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Studies on the exoerythrocytic stages of Plasmodium gallinaceum during the “transitional phase”
Studies on the Exoerythrocytic Stages of P2usmodiz.m gallinaceum during the “Transitional Phase” Clay G. Huff* Prom
the Naval
Medical
Research
Znstitute,
Bethesda,
Md.
The discovery that parasitemia, in the case of certain mosquitoinduced malarial infections, is preceded by a phase of development in fixed tissue cells has raised many new questions. Some of the most interesting of these questions have to do with what may be called the “transitional phase” or the period during which the infection transfers from the fixed tissues to the erythrocytes. It is now known that in sporozoite-induced infections with Plasmodium cathemerium (Porter, 1942) and P. gaEZinaceum (Huff and Coulston, 1944) the earliest preerythrocytic stages are found predominantly in cells of the lymphoidmacrophage system and that there is then a shift in cell preference to the endothelial cells of blood vessels, particularly of the capillaries. There is, next, a shift from the endothelial cells to the erythrocytes that spectacularly manifests i&elf by an increase in parasitemia of some hundredfold within the space of a few hours. This latter phenomenon was described by Huff and Coulston (1944), who referred to it as the “flooding effect.” This paper will be concerned with the following matters pertaining to the “transitional stage” of infections with P. gaZlinaceum: (1) relative numbers of different stages of exoerythrocytic development in the various organs of the host; (2) the distribution of exoerythrocytic stages during the “flooding effect,” (3) the significance of macro- and micro-schizonts; and (4) degenerative changes in the exoerythrocytic stages during the acute parasitemia. MATERIALS Most mod&m
of the data gallinaceum
in this (strain
paper 8A).
AND METHODS
were obtained However, for
from experiments involving extension of, and addition
Plasto, the
* Grateful acknowledgement for assistance is made to Mrs. Dorothy F. Marchbank, Mrs. Agnes H. Saroff, Mr. Paul W. Scrimshaw, and Miss Tsugi Shiroishi. The opinions and statements contained herein are the private ones of the writer and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 392
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
GALLINACEUM
393
results obtained, I have drawn freely upon work done on P. lophurae, P. jallax, P. cathemerium, and P. relictum, extending over a period of ten years. The methods used in growing, infecting, and dissecting mosquitoes, in the inoculation of sporozoites, fixation, embedding, cutting and staining of sections have been described in a previous publication (Huff and Coulston, 1944). The measurement of merozoite size was carried out by a method recommended by Dr. Marshall Hertig. Drawing paper was ruled off with lines corresponding wit-h the lines on the ocular micrometer disc and representing a magnification of 10,000 times. Merozoites lying parallel to the line of vision were so chosen that an optical cross section could be seen. By moving the slide the merozoite to be measured was placed so that its image lay between the lines on the ocular micrometer. The merozoites were then drawn between the ruled lines on the drawing paper in the proportions observed. It was then possible to measure the drawing and convert the readings to microns. RESULTS
I The Onset of Parasitemia
in Sporozoite-Indticed
Infections
In infections of chicks induced by the inoculations of 100 or more pairs of salivary glands containing sporozoites of P. gallinaceum Huff and Coulston (1944) observed that the parasitemia was visible in stained smears taken 4% or 5 days after the inoculation. On the 6th to 8th post-inoculation days an increase of the number of erythrocytic parasites of approximately 200 times occurred within a 24-hour period. Since this increase was many times what could be accounted for by the multiplication of the existing erythrocytic stages it was necessary to conclude that there was a period of sudden transition from tissue- to bloodhabitation on the part of the parasites. This phenomenon was referred to as the “flooding effect.” Since that time it has been observed many times. In spite of numerous attempts, however, to observe the parasitemia and then to kill the bird in order to study the corresponding infections in the tissues, only two infections have been suitable for the combined study. These two infections (birds 331 and 333) serve as the basis for the data presented herein. Chick B331 was inoculated intravenously with the salivary glands from 70 Aedes aegypti infected with P. gallinaceum. The prepatent period was 88 hours and the bird died 69 f 3 hours after the first observed parasitemia, or 157 f 3 hours after inoculation. The salivary glands from 40 infected Aedes aegypti were intravenously inoculated into chick # 333. The prepatent period was 108-36 hours. The bird was killed 53 hours after the first observed parasitemia, or 161-g hours after inoculation. Figure 1 represents the parasite counts from smears taken at 6-hour intervals
394
CLAY
G.
HUFF
During the final 24 hours that chick 331 lived there was a threefold increase in the numbers of parasites in the blood. Inasmuch as it died when the ratio of parasites to erythrocytes was low (57/10,000) it appears probable that its death was caused by the exoerythrocytic stages. On the other hand the number of parasites in the blood of chick 333 underwent an increase of 87-fold during its last 24 hours of life. It was killed when the parasite-erythrocyte ratio was 3549/10,000. It would seem
FIG. 1. Parasitemia following inoculation of large numbers of sporozoites P. gallinaceum intravenously into chicks ( #331 and S333).
of
reasonable to assumethat chick 331 died at the beginning of the “flooding” stage whereas chick 333 was killed near the peak of this stage. These possible conditions need to be kept in mind in interpreting the observations made on the tissues of the two animals (Fig. 1). Distribution of Exoerythrocytic &yes in the Organs The organs of the two birds mentioned above were fixed in Zenker form01 fixative, embedded in celloidin and stained with Maximov’s hematoxylin-eosin-aeur stain. The numbers of exoerythrocytic parasites per square millimeter of tissue are shown in Table I. Owing to the great differences which exist in the structure and degree of heterogeneity of
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
395
GALLlNACEUM
the organs the significance of the relative numbers of exoerythrocytic stages counted per square millimeter is difficult to assess. However, the order of the relative numbers in the various organs is fairly consistent in the tissues of the two birds studied except in kidney and spleen. Relative Numbers
of D@erent Exoerythrocytic Stages
Counts were made of the numbers of the various exoerythrocytic stages in the various tissues of the same two birds but without regard to abundance per unit of area. At first, tabulations were made for several categories of stages, i.e., (1) small trophozoites, (2) medium schizonts, TABLE Distribution
of
Exoerythrocytic Chicks Infected
Stages by
Means Bird
Parasites per mm2 of section
Organ
Kidney, ........... Lung. .............. Heart. ............. Liver ............... Spleen ............. Brain. ............. Testis. ............. Intestine. .......... Thymus............
.. . . .. ..
I
of Plasmodium of Large Doses
gallinaceum of Sporozoites
#331
Bird
Relative order of frequency
17 46 53 11 60 9
4 3 2 5 1 6
3
7
..
