TPC
NEWS, Vol.10, No.1,
Fall, 1991 (Whole Number 18)
(English Summary)
Page-3
Message from the Director-General of NIH, Japan
Dr. Tokunaga was inaugurated this spring
as the director-general of NIH, Japan. The editorial board of TPC News
requested him to write a manuscript on his hope for TPC, a branche of NIH.
Here is a summary of his message.
In the first place, he expresses his deep
appreciation to those who have made a
large contribution to TPC, including Dr. Honjo ( the first director), Dr.
Sugahara( the second director) and many other people concerned.
He states that NIH and TPC are now on the turning point to step forward
to a new era. NIH is going to move in a new site,
Toyama, near Shinjuku, with the National Institute of Health and Nutrition and
the National Institute of Medical Care and Hospital Control.
This new area, therefore, will become a
big center of
health and medical
science along with the neighboring institutes, the National Hospital and
the National Medical Center. Dr. Tokunaga is sure that this center will
yield a new demand for laboratory primates.
At Murayama division, to which TPC's monkeys have been sent for a safety
test of vaccines, the building for the Departments of General Biological Product
Control is now under construction. The importance
of the primate will
increase to assure the
safety of the biological products. The system of NIH being supported by
the triangle made of Toyama, Murayama and Tsukuba(TPC) is extremely valuable.
The needs of the primate for
experimentation have increased in research fields,
such as senility, neuro-science, pharmacology and so on. The Ministry of
Health and Welfare
organized a committee on "The Supply System of
Laboratory Animals for Research."
The report from the committee will soon be released.
This spring,
Dr. Yoshikawa
was appointed
TPC's new
director. There
are new administrative members also. Dr. Tokunaga expects TPC to tackle
various problems and continue making steady progress toward the 21st century.
Page-4
Message of TPC's director
Thirteen years have passed since Tsukuba Primate Center for Medical
Science (TPC)
was established.
A large-scale indoor breeding of nonhuman primates was begun
at TPC to respond to the needs of the time: conservation of wild animals,
the demand for laboratory primates of higher quality and the stable supply of
nonhuman primates to national research institutes.
TPC's strategy to meet these needs at a time was the self-sustaining
production system in which laboratory-bred animals are basically involved as a
breeder. It
has been a unique attempt in the world. Therefore, the experience of TPC itself
has been a large scale experiment. After thirteen years, the
ratio
of the number of wild-originated monkeys for whole monkey population has become
less than 10%.
The initial plan has been steady-going.
At the same time, however, new problems such as the deterioration of the
laboratory-bred mother's breeding ability, latent virus control in
indoor-colonies and establishment of the long-term stable equilibrium for
reproduction, have emerged.
In the field of medicine, as the society of the aged has come, the
diseases which had not been serious so far, such as dementia by age, Parkinson
disease, and opportunistic infections among the aged, etc., have become serious
problems. Complicated
social structures increase mental disorders and psychoneurosis.
Moreover, intractable diseases like AIDS and slow-virus disease should be
controlled. It
is demanded that animal models, in particular, those of the primate which is
human's close relatives, are developed to clarify causes, to diagnose, to
prevent, as well as to develop therapeutic medicines.
In January 1991, I arrived at my post of the new director of TPC.
I am burdened the second period of TPC.
The one of the main work of TPC as a branch of NIH of Japan so far, has
been to breed and supply of good laboratory primates for the national safety
test of vaccines.
It will be needed in the future, too.
On the other hand,
as the disorder in the medical field has changed its phase as mentioned
above, the laboratory primate for medical science has to be changed: from those
which are simply microbiologically and genetically controlled to the ones of
deversity, for example, mutants, aged animals and the models of infectious
disease. In
the present state and also in the future, I think that TPC can be the core of
the organizations for the research of laboratory primates, although its present
research activities, manpower, facilities and equipment are not efficient.
Recently, the movement to establish the national center for research and
supply of the primate for medical science has resulted in starting several
special committees by the
government and the private enterprises.
Those committees indicate the necessity of the public research and supply
center of laboratory primates.
In order to become the real center of research and supply of laboratory
primates, TPC should have originality, and the primates supplied by TPC can meet
the demand in the future. Therefore, development of
the mutant strains
for disease
models, preservation of genetic resources and techniques of reproduction
should be strengthened.
One research
project on
the primate
requires about
ten years.
Because their alteration of generation takes ten
or more years. I would
like to ask everyone to cooperate with us to make TPC develop as a unique
national institute which promotes health sciences and disease control.
Page-5
Announcement about PRIMATE TALK
TPC News editor received a
letter from Dr. Lawrencer
Jacobson of the Wisconsin
Regional
Primate Research Center (WRPRC) asking to introduce PRIMATE TALK
-- A Discussion Forum for Primatology in TPC News.
It is an new electronic mail listserver open to electronic mail users
world-wide with an interest in primatology.
For further information, please contact with Dr. L. Jacobson
Page-6
<TPC's Laboratory-Bred Cynomolgus
Mmonkeys > Pregnancy
rate and successful
nursing rate
On its opening, TPC began to establish
a self-sustaining breeding colony of the cynomolgus
monkey,
ie. wild-originated
animals
imported
from Malaysia,
the Philippines and Indonesia were introduced as breeders. We thought
that this breeding system was essential to supply laboratory primates of good
qualities and to conserve the wild primate population. In 1983, the first filial
generation (F1) breeders were introduced for the first time into the breeding
colony. The alteration of generation has steadily
progressed.
At the end of 1990, the number of laboratory-bred (lab-bred) animals
occupied for 77
percent of all animals
in the breeding colony.
