Cytochemical study of nucleic acids in relation to the polymorphism of the trypomastigote forms of Trypanosoma cruzi

Four morphological types of T. cruzi trypomastigotes are distinguished in mouse blood. These differ in RNA contents, in the distribution pattern of RNA in the cytoplasm, in the intensity of the Feulgen reaction and the topography of DNA in the nucleus, and in the contents and distribution of both the nucleic acids in the kinetoplast. Among the trypomastigotes examined, forms C and S differ at a lesser degree, than their slender and middle variants differing much stronger. The early slender trypomastigotes are characterized by poor and diffuse RNA in the cytoplasm, by a homogeneous distribution of DNA in the nucleus and by a low content of DNA (sometimes RNA) in the kinetoplast. The middle trypomastigotes, dominating at the final step of the infection, are rich in granular RNA, differ (despite their inability to divide) in their nuclear organization mostly characterized by a large karyosome and uneven distribution of chromatin at the periphery; the kinetoplast is rich in DNA and often contains RNA. The peak of trypomastigotes with the kinetoplast deprived of obviously stained RNA precedes the impetuous increase of parasitemia. It coincides with the decrease in the number of destroyed parasites, and with the active substitution of slender variants by the middle ones within both C- and S-forms. Thus, changes in the nucleus and kinetoplast are involved in the trypomastigote transformation and in the development of infection.     

Cryptobia cataractae from the blood of Semotilus atromaculatus: structure and division in the fish

A cryptobiid was found in the blood of 2 of 9 Semotilus atromaculatus from a tributary of the Saugeen River in Ontario, Canada. Blood inoculation produced an infection in 2 uninfected S. atromaculatus but not in any Oncorhynchus mykiss, Catostomus commersoni, or Carassius auratus. The flagellate was identified as Cryptobia cataractae, based on host restriction. Cryptobia cataractae occurred as slender and broad forms (body width 3.0-8.7 microns). The length of the anterior flagellum was equal to body length, whereas that of the free recurrent flagellum was half body length. Cryptobia cataractae divided by equal binary fission that produced elongate, slender daughter cells.     

Trypanosoma brucei: fluxes of the morphological variants in intact and X-irradiated mice

The number of dividing, slender, intermediate, and stumpy forms of Trypanosoma brucei in the blood of inbred mice changed daily. In both intact mice and mice which were exposed to whole body X-irradiation before infection, slender and dividing forms predominated during the first 72 hr of infection, and the number of intermediate and stumpy forms increased to a maximum between 72 and 140 hr. The rate at which stumpy forms accumulated in the blood and the number of these forms which eventually circulated were the same in both groups. However, the fluxes in the number of slender and dividing forms differed in intact and X-irradiated mice. In intact mice, the number of dividing forms in the blood decreased between 72 and 140 hr, and the number of slender forms decreased between 96 and 140 hr. In X-irradiated mice, the number of both these forms increased throughout infection. Electrophoresis of serum proteins and agglutination tests showed that X-irradiation severely depressed the ability of the mice to make antibody. Homogenates of spleen and bone marrow of intact mice contained many dividing forms throughout the infection. It is concluded that although host antibody does not directly induce the transformation of slender forms into stumpy forms, it may influence the morphological composition of the peripheral blood population of trypanosomes in several ways.     

Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi

The origin of Trypanosoma cruzi slender and broad forms found in the circulation of the mammalian host has remained obscure and, unlike what has been proposed for African trypanosomes, no precise form-function relationship has been ascribed to them. We show here that parasites circulating in the blood of infected animals display a high degree of polymorphism. Around 10% of the forms found circulating in mice during the acute phase of infection were amastigotes, and the other 90% included slender and broad trypomastigotes and intermediate forms between amastigotes and trypomastigotes. Slender trypomastigotes, from blood or cell culture, undergo extracellularly morphological rearrangements in which the parasites become gradually broader and transform into amastigotes. By scanning electron microscopy a progressive internalization of the flagellum and reorganization of the cell shape in a helical fashion were observed in parasites undergoing transformation. After 48 hr of extracellular incubation the parasite population consisted exclusively of amastigotes with a short protruding flagellum. The morphological changes were associated with the expression of different surface antigens defined by monoclonal antibodies: the trypomastigote-specific antigens Ssp-1 (a 100-120-150-Mr glycoprotein), Ssp-2 (a 70-Mr glycoprotein), Ssp-3 (undefined), and Ssp-4, an amastigote-specific surface antigen. Ssp-4 was also detected on intracellular amastigotes (in vitro and in vivo). We conclude that trypomastigotes are programmed to develop into amastigotes whether or not they enter cells, and that the differentiation can occur in the blood of the vertebrate host. These findings raise some questions regarding conventional views on the life cycle of T. cruzi.     

Transmission stages dominate trypanosome within-host dynamics during chronic infections

Sleeping sickness is characterized by waves of the extracellular parasite Trypanosoma brucei in host blood, with infections continuing for months or years until inevitable host death. These waves reflect the dynamic conflict between the outgrowth of a succession of parasite antigenic variants and their control by the host immune system. Although a contributor to these dynamics is the density-dependent differentiation from proliferative "slender forms" to transmissible "stumpy forms," an absence of markers discriminating stumpy forms has prevented accurate parameterization of this component. Here, we exploit the stumpy-specific PAD1 marker, which functionally defines transmission competence, to quantitatively monitor stumpy formation during chronic infections. This allows reconstruction of the temporal events early in infection. Mathematical modeling of these data describes the parameters controlling trypanosome within-host dynamics and provides strong support for a quorum-sensing-like mechanism. Our data reveal the dominance of transmission stages throughout infection, a consequence being austere use of the parasite's antigen repertoire.     

Plasmodium hegneri n. sp. from the European Teal Anas c. crecca in Taiwan*

SYNOPSIS. Plasmodium hegneri n. sp. is described from the European teal duck, Anas c. crecca, from Taiwan. The blood stages, so far the only ones seen, are distinguished mainly by the elongate character of the gametocytes, which closely resemble Haemoproteus (though the pigment tends to be finer and less abundant), and the failure of both asexual and sexual forms to displace the nucleus or otherwise alter the host cell. Merozoites average 13.4 ± 2.2 (range 10–19) per segmenter. Trophozoites often adhere to the host cell nucleus, and may have a large vacuole and a remarkable long, slender pseudopodium. The species so far has been seen only in the European teal, although blood films from 194 species and over 1200 birds have been examined.     

Plasmodium octamerium n. sp., an Avian Malaria Parasite from the Pintail Whydah Bird Vidua macroura*

SYNOPSIS. A new species of avian malaria parasite is described from the pintail whydah Vidua macroura, a very small African finch of the weaver bird family (Ploceidae). Its structure has been studied chiefly in the canary, to which it is easily transmissible by blood inoculation. Since the segmenters most often produce 8 merozoites, the name Plasmodium octamerium n. sp. is proposed. Other characteristics include sexual stages which are usually elongate, often slender, and do not displace the host cell nucleus, and gametocytes indistinguishable from those of many species of Haemoproteus. Erythrocytes are the only blood cells parasitized. The new species resembles Plasmodium fallax in many respects, but gives rise to fewer merozoites and the asexual forms are smaller. Blood-induced infections are also of strikingly different type in some host species. Among susceptible host species are several kinds of finches, pigeons, quail, young chicks, chukars, tree and song sparrows. In most of these hosts infections are mild, but some tree sparrows die as the result of blood infection, and chukars usually die because of massive invasion of the capillary endothelium of the brain by exoerythrocytic forms. These are of the gallinaceum type and may be quite large, producing hundreds of merozoites. Exoerythrocytic stages were sought but not found in other host species.     

Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins

Sindbis virus 26S RNA has been translated in a cell-free protein-synthesizing system from rabbit reticulocytes. When the system was supplemented with EDTA-stripped dog pancreas microsomal membranes, the following results were obtained: (a) Complete translation of 26S RNA, resulting in the production, by endoproteolytic cleavage, of three polypeptides that are apparently identical to those forms of C, PE2, and E1 that are synthesized in vivo by infected host cells during a 3-min pulse with [35S]methionine. (b) Correct topological deposition of the three viral polypeptides--in vitro-synthesized PE2 and E1 forms are inserted into dog pancreas microsomal membranes in a orientation which, by the criterion of their limited (or total) inaccessibility to proteolytic probes, is indistinguishable from that of their counterparts in the rough endoplasmic recticulum of infected host cells; in vitro-synthesized C is not inserted into membranes and therefore is accessible to proteolytic enzymes, like its in vivo-synthesized counterpart. (c) Core glycosylation of in vitro-synthesized PE2 and E1 forms, as indicated by binding to concanavalin A Sepharose and subsequent elution by alpha-methylmannoside.     

Trypanosoma brucei: in vitro propagation of metacyclic forms derived from the salivary glands of Glossina morsitans

1 Metacyclic forms of Trypanosoma brucei obtained from the salivary glands of the tsetse fly, Glossina morsitans have been cultured for the first time in their infective forms for more than 200 days in continuous culture. The parasites were grown at 25 C and 30 C on a bovine embryonic spleen (BESP) feeder layer in buffered RPMI 1640 medium supplemented with 20% heat-inactivated bovine fetal serum (BFS) and 5% lactalbumin hydrolysate. Initial growth rate was enhanced when normal, noninfected, salivary glands were added to the cultures. The parasites thus cultured appeared like slender or intermediate blood stream forms which were infective to rats and mice. Addition of rat anti-T. brucei specific antiserum to the cultures caused agglutination of the parasites and rendered them noninfective. This study opens up new areas of investigating sleeping sickness. The cultured metacyclic parasites have the potential of being applied as antigens for controlling African trypanosomiasis.     

Morphological changes in trypanosoma brucei rhodesiense following inhibition of polyamine biosynthesis in vivo

The effect of α-difluoromethylornithine (DFMO) treatment on the morphology of African trypanosomes was investigated. For this purpose inbred mice were immunosuppressed and infected with a clone of the protozoan blood parasite Trypanosoma brucei rhodesiense. The mice were then treated with DFMO, an irreversible inhibitor of ornithine decarboxylase, which inhibits polyamine synthesis. DFMO treatment in the absence of host immunity resulted in arrest of cytokinesis of the trypanosomes and many binucleated cells could be seen in blood smears. If mice were infected with a highly virulent trypanosome clone (ETat 1.10), which does not normally transform from long slender (LS) to short stumpy (SS) forms, DFMO treatment caused SS transformation to occur on days 3–4. This morphological SS transformation was substantiated by the presence of diaphorase activity and nuclear and mitochondrial changes. The results suggest a possible involvement of polyamines in the transformation from LS to SS forms.     

Symbiotic innovation in the oxymonad Streblomastix strix

Streblomastix strix is an enigmatic oxymonad found exclusively in the hindgut of the damp-wood termite Zootermopsis. Streblomastix has a number of unusual morphological characters and forms a complex but poorly understood symbiosis with epibiotic bacteria. Here we described the ultrastructure of S. strix, with emphasis on the axial cytoskeleton and cell-cell associations, in its normal state and when treated with antibiotics. In untreated cells, epibiotic bacteria were orderly arranged end-to-end on six or seven longitudinal vanes, giving S. strix a stellate appearance in transverse section. The epibiotic bacteria were unusually long bacilli of at least three different morphotypes. Bacteria adhered to the oxymonad host by distinct cell-cell junctions that protruded between the poles of adjacent epibiotic bacteria. Treating termites with the antibiotic carbenicillin led to the loss of most (but not all) of the bacteria and the transformation of S. strix from a long slender cell to a teardrop-shaped cell, where the axostyle was compacted and became bifurcated near the posterior end.     

