Influence of dietary cod liver oil upon Trypanosoma congolense, T. cruzi, T. vivax and T. brucei

EXPERIMENTAL

7, 255-268 (1958)

PARASITOLOQY

Influence

of Dietary

Cod Liver

Oil upon

Trypanosoma congolense, T. cruzi, T. vivax and T. bruce? D. G. Godfrey’ National Institute for Medical Research, Mill Hill, London, N. W. 7., England

(Submitted

for publication,

25 March 1956)

In 1953 Keppie reported that Trypanosoma congolense failed to develop normally in mice given a diet composed of casein, starch, salts, margarine, dried yeast, and cod liver oil. The purpose of the present paper is to report confirmation of this observation (although the suppression was not of the same high degree reported by Keppie), and to show that the rate of multiplication of T. congolense is determined by the amounts of cod liver oil and vitamin E in the diet; this demonstration parallels the results obtained with Babesia rodhaini and Plasmodium berghei (Godfrey, 1957a, b). Information is also given about the effects of dietary cod liver oil and vitamin E upon T. cruzi, T. vivax and T. bnlcei. MATERIALS

AND METHODS

The general experimental techniques and conditions were described in an earlier paper (Godfrey, 1957a). The diets used in the present work were based on the following: Basic-Diet

Casein, Light, White, Soluble Sucrose (Domestic Granulated Sugar) Glaxo Salts Mixture

20%

71% 4%

1 The work described forms part of a thesis approved by the University of London for the degree of Ph.D. and was carried out whilst in receipt of a Colonial Medical Research Studentship. * Present address: Protozoology Section, West African Institute for Trypanosomiasis Research, Vom, Plateau Province, Northern Nigeria. 255

256

GODFREY

1.5%

Dried Brewers’ Yeast Margarine (670-900 I.U. vit. A) 90 I.U. vit. D) Cod Liver Oil, “Solvitax”

3% 0.5%

Semi-Synthetic Diet with 5 % Cod Liver Oil 20% 71% 4% 5%

Casein Sucrose Glaxo Salts Cod Liver Oil

per kilo of diet

plus

5w

Thiamine Riboflavin Pyridoxine Calcium pantothenate Choline chloride Nicotinic acid p-Aminobenzoic acid Inositol

5w 5w 30 mg 500 mg 50 mg 40 mg 1 gram Control Die!

In each group of experiments there was a control group of mice fed on the Cube Diet ‘41’ as normally used in this Institute (Bruce and Parkes, 1949). Vitamin E Vitamin olive oil.

E was given orally

as a-tocopheryl

acetate, dissolved

in

Animals Albino mice of either the Parkes, Schneider or Swiss strains were used. The pre-inoculation data of each experimental group are given in Table I. The Parasites T. congolense. The strain of this trypanosome was originally isolated by Davey and it has been maintained in this laboratory for many years by syringe-passage. Inocula of 30,000 to 60,000 trypanosomes per mouse

T. brucei

T. vivax

T. cruzi 30 diet diet +

diet diet +

diet diet +

1. Cube diet ‘41’ 2. Synthetic C.L.O. 3. Synthetic C.L.O. vit. E

1. Cube diet ‘41’ 2. Synthetic C.L.O. 3. Synthetic C.L.O. vit. E

1. Cube diet ‘41’ 2. Synthetic C.L.O. 3. Synthetic C.L.O. vit. E

7

6

5

23

1. Cube diet ‘41’ 2. Synthetic C.L.O. diet 3. Synthetic C.T,.O. diet + vit. E

4

36

37

23

1. Cube diet ‘41’ 2. 5.5% C.L.O. diet 3. 5.5% C.L.O. diet + vit. E

3

23

28

1. Cube diet ‘41’ 2. 5.5% C.L.O. diet 3. Basic diet without C.L.O.

1. Cube diet ‘41’ 2. Basic diet

3,000

ca. 1 X 106

ca. 80,000

40,000

30,000

40,ooo

12,500

Inoculum (No. of Tryps.)

