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Quantitative studies on heterologous sera inducing development of Trypanosoma lewisi in mice
PARASITOLOGY 11, 68-76
EXPERIMENTAL
(1961)
Quantitative Studies on Heterologous Sera Inducing Development of Trypanosoma Zewisi in Mice1s2-R David Richard Lincicome and Earlie Hill Francis Department
Laboratory of Parasitology, of Zoology, Howard University,
(Submitted
THE The with
use
of
homologous
an
for publication,
host
tissue
host
supplIecl
fractions
10 December
D.C.
1960)
an heterologous mouse environment. This in turn has provided a quantitative basis upon which to study comparatively other sera. The present report presents: 1) the experimental work establishing the daily minimal quantities of rat serum necessary to maintain Trypanosoma lewisi in the mouse; and 2) a comparative quantitative study of several sera for their ability to promote the growth of the rat trypanosome in the mouse. A preliminary account of this work has been presented before the 35th meeting of the American Society of Parasitologists (Lincicome and Francis, 1960).
PROBLEM
heterologous
Washington,
has
been advanced as an experimental tool for the study of the molecular biology of parasitism (Lincicome, 1958). Evidence collected by use of the system (the heterologous mouse plus the rat trypanosome, Trypanosoma lewisi) has indicated that the parasitic cell requires a substance in the serum of its homologous host (Lincicome 1955, 1957, 1958). The use of the identical test system on a serial basis over a period of several years has further shown that the parasitic cell undergoes no fundamental change in its chemical requirements of sufficient magnitude either to prevent it from developing in its normal, homologous environment (Lincicome, 1959 a and b), or to permit its growth without serum in the heterologous host.
SUMMARY
OF
RESULTS
The minimal daily volume of normal rat serum that will support the development of Trypanosonza lewisi in the heterologous mouse host was established between 0.05 and 0.01 ml. Since a volume basis appeared not wholly desirable due to variations of serum components, the minimal daily values of rat serum were also determined in terms of volumes adjusted on the basis of protein concentration assuming a parallel relationship between it and that of the trypanosome growth factor. Using this criterion the minimal values were those volumes having between 2.5 and 2.0 mg of protein equivalents. Using these minimal values the sera of the horse, man, rabbit, guinea pig, hamster, chicken, chinchilla, pig, dog, and cow were compared qualitatively and quantitatively with rat serum. Only sera of the hamster, guinea pig and rabbit had trypanosome growth promoting properties comparable to those of rat serum. With rat serum as the
It is of interest, therefore, to determine the minimal daily requirement of homologous serum that will support the trypanosome in 1 The organization of this paper is experimental. It was deliberately designed to make the results of the investigation more readily accessible to the reader by putting the relatively lesser important supporting information in its rightful place. The senior author invites readers’ comments, favorable or unfavorable, criticisms, or other ideas. 2 Part of this work was done while the senior author was a Guest Scientist in the Division of Parasitology, Naval Medical Research Institute, Bethesda, Maryland. Appreciation is expressed to the Commanding Office of the Institute for the privileges extended during this work. a Supported in part by a research grant to the senior author from the National Institutes of Health. 68
SERA
INDUCING
DEVELOPMENT
standard it was determined that hamster serum was potentially 78% as effective, guinea pig 53F and rabbit 42%. SIGNIFICANCE
The work reported here has shown that small volumes of rat serum daily are sufficient for the development of Trypanosoma lewisi in the heterologous mouse host. The titrations using a volume basis indicated that the endpoint lay between 0.05 ml and 0.01 ml. A volumetric basis for measuring quantitatively the effectiveness of homologous serum in maintaining this trypanosome in its heterologous environment did not appear to be wholly satisfactory. In view of the well known variations of serum components from animal to animal and even in the same animal from time to time, it was considered necessary to secure a basis of comparison less subject to variations. In the absence of specific information concerning the identity of the factor or factors present in rat serum that are responsible for the growth of T. lewisi in the mouse it was impossible to select the proper criterion for measurement of serum. Total protein content was, therefore, selected as the basis and, assuming a parallel relationship between these unknown trypanosome growth factors and serum proteins, further titrations were made employing volumes of serum containing predetermined protein concentrations which are hereinafter referred to as protein equivalents. The endpoint of these titrations was between 2.5 and 2.0 mg of protein equivalents. The trypanosome failed to survive and grow in the mouse when a 2.0 mg protein equivalent volume of rat serum was employed daily, but good growth was obtained at 2.5 mg. No further testing between 2.5 and 2.0 mg was done. The small quantity of serum required for this phenomenon suggests that the active factor might be protein in nature. This is interesting speculation, and offers a promising basis for further examination of rat serum chemically to determine the exact nature of this factor. We have tentatively assumed that the responsible substance might be protein in nature, and are now engaged in pre-
OF
TRYPANOSOMES
IN
MICE
69
liminary chemical characterization of the fraction by dialysis, metal recombination, and preparative fractionation electrophoretically (Evans, 1959). The establishment of 2.5 mg of protein equivalents as the minimum with which good populations of T. lewisi would develop, provided a method for testing quantitatively other sera that had been shown qualitatively to be effective in supporting the trypanosome (hamster, guinea pig, and rabbit sera) in the mouse. Titrations of these have shown that although hamster, guinea pig and rabbit sera possess the factor or factors that make it possible for the trypanosome to grow in the mouse, they do not have equally effective concentrations as rat serum. Calculation of effective indices for hamster, guinea pig, and rabbit sera based upon maximal parasitemias has shown that hamster serum has a potential only three-quarters that of rat serum. Guinea pig serum is only about half and rabbit only 40% as effective as the homologous rat. Assigning rat serum a potential value of one, it is clear that volume for volume hamster, guinea pig and rabbit sera are considerably inferior quantitatively, and that theoretically comparable trypanosome population responses might be obtained with them were the volumes of these heterologous sera increased proportionately. This, though interesting, has not been done. Human, chicken, cow, dog, swine, chinchilla, and horse sera have been shown to be inactive with respect to factors that support the development of T. lewisi in an heterologous environment. Cow, swine, and chicken sera had marked aggulutinating antibodies that clumped trypanosomes and mouse red blood cells. These sera were heat-inactivated and tested again for their ability to promote the growth of the trypanosome without success. The promotion of growth of Trypanosoma lewisi by rat, hamster, guinea pig, and hamster sera raises the question of the specificity of the phenomenon. The possession of the property by all four sera does not support the view earlier held that the factor was specific for rat serum. Were this absolute it would provide interesting possibilities of ex-
70
LINCICOME
plaining
AND
the development of the uniqueness
of the association between a parasitic cell and its sheltering host on a chemical basis. Whatever the nature of this property it is clear from the data presented in this paper that it is shared by four potential host animals. There is little evidence in the literature that T. lewisi can grow in any host except the rat. French workers (see Lincicome, 1958, for references) have reported survival of the
trypanosome in mice, hamsters and other rodents after injection of large numbers of parasites in significant amounts of homologous blood. Coventry (1929) reported that T. Zewisi will exist for a limited period in the guinea pig and Laveran and Roudsky (1914) had a similar but more limited experience. In view of the sharing of this growth promoting factor by rat, hamster, guinea pig and rabbit sera, it is not surprising that this trypanosome is able to become established on a limited basis in guinea pigs and rabbits. The final chemical identification of the substance
or substances
may well show that
these are not absolutely identical in all these animals
even though
they
possess a degree
of common action. Speculatively, these factors may all share a common cyclical protein base that differs among them in the possession of side chains or specific groups that metabolically antagonize the common base, thus accounting for the varying potentials that have
been noted in this paper. Though we have shown here that the earlier view of the specific nature of this phenomenon can not now obtain, it may be of greater theoretical interest biologically that the sera of the four animals share a similarly
active
factor
that
enables
investigation
have
therefore
broad
implications in establishing the ground work for understanding the ultimate molecular host relationships of a foreign but relatively autonomous
cell
as it
establishes
itself
in
its tissue environment. RESULTS
Titration titrations
of
rat serum. Four preliminary
of rat serum
employing
0.25 and 0.1 ml quantities indicated:
1.0, 0.5,
of normal
serum
1) that the serum was active in supporting the growth of Trypanosoma lewisi in the heterologous mouse host at the concentrations used. 2) that heavier mice did not support the trypanosomes as well as younger mice. Tables I and II present data from two of these experiments. The results of four quantitative assays of TABLE
Populations
I
of Trypanosoma lewisi Developing in 2.5 g C3H mice
Average hemacytometer count of trypanosome$ Normal rat serum (ml) Days after inoculation
1
0.5
0.1
(3)b
(3)
(3)
3 5 7
10 107 331
4 4 12
+ -d
8
+
+
-
+"
0 All figures rounded to nearest whole integer. b Indicates number of hosts employed. c Indicates trypanosomes were present but not in sufficient quantities to be estimated in the hemacytometer. d Indicates no trypanosomes were found. TABLE
II
Populations of Trypanosoma lewisi Developing in C3H Mice Weiahina 15~ or Less
Average hemacytometer count of trypanosomes
a para-
sitic cell to establish itself in a sheltering host. The work, technics, and results of the present
FRANCIS
Days after inoculation 3 4 6 8 10 12
Normal rat serum (ml) 1 0.5 0.1 (3)”
(3)
(3)
+”
+
+
7 206 499 1069
6 123 584 807
2 27 132 277
Dead
Dead
Dead
5 Indicates number of hosts used. b Indicates insufficient numbers to be recorded in hemacytometer.
SERA
INDUCING
DEVELOPMENT
rat serum employing lo-15 g mice are given in Tables III and IV. The endpoint of these titrations lies between 0.05 and 0.01 ml volumes. The titrations of normal rat serum in terms of protein equivalents are summarized in Table V. The endpoint of these tests lies between 2.5 and 2 mg of protein equivalents. Hamster serum assay: A preliminary experiment testing hamster serum employing one milliliter aliquots of sera showed that hamster serum was effective in promoting the growth of the parasite. Since the parasitemias were expressed on an arbitrary scale it was not possible to distinguish subtle difTABLE III Populations of Trypanosoma lewisi Developing in lo-15g Mice Supplemented with 1.0, 0.1, and 0.01 ml of Normal Rat Serum Average hemacytometer of trypanosomes Normal
3 4 5 6 7 8
a Indicates
Average hemacytometer of trypanosomes Normal 0.01 1
Exp. Ko. 2
1
2
4 7 11 17
2 75 33 9.5
3
2 9-15 19
-(I
-
-
-
16 15
94 -
8
28 _
_
-_
5 9
8
no trypanosomes
1
Days after inoculation
2
found.
rat serum (ml) 0.01
Exp. No. 3
43
2
3 4 5 6
32 34
101
7
-
98
4
19 18 78
7
3 6 11 8
7 7 12
-
10
(* Indicates no trypanosomes
Average hemacytometer
count
0.05
TABLE V of Trypanosoma lewisi Developing in Mice Supplemented Quantities of Normal Rat Serum Protein Equivalents
Populations
71
MICE
TABLE IV Populations oj Trypanosoma lewisi Developing in Mice Supplemented With Varying Volumes of Normal Rat Serum
count
0.1
1
IN
ferences between the population responses afforded by the hamster serum and those by the control rat serum. The parasitemia developing from the hamster serum was heavy and continued maximally (4+) for 5 days. The overall parasitemic period was 11 days. The control animals showed 4+ parasitemias for 5 days but the whole parasitemic period lasted only 9 days. Experiments 8, 9 and 10, testing hamster serum quantitatively with protein equivalents as the basis for comparison, are summarized in Tables VI and VII. The volume of serum containing 50 mg of protein equivalents used in experiment 9 (Table VI) approximated the 1.0 ml volumes
rat serum (ml)
1
Days after inoculation
OF TRYPANOSOMES
3
3
4
-a
-
-
-
-
-
-
-
found.
With Variable
count of trypanosomes
Normal rat serum (mg of protein equivalents) Days after inoculation 3 4 5 6
7 8
(1 Indicates
50 5 8 62 58 75 88 87
2.5
6
7
5
-a
-
4 39 49 70 78
6 7 22 38 43
3 9 15 15 18 12
no trypanosomes
7
6
were found.
0.5
2.0
4 8 8 10 8
Exp. No. 6
7
5
5
6
0.1
7
5
6
-
-
-
-
-
-
-
-
-
7
72
LINCICOME
AND
of the maximum parasitemia observed for hamster serum divided by that observed with control sera, it is apparent that hamster serum is only half as effective as rat serum (Table VI). If the efficiency ratio is calculated on a day to day basis (Table VI) there is a variable pattern of efficiency. In the initial phases of the development of the parasitemia the index is high but falls rapidly to essentially the level of the overall index calculated above. Comparison of similar indices calculated from parasitemias observed when hamster serum is titrated (Table VII) reveals a daily pattern that is not wholly unlike that observed with 50 mg equivalents (Table VI). The chief difference lies in the maximum parasitemia effective indices which were essentially identical for both experiments 9 and 10 (Table VII). It is possible, therefore, that hamster serum contains about 75 to SOc/r of the growth-promoting properties of homologous rat serum. Volumes of serum beyond the minimal effective quantity are not necessarily associated with linear growth patterns of the trypanosomes. Assay of cow, swine, chicken, dog, horse,
that have been frequently employed in this and other work. If the efficiency of this quantity of the two sera is calculated as the ratio TABLE VI Populations of Trypanosoma lewisi Developing in lo-15g Mice Supplemented With Rat and Hamster Sera (Experiment 8) Average hcmacytometer count
Days after inoculation
50 mg protein equivalents
3 4 5 6 7 S
6 13 28 68 86 99
Effective
Index =
trypanosome
Normal hamster serum
Normal rat serum
-
SOmg protein equivalents
Efficiency relative to control (%)
6 11 15 38 53 4.5
100 85 54 56 62 4s
Max. Parasit. of Exp. Max. Parasit. of Cont. =-Ix
53
54%
99
Populations
FRANCIS
TABLE VII of Trypanosoma lewisi Developing in lo-15g Mice Supplemented Varying Quantities of Rat and Hamster Sera Using Protein Equivalents as a Basis of Measurenzrnt Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.5 2.0 Days after inoculation
2 5 9 11
10
9
10
2 5 ‘3 10 11
10
-u --
-
-
-
Index = 9
for Exp.
