ROOE‘A USE ONLY GANETOCYTE PERIODICITY IN LEUCOCYTOZOON SIMONDI MATHES AND LEGER INFECTIONS AND ALTERATIONS NOTED AFTER PASSIVE IMMUNIZATION By RICHARD H. KOCAN A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1965 ACKNOWLEDGMENTS The author wishes to extend his sincerest gratitude to the follow- ing people: To Dr. David T. Clark, for his guidance, criticism and especially for his contagious enthusiasm toward research; To my dearest wife, whose help as a technician and understanding wife made this endeavor the success that it is; To Mr. R.D. VanDeusen and the staff of the H.K. Kellogg Bird Sanctuary whose cooperation aided in completing this work in record time; To Dr. A.H. Stockard, Director of the University of Michigan Bio- logical Station, for allowing the author to use the Station's facilities for temporary housing of experimental animals. TABLE OF CONTENTS Page INTRODUCTION.................................................. l LITERATURE SURVEY............................................. 3 MATERIALS AND METHODS......................................... l8 Set I: Early Summer Exposures 18 Set ll: Midsummer Exposures 2] RESULTS....................................................... 25 Set I: Early Summer Exposures 25 Set Il: Midsummer Exposures 25 DISCUSSION AND CONCLUSIONS.................................... 4] SUMMARY 65 LITERATURE CITEDOOOOOOOOOOOOOIOOOOOOOOCOOOOOOOOOOOOOO0.......0 67 Figure 4a. hb. 7a. 7b. IO. ll. l2. LIST OF FIGURES Cellulose Acetate strip electrophoretic analysis of whole pooled immune duck serum compared with the purified gamma globulin.......................... Photomicrograph of a round and an elongate gametocyte from a duck infected with Leucocytozoon simondi...... Photomicrograph of immature gametocytes of Leucocy- tozoon simondi in unidentified host cells............ Summary graph of Table I showing mean gametocyte counts of control birds.............................. Summary graph of Table II showing mean gametocyte counts of control birds kept out of direct sunlight.. Summary graph of Table III showing gametocyte den- sities of four birds apparently protected by pre- infection immunization............................... Summary graph of Table V showing mean gametocyte densities of birds immunized at beginning of gameto- cyte Patency....0.0..00..OOOOOOOOOOOOOOOOOOIOOOOO...O Graph comparing gametocyte density fluctuations with erythrocyte counts of infected ducks................. same as 78.0.0000000000000000000000000000000000000IOO Graph showing the percentages of the three “stages” of gametocytes at various times during the verte- brate phase of infection............................. Photomicrographs of round and polygonal gametocytes in ”elongate” host cel15............................. Photomicrographs of apparent erosion of parasitized hOSt Ce]150.000.00.00OOOOOOOOOOOOOOCOOOO00.0.00....00 Fallis' graph of gametocyte counts based on one minute counting intervals............................ Author's hypothesized routes of Leucocytozoon simondi development in duck host............................. Pane 23 27 3O 32 35 40 45 50 LIST OF TABLES Table Page I. Table of individual gametocyte counts of control birdSOOOOOOCOOCOOOCOOCCOOOOCOOOOOOOOIO...I...0.00.... 23 II. Table of individual gametocyte counts of control birds kept out of direct sunlight.................... 29 III. Gametocyte densities of the four birds who demon- strated an apparent beneficial effect from being immunized with immune gamma globulin prior to infGCtionOOOOOOOCOOOOCOOO'COOOOOCCCOOCOOOOOOOC0...... 3] IV. Four birds from the group immunized prior to infec- tion which showed no apparent effect from the immune garlma QUObU]inOO00......0000000000......OCOOOOOOOOOOO 3] V. Table of individual gametocyte densities of birds immunized at beginning of gametocyte patency......... 33 VI. Chernin's table of variations in parasitemia dependent on dates Of e>(posureoOOOOCOOOOOOOOOOOOOOOOCOO0.0.0... [+2 VII. Chernin's table showing consistent appearance of elongate gametocytes on the fifth day of patency in ducks exposed for 24 hours........................ 47 INTRODUCTION Since its discovery in l890 by Danilewsky, ngcocytggggn has remained an incompletely understood protozoan parasite. O'Roke's early descrip- tion (l93#) of the life cycle of Lgycocytozoon simondi Mathes and Leger was relatively accurate for the sexual cycle in the definitive host, the vector. His description of the asexual cycle in the duck has been proved inaccurate. The literature today has numerous descriptions of the asex- ual cycle, but due to the conflict among them, the exact cycle is still poorly understood. The work being reported here is an attempt to more accurately de- scribe the cycle of L, simondi in the domestic duck,‘AgggrpLatyrhynchos Linneaus, and to determine some of the defense mechanisms of the host. Standardization of techniques employed for the exposure to vectors and determination of parasite density was attempted. An attempt is also made to explain some of the observed phenomena of the infection in terms of immunity. There are both economic and academic reasons for studying this or- ganism, although one can not always separate the two. Both duck ranchers and sportsmen are interested in the control of.g, simondi. Hatcheries can be located in nonendemic areas to prevent transmission, but the sportsman is concerned because the organism is pathogenic to both ducks and geese of many species in their breeding grounds. At the time of nesting, the adult birds relapse if they have been previously infected, making the parasite readily accessible to the vector and subsequently to the young birds who are extremely susceptible. Today taxonomists relate W to the genus W. If the relationship is correct this organism affords an excellent form with 2 which to study methods of immunization and treatment which might aid in the problem of human malaria eradication. This problem has been under considerable study ever since malaria control was accomplished a number of years ago. The line of attack under investigation here is the host's defense mechanisms. This has been complicated in Plasmodium research by the occurrence of gametocytes and schizonts in the host's erythrocytes. Leucocytozoon, however, affords a better opportunity to study each stage separately with respect to immunity since only the gametocytes occur in the peripheral circulation while the asexual schizonts occur in the inter- nal organs. LITERATURE Lgycocytozoon simondi infections are described in textbooks as the cause of a blood protozoan disease of waterfowl, initially acute with a gradually decreasing parasitemia developing into a chronic infection which relapses regularly each spring during the breeding season. Trans- mission is biological, through various species of Simulium and possibly Culicoides (Fallis and Bennett, l96l). The vector is also the defini- tive host with the parasite exhibiting developmental stages very similar to those of Plasmodium in the mosquito. Transmission occurs in the north- ern breeding grounds of the ducks and geese which overlaps the endemic area of the black fly. Since no transovarial transmission has been re- ported to occur in the vector, the flies are believed to become infected from relapsing parent birds who have recovered from previous Leucocyto- gggn_infections and have returned to the breeding grounds. The organ- ism is then transmitted to the newly hatched ducklings and goslings. The course of the infection in the vertebrate host is vaguely out- lined in most texts. It is described as having a prepatent period of 6 to lh days after which time the gametocytes appear in the blood. The gametocytes reach their maximum number by the eighth to tenth day, and if death does not occur the gametocyte number drops to near zero by the thirtieth day and remains at a low level or latent condition until the following spring. This latent condition or lack of total recovery is believed to be the result of multiplication of the tissue stages of this organism. These stages become apparent, upon autopsy of the infected bird, in the liver, spleen, lungs, brain and most organs with lymphatic tissue. Tissue stages are present prior to the appearance of the game- tocytes in the blood and are present, as far as is known, for the 3 1+ remainder of the life of the host. Morphologically, stages in the vertebrate host are described as having two distinct gametocyte types and two distinct schizont types. The predominant gametocyte type is reported to be round, but dimensions are seldom stated. The other type of gametocyte reported is the elongate form. This measures IA to 22u long. In either case the microgametocytes are slightly smaller than the macrogametocytes. The gametocytes are contained within a host cell which has not as yet been definitely identi- . fied. It is believed to be either a cell of the lymphocytic series or an erythrocyte in a greatly distorted condition (Figure 2). The tissue stages commonly reported are the liver form or “hepatic schizont”, meas- uring II by l8u and the lymphoid or “megaloschizont”, measuring 60 by l64u. Both of these forms are in the organ cells and are never seen in the peripheral blood. Description of the symptoms and pathogenicity are essentially in accord with one another. The course of infections is consistent regard- less of the sex of the infected bird. The most susceptible birds are the newly hatched, under the age of four weeks. An outbreak of Leucocy- tozoon infections in ducks is characterized by its suddenness of onset, listlessness, rapid breathing, and lack of interest in feed. Death occurs by the l0th or llth day of infection if recovery is not complete. Fallis et al. (I951) and O'Roke (I934) have clearly shown that Leucocytozoon is transmitted to the duck by means of sporozoites which have developed in the black fly. After introduction of the sporozoites into the duck by black flies, there occurs a prepatent period of approximately 6 to IA days, depending on the method of exposure used by the investigator. Fallis et al. (ISSI) 5 stated that a greater cyclic synchrony of parasitemia and patency occurred among birds injected with macerated black flies than occurred with nat- ural exposures. Their periods of exposure varied from one to several days. They also suggested that there is probably one asexual generation in the internal organs of the duck prior to gametocyte patency, and that this cycle is not necessarily synchronous among all schizonts and that the number of Sporozoites introduced may affect the prepatent period. In a later work Fallis et al. (I956) reported the finding of Dr. Ritchie of the Department of Pathology, University of Toronto, in which he found hepatic schizonts in the Kupffer cells of the liver on the second day of the infection. From this observation Fallis et al. reconsidered their first suggestion and postulated two possible asexual cycles prior to game- tocyte patency--the first generation in the Kupffer cells producing the second generation which develops in the spleen and other lymphoid tissue where they become megaloschizonts. These then produce the gametocytes which appear in the circulation. These findings correlate nicely with their observations of I95] in which they noted that the maturation time of a gametocyte is approximately 48 hours or less. Cowan (I955), using birds exposed for five days for a study of the tissue schizonts, concluded that the megaloschizont is the final prepa- tent generation resulting from some previous asexual cycle, and is the sole contributor of gametocytes to the peripheral blood. This is in agreement with the views of Fallis et al. (1956). Huff (l9h2) put forth three possible explanations for the occur- rence of two types of schizonts: l) hepatic schizonts may represent a first generation followed by megaloschizonts, 