TPC NEWS Vol. 5 No. l Spring (Whole Number )
( English Summary)
Page-3 Essay: - Our African Green monkeys
Five years ago, on January 2, I received a wonderful news of the first newborn case in our African green monkey colony. The news brought me a great pleasure and moved me profoundly. I had already stated about this experience in this column, (See, Vol. 2, No. 1, Spring 1983.)
The scale of our green monkey colony is not yet so large. But it is developing steadily owing to a constant effort of our young active animal technicians. By the end of November in 1985, we have produced 100 offsprings. Four males and six females of them have already grown into candidate breeders. The life history of each animal has precisely been recorded preparatory to his (or her) experimental use---this is the way of our center. So, I inwardly think that there have ever been very few laboratory primate facilities such as our center, which made a sound progress on the breeding of the green monkey.
As every one knows, green monkeys have very frequently been utilized as an animal resource of the kidney tissue in the production and testing of polio vaccine for the past twenty years or more, In addition, their usefulness as an animal model of various kinds of human infectious diseases such as cholera is widely recognized. Nevertheless, we have few scientific books and papers on research of their breeding. I don't know why. But I guess, the number of the facilities where a large scale breeding is carried out is very small. The Institute of Primate Research, IPR, in Kenya (Director: Dr. J.E. Else), is one of those facilities. And it systematically develops the breeding of green monkeys, making its advantages of geographical location. We are expecting much from it.
In our center, some characteristics of green monkeys, which had not been known before, have gradually been revealed in the course of successful breeding and rearing. As for the behaviors of this species, we have found many prominent features, in comparison with the cynomolgus monkey that is the main species in TPC. For example, the cynos make big noise and act violently at feeding time, whereas the green monkeys are almost always calm. The infant cynos are so mischievous that they soil their cages with diets and their own stool. But the green monkeys, regardless of adulthood and infancy, keep their cages clean. Injuring among the infants hardly occurs. The infant green monkeys suck mother's milk from both right and left nipples at a time, while the infant cynos never do so. The green monkeys are noble, clean and lovely. This is my impression with our African green monkeys.
Recently, one of our research staffs and his group have revealed some metabolic characteristics such as the presence of hypoproteinemia, hypocalcemia and insulin deficiency in the green monkeys. Moreover, as we see in the study of Simian T-cell leukemia, the usefulness of green monkeys as an animal model of serious viral diseases of man is worthy of attention.
Any way, a number of biological characteristics in the green monkey have gradually been revealed in the successful process of breeding and rearing works at TPC. I think, this fact means how important the effort of consolidating the bases is to develop studies.
Page-4 Breeding Topics:- Changing fetal presentation
Abnormal presentation of fetus is one of the most direct causes for naturally occurring stillbirth, (See, TPC NEWS Vol. 3, No. 1, 1984). We have looked for the way to save the life of the fetus showing breech presentation in the last days of gestation period. So, we attemped to perform a procedure of external version on monkey fetuses, getting a hint from human cases.
Before performing the version, it is important to grasp general. conditions of the fetus and mother monkey, for example, life or death, behavior, position of the fetus and the placenta, amount of the amniotic fluid and so on. Two animal technicians hold a mother monkey. The operator puts his palms on a part of the abdominal wall through which the head of the fetus, especially the occipital part, is felt. He pushes the breech up, as he pushes the head down, (See, the Figure.) This procedure is repeated slowly and gently. Then, in many cases, the fetus changes his/her presentation from breech to transverse one. Passing transverse presentation, the fetus spontaneously changes his presentation to cephalic.
After finishing the external version, the stethoscopic examination on the fetal heart tones is necessary. And every day examination should be done to confirm whether the cephalic presentation is being kept or not.
We have changed 28 breech presentation of cynomolgus monkeys, 4 of African green monkeys and 6 of squirrel monkeys to cephalic presentation. Twenty-six of the 28 cynomolgus monkey cases and all cases of green and squirrel monkeys resulted in normal birth. One case of the cynomolgus monkeys was stillbirth. Another case was anintra-uterine fetal death, on which Cesarean operation was performed.
At TPC, we are carrying out everyday-check-ups of the fetal presentation on pregnant cynomolgus monkeys who are over 155-gestation-days, and perform changing fetal presentation on the breech cases.
