TPC NEWS

TPC NEWS, Vol.11 No.1, Spring, 1992 (Whole Number 19 )

(English Summary)

Page-3 Work of the director

One year has passed since I was appointed the director of TPC. Now I have little time to carry out experiments by myself. I keenly feel that doing experiments by my own hands is essential in order to not lose the sense of experimenting.　 Therefore, I will make time for myself this year.

Looking back on this past year, I realize that my life-style has been changed fundamentally. In the first place, I had to make a resolution to take heavy responsibility for the future of TPC. A failure of an experiment in a laboratory can be attributed to the disability of its performer, and is a matter of personal responsibility. But for the present, as there is no other primate research center like TPC in Japan, the progress of laboratory primate medical science depends on TPC's effort. Which means that TPC's director and staff members are immediately responsible for whether laboratory primate science in Japan will develop or not. I think that there is no more than that we make steady progress not to be crushed out by this heavy responsibility, having a long-term vision. For the next few years, TPC's future course should be clarified after researching various possibilities.

In the second place, my laboratory life had basically showed a loop-structure, in which I set forth a hypothesis, carried out experiments, analyzed data, discussed problems, and again set forth a new hypothesis on newly emerged problems. On the other hand, director's work can be said to be social scientific. It patterns an inchworm. For example, my ideas and attempts are influenced by others' judgments and strategies. It is, therefore, impossible that the same situation can ever be set up again for what once was done. I have realized that collection of correct information without prejudice, harmonious human relationships such as "nemawashi," and objectivity with which we can understand other people's situations are essential for the inchworm which goes on progress without falling from a branch.

In the third place, I had to reconsider the concept of natural science. I was given a point of view through which I see relatively the natural science itself on which I had been based a priori. I have experienced that the presence of the people, those who oppose the animal experiments, those who don't deny animal experiments but demand some regulation on it and those who are expressing strong anxiety and opposition to the progress of natural science.

I would like to make progress step by step in this second year.

Page-4 Natural infections with various pathogenic agents

Bacteria

TPC's African green monkey colony was founded on the wild African green monkey populations imported from Kenya from 1979 to 1981. Mrs. Kohno and Dr. Takasaka report on the state of natural infections with bacteria in the wild-originated and laboratory-bred African green monkeys in our colony.

(1)Shigella and Campylobacter

Table 1 shows the infection rate of Shigella. In the quarantine period, Shigella was isolated from 4 of 65 wild-monkeys (6.2%) imported from 1979 to 1981. Its types were Shigella flexneri3b (2 cases) and Shigella flexneri 4b (2 cases). Antibiotics were administrated to the infected monkeys. After that, the breeding colony was established. Until 1982, scattering outbreak of shigella had been seen, since the positives were still involved in the colony. During this period, Shigella was isolated from 21 wild-animals and from 17 lab-bred animals (34 cases were S. Flexneri Y and 4 cases were S. flexneri 4a).

A total of 146 animals (37 wild-born and housed at TPC for 8-10 years, and 109 lab-bred animals) were examined for Campylobacter. As no Campylobacter were isolated from the animals, we carried out IgG antibody examination by ELISA to know the past infection with Campylobacter Jejuni. All wild-originated animals (36 cases) of quarantine period were positive, but 10 of the 37 animals housed for 8-10 years after importation were negative. All of the lab-bred animals were negative.

(2) Tuberculosis

Although we have found no case of tuberculosis in spontaneously dead African green monkeys, some pseudopositive animals have been seen since 1989. Further study is now in progress.

Page-5 Virus

We present results of our serological surveys on viral infections in newly imported and laboratory-bred monkeys in our African green monkey colony. Concerning the herpesvirus, we use HSV-1 CF antigen in order to estimate SA8 infection. As shown in the table, positive ratio for antibodies against HSV-1 in newly imported monkeys was 40.6% and 0% in laboratory-bred monkeys, suggesting that our breeding and rearing system exclude SA8. Wild originated animals kept in our colony as the breeder for 8 to 10 years seem to be infected with this virus from the positive ones, because seropositive rate for this virus increased from 40.6% to 87%. All the wild originated animals are positive for SA6 (vervets CMV), while 70.1% of the laboratory bred monkeys were seropositive for this virus. Because we had the outbreak of simian varicella like herpesvirus infection in our cynomolgus monkey colony in November 1989, the incidence of antibodies to this virus and Delta herpes virus was monitored on African green monkeys in our colony. All the animals were seronegative to these viruses. It suggests that the outbreak of the epidemic in our cynomolgus monkey colony was not caused by the virus from African green monkeys. As for the antibodies to paramyxoviruses (measles virus and SV-5), bred and reared monkeys were all seronegative. With regard to the serology on retroviruses (STLV-I, SIV), some of the bred monkeys ( 6/114, 11/114, respectively ) are still seropositive for both retroviruses, indicating that these viruses are transmitted vertically at some rate.

