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Stress and parasitism. I. A preliminary investigation of the effects of stress on ground squirrels and their parasites
EXPERIMENTAL
PARASITOLOGY
11,
63-67
(1961)
I. A Preliminary Investigation Stress and Parasitism. of the Effects of Stress on Ground Sqnirrels and Their Parasites’ Glenn California
Slate Polytechnic
A. Noble College, San Lois Obispo, California
(Submitted for publication,
11
October 1960)
The Uinta Ground Squirrel, Citellus arnzatus, in Jackson Hole, Wyoming, was selected for a study of stress and parasitism during the summers of 1959 and 1960. TWO hundred and twenty-six squirrels were live-trapped; 72 of the animals were used as controls and 155 were subjected to the stress of heat and light, noxious stimulants, noise, crowding, darkness, hunger, annoyance, extreme confinement or caging without added stimuli. A carefully controlled dilution technique was devised to give a representative count of the cecal protozoa, mostly Trichontonas. Over the period of two summers there was an average increase of 48% in numbers of protozoa in the stressed animals as compared to the controls. The white blood cell count decreased as the numbers of protozoa increased. The weights of the adrenal glands, however, also decreased in the stressed animals. .4 continuing study will bc made of these and of related problems. was made at the Biological Research Station, Moran, Wyoming. The station is operated by the University of Wyoming under the direction of Dr. L. Floyd Clarke. Able assistance was given this project by Dr. Robert B. Tesh. This study was of an exploratory nature only. The objective was to find out whether research along the lines followed would show promise for further work and, if so, what specific approach should be continued.
Stress in vertebrates is followed by complex hormonal changes which are associated with a decrease in resistance to bacterial infection. This decrease is due in part to the production of adrenocorticotropic hormones (ACTH) from the pituitary gland and consequent release of adrenal glucocorticoids which, among other things, diminish normal inflammatory responses. Studies by Davis and Read (1958), Christian (1956, 1959), Louch (1956), Sayers (1950), Selye (1946a,b, 1950, 1959), Siegel (1960), Southwick (1958, 1959), and many others show clearly that stress is also intimately related to population density, sexual cycles, changes in blood picture, antibody formation, metabolism of basic foodstuffs, tissue damage, and a host of other bodily activities and reactions. Few studies, however, have been made concerning the effect of stress on parasitic infections. During the summers of 1959 and 1960 a preliminary study of stress and parasitism
MATERIALS
AND
METHODS
The Uinta Ground Squirrel, Citellus urmztus, was selected as the host animal, the cecal flagellate Trichomonas (Tritrichomonas) chosen for special attention. During July and August of 1959 one hundred and twelve ground squirrels were live-trapped. All of the animals were brought to the laboratory and 33 of them were examined immediately as controls. The remaining 69 were treated variously as indicated below. During June, July and August of 1960, one hundred and fourteen squirrels were live-trapped at various localities. Thirty-nine were sacrificed immediately in the field. Blood smears were
1 This investigation was supported in 1959 by a research grant from the New York Zoological Society and in 1960 by a PHS research grant E-2681 from the National Institute of Allergy and Infectious Diseases, Public Health Service. 63
64
NOBLE
made and cecal samples were collected to be examined later in the laboratory. Seventyfive squirrels were brought alive to the laboratory and subjected to the stress of caging and crowding. The 1959 group of 69 animals were fed daily with dandelion plants and occasionally with commercial rabbit pellets. Fresh water was present at all times. Almost every day for a month at least one caged squirrel and one newly trapped field animal were examined. Each animal was killed with an jection of 5 ml of a lo? solution of nembutal. A record was made of its sex and weight, a blood smear was taken and a differential blood count was ma?e. Five hundred milligrams of the cecal contents were weighed and mixed with 10 ml of normal saline. A drop of this mixture was placed on a slide, mixed with one drop of D’Antoni’s iodine solution, covered with a 22-mm square coverslip and examined under objective of a microscope. the “high-dry” Five equally-spaced areas on each slide were counted and two slides were made from each squirrel. The ten counts were averaged and this number remained as the protozoa count for any one animal. Stimuli employed were as follows: 1) Heat and light. Fifteen squirrels were placed in a large (20 x 22 x 15 inch) cage in which a shielded 60-watt electric bulb was hung. The temperature ranged from 20” C to 32” C as compared to the outside range of about 15” C to 23” C. 2) Noxious stimulants. Fourteen animals were kept in a cage of similar size (see 1 above) and subjected to fairly constant fumes of formalin, ammonium hydroxide or glacial acetic acid. One of these substances was placed in a protected glass container in the cage for a day or two and then replaced by one of the others. 