‘Msu LIBRARIES “— RETURNING MATERIALS: Place in book drop to remove this checkout from your record. .[lfl§§ will be charged if book is returned after the date stamped below. INTERACTIONS OF COLD STRESS AND PASTEURELLA HAEMOLYTICA IN THE PATHOGENESIS OF PNEUMONIC PASTEURELLOSIS OF CALVES By Ronald F. Slocombe A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1982 ABSTRACT INTERACTIONS OF COLD STRESS AND PASTEURELLA HAEMOLYTICA IN THE PATHOGENESIS OF PNEUMONIC PASTEURELLOSIS OF CALVES By Ronald F. Slocombe The objectives of this research were to identify factors of probable significance in the pathogenesis of pneumonic pasteurellosis caused by Pasteurella haemolytica in calves. Fifteen healthy calves were chilled with cold water and had a focal tracheitis induced by spraying the tracheal mucosa with a 5% acetic acid solution. Blood samples and pulmonary function tests were taken before the first stress, immediately following a second chilling and then for 12 hours subsequent to intratracheal injection with saline (control group n = 6) or saline con- taining 2 x 109 live 3. haemolytica organisms (Pasteurella group n = 7). All calves were examined grossly and histologically (including 2 calves exposed to E. haemolytica but not tested for pulmonary function). Control calves had focal areas of atelectasis and a few scattered inflammatory cell infiltrations in the lungs. These did not cause any alterations in pulmonary function tests nor in leukograms or hemograms. Cold stress in both groups of calves was associated with increased serum cortisol, 02 consumption, C02 production, tidal volume, and alveolar ventilation. These alterations may facilitate pneumonia by steroid induced immunosuppression and increased exposure of alveolar surfaces to inhaled pathogens. Ronald Francis Slocombe Alterations in pulmonary function occurred within 1 hr of Pasteurella exposure. Increases in minute ventilation were associated solely with increased dead space ventilation and by 3 hrs post inocula- tion decreases in dynamic compliance accompanied gas exchange impair- ment. The mechanism for these early changes in breathing pattern and gas exchange function remains uncertain but probably involves stimula- tion of intrapulmonary J receptors and local disturbances of ventilation-perfusion matching within damaged portions of lung. The data clearly indicate that the initial injury was in the lung parenchyma not in the bronchial tree. By 12 hours post inoculation calves had extensive pneumonia, with alveolar wall necrosis with edema and hemorrhage and accumulations of inflammatory cells occurring as apparently 2 independent but concurrent processes. Calves were also neutropenic and had increased serum cortisol levels. The data indicate that mast cell mediators and bradykinin do not contribute to pulmonary injury and suggests that pulmonary injury is most likely due to the com- bined effects of bacterial and inflammatory cell products. ACKNOWLEDGEMENTS My appreciation is extended to the members of my committee for their encouragement, support and advice on the many diverse aspects of this research project. Drs. Trapp and Langham provided the expertise necessary in the area of histOpathology, Dr. Robinson served in a major capacity in the develOpment of overall experimental design and in pulmo- nary function testing, Dr. Echt was an invaluable consultant on pulmo- nary microanatomy and Dr. Hhitehair provided a vital input in the areas of general pathology and in the overall implementation of the project. Special thanks to Dr. Mather, Large Animal Clinical Sciences Department; Dr. Krehbiel, Pathology Department and Dr. Keahey, Animal Health Diagnostic Laboratory for their support of this research. To my colleague, Dr. Derksen, for his invaluable assistance in data collection, I extend my sincere thanks. I extend my appreciation to Robert Ingersoll, Roberta Milar, Dr. Esther Roege, Mae Sunderlin and Frances Whipple for their skilled technical assistance. Lastly, to my wife, Judy, and children. Their support for my involvement in this research project greatly facilitated its completion and helped make it an enjoyable experience. ii TABLE OF CONTENTS Page LIST OF TABLESOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO V LIST OF FIGURES.0.0.00.0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... v1. I. CHAPTER 1: A review of factors of possible significance in the pathogenesis of initial and develOped lesions of Pasteurella haemolytica pneumonia...................... 1 A redirection in bovine reSpiratory disease complex research............................................ 2 Pneumonic pasteurellosis............................... 4 Defense mechanisms against pulmonary pasteurellosis.... 5 Stress as a predisposing factor of pneumonic pasteurellosis...................................... 7 Normal respiratory structure and function and its reSponse to injury.................................. 8 Host mediated responses of possible importance in the pathogenesis of P. haemolytica pneumonia............ 12 Experimental rational.................................. 24 References............................................. 27 II. CHAPTER 2: Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: method of induction and changes in the cirulating blood constituents during the onset of pulmonary injury.......................... 48 Summary................................................ 49 Introduction........................................... 50 Materials and methods.................................. 51 Results................................................ 57 Discussion............................................. 78 References............................................. 87 TABLE OF CONTENTS--continued III. IV. V. VI. VII. CHAPTER 3: Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: Changes in pulmonary function with cold stress and during the development of pasteurEIIOSiSOOOO000.000.000.00...OOOOOOOOOOOOOOOOOCO. Summary................................................ Introduction........................................... HethOdSCOOOOOOOOOOOOOOOOOOOOOOOOOOOO00.000000000000000. Results................................................ Discussion............................................. REferenceSOO0.00000000000000.0.0.0....0.00.00.00.00.0.. CHAPTER 4: Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: gross and microsc0pic IeSionSOOOOOOOOOOOOOOO0..0..OCOOOOOOOOOOOOOOOCOOOOO0.0. Summary................................................ Introduction........................................... Methods................................................ Results................................................ Discussion............................................. ReferenceSOOOOOOOOOOOOOO0.0.0.000...OOOOOOOOOOOOOO0.0.. CHAPTER 5: COnCIUSionSOOOOOOIOCOOOOOOOOOOOOOOOOOOOOOOOOOOO EffeCtS 0f COId StrESSOOO0.0000000000IO0.0.0.0.0000...O Effects Of Pasteurella haemOIyticaOOOOOO0.00.0000....0. APPENDICES A. Equations used for calculation of gas exchange variableSOOOOOOOOOOOOOOO0.0.0.0.0000000000000000000 8. Method of fixation of lung samples for histologic StUdieSOOOOOOOOOOOO...0.0I.OOOOOOOOOOOOOOOOOOOOOOOO VITAOOCOOOOOO0.0...0....0.000COOOOOOOOOOOOOOOOOOOO000...... iv Page 93 94 95 96 99 132 139 143 144 145 148 152 206 215 220 221 223 226 228 231 LIST OF TABLES TABLE Page 4-1. Summary of experimental protocol for control (C) and 149 Pasteurella-exposed (P) calves............................. FIGURE 2'1. 2'2. 2'3. 2‘4. 2‘5. 2‘6. 2'7. 2‘8- 2‘9. 3‘1. LIST OF FIGURES Alterations in blood total white cell count in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure................ Alterations in the blood segmented neutrOphil count in two groups of calves; cold stress alone and cold stress combined with g, haemolytica exposure........ Alterations in plasma total solids in two groups of calves; cold stress alone and cold stress combined with f, haemolytica eXposure........................ Alterations in blood hematocrit in two groups of calves; cold stress alone and cold stress combined with P, haemolytica exposure....................... Alterations in blood erythrocyte count in two groups of calves; cold stress alone and cold stress com- bined with P, haemolytica exposure ................. Alterations in blood hemoglobin content in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure............... Alterations in serum triiodothyronine (T3) in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure............... Alterations in serum thyroxine (T4) in two groups of calves; cold stress alone and cold stress combined With E: haemolytica exposurEOOOOOOOOOOOOOO0000...... Alterations in serum cortisol in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure........................ Alterations in tidal volume in two groups of calves; cold stress alone and cold stress combined with P, haemOIytica exposureOOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOO vi Page 59 61 63 65 67 69 72 74 76 101 LIST OF FIGURES--continued FIGURE 3’2. 3'3. 3-4. 3’5. 3'6. 3'7. 3’8. 3'9. 3'10. 3‘11. 3-120 3’13. Alterations in respiratory rate in two groups of calves; cold stress alone and cold stress combined with_fl. haemolytica exposure........................ Alterations in minute ventilation in two groups of calves; cold stress alone and cold stress combined With -P-O haemOIJtica exposureOOOOOOOOOOOOOO0.0.000... Alterations in alveolar-arterial oxygen difference in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure........ Alterations in arterial oxygen tension in two groups of calves; cold stress alone and cold stress com- bined "it" £0 haemOTJYtica exposurEOOOOOOOOOOOOOOO0.0. Alterations in arterial carbon dioxide tension in two groups of calves; cold stress alone and cold stress combined with P, haemolytica exposure............... The effects of cold stress and intratracheal exposure to saline or P. haemoLytica infected saline on C02 production (VCOz) and oxygen uptake (V02)........... The effects of cold stress and intratracheal exposure to saline or P. haemolytica infected saline on a1ve01ar ventTlationOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. Alterations on dead space ventilation in two groups of calves; cold stress alone and cold stress combined With E. haemOIthica exposureoOOOOOOOOO0000.00.00.00. Alterations in dead space/tidal volume ratio in two groups of calves; cold stress alone and cold stress combined with_P. haemolytica exposure............... Alterations in respiratory exchange ratio in two groups of calves; cold stress alone and cold stress combined with P. haemolytica exposure............... Alterations in dynamic compliance in two groups of calves; cold stress alone and cold stress combined with P. haemoLytica exposure........................ Alterations in total pulmonary resistance in two groups of calves; cold stress alone and cold stress combined with_P. haemolytica exposure............... vii Page 103 105 108 110 112 115 117 119 121 124 126 128 0" LIST OF FIGURES--Continued FIGURE 3.14. 4-1. 4‘2. 4'3. 4'4. 4'5. 4'6 0 4‘7. 4'8. 4-90 4-10. 4-11. 4'12. 4-13. 4-14. Effects of cold stress and following exposure to f, haemolytica on forced oscillating resistance in CdTVESO...’0....00.000.00.000...OCIOOOOOOOOOOO0.0.0.. Schematic diagram of calf lungs illustrating the sampling sites for lung and airway tissue samples taken for histologic evaluation...................... Focal tracheitis resulting from acetic acid exposure. Macrosc0pic appearance of normal calf lungs.......... Macrosc0pic appearance of pneumonic Pasteurellosis in a calfOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Subgross micrograph of lungs affected with pneumonic PaSteurEIIOSTSoooooocan.ooooooooooooooooooooooooooooo Extensive pneumonia and pleuritis in a calf infected With E: haemOIytica for 36 hrSOOOOOO0.00000000000000. Micrograph of the tracheal lesion of a calf follow- ing acetic acid Spray into the tracheal lumen........ Micrograph of lung tissue from a control calf illus- trating focal areas of atelectasis associated with bronChia] ObStrUCtionoooooooooooo0.000000000000000... Effect of lung fixation by perfusion of airways with fixative under pressure on normal calf lungs......... Macrograph of normal calf lungs illustrating “edema“ artifact resulting from airway pressure-perfusion With fixativeOOOOOOOOOO0......OOOOOOOOOOOOOOOOOOOOOOO Photomicrograph of a focal lesion in the pulmonary parenchyma of a control calf......................... Photomicrograph of the small intestine of a calf with subacute enteritis associated with CryptOSporidium éEfOOOOOOOOOOOOOOOO0.0.00....OOOOOOOOOOOOOOOOOOOOOOOO Photomicrograph of the histologic lesions of lungs infected with Pasteurella haemolytica................ Photomicrograph of a necrotizing alveolar lesion from a calf infected with Pasteurella haemolytica......... viii Page 131 149 156 158 160 162 165 168 170 172 174 176 179 181 183 LIST OF FIGURES--continued FIGURE 4-15. 4-16. 4-17. 4‘18. 4-19. 4-20. 4-21. 4'22. 4-23. Appendix B Photomicrograph of lymphatic distension with edema associated with pneumonic Pasteurellosis............. Photomicrograph of lobular pneumonia in a calf exposed to Pasteurella haemolytica................... Photomicrograph of well developed pneumonic lesions in a calf inoculated with Pasteurella haemolytica.... Photomicrograph of well develOped pneumonic lesions in a calf inoculated with Pasteurella haemolytica.... Photomicrograph of acute necrotizing lesions in the pulmonary parenchyma of a calf inoculated with PaSteurEIIa haemOIIticaOOO0.0000000000000IOOOOO0.0... Comparison of the histologic appearance of bron- chiolar exudates of control and Pasteurella eXposed caIVESOOOO..00.00.00000000000000000000000000......0.. Photomicrograph of the inflarmiatory response to Pasteurella haemolytica for an established pulmonary TESfonOOOOOOOOOOO000......00.000.000.000....00...O... Photomicrograph of subpleural and peribronchiolar hyperinflation in otherwise normal lung tissue of a calf inoculated with Pasteurella haemolytica......... Acute suppurative lymphadenitis of mediastinal lymph node in a calf inoculated with Pasteurella haemonticaOOOOO0.0.0000......OOOOOOOOOOOOOOOOO0...0. Inflammatory changes in the liver of a calf illus- trating focal areas of necrosis and periportal inflamationOOOOO0.0.0......OOOOOOOOOOOOOOOOOOOO0.00. Schematic diagram of the apparatus used in the fixation Of lung salnpleSOOOIOIIOOIOOOOOOOOOOOOOOOOOOO ix Page 185 188 190 192 194 196 198 200 203 205 231 CHAPTER 1 A review of factors of possible significance in the pathogenesis of initial and develOped lesions of Pasteurella haemolytica pneumonia INTRODUCTION Shipping Fever describes a disease syndrome of cattle and less com- monly of horses and sheep which has been recognized for many years.1‘3 Because the causes and pathogenesis remain incompletely understood, this has contributed to the wideSpread use of “Shipping Fever“ as an all- encompassing term to describe the illness.1 The disease in cattle is a fulminating illness with high fever, anorexia, dyspnea and coughing, following a period of stress. Stress is also a term which lacks clear definition, but in the context of predisposing to Shipping Fever, stress associated with transportation is well recognized.2 Pathologic descrip- tions of Shipping Fever are principally limited to those animals that become moribund or die of the disease and the lesions are typically those of severe fibrinous pneumonial, bronchOpneumonia2 and fibrinous pleuritis. The appearance of lungs from acutely affected animals is often described as hepatized1'4, where the lungs become liver like in consistency and color resulting from fluid, exudate and blood accumulation in the lung parenchyma.3'4 “Consolidation" is often used interchangeably with the term 'hepatization“4, but if used in the strictest sense implies pulmo- nary fibrosis and thus is incorrect for acutely develOped lesions. The difficulty with eliminating “Shipping Fever“ as a problem of cattle has been due in part to the inability to identify a causative agent responsible for the lesions. Although numerous microbiologic organisms have been identified in diseased cattle affected with the cli- nical and pathologic changes canpatible with Shipping Fever, Koch‘s postulates have not been fulfilled; that is, exposure of normal cattle to suspected etiologic agents fails to consistently reproduce the clinical and pathologic observations regarded as characteristic of the naturally occurring disease. The rewards of such investigations have been to identify and classify numerous bacteria, viruses, chlamydia and mycoplasmas, that, under certain circumstances when given by themselves, may cause injury to the respiratory system of cattle. Over the last decade, several breakthroughs in the understanding of various aspects of “Shipping Fever" have led to an alteration in the phiIOSOphy of research involving this important disease of cattle. It became apparent that Shipping Fever outbreaks were frequently associated with a variety of stressful stimuli, not simply transportation.2 An increased understanding of natural and vauired mechanisms of resistance, particularly relating to respiratory tract disease supple- mented an increasing knowledge of how stressful stimuli might alter immune reSponses.5 Consequently the pursuit of a single pathogenic agent has waned and rightly so, for the disease of Shipping Fever is now seen as a complex interaction of environmental stresses, competency of host defense mechanisms and exposure to a variety of infectious agents.1’2a5'13 Hence the terminology of bovine respiratory disease complex has largely replaced Shipping Fever in order to better accom- modate these aspects of this disease. Another important development over the last decade has been the realization of the central role Pasteurella s3. has in causing the pulmonary damage associated with Shipping Fever. The develOpment of serologic tests for the various strains of Pasteurella and their sub- sequent association with many disease outbreaks of shipping fever clearly indicate that Pasteurella haemolytica serotype 1A is the prin- ciple agent involved, particularly in cases where extensive pulmonary destruction occurs.1'3 Pneumonic Pasteurellosis and P. haemolytica Pasteurella sp. are not the only cause of severe pulmonary lesions associated with Shipping Fever (Bovine Respiratory Disease Complex), nor are the lesions of pasteurellosis restricted to the lungs. In cattle, a variety of bacteria may cause similar severe lesion58a11:13:14 and bovine respiratory syncitial virus is quite pathogenic for sheep and goats as well as cattle.15'17 In species other than ruminants, including companion animals, persons and birds, P. multocida is of greater importance than R. haemolytica. In most instances in mammals other than ruminants, lesions due to P, multocida are sporadic in nature and variable in the organs affected.18’24 Species of Pasteurella other than B. multocida and P. haemolytica are important causes of morbidity and mortality in birds.24 Pasteurella multocida is an important patho- gen of cattle, sheep, pigs and water buffalo as a cause of pneumonia and hemorrhagic septicemia.2:3»24'25 Pasteurella haemolytica is also an important cause of septicemia and pneumonia limited to domesticated sheep27'33, pigsz3 and cattle1'4:9‘14 and of the various strains of P. haemolytica, only type 1A has such a profound influence on the health of cattle raised in EurOpe and North America.34»35 Therefore 3, haemoly- tica is considered preeminent as the cause of the severe, life threatening lesions of Shipping Fever, although other factors influence the initiation of pneumonia and facilitation of P. haemolytica survival in the lungs. The understanding of the importance of P. haemolytica in this regard has largely resulted from the work of Thomson and coworkers who succeeded in reproducing lesions compatible with field cases of Shipping Fever1»7:35'43 by aerosol exposure or by direct instillation of cultures into the airways. Others have also been successful in inducing lesions similar to field cases of Shipping Fever using P, haemolytica alone”.45 or combined with viruse515-17.27-29.31-33, by septic emboli- zation of the lung30 and using pretreatments with cold stress.45»47 A number of investigators had limited success in inducing pneumonia with P. haemolytica1»43-5O and there are also reports where the disease could not be experimentally induced deSpite administration of organisms via a number of different routes.51‘53 As stated by Rehmtulla and Thomsonl, the lesions in some of the early studies on transmission of Shipping Fever to healthy animals must be questioned, since many investigators did not investigate Specific pathogens but gave inoculations of homoge- nized pneumonic material to healthy calves by various routes.1:54:55 However, Carpenter and Gilman (1921) were probably suc- cessful in reproducing pasteurellosis in a calf after intratracheal challenge of an organism then known as Pasteurella boviseptica.56 The reasons why such diverse methods of pneumonic pasteurellosis induction are successful while others have failed is not totally understood, but may relate to the concentration of organisms delivered to the lung, since most successful studies report pulmonary exposures of greater than 108 organisms. Such differences undoubtedly reflect the cmnplex interactions between host defense mechanisms and the pathogenic effects of_P. haemolytica. Defense Mechanisms Against Pulmonary Pasteurellosis Pasteurella haemolytica is considered a non-pathogenic resident of the nasal cavity of healthy cattle.10 During breathing, aerosols con- taining these bacteria are generated and deposited in the lungs.57-58 Normally cattle apparently c0pe with this low level bacterial exposure and several pulmonary defense mechanisms are likely to be involved; clearance of bacteria by the mucociliary escalator59'50; phagocytosis by resident macrophages and migrating leukocytes39s40:59‘53; inhibition of growth by local tissue factors39:64; and maintenance of suboptimal bac- teria growth conditions. Tipping the balance in favor of bacterial sur- vival is apparently not difficult in cattle. Experimental induction of disease by exposure to large numbers of micro-organisms either by direct intrapulmonary injection, or by inhalation challenge may simply overwhelm the phagocytic capacity of readily available leukocytes.1a7:10 Viruses and irritant chemicals disturb the mucoci- liary escalator and thereby nullify a defense mechanism.7:9:10.13.39:40:43 Septic embolization may place organisms in a microaerophilic environment, rich in nutrients required for growth24 but sequestered away from efficiently functioning phagocytes. Stresses, including cold stress, may impair phagocytic responses by a variety of immunosuppressive mechanisms5 and may facilitate growth and seeding of bacteria from the nasal cavity.57:58a55 Experimentally induced pneumonic pasteurellosis may therefore arise by compromise of defense mechanisms in a multiplicity of ways. The relative importance of each mechanism in defending against pasteurellosis is in doubt, but Thomson and others have suggested macrophages have an important role.1.59 Naturally occurring 3. haemoLytica pneumonia is facilitated by concurrent or prior virally-induced injury to the reSpiratory tract.13 It has only recently been recognized that stress induced by management practices and environmental conditions is of critical importance.5:66 Anecdotal evidence for this view is that E. haemolytica serotype 1A has a world-wide distribution yet the disease of pneumonic pasteurellosis is confined principally to North America and parts of Europez, where cattle are intensively raised. Stress As A Predisposinngactor of Pneumonic Pasteurellosis Stress defined in the broadest sense by Seyle (1976) is that nonspe- cific response by the body to any demand.67 While physical stresses (stressors) are well recognized, only recently has information regarding psychological stresses of animals become available.5 The effects of stress on immune function of homeotherms was recently reviewed by Kelley (1980).5 However, the effects of such environmental influences may not be restricted to the hwnune system. For example thermal stresses, par- ticularly with associated alterations in humidity, predispose to reSpiratory disease in cattle.5’7.10:11a48'50s55a55:57'72 Not only do these climatic conditions appear to alter the capacity of the immune system for both cell mediated and humoral reSponses5 but such changes may enhance bacterial survival in the environment65 as well as in the body and may alter the cellular structure of the airways.72 Not all evidence suggests that such climatic changes are immunosuppressive73, but at least for calves, Kelley suggests that evidence to the contrary is somewhat suspect.5 Crowding, isolation, mixing of animals socially naive to one another, weaning, food deprivation, noise, restraint and excessive physical demands may lead to immunosuppression.5 Clearly, many of the husbandry practices in cattle rearing, especially transportation of livestock for long distances, are combinations of several stressful influences.1‘10 In addition crowding, enclosure in unsanitary environ— ments, particularly with limited ventilation, and exposure to microbial pathogens foreign to the peer group may greatly increase the load of pathogens inhaled into the lungs, even if immune competence is maintained.58:71:74'77 The mechanisms involved in body reSponses to stress are not fully understood. Some events, such as hnnunosuppression appear related to the increase in corticosteroids associated with a variety of stresses, including transportation of cattle.78:79 Corticoid related alterations in hmnunoglobulin absorption from the gut and corticosteroid related thymic and lymphoid atr0phy are two known effects of stress in calves.80"82 However, pharmacologic doses of hydrocortisone to calves do not prevent leukocyte migration or phagocytosis in pneumonic areas of lungs infected with g. haemolytica39 or prediSpose to the development of pneumonia.51 Although no assessment of phagocytic killing of bacteria was made in the former study39, these data indicate that corticosteroids may not be entirely responsible for the various aSpects of immune suppression summarized by Kelley. There is no information regarding the effects of stress on other defense mechanisms in the lung83 or on pulmonary function. Alterations in pulmonary structure and function by stressful stimuli may be a signi- ficant influence in the predisposition of stressed animals to pneumonic pasteurellosis. Normal respiratory system structure and function and its response to m The prime function of the respiratory system is gas exchange. Pulmonary structure is arranged for efficient uptake of oxygen and the excretion of gaseous wastes, principally C02. The cessation of respira- tion is life threatening within minutes and so elaborate mechanisms of control by both neural and humoral mechanisms allow prompt adjustments to maintain adequate gas exchange.84 Since P. haemolytica pneumonia eventually leads to hypoxemia and reSpiratory failure, it is likely that these controlling influences are activated and/or modified by pasteurellosis. The effects of pulmonary pasteurellosis have not been determined so the importance of these events is Speculative. In cattle, other respiratory system functions of olfaction and meta- bolism of polypeptides by the pulmonary vasculature are less well docu- mented, even under normal circumstances, and the effect of pasteurello- sis on these functions is also unknown. The structure of the bovine respiratory system, in health and with pasteurellosis, has received more attention than its function. The bovine lung is a completely lobulated organ so that each lobule has only one pathway to supply ventilation85.86 (that is, a complete lack of collateral ventilatory pathways). As a result, airway obstruction in cattle may profoundly affect gas exchange87 since no alternate pathways for gas to enter obstructed lobules exists. Other structural properties of the bovine lung may also influence function and predispose to pneumo- nic pasteurellosis.88 Cattle have a relatively small gas exchange sur- face in relationship to their metabolic rates and minute ventilation.88 This may not only facilitate exposure to environmental hazards but may je0pardize gas exchange with relatively minor disturbances in reSpira- tory structure. The bovine lung has relatively few alveolar macrOphage585»89 and these are thought to require oxygen for normal