M N 1' W H W éfié \I‘WWMW EXPERSMENTM. TRANSMISEMN {1537 THE? “3m” STRAEN m NASMQEMUM EATHERMERMM 7.7% 'E'Hii BUCK AM} W3 CHEMQWiEEA‘PEWEC EUREKEHLE'F‘A‘ 513R Ri‘lim'ia‘vfi fiNTEM/fifiiflrfi. SEREENWG Wasfia fw- é‘ém Emma 1:? iii; g! WEI-Name? E‘EATE C€3LLE€EE fiamid Lesa flush 2&4?" A! 2.3 ”xv" A 431* .51.; EA. 5:33? ‘ «or: 1’ :fi'qflr 3"?! 5.) - ‘lfif“ W, (1‘ —k“-;;‘ 1:; ‘; ~- ‘ Eag3§§_ in A in}, " _,,;5zg .. ‘ 4 a“ so a; i M y."'» I: It I 2‘??? i i ”‘4 F s Y"—-9E=:-:“'L “Ag 0 ' ‘5: 'Fgg I" L. .1 ; 1311 .4 ‘ hi :1 ‘rw ‘ ‘9: “J'- .I 'q ,..",‘ ', f ‘ l.‘ {5?}. ti“: 3‘23- Z‘L.‘ at «"“55 '.. a‘. $‘§§\§‘ This is to certifg that the thesis entitled Experimental Transmission of the "5m" Strain of Plasmodium cathermerium to the Duck and its Chemotherapeutic Suit- ability for Routine Antimalarial Screen- Date plegéfied by Donald Lee Bush has been accepted towards fulfillment of the requirements for MrS~——d°9r00 iii—Parasitology December 15,1947 Major professor ‘ r\ ‘ V y _. ’ 1. ‘1 ._“‘ H I ' l. " , 3 ' .fn‘ ‘Ta'u' H 1 V m ‘ 1', ."0 EXPERIMENTAL TRANSMISSION OF THE "an“ STRAIN OF PLASMODIUM CATHERMERIUM TO THE DUCK AND ITS CHEMOTHERAPEUTIC SUITABILITY FOR ROUTINE ANTIMALARIAL SCREENING BY DONALD LEE EUSH A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 1947 THE$;$ H\Hw& (A ’ Table of Contents Introduction Purpose Materials Nethods Experimental Hosts Method of Counting Parasites Method of Obtaining Infective Blood Method of Transfusing Malarial Blood Standardization of the Parasitemia Experimental Data Parasitological Periods Peak of Infection Periodicity and Synchronicity ‘ Nunber of Merozoites Produced per Schizont ExoerythrocytichStages Induced "6M" strain of 2} cathermerium in Chicks Chemotherapeutic Experiments Discussion Summary and Conclusions References Page 16 16 26 29 61 Coatney and Roudabush (1937) in the fall of 1936 made a survey of various wild birds in Nebraska by means of blood films and subinoculations, and noted the incidence of red blood cell parasites of birds. A magpie (Eigg pig; hudsonia) was found by them to be heavily parasitized with plasmodiae. Two types of gameto- cytes were noted in the blood smears, one was round and the other was of the elongate type. The blood from this magpie was subinoeulated into a canary withfgrevious infection, and the only type of parasite to appear was that of the round ganetoeyte type. This strain of plas- media. was tentatively identified as P];smogjflg 333!- m, which was later continued by Huff of the University of Chicago. This strain was reported to be decidedly pathogenic for the canary, and has hence been designated as the "5!" strain by the Committee on Terminology of Strains of Avian Malaria. Purpose The purpose of this thesis is: (1) to show the method of passing the "3M" strain of giggmggxgg.gsgggg- [gerigg from the canary to the duck, (2) to determine if it is too virulent for the duck, (s) to look for exo- erythrocytic stages, (4) to standardize the parasitenis so a routine high percentage of the red blood cells will be parasitized, (5) to determine if the "5M" strain of Plgsgogiun egthgrmeriun in ducks is a muitable malaria parasite for the chemotherapeutic screening of anti- malarials, (6) to determine the presence or absence of a filterable lethal agent for ducks associated with Plgsmodium cathermerium (Dearborn 1946), (7) to make observations on the periodicity, synchronicity, course of infection and anemia produced, and (8) to determine 1: the ":5!" strain of Plasmodium._c_gthermeriu_m could be established in the chick. Materials Laboratory hosts consisted of adult female canaries (nginns cgnarius) weighing about 15 grams, and white Pekin ducks obtained when one day old, and raised until they were about 100 grams. The strain birds used were of a much greater weight, usually 300-300 grams. This insured an ample supply of infective blood when it was needed. Chicks used were 3-5 day old white Leghorns weighing 40-50 grams. All feed for both chicks and ducks was fortified broiler mash. Canaries received the usual mixture of seeds. A rotary feed mixing machine was used to mix the drug diets (Litehfield 1939) employed in the chemotherapeutic study. Thorough mixing was achieved in 30 minutes. Previous tests were made with water soluble dyes added to the feed and aliquot portions of feed taken out and com- pared on a Lumetron colorimeter with a known standard of the same