TPC NEWS Vol.3, No.2 Fall 1984  (Whole Number 6)

( English Summary )(Revised Edition)

Page-3 Essay:  What I think of in changes of the seasons at TPC

     Our primate center (TPC) is located in an extensive ground, where trees, grasses and flowers of each season are very beautiful.  The trees and flowering plants trimmed moderately are giving pleasure to us through the year.  The natural environment and the artificial modern buildings of TPC are smoothly harmonizing with each other and bringing a comfortable atmosphere which permits us to work with joy.

     In the noon recess, I sometimes take a walk in the yard.  In spring, our center is filled with colorful azalea flowers. Treetops of fresh young leaves glisten in the beginning of summer. The ground is overspreaded with hot air of thick green lawn under the glaring sunshine in midsummer. Pretty Japanese bush clovers (Lespedeza bicolor) bloom gently in the autumn breeze.  Tinged autumnal leaves shinning in the clear blue sky are magnificent in late autumn.  In winter, deciduous trees without leaves are standing against the cold sky.  We sometimes find out droppings of hares and the nests of skylarks in the growth of orchard grasses.  Grasshoppers jump now and then.  Someone talks about his encountering a pheasant in the yard. Various tiny wild flowers are blooming calmly and modestly in the undergrowth of thin wood of pine trees, there we can also find some exits of mole’s tunnels and little green tree frogs being drenched with dewdrops..., we had unusually much snow in this winter.  A view of the center covered with snow was extraordinarily clear, limpid and even forceful.  A scene of people removing the snow from the pavement in the yard was the very picture exhibiting co-existence of man with nature, being vigorous and so fresh.

     Well, everyday we go into TPC buildings for our works with monkeys, looking at the change of seasons such as mentioned above.  Almost nothing to express the change of seasons but the dresses of people can be seen in these buildings isolated from the outer world by thick concrete walls.  Especially in the buildings for monkeys who are as dear as our own life itself, the temperature and humidity are automatically conditioned night and day.  There various works are actively carried out according to the regular procedures by the people wearing uniforms all the year.  That is; it is an artificial environment almost perfectly constant.  To keep the environmental conditions of animal rooms as constant as possible is the important one of the modern principles for care and management of laboratory animals.  After we have filled these conditions we can obtain good laboratory animals which bring us stable experimental results.

    By the way, are the monkeys who are born and raised under the artificial environment of this center happy?  I often ask myself.  I wonder if this question is much sentimental.  And to my regret, I have no definite answer as yet.  But, for me, there are two excuses rather than answers.  The first one is that in our monkeys's original habitats (South-east Asia, Africa and Northern part of South America) changes of the seasons are not so remarkable as in Japan.  And another one is that the people who work with monkeys and live in changes of the seasons may be able to bring unconsciously some effect of the seasons onto our monkeys through their own delicate attitude in daily works. Needless to say, the latter is only my supposition.  Or rather such a seasonal effect should be extinguished, from the current view point of laboratory animal science.

     At any rate, it seems fairly interesting to know that how much degree and by what means the change of four seasons gives influence to our monkeys, doesn't it?  On the other hand I think it may be true that human sensitivity and behaviors are, more or less, influenced by some seasonal factors.  How do we prove this?  Our monkeys will be able to be a good model of man in the experimental studies of this kind, too.

     I have mentioned about what I think, giving my best regards to our dear monkeys kept in a constant artificial environment without beautiful change of four seasons.

Page-4  Breeding Topics: The first successful foster nursing in the green monkey colony

     Recently we have succeeded in getting the first case of foster nursing at our green monkey colony.  In the morning of May 9, we found a rupture of bag mixed with a little blood from a female green monkey whose expected date of parturition was May 14.  Immediately we examined her with an ultra-sound apparatus and found that the fetus showed the breech presentation in a very little amount of amniotic fluid, though its heart beat was normal.  We performed cesarean section to her and took out a male newborn weighing 330 g. we gave the baby to his mother monkey who had recovered from the anesthesia, but she rejected to receive and nurse him.  Then, we put him in a incubator in the meantime

     By the way, another female green monkey safely gave birth to her baby just on the next day.  So we decided to try to make her foster-nurse the baby who was in the incubator, just like we always perform at the cynomolgus monkey colony.  The newborn was put in his new mother monkey’s arms, and was accepted.

    Until now, two months of nursing period have passed.  The foster child and her own child are both well, however they are retarded in their physical growth than the monkeys reared by single nursing.  We wish to wean them as early as possible, giving them an artificial milk as a supplementary diet.

Page 5 Virus isolations from abortion and stillbirth cases of cynomolgus monkeys

    The rate of abortion in our breeding colony is about 5% of total pregnant cases, and that of stillbirth is about 10%.  We are endeavoring day and night to make clear the causes of those disorders in our special fields.  It has already been found that unusual fetal presentation is important as the direct cause of stillbirth (See, TPC NEWS Vol. 3, No. 1, p.4).

     By the way, there have been many investigations on the relationships between virus and abortion or stillbirth in human.  Measles, rubella, herpes and cytomegalo viruses have been listed as typical examples of viruses which cause abortion or stillbirth in human.

In animals also, various viruses which seem to have something to do with abortion or stillbirth have been reported.  Especially for nonhuman primates, there are a few reports which dealt with natural infections of measles virus resulting in abortion or stillbirth.  Science 1979, we have tried to isolate viral agents from the materials of both mother monkeys and dead fetuses or newborns.  This paper describes the results of the isolation works in comparison with the results in normal delivery cases.