function.90:91 If terminal bronchiolar obstruction occurs in cattle lungs, the phagocytic function of macrophages, which is likely to be limited by their small numbers, will probably be further compromized by 10 local hypoxia.88 Hypoxia may also depress the normal activity of muco- ciliary transport from these regions.92 That these effects of hypoxia are important in calves is supported by the finding that reduced bac- terial clearance rates occur in the lung regions of calves where oxygen tension is thought to be least.63 The lobular structure of the bovine lung may also facilitate atelectasis subsequent to airway obstruction because the loose connective tissue surrounding each lobule may reduce forces of interdependence that would otherwise assist in preventing collapse of the lobule.93 This appears to be the case for the pig94,a Species with lobulated lung similar to cattle.85 Bovine lungs have large numbers of mast cells35, and mediators from these cells may cause airway and vascular modifications so that gas exchange is impaired.87 These aSpects and other functional properties (a pronounced hypoxic vasoconstrictor response95 and an apparent lack of lysozyme in pulmonary secretion588) appear to place cattle in a disadvantaged position in coping with pathogens which arrive in the lung. Veit and Farrell88 suggest that such structural disadvantages have an important role in predisposing to Shipping Fever. The response of the respiratory system to injury clearly has a wide variety of manifestations, dependent in part on the nature of the injurious agent, and the response of host tissues. Direct effects of the injurious agent are readily appreciated with physical and thermal injuries and with the effects of viruses, and often result in cell necrosis.13:15'17a31'33:39:40a43:95 It is not known whether 3. haemoly- ‘3123 has any direct injurious effect on bovine respiratory epithelial cells when the bacteria remain intact. However, ample evidence exists for the release of toxic products from E. haemolytica. Pasteurella 11 haemoLytica is a potent source of endotoxin97, a component normally found in the bacterial cell wall and liberated upon cell destruction, and of cytotoxins.7:97"99 In view of the known potent effects of these 2 bacterial products on leukocyte function98:99 and of the effects of endotoxin on pulmonary vasculature97»100'105 and pulmonary surfactant39s105, it is likely that these products contribute signifi- cantly to functional and structural derangements that ultimately occur in animals dying of pneumonic pasteurellosis. In addition to the effects of bacterial products, the presence of inflammatory exudate may alter lung structure and function. This exu- date which deveIOps in response to P. haemolytica, may not only destroy the bacteria but damage lung tissue by the release of phagolysozymal enzymes, biologically active lipids and mediators into the lung. This aspect is discussed in detail below. Alterations in pulmonary structure associated with naturally occurring and experimental P. haemolytica pneumonia were reviewed by Rehmtulla and Thomson (1981)1, who pointed out the need to determine the early lesions of E. haemolytica pneumonia, since the pathogenic pro- cesses involved in the conversion of healthy pulmonary tissue to those with pneumonia of life threatening severity are uncertain. In cases of severe pneumonia, Rehmtulla and Thomson noted discrepancies were present in the reported degree of airway involvement, of the extent of vascular thrombosis and in the nature of the inflammatory exudate.1 Although mononuclear “swirly” or streaming cells are a characteristic of the exu- date in develOped cases of pneumonia, the derivation of these cells is unclear although Thomson says that they originate from macrOphages.1 Clearly, to clarify the reasons for the discrepancies in the descriptions 12 of the appearance of pneumonic pasteurellosis lesions, further structural studies are needed, particularly in the early stages of the disease. Only one study has addressed the lesions caused by pulmonary .E° haemolytica during the early phases of infection. Gilka et al (1974)39,4O described alveolar edema, loss of surfactant lining film and changes suggestive of increased numbers of macrOphages, 4 hours after bacterial challenge by aerosol exposure. The mechanism of formation of pulmonary edema remains undetermined and as yet the processes that result in the transition of these early lesions to those of acute necro- tizing and fibrinous pneumonia are unknown. Of interest, many years prior to the study of Gilka et al (1974), Edington (1930) described edema, hemorrhage and congestion of alveolar walls, with exudation of serum into alveolar lumens of field cases of pneumonia from which he consistently isolated P. boviseptica.107 Tweed and Edington considered these changes as the early lesions but this claim was not substantiated.107 Host mediated regponses ofppossible importance in theppathogenesis of P. haemolytica pneumonia. a. Hormonal influences I. Corticosteroids. As previously described, corticosteroids are probably liberated in reSponse to stressful stimuli, even in neonatal calves.5»78-79:108 Disease itself may pose a stress which results in further steroid release, as evidenced by increased corticoid levels of young calves with diarrhea.109 It is not known whether_E. haemglytica pneumonia stimulates the II. 13 release of corticosteroids, nor is it apparent from previous studies whether such a response is likely to be beneficial or detrimental to the animal. Steroids may aid in stabilization of cell membranes in response to injuryllo, may reduce pulmonary edema, improve ingested immunoglobulin uptake5»80:81, stimulate surfactant111 and may enhance interferon production.112 However, immunosuppression5:78a79:82 and possible detrimental effects on pulmonary alveolar type II cells39940, pulmonary growth111 and histamine metabolism113 may be of greater impor- tance than the above beneficial effects. Evidence regarding the detrimental effects of corticoid in calf lungs is conflicting39:40»51 even when given in pharmacological doses. Thyroid hormones. Thyroid hormones may play a role in the host responses to P. haemolytica for the following reasons. The hypothyroid state in human neonates increases the incidence of pulmonary disease114 and small alterations in thyroid hormone levels appear to indirectly affect histamine metabolism.113 Such a "permissive effect" may be a nonspecific action of thyroid hormone since thyroid hormones appear to exert such a "permissive“ effect on the actions of other hormones for a wide range of host tissuesllS; there is a seasonal incidence of pneumonia, illness in calves being unst common in winter and spring.59:70:74 Although it appears likely that the prediSposi- tion to pneumonia is based on rather abrupt changes in climatic condition559:70, more rapid than that possible for changes in thyroid hormone to normally occur,115 the influence of seasonal changes in thyroid hormone levels and the predisposition to 14 pneumonia remain to be proved. Since_fi. haemolytica is unlikely to directly affect thyroid function, it also seems unlikely that deficiency of thyroid hormones contributes to the disease because reduced thyroid hormone levels occur at the time of year when the incidence of Shipping Fever is least.69:70:115 III. Other hormones. The lungs are influenced by a variety of other hormones. Stress, particularly that associated with fear, exci- tement or cold, probably exposes the lung to increased amounts of catecholamines liberated from the adrenal medulla. If influenza and parainfluenza viral infections alter the reSpon- siveness of the lung and leukocytes to cholinergic and adre- nergic stimuli, as they are reported to do in guinea pigs117 and personslla‘llg, catecholamines may have modified and perhaps adverse effects in calves developing pasteurellosis with con- current or previous Parainfluenza 3 virus exposure. It is unclear whether the sex hormones have any influence on the develOpment of Pasteurella pneumonia. Pretreatment with estrogens did not facilitate the development of pneumonia in cattle51, but the success rate in experimental pneumonia induc- tion was very poor exclusive of estrogen pretreatment, in this study sex differences that exist with other diseases and possibly with pasteurellosis may relate to different social pressures placed upon male and female animals, rather than a direct hormonal effect.5 b. Humoral Factors I. Bradykinin. Bradykinin may be a mediator of pulmonary damage in cattle, as has been suggested by studies of bovine 15 anaphylaxis.120-122 Studies 13 1119 indicate that the effects of bradykinin are most likely limited to the vasculature as intravenous and aerosol bradykinin exposure of calves does not result in alteration of pulmonary mechanical or gas exchange properties.123 In studies where ventilation is maintained during bradykinin administration, bradykinin causes systemic and pulmonary hypotension123; in other studies where normoxia may not have been maintained, pulmonary vasoconstriction was attri- buted to bradykinin.121.122.124-127 The generation of bradykinin within the vasculature results from activation of plasma kallikrein, an enzyme which cleaves bradykinin, a nonapeptide, from circulating proteins called kininogens.128a129 Increased intravascular levels of bradykinin are generally described to cause smooth muscle contraction, vasodilation, increased vascular permiability, pain and the che- motaxis and modulation of leukocyte reSponse5129-132 and so have been thought to contribute to the vascular damage, leukocyte infiltration and pulmonary edema associated with bovine anaphy- laxis. In our research studies where ventilation was controlled, bradykinin administration did not cause pulmonary edema.123 Since respiratory acidosis may activate mechanisms of bradykinin generation133 and since hypoxemia limits bradykinin breakdown134 (a normal function of angiotensin converting enzyme found bound to the pulmonary vascular endothelium134a135) the detrimental effects of pasteurellosis on gas exchange and vascu- lar integrity may lead to the accummulation of bradykinin in the lungs and cause pulmonary edema and inflammation. In addition, II. III. 16 P, haemolytica may release endotoxin that activates Hageman factor135a137, and activated Hageman factor is a potent promoter of bradykinin formation.128:129:138 Pasteurella sp. could also activate bradykinin by pathways independent of Hageman factor, by liberation of plasminogens, kallikreins and the direct effect of endotoxin on leukocytes which ultimately leads to leukocyte secretion of kallikreins.137 Complement. Endotoxin, Hageman factor activation, and bradyki- nin activation all lead to the stimulation of the complement enzyme cascade system ultimately causing the generation of C3 and CS fragments which are potent chemotactic factors for neutrOphilS and also result in pulmonary congestion, hemorrhage and edema when injected in lungs.137:139"141 It is not known whether complement fragments are generated during 3, haemolytica pneumonia of cattle, and if so what effects they have on bovine lung structure and function. Hageman factor and components of the clotting system. Pasteurella haemolytica may cause endothelial damage via the effects of endotoxin and cause blood coagulation because of exposure of the vascular clotting factors to tissue thromboplastins.135-138.142.143 In addition, as previously men- tioned, Pasteurella haemolytica may activate Hageman factor by both endotoxin-dependent and endotoxin-independent mechanisms leading to stimulation of blood coagulation by the intrinsic clotting pathway.135:137’142:143 In calves, as in other species endotoxemia may result in disseminated intravascular coagulo- pathies, in part due to activation of blood coagulation and in IV. V. 17 part due to platelet aggregation.100'105:144'146 Although such pathogenic mechanisms may explain the devel0pment of large areas of coagulation necrosis in well developed lesions of Pasteurella pneumonia due to vascular thrombosis and infarction, it is not clear why vascular thrombosis is not a consistently reported lesion of pneumonic pasteurellosis.1 Histamine. Circulating histamine may arise from several sour- ces. The major pr0portion of body histamine stores resides in mast cellsl30s135, with lesser contributions from non-mast cell sources such as platelets and circulating blood granulocytes, and from the intestinal tract.130s145'148 Endotoxin may trigger platelet149 and granulocyte degranulationslso‘152 and certain viral153 and bacterial infection5154:156 also appear able to cause increased histamine secretion as well as increased pulmo- nary sensitivity to histamine.154 Complement fragments may also cause mast cell degranulation.139.140 Histamine has broncho- constrictive and hypotensive properties in calves87 and if it is liberated in significant amounts during Pasteurella infec- tion it is likely to contribute adversely to pulmonary function. Immunoglobulins. Immunity to P, haemolytica can be stimulated and circulating hmnunoglobulins appear to be important in establishment of immunity37.157, although other studies have emphasized the role of the pulmonary macrophage.44.45.54 Immunity to P. haemolytica endotoxin prevents pneumonic pasteurellosi5103.158-160, but this is not surprising since endotoxin is part of the bacterial cell wall and may act simply as a surface antigen for immunoglobulin attachment.103 VI. 18 Stimulation of humoral immunity may not be without adverse con- sequences, as vaccination under certain circumstances (aerosol inhalation challenge with adjuvants combined with subcutaneous vaccination) appears to make the disease worse.36 It is not known whether circulating hnnunoglobulins contributed to the development or retardation of lesions in naturally occurring cases of P. haemolytica pneumonia or in the above study.36 Certainly, immune mediated pulmonary diseases are well recognized in other species, but to date there is no evidence to indicate that a Specific syndrome exists which is associated with E. haemolytica infection. Other humoral factors. Through the effects of endotoxin, and perhaps by other mechanisms, platelets, mast cells and cir- culating leukocytes may liberate a variety of substances that circulate in the blood. These may include ADP, serotonin, dopa- mine, prostaglandins and leukotrienes, and in addition, fragments of fibrin degradation products, complement and kinin fragments.128438.142445»149"151 There are complex inter- relationships between the activities of many of these compounds on the tissues they effect. The importance of these factors in the pathogenesis of pasteurellosis has not been evaluated. c. Cellular factors I. Platelets. Platelets may become involved in pneumonic pasteurellosis through a variety of mechanisms. Vascular damage, the disturbance of endothelial prostaglandin synthesis, the presence of vasoactive agents such as histamine, bradykinin activated Hageman factor, clotting and complement fragments and II. 19 the effects of endotoxinS and cytotoxins from Pasteurella haemo- ‘lypigp may cause platelet thrombus formation and the further release of vasoactive agents via platelet degranulation.129.130. 135-138,144-146,149-152,160,161 Fibrinous thrombosis of the pulmonary lymphatics is one of the hallmarks of well developed lesions of pneumonic pasteurellosisl'3, yet vascular thrombosis is not a consistent finding. Therefore, the contribution of platelets to the pathogenesis of Pasteurellosis is in doubt. In cases where vascular thrombosis is a feature of the disease, it would seem likely that platelets are involved in some way. NeutrOphils. The role of neutrophils in the development of pneumonic pasteurellosis is in doubt. Structural studies of well develOped pulmonary lesions do not consistently describe an inflammatory reSponse dominated by neutrophils and some investi- gators claim that purulent exudate which consists mainly of neutrOphils is atypical for pneumonic pasteurellosis.1 Furthermore, there is no evidence to date of the functional effects of neutrophil aggregation in the lungs apart from the physical effects of airway obstruction with exudates. It seems unlikely that neutr0phils are excluded from the inflammatory response to P. haemolytica in the bovine lung, since in other Species, Pasteurella sp. elicit a suppurative inflammatory response.3:18'22 A special circumstance may arise, however, when inhaled bacteria are readily cleared by normal pulmonary defense mechanisms.39a59s50»52 With bacterial per- sistence, it does seem likely that neutrophils would be recruited.62 Several previously discussed mechanisms may serve 20 to recruit neutrOphilS. Antigens including endotoxin are chemo- tactic for neutrophils and 1p_!i!p endotoxin causes sequestra- tion of neutrOphils within pulmonary capillary beds.102,137a144a145,150'152 Additional factors released from macrophagesle, plus the possible effects of kinin129'132, complement139"141 and fibrin fragments may serve to heighten chemotaxis and lead to further accumulations of neutrophils within the lungs. Obviously, such a response would optimisti- cally lead to the destruction of the bacteria and prevent further pulmonary damage. All too frequently the outcome is lethal to the animal and there is no compelling evidence to suggest that a heightened neutrophilic response and subsequent infiltration of the lung is beneficial to the animal. Indeed, the possibility exists that through cell degeneration and lysis, perhaps facilitated by the bacterial release of endotoxin and cytotoxin97'105, neutrOphils may further damage the lung through the release of a variety of chemicals, including leukotrienes and hydroperoxy fatty acids, neutral proteases and a variety of oxidative radicles including hydroxyl, hydrogen peroxide and superoxide anions.153"166 Evidence in support of the potential for neutrophils to exacerbate tissue injury in the lungs was demonstrated in sheep, where neutrophil depletion prevents the pulmonary edema otherwise associated with endotoxin injections.167 In addition neutrOphils and their products cause tissue inflammation in organs other than lungs when injected in the tissues of cattle.165 III. 21 Mononuclear cells. There is no doubt that mononuclear cells are involved in the response to P. haemoLytica pneumonia. Alveolar macrOphages are the first line of defense, and represent an important mechanism of bacterial clearance from the lungs.1a39»59'52»54 Lymphocytes and plasma cells are respon- sible for immunity which under certain circumstances prevents the disease.37-157 Aside from phagocytic prOperties macrophages can secrete a large array of substances, including enzymes, complement components, enzyme inhibitors, nucleotides, endoge- nous pyrogen, oxygen radicles, bioactive lipids, chemotactic factors and factors controlling the rate of replication and function of other inflammatory cells.162 As with neutrOphils, some of these secretory products have the ability under certain circumstances to cause tissue injury.152»158:159 Another interesting aspect of the involvement of lymphomononuclear cells in pneumonic pasteurellosis is that these cells probably are the source for the so called "swirly" or “streaming“ cells charac- teristically seen in developed lesions of E. haemolytica pneumo- nia. These cells appear to be degenerating mononuclear cells which have baSOphilic smudged nuclei and basophilic streaming cytoplasm to form fusiform shapes with frequently no clear limiting membrane. This reaction appears peculiar to pasteurellosis in cattlela3 and so the discovery of the mecha- nisms which lead to the formation of “swirly cells" may have important implications with regard to understanding the pathoge- nesis of pasteurellosis. IV. 22 Mast cells. In the intact animal, mast cell degranulation may be effected by a wide variety of entities, including immunoglo- bulins, principally IgE and 1964169'174, complement fragmentsl39:140, proteolytic enzymesl75»175, phospholipidsl77, prostaglandinsl78, catecholaminesl79, kinins, serotonin180, and hypoxia.181 It is not known whether mast cell degranulation occurs as a result of exposure of cattle to P. haemolytica, but there would seem to be ample Opportunity for this to occur. Alveolar epithelial cells. Membranous (type I) alveolar epithe- lial cells are metabolically active cells which may be sensitive to noxious stimuli such as hypoxia, bacterial products or pro- ducts from inflammatory cells. These cells are united by tight junctions so that the alveolar epithelial barrier normally is extremely competent at preventing fluid loss from the intersti- tium into the alveoli.182 Under conditions of vascular damage and lymphatic thrombosis, compromise of the barrier occurs and may explain the formation of alveolar edema in cases of pasteurellosis. However, the propensity for alveolar fluid for- mation may be greatly exacerbated if P. haemolytica had Specific cytotoxic effects on membranous alveolar cells. DevelOped lesions of pneumonic pasteurellosis often Show extensive necro- sis of alveolar walls1 suggesting that by some mechanism, membranous alveolar cells are damaged. Cuboidal (type II) alveolar cells are primarily responsible for surfactant production in the lung. Gilka et al (1974) reported that some of the earliest lesions of pasteurellosis in calves (4 hrs after aerosol challenge) were related to pulmonary VI. 23 edema and the loss of surfactant from the lungs with subsequent atelectasis.39a4O In addition it was noticed that P, haemoly- .tigg organisms were frequently deposited on the surface of type II alveolar cells.40 Whether this simply represents physical trapping of bacteria against the microvilli of these cells com- pared to the smooth surfaces of type I alveolar cells or was due to a Specific adherence of bacteria to these cells is unknown. Furthermore, type II alveolar cells appear to be of considerable importance hmnunologically, at least in the stimulation of alveolar phagocytes after viral infectionsl33, and alveolar type II cell-macrophage membrane connections have been observed in calves (Slocombe and Echt, unpublished observations). Therefore, importance of alveolar type II cells in the pathoge- nesis of pasteurellosis appears likely, both through alterations in surfactant production, by the possible toxic effects of the bacteria on the cells106 and by increased surfactant require- ments as a result of alveolar edema, and for the type II cell's possible role in the immune response against E, haemolytica. Airway epithelial cells. Although many of the structural characteristics of the airway epithelial types are well described184'189, alterations in function of these cells in response to injury190'195, and the subsequent effects of their alterations on respiratory function are poorly understood. Influences such as hypoxia195, exposure to noxious gases, cholinergic stimulation and the presence of mediators may affect the function of ciliated, mucogenic (goblet), serous, Clara and APUD (amine precursor uptake and decarboxylation) cell5191'195 24 and result in alterations in mucociliary clearance and neural control of airway caliber. Necrosis of airways and in some cases hyperplastic epithelial reSponses are reported in pasteurellosisl, but whether these changes contribute to the pathogenesis of pneumonic pasteurellosis is unknown. VII. Pulmonary connective tissue. Although pulmonary connective tissues have received considerable attention and were recently reviewed (Hance and Crystal, 1975)197, the mechanisms for responses of connective tissues in the lung to infectious pro- cesses is not known. Secretions from macrOphages, including acid hydrolases, glycosidases and proteases may damage the con- nective tissue.152’168:169 MacrOphage involvement is extensive in the well develOped lesions of P. haemolytical:3 when irregu- lar necrotizing foci are commonly observed. These foci often extend between lobules with no apparent confinement by the lobu- lar connective tissue.1:3 Perhaps macrOphage products are involved in the devel0pment of the connective tissue necrosis in these lesions. Experimental Rationale Shipping Fever remains an economically important disease of North American cattle. The pathogenesis of Shipping Fever remains poorly understood, although the etiologic agent Pasteurella haemolytica is now known to have a central role in the rapid destruction of pulmonary tissue of cattle suffering from the disease. In addition, although a variety of stressors have been identified which predispose to Shipping Fever, the 25 mechanisms by which this prediSposition develops are incompletely understood. Shipping Fever lesions associated with pneumonic pasteurellosis represent a well defined disease in terms of the patholo- gic state of the lungs at the time the animal becomes moribund or dies from the disease. However, descriptions of the early lesions of P. haemolytica pneumonia are inconsistent with the lesions that occur later in the course of the illness, and the extent of current knowledge regarding the mechanisms of pulmonary injury by P. haemolytica limited by a lack of information on the changes that occur with the initial injury to the lungs. For these reasons, an experimental series was designed to address the functional and structural changes that occur in bovine lungs following a known important stressor (cold stress by chilling) alone, and in combination with experimentally induced P, haemolytica pneumonia. A variety of hormonal, humoral and hematologic variables were measured in order to identify factors of possible significance in establishing pulmonary injury. Because of the many humoral factors that could possibly contribute to the disease, two were selected to represent sour- ces of activation. Bradykinin was measured as an intravascular "trace“ because its activation is coupled with the clotting, plasmin and comple- ment cascades. Histamine was chosen to represent stored mediators released by mast cells. Previously, the effects of both these mediators on lung function had been determined. To monitor pulmonary function gas exchange and pulmonary mechanical pr0perties were measured. The latter were measured in order to localize the functional site of injury. Briefly, dynamic compliance measurements reflect pulmonary elastic prOperties and small airway caliber. The status of large airways is measured by determination of airway resistance. Because the stage of lung inflation can influence these measurements, it was necessary to determine lung volume at the point of measurement of resistance and compliance. This was achieved by the determination of functional residual capacity by Helium dilution. Pulmonary structural changes were determined using routine histo- pathologic techniques, and structural findings correlated with the hema- tologic and pulmonary functional studies. In order to control the state of pulmonary inflation in structural studies, lung samples were fixed by airway perfusion under a constant pressure. 27 References 1. Rehmtulla AJ, Thomson RG: A review of the lesions in Shipping Fever of cattle. Can Vet J 22:1-8, 1981. 2. Blood DC, Henderson JA, Radostits OM: In Veterinary Medicine, ed 5. Philadelphia, Lea and Febiger, 1979, pp 492-498. 3. Jubb KVF, Kennedy PC: In Pathology of Domestic Animals, ed 2. New York, Academic Press, 1970, vol 1, pp 189-203. 4. Smith HA, Jones TC, Hunt RD: In Veterinary Pathology, ed 4. Philadelphia, Lea and Febiger, 1972, pp 1100-1105. 5. Kelley KW: Stress and immune function: A bibliographic review. Ann Rech Vet 11:445-478, 1980. 6. Thomson RG, Chander S, Savan M, Fox ML: Investigation of fac- tors of probable Significance in the pathogenesis of pneumonic pasteurellosis in cattle. Can J Comp_Med 39:194-207, 1975. 7. Collier JR: Pasteurellae in bovine respiratory disease. .QAVMA 152:824-828, 1968. 8. Ishino S, Oka M, Terui S, Ikeda S: Pathological and micro- biological studies on calf pneumonia occurring in mass rearing facili- ties. Nat Inst Anim Hlth Quart 19:91-103, 1979. 9. Thomson RG: Pathology and pathogenesis of the common diseases of the respiratory tract of cattle. Can Vet J 15:249-251, 1974. 10. Lillie LE: The bovine respiratory disease complex. Can Vet J 15:233-242, 1974. 11. Bryson DG, McFerran JB, Ball HJ, Neill SD: Observations on outbreaks of respiratory disease in housed calves (1) Epidemiological clinical and microbiological findings. Vet Rec 103:485-489, 1978. 28 12. Bryson DG, McFerran JB, Ball HJ, Neill SD: Observations on outbreaks of respiratory disease in housed calves - (2) Pathological and microbiological findings. Vet Rec 103:503-509, 1978. 