The history of germ-free life goes back to Pasteur. In 1885, Louis Pasteur felt that the need for germ-free life was very definite, but he was also very definite in his statement when he said that it was his opinion that life without bacteria would be impossible.

In the succeeding years, from 1885 on, a total of about six investigators tried this experiment. None of the investigations lasted for any period of time. The technical difficulties of getting an animal germfree and keeping it germ-free are tremendous.

So, in 1914, the last work was done in Germany, it lasted for a period of 2 years and then was abandoned. In 1928, I picked this work up at the University of Notre Dame and laid out a long-range program which the university accepted in 1930. This was a 50-year program. It is now 28 years since I started it. The purpose of the program was simply to obtain germ-free animals, to produce them for use as a tool in experimental biology and medicine. Today we are able to do that.

It is the only place, so far as I know, in which animals have been reared through successive generations germ-free and have been used in many different problems of medical importance. It was a very humble start, as you can well imagine.

Today I think the need for germ-free life and the techniques is very well recognized. However, there are only these installations, which are very small. There is one at the Army Medical School at Walter Reed, and one which will go into effect about the end of April at the National Institutes of Health. There is one we also set up in Japan. These constitute at the present time the three known small laboratories other than the Lobund Institute in which germ-free life is done.

As I say, there is a tremendous demand for the animals. We have a backlog of about 185 to 190 requests from leading scientists all over the world and in this country for the use of these animals or products of these animals in their own experimental research. We can fill very few of those. I think that we can activate out of the 185 not more than 10 a year. That is about all we can do.

The need has been recognized further by reports from NIH and from the Army. I think that the problem now before us is to teach the technique and to make it more available.

In going over this with the scientists from various institutes, and in particular those within the Federal Government, such as the National Institutes of Health, the Office of Naval Research, and so forth, the opinion is, and I think quite properly, that there should be a few installations large enough to rear these animals, to breed the colonies that you need, and then any number of needed small installations to which you can ship from the central source these very valuable tools.

I suppose I might very briefly illustrate just a few areas in which the demands exist. For example, the question of infectious hepatitis, which is a rather common disease, and for which we have no experimental animal. In this instance, the germ-free animal is simply used because it has no immunity reactions already mounted within itself, and there are no other organisms within the animal, so any effects you have from putting in blood series or feces from infectious hepatitis cases, any effects you have there are those that are due to the agent that causes this disease.

In the case of dental caries, or cavities, we have always wondered as to whether they have been due to bacteria, diet, or heredity, or a combination of these things.

Working with the University of Chicago, I think we have been able to settle the first phase of this. In the absence of bacteria there are no dental caries.

Your next problem is which of the microbe organisms which are commonly found in the mouth cause dental caries. You can go to the animal and add a single organism to the animal, and then feed it the experimental diets.

To make that problem short, we found among a few others that the intercoccus is a very common organism and is one of the chief

causes.

Problems of this kind then are the kind for which the germ-free animal is used. In the case of cancer, the simple question of whether or not cancer occurs in germ-free animals has yet to be answered. Due to this, you have a strain of animals, mice, in which cancer develops readily and spontaneously, and you have to be able to rear them by hand up to the point where they are weaned and carry them from that point on through successive generations and through their entire life of several years under observation, just simply to answer the question of whether or not tumors occur spontaneously in this strain of animals in which, under normal conditions, it occurs at a very high rate. So, you see, there are many problems of this kind.

It is quite common in our work, for example, to partition an animal. Thursday of this week we sacrificed 6 of these animals, and these 6 animals are to be divided between 10 investigators spread around the country. That is how valuable the parts are and how important the technique is.

What is the problem I want to bring before you? It is simply this: That we do have the technique. We have the know-how. We have the strains of animals. We don't have the facilities to provide widely for the need, which is already apparent. I think that this is a Federal problem. I see no way possible for an installation of this kind, which corresponds in its way very closely to the reactors that you have in AEC, being handled or developed by any private sources. I think it is a national problem because there are many diseases of national importance, and many problems of importance to the Federal research laboratories, Federal research programs, which can be worked on with the germ-free animal. I think this is truly a Federal problem. What I propose is for someone, this group, or some other group, to look into this problem more thoroughly to find out how it can best be organized.

