Chapter VIII: Appendix: “Rhizopods in Poliomyelitis acuta.” (2)

About the same time attention was devoted to the disease of West
African negroes known for a century as “sleeping sickness.”
Castellani (1903) was the first to succeed in demonstrating the
presence of trypanosomes (at first called _T. ugandense_) in
centrifugalized cerebro-spinal fluid obtained by puncture from cases
of sleeping sickness in Uganda. Similar discoveries were made by
Bruce, who also found trypanosomes in the blood of those attacked
with sleeping sickness. Sambon regarded a species of _Glossina_ as
the transmitter. From consideration of the geographical distribution
of the disease Christy regarded _Glossina palpalis_ as the
transmitter. Brumpt first thought it was _G. morsitans_, but, later,
supported the view of _G. palpalis_. Bruce, Nabarro and Greig also
named the same insect as the transmitter, not only for geographical
reasons but also because healthy apes became infected by the bite
of certain _G. palpalis_. The inoculation of cerebro-spinal fluid
from subjects of sleeping sickness into the spinal canal of apes
(_Macacus_) had the same result.

Just as the discovery of the malarial parasites called forth a whole
flood of research memoirs which were followed by a second series on
the relation of the mosquitoes to malaria, so a similar outpouring
occurred after the discovery of the pathogenic trypanosomes of
mammals and men. In both cases the inquiry was not limited to the
stages in man and other vertebrate hosts, but the fate of the
parasites in the intermediate (invertebrate) hosts was investigated,
and allied species were obtained from many different hosts.

Novy and MacNeal (1903) were the first to cultivate trypanosomes in
artificial media (blood-agar).

In 1910 Stephens and Fantham recorded the presence of another human
trypanosome, _T. rhodesiense_, from a case of sleeping sickness
in Rhodesia, where _G. palpalis_ was absent. Kinghorn has since
demonstrated that _T. rhodesiense_ is transmitted by _G. morsitans_.
Kinghorn and Yorke believe that big game (_e.g._, antelope) is the
reservoir of _T. rhodesiense_.

The output of literature on trypanosomiasis in men and animals is
enormous. To cope with it the Sleeping _Sickness Bureau Bulletin_
was founded in 1908, and it is now (since November 1912) continued
as a section of the _Tropical Diseases Bulletin_, wherein current
literature is reviewed.

GENERAL.

Trypanosomes occur in the blood of representatives of all the vertebrate classes. Often the trypanosomes occur so scantily in the blood that they are overlooked on examination. A useful aid in detecting the flagellates in such cases consists in the use of cultures of the blood of the host on artificial media. Stimulated by the medium multiplication occurs, and hence the parasites are more easily detected. [For the composition of such culture media see Appendix.]

There is a periodicity in the appearance of the trypanosomes in the peripheral blood of the host, due to alternating phases of multiplication and of rest on the part of the parasites. Such periodicity has been established both by biological and enumerative methods. Again, a seasonal variation has been observed in the occurrence of certain trypanosomes in the peripheral circulation of the hosts; for example, some trypanosomes (_e.g._, _T. noctuæ_ in birds) are found only in the summer in the blood, while in the winter they occur in the internal organs.

Recent cultural researches have established that trypanosomes, _e.g._, _T. americanum_, may be present in very small numbers in hosts, such as cattle, which are quite unharmed by them, and in which the presence of these flagellates formerly was never suspected (“cryptic trypanosomiasis.”) However, the majority of the trypanosomes occurring in domestic animals are usually deleterious or even lethal to their hosts. Many wild animals, such as various species of antelope, harbour trypanosomes without being injured thereby. In such cases it is probable that the vertebrate hosts have been so long parasitized in the past, that they have become tolerant and immune to the effects of the flagellates. Should such trypanosomes of wild animals be transmitted to domesticated stock or man, they may re-acquire their initial virulence and become pathogenic to the new host. As a general statement, the newer a parasite is to its host the greater is its virulence. For example, _T. gambiense_, _T. rhodesiense_ and _T. brucei_ are innocuous to big game in Africa, but are pathogenic to man and domestic animals respectively. Pathogenic trypanosomes appear to have a wider range of hosts, that is, to be less limited to one specific host than non-pathogenic forms. Thus, _T. rhodesiense_ is pathogenic to man and all laboratory animals, while it is non-pathogenic to antelopes and their kind.

_Morphology._

The general structure of the various trypanosomes shows much uniformity, though variations in size and shape occur. Typically the body is elongate and sinuous. The flagellar end tapers gradually to a point, the aflagellar extremity usually being rounded or more blunt. In some trypanosomes there is much diversity in size, the organisms varying from long, slender forms to short, stumpy ones; in other species relative constancy of size is maintained. The former are known as polymorphic trypanosomes, the latter as monomorphic forms.

Two nuclei are present. The main or principal nucleus, sometimes termed the trophic nucleus, is often situated towards the centre of the body; it is frequently of the vesicular type, containing a karyosome. The blepharoplast or kinetic nucleus is posterior to the nucleus, and usually is rod-like. The flagellum arises close to the blepharoplast, and forms an edge to the undulating membrane. It may or may not extend beyond the limits of the undulating membrane. If it does so, the unattached part is known as the free flagellum. Sometimes a small granule is found at the origin of the flagellum. This is the basal granule, and is considered by some to function as the centriole of the kinetic nucleus.

The undulating membrane is a lateral extension of the ectoplasm or periplast, and is the main agent in locomotion. It is edged by the flagellum, which forms a deeply stainable border to it. Within the membrane substance, often arranged parallel with its edge, are a number of fine contractile elements, the myonemes. These contractile elements may also occur on the body of the trypanosome. They are easily seen in some large trypanosomes, but are difficult of demonstration in others, owing to their great fineness.

Multiplication of trypanosomes in the blood is brought about by binary longitudinal fission (fig. 26). Division is initiated by that of the blepharoplast and nucleus. The division may be equal or subequal, whereby differences in size of individuals partly arise. Multiple division by repeated binary fission, without complete separation of the daughter forms, is known in some trypanosomes (_e.g._, _T. lewisi_), and rosettes of parasites thereby are produced (fig. 27).

The classification of trypanosomes is very difficult. Laveran (1911)[54] has suggested the examination of the relative length of the flagellum as a diagnostic character, and so arranged these flagellates in mammals in three groups. The first group included those trypanosomes always having part of the flagellum free (_e.g._, _T. evansi_, _T. vivax_); the second group comprised forms without a part of the flagellum free (_e.g._, _T. congolense_), while the third group included forms some members of which have free flagella, while others have not (_e.g._, _T. gambiense_). Bruce[55] (1914) and Yorke and Blacklock[56] (1914) have also devised classifications.

