«MIMI l 1 MI» I IN § t M l | I \ \ £ 1 ‘ MW '-I_._. IN-A 0le 11% EFIFECT 01-9 WFFERENT ROUTES GP INC3€3LI£ATIC3N ON THE ADAPTATION O’x‘ INFECTIGUS- WRONCHITIS WRUS TD EMBRYQNATH‘MB CHXCKEN EGGS Thwis far fiw Dagme. cf M. :53. MICHEGAN STATE? CGLLESE Mary H. fianas “€95.31 masts , ' This 1. to certify that the thesis entitled The Effect of Different Routes of Inoculation on the Adaptation of Infectious Bronchitis Virus to Embryonating Chicken Eggs. presented by Mary H. Jones lies been accepted towards fulfillment of the requirements for Major professor New / 1. /7.r/ . >..’ 1‘.; f .' p’ 6.1! '13“ f, ‘\.I '7‘ ‘-'~\": *p‘? . fivv 44 r I'-‘ _.. ‘,.\- .Ju. ,‘ 44,-» ,-‘- we ‘4’.’ _».«-e»r.<.:.m:--.- _ . a w. - '¢ ' .‘ ‘ . ‘ A ' ' - I I" a : I. 1 ii“ 1' 'ba"‘ v t. ._ ' 1"“) ~ 0.: ‘. ‘fi-‘JF“AM-f"(% ("b ' .' fir. , .' n ' o '6" ‘. “Far I»: ‘— . . . 3, firdtg-sasuc ,- ‘ -~:' ’ - s . .‘ ‘ ' \ . | V \ . O O U ‘f I . ’ I l l . I D . ‘ . THE EFFECT OF DIFFERENT ROUTES OF INOCULATION ON THE ADAPTATION OF INFECTIOUS BRONCHITIS VIRUS TO EMBRYONATING CHICKEN EGGS BY MARY H. _J_C_>_NEs 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 MASTER OF SCIENCE Department of BacteriOIOgy and Public Health 1951 THESIS ACKNOWLEDGMENTS The author wishes to express sincere ap— preciation to Dr. C. H. Cunningham, Associate Professor of Bacteriology and Public Health, Michigan State College, for his kind guidance and advice, and to Mrs. M. P. Spring for her assis- tance and c00pe ration. 2637107 TABLE OF CONTENTS REVIEW OF LITERATURE A. Infectious Bronchitis 1. Characteristics of the Disease 2. GeOgraphicaI Distribution 3. Eti010gical Agent 4. Symptoms 5. Lesions 6. Transmission and Immunity Studies. 7. Control Measures and Immunization Studies 8. Diagnosis B. Cultivation of the Virus in Embryonating Chicken Eggs MATERIALS AND METHODS A. Inoculum Used for the First Passage of the Virus B. Routes of Inoculation C. Techniques of Egg Inoculation Page 10 ll 13 20 20 21 22 l. Allantoic Cavity Inoculation 2. Chorioallantoic Membrane Inoculation 3. Amniotic Cavity Inoculation 4. Yolk Sac Inoculation D. Harvest and Preparation of Material for Subsequent Egg Passage 1. Collection of Allantoic Fluid 2. Collection of Chorioallantoic Membranes 3. Collection of Amniotic Fluid 4. Collection of Yolk E. Examination of Embryos and Chorioallantoic Membranes RESULTS AND DISCUSSION . SUMMARY . REFERENCES iv Page 23 24 27 29 30 31 32 34 35 36 37 47 , 49 REVIEW OF LITERATURE A. Infectious Bronchitis 1. Characteristics of the Disease Infectious bronchitis is a respiratory disease of chickens, which has, thus far, not been reported as infectious for other fowl. Originally, the disease was considered to be confined to . 39 . . . chicks, but has Since been shown to infect chickens of all 16 . . . . . . . ages. The morbidity is high, the disease spreading With marked rapidity and sometimes infecting the greater part of a 3 flock over a short period of time. In chicks, the mortality may be as high as 80 or 90 per cent, but in birds over six 2.3.4.16,1 ,28,3 weeks of age the mortality is negligible. 7 9 In— fectious bronchitis presents a serious economic problem in view of the frequency with which it has been noted in chicks and the marked decrease in egg production which usually re— 18 sults when a laying flock becomes infected. 42 The incubation period may vary from 1 day to 6 or 7 days.5 The duration of the disease is relatively short, recovery occurring usually within 10 days to 21 days,5’18'42 In some cases, the disease may follow a protracted course. 42 . Van Roekel _e_t_ §_l_. observed that age has no influence on the course of the disease, as no difference was noted between chicks and sexually mature birds. 2. Geographical Distribution Infectious bronchitis was first reported and described . 39 . . in 1931 by Schalk and Hawn, who had encountered it in North Dakota in 1930. By 1933 the disease had been reported in 8 . . 24 Kansas by Bushnell and Brandly, in Massachusetts by Gibbs, and in California by Beach.3 It was soon found that the disease was nationwide in its distribution. Recently, infectious bron— l chitis has been reCOgnized in England, in the Netherlands and in Canada. 3. EtiOIOgical Agent The causative agent of the disease is a virus which has been shown to be capable of passing through all grades of Berke— 2,4,8,16,17,28,3 feld, preliminary Mandler and Seitz filters. 9 3 The virus has been shown to be air-borne for a distance of five feet.34 a. Morphology. By means of electron microsc0py the virus particles . . . . 37,38 were shown to have round heads With filamentous prOJections. In the initial study the heads were observed to have a mean 3 diameter of 90 millimicrons, 7 but improved technique has demonstrated a mean diameter of 70 millimicrons. b. Distribution in the body. The virus has been found most abundantly in tissues and exudates of the respiratory tract, and has also been isolated . . . 