M WEE-i “ears. mane“ asaca Lam OF A swov 06 ma seas TEST ma maacaouaga a catcaaaa -. “m“ ‘0' the Beam 65%. D. W sun came: Calvin Ame: Page 1954 71.45315 This is to certify that the thesis entitled A Study of the Serum Neutralization Test for Infectious Bronchitis of Chickens. presented by Calvin Amos Page has been accepted towards fulfillment of the requirements for ML—_ degree in Mlogy z/‘f/amtf / Major platessor Date __ M 0-169 .A STUDY OF THE SERUM NEUTRALIZATION TEST FOR.INFECTIOUS BRONCHITIS OF CHICKENS By CALVIN AXES ggca 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 1954 THESIS raw!“ TO MY WIRE, LAURIE 3., THIS MANUSCRIPT IS MOST AFFECTIC-NATELY DEDICATED \' 01" 4‘: l‘}"~p‘,-}' khglja}, r Galvin;Ames:Page candidate for the degree of Doctor of Philos0phy Final Examination: April 16, 1954, 9:00 ANM., Room 101, Giltner Hall Dissertation::.A'Study of the Serum Neutralization:Test For Infectious Bronchitis of Chickens Outline of Studies:' Major subjects: Bacteriology, Virology Minor subjects. Physical Chemistry Biographical Itemst: Born, November 8, 1922, Minneapolis, Minnesota Undergraduate Studies, University of Minnesota, 1945-47 University of Colorado, 1947-48 Michigan.State College, 1948-49 Graduate Studies, Michigan State College, 1950-54 Experiences: Technical Sergeant, United States.Air Force, 1941-1945. Decorated with Distinguished Flying Cross, Air Medal with two Oak Leaf Clusters. Associate Professor of Biology, Southwestern Louisiana Institute, Lafayette, 1951 - _ Society Affiliations: Society of American Bacteriologists, The Society of Sigma Xi, Louisiana Academy of Sciences. TABLE OF CONTENTS . P389 INTRODUCTIONnmom oe-eoe ere..e~e.e.e-e COO-O-QO-O-OO-Ooo e e e e e .0. HISTORICAL! REVEWO-Onfioao-O «0.0.0.0.... 0 0 e 0.00 O 00 O 000 00 EXPERMTAIJ PROCEDURESO-OOO00000000000000.0000 ESWTSOQOCOOOOOOOOOOOOOOOOOOOOOOOO...0.0.0... Thermolability of 1nfectious bronchitis virus at 4,, 22-25,, 37 and 56°C . SOM‘ neutralization teats to e e e e e e e e e-e e e 0 Effect of storage at different temp- eratures on the neutralizing cap- acities of infectious bronchitis immune gemstone-0eeeeeeeeeeeeeee Effect of using different numbers of eggs on virus titer and serum neutralizing indicess...... Effect of constant prOportions but different volumes on serum neutralization;tests............. Effect of amount of inoculum on the neutralizing capacity of infect- ious bronchitis immune serum»...e Effect of dilution-and diluent on the neutralizing capacity of infectious bronchitis immune serumbeeee-eeeeeeeeeeeeeeeeeeeeeee Effect of time and temperature of incubation.onathe serum;neutra1- 123131011 teateeee-eeeeeeeeeeeeeeeee Time-rate of virus neutralization,.. DISCUSSIONOOO-‘OOO000......00.0.0000000COOOOOOO S‘WOOOOOOCOO'OOO0.0.0.0.000...0.0.0.0000... LITERATUE CITEDOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 14 14 26' 26 32 36 38 5O 56 58 59 .‘O....0.0'...IO ACKNOWLEDGEMENTS I wish to express‘my sincere appreciation to Dr. Henrik J .. Stsfseth,, Head of the Department of Bacteriology and Pub- lic Health, and Dr.. Charles H.. Cmningham, Associate Professor of Bacteriology and Public Health, for their unfailing assist- - anoe and encouragement; to the Agricultural Experiment Station, Michigan State College,, for supporting; this investigation in parts to the United States Regional Poultry Research Lab- oratory, East Lansing, Michigan, and Professor Leo Hebert, Poultry Department,, Southwestern Louisiana Institute, Lafay- ette,, fer-supplying cockerels suitable for use in these exp- eriments; to Salsman’s Hatchery,, Lafayette, Louisiana for their aid in supplying fertile eggs of an approved quality; and to Dr. W.D.,Baten,. Professor of Mathematics,, Statistician, Agricultural Experiment Station,, Michigan; State College. INTRODUCTION Infectious bronchitis of chickens is an economically important disease to the poultry industry. The serum neutralizationttest is the only serological procedure that may be employed for diagnosis of the disease. The object of the present study is to obtain.funda- mental knowledge of antibody-antigen reactions with respect to their practical applications to the serum neutralization test for infectious bronchitis. HISTORICAL REVIEW Infectious Bronchitis g; Chickens In 1931, Schalk and Hawn76 described a new respiratory disease occurring in baby chicks and suggested the name "infectious bronchitis". Since that time the disease has been reported throughout the United States5v5:19:43, Canada7, England3'8, and the Netherlands79. Etiological‘ggggt Infectious bronchitis (IB) is caused by a distinct filterable virus capable of passing through all grades of Berkefeld and Seitz filter39'12'34'35'45'76 and the Handler preliminary filter34. The disease is apparently limited to chickensBB, and the virus is found primarily in the tissues and exudates of the respiratory system9. Komarov and Beau- dette58 were unable to find the virus in the liver, spleen, kidney or blood of infected chichens. However, Bushnell and Brandly19 reported successful transmission of the dis- ease using those tissues. Electron micrographs of infect- ious bronchitis virus (IBV) indicate that the virus is round with filamentous projections, having a mean diameter of 70 millimicrons73'74. The virus will remain viable for 180 days if lyophilized and stored at 4°C, and for 80 days if glycer- olized and refrigerated at 4009' ‘Transmission 0 . IBV is highly infectious for chickens and can produce the disease in all ages within 24 to 48 hour35'31'34'83. Aerosol transmission cannot be controlled by ultraviolet irradiation of the air6l. The virus can be readily trans- mitted by intratracheal and intranasal inoculation12'34, but subcutaneous and intramuscular inoculations fail to pro- duce the diseaselz. The virus can be isolated from the yolks .of eggs laid between the second and thirty-sixth post-inocul- ation day, and it can be recovered from tracheal swabs as late as four weeks after inoculationao. Komarov and Beau- dette58 found carriers of the virus 43 days after an outbreak. Delaplane and Stuart34 reported that recovered chickens can be carriers for at least two months. Hofstad45'48 demon- .strated that chickens could transmit the disease for 35 days after recovery. I ‘ Symptoms Characteristic symptoms of IE include gasping, sneezing, coughing and tracheal rales9'12’31v34v75. Nasal discharges are noted in 30 to 50 per cent of the cases31. The outstand- ing lesions are mucous, catarrhal and purulent accumulations in the trachea and bronchi accompanied by congestion and edema of the lung331'34'45'83'84- Edema of the facial sin- uses may be found in chicks under two to three weeks of age. There is no hemorrhage or significant changes in the liver, spleen and kidney and inclusion bodies are not found45. The severity of the symptoms is dependent upon such predisposing factors as environmental conditions and nutritional defic- iencie983. The morbidity of IE is high. The highest mortality rate, ranging to as much as 90 to 100 per cent, occurs in young chicks9'12'31'34'75. In laying flocks there is a marked decline in egg production that may persist from four to nine weeks31'34’83. The first few eggs laid when the flock is returning to production may be misshapen, rough, thin-shelled with watery albumen83. Diagnosis Diagnosis is based on history, clinical symptoms and laboratory tests such as isolation and identification of the virus in embryonating chicken eggs and serum neutrali- zation tests, in conjunction with the characteristic alter- ations of the chicken embryoll'12'24'34'35’37'38'63'83. In addition, IBV does not possess the ability to agglutinate chicken red blood cells as does Newcastle disease virus, and this test may be used for differential diegnoeis38o47. The serum neutralization test is useful in evaluating flock immunity. Normal chicken serum would not be eXpected to have more than 36 neutralizing dose325'39’70'84. A min- imum of one hundred doses is considered as a positive test, and this is usually obtained with serum collected about three weeks following exposure to the viru839v70. Cultivation g; the Virus in Embryonating Chicken Eggs It was found by Beaudette and Hudson12 that the IBV could be prOpagated in embryonating chicken eggs via the chorioallantoic membrane. After a few passages, death of some of the embryos resulted from virus inoculation. After the fourteenth passage in embryos, the virus was still ins fectious for chickens.. Delaplane and Stuartyl'35 reported similar results and noted that with each succeeding passage the virus became more virulent for the embryo.. By the sixty- fifth passage, the virus was completely egg-adapted with respect to embryo mortality and no apparent change was noted with subsequent passage.. At the ninetieth passage, the virus had lost its pathogenicity and antigenicity for chickens. According_to Beaudettelo, the virus had lost its pathogen» icity but not its antigenicity for the natural host after the seventieth passage in eggs.. Neutralizing antibodies could be demonstrated fourteen days after inoculation. Adapt- ation of the virus to embryonating chicken.eggs can be accomr plished earlier by inoculation via the allantoic cavity rather than the chorioallantoic membrane27’32. The outstanding gross alterations of an embryo following inoculationtwith chicken-prepagated virus is curling and dwarfing of the embryo to as much as one-half the normal size. Thinning of the chorioallantoic membrane, thickening of the amnionic membrane, hemorrhage and congestion of the liver, and swelling of the kidney and spleen.are observed11'12'34'35'37' 53:83. In addition, microsc0pic alterations, as reported by Loomis gt _a__l_53, include proliferation of mesodermal and ectodermal cells, edema of the chorioallantoic and amnionic membranes, pneumonia and marked serous exudation, inter- stitial nephritis and necrosis, splenic congestion and con- gestion of brain capillaries. Cunningham and Stuart30 reported that the completely egg-adapted virus is capable of killing all embryos within a 48-hour post-inoculation period. The highest concentration of the virus, following inoculation via the allantoic cavity, was found in the chorioallantoic membrane, followed in order by the allantoic fluid, amnionic fluid, and the liver ac- cording to Cunningham and E1 Dardiry26. The highest titer was obtained at the thirty-sixth hour post-inoculation from living embryos since the virus was thermolabile in eggs in- cubated at 99°F for eight to twelve hours after the death of the embry026. Groups42 demonstrated that infected eggs stored for 24 hours at 36°C after the death of the embryo contained a non- infectious material which adsorbed to and interferred with the infectivity of the virus. According to Groups and Pugh 43, embryos inoculated with egg-avirulent IBV or influenza A virus would be protected against subsequent inoculation with embryo-lethal IBV. The egg-adapted strain of IBV has a greater stability in an acid medium than in an alkaline medium during the first sixty days of storage. After the sixtieth day, the virus is more stable in an alkaline medium29. The virus is in- activated by l per cent phenol, l per cent liquor cresolis saponatus, l per cent metaphen, l:l0,000 KMnO4, 1:1,000 HgCla, 95,70,40 and 25 per cent ethanol, 1:1,000 tincture of Zephiran, l per cent Lugol's iodine, 1:20 NaOH, 5 per cent Neoprontosil and 1 per cent formalin in three minutes or lessza.. Immunity 32 infectious bronchitis Chickens recovered from the disease are immune, and neutralizing antibodies can be demonstrated in the blood 5'12v34'46'55. This immunity persists for at least one year34, but occasionally flock immunity is inadequate to prevent a natural outbreak83. Jungherr and TerrellS5 have reported a naturally acquired passive immunity which may persist in chicks for as long as five weeks after hatching. However, Hofstad and Kenzy49 reported that four, six, seven and tenrday old chicks hatched from eggs laid by immune hens could be infected by overwhelming challenges with IBV. Control A program of active immunization with commercially pre- pared egg-adapted antigenic virus is used in many regions to control the disease10'31'33. Chickens from four weeks to four months of age can be inoculated without untoward results 10,31,33,62,81,82. However, the best age is from ten to fourteen weeks with exposure being made during June, July and August when the flock is on range and conditions are most favorable for recovery82. The program is generally not started unless 75 per cent of the poultrymen in the community approve the plan. About one per cent of the flock is inoculated and the disease spreads naturally in about three to six days64. EXPERIMENTAL PROCEDURES The study was divided into two parts: (1) thermola- bility of IBV, and (2) certain factors involved in the serum neutralization test for IS. In all thermolability and serum neutralization exper- iments, egg-adapted IBV, strain Vll4D, was used. This strain of the virus was capable of killing all embryos within 48 hours following inoculation. It was supplied by Dr. Charles H. Cunningham, Department of Bacteriology and Public Health, Michigan State College, East Lansing, Michigan. IBV, strain VR, in the form of infected allantoic fluid from the first passage of the virus in embryonating chicken eggs was used to inject adult Single Comb White Leghorn cockerels by the intratracheal and intranasal rou- tes for the production of specifically immune serum. This strain was originally isolated by Dr. H. Van Roekel, Univ- ersity of Massachusetts. Serum was collected by cardiac puncture between the sixth and eighth weeks after inoculation. For studies of time-temperature relations of thermo— lability of IBV, virus—infected allantoic fluid collected from living embryos at 26 hours post-inoculation was employed. The allantoic fluid was pooled and frozen at -45°C for about one day. The preparation was then thawed at room temperature and centrifuged at 3,5000 r.p.m. at 4°C for 30 minutes to sediment the insoluble precipitate formed by freezing and lO thawing. The clear supernatant fluid was removed and dis- tributed into 30 m1. screw cap vials which were then stored at ~45°C until ready for use. For uniformity, one vial of fluid was used for each study of thermal inactivation. At the time of use, the virus was thawed at room tem- perature and centrifuged. The clear supernatant fluid was distributed into thin-walled, long neck serum ampoules which were sealed by flame. For all studies with the exception of that at 56°C, 2.0cc ampoules containing 1.7cc of virus suspension and 1.000 ampoules containing 0.7cc of virus sus- pension were prepared. For the studies at 56°C, only 2.0cc ampoules were prepared. A11 ampoules for a particular thermolability test were submerged in a water bath thermostatically controlled at the respective temperatures. At certain time intervals, two ampoules were removed. With the exception of those subjected to 4°C, all ampoules were immersed in an ice bath to stop thermal inactivation of the virus. One ampoule was used for the qualitative infectivity test and the other was stored at -45°C until used for the quantitative infectivity test. Qualitative infectivity tests were performed at 56, 37, and 22-25°C exposures as screening tests to ascertain the maximum exposure period during which the virus retained some degree of infectivity in order to select suitable 11 samples for quantitative infectivity tests.. The qualitative infectivity tests were made by injecting 0.1cc of the respect- ive virus samples into ten 10-day embryonating chicken eggs. The criterion for inactivation of the virus was failure of the virus to kill the embryo within five days after inoc- ulation.. Quantitative determination;of viral infectivity for the thermolability tests was accomplished by preparing serial ten-fold dilutions of the virus-infected allantoic fluid in nutrient broth using separate pipettes perldilution. Five eggs were employed per dilution and each was inoculated with 0.1cc via the allantoic cavity..22 The eggs were in- cubated for five days following inoculation.. Mortality rates were used in computing the titer which was expressed as the lethal doseso (l.d.50) according to the method of Reed and Muench75.. The l.d.50 was calculated to the centile and rounded-off to the decile.. The number of lethal doses of virus was considered to be the antilog of the reciprocal of the 1.d.50.. ‘ All incubation of eggs was.at 99-99.5°F (85-88°F wet bulb) in an electric, forced-draft incubator. Eggs were candled daily.. Embryo mortality during the first 24 hours ‘was considered to be due to nonspecific causes and these eggs were not included in the final results. Statistical interpretations of the results were made according to the procedures of Baten4, Croxton.22 and Dixon36. 12 The results of the thermolability tests best fit a sec- ond-degree parabolic. curve ofthe equation I s a + bx 4 7-0 1 5 5 3* 4 3 >6.7 2 5 5 4 3 1 6.2 4 5 5 5 3 o 6.2 6 5 5 2 1 o 4.8 8 5 5 2 o 4.5 10 5 5 5 1 o 4.2 12 5 5 5 4 2 o 3.6 14 5 5 5 2 o 2.8 16 5 5 5 1 o 2.6 20 5 5 3 l o 2.2 *deaths per four embryos inoculated (These footnotes apply to Tables II, III, IV, V, VI, VII, IX, X, XI, XII, XIII, XIV XV, XVI, XVII) 16 1.d. 50 FIGURE 1 THERMOLABILITY OF.INFECTIOUS BRONCHITIS VIRUS AT 3-4°cr e! no t» -s \m 0\ pl CD O 2 4 6 8 10 12 14 16 18 20 Time - weeks 17 TABLE II THERMOLABILITY or INFECTIOUS BRONCHITIS VIRUS AT 22-25°c 4*— 1 TIEG Qual- log of virus dilutions in In" C -1 2 -3 -4 -5 -6 -7 days teat# 10 10 10' 10 lo 10 10 lo 1.d.so 0 10 5 5 4 3 7.0 3 10 5 5 1 o 5.6 5 9 5 5 2 o 4.8 7 10 5 5 3 o 4.2 9 9 5 5 5 2 O 3.8 11 10 5 5 2 o o 2.8 13 8 5 3 o o 2.2 15 10 4s 4 o o 2.4 17 10 5 2 o o 1.8 19 3 3# 2 o o 0.5 21 7 7# o o o 0.7 23 6## 6V o o o 0.7 25 3 3 0 o - A_#deaths per ten embryos InocuIated ##deaths per nine embryos inoculated (These footnotes.apply to Tables III and IV) 19. FIGURE 2 THERMOIABILITY OF INFECTIOUS BRONCHITIS VIRUS AT 22-25%" H ' ' I a 6.921 + (-o.4336)x 4 0.00672:2 ledeso l-‘NUI-P'U'IO'QCD 01 3 5 7 9 11131517192123 Time-days 20 C} Thermolability at 37°C.. Four hour exposure intervals were used for a period of 60 hours.. Qualitative infectivity tests showed that at 36 hours the virus was capable of killing all embryos. From the thirty-sixth to the fifty-sixth hour there was a slight red- uction in.infectivity, with seven-of tenlembryos being killed by the virus at 56 hours. Three of ten embryos were killed at 60 hours. The l.d.50 decreased from 105'8 at the start of the experiment to 100’28 at 56 hours and to almost complete inactivation at 60 hours. Table III. These data were best fit by the equation'Y a 7.045 . (-0.212)X‘o 0.0016x2.. Figure 3. D.- Thermolability at 56°C} Five minute intervals were used for a total period of 30 minutes. Qualitative infectivity tests showed that at five minutes after exposure, five of ten embryos were killed by the virus, but at ten minutes the virus was completely inactive. The 1.d.50 decreased from 105’8 at the beginning of the experiment to lO°’5.at five minutes and to zero at ten minutes. Table IV. The equationsY s 5.8 s (-1.54)X + 0.096x2 best fit these data.. Figure 4. E. Comparison of Results. Summation of the results of the thermolability studies indicate the following rates of inactivation of IBV expressed as temperature/lethal doses/time interval: 4°C/100'22/week, 22-25°C/10°‘27/24 hours,,37°C/lO°'11/hour, Figure 5,.and 5500/ 101°05/minute. TABLE III THERMOLABILITY 0F INFECTIOUS BRONCHITIS VIRUS AT 37°C Time Qual. log of virus dilutions in Inf. 0 4 hours test# 10 10"1 10-2 10-3 10- 10-5 10"6 10-7 l.d.50 0 10 5 5 4 2* 6.8 4 10 5 4 2 2 6.0 8 lO 5 5 2* O O 5-0 12 10 5 5 5 0 0 4.5 20 9 5 4 4 2 0 0 3.5 24 9## 5 5 2 4 3 4-5 28 9## 4* 5 0 0 0 2.5 32 10 5 5 2 0 0 1.8 36 10 5 4 2 l O 1.8 40 8 4 1 0 0 0.5 44 9 4 2 0 0 0-7 48 9 4 0 1 0 0.5 52 8 4. 0 1 0 0.5 56 7 7# O 0 0 0.3 60 3 3# 0 O - lOdOSO FIGURE 3 THERMOLABILITY 0? INFECTIOUS BRONCHITIS VIRUS AT 37°C HMU-fi'U‘lChNICD O 4 8 16 24 32 4O 48 Time - hours 22 23 TABLE IV THEREOLAEILITY OF INFECTIOUS BRONCHITIS VIRUS AT 56°C Time Qual. log of virus dilutions in Inf. 0 1 2 4 6 min. test# 10 10’ 10‘ 10-3 10' 10"5 10- 10-7 1.0.50 0 10 5 4 2 1 5.8 5 5 5# 0 0 0.5 10 0 - 15 0 - 20 0 - 25 O - 30 O .. 1.d.50 FIGURE 4 THERMOLABILITY OF INFECTIOUS BRONCHITIS VIRUS AT 56°C Y . 5.8 . (-1.54)x . 0.096X2 HNUJS'U'IO‘NCD O 5 10 Time-Minutes 24 25 Oh mm mm mm dm chad I oawa om m¢ Nfi mm ¢m on mm mm mH ¢H OH m N 0...... u.«m NNO +_Hp + d H H .oebw dad comm! mm .006 adeaHAHmdAOEMHmB bmH _ m fiMDuHh 1_e .d.H 26 II. Serum‘Neutralization.Test, .A3. The Effect of Storage at Different Temperatures on the gentralizing;0apacitiesof Infectious Bronchitis Immune brune- 14.16.18.53.54.55.65.B7 Several workers have reported that immune serum will retain-its initial neutralizing cap- acity for as long as one year when stored at 4-6OC.. Olitsky 69 and Murphy found that poliomyelitis immune serum had a 2‘3 after 20 years storage at 4-5°G5. The 64 found l.d.50NI of 10 original titer was not known.. Melnick and Ledinko that.neutralizing antibodies against Coxsackie virus were stabile at 65°07f0r 30 minutes but not at 80°C.. The following experiments were designed to determine the effect of storage of immune serum at 4°03,22-25°C and 37°C for certain time intervals prior to use in the serum neutralization test.. The serum was collected the day before it was subjected to the storage temperature. Serum was distributed into thin-walled ampoules, 2.0cc per ampoule, heat-sealed and submerged in a thermostatically controlled water bath. The results indicated that there was no significant change in the neutralizing capacity of IBV'immune serum.of 6 l.d.50NI 10 '5 following an eight week exposure at 4°C; Table V, or a seven day exposure of IBV immune serum of l.d.50NI 10A'4 at 22-25°C; Table VI. At 37°C; a ten-fold decrease in 6.8 neutralizing capacity from l.d.SONI 10 to 105'8 occurred during a 56 hour exposure. Table VII. T ABLE V EFFECT OF STORAGE 0N INFECTIOUS BRONGHITIS IMMUNE SERUM AT 4°C time in- weeks 100 10-1: loggof virus dilution; 10'? 10"3 10" 10-5 lo“6 10'7 1650 ld5oNI 27 Virus SN' Virus SN“ Virus SN Virus Virus SN‘ 1 0 0 0000 O\O\ #«P MN 00 O O O 5 5 4 4. 4 1 >6..5 0.0 )6.5 0.0 )6.3 0.0 >6.8 0.0 )6 .5 0.0 )6.5 >6.5 )6.3 )6.8 >6.5‘ TABLE VI EFFECT OF STORAGE ON'INFECTIOUS BRONCHITIS IMMUNE SERUM AT 22-25°c“ * time log_0f virus dilution n days 100 10"1 10'2 10'3 10'“ 10'5 10‘5 10"7 ldSO ldsoNI Virus 0 S 4 2 1 5.8 SN 0 5 4 O O 1.4 4.4 Virus 1 5 5 3 2 6.5 SN‘ 1 5 5 l O 1.6 4.9 Virus 3 5 5 3 2 6.5 SN 3 5 4 3 O 2.0 4.5 SN 5 5 4 1 0 1.5 4.3 Virus 7 S 5 2 0 5-8 SN 7 5 l O O 0.6 5.2 29 For assessment of the results Obtained with the serum neutralization tests of serum stored at 37°C, certain factors must be considered. The virus had an initial titer of l.d.50 107'0. During the interim between each serum neutralization test the virus was frozen. This resulted in seven cycles of freezing and thawing during the entire experiment. In ad- dition to this effect on the virus, the cumulative exposure of perhaps ten hours at room temperature, for the entire experiment, must be considered. Using the data previously obtained in thermolability studies of the virus at 22-25°C, Table II, Figure 2, exposure of the virus at this temperature for ten hours would result in a calculated 100‘2 decrease in l.d.50. The titer of the virus at the termination of the experiment, the fifty-sixth hour test period for the serum, was 106'5. Based on previous experience that repeated freez- ing and thawing was deleterious to the virus, it was assumed that a 100’3 decrease could be attributed to this factor. This would mean a total decrease of 100’5 as reflected by the 1.d.50 106'5 of the virus at the termination of the ex- ‘periment. In order to compensate for these combined effects on the l.d.5o of the virus at eaCh test period, the initial l.d.50 may be assumed to decrease by kN, where k : A 1.6..50/n, n = total number of freezing cycles, and N : (test period-l). Example: k 100 5 - 100'07 7 sta 100'07 x 5 = 100'35 TABLE VII EFFECT OF STORAGE ON INFECTIOUS BRONCHITIS IMMUNE SERUM AT 37°C“ I log of virugdilution hours 10° 10"1 10'2 10"3 10'4 10"5 10'5 10-7 1dSO ldsoNI W time in- Virus 0 SN 0 SN 8 SN 16 SN 24 SN 32 SN 40 SN 48 SN 56 Virus 56 bl NWUUU‘UU [OI-'NNOHl-‘O HHI—‘OI—‘OOO 0 0000 5 5 4 3 7.00 0.17 0.35 0.35 0.32 0.50 0.67 0.59 0.67 6.50 U1U1U'|O\O\O\C\O\ e e e e e e e mWWN-P’UTUTCD U‘Omm‘qpmu At the sixth test period the initial l.d. 5 7.0 O 10 for the virus would be considered to have decreased by 100'35 to 105‘65 which was then used in the calculation of the l.d.SONI at this test period. 31 32 B.. The Effect of Using Different Numbers of Eggs on Virus Titer and Serum neutralizing Indices. In studies of the reproducibility of titrations of PR8 strain of influenza virus, Knights7 found that with thirty samples the l.d.so varied from 1012'0 to 1014'3 with CF: 3 100°132,5and v.- 100’017. von.Magnu385, using ten repeated titrations of influenza PR8, with the 1°d°50 varying from 108'5 to 109°1, reported 0”. g 100'”. Knight57 showed by statiétical analysis that when ten embryos per dilution were used, three-fold differences in the concentration of the virus could be readily detected. When five embryos per'dilution were used, differences closer than five-fold could not be detected with accuracy.7 The purpose of the following experiments was to deter- mine if a significantly greater accuracy of virus titrations and serum neutralization tests could be obtained by using from five to as many as ten eggs per dilution. 1. Procedure and Results Serum neutralization tests were performed using one, two, and four hour incubation periods at 22-25°C. The virus was titrated only once for this experiment since previous results has shown that there was no significant thermal effect on the virus during a 24 hour exposure period. Table II. Ten eggs were used per'dilution. Following inoculation, five eggs per dilution for virus titration as well as for serum neutralizat- ion were randomly marked with an x, three with a Y, and two 33 were left unmarked. The results were then recorded accord- ing to the following procedure. For calculations involving ten eggs per dilution, all eggs were counted. For calculat- ions involving eight eggs per dilution, the eggs marked X and Y were used. The eggs marked X and those unmarked were used for calculations involving seven-eggs per'dilution. For calculations involving five eggs per dilution, one group‘ consisted of those eggs marked with an X, and the other group consisted of those eggs marked with a Y and those unmarked. Table VIII. With the two virus titrations involving five eggs per dil- ution, the l.d.50 differed by 100'2 (lo5~4 and 105°2). For jpractical purposes, the arithmetic average (i),.106'3 , was used as a basis for calculation of the l.d.5oNI when five embryos per’dilution were considered. For the entire series of titrat- ions in which five, seven, eight and ten eggs per dilution were considered, i a 106’3.. From these results, it is evident that for virus titrations using serial ten-fold dilutions, equal accuracy is obtained when five to ten embryos are employed per dilution. From these data, 0'. g 100'07 which compares favor- ably with those of Knight57 and von Magnus85 for titration of influenza virus in embryonating chicken eggs. For the serum neutralization test in whiCh two groups of five eggs per dilution were involved, the l.d.soNIs showed greater differences than those obtained with the virus titra- I r 34 TABLE VIII EFFECT OF NUMBERS 0F EGGS ON VIRUS TITRATION ’ AND SERUM NEUTRALIZATION TEST A. Virus Titration Numbers 10g of virus dilution of eggs -— _4 _5 -6 _7 per dilution 10 10 10 10 l.d.50 10 lo 9 7. ’1 6.37 8 8 8 6 0 6. g E 2 2 3, 2-2 5 5 4 3 1 6:2}6'3 B. Serum Neutralizations Number time 195 of virus dilution of eggs in per'dil. hours 100 10"1 10"2 10"3 10“ l.d.so l.d.5ONI 10 ' 1 . 10 5 4 0 1.4 4.9 8 1 6 3 3 0 0.9 5.4 g 1 6 i g 0- 1.2 3.1 1 5 0 . . 10 2 8 4 2 0 0.8 5.5 8 2 6 3 1 0 0.7 5.6 7 2 i 4" 2 0 1.2 5.1 5 2 1 1 0 0.7 5.7 5 2 4 5 1 0 1.2}1°° 5.0'3'5'4 10 4 8 4 2 3 0 1.1 5.2 8 4 6 5 l 2 0 0.9 5.4 7 i 2 3 1 2 0 1.; 3.2 5 2 O 1 0 0. . 5 4 4 2 2 2 0 1.43‘1'1 4.8}5'2 35 tions. For the one hour incubation period, the difference was 100'8 (100'9 and 101‘7), for the two hour period 100'5 (100'7 and 101'2) and for the four hour period 100‘6 (100‘8 and 101'4). The'X,.respectively, was 101°3, 101'0 (100.95) and 101'1). The 2 l.d.soNI for the entire series of serum neutralization tests was 105‘2, a-g 3 100°21 100.043 ,andv: .. From these data, it is evident that a significantly greater accuracy was not obtained using more than five eggs- per dilution in serum neutralization tests. 36 G}. The Effect on the Serum Neutralization.Test of Using, Gbnstant Pr0portions-but Different Volumes of Serum and Virus.. It was Shown by Bryan and Beard17 that the amount of purified papilloma virus neutralized by serum depended on the preportions used. In addition, Melniok and Ledink064 demonstrated that as the quantity of Coxsackie virus was increased, the quantity of serum required for neutralization was also increased. The following experiments were performed to determine if there was any effect on the neutralizing capacity of immune serum when different amounts of virus and serum were mixed. . The serum neutralization test was done by mixing 0.5 ml., 1.0 ml., and 2.0 ml., respectively of each virus dilution with an equal amount of immune serum and incubating at room temperature for 45 minutes prior to egg inoculation. The results, Table IX, show a similar l.d.SONI for each of the three tests.. However, because of the high antibody level, the serum neutralizationaend-point could not be calculated, and definite conclusions could not be established. TABLE IX THE EFFECT ON THE SERUM NEUTRALIZATION TEST OF CONSTANT PROPORTIONS BUT DIFFERENT VOLUMES OF SERUM AND VIRUS 37 log of virus dilution. Vol. 10° 10"1 10"2 10-3 10'4 10"5 10'5 10"7 1d50 1d50NI Virus - SN 00500 0 SN‘ 1.000 0 SN 2.000 1 5 5 4 1 6.5 O O - 1* O - 1* 1 - sax U'IU'IU'! 38 D.. The Effect of Amount of Inoculum on the Neutralizing Capacity of Infectious Bronchitis Immune Serum. Serum neutralization tests were performed using inoc- ulums of 0.0500, 0.100 and 0.200 per embryo for the serum- virus mixtures and 0.100 for the virus dilutions. The data indicate that the maximum 1’d°50NI was obtained when equal inoculums were employed for both titrations. In halving 0r doubling serum-virus inoculums with respect to the virus-dilution.inocu1um, the serum titer was increased. Table x.. TABLE X THE EFFECT OF AMOUNT OF INOCULUM ON THE NEUTRAL- IZING CAPACITY OF INFECTIOUS BRONCHITIS IMMUNE SERUM 39 10g of virus dilution Inoc. 10° 10"1 10"2 10"3 10" 10"5 10"6 10"7 1d50 ldSONI SN 0.05 2* 2 O 0 Virus 001 5 5 4 SN 0.1 1 1* 1 Virus 0.1 5 5 3 SN 0.2 3 1 1 0 2. 6.7 0.4 6.5 6.5 0.5 6.3 )6.5 6.0 .1: §.!.V 1 ‘l.r tallfl! .3... ”I! 1 I: E.. The Effect of Dilution and Diluent on the Neutralizing Capacity of Infectious Bronchitis Immune Serum. By diluting influenza immune serum one in five, Hirst44 found that a tenbfold decrease in neutralizing capacity 17 occurred. Bryan and Beard reported that neutralization of purified papilloma virus by immune serum varied directly with serum dilution.. A ten to one hundred-fold decrease in neutral- izing capacity of Newcastle disease immune serum diluted one in five was reported by Brandly gt §;}5.. A one hundred-fold decrease was observed when the serum was diluted one in ten. Rached72 reported that dilution of Newcastle disease immune serum one in ten decreased the neutralizing capacity from 86 ten to five hundred-fold. Whitman , working with western equine encephalomyelitis virus” reported a decrease in 2.2 neutralizing titer from 105'2 to 10 when human convalescent serum was diluted ten-fold.. Page70 reported that IE immune serum diluted one in five showed a ten to fifteen-fold decrease in.neutralizing capacity.. Dilution as high as one in twenty did not appreciably alter the neutralizing capacity beyond that of a one in five dilution. ' Bell13 reported that physiological saline, distilled water and ten per cent normal monkey serum used as diluents had no effect on comparative titrations of poliomyelitis immune serum. with the viruses of poliomyelitis13o53, Newcastle disease80, influenza A50’86, western equine enceph- 41 alomyelitis55, pneumonia of mioesl, and myxoma71” a linear exponential relationship has been found to exist between.the serum dilution.end-point and the amount of virus neutralized. With 0.1M phosphate buffer,.0.85 per cent NaCl, and nutrient broth diluents, the l.d.SONI ranged for serum un- 509 Oe2 502 diluted and diluted through 10'”4 from 10 to 10 , 10 1.8 5'4 to 10 respectively.. Table XI. From to 101'6 and lo the average l.d.5ONI of the three diluents at the different concentrations of serum employed,, Y : 5.5 4 (-1.11)x and Sy : 1 0.66.; Figure VI.. TABLE XI THE EFFECT OF DILUTION AND DILUENT CN THE NEUTRAL- IZING CAPACITY OF INFECTIOUS BRONCHITIS IMMUNE SERUM A. Virus titration 10g of virus dilution 10-4 10'5 10"6 10'7 l.d.50 5 5 4 0 6.4 B. Serum Neutralization Diluent: Bhosphate buffer, 0.1M log of virus dilution Dil- ution 10° 10'1 10'2 10'3 10'4 10-5 10"6 10-7 l.dso l.dSONI 10° 5 0 0 0 0 0.5 5.9 10-1 3* 2 1 0 1.8 4.6 10-2 5 3 2 1 0 2.7 3.7 10-3 5 4 4 1 o 4.4 2.0 10-4 5 5 5 3 0 6.2 0.2 Diluent: 0.85 per cent NaCl 100 5 2 2 0 1.2 5.2 10"1 5 2 1 l 0 2.3 4.1 10-2 5 2 1 0 0 3.0 3.4 10-3 5 4 0 0 0 4.6 1.8 10'4 5 1 1 O 4.8 1.6 Diluent: Difco nutrient broth 10° 5 2 1 0 0 1.0 5.4 10'1 5 l 0 0 0 1.6 4.8 10-2 5 4 0 0 O 3.6 2.8 10-3 5 3 0 0 0 4.2 2.2 10"4 5 1 O 0 4.6 1.8 M 42 43 FIGURE 5 THE EFFECT OF DIIUTION AND DILUENT ON THE NEUTRAL- IZING CAPACITY OF INFECTIOUS BRONCHIIIS IMMUNE SERUM l.d.50NI 10° 10'1 10'2 10'3 10-4 Serum Dilution A- phosphate buffer 0- 0.85 per cent NaCl D- nutrient broth 44 F.. The Effect of Time and Temperature of Incubation on. the SerumiNeutralization.Test., Andrewes2 reported that neutralization of Rous virus by immune serum was more complete when mixtures were incu- bated at 37°C than at room temperature.: However, Olitsky and Casa1s68,.using poliomyelitis virus, did not find any enhancement of neutralization when;the serum-virus mixtures were incubated for two hours at 37°C prior to inoculation, . as compared to 15 minutes.. B01113, using poliomyelitis virus, found that variation.in the time and temperature of incu- bation prior to inoculation produced only slight differences in the end-points.. 1.. Procedure and Results . Serum neutralization tests were performed at 4°C, 22- 25°C and 37°C.. In all tests, the ingredients were at thermal equilibrium at the time of mixing and were maintained in: water baths at the respective temperaturelsthroughout the incubation period.. The tests were made at one, two, four, eight and sixteen hours after incubation at 4°C and 22-25°c. Tables XII, XIII.. The virus was titrated only at the first test period. Using data previously obtained, Table I, Figure 1, exposure of the virus at 4°C for sixteen hours would have no significant effect on the titer. However, exposure of the virus to 22-25°o for eight hours would result in.a cal- culated 100'2 decrease in 1'd°50’ and for sixteen hours almost a lOO’A decrease, Table II, Figure 2. This thermal effect on the virus must be considered at 22—25°Cs 45 TABLE XII EFFECT OF TIME AND TEMPERATURE OF INCUBATION ON THE SERUM NEUTRALIZATION TEST AT 3 - 4°C: Inigpo log of virus dilution hours 10° 10"1 10-2 10'3 10" 10"5 10"6 10"7 1d50 1d50NI Virus 1 5 5 2 0 5.8 SN 1 2 2 0 0 0 0.2 5.6 SN 2 4 1 0 0 0.5 5.3 SN 4 4 0 1 0 0.5 5.3 SN 8 2 1 0 1 0.3 5.5 SN 16 2 l 1 0 0.3 5.5 TABLE XIII EFFECT OF TIME AND TEMPERATURE OF INCUBATION ON THE SERUM NEUTRALIZATION TEST AT 22-25906 Inggb. log_of virus dilution hours 100 10"1 10"2 10'3 10-4 10-5 10"6 10"7 ld5o 1d50NI Virus 1 5 5 4 0 6.4 SN 1 4 1 2 0 0.8 5.6 SN 2 4 1 1 0 0.6 5.8 SN 4 4 2 0 l 0.8 5.6 SN 8 2 1 0 0 0.0 6.2 SN' 16 2 1 0 0 0.0 6.4 47 At 37°C, virus titration and serum neutralization tests were made at one, two, four and eight hours after incubation. Table XIV. The virus was titrated at each test period because of its thermolability at this temperature as shown by previous studies. Table III, Figure 3. The l.d.SONI at 4°C varied irregularly from 105'5 at the one hour test period to 105'5 after sixteen hours. At 22-2506, the l.d.SONI increased slightly but irregularly from 105'8 at one hour to 106'1 after sixteen hours. At 37°C, the l.d.50NI irregularly declined from 105’7 at one hour to 105’4 after eight hours. The sharp decrease to 104‘3 which occurred at two hours was Considered to be due to technical variations in the test.1 Disregarding the results for this one test period, all data indicate that prolonged incubation at the various“ temperatures did not significantly influence the l.d.SONI. Figure 7. TABLE XIV EFFECT OF TIME AND TEHEERATURE 0F INCUBATION ON THE SERUM NEUTRALIZATION TEST AT 37°C' 48 Tnsfib. 