Title: Studies on the in vivo and in vitro growth of trypanosoma cruzi
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Title: Studies on the in vivo and in vitro growth of trypanosoma cruzi
Physical Description: v, 63, 1 leaves. : illus. ; 28 cm.
Language: English
Creator: Hynes, Jacqueline Adams, 1930-
Publication Date: 1958
Copyright Date: 1958
 Subjects
Subject: Trypanosomiasis   ( lcsh )
Trypanosoma   ( lcsh )
Biology thesis Ph. D
Dissertations, Academic -- Biology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis -- University of Florida.
Bibliography: Bibliography: leaves 58-62.
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General Note: Vita.
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Bibliographic ID: UF00098014
Volume ID: VID00001
Source Institution: University of Florida
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Resource Identifier: alephbibnum - 000572496
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STUDIES ON THE IN VIVO AND IN VITRO

GROWTH OF TRYPANOSOMA CRUZI

(CHAGAS, 1909)










By
JACQUELINE ADAMS HYNES


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY











UNIVERSITY OF FLORIDA
August, 1958









ACITI01 LED3GI ;TS


I rish to thank Dr. E. C. Bovce for initially suugestinC a por-

tion of this problem and for his constant aid and cncourzceccnt during

the course of the investigation. I am very gratefull for the opportunity

of sc:.'vi as his research assistant on work supported by national Insti-

tutes of Health Grant E-l158.

I am grateful to Doctors Levis Berncr, R. II. DeUitt, R. P. Hussey,

J. R. Redoond, T. 1. Stearns, and H. II. Uallbrunn for helpful suges-

tions concerning ry research, for the loan of equip~ncnt, and for read-

ing this dissertation. I especially wish to tank Mr. William C. Sloan

for the valuable ideas which he contributed while discussinG with no

problems which arose during this investigation.

Some equipment used in this research vas purchased irith funds

Created by the Florida Acadeji/ of Science.










TABLE OF CONTETS


IIRODUCTION .. . . ..... . . . 1

MATERIAIAIS AIMD METHOD ............ . ........ 5

Organifsms and Their Maintenance . . . . . . 5
In vivo Experiments ....... ............ 7
In vitro Experimentz .................... 14

RE ULT . . . . . . . . . . . . 20

In vivo LcEx riments .... ................ 20
In vitro Experiments ........ . . . . 44

DISCLUSIG0 ............... . ........

SIt6IARY ADID COICLUIIO3S. . . . . . ... 57

LITUIATURE CITED . . . . . . . . . . . 58

BIOGRAPHY . . . . . . . .......... 63


iii








LIST j0 TABLE3


Table Page

1 Mortality of 21-Day-Old Mice Injected with
Trypanozoncs and/or Cortisone. ... . . .. 8

2 Bazol Kediua . . . . .. . . . . . . 17

3 diu 3 . . . . . . . . . . . .. 19

4 Mortality of 17-Day-01d Mice Injected with
Trypanosomes and/or Cortisone. . . . . . . . 22

5 Mortality of 17-Day-Old lice Injected with
Trypanosomes, Cortisone, and Adenosinc Triphosphate.. . 27

6 Mortality of 17-Day-Old Mice Injected with Trypanosomes
and Cortisone, Treated with Thiamine . . . . .. 30

7 Mortality of 17-Day-Old Mice Injected with Trypanosomns,
Cortisone, Thinaine, Mi esiur and Adenosine Triphosphate 34

8 Mortality of 17-Day-Old Mice Injected with Trypanosomes,
Cortisone, Adenosine Diphosphate, and Inorganic Phosphate 38

9 Paraplegia in Infected ce. . . ... . . . .. 43

10 Growth in the Partially Defined Medium of Citri and
Grossovicz (1955a) . . . . . . . . . 45

11 Grawth of Costa Rican Strain in Experimental Media . . 47








LLT 0? FIGURlE


Figure Pcgo

1 Additive Growth Rates of 17-Day-Old Mice Injected
with Trypano3ones and/or Cortisone . . . . . 24

2 Additive Growth Rates of 17-Day-Old Mice Injected
vith Trypanosomes, Cortisone, and Adenosine Triphosphate 28

3 Additive Growth Rates of 17-Day-Old Mice Injected with
Trypanosomes and Cortisono, Treated rith Thioaine. . . 32

4 Additive Growth Rates of 17-Day-Old icec Injected with
Trypanosoaes, Cortisone, Thiamine, Magnesium, and
Adenosine Triphosphate . . . . . . . ... 35

5 Additive Growrth Rates of 17-Day-Old Mice Injected with
Trypanosomes, Cortisone, Adenosine Diphosphate, and
Inorganic Phosphate. ................... 39








IITRODUCTIOU


Trypanosoma cruzi vas first described and identified as the

causative cent of American trypanosomiasis or Chags' disease by Chagas

in 1909. The disease, once believed limited to the state of Minas

Gerais, Brazil, is now known to affect millions of persons from Mexico

to Argentina (Dias and Laranja, 1948). Naturally infected triatomids

and mammals have frequently been found in Texas, California, and Arizona

(Hall, 1953). The first indigenous case of human Chagas' disease in

this country was reported in 1955 from Corpus Christi, Texas (Woody and

Woody, 1955).

I. ile information concerning its distribution continues to increase,

knowledge of other aspects of the disease remains scanty. There is still

no treatment for the infection (Goble, 1956; Hawking, 1953). Nor is the

manner in which the parasite harms the host known. The idea that the

tryponosome injures its host through the consumption of large amounts of

blood glucose has been discarded (von Brand et al., 1949). Evidence con-

cerning the production of endotoxins by T. cruzi is highly contradictory

(von Brand, 1951). Cameron (1956) believes that the effects of this

parasite are due chiefly to mechanical injury to host tissues. It is un-

likely that solutions to these problems of treatment and causation will

be found until much more basic information on the metabolism and nutri-

tional requirements of T. cruzi has been gained. The present study was

undertaken in an effort to gain some further insight into the physiology

of this organism.









Several excellent reviews of our present day knowledge of the

physiology of T. cru.i have recently appeared (von Brand, 1951, 1952;

LDoff, 1951; Hutner and Provasoli, 1955). It is well established that

culture forms of T. cruzi, which correspond to the form found in the in-

vertebrate host, utilize glucose (von Brand et al., 1949). Ryley (1956)

demonstrated clucose consumption by blood stream forms. Baornstein and

Rees (1952) found aldolase in culture forms of the parasite, and Baern-

stein (1953b) demonstrated tie presence of isomerase and triosephosphate

dehydrogenase. From this, he concluded that T. cruzi probably has the

classical Embden-~'jyrhof-Parnas gljcolytic scheme.

Considerable evidence for the presence in the parasite of a func-

tional tricarboxylic acid cycle has been accumulated. Succinic dehydro-

cenase activity has been shown by Seaman (1953), Agosin and von Brand

(1955), and Ryley (1956). Ialic dehydrogenase and fumarase were demon-

strated by Bacrnstein (1953a), and isocitric dehydrogenase by Agocin and

Weinbach (1956). Von Brand and Agosin (1955) found that the respiration

of T. cruzi was stimulated by the addition of various Krebs cycle inter-

rcediates to the medium. Ryley (1956) detected small amounts of citric

acid in media containing T. cruzi. He suggested that the tricarboxylic

acid cycle was operating in these organisms and that traces of this key

metabolite leaked from the cells. Ryley found no significant differences

in glucose metabolism between blood stream and culture forms. Therefore,

the above findings on culture types of T. cruzi are probably applicable

to the blood stream forms also, and suggest that aerobic metabolism of

the organism is carried out via the tricarboxylic acid cycle.









In view of these data, the report that injection of adenosine tri-

phosphate reduced the mortality of cortisone-injected mice infected with

T. cruzi (Adans, 1954) appeared to be highly significant.

Different strains of T. cruzi vary widely in their pathoenicity

for mice, ranging from a complete avirulcnce to an .etrcem virulence

(HEasch!a, 1947). Since 1951 when Jarpa et al. found that the adtLinis-

tration of cortisone acetate to mice infected with T. cruzi increased

the parasitemia and the mortality rate, this compound has been used to

enhance the effect of the less virulent strains on host organisms.