13. Carter GR: Pasteurella infections as sequelae to respiratory viral infections. .QAVMA 163:863-864, 1973. 14. Gourlay RN, Mackenzie A, Cooper JE: Studies of the micro- biology and pathology of pneumonic lungs of calves. J Comp Path 80:575-584, 1970. 15. Al-Darraji AM, Cutlip RC, Lehmkuhl HD, Graham DL, Kluge JP, Frank GH: Experimental infection of lambs with bovine respiratory syn- cytial virus and Pasteurella haemolytica: Clinical and microbiologic Studies. 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Gilmour NJL, Thompson DA, Snith WD, Angus KW: Experimental infection of lambs with an aerosol of Pasteurella haemolytica. Res Vet 'Sgi 18:340-341, 1975. 30. Gilmour NJL, Angus KW, Sharp JM: Experimental pulmonary infections of sheep caused by Pasteurella haemolytica biotype T. .Vgp Egg 106:507-508, 1980. 30 31. Wells PW, Sharp JM, Rushton B, Gilmour NJL, Thompson DA: The effect of vaccination with a Parainfluenza type 3 virus on pneumonia resulting from infection with Parainfluenza type 3 virus and Pasteurella haemolytica. J Comp Path 88:253-259, 1978. 32. Sharp JM, Gilmour NJL, Thompson DA, Rushton B: Experimental infection of specific pathogen-free lambs with Parainfluenza virus type 3 and Pasteurella haemolytica. J Comp Path 88:237-243, 1978. 33. Rushton B, Sharp JM, Gilmour NJL, Thompson DA: Pathology of an experimental infection of specific pathogen free lambs with Parainfluenza virus type 3 and Pasteurella haemolytica. J Comp Path 89:321-329, 1979. 34. Wray C, Thompson DA: Serotypes of Pasteurella haemolytica isolated from calves. Br Vet J 127:66-67, 1971. 35. Fox ML, Thompson RG, Magwood SE: Pasteurella haemolytica of cattle: Serotype, production and beta-galactosidase and antibacterial sensitivity. Can J Comp Med 35:313-317, 1971. 36. Friend SCE, Wilkie BN, Thomson RG, Barnum DA: Bovine pneumo- nic pasteurellosis: Experimental induction in vaccinated and non- vaccinated calves. Can J Comp Med 41:77-33, 1977. 37. Wilkie 8N, Markham RJF, Shewen PE: Response of calves to lung challenge exposure with Pasteurella haemolytica after parenteral or pulmonary immunization. Am J Vet Res 41:1773-1778, 1980. 38. Stockdale PHG, Jericho KWF, Yates WDG, Darcel C le Q, Langford EV: Experimental bovine pneumonic pasteurellosis. II. Genesis and Prevention. Can J Compred 43:272-279, 1979. 31 39. Gilka F, Thomson RG, Savan M: The effect of edema, hydrocor- tisone acetate, concurrent viral infection and hmnunization on the clearance of Pasteurella hemolytica from the bovine lung. Can J Comp .Mgg 38:251-259, 1974. 40. Gilka F, Thomson RG, Savan M: Microscopic lesions in the lungs of calves aerosolized with Pasteurella hemolytica and treated to alter pulmonary clearance. Zbl Vet Med 21:774-786, 1974. 41. Friend SC, Thomson RG, Wilkie 8N: Pulmonary lesions induced by Pasteurella hemolytica in cattle. Can J Comp Med 41:219-223, 1977. 42. Stockdale PHG, Langford EV, Darcel C le Q: Experimental bovine pneumonic pasteurellosis. 1. Prevention of the disease. 5331;; Comp Med 43:262-271. 1979. 43. Jericho KWF, Langford EV: Pneumonia in calves produced with aerosols of bovine Herpesvirus I and Pasteurella hemolytica. Can J Comp Med 42:269-277, 1978. 44. Walker RD, Corstvet RE, Lessley BA, Panciera RJ: Study of bovine pulmonary response to Pasteurella haemolytica: Specificity of hmnunoglobulins isolated from the bovine lung. Am J Vet Res 41:1015-1023, 1980. 45. Walker RD, Corstvet RE, Panciera RJ: Study of bovine pulmo- nary response to Pasteurella haemolytica: Pulmonary macrophage response. Am J Vet Res 41:1008-1014, 1980. 46. Breeze RG, Magonigle RA: A long acting tetracycline for treatment of Pasteurella pneumonia in calves. Bovine Practitioner 14:15-17, 1979. 32 47. Breeze RG, Laverman LH, Schmitz JA, Magonigle RA: Evaluation of a long acting oxytetracycline for treatment of Pasteurella pneumonia in calves. Bovine Practitioner 15:96-98, 1980. 48. Hamdy AH, Trapp AL: Immunization of cattle against Shipping Fever: Experimental exposure. Am J Vet Res 25:1648-1652, 1964. 49. Hamdy AH, Trapp AL, Gale C, King NB: Experimental transmission of Shipping Fever in calves. Am J Vet Res 24:287-294, 1963. 50. Hamdy AH, Trapp AL, Gale C: Further preliminary studies on transmission of Shipping Fever in calves. Am J Vet Res 25:128-133, 1964. 51. Gale C, Smith HR: 1. The experimental exposure of cattle with various cultures of Pasteurella. Am J Vet Res 19:815-817, 1958. 52. Heddlestone KL, Reisinger RC, Watko LP: Studies on the transmission and etiology of bovine Shipping Fever. Am J Vet Res 23:548-553, 1962. 53. Carter GR: Observations on the pathology and bacteriology of Shipping Fever in Canada. Can J Comp Med 10:359-364, 1954. 54. Gale C, King NB, Sanger VL: Attempt at transmission of Shipping Fever. Cornell Vet 51:219-234, 1961. 55. Jennings AR, Glover RE: Enzootic pneumonia in calves. J Comp Path 62:6-22, 1952. 56. Carpenter CM, Gilman HL: Studies in pneumonia in calves. Cornell Vet 11:111-126, 1921. 57. Pass DA, Thomson RG: Wide distribution of Pasteurella haemo- lytica type 1 over the nasal mucosa of cattle. Can J Comp Med 35:181-186, 1971. 33 58. Grey CL, Thomson RG: Pasteurella haemolytica in the tracheal air of calves. Can J Comp Med 35:121-128, 1971. 59. Thomson RG, Gilka F: A brief review of pulmonary clearance of bacterial aerosols emphasizing aspects of particular relevance to veterinary medicine. Can Vet J 15:99-107, 1974. 60. Lillie LE, Thomson RG: The pulmonary clearance of bacteria by calves and mice. Can J Comp Med 36:129-137, 1972. 61. L0pez A, Thomson RG, Savan M: The pulmonary clearance of Pasteurella hemolytica in calves infected with bovine Parainfluenza -3 virus. Can J Comp Med 40:385-391. 1975. 62. Jackson AE, Southern PM, Pierce AK, Fallis BD, Sandford JP: Pulmonary clearance of gram-negative bacilli. J Lab Clin Med 69:833-841, 1967. 63. Veit HP, Farrell RL, Troutt HF: Pulmonary clearance of Serratia marcescens in calves. Am J Vet Res 39:1646-1650, 1978. 64. Markham RJF, Wilkie BN: Influence of bronchoalveolar washing supernatants and stimulated lymphocyte supernatants on uptake of Pasteurella hemolytica by cultured alveolar macrophages. Am J Vet Res 41:443-446, 1980. 65. Jericho KWF, Langford EV, Pantekoek J: Recovery of Pasteurella hemolytica from aerosols at differing temperature and humi- dity. Can J Comp Med 41:211-214, 1977. 66. Reggiardo C: Role of BVD virus in Shipping Fever in cattle. AAVLD 22:316-320, 1979. 67. Seyle H: In Stress in Health and Disease. Boston, Mass., Butterworths, 1976. 34 68. Jensen R, Pierson RE, Braddy PM, Saari DA, Laverman LH, England JJ, Keyvanfar H, Collier JR, Horton DP, McChesney AE, Benitez A, Christie RM: Shipping Fever pneumonia in yearling feedlot cattle. .JAVMA 169:500-506, 1976. 69. Parker WH: Respiratory disease (Epizootic bronchitis) in housed calves. The Veterinarian 3:235-242, 1965. 70. 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Thorax 29:147-163, 1974. 197. Hance AJ, Crystal RG: The connective tissue of the lung. .Am Rev Resp Dis 112:657-711, 1975. CHAPTER 2 Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: method of induction and changes in the circulating blood constituents during the onset of pulmonary injury 48 49 SUMMARY Six healthy neonatal calves were chilled with cold water and had a focal tracheitis induced by spraying of 5% acetic acid into the tracheal lumen. The effects of these stresses on total and differentiate white cell counts; plasma cortisol, thyroxine, triiodothyronine, histamine and bradykinin; hematocrit, total plasma solids and indices of the erythro- cyte size and hemoglobin content were determined over the subsequent 12 hrs. Cold stress increased plasma cortisol values for less than 1 hr, but did not alter any other variable. This group of calves served as a control group for a second series of neonatal calves (n = 7) which received 2 x 109 organisms of E. haemolytica intratracheally immediately following an identical period of chilling and 5% acetic acid exposure. Calves receiving P. haemolytica became neutropenic. There was a trend toward increased numbers of circulating band neutrophils by 12 hrs post exposure, and plasma cortisol levels were maintained at the same or greater than cold stress levels for all measurement periods subsequent to exposure. Contrary to previous reports, this data suggests a role for the neutrophil in the pathogenesis of early lesions of pasteurello- sis. While the association of corticosteroid release with stress and subsequent infection is clear, our data provide no evidence to support the contention that histamine or bradykinin are involved in the pathoge- nesis of the acute lesions of Pasteurella pneumonia. INTRODUCTION Although Pasteurella Species have been incriminated in Bovine Respiratory Disease complex (8RD) for many years, the mechanisms by which they induce pulmonary injury and the pathophysiology of pneumonic 50 pasteurellosis have not been investigated. In this series of papers we investigate changes in the circulating blood and the lungs during the onset of experimental P. haemolytica pneumonia. A variety of methods are used to induce pneumonic pasteurellosis in cattle.1 In normal animals, instillation of viable bacteria into the lungs generally results in rapid clearance and fails to produce lesions typical for BRD associated with g. haemolytica.2'5 The method we used was initially described by Breeze and Magonigle (1976)6 and does not require the prior exposure of the reSpiratory tract to viruses, yet pro- duces lesions that are very similar to Spontaneously occurring pasteurellosis. Although this method, which utilizes a brief eXposure to cold stress before pathogen exposure, is well documented as a suc- cessful method for induction of E. haemglytica pneumonia, the specific effects of cold stress on the reSpiratory system that may influence establishment of bacterial colonization are not understood. In this report we describe the methods used to initiate Pasteurella pneumonia in calves, and identify alterations in circulating blood constituents including plasma, bradykinin and histamine, serum cortisol, Triiodothyronine (T3) and thyroxine (T4), total solids, hematocrit, the leukogram and hemogram, which result from cold stress and from intratracheal inoculation of P. haemoLytica. MATERIALS AND METHODS Studies were performed on 13 clinically healthy, male neonatal Holstein calves obtained from a local dairy. Calves were deeply sedated 51 with xylazinea (0.1 mg/kg IV) and transported to the MSU Veterinary Hospital where they were allowed to recover. Care was taken to avoid either heat or cold stress to the animals during the transportation pro- cess and during recovery from sedation. Each calf was anesthetized for placement of catheters in the carotid artery and external jugular vein and to make a tracheostomy incision. Anesthesia was induced with halothaneb using a face mask and once the tracheostomy incision was made and endotracheal tube placed, anesthesia was maintained with a halothane-oxygen mixture using an in-circle system. Four hours after recovery from anesthesia, baseline data were collected as outlined below. Following baseline data collection, calves were then stressed by hosing with cold water (approx. 11 C) for 20 minu- tes on two occasions, 12 hrs apart. In addition, each calf was given 0.5 ml of a 5% acetic acid solution in sterile saline, delivered as a Spray to the tracheal mucosa a few centimeters distal to the tracheostomy site. The spray was given twice, immediately following each period of cooling. All variables were measured immediately following the second period of chilling with water and exposure of the trachea to acetic acid. This measurement period was designated “stress“. Calves were then exposed to 20 ml of sterile saline injection (control group n = 6) or to 20 ml of P. haemolytica broth consisting of a suspension of 108 organisms/ml in saline (Pasteurella group n = 7). Instillation was performed rapidly via a large bore catheter and was a Rompun, Haver Lockhart, Shawnee Mission, Kan. b Halothane, Halocarbon Laboratories Inc, Hackensack, NJ. 52 begun at the onset of inspiration. All variables were measured imme- diately following instillation of saline (control group) or of the P. haemolytica suSpension intratracheally and this measurement period designated as T = 0. Measurements were repeated at 1, 2, 3, 6 and 12 hrs after tracheal instillation. The P. haemolytica was obtained from a field isolate of BRDc and was of serotype 1A.d The original isolate was supplied as a pure culture in ovine liver and grown in enrichment brothe for 12 hrs at 37 C. A 1 ml aliquot of this broth was transferred to a second Similar broth and incubated for 6 hrs at 37 C. This second inoculum, totally 50 ml, was centrifuged and washed twice in saline before resuspension in saline. The concentration of 108 organisms/ml was achieved by matching the Opti- cal density of the culture to that of known standard bacterial suspen- sions. Viability of the organisms was confirmed by a quantitative plate count. At each measurement period, blood was withdrawn into siliconized syringes from siliconizedf polyethylene9 catheters placed in the jugular vein and carotid artery. Samples of arterial blood were placed in glass tubes containing sodium EDTA“ for the determination of total and dif- ferential white cell counts, erythrocyte count, hemoglobin content, c The isolate was supplied by Dr. C. Smith, Ohio Agricultural Research and Development Center, Dept. of Veterinary Science, Wooster, Ohio. Typing of the isolate was by Dr. C. Smith, Ohio Agricultural Research and DevelOpment Center, Dept. of Veterinary Science, Wooster, Ohio. Dehydrated BHl, Difco Laboratories, Detroit, Mi. Siliclad, Clay Adams Division, Becton Dickinson and Co, Parsippany, NJ. PE tubing, Clay Adams Division, Becton Dickinson and Co, Parsippany, NJ. EDTA vacutainer, Becton-Dickinson Division, Becton Dickinson and Co, Rutherford, NJ. (3. 3" 40"th 53 packed cell volume and plasma total solids. Characteristics of the erythrocytes (Size and hemoglobin content) were determined using a Coulter counter.i Bradykinin Assay. Paired arterial and venous samples were collected into chilled 10 ml polypropylene tubes which had been previously silico- nized and which contained 3.6 mg of hexadimethrinej and 9.0 mg of sodium EDTA.k Each sample was gently mixed, then centrifuged at 2600 rpm using a chilled centrifuge.1 A 5 ml aliquot of plasma from each sample was precipitated with 0.25 ml of 20% trichloroacetic acid.m After centrifugation, the supernatant and a single rinsed supernatant from the original precipitated pellet were combined and rapidly frozen. These were subsequently analyzed for bradykinin content by radioimunoassayja" Briefly, the thawed supernatant was added to an Amberlite CG-50 column, washed with 0.1 N acetic acid and bradykinin eluted with 50% acetic acid. The eluate containing bradykinin was freeze dried and then resuSpended in sodium barbital buffer. A 16% solution of polyethylene glycol0 was used to separate from free bradykinin. 125I labelled bra- dykinin was used to validate the assay system (recoveries in the initial study were from 90% to 105%, with intra-assay coefficients of variabi- lity of 1 10% and inter-assay coefficients of variance of :_18%). i Coulter counter Models SSR and MHR, Coulter Electronics Inc, Hialeah, Fla. j Polybrene, Polysciences, Inc, Warrington, Penn. k Sodium EDTA, Mallinckrodt, Inc, Paris, Ky. l Model PR-6 Refrigerated centrifuge, International Equipment Co, Needam Heights, Mass. m Trichloroacetic acid, Mallinckrodt, Inc, Paris, Ky. n Radioimmunoassay for bradykinin was performed in the laboratory of Dr. G. Williams, Brigham Young HOSpital, Boston, Mass. 0 Carbowax 6000, Union Carbide Inc. 54 Histamine Assay. Paired arterial and venous samples were collected into chilled, heparin coated glass test tubes.P Samples were centri- fuged at 2600 rmp for 10 minutes and the plasma separated and rapidly frozen. Histamine content was determined by fluorometric assay.3iq The thawed samples were dialyzed against 30% NaCl and the histamine extracted after reacting with NaOH containing 1 x 10‘3M EDTA and N- butanol. The histamine, still in aqueous phase was acidified with 0.1 N HCl and N-heptane added. The aqueous phase, again containing histamine, was extracted, alkalinized and O-phthalaldehyde (OPT) reacted with hista- mine. After a Suitable period for reaction had elapsed the reaction was stopped by the addition of 0.73 M phosphoric acid and the amount of histamine bound to OPT determined with a fluoronephlometer with exciting filter peak transmission at 350 nm.r Sensitivity of the assay was in the order of 100 pg/ml. Assay for Thyroxine (T4),Triiodothyronine_(T3)and Cortisol. Paired arterial and venous blood samples were collected into glass test tubes and allowed to clot. The samples were centrifuged and the serum collected and frozen for subsequent analysis of T3, T4 and cortisol by radioimmunoassay. Serum samples for T3 analysis were assayed using a modified commer- cially available 1251 labeled T3 radioimmunoassay.q The procedure was modified by reconstituting the antibody with a greater volume of buffer (8 vs 5 ml) and by combining 1251 T3 tracer with buffer before mixing p Heparin vacutainer, Becton-Dickinson Division, Becton Dickinson and Co, Rutherford, NJ. q Radioimmunoassay for histamine was performed in the laboratory of Dr. W. Hook, National Institute of Health, Bethesda, Md. r Technicon Corp., Tarrytown, NY. 55 with the sample. The incubation procedure was also modified so that initially the sample stood at 37 C for 30 minutes, was chilled to 0 C for 10 to 15 minutes and then left overnight at 4 C. Samples were eluted against 0.5 ml of cold charcoal solution for 10 minutes. Following centrifugation for 10 minutes at 3200 rpm, each tube (which contained 100 ml of 1251 tracer solution) had 75 mg of 8-anilino-1-naphthalene sulfonic acidS (ANSA) added. Sensitivity of the assay at 90% of total of the standard curve was 0.28 ng/ml. Intra-assay coefficient of variability of the T3 test with bovine serum was :_6.5% and inter-assay coefficient of variability was :_1.3%. Serum T4 was assayed using a modified commercially availablet solid phase 1251 labeled T4 radioimmunoassay. A sample of 30 ul was used to which 0.23 ng of ANSA was added, in order to increase sensitivity. Incubation took place over 2 hrs at 37 C before decanting the tubes. The sensitivity of the T4 assay was 1.5 ng/ml at 90% of the standard curve. Intra-assay coefficient of variability was i 4.3% and inter- assay coefficient of variation 1 5.1%. The assay had complete cross reactivity (100%) with thyroxine isomers but negligible cross reactivity to DL-Thyronine (0.04%). Reverse T3 had 14% cross reactivity. Cortisol was assayed using a modified commercially availableu solid phase 1251 labeled cortisol radioimmunoassay (RIA). The procedure was modified for use with bovine plasma by taking larger samples volumes (20 pl), adding an additional 0.2 mg 8-anilino-1-naphthalene sulfonic acid s ANS, Sigma Chemical Co, St. Louis, Mo. t T4 solid phase radioimmunoassay, Becton-Dickinson Immunodiagnostics, Orangeburg, NY. u Clinical Assays, Cambridge, Mass. 56 (ANSA) per sample tube, and incubating at 37 C for 2 hours before decanting the tubes. Specificity tests of the antiserum indicated 65.8%, 3.8% and 2.1% cross reactivity for Prednisolone, Prednisone and Corticosterone, respectively. Cross reactivity was (1% for Cortisone, Deoxycorticosterone, Dexamethasone, Progesterone and Betamethasone. Precision on replicated quality control samples indicated (10% intra and interassay coefficients of variation. Sensitivity, as calculated from the standard curve at 90% of total trace binding was 3.8 ng/ml and calculated from 0‘: 2 s.d. was (1 ng/ml. Dilution and recovery studies indicated results which supported the validity of the assays for T3 T4 and cortisol.V Serum Specifity of Antibodies Against P. haemolypica. A Single serum sample was taken from each animal during the baseline data collec- tion period in the Pasteurella group. These serums were analyzed for cross reactivity to somatic and capsular antigensw of the strain of P. haemoLytica used to challenge the calves. Titers to capsular antigens were determined by hemagglutination and those to somatic antigens deter- mined by agglutination to specific rabbit antibovine globulin. v Radioimmunoassay for T3 T4 and cortisol was performed in the labora- tory of Dr. R. Nachreiner, Michigan State University, East Lansing. w Courtesy of Dr. C. Smith, Ohio Agricultural Research and Development Center, Dept. of Veterinary Science, Wooster, Ohio. 57 RESULTS There was no alteration in the total white cell count (WBC) in the control group of calves during the course of the experiment. In the Pasteurella group, W8C had decreased significantly by T = 3, and was less than the control group at T = 2, T = 3 and T = 6 hrs. (Figure 2-1) The decline in WBC in the Pasteurella group was caused by a significant decrease in numbers of segmented neutrophils. In the control group of calves, segmented neutrOphil numbers remained constant. (Figure 2-2) Nonsegmented neutr0phil numbers were not significantly different in the control calves compared to the calves in the Pasteurella group. Both groups had significantly increased by T = 6 compared to stress but no significant differences between baseline measurements and any other measurement period were found. There were no significant differences between groups of calves or over time for monocytes, mean :_SEM 277 1.357 cells/mm3), eosinophils (2 1 1 cells/m3) baSOphils (9 1 3 cells/m3) or lymphocytes (2940 1 951 cells/mm3). Plasma total solids (Figure 2-3), hematocrit (Figure 2-4), total erythrocyte count (Figure 2-5) and hemoglobin content (Figure 2-6) had significant and similar declines during the course of the experiment in both groups of calves. No effect of Pasteurella exposure was found. Mean corpuscular volume (mean :_SEM = 36.7 1 2.5 u3), mean corpuscular hemoglobin (mean :_SEM = 13.1 1 0.7 mg) and mean corpuscular hemoglobin concentration were unchanged during the course of the experiment but mean corpuscular hemoglobin concentration was slightly but significantly less in the Pasteurella group of calves (35.5 g/dl cmnpared to 37.2 g/dl). The differences between the calf groups were not significant for any individual measurement period, therefore suggesting minor 58 .Pm>m— mo.o ozu pm asocm acmEuomcu a cow m:_~immmn ucm muo_cma acmsmcammwe cmmzuwn mmocmcmwwwc ucmuwmwcmwm 8.8ch4 Acaa_oo umwocm x—chomm_u ucoommv maaocm ucmsucmcu :mwzumn comwgmasou Low new “can—cu umuezm x__mocmc_u umc_wv aaocm m__mc=mumee we“ to» Assn—cu vacuum; wcmzcmv quota mmmcum uFou mcp Low .uzmve o» “mm— soc» Lento :_ umumcumsp—w m? mvo_cmq ucosmgzmeme :mwzwmn com_cmaeoo to» ovumwuepmae mxmxzh newton mesmoaxm on» cmpww meson m>_m3u u L; NH vo_cwa mgzmoqu any cmuwo meson xwm n L; m vo_cma mesmoaxw may Lmuwm mczoc omega u L; m newton mcamoaxw mzu Lmuwe meson oz“ n L; N vo_cwa mcamoaxm ecu cmute Lao; mac n L; H Ansocm oppoeamumeev mcwpmm cw cmucmamzm eu_uonemez .m.co Aaaoem mmmcum nPoov mc__om mp_coum ;u_z cowumpsoo:_ mecumcumcu:_ Lmumm »_muo_vmeew u mesmoaxm .u_um o_umum :u_x maggot“ asp mo m:_xecam use Lopez u_ou ;p_z mew—F_;u mcwzo_—o» x_muo_umesw n mmmcum mucmsmcammme m:_~mmmn _owp_:w u mew-omen i. "mew muowcma “cosmcamewe we» .AmcE:pou emcouo; »__ecomc_nv mesmoqu muwuxgosme; .a :u_z vmcwaeou mmmcum v—oo ecu AmcE:_oo vacuum; mcesamv mcopo mmmepm vPou mmm>Pou mo masocm oz» :_ uczou P—mu mu_;x Page» voopa :_ mcowumcmpF< .Him mczmwe 59 :ii;igiiiiiiiiigfiiiiiii?iiii; €- 40 0'1 up -' o 85 g E” 5 {3 o E: ”o 0" ‘- l-0 3 Figure 2-l 60 .—w>m_ mo.o as» no aaoem acmEuwmep a com m:_Pimmmn new muo_cma ucmEmgzmwos :mmzpmn mmocmcmwm_u ucmuwmwcmwm mmpocoow A:E:_oo umcezm »__m:ommwc ucoowmv masocm acmEpowLu :mwzumn comwcmasou Low can “can—cu umvmgm xppocommwu umcwmv quota wppwcampmwe mzu Low Assn—cu vacuum; mcmzcmv qzoem mmmepm epoo asp to» .u;m_e o“ ummF seem gouge cw umumcpm:___ we muowcwa pcmsmcsmmms :mozumn com_coqeoo Low ovum_uopmxe mawxzk cowcma meamoqu asp emuwo meson m>Pw2u u L; NH newton mesmoqxw we“ tween meson xwm u L; m newton mgzmoqu we“ Lmuem meson omen» n L; m cowemq mezmoaxm men Lmuwm mczo: oz» u L; N vowemq mesmoaxw asp mewm Lao; oco u L: H .Aasoem m_FmL:mummev mcwpem :_ umucmqmzm mowHN-oamm; .m.co Ansocm mmmtpm u_ouv m:__mm m_wcmpm sue: :o_ue~=uo:_ mezumcumcuc_ memm meum_umee_ n mesmoaxm .vwom uwpmum ewe: mmzuoep we“ we m:_cham use Lopez w-oo sue: mcwppwsu mcwzoFPow waum_umEEw u mmmcpm mucmemgsmems mew—omen —mwuw:w u mewpmmma i. "mew meowcma acmsmczmmma we» .Amcespou umgopm; x_Fe=omewvv meamoqu muwpxposme; .e cpvz umcwneoo mmmcum upoo use Ance=_oo umsoum; memzcmv meo_e mmmepm UPoo mmm>Fmo mo mazocm 02p cw pczoo ngaocuam: umucmEmmm voopn mzu cw meowpwcmpp< .mim mczmwm 61 ............................ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Fu'i Segmented Neutrophlle |.. log .0 Absolute Counts Baseline Figure 2-2 62 .Hm>mH mo.o men He qzoem ucmspewcp a Low mCHHimmen use muowcmq “cosmeamoms cmmzumn mmocmemmmwv acmu_mH:mHm monocmne HcsaHou umumzm AHHecomoHu ecoummv masocm HamEHemcu comzumn comHLMQeou Low use HceaHou emuocm HHHmcomeHv HmLHGV qaoem oHHmezmpmee 059 com .H253Hoo vagoue; acmscmv quota mmmeum uHoo on» com .pzmHL op HemH soc» cmuco :_ umueepmsHHH mH muowemq pcmsmtsmmme cmmzumn comHLmqeou to» QHHmHHmume. mxmxzb onLoa wcsmoqu mg» cmpwe meson m>Hmzu u L; NH coHch mesmoqu we» Lmumm meson me n e; o vowemq mesmoqu meg cmpmm meson omega n L: m cowemq mgzmoaxm we» Lmumw mezo; 03“ u L; N noHemq meamoaxm wcu empwm Lao; mac n L; H anocm wHngsmummmv mcHme cw umucmamzm euwuxgoemm; .m.co quocm mmmegm uHouv mcHHmm mHHLmum saw: =o_HmH:uo:_ Hmmsumcumeu:_ memm meHvawEEH n mczmoqu kue uHHwom cpwz manomeu we“ to mcmecam new empe: uHou saw: mew—HHsu mcwonHom meHmHumEEH n mmmgum mpcmsmeameme mcHHmmwn HmHHH:H u m:_meen i. "mew meowcma acmsmczmmme we» .Hm:E:Hoo um50Hm; xHHecommHuv mesmoqu eoHHNHoEmm; .a zqu um:_neoo mmmcpm uHoo use HmceaHou vacuum: meezomv mcon mmmcpm uHou Hmm>Heu mo mazoem oz» cw muHHom Heuop mamqu CH mcoHHeLmuH< .m-N mcamwu 63 in 23 (7) § § tn 64 .Ho>mH mo.o mzu Ho ooocm “coauomcu o com ocwHiomoo oco mooHLoa ucosoeomooe :oozuoo moococomeo HooUHHHcmHm mopocooe AesoHoo oooozm HHHocomoHo ocooomv masocm “cospooeu coozuoo comwcoosoo com oco HceoHoo oooocm HHHocomoHo umg_wv oooem oHHoeooumoo oz“ cow .H:E:Hoo oosouo; ocooomv oooem mmoepm oHoo we» to» .HzmHL op uon Eocm Looco :H oooocpmoHHH mH moowcoo ucosoeomoos coozuoo cochooEoo to» oHHmHHon.e mxosz oowcoa mcomooxo ocu emote meson o>Hoxp u L: NH oowcoo meomooxo asp Lopmo mono; me u L; m ooHLoo ocomooxo osp Loumo meson oocsu u L; m ooHLoo ocomooxo on» Lopmo meson oz» u L; N ooHLoo ocomooxo on» Lopwo Loo; oco n L; H Hoooem oHHocoopmoov oCHHom :_ ooocoomom ooHHHHoEoo; .m.eo Hooocm mmoeom oHoov oCHHom oHHLopm csz :oHuoHooo:_ HoocoocpoeocH Loumm xHoHoHooEEH u ocomooxo owuo oHHooo ;p_z oogooep oso Ho mCHHoeom new Loom: oHoo csz mew—ngo mcwonHow xHoHoHooEEH u mmoeum mpcosoeomooe ocwHomoo Howuwcw n o:_Homoo i. ”moo mooHeoo Hoosocomooe ogp .Hm:E:Hoo oosopo: HHHocomoHUV oeomooxo ouHuxHoEoo; .o ;p_3 oo=HDEoo mmocpm oHoo oco AwesoHoo oosouo; oeooomv ocoHo mmoepm oHoo Hmo>Hoo wo monocm ozH cw HHLoouoEo; oooHo :H mooHHoLoHH< .oiN oeom_m 65 ///////// 777’7777"'.f,"'_:',’:’.’."7//*""77”77 // // [IV/74111777147 7/u/juxigz'u ///// ooooouoo..-oo 0......o..-o..o..-..¢.......-+ beer...-uuoouoooooon.e...-oov--.o....ooo--4o /7/'7777"'7/ 777t'7'f"7-'- ”"7".“ *5 7777/7777 / ' , ./ :7: 71,-;7;..,-/"- ,/ 77.7 ,7 // //I I, I . 7 '7’ _: v . N," / -’4,7'/ I l/ 1‘ 1’7 1 V 1 ,7 ,4 . . s v, .7 1'. .' , J .7 ,717‘4 11A / ............................................ 64 ........................................... . . 4 m ........................................... 01 A ELL 1 tfflUU/vv—vvwrv‘vv-vuuuuuuf vvvvvvvv ............................................. {{/// ;{// /////,//77//IZ/r/rk/I/IZ’rr‘;;7/77’//’////// ///1':/11/////JA//1 [4411' 411417444'1144144111S / ///7 /// /7/77/7 // /;7/7 eff/"W ///7’” , 7777/77 7/ 7 7 //////L/Mu.1441141/l/:'1///u 4’1'444/444444444A4L44L/4444 ........................................... * ................................. o ........... .094 A . I ///////Z 7%//7/‘/// 7?”2// ooooooooooooooooooooooooooooooooooooooooooooooooo ................................................. y. ................................................. 4 .o. . . ‘ -‘ U11L11L2L1LL212112 22111 1‘21211‘211’,’ IW/llf/rlfrvvfffi7/’",/,”’A.//_' ."’