10g of virus dilution in hours 100 10'1 10"2 10'310‘4 10"5 10'5 10'7 ldso ldSONI Virus 1 5 5 4 0 6.4 SN 1 3 2 1 0 0.7 5.7 SN’ 2 4 3 1 0 1.2 4.3 Virus 4 5 4 2 0 507 SN 4 2 1 0 1 0.2 5.5 Virus 8 5 4 0 0 5.4 SN 8 2 1 0 0 0.0 5.4 l.d. 49 FIGURE 7 EFFECT OF TIME AND TEMPERATURE OF INCUBATION ON THE SERUM NEUTRALIZATION TEST AT 4°C, 22-25°C and 3700' 50 NI 7 6 ......__. -__....—-"".‘“""“'"'------O 5 W_ _ - _' — —o 4 e 3 2 1 O l 2 4 8 16 Time - hours ~——-wo no- can--- 22‘2500 37°C 50 0.. The Time-Rate of Virus Neutralization 'Bushnell and Erwin20 found that maximum adsorption of Newcastle disease virus on chicken red blood cells took place within two and one-half to five minutes after mixing the cells 21 reported that maximum neutralization of and virus. Crawley equine encephalomyelitis virus by immune serum was obtained within a few seconds after mixing the virus and the serum. The following time-rate studies were made to determine the rate of neutralization of infectious bronchitis virus by immune serum. 1. Procedure and Results Time-rate studies were made at 4°C, 22-25°c,_and 37°C in thermostatically controlled water baths. All ingredients were allowed to reach thermal equilibrium prior to combining for the test. Throughout the entire experiment, the virus-serum mixtures were inoculated immediately after preparation and at five minute intervals for a total period of 45 minutes. The virus was titrated last in all instances to take into consideration any possible deleterious effect of incubation at the various test temperatures. At 4°C, the l.d.5ONI increased from 105‘3 at zero time to an approximate maximum of 105'2 after 15 minutes. Accept- ing 1.d.5ONI 105'2 as maximum, 84 per cent of the infectivity neutralizing potential of the serum was utilized at the zero sampling period. Table XV, Figure 8. TABLE XV TIME-RATE 0F VIRUS NEUTRALIZATION AT 4°C 51 time log‘ of virus dilution " in min.. 10° 10‘1 10"2 10'3 10" 10‘”5 10'5 10'7 1d50 1d50NI Virus 55 5 5 5 2 6.8 - SN‘ 0 5 3 2 0 1.5 5.3 SN ' 5" 5 3 0* 0 1.2 5.6 SN 15 4 0 2 0.6‘ 6.2 SN 20 4 1* 0 0.5 6.3 SN: 25 3 1* O 0.3 6.5 SN 30 4 1* o 0.5 6.3 SN 35? 5 1 0 0.6 6.2 SN? 40 5 O 0 0.5 6.3 SN‘ 45 2 2 0 0.6. 6.2 TABLE XVI TIME-RATE 0F VIRUS NEUTRALIzATION AT 22-25°C 52 time .____ loggof virus dilution in- min.. 10° 10"1 10'2 10"3 10"4 10’5 10'5 lo"7 1d50 1&50NI virus 55 5 5 2* 2 6.4 - SN' 0 5 3 0 1.2 5.2 SN" 5 4 2 2 1.0 5.4 SN 10 5 l 2 0.9 5.5 SN 20 4* 0 0 0.7 5.7 SN 25 5 3* 1“ 1 .5 4.9 SN‘ 30 4* 2 2* 1.2 5.2 SN 35 4 0 2 0.6 5.8 SNW “5 5 0 0 0.5 5.9 *‘deaths per three embryos inocfiIEted eeeeeeee OOOOOOOO 53 At 22-2500, the initial l.d.SONI increased from 105"2 to an approximate maximum of 105'? after twenty minutes of incub- ation. Table XVI.‘ Figure 8. In this experiment, there was considerable fluctuation in end-points because of the high evidence of non-specific embryo mortality. In addition,” results were not obtained at two time intervals due to tech- nical difficulties. However, accepting l.d.SONI 105’7 as the maximum, 91 per cent of the infectivity neutralizing potential of the serum was utilized at the zero sampling period. The initial l.d.soNI of 105’9 was increased to an approx- imate maximum of 106'7 after ten minutes incubation at 37°C. Accepting 106’7 as maximum l.d.SONI, 86 per cent of the inf- ectivity neutralizing potential of the serum was realized at the zero time sampling period. Table XVII, Figure 8. These data indicate that between 84 and 91 per cent of the virus neutralizing capacity of immune serum is realized immediately after preparation of the serum-virus mixture. Maximum neutralization occurred 15 and 20 minutes after preparation at the several temperatures employed. TABLE XVII TIME-RATE 0F VIRUS NEUTRALIZATION AT 37°C timo loggof.virus dilution n. 0 -1 -2 -3 -4 -5 -6 -7 min.. 10 10 10 10 10 10 10 10 ldso IEQQNI Virus ‘55 4a 5 4 2 6.7 - SN 0 2* 3 0 0.8 5.9 SN’ 5 3* O 0 0.3 6.4 SN 10 1* 2 O 0.0 6.7 SN 20 2* 0 1 0.3 6.4 SN 25 2 O 1 0.0 5.7 SN ~30 3 O O 0.2 5.5 SN 35 4* 1* 1 0.8 5.9 SN' 40 2* O O 0.0 6.7 SN 45 1* 1 1 0.0 6.7 54 l.d. 50NI FIGURE 8 TIME-RATE STUDY AT 4°C, 37°C, AND 25°C. .fl.\ ‘ .—. /-:.,—---.s—w-.. ,e ‘0..---..‘ a! A) \N -p \fi 0\ ‘4 0 5 101520 2530354045 Time - minutes cannons-nn- - 22-2506 - 37°C 55 56 DISCUSSION? The data obtained from this study of the serum neutral- ization test for infectious bronchitis of chickens offer certainspractical applications for the use of the test as a diagnostic procedure.. Infectious bronchitis virus was more stabile at 4°C than at 22-25°C. 37°C or 56°C for extended periods of time. While the quantitative results did not show marked variation, incub- ation at 4°C for*the serum neutralization.test would be more desirable than.at higher temperatures in order to minimize any possible deleterious effects of temperature. Serum.may be stored at 4°C for as long as eight weeks and at 22-25°C for as long as seven days prior to testing without any change in the neutralizing capacity.. At 37°C;.a ten-fold decrease in neutral- izing capacity occurred after 56 hours storage.. These results indicate that 4°C should be routinely employed for storage of serum, but that no deleterious effect would be expected at 22- 25°C such as would be experienced in shipping serum to the laboratory from the field.. No advantage in accuracy of the serum neutralization.test would be expected when more than five eggs are used per serial tenrfold dilutions for titration.and evaluation of the results by the 50 per cent end-point method.. This offers a considerable saving in.the cost of conducting the test. The data obtained indicate that inoculation of 0.1 cc of a mixture containing an equal part of serum and virus per egg ‘would be expected to yield the most satisfactory results. 57 Decimal dilution of serum followed a linear exponential regression equation when either phosphate buffer, 0.85 per cent NaCl or nutrient broth was used as diluents. Either of the diluents could be used with expected replication of results. The lad-SONI for diluted serum could be multiplied by its. dilution factor to obtain the titer expected for undiluted serum.. There were no apparent benefits to be derived from pro- longed incubation of serum-virus mixtures at 4°C, 22-25°G or 37°C prior to egg inoculations. The results.showed that maximum neutralization of the virus by specifically immune serum occurred from 15 to 20 minutes after mixing of the ingredients.. Consideration.of these data would indicate that a stand- ard procedure for conducting the serum.neutralization test of infectious bronchitis of chickens would consist of using 4°C incubation as a routine measure, although for Short ‘periods of time, incubation could be at 22-25°c.. Five eggs 'per dilution, with either'phosphate buffer, 0.85 per cent NaCl or nutrient broth as diluent, equal parts of serum.and virus, inoculum of 0.1 00 per egg and incubation for about 20 minutes before inoculation of eggs with the serumrvirus mixtures should be employed in.serum neutralization:tests. 'Virue titrations should be made last to take into consider- ation any possible deleterious effects on the virus during the incubation period. 