Cortisone has this same effect on various other spontaneous and experi-

mentally induced infections, including tuberculosis, poliomyelitis, and

certain kinds of malaria. The mechanics of this action is not definitely

!-norn, but there seems to be rather general agreement that cortisone

depresses host resistance to infection rather than having any direct ef-

fect on the infectious agent. It ha been found that the hormone affects

metabolism through its action on various enzymes and that it acts on

lymnpoid tissue, reducing capillary permeability and inhibiting phago-

cytosis, antibody formation, and reticulo-endothelial activity. Any or

all of these effects may be related to its infection-enhancing ability.

This subject has been reviewed in a series of papers edited by Shwartzman

(1953).

The nutritional requirements of T. cruzi are still unknown. The

chief obstacle to the determination of these grort-h needs and to the

study of their metabolism has been the need for a defined synthetic

medium in which to grow the organism. Until quite recently it was be-










lived that T. cruzi could not be grorn in the absence of erythrocytes

(Little and Olecon, 1951) or scrum (Liuoff, 1951). Citri end Grossowicz

(1954) then deviscd a liquid medium in which cr stalline serum aolbuin,

hcomtin, and tomato juice co.mpletely3 replaced blood. They refined the

nodiu. further by szubstituting ikojom gyoirth factors for the tomato

juice (1955a). This modiuu supported satisfactory growth of the Cul-

burtson strain of T. cruzi. Sea an (1957) suc&ested that their medium

might be rather selective for this strain, since several other strains

did not grow wcll in it.

The aims of the present study rsrc: (1) to repeat Adams' (1954)

experiments vrith adenosine triphosphate, (2) to extend his workl to in-

clude the study of the effects of other compounds involved in carbohy-

drate etabolism on trypanosome-infected mice, (3) to test the ability

of Citri and Grossovics's medium (1955a) to support crorth of T. cruzi

strains other than the Culbertson strain, (4) to define further the

partially defined medium of Citri and Grossowicz.









ATER IALS AND MTTDS


Organisms and Their Maintenance


The "Brazil" and "Costa Rica" strains of Trpanosoma crusi were

used for in vivo studios. In addition to these two strains the Culbert-

son strain ~ras utilized in the culture work for comparative purposes,

since it u s for this strain that Citri and Grossorics (1954, 1955a)

devised their media. Axenic cultures were used throughout this study.

The Brazilian strain was obtained from Dr. R. G. Yeager (Tulane

Iledical Center) who had maintained it in a diphasic medium consisting

of Difco brain-heart infusion agar plus 25 per cent defibrinated rabbit

blood overlaid with 2 to 3 ml. of sterile Locke's solution. This

strain was originally isolated from a Brazilian patient in 1942 (Goble,

1951) and has been maintained in several laboratories in this country

since that time.

The Costa Rican strain was secured in Costa Rica by Dr. Herbert

Johnstone of the University of California IMedical Center (Noble et al.,

1953) and was given to me by Dr. E. R. Noble (University of California,

Santa Barbara College). The latter grew the organisms in a medium com-

posed of glucose, NaC1, and peptone to which was added red cell coagulum

or hemoglobin coagulum (McRary et al., 1953).

Dr. N. Grossoricz supplied a culture of the Culbertson strain

which had been maintained at Hebrew Lkiversity, Jerusalem, on Adler's

medium (Adler and Theodor, 1926) since 1949. The early history of this

strain is incomplete (Hauschka, 1947).










All three strains grov tvll in a modification of the medium used

by Younger. This modification is prepared as follows:

Difco brain-heart infusion broth 3.7 ga,
Bacto-agar 1.5 gm.
Pyrex-distilled water 100 ml.

Thio was autoclaved for 15 minutes at 15 p.o.i., ccoled
to approximately 460 C., and to it was added:

Whole blood 10 ml.

Citrated (ACD) blood discarded by the J. H. Thomas Memorial Blood Bank,

Gainesville, Florida, was used and found to produce highly satisfactory

Growth. Each slant of this medium was overlaid with 5 ml. of sterile

Locke's solution (NaC1, 8.0 go.; KC1, 0.2 gm.; CtC12, 0.2 gm.; KH2PO4,

0.3 gn.; dextrose, 2.5 gm.; Pyrex-distilled water, 1000 ml.) to which

were added penicillin (60 ag./ml.) and streptomycin (100 ag./ml.) to

decrease the risk of bacterial contamination. Stock cultures of all

strains were grown in this medium at 270 C., and subcultures were made

every 2 to 3 weeks.

All of the mice used in this study were bred in the laboratory

from a small initial stock of white mice, CF Ho. 1 strain, obtained from

Carworth Farms, Inc., New City, Naw York. Although this procedure

limited the number of mice available for experimentation, it did as-

sure that the nice used were in good health and that they were of th=

desired age. The mice were permitted to feed ad libitun on Purina

Laboratory Chow, and a constant supply of water was provided. Young

mice were weaned at 28 days of age.










In vivo Experiments


1. Injection of mice. Mice to be infected were injected with

both cortisone acetate and trypanosomes, since the Brazilian and Costa

Rican strains of T. crusi are only moderately virulent. Five g. of

cortisone acetate (saline suspension, Cortone Merch) wer injected

subcutaneously. One-half ml. of trypanosome-containing overlay from

two- to three-ueek-old stocl- cultures was injected subcutancously or

intraperitoneally. The split litter technique, with consideration for

sex, was used in all experiments.

At the beginning of this study, it was felt that it night be de-

sirable to inject the mice after they had been weaned. The earliest age

suggested fo weaning is 21 da:s, and 28 days is the recommended time

(Snell, 1941). Culbertson and Kessler (1942) found that when mice under

25 days of age were inoculated with T. cru.i (strain not indicated)

they acquired an infection of greater intensity than that exhibited by

older mice and Generally died, while mice above this age usually sur-

vived. Therefore, three litters of weaned 21-day-old mice were injected

to determine whether infections could be produced at that age. Each

litter was divided into the following treatment groups: (1) no injec-

tion (no inj); (2) cortisone (cort); (3) trypanosomes, Brazilian (Br) or

Costa Rican (CR) strains; (4) trypanosomes and cortisone. At the end of

60 days only two mice (and one of these not infected) had died (see

Table 1). With such a low mortality rate, it would be difficult to

determine rwether injections of adenosine triphosphate or other compounds




8





TABLE 1


MORTALITY OF 21-DAY-OLD MICE INJECTED UIT TRYPAH'OSOMS AThD/OR CORTISOME


Treatment

Io inj

Cort

Dr

Br + court

CR

CR + court


No. mice
injected

2

5

4

5

4

5


Deaths


0

20.0

0

40.0

25.0

4o.o


io. days between
injection and death


9



155, 321

78

43, 312


11111


I


--










reduced the number of deaths resulting from the disease. For this

reason, 17-day-old mice, which are more susceptible to infection with

T. cruzi, were used for all of the experiments in this study, following

the practice of Adams (19514).

IIaeoocytomctr counts of trypanosomes used for injection were

made by diluting the culture with a 1:10 dilution of neutral formalin

(3641 formaldehyde) in distilled water. or i were counted in

80 squares (as recommended for crythrocyte counts) with a magnification

of 430X. Duplicate counts were made in each case and the results of the

two cveraced to Give the number of organisms per ml. of sample.

Other injections were administered as indicated in the description

of the individual experiments.

LExprimental mice wore weighed on the day of injection and eery

other day thereafter for 1 month. Veifits were taken every fourth day

for a second month.


2. IIistoloical techniques. All dead mice were autopsied, and

macroscopically visible changes in internal organs noted, as soon after

death as the animals were discovered. Tissues for histological study

were irr-ediately fixed in Bouin's fluid, and 8, paraffin sections were

stained in Harris' haematoxylin and counterstained in a saturated solu-

tion of orange G in 95 per cent ethanol. Spinal and brain smears were

fixed in methanol and stained with Wright's blood stain. Fresh tissues

to be examined w~re teased or macerated in a drop of Neff's (1955) basic

salt solution (NaC1, 120 nZ.; .IC12.6120, 3 mg.; CaCl2, 3 rag.; FcS04.7T 0,










3 ma.; 0.1.1 KirP04 and 0.2O1I IIa'q, 20 ril. to adjust pH to 6.5; Pyrex-

distilled water to 1000 ml.) and were examined under variable phase con-

trast at a magnification of 400X with an AO-Bakcr Interference licroocope.


