EMZGQTIOLGQY 0? AN ISGIATE CM: ENFECTEG‘US BRQNCHITES VIRUS AND 171% F‘ATHGGEMCSTY IN GWETECARY EEWEE'EN? CHICKEN EMBRYOS Thezzés Es? fiha Degms of M. S. MlCHIGM! STATE UNWERSYW Harvey ‘Gmham Pumhase 3965 THESIS LIBRARY Michigan State University ABSTRACT EPIZOOTIOLOGY OF AN ISOLATE OF INFECTIOUS BRONCHITIS VIRUS AND THE PATHOGENICITY IN GENETICALLY DIFFERENT CHICKEN EMBRYOS by Harvey Graham Purchase A mild strain of infectious bronchitis virus was isolated from chickens with respiratory symptoms at the U. 8. Regional Poultry Research Laboratory. Confirmation of identity was based on symptoms, histopathology, fluorescent antibody, lesions in chicken embryos and lack of lesions on the chorioallantoic membrane, hemagglu— tination with trypsin—treated virus, and lack of hemagglu- tination inhibition. Using serum-virus neutralization tests in embryos, the virus was more closely related to the Iowa 97 serotype of Hofstad (1958) than to any of the other six serotypes described in the literature. The epizootiology indicates that the Virus is main- tained in the nonisolated breeding stock as an inapparent infection. Periodically there is a break in the quaran— tine and the disease occurs in the isolated area in more mature birds. The 23rd chicken embryo passage of virus can be used to immunize immature chickens to prevent a possible drop in egg production if the birds are exposed during the laying period. HARVEY GRAHAM PURCHASE The candling method of determing positive responses was almost as accurate as examining the embryos for lesions. There was some correlation between the con— stant serum-—decreasing virus and the constant virus—- decreasing serum methods of determining antibody. The latter method is useful for screening large numbers of sera, is less expensive, and is of sufficient accuracy for epizootiologic studies. Passage in the allantoic sac of embryonating chicken eggs increased the pathogenicity of the virus for line 7 embryos significantly but the pathogenicity for line 6 embryos remained unchanged. Inoculation of embryos from a diallel cross mating of inbred lines 6, 7 and 151 demonstrated that line 7 embryos die significantly later than line 6 embryos. Like 151 is intermediate between these two. Some matings had significantly more or less mortality than would be expected from a consideration of the lines themselves. The reasons for this are obscure. The difference between reciprocal crosses is significant at 7 days postinoculation. The effect of the line 7 male on delaying the mortality is greater than that of the female. This may be due to one or more sex—linked genes for delaying mortality in line 7 or to a maternal effect in line 7 females causing earlier mortality. EPIZOOTIOLOGY OF AN ISOLATE OF INFECTIOUS BRONCHITIS VIRUS AND THE PATHOGENICITY IN GENETICALLY DIFFERENT CHICKEN EMBRYOS BY Harvey Graham Purchase A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1965 (\ ACKNOWLEDGEMENTS I wish to express my sincere thanks to Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, and to Dr. Ben R. Burmester, Director of the U. 5. Regional Poultry Research Laboratory, for their encouragement and assistance in the research work and preparation of this thesis. I would also like to express my appreciation*to Dr. Jack J. Stockton, Chairman of the Department of Microbiology and Public Health, and to all others connected with Michigan State University and the U. 8. Regional Poultry Research Laboratory who aided my efforts. I acknowledge, with gratitude, the advice on statistical methods given me by Dr. Lyman B. Crittenden, Geneticist at the U. 5. Regional Poultry Research Laboratory. 11 TABLE OF CONTENTS ACKNOWLEDGEMENTS . . . . . . . . . . . TABLE OF CONTENTS . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES , . . . . . . . . . . INTRODUCTION 0 O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . NA TERIALS AND METHODS . . . . . Etiological Agent . . . Incubation and Symptoms PathOIOgy’ e e o e e e e EpiZOOtiOlogy e e e e 0 Virus Isolation and Pass Hemagglutination. . . . Immunity. e e e e DiagnOSLS e e e e SerOIOgy e e e 0 Genetic Studies . g .. . o. m .. e o o o o o 0 (Do 0 o O .. . .. . .. . . .. . .. . to e o .. . .. . 00 e e Chickens and Eggs . . . . . Allantoic Sac Inoculation . Chorio-allantoic Membrane In 1 Diluent e e e e e e e e e 0 Virus Isolation . . . . . . Passage of the Virus. . . Preparation of Virus Stocks Hemagglutination. . . . . . Hemagglutination Inhibition PathOIOgy e e e e e e e e Fluorescent Antibody. . . . Serum-virus Neutralization Tes Constant Serum Method. Constant Virus Method. Calculation of Titers . . . Immunization. e e e e e e 0 Collection of Serum . . . . Studies on the Genetic Resistanc Statistical Analysis. . . . . . 000000.000. eased-000000.020. e e a e e C e o e e e e e e e e e e 0 iii oooooeooooooooeepeo 00000000000000.0300 Page ii iii vii I-‘CDOJVVWC'C‘MJQ U ta H \o \JUJNTOAJNIOADNIOADHFHFIththFH n3ocnaumuzcwonamrauamsaounchw» TABLE OF CONTENTS - Continued RESULTS 0 O O O O O O O O O O C O O O O O 0 Symptoms and Pathology. . . . Hist0path010gy. . . . . . . . Virus Isolation and Passage . EffeCt Of Passage 0 e e e e e PrOpagation and Storage . . . Candling NethOdSe e e e e e 0 Identification of the Virus . n t V (n Constant Serum versus Consta EpiZOOtiOlogy e e e e e e e DevelOpment of a Vaccine. . Genetic Resistance. . . . . 0 00050000000 coop-coco... '1 DISCUSSION AND CONCLUSION. . . . . . . . . Identity of the Reapiratory Disease . Passage Of the VirUS. e e o o o o e e Titration and Neutralization Procedures EpiZOOtiology e e e o e e e e e e e e vaCCleation e e e e e e e e e o e e 0 Genetic Studies . . . . . . . . . . . SUMRY. e e e e e e e e o e e e e e o e 0 LITERATURE CITED 0 O O O O O O O O O O O 0 iv LIST OF TABLES TABLE Page 1. SiXoserotypes of IE and related viruses with isolates falling under each SerOtype e e e e e e e e e e e e 10 2. Source of virus for trypsin treat- ment and hemagglutination . . . . . . 20 3. Criteria for constant virus screen me thOdO 0 O O O O O O O O O O O O O O 27 h. Mortality pattern on isolation and passage of RPL-IBV. . . . . . . . . . 38 5. PrOpagation of virus in the allantoic sac o e e e e e e e e e e o e e e e e no 6. Loss of virus on storage at -75°C . . no 7. Comparison of endpoints of a serum- virus neutralization using different methods of reading positive virus reaponses . . . . . . . . . . . . . . A2 8. Fluorescent antibody results on tracheal scrapings of two birds “Tith Signs Of 18. g Q - y 0 O O 0 0 o o “’2 9. Hemagglutination using trypsin treated RPL‘IBV e e e e e e e e e o 0 ha 10. HI titration of sera from survivors of RPL-IBV infection using 10 HA units of killed NDV (USDA). . . . . . A6 11. Neutralization of RPL-IBV by sera from various sources, , , . . . . . . g7 12. Constant serum versus constant Virus titrationS. e e e e e e e e e e “9 13. Screen for antibodies to RPL-IBV. . . 50 LIST OF TABLES - Continued TABLE 1h. 15. 16. 17. 18. 19. Page Neutralization of RPL-IBV by sera from birds vaccinated by various routes with different passage levels of the hom010gous virus. . . . . . . . 5h Titration of RPL—IBV in line 6 and line 7 embryos e e e e o e e e e e e e 56 Titers and statistical analysis of RPLPIBV titrations in experiment I . . 60 Titration of RPL-IBV in diallel cross matings. O O I O O O O O O O 0 O 0 0 0 61 Results of statistical analysis of diallel cross matings. . . . . . . . . 67 Titers of RPL-IBV in embryos of diallel cross matings. . . . . . . o . 58 vi FIGURE 1. 2. 