THE EFFéCT OF TEfifii‘ézfi‘r‘Cif‘é: ON THE ERYTHROCYTES {2.3: L3; C09. ”@539 “-" ?HE BLGC‘JQ C? “*5 ALEENQ RA? 1395!: 5:2: The 533;?” cf EVE. S. ‘F‘ECREQAH STAKE COLLEGfi “ :: {Ea a . (Swag; i u arms W375 THEgrc This is to certifg that the thesis entitled The effect of terramycin on the erythrocytes and leucocy‘tes of the blood of the albino rat presented bg George E. Garske has been accepted towards fulfillment of the requirements for LEL— degree in _Znnlag3c_ ”(difl Major professor Date May 20, 19550 THE EFFECT OF TERRAMXCIN ON THE ERYTHROCYTES AND LEUCOCYTES OF THE BLOOD OF THE ALBINO RAT By George E. garake AN ABSTRACT 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 0F 80 IENCE Department of Zoology 1955 Approved Wfflb " F513 George E. awake THESIS ABSTRACT Due to the scarcity of information regarding the hematopoietic effects of terramycin an experiment was con- ducted to try to determine what effect, if any, terramycin had on the erythrocytes and leucocytes using the albino rat as an experimental animal. Rats were divided into two groups, experimental and control, and placed on identical diets except that the ration of the experimental group contained 500 grams of terramycin per ton of feed. Litters from.the experimental group were used to check placental transmission and milk carryover of terramycin and to determine the average values for erythrocytes and leucocytes in immature animals. _ Assay data revealed that terramycin was supplied to the young in utero via the placenta and in the milk to suckling young. ' Results of counts made on 12 experimental and 7 con- trol animals showed no marked differences in erythrocyte and leucocyte values for the two immature groups. The experimental breeding colony of 5 animals gave results similar to the control breeding group of h animals in erythrocyte and leucocyte counts and in differential formula. George E. Garske Fbur experimental and 2 control animals 73 days old showed no marked difference in differential formula. All values found for erythrocytes, leucocytes and differential counts fell within the normal range of variation for these elements in the albino rat. Frem.the results obtained it can be concluded that at a level of 500 grams per ton of feed terramycin has no appreciable effect upon the erythrocytes and leucocytes of the albino rat. THE EFFECT OF TERRAMYCIN ON THE ERYTEROCYTES AND LEUCOCYTES OF THE BLOOD OF THE ALBINO RAT By George E. Garske 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 Zoology 1955 I ” yg’Z’Z” 9 5 _ m/ 'K g ACKNOWLEDGEMENTS The author wishes to thank Dr. Karl A. Stiles for his many suggestions and aid in the preparation of this paper. Considerable thanks are due Dr. Roger F. Keller for suggesting this problem and for valuable assistance and extensive information regarding terramycin. The author is grateful to Mr. Jack Leighton for much valuable information about hematological methods and for assistance in.making differential counts. Without the kindly concern and assistance of Mrs. Dale Henderson (Miss Mac) and the encouragement of the author's wife, Susan, this thesis might never have been written. The author is greatly indebted to Chas. Pfizer &.Co., Inc. for supplying the terramycin and for making available the facilities of their Agricultural Research and Develop- ment Farm.in Terre Haute, Indiana. The author also wishes to express his gratitude to the members d? the staff of Chas. Pfizer & Co., Inc. who furnished assistance and adv1ce e t 1' k“. } Cu? is Afr” O... \u TABLE OF CONTENTS PAGE INTRODUCTION OOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOO MATmIA-IS ANDBIETHODS .00....00.0.0.0...OOOOOOOCOCOOOOOO DATA COO...0.0.000...