1. In addition to the parasite of East Coast fever three other organisms were observed. The first of these is a “symbiont” and was found in the Malpighian tubules and ovaries of all the ticks examined; the second, a protozoan, was observed in about 50 per cent. of our ticks in the process of multiplication in the epithelial cells of the gut and in the large phagocytic cells of the body cavity; the third, from its very active movements, probably a ciliate, was seen only in dark field preparations of a few ticks recently engorged. Once recognised these organisms were always easily distinguished from Theileria parva. We were unable to confirm the assumption of Nuttall and Hindle (1913) that practically 100 per cent of ticks fed on blood containing parasites become infective and are thus capable of transmitting the disease. Approximately accurate figures on the survival of ingested parasites can only be secured from sections. Of the 290 ticks of our standard series examined in very complete serial sections, parasites were found in 178, or 60·1 per cent. In the first series of infective nymphae, in which we kept records of sex, 33 per cent. of the ticks containing parasites were females and 66 per cent. were males.
2. In the erythrocytes of the animals suffering from East Coast fever on which the ticks engorged large and small parasites were distinguishable (Figs. 29 and 30 and Fig. I, p. 32). Both showed signs of division.
3. Intra-erythrocytic parasites were observed in the gut of the ticks only up to the sixth day after engorgement (Table V). Liberation of parasites takes place at a different rate in different ticks.
4. Parasites were seen free in the lumen of the gut from the time of engorgement up to 10,13,14 and 8 days, as indicated in Tables I–IV. Beginning about 2 days after engorgement and until they disappear from the gut there is a marked tendency for the parasites to be arranged in clumps, the small ones together and the large ones together (see Fig. I at 2). But the small and large parasites are sometimes scattered indiscriminately. Both types tend to accumulate on the surfaces of the epithelial cells lining the gut (Fig. I at 3).
5. Many parasites are taken up by epithelial cells which stretch further into the lumen than their neighbours and are called “pedunculated.” They are accompanied by lymphocytes, leucocytes, platelets and a variety of foreign materials. Parasites were observed in this situation from engorgement to 14 days (Table I), from the second to thirteenth day after engorgement (Table II), from the sixth to the twenty-first day after engorgement (Table III), and from the first to the eighth day after engorgement (Table IV). The examinations listed in Table III began on the sixth day. The fact that parasites were not seen earlier in this series in the pedunculated cells does not mean that they were absent. On each day during these rather long periods large and small parasites were observed which stained brilliantly and some of which appeared to be dividing. Other individuals of both types stained but feebly and from their irregular outlines were clearly degenerating. How long individual parasites remained could not be determined. Table I shows their presence in pedunculated epithelial cells 4 days after disappearance from the lumen, and Table III, 5 days after. But in the ticks recorded in Tables II and IV they disappeared from the pedunculated cells on the same day that they could no longer be seen in the lumen.
6. It is the parasites which enter non-pedunculated epithelial cells which carry on the life cycle. They enter in smaller numbers and more slowly. Tables I, II and IV record their appearance in this commoner type of epithelial cells just 7 days after they were first noted in the pedunculated cells. They remain intra-epithelial for as long as 20 days (Table I), 25 days (Table II), 22 days (Table III), and 16 days (Table IV). As a rule they are not accompanied by leucocytes and different sorts of debris. The small forms soon disappear. Large forms like those seen in the lumen are at first present in considerable numbers and give rise to a parasite distinctly different from any previously seen, which we call a “zygote,” although we have been unable to demonstrate the process of fertilisation. These zygotes have rather irregular outlines, stain blue by Giemsa's method and are for a time without red-staining nuclei. In this condition they are represented in Figs. 1–5, 40 and 41 (a–d); also in Fig. I at 4 and 5. In this phase of the zygote the parasites have to resist the digestive action of the cell containing them and many succumb, but some grow; their diameter increasing 300 or 400 per cent. (compare Figs. 1 and 5).
The surviving ones begin to exhibit once more characteristically red-staining nuclei about 18 days after engorgement and at approximately the time of moulting (Figs. 6, 41, e–h). Coincident with this change their outlines become smooth and they are evidently less susceptible to digestion. Oökinetes develop very rapidly within their interior (Figs. 42–49 and Fig. I at 6). All stages in the formation of oökinetes from blue-staining zygotes to others which are fully differentiated and have escaped from the epithelial cells of the gut into the body cavity are frequently seen in a single tick. Oökinetes were observed from the first to the eighth day after moulting in sections and to the ninth day in smears (Table II), from the first day before moulting to the eleventh day after (Table III), and from the second day before to the ninth day after (Table IV). They are illustrated in Figs. 7, 11–14 and 49.
7. The oökinetes begin to enter the salivary glands about the time of moulting. Our failure to detect them in this situation in our larval series (Table I) until 11 days after moulting is a negative observation, possibly due to technical difficulties, and of little significance compared with their definite discovery in the salivary glands 1 day after moulting (Table II), 1 day before moulting (Table III), and 1 day after moulting (Table IV). But this entry is probably not restricted to the days mentioned, for typical ookinetes were observed 9 days (Table II), 12 days (Table III) and 10 days (Table IV) after they were first noted in the salivary glands. It is therefore probable that successive crops of oökinetes enter the salivary glands over a relatively long period and that this explains the discovery of parasites in different stages of development in the salivary glands of individual ticks. Soon after penetration they round up but can be recognised at least until 5 days after moulting by their intensely blue-staining cytoplasm and red-staining nuclei (Figs. 15, 50, 51).
