PATHOLOGY OF THE CH1CK EMBRYO (NFECTED WITH iNFECTiOUS BRONCHITIS VIRUS Thesis for fhe Degree of M. 5. MECHIGAN STATE COLLEGE Ladd Nixon Laamis 1949“ l-‘II.I.KPIIIv to .- PATHOLOGY CF THE KICK EXERYO HFECTED RITE IZFLCTICUS 313IZICIrl-{IT‘IS VIRUS By Iadd Eixon Loomis .TT‘P‘t' f‘! 3‘). :1 malt) Submitted to the Schoal of Grufiuate Stuaies of Kichigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of KASTli OF SCIEHCE Department of Bacteriologv and Eublic Health 1949 7613-49 L263 TABLE OF CUETVETS Acknowledgment Introduction Review of Literature Enterials an Kethods Tissue Tochnic for Preparation of Specimens Results Candling Observations Gross Pathological Aopearance of Bubryos Gross Les ens of Embryo Organs MicrOSCOpic Lesions of Embryo Organs Discussion Summary a‘a "onclusions References Photographic Plates AC 1*‘1'051 I‘mc t» 1"??? AMLi H ' o. “.14.": The writer wishes to express his sincere ap- preciation for the advice and guilrncc of D“. C Cusninghnm, Dr. Fr n3 Thorp Jr.. Dr. H. J. Stafsoth an- Dr. R. A. Runnells; also to my. K. . Greg for taking the photographs. IHTRCDUCTIOH Infectious bronchitis is on important virus resyirh- tory fiisesse of chickens characterized by a rapid sprexd and short duration with typical symptoms of sneezing, coughing and respiratory roles. Gross lesions consist of congestion and edema of the lungs, mucus in the bronchi A and lower trachea and, in some cases, cloudiness of the air sec membranes with accumulations of cheesy materiel. Facial swelling may be observed in young chicks. In some chickens a nasal exudate may be gresent. The morbidity rate is usually high but the mortality rate varies with the age of the affected chicken. In chicks the mortality rate mtg be as high as 60 to 70 per cent while in oomi-msture and adult chickens it is negligible. In laying flocks a lowered egg production may persist for as long as two months before returning to the previous level of :roiuetion. In. Chickens recovered from the cleanse ere refrectory to subrequent infection with the virus. naturally acquired passive immunity may be present for as long as four weeks in chicks hatched from eggs layed by immune hens. Inocula- tion of embryonsting chicken cage with suspensions of in* fected materials from chickens suspected of having the disease is one method for isolation end identificrtion of the virus. This study was undertaken as fundamental research on the pathology of the chick-embryo as e possible aid in the differential diagnosis of avian respiratory diseases. C}! REVIEW OF LITERATURE Schulk und‘ggggl(1931) in North Dakota were the first to describe this diseese from observotions made of approximately 25,000 chicks. forbidity rates Were from 25 to 75 per cent and mortality rates were from so to 90 per cent. Gross lesions of the lungs were described as acute congestion with s care-nucoid liquid exudetc. A similar exudate was present in the sinuses. Berkefeld V. d end s filtretes of blood tissues and exudetes of the respirrtory tract of infected chickens were capable of 4roducing the disease. Transmission esperi- ments were complicated by infection of control chickens. This was overcome by housing the control birds in separate buildings and using separate attendants. Bench and Schelm (1936) reported tint Berkefcld V. N and W filtrates of nasal, tracheal and bronchial cxudntes were cupoble of causing infection. Chickens recovered from the discuss were refractory to further infection but no cross-immunity existed against coryze end luryncotrecheitis. Besudette and Hudson (1937) were successful in prepegeting infectious bronchitis virus on the chorioula lantoic membrane of cmbryonsting chicken eggs. The virulence of the virus increased by cdnptetion end embryo desths 1hr creased with such successive pessnge especielly from the seventh passage on. Infected embryos weighed one half as much as normal embryos of the some age. The virus did not produce peck or plscque lesions on the choriosllnntoic membrane. The euthors reported that infected chorioellantoic membranes were thinner and more adherent to the inner shell membrane than normal chorionllontoic membranes. Yelk materiel was semi-solid. Deleplone end Stuart (1939) reported certain similari- ties in the symptoms of infectious bronchitis and lsryngo~ treeheitis. The coughing, sneezing and respiratory dis- tress sere similar but in semi-mature and adult chickens infectious bronchitis did not cause the hemorrhage end fulse membranes in the larynx such us were noted in laryngotrecheitis. Chickens recovered from infectious bronchitis sore immune to further infection. Ho crossaimnunity existed between infectious bronchitis. leryngotrecheitis or in- fectious coryzs. Bronchitis immune serum was capable of neutralizing infectious bronchitis virus so demonstrated by infectivity tests with chickens. The authors reported that one adult chicken was a carrier for as long as eight weeks. thus suggesting one phase of the epidemiology of the disease. The virus was propagated successfully on the chario- sllentoic membrane of cmbryoneting chicken eggs. 30 specific lesions of the membrane were noted. Dcleplsne and Stuart (1941) presented detailed protocols for the modification of infectious bronchitis virus when prepsgsted on the chorioellsntoic