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOO \OOO‘H Erythrocytes .................................... Leucocytes ...................................... l3 Differential Counts ............................. 17 Terramycin Assay ................................ 22 Placental Transmission .................... 22 Milk Carryover ............................ 22 DISCUSSION ............................................. 2h SUMMARY ................................................ 26 LITERATURE CITED ....................................... 27 LITERATURE NOT CITED ................................... 29 INTRODUCTION Terramycin, a broad spectrum antibiotic, was dis- covered by Finlay and associates (1950) in the Biochemical Research Laboratories of Chas. Pfizer Br. 00., Inc. The result of a planned research program, terramycin is a pro- duct of a new actinomycete, Streptomyces rimosus, and is amphoteric forming both the crystalline hydrochloride and the sodium salt. Terramycin is effective against a wide variety cf micro-organisms including many Gram-negative and Gram-positive bacteria, the rickettsia and certain of the large viruses . Terramycin is closely related to aureomycin and tetracycline both in structure and in activity, tetracycline being the base for each of the other two. Aureomycin is modified by having a Cl atom at position 16 and terramycin by having a hydroxyl group at position 12. ' Pan (1950) reported that some dogs receiving varyirg amounts of terramycin intramuscularly showed mild degrees of anemia but believed that the anemia was secondary to chronic ulceration at the site of injection rather than due to the toxic effect of terramycin. The earliest reports that antibiotics might have an adverse effect upon hematopoiesis in man concerned chloram- phenicol (Volini 1950, Gill 1950, Rich 1950). As increasing numbers of reports were received Lewis and his associates (1952) of the United States Food and Drug Administration made a special nation-wide survey of blood dyscrasias in man in relation not only to chloram- phenicol but also to other drugs and antibiotics. The blood dyscrasias involved included aplastic anemia, leukopenia, thrombocytopenia purpura, and granulo- cytopenia. A total of 539 cases studied were grouped into four classes as follows: A. Chloramphenicol alone involved B. Chloramphenicol with other drugs C. Chloramphenicol not involved D. Unclassified Some of the other compounds involved in classes "B" and ”C" included other antibiotics, sulfa drugs, arsenicals, analgesics, anticonvulsants, barbiturates, and antipyretios. In class "C" cases involving broad spectrum antibiotics only, of which terramycin is one, totaled 15. Information as to the types of abnormalities concerned was not given. Following this special survey Welch and his associates (19514.) made a second survey which was much more comprehensive than the first survey and which placed greater stress on human blood dyscrasias associated with drugs other than chloramphenicol. The categories used in this survey were the same as those in the first. Of a total of l,l|.lt8 cases reviewed in this survey 59 involved the use of terramycin. Eighteen of these cases fell into group "B" and on L; occasions terramycin was the only drug involved besides chloramphenicol. The other 14.1 cases did not involve chloramphenicol. A further breakdown of these 14.1 cases by dyscrasias showed the following: 11 cases of aplastic anemia - 2 in which terramycin was the only drug used. 2 cases of anemia. 13 cases of thrombocytopenia purpura - it in which terramycin was the only drug used. 11 cases of granulocytopenia or agranulo- cytosis - 1 in which terramycin was the only drug used. 3 cases of leukopenia - all of which in- volved only terramycin. 