Anisomorphic cell division by African trypanosomes

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic – showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.     

Trypanosoma brucei brucei: In Vitro Production of Metacyclic Forms

ABSTRACT An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei . Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27° C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27° C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Trypanosoma brucei brucei: in vitro production of metacyclic forms.

An in vitro method has been established to obtain metacyclic form populations of Trypanosoma brucei brucei. Trypanosome populations containing more than 98% of metacyclic forms were obtained from cultures which were: 1) initiated with bloodstream forms in primary cultures in the presence of Microtus montanus embryonic fibroblast-like cells (feeder cell layers); 2) maintained in glucose-free Eagle's minimum essential medium supplemented with 10 mM L-proline, 2 mM L-glutamine and 20% (v/v) fetal bovine serum at 27 degrees C without medium change for five days; 3) subcultured in the absence of the feeder cell layers but in the presence of Cytodex 3 beads; 4) maintained for an additional nine days with medium changes on days 5, 8 and 11; and 5) harvested on day 14 by means of diethylaminoethyl cellulose column chromatography prior to the appearance of other infective forms. Most of the trypanosomes obtained under these conditions were morphologically similar to metacyclic forms derived from tsetse fly vectors, coated with variable surface glycoprotein and were infective for mice. In the primary cultures procyclic forms, epimastigotes and metacyclic forms appeared by day 8. When the duration of the subculture was prolonged to 17 days or more at 27 degrees C, the metacyclic forms decreased in number while short trypomastigotes, long slender epimastigotes, and long slender trypomastigotes increased in number. These forms in such long-term cultures also appeared in diethylaminoethyl cellulose-isolated populations along with metacyclic forms.     

Trypanosome alternative oxidase as a target of chemotherapy

Parasites have developed a variety of physiological functions necessary for their survival within the specialized environment of the host. Using metabolic systems that are very different from those of the host, they can adapt to low oxygen tension present within the host animals. Most parasites do not use the oxygen available within the host to generate ATP, but rather employ systems anaerobic metabolic pathways. The enzymes in these parasite-specific pathways are potential targets for chemotherapy.Cyanide-insensitive trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms of the African trypanosome, which causes sleeping sickness in human and nagana in cattle. TAO has been targeted for the development of anti-trypanosomal drugs because it does not exist in the host. Recently, we found the most potent inhibitor of TAO to date, ascofuranone, a compound isolated from the phytopathogenic fungus, Ascochyta visiae.     

Gametocyte Maturation,Exflagellation and Fertilization in Parahaemoproteus (=Haemoproteus) velans (Coatney & Roudabush) (Haemosporidia: Haemoproteidae): an Ultrastructural Study*

SYNOPSIS. The structural changes in macro and microgametocytes of Parahaemoproteus velans following removal of infected blood from the avian host were studied in the light and electron microscope. Gametocytes of both sexes round up and soon escape from their host cells. Shortly thereafter they assume a dumbbell shape. The microgametocyte undergoes exflagellation forming 8 slender microgametes. During fertilization the entire microgamete appears to enter the female. The most striking ultrastructural change in the formation of the macrogamete is the condensation and enclosure by a membrane of abundant amophorus dense material seen in the cytoplasm of the immature gametocyte. Maturation of the microgametocyte begins prior to its escape from the host cell. Axonemes are present in the cytoplasm and nuclear reorganization occurs while the parasite is intracellular. Bundles of microtubules associated with condensed chromatin are found in the peripheral cytoplasm of maturing forms and apparently participate in the formation of small compact microgamete nuclei. Each of these filiform structures consists of a dense, centrally located nucleus and a single axoneme lying in flocculent cytoplasm. The nucleus and axoneme of the microgamete are seen free in the cytoplasm of a fertilized macrogamete.     

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL-14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH(4)Cl, which releases Ca(2+) from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion.