SPECTROPHOTOMHRIC AND SERUM NEUTRALIZATION STUDES OF SERA FROM CHICKENS EXPOSED TO INFECTIOUS BRONCHiTIS VIRUS Thesis for flu Dom» 9! Ph. D, MICHIGAN STATE UNIVERSITY Osman. Hipélifo 1955 TH 52518 This is to certify that the thesis entitled Spectrophotometrio and Serum Neutralization Studies of Sara from Chickens Exposed to Infectious Bronchitis Virus presented by Osmane Hipolito has been accepted towards fulfillment of the requirements for _M2__degree in_____M1°r°b1°1-°U and Public Hes 1th Major professor ; Date August 26, 1955 0-169 SPECTROPHOTOMETRIC AND SERUM NEUTRALIZATION STUDIES OF SERA FROM CHICKENS EXPOSED TO INFECTIOUS BRONCHITIS VIRUS BY OSMANE {npémro A THESIS Submitted to the School of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1955 ABSTRACT Infectious bronchitis of chickens (IB) is an acute and highly Contagious disease of great economic significance to the poultry in- dustry. The purpose of the present investigation was to study the re— lationship of spectr0photometric analyses of serum as compared to antibody response measured by the serum neutralization test follow- ing inoculation of susceptible birds with infectious bronchitis virus. Adult, single-comb, White Leghorn cockerels were used as experimental chickens. ' Two strains of virus were used. Strain V114D, an egg—adapted strain, was employed in all the serum neutralization tests. Strain VL was isolated from field cases of the disease and was used to infect susceptible birds. Chickens were bled by intracardiac puncture immediately prior to exposure to IBV and then after three, five, seven, ten, twelve, sixteen, and twenty weeks. Serum samples were stored at -30°C until used. Sera of individual birds were analyzed with the Beckman Model B spectrophotometer before and after chemical fractionation by the ii method of Wolfson M. The following values were determined: total protein, albumin, and alpha, beta, and gamma globulins. Sera from individual birds bled at the same time interval were pooled in equal portions and analyzed spectrophotometrically. Serum neutralization tests and macro-Kjeldahl determinations were also performed on these pools. A close agreement was observed in the total protein deter- minations by the Kjeldahl and the spectrophotometric methods although the values obtained with the former method were slightly higher. Results obtained by spectrOphotometry after chemical frac- tionation showed a fair agreement with those found by several inves- tigators with the use of the electrophoresis apparatus. There was a close agreement of the results obtained with sera of individual chickens and those of pooled sera with respect to total protein, albumin, total globulin, gamma globulin, and A/G ratios. Alpha and beta globulin results showed a marked variation. A. decrease in albumin values and consequently an inversion of the A/G ratios was observed after IBV inoculation. Alpha and beta globulins were specifically responsible for the increase in globulins. Gamma globulin had no influence on this change since its values were almost uniform during the course of the investigation. iii In an endeavor to explain the results obtained it must be em- phasized that a large amount of blood (20 m1., or approximately one- fourth of the total blood volume) was removed from each bird at every bleeding period. Although the intervals between bleedings were never less than two weeks, it is possible that this interval might not have been sufficient for the complete restoration of the original balance of the serum components. Serum neutralization tests conducted on pooled sera showed typical response with infection by IBV. A maximum titer in antibodies was found between the seventh and twelfth weeks after inoculation. There was no correlation between the changes in serum com- ponents and antibody content. When the serum components had re— turned to preinfection levels the antibody content was at the maximum. iv To MY FAMILY this manuscript is mo 5 t affectionately dedic ated ACKNOWLEDGMENTS The author is grateful for the generous help, guidance, and instruction given to him by Dr. Charles H. Cunningham, Professor of MicrobiolOgy and Public Health. He is also indebted to Dr. Erwin J. Benne and the other staff members of the Department of Agricul- tural Chemistry, responsible for the protein nitrogen analyses. Grateful acknowledgment is also due to Mrs. Martha P. Spring. The writer deeply appreciates the financial support of the Universidade Rural do Estado de Minas Gerais (Brazil) and of The Rockefeller Foundation, which made it possible for him to complete thi s inv e s tigation. vi Osmane Hipolito candidate for the degree of Doctor of Philosophy Final examination: August 26, 1955, 10:00 a.m., Room 101, Giltner Hall. Dissertation: Spectrophotometric and serum neutralization studies of sera from chickens exposed to infectious bronchitis virus. Outline of studies: Major subjects: BacteriOIOgy, Virology. Minor subject: Animal Pathology. BiOgraphical items: Born, January 30, 1920, Uba, Minas Gerais (Brazill. Undergraduate studies, Ginasio Raul Soares, Uba, Minas Gerais. Escola Superior de Agricultura e Vete- rinaria, Vicosa, Minas Gerais, 1935-39. Graduate studies, Michigan State University, 1952-55. Experience: Assistant professor, Escola Superior de Agri- cultura e Veterinaria, Minas Gerais, 1940-42; Assistant professor, Escola Superior de Vete- rinaria, Belo Horizonte, Minas Gerais, 19:12:30; Professor adjunto, Escola Superior de Veteri- naria, Belo Horizonte, Minas Gerais, 1950- Society affiliations: The Society of American Bacteriologists, The Society of Phi Zeta, Sociedade Bra- sileira para o Progresso}: Cié‘ncia, member. TABLE OF CONTENTS Etiology ................................ Transmission ............................. Resistance of Virus to Physical and Chemical Agents ................................. Diagnosis ............................... Isolation of the virus ..................... Serum neutralization test (SN) ............... Control .................................. Expe rim ental Animals ......................... Virus Antigens .............................. Serum Studie s .............................. 10 12 12 12 15 15 15 Determinations ........ Spectrophotometric Analyses Standardization ........ Preliminary Experiments . . . . Storage of Serum Samples . . Wavelength . . .......... Sodium Sulfite Concentration Experimental Results ....... Individual Sera .......... Spectrophotometric results Total protein ....... Albumin .......... Total globulin ...... Alpha globulin ...... Beta globulin ....... Gamma globulin ..... A/G ratio ......... Pooled Sera ............ Spectrophotometric results ix OOOOOOOOOOOOOOOOOO oooooooooooooooooo OOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOO IIIIIIIIIIIIIIIIII OOOOOOOOOOOOOOOOOO oooooooooooooooooo OOOOOOOOOOOOOOOOOO IIIIIIIIIIIIIIIIII nnnnnnnnnnnnnnnnnn oooooooooooooooooo ooooooooooooooooo 22 23 27 27 27 28 28 3O 32 32 32 43 44 44' 45 46 47 47 47 Total protein ......................... Albumin oooooooooooooooooooooooooooo Total globulin ........................ Alpha globulin ........................ Beta globulin ......................... Gamma globulin ....................... A/G ratio ooooooooooooooooooooooooooo Serum neutralization tests .................. DISCUSSION ....... SUMMAR Y ................................... OOOOOOOOOOOOOOOOOOOOOOOOOOO 52 52 52 52 53 53 57 64 66 LIST OF TABLES TABLE Page 1. Absorption of Light at Different Wavelengths ...... 29 II. Spectrophotometric Values Found for Chicken 464 in Different Periods of Time .............. 33 III. Spectr0photometric Values Found for Chicken 466 in Different Periods of Time .............. 35 IV. Spectrophotometric Values Found for Chicken 468 in Different Periods of Time .............. 37 V. Spectrophotometric ValuesFound for Chicken 469 in Different Periods of Time .............. 39 VI. Spectrophotometric Values Found for Chicken 470 in Different Periods of Time .............. 41 VII. Spectrophotometric and Macro-Kjeldahl Determinations on Pooled Sera ............... 48 VIII. Serum Neutralization Tests on Pooled Serum Samples ............................... 54 xi Figure 10. LIST OF FIGURES Comparison of total protein of human albumin and pooled chicken serum by the biuret reaction ...... Selection of sodium sulfite concentration for albumin determination ...................... Spectrophotometric chicken 464 . . . . Spectrophotometric chicken 466 . . . . Spectrophotometric from chicken 468 SpectroPhotometric from chicken 469 Spectrophotom etric from chicken 47 0 Spectrophotometric values for sera from ooooooooooooooooooooooooo 0000000000000000000000000 values for pooled sera ....... Values found for AlG ratio of pooled sera ....... Serum neutralization test of pooled sera ......... xii Page 24 31 34 36 38 40 42 49 50 55 INTRODUCTION Infectious bronchitis of chickens is a disease of great economic significance to the poultry industry. The purpose of the present investigation was to study the re- lationship of spectrOphotometric analyses of serum as compared to antibody response measured by the serum neutralization test follow- ing inoculation of susceptible chickens with infectious bronchitis virus. REVIEW OF LITERATURE Infectious Bronchitis of Chickens Infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens first reported by Schalk and Hawn (85) in North Dakota in 1931. The infection has subsequently been observed throughout the United States (3, 5, 8, 9, 17) and in England (1, 5), Canada (5), and Holland (87). The disease is apparently Specific for chickens and affects all ages, breeds, and sexes. The morbidity is high but the mortality is variable. In chicks the mortality may be as high as, 90 per cent, but in birds over six weeks old the loss is generally negligible. In— fectious bronchitis constitutes a serious economic problem in view of its rapid Spread and the marked decrease in egg production of af- fected laying flocks (34, 88). Etiology Infectious bronchitis is caused by a virus, Tarpeia pulli. This species designation was suggested by Packer (73) in 1950. Infectious bronchitis virus (IBV) is spherical, ranging from 65 to 135 millimicrons with an average of 70 millimicrons (80, 81). 