dye. In this way it was determined that proper mixing was accomplished in 30 minutes. For the periodicity experiment, the birds were kept on a 12 hour light and a 12 hour dark cycle in a regular type brooder pen; 4 birds were used in this experiment. Separate containers for running water and food were designed to accomodate one group of 3 ducks for the chemotherapeutic tests. Thus it was impossible for the running water and the feed to become mixed with that of another group of ducks on test. Ten groups of ducks were put on test at one time. The duck pens were placed on metal racks with one-half inchhardware cloth floors, thus allowing the fecal material to fall on a metal apron. The fecal material automatically passed down an outlet to the sewer by the action of the contin- uously running water, which was the overflow from the drink- ing troughs . Birds that were kept for relapse studies were put in finishing batteries also equipted with running water. The Lumetron colorimeter, calibrated against an Evelyn hemoglobinometer, was used to determine the hemoglobin as oxyhenoglobin. Methods Engrimgntal Hggtg Adult female canaries have been used routinely as experimental hosts for the "3!" strain of Plggmggigg cathermeriun. However, this strain is virulent for the canary and initial infections or relapses result in deaths in many cases. With this in mind, and, also, the report by Dearborn (1946) that an agent lethal for ducks is associated with the "ST” strain of Plasmodigg.gg§nermerium in ducks, it was decided to determine the susceptibility of white Pekin ducks to the "3H" strain; and to possibly make available another.strain in the duck which would nave possibilities for routine antimalarial screening. Canary no. 80 was sacrificed at the peak of infect- ion and 1 ml. of blood obtained from the neart. 0.5 ml. of the olood containing 40 x 106 parasites was quickly frozen in small brown glass tubes with very thin walls by immersing them in a mixture containing dry ice and acetone. This was later placed in a deep freeze unit at -70degrees F. The remainder of the blood containing 40 x 106 parasites was given to two day old ducks via the tarso-metatarsal vein, each duck receiving 20 x 106 parasites. These ducks were designated 10 and 2C. Blood smears were made daily but the parasites were not sufficiently numerous to count. The third day after inoculation 1 ml. and 2 ml. of blood, respectively, were inoculated into two uninfected ducks from duck 20 and the course of the parasitemia was observed in ‘duck 10 to determine the best time to transfer the mal- aria blood. Definite dosages were not attempted until the 5th transfer when 145 x 106 parasites were given to a 100 gram duck. The peak parasitemia was reached on the 4th day with 38% of the erythrocytes parasitized. These preliminary observations suggested that the duck would be a suitable host, and therefore, the strain was maintained by subinoculations every 4th day into young ducks.” After the transmission of the "3M" strain of Plgpmodigg cathernerium to ducks and its adaptation to that host, larger birds were used for maintenance of the strain. Method of Counting Pargsites The method of counting the number of parasites in relation to red blood cells is that calculated by Hartman (1927a) and Gingrich (1932) according to the following formula: N 3 45.954,£;§ where N is the number of red cells to be counted, P is the'number of parasites per sample and I is the sample unit (10,000 RBC). This gives a parasite - red cell ratio which has a probable error of 10% of the observed values. A second method was used which gave a probable error of 20% of the observed values with 10,000 red blood cells used as a sample unit. The formula is as follows: N = 11.373 2:2; letters indicate the same as .the above. P A third method used in counting was that of deter- ‘nining the number of parasites per field when the average .field contained 100 red blood cells. 