107 334 DISTRIBUTION OF THE VIRUS OF INFECTIOUS BRONCHITIS OF CHICKENS IN EMBRYONATED CHICKEN EGGS Thesis for the Dogma of M. S. MICHIGAN STATE COLLEGE Ahmad Hamid EI~Dardiry I947 IHESIS 'l 5. j, t It .;‘ -7 I .' . .' f i}, ‘ I p.. 'I K I "J I. I. I I x o' 'I I I I ‘54 -l I :4 t} . “I .ij .'\:', ’4'. .4 .. . . t . ~ ,- p - Q, .'..‘ I. - o"_,<-4-_.- _ ‘ v' 44 4. - .-.; "few-$795; -‘.7?<.‘:2'-:t‘ 1" ‘ ‘ .. “ A- 1-; f. Thisistoeertifgthatthe thesis entitled The Distribution of the Virus of Infectious Bronchitis of Chickens in Inbryonated Chicken 8638 presented In] Ahmad Hamid. 31 brdiry has been accepted towards fulfillment of the requirements for _1L_a.___degree Wag DISTRIBUTION OF THE VIRUS 0F INFECTIOUS BRONCHITIS 0F CHICKENS IN EMBRIDNATED CHICKEN EGGS Ahmed.HhmidfiElfiQ§rdiry A THESIS submitted to the School of Graduate Studies of Michigan State College o£.Agriculture and.Applied Science . in Partial fulfillment of the requirements for the degree of MASTER.OF’SCIENGB Department of Bacteriology 19h? THESIS INTRODUCTION This study was undertaken in an endeavor to ascertain the activity and distribution of an egg adapted strain of the virus of infectious bronchitis in the extra-embryonic fluids and certain tissues of embryo- nated chicken eggs. Since the chicken embryo serves a useful means for viral diagnostic purposes in the laboratory, the results su‘mitted nay direct attention to suitable procedures for the production of high potency virus. I I86809 2. REVIEW OF LITERATURE Infectious Bronchitis Schalk and Han first described the disease in 1931 in North Dakota. By 1933 it had been recognised in several of the nidsestern states and in California and since that time it has been reported in other states. The disease is infectious only to chickens. Etiolog: Infectious bronchitis is caused by a virus which is found most abundantly in the exudate and tissues. of the affected respiratory organs and it has been also demonstrated in the liver, spleen, kidney and blood. mm and lesions: The most characteristic symptom is gasping although convulsive coughing may also be seen. Depression and weakness are seen in advanced stages of the disease. Nasal discharge and swollen sinuses are frequently observed. Chicks less severely affected exhibit such sylptons irregularly. Infected laying birds shew symptoms similar to those observed in chicks. At autopsy, varying amounts of clear or turbid liquid or viscid exudate are found in the lower trachea and bronchi. It is rarely of the thick yellowish and bloody character which is found in laryngotracheitie. Lungs may she's variable congestion and air sacs frequently are opaque in appearance. - The disease spreads rapidly and usually occurs most frequently in chicks under 3 or I; reeks of age although it has been reported in chicks as young as 2 days old. Beach and Schaln (1936) observed that birds front 10 to 1.12 days old were equally susceptible to artificial infection. 3. Beaudette and Hudson (1937) rePorted the spread of the disease from chicks to adult birds on the same premises. Delaplane and Stuart (1939) observed the disease in semi-mature and older chickens as well as chicks. In young chicks the mortality has been reported to be as high as from 70 to 80 per cent, wkereas in semi-mature and adult birds the. mortal- ity is nominal in most outbreaks. Retardation of growth in send-mature birds and decreased egg production mich persists for several weeks in laying flocks are a considerable economic loss. .Diaflsis: History of the case, clinical symptoms and antepsy findings are usually mfficient for recognition of infectious bronchitis. Transmission, cross insanity, or sense neutralization tests are often required for definite identification of the disease present. Transmission and immunisation tests: The disease may be transmitted by exposure of the respiratory system to infected tracheal exudate and lung material, with a usual incu- bation period from 18 to I48 hours. Symptoms are frequently noted in from 21; to I48 hours. Subcutaneous and air sac inoculation will incite the disease. Delaplane and Stuart (.1939) observed that birds which had recovered from infectious bronchitis developed an imunity which completely protected them against subsequent contact or artificial exposure under field condi- tions. One bird was nomal in every respect except for an almost unnotice- able meal discharge which had persisted for 2 months from the time of infection. During this time the nasal exudate from the bird incited regularly the disease in susceptible chickens. The washing of the nasal passages from this bird after cessation of the exudate was no longer infective. h. Neutralization 