8 . . from the liver, kidney tissues and blood. According to Fabri— 22 . . . cant, the Virus can usually be isolated from chickens through- out the respiratory phase of the disease. c. Effect of physical agents. Beach and Schalmz demonstrated that the virus in tracheal exudate, when frozen, dried, and stored in the refrigerator for 4 180 days, could incite the disease. Virus stored in 50 per cent glycerin remained viable for 80 days. 17 , Delaplane and Stuart reported that egg—prOpagated Virus was viable after storage in the freezing compartment of a re— frigerator for 4—1/2 months but not for 5-1/2 months, as indi- cated by egg tests. It survived at room temperature for 5 to 7 days but not for 14 days, and at 500 C. for 15 minutes. , 10 . . Cunningham and Stuart found that infected allantOic o fluid, stored at -25 c. and at .70° 0., produced a higher virus titer than infected allantoic fluid stored at ~10° C. Allantoic fluid when dried from the frozen state, _i__r_1__ vacuo, and stored at 40 C. for 7 days showed a hundred-fold decrease in virus activity when restored to volume. Freezing and thawing pro— duced no effect on virus activity. , 34 . . LeVine and Hofstad showed that air—borne Virus could be destroyed by ultraviolet irradiation but concluded that under ordinary field conditions such treatment would be of no practical value in controlling the disease. d. Effect of chemical agents. Cunningham and Stuart9 studied the effects of certain chemical agents on an egg—adapted strain of the virus. Infected allantoic fluid was subjected to the action of each agent for a period of 3 minutes, then injected via the allantoic cavity into embryonating chicken eggs. Inactivation of the virus was indi— cated by survival of inoculated embryos. The following reagents were found to inactivate the virus: phenol, 3 per cent and 1 per cent; liquor cresolis saponatus, 3 per cent and l per cent; tincture of metaphen, undiluted and 1 per cent; potassium permanganate, 1:1000 and 1:10,000; ethyl alcohol, 95, 70, 40 and 25 per cent; tincture of zephiran, 1:1000; Lugol's solution, 1 per cent; sodium hydroxide, 1:20; neOprontosil, 5 per cent, and formalin, l per cent. Tincture of iodine, 0.01 per cent, and boric acid, 4 per cent, were without effect on the virus. e. pH stability. The pH stability of the virus was studied by Cunningham and Stuart. 11 For the first 60 days the virus was more stable in an acid medium, but from the 60th day to the 170th day it 6 showed greater stability in an alkaline medium. The virus re- mained active for 170 days in a phOSphate buffer (.05 M) at pH 7.79, and in allantoic fluid at pH 7.80 for 100 days. 4. Symptoms Sneezing, coughing, gaSping and reSpiratory rales are the symptoms most characteristic of the disease. Nasal dis— charge and swollen sinuses may be present in chicks. Severely— affected birds may show depression, weakness and decrease in feed consumption. 2,3,16'18’39 A marked decline in egg production is evident when lay- ing flocks contract the disease.2'16'l8'28 Losses as high as one dollar per bird may result from decreased egg production and abnormal egg quality, both of which frequently persist for a considerable length of time. Egg production may dr0p to as low as 2 to 3 per cent, and, although it increases on recovery, preinfection levels of production are seldom regained. Eggs are often misshapen, rough and thin-shelled with watery albu— 41 men. 5. Lesions Gross lesions are found mostly in the lungs, the bronchi and the lower trachea. These lesions consist of congestion and edema of the lungs, and serous or mucous exudates in the trachea and bronchi, the latter sometimes containing caseous plugs. In some instances, the air sacs may appear cloudy and contain accumulations of caseous, yellow exudate. In chicks, pericarditis, and coating of the nasal mucosa with a heavy, sometimes purulent, mucoid exudate may be found. Splenic changes are not well defined.2'4'7'16’26’39 The histOpathOIOgic lesions, as reported by Hofstad,28 include thickening of the tracheal mucous membrane and sub- mucosa, due primarily to edema and diffuse, leucocytic infil— tration, with little evidence of tracheal and bronchial desqua_ mation and complete absence of gross hemorrhage. No inclusion bodies were noted and no significant changes were observed in sections of liver, spleen and kidney. Beaudette stated that infectious bronchitis produces no changes in nervous tissue. 6. Transmission and Immunity Studies The disease is Spread through direct exposure of sus- ceptible chickens to infected chickens or to ”carrier" birds. Contaminated equipment or brooder houses may serve as in— direct foci of infection. It is considered that infection is introduced through purchase of infected chicks or carrier birds. Delaplane18 has suggested that other birds or animals may serve as carriers of the infection. The disease has been artificially induced by inoculation of respiratory exudates intranasally, intratracheally and intra— peritoneally.4’8’16’359 Symptoms usually deve10p within 48 hours in birds inoculated by the above routes. Intratracheal inoculation has produced the disease as early as 18 hours. The disease has also been produced, although not so quickly, by inoculation via the larynx, air sacs, air Spaces of bones and the thymus. l6 Subcutaneous and intramuscular inoculations were either non—infective or produced the disease after long incubation. Birds recovered from the disease are generally consid— 1,2,4,l6,18,29,3l ered to be refractory to subsequent infection 42 although Van Roekel _e_t a_l_. reported a few instances in which birds that were supposedly immune contracted the disease. Some recovered birds may continue as carriers of the dis— 16,18,31 . Although Hofstad31 was unable to demonstrate ease the persistence of the carrier state for as long as 80 days, Delaplane and Stuart16 reported one instance in which a bird remained a carrier for as long as 8 weeks. Serum from recovered birds is capable of neutralizing the virus. 