Z. Parasitenkunde 45, 281--290 (1975) © by Springer-Verlag 1975
Trypanosoma rhodesiense : Folate Levels in Sera and Tissues of Normal and Folic Acid-Deficient Rats G e o r g i a n a F . Aboko-Cole a n d Clarence M. Lee Department of Zoology, Howard University, Washington Received June 25, 1974
Summary. An experimental model composed of the folie acid deficient SpragueDawley rat and Trypanosoma rhodesi~nsewas used to study folato levels in sera and tissues. Serum folate levels in well fed rats inoculated on Day 21 averaged 21 4- 3 ng/ml; weil feed normal rats averaged 184- 4 ng/ml. In rats given the pair-fed control diet, serum folate levels averaged 17.24- 4 ng/ml for trypanosome-free animals and 20.24- 3 ng/ml for infected ones. In rats given the folie acid-deficient diet, serum folate levels averaged 8.6 4- 2 ng/ml for noninfeeted control animals and 9.3 4- 2 ng/ml for trypanosome-infected ones. Regardless of diet, the infected animals inoculated on Day 56 had higher serum folate levels over the controls on the last day of observation (Day 5 of infection). Livers from rats red complete and pair-fed diets and inoculated on Day 21 showed no significant differences in folate content when compared to control animals. However, livers of rats on a deficient diet showed significantly more folate when compaxed with uninfeeted controls, reaching a maximum of 362% at Day 25. Liver folate levels of rats (regardless of the dietary regimen) inoculated on Day 56 showed signifieantly higher values than the controls on Day 60. Irrespective of the time of inoculation or diet, brain and spinal cord of T. rhodesiense-infected rats had significantly higher folate values than their controls on Day 5 of infection. The fola~e level of the brain and spinal cord, at this time, ranged up to 58 and 107 % respectively. Introduction P r o t o z o a l infections h a v e b e e n shown t o be m o d i f i e d b y a l t e r a t i o n s in t h e v i t a m i n c o n t e n t s of t h e h o s t (Trager, 1943; E r a s m u s , S c o t t a n d Levine, 1960; Caldwell a n d Gyorgi, 1943, 1947 ; Y a e g e r a n d Miller, 1960a, b, c, d ; Lee a n d Aboko-Cole, 1973). T h e p u r p o s e of this p a p e r is to r e p o r t on t h e folie a c i d levels in sera a n d tissues of r a t s infected w i t h Trypanosoma rl~tesiense a n d red complete, folie acid-deficient a n d pair-fed control diets.
Materials and Methods History of Parasite Trypanosoma rhodesiense has been maintained by continous weekly subpassage in rats since about 1962. T. rhode,len,e, a pathogen, usually kills albino rats within 5 to 7 days.
282
G. F. Aboko-Cole and C. M. Lee
Experimental Hosts Three hundred and seventy-eight (378) female albino rats (Sprague-Dawley) were used in six separate protocols. Table I shows the distribution, number, and initial body weights of rats used together with the several dietary grouping forming the structure of each experiment.
Experimental Diets The vitamin B eomplex eomplete and the folie acid-deficient diets were obtained from Nutritional Bioehemica]s, Cleveland, Ohio.
Housing and Feeding o/Rats Rats were housed individually in wire-bottomed cages and fed the appropriate diets from metal feeding cups. All rats in the eontrol diet groups and the folic aciddeficient group were allowed to feed freely. Rate in the pair-fed groups were given the control diet daily in amounts equal to the food eonsumed by their folie aciddeficient paired mates. The daily intake of eaeh rat was determined by subtracting the amount of food remaining in a tared cup from the amount given the previous day. The rats were provided water continuously. Water bottles and feeding cups were cleaned daily and cages were cleaned frequently to minimize algal and bacterial contaminations.
Inoculation o/Rats wlth Trypanosomes Twenty-one days after initiation of a dietary regimen, one half of the rate in each dietary group was injeeted with 100 T. rhodesiense eells in experimente 1, 2 and 3. Fifty-six days after initiation of a dietary regimen, one half of the rats in eaeh dietary group was injeeted with 100 T. rhodesiense cells in experiments 4, 5 and 6. In all experiments, equal numbers of rate served as eontrols. Trypanosomes were collected, washed, and suspended in physiologic saline for inoeulation (Lineicome and Watkins, 1963).