1 case of non-thrombocytopenia purpura. Mirone (1953) reported that a level of 150 mg. of terramycin per kilogram of feed had no adverse effects on red, white or differential counts in mice over a period of nine months. Additional data (unpublished) furnished by Chas. Pfizer 8: Co., Inc. on bone marrow studies in chickens fed varying amounts of terramycin indicate that at a 200 gram per ton level the blood forming tissues remain essentially normal with a slight polycythemia appearing after six weeks and then.disappearing shortly afterwards. At a 2,000 gram. per ton level degeneration of blood forming tissue occurred accompanied by deficient blood cell formation and.production of abnormal cells. The most recent evidence presented by Nelson and Radomski (l9Sh) who worked with dogs showed that terramycin administered orally for a period of approximately three months had no hematopoietic effects. Since terramycin is widely used as a therapeutic agent in man and other animals and as a growth supplement in animals it is important to know of any adverse effects which it might produce. The purpose of the problem undertaken for this study was to try to determine what effect, if any, terramycin had in.the production of these blood dyscrasias. It was decided to try to produce any or all of these effects using the »rat as an experimental animal. Although it was originally planned to administer terramycin.su3pended in mineral oil subcutaneously to new- born rats it was subsequently learned that it is difficult to give a sufficiently large dose and that a constant blood serum level is hard to maintain by this method. Therefore plans were altered and the research.extended to include placental transmission and milk carryover by giving the terramycin orally as a part of the food supply. A level of 500 grams per ton was chosen as this was greater than most commercial feed levels and would assure a constant exposure to the antibiotic. MATERIALS AND METHODS The rats used came from the Chemistry Department of Michigan State College and were highly inbred - no outside animals having been introduced for a period of 15 years - although no planned mating procedure was followed. The rats were divided into two groups - experimental and control - and were placed upon identical diets except that the diet of the experimental group contained 500 grams of terramycin per ton of feed. The terramycin was supplied in the form of Terramycin 10, a feed supplement containing 10 grams of terramycin per pound. Composition of the diet is shown below: Yellow Corn 57.0 % Dried Skim Milk 20.0 Linseed Oil Meal 12.0 Soybean Oil 5.0 Crude Casein 3.7 Alfalfa Leaf Meal 1.5 Iodized Salt .1; Calcium Carbonate .14. 100.0 % Blood was obtained by cutting the tail and bleeding was controlled by using silver nitrate applicators. Blood counts were made in a certified improved Neubauer hemacyto- meter using Bayemfls solution as diluent for red cell counts and 3% acetic acid as diluent for white cell counts. Differential counts were made on both groups of rats at the end of the experiment using blood drawn from.the tail and wright's stain for preparation. Blood for terramycin assay purposes was drawn direct- ly from.the heart by heart puncture, allowed to hemolyze, centrifuged and then the serum.was sent in for assay to the assay department of Chas. Pfizer &.Co., Inc. In very young (newborn) rats blood was obtained by decapitating the animal and allowing the blood along with.small amounts of body fluids to drain into a test tube. Several animals were necessary to obtain sufficient blood for assay and usually a whole litter was sacrificed. Milk was sent to the assay department of Chas. Pfizer & Co., Inc. for terramycin assay to try to determine