W.E. Ormerod

The occult visceral phase of mammalian trypanosomes with special reference to the life cycle of Trypanosoma (Trypanozoon) Brucei

Abstract 1. 1. We have reviewed the indirect evidence for suspecting the existence of an occult visceral phase in Trypanosoma (Trypanozoon) brucei . This evidence consists of:— 1.1. a) Inoculated trypomastigotes of Botswana strains (unlike “laboratory strains” adapted to rats) do not begin to divide and multiply immediately they reach the blood; neither do their numbers affect the height of infection nor the length of prepatent period. They are removed from circulation and are replaced by a new population with different morphology and behaviour. 1.2. b) This new population consists of agranular trypomastigotes which are flushed into the blood at 72 hours; they do not divide until they have been in the blood for some hours. 1.3. c) Trypomastigotes of the first flush (72 hours) and of the relapse (192 hours) we call “long-narrow” forms, they are succeeded by “long-flat” forms which are also agranular under phase contrast, but undergo binary fission. 1.4. d) The long-flat forms develop granules and are removed from the blood. 1.5. e) Multinucleate forms are seen occasionally in the peripheral circulation but their numbers are increased by immuno-suppression, they are also found in smears of internal organs. 1.6. f) Amastigotes and sphaeromastigotes (the “latent bodies” of earlier workers) have been seen in smears of internal organs; most of them show signs of degeneration. 1.7. g) The evidence of headache and associated hydrocephalus in sleeping sickness, also the swelling of the choroid plexus in experimentally infected rats, is reviewed. 2. 2. We have also reviewed the direct evidence of occult stages in different species of the genus Trypanosoma . In all subgenera of mammalian trypanosomes, with the exception of Duttonella and Nannomonas , amastigote and/or sphaeromastigote forms have been found. In Schizotrypanum these forms are intracellular, in Herpetomonas and Megatrypanum they are intravascular. In Trypanosoma (Trypanozoon) brucei the amastigote forms are found in large masses in the choroid plexus. 3. 3. We have proposed, as a working hypothesis, the life cycle shown in Fig. 3 for T. brucei in the vertebrate host. On this hypothesis we base our explanation of the synchronism of the initial infection and of the remission, also our view of the fundamental difference between Rhodesian and Gambian strains. 4. 4. In conclusion, we have discussed the following points:— 1. a) We think that the choroid plexus is the most important but not necessarily the only site for the formation of an occult phase in T. brucei infection; the lung is also a candidate. 2. b) Penetration of the cerebrospinal fluid by the trypanosome is probably not related to the occult phase. 3. c) The secretion of antigen by the granular forms and the massive response of antibody that it elicits might inhibit cell mediated immunity against the amastigote phase. 4. d) Existing chemotherapeutic agents are probably active against the occult visceral phase of T. brucei , but this question needs to be investigated specifically.

The Epidemic Spread of Rhodesian Sleeping Sickness 1908–1960

1. 1) The first recognized cases of T. rhodesiense sleeping sickness occurred in Northern Rhodesia, Nyasaland, and Southern Rhodesia between 1908 and 1912. At this time sporadic cases only occurred in the Rhodesias, but there was an epidemic in Nyasaland. 2. 2) The epidemic spread northwards reaching Central Tanganyika in the 1920s, and Uganda and Kenya in the 1940s. With each extension, the disease has become more acute and the strains more rapidly lethal to experimental animals. 3. 3) In Southern Rhodesia the disease remains sporadic and relatively chronic, with the occurrence of “healthy carriers.” 4. 4) In Northern Rhodesia epidemics have occurred, but the disease remains relatively chronic as compared with the epidemic areas of East Africa. 5. 5) In Ngamiland and the Chobe districts of Bechuanaland sporadic cases have occurred since 1934 (possibly since 1909), but since 1957 minor epidemic conditions have appeared. 6. 6) In areas which have been affected by epidemic Rhodesian sleeping sickness, after the disease has been controlled, sporadic cases continue to occur, and these cases in time gradually become less acute. 7. 7) T. brucei was present in Tanganyika before 1914. Epidemic T. rhodesiense was absent, but invaded the Territory during the first World War: it is not surprising, therefore, that there is a wide distinction between these two “species” in terms of infectivity to man. 8. 8) T. brucei in domestic stock and T. rhodesiense in man both occur sporadically in Southern Rhodesia. Since the time of European settlement there has been no recorded epidemic of sleeping sickness: it is unlikely, therefore, that such a marked distinction in human infectivity exists as in Tanganyika; i.e., the theories of Kinghorn and Yorke (1912a) and of Bruce (1915) on the identity of T. brucei and T. rhodesiense still await experimental proof. 9. 9) Several cases of sporadic trypanosomiasis caused by T. rhodesiense (as here defined) have occurred in man in areas outside the normal “rhodesiense area.” 10. 10) In the better authenticated instances the strains from these sporadic cases from outside the “rhodesiense area” might have been derived either from T. brucei or T. gambiense, since both exist in the areas concerned. The original strain, from which the epidemic form of T. rhodesiense has been derived, arose in the Zambezi basin, this is so far removed from any known focus of T. gambiense as to make it unlikely that the strain was derived from anything other than T. brucei.