100 fields were counted. The probable error was estimated at less than 20%. ° A Howard disc was placed in the left ocular of a Bausch and Lomb binocular microsecpe to facilitate counting. When blood was pooled in sterile rubber stoppered bottles, the parasite count was done directly from the bottle by making a blood smear in the usual manner and then, at the same time, a sample was taken for the red blood cell count. Smears were stained individually on a staining rack with Giemsa's solution. Mgthod g; Obtaining infective Blood The method of obtaining malaria blood from canary 80 was intracardially. The boundaries for the insertion of the needle was at a 45 degree angle between the clav- icle and centrally into the fossa formed by the clavicle. This method was also used in obtaining blood from chickens. The method of obtaining malaria blood from ducks consisted of palpation over the sternum until the strongest heartbeat was felt, and then, merely insert- ing the needle through the sternum, using gentle traction on the plunger of the syringe until the blood appeared in the syringe. Methodgf Transfusing Malaria Blggg Venous transfision was used in every instance. Chicks used were 5 days old, and easily handled. The site of injection was midway between the proximal and distal end of the metatarsus Just above the bony core of the spur. Approximately one chick can be inoculated every 30 seconds. Venous transfusion was less difficult in ducks. The same vein was used and would appear quite prominent even in day old ducks. Stgndardiggtig; g; the Pagggitemia Once the strain was well established in the duck initial experiments were carried out using 1 x 106 parasites per gram of body weight. This produced a sufficiently high parasitemia for a periodicity study, but was considered low for chemotherapeutic studies. After several experiments it was decided to use 1.5 x lo6 parasites per gram of body weight for the chemothera- peutic studies. This size dosage produced a parasitemia with a range of 3800-6500 parasites per 10,000 red blood cells. After 300 transfers the virulence does not seem to have been increased when standardized dosages of parasites were given. Experimental Data Parasitological Period! Parasitological periods (Hewitt 1940) pertains to the relationship between the parasite and its host, and may be divided as follows: (1) prepatent period, which is the period of entrance of the malarial parasite into the body until it can be demonstrated by ordinary diagnostic means, (2) patent periods, 1; the time interval is the time interval when the parasite can be demonstrated 7 in the blood, and (3) the subpatent period when par- asites are not demonstrable by the usual means, but may be present in minute numbers and could be demonstr- ated by subinoculations. In standardized doses of 1.5 x 106 parasites per gram of body weight, the prepatent period is completely eliminated, thus, making the infection available for immediate study (Boyd 1925). ' .ngk'gf Infection .The peak of infection was determined in the follow- ing manner. Slides were made at two hour intervals during the day in order that the periodicity could be determined at the same time. Counts were made by random sampling, and the size of file sample unit for the probable error to equal 10% of the observed values was maintained throughout. Four ducks were used in the combined peak of infection and periodicity study. The ducks were injected with l x 106 parasites per gram of bodvaeight at 11300 Acne and smears were taken at 2:00 P.M. the same day and every 2 hours until 10:00 P.M. and in some instances 11:00 P.M. Initial counts showed that all the birds had received desirable amounts of parasites and that the parasitemia of the various birds were coming up with a close correlation. However, due to 'individual response to malarial infection and biological variations in phagocytosis and immunity, it was decided that the geometric mean gave a better overall picture because it is a calculated value and is dependent upon the size of all the values. The advantages of the geometric mean are several (Arkin and Colton 1946); it is less affected by extreme items, it is a more typical average than the arithmetic mean, since it is less affected by the extremes and it can be manipulated algebraically. The formula when reduced to its logrith- nie form is: Log. Ga 3 Log X1 + log X2 + log X3 + . . . . . log XI divided by N. Graph 1 represents the mean course of the infection in 4 ducks. The peak parasitemia was reached the third day at 10:00 p.s. following the injection of l x 106 parasites per gram of body weight in all the ducks studied. The blood used in the experiment was from ducks which were on a‘l2 hour light and a 12 hour dark schedule (Boyd 1929). These data correspond with the results Wolfson (1943) found with the "3T" strain of Plasmodium cathermerium in the duck. However, Wolfson (1943) states . . . "that further rapid passage of this strain through the duck is likely to increase the degree of parasitemia, to shift the peak to a later time in the infection, and to cause more frequent deaths of the host.I It has been found by the author that if the number of parasites injected remains constant, there was no increase in the degree of virulence of the '3M" strain of Plasmogium cathermerium in over 300 transfers. The degree of virulence appears to be, to a certain PARASITES FER IGOOO R.B.C. 5000 as] am... i? H? #7 ’i 3 ‘0.