  As for the mother monkeys, their throat and vaginal swabs, stool, placenta and amniotic fluid were examined.  As for the dead fetuses and newborns, virus isolations were done with various internal organs.  Each material for those examinations was emulsified and inoculated to three kinds of sell cultures: JINET, R-66 and VER0.

     The results obtained are shown in the table.  Concerning the throat swabs of mother monkeys, viruses were isolated only from abortion cases.  As regards the vaginal swabs, the isolation rate was 3% in abortions and 1% in stillbirths.  Compared with the isolation rate in normal deliveries, the rate in abortions was nearly the same but the rate in stillbirths seemed to be lower.  From the stool, viruses were isolated at a rate of about 10% in both abortions and stillbirth.  It was the same rate as that in normal deliveries.

From the placenta and amniotic fluid, no viruses were isolated.

     The results of virus isolation from the internal organs of 14 aborted fetuses and 522 dead newborns were all negative.  The viruses isolated from abortions and stillbirths were identified to be mainly adeno and entero viruses not only by their CPE conditions but also by the electron microscopic findings.  Two strains of them were identified as SV 15.  The viruses isolated from normal deliveries were also identified to be adeno and entero viruses.

     As I mentioned above, the rates of the virus isolation from abortions and stillbirths were very low and almost the same as those from normal deliveries.  So I suppose that the viruses have no significant roles as a cause of abortion and stillbirth in our breeding colony.  Although the number of cases is small, the isolation rate in abortions was higher than that in stillbirths.  As for the abortions, we think that it is necessary to increase the number of examined cases.  Now we are doing the identification experiments about the isolated strains.  I will report the detailed results in this news again.

Page-6 Introduction of the Equipment : Computer system for care and management of laboratory primates

     The computer adopted here, TPC, is NEC 100/85.  Its configuration is as follows: a central processing unit (CPU) having control memories of 256 KB (0S) and 384 KB (user), a fixed magnetic disk unit (64 MB), a magnetic tape unit (1600RPIx800), a floppy disk drive (1MEx2), a line printer, a system console with display and a work station.  In addition, five wireless data-transceivers are used in animal rooms for sending daily inspection data.  Most of the soft wares are our original, which are applied mainly to the works of animal care, breeding and rearing by the on-line, as well as, to the processing works of the automatic blood chemical data and the serum bank management by the off-line.

     Every morning the observers in animal rooms transmit daily inspection data of all animals to the host computer through transceivers.  The data are processed and printed out by forty or more programs.  For example, the data of menstrual bleeding are processed as follows.  When we find a female breeder who shows menstrual bleeding, the computer checks her menstrual cycle for its regularity, based on the last and before-last records of bleedings.  If the cycle is judged to be regular, the mating date is fixed and the breeder is mated with a male breeder whom the computer indicates.  Following mating, the record of the mating is input.  The record is automatically transferred to the pregnancy diagnosis schedule.  According to the schedide, the pregnancy diagnosis is performed .  In case of pregnancy, the regular physical examination schedules are decided.  When she has given birth to her baby, the delivery record is input on that day.

Then the schedules of physical examination for the newborn and the mother are printed out.

     As I mentioned above, this computer prints out daily, monthly and yearly data of all animals and necessary works .  Now, it has become our indispensable partner.

Page-7 Microbiological survey on cynomolgus monkeys during the quarantine period

     The data we obtained with 160 cynomolgus monkeys imported from South-east Asia between September and October in 1983 are shown in three tables.

(See the attached page 5) .

     As compared with the previous data, the remarkable points are as follows:

(1) The number of natural death cases was quite few.

(2) There were no infections with measles viruses after arrival at TPC.

Page-8 On the Techniques of Care and Management of Cynomolgus Monkeys: Examination of fetal position

     It seems that the fetus is trying to turn.  Moving both arms, the fetus is giving kicks to the mother's abdominal wall. I can see several unevenness on the surface of pregnant big abdomen of mother monkey.  The fetal head which was situated near the navel of the mother is moving clockwise to the underbelly.  The fetus finished to change his (or her.) position from the breech presentation to the cephalic one.  The time required was only ten seconds.

     It has been reported in the "Breeding Topics' of the last issue of TPC News that the breech presentation on the day before delivery results in stillbirth at a very high rate.

So I will mention here about the way of examination of fetal position.

     The examination of fetal position is carried out for all pregnant monkeys by two persons without

using anesthesia on 12th, 16th, 20th weeks of gestation age and every day after 155-days to the day before delivery.  First, an animal technician catches a pregnant monkey in the cage with catching net, and grasps her by the arms with his one hand, holding her back-neck with his another hand.  Then another animal technician holding monkey's feet in his hand palpates the monkey with another hand.  The fetus can be palpated easily through the abdominal wall.  The fetal head is felt to be round and hard, but the buttocks are rugged and angular.  That is, the palpation is carried out gently with a thumb and other four fingers as if you hold an egg in your hand.  The time needed for the examination less than one minute per monkey.  The rate of cephalic presentation to 100 or more pregnancies we examined increases evidently with the progress of pregnancy, being 43% at 12-weeks of gestation age, 53% at 16-weeks, 70% at 20-weeks, 85% at 22-weeks and 90% on the day before delivery.

     At the time when we perform the palpation of fetal presentation, we also carry out the examination of cardiac sound of fetuses by the use of an ultra-sound apparatus, "Doppler", to confirm the survival of the fetus.

