Camp. Biochem. Physiol., 1975, Vol. 51A, pp. 207 to 211. Pergamon Press. Printed in Great Britain

TRYPANOSOMA LEWZSZ: BODY WEIGHT GAINS AND FOOD CONSUMPTION OF RIBOFLAVIN-DEFICIENT RATS GIVEN LIVING CELLS, CELL HOMOGENATES AND CELL

METABOLIC PRODUCTS

CLARENCE M. LEE AND GEORGIANA ABOKO-COLE

Department of Zoology, Howard University, Washington, D.C. 20001, U.S.A.

(Received 15 January 1974)

Abstract-l. Riboflavin deficiency was studied in rats infected with Trypanosoma lewisi. 2. Body weight gains in rats on complete, riboflavin-deficient or pair-fed control diets and given

living cells or homogenate of T. Iewisi showed significant increases over uninoculated controls. 3. Irrespective of dietary group, animals receiving living cells or homogenate of T. lewisi ate more

food than the control animals beginning 12 days after inoculation. 4. Regardless of diet, no differences in weight gain or food consumption were seen in animals

inoculated with physiological saline or metabolic products of T. lewisi when compared with un- inoculated controls.

INTRODUCTION consumption of rats fed a riboflavin-deficient diet

PAFWITISM is a universal phenomenon which results and inoculated with Trypanosoma lewisi.

in graded alterations of the host’s biochemistry, morphology or physiology. Present evidence , sug-

MATERIALS AND METHODS

gests that dependent cells produce their adverse Experimentalhosts effects through impairment of the host’s nutrition (Chandler, 1953; Geiman, 1958).

Two hundred and seventy female albino rats (Sprague- Dawley) weighing 49-51 g were used in three separate

We report here on the body weight gains and food experiments. Table 1 shows the distribution, number

Table 1. Average initial body weight &SD. of rats fed a complete riboflavin deficient and pair- fed control diets and inoculated with physiological saline, Trypanosoma fewisi, homogenate and metabolic products of Trypanosoma fewisi (number of animals employed in parentheses)

Experiment no. L H M P N

A 1 49k 1 (6) 50+ 1 (6) 49+0*9 (6) 5OLtI: 1 (6) 5Ok O-9 (6) 2 50+1 (6) 49+1 (6) 51+1 (6) 50+ l(6) 51+1(6) 3 50+ 1 (6) 5Ok 1 (6) 5Ok 1 (6) 49&-l (6) 50+1 (6)

B 1 49+ 1 (6) 51kl (6) 50+ 1 (6) 49+ 1 (6) 50+@8 (6) 2 5Ok 1 (6) 5OAO.8 (6) 51+1 (6) 50+1 (6) 50 + 0.9 (6) 3 51kl (6) 5Ok 1 (6) 50+ 1 (6) 50+1 (6) 4921 (6)

C 1 50+ 1 (6) 51kl (6) 49+ 1 (6) 50 f 0.7 (6) 50+1 (6) 2 50+@8 (6) 50+ 1 (6) 50+ 1 (6) 50+ 1 (6) 49+1 (6) 3 51+@9 (6) 5Ok 1 (6) 50+ 1 (6) 51kl (6) 50+ 1 (6)

A Complete diet group. H Homogenate (Triturated trypanosomes). B Ribollavin deficient diet group. M Metabolic products. C Pair-fed diet group. P Physiological saline. L Living trypanosomes. N Non-infected.

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208 CLARENCE M. LEE AND GEORGIANA ABOKO-COLE

and initial body weights of rats together with the several dietary groupings forming the structure of each experi- ment.

Experimental diets

The vitamin B complex complete and the riboflavin- deficient diets were obtained commercially from Nutri- tional Biochemicals, Cleveland, Ohio.

Housing and feeding of rats

Rats were housed individually in wire-bottomed cages and fed the appropriate diets from metal feeding cups designed to minimize spillage of food. All rats in the control-diet group and riboflavin-deficient group were allowed to feed at will. Rats in the pair-fed group were given the control diet daily in amounts equal to the food consumed by the riboflavin-deficient paired mates.

The daily food intake of each rat was determined by subtracting the amount of food remaining in the tared cup from the amount given the previous day.

The rats were provided with water continuously. Water bottles and feeding cups were cleaned daily and cages were cleaned frequently to minimize algal and bacterial contamination.

