PATHOLOGIC ALTERATIONS IN THE SYNQVEAL FLUID AND JOINTS 0F CATTLE Thesis for fin chru of DH. D. HEEEEAN STHE BNEVERSETY Rollo Winslow Van Pelt, Jr. 1965 {J mem ' Micl‘iigrgm $2133?) Univ-3133: é . W‘nr w an: 2‘. i m “wow; 1 llllllllmlllllllllllflllll 23 01083 5027 This is to certify that the thesis entitled ’ PATHOLOGIC ALTERATIONS IN THE SYNOVIAL FLUID AND JOINTS OF CATTLE presented by Rollo Winslow Van Pelt, Jr. has been accepted towards fulfillment of the requirements for JILL— degree in lathology 2’36, (.1451 -IL 772:0 L:7”41 c2- ,1... Major professor Date February 17. 1965 O~169 PLACE It RETURN BOX to romovo this ohookout from your rooord. TO AVOID FINES return on or botoro doto duo. DATE DUE” DATE DUE DATE DUE usu IoAnNflrmottvo Action/Equal Opportunity Instituion WWI ABSTRACT PATHOLOGIC ALTERATIONS IN THE SYNOVIAL FLUID AND JOINTS OF CATTLE by Rollo Winslow Van Pelt, Jr. Synovial fluid specimens from 117 joints of 82 cattle of various breeds, ages, and sexes affected with a wide variety of joint diseases were investigated. Where applicable, syno- vial fluid values were compared with their reSpective whole blood or serum values. Pathologic synovial effusions were classified on the basis of anamnesis, clinical symptomatology, etiology, patho- genesis, arthrographs, laboratory analyses, and bacteriologic studies. Hematologic studies were employed to ascertain the systemic effect, if any, exerted by the various types of joint disease. Group I——Synovial Effusions that Resemble Normal Synovial Fluids: Synovial effusions in this group were transudative in nature. Effusions from cattle affected with degenerative joint disease were slightly in excess of normal volume, whereas effusions from cattle affected with tarsal hydrarthrosis were greatly in excess of normal volume; how- ever, these effusions most closely paralleled normal syno- vial fluid with respect to their various constituents in Rollo Winslow Van Pelt, Jr. contrast to effusions from cattle affected with acute and chronic traumatic arthritis. Synovial effusions from cattle affected with acute and chronic traumatic arthritis were slightly in excess of normal volume and generally serosang- uineous to hemorrhagic in nature. Relative viscosity values for the group fell slightly below normal. Tests for quality of mucinous precipitate indicated a hyaluronic acid content with a relatively high degree of polymerization. Synovial fluid sugar levels closely paralleled blood sugar levels for cattle affected with degenerative joint disease and tarsal hydrarthrosis, but exceeded their blood sugar levels for cattle affected with acute or chronic traumatic arthritis. Synovial fluid alkaline phOSphatase activity with the exception of cattle affected with tarsal hydrarthrosis, exceeded that of its serum counterpart. Lactic dehydrogen- ase activity, glutamic oxalacetic and glutamic pyruvic trans- aminase activity for synovial fluid, was found to be less than the activity of their serum counterparts for cattle affected with degenerative joint disease and tarsal hydrar- throsis. Total synovial fluid leukocyte counts for all cattle in this group were slightly higher than normal valhes. Group II--Intermediate Synovial Effusions: Synovial effusions from cattle affected with an idiopathic arthritis were exudative in nature. Attempts to culture bacteria from the effusions met with no success. Total volume was in excess of normal, relative viscosity was reduced, and tests Rollo Winslow Van Pelt, Jr. for mucinous precipitate indicated a low degree of hyaluronic acid polymerization or a low ratio of hyaluronic acid to unit volume. Mean synovial fluid sugar levels were found to be 32.7% lower than their blood sugar levels. Synovial fluid lactic dehydrogenase activity, glutamic oxalacetic and glutamic pyruvic transaminase activity were higher than reported normal synovial fluid values for cattle. Total leukocyte counts and the absolute number of neutrophils were greatly increased. Group III--Septic Synovial Effusions: Synovial effu-’ sions from joints with infectious arthritis were exudative in nature. Total volume was greatly in excess of normal, relative viscosity was reduced and tests for mucinous pre— cipitate indicated a hyaluronic acid content of an extremely low degree of polymerization or almost complete absence of the hyaluronic acid complex. Synovial fluid sugar levels were 48% lower than their blood sugar levels. Alkaline phosphatase activity for synovial fluid was in excess of its serum counterpart, but did not attain the mean level for normal cattle. Total leukocyte counts and the absolute num- ber of neutrOphils were greatly increased. Infectious arthritis was reflected systemically by a granulocytosis in the peripheral blood. Gross pathologic and histopathologic studies were performed on 76 joints of 32 cattle. Joint Specimens were obtained at necropsy or by punch-biopsy. Rollo Winslow Van Pelt, Jr. The most significant gross lesions in degenerative joint disease were confined to the articular cartilages. The most characteristic findings were a yellowing and thin- ning of the articular cartilages, with irregular depressions, flaking, pits, and linear grooves. Microscopically, there was a loss of the hyaline appearance of the cartilage, a reduced number of chondrocytes, and fibrillation of the matrix. The principle gross pathologic lesions in chronic traumatic arthritis were confined to the synovial membrane and consisted primarily of subintimal hemorrhages. Micro— scopically, subintimal hemorrhage and edema were the most consistent findings. The joints of cattle affected with an idiopathic synovitis or arthritis were found to resemble infectious arthritis both grossly and microscopically. Streptococcus viridans was isolated most frequently from joints of cattle with infectious arthritis. Gross and histopathologic lesions varied directly with the virulence of the microorganism. The most severe joint infections were associated with chronic infectious arthritis due to Corynebacterium pyogenes. Attempts to culture bacteria from the joints of cattle affected with a polyarthritis associated with Rollo Winslow Van Pelt, Jr. primary systemic infections met with no success. Hematogen— ous Spread of bacteria from primary sites of infection or hypersensitivity to the bacterial infections were considered the exciting cause in the production of polyarthritis. PATHOLOGIC ALTERATIONS IN THE SYNOVIAL FLUID AND JOINTS OF CATTLE By Rollo Winslow Van Pelt, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology 1965 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Dr. R. F. Langham for his guidance and assistance through- out this investigation. My sincere thanks and appreciation is expressed to Dr. G. H. Conner, Department of Surgery and Medicine for his generous assistance with various aspects of this investiga- tion. The author wishes to express his appreciation to Dr. C. C. Morrill and Dr. S. D. Sleight for their critical reading of this manuscript and to various members of the Department of Pathology for their assistance with various aSpectS of this investigation. Thanks and appreciation are expressed to Mrs. Carolyn R. Easter and Mrs. Joyce A Wilfong, Department of Surgery and Medicine for their assistance with the labora- tory analyses of Specimens. To Mrs. Marian V. Bennett, Department of Microbiology and Public Health for advice and assistance with Specimens for bacteriologic studies. For awarding a Postdoctoral and Special Fellowship my sincere appreciation and thanks is extended to the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland. ii Dedicated to Karen TABLE OF CONTENTS Chapter Page I. INTRODUCTION . . . . . . . . . . . . . . . . . 1 II. REVIEW OF THE LITERATURE . . . . . . . . . . . 3 Synovial Fluid . . . . . . . . . . . . . . . . 3 Joint Tissues . . . . . . . . . . . . . . . . 14 III. MATERIALS AND METHODS . . . . . . . . . . . . 24 Blood, Serum, and Synovial Fluid Studies . . . 24 Gross and Histopathologic Studies . . . . . . 38 IV. RESULTS . . . . . . . . . . . . . . . . . . . 4O Synovial Fluid Analyses . . . . . . . . . . . 40 Group I--Synovial Effusions that Resemble Normal Synovial Fluids . . . . . . . . . . 4O Degenerative Joint Disease . . . . . . 4O Traumatic Arthritis . . . . . . . . . 55 Hydrarthrosis . . . . . . . . . . . . 69 Hematologic Data . . . . . . . . . . . 71 Group II-—Intermediate Synovial Effusions. 75 Idiopathic Arthritis . . . . . . . . . 75 Hematologic Data . . . . . . . . . . . 85 Group III-—Septic Synovial Effusions . . . 90 Infectious Arthritis Due to Bacteria . 90 Hematologic Data . . . . . . . . . . . 102 iv Chapter Page Anamnesis and Clinical, Gross and Histopathologic Observations . . . . . . . . 104 Degenerative Joint Disease . . . . . . . 104 Anamnesis and Clinical Observations . . . . . . . . . . . . 104 Gross Pathologic Observations . . . 104 Histopathologic Observations . . . . 108 Traumatic Arthritis . . . . . . . . . . 112 Anamnesis and Clinical Observations . . . . . . . . . . . . 112 Gross Pathologic Observation . . . . 115 Histopathologic Observations . . . . 115 Idiopathic Synovitis and Arthritis . . . 116 Anamnesis and Clinical Observations . . . . . . . . . . . . 116 Gross Pathologic Observations . . . 119 Histopathologic Observations . . . . 120 Infectious Arthritis . . . . . . . . . . 128 Anamnesis and Clinical Observations . . . . . . . . . . . . 128 Gross Pathologic Observations . . . 130 Histopathologic Observations . . . . 131 Polyarthritis Associated with Primary Systemic Infections . . . . . . . . . . 140 Anamnesis and Clinical Observations . . . . . . . . . . . . 140 Gross Pathologic Observations . . . 143 Histopathologic Observations . . . . 143 Chapter V. DISCUSSION Synovial Fluid Analyses Group I--Synovia1 Effusions that Resemble Normal Synovial Fluid Group II—-Intermediate Synovial Effusions Group III--Septic Synovial Effusions Gross and Histopathologic Observations Degenerative Joint Disease Traumatic Arthritis Idiopathic Synovitis and Arthritis Infectious Arthritis Polyarthritis Associated with Primary Systemic Infections VI. SUMMARY AND CONCLUSIONS REFERENCES vi Page 149 149 149 155 157 160 160 160 161 162 163 165 170 Table LIST OF TABLES Page Summary of Selected physical, cytologic, and chemical properties of synovial fluid from the normal bovine tarsus . . . . . . . . . . . 