As a summary, all the bred monkeys are seronegative except for SA6 and retroviruses. We will continue periodical sero-check from now on.

Page-6 Parasites

We use Negvon (Bayer) for eradication of ectoparasites such as lice, and thiabendazole for that of helminths. No administration has been taken for protozoa. Table 1 shows the infection rate of parasites in the newly-imported wild African green monkeys from Kenya through 1979 to 1981. Strongyloides sp., Oesophagostomum sp., Trichuris trichiura, and Bertiella were found. The infection rate of Genus Entamoeba including Entamoeba histolytica was 54.7%.

Table 2 shows the infection rate of parasites in wild-originated and laboratory-bred African green monkeys. The wild-originated monkeys were housed at TPC for more than 3 years, and the laboratory-bred monkeys were more than 1 years old. The former experienced three thiabendazole treatment regimes, and the latter did once before the examination.

Page-7 Report from the African green monkey colony

Mr. Hiyaoka has been taking care of the African green monkey colony since its

beginning. He describes an outline of the history and problems of the breeding

colony, expressing his affection toward this species.

This report is illustrated with a lot of humorous drawings by Mr. K. Someya.

Page-11 Breeding African green monkeys

Tsukuba Primate Center for Medical Science (TPC) imported African green monkeys three times from 1979 to 1981, and started indoor reproduction of this species using 36 females and 9 males in 1981.

Some reports on menstruation of African green monkeys have been published so far,

in which the evaluation of menstrual charge almost always depends on vaginal swabbing or smears because of their small quantity of menstrual flows. It is inevitable to confirm accurate menstrual cycles beforehand when the one to one timed mating for three days is successfully carried out. In our center, menstrual flow was observed macroscopically with 30 of the 36 females before the first mating trial. But, only 3 females were fertilized for the first time by a total 29 trials of the one to one timed mating for three days, which was based on regular menstrual cycles (Table 1-a). The remaining 33 females were fertilized for the first time by the every other day mating, which was carried out regardless of their menstrual cycles (Table 1-b). However, the females which were once fertilized and experienced parturition became to draw regular menstrual cycles, and were frequently subjected to the one to one timed mating for three days. Accordingly, the number of pregnancies by the one to one timed mating for three days exceeded the one by the every other day mating.

Some females, however, did not draw regular menstrual cycles even after the second parity and were consequently subjected to the every other day mating. Thirty of 48 females (62.5%) were fertilized within 10 weeks from the start of the every other day mating, in which 16 weeks were designated as one term of the mating (Fig. 1). The fact that the 30 females were to be at 5th week of pregnancy when they were diagnosed 10 weeks after the start of mating suggests that the females were fertilized 5 weeks after the start of the mating, by the time of their second ovulation at latest. It can be said therefore that the pregnancy rate by the every other day mating is quite high. Last but not least, four females were diagnosed to be pregnant 21 weeks after the start of the mating, that is, five weeks after the separation.

Table 1-c shows the number of birth of African green monkeys at TPC. Although the stillbirth rate (3.7%) was rather low, the rate of fetal loss including aborted fetuses became 17.7%, a rate which cannot be overlooked. One of our problems to be solved in the future is to decrease the rate of abortion.

At TPC, F1 males and females serve as a breeder from the ages of five and four years, respectively. As shown in Table 2, three of the 17 F1 females aged 4 showed regularly cyclic menstruation according to macroscopic observation. The remaining 14 females were judged to be irregularly cyclic. Occult blood test, however, revealed that 11 of the 12 females were regular cyclic, except for one female of irregular cycles. The menstrual cycles observed by our regular observations averaged 33.4+7.1 days and those confirmed by the occult blood tests averaged 31.4+4.1 days. The average age of menarche of the 12 females was two years and eight months, the earliest was two years and three months and the latest was three years and four months.