3. LIST OF FIGURES Diagram of the buildings and isolation areas at the RPRL . . . . Per cent reSponse of embryos of two inbred lines of chickens to RPL-IBV Cumulative per cent response of two inbred lines of chickens to two passage levels of RPL-IBV . . . . . Per cent reSponse of embryos of diallel cross matings to RPL-IBV. . Cumulative per cent reSponse of embryos of diallel cross matings to RPL“ IBV e e e o e e o o o e e e o 0 vii Page 51 57 59 6h 66 INTRODUCTION Infectious bronchitis (18) was first detected at the U.S.D.A., Regional Poultry Research Labora- tory (RPRL) in 1955 by virus isolation and serum neutralization tests performed by Dr. C. H. Cunningham, Department of Microbiology and Public Health, Michigan State University. A diagnosis of Newcastle disease (ND) was made by Dr. C. F. Hall, Department of Veterinary PatholOgy, Michigan State University in 1957 on the basis of a serum hemagglutination inhibition (HI) titer. Since then there have been periodic occurrences of res- piratory symptoms and several laboratory diagnoses of IE and ND have been made. Reapiratory symptoms were often noticed in 2-week-old chicks being in- oculated with avian tumor viruses. Sometimes they occurred among the line 15 isolated (151) breeding stock and occasionally among chickens in other isolated areas. In these cases signs of the disease were often noticed in more mature birds. A mild respiratory disease occurred in January 1963, in birds on a leukosis experiment. A positive HI test was obtained with serum of 2 birds submitted to Dr. J. P. Newman, Department of Microbiology and Public Health, Michigan State University, on January 28, 1963 but virus was not isolated. An additional 6 samples submitted on January 30 were negative. The purpose of the present study was to isolate and identify the agent (designated Regional Poultry Laboratory infectious bronchitis virus (RPL-IBV) causing the respiratory disease, to study the source and spread of infection and to institute prophylac- tic measures. Every consideration had to be given to the nature of the other research being conducted at the laboratory. LITERATURE REVIEW Etiological Agent Infectious bronchitis is an acute, highly in- fectious and sometimes fatal viral disease of chickens. It is widespread throughout the world (Hofstad, 1959; Kawakubo 33_g;., 1961). The virus is 80-120 my in diameter and is related to the in- fluenza group (Cunningham, 1957; Hofstad, 19593 Berry et al., l96h; Tevethia, 196“). Incubation and Symptoms The incubation period is usually 18-36 hours but may be as long as 15 days depending on the strain of virus, dose, passage level and amount of inoculum (Raggi and Bankowski, 1956). The characteristic symptoms are a nasal discharge, watery eyes, gasping, rales, coughing and sneezing. The mortality may be high. In laying flocks there is a sudden decline in egg production and the shells may be misshapen, rough or soft. The duration of the disease is usually 1-2 weeks but this depends on the environmental conditions and secondary complications such as chronic res- piratory disease (Cunningham, 1957; Hofstad, 1959). Pathology Post mortem findings include a serous or catarhal tracheitis and catarrhal or fibrinous aerocystitis. There is often a focal pneumonia localized around the bronchi. In chicks there is usually a catarrhal rhinitis, sinusitis and con- junctivitis. Hist010gically there is a non-purulent catar- rhal tracheitis, bronchitis and broncheolitis. Inclusion bodies have not been reported. The microSCOpic changes have been regarded by some authors as pathognomonic (Jungherr 3£_gl., 1956). Other authors consider they are only useful for susceptibility studies (Hofstad, l9h5a). The temporal changes after artificial exposure were described by Jungherr g£_gl. (1956). Epizootiology The epizootiolOgy of the disease is not well understood. The chicken is the natural host and, as far as is known, no other avian Species are affected (Van Roekel, 1955). Air-borne trans- mission is probably the most common route in nature (Hofstad, 1959). The virus can be easily transmitted artificially and the intratracheal route is most commonly employed (Cunningham, 1957). The virus can be isolated readily from lung and tracheal material taken throughout the respira- tory phases of the disease (Hofstad, 19h7; Fabricant, 19U9; Fabricant, 1950; Fabricant and Levine, 1951; Kawakubo 23.913, 1958) and from eggs laid 2 to #3 days after infection (Fabricant and Levine, 1951). Delaplane and Stuart (19h1) showed that chickens could carry the virus for at least 8 weeks. Chicks can transmit the virus as long as 35 days after the appearance of symptoms but only if infected within the first few weeks of life (Hofstad, 19u5b; 19b7). Virus Isolation and Passage. Infectious bronchitis virus can be pr0pagated readily in embryonating chicken eggs. The more usual route of inoculation is via the allantoic cavity but other routes can be used (Cunningham and Jones, 1953; Kawakubo 23.31., 1958). Characteristic lesions in chicken embryos which are employed as diagnostic criteria have been described by many workers (Loomis 22.31,, 1950; Hofstad, 1956b; Hofstad, 1959). Embryos may be dwarfed as much as one-half and are ”curled” into a firm compact ball with the feet compressed over the head. There is less amniotic fluid and the allanto-amnionic membrane is often thickened. Living embryos are sluggish in their movements. Edema, necrosis of the kidneys and a persistent mesonephros containing urates are often found. Hemorrhage and necrosis of the liver, pneumonia, and dermal petechiation have also been reported. Embryo mortality is not a constant finding (Cunningham, 1957). More than one serial passage may be necessary before a diagnosis can be made (Fabricant, 19h9). Serial passage of the original (0) isolate in embryonating eggs is accompanied by an increase in virulence of the virus for embryos and develop- ment of the derivative (D) or egg adapted phase. Concurrently there is usually a decrease in viru- lence for chickens and a loss of antigenicity and immunogenicity (Delaplane and Stuart, 19h1; Larose and Van Roekel, 1961), though this is not always the case (Kawakubo 35 31., 1961). The 0 phase is more thermostable than the D phase (Singh, 1957; Singh, 1960). The embryo-adapted virus is speci- fically neutralized by anti-IB serum (Fabricant, 1951). Hemagglutination The virus does not agglutinate chicken eryth- rocytes unless modified with trypsin (Corbo and Cunningham, 1959; Mu1doon, 1960). Specific in- hibition of hemagglutination (HA) has not been demonstrated. Anti-IB sera do not have HI titers of greater than 160 against Newcastle disease virus (NDV) (Fabricant, 1950). Immunity Neutralizing antibodies are first detectable about 2 weeks after infection, they can be reliably demonstrated at 3 weeks and their titer continues to rise until about 5 weeks after infection (Fabricant, 1951; Page and Cunningham, 1962). A serum-virus neutralization index (NI) greater than 2.0 is indicative of previous exposure to IE (Cunningham, 1951; Fabricant, 1951). The serum antibody level of day-old chicks is almost identical with that of their respective dams but this maternal antibody declines rapidly until it is lost by h weeks of age (Jungherr and Terrell, 19h8). Even chicks with a high maternal antibody level are not necessarily protected from showing symptoms when challenged during the first week after hatching (Hofstad and Kenzy, 1950). Diagnosis A diagnosis of IE is dependent on the demon- stration of the following: 