3. Trypanosomcs and/or cortisone. A series of experircnts was

performed to determine what percentage of injected mice died, the number

of days clapsing between time of injection and death,and the Growth pat-

tern (based on weight) of injected nice. These data provide a basis

cainst which to compare the results of later series of experiments in

which the mice =erc, in addition, injected with nucleotides or thiamine,

and also serve for comparing the effects of the Brazilian and Costa

Rican strains of T. crazi.

Ten litters of 17-day-old mice were used in these experiments.

rEac litter was divided into groups, usually four, each of which was

treated in one of the following ways: (1) no injection, (2) cortisone,

(3) trypanosomes, (4) trypanosomes and cortisone. Occasionally, large

litters wero divided into more groups in order to test the two strains

of trypanoscmes on members of the same litter.


4. Adenosine triphosphate (ATP). Each litter was divided into

groups which were injected as follows: (1) trypanosones and cortisone,

(2) trypanosomes, cortisone, and ATP, (3) cortisone and ATP. Later,

same of the litters were further subdivided to test the effects of dif-

ferent counts of ATP on members of the rsme litter. The trypanosomes

and cortisone were injected as previously described, and 8 hours later

the ATP solution was injected subcutaneously. Nine litters of mice were










used in these experiments.

The disodium salt of ATP was dissolved in sterile Locke's solu-

tion to give the desired concentration. The solution vas prepared im-

mediately before it was to be injected, for ATP bre-ks down rapidly in

solution.

To repeat Adams' (1954) worh, 5.0 mg. of ATP were given to each

mouse. When it was found that more of the infected mice treated in this

way were dying than those untreated, the amount was reduced to 2.5 mg.

of ATP.


5. Tliamine. Several mice in the preceding experimental series

7hich had been injected with Brazilian strain and cortisone displayed

symptoms similar to those of thiamine deficiency (Morris, 1947; Woolley

and White, 1943). They were unable to retain their balance and rolled

over and over; they went into convulsions when held by the tail; and

spasticity of the legs and feet was apparent. These observations, coupled

with the reports that culture forms of T. cruzi may produce appreciable

counts of pyruvic acid (Chang, 1948) and that blood stream forrs pro-

duce at least small amounts of it (Ryley, 1956), suggested that a thi-

amine deficiency might be involved in the course of the infection, since

thinmine is required for the metabolism of pyruvate via the Krebs tri-

carboxylic acid cycle. It seemed possible that: (1) the trypanosomes

were using thiamine in amounts that caused the host to become deficient,

(2) the trypanosomes ere producing pyruvic acid to such an extent that

the mouse vas unable to metabolize it, and so the accumulated pyruvate










proved toxic (Robinson, 1951; Grant and Fulton, 1957), or (3) an inde-

perndntly occurring thiamine deficiency might exist in scae mice, per-

haps making them more susceptible to infection. Mice were, therefore,

treated with thiamine to see what effect it might have on the infection.

Each litter was divided into three treatment groups: (1) try-

panocomes end cortisone, (2) t-rpanosomes, cortisone, and thiamine, (3)

thiamine. Ilice from two litters were given 60 "g. of thiamine hydro-

chloride (amount adopted from Woolley and MUhite, 1943) orally on the day

following injection and every other day thereafter for 4 xec!e, giving

a total of fourteen doses. Mice from five litters were injected sub-

cutancously with 25 rg. or 50 *U.. of thiamine hydrochloride on the day

following the trypanosome injection and every other day thereafter for 4

weeks. The thiamine solution was prepared by dissolving thiamine hydro-

chloride in Locke's solution and was sterilized by filtration.


6. Thiaminc, mapgnesiin, and adenosine triphosphate. In the con-

version of pyruvate to acetyl coenzyme A, thiamine is in the form of

thiamine pyrophosphate or cocarboxylase. It has been 1nown for some time

that ATP is necessary for the phosphorylation of thiamine in vitro (Glasa,

1951), and it has recently been demonstrated (Rossi-Fanelli et al., 1954)

that this reaction also occurs in the living animal. Within 1 hour of in-

jecting thiamine and ATP into intact rats, phosphoric groups are trans-

ferred to thiamine, producing di- and triphosphothiamine. Mecnesium ions

ar2 probably necessary for the phosphorylation of thiamine (Glass, 1951;

lieilands and Stumpf, 1955).










Therefore, in conjunction with the thiamine experiments, four

litters of mice were treated with thiamine, I4++*, and AMP. The injec-

tion solution ias prepared to give a 1:1:1 molar ratio of the components.

The desired amounts of thiamine hydrochloride aid MzCl2.6 12 vere dis-

solved in Pyrex-distilled water and sterilized by filtration. The ATP

was dissolved in this solution just before the mice ircro injected. Each

litter was divided into these treatment groups: (1) trypanosomes and

cortisone, (2) try panoso!mes, cortisone, thiamine., M ++ and ATP, (3)

thiamine, MC++, and ATP. On the day following injection of the trypano-

somes and every other day thereafter for 4 weeks, the nice were given sub-

cutaneous injections of thiamine hydrochloride, 200 pg.; MC12.6i20,

120.6 .; and ATP (disodium salt), 370 ag.


7. Adenosine diphosphate and inorganic phosphate. Under physio-

logical conditions the coupling of oxidation and phosphorylation appears

to be obligatory, and both reactions are dependent upon the supply of

adenosine diphosphate (ADP) and inorganic phosphate (iP) (Krebs, 1957).

In other irords, phosphate and/or phosphate acceptors are the rate-limit-

ing factors in oxidation under different conditions u-rich have been

studied (Lardy, 1956), ad I2rcbs (1957) has sucgcsted that both are

probably important in vivo. Since, in most tissues the available con-

centrations of ADP and iP are below; the critical level, the rate of oxygen

consumption depends upon the rate at which ATP splits to form ADP and iP

(Irebs, 1956).

In vicrr of these facts, it seemed possible that whatever effect










ATP had upon the trypanosorm-infectcd mice might be hastened and/or

enhanced by supplying ADP and iP rather than ATP. Five litters of mice

were divided into those treatment groups: (I) trypanosomes and corti-

sone, (2) trypanoscms, cortisone, DP, cand iP, (3) cortisone, ADP, and

iP. The injection solution was prepared so that an amount of ADP equiv-

olent in moles to 2.5 M3. of ATP would be administered and with the

molar ratio of ADP to iP 1:1. The desired amount of I:aHPO4.12H20 wva

dissolved in Pyrex-distilled uater and the solution sterilized at 15

p.a.i. for 20 minutes. The ADP (sodium salt) was dissolved in the

phosphtc solution immediately before injection. Eicht hours after

receiving trypanosome injections, the mice received 2.4 m-. of ADP and

1.8 mg. of Ia2HPO4.12 1120 ubcutaneously.


In vitro Experiments


1. Inoculation of cultures. To establish initial cultures of

trypanosomes in the partially defined medium of Citri and GrossoVicz

(1955a), several stock cultures of each strain were pooled and centri-

fuied at 1400 rpm (r = 15.0 cm.) for 10 minutes. The organisms were

washed twice in 0.85 per cent NaCl solution and finally suspended in 5

Ml. of this. This suspension was used to inoculate tubes of Citri's

medium, and samples were taken for haenocytometer counts.

Other experimental media were inoculated with washed organisms

grown in Citri's medium.

Cultures were grown in 15x125 mm. Pyrex culture tubes, each con-

taining 5 ml. of medium. These were incubated at 270 C. in a slanted










position. Both cxperinental and stock cultures rere routinely checked

for bacteria, using nutrient agar pour-plates.

Population densities were determined by making direct haemocyto-

meter counts as previously described. The counts from duplicate tubes

were averaged to give a measure of growth for each experiment. All ex-

perimnnts were run at least in triplicate.


2. Preparation of media. water rodistilled in a Pyrex glass

still s used in compounding all media. Vitamins, amino acids, and

henin were obtained from Nutritional Biochemicals Corporation. Concen-

trated stock solutions of vitamins and metal mixes were prepared and

stored in the refrigerator. Because the activity of folic acid deteri-

orates during storage (Tracer, 1957), solutions of it were used within

2 months of preparation. The solution was prepare- by dissolving the

folic acid in a few drops of 0.01U IIaOH, then adding water and heating

Gently. Heoin was dissolved in a mall amount of 511 HI0H, then made

up to volume with Pyrex-distilled vater. Solutions of glucose, henin,

cerun albumin fraction V, thiamine, and riboflavin were sterilized by

filtration through a Pyrex brand fitted glass filter disc of ultra-

fine porosity and added aseptically to the autoclaved medium.