//, /// ///11L/1//(1/1414114u144/41//114/144‘1.4"A.14.;4 ‘ /////// ”’////oH mo.o mg» po ooocm Hoosuooep o Low ocwHiomon oco mooHLoo Homeocomooe coozpon moococowwwo pcoowwwcmwm mopocooe HceoHoo oooozm AHHocomoHo ocooomv moooem ucoeuoocu coozpoo comwcooeoo tom oeo AssoHoo oooozm HHHocomoHo pme_wv oooem oHHocooumoo mew com .H:E=Hoo oogouo; ocooomv ooocm mmoeum oHoo oz» cow .ocmHL ou pmoH Eoem Looco :H oouoeumoHHH mH mooHLoo pcoaocomooe coozpoo comweooeoo Low oHumHuon.s mxoxah oowcoo oeomooxo ocp Lopmo meson o>HozH u L; NH ooHLoo ocomooxo osu emote mcooc me n L; m oowcoo oeomooxo ocp Lopeo meson omega u L; m ooHLoo ocomooxo ogu emote meson ozp u L; N oowcoo ogomooxo on» Louwo Loo; mac n c; H Hooocm oHHoeooumoav m:_Hom :_ ooocoomom ooHHNHoeoo; .m.co Hoaocm mmoeum oHooV o:_Hom oHHEon cow: :oHHoHooocH Hoozoocuogucw Loumo xHouoHooEEH u oesmooxo oHoo oHHooo saw: oozooep osu mo m:_xoeom oco Logo: oHoo saw: mcwHszo mcwonHom aHoHoHooEEH u mmoeum mpcmEoczmooe o:_Homoo HoHHH=H u o:_Homoo i. "moo moowgoo ucoeoeomooe on» .Hm:E:Hoo oozouo; AHHocomoHov ocomooxo ooHHxHoEoo; .o ;H_z oocHneoo mmocpm oHoo oco HmchHoo oosopo; oeosomv ocoHo mmoeom oHoo mmo>Hoo mo masocm oz» :H pcooo ouxooecuxco oooHo cw meoHHoLoHH< .miN oeomwm 67 2hr. I h\r. Stress Exposure /7 -_ H -o-o an o ._._._. ............... l lHHHHHHHHa34». ” ..4 “HAW. ..4 eeeetirltrvtttttx‘fiilfilttti ......................... o 504 40 3.0-4 20 IO 0 x IO‘cells Erythrocytes mm Figure 2-5 68 .Ho>oH mo.o ago we oooem pooEHooLH o co; ocHHiomoo oco moowcoo ucosoeomooe coozpon moocoeomwwo pcoowwwcmwm mouocoo4 AesoHoo oooosm HHHocomoHo ocoommv moooem pooEHooLu coozuoo comwcooeoo com com AesoHoo oooozm HHHocomoHo Hmc_wv ooocm oHHocoopmoe «so com .HcE=Hoo oosouo; mcooomv ooocm mmmepm oHoo on» Low .ugmHL on HmoH soc» Looco :_ oouocpmoHHH mH mooHLoo Hcosocomooe coozuon comwcooaoo com oHHmHHonAe whoxoh ooHLoo ocomooxo any Loumo meson m>Hozp u L; NH oowcoo oeomooxo ogu Loumo meson me u L; o ooweoo oeomooxo mg» Louwo mono; omega u L; m ooHLoo oeomooxo map Loumo meson oz» u c; N ooHEoo oczmooxo ozu Loumo Loo; oco u L; H Hoooco oHHocooumoav ocHHom :H ooocoomom ooHumHosmo; .m.go Hooocm mmoepm oHoov o:_Hom oHHLoom ngz :oHHoHooocH Hoogooeuocucw Lopmo xHopoHoosEH u ogomoqxo oHoo owuooo 5HHz oosoocu ogp mo mcwxocom oco Loam: oHoo saw: mow—H_;o mcwonHow xHouoHooEEH n mmmcpm mucosocomoos ocwHomoo HoHHHcH n ocHHomoo i. "moo moowcoo pcosocomoos on» .Hm:E:Hoo oogopo; HHHocomoHov oeomooxo ooHuxHoeoo; .m spH: oocwoeoo mmocpm oHoo oco HmcsoHoo oogouo; oeooomv oeoHo mmocum oHoo Hmo>Hoo mo monogm ozu :H pcoueoo :Hnonoeo; oooHo cw mcowuogopH< .oiN ocomwe 69 Iz'nt I2- Blood IO- Hemoglobin Content 9 MI Tukey's ta Figure 2-6 7O differences in hemoglobin concentration existed between the two groups of calves, unassociated with the develOpment of pasteurellosis. Serum triiodothyronine (T3) was significantly larger in the Pasteurella calves than the control group even before exposure to Pasteurella haemolytica. (Figure 2-7) Both groups of calves had decreases in serum T3 during the course of the experiment. No specific effect of P. haemolytica on T3 serum concentration was detected and there was no difference between arterial and venous serum T3 levels. Taken on an individual measurement period basis, T3 differed signifi- cantly between control and Pasteurella calves only at the stress period being greater in the Pasteurella group. Serum thyroxine levels (T4) were altered similarly to T3 over the course of the experiment (Figure 2-8) and were also greater in Pasteurella calves even during baseline measurements. As with T3, dif- ferences in T4 levels between groups of calves were not different for the latter half of the study. Serum cortisol levels are illustrated in Figure 2-9. Serum cortisol levels of control and Pasteurella calves were not significantly dif- ferent under baseline conditions. In the control group, stress was associated with a rise in serum cortisol levels, not significant by either LSD or Tukeys' test. Serum cortisol rose significantly with stress in the Pasteurella group. The difference in cortisol levels bet- ween calf groups at the stress measurement period was not significant. In control calves, cortisol steadily decreased from stress values over the remaining portion of the experiment. However, Pasteurella exposed calves maintained cortisol levels at or greater than stress levels for the remainder of the experiment. 71 .Ho>oH mo.o oga ao ooocm acoanoea o Low ocwHiomoo oco mooacoo acoeoeomoos coo2aoo moococowmao acooawacmam moaocoo« HogaHoo oooosm HHHocomoHo ocooomV masocm acoanoLa coozaoo comacoosoo Low oco Haas—co oooocm HHHocomoHo amgHmv ooocm oHHocooamoe oza cow .Hce:Hoo oosoao; oeooomv oooem mmoeam oHoo oca to» .acch oa amoH Eoea Looeo :H ooaocamoHHH ma mooHLoo acoeoeomooe coozaon comacoosoo to» oHamHaoam.e maoxoh ooHLoo ocomooxo oca goaao meson o>Hoza n g; NH ooacoo ocomooxo oga coaoo meson xam n L; m ooHLoo ocsmooxo oca Loaoo meoo; oocza u L; m ooacoo ocomooxo osa eoawo mono; oza n L; N ooHLoo oeomooxo oga coamo Loo; oco u L; H Hoooem oHHocooamoov ocwHom ca ooocoomom oowaxHoEoo: .m.Lo Hooocm mmoeam oHoov ocaHom oHHLoam saw; :oHaoHoooca Hoogooeaoea:_ Loaao xHoaoHooeea n oeomooxo oaoo oaaooo saaz oozooea oza mo mconLom oco Loam: oHoo caaz mcwHngo mcwonHow aHoaoHooEEH n mmoeam manoeocomoos ocaHomoo HoHchH u ocwHomoo i. "oco moowcoo acosoeomooe on» .Hmce:Hoo oocoao; HHHocomoHov ogomooxo ooHaHHosoo; .e :aH3 oocwnsoo mmocam oHoo oco AwesoHoo oozoao; otooamv ocoHo mmoeam oHoo mmo>Hoo mo masocm oza ca Hmhv ocacoexzaoooaaea Eoeom ca mcoaaocoaH< .NiN oeomau 72 ? ' c3 6 ' T V: 8 co 9! 8 8 N N —' - ' ' I O '32- E __ I: .21: z. 1: an e: i-‘E Figure 2-7 73 .Ho>oH mo.o oga ao oooem acosaooea o Low ocaHiomoo oco mooacoo acosocomoos coozaoo moocoeommao acooamacmHm moaocoo« H:E:Hoo oooozm HHHocomoHo ocooomv monocm acoanoLa coozaoo comacooeoo com oco AesoHoo oooogm xHHocomoHo ameamv ooocm oHHogooamoa oza to» .H:E:Hoo oosoao; ocooomv ooocm mmoeam oHoo oga cow .agmae oa aon soc» Looeo :H ooaocamaHHH ma mooHLoo acosocomoos :oozaoo comHLooeoo cow oaamaaoam 3 mxoxoh ooHLoo ocomooxo oza Loamo mono; o>Ho3a u L; NH ooacoo oeomooxo oga Loaao mcoo; me u L; m ooHLoo ocomooxo oga Loaoo meson ooeza u L; m ooHLoo ocomooxo oga Loamo meson oza n L: N ooaeoo ocomooxo oca Loamo Loo; oco u L; H Hoooem oHHocooamoev ocHHom :H ooocoomom ooHewHoeoo; .m.co Hoooem mmoeam oHoov ocaHom oneoam saaz :oHaoHooocH HoocoocaocacH Loamo aHoaoHooEEH u oeomooxo oaoo oaaooo caaz oocooea oga to mcaxoeom oco zaaz mcHHHaso acaonHom HHoaoaooEEH n mmoeam macosocomooa ocaHomoo Howaacw n ocHHomoo "oeo mooHLoo acoeocomoos och .Hmc53Hoo HHHocomoaov oezmooxo ooHaxHoeoo; .m.;aH: oocHoEoo mmoeam oHoo oco AwesoHoo oosoao; ocooomv ocoHo mmoeam oHoo Hmo>Hoo Ho quoem oza :H Hohv ocaxocxza Eoeom :_ mcoaaocoaH< .wiN ooomau 74 1? /'§ 13:. i; -—- l- ooo-oowoooo.¢.~o~o4 WW ................. 6hr. ooc~a .o.....¢--.. .................. oooooooooooooooo Figure 2-8 75 .Ho>oH mo.o oga ao ooocm acoanoLa o to» ocHHiomoo oco mooacoo acosocsmooe coozaoo moococommao acooHHHcmHm moaocooe HcE:Hoo oooosm HHHocomoHo ocooomv masocm acoanoea coozaoo comHLooEoo now use Hess—co oooogm HHHocomoHo ameva ozoem oHHoeooamoo oga goo .H:E:Hoo oogoao; ogooomv oooem mmocam oHoo oga Low .azmae oa amoH soot eooco ca ooaocamsHHH ma moowgoo acoEoeomooE coozaoo comHLooeoo Low oHamHaoamze mxoxoh ooaeoo ocomooxo oca Loamo meson o>Hoza u L; NH ooHeoo ogomooxo oza Loaao meoo; me u e; m ooacoo ocsmooxo oca Loamo meson oocca u L; m ooacoo ocomooxo oga Loamo meso; oza u L; N ooHLoo ogomooxo oza Loamo Loo; oco u g; H Hoooem oHHocooamoev ocwHom :H ooocoomom ooHaNMosoo; .m.eo Hooocm mmoeam oHoov ocaHom oHHLoam :aHz :oHaoHooo=H HoozoocaocacH coamo HHoaoaooEEH n ocomoqxo oHoo oHaooo gaaz oozooea oza Ho acaxoeom oco saw: acaHHH;o acaonHom HHoaoHooEEH u mmocam macoeoeomooe ocaHomoo HoHchH u ocHHomoo i. "oeo moowgoq acosocomooa osh .Hm:E=Hoo oogoao; aHHocomoaov ocamooxo ooaaxHoEoo; .a caHz oocaoeoo mmoeam oHoo oco Hm:E=Hoo oocoao; ocooomv ocoHo mmocam oHoo Hmo>Hoo mo monogm oza :H HomHaeoo Ezeom ca mcoaaoeoaH< .miN oeomao 76 :2 -: ......... 4.. ............... A....'.""f- ............ lhn line isitess qulosure -' Q Q (0 ¢ N o_ - ° E £21; ¢: 0-0 U F1'gure 2-9 77 There was a significant difference between arterial and venous plasma histamine, with venous samples having greater values (3.09 i 0.12 ng/ml and 2.89 i 0.11 ng/ml for venous and arterial samples respectively). There were no significant differences between groups of calves and no detected changes in histamine levels associated with P. haemolytica exposure. Both groups had small declines in histamine levels with a minimum value reached by T = 2. Plasma histamine rose after T = 3 so that peak histamine levels were present in both groups of calves by T = 12. Plasma bradykinin levels were only determined in the Pasteurella exposed group of calves. There were no arteriovenous differences and no significant effects of exposure to P, haemolytica (mean :_SEM = l.70;: 0.86 ng/ml). Serological titers to the capsular antigens 0f.E' haemolytica were determined for the Pasteurella calves by hemagglutination test. All titers were 1/8 or less and were considered negative. Serologic titers for the somatic (0) antigens were also determined in the Pasteurella calves by agglutination to specific rabbit antibovine globulin. Low level positive titer (1:2 or less) were obtained in 3/7 calves, with remaining calves having negative titers. DISCUSSION During the course of the experiment, an estimated 20 to 25% of blood volume was removed for the various samples and was replaced with normal saline. Because of the removal of blood cells and dilution of plasma crystalloids with saline, we anticipated a reduction in hematocrit, total solids, erythrocyte and leukocyte counts and hemoglobin 78 concentration. With the exception of total white cell count, the changes in these hematologic variables were identical for both groups of calves, indicating that the changes were due to sampling and that intra- pulmonary challenge with_P. haemolytica does not result in erythrocyte damage or loss of plasma proteins. The lack of a decrease in total white cell counts in control calves indicates sufficient leukocyte mobilization into the circulating pool to replace those lost by repetitive blood sampling. In contrast, calves exposed to P. haemolytica had decreased total leukocyte numbers exceeding anticipated sampling effects. Pasteurella infection might cause this change by either increased utilization of leukocytes or by failure of their replacement in the circulating pool. Our data suggests that the decline in leukocyte numbers is caused by a decrease in cir- culating neutrOphils and from our histologic studies (see Chapter 4) indicates that these cells aggregate in very large numbers in the lungs. There was an increase in band neutrOphils in the circulating blood of Pasteurella calves by T = 12 but this response was insufficient to replace granulocytes lost from the circulation. Lymphocyte, monocyte, eosinOphil and basophil numbers failed to change significantly from baseline levels in both groups of calves suggesting that these cells are less important in the initial reSponse to P. haemolytica than are neutrOphils. This suggestion is supported by histologic findings, since the former cells are not consistently featured in the inflammatory response noted in the lungs of calves exposed to P. haemolytica.1 The normal neonatal calf leukogram and hemogram have been well described and are reviewed in current veterinary texts of clinical 79 pathology.9:10 Baseline values of the leukogram and hemogram of the calves in our study are similar to those reported in these reviewsgslo, with large numbers of circulating leukocytes, primarily neutrOphils when compared to adult cattle. Routine perinatal stresses are cited as responsible for causing this leukogram.9 These stresses are throught to release corticosteroids which, from inference to studies in older cattle, depress lymphocyte and eosinOphil numbers and increase neutrOphil numbers.9o10s11 If the leukograms of neonatal calves are indeed dictated by corticosteroid levels, it is surprising that increased plasma cortisol levels induced by cold stress were not asso- ciated with changes in either the differential or total white cell count. Our data suggests that the bovine neonatal leukogram does not respond in a linear dose-response relationship to cortisol eXposure. This hypothesis is supported by failure of repeated doses of ACTH to have additive effects on the leukogram of young calves.11 Also, adult cows which were injected with corticosteroid doses ranging from 20 to 500 mg have similar alterations in their leukograms9, deSpite 25 fold differences in steroid dose. Since the alterations in serum cortisol of neonatal calves did not produce associated changes in the leukogram, the neutropenia observed in calves with Pasteurella infection is probably not due to cortisol related effects. Endotoxin, a component of gram negative bacterial cell walls, causes leukOpenia, pulmonary edema, fever and accumulation of leukocytes in the lungs of calves injected with E. gpli endotoxin.12:13 Since each calf in our study received 2 x 109_P. haemolytica organisms, this represents a considerable load of endotoxin even if no further bac- terial replication occurred. It is likely that endotoxin from 80 Pasturella haemolytica contributes to the pathogenesis of pasteurellosis by stimulating rapid migration of leukocytes into the lungs and causing vascular damage. To date the extent of that contribution is unknown. We Speculated that thyroid hormones might exert an influence on the pathogenesis of pulmonary injury in calves because hypothyroidism is associated with neonatal pulmonary disease in human infants14 and because thyroid hormones may indirectly affect the metabolism of histamine.15 In other Species thyroid hormones increase in response to chronic rather than acute deviations in environmental temperature so it was therefore not surprising that plasma T3 and T4 of calves remained unchanged after cold stress.16 Pasteurella infection had no effect on plasma T3 and T4. Since plasma T3 and T4 levels do not necessarily reflect thyroid hormone activities in peripheral tissuesl7418, the failure to detect changes in plasma T3 and T4 levels from nonnal sub- sequent to Pasteurella infection does not exclude their role in the pathogenesis of Pasteurella injury. There were significant differences between the T3 and T4 levels of the two groups of calves unrelated to g. haemolytica exposure. These differences may relate to seasonal variations in thyroid hormone levels, since the Pasteurella group of calves was studied over the winter months, when hormone levels are greatest, and the control group of calves was studied in early spring, when thyroid hormone levels repor- tedly decrease below winter levels.19 Although adaptation to cold stress is generally cited as an explanation for maximal thyroid hormone levels in winter, calves in this study were not exposed to winter tem- peratures. Bobeck et al (1980) described peaks in thyroid secretion of calves born in winter but never exposed to outside environmental 81 temperatures, indicating as in our study, that plasma thyroid hormone levels are affected by seasonal factors other than temperature.19 Serum T3 and T4 levels decreased significantly during the course of the experiment in both groups of calves. Since baseline, stress and T = 0 measurements were taken in the morning, with remaining measurement periods extending into the evening, diurnal variations in serum T3 and T4 may account for the declining thyroid hormone values observed over the course of the experiment. Diurnal patterns of thyroid hormone secretion have not been determined in neonatal calves, and although dif- ferences occur with the extent of diurnal rhythm between Species, they are certainly present in older cattle, persons and rats.20'22 Interpretation of our data is further complicated by the influence of plasma dilution during sampling and by declines in serum T3 and T4 levels which occur from the time of birth up until approximately one week of age when values approach adult levels.23:24 Cold stress in calves resulted in increased serum cortisol. This was expected since a number of researchers have demonstrated the respon- siveness of the young calf adrenal to various stressful stimuli.11a25‘27 The decline in cortisol levels subsequent to cold stress in control calves is probably the result of the interaction of the following factors; decline in baseline cortisol secretion rates which occur in the first few weeks of neonatal life28429; dilution of plasma during blood sampling; removal of the stressful stimulus; diurnal variations in the secretion of corticosteroids.30»31 These influences would similarly affect calves in the Pasteurella group and yet this group of calves had cortisol levels maintained at or above stress measurement levels. Acute P. haemolytica infection therefore stimulates .fi 82 cortisol secretion in some manner. "Stress" through disease, resulting in elevated cortisol levels is similarly described for neonatal calves suffering from diarrhea.29 Although the increased cortisol levels may assist indirectly in recovery from the infection by facilitation of uptake of ingested immunoglobulin32 and in stabilization of cell membranes in response to injury33, corticosteroid increases may cause adverse sequelae through hnnune suppression and loss of the ability to efficiently dispose of bacteria in the lungs. The exact role of cor- ticosteroids in modulating immune function of calves is unclear. For example, administration of corticoids to calves is associated with lymphopenia but also with improved interferon responses to viral exposure34, even though interferon is thought to be produced by lympho- cytes. The effect of postnatal corticosteroid exposure on pulmonary growth and differentiation in calves is unknown but may be of impor- tance. The administration of corticoids to premature human neonates aids survival by stimulating surfactant production, but postnatal admi- nistration may be detrimental by reducing pulmonary growth and by other Steroid related changes in the body.35 In other species, infectious disease processes may involve the so- called "mediators of anaphylaxis" in pulmonary injury.35:37 We were curious to determine whether the mediators histamine and bradykinin contributed to the pulmonary injury induced by pasteurellosis. Pasteurellosis was not associated with an increase in plasma hista- mine. The interpretation of plasma histamine determinations as they relate to important physiologic events is more difficult than for brady- kinin for the following reasons; plasma histamine does not represent histamine kinetics from a single body source or compartment. Most 83 histamine is synthesized in connective tissue mast cells and only a pro- portion of histamine released from this source reaches the vascular pool. Since the biological effects of extravascular histamine may be pronounced, it is unclear whether biological effects of histamine arising from mast cell degranulation is adequately reflected by changes in plasma histamine concentrations. A second source of histamine arises from the non-mast cell sources. It is produced in association prin- cipally with exocrine gland function and increased plasma levels occur as a result of cholinergic stimulation or decreased corticosteroids in plasma.38 Alterations in this plasma pool may occur rapidly in response to cholinergic stimuli, but may not necessarily be associated with an increase in tissue histamine levels within the lung. Lastly, the biolo- gic significance of a certain level of plasma histamine may differ, depending on the presence or absence of other mediators, autonomic influences and tissue viability. Therefore, the failure to detect significant increases in plasma histamine associated with E. haemolytica pneumonia does not exclude a possible role for pulmonary mast cells in the pathogenesis of infectious pneumonia, but does exclude the influence of circulating histamine originating from body sources outside the lungs. Assuming pulmonary mast cells do expose the lungs to high local tissue levels of histamine during pasteurellosis, it remains unlikely that administered antihistamines, which must diffuse down a con- centration gradient from the vascular space into the lung tissue, can reach Significant concentrations to prevent the noxious effects of histamine. Therefore, any therapeutic value to antihistamine admi- nistration in cattle affected with pasteurellosis is probably unrelated to antihistaminic drug action. 84 There was a significant arteriovenous difference in plasma histamine across the lung, with samples of blood from the vena cava having greater values than carotid arterial blood. This finding does not necessarily imply histamine catabolism by pulmonary tissues, since the AV difference could arise as a result of intravascular catabolism from normally present intravascular histaminase. In addition, since afferent blood to the lungs contained greater numbers of leukocytes than efferent blood, and Since bovine leukocytes contain histamine which would be released into the plasma by cell lysis during processing, the arteriovenous dif- ference may in part be due to cell sequestration within the lungs. As a corollary to this, if sequestration is an important component of main- tained AV differences, then Pasteurella calves should have wide AV dif- ferences compared to control calves. Since AV differences were similar in both groups of calves, either histamine release from the lungs increased in order to compensate for leukocyte losses from the vascula- ture in Pasteurella calves, or alternately, leukocyte contributions to the AV histamine difference in neonatal calves are trivial using the assay method we describe. The role of bradykinin in the genesis of pulmonary pasteurellosis was easier to identify than that of histamine since there are no stored sources of the mediator in tissues and in calves the effects of bradyki- nin on the lungs are restricted to the vasculature when bradykinin is given intravenously. Aerosol exposure with bradykinin has no effect on airways, gas exchange or vessels.39 Because pasteurellosis is not asso- ciated with increased intravascular bradykinin, our data indicates that bradykinin is unimportant in the genesis of Pasteurella lesions in cattle. 85 We anticipated that bradykinin levels would rise as a result of pasteurellosis, because the following mechanisms of kinin activation and persistence seemed to apply to the situation in calves: endotoxin release from Pasteurella microorganisms leading to the activation of Hageman factor and the subsequent promotion of kinin formation40‘42, possible liberation of bacterial plasminogens and kallikreins by Pasteurella spp.41; liberation of leukocyte kallikrein caused by endotoxin41; loss of vascular integrity during pasteurellosis which limits bradykinin breakdown by angiotensin converting enzyme found in intact pulmonary vascular endothelium43a44; activation of bradykinin generation by hypercarbic acidosis45; impairment of pulmonary bradykini- nase (angiotensin converting enzyme) by hypoxemia.46 The absence of an alteration in bradykinin levels indicates that possibly the bovine responses to endotoxin are different from those of other species and that kinin metabolism is not affected by gas exchange impairment as reported in other species. There are species differences in the response to endotoxin4li47:48 and in preliminary investigations in this laboratory using an inspired gas mixture of 13% 02, 13% C02 in calves exposed to bradykinin, we have not been able to identify any poten- tiating effect of hypoxia or hypercarbia (Slocombe, Robinson, Derksen and Ingersol, unpublished data) on pulmonary or vascular responses to bradykinin. We determined serologic titers to P. haemolytica in Pasteurella challenged calves in order to determine if any failures in producing the disease were due to already present immunity. All Pasteurella challenged calves develOped severe pasteurellosis, indicating that the low levels of serologic titers against 3, haemolytica in these calves 86 was not protective. The antibodies responsible for this cross reactivity are probably the result of nonspecific reactivity arising from colostrally acquired immunoglobulins. While obviously not protec- tive, one would h0pe that such nonspecific hnnunity would augment other defense mechanisms to facilitate recovery. There remains no compelling evidence to suggest such a function and under certain circumstances, antibody against Pasteurella Spp. appears to have exacerbated pulmonary injury through immune mediated mechanisms.49 87 REFERENCES 1. Rehmtulla AJ, Thompson RG: A review of the lesions of shipping fever of cattle. Can Vet J 22:1-8, 1981. 2. Lillie LE, Thompson RG: The pulmonary clearance of bacteria by calves and mice. Can J comp Med 30:129-137, 1972. 3. LOpez A, Thomson RG, Savan M: The pulmonary clearance of Pasteurella haemolytica in calves infected with Bovine Parainfluenza-3 Virus. Can J comp Med 40:385-391, 1976. 4. Thomson RG, Gilka F: A brief review of pulmonary clearance of bacterial aerosols emphasizing aspects of particular relevance to veterinary medicine. Can Vet J 15:99-107, 1974. 5. Gilka F, Thomson RG, Savan M: The effect of edema, hydrocor- tisone acetate, concurrent viral infection and immunization on the clearance of Pasteurella haemolytica from the bovine lung. Can J comp ‘Mpg 38:251-259, 1974. 6. Breeze RG, Magonigle RA: A long acting tetracycline for treat- ment of Pasteurella pneumonia in calves. Bovine Practitioner 14:15-17, 1979. 7. Williams GH, Hollenberg NK: Accentuated vascular and endocrine response to 3020881 in hypertension. N Engl J Med 297:184-188, 1977. 8. Siraganian RP: Refinements in the automated fluorometric histamine analysis system. J Immunol Methods 7:283-290, 1975. 9. Schalm 0W, Jain NC, Carroll EJ: Veterinarnyematology, ed 3. Philadelphia, Lea & Febiger, 1975, pp 122-144. 10. Coles EH: Veterinapy Clinical Pathology, 2nd edition. Philadelphia, W.B. Saunders Co, 1974, pp 43-45, pp 119-148. 88 11. Simensen E, Laksesvela B, Blom AK, Sjaastad 0V: Effects of transportations, a high lactose diet and ACTH injections on the white cell count, serum cortisol and immunoglobulin G in young calves. “Apps Vet Scand 21:278-290, 1980. 12. Musa BE, Conner GH, Carter GR, Gupta BN, Keahey KK: Physiologic and pathologic changes in calves given Escherichia coli endotoxin or Pasteurella multocida. Am J Vet Res 33:911-916, 1972. 13. Wray C, Thomlinson JR: The effects of Escherichia coli endo- toxin in calves. Res Vet Sci 13:546-553, 1972. 14. Jacobsen BB, Peitersen B, Hummer L: Serum concentrations of thyrotrOpin, thryoid hormones and thyroid binding proteins during acute and recovery stages of idi0pathic respiratory distress syndrome. Acta Pediatr Scan 68:257-264, 1979. 15. Csaba B, Yussupova S, Kassay L: The effect of thyroid gland on the histamine and S-hydroxytryptamine level of dog, rabbit and rat tissues. Acta Physiol Acad Scientarium Hungar, Tomus 42:41-47, 1972. 16. Lambertsen JC: In Mountcastle VB (ed): Medical Physiology, ed 14. The CV Mosby Company, 1980, pp 1511-1512. 17. Reimers TJ, Cowan RG, Davidson HP, Colby ED: Validation of radioimmunoassays for triiodothyronine, thyroxine and hydrocortisone (cortisol) in canine, feline and equine sera. Am J Vet Res 42:2016-2020, 1981. 18. Larson PR: Thyroid-pituitary interation. New Engl J Med 306:23-32, 1982. 89 19. Bobek S, Kacinska M, Zapletal P: Thyroxine and Triiodothyronine concentration in the serum of bull calves and its dependence on season of birth and relationship to body weight gain. _;pl Vet Med A 27:697-701, 1980. 20. Lucke C, Hehrmann R, von Mayersback K, von zur Muhlen A: Studies on the circadian variations of plasma TSH, thyroxine and triiodothyronine in man. Acta Endocrinologica 86:81-88, 1977. 21. Rookh HV, Azukizawa M, Distefano JJ, Ogihara T, Hershman JM: Pituitary-thyroid hormone periodicities in serially sampled plasma of unanesthetized rats. Endocrinology 104:851-856, 1977. 22. Refsal KR, Hasenau JJ, Nachreiner RF, Convey EM: Abstract in Proceedings of the 72nd Annual Meeting of the American Society of Animal Science, Cornell University, Ithaca, NY, 1980, p 320. 23. Hernandez MV, Etta KM, Reineke EP, Oxender WD, HafS HD: Thyroid function in the prenatal and neonatal bovine. J Animal Sci 34:780-785, 1972. 24. O'Kelly JC, Wallace ALC: Plasma thyroid hormones and cho- lesterol in the newborn of genetically different types of cattle in a tropical environment. Biol Neonate 36:55-62, 1979. 