EliTÎCTRO ïHO HETIG AND SÎ5RXBÆ N B U TR A LIZA TIO IT STU D IES OF SERA FROM GHICKS3JS EXPOSED TO IN F E C T IO U S B R O N C H ITIS V IR U S by George T, Dimopoullos A Thesis Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health 1952 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. é EIÆ1CTH0PH0HE3TIC AND SERUM DEUTRALIZATIOir STUDIES OP SERA PROM CHICKSRS EXPOSED TO INPB0TI0U8 BRORC3H3TIS VIRUS ■by George T* Dimopoullos AR ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Bacteriology and Public Health 1952 Approved8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. G eorge T , D im o p o tillo e This study was undertaken to ascertain any possible correlations between electrophoretic and serum neutralization analyses of sera from chickens exposed to infectious bronchitis virus (IBV). Adult Single Comb White I»eghorn cockerels were divided into four groups as follows: Group I - Controls-bled at weekly and monthly intervals• Group II - Birds inoculated with IBV and bled immediately prior to inoculation and then at one, two, three, four, six, eight, ten, 12, 16, and 20 weeks. Group III - Birds treated as those in Group II but challenged at the twelfth week and bled one, three, five, and seven weeks after challenge. Group rVa - Birds inoculated with a normal lung and tracheal suspension and bled immediately prior to inoculation and then at one, two, and three weeks. Group rVb - Birds subjected to scarification of the trachea and bled immediately prior to scarification and then at one, two, and three weeks. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. George T, Dîmopoullos 2 Sera were diluted to a final protein concentration of two per cent (5), dialyzed (4), and analyzed electroplioretically in veronal "buffer (2) at pH 8.6, 0,1 ionic strength, using a potential gradient of 6,5 volts/cm^ for 7,600 seconds. The results are expressed as relative per cent serum protein component. For the serum neutralization test (l) equal portions of ten-fold dilutions of XBV and undiluted serum were mixed, lncu"bated and inoculated into embryonating chicken eggs. Results are expressed as the Xethal Pose^Q neutralization Indices (LD50 His) (3), Sera from normal "birds "bled at weekly intervals showed an increase of approximately 0,20 from the initial al"bumin/glo"bulin ratio of 0,85 during the first and second weeks. After this period the ratios decreased 0,40 to 0,60 of the initial value. Sera from normal "birds "bled at four-week intervals showed no significant changes in the relative per cent distribution of serum protein components. Birds exposed to IBV showed marked decreases in the alhumin/glo"bulin ratios to an average value of 0,45 during the first and second weeks. The ratios steadily increased after this period and returned to normal at the twelfth week. The normal values of 0,85 persisted for eight additional weeks. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. George T, Dîmopoulloe Th-e IZDgQ nis increased slowl^r at fhe first and second weeks after exposure. After this period a marked increase occurred. Maximum Els values of 10® were reached between the sixth and eighth weeks and decreased after this period to approximately 10^ at the twentieth week. Uo correlation was observed in the changes of electrophoretic patterns and changes in the îTIs. When electrophoretic patterns had returned to normal the IDgg Els were at their maximum values of 10®. Birds challenged at the twelfth week did not show any significant changes in their electrophoretic patterns but showeid increases in the Els to maximum values of 10*^. The changes in electrophoretic patterns were not considered specific for IBV since normal birds bled at weekly intervals showed similar changes. Sera obtained from birds inoculated with a normal lung and tracheal suspension and birds subjected to a scarification of the trachea showed varying results in the changes of albumin/globulin ratios. Results obtained did not give evidence that these treatments alone were responsible for the changes observed. There were no changes in the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Els, George T. Dimopoullos 1. 4 Cunningli^, C, H, A Laboratory Guide in Virology» Burgess Publishing Comparer, Minneapolis, 1948 2. Longs-worth, L. G. Recent Advances In fhe Study of Proteins by Electrophoresis, Ghem. Rev., 3. 323-340, (1942) Reed, L. J . , and Miench, E. A Simple Method of Estimating Eifty Per Cent Endpoints. Am. J. Hyg., 4. 493-497, (1938) Reinex', M . , and Eenichel, R, L. Dialysis of Protein Solutions for Electrophoresis. Science, 108: 164-166, (1948) 5. Sanders, E . , Huddleson, I. P., and Schaible, P.J. An Electrophoretic Study of Serum and Plasnm. from Normal and leucosis-affected Chickens. J. Biol. Ghem., 155: 469-481, (1944) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To "Boss" Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. George T. Dlmoi^oullos candidate for the degree of Doctor of DhilosoplQr Final Examinât ions September 15, 1952, Room 101, Giltner Hall Dissertations Electrophoretic and Serum Neutralization Studies of Sera from Chickens Exposed to Infectious Bronchitis Virus Outline of Studiess Major Suhjects- Bacteriology, Virology Minor Subjects- Biochemistry, Biophysics Biographical Items s Born- Novemher 24, 1923, Flushing, New York Uhdergraduate Studies- The Pennsylvania State College, 1942-1943, 1946-1949 Graduate Studies- The Pennsylvania State College, 1949-1950, Michigan State College, 1950-1952 Experience : U.S. Army, 1943-1946, Graduate Assistant, The Pennsylvania State College, 1949-1950, Michigan State College, 1951, Alumni Predoctoral Fellow, Michigan State College, 1951-1952 Society Affiliationss The American Association for the Advancement of Science, The New York Academy of Sciences, The Society of American Bacteriologists, The Society of the Sigma Xi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACIdîOTOEIDGEIMEïïTS 1* The author wishes to escpress his gratitude to Michigan State College for awarding him an Alumni Predoctoral Fellowship for the academic year 1951-1952 and supplying other funds for materials Which made it possible to conduct and complete this investigation. 2. He is also indebted to Dr. Charles H. Cunningham and to Dr. Henrik J. Stafseth for their spiritual and material aid and suggestions during the investigation and in the preparation of the manuscript. 3. To Mr. John S. lynch, he is also indebted for his aid during the drawing of the blood samples from the experimental birds. 4. For the sincere cooperation of Dr. Erwin J. Benne and the other staff members of the Department of Agricultural Chemistry responsible for the protein nitrogen analyses, the author is deeply grateful. 5. The author also wishes to express his thanks to Dr. Helson F. Waters and Dr. George S, Cottral for their kind cooperation in supplying the experimental birds. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. â TABIÆ OF C0Î3TFÏÎTS I Introduction ..... 1 II HiBtorical Review ............. 3 . . ......... 3 A. Fleet rophoresie B. Infectious Bronchitis ofChickens III .......... Materials and Experimental Procedures ........ 14 18 A. Experimental Birds ........ 18 B. Virus Antigens ......................... 21 C. Serum Neutralization Test ........ S3 ................ 25 B. Electrophoresis E . Experimental Exposure ............... 32 IV Results ............................................... 36 V Discussion VI Summary Bibliography ........ 53 .............. 63 ........... 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I rN T R O D U C T IO N The moving 'boundaiy method of electrophoresis has, in recent years, contributed greatly to a better understanding of the characteristic properties of plasma and serum and of their respective components. It has become a valuable tool in the study of the change and distribution of plasma and serum components in disease and experimental immunology. The method has been applied in the study of bacterial diseases and to a lesser degree to viral diseases. 5aowledge of antiviral sera has been limited due to their comparative poverty in quantity, and in many cases, to their distinguishing characteristic of exhibiting little or no definite change even though the anti­ viral sera are drawn from subjects with demonstrable increased antibody activity. Results obtained in the study of the relative percentage distribution of plasma and serum components in anti­ viral sera have also been subjects of controversy as compared to results obtained with most antibacterial sera* For the above reasons it was decided to conduct a physical and biological study of infectious bronchitis of chickens. This disease, which is of virus etiology, is of great importance in the poultry industry, causing great losses and morbidity in infected flocks. It was also thought that possibly a contribution to the differential diagnosis of the disease and to fundamental virus research could be given through Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the combination of these studies* Therefore, the investigation was directed toward the study of the change in serum electro­ phoretic patterns and the change in Lethal DosegQ Neutrali­ zation Indices (LD^Q NTs) after primary exposure and challenge to a tissue suspension of a chicken-propagated strain of in­ fectious bronchitis virus. Results obtained early in the study prompted further investigations on the effects of normal tissue inoculations and tracheal injuries on the serum electrophoretic patterns and LD^g NIs, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II HISTORICAL REVIEW A* Electroph-oresis Various substances in the colloidal state, when sus­ pended in an aqueous medium, have a characteristic electric charge, the sign of which depends upon the nature of the particle and the suspending medium* Similarly, protein mole­ cules possess charged groups on their surfaces when in the colloidal state suspended in an aqueous medium. The number of these charged groups determines the net charge density of the protein molecules. The net charge density of the protein mole­ cules determines their mobilities in an applied electric field* Mobility, which is independent of size and shape, may be changed "by varying the ionic atmosphere of the charged particles through change of the pH or ionic strength* This migration of an electrically charged particle in an applied electric field is termed electrophoresis* The application of electrophoresis in the study and analysis of plasma and serum proteins has become a valuable tool during the past 15 years* The development by Tiselius (155) of a standard electrophoresis apparatus and its modifications and improvements (103) have directed investigators in the field of protein chemistry to a more comprehensive knowledge of plasma and serum and their respective components. It has been applied in the study of the changes of plasma and serum components in bacterial diseases and experimental immunology i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (1, 13, 15, 35, 43, 45, 58, 66, 80, 91, 102, 109, 112, 114, 117, 143, 146, 147, 148, 157, 158, 161, 162, 163), in viral diseases (14, 29, 67, 69, 70, 83, 95, 116, 128, 140, 142, 144, 153, 165, 172), in rickettsial diseases (46), to nutritional deficiency in relation to antibody production (4, 11, 12, 22, 48, 50, 97, 117, 159), in tbe study of normal and immune plasma and serum proteins and other body fluids and tissue proteins (2, 17, 19, 21, 27, 28, 31, 41, 42, 44, 58, 60, 62, 63, 64, 68, 81, 82, 88, 90, 99, 100, 102, 104, 106, 107, 108, 109, 120, 121, 122, 126, 129, 134, 144, 150, 151, 152, 155, 156, 157, 158, 160, 167, 175), in the study of the effects of injury and traums.tic shock (22, 62, 63, 64, 124, 125), in the study of nutritional deficiency in relation to serum and plasma protein depletion and regeneration (21, 23, 24, 25, 26, 49, 51, 52, 96, 141, 166, 173, 174), and in non-specific diseases ( 59, 66, 111, 112, 114, 140, 148, 149, 151). In general, results obtained in the a,bove studies show definite qualitative and quantitative changes in plasma and serum components. There are instances vhere little or no change is observed in the serum electrophoretic patterns in Western equine encephalonyelitis (95, 128), influenza (172), and Japanese B encephalitis, and Venezuelan equine encephalonyelitis (95). The alterations observed are not considered to be specific for any one disease since these changes only measure the current status of the subject under study. The outstanding changes of the serum and plasma proteins are a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduction in altsumin, an increase in gamma-globulin, and a moderate increase in alplia-glc^bulins and "beta-globulin (114), In 1937 Tiselius (155) made hie first attempt to analyze "blood serum using h.is improved te clinique and apparatus •whichi utilized the principle of the Poucalt-Toepler schlieren optical method. The new, flat, narrow, multiple-cor^artment electrophoresis cell had the advantages that higher potential gradients could "be utilized. Since the cell contained more surface area, more efficient cooling in the ice-"bath could also "be obtained during the passage of current. Isolation of various components after eeparjation could also be accomplished in this cell* His first analysis demonstrated that serum was composed of four components, or boundaries, which migrated at different and characteristic mobilities under specified conditior of pH, ionic strength, and potential gradient. This unique feature of characteristic mobility for a specific component has aided in the idsntification of various fractions. The four components were designated by Tiselius as albumin, two globulins, and the remaining component was unnamed. During the same period (156, 157), while experimenting with purified horse serum globulin in the electrophoresis apparatus, Tiselius named the components of the globulin mixture alpha-, beta-, and gamma-. These notations are in general use today. Tiselius* method of analysis, although enabling one to observe visually and record photographically the number Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and the migration of the "boundaries, did not yield itself to the interpretation of quantitative data on the relative concentration and homogeneity of these "boundaries. It was not until Longsworth’s (103) modifications and adaptation of the Poucalt-Toepler schlieren method and the development of the schlieren technique that quantitative data could he obtained. The theoretical basis as explained "by Longsworth (103) is as followss The angular deviation of a pencil of light in a boundary present in the glass electrophoresis cell Is pro­ portional to the refractive index gradient and the horizonta.l breadth of the boundary. The displacement of the schlieren dlaphra,gm requited to intercept the pencil of light deflected do^'mward "by the "boundasy is also proportional to the distance betvreen the cell center and the diaphragm. l%;on raising the horizontal schlieren diaphragm the first light beams to be intercepted will naturally be those which are due to the steepest refractive index gradients in the cell, i.e., the center of the boundary. "When a series of photographic exposures are made while raising the diaphragm before each exposure the resulting photographic record gives an indi­ cation of the variation of the refractive index throughout tlie electrophoresis cell. These adjustments are accomplished mechanically in a continuous fashion in the schlieren scan­ ning method and the resulting diagram is recorded photograph­ ically, longsworth *s excellent paper should be consulted for a more detailed description (103). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Longsworth scanning modification of the schlieren technique is unexcelled for obtaining permanent records of electrophoretic patterns but does not allow for direct visual observation of the patterns. If a visual inspection of the entire pattern is desired, use is made of the diagonal schlieren diaphragm method of Thovert in one of its modifi­ cations, incorporating the cylindrical lens. For a detailed description of this method Longsworth*s papers (104, 105) should be consulted. In a study of various buffers for use in electrophor­ esis Longsworth (104) observed that an additional component in human plasma could be resolved from the albumin when employ­ ing sodium diethylbarbiturate buffer (veronal buffer) at pK 8.6 and 0.1 ionic strength. This component, a globulin, was present between the original alpha-globulin and albumin and was designated as alpha 1-globulin, The original globulin was named alpha 2-globulin, This buffer also had the advantages of causing more efficient separation of the gamnra.-globulin at salt-protein boundaries and also giving better symmetry between the patterns obtained in the ascending limb and those of the descending limb. The conponents of horse plasma do not separate as well in Longsworth*s buffer as they do in phosphate buffer at pH 7,7 suggesting that the proper buffer for the analysis of a given type of plasma or serum varies with the species and should be determined experimentally. In additional studies (72) on the effects of various buffers on the resolution of human serum components it has been Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 slio-wn fhat pliosphate buffer at pH 8.8 and 0.15 ionic strength is as good as Longsworth*s buffer (104) in the resolution of the serum components. In veronal buffer, 0.1 ionic strength, at pH values between 8.S and 8.6, there is little change in relative percentage composition of huiiian serum (99). A comparison of results obtained with bovine serum and plasma in veronal, verchal-s odium chloride, and veronalcitrate buffers (79) showed unsatisfactory resolution of the gamma-globulins and fibrinogen. Phosphate buffer was superior for the separation of beta-globulin from fibrinogen. Veronal and veronal-citrate buffers resolved two beta-globulins. Veronal-sodium chloride buffer was found more efficient for the separation of total alpha-globulins from albumin. The most satisfactory resolution of alpha 1- and alpha 2-globulins was found in veronal and veronal-citrate buffers. Resolution of the beta-globulin, fibrinogen, and gamma-gl obul in of swine plasma in phosphate buffer, 0.2 ionic strength was superior to veronal buffer. The resolution of the alpha-globulins and albumin was superior in veronal buffer. Also, in veronal buffer, two alpha-globulins and two betagl obul ins were resolved in comparison to one alpha-globulin and one beta-globulin in phosphate buffer (92). It is evident that the composition of the buffer (79, 92, 104), its pH (2, 92, 104), and ionic strength (2, 92, 104) affect the relative percentage distribution and resolution of the individual components under study. In addition, the degree Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of hemolysis (126, 149), storage conditions (126), the method ot area measurement (73, 126), and the period of dialysis of the diluted sample (126) also affect the rela­ tive percentage distribution of the serum and plasma com­ ponents* Mohility values of the individual components are also affected by the length of dialysis (59), the concen­ tration of protein (34, 101, 132), the pH (34, 59, 99), the ionic strength (34, 93, 136), and the composition of the buf­ fer (59), Chicken serum and plasma and their respective fractions have been extensively studied (17, 27, 41, 43, 100, 115, 121, 122, 129, 143, 144). In a study of the effect of age on the electrophor­ etic patterns of chicken serum and plasma it was shown that there is a relative decrease in albumin and an increase in globulins as the bird matures (17, 122), Prior to sexual maturity chickens were shown to possess low gamma-globulin levels and total serum proteins (17, 144). As the birds matured gamma-globulin levels and total serum proteins in­ creased with no significant changes in the beta-globulins (17, 144), The explanation given by the authors (17) is that this decrease in albumin/globulin ratio may have been the result of a normal development as the bird matured. An additional explanation (43) states that this change may have possibly been due to foreign antigen contacts which stimulated Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 the production of antihody globulins Which in turn was ob­ served as an Increase in total gamma-globulin as measured by ele ctrophores is• Antibodies (43, 129, 143) and possibly antibodyrelated substances (144) have been shown to be associated with semim gamma-globulin in chickens* Antibodies have also been shown to be associated with gamma-globulins in other studies (147, 150, 153, 157, 161, 162, 165). All gamma-globulin is not antibody (87, 91) neither are all antibodies contained exclu­ sive ly in the gamma-globulin fraction (35, 53, 128, 158, 162). The electrophoretic increase or decrease in antibody in chickens immunized with hurmn serum gamma-gl obul in parallels the increase or decrease in gamtœi-globulin (43). This parallelism has also been confirmed in other studies (1, 13, 15, 45, 83, 163, 172). Still other studies have shown that this is not necessarily the case (97, 117, 172). Very characteristic sex differences have been shown to appear in the electrophoretic patterns of chicken serum after sexual maturity at about the fourth or fifth month of life (121, 122). Prior to this time sex differences as observed elsctrophoretically are insignificant. One case is cited in which normal, adult male and fe­ male White Leghorn chickens only showed significant changes in the alpha-globulin and gamma-gl obul in (41). Neither the exact age nor the state of egg production of the birds was mentioned. It has been shown that laying hens possess a greater quantity of gamma-globulin than non-laying hens (17, 100, 122) and adult males (17, 122). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. à 11 The effect of contra-sex hormones in chickens and the resulting electrophoretic patterns have been studied (27, 122), It has been found that electrophoretic patterns of sera change into patterns typical of the opposite sex upon administration of these hormones, Prom the above data it may be seen that many factors will affect the relative composition and mobilities of chicken and other animal sera and plasma and their respective compo­ nents, depending upon the sex and age of the animal and the general composition and properties of the buffer used as the solvent, ITormal electrophoretic values for chicken serum and plasma studied under a variety of conditions have also been reported. Using phosphate buffer at pH 7,7, 0.2 ionic strength, San Clemente (143) demonstrated that normal chicken serum had the follo'wing relative percentage composition; albumin 35 per cent, alpha-globulin - 15 per cent, beta-globulin five per cent, and gamma-globulin - 45 per cent. The average albumin/globulin ratio was 0.54. A beta-globulin anomaly was present in the pattern of the descending limb. The presence of this anomaly has also been reported elsewhere by Deutsch and Goodloe (41), Sex or age of the birds were not mentioned. Electrophoretic mobilities (cm/sec/volt/cm x 10"*^) of the components were calculated as follows; albumin - 5.3, alphagl obul in - 3.9, beta-globulin - 3,0, and gamma-globulin 2,0, These values agreed essentially with the results obtained by Moore (122). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12 Tîie electroplioretic analysis of mature* male olnicken serum in phosphate buffer at pH 7.4 by Moore (122) shoTP^ed an average relative percentage composition as follows : albumin - 43 per cent, alpha-globulin - 21 per cent, betaglobulin - 7.1 per cent, and gamma-globulin - 28.9 per cent in contrast to San Clemente’s study (143). Veronal-citrate buffer at pH 8.6 has been used for the analysis of the plasma of male chickens of undetermined age. The average relative percentage composition of the plasma samples was as followss albumin - 38,2 + 1.3 per cent, alpha 1-globulin - 15.8 + 0.6 per cent, alpha 2-globulin 7.7 + 0.5 per cent, beta-globulin + fibrinogen + gamma-globulin + 37.5 ^ 1.3 per cent. Separation of the three components of lowest mobility was incomplete. Electrophoretic mobilities were also extremely high in this buffer (41). In the analysis of sera from chickens of undetermined sex and age, in veronal buffer at pH 8.6, 0.1 ionic strength, the average relative percentage composition was as follows: albumin - 46 per cent, alpha-globulins - 22 per cent, betaglobulin - 8 per cent, and gamma-globulin - 24 per cent (43), An excellent study of the electrophoretic distribution of nornfâ.1 serum and plasma components of 15 to 18 week old Single Comb White Leghorn chickens of unknown sex was conducted by Sanders, et aL (144) during a study of leucosis. Veronal buffer at pH 8.6 , 0.1 ionic strength was used with a potential p gradient of 6 to 7 volts/cm . These conditions gave excellent Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 resolution of the sérum and plasma components. The following average values for mobilities (cm/sec/volt/cm x 10"®, cal­ culated from the descending limb) were found: Component Average albumin 5.9 5*7-6.0 alpha-gl obul ir^ 4.7 4.5-5.0 beta-globulin 3.5 3.5— 3.6 fibrinogen gamma-globulin Range 2.5 2.5 2.0 X.9-2.1 Average relative percentage composition values, cal­ culated from the descending limb were as follows: Component Average (per c e n t ) albumin 46.8 Range (ascending limb, uncorrected) (per cent) 41.7-47.2 alpha-globulins 17.9 9.0-16.3 beta-globulin 11.3 10.9-14.8 fibrinogen gamma-globulin 14.1 13.5 19.4 13.7-32.5 1.00 0.72-1.34 albumin/globulin ratio Total normal serum protein values were found by Sanders, et al (144) to vary from 2.19 to 3.74 gm/lOO ml of serum in these 15 to 18 week old birds, Brandt, et al (17) have shown values of 4.63 0.29 for four month old cockerels and 4.49 for four month old pullets. In general, total serum protein values increase with maturity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.37 14 B. Infect Î O U 8 Bronch.ltle of CThickens InfectiouB hroncliitis of chicken s was first described "by 8chalk and Hawn (145) in 1931 as an acute respiratory disease of chicks prevalent in the Midwestern States. Since that tiniB the disease has he en reported throughout the United States (5, 8 , 9, 18, 61) and has also h e en ohserved in England (3, 9), Holland (154), and Canada (8, 9). The virus etiology of the disease has heen established hy filtration experiments microscopy (7, 10, 18, 39, 145) and electron (136, 137). The disease was originally thought to he confined solely to chicks (18, 145) hut it has heen reported and is recognised in chickens of all ages (5, 10, 39, 55, 86). The morbidity and mortality rates may he as high as 90 per cent in Infected chicks (18, 39, 145). In mature chick­ ens the mortality rate is negligible (164), although the mor- hidity rate may he high (164). In laying flocks there may he a temporary cessation of egg production (39, 164). After production returns the first few eggs are abnormal in quality (65, 164). symptoms in chickens of all ages are similar hut in adult chickens the eyraptoms are less severe than those observed in chicks (164). Characteristic symptoms of sneezing, gasping, tracheal rales, and anorexia are observed (7, 18, 145, 164), The incubation period varies from 24 to 48 hours (8, 9 , 164), although in some cases symptoms have not been Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 oTDserred, for as long as six days after exposure (164). The symptoms persist for approximately one week (164)* Tlie îiistopathologic alterations resulting from infectious "bronch.ltis in chickens have "been descri"bed (74), These alterations consist of leucocytic infiltration and edema of the mucous membranes and submucosa of the trachea. The liver, spleen, and kidneys do not show any significant lesions which can be ascribed to the disease (74), Gross lesions include accumulation of mucus in the trachea