membrane of embryonnting chicken eggs. During the first eight passages the virus failed to produce characteristic lesions of either the choriosl- lentoic membrane or of the embryo. None of the embryos were killed by the virus. With such succeeding pessnge the virus became mare pathogenic for the embryo end at the 70th passage the virus was so adapted that all embryos were killed by the end of the second day. Gross lesions reported were whitish feel on the liver and congestion and swelling of the kidneys. hhitish Opaque lesions of the choriosllantoic membrane were observed. The virus survived storage in the fresh frozen state in the freezing compartment of an electric refrig- erator for four and one-helf months and at raom tempera- ture for five to seven days. Delsplsnc (1945) discussed the differential disg- noses of avian respiratory diseases and stated that the principal mode of dissemination of infectious bronchitis was through contact exooeure with infected or "carrier” chickens and with contaminated equipment. Typical lesions consisted of mucus. ccterrhel end purulent accumulations in the trachea and bronchi with marked congestion and edema of the lungs. The air sacs might be clouded or show eccumuletionsof a cheesy materiel. but in.moet cases no changes were noted. Facial swelling might be observed in young chicks. Thirty to fifty per cent of infected chickens showed a serene or ceterrhel nasal exudate after the sixth day of infection. Chickens recovered from the disease were immune but individual "carriers" might exist. Hofsted (1945) reported that he was unable to detect carriers of infectious bronchitis by housing sue- ccptiblc and recovered chickens in the some pen for three weeks or longer. Further experiments by Hofsted (1947) showed that chickens recovered from infectious bronchitis could be carriers. gunninghem end Stuort (1946} reported the effects ,of certain chemical agents on.en egguedapted etrein.o£ infectious bronchitis virus. Virus-infected ellcntoic fluid was mixed with the chemical agent and after a reac- tion period of three minutes the mixture was injected into eggs by the clientele soc route. Survival of the embryo was used as the criterion for the inoctivotion of the virus. The following agents were effective against the virus; phenol. 5 per cent; liquor eresolis seponotus, 3 per cent and one per cent; tincture of notephcn, undi~ luted end one per cent; potassium permanganate, 121000 and 1:10.000; ethyl alcohol. 95, 70, 40 end 25 per cent; mecuric chloride. 1:1000; tincture of sepherin, 111000: Lugol's solution, 1 per cent; sodium hydroxide, 1:20: formalin, l per cent. Boric acid (4.0%) and sodium hydroxide (.Ols) were without effect. Cunningham and Stuart (1947) reported that freezing and.thawing of infectious bronchitis virus-infected el- lontoic fluid produced two types of precipitates, one soluble at room tempereturc end the other insoluble at room temperature. The insoluble precipitate could be sedimented by centrifughtion. The virus was more stable when stored at ~25 c or ~70 C than at ‘10 C. No significant differences were observed when Difco nutrient broth. Difco tryptoee phOSphste broth, 0.85 per cent sodium chloride. or H710 phosphate buffer (pH 7.0) were used as diluents for titration of the virus. Agelgplene (19s?) found the nllcntoic sec route superior to the chorioellnntoic membrane route for initial isolation of the virus, as evidenced by dwerfing of em- bryos on the first pessnge. Streptomycin (0.25 gram per one ml.) decreased bacterial conteminetion in tracheal exudates used to in, oculete embryonnting chicken cage for isolation of the virus. Levine and Bofstod (1947) showed that infectious bronchitis virus could be sir-born for as for as five feet. Ultra-violet irradiation for sterilization of the sir proved to be of little or no volue for control of the disease. Rcsflgg, Houser. Lillie end Creire (1948) demonstrated by electron microscopy that the infectious bronchitis virus was round in shape with o neon diameter of 90 mu. A file- nentous projection similar to that of hencestle diocese virus was present. Jugflherr and Terrell (1948) demonstrated by serum neutralization tests that naturally acquired passive in- munity to infectious bronchitis could be present in chicks hatched from eggs laid by hens recovered from either the naturally or artificially induced diocese. Reutrslizing antibodies in the yolk of embryonuting eggs were found to decline after the eleventh day of inn cubution, whereas after sixteen days 3 rise in neutraliz- ing antibodies was observed in the egg content minus the yolk and albumen. The neutralizing antibody level in chicks was high for the first two week after hatching but then declined rapidly and at the fifth seek the chicks were susceptible to infection. Subsequent cryosure of the chicks to the virus resulted in a marked increase of antibody. Febricent (1949) examined 6,000 embryos infected with the virus and emphasized the "curled” epneernnce of the embryo and the characteristic "dwarfing". Dwerfing end curling appeared in 46 yer cent of the infected embryos on the first yessnge, 33 per cent on the second. 