-
9(Q) 8(80)
9 = 82% Av. =
for Exp. 10 =
785%
73%
11
R Indicates that no trypanosomes were found. 7~Efficiency relative to Rat Serum for corresponding
day in 70.
9
10
--(O) f/
3(60) 8(100) 5W) X(73)
4(50)
11
Index = 5
9 l(50) 3(60) 6(67)
8
8
Effective
count
Normal hamster serum (mg of protein equivalents) 2.5 2.0 Exp. No.
9
3 4 S 6 7
Effective
With
-
10
-
SERA INDUCING
DEVELOPMENT
man and chinchilla sera. In none of the experiments utilizing any of these sera was there development of the trypanosome. Volumes of human and chinchilla serum were insufficient to replicate observations. COW. chicken, and swine sera frequently were associated with in vitro agglutination of both trypanosomes and mouse erythrocytes. Heat inactivation did not alter this property. Guinea pig serum assay. The two preliminary assays based on volumes of guinea pig serum showed that this serum was potentially as effective as homologous rat serum in supporting the growth of the trypanosomes. Since these tests were exploratory, two additional protocols were developed (Table VIII). Here, the effective index of guinea pig serum based upon the maximum parasitemias was 5354 (average of both experiments) indicating that whatever the nature of the growth promoting properties present in this serum, the quality is only about half that found in rat serum. The efficiency of guinea pig serum on a day to day basis fell from an initial high in a pattern similar to that observed for hamster serum. Rabbit serum assay. Although there was little evidence that rabbit serum was effective in supporting the development of Trypano-
OF TRYPANOSOMES
73
IN MICE
soma lewisi in the heterologous mouse in a preliminary trial, subsequent work showed that rabbit serum would support the parasite. Table IX presents data from a test of rabbit serum in young mice. TECHNICAL
PROCEDURES, PROTOCOLS AND REFERENCES
Procedures Thirty (30) experiments were completed in which 397 mice were used.” Of these, 373 were female albino ranging in weight from lo-26 g. The others were male C3H of the same weight range. All protocols employed a strain of Trypanosoma lewisi that had been maintained in the laboratory by serial blood passages in albino rats for more than 10 years. Sera of man, hamster, guinea pig, rabbit, chicken, swine, cow, dog, horse, rat and chinchilla were studied. Those of the hamster, guinea pig, rabbit, rat and dog were collected by cardiac puncture. Bloods were allowed to clot under refrigeration (2-4” C) for a variable time, and the sera removed by 4 .4nimals were obtained laboratory of the National Bethesda, Md.
through Institutes
the animal of Health,
TABLE VIII Populations of Trypanosoma Iewisi Developing in lo-15g Mice Supplemented With Varying Quantities of Rat and Guinea Pig Sera Using Protein EquivaJents as a Basis of Measurement Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.5 2.0 Days after inoculation
Exp. No.
3 4 5 6 7 8
11
12
11
12
3 5 8 9 9 6
1 5 9 10 10 7
-0 --
-
11 2(67)b 3(60) j(63) j(56) j(S6) 5(33)
-
(r Indicates no trypanosomes were found. f~ Efficiency Index in percent relative to control of corresponding Effective
count
Normal guinea pig serum (mg of protein equivalents) 2.5 2.0 12 l(100) 4(80) j(56) S(50) 5(50) S(71)
day.
Max. Parasitemia
of Exp.
= +
= 56% for Exp. 11
Max. Parasitcmia
of Control
= $
= 50% for Exp. 12
Index =
11
12
-
-
-
74
LINCICOME
AND
FRANCIS
TABLE IX Populations of Trypanosoma lewisi Developing in IO-15g Mice Supplemented With Varying Quantities of Rat and Rabbit Serum Using Protein Equivalents as a Basis of Measurement Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.0 2.5
Normal rabbit serum (mg of protein equivalents) Exp. No.
13
14
13
14
13
14
13
14
-
-
-
-
-
2(50) l(14)
-
-
4(44) 4(40) 2(33)
-
-
3 4
1
3
-b
4
4
-
-
l(2S)C
5 6
7 7
7 9
-
-
l(14)
7 8
5 3
10 6
-
-
3 (60) l(33)
0 Effective
2.0
2.5
Days after inoculation
count”
2(28)
Index = t
= 43% for Exp. 13 and 4 = 40% for Exp. 14. Av. Index = 42%. 10 b Indicates no trypanosomes were found. c Efficiency in perce.lt relative to control of corresponding day.
centrifugatinn. Human serum was obtained from the ch / -al ser;-ices of the Naval Hospital at the P ational Naval Medical Center, Bethesda, Ma-yland. The sera of the chicken (antibiotic fr :e) and chinchilla were gifts.” All other ser, were purchased commercially. Inactivation of sera was carried out in a water bath at 60” C for one hour to destroy natural agglutiriating antibodies for Trypanosoma lewisi. All sera were inoculated intraperitoneally in amounts varying in the several protocols used. Details may be found in the results section in appropriate context. Experimental
infections
of
Trypanosoma
lewisi in control and test mice were initiated by intraperitoneal inoculations of physiologic saline suspensions of blood drawn either from the tail or heart of donor animals with Trypanosoma lewisi not more than 7 days previously (to minimize the carry-over of antibodies from donor to experimental animals). A few crystals of sodium citrate were always 5 Chicken serum was given by Dr. Virginia L. Blackford, then of the Naval Medical Research Institute, Bethesda, Maryland; the chinchilla serum was collected and given by Dr. Leslie A. Stauber of the Department of Zoology, Rutgers University, New Brunswick, New Jersey.
added to avoid clotting of the suspension. The trypanosomes were distributed throughout the blood-saline suspension by repeated drawing into and expelling the suspension from a 10 ml syringe over a period of 60 seconds at the beginning of a series of inoculations. Between withdrawals of the suspension for inoculation of individual animals the homogeneity of the trypanosome distribution was maintained by a single drawing and expelling from the 10 ml syringe. Numbers of trypanosomes inoculated varied from 3-7 x 106. A red blood cell pipette (Thoma) and an hemacytometer” with Toisson’s fluid7 were used for quantitative estimation of the populations of trypanosomes in peripheral tail bloods of experimental hosts and in bloodsaline suspensions used for initiating infections. All quantitative estimations of parasite populations in tailbloods were in duplicate daily or on alternate days beginning usually the third day after inoculation. The data are all expressed in the tables as averaged counts a Spencer Bright Line, pany, Rochester, N.Y.