1'2’4'16 Jungherr and Terrell33 found that neutral— izing antibodies were present in eggs laid by hens recovered from the disease, in the yolk through the 11th day of embry— onation and in the tissues and serum after the 16th day. Chicks hatched from such eggs maintained high antibody levels for 2 weeks after hatching and lost them by the end of the 4th week. In contrast to this, Hofstad and Kenzy 2 were able to produce the disease in 4, 6, 7 and 10 weeks—old chicks hatched from eggs laid by immune hens. These observations would indicate that natural passive immunity is not sufficient to protect the chicks against a challenge dose of the virus. No cross immunity exists with laryngotracheitis, New—- l,2,4,l6 castle disease or fowl pox. 10 7. Control Measures and Immunization Studies No medicinal agents have been found that are of any value in preventing or controlling the disease. 18 Thorough cleansing and disinfection of houses and equipment after an outbreak, prevention of contact between recovered and suscep— tible birds, and purchase of stock only from "clean" sources are precautionary measures suggested by Beach. Formalized virus has failed to incite immunity. Inoc— ulation of the mucous membrane of the cloaca and the bursa of Fabricius produced immunity but not sufficiently soon to prevent infection via the reSpiratory tract. 16 Beach3 states that applying virus to the cloaca can produce infection 0f the reSpiratory organs and therefore is not applicable as a means of immunization. In New England, a program of immunization has been ad0pted whereby infectious bronchitis is artificially induced in young birds at an age when the disease produces the least ob— jectionable results, i.e. , during the growing stage, beyond the period of high mortality but before the beginning of egg produc- tion. Two to 5 per cent of a flock of healthy birds not under 6 to 8 weeks and not over 16 weeks of age are inoculated ll intratracheally with a chicken—prOpagated strain of the virus. The disease is spread by the inoculated birds to the remainder of the flock. The best results have been obtained with chicks from 10 to 14 weeks old. Little or no retardation in growth has been observed. In an occasional flock, insufficient immunity 41,42 has been produced to prevent a subsequent natural outbreak. In Spite of the relative success of this method in providing protection against the disease during the laying season, Dela— 19 . . plane recommended the exposure of grow1ng pullets to infec- tious bronchitis only where poultry farms are in close proximity as in New England. Egg-prepagated virus has been used to produce the dis— ease in chicks 6 to 8 weeks of age. As with chicken—pr0pagated virus, this procedure has afforded protection to pullets during . 5,18 7 . the laying season. Beaudette suggested that the duration of immunity produced by embryo-modified virus would not be so lasting as that produced by a fully virulent strain. 8. Diagnosis The virus of infectious bronchitis does not cause agglu— tination of chicken red blood cells, and it does not inhibit 12 . . . , 1,30 hemagglutination by Newcastle disease Virus. These phe- nomena have served to differentiate the two viruses. The clinical history, symptoms and lesions found at autOpsy are sometimes sufficient for diagnosis of infectious 2,1 ,1 , 8 bronchitis. 6 8 2 Serum neutralization tests in embryonating chicken eggs are of value in diagnosis of the disease if used not earlier . . 23.36 . than 3 weeks follOWing exposure to the Virus. According . 14 . . . . to Cunningham the serum neutralization titers of normal birds not previously exposed to infectious bronchitis would not be ex— . . . 23 pected to exceed 36 neutralizing doses. Fabricant concluded that a titer of 100 neutralizing doses or greater is diagnostic . . 21 42 for the disease. Fabricant and Van Roekel _e_t _a_l. have used bird inoculation tests in combination with serum neutral— ization tests to substantiate diagnosis by the latter method. Susceptible birds inoculated intratracheally with tracheal exu— dates from suSpected birds will show symptoms of the disease within 18 to 36 hours if the virus is present. Diagnosis is frequently based on characteristic lesions produced in embryonating chicken eggs when respiratory tissue suspensions or exudates from suspected birds are inoculated via 13 . . 20.21 . the allantOic caVity. Hitchner, Reising and Van Roekel26 suggested that diagnosis of infectious bronchitis by this method should be substantiated by the absence of hemagglutination by the allantoic fluid . B. Cultivation of the Virus in Embryonating Chicken Eggs Beaudette and Hudson4 were the first to study the cul— tivation of the virus in embryonating chicken eggs. Inoculation of the virus on the Chorioallantoic membrane proved to be rel- atively nonlethal to embryos in the first few passages. In subsequent passages the virus produced death in the majority of embryos. Gross lesions included dwarfing of the embryos to about one—half normal weight, and congestion of the liver. The yolk fluid was quite solidified, blood vessels were injected, residual albumen was watery and the Chorioallantoic membrane appeared thinner than normal and was adherent to the inner shell membrane. No pock or plaque lesions were observed, but occasionally the membrane showed a few turbid areas. Delaplane and Stuart17 confirmed the observations of Beaudette and Hudson4 with regard to the dwarfing of embryos and the thinness of the Chorioallantoic membrane after several 14 passages of the virus via the Chorioallantoic membrane. The virus increased in virulence for embryos with each succeeding passage until all embryos were killed at the 70th passage. Correspondingly the virus decreased in virulence for chicks so that saline washings of the Chorioallantoic membranes from the 89th passage failed to incite the disease. Gross lesions consisted of whitish foci on the liver, congestion