25. Massip A: The relation between the type of delivery and the acid-base and plasma cortisol levels in the newborn calf. Brit Vet J 136:488-491, 1980. 26. Boyd JW, Hogg RA: Field investigations on colostrum com- position and serum thyroxine cortisol and immunoglobulin in naturally suckled dairy calves. J Comp Path 91:193-203, 1981. 90 27. Wieringa FL, Curtis RA, Willoughby RA: The influence of pre- conditioning on plasma corticostearoid levels, rectal temperatures and the incidence of Shipping fever in beef calves. Can Vet J 17:280-286, 1976. 28. Cabello 0: Neonatal changes in the plasma levels of cortisol, cortisone and aldosterone in the calf. Biol Neonate 36:35-39, 1979. 29. Cabello G: Plasma cortisol and aldosterone levels in healthy and diarrhoeic calves. Brit Vet J 136:160-167, 1980. 30. Fulkerson WJ, Sawyer GJ, Gow CB: Investigations of ultradian and circadium rhythms in the concentration of cortisol and prolactin in the plasma of dairy cattle. Aust J Biol Sci 33:557-561, 1980. 31. Thun R, Eggenberger E, Zerobin K, Luscher T, Vetter W: Twenty- four-hour secretory pattern of cortisol in the bull: evidence for epi- sodic secretion and circadian rhythm. Endocrinology 109:2208-2212, 1981. 32. Johnson NE, Oxender WD: Effect of altered serum glucocorticoid concentrations on the ability of the newborn calf to absorb colostral immunoglobulin. Am J Vet Res 40:32-35, 1979. 33. Yates FE, Marsh DJ, Maran JW: In Mountcastle VB (ed): Medical Physiology, ed 13. The CV Mosby Company, 1974, pp 1696-1740. 34. Cummins JM, Rosenquist BD: Leucocyte changes and interferon production in calves injected with hydrocortisone and infected with Infectious Bovine Rhinotracheitis Virus. Am J Vet Res 40:238-240, 1979. 35. Hitchcock KR: Lung develOpment and the pulmonary surfactant system: hormonal influences. The Anatomical Rec 198:13-34, 1980. 91 36. Welliver RC, Wong DT, Sun M, Middleton E, Vaughn RS, Ogra PL: The develOpment of respiratory syncitial virus-specific IgE and the release of histamine in naSOpharyngeal secretions after infection. .N Engl J Med 305:841-846, 1981. 37. Prasad M, Chandra O, Singhal KC: Plasma histamine levels in pulmonary tuberculosis. Indian J of Med Sciences 25:873-876, 1971. 38. Johnson HL, Beaven MA, Erjavec F, Brodie BB: Selective labelling and release of non-mast cell histamine. Life Sciences 5:115-123, 1966. 39. Slocombe RF, Latter W, Derksen FJ, Robinson NE: The pulmonary response of neonatal calves to intravenous and aerosol bradykinin. .Ap_p Vet Res (in press). 40. Cochran CG: The Hageman factor pathways of kinin formation, clotting and figrinolysis; in Beers RF, Basset E (eds): The Role of Immunological Factors in Infectious, Allergic and Autoimmune Processes. New York, Raven Press, 1976, pp 237-246. 41. Miller RL, Reichgott MJ, Melmon KL: Biochemical mechanisms of generation of bradykinin by endotoxin. J Infect Dis 128:3144-S156, 1973. 42. Kaplan AP, Austen KF: Activation and control mechanisms of Hageman factor-dependent pathways of coagulation, fibrinolysis and kinin generation and their contribution to the inflammatory response. .2 Allergerlin Immunol 56:491-506, 1975. 43. Junod AF: Hormones and mediators in the lung. Am Rev ReSpir Qis_112:94-108, 1975. 44. Bakhle YS, Vane JR: Pharmacokinetic function of the pulmonary circulation. Physiol Reviews 54:1007-1045, 1974. 92 45. O'Brodovich HM, Stalcup SA, Pang LM, Lipset JS, Mellins RB: Bradykinin production and increased permeability during acute reSpira- tory failure in unanesthetized Sheep. J Clin Invest 67:514-522, 1981. 46. Stalcup SA, Lipset JS, Legant PM, Levenburger PJ, Mellins RB: Inhibition of converting enzyme activity by acute hypoxia in dogs. .p_ Appl Physiol 46:227-234, 1979. 47. Kuida H, Gilbert RP, Hinshaw LB, Brunson JG, Visscher MB: Species differences in effect of gram-negative endotoxin on circulation. Am J Phsiol 200:1197-1202, 1961. 48. Hall RC, Hodge RL, Irvine R, Katic F, Middleton JM: The effect of aspirin on the response to endotoxin. .AQEQAK 50:589-601, 1972. 49. Friend SCE, Wilkie BN, Thomson RG, Barnum DA: Bovine pneumonic pasteurellosis: experimental induction in vaccinated and nonvaccinated calves. Can J comp Med 41:77-83, 1977. CHAPTER 3 Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: Changes in pulmonary function with cold stress and during the develOpment of Pasteurellosis 93 94 SUMMARY Thirteen healthy neonatal Holstein calves were cold stressed by hosing with cold water for a period of 20 minutes, on two separate occa- sions twelve hours apart. At the end of each chilling, calves were injected with 0.5 ml of 5% acetic acid intratracheally. Measurements of the pattern of ventilation [tidal volume (VT) reSpiratory frequency (f), minute ventilation (YMIN) and functional residual capacity (FRC)], gas exchange properties of the lungs [reSpiratory quotient (R0), alveolar ventilation (VA) oxygen uptake (V02), C02 production (VC02) dead space ventilation (VD), dead space/tidal volume ratio (VD/VT) arterial oxygen tension (Paoz), arterial C02 tension (PaCOZ) and alveolar-arterial oxy- gen difference (Aa002)] and of the mechanical properties of the pulmo- nary system [dynamic compliance (Cdyn). pulmonary resistance (RL) and total respiratory system resistance (RR5)] were taken at the times described in Chapter 2. Calves responded to chilling by increasing V02 and YCOZ and VA This was accomplished by increasing VT with reciprocal decreases in f so that YMIN remained constant. There was no change in Cdyn. RL or AaDOz. Seven of these 13 calves were then inoculated intratracheally with of 2 x 109 organisms of_g. haemolytica, the remaining calves serving as controls. Within one hour of exposure, calves exposed to E. haemolytica had increased VMIN: f: VD/VT: PPd VD. There was a decrease in Pa02 associated with increased AaDOz, but no change in PaC02, Cdyn or RL. By 3 hours post infection there were pronounced changes in Pa02 and AaDOg, and Cdyn was reduced below baseline values. By 12 hours post 95 infection, calves infected with E. haemolytica had in addition to the above changes, increased RL and RRS and PaCOZ. Data from Pasteurella exposed calves indicates that gas exchange impairment and peripheral lung injury occurs very rapidly, and that increases in airway resistance only develOp relatively late in the injury. The changes described probably arise from ventilation- perfusion mismatching in injured segments of lung. The alterations in breathing pattern associated with the development of gas exchange impairment are likely of reflex origin. Pasteurellosis in calves was clearly initiated from the parenchyma and small airways and was not the result of extension of lesions from conducting airways. INTRODUCTION In a companion article we describe the effect of "stress" and of Pasteurella haemolytica challenge on the circulating blood constituents of neonatal calves over a period of 12 hours following bacterial challenge. The purpose of this article is to describe changes in pulmo- nary function that occur following the same treatments. Alterations in pulmonary function caused by E. haemolytica have not been described in animals. The documentation of alterations of pulmonary function in humans affected with Pasteurellosis is scanty and in persons Pasteurellosis is a sporadic problem1'4 with a different clinical and pathologic course from the typical pneumonic lesions which develop in cattle5, sheep6 and pigs.7 To date, only one study has reported changes in pulmonary function associated with one of the etiologic agents that contribute to infec- tious bovine respiratory disease.8 Kiorpes et al (1978) described an 96 increase in respiratory resistance of calves infected with bovine Herpes 1 virus (18R) and reported alterations in gas exchange consistent with obstructive respiratory disease resulting from virus induced damage to the major airways.8 Because 3. haemolytica can induce pulmonary disease without the influence of other infective agents, and because it has a dominant role in the injury of bovine lungs even when other infectious agents are present5, it was felt that investigation of the role of E. haemolytica in inducing alterations in bovine respiratory function was warranted. METHODS Male neonatal calves weighing between 40 and 46 kg were anesthetized for placement of arterial and venous catheters and for tracheostomies as described in the companion report. Following recovery from anesthesia, baseline measurements of pulmonary function were made in the sequence described below. Calves were restrained by leg hobbles and back straps so that they lay in sterhal recumbency. All animals tolerated restraint well without sedation. Mechanics of Ventilation. An esophageal balloon cathetera was passed via the ventral nasal meatus into the midthoracic esophagus. Catheter construction and response times have been previously described.9 Transpulmonary pressure (PL), the difference between pressure measured at the proximal end of the endotracheal tube and eSOphageal balloon pressure, was determined with a differential pressure transducer.b Air a Uc 516 latex balloon, Anode=Rubber Plating Co, Boling, Tex. b Model PM-131, Statham Instruments Inc, Hato Rey, Puerto Rico. 97 flow rates were measured with a pneumotachographC-transducer systemd attached to the endotracheal tube. Frequency responses of the transpulmonary pressure and air flow catheter-transducer systems were matched to.: 12 Hz. Tidal volumes were determined by electronic integration of the flow Signal.e Transpulmonary pressure, air flow and tidal volume were photographically recorded.e Dynamic compliance (Cdyn) and total pulmonary resistance (RL) were calculated graphically according to the method of Amdur and Mead.10 Respiratory rate was measured from the recording of tidal volume and minute ventilation calculated as the product of frequency and tidal volume. Dynamic compliance and RL were measured during Spontaneous ven- tilation and during a period of controlled ventilation at 15 breaths/minute with a tidal volume of 750 ml.f The initial part of the recording made during controlled ventilation was always discarded because calves resisted forced ventilation for the first few breaths. Total respiratory resistance was also measured by a forced oscillating technique. At end-exhalation, the pneumotachograph- transducer system was connected to a Speaker in box systemg driven by a sine wave generatorhti , and sinusoidal flow oscillations were applied to the reSpiratory system. Endotracheal tube pressure vs. flow was plotted on an oscillosc0pe.e The oscillation frequency was adjusted until the Fleisch No. 1, Dynasciences, Bluebell, Penn. Model PM-5, Statham Instruments, Inc, Hato Rey, Puerto Rico. Model VR-6 Recorder, Electronics for Medicine, White Plains, NY. Harvard Apparatus Co, Inc., Dover, Mass. Acoustic Research 305 cm speaker, Teledyne Acoustic Research, Norwood, Mass. Crown 0 150 R Amplifier, Crown International Inc., Elkhart, Ind. 2005 Function Generator, Continental Specialties Corp., New Haven, Conn. “'3' CD-ht‘DQO 98 pressure-flow loop closed, the closed loop was photographed and the resistance determined as the slope of the closed l00p. Pulmonarnyas Exchange. Expired gases were collected into a Krogh SpirometerJ for 3 minutes and simultaneously arterial blood samples were k and car- collected. Expired gas composition was determined with oxygen bon dioxide1 analyzers. Blood gas tensions were determined with a pH/blood gas analyzer.m Alveolar gas tension, dead space-tidal volume ratio (VD/VT), respiratory exchange ratio (R), alveolar-arterial oxygen difference (AaOOg), C02 production (V502), 02 consumption (V02) alveolar ventilation (VA) and dead space ventilation (VD) were calcu- lated using standard respiratory gas equations.11.12 (Appendix A) Functional Residual Capacity. Functional residual capacity (FRC) was determined by Helium equilibration. One liter of a 15.3% He-air mixture was added to the respiratory system, at the point of passive end- exhalation, using a super syringe.n The gas in the syringe was equilibrated with the gas in the lungs by slowly withdrawing and then reinjecting the 1 liter volume of gas in the syringe 10 times. The concentration of He was determined0 from gas obtained from the 10th refill of the syringe. FRC was calculated as FRC = (Heinitiai/HEfinail-l- Krogh Spirometer, Warren E. Collins Inc., Braintree, Mass. Beckman 0M-14 Oxygen Analyzer, Beckman Instruments Inc., Fullerton, Calif. l Beckman LB-2 C02 Analyzer, Beckman Instruments Inc., Fullerton, Calif. m Model 1L-713 Digital pH/Blood gas analyzer, Instrumentation Laboratory, Inc., Lexington, Mass. n Model 86303 Supersyringe, Hamilton Co., Reno, Nev. 0 Collins Helium Analyzer, Warren E. Collins Inc., Braintree, Mass. KC.“ 99 Experimental Protocol. The measurements described above were made during a baseline period, following "stress" and at O, 1, 2, 3, 6 and 12 hours subsequent to challenge either with sterile saline or with_[. haemolytica suspended in saline. The methods of "stress“ and of challenge at 0 hour are described in a companion paper in detail. Briefly, calves were stressed by exposure to cold water hosing twice, for two periods of 20 minutes, l2 hours apart. At the end of each period of chilling calves received 0.5 ml of 5% acetic acid intratracheally. Calves were either challenged with saline containing .3. haemolytica or with sterile saline, by intratracheal injection. Recordings of PL, air flow and tidal volume were collected at the start of each measurement period, during spontaneous ventilation. Expired and blood gas samples were collected during this time for eva- luation of gas exchange. Air flow, tidal volume and PL were recorded again during controlled ventilation and finally FRC was determined. Statistical Analysis. Data were analyzed using factorial analysis of variance. If F values were significant at p < 0.05, means from each measurement period were compared using Tukey's w and LSD tests.13 RESULTS Tidal volume (VT) was not altered by Pasteurella exposure. Tidal volume was increased in both groups of calves during the stress measure- ment period, remained increased for the T=O measurement, but had returned to baseline by T=1. (Figure 3-1). Respiratory rate (f) was decreased during the stress time period but had returned toward baseline levels by T=0. Respiratory rate remained at baseline levels for the 100 .Ho>oH mo.o oga ao ooogm acoanoLa o Loo ocaHiomoo oco mooacoo acosocomooa coo3aoo moocogoumao acooaaacmwm moaocooor AssoHoo oooozm HHHocomoHo ocooomv moooem acoanoLa coozaoo comwgooeoo to» can HogaHoo oooocm AHHocomoHo amcaav osocm oHHocooamoe oza goo .HcaoHoo oosoao; oeooomv oooem mmogam oHoo oca coo .acmHL oa aon sot» Looco :_ ooaoeamoHHH ma mooHLoo acoEogsmooE coozaoo comwcooeoo Low oHamHaoam 3 mxoxoh ooatoo oeomooxo osa coawo meoo; o>Hoza u L; NH ooHLoo oeomooxo oca Loaoo mono; me u L; o ooHLoo oeomooxo oza coamo meson ooeca u L; m ooHLoo oeomooxo oza Loaao meson oza u L; N ooHcoo oeomooxo oga coamo Loo; oco u L; H Hoooem oHHoeooamoov ocaHom ca ooocoomom ooHaHHoEoo; .m.eo Hoooem mmocam oHoov ocaHom oHHLoam saw; :oHaonoocH Hoozoocaoeaca Loawo aHoaoHoosea n oczmoaxo oaoo oaaooo caaz oosooea oca to mcaaoeom oco Loam; oHoo caHz mcHHHH5o mcaonHow HHoaoHooEEH u mmoeam manoeoeomoos ocHHomoo HoHchH u Hocacoo i. ”oco mooHeoq acoeoeomoos on» .HmcaoHoo oosoaog HHHocomoHov oeomooxo ooHaxHoEoo; .o zaaz oocHano mmocam oHoo oco AwesoHoo oosoao; ocooomv ocoHo mmoeam oHoo mmo>Hoo mo monocm oza :H oono> HooHa :_ mcowaocoaH< .Him oeomam l0l o 5 o 6 o o o O O O O 0 ct IO N " Tidal Volume ml Figure 3-l 102 .Ho>oH mo.o oca ao ooocm acosaooea o Low ocaHiomoo oco mooHLoo acosocomooa :oozaon moococowaao acooamacmam moaocoo« HcsoHoo oooozm HHHocomoHo ocooomv moooem acoanoca coozaoo comacooeoo to» new HessHoo oooocm HHHocomoHo amgamv ooocm oHHocooamoe oca com .H:EoHoo oogoao; ocooomv osoem mmocam oHoo osa Low .azmHL oa aon Eoem Looco :H ooaocamoHHH ma mooacoo acosocomooe coozaoo comHLoosoo Loo oHamHaoam 3 mxoxok ooweoo oeomooxo oga Loawo mono; o>Hoza u L; NH ooacoo oeomooxo oga Loawo mono; xam u E; m ooHLoo oeomooxo oga Loamo meson ooeza u L; m ooaeoo ocomooxo oga Loamo mono; oza u L; N ooHcoo oeomooxo oga Loamo coo; oco u c; H Hozocm oHHocooamoav ocHHom :H ooocoomom ooaaxfloeoo; .m.Lo Hooocm mmocam oHoov ocHHom oHHLoam saH: :oHaoHoooCH Hoozooeaocaow Loaeo xHoaoHooEEH n ocomooxo oaoo oHaooo saw: cocooca ona mo mcaxocom oco coao: oHoo saw: mcHHHaso mcwonHom xHoaoHooEEH u mmocam macoaoezmooe ocaHomon HoHaH:H u Hocacoo i. ”oeo mooagoo acoeoeomooa on» .HmcaoHoo oosoao; HHHocomoHov oeomoqxo ooHaNHoeoo; .o saw: oocaoEoo mmocam oHoo oco HmcaoHoo oosoao; ocooomv ocoHo mmoeam oHoo Hmo>Hoo mo masocm oza :H oaoc Hgoaocaomoc :H mcoaaoeoaH< .N-m ocomam 103 Tukey's w '2 hr. 80+ 60 4O 20- 0 IOO. R°3Pirat0ry Rate bum," /mlnute Figure 3_2 104 remainder of the experiment in the control group but was significantly increased at T=1, T=2 and T=3 measurement periods in the Pasteurella exposed calves compared to the stress measurement period. (Figure 3-2). As a result of these changes in f and VT, minute ventilation was unaltered during stress in either group of calves, and remained at base- line values for the entire experiment in the control group. In contrast, the Pasteurella group increased minute ventilation signifi- cantly at T=1 compared to the control group. Although differences in minute ventilation from baseline remained large at T=2 and T=3, the increase above baseline was not significant. (Figure 3-3). Alveolar-arterial oxygen difference (AaDOz) remained unchanged in the control group of calves. In the Pasteurella exposed group a trend toward increasing AaDOg was noted by T=2 (significantly different from baseline on LSD but not Tukey's uitest) and by T=3, AaDOz was Signifi- cantly greater than baseline. Differences from baseline were signifi- cant at T=6 but by T=12, AaDOg had decreased so that it was no longer significantly different from baseline values, yet remained increased enough above baseline that it was not significantly different from T=6. (Figure 3-4). Changes in Pa02 reflected the alterations in AaDOz. There were no changes in the control calves and in the Pasteurella group of calves the decrease in Pa02 was significantly different from baseline by T=l. Further declines in PaOz over the remainder of the experiment were small so that measurements after T=1 were not significantly different from T=1, but remained different from baseline. (Figure 3-5). Arterial C02 tension remained unchanged in the control group over the course of the experiment. In the Pasteurella group, PaCOz remained .Ho>oH mo.o oga ao ooaeoo acoeocomoos oEom osa to» moooem coozaon moococoawao aooonHcmam moaocoow HcsoHoo oooocm HHHocomoHo ocooomv monocm acosaooca coozaoo comHLooEoo com com HogaHoo oooosm HHHocomoHo ameamv oooem oHHoeooamoo oza to» .Hce=Hoo oogoao; ocooomv oooem mmoeam oHoo oga to» .acmHL oa amoH sot» Looco :_ ooaoeamzHHH ma mooaeoo acoeotswooa :oozaoo cochoosoo com oaamHaoam 3 axoxoh ooaeoo ocomooxo oga Loawo meson o>Hoza u c; NH ooHLoo oeomooxo osa Loawo mono; me u c; m ooHLoo oeomooxo oca Loawo meson oogza u e; m ooHLoo ocomooxo oga eoawo mono; oza u L; N no ooweoo oeomooxo oza Loamo Loo; o:o u L; H O 1. Hosoem oHHoeooamoov ocaHom :H ooocoomom ooHaxHoEoo; .m.co Hooocm mmoeam oHoov o:_Hom oHHcoam caHz coHaonoocH HoocooeaocacH coaao aHoaoHooEEH u oeomooxo oaoo oaaooo saw: oosooea oea to meaxoeom oco Loam: oHoo saHz mow—Haco mcHonHow HHoaoHooeeH n mmoeam macosoeomooe ocHHomoo HoHaaca u Hoeacoo i. "oco moowcoo acoeoeomooe one .Hm:E:Hoo oogoao; HHHocomoHov oeomooxo ooHaHHoEoo; .m gaaz oocaneoo mmocam oHoo oco AwesoHoo oosoao; oeooomv ocoHo mmoeam oHoo mmo>Hoo mo monocm oza ca coHaoHHaco> oaocaa :H mcoaaocoaH< .mim ocomam l06 \\\\ ////////// Tukey's/ or l2 hr 2hr. Stress Exposure ................... Control .................... ................. 20- IO 5. 0 Minute '5' Ventilation liters /mm Figure 3-3 107 .Ho>oH mo.o osa ao osoem acoanoea o eoe ocaHiomoo oco mooaeoo acoeoeomooe coozaon moocoeoeeao acooawacmam moaocooe HosaHoo oooocm HHHocomoHo ocooomv moooem acoanoea :oozaoo comHeooEoo eoe oco HosaHoo oooocm xHHocomoHo ameaev ozoem oHHoezoamoa oza eoe .HcaoHoo oocoao; oeooomv oaoem wmoeam oHoo ona eom .acmae oa aeoH Eoee eooeo :H ooaoeamoHHH ma mooaeoo acosoeomooe :oozaoo :omHeoosoo eoe oaamaaoam 3 maoxzh ooHeoo oeomooxo oza eoaeo meooc o>Hoza u e; NH ooaeoo oeomooxo osa eoaeo meson xam u e; o ooHeoo oeomooxo oga eoamo meoo; ooega u e; m ooaeoo oeomooxo oga eoaeo meso; oza u e; N ooaeoo oeomooxo oga eoaeo eooc oco u e; H Hoooem oHHoeooamoev ocHHom :_ ooocoomzm ooHaHHosoo; .m.eo Hasoem mmoeam oHoov ocHHom oHHeoam saw: coHaoHaoocH HoogooeaoeaeH eoaeo aHoaoHooEEH i oeomooxo oHoo oaaooo saw; oogooea oga mo mcaxoeqm oco eoaoz oHoo zaHz mcwHHHgo mcwonHoe HHoaoHooEEH mmoeam macoeoeomooe o=HHomoo HoHaaca Hoeacoo i. ”oeo mooHeoo acosoeomooa och .HmcsoHoo oosoao; HHHocomoaov oeomooxo ooaaaHosoo; .o saHz oocHosoo mmoeam oHoo oco AwesoHoo oozoao; oeooomv ocoHo mmoeam oHoo mmo>Hoo eo moooem oza :H oocoeoeewo comxxo HoHeoaeoieoHoo>Ho :_ mcoaaoeoaH< .aim oeomae l08 \\\\\\\\\\\\\\\\\ i3 ////////"’ *75 c3 0 6 o 0 Q? F) 00 - t 3 "55¢ a? sea; <1 cv":: o Figure 3-4 109 .Ho>oH mo.o oga ao oooem acosaooea o eom ocwHiomon oco mooHeoQ acoeoeomooe :oo2aoo moocoeoamao acooawacmam moaocooe AesoHoo oooozm AHHocomoHo ocooomv moooem acoanoea coozaoo comaeooeoo eoe oco HessHoo oooozm HHHocomoHo ameaev oooem oHHoeooamoe oza eoe .H:E:Hoo oogoao; oeooomv oooem mmoeam oHoo oga eoe .acmHe oa aeoH Eoee eooeo ea ooaoeamoHHH ma mooHeoa acosoeomoos coozaon comHeooEoo eoa oHamHaoam 3 mxoxok ooHeoo oeomooxo oza eoaeo meso; o>Hoza u e; NH ooHeoo oeomooxo osa eoaeo meson xam u e; m ooHeoo oeomooxo oca eoaeo meooc ooesa u e; m ooHeoo oeomooxo oga eoaeo meooz oza n e; N ooaeoo oeomooxo oca eoaeo eooz oco u e: H quoem oHHoeooawoov oeaHom :H ooocoomom ooHaxHoEoo; .m.eo Hoooem mmoeam oHoov ocaHom oHHeoam :aHz coHaoHoooca Hoosooeaoeaca eoaeo aHoaoHooesw n oeomooxo oHoo oaaooo caHz oogooea oca mo m:_xoeom oco eoao: oHoo zaH; mcaHH_;o mcaonHow HHoaoHooEEH u mmoeam mazoEoezmoos ocwHomoo HoHaH=H u Hoeacoo i. "oeo mooaeoa acosoeomooa on» .Hmc53Hoo oosoao; HHHocomoHov oeomooxo ooHaxHoEoo; .e gaHz cocaoeoo mmoeam oHoo one HosanHoo oogoao; oeooomv oooHo mmoeam oHoo Hmo>Hoo mo moooem oza :_ coamcoa comxxo HoHeoaeo ca mcoaaoeoaH< .mim oeomae llO N\\\\v\\\ 7 537/? 77/ 111/11 1 11"/11 ............ ,rfrrrrfrrrrr—rv—YVr—rvyrvyrrvr.rr rrr/rrrr7/ xx." 7 7 ’. .‘ 7 / Tukey's w ; //////1‘14411AAAAA4AAAAAALAAAAAAA“A_AAA‘A_4A1/1// ................ 12hr. * ///’* 7’":’: ”77/ ////A///LJJ/A A‘4111A11/111 AA1A‘AAA1155 AAAAA ///11// / ~36 7.11:5”:54413 A1111111L11ALA111111111111 11A 141111111'11114111’ 6hr. ................................................ ,,,,,,,, / /’/// // , .17 / 1717.141???7;;2335'5' / // /// . . //////1/1/ 1111171111111 ~'«‘ 5 7 3hr. ....................................................... ................................................ ............ . 7 77'- 7,777 77 /'/ /'.7’ ,1, / /7// /’ ' //7’ '_,,i7’-'./-jf7, 7. 7/Z AAA1A'1AA1'11L1'1A11A11 11 111A 111.1111111111/11.11121 * V ////," 7//////7’//7rT/f7 ' 5 " ’7 7////// " ’////7. /////// 7 7 x ................................................... .......................................... ,/ //7 7/777// /77/ 7'77 777 77 777 7777/77 77/777 //77 ///// // //1/111/AA11111111111//1111A11A///14/11/1 111/1/11/111/// EZ/fl’77777"737"'i*"f77'57*"7777777"7%7;7 /////// .7,,,;;»::;.'.~' ’ ’7 ” . . . // //1/11/11AA.“A;111.; ............ ...... - 1111-71115’7/111/7 // ................................................ ........................................................ ....................................................... 80- 2 hr. lhr. Stress Exposure Control Figure 3-5 111 .—m>m~ mo.o mg» um azogm pcmspmmgu a gas m:__-mmmn ucm muowgwq pcwemgammme :wmzpmn mwucmgowmwu pcmo_mw:mwm mmpocmv« Assapoo umumsm appmcomc_v accommv mazogm pamemmgu :wmzumn comwgmano Low vac Assn—co umuwzm a__m=omm_v pmgwwv aaogm w_—mL:mpmm¢ mzu Low .A:E=Poo vacuum; mgwscmv azogm mmmgum vpoo on“ Low .uzmwg op gmmF 50;; gauge :_ vmumgpmap_w m? mvowgma pcmemgamama :mozgmn comwgmaeoo Low uwumwumpm.s mxmxzh nowgmq mgzmoaxm on» memm mgzo; m>_mzu u L; NH uo_goa mgzmoaxo asp gmpom mgzo; xwm u L; m vowgma «Lamoaxo mg» Lmuwm mgzo; mugs“ u L; m vowgma mgzmoaxo as» Lopvm mgso; oz» u L; N vowgoa mgzmoaxm mgu Lopom L30: «:0 u L; H Aaaogm uppmgampmmav m:__mm :_ umucwamzm muwpxpoeom; .m.Lo quogm mmmgpm u—ouv m:__mm m__gmum cpwz cowumpzoocw Pmmcumgumguc_ mewm x_mpmwumes_ u mgzmoaxm u_om ovumom Suva monomgu mcp we mcwxmgqm vcm Loam: u_oo cpw: m:_p—P;o mcwzoppoy xpmum_umaew u mmmgum mpcmsmgamwws mcwpmmmn meuwcw u Fogucoo I. ”use mvowgmq pcmsmgzmmms as» .Amcas—oo uogopm; A_chommwuv «Lamoaxm cowuwwosmm; .m saw: vmcwneoo mmmgum upou new “masspoo uozupm; mgmacmv mco_m mmwgum vpou mmm>_mu mo mnzogm oz» cw :owmcmu mvwxovu :ongou megmpgm cw mcowumgmpp< .mum mL=m_u 112 Figure 3-6 113 unchanged until T=12 when PaCOz increased 14 mmHg above baseline levels. (Figure 3-6). Production of C02 changed by similar amounts in both group of calves, increasing significantly during stress and T=0 measurement periods. (Figure 3-7). Exposure to f, haemolytica had no effect on VCOZ- Utilization of 02 had similar changes, with 902 only increasing above baseline levels in both groups of calves during stress and T=D measurement periods. (Figure 3-7). Alveolar ventilation increased in both groups of calves at stress and T=D, returning to baseline levels at T=1. There was no effect of g. haemolytica exposure. (Figure 3-8). Dead space ventilation remained constant in the control calves and was unaffected by stress. In the Pasteurella group VD was significantly increased above baseline values at T=1, T=2 and T=3. At T=6 VD had returned to baseline levels but during T=12, 90 was marginally increased above baseline levels (significant on LSD test but not Tukey's test). Dead Space ventilation at T=12 was not significantly different from T=3 or T=6 measurement periods using Tukey's test, but differed from T=3 using the LSD test. (Figure 3-9). The dead Space/tidal volume ratio decreased significantly in both groups of calves during the stress measurement period and returned to baseline levels at T=D. In the control calves, VD/VT did not change from baseline levels during the remainder of the experiment. The dead space tidal volume ratio in the Pasteurella group at T=2, T=3 and T=12 hours was greater than baseline levels and was significantly larger than VD/VT of control calves compared at the same measurement periods. (Figure 3-10). 114 .Fm>m— mo.o ng um aaogm pamEummLp u now mcwp-mmwn vcm mno_gma pcmsmgsmmms :mwzumn mmucmcmwmwv ucmuwwwcmwm mogococx Assn—co umvmcm »—_w:ommwc ucoommv maaogm acoEummcu cmmzpoa comwcmqeoo go» new fleas—cu nmuwcm ap—mcomm_c umg_wv qzocm m__mg:mumma on» Low .AcE:_ou vacuum; wcmzcmv azoem mmmcum v_oo asp Low .p;m_g op ammF socw Lance c? acumepmsp_w m? muovgmq pcmsmcammme coozuwa comvcmqsoo go» o_pmwpcum 3 mxmxsh cowcma mczmoqxo mg» mewm mono: m>~mzu u L; NH uo_gmq wezmoqxo mgu Lmumm meson xwm u L; m vowcma mgzmoaxm mg» empem meson omen» u L; m vowgmq mesmoaxm on“ cause meson oz» u L; N uo_cma mgzmoqxo mgu cmpmm Lao; mac n L; H Aqaocm m_PmL:mpmmmv «awrmm :_ umwcmqmsm muwuwwoemm; .m.co Ansocm mmwcum v_oov m:w_mm m__empm sp.: cowumpzuocw Pwmgumcumepcw cmpwm a_m»m_umesr u mgzmoaxm uwom u_umow ;p_z mmzomeu asp mo m:_xmcgm new Loam: v—ou ;p_z mcwp__cu mcwzoppom xpmumwvmsew u mmmgum mucmsmgzmwms mcwpmmma Powuwcw u m:__mmcn ”mom muovcma pcmswwcmmme on» .ANo>v oxmua: cwmxxo ecu ANoo>v co_uu=uoga Nou co wcwpmm empomm:_ mu_px_oemm; .a co mcwpmm op mesmoaxm megomgumcucw ucm mmmeum n_ou we muomwew one .Num mczmwu T-l2 hr. Tukey's ca . TIGht liters/min Figure 3-7 116 mooo ms» #0 QJOLm #:mEumeH m Lou... GCFFImmmn Ucm mUowLmQ ucmEmLzmme cmwzfiwn mmucmmewa u .czocm m_ mvoHLaQ pcaeacsmaae :aazpan mcaas mo comHLaaeoa Low .Ha>aH cauwwwcmwm mapocauy UHHmHuaHmfis mmaxzp cowcaa acamoaxa asp capwa meson a>Hazp u L; NH uoHLaa agzmoaxa asp Lapwa mono; me u L; m uoHLaa agsmoqxa acu Lapaa maze; aaccg u a; m nowgaq aczmoqxa as“ Lapse mczoz 03H u L; N vowgaq aczmoaxa as» Lauma Lao; aco u L; H Ansocm aHHacaaHmamv aCHHam :H caucaqmam auHHNAoEaa; .M.Lo quocm mmacum uHoov a:_Ham aHHLaum cur; :oHHaquocw Haazuacuagucw Lauma xHauaHuaesw u aesmoaxa uwua owpaua cpwz aacaagp asp mo mCHHaLQm uca Lapaz vHou guH3 mew—cha mcwonHom aHapa_uaeew u mmaapm mucasagsmaae a:_Haman Ha_pwcw u acHHamaa ”ace muowcaq pcaeacsmaas asp .cowuaHHH:a> LaHoa>Ha :o acwHam uagoaecw MUHHNHosaa; .m.Lo acwHam op acsmoqxa Haasoacumcuc_ new mmacum uHou we muuawaa ace .wum aczmwm ll7 7' / 1 .7 , . / 447/ 725/ /// * %//1////5/ //// .7 ,7/ 4%;2g??7{ r':/// , . 