American
Optical
Com-
7 Toisson’s Fluid: lg NaCl; 8g Na,SO, ; 30 ml glycerine; lo-25 mg crystal Violet; 160 ml of dist. HOH.
SERA
INDUCING
DEVELOPMENT
OF TRYPANOSOMES
IN
MICE
75
trol animals each received a daily inoculum of 1.0 ml of normal rat serum. Twelve ( 12) experimental animals each received a daily inoculum of 0.1 ml of normal rat serum; a further series of twelve (12) each received 0.01 ml. 2. Experiments 3 and 4. Twelve ( 12) control mice each received a daily supplement of 1.0 ml of normal rat serum. Twelve ( 12 ) experimental mice received a daily inoculation of 0.05 ml of normal rat serum. Twelve ( 12) others each received 0.01 ml of normal rat serum. The next studies were conducted with rat sera whose total serum protein contents had been assayed. The volumetric basis of titration adopted in experiments 1 and 2 was abandoned and the quantity of serum to be inoculated was expressed in terms of protein equivalents as follows. The total protein content was established at 5.0 g/100 ml. Weights of the mice ranged from lo-15 g. Experiment 5. Six (6) con’ A mice received each a volume normal rat serum having a protein Jquivalent of 50 mg. Six (6) mice rev-eived 2.5 mg and six (6) others each rt -eived 0.5 mg. Experiment 6. Six (6) r’ ;rol mice each received a daily inoculation of normal rat serum containing 50 mg of protein equivalents. Of twelve (12) experimental mice half received 2 mg each and half received 1 mg each of protein equivalents. Experiment 7. Six (6) control mice were each given a daily supplement of 50 Experimental Protocols mg of protein equivalents of normal rat serum of 12 experimental mice half reTitration of rat serum. Four preliminary experiments were performed in which the ceived each 2.5 mg and half 2 mg of populations of trypanosomes were studied in equivalents. 15 to 25-g mice supplemented with daily Hamster serum assay. One preliminary volumes of rat serum ranging from 1.0 ml to test of hamster serum employed 1.0 ml daily 0.05 ml. From these experiments it was de- volumes in mice. Parasitemias were followed cided to titrate rat serum in mice weighing daily until the 12th post inoculation day. 10 to 15 g. The following protocols were thereThree quantitative protocols were used as fore adopted: follows : 1. Experiments 1 and 2. Twelve ( 12) con1. Experiment 8. Six control mice (6) each received a daily inoculum of normal 8 Purina Laboratory Chow, Purina-Ralston Co., rat serum containing 50 mg of protein St. Louis, Missouri. equivalents. Twelve ( 12) experimental 9 Bausch and Lombs Spectronic 20, Rochester, N.Y. mice each received daily a quantity of
p i hemacytometer square. To convert these &;ures to numbers of parasites per cu mm ol blood a factor of 2000 should be applied. In some experiments trypanosome populations in tailbloods were expressed on an arbitrary scale of one to four plus previously defined (Lincicome, 1958) as representing a range of a minimum to a maximum number. Food was withheld from all mice for the first twenty-four hours of each experiment. Thereafter all animals received one gram/day. In some early experiments starvation preceded the initiation of the experiment. The food was a commercially available, balanced laboratory chowR that had been ground in a mechanical mortar. The titration of the quality of serum in promoting the growth of Trypanosoma Eewisi was initially on a volumetric basis. In view of the quantitative differences in serum components from animal to animal and from time to time it appeared desirable to standardize the quantity of serum used on the basis of protein equivalents. Total protein determina.tions were not made on those sera that possessed naturally occurring antibodies to the trypanosomes since preliminary observations had indicated that regardless of the volume employed these sera would not support the development of the trypanosomes. Total serum protein determinations were made by the biuret reaction. Optical density was measured with an electric calorimeter” employing the method of Kingsley (1939) and Gornall et al. (1949).
76
LINCICOME
hamster sera having 50 mg protein equivalents. 2. Experiments 9 and 10. Half of the control group of twelve animals received each a daily rat serum supplement having 2.5 mg of protein equivalents and the other half 2.0 mg per animal. Half of the mice in an experimental group of 24 animals received each 2.5 mg of hamster serum protein equivalents while the other half received only 2.0 mg per animal. Assay of cow, swine, chicken, dog, horse, man and chinchilla sera. A series of tests employing 1.0 ml volumes of rat serum and the sera under assay was completed. From three to six animals were used in both control (receiving rat serum) and experimental groups. No quantitative tests were done since there was no evidence of any activity supporting the growth of trypanosomes. The sera of the cow, chicken, and swine were heatinactivated and tested along with fresh, uninactivated sera. Guinea pig serum assay. Two preliminary volumetric tests using 14 mice gave evidence of growth-promoting properties for the trypanosomes. Two quantitative assays were then designed as follows: Experiments 11 and 12. Sixteen (16) control mice received each a daily inoculum of normal rat serum: half received 2.5 g of protein equivalents per animal, and half, 2.0 mg each. Sixteen ( 16) experimental mice each received a daily inoculum of normal guinea pig serum: half got 2.5 mg of protein equivalents and half 2.0 mg per animal. Rabbit serum assay. Two quantitative assays of rabbit serum were done, although one preliminary test using a dozen animals indicated little or none of the activity sought. Experiments 13 and 14. Sixteen (16) control mice received each a daily inoculum of normal rat serum: half of this group
AND
FRANCIS
received 2.5 mg of protein equivalent; half, 2.0 mg of equivalents. Sixteen (16) experimental mice received a quantity of fresh rabbit serum comparable to the control groups : half received 2.5 mg of protein equivalents; half received but 2.0 mg. REFERENCES COVENTRY, F. A. 1929. Experimental infections with Trypanosoma lewisi in the guinea pig. American Journal of Hygiene 6, 247-259. EVANS, A. S. 1960. Immunophysical methods in parasitic infections: A continuous electrophoresis apparatus for preparative fractionation of protein systems. Experimental Parasitology 6, 105112.
GORNALL, A. G., BARDSWILL, 0. J., AND DAVID, M. 1949. Determination of serum protein by means of the biuret reaction. Journal of Biological Chemistry 177, 751-766. KINGSLEY, G. B. 1939. The determination of serum total protein by the biuret reaction. Journal of Biological Chemistry 131, 197-200. LAVERAN, A., AND ROUDSKY, D. 1914. Contribution a l’etude de la virulence du Trypanosoma lewisi et du Tr. duttoni pour quelques especes animales. Bulletin de la sock%! pathologique exotique 7, 528-535. LINCICOME, D. R. 1955. Growth factor in normal rat serum for Trypanosoma lewisi. Journal of Parasitology 41, supplement, 1.5. LINCICOME, D. R. 1957. Growth of Trypanosoma lewisi in the heterologous mouse host. American Journal of Tropical Medicine and Hygiene 6, 392. LINCICOME, D. R. 1958. Growth of Trypanosoma lewisi in the heterologous mouse host. Experimental Parasitology 7, 1-13. LINCICOME, D. R. 1959a. Observations on changes in Trypanosoma lewisi after growth in calorically restricted and in normal mice. Annals of Tropical Medicine and Parasitology 53, 274-287. LINCICOME, D. R. 1950b. Serial passage of Trypanosoma Zewisi in the heterologous mouse host. I. calorically-restricted hosts. Development in Journal of Protozoology 6, 310-315. LINCICOME, D. R., AND FRANCIS, E. H. 1960. Induction of development of Trypanosoma lewisi in the mouse by heterologous sera. Journal of Parasitology 46, S(2), 42.
