Morphological and taxonomic studies on mammalian trypanosomes

333 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. X X X I I . No. 3. N o v e m b e r , 1938.

COMMUNICATIONS. M O R P H O L O G I C A L AND T A X O N O M I C S T U D I E S M A M M A L I A N TRYPANOSOMES. V.~THE

DIAGNOSTIC

VALUE OF T H E

ON

KINETOPLAST.

BY

CECIL A. HOARE, D.Sc.

From the Wellcome Bureau of Scientific Research, London. Before proceeding with the substance of this paper a few remarks are necessary regarding the terminology employed. In referring to the prominent dark-staining structure at the base of the flagellum in trypanosomes I have at one time used the term parabasal, later changing to kinetonucleus, while the name ln'netoplast has been employed to denote the complex k~netonucleus (or parabasal) + blepharoplast (or basal granule). This vacillation on my part was dictated by the changing views regarding the nature of the structure in question. While the conception of the nuclear nature of this element (hence "kinetonucleus ") has been discarded long ago, considerable doubt has also been thrown on its interpretation as a parabasal body. It is now regarded as an organ of unknown origin, affinities and function, and should, accordingly, be described under a neutral, non-committal name, the most suitable one being undoubtedly kinetoplast, proposed by ALEXEIEFF (1917). Another advantage in the employment of ALEXEIEFF'S name is its conformity with blepharoplast (basal granule), the second element lying at the base of the flagellum, in close association with the kinetoplast. In some of my recent publications and in the present paper the term la'netoplast has accordingly been employed in its original sense, to denote the kinetonucleus alone (without the blepharoplast). It is well known that the kinetoplast of trypanosomes varies in size and shape and in the position it occupies in the body of these flagellates. Though these particulars are sometimes mentioned in text-books on protozoology and in special publications dealing with these parasites, the reference is usually casual and no attempt has ever been made to co-ordinate these characters or to consider their possible bearing upon the identification of the trypanosomes. It is true, some authors have drawn attention to one or two peculiarities of the kinetoplast in certain groups or species of trypanosomes ; but, in the absence of comparative data--and especially of numerical ones for other groups, such observations are mostly of little value. Exception must be made for some recent publications, by JACONO (1935, 1938), SWARTZWELDER(1938) and SCHWETZ(1938), in which the position of the kinetoplast is employed for the differentiation of groups, genera and species of

884

THE KINETOPLAST IN TRYPANOSOMES.

some mammalian trypanosomes. These publications, t h e last two of, -hiCh appeared after the manuscript of the present paper was ready for the press, will be discussed below. To anyone familiar with the various mammalian trypanosomes it is evident that the kinetoplastic characters alone afford no criteria for the diagnosis of species, and this is probably the reason why they have in most cases been left out of consideration. There can be no doubt, however, that the kinetoplast provides valuable supplementary characters for the differentiation o f certain groups, especially in circumstances where some doubt may arise regarding the identity of a trypanosome, as is frequently the case with poor films, made under field conditions or by inexperienced workers. In the present paper an attempt is made to bring together and to correlate the data concerning the features presented by the kinetoplast in various species of mammalian trypanosomes. Since this question has not been dealt with in a comprehensive manner in previous works on trypanosomes, and the gleaning of disconnected data scattered in the literature did not appear to be promising, I decided to study the question independently, from the material at my disposal. This consisted of a collection of blood films stained by one of the Romanowsky methods (Giemsa or Leishman) and comprising fourteen species of mammalian trypanosomes in which all the recognized groups (Lewisi, Evansi, Vivax, Congolense and Brucei) were represented. The characters of the kinetoplast examined were its shape, size and position in the body. As regards shape, the kinetoplast usually appears to be either rounded (circular, oval or elliptic in outline) or in the form of a rod. According to MINCHm (1909) : " With regard to the two forms of the kinetonucleus, it is possible that these may be simply two views of the same thing, a body having the form of a disc which, seen edgeways, would appear rod-shaped, and in surface view would present a circular contour." Recent cytological investigations of this organ seem to confirm this view (cf. LworF, A. & M., 1931), which provides a satisfactory explanation of the observed variation in the appearance of the kinetoplast. It should, therefore, be understood that in distinguishing between " rounded " and " rod "-shaped kinetoplasts all that is meant is that they are orientated in the body of the trypanosomes in such a way as to present this or that aspect to the observer. However, as will be shown below, this apparent " shape " (i.e., orientation) is characteristic of certain groups of trypanosomes. In some cases it was difficult to specify the elongated kinetoplasts on account of their small size : they were regarded as elliptic in outline if the sides appeared to be convex, and as rod-shaped if these were straight. The measurement of the size of the kinetoplast is beset with great difficulties, especially in the case of preparations stained by the Romanowsky methods, for it is well known that these stains tend to deposit around the structures they colour, with the result that these appear much larger than their natural size

CECIL A. HOARE.