[54] _Ann. Inst. Pasteur_, xxv, p. 497.

[55] _Trans. Soc. Trop. Med. & Hyg._, viii, p. 1.

[56] _Annals Trop. Med. and Parasitol._, viii, p. 1.

Resting stages of some trypanosomes have been found in the internal organs of their vertebrate hosts. The formation of these oval, Leishmania-like bodies will be noted in individual cases later. Similar small oval bodies form an important phase in the life-history of _T. cruzi_, which multiplies normally by multiple fission or schizogony into these oval, daughter elements, and not by binary longitudinal fission in the circulating blood.

Polymorphism in trypanosomes (_e.g._, _T. gambiense_, _T. rhodesiense_) is now interpreted as a phenomenon resulting from growth and division.[57] Long, thin forms are those about to divide. Fully mature forms are shorter and broader. Various intermediate types occur and represent growth forms. Formerly, polymorphism was interpreted in terms of sex, thin forms being regarded as males, broad forms as females, while the intermediate types were termed indifferent. Conjugation was not observed, and there is no evidence in support of the sexual interpretation.

[57] Robertson (1912), _Proc. Roy. Soc._, B, lxxxv, p. 527.

The transmission of trypanosomes from one vertebrate host to another is usually accomplished by the intermediation of some biting arthropod in the case of terrestrial animals, while leeches are usually considered to act as transmitters in the case of the trypanosomes occurring in aquatic animals. Developmental phases of the life-histories of trypanosomes occur in the invertebrate transmitters, and will be considered in individual cases.

*Trypanosoma gambiense*, Dutton, 1902.

Syn.: _Trypanosoma hominis_, Manson, 1903. _Trypanosoma nepveui_,
Sambon, 1903. _Trypanosoma castellanii_, Kruse, 1903. _Trypanosoma
ugandense_, Castellani, 1903. _Trypanosoma fordii_, Maxwell Adams.

In vertebrate blood _Trypanosoma gambiense_ is polymorphic, for long, thin forms may be seen in contrast with short, stumpy forms, as well as intermediate forms (fig. 29, _a_--_c_). This polymorphism has been interpreted in terms of sex, especially by German investigators, following Schaudinn (see above). However, there is no evidence of conjugation, and the polymorphic forms are more easily interpreted in terms of growth and division, for the long thin forms are potential dividing organisms, and the stumpy or short parasites, with little or no free flagellum, are the adult individuals.

_Morphology of T. gambiense in the Circulating Blood._

_T. gambiense_ varies from 13 µ to 36 µ in length, its average length being 24·8 µ, as was determined in 1913 by exact biometrical methods by Stephens and Fantham.[58] Three forms of parasite occur. According to Miss Robertson,[59] the relatively short forms from 13 µ to 21 µ long may be regarded as the mature or “adult” type of parasite in the blood. They carry on the cycle in the vertebrate. From them intermediate forms, which are longer than the “adult” but at first have the same breadth, arise by growth. They possess a free flagellum. The intermediate forms grow into long individuals, which are those about to divide. The products of division give rise, directly or indirectly, to the adult forms.

[58] _Annals Trop. Med. and Parasitol._, vii, p. 27.

[59] _Phil. Trans._, B (1913), cciii, pp. 161–184.

The organism has an elongate body with an anterior or flagellar end and a blunter posterior or non-flagellar end. The protoplasm is finely granular, large inclusions being rare. The central nucleus is oval and large, often containing most of its chromatin concentrated as a karyosome, with small granules only scattered near or on the fine nuclear membrane. The blepharoplast is either rounded or rod-shaped. The undulating membrane is thrown into folds and is bordered by the flagellum. A small basal granule may be present near, or at the actual origin of the flagellum.

_Multiplication_ in the vertebrate is brought about by longitudinal division. According to the recent account of division by Miss Robertson, the blepharoplast doubles, then the flagellum splits for the greater part of its length, and the daughter flagella separate, one being shorter than the parent flagellum. The nucleus often shows two well marked dark granules on the membrane at opposite poles, and these appear to act as centrosomes. Nuclear constriction occurs and the halves gradually separate. Finally the two daughter organisms become free, the aflagellar end splitting last. The products of division may be equal or unequal. Repeated division goes on in the general circulation until the blood swarms with parasites. Then the trypanosomes gradually disappear, and a period occurs when it is practically impossible to demonstrate the parasite in the blood. At such a period, trypanosomes can be obtained by puncture of the enlarged lymphatic glands or of the spinal canal, or can be found in the internal organs, more particularly in the spleen, lungs, liver and bone-marrow. In the latter organs, latent bodies are produced (fig. 29, _d_--_f_) which are capable of again becoming flagellates and entering the general circulation. Their formation was described by Fantham (1911).[60] The parasite contracts, the blepharoplast migrates towards the nucleus, a very thin coat differentiates around the two nuclei and a certain amount of cytoplasm, and the parts exterior to the coat disintegrate, leaving a small, oval body behind. Fuller details are given in connection with _T. rhodesiense_. Laveran (1911)[61] considers that latent bodies are “involution” forms, but acknowledges that they can flagellate and become infective in fresh blood.

[60] _Proc. Roy. Soc._, B, lxxxiii, p. 212.

[61] _C. R. Acad. Sci._, 153, p. 649.

No multiplication of the trypanosomes within the cells of the lung, liver or spleen of infected monkeys was found by Miss Robertson in her recent researches.

There appear to be negative periods in infected monkeys, since, although trypanosomes may occur in their blood at such times, they are not infective to _Glossina_.

_Development in Glossina palpalis._--The principal accounts are those by Sir D. Bruce and his colleagues (1911),[62] and by Miss Robertson[63] (1912), whose results will be followed. According to the latter investigator _T. gambiense_ never enters the body cells of the fly (_G. palpalis_), nor does it penetrate the gut wall into the body cavity. Practically no crithidial stage occurs in the fly’s main gut, but a trypanosome facies is retained therein.

[62] _Proc. Roy. Soc._, B, lxxxiii, p. 513.

[63] _Proc. Roy. Soc._, B, lxxxvi, p. 66.

After the trypanosomes are ingested by the fly during a meal of infected blood, sooner or later multiplication occurs. This development usually begins in the middle or posterior part of the mid gut, and trypanosomes of varying sizes are produced. After the tenth or twelfth day, many long, slender trypanosomes (fig. 30, _a_) are found, which gradually move forwards into the proventriculus. Such long, slender forms represent the limit of development in the lumen of the main gut. The proventricular type, developed about the eighth to the eighteenth or twentieth day, is not infective; it may occur in the crop, but is not to be found permanently there. Between the tenth and the fifteenth days multinucleate forms of trypanosomes are found, and may be styled multiple forms (fig. 30, _b_). Some of these latter may be degenerative.

