Addendum to the question of the transmission of human tapeworms*
In my last communication on Ascaris, I overlooked mentioning Grassi, a successful attempt at transmission (Centralblatt für Bakteriologie und Parasitenkunde, v.I, 1887, p.131). His were the first to have yielded positive results, and as they are opposed by a larger number of negative results (Leuckart, Mosler, and other authors), it would not be surprising if - despite the author's well-deserved authority - they did not meet with universal acceptance. There is no satisfactory explanation for these contradictory results and the great number of unsuccessful experiments is overwhelming. Nevertheless, such an explanation can perhaps be found through careful research.
In the communication mentioned, I expressed the conviction, reached quite independently by me, that transmission does occur through the eggs and that the negative results have been due to a source of error during culturing or transmission. On the other hand, Leuckart1 has noted that any discussion is of course invalidated by the argument that this contradiction derives from certain as-yet unknown conditions regarding transmission of Ascaris. This has put me in a favorable position in as much as I have been led to a possible source of error by the very words of the prominent helminthologist.
Examination of Leuckart's figures on development of Ascaris in the work mentioned above (figs. 154, 156) shows that all lost their outer layer, known as the albuminous coat. Leuckart really considers this such a necessary condition for development that he states: “Davaine's figures (Traité des entozoaires Syn. LIX), which allow this coat to remain throughout the period of development, displaying it even in eggs that contain a perfectly formed embryo, are unlikely to have been taken from nature.”
In the same place he writes also: “The albuminous layer described for the ripe eggs is entirely lost through the action of the surrounding fluid, generally before the first division.”
In a series of experiments that I undertook during the last four years with eggs taken from the genital organs of female Ascaris or from the stools of hosts, I obtained the same results as Leuckart: The albuminous layer was lost, development of the embryos proceeded slowly, and many eggs were late, while others did not develop at all. All these cultures were made in water, which was replenished if the culture began to dry out.
In a new culture, I have recently had the opportunity to see for myself that the figures of the French observer may very well correspond to natural conditions. As this point seems to me of utmost importance, I prefer to give a short description of the conditions of my latest experiment.
Under natural circumstances, the eggs of Ascaris develop on the surface of the ground, where air is in abundant supply; they are moistened only sporadically, when it happens to rain. Consequently, I tried to follow these conditions as closely as possible, which had not been the case in the earlier cultures.
For this purpose, in early November, I put a small portion of diarrhea feces from a child infected with Ascaris into an empty 200-cc flask. They were mixed with a very small amount of water so that the resulting fluid (a few cubic centimeters) formed a shallow layer covering only the bottom. The flask was then laid on its side and rolled around so the liquid was distributed over the entire inner surface, to which large numbers of damp eggs adhered. During a long period - until the yolk had contracted entirely-the flask was rolled several times a day and sufficient renewal of air was provided for. The flask was kept at room temperature, in the shade, and a certain degree of humidity was always maintained; this was all the easier as the liquid did not show much tendency to dry out. The temperature was relatively high, since it was summer. The atmosphere was dry for a while and then very damp for a long time. A marked odor of putrefaction never developed inside the flask, and the process of decomposition seemed finished in a few weeks. From then on, rolling and renewal of air were spaced at longer intervals.
Now, less than two months later, all eggs are in a more or less advanced state of development (excepting those that could be already recognized as sterile in the stool, owing to certain peculiarities). Not a single egg has lost its albuminous coat; on the contrary, this is as distinct and sharply delimited in all of them as it was at the time the eggs were eliminated, nor has the coat shown any signs of disintegration, though more than half the eggs contain a perfectly developed embryo in the characteristic position.
In the “mulberry eggs” (as I call those that display an outer coat), the embryo displays only limited mobility; mostly no movement is seen, and sharp stimulation is required to shake the embryo out of its torpor. When this happens, the embryos move about in quite lively fashion, and, according to the shape photographed by Leuckart, they change position completely, acquiring a coiled form similar to that of a cornet-shaped mollusk shell. Embryos were only found outside the egg after rather energetic actions. One of them still displayed signs of life, while the others had probably died in the fluids added (e.g., solutions of caustic soda or ammonia) In many instances, the embryo did not yet show any invagnation through to the formation of the second cuticle; a distinct perforating organ was also not seen, even under the highest magnification. In a general way, these larvae resembled Davaine's figures and probably represent the earliest period of life. It is probable that in their case hatching was due to mechanical prompting, preceded by softening of the coats; they hatched through a small opening between the pole and the equator, generally with the head emerging first, but in one specimen the tail came out before it. In the case of one egg, I observed the loose, folded inner membrane protruding through the empty eggshell.
