Anemia in ducklings treated with malarious plasma

EXPERIMENTAL

PARASITOLOGY

Anemia

18,

281-289

(1966)

in

Ducklings

Robert

M.

Department

Treated

(Submitted

of

University for

Malarious

and R. Barclay

Corwin’

of Zoology,

with

publication,

Plasmal

McGhee

Georgia,

Athens,

27 August

1965)

Georgia

CORWIN, R. M., AND MCGHEE, R. B. 1966. Anemia in ducklings treated with malarious plasma. Experimental Parasitology 18, 281-289. Plasma from ducklings infected with Plosmodium lophurae was filtered to insure the removal of parasites and donor red blood cells; this was administered intravenously in a series of three inocula per recipient duckling. Birds receiving the plasma underwent an anemia and a radical loss of polychromatophil erythroblasts. These manifestations were similar to those observed in ducklings with low-grade infections. In one of three groups of experimental ducklings which received no further treatment, the total red blood cell count reached a mean of less than 1.00 million per cubic millimeter. The birds became moribund and died or were killed. In the other two groups, there was a replacement of red blood cells by the influx of basophil erythroblasts. This alleviated the anemia and recovery followed. From a review of the literature and examination of our own work, we believe that we are observing the effects of one or more soluble antigens in the malarious plasma and of corresponding antierythrocytic antibodies.

The pathological processof malaria, whetxher in birds or in human beings, is marked by anemia. In both, the anemia may be disproportionate to that expected due to cell destruction by the parasites present. Anemia in excess of what might have been expected was observed by Terzian ( 1941) in the domestic chicken infected with the parasite, Plasmodium lophurae. Since parasitemiaswere low, he felt that some factor other than the parasites might be producing hemolysis of unparasitized cells. Gear (1946) proposed that the host erythrocyte might enter into combination with the malaria parasite to produce an autoantigen. The antibody formed would react with uninfected and infected red blood cells and would result in hemolysis and anemia. Excessive loss of erythrocytes has been studied most conveniently in low-grade infec1 This work was supported in part by grant AI 00924 from the National Institutes of Health. 2 Present address: St. Ambrose College, Davenport, Iowa. 281

tions so that direct parasitic hemolysis would not mask other causative factors. McGhee (1960) found that ducklings inoculated subcutaneously with duck embryo-adapted P. lophurae sustained light infections but underwent severe anemias. This same condition was obtained when infections were controlled by quinine dihydrochloride (McGhee, 1964). Similar results were obtained with infections of Plasmodium berghei in rats (Zuckerman, 1960a) and P. Zophuraein chickens (Zuckerman, 1960b). Both McGhee and Zuckerman have suggestedthat their findings involve an autoimmune response. In an extensive review regarding the possible role of autoimmunization in protozoan infections, Zuckerman (1964) contended that malaria parasites, being intracellular, were well placed to alter or modify host cell contents and/or products of these cells and thus become antigenic. Other explanations of the apparent autoimmune state, according to this author, might be the coating of the host cell with parasitic antigen or antiparasitic anti-

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body or the infective agent sharing an antigen or antigens with host cell components. Several workers have demonstrated soluble antigens elaborated by protozoan parasites which might coat the surface of the host target cells. Trypanosoma brucei has an exoantigen on its surface which also is found in the serum and is characterized electrophoretically as a slow-moving substance; this antigen is adsorbed to the surface of red blood cells (Weitz, 1960). Soluble antigens which increasedin direct proportions with an increase in parasitemia have been found in the serum of monkeys infected with Plasmodium knowlesi (Eaton, 1939) and in the serum of white Pekin ducklings infected with P. lophurae (Davis, 1948). If soluble antigens are present in the plasma, they might attach to the surface of host erythrocytes and render them susceptible to defensemechanismsof the host. The present work was designed to determine whether the injection of malarious plasma into ducklings might produce those conditions seen in infected animals, viz., anemia and polychromatophil erythroblast reduction. MATERIALS