and bronchi, a congestion of the lungs, and clouding of the air sac membranes (5, 8, 9, 36, 37, 47, 61, 74, 145, 164), The virus is found most abundantly in tracheal ex­ udates and in the lungs (94, 164) and can be transmitted "by infected tissue suspensions to cTnlckens by the intranasal and intratracheal routes (7) and "by the subcutaneous and intraperitoneal routes (39), The carrier problem has also been extensively studied. Chickens recovered from the disease may continue to discharge virus from the upper respiratory tract and serve as potential reservoirs of infection (75, 77, 94), Birds recovered from the disease develop a specific immunity to subsequent infection with the virus (7, 10, 36, 39, 78, 86), Birds that have become infected naturally or experimentally are capable of producing neutralizing antibodies (7 , 57, 130), naturally acquired, passive immunity in chicks has also been demonstrated (78, 86), Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. à 16 The serum neutralization test has been the only satisfactory serological test used to demonstrate the pre­ sence of neutralizing antibodies to infectious bronchitis virus (32, 57, 150, 164), The virus is incapable of agglu­ tinating red blood cells as is characteristic with Newcastle disease virus (Sj 16, 54, 76, 113) and fowl plague virus (113), Clinical history, lesions, symptoms, and Isolation of the virus with the production of characteristic pathologic alterations of embryonating chicken eggs may also be used as a method of diagnosis (5, 7, 18, 36, 39, 56, 74, 164)* The cultivation of infectious bronchitis virus on the chorio-allantoic membranes of embryonating chicken eggs was first reported by Eeaudette and Hudson (10). Early passages of the virus produced no noticeable changes in the embryo, although succeeding virus transfers produced definite mortal­ ity (10, 39, 40), The virus became increasingly virulent for the embryo and less virulent for the chicken. After the nine­ tieth passage the virus had lost all its virulence for the chicken and became incapable of inciting the production of antibodies, while becoming fatal for all inoculated embryos (10, 40), This egg-adapted virus is in general use today as the antigen in the serum neutralization test (33, 57, 130), The virus produces definite alterations in embryorating chicken eggs (llO), although Hitchner, et al (71) have shown similar changes in embryos inoculated with the B 1 strain of Newcastle disease virus. The changes produced Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1*7 "by infectious "bronchitis virus inoculated via the chorio­ allantoic cavity of emhryonating chicken eggs have "been dis­ cussed "by Delaplane (40), Fabricant (55), and Loomis, e^ al (110). Jones (85) observed that the highest chicken embryo mortality could be produced "by amniotic inoculation followed in decreasing order by chorio-allantoic cavity and chorio­ allantoic membrane inoculations. Low mortality followed yolk inoculations. Chorio-allantoic cavity inoculations were the most desirable because of their great convenience and simpli­ city. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 III KATEKIAIiS AKD EXFERIMEHTAl PROCEDURES Incoii^lete Icnowle&ge of certain physical and “biologi­ cal properties of antiviracl sera prompted this investigation. The study was directed toward an analysis of the changes in serum electrophoretic patterns and the changes in hD^Q îTIs In chickens after exposure to infectious "bronchitis virus. A. Experimental Birds Twenty-four Single Comb White leghorn cockerels ranging in age from five months, seven days to seven months, twenty-four days were obtained from the U. S, Regional Poultry Research laboratory. East Lansing, Michigan on November 15, 1951* These birds were originally used for genetic studies and were from healthy flocks of lines, some resistant and some susceptible to lymphomatosis# They were maintained in batteries under strict sanitary and quarantine conditions in previously unused isolation quarters. The susceptibility and resistance of the various lines to lymphomatosis were as follows: Line 7 - susceptible to lymphomatosis Line 9 - susceptible to lymphomatosis Line 10 - resistant to lymphomatosis Line 14 - resistant to lyuphomatosis Line 15 - susceptible to lymphomatosis Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. é 19 TTie "birds were dlTided into four groups as follows: TTae letter and number designations of the birds are those which were given to each bird by the U.S. Regional Poultry Research laboratory* Tlie number following the hyphen was given to each bird for convenience in Conducting the experiment. Group I - Control birds ÎI727S -1 line 15 M541B2 -2 line 10 M400D2 -4* line 9 M362V -5 line 7 M347B2 -6* line 7 Group II - Birds receiving a primary inoculation of infectious bronchitis virus M716G2 -7 line 15 M34702 -S line 7 ÎI735R -9 line 15 M349T -10 line 7 M538H -11 line 10 1Æ4320 -12 line 9 Group III - Birds receiving a primary inoculation of infectious bronchitis virus and challenged in the twelfth week after primary inoculation * Inoculated with a normal tissue suspension during the seven­ teenth week of this study* Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 M414Q -13 Line 9 M400U -14 Line 9 M559tr -15 Line 10 M727B2 -16 Line 15 M3610 -17 Line 7 M369ÎT -18 Line 7 M541G2 -19 îroup IVa Line 10 - Birds : trachea M346B -21 Line 7 M673V -22 Line 14 M645H -23 Line 14 Group IVb «• Birds subjected to a tracheal injury, virus not introduced M542A -24 Line 10 1Î333H -25 Line 7 M673U -26 Line 14 The birds were maintained on a commercial growing mash* containing not less than 20 per cent protein, not less than 3,50 per cent f a t , and not more than 5,50 per cent fiber. The mash was top-dressed two or three times weekly with a coTmnercial * Michigan State Growing Mash, manufactured by A, X. Zinn & 0o*, Battle Greek, Michigan Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. à 21 BcratcH feed* containing not less than 9 per cent protein, not less than 2 per cent fat, and not more than 5 per cent fiher. TITater was allowed lihitum, B. Virus Antigens Two different strains of infectious bronchitis virus were used. Strain V114D**, a chicken-embryo-adapted strain, was capable of killing all embryos inoculated via the allantoic cavity within 48 hours and was used as the antigen in the serum neutralization tests. This strain is capable of entering into specific combination with antibodies against infectious bron­ chitis virus (32, 33, 57, 130). It is prepared by inoculating nine-to 11-day— old embryonating chicken eggs with undiluted, virus-infected allantoic fluid via the allantoic cavity, in­ cubating for 24 to 30 hours, and harvesting the allantoic fluid of living embryos (32). Strain V R (Lot 285)*** was a chicken-propagated strain which was supplied as lyophilized, infected tracheal washings. This strain was used for all inoculations of the experimentallyinfected groups* In order to have a large volume of inoculum and to check the virulence of the virus for chickens this preparation was resuspended to volume with Difco nutrient broth. * Z i n n *8 Climax Scratch Peed, manufactured by A. %. Zinn & Co., Battle Creek, Michigan ** Strain V114D has been maintained in the laboratory for at least 150 passages in embryonating chicken eggs. *** Supplied by Dr. Henry Van Roekel, Department of Veterinary Science, University of Massachusetts Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2S The resulting suspension was instilled in 0.2 ml amounts intratracheally and 0.05 ml amounts intranaeally in three normal six-week-old chickens. Six-week-old chickens were used hecause it has been shown (78, 86) that naturally acquired, passive antibodies against infectious bronchitis virus are at a low or negligible level at this age and are incapable of protecting young birds against a subsequent inoculation with the virus* The tracheas were scraped with sterile cotton-tipped applicator sticks to facilitate mens intimate contact of the virus with the tracheal epithelium (32). Characteristic symp­ toms of infectious bronchitis (164) were observed in these birds 24 hours after inoculation. The birds were killed 72 hours after inoculation at a period in which syn^toms were at their greatest. The tracheas and lungs were harvested, pooled, ground with sand using a mortar and pestle, and made up to a 20 per cent suspension with Difco nutrient broth. The suspension was then treated with 10, 000 units each of penicillin and strepto* mycin per ml (32, 38) and centrifuged to sediment sand and tis­ sue debris. The supernatant fluid was stored at -40 C for further use. This process of inoculating six-week-old chickens with the chicken-propagated strain of infectious bronchitis virus was repeated and the resulting highly potent virus was used as the antigen for the experimentally-infected groups. One-tenth ml of this suspension was inoculated into five nine-day-old embryonating chicken eggs via the allantoic Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 cavity. The inoculum produced characteristic gross lesions of dwarfing and curling of the embryos (llO) by the third and fourth post-inoculâtion days. Hitchner, et a% (71) showed that the B 1 strain of Newcastle disease virus also may produce lesions, dwarfing, and curling which are similar to those produced by infectious bronchitis virus in the chicken embryo. Therefore, an henfâ-gglutination test was conducted on the harvested allantoic fluid to determine the presence or absence of Newcastle disease virus, lack of agglu­ tination of red blood cells indicated the absence of Newcastle disease virus. Abnormal embryos were not observed* C, Serum Neutralization Test (32) Nine-day-old embryonating chicken eggs were used in the serum neutralization tests. The eggs were maintained in an electric, rorced-draft incubator* at 99,5 F (88 F wet-bulb thermometer). The site for inoculation via the allantoic cavity was determined by trans-illuminâtion of the egg. An area devoid of large blood vessels, approximately two mm below the base of the air cell, and at a side opposite to the embryo was selected, A small hole was drilled through the shell without piercing the shell membrane by means of an electrically-driven drill. An additional hole was drilled directly above the air cell to serve as an àir vent in equilizlng the pressure produced by the injection of the inoculum into the egg. Both holes were painted with tincture of metaphen and the shell membrane above the air cell wa# pierced with a sterile teasing needle, *Model 252, manufactured by Jamesway îÆanufacturing Company Fort Atkinson, Wisconsin Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 Serial ten-fold dilutions of V114D virus-infected allantoic fluid were prepared with. Difco nutrient broth in the proportions of 0.5 ml of virus-infected allantoic fluid to 4.5 ml of diluent. Serum-virus mixtures were prepared separately by mixing equal portions of each virus dilution and test serum. The sera were usually used undiluted, al­ though Occassionally it was necessary to dilute sera IslO or more with Difco nutrient broth to give sufficient volume for the test and for the electrophoretic analysis. In some instances the sera completely neutralized undiluted virus and in order to establish an endpoint for calculation of the IDgQ N I b it was necessary to dilute the sera 1:10 or more* The virus dilutions were mixed with equal portions of Difco nutrient broth to obtain a quantitative estimation of the virus titer* Three-tenths ml of the virus dilutions were mixed with 0*3 ml of the serum preparations. All mixtures were incubated in an ice-bath at 4 C for 30 minutes* Dilution of sera in the serum neutralization test for infectious bronchitis does not significantly affect the ID^Q ÎTIs (131). Pive eggs were used per dilution and each egg re­ ceived an inoculum of 0.1 ml using a one-ml B-D Yale tuber­ culin syringe fitted with a 27 gauge, one-half inch needle* Tlie eggs for the quantitative virus titrations were inoculated last to make provisions for any possible deleterious effect of incubation of virus. Page (131) showed that the infectious bronchitis virus ID^g titer does not significantly up to 16 hours of incube.tion at 4 C. After ct]ange inoculation Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25 tlie boles were sealed witb melted paraffin and tbe eggs were reincubated and candled d a i l y for five days. Deatb of the embryos during the first 18 hours was attributed to trauma or other non-specific causes and these eggs were not in­ cluded in the calculations of the final results. The results of the serum and virus titrations were evaluated according to the 50 per cent endpoint formula of Reed and Muench (138) and expressed as the ID^ q . The difference between the reciprocal of the virus titer and the reciprocal of the serum titer was designated as the NX, The antilog of the XDgQ ÎJI represented the numbers of serum neutralizing doses. In cases where the sera were diluted the hDgo ^^.s calculated by multiplying the difference between the virus titer and the serum titer by the dilution factor* The procedure for the neutralization test for infect­ ious bronchitis has been thoroughly described by Cunningham (32) and should be consulted for further details. D, Electrophoresis All sera obtained were analyzed for protein nitrogen content using the macro-Kjeldahl technique (conversion factor, 6.25) previous to dilution for dialysis. A two per cent final serum protein concentration was used in all electrophoretic analyses. It he.s been shown that the protein concentration greatly affects the mobility (34, 101, 136) and relative percentage distribution of serum protein components (132). A high protein concentration may also produce boundary Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 disturbances (125). Sanders, et aJL (144) used a final two per cent concentration of serum protein and obtained excellent results in the resolution of oblcken serum and plasma compo­ nents • A standard Teronal buffer (104) of 0.1 ionic strength and pH 8.6 as measured with the glass electrode ( Beclcman, Model Gr pH meter) at 25 C was used in all dialyses and dilutions of serum samples. It was composed of 0.0152 M diethylbarbituric acid (2.797 gm/liter) eind 0.1 M monosodium salt of diethylbarbituric acid (20.6 gm/liter). This buffer has been shown to be excellent for use in the electrophoretic analysis of chicken serum (43, 144). Dialysis of serum samples previous to electrophoretic analysis is necessai'y to equilibrate the sample and buffer with respect to electrolytic conductivity. The stirring dialysis method of Reiner and Penichel (139) was used for each serum sample. Tlie diluted serum sample was placed in a bag made of seamless cellulose tubing* which was previously tested for leaks. It was dialyzed against a one-hundred-fold volume of buffer for two hours @,t room temper­ ature. These conditions gave excellent equilibration when electrolytic conductivities of both buffer and sample were measured at 0,5 G using a conductivity cell especially designed * Hojax-Visking Cellulose Sausage Casing (unknown pore size), manufactured by the Vi sking Corporation, Chicago, Illinois Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 for u»e with, the Model 38, Perkin-Elmer Tiselius Electrophoreois Apparatus* and an electrolytio conductivity bridge*** Conductivity measurements are necessary to determine equilibration in electrolytic conductivity since inadequate equilibration may result in boundary disturbances (104, 107, 133). The specific conductance of the dialyzed sample also is needed to determine potential gradients across the cell and mobilities of the individual serum components (104) The specific conductance (Ksp) of each sample was calculated as follows % Esp = ^ (l) R •where Kc is the conductivity cell constant and R is the meas­ ured resistance* The Kc must be determined for each cell since the value varies with the distance between the electrodes* This was determined as follows : Using standard 0.01 IT KCl, C.P. (0*7455 gm/lOOO gm double-distilled, COg -free water) the measured resistance of this solution in the conductivity cell was 1080 ohms (R) at 0 C, The specific conductance of this solution at 0 C is 0*0007728 mhos (^) (98), Substituting in the formula, Ecs j'R (II), a value of 0*8346 was obtained* Substituting the value for Ec * Manufactured by the Perkin-Elmer Corporation, TTorwalk, Conn* ** Leeds and îTorthrup #4960 Electrolytic Conductivity Bridge, manufactured by Leeds and ITorthrup Company, Philadelphia, Pa, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. d 28 in fonmila (I), and measuring the resistance dialyzed sample the specific conductance (R) of the (Ksp) can he determined* From previous studies in this laboratory equilibration of diluted serum samples and buffer can be attained in one and one-half hours at room temperature. Therefore, In order not to disturb the electrophoretic experiments, the samples were dialyzed for two hours at room temperature and placed at 4 C overnight• In this way the samples can be kept cold for use the following day. It was also observed that cold samples and cold electrophoresis cells minimize the formation of air bubbles in the cells during the cell-filling process* Air bubbles greatly disturb the boundaries formed in the cell during the experiments* Conductivities were then measured at the end of the day after all electrophoretic analyses were completed* The Model 38, Tiselius Eledtrophoresis Apparatus was used for the electrophoretic analyses of all serum samples* The apparatus has been described by Moore and TÎShite (127) but for a more detailed description and operational procedure the reader should consult the instruction manual (133)* The Toepler schlieren method has been adopted with certain improvement;s such as the schlieren lens system being replaced by two separate lenses in conjunction with a smaller aperture allowing for a greater reduction in the length of the optical path. A scanning modification (103) of the schlieren system is also employed but with a two-knife-edge diaphragm which gives two similar patterns* Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 A two-ml capacity electrophoresis cell haring a crossp sectional area of ahout 0*30 cm is used with the apparatus (28, 127, 133). A resumé of the operation of the instrument is g Iren as follows: Both sides of the ice compartment of the water-hath are filled with ice. The water-hath. is filled with cold water and the stirrer is turned on until the water-hath reaches a uniform temperature of 0*5 C hy the time the cell is filled* The circulating air pump is started to remove water condensate from the internal windows and len# surfaces hy passing air through a calcium sulphate drying tower. There are also two 75 watt heating elements near the external lens surfaces which heat the lenses and prevent moisture from col­ lecting on them* The cell is greased and assemhlsd according to instruc­ tions and kept at 4 0 for 12 to IB hours in order to have it cold for the following day's experiments* Tiie cell is placed in position in the cell holder, filled, disaligned, and the two buffer hottles are connected* The electrodes are inserted and buffer is added up to the level of the side arms of the assembly* This is done as rapidly as possible to prevent the samples and cell from becoming warm* The complete assembly is placed in the water-hath, clamped into position and the level-equilizing gate is raised. The electrodes are connected to the leads and 15 ml of cold, one-third satu­ rated KCl, C.P* is layered beneath the buffer in each buffer Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30 "bottle through the electrode capillaries hy means of a syringe fitted with a four-inch needle• The cover of the water-hath is replaced and more ice is added through the ioe-ohimney# "When ten^erature equi­ librium is reached (ten to 15 minutes) the level equilizlng gate is lowered gently and the cell channels are aligned hy means of the shifting rod. Buffer is gently flowed into the left-hand buffer bottle to bring the boundaries into view (approximately three ram) by moans of a mechanically-operated compensator*. The boundaries are originally behind the flange plates of the cell. By advancing the boundaries no more than three m m the current can be applied for a longer period of time and opti­ mum resolution of the serum components is usually obtained* The compensator is stopped and the beginning boundaries of the ascending and descending limbs are photographed. Either Kodak M or Kodak Process Panchromatic x 4r^ inch plates are used and are exposed for five seconds. The current is adjusted to six milliamperes and the voltage ifi maintained at approximately 107 volts, giving Do65 watt heat dissipâtion* During the electrophoretic run the separation and migration of the serum components are observed by means of the * Manufactured by the Perkin-Elmer Corporation, Korwalk, Conn, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 cylindrical lens and diagonal schlieren diaphragm attachment of Thovert (104, 105)• When the fastest component has migrated approximately two-thirds of the distance across the cell (7,600 seconds) the current is stopped and scanning photographs of the ascending and descending limbs are taken* These photo­ graphs are developed in total darkness in Kodak D-19 devel­ oper for five minutes and fixed in Kodak Acid Fixer for 15 minutes* After fixing the photographs are washed In running water for 30 minutes and allowed to diy. The pattern from the descending limb (104, 106, 107) is projected with a photographic enlarger and traced on paper at a linear enlargement of double size. The areas attributable to the various serum components are defined by the method of Tiselius and Kabat (158) in which ordinates are drawn from the lowest point between two components to the base line* Each area is measured with a precision disc, compen­ sating polar planlmeter* in arbitrary units and the relative concentrations in per cent are determined by dividing the area of each component by the area of the entire pattern, excluding the area of the epsilon-boundaiy* The epsilon-boundary is a buffer salt-protein interaction complex* Mobilities are calculated on the descending patterns as advocated by Longsworth and Ifeclnnes (107), The distance between the beginning boundary and the ordinate dividing the * Model 4236, manufactured by Eeuffel & Ssser Company, Kew York Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 respective area in half is used as a measure of the distance migrated hy each component* The formula of Longsworth (108) is used in the calcu­ lation of mobilities and is used as follows : u / (caa/sec/volt/cm x 1 0 . — â___— SL— i t m where d is the distance migrated in cm, q is the cross2 sectional area of the descending limb in cm , Ksp is the specific conductance of the sample, t is the time of migration in seconds, i is the current in amperes, and m is the en­ largement factor* The potential gradient (F) in volts/cm^ , which averaged 6,5 volts/cm^ is determined as follows: q Ksp where 1 is the current in amperes, q is the cross-sectional area of the descending limb in cm^, and Ksp is the specific conductance of the sample, E, Experimental Exposure All birds were bled by cardiac puncture one week after they were obtained. The sera collected were analyzed for anti­ bodies specific for Newcastle disease virus by means of the heiaagglutination-inhibition test (54) and for antibodies specific for infectious bronchitis virus by means of the serum neutral­ ization test (32). Infectious bronchitis virus is incapable of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 of agglutinating red blood cells and the serum neutralization test is the only method available at the present time for the detection of antibodies specific for infectious bronchitis virus. All sera tested were considered to be negative according to established standards (33, 54, 57). On January 19, 1952 the investigation was begun. All birds were fasted for 18 to 24 hours prior to bleeding to de­ crease the quantity of serum lipids (134, 144). Zeldis, et bX (17 5) showed that lipids become additive in the beta-globulin of human plasma and in the alpha-globulin in dog plasma. Birds in Groups I, II, and III were bled by cardiac puncture. Birds in Groups II and III were inoculated with the chickenpropagated strain of infectious bronchitis virus by depositing with a syringe, 0.2 ml in the upper trachea and 0*05 ml into the nares. A cotton-tipped applicator stick was used to scarify the lumen of the trachea to ensure more intimate contact of the virus with, the epithelium. Characteristic symptoms of infectious bronchitis (164) appeared in all inoculated birds within 24 to 36 hours after inoculation. The symptoms persisted for eight days and consisted of dyspnea, sneezing, tracheal rales, nasal discharge, and a slight anorexia. Birds in Group II were bled immediately prior to in­ oculation and then at one, two, three, four, six, eight, ten, 12, 16, and 20 week intervals. Birds in Group III were also bled Immediately prior to inoculation and then at one, two, three, four, six, eight, ten, and 12 week inteorvals. At the 4 Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. 34 twelfth, week the hirds in G-roup III were challenged with the chicken-propagated strain of infectious bronchitis virus and hied again at one, three, five, and seven weeks after chal­ lenge • The control hirds in Group I were hied according to the schedule of Group II, hut unfortunately many of them died due to internal hemorrliage caused hy the experimental bleedings. Bird IT727S -1 was hied at four, eight, IS, and 17 week intervals. Birds M541B2 -2 and 1Ï365V -5 were bled at the first bleeding period as hirds in Groups II and III and then at one, two, and three week intervals. Bird M400D2-4 was also bled at the first experimental bleeding period as birds in Groups II and III and then at one, two, three, four, eight, 12, and 17 week intervals. In order to determine if inoculations of normal tissue suspensions could cause changes in serum electrophoretic patterns or changes in IDg^ FIs bird M400D2 -4 wa.s inoculated with a normal lung and tracheal suspension via the intratra.cheal and intra­ nasal routes during the seventeenth week and hied one and two weeks after inoculation. Bird ÎÎ347B2 -6 was also hied at the first experimental period as birds in Groups IT and III and then at one, two, three, four, eight, and 17 week intervals. At the seventeenth week this bird was also inoculated with a normal lung and tracheal suspension and bled at one, two, and three weeks after inoculation. Birds of Group IVa were bled on June 14, 1952 and inoculated with a nonrfâ-1 lung and tracheal suspension followed Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 by a Bcarification of the trachea using a cotton-tipped applicator stick. The birds were bled at one, two, and three week intervals after inoculation. It has been shown that normal homologous tissue inoculations in rabbits produce microscopic manifestations of inflamnation (118, 119). Inflammation caused by intradermal injections of turpentine or other irritants can alter the serum electrophoretic patterns (63, 64). It was thought that possibly a relationship existed between inflammation, inoculations of normal tissues, and changes in serum electrophoretic patterns. Traumatic injury has also been shown to alter the, serum electrophoretic pattern (22, 62, 124, 125). Therefore, birds in Group IVb were bled and subjected to a tracheal injury by scarification using a cotton-tipped applicator stick* These birds were then bled one, two, and three weeks after injury. The presence of Newcastle disease virus or infectious bronchitis virus in the normal tissue suspension was eliminated by inoculating 0.1 ml into nine-day-old embryonating chicken eggs via the allantoic cavity. The embryos were candled daily for five days and the allantoic fluid was harvested. This was repeated for four passages. Ho alterations in the embiyos were observed. The allantoic fluid also did not agglutinate red blood cells* Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 IV KSSULTS A. Group I 1* Electroplioretic Analyses Electroplioretic analyses of sera from this control group showed varied results. Bird 15727S -1 was hied at the four, eight, 12, and 17 week interval, Keither the relative percentage distribution of the serum components nor the alhumin/glohulin ratio changed significantly during this period. (Table 1, Figure I) Bird M541B2 -2 was bled at the first experimental period and then at the one, two, and three week interval. Unfortunately the bird died at the third week because of internal hemorrhage due to cardiac puncture. An increase in the relative percentage of albumin was observed at the first and second weeks. There was a definite decrease in albumin at the third week. Gamma-globulin levels decreased at the first week interval and increased steadily up to and including the third week. Albumin/globulin ratios followed the same general trend as the albumin values. Alpha 1-globulin values changed slightly as did the beta-globulin values. Alpha 2globulin values decreased at the first week interval and steadily increased up to and including the third week. (Table 2) Bird 3V562V -5 was bled at the first experiments,! period and then at the one, two, three, and four week interval. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1 Bird 13727 S -1 Group I-Control Electropboretic /uialyses Period Relative Per Gent Serum Gonponent Weeks Per Cent Globulins Albumin Serum Alpha 1- Alpha 2- Beta- Garama- Prote i] 4 50.7 5.8 6.2 10.5 26.8 1.03 3.43 8 51.6 6,1 5.2 11.7 25.4 1.07 3.43 12 48.9 8.9 6.8 11.4 24.0 0.96 3.43 17 50.8 6.5 8.7 10.8 23.2 1.03 3.60 A/G Albumin/Globulin Ratio Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure I Bird ÎS727S-1 Group I-Control ElectropTaoretic Pat t erne A O t WEEKS A ASCENDING LIMB D DESCENDING LIMB 17 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure I (cont #) A D IZ WEEKS A D 17 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TaTale 2 Bird M541B2 -2 Group I-Control IfSlectroplioretie Analyses Period Helatlye Per Cent ;3erum Component A/G Weeks * Per Ceni Serum AlTDtimin Alpha 1- Alpha 2- Beta- Garnma- 42,3 10.6 9.0 8.5 29.6 47.2 , 12.4 4.7 10.3 25.4 46.7 9.6 5.3 9.6 28.8 0.86 4.04 39.2 12.5 6,0 10.1 32,2 0.65 3.78 Protein 0.73 3,73 3.73 First “bleeding period corresponding to pre-e%posure bleeding period of infected bird Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 This 'bird died at the fourth week period because of internal hemorrhage due to cardiac puncture. A slight increase in the relative percentage of albumin was observed at the second week interval. Albumin decreased after this period up to and including the fourth week* The gamraa-globulin levels de­ creased at the first week but steadily increased up to and including the fourth week. The albumin/globulin ratios followed the same general trend as the albumin values. Alpha 1-globulin values increased at the first week, decreased at the second week, and increased again at the third and fourth weeks. Alpha 2-globulin did not change significantly during the experi­ mental period. Beta-globulin levels showed a slight increase at, the end of the experiment. (Table 3, Figure II) Equal portions of sera from birds K400D2 -4 and MS4732 -6 were pooled and analyzed electrophoretically as one sample at the first experimental period and then at the one, two, three, four, and 12 week interval. A slight increase in the relative percentage of albumin was observed at the first week interval. After this period there was a general decrease in albumin up to and including the twelfth week. Garama-globulin values decreased at the first week but generally increased after this period up to and including the twelfth week. Alpha 1-globulin levels did not significantly change up to the fourth week but decreased greatly at the twelfth week. Alpha 2-globulin levels steadily increased vhile betaglobulin values did not change appreciably. Albumin/globulin ratios varied slightly except that there was a significa.nt Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta"ble 3 Bird im62Y -5 Groiip I-Gontrol Electroplioretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Albumin Alpba 1- Alpba 2- Per Cent Serum Beta- Gamma- Protein 0* 45 •6 7.7 5.5 8.8 32.4 0.84 3.91 1 45.7 12.6 6.0 7.5 28.2 0.84 3.60 2 47.5 4.4 6.8 9.7 31.6 0.91 3.51 3 40.5 9.6 5.4 10.0 34.5 0.68 4.04 4 39.9 9.5 5.3 10.1 35.2 0.66 3.86 * Pirst "bleeding period corresponding to pre-exposure "bleeding period of infected bird Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure II Bird 38362V -5 Group I-Control Electroph-oretic Patterns D O WEEKS FIRST BLEEDING PERIOD I WEEK Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure II {cotitm) J A D Z WEEKS A D 5 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure lï (cont.) A D 4 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 increase at the first week interval following the change in alhnmin. (Tahle 4) At the eighth and seventeenth weeks sera from the ahove hirds were analyzed individually. Bird M400D2 -4 showed a low alhumin value and a high gamma-glohulin value. The other globulins were apparently unchanged. (Table 5) Bird M347B2 -6 showed a significant increase in al­ bumin and a decrease in gamma-globulin at the seventeenth week interval as compared with results obtained at the eighth week. The other serum globulins did not change appreciably. (Table 6, Bigure III) In order to determine if normal tissue suspensions could alter the serum electrophoretic patterns or the IDgg NIs, these two birds were inoculated with s. normal lung and tracheal suspension ©,t the seventeenth week. Bird M400D2 -4 and bird M347B2 “6 were bled one and two weeks after inoculation. (Table 5) Bird M347B2 -6 was also bled three weeks after inoculation. (Table 6, Figure III) Bird M400D2 -4 showed a slight decrease in albumin and a rise in garama-globulin. Alpha 1-globulin values increased appreciably. Alpha 2-globulin and beta-globulin levels did not change significantly. (Table 5) Bird M347B2 -6 showed a great reduction in albumin one week after inoculation. There was also a slight increase in gamma-globulin which decreased slightly up to and including the third week after inoculation. The relative percentage of alpha 1-globulin increased slightly throughout this period. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. T a b le 4 Pool of Birds M400D2 -4 and M347B2 -6 Group I-Control Electropboretio Analyses Period - R e l a M w e Per Gent Serum Component Weeks A/G Globulins Albumin Alpba 1- Alpha 2- Per Cent Serum Beta- Gamma- Protein 0* 40.7 10.6 3.7 11.2 33.8 0.69 3.78 1 43.5 11.1 4.7 9.3 31.4 0.77 3.91 2 39.5 9.6 5.0 10.0 35.6 0.66 4.13 3 38.9 8.2 7.7 9.6 35,6 0.64 4.21 4 41.1 9.6 6.9 9.6 32.8 0.70 3.86 12 38.2 5.8 9.0 12.2 34.8 0.62 4.13 * First bleeding period corresponding to pre-exposure bleeding period of infected bird Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tatle 5 Bird ÎI400D2 -4 Group I-Control EleotropTioretic Analyses Period Relative Per Cent Serum Coarponent Weeks A/G Globulins_______ Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- * Per Gent Prote i] 8 35.9 11.5 4.8 13.4 34.4 0.54 4.21 17* 31.9 14.7 7.8 10.8 34.8 0.42 4.65 1 29.1 16.8 8.2 10.2 35.7 0.41 4.30 2 28.9 13.6 9.7 9.7 38.1 0.40 4.65 Inoculated with normal tissue suspension Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Alplia 2-glo"bulin increased and "beta-globulin renmlned approxiîïKitely at tTie same level as the pre-inoculât ion value* (Tahle 6, Figure III) There was wide variation between different birds in the relative percentage distribution of the individual serum oomponents even at the first bleeding period* Sanders, et al (144), beutsch, et al (43), and Moore (122) found tloe same to be the case in sera of normal chickens* The sera obtained at the first experimental period from the birds in this control group showed fair correlation with the values obtained by Sanders, ^ (144). Gamma­ globulin levels were lower in Sanders* study since 15- to 18week-old chickens were used. It has been shown that garamaglobulin levels in chickens increase with maturity (17, 46, 144). The electrophoretic mobility values of the individual serum consonants of this group and of the other groups closely approximate the values obtained by Sanders, et al (144), when veronal buffer at pH 8.6, 0*1 ionic strength (104) was used* The following average values were found (cm/sec/volt/cm x 10**'^ ) which were calculated from the patterns of the descending limb: albumin 6*0 alpha 1-globulin 5,2 alpha 2-globulin 4.4 beta-globulin 3*6 gamma-globulin 2*0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta”ble 6 Bird 1Æ347B2 -6 Gr oup I-Gont roi Electroplioretic Analyses Period Relative Per Gent Serum Component A/G Globulins Weeks Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- * Per Gent Prote in 8 39.0 10.5 6.9 8.3 35.3 0.64 3.51 17* 43.1 9.0 7.6 10.0 30.3 0.76 3.51 1 33.8 11.3 10.8 10.8 33.3 0.51 3.95 2 41.5 9.3 8.2 9.8 31.2 0.71 4.13 3 41.5 11.0 8.5 10.0 29.0 0.71 3.95 Inoculated with normal tissue suspension Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure III Bird ia347B2 -6 Group I-Control Electroph-oretic Patterns D 8 WEEKS 17 WEEKS fw n ru iffrrh u m i w arm ai Ti?L 5 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 A eignifleant difference in mobility values of serum compo­ nents of inoculated birds and control birds was not observed. The results obtained in this group clearly demonstrate tbat frequent bleeding changes the serum electrophoretic pat­ tern by reversing the albumin/globulin ratio. Bird M727S -1, ■which was bled at four or more weekly intervals did not show this change (Table 1# Figure I) while the other birds in the control group showed a marked change and a decrease in the p> albumin/gl obul in ratio when they were bled at weekly intervals. The change in per cent serum protein did not appear, to be significantly important during the experiment. 2. lethal Bose gQ Neutralization Indices The serum H>gQ NIs of the birds in this group showed variations between periods but remained below the accepted normal values (33, 57). (Tables 7, 8, 9, 10, 11, 12, 13, Figures TV, V, VI, VII, VIII, IX) Page has also observed this type of variation (130). Hemagglutination-inhibition titers for Newcastle disease were negative at each period. I^on examination at necropsy all birds were found to be normal except bird M400B2 —4 which showed a non-specific enteritis. Observations for coccidia and attempts at bacterial isolation proved to be negative. B. Group II 1. Electrophoretic Analyses Electrophoretic analyses of sera from birds of this group showed the same general trend as the birds in Group I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A Table 7 Bird If/27 8 -1 Group I-Control Serum Neutralization Tests Period Weeks Virus Dilutions Virus# Titer 1 Serum 10"^ 10"^ 10-5 10-® 10-’^ Titer# NX 4,38 1,12 4 5.50 5 5 4 0 8 6.32* 4 5 5 1 0 4.52 1.80 12 6,50* j 5 4 3 0 5.00 1.50 17 6.32* 5 5 5 3 1 5.32 1.00 These footnotes apply to serum neutralization test tables for Groups I, II, and III: * Serum diluted 1:10 ** Serum diluted 1:100 # Reciprocal of negative exponent log base 10 t One embryo out of five inoculated died due to trauma ## Number of embryos dead out of five inoculated per dilution, Deatb due to virus. NT Neut rali zat i on index Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. S M f h S o ® u u «0 O o K Q \9 T O S 3 8 3 OI 3 S V 8 % n J - N 3 KIOd5<3 X3GNI K i o i i v z n v y i n 3 N ° ^ o i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O o‘ Table B Bird IX41B2 ~2 Group I-Control Serum Neutralization TeAt8 Period Y/eeks * Virus Dilutions Virus# Titer 10-2 10-2 10"^ 10’^ io“ ## Serum io“'^ Titer# NI Of 6.54 5 5 5 5 0 5.50 1.04 1 6.50 5 5 4 3 0 5.00 1.50 2 5.63 5 5 5 3 1 5.32 0.31 5 6.38* 5 5 4 2 4.68 1.70 First bleeding period corresponding to pre-exposure bleeding period of infected bird Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. s LU UJ O Q P q 9. Q Q o 0\ 3 S V Q DOT J . N 3 M 0 d X 3 X3QNI Noavznm naN “ on Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Æ Table 9 Bird ÎI362V -5 Group I-Gontrol Serum Feutraiiza-tion Tests Period Weeks * Virus# Titer Virus Dilutions 10-^ 10 # Serum. 10"^ 10-^ 10“^ 10-7 Titer# FI 0* 6.54 4$ 5 5 4 2 5.68 0.86 1 6.50 5 5 5 4 0 5.38 1.12 2 5.63 5 4 3 0 0 4.00 1.63 3 6.38* 5 5 4 1 4.50 1.88 4 5.50* 5 5 4 3 5.00 0.50 0 First bleeding period corresponding to pre-exposure bleeding of infected bird Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. W) it: 111 111 5 o Q Q 01 aswa Do-i j.NaNOdX 3 X3QNI NoiivznvbinaN Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta-ble 10 Pool of Birds H400B2 -4 and I347B2 -6 Group I-Control Serum neutralization Tests Period Weeks Virus# Titer Virus Dilutions Serum 10“^ 10-3 10“^ 10-5 10“^ 10“'^ ## Titer# m 1 6.50 5 5 5 1 0 4.63 1.87 2 5,63 5 5 4 1 0 4,50 1.13 3 6.38* 5 5 5 1 4.63 1.75 4 5.50* 5 5 2 0 3.83 1.67 12 6.50* 5 5 4 3 5.16 1.34 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o o « I 82 0» lil III o O Q et 01 a S V Q 001 J . N 3 N 0 d X 3 X30NI N o iiv z n v ^ in B N ® *a i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O Q Ta"ble 11 Bird M400D2 -4 Group I-Control Serum Neutralization Tests Period Weeks * Virus Dilutions Virus# Titer Serum 10-& 10-3 10-^ 10"^ 10"G 10-'^ Titer# NI 8 6.32* 5 5 4 3 0 5.00 1.32 17* 6.32* 5 5 5 5 1 5.63 0.69 1. 