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Note on a cattle trypanosomiasis of Portuguese East Africa
GI': N~; RAL
ARTICLES.
NOTE ON A CATTLE TRYPANOSOMIASIS OF PORTUGU ESE EAST AFRICA.
By WALTER JOWETT, F.R.C.V.S., D.V.H. (Liv.), Department of Agriculture, Cape Town. DURING the past few months-to be exact, during May and June last-an expedition was despatched to Portuguese East Africa with the object of making a general veterinary survey of the animal diseases prevalent in certain districts of that country, and especially to stu,dy cattle trypanosomiasis. I t is hardly necessary to state that a knowledge of these matters would be of considerable importance to those interested, both in the Portuguese territory and the neighbouring British colonies. The expedition, it should be mentioned, consisted of the following members: P. Conacher, Esq., M.R.C.V.S., Chief Veterinary Surgeon, Province of Mocambique; C. V./. Howard, Esq., B.A., F.E.S., Chief of the Entomological Section, Department of Agriculture, Province of Mocambique; and the present writer, who acted as veterinary bacteriologist to the expedition. The members of the expedition have already issued a preliminary report to the various Governments concerned in inaugurating the investigation. At present operations have been suspended until a season more suitable for the further study of trypanosomiasis. Information in regard to the particular biting fly or flies concerned in the transmission of the causal parasite (trypanosome) from animal to animal is particularly required, and it is proposed to re~;ume the investigation into this part of the sU,bject some time between November and March next, when biting flies should be more in evidence. In the present note it is intended merely to give an account of the general morphological features, together with some of the animal reactions, of the trypanosome to which reference has just been made. This part of the subject has been studied and worked up by the writer since his return to the Cape, the material for the research having been furnished by experimental animals which accompanied the expedition, and were infected (by inoculation of blood from cattle, the subjects of trypanosomiasis) at the different centres of the disease visited. The infected experimental animals were forwarded in the first instance to Dr Theiler's laboratory at Pretoria, and thence the same (or, if these had died meanwhile, others sub-inoculated from them) were ultimately despatched to the writer at the Rosebank Experimental Station, Cape Town. The regions visited, and at which the experimental animals were infected, were: (I) various stations on the Buzi River in the neigh. bourhood of Beira; and (2) certain localities in the coastal areas immediately and for some little distance to the south of the area just mentioned,-Masanzane,' Sofala, Mombana, etc., the last-mentioned, of course, being on the Sabe (or Save) River, and all in the territory of the Companhia de Mocambique.
GEN'ERAL ARTICLES.
Tlte Discils,' in Cattll!. In the field the disease was studied only in cattle. In the areas visited horses ",:ere scarce; mules and donkeys too are only occasionally met with. At certain stations goats (and, in one instance, mules) were found in close contact with infected cattle, but microscopical examination of the blood of these in-contact animals failed to demonstrate the presence of trypanosomata therein. Examinations were also made of the blood of dogs and other animals (buck) which it was thought might have had an opportunity of acquiring the disease, but only in the case of cattle was it possible to detect the presence of trypanosomata by the aid of tbe microscope alone. Inoculation experiments with the blood of. these in -contacts were not made for several reasons. In the first place the expedition was constantly on the move, and one had to consider the difficulties of transport; and, again, it was necessary to maintain the experimental animals under "fly-proof" conditions, not always an easy matter when constantly on the m0ve. One had, moreover, to keep a supply of animals in reserve for any transmission experiments which it might be necessary to institute, and it was desirable to obtain as many different strains of the trypanosome as possible for further laboratory investigation . . Since our return we have found that such animals-or, at any rate, goats, sheep, and dogs-may be readily infected with the disease experimentally, and it seems reasonable to assume that under natural conditions they may acquire the disease in the same way as cattle, and, further, may, together with other animals, serve as hosts and reservoirs of the virus from which it may be transmitted by the appropriate vector to cattle and to other susceptible subjects. The disease in cattle is apparently never very acute, pursuing in practically all cases a sub-acute or chronic course, the latter being the more common. The most noticeable symptoms are dulness and general malaise, an unthrifty appearance, and, in advanced cases, symptoms of an~emia and emaciation; these, when the disease is well established. are sometimes very marked. Added to these symptoms, or rather associated with them, is hyperthermia-intermittent or remittent fever. Some of the infected cattle which we examined were in quite good condition, and there was nothing in their general appearance to lead one to suspect that anything was amiss with them. Such cases, of course, were in the early stages of the malady. Later, one may say that emaciation, dulness, and general symptoms of an~mia are all more or less constant. CEdematous swellings, although carefully looked for, were never seen on any part of the body; neither were eye lesions, shedding of the hair. nor enlargement of any of the superficial lymph glands noticed in any of the animals exal,llined. Far advanced cases may be found in decubitus, and such mar decline to make any effort to rise, but the condition here is one of general marasmus; and paralysis, in the true sense of the term, was never seen in any of the infected cattle. Some of our experimentally
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253
infected rabbits and guinea-pigs, however, have shown paralysis of the hind extremities prior to death. The symptoms, then, are merely those of intermittent fever associated with amemia and emaciation. As a matter of fact, there is nothing characteristic in the clinical picture, and for diagnosis one has to rely on the result furnished by the microscopical examination of the blood. The trypanosome, the causal agent of the disease under consideration, is not constantly present in the blood of naturally infected animals. The parasites appear in the blood in crops, and alternating with these periods are others at which microscopical examination may fail to demonstrate their presence. The blood, of course, is infective on inoculation into susceptible subjects at either period. I n the course of our investigations we failed to notice any very close relationship between the body temperature and the number of parasites present. Suspects with a temperature of 104° or 105° were occasionally examined, in which it was impossible to demonstrate the presence of trypanosomes by microscopical means, whilst at other times the parasites were found quite easily in the blood of cattle whose rectal temperature was normal. Rarely are the parasites very numerously present in the blood of naturally infected cattle. Occasionally one found as many as one parasite to each microscopic field; but, on the other hand. in very many instances only from one to half-a-dozen parasites were found in an entire large blood film.
Post-mortem .dppcarances. Although many deaths had occurred at some of the stations prior to our visit, it so happened that none of the infected subjects succumbed to the disease in natural .course whilst we were at hand. A description of the post-mortem lesions is therefore based on the appearances presented by the few well - marked cases which we caused to be slaughtered. The following notes were taken at the autopsy of one such case:SubJect.-Red and white ox in fair condition. Diagnosis by microscopical examination of blood, four to five parasites present per field. No very well-marked symptoms; dull, listless, and not feeding welL Temperature 104°. HistoYJI.-Came on the station four months previously together with other cattle. Judging from the animal's appearance the disease had not been long existent, but had appeared since the advent of these cattle. Post-mortem Extllllillati07l.-Carcase fairly well nourished; no evidence of subcutaneous cedema or hcemorrhage. On opening the abdomen a small quantity of clear fluid was found in the peritoneal cavity. Spleen: there was distinct hypersplenia, the organ weighing 6t lbs. The consistence was firm, the colour mottled red, and the malpighian bodies were more prominent than is usual. The liver was swollen and congested, and there was distinct evidence of fatty changes. The gall bladder and the bile were normal in appearance. The stomach and intestines showed nothing unusual. Amphistomes, which appear to be common in the rumen in this district, were not found in the present instance. The kidneys were rather large, pale.
GENERAL ARTICLIcS.
and fatty. The lungs were healthy ill appearance. The pericardial sac contained about 3 or 4 ounces of clear yellow fluid. The heart muscle was rather pale, but the endocardium was free from h
JIOljJ/LOlogical Clwracters of the Cwsal Trypanosome. The present note is more particularly concerned with the morphology and animal reactions of the causal parasite, or, in other words, the form and appearance of the trypanosome, and the manner in which the various experimental animals of the laboratory react to the inoculation of material containing it. Before entering into a consideration of this part of the subject it may, perhaps, be advisable to mention the methods which one has utilised in the study of the parasite. Fresh blood preparations were examined under a cover-glass in the usual manner, the cover-glass being ringed with vaseline in some instances with the object of retarding evaporation. It is well recognised that preparations which have been allowed to dry, either before or after fixation and staining, present but a distorted picture of the true form of parasites such as the h
255 Staillirtg.-After fixation by the method first mentioned, the film was passed through grades of alcohol into distilled water, then stained in Giemsa's solution (or other modification of the Romanowsky method), washed, if necessary differentiated in orange-tannin, dehydrated rapidly with acetone, cleared in xylol, and mounted in cedarwood oil or in Canada balsam. A few good preparations were obtained in this way, but the weak point in this process lies in the fact that dehydration has to be effected with extreme care and rapidity, otherwise the decolorisation is carried too far. So markedly is this the case that one can never be quite certain that a given specimen wiJI turn out satisfactorilyat least that is the writer's experience. This is all the more unfortunate, since it is possible to obtain beautifully coloured specimens by this process. One can entirely eliminate these defects, of course, by drying the film after staining and washing it-either examining it in this condition under the oil immersion lens, or after the subsequent addition of balsam or oil and a cover-glass. But, unfortunately, the fact that such a preparation has been allowed to dry renders it untrustworthy for the minute study of any parasites which may be present in the preparation. True, wet fixation followed by drying of the stained film is preferable to allowing the film to dry before fixation and staining as weIl as afterwards; but the fact remains that for an exact study of parasites, such as those now under consideration, the blood film must not be allowed to dry from the time of its preparation until it is finally mounted under a cover-slip. (2) Staining after" wet fixation" in Flemming's strong solution (chromo - ace to - osmic acid) or in Schaudinn's fluid (sublimatealcohol). A trial was made of Giemsa's recently introduced wet film method (fixation in sublimate-alcohol, followed by immersion in a solution of iodine, bleaching in a solution of sodium thio-sulphate, staining, washing, dehydration in acetone, clearing in xylol, and mounting in Canada balsam). The writer has not found this method superior in any respect to the one already mentioned. On the other hand, however, very satisfactory results were obtained by the Heidenhain-Breinl process (fixation of the wet film in Flemming's strong solution, using iron alum as a mordant and to differentiate; h;:ematoxylin is used for the stain, alcohol for dehydration, xylol for clearing, and the specimen is then mounted in Canada balsam). A comparison of Romanowsky-coloured films with those stained by the Heidenhain-Breinl process is very instructive. The former method exaggerates very markedly the chromatin staining; for instance, the size of the nucleus (and the kineto-nucleus even more so) is invariably very much greater in the Romanowsky-stained preparations, no matter whether Giemsa's or any of the other modifications of this method have been used. In order to form an accurate idea of the size and relationship of the various details of an organism under examination it would seem inadvisable to trust to Romanowsky-stained specimens alone, but one should contrast and control specimens so stained with others stained by some other process, and the Heidenhain-Breinl method is doubtless as reliable and precise as any. FinalIy, one should compare the stained specimens with the living
GENIl:RAL ARTICLES.