2) the difference may be a manifestation of the host tissue involved and 3) the two forms may 6 represent stages of different parasites. He believed the first explana- tion to be the most probable. The gametocytes appear in the blood approximately seven days post exposure, marking the start of patency. Patency is immediately preceded by an increase in the leucocyte count (Fallis et al.,.I95l). The exact time of gametocyte appearance varies somewhat from author to author and seems to depend on the duration of exposure to the vectors. Chernin (l952c) reported that prepatency ended with the appearance of immature stages or trophozoites in the blood. He described these as the very small stages and the large round stage. Chernin also reported that only during the first two days of patency are multiple infections noted. Sim- ilar superinfections are also reported by Cook (I95H). No reports of multiply infected host cells past this time have been found. Hartman (I929) claimed that the earliest forms seen in the circu- lation occurred in erythrocytes. Cook (I954) and Ramisz (I962) agree with this view. Cook states that lightly infected birds had parasitized erythrocytes only. In heavier infections she reports that lymphocytes were also noted to be infected but that erythrocytes outnumbered the lymphocytes as carriers of the parasites. She differentiated erythro- cytes from lymphocytes by using benzidine-peroxide as an indicator of hemoglobin. She also reported finding two-thirds of the erythrocyte in- fecting trophozoites to be in reticulocytes. With the same indicator she also found hemoglobin in the host cells of the large round gametocytes, but no hemoglobin was found in the elongate host cells. This she attri- butes to the assumption that the hemoglobin has been altered or lost as the parasitized cell grows, thereby losing the ability to stain. Huff (I9h2) recalled Mathes and Leger's proposal that the rounded 7 forms were invaders of mononuclear leucocytes while pyrlform or elon- gate parasites were in erythrocytes or erythroblasts. Huff's own work indicates that the smallest stages occurred in myelocytes, late poly- chromatophil erythrocytes, lymphocytes, monocytes and macrophages. There was no evidence, however, that any form developed past the ear- liest stages in cells other than monocytes or macrophages. He also re- ported finding growth stage series only in lymphocytic cells and con- cluded that erythrocytic infections never developed to maturity. Fallis et al. (l95l) found what they believed to be erythrocytes and cells of the lymphocytic series to be infected. They also concluded that there was possible lymphocytic involvement since blood passage would sometimes transmit the infection. Injections of heart, lung, liver and spleen also produced infections. They considered lymphoid cells as carriers of infective stages which later lodged in some tissue where fur- ther development occurred. Savage and Isa (I959) concluded that although small lymphocytes could be parasitized,tflwy presented a physical impossibility to becom- ing gametocyte carriers, whereas Huff (I963) proposed the rapid growth of small lymphocytes after invasion. They postulated that monocytes or macrophages were the most probable carriers of gametocytes on the basis of: l) volume of the cell, 2) the hyperparasitism known to occur in these cells, and 3) a residual differential count of white cells. Fallis et al. (l95i) observed that ducks exposed for a single day showed predominantly small and large round forms of the gametocyte with elongate fonms appearing later in the infection. However, birds which they exposed to continuous infection showed more numerable small and large round forms. In comparison, Chernin (l952c) showed that one day 8 exposures produced a consistent appearance of elongate gametocytes on the fifth day after patency while eight day exposures showed a great deal of variation as to when elongate forms appeared. The range was from the first to the twelfth day of patency. Although he did not interpret his data in this way it can clearly be seen from his table (Table VII). He also reported that there was a decline in immature forms on day four of patency and suggested that this reflected a halt of schizogony. Seven days later rapid clearance of the gametocytes was noted. This would correspond with the findings of Fallis et al. (l95l) in which they noted that the normal life span of a gametocyte was about six days in a recipient host. Cowan (I955) states that during the first several days of patency merozoites were released into the host's tissue upon rupture of the schizont wall, and due to the hyperemia associated with the infection the merozoites would be in direct contact with blood. Fallis et al. (I956) observed that the maximum number of megaloschizonts in the spleen occurred 7 to 12 days post exposure (day l to 5 of patency). No exposure duration was mentioned. This agreed with his earlier assumption that there were more numerous megaloschizonts in birds which may have gone through more than one schizogony, and that a complete schizogony may take only 2 to 9 days. Several workers have reported changes in the number of schizonts in infected organs with respect to the time of the infection. Cowan (I955, I957) noted that on the first and second day of patency every organ was infected. On the third day only the heart and brain had vis- ible schizonts and by the fourth day the brain alone showed schizonts. He noted that schizonts failed to mature in the brain and assumed that 9 this was an unsuitable site for development. Fallis et al. (l95l), however, reported that from day four to day nine post-infection, the spleen continued to show megaloschizonts. On day ten to eleven the spleen, lung, brain and lymphoid tissue all contained these schizonts. Upon reaching maturity the megaloschizonts undergo a process of septa formation as described by Cowan (I955). This occurred only after maximum schizont size was attained but before internal differentiation ceased. He noted that the septate schizonts usually have the same type of cytomere in each compartment but that occasionally omaoccurred in which the compartments had cytomeres in various stages of development. He is also of the opinion that maximum growth of the schizont is limited by encapsulation or by limits of the tissue elasticity. This limit on the cell, he concluded, produces a pressure on the central body by the proliferating cytomeres and internal fluids of the schizont, causing rupture of the schizont and the release of the merozoites. This inter- pretation of the mechanism of schizont rupture is a result of his belief that the central body is a primordium from which primary cytomeres are cut. These then produce secondary cytomeres and so on until the schi- zont is fully mature. This is in direct opposition to the belief of Huff (I942) and Ningstrand (I947) who believe that the central body is the remnant of an enlarged and distorted host cell nucleus. Agreement among workers as to the exact length of prepatency is not consistent due to the various durations of exposure time. Their views on the course of the infection once patency has begun are more vague than in disagreement. Fallis et al. (I956) reported that maximum spleen size corresponds with the peak of gametocyte density as determined by counting gametocytes per unit time. This peak occurred between the l0 tenth and fourteenth days post-exposure (3 to 7 days into latency). Chernin (l952c) felt that the gametocyte peak was between the third and ninth day post infection. The two authors used exposure times vary- ing between I and 8 days. There is agreement among several authors, however, that the parasite numbers rapidly reach a peak and then gradu- ally decrease (Fallis et al., l95l, I956; Huff, I942; and O'Roke, l93h). Chernin and Sadun (I9A9) expressed the possibility that a fluctuation in gametocyte levels occurred during the first thirty days of the primary infection. This was based on counting parasites per microscopic field. Chernin (l952c) later discounted his belief and conformed to the opinion of others. At this time he also attempted to correlate the presence of round and elongate gametocytes in the host. His definition of im- mature gametocyte was equated with the large round forms as well as the small trophozoites. Since this was later proven to be an incor- rect assumption by Fallis et al. (I95l) and Rawley (I953) the forms will be referred to here as round and elongate since it is difficult to tell exactly how many trophozoites he counted. Chernin's table of peak densities of round and elongate forms are reproduced in Table VII. Chernin interpreted the two curves to indicate that no correlation be- tween round forms to elongate forms could be made. Fallis et al. (I951) using one minute counting periods show a graph of a “typical" parasite- mia (Figure II). It is interesting to note the three peaks shown on the graph. Fallis refers to these peaks as being regular in occurrence but claims that the gametocyte cycle is completely asynchronous or irreg- ular. Briggs (I960) reported that his studies supported the views of Chernin; that there is no correlation between round and elongate ll gametocytes in individual ducklings. Martin (I932) claimed that the elongate forms could not take part in gametogenesis and therefore the round forms were the only functional form. He postulated that the elon- gate gametocytes were decadent individuals or “overmature”. The work of Fallis et al. (1951) and Rawley (1953) shows that both round and elongate forms of gametocytes can exflagellate. This invali- dates Chernin's supposition that the round forms are immature. Three possibilities were put forth by Chernin (l952c) as to what caused the pleomorphic gametocyte forms: I) fundamental host differences, 2) manifestations of specific immune responses and 3) strain differences in the parasite itself. Other commonly suggested reasons are the type of host cell invaded (Fallis et al., l95l) and two species of parasites (Cook, I954; Fallis et al., I95l). Cook points out that if host cell invasion made the difference then both round and elongate forms should appear at the same time due to their simultaneous release from the schizonts (assuming that there was a simultaneous releaSe). She also notes that although Chernin (l952c) observed four birds which showed no elongate forms during the entire course of the primary parasitemia, that no cases of elongate forms only have been reported for‘L. simondi. This would be highly improbable if two species of parasites were involved. Fallis et al. (l95l) suggested that this phenomenon might be due to the inability of the host cell to change shape when infected with a large parasite, while Huff (I963) suggested that lymphocytes may be stimulated to rapid development after invasion. In looking for a relationship be- tween the two forms, Rawley (I953) reported finding no transitional stages between the two, though she did see young stages in what appeared to her to be elongate host cells. She also reported seeing elongate I2 forms I'round up“ while she observed them alive, and seeing round forms become elongate just prior to exflagellation; but not in all cases of exflagellation did this occur. Briggs (I960) compared the parasitemias in the Pekin with those in Muscovy ducks in an attempt to elucidate the confusion concerning the two forms of gametocytes. He noted that the average of the two forms in the Pekin was about equal, while Muscovies seldom showed as many as 5% elongate forms. His graphs resemble very closely those of Chernin for the appearance and density of the gameto- cytes in the Pekin. The spring relapse which occurs in birds with latent infections is also not fully explained. Huff (I942) postulated that the increased number of gametocytes in the