AND

METHODS

The 12A strain of the avian malaria parasite P. lophurae was used for all infections. White Pekin ducklings obtained from a commercial hatchery immediately after hatching were used as experimental hosts. All birds were kept in wire cagesand given no medication prior to infection. Ducklings which were to serve as donors of malarious plasma were given intravenous inoculations of infected blood. Quinine dihydrochloride, when used as a control regimen, was suspendedin 0.8570 saline and administered intraperitoneally in amounts of 3 mg of quinine per 100 gm of body weight. Blood was withdrawn from the right jugular vein of the donor birds. A solution of 27 mg of heparin in 100 ml of 0.85% saline in the ratio of one part per nine parts blood wa.~ used as an anticoagulant. The plasma super-

MCGHEE

natant obtained after centrifugation was filtered by meansof a Seitz filter with a micropore filter pad having pores 0.1 p in diameter. Plasma was injected intravenously into ducklings previously uninfected and untreated. Control groups received plasma from ducklings with no infection. Blood samples for total red blood cell counts, packed cell volume (hematocrit) and blood films were taken from a small puncture of a vein in the shank region. Total red blood cell counts were made with a red cell diluting pipette and a Spencer bright-line hemacytometer. Hayem’s solution was used as a fixative diluent. Counts were calculated as the number of erythrocytes per cubic millimeter. A variation of more than 10% from the mean of the group predicated a recount of the individual. Packed red blood cell volume was determined with heparinized capillary tubes. Two sampleswere taken from each individual per reading. The tubes were centrifuged in an International Micro-Capillary Centrifuge, Model MB, and the volume was read on a microcapillary scale. Blood films were fixed in methyl alcohol for 2 minutes and stained for 30 minutes with a 1: 15 dilution of Giemsa’s stain. Sufficient numbers of stained red blood cells were counted to determine both parasitemia and the percentage of polychromatophil erythroblasts. Classification of the polychromatophil erythroblast follows that of Hewitt (1942). RESULTS

Anemia

in Ducklings

Given

Infected

Plasma

Experiment I. Three ducklings, 3 weeks of age, were injected on days 0, 2 and 5 with 0.2 ml of pooled plasma from donor birds with 5500, 4800, and 6100 parasites per 10,000 erythrocytes. Three ducklings, serving as controls, were injected on the same days with 0.2 ml of pooled plasma from uninfected donor ducklings (Fig. 1). Comparison of the test plasma with the uninfected plasma indicat.ed snme hemolvsis in the former. Al_~__._.. --~~

MALARIAL

ANEMIA

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1. Blood cell counts and hematocrit values ducklings given plasma. A, Mean values for total erythrocyte counts per cubic millimeter. B, Mean values for hematocrits. C, Mean values for the percentage of polychromatophil erythrohlasts. “D” indicates death. FIG.

from

though blood films were made daily, no parasites were observed in this or subsequent experiments. In birds treated with infected plasma, the mean number of red blood cells fell below 2.00 million per cubic millimeter by day 8 and below 1.OO million by day 18. The total count in one duckling was 0.29 million per cubic millimeter on day 24 with a hematocrit reading of 6%. The erythron of the second duckling fluctuated somewhat. Its count dropped to 1.19 million erythrocytes per cubic millimeter on day 18, rose to 1.40 million on day 20, fell again to 1.16 million on day 22 and then increased to 1.36 million on day 24. Its hematocrit reading was 18% on day 24. In the third duckling, a steady decline in numbers of erythrocytes occurred with a