“ COURSE OF. INFECTION ”W" '33 ”3». s ‘4'. " .t’c... gm ‘3? 3- 0 DAY IS? DAY tut DAY 3” DAY 4m DAY Sm DAY 6m MY TIuDAY DAYS AFTER INOCULATION extent, proportional to the number of parasites injected intravenously when the hosts are in good condition and the parasites are obtained from a donor before the peak of the parasitemia is reached. Periodicity 53g Synchronicity Periodicity of the "3H" strain of Plasmodium eathgrmerium was first studied by L. G. Taliferro (1925) in the canary. She discovered that 2, eathermerium, "3H" strain, had a 24 hour periodicity with schizogony occurring largely from 6300 to 9:00 P-M- This was later confirmed by Bronsky and Hegner (1926), Boyd (1929), Hartman (1927) and others. Wolfson (1958) first maint- ained a strain, designated as the "ST" strain of‘fio eathermerium in the duck and Wolfson (1940) reported further studies of that strain in the duck. She found that the period of greatest segmentation occurred between 8:00 P-M- and midnight daily, and the length of the asexual cycle to be about 24 hours in length. The peak of old sehizonts occurred at about 8:00 or 10:00 P.M. daily. The method of making the present periodicity study was as follows; smears were made at 2 hour intervals, and in some instances at one hour intervals, and stained with Giemsa's solution. During the period between 4:00 ‘P-M- and 8:00 P.M., smears were usually made every hour. Fro-.a random sample of 100 parasites from each blood mnear the percentage of each of the following groups 10 was determined: (1) stages containing 5 or more nuclei were designated as old schizonts, (2) microgametocytes, and (3) macrogametocytes. There was a great deal of similarity in the 4 birds studied and graph 2 shows the results of the periodicity study. The periodicity was essentially 24 hours in length. 0n the day of transfusion, the maximum number of schizonts with 5 or more nuclei occurred at 6:00 P.M.; the day after inoculation, or the first day of the infection the maximum number of these schizonts occurred at 5:00 P.M. The second day of the infection the peak becomes flat- tened somewhat and the maximum number of old schizonts occurred at 2:00 P.M., and the third day was like that of the second day. Segmentation occurring earlier in the afternoon in the peripheral blood was noted by Wolfson (1958), by Redmond for the "3A" strain, and by Coatney for the "3M" strain (unpublished) and by Porter (unpublished) for the "5M" strain. This observation of earlier segmentation for the various strains mentioned were infections in the canary. Periodicity of the gametocytes was also studied and recorded on graph 2. The male and female gametocytes appeared in the peripheral blood circulation simult- aneously with the asexual forms which might be expected with the use of a large inoculum. The gametocytes were observed to show a periodicity quite similiar to that of the asexual forms. However, the maximum number of 11 PARASITES PER IQOOO RED BLOOD CELLS g COURSE OF INFECTION -“--' "" ASEXUAL REPRODUCTION- “" SEXUAL REPRODUCTIONo-----e . - / 2‘. I/l’l ——"(’I g "" I I g I, sod 1.0 ‘8‘} it ;\ On I ‘( n 1 '| :1“ I a b—v” : Ia 94 fl. 7. 6. 5. p 4. R I“ ' I) /\ 0'" / i T“ . e :6.— aen 7mm 2 S's—c I01 ’I: "5‘ W A" 2 4'?