After ex-perimnting vith various modifications on Citri's medium,

it vas found that a Iknom mixture of amino acids, the composition of

which is based on an analysis of p-lactoglobulin (Tristram, 1953), could

be substituted for the casein hydrolysate.' Serum albumin fraction V was

omitted from the medium, various metals were added, and slight changes










;ere made in the concentrations of some other ingredients. The growth

factors and their concentrations terc not altered. The composition of

the medium is shoun in Table 2.

The amino acids were dissolved in three-fifths of the final volume

of boiling water, then the salts were added, and the mixture cooled.

TJeen 80 (polyoxycthylene sorbitan monooleate), cytidylic acid, creatine,

creatinine, and ribonucleic acid were added to this solution, and 1.011

IHaOll was used to increase the pH before the vitamins were added. The pH

was finally adjusted to 8.0, the volume made up with redistilled water,

and the medium autoclaved at 15 p.s.i. for 20 minutes. The remaining in-

gredients, as indicated in the table, were added aseptically to the

cooled, sterile medium.

In Medium 1 the amount of hemin was increased to 2.5 ma./100 ml.

of mndium. Medium 2 was identical to Medium 1 except that the hemin was

added before autoclavinG.

Medium 3 differed from the basal medium in several ways. IHemin

polymerizec in al-maline solution so that part of it may not be avail-

able for use by the organisms; therefore, the heain for this medium was

dissolved in triethanolamino (5 nm. hemin/ml. 50 per cent (v/v) aqueous

triethanolamine) as suSgested by Corrperthwaite et al. (1953). The

amount of triethenolamine added to the medium with the hemin was only

half the total amount to be included; the remainder was added as the 50

per cent (w/v) aqueous solution. The triethanolamine also served as a

buffer in the medium. The concentrations of CaC12, WMSO4.7H20, and











TABLE 2

BASAL MEDIU.1


L-Alanino
L-Arginine
L-Aspartic acid
L-Cystine
L-Glut--mic ccid
Glycine
L-IIistidinc
DL-Isolcucine
L-Leucine
L-Lysinc. HC
DL-1othionine
DL-RTcny alnaninc
L-Prolino
DL-Scrinc
DL-Thrconine
L-Tryptophane
L-'Tosine
DL-Valine


2IIOIIP04.12H20
IGI2P04
CaClg2*

IMtal mi B*-* +


mg./100 ml.
70.0
30.0
115.0
20.0
190.0
15.0
15.0
60.0
155.0
110.0
30.0

50.0
40.0
50.0
20.0
35.0
55.0


200.0
300.0
50.0


mg./100 ml.
Paxa-cainobnzoic acd 0.01
Biotin 0.02
Cholinc.C1 0.3
Folic acid 0.25
Inositol 15.0
IIicotina7Aid 1.5
Pyridoxinc. Hl 0.2
Pyrido::al.HC1 0.2
Iyridoxamine. 2HC1 0.2
Riboflavin* 0.1
Thiamine.HCl* 0.1
Cobalanin 0.00001


Ribonucleic acid
Cytidylic acid


Creatine
Creatinine

Heain*
Glucose*
Tween 80


8.0
2.0

2.0
2.0


1.0
200.0
1.0


1.3
50.0
0.2 ml.


S-toci solution sterilized by filtering throuGh ultra-fine sintered
glass filter and added Cseptically to the autocl3vod medium.

i*Stoc;- solution autocl2ved at 15 p.s.i. for 15 min. and added aseptically
to the autoclaved medium.

-l ml. contains: II BO,, 0.057 mr.; CoS04.7T1,0, 0.238 =r;.; CuSO .5HiO,
0.394 G.; Fe So04.7 0 5.0 me.; MnSO.H20, 1.54 r~.; ZnSO.71H, JI.0
zC. (Iatthan and Covpurtlhaite, 1955).




18




glucose were chn a~d, and ethylencdicnmine-tctra-acctic acid (EDTA) ad

FcS04 .1120 vere added to tie mEdiut.i. Netal mix A vws used instead of
me.tal mix B. These chanZgs are surnmmized in Table 3.










T2AIL 3

I EDIU" 3


BaEal Ilcdiun vith Follo ing Changes and Additions


mn./100 ul.


Before autoclvving add:


Triethanola!iinc

Uemoin



Fc04.7 20

LMetal nix A+

CaC32

H8e3o04T7R2O


500.0

2.5

60.0


0.5

2.5 ml.

0.55

40.0


After autoclavinZ add:


Glucose*


500.0


*Stock solution sterilized by filtering through
ultra-fine sintered glass filter and added
aseptically to the autoclaved medium.

+1 ml. contains: H3BO3, 0.023 rg.; CoSO4.7T20,
0.095 mi.; Cu SO*.51Lo, 0.039 ag.; MnSO*.1O20,
3.076 mr.; ZnSO4.7I20, 4.398 mg.; Nal, 0.001 mg.;
EDTA, 1.0 Lg. (Nathan and Cowperthrmitc, 1954).










RESULTS


In vivo Experiments


Before the results of these experlucnts could be cumraxzcd, it

:.as necessary to dctor-iiinc hcethcr there Iercr statistically significant

differences aong2 litters ~hiich would ha'e an effect upon c::perimental

results. For this pu-ipos, an analy-sis of variaice i~; carried out on

all the litters (14) which contained at least threc mnice injected with

Lrazilian strain can cortisonc. The per cent rcicht change fromn the

day of injection to the trclfth day after injection in Dr + cort-in-

jccted mice was the chLracter selected for analysis.


Analysis of Variance

Sourcc of CLu of
v2Xil nce D.f. squares Meaan square F ratio

Ectw.een litters 13 60,460.89 4,650.C 4 F = 4650.84/2780.616
= 1.6725
Within litters 23 77,857.25 2,780.616 F (13, 28) is
between 2.12 and
2.06
Total 41 138,318.14


Therefore, we accept the null hypothesis: the litter means are not cic-

nificantly different. Litter differences, with respect to a basic physi-

ological characteristic, growth rate, are not such as to affect experi-

mental results significantly, and data from similar tests on different

litters may be pooled.










For each series of e:peri.i-'nts a mortality table covering the

O--lday period following injection is presented. A graph portraying ad-

ditive grorbth rate curves of each experitrental group for this period is

also includc-1. Each point on a graph represents the me~ per cent

chenSe in weight on a given day- fro: that ?: days earlier,. ;ith the

weight at the time of injection taken as 100 per cent. Only the weights

of mice whiich rsurvivcd for at least 60 day-s oere used in ccmputing these

curves. The mortality tc-olcs may then be considered to show the effect

of acute trypanosomiazsi, and the growth cures, that of the chronic

disease.

Statistical iaalyscs of results presented in the mortality tables

rrec performed using Fisher's exact treatment of the 2x2 table. Selected

points (those fich appeared most lilhly to show differences) on growth

curves emre analyzed using the t-test for the comparison of 2 means

(:;ith pooln- var-iances). The 0.05 level of significance was selected.


1. Trypaniosarmes and/or cortisone. The results of these experi-

rnnts are presented in Table 4.

There was no statistically significant difference in mortalities

between the follo.rin treatment Groups: lo inj vs. cort (P = 0.22);

cort vs. Br -- cort (0.25); cort vs. CR + court (0.15); Br vs. Br + court

(0.13); CR vs. CR + court (0.63). The probability indicated is that of

obtaining values at least this widely separated by chance alone. The

differencesin mortalities between the following groups were statisti-

cally significant: no inj vs. Br. + court (0.0478) and no inj vs.

CR + court (0.028); but that between Br + cort vs. CR + cort (0.82)was











TALLZ 4

MORTALITY OF 17-DAY-OLD MICE I~ JECTED
ITTH TRYPAIUSO;IES AID/OR CORTISONE


TreatTent

No inj

Cort

Br

Br + court

CR

C0 + court


I~~~o. nPc Dah


lHo. mice
in.lccted

15

24

9

20

4

12


Deaths
No.

0 0


12.5

0

25.0

25.0

3 .' .


No. days
injection
lMean


16.0



11.2.

41.0

13.25


between
and dcath
Ranch


5 33



8 17



7 22


- -----


--...










not. Vc therefore reject the null h2 othesis of no effect. for injec-

tion of the mice with tr;panosoics and cortisone does affect their

sur-vival. It may elso be observed in Tibll 4 that the mean numbers of

days cl31sin; betucon injection anl death for the latter two Cgoups

are vor- close.