parasite in fresh blood preparations; this, it is perhaps hardly necessary to state, is absolutely essential. In studying the trypanosome, which we shall now endeavour to describe, the foregoing methods have been employed, and the description of the parasite is based on the knowledge so obtained. MOlpholog)'.-Although one inclines to the belief that only one species of trypanosome was encountered in the infected cattle examined, it will be convenient to describe the forms seen under two separate headings : (I) Those seen in all the infected cattle; and (2) Forms encountered only in the case of one of the infected subjects. Group I., or the more commonly observed trypanosome : When examined in the living state this parasite is very active, but its movements, if one may apply the term, are always more or less localised. As one authority observes in respect of trypanosoma nanum (to which parasite the one now under consideration shows much resemblance), "the p8rasite seems to take a delight in butting and boring at the erythrocytes." At times it would almost seem to be entangled in the red cells surrounding it, and one might fancy that by its vigorous twisting, turning, and lashing movements the parasite was making an attempt to free itself. But even when in a field in which the blood cells are scanty the organism always remains in their vicinity. At times the trypanosome may be hidden under a group of erythrocytes, and one is then aware of its presence only by the violent agitation which it communicates to the cells in its neighbourhood. Occasionally it may leave one cell or group of cells and pass to another, but rarely does it move far from one place, and practically never does it move out of the field of vision. Viewed in fresh preparations, the body of the parasite is homogeneous, and the presence of an undulating membrane is not very evident. One can sometimes just manage to distinguish the kinetonucleus as a refractile granule near one extremity (the posterior). Otherwise the cytoplasm appears free from granular matter. Ft:red and Stained Specillle1ls. - The appearance presented by the organism after fixation and staining is illustrated in Figs. I and 2. The specimens from which these two figures have been drawn were prepared from two naturally infected cattle, each from It should be noted that these are composite a different estate. drawings. One never finds the trypanosomes so numerously present in the blood of naturally infected cattle as a hasty glance at these figures might lead one to suppose. Reference to the illustrations will show that the trypanosome is stumpy in shape, being widest at its posterior portion and tapering gradually to the other extremity. The hind end is, as a rule, bluntly angular or conical; only very occasionally are forms encountered with distinctly pointed hinder ends, and even in these the hind end is never sharply pointed, as is the case in other trypanosomestrypanosoma lewisi, for example. At the anterior extremity there is no free flagellum, but the protoplasm gradually tapers to the extremity of this structure. The cytoplasm is homogeneous, and in most cases quite free from
GENERAL ARTICLES.
FIG. I.
257
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FIG. 2.
259
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granules, In some instances (especially in animals in which the disease pursues a rapid course and in which the organisms, multiply freely, such as rats and mice) one may occasionally observe a few metabolic granules in that portion of the parasite anterior to the nucleus, but these are unusual in cattle, Vacuoles are not seen in well-fixed specimens, The nucleus is rather large, oval in some parasites (the majority), more rounded in others, It is situated about the centre of the organism, The blepharoplast, kineto-nucleus, or extra-nuclear centrosome, is terminal or sub-terminal. It is prominent (especially in Romanowsky-stained specimens), and, as a rule, placed laterally, i,e" quite close to the wall. Generally it is rounded in form, but in some parasites it is distinctly rod-shaped, One cannot distinguish a clear area surrounding the blepharoplast. Flagellum: From the blepharoplast starts the flagellum, and this, after bordering the'undulating membrane, ends at the anterior extremity of the parasite. There is no free portion. The undulating membrane is but little noticeable. In many cases it is straight and appears to be closely applied to the body of the parasite, but in others it is thrown into two or three folds. Forms in which the blepharoplast has divided, and also those in which there are two nuclei and flagella, are frequently seen. These forms are often slightly longer 'and broader than are those in a resting condition. One occasionally encounters parasites somewhat swollen, and in which, although the flagellum, nucleus, and blepharoplast may stain normally, the cytoplasm stains very faintly indeed. We look upon these merely as involution forms. Measurements (in micra) are given in the following table : Animal,
I
Stain,
Aver(/ge,
Remarks,
17'5
13'4
16 14 15'5
Spontaneously infected.
13'3 12'4 13'5.
" death. Day of
16
11'3
~inth
14
12
17'5
13'6
Day of death-acute and rapidly fatal infection. An unusual case. Twenty-eighth day of disease.
lJIinimu1fb, llIaxim"1n, , -~---
Ox
Lellgth I
Ox 2 Guinea-pig, Rat
Rat Sheep.
Goat Breadth Ox I OX2 Rat
Giemsa
" " " HeidenhainBreinl Giemsa
"
I
9'5 10 10'5 II
I I I I
I I
II
10
1I
I II I
I
"
" "
day of infection,
(at widest pa rt, and inlcluding undulating membrane). Spontaneously inI'S Giemsa 2'5 I'5 I fected. 1 2 1'5 " " 2"2 Ninth" day of disease, Heidenhain2'5 1'5 Breinl
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260
Group II.~" Long" and" short" forms seen together in one animal (which for convenience we will refer to as Ox 48). On examining the blood of an animal at one of the stations visited . we encountered a trypanosome differing in certain respects from that just described. Even in fresh preparations it was quite apparent that one was dealing here with two distinct forms of trypanosome. One could readily distinguish (I) short forms, which, like those already described, remained in the neighbourhood of the red corpuscles twisting, turning, and lashing, but never moving far from one place; and (2) forms considerably longer, passing across the microscopic field with a steady undulating movement and causing but little displacement of the red cells in their passage. The movement of these forms was not rapid; by moving the field from time to time one could easily keep any particular specimen under observation, and also make out its general shape. The blood, one may mention, was examined immediately after its withdrawal from the animal. or, rather, after a delay merely sufficient to enable one to walk a dozen yards or so, from the animal to the microscope. In stained specimens the two forms of parasites were even more readily distinguished. At first it seemed that the long form possessed a free flagellum, but after examining a number of examples we came to the conclusion that such was not the case, but that in every instance the protoplasm of the body was prolonged to the extremity of the flagellum. Fig. 3 illustrates the appearance presented by these two forms of trypanosome in stained films, and the following table gives the measurements (in micra):i
! I
Animal.
Lengtll. Ox 48. Ox 48. Breadtlt Ox 48. Ox 48.
i ,
, •
, I
Stain.
.Minimum. Maxi1mlm . At'e,·uge. "----
---
Giemsa "
20 II
24 15
2~
I
Remarks.
Long forms. Short forms.
-
I"'"'" .)
I
:(at w,ic1est pa rt, and in!clUding u ndulating membrane). I Glemsa 1'5 2 I '6 Long forms. 2 Short forms. 1 '5 1'5 I
I
"
In a rabbit, guinea-pig, and mouse inoculated with blood from this ox only the short form of trypanosome appeared. Rats, rabbits, and guinea-pigs sub-inoculated from the rabbit just mentioned have like,.. wise shown only the one (short) type. A heifer, goat, sheep, and rabbit inoculated with this strain are still alive. and so far only the short form of parasite has appeared in the blood of any of these animals. In the goat, however, at the twenty-eighth and twenty-ninth day of the experimental disease many forms measuring 17'5 I-' were seen; some (dividing parasites) in fact. attained a length of 18 I-' or slightly over this. But long forms of 20 to 24 1-', such as we saw in the naturally infected ox, have not yet been seen in any of the sub-inoculated animals. It may be that they will yet appear in one or more of the sub-inoculated animals at a later stage of infection.
GENERAL ARTICLES.
FIG. 3.
