Observations on Hæmatozoa in Ceylon

Since the publication of our preliminary note (‘Spolia Zeylanica,’ vol. ii, part 6, August, 1904, pp. 78-92) a few more cases have come under our notice, and as there is no immediate prospect of amplifying our investigations owing to the pressure of other duties, we deem it advisable to publish our results in full without further delay. The discoveries of Ross, Grassi, Schaudinn, and others, have rendered it clear that researches on blood parasites must be undertaken with a single aim, and with undivided attention, in order that they may be cyclically complete.

It seems that the experimental difficulties which beset the investigation of the life-cycle of the hæmogregarines and trypanosomes of cold-blooded animals are great; and the ignorance as to the means of transmission of these parasites, and with regard to the order of succession of their different phases, leaves abundant room for future discoveries of biological importance.¹

Schaudinn’s memorable discovery, that the well-known hæmatozoon of birds called Halteridium is a phase in the life-history of species of Trypanosoma, has considerably modified the views as to the generic autonomy of the various protozoon parasites of the blood, besides apparently demonstrating a close affinity between the Sporozoa and the Flagellata. The name Halteridium will, however, doubtless survive to characterise the particular phase referred to, and the same use may probably be made of the term Hæmo-cystidium, which we have introduced to denote a well-marked endoglobular parasite observed by us in a gecko, an analogous phase having been previously described by Dr. Simond from a Trionyx.

In the course of our examination of numerous samples of blood collected in different parts of the island, very many negative results, as was to be anticipated, have been obtained. So far as our observations have extended, they seem to point to the conclusion that the percentage of infected cases of all kinds is greater in the neighbourhood of centres of population than in outlying districts. This fact may indicate that the conditions of nutrition of the Hæmatozoa are more suitable under the circumstances of sanitation peculiar to towns, and that the incidence of blood-parasitism may be directly or indirectly assignable to the far-reaching influences of domestication.

There seems to be a marked contrast between the distribution of the Hæmatozoa and that of the majority of Entozoa or parasites of the digestive tract, the latter being at least equally frequent in regions remote from human habitations as in areas of cultivation.

From the point of view suggested by these remarks, it is interesting to record the occurrence of peculiar parasitic bodies in the blood of birds and man, for which, so far as we can ascertain, no provision has been made in any of the published accounts of the life-histories of the Protozoa of the blood.

In the case of birds these bodies occur in company with Halteridium, and in man associated with the symptoms of malaria. They are crescentic organisms, rather exceeding the length or diameter of the blood-corpuscles, characterised by the absence of pigment and by the presence of vacuoles, and they are very rare in such preparations as we have examined. Their form and size relatively to the blood-cells are shown on Pl. 24, figs. 1—3.

In the blood of a bird, the common babbler, Crateropus striatus (Swainson), taken on July 20th, 1904, Halteridium was present in the films, though scarce, and, in addition, there were some rare parasites of an elongated shape, with two or three vacuoles, free in the plasma. In fresh preparations they appeared to be non-motile, though a slight change of shape could be detected, the parasite becoming slightly broader after a time.

Halteridium only was found in the spleen. After staining by Leishmann’s method, the bodies were coloured deep blue, and did not show chromatin. Sometimes there is the appearance of a clear halo surrounding the body (Pl. 24, fig. 2). In a preparation which had become partly decolorised through lapse of time there appears to be a definite nucleus in the centre of the body.

In the blood of an Indian crow (Corvus splendens) similar bodies were found, but of smaller size, some of them appearing granular without vacuoles (Pl. 24, fig. 3); these would seem to be specifically distinct from the parasites of the babbler.

Finally, analogous bodies, exceedingly rare, have been observed by us in the finger-blood of native fever patients. Their general outline and size might seem to suggest some relation, whether of growth or degeneration, with the ordinary crescents of tropical malaria; but the absence of pigment, the presence of vacuoles, and the deeply staining protoplasm exclude such an assumption, especially in view of their similarity to the bodies observed in birds. In the true crescents, as is well known, there is an aggregation of pigment granules interspersed with chromatin at the centre of the body; the protoplasm stains uniform pale blue, there are no vacuoles, and there are frequently traces of the original blood-cell in which the crescent developed adhering to it.