3) Noise. Seven animals were kept in a large cage and frequently, throughout the day, subjected to sudden noises such as beating on the cage. Almost once a day the cage was placed near a small dog which barked excitedly around it. 4) Crowding. Ten animals were placed in a small cage (9 x 15 x 9 inches). Space was
so limited the squirrels were in constant contact with one another. 5) Darkness. Seven animals were placed in a large cage which was completely covered with several layers of old tenting. Care was taken to permit air to enter the cage but there was no light within. 6) Hunger. Seven animals in a large cage were given water at all times but food was limited to about half of that given to other groups of seven animals. 7) Annoyance. Seven squirrels in another large cage were fed at irregular hours, water was placed in new areas each day and the cage was tilted at different angles for varying periods of time. Dandelions were frequently placed on top of the cage where the animals had to exert special effort to get food. 8) Confinement. Two squirrels were each placed in closely confining wire mesh so that they could not move from one place to another. 9) Caged animals without added stress. Ten animals were placed in a special wire cage with a floor measurement of 20 x 40 inches and an arched roof of 20 inches in maximum height. Sufficient bedding for tunnels was provided. All these squirrels became tamer than did the others. The 1960 group of 7.5squirrels was divided into several groups. Some were fed dandelions only, some received rabbit pellets only, others were fed dandelions in the morning and pellets in the afternoon, and one group was fed both types of food but was kept in constant light and increased temperatures. The method of killing and counting protozoa was the same as employed in 1959. Increase in the weight of the adrenal glands is often considered an indication of stress. These organs from the 114 squirrels collected in 1960 were weighed individually at the time the animals were sacrificed for protozoa counts. .The analytical balance used was an Ainsworth Type LC with a sensitivity to 0.1 w. RESULTS
The average number of Trichomonas in the 33 field animals of 1959 was 92 whereas the average number in the 39 field animals of 1960 was 66. Since these 39 squirrels were
STRESS
AND
examined in the field, as soon as caught, 66 is probably the more accurate figure to use as representative of the controls. Environmental conditions for the protozoa during 1959, however, might have been different than during the following year so the figure of 92 is retained as the control number for the experimental animals of 1959. Table I summarizes the study of Trichomonas in squirrels trapped in 1959. Detailed TABLE I Reszdts of First Summer’s Study of Stressors and Numbers of Trichomonas in Squirrels
Stressors
Per cent -4verage number increase of protozoa per over controls field of view
Crowding Extreme confinement Light and heat Noise Annoyance Koxious stimulants Darkness Hunger
177 160 155 151 147 139 116 86
Caged without added stress
155
92 74 69 69 60 51 26 A decrease of 6.5 per cent 67
data on the studies of 1960 are shown in Tables II, III, IV, and V. The weight of the adrenal glands of the 39 control animals was 28.5 mg, whereas in the 69 caged animals it averaged 22.5 mg-a 21yC decrease. The per cent weight ratio (adrenals/body weight) in the field animals averaged .0095, whereas in the caged animals it was .0090-a decrease of about 5%. DISCUSSION
Hungry squirrels were apparently unable to support their intestinal trichomonads. In almost all other animals the numbers of protozoa increased for about two weeks, then decreased. The squirrels might have become adjusted to stress, or, in preparation for hibernation, the animals might normally have experienced a reduction in protozoa numbers. In some animals, the number of protozoa increased again after the two-week period. Since the squirrels that were not subjected to special stimuli showed an increase in numbers of Trichomonas, which was about the same as the increase in specially treated squir-
PARASITISM.