2 The virus may be passed through all grades of Seitz (34) and Berke- feld filters (6, 10, 34). The etiologic agent can be isolated from the lung and trachea throughout the respiratory phase of the disease (43). Transmission Transmission is either by contact or aerosol with a rapid spread from infected to noninfected chickens. Experimentally the disease can be readily transmitted by in- tratracheal and intranasal inoculations (10, 34), but not by subcutane- ous or intramuscular inoculation. The virus can be transmitted throughout the respiratory stage of the disease and has been recovered from infected chickens for as long as four weeks after inoculation (45). Carriers may be capable of transmitting the virus as long as forty-three days after an outbreak (65), and in some instances as long as two months (34). Hofstad (52) was unable to demanstrate the existence of car- riers of IE among recovered birds by using the direct contact method. Symptoms Symptoms usually appear twenty-four hours after intratracheal inoculation, but in some cases it may be several days before they can be observed. The majority of affected birds manifest symptoms from one to eleven days after inoculation. P Severity of the infection varies with the virulence of the virus, 4 conditions of the flock, and individual susceptibility. _c. Gasping, coughing, sneezing, and tracheal rales are the most prominent symptoms (6, 8, 33, 53, 85). Fluidlike feces appear in some cases (88). The most important aspect of the disease in laying flocks is the marked decline in egg production which may persist for many weeks. Eggs produced by hens recovered from the disease are mis- shapen, rough, soft-shelled, and of poor quality (16, 70, 89). In many instances several months may elapse before a flock returns to the preinfection level of egg production. Many hens suffer perma- nent ovarian damag e . Le 5 ions Lesions are generally confined to the lungs and bronchi (17, 85). These alterations consist of pulmonary congestion and a serous or catarrhal exudate in the trachea. Pleuritis and pericarditis may also be observed. In young chicks pathologic alterations are variable, but edema of the mucosa of the trachea and bronchi and a serous or purulent exudate are uniformly observed. Coagulation of this exudate forms plugs that occlude the lumen of the larynx and produce death by as- -phyxia. Other lesions are an edematous thickening of the tracheal mucosa and submucosa and diffuse, leucocytic infiltration with little desquamation and complete absence of gross hemorrhage. Neither inclusion bodies nor pathognomonic lesions were found in experimental and field cases of the disease (51). In semimature chickens tracheitis is the most constant lesion. In mature birds the tracheal reaction may be more severe and ex- tensive (88). A noncellular film may be found by microscopic exam- ination of the tracheal mucosa of some birds (51). Lesions in the nervous system have not been observed (8). Resistance of Virus to Physical and Chemical Agents Tracheal exudate when frozen, dried, and stored at 4°C. re- mains infective for 180 days. The virus retains its virulence for eighty days when stored in 50 per cent glycerin at 4°C. (6). At room temperature virus-infected allantoic fluid remains viable for five to seven days, but not for fourteen days. At 50°C. the virus is viable for fifteen minutes (35). Freezing and thawing has no harmful effect on the virus. A hundred fold decrease of infective doses occurs in 1y0philized sam- ples after seven days' storage at 4°C. This decrease may be due to incomplete drying of the virus preparation (28). The virus is more stable at 4°C. in an acid medium than in an alkaline medium for sixty days. The reverse is observed from sixty to 170 days with an optimum of pH 7.80 (27). Inactivation of the virus is achieved in three minutes or less by 1 per cent phenol, 1 per cent liquor cresolis saponatus, 1 per cent metaphen, 1:10,000 KMnO 121,000 HgClz, 95, 70, 40, and 25 4’ per cent ethanol, 1:1,000 tincture of Zephiran, 1 per cent Lugol's solution, 1:20 NaOH, 5 per cent Ne0prontosil, and 1 per cent for- malin (26). Diagnosis Clinical symptoms and history may be employed only for a presumptive diagnosis. Definitive identification of infectious bronchitis can be accom- plished in the laboratory by: (1) isolation of the virus in embryonating chicken eggs, or (2) serum neutralization tests for the identification of specific antibodies (6, 10, 34). Isolation of the virus Cultivation of IBV in embryonating chicken eggs was first reported in 1937 by Beaudette and Hudson (10), who used the chorio- allantoic membrane route of inoculation. Beaudette and Hudson (10) and Delaplane and Stuart (35) found that the first few passages of the virus in embryonating chicken eggs by the chorioallantoic membrane route there was but slight embryo mortality. With successive passages, virus increased in virulence for the embryo. With some strains complete adaptation of the virus to the embryo with respect to the mortality occurred by the sixty- fifth passage. With other strains this was observed at the ninetieth and one hundred twenty-fifth passages. Completely egg-adapted strains produced 100 per cent mortality on the second day after inoculation. Delaplane and Stuart (35) reported later that IBV adapted earlier to embryonating chicken eggs by inoculation via the allantoic cavity rather than the chorioallantoic membrane. IBV can be readily isolated and propagated in embryos by the chorio- allantoic and amnionic and allantoic cavity routes of inoculation (29, 59). Increased virulence of the virus for embryo by successive passages is accompanied by a decrease in pathogenicity for chickens. Completely egg-adapted virus is nonpathogenic and nonantigenic for chickens (53). Cunningham and El Dardiry (25) studied the distribution of the virus in embryonating chicken eggs following inoculation via the allantois and found a higher concentration in the chorioallantoic mem- brane followed in decreasing order by allantoic fluid, amnionic fluid, and liver. Yolk material was innocuous. A decrease in titer was observed when eggs were left in the incubator after death of the embryo. An interfering substance in the allantoic fluid of such eggs was detected by Groupé (49). Curling and dwarfing of the embryo are the outstanding gross alterations following inoculation with virus initially isolated from chickens (10, 34, 42, 70). These lesions are pathOgnomonic of in— fection with IBV (70). Thinning and adherence of the chorioallantoic membrane to the shell membrane, fibrosis of the amnion, sluggish and weak embryos, and immaturepfeather development are also ob- served.- Microscopic alterations include proliferation of mesodermal and ectodermal cells, edema of the amnionic membrane, necrosis and hemorrhage of the liver, interstitial nephritis, congestion of the spleen, and slight capillary congestion of the brain. Inclusion bodies are not found. Serum neutralization test (SN) Ability of anti-infectious bronchitis serum to neutralize IBV was first reported by Beach and Schalm (6). Mixtures of virus and serum were not infectious for susceptible chickens. This method was later used with embryonating chicken eggs. It was found by Cunningham (22) that the serum neutraliza- tion titers of normal birds not previously exPosed to IBV would not be expected to exceed 10151.7 i 100'0376, or thirty-six neutralizing doses. Fabricant (44) suggested a minimum of one hundred neutral- izing doses for the diagnosis of the disease. This level is usually obtained in serum collected from chickens three weeks after infec- tion (79). Jungherr and Terrell (60) and Fabricant (44) found that the SN test is reliable when the virus containing at least 104 to 10 embryo infective doses is used and if certain technical details are observed. According to Page (79), the results obtained with the serum «1 -2 neutralization test employing serum diluted 10 and 10 may be adjusted with the respective log unit to give an accurate assessment 10 of the results that would have been obtained by using undiluted serum. IB-immune serum undergoes no significant change in neutral- izing capacity following exposure to 4°C for eight weeks, seven days at 22°-25°C., and at 37°C. a tenfold decrease occurs after fifty-six hours (79). The LDSONI of pooled sera varied approximately 0.4 to 1.0 log unit from the average LD N1 of the individual serum samples 50 comprising the pooled samples. No explanation was offered (38). IBV fails to agglutinate chicken red blood cells and the he- magglutination-inhibition test cannot be used to identify either the virus or specific antibodies (4, 24, 30, 32, 41). Control Birds recovered from infectious 'bronchitis are immune for at least one year, but. in some cases an overwhelming infection may re- establish the disease (69). Naturally acquired passive immunity in chicks was first de— scribed by Jungherr and Terrel (60). Hofstad and Kenzy (54) have shown that five-day-old chicks with high SN titers were susceptible to the disease. 11 Healthy young birds can be successfully immunized by expo- sure to chicken-propagated IBV with little or no retardation in growth (7, 30, 34, 68). Immunization of commercial flocks was first based upon this principle. Young birds preferably between ten to fourteen weeks of age were inoculated intratracheally with chicken-prepagated IBV. About 1 per cent of the flock was inoculated and the disease spread to the rest of the flock in a few days (31, 88). This program had limitations such as the lack of a standardized virus, the possibility of transmission to susceptible flocks, the failure of ”takes" in some birds, and the appearance of concomitant infections (88). Commercially available live—virus infectious bronchitis vac- cines contain virus cultivated in embryonating chicken eggs through a sufficient number of passages to decrease the virulence of the virus but to retain sufficient immunogenicity. These vaccines may be ad- ministered i'ntranasally to individual birds, incorporated in the drink- ing water for autoinoculation, or applied as a dust for inhalation inoc ulation . MATERIAL AND METHODS Experimental Animals Eleven adult, single-comb, White Leghorn cockerels, obtained from the United States Regional Poultry Research Laboratory, East Lansing, Michigan, were used in the experiment. These birds were maintained in batteries in previously disin- fected quarantine quarters. . . 