4 Incidence and pathologic classification of Synovial effusions from the joints of cattle affected with degenerative joint disease . . . 41 Total volume and gross appearance of synovial fluid obtained from the joints of cattle affected with degenerative joint disease . . . 43 Relative viscosity and mucinous precipitate quality of synovial fluid from the joints of cattle affected with degenerative joint disease . . . . . . . . . . . . . . . . . . . . 45 A comparison of synovial fluid and blood sugar values for cattle affected with degenerative joint disease . . . . . . . . . . . . . . . . . 46 A comparison of synovial fluid and serum alkaline phosphatase activity values for cattle affected with degenerative joint disease . . . . . . . . . . . . . . . . . . . . 48 A comparison of synovial fluid and serum lactic dehydrogenase activity values for cattle affected with degenerative joint disease . . . 49 A comparison of Synovial fluid and serum glutamic oxalacetic transaminase activity values for cattle affected with degenerative joint disease . . . . . . . . . . . . . . . . . 50 A comparison of Synovial fluid and serum glutamic pyruvic transaminase activity values for cattle affected with degenerative joint disease . . . . . . . . . . . . . . . . . . . . 52 vii Table 10. ll. 12. l3. 14. 15. 16. 17. 18. 19. 20. Total numbers of erythrocytes and leukocytes in synovial fluid from cattle affected with degenerative joint disease Absolute numbers of various leukocytes in synovial fluid from cattle affected with degenerative joint disease . . . . Incidence and pathologic classification of synovial effusions from the joints of cattle affected with traumatic arthritis Total volume of synovial fluid obtained from the joints of cattle affected with acute or chronic traumatic arthritis Gross appearance of synovial fluid from the joints of cattle affected with acute or chronic traumatic arthritis Relative viscosity and mucinous precipitate quality of synovial fluid from the joints of cattle affected with acute or chronic traumatic arthritis A comparison of synovial fluid and blood sugar values for cattle affected with acute or chronic traumatic arthritis A comparison of synovial fluid and serum alkaline phosphatase activity values for cattle affected with acute or chronic traumatic arthritis Total numbers of erythrocytes and leukocytes in synovial fluid from cattle affected with acute or chronic traumatic arthritis . . . . . . . . Absolute numbers of various leukocytes in synovial fluid from cattle affected with acute traumatic arthritis . . Absolute numbers of various leukocytes in synovial fluid from cattle affected with chronic traumatic arthritis viii Page 53 54 56 58 59 61 63 64 66 67 68 Table 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Incidence and pathologic classification of Synovial effusions from the joints of cattle affected with tarsal hydrarthrosis Summary of physical and chemical data for blood, serum, and synovial fluid for cattle affected with tarsal hydrarthrosis . Total numbers of erythrocytes and leukocytes, and absolute numbers of various leukocytes in synovial fluid from cattle affected with tarsal hydrarthrosis Hematologic data from cattle affected with degenerative joint disease, acute or chronic traumatic arthritis, or tarsal hydrarthrosis Incidence and pathologic classification of Synovial effusions from the joints of cattle affected with an idiopathic arthritis Total volume and gross appearance of synovial fluid from the joints of cattle affected with an idiopathic arthritis Relative viscosity and mucinous precipitate quality of synovial fluid from the joints of cattle affected with an idiopathic arthritis . . . . . . . . . A comparison of synovial fluid and blood sugar values for cattle affected with an idiopathic arthritis . . . . . A comparison of synovial fluid and serum alkaline phOSphatase activity values for cattle affected with an idiopathic arthritis A comparison of synovial fluid and serum lactic dehydrogenase activity values for cattle affected with an idiopathic arthritis A comparison of synovial fluid and serum glutamic oxalacetic transaminase activity Values for cattle affected with an idiopathic arthritis ix Page 70 72 73 74 76 77 79 8O 82 83 84 Table 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. A comparison of synovial fluid and serum glutamic pyruvic transaminase activity values for cattle affected with an idiopathic arthritis Total numbers of erythrocytes and leukocytes in synovial fluid from the joints of cattle affected with an idiopathic arthritis . . . . . . . . . . . . . . Absolute numbers of various leukocytes in synovial fluid from cattle affected with an idiopathic arthritis Hematologic data from cattle affected with an idiopathic arthritis Pathologic classification of Synovial effusions and identification of micro— organisms isolated from the joints of cattle with infectious arthritis due to bacteria Total volume and gross appearance of synovial fluid obtained from the joints of cattle affected with infectious arthritis Relative viscosity and mucinous precipitate quality of Synovial fluid from the joints of cattle affected with infectious arthritis A comparison of synovial fluid and blood sugar values for cattle affected with infectious arthritis A comparison of synovial fluid and serum alkaline phosphatase activity values for cattle affected with infectious arthritis Total numbers of erythrocytes and leukocytes in synovial fluid from the joints of cattle affected with infectious arthritis Page 85 87 88 89 91 95 96 98 99 100 Table 42. 43. 44. 45. Absolute numbers of various leukocytes in synovial fluid from the joints of cattle affected with infectious arthritis Hematologic data from cattle affected with infectious arthritis Pathologic classification and identification of microorganisms isolated from the joints of cattle with infectious arthritis due to bacteria Polyarthritis associated with primary Systemic infections in cattle xi Page 101 103 129 142 Figure 10. 11. LIST OF FIGURES Holstein-Friesian cow with acute infectious tenosynovitis of the peroneus longus and extensor digiti resulting in an acute secondary arthritis of the left tarsus The left femoropatellar joint of a 5-year—old Shorthorn bull . . . . . . . . . The left tarsal joint of a 5—year-old Shorthorn bull . . . . Punch—biopsy Specimen from the left tibiotarsal synovial sac of an ll-year-old Aberdeen-Angus bull Section of synovial membrane from the left tibiotarsal synovial sac of a 2-year-old Holstein—Friesian cow Focal hyalinization of the synovial membrane from the left tibiotarsal synovial sac Hyalinization of the Synovial intimal cell layer Synovial membrane from the right tibiotarsal synovial sac of a 2.5-year—old Holstein— Friesian cow . . . . . . . . Articular cartilage from the left patella of a 5—year—old Shorthorn bull . . . . Fibrillation and splitting of the articular cartilage in degenerative joint disease Vertical Splitting and fibrillation of the articular cartilage in degenerative joint disease xii Page 94 105 107 109 109 110 110 111 111 113 113 Figure Page 12. Focal calcification in the subchondral bone in degenerative joint disease . . . . . . . . 114 13. Synovial membrane from the right tibiotarsal synovial sac of a 5-year-old Holstein— Friesian cow affected with traumatic arthritis . . . . . . . . . . . . . . . . . . 114 14. Giant cell (arrow) in the stratum synoviale of the tibiotarsal Synovial sac in chronic traumatic arthritis . . . . . . . . . . . . . 117 15. Synovial membrane from the right tibiotarsal Synovial sac of a 5-year-old Holstein- Friesian cow . . . . . . . . 117 16. Synovial membrane from the left tibiotarsal Synovial sac of a l—year-old Hereford steer affected with chronic traumatic arthritis . . 118 17. Punch-biopsy Specimen from the left tibiotarsal synovial sac of a 5—year—old Holstein-Friesian cow affected with an idiopathic synovitis . . . . . . . . . . . . 118 18. Synovial villus from the right femoropatellar joint of a l-year-old Hereford steer . . . . 121 19. Chronic idiopathic synovitis with follicle- like collection of lymphocytes around a central arteriole . . . . . . . . . . . . . . 121 20. Punch—biopsy Specimen from the right ' tibiotarsal synovial sac of a 10-months-old Guernsey heifer . . . . . . . . . . . . . . . 122 21. Diffuse lymphocytic infiltration and edema of the stratum synoviale . . . . . . . . . . 122 22. Punch—biopsy Specimen from the left tibiotarsal synovial sac of a 5—year-old Holstein-Friesian cow . . . . . . . . . . . . 123 23. Extreme medial hypertrophy of an artery in the stratum synoviale . . . . . . . . . . . . 123 xiii Figure Page 24. Pannus formation has destroyed the articular cartilage and subchondral bone . . . . . . . . 125 25. Early pannus invasion of the articular cartilage in chronic idiopathic arthritis . . . 125 26. Dissolution of the articular cartilage by pannus formation in chronic idiopathic arthritis . . . . . . . . . . . . . . . . . . . 126 27. Invasion of the articular cartilage by subchondral granulation tissue in idiopathic arthritis . . . . . . . . . . . . . . . . . . . 126 28. Granulation tissue invading the articular cartilage . . . . . . . . . . . . . . . . . . . 127 29. Synovial membrane from the femoropatellar joint of a 2-day-old Aberdeen-Angus heifer . . . . . 127 30. Synovial intimal cells have been replaced by a fibrinous exudate . . . . . . . . . . . . . . . 132 31. Synovial membrane from the left tibiotarsal joint of a 2—week-old Holstein—Friesian heifer . . . . . . . . . . . . . . . . . . . . 132 32. Proliferating capillaries are surrounded by lymphocytes, macrophages, and a few neutrophils . . . . . . . . . . . . . . . . . . 