T'e additive co;th r'.tes for each e::porin::ntal group are shown

in FiCCre 1. The siril.aritics of the cro.tlh patterns shou'n are evident.

Tnceo are especially intcres'tin, since some of the mice represented in

the d~at of Graphs b, e, and f of FiC're 1 had chronic trypTanosomniasi

as cvi.denced by their subsequent deaths ans the appearance of hce,-ily

parasitis. oranms upon cutopsy.

On day 4 only, statistically significant differences exist be-

treen the ric'n per cent vcicht changes of the following groups: no

inj vs. Br + court (0.05 ,P 0.02); Br vs. Br + court (0.01>P 0.001);

no inj v,. CR + court (0.01 P 0.001); CR vs. CR + court (0.01 >P >0.001).

The probability given is that of the differences bctrrcn the means be-

in3 this great by chance alone. By day 8 this difference is made up and

is no longer statistically significant. Differences on subsequent days

are not statistically siCnificant. CR + cort and Br + cort groups dif-

fer significantly only on day 12 (0.02 >P >0.01). The mean values for

other Croups showed such slight differences upon inspection that analyses

ecre not carried out on them. Actual heights of some of the above Croups

were also compared (t-test for comparison of two means) for the days on

thich they rwre most different; no statistically significant differences

were found.











Figure 1. Additive Groirth rates of 17-day-old nice injected with
trypanosomez and/or cortisone.

a. ITo injection

b. Brazilian strain + cortisone

c. Brazilian strain

d. Cortisone

e. Costa Rican strain + cortisone

f. Cocta Rican strain























iS 32 48 44
a


1I 32 41 44


K"


1I 32 46 44
C
0AYS AFTER INJECTION


Picurc 1.


/A


II 3t
e


418 4


12-










2. Adenosine triphosnhate. The results of these experiments

are summarized in Table 5.

The difference in mortalities between groups of nice injected

with cort + 5.0 mg. ATP and court + 2.5 mr. ATP iwn not statistically

significant (P = 0.49). The mortality difference between Br + cort-

and Br + court + 2.5 ag. ATP-treated mice is significant (0.016), as is

that for Br + cort + 2.5 mg. ATP- and Br + court + 5.0 ms. ATP-treated

ones (0.000); while that between Br + cort- and Br + court + 5.0 ag. ATP-

treated ones is probably biologically significant, although it is not

significant statistically (0.052). No statistically significant dif-

ference exists between mortality rates of CR + cort- and CR + cort +

2.5 me. ATP-treated groups (0.78).

Additive growth rate curves for these groups are shown in Figure

2. Again, statistically significant differences in mean per cent weight

gains occur only on single isolated days. The differences between the

means for the court + 2.5 rag. ATP- and court + 5.0 mg. ATP-treated groups

are significantly different on day 24 (0.01 P > 0.001), as are those

for Br + cort- and Br + court + 2.5 mg. ATP-injected ones on day 32

(0.05 > P > 0.02).


3. Thiamine. Results of these experiments are shown in Table 6.

Because the numbers of mice in some of the experimental groups

were small, the mortality values for all mice treated with thiamine were

combined, as were all those for mice treated with BR + cort + thiamine

(differences in mortality rates among the pooled groups were not











TABLE 5

MORTALITY OF 17-DAY-OLD MICE INJECTED
WITH TRYPAVTOSOMES, CORTISOH E,D D ADEHOSEIIE TRIPHOSPIPAE


--o ic I II


Treatment

Cort + 5.0 g. ATP

Cort + 2.5 m3g. ATP

Br + court

Br + court + 5.0 mg. ATP

Dr + court + 2.5 mn. ATP

CR + court

CR + cort + 2.5 riG. ATP


o. mice
injectedd

11

18

20

15

11

9

11


Deaths
Io.

2 18.2

2 11.1

8 40.0

11 73.3

0 0

2 22.2

2 18.2


Ho. days between
injection and death
Me an Range

9.0 7 -11

12.0 3 21

11.5 6 30

10.8 5 39



9.0 4-14

17.0









Figure 2. Additive growth rates of 17-day-old mice injected with
trypanosco-es cortisone, and adenosine triphosphate.

a. Brazilian strain + cortisone + 5.0 rag. ATP

b. Brazilian strain + cortisone

c. Brazilian strain + cortisone + 2.5 mg. ATP

d. Cortisone + ATP
S_ = 2.5 ms. ATP)
x.-.,----x = 5.0 me. ATP

c. Costa Rican strain + cortisone

f. Costa Rican strain + cortisone + 2.5 rG. ATP


































































DAYS AFTER INJECTION
f










TAPML 6


MiOtTALITM OF 17-DAY-OLD I. ITCT -TECTD ITHI
TRYPfAOS0!tOS AND CORTISOT, TREATED WITTH THIAIMS


lio. da, s bet rccn
INo. nice Dc.ths injection and death
Src- atcnt jeinjcctd 1o. 1ln TRa


]:o inj


Th1iainc,
Thiniinc,
Thiamine,;


60 A.G
25 0C.>
50 ,c.,


oral
inj
inj
Total


Br + court


Lr + cor + tiliiaingel
60 rcg. oral
Er + cort + thlioino,
25.c., inj
Er + cort + thicminc,
50 C., inj
Total


CR + court


3 21.4


3 50.0


0


35.7


1 20.0


CR + court + thiiminc,
50 .G- ianj


5 0 0


16.3



16.7


19.5


13 21



13 22


11 23


51.0










statistically significant) for statistical analyses. lo statistically

significant differences in mortality rates were found between the cxperi-

=cntal groups j this series: no inj vs. Br + cort (P = 0.32); Br + court

vs. Br + court + thiamine (0.34); no inj vs. CR + cort (0.45); CR + court

vs. CR + court + thimnine (0.O0). There is an indication that cortisone-

injected mice infected iith the Costa Rican strain ma react differently

to thiamine treatnt than do those infected with the Brazilian strain.

Grorth curves for this series of experiments are presented in

Figur' 3. Values for all mice treated wirth thiamine and for those given

Br + court + thiamine were also pooled here. Moan per cent weight change

values for Br + court + thiecine-treated mice are never significantly

different from those for Br + cort-injected ones, nor are those for the

CR + cort + thiamine and CR + court groups. The difference in means on

day 12 of the no inj and thimine-treated groups is statistically sig-

nificant (0.02 p P 0.01).


4. Thiamine, naGnesium, and adonosine triphosphate. The results

of these e:.xriments are shoim in Table 7, and the grmoth curves in

Figure 4. The number of litters used in this set of experiments and in

the next series is so small that it was not felt that extensive statis-

tical analyses would be meaningful. The differences betuean mortali-

ties in Br + cort- and Br + court + thiamine, Mg+ ATP-treated mice

(P = 0.45), and in CR + cort- and CR + court + thiamine, M14++, ATP-

treated ones (0.27) are not statistically significant. Growth rate

curves for treated and untreated animals in these two groups also appear

to be very similar.









Figurc 3. Additive crcnrth rates of 17-day-old nice injected with
trypanosozoa and cortisone, treated irith thiamine.

a. io injection

b. Brazilian strain + cortlcone

c. Drazirilan strain + cortisone + thiamine

d. Thiamine

c. Costa Rican strain + cortisone

f. Costa Rican strain + cortisone + thiemine






































Q ci


1 10 130

110 \ 10
10 "\ /
t o no


90


16 32 4t0 4


b


140 140




120 Ito

110 10




0 t st 40onC
I I StFTE 48 84 CTI
OAVS AFTEN INIECTION


Per-'0c 3"










TABLE 7

TOCRTALITY OF 17-DAl-0LD IICE IIJECTED 1UIT1
TRYPAIIOSOIES, CORTISOIS0, THiiLAiiE, I .AGI-IESIUi'I, AID ADQIOSEIE TRIPHOSPHATE


llo. days between
Ilo. mic2 Dcaths injection and death
Treatrannt injectcad Ho. i Mean Ran-e

Tlcnimic, M-++H, ATP 10 0 0

B + court 5 1 20.0 3.0

Dr + cori + thiamine, 5 0 0
11++, ATP

CR + court 6 1 16.7 24.0

CR + court + thicainc,
M3-, ATP 6 3 50.0 38.3 11 56










Trijze 4. Additive c-orLth rates of 17-day-old mice injoctcd with
trypanos cec, cortisone, thi'annc, magnesium, and adenosine triphosphate.

a. Tiaisine, n;ium, ad ATP

b. EBr'ilian strain + cortisone

c. Brarilic. strain + cortisone + thiamine, rnagcsiun, and ATP

d. Costa Rican strain cortisone

e. Costa Rica~ strain + cortisone + thiamine, magnesiiu, and ATP



















140

130

It0

110




90 t 3t 46 64

a



0, No

I. 1- 130




S110




,o -s--------- 90 me I s t 41 4

b d


"To AFTER HUISTON
c e


Figure 4.