and swelling of the kidneys and occasional hemorrhages on the embryo's skin. Whitish, Opaque circular lesions were present on the Chorioallantoic membrane at the point of inoculation. Beau- dette5 suggested that Delaplane and Stuart must have used parentally—immune chicks since they had been able to produce the disease in older chickens, i.e., birds old enough to have lost any parental immunity which they might have had at hatching. Delaplane20 reported that the allantoic cavity route of inoculation was superior to the Chorioallantoic membrane route for isolation of field strains of the virus, inasmuch as inocula— tion by the former route produced dwarfing on the first passage. He suggested the use of streptomycin to eliminate bacterial contamination of reSpiratory exudates, thus permitting their use for isolation of the virus in embryonating chicken eggs. 15 . l . . . . ASplin, studying the English strain of the Virus, noted few deaths in early passages. After the 14th passage the ma- jority of embryos died within 2 to 7 days. Dwarfing was the only change observed in the embryos. He confirmed previous 2 observations in the United States that the virus produced a milder effect in chicks with successive passages in eggs. , 21 , . 20 Fabricant confirmed the observations of Delaplane that lesions were produced more rapidly when the virus was inoculated via the allantoic cavity than by the Chorioallantoic . . 4 membrane, and the findings of Beaudette and Hudson that the yolk was of a thicker consistency than normal. Fabricant ob— . . , 4,17 served that, as in preVious studies, embryos were dwarfed and the Chorioallantoic membrane appeared thinner than normal and adherent to the inner shell membrane. In addition, he noted a tendency for the embryos to be tightly curled in a ball—like form due to a bending of the longitudinal axis of the embryo around its ventral surface. Curling usually preceded dwarfing and sometimes occurred in infected embryos of normal size. Curling of the embryo was associated with a decrease in size of the amnion and a decrease in the amount of amniotic fluid. Occasionally the amnion was thickened, cloudy and tightly 16 adherent to the embryo. Curling was considered to be the significant lesion produced by the virus and was sometimes the only change evident in the first three passages. On this basis, virus isolation was successful in 92 per cent of cases with a typical clinical history of infectious bronchitis. Of 116 virus isolations, 46 per cent were diagnosed on the first passage, 33 per cent on the second passage, 20 per cent on the third pas- sage and l per cent on the fourth passage. Loomis, Cunningham, Gray and Thorp35 studied the path— olOgy of chicken embryos inoculated by the allantoic cavity route with a chicken-prOpagated strain of the virus. Their observations confirmed those of Fabricant21 with regard to gross lesions produced by the virus in embryos. In addition, they noted that living embryos were sluggish in their movements, curled embryos had wry necks with lateral curvature, and de— formed feet curled over the head. Feathers were drier than normal and immature in development. Jaundice was observed in about one—third of the embryos and dermal petechiae were occasionally present. In about 25 per cent of the embryos the cloaca was distended with white, fat—like drOplets. Bones were retarded in deveIOpment and softer than normal. Leg bones 17 and tarsal joints were deformed. Incomplete closure of the abdomen, due to retardation of deveIOpment of the body walls, was observed. Proliferation of islands of tissue in the chorio- . . l7 allantOic membrane, described by Delaplane and Stuart, was not observed. Livers were abnormally dark and showed hemor- rhagic and necrotic areas. Kidneys were swollen and edema.— tous with some necrotic foci. Hearts and lungs were markedly smaller than normal, the latter being abnormally pale and soft in consistency. Spleens were twice normal size. Microsc0pic examination revealed edema of the Chorioallantoic and amniotic membranes. Proliferation of mesodermal and ectodermal cells was observed. Evidence of pneumonia, with serious exudation and granulocytic and lymphocytic infiltration was found in the lungs. Perivascular “cuffing,” hepatic hemorrhage, necrosis and abscess formation were observed in the liver. The kid— neys showed interstitial nephritis and necrosis. . . . 27 Hitchner, ReiSing and Van Roekel observed that a strain of infectious bronchitis virus, which has apparently lost its identify as such and is now designated as the B1 strain of . . 26 . . Newcastle disease Virus, caused curling, dwarfing and dryness of embryos, associated with tight adherence of the amnion to 18 the embryo, a marked decrease in amniotic fluid, an increase in the amount of allantoic fluid and thickening of the yolk. This . . . 21 . eVidence seems to refute Fabricant's observation that curling of embryos is pathognomonic for infectious bronchitis. These workers suggested that curling and dwarfing may not be due primarily to growth of virus in the tissue, but to a secondary change due to dehydration as a result of a flow of fluids from the amniotic sac, embryo and yolk sac into the allantoic cavity. . . . 7 A Similar explanation was expressed by Beaudette who sug— gested that curling of the embryo may be caused by a shrinking of the amniotic membrane due to loss of fluid. The prOperties of egg—adapted virus have been studied extensively. A strain of the virus which has been completely . . . . 10 _ egg—adapted Will kill all embryos Within 48 hours. Dwarfing of the embryos and congestion and swelling of the kidneys are . 