133 33. Granulation tissue and a necrotic debris have completely replaced the articular cartilage and subchondral bone of the proximal end of the tibiotarsal bone . . . . . . . . . . . . . 133 34. Punch-biopsy SpeCimen from the left tibiotarsal synovial sac of a l3—day-old Holstein-Friesian heifer . . . . . . . . . . . 134 35. Subintimal hemorrhage and edema in the stratum synOviale of the right femoropatellar joint . . 134 36. Synovial villus from the left coxofemoral joint of a 6-day—old Holstein-Friesian bull . . . . . 136 xiv Figure Page 37. Organized thrombus (A) in a subsynovial artery . . . . . . . . . . . . . . . . . . . 136 38. Subchrondral fibrosis in the area of the epiphysis . . . . . . . . . . . . . . . . . . 137 39. Calcifying bony spicule in the area of subchondral fibrosis . . . . . . . . . . . . 137 40. Degenerating articular cartilage with a focus of chondroplasia (A) . . . . . . . . . 139 41. Focus of chondrocytes in degenerating articular cartilage . . . . . . . . . . . . . 139 42. Giant cells in the subchondral fibrous tissue of the proximal end of the tibiotarsal bone . 141 43. Synovial membrane from the left femoropatellar joint of a l-year—old Holstein-Friesian heifer . . . . . . . . . . 141 44. Synovial villus from the right carpometa- carpal joint of a 6-week-old Hereford bull . 145 45. Degenerating inflammatory cells and adipose tissue in the subsynovium . . . . . . . . . . 145 46. Pannus formation on the articular Surface of the proximal trochlea of the tibiotarsal bone . . . . . . . . . . . . . . . . . . . . 148 47. Calcium deposition on the surface of the pannus . . . . . . . . . . . . . . . . . . . 148 48. Thrombus in a pannus vessel . . . . . . . . . 149 XV I. INTRODUCTION The subject of arthritis has received only limited attention in veterinary medicine. Thus any system of clas— sification and nomenclature applied to the various arthri— tides of domestic animals has not been clearly or adequately defined. With this in mind, this investigation was con- ducted with the purpose of not only evaluating whole blood, serum, and synovial fluid constituents, and joint tissues of cattle affected with a wide variety of joint diseases, but to develop a system of classification and nomenclature based on etiology, pathogenesis, pathognomy, and pathologic find— ings. The synovial fluid of diarthrodial articulations Serves not only as the primary source of nourishment for the articular cartilages, but also serves a mechanical function in the nature of a lubricant to the joint surfaces due to its mucopolysaccharide content. The various constituents of normal synovial fluid therefore serve to reflect a joint that is functioning in a normal physiological and mechanical manner. Pathologic synovial effusions reflect changes with the onset of joint disease; such effusions in turn are a reflection of the pathologic processes taking place in the Synovial membranes and articular cartilages, thus in turn producing a disturbance in normal intra—articular metabolism. Analyses of pathologic synovial effusions Should then reveal: (1) an abnormal exchange between blood, lymph, and the synovial fluid; (2) pathologic alterations in intra- articular metabolism; and (3) variations in the degree and duration of the various types of joint disease in cattle. Essential to a basic understanding of pathologic joint effusions is a fundamental knowledge of normal synovial fluid and intra-articular physiology. The objectives of synovial fluid analyses as supported by comparative studies of whole blood and serum analyses in this investigation were to establish: (1) etiology; (2) diagnosis; (3) prognosis; and (4) determine the degree and duration of the pathologic effusion. Anamnesis, clinical observations, gross pathologic observations, and histopathologic observations of cattle affected with various types of joint disease were conducted to establish a greater degree of correlation among the vari- ous parameters of a Specific type of joint disease. Only after careful evaluation of the various findings can a System of classification and nomenclature for arthritis in cattle be developed. 11. REVIEW OF THE LITERATURE Synovial Fluid Cornelius (1963) pointed out that articular afflic— tions rank high among the crippling diseases of domestic animals. However, he observed that little or no attention had been given to the cellular and chemical analyses of pathologic joint effusions in veterinary medicine. Van Pelt and Conner (1963a, 1963b, and 1963c) in- vestigated the cytologic properties, relative viscosity, quality of mucopolysaccharide, and the sugar level of syno- vial fluid from the tarsal joints of normal cattle (Table 1). Davies (1944) observed that normal synovial fluid from the tibiotarsal joints of cattle often varied from a pale-yellow to a deep-yellow in color. However, he made no attempt to determine the nature of the pigment or pigments present. The relative viscosity of normal synovial fluid from the tibiotarsal joints of cattle has been reported by various investigators employing a wide variety of viscosim- eters. Ropes 33 31. (1939), employing either a Hess or Ostwald viscosimeter, reported a mean relative viscosity of 3.72 at 25 C., while Spector (1956) reported a value of 5.0 TABLE 1. Summary of Selected Physical, Cytologic, and Chemical Properties 2f Synovial Fluid from the Normal Bovine Tarsus Range Mean Volume (cc./joint) 4 to 20 12.6 1 0.5 Color - colorless to pale-yellow, and clear (an occasional sample may contain some minute flocculent debris) Relative viscosity at 38.6 C. 1.63 to 13.01 3.79 1 0.28 Total leukocytes/cmm. 0 to 725 103.50 : 14.23 Differential count (7.) Neutrophils 0 to 69 6.00 i 1.24 Lymphocytes 2 to 92 49.08 i 2.77 Monocytes 2 to 86 38.22 i 2.47 Macrophages 0 to 48 5.93 I 1.38 Eosinophils 0 to 9 0.77 i 0.22 Sugar - approximately the same level as whole blood Alkaline phOSphatase activity 0.00 to 12.90 5.07 i 0.47 (Sigma Units/m1.) Lactic dehydrogenase activity 0 to 905 209 i 22 (LDH Units/m1.) Glutamic oxalacetic transaminase activity 0 to 88 30.36 i 2.53 (Sigma—Frankel Units/ml.) Glutamic pyruvic transaminase activity 0 to 75 15.98 i 1.79 (Sigma-Frankel Units/ml.) Fibrinogen 0 0 *Van Pelt and Conner (1963a, 1963b, and 1963c) and Van Pelt (1964). as determined with a Hess viscosimeter at 20 C. Van Pelt and Conner (1963b) found the relative viscosity of Synovial fluid from the tibiotarsal joints of 73 normal cattle to be 3.79 I 0.28 at 38.6 C. (Table l), employing either a Cannon— Fenske routine viscosimeter or a Cannon-Ubbelohde semi—micro viscosimeter. Fessler e_ _1. (1954) pointed out that the viscosity of human synovial fluid was due to the presence of hyalu— ronic acid and that slight changes in the hyaluronic acid molecule noticeably affected the velocity gradient. Levine and Kling (1956) found the rheologic behavior of synovial fluid to provide valuable information concerning the physio— logic and pathologic nature of the joints. Gardner (1953 and 1959) determined that the viscos- ity of synovial fluid decreased exponentially as the syno— vial fluid was diluted with water. Jebens and Monk—Jones (1959) found that synovial effusions from traumatic knee joints evidenced a reduction in viscosity. They attributed reduced viscosity to transudation of plasma from subsynovial blood vessels into the joint cavity in response to injury, the transudative nature of the effusion acting as a diluent to the synovial fluid. They concluded that only a rapid and simultaneous increase in the production of hyaluronic acid could prevent such a transudate from acting as a diluent. Sunblad (1953) showed that the concentration of hyaluronic acid in effusions from traumatized joints varied approximately in an inverse ratio to the volume of the synovial effusion. Ropes and Bauer (1953) stated that the concentration of hyaluronic acid in joint disease was related to the type, severity, and duration of the disease and the duration of the synovial effusion. The lowest concentrations of hyalu- ronic acid were in severe cases of infectious arthritis or rheumatoid arthritis. Meyer (1947) and Ropes _£ _1. (1947) were not suc- cessful in their attempts to demonstrate the presence of a mucinase or hyaluronidase, or similar enzyme in pathologic synovial effusions. Robertson gt _1. (1940) allowed sterile normal and pathologic synovial effusions to stand for vary— ing periods of time at 25 C. and 37 C., and observed no decrease or alteration in the hyaluronic acid content. The addition of anaerobic cultures of Clostridium perfringens to the Synovial effusions resulted in depolymerization of the hyaluronic acid. Carpenter (1959) stated that hyaluronidase was formed by 91. perfringens, streptococci, pneumococci, certain micrococci, and other microorganisms. Moffett (1954) demonstrated that the permeability of the synovial membrane allowed the passage of fluids and col- loids in either direction between the joint cavity and the Synovial blood vessels, lymphatics, and surrounding tissues. An increase in the effusion of a traumatized joint or joints, according to Collins (1936), was not always due to hemorrhage into the joint cavity. In many cases the increased volume was due to transudation of blood plasma from the subsynovial vessels. Bauer gt 31. (1940) pointed out that experimental studies with animals demonstrated the permeability of the barrier between blood vessels and the joint cavity, not only to diffusible substances and proteins, but to bacteria as well. Permeability of the synovial membrane was greater than that of membranes in other body cavities. Balboni gt _1. (1945) found that penicillin passed readily into the synovial fluid following its intramuscular administration. DeGara (1943) investigated the bactericidal prop- erties of synovial fluid and found that the bactericidal activity for Gram-negative bacteria was closely related to the complement content of the synovial fluid and that bacte- ricidal activity may therefore be expected in effusions con- taining Sufficient amounts of complement. Ropes and Bauer (1953) determined that variations in