Justus F. Mueller

IN VITRO CULTIVATION OF SPIROMETRA MANSONOIDES (CESTODA) FROM THE PROCERCOID TO THE EARLY ADULT.

Spirometra mansonoides has been grown in vitro from the procercoid to the young adult using the methods of Mueller (1959) and Berntzen (1961, 1962, 1964). The process involves scolex differentiation, shedding of the larval body, growth from 0.1 mm to 64 mm in length, and segmentation to form the strobila. Successful cultures had a gas phase of 95% N2/5% CO2 or 90% N2/10% CO2 and were preceded by an evaginating solution containing bile salts and trypsin. The medium is complex but does not contain chick embryo extract. Incubation temperature was 39 C. Oxygen is toxic to the cultures. Lack of C02 or omission of the evaginating solution resulted in no growth. Essentially twothirds of the life cycle of this tapeworm has now been accomplished in vitro in the absence of the normal hosts: snake or mouse, and cat. It has been demonstrated that the tapeworms, Hymenolepis diminuta and H. nana, can be grown from the larval to the ovigerous adult stage in vitro using culture apparatus and media described by Berntzen (1961, 1962, 1964). Mueller previously succeeded in culturing the plerocercoid larva of Spirometra mansonoides in vitro from the procercoid stage using methods described in a series of papers (Mueller, 1958, 1959a, 1959b, 1959c). In vitro-reared plerocercoids proved to be infective for cats, and adult worms resulting from them were normal in every detail. Furthermore, the culture system could be made to serve as a surrogate host to the plerocercoid Received for publication 28 May 1964. *Supported by Research Grants Numbers AI04175-01 and AI-01876-06 from the United States Public Health Service, National Institutes of Health, Bethesda, Maryland. t Department of Medical Microbiology and Immunology. t Department of Microbiology. over several successive generations with no indication of impairment to the worm. Since Hymenolepis is a cyclophyllidean genus, the question arose whether the culture methods developed by Berntzen would prove equally successful for rearing a pseudophyllidean cestode from the plerocercoid to the adult stage in vitro. Smyth (1946, 1949) succeeded in bringing about the differentiation to sexual maturity and oviposition of several larval pseudophyllideans (Ligula, Schistocephalus) in vitro, but these large plerocercoids, from the body cavity of fishes, have abundant amounts of stored energy, and little or no increase in mass is involved. In these larvae the primordia of the genitalia are already present, and in Schistocephalus even segmentation is complete. Smyth (1959) also succeeded in inducing differentiation of a small plerocercoid from fishes (Diphyllobothrium dendriticum ?) in vitro. In this case he obtained segmentation and development of genital primordia, but there was no increase in mass, and the resulting

The laboratory propagation of Spirometra mansonoides (Mueller, 1935) as an experimental tool. VII. Improved techniques and additional notes on the biology of the cestode.

Improvements are given for techniques originally described by the author in 1959. These have to do with collecting and hatching the eggs, rearing of copepods, the optimum time of harvesting and feeding out parasitized copepod cultures, prevention of fungus contamination of in vitro sparganule cultures, and an improved two-step method for implanting spargana into experimental animals. Incidental observations are presented suggesting that susceptibility of Cyclops vernalis to procercoid infection has a genetic basis, and that resistance is of two types. It is also reported that copepod cultures maintain themselves largely by cannibalism of the early nauplii by the older larval and adult forms. In a series of papers, Mueller (1959a, b, c) described methods for the laboratory propagation and manipulation of Spirometra mansonoides. Six years of additional work with this worm system have proved the essential soundness of the methods originally developed. Nevertheless, as might be expected, continuing experience has resulted in improvements and simplifications. Since this helminth is being used by an increasing number of independent workers, it seems appropriate to describe these technical advances.

GROWTH STIMULATION INDUCED BY INFECTION WITH SPIROMETRA MANSONOIDES SPARGANA IN PROPYLTHIOURACIL-TREATED RATS*

Male rats rendered hypothyroid by dosing with propylthiouracil (PTU) from infancy grow more slowly than normal animals and suffer an arrest at a level of approximately 160 to 170 g, but undergo a new cycle of growth when injected subcutaneously with small numbers of spargana of S. mansonoides. Such rats attain a weight of approximately 231 g as compared to 178 g for controls on PTU only. The weight gain reaches a maximum at 5 to 6 weeks and then slowly declines. The effect is almost immediate, giving clear-cut results in the first week, whereas controls hold their level or in- crease only very slowly. The effect is not just an average, but occurred in each rat tested so far. The mechanism involved is not as yet clear, but the weight gain appears to be distributed throughout all tissues and organs. Such heavy rats are larger, entirely normal in behavior, and in every way seem to be more rugged and superior animals than their controls.

FURTHER STUDIES ON PARASITIC OBESITY IN MICE, DEER MICE, AND HAMSTERS.

