- Email: [email protected]
The pathology of African Trypanosomiasis
797
(The previous number of these Transactions, Vol. 64, No. 5, was published on 30th September, 1970).
ROYAL SOCIETY OF TROPICAL M£DICINE AND HYGIENE,
ORDINARY MEETING
Manson House, Thurdsay, 15 October, 1970.
The President, PROFESSOR B. G. 2~EGRAITH~ C.M.G., M.B., B.S., D.SC., D.pHIL., F.R.C.P., r.R.C.P.E, in the Chair
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS L. G. G O O D W I N Nuffidd Institute of Comparative Medicine, The Zoological Society of London, Regent's Park, London, N. IV. 1 I must, at the beginning of this paper, acknowledge the immensely valuable support given to work on all aspects of trypanosomiasis by the British Ministry of Overseas Development. From colonial times, the authorities have recognized the threat of this dangerous disease to man and his domestic animals, and also the fact that its study involves the disciplines of human medicine, veterinary medicine, mammalogy and entomology in equal measures. The Ministry has continued to initiate and to support research projects in Britain and overseas and it holds seminars at which progress is assessed. One of its latest achievements has been to sponsor a book, The African Trypanosomiases edited by Colonel H. W. MULLIGANand Mr. W. H. POTTS (1970), into which the accumulated field and laboratory experience of British workers in all aspects of trypanosomasis has been distilled. It is on the brink o f publication and contains so much wisdom that it is certain to become an 'instant' classic. It would therefore seem somewhat pretentious for me to present a paper, at this particular time, on any aspect of the disease. But trypanosomiasis has not been considered by a full Meeting of this Society for several years, and I hope that this evening's discussion may serve as an aperitif to whet your appetites for what is to come. The ancient dragon of Africa, the trypanosome, is firmly entrenched. Like Tolkien's Smaug, it sometimes slumbers and smoulders quietly for long periods but at any moment it may awaken and cause widespread loss of life to man and his domestic animals. As NODFNOT (1958) says: "the fire seems to be out, but the embers glow under the ashes". Trypanosomiasis remains a serious menace to man and his domestic animals and to the wild game of Africa. The fact that epidemics of human sleeping sickness occur at all is of the greatest interest and you will no doubt remember Dr. A. J. DUGGAN'S (1962) masterly account of the disease in Northern Nigeria. Extensions of the primordial foci depend upon This work was supported by grants from the Ministry of Overseas Development. I wish to acknowledge with thanks the technical assistance of Miss S. V. M. Hook Mr. M. W. Guy and Mr. D. G. Taylor, and the help of my colleagues, especially Dr. P. F. L. Boreham, Dr. C. M.'Hawkey and Dr. A. Voller. A*
798
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
many variables, such as human population density and movement, that influence the degree of man-fly contact. Interactions between the parasite, its vectors and its various hosts can give rise to clinical disease ranging in type from asymptomatic 'premunition' (BLAIR, 1939) to rapidly progressive fatal disease. As Duggan says: " . . . we should not become too concerned with attempts to classify the sleeping sickness of the Lake Chad basin strictly in terms of Gambian and Rhodesian. These polymorphic trypanosomes of man are adapting themselves to their host and therefore variations in clinical and epidemiological behaviour are to be expected". A similar situation exists with regard to T. brucei in cattle. In his original report, BRUCE (1897) describes nagana as " . . . a specific disease which occurs in the horse, mule, donkey, ox, dog, cat and many other animals, and varies in duration from a few days or weeks to many months. It is invariably fatal in the horse, donkey and dog, but a small percentage of cattle recover". Long before this, that acute observer LIVINGSTONE(1861) had lost all of his 43 oxen from nagana during his travels in the Zambesi basin in 1850. He gives a beautifully accurate description of the disease: " . . . the eye and nose begin to run, the coat stares, a swelling appears under the jaw, and sometimes at the navel; and, though the poor creature continues to graze, emaciaat-ion commences, accompanied with a peculiar flaccidity of the muscles. This proceeds unchecked until, perhaps months afterwards, purging comes on, and the victim dies in a state of extreme exhaustion. The animals which are in good condition often perish soon after the bite is inflicted with staggering and blindness, as if the brain were affected. Sudden changes of temperature produced by falls of rain seem to hasten the progress of the complaint; but in general the wasting goes on for months". He also noted that the bite of the tsetse in that area was harmless to man and wild animals, and to calves so long as they continued to suck the cows. "Our children were frequently bitten, yet suffered no harm: and we saw around us numbers of zebras, buffaloes, pigs, pallahs and other antelopes, feeding quietly in the very habitat of the fly. There is not so much difference in the natures of the horse and zebra, the buffalo and the ox, the sheep and the antelope, as to afford any satisfactory explanation of the phenomenon. Is not man as much a domestic animal as a dog?" And, years later, there is an echo from BRUCE(1897): "Why should the wild animals be spared whilst tame animals suffer? What is it in domesticity that removes immunity? Can the domestic animal be supplied with what is present in the wild? Can this something be discovered?" To this day, it is difficult to answer the questions; but Livingstone was lucky. Perhaps the trypanosome he encountered was not T. brucei, but T. vivax or T. congolense. I f so, his observations on the infected oxen could have been much the same (Fig. 1). But if he had come across an organism of the kind transmitted from a bushbuck to man by HEISCH, MCMAHON and MANSON-BAHR(1958), the effects on the human members of his expedition might have been less happy. It has long been known that T. rhodesiense from man can be passaged through bushbuck, fowl, cattle, pigs, monkeys and other animals without losing infectivity for the human species (DUKE, 1935; CORSON, 1935, 1936, and others). Moreover, WILDEand FRENCH(1945) showed that in oxen inoculated with Corson's Kahama strain of T. rhodesiense, the parasite appeared from time to time in the peripheral blood for at least 6 months. The infection was of low pathogenicity and the authors thought it reasonable to assume that cattle could act as true carriers of the disease and present a source o f danger to man. More recently, ONYANGO,VAN HOEVEand DE RAADT(1966) demonstrated the soundness of Wilde and French~s warning by isolating a trypanosome of the T. brutal subgroup from one of a random group of
L. G. GOODWIN
799
cattle surveyed in the Alego Location, Central Nyanza, Kenya during the outbreak of human sleeping sickness that began there in 1963. The organism caused a typical T. rhodesiense infection in a human volunteer. From the 203 cattle examined, 43 isolates were made and although not all were tested for infectivity to man, it was estimated that in every African homestead in the area there were at least 2 animals that carried T. bruce/subgroup trypanosomes.
I
Feb. I Mac
!
I I°4°FBull II. -102
i Feb. i Mar.
104°F
Control.
i
Bull 17.
May
Jun.
Control.
LA
~ 8 ~
/ Dead
~ Feb.
i Apr.
i
Mar
i
,o4"~ /~
Apr.
i
May.
T i
r
y
p
s
.
dun.
IS.
Control.Bull
98 Tsetse
I
l • I
Flies
I
l I
Fed
I I Tryps.fin blood.
FIG. 1. Temperature charts of three cattle infected with T. ~vax by the same batch of wild-caught tsetse flies at Vom, N. Nigeria in 1951. No. 11 died in a few days with an overwhelming blood infection. No. 17 died after 3 months of progressive disease, showing bouts of fever and a series of peaks of parasitaemia. No. 15 was killed in good condition, a year after infection; trypanosomes were still present in the blood. In the light reflected from the many facets of this complex host-parasite-vector relationship by the thousands of pages of scientific print written about it over the past century, there is only one conclusion to be drawn. The vast, swarming world population of the T. brucd subgroup trypanosomes is like the world population of man--all of much the same shape and size, but adapted and adaptable to widely different environments. A group of people from Mayfair would stand little chance if injected into the Australian outback, but might thrive in Manhattan or Caracas. Australian aborigines would have a tough time in the Arctic, but might do better there than in Soho. A trader from the Middle East, much to his credit, would be likely to survive and to flourish anywhere. Perhaps the test that uses the sensitivity of T. bruce/to human serum, devised by RICKMAN and ROBSON (1970) may help to identify the strains potentially
A**
800
T H E PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
dangerous to man. Perhaps biochemical differences will be discovered which separate the strains that are capable of infecting man from those that are not (JAFFE, VOORHEIS and McCORMACK, 1969). But a good case can be made for regarding the T. brucei subgroup as a single species containing subspecies or strains of varying host-specificity and virulence, the important factor to man being the existence of strains which in certain circumstances are able to cause disease and death when transmitted to him by bloodsucking flies. HOARE (1970) comes to the same conclusion, and suggests that the brucei-rhodesiense-gambiense complex can be regarded as subspecies of T. brucei, and designated as T. brucei brucei and T. brucei gambiense. He regards T. rhodesiense as a virulent nosodeme of T. gambiense. A sensible parasite does not kill its host too quickly; if it does so, its own life is in jeopardy and its future outlook is poor indeed. The best adapted organisms do their hosts little harm and thus ensure the continuance of their life cycles and the opportunity of occupying new territory. This is the balanced state of affairs that appears to exist •between the African trypanosomes and their wild animal hosts, and that caused Livingstone and Bruce to ask their questions. It is likely that the balance may be more finely set than at first appears. ASHCROFT, BtmTT and FAIRBAIRN (1959) thought that some wild animal species are spared because they are seldom fed upon by tsetse in nature. The balance could easily be upset by removal of the preferred food source by natural disaster or human interference. A really hungry tsetse will usually have a go at anything; it has its own posterity to consider. We have little knowledge of the mortality caused by trypanosomiasis to young wild animals but CUNNrNGHm~ (1968) and McCtrLLOCH (1967) think that it may be considerable. The new-born may be protected for a time by maternal antibody; even Livingstone's domestic calves survived until they were weaned and FIENNES (1952) showed that it was possible to rear calves in tsetse bush if the mothers were protected with chemotherapeutic drugs. The balance can be upset by stress, as shown by ACH~.D'S attempt to establish a breeding colony of rhinos on one of the islands in Lake Victoria. Although the animals were transported with great care, it was necessary to treat them with diminazene (Berenil); otherwise they died shortly after arrival from an acute upsurge of a previously asymptomatic T. congoleme infection (McCtrLrocrI and ACblARO, 1965; ACl~.ARV, personal communication). I find that in this long preamble to pathology I have already mentioned 5 species of trypanosome and at least 16 host species and I shall no doubt be criticized for confusing the issues. But I wish to point out similarities rather than differences and to show that a basic pattern of pathology underlies all forms of African trypanosomiasis.