Wild-originated animals will
not be seen
in our
colony in
the near
future. Thelab-bred
animals, however,
differ from
wild-originated
animals in
some breeding results.
There are
still several
problems to
be solved
to establish
a complete self-sustaining breeding colony.
TPC's monkeys are basically kept under indoor individually-caged
conditions. All
animals are checked for their health conditions every morning.
As for female breeders, presence or absence of menstrual discharge on a
dropping tray set under the cage is carefully examined.
After two or more successive regular menstrual cycles are ascertained, a
female breeder is introduced into a male breeder's
cage to mate with him during the anticipated period of ovulation (for 72
hours)in the following menstrual cycle: the one-to-one timed mating system.
Afterward, the female breeder is moved to
her own cage, and undergoes pregnancy diagnosis four weeks later.
Pregnant females' health conditions are checked, as well as the
development of their fetuses, with an ultrasound diagnostic device.
After the 155th day of pregnancy, fetal position is examined every day.
If an abnormal fetal position is found, we change the fetus with our
hands to a normal position so that the rate of stillbirths decrease.
The average pregnancy period of the cynomolgus monkey is 165+7 days.
On the day of delivery, a new born baby is checked for the birth
registration (body weight, body length, sex, the presence or absence of
malformation, etc.).
In the case in which the mother monkey refuses to nurse her baby or shows
some problematic nursing behaviors, artificial nursing or foster nursing
procedures are
employed.
The number of deliveries by wild-originated and lab-bred (F1) cynomolgus
monkeys from 1978
to 1990
are shown
in Fig.1.
The total
number of
deliveries by
the wild-originated through the period was 2,435, and that by the lab-bred was
619. The delivery number by the F1 animals exceeded that by the wild-originated
in 1989.
Pregnancy rate by the 72 hours' timed mating system of the
wild-originated and F1animals are shown in Table 1.
These rates show reciprocally the
times of mating
by which
an animal becomes
pregnant according to
the order of
parity. For example,
a wild-originated female
animal required mating of
2.5 times (0.40=1/2.5)
for the first
pregnancy in TPC.
While, a F1
female needed
6.9 times
(0.14=1/6.9). But
F1's rates
of the second parity was about the same
with that of the wild-originated
and the results in the third and fourth
parities were
better. These facts
indicate that
in our rearing system of F1 animals, there are some problems which
suppressed mating individuals.
A solution is urgently needed to improve the pregnancy rate of the first
parity.
Table 2 shows the state of deliveries.
No significant difference was seen between the wild-originated and F1
animals. The
rates of abortions and stillbirths at our center were lower than those reported
by other primate breeding facilities.
It might be significant that we examined fetal position in the late
pregnancy period, and performed changing fetal position as mentioned above.
Table 3 shows the incidence of well nursing in terms of the order of
parity. Of 2033 wild breeders who had given live births to their babies, 1973
animals (97.0%) nursed well their babies.
The causes of unsuccessful nursing (3.0%) were mother monkeys' health
problems such as obesity, prolonged diseases, etc..
As for the F1 breeders, the incidence of well nursing in the total
number(539) was 71.0%, however, the incidence of the first parity was extremely
low (62.2%). Even
though the incidences increased as the parity progressed, they were much lower
than the wild-originated breeders'.
Among F1 breeders who were counted as well nursing mothers, there were
some animals who showed defective nursing behavior, such as holding babies
upside-down. Mother's
age, period of pregnancy, infant's sex and birth weight had no obvious effects
on nursing behavior.
The average age at the first delivery of F1 breeders was about six years.
Therefore, it is one of the solutions for improving the incidence of good
nursing that F1 breeders experience the first delivery at the age as young as
possible. Every
two days mating, group mating, long term mating as well as one-to-one mating are
now in operation.
Page-8
<TPC's Laboratory-Bred Cynomolgus Monkeys> Mother-infant
interactions
Unlike wild-born macaque breeders, some laboratory-bred (lab-bred)
cynomolgus monkeys
are defective in mating and nursing behavior.
From the
ethological view
point, the author, Dr. Nakamichi, describes the characteristics of
nursing behavior of lab-bred mother monkeys (lab-mother), comparing them to
those of wild-born mother monkeys (wild-mother).
The subject animals were wild-born and lab-bred mother monkeys and their
offsprings. The wild-mothers had been kept individually at TPC for
at least five years, experiencing
parturition and
successful
nursing during
this period.
The lab-mothers had been kept with age-mates until they physically matured and
were moved to individual
cages. Sixty-five
percent of
the lab-mothers
were parous
and the remaining 35% were nulliparous. Ten mother-infant pairs of each
group were observed cross-sectionally every other week until the 14th week after
birth.
Fig.1 shows the frequency rates of "Held (Mother holds infant to her
ventrum with one or both of her arms)". "Held" was significantly
more frequently observed in the wild-mothers than in the lab-mothers. Although
"Held" was not necessarily needed when infants suck milk, the
wild-mothers frequently showed this behavior. Sometimes they held their infants
as if they were trying to restrain the infants.
On the other hand, the lab-mothers more frequently took postures of open
chests, grasping the wire mesh of the cages
with both hands, while
their infants were
sucking milk or were sleeping. As for
"Grooming infant
by mother", there
were no
differences in both quality and quantity.
Fig. 2 shows the frequency of "Locomotion ( Infant or mother moved
more than one body length)".
It shows that the infants of both groups moved actively as they grew. The
frequencies of locomotion of the lab-mothers and the infants were significantly
higher than those of the wild-mothers.
Differences were also seen in the quality of behaviors.
While wild-mothers moved slowly in the cage, the lab-mothers moved
suddenly and quickly.