65
I
rels, it might be assumed that capturing and caging, even under what appears to be optimum conditions, produced sufficient stress to be reflected in the parasite population. TABLE II Protozoa Coztnt in Animals Fed Dandelions Rabbit Pellets Squirrel 112 107 110 52 19 108 27 1 113 114 111 98 2 109 58 29 83 54
0 0 6 d d 0 0 0 d d $ 0 P 6 6 d 0 P 86 8 97 0 71 0 8 6 30 d 53 d 101 6” 31 P 25 0 100 P 102 p 22 6 80 d 75 0 76 8 51 0 33 0 35 Squirrels .4verage 99 protozoa 50% increase
Days in Cage 1 3
3 4 4 5 6 6 6 7 7 8 8 9 9 9 10 10 10 10 12 13 14 15 15 16 17 18 19 20 23 24 29 29 32
and
Protozoa Count 129 115 94 140 85 150 58 52 102 92 59 61 50 106 36 37 163 98 287 221 190 144 105 94 41 40 69 113 55 188 94 108 108 252 163
The blood picture was variable but, in general, in both field and caged animals, when the number of cecal protozoa was high, the number of white blood cells was low, and vice versa. These cells were almost entirely lymphocytes and leucocytes, the numbers of eosinophiles being extremely low in all ani-
66
NOBLE
mals examined. The decrease agrees with the findings of other workers who have reported that a low white blood cell count is associated with stress.
TABLE V Protozoa Count in Animals Fed Both Foods and Subjected to Light and Heat
TABLE III Count in Animals Fed Pellets Only
Protozoa
Animal 79 8 90 0
16 8 72 8 106 0 7 0 17 6 18 0 94 0 70 0 95 8 96 8 14 0 15 8 92 0 93 8 16 Squirrels Average 118 Protozoa 79% increase
Protozoa
of adrenal medullary secretions and other factors must be considered.
Days in Cage 4 2 7 10 11 12 12 13 15 17 19 21 30 35 37 42
Protozoa Count
6 0 8 8 0 8 0 0
46 8 85 0 41 P 82 0
4 7 8 10 10 11 13 13 14 24 33
61 0 105 0 103 p
128 46 9s 66 60 71 143 57 96 190 170 160 103 86 226 94
TABLE IV Count in Animals Fed Dandelions 74 35 57 43 4s 37
60 P 104 0
62 0 63 0 89 0 64 0
65 8 84 0
66 8 67 0
5 5 11 11 12 13 14 18 19 21 23 26 28
98 66 111 167 114 142 122 92 176 126 100 108 72
13 Squirrels Average 114 protozoa 73% increase
Only
144 101 105 98 37 61 74 93 45 53 84
11 Squirrels Average 76 protozoa 1.5% increase
Decline in adrenal weight was characteristic of almost all experimental animals regardless of how long they remained crowded or subjected to other stressors. It would be hazardous, however, to draw conclusions from this loss of weight without considerably more analysis. All adrenocortical responses cannot be equated with stress, and the effects
The two most impressive aspects of studies so far are (1) the definite increase in numbers of cecal protozoa in caged squirrels and (2) the complexity of the problem. Ten squirrels in a cage are not all stressed alike, neither internally or externally. The number of variables which are operative, is unknown, and many are not measureable. It is too early to present nice statistical results. A great many problems are raised and many questions are left unanswered but the future looks promising. REFERENCES and reproductive responses to population size in mice from freely growing populations. Ecology 37, 258-273. CHRISTIAN, J. J. 1959. Control of populations in rodents by interplay between population density and endocrine physiology. Wildlife Disease 2, l-38. DAVIS, D. E., AND RE.\D, C. P. 1958. Effect of behavior on development of resistance in trichinosis. Proceeding of the Society for Experimental Biology and Medicine 93, 269-272. LOUCH, C. D. 1956. Adrenocortical activity in relation to the density and dynamics of three confined populations of Microtus Pennsylvanicm. Ecology 37, 701-713. SAYERS, G. 1950. The adrenal cortex and homeostasis. Physiological Reviews 30, 241-320. SELYE, H. 1946a. The general adaptation syndrome and the diseases of adaptation. Jo?lrnal of Allerev-_ 17. 231-247 and 358-398. CHRISTIAN,
J. J. 1956. Adrenal
STRESS
AND
H. 1946b. The general adaptation syndrome and the diseases of adaptation. Journal of Clinicd Endocrinology 6, 117-230. SELYE, H. 1950. Stress. Acta Endocrinologica, Inc. Montreal, Canada. 822 pp. SELYE, H. 1939. Perspectives in stress research. Perspectives in Biology and Medicine 2, No. 4, 403416. SIEGEL, H. S. 1960. Effect of population density on SELYE,
PARASITISM.
I
67
the pituitary-adrenal cortical axis of cockerels. Poultry Science 39, 500-510. SOUTHWICK, C. H. 1958. Population characteristics of house mice living in English corn ricks: density relationships. Proceedings of the Zoological Society of London 131, 163-175. SOUTHWICK, C. H. 1959. Eosinophil response of C57B mice to behavioral disturbance. Ecology 40, 196-197.