whether or not terramycin was transmitted from.female to young via the milk and if so, the ammunt. This milk was obtained by sacrificing suckling young and removing the contents of their stomachs. To gain information on placental transmission of terramycin newborn litters were decapitated and the blood and body fluids were drained off and sent in to be assayed. The experimental group consisted of 1 male and h fe- males plus several young born prior to the start of the experiment. All were placed on the diet containing terra- mycin. The young born to the breeding colony were utilized, by litters, to check placental transmission of terramycin, milk carryover of terramycin and for red and white cell counts. Blood counts were started at the time of weaning and taken approximately every three days throughout the length of the experiment. The procedure for the control group was the same except that no assay work was performed on them. This group consisted of 1 male and 3 females plus some younger animals that had been born before the start of the experiment. Red and white cell counts and differentials were done on the two breeding colonies near the end of the experiment to check the effect of terramycin after prolonged expOsure to the drug. In addition to the breeding colonies a total of 25 animals was used in the experiment. Nineteen of these, 12 experimental and 7 control, were used to determine red and white cell averages in the immature animal. Differential counts were made on the remaining 6 animals, h experimental and 2 control, at age 73 days. DATA Erythrocytes The erythrocyte picture in newborn and young rats shows a gradual increase from about 3 million per cubic mm. at birth to the normal adult level of 9 million.per cubic mm. at approximately four months of age (Creskoff 19h9). Jolly (1909) gives a series of values for erythrocytes at various ages as shown below in Table I. Table I AVERAGE VALUES FOR ERYTHROCYTES AT VARIOUS AGES :{: “5:: Age number of Erythrocytes in da 8 (in millions per cu, mm,) 21 lull. 30 5.5 ha 5.6 60 6.7 Ehrgot (MS. 1916) gives the values for erythrocytes at the various ages as shown in Table II. 10 Table II AVERAGE VALUES FOR ERYTHROCYTES AT VARIOUS AGES L *‘J 1.“ng . , gfimbmi’ilifiamifi? 233.1 28 6.5 us 9.7 60 l 8.8 70 8.8 The values as determined for the rats in this study are shown in Table III. As will be observed from these values there is a tendency for rather marked fluctuations in number at all ages with a gradual rise in the number of cells with age. In the adult rat the erythrocyte count averages 9.35 million per cubic m. with very little difference between the sexes, the normal range being between 7 and 10 million per cubic mm. (Creskoff 1919). Rivas (MS. 19114) gives values in millions per cubic mm. of 8.7, 7.9, and 8.0 for the normal albino rat in three series of determinations. Jolly (1909) gives values in millions per cubic mm. of 8.9 at 365 days of age and 8.8 at 1,095 days of age. 11 Table III AVERAGE VALUES FOR ERYTHROCYTES AT VARIOUS AGES EXPERIMENTAL E CONTROL Asa. Assess. 822225 5 Ass. flasher. 832225 21. 5.2 5.0 - 5.1. ; 211 1le 3.5 - 5.1 25 5.2 u.9 - 5.5 : 27 5.0 u.5 - 5.2 26 h.h 3.0 - 5.0 : 33 5.6 5.u.- 5.9 29 5.1 h.6 - 5.7 u 36 6.0 5.5 - 6.6 36 6.9 6.0 - 8.h E 39 5.8 5.3 - 6.h 37 5.6 5.5 - 6.0 : he 5.7 u.8 - 6.2 no 5.9 5.5 - 6.2 . : us 6.0 5.7 - 6.5 h3 6.8 1.7 - 7.h ; 53 7.6 7.5 - 7.8 no 6.7 6.3 - 7.3 : 56 7.2 6.8 - 7.5 u? 5.7 5.0 - 6.u : 59 6.8 6.5 - 7.1 M9 5.9 8.5 - 7.0 : 62 6.9 6.3 - 7.6 52 6.h. 5.5 - 7.h S ' 55 6.9 6.5 - 7.9 i ! *1 Age in days, number and range in millions per cubic mm. 12 Average counts of the experimental and control breed- ing colonies gave the results shown in Table IV. Table IV AVERAGE ERYTHROCYTE VALUES IN BREEDING COLONIES I EXPERIMENTAL ‘ CONTROL t __ ' ' Number Ranges 9 Number Rangefi- ! 