283

count of 0.66 million per cubic millimeter and an hematocrit reading of 10% on day 20. The mean total erythrocyte count for the control ducklings ranged between 2.92 million per cubic millimeter on day 6 to 2.49 million on day 15. The mean hematocrit readings paralleled the fluctuation of the total erythrocyte counts. The percentage of packed cells was 31 on day 16 and 35 on day 24. The polychromatophil erythroblasts of the experimental birds decreased to a mean low of 1% on day 12. Basophil erythroblasts were seen first on day 14. On the same day there was an increase in numbers of polychromatophi1 erythroblasts until a mean of 22% was reached on day 18. A somewhat erratic pattern was observed then as counts of the polychromatophil erythroblasts in each bird fluctuated as follows: In duck 1, less than 1% of the erythrocytes were polychromatophil erythroblasts on day 20 with a sudden increase to 13% on day 24. The percentage of polychromatophil erythroblasts was 40 on day 20 in duck 2 but only 2 on day 24. Polychromatophil erythroblasts increased in duckling 3 until day 18 at which time 16% of the cells were immature. However, this was followed by a sudden decline to 45% on day 20. In the control ducklings, the mean percentage of polychromatophil erythroblasts fluctuated between a low of 8 on day 3 and 17 on days 11 and 12. During the early stages of reduction in relative numbers of polychromatophil erythroblasts, an aberrant cell appeared (day 7) and persisted throughout the experiment (Fig. 2). Its nucleus was round in contrast to the ellipsoidal shape of nuclei in typical polychromatophil erythroblasts. The nucleocytosomal ratio appeared to increase in contrast to that observed in immature red blood cells in uninfected ducklings; frequently, the cytosome was reduced to a fringe of matter about the periphery of the nucleus. The cytoplasm always stained a dark metallic gray in contrast to the lighter bluish-gray coloration assumed by the typical polychromatophil

284

FIG.

CORWIN

2.

Stained

blood

films

from

experimental

AND

(A)

erythroblasts. The shape of the cell ipse varied from round to oval, whereas immature red blood cells in the peripheral circulation are ellipsoidal. This aberrant cell will be referred to as an “anemocyte” throughout the remainder of this report, since its presence always presaged anemia. The three ducklings manifested a moribund state during the third week: they were immobile, the joints of their legs were swollen and edematous, their eyes were sealed with a viscous lacrimal fluid and their feathers were unkempt and without their usual sheen. Duckling 3 died on day 20; the other two were killed on day 24 because of their continuing poor condition. The anemias seen above were marked and comparable to those observed by McGhee (1960), although in the present case this loss of red blood cells was not as rapid, i.e., the lowest count was reached some 2 weeks later than that observed by McGhee (1960). The decrease in percentages of polychromatophil erythroblasts, although occurring somewhat more slowly, approximated that seen in duck-

and

MC GHEE

control

(B)

ducklings.

Arrow

indicates

anemocyte.

lings infected by subcutaneous injection of embryo-adapted malaria (McGhee, 1960). Efiect of Plasma from Three Levels of Infection Experiment II. Nine ducklings, 4 weeks of age, were injected with plasma from ducklings representing three levels of infection. Three birds (Group I) received three intravenous inoculations of 2.0 ml plasma on alternate days from ducklings with low levels of infection, viz., 1400 to 2000 parasites per 10,000 erythrocytes; this infected plasma was collected the third day of the infection of the donors. Another three ducklings (Group II) received three intravenous inoculations of 2.0, 1.O, and 2 .O ml, respectively, on alternate days from ducklings undergoing moderate infections, viz., 4200 to 5500 parasites per 10,000 erythrocytes; this plasma was collected the fourth day of infection. The remaining three ducklings (Group III) were given three intravenous inoculations of 2.0 ml plasma from ducklings with high infections, viz., 12,900 to 19,000 parasites per 10,000 erythrocytes

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FIG. 3. Blood cell counts and hematocrit values from ducklings given plasma from three levels of infection and from ducklings given uninfected plasma. A, Mean values for total erythrocyte counts per cubic millimeter from three groups of ducklings given infected plasma. B, Mean values for hematocrit readings from the same three groups. C, Mean values for the percentage of polychromatophil erythroblasts from the same groups. D, Mean values from the control group.