5. Ader.osi c diphosphato and Lnorganic phosphatc. The rcuults

of this e:xperinmntal treatnJnt are presented in Table 8 and Ficure 5.

There was no statistically significant difference in mortalities of

treated and untreated CR + cort-injected mice, but the difference be-

turcen the two Br + cort-injected ones was close to the significance

level (P = 0.077). Here again there appears to be a difference in

reaction to treatment between mice infected with the Costa Rican and

Brazilian strains. Gr!.th curves for the treated and untreated groups

are quite csiilar.

If the mortality rates of all the mice injected with Br + court

and those injected with CR + court from all experiments are compared, the

difference between them is not statistically significant (P = 0.24).

Trenty-nine per cent of the mice injected rith Br + court died within

60 days after injection, as compared to 21 per cent of the CR + cort-

injected ones.

When the numbers of days elapsing between injection and death for

these same two groups are compared, it is found that the two variances

are significantly different (P e 0.02 of obtaining an F-value as large

as that found by chance alone). The means cannot, therefore, be

analysed using the usual t-test with pooled variances. However, when

the mcans are compared by the approximate method of Cochran and Cox

(Snedecor, 1946) with no hypothesis about the variances, it is found

that the means probably are not significantly different (P>0.05 of ob-

taining the t-value calculated). The mean number of days elapsing be-

tween injection and death of Br + cort-injected nice is 10.7 (3 30);










TAfLE 8

MIORALITL OF 17-DAY-OLD MICT: I-JECTED WITH
TRYPANOS(~OES CORTISOIE, ADEMOSNE DIPIIOSPIIATE, AIMD INORCGAJIC PHOSPHATE


Tr-iatmnnt

Cort + ADP, iP

r'r + court

Br + cort + ADP, iP

cn + court

CR + court + ADP, iP


I.o. nice
in,'cctcd

12

9

9

6

7


Deaths
Io.

1

3 3:

7 7

0

0


,D 9


3.3

7.8


0


i;o. dv, betwccn
injection and death
Moan ERange

3.0

4.3 3 6

19.0 6 31




39




Figure 5. Aviditiva CrovTh ratoe of 17-2:v.-old nice injected with
tryp3aosomns, cortisone, otdenosino diphosphate, and inorgcnic phosphate.

a. Cortisone + ADP nd iP

b. irazilion strain + cortisone

Brazilian train + cortisone + ADP and iP

d. Costa Rican strain + cortisone

e. Costa Rican strain + cortisone + ADP and iP









































110 46 6
o16 3 4W 64


110, / \.

100

90


Is 32
c


46 4 .LC
0AVS AFTER mt.GIoImN


F'1o-o 5,


- A
IO
30 2 46 64

d


s HC
C


46 64


'"'


d"










for CR + cort-!njectei ones, 13.2;. (4 51).


r. JHistolo-y. ITi.stoloLical ex-mination of tissues from mice

infected with both Brazilian sand Costa Rican strains showed the follow-

ing organs to be parazitized: esophjausz stomach, sEa=ll intestine, heart,

liver, lung, spleen, urinary bladder, brain, and spinal cord. Leishmin

bodies 'wre not observed in the Iidney or pancreas. These findings

compare with those of other workers (HauschI-a, 1947; von Brand et al.,

1949) using the Brczilian and different strains of T. cruzi.

In about half of the nice dying of the acute disease the stomachs

trere enormously distended. They were filled with partially digested

food, and the material at the pyloric end of the stomach soaetimcs con-

tained blood. Gray to blacl spots were frequently visible macroscopi-

cally on tie external stomach wall. Microscopic examination of sec-

tions through these regions and of the fresh tissue macerated in a

drop of saline solution showed these spots to be dense concentrations

of leishnan bodies. In several cases the stomachs were actually per-

forated at these points.

This observation of stomach infections confirms the findings of

Lesser and Luleman (1957) who have presented the only previous report

of stomach involvement. Their studies were done with the Pilcher strain

of T. crusi.


7. Priapism. Tro cases of priapi m ere observed, one in a

nouse injected with CR + court + thianine, b,++, and ATP, the other in

one injected with Br + cort + thiamine. The condition in each lasted











for five days and uac follovwd by paraplegia in one of the anirzals.

This phcnoacnon has been reported in young rats suffering from

a cortisone-vLcravated thimaine deficiency (Uilrcrth and Mcites, 1953).

hcn those authors treated the a~nials vith subonptial mounts of thi-

aoino, the priapisM disappccrol although cortisone injections were

continued.


8. Paraplegia. Ten nice wrc afflicted wirth paraplegia. In-

formation concerning these cases is suanarized in Table 9. The mice

seemed to fall rather definitely into two groups: these which died

very soon after the onset of paralysis, and those which lived for a

long time afterward. In the latter group the condition of two of the

mice alternately improved and regressed, but in no case did a mouse

completely recover from the paralysis. Even when some improvement was

shown here, the mice remained thin. The female infected with Brazilian

strain also developed visual troubles several weeks before its death,

one eye finally becoming completely opaque.

Spinal smears of all the paraplegic mice which died showed leish-

man bodies, but never in great numbers. The parasites rwcr also ob-

served in fresh material from the spinal cord which was macerated in a

drop of saline solution. Brain smears were prepared for two mice, and

the brains of four others were sectioned. One brain smear was positive

for leishman bodies, and throe of the sectioned brains showed then.


9. Chronic infections. During the course of this study 15

deaths resulted from chronic trypanosomiasis. These mice died from











TABLE 9

PARAPLEGIA IN flFCTID MICE


SCx Treatuent

F Br

M CR + cort

M.I CR + court

M CR + court

M2 CR + court

F CR + court

II CR + court

M CR + cort +

M CR + court +
14C++ ATP

II CR + court +
McI', ATP


thiamine

thiamine,


thiarnine,


Io. da..s after injection
Flrzt appearance
of psralysio Died

153 331-

56 326

61 82

63 83

48

48 51

79 **

75 81


Lic -manu bodies
present:
Spinal
cord Brain


+


-
+


5Gtill living 124 days after injection.

**Still living 114 days after injection.


---------- ------------------ --------------


------------










73 45h days (mrn-n = 216) after injection. During thin; period no un-

injected mice of the name eges died. Chronic infections vcre found in

mice urith the following injections: (1) Br, (2) Br + court, (3)

Br + court + ATP, (4) Br + cort + thica.ine, (5) CR, (6) CR + court, (7)

CR + cort + thiamine, uith the larCest number of casos (five) occurring

in the CR + cort-injected group. Since not all of the mice used in ex-

periments could be hept indefinitely, no statement can be made conccrn-

ing the total number of deaths which might have occurred.

Upon histological examination the same organs mentioned previ-

ously were found to be parasitized. Hearts, spleens, and livers were

frequently onlarcgd. Stomachs wcre not distended rlith food as in the

acute cases although leishmen bodies were frequently present in the

tissues. Liver, lung, and urinary bladder, in about half of these cases,

contained unusually large numbers of leislman bodies.


In vitro Expcriments


1. Growth in Citri's pnrtiall.y defined medium. It has been

found that the Erazilian and Costa Rican strains of T. cruzi Goir as

well as, or better than, the Culbertson (C) strain in Citri and Gros-

sovicz's partially defined medium and that this growth is steadily main-

tained through at least eight subcultures. Counts from a representative

series of samples are presented in Table 10.

These population densities arc just slightly lower than those of

the same strains in the diphasic medium. For the Bra.ilian strain the

mean of counts from 26 4-week--old cultrros in diphasic medium was










TABLE 10

GROWTHi El THE PARTIALLY DIIfED (1DIU.I
OF CTRI ALID IGOSSOWICZ (1955a)


Organisms per ml. (x 106)
train At tine of Alter After After
inloculattion 2 wcol:z 3 vcecs :-. eehl
C 0.23 1.7 (6x) 11.0 (39.:) 19.4 (69x)

CR 0.06 5.7 (95x) 20.9 (348x) 29.6 (493x)

Br 0.03 7.9 (263x) 19.2 (640o:) 28.2 (940x)










31.6 x 10i organism per ml.; for the Costa Rican strain, the mean from

19 cultures of the same age ran 42.7 x 10 .