1 . . A commerc1a1 growmg mash containing not less than 18 per cent protein, not less than 3.50 per cent fat, and not more than 6.50 per cent fiber was fed ad libidum. Virus Antigens Two different strains of IBV were used. Strain V114D, embryo-adapted IBV, capable of killing all em- bryos inoculated via the allantoic cavity in twenty—four hours was used as the antigen in all in vitro serum neutralization tests. This strain was originally isolated by Beaudette and has been maintained . in this laboratory for innumerable passages in embryonating chicken eggs. Michigan State Growing Mash, manufactured by A. K. Zinn 81 Co., Battle Creek, Michigan. 12 13 Strain VL, chicken-prepagated IBV, in the form of infected lung and trachea, was used for inoculation of all experimental chick- ens. Within one week prior to initiation of the eXperiment, inocu- lum was prepared by grinding the lung and tracheal material with sterile sand and saline with a mortar and pestle. The suspension was passed through sterile cotton and cheese cloth and the filtrate was centrifuged at low speed to further separate the tissue particles. Approximately 0.2 ml. of the supernatant fluid was used to infect four six-week-old chickens by intranasal and intratracheal instilla- tion. The tracheas were scraped with sterile cotton swabs to facili- tate more intimate contact of the virus with the tracheal epithelium. On the second day after inoculation typical symptoms were observed. The birds were killed and lungs and tracheas harvested and stored at -30°C. This material was then prepared as previously described and was used for inoculation of the'experimental chickens. For further identification of strain VL, five nine-day-old embryonating chicken eggs were inoculated with the virus via the allantoic cavity. Characteristic dwarfing and curling of the embryos was observed. Allantoic fluid harvested from the embryos failed to show hemagglutination activity. 14 Blood samples for SN tests and Spectrophotometric analyses were obtained immediately prior to infection of the experimental chickens and at three, five, seven, ten, twelve, sixteen, and twenty weeks' postinoculation intervals. The chickens were fasted for eighteen to twenty-four hours before each bleeding to decrease the amount of lipids in the serum. Intracardiac puncture was employed by using sterile 20 ml. syringes fitted with 18 gauge, two-inch needles. Between bleedings the syringe and needles were washed with sterile saline. About 20 ml. of blood was collected from each bird, trans- ferred to a large test tube and allowed to coagulate in an inclined position. The coagulum was loosened by a sterile applicator stick and allowed to stand for twelve to sixteen hours at room tempera- ture. Approximately 7 to 10 ml. of serum was obtained from each sample. The serum was centrifuged at 2,500 r.p.m. for ten to fifteen minutes, transferred to a sterile screwcap vial, and stored at -30°C. to prevent any deleterious changes prior to use. In some instances this period was as long as seven to eight weeks. Since the amount of blood collected was approximately one- fourth of the total amount of blood in a chicken, bleeding intervals of not less than two weeks were employed to prevent protein deple- tion. At the end of twenty weeks only two out of eleven chickens 15 were alive. Fatality of the others was attributed to collection of blood and perhaps to severity of the disease. S e rum Studie s All individual sera were analyzed with the spectr0photometer before and after chemical fractionation. The following values were determined: - total protein, albumin, and alpha, beta, and gamma globulins. In addition, sera collected at the same time interval were pooled in equal portions and analyzed as described above. Serum . . . 1 . . neutralization tests and macro-Kjeldahl determinations were also performed on the pools. Chemical Fractionation The procedure of Wolfson et a1. (91) was employed as out- lined below: Reagents 1. Twenty—three per cent sodium sulfate solution: Dissolve exactly 23.0 grams of anhydrous sodium sulfate in distilled water at l Wilfarth‘Gunning modification of the Kjeldahl method. 16 37°C. Make up to 100 ml. with distilled water and store in an incu- bator at 37°C. 2. Twenty-eight per cent sodium sulfite solution: Dissolve exactly 28.0 grams of anhydrous sodium sulfite in distilled water at 28°C. It is difficult to make the salt soluble, but it can be dissolved with sufficient shaking. Make up to 100 ml. with distilled water and store at room temperature. 3. Saline ammonium sulfate solution: In 1 liter volumetric flask, dissolve 193 grams of ammonium sulfate in about 500 m1. of distilled water. Add 40 grams of sodium chloride, dissolve and make up to 1 liter with distilled water. Store at room temperature. 4. Biuret reagent,.Weichse1baum (90): Prepare an accurately titrated 0.2N NaOH solution. Dissolve 90 grams of Rochelle salt in about 400 m1. of this solution. Following solution, add 10 grams of CuSO4-5HZO. When the c0pper sulfate has entirely dissolved add 10 grams of potassium iodide and make up to 2 liters with 0.2N NaOH. Store in a rubber-stoppered waxed glass bottle. 5. Span-ether reagent: Mix 1 m1. of Span 20 (Atlas Powder c30.. Wilmington, Delaware) with 99 ml. of ether, U.S.P. Filter through a moderately fast paper into a 100 ml. graduated cylinder E3-1’1d make up to 100 ml. with ether. Store in a tightly corked bottle. 6. Ether, U.S.P. l7 {Determinations 1. Total protein: (a) Pipette 0.2 ml. of serum into a 10 m1. graduated cylinder and dilute to 5 ml. with distilled water. has Mix well by inversion. Am... -_c.-o..-,‘r (b) Transfer 3.0 ml. to a cuvet, add 3.0 ml. of biuret reagent and mix well by shaking. .’_ (c) Prepare the blank with 3.0 m1. of distilled water and 3.0 ml. of biuret. Save this blank for use in the de- termination of albumin plus alpha globulin (Step 2f) and in the determination of gamma globulin (Step 8g). ((1) Let the solutions stand at least 30 minutes. (Since the color is quite stable, readings may be deferred for as much as 24 hours.) Read on the photoelectric calorimeter or spectrophotometer at a wavelength of 540 millimicrons. 2. Albumin plus alpha globulin: (a) Place 2.3 ml. of 23.0 per cent sodium sulfate solu- tion in a test tube. This solution is best not pipetted since it has a definite tendency to crystallize, par- ticularly on cold pipettes. We have found it convenient 18 to use small calibrated tubes marked at 2.3 ml. and to fill these rapidly from a large burette, preparing enough at one time for a day's determination. (b) Pipette in 0.2 ml. of serum and mix thoroughly by inversion. (c) Add approximately 1.0 m1. of ether and shake vigour- ously for 30 seconds. Centrifuge for 5 to 10 minutes at 1,500 to 2,000 r.p.m. (d) After centrifugation, carefully insert a pipette through the ether layer and beneath the packed globulin, slant- ing the tube to separate the precipitate from the wall of the tube. (e) Withdraw 1.5 ml. of clear supernatant fluid and trans- fer to a cuvet. Add 1.5 m1. of distilled water and 3.0 of biuret reagent. Mix well by shaking. (f) The blank is that prepared in Step 1c. (g) After standing at least 30 minutes, read on the photo- electric colorimeter at ‘a wavelength of 540 millimi- crons. Albumin: (a) Place 4.8 m1. of 28.0 per cent sodium sulfite solution in a test tube. It is suggested that a procedure (C) (d) (e) (g) 19 similar to that advised in Step 2a be used in pre- paring these tubes. Pipette 0.2 ml. of serum and mix thoroughly by in- version. Add about 1.0 ml. of Span-ether reagent and invert gently 5 to 10 times. Centrifuge for 5 to 10 minutes at 1,500 to 2,000 r.p.m. After centrifugation, carefully insert a pipette through the Span-ether layer and beneath the packed globulin, slanting the tube to separate the precipitate from the wall. Withdraw 3.0 ml. of the clear supernatant fluid and transfer to a cuvet. Add 3.0 ml. of biuret reagent and mix well by shaking. Prepare the blank with 3.0 ml. of sodium sulfite so— lution and 3.0 ml. of biuret reagent. After standing for at least 30 minutes, read on the photoelectric colorimeter at a wavelength of 540 mil- limicrons. 4.3 Total globulin: Subtract the value for albumin obtained in Step 3g from the value for total protein obtained in Step 1d. 20 5. True albumin/globulin (A/G) ratio: Divide the value for albumin obtained in Step 3g by the value for total globulin obtained in Step 4. 6. Alpha globulin: Subtract the value for albumin obtained in Step 3g from the value for albumin plus alpha globulin obtained in Step 2g. 7. Beta globulin plus gamma globulin: Subtract the value for albumin plus alpha globulin obtained in Step 2g from the value for total protein obtained in Step 1d. 8. Gamma globulin: (a) Pipette 9.6 ml. of saline ammonium sulfate into a sturdy 15 ml. thick-walled glass or plastic test tube. Layer 0.4 ml. of serum on top of the saline ammo- nium sulfate. Mix the two components by careful, slow, repeated inversion. Continue mixing until, within a minute or two, the gradually develOping visible turbidity has reached an apparent maximum. (b) Remove 1.0 ml. of the mixture with a pipette and discard. (c) Cork the tube securely and centrifuge at 2,250 to 2,750 r.p.m. for 30 minutes. If, at the end of this time, the supernatant is found to be somewhat turbid, (d) (g) (h) 21 cool the tube for a few minutes under the cold water tap and centrifuge again. Accurate results are ob- tained only when the supernatant is crystal clear. Being extremely careful not to disturb the precipitate, gently turn the uncorked tube on its side and permit the supernatant fluid to run off. No attempt should be made to insure complete removal of the fluid at this time. Centrifuge the uncorked tubes at 2,250 to 2,750 r.p.m. for five minutes. Slowly invert the tubes and let them stand in this position on a layer or two of paper toweling or filter paper for a few minutes. Add 3.0 m1. of biuret reagent and 3.0 m1. of distilled water to the tubes and shake briskly for 30 seconds. Let stand 15 minutes. Centrifuge to deposit the slight precipitate and decant the supernatant fluid into a cuvet. Prepare the blank with 3.0 m1. of biuret reagent and 3.0 ml. of water. After the sample has stood at least 30 minutes, read on the photoelectric colorimeter. Divide the protein value obtained by 3 to obtain the globulin concentration. 22 9. Beta globulin: Subtract the value for gamma globulin ob- tained in Step 8h from the value for beta globulin plus gamma globu- lin obtained in Step 7. 