TPC News Vol.14, No.1 Fall 1995 (Whole Number 24)

                      (English summary)

Page-3 Message from the Director of the TPC: In relation to the "Hot Zone"

                   Dr. Yoshikawa, director of the TPC, describes his thought, in relation to the words "Hot Zone," "emerging virus," and "crisis management,"  from a view point of a primatologist.  Reviewing the great earthquake disaster in Hanshin district,  murders by saline  in  subway, and ebola outbreaks in Zaire,  He is expressing his anxiety against the present state of the crisis management in Japan.  He suggests that  the quarantine system for primates including pet monkeys should be established and the  manuals  for crisis and disaster management based on a national agreement should be made immediately .  

       He quotes, "Providing is preventing. "

       The latter half is a letter that Dr.Yoshikawa sent to the people concerned with the primate transportation, in order to inform them the difference between simian ebola virus and ebola virus.  

Page-5 Breeding topics: Management of pre-weaned cynomolgus monkeys in the TPC

                                                         　　　　　     Takahiro ONO and Fumiaki CHO

                   In the TPC, newborn babies are handled and managed as follows:

Registration at Birth:  The mothers and new born babies are weighed separately.  The mothers have been examined for nursing conditions: hemorrhagic volume at parturition, lactation and uterus size immediately after parturition.  The babies are  checked for sex, hair conditions, tooth eruption, injuries, malformation and defecation, and after that the umbilical cord is treated.  Babies whose mothers reject nursing are nursed by  foster mothers or  animal technicians. 

Fosterage: Foster mothers are selected from female monkeys being  experienced in nursing.  The females whose offspring have just been weaned are suitable candidates for the foster mother. 

Artificial Rearing: From 4 weeks of age, the infants are trained to drink milk from a bottle by themselves and to  eat  solid food with apples and oranges.  When the body weight  becomes twice that at birth, infants are weaned. 

Body Weight Measurement: Infants are weighed once a week until 3 weeks of age.  If the body weight gains go well, the infants are weighed once every 3 weeks. 

Deciduous Tooth Eruption: At about 30 weeks of age, after the second molar teeth eruption,  20 deciduous teeth come all out .  Therefore, at weaning, the infants have a total of 16 teeth. 

Health Check: It is hard to observe the face of the infant in mother's arms.  To know the health conditions of the infants, their postures (whether they are clinging to mothers firmly or limply) are observed. 

Seizing: Seizing should be done when the infants are caught, because they move vividly and sometimes may bite care takers.  

Weaning: Infants are weaned when their food ingestion, tooth eruption, body weight, behavior and age in days have fulfilled the requirements. 

Individual Identification: Tattoo marks by numbers are given to the face at weaning. 

Injuries: The infants sometimes receives a serious injury to limbs, which are caught in the netted floor or feeder while they are playing.  Very rarely, the infants also are bitten by mother monkeys, probably because the infants bite a nipple accidentally. 

Diarrhea: The infants who have intestinal disorders are given medicine,  and  received stool examination.  Depending on the results of the examination, antibiotics or vermicide are given.  To prevent dehydration is  important.  

Anesthesia: The infants are anesthetized with ketamine hydrochloride, "Ketalar," (10mg/kg). 

Blood Collection: Blood is collected under anesthesia.  The standard volume of blood to be collected is about 5% of the whole blood volume.

     Being underdeveloped morphologically and functionally, pre-weaned animals should be handled and managed with greatest possible care.    

Page-8  Laboratory Tests: Virological  survey on a cynomolgus monkey suspected HHV-6 like virus infection

                   　　　　　　　　　　　　　　　Toyoko NARITA,  Ryozaburo MUKAI

                   Here we report on the results of a serological survey conducted on an infant cynomolgus monkey (10-month old ) which had been suspected of HHV-6 infection (See, p25).

                   Table 1 shows the result  of the time sequential survey on the prevalence of  antibodies to several viruses which might cause  rash in  the cynomolgus monkey.

                   Figure 1 shows the antibody response to HHV-6 in this  case with passage of time.  Antibody titer at 3 weeks after the rash became 4o times higher than that of  the day of  rash observation, and continued rising until

after 6 weeks.  The antibody response  to HHV-6  and the symptom  (rash) of this case closely resembled those of human HHV-6 infection (Compare with Fig.2).   This case, accordingly, was suspected to be  infected with HHV-6 like virus.

                   We tried to detect  DNA of HHV-6 virus by PCR method, in cooperation with Dr. Yamanishi of the Research Institute for Microbial Diseases, Osaka University, using both frozen and fresh lymphocytes of the peripheral blood of this case,  but we have not been successful in the detection yet.  Then we tried virus isolation from this cynomolgus monkey, using cord blood lymphocytes.   During one month incubation  period,  no cytopathic effect was observed and virus isolation was negative.