EXPERIMENTAL
(1961)
Quantitative Studies on Heterologous Sera Inducing Development of Trypanosoma Zewisi in Mice1s2-R David Richard Lincicome and Earlie Hill Francis Department
Laboratory of Parasitology, of Zoology, Howard University,
(Submitted
THE The with
use
of
homologous
an
for publication,
host
tissue
host
supplIecl
fractions
10 December
D.C.
1960)
an heterologous mouse environment. This in turn has provided a quantitative basis upon which to study comparatively other sera. The present report presents: 1) the experimental work establishing the daily minimal quantities of rat serum necessary to maintain Trypanosoma lewisi in the mouse; and 2) a comparative quantitative study of several sera for their ability to promote the growth of the rat trypanosome in the mouse. A preliminary account of this work has been presented before the 35th meeting of the American Society of Parasitologists (Lincicome and Francis, 1960).
PROBLEM
heterologous
Washington,
has
been advanced as an experimental tool for the study of the molecular biology of parasitism (Lincicome, 1958). Evidence collected by use of the system (the heterologous mouse plus the rat trypanosome, Trypanosoma lewisi) has indicated that the parasitic cell requires a substance in the serum of its homologous host (Lincicome 1955, 1957, 1958). The use of the identical test system on a serial basis over a period of several years has further shown that the parasitic cell undergoes no fundamental change in its chemical requirements of sufficient magnitude either to prevent it from developing in its normal, homologous environment (Lincicome, 1959 a and b), or to permit its growth without serum in the heterologous host.
SUMMARY
OF
RESULTS
The minimal daily volume of normal rat serum that will support the development of Trypanosonza lewisi in the heterologous mouse host was established between 0.05 and 0.01 ml. Since a volume basis appeared not wholly desirable due to variations of serum components, the minimal daily values of rat serum were also determined in terms of volumes adjusted on the basis of protein concentration assuming a parallel relationship between it and that of the trypanosome growth factor. Using this criterion the minimal values were those volumes having between 2.5 and 2.0 mg of protein equivalents. Using these minimal values the sera of the horse, man, rabbit, guinea pig, hamster, chicken, chinchilla, pig, dog, and cow were compared qualitatively and quantitatively with rat serum. Only sera of the hamster, guinea pig and rabbit had trypanosome growth promoting properties comparable to those of rat serum. With rat serum as the
It is of interest, therefore, to determine the minimal daily requirement of homologous serum that will support the trypanosome in 1 The organization of this paper is experimental. It was deliberately designed to make the results of the investigation more readily accessible to the reader by putting the relatively lesser important supporting information in its rightful place. The senior author invites readers’ comments, favorable or unfavorable, criticisms, or other ideas. 2 Part of this work was done while the senior author was a Guest Scientist in the Division of Parasitology, Naval Medical Research Institute, Bethesda, Maryland. Appreciation is expressed to the Commanding Office of the Institute for the privileges extended during this work. a Supported in part by a research grant to the senior author from the National Institutes of Health. 68
SERA
INDUCING
DEVELOPMENT
standard it was determined that hamster serum was potentially 78% as effective, guinea pig 53F and rabbit 42%. SIGNIFICANCE
The work reported here has shown that small volumes of rat serum daily are sufficient for the development of Trypanosoma lewisi in the heterologous mouse host. The titrations using a volume basis indicated that the endpoint lay between 0.05 ml and 0.01 ml. A volumetric basis for measuring quantitatively the effectiveness of homologous serum in maintaining this trypanosome in its heterologous environment did not appear to be wholly satisfactory. In view of the well known variations of serum components from animal to animal and even in the same animal from time to time, it was considered necessary to secure a basis of comparison less subject to variations. In the absence of specific information concerning the identity of the factor or factors present in rat serum that are responsible for the growth of T. lewisi in the mouse it was impossible to select the proper criterion for measurement of serum. Total protein content was, therefore, selected as the basis and, assuming a parallel relationship between these unknown trypanosome growth factors and serum proteins, further titrations were made employing volumes of serum containing predetermined protein concentrations which are hereinafter referred to as protein equivalents. The endpoint of these titrations was between 2.5 and 2.0 mg of protein equivalents. The trypanosome failed to survive and grow in the mouse when a 2.0 mg protein equivalent volume of rat serum was employed daily, but good growth was obtained at 2.5 mg. No further testing between 2.5 and 2.0 mg was done. The small quantity of serum required for this phenomenon suggests that the active factor might be protein in nature. This is interesting speculation, and offers a promising basis for further examination of rat serum chemically to determine the exact nature of this factor. We have tentatively assumed that the responsible substance might be protein in nature, and are now engaged in pre-
OF
TRYPANOSOMES
IN
MICE
69
liminary chemical characterization of the fraction by dialysis, metal recombination, and preparative fractionation electrophoretically (Evans, 1959). The establishment of 2.5 mg of protein equivalents as the minimum with which good populations of T. lewisi would develop, provided a method for testing quantitatively other sera that had been shown qualitatively to be effective in supporting the trypanosome (hamster, guinea pig, and rabbit sera) in the mouse. Titrations of these have shown that although hamster, guinea pig and rabbit sera possess the factor or factors that make it possible for the trypanosome to grow in the mouse, they do not have equally effective concentrations as rat serum. Calculation of effective indices for hamster, guinea pig, and rabbit sera based upon maximal parasitemias has shown that hamster serum has a potential only three-quarters that of rat serum. Guinea pig serum is only about half and rabbit only 40% as effective as the homologous rat. Assigning rat serum a potential value of one, it is clear that volume for volume hamster, guinea pig and rabbit sera are considerably inferior quantitatively, and that theoretically comparable trypanosome population responses might be obtained with them were the volumes of these heterologous sera increased proportionately. This, though interesting, has not been done. Human, chicken, cow, dog, swine, chinchilla, and horse sera have been shown to be inactive with respect to factors that support the development of T. lewisi in an heterologous environment. Cow, swine, and chicken sera had marked aggulutinating antibodies that clumped trypanosomes and mouse red blood cells. These sera were heat-inactivated and tested again for their ability to promote the growth of the trypanosome without success. The promotion of growth of Trypanosoma lewisi by rat, hamster, guinea pig, and hamster sera raises the question of the specificity of the phenomenon. The possession of the property by all four sera does not support the view earlier held that the factor was specific for rat serum. Were this absolute it would provide interesting possibilities of ex-
70
LINCICOME
plaining
AND
the development of the uniqueness