“ I I0 0 DAY [51' DAY 2'") DAY 3!. DAY DAYS AFTER INOCULATION 70 of mus/res with for mm was) ’10 of‘ 6ANETUCYTE$ gametocytes appeared at a later hour than the 01d schi- zonts. This would tend to indicate that the schizonts and gametocytes reached maturity at nearly the same time. These observations were in agreement with those of Shah (1954). Criteria for distinguishing pregameto- cytes, microgametocytes, and macrogametocytes were based on the cytological characteristics offered by Gambrell (1937). Synchronicity appeared to be quite high dispite the fact that segmentation occurred at an earlier hour on the third and fourth days of the infection. The synchronicity was much sharper on the first and second day of the infection with the synchronicity appearing to be low near the peak of the infection. Following the peak of the infection, synchronicity was nearly broken up completely, and multiple parasites were found in the young red blood cells, the polychromatOphilic erythroblasts, (Hegner and Hewitt 1938),(Hegner 1958a), (Hewitt 1959a) more than at any other time. Number gfymerozoites Produced per Schizont Thirty segmenters were counted in each of the 4 slides representing ducks 16C, 17C, 180, and 190. Blood smears chosen were 77 hours after the ducks were injected intravenously with the malaria blood. The method of choosing the segmenters was by random sampling. Only those segmenters were counted which showed complete division of the cytoplasm and no evidence 12 of further division. The merozoite counts were not made during the first and second days of the infection for several reasons which were; the first day counts may be unduly influenced by such factors as nutrition, ete., imposed upon it by the donor bird, and immunity near the peak of the infection in the donor bird, all of which might be reflected in the recipient at such an early date. The following table shows the results obtained: M No. :1 RANGE 51-31331. STJ.ER. C . .M __LLC ”.00 f-M 1.79 10.3.1 1. °/° 1'34 17c; 10.23 (or/6 9.73 10.97 .o“/° 13.4 r_LE(i 13.17 Kati/#195 m7; 3?.0‘7’ 1 +0 13C 1.1.37 é-If 3.); $0.54. 25.07" 13.4 The merozoite mean in the 4 ducks ranged from 10.85 to 15.17, and the actual numbers ranged from 6 to 24. The average number of merosoites for the 4 birds was 11.84 ‘1 0.29 with a standard deviation of 5.15. Statistical formulae (Arkin and Colton 1946) used to determine the arithmetic mean, standard deviation, standard error of the mean, coefficient of variation, and standard error of the cocfrifient of variation were as follows: arithmatic mean (ungrouped data) $2199 (where 2 equals the arithmatic mean,§2'equals the "sum of," x equals the data expressed as individual items, and.N is the number of items; standard deviation formula ”-0 grouped data is 6: ‘37:”) where 6 equals the standard 13 deviation,‘F the frequency, and x the deviation of individual values from its arithmatic mean; standard error of the arithmatic mean,di=:%vr‘where67 is the stand- ard error of the mean and o is the standard deviation of a sample; the coefficient of variation formula, where V’ is the coefficient of variation,<§ is the standard deviation, andx is the arithmatic mean, is vz-g— 100 This formula was used to relate the measure of dispersion to the average, and to convert it to the percentage form, thus, solving the problem presented by the differing units; the standard error of the coefficient of varia- tion formula is, 6v = fiéfi' ”Egg/1%: The destruction rate of the parasite has been deter- mined by Boyd (1959) for the "H" strain of £3 cathermer- $35 in the canary. He found that at the peak of infec- tion the destruction rate was around 90%. The destruction rate was determined for the "5M" strain of £- cathermer- $33 in the duck in the following manner. The number of parasites per 10,000 red blood cells was determ'ned from smears made at 2:00 P.M. on the third day of the infect- ion. flerozoite counts were made at 4:00 P.M. on the same day, and the two multiplied together. This should give the population per 10,000 red blood cells which should theoretically exist in the next 24 hour period. The number of parasites determined for ducks 160,.170, 180, and 190 was 1400, 2505, 5082, and 1449 respectively. The number of merozoites has been reported for these 14 ducks. Hence, the per cent of destruction was as follows: duck 16C, 90%; 170, 90.1%; 180, 96.7%; and 19C, 59%. This high ratio of parasite destruction indicated the extent of acquired immunity at the crisis of the infection. Exogrzthrocytic Stages Corrodetti (1940) drew the conclusion that the exoerythrocytic cycle was a test of the degree of adapt- ation reached by each species in its relation to its respective host. Hewitt (1940) stated that, "if an exoerythrocytic schizogonic cycle does form a part of the life cycle of some strains of avian plasmodia, the factors which govern the appearance of such a cycle are biologically unstable." Wolfson (1940a) observed that exoerythrocytic stages