I want to leave this thought in your mind, that it is very rare in scientific research that an opportunity exists which is as clear cut as this one is with respect to what is needed for the future and how it can be expanded.

As I said before, there is no place in the world where this work has been developed, where the animals exist except at Lobund Institute. This being the case, it is very possible, with wise guidance from this point on, to make this tool available at a minimum of cost and most widely to the research scientists in the country. Thank Thank you very much.

Mr. FOGARTY. Thank you, Doctor. I think it is important enough that this committee should take a trip out there and take a look at it sometime, some weekend, when we can get away from the pressing business here.

As I understand it, it is the only place in the world at the present time where you have strains of animals that are germ-free? You have had them for some generations?

Dr. REYNIERS. The rats are through the 10th generation into the 11th, and the mice are through the 6th generation into the 7th on these special strains.

Mr. FOGARTY. What other animals to you have?

Dr. REYNIERS. We have reared guinea pigs, rabbits, dogs, cats, monkeys, the whole gamut of animals. We haven't bred them all because we don't have the equipment to hold them the length of time, for example, to breed a monkey. But for the small mammals, we breed them and carry through the successive generations. For the larger ones, like the monkey, we use them from the hand-fed stage. Mr. FOGARTY. Tell the committee a little about your technique. Dr. REYNIERS. You have to have rather elaborate machinery, as you can imagine. I didn't know this was a committee of this kind where I could have presented some photographs. It is rather large apparatus in which the animal passes its entire life.

What you do to start with is take advantage of the fact that, in the case of mammals, while the baby is growing, the mother protects that animal against contamination. What we do is take the animal at term and do a Caesaren operation inside this equipment and take the baby out. At this point we have to start handfeeding it.

Since there has been no precedence to work from, this was practically all pioneering work, you have to hand-feed them diets which are sterilized and artificial milks. You feed them every hour day and night through the period of suckling through to weaning.

After they are weaned, you continue to grow them, and then breed them and reproduce and they suckle their own young, in the case of the animals that I mentioned to you.

This is not an easy task at all. You are in unknown areas with respect to the nutritional requirements of the suckling young. When I tell you that in the case of the C3H mice that we had to handle 480 of these, of which we only reared 2 through weaning, and that this represented a period of 24 hours a day, 7 days a week for more than 22 years, I think you can realize the importance of the animal when you have it at that point and the need to continue the strain.

Those two animals have young growing. We have a total from those 2 animals somewhere in the neighborhood of 63 or 64 animals. They will continue producing. This becomes increasingly important for scientists. Unless you are in a position, not to try to rear these by hand, but in a position to breed these through to pure strains, your tool loses its value. That is why there has to be some sort of central organization in which this can be done, and then the animals under very careful condition shipped from that to centers like NIH where they can use them.

It is a perfectly foolish operation, in my estimation, from long experience, to try to set up an installation and have them rear their own animals, because about 95 percent of their time is in the mechanics of

that and 5 percent or 10 percent of their time is in the actual research. Whereas, if they can get the animals shipped to them, it is just the

reverse.

Mr. FOGARTY. I understood you to say that until recently there was no known method of transporting these animals. You have developed a way of transporting them?

Dr. REYNIERS. Yes, Mr. Chairman, we have. Again, of course, we can transport them. We have developed that. But at the other end, the place where they are to be worked on, there must be an installation capable of receiving them, because as soon as you open a pinhole in a glove, they are contaminated. That may be worked out.

We will be able to ship, for example, to these two installations, Walter Reed and the NIH, when they are fully in operation.

Mr. FOGARTY. Your operation out there now is open to everybody? It isn't just a Notre Dame operation?

Dr. REYNIERS. No; it is not. We have a basic program of our own which gave rise to all of this. We have collaborators all over the country. We also have people within the limits of our ability to handle them there for study.