The Study of Volutin Granules in Trypanosomes.

Abstract 1. 1) Refractile inclusion bodies in trypanosomes visible by phase contrast microscopy, are described. These are the “Volutin Granules” which occur naturally, and “Chemotherapy Granules” which are produced by drugs. 2. 2) The relationship between these granules is considered. 3. 3) Their significance in relation to, (i) drug resistance; (ii) the course of infection; (iii) geographical distribution of strains of Trypanosoma rhodesiense causing chronic and acute types of disease; (iv) antibody formation, is discussed.

An amastigote phase of the sleeping sickness trypanosome

Abstract 1. 1. We have found amastigotes closely packed together with “white thrombus” in capillaries, in sections of the choroid plexus of rats which had been infected with a strain of Trypanosoma brucei isolated from man in Botswana. 2. 2. Amastigotes were found at 48 hours during the prepatent period, also when the infection was fully established after the relapse. 3. 3. Amastigotes and sphaeromastigotes, also trypomastigotes in different stages of “unrolling” were found in smears of the choroid plexus. 4. 4. Similar forms in smears of other organs tended to show signs of degeneration. 5. 5. We believe that the choroid plexus is an important, probably the most important site of formation of an occult visceral phase of the sleeping sickness trypanosome.

Intracellular enzymes and their localization in slender and stumpy forms of Trypanosoma brucei rhodesiense

Abstract Acid phosphatase and cathepsin D activity is greater in stumpy than in slender forms, especially when estimations are made on a ‘light lysosomal’ fraction. Acid phosphatase is localized in the region of the flagellar pocket in slender forms but is present in lysosomes, phagolysosomes, autophagosomes and rough endoplasmic reticulum of stumpy forms. In stumpy forms dense bodies develop the macrocrystalline structure of peroxisomes. Intense enzyme activity in stumpy forms appears to be associated with absorption of lipid from the plasma by a mechanism similar to but of greater intensity than that described in atheromatous rabbit aorta.

The ventricular ependyma of mice infected with Trypanosoma brucei

The fine structure of ependymal cells lining the cerebral ventricles of normal mice and of mice infected with Trypanosoma brucei was examined by transmission electron microscopy. Most of the ependymal cells had been stripped from the ventricular surface of the brain in infected animals but some of the remaining ependymal cells contained intracellular trypomastigotes. The same process of stripping had occurred in a single human brain that was included in the series, but intracellular forms were not found. The significance of the intracellular forms and the implication of the stripping of ventricular ependymal cells are discussed in relation to the pathogenesis of sleeping sickness.

Interaction between Trypanosoma brucei and the ependymal cell of the choroid plexus

The fine structure of the normal choroid plexus of rats and mice and of those infected with Trypanosoma brucei was examined by transmission and scanning electron microscopy: extracellular trypomastigotes in the perivascular stroma predominate but the evidence presented suggests that they are derived both from stages in the blood and from others undergoing division within ependymal cells, a process which results in destruction of a large proportion of ependymal cells in the parts of choroid plexus affected. The choroid plexus maintains its integrity by regeneration of an outer layer of ependymal cells.

Granules and tubules in the cytoplasm of the sleeping sickness trypanosome: An electron microscope study.

Abstract A method is described for selecting a single trypanosome so that it can be studied under phase contrast and then by electron microscopy in serial section. Illustrations are given of 18 serial sections through a short-stumpy trypanosome. Ribosomal particles which lie free in the cytoplasm of the long-thin form aggregate into polyribosomes; analogy with other cell types indicates active synthesis of intracellular protein. Larger ribosomal particles are attached to the outer surface of the endoplasmic reticulum. Similar analogy indicates the synthesis of protein for secretion. In the short-stumpy form the endoplasmic reticulum is distended with amorphous material. Similar material is present in “vacuoles” in the cytoplasm which we have shown to correspond to the Type II (phase positive) granules, the appearance of which precedes a crisis in the infection. Type I (volutin) granules which stain with Giemsa have been demonstrated, and have the appearance of lysosomes. The short-stumpy form carries a variable structure between the nucleus and the basal vacuole (an area associated with pinocytotic activity) which may consist of vesicles or distended tubules. We discuss these differences in the ultrastructure of T. brucei in relation to its cycle of development in the blood and to the excretion of exo-antigen.