Trypanosomes in the tissues A thoughtful review of the pathogenesis and pathology of human sleeping sickness has recently been prepared by OR~amOD (1970) and, although I must make reference to this, I shall try not to cover too much of the same ground. Ormerod draws attention to the fact that in man the growth of the parasite occurs mainly in the tissues. This is often forgotten if one is used to dealing with laboratory strains adapted to cause an overwhelming parasitaemia that kills a mouse in a few days. The tsetse fly inoculates the organism with its saliva into the subcutaneous pool of blood on which it feeds. WILLEXT and GORDON (1957) demonstrated that, in some of the rabbits, organisms inoculated by the fly may enter the blood stream directly but that the majority become entangled in the tissue spaces. A local chancre, develops, from which fluid containing trypanosomes can be drawn. The connective tissue, in which the organisms find themselves, is no
L. G. GOODWIN
801
barrier to their spread. ANGEVINE(1959), who has made an extensive study of connective tissue says: "A good working concept of this tissue is to consider it as a system of intercommunicating protein-like fibrils with an immense s u r f a c e . . . The amount of albumin in this space, which can be assumed to correspond roughly with the extracellular space, represents twice the amount contained within the intravascular space" The trypanosomes take to this environment like ducks to water. They multiply, and spread through the tissue spaces and the lymphatics and are soon to be found in swollen lymph nodes, not only in the drainage area of the chancre, but throughout the body. I have discussed elsewhere (GooDwm, in press) some of the writings of earlier observers such as ELMASSIANand MZGONE(1903), MORaX(1907), YORKE(1911 ), WOLBACHand BnqGlm (1912), WATSON (1920) and HORhIB¥ (1949), that show the predilection of trypanosomes of the whole T. brucei group for connective tissue. To quote just one of them, WaRRINGTOlq YORKE (1911) noted that T. rhodesiense multiplied vigorously in oedematous areas in the skins and the corneas of goats and rabbits. He says: " I n early lesions trypanosomes are present in the tissues and as a result there is an oederrmtous condition of the part and a more or less marked degree of leucocytic infiltration. Later, as the number of parasites increases the morbid condition becomes more accentuated. Large numbers of leucocytes are poured into the tissue and new vessels develop. After a time the parasites disappear and with their disappearance there is a tendency on the part of the tissue to recovery. The fact that trypanosomes can multiply so readily in the tissue spaces and at the same time be either entirely absent from the blood, or present in very small numbers only, is one of considerable interest, although the explanation is not very obvious. Perhaps the tissue juices form a more favourable nidus for the growth of the parasites, or possibly in these situations they escape to some extent the action of certain antibodies which have been shown to exist in the blood. Whatever the cause may be, the observation illustrates in what manner it is possible for an animal to be heavily infected and at the same time present no parasites in the peripheral circulation".
T. brucei in rabbits In my own laboratory, we have for the past few years been studying the effects of syringe-transmitted T. brucei infections in rabbits. The strains we used (Lister 42 and 427) produced chronic infections lasting from 2 to 8 weeks; most of the animals died after about 4 or 5 weeks. The animals continued to eat throughout the infection but became wasted, oedematous and scabby and the ears flopped. Parasites were visible in small numbers in smears of the peripheral blood at irregular intervals until the last few days, when the parasitaemia usually increased in intensity. Infectivity titrations in mice showed that trypanosomes were present in the blood within 1 or 2 days of inoculation, and fluctuated but gradually increased in numbers during the course of the infection. The body temperature of the rabbit, measured continuously with the telemetric device demonstrated to this Society by VOLLERand WELLER (1969), rose abruptly on the second day of infection. Unlike the monkey, the rabbit has no marked circadian temperature rhythm. During the first 12 days of the infection, 2 or 3 distinct peaks of temperature occurred. And then something happened which allowed the appearance of more trypanosomes in the circulating blood; the temperature recording remained elevated and ceased to show definite peaks (Fig. 2). Miss Hook and I noted the effect of the disease on the rabbit's ears, which became pale and oedematous during the 3rd week of infection. We studied the vessels by X-ray angiography and showed that the main artery became irregularly constricted and that the arterial arcades, by which the rabbit normally adjusts the blood flow through its ears and uses as radiators to get rid of excess heat, were almost completely shut down. The
802
T H E P A T H O L O G Y OF AFRICAN TRYPANOSOMIASIS
circulation in some parts of the ear tissue was static, and contrast medium collected and persisted in these areas. We also noted that the linings of veins and venules were cluttered with phagocytic cells that took up Indian ink particles injected into the circulation (GoODWlIq and HOOK, 1968). 33
39 338 37 34 4O 39
Oc 38 337 •
35
4O 339 338
0
1
2
~3
4
WEEKS
FIG. 2. Temperature charts of three rabbits infected by syringe with T. brucei. The temperature was recorded continuously by a telemetric device. The black areas indicate the numbers of parasites counted in blood films. Inoculation with trypanosomes at arrow. We followed these observations by a study of the effect of T. brucei infection on the regenerating vessds that can be observed from day to day in rabbit ear chambers (GOODWin, in press). Changes became obvious around the 14th day of infection. The extravascular tissue was heavily infiltrated with mononudear ceils, and phagocytes collected on the endothelium of the venules, leading to obstruction, stasis and eventualy to disintegration of the vasculature. Polymorphs were never much in evidence. In the course of many hours of observation, we never saw a trypanosome swimming freely in the blood running through the vessels, but the perivascular tissue was frequently swarming with parasites. And from time to time the host's phagocytes could be seen mopping up living trypanosomes in the tissue spaces. When the orgy of destruction was over, parasites usually disappeared from the ear chambers for about 24 hours and then the whole process recurred. Once we saw a phagocyte attached to the endothelium of a venule devouring a living trypanosome which it had, no doubt, trapped from the blood streanl.
We were, of course, observing the defence mechanisms of the host attempting to deal with the succession of antigenic variants that arise from the original trypanosome
L. G. GOODWIN
803
strain. GRAY(1962) demonstrated that antibodies develop to a series ofimmunologicaUy distinct trypanosome variants in the serum of rabbits inoculated with T. brucei. Each rise in antibody titre coincides with the disappearance of the homologous variant and when this has been eliminated, a new antigenic type immediately arises. The production of antigenicaUy variable trypanosomes appears to depend upon the ability of the host to produce antibody, and when the defences fail, the final variant multiplies unchecked (WEITZ, 1962). I would like to remind you of the demonstration 60 years ago by Ross and THOMSOI~ (1911) that a succession of variants also occurs in human sleeping sickness. These authors counted the trypanosomes in the blood of their patient every day; a succession of 19 peaks of parasitaemia occurred at 3-4-day intervals, each of which must have represented the emergence of a new variant. They observed that while parasitaemia was increasing, the parasites frequently showed double nuclei and blepharoplasts; these were not seen when the numbers were on the wane. Each wave of parasitaemia was accompanied by fever and was followed by a raised leucocyte count, the increase being due to monocytes, not polymorphs. Immediately after a fall in the numbers of parasites, Giemsa-stained blood films showed that the monocytes were full of reddish d6bris, were vacuolated and of great size. It is of interest that in rabbits a similar sequence of events occurs. Measurements of non-granular leucocytes in stained blood films show that when a T. brucei infection is established, a population of large cells, about 12~ in diameter, and containing much d6bris, replaces the normal mononuclear population which has a modal diameter of 8-2~ (Fig. 3) (GOODWlN and GuY, unpublished). Ross and Thomson's patient, who
l •
60
Before infection.
40
20 o ea) O" U.
4O
After 17 days o f infection (T. brucei).
20
rl r~l
m
4
8
12
16
] I 20
Diameter (Iu).
Fro. 3.
The diameter of non-granular leucocytes in the blood of a rabbit infected with T. brucei. The normal population (mode: 8.2~) is replaced by a population of large mononuclear cells (mode: 125) containing much debris.
804
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
contracted his infection in N.E. Rhodesia in 1909, became drowsy and his mental powers failed; he died with pleurisy and pneumonia 9 months later. He was treated unsuccessfully and blinded with atoxyl and was given vaccines prepared from his own trypanosomes, grown in rats. We now know this course to have been valueless because the vaccine was always at least one step behind the development of a new variant. The enormous load placed upon the immune response of the host by trypanosomes, and by parasitic protozoa in general, is mirrored in the pattern of the serum globulins. Trypanosomiasis stimulates the output of large quantities of immunoglobulin, most of it--and this appears to be the secret of the parasite's survival--is non-specific IgM and has no detectable affinity for the infecting organism (NEAL, GARNHAMand COHEN, 1969). It has been shown by FREEMAN, SMITrmRS, TARG~-TTand WALKER(1970) that more than 95% of an immune IgG preparation from the serum of monkeys (Macaca mulatta) infected with T. brucei is not parasite-specific antibody. The emergence of each new variant, which possibly arises by selection of mutants in a heterogeneous population, elicits an immune response from the host which is out of all proportion to its efficacy in controlling the parasitic population explosion. We now, perhaps, have a clue to this mystery. CLINTON, STAUB~,Rand PALCZUK (1969) showed that hamsters with well developed Leishmania donovani infections, when injected with chicken ovalbumin, responded with a significantly lower antibody titre to the ovalbumin than did normal hamsters, although the serum gamma globulin concentration in the infected animals was high. GREFX',VOOD,I-IERRtCKand VOLrER (1970) found that infection with Plasmodiurn berghei yoelii greatly delayed the appearance of haemolytic anaemia or renal disease in strains of mice that were genetically susceptible to autoimmune disease. And SALAMAN, W~-DD~aBURN and BRUCE-CHWATT (1969) demonstrated that P. bergheiyoelii severely depressed the immune reactivity of mice to sheep erythrocytes for a short period at the height of parasitaemia. We have now shown that chronic T. brucei infections in mice and rabbits also produce profound immunosuppressive effects (Fig. 4, GOODWIN,GuY, GREENandVOLL~, unpublished). The trypanosomes appear to make it difficult for the host to produce antibody to "large" antigens which are believed to require the mediation of macrophages before the antibody-producing cells can be alerted to go into action. As SALAMAN (1970) says, the implications ofimmunodepression by viruses and protozoa are interesting and rather alarming. Imrnunodepression "may predispose to other infections, or increase their severity. It may also perhaps facilitate proliferation and metastasis of neoplastic cells". The parallel with neoplasia is also drawn by CLINTON, STAUBERand PALCZUK(1969) with regard to leishmaniasis, and by ORV~ROD(1961) with regard to the terminal stages of human trypanosomiasis. T. brucei infections in rabbits give rise to intense proliferation of macrophages and these are not so occupied with parasite d6bris that they are unable avidly to collect Indian ink particles from the circulation. They are therefore presumably able to collect particulate antigens such as sheep cells and the important question is why, therefore, are they incapable of performing their function of passing on the message to antibodyforming cells?
Release of pharmacologically active substances The occurrence of a succession of antigen-antibody reactions, each more demanding than the last, may contribute to the tissue damage and physiological disturbance sustained by the host during a trypanosome infection. Antigen-antibody reactions are known to
L. G. GOODWlN
805
be accompanied by the release of a variety of physiologically active substances such as kinin, histamine and "slow-reacting substance A", My colleagues and I have tried to assess the part played by such substances in protozoal disease, and I need not remind you that this interest is shared by the President and his team at Liverpool. MaFGRaIWH, DP-VAKUL and L~iTrm~ (1956) showed that a dramatic temporary recovery occurred when monkeys infected with P. knowlesi were injected with noradrenaline. A similar effect was produced by noradrenaline in puppies in a state of shock caused by Babesia canis infection (M~GP,AITH, GILLES and D~-VAKtrL, 1957). They concluded that the factors that initiated shock might be physiologically active soluble substances of a relatively simple nature derived from the parasite, or as a consequence of host-parasite reaction or tissue damage.
