ON THE TRANSPLANTABILITY OF THE LARVA OF TEh'IA CRASSICOLLIS AND THE PROBABLE R~LE OF THE LIVER IN CYSTICERCUS DISEASE OF RATS E'. 1). BULLOCI< AND M. 1%. CURTIS (Prom Coltil?lhin Uiiroersity, Iiistitute o j Cancer Research, P.C. Wood, Direcfor) In the experimental production of sarcoma of the rat liver through the agency of Cysticercus fasciolaris, the larva of the cat tapeworm, Tenia crassicollis, rats are infested by feeding them with feces from cats accidentally or experimentally infested with the tapeworm (1). The shells of the Tenia eggs which are present in the cat feces are dissolved by the digestive juices of the rat and the freed larvae pass through the walls of the intestinal blood vessels into the portal circulation and are carried to the liver, where they lodge in the capillaries. Many of them become encysted (2) and continue to develop apparently as long as the host lives. The question naturally arises whether the Cysticercus larvae settle in the liver because of some specific property possessed by that organ upon which larval growth and differentiation depends, or merely beacuse they are barred from passing through by the narrowness of the liver sinusoids. However that may be, in the thousands of infested rats which have died and been autopsied since the beginning of the experiments, the parasites had established themselves in the liver and in every animal but one they were confined to that organ. In the exceptional case there were two ordinary cysts in the liver, but there was also a cyst containing a healthy Cysticercus larva 40 cm. long imbedded in the ileocolic mesentery. To account for the presence of the parasite in this unusual situation, three alternative hypotheses are suggested : First: The young larva failed to reach the portal vein, became encysted in the mesentery, and developed there in much the same way as did the two parasites which reached the liver. 444
TRANSPLANTABILITY OF LARVA OF TENIA CRASSICOLLIS 445 Second : This larva passed through the liver capillaries, which stopped the other larvae, and also through the capillaries of the lungs into the systemic circulation, through which it returned to the mesentery. Third: The cyst found in the mesentery started its development in the liver, from which it became first pedunculated and eventually disconnected, acquiring a secondary relationship with the mesentery either before or after its complete separation from the liver. The latter origin does not seem improbable, since pedunculated liver cysts are not infrequently observed and sometimes the pedicles are very long and slender. In the case under consideration there was no visible evidence that there had ever been any connection with the liver. Histological examination of the cyst wall failed to demonstrate the presence of liver structures such as bile ducts or liver cells, which are frequently, but not always, found in the walls of liver cysts. While their presence would have established the hepatic origin of the cyst, their absence does not preclude that possibility, since the structure of this cyst wall was identical with that of many benign liver cysts. The evidence is therefore insufficient to determine how and when the Cysticercus larva reached the mesentery. If either of the first two hypotheses be true, it is evident that the larvae may encyst and develop in other than liver tissue. If the cyst were of hepatic origin, the parasite was able to survive in its new environment, since it was as healthy and well-developed as those from the two liver cysts. The occurrence of this extra-hepatic cyst was an added impetus to proceed with experiments already planned to determine the r6le played by the liver in Cysticercus disease of rats. After unsuccessful attempts had been made to free the young larvae from the liver by perfusing the organ with normal saline, it was decided to transplant to other sites of the body larvae excised from the liver at a time when they lie free in the liver tissue unencompassed by cyst walls. Cell proliferation, the initial stage in the formation of the cyst wall, does not begin until