261
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In addition to the animals mentioned a dog has also been experimentally infected with this strain, but a sufficient interval has not yet elapsed to enable one to make any statement in regard to the subject. What is the significance of the two forms of parasite encountered in the spontaneously infected ox? Do they represent merely different forms of the one species of parasite, or is one dealing here with representatives of two distinct species, the long trypanosome being non-pathogenic for the experimental animals into which we have inoculated it? After due consideration one is inclined to vote against the last-mentioned proposition. The history of Ox 48 is rather against the theory of a double infection. On the particular station on which we found this animal the disease had not made its appearance until after the arrival of ten head of cattle some four months prior to our visit. To reach this station these ten animals travelled a considerable distance by road, and, presumably, became infected en route or in the locality from which they came. Ox 48 was a local animal. He had been on the station for eight years, and so he must have become infected from one or the other of the introduced cattle. But of the fifty-two head of cattle on the place, all the infected ones (including those which had recently arrived) excepting Ox 48 showed the presence only of the short form of trypanosome. Ox 48 was the only one in which the long form was encountered. Now if the cattle which were introduced to the station from without (and which brought the disease with them) had been reservoirs of more than one species of trypanosome surely they and some of the other cattle on the estate should have given evidence of this. vVhy Ox 48 should shO\\' both long and short forms, whilst in the remainder the parasite should assume only the short type, is not very clear. Perhaps it is only under certain conditions of environment, or at a certain stage of the infection, that the long forms make their appearance. Needless to say, the blood of the animals which have been experimentally infected with the strain from Ox 48 will be submitted to daily examination. It may be that conditions suitable for the appearance of the long forms will be shown in one or other of these at a later stage of the infection. It is hoped to furnish a further report at a later date; meanwhile, as already mentioned, one inclines to the belief that but one species of parasite was encountered in the cattle we examined, this approximating, as we shall presently endeavour to show, to the type trypanosoma dimorphon (sensu Laveran) more closely than to any of the other known species. A 1tiJ/lal Reactiolls. Six different strains of the parasite described under Group I. were brought back in rabbits, guinea-pigs, and mice. From these animals others have been sub-inoculated. Our work on this part of the subject is still incomplete, but the results, so far as we have progressed, are embodied in the table on opposite page. Experimental infection with the parasite gives rise to an acutely
(;ENERAL ARTICLES.
fatal disease in rats, mice, dogs, and guinea-pigs. In rabbits, as well as in cattle, goats, and sheep, the course is sub-acute or chronic.
Animal.
Source of Virus.
Duration of Disease to Result Period Date from of of First ApInfection. Incu/Jation. pearance of ParaBites in Blood.
Remarks.
- - - - - - - - I- - - - - - - 1 - - - - - - - - - - -
Rabbit Z " NO.7
"
Ox
"
No. I NO·3 NO·4
Rabbit
No.2b
"
(;uinea-pig No. I " . NO.2. " NO·5· No.2b No.6.
9 days 10
"
"
Rabbit
White Rat No. I No.2 NO·5 No. lb. NO.2b. NO·7
" Sheep Guinea-pig
Coat No.
I
No.2 Sheep No.2 NO·3 NO·4
Heifer NO.7.
" Guinea-pig Rat Rabbit Rat Rabbit Goat Guinea-pig Sheep
Rabbit
6
,. days
53
8
10
II
"
Rabbit
"
'I II
Rat
Dog
9 13 15
I
89 days I07 "
"
,.
Death Still alive
" " "
" Death Still alive
12
"
"
21
days "
Death
22
13
8 6
I~
12
"
"
"
"
~,
"
"
7 days
4 days
Death
7 clays
12 days 12 6 " 6 " 8 ".,
Death
25 days
Still alive
6
7 8 -1-
6
"
" "
10
37
8 days 10
7
"
" ~
25 days
1\
3
"
(, days:
21
1
I
S till ali ve Death Still alive
I 14 days
Parasites still present in blood. Do.
At autopsy ipleen weighed 30'5 grammes. Chloroformed when paralysed ill extrentis. At autopsy spleen weighed 25 grammes.
" " " "
"
7 days
Parasites still present in blood. Do.
I
Still alive
IJarasites still present in blood. Do. Parasites still present in blood. Parasites still present in blood. Parasites still pre-
_________--'-______~I ______ ~ ______ I. _____"___
s_en_t_in_b_lo_Od._
In the case of one sheep, as reference to the table will show, the malady assumed an acute and rapidly fatal course. This sheep,
GENERAL ARTICLES.
however, had been used previously for other experimental work, and an attack of heartwater, from which it had but recently recovered, may have lowered its resistance. In rats and mice, from the time of their first appearance in the blood the trypanosomes multiply steadily and progressively until death, when they may be quite numerous. Only in one instance in these animals has any departure from this course been observed. In this rat there was a distinct periodicity in the appearance of the parasites in the blood, these being more numerous on some days than on others; still, it was always all easy matter to demonstrate their presence by microscopic means alone. In experimentally infected guinea-pigs the trypanosomes arc as a rule constantly present in the blood from the time of their first appearance until the death of their host. I n these animals, however, one may observe oscillations in the number of parasites present in the blood stream, they being on some days more plentiful than on others. The parasites may be numerous at death; but, on the other hand, they may decrease in number in the blood shortly before that event-such trypanolytic crisis, in fact, would seem to .hasten death (due to the liberation of an intracellular toxin ?). In the case of infected guinea-pigs it is not unusual for the animal to succumb somewhat suddenly, and on post-mortem examination of such cases one encounters an enlarged and ruptured spleen with a sanguineous effusion into the peritoneal cavity. We have encountered spleens weighing 25 and 30 grammes even in small and medium sized guinea-pigs. In such spleens the malpighian bodies are invariably enlarged and unduly prominent. In rabbits the parasites make their appearance in the blood in crops, but it is very rare indeed to find them present in these animals in anything but scanty numbers. Alternating with days on which microscopic examination may reveal the presence of a few trypanosomes in the blood occur others in which it is impossible to demonstrate their presence by this means, but even during an apparently trypanosome-free period the blood is still infective when inoculated into susceptible subjects. In the one dog we have so far infected the disease assumed a rapid course. Trypanosomes were constantly present in blood films from the time of their first appearance until shortly before death. Just prior to the occurrence of this event the parasites had considerably diminished in numbers. Trypanolysis, here again, seemed to hasten death. Prior to death in this animal there was well-marked cedema of the skin on the face and limbs. During his last hours the dog lay semi-comatose. In sheep and goats under ordinary conditions the disease seems to run a sub-acute or even chronic course. So far, of course, we have only been able to study the disease in these animals in the early stage. During this period, at any rate, the parasites are always demonstrable on microscopical examination, but the numbers vary from day to day. They are never numerous, and seem to be rather plentiful at the commencement of the infection, tending to become more scanty later. Associated with the period of their decline in the blood stream we have observed, in the case of two goats, a well-marked leucocytosis, the mononuclears being markedly in
GENERAL ARTICLES.
excess. rln
The ldmtity of tlte Trypanosome. Truly, as Sir David Bruce observes, the classification of the trypanosomata is in a state of chaos, and one can attempt the identification of an organism such as the one we have endeavoured to describe only with much uncertainty. Following the custom of some modern writers, one may pass in review the various known species with the object of determining the one to which the parasite under consideration bears most re;;emblance. On morphological and other grounds one may rule out trypanosoma evansi, trypanosoma brucei, trypanosoma theileri, trypanosoma ingens, trypanosoma equinum, and trypanosoma equiperdum. Trypanosoma cazalboui and trypanosoma vivax may also be dismissed since they differ from the organism we have encountered, not only in morphological features but also in their animal reactions, and, if much attention can be paid to a factor so variable, in the degree of the motility they show in fresh preparations. The following species, however, cannot be passed over so lightly, since they each resemble the Beira trypanosome in some degree: trypanosoma dimorphon, trypanosoma congolense, trypanosoma nanum, and a trypanosome described by Theiler from the mouth of the Zambesi and the Chai-Chai districts of Portuguese East Africa. Taking these various organisms in the order in which we have named them, en passattt one may refer to trypanosoma pecaudi. It wiII be sufficient to state that in the case of this parasite two forms are encountered in the blood of infected animals: (a) long and thin forms similar to trypanosoma evansi and possessing a long free flagellum, and (b) short thick forms without free flagellum. The long and short forms persist in the blood of animals infected with this parasite, and are always observed conjointly. Laveran and Pecaud never succeeded in separating the two forms. We are more concerned with the species which follow. I. Trypanosoma Dimorpllolt.-Dutton and Todd, who discovered this parasite ill the Gambia, described three forms of the parasite: (a) tadpole forms with no free flagellum, (b) stumpy forms similar to the last but thicker, and (c) long forms possessing a long free
GENERAL ARTICLES.
flagellum. 'bbservers ill Europe who have subsequently worked with the strain brought back to Liverpool by Dutton and Todd have failed to distinguish the three forms which these two investigators described. Laveran and Mesnil have been able to distinguish only two forms of the parasite in the strain supplied to them from Liverpool, namely, (a) long forms resembling trypanosoma evansi or trypanosoma brucei, differing from the latter only in" that the anterior part of the body is more slender, the protoplasm being continued along the flagellum, almost, if not quite, to the end; and (/I) short forms also without free flagellum, these corresponding to Dutton and Todd's tadpole forms. Regarding the strain of trypanosoma dimorphon at the Liverpool School of Tropical Medicine, Thomas and Brienl make the following statement: "In no instance has the long form described by Dutton and Todd, and possessing a long thin body and a free flagellum, been seen; in this we are in accord with Laveran and Mesnil." In the same connection Brienl, in a communication to, and quoted by, Sir David Bruce, remarks: "With regard to trypanosoma dimorphon you are aware that some remarkable change has occurred in the strain between the time Doctors Dutton anrl Todd brought It back from Africa and we started work on it here (Li\·erpool). \Vhereas Doctors Dutton and Todd describe the long flagellated forms with free flagella, Thomas and myself, and Laveran and Mesnil, could not see these forms with a thin body and long flagellum." The statement has since been made that the trypanosome which Dutton and Todd described did not come from the same animal as that which was the source of all the laboratory strains of trypanosoma dimorphon since worked with, and which have created so much discussion. Fig. 4 is a composite drawing, prepared by the writer from the blood of a rat infected with the Beira trypanosome, whilst Fig. 5 is likewise a composite drawing, but from a stained specimen of trypanosoma dimorphon from the RUl1corn Research Laboratories, Liverpool School of Tropical Medicine. Comparison of these two figures will show that, at any rate in so far as the morphological features are concerned, the two parasites closely resemble each other. 2. Trypanosoma Congo!ense.-Broden first drew attention to this parasite-a short trypanosoma measuring not more than 171', and resembling trypanosoma nanum and the short or tadpole forms of trypanosoma dimorphon; there is no free flagellum. These characters are retained in sub-inoculated animals. Broden, l~odhain, and Dutton and Todd incline to the "iew that trypanosoma congolense belongs to the same species as trypanosoma dimorphon. Laveran, on the other hand, considers that trypanosoma congolense constitutes a series separate and distinct from trypanosoma dimorphon. This investigator found that "a goat which had recovered from trypanosoma congoJense infection and seemed immune to that parasite succumbed to a subsequent inoculation with trypanosoma dimorphon." "But," says one writer in referring to this subject, "we do not follow him (Laveran) entirely in these views, as we consider neither slight variation in size, course, nor apparent immunity to be relied on."