were prevented in the canary by passage through ducks, and no exoerythrocytic stages were found in the duck associated with the "6T" strain. Porter (1942) found a similarity between strains "6M", "3A", and "3T" of Plasmodium cathermerium, as all showed exoerythrocytie schizogony during blood passage after recent passage through mosquitoes, and all were markedly virulent for canaries. In the present search for exoerythrocytic stages, 24 birds have been examined. The organs routinely ex- amined were the brain, heart, lung, liver, spleen, bone marrow, and occasionally the muscle. No exoeryth- roeytic stages were found. However, many parasites were seen outside the red blood cells, and in the brain cs- 15 pecially. The parasites seen were usually immature schizonts and gametocytes. These were undoubtedly the result of rupture of the red blood cells, because they contained malarial pigment. These were stained as bright- ly by Giemsa's solution as were parasites in the red blood cells, and did not appear to be undergoing any degenerative changes when observed. mflmnwlw Wt m *1 ism Six chicks were used in the experiment, ranging from 40 to 50 grams. The dosage of parasites ranged from 1 x 106 parasites per gram of body weight to 450 x 106 .per gram of body weight. In all cases the results were the same. Parasite counts remained nearly constant in all of the chicks for 3 days; thereafter, there was a marked decline until their disappearance. This evidence of the resistance of chicks to the "5M" strain coincided with the results\of Manwell (1966a), and Hegner and west (1941b) using various other strains of 2, ggthgn- acting. Shenaihcrsncaiia.Ezearincnic Possible methods available for testing antimalarial drugs are three in number; (1) in vitro, (2) in humans, and (6) in animals. The first named method has not been adapted for routine screening of antimalarial compounds at the present time. Testing in human beings may be done in induced malarial infections on paretics or on natives where the natural infections are found. It would be an 16 impossibility to screen all new drugs against human malarials; therefore, some experimental animal is necessary as a test for the assay of possible antimalar- ial drugs. Routine screening for antimalarials using canaries, chicks, and ducks, has a disadvantage in that both the host and parasite are different from human malarias. A better strain of malaria with which to test drugs would be one that would infect the rat or some other economical mammal. Moreover, the problem of proper evaluation of host and species specificities.is one of the most serious connected with reaching a solution to malaria problems (Elderfield 1946). From the laboratory vieWpoint, the parasitemia produced in the avian host is much greater and much more easily followed that in the human. Drugs on test were made up in the feed (Litchfield 1939). As the ducks ate only during periods of light, a 4 hour light and dark schedule was followed. The effect of this method of feeding was similar to taking doses of drugs every 4 hours in the human, and uniform blood levels may be maintained in this manner. The time of initiation of the treatment was 24 hours before infection. The duration of treatment with the "5M" strain of £3 ggthermerium was 5 days. The dosage of drug was a maximum of 0.4% in the diet for new drugs and less for small drug samples in the routine screening of new ngSe 17 The size of the inoculum was 1.5 x 106 parasites per gram of duck, which was given intravenously by the method described. The blood smears were made on the third and fourth days after injection of the parasites. From 50% to 60% of the cells were parasitized in the untreated controls. The arbitary criteria of drug activity was 50% parasite suppression at the peak of the parasitemia on the third or fourth day after the inoculum was given. The minimum effective diet per cent of quinine was 0.0125% of the diet figuring on the base of the drug in every instance. Then, the ratio of the test drug to that of quinine producing a comparable suppression of the parasit- emia was designated as the quinine equivalent. The present chemotherapeutic study was carried out by using 50 ducks weighing 84 to 142 grams with a mean weight of 119 grams. The hemoglobins were determined on a Lumetron colorimeter by the oxyhemoglobin method (Coffin 1946). This method of determining the hemoglobin