the concentration of sugar, in contrast to those of other nonelectrolytes, are of diagnostic value in joint diseases. They reported lower sugar values for hemorrhagic effusions from the knee joints of man as the result of an increased glycolytic enzyme activity associated with large amounts of blood in the joint. Fury g3 31. (1959) stated that normal sugar levels for man should not exceed that of their reSpective normal Synovial fluid sugar levels by more than 20 mg./100 ml. in the fasting state. Any difference greater than this indi— cated a pathologic process in the joint. Cajori and Pemberton (1928) demonstrated that the glycolytic activity of cell-free synovial fluid was much less marked than uncentrifuged synovial effusions contain- ing a large number of leukocytes. The presence of glycoly- tic enzymes in Synovial effusions was found by Hubbard and Porter (1943) to be confined almost entirely to the neutro- philic leukocytes. Levene and Meyer (1912) found that a sugar solution will lose a portion of its reducing power under the influence of high leukocyte counts. On the other hand, Collins (1936) found no correlation between total leukocyte counts and the sugar level of Synovial effusions. In man, Ropes E£.3l- (1960) showed that the degree of ini- tial lag and the reduction in subsequent rate of sugar entrance into the joint cavity increased with the severity of the joint disease. Meyers g1 gi. (1934) noted that Syno— vial effusions from joints affected with gonococcal arthri- tis contained high total leukocyte counts, and that the higher the total cell count, the lower the sugar level. The presence of microorganisms in synovial effusions also pro— duced a lower sugar level in spite of low total leukocyte counts. ROpes and Bauer (1953) found the alkaline phOSpha— tase activity of normal cattle Synovial fluid to be much higher than the alkaline phOSphatase activity of the serum. They determined a mean value of 13.8 Bodansky units/100 m1. of synovial fluid in contrast to 4.6 Bodansky units/100 ml. of serum. Van Pelt (1964) determined a mean alkaline phos- phatase activity of 5.07 i 0.47 Sigma units/m1. for synovial fluid and 1.67 I 0.14 Sigma units/ml. for serum from dairy cattle ranging in age from 1 to 10+ years (Table 1). Enzyme activity in the synovial fluid and serum decreased with advancing age. Allcroft and Folley (1941) observed that serum alkaline phosphatase activity in cattle progressively decreased with advancing age until maturity was reached. Thereafter cattle had little or no alteration in their serum alkaline phOSphatase activity. They could establish no cor— relation between milking capacity and serum alkaline phos- phatase activity in dairy cows. Working with Zebu cattle, Garner (1952) observed little variation in serum alkaline phosphatase activity after two years of age. Negi (1960) attributed variations in serum alkaline phosphatase activity of cattle to varying nutritional and physiological condi— tions. Robertson g3 g1. (1940) demonstrated lg XEEEQ that serum alkaline phosphatase will Slowly hydrolyze Synovial mucin. They obtained serum with a high alkaline phosphatase activity by ligation of the common bile duct in a dog. 10 Studies in man by Lehman g3 gl. (1964) demonstrated that there are no significant differences in Synovial fluid alkaline phosphatase levels between the inflammatory and noninflammatory arthritides. The alkaline phOSphatase activ- ity of the Synovial fluid was lower than that of the serum. The source of alkaline phosphatase activity in syno— vial fluid is apparently derived from several sources within the joint. Shaw and Marten (1962) were unable to demonstrate the presence of the enzyme in either synovial intimal cells or the intercellular fibers of the synovial membrane, or in the precipitated synovial fluid from a variety of mammalian knee-joint tissues. They demonstrated enzyme activity in the hypertrophic cartilage adjacent to areas of bone forma— tion. This reaction was localized in the enlarged chondro- cytes, the extracellular material in the lacunae, at the lacunar margins, and in the cartilagenous matrix around the lacunae. Intense cytoplasmic reactions were obtained in the osteoblasts and in the heterophils of guinea pigs. Studies on the presence of alkaline phosphatase activity in the leukocytes of pathologic joint effusions from cattle were made by Van Pelt (1961b). The synovial fluid from septic joints contained many' alkaline phos- phatase-positive polymorphonuclear neutrophils in conjunc- tion with a high Synovial fluid alkaline phosphatase activ- ity. Wiltshaw and Moloney (1955) observed that in pyogenic 11 infections the leukocytes showed greatly increased alkaline phOSphatase activity. Kenny and Moloney (1957) reported that prolonged incubation of neutrophils resulted in an increased alkaline phosphatase activity within the cell. Schajowicz and Cabrini (1954) found increased alkaline phOSphatase activity in osteogenic areas and in cartilage undergoing calcification. Hypertrophied cartilage cells and the cells which produce the intracellular substances of bone (osteoblasts) produced large amounts of alkaline phOSphataSe activity, according to Ham (1957). Kleiner (1954) has pointed out that elevated serum alkaline phos— phatase activity may be due to overproduction of the enzyme in bone in order to compensate for various lesions, or to forced extrusion of the enzyme from injured bony tissues. Septic tissues and tissues surrounding areas of sepsis were found by Steward and Beckett (1959) to contain increased amounts of alkaline phosphatase. West g3 31. (1963) reported that the lactic dehydro- genase activity of synovial fluid from human knee joints affected with degenerative joint disease was comparable to that of normal human Synovial fluid. There was a signif— icant correlation between the number of leukocytes in Syno— vial effusions and the lactic dehydrogenase activity of the effusion. It was concluded that increased lactic dehydro- genase activity in synovial effusions might have resulted 12 from: (1) release of the enzyme from leukocytes in the synovial effusion; (2) release of the enzyme from necrotic or inflamed Synovial tissues; or (3) production and release of an increased amount of enzyme by altered synovial tis- sues. Vesell g_ g1. (1962) found the lactic dehydrogenase activity of synovial effusions from patients affected with rheumatoid arthritis to exceed that of their reSpective serum values. They postulated that elevated lactic dehydro— genase activity in synovial effusions may have its source either in the leukocyte, or the synovial membrane, or both. The synovial fluid glutamic oxalacetic transaminase activity for patients affected with degenerative joint dis- ease was found by West gt 31. (1963) to be within normal limits for Synovial fluid. Abnormal values were found in approximately 50% of the patients affected with rheumatoid or pyogenic arthritis. Cornelius £2.3l- (1959) reported a mean value for serum glutamic oxalacetic transaminase activ— ity of 43.8 i 5.7 Sigma—Frankel units/ml. for Holstein— Friesian cattle ranging from 2 to 10 years of age. They also reported a mean serum glutamic pyruvic transaminase value of 19.7 1 12.6 Sigma-Frankel units/ml. for the same group of cattle. Wheat (1955) made observations on synovial fluid obtained from cattle and horses and noted that if aSpirated synovial fluid was clear and viscid, it was usually sterile 13 and the effusion was generally due to trauma or strain. Stained Smears of synovial fluid from these joints had few leukocytes, usually 100 or less per cubic millimeter and of the mononuclear type. Joint infection was SUSpected when the Synovial fluid was cloudy and flocculent and had less than normal viscosity. Gram—stained smears of this type of Synovial fluid revealed counts of 5,000 to 10,000 or more leukocytes per cubic millimeter, with an increase in the absolute number of neutrophils. In such cases, he advised a bacteriologic examination of Synovial fluid. His observa— tions revealed that Streptococcus 522. were commonly en- countered in infected joints. Lange (1961) found the use of synovial fluid cyto— logic studies in joint diseases of cattle of value as a diagnostic aid, as well as a means of making a prognosis regarding the particular joint condition. Bauer t l. (1930) reported that 90 to 95% of all nucleated cells in thgusynovial fluid of cattle were phago— cytic for particulate matter, cells, and fragments of cells. They demonstrated by supravital techniques that the function of phagocytic synovial cells was the removal of products of wear and tear from the articular cartilages and synovial membranes. 