_Invasion of the Salivary Glands of the Fly._--Long, slender trypanosomes from the proventriculus pass forward into the hypopharynx. They then pass back along the salivary ducts, about sixteen to thirty days after the fly’s feed. The trypanosomes reach the salivary glands as long, slender forms. In the glands they become shorter and broader, attach themselves to the surrounding structures, and assume the crithidial facies (fig. 30, _c_, _d_). As crithidial forms they remain attached to the wall and multiply in the glands. These crithidial stages differentiate into the short, broad trypanosome forms, capable of swimming freely (fig. 30, _e_).

Miss Robertson considers the development in the main gut to be indifferent multiplication, and that salivary fluid seems necessary to stimulate trypanosomes to the apparently essential reversion to the crithidial type. The second development in the salivary gland is the essential feature. The short, stumpy forms of trypanosomes (fig. 30, _e_) finally produced in the salivary glands are alone infective. No conjugation of trypanosomes occurs in the fly. Only about 5 per cent. of captive tsetse flies fed on trypanosome-infected blood become infective, but they probably remain infective for the rest of their lives.

J. G. Thomson and Sinton (1912)[64] have obtained in cultures the various trypanosome forms of _T. gambiense_ seen in the fly’s main gut.

[64] _Annals Trop. Med. and Parasitol._, vi, p. 331.

Duke (1912)[65] found _T. gambiense_ in a species of antelope, the situtunga (_Tragelaphus spekei_), on Damba Island in Victoria Nyanza. Wild _G. palpalis_ could be infected therefrom. The antelope may then act as a sleeping sickness reservoir in that district, but men are apparently the chief reservoir.

[65] _Proc. Roy. Soc._, B, lxxxv, pp. 156, 483.

*Trypanosoma nigeriense*, Macfie, 1913.[66]

Macfie has recently (August, 1913) described a human trypanosome from the Eket district of Southern Nigeria. It is common in young people. The disease produced does not seem to be of a virulent type in Nigeria, and does not occur in epidemic form. In the early stages the glands of the neck are enlarged. In the later stages--cases of which are rarer--lethargy appears. The parasite is a polymorphic trypanosome, morphologically almost indistinguishable from _T. gambiense_, though it may be slightly shorter. Macfie recorded the occurrence in his preparations of a few trypanosomes appearing to have a flagellum free during their whole length. Some of the parasites, as seen in a sub-inoculated guinea-pig, are very small (8 µ long). Other trypanosomes have their nuclei displaced somewhat anteriorly. This parasite may only be a variety of _T. gambiense_. The parasite is perhaps spread by _Glossina tachinoides_.

[66] _Annals Trop. Med. and Parasitol._, vii, p. 339; viii, p. 379.

*Trypanosoma rhodesiense*, Stephens and Fantham, 1910.

The parasite was found in the blood of a young Englishman who had contracted sleeping sickness in the Luangwa Valley, North-eastern Rhodesia, in the autumn of 1909. The patient had never been in an area infested with _Glossina palpalis_.

(1) _Morphology._--The morphology of the parasite in man and sub-inoculated rats was studied by Stephens and Fantham in 1910.[67] They pointed out a morphological peculiarity in the presence of certain trypanosomes with posterior nuclei in sub-inoculated animals, that is, parasites in which the nucleus (trophonucleus) was situated towards the posterior or aflagellar end, close up to or even beyond the blepharoplast or kinetic nucleus (fig. 31, _4_, _5_). When the nucleus was beside the blepharoplast, the former was seen to be kidney-shaped (fig. 31, _4_). The posterior nuclear forms were of the stout and stumpy variety, and about 6 per cent. of the stumpy forms were found to have their nuclei displaced from the centre. The anterior or flagellar end of these trypanosomes often contained chromatoid granules. _T. rhodesiense_ varies in length from 12 µ to 39 µ[68]; short stumpy forms vary from 13 µ to 21 µ, intermediate forms from 21 µ to 24 µ, and long, slender forms from 25 µ onwards. The average length is 24·1 µ.

[67] _Proc. Roy. Soc._, B, lxxxiii, p. 28.

[68] Stephens and Fantham (1912–13), _Proc. Roy. Soc._, B, lxxxv, p. 223, and _Annals Trop. Med. and Parasitol._, vii, p. 27.

Certain regular periods occur in the course of the trypanosomiasis when few or no flagellate trypanosomes are found in the peripheral blood of the patient or of the sub-inoculated animal. These periods can be explained in terms of morphology, for the trypanosomes are capable of assuming a non-flagellate form in the internal organs of the host, particularly in the lungs and in the spleen. Such forms are known as “latent” or “resting” forms. The term “latent body” was first used by Moore and Breinl in 1907[69] in connection with _T. gambiense_. Fantham[70] (1911) has described the process of formation of latent from motile forms and the reconversion of the latent bodies into active flagellates. Fresh preparations of splenic blood or lung blood containing trypanosomes were made. A trypanosome gradually withdrew or cast off its flagellum, concentrated its cytoplasm, and became more or less elongate oval. Nucleus and blepharoplast approached one another and came to lie more or less side by side. Then an opaque line often made its appearance around the nuclear area and differentiated as a slight envelope or covering, the cytoplasm external to this merely degenerating. The small, oval, refractile body (fig. 29, _d_--_f_) thus formed was a non-flagellate latent body, 2 µ to 4 µ in diameter, like _Leishmania_ or the non-flagellate, multiplicative forms of _T. cruzi_ (fig. 34), and remains temporarily inactive in the internal organs of the host. After this period of inactivity, the non-flagellate body, recuperated by its rest, begins to elongate again. The nuclei separate. From a small vacuole-like portion the flagellum differentiates and forces out the ectoplasm, which assumes the form of the undulating membrane with its flagellar border. Subsequent growth results in the production of the typical trypanosome form, which re-enters the circulating blood and multiplies by longitudinal binary fission. Division of the parasite prior to the formation of a latent body may occur and division of the latent forms themselves is known, though less common. Consequently latent bodies, like the flagellate forms themselves, show diversity in size. The blepharoplast of the latent bodies is sometimes less well marked than in _Leishmania_ (see fig. 29, _d_-_f_). Laveran’s views on these bodies have already been given on p. 74.

[69] _Annals Trop. Med. and Parasitol._, i, p. 441.

[70] _Proc. Roy. Soc._, B, lxxxiii, p. 212.

(2) _Animal Reactions._--The posterior nuclear trypanosomes were found in all sub-inoculated animals, such as rats, guinea-pigs, dogs, mice, Macacus, rabbits and horses, but were not seen in the human patient, as few trypanosomes occurred in his peripheral blood. R. Ross and D. Thomson[71] found a periodic, cyclical variation in the number of the parasites in the patient’s blood from day to day, the cyclical period being about a week (fig. 32). Fantham and J. G. Thomson[72] (1911) found a similar periodic, cyclical variation in the trypanosomes in the blood of sub-inoculated rats, guinea-pigs and rabbits. On counting the parasites in the blood of similar animals inoculated with _T. gambiense_, they established, by enumerative methods, that _T. rhodesiense_ was more virulent than _T. gambiense_, while Yorke also showed this marked virulence of _T. rhodesiense_ in practically all laboratory animals. In other words the duration of infection in the case of _T. rhodesiense_ was shorter. It was also found that _T. rhodesiense_ was resistant to atoxyl. The patient, from whom the original strain was obtained, died about nine months after the probable date of infection. Some patients infected with _T. rhodesiense_ have died in an even shorter period, such as four or five months.

[71] _Proc. Roy. Soc._, B, lxxxii, p. 411.

[72] _Annals Trop. Med. and Parasitol._, iv, p. 417.

In sheep and goats _T. rhodesiense_ causes an acute disease, marked by high fever, œdema of the face, and keratitis, as shown by Bevan and others, death resulting after a relatively short period. _T. gambiense_ gives rise, in these animals, to no symptoms except fever, which may be overlooked. _T. rhodesiense_ produces keratitis in dogs.

Stannus and Yorke (1911) observed _T. rhodesiense_ in animals inoculated from a case of sleeping sickness in Nyasaland. Sir D. Bruce and his colleagues[73] have shown (1912) that _T. rhodesiense_ is the parasite usually found in man and in animals sub-inoculated from cases of sleeping sickness in Nyasaland. It has since been found in German East Africa and Portuguese East Africa, while Ellacombe has described a case from North-western Rhodesia.

[73] _Proc. Roy. Soc._, B, lxxxv, p. 423.

(3) _Serum Reactions._--Interesting experiments on this subject were performed during 1911 and 1912 by various French investigators.

(_a_) _Action of Immune Serum_ (Mesnil and Ringenbach)[74]: (1) A goat was infected with _T. rhodesiense_. Twenty-two days later its serum mixed with _T. rhodesiense_ was injected into a mouse. Result: Protection. (2) The serum mixed with _T. gambiense_ was injected into a mouse. Result: Infection.

[74] _C.R. Soc. Biol._, lxxii, p. 58.

(_b_) _Action of Baboon Serum._--Contrary to _T. gambiense_, _T. rhodesiense_ is very susceptible to human and baboon sera. Mesnil and Ringenbach[75] showed that a dose of 1 c.c. of baboon (_Papio anubis_) serum cured mice infected with _T. rhodesiense_. In the same dose it acted very feebly on _T. gambiense_.

[75] _C.R. Acad. Sci._, 153, p. 1,097.

(_c_) _Action of Human Serum._--_1 c.c._ of human serum cured _T. rhodesiense_ mice in three out of four cases; on _T. gambiense_ mice there was no appreciable effect.

Laveran and Nattan-Larrier[76] have shown the same, namely, that human sera act on _T. rhodesiense_, but are quite without action on _T. gambiense_.

[76] _C.R. Acad. Sci._, 154, p. 18.

(_d_) _Trypanolytic Reactions._--Mesnil and Ringenbach[77] have also shown that the sera of animals (man, monkey and guinea-pig) infected with _T. gambiense_ are trypanolytic for the homologous trypanosome, that is, _T. gambiense_, but have no action on the heterologous trypanosome, that is, _T. rhodesiense_.

[77] _C.R. Soc. Biol._, lxxi, p. 609.

(4) _Cross Immunity Experiments._--(_a_) Mesnil and Ringenbach[78] immunized a monkey (_Macacus rhesus_) against _T. gambiense_. It was inoculated with _T. rhodesiense_ on June 7, 1911; on June 27 trypanosomes appeared, the infection being slight; on July 4 it died. A control died in ten and a half days.

[78] _C.R. Soc. Biol._, lxxi, p. 271.

(_b_) Laveran[79] immunized a goat and mice against _T. gambiense_. When they had acquired a solid immunity, they were inoculated with _T. rhodesiense_. They became infected like the controls.

[79] _Bull. Soc. Path. Exot._, v, pp. 26, 241.

(_c_) Laveran and Nattan-Larrier[80] immunized a ram against _T. brucei_, it subsequently became infected with _T. rhodesiense_.

[80] _C.R. Acad. Sci._, 154, p. 18.

(_d_) Laveran[81] immunized a ram and a sheep against different strains of T_. brucei_. Inoculated with _T. rhodesiense_ they both acquired acute infections and died. Conclusion: _T. rhodesiense_ is not _T. brucei_.

[81] _Bull. Soc. Path. Exot._, v, p. 101.

When the converse set of experiments is tried, namely, immunizing an animal against _T. rhodesiense_, and then inoculating with _T. gambiense_, the difficulty immediately arises that it is impossible to immunize an animal against _T. rhodesiense_, owing to its virulence. But a partial and transitory immunity to _T. rhodesiense_ can be obtained by treating the infected animal with drugs, such as arsenophenylglycin. The results, so far as they go, seem to show that an animal immunized against _T. rhodesiense_ is immune not only to _T. rhodesiense_, but also to _T. gambiense_, a fact which, according to Mesnil and Léger, does not invalidate the specificity of _T. rhodesiense_, but tends to show that the two trypanosomes are closely related.

(5) _Mode of Transmission and Reservoir._--Kinghorn has shown that _T. rhodesiense_ is transmitted by _Glossina morsitans_ in which it undergoes development. Kinghorn and Yorke[82] found that about 16 per cent. of the wild game examined in Northern Rhodesia was naturally infected with _T. rhodesiense_. The wild game examined included waterbuck, hartebeest, mpala, bushbuck and warthogs. One native dog near the Nyasaland border was found infected, but not domestic stock. Taute doubts whether _T. rhodesiense_ really occurs in wild game. Approximately 3·5 per cent. of the tsetse flies fed on infected animals may become permanently infected with _T. rhodesiense_, and capable of infecting clean animals. Furthermore, a tsetse fly when once infective probably remains infective for the rest of its life.