Parasites per mm’ of section
64 62 26 20 19 10 4 3 2
in
Organs
of
1333 Relative order of frequency
1 2 3 4 5 6 7 8 9
(3) large schizonts with no evidence of incipient merozoite formation, (4) schizonts with evidence of incipient merozoite formation, (5) segmenters with macromerozoites, (6) segmenters with intermediate merozoites and (7) segmenters with micromerozoites. It was soon discovered that accurate separation of parasites could not be made into all of these categories because of the absence of definite criteria for such separations. Consequently, the data are presented here in only three categories: (1) small trophozoites (uninucleate) ; (2) large schizonts (without division of cytoplasm) ; and (3) segmenters (with evidence of cytoplasmic division). These data are presented in Table II, together with the percentages of corresponding erythrocytic stages in the peripheral blood at the time of death of the host and fixation of its tissues. Analysis of these data will be presented in the Discussion. It will
396
CLAY
G.
HUFF
suffice at this point to call attention to (1) the low percentages of small schizonts in the organs as compared with the blood, (2) the fairly equal distribution of the larger schizonts and segmenters among the different organs, and (3) t,he large percentage of small schizonts in the lung of bird B 333 as compared with the percentages in other organs. TABLE Relative
Numbers of Various in the Organs of Chicks
II
Exoerythrocytic Stages during “Transitional” Bird
Brain, . . Lung, _. . . . . . Kidney.. . .. .. Liver. . . . Spleen. . . . .. Heart. .. . Thymus. . Testis . Intestine.............................. All
.. . .. . . .. . ... . . .. . .. . .
.
.. .
.. .
organs.............................
Erythrocytic stages death of host.
at time
rY331; percentage
Small trophozoites
Organ
of Plasmodium gallinaceum Phase of the Infection of:
Large schizants _~
Segmenters
2 2 0 0 0 0 0
68 24 49 63 61 57 72
30 74 51 37 38 43 28
0.4
55.7
44.1
48.0
39.0
13.0
Bird
~333; percentage
Sld: trophozoites -.~
of:
Large schizants
Segmenter-
63 31 64 36 60 81 68 63 62
31 37 35 64 37 27 30 37 38
8.0
56.9
35.1
99.0
1.0
6 32 1 0 3 2 2 0 0
of
Degenerative Changes in Exoerythrocykic
0
Stages during Acute Parasitemia
In the careful study of individual exoerythrocytic stages necessary to counting and categorizing them it was noticed that a certain portion of them appeared to be undergoing degenerative changes. Since the cytological appearance of the normal exoerythrocytic stages is highly variable and since all degrees of intermediate condition between the completely normal and the obviously dead micro-organism exist, any separation into two categories, normal and degenerating parasites, is subject to observational error. In making this separation I had the advantage of having in the past examined many thousands of exoerythrocytic stages of several avian malarial parasites: (1) in susceptible as well as relatively naturally immune hosts; (2) under the influence of immune substances elaborated in the course of infection; and (3) in hosts which had been sub-
EXOERYTHROCYTIC
STAGES
OF. PLASMODIUM
397
GALLINACEUM
jetted to treatment with various antimalarial drugs. To minimize the possibility of subjective error I set up the following criteria based on this experience for evidence of degenerating parasites. In the merozoites the normally elongate shape may change to spherical, and the normally distinct margins may become indistinct. In the large schizonts the vacuoles representing nuclei, normally regular in size and discrete in arrangement, may become irregular in size and confluent. The granular cytoplasm may come to contain numerous irregular, dark masses. Some of the criteria for degeneration of the segmenters are: (1) merozoites packed into a masssurrounded by a large clear area of fluid; (2) color of TABLE Percentage
of Degenerating
Exoerythrocytic of Chick
III Stages %SS3
.. .
... .
in the Tissues
Percentage of degenerating parasites
organ
Kidney. . Spleen. Thymus. Lung...... Liver...... Heart. Testis. Brain. Intestine.
of P. gallinaceum
63 58 53 44 42 36 23 17 12
merozoites gray or dirty blue instead of bright azure; (3) a dark conglomerate masswith irregular dark blue massesrepresenting merozoites; and (4) entire parasite broken into fragments with indistinct edges. Since the exact hour of death for chick # 331 was not known no counts of relative numbers of norma and degenerating parasites were made from its tissues,for in it we could not distinguish between degenerative changes taking place in the parasites in the living host and degenerative changes presumably due to post mortem change in the host itself. Table III presents the data from chick B 333. Despite the possibility of subjective error in determining the percentage of parasites actually undergoing degeneration it is probable, that the chances for normal growth of exoerythrocytic stages vary in the different organs. It is difficult, however, to reconcile the findings in the various organs with anything now known or assumed about (1) the relative nutritive value to the parasites of the
398
CLAY
Q.
HUFF
cells of the various organs or (2) the relative importance of the different organs in the production of humoral antibodies against malaria. Obviously this study should be extended to other parasite-host combinations and to the entire course of infection before the significance of the differences in the degenerative changes occurring in the various organs can be determined. Significance of Dimorphism
of Xchizonts
Reichenow and Mudrow called attention in a paper published in 1943 to the presence of a dimorphism in the kinds of merozoitcs produced in I’. praecoz (= relicturn) in canaries. They applied the terms, macromerozoites and micromerozoites, to two categories of merozoites based upon whether they were large or small; and the terms, macroschizonts and microschizonts, respectively, the mature schizontjs giving rise to the two types of merozoites. The same phenomenon was observed in 1942 in P. gallinaceum by Huff and Coulston whose publication was delayed until 1944 by security classification. Reichenow and Mudrow (1943) and Mudrow and Reichenow (1944) interpreted this dimorphism as indicating that the macromrrozoites were destined to produce other exoerythrocytic stages while the micromerozoites would produce erythrocytic stages.. Since the explanation of the nature and significance of these two types of schizonts and their corresponding merozoites is likely to be important in our understanding of the interrelations of erythrocytic and exoerythrocytic stages, of the phenomena of relapse and latency, and the efficacy of chemotherapy, attempts have been made to study them from various points of view. It was stated above that difficulty was encountered in making accurate counts of the various types of segmenters. Therefore it became necessary to examine carefully the concept that two categories of segmenters exist. Actual measurements were made of the diameters of five merozoites from each segmenter found in exploring the microscopic fields while studying the various organs of the host. These findings are given in ocular micrometer units in Table IV and the collected data are shown in Fig. 2. It is at once obvious that no dimorphism exists in the merozoites but that the groups referred to as macro- and micromerozoites merely constitute the extremes of a population showing the normal amount of variation. This condition existed in each organ of the .host studied as well as for the combined results. The differences in the mean diameters of the merozoites in the various organs are within the observational error. It should he noted that the data in Table IV are expressed in ocular micrometer
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
TABLE Diameters
of Merozoites
from
Number organ
* In ocular
micrometer
a diameter*
2
52 29 34 55 42 57 19 32 34
14 10 16 34 11 38 21 42 15
4
11 14 3 6 12
34 23 61 24 22 17 18 17 11
1 1 1 10 9 7 1
7
111
227
354
201
34
units.