~85

(cf. MINCHIN, 1909). Moreover, the degree of enlargement also differs according to the conditions under which the slides were made. In the case of the blood films at my disposal it was impossible to ensure absolutely identical Conditions of preparation, since a number were received from workers in different parts of the world, at different periods, some stained on the spot and others on arrival, while many of the films were made by me from strains maintained at this laboratory. However, a comparison of various strains representing the same species of trypanosomes, but of different provenance, showed no appreciable difference in the size of the kinetoplast. This is probably due to the fact that the films were made by workers who are not concerned with the finest cytological details and employ a more or less standardized technique, viz., films dried in the air, fixed for about 5 minutes in methyl or absolute alcohol (in the case of Giemsa's stain), stained for about 30 minutes, and washed in tap-water, without differentiation. This method was used for the films which I stained myself and was recommended to those workers who kindly supplied me with stained ones. In the case of some trypanosomes, strains of which are kept in this laboratory ( T. lewisi, T. cruzi, T. congolense, and members of the Brucei group) blood-films stained by Giemsa's method were compared with those treated by the " wet " method, viz., by fixing in Schaudinn's fluid and staining with Heidenhain's iron haematoxylin. In the " wet " preparations, in which the above-mentioned defects of the Romanowsky staining were eliminated, the relative dimensions of the respective kinetoplasts were found to tally with those in the trypanosomes stained by Giemsa's method. In view of what has been said above, it would seem that the results obtained by the measurement of the kinetoplasts in the dry-fixed preparations, though not exact, nevertheless provide data of relative value for comparing the dimensions of this organ in various trypanosomes. The actual measurement of the kinetoplasts was made by projecting the trypanosomes with the aid of a camera lucida and drawing a line (at × 2,500) corresponding to the longest dimension of the kinetoplast, the resulting figure, divided by 2,500, being the diameter of this organ. The diameter served for the calculation of the area ( = ~rr"~) of the kinetoplast, both measurements being based on the assumption that this organ is disc-shaped. AS regards the position occupied by the kinetoplast, it may lie at the posterior extremity, in which case it is said to be terminal (Figs. 5, 6) ; it may be a short distance in front of the posterior end, or subterminal (Fig. 10 to 12) in position ; or it is disposed nearer to the middle of the body, sometimes not far behind the nucleus (Fig. 1), and can then be called subcentral, for want of a better term. Sometimes, however, it is difficult to decide whether the localization should be termed subcentral or subterminal (Figs. 2, 3). A kinetoplast occupying one of these positions usually lies some distance from the edge of the body (Figs. 9, 10, 12), but in some trypanosomes it is situated at the very edge (Figs. 7, 8) : this position is referred to as marginal or lateral.

386

THE KINETOPLAST IN TRYPANOSOMES.

TABLE. DIFFERENTIAL

CHARACTERS

OF T H E

KINETOPLAST

IN M A M M A L I A N

TRYPANOSOMF.~.

Characters of Kinetoplast. Shape.

Size. Group.

Position.

Species. Rounded.

i

i

DiaOval or i[ meter. Area. ~Circularl Elliptic. ! R o d . !(% (#) (~2)

(%)I(%)

Subcentral.