Stereotyped movements, such as turning round and round, were observed in
the lab-mothers. The infants of lab-mothers tended to move, responding to their
mothers' movement.
Therefore, it is considered that their frequencies seem to become
significantly high.
Fig.3 shows the frequency of "Break contact ( Mother or infant move
to break physical contact )" per 30 min.
The frequencies of "Break contact" of the infants increased as
they grew, but those of the mothers' remained low.
The lab-mothers' frequencies were significantly higher than those of the
wild-mothers. This
was also true for "Make contact ( Mother or infant move to make physical
contact)".
Fig.4 shows the
frequency of "Maternal
aggression (
Mother directs aggression toward infant such as gazing with opening
mouth, clasping, slapping, biting, etc.), which began when the infant became
about two-month old. The lab-mothers' frequencies
were comparatively low. Seventy
percent of "Maternal
aggression were derived from the infants' "Playful contact with
mother (Infant movements accompanied by physical contact with mother such as
jumping on and off mother's back or head, climbing on her back, etc.). These
behaviors, however, are rarely seen in outdoor colonies or group
housing animals. It seems that the infants sometimes made contact with
their mothers as their playing objects
in the physically and socially poor environments.
These results indicated that the lab-mothers were less active in
mother-infant interaction, comparing with the wild-mothers.
The difference might partly be produced by the fact that 35 % of the
lab-mothers were nulliparous.
It is reported that macaque mothers interact very closely or,
contrariwise, insufficiently with their first babies.
The author analyzed the lab-mothers, dividing them into the parous and
the nulliparous.
No significant differences have not been found so far.
At TPC, the infants experience a short time mother-infant separation
whenever they undergo periodical
health check
ups. The
author wishes
that this
artificial
intervention would not have any negative influences on the behavioral
development of the infants.
In this
environment, almost
all wild-mothers
nursed their
infants successfully. As for the
lab-mothers, 35.9 %
of the nulliparouses
and 10 to less than 20 % of the parouses rejected
nursing immediately after delivery (2), but the remaining, about 70 %,
nursed infants successfully.
The lab-mothers spent less time on nursing than the wild-mothers did. The
author hinks,
however, that this change is not necessarily a negative change in nursing, as
long as their infants
are growing well.
Clarifying the changes of nursing in the succession of generations and
decreasing the number of mothers who reject nursing are important tasks to be
solved in future.
Page-10
Errata
An Outbreak of Nonhuman Primate Varicella-Like Herpesvirus Infection in
the Established Breeding Colony of Cynomolgus Monkeys
TPC
NEWS, Vol. 9, No.2, Winter, 1990
(the previous issue)
The
numbers should be corrected as follows:
Page
6, left
half, 11th
line from the top, 35 -> 36.
12th line from the top, 76 -> 75.
Page
7, right
half, 3th line from the bottom, 6 -> 5.
4th line from the bottom, 100 -> 103.
7th line from the bottom, 36% -> 28%.
Page-11
<TPC's Laboratory-ABred Cynomolgus Monkeys> Report from the animal room
for young cynomolgus monkeys
Mr. Hanari is an animal technician, who has been taking charge of the
building for
rearing
young cynomolgus
monkeys (
Building 2
)for seven
years. He
reports the rearing system of cynomolgus monkeys from weaning to the time
they become a breeder, comparing each stage to the education system in Japan.
After being weaned, the infant monkeys are moved into Building 2 from the
building for breeding ( Building 1 ). This step may correspond to that of human
nursery school and primary school. The infants who are more
than three months old, weigh over 700g, have the first deciduous molar
and can eat food by themselves are weaned. Weaning and separation from mothers
cause the
infants a great deal
of stress.
Many of the infants are suffering from stress related disorders, such as
diarrhea, losing weight and appetite. Therefore, we
have adopted a nursing-mother
system: an adult female monkey (nursing mother),
who has
no kinship
with the
infants but
experience of maternity, is made to live with four infants in a cage for
one month after weaning.
This system is considerably effective to reduce those disorders. Three
months later, under anesthesia
the infants
are tattooed
on the
inside of
their thigh
with identification numbers.
The next step would
be compared to that
of primary and
junior high schools of human children. Troubles
caused by bullying
are sometimes seen.
When the monkeys become two or
two and
a half years
old, they
are divided
into two groups,
the candidate of breeders
and the
others to be
used in
vaccine safety tests
in the Murayama division of
NIH Japan.
Every monkey
at TPC undergoes
periodical health examinations, such as
weight measurement, hematological,
biochemical, bacteriological, and
virological
examinations.
Only animals
who passed
these examinations are sent to
Murayama.
When the candidate breeders become
sexually matured, they are moved into an
individual
cage. Menstruation
and its regularity are observed in the female monkeys.
The
male monkeys are carefully checked for the size of the testis and undergo a
mating test: a male and female live together for six months, pregnancy diagnose
and weight
measurement are carried out once a month during this period.
Adult male monkeys have very dangerous big and sharp canine teeth.
We cut the teeth according to the dental treatment of humans.
The monkeys who have been
proved to be fertile are registered as breeders and sent to Building 1
for breeding.
TPC has adopted
a breeding
system called
the "rotation line
breeding system" (RLBS) for
the purpose
of genetic
control
and avoiding
the rise
of inbreeding
coefficient. Breeding work is performed mainly in
Building 1, but some animals who are not involved in the rotation
line stay in Building 2,
and five to six animals become pregnant per month.
All of them
are laboratory-bred
and nulliparous,
therefore, about half of
them are unable
to nurse their
infants successfully. We
have adopted artificial
nursing to rear
the infants.