8.6 6.0 "' 9.8 ' 7.0 (+08 "" 8.7 t t it Number and range given in millions per cubic mm. me average of the control group was lowered somewhat by the fact that during the period counts were being made, one female gave birth to a litter of young. It was noted that the red cell count dropped about 2.5 million per“ cubic mm. but returned to normal in a few days. It must also be noted that the breeders of the ex- perimental group had higher individual red cell counts and therefore higher average counts at all times during the period counts were being made. 13 leucocytes Creskoff (l9L19) states that the newborn rat has a leucocyte average of 3,200 per cubic mm. and that this reaches the adult level in approximately the third week of life. According to Jolly (1919) the number of leucocytes tends to increase with age as shown in Table V. Table V AVERAGE NUMBER OF LEUC OCYTES AT VARIOUS AGES “£21.; “”722: fiibfififfyf” 1 2,766 ' 8 3.2h0 15 5 , 666 30 7,1400 60 6 , 866 365 10,000 target (15. 1916) gives the average values at various ages as shown in Table VI. Table VI AVERAGE NUMBER OF LEUC OCYTES AT VARIOUS AGES Age Number of Leucoc tea (in days) (per cubic mm.) 28 7.102 A6 10,025 60 9.183 70 8,1180 Values found in experimental animals are given in Table VII. ' The average value of leucocytes in the adult rat is 9,000 per cubic mm. with a range of 6,000 to 18,000 par cubic mm. (Creskoff 19119). Rivas (MS. 1911+) gives values of 8,133, 8.1}.67.Iand 11,110? per cubic mm. for normal albino rats in three series of determinations. ‘ Tabla VII AVERAGE NUMBER OF' LEUCOCYTES AT VARIOUS AGES f 15 fl 9 EXPERIMENTAL s CONTROL Ass. 152222. 322525 3 Ass. flashes. Eases? zu h,115 u,080 - n,150 1 an 3,690 2,000 - 6,120 25 3.130 2.550 - 3.600 l 27 8.230 3.850 - n.610 26 2,160 1,275 - 3.072 : 33 5,080 3.n50 - 8,930 29 2,830 2,300 - 3,800 I 36 3.750 3,200 - 1.500 36 6,350 2.950 - 10.950 1 39 6,000 5,600 - 6,210 37 3.uu0 2.0h8 - 1.000 : AZ n.800 3.650 - 6.850 no 3,960 2,200 - 5,700 3 as 1,500 2,650 — 5,630 #3 5.250 2,100 - 12,150 I 53 5,350 u,500 - 6,200 116 11,660 2,800 - 7,1750 : 56 10,800 8,1110 - 13,300 1.7 3.170 2.850 - 1.750 S 59 7,600 5.500 - 9.850 A9 7,750 h,600 - 13,260 1 62 9,650 7,500 - 13,300 52 8.730 2,500 - 6,750 5 ' 55 3.550 1.750 - 6,900 l I * Age is given in days, number and range per cubic mun 16 The values found for the experimental and control breeding colonies in this study are shown in Table VIII. Table VIII AVERAGE LEUCOCYTE NUMBERS IN BREEDING COLONIES to. ‘ ‘ EXPERIMENTAL I CONTR 01. I 3 2 3 2 ' in- .1 Number Ranges ' Nwmber Ranges I 6,310 3,750 - 15,100 ' 8,2u0 u,1oo — 1h.900 ' t t *1 Number and range per cubic mm. Several factors contribute to errors in leucocyte number determinations. One of these is the fact that there is a decided leukopenia in the female rat at the time of estrus (Farris l9h2). Since more females were used for these counts than males and since the estrous cycle in the rat is only h to 5 days in length, the possibility of this occurring is present at all times. Another cause of error is the fact that any emotional stress is reflected by an immediate leukocytosis (Farris 1938). The rat holders used to hold the animals while withdrawing blood consisted of tubes of varying diameters into which the rats were supposed to crawl. At times the animals were not as cooperative as 17 could be desired and had to be forced to enter the holder. Although.no record was kept of such cases, they could be expected to cause an increase in the leucocyte count. This would also be true of the younger animals considered in the preceding section. However this could be expected to occur in