Unfortunately, the literature at our disposal does not permit us to say whether the bodies we have described are, or are not, identical with, or related to, the pseudo-vermicules described from certain birds by Danilewsky, Kruse, L. Pfeiffer, and MacCallum.¹

In the ‘Annales de 1’Institut Pasteur,’ tome xv, 1901, p. 338, Dr. P. L. Simond described, under the name Hæmamœba metschnikovi, a parasite of the blood of the freshwater tortoise, Chitra indica (= Trionyx indiens), which appears at a certain phase of its life-history in the form of an amoeboid and eventually rounded organism containing pigment granules. He says that the chelonian host is very common in the rivers of India, especially in the Ganges and its tributaries, and that it plays the part of a scavenger in the waters of the Ganges, devouring, in addition to its natural diet of fishes and other aquatic animals, the remains of the corpses of Hindus which are thrown into the river after an incomplete cremation owing to the paucity of firewood in certain regions. On this account it is greatly venerated by the Hindus.

In all the adults of this species examined by Dr. Simond, more than twenty in number, he observed the pigmented stages of the Hæmatozoon, and, in addition, he found nonpigmented parasites of the Hæmogregarine type in constant association with the pigmented Hæmamœboids.

On this account, as well as from analogy and in view of the known fact that it is impossible to recognise sexual differences among true Hæmogregarines, whereas such differences are clear among the Hæmamœboids, Dr. Simond inclined to the opinion that the two forms of endoglobular parasites observed by him belonged to one cycle of development.

With regard to the life-history and mode of transmission of these parasites, the author points out that experiments in inoculation have hitherto proved futile, and the only method of dealing with them at present is the morphological method.

The scavenging activity of the tortoise Chitra indica¹ is of some interest in view of the remarks which we have made above and of some which are to follow.

Referring to Dr. Simond’ s discoveries, Professor E. A. Minchin makes the following comment in his work on the Sporozoa in Professor Lankester’s ‘Treatise on Zoology/1903, p. 270:

Hæmamœba metschnikovi “occurs as a minute pigmented endoglobular amcebula resembling the malarial parasites of birds and mammals. Its presence in a cold-blooded animal is, therefore, remarkable and quite exceptional…. Further investigations of this interesting form are required, and Laveran admits it only with some reserve to rank in his genus Hæmamœba.”

In the blood of a tree-dwelling gecko, Hemidactylus leschenaultii, taken at the village of Mamadu, near Vavu-niya, in the Northern Province of Ceylon in April, 1904, we observed a pigmented endoglobular parasite which did not appear to fall within the definition of any hitherto described genus of Hæmamœbidæ. We accordingly named it Hæmo-cystidium simondi.¹ No Hæmogregarine forms were found accompanying it. At the earliest stage observed it consists of a small irregular body with a belt of pigment granules across the centre, occasioning a slight displacement of the nucleus of the corpuscle.

With increase of size of the parasite the nucleus of the host-cell becomes pushed to one end of the corpuscle (Pl. 24, fig. 4).

The parasite was not observed in the fresh state, but in preparations stained by Leishmann’s modification of Romanovsky’s method two distinct kinds are found, resembling one another in size and form, but differing in their reactions to the stain.

These, no doubt, represent sexual differences, as in Halte-ridium. In the male type the body is seen to be faintly granular, the protoplasm is stained a delicate pale-blue, with numerous small pigment granules scattered round the periphery and in the substance of the oval or discoidal organism. In the female type the body is stained dark-blue, and the pigment granules are numerous, though appearing on the whole rather larger than in the male trophozoite. The female trophozoite differs further from the male by the constant presence of vacuoles, varying in number and size (Pl. 24, fig. 4).