3
10-O
•
102-
o _1
10-
CHALLENGE
Befo •
infection,
ks after infection., after infection, after infection
FIG. 4. Haemagglutination titres of mice infected with T. brucei and given an injection of sheep erythrocytes. The titres were determined 1 week after the injection of sheep cells. There is a steady decrease in the ability of the mice to produce antibody. In 1960 Richards and I demonstrated the presence of pharmacologically active peptides of the kinin type in the urine of mice infected with B. rodhaini, T. brucei, P. berghd and other organisms (GOODWIN and R:CHARDS, 1960), and RICH~a~S (1965) showed that kinins were present in the blood of mice infected with T. brucei. Meanwhile, TELLAand M~Ge, AITH (1962) had shown that shock in monkeys with P. knowlesi infection was also related to the liberation of kinins. I(inins are extremely active shortchain peptides derived from a precursor, ldninogen, in the plasma protein by the action of an enzyme (kininogenase). They are destroyed very rapidly by other enzymes (kininases), and are therefore difficult to detect. T h e i r chemical structure is known, and one of their more important properties is their ability to increase the permeability of blood vessels. Kinin is thought to act as a local hormone in the normal salivary gland, and to mediate in the production of salivary secretion. It is also probably con-
806
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
cerned with the more chronic aspects of local inflammation and may be responsible for vascular changes and oedema after the immediate effects of the histamine response have faded. I have little doubt that the release of kinin, probably as a result of antigen-antibody reactions, plays a part in the pathogenesis of trypanosomiasis. BOP,~HaM (1968a) showed that in cattle infected experimentally with T. brucei, a massive release of kinin occurred just after the second peak in parasitaemia, a time at which the common trypanosome antibodies (precipitins and fluorescent antibodies) first appeared in the serum. During the infection the blood kininogen level fell, but when the trypanosomes were killed by an injection of diminazene aceturate (Berenil), the kininogen concentration immediately rose to a level of about twice the normal value. This suggests that the infection is accompanied by a massive turnover of kinin. A similar picture is seen in rabbits infected with T. brucei, a release of kinin occurring at about the 8th day of infection, shortly before obvious changes in the circulation can be detected in ear chambers. Kinin release has also been detected in human trypanosomiasis (Bo~H~I, 1970). Studies of the components of the kinin-releasing mechanism suggest that trypanosomes activate the precursor of the enzyme (kininogenase) that releases the peptide from the plasma protein (Bo~HAM, 1968b). Activation probably follows antigen-antibody reactions as a result of the absorption of Hageman' Factor on to the surface of the complex (Bomm~l and GOODWIN, 1969). In a study of P. knowlesi infection in monkeys, ONABANJOand .~IAJ~GRAITH(1969) found a significant increase of kallikrein (kininogenase) in the blood, resulting in a clinical condition resembling acute pancreatitis, the classical situation in which proteolytic enzymes are free in the circulating blood (SARD~.SAtand T H e , 1966). Histamine is another highly active substance with an effect on blood vessels, that is associated with the inflammatory process and is known to be liberated by antigenantibody reactions. It was present in increased amounts in the blood of Onabanjo and Maegralth's monkeys, and also in the blood of mice infected with T. brucei (RIc~uatos 1965). However, more recent extensive work by YATFS (1970) has failed to detect significant changes in histamine, or in histidine decarboxylase, the enzyme that forms it, in the blood or tissues of mice and rats infected with T. brucei. Whatever the explanation of this difference, it appears that histamine need not be greatly implicated in the pathogenesis of the disease. The effectiveness of noradrenaline in alleviating the condition of shock that accompanies protozoal infections suggests that catechol amine metabolism is likely to be disturbed. Failure of the adrenal medulla could be responsible for cardiovascular collapse and YATES(tO be published) has made a study of the catechol amine content of the adrenals, hearts and urine from animals infected with T. brucei. In rabbits with chronic infections there is an undoubted change in the metabolic pattern.
Connective tissue damage I have already emphasized that parasites of the T. brucei group multiply in the connective tissue. It is surprising how much damage they do. The bundles of collagen fibres are disruptedman observation made Iongago by WOLBACHand BINGER(1912)m and the fibroblasts are destroyed. The damage is especially severe in the perivascular connective tissue. The vessels of a normal rabbit are not particularly robust structures, but in an animal infected with T. brucei the walls of the veins have the consistency of wet blotting paper; it is surprising that they hold together. Electronmicrographs show that much of the connective tissue is reduced to a nondescript necrotic pulp (GoODWlN,
L. G. GOODWlN
807
in press). The fibres of voluntary muscle also suffer; the mitochondria are degenerate, the fibrils are disrupted and wasted. Muscular wasting is characteristic of all forms of chronic trypanosomiasis. The apparent disintegration of the connective tissue collagen suggested to us that we might be able to obtain a quantitative estimate of the damage by determining the output of hydroxyproline in the urine of infected rabbits. Hydroxyproline is an aminoacid that forms one of the essential building blocks of collagen and little is found elsewhere. The result of one of these studies, carried out by Mr. M. W. GuY, is shown in Fig. 5. Fluid intake and output fell as the disease advanced, and towards the end the
ml
mg"2
1
FIG. 5. Daily fluid balance, hydroxyproline output in the urine and parasitaemia in a rabbit infected with T. brucei. Towards the end of the infection, fluid output exceeded the intake, but at no time was there an increase in hydroxyproline output. Innoculation of trypanosomes at arrow.
808
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
animal went into negative balance. The 24-hour output of hydroxyproline was, however, virtually constant; it certainly did not increase. More work is needed before the effect of the parasite on the structure of connective tissue can be explained. The presence in the tissue spaces of phagocytes that have become overblown and have perished after engulfing and digesting trypanosomes must contribute powerful proteolytic enzymes to the tissue fluid. If the tissues are also short of oxygen as a result of vascular damage, the local pH is likely to fall and free lysosomal enzymes that function in an acid medium may cause further destruction of host cells. The suggestion of SEFDand G ~ (1967) that destruction of host tissues by T. gambiense in rabbits leads to the development ofanto-antibodies also deserves consideration. However, the immediate cessation of the pathological process and the rapid repair that occurs in ear chambers of rabbits cured by trypanocidal drugs, suggests that auto-immunity is unlikely to play an important role in pathogenesis in this species (GOODWlN and Guy, unpublished). In human sleeping sickness, tissue destruction also occurs in the brain and meninges. The capillaries of the central nervous system are free from the connective tissue matrix that surrounds them in other organs; instead, they are invested by the interlocking lamellar processes of the astrocytes (WOLFF, 1963). A great deal has been learned about the astroglia in recent years, although its functions--mechanical, transporting or isolating --are still obscure (WOLFF,1970). It would well repay re-examination in cerebral trypanosomiasis, where there is a great increase in numbers of glial cells throughout the central nervous system, and the cerebral perivascular spaces are 'cuffed' with mononuclear cells. The site of origin of the inflammatory mononuclear cells has always been a mystery. None appear to be in mitosis and there is some evidence that, in rabbits, they may migrate from the spleen, bone marrow and lymph nodes (GoODWlN and GuY, 1970).
Blood changes in trypanosomiasis Much also remains to be learned of the causes of changes in the composition of the blood. Trypanosomiasis usually produces a moderate degree of anaemia, and B o ~ I ~ M (1968c) has shown that, in the rabbit, there is a brisk and well sustained reticulocytosis. There is no haemoglobinuria and no change in the fragility of the erythrocytes, although these have a curious crenated appearance in drawn blood, and the spleen and liver contain deposits of haemosiderin. Erythrocyte enzyme estimations, carried out for us by Dr. C. W. PARR, merely showed evidence of a population of young erythrocytes. Determinations of clotting factors and fibrinolytic activity in the plasma of infected rabbits, carried out by Dr. C. M. HAwr,~v, indicated that in advanced infections the calcification time of the blood was prolonged and the fibrinogen content increased. These are signs of diffuse intravascular coagulation, which is not surprising in view of our observations in the ear chambers. FIENNES (1946) in a study of the pathology of T. congolense infections in cattle also reported the occurrence of thrombosis in small vessels.
The trypanosome Thus far, we have given little consideration to the trypanosome itself. We know that it consumes an enormous amount of glucose, and that heavy parasitaemia must constitute a load on the host's carbohydrate metabolism. Voo~r-~Is (1969) thinks that heavily parasitaemic rats develop a condition akin to diabetes, although the blood sugar remains low because of the metabolic activity of the parasites. However, as
L. G. C,OODWlN
809
VON BRAND (1966) points out, the organisms in the vertebrate host do not complete the breakdown of glucose, but return it to the blood as pyruvate which the host can use, so it is not all lost. It is difficult to estimate what effect parasites in the tissue spaces may have on the economy of the host. The withdrawal of carbohydrate and other nutrients from the tissue fluid may well have a deleterious effect on the cells of the host that need them, and it does not seem justifiable to assume with VON BRAND (1966) that because the parasitaemia in the rabbit is never very intense, the influence of the organisms in this respect is negligible. A great deal of work has been done, with little success, in a search for a toxin or toxic metabolite produced by, or contained in, trypanosomes. KAWAMITSU (1958) injected emulsions of heat-killed T. gamtn'ense into rats and observed hypoglycaemia and a lowering of the liver glycogen reserves 3-5 hours later. This did not occur with emulsions of T. lewisi. More recently, SEED (1969) prepared a protein fraction from homogenized T. gambiense that caused oedema and increased vascular permeability when injected intradermally into guinea-pigs or rabbits. He suggested that the fraction might be related to trypanosome exoantigen (WFITZ, 1960). It could also be the substance that activates kininogenase and causes liberation of kinin (BoRErIAM, 1968b). So far, there has been no satisfactory explanation of the disruptive effect of trypanosomes on the structure of connective tissue. HORNBY(1949) regarded the parasite as a malevolent gimlet that bored its way into the tissues: "T. brucei injures the walls of capillaries, causing intravascular clotting and perivascular oedema. It passes through the injured walls and multiplies freely in the oedema. Wherever it goes it does damage, and one lesion accentuates another". Mechanical damage as a result of the motility of the organism would seem to be unlikely. Tissue cells must be quite accustomed to the constant movement between them of migrating leucocytes and wandering macrophages. But the surface of the organism may well play an important part in determining whether the host dies rapidly, dies after a chronic illness, survives in a state of premunition, or recovers completely. BROOM, BROWN and HOARE (1936) made the observation that T. brucei trypanosomes carry a negative electric surface charge. This property has been brilliantly exploited by Sheila Lanharn at the Lister Institute, who has shown that by the use of suitable synthetic anion-exchange columns, trypanosomes can be separated cleanly not only from erythrocytes, leucocytes and platelets but from other species of trypanosome, by virtue of the degree of negative charge they carry (LANHaM, 1968a, 1968b). This elegant technique has proved a most valuable tool for trypanosomiasis research. GODFREY and TAYLOR (1969), also at the Lister Institute, studied the surface membranes of trypanosomes and the effect on them of cobra venom, which contains phospholipase A and a lyric factor. They showed that T. brucei has a trilaminar plasma membrane with a thick uniform external surface coat that is removed by the action of the venom. VIC~R~ N (1969) reported that the outer coat of T. rhodesiense was removed by proteolytic enzymes and was lost when the parasite entered the gut of the tsetse fly. The outer proteinaceous coat, among other functions, may be of importance in determining the antigenic identity of the organism (VICKERMANand LUCKINS,1969) and may perhaps assist it in escaping from the host's defence mechanisms. T. lewisi, which evokes a prompt and effective immune response from its host, does not possess a thick covering of protein. It is almost impossible to avoid jumping to the conclusion that, like schistosomes, pathogenic trypanosomes might put on jackets of host protein as a disguise. But this remains to be seen.
