Hymenolepis diminuta: Lack of pathogenicity in the healthy rat host

EXPERIMENTAL

PARASITOLOGY

Hymenolepis

GABLE

39, 351-357

(1976)

diminuta: Lack of Pathogenicity Healthy Rat Host DRANCH

INSLIZFI

AND

LARRY

S.

in the

ROBERTS

Department of Zoology, University of Massachusetts, Amherst, Massachusetts 01002, U.S.A. (Accepted

for pubhcation

30 June

1975)

INSLER, G. D., AND ROBERTS, L. S. 1976. Hymenolepis diminuta: Lack of pathogenicity in the healthy rat host. Experimental Parasitology 39, 351357. Whether Hymenolepis diminuta (Cestoda: Cyclophyllidea) might affect adversely the growth of its host under normal conditions was studied. Rats were divided into four experimental groups: ( 1) rats infected with the tapeworm and fed ad lib&urn, (2) uninfected rats fed ad libitum, (3) and infected rats fed isocalorically with (4) uninfected rats. Growth rates of infected rats did not differ from infected animals. Infected, meal fed rats Iimited to 15 g synthetic diet/day grew as rapidly as their uninfected counterparts, and infected rats fed ad libitum did not consume more food than the comparable infected group. There were no significant differences in consumption or in excrement produced between groups ( 1) and (2) and groups (3) and (4). Weights attained by the worms were not affected by mode of host feeding (ad libitum or meal fed), whether expressed as wet or dry weight. Since H. diminuta appears not to affect nutrient utilization or consumption in a healthy, unstressed host, at least on a gross level, it probably should be considered an endocommensal. INDEX DESCRIPTORS: Hymenolepis diminuta; Pathogenicity; Rat; Growth; Nutrition.

The term “parasite” is not always used rigorously, and its definitions may emphasize some manner of damage or expense to the host (Henry 1966; Read 1970), an intimate association involving metabolic dependence (Noble and Noble 1971; Cheng 1973), or a phenomenon whereby the parasite feeds directly off the host’s tissues and fluids (Sprent 1963; Dogiel 1966). Definitions ignoring pathogenic effects tend to become blurred into the concept of commensalism. Although harm to the host need not be the sole criterion for parasitism, it can be used most beneficially in conjunction with the newer concepts of metabolic dependence. Read (1970) specified that parasitism is a symbiosis in which one of the associates “profits significantly” at the expense of the other. He pointed out that “parasitologists have tended to claim certain groups of

symbiotes as parasites, although in many of the cases the clauses in the (profit-loss) contracts are not known.” Rees ( 1968) held that pathogenic@ by adult tapeworms is often lacking because they normally have an overabundance of food available and rarely stimulate the host’s defense mechanisms. Even damage to the host’s intestinal lining due to hooked scolices embedded deep in the mucosa is usually localized and minor. With regard to the rat tapeworm, Hymenolepis diminuta, there has been little systematic investigation of the gross effects of the symbiote on the host. Comparisons of such basic physiological parameters as growth rates, food consumption, water intake, and waste production between infected and uninfected rats have not been previously studied. Bailey ( 1970) observed that there were no differences in the 351

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0 1976 by Academic Press, Inc. of reproduction in any form reserved.

352

INSLER AND ROBERTS

amount of food consumed by 49 infected males and 10 uninfected male rats in a 24 hr feeding period. The observations were peripheral to Bailey’s study of circadian movements of H. diminuta in the rat host, were for a short length of time, and involved only single worm infections. He gave no data on the host weights, and the worms were young (9 days old) at the time of measurement. Mettrick (1971) investigated the microbial flora, nutritional gradients, and physicochemical characteristics in the small intestine of rats infected with H. diminuta compared with these parameters in uninfected rats. He found (Mettrick 1972) only about half as much glucose (TCA soluble carbohydrate) in the intestine of infected rats as in uninfected ones following a 1 g glucose test meal preceded by 18 hr starvation. Podesta and Mettrick (1974) investigated changes in oxygen tensions and acid-base balance in the gut lumen and mucosal absorption and accumulation of fluid and glucose, which apparently were induced by the presence of H. diminuta. None of these papers, however, directly addressed the question of whether H. diminuta might affect food consumption and utilization by the whole rat under normal conditions. The present work attempts to determine the balance in the ‘profit-loss contract” by observing gross parameters related to the host’s physiology, i.e., whether, in fact, H. diminuta is living at the “expense” of the healthy rat host.