Collection o/Serum, Liver, Brain and Spinal Cord Rats were killed by dislocation of the vertebrae. T. rhodesiense infeeted rats and controls of each dietary group were killed on days 1 to 7 of infeetion. Blood was colleeted by aseptie heart puneture and serum was extracted after the blood had cooled to room temperature. Five grams of liver, two grams of brain, and 1 gram of spinal cord were collected. Serum, liver, brain, and spinal cord samples were put in separate labelled vials. These were sealed, frozen in dry ice alcohol and stored at --20 degrees Celsius. Tissue homogenates were prepared according to the method of Bennett et al. (1964).
Microbiological Assay o/Folic Acid Folic aeid was determined by measurement of the growth stimulation of Lactobacillus casei by sera and tissue homogenates (Baker et al., 1959; I-Ierbert, 1966).
Statistical Interpretation All like d a t a in E x p e r i m e n t s 1-3 w e r e p o o l e d f o r s t u d y , as w e r e t h e like d e t e r m i n a t i o n s in E x p e r i m e n t s 4 - 6 . S t a n d a r d d e v i a t i o n s w e r e
Trypanoaoma rhodesiense: Folate Levels in Sera and Tissues of Rats
283
Table 1. Average initial body weight (Gin. :~ S.D.) of rats red a eomplete, folie aeid-deficient and pair-fed diets and inoculated with Trypanosoma rhodesiense (number of animals employed in parentheses) Dietary group
1
2
50q-1 (7)
51 4-1 51~-1 50~:1 50q-1 50q-1 (7) (7) (14) (14) (14)
63
50B1 (7)
51q-1 (7)
51±1 (7)
50B1 (14)
50q-1 (14)
50B1 (14)
63
2. Folie acid-deficient Noninfeetedrats 50~1 (7)
51~1 (7)
51~1 (7)
50~:1 50~1 (14) (14)
50:~1 (14)
63
51q-1 (7)
51il (7)
50±1 (14)
50±1 (14)
63
1. Control Noninfectedrats Infectedrats
Infeetedrats 3. Pair-fed Noninfected rats InIectedrats Total number of rats
50±1 (7)
3
4
5
50-F1 (14)
6
Total number of rats
50 -F 1 50-~ 1 51 :t: 1 50 :t: 1 50 ~ 1 50 :t: 1 (7) (7) (7) (14) (14) (14)
63
50q-1
50q-1
51~:1 50q-1
50q-1 50 -4-1
63
(7)
(7)
(7)
(14)
(14)
(14)
42
42
42
84
84
84
378
calculated and the Student " t " test was applied to dcterminc the significance of düferences (Snedecor, 1959). Statistical significanee was assig-ned to all diIferences of means having a probability scale of 1% or less. The development of parasitemia in rats inoculated with T. rhodesicnsc cells was described earlier (Aboko-Cole and Lee, 1974a). Fig. 1 A, B show the folie eontent in serum of rats fed eomplete, folie acid deficicnt and pair-fed diets and inoeulated with T. rhodesiensc on Days 21 and 56, respectively. The average level of folate in adequately fed control rats was 18 q- 4 ng]ml as opposed to 21 q- 3 ng/ml for trypanosome-infeeted rats on the same diet. The irffected animals fed the completc diet had higher folate levels over the controls on the last day of observation. Serum folate levels in rats deprived of folate were about one-half those of animals with aa adequate supply of the vitamin. Serum folate levels in rats red a deficient diet were on the average 7 % greater in deficient inIected rats. A significance of 178 % was evideneed by D a y 25 of the experiment. Serum levels of folate in infected pair-fed eontrol rats were significantly higher (on D a y 25) than noninfected ones red the same diet.