6.93* 5 5 5 4 2 5.68 1.15 2 7.17* 5 5 5 4 2 5.68 1.49 Inoculated witN normal tissue suspension Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o o. <3 O Q Q O O Q O OI aSVQ OOl J.N3N0dXa X30NI N O iiv z n v m n a N “ J541B2- 2 M400D2 -4 1I362V -5 M347B2 -6 1 4 Pool M400D2 -4 13347B2 -6 1.04 0.86 0.91 1.50 1.12 1.87 0.31 1.63 1.13 1.70 1.88 1.75 0.50 1.67 1.12 6 8 1.80 1.32 0.64 10 12 1.50 1.34 16 17* 0.69 1.82 1 1.15 1.50 2 1.49 1.39 1.00 3 *# 0.49 Pirst bleeding period corresponding to pre-exposure bleeding of infected bird * Inoculated witb norrml tissue suspension Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 wTiich were “bled at weekly intervals. The change varied de­ pending upon the pre-exposure levels of the individual serujn components. Bird TÆ347C2 -8 was hied immediately prior to inoc­ ulation and then at the one, two, three, four, six, eight, ten, 12, 16, and 20 week interval. The relative percentage of alhumin decreased greatly one week after inoculation with infectious bronchitis virus while the gamma-glohulin level increased. From this point on, and up to and including the twentieth week, the alhumin generally increased and the gammaglohulin decreased. There were varying changes in the other globulins with a slight decrease in the alpha 1-glohulin. The alhumin/glohulin ratio followed the same general trend as the change in alhumin. (Table 14, Figure X) Bird 1Î349T -10 was bled according to the schedule of bird M347G2 -8 with essentially the same results although the albumin level was lower at the pre-exposure period. The final albumin level was higher than the pre-exposure level while the gamma-globulin level was slightly lower than the pre-exposure value. (Table 15), Bird M538H -11 was bled immediately prior to inoc­ ulation and then at the one, two, three, four, and six week interval. It died at the six week period from Internal hemor­ rhage due to cardiac puncture. Electrophoretic analysis of preexposure serum samples from this bird showed an extrenely low albumin level and a high gamma-globulin level. This trend increased even more following the period after inoculation with virus. (Table 16) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta"ble 14 Bird ÎÆ347G2 -8 Group 11-Primary Exposure Electropb-oretic Analyses Period Relative Per Cent Serum Component ■^eeks A/G Per Cent Serum Glo'bulins Alhumin Alpha 1- Alpha 2— Beta— Gamma- Prote i3 0* 43.3 11.2 4.8 9.1 31.6 0.77 4.17 1 32.4 13.0 8.5 9.7 36.4 0.48 3.86 2 36.6 9.5 4.7 10.4 38.8 0.58 4.83 5 36,7 10.3 6.4 9.4 37.2 0.58 4.65 4 40.1 10.3 4.8 9.5 35.3 0.67 4.21 6 36.9 9.3 4.4 8.9 40.5 0.59 4.48 8 39.9 10.8 3.9 10.8 34.6 0.66 4.21 10 40.4 12.2 5.9 10.1 31.4 0.68 4.39 12 42.0 9.3 6.5 10.5 31.5 0.73 4.21 16 42.5 9.9 6.4 11.0 30.2 0.74 5.00 20 42.1 9.5 6.8 10.0 31.6 0.73 4.21 * Pre-exposure "bleeding, inoculated with, virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure X Bird M347C2 -8 Group II-Primary Exposure Electrophoretic Patterns A D O WEEKS PRE-EXPOSURE A D I WEEK Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plgure X (eont•) Z WEEK A D 3 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure X (oont.) A 0 4-WEEKS 6 WEEKS 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F ig u r e X ( c e n t . ) A D 8 WEEKS A 0 10WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure X (cont«) A D liWEEKS A -D 16 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pigure X (cont.) A D 10 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 15 Bird M349T -10 Group 11-Primary Exposure Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Glo'bulins Per Cent Serum Al'bumin Alpha 1- Alpha 2- Beta- Gamma- Prote it 0* 38.2 10.9 8.1 8,1 34.7 0.62 4.30 1 26.7 12.3 7.7 11.8 41.5 0.36 4.83 2 32.6 10,9 7.5 9.0 40.0 0.48 5.00 3 34.1 12.6 5,3 8.1 39.9 0.52 4.65 4 39.5 7.5 5.8 9.8 37.4 0.65 4.39 6 37.1 9.7 7.8 8.3 37.1 0.59 3.86 8 39.4 10.5 7.5 11.0 31.6 0.65 4.04 10 40.6 10.8 5.4 11.3 31.9 0.68 4.13 12 41.3 9.0 31.7 0.71 3.86 16 43.4 8.4 30.0 0.77 4.13 20 42.2 8,6 31.0 0.73 3.16 * 18.0 7.4 10.8 18.2 Primary "bleeding, pre-exposure, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 16 Bird M538H -11 Group Il-Prlmary Exposure Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Serum Albumin Alpha 1- Alpha 8- Beta- Gamma- * Per Cent Prote in 0* 31,4 10.5 8.7 11.6 37.8 0.46 5.08 1 31,3 8,6 9.6 14.7 35.8 0.46 4.83 2 18.1 14.4 6.9 13.4 47.2 0.22 5,35 3 23,8 13.6 7.7 11.1 43.8 0.31 4.56 4 25.8 14.4 6.1 11.8 41o9 0.35 5.00 6 25,6 11.6 5.8 11.6 45.4 0.34 4.65 Pre-exposure bleeding, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 42 Equal portions of sera from birds I3716G2 -7, M735R -9, and M4320 -12 were pooled and analyzed as an individual sangle Immediately prior to inoculation and then at the one, two, and three week interval. (Table 17 ) At the third week period bird Î5716G2 -7 died because of hemorrhage due to cardiac puncture. Therefore, samples from birds M735R -9 and M4320 -12 were pooled and analyzed at the four, six eight, ten, 12, 16, and 20 week interval, (Table 18) Individual samples coTTçprising this pool were also analyzed separately as follows: bird l'f735H -9- six, eight, ten, (Table 19) bird 1T4320 -12 - 16, and 20 week interval; ten, 16, and 20 week interval, (Table 20). There wzas a decrease in the relative percentage of albumin at the first and second weeks after inoculation with the virus. After this period a general increase in the albumin followed until the end of the experimental period when a general maximum was reached. Oamma-globulln levels reached a maximum at the second week after exposure and decreased gen­ erally until a minimum v^as reached at the end of the experiment. The other globulins showed varying changes although alpha 1globulin increased at the first week and remained at an ele­ vated level throughout the experiment. The values obtained in pre-exposure serum electro­ phoretic analyses closely approximated the results obtained by Sanders, et aa (144), although the gatnma-globulin values were lower in Sanders* study. Birds M349T —10 (Table 15) and M538H -11 (Table 16) showed low pre-exposure albumin values. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 17 Pool of Birds 1Ï716G2 -7, Ï/1735R -9, and M4320 -12 Group II-Primary Ibqposure Electropboretio Analyse© Period Relative Per Cent Serum Coirponent Weeks A/G Globulins Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- * Per Cent Protein 0* 45.3 9.5 7.9 8.9 28.4 0.83 3.47 1 34.0 16.7 7.1 13.2 29.0 0.52 3.95 2 37.7 10.8 6.4 10.3 34.8 0.61 4.04 3 40#0 9,8 5.6 11.2 33.4 0.67 4.04 Pre-exposure bleeding, inoculated witb virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 18 Pool of Birds M735H -9 and M4320 -12 Group Il-Priinary Exposure Electropîioretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote 4 40.0 12.2 6,6 9.6 31.6 0.67 3.69 6 43*6 7.3 6.5 11.6 31.0 0.77 3.51 8 41.2 14.0 5.1 11.7 28.0 0.70 3.69 10 42.0 13.0 8.0 11.0 26.0 0.72 3.43 12 44.5 9.3 6.7 9.9 29.6 0.80 3.78 16 42,4 11.9 4.2 10.1 31.4 0.74 4.39 20 44.4 12.3 6.6 9.2 27.6 0.80 3.95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tatle 19 Bird 1Î735R -9 Group Il-Primary Exposure ElectropTioretic Analyses Period Relative Per Cent Serum Component Weeks A/G Glo'bulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote il 6 43.2 11.7 5.4 9.2 30.5 0.76 3.07 8 44.7 7.8 6.5 12.9 28.1 0.81 3.07 10 49.6 12.4 5.2 11.4 21.4 0.98 2.89 16 50.6 10.0 6.3 11.2 21.9 1.04 3.78 20 52.8 9.6 6.5 10.5 20.6 1.12 3.34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta"ble 20 Bird 114320 -12 Group Tl-Primary Exposure Electroplioretic Analyses Period Relative Per Cent Serum Conç>onent Weeks A/G Glo'bulins Serum Albumin Alpha 1- Alpha 2- Beta- Gamma— 10 37.8 16 38.7 20 42.9 24.2 14.1 6.5 21.2 Per Cent Protein 7.6 30.4 0.61 3,86 8.5 32.2 0.63 4.13 7.6 28.3 0.75 4.13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 Results obtained, on the relative percentage distrihution of serum components of pooled serum samples and samples comprising the pools were favorably comparable. In some in­ stances there were slight differences which were not signif­ icant • A study of the changes of per cent serum protein did not reveal any significant findings, 2, Lethal L o s b ^q Neutralization Indices The serum LD^Q NIs of this group showed a general pattern as follows: There was a slight increase in the ND50 NIs of all sera between one and two weeks after inoculation. After this period a marked increase occurred. The maximum LB50 were reached between the sixth and tenth weeks, decreasing after this period until the end of the experiment, (Tables 21, 22, 23, 24, 25, 26, 27, 28, Figures XI, XII, XIII, XIV, XV, XVI, XVIl) Variations in individual neutralizing antibody responses were also observed (130, 131). Pre-exposure LD5Q NIs were considered normal according to accepted standards (33, 57), Upon examination at necropsy, all birds were normal except birds 1Î349T -10, and M538H -11 which showed visceral lymphomatosis and neural lymphomatosis, respectively, Sanders, at al (144) observed a new component, which was designated as the L component, in sera from chickens inoculated with leucosis-containing tissue suspensions. The L component was not observed in the serum electrophoretic patterns of birds showing manifestations of lymphomatosis in this stu(%r. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 21 Bird M347C2 -8 Group II-Primary Exposure Serum îTeutralization Tests Period Weeks * Virus Dilutions Virus# Titer Serum 10® 10”^ 10-s 10"^ 10"4 10-5 10-5 ## Titer# m 0* 6.54 4# 5 5 4 2 5.68 0.86 1 6,50 5 5 5 1 0 4.63 1.87 2 6.32* 5 4 4 1 0 3.37 2,95 3 6.32* 5 4 2 0 2.68 3.64 4 5,50* 5 4 0 0 0* 1.38 4.12 6 6.72* 5 4 2 0 1 1.84 4.88 8 6.32* 5 3 1 0 0 1.32 5.00 10 6.17* 3 2 0 0 0 0.50 5.67 12 6.50* 5 5 3 0 0 2,17 4.33 16 6.68* 5 5 1 0 0 2.63 4.05 20 6.83* 5 3 3 0 0 2.78 4.05 5 5 Pre-exposure bleeding, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. g s & $ % a OI asve 00*1 jjgsNOdxa X30NI NoiivznvbinaN “ ai Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aim Table 22 Bird 349T -10 Group II-Primary Exposure Serum neutralization Tests Period * Virus Dilutions Virus# Serum loO 10“^ 10~2 10“^ 10“'^ 10-5 10-5 # Weeks Titer 0* 6.54 5 5 4 5 1 5.52 1.02 1 6.50 5 5 5 4 1 5.50 1.00 2 6,32* 5 5 5 1 0 3.63 2.69 3 6.32* 5 5 3 1# 3.33 2.99 4 7.32* 5 5 5 4 3 4.00 3.32 6 6.72* 4 5 3 1 0 2.16 4.56 8 6.32* 5 5 0 2 0# 1.70 4.62 10 6.17* 5 2 1 0$ 0 1.00 5.17 12 6,50* 5 5 3 1 0 2.32 4.18 16 6.68* 5 5 1 1 2.75 3.93 20 6.68* 5 4 3 2 3.33 3. 35 5 5 5 0 0 Pre-exposure bleeding, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Titer# ni ul Ul CL O o o o Q S Ol 3SW9 901 J>iaNOdX3 x a o N i NOlivznvbinaN ®®ai Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. O O -H .O ce CL I (il O Q O Q S 01 aswg 901 xNSNodxa X3QNI Noiivznviun3N Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o O Table 23 Bird l'ISSSH -11 Group II-Primary Exposure Serum Neutralization Tests Period Weeks * Virus# Titer Virus Dilutions Serum 10"^ ID” 2 10-® 10-4 10-5 10-5 ## Titer# m 0* 6«54 5 5 5 3 0 5.17 1.37 1 6,50 5 5 4 2 0 4.68 1.82 2 5.63 3 0 1 0 0 2.31 3.32 3 6.32* 5 3 1 0 2.32 4,00 4 7.32* 5 4* 5 2 0 2.83 4.49 6 6.72* 5 5 2 1 0 1.00 5.72 5 0 Pre-exposure bleeding, inoculated witb. virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in ÜJ CL bJ Ul $ O Ol 3SV9 ocn J.NaNOdX3 X3QNI N0iivznv^in3N °*ai Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ta"ble 24 Pool of Birds M716G2 -7, 1J735R -9, and M4320 -12 Group II-Primary Exposure Serum Neutralization Tests Period * Virus Dilutions Virus# 10"^ 10"3 10“4 10“5 10“6 ## Serum Titer# NI 0 5.38 1.16 1 0 4.50 2.00 5 1 0 3.63 2.69 5 1$ 3.67 2.65 Weeks Titer 0* 6.54 5 5 5 4 1 6.50 5 5 4 2 6.32* 5 5 3 6.32* 5 5 Pre-exposure bleeding, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m OL %n LU m $ s S o o. S O OI 3 S W 0 D O T Q O J.W3NOdX3 x a a N i M0liVZnV^lfl3N Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O Q Table 25 Pool of Birds 11735R -9 and M4320 -12 Group II-Primary Exposure Serum ÏTeutralization Tests Period Weeks Virus Dilutions Virus# Titer Serum 10® 10"^ 10"2 10"^ 10"^ 10-^ 10-6 Titex# m 4.53 2.79 0 3.17 3.55 1 1 1.20 5.12 3 0 0 2.00 4.17 5 5 1 0 2.63 3.87 5 4 2 1 2.84 3.84 5 4 3 0 3.00 3,68 4 7.32* 5 5 4# 5 4 6 6.72* 5 5 5 3 8 6,32* 5 5 2 10 6,17* 5 4 12 6.50* 5 16 6.68* 20 6.68* 5 5 1# 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ul H caco1 «n O CL X UJ to S o z < o1 M < in w R (T S 3' O c. in IL 1 o 2 5 Lo Id liS CC t=t 3 -o CL u. 3 o -I o CC o o a -T" nr o K 2 q -T" q $ O H 01 aSVQ ocn JLN3N0d)@ X3QNI NoiivznvainaN ®‘oi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. "T" o s B» Table 26 Bird H735R -9 Group II-Primary Exposure Serum Neutralization Tests Period Weeks Virus Dilutions Virus# Titer Serum 10“^ 10“^ 10-3 10-4 10-5 10-5 10-7 # Titer# NI 6 6.72* 5 5 5 3 2 3.50 3.22 8 6.32* 5 5 5 5 0 3.50 2.82 10 6.17* 5 5 5 4 1 3.50 2.67 16 6.68* 5 4 3 0 4.00 2.68 20 6.68* 5 4 3 1 4.16 2.52 5 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■8 S % S w 0\ à < I CC II lo K> rs. 5 s Û s U1 fill i7 I cC •CO ) § O "T" — r- o K Q q H 5 o OI 3SV9 301 J.N3N0ciX3 X3QNI N o i ± w i m i n 3 N “ 0 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o d Table 27 Bird K4320 -18 Group II-Primary Exposure Serum Neutralization Tests Period Weeks Titer Serum Virus Dilutions Virus# 10^ 10”^ 10"^' 10"^ 10"' 10"5 ## Titer# ITI 10 6,17* 5 5 3 1 0 2,32 3,85 16 6 ,68* 5 3 3 0 0 2,78 3,90 20 6 ,68 * 5 5 5 1 0 2.63 4,05 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. .o ul cacr 3 ifi O o. 5 X d I I lu Z 5 •2 Û (Ï IL. • £ 01 £ w CL 3 s L9 -r o K -T" nr O Q : $ OI 3S09 ocn lN3NOdX3 X30NI N o a v z n v a i n 3 N “ o i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 28 Summation of U) 50 n i 8 Group II-Primary Exposure Period We eks ™50 Pools M735RM716G2-7 ¥4320-: ¥735R-9 ¥34702-8 M755R-9 M349T-10 M538H-11 ¥4320-12 ¥4320-12 Of 0.86 1.02 1.37 1.16 1 1.87 1.00 1.82 2.00 2 2.95 2.69 3.32 2.69 3 3.64 2.99 4.00 2.65 4 4.12 3.32 4.49 2.79 6 4.88 3.22 4.56 5.72 3.55 8 5.00 2.82 4.62 10 5.67 2.67 5.17 12 4.33 16 4.05 2.68 3.93 3.90 3.84 20 4.05 2.53 3.35 4.05 3.68 5.12 3.85 4.18 . 