With Plate 18, and 44 Text-figures.

Some time ago, while looking at a preparation of Piro-plasma bigeminum lent to me for examination, I was at once somewhat surprised to find that many of these very small parasites distinctly exhibited what has been called “nuclear dimorphism,” for quite typical and non-dividing forms showed the presence of more than one “nucleus” or chromatin mass. This feature of “nuclear dimorphism” is of groat interest at present in elucidating the probable close affinities of the Hæmosporidia and Hæmoflagellates.

As is well known, the micro-organism Piroplasma bigeminum (Babesia bovis) is the pathogenic agent of Texas Fever (Red-water) in cattle. The preparation examined and now described was a blood-smear, made immediately after the death of the Bovine, labelled “scraping from heart-muscle,” and came from Australia. There have been serious ravages among cattle in Queensland, due to Red-water. A few other preparations of the parasite were also examined. ‘

In this particular blood-film from heart-muscle about 90 per cent, of the red corpuscles were infected, one corpuscle usually containing two pyriform parasites, especially characteristic of this, the type-species of Piroplasma, and which led to the specific name bigeminum (Smith and Kilborne).

All members of the genus Piroplasma are small, usually occurring in pairs inside various mammalian erythrocytes, so that the diameter of the host-cells only averages 7 μ, and the parasite is considerably smaller, about 3 μ by 1·5 μ. The minute character of the parasite has been a source of great difficulty in the present research. Great care has been necessary with the illumination, and high magnifications have been used.

For the photo-micrographs, from which the figures in Plate 18 were drawn, I am greatly indebted to Dr. N. H. Alcock, Lecturer on Physiology at St. Mary’s Hospital Medical School, assisted by Mr. G. R. Lynch. To these gentlemen I would tender my best thanks for the trouble they have taken in photographing this exceedingly difficult object. Since it was found that the photo-micrographs, which clearly exhibited the “nuclear dimorphism” in question, were too delicate to sustain clear reproduction in the number of copies required for circulation, the photographs have been drawn (twice the size of the original) by the lithographic artist.

The blood-smear examined had been fixed and stained by the Romanowsky method. The resulting film was a thin, even, and good one, and was covered and mounted in Canada-balsam. The stain of the host-cells (red blood-corpuscles) was somewhat faint, which was afterwards found to be a distinct advantage, but the parasites were still perfectly well stained, and exhibited the red or purplish-red colour in the chromatin and blue in the cytoplasm characteristic of the Romanowsky coloration.

In order to eliminate, as far as possible, sources of error incidental to stained preparations—errors more especially applicable, however, to the shape and structure of the cytoplasm of the parasite—the slide was examined under various kinds of illumination. These, in brief, were—first, critical illumination, using as a source of light the sharp edge of a paraffin flame ; second, monochromatic light (green or yellowish-green was best) ; and thirdly, less critical illumination from a Welsbach burner or an electric lamp. The last mentioned should, on the whole, be rejected as somewhat untrustworthy and uncomfortable in a research of this kind, where so much depends on the correct determination of the colour of the chromatin masses. The monochromatic light is comfortable and useful for structural detail, but a source of white light, such as is obtained from a paraffin flame, is necessary for the determination of colour, especially of the looser chromatin. In all cases, however, the chromatin could be distinguished, either absolutely or relatively, from the cytoplasm of the parasite, but with very bright (white) sources of light, the relative sizes of the chromatin masses to each other were sometimes misleading, and there was, in such cases, a lack of finer detail. Wrong impressions were also apt to be obtained with the very powerful sources of light, concerning the size and condition of the vacuoles mentioned hereafter. Daylight was also used when possible (winter).

The objectives employed were Zeiss’ 2 mm. and 3 mm. apochromatics, aperture 1’40, and compensating oculars 8, 12, and 18, the various combinations of these all giving, in the main, precisely similar results, with little or no increase in detail, for a maximum amount of detail is obtained with compensating ocular 8 and 3 mm. apochromatic homogeneous immersion objective.

Lastly, it seems quite certain that relatively pale-stained preparations are much to be preferred to more deeply stained ones. Nuttall and Graham-Smith (8, p. 588) lay stress on the necessity of differentiation with methylated spirit after coloration with comparatively dilute solutions of Giemsa’s stain. It seems to me probable that, in the past, the frequent deep-blue staining, together with the more or less blue coloration of the enclosing blood-corpuscle, usually obtained by the various modifications of the Romanowsky method, using strong solutions, after one quarter to half an hour’s staining, has perhaps obscured the finer chromatic details and masked the looser chromatin in such small endoglobular forms as Piroplasmata, and has also hidden the finer structural details of the cytoplasm, vacuolated or otherwise.

The parasite of Texas fever was discovered by Smith and Kilborne, and carefully described by them in 1893 (10), though they saw the parasite in 1889 and apparently “noticed [it] in the spleen of a case as early as 1886” (10, p. 213). Their classical monograph is perhaps, nowadays, hardly consulted as muchas it still deserves to be, though the memoir is not always easy of access, unless one procures a copy direct from Washington. The chief stain used by the American investigators was the alkaline methylene blue of Löffler, and the presence of a nucleus in the parasite is not specifically mentioned by them. However, there can be little doubt that the nucleus, as described later by Laveran and Nicolle (3), was really seen by Smith and Kilborne, for on page 215 of their monograph they remark, regarding fresh specimens, that : “The smaller forms [of the parasite] are as a rule homogeneous ; the larger forms are very frequently observed to be provided, in the rounded end of the pyriform body, with a very minute spherical body probably not more than 0·1 to 0·2μ in diameter, which contrasts dark with the body itself …. In the largest pyriform bodies there was seen in the centre of the enlarged end a somewhat larger round or oval body which seemed to take the place of the smaller body or else be associated with it. This second body was from 0·5 to 1 μ. in diameter. It changed its appearance with the focus …. One or both of these bodies were observed in some of those forms undergoing amoeboid changes.” Judging by the latter part of this quotation Smith and Kilborne even may have seen “nuclear dimorphism” in parasites occurring “in fresh blood of the acute disease during life.”