Page-5
Outbreak of Salmonellosis in Newly Imported Cynomolgus Monkeys
Salmonellosis in nonhuman primates is not always considered to be so important. However, we recently experienced an outbreak of serious salmonellosis in our quarantine facility for newly imported cynomolgus monkeys.
From September to October in 1985, 130 wild cynomolgus monkeys born in Mindanao, the Philippines, were imported in three lots. No dysentery bacilli were found in any monkeys. But salmonella was detected in some monkeys in every lot. On arrival at TPC, 7 of the 130 were positive. At the end of quarantine period, the number of the positives totaled to 30 (23%).
Serological examination revealed that all the isolated strains were agglutinated with the antisera against 0-antigen of salmonella B group, and 27 strains identified so far were Salmonella typhimurium.
Main clinical symptom of the infected monkeys was diarrhea. Twenty- eight of the 30 monkeys showed watery or bloody mucous stool as well as the loss of appetite and activity. The remaining two animals exhibited no symptoms and the bacilli disappeared naturally.
Seven animals among the 28 monkeys which showed diarrhea fell into a moribund state and some of the seven monkeys died within 4 to 25 days after the occurrence of diarrhea.
The autopsy findings of these animals were as follows: 4 of 7 animals showed congestion and edema in the ileum, caecum and colon. Salmonella was isolated from the intestines, gall and lymph nodes of these animals.
The results of the drug sensitivity test on the 30 isolated strains were as follows: (1) all strains and 28 showed resistance to chloramphenicol and anpicillin, respectively. (2) All strains were sensitive to colistin and cefazolin. (3) To rifampicin, 29 had sensitivity.
We administered each of these drugs singly or in combination to 22 monkeys suffering from diarrhea. Nineteen of the 22 animals recovered from diarrhea but the remaining three died or were euthanasized. As for 6 ill animals to which no drug was administered, 2 recovered naturally but 4 animals died or were sacrificed. Regardless of the use of drugs, salmonella was still detected in most of the monkeys who recovered from diarrhea. Six silent carrier monkeys were found even at the end of the quarantine period of 9 weeks.
We have never experienced such a serious outbreak of salmonellosis. We should pay more appropriate attention to this bacterial agent found in imported cynomolgus monkeys.
Page
6 Introduction of Facilities: Kitchen for laboratory utensils
Kitchen is an important facility to support our daily research works. From the view point of biohazard control, we have to thoroughly sterilize solid laboratory-utensils, by heating, So, a big autoclave with both side doors is set in our kitchen. However, the utensils for which heat-treatment is impossible are disinfected with appropriate disinfectant in each laboratory, then they are brought into the kitchen.
The disinfected utensils are washed by an automatic washer. The washer can complete washing process in 90 minutes, but it is not available to big-size utensils. Those utensils are soaked in a detergent solution (60' C) for an hour and washed up by hands. Pipettes are washed by an ultra-sound washer and rinsed with pure water. Washed utensils are dried in a dryer (90x70x65 cm) with four infrared ray heaters then stored in cabinets.
When sterilized utensils are needed in laboratories, they are prepared by the use of either a heating sterilizer or an ethylene-oxide gas-sterilizer. This kitchen is functioned by three personnels. The area of the room is 106.6 me, with 3.7 m height. There are an air conditioning system and a rest room for the workers in the kitchen.
Page-7
On the Technique of Care and Management of Cynomolgus Monkeys: Treatment and
artificial nursing of a baby monkeys with fractured mandible
Usually at TPC, infant monkeys are nursed by their mothers. For some cases, such as defective nursing, ill health of either mother or infant and injury, we adopt procedures of artificial nursing.
Artificial nursing for infant monkeys is carefully conducted according to weight growth, body temperature, condition of excrement and so on. Dried milk for human babies is given by using a nursing bottle or an injector. For the newborns, 30-40 m1 of 6-8% milk are given daily. After five days of age, 70-150 m1 of 14% milk are given.