14 Joint Tissues Bennett and Bauer (1931) investigated degenerative joint disease in cattle. They concluded that areas of degeneration in the articular cartilages of the carpometa- carpal articulations of all cattle over two years of age was apparently an adequate explanation for the differences observed between the Synovial fluid from this joint and that of the tibiotarsal (astragalotibial) joint. They further pointed out that the types of articular cartilage lesions observed were not wholly Similar to any of the joint lesions described for degenerative joint disease of man. Evidence of degenerative joint disease (osteoarthri- tis) in the femoropatellar and femorotibial articulations (gonitis) was observed by Frank (1953) to be indicated by a distention of the joint capsule. Severe claudication and distention of the joint capsule were associated with exten- sive ulceration of the articular cartilages. Old bulls with impaired fertility were found by McEntee (1958) to have degenerative changes in the artic- ular cartilages of various joints. Degenerative joint lesions and vertebral exostoses were regarded as the cause of a certain amount of locomotor disturbances. Fowler and Kingrey (1956) reported that degenerative joint disease (osteoarthritis) of the bovine tarsus may be associated with the degenerative changes of advanced age. 15 They reported degenerative joint disease to be fairly common in aged bulls, probably in part because of their great body weight and sometimes because of limited exercise. Roughen— ing of the articular surfaces and periarticular lipping (osteophytosis) were frequently encountered. Radiographs of the tarsus in old bulls revealed a considerable amount of this type of lesion, even in animals showing no evidence of claudication. As the development of osteophytes advanced, however, claudication and gross enlargement of the tarsus resulted. They related faulty limb alignment to the develop— ment of degenerative joint disease in early life. Younger animals so affected revealed on radiographs an overextension of the tarsal joint. They suggested that this condition could be congenital or result from a mild case of rickets when the animal was young. Shupe (1959) reported that, in general, degenerative joint disease in cattle was unaccompanied by Systemic mani- festations. Clinically, degenerative joint disease was characterized by pain on locomotion and standing, and was relieved by rest (ventral recumbency). It occurred chiefly in older cattle and was most often observed in the larger, more freely movable joints. In advanced cases, there was an increase in the amount of synovial fluid which contained increased protein, masses of fibrin, Sequestra, an increase in the nucleated cell count, and joint bodies (corpora libra). 16 Shupe (1961) also observed that some of the causes of degen— erative joint disease in cattle were secondary in nature and resulted from disuse of the malfunctioning joint or joints. The onset of degenerative joint disease in aged bulls was reported by Neher and Tietz (1959) to be frequently insidious in nature; however, it appeared to be associated with trauma to a Specific joint and later had a tendency to become polyarticular in distribution. Minor remissions and exacerbations were characteristic of the condition, although marked remission of symptoms was frequently observed coinci- dental with improved climatic conditions. Neher (1960) further observed that Synovial effusions in approximately one-third of the cases of degenerative joint disease in cattle were in excess of normal volume and that the viscosity of the effusion was less than normal. Vaughn (1960) reported that degenerative joint dis- ease (osteoarthritis) in cattle was initiated in the same way and followed the same course as in man. The rate of progress of the disease, however, was more rapid in cattle than in man, because from the time of the initial injury to the joint, the limb was never allowed to rest, and the mechanical forces at play were enormous, particularly in the femoropatellar and femorotibial joints. Bulls with Spastic contraction of the hindlimbs (stretches) were found by Townsend (1961) to have lesions 17 of degenerative joint disease which he called osteoarthritis. He reported no cure for the condition; however, the intra— articular injection of hydrocortisone gave temporary relief from pain. He concluded that the best control was through prevention, and that bulls with a family history of this condition Should not be used in breeding programs. Good management of the individual bull was considered an important factor, making sure that proper exercise was taken and ade- quate bedding was available, and that physical action which could possibly result in further joint damage be avoided. Degenerative joint disease, which they called degenerative arthropathy, was described by Blood and Hender- son (1963) as occurring unassociated with obvious defects of the bones, particularly in beef bulls and aged dairy cattle. They observed that, when the limb joints were affected, there was evidence of claudication in severe cases, crepitus, and distention of the joint capsule with Synovial fluid. Defi— ciencies or mineral imbalances, particularly of calcium and phosphorous, were suggested by these workers as possible etiologic factors. Boyd (1961) attributed the reduction and eventual loss of intercellular ground substances with subsequent fibrillation of the remaining cartilage to a loss of chon- droitin sulfate from the ground substance. According to Moore (1952), further articular attrition resulted when joint movement destroyed the soft, partially necrotic 18 articular cartilage, thus exposing the subchondral bone to grooving and eburnation. Prior to cartilaginous erosion, osteoblasts in the area of the subchondral bone had under- gone proliferative changes, with the formation of new bony trabeculae, narrowing of the Haversian canals, thickening of existing subcortical trabeculae, and an invasion of the articular cartilage by the newly formed bone. Such degenera- tive changes resulted secondarily in the formation of mar- ginal osteophytes in response to increased periarticular stress. Johnson (1962) pointed out that marginal osteo— phytosis resulted in peripheral or circumferential remodel- ing of the joint, which resulted in an increased diameter at the ends of the bone in the area of the periarticular margins. Progressive changes consisted of periosteal elevation, and chondrification followed by ossification of the ligaments, tendons, or joint capsules attached to the bone in this area. Rodnan gt'_l. (1960) observed in man a sclerotic atrophy of the synovial membrane, which consisted of a flatten- ing and associated decrease in the number of synovial intimal cells. In some Specimens of synovial membrane they observed a complete absence of the Synovial intimal cells, with hyalinization of the stroma. Hyalinization of the stroma was generally homogeneous in nature, while in some Specimens the hyalinized stroma was arranged in broad bands that ran parallel with the surface of the stratum synoviale.y Asso- ciated with sclerotic atrOphy of the synovial membrane was 19 a hyalin thickening of the capillary and arteriolar walls. Mikkelsen gg gl. (1958) observed a minor hyper— plasia of the synovial intimal cells, with a scattering of inflammatory cells in the stratum synoviale of the Synovial membrane Specimens obtained from the knee joints of man. Schwartz and Cooper (1961) and Collins (1949) observed a scattered Subintimal infiltration and associated perivas- cular cuffing with lymphocytes in Synovial membrane Specimens from joints affected with degenerative joint disease in man. Hutyra £3.2l- (1945) described a puerperal arthritis or synovitis associated with parturition in cattle. The joint disease produced a serous, fibrinous, or purulent synovial effusion 6 to 8 days following parturition or, in some instances, later, usually in conjunction with putrefy- ing material in the uterus. The tarsal or carpal joints were most frequently involved. Glattli (1957) stated that puerperal arthritis occurred frequently in the absence of necrotic lochia. He suggested that a saphrophytic contamina- tion of the residual uterine contents occurred and that hypersensitivity to the saphrophytes produced the puerperal arthritis. Sokoloff (1960) pointed out that puerperal arthritis was a relatively common affliction of cattle and regarded the syndrome as being due to dissemination of bac- teria from infected decidual sinuses. He concluded that the condition had no pathologic characteristics that would 20 distinguish it from other types of infectious polyarthritis. A severe, painful form of arthritis or Synovitis of a Sim- ilar nature has been described by Boddie (1962), involving particularly the tarsus in association with septic metritis and mastitis in cattle. Mikkelsen g3 g1. (1958) described follicle-like collections of lymphocytes and plasma cells in perivascular formations in synovial membrane Specimens from patients affected with rheumatoid arthritis; however, they pointed out that no single finding was diagnositic of rheumatoid arthritis. Boyd (1961) attributed the formation of sub- chondral granulation tissue in rheumatoid arthritis as a reSponse by the epiphyses to the inflammatory processes in the joint. Fowler and Kingrey (1956) noted an apparent selec— tive localization of pyogenic microorganisms for the tendo- vaginal sheaths and joints of cattle. Usually the most pronounced involvement was found in the tarsus. Infectious arthritis was especially common in suppurative metritis, suppurative mastitis, and omphalophlebitis. Crawford and Frank (1940) described a synovitis in yearling bulls following the intravenous injection of viable avian tubercle bacilli (Mycobacterium avium) recovered from swine. Joint swellings developed in 4 of 10 animals, approximately 8 months following the initial intravenous 21 injection. Avian tubercle bacilli were isolated from the Synovial effusions of the affected joints. A septic arthritis of the tibiotarsal, femoropatel- lar, femorotibial, and carpal joints of two 8—months-old Hereford steers due to Erysipelothrix insidiosa (rhusiopath- ggg) was investigated by Moulton g3 gl. (1953). Post mortem lesions were restricted to the joints and large quantities of Synovial fluid were observed; there was a marked prolifera- tion of the synovial membranes (extensive villous prolifera— tion), in addition to extensive erosions of the articular cartilages. The ankylosis observed in association with polyarthritis of swine due to E. insidiosa (rhusiopathiae) infections as reported by Sikes (1959) was not observed in either of the two steers. Smith and Jones (1961) described an acute suppura— tive or an acute fibrinous arthritis due to wounds or as a result of a generalized septicemia or pyemia. The most common form of infectious arthritis was pyosepticemia of the new-born (pyosepticemia neonatorum) resulting from infection of the umbilicus at birth. The most common etiologic agents of infectious arthritis in cattle were listed by Jubb and Kennedy (1963) as Streptococcus $22., Escherichia coli, Corynebacterium pyogenes, and Salmonella spp. Streptococcal and coliform polyarthritis was generally considered neonatal in origin, while infectious arthritis due to Q. Eyogenes and Salmonella Spp. occurred at any age. 22 Van Pelt (1962a) isolated