TPC NEWS Vol. 17, No. 1 Spring (Whole Number 28)

(English Summary)

Page-3  Message from the director of the Tsukuba Primate Center (TPC): Circumstances surrounding our new facility for collaborative studies using non-human primates.

Akio YAMADA

     Although the construction of the new facility building has been completed, it seems difficult to predict whether it would be operated without cumbersomeness.  Because of the Government's decision financial support for our center will be drastically reduced from next fiscal year. We will make every effort to keep our research activities despite such bad circumstances.

Page-4  Laboratory Tests: Examination on Entamoeba histolytica by polymerase chain reaction (PCR)

Rieko KOBAYASHI

     In January last year, the WHO/Pan American Health Organization/UNESCO Expert Consultation on Amoebiasis identified that E. histolytica ( an amoebae of pathogenic) and E. dispar are different species .

     We investigated E. histolytica and E. dispar in our monkeys by using polymerase chain reaction (PCR) method, although we had surveyed these amoebae by using zymodeme analysis and monoclonal antibody so far.  Results are shown in the table.             No E. histolytica were identified, agreeing with the results by our previous zymodeme analyses.  PCR method, which is easier and simpler than zymodeme analysis, would be available for daily investigations of many samples to discriminate E. histolytica and E. dispar.  We should be always fully aware of the infection with E. dispar in laboratory primates, because its influence has not been yet known.

Page-5  Case Report: Endometriosis in a cynomolgus monkey

Ippei SAKAKIBARA

     Endometriosis is ectopic growth of endometrial tissue, and according to the affected region, it is roughly classified into internal endometriosis/adenomyosis: the growth in the myometrium of the uterus, and exsternal endometriosis/endometriosis: the one outside the uterus.  Both types of endometiosis have been seen in our cynomolgus monkeys.

     It is said that that endometriosis is a cause of 10 to 20 % of the women suffering from sterility, but this causal relation has been unknown with nonhuman primates. 

     This case died of intrarectal ileus induced by endometriosis.

Case: A cynomolgus monkey of wild origin, more than 18 years old (estimated age).  She had been pregnant 6 times, giving 4 nomal births and 2 births by cesarean section.  Her menstural cycle was 28 days of about five days’ duration.  Abnormal bleeding were observed twice.

Clinical findings: The colon contained a large amount of gas and feces.  Laparolomy was carried out.

Autopsy findings:  Cyst of about 3cm was present at the dosal wall of the tunica serosa uteri, causing adhesion and stricture of the rectum.  The cyst contained brownish liquid.

Histological findings: A large cyst is seen in the tunica serosa uteri (Fig. 1).  The epitherial cells of the tunica intima were similar to those of the uterus, having a round nucleus, cuboidal and monolayered or stratified, and some of them had cilia (Fig. 2).  Stroma were abundant, in which macrophages englobing yellow-brown glanular pigments were sporadically seen. Papillary growth was seen in the ectopic growth of endometrial tissues formed in the wall of large cysts, in which exudate was contained.

Page-6 Message from the author of the Case report

     Dr. Ippei Sakakibara, who has been the auther of “Case Report” since the first number of the TPC News, here roughly looked back his reports:

     He has autopsied more than 2,000 monkeys at the TPC so far.  In the early days of  of the TPC, the main casue of the death in the monkeys were infectious diseases.    However, in theses days, the cause has been diversified, involving infectious diseases, tumors and metabolic diseases.

     The TPC’s large scale indoor breeding system itself is an enormous experiment never done before.  Therefore, some cases, such as rickets or osteomalasia in the cynomolgus monkeys, were suggestions to improve the system.

     Once the TPC experienced an outbreak of nonhuman primate varicella-like herpesvirus infection.  The pathogen was not biosafety level 4, fortunately, and the outbreak ended by proper countermeasures.  We must be always aware of the risk that the monkey might have.

