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I . . 3 3 . . .3 3 . .......Z.......3. . . . 3 . . . 3. 3 3. . . 3 . ..3 3 . .3.“...2 . . . . 4 . 3 .. . 4 . . 3 .. . . . . 3 . . .3 l . 4. 4 b4 .3 . . . . 33 3 . 3 . . 3 3 . . 3 . «.44.4...3.........34 . 3.3,. 3.43.. . .143 ..4. x u. 33 . .3 1.43 3 .32.}. ...... 431...... .Nr...n3.x.~r1....¢3r .... 46...: 44 Tull. . . ...... 5.5.34.4...4394 >4. .....3. ...; 34%....41...“ ....wnflfli . .3....3333..... 33...v.. .. .3.. ....h: ...... ‘rnEim This is to certify that the thesis entitled _ _ ' THE EFFECT OF PASSIVELY TRANSFERRED IMMUNOGLOBULINS ON MAREK'S DISEASE presented by George Harvey Burgoyne has been accepted towards fulfillment of the requirements for __Ell._11.._degree in WP lant Pathology V” {Cg (527 g): ' "Ur/gut? Major professor ' 0-7639 ABSTRACT THE EFFECT OF PASSIVELY TRANSFERRED IMMUNOGLOBULINS ON MAREK'S DISEASE BY George Harvey Burgoyne The effect of passively transferred antibodies, present only during the first three weeks after hatching, on the susceptibility of newly hatched chicks to Marek's disease (MD) was studied by comparing the mortality, appearance and incidence of lesions, and levels of recoverable virus in chicks with and without antibody. Maternal antibody was found to reduce the sus— ceptibility of newly hatched chicks to Marek's Disease virus (MDV) infection. In each of three lines of chickens, antibody positive progeny had lower mortality, fewer lesions, lower levels of recoverable virus and higher incidences and titers of actively acquired antibody than maternal antibody negative progeny. Maternal antibody was found to provide in Kilo neutralization indices (NIs) of approximately 1.0 and 2.0 against cell associated and cell-free MDV respectively. George Harvey Burgoyne Similar levels of protection could be obtained by the administration of immune gamma globulin. The pathogenesis of MDV was examined in antibody positive and antibody negative chicks. Antibody was found to delay the onset of lesions and assert a protective effect on development of lesions in the bursa of Fabricius. Virus appeared in various tissues at the same time, however, mean virus levels were always lower in antibody positive birds. Hyperimmunization of dams did not increase anti— body titers in these dams nor did it reduce the suscepti- bility of progeny. The administration of antibody to the herpes virus of turkeys (HVT) was found to confer protection against MDV infection in newly hatched chicks. The progeny from HVT vaccinated dams were less susceptible than progeny of non—vaccinated dams, however, this decrease in susceptibility could not be accounted for by humoral antibody. * EFFECT OF PASSIVELY T IMMUNOGLOBULINS ON MAREK'S DISEASE BY George Harvey Burgoyne A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1972 To my wife Eleanor ii ACKNOWLEDGMENTS I wish to express my sincere thanks to Dr. Richard L. Witter, Associate Professor of Pathology, Dr. Ben R. Burmester, Professor of Microbiology and Director of the United States Regional Poultry Research Laboratory, and Dr. Everett S. Beneke, Professor of Mycology for their encouragement and assistance in the research work and preparation of this thesis. I would also like to express my appreciation to Dr. William Drew, Chairman of the Department of Botany, Dr. Clifford Pollard, Professor of Plant Physiology and Dr. H. Graham Purchase, Assistant Professor of Microbiology for their aid and advice in this work. I wish to thank Mr. Howard A. Stone, Geneticist at the United States Regional Poultry Research Laboratory and to all members of the genetics department for providing me with the experimental lines of chickens used in the work. I wish to thank Miss Linda Offenbecker, Mrs. Terri Fewless, Mrs. Cecyl Fischer, Mrs. Elizabeth Hood, and Mr. William Payne for their technical assistance. Use of the Michigan State University computing facilities was made possible through support, in part, from the National Science Foundation. iii DEDICATION . ACKNOWLEDGMENTS LIST OF TABLES . LIST OF FIGURES TABLE LIST OF ABBREVIATIONS INTRODUCTION LITERATURE REVIEW Etiology . Gross Pathology Microscopic Pathology Host Range Transmission Systems for Virus Isolation Serology Immunity . MATERIALS AND METHODS Experimental Chickens Cell Cultures Virus Isolation . Assay of MDV in CK Cell Cultures . OF 0 C ONTENTS and Assay o o o Assay of MDV in DEF Cell Cultures . Viruses . Hyperimmunization o Antibody Determinations Gamma Globulin Preparations Diagnosis of Marek's Disease Statistical Analysis EXPERIMENTAL DESIGN Experiment Experiment Experiment Experiment I . II III IV iv Page ii vi viii Page RESULTS . . . . . . . . . . . . . . . 