4.17 3.87 Pre-exposure bleeding, inoculated with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 44 The U)gQ ITïe of the pooled sera varied approxinjately 0*4 to 1.0 log unit from the îTIs of the average of the individual serum samples comprising the pooled samples* Hemagglutination-inhibition titers for Newcastle disease were negative at each period. C. Group III 1. Electrophoretic Analyses This group of birds was cliallenged with infectious bronchitis virus at the twelfth week after initial inoculation. The birds were bled at the following time schedule* birds M414Q -13 a»d M400U -14 - one, three, five, and seven weeks after challenge; M539XJ -15 - immediately prior to prinaiy inoculation, one, two, and three weeks (died at third week due to internal hemorrhage); ÎE727B2 -16 - four, six, eight, ten, and 12 weeks after primary inoculation (challenged but not tested); pool of sera from birds M3610 -17 and M369N -18 immediately prior to primary inoculation, one two, three, four, six, eight, and ten weeks; M3610 -17 - six, ten, and 12 weeks? then at one, three, five, and seven weeks after challenge; H369N -18 - six and ten weeks; M541G2 -19 - immediately prior to primary inoculation, one, two, three, four, six, eight, ten, and 12 weeks; then at one, three, five, and seven weeks after challenge. The relative percentage of albumin decreased slightly one and three weeks after challenge in bird M414Q -13. Gamma­ globulin levels increased slightly and then decreased. The other globulins varied slightly during the experiment. (Table 29) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 29 Bird M414% -13 Group III - Challenge Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Protein 12* 47.8 12.2 5.8 9.7 24.5 0.92 3.78 1 44.5 14.6 5.6 8.5 26.8 0.80 3.69 3 43.4 13.2 7.4 6.9 29.1 0.77 3.86 5 42.8 12.8 8.4 8.9 27.1 0.75 4.13 7 48.1 12.1 5.3 7.3 27.2 0.93 3.86 * Challenge with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 Sera from "bird H400U «14 sTiowed a slight decrease in alhumin at the first week after challenge hut increased again after this interval, Gamna-glohulin increased slightly during the experimental period. Other globulins did not change sig­ nificantly, (Table 30) Sera from bird 1B39TJ -15 showed a marked decrease in albumin one week after primary inoculation and began to increase at the second week. The gamma-globulin level increased greatly at the first week and began to decrease after this period, (Table 31) Sera from bird Î3727B2 -16 showed a definite increase in albumin during the experimental period. There was a slight decrease in the gamma-globulin level. Alpha 1-globulin de­ creased greatly at the twelfth week, while alpha 2-globulin and beta-globulin remained relatively unchanged, (Table 32) Sera from birds 153610 -17 and M369E -18 were pooled and tested as one sample immediately prior to primary inoc­ ulation and then at the one, two, three, four, six, eight, and ten week interval. (Table 33) The sera of these birds were also tested individually as follows: bird 1/E3610 -17 - six, ten, and 12 weeks, and then at one, three, five, and seven week intervals after challenge; (Table 34) bird M369ïr -18 six and ten weeks, (Table 35) The electrophoretic analyses of pooled serum samples from birds M3610 -17 and M369B" -18 showed the characteristic pattern as before. A sharp decrease in albumin and an increase in the relative percentage of gamma-globulin was observed one Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 30 Bird M400U -14 Group III - Challenge Electrophoretic Analyses Period Relative Per Cent Serum Component A/a Globulins Weeks Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote il 12* 45.3 8.2 8.2 10.7 27.6 0,83 3.86 1 43.0 10,2 7,8 11.2 27,8 0,75 3.43 3 45,4 9.6 6,0 11,0 28.0 0.84 3.69 5 43,4 9.8 7.4 9,8 29.6 0,77 3.86 7 47,0 5.0 8,5 9.0 30.5 0.89 3.78 * Challenge with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tatle 31 Bird M539U -15 Group III - Challenge Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- * Per Cent Proteii 0* 48.0 7.2 7.2 10.4 27. 2 0.92 4.13 1 30.3 9.5 7.9 11.8 40.5 0.44 4.56 2 35.0 6.0 12.5 10.8 35.7 0.54 4.48 3 37.8 10.8 7.9 10.3 33.2 0.61 4.30 Pre-exposure bleeding, primary inoculation with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. É Tatle 32 Bird M727B2 -16 Group III - Challenge Electrophoretic Analysée Period Relative Per Cent Serum Component Weeks A/G Globulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Pro te il 4 46.7 8.0 6.2 10.7 28.4 0.88 3.69 6 42.5 11.9 6.0 9.4 30.2 0.74 3.51 8 46,4 9.9 5.4 11.3 27.0 0.87 3.60 10 48.3 10.2 7.8 9.3 24.4 0.93 3.51 12* 51.1 5.1 6.5 11.3 26.0 1.05 3.60 * Challenge with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tatle 32 Bird 15727B2 -16 Group III - Challenge Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote it 4 46.7 8.0 6,2 10.7 28.4 0.88 3.69 6 42.5 11.9 6.0 9.4 30.2 0.74 3.51 8 46,4 9.9 5.4 11,3 27.0 0.87 3.60 10 48.3 10.2 7.8 9.3 24.4 0.93 3.51 12* 51.1 5.1 6.5 11,3 26.0 1.05 3.60 * Challenge with virus Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. T a b le 33 Pool of Birds 1C3610 -17 and TkI369ir -18 Group III - Challenge Electrophoretic Analyses Period Relative Per Gent Serum Component A/G Globulins Weeks Serum Albumin Alpha 1- Alpha 2- Beta- Gamraa- * Per Cent Protein 0* 40.S 10.9 4.8 10.5 33.0 0.69 3.73 1 34.9 11.5 9.2 9.6 34.8 0.54 3.73 2 33.2 12.4 6.0 9.7 38.7 0.51 4.04 3 39.0 7 ,5 6.1 10.1 37.3 0.64 4.39 4 38.5 9.5 6.3 9.5 36.2 0.63 3.78 6 38.9 8.8 6.9 9.3 36.1 0.64 4,13 8 36.4 11.7 6.0 10.4 35.5 0.57 3.86 10 41.3 12.3 6.6 10.7 29.1 0.70 4.04 Pre-exposure bleeding, primary inoculation with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 34 Bird M3610 -17 Group III - Challenge Electropboret le Amlyses Period Relative Per Cent Serum Component A/Q Globulins WeelcB Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote 6 38.6 12.4 6.4 8.4 34.6 0.63 4.04 10 41.4 11.7 6.1 11.2 29.6 0.71 3.95 12* 38.9 10.7 7.9 9.7 32.8 0.64 3.78 1 40.9 7.9 6.4 11.3 33.5 0.69 4.13 3 35.6 8.6 6.7 11.0 38.1 0.55 4.21 5 38.3 8.3 8.7 9.7 35.0 0.62 4.48 7 38.2 11.8 8.2 8.2 34.6 0.62 3.78 Challenge with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tatle 35 Bird M369IT -18 Group III - Challenge ElectropTioretic Analyses Period Relative Per Cent Serum Component Weeks A/G Glotulins Serum Albumin Alpha 1- Alpha 2- Beta- Gamma6* 10 * Per Cent Protein 35.6 10.4 7.4 10.0 36.6 0.55 3.95 43.1 9.7 7.6 8.6 31.0 0.76 4.04 After Primary Inoculation Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and two weeks after primary exposure* An increase in albumin occurred at the third week and at the same time gamma-^ohulin levels began to decrease* This continued until the end of the experimental period of ten weeks* Beta-globulin levels did not change significantly during the experiment * Alpha 1-globulin decreased early during the experiment but increased again at the tenth week. Alpha 2-globulin changed at the first week but re­ mained constant during the rest of the experiment* (Table 33) The electrophoretic analyses of sera from bird M3610 -17 showed a decrease in albumin and an increase in gamma-globulin during the third week after challenge.This condition was reversed after this period up to and including the seventh week* Alpha 2-globulin levels did not change significantly during the experimental period* Alpha 1-glob­ ulin decreased one week after challenge but Increased steadily to a final high value. Beta-globulin decreased during the chal­ lenge period. (Table 34) The analyses of sera from bird ]l069ir -18 during the sixth and tenth weeks showed a great increase in albumin and a decrease in gamma-globulin. Values for the other globu­ lins were relatively unchanged. (Table 35) The same general trend followed in the electrophoretic analyses of sera from bird 3Æ541C2 -19, There was a sharp de­ crease in the relative percentage of albumin at the first week after primary exposure. Gamma-globulin levels increased at this period, decreased at the fourth week and were at a Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 minimum value at the twelfth week. There was a general in­ crease in alpha 1-globulin at the first and second weeks with a decrease in this fraction up to and including the twelfth week. Alpha 2-glohulin decreased slightly during the twelve weeks, while heta-glohulin remained relatively unchanged except that a rise was observed at the first week which re­ mained at this level for twelve weeks. Turing the challenge period there was little change in the relative percentage distribution of the serum compo­ nents, (Table 36, Figure XVIII) The changes in serum electrophoretic patterns after challenge with infectious bronchitis virus was not as marked as the changes observed in the electrophoretic patterns after primary exposure. The same general trend in the change of serum electro­ phoretic patterns in Group II was observed in Group III prior to challenge. After challenge there was relatively little change in the relative percentage distribution of the serum cor^onents. A comparison of results obtained with pooled serum samples and individual serum samples of the pool showed very close correlation in the relative percentage distribution of the serum components, The changes in percent serum protein were not con­ sidered significant. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TaTjle 36 Bird M541C2 -19 Group III - Challenge Electrophoretic Analyses Period Relative Per Cent Serum Component Weeks A/G Globulins Per Cent Serum Albumin Alpha 1- Alpha 2- Beta- Gamma- Prote in 0# 39.6 8.4 9.0 8.4 34.6 0.66 3.99 1 27.8 12.1 6.6 13.6 39.9 0.39 4.04 2 34.0 11.5 5.0 11.9 37.6 0.52 4.30 3 40.9 7.4 6.1 10.0 35.6 0.69 4.04 4 39.8 9.5 4.1 11.8 34.8 0.66 4,13 6 38.0 9.8 6.1 11.0 35.1 0.61 3,69 8 41.8 6.8 7.3 10.5 33.6 0.72 3.95 10 43.0 10.5 6.0 12.0 28 0.75 3.69 12* 44.0 8.0 6.7 11.7 29.6 0.79 3.69 1 43.2 8.3 6.8 11.5 30.5 0.76 3.86 3 46.4 7.8 5.6 11.2 29.0 0.37 3.34 5 44.0 8.9 7.3 10.5 29.3 0.79 3.95 7 43.0 10.7 7.3 9.8 29.2 0.76 3.51 # Pre-exposure Bleeding, Inoculation with Virus * Challenged with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure XVIII Bird ¥5410 2 -19 Group Ill-Challenge Electroplioretic Patterns A 0 O WEEKS PRE-EXPOSURE A Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plgure XVTII (cont.) < WEEKS A D 3WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. figure XVIII (cont.) A 4 WEEKS A 0 D 6 WEEKS Reproduced with permission o f the copyright owner. Further reproduction prohibited without permission. Pigure XVTII (cont.) A D 8 WEEKS A D 10 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pigure XVIII (cont#) A D IZWEEKS challenged A D IWEEK Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure XVIII (cont.) i A 5 WEEKS A D D 5 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure XVIII (cont*) A D 7 WEEKS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 2* Lethal Dose^Q neutralization Indices The serum LD^g nis of this group followed a general pattern as in Group II, There was a slight increase in LD^^ His "between the first and second weeks after primary exposure. After the second week a marked rise occurred and a maximum was reached "between the third and eighth weeks. The maximum lD_rt His that were reached varied with the individual "birds 50 as follows: "bird 1S727B2 -16 - fourth week; "bird 153610 -17 sixth week; "bird 1Î369H -18 - sixth week; bird M541C2 -19 - eighth week* After challenge the LD^g His were higher than the maximum values reached after primary inoculation. The max­ imum LDgg HiB after challenge were reached between the third and fifth weeks, (Tables 37, 38, 39, 40, 41, 42, 43, 44, 45, Figures XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI) Pre-exposure HTs were considered to be normal (33, 57). Hecropsy examination failed to reveal any abnormal­ ities in birds of this group, Hemagglut inat ion-inhibit ion titers for Hewcastle disease were negative at all times, D. Group IVa 1, Electrophoretic Analyses In order to determine if inoculations of a normal lung and tracheal suspension could affect the serum electro­ phoretic patterns or LDgQ His birds M346B -21 and M673V -22 were bled immediately prior to inoculation and then at the one, two, and three week interval. Sera from bird M645H -23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. É Ta-ble 37 Bird m i 4% -13 Group Ill-Challenge SeriJim ITeutralization Tests Period Weeks Titer 12f 6.50* 1 6.38* 3 * Virus Dilutions Virus# Serum 10® 10”^ 10"^ 10"^ 10-4 10"5 ## 5 5 3 2 5 5 5 4 6.00* 5 4 1 5 6.32* 5 3 7 6.50* 5 4 Titer# m 2.67 3.83 0 2.38 4.00 0 0 0.50 5.50 3 1 0 1.14 5.18 3 2 0 1.33 5.17 1 1 Challenge with virus Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. â r o In I Ul c. lU « g «X X u o u I Z t il n _ fe o a. g «I bJ i i s lU lU I 2 3 O o H GC u. ” Ü OL (D 3 O oc a T o K T " nr o ^ : 0( 3GW0 ocn J.N3NOdX3 I 8 X3QNI NOiivznv^iniN Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o Table 38 Bird M400U -14 Group III-Challenge Serum îTeutralization Tests Period Weeks Virus Dilutions Virus# Titer 12* 6.50* 1 6,38* 3 Serum 10^ lO"! 