1. A typical history and symptoms 2. Characteristic histo-pathologic changes 3. Positive fluorescence with a fluorescein labelled anti-IB serum but not with other labelled antisera. h. Lack of HA except with modified virus 5. A negative HI titer using Newcastle dis- ease virus antigen 6. Virus isolation and the demonstration of typical lesions in embryos 7. A NI greater than 2.0 against an isolate of IE 8. Lack of neutralization of other viruses SerolOgy Jungherr 23‘31. (1956) reported that an isolate from Connecticut (Conn) differed from an isolate from Massachusetts (Mass). These were the only two sera-types rec0gnized until 1958 when Iowa 97 and Iowa 609 were,shown to be serologically dif- ferent from the Mass and Conn types (Hofstad, 1958). Then two additional and unrelated isolates, Holte and Grey, were reported by Winterfield and Hitchner (1962). Yates and Fry (1957) and DuBose and Grumbles (1959) showed the chicken embryo lethal orphan virus (CELO) to be similar to the quail bronchitis virus described by Olson (1950). Hitchner g£_gl. (196A) showed conclusively that the above viruses could be grouped into 6 groups or serotypes using cross serum-virus neutralization tests. According to the information presently available at least 28 isolates have been classified according to serotype (Table 1). There is still some confusion as to the im- portance of these serotypes and the exact relation- ship of one type to another since there are weak cross reactions. Immunization with one virus will in many cases afford protection against challenge with another virus of a different serotype even though there are no demonstrable antibodies to the challenge virus. This has lead to the feeling that a cellular and not a humoral immunity is responsible for the protection (Hofstad, 1961; Hitchner 32 31., 196“). The differences among serotypes and im- munologically different strains needs additional research. 10 TABLE 1. Six serotypes of 18 and related viruses with isolates falling under each serotype Serotype Virus Isolates Reference ‘— Mass Massachusetts Jungherr et al. 1956 No. 82828 Jungherr et a1. 1956 IBV #1 IB Virus Repository* Beaudette Beaudette 5 Hudson 1937 No. 66579 Jungherr et al. 1956 Strain 20 Hofstad 1956a IBV #2 IB Virus Repository A 36896 (18 D G S) Jungherr et al. 1956 Iowa 33 Hofstad 1956a Iowa 76, 10A, 132, 31h, 346. 433. 581. 619. 630, 661 Hofstad 1958 Iowa 70“: 7269 7279 730. 758 Hofstad 1961 Conn Connecticut Jungherr et al. 1956 No. A 5968 Jungherr et al. 1956 L-Z Hofstad I961 IBV 46 IB Virus Repository Iowa 97 Iowa 97 Hofstad 1958 Iowa 609 Iowa 609 Hofstad 1958 Gray 5 Holte Gray Hinterfield 8 Hitchner 1962 JMK Winterfield et al. 196A Holte Winterfield 5 Hitchner 1962 CELO Chicken Embryo Lethal Orphan (CELO) Quail Bronchitis Virus (QBV) 1750 Yates & Fry 1957 Olson 1950 Hitchner et al. 196% n * Department of Microbiology 8 Public Health, Michigan State University, East Lansing, Michigan. 11 Genetic Studies Fowls differ genetically in their ability to resist invasion by many parasites including viruses, bacteria, fungi, protozoa, and helminths. Genetic differences have been demonstrated between breeds, strains of the same breed and even between families (Waters and Burmester, 1963; Hutt, 1958). Numerous factors have an influence on the expression of the genetic resistance. Genetic differences must be distinguished from physiological effects, e.g. maternal antibody. By studying the reciprocal crosses of the resistant and susceptible lines, genetic resistance to coccidiosis (Rosenberg g£_gl,, 1954), pullorum disease (Roberts and Card, 1935), and ND (Reta 33.21., 1963) has been demon- strated. The effect of the sire was greater than that of the dam suggesting possible sex linkage but the differences were not statistically sig- nificant. The decrease in the weight of the body and the visceral organs due to vaccination with ND is different for different strains of birds (Francis 22'31., 196A). In many instances animals highly resistant to one disease are susceptible to another. In very few instances has the actual mode of inheri- tance to disease been demonstrated. In mice the 12 gene for resistance to some arboviruses is dominant and autosomal, whereas in mouse hepatitis the gene for susceptibility seems to be dominant. The sus- ceptibility to hepatitis is of a cellular nature since results similar to the above were obtained in cell culture (Goodman and Kaprowski, 1962; Kantoch 35 11,. 1963). In fowls the mode of inheritance has been established for resistance to Rous sarcoma virus (Waters and Burmester, 1961; Crittenden 2£H31., 1963) and erythroblastosis virus (Waters and Burmester, 1963). In both instances inheritance is by a single autosomal gene. In the former susceptibility is dominant whereas in the latter resistance is dominant. Line 7 is homozygous for resistance and Lines 6 and 151 homozygous for susceptibility to Rous sarcoma virus (Crittenden, 1965). The resistance in Line 7 has been proven to be a cellular phenomenon since it occurs in embryo cell culture, embryos and hatched chicks (Crittenden 33.31., 1963). MATERIALS.AND METHODS Chickens and Eggs. All eggs and chickens were produced at the RPRL from one or more of the six highly inbred lines of Single Comb White Leghorns. Birds from this closed flock were considered to be free of pathogenic pleurOpneumonia-like organisms (PPLO) since lack of symptoms and repeated serologic and cultural tests have failed to reveal evidence of PPLO infection. All birds were maintained on commercial feed with a coccidiostat and were placed in semi-isolated pens on corn-cob litter. Eggs were collected and saved for two weeks before setting and were maintained at 15°C. They were incubated at 37.500 in a forced draft, thermal and humidity controlled incubator* with trays that mechanically turned the eggs approximately once every two hours. Line 151 chickens are highly susceptible to ne0plasms caused by viruses of the avian leukosis complex and have been reared in strict isolation and inbred for 26 years. This line has a very low incidence of natural lymphomatosis and is free of LI. *James Manufacturing Company, Model 2528 13 1U agents having "resistance inducing factor" (RIF) activity (Rubin, 1960). Any vaccine used in these birds would have to be RIF-free. Therefore all virus isolation, passage of the virus and preparation of stock virus were done in RIF-free line 151 eggs. As the supply was limited, eggs from line 6 hens mated with line 151 cockerels were used for all serum neutralization tests. Allantoic Sac Inoculation The eggs were candled on the 9th or 10th day of incubation. A mark was made in an area distant from the embryo and amniotic cavity, on the shell over the air cell about 2-3 mm above the base of the air cell. Care was taken not to locate the mark directly over a blood vessel. After cleansing with 70% alcohol a hole was punched at the site using a dentist's probe sharpened to a pyramidal point. By rotating the probe the hole could be enlarged. The punch was dipped in 70% alcohol and flamed after punching every 30 eggs. The inoculum was deposited in the allantoic sac using a separate 26 gauge % inch needle, and 1 cc disposable tuberculin syringe for each lot. The hole was sealed with adhesive and sealing compound.