both groups with the same frequency. A third factor is that during the birth process while the red count drops markedly, the white count rises sharply. The control breed- ing group, as was previously mentioned, contained a female who gave birth to a litter during the period counts were beinggmade. The white count of this animal during this period rose from 6,000 per cubic mm. to 15,000 per cubic mm. and then drOpped to about 7,000 per cubic mm. two days later. Differential Counts The distribution of the various elements composing the white cells of the blood of the normal albino rat as reported by Rivas (MS. 19lh) is shown in Table IX. NOwrey (MS. 1921) gives a breakdown by sexes as shown in Table I. 18 Table IX DIFFERENTIAL FORMULA OF LEUCOCYTES-n- Neutrophijlg Lmhocyt e s Eos inoph 113g Bas Ophils 57.00 h1.60 1.12 .28 52.50 no.60 .60 .30 55.90 n3.00 .90 .20 e- Percentages determined in three separate series of rats. Table I DIFFERENTIAL FORMULA OF LEUCOCY‘I'ES BY SEXES 1 4* ~ 3:..- NeutrOphili Mocytes Monocytgg , Eosinophils Basophils M. 62.07 32.00 1. 83 1.511 .112 F. 55.71 3h.00 2.u9 n.00 .15 Creskoff (19319) states that there is no important difference in differential formula between the sexes and gives average values as shown in Table XI. 19 Table XI AVERAGE DIFFERENTIAL FORMULA Average_ 33253, Neutrophils 20 8 - 39 Lymphocytes 78 55 - 96 Mbnocytes less than 1 0 - 3 Eosinophils 2 O - h Basophils less than 1 O - 0 In.rats conditioned for emotional studies the values given are shown in Table XII (Creskoff l9h9). Table XII AVERAGE DIFFERENTIAL FORMULA IN RATS CONDITIONED FOR EMOTIONAL STUDIES W Avergge £5952, NeutrOphils 35 2h - h? Lymphocytes 60 #7 - 73 Mbnocytes 2 0 - 3 EosinOphils 2 0 - BasoPhils l O - 1 20 The difference between these values can be explained by the fact that emotional stimulation causes an average increase of 12.08% in lymphocytes (Farris 1938). Table XIII gives values found in the rats of the experimental and control breeding colonies. Also shown are values of 73 day old experimental animals and their con- UPOISe AVERAGE DIFFERENTIAL COUNT VALUES FOR EXPERIMENTAL AND CONTROL ANIMALS Table XIII 21 Breeders: EXPERIMENTAL CONTROL 1 Average Rgggg. : Average figggg_ Neutrophils 30.5 15 - 56 ' 3A.? 26 - no Lymphocytes 67.5 RD - 83 l 61.3 56 f 67 Rbnocytes 1.5 0 - 3 l 3.3 0 - Eosinophils O 0 l 1.0 0 - 2 Basophils .5 0 - l l 0 0 g : 73 Days Old: EXPERIMENTAL CONTROL *— I Aver age Ragga ' Average M Neutrophils 18.75 6 - 30 : 12.0 8 - 16 Lymphocytes 80.00 68 - 9h : 86.0 80 - 92 Monocytes 1.00 0 - 2 l 2.0 0 - h Eosinophils .25 0 - 1 l O 0 Basophils 0 0 E O 0 c 22 Terramycin Assay Terramycin assay determinations were done by the staff of the assay department of Chas. Pfizer & 00., Inc. at the Agricultural Research.and Development Fhrm.in Terre Eaute, Indiana. The spot plate methOd of assay was used. on the Whole, assay data was unsatisfactory due to the contradictory results obtained. glacental Transmission Schoenbach (1950) states that the fetal blood serum. level of terramycin is approximately one-quarter that of the maternal level. Harrell (1950) finds that terramycin readily traverses the placental barrier. The assay data in one case yielded results of .2 gamma per ml. in maternal blood and 1 gamma per ml. in the newborn animals. This is higher than is to be expected in the offspring. In other cases both.maternal and newborn sera showed levels of less than .1 gamma per m1. An explanation for these differences cannot be given. Milk Carryover The contents of the stomachs of several litters of suckling rats of various ages gave results upon assay' ranging from greater than .1 gamma per ml. to less than .1 gamma per ml. of terramycin. 