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Physiological and environmental factors affecting the immune reactivity of captive Rocky Mountain bighorn… Emslie, Dorothy Ruth 1977

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PHYSIOLOGICAL AND ENVIRONMENTAL FACTORS AFFECTING THE IMMUNE REACTIVITY OF CAPTIVE ROCKY MOUNTAIN BIGHORN SHEEP (OVIS CANADENSIS CANADENSIS, SHAW, 1804) DOROTHY RUTH EMSLIE B.Sc.(Agr.)» University of British Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1977 ( Q \ Dorothy Ruth Emslie by MASTER OF SCIENCE i n In present ing th is thes is in p a r t i a l fu l f i lment o f the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS ABSTRACT Populations of bighorn sheep have been severely depleted by respiratory disease during the past one hundred years. Although numerous pathogens contribute to the complex etiology of t h i s disease, p h y s i o l o g i c a l and environmental factors may also contribute to the frequency and sev e r i t y of t h i s disease. Therefore, the primary objectives of t h i s study were to (a) assess immune competence and p h y s i o l o g i c a l and p a r a s i t o l o g i c a l status of healthy and diseased bighorn sheep and (b) determine which p h y s i o l o g i c a l and environmental factors contribute to immune competence. Since lymphocyte transformation appears to be a s e n s i t i v e index of immune competence, . i n a second part of t h i s study attempts were made to miniaturize an e x i s t i n g macroleukocyte culture technique. I f simple enough, such a technique could be used to evaluate the immune competence of free ranging animals. Results of t h i s study i n d i c a t e that immune competence i s v a r i a b l e between i n d i v i d u a l s and may o s c i l l a t e preceeding episodes of disease. However, low immune r e a c t i v i t y , depressed n u t r i t i o n a l status and increased l e v e l s of general inflammation appear to occur during episodes of disease. Although numerous variables contribute to immune competence, high l e v e l s of seromucoid were associated with low immune r e a c t i v i t y . / - i i -In a second part of t h i s study suggestions were made for the development of a microlymphocyte culture. Although the data c o l l e c t e d i n t h i s preliminary study suggest that t h i s technique may be possible to assess immune r e a c t i v i t y i n the f i e l d , further i n v e s t i g a t i o n s are necessary to standardize t h i s method. - i i i -TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i i i LIST OF FIGURES v i LIST OF APPENDIX TABLES v i i i ACKNOWLEDGEMENT x i i PART I FACTORS AFFECTING THE IMMUNE REACTIVITY OF ROCKY MOUNTAIN BIGHORN SHEEP A. INTRODUCTION 1 B. MATERIALS AND METHODS 14 1. Animals 14 2. Sampling Methods 15 3. Leukocyte Culture Techniques 16 a. Mitogens 16 b. Preparation of Leukocyte Cultures 16 3 c. R a d i o l a b e l l i n g with H-Thymidine 17 3 d. Processing of H-Thymidine Labelled Leukocytes- • • 17 e. Measurement of R a d i o a c t i v i t y 18 4. Parasite Counts 18 5. C l i n i c a l Chemical Analysis 19 6. Serum Protein Electrophoresis 19 C. RESULTS 21 - i v -Page. D. DISCUSSION 34 1. The e f f e c t of episodes of disease and sampling date on (a) immune r e a c t i v i t y ; (b) n u t r i t i o n a l status; (c) degree of inflammation, and (d) l e v e l of parasitism. 35 a. Immune r e a c t i v i t y 35 b. N u t r i t i o n a l s t a t u s . 38 c. Degree of inflammation 41 d. Level of parasitism 41 2. Association of n u t r i t i o n a l status, inflammation and parasitism with estimates of immune r e a c t i v i t y 43 E. CONCLUSION 45 PART II A MICROTECHNIQUE FOR IN VITRO TRANSFORMATION OF OVINE LEUKOCYTES A. INTRODUCTION 46 B. MATERIALS AND METHODS 48 1. Animals • «48 2. Leukocyte Culture Techniques 48 a. Whole Blood Cultures 48 b. P u r i f i e d C e l l Cultures. 48 3 c. Rad i o l a b e l l i n g with H-Thymidine . . . . 49 d. Harvesting of Leukocyte Cultures . . . . 49 e. Measurement of R a d i o a c t i v i t y 49 - V -Page C. RESULTS 5 1 1. Whole Blood vs. P u r i f i e d C e l l s 5 1 2. The E f f e c t of Serum Concentration  3. The E f f e c t of Temperature and Duration of Storage . . . . 51 4. The E f f e c t of Mitogen Concentration J O 5. The E f f e c t of Culture Time and Length of Pulse 5 8 D. DISCUSSION 6 3 1. Whole Blood vs. P u r i f i e d C e l l s 6 3 64 2. The E f f e c t of Serum Concentration  3. The E f f e c t of Temperature and Duration of Storage . . . . 65 4. The E f f e c t of Mitogen Concentration 66 5. The E f f e c t of Culture Time and Length of Pulse 6 7 E. CONCLUSION 7 1 LITERATURE CITED 7 2 APPENDIX TABLES I-XXII 8 4 - v i -Figure LIST OF FIGURES Page 3 1 H-Thymidine incorporation i n co n t r o l non-stimulated leukocyte cultures (log^dpm/10^ c e l l s ) during health and disease 23 3 2 H-Thymidine incorporation i n phytohemagglutinin (PHA) stimulated leukocyte cultures (log edpm/10 c e l l s ) during health and disease 25 3 Rela t i v e serum seromucoid concentration (% of control serum) during health and disease 28 3 4 Relationship between the H-thymidine incorporation of control non-stimulated and phytohemagglutinin (PHA) stimulated leukocyte cultures (log^dpm/10 c e l l s ) and episodes of disease 31 3 5 Relationship between the H-thymidine incorporation of phytohemagglutinin (PHA) stimulated cultures (log edpm/10^ c e l l s ) , r e l a t i v e seromucoid concentration (% of control serum) and episodes of disease • • 3 6 H-Thymidine incorporation i n phytohemagglutinin (PHA) stimulated whole blood and p u r i f i e d leukocyte cultures (cpm/culture) 53 - v i i -Figure Page 3 7 E f f e c t of serum concentration on the H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture) 55 8 E f f e c t of temperature and duration of storage 3 on H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture) 57 9 E f f e c t of phytohemagglutinin (PHA) concentration 3 on the H-thymidine incorporation of whole blood cultures (cpm/culture) 60 10 E f f e c t of culture time and length of pulse on 3 H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture) 62 - v i i i -LIST OF APPENDIX TABLES Table Page 3 I H-Thymidine incorporation by leukocytes of healthy and diseased Rocky Mountain bighorn sheep i n mitogen stimulated and control non-stimulated cultures (log^dpm/10 c e l l s ) 85 3 II H-Thymidine incorporation of Rocky Mountain bighorn sheep leukocytes i n phytohemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and endotoxin (Endo) stimulated and control (Cont) non-stimulated cultures (log edpm/10 6 c e l l s ) . 85 II I E f f e c t of condition of Rocky Mountain bighorn 3 sheep on the H-thymidine incorporation of phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and endotoxin (Endo) stimulated and control (Cont) non-stimulated leukocyte cultures (log^dpm/10 c e l l s ) 86 IV E f f e c t of condition of Rocky Mountain bighorn sheep on blood urea nitrogen (BUN) l e v e l s (mg/100 ml) and r e l a t i v e serum concentrations of albumin and t r a n s f e r r i n (% of control serum) 86 V E f f e c t of condition of Rocky Mountain bighorn sheep on serum l e v e l s of lysozyme ^ig/ml) and r e l a t i v e serum concentration of alpha-1 gl o b u l i n and seromucoid (% of control serum) 87 VI E f f e c t of condition of Rocky Mountain bighorn sheep on f e c a l p arasite counts ( -^0.02 eggs/gm) 87 - i x -3 VII H-Thymidine incorporation i n c o n t r o l unstimulated Rocky Mountain bighorn sheep leukocyte cultures ( l o g e disintegrations/min (dpm)/10^ c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 88 3 VIII H-Thymidine incorporation i n phytohemagglutinin (PHA) stimulated Rocky Mountain bighorn sheep leukocyte cultures (log disintegrations/min (dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972).. Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks af t e r s t a r t ) 89 3 IX H-Thymidine incorporation i n pokeweed mitogen stimulated Rocky Mountain bighorn sheep leukocyte cultures (l°g e disintegrations/min(dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 90 3 X H-Thymidine incorporation concanavalin A (ConA) stimulated Rocky Mountain bighorn sheep leukocyte 6 cultures (l°g e disintegrations/min(dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) .91 3 XI H-Thymidine incorporation i n endotoxin (Endo) stimulated Rocky Mountain bighorn sheep leukocyte cultures (log disintegrations/min (dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 92 - x -XII Relative serum albumin concentration (% of control serum) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) .93 XIII Relative serum beta-2 g l o b u l i n ( t r a n s f e r r i n ) concentration (% of control serum) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 94 XIV Replicate analyses of blood urea nitrogen (BUN) concentration (mg/100 ml) determined by the method of Chaney (1962) of i n d i v i d u a l animals (1-9), at each sampling period (weeks a f t e r s t a r t ) throughout the study period .95 XV Serum lysozyme concentration (jig/ml) determined by the method of Litwack (1955) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) throughout the study period . . . . 96 XVI Relative serum alpha-1 g l o b u l i n concentration (% of con t r o l serum) of i n d i v i d u a l animals (1-9) at each sampling period (weeks af t e r s t a r t ) .97 XVII Relative serum seromucoid concentration (% of con t r o l serum) determined by the method of Pryce (1967) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) .98 XVIII Relative serum alpha-2 g l o b u l i n concentration (% of control serum) of i n d i v i d u a l animals (1-9) at each sampling period (weeks af t e r s t a r t ) .99 - x i -XIX Re l a t i v e serum beta-1 g l o b u l i n concentration (% of con t r o l serum) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 100 XX Fecal lungworm counts ( 0.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 101 XXI Fecal i n t e s t i n a l nematode counts ( -^'0.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 102 XXII Fecal c o c c i d i a counts ( 0.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) 103 - x i i -ACKNOWLEDGEMENTS I am indeed fortunate to have experienced the leadership and d i r e c t i o n of Dr. R.J. Hudson, currently i n the Department of Animal Science at the Un i v e r s i t y of Alberta. His d i l i g e n t and patient manner and enthusiastic support have greatly influenced the d i r e c t i o n of my academic achievement. A s p e c i a l thanks i s extended to Dr. D.M. Shackleton, Department of Animal Science and Dr. R.C. Fitzsimmons, Department of Poultry Science, U n i v e r s i t y of B r i t i s h Columbia, who took time out from t h e i r busy schedules to make constructive c r i t i c i s m s and suggestions on the presentation and content of t h i s t h e s i s . I am also g r a t e f u l to Dr. CR. Krishnamurti, who managed my academic i n t e r e s t s during the absence of my supervisor. I appreciate the concern shown by Dr. W.D. K i t t s , currently Dean of the Faculty of A g r i c u l t u r a l Sciences, U n i v e r s i t y of B r i t i s h Columbia, who remained on my committee even though deluged by numerous administrative duties. Dr. R.M. T a i t , Department of Animal Science, U n i v e r s i t y of B r i t i s h Columbia, very kin d l y provided the domestic sheep used i n t h i s study and p a t i e n t l y taught me simple methods of handling these elusive beasts. - x i i i -Dr. R.G. Peterson, Dr. R.M. Beames, Mrs. M. S t r i k e r and Mr. D. J e f f r i e s , Department of Animal Science and Dr. CW. Roberts, Department of Poultry Science, Un i v e r s i t y of B r i t i s h Columbia instructed me on the use and abuse of s t a t i s t i c s and on the pe c u l i a r i e s of computers. Throughout t h i s study, expert t e c h n i c a l assistance and advice was offered by Mr. G. Galzy, Mrs. L. Mather and Mr. A. Basford. Mr. J . Richter, Department of Microbiology, U n i v e r s i t y of B r i t i s h Columbia, was the craftsman who designed and made the microculture harvester and was therefore instrumental i n the development of the microculture techniques. Mr. R. McGregor, the l a t e J . C (Barney) McGregor and many A g r i c u l t u r a l Science s t a f f and students were extremely h e l p f u l i n the management of the bighorn sheep herd. I wish to thank Dr. P.E. Reid, Department of Pathology, U n i v e r s i t y of B r i t i s h Columbia, for h i s h e l p f u l suggestions and e d i t o r i a l comments during the preparation of t h i s manuscript. Ms. C.A. Paulson, Communications O f f i c e r , Faculty of A g r i c u l t u r a l Sciences, University of B r i t i s h Columbia, as my f r i e n d and mentor also made innumerable organizational suggestions. The i l l u s t r a t i o n s i n Figures 1 to 5 were drawn by Dr. D.M. Shackleton, Department of Animal Science, U n i v e r s i t y of B r i t i s h Columbia and I wish to thank him for h i s care and s k i l l i n t h i s task. - x i v -Mrs. S. Chan was responsible for the preparation of t h i s manuscript and I am g r a t e f u l f or the speed and accuracy by which she f u l f i l l e d t h i s task. I would also l i k e to acknowledge the continued support and encouragement of my family, without which t h i s study may never have been completed. To these and many more I extend my sincere thanks. D.R. Emslie - XV -To my Dad who taught me patience and perseverance. - XVI -Facts are l i k e v e n t r i l o q u i s t ' s dummies. S i t t i n g on a wise man knee they may be made to utter words of wisdom; elsewhere they say nothing, or t a l k nonsense. Aldous Leonard Huxley (1894-1963) Time Must Have a Stop 1945 (London: Chatto and Windus) ch 30. - 1 -PART I FACTORS AFFECTING THE IMMUNE REACTIVITY  OF ROCKY MOUNTAIN BIGHORN SHEEP A. INTRODUCTION North American bighorn sheep populations have declined markedly since the beginning of the nineteenth century (Buechner, 1960). Seton (1929) has estimated a p r i s t i n e bighorn population of 1,500,000 to 2,000,000 animals. Recent estimates (Trefethen, 1975) suggest that only 36,000 - 41,000 bighorn sheep now e x i s t i n North America, that i s , approximately two percent of t h e i r previous abundance. Epizootics of scabies, hemorrhagic septicemia and verminous pneumonia have been proposed to be responsible for s i g n i f i c a n t declines i n bighorn sheep numbers during the past century (Seton, 1929; Buechner, 1960; Stelfox, 1971). Scabies, a p a r a s i t i c disease caused by mites (Sweatman, 1958) and possibly introduced by domestic sheep (Ovis aries) ( S h i l l i n g e r , 1937), was believed to have caused high rates of mortality among bighorns during the l a t e nineteenth and early twentieth centuries (Ward, 1915; M i l l s , 1937). Other epi z o o t i c s , occurring during the 2nd and 3rd decades of the 20th century, were thought to be due to a Pasturella-induced hemorrhagic septicemia ( M i l l s , 1937; Potts, 1937). However, t h i s diagnosis was l a t e r challenged when large numbers of lungworm parasites (Protostrongylus spp.) were i s o l a t e d from bighorns e x h i b i t i n g s i m i l a r symptoms (Marsh, 1938). Investigators now suggest that verminous or lungworm pneumonia has - 2 -been responsible f o r the majority of recent large-scale d i e - o f f s of bighorn sheep (Buechner, 1960; Forrester, 1971). However, there appear to be numerous pathogens and predisposing factors which contribute to the complex eti o l o g y of verminous pneumonia (Buechner, 1960; Forrester, 1971; H i b l e r , 1974). Numerous parasites have been i s o l a t e d from both captive and free-ranging bighorn sheep (Cowan, 1951; Becklund and Senger, 1967; Blood, 1963; Forrester and Senger, 1967; Uhazy and Holmes, 1971), but the lungworm par a s i t e Protostrongylus s t i l e s i (Dikmans, 1931) appears to have a predominant influence on the incidence of respi r a t o r y disease i n bighorn sheep (Buechner, 1960; Forrester, 1971). The eggs, larvae and adults of t h i s parasite have been found within dis c r e t e nodules scattered throughout the a p i c a l , cardiac and diaphragmatic lobes of the lungs of diseased animals (Forrester, 1971). Although s t i l l not completely understood, the l i f e cycle of t h i s parasite appears to follow c l o s e l y that of other members of the family Metastrongyloidea (Soulsby, 1965). Eggs deposited i n the lungs, by adult females, develop into motile, f i r s t stage larvae within the t i s s u e . Coughing by the infec t e d animals r e s u l t s i n these larvae e i t h e r being deposited d i r e c t l y on the ground or swallowed and then voided with the feces. Under appropriate environmental conditions, these larvae can then penetrate the foot tissues of land s n a i l s of the fa m i l i e s P u p p i l l i d a e , Vallonidae and Zonitidae (Buechner, 1960; Forrester, 1971). They then undergo two successive moults before becoming the third-stage larvae which i n f e c t bighorn sheep. Although there i s some question as to the - 3 -necessity f or the molluscan intermediate host (Buechner, 1960), most investigators consider that the l i f e cycle of the parasite i s completed by the ingestion of an inf e c t e d s n a i l by a bighorn sheep. It i s thought that larvae l i b e r a t e d into the digestive t r a c t of the sheep, penetrate the gut w a l l and are transported to the lungs i n the blood and/or lymphatic systems. However, t h i s portion of the l i f e cycle of the parasite, i s i n dispute and requires further study. Monson and Post (1972) induced a lungworm i n f e c t i o n i n captive mouflon-bighorn hybrids (0. musimon x 0. canadensis) by feeding them s n a i l s i n f e c t e d with the par a s i t e . Since the l e v e l s of i n f e c t i o n were much smaller than those found i n wild populations, ingestion of s n a i l s may not be the only method of i n f e c t i o n . H i b l e r (1972, 1974) suggested that transplacental i n f e c t i o n may be responsible for the i n i t i a l exposure of young lambs to P_. s t i l e s i . Although there has been much circumstantial evidence to support t h i s hypothesis (Buechner, 1960; Forrester, 1971), only recently has more d e f i n i t i v e evidence been obtained. Hibl e r (1972) i s o l a t e d lungworm larvae from both the placental tissue (cotyledons) of pregnant females and from the l i v e r s and the lungs of f o e t a l and neonatal bighorn sheep. This suggests that towards the end of pregnancy, dormant t h i r d stage larvae, sequestered within the tissues of the ewe, possibly i n the lungs, may become active and migrate across the placental membranes to the l i v e r of the fetus. Post partum these larvae may migrate from the l i v e r to the lungs where they mature. Ewes apparently may r e t a i n t h e i r a b i l i t y to - 4 -i n f e c t the fetus a f t e r removal from t h e i r source of i n f e c t i o n , since in f e c t e d lambs have been born i n c a p t i v i t y under circumstances where the r e - i n f e c t i o n of the ewe or lamb was u n l i k e l y (Forrester and Senger, 1964). Numerous b a c t e r i a l , v i r a l and mycoplasma species may also contribute to the verminous pneumonia of bighorn sheep (Buechner, 1960; Howe, Woods and Marquis, 1966; Forrester and Wada, 1967; Woolf, Kradel and Bubash, 1970; Forrester, 1971; Post, 1971; Parks e_t a l . , 1972; Parks and England, 1974). However, t h e i r presence i n both healthy and diseased animals suggests that, these organisms may e x i s t as a part of the normal f l o r a of bighorn sheep and that they may possibly act as opportunists. Although the actual symptoms of the verminous pneumonia of bighorn sheep may be caused by a v a r i e t y of pathogens, numerous environmental and p h y s i o l o g i c a l factors may contribute to the frequency and severity of t h i s disease. T r a d i t i o n a l l y , bighorn sheep u t i l i z e f a m i l i a r ranges rather than seeking new more suitable areas (Geist, 1971, 1974; H i b l e r , 1974). Since these animals appear to prefer high q u a l i t y forages (Demarchi, 1968, 1973) they are acutely susceptible to n u t r i t i o n a l changes r e s u l t i n g from competition, overpopulation, drought or severe winters. Poor n u t r i t i o n may cause the immune status of the e n t i r e population to be depressed, and thus make i t susceptible to death from lungworms, b a c t e r i a , viruses or mycoplasmas (Buechner, 1960; H i b l e r , 1974). - 5 -Attempts have been made to f i n d e a s i l y obtainable parameters which can be used to evaluate the health and condition of f r e e -ranging bighorn sheep (Franzmann, 1972). In order to be p r a c t i c a l , these parameters must (a) be s e n s i t i v e to s u b c l i n i c a l changes; (b) be able to detect seasonal changes; (c) involve tissues e a s i l y sampled; and (d) require only b r i e f handling of animals at a si n g l e capture. P h y s i o l o g i c a l and hematological data has been reported for captive (Woolf and Kradel, 1970, 1973; Woolf, Nadler and Kradel, 1973) and wild bighorn sheep (Franzmann and Thome, 1970; Franzmann, 1971a, 1971b; Franzmann and Hebert, 1971). Such data provide useful information about the health and condition of animals which i s d i r e c t l y r e l a t e d to the condition of the environment (Franzmann, 1972). P h y s i o l o g i c a l and hematological parameters which may be of p a r t i c u l a r importance i n assessing the health and condition of animals include estimates of blood urea nitrogen (BUN), blood proteins, serum glutamic oxalacetic transaminase (SGOT), packed c e l l volume (PCV), hemoglobin concentration, leukocyte count (WBC), erythrocyte count (RBC), and d i f f e r e n t i a l count of leukocytes (Le Resche et al.,1974). Estimates of parasite a c t i v i t y may also i n d i c a t e the q u a l i t y and condition of bighorn sheep populations (Holmes and Samuel, 1974) since the l e v e l of parasitism i n large overpopulated herds i s generally thought to be high (Buechner, 1960). Although the e f f e c t of multiple parasitism has s t i l l not been c l e a r l y elucidated (Uhazy and Holmes, - 6 -1971), the evaluation of lungworm a c t i v i t y may be more valuable i n assessing the condition of bighorn sheep. Lungworm parasites appear to be d i r e c t l y involved i n lungworm-pneumonia and t h e i r a c t i v i t y can be e a s i l y and accurately determined by analyzing the f e c a l p e l l e t s of bighorn sheep f o r lungworm larvae. Hudson, K i t t s and Bandy (1970), have suggested that serum seromucoid l e v e l s may be of value i n detecting s u b c l i n i c a l disease i n free-ranging animals. The t o t a l seromucoid f r a c t i o n has been known to be associated with induced inflammationy. pregnancy and various unrelated disease states i n c l u d i n g cancer, pneumonia and rheumatoid a r t h r i t i s (Putnam, 1975). Elevation i n various seromucoid f r a c t i o n s have also been noted during both the Spring and F a l l r i s e i n lungworm a c t i v i t y of bighorn sheep (Hudson e_t a l . , 1970) . In normal i n d i v i d u a l s elevation i n t h i s alpha-1 g l o b u l i n have also been found a f t e r major surgery and exercise (Putnam, 1975). Although the r o l e of these glycoproteins i s s t i l l not known, they appear re l a t e d to high c e l l p r o l i f e r a t i o n . Lysozyme, a b a c t e r i o l y t i c enzyme, can be found i n the serum, urine and tissues of man and other animals i n a v a r i e t y of normal and diseased states (Currie, 1976). This enzyme i s present i n high concentrations i n the cytoplasmic granules of polymorphs and some types of macrophages (e.g. those i n alveolae), and i s r e a d i l y released from these c e l l s by i n j u r y . As the a c t i v i t y of t h i s enzyme i s directed against mucopeptides present i n b a c t e r i a l c e l l walls, serum lysozyme concentrations may be high i n bighorn sheep affected with verminous pneumonia. Therefore, t h i s parameter may be u s e f u l - 7 -i n assessing and monitoring the phagocytic host response. Many authors suggest that inadequate immune processes are responsible f o r the resultant large-scale bighorn m o r t a l i t i e s (Forrester, 1971; H i b l e r , 1974). However, few studies have been performed to assess the immune competence of bighorn sheep subject to various environmental and p h y s i o l o g i c a l conditions (Hudson, 1973).' Although numerous immunological techniques have been u t i l i z e d to assess immune r e a c t i v i t y i n humans and laboratory animals, r e l a t i v e l y few studies have been performed with domestic l i v e s t o c k or w i l d l i f e (Ling, 1975). In order to demonstrate how immunological techniques could be used to evaluate the health and condition of free-ranging ungulates, a b r i e f introduction to the components, function and techniques used f o r the assessment of immune processes i s presented here. Hess et^ a l . (1975) state that " 'Immunity' i n a h i s t o r i c a l sense designates an e i t h e r g e n e t i c a l l y determined or acquired state of the macro-organism which assures maintenance of i t s function despite a p o t e n t i a l l y harmful microbial environment." Immunocompetence appears to be p r i m a r i l y mediated by numerous, a c t i v e l y metabolizing, lymphoid c e l l s (Park and Good, 1974). Although neutrophils, basophils, eosinophils, monocytes and macrophages appear to play important accessory r o l e s , the lymphocyte appears to be the most important i n the s p e c i f i c i t y , memory and recognition functions of the immune response (Weiss, 1972). - 8 -Two types of lymphocytes appear to p a r t i c i p a t e i n immune reactions (Raff, 1971; Good, 1974): (i) Thymus-derived lymphocytes ( T - c e l l s ) , which mediate t h e i r a c t i v i t y through d i r e c t c e l l contact or through the l i b e r a t i o n of soluble mediators, are responsible for most cell-mediated reactions. They i n i t i a t e delayed a l l e r g i c and graft-vs-host reactions, and p a r t i c i p a t e i n " k i l l e r " function against tumor c e l l s , i n s o l i d t i s s u e a l l o g r a f t r e j e c t i o n and i n immunosurveillance against cancer. The T - c e l l s also act as "helper" c e l l s to antibody producing c e l l s and provide primary protection against c e r t a i n v i r u s e s , fungi and f a c u l t a t i v e i n t r a c e l l u l a r b a c t e r i a l pathogens(Park and Good, 1974). ( i i ) Thymus independent, bursal dependent lymphocytes ( B - c e l l s ) , secrete immunoglobulins and antibodies, produce surface immunoglobulins i n the form of s p e c i f i c antibodies for receptor d i s t r i b u t i o n , detoxify proteins, polysaccharides and other toxins, and may be responsible for i n i t i a l antigen recognition.. These B - c e l l s provide a major l i n e of defense against both high-grade encapsulated b a c t e r i a l pathogens and c e r t a i n v i r u s i n f e c t i o n s and also prevent t h e i r recurrence (Good, 1974). The fact that the lymphocyte appears to be of primary importance i n contributing to immune r e a c t i v i t y "has led to many studies being performed to assess the morphological and functional c h a r a c t e r i s t i c s of t h i s c e l l (Elves, 1972; Greaves, Owen and Raff, 1973; WHO, 1973). Numerous i n vivo and i n v i t r o techniques have been u t i l i z e d to assess immune competence'.(Bloom and Glade, 1971; Bloom et al.,1973; - 9 -Rocklin, 1974). Frequently, i n vivo methods, such as monitoring antibody production or delayed h y p e r s e n s i t i v i t y reactions i n response to antigenic challenge, are now supplemented with various i n v i t r o methods which are simple, r e a d i l y standardized and do not endanger the i n d i v i d u a l . The r a t i o of T and B lymphocytes i s thought to be a l t e r e d during disease (Williams and Messner, 1975), and therefore monitoring the absolute number, percentage, or functional capacity of these c e l l s would be useful i n detecting immunologic abnormalities. However, as lymphocytes are not e a s i l y distinguished on the basis of s i z e , l i f e span or r e c i r c u l a t o r y properties, e f f o r t s have been made to d i s t i n g u i s h these c e l l s by other morphological and f u n c t i o n a l c h a r a c t e r i s t i c s (McCluskey and Leber, 1974). These include u l t r a s t r u c t u r e , c e l l surface receptors for complement and lymphocyte antigens, and by numerous i n v i t r o assays including lymphocyte transformation, c y t o t o x i c i t y , l i b e r a t i o n of soluble mediators and production of v i r a l plagues. In v i t r o lymphocyte transformation has been u t i l i z e d extensively i n experimental studies i n immunology, c e l l biology and genetics (Hirschhorn and Hirschhorn, 1974). Numerous non-specific mitogens are capable of stimulating s p e c i f i c populations of lymphocytes causing them to enlarge and divide i n a manner thought to be s i m i l a r to the response to the i n vivo antigenic challenge (Oppenheim, 1968; Greaves et a l . , 1974). Q u a n t i f i c a t i o n of t h i s test allows not only a study of c e l l a c t i v a t i o n and d i f f e r e n t i a t i o n but also o f f e r s a - 10 -t o o l f o r assessing the o v e r a l l p r o l i f e r a t i v e and f u n c t i o n a l capacity of various lymphocyte populations during health and disease (Janossy and Greaves, 1971). Rocklin (1974) and Herberman (1975) have used lymphocyte transformation as a diagnostic t o o l to detect congenital and acquired immunologic d e f i c i e n c i e s , s e n s i t i z a t i o n caused by either i n f e c t i o n s agents or some autoimmune disease, and to monitor the e f f e c t s of various immunosuppressive