20 per cent on the third end 1 per cent on the fourth passage. Grunge (1949) found a thermoetoble materiel present in the allentoic fluid from infected embryos which.hsd been stored at 56° c for 24 hours after death. This material interfered with the multiplication of infectious bronchitis virus in subsequent egg peseoges. Dilutions of the materiel caused a corresponding increase in chick embryo death rates. Inoculation of meet-inactivated ellentoic fluid containing interfering materiel delayed embryo deaths when inoculated into the clinntoic sec 30 ninutes before injection of ective infectious bronchitis virus. 10 The author see unsuccessful in reproducing the interference phenomenon by the following methods: first. storage of infected ellentoic fluid in 3.4333 for 24 hours at 36 0: second. by storage of infected silentoic fluid in 3.3.33.9. in the presence of normal chorioellentoio men- brenes for 24 hours at so 0; end third, by dilution of infectious bronchitis virus in silentoic fluid harvested from normul thirteen day old embryos killed b; chilling' and subsequently stored at 36 C for 24 hours before hor- VCSto EATERIALS db HE RODS The virus used was s strain of infectious bronchitis virus designated as Lot 258 and supplied by Dr. Henry Von Roekel, Department of Veterinary Science, University of hhssuchusetts. This stroln has been used for voccin» etion seeinst infectious bronchitis in the Hessnchusetts control pregrun. (Van Roekel, 1949) Lot 258 hod been isolated in hey, 1941 from inborn- tory birds that had contracted a natural infection. Since that time the virus hnd been.periodicelly prepu,uted by passcge in susceptible chickens, but had not been prepegntod in cmbryoneting avian seas. The virus sample use received as a saline suspension of washings from the trachea and bronchi of on adult chick- en shouing typical symptoms of the disease on the third day after inocul tion. Two passages in susceptible chicks were mode using intrenesel end intrntruchenl routes of in- oculation. The supernotent fluid of pooled lung and tracheal tis:ue suspensions from three chicks of the second passage were used for the initial egg inoculations. Prior to inoculation the inoculum.wns treated'with penicillin (10,000 units per ml.) and streptomycin (0.01 gm. per ml.). The inoculum.wes 0.2 ml per 10-day embryoneting egg via the sllentoic sec. Infected cllnntoic fluid was collected .on the third post-inoculation day and used as inoculun for the next pessege. This procedure was followed for sub¢ sequent passages of the virus. Tenpdny embryos were used throughout the study. Inoculation see via the ellentoic soc. Eggs were trans. illuminated for selection.of en arcs of the chorioollentoic membrane free from large blood vessels about 3 mm. below the sir cell. A small hole was drilled through the shell, without piercing the shell membrane, by moons of s smell drill attached to the chuck of on electric motor. Ann other hole was drilled through the shell over the top of the air cell. Tincture of metnphen.wes painted over the holes and allowed to dry. The shell membrane over the top of the sir cell see punctured with a sterile teasing needle to allow equalization of pr ssurc within the egg when the ineculum was injected into the silentoic see and to prevent leakage of the inoculun from the site of injection. After injecting the inoculun, using c B~D Yule l-cc. capacity tuberculin syringe. fitted with e 27 guuge. fi-inch needle. the holes in the shell were sealed with melted paraffin and the eggs returned to the incubator. All incubation was at 99° F. in an electric forced-draft incubator. At the time of collection of materials the shell over the sir cell was painted with tincture of notephen and then crooked and removed with forceps. The ellnntoic fluid was collected with a 5-ml. syringe and needle. The chorioallentoic membrane was then ruptured end the egg in- verted to deposit the embryo in a Petri dish. The slinntoic l3 fluid.waa collected on the third day after inoculation and 0.2 cc. of this fluid was used per egg for the next series of egg inoculations. Lactcrial sterility tests of inoculums were negative; l4 TISSUE TECHEIC 'CR EREIARLTION CF SPLCIEENS Hormel and pathological specimens were processed in an identical manner to insure uniformity for histological examination. Specimens were collected from living embryos only and fixed immediately to avoid post-marten changes. The method used was to candle the eggs and select living control embryos and living infected embryos. The embryos were removed from the shells and placed in Petri dishes for comparison and for photography. The abdominal cavity was then.0pened to insure penetration of the fixa- tive and the embryos were drapped into labeled fixation Jars containing Zenker's fixative. Formaloealine, Zenker's- formal (Kelly's) and Bouin's solution were also used. In an attempt to obtain Optimum conditions, tissues were fixed for 4 to 34 hours and washed for 12 to 36 hours. The tissues were dehydrated by successive transfers in ethyl alcohol dilutions, ranging from 30 to 80 per cent; followed by 95 per cent and absolute ethyl alcohol. Cedar oil was used for 24 to 48 hours with one change. Tissues were embedded and sectioned in the usual manner. The egg albumen method for tissue section fixation on the microscOpe slide was used. The staining procedure described by'ETllorz (1937) 15 was the standard