6
10 13 42
The factor
X 0.116 is needed
to convert
the Organs
I
9
5
seen in
of:
8
5
Totals.
having
Segmenters
7
4
Spleen. Liver. . Brain. . . Heart. . Kidney. Lung. Thymus Intestine Testis.
IV
Randomly Selected of Chick #933 -
of merozoites
399
GALLINACEUM
10
I Number measured
Meall size*
114 75 154 114 87 152 74 110 75
.67 .65 .62 .66 .66 .72 .74 .74 .67
11 4 6
21
I
955
1
.68
top.
3s 3*
301 J-
25l I-
20( )-
f 5
l5( I-
% I: IOC)-
tic I-
II4
-
6 5 Oiometer of Merozoites (in ocular micrometer umts 1
FIG. 2. Frequency in all organs of chick the factor: 0.116).
distribution of diameters of exoerythrocytic # 333. (To convert to p multiply ocular micrometer
merozoites units by
400
CLAY
G. HUFF
units which may be converted to microns by multiplying the former by 0.116. This gives a mean diameter for the merozoites from the collected data of 0.795 f 6004 I*. DISCUSSION
Although the first generation of pre-erythrocytic stages (cryptozoites) appears to be restricted to the cells of the lymphoid-macrophage system and, therefore, to be encountered most often in the organs well endowed with these cells, Coulston, Cantrell, and Huff (1945) showed that the distribution of pre-erythrocytic stages of P. gallinaceum after 36 hours was not an indication of sporozoite localization. These authors tested the presence of pre-erythrocytic stages in organs of chicks which had been intravenously inoculated with large doses of sporozoites by removing tissues from the inoculated animals, placing them into the body cavity of normal chicks, and studying the latter for the development of infections. During the first 36 hours following inoculation of sporozoites the spleen, lung, kidney, liver, and pancreas of the inoculated animals contained parasites capable of producing infection in the subinoculated animal. Since the circulating blood of such animals was free from parasites between the 36th and 70th hours after sporozoite inoculation, experiments involving subinoculation of tissues were valid. All tissues tested except brain and bone marrow (i.e., heart, spleen, kidney, liver, thymus, pancreas, lung, and intestine) indicated that infective parasites were present. No real significance can probably be attached to the fact that the brain and bone marrow in those experiments failed to produce infection in subinoculated animals beyond the fact that parasites were probably scarce and, therefore, that success would be realized in a low percentage of trials with them. The results in the present study indicate that the brain, testis, intestine, and thymus (the bone marrow was not studied) contained small numbers of parasites as compared with kidney, lung, heart, liver, and spleen. Although the relative order of the degree of infection in the organs is fairly consistent between the two birds studied, a much larger study would be required before significance could be attached to the differences observed. In common with Porter’s (1942) observations of P. cathemerium, there are no indications in the present study or in any of my previous studies on P. gallinaceum that the brain is the preferred site of localization of exoerythrocytic stages. This belief has probably arisen from two facts: (1) that some investigators study the exoerythrocytic stages
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM.
GALLINACEUM
401
mainly by the use of smears from brain which can be more successfully made than from most other organs, and (2) that the birds with extreme brain involvement are more likely to die and thus to be seen at the height of the involvement of the brain capillaries. Since we are concerned here with the behavior of exoerythrocytic stages during the transition from tissue parasitism to parasitemia, an evaluation of the relative numbers of different stages in fixed tissues of the various organs and in the blood is a possible approach to the solution of some of the problems relating to that period. In each of the two birds studied the parasites in the peripheral blood were rather remarkably out of phase with the exoerythrocytic stages of the organs. The large percentage of young trophozoites in the peripheral blood is in contrast to the small percentage of young exoerythrocytic trophozoites of the tissues and similarly the large percentage of exoerythrocytic schizonts in the tissues contrasts with the very low percentage of erythrocytic schizonts. This would appear to indicate that not only are large numbers of exoerythrocytic stages changing to erythrocyte-inhabiting forms but that relatively few of them are continuing as ‘exeerythrocytic stages. Some of the explanation for the small numbers of young exoerythrocytic stages may reside in the difficulty of finding these stages in sections since they present no contrast in staining to that of the cytoplasm of host cells. However, in certain tissues such as brain and kidney, the chances of overlooking these small forms are much less than in spleen, liver, or heart, yet this fact is not reflected in a correspondingly higher number of small forms counted in brain and kidney. The relatively large percentage of small exoerythrocytic stages in the lung of bird I333 is of interest in that it may indicate a possible predilection for lung tissue on the part of the parasites. In this connection it should be noted that Coulston et al. (1945) found the lung more consistently infected between 48 and 79 hours following mosquito bites, than any of the other organs. It would seem, however, that this point requires much more study before it can be clarified. There was no evidence of localization of the schizonts in any particular organ. The presence of exoerythrocytic stages undergoing degenerative changes has previously been noted (1) in primary infections of P. Zophuraein chickens, ducks, turkeys, and guinea fowl (Huff, Coulston, Laird, Porter, 1947); (2) in primary infect,ions of P. relicturn, and P. gallinaceum in ducks (Huff, 1951); and (3) in P. gallinaceum in chickens which were treated with antimalarial drugs (Coulston and Huff, 1948).
402
CLAY
G.