                   Recent serological survey in the cynomolgus monkeys in our center revealed some interesting  results as follows:

                   In the sera of 1-2 years old cynomolgus monkeys, including this case, the positive rate of the antibody to HHV-6 type A was 17% (5/29 animals), whereas, unlike in humans, that of the antibody to HHV-6 type B was 7% (2/29 animals).   The positive rate of the antibody to human herpesvirus -7, of which antigenicity is resemble to that of HHV-6, in the sera of the infants cynomolgus monkeys aged less than 1 year old was 19% (3/16 animals).  The ratio and its antibody titer were higher than those of the antibody to HHV-6.

                   This time, although  we could not isolate HHV-6 virus from this case,  it was suggested that the cynomolgus monkeys had HHV-6 and HHV-7 like viruses being different from those of humans.  To isolate these viruses  is our new task hereafter, and we expect the cynomolgus monkeys to be animal disease models of  HHV-6 and HHV-7 virus infection.

Page-10  Development of the animal model of nonhuman primates for senile diseases

     I. Summary of the 1st stage of longevity science at the TPC

Yasuhiro YOSHIKAWA

                   We, as a  longevity science research project team, have conducted research  on the primate for three years.  Here I describe the summary of the first stage of our research.   Detail of each research should be present in the future issues.

Purpose of Research

                   This project team aims developing the animal disease model using nonhuman primates for human senile diseases, such as senile plaque, diabetes, hyper lipemia, osteoporosis and retinal (macular) degeneration.

                   This research group tries to establish  information network on laboratory primates in the research on longevity,   developing databases on aged monkeys.  Information should be open to public through PC online network.   This group also makes videotapes on the techniques of rearing and management of aged monkeys for animal technicians.                   

Abstract of research methods, results, and Discussion

A) Development of animal disease models:

                   We conducted  biochemical examination and analyses on the blood  of about 1,500 colony-bred cynomolgus monkeys, including 20 monkeys aged over 20 years, 150 African green monkeys, and 120 squirrel monkeys to investigate changes in physiological and metabolic functions with ages.

                   In the aged monkeys, since there was a  tendency to increase in occurrence of senile diseases such as diabetes, cataract, retinal degeneration, hearing loss, and bone disorders, we examine these monkeys by using the methods developed for humans to know the usefulness of the aged monkeys as animal models.

                   Diabetes was seen in half of the monkeys  aged more than 20 years.   Langerhans island had degenerated or disappeared in the cases which died spontaneously after rapid emaciation.

                   As for senile plaques, 37  cerebra of cynomolgus monkeys were examined histopathologically and immunohistochemically, and 11 out of 14 aged over 20 years old monkeys were positive.   Senile plaques belonging to classical and primitive types were more frequently observed than diffuse type.  These senile plaques were often seen in the cortex of temporal lobes especially superior and inferior gyri.  Since classical and primitive plaques were seen frequently around vascular amyloid, and no amyloid angiopathy was detected in the areas without senile plaques, the close relation between senile plaques of mature type and amyloid angiopathy might be suggested. 

                   Gene analysis and the investigation of genetical polymorphism of the b-amyloid and apolipoproteinE genes are now in progress. 

                   In the study on hyperlipemia using primary cynomolgus liver cell culture,  it

was revealed that expression of apolipoprotein (a) in the liver cells was positively controlled by several lymphokines such as IL-1b, 1L-6 and TNF- a.

                   As the model of osteoporosis, bone mineral density (BMD) of the African green monkey's (AGM) lumber vertebrae L3-L5 was scanned by a dual energy X-ray absorptionmetry  (DPX-a, USA).

                   For the half year interval analysis of kinetics on lumber vertebrae in both young and aged monkeys, bone area, mean width and bone mineral content (BMC) of L3-L5 were measured, and bone mineral density (BMD) was calculated.

                   Although no remarkable change in BMD of laboratory- bred animals aged less than two years was observed, significant decreases in BMD were detected in laboratory-bred animals aged over 6 years as well as in aged wild animals.

                   Serum biochemical values were compared among three groups, one male group  and two groups of female, by  a canonical discriminate analysis.  As the result of the canonical discriminant analysis, close relationship between serum biochemical properties and BMD was demonstrated.

                   We tried to make a database on the eye diseases in the primate.  As a result, it was revealed  that 1)  types of abnormalities in our colony-bred  cynomolgus monkeys were 17 in number and the incidence was 19%, 2) types of abnormalities in  the wild-originated monkeys were 16 in number and the incidence was 21%,  and 3) 244  abnormalities were observed in 225 out of 1162 animals .

                   We think that this database is an epoch-making achievement , and we expect the database to be useful in the research to develop animal disease models hereafter.

                    Two types of macular degeneration were observed in our monkey colony.  One is senile macular degeneration like that of humans', and the other is genetical one. 