10. Normal values: Obtained from pooled serum samples. Spectrophotometric Analyses 1 A Beckman Model B Spectrophotometer was used for all anal- yses. Standardization Standard curves were calculated for human albumin.2 and nor— mal chicken serum. Protein concentrations were determined by the macro—Kjeldahl method. Using 99.0 per cent pure human albumin (12) approximately 0.8 gram was weighed and diluted to 10 ml. with distilled water. From this solution several dilutions of known protein concentration Manufactured by Beckman Instruments, Inc., South Pasa- dena, California. 2 Obtained through the courtesy of Dr. Keith McCall, Michi— gan Department of Health, Lansing, Michigan. 3 All Kjeldahl analyses were done at the Agricultural Chem- istry Laboratory, Michigan State University. 23 were prepared and analyzed with the spectrophotometer. The results obtained by plotting optical density against protein concentration were used to prepare the standard curve. A. standard curve for pooled normal chicken serum was pre- pared in a similar manner and used for all readings. The results obtained for the albumin and pooled chicken serum fit the straight— line equation: Y = a + b(X) (Figure 1). Se rum Neutralization T ests All in vitro serum neutralization tests were performed ac- cording to the technic described by Cunningham (23). Nine-day-old embryonating chicken eggs maintained in an elec- 1 tric forced-draft incubator at 99.5°F. (88°F. wet—bulb thermometer) were used for this test. The eggs were examined by transillumination in order to de- termine the site of inoculation. An area devoid of large blood vessels <>pposite to the embryo and approximately 2 m1. below the base of the air cell was chosen. A hole was drilled through the shell by IT‘ieans of an electric drill without piercing the shell membrane. A Second hole was drilled above the air cell. K Manufactured by James Manufacturing Company, Fort At— kinson, Wisconsin. Concentration (Gm. 70) 7.0 5 6.0 l i 5.0 , 4.0 i l 3.0 l I l I 2.0 1.0 , '0 Pooled chicken serum 0 0 -- --- Human albumin 3 . l 0 0.1 0.2 0.3 0.4 0. Optical Density Figure 1. Comparison of total protein of human albumin and pooled chicken serum by the biuret reaction. 24 25 Tincture of metaphen was used to paint both holes. The shell membrane under the hole above the air cell was pierced by a sterile teasing needle just before inoculation in order to equalize the pres- sure produced by the injection of the inoculum into the egg. The frozen virus‘infected allantoic fluid was thawed and cen- trifuged for five to ten minutes at 2,500 r.p.m. to deposit the amor- phous urate material. From the clear supernatant fluid serial ten- fold dilutions were prepared in Difco nutrient broth1 in the proportion of 0.5 ml. of the virus to 4.5 ml. of the diluent. The serum was passed through a Swinny filter.2 Serum virus mixtures were prepared separately by mixing equal portions of each Virus dilution and undiluted serum. Quantitative virus titrations were prepared by mixing equal POrtions of each virus dilution with Difco nutrient broth. The mixtures were allowed to stand for ten minutes at room temperature and then inoculated into the eggs using 0.1 ml. per egg and five eggs per dilution. The eggs for the quantitative virus titration were inoculated last to minimize any possible deleterious effect of incubation on the virus. ‘__ 1 Difco Company, Detroit, Michigan. Manufactured by Becton, Dickinson Company, Rutherford, New Jersey. 26 After inoculation the holes were sealed with melted paraffin and the eggs reincubated and candled daily for five days. Death occurring during the first eighteen hours was attributed to trauma or other unspecific causes. These eggs were not counted in the final calculations. The 50 per cent endpoint formula of Reed and Muench (82) The LD was used to evaluate all titrations. 50 expressed as LD50 neutralization index (LD5ONI) was the difference between the recip- rocal of the virus and the serum titers. The antilog was the num- ber of neutralizing doses. RESULTS Preliminary Experiments Preliminary experiments were conducted in order to obtain mo re-detailed information about the technic to be used as well as to adjust the method to the present investigation. Storage of Serum Samples To provide for replication and further experiments it was de- sired tostore the serum samples for as long as possible without any deleterious change. Storage at -30°C. was chosen to minimize the effects of any possible bacterial contaminatiOn that would occur in Processing the samples, thus making unnecessary the use of aseptic teczhnics. To observe any modification that would occur following freez- ing and thawing, as stated by Moore (76), one portion of the normal Serum was frozen and the other portion stored at 4°C. Both sam- Ples were analyzed by the spectrophotometer for total serum protein, and no significant differences were observed. 27 28 Wavelength In order to observe if the wavelength suggested by Wolfson et a1. (91) fit the present experiment a pooled sample of-serum from normal chickens was examined for total protein at different wave- lengths . The results showed that the greatest absorption of light oc— curred at 540 millimicrons (Table I). Sodium Sulfite Concentration In choosing the technic of Wolfson £231. (91) for fractionation of the serum samples in this investigation, some facts were taken into consideration (20, 21, 64, 74). The method of Howe (55) em- pIOyed slow filtration which required more than twelve hours. Sep- aration of the fractions in the chosen technic was made by centrifug- ation as a time-saving procedure. The principle of Kingsley (63), Who employed ether to decrease the density of globulin precipitated by sodium sulfate, was used. The application of the same principle in the use of sodium sulfite for determination of albumin was of no Value. Addition of a surface active agent such as Span 20 permitted separation of globulin and did not affect the albumin values. TABLE I ABSORPTION OF LIGHT AT DIFFERENT WAVELENGTHS Optical Density Wavelength (millimicrons) Serum 11 Serum 11 400 0.14 0.25 450 0.08 0.13 500 0.17 0-33 525 0.22 0-43 540 0.23 0.45 560 0.23 l 0.43 600 0.17 0.31 625 0.11 0-40 540 0.23 0.45 x .. A Pooled chicken serum: Kjeldahl, 4.26 gram per cent; spectrophotometry, 4.40 gram per cent. Human serum: Kjeldahl, 8.50 gram per'cent; spectro- Photometry, 8.85 gram per cent. 3O Wolfson et a1. (91) modified this original method for gamma globulin by diluting the serum with ammonium sulfate 1:25. In this manner a higher yield of gamma globulin was obtained as compared to using undiluted serum. No correlation was found between immuno— logic and electrophoretic estimation of gamma globulin (56). Jager et a1. (58) found sodium sulfite the best method for albumin determi— nation after magnesium sulfate. Leland (67) applied the same technic and found that with rat serum the best concentration of sodium sulfite for albumin determina— tion was 24.0 per cent instead of 28.0 per cent, as suggested by Wolfson et a1. (91). This was determined by electrophoretic meas- Using a pooled sample of normal chicken serum the percent- age of albumin was determined with concentrations of sodium sulfite varying from 20.0 to 30.0 per cent. The same serum was analyzed elect1‘0phoretically and it was observed that the solution suggested for human serum was of equal value for chicken serum (Figure 2). Experimental Results In order to minimize the variations encountered in the serum COmponents at the several sampling periods when expressed as grams Per cent, all results have been analyzed on the basis of per cent total Albmnin (Gm. 70) 31 2.00 1.80 1.60 I Electrophoresis 1—- m__._._.._ ....2 . ..__._.._.,_,--L . ._, 1.40 ; Albumin l 23 1.20 T; t '1 t i 1 \ 1.00? \., I "a I I 9-80L_ ---- _ .. _._--__--,-__-_-_..---_- 20 22 24 26 28 30 Per Cent Sodium Sulfite Figure 2. Selection of sodium sulfite concentration for albumin determination. 32 protein of the sample. This permits a more valid interpretation of the relative per cent serum components. Individual Sera Spe c t rophotom etric re sults Spectrophotometric results obtained for the individual sera can be seen in Tables II, III, IV, V, and VI, and in Figures 3, 4, 5, 6, and 7. Total protein. The results obtained with sera from chickens 464 and 469 showed a slight increase in total protein during the pe- riod Of their participation in the experiment. The others showed a marked variation and with chicken 470 a decrease was observed. The initial values for all chickens ranged from 3.60 to 3.95 gram S per cent protein. At the seventh week the total protein had increased to 3.87 grams per cent with chickens 464 and 466. These Values remained constant at the twelfth and twentieth weeks. With Chi-Cken 469 the grams per cent. total protein was 3.95 at the seventh Week and 4.60 at the sixteenth and twentieth weeks. The reverse oc- C\lrred with chickens 468 and 470, in which the initial protein values Were 3.95 and 3.70 grams per cent, respectively. At the seventh week 1illese values had decreased to 3.58 and 3.50 grams per cent protein. 33 TABLE II SPECTROPHOTOMETRIC VALUES FOUND FOR CHICKEN 464 IN DIFFERENT PERIODS OF TIME I Jb I I I A_f 1: Time Interval (weeks) Item “w 0 3 5 7 10 12 Grains Per Cent Protein Total protein ........ 3.60 3.60 3.14 3.87 2.90 3.87 Albumin ............ 2.12 1.85 1.30 1.65 1.30 2.03 Total globulin . . . ..... 1.48 1.75 1.84 2.22 1.60 1.84 Alpha. globulin . ...... 0.38 0.65 0.93 1.10 0.55 0.55 Beta globulin ........ 0.30 0.24 0.11 0.41 0.37 0.55 Gamma globulin ...... 0.80 0.86 0.80 0.71 0.68 0.74 A/G ratio .......... 1.43 1.06 0.71 0.74 0.81 1.10 Egaflve Per Cent Serum Components Based On Total Protein Albumin ............ 58.8 51.4 41.4 42.6 44.8 52.4 Total globulin ........ 41.2 48.6 58.6 57.4 55.2 47.6 Alpha globulin ....... 10.6 18.0 29.6 28.4 18.2 14.2 Beta g1obulin ........ 8.4 6.6 3.5 10.7 12.7 14.2 Gamma globulin ...... 22.2 24.0 25.5 18.3 24.3 19.2 §: 34 .wov coono Eopm whom he musing owfioagozaopfiooom .m unawflh NH 2 N. m m o 11.0 \olol. . \O 0 ’9’01104‘1 \0 ololo ll. nfidaoHU ovum all... all... 1... 6 II . 1|. 0 \\ \w 5:530 £6211 n 7 11 1 2 - l i m sfldaofiu $8930. /7 I III 4 \ .. \y I./ \\ 1 Mom k, \ L I b I \ \ Ill. [II i IL 7‘ [/1] >/Vl|m1\ru\nlli.|uov|.l.vll.llllli .x. H 111 I 111 l\\ M 0% 52530 aduohi magnifies {-3 o.m £30.” nu ~30 H. ko Per Cent Value 5 Protein (Gm. ‘70) TABLE III 35 SPECTROPHOTOMETRIC VALUES FOUND FOR CHICKEN 466 IN DIFFERENT PERIODS OF TIME % r 1 v j L j I Time Interval (weeks) Item 0 3 5 7 10 12 20 Grams Per Cent Protegl Total protein 3.80 3.70 3.50 3.87 3.21 3.70 3.87 Albumin ....... 