and immunotherapeutic manipulations. This test also appears to be s e n s i t i v e to the day-to-day changes within an i n d i v i d u a l and may demonstrate continual changes i n the v immune competence of an i n d i v i d u a l (Dionigi e_t a l . , 1973) . "Nonspecific" mitogens can transform 60% to 90% of the lymphocytes from a l l normal subjects independent of immunization (Oppenheim and Schecter, 1976). However, antigens stimulate lymphocytes only from s e n s i t i z e d primed donors. Antigen induced lymphoproliferation represents a secondary response i n v i t r o and therefore activates only a small percentage of the s t a r t i n g lymphocyte population. A f t e r longer periods of incubation, 5% to 35% of antigen stimulated lymphocytes transform. Stimulants are thought to bind to s p e c i f i c receptors present on the surface of a l l lymphocytes i n the case of mitogens or to only a small number of lymphocytes i n the.case of antigens. Lymphocytes are then activated by some unknown mechanisms and transform into " b l a s t " c e l l s . Stimulants can be further c l a s s i f i e d by whether they stimulate p u r i f i e d T, but not B c e l l s , of whether - 11 -they stimulate p u r i f i e d B and not T c e l l s (Anderson e_t a l . , 1972). In the usual heterogenous mixtures of T and B lymphocytes, T - c e l l stimulants a c t i v a t e both lymphocyte populations ( P h i l l i p s and R o i t t , 1973), presumably because they stimulate T c e l l s to make factors that enhance the reactions of B c e l l s (Janossy et a l . , 1973; Chess, McDermott and Schlossman, 1974). However, B - c e l l mitogens w i l l a c t i v a t e only B c e l l s even i n mixtures of B and T c e l l s . Stimulants commonly used include phytohemagglutunin (PHA) and concanavalin A (Con A), both T c e l l stimulants; pokeweed mitogen (PWM) which stimulates both B and T c e l l s ; and lipopolysaccharide endotoxins and a n t i -immunoglobulin antibodies which activ a t e B c e l l s (Gery, Kruger and Spi e s e l , 1972). However, these l a t t e r two antigens are often too l i m i t e d i n potency to permit evaluation of B lymphoproliferative r e a c t i v i t y i n man. Antigens commonly used to evaluate lymphocyte transformation are a l l thymus-dependent and activ a t e predominantly T c e l l s . They include p u r i f i e d protein d e r i v a t i v e (PPD), s t r e p t o l y s i n - 0 , Candida albicans and tetanus toxoid. Some investigators have proposed that a " c o c k t a i l " , c o n s i s t i n g of a pool of antigens, be used to evaluate lymphocytic r e a c t i v i t y i n v i t r o (Leguit et^ al., 1973). Hudson (1973), u t i l i z i n g the i n v i t r o transformation technique, demonstrated a marked immunosuppression i n bighorn sheep subject to the stress of capture and transport. Although i n i t i a l l y depressed, immune r e a c t i v i t y became elevated when animals appeared to have adapted to t h e i r environment. Since immune responsiveness appears to be greatly influenced by numerous environmental and p h y s i o l o g i c a l factors - 12 -(Hudson e_t a l . , 1974) , measurement of immune competence may be an accurate estimate of the q u a l i t y and condition of both free-ranging animals and t h e i r environment. The objectives of t h i s present study were to; (a) investigate the e f f e c t s of disease and season on various immunological, p h y s i o l o g i c a l and p a r a s i t o l o g i c a l parameters i n bighorn sheep; (b) determine which parameters were c l o s e l y associated with immunological changes; and (c) develop a method which could be used to assess the immune competence of free-ranging animals. To these ends estimates of immune competence, n u t r i t i o n a l status, degree of inflammation and p a r a s i t e load were monitored at i r r e g u l a r i n t e r v a l s , i n nine captive Rocky Mountain bighorn ewes for a period of ten months. During the study animals were on occasion, observed to show v i s i b l e symptoms of disease. Values of the above parameters c o l l e c t e d at each sampling period were displayed for i n d i v i d u a l animals i n order to show the influence of disease or seasonal or c y c l i c trends i n the various parameters. Plots of various parameters were made for i n d i v i d u a l animals i n order to determined which parameters were c l o s e l y associated with immunological changes and episodes of disease.Lymphocyte transformation to mitogens was u t i l i z e d to monitor immune r e a c t i v i t y ; blood urea nitrogen and serum protein l e v e l s to measure n u t r i t i o n a l status; serum lysozyme, alpha-1 g l o b u l i n and seromucoid to indic a t e inflammation; and f e c a l parasite counts to - 13 -estimate the l e v e l of parasitism. As animals had been held i n c a p t i v i t y f o r a year p r i o r to t h i s study the e f f e c t s of capture stress were assumed to be minimal. During the second part of t h i s study an attempt was made to develop a microlymphocyte culture technique which would be suitable for measuring the immune responses of free ranging bighorn sheep. Such a technique would be of use i n the f i e l d to estimate the immune competence of wi l d populations of bighorn sheep subject to a complex array of environmental and p h y s i o l o g i c a l f a c t o r s . - 14 -B. MATERIALS AND METHODS 1. Animals Nine Rocky Mountain bighorn ewes (Ovis canadensis canadensis, Shaw) were a v a i l a b l e at the i n i t i a t i o n of t h i s study i n October, 1972. This herd was composed of an adult ewe captured on the Stoddart Creek Range south of Ba n f f , A l t a . , i n 1965; two adult ewes captured on the Morrisey-Wigwam Range near Cranbrook, B.C., i n March 1969; and s i x younger adult ewes captured i n Jasper National Park, A l t a . , i n November 1971. A l l animals were housed together at the University W i l d l i f e Unit and maintained on an ad li b i t u m d i e t of a l f a l f a hay supplemented with barley or a balanced p e l l e t e d r a t i o n (UBC-36-57, Wood e t a l . , 1961), and unlimited access to water. During the study, episodes of i n t e s t i n a l and respi r a t o r y d i s t r e s s were frequently encountered. Symptoms such as scouring and coughing were often accompanied by a nasal discharge, anorexia, weakness and voluntary segregation from the rest of the herd. Intramuscular i n j e c t i o n s of a p e n i c i l l i n - s t r e p t o m y c i n preparation^" usually a l l e v i a t e d these symptoms. 2 In September 1972, p r i o r to the study, a coc c i d i o s t a t - (sodium s u l f a 3-quinoxaline) was added to the drinking water of a l l the animals to c o n t r o l the increasing numbers of several species of i n t e s t i n a l 1 2 Derafort, Ayerst, Montreal Vi-o x a l i n e , 12-8, VioBin Vet Prod. Ltd., St. Thomas, Ontario. - 15 -protozoa (Eimeria spp.), found by f e c a l examination. Drinking water was medicated i n t e r m i t t e n t l y using the 3:2:3 method suggested by Davies and Kendall (1954). This method consisted of two medication periods, each of three days duration, divided by a two-day period when normal drinking water was provided. No data were c o l l e c t e d during t h i s treatment period, nor f o r the following week. The death of three animals (1, 3, and 5) during the course of t h i s study, was p r i m a r i l y due to re s p i r a t o r y and digestive disturbances. Autopsies revealed mild to severe lungworm i n f e s t a t i o n s . Serum v i r a l t i t r e s from two animals which displayed chronic r e s p i r a t o r y symptoms were tested (Lethbridge Animal Disease Research I n s t i t u t e ) , and found negative for i n f e c t i o u s bovine r h i n o t r a c h e i t i s (IBR), bovine v i r a l diarrhea (BVD) and para-influenza-3 v i r u s (PI-3). 2. - Sampling- Methods At approximately 3-week i n t e r v a l s f o r a period of 10 months, each bighorn sheep was captured i n a handling net, b l i n d f o l d e d , : b l e d and f e c a l samples collected.' Jugular venous blood (40-60 ml) was c o l l e c t e d f o r ti s s u e culture and c l i n i c a l chemical analysis. Fecal samples c o l l e c t e d per aniim or from observed defecations, were used for parasite counts. If a f e c a l sample could not be obtained at the same time as the blood, samples c o l l e c t e d either one week p r i o r to or one week following t h i s sampling date were used for the ana l y s i s . - 16 -3. Leukocyte Culture Techniques a. Mitogens The cultures were stimulated with the following mitogens, i . Phytohemagglutinin (PHA-M, Difco) A 5 ml v i a l was reconstituted with d i s t i l l e d water as directed by the manufacturer. Aliquots (0.1 ml) of t h i s s o l u t i o n were used to stimulate each t r i p l i c a t e culture. i i . Concanavalin A (Con A, Pharmacia.). A 1:10 d i l u t i o n of a stock s o l u t i o n containing lmg/ml was made with d i s t i l l e d water and aliquots of 0.1 ml were added to each t r i p l i c a t e culture, i i i . Pokeweed Mitogen (PWM, GIBCO). A 1:100 d i l u t i o n of a 10 ml. v i a l was made with Medium 199 (Difco) as directed by the manufacturer, and aliquots of 0.2 ml were added to each t r i p l i c a t e culture. i v . Endotoxin (LPS-W, D i f c o ) . A 1:10 d i l u t i o n was made of a stock s o l u t i o n containing 2 mg/ml, with d i s t i l l e d water and aliquots of 0.1 ml were added to each t r i p l i c a t e culture. b. Preparation of Leukocyte Cultures Aliquots of 1.6 ml of the p u r i f i e d c e l l suspensions were d i s t r i b u t e d i n t o 5 ml (12 x 75mm) culture tubes (Falcon P l a s t i c s , Oxnard) containing 0.4ml of pooled domestic sheep serum. T r i p l i c a t e cultures^ were stimulated with the appropriate quantities of the various mitogens; cultures containing a d d i t i o n a l medium instead of mitogen - 17 -served as controls. A f t e r the tubes were t i g h t l y capped they were incubated at 37°C f or 48 hours. 3 c. R a d i b l a b e l l i n g with H-Thymidine Cultures were t r i t i a t e d with one microcurie of t r i t i a t e d thymidine (Thymidine-6-H3, sp.act. 5Ci/mmole, Amersham/Searle), i n 0.1ml of media, 48 hours a f t e r the i n i t i a t i o n of culture. A f t e r a further incubation for 16 hours, cultures were stored at -20°C. Previous experiments have shown no e f f e c t of short term freezing on subsequent cpm of each culture (Shons, Etheredge and Najarian, 1972). 3 d. Processing of H-thymidine Labelled Leukocytes Cultures were analysed by a modification of the method of Caspary and Hughes (1972). A f t e r removal of the supernatant medium, the c e l l p e l l e t s were washed with 4ml portions of both three percent ac e t i c acid and phosphate buffered s a l i n e , two 2ml portions of f i v e percent t r i c h l o r o a c e t i c acid and one 2ml portion of methyl hydrate, re s p e c t i v e l y . The c e l l p e l l e t s were then incubated i n 0.5ml of Hyamine Hydroxide (New England Nuclear) at 56°C for one hour or u n t i l complete d i s s o l u t i o n had occurred. The digests were transferred to s c i n t i l l a t i o n v i a l s by two successive washes of the culture tube with s c i n t i l l a t i o n f l u i d (5 gms 2, 5-diphenyl oxazole (PPO), 0.3gm 1, 4-bis-2-(5-phenyl-oxazolyl)-benzene (POPOP) i n 1 l i t r e of toluene). - 18 -e. Measurement of R a d i o a c t i v i t y A f t e r the v i a l s had been shaken and allowed to e q u i l i b r a t e i n the cold and dark f o r one hour, they were counted i n an Isocap 300 (Nuclear Chicago) s c i n t i l l a t i o n counter for 10 minutes. Results were corrected f o r e f f i c i e n c y and quench (Bruno and C h r i s t i a n , 1961; Bush, 1963) and recorded as di s i n t e g r a t i o n s per minute (dpm) per m i l l i o n c e l l s . These corrected values were subjected to a logarithmic transformation and expressed d i r e c t l y as gross stimulations. 4. Parasite Counts Fecal samples were a i r dried and stored at room temperature u n t i l they could be analyzed (5-6 mos). Parasites were then evaluated by a modification of the Gordon-Whitlock Technique described by Bodie (1969). Fecal samples (2 gm), softened overnight i n a small amount of water (5 ml), were crushed and suspended i n Sheather's sugar f l o t a t i o n medium (Sloss, 1970) to a t o t a l volume of 60 ml. A f t e r vigourous a g i t a t i o n , duplicate samples of t h i s f e c a l suspension were removed and transferred to both chambers of moistened McMaster s l i d e s . A f t e r approximately 15 minutes, the eggs and larvae which had r i s e n to the top of the chambers, were counted and d i f f e r e n t i a t e d m i c r o s c o p i c a l l y . C a l c u l a t i o n of numbers of eggs per gram of feces was performed as described by Bodie (1969). Larvae and ova were i d e n t i f i e d with the aid of photomicrographs, drawings and measurements (Soulsby, 1965; Sloss, 1970). Results were expressed by transformed counts (*\/Q.02 eggs/gm)as performed by Whitlock (1961). - 19 -5. C l i n i c a l Chemical Analysis Blood urea nitrogen was determined by the Hyland phenate-hypochlorite (UN-TEST) method (modified Berthelot reaction, Chaney, 1962). A l l reagents and procedures used followed manufacturers i n s t r u c t i o n s . Serum seromucoid was measured using a modification of the method of Pryce (1967). Aliquots (1ml) of 1..2M p e r c h l o r i c acid were added to centrifuge tubes containing 0.5 ml of the test serum and the solutions mixed. . After c e n t r i f u g a t i o n at 15,000 rpm i n a r e f r i g e r a t e d centrifuge f or 15 minutes, the supernatants obtained were added to tubes containing 0.5 ml stock biuret reagent. A f t e r a further 30 minutes, the absorbence was read at 550 nm i n a Bausch and Lomb Spectronic 20 Spectrophotometer. Serum lysozyme was assayed using a Lysozyme Assay Set (Worthington Biochemicals) according to the method of Litwack (1955). 6. Serum Protein Electrophoresis Serum protein f r a c t i o n s (albumin alpha-1, alpha-2, beta-1 and beta-2) were separated by agarose g e l electrophoresis using the ACI Agarose Film/Cassette system ( A n a l y t i c a l Chemists Inc.). Reagents and procedures were as s p e c i f i e d by the manufacturer except that electrophoresis was conducted at 4°C for 45 minutes, instead of 25°C for 35 minutes to r e s u l t i n greater r e s o l u t i o n of the bands. The use of agarose films allowed simultaneous electrophoresis of one standard and seven test serums. A f t e r densitometry (Densicord, Photovolt) of agarose f i l m s t r i p s stained i n Amido Black, f r a c t i o n s were a r b i t r a r i l y designated albumin, alpha-1, alpha-2, beta-1, and - 20 -beta-2 i n order of f a s t e s t to slowest m o b i l i t i e s respectively. Results were expressed as the r a t i o of the peak heights of the t e s t serums to those found i n a standard serum. Because t r a n s f e r r i n appears to be a s a t i s f a c t o r y c o r r e l a t e of n u t r i t i v e status (McFarlane e_t a l . 