Hematoxylinpeosin method, using Harris' aqueous alum hematoxylin.1 per cent and acidified aqueous eosin l per cent. The regressive staining procedure using acid alcohol (ethyl alcohol 95 per cent, HCl 1 per cent) was used. Also, Wolbsch's variation of the Giemsa stain (Lillie, 1948) was used cepecially to detect myelogenous tissue. However, the sharper hematoxylin—eosin stain was preferred for routine tissue examination. A rapid acetone method of tissue preparation.uas used as follows: formalin one hour, three changes of see- tone in One hour and one change of paraffin in one hour (all in the paraffin oven). No difference in staining quality could be detected by the writer between the standard hematoxylinpeosin method and the rapid acetone hematoxylin- eosin methods 16 RESULTS ‘zgensilluminetionigg Infected Enhrgonetinfi Efits. Gross alterations of the infected embryos could be detected easily by cendling the egg. The infected embryo had a typical curled position and did not have normal free motion when the egg was sharply rotated. Large ves- sels of the extra-embryonic membranes were more prominent than those of normal embryos. The infected ohorioel— lentoic membranes had en anemic eppeernnce, which was possibly due to impaired capillary circulation; This change was most noticeable on the third or fourth day after inocula- ti on. Gross Alterations‘gg Infected Embryos. In general the gross alterations agree with the de- scription of geleglene and Stuart (1941) and Beeudette and Endson (1957). The most marked characteristic was the curled position or the embryo and its smell size as com- pared with the normal embryo of the some age. Kevements of the infected embryo were noticeably slower and seeker. The embryo was drawn up with its feet over its head into a firm round shape and Wes dwarfed in size, to 50 per cent the size of the normal embryo. The thickened emnionic membrane adhered to the 17 embryo.end resisted renovel‘ When the amnionic membrane was removed a dry fibrotic surface was left on the inside of the amnionie membrane and the feathers of the embryo were drier than normal. Some embryos showed Jaundice end all embryos showed decreased feather develoyment. Infected embryos exhibited deformed feet that were congressed over the herd and also a wry neck which had a characteristic lateral curvature. The organs remained outside the abdominal cavity with greater frequency when the embryo was infected. About 33 yer cent of the embryos showed excess bile deposits. Ten per cent of the embryos had a distended white clones filled w th fat draplete. Approximately one to two per cent of the embryos were resistant to infection with this strain of infectious bronchitis virus. These embryos resembled the normal embryos in every respect. Ho abnormal odor woe noticed from the infected embryos. Occasionally smell petechiel hemorrhages were noted in the skin of a few infected embryos, as mentioned by Dclnplene and Stuart (1941). lo 5. 6. 7. 8. 9. 10. 18 SUKHARY CF GRCSS PATH~LCGICAL ArrEARAHCE CF IEFECTED EILEEYOS.’ The chorioellentoic membrane was sdhorent to the inner shell membrane and egpeored thinner than norms 0 Living infected embryos were sluggish end week. The embryos were dwarfed as much as one half normal size. The embryo essuned e ballalih shape characterized by e curled position with the feet deformed and compressed over the head. The dry fibrotic onnionic membrane resisted removal from the embryo. Ferthers of the embryo were not as moist as normal. The feather develctnent was immature. . 7 MAJ“ A--"\Jl._r-' The embryo skin endzfieethersrscre icteric. Residual yolk material was or greater volume and of firmer consistency then normal. The cloeca was distended with fot droylets, which caused a white appearance. This occurred in ebout 25 per cent of infected embryos. A eherect ristic wry neck was observed in all infected embryos. / ' 19 JTH"‘ ’ 11.5”Incompletevinvolution ot“nbdomincl"orge1s occur ed ‘in ebout 4 per cent of the infected embryos. The organs remained external to the abdominal c vity. 12. he abnormal odor was detected. 13. Gross lesions of internal organs: Livers were either hemorrhagic, iatcric, or contained necrotic foci: kidneys were usually swollen end some showed foci of necrosis. or ieterns; lungs were small, pole. and viscid; hesrt developed to half normal size: 14. A proximately 33 per cent of the infected embryos showed bile discoloration of liver and kidneys. 15. Apyrorisetely one to two per cent of the embryos did not exhibit pathological alterations. 20 GROSS LESICHS CF EEERYU ORGARS The heart in all infected embryos was noticeably decreased in size. Ho other lesions were observed either grossly or microscopically. nee Extensive pathological lesions of the liver were noticed in every case. Grossly the linr showed on ex» tensive hepatitis with hemorrhages in the tissue or under the screen. Excess bile production and or bile obstruction [ere seen. About :3 per cent or the lirers exhibited.marked biliary discoloration. Instead of a normal yellowish fatty appearance the livers were usually dsrk red to purple. Oneathird of the livers examined showed varying shades of green, either at the edges or throughout the liver. especial- ly in the ventral area adjacent to the gall bladder. Whitish or yellowish necrotic areas alternating with hemorrhagic areas were noticed on the surfaces of some livers. Lungs V Long develoencnt was markedly retarded. Infected lungs were of soft consistency and pale pink in color. About 70 per cent of the lungs were found adherent to the thoracic walls by fibrotic strands that increased the difficulty or removing them from the embryo both when.tresh or fixed. 