Control of losses in freshly-imported laboratory primates during the acclimatization period.код для вставкиСкачать
Control of Losses in Freshly-imported Laboratory Primates during the Acclimatization Period P. F. LEWIS C o m m o n w e a l t h S e r u m Laboratories, Parkville, Victoria, Australia ABSTRACT Observation of large numbers of freshly-trapped rhesus and crab-eating macaques transported by air from Asia to a n Australian laboratory has shown that severe losses may occur during the first few weeks following importation and that it is up to 12 weeks before the animals become properly acclimatized to the new environment. Deaths which occur during this period are primarily due to enteric and/or respiratory tract bacterial pathogens. In addition, endoparasitic infestations are almost invariably present; and viral infections may enhance the virulence of other pathogens. Measures which have been taken to reduce losses during the period of stabilization involve good husbandry, the treatment of parasites, and the prophylactic use of antibiotics. However, greatest success has been achieved by partly acclimatizing monkeys to captivity in the country of origin, during which period they are treated for intestinal helminths. Only robust, adolescent animals are selected for dispatch to Australia by air in small consignments. The provision of healthy animals is of great importance to all whose work involves the use of laboratory primates. In general, the user can pursue one of two courses. He can ( a ) import directly or indirectly from the country in which the desired species occurs naturally and can hold these animals through a period of quarantine and observation until satisfied that they are in good health; or he can ( b ) obtain animals that have been reared in a laboratory as are other species of laboratory animals. Without doubt, the latter is to be preferred; but there are comparatively few laboratories with facilities and funds to breed their own required monkeys, especially when large numbers are needed. The importation of freshly-trapped, jungle-reared primates is always troublesome. Regardless of the care taken to select apparently healthy animals, the stress of trapping and transportation and the changes imposed by the new environment serve to activate latent infections. Illness of many animals and the death of some appears to be almost inevitable, especially when they are received in large consignments. These problems are described in published work from many sources, e.g., AM. J. PHYS. ANTHROP.,38: 50s-510. Carpenter ('40), Goffe ('56), Habermann and Williams ('57), de Valois ('60), Sauer and Fegley ('60), Greening ('62), Roth ('65), Honjo ('66), Good, May and Kawatomari ('69), Coid ( ' 7 0 ) , Geldenhuys et al. ('71), Kalter ('71). In this paper the background of Australian experiences is used to establish the major epidemiological features of illnesses occurring during the acclimatization period and thence to deduce the action needed to ensure a supply of healthy monkeys. METHODS In 1955 the Commonwealth Serum Laboratories, Melbourne, commenced work on production of Salk-type poliomyelitis vaccine for use throughout Australia. Production and testing of this vaccine involved the use of large numbers of rhesus macaques (Macaca mulatta) and crabeating macaques ( M . fascicularis). Importation of monkeys from Asian countries, almost entirely by air, commenced in 1955. Initially, consignments of about 200 or 300 animals were obtained. As the project progressed, however, it was necessary to charter a freighter aircraft to carry numbers ranging from 1,000 to 1,800 at one time. From the year 1962 the demand for 505 506 P. F. LEWIS monkeys was greatly reduced, and the Laboratories reverted to the importation of small consignments carried by regular airline services. This arrangement continues. In all, over 50,000 monkeys have been imported. The great majority of these have been held in quarantine for at least three months, preferably up to six months, before use. Some were kept as reserve stock for periods in excess of two years, and a small breeding colony was maintained for four years to investigate the economics of breeding these animals in the laboratory. Complete records have been kept of environmental changes, signs of illness, medical treatment, and numbers of animals that died or were issued each week from each consignment. Post mortem examinations were carried out on all carcasses (except from