Page-7  Clinography: Research support from clinical science

Fumiko ONO

      Along with the opening of the new joint-use facility, the research at the TPC has to be diversified from those related to breeding and rearing monkeys, involving the studies of gene therapy, longivity science, and nuro-science.  We, who have mainly been in charge of health management of the monkeys so far, has to change ourselves so that we can support those new research and implement new techniques demanded.

1. Management of a gene transfer program in nonhuman primates

     When Dr. Robert E. Donahue, who is an authority on the gene transfer into the bone marrow of myeloblasted nonhuman primates was invisited to Japan by the Science and Technology Agency of Japan in September 1996, he advised us on technical matters of performing gene transfer into the bone marrow of nonhuman primates and on the design of the new facility.  On that time, he invited me to his facility, the 5 Research Coat of National Institutes of Health (NIH), and there I could learn the techniques of bone marrow harvest and animal care in gene transfer program.

     Animals for gene transfer program should be serologically negative for the simian immunodeficiency virus, simian AIDS-related type D virus, and herpes-B virus by strict quarantine and dry bedding system, since the animals are immunosuppressed and virus vectors are used.  Therefore, in Dr. Donahue’s facility, the subjected animals were under careful management in a intensive care unit (ICU), where daily care such as an intravenous drip injection, and monitoring and controlling microorganisms were carried out.  In the ICU, positive pressure isolators were used to prevent strictly the animals from exposure to pathogens.

   We would like to establish ICU care system for our new joint-use facility with reference to these knowledge obtained.

2. Management of the anesthesia

     In the TPC, we use ketamine as an anesthetic agent for restraint and treatment for  our monkeys.  In future, improvement of anesthetic techniques should be demanded from the various experiments at the new facility.  It is important to appreciate that poor anesthetic technique can make a serious adverse effect on the quality of  the results obtained from animal experimentation, and that safe and effective anesthetic techniques is indispensable from the view point of laboratory animal welfare.

    We have started using isoflurane as inhalational anesthetics for experimental operation from last year.  Atropine (0.05mg/kg) and ketamin (7.5mg/kg) as pre-anesthetic medication were given to the cynomolgus monkeys, then endtracheal tube (size; 4-4.5 Fr. for adult mail, 3-3.5 Fr. for female) was inserted to the trachea sprayed xylocain beforehand.  Following the administration of the anesthetic, general conditions such as SpO2, electrocardiogram (E.C.G), heart rate (HR), blood pressure (BP) and body temperature (BT) should be monitored.  SpO2 was maintained over 98%, heart rate was 140 to 160 beats/min. at the start of anesthesia and it decreased gradually.  B.T. decreased from 38C to 35C, although monkeys were heated by a water mat.  The systolic blood pressure was 81.1mmHg (SD;18.16, n=10) and diastolic blood pressure was 39.8mmHg (SD;16.38, n=10) under isofuluren anesthesia.  The data were lower by about 10-20% than those of ketamine anesthesia.  The figure showed a case of changes of vital sings under isoflurane inhalation anesthesia.

Page-9  Developmental biotechnology in primates

Tadashi SANKAI

    Our research group at the Tsukuba Primate Center, NIID has carried out　basic research to establish developmental biotechnology techniques and to elucidate mechanisms of fertilization and preimplantation embryo development in primates.  Here, we summarize our results on the quality of fresh and frozen-thawed spermatozoa, in vitro maturation of germinal vesicle stage oocytes, in vitro fertilization, microfertilization, and embryo transfer.  We also discuss related problems in such research that need to be solved.

Page-14  Past, present and future of behavioral and psychological study in the TPC

Katsuyoshi KAWASAKI, Takamasa KOYAMA and Keiji TERAO

Page-17  Establishing an Specific Pathogen-Free (SPF) colony of cynomolgus monkeys

Masao TAKASAKA,  Toyoko NARITA  and  Ryozaburo MUKAI

     In 1978, the Tsukuba Primate Center was established to breed and rear cynomolgus monkeys as laboratory animals under the artificial environment, and breeding colonies mainly consisting of feral cynomolgus monkeys imported from South East Asian countries were made up.  However, since these animals had been infected with various pathogens, it was needed to eradicate the pathogens and produce SPF cynomolgus monkeys.