C. pyogenes, Staphylococ— cus aureus, S. fecalis, and E. coli from the synovial effu- sions of infected joints of cattle of various ages, breeds, and sexes. These microorganisms were introduced to the joint cavity by foreign body penetration, extension from an area of infection adjacent to the joint cavity, associated with a septicemia, or had metastasized from a focus of infec- tion in some other part of the animal's body. The recovery of Mycoplasma Spp., pleurOpneumonia—like organisms (PPLO), from the joints, kidneys, and Spleen of a calf showing evidence of a severe arthritis and bronchopneu— monia was reported by Moulton g3 31. (1956). The organism was experimentally inoculated into young cattle and resulted in clinical Signs of stiffness, claudication, and corneal keratitis. Agglutinins for Myc0p1asma Spp. were demonstrated in the serum from these animals. Cordy (1959) reported the occurrence of arthritis in ruminants due to Mycoplasma Spp. (PPLO), and recovered large numbers of the causative agent from the synovial effusions of infected animals. The atlan- to-occipital and larger diarthrodial articulations were most commonly involved. The synovial effusions were yellowish and turbid, but only moderately increased in quantity. Frequent- ly the Synovial effusions clotted immediately following collection. AS expected, the more severe cases contained large amounts of fibrin, which upon microscopic examination 23 revealed extensive masses of compacted fibrin interlaced with many disintegrating neutrophils. Simmons and Johnston (1963) reported an arthritis in calves also due to Mycoplasma £22. (PPLO) which, in the initial stages of the disease, was characterized by a fibrino—purulent synovial effusion that became fibrinous in the latter stages of the disease. Arthritic lesions had been completely resolved in the case of one of the calves when a post mortem examination was conducted 5 months following the initial infection. lull'." . I Coggeshall g£__1. (1941) investigated synovial fluid and synovial membrane Specimens obtained post mortem from patients who had died as a result of a generalized infection and found that the tissues occasionally Showed an inflamma— tory reaction which was reflected by the cytology of the synovial effusion even though microorganisms were not isolated or demonstrated in cultures or histologic sections. Recently, Wilske and Decker (1965) reported that ulcerative colitis, regional enteritis, and Whipple‘s dis— ease of man frequently had in common an intestinal disorder, an asymmetrical nondeforming large—joint arthritis and an increased incidence of Spinal joint involvement. III. MATERIALS AND METHODS The nomenclature and classification of the arthri— tides, arthroses, and synovites studied in this investiga— tion were based on the work of Blumberg g3 31. (1964) as established by the Nomenclature and Classification Committee of the American Rheumatism Association. Blood, Serum, and Synovial Fluid Studies Samples of Synovial fluid were collected from a total of 117 joints of 82 cattle of various breeds, ages, and sexes. The various types of arthritis, arthrosis, and syno— vitis encountered in the course of this investigation were diagnosed and classified with the aid of the following criteria: (1) etiology; (2) pathogenesis; (3) pathology; (4) anamnesis; (5) clinical signs; (6) arthrographs; (7) synovial fluid analyses; and (8) bacteriologic studies. In addition to Synovial fluid analyses, a complete hemogram was made on the peripheral blood of each animal. Classification of Pathologic Synovial Fluids On the basis of the preceding criteria and the results of Synovial fluid analyses, the synovial fluids '24 25 analyzed in this investigation have been grouped into three major categories based on the work of Ropes and Bauer (1953). Group I--Synovial Effusions that Resemble Normal Synovial Fluids These effusions are primarily transudative in nature. Pathologic joint effusions included in this group are the result of degenerative joint disease, osteochondromatosis, osteochondritis dessicans, hydrarthrosis, and traumatic arthritis. Group II--Intermediate Synovial Effusions These effusions possess characteristics of both Groups I and III, i.e., they may be transudative to exuda- tive in nature. Pathologic conditions included in this group include acute, subacute, or chronic synovitis and arthritis, arthritis associated with disturbances in metab— olism (gout), and hemophilic arthropathies. Group III-~Septic Synovial Effusions These effusions are exudative in nature and are the result of a Specific infectious agent in the joint as iden- tified by bacteriologic examination of the synovial fluid. The effusions may be of variable duration and may be primary (due to penetration of a foreign object into the joint cavity), secondary (extension from an area of infection adjacent to the joint cavity), or tertiary (associated with a septicemia or metastasis from an area of infection in some 26 other part of the body not adjacent to the joint cavity). Blood Serum Samples Whole blood samples for determination of serum alkaline phosphatase activity(ALP),1actic dehydrogenase activity (LDH), glutamic oxalacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) levels were obtained from the jugular vein by venipuncture prior to arthrocente— sis of the respective joint or joints. The clot was allowed to retract at 5 C., and the sample then centrifuged* at 2,500 revolutions per minute (r.p.m.) for variable periods of time at 5 C. in order to obtain the clear supernatant serum. All alkaline phosphatase activity determinations were made on the day blood was collected. Serum for lactic dehydrogenase activity, glutamic oxalacetic transaminase, and glutamic pyruvic transaminase levels was frozen follow— ing centrifugation at —25 I 2 C. and then stored at —70 I 5 C. until analyses were conducted. Hemolyzed serum samples were excluded from the various enzymatic determinations. Synovial Fluid Samples Synovial fluid was obtained by arthrocentesis as described by Van Pelt (1961a and 1962b) from the following joints: (1) radiocarpal; (2) intercarpal; (3) metacarpo— phalangeal; (4) femoropatellar; (5) tibiotarsal; and (6) *Model PR-2, International Portable Refrigerated Centrifuge, International Equipment Company, Needham Heights, Mass. 27 metatarsophalangeal. Synovial fluid was collected from the various individual joints and in some instances from the correSponding joints of the opposite limb. All glassware and needles had been previously cleansed and rinsed with distilled water prior to air—drying in a hot oven. ASpira— tion of synovial fluid was made with sterile 16- or l8-gauge needles of various lengths. Sterile, dry, 5-, 10-, or 20-cc. metal—tipped glass Syringes were employed for withdrawal of the Synovial fluid. Samples were then transferred immediate- ly to dry, screw-capped vials, one containing no anticoagu- lant, and the other containing an anticoagulant. That por- tion of a sample intended for bacteriologic studies was transferred immediately to a sterile vial. Gross appearance and total volume of the synovial effusion were observed and recorded at the time of collection. A minimum quantity of 3.0 ml. was required for a complete analysis. With the exception of that portion of the sample employed for total erythrocyte and leukocyte counts, and bacteriologic Studies, all samples were centrifuged at 2,500 r.p.m. for variable periods of time at 5 C. in order to obtain a supernate free of cellular material and debris. All synovial fluid alkaline phOSphatase activity determinations were made on the day the sample was obtained. Synovial fluid for lactic dehydrogenase activity, glutamic oxalacetic transaminase and glutamic pyruvic transaminase levels were frozen following centrifuga- tion at -25 1 2 C. and then stored at —70 i 5 C. until analyses were conducted. 28 Hemolyzed samples were excluded from the various enzymatic determinations; however, samples that were xanthochromic in nature were employed for enzyme analyses. Gross Appearance of the Synovial Fluid The gross appearance of the Synovial fluid was classified as follows based on the investigations of Van Pelt and Conner (1963a) and Van Pelt (1963d): N NF NO NOF YF YO YOF AF AO AOF TY TYF normal (colorless and clear) colorless and clear, with some flocculent material colorless and opaque colorless and opaque, with some flocculent material pale yellow pale yellow and and material pale yellow pale yellow and and material amber amber amber amber and clear and clear, and opaque clear clear, with some flocculent opaque opaque, with some flocculent with some flocculent material and opaque, with some flocculent material turbid and yellow turbid and yellow, with some flocculent material 29 SS serosanguineous 11‘. ll hemorrhagic Blood and Synovial Fluid Anticoagulants Two anticoagulants were employed in this investiga- tion to prevent clotting of whole blood and pathologic Syno- vial effusions. Initially in the investigation a 20% aqueous solution of ammonium oxalate and potassium oxalate (APO) (approximately 2 mg./ml. of blood or synovial fluid) was employed. In the latter portion of the investigation, a 10% aqueous solution of dipotassium ethylenediaminetetra- acetate* (EDTA) was used to prevent clotting of blood and synovial fluid (approximately 2 mg./ml. blood or synovial fluid). Vials containing the anticoagulant were prepared prior to time of sample collection and the respective anti— coagulant solutions evaporated to dryness at 68 C. Ammo- nium oxalate and potassium oxalate was employed in the latter aSpects of the investigation for the collection of synovial effusions intended for alkaline phOSphatase activity determinations when the total quantity of synovial fluid was Such that there would be an insufficient amount of super- natant following clot retraction and Subsequent centrifuga- tion to permit enzymatic analyses. *Cambridge Chemical Products, Inc., Dearborn, Mich. 30 Blood and Synovial Fluid Sugars Whole blood sugar values were determined and eval— uated in relation to their reSpective synovial fluid Sugar levels. These values were determined by a modification of the original work of Folin and Wu (1920) and Folin (1926). When quantities of synovial fluid were insufficient to permit analyses by macromethods a micromethod modification of the original technique as described by Hepler (1957) using 0.1 ml. quantities was utilized. Samples were read at 430 mu in a Spectrophotometer* employing 19 x 105 mm. cuvettes for the macromethod and 12 x 75 mm. cuvettes for the micromethod. A standard glucose solution (0.1 mg. of glucose per 1 m1. of a 0.25% benzoic acid solution) was employed for each series of sugar determinations. The sum total of reducing agents in the whole blood and synovial fluid was reported and referred to as Sugar in view of the fact that they do not represent glucose pg; gg, as reported by Benedict (1931), Fashena (1933), Fashena and Stiff (1941), Folin and Wu (1920), Folin (1926), and Hepler (1957). All sugar values were expressed in milligrams per 100 ml. of whole blood or synovial fluid. All animals had free access to feed immediately prior to the time of sample collection. *Model 6-A, Junior Spectrophotometer, Coleman Instruments, Inc., Maywood, I11. 