Japan. J. Med. Sci. Biol., 22, 117-121, 1969 NOTES SEROLOGIC SURVEY WITH THE SERA OF MONKEYS IN REGARD TO THEIR NATURAL INFECTION WITH MEASLES VIRUS High incidence of natural infection of the monkeys with measles virus is an important problem in the study of measles virus in the monkey which is the only laboratory animal having a high susceptibility to this virus. The causative agent which is responsible for the development of measles antibody in the sera of monkeys is considered to be of human origin, although a possibility of an involvement of virus of monkey origin has not been completely excluded as yet. The present paper describes the results of serologic surveys on the sera of 988 cynomolgus monkeys imported from countries in southeastern Asia from 1960 to 1968. Involvement of measles virus of human origin in the natural infection of those monkeys was suggested by comparing the results of experimental infection with those of serelogic surveys on the imported monkeys. The probable mode of spread of measles infection among the group of monkeys during quarantine has also been discussed. Details of the procedures for virus neutralization and hemagglutination-inhibition (HI) tests have previously been described (Shishido et al., 1967). Complement fixation (CF) test was conducted on plastic trays by a microtechnique. Routine procedure for the quarantine and maintenance of monkeys has been described by Takasaka et al. (1964). For the inoculation experiment, monkeys were kept in strict isolation to eliminate the natural infection by measles virus. Results and Discussion: A total of 988 cynomolgus monkeys consisting of 37 groups has been imported from Malaya, Vietnam, the Philippines, and Cambodia from 1960 to 1968. Each group of monkeys was bled on the day of arrival in our laboratory. Sera were examined for measles antibody by the CF test from 1960 to 1964, and by the HI test after 1965. The results are summarized in Table 1. Variation in the ratio of prevalence of measles antibody was observed among the countries. With the monkeys from Malaya, 3 out of 178 monkeys in 5 groups were found to have antibodies. Thirteen out of 120 monkeys in 3 groups from Vietnam were antibody positive. Until 1966, the Philippines was the major source of monkeys for our laboratory. Among a total of 339 monkeys in 15 groups, 42 were found antibody positive. Since 1967 we have been using monkeys from Cambodia exclusively for measles experiment. Among a total of 351 monkeys in 14 groups imported from this country during the past 9 years, none was found to have measles antibody. The reason for the apparent absence of measles antibody in the monkeys from Cambodia could not be explained at present. 117
118 NOTE Vol. 22 Table 1. Prevalence of measles antibody in monkeys immediately after arrival * From 1960 to 1964, sera were examined by CF test. After 1965, HI test was conducted. In total, 930 monkeys which correspond to 94.1 % of the total monkeys from these 4 countries were demonstrated to be antibody free. Since the measles antibody in monkeys has been considered to persist for a long time, probably through life as in the case of human infection, those monkeys examined in the present survey can be considered to be free of measles antibody in their natural habitats. In spite of the absence of antibody in the majority of monkeys on their arrival in our laboratory as shown in Table 1, most of the antibody-free monkeys acquired antibody during our routine quarantine for 6 weeks. Ten groups of the monkeys were examined serologically at intervals during quarantine by our routine procedure. As summarized in Table 2, almost all the monkeys remained antibody free for the first week. From the second week, the number of monkeys having measles antibody gradually increased and finally all the monkeys acquired antibody by 5th to 6th weeks. Experimental inoculations were conducted subcutaneously with TYCSA and
1969 NOTE 119 Table 2. Development of measles antibodies in monkeys during quarantine by routine procedure * Not tested. Sugiyama strains of the attenuated type and Tanabe strain of the virulent type. A group of 4 to 5 monkeys was employed for each virus and the sera were collected weekly. Similar results were obtained with all the three strains. Development of virus neutralizing, HI and CF antibodies were observed in 2 weeks after inoculation. Peebles et al.(1957) inoculated monkeys with measles virus by the intraveneous, intranasal, and intramuscular routes, and observed the development of CF antibodies in 9 to 15 days. Although there might be some differences in the infection route, virus dose, or virulence of virus between experimental inoculation and natural infection, it may be reasonable to estimate an approximate latent period before appearance of antibody roughly to be 2 weeks, in the present survey. Thus, the monkeys which showed antibody rise later than 3 or 4 weeks after arrival were suggested to be infected during quarantine in our laboratory and not before the time of their arrival. The monkeys which showed antibody rise within 2 weeks after arrival may probably be infected before or during the shipment. There are many reports on the prevalence of measles antibody in the sera of monkeys (Bhatt et al., 1966; Kalter et al., 1967; Meyer et al., 1962; Shishido, 1966). Generally the sera of monkeys have been reported to be free of measles antibody at the time of the capture in jungle areas, and a subsequent acquisition of measles antibody after contact with man has been suggested. The absence of antibody in the majority of the sera of monkeys collected on the day of arrival in our laboratory also supports this idea. No evidence for the presence of virus having close serologic relationship with measles virus of monkey origin has been reported as yet. MINIA is a virus isolated directly from the primary cultures of, monkey kidney cells. However, no serologic or biologic difference from measles