* g *Prince Company-cellulose nitrate base. 15 Chorio-allantoic Membrane Inoculation Uncandled lO-day incubated eggs were cleansed over the air cell with 70% alcohol and a hole was punched through the shell and enlarged to allow free air flow. The egg was then candled and if infertile, cracked, or if the embryo was dead it was discarded. A rubber tube was attached to the housing of the candler. Oral suction on the tube drew air from around the light source. When an egg was placed with the Opened air cell against the Opening of the candler, a negative pressure was created within the air cell. An area, not directly over the embryo, but having a fairly good blood supply, was chosen. It was disin- fected with alcohol and a small hole was then punched between two blood vessels. The punch was rotated gently so as to enlarge the hole through the shell and then pierce the shell membrane. Simultaneously suction was applied to the original air cell and as soon as the shell membrane was pierced the chorio-allantoic membrane (CAM) dropped. Suction was continued until the original air cell was obliterated. The punch was dipped in 70% alcohol and flamed between each egg. With practice 200 eggs per hour could be handled in this way and losses due to faulty drapping of the l6 CAM amounted to 1 to 2%. The inoculum was deposited on the CAM with a % inch 26 gauge needle and 1 ml tuberculin syringe. The holes were then sealed with adhesive compound taking care to allow the adhesive over the artificial air cell to dry before sealing the hole in the original air cell. The eggs were either placed upright in incubator trays and not allowed to turn, or were placed in egg flats and stacked 2 to 3 flats deep. Diluent In all experiments, unless otherwise stated, the diluent was either Simm's balanced salt solution with 2% horse serum or cell culture medium 199 with 2 to 3% calf serum. In either case 100 to 500 units potassium penicillin G and 0.1 to 0.5 mgm dihydrostreptomycin sulfate were added per m1. Virus Isolation A bird with acute symptoms was killed. The trachea and bronchi, from the larynx to the beginn ning of the bronchioles, were removed aseptically and then out in half longitudinally. For virus isolation one-half the trachea was weighed to the nearest 0.1 g and then cut into pieces no larger than 5 mm in diameter. The pieces were placed in a "Ten-broek” grinder, kept in crushed 17 ice, to hhiCh enough cold diluent without anti- biotics was added to make a 20% weight/volume ex- ract of trachea. After grinding periodically until there were no visible pieces of trachea, the homagenate was centrifuged at about 1000 X g in a refrigerated centrifuge* at 2°C for 30 minutes. The clear supernatant fluid was removed and 1.5 ml portions were placed in soft glass ampoules kept on ice. The ampoules were flame-sealed and stored at -75°C. At the time of use, 2 ampoules were thawed rapidly under cold running water at about 20°C. To one was added 1 m1 of diluent with anti- biotics while the second was left untreated. Both preparations remained on ice for 10 to 15 minutes by which time the eggs were ready for inoculation. Six eggs were used for each lot and 0.2 ml was inoculated into the allantoic sac of each egg. All eggs were candled daily and dead embryos were examined for lesions. Passage of the Virus 0n the third or fourth day after inoculation the allantoic fluid was harvested from two embryos taken at random. The embryos were alive or may have died within the previous 2h hours. The P *International Equipment Company, Model PR—2 18 allantoic fluid was pooled and immediately injected into other eggs without freezing. Aseptic tech- niques were used throughout. Preparation of Virus Stocks Virus, 0.2 ml, was inoculated into the allantoic sac of each of a group of 10-day-old embryonating eggs. After 3 days incubation, the allantoic fluid from each egg was harvested and pooled in a flask kept in crushed ice. The pool was then divided into 10 ml portions in soft glass ampoules in ice, sealed and stored at -75°C. The vials were periodically subdivided into smaller portions suitable for in- dividual experiments. In this case they were thawed rapidly by vigorous agitation under cold running water at about 20°C. As the last piece of ice melted the ampoule was placed in a container of crushed ice, the tap was cracked open and 1.5 ml portions were transferred to smaller ampoules also in the ice bath. These were then sealed and refrozen. Before use and as close as possible to the time of inoculation, an ampoule was thawed rapidly as above. The allantoic fluid was then centrifuged at about 1000 X g for 30 minutes at 2°C. All the precipitate which had appeared on freezing and thawing was sedimented by this treatment. Only the clear supernatant fluid was used. 19 Hemagglutination The 7th and 10th passages of RPL—IBV were inoculated into the allantoic sacs of 10—day-old embryonating eggs. 0n the 3rd and 6th days post- inoculation, allantoic fluid was harvested from one or two living embryos and the remaining eggs were incubated to determine the effects of the virus (Table 2). The allantoic fluids were frozen, thawed and then centrifuged at about 1000 X g for 30 minutes at 2°C. Part of each sample was treated with trypsin by adding 0.25 ml of 1% trypsin (1,250.) to 0.5 ml of the supernatant fluid. The mixture was incubated at #800 for 30 minutes and then at 56°C for 10 minutes. Egg white trypsin inhibitor, 0.25 ml, was then added and the mixture was agitated intermittently and incubated at room temperature for one hour. Six m1 of fresh chicken blood was collected in 0.6 ml of 2.5% pOtassium citrate. The mixture was centrifuged at about 1000 X g in a clinical centrifuge** and the plasma removed. The erythro- cytes were washed six times in Hemagglutination L *Nutritional Biochemicals Corporation **International Clinical Centrifuge 20 TABLE 2. Source of virus for trypsin treatment and hemagglutination Harvested By 10th day p.i. Virus No. 3rd day 6th day No. No. with Passage Eggs p.i.* p.i. dead lesions 7 l8 2 13 IA 10 12 l l 10 10 Uninoc. 8 l 1 0 0 * post inoculation. 21 buffer* and resuspended in the buffer, 0.5% cells by volume. Two-fold dilutions of the untreated virus and of the trypsinized virus (T) were made in buffer. An equal quantity of washed erythrocytes was added to each tube (thus giving a a dilution of virus in the first dilution tube). The mixtures were then in- cubated at room temperature for 2 hours and ob- served for agglutination. They were graded and recorded from - (no agglutination) to ++++ (full agglutination). The end-point was the highest dilution with a ++ or greater agglutination. Hemagglutination-Inhibition Sera were collected from 21 infected birds 3 weeks after the initial reSpiratory disease. Two- fold dilutions of the sera were titrated against 10 HA units of killed USDA Newcastle disease virus antigen by the method described by Cunningham (1953). A known positive (accession 2709) and a known negative (accession 1339) serum obtained from Dr. J. P. Newman, Department of MicrobiOIOgy, Michigan State University, were used as controls. A; *Baltimore Biological Laboratory Ltd. 22 PatholOgy All birds dying at the RPRL are routinely necrOpsied. Where necessary, samples of affected organs are fixed in 10% formalin buffered with calcium carbonate. Sections are stained with hematoxylin and eosin for microsc0pic examination. Fluorescent Antibody The half of the trachea not used for virus isolation was used for fluorescent antibody studies. Using a scalpel, the mucous membrane near the bi- furcation of the trachea was scraped several times. The mucus and dislodged cells were Spread thinly onto 3 glass slides. They were allowed to dry at room temperature for 30 minutes and then placed in a humidity chamber. Two draps of fluorescein- 1abelled antiserum against NDV, infectious laryngo- tracheitis virus (ILT) and IB virus obtained from M. O. Braune, Pennsylvania State University, were placed onto each reSpective slide and they were allowed to incubate for 30 minutes at room temperature. The slides were then blotted dry, placed in a rack and submerged in continuously agitated phosphate buffered saline (pH 7.2) for 30 minutes. The slides were then mounted using a 9:1 glycerinezbuffer mixture and examined under a 23 Leitz fluorescence microsc0pe. The brightness and specificity of fluorescence were graded from - to ++++. Serum-virus Neutralization Tests. The purposes of using neutralization tests in these studies are the following (Cunningham, 1963): 1. To identify the virus and show its relation to other strains of IBV. 2. To show the presence of circulating anti- bodies as a measure of immunity after inoculation. 