immunized with irradiated and lyophilized stages of Trypanosoma cruzi to infections with metacyclics. International Journal for Parasitology12: 251–256. BALB/c mice were immunized with either irradiated or lyophilized metacyclic, epimastigote or bloodstream forms of Trypanosoma cruzi in three weekly injections of 1 × 10⁸ trypanosomes/injection. The lyophilized trypanosomes were emulsified in equal quantities of Freund's complete adjuvant. Two weeks following the final immunization, the mice were challenged subcutaneously with metacyclics obtained from either culture or the vector Triatoma infestans. The mice challenged with metacyclics from culture included groups of mice immunized with each of the three stages, while those challenged with metacyclics from the T. infestans included mice immunized with the epimastigotes or metacyclics. Mice immunized with the irradiated epimastigotes, metacyclics and blood-stream form trypomastigote challenged with metacyclics from culture exhibited reduced parasitemias compared to mice of the control groups. Parasitemias were lowest in those mice immunized with irradiated metacyclics. The parasitemias terminated in the immunized mice before those of the control animals. No protection was detected in the mice inoculated with lyophilized trypanosomes and challenged with culture metacyclics. Groups of mice injected with either irradiated or lyophilized epimastigotes or metacyclics and challenged with metacyclics from T. infestans exhibited resistance both by reduction of the parasitemias and the duration of the parasitemias when compared to the infected control animals. This study demonstrated the comparative effectiveness in mice of irradiated and lyophilized vaccines produced from either metacyclics, epimastigotes or bloodstream forms when challenged with metacyclics obtained from culture and the vector.     

Effect of non-complementary nucleotides on the rate of helix formation: kinetics of formation of poly (I)-poly (C,I) and poly (I)-poly (C,U) complexes

Apparent second-order rate constants for complex formation between poly (I) and poly (C) and copolymers of C containing non-complementary I or U residues have been determined spectrophotometrically. The rate constants decrease as the concentration of either I or U in the C strands increases–the effect seems insensitive to the species of residue involved, when differences in the thermal stabilities of the poly (I) poly (C,I) and poly (I). poly (C,U) complexes are taken into account. These results suggest that low concentrations of relatively stable defects can alter the apparent kinetic “complexity” of polynucleotides as determined by hybridization methods (C₀t analysis).     

Antigenic analysis by immunofluorescence of in vitro-produced metacyclics of Trypanosoma brucei and their infections in mice

The antigenic types in populations of Metacyclic trypanosomes of Trypanosoma brucei isolated from Glossina morsitans head-salivary gland trypanosome cultures and bloodstream forms in the early parasitemias produced from whole culture supernatant fluids containing metacyclic forms, were analyzed by the indirect fluorescent antibody test using clone-specific antisera. Metacyclic trypanosomes in cultures initiated with cloned bloodstream forms with heterogeneous with respect to their variable antigenic type (VAT). Trypanosomes comprising early parasitemias in immunosuppressed mice infected with metacyclics produced in cultures also had a range of VATs. Three of the VATs detected in the early parasitemias in mice have also been identified by other investigators in tsetse fly-transmitted populations of the same stock.     

The nature of different influence of Cd<Superscript>2+</Superscript> ions on the conformational equilibrium of triple-stranded polyribonucleotides poly(U)•poly(A)•poly(U) and poly(I)•poly(A)•poly(I) in aqueous solutions

The influence of Cd²⁺ ions on the conformational equilibrium of single-stranded (poly(U), poly(A), poly(I)) and triple-stranded polyribonucleotides (A2I, A2U) in aqueous solutions (0.1 M Na⁺ pH 7) has been investigated using difference UV spectroscopy and thermal denaturation. Analysis of the shape and intensity of the DUV spectra of poly(A), poly(I), and A2I has revealed the presence of two types of complex formed as a result of (i) interaction between Cd²⁺ and the N7 atoms of purines, producing macrochelates; and (ii) binding of Cd²⁺ to the N1 atoms of poly(A) and poly(I). Since Cd²⁺ ions are not bound to heteroatoms of the bases in A2U, the conformation of the structure remains stable up to 0.02 M Cd²⁺. There is a critical Cd²⁺ concentration (～1.5?10^?4 M) above which A2I assumes a new helical conformation with lower thermal stability. It is supposed that, upon the formation of the “metallized” A2I triplex, the Cd²⁺ ions are located inside the triple helix and form bridges between the hypoxanthine and adenine of the homopolynucleotide strands.     