Although artificial nursing needs a large amount of time and more days
until weaning than maternal nursing, most of the infants
are growing well.
Some mother monkeys
refuse to
nurse. A rearing system to correct this problem is urgently needed.
Page-15
<TPC's Laboratory-Bred Cynomolgus Monkeys>
Natural Infections with Various
Pathogenic Agents in Laboratory-Bred Cynomolgus Monkeys
Viruses
Since its opening, TPC has aimed at producing laboratory primates of good
quality. Mrs. Narita presents the results of virological survey on some viruses
in TPC's lab-bred monkeys, comparing with those of the wild-originated monkeys.
It is one of our successful results that we have established a measles
virus free colony.
Table 1
shows the
incidences
of
antibody
to measles
virus by
hemagglutination inhibition
test (HI)
in imported
wild (from
1979 to
1987) and laboratory-bred cynomolgus monkeys. The average positive rate
for the imported monkeys on arrival was
45 %. Nine weeks
later, it became
more than 80%. However, 1518 lab-bred
monkeys were
all negative.
As monkeys
are susceptible
to measles virus, it is very difficult to obtain measles free wild
monkeys. It is valuable that TPC's lab-bred cynomolgus monkeys have been
negative to this virus.
Herpes virus simiae (Herpes B virus which is indigenous to macaque
species) has cross-antigenicity to HSV-1.
Now we can not use Herpes B virus since it is categorized as Class IV by
the criteria for biohazardous microorganisms.
We, therefore, have measured the antibody titer of Herpes virus simiae
by complement fixation test (CF), using HSV-1 antigen. Table 2 shows the
incidence of antibody to HSV-1 in newly-imported wild and lab-bred cynomolgus
monkeys. The
mean positive rate of the imported monkeys on arrival and
nine weeks later was about 50 %, whereas that of the lab-bred monkeys was
only 3 %. In
addition to CF
test we recently surveyed them by
enzyme-linked assay (EIA).
The survey revealed that 80 % of the imported monkeys were positive.
Most of their antibodies are thought to be derived from B virus
infection.
Three percent positive cases in lab-bred monkeys were the animals bred in
a gang cage of group-feeding, which had been attempted exceptionally at TPC. It
seems that infection from positive imported animals to lab-bred animals
repeatedly occurred in the cage.
All cynomolgus
monkeys bred by
TPC's standard
breeding
system were
negative. Therefore, it can be said that
we have eradicated Herpes virus simiae as well as measles virus.
Figure 1 shows the
positive rate of
antibody to simian
cytomegaro virus (SA-6 virus) in our lab-bred cynomolgus monkeys.
Although the mean positive rate was about 85 %, the rate came to 100 % as they
grew older.
Fig. 2 shows the positive rate of antibody to Cyno-EBV in our lab-bred
cynomolgus monkeys.
These results indicate that TPC's present breeding system is unable to
produce CMV and EBV free monkeys.
Last year, when an outbreak of nonhuman primate varicella-like
herpesvirus (TSVV) infection occurred, we had stoped some of routine rearing
work. As
a result, a number of negative cases to these viruses were found among the
infants aged over two.
They were the animals which had lived with their mothers for one year and
seven months after birth without weaning.
These negative cases suggest that horizontal transmission is the main
cause of the infection of CMV and EBV at TPC.
Table 4 shows the incidence of antibody to TSVV measured by an indirect
immunofluorescent assey (IFA) in the animals of Room 1 to Room 3 after the end
of the epidemic.
The positive rate was about 13 % in both imported and lab-bred animals in
Room 1, where the epidemic occurred. The survey on the sera of these positive
animals revealed that they had already had the antibody before the outbreak.
All animals in Room 2 were negative.
One lab-bred animal in Room 3 was positive.
This animal was born in Murayama Division of NIH not TPC, and its sera
taken immediately after the arrival at TPC (1979) was already positive.
There might be some latently infected animals which could not be detected
by the sensitiveness of IFA.
It is one of our tasks to establish TSVV free colony.
The fact that some of the imported animals were positive already on their
arrival at TPCmay become one of the clues to clarify the source of infection.
Table 6 shows the incidence of antibody to STLV-I and SIVmac in
wild-originated and laboratory-bred cynomolgus monkeys by IFA.
It demonstrates that the positive rate of cynomolgus monkeys at TPC
considerably low regardless of their origin.
Bacteria
Mrs. Kohno
reports the
result of
the bacteriological
survey in
lab-bred and imported cynomolgus monkeys.
Table 1 shows the infection rate of enteropathogenic bacteria in
newly-imported wild (imported from 1979 to 1987) and lab-bred ( surveyed from
1986 to 1990) cynomolgus monkeys.
The infection rates of Shigella and Salmonella in
the imported wild monkeys were 10.2 and 2.5 %, respectively; whereas, no
positive cases were found in the lab-bred monkeys.
Eight types of Shigella including Shigella flexneri 2a, S. sonnei, and
provisional serotype 1621-54 of Shigella were isolated.
Eight of Salmonella including Salmonella weltevreden and S. typhimurium
were isolated.
The infection rate of Campylobacter in the wild monkeys was 36.3 % and
that in the lab-monkeys was 26 %; the lower their age, the higher the rate.
The species of Campylobacterisolated are shown in Table 2.
Table 3 shows the infection rate and species of Campylobacter isolated
from long-term diarrheal monkeys (1985-1986).
TPC has monitored tuberculosis infection in monkeys by tuberculin tests,
hematological and pathological examinations, and identification tubercle
bacillus. According to the survey carried out from 1979 to 1987, only one case
infected human tubercle bacillus has been found in 2,484 imported animals.