63 Status of Experimental Dams and Chicks in Experiments I and II . . . . . . . . . 63 Experiments I and II: Susceptibility of Chicks from Naturally Exposed, Hyperimmunized and Control Dams to Mortality and Lesions of MD . . 67 Influence of Parental MDV Exposure on Mortality and Lesions in Chicks after IA or Air Challenge . . . 67 Experiment III: Simulation of the Protective Effect of Maternal Antibody by the Administra- tion of Immune Gamma Globulin . . . . . 76 Development of a Short Term in Vivo Assay for MDV . . . 81 Experiment III: Protection of Chicks by Maternal Antibody and Administered Immune Gamma Globulin . . . . . . . . . . . . . 84 Experiment IV: Influence of Antibody on Various Host Responses to Infection . . 88 Experiment IVa: Comparison of Chicks with and without Maternal Antibody . . . 88 Experiment IVb: Influence of Administered Immune Gamma Globulin on MDV Infection . . . 103 DISCUSSION . . . . . . . . . . . . . . 113 Influence of Maternal MDV Antibody on MDV Susceptibility of Progeny . . . . . . 113 Hyperimmunization of Dams as a Means of Decreasing the Susceptibility of Progeny . . . 117 Influence of HVT Vaccination of Dams on the Susceptibility of Progeny . . . . . . . . ll9 SUMMARY . . . . . . . . . . . . . . . 121 LITERATURE CITED . . . . . . . . . . . . 123 APPENDIX . . . . . . . . . . . . . . I. Table 1. 10. 11. 12. 13. LIST OF TABLES Relation of experiments to thesis objectives . . . . . . . . . . . Designation of 72 and 15I parental groups of Experiment I . . . . . . . . . . Basic design of Experiment I . . . . . . Designation of line B parental groups of Experiment II . . . . . . . . Basic design of Experiment II . . . . . Basic design of Experiment III . . . . . Design of Experiment IVb . . . . . . . Antibody status of experimental dams and progeny of lines 72 and 151 . . . . . Antibody status of experimental dams and progeny of line B . . . . . . . . Mortality, gross and microscopic lesions in progeny of 72 and 15I dams which received different MDV exposures . . . Least squares analysis of variance of mortality, gross and microscopic lesions in progeny of 72 and 151 dams of different virus exposures . . . . . . . Comparison of virus isolation and antibody titers in progeny surviving 12 weeks from 15I dams of different Virus exposures . . Mortality and gross lesions in progeny of line B dams receiving different virus exposures . . . . . . . . . . vi Page 46 51 52 56 57 60 61 64 66 68 70 71 73 Table 14. 15. 16. _17. 18. 19. 20. 21. 22. 23. 24. Least squares analysis of mortality and gross lesions of progeny from line B dams of different virus exposures . . . Statistical evaluation of differences between treatment groups of line B progeny . . . Induction of serum antibody levels by the administration of immune gamma globulin . Development of an in Vivo quantitative assay for MDV: EvaluaEIon of sacrifice time, response and source of chicken . . . . Comparative sensitivity of candidate methods for the in Vivo quantitation of MDV . . . Susceptibility of line 100 chicks to graded doses of cell associated MDV following administration of immune gamma globulin . Susceptibility of line 100 chicks to graded doses of cell—free MDV following administra- tion of immune gamma globulin . . . . . Sequential observation of microscopic lesions in MDV exposed chicks of different parental antibody status . . . . . . Sequential observations of virus and antibody in MDV exposed chicks of different parental antibody status . . . . . . . . . Sequential observation of microscopic lesions in MDV exposed chicks with natural antibody, administered antibody and without antibody. Sequential observation of virus isolation, antibody and viral antigens in MDV exposed and unexposed chicks with natural antibody, administered antibody and without antibody . . . . . . . . . Page 74 75 77 82 83 85 87 89 104 106 107 Figure l. 10. 11. 12. 13. 14. 15. 