HUFF
The report herein of similar degenerative changes in primary infecticns of P. gallinaceum in chicks, which represent one of the most, highly susceptible of hosts, indicates that the mechanisms of acquired immunity probably begin to exert their effect on exoerythrocytic st.ages fairly early in infections induced by large inoculums. Since the initial observations of a dimorphism in the schizonts of exoerythrocytic stages of P. praecox (‘= relicturn) (Reichenow and Mudrow, 1943) and of P. gaZEinaceum (Huff and Coulston, 1944) there has been a general tendency on the part of many authors to claim that macromerosoites are destined to continue the exoeryt)hrocytic cycle while micromerozoites enter red cells and initiate the parasitemia. This belief derived from (1) the fact that the pre-erythrocytic stages were predominantly of the macroschizont type, (2) the observed shift during the course of the infection from a preponderance of macroschizonts to a preponderance of microschizonts, and (3) the similarity between micromerozoites and the merozoites in erythrocytic schizont’s. If the findings herein reported that two categories of schizonts do not exist prove to hold for other species of malarial parasites and for other stages in the infection, a change of viewpoint will be required in attempting to explain the transformations which are known to occur from exoerythrocytic parasitism to pa.rasitemia and vice versa. In the light of the result of this study it would appear that the macromerozoites and micromerozoites merely represent the extremes in size of a population of merozoites of varying sizes in which there is a complete series of connecting forms. The latter are .probably usually neglected in examination of sections of infected Organs because one is not faced with making quantitative separation of the merozoites into categories. Both Mudrow and Reichenow (1944) and Geigy and Britschgi (1950) observed (the former authors in P. praecox; the latter in P. gallinaceum) that some microschizonts mature at an early stage and produce a number of merozoites barely larger than the number produced by macroschizonts. The latter authors made a quantitative study of the number of nuclei in the various sizes of macro- and microschizonts. Their macroschizonts had a high proportion which were small in size (l-20 merozoites), a medium proportion medium (21-40 merozoites), and a small proportion which were large (over 40 merozoites), whereas these proportions were almost exactly reversed in the microschizonts. These findings can be briefly summarized by the statement that the merozoites tend to be large in small-sized schizonts and small in large-sized schizonts. However, they also indicate
E~~ER~THRocYTw
STAGES
OF
PLASMODIUM
GNAJNACEUM
403
that the authors were dealing with a population with continuity of valiation in size which they arbitrarily separated into two components. Nothing in the present observations would conflict with the observed facts that macroschizonts are predominant in the pre-erythrocytic stages of the infection while microschizonts are predominant in the later phanerozoic stages of infection if we substitute for the term macroschizont the expression “schizonts with larger mean size,” and for the term “microschizonts” the expression “schizonts with smaller mean size.” The simpler statement would be that merozoite size decreases as a sporozoite-induced infection progresses to the stage in which parasitemia predominates over tissue parasitism. The argument that microschizonts produce erythrocytic stages because of the similarity of size may or may not be pertinent (Mudrow and Reichenow, 1944). I know of no study which shows that the youngest erythrocytic stages are of uniform size throughout the course of a sporozoite-induced infection, or in other words that larger pre-erythrocytic merozoites do not produce erythrocytic infections. In fact, if the larger merozoites should enter erythrocytes and thus appear as larger than the usual first stage erythrocytic trophozoites it seems likely that they might be mistaken for erythrocytic parasites originating from smaller merozoites that had been living in the erythrocytes for several hours. If one assumes that micromerozoites (i.e., the smallest of the merozoites) produce erythrocytic stages and macromerozoites (i.e., the largest merozoites) continue the exoerythrocytic cycle one is left in the dilemma of having to account for the fate of the merozoites of intermediate size which make up fully 80% of the total population. At this stage in the development of our knowledge it is not possible to determine the causes for the change in average size of merozoites as a sporozoite-induced infection progresses. Since, however, we know that there is a marked shift in frequency of occurrence of the exoerythrocytic stages in cells of the lymphoid macrophage system to occurrence in cells of the true endothelium (Porter, 1942; Huff and Coulston, 1944) it would seem most likely that the type of cell inhabited has some effect on the average size of the merozoites. It would then be only a fortuitous circumstance that the so-called micromerozoites happen to be most prevalent at the time that erythrocytic parasites become plentiful enough to study. Such an argument would not be overthrown by the possibility that occasional small merozoites might be found in macrophages or fibroblasts, or large merozoites in endothelial cells, since these might be
404
CLBY G. HTJFF
simply the merozoites on the extremes of size in populations with normal variabilities. The disproof of the existence of a true dimorphism in exoerythrocytic schizonts would not bring us much closer to the explanation of the causes for the shift from tissue parasitism to parasitemia, except to remove a spurious clue. The real explanation probably is as elusive as the explanation of what determines whether a given erythrocytic trophozoite will produce a gametocyte or a schizont. SUMMARY
Studies made during the transitional stage infection of P. gallinaceum ir chicks (i.e., the period of transition from tissue parasitism to parasitemia) have yielded the following results: 1. The relative frequency of exoerythrocytic stagesexpressed as number of parasites per square millimeter of section is fairly consistent for the two birds examined except for the kidney and spleen. The brain ranked low in relation to kidney, lung, heart, and liver as the organ of localization. 2. At the time when the preponderance of parasites in the fixed tissue consisted of large schizonts and segmentersthe preponderance of erythrocytic parasites consisted of young trophozoites. The larger schizonts and segmenterswere fairly equally distributed among the different organs whereas in one of the birds 32ye of the exoerythrocytic stages of the lung were small trophozoites. It would appear that not only were large numbers of exoerythrocytic merozoites changing to erythrocytic stages but that relatively few of them were continuing as exoerythrocytic stages. 3. Degenerative changes in the exoerythrocytic stages, possibly due to the development of antibodies by the host, were seen as early as 53 hours after the first observed parasitemia. The type of degenerative change in the parasite was similar to that observed previously as the result of the action of natural immunity and of antimalarial drugs. 4. So-called macromerozoites and micromerozoites were found to be only the extremes in size of a population of merozoites with normal variation in size. Thus, no dimorphism exists in the schizonts of this species. 5. It is believed that, although no dimorphism of the schizonts exists, the average size of the merozoite decreasesduring the transitions from a condition in which the host cells are predominantly of the lymphoidmacrophage system to the condition in which they are predominantly
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
GALLINBCEUM
405
endothelial in type, and then to the stage where the erythrocytes are the cells which are predominantly inhabited. 6. The prevalent view that the smaller merozoites are destined to produce erythrocytic stages while the larger merozoites are destined to continue the exoerythrocytic cycle has not been adeqrately proved. REFERENCES
W., AND HUFF, C. G. 1945. The distribution and localiand pre-erythrocytic stages in infections with PlasmoJ. Infectious Diseases 76, 226238. COULSTON, I?., AND HUFF, C. G. 1948. Symposium on exoerythrocytic forms of malarial parasites. IV. The chemotherapy and immunology of pre-erythrocytic stages in avian malaria. J. Parasitol. 34, 290-299. GEIGY, R., AND BRITSCHGI, H. 1950. Untersuchungen tiber die E-formen vor Plasmodium gallinaceum in Organen des Htihnchens am 9/10. Infektionstag. Rev. suisse 2001. 67, 526-532. HUFF, C. G. 1951. Observations on the pre-erythrocytic stages of Plasmodium relictum, P. cathemerium, and P. gallinaceum in various birds. J. Infectious Diseases 88, 17-26. HUFF, C. G. AND COULSTON, F. 1944. The development of Plasmodium gallinaceum from sporozoite to erythrocytic trophozoite. J. Znjectious Diseases 76, 23249. HUFF, C. G., COULSTON, F., LAIRD, R. L., AND PORTER, R. J. 1947. Pre-erythrocytic development of Plasmodium lophurae in various hosts. J. Infectious Diseases 81, 7-13. MUDROW, L., AND REICHENOW, E. 1944. Endotheliale und erythrocytiire Entwicklung von Plasmodium praecox. Arch. Protistk. 97, 101-170. PORTER, R. J. 1942. The tissue distribution of exoerythrocytic schizonts in sporozoite-induced infections with Plasmodium cathemerium. J. Infectious Diseases 71, I-17. REICAENOW, E., AND MUDROW, L. 1943. Der Entwicklungsgang von Plasmodium praecox im Vogelkorper. Deut. Tropenmed. 2. 47, 289-299. COULSTON,
zation dium
F.,
CANTRELL,
of sporozoites gallinaceum.
the Naval
Medical
Research
Znstitute,
Bethesda,
Md.