Page-9 A Special Article: A valuable experience -An experiment of immuno-suppression using colony-bred monkeys-

     When we use wild-originated monkeys as laboratory animals, someone may have an idea that contaminated state of animals with various pathogens is rather preferable for studying human diseases since man must be contaminated most highly among many species of animals.  But, it is evident that pathogen-free monkeys who are not infected with pathogens are needed in "scientific" experiments which should be done with high precision.  Recently I just experienced both of these pressing problems .

     Several years ago, the vaccine of vesicular exanthema virus (VZV) was produced.  And the vaccine strain was required to be judged of its virulence by an animal experiment.  Such viruses belonging to B- subgroup of herpes virus as VZV and cytomegalovirus (CMV) hardly infect other animals than their specific hosts unless they are in vitro adapted to cells of the animal concerned because their host-specificity is very strict.  But how about the susceptibility of the monkeys immunosuppressed beforehand?  At any event, I started on a new experiment.

      On the basis of my experiences and some literature, a combined dose of anti-monkey thymocyte globulin, cyclophosphamide and cortisone acetate was administered to cynomolgus monkeys of wild origin to evoke marked immunosuppression.  Experimental injections with three VZV strains ---- a fresh strain passaged 6 times on human embryonic lung cells, an old strain passaged 18 times on human embryonic lung cells, and a vaccine strain passaged 7 times on guinea-pig embryonic cells following 11 passages on human embryonic lung cells were carried out using several tens monkeys for the experimental group and other 12 monkeys for the control group.  The experiment was repeated four times for three years with marvelous results.  Almost all the monkeys of the experimental group autopsied three weeks after the viral inoculations exhibited systemic infection.  But all of the control monkeys gave negative result, including six monkeys autopsied within two weeks.  Also the difference in infectivity depending on the difference of virus strain was noticed.  We tried the experiment again and again to make confirmation of reproducibility of the results.  Histopathological examination revealed the lesions involving cells with a specific intra-nuclear inclusion body.  Furthermore I recognized the viral particles peculiar to herpes B-subgroup by the electron microscope and could prove the presence of specific viral antigen by the fluorescence antibody technique.  I was very pleased with the result.  However it was, in effect, my great mistake.  The fact was the infection with CMV.

     VZV and CMV closely resemble each other, having common characteristics in their electron microscopic features. To identify each of them, we must detect the difference in their antigenic characters by the florescence antibody technique and so on.  First I wasted one ampule of anti-VZV rabbit serum without obtaining any clear result.  As a matter of fact, however, this result was correct.  Next, using convalescent sera from human cases of herpes zoster ( It's anti-MW1 titer was more than 1:1024, anti-HSV titer was negative, and anti-CIA7 titer was not-tested ), I recognized pale but clear specific fluorescence in the intra-nuclear inclusion bodies in the lesions observed and could take the pictures of the inclusions well enough.  At this point of time, I reported the results at the general meeting of Japanese Pathological Society and attracted all eyes.  The next year I repeated the experiment to establish the conditions for the successful infections.  As there was no contradiction in the experimental results I had written two papers about this work.  Though I was suggested of the necessity to deny the possibility of infection with viruses of other herpes groups in some related comments, I did not pay any special attention to it.  I asked an associate professor of The Institute for Medical Science, University of Tokyo, Dr. Kurata, to examine about H-CMV, HSV and EBV etc.  After a few days, he called me and said, " That is the infection with Cytomegalo virus, Dr. Ohtaki."  I was shocked to hear his words, but I could not believe it.  So I told him, "I have tried the experiment again and again, and the monkeys were never infected with VZV".  At all events, I decided to make the experimental results sure.  After several days, his advice was proved to be correct by the fact that the lesion which had shown clear fluorescence reaction with anti-CMV did not react to anti-VZV rabbit serum at all.

     My mistakes must have been related with the following three possibilities.

(1) The serum used for the identification had the anti-CMV antibody of high titer as well as the anti-VZV antibody.  Science human sera usually contain various antibodies, I had to dilute the serum more than ten-times in order to make other antibodies than VZV antibody undetectable, even though the serum contained anti-VZV antibody of such a very high titer as 1:1,000.  Nevertheless, anti-CMV titer of the serum used was still high enough so that the FL-positive picture could be taken.  In this regard, the positive rate of H-CMV antibody in sera of human adults is 95% and almost all of the positive sera show the latent infection with CMV.

(2)  The origin of CMV inoculated into monkeys:

     If the virus inoculated into the monkeys (human embryonic lung cells) had been contaminated with CMV, all the three virus strains used would have been positive for CMV.  However, the strain which showed the highest fluorescence reacted well anti-VZV in vitro.  So there seemed no problem regarding the contamination of the inoculated strains.

(3)  A possibility of natural infection with CMV in the monkeys used:

     If the serum samples taken just before the experiment are examined for the presence or absence of anti-CMV antibody, this problem can be solved.  Of course, the monkeys were negative for anti VZV antibody.  How about CMV antibody?  In this paper, I do not announce the data concerned in detail.  Generally they were positive for CMV.  Identifying the CMV with either Simian CMV or H-CMV is now in progress at the laboratory of Dr. Motofuji of The Institute for Medical Science, University of Tokyo.