3.33.353’ It has been shown by Delaplane and Stuart (1939) that when serum from birds which were imne to infectious bronchitis was mixed with the virus and incubated for 1 hour at 37 degrees 0, the mixture did not incite the disease when injected into air sacs of susceptible birds. This indi- cated neutralization of the virus by the serum. Birds that were injected subcutaneously with 0.25 cc of serum followed after 1 day by artificial inoculation of virus intratracheally developed the disease indicating no protection from the quantity of serum employed although this serum neutralized the virus in vitro. I Imunisation studies: Beaudette and Hudson (1937), and Delaplane and Stuart (1939) investigated the possibility of active immunization of chickens by ino- culating the cloacal mcous membrane and the bursa of Fabricius with infectious tracheal exudate. Mild "takes" were noted within 2h hours to I48 hours thich completely subsided after 72 hours following inoculation. The latter authors showed by transmission tests that the virus had grown in the cloacal mucous membrane. This did not develop immunity against. the disease in the respiratory tract in 3 to 5 days after inoculation. The failure to do so was attributed to insufficient production of antibodies to prevent infection through the respiratory system. Delaplane and Stuart (1939) were unsuccessful in producing imun- ity with a vaccine consisting of 20 per cent lung and tracheal exudate suspension in 0.85 No.01 and formalised to 0.5 per cent. This vaccine was inoculated in 2 cc amounts either subcutaneously or in the air sacs. The vaccimted birds were inoculated with the virus in the larynx and trachea 2 weeks later and developed the disease. Cultivation of the Virus in Dnbryonated Chicken Eggs During the first few egg passages of field strains of virus ob- tained from tracheal exudate from infected birds, there were no deaths or distinctive or characteristic lesions of the embryo. Dehplane and Stuart (1939) demonstrated growth of the virus in the embryo at this stage by the appearance of typical symptoms of the disease in chicks hatched from inoculated eggs. With each successive transfer the virus became more virulent to the embryo. From the fifth to the tenth transfers ‘ some embryos died 5 to 6 days following inoculation. As egg passages were continued anbryos died at the fourth, then the third day, and finally at about the 60th passage the bulk of the embryos died between the second and third days. Apparently the ability of the virus to kill the embryo became fixed. Delaplane and Stuart (19141) studied the modification of the Rhode Island strain and Beaudette's strain of virus in embryonated eggs, and did not note any marked difference in the effect on embryos. The Rhode Island strain at the 89th, 90th and 91st egg passages was avirulent for baby chicks exposed by spraying of the respiratory tract or the inoculation of bursa of Fabricius. Beaudette's strain of virus retained its virulence and incited typical symptoms of the disease in baby chicks at the 120th to 125th transfers during the winter months but only irregularly so daring the smmer months. This would indicate that seasonal variations as well as different strains of the virus have an influence on the virus activity. . Beaudette's embryo propagated strain (117th passage) incited symptoms in chickens. When re-isolated and passed in eggs, little change in unbryo death rate was noted which would indicate that such a chicken passage failed to return the embryo-adapted virus to the field type. 5. Effect of Physical Agents Beach and Shah (1936) showed that tracheal exudate dried after freezing and stored in a refrigerator remained viable for 180 days. Under the same conditions, the virus when kept in 50 per cent glycerin, was viable for 80 days. Delaplane and Stuart (191:1) found that the embryo propagated virus survived storage in the fresh frozen state for 1;} months but not for 5 months. It survived room temperature for 5 to 7 days, and at 50 . " degrees C for f, 10 and 15 minutes as indicated by egg tests. Cunningham and stuart (191;?) reported that infected allantoic fluid which had been dehydrated from the frozen state at 0.15 m mercury pressure for 8 hours, sealed in vacuo, and stored at 1; degrees 0 for 7 days showed a 100 fold decrease in activity when restored to volume. Freezing and thawing were without effect on the virus and loss of virus activity was probably due to dehydration. 