The discovery that parasitemia, in the case of certain mosquitoinduced malarial infections, is preceded by a phase of development in fixed tissue cells has raised many new questions. Some of the most interesting of these questions have to do with what may be called the “transitional phase” or the period during which the infection transfers from the fixed tissues to the erythrocytes. It is now known that in sporozoite-induced infections with Plasmodium cathemerium (Porter, 1942) and P. gaEZinaceum (Huff and Coulston, 1944) the earliest preerythrocytic stages are found predominantly in cells of the lymphoidmacrophage system and that there is then a shift in cell preference to the endothelial cells of blood vessels, particularly of the capillaries. There is, next, a shift from the endothelial cells to the erythrocytes that spectacularly manifests i&elf by an increase in parasitemia of some hundredfold within the space of a few hours. This latter phenomenon was described by Huff and Coulston (1944), who referred to it as the “flooding effect.” This paper will be concerned with the following matters pertaining to the “transitional stage” of infections with P. gaZlinaceum: (1) relative numbers of different stages of exoerythrocytic development in the various organs of the host; (2) the distribution of exoerythrocytic stages during the “flooding effect,” (3) the significance of macro- and micro-schizonts; and (4) degenerative changes in the exoerythrocytic stages during the acute parasitemia. MATERIALS Most mod&m
of the data gallinaceum
in this (strain
paper 8A).
AND METHODS
were obtained However, for
from experiments involving extension of, and addition
Plasto, the
* Grateful acknowledgement for assistance is made to Mrs. Dorothy F. Marchbank, Mrs. Agnes H. Saroff, Mr. Paul W. Scrimshaw, and Miss Tsugi Shiroishi. The opinions and statements contained herein are the private ones of the writer and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 392
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
GALLINACEUM
393
results obtained, I have drawn freely upon work done on P. lophurae, P. jallax, P. cathemerium, and P. relictum, extending over a period of ten years. The methods used in growing, infecting, and dissecting mosquitoes, in the inoculation of sporozoites, fixation, embedding, cutting and staining of sections have been described in a previous publication (Huff and Coulston, 1944). The measurement of merozoite size was carried out by a method recommended by Dr. Marshall Hertig. Drawing paper was ruled off with lines corresponding wit-h the lines on the ocular micrometer disc and representing a magnification of 10,000 times. Merozoites lying parallel to the line of vision were so chosen that an optical cross section could be seen. By moving the slide the merozoite to be measured was placed so that its image lay between the lines on the ocular micrometer. The merozoites were then drawn between the ruled lines on the drawing paper in the proportions observed. It was then possible to measure the drawing and convert the readings to microns. RESULTS
I The Onset of Parasitemia
in Sporozoite-Indticed
Infections
In infections of chicks induced by the inoculations of 100 or more pairs of salivary glands containing sporozoites of P. gallinaceum Huff and Coulston (1944) observed that the parasitemia was visible in stained smears taken 4% or 5 days after the inoculation. On the 6th to 8th post-inoculation days an increase of the number of erythrocytic parasites of approximately 200 times occurred within a 24-hour period. Since this increase was many times what could be accounted for by the multiplication of the existing erythrocytic stages it was necessary to conclude that there was a period of sudden transition from tissue- to bloodhabitation on the part of the parasites. This phenomenon was referred to as the “flooding effect.” Since that time it has been observed many times. In spite of numerous attempts, however, to observe the parasitemia and then to kill the bird in order to study the corresponding infections in the tissues, only two infections have been suitable for the combined study. These two infections (birds 331 and 333) serve as the basis for the data presented herein. Chick B331 was inoculated intravenously with the salivary glands from 70 Aedes aegypti infected with P. gallinaceum. The prepatent period was 88 hours and the bird died 69 f 3 hours after the first observed parasitemia, or 157 f 3 hours after inoculation. The salivary glands from 40 infected Aedes aegypti were intravenously inoculated into chick # 333. The prepatent period was 108-36 hours. The bird was killed 53 hours after the first observed parasitemia, or 161-g hours after inoculation. Figure 1 represents the parasite counts from smears taken at 6-hour intervals
394
CLAY
G.
HUFF
During the final 24 hours that chick 331 lived there was a threefold increase in the numbers of parasites in the blood. Inasmuch as it died when the ratio of parasites to erythrocytes was low (57/10,000) it appears probable that its death was caused by the exoerythrocytic stages. On the other hand the number of parasites in the blood of chick 333 underwent an increase of 87-fold during its last 24 hours of life. It was killed when the parasite-erythrocyte ratio was 3549/10,000. It would seem
FIG. 1. Parasitemia following inoculation of large numbers of sporozoites P. gallinaceum intravenously into chicks ( #331 and S333).
of
reasonable to assumethat chick 331 died at the beginning of the “flooding” stage whereas chick 333 was killed near the peak of this stage. These possible conditions need to be kept in mind in interpreting the observations made on the tissues of the two animals (Fig. 1). Distribution of Exoerythrocytic &yes in the Organs The organs of the two birds mentioned above were fixed in Zenker form01 fixative, embedded in celloidin and stained with Maximov’s hematoxylin-eosin-aeur stain. The numbers of exoerythrocytic parasites per square millimeter of tissue are shown in Table I. Owing to the great differences which exist in the structure and degree of heterogeneity of
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
395
GALLlNACEUM
the organs the significance of the relative numbers of exoerythrocytic stages counted per square millimeter is difficult to assess. However, the order of the relative numbers in the various organs is fairly consistent in the tissues of the two birds studied except in kidney and spleen. Relative Numbers
of D@erent Exoerythrocytic Stages
Counts were made of the numbers of the various exoerythrocytic stages in the various tissues of the same two birds but without regard to abundance per unit of area. At first, tabulations were made for several categories of stages, i.e., (1) small trophozoites, (2) medium schizonts, TABLE Distribution
of
Exoerythrocytic Chicks Infected
Stages by
Means Bird
Parasites per mm2 of section
Organ
Kidney, ........... Lung. .............. Heart. ............. Liver ............... Spleen ............. Brain. ............. Testis. ............. Intestine. .......... Thymus............
.. . . .. ..
I
of Plasmodium of Large Doses
gallinaceum of Sporozoites
#331
Bird
Relative order of frequency
17 46 53 11 60 9
4 3 2 5 1 6
3
7
..