Accelerated weight gain caused by infection with spargana of Spirometra mansonoides occurs in male mice as well as females, and in older mice approximating 40 g starting weight, as well as in younger animals. Below a starting weight of 10 g the effect is greatly reduced and delayed if seven spargana per mouse are used. Deer mice, Peromyscus, show the same response. The oriental worm, S. ranarum, has an even greater stimulating effect than mansonoides, but is more antigenic, becoming encapsulated in the first 2 months, after which the effect is lost. The weight gain effect is additive, increasing with the number of spargana, but reaches a plateau at about 12 worms per 20-g mouse, after which either no further gain occurs, or gains are offset by increased mortality of the mice. Hamsters show an even more pronounced effect. Both males and females respond. In this case the infected animals grow at a very similar accelerated rate, while the controls show wide variation. True growth as well as obesity is involved, since infected hamsters are not only heavier but larger as shown by inspection and by X-ray. Small numbers of spargana appear to have no effect on longevity; a group of the original experiments allowed to die off naturally showed similar mortality rates for both experimental and control mice. Nor is there any effect on reproduction. Infected mice and hamsters breed as well as controls if given the opportunity. Mueller (1963a) described accelerated weight gain in mice infected with small numbers of plerocercoids of Spirometra mansonoides. In the original experiments only female mice were used at an average starting weight that varied from 11.7 to 21.4 g. The numbers of spargana per mouse varied from seven to eight. For smaller numbers of worms the effect was shown to be additive. The obesity effect was not dependent upon the strain of mouse since white mice from various suppliers reacted similarly. Gains of experimental mice over controls at 20 weeks averaged 17.60%, the larger gains tending to occur in mice of greater starting weight. Mice infected at a starting weight of 11.68 g showed an initial lag, but in the 2nd week overtook and passed the controls, thereafter increasing their advantage. ADDITIONAL EXPERIMENTS ON MICE This phenomenon has now been examined in greater detail, with preliminary results published in abstract (Mueller, 1963b, 1965). In five experiments on male mice (21 experimental, 20 control), at starting weights ranging from 9.5 to 38.75 g, and continuing for periods of 2 months or more, four experiments showed marked acceleration of the infected animals over the controls (Fig. 1), the maximum occurring in one experiment (E-93) where starting weight was 22.5 g. In male Received for publication 23 February 1965. mice with starting weight of 9.5 g (E-90) the experimentals lagged behind the controls for the first 4 weeks and then surpassed them, achieving an advantage in weight increase of 11% at the end of 12 weeks, but with the differential diminishing from 14 to 29 weeks (Fig. 2). Twenty-two further experiments have been done on the effect in female mice (185 experimental, 114 control animals1) ranging in weight from 10 to 39 g. With females of 10 and 11 g starting weight (E-95 AB, CD) there occurred only minimal gains over controls (5 and 12% respectively at 11 weeks) and then only after an initial period of several weeks in which the experimental animals lagged behind the controls (Fig. 3), as in the case of young male mice. It is thus clear that any advantage in weight gain bestowed on mice by this type of parasitism is minimal if the animals are infected at or below 10 g in weight, and it seems probable that below this weight the effect disappears altogether, or could be demonstrated only with a smaller number of worms. Contrary to expectation, a weight gain was also produced in older female mice (E-117, E-105) of 33.5 and 39 g starting weight, respectively (Fig. 4). The gains over controls The numerical asymmetry results from the fact that some of the experiments had multiple groups of mice, infected with different numbers of worms, with only a single set of controls.

The Laboratory Propagation of Spirometra mansonoides (Mueller, 1935) as an Experimental Tool. III. In vitro Cultivation of the Plerocercoid Larva in a Cell-Free Medium.

As previously reported in abstract (Mueller, 1958), the procercoid of Spirometra inansonoides has been grown in a cell-free medium to the plerocercoid stage, in the complete absence of the second intermediate host from the life history. Such cultured plerocercoids regularly produce infection with the adult worm when fed to cats (Mueller, 1959c). Since the medium employed contains only standard tissue culture ingredients, the problem of culturing these larvae, once solved, turns out to be one of laboratory manipulation, rather than of satisfying any unusual metabolic requirements on the part of the worm. Previous papers have described the technique of collecting and hatching the ova (Mueller, 1959a) and of propagating and infecting the copepod host in laboratory culture, and harvesting the procercoid larva in mass (Mueller, 1959b). The axenic procercoid, collected by the method described, forms the starting point for culture. This paper will describe the technique of maintaining and manipulating these cultures.

A New Species of Trichodina (Ciliata) from the Urinary Tract of the Muskalonge, with a Repartition of the Genus

In 1932 the author described Trichodila renicola from the urinary tract of the pickerel, Esox iiger, in Oneida Lake. This was the first record of a Trichodina from the urinary bladder of a fish. On numerous subsequent occasions the urinary tract of Esox lucius from various localities was examined for related ciliates, but with negative results. An added interest therefore attaches to the discovery of a similar parasite in the urinary passages of the muskalonge, Esox masquinongy, of Chautauqua Lake, N. Y. This parasite was present in all adult muskalonge examined.