810
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
I have covered a great deal of ground very sketchily, and have made only passing references to the biochemistry of the parasite, and none at all to the interesting phenomenon of "natural immunity" shown by some hosts, or to the possibility of there being a further stage in the life history of T. brucei subgroup trypanosomes. Nonflagellate, latent bodies were described in the vertebrate host by FANTImM (1911) and he fought a long, losing battle for them with a fierce, missionary zeal. They are now again in the news, and it is hard to say what influence their resurrection may have on our picture of the pathology of trypanosomiasis. But~!when pathogenesis occurs, it seems likely that allergic reactions to successive antigenic variants are at the root of the trouble. There is no lack of mobilization of the host's defence mechanisms. The tissues are packed with cells that, from their appearance and behaviour, should be competent to deal with all invaders, but the defences seem to lack direction and control. [_The polymorphs take peculiarly little interest in the cataclysms that occur in the tissues.] Mononuclears swarm everywhere and their lysosomes snap, crackle and pop so that sober cellular citizens become casualties, important structural edifices crumble, and poisonous ingredients of the necrotic soup leak back into the circulation to play hell with the host's physiology. Vast quantities of immunogiobulin are produced by the host and most of this crippling effort is useless. A small antigenic variation is enough to confuse the intelligence services, the disguised invaders multiply and bring about a dismally inevitable series of ill-directed steam-hammer blows that end in the exhaustion of the host's reserves. It's all rather like a T o m and Jerry cartoon, with a monstrously inept cat pulling the place down in his efforts to pulverise a diminutive, agile and highly resourceful mouse. I n h i s recent review of Ronald Hare's book, The Birth of Penicillin, PtaI~ (1970) says that research workers: " . . . hold in their minds a mass of half-formulated impressions and ~incompletely remembered observations. Out of this untidy chaos, decisions on the probable relevance of the various factors finatly emerge and notes on these factors are made. But there is no infallible way to distinguish at the start the relevant from the irrelevant factors. This difficulty obtrudes itself increasingly the more novel the phenomenon and the younger the experimental discipline. Experience is therefore now a surer guide in physics than in biology." As Mach pointed out, it is experience rather than logic that leads us to conclude that the colour of the weights on a balance is irrelevant. The most difficult part of research is finding out how to do the experiments--that is to say, learning what to control, what to observe and what to measure. When this has been done, almost anyone can get useful results in the domain that has been pioneered. It is harder to see what should be controlled while experience is still being integrated into a testable point of view. This is what makes the beginnings of research look like 'sleepwalking' to non-scientists". I don't think Pirie would disagree if I added that it often looks suspiciously like sleepwalking to the research workers themselves--especially in the field of sleeping sickness. REFERENCES ANGEVIN,, D. M. (1959). In Modern Trends in Pathology. Ed. Collins, D. H. p.47. London: Butterworth. ASHCROFT,M. T., BURTT,E. & FAIRBAIRN,H. (1959). Ann. trop. Med. Parasit., 53, 147. BLAIR, D. M. (1939). Trans. R. Soc. trop. Med. Hyg., 32, 729. BORF.HAM,P. F. L. (1968a). Br. J. Pharmac. Chemother., 32, 493. (1968b). Ibid., 34, 598. (1968c). Thesis, University of London. (1970). Trans. R. Soc. trop. Med. Hyg., 64, 394. ~-& GOODWIN, L. G. (1969). Pharmacol. Res. Comm. 1, 144. -
-
L. G. GOODWlN
811
BROOM, J. C., BROWN,H. C. & HOARE,C. A. (1936). Trans. R. Soc. trop. Med. Hyg., 30, 87. BRUCE, D. (1897). Further Report on the Tsetse Fly Disease of Nagana in Zululand, Ubombo, 1896. London: Harrison. CLI~rrON, B. A., STAtmER, L. A., & PALCZtrK,N. C. (1969). Exp. Parasit., 25, 171. CORSON, J. F. (1935). ft. trop. Med. Hyg., 38, 9. (1936). Ibid., 39, 125. CtYNNING~M, M. P. (1968). E. Afr. agric, for. J., 33, 264. DUGGAN, A. J. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 439. DUKE, H. L. (1935). Parasitology, 27, 46. ELMASSXAN,M. & MmONE, E. (1903). Ann. Inst. Pasteur, 17, 241. FANTnAM, H. B. (1911). Ann. trop. Med. Paras#., 4, 111. FIENNES, R. N. T-W- (1946). ft. comp. Path., 56, 28. (1952). Vet. Rec., 64, 423. FREEMAN, T., SMITn~RS, S. R., TARGETT, G. A. T. & WALKER, P. J. (1970). J. infect. Dis., 121, 401. GODFREY, D. G. & TAYLOR, A. E. R. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 115. GOODWIN, L. G. Ibid., (in press). & Gtr% M. W. (1970). Ibid., 64, 470. & HOOK, S. V. M. (1968). Br. J. Pharmac. Chemother., 32, 505. & RmnA~s, W. H. G. (1960). Ibid., 15, 152. GRAY, A. R. (1962). Ann. trop. Med. Paras#., 56, 4. GREENWOOD,B. M., HERRICK,E. M. & VOLLER,A. (1970). Nature, Lond., 226, 266. HEISCH, R. B., McMArlON, J. P.& MANSoN-BAHR,P. E. C. (1958). Br. reed. ~., 2, 1203. HOARE, C. A. (1970). In The African Trypanosomiases, p. 51. Eds. Mulligan, H. W. & Potts, W. H. London: Allen & Unwin. HORNBY, H. E. (1949). Animal Trypanosomiasis in Eastern Africa. H.M. Stationery Office, London. JAFFE, J. J., VOORHEIS, H. P. & McCORMACK, J. J. JR. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 118. KAWAMITStl, K. (1958). Nagasaki Igakkai Zassi., 33, No. 11. Suppl., 22. LANI-IAM,S. (1968a). Trans. R. Soc. trop. Med. Hyg., 62, 4. (1968b). Nature, Lond., 218, 1273. LIVINGSTONE,D. (1861). A Popular Account of Missionary Travels and Researches in South Africa. London: Murray. McCULLOCH, B. (1967). Ann. trop. Med. Parasit., 61, 261. & ACHARD,P. L. (1956). Oryx, 8, 131. MAEGRAITH, B. G., DEVAKUL, K. & LEITHEAD, C. S. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 311. GILLES, H. M. & DEVAKUL,K. (1957). Z. tropenmed. Parasit., 8, 485. MORAX, V. (1907). Ann. Inst. Pasteur., 21, 47. MULLIGAN, H. W. & POTTS, W. H. (1970). Eds. The African Trypanosomiases. London: Allen & Unwin. NEAL, R. A., GARlqI-IAM,P. C. C. & COHEN, S. (1969). Br. reed. Bull., 25, 194. NODENOT, L. (1958). Proc. VII Int. Sci. Comm. Tryp. Res. Brussels. 207. ONABANJO, A. O. & MAEGRAITH,B. G. (1969). PharmacoL Res. Comm., 1, 179. ONYANGO, R. J., VAN HOEVE, K. & DERAADT, P. (1966), Trans. R. Soc. trop. Med. ttyg., 60, 175. ORMEROD, W. E. (1961). Ibid., 55, 313. - (1970). In The African Trypanosomiases p. 587. Eds. Mulligan, H. W. & Ports, W. H. London: Allen & Unwin. PIRIE, N. W. (1970). Listener, 84, 154. RICI-IARDS,W. H. G. (1965). Br. ~. Pharmacol., 24, 124. RICKMAN, L. R. & ROBSON, J. (1970). Bull. Wld Hlth Org., 42, 650. Ross, R. & THOMSON, D. (1911). Ann. trop. Med. Parasit., 4, 395. SALAMAN,M. H. (1970). Proc. Roy. Soc. Med., 63, 11. , WEDDERBURN, N. & BRUCE-CHWATT,L. J. (1969). o7. gen. Microbiol., 59, 383. SARDESAI,V. i . & THAL, A. P. (1966). In Hypotensive Peptides, p. 463. Eds. Erdos, E. G., Back, N., Sicuteri, F. & Wilde, A. F. New York: Springer. SEED, J. R. (1969). Exp. Parasit., 26, 214. & GAM, A. A. (1967). ~. Parasit., 53, 946. TELLA, A. & MAEGRAITH,B. G. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 6.
812
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
K. (1969). ft. Cell. Science, 5, 163. & LUCKINS,A. G. (1969) Nature, Lond., 224, 1125. VOLLER, A. & WFLLER, C. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 418. VoN BRAND,T. (1966). In Biochemistry of Parasites. p. 160. New York & London: Academic Press. VOORHEIS, H. P. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 122. WATSON, E. A. (1920). Dourine in Canada 1904-20. History, Research & Suppression. Dept. Agric. Health, Anita. Branch. Dom. Canada 1. WF.XTZ,B. (1960). ft. gen. Microbiol., 23, 589. (1962). In Drugs, Parasites & Hosts. p. 180. Eds. Goodwin, L. G. & Nimmo-Smith, R. H. London: Churchill. WADE, J. K. H. & FR~NCI-I,M. H. (1945). J. comp. Path., .%5,206. WILLETT, K. C. & GORDON,R. M. (1957). Ann. trop. Med. Parasit., 51, 471. WOLBACH, S. B. & BINGER, C. A. L. (1912). J. reed. Res., 27, 83. WOLFF, J. R. (1963). Z. Zellforsch., 60, 409. (1970). Triangle, 9, 153. YATES, D. B. (1970). Trans. R. Soc. trop. Med. Hyg., 64, 167. YORKE, W. (1911). Ann. trop. Med. Parasit., 4, 385. VICI~RMAN,
(The previous number of these Transactions, Vol. 64, No. 5, was published on 30th September, 1970).