17 25 usually the only 1eSions observed. Groupe reported the presence of a thermostable interfering material in allantoic fluid from infected dead embryos which had been stored at 360 C. for 24 hours after death. This material produced a markedly slower death rate in embryos. 19 When an egg—adapted strain was inoculated into embry— onating chicken eggs via the allantoic cavity, the highest con- centration of the virus was found in the Chorioallantoic membrane, followed in decreasing order by allantoic fluid, amniotic fluid, . . l3 and liver. Yolk was innocuous. Egg-adapted virus will not incite the disease in chicks 33 but can be neutralized by immune serum. MATERIALS AND ME THODS This study was undertaken with the purpose of determin- ing the effect of different routes of inoculation 0n the adapta- tion of infectious bronchitis virus to embryonating chicken eggs in the hOpe that it might contribute to general knowledge con- ce rning the vi rus. A. Inoculum Used for the First Passage of the Virus The strain of infectious bronchitis virus used, designated as Lot 290, was supplied by Dr. Henry Van Roekel, Department of Veterinary Science, University of Massachusetts. The virus was received as a saline suspension of traCheal washings from infected chickens. This was a sample of a chicken—prepagated strain which had not been cultivated in embryonating chicken eggs. In order to render the suSpension bacteria free, peni— cillin and streptomycin were added in amounts of 10,000 units each, per milliliter of suspension. The suSpension was centri— fuged in a clinical centrifuge (International Equipment Co.) for ten minutes and the supernatant fluid was used for inoculum. For eggs inoculated via the allantoic cavity, Chorioallantoic 1....“ 21 membrane and yolk sac routes, 0.1 cc. of the undiluted virus suspension was injected per egg. The suspension was diluted with saline, one in five, for inoculation via the amniotic cavity, and 0.1 cc. was injected per egg. The dilution of the virus susPension was necessitated by a limited amount of the original material. Also, dilution was considered a means of avoiding the possibility of injecting an overwhelming dose of the virus into the amniotic cavity. B. Routes of Inoculation Single Comb White Leghorn embryos were used through- out the study. Eggs were inoculated via four routes: (1) a1- lantoic cavity, (2) Chorioallantoic membrane, (3) amniotic cavity and (4) yolk. With the exception of the first passage, for which a suSpension of tracheal washings was used, inocula used for all subsequent passages corresponded to the route of inocula—- tion, i. e. , allantoic fluid was injected into the allantoic cavity, amniotic fluid into the amniotic cavity, etc. Seven serial pas— sages of the virus were carried out by each route. 22 C. Techniques of Egg Inoculation Asepsis was maintained throughout all procedures. Teasing needles were flamed to a red heat then cooled for about thirty seconds before use. Fine, curve—tipped forceps were kept in a C0plin jar of ethyl alcohol and flamed just be— fore use. Syringes and needles were sterilized by autoclaving. Vials of inocula were kept in cracked ice in “Thermos" lab— oratory vessels during the process of inoculation in order to minimize thermal inactivation of the virus. An inoculum of 0.1 cc. per egg was used for the first passage of the virus due to the limited amount of the original virus suspension. For all subsequent passages, an inoculum of 0.2 cc. per egg was used since that amount had been em— . . . . 11,35 , _ ployed in preVious studies of the Virus. A sterility test was conducted on a sample of each inoculum. Each sample was seeded on a nutrient agar Slant (Difco), which was then incubated at 990 F. for at least 48 hours. Thirty eggs were usually injected by each route of inoculation. From those, ten eggs, selected at random after inoculation, were marked for subsequent harvesting. Where less than thirty eggs were used, as occurred in the first passage, a corresponding prOportion of 23 “harvest" eggs was marked. Uninoculated embryos were used as controls, a range of 3 to 12 being used per route of inocula— tion per passage . All incubation was at 99° F. (wet bulb 86—880 F.) in an electric, forced—draft incubator (Jamesway, Model 252), in which the eggs were turned automatically every two hours. 1. Allantoic Cavity Inoculation Thirty ten—day embryos were inoculated for each pas— sage. To determine the site for injection into the allantoic cavity, eggs were transilluminated for selection of an area of the Chorioallantoic membrane, on the side Opposite the embryo, about 3 mm. below the air Space and free from large blood This point was marked with a pencil. A similar vessels. By means of mark was made on the shell over the air cell. a small drill (Handee Speed Drill, No. 10, Chicago Wheel and Mfg. Co. ), attached to the chuck of an electric motor (Deluxe Handee Model, Chicago Wheel and Mfg. Co.), a hole, just large enought to allow passage of the inoculating needle, was drilled at each pencil—mark, without damaging the underlying shell membranes. With the eggs supported, air cell uppermost, in 24 cardboard flats, tincture of metaphen was applied to the holes and allowed to dry. With a teasing needle, the outer shell membrane over the air cell was pierced to serve as an air vent, permitting equalization of pressure produced by injection of inoculum, and preventing leakage of the inoculum and embry— onic fluid from the site of injection. A 27—gauge, 1/2—inch needle, attached to a B—D Yale, l—cc. capacity tuberculin syr— inge, was inserted, to a