21 The infected lungs were of a sticky texture which was easily detected when they were glsced on a glass plate. A thin tenacious serous exudate we .8 ins m ably present. A 50 per cent decree ec in the size of ti 8 lung was of 2 common occurrence in i ‘ected enhr; vos. Kidnez The kidneys of infected embryos were swollen and exhibited yellowish foci; they were so newhjt firmer tnenr A'W normal kidneys. The swellim f5 were duelto odes; a cellular infilt etionSof syclogenous cells. Part of the yellowish deposits were considered te be urete crystals. Some kidneys were dark green in color, due to excessive bile. The limers of these embr;ros were else bi 10 st ined end were dark green in color. ChorionllentrieEEmhrane The chorioellenieic membr ne was thickened by pro- liferetion of mnny layers of mesoderm. Edema of areas of Junction of the ellentoic membrane to the smnienic nem- brene :Iere noticed. The marked pxoliferetion of iele.nd8 of tissue that could be produced by strains of virus isolated by Delnylene and Stuart (1941) were not seen in tissues irfected with this strain of virus. Despite the appearance of thinness and its tenacious¢ mess to toe sh oil. the iniectcd chorioelle.ntoie mezbrnn gas thicker and more Opaque when fixed then the normal. 22 3.1.13 £53. fiercbrene The outstanding gross feature was extreme frisbility or the yolk see which invariably ruptured when the embryo was removed. This was true also in the normal embryo but to a lesser degree. Amnionic Kembrsne "" "m‘ f" x. " " This appeared to be thickened. Opaque end to-cefii strict embryo movement. The bones of the infected embryo were softer and uniformly appeared to be several days behind the normal in length, rigidity and diameter. Brain No lesions were observed. GO XIW LOSCCI IC LESICI IS CF EZBRYO ORCAES Lung Pneumonia characterized by congestion. granulocytic and nonocytic infiltrstion, together with a serous exudate in the bronchial once was found in all infected lungs.. These reactions were aistributed evenly throughout the entire lung tissue and the condition.ses apparent from as early as the fifth day of infection.- While moderate dosquhmstion of enithelisl cells of the bronchiel secs occurred, no areas of extensive ne- crosis or abscess tion‘ were found. The infiltration of riveaeul‘r‘elehents, serous eruurte one desquenetion, progress- ed in severity as hatching ego was reached but without the occurrence of the severe tissue reaction observed in the liver and kidneys. A pulmonary tissue-virus equilibrium seemed to be established in the lungs. The bronchioles and bronchi contained a serous exudste which on s com;>esed of fine yienulsr eosinophilic particles. grenulocytos, monocytes and epithelial cells. No diphtheritio membrane, extensive necrosis or abscess formation was seen in any bronchioles or bronchi examined. The smooth muscle cells at the edges of the bronchial sues were hyelinizcd to e uniform and extensive degree esyecielly after the sixth or seventh day. Later, these hyelinized erees were infiltreted with arc-nulecytes 9.nd menocytes. Interstitial edema was not detected. 92.1.9.9. Early manifestations of virus netivitv were pro- duced on the hepatic vesculer sys tom 1 Severe concest ion with perivescular "cufiing" Hes seen thronghout the liver by the sixth day. At this time pyknosis end koryorrhexis of the nuclei of hepatic cells began to 9;;eer. Congole- tficn necrosis with nucleolysis was present on about the eighth day. This condition was observed to involve whole lobes of the liver. By the nineteenth any extensive abscesse- tion occurred in many embryo livers. The periphery of those abscesses was ringed by grenalocytie end monocytic cells, nearby areas exhibited hemorrhage and also congested hepatic arterioles and capillaries. Some livers became extensively stained with bile pigmentsAbut the nicroscooic lesionSof this type of liver advanced.with the sens degenerative steges es the hemorrhagic reaction.nith the exceetion that much more bile pigment was present in the tissue sec- 1310110 Eormel fatty chenges of the liVCr did not take 31906 in the majority or infected livers. Fat vacuoles present in the normal hepatic cells were noticeably absent in the nstholOQicel hepatic cells. Extensive hemorrhage beneath Glisson's capsule was frequently noted. -. .1 '1‘ .; .Ci: 0% J. XI“ The interlobuler bile.eepillerics underwent the see degenerative changes. and at the some time, as the hepatic cells. The reaction of the reticular-endotheliel system (Kupffer cells) was masked by the severe blood vascular reaction. Kidne Interstitial nephritis with edema and diet ntion of the yroy'mel convoluted tubules with lerge hemoglobin casts were the early lesions observed. East changes occurred in the metenephroe. Due to the immaturity of the tissue, the granular appearance of some tubular eeithelium ees d1££icult to evaluate: while in