ROYAL SOCIETY OF TROPICAL M£DICINE AND HYGIENE,
ORDINARY MEETING
Manson House, Thurdsay, 15 October, 1970.
The President, PROFESSOR B. G. 2~EGRAITH~ C.M.G., M.B., B.S., D.SC., D.pHIL., F.R.C.P., r.R.C.P.E, in the Chair
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS L. G. G O O D W I N Nuffidd Institute of Comparative Medicine, The Zoological Society of London, Regent's Park, London, N. IV. 1 I must, at the beginning of this paper, acknowledge the immensely valuable support given to work on all aspects of trypanosomiasis by the British Ministry of Overseas Development. From colonial times, the authorities have recognized the threat of this dangerous disease to man and his domestic animals, and also the fact that its study involves the disciplines of human medicine, veterinary medicine, mammalogy and entomology in equal measures. The Ministry has continued to initiate and to support research projects in Britain and overseas and it holds seminars at which progress is assessed. One of its latest achievements has been to sponsor a book, The African Trypanosomiases edited by Colonel H. W. MULLIGANand Mr. W. H. POTTS (1970), into which the accumulated field and laboratory experience of British workers in all aspects of trypanosomasis has been distilled. It is on the brink o f publication and contains so much wisdom that it is certain to become an 'instant' classic. It would therefore seem somewhat pretentious for me to present a paper, at this particular time, on any aspect of the disease. But trypanosomiasis has not been considered by a full Meeting of this Society for several years, and I hope that this evening's discussion may serve as an aperitif to whet your appetites for what is to come. The ancient dragon of Africa, the trypanosome, is firmly entrenched. Like Tolkien's Smaug, it sometimes slumbers and smoulders quietly for long periods but at any moment it may awaken and cause widespread loss of life to man and his domestic animals. As NODFNOT (1958) says: "the fire seems to be out, but the embers glow under the ashes". Trypanosomiasis remains a serious menace to man and his domestic animals and to the wild game of Africa. The fact that epidemics of human sleeping sickness occur at all is of the greatest interest and you will no doubt remember Dr. A. J. DUGGAN'S (1962) masterly account of the disease in Northern Nigeria. Extensions of the primordial foci depend upon This work was supported by grants from the Ministry of Overseas Development. I wish to acknowledge with thanks the technical assistance of Miss S. V. M. Hook Mr. M. W. Guy and Mr. D. G. Taylor, and the help of my colleagues, especially Dr. P. F. L. Boreham, Dr. C. M.'Hawkey and Dr. A. Voller. A*
798
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
many variables, such as human population density and movement, that influence the degree of man-fly contact. Interactions between the parasite, its vectors and its various hosts can give rise to clinical disease ranging in type from asymptomatic 'premunition' (BLAIR, 1939) to rapidly progressive fatal disease. As Duggan says: " . . . we should not become too concerned with attempts to classify the sleeping sickness of the Lake Chad basin strictly in terms of Gambian and Rhodesian. These polymorphic trypanosomes of man are adapting themselves to their host and therefore variations in clinical and epidemiological behaviour are to be expected". A similar situation exists with regard to T. brucei in cattle. In his original report, BRUCE (1897) describes nagana as " . . . a specific disease which occurs in the horse, mule, donkey, ox, dog, cat and many other animals, and varies in duration from a few days or weeks to many months. It is invariably fatal in the horse, donkey and dog, but a small percentage of cattle recover". Long before this, that acute observer LIVINGSTONE(1861) had lost all of his 43 oxen from nagana during his travels in the Zambesi basin in 1850. He gives a beautifully accurate description of the disease: " . . . the eye and nose begin to run, the coat stares, a swelling appears under the jaw, and sometimes at the navel; and, though the poor creature continues to graze, emaciaat-ion commences, accompanied with a peculiar flaccidity of the muscles. This proceeds unchecked until, perhaps months afterwards, purging comes on, and the victim dies in a state of extreme exhaustion. The animals which are in good condition often perish soon after the bite is inflicted with staggering and blindness, as if the brain were affected. Sudden changes of temperature produced by falls of rain seem to hasten the progress of the complaint; but in general the wasting goes on for months". He also noted that the bite of the tsetse in that area was harmless to man and wild animals, and to calves so long as they continued to suck the cows. "Our children were frequently bitten, yet suffered no harm: and we saw around us numbers of zebras, buffaloes, pigs, pallahs and other antelopes, feeding quietly in the very habitat of the fly. There is not so much difference in the natures of the horse and zebra, the buffalo and the ox, the sheep and the antelope, as to afford any satisfactory explanation of the phenomenon. Is not man as much a domestic animal as a dog?" And, years later, there is an echo from BRUCE(1897): "Why should the wild animals be spared whilst tame animals suffer? What is it in domesticity that removes immunity? Can the domestic animal be supplied with what is present in the wild? Can this something be discovered?" To this day, it is difficult to answer the questions; but Livingstone was lucky. Perhaps the trypanosome he encountered was not T. brucei, but T. vivax or T. congolense. I f so, his observations on the infected oxen could have been much the same (Fig. 1). But if he had come across an organism of the kind transmitted from a bushbuck to man by HEISCH, MCMAHON and MANSON-BAHR(1958), the effects on the human members of his expedition might have been less happy. It has long been known that T. rhodesiense from man can be passaged through bushbuck, fowl, cattle, pigs, monkeys and other animals without losing infectivity for the human species (DUKE, 1935; CORSON, 1935, 1936, and others). Moreover, WILDEand FRENCH(1945) showed that in oxen inoculated with Corson's Kahama strain of T. rhodesiense, the parasite appeared from time to time in the peripheral blood for at least 6 months. The infection was of low pathogenicity and the authors thought it reasonable to assume that cattle could act as true carriers of the disease and present a source o f danger to man. More recently, ONYANGO,VAN HOEVEand DE RAADT(1966) demonstrated the soundness of Wilde and French~s warning by isolating a trypanosome of the T. brutal subgroup from one of a random group of
L. G. GOODWIN
799
cattle surveyed in the Alego Location, Central Nyanza, Kenya during the outbreak of human sleeping sickness that began there in 1963. The organism caused a typical T. rhodesiense infection in a human volunteer. From the 203 cattle examined, 43 isolates were made and although not all were tested for infectivity to man, it was estimated that in every African homestead in the area there were at least 2 animals that carried T. bruce/subgroup trypanosomes.
I
Feb. I Mac
!
I I°4°FBull II. -102
i Feb. i Mar.
104°F
Control.
i
Bull 17.
May
Jun.
Control.
LA
~ 8 ~
/ Dead
~ Feb.
i Apr.
i
Mar
i
,o4"~ /~
Apr.
i
May.
T i
r
y
p
s
.
dun.
IS.
Control.Bull
98 Tsetse
I
l • I
Flies
I
l I
Fed
I I Tryps.fin blood.
FIG. 1. Temperature charts of three cattle infected with T. ~vax by the same batch of wild-caught tsetse flies at Vom, N. Nigeria in 1951. No. 11 died in a few days with an overwhelming blood infection. No. 17 died after 3 months of progressive disease, showing bouts of fever and a series of peaks of parasitaemia. No. 15 was killed in good condition, a year after infection; trypanosomes were still present in the blood. In the light reflected from the many facets of this complex host-parasite-vector relationship by the thousands of pages of scientific print written about it over the past century, there is only one conclusion to be drawn. The vast, swarming world population of the T. brucd subgroup trypanosomes is like the world population of man--all of much the same shape and size, but adapted and adaptable to widely different environments. A group of people from Mayfair would stand little chance if injected into the Australian outback, but might thrive in Manhattan or Caracas. Australian aborigines would have a tough time in the Arctic, but might do better there than in Soho. A trader from the Middle East, much to his credit, would be likely to survive and to flourish anywhere. Perhaps the test that uses the sensitivity of T. bruce/to human serum, devised by RICKMAN and ROBSON (1970) may help to identify the strains potentially
A**
800
T H E PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
dangerous to man. Perhaps biochemical differences will be discovered which separate the strains that are capable of infecting man from those that are not (JAFFE, VOORHEIS and McCORMACK, 1969). But a good case can be made for regarding the T. brucei subgroup as a single species containing subspecies or strains of varying host-specificity and virulence, the important factor to man being the existence of strains which in certain circumstances are able to cause disease and death when transmitted to him by bloodsucking flies. HOARE (1970) comes to the same conclusion, and suggests that the brucei-rhodesiense-gambiense complex can be regarded as subspecies of T. brucei, and designated as T. brucei brucei and T. brucei gambiense. He regards T. rhodesiense as a virulent nosodeme of T. gambiense. A sensible parasite does not kill its host too quickly; if it does so, its own life is in jeopardy and its future outlook is poor indeed. The best adapted organisms do their hosts little harm and thus ensure the continuance of their life cycles and the opportunity of occupying new territory. This is the balanced state of affairs that appears to exist •between the African trypanosomes and their wild animal hosts, and that caused Livingstone and Bruce to ask their questions. It is likely that the balance may be more finely set than at first appears. ASHCROFT, BtmTT and FAIRBAIRN (1959) thought that some wild animal species are spared because they are seldom fed upon by tsetse in nature. The balance could easily be upset by removal of the preferred food source by natural disaster or human interference. A really hungry tsetse will usually have a go at anything; it has its own posterity to consider. We have little knowledge of the mortality caused by trypanosomiasis to young wild animals but CUNNrNGHm~ (1968) and McCtrLLOCH (1967) think that it may be considerable. The new-born may be protected for a time by maternal antibody; even Livingstone's domestic calves survived until they were weaned and FIENNES (1952) showed that it was possible to rear calves in tsetse bush if the mothers were protected with chemotherapeutic drugs. The balance can be upset by stress, as shown by ACH~.D'S attempt to establish a breeding colony of rhinos on one of the islands in Lake Victoria. Although the animals were transported with great care, it was necessary to treat them with diminazene (Berenil); otherwise they died shortly after arrival from an acute upsurge of a previously asymptomatic T. congoleme infection (McCtrLrocrI and ACblARO, 1965; ACl~.ARV, personal communication). I find that in this long preamble to pathology I have already mentioned 5 species of trypanosome and at least 16 host species and I shall no doubt be criticized for confusing the issues. But I wish to point out similarities rather than differences and to show that a basic pattern of pathology underlies all forms of African trypanosomiasis.