[82] _Annals Trop. Med. and Parasitol._, vii, p. 183.

Kinghorn and Yorke, however, have shown that climatic conditions, namely, those of temperature, also affect the infectivity of the tsetse fly, as the ratio of flies capable of transmitting _T. rhodesiense_ to those incapable of transmitting the virus is 1 : 534 in hot valley districts (_e.g._, Nawalia, Luangwa Valley, temperature 75° to 85° F.), while on elevated plateaux (_e.g._, Ngoa, on the Congo-Zambesi watershed, temperature 60° to 70° F.) the ratio falls to 1 : 1312.

Mechanical transmission by the tsetse fly does not occur, if a period of twenty-four hours has elapsed since the infecting meal.

_Developmental Cycle in the Fly._--The period which elapses between the infecting feed of the flies and the date on which they become infective varies from eleven to twenty-five days in the Luangwa Valley, according to Kinghorn and Yorke. Attempts carried out at laboratory temperature on the Congo-Zambesi plateau, during the cold season, to transmit _T. rhodesiense_ by means of _G. morsitans_ were always unsuccessful. The developmental cycle of the trypanosome in the fly is influenced by the temperature to which the flies are subjected (as stated above). The first portion of the developmental cycle proceeds at the lower temperatures (60° to 70° F.), but higher temperatures are necessary for the completion of the development of the trypanosome. Kinghorn and Yorke found that the trypanosomes may persist in the fly, at an incomplete stage of their development, for at least sixty days when the climatic conditions were unfavourable.

The first portion of the developmental cycle of the trypanosome takes place in the gut of the fly. Invasion of the salivary glands of the tsetse is secondary to that of the intestine, but is necessary for the infectivity of the fly. A relatively high mean temperature, 75° to 85° F., is essential for the passage of the trypanosomes into the salivary glands and the completion of their development therein.

Kinghorn and Yorke[83] state that the predominant type of trypanosome in the intestine of infected _G. morsitans_ was a large broad form, quite different from that which is most common in the salivary glands. The trypanosome in the glands resembles the short form seen in the blood of the vertebrate host. The authors quoted state that both the intestinal and salivary gland forms of infective _G. morsitans_ are virulent when inoculated into healthy animals.

[83] _Annals Trop. Med. and Parasitol._, vii, p. 281.

Bruce and colleagues[84] have quite recently (June, 1914) published an account of their investigations of _T. rhodesiense_ in _G. morsitans_ in Nyasaland. (Incidentally it may be remarked that Bruce considers _T. rhodesiense_ to be identical with a polymorphic strain of _T. brucei_--see pp. 83, 94). The development of _T. rhodesiense_ takes place in the alimentary canal and salivary glands, not in the proboscis, of the tsetse fly. In feeding experiments with laboratory bred flies, as well as with a few wild flies, fed on infected dogs or monkeys, only 8 per cent. of the flies were found to be infected on dissection. Of such infected flies, however, only some allow of the complete development of the trypanosomes within them, in other words only about 1 per cent of the flies become _infective_. The length of time which elapses before a fly becomes infective varies from fourteen to thirty-one days, averaging twenty-three days, when kept at 84° F. (29° C.). The dominant intestinal type of flagellate in the fly is that seen in the proventriculus, which contains many long, slender trypanosomes. These proventricular forms find their way to the salivary glands, wherein crithidial and encysted forms are seen. They change into “blood forms,” which are short, stumpy trypanosomes and are infective. “The infective type of trypanosome in the salivary glands--corresponding to the final stage of the cycle of development--is similar to the short and stumpy form found in the blood of the vertebrate host.” The cycle is thus very similar to that of _T. gambiense_ in _G. palpalis_ (fig. 30).

[84] _Proc. Roy. Soc._, B, lxxxvii, p. 516.

CULTURE.--J. G. Thomson (1912),[85] and subsequently Thomson and Sinton, succeeded in cultivating _T. rhodesiense_ in a modified Novy-MacNeal medium. The development obtained resembled that of the trypanosome in the intestine of _Glossina_.

[85] _Annals Trop. Med. and Parasitol._, vi, pp. 103, 331.

GENERAL NOTE ON TRYPANOSOMES WITH POSTERIOR NUCLEI.

Posteriorly placed nuclei have been found to occur not only in _T. rhodesiense_ by Stephens and Fantham (1910), but also in _T. pecaudi_ by Wenyon (1912), in _T. brucei_ by Blacklock (1912), and in _T. equiperdum_ by Yorke and Blacklock (1912).

Recently Stephens and Blacklock (1913)[86] have shown that two trypanosomes, different morphologically, have been confused under the name _T. brucei_. One of these is polymorphic (_i.e._, it exhibits long and slender as well as short and stumpy forms) and came from Uganda, while the other is monomorphic and is the original Zululand strain described by Bruce from cattle suffering from “nagana.” Bruce (1914) considers that morphological change has occurred in _T. brucei_ in its passage through laboratory animals, and thus explains the diversity of views. The posterior nuclear forms described by Blacklock occurred in the Uganda strain of _T. brucei_. (See p. 95.) Similarly, a posterior nuclear form, _T. equi_, has been separated from _T. equiperdum_. (See p. 98.)

[86] _Proc. Roy. Soc._, B, lxxxvi, p. 187.

Again, Bruce and his colleagues on the Royal Society Commission investigating sleeping sickness in Nyasaland, have stated (April, 1913) that “evidence is accumulating that _T. rhodesiense_ and _T. brucei_ (Plimmer and Bradford) are identical.” The exact identity of trypanosomes showing posterior nuclei is, then, far from settled, although Laveran by cross immunity tests has declared that _T. brucei_ is distinct from _T. rhodesiense_. No one has yet seen posterior nuclei in _T. gambiense_.

*Trypanosoma cruzi*, Chagas, 1909.

Syn.: _Schizotrypanum cruzi_, Chagas, 1909.

The trypanosome was discovered by Chagas[87] in the intestine of the bug, _Triatoma_ (_Conorhinus_) _megista_, in Brazil, and then in the blood of a small monkey bitten by the bug. A little later it was found in the blood of a child, aged two years, suffering from irregular fever, extreme anæmia and enlarged glands in the State of Minas Geraes, Brazil. Chagas found that he was able to infect many of the usual laboratory animals with the trypanosome, by allowing the bug to bite them. He was also able to culture the parasite on blood agar.

[87] _Mem. Inst. Oswaldo Cruz._, i, p. 159.