peripheral blood following a low winter parasitemia was due to a true relapse since no vectors were available. Birds which are held in an endemic black fly area showed the same blood picture as did those kept in an area of no transmission (Barrow, unpub- lished). Huff's results showed that young stages of Lgucocytozoon and Haemoproteus became apparent in April. In accord and in contrast to this, Chernin (l952b) reported the same occurrence of gametocyte increase in the spring of the year but that immature forms did not become evident until one month after the relapse, whereas they constituted 20% of the gametocyte population during the winter months. His reference to imma- ture forms includes the round forms. From his data he concluded that schizogony in the tissues probably occurs throughout the winter months, which explains the immature gametocyte population during that time. Due to the correaltion among relapse, increased day length, and egg production or breeding, Chernin (I952b) carried out experiments which were intended to clarify the relationship among the three I3 occurrences. He found that if the light on the birds was increased from l0 hours to I6 hours between November and December, that egg lay- ing and relapse occurred in January. Eleven to 60 days post-egg—pro- duction the parasitemias increased 8 fold as compared to pre- egg-pro- duction. All relapses lasted not less than three months. Interestingly enough, males also relapsed during the egg laying period. Relapses occurred among females isolated from males, males isolated from females and males and females together during this period. It has been pointed out by Clark (I964) that a species of £3222: cytozoon in the Magpie showed gametocytes in the tissues throughout the year, even when the blood was negative for them. Briggs (I960) reported that anemia, due to the Leucocytozoon infec- tion, was an important factor or the primary cause of death in the Pekin. He also noted that severely infected birds died before the ap- pearance of elongate gametocytes and before the peak of round forms. Cook (I954) relates anemia to destruction of erythrocytes by the inva- ding parasites. Huff (l963) suggested the possibility that autoimmune responses similar to those reported for malaria may account for the anemia. Chernin (I9523) made several observations on mortality as a result of.L. simondi infections. He remarked that the greatest losses were among ducks less than four weeks old, and that indigenous birds suffered much lower losses than did imported birds exposed for brief periods (8 days). The highest mortality occurred between IO and I9 days post exposure, with a gradually decreasing number of deaths occurring until latency. Premunition is a commonly reported phenomenon among organisms which .wv {)0 SC h'C ill.’ H K'- hlIIIIrLin O? I I4 leave a residual population after the host has reduced the parasite numbers and symptoms of the disease have disappeared. This results in the lifetime immunity of the host. Malaria is reported to produce just such a response, and Leucocytozoon, supposedly related to Plasmodium, would be expected to produce such a lasting immunity in the host. It is not uncommon to see reports of birds showing relapses for a number of years after their first exposure to the organism, which indi- cates that the organism is still present and being kept in check by the host. Indeed, it has been reported by several authors (Fallis et al., l95l; Chernin, I952c) that recovered birds do exhibit premunition. There is, however, an interesting finding by some authors concerning this phenomenon. Fallis et al. (l95l) reported that recovered ducks exposed one year later succumbed to the second exposure, supposedly due to a lack of immunity. Further experiments showed that birds exposed constantly throughout the summer became immune to reinfection the fol- lowing year. This work is in agreement with the supposition of Hartman (I929). He reported that evidence pointed to little resistance as a result of low infections. Chernin (l952c) postulated that immunity probably developed very rapidly during primary infection, evidenced by the steady decrease in number of gametocytes after the initial peak. Fallis et al. (l95l) reported that there was no difference between splenectomized and whole ducks which were xposed for five days. They did, however, note that there was a higher level of parasitemia and longer duration of parasites before latency, in birds splenectomized after exposure. He concluded that the spleen must be an important defense organ and that this might account for the larger number of IS schizonts seen in the spleen after the initial cycles. Briggs (I960) reported that in addition to the low percentages of elongate forms seen in the Muscovy ducks that they had much lower para- sitemias and died later, if at all, than did the Pekins. He attributed this to a natural resistance in the Muscovies. Cowan (I957) did an extensive histological study on the host's re- action against the megaloschizonts. He noted five visable host reactions: I) encapsulation or possibly walling off by the host, 2) phagocytosis, 3) necrosis, 4) phagocytosis and necrosis, and 5) destruction by inflam- matory cells. These reactions were noted only in cells which had reached their maximum dimensions. Heterophils and macrophages were the most active phagocytic components, but in cases where only a few host cells were present they were heterophils and occurred near the outer margin of the schizont. He noted that as parasite removal proceeded there was an increase in the number of macrophages. There were, however, only inflammatory cells observed in the forms found in the brain. These forms, he noted, did not reach maturity regardless of whether or not there was a reaction by the host. There were some schizonts which ex- hibited both phagocytosis and necrosis at the same time. Others showed necrosis without phagocytosis, which in some cases extended inward from the limiting membrane of the parasite and could be interpreted to indi- cate that humoral immunity of some sort exists. Hhich of these mech- anisms occurred first, necrosis or phagocytosis, could not be determined by him. He does, however, postulate on the findings of other workers concerning premunition. He believes that constant reinfection keeps cellular elements at a high level thereby conferring a high degree of resistance, while one exposure with subsequent recovery does not l6 stimulate high cellular activity. The role of gamma globulin in malarial infections has just recently come to the fore. Work by Coggeshall and Kumm (I937) demonstrated that gamma globulin from recovered monkeys conferred a partial resistance to newly infected animals of the same species providing that the infection was of low intensity. They also demonstrated that there is a Species specificity of this globulin by attempting immunization of monkeys in- fected with Plasmodium inui with globulin from monkeys recovered from 2, knowlesi. Monkeys infected withufi. knowlesi showed an immunity upon being transfused with the recovered globulin while those infected with the milder E._ngi showed no immunity when treated with the same glob- ulin. Cohen and McGregor (I963) reported that the antibodies responsible for humoral immunity to malaria are in the 7S fraction of the gamma globulin. Globulin from uninfected patients showed no activity against malarial infections. Working with E. falcipardm infections in Gambian, African children, Cohen and McGregor (I963) showed that gamma globulin from hyperimmune adults drastically reduced the number of circulating parasites in newly acquired infections, providing the treatment was begun when the earliest stages of the parasite were observed in the blood. There was no immed- iate reduction in numbers but following the second schizogony after treatment began, the parasite density fell to below 1%10f its original value. Along with the reduction of parasite numbers there was an alle- viation of all symptoms except pyrexia. This continued even after ap- parent recovery which indicates that some mechanism other than the parasite's presence in the peripheral circulation causes the elevated 17 temperature. The protection afforded by this globulin, being passive in nature, lasted only up to twelve weeks, after which time the patient was again fully susceptible to further infection. These authors suggested that the action of the globulin was against the mature intracellular parasites or on the newly released merozoites. The latter appeared to be the most probable and helped to explain the apparent low level of protection of the malaria antibody, since it had access to the merozoites for only limited times and for short duration. The authors were not able to explain the few remaining parasites which persisted after the initial rapid clearance. It would seem that if the immune globulin caused the clearance of several million parasites that it would also remove the remaining few. Their work also contradicted the belief that it was necessary for the reticulo-endothelial system to be stimulated for a prolonged period before clearance was accomplished. This was noted when infant's blood was rapidly cleared of parasites during an initial infection when treated with immune globulin. MATERIALS AND ME H003 Set I: Early Summer Exposures For the pilot experiment, blood was collected from a single one- year-old mallard, Anas platyrynchos, who had recovered from Leucocytozoon infection, at a rate of 30 ml per week until 90 ml of serum was obtained. The serum was then mixed with saturated (NH4)2504 solution equal to one- half the serum volume, giving a 33% saturated mixture, which precipitated gamma globulin. The precipitate was then redissolved in 0.85% NaCl and the pH adjusted to 7.3. The entire procedure was repeated three times to increase the purity of the product (Figure I). This procedure is described in Campbell et al. (I963). Globulin precipitated in this man- ner is reported to contain both 75 and I95 gamma globulin. No attempt was made to separate these further. The precipitate from the third (NHn)2504 treatment was dissolved in borate buffered saline, pH 8.4, and dialysed against the same at 40C until sulfate ions could no longer be detected upon treatment of the dialysate with BaClZ. The product was then treated with l0 mg/ml peni- cillin-streptomycin and stored at -200C for four weeks. Thirty, two-week-old Pekin ducks were exposed to the vectors of Leucocytgzoon simondi on June l6, I964, at Indian River, Michigan. This is approximately thirty miles south of the Straits of Mackinac, and a known transmission area for Leucocytozoon. The exposure time was twenty- four hours in the endemic area. This timing was to insure a twenty-four hour or less difference in parasite deveIOpment. On June I7, twenty- four hours after exposure, all birds were returned by car to the W.K. Kellogg Bird Sanctuary in Battle Creek, Michigan, an area where transmission of Leucocytozoon to waterfowl does not occur. They were l8 Figure l. Cellulose Acetate strip electrophoresis of whole pooled duck serum compared to purified gan'ma globulin from the same source. Figure 2. A round and an elongate gametocyte of Leucocytozoon simondi i" the blood of an infected duck. (Giemsa). 