GENERAL ARTI(:LES.
3. Trypanosoma iVa1l1t1J1 (the dwarf trypanosome).-Laveran described this parasite from the study of stained preparations as short, being IO to I4,u long and 1·5 to 2,u broad; there is no free flagellum.
FIt;. 4.
\Vith regard to its animal reactions, this parasite is without effect on inoculation into rabbits and rats. Dutton, Todd, and Kinghorn suggested that trypanosoma nanum
GENERAL ARTICLES.
'.
/.
IJ..
FIG. 5.
GENERAL ARTICLES.
is merely the short and tadpole form of trypanosoma dimorphon. Balfour and 'Wenyon do not accept this version. It is noteworthy that Wenyon, who has recently studied trypanosoma nanum in the Anglo-Egyptian Soudan, has met with the parasite in two forms: (1) short forms without free flagella, and resembling the tadpole forms of trypanosoma dimorphon; and (z) longer ones measuring about ZOP-, "of which 5 P- are taken up by a free flagellum." Intermediate forms between (1) and (z) were also seen. 4. Tlte Trypanos01lle .found ill Cattle mar tlte moutlt o.f tlte Za11lbesi and in tlte Cllai-Cllai districts o.f Portuguese East A.frica.- This trypanosome is short, resembling trypanosoma congolense, but when guinea-pigs were inoculated with blood containing this parasite it failed to infect them; and, further, animals of great susceptibility (rats) when injected with the blood of such guinea-pigs failed to become infected. Theiler's trypanosome is therefore apparently non-pathogenic for guinea-pigs. Through the courtesy of Dr Theiler, the writer was furnished some time ago with two rabbits infected with the Chai-Chai strain. From a study of the parasite in these animals, and in others sub-inoculated from them, it is evident that the Chai-Chai parasite bears a close morphological resemblance to the short form of trypanosoma we have encountered in the Beira cattle. The latter, however, has proved itself markedly pathogenic for guinea-pigs. In this respect, then, the two parasites differ. Moreover, in the Beira cattle a long form of trypanosoma was met with, at any rate in one instance, together with the short forms; whilst one understands that in the case of the Zambesi and Chai-Chai cattle only short forms of trypanosoma (resembling trypanosoma congolense) were seen, either in the spontaneously infected cattle or in the experimental animals which were inoculated from them. Do the Chai-Chai, Zambesi, or Beira cattle trypanosomes, then, constitute different species, or do they represent merely different strains of the one species, these different strains varying in their degree of virulence for c.ertain animals? At the present stage of the investigation one cannot express a decided opinion on this point. In so far as concerns the trypanosoma we have encountered in cattle in the Beira district, it may be that, as a result of further investigation, information may be gleaned which will either confirm or cause one to alter any opinion expressed at the present juncture; meanwhile it seems to the writer that the trypanosome now referred to resembles trypanosoma dimorphon (Se1ZSU Laveran) more closely than any of the other known species of the trypanosomata.
Transmitting Agent.-With regard to this part of the subject, one cannot do better than quote the following conclusions which were formulated in the preliminary report recently issued by the members of the expedition : 1. "In all cases outbreaks of the disease have occurred in a herd after the introduction of new members brought from a distance and which may (although there is no conclusive evidence on this point) have been subjected en route to the attack of one or other species of glossina."
GENERAL ARTICLES.
2. "In all cases it seems clear that, following the advent of the imported cattle, locally bred animals of mature age, which have never previously shown symptoms of illness, have contracted the disease in localities in which there is no evidence of the presence of glossince." 3. "Further research is needed to determine the species of biting insect capable of transmitting the parasite (trypanosome) from animal to animal."
Otlter PatllOgenic Proto::;oa encoulltered in tlte Beira District. The only other pathogenic protozoa encountered during the recent travels of the expedition were: piroplasma bigeminum in cattle and piroplasma canis in dogs. ADDITION .\LNOTE.
Since the foregoing article was written, and, indeed, whilst it was on the point of being despatched for publication, the writer has seen an abstract of a paper which Bruce, Hamerton, Bateman, and Mackie have recently issued. This paper has an important bearing on the subject we have considered in our present note, as the following brief extract will show:The authors state, "As far as our present knowledge goes, the morphology of trypanosoma dimorphon, trypanosoma congolense, the Uganda, Zanzibar, Chai-Chai, Zambesi, Zululand, and Rhodesian strains is identical; these trypanosomes affect the same important domestic animals; . . . these various forms should, for the present, be grouped under one name, and ... we propose the name trypanosoma pecorum for this group." Elsewhere they say, "The name dimorphon is a misleading one, and can only be accounted for by Dutton and Todd describing under one name two species of trypanosomes occurring in the same animal. . . . The term trypanosoma .dimorphon must therefore disappear, since it was born of a misapprehension." The authors conclude : I. "Trypanosoma pecorum is an important trypanosome disease of .domestic animals in Uganda." 2. "It is similar in morphology, action on animals, and cultural characters to the trypanosoma dimorphon described by Laveran and Mesnil, . . . except that trypanosoma pecorum is not pathogenic to guinea-pigs." 3. "The carrier is unknown, but is probably a tabanus, and not stomoxys." I have not yet had an opportunity to consult this important paper in the original. DESCRIPTION OF FIGURES. The accompanying drawings were made with a Leitz achromatic objective, ocular No. S, draw tube 170 millimetres. The magnification is approximately 1680 diameters. Each of the figures is a composite drawing from several different fields. Fig. I. From a naturally infected ox. Strain I. Fig. 2. From a naturally infected ox. Strain 2. One of the erythrocytes .contains two piroplasmata (piroplasma bigeminum).
GENERAL ARTICLES.
Fig. 3. From a naturally infected ox (Ox 48). This sketch shows both the long and short forms of trypanosoma encountered in this one animal. Fig. 4. From an experimentally infected rat shortly b~fore death. Strain 2. Fig. 5. Trypanosoma dimorphon. The preparation from which the drawing was nlade was obtained from the Runcorn Research Laboratories (Liverpool School of Tropical Medicine).
FEEDING EXPERIMENTS WITH THE F.lECES OF TUBERCULOUS COWS.
By A. M. TROTTER, l\I.R.C.V.S., Glasgow. NU:-"lEROUS investigations have been made to determine the presence of tubercle bacilli in the excreta and secretions of cows as a reliable means of diagnosis, but it is even more important to ascertain the manner in which the bacilli leave the body, because this furnishes scientific data upon which preventive measures may be based. Cases in which there is breaking down of the lesions and discharge of bacilli are designated open, those not marked by such conditions being known as closed. During the act of coughing, which is one of the symptoms of pulmonary tuberculosis, mucus and other material from the bronchi are ejected into the mouth, where it in great part remains until eventually swallowed. If the material contains tubercle bacilli these may become arrested and develop in some part of the alimentary tract, or they may be expelled along with the fceces. Schrceder and Cotton 1 found by microscopic examination that out of seven cows affected five were passing bacilli in that way, and when test animals were inoculated with the fceces of four of these three were found infective. Three cows had cultures of tubercle bacilli administered in their food, with the result that in their fceces. equally distributed throughout, were found bacilli which inoculation and feeding tests proved were those of tubercle. Taking the average daily weight of fceces per cow as 30 lbs., and estimating this sufficient to make 6,300,000 preparations showing an average of six bacilli each, these observers thus calculated the number of microscopically demonstrable tubercle bacilli voided by each cow daily as no less than 37,800,000. \vhich strikingly demonstrates the danger to other animals from this source of infection. Schrceder in a later publication 2 records the results of his experiments with thirty cows, twenty-four of which were removed directly from dairy herds, the other six being known to have been affected with tuberculosis for three years or more. Ten of the former and all the latter were found to be expelling tubercle bacilli. Many of these appeared in good health. The Royal Commission on Tuberculosis recently 3 published the results of feeding experiments with the fceces of two cows which on post-lIlortem examination were found extensively affected with tuberculosis. In both the disease had invaded, among other parts, the 1 Uniteu States Department of Agriculture. Bureau of Animal Industry, Bulletin No. 99 : ,. The danger from tubercle bacilli in the environment of tuberculous cattle." 2 United States Department of Agriculture, Bureau of Animal Industry, Circular 118, 1907 : "The Unsllspecte(l but Dangerously Tubercular Cow." 3 "Thirtl Interim Report of Royal Commission 011 Human and Bovine Tuberculosis," 1!l0!l.
ARTICLES.