was used for several reasons; (1) hemoglobins are determined quickly and by simple methods, and, thus, large numbers of samples c:n be run in a rew minutes time, (2) color changes form almost immediately, avoiding delay and inherent error in the slow color development of acid hematin methods, (5) elimination of the personal factor in color matching and, (4) a correction factor does not have to be worked out with the oxyhemoglobin method, as it does in the acid hematin method due to the 18 nucleated red blood cells; therefore, acid hematin values give higher results which are erroneous and of a relative value only. The mean hemoglobin values for the 50 ducks was 9.7 gms.%, with a range of 8.9 to 11.87 gms.%. Six ducks (groups 1 and 2) were used as controls, and the 24 remaining ducks were placed 5 to a pen and put on the following drug diets: .BJJLQ 9.22.2.9. £20 93“.; gigs-Diet 2 1. control 0 2. control 0 5. quinine 0.0125 4. quinine 0.025 5. quinine 0.05 6. . PAM 25* 0.0125 7. Sulfadiazine 0.8 8. PAN 2-4-2* 0.0008 9. ' PAM 5-4-b* 0.00078 10. Atebrin 0.0014 Hemoglobin readings were taken before inoculation and on the third, fourth, fifth, sixth, seventh, and twelth day of the infection at 10.00 A.M. 'Thin blood smears were made at 8.00 A.M. on the first, third, fourth, and fifth days after injection of 1.5 x 106 parasites per gram of body weight. Groups 1 and 2 (controls) reached a parasitemia peak on the fourth day of the infection with a count * Confidential Parke, Davis Antimalarials 19 of 5552 and 5705 parasitesper 10,000 erythrocytes. The geometric mean of the two groups was 5524. Groups 5,4,and 5 (quinines) reached a peak on the third day. Group 3 ducks 0n 0.0125% drug diet (minimum effective dose) had a parasitemia of 1695 parasites per 10,000 red blood cells. Group 4 receiving 0.025% quinine had a peak of 1409 parasites, while group 5 on 0.05% quinine had a peak of 459 parasites per 10,000 red blood cells. Ducks on group 6, PAM 25, 0.0125%, reached a peak on the fourth day, as did the controls with a peak parasite count of 4051. This dosage of drug was apparent- ly a subeffective level. Group 7 on sulfadiazine reached a peak on the fourth day with a count of 2059 parasites, although the peak tended to be flattened out. Sulfadiazine was put on test as a check to determine if this strain of parasite was sulfa susceptible. 1 Group 8 on PAM 2.4.2 received 0.0008% drug in the diet. This group of ducks reached a peak parasitemia on the fourth day of the infection with 2154 parasites. fiarked morphological changes were noted in the parasites, many appearing "punched out" and dark in appearance. The number of merozoites were noted to be fewer in number and there appeared to be good gametocidal action. The dosage of this drug was the minimum effective level. The counts were suppressed 50% that of the controls 20 and the quinine equivalent was 15, when the ratio of PAH 2.4.2 was compared to that of quinine (MED). Group 9 on PAM 5.4.b, 0.00078% drug never reached a peak. The amount of drug employed completely suppress- ed the infection, preventing a peak of any kind. From this preliminary test, however, the drug had a Q64+. Gametoeidal and schizonticidal action was most apparent. Gametocytes and schizonts appeared as dark shrunken spots in the red blood cells. Morphological characteris- ties were obliterated. The maximun number of merOZoites discernible was 4 or 5, and this number could be consider- ed as only approximately accurate as the schizonts were so dark. Ducks in group 10 receiving 0.0014% atebrin reached a peak on the fourth day with 1240 parasites per 10,000 red blood cells. Thus a Q17 is in the range reported by other workers. This drug was put on test as merely a drug control, the same as sulfadiazine, to determine if there were any manifestations of hypersensitiveness or idiosynehrosis to drugs already reported on. Worthy of mention was the ducks on PAM 25, sulfa- diazine, PAM 2.4.2, atebrin, and the controls all reached a peak on the fourth day of the infection and all show- ed the same type of parasitemia curve. That is, the parasitemia progressed to the third day with a straight line approach and then flattened out before the peak and dropped rapidly. In contrast, the quinine curves 21 showed a rapid rise to a peak and then flattened out on the other side of the peak, The characteristics of the two types of parasitemia