A Diphyllobothrium from cats and dogs in the Syracuse region.

On March 7, 1933, two cats from the Humane Society of the City of Syracuse were autopsied in this laboratory, and found infested with a species of Diphyllobothrium. One of these cats harbored a single worm, the other two. The smallest of these worms was about 20 cm. long, the largest 48 cm. Fifty additional cats from the same source were examined subsequently at different times up to the middle of June, but no more of the worms were discovered.

The laboratory propagation of Spirometra mansonoides as an experimental tool. I. Collecting, incubation and hatching of the eggs.

Although parasitic helminths in general are increasingly the subject of biochemical, physiologic, and immunologic investigation, comparatively few species are employed in such studies due to the difficulty of propagating the worms in the laboratory. The only tapeworms which have received extensive experimental attention are the small cyclophyllideans Hymenolepis nana, H. diminuta and, to a lesser extent, Taenia crassicollis, Echinococcus granulosus, and E. multilocularis. The plerocercoids of certain pseudophyllidean cestodes (Ligula, Schistocephalus, Dibothriocephalus latus, and Diphyllobothrium sp.) have been used for similar investigations, but require access to particular lakes where naturally infected fishes occur. Cyclical propagation of these forms under artificial conditions is difficult or impossible, because of the wild hosts involved. Since pseudophyllidean cestodes are more primitive than their cyclophyllidean relatives, have a more expanded life history, and show important differences of vitamin B12 content, it would appear that a pseudophyllidean which can easily be propagated in the laboratory should offer a fertile field for comparative studies. A variety of forms are included in the genus Spirometra. Some of these require wild hosts, and most of them are so intergrading and poorly characterized that species definition is an unsolved problem (Iwata, 1933). S. mansonoides (Mueller 1935) is unique in having constant, well-defined characters (Mueller, 1936), and thrives in hosts that are easily maintained under artificial conditions. In the spring of 1956, the author reactivated work on S. mansonoides with the primary purpose of attempting to culture the plerocercoid, and possibly other stages, in vitro. Since it required an abundant supply of all life history stages, practical methods had to be developed for the massive propagation of the worm. These methods have now been perfected and simplified to a point that could not have been anticipated. The Syracuse focus of natural infection in domestic cats with S. mansonoides, originally reported by Mueller (1935), still exists, as does also enzootic infection in local water snakes (Mueller, 1938). Since incidence of natural infection in cats is low (2 or 3 percent), spargana were collected from water snakes. A collection of 7 Natrix, from Big Bay Creek, at the western end of Oneida Lake, taken on May 25, 1956, yielded 2 small spargana lying under the skin of a single snake. These spargana were fed to 2 worm-free cats on May 29. Both cats showed ova in the feces 12 to 13 days later. These 2 cats were the source of our present experimental

The laboratory propagation of Spirometra mansonoides as an experimental tool. V. Behavior of the sparganum in and out of the mouse host, and formation of immune precipitates.

other experimental animals (Mueller, 1959). In mice infected early in life, at a weight of 10 to 15 g with procercoids per os, as many as 150 larvae may develop in a single mouse, reaching a length at the end of a year of 200 to 400 mm per worm, and forming as much as 17 percent of the weight of the animal (Mueller, 1958). When such mice are posted, the subderlnal spaces and intermuscular fascial planes are cluttered with great masses of spargana. Usually these worms have only very light encapsulation, insufficient to prevent migration. At times it appears that a wall of host tissue is entirely lacking. Nevertheless the worms lie in small piles, often discrete, but also frequently touching (figs. 1, 2). However, they never become knotted or tangled in the mouse. It is therefore easy to remove them individually with a fine forceps to a dish of saline for counting, measuring, etc. Outside the body of the mouse, whether in saline or culture medium, at room temperature or 37 C, the worms show constant slow peristalsis, with waves of contraction and extension passing along the body from anterior to posterior. If a number of worms are left in the same

Food intake and weight gain in mice parasitized with Spirometra mansonoides.

Six experiments were performed using 54 female mice (27 infected, 27 uninfected) in an attempt to determine whether relative food intake is involved in the accelerated weight gain observed in small laboratory animals infected with small numbers of spargana. In five experiments the infected mice ate more and gained more, and in one experiment they ate less and gained less than their respective control groups on an absolute basis. Total amount of food consumed by the experimental animals was 6.37% greater than that consumed by the controls. However since some of these mice had been infected several weeks previously they were already heavier than their controls at the beginning of the experiment, and in relation to weight, the food intake of the two groups of animals was essentially the same. It seems clear this type of weight gain is not explainable on the basis of any appreciable difference in relative food intake of the two groups. A tendency on the part of the controls to scatter their food suggested that they found the oily diet less acceptable than did the experimental animals. Basic to understanding the physiology of accelerated weight gain in mice and hamsters parasitized by spirometrid spargana (Mueller, 1963, 1965) is the question of whether such animals eat more than their controls, or whether they gain weight because they put out less energy on the same caloric intake. The present work was undertaken in an attempt to answer this question.