NOTE ON A CATTLE TRYPANOSOMIASIS OF PORTUGU ESE EAST AFRICA.
By WALTER JOWETT, F.R.C.V.S., D.V.H. (Liv.), Department of Agriculture, Cape Town. DURING the past few months-to be exact, during May and June last-an expedition was despatched to Portuguese East Africa with the object of making a general veterinary survey of the animal diseases prevalent in certain districts of that country, and especially to stu,dy cattle trypanosomiasis. I t is hardly necessary to state that a knowledge of these matters would be of considerable importance to those interested, both in the Portuguese territory and the neighbouring British colonies. The expedition, it should be mentioned, consisted of the following members: P. Conacher, Esq., M.R.C.V.S., Chief Veterinary Surgeon, Province of Mocambique; C. V./. Howard, Esq., B.A., F.E.S., Chief of the Entomological Section, Department of Agriculture, Province of Mocambique; and the present writer, who acted as veterinary bacteriologist to the expedition. The members of the expedition have already issued a preliminary report to the various Governments concerned in inaugurating the investigation. At present operations have been suspended until a season more suitable for the further study of trypanosomiasis. Information in regard to the particular biting fly or flies concerned in the transmission of the causal parasite (trypanosome) from animal to animal is particularly required, and it is proposed to re~;ume the investigation into this part of the sU,bject some time between November and March next, when biting flies should be more in evidence. In the present note it is intended merely to give an account of the general morphological features, together with some of the animal reactions, of the trypanosome to which reference has just been made. This part of the subject has been studied and worked up by the writer since his return to the Cape, the material for the research having been furnished by experimental animals which accompanied the expedition, and were infected (by inoculation of blood from cattle, the subjects of trypanosomiasis) at the different centres of the disease visited. The infected experimental animals were forwarded in the first instance to Dr Theiler's laboratory at Pretoria, and thence the same (or, if these had died meanwhile, others sub-inoculated from them) were ultimately despatched to the writer at the Rosebank Experimental Station, Cape Town. The regions visited, and at which the experimental animals were infected, were: (I) various stations on the Buzi River in the neigh. bourhood of Beira; and (2) certain localities in the coastal areas immediately and for some little distance to the south of the area just mentioned,-Masanzane,' Sofala, Mombana, etc., the last-mentioned, of course, being on the Sabe (or Save) River, and all in the territory of the Companhia de Mocambique.
GEN'ERAL ARTICLES.
Tlte Discils,' in Cattll!. In the field the disease was studied only in cattle. In the areas visited horses ",:ere scarce; mules and donkeys too are only occasionally met with. At certain stations goats (and, in one instance, mules) were found in close contact with infected cattle, but microscopical examination of the blood of these in-contact animals failed to demonstrate the presence of trypanosomata therein. Examinations were also made of the blood of dogs and other animals (buck) which it was thought might have had an opportunity of acquiring the disease, but only in the case of cattle was it possible to detect the presence of trypanosomata by the aid of tbe microscope alone. Inoculation experiments with the blood of. these in -contacts were not made for several reasons. In the first place the expedition was constantly on the move, and one had to consider the difficulties of transport; and, again, it was necessary to maintain the experimental animals under "fly-proof" conditions, not always an easy matter when constantly on the m0ve. One had, moreover, to keep a supply of animals in reserve for any transmission experiments which it might be necessary to institute, and it was desirable to obtain as many different strains of the trypanosome as possible for further laboratory investigation . . Since our return we have found that such animals-or, at any rate, goats, sheep, and dogs-may be readily infected with the disease experimentally, and it seems reasonable to assume that under natural conditions they may acquire the disease in the same way as cattle, and, further, may, together with other animals, serve as hosts and reservoirs of the virus from which it may be transmitted by the appropriate vector to cattle and to other susceptible subjects. The disease in cattle is apparently never very acute, pursuing in practically all cases a sub-acute or chronic course, the latter being the more common. The most noticeable symptoms are dulness and general malaise, an unthrifty appearance, and, in advanced cases, symptoms of an~emia and emaciation; these, when the disease is well established. are sometimes very marked. Added to these symptoms, or rather associated with them, is hyperthermia-intermittent or remittent fever. Some of the infected cattle which we examined were in quite good condition, and there was nothing in their general appearance to lead one to suspect that anything was amiss with them. Such cases, of course, were in the early stages of the malady. Later, one may say that emaciation, dulness, and general symptoms of an~mia are all more or less constant. CEdematous swellings, although carefully looked for, were never seen on any part of the body; neither were eye lesions, shedding of the hair. nor enlargement of any of the superficial lymph glands noticed in any of the animals exal,llined. Far advanced cases may be found in decubitus, and such mar decline to make any effort to rise, but the condition here is one of general marasmus; and paralysis, in the true sense of the term, was never seen in any of the infected cattle. Some of our experimentally
GENERAL ARTICLES.
253
infected rabbits and guinea-pigs, however, have shown paralysis of the hind extremities prior to death. The symptoms, then, are merely those of intermittent fever associated with amemia and emaciation. As a matter of fact, there is nothing characteristic in the clinical picture, and for diagnosis one has to rely on the result furnished by the microscopical examination of the blood. The trypanosome, the causal agent of the disease under consideration, is not constantly present in the blood of naturally infected animals. The parasites appear in the blood in crops, and alternating with these periods are others at which microscopical examination may fail to demonstrate their presence. The blood, of course, is infective on inoculation into susceptible subjects at either period. I n the course of our investigations we failed to notice any very close relationship between the body temperature and the number of parasites present. Suspects with a temperature of 104° or 105° were occasionally examined, in which it was impossible to demonstrate the presence of trypanosomes by microscopical means, whilst at other times the parasites were found quite easily in the blood of cattle whose rectal temperature was normal. Rarely are the parasites very numerously present in the blood of naturally infected cattle. Occasionally one found as many as one parasite to each microscopic field; but, on the other hand. in very many instances only from one to half-a-dozen parasites were found in an entire large blood film.
Post-mortem .dppcarances. Although many deaths had occurred at some of the stations prior to our visit, it so happened that none of the infected subjects succumbed to the disease in natural .course whilst we were at hand. A description of the post-mortem lesions is therefore based on the appearances presented by the few well - marked cases which we caused to be slaughtered. The following notes were taken at the autopsy of one such case:SubJect.-Red and white ox in fair condition. Diagnosis by microscopical examination of blood, four to five parasites present per field. No very well-marked symptoms; dull, listless, and not feeding welL Temperature 104°. HistoYJI.-Came on the station four months previously together with other cattle. Judging from the animal's appearance the disease had not been long existent, but had appeared since the advent of these cattle. Post-mortem Extllllillati07l.-Carcase fairly well nourished; no evidence of subcutaneous cedema or hcemorrhage. On opening the abdomen a small quantity of clear fluid was found in the peritoneal cavity. Spleen: there was distinct hypersplenia, the organ weighing 6t lbs. The consistence was firm, the colour mottled red, and the malpighian bodies were more prominent than is usual. The liver was swollen and congested, and there was distinct evidence of fatty changes. The gall bladder and the bile were normal in appearance. The stomach and intestines showed nothing unusual. Amphistomes, which appear to be common in the rumen in this district, were not found in the present instance. The kidneys were rather large, pale.
GENERAL ARTICLIcS.
and fatty. The lungs were healthy ill appearance. The pericardial sac contained about 3 or 4 ounces of clear yellow fluid. The heart muscle was rather pale, but the endocardium was free from h
JIOljJ/LOlogical Clwracters of the Cwsal Trypanosome. The present note is more particularly concerned with the morphology and animal reactions of the causal parasite, or, in other words, the form and appearance of the trypanosome, and the manner in which the various experimental animals of the laboratory react to the inoculation of material containing it. Before entering into a consideration of this part of the subject it may, perhaps, be advisable to mention the methods which one has utilised in the study of the parasite. Fresh blood preparations were examined under a cover-glass in the usual manner, the cover-glass being ringed with vaseline in some instances with the object of retarding evaporation. It is well recognised that preparations which have been allowed to dry, either before or after fixation and staining, present but a distorted picture of the true form of parasites such as the h
255 Staillirtg.-After fixation by the method first mentioned, the film was passed through grades of alcohol into distilled water, then stained in Giemsa's solution (or other modification of the Romanowsky method), washed, if necessary differentiated in orange-tannin, dehydrated rapidly with acetone, cleared in xylol, and mounted in cedarwood oil or in Canada balsam. A few good preparations were obtained in this way, but the weak point in this process lies in the fact that dehydration has to be effected with extreme care and rapidity, otherwise the decolorisation is carried too far. So markedly is this the case that one can never be quite certain that a given specimen wiJI turn out satisfactorilyat least that is the writer's experience. This is all the more unfortunate, since it is possible to obtain beautifully coloured specimens by this process. One can entirely eliminate these defects, of course, by drying the film after staining and washing it-either examining it in this condition under the oil immersion lens, or after the subsequent addition of balsam or oil and a cover-glass. But, unfortunately, the fact that such a preparation has been allowed to dry renders it untrustworthy for the minute study of any parasites which may be present in the preparation. True, wet fixation followed by drying of the stained film is preferable to allowing the film to dry before fixation and staining as weIl as afterwards; but the fact remains that for an exact study of parasites, such as those now under consideration, the blood film must not be allowed to dry from the time of its preparation until it is finally mounted under a cover-slip. (2) Staining after" wet fixation" in Flemming's strong solution (chromo - ace to - osmic acid) or in Schaudinn's fluid (sublimatealcohol). A trial was made of Giemsa's recently introduced wet film method (fixation in sublimate-alcohol, followed by immersion in a solution of iodine, bleaching in a solution of sodium thio-sulphate, staining, washing, dehydration in acetone, clearing in xylol, and mounting in Canada balsam). The writer has not found this method superior in any respect to the one already mentioned. On the other hand, however, very satisfactory results were obtained by the Heidenhain-Breinl process (fixation of the wet film in Flemming's strong solution, using iron alum as a mordant and to differentiate; h;:ematoxylin is used for the stain, alcohol for dehydration, xylol for clearing, and the specimen is then mounted in Canada balsam). A comparison of Romanowsky-coloured films with those stained by the Heidenhain-Breinl process is very instructive. The former method exaggerates very markedly the chromatin staining; for instance, the size of the nucleus (and the kineto-nucleus even more so) is invariably very much greater in the Romanowsky-stained preparations, no matter whether Giemsa's or any of the other modifications of this method have been used. In order to form an accurate idea of the size and relationship of the various details of an organism under examination it would seem inadvisable to trust to Romanowsky-stained specimens alone, but one should contrast and control specimens so stained with others stained by some other process, and the Heidenhain-Breinl method is doubtless as reliable and precise as any. FinalIy, one should compare the stained specimens with the living
GENIl:RAL ARTICLES.