                   A funduscopical examination including FAG was also done across several generations.

B) Establishing information net-work:

                   We have exchange information with several primate research centers in the U.S.A., and opened a private online forum on the primates in a domestic net-work.  

                   Making a database on aged monkeys and a video cassette on handling aged monkeys and examination techniques are now in  progress.

Summary of result

                   From our research for three years, as I  previously mentioned,   primates are very  useful as  animal disease models in the research of longevity science.   We should progress these research, in cooperation with the researchers in basic or clinical medicine.

Page-12 Research Project at the TPC: Nonhuman primate models for senile diseases

       II: Measurement of lumbar vertebrae and whole body mass in primate species: Bone mineral density regarding obesity and emaciation

Takashi YOSHIDA

                   The population of the aged people in Japan is  increasing rapidly,  and senile diseases have become one of the most important social problems in Japan.

                  The project of the 1st stage of longevity science is now being developed at the TPC under the support of Ministry of Health and Welfare.

                   To establish the animal model of osteoporosis in the aged people, we measured monkeys' lumber vertebrae.   The lumber vertebrae were measured by the anteroposterior projection as well as the lateral projection by a dual energy X-ray absorptiometry (DXA) . 

                 Age related changes in lumbar vertebrae were demonstrated morphometrically (Fig. 1).  Pregnancy related changes in bone mineral density of lumbar vertebrae by the measurement of the lateral projection in the African green monkeys were demonstrated  (Fig.3). 

                      Furthermore, we applied the DXA to whole body measurements in the cynomolgus monkeys.  Total body bone mass and total soft tissue (lean and fat) mass were measured successfully, and  values of % Fat ( a ratio of fat mass to soft tissue mass ) were calculated.  The value of % Fat was judged to be a good index of obesity or emaciation in the cynomolgus monkeys (Fig. 4). 

                      Obesity and emaciation in cynomolgus monkeys and those in the teenagers  in Japan were compared and discussed (Fig.5).

                      Usefulness of the DXA in not only bone measurement but also whole body measurements in primate species was demonstrated.

Page-17  Development of a system of behavior analysis using neuro-computer

Katsuyoshi KAWASAKI

                   Dysfunction of the brain induces abnormal behaviors.  This project aimed to develop neural net-helped computer system for automatically calcifying the non-human primate behaviors.   At the first step, 24 hour sequential recording VTR system has been developed, and the behavior of a cynomolgus monkey was recorded completely for 1 week.

                   We successfully developed the computer programs for locomotive image analysis, which consists of frame subtraction, blend, optical-flow, snake and background manipulation programs for DVI (digital video interactive) system in a workstation.

                   By using these programs, especially by the frame subtraction program, behaviors of the monkey were classified into four basic categories: rounding, rearing, standing and sitting.  Furthermore, time sequential analysis of the parameters for the length of x and y axes,  the movement of the center of gravity and the image area  of the object gave useful information to differentiate looking,  drinking, and exploring behavior with 2 or 4 legs.

                   Voice analysis disclosed that the juvenile male monkeys from 2 to 3 years old had higher-pitched voice than the female of the same age, and that  male monkeys  of 3 to 4 years old  break  to the lower tone of matured males.   However, in the females, voice tone became lower gradually with age.

        We are planning  that  we establish  a computer system for detection and estimation of abnormal behaviors  by using our neuro-computer installed and trained by the parameters obtained from  these  images and voice analyses.

Page-24   Case Report: Staphylococcal dermal granuloma with surfer                    granule in a cynomolgus monkey                            

Ippei SAKAKIBARA

                   In general, infection of Staphylococcus aureus  in cynomolgus monkeys progresses rapidly, resulting in abscess formation at  the infected area or in septicemia.   But,  the case here I report  had granuloma and abscesses in the skin through about ten years.

                   It is said that surfer granule/druse is a useful pathological finding of fungal diseases.  However, when  chronic infection of a bacterium causes abscess or granuloma, sulfur granules sometimes are formed, as you see in this case.

Case: A12 years old male cynomolgus monkey, born in 1981 at our Center.

Onset and Clinical findings :  When this animal was one and half years old, concrete nodules  were found in the dermal tissue for the first time.  During  10 years from the onset, nodules had spread to the  back and breast .  Although antibiotics, such as aminobenzyl-penicillin and cephazolin had been  administrated  during this period, the animal died.

Causative agent: Staphylococcus arureus  and Streptococcus sp. were isolated from the dermal nodules, but no fungi.

Autopsy findings:  Hair　had widely fallen out.  Diffused or clustered nodules of red-been size were seen under the skin of the back and breast.  The nodules were upheaval on the skin surface with scab.  Some of the nodules were dark brown in color and bleeding.  From the section of the nodules, pus of yellow in color flowed out .   Nodules were also seen in the breast muscle.  The surface of the nodules were congested,  and pale yellow pus flowed out from the section.