1969) , i t was necessary to further i d e n t i f y t h i s f r a c t i o n . Preparation and electrophoresis of an ethacridine l a c t a t e soluble f r a c t i o n of serum, i n which t r a n s f e r r i n was soluble (Hudson, 1971), yie l d e d a peak i n the beta-2 mobility range. Therefore, the beta-2 peaks on a l l serum p r o f i l e s were used as a q u a l i t a t i v e measurement of the t r a n s f e r r i n contained i n the serum samples. - 21 -C. RESULTS I n i t i a l l y , analyses of variance were used to compare the apparent health and disease of animals u t i l i z i n g indices of immune r e a c t i v i t y , n u t r i t i o n a l status, l e v e l of general inflammation and parasitism (Appendix Tables I-VI ). However differences (P<j.0.05) were only found i n the leukocyte response to a l l mitogens, c o l l e c t i v e l y , and l e v e l s of blood urea nitrogen (BUN) between apparently healthy and diseased animals (Appendix Tables I and I V ) . Data of each of the immunological,physiological and p a r a s i t o l o g i c a l parameters c o l l e c t e d from i n d i v i d u a l animals at each sampling period are shown i n Appendix Tables VII-XXII . Data c o l l e c t e d from apparently diseased animals are i d e n t i f i e d . Immune r e a c t i v i t y , determined by i n v i t r o lymphocyte response to the pressure or absence of non-specific mitogens i n PHA, Con A, Pokeweed Mitogen and Endotoxin stimulated cultures and non-stimulated control cultures fluctuated i n most animals throughout the experimental period (Appendix Tables VII-XI ). Figure 1 and 2 show that episodes of disease were frequently preceeded by high l e v e l s of i n v i t r o leukocyte a c t i v i t y . However, depressed leukocyte responses were frequently associated with v i s i b l e symptoms of disease. Although r e l a t i v e serum concentrations of albumin and t r a n s f e r r i n , indices of n u t r i t i o n a l status, remained r e l a t i v e l y constant throughout the study, (Appendix Tables XII & XIII),serum l e v e l s of BUN varied considerably (Appendix Table XIV ). Many of the lowest values of BUN were found between weeks 16 and 28 (January through to A p r i l ) . Low - 22 -Figure 1. "'H-Thymidine incorporation i n c o n t r o l non-stimulated leukocyte cultures (log^dpm/10^ c e l l s ) during health and disease. Complete ( f t ) and incomplete © ) observations from i n d i v i d u a l animals (1-9), throughout the study period. Observations within the s t i p l e d areas are from animals with v i s i b l e symptoms of disease. M i n d i c a t e s m o r t a l i t y . - 23 -121 T I M E ( w e e k s a f t e r s t a r t ) - 24 -Figure 2. H-Thymidine incorporation i n phytohemagglutinin (PHA) stimulated leukocyte cultures (log edpm/10^ c e l l s ) during health and disease. Complete (• 9) and incomplete (0 © ) observations from i n d i v i d u a l animals (1-9) throughout the study period. Observations within the s t i p l e d areas are from animals with v i s i b l e symptoms of disease. M i n d i c a t e s m o r t a l i t y . - 25 -- 26 -concentrations of BUN were sometimes associated with episodes of disease except i n animals No. 3 and No. 5 where extremely high values were found p r i o r to death, and throughout chronic i l l n e s s , r e s p e c t i v e l y . Concentrations of serum lysozyme, r e l a t i v e concentrations of serum alpha-1 gl o b u l i n and seromucoid were u t i l i z e d as indices of inflammation. Serum lysozyme was found to ex i s t only i n extremely low quantities throughout the study (Appendix Table XV ) and was therefore thought not to be a good index of the inflammatory processes occurring i n bighorn sheep. Alpha-1 and seromucoid r e l a t i v e concentrations appeared to vary only s l i g h t l y throughout the experimental period (Appendix Tables XVI & XVII). However, elevated amounts of seromucoid were often associated with disease processes (Figure 3). Alpha-2 and Beta-1 glo b u l i n f r a c t i o n s also fluctuated i n some animals throughout the study (Appendix Tables XVIII & XIX) and may have become elevated during episodes of the disease. However, as these serum f r a c t i o n s were composed of wide v a r i e t y of proteins, the s i g n i f i c a n c e of these r e s u l t s were not interpreted. A c t i v i t y of lungworms, i n t e s t i n a l nematodes and i n t e s t i n a l protozoa was determined by f e c a l exmaination f or larvae, eggs and oocysts of these p a r a s i t e s . However, the a c t i v i t y of these parasites remained low i n most animals throughout t h i s study (Appendix Tables XX-XXII). Lungworm a c t i v i t y appeared to be maximal during the 20th to the 28th week of the experiment (February - A p r i l ) except i n animal No. 5 where peaks were found i n weeks 15 and 26 (January and March). Levels of i n t e s t i n a l nematodes and cocc i d i a were also - 27 -Figure 3. Rel a t i v e serum seromucoid concentration (% of co n t r o l serum) during health and disease. Complete ( ) and incomplete ) observations from i n d i v i d u a l animals (1-9) throughout the study period. Observations within the s t i p l e d areas are from animals with v i s i b l e symptoms of disease. M i n d i c a t e s m o r t a l i t y . - 28 -- 29 -elevated i n animal No. 5 throughout the study. Only c o c c i d i a l e v e l s were high i n other animals although not always associated with episodes of disease. High l e v e l s of a l l parasites may have contributed to the gradual wasting of animal No. 5. Figure 4 demonstrates the r e l a t i o n s h i p between the leukocyte responses i n PHA stimulated and control cultures. During episodes of disease, PHA responses were frequently reduced and approached responses found i n control non-stimulated cultures. This demonstrates a loss i n a c t i v i t y i n PHA responsiveness during episodes of disease. Losses i n PHA responsiveness frequently accompanied high concentrations of serum seromucoid (Figure 5). Episodes of disease frequently accompanied low PHA responses and high l e v e l s of serum seromucoid. - 30 -Relationship between the °H-thymidine incorporation of c o n t r o l non-stimulated and phytohemagglutinin (PHA) stimulated leukocyte cultures (log edpm/10^ c e l l s ) and episodes of disease. Complete ( #_ 9 ) and incomplete (• 6 ) observations from i n d i v i d u a l animals (1-9) throughout the study period. Observations w i t h i n the s t i p l e d areas are from animals with v i s i b l e symptoms of disease. • PHA response. ir Control response. M i n d i c a t e s m o r t a l i t y . - 31 -- 32 -Figure 5. Relationship between the H-thymidine incorporation of phytohemagglutinin (PHA) stimulated cultures (logedpm/10* c e l l s ) , r e l a t i v e seromucoid concentration (% of control serum) and episodes of disease. Complete (» 9 ) and incomplete (© © ) observations from i n d i v i d u a l animals (1-9) throughout the study period. Observation wit h i n the s t i p l e d areas are from animals with v i s i b l e symptoms of disease. e PHA response. •ic seromucoid response. M i n d i c a t e s m o r t a l i t y . - 34 -D. DISCUSSION A v a r i e t y of i n t e r r e l a t e d events occur i n response to the invasion of host tissues with foreign and often pathogenic micro-organisms. M o b i l i z a t i o n of phagocytes, development of l o c a l i z e d inflammation and the a c t i v a t i o n of immunogenic mechanisms are c h a r a c t e r i s t i c host responses to i n f e c t i o n . Numerous metabolic responses may also contribute to host defense mechanisms although these responses are at present only poorly understood (Beisel , 1975). In t h i s present study, few differences were found i n the immune r e a c t i v i t y , degree of inflammation, l e v e l of parasitism and n u t r i t i o n a l status of apparently healthy and diseased animals (Appendix Tables I-VI). However, because many metabolic changes may occur p r i o r to the onset of c l i n i c a l symptoms of disease, the method of i d e n t i f y i n g diseased animals i n t h i s study by overt c l i n i c a l symptoms" o f disease may have been inadequate for the detection of s u b c l i n i c a l disease. Numerous p h y s i o l o g i c a l changes may preceed the c l i n i c a l symptoms of disease. Metabolic changes may be influenced by the severity and duration of the i l l n e s s , the e f f e c t s of therapy, by preexisting factors i n the genetic, immunological and n u t r i t i o n a l status of the host and even by the time of day that an i n f e c t i o u s process i s i n i t i a t e d ( B e i s e l , 1975). Therefore, i n t h i s present study, the data from i n d i v i d u a l animals was examined i n an attempt to determine (1) the e f f e c t of episodes of disease and sampling time on various immunological, p h y s i o l o g i c a l and p a r a s i t o l o g i c a l parameters, and - 35 -(2) the e f f e c t of various p h y s i o l o g i c a l and p a r a s i t o l o g i c a l factors on i n v i t r o lymphocyte responses of bighorn sheep. 1. The e f f e c t of episodes of disease and sampling date on (a) immune r e a c t i v i t y , (b) n u t r i t i o n a l status, (c) degree of  inflammation, and (d) l e v e l of parasitism, a. Immune r e a c t i v i t y In v i t r o lymphocyte r e a c t i v i t y has been used as an index of both humoral and c e l l u l a r immune r e a c t i v i t y (Benezra, Gery and Davies, 1969). However, when data from the present study was submitted to an analyses of variance with si n g l e degree of freedom contrasts between mitogen responses of\healthy and diseased animals, no differences were found between the s p e c i f i c mitogen responses of leukocytes from apparently healthy and diseased animals. However, v i s u a l appraisal of mitogen responses of s p e c i f i c animals demonstrated that episodes of disease were frequency preceeded by elevation i n mitogen responses; whereas, the occurrence of v i s i b l e symptoms of disease were frequently accompanied by depressed responses to mitogens. Diongi et^ a l . , (1973) also noted a depression i n the i n v i t r o lymphocyte response to PHA i n an i n d i v i d u a l developing an upper r e s p i r a t o r y i n f e c t i o n . However, whether the v a r i a t i o n s seen have any r e l a t i o n s h i p to the i n v i t r o lymphoid response to antigen,or are merely a r t i f a c t s detectable only i n the i n v i t r o system, i s s t i l l not c l e a r l y known. The presence of high l e v e l s of leukocyte r e a c t i v i t y , f o l l o w e d by a depression of t h i s response with the onset of v i s i b l e symptoms of disease,suggests that immune responses i n i t i a t e d against the pathogen may have been - 36 -overwhelmed thus causing c l i n i c a l symptoms of the disease to be produced. Although the disease causing organism encountered i n the present study was not i s o l a t e d , i t i s now known that b a c t e r i a l and v i r a l i n f e c t i o n s may .• i n t e r f e r e with both the non-specific and s p e c i f i c defenses present i n the host (Smithy 1975). - Although le s s i s known•about b a c t e r i a l mechanisms, i t i s postulated that viruses may delay or reduce the., protective e f f e c t of antibody by (i) being present i n large amounts and thus "swamping" antibody; ( i i ) being "bad" antigens for inducing antibody; ( i i i ) having a large degree of antigenic v a r i a t i o n , and (iv) i n f e c t i n g and i n h i b i t i n g the function of antibody-forming c e l l s . Although c e l l u l a r immune reactions, as judged by graft r e j e c t i o n or delayed h y p e r s e n s i t i v i t y reactions, are depressed i n many vi r u s i n f e c t i o n s (Notkins, Mergenhagen and Howard, 1970) the mechanism of t h i s depression of c e l l u l a r immunity against the i n f e c t i n g v i r u s has not been examined. Numerous environmental and p h y s i o l o g i c a l factors appear to influence immune r e a c t i v i t y (Hudson et^ a l . , 1974). Hormonal balance, n u t r i t i o n and climate are only a few of the factors which may influence both s p e c i f i c and non-specific defense mechanisms. O s c i l l a t i o n s i n lymphocyte r e a c t i v i t y occur continually i n healthy i n d i v i d u a l s (Diongi e_t aJL. , 1973; A l l e n , 1974). These changes may be correlated to environmental changes. Depressed leukocyte a c t i v i t y may be - 37 -associated with a high degree of disease s u s c e p t i b i l i t y . Montgomerie et a l . , (1969) noted that immunosuppressed renal transplant patients were p a r t i c u l a r i l y susceptible to i n f e c t i o n s i n v o l v i n g herpes simplex virus, an opportunistic organism. As v i r u s r e p l i c a t i o n and destruction may exist i n a dynamic equilibrium, any natural or a r t i f i c i a l immuno-suppression may r e s u l t In the production of disease. In the present study, PHA and control responses were s i m i l a r i n animals showing v i s i b l e symptoms of disease (Figure 4). Some authors express t h i s r e l a t i o n s h i p as a r a t i o or stimulation index (SI) where _ mean dpm with stimulus  mean dpm without stimulus (Graf and Mather, 1972). This r a t i o may be useful when one wishes to compare the r e s u l t s of d i f f e r e n t i n d i v i d u a l s . Lymphocytes of diseased i n d i v i d u a l s may e i t h e r be incapable of further stimulation due to i n t e r a c t i o n with the pathogen or may no longer be ava i l a b l e f o r stimulation with non-specific mitogens. There may be a pool of mu l t i p o t e n t i a l lymphocytes a v a i l a b l e f o r i n t e r a c t i o n with various antigens (Metcalf, 1971), but t h i s pool may become reduced during disease. ' Williams and Messner (1975) have attempted to demonstrate a l t e r a t i o n s i n T and B c e l l s during disease. These authors expressed T or B c e l l s i n terms of percentage of lymphocytes and as absolute numbers. These methods give i n s i g h t into the a l t e r a t i o n s i n the balance of c e l l s a ctive i n c e l l u l a r and humoral immunity and gives an i n d i c a t i o n of the magnitude of t h e i r p o t e n t i a l l e v e l s . - 38 -In the present study, the mitogenic responses of T and B c e l l s appeared to be reduced during disease (Figures 1 and 2 and Appendix Tables VT-XI). This may in d i c a t e that there are perhaps detrimental e f f e c t s on both the humoral and cell-mediated immune r e a c t i v i t y during disease processes. Lymphocyte responses of animals not e x h i b i t i n g v i s i b l e symptoms of disease were also depressed towards the end of the study period. As these depressions were associated with l a t e Winter and early Spring, host immune responses may have been influenced by numerous environmental changes. b. N u t r i t i o n a l status An analysis of variance comparing n u t r i t i o n a l status of apparently healthy animals with that of animals displaying v i s i b l e symptoms of disease, indicated that blood urea nitrogen values (BUN) were higher i n apparently healthy animals than diseased animals (Appendix Table IV ). However, no differences were noted i n the r e l a t i v e concentrations of albumin or t r a n s f e r r i n (Appendix Table IV ). In ruminants, the l e v e l of BUN has been used as an index of protein q u a l i t y and intake (Lewis, 1957). Results of the present study may in d i c a t e that diseased animals v o l u n t a r i l y r e s t r i c t e d intake or that inadequate n u t r i t i o n a l balances caused animals to become predisposed to disease. Although l e v e l s of albumin and t r a n s f e r r i n can be used as more s p e c i f i c indices of the animal's t o t a l n u t r i t i o n a l condition and are affected l e s s by protein intake (McFarlane et^ a l . 