Parasites per mm’ of section
64 62 26 20 19 10 4 3 2
in
Organs
of
1333 Relative order of frequency
1 2 3 4 5 6 7 8 9
(3) large schizonts with no evidence of incipient merozoite formation, (4) schizonts with evidence of incipient merozoite formation, (5) segmenters with macromerozoites, (6) segmenters with intermediate merozoites and (7) segmenters with micromerozoites. It was soon discovered that accurate separation of parasites could not be made into all of these categories because of the absence of definite criteria for such separations. Consequently, the data are presented here in only three categories: (1) small trophozoites (uninucleate) ; (2) large schizonts (without division of cytoplasm) ; and (3) segmenters (with evidence of cytoplasmic division). These data are presented in Table II, together with the percentages of corresponding erythrocytic stages in the peripheral blood at the time of death of the host and fixation of its tissues. Analysis of these data will be presented in the Discussion. It will
396
CLAY
G.
HUFF
suffice at this point to call attention to (1) the low percentages of small schizonts in the organs as compared with the blood, (2) the fairly equal distribution of the larger schizonts and segmenters among the different organs, and (3) t,he large percentage of small schizonts in the lung of bird B 333 as compared with the percentages in other organs. TABLE Relative
Numbers of Various in the Organs of Chicks
II
Exoerythrocytic Stages during “Transitional” Bird
Brain, . . Lung, _. . . . . . Kidney.. . .. .. Liver. . . . Spleen. . . . .. Heart. .. . Thymus. . Testis . Intestine.............................. All
.. . .. . . .. . ... . . .. . .. . .
.
.. .
.. .
organs.............................
Erythrocytic stages death of host.
at time
rY331; percentage
Small trophozoites
Organ
of Plasmodium gallinaceum Phase of the Infection of:
Large schizants _~
Segmenters
2 2 0 0 0 0 0
68 24 49 63 61 57 72
30 74 51 37 38 43 28
0.4
55.7
44.1
48.0
39.0
13.0
Bird
~333; percentage
Sld: trophozoites -.~
of:
Large schizants
Segmenter-
63 31 64 36 60 81 68 63 62
31 37 35 64 37 27 30 37 38
8.0
56.9
35.1
99.0
1.0
6 32 1 0 3 2 2 0 0
of
Degenerative Changes in Exoerythrocykic
0
Stages during Acute Parasitemia
In the careful study of individual exoerythrocytic stages necessary to counting and categorizing them it was noticed that a certain portion of them appeared to be undergoing degenerative changes. Since the cytological appearance of the normal exoerythrocytic stages is highly variable and since all degrees of intermediate condition between the completely normal and the obviously dead micro-organism exist, any separation into two categories, normal and degenerating parasites, is subject to observational error. In making this separation I had the advantage of having in the past examined many thousands of exoerythrocytic stages of several avian malarial parasites: (1) in susceptible as well as relatively naturally immune hosts; (2) under the influence of immune substances elaborated in the course of infection; and (3) in hosts which had been sub-
EXOERYTHROCYTIC
STAGES
OF. PLASMODIUM
397
GALLINACEUM
jetted to treatment with various antimalarial drugs. To minimize the possibility of subjective error I set up the following criteria based on this experience for evidence of degenerating parasites. In the merozoites the normally elongate shape may change to spherical, and the normally distinct margins may become indistinct. In the large schizonts the vacuoles representing nuclei, normally regular in size and discrete in arrangement, may become irregular in size and confluent. The granular cytoplasm may come to contain numerous irregular, dark masses. Some of the criteria for degeneration of the segmenters are: (1) merozoites packed into a masssurrounded by a large clear area of fluid; (2) color of TABLE Percentage
of Degenerating
Exoerythrocytic of Chick
III Stages %SS3
.. .
... .
in the Tissues
Percentage of degenerating parasites
organ
Kidney. . Spleen. Thymus. Lung...... Liver...... Heart. Testis. Brain. Intestine.
of P. gallinaceum
63 58 53 44 42 36 23 17 12
merozoites gray or dirty blue instead of bright azure; (3) a dark conglomerate masswith irregular dark blue massesrepresenting merozoites; and (4) entire parasite broken into fragments with indistinct edges. Since the exact hour of death for chick # 331 was not known no counts of relative numbers of norma and degenerating parasites were made from its tissues,for in it we could not distinguish between degenerative changes taking place in the parasites in the living host and degenerative changes presumably due to post mortem change in the host itself. Table III presents the data from chick B 333. Despite the possibility of subjective error in determining the percentage of parasites actually undergoing degeneration it is probable, that the chances for normal growth of exoerythrocytic stages vary in the different organs. It is difficult, however, to reconcile the findings in the various organs with anything now known or assumed about (1) the relative nutritive value to the parasites of the
398
CLAY
Q.
HUFF
cells of the various organs or (2) the relative importance of the different organs in the production of humoral antibodies against malaria. Obviously this study should be extended to other parasite-host combinations and to the entire course of infection before the significance of the differences in the degenerative changes occurring in the various organs can be determined. Significance of Dimorphism
of Xchizonts
Reichenow and Mudrow called attention in a paper published in 1943 to the presence of a dimorphism in the kinds of merozoitcs produced in I’. praecoz (= relicturn) in canaries. They applied the terms, macromerozoites and micromerozoites, to two categories of merozoites based upon whether they were large or small; and the terms, macroschizonts and microschizonts, respectively, the mature schizontjs giving rise to the two types of merozoites. The same phenomenon was observed in 1942 in P. gallinaceum by Huff and Coulston whose publication was delayed until 1944 by security classification. Reichenow and Mudrow (1943) and Mudrow and Reichenow (1944) interpreted this dimorphism as indicating that the macromrrozoites were destined to produce other exoerythrocytic stages while the micromerozoites would produce erythrocytic stages.. Since the explanation of the nature and significance of these two types of schizonts and their corresponding merozoites is likely to be important in our understanding of the interrelations of erythrocytic and exoerythrocytic stages, of the phenomena of relapse and latency, and the efficacy of chemotherapy, attempts have been made to study them from various points of view. It was stated above that difficulty was encountered in making accurate counts of the various types of segmenters. Therefore it became necessary to examine carefully the concept that two categories of segmenters exist. Actual measurements were made of the diameters of five merozoites from each segmenter found in exploring the microscopic fields while studying the various organs of the host. These findings are given in ocular micrometer units in Table IV and the collected data are shown in Fig. 2. It is at once obvious that no dimorphism exists in the merozoites but that the groups referred to as macro- and micromerozoites merely constitute the extremes of a population showing the normal amount of variation. This condition existed in each organ of the .host studied as well as for the combined results. The differences in the mean diameters of the merozoites in the various organs are within the observational error. It should he noted that the data in Table IV are expressed in ocular micrometer
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
TABLE Diameters
of Merozoites
from
Number organ
* In ocular
micrometer
a diameter*
2
52 29 34 55 42 57 19 32 34
14 10 16 34 11 38 21 42 15
4
11 14 3 6 12
34 23 61 24 22 17 18 17 11
1 1 1 10 9 7 1
7
111
227
354
201
34
units.
6
10 13 42
The factor
X 0.116 is needed
to convert
the Organs
I
9
5
seen in
of:
8
5
Totals.
having
Segmenters
7
4
Spleen. Liver. . Brain. . . Heart. . Kidney. Lung. Thymus Intestine Testis.