Laveran and Nicolle, apparently, were the first definitely to describe and figure1 the nucleus of P. bigeminuin in 1899 (3), from stained specimens treated with Borrel blue and eosin, according to Laveran’s modification of the Romanowsky method, after fixation by heat and corrosive sublimate. In twin intra-corp oscular forms they found a spherical or oval karyosome at the blunt end of the parasite, measuring 0·7 μ to 0·9 μ. in diameter, with a clear zone surrounding the karyosome, which clear area they regarded as the peripheral part of the nucleus. Laveran and Nicolle also mention the frequent presence of numerous, easily stained granules at the pointed extremity of the parasite. These granules, they state, might easily be mistaken for a second karyosome in deeply stained specimens, but, on decolourising a little, are seen to be really only “agglomerations of granules.” Personally, I consider that at the pointed end of the parasite loose chromatin is not infrequently seen (see p. 303).

It should also be noted that Ziemann (12), in 1898, very briefly mentioned the occurrence of chromatin in P. bigeminum without figuring it.

Schaudinn (1904), in a brief note at the end of his remarkable memoir on Trypanosoma noctuæ and Spirochaeta ziemanni (9, p. 438), points out the presence of nuclear dimorphism in P. canis and P. bigeminum. Lühe (4, 5), in 1906, confirmed the observation as regards P. canis, and further considered the question. Schaudinn and Lühe described a large mass of chromatin, the “principal” nucleus, and a smaller, dense, punctiform mass of chromatin, which Schaudinn suggested was homologous with the blepharoplast of a Trypanosome, to which Lühe agrees.

Lastly, Nuttall and Graham-Smith (8), in October, 1906, also saw these two chromatin masses in P. canis, and, in addition, a third mass of loose chromatin, described by them for the first time.

My own observations on the chromatin of P. bigeminum, as seen more especially in blood from the heart-muscle, may now be set forth.

The observations to be described were always made on those parts of the preparation which appeared well fixed and properly stained, especially in so far as the parasite was concerned.

It may be stated at the outset that to give a general account of the distribution of the chromatin, which might be taken as the average or type, is difficult, as considerable variation was noticed. This variation has been already notified by Nuttall and Graham-Smith (8) in the case of the chromatin of P. canis, a species larger than P. bigeminum. It would seem best, then, to illustrate, by accurate diagrams, as many different forms as possible, at the same time noting their relative frequency of occurrence.

(α) The Chromatin in Pairs of Pyriform Intra-corpuscular Parasites

These were the most numerous forms observed, and often in each parasite two distinct masses of chromatin were seen, situated on the sides of the body of the micro-organism at about the middle of its length (text-figs. 1, 2,3). For convenience, this position may be termed “the equatorio-lateral”— a somewhat cumbrous term, perhaps, and not very correct geometrically—but the position of the chromatin masses in ovoid forms has also to be considered, and in such cases the term will be rather useful. Of these lateral chromatin masses one is nearly always larger than the other (text-figs. 1, 2, 3, etc.), though it cannot always be stated that the smaller chromatin mass is merely “punctiform,” as it often appears relatively larger than the idea of size conveyed to my mind by such a term, and may be surrounded by a somewhat deeply-staining area of cytoplasm. Sometimes the lateral chromatin masses are very nearly equal in size (text-fig. 13). Besides these lateral chromatin masses there is not infrequently a third rather loose mass of chromatin, more mesh-like or woolly in character, often situated at the pointed end or apex of the parasite in the midst of rather deeply-staining granulai’ cytoplasm (text-fig. 2). Laveran and Nicolle (3) mentioned the occurrence of numerous granules, easily stained, at the pointed end of the parasite, as we have already briefly noted. They did not consider that these granules formed a “second karyosome”—that is, that they were composed of chromatin, as at first sight appeared, because after slightly decolourising the preparation —which, they state, appeared too deeply stained at first—it could be seen that this apical mass consisted only of granules (“une agglomération de granulations”). It seems to me likely that, in decolourising the preparation a little, the characteristic colour-reaction of the chromatin became very faint or was lost, for among these granules of cytoplasm, situated at or near the pointed end of the parasite, I feel sure that there is also chromatin present, sometimes perhaps only loosely packed—at any rate in many cases (Pl. 18, figs. 1, 2, and 4 show position of this). Further, Koch depicts chromatin as occurring at the apex of P. bigeminum from Africa (1, taf. 1, figs. 1 to 4), and Nuttall and Graham-Smith figure masses of apical chromatin in P. canis (8, Diagram 1, figs. 3, 4, 14, 20; Diagram 3, figs. 4, 6, 8, 9, 13, 16, 19).