Trypanosomes in the tissues A thoughtful review of the pathogenesis and pathology of human sleeping sickness has recently been prepared by OR~amOD (1970) and, although I must make reference to this, I shall try not to cover too much of the same ground. Ormerod draws attention to the fact that in man the growth of the parasite occurs mainly in the tissues. This is often forgotten if one is used to dealing with laboratory strains adapted to cause an overwhelming parasitaemia that kills a mouse in a few days. The tsetse fly inoculates the organism with its saliva into the subcutaneous pool of blood on which it feeds. WILLEXT and GORDON (1957) demonstrated that, in some of the rabbits, organisms inoculated by the fly may enter the blood stream directly but that the majority become entangled in the tissue spaces. A local chancre, develops, from which fluid containing trypanosomes can be drawn. The connective tissue, in which the organisms find themselves, is no
L. G. GOODWIN
801
barrier to their spread. ANGEVINE(1959), who has made an extensive study of connective tissue says: "A good working concept of this tissue is to consider it as a system of intercommunicating protein-like fibrils with an immense s u r f a c e . . . The amount of albumin in this space, which can be assumed to correspond roughly with the extracellular space, represents twice the amount contained within the intravascular space" The trypanosomes take to this environment like ducks to water. They multiply, and spread through the tissue spaces and the lymphatics and are soon to be found in swollen lymph nodes, not only in the drainage area of the chancre, but throughout the body. I have discussed elsewhere (GooDwm, in press) some of the writings of earlier observers such as ELMASSIANand MZGONE(1903), MORaX(1907), YORKE(1911 ), WOLBACHand BnqGlm (1912), WATSON (1920) and HORhIB¥ (1949), that show the predilection of trypanosomes of the whole T. brucei group for connective tissue. To quote just one of them, WaRRINGTOlq YORKE (1911) noted that T. rhodesiense multiplied vigorously in oedematous areas in the skins and the corneas of goats and rabbits. He says: " I n early lesions trypanosomes are present in the tissues and as a result there is an oederrmtous condition of the part and a more or less marked degree of leucocytic infiltration. Later, as the number of parasites increases the morbid condition becomes more accentuated. Large numbers of leucocytes are poured into the tissue and new vessels develop. After a time the parasites disappear and with their disappearance there is a tendency on the part of the tissue to recovery. The fact that trypanosomes can multiply so readily in the tissue spaces and at the same time be either entirely absent from the blood, or present in very small numbers only, is one of considerable interest, although the explanation is not very obvious. Perhaps the tissue juices form a more favourable nidus for the growth of the parasites, or possibly in these situations they escape to some extent the action of certain antibodies which have been shown to exist in the blood. Whatever the cause may be, the observation illustrates in what manner it is possible for an animal to be heavily infected and at the same time present no parasites in the peripheral circulation".
T. brucei in rabbits In my own laboratory, we have for the past few years been studying the effects of syringe-transmitted T. brucei infections in rabbits. The strains we used (Lister 42 and 427) produced chronic infections lasting from 2 to 8 weeks; most of the animals died after about 4 or 5 weeks. The animals continued to eat throughout the infection but became wasted, oedematous and scabby and the ears flopped. Parasites were visible in small numbers in smears of the peripheral blood at irregular intervals until the last few days, when the parasitaemia usually increased in intensity. Infectivity titrations in mice showed that trypanosomes were present in the blood within 1 or 2 days of inoculation, and fluctuated but gradually increased in numbers during the course of the infection. The body temperature of the rabbit, measured continuously with the telemetric device demonstrated to this Society by VOLLERand WELLER (1969), rose abruptly on the second day of infection. Unlike the monkey, the rabbit has no marked circadian temperature rhythm. During the first 12 days of the infection, 2 or 3 distinct peaks of temperature occurred. And then something happened which allowed the appearance of more trypanosomes in the circulating blood; the temperature recording remained elevated and ceased to show definite peaks (Fig. 2). Miss Hook and I noted the effect of the disease on the rabbit's ears, which became pale and oedematous during the 3rd week of infection. We studied the vessels by X-ray angiography and showed that the main artery became irregularly constricted and that the arterial arcades, by which the rabbit normally adjusts the blood flow through its ears and uses as radiators to get rid of excess heat, were almost completely shut down. The
802
T H E P A T H O L O G Y OF AFRICAN TRYPANOSOMIASIS
circulation in some parts of the ear tissue was static, and contrast medium collected and persisted in these areas. We also noted that the linings of veins and venules were cluttered with phagocytic cells that took up Indian ink particles injected into the circulation (GoODWlIq and HOOK, 1968). 33
39 338 37 34 4O 39
Oc 38 337 •
35
4O 339 338
0
1
2
~3
4
WEEKS
FIG. 2. Temperature charts of three rabbits infected by syringe with T. brucei. The temperature was recorded continuously by a telemetric device. The black areas indicate the numbers of parasites counted in blood films. Inoculation with trypanosomes at arrow. We followed these observations by a study of the effect of T. brucei infection on the regenerating vessds that can be observed from day to day in rabbit ear chambers (GOODWin, in press). Changes became obvious around the 14th day of infection. The extravascular tissue was heavily infiltrated with mononudear ceils, and phagocytes collected on the endothelium of the venules, leading to obstruction, stasis and eventualy to disintegration of the vasculature. Polymorphs were never much in evidence. In the course of many hours of observation, we never saw a trypanosome swimming freely in the blood running through the vessels, but the perivascular tissue was frequently swarming with parasites. And from time to time the host's phagocytes could be seen mopping up living trypanosomes in the tissue spaces. When the orgy of destruction was over, parasites usually disappeared from the ear chambers for about 24 hours and then the whole process recurred. Once we saw a phagocyte attached to the endothelium of a venule devouring a living trypanosome which it had, no doubt, trapped from the blood streanl.
We were, of course, observing the defence mechanisms of the host attempting to deal with the succession of antigenic variants that arise from the original trypanosome
L. G. GOODWIN
803
strain. GRAY(1962) demonstrated that antibodies develop to a series ofimmunologicaUy distinct trypanosome variants in the serum of rabbits inoculated with T. brucei. Each rise in antibody titre coincides with the disappearance of the homologous variant and when this has been eliminated, a new antigenic type immediately arises. The production of antigenicaUy variable trypanosomes appears to depend upon the ability of the host to produce antibody, and when the defences fail, the final variant multiplies unchecked (WEITZ, 1962). I would like to remind you of the demonstration 60 years ago by Ross and THOMSOI~ (1911) that a succession of variants also occurs in human sleeping sickness. These authors counted the trypanosomes in the blood of their patient every day; a succession of 19 peaks of parasitaemia occurred at 3-4-day intervals, each of which must have represented the emergence of a new variant. They observed that while parasitaemia was increasing, the parasites frequently showed double nuclei and blepharoplasts; these were not seen when the numbers were on the wane. Each wave of parasitaemia was accompanied by fever and was followed by a raised leucocyte count, the increase being due to monocytes, not polymorphs. Immediately after a fall in the numbers of parasites, Giemsa-stained blood films showed that the monocytes were full of reddish d6bris, were vacuolated and of great size. It is of interest that in rabbits a similar sequence of events occurs. Measurements of non-granular leucocytes in stained blood films show that when a T. brucei infection is established, a population of large cells, about 12~ in diameter, and containing much d6bris, replaces the normal mononuclear population which has a modal diameter of 8-2~ (Fig. 3) (GOODWlN and GuY, unpublished). Ross and Thomson's patient, who
l •
60
Before infection.
40
20 o ea) O" U.
4O
After 17 days o f infection (T. brucei).
20
rl r~l
m
4
8
12
16
] I 20
Diameter (Iu).
Fro. 3.
The diameter of non-granular leucocytes in the blood of a rabbit infected with T. brucei. The normal population (mode: 8.2~) is replaced by a population of large mononuclear cells (mode: 125) containing much debris.
804
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
contracted his infection in N.E. Rhodesia in 1909, became drowsy and his mental powers failed; he died with pleurisy and pneumonia 9 months later. He was treated unsuccessfully and blinded with atoxyl and was given vaccines prepared from his own trypanosomes, grown in rats. We now know this course to have been valueless because the vaccine was always at least one step behind the development of a new variant. The enormous load placed upon the immune response of the host by trypanosomes, and by parasitic protozoa in general, is mirrored in the pattern of the serum globulins. Trypanosomiasis stimulates the output of large quantities of immunoglobulin, most of it--and this appears to be the secret of the parasite's survival--is non-specific IgM and has no detectable affinity for the infecting organism (NEAL, GARNHAMand COHEN, 1969). It has been shown by FREEMAN, SMITrmRS, TARG~-TTand WALKER(1970) that more than 95% of an immune IgG preparation from the serum of monkeys (Macaca mulatta) infected with T. brucei is not parasite-specific antibody. The emergence of each new variant, which possibly arises by selection of mutants in a heterogeneous population, elicits an immune response from the host which is out of all proportion to its efficacy in controlling the parasitic population explosion. We now, perhaps, have a clue to this mystery. CLINTON, STAUB~,Rand PALCZUK (1969) showed that hamsters with well developed Leishmania donovani infections, when injected with chicken ovalbumin, responded with a significantly lower antibody titre to the ovalbumin than did normal hamsters, although the serum gamma globulin concentration in the infected animals was high. GREFX',VOOD,I-IERRtCKand VOLrER (1970) found that infection with Plasmodiurn berghei yoelii greatly delayed the appearance of haemolytic anaemia or renal disease in strains of mice that were genetically susceptible to autoimmune disease. And SALAMAN, W~-DD~aBURN and BRUCE-CHWATT (1969) demonstrated that P. bergheiyoelii severely depressed the immune reactivity of mice to sheep erythrocytes for a short period at the height of parasitaemia. We have now shown that chronic T. brucei infections in mice and rabbits also produce profound immunosuppressive effects (Fig. 4, GOODWIN,GuY, GREENandVOLL~, unpublished). The trypanosomes appear to make it difficult for the host to produce antibody to "large" antigens which are believed to require the mediation of macrophages before the antibody-producing cells can be alerted to go into action. As SALAMAN (1970) says, the implications ofimmunodepression by viruses and protozoa are interesting and rather alarming. Imrnunodepression "may predispose to other infections, or increase their severity. It may also perhaps facilitate proliferation and metastasis of neoplastic cells". The parallel with neoplasia is also drawn by CLINTON, STAUBERand PALCZUK(1969) with regard to leishmaniasis, and by ORV~ROD(1961) with regard to the terminal stages of human trypanosomiasis. T. brucei infections in rabbits give rise to intense proliferation of macrophages and these are not so occupied with parasite d6bris that they are unable avidly to collect Indian ink particles from the circulation. They are therefore presumably able to collect particulate antigens such as sheep cells and the important question is why, therefore, are they incapable of performing their function of passing on the message to antibodyforming cells?
Release of pharmacologically active substances The occurrence of a succession of antigen-antibody reactions, each more demanding than the last, may contribute to the tissue damage and physiological disturbance sustained by the host during a trypanosome infection. Antigen-antibody reactions are known to
L. G. GOODWlN
805
be accompanied by the release of a variety of physiologically active substances such as kinin, histamine and "slow-reacting substance A", My colleagues and I have tried to assess the part played by such substances in protozoal disease, and I need not remind you that this interest is shared by the President and his team at Liverpool. MaFGRaIWH, DP-VAKUL and L~iTrm~ (1956) showed that a dramatic temporary recovery occurred when monkeys infected with P. knowlesi were injected with noradrenaline. A similar effect was produced by noradrenaline in puppies in a state of shock caused by Babesia canis infection (M~GP,AITH, GILLES and D~-VAKtrL, 1957). They concluded that the factors that initiated shock might be physiologically active soluble substances of a relatively simple nature derived from the parasite, or as a consequence of host-parasite reaction or tissue damage.