Parasites
Dr. Takasaka documents the present state of parasitic control in newly
imported, wild originated and lab-bred cynomolgus monkeys.
We use Negvon (Bayer;O,O-dimethyl
2,2,2,-trichloro-1-hydroxyethyl-phosphonate) to eradicate ectoparasites such as
lice, and the monkeys harboring helminths are treated with thiabendazole.
However, no measure is presently adopted to control protozoa.
Table
1 shows the infection rate of parasites in imported (1979-1986) wild cynomolgus
monkeys.
The species of
Helminths found were Strongyloides, Oesophagostomum, Stretopharagus, and
trichuris trichiura, Bertiella.
Table
2 shows the infection rate of parasites in wild-originated and laboratory-bred
cynomolgus monkeys (1985-1986). The wild-originated monkeys experienced
three times thiabendazol treatment regimes , and the lab-bred monkeys
experienced the regime twice before the examination.
As for protozoa, they were found in the feces of both wild-originated and
lab-bred animals. The infection rates of the lab-bred animals were lower, as a
whole, than that of the wild-originated ones.
All animals, except one lab-bred one, were negative to Helminths.
Measures to eradicate Protozoa is to be needed hereafter.
Page-20
TPC's New Mark
We announce with pleasure that
TPC's new mark was decided.
It was designed by Dr.Yoshida's brother, Hiroshi Yoshida, oil painter. It
is a symbol of our center specializing in primates.
Page-21
< TPC's Laboratory-bred cynomolgus monkeys > Hematological and serum
biochemical values
Age-related change of hematological and serum biochemical value in our
lab-bred cynomolgus monkeys were presented by Dr. Yoshida.
Significant changes of hematological values were observed in red blood
cell count (RBC) and mean corpuscular volume (MCV). The value of RBC decreased
with increasing age of animals
(Table 1). Regarding
the age-related changes
in serum biochemical values, values of serum protein
(total protein concentration: TP, albumin-globulin ratio: A/G), lipid
(high density lipoprotein concentration : HDLCHO, non-esterified fatty acid
concentration: NEFA, free cholesterol concentration: FCHO), carbohydrate
(glucose: GLU)
and some
enzyme activities
(glutamic pyruvic
transaminase: GPT, alkaline phosphatase: ALP) correlated with the age
of animals in both sexes (Table
2). There was a close
relationship between serum ALP and
body size changes in the
laboratory-bred animals. It is a matter of general knowledge that serum
ALP reflects bone-growth in the animal.
Furthermore, serum biochemical values between lab-bred
animals aged from one to over 12 years ( nine age groups: 1 year, 2
years,..., 7-8 years, 9-11 years, and over 12 years )and wild originated animals
(W group) reared under indoor conditions for 5 to 6 years were analyzed by
canonical discriminant analysis (discriminant analysis with reduction of
dimensionality). Discrimination on
two-dimensional plane, Z1-Z2
between the laboratory-bred animal
groups and the wild
originated animal group in males (Fig. 1) and females (Fig. 2) was
demonstrated.
Page-23
<TPC's Laboratory-Bred Cynomolgus Monkeys>
Endocrinological characteristics
Age-related
changes in serum testosterone level in males (Fig.1) and progesterone level
in females
(Fig. 2)
were demonstrated.
The age
at puberty
of the
laboratory-bred cynomolgus monkey was judged to be 3 - 3.5 years in males
and 2 - 2.5 years in females.
Serum growth hormone (GH) levels were determined by using a newly
developed human immuno-radiometric assay kit. Serum GH concentrations of the
animals over ten years old showed less than 0.4 ng/ml regardless of their age.
The higher value of serum GH was obtained from the animals less than 5 years old
in females (Fig.3, upper panel) and less than 8 years old in males (Fig.3, lower
panel).
Effects of aging on the pituitary-thyroid function in the cynomolgus
monkeys were analyzed. The age-related changes in serum levels of
thyrotropin(TSH), triodothyronine (T3) and thyroxine were investigated (Fig.4).
Although age-related decrease in the serum TSH concentration was not
observed in the females, decreases in T3 and T4in the serum were observed.
In the males, age-related decrease in TSH, T3, and T4in the serum was
observed. After
IV injection of TSH-releasing hormone(TRH), significant increases in the serum
TSH concentrations were detected.
The oldest group (16 years old) showed the highest response among the
five different age groups tested (Fig. 5, upper panel).
The highest responses of T3 and T4 release from the thyroid gland after
TRH injection were obtained from the 10 year old group.
The results suggest that the sensitivity of the thyroid gland to TSH
and/or the productive or releasing capacity of T3 and T4 in the thyroid gland
gradually decrease on and after the peak of the tenth year.
A part of endocrinological characteristics of the laboratory-bred
cynomolgus monkeys was presented.
Page-26
Laboratory-bred monkeys in immune studies
The use of laboratory-bred (lab-bred) monkeys in biomedical studies has
several advantages as compared to wild-originated ones.
We are going to show here three results obtained in our experiments with
lab-bred cynomolgus monkeys for demonstrating the usefulness of lab-bred monkeys
in immunological study.
[1]
Clearly known age:
Nonhuman primates are the most useful laboratory animals to determine the
age-related change of immune functions in human, because they are long-lived and
have similar immune system and functions to human. However, it is needed to use
lab-bred monkeys whose ages are clearly known in
this experiment.
We were studying age-related change
of immunoglobulin
levels to determine the change of humoralimmunity in cynomolgus monkeys.