of the association between a parasitic cell and its sheltering host on a chemical basis. Whatever the nature of this property it is clear from the data presented in this paper that it is shared by four potential host animals. There is little evidence in the literature that T. lewisi can grow in any host except the rat. French workers (see Lincicome, 1958, for references) have reported survival of the
trypanosome in mice, hamsters and other rodents after injection of large numbers of parasites in significant amounts of homologous blood. Coventry (1929) reported that T. Zewisi will exist for a limited period in the guinea pig and Laveran and Roudsky (1914) had a similar but more limited experience. In view of the sharing of this growth promoting factor by rat, hamster, guinea pig and rabbit sera, it is not surprising that this trypanosome is able to become established on a limited basis in guinea pigs and rabbits. The final chemical identification of the substance
or substances
may well show that
these are not absolutely identical in all these animals
even though
they
possess a degree
of common action. Speculatively, these factors may all share a common cyclical protein base that differs among them in the possession of side chains or specific groups that metabolically antagonize the common base, thus accounting for the varying potentials that have
been noted in this paper. Though we have shown here that the earlier view of the specific nature of this phenomenon can not now obtain, it may be of greater theoretical interest biologically that the sera of the four animals share a similarly
active
factor
that
enables
investigation
have
therefore
broad
implications in establishing the ground work for understanding the ultimate molecular host relationships of a foreign but relatively autonomous
cell
as it
establishes
itself
in
its tissue environment. RESULTS
Titration titrations
of
rat serum. Four preliminary
of rat serum
employing
0.25 and 0.1 ml quantities indicated:
1.0, 0.5,
of normal
serum
1) that the serum was active in supporting the growth of Trypanosoma lewisi in the heterologous mouse host at the concentrations used. 2) that heavier mice did not support the trypanosomes as well as younger mice. Tables I and II present data from two of these experiments. The results of four quantitative assays of TABLE
Populations
I
of Trypanosoma lewisi Developing in 2.5 g C3H mice
Average hemacytometer count of trypanosome$ Normal rat serum (ml) Days after inoculation
1
0.5
0.1
(3)b
(3)
(3)
3 5 7
10 107 331
4 4 12
+ -d
8
+
+
-
+"
0 All figures rounded to nearest whole integer. b Indicates number of hosts employed. c Indicates trypanosomes were present but not in sufficient quantities to be estimated in the hemacytometer. d Indicates no trypanosomes were found. TABLE
II
Populations of Trypanosoma lewisi Developing in C3H Mice Weiahina 15~ or Less
Average hemacytometer count of trypanosomes
a para-
sitic cell to establish itself in a sheltering host. The work, technics, and results of the present
FRANCIS
Days after inoculation 3 4 6 8 10 12
Normal rat serum (ml) 1 0.5 0.1 (3)”
(3)
(3)
+”
+
+
7 206 499 1069
6 123 584 807
2 27 132 277
Dead
Dead
Dead
5 Indicates number of hosts used. b Indicates insufficient numbers to be recorded in hemacytometer.
SERA
INDUCING
DEVELOPMENT
rat serum employing lo-15 g mice are given in Tables III and IV. The endpoint of these titrations lies between 0.05 and 0.01 ml volumes. The titrations of normal rat serum in terms of protein equivalents are summarized in Table V. The endpoint of these tests lies between 2.5 and 2 mg of protein equivalents. Hamster serum assay: A preliminary experiment testing hamster serum employing one milliliter aliquots of sera showed that hamster serum was effective in promoting the growth of the parasite. Since the parasitemias were expressed on an arbitrary scale it was not possible to distinguish subtle difTABLE III Populations of Trypanosoma lewisi Developing in lo-15g Mice Supplemented with 1.0, 0.1, and 0.01 ml of Normal Rat Serum Average hemacytometer of trypanosomes Normal
3 4 5 6 7 8
a Indicates
Average hemacytometer of trypanosomes Normal 0.01 1
Exp. Ko. 2
1
2
4 7 11 17
2 75 33 9.5
3
2 9-15 19
-(I
-
-
-
16 15
94 -
8
28 _
_
-_
5 9
8
no trypanosomes
1
Days after inoculation
2
found.
rat serum (ml) 0.01
Exp. No. 3
43
2
3 4 5 6
32 34
101
7
-
98
4
19 18 78
7
3 6 11 8
7 7 12
-
10
(* Indicates no trypanosomes
Average hemacytometer
count
0.05
TABLE V of Trypanosoma lewisi Developing in Mice Supplemented Quantities of Normal Rat Serum Protein Equivalents
Populations
71
MICE
TABLE IV Populations oj Trypanosoma lewisi Developing in Mice Supplemented With Varying Volumes of Normal Rat Serum
count
0.1
1
IN
ferences between the population responses afforded by the hamster serum and those by the control rat serum. The parasitemia developing from the hamster serum was heavy and continued maximally (4+) for 5 days. The overall parasitemic period was 11 days. The control animals showed 4+ parasitemias for 5 days but the whole parasitemic period lasted only 9 days. Experiments 8, 9 and 10, testing hamster serum quantitatively with protein equivalents as the basis for comparison, are summarized in Tables VI and VII. The volume of serum containing 50 mg of protein equivalents used in experiment 9 (Table VI) approximated the 1.0 ml volumes
rat serum (ml)
1
Days after inoculation
OF TRYPANOSOMES
3
3
4
-a
-
-
-
-
-
-
-
found.
With Variable
count of trypanosomes
Normal rat serum (mg of protein equivalents) Days after inoculation 3 4 5 6
7 8
(1 Indicates
50 5 8 62 58 75 88 87
2.5
6
7
5
-a
-
4 39 49 70 78
6 7 22 38 43
3 9 15 15 18 12
no trypanosomes
7
6
were found.
0.5
2.0
4 8 8 10 8
Exp. No. 6
7
5
5
6
0.1
7
5
6
-
-
-
-
-
-
-
-
-
7
72
LINCICOME
AND
of the maximum parasitemia observed for hamster serum divided by that observed with control sera, it is apparent that hamster serum is only half as effective as rat serum (Table VI). If the efficiency ratio is calculated on a day to day basis (Table VI) there is a variable pattern of efficiency. In the initial phases of the development of the parasitemia the index is high but falls rapidly to essentially the level of the overall index calculated above. Comparison of similar indices calculated from parasitemias observed when hamster serum is titrated (Table VII) reveals a daily pattern that is not wholly unlike that observed with 50 mg equivalents (Table VI). The chief difference lies in the maximum parasitemia effective indices which were essentially identical for both experiments 9 and 10 (Table VII). It is possible, therefore, that hamster serum contains about 75 to SOc/r of the growth-promoting properties of homologous rat serum. Volumes of serum beyond the minimal effective quantity are not necessarily associated with linear growth patterns of the trypanosomes. Assay of cow, swine, chicken, dog, horse,
that have been frequently employed in this and other work. If the efficiency of this quantity of the two sera is calculated as the ratio TABLE VI Populations of Trypanosoma lewisi Developing in lo-15g Mice Supplemented With Rat and Hamster Sera (Experiment 8) Average hcmacytometer count
Days after inoculation
50 mg protein equivalents
3 4 5 6 7 S
6 13 28 68 86 99
Effective
Index =
trypanosome
Normal hamster serum
Normal rat serum
-
SOmg protein equivalents
Efficiency relative to control (%)
6 11 15 38 53 4.5
100 85 54 56 62 4s
Max. Parasit. of Exp. Max. Parasit. of Cont. =-Ix
53
54%
99
Populations
FRANCIS
TABLE VII of Trypanosoma lewisi Developing in lo-15g Mice Supplemented Varying Quantities of Rat and Hamster Sera Using Protein Equivalents as a Basis of Measurenzrnt Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.5 2.0 Days after inoculation
2 5 9 11
10
9
10
2 5 ‘3 10 11
10
-u --
-
-
-
Index = 9
for Exp.