Some experiments have shown me that the gibbous outer coats are very resistant and not perceptibly affected by digestive juices. Thus, twelve hours after feeding the ‘mulberry’ eggs to a mouse, they were recovered from the small intestine and the appendix, either with a ball of yolk or even with embryos in them (which were still alive). (Leuckart observed something similar in eggs without a mulberry shell.) I obtained the same results when I put part of the culture together with artificial gastric juice in a small closed flask, which was kept for 20 hours at the temperature of the digestive tract. Although the solution of pepsin and hydrochloric acid digested flesh vigorously, I found the coats well preserved, even with living embryos inside the eggs. Finally, I swallowed a little bag of parchment paper like those used for dialysis, about the size of a cherry, into which I had previously put eggs with mulberry coats. After two and a half hours, the bag was drawn out by the thread to which it had been attached. The coats were intact and there were no hatched embryos.
Final Part2
Solutions of Fel tauri inspissat., with or without the addition of pancreatin, acting for three to four hours (at body-temperature), presented no results other than the impression that the ovular membrane had lost some of its resistance. A more pronounced alteration through bile, pancreatic juice, or intestinal juices was hardly to be expected, because the eggs had of course been subjected to their effects even before their passive immigration, without showing changes beyond a permanent darkening of the outer coat (caused by the gall pigments).
These results allow one to conclude that the mulberry eggs resist digestion. (We must now ask ourselves whether it is correct to use the term “albuminous coats,” since its their behavior in relation to digestive juices is in no way analogous to that of albumen). The liberation of the embryos must be due to their own efforts and (in the mulberry-like eggs) will probably not occur before the intestine. Supposing, on the contrary, that in eggs displaying only a thin, smooth shell, the embryos either die inside the egg or are eliminated in the stomach, or hatch there and are killed by the gastric juices, we then have a plausible explanation for the unsuccessful attempts at transmission.
This presupposes, of course, that Ascaris lumbricoides behave differently from Ascaris mystax, the embryos of which, according to Leuckart, start developing in the stomach. However, I would like to point out that the adults of the latter are often found in the stomach of the host (according to several observations of my own), whereas the adults of the former are only tolerated in the stomach for a short time, causing considerable discomfort. Perhaps the eggs of the former can only tolerate stomach secretions if they are gradually put in contact with them through the mulberry coats, while they perish when this is absent, as in Leuckart's experiments. Such a possibility may also occur in human Ascaris.
Davaine, who probably experimented with mulberry-coated eggs (to judge from his drawings), was also the only one to obtain hatched living embryos after passage through the intestinal tract (of the rat in this case). In other experiments, which were probably all conducted using eggs without coats, either the worms did not hatch (mice, sick puppies) or they were killed or digested (dog and probably man; see Leuckart loc. cit.).
I obtained a different result in an experiment on an adult, medium-sized dog, into whose stomach I introduced mulberry-coated eggs at different stages of development, enclosed in a small parchment bag. Evacuation took two days and was eventually induced by calomel. Examination could only be made after several hours of cooling, with the following findings: The mulberry-like coats were intact in all eggs. Some eggs contained embryos in an early stage of development. Two eggs had a half-hatched embryo in the vicinity of the pole. Preliminary examination revealed four well-preserved embryos with a pointed, conical head (so-called perforating tooth) but without evident duplication of the cuticle. On warming up the preparation, the more or less curved embryos showed some movement, which could not with certainty, however, be interpreted as signs of life. In this case there can be no doubt that the embryos spontaneously broke through the eggs (which perhaps had become somewhat softened already); part of them survived hatching, at least for a short period.
Experiments with mulberry-coated eggs on human beings are now needed and I hope to report on them soon. Meanwhile, it would be interesting to know if in Grassi's experiments, either all or some of the eggs still had the so-called albuminous coats.
It seems not improbable that the loss of the outer coats, thanks to long maceration and under the influence of certain ferments, caused the failure of earlier experiments on human beings. The rapid development and the persistence of the gibbous coat may be seen as the most favorable or normal condition. However, since the outer shell is not necessary to development in the outside environment, as experience has shown, its presence is probably significant only inside the digestive tract. Otherwise, it would have to be regarded merely as a rather superfluous organ and, teleological considerations aside, this does not agree with biological experience. The embryo probably has to attain a certain development inside the egg before it is able to break out.
We may probably conclude further that in cultures without constant immersion but with sufficient humidity, conditions are not only just as favorable or even more favorable than the original methods, as Leuckart found in his little dew chamber, but rather that humidity allied with sufficient ventilation imitates natural conditions and furnishes material fit for transmission.
Further research on the topic is underway.
S. Paulo, Jan. 7, 1888.