20 maintained there for the remainder of the experimental period. All birds were individually marked by wing bands and housed in wire pens of approx- imately I0 ft x 50 ft with a shed for shelter at one end measuring l0 ft x l0 ft. The exposed ducks were allotted to four groups, to be sampled every 2 to 3 days. Group No. l consisted of four birds selected prior to ex- posure, who received, via the tibial vein, an injection of the above des- cribed purified gamma globulin equivalent to that in 5.5 ml of whole re- covered duck serum. Group No. 2, also four birds, was given identical treatment as Group No. I on the first day of patency based on the first appearance of blood forms. This occurred on June 30th, fourteen days post-exposure. Group No. 3, four untreated birds served as infected con- trols. Group No. 4 consisted of three unexposed birds who served as un- infected controls. All birds were controls to check transmission of other vector borne parasites which might complicate the course of the organism under study. Examinations were made every 2 to 3 days between 7:00 and l0:00 P.M. Hemocytological procedures consisted of red blood cell counts made on an A.0. Spencer “Bright-Line” hemocytometer. Blood samples for counts were collected from a puncture wound made in a web vein with a sharpened dis- secting probe. The first drop to appear above the surface of the web was touched to a clean microscope slide, smeared, air dried, fixed in methanol and later stained with Giemsa stain. The second drop of blood was drawn up into a red blood cell diluting pipette and diluted with standard Hayem's diluting fluid. (As each sample was collected the pipette was placed on a pipette shaker. When samples from six birds were taken, they were counted before the next birds were sampled. This was done to insure a 2l standard elapsed time between the first and last birds sampled and the time when the red cells were counted. Gametocyte density was calculated from the red blood cell counts and the parasite counts from the stained slides. All blood samples collected for future serum extraction were drawn from the jugular vein into a l ml tuberculin syringe. The samples were then placed in a water bath at 370C for one hour, after which time they were placed in the refrigerator at 40C overnight to retract the clot. The following morning the sera were poured off the clots and stored at 4°C for future use. Set II: Midsummer Exposures In an attempt to duplicate and clarify the results of the pilot experiment, a second set of young ducks was taken to Indian River on July l6, I964. This set consisted of thirty-two birds who were exposed for twenty-four hours. At the end of that time they were returned to the Kellogg Bird Sanctuary and housed in a manner identical to that described for Set I. The birds were two-weeks-old at the time of exposure, except two adult birds from the clean controls of Set I. Five birds were designated as infected controls and kept in the pen with the rest of the experimental birds. Another group of five birds was also kept as controls but was maintained in a 5 ft x 3 ft x 3 ft cage entirely covered by canvas except for a six inch strip along the bottom of each side. This allowed for a normal day-night photoperiod but excluded any direct sunlight. These will be referred to as dark controls. Group No. l corresponded to Group No. I of Set I. This group consisted of IO birds, each of which received gamma globulin 22 equivalent to that in 5 ml of Pekin serum recently recovered from Lgygf ocytozoon. This was administered via the tibial vein immediately prior to exposure to the vectors. Group No. 2 corresponded to Group No. 2 of Set I and consisted of IO birds. On July 24, eight days post exposure, the first day blood forms were evident, these birds were given the same globulin treatment as was Group No. I. The globulin used on birds in this set was obtained from the un- treated birds from Set I who had recovered from‘Leucocytozoon. All serum was pooled, and gamma globulin was prepared as described above. Group No. I of this set received globulin from serum collected on July l0, five days after the final gametocyte crisis; and Group No. 2 received globulin from serum collected from the same birds on July 20, fifteen days after the last crisis. This was done because of the limited number of recovered ducks aVailable and the tremendous volume of serum needed to immunize twenty ducklings. Blood collection and sampling was carried out in exactly the same way as was described earlier for Set I. The number of parasites per mm3 was also determined in the same way. Determination of the per cent of immature, mature and elongate game- tocytes in the peripheral blood was determined. Classification was based on the size and shape of the parasite. The elongate forms were obvious in most cases, but all forms which showed a pyriférm host cell were con- sidered elongate. Round forms included all mature microgametocytes and macrogametocytes with a round or polygonal shape. The immature gameto- cytes were only those which were smaller than the differentiated gameto- cytes. This classification differs from that of some other investigators. Figures 2 and 3 show those forms included in the classification. Figure 3. Two immature gametocytes of Leucoc tozoon simondi within unidentifiedblood cells of a duck. (Giemsa). 24 One hundred parasites were counted and the per cent of each type was calculated. In low level parasitemias, 0.3 to 0.7 per l03 red cells, only fifty parasites were counted and in extremely low infections, less than 0.3 per IO3 cells, only twenty-five parasites were counted. *Two of the birds exposed with this set were the full-grown unexposed controls from Set I. Blood samples were taken at random intervals and the Coombs anti-globulin test performed on their washed red cells. The procedure consisted of washing the cells of the infected ducks four times with 0.85% NaCl and treating with 0.5 ml of a I% suspension of these cells with rabbit anti-duck globulin serum. This serum was inactivated at 56°C for 30 minutes and adsorbed with normal duck erythrocytes prior to its use in the test. After the cells and anti-serum were mixed they were placed in a water bath at 37°C for thirty minutes. At the end of this time they were centrifuged for one minute in an International Clinical Centrifuge and the button at the bottom checked for agglutination. Con- trols consisting of cells from uninfected ducks were run in the same manner. RESULTS Set I: Early Summer Exposures Thirty-five per cent of the thirty birds exposed on June I6th be- came infected as determined by blood examination. The prepatent period, judged by gametocyte appearance, lasted from the I6th to the 30th of June, a total of I4 days. Patency lasted l0 days; until on July 9th only rarely could blood forms be seen. A single peak in gametocyte numbers was noted between July 2nd and July 5th as determined by calculating the number of parasites per mm of blood. In the control group which showed 3 out of 4 infected birds, the average number of parasites per mm3 of blood at the peak count was 5,l52 with an accompanying erythrocyte count of l.3 x l06 per mm3. This is a 3 loss of approximately 7 x l05 cells per mm when compared to the normal controls who showed about 2.] x I06 per mm3. Group No. I, which received globulin prior to exposure, showed only 2 out of 4 birds infected. In these a parasite density of 3,400 per mm3 was noted and a blood count of 1.8 x I06 per mm3, 2 x IO5 red cells less per mm3 than that of normal birds. Group No. 2, the post-exposure treated birds, showed only one infection out of four birds. This bird had less than l,000 parasites per mm3 of blood 3 and a red cell count of 2.l5 x l06 per mm , normal for a healthy duck. Set II: Midsummer Exposures Twenty-two of the 32 birds exposed on July I6th, a total of 69%. demonstrated an infection with a prepatency of 7 days. Gametocyte paten- cy lasted until August l5th, a period of 2l days. During this time 25 26 calculations showed a fluctuation of parasite density which had at least two peaks and in II birds three peaks were evident. These occurred on the 4th, 8th to l0th and l4th to l6th days of patency. Two birds showed some variation to this cycle and will be discussed below. The controls held in the dark showed identical fluctuations and similar parasite densities as compared to the controls who were kept outside and used as a base line for comparison of all experimental birds. Figures 4a and 4b and Tables I and II show gametocyte counts for the controls and dark controls as an average of each and as indiViduals in the table. Due to their obvious similarity they will both be considered one control group when comparisons are made. Eight of the IO birds exposed in Group No. I showed an infection. Of these, four had fluctuations in parasite densities similar to the controls. The other four showed only one peak, which corresponded to the first peak of the controls. Of these four, three had parasite den- sities of approximately I,500 per mm3. The fourth showed a density of 2,250 per mm3. All but one bird in this group had less acute density peaks than did the controls (Figure 5 and Tables III and IV). Group No. 2 consisted of 7 infected birds out of IO exposed. Six of these had parasite densities and blood counts comparable to the con- trols. #2665 exhibited a single peak that appeared to be a blending of the first and second peaks of the controls (Figure 6 and Table V). By August I5th all of the birds were recovered or showed extremely low parasite densities. All birds maintained a normal red cell count of I.9 x IO to 2.2 x I06 per mm3 during the prepatent period. On the first day of patency several of the infected birds showed a slight drop in count. .1il l Figure 4a. Summary graph of Table I showing the mean gametocyte densities per mm) blood of the control birds. 27 94 12,400 15,840 0' “b” amt/\a-fi' l 9’ o 0 .> fa .1!" 00' O _< _.I I'll mm UC.) m I) Z $2; X 5 o: '6 .0 .1 04—— —_ 4 6 IO I2 I4 I6 l8 2| 23 25 27 Figure lla. DAYS POST EXPOSURE Figure 4b. Summary graph of Table II showing the mean gametocyte densities per mm3 blood of the birds kept out of direct sunlight. .. ., _.....—— ._-_..— .w_-— 28 I0370 9'2 . ” 0'2 0'3 9'; QOI X SHAW 83d SBLAOOLBWVS O'I 9'0 O 4 6 IO 12 14 I6 15 21 23 25 Figure 4b. DAYS POST EXPOSURE 27 29 poo—n mEE Lea mou>00uoEmmk o m_e_ omo_ ~_m omNN o~mo. omma o_k~ o_om m_: emete>< o o o m_m ooa~ om~o_ oo_m owe. owNN om: mama o ommm comm mom. oo_~ omSo. com. osmm os~m «0.: mmo~ .xmoa pcooom as» c_ co_um_cm> acm__m m >_co gum: .>_eo_c pcoamoccoo mxmoa omega ecu “as“ meow eco mpc_n _oLucoo on“ cu_3 mo_u_mcep xmma mc_cma50u .mc_ucm__ u£m_c u>mp .mELOC ooca_coaxo ob nozo__m use can xcmp m cm uqox mpc_n mo mucaou ou>00uoEmm _m:v_>_pc_ .__ e_nmh o mom oo__ mON mkom ommm oemm_ w~mm scam. wou emete>< o o o o oeo_ ommw omm.~ ooom o o omew o omo ox.~ om: m_m_ owe. oo_~_ ommm cams. mo. . mmom o o em__ new. omwm kmmm ekmm_ 0mm: ONMNN team mmea km ma mu _N m_ a. a. N. o_ m .02 et_m ocsmoaxmuumom m>mo .mpcmn cacao one ob .mo_ucop_ ommZLecuo m_ use xmoa umc_m esp >_co mxom_ 6L_n 6L_;u ash .mcsmonoiumoa >mp pc_;u 1>uce36 pcm .LucoouL30m .cucou exp :0 mc_ccaooo mxmoa >u_mcop ou>00uo5mm omega 305m mpc_n 03H .mpe_n .mucoe 1_Loaxo as“ mo omega OH .mo_ucop_ mco_u_vcou Lope: ueox m_oLucou pouoomc_ c_ mouxooumEmm mo embasz ._ o_nmh I' III!!! III I‘ll-I'll. 1 Figure 5. Average gametocyte density of four birds in Table III showing an obvious lack of the second and third peaks of gametocyte density. #— 30 g» 4,427 °‘ I l 9‘ O G > s .4!" 001 O _< ._I m mm -00 {TI :0 Z $2; X 5 o: '5 .0 ml 0% J 1. 4 6 IO I2 l4 l6 l8 2| 23 25 27 Figure 5.. . DAYS POST EXPOSURE poo—n mes Lea mou>oouoEmmk mm omo moo. :om Room Noam. .kmm. ooam momm. .mN eoeee>< o oms omm o omsm. oowsN a..m. ooom oNNN. o mooN o oNo. ooo. moo. oon. ommo. oone oemN oeso. on oooN o o o.o o oNNN oooo omom oomm oo.m omN mooN .mm oko. ooo. mo.. ooom oom.. o¢.o. oooo. on.N eomm oooN .mnc_n mo masoLm Lozuo exp 06 mm m>mo oEmm exp :0 Laooo mo_u_mcon ou>00uoEmm xmoa och .c__:no_m mEEmm oases. as“ EoLm Noommo ucocmqam o: bozozm £0.53 co_uoowc_ op Lo_ca 6o~_c:EE_ QuoLm ecu Soc» an_n Loom .