John E. Larsh

The relationship in mice of intestinal emptying time and natural resistance to Hymenolepis.

Mice have a strong natural resistance to H. nana var. fraterna as demonstrated by the development, usually, of much less than five per cent of the eggs administered (Shorb, 1933; Hunninen, 1935; Larsh, 1944). Although experimentally this resistance can be modified to some extent, little is known of its mechanism. There is a suggestion, however, that intestinal emptying time may be involved, since 35 per cent or more of the eggs, many still viable, pass unaltered through the digestive tract (Hunninen, 1935). If this is due to a rate of passage through the body so rapid that many of the eggs do not have time to hatch, then slowing this passage should result in the hatching and, perhaps, development of larger numbers. Since opium and morphine are utilized widely in slowing intestinal motility under certain conditions (Goodman and Gilman, 1941), it seemed desirable to test the effect of these drugs in mice to determine whether they would slow the intestinal emptying time, and, if so, whether this per se would increase the rate of development of Hymenolepis.

The effect of thiouracil and thyroid extract on the natural resistance of mice to Hymenolepis infection.

White mice about 2.5 months old appear to be the most susceptible to an initial experimental infection with Hymenolepis nana var. fraterna, whereas those past 5 months of age demonstrate the greatest resistance (Shorb, 1933; Hunninen, 1935; Larsh, 1944.) When Holtman (1946) showed that an altered metabolic rate in mice affected their natural resistance to a certain virus, it suggested a possible explanation for the above difference in response to Hymenolepis as the metabolic rate differs greatly for mice of these separate age groups. The present experiments were designed, therefore, to test the effect on natural resistance to Hymenolepis of depressing the normal high metabolic rate of young mice with thiouracil injections, and elevating the normal low rate of old mice with thyroid extract injections; these results were reported earlier in abstract form (Larsh, 1947a). As far as known, this approach differs from that used in related studies, as in these cases the normal rate of young animals alone was depressed or elevated to determine the effect on natural resistance (Todd, 1948, 1949; Wheeler et al, 1948; Whitlock, 1949).

THE EFFECT OF CONCURRENT INFECTION WITH NIPPOSTRONGYLUS ON THE DEVELOPMENT OF HYMENOLEPIS IN MICE

A few observations have been made but little or no specific work has been done to show what effect the presence of one species of parasite has on the development of a second. Brumpt (1933) noticed that the resistance of one mouse to reinfection with Hymenolepis nana var. fraterna was broken down by a secondary infection with Strongyloides. Apparently there are no reported observations on the mutual effect of two different parasites following simultaneous entry into a host. It was to obtain such information that the present study was undertaken, since the nematode, Nippostrongylus muris, and the cestode, H. nana var. fraterna, were both available in the laboratory at the same time. METHODS Quantitative doses of Hymenolepis eggs isolated from host feces which had been stored 48-72 hours were administered to mice by stomach tube, and 93-hour cysticercoid counts were made after preparing the intestine free from mucus (Larsh, 1942). Infective Nippostrongylus larvae were isolated from 10-20 day old cultures, washed by centrifugation in normal saline, and counts made by a modification of Scotts (1928) dilution count. Known numbers of larvae were injected subcutaneously into mice in the abdominal wall just medial to the flank. EXPERIMENTAL DATA

Host-parasite relationships in cestode infections, with emphasis on host resistance.

For adequate coverage of host-parasite relationships, attention must be directed not only to the many relations between host and parasite during infection, but also during the period when transmission takes place. In addition, the fascinating problems of host-parasite specificity and host-parasite adjustments should be considered. Therefore, by necessity, the present discussion of these relationships is quite incomplete, since the major emphasis will be on host resistance. As a matter of further expediency, no attempt will be made to review all of the literature, but rather selected references will be used for illustration. These deal chiefly with Hymenolepis in laboratory animals, not because of a desire to impart undue importance to this species, but because our knowledge of resistance to this cestode is fairly complete. This is no doubt due to the fact that it is the exception among cestodes, having in the same host a parenteral phase (cysticercoid) and an intestinal lumen phase (adult) permitting the study of all types of resistance. There is an added advantage of limiting this discussion for the most part to one host-parasite combination, since the various relationships are quite involved.

Comparative studies on a mouse strain of Hymenolepis nana var. fraterna, in different species and varieties of mice.

As a result of the many studies on Hymenolepis nana var. fraterna in white mice, much information has been presented on the relations between this parasite and host. Few data have been published, however, on any of these relations in other species and varieties of mice. Hunninen (1935) compared the percentage development of the parasite in white mice with that in deer mice (Peromyscus maniculatus) and other rodents, but he had opportunity to test only a small number of the wild mice. Therefore, when the writer was presented with several mating pairs of deer mice and house mice (Mus musculus), it seemed worth while to make further tests with these hosts. In addition, a series of other comparisons were made between white and dilute brown laboratory mice.

The relationship between the intestinal size of young mice and their susceptibility to infection with the cestode, Hymenolepis nana var. fraterna.