TABLE I Gains and Deaths

Pre-inocul’n feeding period in days

2

T. congolense 1

Experiment

Weight

5 10 10

5 10 10

10 10 10

10.9

10.6 10.6

11.5 12.0 11.6

11.18 13.3 12.5

14.2

14.0 14.6

14.0

10

10 10 10

15.0 14.2

14.4 14.7 14.5

14.2 14.5

Average wt. in grams

10 10

10 10 10

16 16

Initial No. of mice

0 4 4

0 1 0

0 1 0

0 1 2

1 2 1

No. of deaths

114 36 53

47 33

103

152 48 63

92 36 37

80 -17 -18

90 -19 -12

84 20

Average 70 incr. in wt. survivors

258

GODFREY

usually killed about half the animals after some days. The experimental animals were inoculated intraperitoneally with a known number of trypanosomes suspended in 0.2 ml titrated saline. T. cruzi. The strain used in this work was obtained from the Wellcome Laboratories of Tropical Medicine. It was originally isolated in Brazil in 1926. The inoculum for the experimental mice was prepared by pooling the heart blood of many heavily infected mice with a minimum amount of titrated saline. Inoculations were made subcutaneously. T. tivaz. The strain was isolated near Ilorin in Nigeria in 1951 and maintained in rats with the aid of a supplement of sheep serum. Virulence of the strain increased until it was possible to dispense with the supplement. The strain was also established in mice at the Wellcome Laboratories of Tropical Medicine and sent to this Laboratory in 1954. The virulence of the trypanosome to mice varied considerably from time to time but the experiments described below took place during a phase of extreme virulence. The experimental animals were inoculated with 0.2 ml of whole blood from an infected mouse with a little titrated saline. T. brucei. The strain was isolated in Nigeria in 1955 and has since been maintained in this laboratory. It is lethal to mice and experimental inoculations were carried out subcutaneously to ensure a reasonable period for counting the trypanosomes in the blood before the mouse died. The inoculum for each mouse was a suspension of infected blood in 0.2 ml titrated saline. Counting. Except in the experiments with T. cruz’1,all counts were made from fixed thin blood films stained in 5% Giemsa for 1 hour. The films were examined under a total magnification of 360 and the numbers of trypanosomes per 20 fields were estimated. In order to avoid errors due to the slight tendency of the trypanosomes to collect in masses towards the edges, counting was always carried out on a series of fields across the width of a film; the extreme edges were excluded. Since the results from groups within an experiment were always interpreted comparatively, it was felt that this technique yielded sufficiently accurate results. Since T. cruzi is a fragile organism and individuals are destroyed by fixation and staining, counts of this organism were made from wet films. Fortunately, the maximum number of trypanosomes seen in one field was low and, this, coupled with the fact that each one revolved more or less about a fixed point, enabled accurate counts to be made. Although the results reported below are quoted in terms of one experiment, each result has been confirmed at least once.

DIETARY SUPPRESSION OF TRYPANOSOMES

259

RESULTS

Trypanosoma congolense Expt. 1. The basic diet. Early in the investigation it was found that a feeding period of four weeks before inoculation of the parasites produced the best suppression; further extension of the period caused the deaths of many mice. In the following typical experiment two groups of male Swiss mice were used. Each group was fed on one of the following diets. 1. Cube Diet 41 2. The basic diet The results are shown in Fig. 1. The suppression of the trypanosomes was distinct, but not nearly as marked as the suppression reported by

CONTROLS

. /

DAYS

AFTER

INOClJLAlION

FICA 1. T. congolenee

..

260

GODFREY

Keppie (1953). Moreover, her mice given the basic diet, gained as much weight as the controls, whereas in the present work, mice given a similar diet gained only a quarter of the weight gained by the controls. Six experiments were carried out with the basic diet but a high degree of suppression was never observed. In fact, on two occasions the diet failed to suppress the trypanosomes at all. Expt. 2. Basic diet with 5.5% cod liver oil. Since experiments with Babesia rodhaini had shown that dietary cod liver oil tended to inhibit the development of the parasites (Godfrey, 1957a), it was decided to test this factor with Trypanosma congolense. Three groups of male Parkes mice were each fed on one of the following diets: 1. Cube diet 41 2. Basic diet with 5.5 % cod liver oil 3. Basic diet without cod liver oil The results are shown in Fig. 2. The trypanosomes did not develop

DAYS

AFTER

llOCULAtlOW

FIQ. 2. T. congolense

DIETARY

SUPPRESSION

OF

TRYPANOSOMES

261

as well in the mice given the high cod liver oil diet as in those fed on the basic diet without cod liver oil; in neither of these groups did the parasites multiply as well as in the controls. This experiment showed that the suppressive action of the basic diet was mainly due to the presence of cod liver oil. Expt. 3. vitamin E. The experiments with Babesia rodhaini had also shown that the suppressive action of the dietary cod liver oil was reversed by vitamin E, hence a number of experiments was carried out to investigate the relationship between these substances and infections of Trypanosoma congoknse. Three groups of male Parkes mice were each fed on one of the following diets : 1. Cube diet 41 2. Basic diet with 5.5 % cod liver oil 3. Basic diet with 5.5 % cod liver oil plus a total of 40 mg of a-tocoph-