with the plasma collected from the fifth day of infection. Control ducklings received three intravenous inoculations of 2.0 ml plasma from uninfected birds. A steady reduction of red blood cells was noted from the onset of the experiment (Fig. 3A). By the end of the second week, the mean red blood cell count in the experimental group was reduced by approximately one million cells, whereas the count for the control group was relatively stable (Fig. 3D). Hematocrit readings (Fig. 3B) indicated similar decreases. Concomitant with the loss of erythrocytes, a sudden drop in relative numbers of polychromatophil erythroblasts occurred (Fig. 3C). By day 10, there was but 1% of this type of cell in the peripheral circulation. Anemocytes appeared on the fourth and fifth

ANEMIA

285

days. This low percentage of polychromatophi1 erythrocytes persisted for several days; then a sharp rise in numbers occurred. This influx of cells coincided with the appearance of basophil erythroblasts which were noted first on days 14 and 15. The mean percentage of polychromatophil erythroblasts in the control group (Fig. 3D) was 10 on day 0 and 8 on day 14, with neither anemocytes nor basophi1 erythroblasts observed. The third week of the experiment was marked by a period of return to conditions which approximated that on day 0. The packed red blood cell volume increased to approximately 35 ‘$ in the three experimental groups and was 36% in the control group. The percentage of polychromatophil erythroblasts reached a mean of 13. Basophil erythroblasts and anemocytes were no longer seen in blood films at the close of the experiments. One of the ducklings in Group II showed a marked decline in numbers of red blood cells so that by day 14 its erythrocyte count was only 1.15 million per cubic millimeter. The count then rose to 1.39 million red blood cells per cubic millimeter on day 16. The percentage of polychromatophil erythroblasts paralleled that seen in the other experimental animals, i.e., there was a rise from 1 on day 10 to 9 on day 16. Nevertheless, the bird died on day 17. The level of parasitemia from which the plasma was taken appeared to have little effect upon the degree of anemia experienced. The mean total erythrocyte counts dropped below 2.00 million per cubic millimeter on day 10 for Group I, on day 11 for Group II and on day 12 for Group III. The mean total loss was approximately 1.00 million red blood cells per cubic millimeter at the end of the second week for each group. Anemocytes appeared sooner in Group III (day 4 vs. day 5) but the mean loss of polychromatophil erythroblasts was not as marked as in the other two experimental groups (2% vs. 1%) and the subsequent percentage increase was not as great (16 vs. 20 and 22 for Groups I and II,

286

CORWIN

AND

MC

GHEE

respectively). These differences are slight, however, and may be attributed to properties of each host animal. Comparison of results obtained from these three experimental groups with those from the first experiment (cf. above) show a similarity until about day 10. The anemia in the first experiment is seen to continue; apparently the loss of red blood cells is occurring more rapidly than can be dealt with by the restorative powers of the hematopoietic centers. In Groups I, II, and III, the influx of early immature red blood cells furnished a replacement for those lost in the immune process. E#ect of Plasma Inocula Days

3.0 2 E f i

2o -CONTROL

9

Given on Successive

Experiment NZ. Four ducklings, 24 weeks of age, were injected with plasma from ducklings with infections of 3000, 4000, and 3600 parasites per 10,000 erythrocytes on 3 successive days. Four control ducklings of the same age were given intravenous inoculations of uninfected plasma on the same days. The mean total erythrocyte count was 3.02 million per cubic millimeter on day 0 and 2.22 million on day 10 (Fig. 4A). There was a sudden rise in numbers of erythrocytes to 2.97 million per cubic millimeter in one bird on day 12. If this figure is not included, the mean total erythrocyte count for the other three experimental ducklings is 2.07 million per cubic millimeter. The last day of the experiment was day 14 when the mean count for the four experimental birds was 2.57 million red blood cell per cubic millimeter. There was a decline in relative numbers of polychromatophil erythroblasts (Fig. 4B) from 1470 on day 0 to 2% on day 6. Anemocytes first appeared on day 4 with basophil erythroblasts seen by day 9. The percentage of polychromatophil erythroblasts increased to 16 on day 14. In contrast, the percentage of polychromatophil erythroblasts remained relatively unchanged in the control group being 14 on day 0 and 10 on days 10 and 14 (Fig. 4B).

TIME

IN

DAYS

FIG. 4.