Enhancement by double-stranded polyribonucleotides of production by cultured mouse peritoneal macrophages of differentiation-stimulating factor(s) for mouse myeloid leukaemic cells.

Mouse peritoneal macrophages release a factor(s) that stimulates differentiation of a mouse myeloid leukaemic cell line into mature granulocytes and macrophages. Treatment of the macrophages with the synthetic double-stranded polyribonucleotides poly(I).poly(C) and poly(A).poly(U) resulted in enhanced release of the factor into the culture medium. The effect was maximal after treatment with polyribonucleotides for 1 h, and the optimal dose of poly(I).poly(C) was 50 microgram/ml. The single-stranded polyribonucleotides poly(I) and poly(C) at the same concentration were far less effective. The differentiation-stimulating factor was detected not only in the cultured medium but also in the cell lysate. Exposure of macrophages to poly(I).poly(C) enhanced the total activity of the factor in both the culture medium and the cell lysate. The effect of this compound was blocked by the presence of cycloheximide. These results suggest that double-stranded polyribonucleotides enhance production of the differentiation-stimulating factor by peritoneal macrophages.     

Trypanosoma musculi infections in complement-deficient mice

Strains of mice (CFW, C57B1/10Sn, B10.D2/nSn, and B10.D2/oSn) were infected with Trypanosoma musculi (Trypanosoma duttoni). The complement-deficient B10.D2/oSn mice showed typical parasitemias similar to those presented by the strains possessing hemolytic complement activity. Peak parasitemias occurred 12 days postinoculation. The highest parasitemias were measured in CFW mice (657 ± 82 T. musculi/30 hi-power fields), while infections in C56BL/10Sn (528 ±44 T. musculi/30 h.p.f.), B10.D2/oSn (502 ± 20 T. musculi/30 h.p.f.) and B10.D2/nSn (512 ± 35 T. musculi/30 h.p.f.) were less severe and quantitatively comparable. The percentages of dividing forms were similar during infections in each of the strains. While parasites were detected in peripheral blood until Day 22 of infection in three of 10 C57BL/10Sn mice, none could be found at this time in blood films of CFW, B10.D2/nSn or B10.D2/oSn mice. Giemsa stained kidney imprints indicated the presence of parasites in animals of each of the strains after 33 days, when trypanosomes could no longer be detected in the peripheral blood of the mice. The minor variations in the parasitemias appeared related to the mouse strain. Complement dependent, antibody mediated immune cytolysis was not indicated as a mechanism for the elimination of T. musculi by the infected mouse.     

A comparison of susceptibility of two allopatric populations of Glossina pallidipes for stocks of Trypanosoma congolense

Abstract. A colony of Glossina pallidipes Austen which originated from Nguruman, Rift Valley Province, Kenya, was significantly more susceptible to infection (19.3%) with a stock of Trypanosoma congolense Broden isolated from G. pallidipes in Nguruman than a colony of the same species which originated from Shimba Hills, Coast Province, Kenya (5.6%). Male G. pallidipes from Nguruman were significantly more susceptible than females to this T. congolense stock whilst the susceptibility of both sexes of G. pallidipes from Shimba Hills did not differ significantly. All six goats on which six infected G. pallidipes fed singly (three tsetse per colony) became infected. Similarly, the G. pallidipes colony of Nguruman origin was significantly more susceptible to infection (16.4%) with a stock of T. congolense isolated from G. pallidipes in Shimba Hills than the colony of Shimba Hills origin (4.9%). The susceptibility of the sexes of G. pallidipes from both the colonies to this stock of T. congolense did not differ significantly. Again, all six goats on which six infected G. pallidipes fed singly (three tsetse per colony) became infected. If the observed differences in susceptibility of the two G. pallidipes colonies reflect transmission of trypanosomes by the two allopatric populations of tsetse in the field, then the epidemiology of congolense-trypanosomiasis in livestock must differ between these two areas of Kenya endemic for trypanosomiasis.     