(%)

i i

1.2 1.0 1.4

--

+

+

+

1.1 1.1

0.9 0.9

30-52 38-54

48-70 46-62

---

---

Congolensel T. congolense T. simiae

0.7 0-8

0.4 0.5

20-45 52

10-40 26

15-70 22

Brueei

L6~vist

Vivax

Evansi

T. vivax T. uniforme

1.1

T. brucei T. rhodesiense T. gambiense

o.6

0.3

25

0.6 0.6

0.3 0.3

ll

T. evansi T. equiperdum

0-7 0.6

0.4 0-3

17-40 25

5

I ;

i

(%)

(%)

r

10O 1OO

--

84

---

+

24-69 26-35

31-76 65-74

16-28 26-31

---

86-93 99

7-14 I

78-89 89

74 74 62

4 3 8

22 23 30

l

1OO 10O

i

i

Marginal. L

15 i 85 76 i 24 7 0 - 8 1 1 I 19-30 84 16

1-0

(%)

i

0.8 0.9 1.1 0.8 1.5

T. lewisi T~ theileri T. eruzi T. evotomys T. melophagium*

SubTerterminal.! minal.

7-12 i

75

--

89

--

95

--

48-76 75

---

I 91-99 100

8

1-9 --

I 1

27-47 39

[

* Only two specimens of this species were available.

The characters of the kinetoplast are set forth in the accompanying Table, the figures for the size (in ~) being based on the mean of ten measurements in each strain (except T. melophagium), while those referring to shape and position were calculated from the examination of one hundred individuals in each strain (of which several were available for most of the species). In drawing conclusions from the table it should be borne in mind that the majority of species can be readily differentiated by their general morphological characters. A notable exception is presented by the species of the Brucei group which are indistinguishable both from each other--and indeed can hardly be regarded as distinct species--and, under certain conditions, from members of the Evansi group.

3.q7

CECIL A. HOARE.

7

8

II

12

SIZE, SHAPE AND P O S I T I O N OF THE K I N E T O P L A S T I N M A M M A L I A N TRYPANOSOMES.

l.--Trypanosoma melophagium ; 2 . - - T . lewisi ; 3 . - - T . theileri ; 4 . - - T . cruzi ; 5 . - T. vivax ; 6 . - - T . uniforme ; 7 . - - T . con¢olense ; 8 . - - T . simiae ; 9, 1 0 . - - T . rhodesiense ; 11.--T. evansi ; 1 2 . - - T . equiperdum. (All the figures were traced from enlarged photo-micrographs and are represented at × 2,000.)

CECIL A. HOARE.

889"

However, sometimes individuals of one species may bear a superficial resemblance to those of another and their diagnosis may present some difficulty. This may occur in light infections, when the classification has to be based on isolated parasites, as well as in poor blood-films. In such cases the kinetoplast, which is the most conspicuous structure present, provides valuable supplementary characters for the identification of the trypanosomes. The differential diagnosis of the various trypanosome groups and species on the basis of the characters of the kinetoplast (as shown in the table) may be summarized as follows :-I.

Lewisi GROUP.

Kinetoplast large (1.0 to 1.4~): shape rounded; position subterminal or subcentral, never terminal. (1) T. lewisi (Fig. 2); kinetoplast typically elliptic in outline; position subterminal, far from posterior end of the body. (2) T. theileri (Fig. 3) : kinetoplast typically circular in outline ; position subterminal, far from posterior end of the body in all and marginal in majority. (3) T. cruzi (Fig. 4) : kinetoplast very large, typically irregularly circular in outline ; position subterminal, near the posterior end of the body. Owing to its size and position, it is in contact with both sides of the body. (4) T. evotomys (from vole): kinetoplast typically circular in outline; position subterminal, in a few marginal. (5) T. melophagium (Fig. 1) (ovine) : (available material too scanty for comparison: included in the table as an illustration of trypanosome with large kinetoplast in subcentral position). II.

Vivax GROUP.

(6) T. vivax (Fig. 5) and (7) T. uniforme (Fig. 6): distinguished from the remaining three groups of pathogenic trypanosomes by the large size of the kinetoplast (1.1/~) ; its shape is always rounded, more or less regularly circular or elliptic in outline; position terminal or subterminal, in a fair number also marginal. III.

Congolense GROUP.

(8) T. congolense (Fig. 7) and (9) T. simiae (Fig. 8) : kinetoplast of medium size (0.7 to 0"8t~) (considerably smaller than in the Vivax group, but slightly larger than in the Brucei group) ; shape may be rounded or rod-like ; position in great majority subterminal and marginal. When the arrangement is marginal, the rhizoplast of the flagellum frequently starts from the posterior part of the kinetoplast (Figs. 7, 8) and the latter, if elongated, usually lies more or less parallel to the long axis of the body (el. Brucei group).

,q~0

THE KINETOPLAST IN TRYPANOSOMES.