rina Lab Chow up to 10 days postinfection. Starting on the 10th day postinfection, all rats were fed a synthetic diet modified from the Low Roughage diet of Roberts and Platzer (1967) to include the Williams-Briggs Modified salt mixture (Cohen et al. 1967). This diet proved adequate for normal development of both rat and worm. The salt mixture was obtained from General Biochemicals and the other dietary components were from Nutritional Biochemical Co. After being fed for 5 days on the synthetic diet (Days 11-15 postinfection) the rats were assigned to four equal groups as follows: (1) infected rats fed ad Zibitum, (2) uninfected rats fed ad Zibitum, (3) and infected rats fed isocalorically with (4) uninfected rats at 15 g per day. These meal fed rats were fed at OS30 hr and always finished their ration during the 24 hr period. The rats were caged singly in metabolism cages ( Hoeltge, Inc. ) for the duration of the experiment. The cages were equipped with calibrated water bottles, food tunnels, and feces/urine separators. The urine collecting tip was fitted with a 3 in. length of rubber tubing leading to the mouth of a 30 ml glass vial. The fecal pellets rolled past the tight fitting tube and down into tared aluminum cups. Any fecal pellets remaining stuck to the collector were removed each day for weighing along with those in the cup. The food tunnels were designed to eliminate contamination of excrement with food. The diet was finely strained so that no large chunks of food could be carried back to MATERIALS AND METHODS the main cage area and be dropped or Cysticercoids of H. diminuta were dis- thrown out. Any food left clinging to the sected from Tribolium confusum reared at rat’s whiskers was automatically brushed 39 C on Quaker Oats. Male Holtzman rats away by the narrow sides of the feeding weighing 90-110 g were lightly anesthe- tunnel and fell into a plastic tray directly sized and given 10 cysticercoids each via below, along with any other food spilled in feeding. The diet remaining in this tray stomach tube. Twelve rats were inoculated and 12 were left uninfected to serve as was weighed each day and added to the controls. Rats were fed ad Zibitum on Pu- rest of the diet not eaten. Therefore, the

PATHOGENICITY

0~ Hymenobpis

amount of food actually eaten was determined with accuracy. At 15 days postinfection, and every day thereafter, until 35 days postinfection, feces produced, water consumed, urine produced, food eaten, and rat weight were recorded. Wet feces was dried for 24 hr at 95 C and weighed. Observations were carried out in the same order each day, so that all the measurements would most closely represent a 24 hr period, even though the actual observations ran from 0800 to 1000. At 35 days postinfection the rats were killed by a blow on the head and the worms recovered as in Roberts ( 1961). Twelve worms were chosen at random from each of the infected groups and weighed both wet (after blotting) and dry (24 hr at 100 C). Differences in results were considered significant if PsO.05 by Student’s t test. RESULTS

No differences were found between the growth rates of the uninfected and infected rats fed ad Zibitum (Fig. 1). The meal fed rats grew at a slower rate than their ad Zibitum counterparts, but infection with H. diminuta did not affect growth rate (Fig. 2). A slight increase in the growth rate of the meal fed rats can be seen between Days 22 and 25 postinfection. On Days 21 and 22 two rats had very small

353

IN RAT

320 -Infected -.--‘-. Uninfected

200

f

240

E

‘ /

200 ,” 8 160

I

’

3

.