In a Trypanosoma, carefully stained by the Romanowsky method, as by Giemsa’s solution, the nucleus appears bright red and the blepharoplast violet-red in colour. In Piroplasma, if the two lateral chromatin masses, more or less unequal in size, be strictly homologous with the nucleus and blepharoplast1 of a Trypanosome, one would expect similar differences in staining reaction. In the specimens which have come under my observation it is difficult occasionally to distinguish in depth and colour of staining between the larger and smaller chromatin masses of this minute parasite, as sometimes the one, sometimes the other, may appear slightly more deeply stained, and a difference in tint, as regards violet, is exceedingly difficult to determine in this case. However some specimens certainly showed the blepharoplast more violet in tint after staining. Furthermore, Piroplasma is generally intra-corpuscular, and relatively not often free in mammalian blood, and is much smaller than any Trypanosome, while the exact method of staining and mounting the preparation, together with its age, are of the utmost importance in determining the finer staining reactions of chromatin.

As regards the structure of the chromatin masses, it is very difficult to make out any details in such small objects. The principal nucleus and blepharoplast appear compact, and separate granules of chromatin cannot be made out in them with any degree of certainty, though, as a rule, the nucleus is rather less dense than the blepharoplast. The loose chromatin varies somewhat in texture in different forms. It often consists of relatively coarse, chromatic granules, closely associated and. touching each other, yet loosely packed compared with the principal chromatin masses, and staining less deeply than these ; and this structure may be well seen under monochromatic light, after one has carefully determined the loose chromatin area under white light.

The foregoing description applies to the distribution and structure of the chromatin masses as seen in the most commonly-occurring parasites (about one-fifth of the whole).

Another fairly common form of distribution of the chromatin in pyriform parasites may be termed the “polar” distribution (text-figs. 5, 6, 7, and 8). In these forms there is a large nucleus near the blunt end of the parasite, and a small mass of chromatin—the so-called blepharoplast—at some distance from it, sometimes near the apical end (text-figs. 5, 6, 7). The position of the nucleus in these parasites corresponds with that often noted in the past before the blepharoplast was observed. Occasionally, however, the relative position of these two nuclear masses may be reversed (text-figs. 11 part,1 14 part), and the larger chromatin mass then appears near the pointed end.

The nucleus and so-called blepharoplast occasionally occur close together (text-figs. 10, 9 part, 18 part).

Forms in which the chromatin masses appear much alike, both as regard size and density, are sometimes found (text-figs. 13, 15).

One of the parasites represented in text-fig. 18 has much chromatin of varying density, along one side (cf. Koch [1; taf. 1, figs. 1, 2, 4]).

Other types, such as are drawn in text-figs. 9, 12, and 16, are rare, especially the latter, where the parasites possessed only a paucity of chromatin.

Smaller, and presumably younger, intra-corpuscular pairs of parasites usually exhibited two nuclei already differentiated; it was somewhat rare to find only one “nucleus” in small forms, and then the single chromatin mass was polar, as figured by Laveran and Nicolle (3).

Having noted the principal types of chromatin distribution in intra-corpuscular pyriform pairs, a few words are necessary regarding the inter-relation of the three chromatin masses already mentioned. There is sometimes a bridge of some-what loose chromatin connecting the nucleus aud blopharoplast (PI. 18, fig. 1 ; and text-fig. 2 part).

Fig. 1—9

In these and following text-figures the dense chromatin masses of a parasite are represented black; the loose chromatin is shaded. The cytoplasm is diagrammatically indicated by small dots, more closely aggregated and deeply marked where the cytoplasm was more deeply stained. Very faintly-stained areas and vacuoles are left clear. Intra-corpuscular parasites are figured inside more or less circular areas, representing the outlines of infected red blood-corpuscles.

Fig. 1—9

In these and following text-figures the dense chromatin masses of a parasite are represented black; the loose chromatin is shaded. The cytoplasm is diagrammatically indicated by small dots, more closely aggregated and deeply marked where the cytoplasm was more deeply stained. Very faintly-stained areas and vacuoles are left clear. Intra-corpuscular parasites are figured inside more or less circular areas, representing the outlines of infected red blood-corpuscles.

In many cases, a marked cytoplasmic band stretches across the parasite equatorially between these two “nuclei” (text-fig. 1 part).

The loose chromatin sometimes surrounds one or other of these denser chromatin masses (text-fig. 3 part, 12 part, 18 part, see also text-fig. 34), or forms a rod in relation with one of these masses (text-fig. 15 part).

Attention may be drawn to the small forms shown in text-fig. 17, where the blepharoplast occurs at the apical end, and the principal chromatin mass stretches across the parasite near the blunt “polar” end. Loose chromatin cannot be definitely distinguished as a separate mass in these small and young forms.

Usually the shape of the principal chromatin mass is ovoid, especially when occurring laterally. At other times, but less commonly, it is round, as in the case of “polar” distribution. Occasionally it is seen to be irregular (text-fig. 10 part). The so-called blepharoplast is usually round, occasionally ovoid. Rarely, the principal chromatin mass is in the form of a curved rod (text-figs. 8 part, 16).

These different forms and appearances, more especially the varying distribution of the chromatin masses, whether “equatorio-lateral” or “polar,” may be explained, at any rate in part, by regarding the parasite as viewed from different aspects, as Christophers has pointed out in connection with the Leishman-Donovan body. It may also be that, as the parasite grows older, the blepharoplast becomes further removed from the nucleus. The intra-corpuscular pyriform parasites are almost certainly flattened to a greater or less extent, as Nuttall and Graham-Smith (8, p. 639) have shown in the case of the larger form, P. can is, from observations on living blood.

These observers also mention the occurrence of an achromatic halo around the nucleus or blepharoplast in some cases (8, p. 592). I have very rarely seen this in P. bigeminum, and then somewhat imperfectly (cf. text-fig. 9, part).