other areas marked extrusion of cytoylesm from the rup- tured epithelial cells into the lumen of the tubules was 86011. The majority of the clemeruli did not seem to be altered. The intreglomeruler space was clear end Bowman's capsule see of normal thickness and was not adherent to the glemcruler tuft. Eowevvr, a few sections of the kidneys showed dilated glomeruler Spaces, some contnining desquemeted epithelial cells surrounded by crennlocytic cells. Other glomeruli scge noticed to be enlarged with swollen efipillery tufts plugg>d with granuloeytcs. Vecuolize- tion of endotheliel cells in these capillaries wrs noted. Also a few sections contained areas of dissolution of the gloneruler tuft that left on intact basemeit membrane sur- rounding tissue detris. This occurred near arena of focal necrosis of tubular epithelium. The rensl vascular system exhibited extreme con- gestion, of both arteries and capillaries. Some large areas of both subcapsulsr and interstitial hemorrhage were observed surrounded by ley rs of grenulocytic and monoeytic cells. Erosion of arterioles in the vicinity was considered to be the cause. Spleen The spleen was enlarged to twice normal size. Exh cess hemoglobin was gresent. Also many hyaline-like clumps of meteris1.were present. He areas of necrosis were seen. but some capillary congestion.ses observed. (The clumps of eosinophlic material indicated excessive debris from crthrocyte destruction) Brain Slight capillary congestion wee the only abnormality observed. Choriosllsntoigemembrsne Marked proliferation of the cells was observed in both mesothelium and ectoderm. Edema was marked. Ho areas of hemorrhage or necrosis were observed. 27 Amnionic Hembrane Edtmatous swelling and proliferation of the endo and mesothelium were seen. Yolk Sec Kcmbrnne The yolk sac membrane sh;wed ceyillery congestion. Eons Ho cellular changes were observed. 28 DISCUSSION ghbricsnt (1949) in a recent report emphasized the “untied" position of embryos infected with infectious bronchitis as well as the dwsrfing mentioned by Besudette and Hudson (1937). Deleplenc end Stuart (1939), end Dela- plnne (1947). This report confirms both alterations and it is the author's opinion that these changes together are potho- gnononic or the disease. Inoculation via the allontoic soc using sllentoic fluid (treated with antibiotics) proved a very satisfac- tory method for virus progegetion. The pathological el- terotion of embryos has noted on the first egg passage. The lesions were considered to be due to the virus since the preparations were bacteria-free. Brundlez,i£hg£2'snd Prickett (1949) reported that intravenous inoculation of normal whole blood or leukotic materiel caused extensive lesions to develoy in.embryos. However, those pathological manifeststions were considered different from those reported in this investigation. While perivsseulnr infiltration did occur in the materiel studied by Brendlcz.g§‘§l the much more severe hepatic lesions caused by infectious bronchitis virus was 29 evident. Erandlez‘ggflgé reported no kidney lesions except a decrease in size; whereas the infectious bronchitis virus infected kidneys were enlarged and showed extensive lesionSu Brandlez'gt.gl found marked bone changes such as enlargement of the shafts of long bones to three times normal d anetero The bones of infectious bronchitis infected embryos ex- hibited a decrease in length and diameter as well as rigidity. The kidney lesions resembled those reported by Junghcg; (1948) for avian nonocgtosis. Enlarge ent of kidneys, uric nephritis. desquanation of the epithelium of the proximal convoluted tubules, dilation of tubules with hyaline-like casts, or plugs of hemoglobin and nrystalloid deposits are all similar findings. However, no thickening of the base- ncnt membrane or protein preeinitate in Bowman's space was seen in the kidneys in this investigation. Extensive necrosis and marked vascular reaction indicated a more sev re tissue reaction, in infectious bronchitis. Although "coffins" of hepatic vessels is compara- tively rare, Yurohr (1916) reported the same reaction from the use of sterile tissues as the inoculum. He concluded that "surfing" was the result of splenic stimulation. ‘His explanation of this reaction was "That grafts of adult spleen. bone marrow, liver, and kidney placed in the outer men rane of a chick embryo cause stimulation of the embryo spleen and lead to proliferation of certain leukocytic elements in the mesoderm, subcutaneous tissues and around vessels in the liver and kidney". The spleens in this report were incree33d to twice normal size but not to the size described by Yarggi and without the mieroecogic cienges aeseribea by Deetgengketg (1920). In this study "cuffing" occurred in approximately thirty per cent of the livers examined. Combined with the cross embryo changes and the other microseoyie lesions discussed, "cuffing" should be considered as an aid in aiagnosis but not as a Specific lesion eetermining a ding» 110555.30 Ho inclusion bodies were found in the tissues examined. (Lucas, l949) Delnglene (1941) mentioned that infected embryos did not live after hatching. This was readily unaerstocd when the extensive l“ 2, liver and kilney changes were evaluated. These tissues would be unable to meet the