>_ a_nmh 3i o o o o o o RN. mo._ eNsa omN eoeLo>< o o o o o o o o om. ,o mooN o o o o o o o.m o.m. ooo. MN. kooN o o o o o o o ooo. ooo. o NooN o o o o o o o ooo. oomo. eon .moN NN mN MN .N m. o. a. N. o. m .02 ot.o ocsmoaxM1umom m>mo .azoLm .ococoo ago 2. xmoa pme_u ecu mm oE_u 05mm 0:» um mcaooo xmoa o_mc_m och .co_uoomc_ cu Lo_ca c__:no_m mEEmm 0:358. :u_3 po~_c:EE_ mc_on EOE» poowmo _m_o_mocoa ucocmdam cm poumcumcoeop 0:3 moc_n Look ecu mo mo_u_mcop ou>00uoEmw .___ 0.6mh Figure 6. Summary graph 8f Table V showing the gametocyte densities per mm blood of the birds immunized at the beginning of patency. 92 O1 2 N 0'2 5'3 0'3 QOI X QWW 83d SBIAOOLBWVD Q'I O'I 9'0 O 1 —- 4 6 I0 I2 I4 I6 I8 2i 23 25 27 Figure 6. DAYS POST EXPOSURE 33 poo—n mEE Loo mou>ooNoEmmk :.N mmm omm :.o ookN oNkm m:m.. amom N.so oom eoete>< o o o oo o.m. omm. omom. omm. oomm ooo. mooN o mNo o.m Noo ooom omo: ONosN oom. osom :oN mooN oom. oomo. omoN owNm oomm omm: omNN. oaoN oomk. owkm oooN o o ook o mNN ommm o.om mN: o o mooN o o o me mos. osoN oomm omm: ooo. ooN NooN o m.m oNo. o oNo. oomm omoq ooN. oooo o .ooN o omo omoN o omNo omom. o..m. mmo: ommo aoNo oooN NN mN MN .N w. o. a. N. o. m .02 ot.o oczmoaXmubmom m>ma .azoLm _oLucoo use we once“ ;u_3 >_omo_o pcoamoccoo moE_u omozh .oczmoaxouumoa xmp pc_;ui>ucozu 6cm .zucoouc30m .zucou 6;“ co uoccaooo neon63c ou>ooNoEmm xmoa 6:» Hosp aaocm oc_uco ecu mo ommco>m ecu eoLm new 6L_n some mo ocoooc ecu 50cm pope: on 6—:ozm u. oczumee_ >66 umc_w ecu co c__:no_m mEEmm oc:EE_ ;y_3 noN_c:EE_ muc_n ecu c_ me_u_mcop ou>00uoEmo .6oo_n ecu c. nmcmoaam mop>ooboEmm .> o_nmh 3.11 3 Very few birds lost more than I x IO6 cells per mm at any time during the course of the parasitemia but all birds demonstrated some degree of anemia, though variable, during the crises. Figures 7a and 7b demonstrate the lack of uniformity in the degree of anemia exhibited; however, they show that there tends to be some fluctuation in erythro- cyte numbers in accordance with the parasite density when it remains low. Ducks with high gametocyte densities showed an anemia which began with the first appearance of gametocytes and lasted until recovery from the primary parasitemia. Those with low gametocyte densities showed low red cell counts which corresponded to the peaks of gametocyte den- sity and recoveries which corresponded to the crises. By the last game- tocyte crisis all birds showed a blood count within the range of the uninfected controls Differential counts of the various forms of gametocytes showed a definite change in forms that corresponded to the changes in peaks and crises of gametocyte density. The per cent of each type of cell is in- dicated in Figure 8. It can be seen on comparison of the form graph and the parasite density graphs that immediately prior to each peak of parasite density there is a greater per cent of immature forms. This is very evident during the first two peaks but becomes somewhat obscured by the third peak. It will also be noted that the highest per cent of round gametocytes occurred at the first peak and decreased continuously as the infection progressed. The elongate forms first appeared in very low percentages twelve days post exposure, at the time of the first <=risls. From this time on their levels became higher until by August ]0th only elongate forms were seen. The four birds in Group No. I which showed low parasite densities Figure 7a. This figure depicts the concurrent but reciprocal fluctuation of gametocyte and erythro- cyte numbers observed in some of the infected birds with low parasite densities. 35 1 / . , /// mm. // We / nw / O \ kW E \\ 4T \ 1% A/ P // S \V um. \\ D \ o. \ \ .6 \\ . lea Idle. 5 .Nfl ale . e. a . . . 0 zoom 6mm 2...... Eco 1.111 m o>2maooma N_ 0_ 1T m» 0204 QZDOm mm3h<22_ O 0. ON O¢ On 00 Oh Om 0m 9%. .w ocsm_u 38 and a single peak, never showed elongate forms even though their para- sites were evident beyond the time when all other birds showed these forms. One of the other four birds from this group, who showed an otherwise normal cycle, demonstrated a delayed appearance of elongate forms. It also showed a relatively low gametocyte density. Close examination of the elongate forms at their high per cent levels showed a number of forms which appeared to be similar to the small immature or undifferentiated forms. Their percentages were not determined due to the uncertainty as to what they were and their dis- torted shape. Also noted was a number of round or polygonal parasites in elongate host cells (Figures 9a, 9b and 9c). On August 8th, twenty- two days after exposure, a change was noted in the appearance of the host cell nucleus of the elongate and some of the round forms (Figure l0). There appeared to be an erosion or breaking apart of the cell nucleus. This same phenomenon occurred in the first set of birds but at the time it went unrecorded because it was believed to be an artifact of stain- ing or fixation. A positive anti-globulin test was obtained two times during the primary infection of the two adult birds. Data as to duration of pos- itive anti-globulin reaction and titer throughout the infection were not obtained due to a contamination noted in the rabbit anti-gamma glob- ulin serum after the first two tests. Figure 9a. I ' " D Figure 9b. ~.' . "_ .I D ‘ Q I G Figure 9c. . J . . 1 ’ 7..o ‘1 0‘ Figures 9a, 9b and 9c. Round and polygonal gametocytes of Leucocy- m simondi in “elongate" host cells. ilote the extensions of the host cell . (Giemsa). . .o 1 b ._ I. “ a I ' '.I a \ ‘ -. . .A I 1‘ 5“ 0 t. .0 7'. » .\ ‘0 Q) I. ‘U > "W., ‘. " '0‘... . .s 4 b. 0‘. " " o a- o a, s'! .‘ 0.. 7 .0 ts ‘. .‘.‘ ‘ a I 9‘-.. ‘0 " Figure I0. Photomicrograph of two gametocytes of LeUCOCVtozoon simondi In host blood cells showing signs of erosion (Giemsa). DISCUSSION AND CONCLUSIONS A careful review of the work done to date on Leucocytozoon simondi reveals that there is a great deal yet to be clarified before a thorough understanding of the organism, in all of its aspects, will be possible. A discussion of the results reported above may serve to clarify some of the vague aspects of the biology of‘L, simondi. In comparing the data obtained from the first set of experimental birds exposed on June I6th with those exposed on July I6th, it was noted that the duration of prepatency and patency were not the same in both groups. This phenomenon has been recorded several times. The first time was during the summer of I963 while the author was working on a Lgucocytozoon problem for Dr. J.H. Barrow at the University of Michigan Biological Station, Pellston, Michigan. The second occurrence was dur- ing the first set of experiments reported in this paper and again in I964 by Dr. Barrow (personal communication). The birds observed at Douglas Lake and those reported by Barrow all showed extended prepatent periods lasting l0 or more days and terminated in the death of the birds. The birds exposed by the author in mid June, I964 showed the same pre- patent times but survived the initial parasitemia and subsequently re- covered. Chernin (l952a) also reports that those birds exposed prior to the first fly feeding episodes and remaining in the endemic area for the duration of the transmission period showed a different parasite cycle in the host than did those birds exposed at a later date. He described this as being low level infection which did not appear until more than ten days post-exposure (Table VI). The extended prepatent period was probably somewhat obscured due to some amount of time in wait- ing for the appearance of the vectors. Only the prepatent period was 4i 42 o o o :NIw Cu w_|m o_ _> 0 mm Om m—lw Ou mlm O— > mm mm om Jim Ou NNIN O— >— 3. :0 mm mmlm Cu m_lm m. ___ mm OO- 00_ m—lm OH ——IN N— __ o .. No . NA 3 No .. . O O 00— leQ Ca O—lm —— ucwcmEme mo_u__mumu m»MWuMh We pouuomc_ x ocamoaxo mo mono: mnc_n mo .02 acme—LoQXm .uo_coa co_mm_EmcmLu ecu unecmsoLzu poem o_Eovco ecu c. c_mEoL ea nozo__o 6cm acmmom ecu c_ >_Lmo bemoaxo mpe_n omega mo po_eoa acoumaoca concouxo 6cm >u__mucoe 3o. any ouoz .LoEEDm ego m:_L:n mes.“ moo—cm> um mo__w xom_n ea vomoaxe mnc_n seem Amwmm_v c_cLo;u >n nouoo__oo mama ..> o_nmh 43 involved in the work reported here since the birds were exposed for only twenty-four hours. To what this extended prepatency could be attributed is open purely to speculation. If the gametocyte numbers are any indication of the number of sporozoites introduced, then the inoculum size could not account for this extended prepatent period. This is supported by comparing the gametocyte densities from the first set of birds with those of the sec- ond set. An attractive explanation for this occurrence is that these infec- tions are produced by the first infected flies of the season. Since the parasite has not been shown to be transovarially transmitted from fly to fly it is probably acquired anew each year by the newly emerged flies. The parasite's long residence in the duck, which is by this time at least a year long, may have attenuated the gametocytes to such an extent as to decrease their virulence and thus one sees an “abnormal” cycling in the first infections of the year. Once the parasite has been rapidly passed from vector to host and back to vector a number of times, which indeed does happen in endemic areas, the virulence of the organism is enhanced and one then sees a “normal“ cycle in the host. Another explanation may lie in the species vector involved in the transmission. During the early weeks of the summer a different species of Simulium may be transmitting the parasite while later in the summer a different Species may be the vector, and indeed the vector at either time may not even be a Simulium but some other arthropod such as the recently suspected Culicoides. The possibility of different vectors finds support in Fallis' extensive work with these vectors which showed many species of Simulium to be capable of transmitting Leucocytozoon to 4L, the Anserorida and Culicoides as being able to transmit Leucocytozoon to other avian Species. The time of exposure also affects the pattern of the disease in the vertebrate. In contrast to early summer exposures, midsummer expos- ures produce a gametocyte density fluctuation during the primary infection. Also necessary for detection of this fluctuation is a reliable counting method for determining gametocyte density in the peripheral blood. A method employed by malariologists, that of calculating the number of par- asites per volume of blood, was used in this work in an attempt to get a truer and more reliable picture of the gametocyte density during the pri- mary attack. As Figures 4a, 4b and 6 and Tables I, II and V show, a fluc- tuation in the density of gametocytes present in the circulating blood was detected. This phenomenon has not to this date been described for ngcocytozoon. It was suspected by Chernin and Sadun (I949) but was later repudiated by Chernin (I952c) on the basis of counting gametocyte- cytes per unit time. Fallis et al. (l95l) presented a graph which is based on a similar counting method which shows three peaks which corre- spond nicely to those described in this paper (Figure ll). However, the degree of rise and fall in numbers is greatly reduced in this graph. This is probably due to the number of erythrocytes present in any given volume of blood varying during the sampling period while the speed at which the observer could count parasites remained essentially the same, except at extremely low and high parasite densities. In the experiments reported here a standard exposure time of twenty- four hours was set and data analyses were based on these exposures. All of the figures showing the course of infection in the Pekin presented here show that the first sign of infection appears on the seventh day Figure II. A graph reproduced from Fallis et al. (l95l) showing three distinct peaks in gametocyte num- bers. These peaks correspond closely to those reported by the author but are based on one minute counts from blood smears. 