depth of about 1/4 inch, through the hole in the Side of the egg, and the inoculum was injected. After inoculation, the holes in the shell were sealed with melted paraffin. The eggs were then returned to the incubator. Chorioallantoi c Membrane Inoculation Thirty ten—day embryos were used for each passage of the virus. The "artificial air cell" method was used, to make sure that all the inoculum was deposited on the Chorioallantoic membrane. Eggs were transilluminated to determine the location of the embryo. A pencil—line was made on the shell directly over the embryo, parallel to the long axis of the egg and equi— distant from the ends of the egg. A mark was made on the 25 shell over the air space. Using a small carborundurn disc (Rubber Cutting Disc, NO. 25, Chicago Wheel and Mfg. Co.) attached to the chuck of an electric motor, a groove, 1 cm. long and about 1 mm. wide, was made along the pencil-line, sufficiently deep to penetrate the shell without piercing the shell membranes. A small hole was drilled through the shell over the air Space to provide an air vent. Tincture of metaphen was applied to the groove cut by the disc, and to the hole Over the air Space, and allowed to dry. The exposed outer shell membrane over the air cell was punctured with a teasing needle. An egg candler with its Opening at one side was placed on the bench and the room was darkened. While held in the hand, with the long axis in the horizontal plane and with groove upper- most, the egg was transilluminated. The bend of an angularly— tipped teasing needle was placed in the groove. While slight downward pressure was applied on the elbow of the bend, the point of the needle was pressed gently downward sufficiently to rupture the Shell membranes without piercing the Chorioal— lantoic membrane. When the pressure thus applied was insuf- ficient to cause the Chorioallantoic membrane to drOp from the shell membranes, as determined by transillurnination, further ill-J1 lift? 26 pressure on the elbow of the teasing needle usually sufficed to produce this desired result, thus creating an artificial air cell on the side of the egg. In a few instances, it was necessary to apply gentle suction, by means Of a medicine drOpper bulb, at the hole over the normal air cell. With the egg supported, groove uppermost, in an egg flat, a 27—gauge, l/Z—inch needle, fitted to a B—D Yale, l—cc. capacity tuberculin syringe, was in— serted diagonally, for a length of about 1/4 inch, through the intact shell membranes over the artificial air cell, and the in... oculum was deposited on the Chorioallantoic membrane. In this way, the rupture made previously in the shell membranes served as an air vent, preventing leakage of the inoculum at the site of injection. Melted paraffin was applied to the groove and to the hole over the normal air Space. After inoculation, the eggs were returned to the incubator, supported in egg flats in the same horizontal position in which they were inoculated, to pre- vent any disturbance in the position of the artificial air cell. After not less than 24 hours in this position, the eggs were placed in the racks of the incubator. 27 3. Amniotic Cavity Inoculation Thirty eight—day embryos were inoculated for each pas— sage of the virus, with the exception of the first passage for which twenty eggs were inoculated. Eggs were transilluminated and the location Of the embryo was marked on each shell. A circle was drawn parallel to and about 5 mm. above the base of the air cell. By means of a small, carborundum disc, the shell was cut through at the circle, without piercing the outer shell membrane. Tincture Of metaphen was not applied to the groove cut by the disc, as it was considered possible that dif— fusion of the disinfectant along the inner shell membrane may have contributed to the death of embryos in previous experiments. With the egg supported blunt end uppermost, the cap Of Shell over the air cell was removed with sterile forceps, care being taken so that fragments Of shell did not fall on the inner shell membrane. A drOp of sterile physiOlOgical saline was placed on the inner shell membrane directly over the amniotic cavity, the position‘ of which had been determined by candling. The point of the 27—gauge needle, fitted to the syringe used to dis.- pense the saline, was placed on the inner shell membrane, below the drOp of saline, at an angle of 45°. Gentle pressure was fi-..._ 4‘ . 28 applied downward and backwards so as to produce a small slit in the membrane without damaging the Chorioallantoic membrane. The saline drOp then passed through the slit, flowed between the inner shell membrane and the Chorioallantoic membrane and caused their separation. With the point of the needle, a small portion of the inner shell membrane was retracted very care- fully to give complete visibility of the route for amniotic cavity inoculation. A 27-gauge, 3/4-inch needle, fitted to a B—D Yale, l—cc. capacity, tuberculin syringe, was inserted, by a short, sharp thrust, through the Chorioallantoic membrane, in an area free of large blood vessels, and through the amnion without piercing the embryo. The presence Of the needle within the amniotic cavity was confirmed by changes in the location of the embryo correSponding to Sidewise movements of the needle. After injection of the inoculum, each egg was capped over the air cell with a sterile souffle cup from which the rim had been removed. The junction of the egg and the cap was sealed and the entire paper cup was covered with melted paraffin to pre— vent evaporation. This procedure provided an advantage over the use of scotch tape or adhesive tape since these materials 29 cannot be sterilized. The eggs were maintained with long axis vertical, in egg flats, throughout the period Of incubation, as previous experiments Showed that this treatment minimized mortality Of embryos. 