7. MATERIAIS AND METHODS Virus A strain of infectious bronchitis virus which had been adapted to cultivation in embryonated chicken eggs us need for inoculum. This virus was capable of killing the majority of the embryos by the end of the second day and the remainder by the end of the fourth post-inoculation day. The virus suspension consisted of pooled allantoic fluid and had a titer of 10-7.6 as indicated in Table I. Procedure Ten-day-old embryonated chicken eggs were used throughout this experiment except in two instances when 9 and 11—day-old eggs were used. Inoculation was via the allantoic route. Eggs were transilluminated for selection of an area on the chorio-a‘llantoic membrane free from large blood vessels, about 3 mm below the air sac, and a mark made for the site of injection. A small hole was drilled through the shell at this point without piercing the inner shell menbrane. Another hole was made through the shell over the top of the air sac. Before inoculation, tincture of metaphen was painted over the holes and allowed to dry. 'me shell mem- brane in the hole at the top of the air cell was punctured. This allowed equalization of pressure within the egg when the inoculum was injected into the allantoic sac and prevented leaking out of the inoculum from the site of injection. Deflection of Material Twenty—five lO-day-old embryonated eggs (were inoculated with 0.1 ml of the virus suspension via the allantoic sac, and the eggs were returned 8. to the incubator for harvest of certain tissues and extra—embryonic fluids from living and dead embryos at 12 hour post-inoculation intervals. The eggs were inoculated at 8 p.m. for convenience of harvesting every 12 hours and with the expectation that the majority of the embryos would die between the 36th and hBth hours, 8 a.m. to 8 p.m., so that the eggs could be candled frequently during this time to select embryos as near to these hours as possible. Eggs were painted with tincture of metaphen over the region of the air cell and the shell over the air cell was cracked and removed. with forceps. Separate sterile instruments were used for each operation. The materials were collected in the following manner and order. Allantoic fluid was removed with a 5 ml syringe and needle. The shell membrane was re- moved with forceps to expose the embryo in the amnionic sac. Using another 5 ml syringe the amnionic fluid was removed. Yolk material was also col- lected in the same manner. The chorio-allantoic membrane was ruptured and the mbryo and fluids poured into a sterile Petri dish. Then the chario- allantoic membrane was moved from the shell and the albumen adhering to the maibrane was washed in physiological saline and drained of excess fluid. The liver was collected from the embryo by dissection with forceps, and the embryo was examined for gross pathological lesions. Harvested materials were placed in separate 10 cc screw cap vials which were labeled with pertinent information and frozen at --35 degrees C. All the materials were bacteriologically sterile as shown by inoculation of agar slants. Titrations Vials containing allantoic fluid, amnionic fluid and yolk were thawed at room temperature, centrifuged at 2500 r.p.m. for 5 minutes and the supernatant fluid used for titration. The chorio-allantoic membranes 9. and livers were thawed at room temperature, weighed and ground with sand in a mortar and pestle. Broth was added to the ground material in the proportions of 9 parts by volume of broth to 1 part by weight of the material. This was considered to be a 10"]- dilution of the material. In two instances the weight of the liver material was so little that a 10'1 dilution would have been insufficient for inoculation purposes. In these cases broth was added in the proportions of 99 parts of broth to 1 part of liver material to make a 10-2 dilution. After thorough emlsification, the fluid was aspirated with a pipette and transferred to a vial. A few drops of the fluid were trans- ferred to agar slants for bacteriological sterility tests and the vial returned to the freezer. In all titrations 5 eggs per dilution were used and the eggs were incubated for h days. This was considered to be a significant rmmber of eggs for detection of viral activity in 10-fold serial dilu- tions. Knight (191th) working with influenza virus reported that when using 5 eggs per dilution, one can scarcely expect to detect with an certainty differences less than h or 5 fold. Procedure: The procedure which has been described was used in all titrations. The titer was considered to be the highest dilution which killed more than 50 per cent of the embryos inoculated (Cunningham and Stuart 1910). Inoculum consisted of supernatent fluid from samples thich had been frozen at ~35 degrees C, thawed at room temperature and then centri- fuged. Difco nutrient broth