PARASITOLOGY
11,
63-67
(1961)
I. A Preliminary Investigation Stress and Parasitism. of the Effects of Stress on Ground Sqnirrels and Their Parasites’ Glenn California
Slate Polytechnic
A. Noble College, San Lois Obispo, California
(Submitted for publication,
11
October 1960)
The Uinta Ground Squirrel, Citellus arnzatus, in Jackson Hole, Wyoming, was selected for a study of stress and parasitism during the summers of 1959 and 1960. TWO hundred and twenty-six squirrels were live-trapped; 72 of the animals were used as controls and 155 were subjected to the stress of heat and light, noxious stimulants, noise, crowding, darkness, hunger, annoyance, extreme confinement or caging without added stimuli. A carefully controlled dilution technique was devised to give a representative count of the cecal protozoa, mostly Trichontonas. Over the period of two summers there was an average increase of 48% in numbers of protozoa in the stressed animals as compared to the controls. The white blood cell count decreased as the numbers of protozoa increased. The weights of the adrenal glands, however, also decreased in the stressed animals. .4 continuing study will bc made of these and of related problems. was made at the Biological Research Station, Moran, Wyoming. The station is operated by the University of Wyoming under the direction of Dr. L. Floyd Clarke. Able assistance was given this project by Dr. Robert B. Tesh. This study was of an exploratory nature only. The objective was to find out whether research along the lines followed would show promise for further work and, if so, what specific approach should be continued.
Stress in vertebrates is followed by complex hormonal changes which are associated with a decrease in resistance to bacterial infection. This decrease is due in part to the production of adrenocorticotropic hormones (ACTH) from the pituitary gland and consequent release of adrenal glucocorticoids which, among other things, diminish normal inflammatory responses. Studies by Davis and Read (1958), Christian (1956, 1959), Louch (1956), Sayers (1950), Selye (1946a,b, 1950, 1959), Siegel (1960), Southwick (1958, 1959), and many others show clearly that stress is also intimately related to population density, sexual cycles, changes in blood picture, antibody formation, metabolism of basic foodstuffs, tissue damage, and a host of other bodily activities and reactions. Few studies, however, have been made concerning the effect of stress on parasitic infections. During the summers of 1959 and 1960 a preliminary study of stress and parasitism
MATERIALS
AND
METHODS
The Uinta Ground Squirrel, Citellus urmztus, was selected as the host animal, the cecal flagellate Trichomonas (Tritrichomonas) chosen for special attention. During July and August of 1959 one hundred and twelve ground squirrels were live-trapped. All of the animals were brought to the laboratory and 33 of them were examined immediately as controls. The remaining 69 were treated variously as indicated below. During June, July and August of 1960, one hundred and fourteen squirrels were live-trapped at various localities. Thirty-nine were sacrificed immediately in the field. Blood smears were
1 This investigation was supported in 1959 by a research grant from the New York Zoological Society and in 1960 by a PHS research grant E-2681 from the National Institute of Allergy and Infectious Diseases, Public Health Service. 63
64
NOBLE
made and cecal samples were collected to be examined later in the laboratory. Seventyfive squirrels were brought alive to the laboratory and subjected to the stress of caging and crowding. The 1959 group of 69 animals were fed daily with dandelion plants and occasionally with commercial rabbit pellets. Fresh water was present at all times. Almost every day for a month at least one caged squirrel and one newly trapped field animal were examined. Each animal was killed with an jection of 5 ml of a lo? solution of nembutal. A record was made of its sex and weight, a blood smear was taken and a differential blood count was ma?e. Five hundred milligrams of the cecal contents were weighed and mixed with 10 ml of normal saline. A drop of this mixture was placed on a slide, mixed with one drop of D’Antoni’s iodine solution, covered with a 22-mm square coverslip and examined under objective of a microscope. the “high-dry” Five equally-spaced areas on each slide were counted and two slides were made from each squirrel. The ten counts were averaged and this number remained as the protozoa count for any one animal. Stimuli employed were as follows: 1) Heat and light. Fifteen squirrels were placed in a large (20 x 22 x 15 inch) cage in which a shielded 60-watt electric bulb was hung. The temperature ranged from 20” C to 32” C as compared to the outside range of about 15” C to 23” C. 2) Noxious stimulants. Fourteen animals were kept in a cage of similar size (see 1 above) and subjected to fairly constant fumes of formalin, ammonium hydroxide or glacial acetic acid. One of these substances was placed in a protected glass container in the cage for a day or two and then replaced by one of the others. 3) Noise. Seven animals were kept in a large cage and frequently, throughout the day, subjected to sudden noises such as beating on the cage. Almost once a day the cage was placed near a small dog which barked excitedly around it. 4) Crowding. Ten animals were placed in a small cage (9 x 15 x 9 inches). Space was