If it is the infection of S-CMV, it is evidently an endogenous infection.  Judging front the experimental results, almost all the monkeys may harbor CMV.  According to Dr. Sasagawa et. al, of TPC, 80% of the cynomolgus monkeys of wild origin are positive for Herpes simplex virus antibody.  Therefore, the positive rate of CMV among wild-originating cynos is probably much higher that that of HSV.

     It may be said that except for a possibility of contamination of the inoculated virus strains, almost all the monkeys used and the serum of patient used for the identification of viral species have been contaminated with CMV.  What should I do, then?

     Just at that time, a good news reached me, informing that monkeys bred and reared at TPC would become to be used for experiments.  Moreover the news told me that the monkeys were free from infections with various pathogenic agents.  They were virtually negative for HSV, Measles and SV5 antibodies.  Thus, the advantages of them are: (1) We can safely carry out the immunosuppression treatment since they are negative for HSV or B-virus.  (2) They seem to be negative for CMV, too.  Thus endogenous infection of CMV will not occur.  (3) On the ground of (1), (2) a strict experiment on VZV infection can be performed.  (4) At the same time, an experiment on the human CMV (No one has succeeded in it so far.) can be made.  If no infection is established with human CMV, the possibility of contamination of the virus strains used for our previous experiment will be denied. 

     The result obtained are shown in the table.  Two of three sera- negative green monkeys of wild origin died of bacteriemia caused by Gram-negative Enterobacteriaceae, not exhibiting any sign of infection with VZV.  Regarding two groups of colony-bred cynomolgus monkeys inoculated with VZV strains (a new standard strain and a previously used strain), CMV infection occurred also in one of the three monkeys of the latter group but no VZV infection occurred in all of them.  Moreover, H-CMV infection was not established either, in any monkeys that were immunosuppressed or not.  Thus it has become clear that human herpes B-subgroup does not have infectivity to monkeys even under such a highly immunosuppressed condition that endogenous CMV infection is evoked without fail.  The fact that CMV infection was detected in one of the colony-bred monkeys inoculated with VZV should be regarded as showing that CMV was not completely excluded even from F₁ monkeys.  Now I am examining the pre-experimental serum for CMV antibody.

     As I mentioned above, fortunately, I could have denied VZV infection in monkeys for the first time by the "scientific" experiment using colony-bred and -reared monkeys.

     On the other hand, what should I learn from the troubles of the experiment using wild originated monkeys?  We can not ignore that CMV infection was detected only in animals inoculated with a certain strain of VZV, but not in monkeys inoculated with no VZV under the same immunosuppressed conditions.  That the difference of virus strain resulted in the differences of experimental data means that the inocula may have played some roles.  Though I don't think easily that the strain I used was contaminated with CMV, it is possible to think that some virologically associating or helping agents were involved in the strain.  But more evidently, I think that the inoculation with materials of human origin might give some influence to monkeys immunologically.

     H-CMV harbors latently in human, causing no active symptoms usually.  However, in recent years, it has been realized that H-CMV may cause serious disease problems in pregnancy, organ-transplantation, blood transfusion and bone marrow grafting, etc.  (96% incidence as for kidney transplantation) .  As regards the condition which activates latent CMV infection, a recent theory maintains that the immune reaction against a different histocompatibility antigen is needed, besides the decline in immune function of host.  In fact there are some reports presenting concrete evidences on mouse CMV.

     Unexpectedly, we have made an experiment on one of the most up-to-date problems, in which xenogeneic stimulation (human material to monkey) caused some conditions that made the opportunistic CMV infection apparent.  I have reported about this work at the General Meeting of Pathological Society in this spring, correcting my mistaken results I had announced last year.

(Table )

   Experimental infections with human CMV and m in colony-bred cynomolgus monkeys who were highly immunosuppressed Monkeys

Page-11 A comment on My Research - Treasure-hunting in the field of pathological studies on the cynomolgus monkey

     When we start an experiment, it is very important what species of animals we select as laboratory animal.  For example we often encounter spontaneous bacillary dysentery in newly imported cynomolgus monkeys.  Contaminated state of wild-originating monkeys such as this is one of the obstacles in scientific experiments using monkeys.  At the same time, this state means that the monkey is a very suitable animal for the study of bacillary dysentery.  However, if we use "monkeys of bad quality" that are not completely quarantined, for the experimental infection with Shigella, it will be an important problem to distinct naturally occurring shigellosis from experimentally produced shigellosis.

     On the other hand, if cynomolgus monkeys are used for an experiment of human viral hepatitis (type B), it would be unable to succeed in the experimental infection itself, since no latent infection with this virus has been reported as yet and there seems no possibility of it.

     By the way, when we establish an animal model using cynomolgus monkeys, it is one of the study subjects to find diseases similar to humans’ in their natural death cases.   I usually carry out pathological studies on spontaneous diseases of nonhuman primates.   In this paper, I'll mention my aims of research, referring to some of the cases in my studies, [Our purpose of research]

(1) Discovery and development of animal models for human disease: We carry out comparative studies on spontaneous diseases of monkeys to utilize the fruits of such studies for researches of human diseases.  Furthermore, we endeavor to find out families in which a certain disease frequently occurs and to develop methods for making laboratory animal models.  For example:

Brain abscess by the infection with Nocardia asteroides (See, TPC News Vol.1, No. 1)

Diabetes                                     (             Vol.1, No.2 )

Meteorism                                   (             Vol.2 No. 2)

Congenital polycystic kidney                     (             Vol.2 No. 3)

(2)  Contributions to the prevention of zoonoses: I have to do pathological studies on zoonoses to establish some countermeasures for preventing personnel and monkeys of TPC from contacting such diseases.  For example:

Anatorichosomeasis                             (See,          Vol. 3 No. 1)  Amebic dysentery                               (See,          present issue )

(3) Contributions to the production of experimental animals with better quality: we conduct pathological studies on the presence of pathogens interfering with experiments and of metabolic diseases, making efforts to remove those diseases from our primate colony.  For example:

Obese syndrome;  Fatty liver with ketosis.