IV
Randomly Selected of Chick #933 -
of merozoites
399
GALLINACEUM
10
I Number measured
Meall size*
114 75 154 114 87 152 74 110 75
.67 .65 .62 .66 .66 .72 .74 .74 .67
11 4 6
21
I
955
1
.68
top.
3s 3*
301 J-
25l I-
20( )-
f 5
l5( I-
% I: IOC)-
tic I-
II4
-
6 5 Oiometer of Merozoites (in ocular micrometer umts 1
FIG. 2. Frequency in all organs of chick the factor: 0.116).
distribution of diameters of exoerythrocytic # 333. (To convert to p multiply ocular micrometer
merozoites units by
400
CLAY
G. HUFF
units which may be converted to microns by multiplying the former by 0.116. This gives a mean diameter for the merozoites from the collected data of 0.795 f 6004 I*. DISCUSSION
Although the first generation of pre-erythrocytic stages (cryptozoites) appears to be restricted to the cells of the lymphoid-macrophage system and, therefore, to be encountered most often in the organs well endowed with these cells, Coulston, Cantrell, and Huff (1945) showed that the distribution of pre-erythrocytic stages of P. gallinaceum after 36 hours was not an indication of sporozoite localization. These authors tested the presence of pre-erythrocytic stages in organs of chicks which had been intravenously inoculated with large doses of sporozoites by removing tissues from the inoculated animals, placing them into the body cavity of normal chicks, and studying the latter for the development of infections. During the first 36 hours following inoculation of sporozoites the spleen, lung, kidney, liver, and pancreas of the inoculated animals contained parasites capable of producing infection in the subinoculated animal. Since the circulating blood of such animals was free from parasites between the 36th and 70th hours after sporozoite inoculation, experiments involving subinoculation of tissues were valid. All tissues tested except brain and bone marrow (i.e., heart, spleen, kidney, liver, thymus, pancreas, lung, and intestine) indicated that infective parasites were present. No real significance can probably be attached to the fact that the brain and bone marrow in those experiments failed to produce infection in subinoculated animals beyond the fact that parasites were probably scarce and, therefore, that success would be realized in a low percentage of trials with them. The results in the present study indicate that the brain, testis, intestine, and thymus (the bone marrow was not studied) contained small numbers of parasites as compared with kidney, lung, heart, liver, and spleen. Although the relative order of the degree of infection in the organs is fairly consistent between the two birds studied, a much larger study would be required before significance could be attached to the differences observed. In common with Porter’s (1942) observations of P. cathemerium, there are no indications in the present study or in any of my previous studies on P. gallinaceum that the brain is the preferred site of localization of exoerythrocytic stages. This belief has probably arisen from two facts: (1) that some investigators study the exoerythrocytic stages
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM.
GALLINACEUM
401
mainly by the use of smears from brain which can be more successfully made than from most other organs, and (2) that the birds with extreme brain involvement are more likely to die and thus to be seen at the height of the involvement of the brain capillaries. Since we are concerned here with the behavior of exoerythrocytic stages during the transition from tissue parasitism to parasitemia, an evaluation of the relative numbers of different stages in fixed tissues of the various organs and in the blood is a possible approach to the solution of some of the problems relating to that period. In each of the two birds studied the parasites in the peripheral blood were rather remarkably out of phase with the exoerythrocytic stages of the organs. The large percentage of young trophozoites in the peripheral blood is in contrast to the small percentage of young exoerythrocytic trophozoites of the tissues and similarly the large percentage of exoerythrocytic schizonts in the tissues contrasts with the very low percentage of erythrocytic schizonts. This would appear to indicate that not only are large numbers of exoerythrocytic stages changing to erythrocyte-inhabiting forms but that relatively few of them are continuing as ‘exeerythrocytic stages. Some of the explanation for the small numbers of young exoerythrocytic stages may reside in the difficulty of finding these stages in sections since they present no contrast in staining to that of the cytoplasm of host cells. However, in certain tissues such as brain and kidney, the chances of overlooking these small forms are much less than in spleen, liver, or heart, yet this fact is not reflected in a correspondingly higher number of small forms counted in brain and kidney. The relatively large percentage of small exoerythrocytic stages in the lung of bird I333 is of interest in that it may indicate a possible predilection for lung tissue on the part of the parasites. In this connection it should be noted that Coulston et al. (1945) found the lung more consistently infected between 48 and 79 hours following mosquito bites, than any of the other organs. It would seem, however, that this point requires much more study before it can be clarified. There was no evidence of localization of the schizonts in any particular organ. The presence of exoerythrocytic stages undergoing degenerative changes has previously been noted (1) in primary infections of P. Zophuraein chickens, ducks, turkeys, and guinea fowl (Huff, Coulston, Laird, Porter, 1947); (2) in primary infect,ions of P. relicturn, and P. gallinaceum in ducks (Huff, 1951); and (3) in P. gallinaceum in chickens which were treated with antimalarial drugs (Coulston and Huff, 1948).
402
CLAY
G.