On the whole, in the case of pairs of parasites, each member of the pair is usually very like its fellow in size, shape, and distribution of chromatin. Variations, however, occur (e. g., text-figs. 9, 15), which are comparatively few in proportion to the large number of parasites examined. Reference has already been made, incidentally, to these variations, whenever it has been necessary to use the word “part” in referring to a figure, thereby indicating variation among the individuals of a pair.

Minute isolated masses or dots of chromatin, other than the three masses already mentioned, were rarely, if ever, seen with certainty in these minute parasites. Further, no really definite cases of the entire absence of chromatin from a parasite were encountered.

Regarding the relative positions of the nucleus and blepharoplast, it may be noted that although the latter is fairly often seen in close proximity to the former, and even connected with it by loose chromatin, yet I have never seen the blepharoplast actually on the nucleus, or directly budded off from it, as stated by Lühe in P. canis. Also, the relative sizes of these two chief chromatin masses can only be determined by careful differentiation and sharp focussing from the granular cytoplasm, sometimes surrounding one or both of them.

(β) The Chromatin in Pairs of Ovoid Intra-corpuscular Parasites

Pairs of strictly ovoid parasites, in which each member of the pair is truly ovoid, are rare. Under this heading it will be convenient to consider cases of pairs of parasites where one member is strictly ovoid in shape while the other is more or less so, yet still shows something of a pyriform contour and may be rather fusiform (text-fig. 24), for the gradation in shape between the two forms is progressive. Such pairs of parasites merit separate consideration as a group after the large number of more strictly pyriform types already discussed.

In these cases we have the same broad distribution of chromatin as before. The “equatorio-lateral” arrangement of the nucleus and so-called blepharoplast preponderates. This distribution of the chromatin, together with the ovoid contour of the micro-organism, recalls, superficially, the appearance of the Leishman-Donovan body.

A few interesting forms showing the connecting bridge of chromatin between the nucleus and blepharoplast were seen (text-fig. 20 part). Three masses of chromatin in each parasite of a pair are shown in text-fig. 19, which masses appeared stained about the same colour and intensity, while the mass which might probably be termed the blepharoplast was in each case triangular in surface view, with the apex tapering towards the centre of the parasite and surrounded by loose chromatin in one case. The principal nucleus of the more pyriform parasite (text-fig. 19) was rather dumb-bell-shaped, suggesting division, but the general facies of this parasite did not lend support to this view. In text-fig. 24, where each parasite is seen to be somewhat pointed at the ends, there were well-marked masses of loose chromatin at one end. In text-fig. 23 two pale-staining parasites are drawn, with peripheral, flattened chromatin. One of them showed a pointed process of cytoplasm, rarely seen in stained preparations apart from spleen-blood. In text-fig. 22 a rather unusual difference in size between the individuals is shown, the larger one being bean-shaped. Both exhibited nuclear dimorphism.

TEXT-FIGS. 19 —24.
TEXT-FIGS. 19 —24.

(γ) The Chromatin of Single Pyriform Intra-Corpuscular Parasites

These forms were not numerous, and no new disposition of the chromatin masses was found. Text-fig. 25 represents a typical parasite, possessing nucleus and blepharoplast, with, in this case, a rather well-marked’ bridge of looser chromatin joining them (cf. preceding text-figs. 2, 20). The presence of such a connecting bridge or strand of chromatin in forms of P. canis is mentioned and figured by Nuttall and Graham-Smith (8, p. 594, fig. 12).

TEXT-FIGS. 25 —26.
TEXT-FIGS. 25 —26.

In text-fig. 26 a large pyriform parasite is represented with less well-marked masses of chromatin, the larger of which is irregular in shape.

The loose mass of chromatin was not always to be found in these single intra-corpuscular forms.

(δ) The Chromatin of Rounded In tra-Corpuscular Parasites

These are not very common, and the parasites may occur either singly or in pairs—usually only one in a corpuscle. They generally exhibit a clear, very faintly stained, or quite unstained, area in the centre, and the chromatin is, in some cases, not easily identified. However, chromatin is always present, and is situated strictly peripherally, the chromatin masses being flattened against the circumference of the parasite. One, two, or three (text-fig. 28) masses of chromatin were seen in various specimens. In text-fig. 27 there are really two masses of chromatin, as a blepharoplast occurs close to the nucleus.

TTEXT-PIGS. 27 AND 28..
TTEXT-PIGS. 27 AND 28..

These parasites are truly rounded forms, not merely pyriform ones seen blunt-end-on, as could be determined by focussing, and from the fact already mentioned, that the central area was clear ; also the corpuscle-hosts are only thin, bi-concave discs. Some of these parasites are ring-like.

(ε) On the Chromatin of Endoglobular Parasites occurring in Groups of More than Two in a Corpuscle

Cases of three or four parasites, more especially four, occurring together within a corpuscle are fairly common. These may result from multiple infection or from two binary divisions after a single infection. All the members of such a group usually exhibit the same or similar distribution of the chromatin elements. In text-figs. 29—31 there are slight differences in the chromatin masses among the members of a group, and it is for this reason more especially that they are figured.

TEXT-FIGS. 29—31.
TEXT-FIGS. 29—31.

Of the group represented in text-fig. 29, one of the parasites only possessed a mass of loose chromatin. In text-fig. 30 two easily distinguished pairs are shown, which would appear almost certainly to have resulted from a multiple (binary) infection. In text-fig. 31 a group of three parasites is represented, all differing to some extent. Such groups are rare.