45 @Z .LNOOO MN I 83d SBLISVHVd 001 0 I4 I6 IE 20 22 4 6 8 IO I2 FISiure ll. DAYS POST PATENCY 46 post-exposure. Chernin's results show clearly that all those ducks exposed for a single twenty-four hour period became patent at the same time; whereas ducks exposed for extended periods show infections in which the beginning of patency varied considerably, although he did not interpret his results this way. A factor in favor of single exposures is seen in the consistent appearance of the pleomorphic gametocyte forms. Although these will be discussed in greater detail later in this paper it should be noted here that when ducks are exposed for a single day the elongate form of the gametocyte appears on the same day in all birds exposed at the same time. On the other hand, birds exposed for more than one day show a parasite picture with variable appearance of the elongate forms (Figure 8 and Table VII). If these forms are a part of the maturation cycle of one species of parasite then it seems logical to expect them all to appear at the same time if their precursors were all introduced into the host at the same time. Another point which also advances the idea that single exposures give truer pictures of the vertebrate cycle is the very synchronous gametocyte fluctuations which occur during the course of the primary infection. The fluctuation in gametocyte levels does not appear to be the result of sequestering of cellular elements with their subsequent re- lease. This is borne out by the observation that in some cases the num- ber of erythrocytes was at a low point while the gametocyte density was at a high point. The possibility of preferential or Specific seques- tering of the parasite from the blood should not be overlooked. In Plasmodium falciparum infections such a sequestering of blood forms 47 ommco>m O KO C ox Ln 4' :1" mNd'Cod'mxoxooomo-‘x mm—mmfimixo:\o mmNm-a' Lnxoa'xomm ocamoaxo >mp w ommco>m Ln \0 LA |'\ 0 Ln (I) M common comm“) LnLnLnLn moczmoaxo >mn oco :I'd'd'm xmoo _mu0u xmoa menace—e moummco_o um. xmoa ocaumEEm we eucmemoaam .mpo_cma vevcouxo Low bemoaxo mpe_n c. mELOm omega mo oocmcmoaam we eE_u o_nm_cm> ecu o» pocmasou mm meso: :N Lo$ oomOQXo mvc_n c. >ucouma mo >mn nuw_m exp :0 mou>o touoEmm mummco_o ecu mo oocmcmoaam acoum_mcoo asp m:_3o;m AONmm_V o_nmu m.c_cco;u .._> 0.364 48 does indeed take place. However, the forms removed are the maturing schizonts, not the gametocytes. Since no circulating schizonts have been shown to occur in L, simondi infections this would be a unique situation if the gametocytes were removed. Another, and more likely explanation, is that gametocytes are being released ggumgsgg from the tissue megaloschizonts at specific times during the course of the infection. The prepatent asexual development proposed by Fallis et al. (I956) consists of two asexual cycles prior to gametocyte release. Since gametocyte patency usually occurs on the 6th to 8th day post-exposure an asexual cycle of 3 to 4 days in length is likely. If this same cycling continued beyond that first appearance of gametocytes it would be expected to produce a high percentage of immature forms and peaks in gametocyte density about 3 to 4 days apart; and indeed that is exactly the picture seen in Figures 4a, 4b, 6 and 8 and Tables I and II. Cowan (I955) stated that he believed the megaloschizonts to be the sole contributors to the gametocyte population seen in the blood. If this is true then the hepatic schizonts in the Kupffer cells of the liver must be the precursors of the first megaloschizonts. The question now arises: Do the megalo- schizonts produce only gametocytes or both gametocytes and more megalo- schizonts? If they do not produce more megaloschizonts, then the hepatic schizonts must survive and divide during the remainder of the infection or are capable of reproducing themselves. If one of these suggested possibilities were not true then it would be difficult to explain the presence of relapse gametocytes more than a year after the initial ex- posure to sporozoites. Another possibility is that the megaloschizonts produce in addition to gametocytes, a merozoite similar to the sporo- zoite which then invades the liver and begins the cycle over from the 49 beginning. Figure I2 shows the hypothetical cycling possibilities which could explain the fluctuation in gametocyte densities and the presence of parasites after recovery from the initial acute infection. If this type of cycling was the case it would be reminiscent of Coccidial infec- tions. Eimeria tenella, a parasite of chickens goes through a develop- ment which is very similar to 'b' in Figure l2. Following infection by sporozoites there occurs a first generation schizont. This produces a second generation schizont which is the precursor to gametocytes and a third generation schizont. The third generation schizont produces game- tocytes and very rarely another schizont generation. This development would be analogous to the hepatic schizont of Leucocytozoon producing megaloschizonts which are the source of gametocytes and other megalo- schizonts. A third explanation for the occurrence of the peaks in gametocyte levels has its basis in the work of Cowan (I955, I957) and Fallis et al. (l95l). These workers demonstrated megaloschizonts in various tissues during the course of the primary infection. As the infection progressed, more and more of the tissues became negative for megaloschizonts indi- cating that they had either ruptured and spilled their merozoites into the host or that the host had destroyed them with some immunological mechanism, as described by Cowan (I957). If they were disappearing due to their rupture upon becoming mature, then it is possible that megalo- schizonts in different tissues mature at different rates and therefore release gametocytes at different times during the infection. The fourth possibility is the presence of two or even three species or strains of Leucocytozoon with the same host range and spec1f1c1ty. ' ould If such a situation did occur it is conce1vable that the vectors w 50 .o_o>o om__ esp unenm muomw czocx mc_um_xo co comma x036 ozu c— ecoEeo.o>ep mucoE_m coo~0u>ooolw4 mo mouse. .mo_ue;u0d>s m.co;u:< .N. ocsm_m muco~_LUm umumeostllllllll muco~_50m o_umao; llllllll mucoN_;6mo_mmms muco~_com o_umam; meu_0NoLoam Au vc_£u muco~_LUmo_mmoE \\\\\\ \\\ acouom muco~_;omo_mmme mou>ooNeEmm mmuxuouoEmm \\\ pmc_m mou>o0uoemm muco~_;umo_mmee Ezo/ mucow_;umo_mmee pcooemillll mou>ooNoEmm mucow_com omumae; nllltmoumouocoam An muco~_20mo_mmme.\\\\\\t mmu>00uesmm \\ umc_m mou>00uosmm muco~_50mo_mmoe \\\ 6L_:u mucowmsomo_mmoe mou>00ueEmm \ pcooom {I 3:323 ozmemclmmtowosqm Am mou>00umEmm muco~_50mo_mmoe \\\\\\t \ at... mou>00uoEmm 5l be infected with two or three of these species or strains and that trans- mission of all of them occurred simultaneously. The report by Chernin (l952c) that four ducklings went through an entire cycle without show- ing a difference in gametocyte morphology is interpreted by some to be an indication of a single species of parasite. If the different species or strains had different maturation times this would account for the gametocyte peaks occurring at different times but in coincidence with each other. The presence of different species or strains in the same host may result in a type of competition and/or inhibition of one by the other. If this were the case then the cycles of these different parasites would be out of phase with one another and their maximum gametocyte densities would be at different times. The reduction in number of circulating gametocytes by the thirtieth day of infection would most likely be the result of the host's immune mechanisms keeping these forms in check. This will be discussed further in the section on immunity. On the other hand, it may be due to a mech- anism similar to that seen in various Eimeria species in which a char- acteristic number of schizogonies occur after which the infection is terminated. The investigators in this area have not made use of the techniques of malarial research even though the two organisms are suspected to be closely related. Standardization of inoculum or exposure time in 5332: ocytozoon research is practically unheard of though it is common prac- tice among malariologists. A second error in results of experiments with this organism lies in the determination of the parasite density in the bird host. This has previously been done by counting the number of parasites in a standard thin blood smear observed during some given 52 period of time. A second commonly used method is to count the number of parasites per microscopic field or per some given number of erythro- cytes. It becomes obvious that these methods must be improved before an accurate picture of the course of the infection in the vertebrate host can be elucidated. It seems improbable that sporozoites introduced into a single host over a period of a week will all be in the same stage of development at the same time. If they all develop at approximately the same rate some will be ahead of others in their maturation, thus a single expos- ure of short duration (l2 hours) would give the most accurate picture of the parasite's course in the vertebrate. Unless exposure and counting techniques are standardized it will be difficult to control other phases of research with this organism. The results of the passive immunity experiments reported above would not have been observed unless these conditions were met. Since reference to immunity to Leucocytozoon is rarely found in the literature, it was necessary to turn to Plasmodium research for ideas on how to attack the problem of humoral protection to Leucocytozoon. The majority of the literature on plasmodial immunity deals with cell- ular immunity since it has been believed for many years that humeral antibody to malaria was nonexistent or at most a nonprotective factor. The work reported by Cohen and McGregor (I963) on passive transmission of humoral immunity to Plasmodium falcjparum in Gambian infants pro- vided an excellent basis on which to develop methods for studying this same phenomenon in Lgycocytozoon infections. The methods used to pas- sively immunize ducks toqLeucocytozoon were similar to those used in the above reported work. The results, however, manifested themselves 53 in quite a different way from those results obtained by the two authors. As mentioned under results, when immune globulin was given to infec- ted ducks at the first Sign of parasites in the blood, there was no ef- fect noted. The infections proceeded in a manner very similar to the in- fections of the untreated control birds (Figure 6). However, when immune globulin was administered immediately prior to exposure to the vectors there occurred a lower number of gametocytes in the peripheral circula- tion and a loss of the second two peaks of gametocyte density in half of the birds as compared to the gametocyte population noted in the controls. There also appeared to be no alteration in the degree of anemia in the birds who showed reduced parasitemias. This point is contrary to the reports of Cohen and McGregor (I963) since they demonstrated the allevi- ation of all symptoms of the disease with the exception of pyrexia. Since the nature of perpetuation of the parasite in the tissues has yet to be elucidated it is impossible to know just where the humoral protection might act. One explanation for the lower gametocyte number is that it was not due to transferred immunity at all, but was a normal variation in the vertebrate cycle of the parasite. It seems unlikely, however, that of all of the birds used this should occur in half of the treated birds in one group and in none of the other birds examined during the experiment. Since other authors used different methods of determining gametocyte density it is impossible to compare other work in this case. If the lowered