3
10-O
•
102-
o _1
10-
CHALLENGE
Befo •
infection,
ks after infection., after infection, after infection
FIG. 4. Haemagglutination titres of mice infected with T. brucei and given an injection of sheep erythrocytes. The titres were determined 1 week after the injection of sheep cells. There is a steady decrease in the ability of the mice to produce antibody. In 1960 Richards and I demonstrated the presence of pharmacologically active peptides of the kinin type in the urine of mice infected with B. rodhaini, T. brucei, P. berghd and other organisms (GOODWIN and R:CHARDS, 1960), and RICH~a~S (1965) showed that kinins were present in the blood of mice infected with T. brucei. Meanwhile, TELLAand M~Ge, AITH (1962) had shown that shock in monkeys with P. knowlesi infection was also related to the liberation of kinins. I(inins are extremely active shortchain peptides derived from a precursor, ldninogen, in the plasma protein by the action of an enzyme (kininogenase). They are destroyed very rapidly by other enzymes (kininases), and are therefore difficult to detect. T h e i r chemical structure is known, and one of their more important properties is their ability to increase the permeability of blood vessels. Kinin is thought to act as a local hormone in the normal salivary gland, and to mediate in the production of salivary secretion. It is also probably con-
806
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
cerned with the more chronic aspects of local inflammation and may be responsible for vascular changes and oedema after the immediate effects of the histamine response have faded. I have little doubt that the release of kinin, probably as a result of antigen-antibody reactions, plays a part in the pathogenesis of trypanosomiasis. BOP,~HaM (1968a) showed that in cattle infected experimentally with T. brucei, a massive release of kinin occurred just after the second peak in parasitaemia, a time at which the common trypanosome antibodies (precipitins and fluorescent antibodies) first appeared in the serum. During the infection the blood kininogen level fell, but when the trypanosomes were killed by an injection of diminazene aceturate (Berenil), the kininogen concentration immediately rose to a level of about twice the normal value. This suggests that the infection is accompanied by a massive turnover of kinin. A similar picture is seen in rabbits infected with T. brucei, a release of kinin occurring at about the 8th day of infection, shortly before obvious changes in the circulation can be detected in ear chambers. Kinin release has also been detected in human trypanosomiasis (Bo~H~I, 1970). Studies of the components of the kinin-releasing mechanism suggest that trypanosomes activate the precursor of the enzyme (kininogenase) that releases the peptide from the plasma protein (Bo~HAM, 1968b). Activation probably follows antigen-antibody reactions as a result of the absorption of Hageman' Factor on to the surface of the complex (Bomm~l and GOODWIN, 1969). In a study of P. knowlesi infection in monkeys, ONABANJOand .~IAJ~GRAITH(1969) found a significant increase of kallikrein (kininogenase) in the blood, resulting in a clinical condition resembling acute pancreatitis, the classical situation in which proteolytic enzymes are free in the circulating blood (SARD~.SAtand T H e , 1966). Histamine is another highly active substance with an effect on blood vessels, that is associated with the inflammatory process and is known to be liberated by antigenantibody reactions. It was present in increased amounts in the blood of Onabanjo and Maegralth's monkeys, and also in the blood of mice infected with T. brucei (RIc~uatos 1965). However, more recent extensive work by YATFS (1970) has failed to detect significant changes in histamine, or in histidine decarboxylase, the enzyme that forms it, in the blood or tissues of mice and rats infected with T. brucei. Whatever the explanation of this difference, it appears that histamine need not be greatly implicated in the pathogenesis of the disease. The effectiveness of noradrenaline in alleviating the condition of shock that accompanies protozoal infections suggests that catechol amine metabolism is likely to be disturbed. Failure of the adrenal medulla could be responsible for cardiovascular collapse and YATES(tO be published) has made a study of the catechol amine content of the adrenals, hearts and urine from animals infected with T. brucei. In rabbits with chronic infections there is an undoubted change in the metabolic pattern.
Connective tissue damage I have already emphasized that parasites of the T. brucei group multiply in the connective tissue. It is surprising how much damage they do. The bundles of collagen fibres are disruptedman observation made Iongago by WOLBACHand BINGER(1912)m and the fibroblasts are destroyed. The damage is especially severe in the perivascular connective tissue. The vessels of a normal rabbit are not particularly robust structures, but in an animal infected with T. brucei the walls of the veins have the consistency of wet blotting paper; it is surprising that they hold together. Electronmicrographs show that much of the connective tissue is reduced to a nondescript necrotic pulp (GoODWlN,
L. G. GOODWlN
807
in press). The fibres of voluntary muscle also suffer; the mitochondria are degenerate, the fibrils are disrupted and wasted. Muscular wasting is characteristic of all forms of chronic trypanosomiasis. The apparent disintegration of the connective tissue collagen suggested to us that we might be able to obtain a quantitative estimate of the damage by determining the output of hydroxyproline in the urine of infected rabbits. Hydroxyproline is an aminoacid that forms one of the essential building blocks of collagen and little is found elsewhere. The result of one of these studies, carried out by Mr. M. W. GuY, is shown in Fig. 5. Fluid intake and output fell as the disease advanced, and towards the end the
ml
mg"2
1
FIG. 5. Daily fluid balance, hydroxyproline output in the urine and parasitaemia in a rabbit infected with T. brucei. Towards the end of the infection, fluid output exceeded the intake, but at no time was there an increase in hydroxyproline output. Innoculation of trypanosomes at arrow.
808
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
animal went into negative balance. The 24-hour output of hydroxyproline was, however, virtually constant; it certainly did not increase. More work is needed before the effect of the parasite on the structure of connective tissue can be explained. The presence in the tissue spaces of phagocytes that have become overblown and have perished after engulfing and digesting trypanosomes must contribute powerful proteolytic enzymes to the tissue fluid. If the tissues are also short of oxygen as a result of vascular damage, the local pH is likely to fall and free lysosomal enzymes that function in an acid medium may cause further destruction of host cells. The suggestion of SEFDand G ~ (1967) that destruction of host tissues by T. gambiense in rabbits leads to the development ofanto-antibodies also deserves consideration. However, the immediate cessation of the pathological process and the rapid repair that occurs in ear chambers of rabbits cured by trypanocidal drugs, suggests that auto-immunity is unlikely to play an important role in pathogenesis in this species (GOODWlN and Guy, unpublished). In human sleeping sickness, tissue destruction also occurs in the brain and meninges. The capillaries of the central nervous system are free from the connective tissue matrix that surrounds them in other organs; instead, they are invested by the interlocking lamellar processes of the astrocytes (WOLFF, 1963). A great deal has been learned about the astroglia in recent years, although its functions--mechanical, transporting or isolating --are still obscure (WOLFF,1970). It would well repay re-examination in cerebral trypanosomiasis, where there is a great increase in numbers of glial cells throughout the central nervous system, and the cerebral perivascular spaces are 'cuffed' with mononuclear cells. The site of origin of the inflammatory mononuclear cells has always been a mystery. None appear to be in mitosis and there is some evidence that, in rabbits, they may migrate from the spleen, bone marrow and lymph nodes (GoODWlN and GuY, 1970).
Blood changes in trypanosomiasis Much also remains to be learned of the causes of changes in the composition of the blood. Trypanosomiasis usually produces a moderate degree of anaemia, and B o ~ I ~ M (1968c) has shown that, in the rabbit, there is a brisk and well sustained reticulocytosis. There is no haemoglobinuria and no change in the fragility of the erythrocytes, although these have a curious crenated appearance in drawn blood, and the spleen and liver contain deposits of haemosiderin. Erythrocyte enzyme estimations, carried out for us by Dr. C. W. PARR, merely showed evidence of a population of young erythrocytes. Determinations of clotting factors and fibrinolytic activity in the plasma of infected rabbits, carried out by Dr. C. M. HAwr,~v, indicated that in advanced infections the calcification time of the blood was prolonged and the fibrinogen content increased. These are signs of diffuse intravascular coagulation, which is not surprising in view of our observations in the ear chambers. FIENNES (1946) in a study of the pathology of T. congolense infections in cattle also reported the occurrence of thrombosis in small vessels.
The trypanosome Thus far, we have given little consideration to the trypanosome itself. We know that it consumes an enormous amount of glucose, and that heavy parasitaemia must constitute a load on the host's carbohydrate metabolism. Voo~r-~Is (1969) thinks that heavily parasitaemic rats develop a condition akin to diabetes, although the blood sugar remains low because of the metabolic activity of the parasites. However, as
L. G. C,OODWlN
809
VON BRAND (1966) points out, the organisms in the vertebrate host do not complete the breakdown of glucose, but return it to the blood as pyruvate which the host can use, so it is not all lost. It is difficult to estimate what effect parasites in the tissue spaces may have on the economy of the host. The withdrawal of carbohydrate and other nutrients from the tissue fluid may well have a deleterious effect on the cells of the host that need them, and it does not seem justifiable to assume with VON BRAND (1966) that because the parasitaemia in the rabbit is never very intense, the influence of the organisms in this respect is negligible. A great deal of work has been done, with little success, in a search for a toxin or toxic metabolite produced by, or contained in, trypanosomes. KAWAMITSU (1958) injected emulsions of heat-killed T. gamtn'ense into rats and observed hypoglycaemia and a lowering of the liver glycogen reserves 3-5 hours later. This did not occur with emulsions of T. lewisi. More recently, SEED (1969) prepared a protein fraction from homogenized T. gambiense that caused oedema and increased vascular permeability when injected intradermally into guinea-pigs or rabbits. He suggested that the fraction might be related to trypanosome exoantigen (WFITZ, 1960). It could also be the substance that activates kininogenase and causes liberation of kinin (BoRErIAM, 1968b). So far, there has been no satisfactory explanation of the disruptive effect of trypanosomes on the structure of connective tissue. HORNBY(1949) regarded the parasite as a malevolent gimlet that bored its way into the tissues: "T. brucei injures the walls of capillaries, causing intravascular clotting and perivascular oedema. It passes through the injured walls and multiplies freely in the oedema. Wherever it goes it does damage, and one lesion accentuates another". Mechanical damage as a result of the motility of the organism would seem to be unlikely. Tissue cells must be quite accustomed to the constant movement between them of migrating leucocytes and wandering macrophages. But the surface of the organism may well play an important part in determining whether the host dies rapidly, dies after a chronic illness, survives in a state of premunition, or recovers completely. BROOM, BROWN and HOARE (1936) made the observation that T. brucei trypanosomes carry a negative electric surface charge. This property has been brilliantly exploited by Sheila Lanharn at the Lister Institute, who has shown that by the use of suitable synthetic anion-exchange columns, trypanosomes can be separated cleanly not only from erythrocytes, leucocytes and platelets but from other species of trypanosome, by virtue of the degree of negative charge they carry (LANHaM, 1968a, 1968b). This elegant technique has proved a most valuable tool for trypanosomiasis research. GODFREY and TAYLOR (1969), also at the Lister Institute, studied the surface membranes of trypanosomes and the effect on them of cobra venom, which contains phospholipase A and a lyric factor. They showed that T. brucei has a trilaminar plasma membrane with a thick uniform external surface coat that is removed by the action of the venom. VIC~R~ N (1969) reported that the outer coat of T. rhodesiense was removed by proteolytic enzymes and was lost when the parasite entered the gut of the tsetse fly. The outer proteinaceous coat, among other functions, may be of importance in determining the antigenic identity of the organism (VICKERMANand LUCKINS,1969) and may perhaps assist it in escaping from the host's defence mechanisms. T. lewisi, which evokes a prompt and effective immune response from its host, does not possess a thick covering of protein. It is almost impossible to avoid jumping to the conclusion that, like schistosomes, pathogenic trypanosomes might put on jackets of host protein as a disguise. But this remains to be seen.