Sometimes the contour of the Hæmocystidium makes an almost perfect circle, and in accordance with the shape of the blood-corpuscle it is probable that the parasite is not spherical but rather shaped like a biconvex lens. Its diameter nearly equals the shorter diameter of the corpuscle, while the elongated forms nearly fill the latter.

In one case of a double infection the growth of the two male trophozoites had caused a deep constriction of the blood-corpuscle, nearly cutting it in two.

The species described by Dr. Simond, which should now be named Hæmocystidium metschnikovi (Simond) resembles the parasite of the gecko in general features but differs markedly from it in point of size, rarely exceeding the half of the corpuscle in diameter, in the smaller number of pigment granules, and in the fact that it does not cause a displacement of the nucleus of the corpuscle, according to Dr. Simond’s figures. Double or even treble infections of a corpuscle by H. metschnikovi do not affect the outline of the latter. It is, therefore, clearly a smaller species than H. simondi.

The natures of the hosts are also widely different, the one an aquatic chelonian, the other a terrestrial gecko living in the driest province of Ceylon.

The same species of gecko whose blood is infected with Hæmocystidium simondi harbours a nematode parasite in its intestine, which has been described by Dr. von Linstow as a new species of Oxyuris, 0. megaloon.¹

The hæmogregarines as a whole appear to favour foulfeeding animals, and there are few fouler feeders than Nicoria trijuga, a tortoise with amphibious habits commonly met with in the ditches and marshy lands round Colombo and in the Colombo Lake.

The larger specimens of this tortoise are generally found to be infected with a hæmogregarine which does not show any particularly striking properties distinguishing it from other similar species. It is, however, important to establish the fact of its almost constant occurrence in a host which derives its nourishment largely from offal.

The tortoise also harbours Nematode parasites in its intestine which have been kindly named and described by Dr. von Linstow as a new species of Oxysoma, O. falcatum.²

When the blood is examined in the fresh condition, the crescent-shaped or reniform body of the endoglobular parasite presents a clear pole, one granular pole and a clear but sharply defined central tract, which in stained preparations proves to contain the nucleus.

Frequently the clear pole is directed towards the displaced nucleus of the blood-corpuscle, but there is no constant orientation.

The granular pole is the growing end of the organism, which becomes bent round upon itself in the manner characteristic of the genus Hæmogregarina.

The doubling of the parasite usually takes place by a very narrow bend, but occasionally a wider bight is produced. Young stages before the bending also came under our observation both in fresh and in stained preparations, and we have seen a double infection of a corpuscle, though this appears to be rare.

The nucleus consists of a more or less diffuse aggregation of chromatin or cyanophil granules which sometimes extend to the recurved limb of the parasite. The length of the bent organism is 0·01 mm. (10 μ). In one corpuscle the parasite had unbent itself and appeared as a long “vermicule,” the corpuscle being enlarged and somewhat distorted.

In a hanging-drop prepared from the blood of a specimen which had been killed some hours previously we have once only observed a motile parasite free in the blood-plasma. It resembled the one which was found unrolled in the corpuscle. The movements consisted of slow revolutions in the direction of the arc of the parasite and also of movements of flexion. The granular pole was directed forwards, while the other pole remained more or less fixed and appeared to be adhesive. Finally, the parasite was attracted by an irresistible chemotaxis to a neighbouring phagocyte, by which it was gradually absorbed.

It may be noted here that we have hitherto not succeeded in finding any parasite in the blood of the herbivorous land tortoise of Ceylon, Testudo elegans.

The unrolling of the parasites would seem to precede its liberation from the corpuscle. This is obviously a critical moment in its life-history, but it is not known what happens next.

Occasionally the bent forms are seen free in the plasma, but this we attribute to accidental rupture or liquefaction of the corpuscle. When the parasite normally becomes free it is probable that the corpuscle undergoes disintegration.