2.58 1.65 1.40 1.42 1.34 1.80 1.00 Total globulin . . 1.22 2.05 2.10 2.45 1.87 1.90 2.87 Alpha globulin 0.27 0.93 1.10 1.33 0.77 0.78 1.80 Beta globulin 0.40 0.27 0.17 0.30 0.35 0.32 0.27 Gamma globulin 0.55 0.85 0.83 0.82 0.75 0.80 0.80 A/G ratio ..... 2.11 0.80 0.66 0.58 0.71 0.94 0.34 Relative Per Cent Serum Components Based on Total Protein Albumin ....... Total globulin . . . Alpha globulin Beta globulin Gamma globulin 67.9 32.1 7.1 10.5 14.5 44.5 55.5 25.1 7.5 22.9 40.0 60.0 31.4 4.8 23.8 36.7 63.3 34.3 7.7 21.3 41.7 58.3 24.0 10.9 23.4 48.6 25.8 51.4 74.2 21.1 46.5 8.7 7.0 21.6 20.7 ' I 36 .034 Goono 80.3 whom Mom moddg oflhpoEoQoflaonpooam .v ohdwfim 3303 on 2 ---.---il.il.NHt..ib.Fs .-:.N. . m .-m..-.i..lllo 1 . N 5:550 noomol. 1. o .1. .I. .71 4‘4 5532 55550 25.6. . / \ \ « 5350 18.3. \ Empoum H.308 64.. Per Cent Values Protein (Gm. %) TABLE IV 37 SPECTROPHOTOMETRIC VALUES FOUND FOR CHICKEN 468 IN DIFFERENT PERIODS OF TIME %a L Time Interval (weeks) Item 0 3 5 7 10 12 Grams Per Cent Protein Total protein ........ 3.95 3.70 3.50 3.58 3.70 3.50 Albumin ............ 2.03 1.65 1.65 1.60 1.45 1.85 Total globulin ........ 1.92 2.05 1.85 1.98 2.25 1.65 Alpha globulin ....... . 0.47 0.75 0.35 0.63 0.95 0.38 Beta globulin ........ 0.80 0.44 0.60 0.55 0.50 0.53 Gamma globulin ...... 0.65 0.86 0.90 0.80 0.70 0.74 AM] ratio .......... 1.05 0.80 0.89 0.81 0.64 1.12 Relative Per Cent Serum Components Based on Total Protein Albumin ............ 51.4 44.5 47.1 44.7 39.1 52.6 Total globulin ........ 48.6 55.5 52.9 55.3 ' 60.9 47.4 Alpha globulin ....... 11.9 20.3 10.1 17.8 25.7 10.8 Beta globulin ....... 20.2 11.8 17.1 15.3 13.5 15.5 Gamma globulin ...... 16.5 23.4 25.7 22.2 21.7 21.1 38 v soono 80pm whom new 930m mofidg oauuoaoobcrmoyoboam .m 6.3th .wo mxoog NH E 1121-11-11; -1. .1.....Nu.11111...11.1am... 1:11..- _ .m . .- 11111.10 0 G.“ o a a I \1 1 . 0: egg H0 g H< "lll. D \O ‘u b ‘0 I o O \\ [’1’ °\\ 0 / \\ \ GSSABO duomo «a 11 1 .I. 0 (o .o\// 161 1 1 a u I \\ II \\ / \ ’I I \ \ \ I \ 1‘ \1 ’\\q / HHHHHflnflOH—HU NEENU X (I i A! K. I in l .9 III x /1 1“ 11‘. ”ON 1‘ \ / .1.“ V . 58.934 17 1. 11111111 1 1 1 1 I \‘ \ ‘ II. I \ \ I 0’ \ \ / \I \ \. / 1 . .oo 53on ago 9/11111111111111111111)./1/1 O M J O ‘1‘ Per Cent Values Protein (Gm. %) TABLE V 39 SPECTROPHOTOMETRIC VALUES FOUND FOR CHICKEN 469 IN DIFFERENT PERIODS OF TIME % j - Time Interval (weeks) Item 3 5 7 10 12 16 20 Grams Per Cent Protein Total pro- tein . . . 3.80 3.40 3.95 3.70 3.87 4.60 4.60 Albumin . . 1.85 1.58 1.65 1.44 2.05 2.03 1.47 Total globe ulin . . . 1.95 1.82 2.30 2.26 1.82 2.57 3.13 .Alpha glob- ulin . . . 0.90 0.65 1.00 1.24 0.90 0.37 1.23 Beta glob— ulin . . . 0.25 0.37 0.53 0.26 0.12 1.40 1.07 Gamma globu- 1in 0.80 0.80 0.77 0.76 0.80 0.80 0.83 A/G ratio . . 0.94 0.86 0.72 0.64 1.12 0.79 0.47 Relative Per Cent Serum Components Based on Total Protein Albumin . . 48.6 46.4 41.7 38.9 53.0 44.1 32.0 Total glob- I ulin . .. 51.4 53.6 58.3 61.1 47.0 55.9 68.0 Alpha glob— ulin . .. 23.7 19.1 25.3 33.5 23.2 8.1 28.4 Beta glob- ulin. .. 6.7 10.9 13.4 7.1 3.2 30.4 21.9 Gamma globu- lin 21.0 23.6 19.6 20.5 20.6 17.4 17.7 a Lost. 40 .mouv C3320 scum whom How 935w modig ofiuuoEOpofiaohuuomm .o 0.32th mxooNS ON a: NH OH N. m m 9 \ I Q /0/ O\0 \ I O 01‘ \ / 10 CH 5 o “88.“ [6" NIL K... I I I I, .H n #0 U \\ A} Kid" -‘III fl -X‘I‘l‘)/\X.\\ ’JL‘\* “3.3530 ovum oloxx o / . \ /u1\.. I 1 1.. \ < ,, \1 a. \ / I \ 536.30 5 3. 8 . , 1 ngndfl £35330 .7308 .1 3.511509% 18.0 .H. o m on m a V .m e C r 0* e P co 0.2% m @1 .m m o «4m P TABLE VI 41 SPECTROPHOTOMETRIC VALUES FOUND FOR CHICKEN 470 IN DIFFERENT PERIODS OF TIME % 4 Time Interval (weeks) Item LL 0 3 5 7 10 Grams Per Cent Protein Total protein ............. 3.70 3.87 - 3.50 3.55 Albumin . . .‘ .............. 1.85 1.65 - 1.30 1.55 Total globulin ............. 1.85 2.22 - 2.20 2.00 Alpha globulin ............ 0.90 0.85 - 1.15 1.10 Beta globulin ............. 0.15 0.42 - 0.23 0.20 G amma globulin ........... 0.80 0.95 - 0.82 0.70 A-JG ratio ............... 1.00 0.89 - 0.59 0.77 Relative Per Cent Serum Components Based on Albumin ................. Total globulin ............. Alpha globulin ............ Beta globulin ............. G amma globulin ........... 50.0 50.0 24.3 4.1 21.6 42.6 57.4 21.9 11.0 24.5 Total Protein - 37.1 43.4 - 62.9 56.6 - 32.8 31.0 - 6.7 5.9 - 23.4 19.7 \-\\ 42 .054 noono 80.5 whom .80 9.30m musing awhpogogfimohuoomm 9‘00? 0H N. m m 51530 «BBQ. 1. o 11 01 o1| 0 .N. whom?” S . e £25330 088.60.. (73.1191 .omtm It I‘Ifllvl .111 a 1! Tito.) 1\ ‘11 ”111‘“ W'ATVI‘J "m \\ 1‘1! 1‘ t 50550 35¢-111-111,111111-11 m C r 0V 6 .1/1 53550 253.---.. 1 1 1.1.11 5.8.80 Eon. /\ '0 co cf w . Protein (Gm. %) 43 A“: the twelfth week there was a further decrease to 3.50 grams per Qelm: for chicken 468. Variations that occurred during the intervening periods did not significantly alter the general trend. Albumin. Initial values for albumin ranged from 50.0 to 67.9 Per cent of the serum total protein. In general there was a decrease in albumin up to the seventh or tenth week followed by an increase at the twelfth week. With bird 464 the lowest value, 41.4 per cent, Was obtained at five weeks. Between the tenth and twelfth weeks an appreciable increase to 52.4 per cent was observed. With chicken 466 a low value of 36.7 per cent for albumin Occurred at the seventh week. For chickens 468 and 469 low values 01' 39.1 and 38.9 per cent, respectively, occurred at the tenth-week p eriod. The initial values for birds 466, 468, and 469 were, respec- tively, 67.9, 51.4, and 48.6 per cent albumin. The increase observed at the twelfth week was followed by a decrease at the sixteenth week for bird 469. Albumin values at the end of the experiment (20 weeks) Were reduced to 25.8 and 32.0 per cent for birds 466 and 469, re- Sjpectively. A. decrease from 50.0 to 37.1 per cent albumin was observed With chicken 470 between the beginning of the experiment and the 44 Saw enth-week period. At the tenth week this value had increased to 43 *4 per cent. Total globulin. With the exception of chicken 469, for which the value for the original sample was not available, all birds showed an increase in total globulin at the third week. This increase was marked with bird 466 for which the total globulin increased from 32.1 to 55.5 per cent. With other birds this increase was not so marked. After reaching a maximum value of 58.6 per cent at the fifth Week, chicken 464 had a decrease in total globulin up to the twelfth Week. With bird 466, a value of 63.3 was obtained at the seventh week, but the maximum of 74.2 per cent occurred at the twentieth week Alpha globulin. Examining the initial results, it was f0und that alpha globulin ranged from 7.1 to 24.3 per cent. Results ob- tained with individual birds showed some variation. With chickens 4 64 and 466 a similar pattern was obtained. An increase up to the fifth- or seventh-week period was noted. These values were 29.6 and 34.3 per cent, respectively. From this point through the twelfth Week a decrease was observed. At the end of twenty weeks the value for bird 466 was 46.5 per cent. This was more than a sixfold in— Q rease from the original value. 45 The same phenomenon was seen with bird 469. After reach- 111g 3. 33.5 per cent value at the tenth week, a decrease to 8.1 per Cent was observed at the sixteenth week. The final result at the end of twenty weeks was 28.4 per cent. Chickens 468 and 470 showed a large variation. Initial values were 11.9 and 24.3 per cent, re- Spectively. At the tenth week chicken 468 reached 25.7 per cent, Whereas the highest value with chicken 470, 32.8 per cent, was ob- t23.:ined at the seventh week. At the tenth week only chicken 470 had a. value higher than at the beginning. Beta g1_obu1in. Results found for beta globulin showed a marked Variation. Initial values ranged from 4.1 to 20.2 per cent of the total p rotein. With bird 464 a decrease was observed at the fifth week when the value was as low as 3.5 per cent. By the twelfth week a sub- Stantial increase to 14.2 per cent was recorded. A similar picture was seen with chicken 466. The only dif- ference was that, after ten weeks, the values were more or less c-‘-<)nstant at about 8 per cent through the twentieth week. A decrease from 20.2 to 11.8 per cent beta globulin was ob- Served between zero and three weeks with bird 468. The results at the subsequent periods showed a regular increase and at the end of twelve weeks a value of 15.5 per cent was found. 46 The highest value for bird 469, 30.4 per cent, was observed at the sixteenth-week period. This was 4.5 times greater than the Q 1‘ i ginal value. With bird 470 a variation occurred. There was an increase from 4.1 to 11.0 per cent between zero and three weeks. After that a. slight decrease through the tenth week was observed. Final value Was 5.9 per cent, slightly higher than at the beginning. Gamma globulin. Initial values for gamma globulin ranged from 14.5 per cent for chicken 466 to 22.2 per cent for chicken 464. There was a general increase between the beginning of the experiment through'the fifth-week period. The greatest increase occurred with bird 466, from 14.5 to 23.8 per cent. With birds 464, 466, and 469 a decrease at the seventh week was observed followed by an increase at the tenth week. A subse- C1uent decrease occurred at the twelfth week and extended through the twentieth week with birds 466 and 469. Values obtained at the last bleeding period for these chickens were 19.2, 20.7, and 17.7 per cent gamma globulin, respectively. Chickens 468 and 470 showed a decrease between three and ten weeks to 21.7 and 19.7 per cent, respectively. Final values for 1Zhese chickens were 21.1 and 19.7 per cent gamma globulin, respec- 1lively. 