16. LIST OF FIGURES Plastic rearing isolators . . . . . . . Stainless steel isolators . . . . . . . AGP test for MDV antibody . . . . . . . Fluorescent antibody test for MDV antibody . Gross nerve lesions associated with MD . . Gross visceral lesions associated with MD . Time relationships between serum collections, immunizations and egg collections using dams of line 72 . . . . . . . . Time relationships between serum collections, immunizations and egg collections using dams of line 151 . . . . . . . . Time relationships between serum collections, immunizations and egg collections using dams of line B . . . . . . . . Decay of passively acquired serum antibody . Normal bursa of Fabricius and bursa with minor MDV lesions . . . . . . . . Normal bursa of Fabricius and bursa with severe degenerative MDV lesions . . . . Comparison of mean bursa weights of progeny from dams with and without antibody . . . Normal nerve and nerve with MDV lesion . . Normal gonad and gonad with MDV lesion . . Normal liver and liver with MDV lesion . . viii Page 22 25 33 37 40 42 47 49 54 79 91 93 95 97 99 101 .-' '4 ‘ V, 1.41II,1'. .- .- _ a. r-.- =._ .-_: _' . “...-x in . Feather follicle of MDV infected chicken 'E’fi’l' . . with lymphocytic infiltration and ' Iii-"ii 5-H; inclusion bodies . . . . . . . . . . 108 --_" 18. Comparison of mean bursa weights of progeny with maternal antibody, antibody from administration of gamma globulin and without antibody . . . . , . . . . . 111 ix LIST OF ABBREVIATIONS AGP - agar gel precipitin Bursa - bursa of Fabricius CAM - chorioallantoic membrane CEF — chick embryo fibroblast CK - ckick kidney CPE - cytopathic effect DMSO — dimethyl sulfoxide DEF - duck embryo fibroblast FA - fluorescent antibody HVT — herpes virus of turkeys IA - intraabdominal line 6 - a Regional Poultry Research Laboratory inbred line of chickens resistant to MD and lymphoid leukosis tumor development but susceptible to virus infection LPD - dose required to produce lesions in 50% 50 . . subjects at r1sk. JM - isolate of MDV from M. Sevoian, Univ. of Massachusetts, Amherst, Mass. MD — Marek's disease MDV — Marek's disease virus nm - nanometers NIs - neutralization indices PBS — phosphate buffered saline PFU — plaque forming unit - a buffer containing sucrose, phosphate, glutamine and bovine albumin - white blood cell - weight/volume xi INTRODUCTION Marek's disease (MD) is economically the most important disease of chickens. Losses due to deaths and ,condemnations have been estimated at $150-200 million annually in the United States. Marek's disease is a neoplastic disease character— 'ized by infiltration and proliferation of lymphoid cells in the nerves, eye, skin, skeletal muscles, and visceral organs. The etiologic agent is a highly cell-associated herpes virus, which can be transmitted from infected to susceptible birds by inoculation of cellular material or by contact exposure. Marek's disease virus (MDV) is highly infectious and chickens in most flocks become infected and develop antibodies which persist for indefinite periods of time. Maternal antibody, i.e., antibody passed from the dam to her progeny, is known to decrease the suscepti— bility of young animals to other diseases. It has been reported that progeny from dams with MDV antibody acquired by either natural infection or multiple inoculation (hyperimmunization) were more resistant to MD than progeny of antibody-free dams but it is not known whether this resistance is actually due to materanl antibody. 1 (b) examine how various aspects of MDV infection and clinical disease differ in birds with and without maternal antibody, (c) determine if hyperimmunization of dams increases the resistance of progeny to MD, (d) determine if infection of dams with a serologically related, non- pathogenic herpes virus from turkeys will provide pro— tection to progeny and (e) seek some insights into the possible mechanisms for the observed differences in susceptibility. LITERATURE REVIEW Marek's disease is a world wide important disease of chickens characterized by neoplastic lesions occurring in the peripheral nerves and visceral organs (Biggs and Payne, 1967). Marek (1907) first described the disease as a polyneuritis in a case report of four adult chickens. Since then it has been referred to as Marek's disease, neural lymphomatosis, fowl paralysis, range paralysis, Marek's