This note deals with a case of a baby cynomolgus monkey with fractured mandible. His mandible was broken vertically by 5 mm between the left and the right first incisors. Its left half was dislocated from the mandibular joint. So when he moved his mouth, the left half was protruded, bleeding from the cut (Photos, 1 and 2). He could not suckle milk either from an injector or from a bottle. We fixed his mandible with a plaster and inserted a catheter into his stomach to give nutrient compulsorily. We kept the catheter inserting into the stomach lest the wounded mandible should be moved. The end of the catheter was entwined around the plaster net (Photo. 3). This monkeys was 19 days old, so he was given 15-20 m1 of 14% milk five times a day. The quantity of the milk given was gradually increased. At 34-days of age, it amounted to 130 m1 a day (Photo. 4). The wound was X-ray examined twice to confirm the recovery. After the plaster was set off, milk was given with a nursing bottle (Photo. 5,and 6).
This infant monkey was sent back to his mother 16 days after he came to the artificial nursing room.
Page-8
Quarantine Report of c Monkeys Recently Imported
Three tables are showing the data we obtained.
Salmonellosis exhibiting diarrhea as a main symptom was found in many monkeys. Details are being described the following pages.
[Table.1] Infection rates with parasites
Chromosome studies of nonhuman primates have been inspired by the remarkable development of human cytogenetics. The majority of works on monkey chromosomes have been concerned with sporadic abnormal cases with marked variation in chromosomal conditions. Systematic population studies of chromosomes, however, have not been made so far, due probably to a difficulty in standardizing the chromosomal complement of nonhuman primates. We made cytogenetic population surveys of cynomolgus monkeys (Macaca fascicularis) of TPC, aiming at elucidating the range of chromosome variations, the incidence of chromosomal abnormalities and the causes and consequences of the deviated chromosomal conditions. We surveyed more than 200 individual monkeys, and found a male with a mosaic sex chromosome constitution (XYY/XY) without any phenotypic abnormalities. Except for this case, there were no other notable chromosomal variations.
Recently, the detection of a new category of chromosome marker, namely, fragile sites, has encouraged the study of chromosomes at the population level. Fragile sites are defined to be the sites prone to be broken nonrandomly under certain culture conditions. More than 20 sites are reportedly determined. These fragile sites on human chromosomes have drawn attention because of their possible association with the origin of chromosome rearrangements in cancer. The incidence of BrdU ( bromodeo-xyuridine ) –requiring fragile site in hetero-zygous condition is reported to be unexpectedly high: 1/50 - 1/200 in the general populations. Are those who carry chromosomal fragile sites assumed to have an increased predisposition to cancer? Do they constitute a high risk population to cancer? To elucidate the biomedical significance of fragile sites, research using experimental animals is needed. However, such sites have not yet been adequately investigated on animal chromosomes. In the present study, blood samples from a total of 150 cynomolgus monkeys, composed of 90 females and 60 males, were cultured under the conditions known to reveal the fragile sites of human chromosomes. One individual was found to show a possible BrdU-requiring ' heritable fragile site on the No. 11 chromosome. This may lead us to expect some genetic homology between the , No.11 chromosome and the human No.10 chromosome, which is known to bear a BrdU-requiring fragile site. However, after banding analysis, this was found not to be the case. In addition to the above-mentioned site, more than ten fragile sites (or hot spots) were found. In particular, the No.16 chromosome showed distinct gaps or breaks under any culture conditions. Probably this exhibits a different class of fragile site. Investigations such as breeding experiments between individuals bearing fragile sites will shed further light on the biomedical significance of fragile sites.
Page-10
Establishment of Primate Society of Japan
On July 20, in 1985, Primate Society of Japan was established, and the number of members exceeded 500 in January this year. The aims of this society are: (1) To contribute to the development of the primate research through mutual understanding among researchers of different scientific disciplines who are engaged in the study of nonhuman primates.
(2) To make an organization which can express opinions on the problems related to the conservation and experimental use of nonhuman primates to the public.
As a main activity of this society, an organ titled "Primate Research" is published. It is written in Japanese and the first number was issued in January, 1986.
Page-11
A Comment on My Research: Development and improvement of the techniques of
breeding and rearing-Establishment of the technique for early pregnancy
diagnosis by an ultrasonic device
Daily works carried out in the animal rooms of TPC are essentially regular and repetitional. Nevertheless, they should be improved according to new results of researches. So it can be said that daily works are being always developed and supported by the fruits of objective researches as well as contrivance on rearing techniques and management procedures.