We have named this species Hæmogregarina nicoriæ.¹ In the present stage of knowledge of the hæmo-gregarines the specific differences are largely a matter of host, or, in other words, of environment. All authorities seem to be agreed that there are different species, and that it would be wrong to call all hæmogregarines H. stepa-novi, following the example of Halteridium, all the forms of which are called H. danilewskyi. It will be seen below that there are, so far as we can tell, extraordinary differences in the life-histories of hæmogregarines infesting different hosts.

The fact seems to be that the parasites are hard to distinguish in the immature, asexual, or hæmogregarine phase.

Finally, with regard to H. nicoriæ, it may be mentioned that although the bent parasite is not more than about one half the length and one third the width of the corpuscle, yet the nucleus of the latter is displaced towards one end of the cell.

In one or two cases the nucleus of the parasite has presented the appearance of having divided into four daughter nuclei, two of which occur at one end and two at the opposite end of the body. We have observed no further indications of sporulation.

The freshwater snakes of the Colombo district seem to be frequently the unconscious hosts of a blood-parasite which in its endoglobular phase offers few characters of distinction. In snakes of this and other species obtained in places remote from Colombo we have searched in vain for Hæmatozoa, although Entozoa are common in all parts of the country.

A young snake of the above-named species, upwards of two feet long, was found to be infected with an endoglobular hæmogregarine of the usual type, all the trophozoites being approximately at the same stage of growth, more or less bent into a U shape. They show slight differences from Hæmo-gregarina nicoriæ. Their size is larger, 12 microns or ·012 mm. in length, the protoplasm stains uniform blue, the reddish-blue stained nucleus is denser, and placed near the pole opposite to the bent portion. The greater density of the nucleus is evidenced, not only by the closer aggregation of its chromatin material, but also by a greater resistance to the staining reagent; in many instances it has remained unstained. If the parasite is artificially liberated from the blood-corpuscle on the slide, its nucleus becomes well stained.

The parasites were kept fresh under observation in a hanging drop for several days, but no change was observed to take place.

It is not known what stimulus provokes the endoglobular hæmogregarines to enter upon a new course of development, and it appears that they may live for several months without undergoing much appreciable change within the blood-cells of their host.

It seems clear, however, that a point is reached eventually when something must happen, and a great change take place analogous to the change which Schaudinn had described in the life-history of Halteridium.

A few days after making the observations described above we were able to examine the blood of a large snake of the same species, nearly four feet in length. This examination revealed the presence of great numbers of hæmogregarines free in the blood-plasma in which they performed active movements, displacing the corpuscles as they glided along. Several hanging drops were prepared as usual, but on the following morning the parasites had all undergone dissolution after the manner of Trypanosoma.

The movements consist of slow gliding and turning, the “vermicide” sometimes actually doubling upon itself. Sometimes it will become fixed by its attenuated hinder end, and will revolve by a slow screw-like motion after the manner of the spores of Sarcocystis. Then may ensue a rapid whirling of the body, causing a disturbance among the neighbouring corpuscles.

Many films of the snake’s blood were prepared and stained, and in most of them we found that, besides the free parasites, many of the corpuscles contained bodies of relatively large size, more or less crescentic in shape. These endoglobular organisms were not at all like the ordinary hæmogregarines, but consisted of a delicate protoplasmic membrane with red-stained granules surrounding a pale-blue stained central body with a densely staining nucleus. The discovery of various stages of development within certain narrow limits enabled us to recognise the enveloping body as the mother-cell of the contained body, and we accordingly described it as a cytocyst.

The single organism or monozoite which the cytocyst produces eventually escapes from the membrane and from the corpuscle, very much as a young Nemertine worm escapes from its pilidium, and becomes the free motile parasite described above. We find the monozoites at all stages of emergence, and it is probable that some of the rapid oscillations of the parasite which were observed in the fresh preparations represented violent efforts to free their hinder extremities from the corpuscles.