paralysis, acute leukosis, ocular lymphomatosis, neuritis and visceral lymphomatosis. Etiology Early workers had difficulty in transmitting MD successfully (Pappenheimer et_al., 1926; Pappenheimer gt_gl., 1929; Blakemore, 1939; Durant and McDougle, 1939; Durant and McDougle, 1945). Not until Sevoian 35431. (1962) and Biggs and Payne (1963 and 1967) showed the need for intra-abdominal inoculation of susceptible one—day- old chicks with intact viable cells did transmission meet with regular success. The independent discovery by Churchill and Biggs (1967), Solomon et a1. (1968), and Nazerian et a1. (1968) of a cell associated herpes Virus associated with Marek's disease was a major breakthrough. Circumstantial evidence accumulated rapidly (Witter et_31., 1969a; Biggs §t_al., 1968; Ahmed and Schidlovsky, 1968; Bankowski e£_§1., 1969; Calnek and Madin, 1969; Eidson gt_31., 1969; Sharma and Kenzy, 1969; Spencer, 1969; Noboru gt_a1., 1969; Naito gt_al., 1969) incriminating MDV as the cause of MD. Experiments showing the site of viral maturation to be the skin, more specifically the epithelium of the feather follicle and transmission of the disease with cell—free extracts (Calnek eE_al., 1970a; Nazerian and Witter, 1970; Purchase, 1970) proved MDV to be the etiological agent of MD. The virus in its enveloped infectious form is 150- 400 nm in diameter (Calnek et_al., 1970c; Nazerian and Witter, 1970). The capsid, 85-100 nm in diameter, is icosahedral and has 162 cylindrical capsomers (Ahmed and Schidlovsky, 1968; Nazerian et_al., 1968; Calnek §E_31., 1970c; Epstein g£_a1., 1968). The nucleoid of the complete particle is a dense, centrally located structure 65 nm in diameter. Virus is seen only rarely in tumors, affected nerves or other tissues (Schidlovsky §E_§1., 1969; Calnek et_31., 19700; Ubertini and Calnek, 1970) of infected chickens but is common in the nuclei and cytoplasmic inclusions of the feather follicle epithelium (Calnek et al., 1970a; Nazerian and Witter, 1970). The highly Cell associated nature of the virus categorizes it as a B type herpes virus (Melnick et al., 1964; Wilner, 1969; Churchill and Biggs, 1967; Nazerian et al., 1968; Churchill, 1968; Lee et al., 1969). Gross Pathology When infection proceeds to disease, the clinical signs (Jungherr and Hughes, 1965; Sevoian and Chamberlain, 1964; Biggs, 1966) typically include asymmetrical, pro- gressive paresis and later complete paralysis of one or more of the extremities. Symptoms vary from bird to bird depending on which nerves are involved. Any or all of the following may be observed: drooping of the wings, lOWered head with torticollis, dilatation of the crop, gasping, ataxia, lameness and paralysis. A characteristic attitude is one in which one leg is stretched forward and the other back as a result of paralysis. Enlargement of the peripheral nerves, spinal roots or ganglia and/or visceral tumors are commonly found on gross examination. Nerve lesions are found in certain autonomic nerves as well as the more obvious nerves and plexuses (Goodchild, 1969) and most cases can be diagnosed by examining the following: celiac, cranial, mesenteric, brachial and sciatic plexuses, nerve of Remak, vagus and the greater splanchnic nerve. The plexuses of the sciatic and brachial nerves are more often enlarged than their respective trunks. Affected nerves lose cross striations, become gray or yellow and swollen. Localized or diffuse enlarge- ments can cause the affected portion to be two, three or more times normal size. Tumors may be found in any of the visceral organs; most commonly affected are the gonads, liver, spleen, heart, and kidney. The lungs, mesentery, adrenals, pancreas, proventriculus, intestine, iris, muscle and skin) may also be affected. Organs may be several times normal size with a diffuse grayish discoloration or focal, nodular lesions may be seen. Microscopic Pathology The lesions found in the nerves are the most con- stant finding in MD. Payne and Biggs (1967) describe nerve lesions of three types: A, B, and C. Type A lesions consist of small, medium, and large lymphocytes, a few plasma cells and degenerating lymphoblasts (MD cells). Large lymphoblasts often predominate and edema, myelin degeneration and Schwann cell proliferation may occur. Type B lesions are characterized by edema with