58 SUMMARY 1. IBV was more stabile at 4°C than at 22-2500, 37°C, and 56°C. 2. IB immune serum undergoes no significant change in neut- ‘ ralizing capacity following exposure of eight weeks at 4°C, seven days at 22-2500, and at 37°C a ten-fold decrease occurs after 56 hours. 3. No significant increase in virus infectivity was detected by increasing the number of eggs used per dilution from five to seven, eight or ten. The i l.d.50 : 105'}, 0" e : 10°-07. For serum neutralization tests using these different number of eggs per dilution, the E l.d.SONI : 105‘2, 0': 1 100’21. 4. Inoculums of 0.100 of equal parts of serum and virus showed maximum neutralizing capacities. 5. Immune serum can be diluted in phosphate buffer, 0.85 per cent NaCl, or nutrient broth without sacrificing accuracy. Decimal dilutions resulted in a decimal decrease in l'd'SONI I 100.66. 6. No increase in neutralizing capacity occurs with pro- longed incubation from one to eight hours at 37°C, or one to sixteen hours at 4°C and 22-2500. 7. Eighty-four to ninety-one per cent of the infectivity neutralizing potential of IE immune serum is utilized immed— iately after serum-virus contact, and maximum neutralization is obtained 15 to 20 minutes later depending upon the tem- perature of incubation. 1. 2.. 3. 7.. 8.. 10.. 11. 12. 13.. 59 LITERATURE CITED Amies, C.R.:, The influence of temperature on the sur- vival of pure suspensions of the elementary bodies of vaccinia.. Brit..J..Exp. Path., 15,: (1934): 180-185.. Andrewes,.C.H.: Some prOperties of immune sera active against fowl-tumor viruses.. J..Path..and Bact., '25,, (1932): 243-249.. Asplin, F.D.: Identification of infectious bronchitis in England. vet..Rec., 69,. (1948):: 485-486. Baten, W.:- Personal communication_ (1954). Beach, J.R.: Poultry diseases; recent discoveries. Proc. 5th. Pac. Sci. Congr.,_4, (1933): 2961-2968. _ : Coryza and other respiratory infections in CEIcEens. Proc.-l2th. Int..Vet..Congr.,‘2, (1934): 144-155. J__: Diseases of Poultry, edited by H.E. Biester and L. DevrIes.. Iowa 8 a s College Press, Ames, Iowa.. (1943):: 415-418.. _:_: Diseases of Poult , 2nd. edition, edited by H.E. BIester and—L.H. c warte.. Iowa State College Press,.Ames, Iowa.. (1948): 475-479. Beach, J.R. and 0.W. Schalm: A filterable virus, distinct from that of laryngotracheitis, the cause of a res- piratogy disease of chicks.. Poult..Sci.,'l§, (1936): 199-20 0. Beaudette, F.R.: Twenty years of progress in immunization against virus diseases of birds. J. Am. Vet. Med. Assoc., 115, (1949): 367-380.. : Infectious bronchitis, differential wCharacteristics from Newcastle disease. Canad. J. Comp. Med. and Vet. 801., 14,. (1950):- 24-27. and 0.3. Hudson:: Cultivation of the Virus of InfectIous bronchitis.. J. Am..Vet..Med..Assoc., 29.9. (1937): 51-60. Bell, E.J.: Experimental studies of variables in neutral- izationitests with Lane Poliomyelitis virus. Am. Jo. Hyg., 5202,. (1948) 3? 3 1-3930 14.. 15. 16.. 17.. 18.. 19.. 20. 21.. 23.. 24. 25.. 26. 60 Brown, 0.0% and T..Francis, Jr.: The virus-neutralizing action of serum from mice infected with poliomyelitis Vimae- Je EXPO Med. ,. .8...]:’ (1945) 3 151-169 e- Brandly, C.A., H.D. Moses,,and E.L. Jungherr: Transmission of anti-viral activity via the egg_and the roll of con- gential passive immunity to Newcastle disease in chickens. Am. J. Vet. Res.,‘1. (1946): 333-342. Brodie, M.: The potency and changes with storage of polio- myelitis serum. J..Immun01., 21,, (1934): 479. Bryan, W.R..and J.w. Beard: Quantitative studies on the neutralization of purified papilloma virus. I.. The relations between sermm,.tota1 virus and free virus. Je- InfOCte. D18. '; fig, (1941): 133'1700 Burnet, F.M..and J. Macnamara: The activity of stored anti-poliomyelitis serum in experimental poliomyelitis. J. Med..Australia'g, (1920): 851. Bushnell, L.D. and C.A..Brandly:r LaryngotraCheitis in chicks.. Poult. 801.,‘lg,. (1933): 55-60. and L.E. Erwin: Studies on Newcastle dis- ease. VII.. The rate of virus adsorption.in the hem- agglutination reaction of Newcastle disease virus. Trans. Kans..Acad..Sci., 22,, (1950): 378-380. Crawley, J.F.: Neutralization tests with eastern.equine encephalomyelitis virus in the chick embryo. .A stat- istical treatment of the results by the probit method. Croxton, F.E.: Elements Statistics with A lications in Medicine. PrenEIce-EalI,.Inc., New YorE, N.Y. (I953). Cunningham, C.H.: A Laborato Guide in Virolo . 2nd. Edition, Burgess PuEIIEEIng 00., MIEneapo s,.Minna : Methods employed in the diagnosis and Inféstigationxof infectious bronchitis and Newcastle disease.. Proc. Book Am. Vet. Med. Assoc. 89th..Ann. Meet.” Alantic City, N.J.. June 23-26, 1952: 250-257. : Neucastle disease and infectious bronr Chitis neutralizing antibody indexes of normal chicken serum. Am. J. Vet. Res., 12,, (1951): 129-133. and AmHe E1 Dardiry: Distribution of the virus oTFIEIectious.bronchitis of chickens in embryon- sting chicken eggs. Cornell vet.,.3§, (1948): 381-388. 27.. 28. 29.. 31.. 32. 33.. 34. 35. 36. 37.. 61 and M.H. Jones:. The effect of different routes of Inoculation onithe adaptation.of infectious bronchitis virus to embryonating chicken.eggs. Prec. Am. vet..Med. Assoc. 90th..Ann. Meet., Toronto, Canada, July 20-23, 1953: 337-342. and H.O..Stuart:. The effect of certain CEEmical agents on the virus of infectious bronchitis offichickens.. Am. J..Vet..Res.,lI, (1946): 466-469. 8 The pH stability of the VIfus of Infectious brondhitIs of chickens. Cornell vet. .. 3 ,. (1947) 5 99-103. : Cultivation of the virus o? InfectIOus bronEhitIs of chickens in embryonated chickenzeggs.. Am. J. vet..Res., 8,. (1947): 209- 212.. Delaplans,,J.P.: The differentiation of the respiratory diseases of chickens. Rhode Island Agr. Exp..Sta., Bull. 288, (1943). : Technique for the isolation of infectious brendhitis or Newcastle virus including observations on the use of streptomycin41n overcoming bacterial contaminants. IProc. of the 19th. Ann..Conf. Lab. Workers Pullorum Dis..Control, Raleigh. N.C. June 11-12-13,.1947. : Panel discussionLon poultry diseases. J. Am. Vet..Med. Assoc., 106,, (1945):; 91-105. and H.0. Stuart: Studies of infectious eronéhitis.. Rhode Island Agr. Exp. Sta..Bull..273. 1939 . : The modificationiof Infectioui—bronchitis virus of Chickens as the result of propagation in embryonated chicken e s. Rhode Island Agr. Exp. Sta., Bull. 284, (194%?: 1-20. Dixon, W.J. and F.J. Massey: Introduction to Statistical Analysis. McGraw-Hill B005 00., Inc.,CI951. Fabricant, J.: Studies on.the diagnosis of Newcastle disease and infectious bronchitis of fowls. II.. The diagnosis of infectious bronchitis by virus isolation in Chick embryos.. Cornell Vet.,I3_, (1949): 414-431.. 38. 39. 41.- 42. 43.. 47.. 48.. 49. 62 .__ : Studies onithe diagnosis of Newcastle diiease and infectious bronchitis of fowls. III. The differential diagnosis of Newcastle disease and §3fzgtious bronchitis.. Cornell vet.,.jg, (1950): 8 Studies on the diagnosis of Newcastle disease and infectious bronchitis. IV.. The use of the serum neutralization.test in the diagnosis of égfgctious bronchitis. Cornell Vet., 41, (1951): - 0. and P.P. Levine: The persistence of Infectious bronchitis virus in eggs and tracheal exudates of infected chickens.. Cornell vet.,‘4;, (1951): 240-246. Gibbs, 0.8.: Bronchitis of baby chicks. Poult. 