446 F. D. BULLOCK AND M. R. CURTIS about the eighth day after the rat is fed Tenia eggs, although by the sixth day the larvae are visible to the naked eye, appearing as small vesicles or spots on the surface of the liver. In these experiments young rats of several different strains were fed large doses of Tenia eggs and six to nine days afterward they were used as the source of larvae for transplantation into other young rats. The recipient of a larva was sometimes of the same strain as the donor and sometimes it was of a different strain. The larvae used from February 7, 1924, to February 6, 1925, were from thirty different feedings and they were transplanted into 978 rats, each animal receiving one to four larvae. The larvae for transplantation were excised by cutting through the surrounding liver tissue with small curved scissors, aseptic precautions being observed. No attempt was made to free the larva from the adherent rim of liver tissue for fear of injuring the delicate organism. Each excised larva was either transplanted immediately after its removal from the liver or it was placed in a Petri dish containing sterile normal salt solution and kept at room or body temperature until the number required for the particular experiment was prepared. A large caliber inoculating needle of the type used for tumor inoculations was used to transplant the larvae. In 18 animals the larvae were introduced into the peritoneal cavity. In all the other 960 animals the larvae were inoculated into one or both groins. Three of the aninials used for intraperitoneal inoculation of the larvae died soon after they were inoculated; nine were killed on the twent,y-fourth day arid were all negative; of the remaining six which were killed on the forty-second day, five were negative, while one showed a young apparently healthy cyst in the peritoneum. On October 1, 1925, there are still living 450 of the 960 rats in which the larvae were introduced into the subcutaneous tissues of the groin. Of the 510 which have died or been killed, autopsy records are complete for 348. Of these 62 (17.8 per cent) showed in the groin one to four apparently healthy encysted larvae in various stages of development. Larvae under two centimeters in length were recorded as early or as young
TRANSPLANTABILITY OF LARVA OF TENIA CRASSICOLLlS 447 growing and were all found in rats dead or killed three to nine weeks after inoculation. Forty-seven larvae from groin cysts were measured. Figure 1 shows the length of each of these larvae plotted to the number of months (days +- 30) which it had spent in the groin of the host. The solid line connects the mean FIG. 1. DIAGRAM SHCJWING THE RELATION BETWI<CEN THE AGE AND THE LENGTH OF THE cysticer( US IAAIlVAE FROM THE GROINS. The open circles represent individual olwervations and the solid dots which are connected by thc line represent the means of thc age groups. length of larvae for each unit of time. The size of the larvae seems to depend chiefly on the time between the transplantation of the organism and the death of the host. The variability as well as the average size of the larvae of any particular age cor-
448 F. D. BULLOCK AND M. R. CURTIS responds in general to that shown by larvae of the same age from liver cysts. The longest larva from a groin cyst was 30 cm. and it had resided in the host 427 days, or fourteen months. Cysts sometimes regress in the groin as they often do in the liver, since 31 (8.9 per cent) of the animals with complete autopsy records showed in the groins atropic cysts or encapsulated remains of dead larvae. FIG. 2. WALL OF A COMMON TYPI'. OF CYSTICE:R(~US CYST OF THE LIVER. X 140 Microscopically the cyst walls which were formed about the organism in the groin tissues resembled the walls of the ordinary liver cysts (see Figs. 2 and 3), except that they were all free from liver cells and bile ducts. The early stages in the development
TRANSPLANTABILITY OF LARVA OF TENIA CRASSICOLLIS 449 of the groin cysts have not been studied and the fate of the liver tissue introduced with the larvae is unknown. None of the groin cysts showed sarcomatous changes and only two showed excessive or atypical cell proliferation. There is, however, no known reason why a sarcoma is not as likely to develop in the wall of a cyst in the groin as in a similar cyst in the liver. Such a tumor may yet appear as a result of these experiments, since nearly half the animals are still alive. FIG. 3. M ALL OF CYSTICEHCUS CYST FROM THE GROIN. X 160 The temporary residence of the larvae in the liver of a rat of one strain did not seem to lessen their chance of survival or affect their power of growth when intoduced into animals of another strain.