so limited the squirrels were in constant contact with one another. 5) Darkness. Seven animals were placed in a large cage which was completely covered with several layers of old tenting. Care was taken to permit air to enter the cage but there was no light within. 6) Hunger. Seven animals in a large cage were given water at all times but food was limited to about half of that given to other groups of seven animals. 7) Annoyance. Seven squirrels in another large cage were fed at irregular hours, water was placed in new areas each day and the cage was tilted at different angles for varying periods of time. Dandelions were frequently placed on top of the cage where the animals had to exert special effort to get food. 8) Confinement. Two squirrels were each placed in closely confining wire mesh so that they could not move from one place to another. 9) Caged animals without added stress. Ten animals were placed in a special wire cage with a floor measurement of 20 x 40 inches and an arched roof of 20 inches in maximum height. Sufficient bedding for tunnels was provided. All these squirrels became tamer than did the others. The 1960 group of 7.5squirrels was divided into several groups. Some were fed dandelions only, some received rabbit pellets only, others were fed dandelions in the morning and pellets in the afternoon, and one group was fed both types of food but was kept in constant light and increased temperatures. The method of killing and counting protozoa was the same as employed in 1959. Increase in the weight of the adrenal glands is often considered an indication of stress. These organs from the 114 squirrels collected in 1960 were weighed individually at the time the animals were sacrificed for protozoa counts. .The analytical balance used was an Ainsworth Type LC with a sensitivity to 0.1 w. RESULTS
The average number of Trichomonas in the 33 field animals of 1959 was 92 whereas the average number in the 39 field animals of 1960 was 66. Since these 39 squirrels were
STRESS
AND
examined in the field, as soon as caught, 66 is probably the more accurate figure to use as representative of the controls. Environmental conditions for the protozoa during 1959, however, might have been different than during the following year so the figure of 92 is retained as the control number for the experimental animals of 1959. Table I summarizes the study of Trichomonas in squirrels trapped in 1959. Detailed TABLE I Reszdts of First Summer’s Study of Stressors and Numbers of Trichomonas in Squirrels
Stressors
Per cent -4verage number increase of protozoa per over controls field of view
Crowding Extreme confinement Light and heat Noise Annoyance Koxious stimulants Darkness Hunger
177 160 155 151 147 139 116 86
Caged without added stress
155
92 74 69 69 60 51 26 A decrease of 6.5 per cent 67
data on the studies of 1960 are shown in Tables II, III, IV, and V. The weight of the adrenal glands of the 39 control animals was 28.5 mg, whereas in the 69 caged animals it averaged 22.5 mg-a 21yC decrease. The per cent weight ratio (adrenals/body weight) in the field animals averaged .0095, whereas in the caged animals it was .0090-a decrease of about 5%. DISCUSSION
Hungry squirrels were apparently unable to support their intestinal trichomonads. In almost all other animals the numbers of protozoa increased for about two weeks, then decreased. The squirrels might have become adjusted to stress, or, in preparation for hibernation, the animals might normally have experienced a reduction in protozoa numbers. In some animals, the number of protozoa increased again after the two-week period. Since the squirrels that were not subjected to special stimuli showed an increase in numbers of Trichomonas, which was about the same as the increase in specially treated squir-
PARASITISM.
65
I
rels, it might be assumed that capturing and caging, even under what appears to be optimum conditions, produced sufficient stress to be reflected in the parasite population. TABLE II Protozoa Coztnt in Animals Fed Dandelions Rabbit Pellets Squirrel 112 107 110 52 19 108 27 1 113 114 111 98 2 109 58 29 83 54
0 0 6 d d 0 0 0 d d $ 0 P 6 6 d 0 P 86 8 97 0 71 0 8 6 30 d 53 d 101 6” 31 P 25 0 100 P 102 p 22 6 80 d 75 0 76 8 51 0 33 0 35 Squirrels .4verage 99 protozoa 50% increase
Days in Cage 1 3
3 4 4 5 6 6 6 7 7 8 8 9 9 9 10 10 10 10 12 13 14 15 15 16 17 18 19 20 23 24 29 29 32
and
Protozoa Count 129 115 94 140 85 150 58 52 102 92 59 61 50 106 36 37 163 98 287 221 190 144 105 94 41 40 69 113 55 188 94 108 108 252 163
The blood picture was variable but, in general, in both field and caged animals, when the number of cecal protozoa was high, the number of white blood cells was low, and vice versa. These cells were almost entirely lymphocytes and leucocytes, the numbers of eosinophiles being extremely low in all ani-
66
NOBLE
mals examined. The decrease agrees with the findings of other workers who have reported that a low white blood cell count is associated with stress.