gametocyte density was due to a passively transferred immunity, the results can be explained. The results obtained in malarial immunity studies have led investigators to speculate that the humoral protection acts against the schizonts or against the merozoites when 54 they are in an extracellular state. The latter suggestion seems most likely since the schizonts remain inside their erythrocyte until rup- ture of the parasite occurs. Based on these postulates one can see that when the earliest forms appear in the circulation in Leucocytozoon in- fections they are the result of previous schizogony and have already entered their host cell and are therefore protected against attack by humoral antibody. It also becomes apparent that the schizonts in nggr ocytozoon infections are even less susceptible to humoral antibody attack than are the erythrocytic schizonts of malaria, since they are entirely within the internal organs. Even when acquired immunity has eliminated essentially all of the blood forms of malaria, the tissue schizonts remain unharmed, as is the case with recoveries from Leucocy- tozoon. The survival of the tissue schizonts could be due to their be- ing nonantigenic -unharmed by the antibody produced against themsaor be- cause they are not in contact with the antibody produced. Clark (I964) showed that the internal organs of the magpie, infected with a species of Leucocytozoon, were positive for gametocytes even when the peripheral blood was negative. This indicates that production of gametocytes or at least survival of them continues throughout the life of the host but the host is capable of suppressing their entrance into the peripheral circulation. From the results of the birds immunized at the first sign of peripheral stages, it can be seen that the immune globulin does not work against the gametocytes. Based on these results it must be assumed that the protection noted in the pre-exposure treated birds must be the result of antibody action on the prepatent stages. This immune action might well be against the tissue stages, but in untreated birds humoral immunity does not manifest itself until patency has first been noted 55 (Cowan, I957). If Cowan is correct in interpreting the results he ob- tained from histological studies of the tissue stages at the onset of patency, then it must be assumed that humoral protection occurred before this time in the immunized birds. If the prepatent asexual cycle pro- posed by Fallis is correct then it is possible to speculate on just where the immunity does work. Starting with the first proposed asexual generation, the immune action may have been toward the entering sporozoites or against the he- patic schizonts themselves. If it was against the sporozoites then there would be fewer or attenuated hepatic schizonts. If the action was against the schizont or against its newly released merozoites, then the megalo- schizont generation would be interrupted. This again might manifest itself in lower numbers or in attenuated gametocytes. The last place that immune globulin might act to alter the course of the infection in the vertebrate host is at the megaloschizont, as Cowan suggested, or on its progeny, the merozoites. Referring back to the postulated tissue recycling, a number of pos- sibilities or combinations of these may represent the susceptible stage in the cycle. If as mentioned above the reduction In numbers of game- tocytes and the lack of the last two peaks in density were a normal var- iation in the cycle of the disease, then further discussion is beyond the scope of this paper. However, if they are the result of the immunization then some discussion as to how these phenomena came about is in order. If the first explanation for the peaks in gametocyte density, that of preferential sequestering, were true, then the immune globulin might well enhance the ability of the host to sequester and retain the game- tocytes within the tissues. Since gametocytes have not been shown to 56 play a role in the pathology of this disease, then this mechanism would not play a protective role in the defense against the parasite. Look- ing at the entire waterfowl population, however, it is obvious that if there were a low pool of gametocytes for the vectors to draw from then transmission should be reduced to where sporozoite numbers would be below a critical level. Such a mechanism is substantiated by the low mortality rate in wild populations. The second proposed explanation for the gametocyte density peaks is an internal cycling which releases merozoites at regular intervals. Any action on the tissue stages or the merozoites which they release would have an effect on the later gametocyte population. If the action were against the merozoites which were destined to produce megaloschizonts then the lower gametocyte numbers would be a direct result of fewer megaloschizonts, and indirectly the result of immune action on their precursors. The possibility of there being different rates of megaloschizont development in different tissues might go hand in hand with the possi- bility of different tissues being more susceptible to infiltration by humoral globulin. If this were true, the gametocyte fluctuation could be explained in this way: Certain of the tissues are invaded by the merozoites which rapidly develop into megaloschizonts while they are relatively well shielded from immune attack. Other megaloschizonts, developing in other tissues, would be maturing more slowly and would be more susceptible to the action of immune globulin. This situation would yield the early gametocyte peak from the rapidly developing and protected megaloschizonts. The gametocyte picture which would result from this series of happenings would be a single peak occurring at its expected 57 time and the inhibition of development of the gametocytes for the suc- ceeding peaks. If the fourth possibility for gametocyte fluctuation were true, that is that more than one species or strain of Leucocytozoon might be involved the possibility arises that one of these strains might be more suscepti- ble to the host's defense mechanisms than the others. If the above pro- posal is correct and the peaks in gametocyte density are due to the dif- ferent rates of development of the various strains or species of the para- site, then when the host's antibody destroys one or more of these the antibody titer against it gradually diminishes until by the time the serum for immunization is collected the titer is insufficient to protect the immunized birds. Based on this possibility, the course of the game- tocytes in the immunized bird would be as follows: The second and third peaks which do not appear in the immunized birds are the peaks produced in the unimmunized birds by the parasites which are not completely elim- inated by the host's defense mechanisms. The result is high antibody titer resulting from premunition, the constant stimulation by the resid- ual parasites. The first peak would represent those parasites which were completely eliminated by the unimmunized host. Since the titer was declining at the time of serum collection there was incomplete pro- tection conferred on the immunized birds, or if the titer was so low as to produce no protection, the low peak may be a result of cross immunity between the species or strains which survived and that one which was eliminated. Birds which survive the primary parasitemia produce a latent infec- tion. This is most likely the result of an acquired immunity obtained during the course of the initial encounter with the parasite. As 58 Clark (I964) reported the number of gametocytes in the tissues far ex- ceeds the number noted in the circulation during periods of latency. This would indicate that there is little or no immune inhibition of the production of gametocytes but that they are being retained within the tissues where they were produced. Whether or not they can survive indef- initely once they have been produced has not been demonstrated. Indica- tions tend to exclude their continued existence beyond some short time. Fallis et al. (I95l) showed that gametocytes transplanted into clean hosts survived in the circulation only 6 to 7 days. Also, if all gametocytes produced during the period of latency were to be retained in a viable state throughout the year then why does the relapse the following spring Show only l/l000 the gametocyte density as did the primary infection? It is possible that gametocytes are produced and destroyed at a constant rate during the latent period and are released into the circu- lation by some mechanism during the relapse noted in the spring. All of the defense mechanisms of the host may not always be to its advantage. Many authors have suggested that in protozoan infections, and in particular, malarial infections, there is the possiblity of an autoimmune mechanism acting in the host. The observation that anemia occurs during L. simondi infections is frequently reported; however, as mentioned above, accurate erythrocyte counts were not made routinely and the infections studied were multi- ple infections by the same organism resulting from long exposure, thus tending to blend and/or obscure any accurate picture of the anemia. Comparing the erythrocyte counts with the gametocyte densities in Figure 4a and Tables I and II it becomes obvious that the high and low peaks of gametocytes correspond inversely or directly. Comparing the 59 anemia with the course of infection in the vertebrate, one can see that those figures referred to for comparison of anemia with parasitemia show a relatively low parasite level. The above described coincident rise and fall in erythrocyte and gametocyte levels could be interpreted to be the result of some common cause. Since it is quite evident that the circulating gametocytes could not cause such a drop in erythrocyte numbers the next most obvious cause would be the tissue stages. There is good evidence, as mentioned above, that the megaloschizonts are responsible in some way for the fluctuations in gametocyte levels, but these stages present no obvious means of caus- ing anemia. As will be recalled from the section on results, a positive anti-globulin test was noted on several occasions during the infection. This will be discussed in greater detail in the next section on immunol- ogy but it might be noted here that this could readily cause an erythro- cyte destruction as the result of an autoimmune response. The tissue stages may be responsible for the autoimmune response and loss of ery- throcytes. This mechanism could account for the seeming coincident fluctuations of gametocyte density and erythrocyte numbers. Zuckerman (I964) put forth a number of possible explanations for such autoimmune responses, some of which would do well to explain the erythrocyte loss in.L. simondi infections. Since it is still unknown as to which type or types of cells are involved as infected host cells in this infection, it is more difficult to postulate just why the erythro- cytes become Coomb's positive. One or more of the following proposals may be the answer: I) The possibility of a common antigen or a similar antigen would explain the positive test. That is to say, the parasite possesses some antigen, which is different enough from those of the host 60 to elicit an antibody response but is still similar enough to the host's erythrocyte antigens to cause the antibody to react with the erythrocytes. A similar situation is seen in the treatment of rabies victims with rab- bit tissue from the CNS. In some cases the patient's immune response to the rabbit tissue also acts on his nervous system in a way which des- troys a portion of the myelin sheath. 