450 F. I). BULLOCK AND M. R. CURTlS In spite of the relatively few growing larvae in the groins, the result of these inoculations does not compare unfavorably with that of many of the feeding experiments. In fact, many of the rats in the transplantation experiments which had been fed Tenia eggs before or subsequent to their inoculation showed no cysts in the liver. While the probability that rats differ in their susceptibility to the infestation must be admitted, it is certain that a very large factor in the fate of the transplanted larva is the viability and vitality of the organism itself. Presumably any young larva having the power to grow progressively in the liver would grow and differentiate if transplanted into the subcutaneous tissues of any other rat of equal susceptibility. The difficulty lies in the uncertainty of predicting the fate of the larva in the liver by its appearance at such an early stage of development, making the selection of larvae for inoculation largely a matter of chance. Unfortunately a very large number of nonviable Tenia eggs were accidently used in these experiments both for the large feedings from which larvae for transplantation were obtained and for the oral administration to the animals used for the inoculation of these larvae. Some of these animals were fed before and some after the subcutaneous inoculations were made, but unless there is a larger proportion of infested animals among those still living than have been found among those already autopsied, this set of experiments may not decide whether or not the presence of Cysticercus cysts in the liver renders the animal immune to inoculations of the parasite in the groins, and uice versa. However, the experiments have already established the fact that the larvae of Tenia crassicollis can encyst and develop normally in the subcutaneous tissue of the groin. SUMMARY 1. During experiments on the production of C'ysticercus sarcoma of the rat liver autopsies have been performed on thousands of rats infested with Cysticercus jasciolaris by feeding them cat feces containing the eggs of Tenia crassicollis.
TRANSPLANTABILITY OF LARVA OF TENIA CRASSICOLLIS 451 2. In every case but one the larvae were confined to the liver. The exceptional case showed two ordinary cysts in the liver and one equally well developed and of similar morphology embedded in the ileocolic mesentery. 3. The absence of liver structures in the wall of this cyst renders doubtful, but does not disprove, its hepatic origin. It may have been a pedunculated liver cyst which became disconnected from the liver, or the young larva may have encysted and developed in the mesentery. 4. Young Tenia larvae which had not yet encysted were excised from the livers of young rats experimentally infested and transplanted subcutaneously into the groins of other rats, each animal receiving one to four larvae. 5. Of the 348 inoculated rats which are already dead, and on which autopsy records are complete, 62 (17.8 per cent) showed in the groins one to four healthy encysted Cysticercus fasciolaris. 6. The size of the larvae from the groin cysts was roughly proportional to the duration of the infestation. The average size and the amount of variation in the length at any particular age was similar for larvae from groin and liver cysts. 7. Morphologically the walls of the groin cysts resembled those of many liver cysts. None of them showed liver structures. 8. In no case did a groin cyst show sarcomatous transformation and only two showed atypical cell proliferation, although there seems to be no reason why a sarcoma is not as likely to develop in a groin as in a liver cyst. Perhaps such neoplasms will occur in some of the 450 animals still alive. 9. A few rats were inoculated intraperitoneally with the young Tenia larvae. The fifteen which survived were killed 24 and 42 days after inoculation and one of them showed an apparently healthy cyst embedded in the peritoneum. CONCLUSIONS These experiments prove that Cysticercus fasciolaris can encyst and develop normally in the subcutaneous and probably also in the peritoneal tissues of the rat. This fact supports the
452 F. D. BULLOCK AND M. R. CURTIS theory that the r6le of the liver in Cysticercus disease of rats is mechanical. That is, it probably acts as a filter through the pores (sinusoids) of which the Tenia larvae cannot pass. REFEKENCEB (1) BULLOCK, F. D, AND CURTIS, 31. 11.: The experimental production of siircoma of the liver of rats. Pror. S. Y. Path. Hoe.. 1920, N. S., xx, 149. (2) BULLOCK, F. D., AND CURTIS, bl. It.: h study of thr reactions of the tissues of the rat's liver to the larvae of Tcmn crasszcollzs and thc histogrnesis of Cyst+ cercus sarcom:i. J. Cancer hs., 1924, viii, 446.