2. Growth in experimental media. A synthetic medium completely

defined except for ribonucleic acid, has bee devised. The Costa Rican

strain crew as vell in each of the modifications of this medium as it

did in control tubes of Citri's medium. The results of those experiments

arc summarized in Table 11.









TABLE 11

CR(OTH OF COSTA RICAN STRAIN III EXPERIM"PTAL MEDIA


Organisms per nl. (x 106)
At time of
tediumm inoculation After 5 eaL-ek

Basal medium 0.9 1.2 (1.33x)
Citri's medium 0.9 0.9 (1.00x)

Kediu 1 0.75 1.4 (1.87:)
Citri's medium 0.75 0.9 (1.20x)

Medium 2 0.75 1.3 (1.73x)
Citri' medium 0.75 1.4 (1.87x)

Media 3 0.9 1.5 (1.67x)
Citri's medium 0.9 1.75 (1.94x)








DISCUSSION


Incomplete oxidation of carbohydrates is characteristic of the

metabolism of all the trypanosomes studied to date. For example, the

pathogenic African trypanosomes break down glucose only as far as pyru-

vato. Both blood stream and culture forms of T. cruzi are able to

oxidize glucose more completely, and about half of the glucose molecule

is converted to carbon dioxide, the remainder being degraded to succinic,

acetic, and lactic acids (Ryley, 1956). Chang (1948) and von Brand et

al. (1949) both demonstrated glucose utilization by culture forms of T.

cruzi, but Glucose consumption was not shown for blood stream forms (von

Brand et al., 1949). Ryley (1956) reported that both forms use glucose

and that they do not differ markedly in their metabolism of it.

Ryley's analyses for organic acids were carried out on two to two

and one-half hour incubates in Ringer-bicarbonate-glucose medium. Chang

(1948), in analyzing 1- to 2-week-old diphasic cultures of T. cruzi,

found appreciable amounts of succinic, pyruvic, and lactic acids and

small amounts of carbon dioxide and formic acid, but no acetic acid.

Unfortunately, no metabolic studies have been carried out on tissue

forms of T. cruzi.

Pyruvate is the only one of these metabolic end-products for which

host tissues have been analyzed. Coleman and von Brand (1957) studied

blood pyruvate levels in rats infected with various trypanosomes. They

found that in all cases, whether the host was infected with T. cruzi or

with one of the trypanosomes producing nuch larger amounts of pyruvic

acid, the host animals were able to maintain normal blood pyruvate










levels. However, they made no mention in their report of how long the

host animals had been infected. In T. cruzi infections there is fre-

qucntly extensive damage to liver and A muscle tissues. It is well known

that with severe liver disorders in man there is a decreased utiliza-

tion, and consequent accumulation in the blood, of pyruvate (Parida and

Karl, 1956). Therefore, maintenance of normal pyruvate levels may very

.Sll depend upon the extent of tissue, especially liver, damage in the

host. As tissue damage progressed it is quite likely that pyruvate

levels would rise, eventually to concentrations toxic to the host.

Under the conditions of the present experiments the treatment of

T. cruzi-infected mice with thiamine or with thiamine, magnesium, nd

ATP apparently had no effect. If, as suggested above, the mice are able

to rmtabolize pyruvate satisfactorily until tissue damage becomes too ex-

tensive, it can be seen why thicmine did not affect the course of the

disease. However, if an animal were suffering from a slight pre-existent

thiamine deficiency, the increased amount of thiamine necessary to

._.tabolize the pyruvate produced during a trypanosome infection might

be enough to meke the deficiency pronounced. In such a case, thiamine

therapy might be effective, at least temporarily. It should be borne

in mind also that even if a thiamine deficiency did occur during a

trypanosome infection and were treated, it is very likely that the

disease has other effects which miGht completely mask any results of

the thiamine therapy.

Grant and Fulton (1957) found abnormally high levels of pyruvate

in the blood of rats infected with T. rhodesiense and showed a positive










correlation to exist b between the pyruvate level and the dejee of para-

citemia. Upon treating the rats with a trypanocidal drug, they noted

that parasites had disappeared from the blood 90 minutes after treat-

ment, and at 120 minutes the lcto-acid levels in these rats were almost

identical to those of control animals. Here there was apparently no ex-

tensive tissue damage. The failure to maintain normal blood pyruvate

levels in this case may have been due to a limiting mount of some co-

factor rcqaired for pyruvate metabolism. The possibility that the para-

site is in some way able to inhibit this metabolic reaction should not

be ruled out.

These ideas presented concerning pyruvate metabolism are probably

applicable also to the utilization by the host of the other organic acids

produced by T. cruzi. The host should be able to maintain normal levels

of cuccinate, lactate, and acetate until tissue damage becomes extensive.

Perhaps in early infections light pre-existing deficiencies of vitamins

or other compounds required for the oxidation of these organic acids may

be aggravated. In this case, treatment of the host with the needed factor

would prevent the accumulation of these acids to toxic levels, staving off

death from this cause and giving the animal time to fiCht off, or adjust

to, the infection. Such deficiencies miCht also occur in chronic cases

of trypanosomiasis. Deficiencies of thiamine, a-lipoic acid, pantothenic

acid, riboflavin, and nicotinamide are among those which might be ex-

pected to occur.

The one factor c oon to the utilization of all these metabolic

end-products of the trypancsome is ATP or ADP and iP. As pointed out










carlier, the rate of cbstra.te dc'raddation is dependent upon the rate

of o::idativc phosphorylation, which is in turn controlled by the rate

at .rhic AiTP is split to ADP and iP.

The results of the present experim~L ts confirm Ada-ms' (1954-) find-

ing that ATP treatmCat reduces the mortality of mice infected lrith the

Brazilian strain of T. cruzi. Failure of the treatment to have the sei

effect on mice infected with the Costa Ricea strain may be attributed

to: (1) the smaller number of experimental animals involved, (2) the

greater variability of the Costa Rican strain; or (3) a real difference

in reaction to the trcatmont btreecn the two strains.

Tlc finding that 2.5 r,. of A TP reduce the mortality of infected

nice ~lile 5.0 mS. increase it was unexpected. It was the latter amount

liichl Admns used in his experiments. The larger amount of ATP was not in

itself toxic, for whcn Given to cortisone-injected mice it van not fatal.

Only ihen the rice had previously been injected with trypanosomes did the

5.0 mr. dosage have an adverse effect. I ha no explanation for the re-

action to this dosage difference, if indeed it is a real thing. It is in-

teresting, haoever, to imrnine that the host may be able to claim for its

own use certain amounts of ATP, but that above a certain level the ATP

is not irrsdiately utilized and becomes available to the trypanosomes.

The manner in which ATP acts to reduce the mortality of trypano-

some-infected mice remains hypothetical. Adams (195)) suggested that it

was used in coRbating a hypoglycemia produced in the host by the trypano-

somes. Since it has been sh .on that animals infected with T. crusi

usually maintain normal blood sucGr levels (von Brand et el., 1949),










thi i~oulI not seen to be the c.planalition. The fact that the ATP Ias

administered only once, very early, in the infection, precludes the

possibility that its enerjr ras available for repair of dama-ed tissues.

It is possible however 'hlat it could be used for synthesis in the produc-

tion of antibodies or pha ocytic cells. The ATP injections rcre adminis-

tered at about the time that the metacyclic trypanosomes injected are

becinn:ing to change to leishman forms (Pere:-Rcyes, 1953), the forms

which reproduce wirthi the vertebrate host. It seems more likely, how-

cver, that the ATP becomes involved directly in the metabolism of

rouse or the trypanosome or both. It may very well be used to provide

phosphate and/or phosphate acceptors neccssry for the oxidation of the

nmtabolic end-products of the trypanosomos, Giving the host tine to ad-

just its metabolic pattern to acco. nodate this new situation.