G. F. Aboko-Cole a n d C. M. Lee
284
45 44 43 42 41 40 39 58 37
2O 19
\
~g 18
~'d
~ m
7
15 12
IO
~X
45 40 35
7
30
6 5
w
4
•~ 2 o o
15
5 2
5
I
\ \ J
5
I0
~
J
l
15 20 25 DAYS A
0
I
5
i
I0
I
15
L I
20 25
~
30 35 4 0 DAYS B
45 50
55 60
Fig. 1. Folic Acid C o n t e n t in S e r u m of R a t s F e d Complete, Folie Acid-Deficient a n d Pair-fed Diets a n d I n o c u l a t e d with Trypanosoma rhodesiense, o averages for r a t s inoculated w i t h Trypanosoma rhodesiense a n d fed a Complete Diet, , a v e r a g e s for u n i n o c u l a t e d r a t s fed a Complete Diet, , averages for r a t s inoculated w i t h Trypanosoma rhodesien~e a n d fed a Folic Acid-Deficient Diet, x averages for u n i n o e u l a t e d r a t s fed a FolicAcidDeficient D i e t , , averages for r a t s inoculated w i t h Trypanoaoma rhodesienae a n d fed a Pair-fed Diet, O averages for u n i n o c u l a t e d r a t s fed a Pair-fed Diet. A ~ r a t s inoculated on D a y 21, B ~ r a t s inoculated on D a y 56
Trypanosoma rhodes~nse: Folate Levels in Sera and Tissues of Rats
285
I0
=.
ùJ
~o i5 ~o ~5 DAYS A
~ 8
6
-:4
m o
_1
5
I0
15 20 25 3 0 35 4 0 4 5 DAYS B
50
55 60
Fig. 2. Folie Acid Content in Liver of Rats l~ed Complete, Folie Acid-Deficient and Pair-fed Diets and Inoculated with Tryl~ano«oma rhodesiense, o averages for rats inoculated with Trypanosoma rhodesiense and red a Complete Diet, « averages for uninoculatedrats red a Complete Diet, o averages for rats inoculated with Tryt)anosoma rhodes/ense and red a Folic Acid Deficient Diet, x averages for uninoculated rats red a Folic Acid Deficient Diet, • averages for rats inoculated with Trypanosoma rlmäe~ens« and red a pair-fed Diet, O averages for uninoculated rats red a pair-fed Diet. A ----rats inoculated on Day 21, B ----rats inoculated on Day 56
Regardless of diet, t h e infected a n i m a l s h a d higher folate levels over t h e controls o n t h e last d a y of o b s e r v a t i o n (Day 5 of infection). O n a percentage basis i n relation to controls, folate level i n deficient animals increased to 673 % a t I ) a y 60; 153 % i n a n i m a l s fed full c o m p l e m e n t diets a n d 85% i n pair-fed control animals. T h e folic acid c o n t e n t i n liver of rats red complete, folic acid deficient a n d pair-fed diets a n d i n o c u l a t e d with T. rhodesiense on D a y s 21 a n d 56 respectively, are s h o w n i n F i g u r e s 2A, B.
286
G. F. Aboko-Cole and C. M. Lee
~IO0
Z
&
õ6o ùx. ~ ~x
-& I
I
,o ,5 2'o ~5 DAYS A
I00 -= Iso x
~,6o \x ~
'~%x\\ .ä20
i.
i
t
I
DAYS B Fig. 3. Folic Acid Content in Spinal Cord of Rats Fed Complete, Folic Acid-Deficient and Pair-fed Diets and Inoculated with Trypanosoma rhodesiense, o averages for rats inoculated with Trypanosoma rhodesiense and red a Complete Diet, Aaverages for uninoculated rats red a Complete Diet, D averages for rats inoculated with Trypanosoma rhodesiense and red a Folic Acid-Deficient Diet, x averages for uninoculated rats red a Folic Acid-Deficlent Diet, v averages for rats inoculated with Trypanosoma rhodesiense and red a Pair-fed Diet, O averages for uninoculated rats red a pair-fed Diet. A = rats inoculated on Day 21, B = rats inoculated on Day 56
Livers of ra~s on c o m p l e t e a n d pair-fed diets an d i n o c u l a t e d on D a y 21 with T. rhodesiense showed no significant differences in folate c o n t e n t when c o m p a r e d to control animals. H o w e v e r , livers of in/ected rats on a deficient diet showed significantly m o r e folate when c o m p a r e d with deficient uninfected controls, reaching a m a x i m u m of 362 % at D a y 25. L i v e r folate levels of rats on complete, folic acid-deficient a n d pair-fed control diets a n d i n o c u l a t e d on D a y 56 showed significantly higher values t h a n t h e controls on D a y 60.
Trypanosoma rhodesiens«: Folate Levels in Sera and Tissues of Rats
287
140
130
120
i
l I00 .=_
~o 9o
80
140
~x
DAYS A
130
120 w= ».