47 A/G ratio. The A/G ratio at the beginning of the investiga- tion ranged from 0.94 to 2.11. A general decrease through the fifth a«TI-c1 seventh weeks was observed. The most marked decrease was S'klown with bird 466, in which the ratio changed from 2.11 to 0.58. After this point an increase in A/G ratios was observed through the tenth and twelfth weeks. With chickens 466 and 469, values as low as 0.34 and 0.47, respectively, were observed at the end of twenty weeks. Pooled Sera Spectrophotom etric re s ults The results obtained are shown in Table VII and Figures 8 and 9. Total protein. At the beginning of the experiment the total protein was 373 grams per cent by the macro-Kjeldahl method and 3 .60 grams by spectrophotometry. These values remained without marked variation up to the tenth week, when they were 3.6 and 3.4 grams per cent, respectively. While there were slight differences in the values obtained by the two methods at the intermediate pe- riods, there was a definite parallelism with the values obtained by 1:he Kjeldahl method being uniformly higher than those obtained by TABLE VII 48 SPECTROPHOTOMETRIC AND MACRO-KJELDAHL DETERMINATIONS ON POOLED SERA Time Interval (weeks) Item 0 3 5' 7 10 12 16 20 Grams Per Cent Protein Total pro— tein (ml. 3.73 4.08 3.86 3.99 3.60 3.82 - 4.20 Total pro- tein (5)2. 3.60 3.87 3.70 3.87 3.40 3.31 4.60 3 87 Albumin . . 1.84 1.75 1.55 1.65 1.47 1.65 2.03 1 40 Total glob- ulin . . . . 1.76 2.12 2.15 2.22 1.93 1.66 2.57 2 47 Alpha glob-t ulin . . . . 0.65 0.83 1.03 1.05 0.46 0.75 — 1.10 Beta glob- . ulin . . . . 0.23 0.43 0.37 0.83 0.79 0.21 - 0.63 Gamma globulin 0.88 0.86 0.85 0.84 0.70 0.70 - 0.74 A/G ratio 1.04 0.83 0.72 0.74 0.76 1.01 0.75 0.56 Relative Per Cent Serum Components Based on Total Protein Albumin. . 51.2 42.7 41.9 42.7 43.4 50.0 44.0 36.2 Total glob- ulin . . . . 48.8 57.3 58.1 57.3 56.6 50.0 56.0 63.8 Alpha glob- ulin . . .. 18.1 21.4 25.2 24.5 13.5 22.6 - 28.4 Beta glob- ulin . . . . 6.3 10.7 10.0 11.1 23.2 6.3 - 16.2 Gamma globulin 24.4 25.2 22.9 21.7 19.9 21.1 - 19.2 § 1 Kj eldahl method. Spectr0photometry. . Flu. JIWNldrrffl if: {‘U .MHMm “0300a How media; owhaoEooaoflm0Apovmm .w ohswfirm 9 3063 4 om 2 2 S -.--!,~...l..im; m oo 0 \o o \o o\0\\ /o ‘O‘OIOI \Q\ 9\9\° \ / 9\0 Q S 53330 dummo\i o\ / \e\ i. t) N m 53330 dfifiooulxiils I. alxiuillilv.\/u.luul.tl.lilx xiiit o a . \nll‘\1.|lui|nl" 11‘1'1'1'!‘\/\*./$||11.\|$.\I|* V l \ t. 1 .t -ititi n £320 £12 - a r fiendfl< mow me. illiilrllli/a \\ \ \ \ ............ oo £320 :33. x 9m 0 m [ox ghaogouOSQOHuommmv .m Eoooem :33. cam r Edwfiommv P 538m 30.5 ii 50 .mnom vofiooa mo oflmh O\< you 930w mogm> mxoog om 3 NH . 0H .0 oudmfim 0"-.. 0N6 .ovd cod ow.o wooé 0N4 ouvA 09H owé oo.~ A/G Ratio 51 spectrophotometry. At the twelfth week there was a variation in the parallelism in that there was an increase in the value obtained by the Kjeldahl method and a decrease in the value found by spectro- photometry. Unfortunately serum was not available for analysis by the latter method at the sixteenth week and a comparison cannot be made to the spectrophotometric analyses which showed a marked in- crease in total protein. At the twentieth week the total protein as analyzed by both methods was 4.20 and 3.87 grams per cent, re- spectively. This was an increase from the beginning of the exPeri- ment of 0.47 grams per cent by the Kjeldahl method and 0.27 grams per cent by the spectrophotometric method. During the course of the investigation there were only two periods at which the total protein was lower than at the beginning. This occurred at the tenth and twelfth weeks as analyzed spectro- photometrically and at the tenth week when analyzed by the Kjeldahl method. Albumin. The initial value for albumin was 51.2 per cent in relation to the total protein value of the serum. At the third week this value decreased to 42.7 per cent and remained constant until the twelfth week when the value was 50.0 per cent. At the end of the twentieth week the albumin value decreased again to 36.2 per cent which was the lowest value during the course of the investigation. ‘3?" m-F—H 52 Total globulin. At the beginning the total globulin was 48.8 per cent. An increase to 57.3 per cent was observed at the end of the third week. This value remained constant until the end of the tenth week when a decrease occurred. There was 50.0 per cent al- bumin at the twelfth week, but this value increased to a maximum of 63.8 per,cent at the twentieth week. Alpha globulin. Initially the alpha globulin was 18.1 per cent followed by an increase to 21.4 per cent at the third week. This value continued to increase to 25.2 per cent at the fifth week and then a decrease occurred. The lowest value was observed at the tenth week when only 13.5 per cent of the total protein was consti- tuted by albumin. A recovery followed and at the end of the twenti- eth-week period alpha globulin reached a maximum value of 28.4 per cent. Beta globulin. From the beginning of the experiment through the tenth week there was an increase of beta globulin from 6.3 to 23.2 per cent. At the twelfth week the values were 6.3 per cent, followed by an increase to 16.2 per cent at the twentieth week. Gamma globulin. Gamma globulin values increased from 24.4 per cent at zero weeks to 25.2 per cent at the end of the third week. 53 Following this period there was a general decrease to 19.2 per cent at the end of the twentieth week. Afi/G ratio. A/G ratios showed a direct correlation with the data obtained for albumin and total globulin. The values for these two components showed an indirect relationship at the various sam— pling periods. The A/G ratio was 1.04 at the beginning of the experi- ment followed by a decrease to 0.83 at the third week. From this point to the tenth week the ratio was rather constant. At the twelfth week the ratio was 1.01, and at the twentieth week it was 0.56 (Fig- ure 9). Serum neutralization te sts The results seen in Table VIII and Figure 10 show that the 0. LDSONI for the preinoculation sample was 10 5, or three neutralizing doses. This was well within the accepted range of normal chicken serum (22). Following inoculation there was a marked increase in antibodies to the seventh week, when the maximum level of LD 6. 10 5, or 3,162,000 neutralizing doses, was obtained. This level was soNl' constant through the twelfth week followed by a decline to LDSONI .6 of 105 , or 398,100 neutralizing doses at the sixteenth week, and .4 LDSONI of 105 , or 251,200 neutralizing doses at the twentieth week. TABLE VIII 54 SERUM NEUTRALIZATION TESTS ON POOLED SERUM SAMPLES e —:=:=::: - —— _L_x Virus Serum T. . . ““6 Titer Titer LD NI Neutrahzmg (weeks) LD LD 50 Doses 50 50 0 10"7‘o 10‘6'5 106’5 3.1 - . -2. 4. 3 10 6 6 10 6 10 6 10,000 - . -1, . 5 10 7 6 10 6 106 6 1,000,000 7 10’6'5 2106 ”5106'5 ">- 3,162,000 10 10‘65 2100 '>_106'5 3‘ 3,162,000 -6. —— _ 6. ._. 12 10 5 < 100 > 10 5 > 3,162,000 - . -1. .6 16 10 6 6 10 6 105 398,100 20 10'6'6 10'1'2 105'4 251,200 l I! I5} ...,. t: a LD5O Neutralization Index Exponent Log Base 10 55 8.0 o 6.0 1 4.0 2.0 I 0 -_-- menu--.“ -.-_.----..._ 0 3 5 7 10 12 16 20 Weeks Figure 10. Sermn neutralization test of pooled sera. 56 The antibody response as shown with the serum neutralization test was typical of infection with this virus. Hemagglutination-inhibition tests of all sera for the presence of Newcastle disease virus antibodies were negative. DISCUSSION Chicken serum and plasma have been extensively investigated (with the use of the electrOphoretic technic (15, 38, 75, 83, 84). The values found fornormal chickens showed a wide variation in the rel- ative per cent distribution of the individual serum components. San Clemente (83), using phOSphate buffer at pH 7.7, 0.2 ionic strength, found the following per cent values: albumin, 35.0; alpha globulin, 15.0; beta globulin, 5.0; and gamma globulin, 45.0. A/G ratio found was 0.54. With veronal buffer at pH 8.6 the electrophoretic analyses of plasma of male chickens were as follows, according to Deutsch and . Goodloe (36): albumin, 38.2 :t 1.3 per cent; alpha l-globulin, 15.8 :i: 0.6 per cent; alpha 2-globu1in, 7.7 :1: 0.5 per cent; beta globulin + fibrinogen + gamma globulin, 37.5 :I: 1.3 per cent. Deutsch M. (37) used veronal buffer at pH 8.6, 0.1 ionic strength, and found the following average per cent composition: albumin, 46.0; alpha globulin, 22.0; beta globulin, 8.0; and gamma globulin, 24.0. Sanders £13.31. (84), using veronal buffer at pH 8.6, 0.1 ionic strength, found for Leghorn chickens the following average per cent 57 58 values: albumin, 46.0; alpha globulin, 13.1; beta globulin, 15.7; and gamma globulin, 21.6. Dim0poullos (38) reported the following values: albumin, 50.5 per cent; alpha, beta, and gamma globulins, 13.5, 11.1, and 24.8 per cent, respectively. A/G ratio found was 1.02. Results found in the present study using the spectrophotometric technic for pooled sera from adult, single-comb, White Leghorn cock- erels prior to infection with IBV were as follows: albumin, 51.1 per cent; alpha globulin, 18.0 per cent; beta globulin, 6.4 per cent; and gamma globulin, 24.5 per cent. A/G ratio was 1.04. These values showed a reasonable agreement with those found by electr0phoresis. Qualitative and quantitative changes in the protein content of serum and its fractions can be induced by physiological as well as pathological conditions such as age, sex, nutritional state, injury, loss of plasma protein, and severity of the disease. Some references will be mentioned to substantiate such assertions. The work of Mad— den and Whipple (71) is a good review on plasma proteins, their source, production, and utilization. Modifications through the age have been shown by Brandt it 31. (15), who reported a decrease in albumin and an increase in glob- ulin as chickens matured. 