IV.

Brucei GROUP.

(10) T. brucei, (11) T. rhodesiense (Figs. 9, 10) and (12) T. gambiense: kinetoplast small (0.6/~), usually rod-shaped; position in the majority subterminal, and in a fair number marginal. In the latter case the kinetoplast, if elongated, usually lies transversely to the long axis of the body, while the rhizoplast in most cases starts from the anterior part of the kinetoplast (Fig. 9). The last two features may help to distinguish the " stumpy " trypanosomes of this group from T. eongolense (v. supra).

V. Evansi GROUP. (13) T. evansi (Fig. 11) and (14) T. equiperdum (Fig. 12) : as regards the kinetoplastic characters, the above members of this group do not differ in any respect from those of the Brucei group, but T. equinum (not shown in the table) differs from all other trypanosomes in the absence of the kinetoplast. DISCUSSION. We can now consider the views expressed in some recent publications regarding the diagnostic value of the kinetoplast and discuss their bearing upon the data obtained in the present paper. Judging from the literature and from inquiries received from time to time, it would seem that observers sometimes find it difficult to distinguish between T. eongolense and T. uniforme, and some authors have adduced the marginal position of the kinetoplast as a differential diagnostic character of T. congolense. Thus SCHWETZ (1938) states that all the trypanosomes of the Congolense group are characterized by the lateral (marginal) position of the kinetoplast, which distinguishes it from the Vivax group in which the kinetoplast is always terminal or subterminal, but never lateral. However, as can be seen from the accompanying table and from the illustrations of T. vivax and T. uniforme appearing in a preceding paper (HOARE and BROOM, 1938: Figs. 2, 8, 12) trypanosomes with a marginal kinetoplast are quite common in these species: in the present study such forms were found in up to 28 per cent. of T. vivax, and in up to 31 per cent. of T. uniforme. It is, therefore, evident that the position of the kinetoplast is not a reliable character and cannot per se serve to differentiate trypanosomes of the Congolense group from those of the Vivax group, though it is true that the marginal situation of the kinetoplast, occurring in about 90 per cent. of trypanosomes of the Congolense group, is one of the characteristic features of the latter. On the other hand, in the case of doubt, 7". uniforme can readily be distinguished from T. congolense by the large size of its kinetoplast. There has recently been an attempt, by JACONO (1935, 1938), to revive CHALMERS' classification of the trypanosomes on a modified basis, while SWARTZWELDER ( 1 9 ~ ) has given an exposition of JACONO'S classification as it

CECIL A. HOARE.

341

applies to mammalian trypanosomes, using the position of the kinetoplast for their separation into the two genera accepted by JACONO : (1) Trypanosoma (with T. rotatorium as type-species), in which it is situated near the nucleus (" non-terminal," according to SWARTZWELDrR'S definition, or subcentral, according to the definition adopted in the present paper), and (2) Castellanella, in which it lies near the posterior end of the body (terminal or subterminal). SWARTZWELDER points out that, in accordance with this scheme, the species belonging to the Lewisi group--which were characterized by HOAREand COOTELEN (1933) as having a non-terminal kinetoplastJshould be retained in the genus Trypanosoma, while those belonging to the other groups (Evansi, Vivax, Congolense and Brucei)--with a terminal or subterminal kinetoplast--should be transferred to Castellanella. From a personal discussion with Dr. J. C. SWARTZWELDERit appears that his suggestion regarding the separation of the Lewisi group from the other groups of mammalian trypanosomes was based mainly on the definition of the position of the kinetoplast in the first-named group as given in HOARE and COUTELEN'S scheme of classification, regarding which there has been some misunderstanding which stands in need of rectification. Though the terms were not explained in our key, the word " non-terminal " was actually employed to indicate that the kinetoplast was not terminal, but not necessarily " near the nucleus," as interpreted by SWARTZWELDER. As a matter of fact, from the data discussed in the present paper, it is evident that though the Lewisi group, as a whole, can be characterized by the absence of forms with a terminal kinetoplast, the actual position of this organ in the body of the trypanosome varies considerably in different species : it is almost terminal in T. cruzi (Fig. 4) ; relatively near the posterior end and far from the nucleus in T. lewisi and T. theileri (Figs. 2, 3) (as well as in many other mammalian trypanosomes of this group) ; and is near the nucleus (or " nonterminal " according to SWARTZWELOER'Sdefinition of this position) only in T. melophagium (Fig. 1) and in similar forms not dealt with here. It has also been shown that in the majority of the mammalian trypanosomes outside t h e Lewisi group the kinetoplast is likewise usually subterminal, in which respect they do not differ essentially from most of the trypanosomes in the Lewisi group (cf. Figs. 11, 12, with Figs. 2, 3 ; and Figs. 7 to 9, with Fig. 4), members of the Vivax group being the only mammalian trypanosomes in which the kinetoplast is typically (though not invariably) terminal. It is thus seen that in the majority of mammalian trypanosomes--irrespectivc of the group to which they b e l o n g J t h e kinetoplast is neither terminal nor subcentral (near the nucleus), but subterminal, varying only in the degree of approximation to the posterior end of the body. The subcentral (" nonterminal") position of the kinetoplast, which differentiates JACONO'S genus Trypanosoma from his Castellanella is found only in a few species of the Lewisi group (e.g., T. melophagium, T. ingens, T. perodictid) which are similar in this