Nochtia nochti; Parasitic disease of stomach by Nochtia nochti .

Pneumonyssus simicola; Lung mite disease by Pnewmonyssus simicola.

     Nonhuman primates are frequently infested with these parasites and mites that usually bring about only light diseases.  However, when primates are used for an experiment of stomach cancer or of respiratory infection, it must be one of the necessary conditions that they are free from infestations of these parasites.

(4)  Services to good uses of autopsied organs : As far as there is no danger of zoonosis and no trouble for pathological examination, I think we had better distribute autopsied organs to those researchers who want to use them, from the view point of good uses.

       Monkeys who naturally died or were experimentally killed should have originally been alive until the end of their natural lives.  I am sure that to utilize completely all of their organs will result in the conservation of nature and will be a great consolation for those animals.

[ Comment on the cases ]

     Some of the interesting cases we found have already been reported in the pages of "Case report in TPC News.  Here, I describe two cases which are different from each other in motives to begin experiments, introducing a part of my thought on research.

(Case 1) Obesity syndrome : Fatty liver with ketosis.

     This syndrome was first found in a monkey died of unknown causes.  We made effort to clarify the cause of the monkey's death and to establish a preventive measure to the occurrence of the syndrome, considering about the cause of the disease.  In the course of this study, we established the criteria for obesity in our cynomolgus monkeys as well as the normal value of ketone bodies in blood.  At present, we judge that the fundamental cause of this syndrome must be the unbalance between amount of high calorie foods and that of monkey's exercise .

     In this case, at first the cause of death was unknown, therefore, we gave a disease name depending on histopathological diagnosis, "severe fatty liver".  There were no special clinical signs except for the loss of appetite.  Vomiting was rarely noticed.  The dead body was so fat that thickness of subcutaneous fat was several times that of healthy monkeys '.  In adipose tissues of the whole body (for example, omentum and epicardium) severe fat deposition was observed. (Fig.1)  The swollen liver was floating in formalin. (Fig.2)

      Large fat globules were observed in almost all hepatic cells . ranging from central to peripheral 20f18 of hepatic lobules.  Nucleus of the cells maldistributed, being pressed by fat globules. (Fig.3) There was no finding of inflammation.  Severe fatty degeneration was also found in epithelial cells of renal tubules of the kidney.

     For the dead case of unknown cause like this, we have tried to take out blood and urine in the bladder as soon as possible after death so as to get a clue for clarifying the cause of death.  But, usually, it is hard to collect urine from the dead body.  After that, we have had three dead cases showing the same clinical and histological findings as the first case.  We were able to obtain a little amount of urine from one of the three dead cases, and found out a large amount of ketone bodies in it.  As the number of cases increased, we have conducted measurements of total ketone and each fraction of ketone in blood, and reported the result as a newly-found disease in cynomolgus monkeys at the Meeting of the Veterinary Pathological Society.

     This syndrome has not been found in newly imported wild-originating monkeys.  Moreover, there seems no report about this case in the cynomolgus monkey in any other laboratories than TPC.  It seems to me that this syndrome is apt to occur under such conditions of care and management as those in this center.

     Judging from these matters, I can not but think that these dead cases are giving warning to the way of thinking of us, Japanese, who get used to glut ourselves with rich foods and modern conveniences.  Can it be my over-anxiety?

(Case 2) An experiment of infection with amebiasis originated from the cynomolgus monkey using kittens

     A species of intestinal protozoa, Entamoeba histolytica being hazardous to human health, was parasite detected at a high ratio among wild imported cynomolgus monkeys at TPC.  Nevertheless, we have no experience of the disease caused by this ameba at all.

This situation motivated me to start an infection experiment using kitten.  As a result of the experiment, we have got new knowledge that the combinations of host and parasite.

    A group of wild-originated cynomolgus monkeys consisting of 100 Indonesian, 100 Malaysian and 100 Philippine animals was examined for the infestation with Entamoeba histolytica (hereafter referred to "the protozoa"), The infestation rates were 4, 7, and 6% with respective country of origin.

     Examining rectal swabs for the presence of the protozoa, we studied how and when the protozoan cysts are excreted by a mother monkey transmitted to her baby.  At the same time, presence or absence of clinical symptoms was carefully observed with both mother and baby monkeys.  The establishment of infection, was recognized in all baby monkeys within 5 to 10 weeks after birth.  However, during this observation period nothing unusual happened with  their health conditions including stool property.  In imported wild monkeys who were positive for E. histolytica, any clinical symptoms that were suspected to be related with the protozoa infection were not detected. These findings gave rise to a question whether the protozoa parasitizing the cynomolgus monkey had an extremely different pathogenicity from the protozoa parasitizing human.  Then I tried to conduct an infection experiment to know exactly the pathogenicity of the protozoa using cysts collected from a latently-infected monkey.