Mr. FOGARTY. You have gathered there today all the scientists that have grown up with this operation?

Dr. REYNIERS. That is correct.

Mr. FOGARTY. You have the only collection in the world of scientists who know how to rear them and raise them and work on them?

Dr. REYNIERS. Yes, and use them. Suppose a man comes to us. Take muscular dystrophy, for example, it is very simple for him to come in and say, "I want to do this particular thing," but unless he has had the experience of our own Dr. Gordon, for example, in the anatomical development, unless he knows what the germ-free animal is like to begin with, whatever he judges is likely to be wrong. You have to have the background, and you have to have people who have that background who can work with other people. This is the only staff that is trained. It is a small organization. I think where we fall down at the present time is that we are trying to supply all of these people from very primitive and handmade equipment.

Mr. FOGARTY. Your problem is one of expansion in order to meet the demands. You have to expand your facilities and, as I understood you to say, it is something that the university cannot do. You think it is important enough that the Federal Government ought to assist, rather than have it go out of existence; is that correct?

Dr. REYNIERS. That is correct. It won't go out of existence. I may have mentioned UNESCO has agreed now to set up a central installation in Europe. I know it is going to go in Sweden and in Holland, that is, the Netherlands. It is going to go in Belgium. It is also going to go in England. The technique is too important for it to disappear. There is a question as to how it is to be handled here in the United States.

I think, as I said before, that here is one opporunity where this can be guided to a definite end. I don't know of any other situation in my experience in science where that position has existed for a group of people, because it is simply at Notre Dame and no other place where this was developed.

Mr. FOGARTY. I think it is important enough for this committee to go out there sometime and take a look at it. I hope that when the Senate committee holds their hearings that you will ask to be heard over there and tell the same story to the similar committee in the Senate.

I think you have made a fine presentation, Doctor. Do you have any questions, Mr. Fernandez ?

Mr. FERNANDEZ. No.

Mr. FOGARTY. Mr. Denton.

Mr. DENTON. Do you have to have sunlight for those animals? Do they live in an enclosure all the time?

Dr. REYNIERS. Yes.

The one question that I thought you just mentioned here, which has been a very important one to us and we have not been able to answer, is how long does a germ-free animal live. It is the question of aging. This is a most important thing.

They are, understand, perfectly healthy animals because we know how to rear them that way. But we don't know, for example, how long a germ-free animal will live. The average animal, as you know, is subject to infection throughout its life. We don't know that because we have never been able, for example, to have the equipment to set aside long enough to find it out. Even for rats that would take a period of 5 years.

Mr. DENTON. They are in an enclosure, I take it?

Dr. REYNIERS. Yes, sir.

Mr. DENTON. Don't they have to have light, or is that given to them artificially?

Dr. REYNIERS. We know how to handle that. We handle that by supplying the necessary vitamins, necessary foods to them. We know how to sterilize those foods with steam under pressure.

There is another side of this that I am not too sure that you appreciate, and that is that the same apparatus and technique used to contain a germ-free animal, can be used in very dangerous experimentation of airborne infection, and has been widely used by installations in this country, like at Frederick, Md., and so on. In other words, the very

same apparatus is used for handling very dangerous pathogens. Mr. DENTON. What would you call that?

Dr. REYNIERS. A germ-free enclosure.

Mr. DENTON. Are they more susceptible to disease than other animals?

Dr. REYNIERS. Yes.

Mr. DENTON. They die very fast?

Dr. REYNIERS. Yes; unless they happen to mount the defense very quickly. That is the thing that interests most of us as scientists. For example, when we take the animal out, as you mention, it has no antibodies. The little white cells that ordinarily go around the body picking up bacteria are not active. They have never learned to do that. The lymphatic system, which is one of the systems in the body which lines the intestinal canal, and so on, is only about one-third or one-fifth that which you find in the normal animal.

This animal is an animal at rest. The intestinal tract is an idling system. All it does is just digest food, whereas the normal tract not only digests food but has bacteria. The first thing that happens