the first 16 consignments) and gross pathological changes were recorded; detailed histopathological and microbiological studies were not undertaken unless specifically indicated. Measures were taken to reduce losses and to improve the health of the animals in each consignment; these were carried out whenever possible on a n experimental basis. Groups of monkeys were treated with antibiotics before, during, and subsequent to being transported. Some were vaccinated before dispatch with a n autogenous Pasteurella vaccine, injected with monkey globulins, or treated with anthelmintics. Care was taken at all times, during flight and after receipt, to control environmental temperature and humidity, to ensure best possible conditions for caging and handling, and to ensure the supply of food and water. To evaluate and compare the success of these efforts, two parameters may be applied to the recorded results. These are the Crude Mortality Rate (C.M.R.) and the Grouped Cause Death Rate (G.C.D.R.). In human epidemiology, the C.M.R. is normally calculated on a time interval of one year. In order to obtain a meaningful figure when applied to monkeys, the C.M.R. is here defined as the percentage of deaths in a week per mean population in that week. Similarly the Cause Specific Death Rate (C.S.D.R.) used in the study of human disease to demonstrate the changes in severity of a disease from year to year must be modified. To arrive at a true C.S.D.R., it is necessary to ensure that all deaths attributed to the disease are correctly diagnosed and that the specified disease is the primary cause of death. It is not possible to define the primary cause of death- for every monkey, nor is it practicable to attempt to isolate and identify the causal organism in more than a small sample of affected animals. Moreover, it is impossible to differentiate between similar diseases on the grounds of gross pathological changes only; however, it is possible to group diseases according to the distribution of lesions in organs or systems. On this basis, all post-mortem examination results have been separated into five broad groups, namely of those animals in which lesions were confined to the thorax, to the alimentary tract, and to both the thorax and alimentary tract, those with no gross lesions; and, finally, a group covering all other causes of death. With post-mortem findings grouped in this manner, a Grouped Cause Death Rate (G.C.D.R.) may be calculated when G.C.D.R. is defined as the percentage of deaths from a grouped cause in a week per mean population during the week. In addition to calculation of the C.M.R. and G.C.D.R. for groups of freshly imported monkeys, the C.M.R. is presented for stabilized colonies. In these, losses are comparatively infrequent; and it is necessary to calculate the C.M.R. on the basis of deaths which occur in the colony in a month, rather than per week. RESULTS T h e crude mortality rate in freshly-imported monkeys Figure 1 shows the mean weekly C.M.R. for 65 consignments totalling 47,216 freshly imported crab-eating and rhesus macaques. These were received over the period from May 1955 to August 1961. The C.M.R. rose to 12.2% in the third week after arrival and then declined to 1.5% by the twelfth week. Thus a period of at least 12 weeks is required for acclimatization and stabilization for freshly-trapped monkeys transported into a new environment. 507 LOSSES IN FRESHLY IMPORTED PRIMATES I9 CONSIONMENTS 13.992 HONKEVS ‘I \,-\ ENTERIC I = THORACIC I---THORACIC LENTERIC O---O NO GROSS LESIONS 0-0 ALL OTHER CAUSES - I 2 Fig. 1 A 6 8 10 WEEKS AFTER ARRIVAL 12 Crude mortality rate T h e grouped cause death rate in freshly-imported monkeys The results of post-mortem examination of all deaths which occurred in 49 consigments, totaling 43,992 monkeys, were grouped as described under Methods; and the weekly G.C.D.R. was calculated for each group (fig. 2 ) . Enteritic infections cause greatest loss in newly-imported monkeys. The G.C.D.R. for enteric infections ranges from 2 % to 6.2% per week for the first nine weeks, with highest losses in the second and third weeks. Respiratory infections are next in importance, but the G.C.D.R. for this group diminishes much more abruptly with time than does that for enteric infections. A substantial percentage of affected animals contract both enteric and thoracic infections concurrently, as can be seen by the curve for the thoracic and enteric group. There is circumstantial