1969; Putnam, 1975), these proteins remained r e l a t i v e l y unchanged - 39 -during the study. These findings may only indicate that animals were not severely stressed nutritionally throughout this study. Nutritional factors are thought to interact with infections in at least two ways: (a) malnourished individuals may be more susceptible to infections, and (b) infections can precipitate malnutrition (Faulk, Demaeyer and Davies, 1974). The relationship i s cyclic as one pathophysiological condition is capable of accentuating the other (Scrimshaw, 1968). The exact mechanism whereby nutrition affects immunity is s t i l l not well understood as data are often contradictory. Many studies have been performed on malnourished subjects, but assessment of nutritional status is often d i f f i c u l t (WHO, 1972). However, persons suffering from protein malnutrition are not able to make circulating antibodies in response to certain bacterial and v i r a l antigens (Brown and Katz, 1966). This capacity is restored when proteins are incorporated into the diet (Mathews et a l . 1972). The degree of restoration has been corre with the amount of protein supplementation (Reddy and Srikantia, 1964). Immunity to v i r a l antigens appears to be less adversely affected by poor nutrition (Faulk, Demaeyer and Davies, 1974) as shown by observations that malnourished children may develop certain types of immunity at a younger age than healthy controls (Brown and Opio, 1966) . Vitamin deficiencies are less common and do not appear to significantly influence antibody synthesis in man. - 40 -Delayed h y p e r s e n s i t i v i t y i s thought to be depressed i n malnutrition as demonstrated by f a l s e l y negative skin test f o r tuberculosis (WHO, 1972). R e a c t i v i t y to other antigens that are characterized by delayed h y p e r s e n s i t i v i t y has also been found to be depressed i n malnut r i t i o n (Smythe et a l . , 1971). Depressed responses to PHA by lymphocytes i n v i t r o , have been described i n protein c a l o r i e malnutrition. The thymus gland, an important regulator of cell-mediated immune reactions, i s commonly atrophied i n malnourished chi l d r e n (Smythe et a l . , 1971). Undernutrition and depletion of protein reserves r e s u l t i n atrophy of l i v e r , spleen, bone marrow and lymphoid tissues from which phagocytes and lymphocytes originate (Schonland, 1972). Leukocytes from malnourished persons appear to be biochemically d i f f e r e n t than normal c e l l s . Their capacity to k i l l phagocytosed. b a c t e r i a i s also l e s s than normal, but t h i s property approaches normal following treatment of the primary malnutrition (Ratram, Selvaraj and Bhat, 1972). In the current study, r e l a t i v e concentrations of albumin and t r a n s f e r r i n did not appear to exhibit c y c l i c trends (Appendix Tables XII and XIII). However, BUN values did appear to be s l i g h t l y reduced during the winter months i n some animals (Appendix Table XIV). Feed intake of a l l animals appeared to be noticably depressed during the winter months (personal observation; Hudson, 1971), however, the metabolic s i g n i f i c a n c e of t h i s observation was not immediately c l e a r . - 41 -c. Degree of inflammation Seromucoid, an alpha-1 g l o b u l i n , and lysozyme have been used to detect inflammatory processes i n man and other animals (Putnam, 1975; Lewis, 1976). In the present study, episodes of disease were frequently accompanied by high seromucoid values (Figure 3). Hudson e_t a l . , (1970) noted el e v a t i o n of serum seromucoid concentrations of bighorn sheep subject to high l e v e l s of parasitism. An increase i n these "acute phase" inflammatory reactants may be due to increased non-specific defense reactions to foreign b a c t e r i a and viruses. .. / Apparent increases i n inflammatory indices i n the Spring may suggest the presence of an increased pathogenic population. d. Level of parasitism Three types of parasites were encountered during t h i s study: lungworms, i n t e s t i n a l nematodes and c o c c i d i a . However, i n most i n d i v i d u a l s , parasite loads were not heavy and did not appear to be d i r e c t l y responsible for symptoms of disease. Uhazy, Holmes and Graham (undated) however, have found a s t a t i s t i c a l l y s i g n i f i c a n t cor-r e l a t i o n (P <C0.05) between t o t a l numbers of helminths and symptoms of disease caused by pathogens other than a metazoan parasites i n bighorn sheep. High l e v e l s of parasite a c t i v i t y were found only i n one animal (No. 5). As t h i s animal was i n poor condition p r i o r to the study, i t was not known whether loss of condition was i n i t i a l l y due to pa r a s i t e a c t i v i t y . Hudson (1973) has noted that parasite i n f e c t i o n s may be accompanied by immunologic exhaustion or immune - 42 -tolerance. Also secretions of the parasites themselves may make p a r a s i t i z e d animals more susceptible to i n f e c t i o n s with other pathogens. Although i n a previous study (Hudson, 1971) 3parasite a c t i v i t y was found to peak i n Spring and F a l l , only the lungworm parasites of animal No. 5 demonstrated t h i s a b i l i t y . This c y c l i c a c t i v i t y i n some nematode parasites may be due to recently acquired worms o r i t i s possible that host responses may influence the fecundity of adult parasites or l i b e r a t e arrested stages of these worms (Michel, 1975). I n a b i l i t y to detect trends i n parasite a c t i v i t y i n the present study was probably due to a lack of p a r a s i t e s . Individual animals appear to vary i n t h e i r a b i l i t y to r e s i s t i n f e c t i o n . Although genetic factors may be important, /the condition of animals may also influence immune mechanisms. More data i s required to assess immune r e a c t i v i t y during disease. Controlled experiments on domesticated animals.infected with various organisms may be b e n e f i c i a l i n determining immune responses during health, i n i t i a l i n t e r a c t i o n with pathogens and during acute and chronic disease. A broad array of immunological tests may more c l e a r l y define immunologic processes occurring during disease. Interactions of numerous environmental and p h y s i o l o g i c a l factors may enhance disease s u s c e p t i b i l i t y . These r e l a t i o n s h i p s must be complex, since the animals u t i l i z e d i n t h i s study which were subjected to only low l e v e l s of parasitism and n u t r i t i o n a l d e f i c i e n c i e s did e x h i b i t symptoms of disease. More s e n s i t i v e indices - 43 -of physiologic condition of animals are required to enable one to evaluate the e f f e c t on condition to disease s u s c e p t i b i l i t y . 2. Association of n u t r i t i o n a l status, inflammation arid  parasitism with estimates of immune r e a c t i v i t y In t h i s study, lymphocyte responses to PHA appear to be negatively associated with high l e v e l s of seromucoid (Figure 5). High l e v e l s of seromucoid were commonly associated with low leukocyte r e a c t i v i t y to PHA and episodes of disease. Inflammatory changes occurring i n the body tissues during chronic or acute disease may f a c i l i t a t e the entry of pathogenic organisms and contribute i n d i r e c t l y to immunosuppression. Recovery from disease may be i n i t i a t e d by numerous metabolic, immunological,environmental and genetic f a c t o r s . Although there i s a f a i r degree of c o r r e l a t i o n between c e l l u l a r immune competence and non-specific T c e l l stimulation by mitogens, r e s u l t s of t h i s study i n d i c a t e that many parameters may be required to assess the health and condition of bighorn sheep. Reduced responses to mitogens were commonly associated with disease but were not always i n d i c a t i v e of c l i n i c a l disease. PHA responses of animal No. 2 did become depressed during the study but t h i s animal did not e x h i b i t c l i n i c a l symptoms of disease. A b r i e f examination of the PHA responses of a l l animals indicated that immune r e a c t i v i t y i s v a r i a b l e during apparent health. However, elevations i n immune r e a c t i v i t y commonly preceeded episodes of disease. Animals may be continually subject to the immunosuppressive e f f e c t s of various p h y s i o l o g i c a l and environmental factors but episodes - 44 -of disease may only occur a f t e r the immune response has f a i l e d to ne u t r a l i z e the pathogen. Although the exact immunological mechanism during disease states i s s t i l l not known, immunologic exhaustion may contribute to increased disease s u s c e p t i b i l i t y . In v i t r o lymphocyte transformation to mitogens may be a useful index of immune competence i n free-ranging animals since only a singl e capture Is required. However, healthy animals demonstrate ' c y c l i c trends i n immune r e a c t i v i t y . Other parameters such as measures of inflammation may be useful to indic a t e whether animals are threatenedby disease or whether immunodepression i s due to transient p h y s i o l o g i c a l and environmental changes. However, cont r o l l e d studies using domestic analogs may be required to i n i t i a l l y assess immunologic and metabolic responses to disease. Although t h i s area i s receiving some attention i n human and laboratory animal studies, very few attempts have been made to apply t h i s data to. domestic or wild species. Since animal health i s of gross economical importance to the l i v e s t o c k industry, one might hope that more d e t a i l e d studies would be performed to assess the environmental and p h y s i o l o g i c a l factdrs a f f e c t i n g both w i l d and domestic ungulates. - 45 -E. CONCLUSION Episodes of disease appear to be characterized by a loss i n leukocyte r e a c t i v i t y to mitogens, depressed l e v e l s of blood urea nitrogen and high l e v e l s of seromucoid. As the l e v e l s of lung and i n t e s t i n a l parasites were low throughout the study, the e f f e c t of parasitism could not be determined. The presence of inflammation processes may have contributed to a large amount of the v a r i a b i l i t y i n leukocyte responses to mitogens. However, further studies are required to more c l e a r l y assess the environmental and p h y s i o l o g i c a l factors i n f l u e n c i n g immune r e a c t i v i t y . - 46 -PART II A MICROTECHNIQUE FOR IN VITRO TRANSFORMATION OF OVINE LEUKOCYTES A. INTRODUCTION Of the numerous i n v i t r o t e s t s developed for the evaluation of cell-mediated immunity ( R e v i l l a r d , 1971; Bloom, 1973), lymphocyte transformation with a v a r i e t y of mitogens and antigens has become a popular technique f o r the assessment of immunocompetence. The procedure i s reproducible, semi-quantitative and appears to be correlated with immune r e a c t i v i t y (Oppenheim, 1976). However, the requirement f o r large numbers of p u r i f i e d c e l l s has l i m i t e d i t s a p p l i c a t i o n to studies 7performed on man and large animals. , Recently, numerous e f f o r t s have been made to develop microculture techniques which u t i l i z e small numbers of p u r i f i e d c e l l s or whole blood, automatic pi p e t t e s , m i c r o t i t e r plates and a multiple sample harvester (Junge et a l . , 1970; Park and Good, 1972; Kaplan and Razanno, 1973; Pellegino et a l , , 1973). These techniques can be performed accurately and allow one to economize on blood, materials, space and labour (Oppenheim, 1976). In the present study, an attempt was made to develop a micro-culture technique which could be used to monitor immune r e a c t i v i t y of free-ranging ungulates i n t h e i r natural environment. If ,simple enough, such a technique would allow studies of bighorn sheep i n t h e i r natural environment where f a c i l i t i e s a v a i l a b l e to an in v e s t i g a t o r are usually minimal. Numerous culture conditions - 47 -are known to a f f e c t the k i n e t i c s and i n t e n s i t y of the lymphocyte response i n v i t r o (Schellekens and E i j s v o o g e l , 1968) and therefore attempts were made i n t h i s study to determine the optimal culture conditions for a reproducible assay. i - 48 -B. MATERIALS AND METHODS 1. Animals Domestic sheep (Ovis aties) ' were p r i m a r i l y used throughout t h i s study, although Rocky Mountain bighorn sheep (Ovis canadensis  canadensis) were also used where indicated. Conditions of management have been described previously (Part I, Section B ( l ) ) . 2. Leukocyte Culture Techniques a. Whole Blood Cultures Aliquots of 25 or 50 j i l of jugular blood were dispensed into the wells of s t e r i l e m i c r o t i t e r plates (IS FB-96-TC, Linbro, New Haven) which contained 150jul of medium (M 199, Difco, Detroit) The medium was supplemented with a d d i t i o n a l glutamine, p e n i c i l l i n and streptomycin (Difco, Detroit) at f i n a l concentrations of 5mM, 100 units/ml and lOOug/ml r e s p e c t i v e l y . Except i n experiments where the e f f e c t of serum was tested, the medium contained 20% pooled domestic sheep serum. Aliquots (50jil) of a 1:2 d i l u t i o n of a stock s o l u t i o n of PHA (Difco, D e t r o i t , reconstituted according manufacturers di r e c t i o n s ) were usually added to t r i p l i c a t e cultures Control unstimulated cultures received 50 jul aliquots of p l a i n medium. Culture plates were sealed with an adhesive mylar f i l m and incubated at 37°C for 48 hours. b. P u r i f i e d C e l l Cultures P u r i f i e d c e l l s were obtained from bighorn sheep jugular blood by g e l a t i n sedementation as described previously (Part I, Section B(2b)). Aliquots (100 j i l ) containing 2, 5 or 10 x 10 5 - 49 -p u r i f i e d leukocytes were dispensed i n t r i p l i c a t e to the wells of m i c r o t i t e r plates containing 100 jul of medium. These cultures were then stimulated with 50 p i aliquots of a 1:2 d i l u t i o n of a stock s o l u t i o n of PHA, sealed with an adhesive mylar f i l m and incubated.at 37°C for 48 hours. 