     Ten kittens were divided into two groups, that is, seven animals of the experimental group and three of the control.  After one-day fasting, fresh stool collected from a monkey latently infected with E. histolytica was given to kittens (two grams of the stool per kitten) for two days with daily foods.  Four of seven kittens of the experimental group began to excrete bloody mucous stool from the fourth, seventh and ninth day after the stool administration, respectively.  Trophozoites of the protozoal species were found in bloody mucous stool.  At autopsy, the lesions of a needle eye size could be found out, being scattered from the ileo-cecal valve to the colonic mucous membrane.  The protozoa which were invading into the tissue adjacent to the lesion as well as the part of erosion or ulcer were found. (Fig.4)

      This experiment revealed that the protozoa harboring in the cynomolgus monkey had strong pathogenicity against the kitten.  In addition, we have learned that we should not easily judge of the pathogenicity of some parasitic, bacterial and viral agents naturally infecting the cynomolgus monkey and being usually considered to have little or no pathogenicity to humans.

[ Afterwards ]

     One of the roles of us who are working with nonhuman primates is to make a study on the animal models of human diseases.  In order to conduct such a study there must be two ways; a passive way in which we analyze naturally occurring cases such as the fatty liver, and an active way like the case of study on amebiasis.  In either case, I depended on the classical method of pathology to conduct the studies, using my own experiences, hands and eyes.

     Why do I like to continue such inefficient works without longing for the newest equipment and the most up-to-date method?  Because I am greatly expecting that the nonhuman primate as a model of man must keep an enormous treasure in their unknown usefulness.

Page-14 A Clinical Commentary b a Cub Veterinarian: On a case of fracture

     One day an animal technician found a monkey whose arm was caught in between two cages, The monkey was tied up and could not move himself at all. Some animal technicians and I pulled out the monkey's arm with great difficulty and found it fractured.  I had the arm soft-X-rayed to make sure the fracture. Both the radius and the ulna were broken.

     As this case was our first experience of fracture at TPC, we had no plaster, and I did not know well how to treat it.  Then I went to a professional bonesetter, Mr. A., and asked his help.  He was so kind as to teach me the way of treatment and spare me some plasters.  I returned TPC and applied the plaster to the monkey just as I was told by Mr. A.

     Three days later, I saw the plaster was broken by the monkey’s own bite.  I bandage it again.  This time I fixed the arm to the monkey's chest not to be crushed by his teeth.  A month had passed, I set off the plaster from his arm, the bones were connected with a little bit of curve.

     The monkeys at TPC are reared in cages all through their lives, different from wild monkeys.  They try to play various kinds of tricks on their cages to kill time, But there had been no serious case which has become a fracture.

     By the way, the monkey I mentioned above is very well now, however his arm curves a little.  He does not put his arm between cages any more, but threats me much more than before he fractured his arm.

Page-15 A Report from the Front of Animal Feeding: Daily works at the breeding facility

     Today I'll show you our daily works at the breeding facility of cynomolgus monkeys with illustrations, TPC has five buildings for keeping laboratory monkeys.  One of them is the building for breeding of cynomolgus monkeys.  It is the second largest building at TPC and available to keep about 800 breeders at a time.  It consists of five animal rooms. The room No, 1 is used for breeders from Indonesia, No. 2 for from the Philippines and No. 3 for from Malaysia.  About 20 male and 130 female breeders are living comfortably in individual cages at each room.  In the room No. 4, female breeders who are at the last stage of gestation as well as mother monkeys nursing their newborns are being kept.  The scenes in the room are similar to those of a maternity hospital.  Mischievous juvenile monkeys and five big families kept by group-feeding are living in the largest room No. 5. 

     The most dramatic events such as encountering companions, love, marriage, conception, parturition and nursing in our human life are seen as everyday affairs in the breeding building.  Now, let's go to the building with me!

     It is nine o'clock in the morning.  Animal technicians have entered animal rooms of which they are in charge.  Here in the room No.1, Mr. 0no, a fresh technician of TPC is observing health conditions of each monkey, sending animal health data such as activity, appetite, stool property, menstrual bleeding and additional remarks through transceivers to the computer set in the administration building.  The most important point in the morning observations is a judgment of sexual cycle, that is, the menstrual bleeding.  Because the mating schedule is decided on the basis of menstrual cycle.  The computer indicates the date of mating and selects a male counterpart, depending on these data collected by everyday observations.

     In the room No. 2, Mr. Someya is carrying out the works for mating, based on the indications from the computer.  First, a female breeder judged to be at the optimal time for mating is caught with a catching net, then she undergoes the strict physical check-ups before mating, whether or not there are any injuries or any troubles with her genitals.  In addition, she is examined for the uterus size and weighed.  There has come the time when she is introduced a cage of a male breeder, 0h, the male looks very anxious.  "Do you help him in the case like this?",  I asked Mr. Someya.  He replied calmly, "No, because time is the best teacher."

     In the next room No.3, you see two apparatuses with which we are familiar in a maternity hospital.  The one is an ultra-sound scanning apparatus and the other is an ultra-sound sthetoscope.  The ultra-sound apparatus was adopted two years ago at TPC and has been a very useful tool for the pregnancy diagnosis.  "We can diagnose a case of pregnancy only three weeks after mating by this apparatus.  Mr. Narita, who examines 20 monkeys a day at the most, explains us with confidence.  The apparatus is also useful for observing the fetal presentations in early stage of gestation.  Well, let's watch the screen of the apparatus.  Everyone who sees the scene of heart beating of a little fetus in the uterus of mother can not help being moved.