The nucleus of the monozoite lies behind the centre of the body both before and after its birth. When fully formed within the cytocyst the hinder end of the monozoite is slightly bent, indicating that some pressure or tension is being exerted within. In the next stage the anterior end is found to have perforated the wall of the cytocyst, and the monozoite begins to push its way out through the opening thus produced. Here and there a corpuscle contains an empty cytocyst from which the monozoite has escaped. In such cases the orifice of exit can still be discerned.

In some cases at the moment of fixation of the blood-film on the slide the monozoite had extended its body as far as the level of the nucleus, which appears constricted in the narrow orifice as if it were being squeezed through. Sometimes only the hinder end remains within the cytocyst and corpuscle, the rest of the body being free.

Occasionally, instead of emerging from the corpuscle the monozoite issues from its mother-cell again into the substance of the corpuscle. This is probably a miscarriage.

Sometimes the cytocyst is difficult to distinguish, and the monozoite appears to lie in the corpuscle without a sheath. In such cases the membrane can often be identified on close inspection, but not always.

The monozoites which emerge from the cytocysts are all of one kind and of one size within the limits of a slight variation.

Among the free-living forms in the plasma of the blood some are found to be about twice as bulky as others, indicating that they continue to grow after becoming free. The staining reactions of all are the same, namely, pale-blue cytoplasm and dense reddish-blue nucleus.

We have been fortunate enough to find stages in the formation of the monozoite within the cytocyst, the substance of the former being only partly differentiated and merging imperceptibly into the protoplasm of its mother-cell, the cytocyst. Such differentiation as has taken place is indicated by the pale-blue staining reaction, but chiefly by the structure of the nucleus, in which the chromatin loops or threads are clearly visible, showing unmistakable signs of formative activity.

With regard to the red-staining granules which we have mentioned above in association with the membrane of the cytocyst, we have interpreted these as belonging to a thin layer of residual protoplasm which is left round the periphery of the mother-cell after the formation of the axial monozoite. The membrane which is left empty within the corpuscle after the birth of the monozite sometimes show a slight crumpling, due to collapse; it clearly belongs to the category of plasmatic membranes.

The provisional assumption of a direct genetic connection between the endoglobular hæmogregarines of the first snake and the cytocysts of the second is not supported by observation, but is based upon analogy, upon the specific identity of the hosts, and upon the proximity of the localities. If it is true, and if Simond’s analogous assumption in the case of Hæmocystidium metschnikovi is also true, the fifehistory of the Hæmogregarines must be highly varied and complicated, and cannot be interpreted until some of the many gaps are filled up.

Another large Tropidonotus piscator was dug up, together with a clump of eggs, from a garden in Colombo, in February, 1905. Its blood was found to be in the same condition as in the previous specimen which was captured in July, 1904—that is to say, infested with cytocysts and free monozoites. No further information was obtained beyond a confirmation of results. The eggs contained young in an advanced stage of development, united to the yolk-sac by a narrow umbilical cord. The examination of the blood of these young incubants was negative in respect to Hæmatozoa.

Since the publication of our preliminary note we have become acquainted with a paper by N. Berestneff (“Über einen neuen Blutparasiten der indischen Frösche,” ‘Archiv. f. Protistenkunde,’ vol. ii, 1903, pp. 343-348), in which a closely similar parasite was described from the blood of Indian frogs. The illustrations in the plate accompanying the paper were executed by the method of micro-photography, with the result that the points are not very clearly brought out. Microphotography appears to have the advantage of absolute fidelity, counterbalanced by considerable obscurity. However, the text makes amends for the shortcomings of the plate.

Dr. Berestneff examined the blood of cold-blooded freshwater vertebrates in the neighbourhood of a plague laboratory and a leper asylum in Bombay. He found Trypanosoma rare in tortoises, no hæmatozoa in lizards, but only exceptionally did he find a frog free from blood-parasites. The Bombay frogs, of which he examined 372, belonged to two species—Rana tigrina and Rana limnocharis, both of which also occur in Ceylon, where, however, we have not yet established the presence of Hæmatozoa.