4. Yolk Sac Inoculation Thirty six—day embryos were used for each passage Of the virus, With the exception of the first passage, for which ten embryos were inoculated. Each egg was transilluminated with the long axis in a vertical plane, and the yolk sac was located. On the side of the egg Opposite the embryo, a pencil—- mark was made Over the yolk sac about half way from the small end of the egg to the apex of the curvature of the shell. A similar mark was made on the shell Over the air Space. A small hole was drilled through the Shell at each mark Without piercing the shell membrane. Tincture of metaphen was ap— plied Over the holes and allowed to dry. The exposed outer shell membrane over the air cell was pierced with a teasing needle. With the long axis Of the egg in a vertical position, a 20~gauge, 19—inch needle fitted to a B—D Yale, l-cc. capacity tuberculin syringe, was inserted, horizontally, to a depth of WWG 30 about 1/2_ inch, through the hole in the side of the egg, and the inoculum was injected. The holes in the egg were sealed with melted paraffin and the eggs were returned to the incubator. For not less than 24 hours after inoculation, the eggs were incubated in flats in the same position in which they were inoc— ulated, after which time they were placed in the racks of the incubator. D. Harvest and Preparation of Material for Subsequent Egg Passage On the third post—inoculation day, from eggs previously marked for harvesting, five living embryos, per route of inocu— lation, were selected for collection of infected material to be used as inocula for the next passage of the virus. Before har— vesting, the eggs were chilled in the refrigerator for not less than four hours. Chilling causes contraction of the yolk and constriction of the blood vessels and thereby facilitates collec— tion of the fluids. Separate sterile instruments were used for the collection of each material. Allantoic fluid, Chorioallantoic membranes, amniotic fluid and yolk were harvested from eggs inoculated by these respective routes. Materials harvested from each route were pooled in sterile, 30-cc. capacity, 31 screw—cap vials, which were kept in “Thermos" laboratory vessels full of cracked ice, to minimize thermal inactivation on the virus. A small sample was removed from each pool for a sterility test performed in a manner similar to sterility tests on inocula. The materials were stored immediately after collection at —450 C. until ready to use as inocula for the next passage. After collection of the desired fluid or membrane, the inner shell membrane was removed with forceps from the upper pole of the Chorioallantoic membrane, which was then ruptured and the egg was inverted to deposit the contents in a Petri dish. The embryo and Chorioallantoic membrane were examined for gross lesions typical of infection with infectious bronchitis virus. 1. Collection of Allantoic Fluid Fluid was collected from the allantoic cavity from eggs inoculated via the allantoic cavity. With the eggs supported vertically, blunt end uppermost, tincture of metaphen was applied to the shell over the air cell, and allowed to dry. The shell 32 in this region was then cracked and removed with forceps to within 5 mm. of the base of the air cell. Care was exercised so that fragments Of shell did not fall on the inner shell mem— brane. A 20—gauge, l—inch needle, attached to a 5—cc. Luer Syringe, was inserted through the inner—shell membrane and Chorioallantoic membrane into the allantoic cavity, and 5 cc. of allantoic fluid was aspirated. Pooled allantoic fluid harvested from the five eggs per passage was stored at -450 C. until ready for the next passage. Just prior to inoculation, the fluid was thawed at room temperature, then centrifuged in the clinical centrifuge for five to ten minutes, in order to remove the pre- cipitate formed by freezing and thawing. 10 The supernatant fluid was used for inoculation. 2. Collection Of Chorioallantoic Membranes Chorioallantoic membranes were harvested from eggs inoculated via the Chorioallantoic membrane. The eggs were supported with the long axis in a vertical plane, air cell upper- most. The shell over the air cell was painted with tincture of metaphen, allowed to dry, and then was cracked and removed with forceps to within 5 mm. Of the base Of the air cell. The 33 inner shell membrane was removed from the upper pole of the chorioallantoic membrane, and the chorioallantois was ruptured. The egg was inverted and the embryo, yolk sac and extra— embryonic fluids were deposited in a Petri dish. In most in— stances, the chorioallantoic membrane adhered to the inner shell membrane and remained in the shell. The albumen was removed with forceps and the chorioallantoic membrane was separated from the inner shell membrane. The chorioallan— toic membrane was "cleaned" of extraneous fluids by holding it with one pair of forceps and "stripping” it with another pair, before being pooled in a screw—cap vial with other harvested membranes. The membranes were prepared for inoculation by grinding them in a mortar with a pestle and sterile sand. The ground membranes and sand were suspended in Difco nutrient broth, 3 cc. Of broth being added for each membrane. The suspension was centrifuged for five to ten minutes in the clinical centrifuge to remove sand and gross tissue particles from the suSpension. A small sample of the supernatant was taken for a sterility test, before the suspension was stored at ~45o C. Just before the inoculation for the next passage, the material was thawed at room temperature. 