59 Bjdrneboe (13) observed that, upon immunization of animals with bacteria, a change in the electrophoretic patterns occurred. There was a direct correlation between globulins and antibody pro- tein. A direct correlation between increased gamma globulin and antibody titers has been shown by several workers (13, 14, 72, 86, 92). All gamma globulin is not antibodies, nor are all antibodies ‘ l encountered only in the gamma globulin fraction (40, 61, 62). J It is known that electr0phoretic patterns are changed from the normal due to protein depletion (39) or by plasmapheresis (11, 19, 93). The antibody-fabricating mechanism is impaired in hypo- proteinemia (11, 18). In an extensive investigation of sera from nOrmal and IB— infected birds as analyzed electrophoretically it was found that, in chickens bled at weekly intervals, signs of protein depletion were evident by the inversion of the A/G ratios. This was not observed when birds were bled at monthly intervals (38). Injury is responsible for some changes occurring in the elec- trophoretic patterns of sera from dogs and goats (47, 48). Leland (67) reviewed this aspect in an investigation of sera from rats im- munized against the nematode, Nippostrongylus muris. An increase in beta globulin was found, but this was attributed to trauma in the 60 lungs due to formation of cysts by the parasite. The lungs were considered to be the probable site of antibody formation and not re- sponsible for the increase in beta globulin. No specific changes in the electrophoretic patterns of sera were found when birds were subjected to tracheal injury similar to that used for inoculation of IBV into susceptible chickens (38). From the results obtained during the course of the present investigation the first point to be considered is the slight increase in the total protein as shown by macro-Kjeldahl and spectr0photo- metric methods. A reasonable agreement was obtained although values by the Kjeldahl method were consistently higher than those obtained with spectr0photometry. Differences between the two methods ranged from 0.17 to 0.51 grams per cent protein, corresponding to 4.5 to 13.3 per cent deviation. Albumin values decreased, and as a result the A/G ratios were lowered considerably at the bleeding periods subsequent to the exposure of birds to IBV. An increase of globulins with decrease of albumin appears to be the characteristic serum alteration in many diseases (50). A rise in globulins can be produced by: (1) forma- tion of antibodies, (2) alteration of the relative production and utiliza- tion of albumin and globulin, and (3) compensatory rise in globulins as an attempt to maintain the osmotic pressure. 61 The first two conditions have already been discussed. Con- sideration must be given to the third condition for increase in globu- lins. Increase in globulins and a decrease in albumin is somewhat a compensatory mechanism. Albumin is responsible in great part for the osmotic pressure. Osmotic pressure depends upon the num- F] I ber of particles and not the size, and it has been proposed that the globulin concentration compensates for any decrease in albumin. 1 .a x- “Ft—M- I ‘_ . A "“1 _ According to quirneboe (13), a definite relationship between albumin and globulin concentration maintains the Optimal osmotic pressure. This hypothesis has not been accepted generally (46). In an endeavor to explain the results obtained it must be emphasized that a large volume of blood (20 m1., or approximately one-fourth of the total blood volume) was removed from each bird at every bleeding period. Although the intervals between bleedings were never less than two weeks, it is possible that this interval might not have been sufficient for the complete restoration of the original balance of the serum components. Results found with pooled sera when compared with those of individual chickens showed a fair agreement for total protein, albumin, total globulin, gamma globulin, and A/G ratios. 62 Alpha globulin values for individual birds were quite variable. Results with pooled sera showed a tendency to increase, except be- tween the fifth and twelfth weeks, when a small decrease was ob- served. Final values were higher than those observed at the begin- ning of the experiment. With beta globulin a marked variation was also found. The tendency shown by the pooled sera was an increase up to the tenth week, followed by a sharp decrease at the twelfth week. Values at the end of twenty weeks were higher than the initial figures. Increase in globulins was due especially to increase in alpha and beta globulins, since with gamma globulin a very small variation was observed. Results obtained by the serum neutralization test of pooled sera showed a close agreement with those found by other investigat- ors (38, 78, 79). The decrease in antibody titer observed by Page (78) in the eighth week after inoculation was not confirmed. The highest antibody titer (1065) occurred at the seventh, tenth, and twelfth weeks after inoculation. Prior to infectionwith IBV the LDSONI was 1005, or three neutralizing doses, confirming results found by other investigators (22, 38, 78, 79). ‘3;— ,: 63 There was no correlation between the changes in serum com- ponents and antibody content. When the serum components had re- turned to preinfection levels the antibody content was at the maximum. This was also observed previously by Dimopoullos (38) with the elec- tr0phoresis apparatus. No definite changes in serum electrophoretic patterns were found in animals immunized with West and Venezuelan equine encephalo- myelitis (77) and Japanese B encephalitis (66). Viruses probably be- have differently from bacteria in the formation of antibodies. Determination of antibody content in the fractions of anti-1B serum seems to be highly desirable. Short bleeding intervals and withdrawal of large amounts of blood should be avoided. “ -“kg‘n SUMMAR Y 1. Spectrophotometric and serum neutralization studies were performed with sera of normal and IBV-infected adult, single-comb, White Leghorn cockerels. 2. Experimental birds were bled by intracardiac puncture im- mediately before inoculation with IBV and at three-, five-, seven-, ten-, twelve-, sixteen-, and twenty-week postinoculation intervals. 3. Sera obtained from these chickens were clarified by cen- trifugation and kept at -30°C. until used. 4. Spectrophotometric analyses were performed with Beckman Model B SpectrOphotometer on individual sera before and after chem- ical fractionation by the method of Wolfson et al., and the following values were determined: total protein, albumin, and alpha, beta, and gamma globulins. 5. Pooled sera from birds bled at the same time intervals were analyzed spectr0photometrically and serum neutralization tests‘ were performed. Macro-Kjeldahl analyses were also made of these pools. 6. A. close agreement was observed in the total protein de- termination by the macro-Kjeldahl and the spectrophotometric 64 65 methods. Values obtained with the former method were slightly higher. 7. Results obtained by the use of the spectrOphotometer after chemical fractionation showed a fair agreement with results found by electrophoresis. 8. A decrease in albumin and a consequent inversion of the A/G ratio was observed after IBV inoculation. Alpha and beta glob- ulins were specifically responsible for the increase in globulins. Gamma globulin had no appreciable influence, as it was of a rela- tively constant concentration throughout the experiment. 9. Serum neutralization tests conducted on pooled sera showed typical results following IBV infection. A maximum titer was obtained between seven and twelve weeks after inoculation. 10. There was no correlation between the changes in serum components and antibody content. When the serum components had returned to preinfection levels the antibody content was at the max- imum . w‘3mw1 LITERATUR E CITED Asplin, F. D. 1948 Identification of infectious bronchitis of chickens in England. Vet. Record, 60: 485-486. Balch, H. H. 1950 Relation of nutritional deficiency in man to antibody production. J. Immunol. 64: 397-410. Beach, J. R. 1934 Coryza and other respiratory diseases in chicks. Proc. XII Int. Vet. Congr., 3: 144-160. Beach, J. R. 1948 The application of the hemagglutination-inhibition test in the diagnosis of Avian Pneumoencephalitis (New- castle disease). J. Amer. Vet. Med. Assn., 112: 85-91. Beach, J. R. 1948 Chapter 19. Diseases of Poultry. Second edition. Edited by H. E. Biester and L. H. Schwarte. The Iowa State College Press, Ames, Iowa. 1154 pp. Beach, J. R., and O. W. Schalm. 1936 A. filterable virus distinct from that of laryngotracheitis the cause of a respiratory disease of chicks. Poult. Sci., 15: 199-206. B eaudette , F R. 1949 Twenty years of progress in immunization against virus diseases of birds. J. Amer. Vet. Med. Assn., 115: 367-377. 66 10. 11. 12. 13. 14. 15. 16. 67 Beaudette, F. R. 1950 Infectious bronchitis. (Differential characteristics from Newcastle disease.) Can. J. Comp. Med., 14: 24-27. Beaudette, F. R 1951 Infectious bronchitis and Newcastle disease. Can. J. Comp. Med., 15: 67-71. Beaudette, F. R., and C. B. Hudson. 1937 Cultivation of the virus of infectious bronchitis. J. Amer. Vet. Med. Assn., 90 (N.s. 43): 51-60. Benditt, E. P., et a1. 1949 Loss of body protein and antibody production by rats on low protein diets. Proc. Soc. Exp. Biol. Med., 70: 240-243. Bernhard, A., and Y. Scher. 1951 Bovine albumin standard for serum protein determina- tions. Science 114: 674. Bjdrneboe, M. 1944 Serum proteins during immunization. Acta Path. Microb. Scand., 20: 221-239. Boyd, W. C., and H. Bernard. 1937 Quantitative changes in antibodies and globulin frac- tions in sera of rabbits injected with several antigens. J. Immunol., 33: 111-122. Brandt, L. W., et al. _ 1951 The effect of age and degrees of maturity on the serum proteins of the chicken. J. Biol. Chem., 191: 105—111. Broadfoot, D. 1., et a1. 1954 Effects of infectious bronchitis on egg production. J. Amer. Vet. Med. Assn., 124 (923): 123-130. 17. 18. 19. 20. 21. 22. 23. 24. 68 Bushnell, L. D., and C. A. Brandly. 1933 Laryngotracheitis in chickens. Poult. Sci. 12: 55-60. Cannon, P. R . 1945 The importance of proteins in resistance to infection. J. Amer. Med. Assn., 128: 360-362. Chow, B. F. 1946 Electr0phoretic studies on the effect of protein deple- tion. Ann. N. York Acad. Sci., 47: 297-316. Cohn, E. J., et al. 1940 Preparation and properties of serum and plasma pro- teins. 1. Size and charge of proteins separating upon equilibration across membranes with ammonium sulfate solutions of controlled pH, ionic strength and temper- ature. J. Amer. Chem. Soc., 62: 3386-3393. Cohn, C., and W. Q. Wolfson. 1948 Studies on serum proteins. 11. A rapid clinical method for the accurate determination of albumin and globulin in serum or plasma. J. Lab. and Clin. Med., 33: 367-370. Cunningham, C. H. 1951 Newcastle disease and infectious bronchitis neutraliz- ing antibody indexes of normal chicken serum. Amer. J. Vet. Res., 12: 129-133. Cunningham , C . H . 1952 A Laboratory Guide in Virology. Second Edition. Bur- gess Pub. Co., Minneapolis, Minnesota. 88 pp. Cunningham, C. H. 1952 Methods employed in the diagnosis and investigation of infectious bronchitis and Newcastle disease. Proc. Book Amer. Vet. Med. Assn., 89th. Ann. Meet., Atlantic City, N. J., June 23-26: 250-257. 25. 26. 27. 28. 29. 30. 31. 32. 33. 69 Cunningham, C. H., and A. H. El Dardiry. 1948 Distribution of the virus of infectious bronchitis of chickens in embryonating chicken eggs. Cornell Vet., 38: 381-388. Cunningham, C. H., and H. 0. Stuart. 