Fig. 1 shows the
change of IgG, IgM and IgA levels from 0 to 20 or
more years old. Trace amounts
of IgM and IgA
are detected in the serum of infant monkeys. Their
levels increase
with age and
reach adult level at around 4 to 5 years old. About
80% to adult level of IgG
can be detected at 0-day old infants. It
rapidly decreases
until 4
to 5 month
of age,
showing that
IgG detected in infant serum must
be maternal IgG which transfer
from mother to fetus
through placenta during gestation. Then, the level of IgG shows
age-related increase and reaches adult level at 4
to 5 years old. Fig.2 shows
the change of anti-A and
anti-B blood
group antibody
levels in
relation
to age.
Both anti-A
and anti-B
antibody titers increase with development and reach the peak at 4 to 5
years of age, after that they decrease with aging. The early increase of
antibody titer shows the development of humoral immune function and the latter
decrease reflects the decline of immune function induced by
aging. We can determine not
only the development of immune functions but also
the immune functions
in aged monkeys
by using lab-bred monkeys.
[2]
Plain immuno-reaction without biasses of infection:
Since naturally occurring allergy induced with the pollen of Japanese
cedar was
found in wild Japanese monkeys, a corroborating study has started to establish
the disease model of cedar-pollen-induced allergy with monkeys.
As the first step, we tried to determine the level of anti-cedar-pollen
IgE antibody titer with pollen-sensitized monkeys.
For this purpose, negative control serum was needed to determine the
cut-off point between antibody-positive and negative monkeys.
We examined the antibody titer with sera obtained from wild-originated
cynomolgus monkeys who lived in Indonesia where no Japanese cedar grows.
As we expected, no anti-cedar-pollen IgE antibody was detected, but very
high levels of total IgE were detected in these monkey sera.
The total IgE levels of Indonesian monkeys were 1.5 times and 6 times as
higher as those in wild Japanese monkeys and in lab-bred cynomolgus monkeys,
respectively. These
high levels of IgE must be caused by parasite infection when they were in the
jungle. In
fact, serum IgE levels decrease in monkeys who were treated with anti-parasite
agents and kept in indoor environment for ten or more years.
In the use of wild-originated monkeys, the latent infection of several
infectious agents can affect the results of the experiment.
[3]
Family analysis:
Major histocompatibility complex (MHC) plays an important role in
interaction between immune cells.
The expectation is to find a MHC homozygote monkey to analyze
immune
functions induced cell-to-cell interaction.
We keep several monkeys who have been born by inbreeding such as
father-daughter or half-sib mating.
There is a possibility to find MHC homozygote in these "inbred"
monkeys.
Because the MHC gene is highly polymorphic, the unrelated mother does not
share the identical MHC gene with the father, so that the MHC homozygote can not
be found in offspring (Family A in Fig.3).
On the other hand, if the mother shares the identical MHC gene with the
father, the possibility of obtaining MHC homozygote offspring is 1/4 (Family B).
The problem is how to detect MHC homozygote monkeys.
The class-II MHC antigens can be detected by mixed lymphocyte culture
(MLC). Then,
we determined MLC response between lymphocytes from family members including
"inbred" offspring.
If offspring is MHC homozygote, they share identical MHC with their
parent, and parent lymphocytes do not respond to offspring lymphocytes in MLC.
As shown in Table 1, all of MLC responses among family members were
positive ( all of the SI value are over 2 ) in Family A.
In Family B, MLC responses of both mother and father to offspring were
negative ( SI is under 2 ).
This offspring was born by father-daughter mating and must be MHC
homozygote.
Keeping successive generations of lab-bred monkeys has an advantage
in using them in family analysis of genetic properties.
Page-29
<TPC's Laboratory-Bred Cynomolgus Monkeys>
Normal findings in ocular fundi of cynomolgus monkeys from birth to 20
years
Mr. Suzuki reports
on the normal
findings in ocular
fundi of
1151 cynomolgus monkeys from birth to 20 years.
The number and age of the monkeys used for the investigation are shown in
Table 1. These monkeys
except the
ones aged
less than
7-day old
were anesthesized
with ketamine-HCl. One
drop of
the mixed
solution of
tropicamide
and phenylephrine hydrochloride was instilled
into each
eye of the
monkeys 20
minutes before the investigation.
Daylight-type color films were used.
1.
Normal findings
Neonates
The color of the retina, including the optic disc, was salmon pink.
Macular was not seen clearly.
Vessels of the chorioidea was transparent, as the pigmentation was not
enough in the retinal epithel.
The diameter of retinal artery around the optic disc was about one-third
of that of the retinal vein.
The veins and arteries extended up and down from the optic disc, drawing
arcs. Retinal
hemorrhages were seen in a number of animals.
3-day-old
The retinal color
was the
same as
the neonates'.
The macula
became a little darker, enabling visualization of its shape.
7-day-old
The macula became darker.
14-day-old
Nerve fibers were seen clearly. Retinal
light reflexes were recorded. The color of the macula was much darker.
But the central pit was not visible.
4-week-old
The color of the fundi changed to blue-green. The optic disk was vivid
orange in color.
The macula was dark.
Retinal light reflexes were observed more clearly.
6-week-old
The findings were not significantly different from those of the
4-week-olds
13-week-old
The color of the fundi had changed from blue-green to steel blue-gray.
Retinal light reflexes were stronger than that of the 6-week-olds.
The diameter of the retinal artery had become about two-thirds of that of
the vein.
1-year-old
The fundus of the 1-year-old monkeys had most of the characteristics of
the mature optic fundus.
The macula and central pit were clearly visible.
3-year-old
The color of the fundi changed from blue-gray to brown. Light reflexes
were less strong than those of the 1-year-olds. Sheathing
of the blood vasculature had begun
from the age of three months.