3. Th compare the constant serum with the con- stant virus method of antibody assay. h. To study the epizootiology of the virus among the RPRL flock. The constant serum-decreasing virus method and the constant virus-decreasing serum method were used. The former was employed for the titration of virus and antibodies and the demonstration of serologic relationships between viruses. It requires large numbers of eggs and more complicated dilution pro- cedures but it gives accurate titers of the sera (Page and Cunningham, 1962) and the virus is titrated simultaneously. The latter method is most efficient for the screening of large numbers of samples for the presence of antibody because fewer 211 eggs are used for each serum. The amount of virus used depends on a previous titration of that virus stock. However, by having adequate controls, one can determine the reliability of the results. Constant Serum-decreasing Virus Method The method outlined by Cunningham was used with the following modifications: 1. All sera were inactivated at 56°C for 30 minutes. 2. A mixer* was used to mix the contents of a tube rather than repeated aspiration and expul- sion by pipette. 3. The prepared dilutions of virus consisted of 10 parts of each tenfold dilution of virus to which was added 8 parts of diluent. Serum, 0.2 ml, was placed in each of the serum tubes and 0.2 ml of diluent in the virus control tube. To each tube was added 1.8 ml of the prepared tenfold dilutions of virus. This gave a £2221_33£gm dilution of 1/10. Thus this is equivalent to adding equal quantities of the respective tenfold dilution of virus to a 1/5 dilution of serum. U. The inoculum was 0.2 ml per egg. This *Vortex Jr. Mixer, Scientific Industries Incorporated. 25 represents 0.1 m1 of serum and 0.1 m1 of the reSpect- ive dilution virus. Constant Virus-decreasing Serum Method A method similar to that of Fontaine 2£_gl. (1963) was followed. 1. Each serum, 0.2 ml, was placed in a tube and inactivated at 56°C for 30 minutes. The tubes containing serum were then placed in an iced bath. 2. The virus was diluted to contain 103 EIDSO per m1 and placed in an iced bath. To every 10 parts of this virus was added a further 8 parts of cold diluent and then 1.8 m1 of the final dilution was added to each tube using a sterile ”Cornwall” continuous pipetting apparatus.* Using the above method there were 100 E1050 of virus in each 0.2 m1 of inoculum and a final dilution of 1/10 of serum. 3. Diluent, 0.2 ml, was placed in the first control tube and 1.8 ml in each of another 5 tubes. To the first tube was added 1.8 ml of the final dilution of virus described above. Four tenfold dilutions were made by transferring 0.2 ml from tube to tube using separate pipettes and mixing L *Beckton, Dickinson and Co. 26 between each transfer. The last tube was left with diluent alone. A. The tubes were incubated at room temperature (ZS-30°C) for 30 minutes and then replaced in an iced bath until inoculation. The inoculum was 0.2 ml into each of 5 or 6 eggs for each lot. 5. In some cases dilutions of the serum were prepared before being added to the tubes. 6. The positive virus responses were detected by candling alone (Table 3). Calculation of Titers The Reed and Meunch (1938) method of calcu- lating 50% endpoints was used wherever applicable. Virus titers are expressed as the positive 10garithm to base 10 (loglo) of the number of 50% egg infective doses (EIDSO) per ml of original allantoic fluid, e.g., allantoic fluid with 106"5 EID50 per m1 would have a titer of 6.5. Virus doses are expressed as the 10g10 of the final dilution plus the 10g10 of the inoculation factor, e.g., if 0.2 ml of a 10"3 dilution is inocun lated the dose is -3.699. They represent the 10g10 of the fraction of 1 ml of original allantoic fluid inoculated. 27 TABLE 3. Criteria for constant virus screen method _L Positive Virus Responses No. of eggs Antibody Antibody per lot positive Doubtful negative A 1 2 or 3 h 5 l 2, 3 or A 5 6 lor2 30r1+ Sor6 28 In the calculation of serum titers it was assumed that one antibody molecule is responsible for neutralization of one virus particle, i.e. the neutralization of virus by serum is a first order reaction (Hirst, 1959). This is an arbitrary as- sumption and does not always hold true. Thus the neutralization index (NI) of the serum using either the constant serum or constant virus method is: The amount ofvirus neutralized in EID50 Serum dilution or the 10g10 of amount of virus neutralized minus the loglo serum dilution. Immunization The 8th and the 23rd passages of RPL-IBV were used in immunization trials. At least 10“ ETD50 were given to each chicken. The intratracheal route was used except where the 6-day-old chicks were reimmunized. In this case the chickens were given one drop on the conjunctiva and two draps intranasally. Collection of Serum When small quantities of serum (1 to 5 ml) were needed, birds were bled from the heart with 29 a ”Peel-a-way"* syringe and either a 20 guage 1% inch or a 22 guage 1 inch needle depending on the size of the chicken. When birds were to be exsanguinated, blood was collected from the heart using an 18 guage 1% inch needle on one end of a piece of polythene tubing. To the other end of the tubing was attached a shorter needle which was inserted through a rubber sleeve stOpper on a 100 m1 prescription bottle. A second piece of polythene tubing attached to another needle was also inserted through the stopper. Negative pressure was obtained in the bottle by oral suction on the second tubing. A piece of glass tubing packed with cotton was used as a mouthpiece and filter on the end of the tubing. The bottle and tubes were sterilized before use. A separate apparatus was used for each bird. All blood samples were allowed to clot for l to 2 hours at room temperature and then the clot was loosened from the glass by shaking. It re- mained at room temperature overnight. The mix- ture of serum and erythrocytes removed from the clot was centrifuged at about 1000 X g for 10 minutes and the clear supernatant serum was placed in plastic ampoules**and stored at -25°C. g *Scientific Products. **Insemikit Company. 