Several workers have noticed that very young mice are more resistant to infection with the cestode, H. nana var. frater,na, than are mice between two and three months of age. Woodland (1924) observed that the smallest and the largest mice are least liable to be infected naturally. Later, in controlled experiments, Shorb (1933) and Hunninen (1935) showed that considerably fewer worms develop in mice about one month old, than in those of the most susceptible age (about two and one-half months old). Although the differences are not as striking, the writer has recently noted this same relationship in a series of experiments involving young mice, most of which were 21 to 25 days old. The percentage development of cysticercoids in these mice was about six compared with about eight to ten in mice two and one-half months old. Of the suggestions offered to explain this difference, Hunninens seems most plausible. In his opinion, the shortness of the intestine and the small size of the villi in the very young mice act as a mechanical disadvantage to the normal hatching and penetrating activities of the onchospheres. If this is really the reason for their greater resistance to infection with this tapeworm, any factor that would increase their intestinal size should make them more susceptible. It has been firmly established that the anterior pituitary of higher animals has a special relation to bodily growth and splanchnomegaly. The only figures that were found which showed a specific action of this gland on the development of intestinal tissue are from experiments with juvenile pigeons (Schooley et al., 1937) in which the response was accredited to the hormone, prolactin. By repeated injections of this hormone after hypophysectomy, the body weight of the pigeons was increased by eight per cent, intestinal length by 15 per cent, empty weight (weight after contents are removed) of intestine by 10 per cent, and villus length by 17 per cent over the corresponding values found in unoperated controls. It seemed desirable to test the effect of this hormone on the development of intestinal tissue in young mice to determine whether a similar increase could be produced, and, if produced, whether it would increase their susceptibility to the tapeworm.

The role of reduced food intake in alcoholic debilitation of mice infected with Hymenolepis.

Previous studies (Larsh, 1945, 1946) have shown that alcohol, especially in high concentrations given over long periods, reduces the resistance of young mice both to initial infection and to reinfection with H. nana var. fraterna. Since the alcoholic animals in these experiments appeared to consume much less food than the controls, there was a suggestion that malnutrition may have been an important factor in producing the weakened resistance. The first objective in the present study was to obtain further evidence on the relation of the reduced food intake produced by alcoholic debilitation of mice to the reduction of their resistance to initial infection with Hymenolepis. The second objective was to see what effect supplemental vitamins would have on the reduced resistance to this parasite produced by alcoholic debilitation.

The Effect of Alcohol on the Development of Acquired Immunity to Hymenolepis in Mice.

High concentrations of alcohol have been shown recently to have a detrimental effect on the ability of mice to resist an initial infection with H. nana var. fraterna (Larsh, 1945). The purpose of the present study was to determine the influence of this drug on the development of resistance to a second infection, i.e., its influence on acquired immunity. Earlier workers have shown that healthy mice are so remarkably resistant to reinfection with this parasite that few, if any, cysticercoids can be demonstrated (Hunninen, 1935; Hearin, 1941). This immunological response is very rapid and appears to be due chiefly to the elaboration of specific antibodies which have been detected both in vivo and in vitro (Hearin, l.c.; Larsh, 1942, 1943, 1944). Therefore, reinfection of mice following debilitation would reflect an interference with the processes involved in the defense of the body. Because of the importance of learning more concerning this mechanism, it seemed of value to determine whether or not alcoholic debilitation offers such interference. Two experimental approaches were used in this study. In the first the animals were immunized by an initial infection before receiving alcohol, and were reinfected after three weeks of treatment. In other experiments, the mice first were treated daily with alcohol for several weeks and then given an initial infection after which reinfection followed from 24 hours to one week later in the various experiments.

A Comparative Study of Hymenolepis in White Mice and Golden Hamsters.

Although golden hamsters, Cricetus auratus, are known to harbor H. nana (Stunkard, 1945), there appear to be no reports giving the percentage development of the parasite or other host-parasite relationships. However, other rodents, including rats, guinea pigs, deer mice, house mice, and brown laboratory mice have been compared with white mice in such respects (Shorb, 1933; Hunninen, 1935b; Larsh, 1944). In the present work with hamsters and white mice a comparison was made of the percentage development of adults, of the length of eleven-day worms, and of the extent of the prepatent and patent periods.

Effects of alcohol on natural resistance to the dwarf tapeworm in mice.

Mice have a strong natural resistance to infection with the dwarf tapeworm, Hymenolepis nana var. fraterna. Following initial infection it is common for less than five per cent of the eggs to develop into mature cysticercoids in the intestinal villi (Hunninen, 1935; Larsh, 1943b). The underlying mechanism responsible for this resistance is undetermined, but certain factors as age, concurrent infection and splenectomy have been shown to have a favorable or unfavorable influence. The present study was an attempt to gather additional information by determining the influence of alcohol on this resistance. Tests with the drug were performed: (1) to determine the effect on resistance of various concentrations (10 to 45 per cent) given for varying periods of time; (2) to compare the effect on resistance of injections given orally with those given by peritoneum; (3) to learn whether or not alcoholic debilitation is permanent; and (4) to determine the effect on resistance of one intoxicating dose. Many workers have found large amounts of alcohol detrimental to the process of resistance to many bacterial infections. This is well illustrated by the work of Koch (1885), Doyen (1885) and Thomas (1893) on cholera; of Laitinen (1900) and Goldberg (1901) on anthrax; and of Rubin (1904) and Pickrell (1938) on pneumococcic infections. In short, numerous investigators using many different infecting agents agree that alcohol in large amounts, especially over long periods, has a definitely harmful effect on the natural defenses of many animals. Although the effect of moderate amounts of alcohol is disputed, most workers believe that small quantities (less than 1.5 cc per kilogram) have little or no effect on resistance (Laitinen, 1900; Kruschilin, 1909; Parkinson, 1909). From a review of experimental work, one concludes that the effect of alcohol on the bodily processes involved in resistance depends in large measure on the concentration of the drug and the number of injections given.