DAYS .AFlER

IWOCULAflOW

FIG. 3. T. congolense

262

GODFREY

eryl acetate in olive oil per mouse given throughout the pre-inoculation feeding period. The results of the experiment are shown in Fig. 3. On this occasion the basic diet with cod liver oil failed to suppress the infection. The mice given the same diet plus vitamin E had infections which were much higher than those in either the control animals or the mice given the unsupplemented high cod liver oil diet. Usually, this high cod liver oil diet suppressed the parasites but this particular experiment is quoted to demonstrate that vitamin E will increase the rate of multiplication even when the oil apparently does not affect the parasites. Expt. 4. Semi-synthetic 6% cod liver oil diet. An experiment was set up to investigate the effect of a diet with a high cod liver oil concentration and most of its vitamin content from synthetic sources only. Three groups of male Parkes mice were each given one of the following diets : 1. Cube diet 41

OAIS

AflLR

lWOclJLATlOW

FIG. 4. T. congolenee

DIETARY

SUPPRESSION

OF

263

TRYPANOSOMES

2. Semi-synthetic diet with 5 % cod liver oil 3. Semi-synthetic diet with 5 % cod liver oil, plus 20 mg a-tocopheryl acetate in 0.2 ml olive oil given to each mouse on the day of inoculation. The results are expressed graphically in Fig. 4. The infections in the mice given the synthetic high cod liver oil diet were markedly suppressed, whereas those in the mice given the same diet plus vitamin E were as intense as in the controls. The experiment showed that T. congobnse infection was suppressed by cod liver oil in a diet of which the main vitamin supply came mainly from synthetic sources,and that the suppression was completely reversed by vitamin E. The suppression by the fortified high cod liver oil diet was constant. Since this was not so for the unfortified diet (see previous experiment), it seemsthat the water soluble vitamins must also influence the rate of multiplication of the trypanosome.

I 2

I 6

I 3

DAYS

AflLR

I 5

lWOCULATlDS

FIQ. 5. T. cruzi

I

I

6

I

264

GODFREY

T. cruzi Expt. 5. Semi-synthetic 6% cod liver oil diet. Three groups of female Parkes mice were each given one of the following diets: 1. Cube diet 41 2. Semi-synthetic 5 % cod liver oil diet 3. Semi-synthetic 5% cod liver oil diet plus 20 mg a-tocopheryl acetate per mouse, given orally in 0.2 ml olive oil on the day of inoculation. The results are shown in Fig. 5. The infections in each group were all of the same order. This experiment showed that a cod liver oil diet had no apparent action upon T. cruzi and that vitamin E had no effect upon the course of infection in mice fed on such a diet.

CONTROLS I-

5-

2-

I.-

-. l . I 5

2 DAYS

AFTER

I 4 INOCULATlOU

FIQ. 6. T. vivax

I 5

.\

SINWFTIC ..Y.,o. DlLl b

DIETARY

SUPPRESSION

OF

TRYPANOSOMES

265

T. vivax Expt. 6. Semi-synthetic 5% cod liver oil diet. Three groups of male Schneider mice were each given one of the following diets: 1. Cube diet 41 2. Semi-synthetic 5 % cod liver oil diet 3. Semi-synthetic cod liver oil diet, plus 20 mg a-tocopheryl acetate given on the day of inoculation. The courses of the infection are shown in Fig. 6. Marked suppression of the parasites occurred in the mice fed on the high cod liver oil diet; the animals given the same diet but with a supplement of vitamin E showed infections which were similar to those in the control animals. A high cod liver oil diet suppressed T. vivax and this suppression was almost completely reversed by vitamin E.

CONTROLS

OAlS

AFTER

. +

INOCULATION

FIG. 7.

T. brucei

266

GODFREY

T. brucei Expt. 7. Semi-synthetic 6% cod liver oil diet. Three groups of male Schneider mice were each fed on one of the following diets: 1. Cube diet 41 2. Semi-synthetic 5 % cod liver oil diet. 3. Semi-synthetic 5% cod liver oil diet, plus 20 mg a-tocopheryl acetate per mouse, given by mouth in 0.2 ml olive oil on the day of inoculation. The results are shown in Fig. 7. The infections in the mice given the diets with 5 % cod liver oil were lower than the controls on only a few days. The infections in the mice given the semi-synthetic diet alone and the same diet plus vitamin E were of the same order. This experiment suggested that T. brucei was probably unaffected by cod liver oil or vitamin E in the diet of the host. Unfortunately, in each of the two experiments carried out with T. brucei a large proportion of the mice died (see Table I). The cause of death was not known. The results from both experiments were very similar, however, but it is possible that they are misleading owing either to the low final numbers or to the unknown factor causing the deaths. DISCUSSION