23 From these assay data it can be seen that terramycin was supplied to the experimental animals via the placenta and also through the mdlk but the amounts cannot be con- sidered as reliable since few determinations were made. 2h DISCUSSION The elements of the blood of the rat have wider normal ranges than those of human blood. This fact tends to make dyscrasias more common in the rat than in.man. Since the total blood volume is small, repeated bleedings may produce anemia. Also repeated bleedings from the same area may produce inflammation and leucocytosis. Health.and nutrition also markedly influence the blood picture (Creskoff 1919). All these facts tend to make the rat a difficult animal on.which to do hematological studies. Concerning terramycin assay data it can be stated that terramycin passes from.female to offspring through.the placenta and also that it is transmitted to the suckling young via the milk. This is important in that it shows that the experimental animals were exposed to the antibiotic from.before birth.until the end of the experiment. The erythrocyte picture in young animals of both the experimental and control groups (Table III) shows similar tendencies to increase with age with much variation at any particular age. This tendency is not as marked in the white cell picture of the young animals (Table VII). Although fluctuations occur from age to age there is a more constant rise in the control group. 25 The red cell averages (Table IV) in the experimental and control breeding colonies are both within the normal range for the albino rat. waever the control group average is lower than would be expected. Considering all the factors tending to cause error in leucocyte counts, the values for the two breeding colonies (Table VIII) are acceptable since they fall within the normal range for the rat. This appears to be even more so when.the differential counts are considered. The differential formulas both for the breeders and for 73 day old animals revealed a similar picture in the experimental and control animals (Table XIII). 26 SUMMARY Data on the average erythrocyte and leucocyte values in immature rats showed similar results in.the 12 experi- mental and 7 control animals. The experimental and control breeding colonies gave average values for erythrocyte, leucocyte and differential counts that were similar. Differential counts made on 73 day old animals (h experimental and 2 control) gave results that were con- sistent for the two groups but that were slightly different from.those of the experimental and control breeders. All average values found for erythrocyte, leucocyte and differential counts were within the range of normal variation of these elements for the albino rat. Terramycin assay data revealed that the antibiotic is supplied to the young in utero via placental transmission and to suckling animals through the milk. From.the results obtained in this study it can be said that at a level of 500 grams per ton of feed, terramycin has no appreciable effect upon the leucocytes and erythro- cytes of the blood of the albino rat. 27 LITERATURE C ITED Creskoff, A. J., T. Fitz-Hugh, Jr., and E. J. Farris. l9u9. Chap. 1h, Hematology of the Rat - Methods and Stand- ards. Farris, E. J. and J. Q. Griffith, Editors. The Rat in Laboratory Investigation. Philadelphia: J. B. Lippincott Co. and Ed. 512 pp. Farris, E. J. 1938. Emotional Lymphocytosis in the Albino Rat. Amer. Jbur. Anat. 63: 325-3h8. 19E2. Leucopenia Associated with Normal Estrqu; in the Albino Rat. Anat. Rec. 82: 1&7-151. Finlay, A. 0., et a1. 1950. Terramycin, a New Antibiotic. Science. 111: 85. Gill, P. F. 1950. AgranulocytOpenia Following "Chloromycetin." Report on Two Cases. Med. Jour. Australia. 1: 768. Harrell, W. E., F. R. Heilman, and w. E. Hellman. 