Chagas found the Reduviid bug, _Triatoma megista_, in the houses of the poorer inhabitants of the Brazilian mining State, and that it attacked the people, more especially the children, at night, biting the face. On this account the insect is called “barbeiro” by the inhabitants. The bite is somewhat painful. The disease has since been found in other parts of Brazil, _e.g._, Matta de São João in Bahia province, Goyaz, Matto Grosso and São Paulo provinces, as well as in Minas Geraes.

_Morphology._--The trypanosome has a large blepharoplast or kinetic nucleus. It is stated to occur both free and in the red blood corpuscles in the peripheral blood. It is about 20 µ long, on an average.

Two forms of the parasite (fig. 33, _6_, _7_) are described in the human blood. In one free form there is a large egg-shaped blepharoplast and the posterior (aflagellar) end of the parasite is drawn out. The blepharoplast (kinetic nucleus) may have a chromatin appendage. The nucleus is oval or band-like, containing a karyosome. The flagellum, starting close to the blepharoplast or its appendage, has a free portion of variable length. The other free form in the blood has a more or less round, terminal blepharoplast, smaller than in the first form, without a chromatin appendage as a rule. The body of this second form is decidedly broader than that of the first mentioned.

The dimorphism has been interpreted sexually, the first mentioned forms being termed males, the second ones females. The correctness of this interpretation is very doubtful.

No sign of longitudinal division was ever seen in the peripheral blood or in the internal organs. The “endocorpuscular” forms may be completely or partially enclosed in the red cell or only attached thereto (fig. 33, _1_-_5_). At the beginning of infection the endocorpuscular forms are the more numerous. Some authorities, however, doubt these stages.

_Life-history in the Vertebrate Host._--Chagas found fluctuations in the number of the parasites in the peripheral blood. He believes the increase of the parasites to be periodic.

The investigations of Chagas and of Hartmann have revealed two types of multiplication which take place in the internal organs of the vertebrate host.

(_a_) The first type--which possibly belongs to another organism, _Pneumocystis carinii_, see p. 90--occurs in the capillaries of the lungs. The flagellate parasite entering the lung capillaries loses its flagellum and undulating membrane. Its body becomes curved, and the two ends fuse, and so an oval mass is formed (fig. 33, _8_-_11_). In some cases the blepharoplast disappears, in other cases it blends or fuses with the nucleus. The nucleus of the rounded parasite then divides into eight by successive divisions (fig. 33, _12_-_15_). Next the body, which is surrounded by its own periplast, also divides, giving rise to eight tiny daughter individuals or merozoites (fig. 33, _15_). The merozoites lie inside the periplast, which acts as a sort of “cyst wall.” The merozoites are said to exhibit dimorphism, and Chagas has interpreted the dimorphism in terms of sex. The daughter forms, produced by the parent trypanosomes which kept their blepharoplasts, themselves have blepharoplasts as well as nuclei, and have been termed “males” or “microgametes.” The merozoites, arising from parent trypanosomes which lost their blepharoplasts, have themselves only nuclei, and have been called “females” or “macrogametes.” In the case of the so-called “female” forms the single nucleus divides into two unequal parts, of which the smaller becomes the blepharoplast, and a flagellum is formed later. The so-called “males” possess early a rudiment of a flagellum. Both kinds of merozoites escape from the parent periplast wall, and enter red blood corpuscles. They grow into flagellates within the corpuscles, and then become free as adult trypanosomes in the blood-stream.

(_b_) The second mode of multiplication is one of asexual reproduction (schizogony or agamogony). It was first described by Hartmann from hypertrophied endothelial cells of the lungs. It has since been found in the cardiac muscle, in the neuroglia of the central nervous system, and in striped muscle (fig. 34). In laboratory animals it has also been found in the testicle and suprarenal capsules. In these tissues the parasite is intracellular, appearing as a small rounded body with nucleus and blepharoplast, without flagellum or undulating membrane. In other words the parasite is _Leishmania_-like in the body tissues, and recalls the organism of kala-azar.

Chagas considers this second mode of multiplication to be strictly asexual. By this means the number of parasites in the vertebrate host is increased, and symptoms are produced. On the other hand the first mode of multiplication, seen in the lung capillaries, is considered by Chagas to be a process of gametogony, in which sexual forms are differentiated. He finds that (1) the adult trypanosomes exhibit a dimorphism in human blood rarely seen in artificially infected guinea-pigs. In these guinea-pigs (infected from guinea-pigs) the so-called gametogony in the lungs is seldom seen. (2) The intermediate host, _Triatoma_ (_Conorhinus_), becomes infective if fed directly on infected human blood, but very rarely so if fed on guinea-pigs. Chagas is led to believe that the occurrence of sexual forms constantly in the blood of man implies a greater resistance to infection on the part of man than on the part of guinea-pigs or other animals, assuming the general hypothesis that the formation of gametes represents a reaction of the Protozoön to unfavourable conditions. In human infection the number of parasites is always less than in laboratory animals, and their presence in the blood is transitory, lasting from fifteen to thirty days in acute cases. In many cases examination of the tissues at death has shown the presence of parasites in patients who did not exhibit them in the general circulation.

_Life History in the Invertebrate Host._--About six hours after the ingestion of infected blood by the bug (_Triatoma megista_), the kinetic nucleus of the trypanosome moves towards the nucleus, and the flagellum is usually lost (fig. 35, _1_-_5_). The parasite becomes rounded and _Leishmania_-like (fig. 35, _3_-_5_), and multiplies rapidly by division. After a time, multiplication having ceased, the rounded forms become pear-shaped and develop a flagellum at the more pointed end. Crithidial forms (fig. 35, 7) are thus produced and pass into the intestine, where they multiply and may be seen in about twenty-five hours after the ingestion of blood. The crithidial forms may also be found in the rectum and fæces. The last stage in the invertebrate is a small, trypanosome-like type, long and thin with a band-like nucleus and conspicuous kinetic nucleus. These parasites are found in the hind gut and in the body cavity. They find their way into the salivary glands, and are the forms (fig. 36) which are transmissible to a new vertebrate host. The development in the bug takes about eight days altogether, after which time the bugs are infective.

There are thus three principal phases in the development of _T. cruzi_ in _Triatoma megista_: (1) A multiplicative phase (_Leishmania_-like) in the stomach of the bug, (2) a crithidial phase, which is also multiplicative, in the hind-gut, and (3) a trypanosome phase, which is “propagative,” and apparently passes through the wall of the alimentary canal into the body cavity and so into the salivary glands.

Brumpt found that _T. cruzi_ could live in _Cimex lectularius_, _C. boueti_, and _Ornithodorus moubata_. The _Cimex_ fæces may be infective. Blacklock found multiplication of the parasite in _C. lectularius_.