810
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
I have covered a great deal of ground very sketchily, and have made only passing references to the biochemistry of the parasite, and none at all to the interesting phenomenon of "natural immunity" shown by some hosts, or to the possibility of there being a further stage in the life history of T. brucei subgroup trypanosomes. Nonflagellate, latent bodies were described in the vertebrate host by FANTImM (1911) and he fought a long, losing battle for them with a fierce, missionary zeal. They are now again in the news, and it is hard to say what influence their resurrection may have on our picture of the pathology of trypanosomiasis. But~!when pathogenesis occurs, it seems likely that allergic reactions to successive antigenic variants are at the root of the trouble. There is no lack of mobilization of the host's defence mechanisms. The tissues are packed with cells that, from their appearance and behaviour, should be competent to deal with all invaders, but the defences seem to lack direction and control. [_The polymorphs take peculiarly little interest in the cataclysms that occur in the tissues.] Mononuclears swarm everywhere and their lysosomes snap, crackle and pop so that sober cellular citizens become casualties, important structural edifices crumble, and poisonous ingredients of the necrotic soup leak back into the circulation to play hell with the host's physiology. Vast quantities of immunogiobulin are produced by the host and most of this crippling effort is useless. A small antigenic variation is enough to confuse the intelligence services, the disguised invaders multiply and bring about a dismally inevitable series of ill-directed steam-hammer blows that end in the exhaustion of the host's reserves. It's all rather like a T o m and Jerry cartoon, with a monstrously inept cat pulling the place down in his efforts to pulverise a diminutive, agile and highly resourceful mouse. I n h i s recent review of Ronald Hare's book, The Birth of Penicillin, PtaI~ (1970) says that research workers: " . . . hold in their minds a mass of half-formulated impressions and ~incompletely remembered observations. Out of this untidy chaos, decisions on the probable relevance of the various factors finatly emerge and notes on these factors are made. But there is no infallible way to distinguish at the start the relevant from the irrelevant factors. This difficulty obtrudes itself increasingly the more novel the phenomenon and the younger the experimental discipline. Experience is therefore now a surer guide in physics than in biology." As Mach pointed out, it is experience rather than logic that leads us to conclude that the colour of the weights on a balance is irrelevant. The most difficult part of research is finding out how to do the experiments--that is to say, learning what to control, what to observe and what to measure. When this has been done, almost anyone can get useful results in the domain that has been pioneered. It is harder to see what should be controlled while experience is still being integrated into a testable point of view. This is what makes the beginnings of research look like 'sleepwalking' to non-scientists". I don't think Pirie would disagree if I added that it often looks suspiciously like sleepwalking to the research workers themselves--especially in the field of sleeping sickness. REFERENCES ANGEVIN,, D. M. (1959). In Modern Trends in Pathology. Ed. Collins, D. H. p.47. London: Butterworth. ASHCROFT,M. T., BURTT,E. & FAIRBAIRN,H. (1959). Ann. trop. Med. Parasit., 53, 147. BLAIR, D. M. (1939). Trans. R. Soc. trop. Med. Hyg., 32, 729. BORF.HAM,P. F. L. (1968a). Br. J. Pharmac. Chemother., 32, 493. (1968b). Ibid., 34, 598. (1968c). Thesis, University of London. (1970). Trans. R. Soc. trop. Med. Hyg., 64, 394. ~-& GOODWIN, L. G. (1969). Pharmacol. Res. Comm. 1, 144. -
-
L. G. GOODWlN
811
BROOM, J. C., BROWN,H. C. & HOARE,C. A. (1936). Trans. R. Soc. trop. Med. Hyg., 30, 87. BRUCE, D. (1897). Further Report on the Tsetse Fly Disease of Nagana in Zululand, Ubombo, 1896. London: Harrison. CLI~rrON, B. A., STAtmER, L. A., & PALCZtrK,N. C. (1969). Exp. Parasit., 25, 171. CORSON, J. F. (1935). ft. trop. Med. Hyg., 38, 9. (1936). Ibid., 39, 125. CtYNNING~M, M. P. (1968). E. Afr. agric, for. J., 33, 264. DUGGAN, A. J. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 439. DUKE, H. L. (1935). Parasitology, 27, 46. ELMASSXAN,M. & MmONE, E. (1903). Ann. Inst. Pasteur, 17, 241. FANTnAM, H. B. (1911). Ann. trop. Med. Paras#., 4, 111. FIENNES, R. N. T-W- (1946). ft. comp. Path., 56, 28. (1952). Vet. Rec., 64, 423. FREEMAN, T., SMITn~RS, S. R., TARGETT, G. A. T. & WALKER, P. J. (1970). J. infect. Dis., 121, 401. GODFREY, D. G. & TAYLOR, A. E. R. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 115. GOODWIN, L. G. Ibid., (in press). & Gtr% M. W. (1970). Ibid., 64, 470. & HOOK, S. V. M. (1968). Br. J. Pharmac. Chemother., 32, 505. & RmnA~s, W. H. G. (1960). Ibid., 15, 152. GRAY, A. R. (1962). Ann. trop. Med. Paras#., 56, 4. GREENWOOD,B. M., HERRICK,E. M. & VOLLER,A. (1970). Nature, Lond., 226, 266. HEISCH, R. B., McMArlON, J. P.& MANSoN-BAHR,P. E. C. (1958). Br. reed. ~., 2, 1203. HOARE, C. A. (1970). In The African Trypanosomiases, p. 51. Eds. Mulligan, H. W. & Potts, W. H. London: Allen & Unwin. HORNBY, H. E. (1949). Animal Trypanosomiasis in Eastern Africa. H.M. Stationery Office, London. JAFFE, J. J., VOORHEIS, H. P. & McCORMACK, J. J. JR. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 118. KAWAMITStl, K. (1958). Nagasaki Igakkai Zassi., 33, No. 11. Suppl., 22. LANI-IAM,S. (1968a). Trans. R. Soc. trop. Med. Hyg., 62, 4. (1968b). Nature, Lond., 218, 1273. LIVINGSTONE,D. (1861). A Popular Account of Missionary Travels and Researches in South Africa. London: Murray. McCULLOCH, B. (1967). Ann. trop. Med. Parasit., 61, 261. & ACHARD,P. L. (1956). Oryx, 8, 131. MAEGRAITH, B. G., DEVAKUL, K. & LEITHEAD, C. S. (1956). Trans. R. Soc. trop. Med. Hyg., 50, 311. GILLES, H. M. & DEVAKUL,K. (1957). Z. tropenmed. Parasit., 8, 485. MORAX, V. (1907). Ann. Inst. Pasteur., 21, 47. MULLIGAN, H. W. & POTTS, W. H. (1970). Eds. The African Trypanosomiases. London: Allen & Unwin. NEAL, R. A., GARlqI-IAM,P. C. C. & COHEN, S. (1969). Br. reed. Bull., 25, 194. NODENOT, L. (1958). Proc. VII Int. Sci. Comm. Tryp. Res. Brussels. 207. ONABANJO, A. O. & MAEGRAITH,B. G. (1969). PharmacoL Res. Comm., 1, 179. ONYANGO, R. J., VAN HOEVE, K. & DERAADT, P. (1966), Trans. R. Soc. trop. Med. ttyg., 60, 175. ORMEROD, W. E. (1961). Ibid., 55, 313. - (1970). In The African Trypanosomiases p. 587. Eds. Mulligan, H. W. & Ports, W. H. London: Allen & Unwin. PIRIE, N. W. (1970). Listener, 84, 154. RICI-IARDS,W. H. G. (1965). Br. ~. Pharmacol., 24, 124. RICKMAN, L. R. & ROBSON, J. (1970). Bull. Wld Hlth Org., 42, 650. Ross, R. & THOMSON, D. (1911). Ann. trop. Med. Parasit., 4, 395. SALAMAN,M. H. (1970). Proc. Roy. Soc. Med., 63, 11. , WEDDERBURN, N. & BRUCE-CHWATT,L. J. (1969). o7. gen. Microbiol., 59, 383. SARDESAI,V. i . & THAL, A. P. (1966). In Hypotensive Peptides, p. 463. Eds. Erdos, E. G., Back, N., Sicuteri, F. & Wilde, A. F. New York: Springer. SEED, J. R. (1969). Exp. Parasit., 26, 214. & GAM, A. A. (1967). ~. Parasit., 53, 946. TELLA, A. & MAEGRAITH,B. G. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 6.
812
THE PATHOLOGY OF AFRICAN TRYPANOSOMIASIS
K. (1969). ft. Cell. Science, 5, 163. & LUCKINS,A. G. (1969) Nature, Lond., 224, 1125. VOLLER, A. & WFLLER, C. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 418. VoN BRAND,T. (1966). In Biochemistry of Parasites. p. 160. New York & London: Academic Press. VOORHEIS, H. P. (1969). Trans. R. Soc. trop. Med. Hyg., 63, 122. WATSON, E. A. (1920). Dourine in Canada 1904-20. History, Research & Suppression. Dept. Agric. Health, Anita. Branch. Dom. Canada 1. WF.XTZ,B. (1960). ft. gen. Microbiol., 23, 589. (1962). In Drugs, Parasites & Hosts. p. 180. Eds. Goodwin, L. G. & Nimmo-Smith, R. H. London: Churchill. WADE, J. K. H. & FR~NCI-I,M. H. (1945). J. comp. Path., .%5,206. WILLETT, K. C. & GORDON,R. M. (1957). Ann. trop. Med. Parasit., 51, 471. WOLBACH, S. B. & BINGER, C. A. L. (1912). J. reed. Res., 27, 83. WOLFF, J. R. (1963). Z. Zellforsch., 60, 409. (1970). Triangle, 9, 153. YATES, D. B. (1970). Trans. R. Soc. trop. Med. Hyg., 64, 167. YORKE, W. (1911). Ann. trop. Med. Parasit., 4, 385. VICI~RMAN,