30 During collection and processing of the sera aseptic technique was used. No difficulty was en- countered with contamination. Studies on the Genetic Resistance of Embryos to RPL-IBV Over the past 26 years several lines of White Leghorns have been maintained at the RPRL. They have been inbred to the point where the coefficient of inbreeding as measured by Wright's standard (F) is greater than 95% (Waters and Burmester, 1961; Waters and Burmester, 1963). This indicates a very high degree of homozygosis. Of the many lines maintained, lines 6, 7, and 151 have been studied most extensively. These lines of chickens vary in their response to different avian tumor viruses (Burmester 33 31., 1960). The purpose of the fol- lowing experiments was to determine whether or not there was any genetic difference in reSponse to RPL—IBV between the different lines. Experiment 1 Lines 6 and 7 were chosen for a pilot experi- ment because of the availability of eggs and the great differences between the lines in all reSpects studied previously. Six eggs were used per lot of line'7 and five eggs per lot of line-6 according to the availability of the eggs. 31 8 of each of the 8th Dilutions from 100 to 10' and 23rd passages of RPL-IBV were prepared and 0.1 m1 of each dilution was injected into the allantoic sac of each egg. Each egg in the tenth lot was inoculated with 0.1 ml of diluent alone. The eggs were identified inconSpicuously by lot number and were placed randomly in the incubator trays. The eggs were candled daily until the 10th day after inoculation. The number of embryos dead in each lot was recorded and the date of death was recorded on each egg. The eggs with dead embryos were re- moved each day and stored at #00. The Spaces in the trays left when eggs were removed were filled using eggs taken from a row at one end of the tray. In this way an additional randomization of the eggs of each lot occurred. On the 10th day the total number of eggs in each lot was rechecked before discarding the eggs. EXperiment II Experiment 1 yielded significant results and a second, more extensive diallel cross experiment was performed. Matings of pure lines 6, 7, and 151, their crosses and reciprocal crosses were prepared. For each of the 9 possible combinations there were 32 two Fens containing 15 to 20 hens and two cockerels each as follows: Males (2 per pen). 6 7 151 Females 6 6X6 7X6* 15IX6 (15—20 7 6X7 7X7 15IX7 fer pen). 151 6x151 7x151 151x151 Ten eggs were used per lot and care was taken to insure that each lot contained the same number of eggs from each of the 2 pens supplying each pure line or cross. Only the 23rd passage of RPL—IBV was used. Details of dilution, inoculation and candling were the same as in the previous experiment. Statistical Analysis In Experiment 1, different numbers of eggs were used for line 6 and 7 and titration of the virus in these eggs yielded similar endpoints. The only difference observed was between latent periods before death of the embryos. Line 6 embryos died earlier than line 7. A statistical analysis was performed using the method of Mantel and Haenzel (1959) since this takes into account the number of *In any cross e.g. 7X6 the male is always given firSte 33 embryos at risk on each day (i.e. includes latent period and titer) and is not influenced by the number of embryos in each lot. In EXperiment II where there was a three-way diallel cross setup, the effects of the crosses and reciprocal crosses were examined as well as the effect of the lines. The number of eggs in each lot was nearly constant. Only those embryos dying within 2h hours after inoculation affected the number per lot. The number of embryos dead before a certain date reflects the latent period, i.e. the shorter the latent period the greater the num- ber of deaths before the selected time and the fewer the deaths afterward. In this experiment the number of embryos dying during the first 5 or 7 days was compared using the method outlined by Griffing for diallel cross systems (Griffing, 1956a and 1956b). RESULTS Symptoms and Pathology On September 19, 1963 severe respiratory symptoms were observed in chickens in house B6 (Fig. l). 1. Immediately on entry into the vestibule of the pen almost continuous coughing and sneezing sounds could be heard with no difficulty. There were approximately 100 ten-week-old birds in the pen. 2. On entry into the pen many of the birds were coughing and sneezing and some were gaSping and appeared cyanotic, especially after being handled. Rales could often be heard. 3. Clinically there was a pronounced con- junctivitis, but no nasal discharge, even when pressure was applied to the infra-orbital sinuses. A. There was less than 9% mortality but the morbidity was estimated at 90-100%. The birds that died during the acute and convalescent stages had a catarrhal tracheitis over the entire length of the trachea with an increase in mucous exudate in the lumen. No changes were seen in the air sacs. 5. On September 20, 1963 the only other pen of birds in the same building had the same symptoms. 3h 35 Fewer birds were so acutely affected. 6. About a week after the first symptoms were observed all birds appeared to have recovered. In one pen, one bird gasped for many weeks afterward. It was thought that the bird had an unrelated res- piratory or circulatory lesion. 7. These birds were not yet laying. However, when the same disease occurred elsewhere on the premises, there was a sudden severe dr0p in egg production which did not return to normal for over one month. No misshapen eggs were layed. A tentative diagnosis of infectious bronchitis was made and material was taken from one bird of this group for virus isolation. Histopathology. The few birds that died during this initial outbreak and during the vaccination experiments had typical histOpathological changes associated with 18. These included: 1. A slightly thickened mucosa with edema and congestion. Sometimes there was disruption of the mucosa and loss of cilia. 2. A marked infiltration of lymphocytes and macroPhages in the sub-mucosa. Occasionally heterOphils were also present. 36 3. Inclusion bodies were not found. Virus Isolation and Passage 1n the initial isolation, all seven embryos inoculated with the tracheal extract alone died within 2h hours due to bacterial contamination. However, of the 8 inoculated with the extract to which antibiotic had been added, 2 embryos had died by the fourth day. At this time 2 embryos were taken as donor material for passage. By the seventh day after inoculation, 2 more had died. The last 2 died on the ninth day after inoculation and had the following 1eSions: 1. They were obviously dwarfed or stunted. This could be seen on candling. 2. The embryos were curled and held in a firm compact ball by the closely adherant and thickened amniotic membrane. 3. The down was underdeveloped, i.e., clubbed. A. On necr0psy, the embryos had a severe generalized congestion of the liver and lungs and an accumulation of urates in the mesonephrons and ureters. The above signs are typical of those encountered when embryos are inoculated with IE virus in low egg passage. 37 The mortality pattern in all passages of the virus is shown in Table A. Between the lhth and 18th passages an attempt was made to ”clone” a more rapidly growing and more lethal virus. For each passage the more stunted embryos were taken for harvest and the allantoic fluid was diluted between -1 10 and 10-3. Passages 19 through 23 gave a more uniform mortality than previous passages. Effect of Passage To confirm the differences between the 8th and 23rd passages noted above these two preparations were used for inoculation of line 6 and line 7 embryos as described under ”Studies on Genetic Resistance to RPL-IBV.” In the cumulative mor- tality graph (Fig. 3), the 23rd passage of virus gave consistently earlier mortality than the 8th passage when assayed in line 7 eggs. When analyzed statistically by the method of Mantel and Haenzel (1959), the difference is significant at P=0.05 when the virus was assayed in line 7 (Table 16). For line 6 embryos, there was no difference be- tween the two virus preparations. The titer of the 23rd passage of virus was slightly higher than that of the 8th passage (Table 16). This effect has also been included in the statistical analysis. TABLE A. M Mortality pattern on isolation and passage of RPL-IBV every 3-A days in 10 day old Line 151 embryos ‘—_ No. lesions/ No. dead/ Passage No. Dilution Total Total 1. 