The next stage, which we refer to as a beginning “sporont,” is represented in Figs. 16, 52, 54 and to the right in Fig. I at 8. The nucleus of the oökinete is lost, but the parasite can be identified within the salivary gland cells by its intense deep blue colour when stained by Giemsa's method, by the clear halo about it, and by its relatively large size. The salivary gland cell containing it is usually considerably distended. This condition persists for a variable time which we estimate at about 3 days. Approximately 8 days after moulting buds appear on the surface of the sporont (Figs. 17 and 53) and spherical masses within its interior (Fig. 55) which constitute the “sporoblasts.” At the same time the central part of the parasite stains less intensely and the halo about it becomes barely distinguishable (Fig. I at 10). A little later on the central part stains still less strongly and the sporoblasts become proportionally more clearly defined (Fig. 18). Red-staining chromatin reappears in irregular masses in the sporoblasts (Fig. I at 11). This, in our experience, is as far as the parasite develops before the ticks containing it are placed on animals with a view to transmitting East Coast fever. During the first 3 days of feeding on susceptible animals the parasites were examined in great detail.
After 1 day a total of ten ticks were studied in serial sections, of which three contained parasites in their salivary glands. All of them were in the sporont stage. The central area of the sporont shown in Fig. 19 is developed beyond the condition illustrated in Fig. 18. It is less dense, it is stained a light bluish green and the sporoblasts have moved from it to the periphery. A careful search was made of the digestive tracts of all three ticks. No blood cells could be detected. The lumina were not sufficiently dilated to suggest the intake of cell-free plasma or lymph.
After 2 days four out of ten ticks examined contained parasites. In three of them sporonts were observed which apparently did not differ from those just described; but in the fourth (an adult female) the sporoblasts were further separated from the parent mass—a change which seemed to be preliminary to the more radical development noted the next day. In this particular tick no blood cells could be detected in the lumen of the digestive tract, which, however, was slightly but distinctly dilated perhaps by fluid from the animal on which the tick fed.
After 3 days a striking alteration occurred in the organisms in all five of nine ticks parasitised as illustrated in Figs. 57 and 58. The red-stained chromatin represented in Fig. 57 which before (Fig. 56) was distributed irregularly is becoming arranged in small masses at the surfaces of the sporoblasts. The differentiation of these chromatin masses is still further advanced in Fig. 58, though it illustrates conditions in the same tick. The masses have in many cases broken loose from the sporoblasts, and each is seen to be made up of some blue-stained cytoplasm in which a single spherical or cup-shaped nucleus is embedded. These are the sporozoites. While this development of sporozoites in association with the sporoblasts was observed in all of the five ticks wellformed sporonts like those illustrated in Figs. 18 and 19 were only seen in one. All of the ticks seemed to contain a slightly increased amount of fluid in their digestive tracts, but in only one could ingested leucocytes be recognised.
The period from 4 to 18 days is divisible into two parts: before engorgement (Figs. 20–25, 60–67) and after engorgement (Figs. 26–28, 68, 69).
Before engorgement the maximum production of sporozoites takes place. At 4 days the process is going on with extreme rapidity. Figs. 66 and 67 show two active sporoblasts with sporozoites radially arranged about their periphery. The morphology of the sporozoites on the fifth day is well shown in Fig. 59. When first formed they are somewhat larger than when later on they are ready for discharge. This difference is illustrated in Fig. 23 and in Fig. I (compare stages 12 and 13).
At 5 days larger sporoblasts were noted (Figs. 60–65), and the sporonts are broken up by the spreading apart of the sporoblasts through the tremendous development of sporozoites (Figs. 20–22). Fig. 24 represents a mass of sporozoites apparently ready for discharge into the lumen of the acinus on the right.
The maximum number of salivary gland cells in any tick containing parasites at any stage in the cycle is about 50 per cent. Such an infestation is, however, very exceptional, occurring in only four or five of the fifty-nine ticks of our standard series possessing parasites in this location. The usual infestation is about 8 per cent. In some ticks, however, parasites are only to be found in one or two cells after long search; that is to say, in a percentage of, say, 0·1 per cent. But when the infestation is so small the individual cells involved may be charged with parasites just as heavily. By far the heaviest cellular charge with parasites, though not the highest percentage infestation, was discovered in five out of seventeen female ticks found naturally attached to an animal in a region in which East Coast fever was very common. The degree of infestation probably depends upon the number of ookinetes which settle in the salivary glands and the individual cellular charge upon adverse or favourable conditions in the salivary glands to the development of parasites.
After engorgement both the percentage infestation and the cellular charge are apparently reduced. The conditions 6, 8, and 11 days after attachment are illustrated in Figs. 27, 28 and 68 and 69. Many of the parasites are obviously degenerating, and the whole mass of parasites may be sloughed off from the cell into the lumen of the acinus (Fig. 29). The last parasites were seen 18 days after the tick containing them was placed on a susceptible animal (Table IV), which is long past the time of probable transmission of the disease. Our experiments do not show how much longer they persist and it is likely that they are soon destroyed, because it has been proved experimentally by other investigators that the ticks are cleansed by feeding.
8. Three of our four series of ticks, which contained parasites in their salivary glands, which were fed on susceptible animals, produced typical cases of East Coast fever. It is probable that the other series (Table II), which possessed parasites in equal abundance, would also have transmitted the disease if the animal infested had not died so soon under chloroform anaesthesia as we were removing a lymph gland for study.