and clinical evidence that many monkeys with no gross lesions initially suffer from enteric infection, and death occurs as a result of dehydration, upset i n ionic balance, or inanition and exhaustion. In these cases it is not possible to establish the cause of death with certainty; hence the special “no gross lesions” grouping in figure 2. It is interesting to note that all other causes of death account for only about 6% of all deaths in rhesus macaques and 2% in crab-eating macaques. Moreover, the weekly G.C.D.R. remains comparatively constant throughout the entire period of observation. This constant rate indicates that causes of death such as wounding, metritis, and pyogenic infections occur sporadically and that epizootics, such as endoparasitic infesta- WEEKS AFTER ARRIVAL Fig. 2 Grouped cause death rate tions, occur without any time relationship to transportation. Losses in stabilized colonies Figure 3 presents i n histogram form the monthly C.M.R. for four consignments totalling 2,820 monkeys that were maintained as reserve stock for a period of 28 months. This figure shows that after acclimatization the C.M.R. stabilizes at about 1% per month. The epidemiological pattern of losses in a colony of young, locally-born cynomolgus monkeys is quite different. Figure 4 shows that three major epizootics occurred over a four-year period causing the mean monthly C.M.R. to approximate that which was experienced in freshly-received consignments. However, between epizootics losses were negligible. The average size of this colony was 112 weanlings. Evaluation of measures f o r improved survival in freshly-imported monhey s ( a ) E f e c t of size of consignment During the years 1955 and 1956,27 consignments were received averaging 216 monkeys per consignment. From 1957 through 1961, 38 large consignments, averaging 1,089 monkeys each were imported. The weekly C.M.R. for these two groups is given in figure 5. Losses of monkeys are significantly less when they are transported in small consignments. However, a period of 12 weeks is still necessary before the monkeys stabilize. Thus in 1962, when large numbers of monkeys were no longer required, the Laboratories returned to importation of small consignments by regular airline services. 508 P. F. LEWIS 25 2 n 201 10 5 /I n 7 5 10 15 20 MONTHS AFTER ARRIVAL 25 30 Fig. 3 Monthly crude mortality rate. Imported, stabilized monkeys. ( b ) Acclimatization before transportation Another procedure which has resulted in significant improvement in the survival of imported monkeys is to maintain the animals in captivity in the country of origin for 12 weeks before transportation. During this time they are treated for intestinal parasites and only healthy adolescent animals are selected for dispatch. The effect of this course of action is demonstrated in figure 6 in which the weekly C.M.R. is plotted for 16 consignments of freshlytrapped monkeys and for seven consignments of acclimatized monkeys, the mean size of consignments in both groups being identical. DISCUSSION It is well recognized that heavy losses occur in freshly-trapped monkeys which are transported into a completely different environment. Roth ('65) stated that a conservative estimate of these losses was 35% of the total intake. The period required for quarantine and acclimatization after transportation is considered to be at least six weeks. Coid ('70) advised not less than 12 weeks, if the monkeys are kept in pairs, and not less than eight weeks if in single cages. Australian experiences confirmed that high mortality rates are to be expected when freshly-trapped monkeys are transported and that a period of 12 weeks must elapse before the animals can be regarded as having become acclimatized. Numerous reports have appeared in the literature of the comparative importance of enteric infections and helminthic infesta- Fig. 4 Monthly crude mortality rate. Cynomolgus weanlings. /\,",: l2. 