801., L2.” (1933) 3 46e- Groupe, V.:- Demonstration of an interference phenomenon. associated with infectious bronchitis virus (IBV) of chickens. J..Bact.,.§§,. (1949): 23-32. and L..Pugh: Interference between influenza 71 virus and infectious bronchitis of chickens. J. Bact., .62” (1952): 295-296.. Hirst,,G.K.: The quantitative determination.of influenza virus and antibodies by means of red cell agglutin- ation.. J..Exp..Med., ‘15, (1942):- 49-64. Hofstad, M.S.: .A study of infectious bronchitis of chickens. I. The pathology of infectious bronchitis. Cornell vet., .25, (1945): 22-31. 8' A study of infectious bronchitis of chickens.. II. Observations on the carrier status of chickens recovered from infectious bronchitis. Cornell Vet., 3 ,. (1945): 32-35.. x: A study of infectious bronchitis of chickens. III. Attempts to utilize the chicken red cell agglutinationitest as-a diagnostic aid in infect- ious bronchitis.. Cornell V0t.,'2§, (1945): 60-61. 8 A study of infectious bronchitis in. chICkens. IV}. Further observations on the carrier status of chickens recovered from infectious bronchitis. Cornell Vet., 31, (1947): 29-34. and 8.0. Kenz : Susceptibility of chicks ' Eitdfiea'from recovered one to infectious bronchitis. Cornell Vet., 2_. (1950): 87-89. 50. 51. 52. 53.. 54.. 55. 56. 57. 58.- 59.. 60. 61.. 63 : Neutralization of epidemic influenza Virus.. The linear relationship between the quantity of serum and the quantity of virus neutralized. J. Exp. Med., 19, (1939): 209-222. and E.C. Gurneu: Studies on pneumonda virus of mice (PVM).. I. The precision of measure- ments in vivo of the virus and antibodies against it. Jo Ems-Meae. Q, (1945): 25-42e and E.H. Lennette: Neutralization of Infiuenza A virus by human.serum.. J. Exp..Med., 13, (1941): 327-333.. Howitt, B.F.: Poliomyelitis. IV. Further studies on the immunization of sheep to the virus of poliomyelitis, with a comparison of neutralization.tests, using the old and a recent strain of virus. J. Infect. Dis., 52. (1933): 145-156. : Deve10pment of neutralizing antibodies to the virus of equine encephalomyelitis (Western Strain) and St. Louis encephalitis in the blood and cerebro- spinal fluid of man and animals together with recovery of the St. Louis virus from the blood of monkeys. J. Immunol.,hflg, (1941): 177-181. : A non-specific heat-labile factor in the serum neutralization test for Newcastle disease virus. J. Immunol., ‘64} (1950): 73-84. Jungherr, E.L. and N.L. Terrell: Naturally acquired passive immunity to infectious bronchitis inzchicks. Allie Je Vet. R030. 2,. (19%): 201-2050 Knight, C.A.: Titration of influenza virus in.chick embryos. J. Exp. Med.,‘zg,- (1944): 487-495. Komarov, A. and F.R. Beaudette: Carriers of infectious bronchitis. Poult. Sci., 11,- (1932): 335-338. Lauffer5,M.A., E.L. Carnelly and E. MacDonald: Thermal destruction of influenza A virus infectivity. Arch. of. Biochem... g, (1948): 321-328. Lauffer,.M.A. and W.C. Price: Thermal denaturation of tobacco mosaic virus.. J. Biol. Chem., 132 , (1940) 1-15 0 Levine, P.P.: Infectious bronchitis. Pros. Ann..Meet. U.S. Livestock Sanitary Assoc., 55, (1951): 183-186. 62.. 63. 64.. 65.. 66.. 67.. 68.. 69.. 70.. 71. 72.. 73. 64 and M.S..Hofstad: Attempts to control air-borne infectious bronchitis and Newcastle dis- ease of fowls with sterilamps.. Cornell Vet., 21, (1947): 204.211. Loomis, L.M., C'.H. Cunningham,.M.L. Gray, and Frank Tharp Jr.: Pathology of the chicken embryo infected with infectious bronchitis virus.. Am..J. Vet. Res., 29... (1950): 245-251. Melnick, J.L. and N. Ledinkox. Immunological reactions of the Coxsackie virus.. I.. The neutralization test: Technic and application. J. Exp..Med.,‘Qg, (1950): 463-482. Monroe, E., W. van Herick, and G. Meiklejohn: Studies on the etiology of primary atypical pneumonia. III. Specific neutralization of the virus by human serum. Je EXPe Made , _83,- (1945): 329-342eo Morgan, 1.: Quantitative study of the neutralization of Western.equine encephalomyelitis virus by its antiserum and the effect of complement.. J. Immunol., 3... (1945): 359-371. Nanavutty, S.H.: The thermal death-rate of the bacterio- phage.. J. Path..and Bact., 22, (1930): 203-214. Olitsk , P.K. and J. Casals:z The effect of incubation at 3 .C on the neutralization.test with various enceph- alitis viruses including Lansing strains of polio- myelitis virus. J. Immunol., ‘_Q,. (1948): 487-496. and L.C. Murphyx‘ Effect of prolonged storage at I to 5°C on the neutralizing antibody of antiserum against poliomyelitis virus. J. Lab. and Clin. Med., 26,, (1950): 163-166.. Page, C1A.: Antibody response of chickens exposed to infectious bronchitis virus.. Thesis, Master of Science Degree, Michigan State College Library, East Lansing, Parker,.R.F..and L.E. Bronson: Neutralization of the virus of Myxoma by specific immune serum. J. Immunol., 49, (1941): 147-152. Reched, S.H.: Antibody response of turkeys vaccinated with formalin-inactivated Newcastle disease virus. Michigan State College Agr. Exp. Sta., Tech. Bull. 215, (1949). Reagan, R.L., A.L. Brueckner, and J.P. Delaplane: Morpho- logical observations by electron microscopy of the viruses of infectious bronchitis of chickens and the chronic respiratory disease of turkeys. Cornell vs., .42., (1950): 384-386. 74. 75.. 76.. 77.. 78.. 79.- 80. 81.. 82. 83. 84. 85.. 65 , J.E. Hauser,,M.G..Lillie, and A.H. CraIge,,Jr.:. Electron.micrograph of the virus of infectious bronchitis of.chicksns. Cornell vet., 2... (1948):. 190-191. Reed, L.J..and H. Muench: A simple method of estimating {égt per cent end-points.. Am. J. Hyg., 21, (193 )3 "' 97o Schalk, A.F..and M.C. Hawn: An apparently new respiratory disease of baby chicks.. J. Am..Vet. Med. Assoc., Zé. (1931): 13-422. Schalm,.0.w. and J.R. Beach: The resistance of the virus of infectious laryngotracheitis to certain ph sical and chemical factors. J. Infect. Dis., 5Q, 1935): 210-223e. Shanan, M.S.: Effect of temperature, phenol and crystal violet on vesicular stomatitis virus.. Am. J. Vet. Swierstra, D.: Bronchitis infectiosa biJ Kippen in deerignd. Tijdschr..Diergeneesk,hzg, (1947): 7 5-7 .. Tyrrell, D.A.J. and E.L. Horsfall,.Jr.: Neutralization of viruses by homologous immune serum. 1. Quant- itative studies on factors which affect the neutral- ization reaction with Newcastle disease, influenza A, aged bacterial virus,, '13. J. Exp. Med.,, 2Q, (1952): 3 7.. . Van Roeke1,.H., E.L. Bullis, 0.8. Flint, and M.K. Clarke: Poultry Disease Control Service. Mass. Agr. Exp. Sta. Ann..RBpt., Bulle 388,. (1942):‘ 99'103e : Poultry Disease‘abntrSI Service. Mass. Agr. Exp. Sta. Anne Rapte, Bulle 428, (1949): 64-66e , M.K. Clarke, O.M. Olesiuk, andvFTG.Sperling: Infectious Bronchitis. Mass. Agr. Exp. Sta., Amherst, Mass., Bull. 460, (1950). : Infectious bronchitis. Am. J:— von.Magnus, P.: PrOpagation of the PR8 strain of influenza A virus in chick embryos.. Acta Path. and Micro.. Scand., 28, (1951): 250-277.. 66. 86. Whitman, L.: The neutralization of Western Equine encep- halomyelitis virus by human convalescent serum. The influence of heat labile substances on the neutrali- zation index. J. Immunol., 56 (1947): 97-108. 87. Young, L.E. and M. Merrell: The mouse-adapted Lansing strain of poliomyelitis virus. II. A quantitative study of certain factors affecting the reliability of ghe neutralization test. Am. J. Hyg., 21, (1943): 0-92 e - RHMEE‘Q u .E ’guflw tray "7|" IIGUIAUINI W W WSW HWIBHWITIB 3 1293 03142 6616