The general distribution of the chromatin in groups of parasites conformed to that already given.

(ζ) The Chromatin of Intra-corpuscular Parasites suggesting Division

Parasites in process of division were not common. In text-fig. 32 is represented a typical, heart-shaped form in process of longitudinal division. Such forms have been figured previously, but without showing the blepharoplast in the partially separated forms. In this dividing parasite two nuclei, “polar” in position, and two blepharoplasts, “apical” in position, were clearly seen.

TEXT-FIGS. 32 —35.
TEXT-FIGS. 32 —35.

In text-fig. 33 nuclear division is depicted in a large, ovoid form. The principal nucleus is here drawn out into a some-what curved rod, with slightly enlarged ends, the outline of the rod. in its middle portion being not quite sharp, but a little irregular. Nothing in the nature of chromosomes could be definitely distinguished, nor reduction division with certainty. The blepharoplast (text-fig. 33) had divided into two dots, lying side by side and still touching, at the periphery of the parasite, away from the dividing nucleus. A vacuole was present near the latter.

Text-figs. 34 and 35 represent ovoid forms, suggesting parasites in process of division. Each contains several chromatin masses of varying size, and more or less peripheral in position. In text-fig. 34 the relative sizes of the four chromatin masses suggest the presence of two nuclei and two blepharoplasts.

(η) Other Intra-corpuscular Forms

In text-fig. 36 is shown a rare form, consisting of two intra-corpuscular parasites, closely approximated at one end, which may be termed rod-shaped, but are really more like a dumb-bell or spoon in outline. Bacillary or rod-like forms of P. bigeminum are known, but from immune cattle (Theiler). The parasites drawn in text-fig. 36 may possibly be pyriform ones seen edge-on. Two masses of chromatin, one larger than the other, were seen in each parasite situated near the ends.

TEXT-FIGS. 19 —24.
TEXT-FIGS. 19 —24.

Irregular forms are very rare. Nuttall and Graham-Smith (8) describe intra-corpuscular parasites showing processes in stained preparations and also in the living condition, and remark (more especially concerning free parasites) that cytoplasmic processes seen in living parasites are often not found in stained preparations, owing to retraction at death or to the preparation of the film or smear and the difficulty of staining such fine processes. I have only seen one good case of a stained parasite with a protoplasmic process (text-fig. 23), already mentioned.

(θ) The Chromatin of Free Parasites

Extra-corpuscular or free parasites are not very numerous, and are found chiefly, as might be expected, at the edges of the smear. Regarding epi-corpuscular forms, emphasised by Lühe (4, 5) in the case of P. canis, I have only seen one or two doubtful cases, not sufficiently definite to merit detailed description, and my observations have been strictly confined to stained specimens.

The free parasites are nearly always more or less pyriform in shape (text-figs. 37 to 42), occasionally rounded (text-figs. 43 and 44), and are usually slightly larger in size than the corresponding intra-corpuscular forms.

The distribution of chromatin in free forms on the whole closely corresponds with that found in pyriform intra-corpuscular parasites. Common cases are shown in text-figs. 37 and 39, where nucleus and blepharoplast are distinguished, and in some cases an apical mass of looser chromatin (text-figs. 37 and 38) is seen. In text-figs. 38 and 39 the strictly pyriform contour is modified, approaching the fusiform.

Occasionally the pyriform outline is seen to be modified in the direction of its long axis. We then have interesting forms like those shown in text-figs. 40 to 42. These are long and thin, and sometimes exhibit a prolongation of the pointed extremity into a short, pointed cytoplasmic process (“flagellum”1 of some authors). Such processes, however, hardly suggest great motility. These forms always possessed a nucleus and blepharoplast, the latter at the pointed end. Rarely, smaller chromatoid granules could, with difficulty, be distinguished (text-fig. 42). These forms bear at least a superficial resemblance to Crithidia (Léger) and some species of Herpetomonas (vide Woodcock [11, p. 268, fig. 38]). Similar forms have been observed in different species of Piroplasma, often without “nuclear dimorphism” having been noticed therein. Such free forms of Piroplasma bigeminum are certainly very interesting, more especially to those parasitologists who attach great weight to the observations of Schaudinn ([9],p. 438) on blood-films prepared by Weber at night from a cow kept in the dark, and dying of piroplasmosis, wherein a small Trypanosome was found accompanying P. bigeminum. Trypanosome-like (trypaniform) bodies were also found in old films of Kossel and Weber made from the contents of the digestive tract of ticks, which had fed on cows suffering from piroplasmosis. However, the inference that Piroplasma possesses a trypaniform stage is not in the least supported by the researches of Koch on P. bigeminum and P. parvum (1) from the gut of ticks, nor by the work of Kleine (2) on culture forms of P. canis. Kleine also carefully searched for flagellate stages in the blood of infected dogs, without success.

TEXT-FIGS. 37 —44.
TEXT-FIGS. 37 —44.

Regarding the chromatin of free rounded forms (text-figs. 43, 44) it closely resembles in distribution that of similarly shaped intra-corpuscular forms, and needs no further comment.