5

7

9 II Days

13 15 17 I9

21

FIG. 2. Growth of rats meal fed (Groups 3 and 4) beginning 15 days postinfection. Points are wt f SE, n=6. () = Infected, mean (- - - - -) = Uninfected.

weight gains; meal portions for all rats were increased to 17 g so that weight loss in any rat as a potential variable might be obviated. By Day 26 all rats were again growing at a satisfactory rate, and meal portions were returned to 15 g. Daily weight gains were continuously monitored, and at no other time did weight loss in any host appear imminent. There were no differences between infected and uninfected groups in the quantity of food eaten per unit weight, even in the ad libitum fed rats, where they were presented with an unlimited food supply (Fig. 3). Feces production as a function of food consumption was measured. Dry feces per 10 g food consumed was between 0.5 and

-Infected -I’-‘_

Uninfected m

160-

g L

I

3

5

7

9 II 13 DOYS

15 17 19 21

FIG. 1. Growth or rats fed ad Zibitum (Groups 1 and 2) beginning 15 days postinfection. Points are mean wt*SE, n=6. () = Infected, (- - - - -) = Uninfected,

4.0 I

3

5

7

9 II Days

I3 15 I7 I9

FIG. 3. Ratio of food consumed to body respect to time postinfection for rats fed turn. Points are mean ratios, n = 6. ( -) fected, (- - - - -) = Uninfected,

21

wt with ad Zibi= In-

354

INSLER

AND

TABLE Ratios

of Dry

Group

1 2 3 4

Feces

to Food

Eaten

Treatment

Infected, ad lib&m fed Uninfected, ud lib&m fed Infected, meal fed Uninfected, meal fed

0.9 g, and there were no discernible differences between groups (Table I). The moisture of the feces fluctuated with no observable patterns or differences according to group (Fig. 4). The moisture content in all groups tended to increase at periods about 1 week apart. These peaks were attributed to an increased humidity of the animal room corresponding to the weekly floor washing. The relationship of water consumption to food eaten was compared between the four groups (Table I). No patterns could be observed, but high individual variation in both food and water intake may have made detection of minor trends (if any) impossible. Water balance on a gross level was examined as a ratio of water consumedlvolume urine produced (Fig. 5). Again, vari-

ROBERTS

I

and of Water

Consumed

Grams food

to Food

dry feces/l0 g mean and SE n = 20 0.71 0.65 0.69 0.67

& f f xt

Eaten Milliliters water/l0 g food mean and SE 72 = 21

0.02 0.01 0.02 0.02

13.5 13.8 14.0 15.3

f f f f

0.3 0.3 0.4 0.3

ability may have obscured differences caused by the presence of H. diminuta. In addition to inherent variation in urine production between rats, time of urine production may have contributed to the fluctuations observed, i.e., more would have been lost by evaporation from urine produced early in the 24 hr period than that passed shortly before collection. Presumably, such factors would be randomized between groups but might help to explain some of the variability. The ratio of water consumption to body weight was calculated for all groups (Fig. 6). Since weights were increasing at a fairly constant rate within each group (ad Zibitum and meal fed), the resulting curves were regular but failed to demonstrate any relationship for the infected rats that was different from the uninfected rats. The only trend detectable was a general decrease in

,

I

3

5

,

7

,

9

, , , , , , , ,

II 13 15 17 19 21

Days

FIG. 4. Percentage moisture in feces for all four groups with respect to time postinfection. Points ) = Inare mean ratios, n = 6 in all cases. (fected, ad Z&turn; (- - - - -) = Uninfected, ud Z&iturn; (- * - * -) = Infected, meal fed and (- - -)

= Vninfected,

meal fed.

FIG. 5. duced for infection. = Infected, Zibitum; (- - -)

Ratio of water consumed to urine proall four groups with respect to time postPoints are mean ratios, n = 6. (---) ad Zibitum; (- - - - -) = Uninfected, ad (- . - * -) = Infected, meal fed and = Uninfected, meal fed.