Blood cell counts from ducklings given infected and uninfected plasma on successive days. A, Mean values for total erythrocyte counts per cubic millimeter. B, Mean values for the percentage of polychromatophil erythroblasts.

The anemia observed in this experiment was not as marked as that seenin the previous ones. There was but a mean loss of 0.80 million red blood cells per cubic millimeter occurring in the first 10 days. The inocula being given on successive days seemed to have a less deleterious effect upon the animals than did those given on alternate days. The age of the ducklings was such that greater susceptibility was expected but, in this experiment, greater susceptibility was not observed. DISCUSSION

The injection of plasma from malarious ducklings into uninfected ducklings resulted in anemia and a loss of polychromatophil erythroblasts. These phenomena have been observed by other investigators in infected animals in which parasitemias were kept at a low level. McGhee (1960) demonstrated anemia incommensurate with level of parasitemia by subcutaneousinoculation of duck

MALARIAL

embryo-adapted P. lophurae into ducklings. Eight days after infection, the mean number of red blood cells per cubic millimeter for these experimental animals was 1.19 million. The mean infection could not be calculated because there were not enough parasites present to make accurate counts. The mean polychromatophil erythroblast percentage dropped from 24 on day 0 to 0.6 on day 8. Similar results (McGhee, 1964) were reported upon control of infection in ducklings by use of quinine dihydrochloride. He concluded that there was an immune response on the part of the host which accounted for the marked anemia and specific removal of polychromatophi1 erythroblasts. Zuckerman (1960a, b) reported disproportionate anemias with P. berghei infections in rats and P. Eophurae infections in chickens. Sloan and McGhee (1965) noted a loss of only mature red blood cells in chickens infected with P. Eophurae. No mention was made in the above works concerning the appearance of atypical red blood cells nor the influx of basophil erythroblasts during the course of infection. In this present work, anemias were induced in the absence of the malaria parasite, P. Zophurae. The loss of red blood cells in one experimental group was as pronounced as that reported from ducklings which had received intravenous injections of infected duck embryo red blood cells (McGhee, 1960). In this latter case, the total red blood cell count decreased to 0.78 million per cubic millimeter by day 5. The mean total red blood cell count for the experimental ducklings which ,received malarious plasma was 0.72 million per cubic millimeter on day 19. This represents a lag of 2 weeks, but this cell loss is without the complication of lysis by the emerging parasite. Subsequent experiments with malarious plasma did not exhibit such dramatic results but losses did amount to 0.80-1.00 million red blood cells per cubic millimeter in a 9to 14-day period. Losses of polychromatophil erythroblasts were as marked and as rapid as those observed by McGhee (1960, 1964).

ANEMIA

287

In two experiments (II, III), there were increases in numbers of erythrocytes between days 10 and 14 with subsequent relief of anemia. A soluble antigen has been extracted from monkeys infected with P. knowlesi (Eaton, 1939). This antigen was found to fix complement, to increase in direct proportions with an increase in number of parasites and to be chemically as well as serologically similar to antigens extracted from parasitized cells. Complement-fixing antibodies reached the highest titer l-2 weeks after the peak of infection and their appearance was accompanied by the disappearance of soluble antigen. A soluble antigen was collected from the serum of ducklings infected with P. lophurae at peak parasitemias (Davis, 1948). This antigen was also capable of fixing complement. Although Eaton (1939) found the antigen in P. knowlesi infections to increase with a rise in parasitemia, we found that there was little demonstrable difference in the effect of plasmas taken from infections of 3, 4, and 5 days duration. At higher infective levels, antigens may be bound to antibodies formed specifically against them, thereby eliminating their effectiveness when transferred to another host. From our findings, it appears that despite possible binding with antibody, there is still a sufficient amount of antigen present to induce severe anemia. One interpretation of the phenomena which we have observed in the above experiments is as follows: Initially the soluble antigen elaborated by the parasite coats the uninfected red blood cell. The host responds with the production of an antierythrocytic antibody with a resultant erythrophagocytosis of the red blood cell-antigen-antibody complex. This view is held by other investigators (Cox, personal communication). The possibility of reactive sites on erythrocytes has been demonstrated with the hemagglutinins of the myxoviruses (Hirst, 194 1) . Since hemagglutinins have been indicated in other works (Schroeder et al., 1965; Zuckerman, 1960a),