parasite in fresh blood preparations; this, it is perhaps hardly necessary to state, is absolutely essential. In studying the trypanosome, which we shall now endeavour to describe, the foregoing methods have been employed, and the description of the parasite is based on the knowledge so obtained. MOlpholog)'.-Although one inclines to the belief that only one species of trypanosome was encountered in the infected cattle examined, it will be convenient to describe the forms seen under two separate headings : (I) Those seen in all the infected cattle; and (2) Forms encountered only in the case of one of the infected subjects. Group I., or the more commonly observed trypanosome : When examined in the living state this parasite is very active, but its movements, if one may apply the term, are always more or less localised. As one authority observes in respect of trypanosoma nanum (to which parasite the one now under consideration shows much resemblance), "the p8rasite seems to take a delight in butting and boring at the erythrocytes." At times it would almost seem to be entangled in the red cells surrounding it, and one might fancy that by its vigorous twisting, turning, and lashing movements the parasite was making an attempt to free itself. But even when in a field in which the blood cells are scanty the organism always remains in their vicinity. At times the trypanosome may be hidden under a group of erythrocytes, and one is then aware of its presence only by the violent agitation which it communicates to the cells in its neighbourhood. Occasionally it may leave one cell or group of cells and pass to another, but rarely does it move far from one place, and practically never does it move out of the field of vision. Viewed in fresh preparations, the body of the parasite is homogeneous, and the presence of an undulating membrane is not very evident. One can sometimes just manage to distinguish the kinetonucleus as a refractile granule near one extremity (the posterior). Otherwise the cytoplasm appears free from granular matter. Ft:red and Stained Specillle1ls. - The appearance presented by the organism after fixation and staining is illustrated in Figs. I and 2. The specimens from which these two figures have been drawn were prepared from two naturally infected cattle, each from It should be noted that these are composite a different estate. drawings. One never finds the trypanosomes so numerously present in the blood of naturally infected cattle as a hasty glance at these figures might lead one to suppose. Reference to the illustrations will show that the trypanosome is stumpy in shape, being widest at its posterior portion and tapering gradually to the other extremity. The hind end is, as a rule, bluntly angular or conical; only very occasionally are forms encountered with distinctly pointed hinder ends, and even in these the hind end is never sharply pointed, as is the case in other trypanosomestrypanosoma lewisi, for example. At the anterior extremity there is no free flagellum, but the protoplasm gradually tapers to the extremity of this structure. The cytoplasm is homogeneous, and in most cases quite free from
GENERAL ARTICLES.
FIG. I.
257
GENERAL ARTICLES.
FIG. 2.
259
GENERAL ARTICLES,
granules, In some instances (especially in animals in which the disease pursues a rapid course and in which the organisms, multiply freely, such as rats and mice) one may occasionally observe a few metabolic granules in that portion of the parasite anterior to the nucleus, but these are unusual in cattle, Vacuoles are not seen in well-fixed specimens, The nucleus is rather large, oval in some parasites (the majority), more rounded in others, It is situated about the centre of the organism, The blepharoplast, kineto-nucleus, or extra-nuclear centrosome, is terminal or sub-terminal. It is prominent (especially in Romanowsky-stained specimens), and, as a rule, placed laterally, i,e" quite close to the wall. Generally it is rounded in form, but in some parasites it is distinctly rod-shaped, One cannot distinguish a clear area surrounding the blepharoplast. Flagellum: From the blepharoplast starts the flagellum, and this, after bordering the'undulating membrane, ends at the anterior extremity of the parasite. There is no free portion. The undulating membrane is but little noticeable. In many cases it is straight and appears to be closely applied to the body of the parasite, but in others it is thrown into two or three folds. Forms in which the blepharoplast has divided, and also those in which there are two nuclei and flagella, are frequently seen. These forms are often slightly longer 'and broader than are those in a resting condition. One occasionally encounters parasites somewhat swollen, and in which, although the flagellum, nucleus, and blepharoplast may stain normally, the cytoplasm stains very faintly indeed. We look upon these merely as involution forms. Measurements (in micra) are given in the following table : Animal,
I
Stain,
Aver(/ge,
Remarks,
17'5
13'4
16 14 15'5
Spontaneously infected.
13'3 12'4 13'5.
" death. Day of
16
11'3
~inth
14
12
17'5
13'6
Day of death-acute and rapidly fatal infection. An unusual case. Twenty-eighth day of disease.
lJIinimu1fb, llIaxim"1n, , -~---
Ox
Lellgth I
Ox 2 Guinea-pig, Rat
Rat Sheep.
Goat Breadth Ox I OX2 Rat
Giemsa
" " " HeidenhainBreinl Giemsa
"
I
9'5 10 10'5 II
I I I I
I I
II
10
1I
I II I
I
"
" "
day of infection,
(at widest pa rt, and inlcluding undulating membrane). Spontaneously inI'S Giemsa 2'5 I'5 I fected. 1 2 1'5 " " 2"2 Ninth" day of disease, Heidenhain2'5 1'5 Breinl
GENERAL ARTICLES.
260
Group II.~" Long" and" short" forms seen together in one animal (which for convenience we will refer to as Ox 48). On examining the blood of an animal at one of the stations visited . we encountered a trypanosome differing in certain respects from that just described. Even in fresh preparations it was quite apparent that one was dealing here with two distinct forms of trypanosome. One could readily distinguish (I) short forms, which, like those already described, remained in the neighbourhood of the red corpuscles twisting, turning, and lashing, but never moving far from one place; and (2) forms considerably longer, passing across the microscopic field with a steady undulating movement and causing but little displacement of the red cells in their passage. The movement of these forms was not rapid; by moving the field from time to time one could easily keep any particular specimen under observation, and also make out its general shape. The blood, one may mention, was examined immediately after its withdrawal from the animal. or, rather, after a delay merely sufficient to enable one to walk a dozen yards or so, from the animal to the microscope. In stained specimens the two forms of parasites were even more readily distinguished. At first it seemed that the long form possessed a free flagellum, but after examining a number of examples we came to the conclusion that such was not the case, but that in every instance the protoplasm of the body was prolonged to the extremity of the flagellum. Fig. 3 illustrates the appearance presented by these two forms of trypanosome in stained films, and the following table gives the measurements (in micra):i
! I
Animal.
Lengtll. Ox 48. Ox 48. Breadtlt Ox 48. Ox 48.
i ,
, •
, I
Stain.
.Minimum. Maxi1mlm . At'e,·uge. "----
---
Giemsa "
20 II
24 15
2~
I
Remarks.
Long forms. Short forms.
-
I"'"'" .)
I
:(at w,ic1est pa rt, and in!clUding u ndulating membrane). I Glemsa 1'5 2 I '6 Long forms. 2 Short forms. 1 '5 1'5 I
I
"
In a rabbit, guinea-pig, and mouse inoculated with blood from this ox only the short form of trypanosome appeared. Rats, rabbits, and guinea-pigs sub-inoculated from the rabbit just mentioned have like,.. wise shown only the one (short) type. A heifer, goat, sheep, and rabbit inoculated with this strain are still alive. and so far only the short form of parasite has appeared in the blood of any of these animals. In the goat, however, at the twenty-eighth and twenty-ninth day of the experimental disease many forms measuring 17'5 I-' were seen; some (dividing parasites) in fact. attained a length of 18 I-' or slightly over this. But long forms of 20 to 24 1-', such as we saw in the naturally infected ox, have not yet been seen in any of the sub-inoculated animals. It may be that they will yet appear in one or more of the sub-inoculated animals at a later stage of infection.
GENERAL ARTICLES.
FIG. 3.
261
GENERAL ARTICLES.