Trypanosoma congolense: Specific transformation in vitro of leukocytes from infected or immunized cattle

An assay to measure the specific proliferation in vitro of peripheral blood leukocytes (PBL) in response to ultrasonicated trypanosomes was adapted for use in cattle. The kinetics of mitosis exhibited by PBL from cattle which had been treated following infection with Trypanosoma congolense paralleled the development of a delayed-type skin reaction elicited with ultrasonicated and Formalin-fixed T. congolense. Responses in both tests were maximal on the fourth day after initiation. Specific proliferation of PBL harvested from cattle which had been immunized with intact, nonviable trypanosomes was also elicited in vitro by trypanosomal antigen. Peripheral blood leukocytes taken from cattle immunized with 50 μg of variant-specific surface antigen (VSSA) from T. brucei and from cattle infected with T. congolense were not stimulated to divide in vitro by ultrasonicated trypanosomes.     

Trypanocidal drugs and the role of kidney forms of Trypanosoma (Herpetosoma) musculi in immunity of mice against reinfection

Adrenals, hearts, kidneys, livers, lungs, and spleens were removed from C3H/Anf mice which had been inoculated with Trypanosoma (Herpetosoma) musculi and no longer exhibited parasitemias. Imprints of each organ were examined microscopically, and each was homogenized and injected into recipient mice. It was confirmed that trypanosomes could be detected only in the donor kidneys. Lampit or Ethidium treatment eliminated bloodstream and kidney forms when administration was initiated after the development of patent parasitemias. However, mice treated with Lampit on the same day they were inoculated with T. musculi developed parasitemias later than animals injected with drug after parasites had appeared in their blood. Both Lampit and Ethidium depressed antibody production as detected in enzyme-linked immunosorbent assays of antisera from animals having parasitemias at the time of treatment. The elimination of kidney forms by Lampit or Ethidium treatment did not reduce the resistance of mice to reinfection by T. musculi 12 weeks or 15 and 22 weeks, respectively, after the initial inoculation of these animals with the parasites. Kidney forms were not required for the sustained protective immunity of the mice against reinfection during the intervals of these experiments.     

Effect of Holothurin on Trypanosoma musculi infection in Three Strains of Mice1

The effect of holothurin (a marine biotoxin) on the resistance of mice to Trypanosoma musculi was measured by studying changes in the parasite population in vivo. Swiss Webster (SW), Beige (BG), and Black (BL) mice treated with holothurin prior to and simultaneously with infection of trypanosomes had lower parasitemias than controls. Higher levels of parasitemia were observed in mice treated after infection with trypanosomes. The timing of administration of holothurin appeared to be an important factor in the observed effect. The minor variations in the parasitemia seemed to be related to the mouse strain.     

Binding of Trypanosoma congolense to the Walls of Small Blood Vessels*

SYNOPSIS The mesenteric microvasculature was studied in rats and rabbits infected with Trypanosoma congolense. By examining vessels in the living animals, trypanosomes were observed to adhere to vessel walls by their anterior ends. It was evident from stained preparations of the vessels that the microcirculation contained 4–1400 times as many trypanosomes as were free in the cardiac blood. Parasites were more numerous in very small vessels than in larger vessels, and they were clustered in groups within the small vessels. The localization of T. congolense in the microvasculature is demonstrated and it is shown that this localization is established by attachment of the organism to the vessel wall.     