We adopted two mating systems for the F1 breeders, the one to one timed mating for three days and the long termed mating for a certain period of time. The regularly cyclic females were subjected to the one to one timed mating for three days as well as to wild-originated breeders. As a result of a total of 25 trials of the one to one timed mating for three days using 8 females, 3 pregnancies were obtained. The pregnancy rate for the total number of trials was rather low, 12% (3/25), and that for the number of the animals was 38% (3/8). The irregular cyclic females were subjected to the long termed mating, in which a male and a female were housed in a cage for a certain long period of time. The pregnancy rates by this system was high, 70% (for the total number of trials) and 92% (for the number of animals).

Table 3 shows the results of the long termed mating of two successive trials. During the first trial, 8 of 19 males fertilized the males, whereas 18 of 24 females were fertilized. The 11 males which had failed in fertilizing females during the first trial were subjected to the second long termed mating using other females, and 4 of the males fertilized females. The remaining 7 males were unable to fertilize females even after the long termed mating for 2 years. The 6 females which had not been fertilized during the first trial were also subjected to the second long termed mating using other males, and 4 of them were fertilized.

The period of time which is needed for a male to fertilize a female was studied with the 8 males which had fertilized the females in the long termed mating. During the first trial of the long termed mating, all of the mails fertilized the females in 4 to 11 months, averaging in 5.4 months, after the start of the mating. They also fertilized the females in 4.3 months and 3.2 months on an average during the second and third trials, respectively. During the fourth trial, 2 of the males were subjected to the long termed mating and they fertilized the female in 3 and 2 months, respectively. These data show that males tended to become better breeders as the trials were repeated. The females which were subjected to the long termed mating should also be studied in the future.

We have dealt with a total of 25 births of F1 African green monkeys since 1985. Of the 25, normal birth totaled 24, and abortion numbered only 1. All babies have been successfully nursed. Compared to the fact that the successful nursing rate of F1 crab-eating monkeys in the first birth in our center was 62%, the high rate of successful nursing with the F1 African green monkeys is quite significance. Nineteen of the 24 babies successfully nursed have been weaned and the remaining 5 are being nursed well by their own mothers.

We will collect further data on the F1 African green monkeys and try to analyze them more minutely in the near future.

Page-13 In vitro fertilization of the African green monkey

Two African green monkeys were subjected to the experiment of in vitro fertilization. Dr. Sankai reports the results:

The sperm were collected by the method using fingers. They were preincubated for about four hours with TYH medium containing caffeine and dB-cAMP, because they showed hyperactivated motion in these conditions.

Hormone administration to stimulate ovary should be done according to the menstrual cycle. We inspected daily menstrual bleeding to confirm the first day of the cycle, inserting an applicator into the vagina under general anesthesia. We administrated PMSG three times from the 3rd 3 to 14th day of the cycle. On the 15th day, hCG were injected, and after 28hs, oocytes were collected by laparotomy. Table 1 shows the results.

Table 2 shows the results of in vitro fertilization using these sperm and oocytes. We implanted eight-cell embryos to two recipients on the 14th and 15th day of the menstrual cycle, respectively, but either cases failed in conception.

Page-14 Blood groups as genetic marker for a management of African green monkey breeding colony at TPC

Blood groups of nonhuman primate species have been investigated as the models to clarify the phylogeny of human blood groups and as the genetic markers to demonstrate intra- and/or inter-specific differences among them. Human-type ABO blood groups are the most extensively studied blood groups in nonhuman primates including African green monkeys. Three phenotypes, A, B and AB, have been found in captive Cercopithecus sp.. But no type-O monkeys have been found in the wild population so far.

The mode of inheritance is analyzed by adjusting the data to the Hardy-Weinberg equilibrium model based on 2-locus(Sand B)- model or 3-locus(A,B and O)-model. Although no type-O animal was in our breeding colony, the presence of O gene was clearly demonstrated by family analyses (Table 1). Offsprings of genotypes AO and BO were born from the parents of genotypes AO x AB and BO x AB, respectively. This result finally confirmed the presence of O gene in African green monkeys.