288

CORWIN

AND

it could be that the same reaction is occurring in the anemias we observed. Schroeder et al. (1965) found autohemagglutinins present in the sera of rats infected with Babesia vodhaini. This factor was correlated closely with erythrophagocytosis. Such sensitizing agents have as yet not been demonstrated in conjunction with P. lophurae, although Zuckerman (1960a) detected an agglutinin in rats infected with P. berghei. Doubt was cast on this finding when the same test produced positive results in pre-bled rats and in those made anemic with phenylhydrazine (Zuckerman and Spira, 1961). Preliminary studies in our laboratories have shown that the inoculation of malarious plasma induces erythrophagocytosis in proportions which parallel those observed by McGhee and Corwin (1964). A sensitization of the red blood cells prior to engulfment seems to be indicated in this stimulation of phagocytosis. Since both mature erythrocytes and polychromatophil erythroblasts appear to be selected for in infected ducklings, the nature of the antibody may not be as specific as that which may be found in chickens infected with P. lophurae. There is also the possibility that two antibodies may be formed, one specific for mature red blood cells and the other for polychromatophil erythroblasts. A second possibility is that an antigen or collection of antigens is acting on the developing cells of the bone marrow and is suppressing the development of polychromatophil erythroblasts. Suppression of erythropoiesis in itself could induce anemia. Erythrophagocytosis would in this instance be a removal of effete cells by the macrophages. The action of antigen plus the later action of antibodies and emergence of parasites in malaria infections could easily account for the profound anemias observed in infections of ducklings with P. lophurae. A final possibility is that the malaria parasite is accompanied by a virus which in itself is responsible for some if not most of the symptoms seen in these studies. Jacobs (1957)

MC

GHEE

did encounter an ornithosis virus in certain of his ducklings infected with P. lophurae. In these ducklings there was an increased resistance to malaria, whereas in our work it apparently resulted in increased pathogenicity. If a virus is present it is capable of passing through a filter with a pore size of 0.1 u thus separating it from the larger ornithosis virus. Nor does it possess the pathogenicity of Trager’s spleen necrosis virus (1959). A virus was suspected in infections studied in the early work of McGhee (1960) concerning the possibility of autoantibody. The blood of ducklings suspected of being infected with a virus was injected into chick embryos, and tests run for the presence of hemagglutinins but without success. Infections in embryos were also studied and it was found that there was no diminution of total red blood cells or in polychromatophil erythroblasts. Although these tests proved negative it may have been that neither test was suitable for demonstrating the contaminating virus. In view of this, attempts to demonstrate virus continue. The appearance of an atypical cell in the peripheral circulation prior to anemia was observed in each case when malarious plasma was introduced. These cells were readily apparent due to the metallic gray coloration of the cytoplasm. The origin of this type of cell needs further study, as does’ its possible role in the circulatory system. There may be some correlation between this cell and the spherocyte seen in macrocytic anemias of mammals. However, blood samples examined microscopically when making total red blood cell counts showed only small flattened cells. Therefore, we have tentatively designated this cell an anemocyte. Its presence should serve as a reliable indicator of ensuing anemia. The appearance of the basophil erythroblast during the increase in numbers of polychromatophil erythroblasts in all probability is due to a feedback mechanism whereby the hematopoietic centers release immature cells in response to their depletion in the peripheral circulation.