10" dilution 6/6 u/6 2. Undiluted 3/6 5/6 3. “ 3/5 4/5 A. ” 2/3 3/3 5. ” All used as donors 6. H lO/Ih IZ/lu 7. ” 19/19 19/19 8.* M 13/16 Ih/Ie 9. “ h/A IO. ” 6/6 11. " 10/10 10/10 12. H 11/12 13. " 8/10 in. 10'3 dilution 17/18 15. 10-2 H 15/18 16. 10'2 " 17/25 17. 10'3 " 7/11 18. 10" H 16/16 19. Undiluted A/A 20. " Ih/Iu 21. ” All used as donors 22. “ 13/13 23.* " 7/7 * Virus stock prepared. 39 Fifteen embryo passages of the virus and some selection has produced a preparation which can kill embryos sooner i.e. it can partially overcome the factor in line 7 embryos which delays the mortality. PrOpagation and Storage For the preparation of a virus pool, ten-day- old embryos were inoculated with decimal dilutions of the 23rd passage of virus. Ten embryos were used for each lot. The allantoic fluid was harvested after 3 days, pooled by lot and later titrated. The highest virus yield came from embryos inoculated with a 3.7 and a u.7 10g dose of virus (Table 5). With higher doses of virus there was a decrease in titer. This pehnomenon was described by Von Magnus (l95b) and reviewed by Hirst (1959) for influenza virus where the input multiplicity was very high. RPL-IBV decreased 0.8 lags in activity during 6.5 weeks at -75°C (Table 6). Candling Methods By the 8th passage it became evident that almost all dead embryos had typical lesions of 18. The number of positive cases was not appreciably increased by examining the embryos that lived until the 20th day of incubation. In addition some of #0 TABLE 5. PrOpagation of virus in the allantoic sac Dose inoculated -0.7 -l.7 -2.7 -3.7 -A.7 -5.7 Yield of virus 7.0* 7.0 7.0 7.6 7.6 7.5 ______________J * log titer. TABLE 6. Loss of virus on storage at -75°C Date of titration 12/17/63 l/3/6h l/ll/6A l/3l/6# No. of weeks stored 0 2.5 3.5 6.5 Titer of virus 7.6 7.1 6.9 6.8 : 4—-__=._-J #1 these survivors were so stunted that the stunting was easily visible on candling and they would certainly have died before hatching. The following arbitrary criterion was employed. Any embryos definitely stunted and lacking motility on candling at the 10th day after inoculation were included with those that died between the lst and 10th days and were considered as positive reSponses. A similar method of reading reSponses was used by Hitchner _£__l. (196A) and gives results equivalent to the embryo inSpection technique used by earlier workers. To test the accuracy of the method of scoring positive reSponses one of the routine constant serum-decreasing virus neutralization tests was utilized. All eggs were candled and the positive responses were recorded. Each egg was Opened and the living and dead embryos were examined. The results are presented in Table 7. Some embryos died without lesions and some embryos had lesions that were not detected by candling alone. The overall agreement was good so the method using candling alone was used for all subsequent experiments. Identification of the Virus 1. Symptoms in affected birds. A2 TABLE 7. Comparison of endpoints of a serum-virus neutralization using different methods of reading positive virus responses =—WW Serufi: Serum Method of reading alone 1 (Mag) 2 (Neg) 3 (Neg) 4 (Pee) Lesions alone 7.5 7.7 7.3 6.6 3.2 Dead and lesions 7.7 7.7 7.8 6.8 3.9 Candling 7.7 7.7 7.5 6.8 3.6 TABLE 8. Fluorescent antibody results on tracheal scrapings of two birds with signs of ID Trachea from: Fluorescent Results Remarks antiserum Bird 1 IB +++ Clear specific ILT + Some non-specific NDV - Negative Bird 2 IB +++ Clear specific 1LT - Negative NDV - Negative 43 2. Fluorescent antibody. Half of the trachea used for virus isolation (Table 8, Bird 1) and the trachea of a second bird that died 2 days later (Table 8, Bird 2) were used for fluorescent antic body studies (which were positive for IE). 3. Lesions in embryos. u. CAM inoculation. The 23rd passage of virus was used to inoculate 26 eggs on the CAM. The absence of pocks on the 8th day post inoculation eliminated the possibility that 1LT or fowl pox were present in the inoculum. 5. Hemagglutination. The virus did not cause direct HA. Trypsin treated virus harvested on the 3rd day from both the 7th and 10th passages ag- glutinated chicken erythrocytes which is a characteristic of IE virus (Corbo and Cunningham, 1959; Cunningham, 1960). Uninfected allantoic fluid treated with trypsin did not cause HA (Table 9). In the 7th passage, third day harvest, there was no agglutination at 1/t and only 2+ at 1/8. This appears to be a "prozone" phenomenon which was not described by Garbo and Cunningham (1959). 6. Hemagglutination inhibition. Serum from one of the 2 birds submitted to Dr. Newman on January 28, 1963 had an HI titer of 1280. However, uh TABLE 9. Hemagglutination using trypsin treated RPL-IBV 1 1 1 1 1 1 Virus Source Treatment A '8 .76 32 6A 128 I I w 7" 3* '** " ' ' 1 0- 3 - - - - 10-6 - - - - not done 0-3 ' ' ' “ 0-6 - - - - 7‘3 T - 2+ 3+ 4+ 3+ + 10“3 . T A+ A+ A+ A+ A+ 3+ 10-6 T - - - - - - 0-3 T - - - - - - 0-6 T - - - - - - Titer of 7-3 is 1:65 and of 10-3 is 1:128. * 7-3. Inoculum was 7th passage of RPL-IBV. Harvested 3rd day post inoculation. ** - No agglutination. + 25% 2+ 50% 3+ 75% h+ 108% agglutination. on the “5 an additional 6 samples submitted later were negu ative. There was no H1 in 21 additional samples (Table 10). This was indicative that the birds had not been infected with ND. NO explanation could be found for the one positive serum. 7. Serum-virus neutralization tests. On the basis of the results Of neutralization tests by both the constant serum and the constant virus methods (Table 11), the following conclusions can be drawn: a. RPL-IBV was not neutralized by hyperm immune sera against ND, 1LT, or avian en- cephalomyelitis viruses. b. The antiserum against "gallus adeno- like" (GAL) virus (Sharpless and Jungherr, 1961; Sharpless 2£_él., 1961) prepared at the RPRL had antibody activity against this agent. On the other hand, the antiserum pre- pared in New York had no activity even though it had a high titer against GAL. Thus RPL~ IBV does not cross-neutralize with GAL virus and the presence of activity in the above serum indicated inadvertent infection with RPL-IBV. At the time the antiserum was pre- pared (1958), RPL-IBV had not been reOOgnized. #6 TABLE 10. HI titration of sera from survivors of RPL-IBV infection using 10 HA units of killed NDV (USDA) (A) Serum tit ration Serum no. Serum dilution 1 __I. .1 .1 .1 .1. _L. .1. ____I ___.I 5 10 20 A0 80 160 320 6A0 1280 2560 1. +* + + + + + + + + + 2. + + + + + + + + + + 3. + + + + + + + + + + A. + + + + + + + + + + 5. + + + + + + + + + + 6. + + + + + + + + + + 7. + + + + + + + + + + 8. + + + + + + + + + + 9. + + + + + + + + + + 10. + + + + + + + + + + 11. + + + + + + + + + + 12. + + + + + + + + + + 13. + + + + + + + + + + 14. + + + + + + + + + + 15. + + + + + + + + + + 16. + + + + + + + + + + 17. + + + + + + + + + + 18. + + + + + + + + + + 19. + + + + + + + + + + 20. + + + + + + + + + + 21. + + + + + + + + + + 1339(-) + + + + + + + + + + 2709(+) - - - - - - - - - + Saline - - (B) Antigen titration Antigen dilution 1 I I .1 .1 I I .1. 