_Culture._--The trypanosome can be cultivated on Novy-MacNeal’s blood agar, and the cultural forms resemble those described in the bug.

_Possible Reservoir._--Chagas thinks that probably the armadillo or “tatu” (_Dasypus novemcinctus_) may be the reservoir of _T. cruzi_. He also thinks that _Triatoma geniculata_ is a transmitter; it lives in the burrows of the armadillo. Other carriers may be _Triatoma infestans_ and _T. sordida_.

_Clinical Features._--The trypanosomiasis of Brazil, produced by _T. cruzi_ and spread by _Triatoma_ spp. has received various names, such as oppilação, canguary, parasitic thyroiditis, and coreotrypanosis. It is also known as the human trypanosomiasis of Brazil, South American trypanosomiasis, and Chagas’ disease.

Chagas[88] reports two principal forms--acute and chronic. The _acute infection_ is rare, and is characterized by increase in the volume of the thyroid gland, pyrexia, a sensation of crackling in the skin, enlarged lymphatic glands in the neck, axilla, etc., while the liver and spleen are increased in volume. Sclerosis of the thyroid gland is found at autopsy and fatty degeneration of the liver. During an attack of fever, trypanosomes are found in the blood. The acute form was only observed in children.

[88] _Brazil Medico_, Nov. 15, 1910. Longer account in _Mem. Inst. Oswaldo Cruz_, iii, pp. 219–275. See _Sleep. Sick. Bull._, Nos. 35 and 40.

_In the chronic form_ Chagas reports several varieties: (_a_) A pseudo-myxœdematous form, occurring in most cases, especially up to the age of 15. There is hypertrophy of the thyroid gland or at least signs of hypothyroidism, general hypertrophy of glands, disturbance of heart rhythm, and nervous symptoms. (_b_) The myxœdematous form is characterized by similar symptoms, especially by considerable swelling of the thyroid body, and myxœdema of the subcutaneous cellular tissue; sometimes there is a true pachydermic cachexia. (_c_) In the nervous form there are motor disturbances, aphasia, disturbances of intelligence or signs of infantilism, athetosis of the extremities and idiocy. There are also paralytic symptoms of bulbar origin, disturbances of mastication, phonation and deglutition, and in some cases convulsive attacks. (_d_) The cardiac form, characterized by disturbance of the heart rhythm. In all these forms the parasite is found at autopsy in the nervous substance, brain, bulb and heart.

Vianna (1911)[89] has studied the histopathology of the disease. Some of the chief points are: in the heart muscle destruction of the sarcoplasm, followed by interstitial myocarditis; in the central nervous system invasion of the neuroglia cells and inflammatory reaction; in the suprarenal capsule invasion of medulla or cortex; inflammatory reaction can also be seen in the kidneys, the hypophysis and thyroid gland.

[89] _Mem. Inst. Oswaldo Cruz_, iii, p. 276.

Recently Chagas states[90] that “schizotrypanosomiasis” has been found in a child 15 to 20 days old, and that _Trypanosoma cruzi_ has also been found in a fœtus--the mother being infected with the trypanosome. The trypanosomiasis can, then, be transmitted hereditarily.

[90] _Rev. Med. S. Paulo_ (1912), xv, p. 337.

*Trypanosoma lewisi*, Kent, 1881.

The trypanosome has a nucleus somewhat displaced anteriorly, about one-third of the way from the anterior (flagellar) end of the body, a relatively straight edge to the undulating membrane, and a rod-shaped blepharoplast (fig. 37, A). It averages about 25 µ long and 1·5 µ broad.

Much attention has been devoted in recent years to the elucidation of the life history of the rat parasite, _Trypanosoma lewisi_. It is usually non-pathogenic to its host. It has been shown that the trypanosome can be transmitted from rat to rat by the rat-flea, _Ceratophyllus fasciatus_, and by _Ctenocephalus canis_ (the so-called dog-flea). (See also p. 92). The flagellate may also persist, but doubtfully develop, in the rat-louse, _Hæmatopinus spinulosus_. These researches may now be summarized.

_Life Cycle in the Vertebrate Host._--After infection of a rat, the trypanosomes usually appear in the animal’s blood in five to seven days. This incubation period applies either to a natural or an artificial infection. The trypanosomes first observed in the rat’s blood are diverse in form (fig. 37), being small, medium and large in size. This diversity is explained by the rapid multiplication taking place. A trypanosome may divide by equal longitudinal fission (fig. 37, C, D), but more commonly multiple fission occurs (fig. 37, G, H), and is unequal. Rosette forms are produced, in which the parent form can be recognized by its long flagellum (fig. 37, H) and attached to it are daughter individuals, smaller in size, from which flagella are growing. Minchin and J. D. Thomson (1912) find that the daughter forms may be set free sometimes with a crithidia-like facies (fig. 37, I), the blepharoplast being anterior but near to the nucleus. The daughter forms, when set free, may themselves divide by binary or multiple fission, in the latter case forming rosettes (fig. 37, K). Rosette forms were described by Moore, Breinl and Hindle in 1908.

Lingard, some years ago, described as a distinct species, _T. longocaudense_, certain forms with markedly elongate posterior ends (fig. 37, E). According to Minchin, “these forms appear to arise by binary fission” (fig. 37, D). These long drawn-out forms “are of constant occurrence and very numerous at a certain stage of the multiplication period.” It is about the eighth or tenth day after infection that the multiplication of _T. lewisi_ is at its maximum in the rat’s blood. About the twelfth or thirteenth day the trypanosomes seen in the blood appear uniform. According to Minchin (1912)[91] the rat “gets rid of its infection entirely sooner or later, without having suffered, apparently, any marked inconvenience from it, and is then immune against a fresh infection with this species of trypanosome.” There is, then, a cycle of development in the vertebrate host. Minchin notes that the records of the pathogenicity of _T. lewisi_ in rats, causing their death, need further investigation.

[91] “Protozoa,” p. 294.

_T. lewisi_ inoculated into dormice (_Myoxus nitela_) and jerboas may become pathogenic thereto.

Carini found cysts in the lungs of rats infected with _T. lewisi_. He thought the cysts were schizogonic stages of the trypanosome, comparable with those found in the lungs of animals sub-inoculated with _T. cruzi_. Delanoë (1912)[92] has found, however, that such cysts, containing eight vermicules, occurred in rats uninfected with _T. lewisi_. Delanoë concludes that the pneumocysts are independent of _T. lewisi_, and represent a new parasite, _Pneumocystis carinii_. The pneumocysts may be allied to the Coccidia, and must be considered when investigating the life-cycle of a trypanosome in a vertebrate host. Some of the stages of _T. cruzi_ may possibly be of this nature.