1950. Some Bacteriologic, Pharmacologic, and Clinical 0b- servations on Terramycin. Ann. N. Y. Acad. Sc. 53: Arte 2, M8‘LL58. Jolly, J. 1909. Variations de 1'hemoglObine, du nombre des globules rouges et de la valeur globulaire aux differentes periodes de la vie, chex 1e rat blanc. C. R. Soc. Biol. Paris. 66: 136-139. I9I9. Sur les modifications morphologiques qui se passent dans le sang des mammiferes au moment de la naissance. C. R. Soc. Biol. Paris. 82: 800-802. Lewis, G. N., et a1. 1952. Chloramphenicol (Chloromycetin) in Relation to Blood Dyscrasias with Observations on Other Drugs. Anti- biotics and Chemotherapy. 2: 601-609. 28 Margot. 1916. Manuscript. Donaldson, H. H., Editor. The Rat. memoirs of the Wistar Institute of Anatomy and Biology. No. 6. Philadelphia. 2nd Ed. 192k. Nflrone, L. 1953. Effect of Aureomycin and Terramycin on Growth, Re- production and Blood Count in Mice. Antibiotics and Chemotherapy. 3: 600-602. Nelson, A. A. and J. L. Radomski. 195R. Comparative Pathological Study in Dogs of Feeding of Six Broad-Spectrum.Antibiotics. Antibiotics and Chemotherapy. h: 117h-1180. Howey. 1921. manuscript. Donaldson, H. H., Editor. The Rat. Memoirs of the Wistar Institute of Anatomy and 31.010ng NO. 6. Philadelphia. and Ede 19214». P'an, S. Y., L. Scaduto, and EL Cullen. 1950. Pharmacology of Terramycin in Experimental Animals. Ann. N. Y. Acad. Sc. 53: Art. 2, 238-252. Rich, EL L», R. J. Ritterhoff, and R. J. Hoffman. 1950. A Fatal Case of Aplastic Anemia Following Chloram- phenicol (Chloromycetin) Therapy. Ann. Int. Med. 33 : 1159. Rivas. “ 191R. Rhnuscript. Donaldson, H. H., Editor. The Rat. Memoirs of the Histar Institute of Anatomy and Biology. No. 6. Philadelphia. 2nd Ed. 192R. Schoenbach, E. B., M. S. Bryer, and P. H. Long. 1950. The Pharmacology of Terramycin in Animals and Man with Reference to its Clinical Trial. Ann. N. Y. Acade SC. 53: Art. 2, 2115-252. Volini, I. F., et al. 7 1950. Hemopoietic Changes During Administration of Chloromycetin (Chloramphenicol). J. A. M. A. 1&2: 1333. welch, H., C. N. Lewis, and I. Kerlan. 195R. Blood scrasias. Antibiotics and Chemotherapy. h: 607- 23. 29 LITERATURE NOT CITED Blake, F. G. 1952. The Present Status of Antibiotic flierapy, with Particular Reference to Chloramphenicol, Aureomycin, and Terramycin. Springfield, 111.: Thomas. 26 pp. Briese, E. 1936. Cytologic Studies of the Blood of the Newborn Rat. Proc. Staff Meet. Rhyo Clinic. 11: (15) 233-236. Burhoe, S. 0. 19110. Methods of Securing Blood from Rats. Jour. Heredity. 31:1145-14118. Dacie, J. V. 1950. Practical Haematology. London: J. 8: A. Churchill. 172 pp. Karel, L. and E. S. Roach. 1951. A Dictionary of Antibiosis. New York: Columbia Univ. PreSS. 293-29’4. Kindred, J. E. and E. L. Corey. 1930. Studies on the Blood of the Fetal Albino Rat' - Total Counts of the Red and White Blood Corpuscles. Anat. ROCe u7: 213-227e Lawrence, J. S. and J. Francis. 1953. The Sulphonamides and Antibiotics in Man and Ani- mals. London: E. K. Lewis 8: 00., Ltd. 150-153. Pratt, R. and J. Dufrenoy. 1953. Antibiotics. Philadelphia: Idppincott. 2nd Ed. 398 pp. Robinson, F. A. 1953. Antibiotics. new York: Pitman Pub. Corp. 132 pp. 'Ihewlis, E. W. and 0. 0. Meyer. 1914.2. The Blood Count of Normal White Rats. mat. Rec. 82: 115-125e Waksman, S. A. and H. H. Lechevalier. 1953. Guide to Classification and Identification of th. Actinomycetes and Their Antibiotics. Baltimore: Williams 8:: Wilkins. 2116 pp. GhESe Pfizer 86 C00, IIICe 1953. Terramycin, Review of Clinical Literature. Brooklyn, New York. U. S. Naval Medical School. l9h5. Hematology. National Naval Mbdical Center. Bethesda, Maryland. 30 W1 2:: v A031 '1 235 ;'- Aug 31 ” ‘ry‘3177 adv“ (Ni 7 Jan ‘| Slta