« ù0 I 0 0
õ .c o
.
Trypanosoma rhodesiense : Folate Levels in Sera and Tissues of Normal and Folic Acid-Deficient Rats G e o r g i a n a F . Aboko-Cole a n d Clarence M. Lee Department of Zoology, Howard University, Washington Received June 25, 1974
Summary. An experimental model composed of the folie acid deficient SpragueDawley rat and Trypanosoma rhodesi~nsewas used to study folato levels in sera and tissues. Serum folate levels in well fed rats inoculated on Day 21 averaged 21 4- 3 ng/ml; weil feed normal rats averaged 184- 4 ng/ml. In rats given the pair-fed control diet, serum folate levels averaged 17.24- 4 ng/ml for trypanosome-free animals and 20.24- 3 ng/ml for infected ones. In rats given the folie acid-deficient diet, serum folate levels averaged 8.6 4- 2 ng/ml for noninfeeted control animals and 9.3 4- 2 ng/ml for trypanosome-infected ones. Regardless of diet, the infected animals inoculated on Day 56 had higher serum folate levels over the controls on the last day of observation (Day 5 of infection). Livers from rats red complete and pair-fed diets and inoculated on Day 21 showed no significant differences in folate content when compared to control animals. However, livers of rats on a deficient diet showed significantly more folate when compaxed with uninfeeted controls, reaching a maximum of 362% at Day 25. Liver folate levels of rats (regardless of the dietary regimen) inoculated on Day 56 showed signifieantly higher values than the controls on Day 60. Irrespective of the time of inoculation or diet, brain and spinal cord of T. rhodesiense-infected rats had significantly higher folate values than their controls on Day 5 of infection. The fola~e level of the brain and spinal cord, at this time, ranged up to 58 and 107 % respectively. Introduction P r o t o z o a l infections h a v e b e e n shown t o be m o d i f i e d b y a l t e r a t i o n s in t h e v i t a m i n c o n t e n t s of t h e h o s t (Trager, 1943; E r a s m u s , S c o t t a n d Levine, 1960; Caldwell a n d Gyorgi, 1943, 1947 ; Y a e g e r a n d Miller, 1960a, b, c, d ; Lee a n d Aboko-Cole, 1973). T h e p u r p o s e of this p a p e r is to r e p o r t on t h e folie a c i d levels in sera a n d tissues of r a t s infected w i t h Trypanosoma rl~tesiense a n d red complete, folie acid-deficient a n d pair-fed control diets.
Materials and Methods History of Parasite Trypanosoma rhodesiense has been maintained by continous weekly subpassage in rats since about 1962. T. rhode,len,e, a pathogen, usually kills albino rats within 5 to 7 days.
282
G. F. Aboko-Cole and C. M. Lee
Experimental Hosts Three hundred and seventy-eight (378) female albino rats (Sprague-Dawley) were used in six separate protocols. Table I shows the distribution, number, and initial body weights of rats used together with the several dietary grouping forming the structure of each experiment.
Experimental Diets The vitamin B eomplex eomplete and the folie acid-deficient diets were obtained from Nutritional Bioehemica]s, Cleveland, Ohio.
Housing and Feeding o/Rats Rats were housed individually in wire-bottomed cages and fed the appropriate diets from metal feeding cups. All rats in the eontrol diet groups and the folic aciddeficient group were allowed to feed freely. Rate in the pair-fed groups were given the control diet daily in amounts equal to the food eonsumed by their folie aciddeficient paired mates. The daily intake of eaeh rat was determined by subtracting the amount of food remaining in a tared cup from the amount given the previous day. The rats were provided water continuously. Water bottles and feeding cups were cleaned daily and cages were cleaned frequently to minimize algal and bacterial contaminations.
Inoculation o/Rats wlth Trypanosomes Twenty-one days after initiation of a dietary regimen, one half of the rate in each dietary group was injeeted with 100 T. rhodesiense eells in experimente 1, 2 and 3. Fifty-six days after initiation of a dietary regimen, one half of the rats in eaeh dietary group was injeeted with 100 T. rhodesiense cells in experiments 4, 5 and 6. In all experiments, equal numbers of rate served as eontrols. Trypanosomes were collected, washed, and suspended in physiologic saline for inoeulation (Lineicome and Watkins, 1963).