was used as a diluent . Serial 10-fold dilutions of the fluid were made in the ratio of 1 part virus (0.5 ml): 9 parts diluent (13.5 ml) in screw cap vials (17 x 65 m) from which 10. serial dilutions were made using a separate pipette (1.0 ml, graduation interval 0.01 ml, to deliver to the tip) for each dilution wish was aspirated into and expelled from the pipette 10 tines. After mixing the virus and diluent, the vial was vigorously shaken 100 times. Progressing from highest to the lowest dilution, using one syringe for the entire operation, one egg per dilution was injected with 0.1 ml of inoculun. Sterility 33333: 13va original specimen used, as well as every dilution, was checked for bacterial contamination. Vials containing the diluted material, as well as inoculated nutrient agar slants ,were kept in the incubator and inspected every day until the fourth day on which inoculated eggs were discarded. RESULTS The distribution of the virus in the tissues and extra-embryonic fluids is shown in Table II. In 12 hour living and 21; hour dead embryos the titer of the virus was 10'5-5; and in 21; hour living embryos it was 10‘5-3. A maximn titer of 10’7'1 was attained in 36 hour living embryos. In 36 hour dead embryos the titer was 1045. Anionic £1_u_i_d_: The titer of amnionic fluid in 12 hour living embryos was 10-3-6, 10-5.8 in 21; hour living embryos after inoculation and 1044.3 in 21. hour dead embryos. In 36 hour living embryos the titer was 10-h.5 and in 36 hour dead embryos it was 104%03. _Ch£r_ig-allantoic membrane: In 12 hour living embryos the titer was 10-5-5. it an hours the titer was 10-6-1 in dead embryos, and 10-6-3 in living embryos. It reached a 10-7o6 titer at 36 hours in living embryos and declined to 10-545 at the time of death. 9.1.2932: The livers showed variable titers. After 12 hours, it was 10-3-1. In dead embryos it was 10'3"5 after 214 hours. In living embryos it was 10'3-7 after 214 hours. The titer attained 10"3°S in living embryos at 36 hours and it reached 1044 in dead ones in the same length of time. Yolk: Viral activity in yolks was insignificant. 12. Virus concentration was higher in the chorio-allantoic membrane and allantoic fluid than in the amnionic fluid and livers at correspond- ing periods. In the yolk virus was scarcely detectable. Maximum titers of the chorio-allantoic membrane and allantoic fluid were obtained in 36- hour living embryos. The low titer obtained from the 36-hour dead embryos may be attributed to the thermolability of the virus. These embryos were kept at the incubation temperature from the time of death to approximately 8 hours before the collection of material. The tendency of the virus to propagate most freely in the chario- allantoic membrane can probably be attributed to the natural tendency of the virus to invade the respiratory organs of chickens. To clarify the point of thermolability of the virus, the experiment was repeated. The original inoculum was retitrated and 25 eggs were inoculated. Chorio-allantoic membranes and allantoic fluids were harvested at 21;, 36, and 146 hours from dead and living embryos. In order to observe the death of embryos and harvest materials between the 2hth and 36th hours, the critical period as previously described, eggs were inoculated at 8 a.m. instead of at 8 p.m. as was done in the first experiment. It was not possible to harvest embryos at the time of death between the 36th and h8th hour. This period of time happened to be between 8 p.m. and 8 a.m. Table I shows the number of inoculated embryos and their death rate in the first and second experiments. In the first experiment it was noticed that the death rate of the embryos was high between the thh and 36th hours after inoculation. In the second experiment, the majority of embryos died after the first 2h hours following inoculation. Eggs were candled every 2 hours from the 2hth to the 36th hour and thosewith dead mbryos were refrigerated until the 36th hour when pooled material was'harvested from the embryos dead Number of Inoculated Embryos and their Death Rate Table I in the First and Second bcperiments 13. . of Embryo No. of Chick fibryos harvested Exp. Orig.Inoc. Titer Dead per Hour 1‘2— 1? 32 he h 36 h Living De Livin Dead Li De Livin Dead 1 All.fluid10'7-6 8 lo - 3 - 3 3 3 - - 2 All.fluid 10- 6.5 12 6 1 - - 3 3 3 - 1 by this time. 36th and h8th hours. 36th hour. were practically the same as in the first experiment. Only one embryo survived which died sometime between the Table III shows the results of the second experiment which was higher in the chorio-allantoic membrane than in the allantoic fluid, the maximum titer being in the 36-hour living embryo. 