31 Relative Viscosity Relative viscosity of the Synovial fluid was deter— mined on the clear supernatant fluid after centrifugation of the sample as described by Van Pelt and Conner (1963b). These determinations were made at 38.6 C. employing either a routine viscosimeter* (A.S.T.M., sizes 100, 150, or 200) for quantities of 5 or 10 ml., or a semi—micro-dilution‘ viscosimeter** (A.S.T.M., Sizes 100 or 150) for 1 ml. quan- tities. The viscosimeters were SuSpended in a kinematic constant temperature water bath*** (A.S.T.M., No. D445-53T) designed for use with capillary viscosimeters. A constant bath temperature was maintained by a mercury, water vapor— type thermoregulator. Distilled water was employed as the reference liquid for relative viscosity determinations be- cause of its complete stability for Such purposes as shown by Cannon (1960). Efflux measurements for synovial fluid were determined with a hand-operated stop watch. *Cannon—Fenske Routine Viscometer, Cannon Instru- ment Company, State College, Pa. **Cannon-Ubbelohde Semimicro Dilution Viscometer, Cannon Instrument Company, State College, Pa. ***Krebs Constant Temperature Water Bath, Krebs Electric and Manufacturing Company, New York, N.Y. 32 Mucinous Precipitate The mucinous precipitate test was performed on the supernatant synovial fluid as described by Van Pelt and Conner (1963b) following centrifugation of the sample. This procedure was performed to determine the degree of polysac- charide polymerization. Clot formation and turbidigenic properties were determined by the addition of synovial fluid in a 1:4.1 ratio to a 2.5% aqueous solution of glacial acetic acid (pH 2.3 at 25 C.). The results were graded as follows: (1) normal (N), a tight, ropy clump in a clear solution; (2) fair (F), a soft mass in a very Slightly turbid solution; (3) poor (P), Small, friable masses in a turbid solution; and (4) very poor (VP), a few flecks in a turbid solution. Serum and Synovial Fluid Alkaline PhOSphatase Activity Serum and synovial fluid alkaline phosphatase activ- ity (ALP) were determined by a micromethod as described by Sommer (1954) employing 0.1 ml. quantities of the respective fluids. Samples were incubated at 38 C. for 30 minutes employing p-nitrophenyl phosphate disodium tetrahydrate* (absorption maximum of 400 mu) as the substrate (buffered with 0.1 M aminoacetic acid to pH 10.5) and the reaction *Sigma Chemical Company, Inc., St. Louis, Mo. 33 stopped by the addition of 10 ml. of 0.02 N NaOH. Samples were then transferred to a 10—mm. rectangular silica cell and read at 410 mu against a Substrate blank (0.1 ml. of double distilled water with less than 1 p.p.m. NaCl was substituted for serum or synovial fluid during the incuba— tion procedures) in a Spectrophotometer* at 25 C. and the per cent transmittance recorded. The inherent color of the blank, serum, and synovial fluid was corrected for by the addition of 0.1 ml. of concentrated HCl to each incubation tube. Samples were then read again, against the blank and the second reading recorded and subtracted from the first to obtain the correct alkaline phOSphataSe activity of each , sample. Alkaline phosphatase activity was reported in Sigmg units per milliliter of serum of synovial fluid as deter- mined by a standardization curve. One Sigmg unit is that amount of alkaline phOSphatase activity which will liberate 1 pM (micromol) of p-nitrophenyl per hour at 38 C. (1 uM = 0.1391 mg.). Serum and Synovial Fluid Lactic Dehydrogenase Activity Serum and synovial fluid lactic dehydrogenase activ— ity (LDH) were determined by a micromethod based on the work of Cabaud and Wr8blewski (1958) employing 0.1—ml. quantities of the reSpective fluids. Serum was diluted 1:5 with *Model D-B Spectrophotometer, Beckman Instruments, Inc., Fullerton, Calif. 34 double-distilled (less than 1 p.p.m. NaCl) water and synovial fluid was utilized in undiluted quantities. In some in- stances serum dilutions of 1:29 and synovial fluid dilutions of 1:5 or 1:29 were required. Samples were incubated at38 C. for 30 minutes in a substrate solution containing 1.0 mg. of gggg-diphOSphopyridine nucleotide; dihydronicotinamide adenine dinucleotide-disodium (b-DPNH)* and 1.0 m1. of phOSphate buffered pyruvic acid (pH 7.6 at 250 C.) and the reaction stopped at the termination of the incubation period by the addition of 1.0 ml. of a 2,4—dinitropheny1hydrazine HCl solu— tion. Color was allowed to develop at room temperature for 20 minutes and 10 m1. of 0.4 N NaOH was added and allowed to stand at room temperature for 5 to 10 minutes before reading the sample. Samples were then transferred to a lO-mm. rectangular Silica cell and read at 470 mu against distilled water as the reference liquid in a Spectrophotometer and the per cent transmittance (%T) recorded. Lactic dehydrogenase activity was reported in LDH units per milliliter of serum of synovial fluid as determined by a standardization curve. Each LDH unit is equivalent to that amount of enzyme that would cause a decrease in O.D. at 340 mu of 0.001/minute in a reaction mixture of a 3.0 ml. volume as established by Wr8blewski and LaDue (1955). *Sigma Chemical Company, Inc., St. Louis, Mo. 35 Serum and Synovial Fluid Glutamic Oxalacetic and Glutamic Pyruvic Transaminase Activities Serum and synovial fluid glutamic oxalacetic trans— aminase (GOT) and glutamic pyruvic transaminase (GPT) activ- ities were determined by a micromethod as described by Reitman and Frankel (1957), employing 0.2 ml. quantities of the reSpective fluids. Samples for serum and Synovial fluid GOT determinations were incubated at 38 C. for 60 minutes in 1.0 ml.quantities of an aspartate—a—ketoglutarate* substrate (buffered to pH 7.5). Samples for serum and synovial fluid GPT activity were incubated at 38 C. for 30 minutes in 1.0 ml. quantities of an alanine-a-ketoglutarate* substrate (buffered to pH 7.5). The reaction for both serum and Syno- vial fluid GOT and GPT activity was stopped at the end of the incubation period by the addition of 1.0 ml. of 2,4- dinitrophenylhydrazine HC1.* Color was allowed to develop at room temperature for 20 minutes, after which 10 ml. of 0.4 N NaOH were added. Samples were allowed to stand for 5 to 10 minutes at room temperature and then transferred to a lO—mm. rectangular silica cell and read at 505 mu against double—distilled water as a reference in a spectrophotometer at 25 C. The per cent transmittance (%T) was recorded and serum and Synovial fluid GOT and GPT activity determined by a standardization curve and expressed in Sigma—Frankel (S-F) *Sigma Chemical Company, Inc., St. Louis, Mo. 36 units per milliliter. One Sigma-Frankel unit of GOT or GPT will form 4.82 x 10‘4 uM of glutamate per minute at pH 7.5 at 25 C. Cytologic Studies Total erythrocyte and leukocyte counts were made from a noncentrifuged portion of the synovial fluid. For total erythrocyte and leukocyte counts, the synovial fluid was diluted 1:10 or 1:20 in a standard white blood-cell pipette with a 1% methyl violet-sodium chloride solution as described by Van Pelt (l962d). Synovial fluid that was serosanguineous or hemorrhagic in nature was diluted in a standard red blood—cell pipette 1:100 or 1:200. Leukocytes and erythrocytes were then counted in Succession in the same counting chamber of a standard hemacytometer (improved Neubauer ruling). Differential leukocyte counts were made from stained Smears of the sediment after centrifugation of the synovial fluid. Smears were made on glass microslides (25 by 75 mm.) previously cleaned with absolute methanol (acetone—free), air-dried, and Stained with Wright's stain* for 1 minute. Phosphate buffer (.045 M at pH 6.33) was then added for 4 minutes, the slide then rinsed with double—dis— tilled water, placed on end and air-dried. One hundred leukocytes were identified as described by Van Pelt and *Gradwohl Laboratories, St. Louis, Mo. 37 Conner (1963a), and counted, and the absolute number of the various cell types determined for each sample of synovial fluid. These cell types included: (1) polymorphonuclear neutrophils; (2) lymphocytes; (3) monocytes; (4) macro- phages; and (5) eosinophils. Synovial lining cells and degenerated leukocytes were occasionally Seen but never in— cluded in either the total or differential leukocyte counts. Bacteriologic Studies Synovial fluid Specimens intended for bacteriologic examination were transferred immediately from the aspirating Syringe to sterile vials. Isolation and identification of microorganisms in septic synovial fluids were made by the Diagnostic Laboratory, Department of Microbiology and Public Health, College of Veterinary Medicine, Michigan State University. 