The results of the present investigation confirm the report by Keppie (1953) that Trypanosma congolensehas a reduced rate of multiplication in mice fed on a certain diet. The marked degree of suppression observed by her, however, was never encountered in this work; and at times, there was no suppression whatsoever. It is unlikely that the strain of Swiss mice used by Keppie was the cause of the difference, since the experiment with this strain also showed a wide range in intensity of infections. The strains of T. congoleme, however, appear to be quite different in their virulence towards mice and this fact might have accounted for the differences between Keppie’s results and those of this investigation. Despite the apparent similarity of the basic diets, the one used by Keppie appears to be the more nutritious for the mice. Her animals had better survival rates and greater gains in weight than the mice in this work. It is possible that her mice were resistant to infection with T. congolensebecause of their good nutritional status whereas the basal diet in the present work was lacking in one or more factors which would enhance defensive mechanisms. In this respect, it is interesting to note that the high cod liver oil diet

DIETARY

SUPPRESSION

OF

TRYPANOSOMES

267

with added vitamins gave consistent suppression in contrast to the inconsistency in results with the unfortified diet. Another factor which might have accounted for the different results, is the concentration of vitamin E in the pre-experimental diet. This point is discussedfully by Godfrey (1957a) and here it suffices to suggest that Keppie’s mice derived and stored little vitamin E from their preexperimental diet. Hence, there was little reversal of the antiparasitic action of the cod liver oil in the basal diet. The converse may be true regarding the mice used in the present work. The difference in Keppie’s results then might have been due to three factors; the high virulence of the strain of T. congolense used in the present work; the better nutritional status of Keppie’s mice at the time of inoculation; and, the low vitamin E reserves of her mice. It is suggested in the earlier papers dealing with the action of the cod liver oil upon Babesia rodhaini and Plasmodium berghei that its unsaturated fatty acids are converted to peroxides by the host (Godfrey 1957a, b). These lipid peroxides may then oxidise the sulphydryl groups of some of the parasites’ enzymes; vitamin E probably prevents the conversion of the unsaturated fatty acids to peroxides. Hydrogen peroxide is toxic to trypanosomes in vitro; the toxicity in vivo is considered negligible since the catalase of the host’s blood would soon destroy it (Reiner and Leonard, 1932; Strangeways, 1937). However, catalase does not act on organic peroxides and it is possible that they are trypanocidal in vivo. Recent work shows that the respiration of T. rhodesiense in vitro is inhibited by peroxide formed during the aerobic oxidation of thiols (Fulton and Spooner, 1956). SUMMARY

(i) Trypanosoma congolense multiplied more slowly in mice given a diet composed of casein, sucrose, dried yeast, salts, margarine and cod liver oil than in mice given a normal diet. This broadly confirms the report of Keppie (1953), except that the high degree of suppression reported by her was never observed in the present work. (ii) Cod liver oil given in diets adequate in water soluble vitamins suppressed the multiplication of T. congolenseand vitamin E reversed this effect. (iii) T. vivax was markedly suppressedby a high cod liver diet and the suppression completely reversed by vitamin E.

268

GODFREY

(iv) A high cod liver oil diet did not suppress T. cruzz’,and it probably did not suppress T. trucei. ACKNOWLEDGMENTS I would like to thank Dr. F. Hawking for his advice and encouragement. My thanks are also due to Miss V. M. Smith and Mr. M. J. Worms for their technical assistance. REFERENCES BRUCE, H. M., AND PARKES, A. S. 1949. Feeding and breeding of laboratory animals. IX. A complete cubed diet for mice and rats. J. Hyg. 47, 202-208. FULTON, J. D., AND SPOONER, D. F. 1956. Inhibition of the respiration of Trypanosoma rhodesiense by thiols. Biochem. J. 63,475-481. GODFREY, D. G. 1957a. The influence of dietary cod liver oil and vitamin E upon Babesia rodhaini in mice. Exptl. Parasitol. 6,465-485. GODFREY, D. G. 1957b. Anti-parasitic action of dietary cod liver oil upon Plasmodium berghei and its reversal by vitamin E. Esptl. Parasitol. 6, 555-565. KEPPIE, A. A. N. 1953. Modified course of T. congolense infection in mice given diets with milk casein. Brit. Med. J. 2, 853-867. REINER, L., AND LEONARD, C. S. 1932. Toxic action of Hydrogen Peroxide on trypanosomes and a note on chemotherapeutic mechanism. Brit. Med. J. 29,951953. STRANGEWAYS, W. I. 1937. Observations on the trypanocidal action in vitro of solutions of glutathione and ascorbic acid. Ann. Trap. Med. Parasilol. 31,

404416.