7 75",." p ’///>, ”I , VII/9’ ///§1”1'11/ 1' Baseline Stress Exposure loT Alveolar Ventilation (5. liters/min 4i :3. 0 Tony‘s or l2 hr 6'hr. 3hr. 2hr. lhr. Figure 3-8 118 .Fm>m~ mo.o mg» um qzogm “cwEummLu a Low warpummmn ucm muowgma pcmsmgzmmms cmmzpmn mmucwgmm»_u pcmupw_:mwm mmpocmu« Acaapoo nmumzm x—Pmcomm_u ucoummv mqsogm “cospmmgu comzuma comwgmaeou no» ucm Agaspoo umuwzm a_Pm:omm_w umg_$v azogm mp_mgzmummm mgu Low .A:E:Poo umgoum; mngamv azogm mmwgpm upon as“ Low .pcmwg cu pme sag; gauge :_ umumgum:—__ m_ muo_gmq pcmsmgsmmma :mmzpwn comwgmanu Lo» u_umwumpm 3 mxwxzh vowgoa mgamoqu an» mewm maze; m>~mzp u L; NH uo_gma «Lamoaxm mg“ gmuwm maze; xwm n L; m vowgoq mgsmoaxm asp Logan mgso; mags» u L; m wowgwq mgsmoaxm any Logan mason ozu u as N uo_LmQ mgzmoqu ms“ gmgam Lao; mac n L; a Aazogm m_—wL=mummav m:__wm :_ umucwqmsm mo_uz_oemmz .m.go quogm mmmgpm uFouv m:_—mm mFPLmum :uw; cowumpauo:_ Pmmguwgumguc_ memo xpmumwvmas_ . mgzmoaxm u_om u_pmom saw: mmsumgu mg» we m:P»mLam ucm Lmumz uFoo zpw: mcw_—P:u mewzoppom »_mumwumes_ mmmgum mpcwsmgammms m:_mema mepv:_ mewpmmmn I. "mam mnowgwa pcmsmgzmmms ugh .Amcazpoo nmgoum; A_chomwwuv wgamoqu cowuxfiosmm; .a ;u_z umcwneoo mmmgum vpoo can Amcsz—oo umcuum; mLmzcmv acon mmmgum u_oo mmm>Fmo ac masogm ozp cw cowumpwpcm> momqm ummu cw mcowpmgmpF< .mum mg:m_u 119 \\\\\\\\\\\\\\ ’/////////// .............. Tm:'\s/ w T!I I2 hr. T86 hr. T'3hf. TIZhr. hr. 1': (D 'I .— -k.uiTT+.HTT., 3: ............. m ”A :t;. :31 TfI- ii .7 H .,._i‘:_:_1¢ o a: _fi N I I I cg 'g CL.— \~ ”’2 a 1D 2: :5 85 -:'-. O> Figure 3—9 120 ._m>m— mo.o asp cc cccgm pcmsuccgu c Loy c:w_-cmcc ccc mccwgwc pcmsmccmccs cmmzucc mccccgcmwwc pcccww_=mwm mmpcccct Acacpcc ccccgm AFPcccmcwc cccccmv mcccgm “cospcmgu ccwzucc ccm_gccscc gca ccc Acacpcc cmcccm a—Pcccmc_c umg_»v cccgm cPFcaccpmcc ccu Low .Acsc_cc coscpc; mgcccmv cccgm mmcgpm cpcc mg» no» .cgmwg cu u»mp Ecgy Lucgc cw cupcgpm=p__ mw mcc_gcc uccsmgcmccs :cwzucc ccm_gccscc Loy ovumwucum 3 mxwxc» cowgmc mgcmccxc ms» gmpwc mgccs c>~czu u L; NH cowgmc cgcmccxc msu Lcuwc mgcc; x_m u L; o cowgcc cgcmccxm mg» scuba mgccg wcgnp n L; m cc_gcc cgcmccxm on“ chmc mgcc; c3» u L; N cc_gcc mgcmccxc cg» Lccwc Lac; mac n L; H Acccgm c__mgccumccv mcw—cm :_ ccccmcmsm ccwuchsmc; .m.gc Acccgm mmcgcm chcv mcwpcm c__gccm cuwz =c_uc_ccccw Pcmgccgpcgccw Lmucc meuc_ccEE_ u cgcmccxc cwcc ovumcc :u_; cczccgc cg“ cc mcwxcgcm ccc Lcucz chc 5pm: mcw—ngc mcwchFcp xpmucwcmeew u mmcgpm mpzcscgcmccs chPcmcc chpwc_ u chcccc I. ”can mccwgmc uccscgcmcce asp .Amcsc_cu vacuum; x__cccmcwcv mgcmccxm cc_ux_csmcc .a cpwz cmcwcscc mmwgum c_cc can amasspcc ccchc; mgcccmv cchc mmmgum cpcc mmc>pcc mc mcccgm czu cw c_ccg cac_c> Fcc_u\ccccm ccmc cw mccwpcgch< .oHum cgcmvm 121 Dead Space / Tidal Volume Ratio 60- Fiqure 3-10 122 Respiratory quotient remained unchanged in the control group of calves, and in the Pasteurella group all measurements did not differ significantly from baseline. However, in the latter group R was increased at the T=6 measurement period when compared to the three measurement periods preceeding it. (Figure 3-11). Functional residual capacity remained unchanged for the entire experiment in both groups of calves (mean :_SEM of all observations 2.21 .1 0.65 liters). Rectal temperature was also unchanged during the course of the experiments (mean :_SEM of all observations = 38.9 1 1.7 C). Dynamic compliance under Spontaneous ventilation was very variable with no significant effects of Pasteurella exposure or stress detected (mean :_SEM of all observations 111.4 :_88.2 ml/cm of H20). There was no difference in Cdyn measured during controlled ventilation for any measurement period in the control group of calves. Although Cdyn had decreased to less than half of baseline values in the Pasteurella group by T=12, these differences were not significant. However, Cdyn at T=3 was significantly less than at stress and T=1 measurement periods by LSD test but not Tukey's test, and Cdyn at T=6 and T=12 were significantly less in the Pasteurella group compared to the control calves. (Figure 3-12). Total pulmonary resistance determined during Spontaneous ventilation was very variable and we were unable to detect any changes associated with stress or Pasteurella infection (mean :_SEM of all observations 7.04 i 7.06 cm of H20/liter/sec). During controlled conditions, RL was significantly greater at T=12 than at baseline and T=2 measurement periods. There were no significant differences between groups of calves except at the T=1 measurement period where RL in the control group of 123 HcEchc cccccm aHHcccmch cccccmv mccccm pccspcccp ccczccc ccmHLccEcc Low ccc Acachc coccsm A—Hcccmch umcwmv ccccm cHHccccumcc czu com .Hcechc cczcuc; cgcccmv ccccm mmmcpm chc any com .pgmHL cu pmc— soc» Lcccc cw cccccpmcHHH m_ mccwccc pccscccmccs :cczucc :cmwcccscc cc» cHHmHucum 3 macxch ccHLcc cccmccxm cg» chwc mecc; c>chc u L; NH ccwccc wccmccxc ccu chmc mecc; me u L; o ccwccc cccmccxc ccp gcpmc mecc; aces» u L; m ccHLcc cccmccxc cgc Lcuwc mecc; czc u L; N ccHLcc cccmccxc cg» cuppa Lac; mac n L; H Accccm cHHmcccpmccv c:_Hcm :_ coccccmcm ccwuchEcc; .m.cc Accccm mmccum chcv cCHHcm cHHLcum saw: :cHHchcc=H HcczcccucgucH chmc chuchcEEH u cccmccxc chc cwuccc guH3 ccscccu ccu cc mcchLcm ccc Lccc: chc saw: mcwHstc mcwxcHHcm chuchcEEH u mmcccm mpccscgcmcce ccchmcc Hc_p_:H n c:_Hcmcc i. “ccc mccwccc pcmscccmcce one .HmcEchc ccgcuc; AHHcccmcHUV cccmccxm ccwuchEmc; .c zur; cmcchcc mmccum chc ccc Am:Echc ccscpc; cccccmv ccch mmcgum chc mmc>Hcc cc mccccm cxu cw cHHcL cmcczcxc chcccwcmcc cw mccHHcccpH< .HH-m cecde 124 Tukey 's or 7 1 A 1 ..................... .avo.........- ....... ..................... ................... .................. ................ .................... ..................... ..................... ................. lhr.! ..................... ...................... ...................... Stress Exposure Baseline |.O« O.5« Respiratory Exc honqe Ratio l2 hr 3hr. 2 hr. Figure 3-ll 125 .Hc>cH mo.o cgp cc ccwccc uccecccmcce cacm cgu com mccccm :cczucc mcccccccmwc ucccwmwcmwm mcpcccce Acachc cccccm HHHcccmch cccccmv mccccm uccEpchu ccczucc :cmwccQEcc cc» ccc Acachc cccccm HHHcccmch umgwmv ccccm cHHccccpmcc cs“ Low .H:E=Hcc ccgcpc; cccccmv ccccm mmccpm chc cg“ cc» .pgmHL cu pmcH Eccw ccccc :_ ccpccpmcHHH mH mccwccc cccscgcmcce ccczpcc ccmwcccscc Low chchcum 3 mxcxcp ccHLcc cccmccxc cgc cccmc mecc; c>chu u L; NH ccHLcc cgcmccxc on» ccpwc meccz me u c: m ccHLcc cccmccxc cgu memc meccg «menu u L; m cchcc cecmccxc cg» ccpmc mecc; czu u L; N ccwccc cccmccxc cs“ Lcuwc Lac; mac n L; H Aczccm cHHchccmccv c=HHcm :H ccccccmcm ccHHchecc; .m.cc Acccem mmcccm chcv cCHHcm cHHchm cur: :cHuchcc:_ Hccsccccccucw ccuwc chchccEEH u cccmccxc crow cwuccc chz cczcccp cs» cc mcHchcm ccc chcz chc :sz mcHHngc mcwchHcm chuchcEEH u mmccum mpccscccmcce ccchmcc HchHcH u c:_Hcmcc I. ”mew mccwccc pccscccmcce ugh .Hm:E:Hcc ccchc; HHHcccmchv cccmccxc ccHuHHcEcc: .c saw: cccwcecc mmccum chc ccc Hm:E:Hcc cccccc; cccccmv mcch mmccum chc mmc>Hcc cc mccccm czp :H coccHHcscc cwsccxc cw mccwucccpH< .NH-m cczmwd 126 \23 . //////// l hr. Stress Exposure i T t l i I i 1 Baseline l 20- Dynarnic '00 ‘ Compliance Figure 3-12 127 .Hc>cH mo.o ccp cc ccccm pccEccccu c cc» cc»Hucmcc ccc mcc»ccc pccscccmccs ccczpcc mcucccc»»»c uccc»»»:m»m mcuccccc Acachc cccczm xHHcccmch cccccmv mccccm accEccccc ccczccc :cchccscc cc» ccc Acachc ccccsm AHHcccmc»c umc»»v ccccm cHHccccpmcc cg» cc» .A:E:Hcc cczcuc; cccccmv ccccm mmccum chc any cc» .ccm»c cu p»cH Ecc» ccccc :_ cccccumcHH» m» mcc_ccc accEcccmcce ccczccc ccm»cccscc cc» cHumeccm 3 mxcxc» cc»ccc cccmccxc cap ccc»c mccc; c>chu u c; NH ccwccc cccmccxc asp ccp»c mccc; xwm u c; m ccwccc cccmccxc mg» ccp»c mccc; ccccp u c; m cchcc cccmccxc cc» ccu»c mccc; c3» u c; N ccwccc cccmccxc cs» ccu»c ccc; mac n c; H Accccm cHHccccumccv c:»Hcm c» coccccmcm cc»uchEcc; .m.cc Accccm mmccpm chcv cc»Hcm cHHcccm cu»: cc»ccH:ccc» Hccgcccpccpc» ccc»c chccwccEEH u cccmccxc chc cHuccc cc»: ccccccc cc“ »c m:»»cccm ccc cccc: chc cu»: mCHHH»:c mcHchHc» HchchcEE» u mmccum mpccscccmcca c:_Hcmcc Hch»:» u ccchmcc l. "ccc mccwccc pccscccmcce cc» .Hmcschc ccscuc; AHHcccmc_cv cccmccxc cc»uchEmc; .c cu»: cc:»c5cc mmcccm chc ccc Am:E:Hcc ccgccc; cccccmv ccch mmccpm chc mmc>Hcc »c mccccm c3» :» coccumHmcc Accccchc Hcccu c» mccHHcccpH< .mH-m cccm»c 128 \\\\\\\\\\\\\\\\ /////////////, ' éoo': J- N-_ U . O -2 9»: 22 g: h gaggié c: £2 m — Figure 3-l3 129 calves was greater than in the Pasteurella group. (Figure 3-13). However, if the total numbers of observations of RL in each group of calves were compared, then RL was significantly greater in the control group (12.5 :_O.8 compared to 8.1 1 0.8 cm of HZO/liter/sec). We do not believe this relates to a significant difference in airway caliber bet- ween groups of calves, since three calves in the control group were fitted with endotracheal tubes of a smaller size. Forced oscillating resistance measurements were only taken in 3/6 control calves, so that statistical comparisons between the control and Pasteurella calves were not possible using multifactorial analysis due to the wide disparity in numbers of observations between each group. ReSpiratory system resistance determined by forced oscillation remained unchanged in the control group of calves (mean :_SEM of control group = 9.0 i 7.2 cm of H20/liter/sec) but in the Pasteurella group resistance had increased four fold by T=6 and was significantly different at T=12 hours from all preceeding measurements with the exception of T=6. (Figure 3-14). 130 .Hc>cH mo.o cgu cc ccccm accEpcccu c cc» ccHanmcc ccc mcchcc pccscccmcca :cczucc mcccccc»»»c Hccc_»»:m_m mcpcccce .czczm m» mcchcc pccscccmcce ccczpcc mcccs »c :cchccEcc cc» cHumHHch 3 macxch cchcc cccmccxc mzu ccu»c mccc; c>chu u c: NH cchcc cccmccxc csp cmp»c mccc; me u c; c cchcc cccmccxc cnu ccu»c mccc; ccccu n c; m cchcc cccmccxc cgc ccp»c mccc; c2u u c; N cchcc cccmccxc cc» ccu»c ccc; mac n c; H Accccm cHHccccumccv ccHHcm :» coccccmcm ccHHNHcemc; .m.cc Accccm mmcccm chcv ccHHcm cHHccum nu»: :cHHchccc» Hccgcccuccpc» ccc»c HchchcEEH u cccmccxc chc cHuccc cc»: ccccccc czc »c m:_xcccm ccc ccucz chc cc»: m:»HH»;c mewxcHHc» chuchcas» u mmccum muccscccmcca cc»Hcmcc Hch»:_ u c:HHcmcc "wmc mcchcc cccEcccmcce cg» .mc>Hcc c» ccccumHmcc mCHHcHHHcmc ccccc» :c ccHuHHcecc; .c cc cccmccxc mCHchHc» ccc mmccum chc »c mucc»»m .cHlm cccch l3l W w 3hr. i %- /" W- 5 é 9 27 m ‘0 «3 o 5% E s as a 6 hr. Tukey's w l2 hr. 2hr. I hr. Exposure Baseline Stress Figure 3-l4 132 DISCUSSION Although cold stress alone is thought to predispose to reSpiratory disease in cattle, clinical and epidemiologic studies suggest that chilling associated with high humidity or wetting facilitates the deve- l0pment and severity of the condition.14'20 The mechanism for this pre- disposition is uncertain in calves, but may involve alterations in respiratory epithelial viability21, in bacterial survivalzz and in immune function.14 In a companion article we demonstrated that cold water chilling of neonatal calves leads to increased plasma cortisol levels and suggested that this may result in immunosuppression. Kelley (1980) indicated that cold stress may also be hnmunosupressive14, but the mechanisms reSponsible for immunosuppression remain unclear. In this article we explored another possible avenue in which the lungs abi- lity to respond to Pasteurella exposure may be compromised as a result of cold stress, namely pulmonary function. Cold stress in calves induced by chilling with cold water results in immediate changes in the clinical appearance of calves so exposed. The animals become subdued, develop a “tucked up“ posture and shivering begins almost immediately. These responses were effective in main- taining body temperature as measured by rectal thermometer, probably by a combination of increased body metabolism through shivering and by peripheral vasoconstriction insulating the body core from the skin, the skin surface becoming cold soon after cold water spraying began. Increased metabolism, due in part to shivering, resulted in increased 902 and 9C02- In order to meet these metabolic demands without changes PaOz or PaCOz calves increased VA while maintaining minute ventilation 133 constant. This was accomplished by increasing the depth of respiration with reciprocal decreases in reSpiratory frequency. As a result VD/VT decreased. Although such reSponses may serve to meet metabolic demands, the alteration in breathing pattern may serve to deposit infec- tive aerosols further into the lungs and predispose to pneumonia. Chilling did not alter the mechanical prOperties of the lungs, (Cdyn and RL) nor did it alter gas exchange as measured by AaDOz, PaOg and PaCOz. It would seem probable that chilling of calves with cold water pro- vides a greater stress than does eXposure to cold environmental temperatures.15'20 If this is so, our data suggests conpromise of pulmonary defense mechanisms may be greater in the former circumstance because of large increases in circulating cortisol levels and increased exposure of gas exchange surface for a given minute ventilation. What are the first changes in lung function caused by E. haemolytica? There were no instantaneous effects of exposure to E. haemolytica but by 3 hours post exposure Pa02 had decreased. Because PaCOz was constant, hypoxemia was not due to alveolar hypoventilation but was due to either ventilation-perfusion mismatching or diffusion impairment as reflected by an increased AaDOz. Increases in respira- tory frequency combined with a tidal volume maintained at baseline levels resulted in increased minute ventilation compared to baseline at this time. The increased minute ventilation was not effective in increasing alveolar ventilation (VA and PaCOz remained unchanged) indi- cating that the increased ventilation was dead Space ventilation (increased V0 and VD/VT were observed). To determine whether this increase in dead space ventilation reflected a shift in gas distribution in the lungs or whether it was merely the result of more rapid 134 respiratory frequency, we calculated the dead space volume (VD) where VD = vD/f at baseline and at T=1, T=2 and T=3. The volume of V0 was greater at T=1, T=2 and T=3 than at baseline by approximately 50%, indi- cating that alterations in 99 are not fully explained by changes in respiratory frequency alone and are due to increases in dead space volume. Increased VD/VT in the early stages of pasteurellosis was accompanied by increased AaDOz and indicates the develOpment of ventilation-perfusion (V/D) inequalities within a few hours of f, haemo- .Lytiga exposure. Two mechanisms may serve to promote the rapid and extensive develop- ment of V/D inequalities. Firstly, the destruction of pulmonary tissues associated with bovine pneumonic pasteurellosis involves principally the cranial lobes. AS a result, anatomic dead space is probably increased as more of the inspired air is directed to caudal lobes away from poorly ventilated diseased cranial lobes. Secondly, gas exchange impairment may be compounded by the per- sistence of perfusion to poorly ventilated, pneumonic regions of lung which were damaged by E. haemolytica. It would appear that, at least in dogs, bacterial products disturb local mechanisms of 9/6 matching, perhaps by impairing hypoxic vasoconstrictor responses.23 Similar mechanisms of bacterial induced 9/6 mismatching may be present in calves. Evidence from histopathologic studies of E. haemolytica infected calves (see Chapter 4) indicates extensive perfusion of pneumo- nic portions of lung. From previous studies in neonatal calves, where AaDOg and PaOz were determined while calves were anesthetized, we suspected that the low PaOz and relatively large AaOz were due to the combined effects of 135 anesthesia and thoracic restriction of motion by placement in sternal recumbency.24 Data from this study, in which animals remained awake but were Similarly positioned, suggests that anesthesia was not a factor, since AaDOg and PaOz are similar to values we previously described for neonatal calves.24 The differences in AaDOz and PaOg between these stu- dies and those of older calves and adult cattle25'28 may therefore relate to age differences or differences in body position during sampling, rather than the effects of anesthesia as we had originally supposed. Increases in respiratory rate develop at the same time that altera- tions in gas exchange occur. Although the decrease in PaOz might stimu- late respiration at this time, it is unlikely, because hypoxemia is a weak stimulus for respiration in calves.28 In healthy persons, PaOz usually only drives respiration at or below a carotid body PaOz of 50 mmngg, a considerably more hypoxemic condition than occurs in Pasteurellosis at T=1, and the hypoxic ventilatory drive of calves is thought to be even less sensitive than that of adult persons.28 Since there was no change in PaCOz, the most likely cause for increasing the respiratory rate is that of receptor stimulation within the lungs. The two types of receptors most likely to be involved in altering the reSpiratory rate in response to pulmonary injury are irritant recep- tors and J receptors.30:31 Irritant receptor stimulation usually leads to reflex brochoconstriction30:31 and was not observed in calves at a time when respiratory rates were elevated (as indicated by no change in RL at this time). The function of J receptors is only partially understood, but one stimulus for receptor activation appears to be pulmonary edema.30"32 136 The severe pulmonary vascular damage associated with Pasteurella haemo- .Lytiga pneumonia may lead to J receptor stimulation and initiate tachyp- nea. Stimulation of pulmonary receptors with vagal afferents is the cause of tachypnea associated with ovalbumin33 and 3 methylindole34 induced pulmonary injury of horses and with tachypnea associated vagal afferent activity of dogs after Ascaris suum antigen challenge.35 From the above findings by ourselves and others33'35, it therefore appears that tachypnea observed in a variety of pulmonary injuries in several Species occurs unrelated to gas exchange impairment or broncho- constriction. we believe that a common mechanism is responsible for tachypnea under these circumstances and that it is probably due to pulmonary receptor stimulation. The maintenance of Cdyn and RL at baseline levels during the deve- lOpment of gas exchange impairment and alternations in respiratory rate suggests that the initial lesions of g. haemolytica pneumonia occur in peripheral lung areas rather than as an extension from central airway lesions. Studies of clearance mechanisms of E. haemolytica from calf lungs after intratracheal inoculation also indicate that lesions asso- ciated with bacterial retention occur chiefly in the pulmonary parenchyma.36 It remains unclear whether initial lesions of naturally occurring 3. haemolytica infections develop in the lung parenchyma since experimentally induced disease may preferentially deposit bacteria in lung parenchyma and therefore by association, cause initial lesions in parenchymal tissues. By 12 hours post infection, calves had develOped profound hypoxaemia and alveolar hypoventilation but AaDOg had improved compared to 6 hours 137 post inoculation. Such a change in AaDOg can be explained if PAOZ declined from 6 to 12 hours post inoculation, since the effects of 9/6 inequalities become less as PA02 approaches the P02 of venous blood. Since there was no change in 902 or 9502 at 12 hours post infection com- pared to baseline values, the increase in PaCOz was the result of decreased 9A. Because minute ventilation was unaltered from baseline, the decrease in VA was due to increased VD/VT, and probably reflects respiratory failure resulting from increased work of breathing due to changes in the mechanical prOperties of the lungs. By 12 hours post infection, gas exchange impairment and hypoxemia were accompanied by a decrease in Cdyn and an increase in RL. Increases in RL may result from central and/or peripheral airway obstruction through either bronchoconstriction and/or physical obstruction of airways with cellular debris, mucus and exudates. Because peripheral airways comprise a small proportion of respiratory resistance, extensive peripheral airway obstruction is necessary to cause measurable increases in RL. From our histologic studies, it would appear that increases in RL are probably caused by extensive peripheral airway obstruction as a result of physical obstruction with purulent exudates and edema fluid. Decreases in Cdyn may occur as a result of increases in FRC, loss of parenchymal elasticity or peripheral time constant inequalities asso- ciated with small airway obstruction. Such processes are likely to exist together in calf lungs. Because bovine lungs have no collateral ventilatory pathways, peripheral airway obstruction in calves should always result in the development of peripheral time constant inequali- ties between pulmonary segments, atelectasis of segments with completely 138 obstructed airways, and hyperinflation of normal segments of lung given that FRC is unchanged. Pasteurella infection was not associated with an alteration in FRC. Because FRC was determined by Helium dilution, it reflects the volume of gas in the thorax in communication with the endotracheal tube. Histopathologic studies on the calves infected with E. haemolytica (see companion paper) indicate that extensive areas of atelectasis, exudative pneumonia and obstructive supporative bronchiolitis had occurred by twelve hours post infection. In order for FRC to remain constant deSpite the develOpment of these lesions, hyperinflation of the remaining portions of lung in communication with the unuth needed to occur. In so doing, the hyperinflation results in decreases in Cdyn below baseline values because tidal breathing must occur at a flat- tened portion of the pulmonary pressure-volume curve where lung compliance is low. Hyperinflation of healthy lung tissue probably contributed to the decrease in Cdyn associated with Pasteurellosis. We describe decreases in Cdyn and gas exchange impairment of calves at 3 hours postexposure to Pasteurella, prior to any change in RL, clearly showing that peripheral airways and pumonary parenchyma are involved in Pasteurellosis well before any possible involvement of central air ways. This situation obviously differs from that caused by IBR virus in calves, where increases in resistance and hypoventilation occur as a result of central airway disease, and are unassociated with gas exchange impairment and alteration in Cdyn-B 139 REFERENCES 1. Beyt BE, Sondag J, Roosevelt TS, Bruce R: Human pulmonary pasteurellosis. JAMA 242:1647-1648, 1979. 2. Furie RA, Cohen RP, Hartman BJ, Roberts RB: Pasteurella multi- codia infection. NY State J Med 80:1597-1602, 1980. 3. Berkmen YM: Uncommon acute bacterial pneumonias. Seminars in Roentgenolggy715:17-24, 1980. 4. Starkebaum GA, Plorde JJ: Pasteurella pneumonia: Report of a case and review of the literature. J Clin Microbiol 5:332-335, 1977. 5. Rehmtulla AJ, Thomson RE: A review of the lesions in Shipping Fever in cattle. Can Vet J 22:1—8, 1981. 6. Al-Darraji AM, Cutlip RC, Lehmkuhl HD, Graham DL: Experimental infection of lambs with bovine respiratory syncitial virus and Pasteurella haemolytica: Pathologic studies. Am J Vet Res 43:224-229, 1982. 7. Bentley 0E and Farrington DO: Evaluation of an induced Pasteurella multocida swine pneumonia model. Am J Vet Res 41:1870-1873, 1980. 8. Kiorpes AL, Bisgard GE, Manohar M, Hernandez A: PathOphysio- logic studies of infectious bovine rhinotracheitis in the Holstein-Fresian calf. Am J Vet Res 39:779-783, 1978. 9. Derksen FJ, Robinson NE: ESOphageal and intrapleural pressures in the healthy conscious pony. Am J Vet Res 4l:l756-l76l, l980. 10. Amdur M0, Mead J: Mechanics of respiration in unanesthetized guinea pigs. Amer J Physiol 192:364-368, 1958. 11. Slonim NB, Hamilton LH: Respiratorerhysiology, ed 4. St. Louis, The CV Mosby Company, 1981, pp 243-272. 140 12. West JB: Respiratornghysiology - The Essentials, ed 2. Baltimore, The Williams and Wilkins Company, 1979, pp 160-165 13. Steel RGD, Torrie JH: Principles and Procedures of Statistics. New York, McGraw-Hill Book Company, Inc, l960, pp. l06-llO. 14. Kelley KN: Stress and hmnune function: A bibliographic review. Ann Rech Vet 11:445-478, 1980. 15. Jensen R, Pierson RE, Braddy PM, Saari DA, Laverman LH, England JJ, Keyvanfar H, Collier JR, Horton DP, McChesney AE, Benitez A, Christie RM: Shipping Fever pneumonia in yearling feedlot cattle. .JAVMA 169:500-506, 1976. 16. Lillie LE: Symposium on immunization of cattle against the common diseases of the respiratory tract. Can Vet J 15:233-242, 1974. 17. Bryson DG, McFerran JB, Ball HJ, Neill SD: Observations on outbreaks of respiratory disease in housed calves ~ (1) Epidemiological, clinical and microbiological findings. Vet Record 103:485-489, 1978. 18. Parker NH: Respiratory disease (Epizootic Bronchitis) in housed calves. The Veterinarian 3:235-242, 1965. 19. Martin SH, Schwabe CH, Franti CE: Dairy calf mortality rate: The association of daily meteorological factors and calf mortality. .932 J comp Med 39:377-388, 1975. 20. Anderson JF, Bates DH: Influence of improved ventilation on health of confined cattle. .JAVMA 174:577-580, 1979. 21. Jericho KNF, Magwood SE: Histological features of respiratory epithelium of calves held at differing temperature and humidity. .Can_J comp Med 41:369-379, 1977. 141 22. Jericho KNF, Langford EV, Pantekoek: Recovery of Pasteurella haemolytica from aerosols at differing temperature and humidity. 'Can_J comp Med 41:211-214, 1977. 23. Light RB, Mink S, Wood LOH: Pathophysiology of gas exchange and pulmonary perfusion in pneumococcal lobar pneumonia in dogs. _J_Appl Physiol 50:524-530, 1981. 24. Slocombe RF, Robinson NE: Histamine H1, H2 receptor effects on mechanics of ventilation and gas exhange in neonatal calves. Am J Vet .333 42:764-769, 1981. 25. Kiorpes AL, Bisgard GE, Manohar M: Pulmonary function values in healthy Holstein-Fresian calves. Am J Vet Res 39:773-778, 1978. 26. Musewe V0, Gillespie JR, Berry JD: Influence of ruminal insufflation on pulmonary function and diaphragmatic electromyography in cattle. Am J Vet Res 40:26-31, 1979. 27. Donawick HJ, Baue AE: Blood gases, acid-base balance, and alevolar-arterial oxygen gradient in calves. Am J Vet Res 29:561-567, 1968. 28. Bisgard GE, Ruiz AV, Grover RF: Ventilatory control in the Hereford calf. J Appl Physiol 35:220-226, 1973. 29. Lambertsen CJ: In Mountcastle VB (ed): Medical Physiology, ed 13. St. Louis, The CV Mosby Company, 1974, vol 2, pp 1423-1495. 30. Sant'Ambrogio G: Information arising from the tracheobronchial tree of mammals. Physiol Reviews 62:531-569, 1982. 31. Paintal AS: Vagal sensory receptors and their reflex effects. Physiol Reviews 53:159-227, 1973. 142 32. Coleridge HM, Coleridge JCG: Impulse activity in afferent vagal C-fibres with endings in the intrapulmonary airways of dogs. Respiration Physiology_29:125-142, 1975. 33. Derksen FJ, Robinson NE, Slocombe, RF: Ovalbumin induced lung disease in the pony: Role of vagal mechanisms. J Appl Physiol (in press). 34. Derksen FJ, Robinson NE, Slocombe RF: 3-Methylindole-induced pulmonary toxicosis in ponies. Am J Vet Res 43:603—607, 1982. 35. Cotton DJ, Bleecker ER, Fischer SP, Graf PD, Gold NM, Nadel JA: Rapid, shallow breathing after Ascaris suum antigen inhalation: Role of vagus nerves. J Appl Physiol 42:101-106, 1977. 