38 Gross and Histopathologic Studies Synovial tissue Specimens were obtained by joint punch-biopsy as described by Van Pelt (l962c) and at necropsy from the various perimeters of the synovial membrane of 76 joints of 32 cattle and fixed immediately in 10% buff- ered neutral formalin for a minimum of 24 hours. Specimens of articular cartilage and subchondral bone were fixed as for Synovium, decalcified,* and washed in running tap water for 2 hours prior to further tissue processing. Gross obser- vations were made of the joint structures and other body organs at necropsy. Where indicated to establish disease processes other than that confined to the joint or joints, representative tissues were collected for histopathologic examination and processed accordingly. Where indicated, Specimens for bacteriologic examination were taken from the joints and other body organs. Sections of joint tissue were cut at 6 microns and stained according to the following procedures as outlined in the Armed Forces Institute of Pathology Manual of His- tologic and Special Staining Technics (1960): (1) Harris' hematoxylin and eosin Y (H & E); (2) periodic acid-Schiff (PAS) reaction and counterstained with Harris' hematoxylin or light green solution; and (3) Von Kossa‘s method for *Decal, Omega Chemical Company, Garden City, N.Y. 39 demonstrating calcium, counterstained with nuclear fast red. The joint capsule was divided into two strata accord- ing to the work of Trautmann and Fiebiger (1957). A stratum fibrosum, made up of dense fibrous tissue immediately blended with the ligamentous structures of the joint and a stratum synoviale (Synovial membrane or synovium) which formed the lining of the joint cavity itself. The stratum synoviale was classified on the basis of the predominant structure of the Subintimal connective tissue aS described by Castor (1960): (l) fibrous; (2) fibro-areolar; (3) areolar; (4) areolo—adipose; and (5) adipose. In the course of histopathologic observations and descriptions, the articular cartilages were divided into four strata based on the work of Barnett E£.2l- (1961). From the articular surface to the subchondral bone, these strata are: (l) the stratum superficialis; (2) the stratum intermedius; (3) the stratum radiatus; and (4) the carti- laginous stratum calcificatum. IV. RESULTS Synovial Fluid Analyses Group I--Synovial Effusions that Resemble Normal Synovial Fluids Dggenerative Joint Disease The joints of 29 cattle (Table 2) of various breeds, ranging in age from 1.5 years to 14 years, with a mean age of 7.4 i 0.8 years, were investigated. Nine of the animals were bulls and 20 were cows. The tibiotarsal joint was most frequently affected, accounting for 76.5% of all joints investigated in this study. Joints of 2 of the animals were sampled twice. Primary degenerative joint disease accounted for 68% of all affected joints. Total volume and gross appearance of the synovial effusions were observed and recorded for each joint (Table 3) at the time of arthrocentesis. An excessive synovial effu- sion was not considered pathognomonic of either primary or secondary degenerative joint disease; however, the gross appearance of the effusion as denoted by variable amounts of flocculent material was considered indicative of the degener- ative processes taking place in affected joints. 4O 41 TABLE 2. Incidence and Pathologic Classification of Synovial Effusions from the Joints of Cattle Affected with Degenerative Joint Disease No. Breed (:5?) Sex Joint(s) C::::;£:_ l Holstein- 14.0 Bull Right Intercarpal Primary Friesian Left Intercarpal Primary Left Tibiotarsal Primary 2 Aberdeen- 6.0 Bull Right Tibiotarsal Primary Angus 3 Holstein- 4.0 Cow Right Tibiotarsal Secondary Friesian 4 Holstein— 10.0 Cow Left Tibiotarsal Secondary Friesian 5 Holstein- 6.5 Bull Left Tibiotarsal Primary Friesian Right Tibiotarsal Primary 6 Shorthorn 5.0 Bull Left Femoropatellar Primary Right-Femoropatellar Primary Left Tibiotarsal Primary Right Tibiotarsal Primary 7 Holstein- 2.5 Cow Left Radiocarpal Primary Friesian Right Radiocarpal Primary Left Tibiotarsal Primary 8 Guernsey 12.0 Bull Left Tibiotarsal Primary Right Tibiotarsal Primary 9 Aberdeen- 8.0 Bull Left Tibiotarsal Secondary Angus Right Tibiotarsal Secondary 10 Holstein- 12.0 Cow Right Tibiotarsal Primary Friesian ll Holstein- 12.0 Cow Right Tibiotarsal Primary Friesian 12 Holstein- 14.0 Cow Right Tibiotarsal Primary Friesian l3 Holstein- 15.0 Cow Right Tibiotarsal Primary Friesian l4 Holstein- 3.5 Cow Left Tibiotarsal Secondary Friesian 15 Holstein- 3.0 Cow Right Tibiotarsal Secondary Friesian formation abnormalities and excessive microtrauma. * . . . . . . Pathologic jOlnt efqulons claSSlfled as follows: (1) primary = ordinary usage; and (2) secondary those associated with aging processes and those due to joint con— 42 TABLE 2—-Continued No. Breed (35?) Sex Joint(s) Ciziiéfil' l6 Shorthorn 1.5 Bull Right Metacarpo- phalangeal Secondary l7 Holstein- 9.0 Bull Left Tibiotarsal Primary Friesian Right Tibiotarsal Primary 18 Holstein- 5.5 Cow Right Tibiotarsal Secondary Friesian l9 Guernsey 6.0 Cow Left Metatarso- phalangeal Secondary 20 Guernsey 5.0 Cow Left Tibiotarsal Primary Right Tibiotarsal Primary 21 Holstein- 8.0 Cow Right Tibiotarsal Primary Friesian 22 Holstein— 6.0 Cow Left Tibiotarsal Primary Friesian 23 Holstein- 16.0 Cow Right Tibiotarsal Primary Friesian 24 Holstein— 4.25 Cow Left Tibibtarsal Secondary Friesian Right Tibiotarsal Secondary 25 Holstein- 8.0 Cow Left Tibiotarsal Primary Friesian Right Tibiotarsal Primary 26 Aberdeen- 8.0 Bull Right Radiocarpal Primary Angus Right Intercarpal Primary Right Metacarpo— phalangeal Secondary 27 Holstein— 3.17 Cow Right Tibiotarsal Secondary Friesian 28 Holstein- 4.0 Cow Left Tibiotarsal Secondary Friesian Right Tibiotarsal Secondary 29 Jersey 4.0 Cow Left Tibiotarsal Primary Right Tibiotarsal Primary 43 .cmme esp mo Houuo pumpcmumrr .Hdfluopme Humanooon meow Eye; .zoaam> bum banana u me new ”Hmfiuoyms pnmasoooaw meow seas .osdmdo new Mensa u m0< mamfiuopme pcoasoooam meow Eva: .Hmoao paw nomad u m< ”Hmfinoume Humanoooaw meow Spa: .osvmdo new 30HH6> mama u mow ”Hmfiumwms pumasoooaw meow spa: .mzdmmo paw mmmauoaoo u moz ”Hmfiuopme Humanoooam meow spa: .Hmoao 0cm mmmauoaoo u mz ”mummao new mmoHHoHoov Hmsuoc u z ”UHSHM Hmfi>oc>m 659 MO mocmummmmm mmouwr 0 H 0 0 0 0 0 00.0 + mN.N 0.0 09 mm.m H Hmmmcmamnmomumwmwmz o o H m HH H NH SS.H + R©.mH o.mq o“ m.m om HmmHmHOHnHH o o o o m o o om.m H om.o o.OH 0» o.m m uaHHmHNQOHOEmm H o o o H o o om.m H mm.m m.m oH o.H m HammqumnaoaumuaHmz o o o H o o m om.o H 00.0 m.o on m.w m HmmuauuchH o o o H m o o «45H.m H om.m m.o ow o.m m HmaHNUOHemm awe mo< m< mow moz m2 2 can: mmcmm .oz musaob rmocmummad< mmouw Apcfl0m\.oov menao> HmpoH I lllll ommomfia HGHOH o>fiumuocmmoa ape: Uopommm< mappmo mo madaOb may Scum bmcfimuno panda Hmfi>oc>m mo moamumoda< mmouw paw mESHo> HmpoH .m mqmpHpcm30 u mzdrkkr .cmoe map 00 Houum Uumvcmpm«¥« .Hoom >Ho> n m> Una ”noon u m ”HHmw m ”Hmsuo: u z ”Umvmuw szHmDU mpmpHQHomHQ msocH632¥r .0 0.00 um owns mun: chHquHEHopov >HHmoomH> o>HHMHmm*_ 0 0 0 H H 026 «arrmzd H Hmowdemamomumpmpmz o 0 HH mm 00 00.0 H mv.0 «0.0 Op 00.H o0 HmmHmHOHDHH 0 H H 0 m SH.0 H v0.0 00.5 0H 00.0 m umHHmHMQOHOEom o o H o H 0.0 H mo.om o H HammcmHHHQOQHmumHmz 0 0 o 0 0 00.00 H 00.00 v0.HHH 0» 0q.o 0 HmmumonuwcH 0 0 H m 0 k**00.mv H oo.v¢ No.00 Op 00.0 m HdQHdUOHUmm m> m m 2 .oz awe: mmcmm .oz mvcHOW mHmHHmwwmmMWMflmeosz ¥>pHmoomH> m>HpmHmm mmmmmHQ HGHOW m>HHmumam0m0 :sz Umuommw< mHHHmO mo mucH00 on“ Scum UHsHm HmH>oc>m mo >HHHmsd onHHQHomHm mnoaHosz can >HHwoomH> o>HpmHem .v mHm umwsm UooHn .mmsHm> umwsm UOOHD cam UHSHM H0H>oc>m cmozpmn Umumpcsoocm was mocmHmmeU pcmonchHm >HHmoHpmempm oz««« .cwoe may mo HouHm UH0©G0~00¥ Una wHan HmH>oc>m .HmEHcm some uom« 0H.m H 00.00 00H.HH00 00*¥«0H.m + mw.m0 mmH.xH0H 00 000H0> H0000 00.0 H 00.00 0 H 00.0 H 00.00 0 H HammcmHmnaomumHmpmz 00.0 H 00.00 00H.HH00 004¥4m0.m H mm.00 mmHHHH0H 00 HmmumHOHnHe 00.0 H 00.00 0 H 00.0m H 00.H0 00 0H mm m anHmpmdoHosmm 00.0 H 00.00 00 00 00 m 00.0H H 00.00 00HAHH00 m HammcmHmnaoaumumumz 00.0 H 00.00 00 00 00 m 00.0 H 00.00 H0 00 00 m HmaumuuopuH 00.0 H 0m.H0 00 00 00 m simm.wH H 00.00 00 00 00 0 H000000H000 cam: 00cmm mmHQEmm :00: 00:00 *mmHQEmm mwcH00 000Hm .He 00H\Hmm:0 00Ho0 .HE 00H\Hmm:0 vOOHm .02 mo .02 HMH>oc>0 .02 mo .02 ommmmHQ chOW m>Hpmumcmme EHHB Umpomww< oprmo How mmsHm> H0030 UOOHm 0cm UHsHm H0H>oc>0 mo somHHmQEoo < .0 mqm ESHmm 0CH©commoHHoo 05“ away Hmpmoum AH00.0 n m0 >HpcmonHcmH0*«« .2068 may mo uouuo vumucmpmrr . 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The preSence of neutrophils was not a consistent feature and eosinuufifiis, with the exception of data collected from the tibiotarsal joints, proved to be non-existent in synovial effusions obtained from joints affected with degenerative Joint disease. 52 .03H0> 83:00 0:H0:oam0::oo :00“ 000H AH00.0 n 00 0HH:0onH:0H0*¥« .:00E 0:9 mo :o::0 0:00:0p0«¥ .00:HE:0H00 0:03 003H0> >HH>Hpo0 000:HE00:0:H oH>3:>Q oHE0H3H0 83:00 0:0 0H3Hw H0H>o:>0 .H0EH:0 £000 :000 00.0 H H0.Hm 00H 00 0 H0 H¥¥0H.m + 00.0H 00 00 0 0m 000H0> H0000 00.0 H 00.00 0 H 00.0 H 00.00 0 H H00000H000 now:0H0H02 00.0 H 00.HN 00H 0» 0 0H 00000.0 H 0H.0H 00 OH 0 H0 H00:0HOHDHH H0.0 H 00.0H 00 00 0 0 0*H0.0 H 00.0 0H 00 0 m HmmwcmHmna 100:000902 00.0 H 00.0 0 H 00.0 H 00.0 0 H H00H000000H 00.0 H 00.0 0 H 00.0 H 00.0 0 H H00H000H000 :00: 00:00 m0HQE00 :002 00:00 000HQE00 0H:H00 53:00 mo 83:00 0H3H0 H0H>omN0 mo H:H00 .HE\0HH:0 H00:0:0I0E0H0 mo .02 .HE\0HH:D H00:0:0u0E0H0 mo .02 00000H0 H:H00 0>HH0:0:000Q 0HH: 00Ho0mm< 0pr00 :00 m03H0> >HH>HHo< 000:HE00:0:H oH>3:>m oHE0p3H0 83:00 0:0 0H3H0 H0H>o:>0 mo :omH:00EoO < .o mqmHH00000: 0:0 :00 m03H0> mo 00:0000 .:00E 0:» mo :o::0 0:00:0900 6.0 60 60 6.0 000.050 00.0 +0000 00.0 +00.0HH 00.0 +0000 60 . 60 80.000H0H00 80.00Hh00000 000.000 00.0 .000 00.000.00.05 00.00 .0000 A00.0mH00.00 A00.000hu00.00 m00.000MH00.00 A00.000HH00.00 v0.0+.H0.H 00.0 1.00.0H 00.Hv 1.0H.00H 00.00 +.00.00H 80.00.0000 80.005.00.03 80.0000.00000000000u000000 00.0+00.0 00.000.00.me 00.007.00.000 2000.00.00 6.0 00.0 +00.0.\. 80.030.00.03 HH.H0 +0000. 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