84~

THE KINETOPLAST IN TRYPANOSOMES.

r~spect to trypanosomes of certain reptiles and amphibia (e.g., T. grayi, T. varani, T. primeti, T. mega : cf. HOARE, 1931). T h o u g h the position of the kinetoplast affords no grounds for separating the Lewisi group, as a whole, from the other groups, SWARTZWELDER'Ssuggestions regarding the extension of JACONO'S classification, if rigorously applied, would necessitate the removal from the Lewisi group of trypanosomes of the melophagium type, which conform to JACONO'S genus Trypanosoma. However, the fact that they possess all the other characteristics--morphological and biological---of the Lewisi group provides a strong argument for their retention in this group. This is not the place to go into the merits and demerits of JACONO'S classification on general grounds, but from the facts adduced above it is evident that it cannot be applied to the mammalian trypanosomes, the two main subdivisions of w h i c h - - t h e Lewisi group, on the one hand, and the other four groups, on the other---cannot be differentiated by the position of the kinetoplast. This being so, the presence among these trypanosomes of species in which the kinetoplast occupies the same situation as in T. rotatorium likewise precludes the adoption of the genus Castellanella for the mammalian trypanosomes. REFERENCES. ALEXEIEFF,A. (1917). Sur la fonction glycoplastique du kin&oplaste ( = kin&onucleus) chez les flagell6s. C . R . Soc. Biol. Paris. 80. 512. HOARE, C.A. (1931). Studies on Trypanosoma grayi. III. Life cycle in the tsetse-fly and in the crocodile. Parasitology. 23. 449. (1936). Morphological and taxonomic studies on mammalian trypanosomes. I. The method of reproduction in its bearing upon classification, with special reference to the Lewisi group. Ibid., 28, 98. --~ & BROOM, J . C . (1938). Morphological and taxonomic studies on mammalian trypanosomes. IV. Biometrical study of the relationship between Trypanosoma uniforme and T. vivax. Trans. R. Soc. trop. Med. Hyg., 31,517. --~ & COUTELEN,F. (1933). Essai de classification des trypanosomes des mammifbres et de l'homme bas6e sur leurs earact6res morphologiques et biologiques. Ann. Parasit. hum. comp., 11,196. J^coNo, I. (1935). Osservazioni sui tripanosomi e proposta di una nuova classifica. Ann. Med. nay. colon., 41 (1). (1938). A further contribution to the proposal of a new classification of trypanosomes. J. trop. Med. Hyg., 41, 53. LWOFF, A., & LWOFF, M. (1931). Recherches morphologiques sur Leptomonas ctenocephali Fanth. (Trypanosomide.) Remarques sur l'appareil parabasal. Bull. biol. 65, 170. MINCHIN, E. A. (1909). The structure of Trypanosoma lewisi in relation to microscopical technique. Quart. J. micr. Sci., 53,755. SCHWETZ,J. (1938). Quelques r6flexions et suggestions pour une future classification des trypanosomes pathog6nes de l'Afrique centrale. Ann. Parasit. hum. comp., 16, 265. SWARTZWELDER,J . C . (1938). Consideration of the classification of trypanosomes, with special reference to the classification of JACONO. y. trop. Med. Hyg., 41, 182.