Trypanosoma brucei: influence of host strain and parasite antigenic type on infections in mice

Inbred mice were infected with cloned populations of Trypanosoma brucei brucei Lister S42 under carefully controlled conditions. The course of infection was found to depend both on host strain and the antigenic type of the infecting organisms. The two antigenic types used, “NIM2” and “NIM6” had differing virulence for (CBA/H × C57BL/6)F₁ mice, and when mice were infected with parasites of one clone, trypanosomes of the other type frequently appeared after the initial population had been eliminated. Different mouse strains had varying susceptibility to clone NIM6. In most cases there was an inverse relation between the survival time and the parasite load during the first peak of parasitemia. The differences in resistance to T. brucei were unrelated to H-2 haplotype: C57BL/6 and (CBA/H × C57BL/6)F₁ were most resistant, CBA/H, BALB/c and DBA/2 less so, and C3H/He most susceptible.     

Interaction of ethidium bromide with synthetic double-stranded polyribonucleotides

The interaction of ethidium bromide (EtBr) with double helical synthetic polyribonucleotides poly(G).poly(C), poly(A).poly(U) and poly(I).poly(C) has been investigated by the method of isothermal microcalorimetry and according to the character of changes on the spectra of circular dichroism, absorption and fluorescence at binding. The calculations showed that at binding of EtBr with poly(A).poly(U) the saturation stechiometry was one EtBr molecule per 2 base pairs with binding constant (2.5 +/- 0.5).10(6) M-1 at 30 degrees C and 0.1 M. NaCl. In the case of binding of EtBr with poly(G).poly(C) and poly(I).poly(C) the saturation stechiometry was not less than 0.2 EtBr molecule per 1 base pair with binding constant (4 +/- 1).10(3) M-1 and (1.5 +/- 0.3).10(4) M-1 respectively, at 25 degrees C and 0.1 M NaCl. The binding enthalpies of EtBr with poly(A).poly(U) and poly(G).poly(C) have been determined to be (-7.5 +/- 0.5) Kcal per 1 mol of bound EtBr in average for both polymers. It has been shown that the observed strong selectivity of EtBr binding with polyribonucleotides is of entropic origin.     

Ultrastructural Studies of Certain Aspects of the Development of Trypanosoma congolense in Glossina morsitans morsitans*

SYNOPSIS The course of Trypanosoma congolense infections in Glossina morsitans morsitans was followed by electron-microscopic examination of ultrathin sections of the guts and proboscises of infected flies. Guts dissected from flies 7 days after infection with culture procyclic forms of T. congolense had heavy trypanosome infections in the midgut involving both the endo- and ectoperitrophic spaces. Trypanosomes were also seen in the process of penetrating the fully formed peritrophic membrane in the central region of the midgut. By post infection day 21, trypanosomes had reached the proboscis of the fly and were found as clumps of epimastigote forms attached to the labrum by hemidesmosomes between their flagella and the chitinous lining of the food canal. Desmosome connections were observed between the flagella of adjacent epimastigotes. Flies examined at postinfection days 28 and 42 had, in addition to the attached forms in the labrum, free forms in the hypopharynx.     

Induction by synthetic polyribonucleotide poly(I) of differentiation of cultured mouse myeloid leukemic cells.

The effects of some synthetic polyribonucleotides on induction of differentiation of mouse myeloid leukemic M1 cells were examined. Poly(I) was found to be a potent inducer; on treatment with 100--200 microgram/ml of poly(I) for 2--4 days, M1 cells differentiated into cells resembling macrophages and granulocytes and developed phagocytosis and locomotive activities, Fc receptors and lysozyme activity. Poly(C) was less effective than poly(I) for induction of phagocytic activity, while the other single-stranded RNAs, poly(U) and poly(A), had no effect. Double-stranded RNAs, such as poly(I) . poly(C) and poly(A) . poly(U), were cytotoxic to M1 cells, and differentiation of the cells could not be detected even at the highest tolerable concentrations of these double-stranded RNAs.