PATHOGENICITY

$ z

6.01,,

, , , , , , , , , , I

3

5

7

9 II Days

, , ,

Hymenolepis

OF

, , , ,

13 15 17 19 21

FIG. 6. Ratio of water consumed to rat weight for all four groups with respect to time postinfection. Points are mean ratios, n = 6. ()= Infected, ad Z&turn; (- - - - -) = Uninfected ad Zibitum; (- - - - -) = Infected, meal fed and (- - -) = Uninfected, meal fed.

the ratio as the rats increased in size, but this was common to all the groups. Weights attained by the worms were not affected by mode of host feeding (ad Zibiturn or meal fed), whether expressed as wet or dry weight (Table II). DISCUSSION

Hymenolepis diminuta appears to have little affect on the nutrient consumption and utilization of its rat host. Neither growth rates, food intake, nor levels of excrement produced were significantly different between infected and uninfected groups. The design of the experiment would have allowed for detection of harmful effects on host growth rate on an isocaloric feeding regime, and if so, whether the effects might be ameliorated by compensatory feeding if the food was presented ad libitum. TABLE Effect

of the Feeding

Group

Ad libitum fed rats Meal fed rats

Regime

on Wet

Wet weight (mg), mean and SE (n = 12) 326.9 298.8

f f

15.9 15.0

IN

355

RAT

Mettrick (1971) also found that infection with H. diminuta did not affect host food consumption. He reported that the food eaten by male rats weighing 140-160 g was comparable over a 24 hr period for infected (10 worm infection of H. diminuta) and uninfected rats, observing 10.9 + 1.0 g/100 g body weight and 11.5 -C 0.9 g/100 g body weight, respectively. The rats used were of “similar size” and were carrying either 13 or 17 day old worms during the 24 hr period of measurement. Thus, Mettrick maintained that various physicochemical differences observed between the infected and uninfected rat small intestines could not be attributed to differing levels of food consumption. The lower levels of total lipid, TCA soluble and insoluble nitrogen, lowered pH and increased pCOZ, as well as reduced glucose and fluid absorption in the infected small intestine (Mettrick, 1971; Podesta and Mettrick, 1974) must be, in light of the present results, of little consequence to the rat. It is also interesting that there was a reduction in the microbial flora in the intestine of infected hosts, both small intestine and colon, to half the usual number of microorganisms, Eschichia coli and other coliforms decreasing most markedly (Mettrick, 1971). Although one might anticipate an effect on rat growth with half the microbial population absent (presumably including some helpful vitamin and amino acid synthesizing bacteria), this apparently does not occur. No direct competition for food between worm and host results from the change in flora or if such a competition does exist, it was too small to affect the II