Inoculation of rats with trypanosomes

Twenty-eight days after initiation of a dietary regimen, one half of the rats in each dietary group was injected with 100 T. Zewisicells in all experiments.

Rats were inoculated intraperitoneally with 1 ml of a suspension of trypanosomes in physiological saline.

Suspensions of trypanosome cell free from host tissue were prepared by the method of Lincicome & Watkins (1963). The number of trypanosomes per unit volume of suspension was estimated with red blood cell pipette, hemacytometer, Toisson’s fluid and a constant dilution factor of 200 x (Liiicicome &Watkins 1963).

Measurement of host growth andfood consumption

Weight gains and food intake were determined for all rats in all experiments; each rat was weighed on a laboratory balance having a sensitivity of 0.05 g. Weights were taken every 5 days, and the gains were expressed as cumulative average percentage relative to initial weights. All computations for food consumption were made as 5-day averages.

Trypanosome metabolic products and triturated cells (homogenate)

Metabolic products and homogenates of T. lewisi were prepared by the method of Thillet & Chandler (1957). Normal physiological saline was incubated and stored under the same conditions as the metabolic products and homogenate.

The inoculation of metabolic products and triturated cells was started on day 28 after the initiation of each experiment at the point where the riboflavin-deficient animals began to show physical signs of the deficiency syn- drome.

In all three experiments, rats on complete, riboflavin- deficient and pair-fed control diets were each given 1 ml of metabolic products of 10s trypanosomes intraperitone- ally at 3-day intervals. A second group of rats fed similar diets were given intraperitoneal injections each of 1 ml of homogenate having lo* triturated trypanosomes per ml of saline suspension.

A third group of rats were inoculated with comparable volumes of physiological saline in the same manner as experimental animals were inoculated with homogenates or metabolic products.

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Fig. 1. Per cent body weight gains and food consumption of rats given a full complement diet. L, Averages for rats inoculated with living cells of T. Zewisi; H. averages for rats inoculated with homogenate of T. lewisi; M, averages for rats inoculated with metabolic products of T. Zewisi; P, averages for rats inoculated with physiological saline;

and N, averages for uninoculated rats.

Riboflavin deficiency in Trypanosoma-infected rats 209

STATISTICAL EVALUATION

The data were studied as averages calculated from all experiments. Statistical treatment of the data was limited to expression of standard deviations and application of Student’s f-test for significance of differences of means having a probability level of 1 per cent or lower.

RESULTS

Figure 1 shows the rates of food intake and body- weight gains in animals fed a full complement diet and inoculated with physiological saline, living cells of T. Zewisi and derivatives of T. lewisi (metabolic products and homegenate). In terms of percentage of control values, the body weight gains of infected animals became statistically significant from day 30 and continued until the last day of observation. The overall difference in percentage gain ranged from 1 to 26 per cent. With respects to control values animals inoculated with homogenate showed signifi- cant increases from day 35. The greatest weight gain, in these group of animals, occurred at day 45 (27 per cent) and declined thereafter to 13 per cent by day 60. Animals inoculated with physiological saline or metabolic products of T. lewisi showed no weight advantage when compared with control rats. Infected animals consumed significantly more food than non-infected rats, beginning 12 days after inoculation. At day 40, infected animals ate 27 per cent more food than controls; by day 60, the differences increased to 30 per cent. In all three experiments, animals receiving the homogenate ate more food than control animals beginning 17 days after inoculation. There were no significant differ- ences in the quantity of food consumed by rats given metabolic products and physiological saline when compared with uninoculated controls.

The rates of food intake and body weight gains in animals fed a riboflavin-deficient diet and inocu- lated with physiological saline, living cells of T. lewisi and derivatives of T. lewisi are graphically illustrated in Fig. 2. In all experiments, the infected deficient rats gained more weight than non-infected control rats. In the latter days of the experiments, the rats experienced weight lost. The deficient rats, in this case, lost less weight than non-infected rats. Greatest weight gains were shown in rats receiving the homogenate. In terms of percentage advantage over controls, growth reached a maximum of 11 per cent by day 50 and declined to 5 per cent by day 60.