TABLE V Protozoa Count in Animals Fed Both Foods and Subjected to Light and Heat
TABLE III Count in Animals Fed Pellets Only
Protozoa
Animal 79 8 90 0
16 8 72 8 106 0 7 0 17 6 18 0 94 0 70 0 95 8 96 8 14 0 15 8 92 0 93 8 16 Squirrels Average 118 Protozoa 79% increase
Protozoa
of adrenal medullary secretions and other factors must be considered.
Days in Cage 4 2 7 10 11 12 12 13 15 17 19 21 30 35 37 42
Protozoa Count
6 0 8 8 0 8 0 0
46 8 85 0 41 P 82 0
4 7 8 10 10 11 13 13 14 24 33
61 0 105 0 103 p
128 46 9s 66 60 71 143 57 96 190 170 160 103 86 226 94
TABLE IV Count in Animals Fed Dandelions 74 35 57 43 4s 37
60 P 104 0
62 0 63 0 89 0 64 0
65 8 84 0
66 8 67 0
5 5 11 11 12 13 14 18 19 21 23 26 28
98 66 111 167 114 142 122 92 176 126 100 108 72
13 Squirrels Average 114 protozoa 73% increase
Only
144 101 105 98 37 61 74 93 45 53 84
11 Squirrels Average 76 protozoa 1.5% increase
Decline in adrenal weight was characteristic of almost all experimental animals regardless of how long they remained crowded or subjected to other stressors. It would be hazardous, however, to draw conclusions from this loss of weight without considerably more analysis. All adrenocortical responses cannot be equated with stress, and the effects
The two most impressive aspects of studies so far are (1) the definite increase in numbers of cecal protozoa in caged squirrels and (2) the complexity of the problem. Ten squirrels in a cage are not all stressed alike, neither internally or externally. The number of variables which are operative, is unknown, and many are not measureable. It is too early to present nice statistical results. A great many problems are raised and many questions are left unanswered but the future looks promising. REFERENCES and reproductive responses to population size in mice from freely growing populations. Ecology 37, 258-273. CHRISTIAN, J. J. 1959. Control of populations in rodents by interplay between population density and endocrine physiology. Wildlife Disease 2, l-38. DAVIS, D. E., AND RE.\D, C. P. 1958. Effect of behavior on development of resistance in trichinosis. Proceeding of the Society for Experimental Biology and Medicine 93, 269-272. LOUCH, C. D. 1956. Adrenocortical activity in relation to the density and dynamics of three confined populations of Microtus Pennsylvanicm. Ecology 37, 701-713. SAYERS, G. 1950. The adrenal cortex and homeostasis. Physiological Reviews 30, 241-320. SELYE, H. 1946a. The general adaptation syndrome and the diseases of adaptation. Jo?lrnal of Allerev-_ 17. 231-247 and 358-398. CHRISTIAN,
J. J. 1956. Adrenal
STRESS
AND
H. 1946b. The general adaptation syndrome and the diseases of adaptation. Journal of Clinicd Endocrinology 6, 117-230. SELYE, H. 1950. Stress. Acta Endocrinologica, Inc. Montreal, Canada. 822 pp. SELYE, H. 1939. Perspectives in stress research. Perspectives in Biology and Medicine 2, No. 4, 403416. SIEGEL, H. S. 1960. Effect of population density on SELYE,
PARASITISM.
I
67
the pituitary-adrenal cortical axis of cockerels. Poultry Science 39, 500-510. SOUTHWICK, C. H. 1958. Population characteristics of house mice living in English corn ricks: density relationships. Proceedings of the Zoological Society of London 131, 163-175. SOUTHWICK, C. H. 1959. Eosinophil response of C57B mice to behavioral disturbance. Ecology 40, 196-197.