Besides Drepanidium monilis, Danilewskya krusei, and Trypanosoma, Dr. Berestneff found a parasite which he believed rightly had not been previously described. He gives numerical details, which we regret we cannot clearly understand. There is one table, from which it appears that out of the 372 frogs 47 contained the new parasite; but he mentions other frogs examined in June and July, 1900, which are not included in the table, so that we are unable to calculate the percentage of infected cases. Dr. Berestneff described the parasite as follows:—In the red blood-corpuscles there is to be seen a strongly refringent colourless capsule like a cylindrical tube, with one end enlarged in a club-shaped manner. The parasite lies within the capsule; the attenuated end of the capsule is empty, both ends are rounded and curved, the whole embracing the displaced nucleus of the corpuscle after the manner of Halteridium.

The author states that when the red corpuscle becomes disintegrated or dissolved the capsule withits contained parasite issues into the blood-plasma and straightens itself out, and under these conditions the parasite only occupies a portion of the capsule and eventually begins to move about inside the capsule, gliding from the wider portion into the narrower portion of the latter. Thereupon the motile parasite perforates the capsule at its narrower end and emerges into the plasma actively moving.

Dr. Berestneff says that the parasite closely resembles the free Danilewskya krusei. It is blunt at one end, acuminate at the other; it moves with the blunt wider end directed forwards and the nucleus lies near to the anterior end. The movements are much slower than those of the Drepanidia.

After a few minutes, while under observation, the movements cease, the parasite becomes hyaline, and finally dissolves in the plasma.

He observed simultaneous infection of a corpuscle by the new parasite and by a Drepanidium. In his modification of the Romanowsky method the nucleus of the parasite stained intensely reddish violet, the protoplasm blue, the capsule reddish-violet.

While encapsuled inside the corpuscle the acuminate posterior end of the parasite lies in the widened end of the capsule.

Dr. Berestneff says that the parasite which he discovered in the Bombay frogs belongs by its structure, form, and movements, to the Hæmogregarinidæ, and shows close affinity to Danilewskya krusei, Labbé (Drepanidium m agnum = Hæmogregari n a magna, Grassland Feletti).

Attempts to transmit the parasite from infected to noninfected frogs by injection into the dorsal lymph sinus gave no result.

From the above description, which we have thought well to quote at some length, it seems certain that Dr. Berestneff’s parasite differs at least specifically from that described by us, as shown, to mention only two points, by the relation of the monozoite to its capsule, and by the position of the nucleus. For facility of future reference we propose to name Berestneff’s parasite Hæmogregarina berestneffi. It will probably not enjoy this name long, since the genus Hæmogregarina will most likely be found to have as little stability as Halteridium.

In the light of the discoveries which Schaudinn has recorded these two parasitic forms should be considered together, although we have not yet found them associated in the same host.

Trypanosoma lewisi (Sav. Kent) is extremely common in the house rats (Mus decumanus) of Colombo. Some times the blood is literally seething with them, and it is known that they may equal or surpass the corpuscles of the blood in number. Like Hæmogregarina, the Trypanosoma is chiefly met with in domesticated or semi-domesticated animals. We have not found it in birds, but have met with it in several species of freshwater fishes occurring in the Colombo Lake, a sheet of water in the heart of the town. One of the principal hosts is the siluroid fish, Sacco-branchus fossilis; most of the individuals of this species in the Colombo Lake, from seven to about twelve inches in length, appeared to be infected with a species of Trypanosoma, which we will name, in accordance with the method adopted by MM. Laveran and Mesnil, T. sacco-branchi. Here, as in the rat, the parasites vary greatly in number in different individuals, from very rare to very numerous. In a fresh hanging drop the parasites appeared two or three in the field of the microscope; about two hours later those near the edges of the drop had begun to slow down, so that the movements of the undulating membrane could be observed. These consisted of a rapid shivering along one side of the body. The movements of the membrane determine the serpentine convolutions of the body, which are perpetual. Sometimes the body rests momentarily, only the flagellum and anterior portion keeping up their movements.

