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Control of losses in freshly-imported laboratory primates during the acclimatization period.

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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.
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