and Dry

Weights Dry

of Hymenolepis

weight (mg), mean and SE (n = 12) 72.42 67.26

f 4.16 f 4.63

Diminuta Dry/wet weight, mean and SE (n = 12) 0.221 0.224

f 0.005 f 0.009

356

INSLER AND ROBERTS

parameters measured in the present ex- plored, but much interesting information periments. on the host-parasite relationship might acMettrick ( 1972) found that following ad- crue from such investigation. ministration of a 1 g glucose test meal, ACKNOWLEDGMENT TCA soluble gut contents, hepatic and sysThis investigation was supported by Research temic blood glucose, and liver TCA-soluble Grant AI-06153 from the National Institute of carbohydrate were lower in rats infected Allergy and Infectious Diseases, Bethesda, Marywith H. diminuta compared with unin- land 20014. fected rats. Liver TCA-insoluble carbohyNote added in proof. Mettrick [1973. Canadian drate was initially lower in infected animals Journal of Public Health 64 (Monograph Suppl.), but was not different 2 hr after the test 701 reported a 20% reduction in growth rate and meal. The rats had been starved for 18 hr an 18% increase in caloric intake per gram increase in weight of young rats infected and fed prior to the test feeding. He concluded that ad Zibitum. Though this observation appears at the degree of deprivation of host nutrients variance with the present report, Mettrick only by the worm questions the assumption that studied the 24 hr period from 15-16 days after intestinal parasites, such as tapeworms, do infection. Our more extended study showed that observations alone are of uncertain not significantly affect host nutrition, but short-term because of the great daily variation. he recognized that the “high level of preda- reliability Because Mettrick (1973) reported host weight tion by the worms on host nutrients was gains without indicating the sample size or stanprobably due to the experimental condi- dard errors, interpretation of his results in relations utilized and the small amount of food tion to ours is even more difficult. fed.” We believe that this also explains REFERENCES why the proportion of dietary calories conG. N. A. 1971. Hymenolepis diminuta: sumed by the worms did not lead to a de- BAILEY, Circadian rhythm in movement and body length crease in growth rate of their hosts in the in the rat. Experimental Parasitology 29, 285present experiments. On the other hand, 291. Mettrick (1972) referred to unpublished CHENG, T. C. 1973. “General Parasitology.” Acaresults showing that H. diminuta caused a demic Press, New York. COHEN, N. L., REYES, P., TYPPO, J. T., AND BRIGGS, 20% reduction in growth rate and an IS% G. M. 1967. Vitamin B-12 deficiency in the increase in the caloric intake per gram ingolden hamster. Journal of Nutrition 91, 482crease in weight of young rats fed ad libi488. turn. There seems to be no ready explanaDOGIEL, V. A. 1966. “General Parasitology.” Acation for this discrepancy with our results. demic Press, New York. Vol. I. Academic The results of this study support Sprent’s HENRY, S. M. 1966. “Symbiosis,” Press, New York. (1963) suggestion that cestodes should be METTRICK, D. F. 1971. Hymenokpis diminuta: regarded as endocommensals. Nevertheless, The microbial fauna, nutritional gradients, and it is possible that H. diminuta could have physicochemical characteristics of the small intestine of uninfected and parasitized rats. Caa deleterious effect on its host under connadian Journal of Physiology and Pharmacology ditions other than those employed here. 49, 972-984. Our object was to determine whether the METTRICK, D. F. 1972. Changes in the distribuworm was living at the rat’s expense under tion and chemical composition of H. diminutu, and the intestinal nutritional gradients of unnormal conditions, i.e., unstressed hosts in infected and parasitized rats following a glugood health and provided with adequate cose meal. Journal of Helminthology 46, 407food and water. Any of a variety of stress 429. D. F. 1973. Conadian Journal of Pubconditions could be applied: feeding nu- METTHICK, lic He&h 64 (Monograph Suppl.), 70. tritionally suboptimal or bare maintenance NOBLE, E. R., AND NOBLE, G. A. 1971. “Paradiets, provoking psychological or pharmasitology. The Biology of Animal Parasites,” 3rd cological distress, etc. These were not exed. Lea and Febiger, Philadelphia,

PATHOGENICITY’

OF

PODESTA,R. B., AND METTRICK, D. F. 1974.Pathophysiology of cestode infections: Effect of Hymcnolepis dimintbta on oxygen tensions, pH and gastrointestinal function. International Journal for Parasitology 4, 277-292. READ, C. P. 1970. “Parasitism and Symbiology.” Ronald Press, New York. REES, G. 1968. Pathogenesis of adult cestodes. Helminthological Abstracts 36( 1) , l-23.

~tjVX?defIi$

density ROBERTS,

IN

357

RAT

on patterns L. S. 1961.

and physiology of growth The influence of population diminuta in the definitive host.

in Hymenolepis Experimental Parasitology

11,

332-371.

ROBERTS, L. S., AND PLATZER, E. G. 1967. Effects of changes in host dietary carbohydrate and roughage on previously established Hymenolepis diminuta. journal of Parasitology 53, 85-93. SPRENT, J. F. A. 1963. “Parasitism.” Williams & Wilkins, Baltimore.