Surprisingly, hardly any studies have been made of the effect of

ATP administration on intact animals. From the few experiments per-

formed it has been learned that there is a direct transfer of phosphate

groups from ATP to thiamine and incorporation of the resultant di- and

triphosrhothiamines into the liver within 1 hour of injecting rats rith

ATP32 and thia-ine (Rossi-Fanolli et al., 1954). Hascitelli-Coriandoli

and Boldrini (1957) found th.t the injection of ATP raises the levels of

ATP-phosphorus, creatine phosphate-phosphioruc, end inorganic phosphorus

to almost normal levels in the heart muscle of rats treated vith thyrox-

inc. Until more is IIown concerning the fate of injected ATP in animals,

and until the nutritional requir!emnts of T. cruzi and the effect of

this parasite upon its host are better understood, no final c=planation










for the effect of ATP on tr'pio l.zis cona be given.

The treatrmant of trypar.nosonc-infectcI uic. with UAP and iP did not

reduce uc e at ortclity rat:e. In view. of the sail number of niec used in

this set of c::pri ments, thisec results cannot be considered to be con-

clusive. With a lage enoui sample, the results of this treat-xct

should be siilr to those obtainid with ATP injection.

The high degree of adaptation bcticen T. cruzi and its host is well

c naplified in the very close similaiaity of growth rates of infected and

uninfected juice. Further evidence of this adaptation is found in the

relatively larc number of infected mice which survived the acute infec-

tion end continued to live rith chronic infections. At least a partial

eplanation for this host-parasite relationship lies in the very nice

nLtabolic exchange between the two. The parasite takes glucoce from the

Iiost, but does not completely nctobolize it, returning at least half of

the moleculc to the host in the form of organic acids uhich the host is

then able to utilize. In this waj both organisCi obtain encercv from the

substrate.

The occurrence of chronic trypanosomiasis in laboratory ;ii'ials

is rarely mentioned in the literature. Johnson (1933) reported that of

19 dos c::perincntally infected with T. crusi, 9 of then developed acute

infections and 11, chronic infections. Coble (1951) und Haucschia (1947)

licce both noted the occurrence of the chronic disease in nice. Since

clhonic trypaosermisis is the moDt frequent form encountered in humans,

it is suggested that nice writh clironic infections should lend themselves










':cll to ezpcrim2ntcal .;'orl concerned itlh the cfiect' of the chronic

disoasc on the host an1 '.with therapy of the disease.

Paraplegia in nice infected with T. cruzi Las been reported, to rLy

1:ncr iZlea, only once before. Do Souse C,.j-os (1925) state that .- of 12

infected nice shzowd parcaplcgia. Thi condition is fairly ccnan. xa in in-

fected dogs (Goble, 1952; Villela, 192n) cnd rabbit.o (do Sousa Corpo;,

1724). De Sousa Cmipos (1925) reported that lesians of the central

nervous system wcre not obscrvlcd in all casos of paraplegia and s gCrcted

that this might depend on the duration of teo infection. The aflaocllated

forao were found in the central nervous system of all tie other para-

plegic cases rcported, although they were never numerous. Inflamiatory

centers formed by infiltration of macrophages .'cre encountercl more fre-

quently during microscopic c aination of brain and spinal cord sections

then iore lciscLG -n bodies. Goble (1952) found Uthat dogs may die within

a few days of becoming paraplegic or may continue to live for scme tine

with varying dcreo of wrcaLness and paralysis. The neurotropic form of

tryprnosomiasis appears to be very similar in dogs land in the mice ob-

sr7red in the present c.prcrimnts. Goble, however, easo reported the

recovery of one paraplegic dog. In view of those reports where leish-

ian bodies could not be demonstrated in the central nervous system and

where there was a complete recovery from paraplegia, paralysis in try-

panoscme-infected mice should not be haphazardly attributed to the

direct effect of the trypanosomc. It might be pointed out that a phos-

phorus deficiency will produce paralysis of the hind legs (Footer ct al.,

1949) in mice. A thiamine deficiency also affects the lces of nice,










-.i'ini':; them so v ncx: thntt thly ',e un:i'le to support t'ih body (Uooll.y:

and M2hite. 1943). Thierfore, it is quite possible that paraplecia in

trypraosa-n-infeetod ice szou.ld not be wholly attributed to a direct

effect of tha pcrasitcs in the central nervous system.

It is no- ::n.ro that the partially defined synthetic nodiu-i of

Citri and Grosrovics (1955a) will support grourth of three strains of

T. crtzi, .s orhwn by the rcesTlto presented earlier in this paper, and

31 different strains of Loishme.nia tropica (Citri and Grossowicz, 1955b).

The latter muthors foundr that the medium did not support gro rth of L.

infentui. L. donovan-. L. braciliensis, ran- L. mae, hou'ver. This

cstuesztz that the Growth requireC-cnts of L. tropical and T. cr--i ere

norc i:211ar than are those of L. tropical cnd the other leis'- .mias of



D1uin1 the tine that gro.wt Y- :oriments in redia containing !mo.-.m

\nino acidc w:cr bein3 conducted, the cultures in this laboratory ~;or

undcrgoinu, one of the une::plained Grcwth depressions ~Iich occasionaCly7

affect tr;'paioso o cultures (Balamuth; 1957). It is for thick reason

that the popu.ltions attained in the excprimental media cn: in the Citri'3

mediun controls were rchi lo-1.r thaen V :ould be extpcto1.. 'Th relative

c-o unt of Crowth obtained in the enGermantal medin as conparcd to that

in the control medii appccars to be soiCificcnt, vnd there is no reason

to believe thcV this rill chCen.ce under improved CroI.th conditions. No

differences in rocrth supporting ability Lerre observed cmoen the dif-

ferent e:pcrirmnntal modia. Future e:;periicnts ere plaUmed in ihich




5C





prince and pyrinidincs or nuclcotidcs irill be substitute for the

ribonuclcic acid in the icdia now bcing usec. If this substitution is

accomrplished, the nodium for T. cruzi will be completely defined.










SUMMARY AID CONCLUSIONS


Injection with adenosine triphosphate reduced the mortality of

mice infected with Trypanosoma cruzi. Treatment of infected mice with

thiamine or with thiamine, magnesium, and adenosine triphosphate appar-

ently had no effect on the course of the disease. With a small number

of mice, adenosine diphosphate and inorganic phosphate appeared to pro-

duce no effect on the infection.

Chronic trypanosomiasis resulted frequently when mice were injected

with trypanosomeo and cortisone. Crowth rates of normal, uninfected mice

wrre not siGnificantly different from those of mice with the chronic

disease.

Leishman bodies were observed in heart, spleen, liver, lung, and

other orGans commonly parasitized by T. cruzi. In addition, the aflagel-

lated forms were found in stomach tissues of about one-half of all mice

dying of acute trypanosomiasis, confirming a single previous report of

stomach infections.

Ten infected mice developed paraplegia. LeishmGan bodies were

present in the central nervous systems of these animals.

The partially defined medium of Citri and Grossowicz (1955a) was

found to support growth of the Brazilian and Costa Rican strains of T.

cruzi as well as it did that of the Culbertson strain for which it was

devised. Modifications of the medium rere made, and ribonucleic acid is

now the only undefined component. A known mixture of amino acids com-

pletely replaced the protein source. Growth of T. cruzi (Costa Rican

strain) was as good in amino acid-substituted media as in the original

medium.









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BIOGRAP I.y


Jacqueline Adam. was born in Ashland, Kentucky, on June 3, 1930.

In 1952 she married Dennis Hynos. She received the Bachelor of Arts

Decree in June, 1952, from MIacalester College, St. PIal, Minnooota, and

the Icastcr of Science Dcgree from the University of IlichiCan in Febru-

ary, 1954. From 1954 to 1956 she iras employed as Assistant in Research

(Instructor) in the Department of Civil Engineering, University of

Florida. She rcCumn-d her graducto studies at the University of Florida

in 195' and while thero held a crEduate acsistentship, tie Dudley

Beaumont Menorial Followmhip, and a research assistantsnip.










Sis dissertation vas prepared under the direction of the

chairman of the camidldato's supervisory committee and has been ap-

proved by all menmbes of that comnittcc. It was submitted to the

Dean of the Collse of Arts and Sciences and to the Graduate Council,

a'd vac approved as partial fulfi.l ent of the requiraem ~ts for the

doercc of Doctor of Phlilosophy.



A--ust 9, 1958 D3'______
Dean, ColloGe of Art eand Sciences



Dean, Graduate School



SUPaTVIS CY COMMITTEE:




^Cama./r




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