-
9(Q) 8(80)
9 = 82% Av. =
for Exp. 10 =
785%
73%
11
R Indicates that no trypanosomes were found. 7~Efficiency relative to Rat Serum for corresponding
day in 70.
9
10
--(O) f/
3(60) 8(100) 5W) X(73)
4(50)
11
Index = 5
9 l(50) 3(60) 6(67)
8
8
Effective
count
Normal hamster serum (mg of protein equivalents) 2.5 2.0 Exp. No.
9
3 4 S 6 7
Effective
With
-
10
-
SERA INDUCING
DEVELOPMENT
man and chinchilla sera. In none of the experiments utilizing any of these sera was there development of the trypanosome. Volumes of human and chinchilla serum were insufficient to replicate observations. COW. chicken, and swine sera frequently were associated with in vitro agglutination of both trypanosomes and mouse erythrocytes. Heat inactivation did not alter this property. Guinea pig serum assay. The two preliminary assays based on volumes of guinea pig serum showed that this serum was potentially as effective as homologous rat serum in supporting the growth of the trypanosomes. Since these tests were exploratory, two additional protocols were developed (Table VIII). Here, the effective index of guinea pig serum based upon the maximum parasitemias was 5354 (average of both experiments) indicating that whatever the nature of the growth promoting properties present in this serum, the quality is only about half that found in rat serum. The efficiency of guinea pig serum on a day to day basis fell from an initial high in a pattern similar to that observed for hamster serum. Rabbit serum assay. Although there was little evidence that rabbit serum was effective in supporting the development of Trypano-
OF TRYPANOSOMES
73
IN MICE
soma lewisi in the heterologous mouse in a preliminary trial, subsequent work showed that rabbit serum would support the parasite. Table IX presents data from a test of rabbit serum in young mice. TECHNICAL
PROCEDURES, PROTOCOLS AND REFERENCES
Procedures Thirty (30) experiments were completed in which 397 mice were used.” Of these, 373 were female albino ranging in weight from lo-26 g. The others were male C3H of the same weight range. All protocols employed a strain of Trypanosoma lewisi that had been maintained in the laboratory by serial blood passages in albino rats for more than 10 years. Sera of man, hamster, guinea pig, rabbit, chicken, swine, cow, dog, horse, rat and chinchilla were studied. Those of the hamster, guinea pig, rabbit, rat and dog were collected by cardiac puncture. Bloods were allowed to clot under refrigeration (2-4” C) for a variable time, and the sera removed by 4 .4nimals were obtained laboratory of the National Bethesda, Md.
through Institutes
the animal of Health,
TABLE VIII Populations of Trypanosoma Iewisi Developing in lo-15g Mice Supplemented With Varying Quantities of Rat and Guinea Pig Sera Using Protein EquivaJents as a Basis of Measurement Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.5 2.0 Days after inoculation
Exp. No.
3 4 5 6 7 8
11
12
11
12
3 5 8 9 9 6
1 5 9 10 10 7
-0 --
-
11 2(67)b 3(60) j(63) j(56) j(S6) 5(33)
-
(r Indicates no trypanosomes were found. f~ Efficiency Index in percent relative to control of corresponding Effective
count
Normal guinea pig serum (mg of protein equivalents) 2.5 2.0 12 l(100) 4(80) j(56) S(50) 5(50) S(71)
day.
Max. Parasitemia
of Exp.
= +
= 56% for Exp. 11
Max. Parasitcmia
of Control
= $
= 50% for Exp. 12
Index =
11
12
-
-
-
74
LINCICOME
AND
FRANCIS
TABLE IX Populations of Trypanosoma lewisi Developing in IO-15g Mice Supplemented With Varying Quantities of Rat and Rabbit Serum Using Protein Equivalents as a Basis of Measurement Average hemacytometer
trypanosome
Normal rat serum (mg of protein equivalents) 2.0 2.5
Normal rabbit serum (mg of protein equivalents) Exp. No.
13
14
13
14
13
14
13
14
-
-
-
-
-
2(50) l(14)
-
-
4(44) 4(40) 2(33)
-
-
3 4
1
3
-b
4
4
-
-
l(2S)C
5 6
7 7
7 9
-
-
l(14)
7 8
5 3
10 6
-
-
3 (60) l(33)
0 Effective
2.0
2.5
Days after inoculation
count”
2(28)
Index = t
= 43% for Exp. 13 and 4 = 40% for Exp. 14. Av. Index = 42%. 10 b Indicates no trypanosomes were found. c Efficiency in perce.lt relative to control of corresponding day.
centrifugatinn. Human serum was obtained from the ch / -al ser;-ices of the Naval Hospital at the P ational Naval Medical Center, Bethesda, Ma-yland. The sera of the chicken (antibiotic fr :e) and chinchilla were gifts.” All other ser, were purchased commercially. Inactivation of sera was carried out in a water bath at 60” C for one hour to destroy natural agglutiriating antibodies for Trypanosoma lewisi. All sera were inoculated intraperitoneally in amounts varying in the several protocols used. Details may be found in the results section in appropriate context. Experimental
infections
of
Trypanosoma
lewisi in control and test mice were initiated by intraperitoneal inoculations of physiologic saline suspensions of blood drawn either from the tail or heart of donor animals with Trypanosoma lewisi not more than 7 days previously (to minimize the carry-over of antibodies from donor to experimental animals). A few crystals of sodium citrate were always 5 Chicken serum was given by Dr. Virginia L. Blackford, then of the Naval Medical Research Institute, Bethesda, Maryland; the chinchilla serum was collected and given by Dr. Leslie A. Stauber of the Department of Zoology, Rutgers University, New Brunswick, New Jersey.
added to avoid clotting of the suspension. The trypanosomes were distributed throughout the blood-saline suspension by repeated drawing into and expelling the suspension from a 10 ml syringe over a period of 60 seconds at the beginning of a series of inoculations. Between withdrawals of the suspension for inoculation of individual animals the homogeneity of the trypanosome distribution was maintained by a single drawing and expelling from the 10 ml syringe. Numbers of trypanosomes inoculated varied from 3-7 x 106. A red blood cell pipette (Thoma) and an hemacytometer” with Toisson’s fluid7 were used for quantitative estimation of the populations of trypanosomes in peripheral tail bloods of experimental hosts and in bloodsaline suspensions used for initiating infections. All quantitative estimations of parasite populations in tailbloods were in duplicate daily or on alternate days beginning usually the third day after inoculation. The data are all expressed in the tables as averaged counts a Spencer Bright Line, pany, Rochester, N.Y.