In this note I will describe the process of establishing the technique for early pregnancy diagnosis. As the technical requirements to pregnancy diagnosis, high reliability, brevity, easiness, economy, harmlessness to both mother and fetus and short time for detection should be considered.
In our timed mating system (one male to one female, for three days) in which a female breeder is mated around the day of ovulation, it is essentially important for the effective use of breeders to judge pregnancy before the next mating time comes. Moreover, the test for teratogenecity of drugs is carried out by using animals of early stage of gestation, for example, of 27-28 gestation days. So it is of fundamental importance to know pregnancy before the test.
We have conducted the uterine palpation to diagnose pregnancy. By the palpation, the size and condition of uterus are judged through rectal wall with a forefinger.
The pregnancy could be diagnosed at the success rate of more than 99% within five weeks after mating by this method which needs no special instruments except some experiences and is capable of judging immediately.
In addition, early pregnancy diagnosis by detecting chorionic gonadotropin (mCG) in the blood and urine of pregnant breeders is also conducted in our laboratory. But this method needs a long time to get the result, and the detectable period of CG in nonhuman primates is limited.
The ultrasonic device (U.S.D.) is one of the medical electronic apparatuses which have remarkably been developed in recent years. We also introduced it for breeding works.
For diagnosis of pregnancy at early gestation period, we examined the size and form of the uterus by the use of U.S.D. As for the fifty female breeders we examined, the earliest case we confirmed pregnancy was of 17 gestation-days (20.4 gestation-days on the average).
When pregnant cases are examined, an image of black round or oval shape (3x3 m0 like a pond can be observed in the uterus. That is the gestational sac (GS). If GS is recognized in the picture, we have to confirm it by scanning the uterus at least four times from four different directions (upward, downward, left and right). An embryo of a grain of rice size appears in the GS after 5.4 days on an average following the appearance of GS, and the heart beat in the embryo can be observed.
Sometimes, an image like a pond is observed also in a non-pregnant uterus. Such an image may appear when the non-pregnant uterus is pressed and distorted by an external pressure. Therefore, such an image changes its shape according to the external pressure. That is not the case with a pregnant uterus.
As a result of the examination mentioned above, the system of the pregnancy diagnosis at TPC was altered from the uterine palpation made five weeks after mating to the use of U.S.D. which is performed three times: 3,4 and 5 weeks after mating.
The advantages brought by this alteration are:
1) pregnancy can be diagnosed earlier than 5 weeks after mating.
2) to confirm life or death of the embryo is possible by observing GS and heart beat.
As for 133 female breeders which became pregnant from January through October in 1985, 35% of them were diagnosed to be pregnant at the 3rd gestation- week, 55% at the 4th and 10% at the 5th gestation-week. Furthermore it has become possible to confirm the time of abortion occurring in early gestation period. We have already confirmed 8 abortions of 40 to 64 gestation-days for the last one year.
Today, this ultra-sonic device is used so much not only for the pregnancy diagnosis but also for the observation of pregnancy course.
Page-12
International Guiding Principles for Biomedical Research Involving
Animals
Early in 1985 the Council for International Organization of Medical Science (CIOMS) published International Guiding Principles for Biomedical Research Involving Animals. This was the culmination of a three-year program initiated in 1982 with the encouragement of the WHO Advisory Committee on Medical Research and the active collaboration of expert staff members of WHO.
The purpose of this Guiding Principles is to provide a conceptual and ethical framework, acceptable both to the international biomedical community and to responsible animal welfare groups, for whatever regulatory measure each country chooses to adopt in respect of the use of animal for scientific purposes. The basic principles of the Guiding Principles are as follows:
III.
Animal experiments should be undertaken only after due consideration of
their relevance for human or animal health and the advancement of biological
knowledge.
IV. The animals selected for an experiment should be of an appropriate species and quality, and the minimum number required, to obtain scientifically valid results.
Page-13 Case Report: Multiple liver abscess in a cynomolgus monkey
Human liver abscess shows high mortality. However, the death rate due to this kind of disease in nonhuman primates is still unknown because of difficulty in diagnosis before dying. Death cases of liver abscess in cynomolgus monkeys are very rare. Few cases have been found in our cynomolgus monkey colony, but two of several hundreds autopsied cases.