34 3. Collection of Amniotic Fluid Amniotic fluid Was collected from eggs inoculated via the amniotic cavity. The eggs were supported, air cell upper- most, in an egg flat, and the paper caps were removed just previous to harvesting. The shell around the air cell was painted with tincture of metaphen and allowed to dry. The allantoic fluid was collected, in the manner described above, and discarded. This was done to prevent mixture of the allan— toic fluid with the amniotic fluid, while the latter was being collected. The inner shell membrane was removed from the upper pole of the chorioallantoic membrane with forceps, and the chorioallantoic membrane was ruptured and reflected. The amnion was graSped with forceps and a portion pulled upward to form a ”tent.“ A 20—gauge, 1-inch needle, attached to a B—-D Yale, 2-cc. capacity tuberculin syringe, was inserted through the amnion and the amniotic fluid was aspirated. A smaller capacity syringe than that used for collecting allantoic fluid was found to be more effective for harvesting amniotic fluid, as less suction was applied by the smaller syringe, thus pre- venting the needle from becoming clOgged with feathers and from drawing the embryo against the lumen. 35 Before inoculation for the next passage of the virus, the amniotic fluid was removed from the freezer, thawed at room temperature, then centrifuged for five to ten minutes in the clinical centrifuge. The supernatant fluid was used as inoculum. 4. Collection of Yolk With the egg supported air cell uppermost, tincture of metaphen was applied to the shell Over the air cell. The shell in that region was then cracked and broken away, with forceps, to within 5 mm. of the base of the air cell. The exposed inner shell membrane was removed and the chorioallantoic membrane reflected. An 18—gauge, l—inch needle attached to a 5—cc. capacity Luervsyringe was inserted vertically into the yolk. About 5 cc. of yolk was drawn into the syringe and ex— pelled into a large sterile test-tube. Test tubes proved more desirable for storing yolk, since they did not crack as did the flat—bottom vials when the yolk was frozen. Yolk was thawed at room temperature before inoculation. No centrifugation was deemed necessary, inasmuch as the thawed yolk material appeared to be homogeneous. Hm 36 E. Examination of Embryos and Chorioallantoic Membranes Eggs were candled twice daily. Embryos dying within 24 hours after inoculation were discarded and were not included in the calculation Of embryo mortality rates, as death was con— sidered to be due to trauma Or other non—specific causes. Thereafter, embryos which died were examined for gross path— OlOgic alterations typical Of infection with the virus. On the seventh post—inoculation day, all surviving and control embryos of the same age were examined and compared. The shell over the air cell was cracked and removed with forceps, the chorio— allantois was ruptured, and the contents of the egg were de— posited in a Petri dish. Inoculated embryos were examined for evidence of gross lesions considered typical Of the action of infectious bronchitis virus. Curling and dwarfing of the embryo and distension Of the cloaca with fat—like drOplets were COnSidered to be the significant lesions produced by the virus. The chorioallantoic membranes were examined for evidence of adherence to the shell membrane, abnormal thickness and the presence of whitish, Opaque lesions. RESULTS AND DISCUSSION The effect of the virus on embryos inoculated by the allantoic cavity route is given in Table I. The mortality showed a tendency to increase gradually from 30 per cent in the first passage to a peak of 95 per cent in the sixth passage. A drOp to a mortality of 85 per cent in the seventh passage was observed, but the difference between these last two pas— sages is probably not Significant inasmuch as one less death in the sixth and one more in the seventh passage would have given a mortality of 90 per cent in both passages. No expla— nation can be offered for the drOp in mortality in the third and fourth passages. The majority of surviving embryos in all passages, when examined on the seventh postinoculation day, showed the gross lesions characteristic of infection with the virus. Marked curling and dwarfing associated with dis— tension of the cloaca with fat—like droplets were the most sig— nificant lesions noted and these occurred as early as the third postinoculation day in all passages. The results of serial passages of the virus by amniotic inoculation are shown in Table II. Inoculation of the virus by 38 .momamo Owfloommlcoo nofio can mason» ob map waffle poets: moaned veamgoonw nonEdZ * o.mw ohms o.m\.. ode Név o.m~. 0.0m 32muu08 ammo Mona poumgooaw .OZ SQ: ON}: ONE: SQE Sb. SQS 8} being; euro so .02 "can.“ thafiguoz mournao m H m w 0” m h: muggy?“ mo Hongsz *UBSOOOGM ON ON om om : om 0N motcefio mo Hongz N. o m “v m N H m Oman mom. Hmwuom WEE/<0 OHOHZ ZOHH<1~DOOZH OZHBOAAOH WEB: WHHJJNHMOE Omrmmzm H Hinged. 39 .momsno oflwoommlao: hosuo van @8593 Ow 36 main .2555: mania Omani—00nd nongz u. w .E b .3 m .5 N am N «m a .1. 53 rsutofi 23 8m Ovumafioonw .OZ 2}: mi: £\2 S}: S\£ 2}: .2} uoHBEu euro so .02 "can.“ unaidauog mo>H£Eo v N m m m a v mnfiwtradm mo pongz *von—MHdoonw M: 2 fl 2 S S 2 uoinEu so sunfisz h o m w m N H mommmmmm 13.3w VHH> ZOHH H: H4mm>u5m mo Hongz $533005 om SN om om om S b moss—Eu do Saga N. o m w. m N H m ommmmmm 13.3w MAO? HER. 43> ZOHHH H4Q