As judged by the distribution of the stain, the cytoplasm is more especially peripheral, and often a clear area is seen in the centre. Such a clear area may be regarded as a vacuole, though one must beware of such appearances, sometimes deceptive, when observations are confined to stained preparations. However, vacuoles have been described in Piroplasmata by various observers while examining the parasites in the living condition. The vacuolated condition, as determined from stained specimens, is well shown in text-figs. 1, 2, 3, 9, 12, 21, 26, 27, and 32. A vacuole with a sharp and definite edge is shown in text-fig. 33. It does not always follow that the clear space inside a parasite, as diagrammatically represented in the text-figures, is a vacuole, for when one examines the preparation under monochromatic light a slight shadow may sometimes appear therein, suggesting a continuous cytoplasm within such parasites, which may be more clear and hyaline in the centre, or less permeable to stain than that at the periphery. The vacuoles, of which more than one may occur in a parasite, usually lie between the chromatin masses, and appear only in the larger parasites. Two vacuoles may occur in a parasite separated by a marked cytoplasmic strand (text-figs. 3 part, 21 part, 22 part).

The cytoplasm shows a granular structure, more especially at the apical end of some forms (text-figs. 3, 12 part). The very finely-granular character of the protoplasm is difficult to represent in drawings. The very small dots in the text-figures, which figures are semi-diagrammatic, only show the limit of the obviously stained area, yet that is almost the limit of certainty in such small and delicate (endocorpuscular) objects as Piroplasmata.

The central clear area seen in most rounded parasites would appear to be a vacuole.

I was not able to obtain any further evidence as to the character of the cytoplasm from an extended examination of the stained free forms.

It may be stated at this point that the dimensions of the larger intra-corpuscular parasites examined was broadly about 3 μ by 1·5 μ, and agreed well with the measurements given by former observers, though variations occur. The nucleus (largest dense chromatin mass) was from 0·3 μ to nearly 1 μ in long diameter in some forms, and exhibited variations in size in different specimens. The so-called blepharoplast was sometimes only about half that in diameter.

As far as evidence goes at present—that is, considering the observations of Schaudinn (9), Lühe (4, 5), and Nuttall and “Graham-Smith (8), as well as those herein set forth, it would appear that there is some justification for the terms “nucleus” and “blepharoplast,” as applied to the larger and smaller dense chromatin masses respectively in Piroplasmata, at any rate, as a “working hypothesis” (Schaudinn), and so suggesting affinities of this rather aberrant Hæmosporidian genus with the Hæmoflagellates.

The loose, reticulate chromatin, it seems to me, might similarly be tentatively compared with the chromatoid granules often recorded in Trypanosomes, though the loose chromatin of Piroplasma appears to be relatively of greater bulk.

Mention has already been made of Schaudinn’s observation on Weber’s blood films taken at night from a diseased cow, and Kossel and Weber’s films from the gut of ticks. It is much to be regretted that the trypanosome-like organisms occurring in these preparations were not subjected to a lengthy and detailed examination and carefully figured. Even then the question of a double infection would not have been disposed of satisfactorily.

Also, it has been mentioned that Koch’s (1) and Kleine’s (2) researches do not at all support, so far as known at present, the hypothesis of a trypaniform phase in the life-history of Piroplasma. However, in Koch’s large pyriform parasites from the gut of ticks, with protoplasmic radiations at the blunt (“polar”) end, there is a larger nucleus at this end and a smaller blepharoplast-like body nearer the other (“apical”) end. Three chromatin masses are seen in the zygote-like-forms, with radiations at opposite poles. It is remarkable that Kleine (2) obtained similar results by cultural methods in the case of P. can is.

The superficial resemblance of Piroplasmata, exhibiting well-marked nuclear dimorphism, to the Leishman-Donovan body is striking. At one time it occurred to me that, perhaps, the loose chromatin of Piro plasma, in some forms of its distribution, might be compared with the “tail” 1 of chromatin in the Leishman-Donovan body, though this “tail,” when present, seems to be always attached to the blepharoplast of the Leishman-Donovan body, and stains rather darkly, and the resemblance to loose chromatin is none too well marked. Laveran and Mesnil would seem to be perhaps justified in placing this body in the genus Piroplasma (as P. dono vani), considering superficial appearances only, but the difference of habitat must be borne in mind, and the Leishman-Donovan bodies are rarely, if ever, intra-corpuscular. The chromatin masses of the Leishman-Donovan bodies, too, arc well defined. Flagellate stages most probably do occur in the life-cycle of these bodies (c f. Rogers, Christophers, Leishman, and others on “cultures.”).

I will venture, however, to make a suggestion on the “nuclear dimorphism” of Piroplasma from a rather different point of view, namely, its relation and interpretation in terms of the distribution of chromatin in the Protozoa generally (vide Mesnil [6]). From this standpoint it is probable that the nucleus (large chromatic body) of Piro-plasma consists of vegetative chromatin, and is, indeed, a trophic nucleus, while the blepharoplast, according to the upholders of the flagellate affinities of Piroplasma, would be a kinetic nucleus—that is, a specialised and separate portion of vegetative chromatin. But the so-called blepharoplast, usually denser and more deeply staining, may really consist of generative chromatin (cf. the micro-nucleus of Paramcccium). The loose chromatin would be a reticulum of extra-nuclear chromatin or chromidia. Whether this loose chromatin consists of tropho-chromidia (chromidia properly so-called) related to the cell-metabolism of Piro-plasma, or whether the loose chromatin consists of idio-chromidia and represents a reserve of generative chromatin, is difficult to say at present. The significance of the so-called blepharoplast at the moment is most problematic, and precludes more definite statements.

Nuttall and Graham-Smith (8, pp. 641-643) consider the various hypotheses which have been advanced at different times regarding the development of Piroplasma. They reject them all, after careful studies on the living parasites.

It would appear, then, premature and useless to discuss further the possible life-cycle and affinities of Piroplasma; there have been, recently, it seems to me, too many “phylogenies” of such problematic organisms, often founded on slender evidence, and the complete life-history of Piroplasma, and even Trypanosoma, has yet to be ascertained.