The case: A two-year-old (estimated age) male cynomolgus monkeys of Philippine origin. On arrival at TPC, he was bled under anesthesia with Ketalar. He died without recovering from the anesthesia.
Hematological and biochemical findings: RBC(347x104, Ht 24.2%, Hb 6.3 g/d1, WBC 12900, TP 5.18 g/dl, A/G 0.52, GOT was too high to measure, CPT 674 Karmen Unit, ALP 41.3 King-Armstrong Unit, LDH 3498 Wroblewski Unit, Alb, 34.4, α-Glob. 23.3, A--Glob. 17.7, β-Glob. 24.4% (by electrophoretic fractionation).
Bacteriological findings: Both gram-negative anaerobic bacilli and hemolytic streptococci were isolated.
Autopsy and histopathological findings: On the surface of the liver, pale yellowish multiple or diffused abscesses of hempseeds-size were found with hemorrhagic surroundings. The right lobe of the liver adhered to a part of the diaghram. When the adhesion was separated, a large amount of pus exuded from the lesion of 3 cm in diameter. The lesion consisted of four zones, central zone of necrotic cellular debris(pus), colonies of bacteria, the zone of infiltrated leukocyte and that of hepatic cells. In the hepatic tissue, sinusoid congestion and partial degeneration of liver cells were observed but no fibrosis. Marked infiltration of small round cells were observed in Glisson's sheath. Satellite cells of Kupffer were activated. From the lesion, two bacterial species were isolated. Gram-negative anaerobic bacilli were judged to be the main causative pathogen. Remarkable changes were noticed in the values of the biochemical tests for this case. Those parameters will be useful for the clinical diagnosis of liver damages of this kind.
Page-14
A Clinical Commentary by a Veterinarian: An African green monkey died of
placenta previa
One morning, a pregnant African green monkey who was at the full term was lying on the cage floor, exhibiting heavy vaginal hemorrhage and prolapse. By these symptoms, she was judged to be suffering from placenta previa. I tried to hear the cardiac sound of her fetus with an ultra-sonic apparatus, but I could not hear it. So I decided to perform Cesarean operation on her. In this case no anesthesia was preferable. But the monkey attacked me so hard that I used 0.2 m1 Ketalar, a half amount of its ordinary dose. I opened her abdomen. No amniotic fluid was existing in the uterus. The fetus had been dead. In addition, staring to say, no placenta was found in the uterus. I looked for the lost placenta tracing back the umbilical cord, It Was incarcerated into the pelvic cavity.
I curetted the wall of the uterus and finished the operation, On the surface of the placenta, diffuse hemorrhages of about 2 cm each were found. The mother monkeys was brought back to her cage but she never showed a sign of getting up. I put a hot-water bottle covered with towel on the cage floor to keep her warm, and observed her through a TV monitor, She still laid herself, breathing regularly.
In the afternoon, I carried out a fluid therapy on her by the method of intravenous drip infusion. Electrolyte fluid was given at a pace of 1 m1/min, But, when 100ml fluid were given, she fell into a labored breathing and her heart stopped.
I recorded her death in our regular manner, reconsidering the diagnosis and treatment I performed on her.
I have shown you the daily works in the building for breeding before (See, Vol.3, No.2, Fall, 1984). This time I will guide you into the building for rearing young cynomolgus monkeys, the next stage of the infant monkey's life.
By the way, I myself have a little son, so I know strong maternal love and that what a painful experience for mothers parting from babies is. Moreover, I remember a story of a mother Japanese monkey in Takasaki-yama troop who held her dead and mummied baby for a year. I asked Mr. H. if there is any trouble in weaning and the separation of mother and infant. He replied, that not a few mother animals lose their appetite and activity on account of depression in spirit, and as for the infants, some of them show diarrhea add weight decrease resulting probably from mental depression.
To avoid these disorders staffs of the rearing section have adopted a unique system of nursing by which for the first one month after weaning every four infants are made to live with an adult female monkey who has an experience of maternity. The adult monkey is just like a teacher of kinder- garden, playing a role of mother or elder sister for the infants. Mr. H. proudly explained me that a considerable number of disorders was decreased as a result of adopting this nursing system. The system was devised from many accumulated experiences of the animal technicians working for this section. During this nursing period the infants learn how to cooperate with and be independent of each other, undergoing health check-ups twice a week.