3 c. R a d i o l a b e l l i n g Leukocyte.Cultures with H-Thymidine A f t e r incubation for 48 hours, the cultures were treated with one microcurie t r i t i a t e d thymidine (Thymidine-t-H3, spec. act. 5Ci/mmole, Amersham/Searle) i n 50 )il of medium (M199)... A f t e r r e s e a l i n g the p l a t e , cultures were usually incubated for a further 16 hours. d. Harvesting of Leukocyte Cultures Cultures were harvested using an automated multiple sample harvestor (Richter S c i e n t i f i c ) which enabled the simultaneous deposition of the contents of 12 microculture wells onto glass f i b e r f i l t e r paper s t r i p s (Reeve Angel). In order to lyse the erythrocytes, p r e c i p i t a t e the n u c l e i c material, and remove the excess r a d i o a c t i v i t y , the c o l l e c t i n g f i l t e r s were washed with d i s t i l l e d water. A f t e r washing, the f i l t e r s were a i r - d r i e d overnight i n glass s c i n t i l l a t i o n v i a l s . e. Measurement of R a d i o a c t i v i t y Aliquots (10ml) of s c i n t i l l a t i o n f l u i d (5 g. of 2, 5 diphenyloxazolyl (PP0) and 0.3 g. of p-bis-2-(5 phenyloxasolyl)-benzene (POPOP) i n one l i t e r of toluene), were added to the s c i n t i l l a t i o n v i a l s containing the f i l t e r s and allowed to adapt to the cold and dark f o r 30 minutes. Sample s c i n t i l l a t i o n s were then - 50 -counted f o r 2 minutes i n an Isocap 300 (Nuclear Chicago) s c i n t i l l a t i o n counter. The r e s u l t s were expressed as counts minute (cpm) per culture. - 51 -C. RESULTS 1. Whole Blood vs P u r i f i e d C e l l s As shown i n Figure 6, 50 jul quantities of whole blood incorporated s i g n i f i c a n t l y larger amounts of t r i t i a t e d thymidine than did the 25 jil quantities (P<5*0.05). Culture of 100 jul quantities of whole blood proved unsuccessful because numerous problems were encountered during the processing of these cultures. Further studies of p u r i f i e d c e l l and whole blood cultures of bighorn sheep indicated that 50 ;ul of whole blood, containing approximately 2.5 x 10^ lymphocytes, responded s i g n i f i c a n t l y greater than p u r i f i e d cultures containing 2, 4 or 10 x 10^ p u r i f i e d c e l l s . 2. The E f f e c t of Serum Concentration Figure 7 demonstrates that cultures grown i n the absence of a d d i t i o n a l serum had a greater response to a 1:2 d i l u t i o n of stock PHA, than did cultures grown i n media containing 20% serum (P <C 0.05). S i g n i f i c a n t differences i n responses of whole blood cultures were observed when serum was added to the medium at d i f f e r e n t concentrations (P ^ 0.05). Optimal responses were observed at a serum concentration of 5%. 3. The E f f e c t of Temperature and Duration of Storage To determine whether the temperature and duration of storage of blood affected lymphocyte r e a c t i v i t y , blood was stored at 4, 25 and 37°C for periods of 5, 8 and 26 hours. Control samples (0 hours) were processed immediately a f t e r c o l l e c t i o n . The r e s u l t s obtained indicated, as shown i n Figure 8, that lymphocyte a c t i v i t y declined markedly regardless of the storage temperatures. Furthermore, most of the - 52 -Figure 6. H-Thymidine incorporation i n phytohemagglutinin (PHA) stimulated whole blood and p u r i f i e d leukocyte cultures (cpm/culture). Points are means of t r i p l i c a t e cultures. A l l cultures stimulated with 50 }il of a 1:2 d i l u t i o n of stock phytohemagglutinin (PHA). Results of three experiments. 2500 2000 1500 H 1000 500 100 n n 25 50 25 50 Whole blood Qui) ( n 2 4 , 10) 5Qul xlO 6 / P u r i f i e d Whole leukocytes blood DOMESTIC SHEEP BIGHORN SHEEP - 54 -Figure 7. E f f e c t of serum concentration on the H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture). Points are means of t r i p l i c a t e c u l t u r e s . A l l cultures were stimulated with 50 jil of a 1:2 d i l u t i o n of stock PHA. Results of three experiments. 25 H - 55 -20 1 n o 0 ft a C o •H 4-1 cfl O U O o io H c •H •H 5 1 EU a 0 20 0 20 0 5 10 20 Serum concentration (%) - 56 -Figure 8. E f f e c t of temperature and duration of storage on 3 H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture). Points are means of t r i p l i c a t e c u l t ures. A l l cultures were stimulated with a 1:2 d i l u t i o n of a stock s o l u t i o n of phytohemagglutinin (PHA). - 57 -1200 1000 H s ft o o •H o ft o a C •H QJ C •H T3 •H 4-1 I K ro 800 -I 600 H 400 -4 200 J Temp.( C) 25 37 4 25 37 4 25 37 4 25 37 5 8 Time (hours) 26 - 58 -a c t i v i t y was l o s t during the f i r s t 5 hours a f t e r c o l l e c t i o n . In these experiments serum was omitted from the culture media. 4. ' E f f e c t of Mitogen Concentration The r e s u l t s of three experiments performed to determine the dose response of ovine c e l l s to various concentrations of PHA are shown i n Figure 9. Optimal doses varied between experiments and although t h i s could represent i n d i v i d u a l v a r i a b i l i t y , i t may also represent differences between batches of PHA. For these reasons i t was not possible to determine the optimal dose of PHA. Therefore, the most convenient d i l u t i o n of 1:2 was selected for further studies. 5. . E f f e c t of Culture Time and Length of Pulse Figure 10 shows the r e s u l t s of an experiment designed to determine the optimal length of culture and the optimal duration of the radioactive pulse i n PHA stimulated cultures containing 5% serum. The responses were highest when cultures were t r i t i a t e d a f t e r a 72-hour growth period and then pulsed for a further 48 hours. However, such time periods would be unacceptable i n the f i e l d . The cultures t r i t i a t e d a f t e r 24 and 48 hours, and pulsed for 16 and 24 hours y i e l d e d s i g n i f i c a n t l y , lower responses (P <. 0.05).. However, these shorter incubation and pulse periods would be more suit a b l e for f i e l d studies. - 59 -Figure 9. E f f e c t of phytohemagglutinin (PHA)' concentration 3 on the H-thymidine incorporation of whole blood cultures (cpm/culture). Results of three experiments. - 60 -Concentration of PHA ( d i l u t i o n of a stock solution) - 61 -Figure 10. E f f e c t of c u l t u r e time and length of pulse on 3 H-thymidine incorporation of phytohemagglutinin (PHA) stimulated whole blood cultures (cpm/culture). Points are means" of t r i p l i c a t e c u l t ures. A l l cultures were stimulated with 50 u l of a 1:2 d i l u t i o n of a stock s o l u t i o n of phytohemagglutinin 3 (PHA). H-thymidine was added a f t e r O 24 m 4 8 CD 72 hours of i n i t i a l culture and pulsed for 16, 24, 48 and 72 hour periods. Pulse i n t e r v a l (hours) - 63 -D. DISCUSSION 1. Whole Blood vs P u r i f i e d C e l l s Even though whole blood cultures probably contain as few as 2.5 x 10^ lymphocytes, the r e s u l t s of t h i s study i n d i c a t e s that 50 u l aliquots of ovine blood can be cultured more su c c e s s f u l l y than 10 x 10^ p u r i f i e d c e l l s . Similar r e s u l t s have been obtained i n studies of the lymphocytes of man (Park and Good, 1974) and other animals (Strong et a l . , 1973). In some i n v e s t i g a t i o n s , the blood was d i l u t e d to f a c i l i t a t e the dispensing of small quantities of whole blood (Eskola e_t_ al_. , 1975) p r i o r to adding i t to the culture. However, Kaplan and Razzano (1973) cultured 7 jil quantities of human blood.They showed that lymphocyte transformation i n these whole blood cultures, which contained approximately 0.14 x 10^ lymphocytes, were twice as great as those found with cultures of p u r i f i e d c e l l s . The requirement of 50 j j l of blood i n t h i s study may r e f l e c t the notable unresponsiveness of ovine c e l l s i n v i t r o (Ling, 1975). Diminished responses of p u r i f i e d c e l l cultures have been noted (Kaplan and Razzano, 1973). P u r i f i e d c e l l suspensions prepared by g e l a t i n sedimentation contain large numbers of v i a b l e lymphocytes. Therefore, the depressed responses noted i n p u r i f i e d preparations when compared to whole blood do not appear to be due to an absence of c e l l s . Loss of a c t i v i t y may occur during the p u r i f i c a t i o n process. Aside from the p o s s i b i l i t y of c e l l damage or membrane a l t e r a t i o n s , some components which exert s y n e r g i s t i c e f f e c t s on lymphocyte - 64 -transformation may be removed (Kaplan and Razzano, 1973). Hypotonic l y s i s , an a l t e r n a t i v e method of p u r i f y i n g lymphocyte populations, appears to remove c e r t a i n lymphocyte populations and to diminish t h e i r responsiveness to PHA (Thomson, B u l l and Robinson, 1966). Although many p u r i f i c a t i o n techniques are based on the p r i n c i p l e of s e l e c t i v e l y removing erythrocytes, t h e i r influence on lymphocyte responsiveness i s s t i l l unclear (Johnson, Smith and K i r k p a t r i c k , 1972). The e f f e c t of monocytes and granulocytes on lymphocyte r e a c t i v i t y i s also s t i l l unknown, although lymphocytes react poorly i f these c e l l s are completely removed (Schellekens and Eij s v o o g e l , 1968). A d d i t i o n a l advantages of using whole blood are: i . an enhancement of the s e n s i t i v i t y and s t a t i s t i c a l accuracy of the lymphocyte transformation t e s t ; i i . many other lymphocyte studies can be performed simultaneously; i i i . the test can be performed simply and safely on both small animals and ch i l d r e n ; i v . i n d i v i d u a l s can be sampled with minimal e f f e c t s to t h e i r blood chemistry; and v. lymphocytes from c e l l u l a r compartments other than blood may be examined (Levinson, Lisak and Zweiman, 1974). 2. The E f f e c t of Serum Concentration Results of t h i s study i n d i c a t e that small amounts of serum may be required to y i e l d optimal responses to PHA, but large quantities appear to i n h i b i t growth. As noted by Ling (1975), there are many factors to consider when serum i s incorporated into culture media: - 65 -i . a p r o t e c t i v e or growth promoting e f f e c t , which may be due to the presence of macromolecules producing t h e i r e f f e c t i n an i l l -defined manner; i i . the presence of small molecules which supply trace nutrients not present i n the media, e.g. nucleosides, vitamins, hormones and co-enzymes; i i i . n e u t r a l i z i n g factors (possibly antibodies) which may combine with the stimulant; i v . the presence of " n a t u r a l " antibodies to antigenic s i t e s on the surface of lymphocytes, and v. the presence of foreign antigens. These factors may be stimulatory or cytotoxic. 3. The E f f e c t of Temperature and Time of Storage The a b i l i t y of lymphocytes to transform i n culture appears to be greatly influenced by the conditions of storage of blood a f t e r i t i s c o l l e c t e d . Results of t h i s study ind i c a t e that lymphocyte trans-formation deteriorates dramatically i n whole blood stored for f i v e hours at 4°, 25° or 37°C. However, other investigators have suc c e s s f u l l y stored whole blood at room temperature for 24 hours (Park and Terasaki, 1974) while others have cryopreserved or stored p u r i f i e d c e l l s at various temperatures with no apparent loss i n f u n c t i o n a l c h a r a c t e r i s t i c s i n c l u d i n g response to PHA, a l l o g e n i c c e l l s and antigens (Osoba et^ a l . , 1975; Weiner, 1976). The reasons for thesed'i ;fferences are unknown and require further study. - 66 -The retention of lymphocyte transf o r m a b i l i t y u n d e r a v a r i e t y of storage conditions would be of great advantage. Numerous samples from the same or d i f f e r e n t i n d i v i d u a l s could be assayed under s i m i l a r or i d e n t i c a l conditions, thus reducing day-to-day experimental error. The e f f e c t s of storage conditions must be studied much more c l o s e l y before our technique can be used i n the f i e l d . 4. The E f f e c t of Mitogen Concentration In t h i s study an optimal concentration of PHA was not found. In most of our experiments 50 j i l of a 1:2 d i l u t i o n of stock PHA was used to stimulate c e l l s and s i m i l a r dosages have been used by others (Eskola et al_. , 1975) . However, as the manufacturer of the PHA used i n t h i s study did not i n d i c a t e absolute q u a n t i t i e s , batches of PHA may contain d i f f e r e n t q u a n t i t i e s of a c t i v e PHA. Comparison with other studies i s d i f f i c u l t . Oppenheim (1976) suggests that mitogens be used at suboptimal doses. In a number of immunodeficiency states the p r o l i f e r a t i v e response to optimal doses of PHA w i l l be normal whereas subnormal r e a c t i v i t y can be detected only i n response to suboptimal doses of PHA (Zie g l e r , Hansen and Penny, 1975). D i f f e r e n t i n d i v i d u a l s may also have d i f f e r e n t optimal doses (Oppenheim, 1976). However, t h i s p r a c t i c e becomes impractical where the c e l l s of a large number of i n d i v i d u a l s are to be cultured. More studies should be performed on a large number of animals to determine an optimal dose of mitogen. ( - 67 -5. The E f f e c t of Culture Time and Length of Pulse The estimation of DNA synthesis by c e l l s i n the S phase i s commonly performed by measuring the uptake of t r i t i a t e d thymidine. As stated by Oppenheim (1976), "The development of quantitative assays f o r the incorporation of radioisotope precursors has l i b e r a t e d the evaluation of the lymphocyte transformation technique from the vagaries of labourious morphological evaluation". This,assay requires that the r a d i o l a b e l l e d precursor be present i n excess and that i t possess a low s p e c i f i c a c t i v i t y i n order to prevent r a d i a t i o n damage to the c e l l s (Sample and Chretien, 1971). Numerous inve s t i g a t o r s have chosen to culture lymphocytes f o r various lengths of time (Hughes and Caspary, 1970). D i f f e r e n t mitogens are reported to produce optimal responses on d i f f e r e n t days. In our study, lymphocytes appeared to incorporate the greatest amounts of t r i t i a t e d thymidine a f t e r a culture period of 72 hours. Whole blood cultures are thought to reach t h e i r peak of a c t i v i t y l a t e r than p u r i f i e d c e l l cultures (Park and Good, 1974). However, high l e v e l s of incorporation were also found to ex i s t a f t e r an i n i t i a l 24 and 48 hour