Four or five hours later bacteria had penetrated the entire drop, but in a portion of the latter where the bacteria were particularly dense the Trypanosomes had collected together, as many as twenty being visible at a time in the field of the microscope, a few only being found elsewhere in the drop.

In their recent monograph on ‘Trypanosomes et Trypanosomiases,’ Paris, 1904, MM. Laveran and Mesnil have shown how difficult, and in some cases impossible, it is to distinguish one species of Trypanosoma from another on morphological grounds. There is the greatest possible similarity of structure and proportions between the Trypanosoma of rats and those of fishes. Such points as the position of the nucleus and the length of the flagellum can hardly be regarded as sufficiently constant to be diagnostic. The length of the tail or portion of the body behind the centrosome or blepharoplast is rather characteristic, but this, again, fluctuates.

It seems hardly worth while to attempt a detailed differentiation of species from different hosts until some method of culture can be devised which will reveal the true properties of each. There is at present no basis of comparison, and. it seems that none is afforded by the mere retention of Trypanosoma under observation for several days in. citrated blood.

In T. saccobranchi the caudal prolongation is obsolescent, the centrosome being placed very far backwards, much as in T. danilewskyi, Lav. Mesn., from Cyprinus carpio.

Dr. Lingard has recorded the presence of Trypanosoma in the blood of several species of freshwater fishes in India, namely, Trichogaster fasciatus, Ophiocephalus striatus, Macrones seenghala,andMacrones tongara.¹ He noted that the fishes that live in mud are more frequently infected than others, and that the parasites appeared in greatest number during the months of May and June.

We have not seen Dr. Lingard’s original Report in which the above observations were published, but it is to be presumed that he obtained his material in Northern India, since only one of the above-named fishes ranges so far south as Ceylon, namely the Ophiocephalus striatus, known locally as the “lulla.” The examination of the blood of one individual of this species from a tank at Mamadu, in the Northern Province of Ceylon, gave negative results. It would be interesting to examine the blood of a large number of specimens from different tanks, an investigation which, merely from the standpoint of distribution and environment, might yield important information.

With regard to Trypanosoma saccobranchi from the polluted Colombo Lake we have found it abundantly in the months of August and September. On the other hand, the examination of the blood of an Ophiocephalus striatus from the Colombo Lake in May, 1905, gave negative results.

We have also found Trypanosoma in the blood of Macrones cavasius (Siluridæ) and in Gobius giuris.

As a mere matter of fact we may mention that we have never observed Trypanosoma associated with endoglobular parasites in the same host, nor have we found Trypanosoma in any species of animals in which we have found endoglobular forms.

Dr. Hanna has described a Trypanosoma in the blood of the Indian Crow (Corvus splendens), and we have seen Halteridium in this species as well as in the Black Crow (Corvus macrorhynchus), both examined in the month of August, also, as mentioned above, in the common babbler, Crateropus striatus, examined in July, and in the common Scops owl, Scops bakkamœna, var. mala-baricus in July. All these hosts are more or less open to the charge of foul-feeding, and all frequent the neighbourhood, sometimes even the precincts, of human habitations.

MM. Laveran and Mesnil have described Trypanosoma soleæ, from Solea vulgaris, in association with Hæmo-gregarina simondi.¹

Besides Filaria immitis in the dog and Filaria vivi-para, v. Linstow, in the Indian crow, we found a Filaria in the blood of the Brahminy lizard, Mabuia carinata, and described it in our preliminary note under the name of Filaria mansoni.² Dr. von Linstow has since pointed out, in a paper which will shortly be published in ‘ Spolia Zeylanica,’ that this name bad already been applied by Cobbold in 1880 to a species of Filaria from the orbit of Gallus gallinaceus, and he accordingly proposes the new name Filaria tuberosa, so that the species now reads F. man-soni, Castellani and Willey (not Cobbold) = F. tuberosa, v. Linstow. Two adult females were found imbedded in the musculature of the body-wall, one in the ventro-lateral abdominal region, showing through the peritoneum, the other in the dorsal wall of the body cavity.¹

The blood-filariæ of Sauropsida are the embryos of adult Nematoda, which are to be found in the peritoneum of the same host which harbours them. We have never found embryos in the blood without adults in the peritoneum; the females are always ovoviviparous and the males appear to be rare. Both in the Indian crow and in the Brahminy lizard only females were found.