The next step for the infants is similar to an elementary and junior high school. They are tattooed with the identification number corresponding to their name on the inside of their thigh.
"Bullying", a serious problem is seen here like in human society. Troubles such as rectal prolapse, drastic loss of weight and dermal injury often result from "Bullying". Everyday, Mr. O. makes careful observations on behaviors of the infants. When cases of "bullying" are found, animal technicians in charge of send for a veterinarian and have a meeting to take some countermeasures for both the bullied and bully.
Any way, after three months from weaning, infants finish the step of junior high school and enter the next stage: the three-year rearing period. During this period, they become an adult. Until three years ago, another way of group-feeding had been adopted. The previous way gave the infants a lot of chance to meet new peer mates and brought often many troubles. Now, four animals grouped in the beginning are being kept through this period in a cage(90x60x60 cm). Weight measurement is carried out every four weeks at early infantile period, thereafter every three months. At the end of this period, the females become 3 kg in body weight and the males 5 kg, and almost all of the permanent teeth finish to erupt.
The males are carefully checked for the size of testes and finally they undergo a mating test, If the tested animals prove fertile, they are registered as a breeder, As for the females, menstruation begins at two years of age at the earliest and at two years two months on an average. I asked Mr.O, the youngest animal care-taker, "Are the girl monkeys anxious about their menstruation?". He answered with hesitation, "I don't know". Well, the matured virgin females whose menstrual cycle has been established are moved to the building for breeding. There, they mate, become pregnant, deliver and nurse as their mother once did.
Only a matter I felt pity for the monkeys in this room is that no international marriage is permitted. From the view point of genetic control, their mating program is strictly designed by the computer system. He/She can not make love to the opposite sex of different country of origin. Is this a destiny of laboratory animals?. But, in spite of this prohibition pregnancies sometimes occur if the period of group rearing is extended for some reasons. Therefore, a strict periodical pregnancy diagnosis is needed for young animals kept in group cages.
The area of this building is 917 m2 divided into 9 rooms. Now, 800 young cynomolgus monkeys are living here."' Those who work with these monkeys are just like monkeys' parents and elder brothers or sisters. They are taking care of, and sometimes, talking with (?) those monkeys. I was very impressed with their earnest effort to develop their works.
Page-18
Sketching from a s of Animal Room: [Morphological Characteristics of Growth in
Infant Cynomolgus Monkeys
When we rear laboratory cynomolgus monkeys, it is necessary for us to recognize the normal process of growth. We measure body weight of our monkeys as a routine health check-ups. So we have accumulated a full and detailed . data of the body weight growth with regard to the cynomolgus monkey.
In addition, we adopted a somatometric method in order to clarify the morphological growth characteristics in the cynomolgus monkey.
As for the infant cynomolgus monkeys before weaning, the growth rate of central parts of the body was lower than that of the distal parts, as was generally recognized. It means that the monkeys' hands and feet grow, comparatively early in their infant days. Regarding the head, the facial part grows earlier than the cephalic part. It can be said that the face of infant cynomolgus monkeys already resembles that of adult monkeys.
Early growth is confirmed also by the behaviors which infant animals exhibit. For example, infant monkeys are always clinging to their mother's breast. Fairly developed hands and feet are needed not to fall down. Now, we have finished analyzing the data of infant cynomolgus monkeys, and are examining the monkeys of more advanced age group.
[On
the Growth of African Green Monkeys at TPC]
TPC began to breed African gem monkeys in 1979. Now, 8 male and 30 female breeders of feral origin are being kept. Until today, 100 Fl keys were born. The oldest Fl monkeys are now five years old, and some of them have been used as breeders.
In this note the author is describing the growth of the Fl monkeys, showing some figures.
Page
20 [Squirrel Monkeys, . . . . The
Six-year History of Rearing]
Common squirrel monkeys are being bred at TPC as the third primate species, following cynomolgus and African green monkeys. They were imported from Bolivia in 1980. They have some marked characteristics. One of them is a seasonality of breeding. We are observing carefully how long the seasonality will continue under the artificial environment such as TPC.
The record and data concerning breeding, weight growth and gestation rate of this species are showed in a table ,and in some figures.