7-year-old
The fundi were
dark. Light
reflexes became
much weaker.
The central
pit and macula were seen clearly.
The blood vascular light reflexes were partly observed.
15-year-old
The fundi were much darker. gray-brown. Nerve fiber light reflexes were
not seen anymore.
The central pit
was clearly visible.
Pigmentation around
the optic disk was evident, making a conus like pattern in a part.
20-year-old
Light reflexes were no longer seen. The choroidal vasculature without
pigments was partly observed. The
central pit
and macula were
evident. Scleral
ring was seen
around
the optic disk.
Characteristic findings of the ocular fundi according to age are shown in
Tables 2 and 3.
2.
Retinal hemorrhages in neonates
Retinal hemorrhages were observed in 66 of 96 neonates born normally.
One to ten hemorrhages in the shapes of spot, line and stain were seen
per ocular fundus.
These retinal hemorrhages disappeared naturally within 3 to 14 days after
birth. Fluoro-funduscopic
examination revealed that these hemorrhages occurred in the retinal strata and
that the bleeding had already stopped.
There was no significant relationship between the retinal hemorrhages and
the sex or birth weight of the neonates, and the mothers' parity. Shown in Table
4, the incidence of retinal hemorrhage of the neonates born by Cesarean
operation was significantly lower than that of those born normally.
3.
Persistence and disappearance of the hyaloid artery
Forty-five newborn cynomolgus monkeys were examined at every three to
four days for the persistence and disappearance of the hyaloid artery. Hyaloid
artery was seen in all of the monkeys. It regressed within 21 to 45 days, 27
days on average, after birth.
Fig. 1 shows the percentage of the regression according to the week.
Page-31
Overseas Topics:
Research Focus-----Understanding Aging
from "Center Line " of the Wisconsin Regional Primate Research
Center
Dr. Cho translated "Center Line" (Vol.1, No.3, Fall, 1990.)
into Japanese.
The newest data revised by
Dr. Uno of the
Wisconsin Regional Primate Research Center are presented.
Page-33
The present state and problem in developmental biotechnology
Dr.
Yoshiro Ishijima, professor
of Tokyo University
of Agriculture, presented a lecture titled "The Present State and
Problem in Developmental Biotechnology" at TPC last year.
We can see the essence of the lecture again on the pages 33, 34 and 35.
Page-35
<PTC's Laboratory-Bred
Cynomolgus
Monkeys> Usefulness
in the
study
of
reproduction
Dr. Sankai demonstrates the
usefulness of the lab-bred
cynomolgus monkeys in a reproductive study( hormone administration ) at
TPC.
The female cynomolgus monkey originally releases an egg from the follicle
during the menstrual cycle. But,
the study of
reproduction needs a
number of eggs. TPC,
therefore, has established the
technique to obtain a
number of eggs
at a time by hormone(PMSG and hCG) administration.
The lab-bred cynomolgus monkeys responded more sensitively to this
hormone administration than the wild-originated ones.
The number of the follicles (mean+SD) obtained from the lab-bred animals
treated with the hormone was 47.8+31.1 (n=23), whereas that of the
wild-originated animals was 16.9+6.7 (n=8).
The numbers of the recovered eggs were 22.3+13.8 (n=19) in the lab-breds
and 10.5+6.4 (n=8) in the wild-originateds (Fig.1).
The ovary of the lab-bred animals reacted very sensitively to the hormone
treatment. Moreover,
the value of blood estradiol of the lab-breds increased extremely above the
normal level, but that of the wild animals did not. The value of blood hormones
in the wild-originated animals showed the change similar to that of the
lab-breds, even though the level was low.
Age is considered
as a cause
of the low
reactivity of the
ovary of
the wild animals. Nonhuman primates
are so valuable
animals. Studies
on the
relationship between age and hormonal response and maturity of eggs in the
primate are needed from the view point of effective use of primates, as
well as, from that of the study of aging.
In these experiment, hormone administration was carried
out 9 times for 13 days before follicular suction. It
might have been
a strong streesor,
which gave some
influence on hormonal changes of the animals.
Therefore, the number of days, which were needed to occur menstrual
bleeding after
the treatment with PMSG and hCG, was examined (Table 1). The lab-bred
animals recovered earlier than the wild-originated animals. They have contacted
with humans since they were born. The influence of the stress by hormone
administration was smaller in the lab-breds
than that
in the wild-originateds animals. The lab-bred animals
have much advantage in reproductive studies.
Page-37
<TPC's Laboratory-Bred Cynomolgus Monkeys>
In vaccine safety tests
Three authors, Drs. Chino, Kobune
and Arita of the NIH
of Japan, who have beenengaging in research work of virus infection
and vaccine safety tests, mention the usefulness of lab-bred monkeys.
(1)
The lab-bred animals at TPC have clear birth date and parentage.
Moreover, it is important that they are free from measles virus and
parasites. In
a measles infection experiments and its vaccine safety tests,
Warthin-Finkeldey-type infected giant cells appear in lymph nodes and the thymus
when the animals subjected are contaminated with measles virus.
The number of the giant cells coefficients with the vaccine virulence.
The appearance of the giant cell is an important barometer of the vaccine
safety.
In the case in which the animals are infected with parasites, foreign
body giant cells
sometimes appear. These giant cells can hardly be differentiated
morphologically from Warthin-Finkeldey-type giant cells.
Therefore, parasite free lab-bred monkeys are useful for vaccine safety
tests as well as experiments.
When animals are positive to the antibody against measles virus, measles
infection can not be
established, and no
giant cells appear.
Most of the
wild animals are positive to the antibody. The laboratory-bred monkeys,
therefore, are indispensable in vaccine safety tests.