only a few small lymphocytes present. This type of lesion occurs mainly in older birds or in chronic cases. The type C lesion is very mild, consisting of a light infiltration of plasma cells and small lymphocytes. Lesions of this type appear in clinically normal birds. An encephalitis with perivaScular cuffing by lymphocytes and areas of gliosis and endotheliosis may be found in the brain.. In the eye mononuclear infiltration of the iris is found. Occasionally the muscles of the eye are also involved. Lymphoid tumors of the visceral organs and skin consist of large accumulations of pleomorphic lymphocytes. The lesions resemble the A type lesions described for nerves. In the liver, infiltration proceeds from the portal tracts replacing large areas of the normal hepatic tissue with lymphocytes. In the heart, lesions may be confined to accumulations on the epicardium or infiltra- tions between the myocardial fibers may occur. Skin lesions may consist of small focal areas in the subcutis or may involve all the layers beneath the stratum basale. Both degenerative and proliferative lesions may occur in the bursa of Fabricius and thymus (Purchase and Biggs, 1967; Jakowski, et_a1., 1969). Degenerative changes in the bursa consist of cortical and medullary atrophy, cyst formation and reticular replacement. In the thymus the cortex may be absent and the medulla may lack thymocytes. Proliferative changes in the bursa and thymus consist of interfollicular infiltrations of pleomorphic lymphoid cells. In the feather follicle, degenerative changes are present in the intermediate and transitional layers (Nazerian and Witter, 1970). Cells are frequently ' vacoulated and contain cytoplasmic and Cowdry type A inclusion bodies. Near the stratum corneum the nuclei of cells are small and basophillic or have undergone karyorrhexis and are not visable. In the most super— ficial layer the cells disintegrate into fragments. Host Range Although chickens are the principal host, natural infection with MDV also occurs in quail (Kenzy and Cho, 1969). Turkey poults were found susceptible to experi- mental inoculation (Sevoian et_§1., 1963) and visceral tumors were produced in turkeys (Witter et al., 1970b) although no virus or antibody could be detected. Lesions resembling those of MD have been reported in the duck, goose, canary, budgeriger, swan and pheasants (Jungherr, 1939; Cottral and Winton, 1953; Wight, 1963). Successful transmission has also been reported in the pheasant (Harriss, 1939; Johnson, 1941) although no agent was identified. Baxendale (1969) could not detect natural antibody in pigeons, starlings, yellow hammers, sparrows or pheasants but did demonstrate antibody and re—isolate virus from ducks after laboratory infection. Attempts to infect mammals have not been successful (Churchill, 1968). Marek's disease Virus propagates well in chick kidney cells (Churchill, 1968; Churchill and Biggs, 1967; Witter §t_al., 1969b) and in duck embryo fibroblasts (Solomon gt_al., 1968; Witter et_al., 1969b). Chick embryo fibroblasts have been reported to be susceptible ~to MDV under certain circumstances (Kottaridis §E_al., 1968; Nazerian, 1968; Nazerian, 1970; Witter §£_§1., 1968b), however, CPE may or may not be produced. MDV has also been reported to grow in pheasant, turkey, goose, and quail embryo fibroblast (Baxendale, 1969; Purchase et_al., 1971; Sharma, 1970). MDV failed to grow in mamalian cell cultures (Calnek gt_al., 1969; Sharma, 1970). Transmission Vertical or egg transmission has been considered as a probable means of transmission of the agent (Biggs, 1966). Sevoian (1968) described isolation of the virus from eggs, but his observations have not been confirmed. Cole (1949) and Cole and Hutt (1951) considered egg transmission as an unimportant avenue of infection and recently overwhelming evidence has been reported (Rispens et al., 1969; Solomon et al., 1970; Witter, 1971; Drury et al., 1969) which suggests that egg trans- mission is rare if it occurs at all. 10 Horizontal transmisSion has been shown to occur readily by direct and indirect contact (Biggs and Payne, 1963; Biggs and Payne, 1967) and by contaminated air (Sevoian 3331., 1963; Colwell and Schmittle, 1968). Infection has been accomplished by oral washings (Kenzy and Biggs, 1967; Witter and Burmester, 