inn creased aemnnds made of them to support the active life or a newly hatched chick. In view of the liver lesions that occur in infeeted embryos, perhaps if virus derived from only liver tissue were used in successive e35 pesscges, o viscerotroyhio adaptation might take place and a liver vaccine for adult 31 chickens might be dzveloped. The physiological reserve of the adult chicken liver might be able to withstand the infection without a high mortrlity ana an.active immunity might be produced: 32 “UTW”RY'A!D CC: ECLUSIO 3 Infectious bronchitis virus caused a characteristic pathological effect on chick cmbr3os. A distinctive curled position and dwarfing was consistently found. Fifty per cent of the embryos died within five days after inoculation via the allantoic soc. Approximately one to two per cent reached hatching age. Approximately one per cent of the embr; 08 were completel3' refrictive to the virus (in 91 ght passages). The first and sioscquent pcsio'cs caused in rt» in; and curling of embryos. 30 increase in mortality rates w:as dete cted curing the eight pesos es. A failure to grow to one-half normal size res regi orly p odueed in the heart, liver, and lungs. The kidney and spleen were enlarged to twice normal size or more. Pneumonia with no rked serous exudation was 9 constant lung lesion. Hemorrhcgic hepatitis merging into necrosis and abscessetion was uniformly observed. Interstitial .nephritis degenerating into necrosis one ebscessotion oc— currei. The spleen exhibited congestion and inure osod activity. The brain tissue was cengested. The gross and.micr0300pic tissue 0 onges combined with the distinctive slteretion of position and size of icick embr3os one of die gno ostic signifies nee in the differ- -entiation of virus activity in avian resgiratory iiseeses. 35 Beech, J. R. and Schelm, 0. W. (1956). A filtertblc irus, distinct from that of Lor3n5otrccheitis, the cause of c resyira- tor3 disease of chicks s. Poul. Sci. 15 3 199- 2060 Besudette, F. B. and Hudson, C. B. (19: 7). Cultivation of the virus of infectious Bl‘OILCEiitiSO 11313.1... §.0.v.:€vo’ 90: 51-60. Beveridge, w. I. B., and Burnct. F. H. (1946). The cultive tion of viruses and ricketsiee in the chick embryo. Fed. Research Council (Brit.). Special Kept. Series, 30. 356. Erundly. C. A.. Thorp, Frank, Jr. and ?ricl:ett, 0.0. (1949). Heeponse of Chicken Lnbr3c s to Tissues of Chickens hffcctcd with the Avian Loukosis Complex and to Tissues of Hormel Birds. Poul. Sci., 23: 486-497. Cunningham, Charles H. and Stuart. H. C. (1946). The Effect of Certain Chemical Agents on the Virus of Infectious Bronchitis of Chick ens. ‘§§.‘£. Vet. Res., 7: d66-469. Cunningham, C. 3., and Stuart, 3. G. {1947). ' Cultivation of the virus of infectious bronchitis of chickens in embryonutcd eggs. 1'1. J. L to PP‘W‘ I'Ch, 8: 209-2 2. Cunninchem. C. 3., and El D.rdir3, A. H. (19 48). Distribution of he virus of inr;ctious bronchitis 01 on. V} ens in exit-1" :«tcd chicken eggs. The Cornell l'rt., 28: €81¢388. Dentschuhoff, Vere (1920). Eyeloid meicplssie of tlze embr3onie essen- chyme in relation to cell ootentist lities end di ffer;nti l fe.c‘ors. C'rnfirie Inn stitution of no. hlnrtIn, Contributi.cns “£g Enhryolrgf, Ho. 49: 3—.s. Delaplfane, J. F. 911d StU.f*‘-.I"t, Ho 00 (19:9). Studies of Infectious Bronchitis. ‘figggg 1513.313. A ”r109 1::{77Cro Sta. Bull. 275. Deleplone, J. P. and Stusrt, H. O. (1941). The sodificetion of Infectious Bronchitis Virus of Chickens as the Result of Preps- getion in Embryoneted Chicken 3558. Rhode Island Aerie. Inner. E239 Bull. 284. Delaplene. J. P. (1947). Technique for the isolation of infectious bronchitis or fleecestle virus including observations on the use of stregtonycin in overcoming beet rial contaminants. Kineo. resort Hineteenth innuel Bullcrum ”(r-""- ‘Q‘ ,. . '- ‘ ‘T—,T r?“ oiseese conierence. no e15n, u. C. ’3une 11-13-13. Fabricont. Julius (1949). Studies on the Diagnosis of flowerstle Disease and Infectious Bronchitis of Fouls. _II. The Diagnosis of Infectious Bronchitis b3 Virus Isolation in Chick Embryos. 222 Cornell we. 39: 4:14-4:31. Groups, Vincent (1949). Demonstration of en Interference Thenomcnon Associated with Infectious Bronchitis Virus (ICE-v.) 0f CiliCkeflBo it 9-;- E-f'aCto. 583 23-310 iiufstild, 1;. S. (194.5). A Study of Infectious Bronchitis in Chickens. The Cornell Vet., 35: 52-35. Hoistod, I. S. (194?). A Stuay of Infectious Bronchitis in Chickens. The Cornell Vet., 37: 29—54. Jl‘l‘frherr, E. Lo, and Terrell, II. In, (1948). Huturally acquired immunit3 to infectious bronchitis in chicks. in. J. Vet. Research. 9: 201-205. Levine, P.P., end Hofsted, E. S. (1947). , Attengts to Control Air-borne Infectious Bronchitis end fiescnstle Disease of F wls with Sterilsmps. The Cornell Vet., 37: 204-211. Lillie, R. D. (1948). Histopsthologicel Technic. The Blskiston 00.. ihiledelphia. Lucas. A. B0 (194:9). Personal communication. Mallory. F. B. (1938). Pathological Technique. w. B. Saunders 00., Philaaelphia. ELLI‘Dhy, Jo 59 (19.1.6). The effect of adult and chicken organ grafts on the chick embryo. J. prer. Ked. 24:: l~5o SChn—lk. A. F. and. Kat-m, Tic Co (19"1). An apyarently new resgiratory &ieense of baby chicks. Jour. A.V.X»A.. 21: 4l3.432. van Rnekel, H. (1949). Personal communication. The curled position of an embry pcthognomonic of infectious bronchitis virus. l7-dey—old embryo. 