In addition to the animals mentioned a dog has also been experimentally infected with this strain, but a sufficient interval has not yet elapsed to enable one to make any statement in regard to the subject. What is the significance of the two forms of parasite encountered in the spontaneously infected ox? Do they represent merely different forms of the one species of parasite, or is one dealing here with representatives of two distinct species, the long trypanosome being non-pathogenic for the experimental animals into which we have inoculated it? After due consideration one is inclined to vote against the last-mentioned proposition. The history of Ox 48 is rather against the theory of a double infection. On the particular station on which we found this animal the disease had not made its appearance until after the arrival of ten head of cattle some four months prior to our visit. To reach this station these ten animals travelled a considerable distance by road, and, presumably, became infected en route or in the locality from which they came. Ox 48 was a local animal. He had been on the station for eight years, and so he must have become infected from one or the other of the introduced cattle. But of the fifty-two head of cattle on the place, all the infected ones (including those which had recently arrived) excepting Ox 48 showed the presence only of the short form of trypanosome. Ox 48 was the only one in which the long form was encountered. Now if the cattle which were introduced to the station from without (and which brought the disease with them) had been reservoirs of more than one species of trypanosome surely they and some of the other cattle on the estate should have given evidence of this. vVhy Ox 48 should shO\\' both long and short forms, whilst in the remainder the parasite should assume only the short type, is not very clear. Perhaps it is only under certain conditions of environment, or at a certain stage of the infection, that the long forms make their appearance. Needless to say, the blood of the animals which have been experimentally infected with the strain from Ox 48 will be submitted to daily examination. It may be that conditions suitable for the appearance of the long forms will be shown in one or other of these at a later stage of the infection. It is hoped to furnish a further report at a later date; meanwhile, as already mentioned, one inclines to the belief that but one species of parasite was encountered in the cattle we examined, this approximating, as we shall presently endeavour to show, to the type trypanosoma dimorphon (sensu Laveran) more closely than to any of the other known species. A 1tiJ/lal Reactiolls. Six different strains of the parasite described under Group I. were brought back in rabbits, guinea-pigs, and mice. From these animals others have been sub-inoculated. Our work on this part of the subject is still incomplete, but the results, so far as we have progressed, are embodied in the table on opposite page. Experimental infection with the parasite gives rise to an acutely
(;ENERAL ARTICLES.
fatal disease in rats, mice, dogs, and guinea-pigs. In rabbits, as well as in cattle, goats, and sheep, the course is sub-acute or chronic.
Animal.
Source of Virus.
Duration of Disease to Result Period Date from of of First ApInfection. Incu/Jation. pearance of ParaBites in Blood.
Remarks.
- - - - - - - - I- - - - - - - 1 - - - - - - - - - - -
Rabbit Z " NO.7
"
Ox
"
No. I NO·3 NO·4
Rabbit
No.2b
"
(;uinea-pig No. I " . NO.2. " NO·5· No.2b No.6.
9 days 10
"
"
Rabbit
White Rat No. I No.2 NO·5 No. lb. NO.2b. NO·7
" Sheep Guinea-pig
Coat No.
I
No.2 Sheep No.2 NO·3 NO·4
Heifer NO.7.
" Guinea-pig Rat Rabbit Rat Rabbit Goat Guinea-pig Sheep
Rabbit
6
,. days
53
8
10
II
"
Rabbit
"
'I II
Rat
Dog
9 13 15
I
89 days I07 "
"
,.
Death Still alive
" " "
" Death Still alive
12
"
"
21
days "
Death
22
13
8 6
I~
12
"
"
"
"
~,
"
"
7 days
4 days
Death
7 clays
12 days 12 6 " 6 " 8 ".,
Death
25 days
Still alive
6
7 8 -1-
6
"
" "
10
37
8 days 10
7
"
" ~
25 days
1\
3
"
(, days:
21
1
I
S till ali ve Death Still alive
I 14 days
Parasites still present in blood. Do.
At autopsy ipleen weighed 30'5 grammes. Chloroformed when paralysed ill extrentis. At autopsy spleen weighed 25 grammes.
" " " "
"
7 days
Parasites still present in blood. Do.
I
Still alive
IJarasites still present in blood. Do. Parasites still present in blood. Parasites still present in blood. Parasites still pre-
_________--'-______~I ______ ~ ______ I. _____"___
s_en_t_in_b_lo_Od._
In the case of one sheep, as reference to the table will show, the malady assumed an acute and rapidly fatal course. This sheep,
GENERAL ARTICLES.
however, had been used previously for other experimental work, and an attack of heartwater, from which it had but recently recovered, may have lowered its resistance. In rats and mice, from the time of their first appearance in the blood the trypanosomes multiply steadily and progressively until death, when they may be quite numerous. Only in one instance in these animals has any departure from this course been observed. In this rat there was a distinct periodicity in the appearance of the parasites in the blood, these being more numerous on some days than on others; still, it was always all easy matter to demonstrate their presence by microscopic means alone. In experimentally infected guinea-pigs the trypanosomes arc as a rule constantly present in the blood from the time of their first appearance until the death of their host. I n these animals, however, one may observe oscillations in the number of parasites present in the blood stream, they being on some days more plentiful than on others. The parasites may be numerous at death; but, on the other hand, they may decrease in number in the blood shortly before that event-such trypanolytic crisis, in fact, would seem to .hasten death (due to the liberation of an intracellular toxin ?). In the case of infected guinea-pigs it is not unusual for the animal to succumb somewhat suddenly, and on post-mortem examination of such cases one encounters an enlarged and ruptured spleen with a sanguineous effusion into the peritoneal cavity. We have encountered spleens weighing 25 and 30 grammes even in small and medium sized guinea-pigs. In such spleens the malpighian bodies are invariably enlarged and unduly prominent. In rabbits the parasites make their appearance in the blood in crops, but it is very rare indeed to find them present in these animals in anything but scanty numbers. Alternating with days on which microscopic examination may reveal the presence of a few trypanosomes in the blood occur others in which it is impossible to demonstrate their presence by this means, but even during an apparently trypanosome-free period the blood is still infective when inoculated into susceptible subjects. In the one dog we have so far infected the disease assumed a rapid course. Trypanosomes were constantly present in blood films from the time of their first appearance until shortly before death. Just prior to the occurrence of this event the parasites had considerably diminished in numbers. Trypanolysis, here again, seemed to hasten death. Prior to death in this animal there was well-marked cedema of the skin on the face and limbs. During his last hours the dog lay semi-comatose. In sheep and goats under ordinary conditions the disease seems to run a sub-acute or even chronic course. So far, of course, we have only been able to study the disease in these animals in the early stage. During this period, at any rate, the parasites are always demonstrable on microscopical examination, but the numbers vary from day to day. They are never numerous, and seem to be rather plentiful at the commencement of the infection, tending to become more scanty later. Associated with the period of their decline in the blood stream we have observed, in the case of two goats, a well-marked leucocytosis, the mononuclears being markedly in
GENERAL ARTICLES.
excess. rln
The ldmtity of tlte Trypanosome. Truly, as Sir David Bruce observes, the classification of the trypanosomata is in a state of chaos, and one can attempt the identification of an organism such as the one we have endeavoured to describe only with much uncertainty. Following the custom of some modern writers, one may pass in review the various known species with the object of determining the one to which the parasite under consideration bears most re;;emblance. On morphological and other grounds one may rule out trypanosoma evansi, trypanosoma brucei, trypanosoma theileri, trypanosoma ingens, trypanosoma equinum, and trypanosoma equiperdum. Trypanosoma cazalboui and trypanosoma vivax may also be dismissed since they differ from the organism we have encountered, not only in morphological features but also in their animal reactions, and, if much attention can be paid to a factor so variable, in the degree of the motility they show in fresh preparations. The following species, however, cannot be passed over so lightly, since they each resemble the Beira trypanosome in some degree: trypanosoma dimorphon, trypanosoma congolense, trypanosoma nanum, and a trypanosome described by Theiler from the mouth of the Zambesi and the Chai-Chai districts of Portuguese East Africa. Taking these various organisms in the order in which we have named them, en passattt one may refer to trypanosoma pecaudi. It wiII be sufficient to state that in the case of this parasite two forms are encountered in the blood of infected animals: (a) long and thin forms similar to trypanosoma evansi and possessing a long free flagellum, and (b) short thick forms without free flagellum. The long and short forms persist in the blood of animals infected with this parasite, and are always observed conjointly. Laveran and Pecaud never succeeded in separating the two forms. We are more concerned with the species which follow. I. Trypanosoma Dimorpllolt.-Dutton and Todd, who discovered this parasite ill the Gambia, described three forms of the parasite: (a) tadpole forms with no free flagellum, (b) stumpy forms similar to the last but thicker, and (c) long forms possessing a long free
GENERAL ARTICLES.
flagellum. 'bbservers ill Europe who have subsequently worked with the strain brought back to Liverpool by Dutton and Todd have failed to distinguish the three forms which these two investigators described. Laveran and Mesnil have been able to distinguish only two forms of the parasite in the strain supplied to them from Liverpool, namely, (a) long forms resembling trypanosoma evansi or trypanosoma brucei, differing from the latter only in" that the anterior part of the body is more slender, the protoplasm being continued along the flagellum, almost, if not quite, to the end; and (/I) short forms also without free flagellum, these corresponding to Dutton and Todd's tadpole forms. Regarding the strain of trypanosoma dimorphon at the Liverpool School of Tropical Medicine, Thomas and Brienl make the following statement: "In no instance has the long form described by Dutton and Todd, and possessing a long thin body and a free flagellum, been seen; in this we are in accord with Laveran and Mesnil." In the same connection Brienl, in a communication to, and quoted by, Sir David Bruce, remarks: "With regard to trypanosoma dimorphon you are aware that some remarkable change has occurred in the strain between the time Doctors Dutton anrl Todd brought It back from Africa and we started work on it here (Li\·erpool). \Vhereas Doctors Dutton and Todd describe the long flagellated forms with free flagella, Thomas and myself, and Laveran and Mesnil, could not see these forms with a thin body and long flagellum." The statement has since been made that the trypanosome which Dutton and Todd described did not come from the same animal as that which was the source of all the laboratory strains of trypanosoma dimorphon since worked with, and which have created so much discussion. Fig. 4 is a composite drawing, prepared by the writer from the blood of a rat infected with the Beira trypanosome, whilst Fig. 5 is likewise a composite drawing, but from a stained specimen of trypanosoma dimorphon from the RUl1corn Research Laboratories, Liverpool School of Tropical Medicine. Comparison of these two figures will show that, at any rate in so far as the morphological features are concerned, the two parasites closely resemble each other. 2. Trypanosoma Congo!ense.-Broden first drew attention to this parasite-a short trypanosoma measuring not more than 171', and resembling trypanosoma nanum and the short or tadpole forms of trypanosoma dimorphon; there is no free flagellum. These characters are retained in sub-inoculated animals. Broden, l~odhain, and Dutton and Todd incline to the "iew that trypanosoma congolense belongs to the same species as trypanosoma dimorphon. Laveran, on the other hand, considers that trypanosoma congolense constitutes a series separate and distinct from trypanosoma dimorphon. This investigator found that "a goat which had recovered from trypanosoma congoJense infection and seemed immune to that parasite succumbed to a subsequent inoculation with trypanosoma dimorphon." "But," says one writer in referring to this subject, "we do not follow him (Laveran) entirely in these views, as we consider neither slight variation in size, course, nor apparent immunity to be relied on."