1967), litter, droppings and feces (Witter and Burmester, 1967; Witter et_al., 1968a), dust and dander (Beasley 23431., 1970) and feathers and extracts of feathers (Calnek et al., 1970b). The darkling beetle, Alphitobus diaperinus, has been shown to act as a mechanical vector of the disease (Eidson gt_al., 1966), but other insects have not (Witter et_a1., 1968a; Brewer et al., 1969). The portal of entry in the host through which the virus gains entry and initiates infection has not been determined, but the respiratory tract appears to be the best candidate. The portal of exit from the host is the epithelium of the feather follicle (Calnek and Hitchner, 1969; Calnek §E_al., 1970a; Nazerian and Witter, 1970; Purchase, 1970). Virus is shed into the environment one or two weeks after infection (Kenzy and Biggs, 1967). Presumably, Virus-containing dead cells, and feathers with adherent virus are shed continuously from the chicken and become part of the dust in the poultry house. Excretion of the e 6..., "07"". ' . L '1'”. l” . ‘1‘? o. 'H.>h I?!) at ”I" ‘ 3 Its 11 virus in this way would provide a convenient and excellent method for airborn transmission. The minimum incubation period for the disease is ‘between one and two weeks. Chicks inoculated with virulent MDV strains start to excrete virus (Kenzy and Biggs, 1967) and develop microscopic lesions as early as two weeks after inoculation. Clinical signs and gross lesions do not appear until the third or fourth week (Sevoian et al., 1962; Biggs and Payne, 1963; Biggs and Payne, 1967; Vickers §E_al., 1967). In chicks placed in a contaminated house, infection was detected after one or two weeks (Witter, et_al., 1970a) and spread rapidly until nearly 100% of the birds were infected. Although nearly all birds become infected, infection does not usually proceed to clinical disease (Chubb and Churchill, 1968a; Witter e£_§1., 1969a). When clinical disease does develop the outcome is usually death, but a few birds may recover. The incidence of clinical disease is influenced by breeding and husbandry procedures. Genetic selection for resistance to Marek's disease can be accomplished in a few generations (Biggs, et_al., 1968b; Cole, 1968). Resistance is a dominant trait and shows no correlation with the various production traits or with resistance to lymphoid leukosis (Cole, 1970). A selective breeding program directed toward Marek's disease resistance can be beneficial but short term results are not often marked. 12 Rearing of chicks in houses equipped with positive pressure and air filtering systems in conjunction with good sanitation and isolation practices, can reduce the incidence of Marek's disease (Drury, et_al., 1969; Witter, 1971). Even in instances where infection is not eliminated, it is delayed and mortality and gross lesions are reduced (Witter, 1971). The practical value of such a program depends on whether birds must be raised indefinitely in these units or if after rearing in these houses for a limited period of time they can be returned to conventional pens. Systems for Virus Isolation and Assay MDV can be propagated and assayed in newly hatched chicks, avian cell culture or in chick embryos. The inoculation of susceptible one—day—old chicks is the most sensitive method, and until recently the only method, for assay of MDV (Sevoian et_al., 1962; Biggs and Payne, 1963; Biggs and Payne, 1967; Witter and Burmester, 1967). Chicks are inoculated or exposed and then held in strict isolation to prevent adventitious infection. After 2—10 weeks the chickens are examined for evidence of infection (Witter and Burmester, 1967). Gross or micro— scopic lesions, virus isolation in cell culture, positive fluorescent antibody test on tissues, or detection of antibody can be used as criteria of infection. 