2) Another explanation may lie in the possible alteration of the host's tissue or possibly the exposure of certain antigens which normally are not in contact with the immune mechanism of the host. In this case the host reacts with his own tis- sue to produce an antibody against a part of its normal antigenic make- up. The alteration or exposure of their antigens would be the result of the parasite's penetration of the host's cells. 3) The third explana- tion is the adsorption of parasite antigen on the surface of the host's erythrocytes. These antigens may be metabolic products of the parasite released into the extracellular spaces of the host or may result from the breakdown of whole parasites. In either case, the antibody response toward these antigens on the surface of the host's cells would appear to be a response against the cells themselves. 4) The altered ability of the host to recognize self from non-self would also produce such an auto- immune response. In this case the host produces antibodies against its own tissue. This possibility may have grounds in the supposition of a number of authors who state that the invaded host cells are cells of the lymphoid series. Since these cells are known to play a role in antibody production it is conceivable that they may be altered to such an extent by the invasion that they react against the host's own antigens. Obviously, from the four possibilities for autoimmune responses mentioned above, there is no one solid definition of an autoimmune response. 6l As it is used here it will be considered the apparent destruction of the host's own tissue as a result of its own immune responses. This autoimmune response usually manifests itself in the form of anemia or in the destruction of some vital tissues of the host. As men- tioned above, a positive anti-globulin test was demonstrated during the course of an.L. simondi infection in an adult Pekin. Since this test was negative for uninfected controls it was assumed that the reaction was the result of the infection. Zuckerman (I964), however, has recently shown that the reticulocytes produced during anemia also exhibit a pos- itive Coomb's test. This makes the above results somewhat doubtful. At least a new test must be employed to clarify the proposed autoimmune response. If an autoimmune mechanism is not involved and some other means of erythrocyte destruction is involved, then the graphs showing gametocyte and erythrocyte fluctuations (Figure 7) might be interpreted to show that the host can compensate for erythrocyte loss very rapidly providing that the parasite burden is not too great. When the gametocyte levels are high, indicating a heavier parasite burden, there appears an initial drop in erythrocyte numbers which remains at a low level until recovery. The host apparently can not compensate for the loss. The suggestion by Cook (I954) that erythrocyte destruction is due to their penetration by merozoites seems completely without grounds. If this destruction was due to parasite invasion, what can account for the loss of one million or more erythrocytes when only 5 to 20 thousand parasites are present in an equal volume of blood. One argument might be that the destruction is occurring in the internal organs. What then would be the mechanism of destruction since it is well established that 62 no schizogony occurs in the erythrocytes and no evidence has been put forth that the gametocytes change host cells during their residence therein. Another argument might be that the stem cells of the erythro- cytic series are being invaded. If this were true one could not explain the rapid recovery from anemia three consecutive times. A single recov- ery would be plausible due to the storage of erythrocytes in the bone marrow, but it is difficult to conceive of three rapid recoveries when the stem cells are being destroyed and are unable to replenish the stor- age cells. Immunity or autoimmunity may also be involved with other cell lines in the host, such as the leucocytes. Although no experiments were designed to elucidate just why there was more than one host cell type and parasite shape, there appeared in the results of this work a phenomenon which may be of some value in clar- ifying this question. In reference to Figure 8, it can be seen that on the sixth day after the onset of patency and at the first gametocyte crisis, there appeared the so-called elongate gametocytes. From that time on they increased in per cent until by the onset of latency they constituted IOO% of the gametocyte population. Figures 2, 9 and l0 show elongate forms which were observed during the course of the blood slide examinations. There appeared to be more than just the elongate gametocyte involved in these spindle shaped host cells. Some are imma- ture gametocytes, others are the commonly observed round or polygonal shaped gametocyte, and the elongate gametocyte. The reports that no transitional stages exist between the round and elongate forms seen in the blood are not borne out by these photographs. There are obviously round gametocytes enclosed in an elongate host cell. 63 Just what causes the pleomorphic forms has not been shown. Some authors suggest the difference to be in the type of host cell invaded, but as Cook (I954) pointed out, if this were true the two forms should appear at the same time. Chernin's suggestion (l952c) that it may be the result of the host's immune response is not supported by the results obtained in the immunization experiments reported above. No difference was noted be- tween the immunized and control birds in the appearance or number of elon- gate forms. One exception to this is noted in the four birds which showed only one low gametocyte density peak. At no time did elongate forms of the gametocyte become evident during the course of the blood examinations of these four. However, if this were the result of the immune globulin acting on the host cell-parasite complex, the same type of reaction would be expected to occur in the birds immunized when the gametocytes first appeared since the antibody titer would be higher in these birds at the time the gametocytes were released. In fact, this lack of elon- gate forms did not occur in any other birds in these experiments. Another suggestion by Chernin (l952c) is the influence of fundamen- tal host differences. This could account for the various forms and could also be responsible for the single peak in gametocytes discussed above. His third suggestion, that of parasite strain differences also presents a likely possibility for explaining the variation in gametocyte forms. Briggs' report (I960), that Muscovy ducks showed less than 5%.elon- gate forms and suffered much less severely from Leucocytozoon infections, compared with the apparent lack of elongate forms in the immunized ducks mentioned above does indeed suggest that some type of immunity may be the cause of this phenomenon. Since good evidence has been presented which rules out humoral influence on this occurrence the possibility arises 64 that cellular sensitization may produce the difference. This could be explained if during the early part of the infection macrophages or other phagocytic cells are becoming sensitized to parasite antigen while phag- ocytizing it, and later when they come in contact or are invaded by a merozoite this elongation response manifests itself. This mechanism is purely speculative but it does have possibilities. SUMMARY Two-week-old Pekin ducklings were exposed for twenty-four hours to infected black files so as not to produce concurrent infections of ngcocytozoon simondi originating at different times which might obscure the true course of a single infection in the duck host. Red blood cell counts and gametocyte densities per mm3 of blood were made during each experiment. Two sets of birds were exposed, the first on June I5th and the sec- ond on July l5, I964. There were obvious differences in the gametocyte picture and duration of the prepatent and patent periods in the two sets of birds. Prepatency in birds exposed early in the season lasted l4 days followed by a steady increase in gametocyte numbers and a subsequent crisis and recovery. The birds exposed in midsummer demonstrated a seven day prepatency and a gametocyte density which was observed to have a periodicity with three peaks, one on the 4th, 8th to l0th and l4th to I6th days of patency. The significance of the peaks is treated in the paper. Another observation which suggested the possibility of a period- icity existing in the vertebrate cycle of this parasite was the appear- ance of the so-called "elongate“ gametocytes. These first appeared in all but one bird on the sixth day of patency. Elongate host cells con- taining round gametocytes were observed and interpreted to be inter- mediate forms possibly resulting from a previous sensitization of the host's cells. An attempt was made to passively immunize ducks against Legcocytozoon using gamma globulin from recently recovered ducks. None of the birds immunized on the first day of patency showed a deviation of parasitemia when compared to control birds. Half of the birds immunized immediately 65 66 prior to infection demonstrated a loss of the second two peaks of game- tocytes and a drastically lower number of gametocytes when compared to the controls. It is suggested that anemia which accompanies infections of Leucocytozoon may be due to an autoimmune response of the host. It has been demonstrated in several instances that fluctuations in gametocyte density and anemia occur simultaneously. LITERATURE CITED Barrow, J.H. I964. (Personal communication). Briggs, N.T. I960. A comparison of Leucocytozoon simondi in Pekin and Muscovy ducklings. Proc. Helminth. Soc. Wash. 21; l5I-l56. Campbell, D.H., J.S. Garvey, N.E. Cremer and D.H. Sussdorf. I963. Methods in Immunology. W.A. Benjamin, Inc. Chernin, E. l952a. The epizootiology of Leucocytozoon simondi infections in domestic ducks in northern Michigan. Ann J. Hyg.‘5§: 39-57. Chernin, E. I9526. The relapse phenomenon in the Leucocytozoon simondi infection in the domestic duck. Am. J. Hyg.‘5§: lOI-llo. Chernin, E. l952c. Parasitemia in primary Leucocytozoon simondi infec- tions. J. Parasitol. 38: 499-508. Chernin, E. and E.H. Sadun. I949. Leucocytozoon simondi infections in domestic ducks in northern Michigan with a note on Haemoproteus. Poultry Sci. 28; 890-893. Clark, G.N. I964. Frequency of infection and seasonal variation of Leucocytozoon berestneffi in the yellow-billed magpie, Pica nuttalli. J. Protozool. ll; 48I-484. Coggeshall, L.T. and H.W. Kumm. I937. Demonstration of passive immunity in experimental monkey malaria. J. Exp. Med. 66; I77-l90. Cohen, S. and I.A. McGregor. I963. Gamma globulin and acquired immunity. lg; Immunity to Protozoa, P.C.C. Garnham, ed., F.A. Davis Co., Philadelphia. pp. l23-l55. Cook, A.R. I954. The gametocyte development of Leucocytozoon simondi. Proc. Helminth. Soc. Wash. 215 l-9. Cowan, A.B. I955. The development of megaloschizonts of Leucocytozoon simondi Mathes and Leger. J. Protozool. 2: l58-l67. Cowan, A.B. I957. Reactions against the megaloschizonts of Leucocyto- zoon simondi Mathes and Leger in ducks. J. Inf. Dis. I00: 82-67. Fallis, A.H., R.C. Anderson and G.F. Bennett. I956. 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Protozoa of the genus Leucocytozoon Danilewsky l890 in birds of the environs of Nraclaw (Poland). Acta. Parasitol. I0: 39-52. '- Rawley, J. I953. Observations on the maturation of gametocytes of Leucocytozoon simondi. Proc. Helminth. Soc. Wash. 21: l27-l28. Savage, A. and J.M. Isa. I959. Note on the blood of ducks with Leuc- ocytozoon disease. Canad. J. Res. (Zool). 31: ll23-Il26. Ningstrand, K.G. I947. On some Haematozoa of Swedish birds with remarks on the schizogony of Leucocytozoon sakharoffi. K. svenska VetenskAkad. Handl . 24: l-3I . Zuckerman, A. I964. Autoimmunization and other types of indirect dam- age to host cells as factors in certain protozoan diseases. Exp. Parasitol.,lfl: l38-l83.