culture i n t e r v a l . In most experiments performed i n t h i s study cultures were t r i t i a t e d a f t e r an i n i t i a l 48 hours of cul t u r e . Longer i n t e r v a l s allow c e l l s to become synchronized. L a b e l l i n g p r a c t i c e s also vary and many inv e s t i g a t o r s used d i f f e r e n t lengths of pulse. One'can usually demonstrate s i g n i f i c a n t incorporation by adding r a d i o l a b e l l e d precursors from 4 to 16 hours p r i o r to the processing of the cultures (Bain, 1970). Although the - 68 -shorter exposure time i s preferable as thymidine may be degraded and high thymidine concentration can only be maintained f o r short periods of time (Sample and Chretien, 1971), overnight l a b e l l i n g i s often used f o r the sake of convenience. Thymidine breaks down slowly on storage and r a p i d l y on incubation with leukocytes at 37°C, y i e l d i n g thymine and dehydrothymine which are not u t i l i z e d as precursors for DNA synthesis (Cooper and Milt o n , 1964; Milton et a l . , 1965). Degradation products have been found within 5 minutes of addition of isotope l a b e l l e d thymidine to leukocytes and are formed at a rate proportional to thymidine concentration (Ling, 1975). Granulocytes and stimulated lymphocytes enhance t h i s degradation process (Wolberg, 1971). These observations may account for the observed low thymidine incorporation when isotope i s added at the i n i t i a t i o n of the culture (Ling, 1975). These factors may be of minor importance when isotope i s added f o r short pulses a f t e r the culture has been established for some time. Thymidine i s not a normal precursor f o r lymphocyte DNA synthesis (Ling, 1975). Any present at the beginning of culture would be destroyed and thymidine nucleotides w i l l normally be synthesized at the nucleotide l e v e l from ribonucleotides. I t i s possible that the rate l i m i t i n g step i n the incorporation of thymidine into DNA could be the uptake or phosphorylation of the thymidine rather than rate of DNA synthesis. The rate of thymidine incorporation by lymphocytes stimulated i n a v a r i e t y of ways increases with the concentration of thymidine added, and saturation occurs only at thymidine concentrations of about - 69 -20ul/ml (Hartog et a l . , 1967; Schellekensand E i j s v o o g e l , 1968; Bain, 1970; Sample and Chretien, 1971). At such concentrations endogenous synthesis of thymidine nucleotides i s probably completely suppressed but saturation can be f u l l y maintained f o r only about 4 hours (Sample and Chretien, 1971). Very high thymidine concentrations i n h i b i t DNA synthesis (Hartog et a l . , 1967) by i n t e r f e r i n g with the synthesis of other deoxyribonucleotides. Many investigators use very low thymidine concentrations. 3 Addition, f o r example, of luCi/ml of H-thymidine at a s p e c i f i c a c t i v i t y of 5Ci/mmole gives a thymidine concentration of less than 0.lug/ml. Most of the thymidine nucleotides entering DNA are derived 3 from the endogenous pathway. The r e l a t i v e rates of H-thymidine incorporation i n DNA by d i f f e r e n t cultures w i l l only indicate t h e i r r e l a t i v e rates of DNA synthesis i f ' t h e proportion of nucleotides entering DNA derived from the l a b e l l e d thymidine i s constant. In f a c t , there i s considerable empirical evidence that the rate of thymidine incorporation does cor r e l a t e quite well with other parameters of lymphocyte stimulation. At these low thymidine concentrations incorporation i s l i n e a r f o r only a few hours (Bain, 1970). The subsequent decrease Is probably due i n part to degradation of the isotope and i n part, to r a d i a t i o n damage to the c e l l s . These e f f e c t s are probably more serious i n highly stimulated c e l l s and may mask rather than exaggerate differences between cultures. At very low thymidine concentration, below 0.02ug/ml, incorporation into DNA i s disproportionately reduced i n d i c a t i n g a threshold concentration needed for the u t i l i z a t i o n of exogenous thymidine. - 70 -Incorporation of t r i t i a t e d thymidine i s only an estimate of p r o l i f e r a t i o n since c e l l s synthesizing DNA i n S phase do not n e c e s s a r i l y go on to divide (Oppenheim, 1976). DNA synthesis can be determined most r e l i a b l y from the t h e o r e t i c a l viewpoint by measurement of the incorporation of high concentrations of thymidine during short pulses. Raising the s p e c i f i c a c t i v i t y of the isotope or increasing the l a b e l l i n g time with low s p e c i f i c a c t i v i t y isotope increases the s e n s i t i v i t y of determination and seems to be j u s t i f i e d e m p i r i c a l l y . Experiments i n which the incorporation of high s p e c i f i c a c t i v i t y isotope i s determined over long periods i n which very low thymidine concentrations are used, or i n which the isotope i s added at the beginning of culture, should be regarded with more suspicion (Ling, 1975). - 71 -E. CONCLUSION The aim of t h i s study was to develop a microculture technique whereby lymphocyte transformation could be measured under f i e l d conditions. The measurement of lymphocyte transformation by the incorporation of t r i t i a t e d thymidine i s a generally accepted immunological procedure which has been found to be c l o s e l y correlated with immune competence (Oppenheim, 1976). However, i n order to perform t h i s technique i n the f i e l d s i m p l i f i e d procedures and the exact s p e c i f i c a t i o n s of culture parameters were required. This study indicated that highest incorporations were observed using 50ul of whole blood, 5% serum i n cultu r e , to be cultured immediately.after c o l l e c t i o n with a culture period of 72 hours followed by a pulse of 16 or 24 hours. These conditions are s i m i l a r to those used by others, but require modification before they w i l l be appl i c a b l e f o r f i e l d use. 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C e l l mediated immunity and resistance to i n f e c t i o n : Report of a WHO s c i e n t i f i c group. Wld.Hlth.Org.Tech. Rept. Series 519:1-64. Zei g l e r , J.B., P. Hansen and R. Penny. 1975. I n t r i n s i c lymphocyte defect i n Hodgkins disease: Analyses of the phytohemagglutinin dose response. C l i n . Immunol. Immunopathol. 3:451-460. - 84 -APPENDIX TABLES I - XXII. Appendix Table I. 3H-Thymidine incorporation by leukocytes of healthy and diseased Rocky Mountain bighorn sheep in mitogen stimulated and control non-stimulated cultures (log dpm/106 cells). Condition* Mean S.D. 6.51 1.63 242 5.96 1.20 110 Healthy Diseased Significant (P 0.05) 3 Appendix Table II. H-Thymidine incorporation of Rocky Mountain bighorn sheep leukocytes in phytohemagglutinin • (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and endotoxin (Endo) stimulated and control (Cont) non-stimulated cultures (logedpm/10^ cells). Mitogen Mean S.D. 0.83 72 1.58 77 1.41 63 1.51 76 1.05 64 Significantly different (V6=. 0.05) from control values when submitted to single degree of freedom comparisons. Cont PHA3 Con A a PWMa Endo 5.37 7.53 6.15 6.76 5.67 - 86 -Appendix Table I I I . Effect of condition of Rocky Mountain bighorn 3 sheep on the H-thymidine incorporation of phytohemagglutinin (PHA), conconavalin A (Con A), pokeweed mitogen (PWM) and endotoxin (Endo) stimulated and control (Cont) non-stimulated leukocyte cultures (log^dpm/10^ c e l l s ) . Condition 3 Mitogen Healthy Diseased Mean S.D. n Mean S.D. Cont 5.41 0.86 50 5.35 0.77 22 PHA 7.74 1.61 55 7.02 1.42 22 ConA 6.32 1.49 41 5.82 1.20 22 PWM 7.00 1.61 54 6.20 1.05 22 Endo 5.79 1.20 42 5.44 0.66 22 a Significant differences (P^0.05) were not found when single degree of freedom comparisons were made between the individual mitogen responses of healthy snd diseased animals. Appendix Table IV. Effect of condition of Rocky Mountain bighorn sheep on blood urea nitrogen (BUN) l e v e l s (mg/100 ml) and r e l a t i v e serum concentrations of albumin and tran s f e r r i n (% of control serum). Condition Parameter Healthy Diseased Mean S.D. n Mean S.D. n BUN (mg/100 ml)* 27.33 7.28 51 22.93 4.11 16 Albumin 100.4 2.79 55 99.4 1.63 18 Transferrin 103.7 30.1 55 90.0 15.8 18 Signi f i c a n t (P^L0.05). Appendix Table V. Effect of condition' of Rocky Mountain bighorn 8heep on serum levels of lysozyme (Jjg /ml) and re l a t i v e serum concentration of alpha-1 globulin and seromucoid (% of control serum). Condition Parameter Healthy Diseased Mean S.D. n Mean S.D. n Lysozyme fcy/ml) 0.58 0.89 47 0.40 0.26 14 Alpha-1 globulin 88.5 24.2 55 88.3 22.4 18 Seromucoid 91.4 24.1 36 99.4 19.1 13 Appendix Table VI. Effect of condition of Rocky Mountain bighorn sheep on f e c a l parasite counts («^ 0.02 eggs/gm). Condition Parasite Healthy Diseased Mean S.D. n Mean S.D. n Protostrongylus sp .1.19a 1.63 46 1.64a 1.73 16 In t e s t i n a l sp.''' 1.26a 1.59 46 1.06a 1.16 16 Coccidia sp. A.08 b 1.72 46 5.32C 2.36 16 Primarily Ostertagia and Nematodirus spp. Means with d i f f e r e n t superscripts are s i g n i f i c a n t l y different (P^=. 0.05) when tested with a Newman-Keul1 s multiple range test. - 88 -Appendix Table V I I . H-Thymidine incorporation i n c o n t r o l unstimulated Rocky Mountain bighorn sheep leukocyte c u l t u r e s (log d i s i n t e g r a t i o n s / 6 ^ min (dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) . Animal No. 3-4 Sampling Period (weeks) 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 6.11 5.62 7.16 6.16 5.64 7.28 5.36 5.99 7.18 6.11 4.14 3.28 5.55 7.47 6.03 3.49 3.38 6.32 6.88 5.55 4.82 3.70 6.14 7.56 6.05 4.50 4.62 5.48 4.72 5.64 4.25 3.74 6.11 . 4.66 5.79 4.15 4.73 7.54 4.81 4.38 5.52 3.84 5.58 8.01 4.06 6.05 4.17 5.26 7.70 4.05 4.50 4.83 7.68 5.51 5.38 5.28 5.12 5.03 4.99 5.33* 5.87* 5.67* 5.12 4.95 5.05 5.68* 4.78* 5.55* 4.90* 5.24* 5.38 5.32 5.21 6.85 6.66 5.15* 6.40 5.48 5.41 5.17 5.43 5.92 5.68 5.40 5.36 5.35 4.21 4.39 4.41 5.13 5.64* 5.61* 5.34* 4.89* 5.12 5.52* 5.46* 5.30* 4.94* 5.23 5.52* 5.37* 5.33* 5.10* 4.10* 6.39* 4.97* 4.15* 4.14* 6.37* 4.91* 4.26* 4.07* 6.18* 5.15* 5.16* — 5.5C> — 5.04* 5.48* — 6.95* — 6.79* — 6.66* — 6.26* 4.65* 6.24* 4.88* 6.40* 5.07* 6.39 6.34 5.82 7.52* 6.86* 4.46* 5.26* 5.58* 7.00* 7.31* 5.00* 4.75* 5.62* 7.54* 7.06* 5.62* 4.85* 6.35* 6.06* 5.73* 5.09* 4.47* 4.58* 5.54* 5.15* A.49* 4.19* 4.98* 4.49* 4.35* 5.98 5.63 5.85 3.70 3.57 3.97 3.90 3.46 5.56 4.16 4.40 4.81 4.66 4.87 7.26 5.21 5.19 5.12 5.21 5.17 5.69 5.72 5.59 5.61 5.70 5,59 5.05 6.63 5.51 5.56 4.63 6.05 2.97 3.21 3.16 4.63 5.08 4.98 4.48 4.76 4.49 4.84 4.50 4.83 5.89 5.68 5.50 4.34 4.78 4.09 5.07* 4.35 5.21* 4.57 5.25* 4.20 4.60 4.47 4.50 5.40 4.29 4.54 3.31 3.49 3.18 3.96 4.16 4.44 4.92* 5.17* 5.35* 5.05 5.90 5.42 5.51 5.53 5.74 5.86 5.16 5.00 5.00 5.05* 5.08* 5.23* 3.88* 3.96* Data from diseased animals. Died i n week 25. Died i n week 40. Died i n week 32. A d d i t i o n a l Data: a b Animal No.1 p r i o r to death i n week 25: 4.92,4.94,4.86 log dpm/l0 6 c e l l s . Animal No.3 p r i o r to death i n week 40: 5.16,4.84,4.83 logedpm/10 c e l l s . Animal No.5 i n week 4: 5.02,5.13,5.86 l o g dpm/106 c e l l s . A p p e n d i x T a b l e VIII. H - T h y m i d i n e i n c o r p o r a t i o n l n p h y t o h e m a g g l u t i n i n (PIIA) s t i m u l a t e d R o c k y M o u n t a i n b i g h o r n sheep l e u k o c y t e c u l t u r e s ( l o g d i s -g e i n t e £ r a t i o n s / m i n ( d p m ) / 1 0 c e l l s ) d e t e r m i n e d by the method o f C a s p a r y and Hughes ( 1 9 7 2 ) . R e s p o n s e s o f i n d i v i d u a l a n i m a l s ( 1 - 9 ) a t e a c h s a m p l i n g p e r i o d (weeks a f t e r s t a r t ) . A n i m a l N o . 3-4 5-6 8-9 S a m p l i n g P e r i o d (weeks) 14-15 17 20-21 23 24-26 29-30 32-34 38 9 . 2 7 8 . 0 4 9 . 2 2 6 . 0 1 * - - - - -9 . 8 5 8 . 7 6 8 . 9 5 6 . 0 3 * 9 . 9 6 7 . 8 8 9 . 1 7 6 . 1 0 * - - - - -9 . 6 3 1 0 . 7 9 6 . 1 9 6 . 2 7 6 . 4 4 6 . 9 7 8 . 1 0 9 . 7 1 1 0 . 9 3 6 .27 6 . 2 2 6 . 0 5 7 .27 8 . 8 8 9 . 7 6 1 1 . 0 6 6 . 2 7 5 . 9 1 5 . 8 9 7 .27 8 . 8 2 7 . 9 6 8 . 1 8 6 . 6 8 * 8 . 0 4 * 6 . 6 9 5 . 7 8 6 .17 8 . 3 2 8 . 6 0 6 . 4 0 * 8 . 4 0 * 6 . 3 9 5 . 6 0 6 . 1 5 9 . 0 3 6 . 8 4 * 8 . 0 3 * 6 . 3 1 5 . 8 0 6 . 2 9 9 . 7 6 6 . 6 9 7 . 1 1 6 . 8 7 * 6 . 7 2 * 5 . 7 6 * 6 . 3 8 * 9 . 5 1 1 1 . 0 7 7 . 2 2 7 . 1 6 * 6 . 2 4 * 6 . 0 4 * 6 . 3 9 * 9 . 9 8 1 1 . 2 9 7 . 2 3 7 . 2 8 * 6 . 6 3 * 6 . 0 0 * 6 . 6 9 * 8 . 9 2 7 . 6 8 5 . 6 9 7 . 3 2 8 . 2 7 8 . 5 1 8 . 9 3 9 . 0 9 8 . 4 3 6 . 3 0 5 . 0 0 6 . 8 8 5 . 0 6 8 . 0 1 5 . 3 9 6 . 9 9 7 . 2 0 7 . 0 3 7 . 2 2 6 . 2 3 6 . 0 6 5 . 9 0 6 . 0 0 5 . 5 5 5 . 1 6 4 . 6 7 1 0 . 2 2 * 6 . 5 4 * 9 . 7 9 * 8 . 8 6 * 6 . 2 9 * 7 . 7 1 * 6 . 4 3 * 6 . 8 6 * 5 . 9 6 * 1 0 . 1 9 * 9 . 8 0 * 5 . 0 8 * 8 . 5 7 * 6 . 7 8 * 7 . 2 4 * 7 . 6 5 * 7 . 0 7 * 6 . 2 9 * 8 . 8 9 * 8 . 3 2 * 6 . 8 9 * 7 . 6 3 * 7 . 7 4 * 6 . 7 7 * 5 . 5 2 * 1 0 . 2 2 6 . 8 9 * 1 0 . 0 5 * 7 . 6 5 * 9 . 3 9 * 1 0 . 9 0 * 7 . 3 7 * 6 . 3 7 * 5 . 7 2 * 5 . 8 5 * 1 0 . 8 5 7 . 2 4 * 9 . 9 3 * 6 . 5 4 * 9 . 2 8 * 1 0 . 5 4 * 7 . 3 4 * 5 . 9 0 * 6 . 1 5 * 5 . 9 7 * 1 1 . 1 9 7 . 6 1 * 1 0 . 0 1 * 6 . r . 6 * 9 . 3 7 * 1 0 . 8 7 * 7 . 7 8 * 6 . 1 6 * 6 . 0 0 * 6 . 5 5 * 8 . 4 0 8 . 3 5 8 . 8 6 5 . 6 1 6 . 3 6 5 . 7 4 5 . 2 3 5 . 7 7 3 . 6 0 6 . 2 7 6 . 9 5 — 6 . 1 1 6 . 6 2 7 .07 7 . 1 1 8 . 4 9 8 . 5 8 8 . 7 0 7 . 3 2 7 . 4 4 8 . 4 9 6 . 8 8 6 .94 6 . 8 3 7 . 0 2 7 . 0 2 7 . 0 9 7 . 7 9 9 . 5 4 8 . 6 4 7 . 8 3 7 . 1 6 6 . 1 5 7 . 6 6 7 . 8 6 4 . 7 8 8 . 2 8 7 .97 7 . 8 0 5 . 5 0 6 . 2 9 6 . 5 2 8 . 3 9 8 . 3 1 8 . 0 5 9 . 5 8 9 . 7 2 9 . 7 6 5 . 5 7 5 . 6 5 5 . 3 8 6 . 3 0 * 6 . 0 8 * 6 . 1 7 * 5 . 1 3 5 . 3 6 5 . 5 2 6 . 3 6 6 . 5 0 6 . 5 5 7 . 1 8 1 1 . 1 1 1 1 . 1 3 5 . 3 5 5 . 6 2 5 . 4 1 7 . 5 6 8 . 0 7 7 .67 6 . 7 1 * 8 . 8 1 6 . 7 6 * 9 . 3 2 7 . 1 4 * 1 0 . 1 4 9 . 3 3 9 . 6 9 9 . 9 2 9 . 5 8 9 . 6 3 9 . 3 9 6 . 1 9 6 . 4 0 5 . 8 5 5 . 2 2 * 6 . 7 4 * 5 . 2 5 * 6 . 9 2 * 5 . 0 1 * 6 . 7 3 * Data f r o m d i s e a s e d a n i m a l s . a D i e d i n week 2 5 . b D i e d i n week 4 0 . c D i e d i n week 3 2 . A d d i t i o n a l d a t a : A n i m a l N o . l p r i o r t o d e a t h i n week 2 5 : 4 . 9 8 , 4 . 7 4 , 5 . 0 5 l o g d p m / 1 0 ° c e l l A n i m a l N o . 3 p r i o r t o d e a t h i n week 4 0 : 7 . 3 1 , 7 . 3 7 , 7 . 5 9 l o g c d p m / 1 0 c e l l A n i m a l N o . 5 i n week 4 : 1 0 . 3 7 , 1 0 . 1 3 , 8 . 