American
Optical
Com-
7 Toisson’s Fluid: lg NaCl; 8g Na,SO, ; 30 ml glycerine; lo-25 mg crystal Violet; 160 ml of dist. HOH.
SERA
INDUCING
DEVELOPMENT
OF TRYPANOSOMES
IN
MICE
75
trol animals each received a daily inoculum of 1.0 ml of normal rat serum. Twelve ( 12) experimental animals each received a daily inoculum of 0.1 ml of normal rat serum; a further series of twelve (12) each received 0.01 ml. 2. Experiments 3 and 4. Twelve ( 12) control mice each received a daily supplement of 1.0 ml of normal rat serum. Twelve ( 12 ) experimental mice received a daily inoculation of 0.05 ml of normal rat serum. Twelve ( 12) others each received 0.01 ml of normal rat serum. The next studies were conducted with rat sera whose total serum protein contents had been assayed. The volumetric basis of titration adopted in experiments 1 and 2 was abandoned and the quantity of serum to be inoculated was expressed in terms of protein equivalents as follows. The total protein content was established at 5.0 g/100 ml. Weights of the mice ranged from lo-15 g. Experiment 5. Six (6) con’ A mice received each a volume normal rat serum having a protein Jquivalent of 50 mg. Six (6) mice rev-eived 2.5 mg and six (6) others each rt -eived 0.5 mg. Experiment 6. Six (6) r’ ;rol mice each received a daily inoculation of normal rat serum containing 50 mg of protein equivalents. Of twelve (12) experimental mice half received 2 mg each and half received 1 mg each of protein equivalents. Experiment 7. Six (6) control mice were each given a daily supplement of 50 Experimental Protocols mg of protein equivalents of normal rat serum of 12 experimental mice half reTitration of rat serum. Four preliminary experiments were performed in which the ceived each 2.5 mg and half 2 mg of populations of trypanosomes were studied in equivalents. 15 to 25-g mice supplemented with daily Hamster serum assay. One preliminary volumes of rat serum ranging from 1.0 ml to test of hamster serum employed 1.0 ml daily 0.05 ml. From these experiments it was de- volumes in mice. Parasitemias were followed cided to titrate rat serum in mice weighing daily until the 12th post inoculation day. 10 to 15 g. The following protocols were thereThree quantitative protocols were used as fore adopted: follows : 1. Experiments 1 and 2. Twelve ( 12) con1. Experiment 8. Six control mice (6) each received a daily inoculum of normal 8 Purina Laboratory Chow, Purina-Ralston Co., rat serum containing 50 mg of protein St. Louis, Missouri. equivalents. Twelve ( 12) experimental 9 Bausch and Lombs Spectronic 20, Rochester, N.Y. mice each received daily a quantity of
p i hemacytometer square. To convert these &;ures to numbers of parasites per cu mm ol blood a factor of 2000 should be applied. In some experiments trypanosome populations in tailbloods were expressed on an arbitrary scale of one to four plus previously defined (Lincicome, 1958) as representing a range of a minimum to a maximum number. Food was withheld from all mice for the first twenty-four hours of each experiment. Thereafter all animals received one gram/day. In some early experiments starvation preceded the initiation of the experiment. The food was a commercially available, balanced laboratory chowR that had been ground in a mechanical mortar. The titration of the quality of serum in promoting the growth of Trypanosoma Eewisi was initially on a volumetric basis. In view of the quantitative differences in serum components from animal to animal and from time to time it appeared desirable to standardize the quantity of serum used on the basis of protein equivalents. Total protein determina.tions were not made on those sera that possessed naturally occurring antibodies to the trypanosomes since preliminary observations had indicated that regardless of the volume employed these sera would not support the development of the trypanosomes. Total serum protein determinations were made by the biuret reaction. Optical density was measured with an electric calorimeter” employing the method of Kingsley (1939) and Gornall et al. (1949).
76
LINCICOME
hamster sera having 50 mg protein equivalents. 2. Experiments 9 and 10. Half of the control group of twelve animals received each a daily rat serum supplement having 2.5 mg of protein equivalents and the other half 2.0 mg per animal. Half of the mice in an experimental group of 24 animals received each 2.5 mg of hamster serum protein equivalents while the other half received only 2.0 mg per animal. Assay of cow, swine, chicken, dog, horse, man and chinchilla sera. A series of tests employing 1.0 ml volumes of rat serum and the sera under assay was completed. From three to six animals were used in both control (receiving rat serum) and experimental groups. No quantitative tests were done since there was no evidence of any activity supporting the growth of trypanosomes. The sera of the cow, chicken, and swine were heatinactivated and tested along with fresh, uninactivated sera. Guinea pig serum assay. Two preliminary volumetric tests using 14 mice gave evidence of growth-promoting properties for the trypanosomes. Two quantitative assays were then designed as follows: Experiments 11 and 12. Sixteen (16) control mice received each a daily inoculum of normal rat serum: half received 2.5 g of protein equivalents per animal, and half, 2.0 mg each. Sixteen ( 16) experimental mice each received a daily inoculum of normal guinea pig serum: half got 2.5 mg of protein equivalents and half 2.0 mg per animal. Rabbit serum assay. Two quantitative assays of rabbit serum were done, although one preliminary test using a dozen animals indicated little or none of the activity sought. Experiments 13 and 14. Sixteen (16) control mice received each a daily inoculum of normal rat serum: half of this group
AND
FRANCIS
received 2.5 mg of protein equivalent; half, 2.0 mg of equivalents. Sixteen (16) experimental mice received a quantity of fresh rabbit serum comparable to the control groups : half received 2.5 mg of protein equivalents; half received but 2.0 mg. REFERENCES COVENTRY, F. A. 1929. Experimental infections with Trypanosoma lewisi in the guinea pig. American Journal of Hygiene 6, 247-259. EVANS, A. S. 1960. Immunophysical methods in parasitic infections: A continuous electrophoresis apparatus for preparative fractionation of protein systems. Experimental Parasitology 6, 105112.
GORNALL, A. G., BARDSWILL, 0. J., AND DAVID, M. 1949. Determination of serum protein by means of the biuret reaction. Journal of Biological Chemistry 177, 751-766. KINGSLEY, G. B. 1939. The determination of serum total protein by the biuret reaction. Journal of Biological Chemistry 131, 197-200. LAVERAN, A., AND ROUDSKY, D. 1914. Contribution a l’etude de la virulence du Trypanosoma lewisi et du Tr. duttoni pour quelques especes animales. Bulletin de la sock%! pathologique exotique 7, 528-535. LINCICOME, D. R. 1955. Growth factor in normal rat serum for Trypanosoma lewisi. Journal of Parasitology 41, supplement, 1.5. LINCICOME, D. R. 1957. Growth of Trypanosoma lewisi in the heterologous mouse host. American Journal of Tropical Medicine and Hygiene 6, 392. LINCICOME, D. R. 1958. Growth of Trypanosoma lewisi in the heterologous mouse host. Experimental Parasitology 7, 1-13. LINCICOME, D. R. 1959a. Observations on changes in Trypanosoma lewisi after growth in calorically restricted and in normal mice. Annals of Tropical Medicine and Parasitology 53, 274-287. LINCICOME, D. R. 1950b. Serial passage of Trypanosoma Zewisi in the heterologous mouse host. I. calorically-restricted hosts. Development in Journal of Protozoology 6, 310-315. LINCICOME, D. R., AND FRANCIS, E. H. 1960. Induction of development of Trypanosoma lewisi in the mouse by heterologous sera. Journal of Parasitology 46, S(2), 42.