Lastly, as definite and further pronouncements1 on the developmental stages of Piroplasmata in ticks may be expected shortly, it is better to wait for these, when we may learn whether a flagellate stage really does occur in the life-cycle of Piroplasma, and what is the true significance of Koch’s forms, so difficult to interpret alone.

(1) All the specimens (stained) of Piroplasma bigeminum, which were examined, showed the presence of a chromatin mass or masses. Usually there was more than one chromatin mass in each parasite (see pl. 18, figs. 1 to 7).

(2) In the pyriform and ovoid parasites there are usually present (α) a rather large and dense chromatin mass—the nucleus ; (β) a second, somewhat smaller, usually denser mass of chromatin—the blepharoplast, which is sometimes only punctiform; and many parasites possess in addition (γ) a rather looser mass of chromatin, of a woolly or mesh-like structure (chromidial reticulum).

(3) Variations occur in the relative positions of these chromatin masses, and less frequently in their relative sizes. These variations may be due in part to the parasite being viewed from different aspects.

(4) The loose chromatin mass is often relatively well-marked in P. bigeminum.

(5) Free parasites were sometimes seen in which the pyriform contour was slightly modified, and the apical end prolonged into a short cytoplasmic process. Such forms possessed a nucleus and blepharoplast, and somewhat resembled Hæmoflagellates (text-figs. 40 to 42).

(6) In round forms nuclear dimorphism also probably occurs, for there is usually more than one chromatin mass present (text-figs. 27, 28, 43, and 44). Amœboid forms were not available for examination.

(7) The cytoplasm of P. bigeminum appears to be vacuolated in character. Unfortunately, the observations were, of necessity, confined to stained preparations.

(8) The possible significance of these results is briefly discussed in the preceding section. More definite pronouncements are premature, pending further knowledge of the developmental stages of Piroplasma in the tick.

December 31st, 1906.

Since writing the foregoing, two papers have appeared relating to Piroplasma (Babesia) canis, one by Christophers (13) on the developmental forms of the parasite in the dog-tick (Rhipicephalus sanguineus) of Madras, and the other by Kinoshita (14) on the forms of the parasite in the dog’s blood and in sodinm-citrate cultures.

The paper by Christophers, though only a preliminary account, is most important. Most of the forms mentioned by Koch in the cases of P. bigeminum and P. parvum in the gut of adult ticks were seen by Christophers, except the markedly radiate forms. Stages in the development of P. canis were traced in the tick-egg, in the nymph, and in the developing adult of the dog-tick. The life history appears to be simple. Loose chromatin was seen in several stages, but no mention of any flagellate form occurs, nor is such figured. Also the presence of a blepharoplast is not mentioned. This absence of a flagellate stage in .the developmental cycle in the tick is most important, and Christophers’ fuller memoir will be awaited with the keenest interest.

Kinoshita’s paper is interesting, though to me it is a little difficult to summarise. The author gives excellent figures of nuclear dimorphism in the comparatively large form, P. canis, the largest species of Piroplasma, and sufficiently large to provide finer morphological detail often invisible in the smaller P. bigeminum. He describes schizogony and gametogony, and considers the blepharoplast to consist of “animal” chromatin. He figures two forms, each with a chromatic appendage arising from a large blepharoplast, which he considers to be microgametes. Kinoshita himself, apparently, does not think that a flagellate stage normally occurs in Piroplasma, though in an editorial footnote, p. 306, additional and independent evidence for the presence of a flagellate stage is set forth.

It is most probable, indeed certain, that a flagellate stage does occur in the life-cycle of the Leishman-Donovan body, and may be expected in the alimentary tract of a blood-sucking Arthropod, namely, the bed-bug, as suggested by Rogers, and now being worked out by Patton (vide ‘Ind. Med. Gaz.,’ 1906, p. 302). On the other hand, a flagellate stage appears to be absent in the case of Piroplasma. The pathogenic agents of kala-azar and hæmoglobinuria would not appear, then, to belong to the same genus. However, the evidence at present available is convicting, and further discussion is premature.

February 14th, 1907.