7 4 l o g d p m / 1 0 6 c e l l s ? - 90 -Appendix Table IX. ""H-Thymidine incorporation i n pokeweed mitogen stimulated Rocky Mountain bighorn sheep leukocyte cultures (log di s i n t e g r a t i o n s / 6 ^ min(dpm)/10 c e l l s ) determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 8.24 7.66 7.08 7.16 5.22* 7.99 8.14 7.82 7.45 5.06* 8.63 7.91 6.92 7.41 5.47* 2 9.03 3.72 7.32 9.40 10.33 5.78 6.36 6.14 6.00 6.97 8.72 5.14 7.84 10.47 9.84 5.44 6.40 6.21 6.07 6.55 8.78 8.13 9.08 9.82 5.40 5.94 6.35 6.07 8.33 3 b 9.46 5.58 5.81 6.81 5.75 6.17* 6.45* 6.54 5.74 4.62 9.03 6.26 5.91 7.49 6.45 6.17* 6.61* 6.91 5.89 4.68 9.26 6.51 5.77 6.54 6.03* 6.55* 6.80 5.79 4.73 4 9.37 8.32 5.33 8.40 ; 9.99 5.55 7.02* 6.12* 5.71* 6.01* 8.85 9.41 5.34 8.69 9.74 5.85 6.60* 6.14* 5.22* 6.22* 9.54 7.03 4.24 8.63 9.49 5.59 6.61* 5.75* 5.34* 6.04* 5 C 9.50* 4.95* 8.83* 8.11* 5.23* 6.76* 7.60* 5.87* 5.44* 10.17* 5.45* 8.84* 6.77* 5.14* 6.48* 7.33* 5.86* 5,?4* 8.17* 7.72* 5.37* 6.63* 7.59* 5.77* 5.31* 6 10.79 7.31* 9.43* 5.18* 6.96* 8.48* 6.76* 5.98* 4.64* 5.19* 10.93 7.05* 9.15* 6.27* 6.38* 8.36* 6.82* 5.47* 4.46* 5.06* 10.88 6.62* 8.38* 5.70* 6.30* 8.56* 6.50* 5.55* 5.10* 5.27* 7 8.63 5.88 3.71 5.88 5.27 7.05 6.35 5.89 6.29 8.60 5.65 3.84 6.09 5.10 7.05 6.24 4.86 6.20 8.62 5.31 3.71 6.13 5.25 7.03 6.01 6.13 6.34 8 9.57 7.74 5.02 6.36 5.68 6.04 7.42 5.60 5.81* 4.56 5.68 8.05 7.95 3.64 7.15 4.89 5.92 6.34 5.21 5.91* 4.18 5.30 7.72 8.21 2.93 6.70 5.85 5.92 5.40 5.91* 4.60 5.58 9 ' 10.11 4.00 5.82 6.36 2.48* 8.31 6.04 5.54 4.85* 5.61* 6.08 4.43 6.80 5.74 2.87* 6.47 7.34 5.23 5.07* 6.02* 8.81 4.66 6.40 5.83 2.70* 6.71 7.51 5.39 4.93* 5.55* * Data from diseased animals. a b Died Died i n week in week 25. 40. c Died i n week 32. » g Additional Data: Animal No.1 prio r to death i n week 25: 5.18,5.30,5.14 log edpm/10 6 c e l l s Animal No.3 p r i o r to death i n week 40: 6.94,6.78,6.97 logedpm/10 c e l l s Animal No.5 i n week 4: 8.12,9.63,9.30 log dpm/106 c e l l s . - 91 -Appendix Table X. H-Thymidine in c o r p o r a t i o n concanavalin A (ConA) stimulated Rocky Mountain bighorn sheep leukocyte c u l t u r e s (log d i s i n t e g r a t i o n s / min(dpm)/lO c e l l s ) . determined by the method of Caspary and Hughes (1972). Responses of i n d i v i d u a l animals (1-9) at each sampling period (weeks a f t e r s t a r t ) . Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 6.36 6.26 8.27 6.28 6.53 8.29 6.78 7.33 7.95 5.12* 5.47* 5.42* 4.99 7.22 7.04 8.98 9.32 9.16 10.05 9.85 10.00 5.16 5.34 5.29 5.16 5.21 5.14 5.38 5.78 5.66 5.57 5.57 5.38 6.10 6.15 5.50 6.73 6.01 6.45 7.11 5.91 5.84 6.68 6.10 6.51 5.73* 6.26* 6.78 5.73* 6.19* 5.48* 6.43* 6.40 6.71 5.48 5.69 5.60 4.48 4.58 4.57 10.63 6.35 6.83 9.76 5.34 6.02* 6.04* 5.50* 5.36* 10.59 6.96 6.58 — 10.05 5.24 6.16* 5.60* 5.09* 5.49* 10.59 5.78 6.77 9.10 5.15 6.01* 5.70* 5.06* 5.37* 5.64* 8.12* 5.24* 8.56* 5.25* 5.06* 5.03* 5.15* 8.30* 5.15* 5.x3* 6.63* 5.88* 5.94* 5.16* 6.69* 5.85* 6.12* 5.44* 6.87* 5.78* 5.88* 5.32* — 6.68* 8.59* — 6.48* 8.H3* - - 6.88* 8.75* 5.85* 6.43* 9.20* 6.03* 5.31* 4.47* 4.86* 6.10* 6.60* 9.27* 6.17* 5.20* 4.59* 4.33* 6.23* 9.21* 5.95* 5.25* 4.47* 4.53* 4.47 3.44 4.51 5.75 5.77 5.69 4.55 5.04 4.51 6.45 6.56 6.64 5.48 5.28 5.32 5.68 5.49 5.47 5.12 5.22 5.31 8.88 8.12 7.84 3.85 4.15 4.87 5.97 6.24 5.15 5.89 6.20 5.39 5.98 5.04 5.12 4.96 5.88* 5.40* 5.77* 4.40 4.26 4.33 4.90 4.79 4.83 5.94 6.03 5.71 — 5.52 — 5.58 — 5.59 5.38* 3.33 5.64* 3.37 5.33* 3.46 6.47 6.69 5.95 7.44 7.31 7.73 5.24 5.01 5.09 4.69* 4.25* 5.10* 4.05* 4.90* 4.06* Data from diseased animals A d d i t i o n a l Data: a Died i n week 25. b Died i n week 40. c Died i n week 32. g Animal No.1 p r i o r to death i n week 25: 4.77,4.74,4.92 log edpm /10 6 c e l l s Animal No.3 p r i o r to death i n week 40: 5.86,5.68,5.69 log dpm/10 c e l l s - 31 -Appendix Table XI. JH-Thymidine incorporation in endotoxin (Endo) stimulated Rocky Mountain bighorn sheep leukocyte cultures (log disintegrations/min (dpm)/10 cells) determined by the method of Caspary and Hughes (1972). Responses of individual animals (1-9) at each sampling period (weeks after start). Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 I 3 5.83 6.72 6.35 5.50* — 5.76 8.22 6.02 5.69* 5.26 7.53 6.09 5.84* 2 4,52 5.96 7.13 5.02 5.31 5.92 4.83 6.30 3.57 6.20 7.00 5.17 5.23 5.82 5.14 6.14 — — — — . — 4.01 6.13 7.46 5.34 5.07 5.88 5.49 6.41 3 b 4.37 5.45 4.46 4.76* 5.59* 6.58 5.40 5.23 4.19 5.66 5.46 4.32* 5.79* 6.58 5.18 5.24 — 4.16 5.48 4.77 4.85* 5.76* 6.27 5.59 4.39 4 10.18 4.06 5.58 7.85 5.45 5.76* 5.57* 4.55* 4.86* • 10.01 4.77 5.67 8.33 5.49 6.04* 5.60* 4.94* 5.03* 10.29 4.26 5.41 7.83 5.85 5.99* 5.61* 4.69* 4.90* 5 C 5.49* 5.84* 4.78* 5.05* 6.56* 6.09* 5.19* 5.68* 4.16* 4.98* 5.03* 6.35* 5.82* 5.12* 4.41* 4.67* 5.1b* 6.48* 5.98* 4.56* 6 6.43* 6.27* 7.02* 5.02* 6.35* 5.67* 5.44* 4.47* 5.53* 6.95* 5.18* 5.74* 5.18* 6.81* 5.75* 5.46* 4.32* 5.62* • 6.93* 6.01*. 6.47* 4.98* 6.24* 5.67* 5.62* 4.53* 5.63* 7 6.58 4.43 5.05 5.27 4.95 5.66 5.63 3.70 4.11 5.03 5.36 5.40 5.25 5.70 3.62 4.54 4.75 4.95 5.02 4.30 5.48 8 5.02 3.37 4.63 4.90 4.37 4.87 5.47* 4.14 4.65 • 8.78 3.28 4.83 5.01 4.79 4.83 5.52* 4.09 4.96 8.64 3.34 4.09 6.69 4.18 4.78 5.51* 4.54 4.91 9 6.00 4.88 5.27* 5.12 5.73 5.59 5.10 5.00* 4.47* 5.88 4.68 5.52* 5.23 5.66 5.67 4.92 5.01* 4.76* . 5.89 4.66 5.44* 5.63 5.74 5.27 4.94* 4.30* Data from diseased animals. a Died in week 25. b Died in week 40. c Died in week 32. Additional Data; Animal No.l prior to death in week 25: 4.95,4.78,4.66 log dpm/10^  cells Animal No.3 prior to death in week 40: 4.80,5.04,4.83 logcdpm/10 cells - 93 -Appendix Table XII. Relative serum albumin concentration (% of control serum) of ind i v i d u a l animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 100.0 90.6 102.1 104.3 101.0 97.9* 2 102.1 105.4 101.0 100.0 104.3 99.0 96.9 99.0 102.2 100.0 3 b 101.1 97.9 99.0 101.1 103.3 95.8* 96.9* 95.8 100.0 100.0 4 101.1 — 99.0 102.1 104.3 100.0 99.0* 99.0* 100.0* 100.0* 5° 101.1* 98.9* 92.7* 94.7* 98.9* 93.6* 95.7* 91.7* 94.8* 92.4* 101.1 104.3 101.0* 98.9* 100.0* 97.9* 100.0* 100.0* 100.0* 103.2 101.1 101.1 100.0 100.0 97.9 100.0 95.8 92.6 103.2 103.2 101.1 JOO.O 103.3 98.9 96.9 100.0* 100.0 101.1 102.2 96.9 103.2 101.1 103.3* 102.2 100.0 99.0 -. 98.9* 100.0* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c Died i n week 32. - 94 -Appendix Table XIII. Relative serum beta-2 globulin (transferrin) concentration (% of control serum) of individual animals (1-9) at each sampling period (weeks, after s t a r t ) . Animal No. Sampling Period (weeks) 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 100.0 58.7 100.0 228.6 75.4 104.2* 2 115.4 125.0 93.5 87.2 131.4 71.9 75.0 90.0 110.3 95.5 3 b 120.5 113.0 93.5 100.0 140.0 64.9* 79.2* 100.0 102.3 93.6 4 189.7 91.3 85.1 128.6 71.9 70.8* 92.0* 82.1* 89.4* 5° 135.9* 159.1* 147.8* 150.0* 140.5* 182.6* 171.7* 114.6* 130.0* 89.7* 6 125.6 131.8 95.7* 78.6* 89.1* 66.7* 96.0* 94.9* 93.6* 7 95.5 87.2 116.7 "3.7 91.3 83.3 109.1 76.6 74.5 8 111.4 163.8 107.1 75.4 145.7 73.9 81.3 109.1* 100.0 93.6 9 97.7 73.9 110.6 116.7 131.4* 131.4 87.0 84.0 93.2* 89.4* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c Died i n week 32. - 95 -Appendix Table XIV. R e p l i c a t e a n a l y s e s of b l o o d urea n i t r o g e n (BUN) c o n c e n t r a t i o n (mg/100 ml) determined by the method of Chaney and Marbach (1962) of i n d i v i d u a l a n imals ( 1 - 9 ) , a t each sampling p e r i o d (weeks a f t e r s t a r t ) throughout the study p e r i o d . Sampling P e r i o d s (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 43.19 27.43 27.45 — 31.70 15.0C 41.64 30.00 27.87 34.04 14.57 2 32.33 22.72 17.87 18.80 16.90 17.25 25.88 21.35 30.19 33.88 20.96 17.87 18.98 17.28 18.00 24.75 19.23 29.05 3 b 32.53 33.84 16.93 24.04 24.87 26.06* 16.50* 26.06 30.00 30.57 35.69 36.28 16.50 25.37 26.58 26.81* 17.25* 27.31 30.38 31.14 4 41.38 32.09 22.72 ~ 18.80 26.81 24.00* 22.59* 17.09* 30.76* 40.09 32.09 21.18 17.47 27.94 26.25* 24.68* 16.71* 27.34* 28.60 33.62* 43.61* 30.64* 51.84* 59.36* 44.08* 67.89* 68.81* 73.08* 94.75* 30.52* 41.86* 28.29* 50.74* 59.57* 47.28* 66.00* 68.81* 69.23* 94.18* 28.45 19.15* — 30.19* 16.54* 20.39* 23.17* 24.68* 29.48 20.43* 27.75* 15.00* 20.96* 24.68* 24.68* 33.36 28.68 26.17 16.90 34.50 23.46 24.68 28.67 33.61 27.13 27.45 16.33 38.62 25.00 22.79 29.24 36.28 20.85 27.45 21.21 22.31 22.50 23.08* 26.77 24.30 36.98 19.57 25.53 21.43 20.81 24.40 21.15* 27.72 25.25 48.84 35.74 34.19 30.21 21.70* 25.71 29.81 27.94 26.20* 25.25* 54.07 36.40 35.07 30.43 21.49* 25.29 30.29 26.25 26.20 25.06 * Data from a d i s e a s e d a n i m a l , a Died i n week 25. b Died i n week 40. c Died i n week 32. A d d i t i o n a l d a t a f o r Animal No. 3 p r i o r to death i n week 40: 104.81, 117.34 mg/100 ml. - 96 -Appendix Table XV. Serum lysozyme concentration (ug/rol) determined by the method of Litwack (1955) of individual animals (1-9) at each sampling period (weeks after start) throughout the study period. Animal No. I 3 0.66 0.30 0.64 5 C 0.74* 1.14* 0.34* 0.81* 0.41* 0.32* 1.38* 0.70* 0.78* 1.54* Sampling Period (weeks) 3 - 4 ^ 5 - 6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 0 . 0 4 2 0.12 0.16 0.10 0.65 0.04 0.40 0.15 0.12 0.08 0.06 3 b 1.44 0.72 0.98 0.08 0.12 0.70* 0.08 0.35 0.38 0.94 _ 0.71 1.07 6.60 — 0.17 0.80 0.08* 0.70* 0.92* 0.50* 0.75 0.42 0.60* 0.12* 0.10* 0.50* 1.17* 0.20* 0.40* 0.70 1.15 0.30 0.49 0.38 0.65 0 < 9 8 o.l2 0.60 1.88 1.46 0.59 0.61 0.72 0.32 0.60 1.18 1.33* 0.50 1.09* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c Died i n week 32. Additional data for Animal No.3 in week 40: 0.38 ug/ml. Appendix Table XVI. Relative serum alpha-1 globulin concentration (% of control serum) of individual animals (1-9) at each sampling period (weeks af t e r s t a r t ) . Sampling Period (weeks) Animal No. — — 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 100.0 45.0 94.7 35.8 90.5 140.0* 2 117.6 100.0 95.0 78.9 : 35.8 71.4 75.0 85.7 87.0 87.0 3 b 117.6 95.0 105.0 89.5 39.6 76.2* 80.0* 95.2 104.3 95.2 4 123.5 80.0 78.9 34.0 71.4 80.0* 109.5* 73.9* 90.5* 5 C 147.1* 121.1* 95.0* 131.3* 168.8* 115.0* 130.0* 100.0* 119.0* 73.9* 6 111.8 105.3 80.0* 93.8* 90.0* 60.0* 95.2* 82.6* 104.8* 7 121.1 94.7 106.3 114.3 95.0 85.0 104.3 123.8 138.1 8 100.0 84.2 106.3 85.7 37.7 85.0 85.0 95.7* 91.3 109.5 9 84.2 70.0 94.7 100.0 32.1* 34.0 85.0 85.7 100.0* 104.8* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c Died i n week 32. - 98 -Appendix Table XVII. Relative serum seromucoid concentration (% of control serum) determined by the method of Pryce (1967) of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. — — — — — 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 93.3 63.8 .89.0 2 86.7 80.0 62.5 80.0 79.5 100.0 85.0 72.1 . 83.3 3 b 106.7 68.8 84.0 100.0* 110.3 200.0 96.0 61.3 80.0 83.6 100.0* 140.0* 117.6* 81.7* 5 C 140.0* 130.7* 60.0* 130.7* 164.4* 188.3* 194.1* 161.8* 6 65.0* 108.2* 86.7* 89.7* 91.2* 91.7* 7 80.0 93 3 125.0 115.0 J J . J 8 86.7 75.0 — ' 103.3 103.3 101.7 . 9 80.0 109.3 101.2 69.3 100.0 80.8 100.0* 120.0* * Data from diseased animals, a Died in week 25. b Died i n week 40. c Died i n week 32. - 99 -e XVIII. Relative serum alpha-2 globulin concentration (% of c of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. :  3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 100.0 42.9 93.3 — — 100.0 193.8* 2 114.3 138.5 114.3 80.0 78.6 75.0 — 3 b 135.7 7.1 100.0 100.0 — 85.7* 68.8* 100.0 4 121.4 107.1 73.3 — 92.9 93.8* 100.0* 5° 171.4* 146.2* 85.7* 133.3* 191.7* 128.6* 150.0* 100.0* 6 7.1 7.7 7.1* 141.7* 114.3* 62.5* 125.0* 7 123.1 — — 80.0 108.3 l i s . 3 100.0 87.5 118.8 62.5 8 138.5 93.3 125.0 107.1 107.1 93.8 — 112.5 9 130.8 71.4 93.3 8.3 100.0 6.3* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c Died i n week 32. - 100 -Appendix Table XIX. Relative serum beta-1 globulin concentration (% of control serum) of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. Anxmaj. ao. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 I 8 100.0 45.5 90.9 96.0 84.8 112.9* 2 69.2 60.0 84.8 60.6 124.0 75.8 83.9 85.7 96.7 87.1 3 b 126.9 93.9 93.9 90.9 140.0 78.8* 83.9* 88.6 103.2 93.9 4 111.5 84.8 81.8 140.0 81.8 77.4* 102.9* 93.3* 87.9* 5 C 150.0* 113.3* 84.8* 96.7* 113.3* 109.7* 116.1* 80.6* 94.3* 76.7* 6 142.3 136.7 97.0* 76.7* 103.2* 77.4* 102.9* 96.7* 103.0* 7 106.7 57.6 103.3 93.9 93.5 96.8 119.4 103.0 72.7 8 76.7 34.6 45.5 106.7 93.9 88.0 83.9 90.3 106.5* 100.0 90.9 9 106.7 78.8 103.0 110.0 140.0* 136.0 71.0 100.0 119.4* 97.0* * Data from a diseased animal, a Died i n week 25. b Died i n week 40. c • Died i n week 32. - 101 -Appendix Table XX. Fecal lungworm counts ( -\/o.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 0* 2.00* 29-30 32-34 38 0 0 0* 0* 0 0 0 0 0 0 0 0 0 0 0 0 2.45 4.24 1.41 2.00 0 0 1.41 2.00 9.38 7.48 5.48* 4.90* 0* 0* 0* 0* 0* 0* 3.74* 1.41* 0* 2.00* 0* 1.41* 0* 2.00* 6:93* 4.90* 8.94* 7.07* 0* 1.41* 6.00* 7.35* 9.27* 7.75* 0* 0* a b c 0 0* 0 0* 0 1.41 2.00 2.00 2.00 2.00 Data from a diseased animal. Died i n week 25. Died in week 40.• Died i n week 32. 0* 0* 2.00 2.45 0 0 4.00* 4.24 5.10* 4.24 1.41 2.00 3.16 2.45 3.16 3.46 2.23* 3.74* 1.41* 2.23* 3.16* 0* - — 2.00 1.41 0 0 4.24 4.69 2.00 0 1.41 0 4.00 4.69 5.48 4.24 0 0 0 0 - 1.41* - 1.41* 0 0 1.41 0 3.46* 2.83* 4.47& 4.47* - 102 -Appendix Table XXI. Fecal Intestinal nematode counts ( ~\/ 0.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. 3-4 5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 l a 0 1.41 0 2.83 2.45 2.45 1.41 1.41 0 1.41 0 0 2 5.66 6.48 1.41 2.45 2.45 2.00 4.47 3.16 2.45 3.46 0 2.00 1.41 2.00 0 0 1.41 1.41 3 b 7.48 6.32 3.46 4.24 4.24 4.90 2.83 2.00 . 3.46 1.41 3.16* 2.45* 0* 1.41* 2.00 2.83 1.41 1.41 0 0 4 2.00 2.00 0 1.41 1.41 0 2.83 0 1.41 0 0* 0* 1.41* 1.41* 0* 1.41* 0* 0* 5 C 3.16* 3.16* 5.83* 4.47* 3.74* 2.83* 8.94* 6.32* 3.16* 1.41* 3.74* 4.24* 4.00* 4.47* 4.47* 4.00* 1.41* 2.00* 4.24* 3.46* 6 1.41 0 2.00* 2.45* 3.32* 4.69* . ' 2.00* 2.45* 1.41* 4.00* 1.41* 1.41* 1.41* 0* 2.83* 0* 7 o o 2.00 2.00 1.41 2.00 0 0 0 0 0 n u 8 n 1.41 n 3.74 3.16 0 0 u o V 0 U 0 0 0 0 0 \j 9 2.00 3.46 0 0 1.41* 0* 0 0 0 0 0 0 0 0 1.41* 0* 1.41* 0* * Data from a diseased animal, a Died i n week 25. b Died in week 40. c Died i n week 32. - 103 -Appendix Table XXII. Fecal coccidia counts ( ~\/o.02 eggs/gm) determined by a sugar f l o t a t i o n technique (Bodie, 1969) of individual animals (1-9) at each sampling period (weeks after s t a r t ) . Sampling Period (weeks) Animal No. : '• ——— 3-4 .5-6 8-9 14-15 17 20-21 23 24-26 29-30 32-34 38 I 8 2.0 2.83 3.74 4.90 2.83 3.16 0 2.00 3.46 3.74 2.45 2.83 2 6.93 3.16 6.00 3.74 6.48 2.45 6.32 2.83 3.16 6.16 3.16 5.83 3.46 8.12 2.83 5.29 4.90 1.41 3 b 4.00 2.83 5.29 5.66 3.16 4.24* 5.29* 2:83 2.45 4.47 6.16 4.47 6.78 5.10 . 2.83 4.00 5.83* 4.47 1.41 4.00 4 3.16 2.83 0 6.48 7.35 3.16* 8.37* 3.74*" 5.66* 4.00 4.00 2.45 4.47 5.83 4.69* 11.22* 3.46* 7.87* 5° 7.75* 4.00* 5.83* 8.94* 8.25* 2.45* 10.39* 21.26* 23.75* 14.56* 5.29* 5.29* 5.66* 4.47* 5.48* 3.74* 8.25*23.32* 22.98* 16.43* — 3.46 4.00* 10.10* • 4.69* 4.24* 1.41* 2.83* 4.47* 4.47 4.24* 8.12* 3.46* 5.66* 3."4* 2.83* 2.00* 0 2.83 2.45 2.00 2.45 2.83 2.00 3.46 2.45 2.83 2.00 2.83 2.83 3.16 3.16 3.16 2.45 6.16 5.48 3.46 — 3.46 2.00 2.00 3.74 2.45 6.32 6.32 3.16 5.10 3.74 5.10* 5.83 8.25 7.35 3.16 4.47* 6.78* 4.24 4.69 6.63* 7..07 8.12 7.75 3.16 4.47* 6.93* * Data from a diseased animal, a Died in week 25. b Died i n week 40. c Died i n week 32. 

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