MALARIAL

Whether the anemias produced by introduction of malarious plasma is the result of an intercurrent virus infection, antigenic action or antibody formation or of a cell-antigen-antibody complex cannot be answered at this time. Other investigators have demonstrated several antigenic components in plasmodial infections (including P. Zophurae infections) by freeing the parasites from their red blood cell hosts (Zuckerman and Spira, 1965; Sherman, 1964). The antigens with which we are concerned in producing anemias may well be the same as those extracted by other workers. Most other investigators have been concerned with examination of the parasite, whereas our work has demonstrated that a soluble antigen or antigens may produce part of the disease pattern found in malaria. Further elucidation of the chemical nature of the substance or substances producing the anemias, its presence in other malarias and the efficacy of its powers to immunize in a more highly refined chemical state are problems yet facing us. REFERENCES DAVIS, B. D. 1948. Complement fixation with soluble antigens of Plasmodium knowlesi and Plasmodium lophurae. Journal of Immunology 58, 269-281. EATON, M. D. 1939. The soluble malarial antigen in the serum of monkeys infected with Pkzsmodium knowlesi. Journal of Experimental Medicine 69, 517-532. GEAR, J. 1946. Autoantigens and autoantibodies in the pathogenesis of disease, with special reference to blackwater fever. The Royal Society of Tropical Medicine and Hygiene. Transactions. 39, 301-314. HEWITT, R. 1942. Studies on the host-parasite relationships of untreated infections with Plusmodium lophurae in ducks. American Journal of Hygiene 36, 6-42. HIRST, G. K. 1941. The agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. Science 94, 22-23. JACOBS, A. R. 1957. Effect of ornithosis on experimental fowl malaria. Proceedings of the Society for Experimental Biology and Medicine 96, 372373. MCGHEE, R. B. 1960. An autoimmune reaction

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produced in ducklings in response to injections of duck embryo blood infected with Plasmodium lophurae. Journal of Infectious Diseases 107, 410-418. MCGHEE, R. B. 1964. Autoimmunity in malaria. American Journal of Tropical Medicine and Hygiene 13, 219-224. MCGHEE, R. B., AND CORWIN, R. M. 1964. Bone marrow dycrasia in malarious ducklings. Jou7ml of Parasitology 50 (supplement), 12. SCHROEDER, W. F., Cox, H. W., AND RISTIC, M. 1965. Erythrophagocytosis and hemagglutinins associated with anemia and recovery from Babesia rodhaini infections of rats. Journal of Parasitology 51 (supplement), 30. SHERMAN, I. W. 1964. Antigens of Plasmodium lophurae. Journal of Protozoology 11, 409-417. SLOAN, B. L., AND MCGHEE, R. B. 1965. Autoimmunity in chickens infected with Plasmodium lophurae. Journal of Parasitology 51 (supplement), 36. TERZIAN, L. 1941. Studies on Plasmodium lophurae, a malarial parasite in fowls. II. Pathology and the effects of experimental conditions. American Journal of Hygiene 33, 23-40. TRAGER, W. 1959. A new virus of ducks interfering with development of malaria parasite, Plasmodium lophurae. Proceedings of the Society for Experimental Biology and Medicine 101, 578-582. of some antigens WEITZ, B. 1960. The properties of Trypanosomu brucei. Journal of General Microbiology 23, 589-600. ZUCXERMAN, A. 1960a. Autoantibody in rats with Plusmodium berghei. Nature 185, 189-190. ZUCXERMAN, A. 1960b. Blood loss and replacement in plasmodial infections. IV. Plasmodium gallinaceum and Plasmodium lophurae in untreated and pre-bled mature chickens and in untreated chicks. Journal of Infectious Diseases 107, 133148. ZUCKERMAN, A. 1964. Autoimmunization and other types of indirect damage to host cells as factors in certain protozoan diseases. Experimental Parasitology 15, 138-183. ZUCXERMAN, A., AND SPIRA, D. 196,l. Blood loss and replacement in plasmodial infections. V. Positive antiglobulin tests in rat anemias due to the rodent malarias Plasmodium berghei and Plasmodium vinckei, to cardiac bleedings, and to treatment with phenylhydrazine hydrochloride. Journal of Infectious Diseases 108, 339-348. ZUCKERMAN, A., AND SPIRA, D. 1965. Immunoelectrophoretic comparison of plasmodial antigens. World Health Organization/Mal/497.65, 1-6.