Collection o/Serum, Liver, Brain and Spinal Cord Rats were killed by dislocation of the vertebrae. T. rhodesiense infeeted rats and controls of each dietary group were killed on days 1 to 7 of infeetion. Blood was colleeted by aseptie heart puneture and serum was extracted after the blood had cooled to room temperature. Five grams of liver, two grams of brain, and 1 gram of spinal cord were collected. Serum, liver, brain, and spinal cord samples were put in separate labelled vials. These were sealed, frozen in dry ice alcohol and stored at --20 degrees Celsius. Tissue homogenates were prepared according to the method of Bennett et al. (1964).
Microbiological Assay o/Folic Acid Folic aeid was determined by measurement of the growth stimulation of Lactobacillus casei by sera and tissue homogenates (Baker et al., 1959; I-Ierbert, 1966).
Statistical Interpretation All like d a t a in E x p e r i m e n t s 1-3 w e r e p o o l e d f o r s t u d y , as w e r e t h e like d e t e r m i n a t i o n s in E x p e r i m e n t s 4 - 6 . S t a n d a r d d e v i a t i o n s w e r e
Trypanoaoma rhodesiense: Folate Levels in Sera and Tissues of Rats
283
Table 1. Average initial body weight (Gin. :~ S.D.) of rats red a eomplete, folie aeid-deficient and pair-fed diets and inoculated with Trypanosoma rhodesiense (number of animals employed in parentheses) Dietary group
1
2
50q-1 (7)
51 4-1 51~-1 50~:1 50q-1 50q-1 (7) (7) (14) (14) (14)
63
50B1 (7)
51q-1 (7)
51±1 (7)
50B1 (14)
50q-1 (14)
50B1 (14)
63
2. Folie acid-deficient Noninfeetedrats 50~1 (7)
51~1 (7)
51~1 (7)
50~:1 50~1 (14) (14)
50:~1 (14)
63
51q-1 (7)
51il (7)
50±1 (14)
50±1 (14)
63
1. Control Noninfectedrats Infectedrats
Infeetedrats 3. Pair-fed Noninfected rats InIectedrats Total number of rats
50±1 (7)
3
4
5
50-F1 (14)
6
Total number of rats
50 -F 1 50-~ 1 51 :t: 1 50 :t: 1 50 ~ 1 50 :t: 1 (7) (7) (7) (14) (14) (14)
63
50q-1
50q-1
51~:1 50q-1
50q-1 50 -4-1
63
(7)
(7)
(7)
(14)
(14)
(14)
42
42
42
84
84
84
378
calculated and the Student " t " test was applied to dcterminc the significance of düferences (Snedecor, 1959). Statistical significanee was assig-ned to all diIferences of means having a probability scale of 1% or less. The development of parasitemia in rats inoculated with T. rhodesicnsc cells was described earlier (Aboko-Cole and Lee, 1974a). Fig. 1 A, B show the folie eontent in serum of rats fed eomplete, folie acid deficicnt and pair-fed diets and inoeulated with T. rhodesiensc on Days 21 and 56, respectively. The average level of folate in adequately fed control rats was 18 q- 4 ng]ml as opposed to 21 q- 3 ng/ml for trypanosome-infeeted rats on the same diet. The irffected animals fed the completc diet had higher folate levels over the controls on the last day of observation. Serum folate levels in rats deprived of folate were about one-half those of animals with aa adequate supply of the vitamin. Serum folate levels in rats red a deficient diet were on the average 7 % greater in deficient inIected rats. A significance of 178 % was evideneed by D a y 25 of the experiment. Serum levels of folate in infected pair-fed eontrol rats were significantly higher (on D a y 25) than noninfected ones red the same diet.