curves may or may not be significant. It must be kept in mind that in the untreated controls the "5M" strain of E; ggthermerium reached a peak on the fourth day irrespective of the size of the dosage of parasites inoculated. Therefore, when ducks reached a peak on the third day of the in— fection on the quinine diet it became apparent that third day counts must be shown consideration in the evaluation of a drug when they are compared by the ratio- method to that of the quinines. Then too, the peak of anemia of the quinine birds occurred on the day after the peak of the parasitemia, even though the peak occurr- ed one day earlier. Also worthy of mention is the variation in the morphology of the avian malaria parasites under drug therapy. The avian malarial parasites in ducks receiving quinine exhibited a definite and consistant chemo- therapeutic damage. That is, they appeared "punched out" and somewhat faded. In contrast, the parasites in ducks receiving PAM 5.4.b showed a different type of damage. Their morphology was altered from the normal in that they were extremely dark and some were black, and appeared to be, what I shall term, "chemotherapeut- ically burned." It is therefore suggested that the morphological alterations of the avian malarial parasite 22 under drug therapy may be.a significant factor both from the chemical and biological viewpoint as; desirable type of drug action,i.e. detrimental to a particular system of the parasite which would be irrever- sible,etc., drug resistance could be noted, and repro- ductive ability, both sexual and asexual could be noted. Ben Harel (1925), Young (1957), Terzian (1941), and Hewitt (1942) demonstrated a fall in red cell number accompanied by a diminutuion in hemoglobin. Hewitt, Richardson, and Seager (1942) conducted experiments involving a considerable number of ducks and concluded that "the fall in hemoglobin following different doses of parasites follows the parasite curve very closely ..... and serves as a supplementary method for estimat- ing the degree of parasitemia." Then too, they found that the sharp drop in hemoglobin on the day after the peak (2. lophgrae) was evidence pg; 33 to denote the day on which the highest number of parasites was reached, even though parasite counts were not made. Anemia and chemotherapeutic experiments run con- currently confirmed the results of Hewitt, Richardson, and Seager using the "5M" strain of‘fi. cathermerium. The anemia and parasitemia curves of the controls andthe ducks on test agreed very closely. The lowest hemoglobin values were on the fifth day of the infection, which was the day following the peak of the infection in all cases except the birds on quinine diets. These quinine 26- birds (groups 5,4,5) reached a peak parasitemia on the third day of the infection, and the greatest anemia occurred on the following day(4th day). In contrast ‘2. lophurae infections in the duck are quite asynchron- ous, and the peak of the infection using 1 x 106 parasites per gram of body weight may be on the fifth or sixth day, while 2. cathermerium was constant in that the peak was always reached on the fourth day. Results of the chemotherapeutic and anemia study are shown on graph no. 5. 0n the fifth day the hemoglo- bins of group l had dropped from 9.54 gms.% to 4.4 gms. %. This represents a hemoglobin loss of 52.9%. Group 2 ducks had a hemoglobin level of 9.45 gms.% before inoeulatiog and on the 111th day it was 4.2 gms.%, a loss of 55.6 gns.%. of the hemoglobin originally present. Group 5 of the quinine dueks (0.0125%) averaged 9.87 gm8.% before inoculation, and at the peak of their anemia on the fourth day the hemoglobin was 4.51 gms.%. A loss of 54.4 gms.% of hemoglobin. Group 4 ducks on 0.025% quinine had hemoglobin values of 9.56 gms.%, and on the fourth day it was 7.15 gms.%, a loss of 25.5%. Group 5 dueks receiving 0.05% quinine lost 15.2% of their hemoglobin. The initial reading before inoc- ulation being 9.45 gns.%, and it was 8.02 gms.% on the fourth day. Group 6 ducks on PAM 23 lost 54.8% of their hemo- 24 _ ___ __ .._.._._—_. WM...“ _ -_n.-. __ _n_ ___. $9.56 260.605... It. Mint 514. cs’fiSULr/‘omzm: GROUP 1 A £100 000 900 00¢ m SW 500 409 M 200 $2.56 2530022.. n)w.aa1..6. a 4. GROUP 2‘" "0 S L— WI TP we a £935 2.00.605... “unwise 2.00.605: u n £6.14 a 4.. M "(nae w A E 0 e? .M I. M” J few 4 OW m m we .. o G .. m m. Wmmmrmmm m m mu...» 5 $m2