36. Gilka F, Thomson RG, Savan M: Microsc0pic findings in the lungs of calves aerosolized with Pasteurella haemoLytica and treated to alter pulmonary clearance. Zbl Vet Med 21:774-786, 1974. CHAPTER 4 Pathogenesis of bovine pneumonia caused by Pasteurella haemolytica: gross and microscopic lesions 143 144 SUMMARY Routine gross and light microscopic studies were performed on two groups of neonatal Holstein calves. The first group of 6 animals served as controls. They were subjected to cold stress and subsequent recording of hematologic and physiologic variables for 12 hrs following injection of saline into the trachea, as described in companion chapters. At the end of this period, animals were euthanatized and a gross and microsc0pic evaluation of each animal was performed, with par- ticular attention being paid to the respiratory tract. Acetic acid spraying of the trachea, administered during the cold stress periods, caused severe focal necrotizing lesions in the trachea. All calves had inhaled necrotic debris, neutrophilic exudate and sloughed portions of large airway mucosa. The majority of the lungs were normal but a few lobules had partially collapsed and had variable amounts of edema, hemorrhage and neutrOphilc exudates present in their lumens. The second group of 9 calves had been exposed to P. haemolytica by intratracheal inoculation, and had died or been euthanatized at various time intervals following challenge. Calves which survived 18 hrs or longer had extensive fibrinous pneumonia with necrotic foci and an inflammatory exudate containing 'swirly“ cells characteristic of P. haemolytica pneumonia. Calves euthanatized at 12 hrs post infection had similar but less severe lesions, which were associated principally with peribronchial alveoli. The lesions were most prevalent in the anterior and cardiac lobes and consisted of atelectasis, alveolar edema, congestion of alveolar vessels and a mixed inflammatory cell infiltrate accumulating principally in the alveoli and respiratory bronchioles. Calves that died at 6 hrs or less after challenge had scattered areas of 145 atelectasis accompanied by alveolar edema and an inflammatory response dominated by macrophages and neutrophils. Data from these structural studies supports the hematologic and phy- siologic studies I described for the same groups of calves. Pasteurella haemolytica causes an acute pneumonia and bronchiolitis characterized by vascular damage, edema and mixed inflammatory cell infiltration. These structural alterations correspond well with the impairment of oxygen exchange, reduced dynamic compliance and with the rapid loss of neutrOphilS from the circulation, reflecting the injury of the pulmonary parenchyma as the initial lesion of pneumonic pasteurellosis. INTRODUCTION The gross and microsc0pic lesions associated with Pasteurella pneumonia of cattle were recently reviewed by Rehmtulla and Thomson (1981).1 Although the lesions observed in lungs of severely ill cattle and those dying of pneumonia are well described, Rehmtulla and Thomson remarked upon the lack of information regarding the development of pulmonary lesions. One objective of the present study was therefore to describe the early lesions of pneumonic pasteurellosis and to correlate these findings with changes in lung function. The previous reportsz:3 describing early lesions were part of a study designed to examine mecha- nisms of bacterial clearance from calf lungs. Lesions described in some calves at 4 hours post inoculation consisted of alveolar edema and there was some evidence of increased numbers of mononuclear cells. Gilka et al (1974) postulated that the initial lesion of pulmonary pasteurellosis was pulmonary edema, which subsequently led to a washout of surfactant, atelectasis and subsequent bacterial overgrowth.2a3 Tweed and Edington 146 in 1940 had speculated that lesions, assumed to be the earliest changes in naturally occurring cases of pasteurellosis, were those of alveolar congestion, edema and desquamation of epithelial cells.4 However, others have suggested that atelectasis and alveolar edema are initially present and that Pasteurella then begin to grow in these particular areas.5 As reviewed by Rehmtulla and Thomsonl, the lesions of E. haemolytica pneumonia are thought to progress from atelectasis and pulmonary edema to those of acute fibrinous pneumonia and pleuritis, with areas of infarction and irregular foci of necrosis. There are, however, other reports of histologic lesions associated with pneumonic pasteurellosis that do not fit this classical description of Shipping Fever pneumonia; one of these lesions, described by a number of different authors as a prominent bronchOpneumonia4’10 associated with Pasteurella infection was thought uncharacteristic of the disease by Thomsonl, hisI coworkers11 and others.5:12:13 Clearly, there is conflicting evidence regarding the nature of lesions of experimental and naturally acquired pasteurellosis, even when the lesions are well developed. Differences in the lesions of experimental pasteurellosis may in part be due to dif- ferences in experimental design, since some methods of inducing pasteurellosis rely on previous or concurrent viral infections, but others do not. This study was designed to describe the gross and microsc0pic lesions of acute_fi. haemolytica infection without concurrent viral expo- sure. Changes in pulmonary structure were correlated with those altera- tions in pulmonary function and blood constituents described in the pre- vious chapters. 147 METHODS Control Calves Six neonatal calves (C1 through C6 respectively), instrumented and exposed to a single intratracheal injection of saline as previously described, were used as control animals. These animals had previously been chilled and acetic acid sprayed into the trachea (see previous chapters). After completion of the physiologic studies, animals were euthanatized by electrocution. The calves were promptly necropsied, with inspection of all organs, before samples of lung, liver and kidney were taken for microbiologic survey as outlined below. With the excep- tion of brain tissue, which was fixed whole in 10% phOSphate buffered formalin, and lung tissue, which was fixed in the manner described below, samples consisting of 1 cm thick slices of body organs were taken for routine histOpathology. A major bronchus from the left and right apical, cardiac and diaphragmatic lobes was cannulated with a tightly fitting PE catheter and these lobes fixed by a combination of submersion and airway perfusion. Airway perfusion pressure was 33 cm of H20 and the fixative was modified Karnovsky's fixative. The apparatus used for fixation is described in Appendix B. Sections of trachea and major bronchi were also fixed by submersion in Karnovsky's fixative. Sample sites for histopathologic evaluation are illustrated in Figure 4-1. Pasteurella Group Five neonatal calves (Pl-P5), instrumented and exposed to a single intratracheal injection of 2 x 109 E. haemolytica organisms were eutha- natized by electrocution 12 hrs after eXposure to_fi. haemolytica. The sixth calf (P6) died at 18 hrs after challenge and a seventh calf (P7) 148 .cchscm cc: me: new ccccUHcc» cc: m» cccH Accmmcccc cc» .H<4v caacaa ccac new Accv cacccaa scaH .Hccv occasmaceaacc ccac .Homv c_caEmac;aa_c ceccc .Hcav cchccc uanc .Hc UHmchum»; cc» :cxcu mchEcm ccmmwu xcchc ccc accH cc» mccwm m:_HcEcm czu mcwcccumcHH» mmccH »Hcc »c Eccmcwc UHHcsczcm .H-e acamcc Hue cccc»c ....H.. t, \s\\\ a K _\.1\\\ WM»... . n v\ x.» “W .. I.“ cm »c :cHHcccxc cg“ cc»: Hes -cc: ccc mmccH cc» .mu »Hcc Ecc» ccc mmccH cch .mmccH »Hcc HcEcc: »c cccccccccc chccmccccz .m-e ccsccc 158 m-s acaccc 159 .spmccH :» ac m.mH m» ccxcce cc» .cmcc asp »c cop cs» ccczcu mcHH cczcccp ugh .mcccH cHHcsmccgcch acch czu ccc Hcc -Hcc cccc .cchccc cgmwc cs» :» cccccccccccc ccc ccc cwccscccc »c meccc ccc mecca xccc cc» .mc »Hcc Ecc» ccc mmccH czh .»Hcc c :_ mHmcHHccccpmcc cwccscccc »c cccccccccc cHQccmccccz .¢-e acamcc 160 Figure 4-4 161 .5c H mcccccccc ccxccz .ccccm cmeccw .cccH» cc»: ccccccc chccz ccc ccccm cchcchcccc ccc ccc Hzccccv ccccccm czc »c mccccccc ccc chc :» cccmccc m» cccccxu .HHc »Hcc »c cccH Hccccc ccmcc ccc Ecc» :cccccm mccc .cgccccc ccnce cgc cc cmcmch mchccH c» ccc>cm cw9= ccc mcccmcH cc» .mHmcHHcccccmcc ccccEcccc cc»: ccccc»»c mmccH »c :cccmccccs mmccmccm .m-e acsmcc 162 rfE‘"""' ‘ 05" u A, 4" *.\ \ Figure 4-5 163 considerable numbers of adjacent lobules were affected. Sectioning through these lesions resulted in the oozing of serosanguineous fluid from the cut surface. An estimated 10 to 15% by volume of pulmonary tissue was affected in each calf. There was no evidence of pleural effusion or pleuritis in these calves but mediastinal lymph nodes were enlarged, congested and edematous. Calf P8 (died at 36 hrs after B. haemolytica inoculation) had an estimated 50% of the lung involved in a severe pneumonic process. There was extensive involvement of the anteroventral aspects of the lungs which were red to purple and very firm. The pleura over the affected regions of lungs was covered with a fibrinopurulent exudate and there was excessive amounts of sero- sanguineous fluid in the pleural cavity which contained fibrinous clots. There were fibrinous adhesions of the visceral pleural to parietal pleura in the anteroventral regions of the thorax. (Figure 4-6). The trachea and lymph nodes had changes similar to those described for other Pasteurella exposed calves. Calf P9 (died at 4 hrs after B. haemolytica inoculation) had changes in the trachea associated with tracheostomy and exposure to acetic acid but the lungs were not different to those in the control group. No other organs were abnormal on gross examination of all Pasteurella exposed calves. Histopathology The severe damage to the tracheal mucosa caused by acetic acid Spray was evident in all calves. Histologic sections taken through the acetic acid exposed area were devoid of mucosa. The luminal surface of these areas was lined by a diptheritic pseudomembrane consisting of necrotic cellular debris, fibrin and a neutrOphilic infiltration. The 164 .Amzcccc chcccv cccH ccccEmccccccc cgmcc mcc »c ccc»ccm Hcccccc ccc :c mccc cccmcccc ccoHcccccccccH» cmccH c ccc Hzcccc chccmv chHmc> mc ccm Hcccccccccc use .czmcc csc ccczcc mccH ccccccc cg» .mcccH chcEmccscch cgc »c ccccmc Hcmccc mcc ccc HHc acc>Hc>cc cccc23ccc c>cmcccxc m» ccczc .ch »Hccv mc; mm cc» cchHHcEcc; .m.;cH: coccc»cc »Hcc c :_ mcccccch ccc ccccEcccc c>cmcccxm .c-¢ acsmcc T65 c-3 acamcc 166 superficial layers of underlying submucosa were also necrotic. The remaining submucosa was edematous and infiltrated with neutrophils. (Figures 4-7). The lesion was limited to the trachea. The mucosal sur- faces of mainstem bronchi and their ramifications were normal. However, relatively small numbers of airways and alveoli had accumulations of necrotic exudate, often mixed with sloughed pseudostratified epithelium, present in their lumens. Occasionally these masses completely obstructed airways and were associated with small areas of atelectasis (Figure 4-8). The majority of pulmonary tissue in the control calf sections was normal. Because of the pressure-perfusion method of fixation, lung tissue was expanded. (Figure 4-9). Sections of lung tissue prepared in this manner had widened zones of connective tissue separation around blood vessels, bronchi and interlobular interstitium. (Figure 4-10). A minor proportion of lobules from lungs of all control calves were abnor- mal. These appeared partially collapsed, had increased numbers of mono- nuclear cells free in the alveolar spaces and in the most severe instan- ces had accumulations of neutrOphilS within alveolar Spaces. In these lobules the alveolar walls were congested and occasionally small areas of hemorrhage and accumulation of proteinaceous material were noted in alveolar lumens and terminal airways. (Figure 4-11). Microsc0pic lesions were noted in the livers and small intestines of control calves. Sections of liver tissue from all control calves had periportal accumulations of mixed inflammatory cells. With the exception of calves C2 and C6, the liver changes were also accompanied by small necrotizing focal lesions in the parenchyma which had a 167 .==_m mcccccccc ccxcca cc» .ccccm Na: .cu »ch .Accmccv chccccccc: »c xHHcccccccc mcccmcmccc mHHcc accccEEcH»:H cccz cccccHH»:c cmccccc cc ccccccsccm ccc cmcc32ccm ccc cccc ccccccxc mccmmcc »c mcmcccc: ccc .ccscH Hccccccc ccc cccc xcccm cccc cccccc accchHc» »Hcc c »c cccmcH Hccgcccc czc »c ccccmccccz .cic cccccc T68 c-e acsmcc 169 .Ec H mmccccccc ccxccz .ccccm ma: .cu »Hcc »c cccH ccccccc c»cc .ccmcc cgc c» Hccccc c» czccm m» ccc Hmzccccv Eccccccccc Hcccmcccmcc cc_»_ccccmccccmc »c mccccccm cccmcccm can cccccxc cchgccccccc »c mcmcmccc cgccccc HcccEccc csc meccccccmcc .cccccce cc» .ccccccccmcc Hccnccccc zccz cccccccmmc mcmcccccccc »c meccc Hccc» accccccmcHHH »Hcc Hcccccc c Ecc» ccmmcc mccH »c zcccmccccz .c-a acamcc l70 c-e ac=c_c 171 .55 m maceccccc caxcaz .ccacm cc: .ac ccoc co ago. occacmacccacc ccccc .mmcac »Hcc Hccccc cc cccmmccc cocc: c>Hccxc» cccz maczccc »c cccmc»ccc Ac cccccxc» mccH »c ccc»»m .mic cccccc 172 Figure 4-9 173 ccxccz .55 H mccccccc» .ccccm Na: .cu »Hcc »c cccc cccccmccccccc ccmcc .c>_ccxc» cc»: cccmc»ccc cccmmccc chccc scc» mccchmcc ccc»»ccc zccccc= mccccccmcHHH mmccH »Hcc Hccccc »c ccccmccccz .cc-e mc=m_c T74 175 .cccccc ooN cccccccc» cccccc .ccccm Na: .cc »ch .mccxcccccxcc ccc mHHcc ccchcccccc cmccc Hccccmcccc ccc: cch ccc «cccccccccc »c cccccc mcccmcmccc mHHcc acccccEcH»cc ccccccc mccacH ccccc ccc ccccH»cc xch» ccc ccc chc>H< .»Hcc Hcccccc c »c ccxccccccc HccccEHcc ccc cc cccmcH Hccc» c »c ccccmcccceccccc .HHic mccmcc 177 dominant neutrOphilic infiltrate. (See Figure 4-24) Sections of small intestine from calves C4, C5 and C6 had areas where villi were shortened and occasionally fused. There was congestion of the tips of affected villi and a mild to moderate increase in lymphomononuclear cell numbers in submucosal areas. In all three calves, numerous protozoal forms characteristic for Cryptosporidiumigp were noted on the epithelial sur- faces of affected villi. (Figure 4-12). The histologic appearance of control calf brain tissue was not con- sidered abnormal, although on first impression there appeared to be some degree of perivascular cuffing. This appearance was attributed to the abundance of connective tissue nuclei normally found in the blood vessel walls of neonatal calves. Similarly, although the width of the Splenic follicles was narrow (100 to 200 pm) this was also considered normal. Small clusters of cells, principally comprised of leukocytes and their precursors, but occasionally containing erythrocyte precursors and mega- karyocytes were noted in the adrenal cortex and Spleen. Similar Small groups of cells were not infrequently observed in liver tissue but were never found to contain megakaryocytes. Calves P1, P2 and P9 had changes in the respiratory tract that were similar to those observed in control calves. In addition, some lobules had mild to moderate amounts of hemorrhage and alveolar edema, and large numbers of inflammatory cells, often dominated by neutrophils, in the alveolar spaces and terminal airways. Affected lobules were atelec- tatic. (Figures 4-13 to 4-15). Calves P3-P7 had more extensive and severe lesions than calves P1, P2 and P9. Major airways had normal mucosal surfaces but occasionally had necrotic cellular debris and inflammatory exudate plugging their 178 .cccccc oH meccccccc ccxccc .ccccm Na: .cu »Hcc »c EccHH .Amzccccv mccc»c:m HHcc Hcmcccc ccc cc cc~chcm_> cc ccc mccc» cchccccc mccccczc Accmccv ccccccc»ccmce ccccccm c< .cmccccccm ccc cc ccccch»cc cccccccccccccccxH c cc ccccc ccc ccccccccm ccc HHHH> .mm.Eccchccmccchu ccc: cccccccmmc mcccccccc cccccccm ccc: »Hcc c »c ccccmmccc HHccm ccc »c ccccmcccccccccc .Nc-¢ acaccc T79 Figure 4-l2 180 .25 N.o mcccccccc ccxcce .ccccm Na: .Nc »ch .Amzccccv cccH» ccccc .cccc ccccccc ccc: ccc—c» ccc HHcc>Hc cecm .chcccccccc »c HHcccE ccccmcm -ccc cccccxc cchHHcc ccccccc ccc ccccH»cc AHHcccccc chc ccc cccc>H< .ccccmccccc ccc »c cccccc ccc cccc cccccccm cc cccccxc cccccccc »c cccccc HHcEm c accccccccc chHcccccc < .ccccchcccc cHHcccccmcc ccc; ccccc»cc mmccH »c mcccmcH ccmchcmcc ccc »c ccccmccuccccccc .mHuc cccmcm Figure 4-l3 182 .22 m.o mccccccc» ccxcce .ccccm Na: .Nc »ch .cccmccc cc cmcccmcc Hcccceccc»c» chcccccccc chc < .ccccmccccc ccc »c cccccc ccc cc cccmccc m» cmcccccccc ccc ccccc .mcmccccc Hch cchc>H< .ccccHHccccc cHHcccccmcc ccc: ccccc»c» »Hcc c Ecc» cccmcH cchc>Hc mcc~ccccccc c »c ccccmccuccccccc .ac-a acsccc 183 vpuc mczmwm {v.3 7 184 .55 m.o mcccccccc ccxccc .ccccm ma: .Nc »ch .Hmzccccv Hcccccce cccc ccccccc ccc» HHcc c ccc: ccccccmcc ccc muccccceaH ccc cccmccc mc ccccc cchcmc>cccc ccc HcccccmccccH .mcm -cHHcccccmcc ccccccccc ccc: cccccccmmc ccccc cc»: cccmcccmcc cccccccxH »c ccccmcccccccccc .mH-e cczmcc m r— I <7 6’ S. 3 O"! ~r- Ll— 186 lumens. (Figures 4-16to 4-19). The bronchioles and alveolar ducts con- tained exudate consisting of a mixed inflammatory cell exudate quite different than that observed in the control calves. (Figure 4-20). The exudate contained numerous basophilic degenerative cell cytoplasms that also had large smudged baSOphilic nuclei. There were also large numbers of degenerating neutrOphils and bacteria, the latter being readily visualized as small Short rods on Giemsa stains (Figure 4-21), and which were gram negative. Many of the mononuclear cells in the exudate had a characteristic fusiform, swirling or streaming appearance to their cytOpIaSms. The alveolar walls of affected lobules had congested blood vessels but accumulations of cells, exudate or thrombi within alveolar walls were not noted. Alveolar lumens nearest major airways seemed most severely involved, but atelectasis of alveoli in otherwise unaffected lobules was also noted. Alveoli often contained much proteinaceous material, with variable amounts of hemorrhage and exudate Similar to that found in the bronchioles. In some areas the proteinaceous material had a fibrillary network, suggestive of fibrin. In some areas, where there was obvious alveolar wall necrosis, there was extensive hemorrhage. (See Figure 4-19). The develOpment of exudate in alveoli and of proteinaceous fluid deposition with or without hemorrhage appeared to occur as independent processes as some alveoli had cellular exudate alone, others had edema alone and yet both occurred together in the majority of alveoli. (See Figures 4-13, 4-14 and 4-16) There were focal areas of subpleural and peribronchial enphysema in areas of lung that were not affected with pneumonia. (Figure 4-22) 187 .Ec H mcccccccc ccxcce .ccccm Na: .mc »Hco .ccccccccccc ccc mcccccc mccccccmccc ccc Hzccccv cccH» csccc cc»: ccccccmcc ccc mocccccea— Hcccccmcmccc .cccmcH chcccccc ccc mH ceccc cchc>Hc ccccz mccccc ccc cccccHH»cH cchHHcc cmccccc »c ccccc mccccccc chccH ccc .ccccHHccccc cHHcccccmcc cc ccmccxc »Hcc c c_ ccccacccc cchccH »c cccccccccsccccc .cc-¢ aczccc Figure 4-l6 189 .55 m.o mccccccc» ccxccs .ccccm Na: .oc »ch .mxczccc ccmccH ccc cc ccccH253ccc ccc cccccxc ccc mHHcc xccccEEcH»cH ccc: ccccccH»»cc ch>cmcccxc cc chccH ccc .ccccchEccc cHHcccccmcc ccc: cccchcccc »Hcc c cc mcccmcH cccc53ccc cccch>cc Hch »c ccccmccccsccccc .mHuc mcsmcc l90 nHiv mccmwm 191 .55 H.o ccccccccc ccccce .ccccm Na: .oc »ch .Amzccccv mHHcc xccccescH»cc ccccccccc c>cc cccc ccscccc mccccccc» cmccH cccc -ccc ccc cccH» ccccc ccc: ccccccmcc chcccm ccc mocccccexH Hcccccmccccc cc» .mcccccH ccc cc cccchccccc ccc cccccxc cchHHcc c>cmcccxc ccc ccmccHHcc ccc ccmmcc cchc>H< .ccccHHcEccc cHHcccccmcc ccc: cccchcccH »Hcc c c» mcccmcH cccce:ccc cccch>cc Hch »c ccccmccccsccccc .mHic cccmcc 192 . . . {L ..e . . .mcrcwmxc \r e '1‘ . he mcuc mccmcc 193 .cc_H.o mcccccccc ccxcca .ccccm ma: .Nc »Hco .cccH» ceccc ccc cccxcccccxcm .mHHcc xccccEEcH»cc .mccccc cccccccc »c mcccmcmccc HHHcccccccc mcccccscc mccccccccm ccc .chccmccmcccmcc ccmccH cc ccc mHch cchc>H< .ccccchcccc cHHmccccmcc ccc: ccccH uccccc »Hcc c »c cexcccmccc xccccEHcc ccc c» mcccmcH chNHccccmc ccccc »c ccccmcccccccccc .mc-e acgmcc T94 m_-3 acsmcc 195 .mcccc_E ON mcccccccc ccxccz .ccccccc mccEccccm c »c ccccmcmmcm cccm cc cccc» .Hmzccccv ccmccsm ccc ccccz »c xccs .mHHcc ccchcccccE »c mcmcmccc ccc Amcv mcmcHHcccccmcc Acccccccc »c mcccmcH cccch>cc Hch ccc: cccccccmmc cccccxc ccc mcccccmcHHc NONuc cccmcc .mcccccccccc »c accccc mcmcmccc ccc Acuv ccccccm »_cc Hcccccc ccc» cccccxc Hccccxc mccccccc» Hcc ccmccxc -cHHcccccmcc ccc Hcccccc »c mcccccxc cchccccccc »c cccccccccc ccmccccmcc ccc »c ccmccccEco .cN-a acsccc 1 1111 197 .mcccccs oH mcccccccc ccxccc .ccccm cmsccu .mc »ch .mccEcH cchc>Hc cc acccchscccc cccccxc ccc c» cccmccc ccc ccccc -ccc mccccccc ccc cccccccc m» mHHcc ccchcccccE cccccmccc» »c mcccccccm .cccmcH xcccccccc cccmchccmc cc cc» ccccchcccc cHHcccccmcc cc cmcccmcc xccccEEcH»cH ccc »c ccccmccccEccccc .HN-e ac=c_c T98 .55 H ccccccccc ccxcca .ccccm Na: .mc »ch .cccccccc ccc mHch cchc>Hc »c ccccccc .cccccH»cccc>c »c mcmcc ccc>cm cmcc ccc cH .ccccchemcc cHHcccccmcc ccc: ccccccccc» »Hcc c »c ccmmcc 199 mccc Hccccc cmczccccc cc cccccH»chcch cchHcccccccccc ccc Hcccmccccm »c ccccmcccccccccc .NNic wccmcu 200 NNI¢ acumHm 64. .v c. 33 ,. I, c c . ’ b . ... e O as .0 .e . . 4 I .4 e . .ee . k . p e a . w e C . 201 There were no lesions in the arterial vasculature but veins were often congested and had pronounced margination of leukocytes, prin- cipally neutrOphils against endothelial surfaces. Capillaries were congested not only in lobules containing exudate but were also congested in the alveolar walls of neighboring lobules. The lymphatics asso- ciated with airways and the interstitium were distended with proteina- ceous fluid but only rarely contained inflammatory cells or fibrinous thrombi. The pleural surfaces of the lungs had similar inflammatory changes as a result of extension from neighboring lobules. Fibrinous thrombi containing small numbers of inflmnnatory cells were present on the pleural surfaces of pulmonary sections from P6. (See Figure 4-18). All calves in the Pasteurella group had a neutrophilic inflammatory reSponse of the bronchial lymph nodes, with large numbers of neutrophils filling medullary and subcapsular sinuses. The lymph nodes were congested and edematous and in calves P2, P4 and P7, the lymphoid tissue in the cortices of the nodes was depleted. (Figures 4-23). Calf P8 had the most severe lesions, with large irregular areas of necrosis that were often confluent between lobules. The inflammatory changes were histologically similar in nature to those previously described for calves P3-P7. Calves in the Pasteurella group had a similar histologic appearance of hepatic and Splenic tissue as the control calves. (Figure 4—24). Calves P2 and P3 had Similar changes in the small intestine as described for calves C4, C5 and C6 except that no protozoal organisms were observed. Calves P1, P2 and P3 had mild lymphomononuclear to mixed inflammatory cellular perivascular cuffing in the brain and in P2 this was associated with a single microabscess in the cerebral gray matter. 202 .52 m mccccccc» ccxccs .ccccccm Na: .oc »ch .Amzccccv cccccccccc mc mcmmcc ccccccxH Hccccccc »c cccccccco .Hccmcc ccmv chcccccccc mccccccc ccccccc ccccz mcmcccm cchmcccccm ccc ace—Hacce »c ccccchc ccc: mcccccccc mc ccch ccc .ccccxmccccc cHHcccccmcc ccc; cccccccccc »ccc c cc cccc ccaxH Hccccmccccc »c mcccccccccexH m>ccccccc=m cccc< .mm-¢ acsccc 203 Figure 4-23 204 .55 H.o mcccc icccc ccxcce .ccccm Na: .Nc »ch .Azccccv cccccc ccc »c cccc Hccccccccc ccc c» cccmmcc cc cmcccmcc HHcc accccsccH»cH ccxca < .cexccccccc cm>cH ccc cc .chcccccccc »c xccccccsccccc ccmcccccc .cccccccc accccEEcH»cc cmcccc» cc ccc mcmccccc »c mccc» ccc .ccccceccH»cc Hccccc icccc ccc mcmccccc »c mcccc Hccc» mccccccmcHH» »Hcc c »c cc>HH ccc cc mcmcccc accccEEcH»cH .3N-a acsmcc 205 vNiv cccmcc 206 A Single thrombus was noted in a small arteriole in the brain of calf P3. Bacteria were not observed in the perivascular cuffs or in the microabscess using H&E, Geimsa and Gram stains. Sections of pituitary, kidney, heart, adrenal, spleen, pancreas, skeletal muscle, tongue and colon were normal in all calves. Sections embedded in glycol methacrylate had little evidence for alteration in mast cell numbers or their degree of granulation, or for changes in the numbers of goblet cells and the staining properties of their mucus. DISCUSSION The respiratory system of the control group of calves was clearly abnormal structurally but was not functionally affected because of the mild nature of the lesions. (See chapter 2). The pulmonary lesions of control calves consisted of atelectasis and foci of edema and neutrOphi- lic inflammation. Several influences may have invoked this reaction. The acetic acid Spray was obviously a potent necrotizing agent in the trachea, where presumably most of the chemical was delivered. The potential for aerosolized dr0plets to be inhaled into the lungs was pre- sent and these may be in part responsible for the lesions observed. In previous studies, that used acetic acid as part of a protocol to infect calves with Pasteurella §pp, adverse effects of acetic acid on the respiratory system were not noted except in one calf14915, that repor- tedly developed tracheal ulceration after submucosal injection with acid. However, acetic acid may have contributed to the histologic changes noted in the lungs. 207 Other than a direct effect of acetic acid, inhalation of necrotic mucosa, exudate and neutrOphils which originate from the acetic-acid- induced tracheal lesion may lead to small airway obstruction, atelec- tasis and local inflmmnatory changes in the lungs. As suggested in the preceeding chapters, cold stress may facilitate the develOpment of pneumonia by immune suppression and increased eXpo- sure of alveolar surfaces to inspired pathogens. Isolation of small numbers of mixed bacterial types was possible from the lungs of all control calves. These small mixed bacterial infections may have contri- buted to the devel0pment of the pulmonary lesions of control calves. Despite the lesions found in control calves, whatever their cause, we believe that the animals fulfilled the role of control animals, since the majority of lung tissue remained normal and respiratory system func- tion remained unimpaired. A structural change occurred in all lung tissues of control (and Pasteurella) calves unassociated with any antemortem tissue injury. This change is the so called "edema artifact“ induced by pressure per- fusion of the airways with fixative and explains the widening of interstitial, peribronchial and perivascular tissue spaces with fixative fluids.16 Antemortem edema of these regions is therefore very dif- ficult to assess unless accompanied by a high protein content. The advantage of lung samples prepared in this manner, was that the size and state of inflation of alveoli could be critically assessed. Previous studies of pneumonic pasteurellosis have not prepared pulmonary tissue in a standardized manner suitable for assessing alveolar Sizes. The aging of acute pulmonary lesions based on histological appearance is difficult and yet, to date, understanding of the mecha- 208 nisms of E. haemolytica induced pulmonary injury have relied on such interpretations. Exceptions are the studies by Gilka et al (1974)-1»4 and Friend et al (1977)11 who recorded the length of time post exposure to E. haemontica till death, during the first days of g. haemolytica pneumonia. Hallmarks of established lesions in other organs may not be valid in lungs since blood, fluid and macrOphages may be pre- sent in alveoli in early as well as devel0ped lesions. Other dif- ficulties with histologic interpretation of pulmonary tissues that have, to date, been ignored are the possible presence of concurrent subclini- cal disease and the failure to fix lung specimens under standardized conditions. In this study I minimized these detrimental influences on histolo- gic interpretation by using neonates tested for pulmonary function as subjects (minimizing the risk of concurrent or chronic disease) and by fixing the lungs in a standardized manner. I also avoided the rationa- lization that less severely affected lobules reflected early changes, since there is no evidence to the contrary to indicate that such lesions are not Simply a reflection of graded responses to injury. From physiologic studies (see Chapter 3) it was predicted that the early lesions of bovine pneumonic pasteurellosis would be associated with pulmonary parenchyma and peripheral airways. The lesions of P7 and P9 examined at 4 and 6 hrs post challenge reSpectively indicated the initial involvement of parenchymal tissues without changes in the major intrapulmonary airways. Similar changes are described in calves 4 hrs after exposure to_g. haemolytica.3 The Spread of inflammatory exudate into small bronchi had occurred by 12 hrs post exposure. In my study, involvement of the major airways 209 either by plugging with exudate or by necrosis and inflammation of the bronchial walls was not a feature of the disease, even in the animal that lived for 36 hrs. In other studies significant bronchitis was absent in calves exposed to B, haemolytica aerosol for at least 18 hrsZ-3:11 but is present in experimental cases after three11 or four8 days. One aim of this project was to determine alterations in alveolar lumen dimensions and alveolar wall thickness in calves 12 hrs after infection with 3. haemolytica. I did not anticipate the extent and severity of lesions found after this time, as they were similar to well devel0ped lesions described in naturally occurring cases and experimen- tal cases that were several days old.1i3:3»11 Because of these severe changes, a morphometric analysis