120 NOTE Vol. 22 virus of human origin was demonstrated, and this virus is considered to have come from monkeys spontaneously infected with measles virus of human origin (Ruckle, 1958; Ruckle-Enders, 1962). Since the monkeys are imported from countries in southeastern Asia where the possibility of infection with rinderpest virus is still remaining among domestic animals and this virus has a close serologic relationship with measles virus (Bogel et al., 1966), it was possible that the monkeys were spontaneously infected with rinderpest virus resulting in an apparent rise of measles antibody. However, the serologic survey on the monkey sera revealed that they contained virus neutralizing antibody to measles virus in a much higher titer than that to rinderpest virus, and no evidence of an involvement of rinderpest infection was demonstrated (Yamanouchi et al., 1969). Thus a virus source of the natural infection of the monkeys after captivity appears to be measles virus of human origin. A probable mode of their natural infection with measles virus is suggested as follows: a few monkeys are infected by a contact with children disseminating measles virus in a village or exporter's compound after captivity. During the holding period in the compound or transportation in an airplane, these infected monkeys serve as a source of transmission to the other non-immune monkeys. In our quarantine unit, possibility of infection from the personnel can be excluded since they all have measles antibody. The natural infection in our quarantine unit is supposed to spread by a direct transmission of virus from monkey to monkey. Therefore, strict isolation of each monkey throughout the holding period in the compound from the time of their capture, during transportation, and also throughout the quarantine in laboratory will be necessary to procure non-immune monkeys for measles experiments. Potkay et al. (1966) reported an epizootic of measles with clinical signs of the disease in rhesus monkeys under quarantine. Thus far, we have never observed such clinical signs in monkeys in our laboratory in spite of the high incidence of natural infection as demonstrated in the present study. In the other experiments (Yamanouchi and Uchida, to be published), we have observed appearance of giant cells in the lymph nodes removed at intervals during quarantine. In all the cases examined, appearance of the lesions in the lymph nodes was followed by a subsequent rise of measles antibody in the next week, indicating that these pathological lesions were produced by natural infection with measles virus. However, no clinical sign could be observed. Apparently the natural infection with measles virus occurred without showing any detectable clinical signs. ACKNOWLEDGEMENT The authors wish to express their appreciation to Drs. S. Honjo and T. Fujiwara for their help in quarantine and maintenance of monkeys. REFERENCES BHATT, P. N., BRANDT, C. D. WEISS, R. A., Fox, J. P. AND SHAFFER, M. F.(1966): Viral infections of monkeys in their natural habitat in southern India. II. Serological evidence of viral infection. Am. J. Trop. Med. Hyg., 15, 561-566. Bð GEL, K., PROVOST, A. AND ENDERS-RUCKLE, G.(1966): Hð magglutinations-hemmungs-
1969 NOTE 121 Reaktion mit Masernantigen bei Rinderpest. Zbl. Bakt. I. Orig., 199, 1-19. KALTER, S. S., RATNER, J., KALTER, G. V., RODRIGUEZ, A. R. AND KIM, C. S.(1967): A survey of primate sera for antibodies to viruses of human and simian origin. Am. J. Epidemiol., 86, 552-568. MEYER, H. M., Jr., BROOKS, B. E., DOUGLAS, R. D. AND ROGERS, N. G.(1962): Ecology of measles in monkeys. Am. J. Dis. Child., 103, 307-313. PEEBLES, T. C., MCCARTHY, K., ENDERS, J. F. AND HOLLOWAY, A.(1957): Behavior of monkeys after inoculation of virus derived from patients with measles and propagated in tissue culture together with observations on spontaneous infections of these animals by an agent exhibiting similar antigenic properties. J. Immunol., 78, 63-74. POTKAY, S., GANAWAY, J. R., ROGERS, N. G. AND KINARD, R.(1966): An epizootic of measles in a colony of rhesus monkeys (Macaca mulatta). Am. J. Vet. Res., 27, 331-334. RUCKLE, G.(1958): Studies with the mokey-intra-nuclear-inclusion-agent (MINIA) and foamy-agent derived from spontaneously degenerating monkey kidney cultures. I and II. Arch. ges. Virusforsch., 8, 139-166;167-182. RUCKLE-ENDERS, G.(1962): Comparative studies of monkeys and human measles-virus strains. Am. J. Dis. Child., 103, 297-307. SHISHIDO, A.(1966): Natural infection of measles virus in laboratory monkeys. Japan. J. Med. Sci. Biol., 19, 221-222. SHISHIDO, A., YAMANOUCHI, K., HIKITA, M., SATO, T., FUKUDA, A. AND KOBUNE, F.(1967): Development of a cell culture system susceptible to measles, canine distemper, and rinderpest viruses. Arch. ges. Virusforsch, 22, 364-380. TAKASAKA, M., HONJO, S., FUJIWARA, T., HAGIWARA, T., OGAWA, H. AND IMAIZUMI, K. (1964): Shigellosis in cynomolgus monkeys (Macaca irus). I. Epidemiological surveys on Shigella infection rate. Japan. J. Med. Sci. Biol., 17, 259-265. YAMANOUCHI, K., FUKUDA, A., KOBUNE, F., HAYAMI, M. AND SHISHIDO, A.(1969): Serologic response in monkeys inoculated with rinderpest and measles viruses. Am. J. Vet. Res. (In press). Department of Measles Virus, National Institute of Health, Murayama-cho, Tokyo 190-12, Japan KAZUYA YAMANOUCHI AKIKO FUKUDA FUMIO KOBUNE MICHIKO HIKITA AKIRA SHISHIDO (Received: February 14th, 1969)