     The pathogens we targeted for establishing  SPF breeding and rearing colonies were B virus, Shigella, Salmonella, and tsubercle bacillus ( Mycobacterium tuberculosis, M. bovis, and M. avium) from the view point of zoonosys; measles virus, retroviruses (STLV-I, SIV, SRV/D, etc.), and parasites, which were obstacle for biomedical researches and vaccine safety tests; and simian varicella-like herpesvirus that is a great threat for cynomolgus monkeys.

     As for the viruses, it is considered that all viruses we targeted have been  eradicated, except for retroviruses which now are in progress.  It is also considered that the bacteria we targeted have been exterminated.  However, the animals in the colony have been infected in relatively high rate with Campylobacter jejuni and C. fetus subsp. fetus, on which there had been few information in those days when the TPC was established, therefore, these pathogens were not targeted.  We would like to eradicate these pathogens, since they are pathogenic for humans, and Campylobacter jejuni is considered as a causative agent of diarrhea in infant monkeys.  As for the parasites, helminths and lice have nearly been eradicated.  However, infections of protozoa; Entamoeba histolytica (No pathogenic strains for humans have been identified in our survey, so far.), Giardia lamblia, etc. have been seen.  The protozoa transmissible to humans should be targeted here after.

     Experiments using nonhuman primates will be diversified in the future, and  microbiologic control suitable for the purposes of those experiments will be required more and more.  We would like to cope with any needs, using the data we accumulated during the past 20 years.

Page-21  Report of the Seminar: Overview of the Seminar ’98 by the Corporation for Production and Research of Laboratory Primates (CPRLP)

Koji FUJIMOTO

     The CPRLP held the Seminar ’98 on February 13, at the Tsukuba Center for Research, in Tsukuba city.   Theme of the Seminar was “Zoonosis, on the year of the revision of Communicable Disease Prevention Low.”  This theme was suitable for the requirements of those who have been working with laboratory animals for a log time. Number of the participants far exceeded the expectations of the organizer.

Page-22  Joint-use facility for research with nonhuman primates

Fumiaki CHO

     The Ministry of Health and Welfare (MHW) has just completed the cnstruction of joint use facilities for research with nonhuman primates in the site of the TPC, Tsukuba.  The facilities shall be used by the researchers from research laboratories under the control of the MHW and those from the National Center for Avanced Medicine.

     The new facilities, the total floor area of 3110 m², were designed so as to be able to carry out the studies of three fields, namely, genetherapy, mechanism of brain and nerves,  and aging.  The first floor (in vivo facility) of the main building contains animal rooms accommodating a total of about 300 monkeys in stainless steel individual cages (W50 x D80 x H80 cm, 42 kg), an animal nursery, laboratories for using live animals for various purposes (physical measurement and functions etc.), an operating theater, an ICU/recovery room, a dissecting room, X-ray rooms, observation rooms, and an RI experiment area with stairs and elevator for exclusive use. 

     The second floor (in vitro facility) contains laboratories for using materials collected from the animals, RNA and DNA laboratories, genetherapy laboratories, a flowcytometry measurement room, a protein/cytochemistry laboratory, rooms for preparation of histopathological specimens, an RI experiment area, a low-temperature incubation room, a laboratory-equipment washing room, a centrifugation room, a central machine room, a meeting room, rooms for visiting researchers and animal technicians, a machine room (510 m²), and a rooftop machine area. 

     The facility also has a service building (260 m² ), which contains an independent electric power plant (100 KVA, 200 V, 50 Hz generated by 125 h.p. engine) with transformer/accumulation of electricity and RI waste fluid reservoirs with three tanks.  It  also has a building as a data center consisting of a seminar room ( 37 m² ), a domitory, and a laundry.  The domitry has five one-person rooms ( 21 m²/room) each with a balcony on the first floor and six rooms on the second floor.