Histological findings: Druse of different shapes and sizes were seen in the subcutis, and they were surrounded by small abscesses or granuloma  (Fig.2).  The center of the druse was stained with hematoxylin, and the surrounding was stained with eosin.  In the

peripheral part of the druse clubs  were seen.   Tuberculoid gramulomatous reactions, such as  lymphocytes, neutrophils and giant cells  of Langerhans-type were seen around the druse.   Foam cells and plasma cells  were also seen. and around the cells,  fibrosis and capillary vessel genesis were observed.  In the tissue of the  central nervous system,  disseminated lesions by this agent, meningitis and demyelination, were seen.

                   An Immunohistological study (ABC stain) revealed that the causative agent of this lesion was Staphylococcus aureus.

Page-25  Clinography: Rash

                                                                                                   Fumiko ONO

                   For the maintenance of breeding colonies, the prevention  of outbreaks of virus infection is a top priority.  In particular, in the breeding colonies like our center (TPC) , which  aims to  establish a clean or SPF primate colony, virus infection  can easily spread for a moment.   Therefore,  when we fined a monkey  with rash, we have to treat it with a  great caution as we do against the cases of acute viral infections.  Here we report on a case of HHV-6 (Human herpes virus-6) like disease.

                   We fined a cynomolgus monkey (10-month old, male)  with a rash in a isolation room for monitoring the antibody titer of TSVH (Tsukuba Simian Varicella-like Herpes virus).   Thereafter, we observed carefully all 29 monkeys in the room, but we did not fined  any other animals with the rash and disorder.  We isolated the monkey and measured its serum antibody titers to the viruses related to  rashes.   At the same time, we strengthened our preventive methods, for example, we sterilized working  wears with iodine and set the working order of the isolation room  last.

                   The virological examination (See, p8, this issue) suggested that the rash  had resulted from   HHV-6 like virus infection. 

                   The clinical findings of the monkey are as follows:  

                  Pink rashes were seen in  the oral cavity, temporal,  neck,  and insides of the limbs.  But the monkey did not have much fever.   Five days after the appearance of the  rash,  erythema was observed at the angles of the mouth  but no  vesicular ulceration .  After 10 days, although the rash still remained, the erythema had crusted.  Lymphoadenopathy was observed, but no other clinical symptoms such as anorexia was observed  The rash on the face disappeared after about 1 month , but lymphoadenopathy persisted through 3 months.  This monkey and his mother were positive for antibody against HHV-6.   However, another monkey, which had been housed  in the same cage with him for three months, was seronegative. These findings suggested that the monkey had infected from his mother by vertical transmission.  The possibility of horizontal transmission was considered to  be weak. 

                   In this case, we could avoid the epidemics of the disease. The reasons were 1) the virus was a weak and latent one in the cynomolgus monkeys, and 2) the group including the  contaminated  monkey could be isolated easily by the antibody checking.

                   For the prevention of outbreak of known or unknown virus diseases, it is essential that we keep on careful observation and taking prompt response in the first stage after we find  any animals with disorders.

Page-26 Report on Symposium, Meeting and Seminar:

                   12th Annual Symposium on Nonhuman Primate Model for AIDS Oct.12-15, 1994, Boston

                                      Dr. Kazuyasu Mori r have been to Boston,  U.S.A. to attend the   12th Annual Symposium on Nonhuman Primate Model for AIDS.   He reports on the symposium in a diary style.

Page-31  Report of "The 16th Annual Meeting of the American Society for Bone and Mineral Research (ASBMR)

     Dr. Yoshida from TPC sat in the 16th annual meeting of the ASBMR at Kansas  City, Missouri, September 9-13,1994.  Current activities of the study in this field are reported  here.  The information  obtained  from this meeting  should be  useful for developing future  study in Japan.

Page-34  Seminar "95" by the Corporation for Production and Research of                     Laboratory Primates (CPRLP)

                   "Seminar "95" by the Corporation for Production and Research of Laboratory Primates " was held on  February 23 and 24 in Tsukuba.  Ms. Kobayashi summarized here the symposium.

                   The theme was "Present state of the understanding of the laboratory primates," and practical techniques in handling laboratory primate were focused this time. 

                   Although the symposium was the first one sponsored by the CPRLP, it was unexpected success.  The  number of the applicants far beyond anticipation suggests  that there is a strong demand for the practical knowledge on handling the laboratory primate.  Next seminar is to be expected.

Page-34  Two items were added to the virus tests offered by the CPRLP

Page-36  Memory of Trainees: What I learned at the TPC

                   A trainee, who was  dispatched from a company,  describes here his experience at the TPC, appreciating the chance given by the TPC.

Page-37  Videocassettes on the techniques of care and management of laboratory primates are now on release