2 1 2 11 ‘3 I6 32 ET 128 256 + + + + + + - - - - * + Greater than 50% agglutination. - Less than 50% agglutination. 47 TABLE 11. Neutralization of REL-18V by sera from various scurres m 1 NO. of Constant Constant Source Host Serum against Sera Virus Serum NI NADL* Turkey NOV 2 - NADL Chicken NOV 2 - NADL Chicken LT 2 - RPRL Chicken GAL 1 NY Chicken GAL l - CONN Chicken AE 3 - NAOL Chicken Mass IBV 2 - ISU Chicken Mass IBV (726) l - 1.2 MSU Chicken Mass IBV (#1) l - <1.8 MSU Chicken Mass IBV (41) l - <1.5 NAOL Chicken Conn IBV l - MSU Chicken Conn IBV (A6) 1 - <1.5 ISU Chicken IBV 609 l - <1.0 ISU Chicken IBV 97 2 i 2.3 RPRL Chicken RPL-IBV l + 5.6 RPRL Chicken RPL-IBV l + 9.9 RPRL Chicken RPL-IBV l + A.3 RPRL Chicken Negative 2 - <1.8 NAOL Chicken Negative 1 - * NADL - National Animal Diseases Laboratory, Ames, Iowa. RPRL - Regional Poultry Research Laboratory. East Lansing,Mich. NY - American Cyanamid, Pearl River, New York. CONN - University of Connecticut, Storrs, Conn. ISU - Iowa State University, Ames, Iowa. GAL - Gallus Adena-like virus. AE - Avian Encephalomyelitis. c. RPL- BV was neutralized by antiserum against Mass IBV (IBV 41 or Iowa 726), Conn IBV (IBV A6), or Iowa 609 isolate (Hofstad, 1958). d. RPL—IBV was neutralized by antiserum against Iowa 97 isolate (Hofstad, 1958) and by homOlOgous antiserum. Thus RPL-IBV is most closely related to Iowa 97. Constant Serum versus Constant Virus To compare these two methods sera from 6 im- mune and 5 non-immune birds were tested in parallel against the same virus preparation by both methods (Table 12). Even though the titers did not exactly agree, all the positive sera were positive by both tests and all the negative sera were negative by both tests. Because the constant virus method was less eXpensive, requiring fewer eggs per serum tested, it was used for the majority of the screening tests. Epizootiology Using the constant virus method, 18h sera were tested for antibody against RPL-IBV (Table 13). The layout of the buildings and isolation areas at the RPRL is given in Figure 1. The results from #9 TABLE 12. Constant serum versus constant virus titrations* Serum No. Status Constant Serum Constant Virus Test I 3143 Immunized 5.7** h.5 3136 ” 4.9 5.1 3137 ” 4.3 3.5 3105 Not immunized <1.8 <3.l 310“ ” . 3 Response 5"——T—‘555_55_Ig_9 €666 . ,00 5555555553 {Izzzggizz r -— * One line 6 embryo died on the 3rd day after a -h log dose of the 8th passage of RPL-IBV was inoculated into the ailantoic sac, etc. 60 i- Line 6 .I>. O T 20% Percent response ——> 60 I- Line 7 fl .5 :9 I 20- 0 Ill-Em 6 1 2 3 4 5 Percent reSponse /% 9 10 Days post—inoculation —>- FIG. 2. Percent response of cmbn'os of two inbred lines of chickens to RPL—IBV (8th and 23rd passages combined). 58 of each passage has been combined and plotted against the days after inoculation. When the cumulative mortality in these lots is plotted against the days post inoculation (Fig. 3), the curve for the mean of the virus preparations in line 6 embryos is straighter than in line 7 em- bryos which is concave upwards. The X2 indicates that the difference between lines 6 and 7, using either the 8th passage or the 23rd passage or combining the data, is highly sig- nificant (Table 16). The 23rd passage of virus killed embryos earlier than the 8th passage, eSpecially in line 7 (Fig. 3), and also had a slightly higher titer (Table 16). These differences have been discussed under "Effect of Passage of the Virus." Experiment II The experimental design and the embryo mor- tality on each day after inoculation is presented in Table 17. The daily mortality in the 6 lots of embryos inoculated with the highest doses of virus has been pooled and is presented in Fig. U. Embryos of line 6X6 died earlier and those of line 7x7 died later. Line 15IX151 was intermediate between these two, some dying early and some late. 100 " Inoculum Passage of RI’L~-1BV 8“ ' 81h 23m . \. . . ., . 7‘ x .\ A In line 6 cmbI) es 0 0 in llnc 7 embryos e (D ' E g. ' I- 9.". E G) E e C. E 40 P- CU E 5 U 20 F X 0 0 0 2 ' 4 6 8 10 Days post-vinoculation —> FIG" 3" (,Zumulutive percent response of two inbred lines of chickens to two passage levels of RPlp-IBV. 60 TABLE I6. Titers and statistical analysis of the RPL-IBV titrations in experiment I (A) Titers of RPL-IBV Virus Line 6 Line 7 8th passage 7.5 7.2 23rd passage 7.6 7.6 (B) x2 for the difference between line 6 and line 7 8th passage _lS.9h* Data Combined 28.76 23rd passage ll.8| (C) x2 for the difference between the 8th and 23rd passages of virus Line 6 0.06 Line 7 5.23 * One degree of freedom in every case. At p.01 x2 - 6.6h At 9.05 x2 ' 3'8“ 61 M ?m ??????m ???m. ???m ?? ???m ?????? N: a an SSESW .o o _ o_o_o_wo_o_ o o N NESSSSS 3.838. . _ _ _o__NN_ NN. _ o. m _ N N N a N N m N m N N _ m .u .0 Na... .NNN. N _ _ am _ ____ .m N _ N __ N w. 0 n N _ __ _ _.m N ow. _ ._ ___ N:N_ mm. U ___m __ .a m _ a .. .n: _. __Nmm_ m __ N M Na _NmN.3 N _ _ _ _ _ _ _ o m-m-N-o-m..:-TN-T o T??? ff??? 38 o m- m- .T o- m- .1 n- N- T o x um. 0:3 o x N2: o x c on“; ll mmcmums anacu _o__m_p cm >m—u4mx no comuMcum» .m. u4mo~uamz to omamnwn puma new up «new me. .u m coumm xmv vcouom ozu co voNu moxcaeo o x o ocN. each « l'l ll?|??.??????2llh ???22?|?m? a o _ win _ .o N _ a o _ o m SmISBW o . _ wIwISSBw. o. «22.8.. _ _ a . N _ N _ N _ _ o. . m . N N m o m a _ _ N a _ _ N m mm s N_NNN_ .Nnm_: N_.N_ mm. s _ N _ _ _ _ _ N N _ N “H w _ _ _ N m _ _ N N o m. N _ . _ _ N _ m mp w _ m N _ _ _ _ _ a m a _ _ _ N _ N _ m _ . _ a m _ _ _ _ N a N _ _ _ a m-N-N-a-m-s-N-N-T o m-m-N-o-m-s-N-N-T 38 o a- N- N- o- m- .1 N- N- .- ~m_ x um. 0:: —m_ x s 0:3 Nm_ x o «cm; Acuacmucoua.N_ m4m

- FIG. 4. Po rcent response of embryos of diallel cross matings to RPL—IBV. 65 The cumulative mortality figures are shown in Fig. 5. In each of the 9 graphs an arbitrary point has been placed in the same position relative to the ordinate and abscissa in each graph. Wherever line 7 is one of the parents of the embryos, the curve passes beneath this point and in all other cases it passes over this point. In the former the embryos died later than in the latter. Statistical analysis by the method of Griffing (1956) yielded the information in Table 18. The difference between the crosses (i.e. between each of the 9 different matings) is significant both at the 5th and at the 7th day post inoculation. The most obvious difference was between the 6X6 and 7X6 matings (Fig. 5). In the former the graph is con- cave downward whereas in the latter it is concave upwards. The_effect of dose was significant as was expected. The higher the dose of virus, the shorter the latent period. The titers of RPL-IBV calculated from mortality to the 10th day post inoculation, are similar in all crosses (Table 19). The general combining ability (GOA) is a measure of the differences between the lines of chickens, irrespective of influences of other lines 66 Line 6 male Line 7 male Line 151 male Line 6 female at * x 0 Line 7 0 0 female il- * 3|: 3 5 100 J‘- 2 ‘5 3. Line 151 g 50 4 female .2 ' *5 «- a: at '5 S O O x/ O O 5 10 Days pos t-inoculation —>- FIG. 5. Cumulative percent response of embryos of diallel cross matings to RPL-JBV. For the symbols *, X and 0 see the text. 57 TABLE l8. Results of statistical analysis of diallel cross matings 5th day Sources Degrees of Freedom F Crosses 8 7.80** Lots (doses) 5 8.ll** Lots X crosses 40