Filariæ occurred also in the blood of a lizard, Calotes versicolor, examined in August, 1904. These were about six times the length of a blood-corpuscle and offered no very striking characters. Two adults, a smaller male anda female about three times larger, were found in the mesentery below the aorta at the level of the testes. Both of them were characterised by the bright lemon-yellow colour of the intestine, and we accordingly proposed the name Filaria flaves-cens, subsequently forwarding the specimens to Dr. von Linstow, who was good enough to confirm the identification, adding further details, which will be duly published.

In the blood of a Scops owl (Scops bakkamœna, var. malabarica) which diedin Colombo in September, 1904, numerous filariæ of an unusually large size occurred,measuring 0·22 mm. in length, the anterior end rounded and sometimes, in the dried film, somewhat withdrawn from the cuticle, the tail acuminate. We name this Filaria scopsiana. Three adults were found in the peritoneum, the largest of which measured 13·25 mm. in length, the smallest less than half this size. The oesophagus was rather indistinct, but appeared to measure between th and th of the total length.

FIG. 1.—Non-pigmented parasite (“pseudoverinicule “) in the blood of a fever case from the Policé Hospital, Colombo [malarial parasites absent; vidal reaction negative]. April 24th, 1905.

FIG. 2.—Another parasite of the same kind; the two dark spots are chromatoid granules. In this case a few ring-forms of tropical malaria were observed, but no crescents. Feb. 17th, 1905.

FIG. 3.—A third example of the same parasite. Stained by Jenner’s method, dark blue near the terminal vacuoles, paler blue in the centre. March 16th, 1905.

FIG. 4.—Analogous “pseudovermicules” in the blood of a common babbler, Crateropus striatus. Sometimes the free parasite appears to be surrounded by a clear halo. Two blood-corpuscles are shown with Halteridium. July 21st, 1904.

FIG. 5.—“Pseudovermicules” in the blood of the Indian crow, Corvus splendens. Colombo, Aug. 9lh, 1904.

FIG. 6.—Hæmocystidium simondi, Castellani and Willey, in the blood of Hemidactylus lescheuaulti.

FIG. 7.—Hæmogregarina nicoriæ, from Nicoria trijuga; one of the corpuscles shows a double infection.

FIG. 8. —Hæmogregarina mirabilis, Castellani and Willey, from Tropidonotus piscator. (a) Normal corpuscle; (b) the pale blue monozoite inside its red sheath; (c) monozoite commencing to issue from the sheath (cytocyst) and corpuscle; (d) monozoite still further emergent, and in this case taking a reddish tinge as though it were coated with a thin film of mucus or residual protoplasm; (e) monozoite nearly free; (f) monozoite free; (g) corpuscle containing an empty cytocyst from which the monozoite has been discharged.

Stained by Leishmann’s modification of Romanowsky’s method.

FIG. 9.—Trypanosoma saccobranchi u. sp. from Saccobranchus fossilis.

FIGS. lOaud 11.—Two embryos of Filaria tuberosa from the blood of Mabuia carinata. The dried blood-film was fixed in absolute alcohol and stained with hæmatoxylin and eosin. The body of the organism is seen to be contracted within its cuticular sheath.

FIG. 12.—Embryo of Filaria flavescens n. sp., from the blood of Calotes versicolor.

FIG. 13.—Embryo of Filaria scopsiana n. sp., from the blood of Scops bakkamosna var. malabarica (Colombo).