[92] _C. R. Acad. Sci._, clv, p. 658.

_Life-cycle in the Invertebrate Host._--This occurs in fleas, and has been investigated in considerable detail by Minchin and Thomson in _Ceratophyllus fasciatus_, and by Nöller in _Ctenocephalus canis_ and _Ctenopsylla musculi_.

When infected rat’s blood is taken up by the flea, the parasites pass with the ingested blood direct to the mid-gut of the Siphonapteran. In the flea’s stomach they multiply in a somewhat remarkable manner, namely, by penetration of the cells of the lining epithelium, and division inside the epithelial cells. Inside these lining cells the trypanosomes first grow to a large size and then form large spherical bodies, within which nuclear multiplication occurs (fig. 38, A-F). Any one of these large spherical bodies contains at first a number of nuclei, blepharoplasts and developing flagella, the original flagellum still remaining attached for a time. The cytoplasm then divides into daughter trypanosomes which are contained within an envelope, formed by the periplast of the parent parasite. Inside the periplast envelope are a number of daughter trypanosomes “wriggling very actively; the envelope becomes more and more tense, and finally bursts with explosive suddenness, setting free the flagellates, usually about eight in number, within the host-cell” (fig. 38, F). The daughter forms escaping from the host cell into the stomach lumen of the flea are fully formed, long trypanosomes.

The trypanosomes (fig. 38, G) pass into the flea’s rectum. The next phase is a crithidial one. The parasites become pear-shaped, in which the blepharoplast (kinetic nucleus) has travelled anteriorly past the nucleus towards the flagellum (fig. 39). The crithidial forms attach themselves to the wall of the rectum, and multiply by binary fission (fig. 39, D). A stock of parasites is thus formed which, according to Minchin and Thomson, “persist for a long time in the flea--probably under favourable conditions, for the whole life of the insect” (fig. 39, A-I).

From the crithidial forms of the rectum, according to Minchin, small infective trypanosomes arise by modification morphologically (fig. 39, J--M). The flagellum grows longer and draws out more the anterior part of the body, the blepharoplast migrates posteriorly, behind the nucleus, and carries with it the flagellar origin. These trypanosomes are small, but broad and stumpy (fig. 39, N), and can infect a rat. Minchin and Thomson formerly considered that the small, stumpy, infective trypanosomes pass forwards from the rectum into the stomach, and “appear to be regurgitated into the rat’s blood when the flea feeds.” However, the small infective trypanosomes were previously described by Swellengrebel and Strickland.[93] They may be found in the flea’s fæces. Nöller (1912)[94] has found that the development of _T. lewisi_ proceeds quite well in the dog flea (_Ctenocephalus canis_) in Germany. Wenyon confirms this, and states that the human flea, _Pulex irritans_, and the Indian rat-flea, _Xenopsylla cheopis_, are also able to serve as true hosts for _T. lewisi_.

[93] _Parasitology_, iii, p. 360.

[94] _Arch. f. Protistenkunde_, xxv, p. 386.

Nöller stated that rats were not infected with _T. lewisi_ by infective fleas biting them, but by the rats licking up the fæces passed by the fleas while feeding. This is not in agreement with Minchin and Thomson’s earlier views of regurgitation, which, apparently, they have now abandoned.[95] Wenyon (1912) confirms Nöller’s experiments. He took a dog flea, containing infective trypanosomes in its fæces, and allowed it to feed on a clean rat. The fæces of the flea, passed while feeding, were carefully “collected on a cover glass and taken up in culture fluid with a fine glass pipette.” The contents of the pipette were discharged into the mouth of a second clean rat. Injury to the rat’s mouth was carefully avoided. The first rat, on which the infective flea was fed, did not become infected, while the second rat, in whose mouth infective flea fæces were placed, became infected in six days.

[95] Report to Advis. Comm. Trop. Dis. Research Fund for 1913, p. 74.

When infective forms of _T. lewisi_ have been developed within the gut of a rat flea, they may enter and infect the vertebrate host by[96] (_a_) being crushed and eaten by the rodent; (_b_) the rat may lick its fur on which an infected flea has just passed infective excrement; or (_c_) the rat may lick, and infect with flea excrement, the wound produced by the bite of the flea.

[96] Nuttall, _Parasitology_, v, p. 275.

The time taken for the full development of _T. lewisi_ in the flea is about six days. The intracellular phase is at its height about the end of the first day; the crithidial phase, in the flea’s rectum, begins during the second day; the stumpy, infective trypanosomes are developed in the rectum about the end of the fifth day.

Wenyon[97] writes that, “the fleas, when once infected with _T. lewisi_, remain infected for long periods, for though many small infective trypanosomes are washed out of the gut at each feed, those that remain behind multiply to re-establish the infection of the hind gut. Further, the infection is still maintained even if the flea is nourished on a human being, so that fresh human blood does not appear to be destructive to the infective forms in the flea.”

[97] Report to Advis. Comm. Trop. Dis. Research Fund, October, 1912, p. 91. See also _Journ. Lond. Sch. Trop. Med._, ii, p. 119.

The best method of controlling fleas during experiments is that due to Nöller. He adopted the method of showmen who exhibit performing fleas, and secure them on very fine silver wire.

Of fleas fed on an infected rat only about 20 per cent. become infective. About 80 per cent. are immune. If fleas are examined twenty-four hours after feeding, trypanosomes will be found in all, so that many of the parasites are destined to degenerate.

It may be of interest to note that Gonder[98] (1911) has shown that a strain of _T. lewisi_ resistant to arsenophenylglycin loses its resistance after passage through the rat-louse, _Hæmatopinus spinulosus_. These experiments suggest that physiological “acquired characters” may be lost by passage through an invertebrate host.

[98] _Centralbl. f. Bakt._, Orig., lxi, p. 102.

*Trypanosoma brucei*, Plimmer and Bradford, 1899.

_Trypanosoma brucei_ was discovered by Sir D. Bruce in 1894 in cattle in Zululand and was named _T. brucei_ by Plimmer and Bradford in 1899 in honour of its discoverer. This trypanosome is of considerable economic importance, as it is responsible for the fatal tsetse fly disease, or “nagana,” in cattle, horses and dogs. The disease is widely distributed in Africa and is transmitted from host to host by the tsetse, _Glossina morsitans_, and other species of _Glossina_. The virus is maintained in nature in certain big game, such as wildebeest, bushbuck and koodoo, which thus act as living reservoirs of disease from which the tsetse may become infected. These reservoir hosts are not injured, apparently, by the presence of the parasites.