G. F. Aboko-Cole a n d C. M. Lee
284
45 44 43 42 41 40 39 58 37
2O 19
\
~g 18
~'d
~ m
7
15 12
IO
~X
45 40 35
7
30
6 5
w
4
•~ 2 o o
15
5 2
5
I
\ \ J
5
I0
~
J
l
15 20 25 DAYS A
0
I
5
i
I0
I
15
L I
20 25
~
30 35 4 0 DAYS B
45 50
55 60
Fig. 1. Folic Acid C o n t e n t in S e r u m of R a t s F e d Complete, Folie Acid-Deficient a n d Pair-fed Diets a n d I n o c u l a t e d with Trypanosoma rhodesiense, o averages for r a t s inoculated w i t h Trypanosoma rhodesiense a n d fed a Complete Diet, , a v e r a g e s for u n i n o c u l a t e d r a t s fed a Complete Diet, , averages for r a t s inoculated w i t h Trypanosoma rhodesien~e a n d fed a Folic Acid-Deficient Diet, x averages for u n i n o e u l a t e d r a t s fed a FolicAcidDeficient D i e t , , averages for r a t s inoculated w i t h Trypanoaoma rhodesienae a n d fed a Pair-fed Diet, O averages for u n i n o c u l a t e d r a t s fed a Pair-fed Diet. A ~ r a t s inoculated on D a y 21, B ~ r a t s inoculated on D a y 56
Trypanosoma rhodes~nse: Folate Levels in Sera and Tissues of Rats
285
I0
=.
ùJ
~o i5 ~o ~5 DAYS A
~ 8
6
-:4
m o
_1
5
I0
15 20 25 3 0 35 4 0 4 5 DAYS B
50
55 60
Fig. 2. Folie Acid Content in Liver of Rats l~ed Complete, Folie Acid-Deficient and Pair-fed Diets and Inoculated with Tryl~ano«oma rhodesiense, o averages for rats inoculated with Trypanosoma rhodesiense and red a Complete Diet, « averages for uninoculatedrats red a Complete Diet, o averages for rats inoculated with Tryt)anosoma rhodes/ense and red a Folic Acid Deficient Diet, x averages for uninoculated rats red a Folic Acid Deficient Diet, • averages for rats inoculated with Trypanosoma rlmäe~ens« and red a pair-fed Diet, O averages for uninoculated rats red a pair-fed Diet. A ----rats inoculated on Day 21, B ----rats inoculated on Day 56
Regardless of diet, t h e infected a n i m a l s h a d higher folate levels over t h e controls o n t h e last d a y of o b s e r v a t i o n (Day 5 of infection). O n a percentage basis i n relation to controls, folate level i n deficient animals increased to 673 % a t I ) a y 60; 153 % i n a n i m a l s fed full c o m p l e m e n t diets a n d 85% i n pair-fed control animals. T h e folic acid c o n t e n t i n liver of rats red complete, folic acid deficient a n d pair-fed diets a n d i n o c u l a t e d with T. rhodesiense on D a y s 21 a n d 56 respectively, are s h o w n i n F i g u r e s 2A, B.
286
G. F. Aboko-Cole and C. M. Lee
~IO0
Z
&
õ6o ùx. ~ ~x
-& I
I
,o ,5 2'o ~5 DAYS A
I00 -= Iso x
~,6o \x ~
'~%x\\ .ä20
i.
i
t
I
DAYS B Fig. 3. Folic Acid Content in Spinal Cord of Rats Fed Complete, Folic Acid-Deficient and Pair-fed Diets and Inoculated with Trypanosoma rhodesiense, o averages for rats inoculated with Trypanosoma rhodesiense and red a Complete Diet, Aaverages for uninoculated rats red a Complete Diet, D averages for rats inoculated with Trypanosoma rhodesiense and red a Folic Acid-Deficient Diet, x averages for uninoculated rats red a Folic Acid-Deficlent Diet, v averages for rats inoculated with Trypanosoma rhodesiense and red a Pair-fed Diet, O averages for uninoculated rats red a pair-fed Diet. A = rats inoculated on Day 21, B = rats inoculated on Day 56
Livers of ra~s on c o m p l e t e a n d pair-fed diets an d i n o c u l a t e d on D a y 21 with T. rhodesiense showed no significant differences in folate c o n t e n t when c o m p a r e d to control animals. H o w e v e r , livers of in/ected rats on a deficient diet showed significantly m o r e folate when c o m p a r e d with deficient uninfected controls, reaching a m a x i m u m of 362 % at D a y 25. L i v e r folate levels of rats on complete, folic acid-deficient a n d pair-fed control diets a n d i n o c u l a t e d on D a y 56 showed significantly higher values t h a n t h e controls on D a y 60.
Trypanosoma rhodesiens«: Folate Levels in Sera and Tissues of Rats
287
140
130
120
i
l I00 .=_
~o 9o
80
140
~x
DAYS A
130
120 w= ».
« ù0 I 0 0
õ .c o
.