GENERAL ARTI(:LES.
3. Trypanosoma iVa1l1t1J1 (the dwarf trypanosome).-Laveran described this parasite from the study of stained preparations as short, being IO to I4,u long and 1·5 to 2,u broad; there is no free flagellum.
FIt;. 4.
\Vith regard to its animal reactions, this parasite is without effect on inoculation into rabbits and rats. Dutton, Todd, and Kinghorn suggested that trypanosoma nanum
GENERAL ARTICLES.
'.
/.
IJ..
FIG. 5.
GENERAL ARTICLES.
is merely the short and tadpole form of trypanosoma dimorphon. Balfour and 'Wenyon do not accept this version. It is noteworthy that Wenyon, who has recently studied trypanosoma nanum in the Anglo-Egyptian Soudan, has met with the parasite in two forms: (1) short forms without free flagella, and resembling the tadpole forms of trypanosoma dimorphon; and (z) longer ones measuring about ZOP-, "of which 5 P- are taken up by a free flagellum." Intermediate forms between (1) and (z) were also seen. 4. Tlte Trypanos01lle .found ill Cattle mar tlte moutlt o.f tlte Za11lbesi and in tlte Cllai-Cllai districts o.f Portuguese East A.frica.- This trypanosome is short, resembling trypanosoma congolense, but when guinea-pigs were inoculated with blood containing this parasite it failed to infect them; and, further, animals of great susceptibility (rats) when injected with the blood of such guinea-pigs failed to become infected. Theiler's trypanosome is therefore apparently non-pathogenic for guinea-pigs. Through the courtesy of Dr Theiler, the writer was furnished some time ago with two rabbits infected with the Chai-Chai strain. From a study of the parasite in these animals, and in others sub-inoculated from them, it is evident that the Chai-Chai parasite bears a close morphological resemblance to the short form of trypanosoma we have encountered in the Beira cattle. The latter, however, has proved itself markedly pathogenic for guinea-pigs. In this respect, then, the two parasites differ. Moreover, in the Beira cattle a long form of trypanosoma was met with, at any rate in one instance, together with the short forms; whilst one understands that in the case of the Zambesi and Chai-Chai cattle only short forms of trypanosoma (resembling trypanosoma congolense) were seen, either in the spontaneously infected cattle or in the experimental animals which were inoculated from them. Do the Chai-Chai, Zambesi, or Beira cattle trypanosomes, then, constitute different species, or do they represent merely different strains of the one species, these different strains varying in their degree of virulence for c.ertain animals? At the present stage of the investigation one cannot express a decided opinion on this point. In so far as concerns the trypanosoma we have encountered in cattle in the Beira district, it may be that, as a result of further investigation, information may be gleaned which will either confirm or cause one to alter any opinion expressed at the present juncture; meanwhile it seems to the writer that the trypanosome now referred to resembles trypanosoma dimorphon (Se1ZSU Laveran) more closely than any of the other known species of the trypanosomata.
Transmitting Agent.-With regard to this part of the subject, one cannot do better than quote the following conclusions which were formulated in the preliminary report recently issued by the members of the expedition : 1. "In all cases outbreaks of the disease have occurred in a herd after the introduction of new members brought from a distance and which may (although there is no conclusive evidence on this point) have been subjected en route to the attack of one or other species of glossina."
GENERAL ARTICLES.
2. "In all cases it seems clear that, following the advent of the imported cattle, locally bred animals of mature age, which have never previously shown symptoms of illness, have contracted the disease in localities in which there is no evidence of the presence of glossince." 3. "Further research is needed to determine the species of biting insect capable of transmitting the parasite (trypanosome) from animal to animal."
Otlter PatllOgenic Proto::;oa encoulltered in tlte Beira District. The only other pathogenic protozoa encountered during the recent travels of the expedition were: piroplasma bigeminum in cattle and piroplasma canis in dogs. ADDITION .\LNOTE.
Since the foregoing article was written, and, indeed, whilst it was on the point of being despatched for publication, the writer has seen an abstract of a paper which Bruce, Hamerton, Bateman, and Mackie have recently issued. This paper has an important bearing on the subject we have considered in our present note, as the following brief extract will show:The authors state, "As far as our present knowledge goes, the morphology of trypanosoma dimorphon, trypanosoma congolense, the Uganda, Zanzibar, Chai-Chai, Zambesi, Zululand, and Rhodesian strains is identical; these trypanosomes affect the same important domestic animals; . . . these various forms should, for the present, be grouped under one name, and ... we propose the name trypanosoma pecorum for this group." Elsewhere they say, "The name dimorphon is a misleading one, and can only be accounted for by Dutton and Todd describing under one name two species of trypanosomes occurring in the same animal. . . . The term trypanosoma .dimorphon must therefore disappear, since it was born of a misapprehension." The authors conclude : I. "Trypanosoma pecorum is an important trypanosome disease of .domestic animals in Uganda." 2. "It is similar in morphology, action on animals, and cultural characters to the trypanosoma dimorphon described by Laveran and Mesnil, . . . except that trypanosoma pecorum is not pathogenic to guinea-pigs." 3. "The carrier is unknown, but is probably a tabanus, and not stomoxys." I have not yet had an opportunity to consult this important paper in the original. DESCRIPTION OF FIGURES. The accompanying drawings were made with a Leitz achromatic objective, ocular No. S, draw tube 170 millimetres. The magnification is approximately 1680 diameters. Each of the figures is a composite drawing from several different fields. Fig. I. From a naturally infected ox. Strain I. Fig. 2. From a naturally infected ox. Strain 2. One of the erythrocytes .contains two piroplasmata (piroplasma bigeminum).
GENERAL ARTICLES.
Fig. 3. From a naturally infected ox (Ox 48). This sketch shows both the long and short forms of trypanosoma encountered in this one animal. Fig. 4. From an experimentally infected rat shortly b~fore death. Strain 2. Fig. 5. Trypanosoma dimorphon. The preparation from which the drawing was nlade was obtained from the Runcorn Research Laboratories (Liverpool School of Tropical Medicine).
FEEDING EXPERIMENTS WITH THE F.lECES OF TUBERCULOUS COWS.
By A. M. TROTTER, l\I.R.C.V.S., Glasgow. NU:-"lEROUS investigations have been made to determine the presence of tubercle bacilli in the excreta and secretions of cows as a reliable means of diagnosis, but it is even more important to ascertain the manner in which the bacilli leave the body, because this furnishes scientific data upon which preventive measures may be based. Cases in which there is breaking down of the lesions and discharge of bacilli are designated open, those not marked by such conditions being known as closed. During the act of coughing, which is one of the symptoms of pulmonary tuberculosis, mucus and other material from the bronchi are ejected into the mouth, where it in great part remains until eventually swallowed. If the material contains tubercle bacilli these may become arrested and develop in some part of the alimentary tract, or they may be expelled along with the fceces. Schrceder and Cotton 1 found by microscopic examination that out of seven cows affected five were passing bacilli in that way, and when test animals were inoculated with the fceces of four of these three were found infective. Three cows had cultures of tubercle bacilli administered in their food, with the result that in their fceces. equally distributed throughout, were found bacilli which inoculation and feeding tests proved were those of tubercle. Taking the average daily weight of fceces per cow as 30 lbs., and estimating this sufficient to make 6,300,000 preparations showing an average of six bacilli each, these observers thus calculated the number of microscopically demonstrable tubercle bacilli voided by each cow daily as no less than 37,800,000. \vhich strikingly demonstrates the danger to other animals from this source of infection. Schrceder in a later publication 2 records the results of his experiments with thirty cows, twenty-four of which were removed directly from dairy herds, the other six being known to have been affected with tuberculosis for three years or more. Ten of the former and all the latter were found to be expelling tubercle bacilli. Many of these appeared in good health. The Royal Commission on Tuberculosis recently 3 published the results of feeding experiments with the fceces of two cows which on post-lIlortem examination were found extensively affected with tuberculosis. In both the disease had invaded, among other parts, the 1 Uniteu States Department of Agriculture. Bureau of Animal Industry, Bulletin No. 99 : ,. The danger from tubercle bacilli in the environment of tuberculous cattle." 2 United States Department of Agriculture, Bureau of Animal Industry, Circular 118, 1907 : "The Unsllspecte(l but Dangerously Tubercular Cow." 3 "Thirtl Interim Report of Royal Commission 011 Human and Bovine Tuberculosis," 1!l0!l.