HUFF
The report herein of similar degenerative changes in primary infecticns of P. gallinaceum in chicks, which represent one of the most, highly susceptible of hosts, indicates that the mechanisms of acquired immunity probably begin to exert their effect on exoerythrocytic st.ages fairly early in infections induced by large inoculums. Since the initial observations of a dimorphism in the schizonts of exoerythrocytic stages of P. praecox (‘= relicturn) (Reichenow and Mudrow, 1943) and of P. gaZEinaceum (Huff and Coulston, 1944) there has been a general tendency on the part of many authors to claim that macromerosoites are destined to continue the exoeryt)hrocytic cycle while micromerozoites enter red cells and initiate the parasitemia. This belief derived from (1) the fact that the pre-erythrocytic stages were predominantly of the macroschizont type, (2) the observed shift during the course of the infection from a preponderance of macroschizonts to a preponderance of microschizonts, and (3) the similarity between micromerozoites and the merozoites in erythrocytic schizont’s. If the findings herein reported that two categories of schizonts do not exist prove to hold for other species of malarial parasites and for other stages in the infection, a change of viewpoint will be required in attempting to explain the transformations which are known to occur from exoerythrocytic parasitism to pa.rasitemia and vice versa. In the light of the result of this study it would appear that the macromerozoites and micromerozoites merely represent the extremes in size of a population of merozoites of varying sizes in which there is a complete series of connecting forms. The latter are .probably usually neglected in examination of sections of infected Organs because one is not faced with making quantitative separation of the merozoites into categories. Both Mudrow and Reichenow (1944) and Geigy and Britschgi (1950) observed (the former authors in P. praecox; the latter in P. gallinaceum) that some microschizonts mature at an early stage and produce a number of merozoites barely larger than the number produced by macroschizonts. The latter authors made a quantitative study of the number of nuclei in the various sizes of macro- and microschizonts. Their macroschizonts had a high proportion which were small in size (l-20 merozoites), a medium proportion medium (21-40 merozoites), and a small proportion which were large (over 40 merozoites), whereas these proportions were almost exactly reversed in the microschizonts. These findings can be briefly summarized by the statement that the merozoites tend to be large in small-sized schizonts and small in large-sized schizonts. However, they also indicate
E~~ER~THRocYTw
STAGES
OF
PLASMODIUM
GNAJNACEUM
403
that the authors were dealing with a population with continuity of valiation in size which they arbitrarily separated into two components. Nothing in the present observations would conflict with the observed facts that macroschizonts are predominant in the pre-erythrocytic stages of the infection while microschizonts are predominant in the later phanerozoic stages of infection if we substitute for the term macroschizont the expression “schizonts with larger mean size,” and for the term “microschizonts” the expression “schizonts with smaller mean size.” The simpler statement would be that merozoite size decreases as a sporozoite-induced infection progresses to the stage in which parasitemia predominates over tissue parasitism. The argument that microschizonts produce erythrocytic stages because of the similarity of size may or may not be pertinent (Mudrow and Reichenow, 1944). I know of no study which shows that the youngest erythrocytic stages are of uniform size throughout the course of a sporozoite-induced infection, or in other words that larger pre-erythrocytic merozoites do not produce erythrocytic infections. In fact, if the larger merozoites should enter erythrocytes and thus appear as larger than the usual first stage erythrocytic trophozoites it seems likely that they might be mistaken for erythrocytic parasites originating from smaller merozoites that had been living in the erythrocytes for several hours. If one assumes that micromerozoites (i.e., the smallest of the merozoites) produce erythrocytic stages and macromerozoites (i.e., the largest merozoites) continue the exoerythrocytic cycle one is left in the dilemma of having to account for the fate of the merozoites of intermediate size which make up fully 80% of the total population. At this stage in the development of our knowledge it is not possible to determine the causes for the change in average size of merozoites as a sporozoite-induced infection progresses. Since, however, we know that there is a marked shift in frequency of occurrence of the exoerythrocytic stages in cells of the lymphoid macrophage system to occurrence in cells of the true endothelium (Porter, 1942; Huff and Coulston, 1944) it would seem most likely that the type of cell inhabited has some effect on the average size of the merozoites. It would then be only a fortuitous circumstance that the so-called micromerozoites happen to be most prevalent at the time that erythrocytic parasites become plentiful enough to study. Such an argument would not be overthrown by the possibility that occasional small merozoites might be found in macrophages or fibroblasts, or large merozoites in endothelial cells, since these might be
404
CLBY G. HTJFF
simply the merozoites on the extremes of size in populations with normal variabilities. The disproof of the existence of a true dimorphism in exoerythrocytic schizonts would not bring us much closer to the explanation of the causes for the shift from tissue parasitism to parasitemia, except to remove a spurious clue. The real explanation probably is as elusive as the explanation of what determines whether a given erythrocytic trophozoite will produce a gametocyte or a schizont. SUMMARY
Studies made during the transitional stage infection of P. gallinaceum ir chicks (i.e., the period of transition from tissue parasitism to parasitemia) have yielded the following results: 1. The relative frequency of exoerythrocytic stagesexpressed as number of parasites per square millimeter of section is fairly consistent for the two birds examined except for the kidney and spleen. The brain ranked low in relation to kidney, lung, heart, and liver as the organ of localization. 2. At the time when the preponderance of parasites in the fixed tissue consisted of large schizonts and segmentersthe preponderance of erythrocytic parasites consisted of young trophozoites. The larger schizonts and segmenterswere fairly equally distributed among the different organs whereas in one of the birds 32ye of the exoerythrocytic stages of the lung were small trophozoites. It would appear that not only were large numbers of exoerythrocytic merozoites changing to erythrocytic stages but that relatively few of them were continuing as exoerythrocytic stages. 3. Degenerative changes in the exoerythrocytic stages, possibly due to the development of antibodies by the host, were seen as early as 53 hours after the first observed parasitemia. The type of degenerative change in the parasite was similar to that observed previously as the result of the action of natural immunity and of antimalarial drugs. 4. So-called macromerozoites and micromerozoites were found to be only the extremes in size of a population of merozoites with normal variation in size. Thus, no dimorphism exists in the schizonts of this species. 5. It is believed that, although no dimorphism of the schizonts exists, the average size of the merozoite decreasesduring the transitions from a condition in which the host cells are predominantly of the lymphoidmacrophage system to the condition in which they are predominantly
EXOERYTHROCYTIC
STAGES
OF
PLASMODIUM
GALLINBCEUM
405
endothelial in type, and then to the stage where the erythrocytes are the cells which are predominantly inhabited. 6. The prevalent view that the smaller merozoites are destined to produce erythrocytic stages while the larger merozoites are destined to continue the exoerythrocytic cycle has not been adeqrately proved. REFERENCES
W., AND HUFF, C. G. 1945. The distribution and localiand pre-erythrocytic stages in infections with PlasmoJ. Infectious Diseases 76, 226238. COULSTON, I?., AND HUFF, C. G. 1948. Symposium on exoerythrocytic forms of malarial parasites. IV. The chemotherapy and immunology of pre-erythrocytic stages in avian malaria. J. Parasitol. 34, 290-299. GEIGY, R., AND BRITSCHGI, H. 1950. Untersuchungen tiber die E-formen vor Plasmodium gallinaceum in Organen des Htihnchens am 9/10. Infektionstag. Rev. suisse 2001. 67, 526-532. HUFF, C. G. 1951. Observations on the pre-erythrocytic stages of Plasmodium relictum, P. cathemerium, and P. gallinaceum in various birds. J. Infectious Diseases 88, 17-26. HUFF, C. G. AND COULSTON, F. 1944. The development of Plasmodium gallinaceum from sporozoite to erythrocytic trophozoite. J. Znjectious Diseases 76, 23249. HUFF, C. G., COULSTON, F., LAIRD, R. L., AND PORTER, R. J. 1947. Pre-erythrocytic development of Plasmodium lophurae in various hosts. J. Infectious Diseases 81, 7-13. MUDROW, L., AND REICHENOW, E. 1944. Endotheliale und erythrocytiire Entwicklung von Plasmodium praecox. Arch. Protistk. 97, 101-170. PORTER, R. J. 1942. The tissue distribution of exoerythrocytic schizonts in sporozoite-induced infections with Plasmodium cathemerium. J. Infectious Diseases 71, I-17. REICAENOW, E., AND MUDROW, L. 1943. Der Entwicklungsgang von Plasmodium praecox im Vogelkorper. Deut. Tropenmed. 2. 47, 289-299. COULSTON,
zation dium
F.,
CANTRELL,
of sporozoites gallinaceum.