We established a new simian-type blood group system (M blood groups), using a hemagglutinating antibody which was developed by alloimmunization. The M blood group system consists of two phenotypes (M and m), and is governed by the two alleles (dominant M and recessive m). At present, we can apply 5 blood group genes (A, B, O, M and m) for genetic control of our green monkey breeding colony. Differences in the phenotypic distribution and the gene frequency of ABO and M blood groups were observed among 3 founder populations imported at different times (Table 2). Based on this result, we formed two breeding populations (1978+1981 and 1980) and carried out closed mating. More than 120 offsprings were obtained during the past 10 years.

There was no difference in the gene frequencies of 5 blood group genes between the parental and the first filial populations. This suggests that genetic variability in the parental population is being maintained in the first filial population in our African green monkey breeding colony.

Page-16 Age related Changes in African Green monkeys Body weight changes

Five growth models were applied to the individual body weight data of African green monkeys to estimate the growth pattern. Body weight was measured continuously from birth to six years of age. The average body weight in female laboratory-bred African green monkeys was 330+15g (M+S.D.) at birth, and 2.71+0.33kg at four years of age (Fig. 1). The body weight of female African green monkeys was judged to reach a plateau about four years from birth. As a result of the adaptation of the five growth models, the most suitable coefficient of determination between the growth data and growth models was obtained by the application of Gompertz equation. Three parameters of Gompertz equation ( mature size, rate of maturation and age at an inflection point) strongly correlated with the age of menarche.

The average body weight in the male laboratory-bred monkeys was 360+25 g at birth, and 4.54+0.23 kg at five years of age, the body weight was judged to have reached a plateau by that time (Fig. 2).

The data of the males were divided into two groups by age. Gompertz model fitted best to the data of the period from births to 2 years and 10 months of age (R2 = 0.982+0.011). The age at the inflection point in Gompertz model corresponded to the age of weaning. Logistic model was most suitable for the data of the monkeys more than 2 years and 10 months old. The age at inflection point of Logistic equation corresponded approximately with the age of sexual maturation.

Page-17 Ophthalmoscopic observations in ocular fundi of African green monkeys

Mr. Suzuki reports on the findings in ocular fundi of laboratory-bred neonatal and infant African green monkeys.

Fifty-three ( 30 males and 23 females) healthy animals aged from neonate to 90 days old were used. They were anesthetized with ketamine-HCl, and instilled with toropicamide and phenylephrine hydrochloride. The characteristic findings were approximately similar to those of the cynomolgus monkeys( See TPC News Vol.10 No.1). Table 1 shows the results. Retinal hemorrhage were seen in 6 of the 30 neonates (20%) born normally (Fig. 1). This rate was lower than that of the cynomolgus monkeys (69%, 66/96). The hemorrhage vanished one week later. Persistence of the hyaloid artery was observed in all of the neonates subjected (31 cases), and it disappeared completely within 21-31 days.

Further observation is needed to know whether the findings of more aged animals differ from those of the cynomolgus monkeys.

Page-18 Immunological study of African green monkeys

Since the African green monkey was regarded as a natural host of lymphotropic retroviruses such as SIV and STLV-I, immunological studies of African green monkeys have become more interesting research theme.

We have prepared the antisera to African green monkey immunoglobulins(Ig), and now are using them for some immunological studies.

Ig was separated from pooled sera of adult African green monkeys. IgG, IgA and IgM were purified by the combined procedure of ammonium sulfate precipitation and column chromatography on DEAE-cellulose, sephacryl S-300 and sepharose 4B conjugated with antisera to human IgG, IgA and IgM. Monospecificity of antisera to IgG, IgA and IgM was confirmed by immunoelectrophoresis in agarose gel(Fig. 1). Species specificity of the antisera was examined by the Ouchterlony test(Fig. 2) using the sera of man, cynomolgus and squirrel monkeys. Cross-antigenicity of IgG between African green monkeys and cynomolgus monkeys, humans, and squirrel monkeys was 82%, 47% and 14%, respectively.

IgG concentration detected at birth was approximately the same level with the adult's. However, its level decreased gradually for the first one month after birth. This indicates that IgG found at birth in the African green monkey is of maternal origin. Low level IgM was detected in all of the newborn African green monkeys, but no IgA was found in any newborns. Serum IgG, IgA and IgM concentrations of the African green monkeys nearly reached the normal adult level by the age of 24 to 36 months.