There were no essential differences in body weight gains of rats given a riboflavin-deficient diet and inoculated with physiological saline and trypanosome metabolic products when compared with control rats. Trypanosome-infected, riboflavin-deficient rats consumed significantly more food than non-infected controls beginning at day 45 of observation. Animals given the homogenate of T. lewisi consumed signifi- cantly more food than non-infected rats, beginning at day 45. There were no significant differences in the

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Fig. 2. Per cent body weight gains and food consumption of rats given a r&of&n-deficient diet. L, Averages for rats inoculated with living cells of T. lewisi; H, averages for rats inoculated with homogenate of T. lewisi; M, averages for rats inoculated with metabolic products of T. Iewisi; P, averages for ratsinoculatedwith physiological

saline; and N, averages for uninoculated rats.

quantity intake of food by animals fed a deficient diet and inoculated with physiological saline and metabo- lic products of T. lewisi when compared with uninoculated controls.

Data on body weight gains in animals fed a pair- fed diet and inoculated pith physiological saline, living cells of T. lewisi and derivatives of T. lewisi are shown in Fig. 3. Trypanosome-infected, pair-fed control rats gained more weight than non-infected animals, reaching a maximum of 22 per cent by day 50. The rats receiving the homogenate gained more weight than non-infected controls. In terms of percentage of control values, the greatest weight gain occurred at day 50 (25 per cent) and declined there- after to 17 per cent by day 60. Although the pair-fed animals inoculated with trypanosome metabolic products and physiological saline showed no statistically significant increases, the differences in body weight gain over the control ranged from 2-l 1 per cent for the trypanosome-metabolic product group and 2-8 per cent for the physiological saline group.

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Fig. 3. Per cent body weight gains and food consumption of rats given a pair-fed diet. L, Averages for rats inocu- lated with living cells of T. lewisi; H, averages for rats ino- culated with homogenate of T. lewisi; M, averages for rats inoculated with metabolic products of T. Zewisi; P, averages for rats inoculated with physiological saline;

and N, averages for uninoculated rats.

DISCUSSION

Body weight gains of control animals showed similar patterns of growth stimulation as have been previously noted in the laboratory (Lincicome et al., 1960, 1963; Lincicome & Shepperson, 1961, 1963). Infected animals given the complete diet showed increments ranging up to 26 per cent (Fig. 1). This was further supported by the fact that animals given the complete diet and inoculated with the homo- genate of T. lewisi showed increments ranging up to 27 per cent (Fig. 1). Experimental animals deficient in riboflavin showed typical patterns of weight loss but the uninfected animals suffered a greater loss than infected ones (Fig. 2).

This agrees with the previous observations of Lincicome & Shepperson (1963) that experimental animals infected with trypanosome grow faster than normal (uninfected) ones. Lincicome & Shepperson (1965) reported evidence of molecular exchanges between a dependent trypanosome cell and its host.

The results of these studies showed that T. lewisi can stimulate growth of the rats by increasing metabolic activities of the host cells. Since the lack of thiamine induced biochemical defects in rat tissue, and the presence of T. lewisi moderated the defect that thiamine alone could correct, theoretically T. lewisi supplied thiamine to the deficient host. In the present study, several additional parameters have been observed: (1) animals given the complete or ribo- flavin-deficient diet and inoculated with the homo- genate of T. lewisi; (2) animals given the complete or riboflavin-deficient diet and inoculated with the metabolic products of T. lewisi.

It is clear from the above discussion that T. lewisi can supply essential growth substances to the host. This factor may well account for the increased rate of growth demonstrated by riboflavin-deficient animals infected with T. lewisi as compared to the lack of growth exhibited by their metabolic product mates.

It is obvious from this study that the nutritional relationship of protozoans to the well-being of the host should be investigated to a greater extent. Critical analyses of parasitic action in specific deficient states may be complicated by inter-relation- ships of other vitamins and/or hormones. Results of this study must therefore be interpreted cautiously especially in the light of information linking ribo- flavin with thiamine, ascorbic acid, vitamin Bra and functions other than its classical role in oxidation- reduction reactions (Ferrebee & Weissman, 1943 ; Cimino, 1947; Whitby, 1954; Lavate & Sreenivasan 1959).

Food consumption in any host should be closely aligned to the efficiency of food utilization. In case of an infected organism, the inter-relationship between the host and parasite is an important factor in food consumption.