of alveolar sizes had to be abandoned since many areas had extensive necrosis of alveolar boundaries. The characteristics of pneumonia by 18 hrs post infection were well sum- marized by Rehmtulla and Thomson (1981)1 who describe interstitial and alveolar edema, interstitial lymphatic thrombosis, fibrinous pleuritis and alveolar wall necrosis. From the data of calf leukograms, where pronounced neutropenia devel0ped, and the pulmonary histologic studies, it is evident that neutrophils have a significant role in the disease both in the early and develOped responses to 3. haemolytica. I describe leukopenia in calves associated with 3. haemolytica pneumonia as has been described elsewhere3»17, and found suppurative lymphadenitis and neutrophilic exudates in alveolar spaces and bronchioles of calves examined histologically 12 hrs after exposure. I therefore differ from the view of Rehmtulla and Thomsonl, that sup- purative responses are atypical of the disease. Others have also noted 210 exudates containing many neutrOphils in cases of experimental E. haemo- lytica pneumonia in calves.8:9»11 Mononuclear cells have been described by Gilka et al (1974) as the initial inflmnnatory cell involved in the responses to_fl. haemolytica exposure. My data suggests that mononuclear cells, principally macrOphages, have a role in both early and developed pulmonary lesions, but that this response is frequently mixed with neutrophilic infil- trates. Thomson and others have attributed the “swirling“ or "streaming“ basophilic mononuclear cells, so characteristic of bovine pasteurellosis, to altered populations of macrophages. I cannot con- finn that macrophages are the only cell type to contribute to the exu- date which contains swirly cells. Degenerative changes in the alveolar type II cells have been observed, resulting in their sloughing into the cellular debris collecting in alveolar lumens.1:3a18 Other mononuclear cells such as bronchiolar cells, and migrating lymphoid cells might also contribute to the exudate. The exudate observed with pasteurellosis is distinctive in part because of the papulation of fusiform “streaming" cellsl:8-11:19a20, yet the cause for the “streaming“ appearance is unclear. Since necrotic debris and exudate from acetic acid induced lesions had a distinctly different appearance to that associated with pasteurellosis, it seems that the streaming effect is unlikely to be related to mechanical stresses placed on collapsed segments of lungs, Since both caused air- ways obstruction and atelectasis. The cause of “streaming“ therefore appears directly related to E. haemolytica. Furthermore "streaming“ was not present in calves P7 and P9 indicating that the response occurs only in well developed lesions. Whether this effect is directly related to 211 some cytotoxic activity of_g. haemolytica or due to the effects of liberated inflmmnatory cell products is unknown. Both mechanisms may act simultaneously.21‘25 In my research, edema formation often occurred independently and separately from Sites of cellular exudate formation and suggests that E. haemoiytica may injure the lung by at least two separate mechanisms. Pulmonary edema has been previously suggested as the initial lesion of E, haemolytica pneumonia2.3, but small numbers of inflammatory cells were also noted in these early lesions. Although the lesions in the lungs tended to have an anteroventral distribution, no differences in the histologic appearance of the lesions were noted suggesting that similar pathogenic processes take place in all lung lobes. The cause of the vascular damage associated with Pasteurellosis is uncertain. Othersl-11 describe the development of fibrinous thrombi in lymphatics and the flooding of alveoli with proteinaceous fluid that contains fibrillary strands suggestive of fibrin, as was the case in this study. From this study, the distension of lymphatics with fluid and edema of alveolar spaces occurs before thrombosis of lymphatics, indi- cating that lymphatic thrombosis is not the cause of alveolar edema. Others have reported blood vessel thrombosis as part of pneumonic pasteurellosis3, but in some studies it was only noted in a proportion of casessa12 while in others it was never observed.11.19.20 One possible cited cause for the vascular injury is endotoxin. Pasteurella .33 are potent sources of endotoxin and in calves its administration is associated with pulmonary hypertension, increased pulmonary vascular resistance, and leukocyte migration into the lungs.25'33 Although spe- cies vary in their reSponse to endotoxin, generally endotoxemia is 212 'Essociated with vascular thrombosis as a result of platelet activation, aggregation and degranulation.34'36 Therefore, endotoxin may account for some of the vascular damage observed in pasteurellosis of cattle. However, the failure to find vascular thrombosis in my study is incom- patible with known actions of endotoxin in cattle. Functional evidence of ventilation-perfusion (V/O) inequalities in Pasteurella exposed calves is the increase in alveolar-arterial oxygen difference that occurred in association with decreased dynamic com- pliance in infected animals. These changes suggest that peripheral air- way and parenchymal injury to the lungs leading to hypoventilation of these regions was not accompanied by decreased blood flow to affected regions. Our structural studies support this conclusion because many lobules were atelectatic, with exudate obstructing the bronchioles and yet had pronounced vascular engorgement of alveolar capillaries. Endotoxin might disturb V/O matching because of its potent effects on nonnal bovine pulmonary vasculature.32 Other factors are likely to be involved; gram positive infections of other Species cause similar V/O mismatching in the lungs37 but gram positive organisms do not synthesize endotoxin. The source for such vascular changes many come from bac- terial products other than endotoxin, such as cytotoxins”:22 and bac- terial kallikreins.38 Other sources for the vascular and tissue injury observed in pulmo- nary pasteurellosis are the cellular products of platelets and leuko- cytes. The release of leukocyte lysozomal products, platelet biogenic amines, prostaglandins and leukotrienes synthesized by macrophages, kallikreins and proteases can cause tissue damage in the lungs of other species and may contribute to the severity of pasteurella induced 213 injury.23’25s34 The develOpment of irregularly Shaped areas of coagulative necrosis in lungs affected with pasteurellosis for 18 hrs or more has been thought the result of vascular thrombosis and infarction.1-8a10a11 The lesions we observed in calves P6 and P8 deve- laped without widely distributed vascular thrombosis but were always associated with an intense infiltrate of macrOphageS and lesser numbers of other inflammatory cell types. Tissue necrosis may therefore be associated in some way with these inflammatory cells. It is uncertain whether these necrotic areas only occur in develOped lesions because of a necessity to accummulate large numbers of inflammatory cells, or whether the response is related to bacterial products which are necro- tizing and which reach toxic levels when bacterial numbers increase suf- ficiently following inoculation. In physiologic studies of pneumonic calves, I was unable to detect alterations in functional residual capacity (FRC) despite atelectasis and hepatization of diseased portions of lung. I concluded that remaining ventilated portions of lung would become hyperinflated in order for FRC to remain constant. Structural studies demonstrated that areas of hyperinflation and emphysema did occur, these being described in some but not all descriptions of'f, haemolytica pneumonia summarized by Rehmtulla and Thompson.1 Emphysematous changes were only detected on histologic evaluations of pneumonic lungs in my study but grossly visible emphysematous bullae have also been described in calves infected with E. haemolytica.39 In a companion paper, I discuss theoretical reasons why plasma histamine levels may not adequately reflect tissue histamine levels if pasteurellosis was to cause mast cell degranulation. Preliminary 214 Surveys of Toluidine Blue stained plastic sections did not reveal marked differences between populations of mast cells associated with the major airways of control and Pasteurella exposed calves, further indicating that histamine is unlikely to be an important mediator of pulmonary injury associated with pasteurellosis. Control calves and Pasteurella eXposed calves had lesions associated with the small intestine and liver. The liver lesions may have deve- l0ped as a result of portal venous or biliary tranSportation of hepato- toxic substances or bacteria from the inflamed intestine. It is not known whether cold stress facilitated the lesions. The finding of mild perivascular cuffing of the brain in 2 calves infected with pasteurello- sis, thrombosis of a small arteriole in the cerebrum of P3 and one calf with a microabscess also in the brain, may have been unrelated to the Pasteurella lesions. No bacteria were observed in specially stained histologic slides of affected regions, but thrombosis of neighboring vessels may account for these lesions. The possibility of a circulating factor released from the lungs of calves with pasteurellosis cannot be excluded, but would seem unlikely since only 2 of 9 calves had the lesion and both calves with brain lesions had relatively mild lung disease. Both groups of calves had some evidence of extramedullary hematOpoiesis and had narrow zones of small lymphocytes occupying the Splenic white pulp. These findings have previously been described in normal neonatal calves40 and therefore were not the results of stress. The same authors describe typical lymph nodes of neonatal calves, which were similar to control calves in this study. 215 REFERENCES I. Rehmtulla AJ, Thomson RG: A review of the lesions in Shipping Fever of cattle. Can Vet J 22:1-8, 1981. 2. Gilka F, Thomson RG, Savan M: The effect of edema, hydrocor- tisone acetate, concurrent viral infection and hmnunization on the clearance of Pasteurella hemolytica from the bovine lung. Can J comp .333 38:251-359, 1974. 3. Gilka F, Thomson RG, Savan M: Microscopic findings in the lungs of calves aerosolized with Pasteurella hemolytica and treated to alter pulmonary clearance. Zbl Vet Med 21:774-786, 1974. 4. Tweed N, Edington JH: Pneumonia of bovine due to Pasteurella boviseptica. J Comngath 43:234-252, 1930. 5. Ishino S, Oka M, Terui S, Ikeda S: Pathological and micro- biological studies on calf pneumonia occurring in mass rearing facili- ties. Nat Inst Anim Hlth Quart 19291-103, 1979. 6. Carter GR: Observations on the pathology and bacteriology of Shipping Fever in Canada. Can J comp Med 28:359-364, 1954. 7. Langham RF, Thorp F, Ingle RT, Scholl LB: Some observations on the pathology of pneumonia in food producing animals. Am J Vet Res 3:139-145, 1942. 8. Stockdale PHG, Langford EV, Darcel C le 0: EXperimental bovine pneumonic Pasteurellosis. 1. Prevention of the disease. Can J comp ‘flgg 43:262-271, 1979. 9. Hamdy AH, Trapp AL, Gale C: Further preliminary studies on transmission of Shipping Fever in calves. Am J vet Res 25:128-133, 1964. 216 10. Collier JR: Pasteurellae in bovine reSpiratory disease. JAVMA 152:824-828, 1968. ' 11. Friend SCE, Thomson RG, Wilkie BN: Pulmonary lesions induced by Pasteurella hemolytica in cattle. Can J comp Med 41:219-223, 1977. 12. Bryson DG, McFerran JB, Ball HJ, Neill SD: Observations on outbreaks of respiratory disease in housed calves - (2) Pathological and microbiological findings. Vet Rec 103:503-509, 1978. I3. Gourlay RN, Mackenzie A, C00per JE: Studies on the micro- biology and pathology of pneumonic lungs of calves. J Comngath 80:575-585, 1970. 14. Breeze RG, Magonigle RA: A long acting tetracycline in treat- ment of Pasteurella pneumonia in calves. Bovine Practitioner 14:15-17, 1979. 15. Breeze RG, Lauerman LH, Schmitz JA, Magonigle RA: Evaluation of a long-acting oxytetracycline for treatment of Pasteurella pneumonia in calves. Bovine Practitioner 15:96-98, 1980. 16. Dungworth DL, Phalem RF, Schwartz LN, Tyler HS: Morphological methods for evaluation of pulmonary toxicology in animals. Ann Rev Pharmacol Toxicol 16:381-399, 1976. 17. Hamdy AH, Trapp AL, Gale C, King NB: Experimental transmission of Shipping Fever in calves. Am J Vet Res 24:287-294, 1963. 18. Jensen R, Pierson RE, Braddy PM, Saari DA, Lauerman LH, England JJ, Keyvanhar H, Collier JR, Horton DP, McChesney AE, Benitez A, Christie RM: Shipping Fever pneumonia in yearling feedlot cattle. JAVMA 169:500-506, 1976. 217 19. Rushton 8, Sharp JM, Gilmour NJL, Thompson DA: Pathology of an experimental infection of Specific pathogen-free lambs with Parainfluenza virus type 3 and Pasteurella haemolytica. J Comngath 89:321-329, 1979. 20. Al-Darraji AM, Cutlip RC, Lehmkuhl HD, Graham DL: Experimental infection of lambs with bovine reSpiratory syncitial virus and Pasteurella haemolytica: Pathologic studies. Am J Vet Res 43:224-229, 1982. 21. Bensen ML, Thomson RG, Valli VEO: The bovine alveolar macrOphage. II. In vitro studies with Pasteurella haemolytica. Can J comp Med 42:368-369, 1978. 22. Markham RJF, Ramnaraine MLR, Muscoplat CC: Cytotoxic effects of Pasteurella haemolytica on bovine polymorphonuclear leukocytes and impaired production of chemotactic factors by Pasteurella haemolytica- infected alveolar macrOphages. Am J Vet Res 43:485-488, 1982. 23. Rinaldo JE, Rogers RM: Adult reSpiratory-distress syndrome. 3L_ Eng J Med 306:900-909, 1982. 24. Nathan CF, Murray HN, Cohn ZA: The macrOphage as an effector cell. N Eng J Med 303:622-626, 1980. 25. Heissman G, Smolen JE, Korchak HM: Release of inflammatory mediators from stimulated neutrophils. N Eng J Med 303:27-34, 1980. 26. Rhoades KR, Heddleston KL, Rebers PA: Experimental hemorrhagic septicemia: Gross and microsc0pic lesions resulting from acute infec- tions and from endotoxin administration. Can J comp Med 31:226-233, 1967. 27. Wray C, Thomlinson JR: The effects of Escherichia coli endo- toxin in calves. Res vet Sci 13:546-553, 1972. 218 28. Musa BE, Conner GH, Carter GR, Gupta BN, Keahey KK: Physiologic and pathologic changes in calves given Escherichia coli endotoxin or Pasteurella multocida. Am J Vet Res 33:911-916, 1972. 29. Heddleston KL, Rebers PA: Properties of free endotoxin from Pasteurella multocida. Am J Vet Res 36:573-574, 1975. 30. Tikoff G, Kuida H, Chiga M: Hemodynamic effects of endotoxin in calves. Am J Physiol 210:847-853, 1966. 31. Thomson GH, McSherry BJ, Valli VEO: Endotoxin induced dissemi- nated intravascular coagulation in cattle. Can J comnged 38:457-466, 1978. 32. Reeves JT, Daoud FS, Estridge M: Pulmonary hypertension caused by minute amounts of endotoxin in calves. J Appl Physiol 33:739-743, 1972. 33. Keiss RE, Hill ON, Collier JR: Skin toxicity and hemodynamic prOperties of endotoxin derived from Pasteurella haemolytica. Am J Vet 333 25:935-942, 1964. 34. Kux M, Coalson JJ, Massion NH, Guenter CA: Pulmonary effects of E. coli endotoxin: Role of leucocytes and platelets. Annals of Surgery 175:26-34, 1972. 35. Bradley SG: Cellular and molecular mechanisms of action of bacterial endotoxins. Ann Rev Microbiol 33:67-94, 1979. 36. Morrison DC, Ulevitch RJ: A review: The effects of bacterial endotoxins in host mediation systems. Am J Path 93:527-617, 1978. 37. O'Brodovich HM, Stalcup SA, Pang LM, Lipset JS, Mellins RB: Bradykinin production and increased permeability during acute respira- tory failure in unanesthetized sheep. J Clin Invest 67:514—522, 1981. 219 38. Miller RL, Reichgott MJ, Melmon KL: Biochemical mechanisms of generation of bradykinin by endotoxin. J Infect Dis 128:5144-3156, 1973. 39. Stockdale PHG, Jericho KWF, Yates NDG, Darcel C le 0, Langford EV: Experimental bovine pneumonic Pasteurellosis. II. Genesis and pre- vention. Can J comp Med 43:272-279, 1979. 40. Hinquist G: Morphology of the blood and hemopoietic organs in cattle under normal and some experimental conditions. Acta Anatomica 22:7-112, 1954. CHAPTER 5 CONCLUSIONS 220 221 CONCLUSIONS Epidemiological studies have clearly incriminated Pasteurella haemo- ‘Lygigg as a prime factor in the injury of the lungs associated with Shipping Fever, and have also identified the importance of stress, par- ticularly thermal stress and that resulting from transportation, in pre- disposing to pneumonia. The mechanisms that act to cause the initial lung injury in cattle with Shipping Fever are unknown. This study was designed to describe the development of initial pulmonary injury by structural and functional means and to test several hypotheses regarding postulated mechanisms of injury. I. Cold Stress Cold stress and Spraying of the trachea with acetic acid resulted in a focal necrotizing tracheitis and isolated lobules of lung had hemorrhage, edema and often cellular infiltrates accumulating in the alveolar lumens when examined 12 hrs after the cold stress. These lesions did not cause any functional changes in the lungs, and may have arisen because of acetic acid inhalation into the lungs or because of early bacterial pneumonia. From the data obtained in this study, it was not possible to determine whether the mixed bacterial types cultured from control calf lungs were the cause of the focal pneumonic areas or were simply isolated from the lungs without exerting pathogenic effects. Cold stressed calves had no change in their hemograms immediately following stress but plasma total solids, erythrocyte numbers, hema- tocrit and hemoglobin content decreased over the remainder of the experiment presumably because of repeated sampling of blood. The leukogram was maintained at baseline levels in the control calves, 222 indicating the addition of leukocytes to the circulating blood occurred in order to replace losses through repetitive blood sampling. There was no change in plasma bradykinin and histamine levels, serum thyroxine or triiodothyronine but serum cortisol was significantly increased by cold stress. These data indicate that thyroid hormones, mast cell products and humoral factors which may activate the kinin system (principally the intrinsic clotting system) are not involved in responses to cold stress. The importance of cold-stress induced cor- ticoid release is unclear Since it did not affect circulating leukocyte numbers. These experiments did not determine leukocyte function so that the Specific effects of cold stress on corticoid-induced immuno- suppression were not determined. Cold stress increased alveolar ventilation because tidal volume was increased while minute ventilation was maintained at baseline levels. The increased alveolar ventilation was necessitated by an increased metabolic demand for oxygen uptake and C02 excretion from the lungs so that no change in arterial 02 or C02 tension occurred. While such respiratory adjustments function well to preserve homeostasis in the face of thermal stresses, they may increase the load of inspired patho- gens delivered into the respiratory exchange area. Since 3. haemolytica is normally present in aerosolized dr0plets generated from the nasal cavity during normal breathing, increased alveolar ventilation may be an important mechanism for predisposing to Shipping Fever during changes in body metabolism induced by thermal stress. 223 II. Effects of Pasteurella haemolytica The reSponse of calves to 3. haemolytica exposure seemed to involve three phases. An initial phase, measurable within one hour of challenge and extending for about 3 hours, resulted in alterations in the pattern of breathing and in gas exchange function but was not accompanied by changes in the hemogram or leukogram. The onset of injury at this time was associated with maintenance of serum cortisol levels above baseline. Alterations in the respiratory rate resulted in increased dead Space ventilation but dead space ventilation increased above the level pre- dicted by changes in frequency alone. In addition, arterial oxygen decreased despite a maintenance of alveolar ventilation. These changes indicate the development of ventilation-perfusion inequalities within the lungs. Since the impairment of gas exchange did not worsen from 3 to 6 hours post inoculation but respiratory rate returned to baseline levels, these data indicate that the initial stimulus for increased respiratory rates was not hypoxemia and probably was reflex in origin. Because irritant receptor stimulation leads to reflex bronchoconstric- tion, and since no functional evidence of bronchoconstriction was pre- sent, it is probable that reflex stimulation of respiration arose through intrapulmonary J receptor stimulation. These receptors are sti- mulated by develOping pulmonary edema, which was a consistent histologic lesion present in all calf lungs exposed to_E. haemolytica. The second phase was ushered in from three to less than 12 hrs post inoculation by decreased dynamic compliance and increased alveolar- arterial oxygen difference. The pattern of breathing had returned to normal but there was an increase in dead space ventilation. These data indicate continued gas exchange impairment and peripheral airway and 224 lung parenchymal injury caused changes in compliance. The histologic findings of calves dying before 12 hrs supports these conclusions. Furthermore, the concentration of pneumonic lesions in the anterior lobes probably resulted in a redistribution of ventilation to the diaphragmatic lobes. This redistribution further increases dead Space and since it did not appear to be accompanied by a redistribution of perfusion away from pneumonic tissues, based on the histologic appearance of pneumonic lung, probably led to exacerbation of ventilation-perfusion mismatching. The third phase of the disease had occurred by 12 hours post inocu- lation, and was characterized by hypoventilation and increased airway resistance in addition to changes previously described. These changes indicate that acute respiratory failure had developed by this time and that the extensive alterations in the pulmonary mechanical properties probably resulted in respiratory muscle failure. Because functional residual capacity (FRC) did not decrease in calves with pneumonia, hyperinflation of the remaining healthy portions of lung occurred. Since these portions of lung then operate at greater inflations than normal, the failure of calves to reduce FRC during the development of pneumonia further exacerbated the decrease in dynamic compliance and probably hastened respiratory failure. Data from the study of hematologic and hormonal variables during this third phase indicated that 3, haemolytica pneumonia resulted in neutrOpenia and increased serum cortisol. Histologic studies support the importance of the neutrOphil in the initial response to Pasteurella haemolytica injury of the lungs. It is unknown whether neutrophils and the other inflammatory cells of the lungs contribute to pulmonary injury 225 in this disease. Histologically, the processes of pulmonary edema and inflammatory cell infiltration into the lungs appeared to occur indepen- dently suggesting that alveolar wall injury can occur independently from leukocyte action. The large irregular zones of necrosis that begin to develop in calf lungs exposed to E, haemolytica for 12 hrs were always associated with cellular exudates and may indicate injury mediated by inflammatory cell products at this time. There was no evidence to support a role of histamine in the pathoge- nesis of pneumonic Pasteurellosis. Histamine was not released into the blood, nor were there physiologic or histologic changes compatible with the generation of histamine. Similarly, the data do not support a role for bradykinin. This was surprising since several possible sources of kinin pathway activation seemed to be present. The principle stimulus for kinin generation is activated Hageman factor, and such activation was thought likely because of endotoxin and should result in vascular thrombosis. Vascular thrombosis was not a feature of this disease, although histologic methods to detect intra-alveolar capillary throm- bosis may have been insensitive. These data therefore suggest that thrombosis and kinin production are not a feature of the disease and that if endotoxin is important in the pathogenesis of pneumonic Pasteurellosis, it has no effect on the bovine kinin system. APPENDIX A The following variables were measured for the determination of pulmonary gas exchange: Expired 02 partial pressure P502 Expired C02 partial pressure PECOZ Expired N2 partial pressure PENZ Inspired N2 partial pressure P1N2 Inspired 02 partial pressure P102 Tidal volume VT Inspired oxygen fraction F102 Expired oxygen fraction F502 Inspired C02 fraction FICOZ Expired C02 fraction FECOZ Barometric pressure P3 Arterial oxygen tension Pa02 Arterial C02 tension PaCOz Respiratory frequency f From these measurements the following formulae were used in order to calculate the aSpects of gas exchange outlined below. 1. Alveolar oxygen tension (PA02) PACO 1-R where PHgO is the water vapor pressure at body temperature and where P CO x P N RE = E 2 I 2 _ PEOZ P102 x PEN2 226 227 The equation is solved by assuming that PACOg is closely approxima- tely by PaCOZ. 2. Alveolar-arterial oxygen difference (AaDOz) A6302 = PA02 - P302 3. Dead space/tidal volume ratio (VD/VT) PACOZ - PECOZO vD/VT g PACOZ This equation is solved by substituting PaCOz in place of PACOZ. 4. Alveolar ventilation (VA) VA = VM1N(1-VD/VT) where IMIN = VT x f 5. Dead space ventilation (V0) V0 = VMIN - VA 6. C02 production (VCOZ) . . [FEC02(1-F102)-F1C02(1-F502)] Vcog = VMIN 1.1-102-FIC02 Assuming that FICOZ = 0 then the equation simplifies to Vcoz = VMIN X FECOZ- 7. Oxygen consumption (V02) V02 = VMIN (F102 - F£02) APPENDIX B The apparatus used for fixation of lung specimens is illustrated (see Figure). A fixation tank, constructed of plexiglass, was made with a centrally positioned manifold containing exit ports for passage of pressurized fixative. Exit ports were made of standard stainless steel catheter connections. Samples of lung tissue, with major bronchi cathe- terized and tied firmly in place, were connected to the exit ports of the fixation tank manifold. The samples were submerged under fixative in the fixation tank and were also fixed by pressure perfusion of the airways via the bronchial catheter. Effluent fixative escaping from the lungs drained from the fixative tank into a sump after passing through a filter. Fixative was delivered to a pressure reservoir from the sump by a continuously running, magnetically driven, corrosion resistant submer- sible pump. The height of fixative in the pressure reservoir was main- tained constant by way of a continuous overflow into a wide base sump return pipe. Fixative entered the manifold in the fixation tank by a connecting pipe from the base of the pressure reservoir, and the pressure at the level of the exit ports in the manifold altered by raising or lowering the pressure reservoir in relation to the fixation tank. By this means lung tissue was fixed by a combination of airway per- fusion under constant hydrostatic pressure head and also by submersion. 228 229 Appendix 3, Figure 1 Schematic diagram of the apparatus used in the fixation of lung samples. "VAT? Reservoir 230 Constant _ Level 1 Fixation, ’ Pressure 91C :31; .... .p.,e.o.a.o..e.o.e-£/§8 .1 3HMiion-Tank-E-E-E.E-E-;§ finnn'n I Filter y'Pump Sump Tank Appendix B Figure l VITA The author was born at Bacchus Marsh, Victoria, Australia, in 1951. He attended small country schools for his primary and secondary school education, matriculating first in his class at Ballarat East High School in 1969. He enrolled in the veterinary curriculum at the University of Melbourne, Melbourne, Australia, in 1970, graduating with first class honors in 1974. After 18 months as an assistant in mixed veterinary practice in central Alberta, Canada, the author joined the faculty at Michigan State University (MSU) in July, 1976, for pursuit of a residency in Food Animal Medicine and also concurrently enrolled in a Masters Degree program with the Department of Large Animal Surgery and Medicine. After completion of these programs in 1979, the author joined the Department of Large Animal Surgery and Medicine as an instructor and the Pathology Department as a graduate student enrolled in a PhD program. The author completed his PhD in Pathology in 1982. During his appointments at MSU, the author was nominated to Phi Zeta, Phi Kappa Phi and Sigma Xi, and was the recipient of a graduate student award in Sigma Xi for his research achievements. 231