Dot, dot, dot....  M Tanaka, a clinical veterinarian is trying to hear the cardiac sounds of fetus in mother monkey who has passed her expected day of parturition.  For some cases, cesarean section is needed.  How about this case?  She is all right.  Well beating sounds are recognized.  She will give birth naturally to her baby.

     The room No. 4 is the delivery and nursing room.  Mr. Shimizu has just finished conducting a postpartum treatment for a female breeder who was delivered of a baby last night.  When we find newborns in the morning observation, we first separate a baby from the mother to check the conditions of bleeding, placenta, mammary glands, milk secretion and body weight with the mother monkey.  Then, with the newborn, the umbilical cord is treated, and birthweight, gender and skin conditions are examined.  The time needed for these procedures for a pair of mother and infant is about ten minutes.  Mr. Shimizu says, "I always hope to return babies to their mothers as quickly as possible."  Being offered her baby, the mother monkey holds it tightly in her arms at once.

The scene is very beautiful.  Baby monkeys are nursed by their mothers for at least three months until weaning, being weighed and checked for their health conditions at 1, 2, 3, 6, 9, 12, 15, 18 and 21 weeks of age.  This period is very important to establish a good mother-infant relationship.  If any trouble happens with both mothers and children, Mr. Shimizu, a young daddy has to play the role of a pediatrician for baby monkeys.  After the weaning, infant monkeys are separated from their mothers and sent to the building for rearing of juvenile cynomolgus monkeys.  Mr. Shimizu sees them off with encouraging words and looks forward to their returning to this room for parturition in future.

     have mentioned above, the breeding works are being carried out smoothly under fine cooperation and good considerations of our staff members.  Each animal has its own individuality and characteristics unlike the laboratory mice or rats.  Special care and treatment are required for efficient breeding.  All the staff members in our breeding facility are experts to perform and support monkey's happy marriage life.

Page-18 The observation of a cross section of endocrinological researches into nonhuman primates in U.S.A.

     It was still cold when I and Prof. Ohshima of the Primate Research Institute of Kyoto University as the members of Japan-U.S. Science Cooperative Program left for America to observe the present situation of endocrinological research on nonhuman primates in U,S.A.  We had a hard schedule, by which we visited the National Institute of Health (NIH) and its related facilities in Washington DC, Yerkes Regional Primate Research Center in Atlanta, Regional Primate Research Center of the Univ. of Washington in Seattle, Oregon Regional Primate Research Center in Beaverton and California Primate Research Center in Davis.

    First of all, we visited the NIH in Bethesda and met an endocrinologist, Dr. R. H. Williams, who had been working with a famous endocrinologist, Dr. Hodgen, till the time just before we arrived.  Dr. William told us his interesting research theme, a study on sterility.  As is well known, anovulation caused by hyperprolactionemia is a serious problem for women.  In female rhesus monkeys also, the level of prolactin in blood is elevated in the last period of pregnancy as well as during nursing period, resulting in anovulation.  However, the prolactin level drops and the ovulation begins again regularly in the period of weaning.  Dr. William was investigating the recovery process of ovulation, measuring gonadotropins and sex-steroid hormones.  He also told us that there might be a relationship between the anovulatory period and the age of mother, and the younger mother seemed to have the shorter period of anovulation post conception.  In human cases the prolactin level is kept high without ovulation throughout the gestation period.  How does the ovary of cynomolgus monkey work during the period of low prolactin level?  This is a very interesting study subject.  Our discussion with Dr. William did not seem to have come to an end.

     Next, we visited Dr. P. K. Chakraborty at his laboratory in Uniformed Service University that was close to NIH.  He, having a chair of clinical endocrinology course of the University Hospital, was studying on the hypothalmo-hypophyseo-gonadal axis.  Several technicians were busily working at his laboratory.  They were operating RIA apparatuses, observing chromatography chamber for hormone refinement.  We were very much surprised with a great variety of the methods used for measuring hormones and with the organizing capacity of Dr. Chakraborty.

     Emory University is a famous private school in the South, to which Yerkes Regional Primate Research Center was attached.  We met Dr. Wilson who acted for Dr. Blank being in charge of the laboratory of RIA of hormones.  He was interested in the stopping and recovery mechanisms of ovulation, like Dr. William of NIH.  He has not yet obtained any relationship between the period of anovulation and the age of mothers.  His laboratory was conducting comprehensive studies on the processes of ovulation, pregnancy, delivery and nursing in nonhuman primates.  That is, they have investigated not only the endocrine mechanisms including serum gonadotropins, prolactin, growth hormone and somatomesin, but also the metabolic processes of hormones in living bodies. 

     Then we went to the Field Station of the Yerkes Primate Research Center by a half hour drive in a misty rain, Pygmy chimpanzees, mangabey, squirrel monkeys were being kept there in a open field for breeding.  Dr. T. P. Gordon was doing endocrinological studies on the effect of the time from sunrise to sunset upon reproductive functions, using rhesus monkeys bred in semi-out door colony, talking about the difficulties of blood taking on a restraining chair and other efforts in his studies.  This phenomenon is called "summer sterility".  However, when fall comes, ovulation begins again in these species.   This phenomenon is considered to be essentially the same as the anovulation and the recovery of ovulation related with pregnancy and nursing.  In Japan, Dr. Nozaki of the Primate Research Institute of Kyoto University and Dr. Torii of Shiga University were endeavoring to make clear the mechanism of summer sterility, using Japanese monkeys as a model of human sterility.