1.
Koch
,
R.
,
1906
. —“
Beitrage zur Entwicklungsgeschichte der Piro-plasmen
,”
‘Zeitschr. f. Hyg.,’
liv
, July 26th, 1906, pp.
1
7
, 3 pls.
2.
Kleine
,
F. K.
,
1906
. —“
Kultivierungsversuche der Hundepiroplasmen
,”
‘Zeitschr. f. Hyg.,’
liv
, pp.
8
16
, 2 pls.
3.
Laveran
,
A.
et Nicolle
,
M.
,
1899
. —“
Contribution à l’élude de Pyrosoma bigeminum
,”
‘C. R. Soc. Biol.,’
li
, pp.
748
751
, 15 text-figs.
4.
LÜhe
,
M.
,
1906
. —“
Zur Kennlniss von Bau und Entwicklung der Babesien
,”
‘Zool. Anzeiger,’
xxx
, pp.
45
52
.
5.
Lühe
,
M.
,
1906
. —“
Die in Blute schmarotzenden Protozoen und Hire nächsten Verwandten
,”
in Mense’s ‘HandbuchderTropenkrankheiten,’ Leipzig, Bd
.
iii
(‘Babesia,’ pp.
193
202
, taf. 8).
6.
Mesnil
,
F.
,
1905
. —“
Cbroinidies et questions connexes
,”
‘Bull. Inst. Pasteur,’
iii
, pp.
312
322
.
7.
Minchhj
,
E. A.
,
1903
. —“
Sporozoa
,”
in Lankester’s ‘Treatise on Zoology,’ pt. i, fase. 2
, pp.
150
360
(‘Piroplasma,’ pp. 269-270, fig. 80).
8.
Nuttall
,
G
, H. F., and
Graham-Smith
,
G. S.
,
1906
. —
“Canine Piro-plasmosis
. —V. Further Studies on the Morphology and Life-History of the Parasite,”
‘Journ. Hygiene,’
vi
, No.
5
, pp.
586
651
, 3 pls.
9.
Schaudinn
,
F.
,
1904
. —“
Generations-und Wirtswechsel bei Trypanosoma und Spirochæte. Vorlaufige Mitteilung
,”
‘Arb. a. d. Kaiserl. Gesundheitsamte,’
vol.
xx
, pp.
387
439
, 20 figs, (see p. 438).
10.
Smith
,
T.
, and
Kilborne
,
F.
,
1893
. —“
Investigations into the Nature, Causation, and Prevention of Texas or Southern Cattle-Fever
,”
‘Eighth and Ninth Ann. Rept., Bureau of Animai Industry,’
pp.
177
304
, with 10 pls. Washington, U.S.A.
11.
Woodcock
,
H. M.
,
1906
. —“
The Hæmoflagellates : a Review of Present Knowledge relating to the Trypanosomes and Allied Forms
,”
part ii, ‘Quart. Journ. Micr. Sci.,’ N.S
.,
50
, ii, June, 1906, pp.
233
-
331
. (See p. 244, footnote 3 ; pp. 253-266 [fig. 37] and p. 269. Also “Bibliography on Leishman-Donovan Bodies,” p. 330.)
12.
Ziemann
,
H.
,
1898
. —“
Nene Untersuchungen über die Malaria und den Malariaerregern nahestehende Blutparasiten
,”
‘Deutsche med. Woclienschr.,’
xxiv
, pp.
123
125
(especially p. 125).

References quoted in Addendum

13.
Christophers
,
S. R.
,
1907
. —“
Preliminary Note on the Development of Piroplasma canis in the Tick
,”
‘Brit. Med. Journ.,’
No. 2402, Jan. 12th, 1907, pp.
76
78
, with 26 text-figs.
14.
Kinoshita
,
K.
,
1907
. —“
Untersuchungen über Babesia canis (Piana und Galli-Valerio)
,”
‘Arch. f. Protisteukunde,’
viii
, pp.
294
320
, tafs. 12 and 13.

Illustrating Mr. H. B. Fantham’s paper, “On the Chromatin Masses of Piroplasma bigeminum (Babesia bovis), the Parasite of Texas Cattle-Fever.”

Figs. 1-7.—Lithograph drawings made from photo-micrographs of Piro-plasma bigeminum (Babesia bovis) from heart-smear, showing chromatin masses and their distribution. These photo-micrographs were taken under monochromatic (yellowish-green) light, using a Thorpe’s grating. Zeiss’s 3 mm. apochromatic homog. immers. objective was used, with compensating oculars 8 or 12.

The lithograph drawings are twice the size of the photographs.

These minute intra-corpuscular parasites were very difficult to focus for photography in order to bring out detail successfully, and so the relative sizes of the nuclear masses do not appear always precisely correct.

The red blood-corpuscles were in all cases only very faintly stained.

FIG. 1.—Shows bridge of chromatin between nucleus and blepharoplast in parasite to left. Note also pair of parasites on right.

FIG. 2.—Pair of large pear-shaped intra-corpuscular parasites, showing nucleus and blepharoplast in each, and loose chromatin at apex of one. Magnification greater than in other figures.

FIG. 3.—Blood-corpuscles showing rather better than in other figures, one pair of parasites distinct.

FIG. 4.—One parasite shows three chromatin masses, and chromatin bridge.

FIG. 5.—One parasite, on right, shows chromatin bridge faintly.

FIG. 6.—Several pairs of parasites, on left, show nuclear dimorphism some-what faintly.

FIG. 7.—Parasites at top left-hand part of field show chromatin masses.

TEXT-FIGS. 10—18

TEXT-FIGS. 19 —24

TEXT-FIGS. 25 AND 26

TEXT-FIGS. 27 AND 28

TEXT-FIGS. 29—31

TEXT-FIGS. 32—35

TEXT-FIG. 36

TEXT-FIGS. 37—44

1

These figures are reproduced in Minchin’s ‘Sporozoa’ (7), p. 269, fig. 80.

2

The term “blepharoplast,” as relating to Piroplasma, is used in this memoir for convenience, and without prejudice to its literal meaning as a nuclear body related to a flagellum. In the present state of our knowledge it is impossible to state definitely whether a flagellate stage occurs in the life-history of Piroplasma or not (vide Sect. VI).

1

“Part” here refers to the fact that only one member of the pair of parasites shows the particular feature.

1

Unfortunately tins term (“flagellum”) is used, in the Protozoa, for both a cytoplasmic process and a chromatic process rather indiscriminately.

1

This “tail,” which is stated to occur in some of the larger forms of the Leishman-Donovan parasite, is perhaps, on the other hand, only the developing flagellum of the flagellate stages first found by Rogers in cultures, though one hardly gathers this from the published accounts of the formation of the flagellum. This chromatin “tail” merits further consideration by workers with the necessary material, for a flagellum does not occur in parasites in the human spleen.

1

See Addendum.