10"^ 10"^ 10-4 10"^ # 5 5 3 0 5 5 3 0 7,00*£ 5 4 2 5 6.32* 5 5 7 6.50* 5 3 1 Titer# HI 2.17 4.33 1 1.31 5.07 0 0 0.68 6.32 0 0 0 0.17 6.15 2 0 0 0.50 6.00 * * Challenge with virus È Endpoint not reached when serum was diluted 1:100. Virus titer arbitrarily fixed at 10^ to calculate Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ITI r a o -lo % lu M â X o III lu 2 lü u VJ Z «»* ^ — -g -W jI l_ I N lO s iu bJ - $ § O o K O 4> "T" O US T" -r : oi 35vg D a w w 3 -T“ o h.alom;'/elitis Produced Experimentally in Rhesus Monkeys, VI. Changes in the Cerehrospinal Eluid Proteins, J, Exp, Med., 9 3 , 89, (.1 9 5 1 ): 615-633 Earzon, D. E , , and Bang, E. B, The Pathogenesis of Infection with a Virulent (CG- 179) and an Avirulent (B) Strain of IJewcastle Disease Virus in the Chicken. II. Development of Antibody, J. 90, Exp. Med,, 9 3 , (1951): 285-296 Kekwick, R. A, The Electrophoretic Analysis of Mormal Human Serum, Biochem, J , , 91, (1939): 1 1 2 2 -1 1 2 9 EelT';iok, R. A., and Record, B, R, Some Physical Properties of Diphtheria. Antitoxic Horse Sera. Brit. J . Exp, Path,, 22, ( 1 9 4 0 ) : 92, 29-44 Koenig, V, B . , and Hogness, K. R, Electrophoretic Analj'-sis of Swine Pla.sma and Serum, Arch, Biochem,, 9, (1946): 119-128 93, Koenig, V, L., Perrings, J. D . , and Hogness, H. R. The Effect of tl\e Variation of Ionic Strength on Electrophoretic Analysis of Bovine Plasma, Arch, Biochem,, il, (1946): 345-361 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the 81 94. Komarov, A., and Beaudette, F. R. Carriers of Infectious Broncîiitis. Poultly Soi., 11, (1932): 335-338 95. Koprovrski, E., Richmond, G., and Moore, D. H. Electrophoretic Study of Antiviral Sera. J. Exp. Med., 96. (1947): 515-530 Erehs, E. G. Depression of Gansna Glohulin in I^poproteinemia due to Mhlnutrit ion. J. Bah. Clin. Med., 31, (1946): 85-89 97. Bars on, D. B . , and Tomlinson, B. J, Quantitative Antihody Studies in Mhn. II. The Relation of the Bevel of Serum Proteins to Antihody Production. J. Bah. Clin. Med., 98. (1952): 129-134 Beeds & Eorthrup Company Directions for Operating B & B Portable Electrolytic Conductivity Bridge Mo. 4960. Hiiladelphla, Pa. 99. Benke, S. E . , and Berger, H. M. Effects of Buffer pH on the Electrophoretic Patterns of Human Serum. Bah. Digest, 15, (1951): 4-6 100. Beyton, G. Bleotroforeeis en el estudio de Antigènes y Anticuerpos. Santiago de Chile, Imprenta Tftiiversitaria, (1948) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. â 82 101. Xippman, R. W . , and Banovitz, J . Influence of Protein Concentration upon Electrophoretic Mobility of Serum Proteins. Fed. Proo., 11* (1952): 250 102. Xoeh, R # F . Plasma Proteins in Health and Disease. Few England J. Med., 224. (1941): 980-987 103. Longsworth, L. G. A Modification of the Schlleren Method for Use in Electrophoretic Analysis. J. Am. Chem. Soo., 104. (1939): 529-530 LongsWorth, X. G. Recent Advances in the Study of Proteins hy Electrophoresis. Chem. Rev., 105. (1942): 323-340 Xongsworth, X. G. Optical Methods in Electrophoresis. Ind. Eng, Chem., Analyt. Ed., 18, (1946); 219-229 106. Xongsworth, X. G, The Quantitative Interpretation of the Electrophoretic Patterns of Proteins, J, Phys. Colloid Chem., 107. (1947): 171-183 Xongsworth, X. G . , and Mhclnnes, D. A. Electrophoresis of Proteins by Tlselius Method. Chem. Rev., 24, (1939): 271-287 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. i 85 108. longsworth, 1. G,, and 15acInnés, I). A. The Interpretation of Simple Electrophoretic Patterns, J. Am. Chem. Soo., 62, (1940): 705-711 109. longsworth, 1, G , , Shedlovsky, T ,, and l&,clnnes, D, A. Electrophoretic Patterns of ÏTormal and Pathological Human Blood Serum and Plasma. J. Exp. Ifed., 70, (1939): 399-413 110. loomle, 1,H , , Cunningham, 0, H,,Gray, M, 1,, and Thorp, P., Jr. Pathology of the Chicken Embryo Infected with Infectious Bronchitis Virus, Am. J, Vet, Res., 111. (1950) : 245-251 luetscher, J . A,, Jr. The Effect of a Single Injection of Concentrated Human Serum Alhumin on Circulating Proteins and Proteinuria in Nephrosis. J. Clin. Invest., 112. (1944): 365-371 luetscher, J, A,, Jr. Biological and Medical Applications of Electrophoresis, Physiol, Rev., 27, (1947): 621-642 113. lush, D. The Chick Red Cell Agglutination Test with the Viruses of Newcastle Disease and Fowl Plague. J, Comp. Path, Therap., 53, (1943): 157-160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 84 114# lîarrack, J. R,, and HocTi* H, Semra Proteins : A Review. J. Clin. Path., 2, (1949); 161-192 115. 3$arshall, î/T. E., and Dent soh* H. P. Distrihution of Egg White Proteins in Chicken Blood Serum and Egg Yolk, J. Biol. Chem., 1 ^ , 116. (1951); 1-9 Martin, R. H. The Components of the Serum Proteins in Infective Hepa­ titis and in Homologous Serum Jaundice (An Electropho­ retic Study). Brit. J. Exp. Path., 117. (1946); 363-368 Met ooff, J . , Darling, D. B., Scanlon, M. H., and Stare, P. J. Eutritional Status and Infection Response. I. Electropho­ retic, Circulsting Plasma Protein, Hematologic, Hemapoietic, and Immunologic Responses to Salmonella tvnhimurium (Bacillus aertiycke) Infection in the Proteindeficient Rat. J. Dah. Clin. Med., 118. (1948); 47-66 Moon, V. H . , and Tershakovec, Gr. A. Dynamics of Inflammation and of Repair. I. The Trigger Mechanism of Acute Inflammation. Arch. Path., 119. (1951); 369-377 Moon, V. H . , and Tershakovec, G. A. Dynamics of Inflammation and of Repair. II- Chemotactic Substances in Hormal Tissues. Arch. Path., 52, (1951): 441-446 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ô5 120. ISoore, D. H, The Effect of Urea on the Electrophoretic Patterns of S e n m Proteins, J. Am, Chem, Soc.» 121. (1942): 1090-1092 Moore » D, H. Species Differences in Sertnn Protein Patterns* J. Biol Chem., 1 6 1 , (1945): 21-32 122. Moore, D, H. Effect of Reciprocal Steroid Treatment on the Electrophoectfc Patterns of Powl Sera. Endocrinology, 123. (1948): 38-45 Moore, D, H,, and Abramson, H, A, Chapter on Electrophoresis, Medical Phvsios. Vol, II, edited by 0, Classer. Year Book Publishers, Inc., Chicago, Illinois. 124. 1950 Moore, D, H,, and Pox, C. L. Correlation of Electrophoretic Studies and Other Pactors in the Syndrome of Secondary Shock. Nature, 1 6 5 , (1950): 872-876 125. Moore, D, H, Nickerson, J, X.,, Powell, A. E , , and Marks, G, A Study of the Transfer of Serum Proteins into Tissue Injured by Tourniquet. Proo* Soo, Exp, Biol, Med,, 77, (1951): 706-709 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. â 86 126. M o o r e , D. H«, Ho'berts , J. B . , Costello» M. » and Sobonberger» T. W. factors Influencing tbe Electropboretic Analysis of Human Serum, J. Biol. Chem.» 180, (194#)* 1147-1158 127. Moore » D. H., and #iit@, J. U. A Hew Compact Tlselius Electrophoresis Apparatus. Rev. Sci. Instruments, 19, (1948): 700-706 128. Morgan, I. M. Quantitative Study of the neutralization of Western Equine Encephalomyelitis Virus by its Anti-serum and the Effect of Complement. J. Immunol.» 50, (1945): 359-371 129. Hichol, J. C. » and Beutscb, H. P. Biophysical Studies of Blood Plasma Proteins. VII, Separation of y -globulin from the Sera of Various Aninals. J". Am. Ghem, Soc.» 70, (1948): 80-83 130. Page, C , A. Antibody Response of Chickens Exposed to Infectious Bronchitis Virus. Thesis, Michigan State College, 131. (1950) Page, C. A. Bhpubl ished Data Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 87 132. Pearlmarm, G. E., and Kaufman, D. The Effect of Ionic Strength and Protein Concentration in Electrophoretic Analysis of Human Plasma* J» Am. Chem. Soc.» 6%» (1945); 638-641 133. The Perkln-Elmer Corporation Instruction îüanual. Portable Tlselius Electro-phoresis Ap-paratus. Model 38* Horwalk, Conn* 134* 1951 Popjàk, G., and lUcCartly» E. E* Osmotic Pressures of Es^erimental and Human Lipaemic Sera* Evaluation of Alhumin/Glohulin Ratios with the Aid of Electrophoresis. Biochem* J . , 135* (1946): 789-803 Putnam, E. W . , Lamanna, C . , and Sharp, D. G. Physicochemical Properties of Crystalline Clostridium hotulinum Type A Toxin* J. Biol. Chem*, 17®f (1948): 401-412 136* Reagan, R. L., Brueckner, A. L*, and Delaplane, J. P. Morphological Observations hy Electron Microscopy of the Viruses of Infectious Bronchitis of Chickens and the Chronic Respiratory Disease of Turkeys. Cornell Vet*, 137* (1950); 384-386 Reagan, R. 1., Hauser, J. E . , Lillie, M. G. , and Craige, A. H., Jr* Electron Micrograph of the Virus of Infectious Bronchi­ tis of Chickens* Cornell Vet*, 38, (1948): 190-191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Æ 88 138. Heed, L. J . , and Miencli, H. A Simple TWkthod of Estimating Fifty Per Cent Endpoints. Am. J. I%rg., 27, (1938): 493-497 139. Heiner, M . , and Fenichel, H. L. Dialysis of Protein Solutions for Electrophoresis. Science, 108, (1948): 164-166 140. Ricketts, W. E . , and Sterling, K. Electrophoretic Studies of the Serum Proteins in Virus Hepatitis* J. Clin. Invest., 28, (1949^: 1477-1486 141. RohscheIt-Rohhins, F. S., Miller, h.L., and Hfhipple, G. H. Maximal Hemoglohin and Plasma Protein Production Under the Stimulus of Depletion. J. Exp. Med., 82, (1945): 311-316 142. Routh, J. I,, and Paul, W. D. Beta Disturbance of Electrophoretic Patterns in Disease. Fed. Proo., 11, (1952): 278 143. San Clemente, C. L. An Electrophoretic Study of Pulloriun-Agglutinating Chicken Serums. Am. J. Vet. Res., 3, (1942): 219-221 144. Sanders, E . , Huddleson, I. F . , and Schaihle, P. J . An Electrophoretic Study of Serum and Plasma from Hormal and Leucosis-affected Chickens. J. Biol. Chem., 155. (1944): 469-481 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. é 89 145. Scfcalk, A. P., and Hawn, M. C, An Apparently Hew Respiratory Disease of Ba"by Chicks. J. Am. Vet. Ked. Assn., 3%, (1931): 413-422 146. Seihert, P. B., and Helson, J. W. Electrophoretic Study of the Blood Protein Response in Tuherculosis. J. Biol. Chem., Ij^, (1942): 29-38 147. Seihert, P. B . , and Helson, J. W. Proteins of Tuherculin. J. Am. Chem. Soc., §5, (1943): 272-278 148. Seihert, P. B . , Seihert, M. V., Atno, A. J . , and Camp­ bell, H. W. Variation in Protein and Polysaccharide Content of Sera in the Chronic Diseases, Tuherculosis, Sarcoidosis, and Carcinoma. J. Clin. Invest., 26, (1947): 90-102 149. Shapiro, S., and Moore, D. E. Electrophoretic Patterns After Dicumarol Medication. Proc. Soc. Exp. Biol. Med., 150. (1948): 501-502 Sharp, D. G., Cooper, G. R . , and Heurath, H. The Electrophoretic Properties of Serum Proteins. I, Hormal Horse Pseudoglohulin GI, J, Biol. Chem., 142, (1942): 203-216 151. Shedlovslqr, T. , and Scudder, J. A Comparison of Erythrocyte Sedimentation Rates and Elec­ trophoretic Patterns of Hormal and Pathological Human Blood. J. Exp. Med., 75, (1942): 119-126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. É 90 152# Shipley, R# A*, S t e m , K. G., and White, A# Electrophoreeie of Anterior Pituitary Proteins. J. E3Cp. Med., 69, (1939): 785-800 153. Sterling, K. The Serum Proteins in Infectious Mononucleosis. Elec­ trophoretic Studies* J. Clin. Invest., 28, (1949): 1057-1066 154. Swierstra, D. Infectious Bronchitis of Chickens in Holland# Tijdschr# Biergeneesk., 7^, (1947): 745-746 Abstract from Vet. Bull., 1^, (1949): 145 155. Tiselius, A. A He w Apparatus for Electrophoretic Analysis of Colloidal Mixtures# Trans# Paraday Soc#, 156# (1937): 524-531 Tiselius, A, Electrophoresis of Serum Globulin. I# Biochem# J. , 3^, (1937): 313-317 157# Tiselius, A. Electrophoresis of Serum Globulin. II. Electrophoretic Analysis of Hormal and Immune Sera# Biochem# J#, 31, (1937): 1464-1477 158. Tiselius, A., and Rabat, E. A# An Electrophoretic Study of Immune Sera and Purified Antibody Preparations# J. Exp# Med#, 69, (1939): 119-131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 91 159. Tobin, J. R . , Jr., Bergenstahl, D . , and Steffee, C. H. Tbe Relationebip of Protein Reserves to the Production of Hyaluronidase and Antihyaluronidase. Areli. Biochem*, 16, (1948): 575-378 160. Treffers, H. P., Moore, D. K . , and Heidelherger, M. Quantitative Experin»nts with Antibodies to a Specific Precipitate. III. Antigenic Properties of Horse Serum Praetions Isolated hy Electrophoresis and hy Ultra­ centrifugation. J. Exp. Med., 75, (1942): 135-150 161. van der Scheer, J. , Bohnel, E., Clarke, P. H . , and ^ c k o f f , R. W. G. An Electrophoretic Examination of Several Antipneumococcal Rahhit Sera, J. Immunol., 44, (1942): 165-174 162. van der Scheer, J., l%rckoff, R. W. G . , and Clarke, P. H, An Electrophoretic Analysis of Several hyperimmune Horse Sera. J, Immunol., 39, (1940): 65-71 163. van der Scheer, J . , %-ckoff, R. W. G . , and Clarke, P. H. The Electrophoretic Analysis of Tetanal Antitoxic Horse Sera. J. Immunol., 164. (1941) : 173-177 Van Roekel, H . , Clarke, M. E . , Bullis, K. P., Olesuik, O. M . , and Sperling, P. G. Infectious Bronchitis. Mass. Agric. Exper. Station, Bull. Ho. 460 i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 92 165. Waldenstrflm, J. , Pedersen, K, O . , Harboe, IF., and Sonek, C. S. TJltracentrifugation, Electrophoresis and Viscometxry of Serum Proteins. 1. Lympbogranuloma Venereum. Acta Medica Soandlnavloa, 141. (1951) : 195-204 166. 'Whipple, G. H., Robscheit-Robbins, E. S., and Miller, L. L. Blood Protein Regeneration and Interrelation. Ann. Hew York Acad. Sci., 167. (1946): 317-326 Wiener, A, S., Berger, H., and Benke, S. Serum Gamma Globulin in Infants (Preliminary Report). Lab. Digest, 14, (1951); 11-12 168. Wissler, H. W. The Effect of Protein Depletion and Subsequent Immunization upon the Response of Animals to Pneumococcal Infection. I. E]cperiments with Babbits. ■ .T. Inf. Dis., 80^(1947): 250-253 169. Wissler, R. W. The Effect of Protein Depletion and Subsequent Immuni­ zation upon the Response of Animals to Pneumococcal Infection. II. Experiments with Male Albino Hats. J. Inf. Dis., 80^ (1947): 264-277 170. Wissler, H. W . , Woolridge, H. L . , Steffee, C. H. and Cannon, P. R. The Relationship of the Protein-Reserves to Antibody Production. II. The Influence of Protein Repletion Upon the Production of Antibody in I^oproteinemic Adult White Rats. J. Immunol., 52, (1946): 267-279 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 93 191# Wohl, M, G,, Reirihold, J. G,, and Rose, 8. B* Anti'body Response in Patients with Hypoproteinemia with Special Reference to the Effect of Supplementation with Protein or Protein Rydrolysate# Arch. Int. Med., 83, (1949): 402-415 172. lü^ckoff, R. W. G . , and Rhian, M, An Electrophoretic Study of an Anti-influenzal Horse Serum. J. Immunol., 173. (1945): 359-363 Zeldis, 1. J., and Ailing, E, L. Plasne, Protein Metaholism-Electrophoretic Studies. Restoration of Circulating Proteins Following Acute Depletion hy Plasmapheresis. J. Exp. Med., 81, (1945): 515-537 174. Zeldis, I». J . , Ailing, E . D . , MoCoord, A. B . , and Kulka, J. P. Plasma Protein 3fetaholism-Electrophoretic Studies. Chronic Depletion of Circulating Proteins During Low Protein Feeding. J. Exp. Med., 175. (1945): 157-179 Zeldis, L. J . , Ailing, S. L.,McCoord, A. B . , and Kulka, J. P. Plasma Protein Ifetaholism-Electrophoretic Studies. The Influence of Plasma Lipids on Electrophoretic Patterns of Human and Dog Plasma, jr. Exp. Med., 82, (1945): 411-430 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.