Voris et al. (1942) found that riboflavin possessed specific growth-promoting effects unrelated to appetite. Mamrering & Elvehjem (1944) observed modem inanition in riboflavin-deficient rats which did not cause death. These investigators indicated that deficient animals maintained relatively high levels of food consumption which often increased before death. Vaughan & Vaughan (1959) suggested that growth response of riboflavin-deficient rats subjected to low environmental temperatures (stress) was a function of appetite and efficiency of food utilization.

As in previous studies (Lincicome & Shepperson, 1965), there was a significant increase in food consumption in infected rats on complete diet over uninfected rats. In the riboflavin-deficient group, the average food consumption by the infected rats ranged from 1.1 to 2.2 g over the uninoculated controls. The food intake by rats inoculated with metabolic products did not show any significant increase over the uninoculated controls in any of the dietary groups. The rats inoculated with T. lewisi

Riboflavin deficiency in Trypanosoma-infected rats 211

homogenate showed significant increase in food consumption in all the dietary groups.

It is possible that a parasitic organism such as a trypanosome can deplete the food supply of the host and in order to compensate for this depletion, the host must increase the intake of food. This line of reasoning seems logical for infected animals but fails to explain the results obtained with rats inocu- lated with homogenate or metabolic products.

The whole concept of stress and infection that was introduced by Lincicome et al. (1963) must be re-examined since animals inoculated with physiolo- gical saline showed no increase in food consumption in any dietary group.

REFERENCES

CHANDLER A. C. (1953) The relation of nutrition to parasitism. J. Egypt. Med. Assoc. X&533-552.

CIMINO S. (1947) Intervitaminic correlations; relation between ascorbic acid and riboflavin. Arch. Sci. biol. 32,37-45.

FERREBEE J. W. & WEISSMAN N. (1943) Riboflavin and thiamine interrelationships in rats and in man. J. Nutr. Z&459469.

GERMAN Q. M. (1958) Nutritional effects of parasitic infections and disease. Vitamins and Hormones 16, l-33.

LAVATE W. V. & SREEE~~~ASAN A. (1959) Metabolic interrelationships of dietary riboflavin and vitamin B,, in the rat. Br. J. Nutr. 13,468-474.

LINCICOME D. R., ROSSAN R. N. & JONES W. C. (1960) Rate of body weight gains of rats infected with Try- panosoma lewisi. J. Parasit. 46,42.

LINCICOME D. R., ROSSAN R. N. & JONES W. C. (1963) Growth of rats infected with Trypanosoma lewisi. Expl Parasit. 14,54-65.

LINCICOME D. R. & SHEPPER~ON J. (1961) Increased rate of growth of experimental hosts with foreign autono- mous cells. The Physiologist 4,65.

LINCICOME D. R. & SHEPPERSON J. (1963) Increased rate of growth of mice infected with Trypanosoma duttoni. J. Parasit. 49,3 l-34.

LINCICOME D. R. & SHEPPERSON J. (1965) Experimental evidence for molecular exchange between a dependent trypanosome cell and its host. ExpZ Parusit. 17, 148- 167.

L~CICOME D. R. & WATKINS R. C. (1963) Method for preparing pure cell suspensions of Trypanosoma lewisi. Am. Inst. Biol. Sci. Bull. 13.53-54.

MANNERING G. J. & ELVEHJEM C. A. (1944) Food utiliza- tion and appetite in riboflavin deficiency. J. Nutr. 28, 157-163.

THILLET C. J. & CHANDLER A. C. (1957) Immunization against Trypunosoma Zewisi in rats by injection of metabolic products. Science, Wash. 125,346347.

VAUGHAN D. A. & VAUGHAN L. N. (1959) The effect of a low environmental temperature on the weight and food consumption of riboflavin deficient rats. J. Nutr. 68,485-493.

Vorus L., BLACK A., Swrm R. W. & FRENCH C. E. (1942) Thiamine, riboflavin, pyridoxine, and pantothenate deficiencies as alfecting the appetite and growth of the albino rat. J. Nutr. 23,555-566.

Wnrrny L. G. (1954) Transglucosidation reactions with flavine. Nature, Lond. 57,390-396.

Key Word Index-Trypanosoma lewisi; growth; ribo- flavin: metabolism; vitimins; rats; food consumption; protozoa.