It is supposed that IgG of nonhuman primates was transferred across the heamochorial placenta from mother to fetus like that of humans. However, transferability of IgG via the placenta in monkeys is rather low than that in humans. The average ratio of IgG level between newborns and their mothers in African green monkeys was 70%, and those of cynomolgus and squirrel monkeys were 85% and 50%, respectively. These results suggest that the transferability of IgG via the placenta in African green monkeys ranks between those of cynomolgus and squirrel monkeys. Further immunological characterization of African green monkeys, including the immunity against an infection with lmphotropic retroviruses, would increase the usefulness of this monkey species as an experimental animal in immunological studies.

Page-20 Do African green monkeys dream of AIDS? -1992

Dr. Y. Murayama comments:

I suppose that the most fascinating but suspicious subject in primatology is the research on evolution. In this field, inevitability and originality are required over the perfect validity. In this paper, I present the possibility that African green monkeys might have conquered AIDS during their evolution. I can draw the following hypothesis from my unpublished experimental results: "CD4 cells of African green monkeys have completed their development after the stimulation of antigens in the periphery, concealing CD4 molecules from the cell surface. As a result, the activated CD4 cells and memory CD4 cells became CD4 negative. African green monkeys developed two types of helper T cells of CD4⁺ and CD4^- during the adaptation to SIV, and became to be able to evade a fatal immunodeficient state by CD4^- helper T cells escaping from SIV infection."

Page-22 Vitamin D3 deficiency in African green monkeys at TPC?

Since 1979, we have maintained a colony of African green monkeys. During the past ten years, we found some animals showing low concentration of serum protein, calcium and sodium, and high concentration of potassium. Serum protein and calcium concentrations were highly correlated. Although the oral administration of vitamin D had no effect on the improvement of serum characteristics, the body weight growth of the infants born from the mother monkeys given vitamin D was apparently improved.

Further studies are required for elucidating an underlining mechanism of a vitamin D deficiency in African green monkeys of wild origin at TPC.

Page-24 Acute gastric dilatation syndrome

Acute gastric dilatation syndrome is one of the most important diseases among our African green monkeys. Since TPC established the breeding colony of this monkeys species, we have bred 151 monkeys, but of which 18 were dead cases. Eleven cases of the 18 (61%) were attributed to acute gastric dilatation syndrome. Their age ranged from two to four years.

All of the cases occurred at night and the monkeys were found being dead the next morning. They had shown nothing abnormal in health conditions before the occurrence. The pathological findings common to all 11 cases were as follows: An extremely distended abdomen, protruding eyes, rectal prolapse, hemorrhages in the oral and nasal cavities and around the anus, and a plenty amount of gas deposited beneath the body skin. Pressing the skin made a snappy sound. When the skin was flayed, the gas with a fermentative odor was emitted. Innumerable air vesicles of chestnut size were

seen in the subcutis. Diffused hemorrhages from very small to Ping-Pong ball size were observed in the skin and muscle, in particular, in the abdomen and inside of the femor. The stomach was distended by a plenty amount of gas and food fermented. Congestion were recognized in the liver, kidney and lung. Post mortem changes of the organs in the abdominal cavity were severe. Gram-positive bacilli were found, however, little inflammatory lesions were seen around them. Staphylococcus, Micrococcus, Streptococcus, and Enterococcus were isolated from aerobic cultures, and Propionibacterium was isolated from anaerobic cultures. It is well known that some bacteria of Clostridium genus cause a disease generating gas in humans, but none of them were isolated at TPC. Lactobacillus sp. which lives in gastric organs were isolated with high ratio from every organ including subcutaneous tissues. This agent seems to be one of the causes of gas generation.

At TPC, this disease have been found in the African green and cynomolgus monkeys so far. Although the occurrence in cynomolgus monkeys was accidental, the occurrence in the African green monkeys concentrated on a certain family (Family A). As shown in the figure, out of 11 dead cases nine were derived from this family. There might be a hereditary factor of predisposition to this disease in the African green monkey.

Page 25, 26 Memory of Trainees

Two trainees who came from Ibaraki University and Ina Research Inc. describe their experience at TPC, showing appreciation for TPC's openness and friendships.