0-027 SMALL No./CONSIGT.CONSIGNMENTS 216 % x. WEEKS AFTER ARRIVAL Fig. 5 Effect of size crude mortality rate. #-I 0-0 of consignment on 18 CONSI6NHENTS(lSO8 MONKEYS) FRESHLY TRAPPED 7 CONS16NMENTS(619 MONKEYS) ACCLIMATIZE0 . WEEKS AFTER ARRIVAL Fig. 6 Effect of pre-flight acclimatization on crude mortality rate. tions as causes of loss i n freshly-trapped monkeys. Shigellosis, salmonellosis, endoparasitism, and amoebic dysentery are particularly common. This situation appears to be generally true for all species of jungle-reared primates and is certainly the case for rhesus and crab-eating macaques (Habermann and Williams, '57). Pasteurella and Diplococcus were the most common bacterial pathogens isolated from pneumonic lungs. An epizootic of pneu- LOSSES IN FRESHLY IMPORTED PRIMATES monia, if it occurs, commences about the second week after arrival; but losses subside after two or three weeks. On the other hand, outbreaks of enteric disease persist over a much longer period. Associated with losses from enteritis there is usually a smaller number of deaths having no gross lesions. The carrier state is known to occur with many of these bacterial infections (Goffe, ’56); stresses caused by transportation, chilling, or changes of diet precipitate outbreaks of disease. Thus heavy losses occur when large consignments are shipped; when large numbers of animals are brought into close contact, the risk of introducing several diseases concurrently is much greater. By providing a period of acclimatization in the country of origin, the monkeys are adjusted to captivity with a minimum of stress from fatigue, or from climatic or dietary change. Some cross infection inevitably occurs, but the monkeys have time to recover from transient illnesses and to develop immunity against reinfection. If transported in small consignments, comparatively few of these acclimatized animals are lost. CONCLUSIONS Freshly-trapped monkeys transported to a new environment require a minimum period of 12 weeks for acclimatization. Losses may be severe during the acclimatization period, particularly if the animals are transported in large consignments. After acclimatization, the “normal” loss in a stabilized colony is about 1 % per month. Mortalities are primarily due to enteric pathogens which cause prolonged epizootics characterized clinically by diarrhoea, dysentery, and dehydration. Epizootics of pneumonia are next in importance and are generally of shorter duration than are outbreaks of enteritis. Control of serious loss in freshly-trapped monkeys has been achieved by arranging for their acclimatization in the country of origin and for transportation in small consignments. ACKNOWLEDGMENTS Acknowledgment is made of the work of 509 numerous colleagues and members of staff of the Commonwealth Serum Laboratories who participated in the procurement and care of the animals and to the Director and staff of the Veterinary Research Institute, Parkville, where most post mortem examinations were undertaken. Special thanks are due to the Director, C.S.L., for permission to present this paper, to Miss H. Woodward for painstaking maintenance of records, and to Mr. M. Chin of Kuala Lumpur for his willing co-operation in carrying out pre-flight acclimatization of recent consignments. LITERATURE CITED Carpenter, C. R. 1940 Rhesus monkey (Macaca mulatta) for American laboratories. Science, 92: 284-286. Coid, C. R. 1970 The management and use of laboratory primates for medical research J. S. Afr. vet. med. Assoc., 41: 157-164. de Valois, D. G. 1960 Problems associated with the transportation of monkeys. Ann. N. Y. Acad. Sci., 85: 752-757. Geldenhuys, J. J., J. H. Groenewald, J. J. W. van Zyl, H. D. Brede and H. W. Weber 1971 Health problems encountered at the University of Stellenbosch Primate Colony. J. Sth. Afr. vet. med. Assoc., 42: 63-65. Groffe, A. 1956 Infections in laboratory monkeys. Collected Papers, Laboratory Animals, Bureau, London, 4: 29-36. Good, R. C., B. D. Mav and T. Kawatomari 1969 Enteric pathogens in monkeys. J. Bact., 97: 1048-1055. ~ . ~~ . . .. Greening, C. L. 1962 The controlled collection, holding, transport and stock housing of monkeys intended for tissue-culture production. In: Proc. 7th int. Congr. microbiol. Stand., London, 1961,edited by Standfast, A. F. B.,D. G. Evans and B. G. F. Weitz, E. & F. Livingstone Ltd, London, 1962, pp. 111-117. Habermann, R. T., and F. P. Williams, Jr. 1957 Diseases seen at necropsy of 708 M. mulatta and M . philippinensis. Am. J. Vet. Res., 18: 419-421. Honjo, S. 1966 Physiologic adaptation of cynomolgus monkeys to the alteration of environmental conditions, Jap. J. med. Sci. Biol., 19: 224-225. Kalter, S. S. 1971 Problems associated with the use of nonhuman primates. Lab. Animal Sci., 21: 997-1001. Roth, T. W. 1965 Editorial. Primates from trap to test tube. Lab. Animal Care, 15: 243-246. Sauer, R. M., and H. C. Fegley 1960 The roles of infectious and noninfectious diseases in monkey health. Ann. N. Y. Acad. Sci., 85: 866-888.