1946 The effect of certain chemical agents on the virus of infectious bronchitis of chickens. Amer. J. Vet. Res., 7: 466-469. Cunningham, C. H., and H. 0. Stuart. 1947 The pH stability of the virus of infectious bronchitis of chickens. Cornell Vet., 37: 99-103. Cunningham, C. H., and H. O. Stuart. 1947 Cultivation of the virus of infectious bronchitis of chick- ens in embryonating chicken eggs. Amer. J. Vet. Res. 8: 209-212. Cunningham, C. H., and M. H. Jones. 1953 The effect of different routes of inoculation on the adaptation of infectious bronchitis virus to embryonat- ing chicken eggs. Proc. Amer. Vet. Med. Assn., 90th. Ann. Meet., Toronto, Canada, July 20-23: 337-342. Delaplane, J. P. 1943 The differential diagnosis of the respiratory diseases of chickens. Rhode Island Agr. Exp. Sta., Bul. 288. Delaplane, J. P 1945 Panel discussion on poultry diseases. J. Amer. Vet. Med. Assn., 106: 91-105. Delaplane, J. P. 1945 Differential diagnosis of respiratory diseases of fowl. J. Amer. Vet. Med. Assn., 106: 83-87. Delaplane, J. P 1949 PrOgress report in infectious bronchitis research. J. Amer. Vet. Med. Assn., 115: 257-258. 77 ' _ . E, O 34. 35. 36. 37. 38. 39. 40. 41. 70 Delaplane, J. P., and H. 0. Stuart. 1939 Studies on infectious bronchitis. Rhode Island State College Exp. Sta. Bul. 273. Delaplane, J. P., and H. O. Stuart. 1941 The modification of infectious bronchitis virus of chick- ens as a result of propagation in embryonated chicken eggs. Rhode Island Exp. Sta. Bul. 284. Deutsch, H. F., and M. B. Goodloe. 1945 An electr0phoretic survey of various animal plasmas. J. Biol. Chem., 161: 1-20. Deutsch, H. F., et a1. 1949 Biophysical studies of blood plasma proteins. XI. Immunological and electrophoretic studies of immune chicken serum. J. Immunol., 63: 195-210. Dim0poullos, G. T. 1952 Electr0phoretic and serum neutralization studies of sera from chickens exposed to infectious bronchitis virus. Unpublished Ph.D. thesis. Michigan State College. 93 numb. leaves, 35 figures. Editors. 1944 Protein reserves and antibody production. Nutrit. Rev., 2: 197-199. Enders, J. F. 1944 Chemical, clinical and immunological studies on the products of human plasma fractionation. X. The con- centration of certain antibodies in globulin fractions derived from human blood plasma. J. Clin. Invest., 23: 510-530. Fabricant, J. 1949 Studies on the diagnosis of Newcastle disease and infec- tious bronchitis of fowls. I. The hemagglutination-inhibi- tion test for the diagnosis of Newcastle disease. Cornell Vet., 39: 202-220. 42. 43. 44. 45. 46. 47. 48. 49. 71 Fabricant, J. 1949 Studies on the diagnosis of Newcastle disease and in- fectious bronchitis of fowls. II. The diagnosis of in- fectious bronchitis by virus isolation in chicken embryos. Cornell Vet., 39: 414-431. Fabricant, J. 1950 Studies on the diagnosis of Newcastle disease and in- fectious bronchitis of fowls. III. The differential di- agnosis of Newcastle disease and infectious bronchitis. Cornell Vet., 40: 39-48. Fabricant, J. 1951 Studies on the diagnosis of Newcastle disease and in- fectious bronchitis of fowls. IV. The use of serum neutralization test in the diagnosis of infectious bron- chitis. Cornell Vet., 41: 68-80. Fabricant, J., and P. P. Levine. 1951 The persistence of infectious bronchitis virus in eggs and tracheal exudates of infected chickens. Cornell Vet., 41: 240-246. Franklin, M., et a1. 1951 Electr0phoretic studies in liver disease. 11. Gamma globulin in chronic liver disease. J. Clin. Invest., 30: 729-737. Gjessing, E. C., et a1. 1947 Fractionation, electrophoresis and chemical studies of proteins in sera of control and injured dogs. J. Biol. Chem., 170: 551-569. Gjessing, E. C., et al. 1948 Fractionation, electrophoresis and chemical studies of proteins in sera of control and injured goats. J. Biol. Chem., 174: 683-696. Groupé, V. 1949 Demonstration of an interference phenomenon associated with infectious bronchitis virus of chickens. J. Bact., 58: 23-32. 50. 51. 52. 53. 54. 55. 56. 57. Gutman, 1948 Hofstad, 1945 Hofstad, 1945 72 A. B. The plasma proteins in disease. Adv. Prot. Chem., 4: 155-250. M. S. A study of infectious bronchitis of chickens. 1. Path- ology of infectious bronchitis. Cornell Vet., 35: 22-31. M. S. A study of infectious bronchitis of chickens. II. Ob- servations on the carrier status of chickens recovered from infectious bronchitis. Cornell Vet., 35: 32-35. Hofstad, M. S. 1952 Chapter 19. Diseases of Poultry. Third edition. Edited by H. E. Biester and L. H. Schwarte. Iowa State Col- lege Press, Ames, Iowa. 1245 pp. Hofstad, M. S., and S. G. Kenzy. 1950 Howe, P. 1921 Jager, B. 1951 Jager, B. 1948 Susceptibility of chicks hatched from recovered hens to infectious bronchitis. Cornell Vet., 40: 87-89. E. The use of sodium sulfate as the globulin precipitant in the determination of proteins in blood. J. Biol. Chem., 49: 93-108. V., and E. L. Smith. Lack of correlation between immunologic and electro- phoretic estimation of gamma globulin in human serum. J. Clin. Invest., 30: 652. V., and M. Nickerson. Clinical application of a single method for estimating gamma globulin. J. Clin. Invest., 27: 231-238. 58. 59. 60. 61. 62. 63. 64. 65. 66. 73 Jager, B. V., et a1. 1950 Comparative electrophoretic and chemical estimations of human serum albumin: an evaluation of six methods. J. Lab. and Clin. Med., 35: 76-86. Jones, M. H. 1951 The effect of different routes of inoculation on the adap- tation of infectious bronchitis virus to embryonating chicken eggs. Unpublished M.S. thesis. Michigan State College. 53 -, numb. leaves. 7 Jungherr, E. L., and N. L. Terrell. 1948 Naturally acquired passive immunity to infectious bron- chitis in chicks. Amer. J. Vet. Res., 9: 201-205. . ‘51 Kabat, E. A. 1943 Review--Immunochemistry of the proteins. J. Immunol., 47: 513-587. Kabat, E. A., and M. M. Mayer. 1948 Experimental immunoche'mistry. First Edition. Charles C. Thomas, Pub. Springfield, Illinois. 566 pp. Kingsley, G. R. 1940 A rapid method for the separation of serum albumin and globulin. J. Biol. Chem., 133: 731-735. Kingsley, G. R. 1941 The direct biuret method for the determination of se- rum protein as applied to photoelectric and visual colorimetry. J. Lab. Clin. Med. 27: 840-845. Komarov, A., and F. R. Beaudette. 1932 Carriers of infectious bronchitis. Poult. Sci., 11: 335-338. Koprowski, H., et a1. 1947 Electr0phoretic studies of anti-viral sera. J. Exp. Med., 85: 515-530. 67. 68. 69. 70. 71. 72. 73. 74. 75. 74 Leland, S. E., Jr. 1951 An electrophoretic and chemical fractionation study of sera from rats immunized against the nematode, Nippostrongylus muris. Unpublished Ph.D. thesis. Michigan State College. 93 numb. leaves, 23 figures. Levine, P. P. . 1951 Infectious bronchitis. Proc. Ann. Meet. U. S. Livest. Sanit. Assn., 55: 183- 186. Levine, P. P., and M. S. Hofstad. 1947 Attempts to control air-borne infectious bronchitis and Newcastle disease of fowls with sterilamps. Cornell Vet., 37: 204-211. ‘ 3‘.“ . Loomis, L. M., et a1. 1950 Pathology of the chicken embryo infected with infectious bronchitis virus. Amer. J. Vet. Res., 40: 245-251. Madden, S C., and G. W. Whipple. 1940 Plasma proteins, their source, products and utilization. Phys. Rev., 20: 194-217. Martin, N. H. 1946 The components of the serum protein. Brit. J. Exp. Path., 27: 363-368. Merchant, I. A. 1950 Veterinary Bacteriology and VirolOgy. Fourth Edition. The Iowa State College Press, Ames, Iowa. 885 pp. Milne, J. 1947 Serum protein fractionation: a comparison of sodium sulfate precipitation and electrophoresis. J. Biol. Chem., 169: 595-599. Moore, D. H. 1945 Species differences in serum patterns. J. Biol. Chem., 161: 21-32. 76. 77. 78. 79. 80. 81. 82. 83. 75 Moore, D. H., et a1. 1949 Morgan, 1945 Page, C. 1950 Page, C. 1954 Factors influencing the electrOphoretic analysis of human sera. J. Biol. Chem., 180: 1147-1158. I. M. Quantitative study of the neutralization of Western equine encephalomyelitis virus by anti-serum and the effect of complement. J. Immunol., 50: 359-371. A. Antibody response of chickens exposed to infectious bronchitis virus. Unpublished M.S. thesis. Michigan State College. 39 numb. leaves, 10 figures. A. A study of the serum neutralization test for infectious bronchitis of chickens. Unpublished Ph.D. thesis. Michigan State College. 66 numb. leaves, 8 figures. Reagan, R. L., et a1. 1948 Electron micrOgraph of the virus of infectious bron- chitis of chickens. Cornell Vet., 38: 190-191. Reagan, R. L., et a1. 1950 Reed, L. 1938 Morph010gical observations by electron microsc0py of the viruses of infectious bronchitis of chickens and the chronic respiratory disease of turkeys. Cornell Vet., 40: 384-386. J., and H. Muench. A single method of estimating fifty per cent endpoints. Amer. J. Hyg., 27: 493-497. San Clemente, C. L. 1942 An electrophoretic study of a Pullorum-agglutinating chicken serum. Amer. J. Vet. Res., 3: 219-221. ‘7' 0“IJ__ ILL. YnflAfii : fl 84. 85. 86. 87. 88. 89. 90. 91. 92. 76 Sanders, E., et a1. 1944 An electrophoretic study of serum and plasma from normal and leucosis affected chickens. J. Biol. Chem., 155: 469-481. Schalk, A. F., and M. C. Hawn. 1931 An apparently new respiratory disease of baby chicks. J. Amer. Vet. Med. Assn., 78 (N.S. 31): 413-422. Seibert, F. B., and J. W. Nelson. 1943 Proteins of tuberculin. J. Amer. Chem. Soc., 65: 272-278. Swierstra, D. 1947 Infectious bronchitis of chickens in Holland. Tijdschr. Diergeneesk, 72: 745-746. Abstr. from the Vet. Bull., 19 (1949): 145. van Roekel, H., et a1. 1950 Infectious bronchitis. Mass. Agr. Exp. Sta., Amherst, Mass., Bul. 460. van Roekel, H., et al., 1951 Infectious bronchitis. Amer. J. Vet. Res., 12: 140-146. Weichselbaum, T. E. 1946 An accurate and rapid method for the determination of protein in small amounts of blood serum and plasma. Amer. J. Clin. Path., 16 (Tech. Sect. 10: 40-49). Wolfson, W. Q., et a1. 1948 Studies in serum protein. V. A rapid procedure for the estimation of total protein, true albumin, alpha, beta and gamma globulin in 1.0 m1. of serum. Amer. J. Clin. Path., 18: 723-730. Wyckoff, R. W. G., and M. Rhian. 1945 An electr0phoretic study of an anti-influenzal horse serum. J. Immunol., 51: 359-363. ’ .' fi-Q a.m:-c—n_r we ‘ e 77 93. Zeldis, L. J. 1945 Plasma protein metabolism. Electr0phoretic studies. Restoration of circulating protein following acute de- pletion by plasmapheresis. J. Exp. Med., 81: 515-517. Jun-T 1 1 .l .41.! ._ 111 fl . .. rtr: :11! It”! 8}! 66"~:..‘,,, 3.6th Ra. M'Tl'l'l'lllltfllll((Mjffllsiiflflttt'ES