Table 1 shows the different susceptibility for neurovirulence test of
polio-virus in monkeys from three different countries of origin, Philippines,
Indonesia and peninsular region (Malaysia and others).
(2)
and (3)
In these articles both authors describe the problems of wild animals and
advantages of lab-bred animals in vaccine safety tests, expecting TPC to produce
better quality lab-bred monkeys.
Page-39
Announcement on the 14th International Primatological Society
XIVth
Congress of the International Primatological Society (IPS)
Date: August 16 to 21, 1992.
Place: The Congress Palast in Strasbourg, France.
The host institution: The Societe Francophone De Primatologie (SFDP).
Official language: English with an eventual translation from and into
French.
For
further information, please contact the organizing committee.
Page-40
<TPC'S Laboratory-bred Cynomolgus Monkeys> Morphological characteristics
of growth
By what is normal growth of cynomolgus monkeys proved? An animal
technician, Mr. Shimizu, describes morphological characteristics of growth
in TPC's
lab-bred cynomolgus monkeys aged from birth
to 9 years.
The biometrical data obtained wereanalized by multivariate allometric and
principal component analyses.
Fig.1 shows relative growth in cynomolgus monkeys from birth to 12 weeks
of age in females(o) and males(o).
The relative growth
rate was standardized
by the growth rate of head breadth(*).
The abbreviation of
traits: A
(total face
length), B
(upper face
length), C (total head height),
D (head
length), E (head
breadth), F
(bi-iliac breadth), G (bi-acromial breadth), H
(anterior trunk length),
I (upper arm
length), J (thigh length), (lower arm length), L (leg length), M (hand
length), N (foot length), and O (tail length).
The growth rates of the trunk and face in
the infants were higher than those of the limbs and arms.
This matter means that
the limbs and
arms have already grown before birth, and the
trunk, on the
other hand, grows
remarkably after birth.
It corresponds to the needs of the infants, who have to cling tightly to
their mothers' chests immediately after birth.
Figs. 2 and 3 shows the result obtained by the multivariate analysis
using principal components (PC 1 and PC 2).
PC 1 is a size factor.
PC 2 means a factor of body proportion.
Shown in the Figs., the values of
PC 1 became constant after the age of five with the males, and after the
age of three with the females.
The values of PC 2 have no difference between the males and female until
the age of three.
During this period, the growth of the limbs and arms was superior to that
of the trunks.
PC 2 of the males showed plus values after the age of sexual maturation (
male---3.5 and female--- 2.5 years old), whereas that of the fe0males were minus
numbers. This
means that the difference of body proportion between males and females began
from the age of sex maturation.
Males are characterized by the stout trunks and long limbs and arms. On
the other hand, females have the plump trunks and comparatively shorter limbs
and arms.
Page-42
<TPC's Laboratory-bred Cynomolgus Monkeys>
Body weight change
An animal technician
Mr. Ohto
reports on
body weight
change in
our lab-bred cynomolgus monkeys.
Judging from a weight management, the cynomolgus monkeys are classified
into five categories: newborns, weanlings,
juveniles of group-rearingperiod
and adults. The frequency of weight measurement differs with the
categories.
Newborns: We used to weigh every newborn every week. However, we noticed
that the growth rate
of this
period was
constant
regardless of
birth weight.
We have decreased the weighing frequency of the animals with a normal
growth rate.
Weanlings: Our monkeys are weaned about 20 weeks after birth.
Weaning (separation from mother ) sometimes results in diarrhea for the
weanlings. Therefore,
they are weighed twice a week and checked their health conditions for five weeks
after weaning, and at the 6th, 7th, 8th, 10th, 12th weeks, subsequently.
Juveniles of group-rearing:
The period
of group-rearing
is three
years. Body weight is measured every three months.
Adults: The female breeders is
weighed
before mating
and at
the times
of pregnancy diagnosis, delivery, lactation, and weaning.
The females which stop breeding are weighed every three months. The males
are weighed every six months.
Fig.4 shows the body weight changes and
coefficient of variances in our monkeys from birth to the age of 500
weeks(10 weeks). The data was obtained from about 3100 lab-bred animals (
F1 - F5
). Its total
number have
amounted to about
120,000, excluding
the data of females during pregnancy and lactation period.
The mean birth weight (M) and standard deviation (S.D.) were 351+59 g
with the males and 325+53
g with the
females. The males
and females drew
the same growth curves until the age
of 160 weeks, after
that the males
showed rapid increase in growth.
The weight of the age of 500 weeks were 6.3 kg (male) and 3.8 kg
(female).
The coefficient of variance (100xSD/M) of the males suddenly changed from
the age of 200 to 400
weeks. It means
that the time
of sexual maturation
depend on each animal. The maximum
value was 36
% at the
age of about
300 weeks. On
the other hand, the weight of females increased gradually and constantly.
What is the normal
weight for the
lab-bred cynomolgus monkeys?
There are some animals of obesity or of diabetes in our colony. We have
to consider this problem in every aspect.
Page-43
Japan-China joint workshop on laboratory animals
The third Japan-China joint workshop on laboratory animals was held on
March 27-28at the Tsukuba Center for Institutes. It was highly successful and
the participants was over 50 in number. Dr. Cho
introduces the programs related to primatology with his impression of the
meeting.
Pages-44,
45, and 46
Memories of Three Trainees
Three senior students of Japan Women's University had been at TPC as a
trainee for a month, studying
behavior of cynomolgus monkeys.
The fruits of their study
are presented in these two
papers titled Age related
difference in response to novel objects and Influence of short period
separation on both mother and infant cynomolgus monkeys.