     In Seattle, we were welcomed by Dr. D. M. Bowden, vice-director of Washington Regional Primate Research Center who is a neurophysiologist.  He kindly introduced us to Mr. G. C. Ruppenthal of the nursery for infant monkeys.  It would be highly possible just in such a colony as this, where a lot of newborns are raised to carry out endocrinological studies on pregnancy or on newborns.  However, we were sorry but we could meet no specialist of endocrinology this time.

    Oregon Regional Primate Research Center gave us the impression that endocrinological researches were most actively conducted among several primate centers we visited.  It was situated in the suburbs of Portland surrounded by farms and woods.

In the laboratory of the director, Dr. V. Critchlow, the investigation on changes of hormone levels in blood with increasing age were being carried out using rhesus monkeys.  And they were trying to cultivate the hypophysis of the rhesus monkey in vitro.  At that time they have not yet come to the cultivation of monkey's hypophysis, while the preliminary experiment using rats was successful.  The rooms for monkeys aged over 20 years were set in the breeding facilities.  We were impressed with their earnest devotions to the study on aging of monkeys.

     Under Dr. H. G. Spies, the mechanism of LH-RH secretion was being investigated by examining the influence of each kind of steroid hormones.  At the same time, studies are being carried out regarding the mechanism of LH -and FSH - secretion induced by LH-RH.  Moreover they were cultivating the cells of hypophysis to study prolactin producing cells.  There, comprehensive studies covering wide fields were being conducted very actively.

     Dr. R. M. Brenner succeeded in producing an experimental system, by which we are able to observe directly the changes of uterine tube caused by sexual cycles. That is, he cut and took off a little piece of the uterine tube of a cynomolgus monkey and autotransplanted it under the abdominal skin so that the influence of the sexual gland was avoided.  Moreover, he isolated and purified estrogen-receptor with which antiserum was produced in order to examine its nature, distribution in the uterine tub-e cell and its changes depending on sexual cycles.

     The researcher who was conducting studies on circadian rhythms of hormone level was Dr. C. A. Ducsay.  He was examining widely the relation between hormone levels of mother and of fetus, the pressure of amniotic fluid and its circadian rhythms.

    Using black Celebes apes, Dr. C. F. Howard was investigating diabetes which was an important problem also in TPC.  His result obtained from black Celebes apes were almost the same as those in cynomolgus monkeys at our center.  He was in charge of the histopathological section of Oregon Center, though his major was originally biochemistry.  His studies were very accurate and academic.

    In the field station, Dr. C. Eaton was doing behavioral studies on Japanese monkeys, administering steroid hormones to pregnant females to observe the influence of the hormones on their monkeys.  Now, I have to tell you what I mentioned above is only a part of various activities in Oregon Primate Research Center.

     The last place we visit was California Primate Research Center.  We had a chance to discuss with Dr. A. G.Hendrickx, Vice Director, seeing 16mm films of the apparatus for blood taking from fetuses in uterus.  There, many efforts were being made also in the technical fields of endocrinological researches. 

     In my brief visit to America, I was deeply impressed with the following two matters. One is that strong cooperation among many researchers are seen in various fields of studies on nonhuman primates.  Today, a researcher can make little results only by himself, great cooperation of many people are absolutely needed for successful works.  The other is that the strong cooperative relationships between each regional primate center and the surrounding medical institutions or medical colleges.  An institution can make little results by itself.  Big cooperation by many institutions are needed.

     Narita Air Port where we came back from warm California was covered with deep snow.

Page-20 Overseas Topics - The situations concerning the international trade of nonhuman primates --- Malaysia totally bans the export of nonhuman primates

     Malaysian newspaper, New Straits Times, dated June 6 said, that Malaysian Government had imposed five-year ban on the export of monkeys to carry out a census of their population and distribution.  For the present, the effect of this treatment on works concerned with laboratory primates in Japan will not be so serious, because the ratio of Malaysian monkeys accounts for no more than twelve percent of all the laboratory monkeys imported from South-east Asia.  But it may be considered that other countries exporting monkeys such as Indonesia and the Philippines will also impose strict regulations on export of monkeys from the view point of animal protection and/or of conservation of natural resources.

Page -21 Sketches from Animal Rooms: Joyful Cynos

     The author, a young animal technician, is introducing some monkeys who have funny characteristics.  For example, a monkey always breaks wind of bad smell against an observer during the morning inspection.  There is a monkey who repeats to pretend to drink warm water sprayed for cleaning cages.  Another monkey takes his diets in his own way.   At TPC, monkeys are fed twice a day, that is, biscuits are given in the morning and fruits in the afternoon.  The monkey never eat his biscuits of the morning and he stock them until fruits are given.  He eats the solid diets and fruits alternately in the afternoon.

Our Team, “Green Monkeys”

     This author, being in charge of a green monkey colony, is a young animal technician.  He is describing about his daily works and the monkeys of which he is taking care, comparing them to members of a baseball team.

     Obscures he is a manager and the monkeys are players.  The Manager always anxious about the health conditions of the players ----- good breeders.  Sometimes, the team must fight enemy teams, some diseases.  The team, Green Monkeys is a fresh and promising team.

I’m Like a Midwife of TPC

     This author is still young but an expert of the treatment of monkey’s delivery.  He is like a midwife of TPC.  He is describing about mother monkeys and newborns with his love.