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HHH 0.3.3. .doavmdaboflH Moped ugmvnH 300 as ache-fl 6009 find @553 H0 16 0 EFFECT OF DILUTED INOCULUM ON TITER INFECTIVE FOR EGGS Working with influenza virus Henle and Chambers reported that when embryos were inoculated with a low concentration of virus the result- ing titer of harvested naterial was higher than that obtained with an im- culum of a high virus concentration. This was confirmed by Miller. Parallel to experiment 2, an additional lot of 25 eggs were ino- culated to study the effect of the concentration of inoculum on the resulting titers of the chorio-allantoic membrane and of the allantoic fluid at the same tine intervals as in experiment 2. A 10-3 dilution of the original inoculmn in experiment 2 was used. The experiment did not indicate any advantage in using this low concentration of virus as the results obtained were, for the most part, nearly the same as those obtained in experiment 2 with the undiluted inoculum. 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The maximum activity of the infectious bronchitis virus in the different tissues and fluids of the chick embryo using the allantoic sac inoculation route was as follows, from the highest titer to the lowest: a. Chorio-allantoic nanbrane b. Allantoic fluid o. Annionic fluid d. Livers e. Yolk 2. The virus reached its maximum titer by the 36th hour after inoculation. 3. Fluids and tissues had a higher titer in living embryos than in dead embryos harvested at the same tine. 1;. lbs low titer of fluids and membranes from dead enbryos that were held for as long as 8 hours after death may be attributed to the thermo- lability of the virus (incubating temperature 99 degrees F). S. Inoculun with low virus concentration yielded results comparable to those obtained with the inoculun possessing high virus concentration. 1. 2. 3. 6. 7. 9. 10. 13. 1h. REFERENCES Beach, J. R.: Corysa and other Respiratory Infections in.Chichens. Proc. 12th International Veterinary Congress, (l93h), iii, pp. lhhr 155 (washington, D. C. 1935). U. S. Government Printing Office. Beach, J. R.: Poultry Disease Recent Discoveries. Proc. 5th Pacific Beach, J. R. and Schalm, 0.‘W.: A Filtrable Virus Distinctive from that of Laryngoteacheitis, the Cause of Respiratory Disease of Chicks. Poultry Science, IV, (1936), No. 3, pp. 199—206. Beaudette, F. R. and Hudson, C. B.x Cultivation.of the Virus of Infectious Bronchitis. Jour. Am. vet. Med. Assn., 90,(l937), pp 0 51-58 0 Bushnell, L. D. and Brandly, C. A.: Laryngotracheitis in Chicks. Poultry Science, xii, (1933), 1, pp. 55-60. Committee Report: Virus Disease of Birds. Jour..mm. Vet. Med. Assn. Nov. 1939, pp. 617-621. Cunningham, C. H. and Stuart, H.O.: Cultivation of the Virus of Infectious Bronchitis of Chicken in Embryonated Chicken Eggs. Am. Jour. Vet. Med. Assn., 8, (191.7), pp. 209-212. Delaplane, J. P.: Differentiation of the Respiratory Disease of Chickens. Rhode Island Exp. Stat. Bull. 288, (l9h3). Delaplane, J. P. and Stuart, H. 0.: The Modification of Infectious Bronchitis Virus of Chicken as the Result of Propagation.in Embryonated Chicken Eggs. Rhode Island.Agric. Exp. Stat. Bull. 28h, 19h1. Delaplane, J. P. and Stuart, B. 0.: Studies of Infectious Bronchitis. Rhode Island Agric. Exp. Stat. Bull. 278, pp. 5—15. Henle, W. and Chambers, L.A.: The Seriologica1.Activity of Extrap EMbryonic Fluids of Chicks Infected'sith Virus Influenza. Proc. Soc. Emp. Biol. and Med., h6, (19h1), 713-717. Hofstad, M. H.: Carrier in Infectious Bronchitis. Cornell Vet., Jana 19h? 0 Knight, C. A.: Titration of Influenza Virus in Chick Embryo. Jour. Exp. made, 79, (19M), h87-h950 Miller, G. L.x A study of Conditions for Optimum Production of PR8 Influenza in Chick Embryos. Jour. Exp. Med., 793 (l9hh), pp. 173-183. ‘t'iifl.' 15. Reed, L. J. and Munich, n.. A Simple Method of Estimating 50 Per Cent Endpoints. The Jour. of Hygiene, 2?, (1938), pp. 1:93-1:95. l6. Schalk, A. F. and Ham, M. 0.: An Apparently new Respiratory Disease of easy Chicks. Jour..Am. vet. Med. Assn., 78, (1931), pp. h13. ACKNOWLEDGEMENT To Dr. C. H. Cunningham the writer wishes to express sincere thanks fer his constant guidance and effective technical advice throughout this work. He also wishes to express his grateful appreciation for ' the kindness and cooperation shown by Dr. _H. J. Stafseth. l ROOM USE ONLY \ TV“ 0 . . J. « .I..V .Qgfifl‘ifiafiy.d.qil.‘lfil \«‘l~.3. 6.1.8.?1 .. q I... x . 7’ \“.l. l |.4‘01AIII.I.(‘..KI W ~19...’ \ o {Lu/34;}... 2 r. 1.3.... 181-11.... Peed... an?! .20. 2!... .t .0.v‘!l§1¥| MICHIGAN STATE UNIVERSITY LIBRARIES lllllllllllHH IIH l 3 1293 03071 2404