7th day of infection» Kodachrome yrint. Fig. 1. 56 Fig. 2. The embryo on the left is e seven- teen—day-old embryo, 7th day of infection. It illustrates th drerfing effect of the virus. fiotice the decreased feather de- veloyment, anemia, the deformed feet. The closes is distended with fat droplets. Hormel embryo of the same age is on the right. Fig. no The two dwarfed embryos are infected. All three are nineteen-dey-old embryos. This illustrates the individual verieu tion in size of the two infected em~ bryos on the left. The alteration of the necks and the compressed feet are typical. The normal embryo of the same age is on the right. Fig. 3'7 F180 4:. Fig. 50- Heert, liver, lungs and iidneys (pelvic girdle) from two normal embryos on the left and from two infected embryos on the right. The difference in organ size is epyarcnt. The group of organs third from the left shows a hemorrhigic hepatitis with focal necrosis of one lobe. Undeve10ped lungs with areas of pneu- monia. The kidneys are Spotted with necrotic erees end are swollen. The group of organs on the extreme right illustrates the bile discolor- ed type of liver reaction. Under. developed lungs and the kidneys contain bile pigments in correlation with the excessively bile stained liver. All embryos are nineteen days Old. Organs (heart, liVer and lungs) of the infected embryo are on the left. Hotice the hemorrhagic hepatitis with a necrotic ares on the tip of the left dorsal lobe. The lungs are pneun manic end the tenacious serous exudete is evident about the lungs. The normal organs on the right illus- trate the norncl fatty appearing liver and the highly vascular lung tissue. Fig- 4 Fig- 5 Fig. 60 Fig. 7. Hormel kidneys above and the in- fected kidneys below. Hotice the bile pigmentation of the infected lower left kidney. The lower right kidney illustrates the necrotic areas seen in some embryos. The kidney is enlarged. The differ— ence in size of the pelvic girdle between nornrl and pathologicel embryo is shown. Photonicrogrsph of a kidney of an infected seventeen-dey-old embryo showing plugging of a proximal con- voluted tubule wi,h s hemoglobin cast. in upper right. Bdenoious tissue with disintegrated tubules in center, and in lower right e di- lated glemerulnr Space Centeining dcsquemeted cells with en enlerged and congested glemerulfir tuft. 1351 Kodachrome print. Fig. 8. Fig. 90 Photomicrogrsph of lung tissue of an infected eighteen-dcy—old embryo. Pneumonia with serous exudation end granulocytic and lymphocytic infil- tration is shown. Congestion of pulmonary arteriole is e:en in the lower right hand corner. The bronchial sees are filled with serous exudate containing granulocytic end desqusmstcd epithelial cells. 90X Kodachrome print. Ehotomicrogrsph of liver tissue of an infected embryo fifteen days old. Perivasculer "cuffing" of cells of the granulocytic series is shown. 90X Kodachrome print. 4-0 Fig. 10. A norm 1 ehoriosllantoic membrrne from s lS-dey-old embryo. lZSX Kodachrcme print. Fig. 11. A pathological ehorionllenteic mem- brane showing the cellular prolifer- ation of the mesoderm and ectoderm, also edema. lESX Kodachrome print. Fig. 10 Fig. ll 41 Fig. 120 13th ds‘. First of a Series of four photo- graphs showing a comp risen of size between an infectious bronchitis virus infected embryo and a control embryo the ssme age. Notice the closes distended with fat droplets. Also the deformed feet of the in— fected embryo. 4-2 F155. 30. 14th day. The characteristic bending and shortening of the neck in rela- tion to the body and head is seen, also the deformed feet. Notice the difference in feather dcveloPo ment. 1'15. 13 43 Fig. 14. A marked difference in size. feather 15th deV. development and foot posture is 11- lustreted. 4-4 Fig. 14 Fig. 15. notice the slt:rhtion of body and 16th day. head size to neck length in the in- fected embr"o, also its size in com- pariSUn to the normal. 45 F160 15 Fig.0 16. 16th day. Fig. 170 16th. day. A comparison of embryo position and size upon removal from on: shell. Pathological Embryo The curled position assumed by the nfectcd embryo. The membranes are more opaque than the normal membranes. The yolk material is of e firmer coup sisteney. Kernel Embryo Shows the transparency of the membranes and the fluid spoeerance of the yolk materiel. Eotice the freedom from constriction as oompered to the pathological em- bryo, also the advanced feather de- velOpment. “-60 1'6 Fig. 180 Conusrieon between embryos just J. broken embryo embryo embryo Opaque out of shell. The top is normal. The bottom is a bronchitis infected showing the thickened and membranes. 4'! Fig. 18 Fig. 19. Photograph in black and white tne same embryo as Fig. l. Fig- 19 48 Fig. 20. Ihotogroph in black and white of some embryos as Fig. 2. Fig. 21. Photograph in black and white of some embryos as Fig. 3. Fig. 20 n8- 21 F160 22. Fig. 23. Photograph in black and white of same organs as Fig. 5. PhotOgraph of normal and pathological kidneys of eighteenpdsy-old embryos. Eormel kidney is on right. Notice the swollen appearance of the infeetn ed kianey, also the minute arses of necrosis. These kidneys had some areas of crystalline (ureete) deposits.