13 All MDV strains can be propagated in cell cultures of duck embryo fibroblasts (Solomon gt_al., 1968; Witter §E_§1., 1969b; Kato g£_§1., 1970) or chicken kidney cells (Churchill and Biggs, 1967; Witter 9111.,196910). Suitable cultures are prepared, incubated at 37 C until cell sheets are confluent and then inoculated. Inocula of choice for primary isolation are white blood cells, tumor cells, trypsinized kidney cells or blood. Infected cultures develop focal lesions which consist of clusters of rounded, refractile, degenerating cells. Foci are approximately 1 mm. in diameter and affected cells may contain two or more nuclei, type A intranuclear and cyto— plasmic inclusion bodies. Large areas of lysis may or may not occur when the CPE is mature. Plaques develop in 5-14 days on primary isolation and are usually counted with a microscope. Continued serial passage in cell culture results in an attenuation of MDV (Churchill, §t_al., 1969; Nazerian, 1970). Although tissue culture methods are less sensitive than the inoculation of day-old chicks for primary isolation of MDV, they are easier and faster (Witter, et_al., 1969). Assay of MDV can also be accomplished by yolk sac inoculation (Von Bulow, 1968) of 4—6 day-old chick embryos and enumeration of the pocks that develop on the CAM. Embryo inoculation and cell culture assay are approximately equal in sensitivity. (NU. ‘2' 5 - .. ‘3 . 9' ' g .. . " ' In :1 «‘14 ‘f "it. 14 Serology Antibodies specific for MDV may be detected by several Serologic tests. The agar—gel precipitin (Chubb and Churchill, 1968a), indirect fluorescent antibody (Purchase, 1969; Purchase and Burgoyne, 1970; Spencer and' _Calnek, 1970), passive hemagglutination (Eidson and Schmittle, 1969) and serum neutralization (Calnek and Adldinger, 1971) tests have been described. The agar—gel precipitin test (AGP) is a double diffusion test in agar medium containing 8% sodium chloride. The antigen can be obtained from heavily infected cell cultures by concentration of the supernatant fluids or disruption of the cells, or from infected birds by the extraction of skin or feather tips. The test requires minimal amounts of reagents and is very easy to perform. Cross reactions with other avian viruses do not occur but laboratory isolates of MDV cross react freely (Chubb and Churchill, 1968a). The precipitin test and the indirect fluorescent antibody test often give closely comparable results (Purchase and Burgoyne, 1970), but probably detect different antibodies (Witter et_al., 1971). In the indirect florescent antibody test (FA), the antigen consists of infected tissue cultures grown on coverslips. It is reacted with a test serum and then reacted with a fluorscein labeled antiserum against chicken gamma globulin. The test is Specific and can detect 15 antibodies against different MDV isolates (Purchase, 1969). The indirect FA test is a more sensitive indicator of infection than the AGP test. By the indirect FA test antibody can be detected earlier and in higher titers (Purchase and Burgoyne, 1970). On the other hand, the indirect FA test requires more manipulations to perform and experience to read than the precipitin test. The passive hemagglutination test (Eidson and Schmittle, 1969) uses extracts of infected cell cultures absorbed onto tanned horse erythrocytes as antigen. The test is specific for MDV and high titers of antibody can be demonstrated, but maternal antibody cannot be detected. Large quantities of antigen are needed and the test is difficult to perform. In the serum neutralization test (Calnek and Adldinger, 1971) four volumes of cell—free virus were mixed with one volume of serum and incubated at 37C for 30 minutes and then assayed on chick kidney (CK) cultures for residual Virus. Serums from MDV—exposed birds neutralized 102 plaque forming units (PFU) while no neutralization was obtained from birds without exposure to MDV. Serums that neutralized virus also reacted in the AGP test. Immunity . i Although ltS exact role in MD is not known, an immune response does occur after exposure to the virus. Antibodies are found in most birds in commercial flocks. Antibodies are also observed in the serum of day-old chicks but these are depleted in about 2—3 weeks. Actively acquired antibodies may appear by four to five weeks of age in exposed birds and these antibodies last for long periods of time (Chubb and Churchill, 1968a; Purchase, 1969; Purchase and Burgoyne, 1970; Witter et_al., 1971). Chubb and Churchill (1969b) report that although chicks with parental antibody were not refractory to infection, the presence of antibody did limit and delay the onset of disease. The administration of serum con- taining antibodies to chicks without antibodies did not confer the same protection to Chicks as natural maternal antibody, but did suggest that antibody may have a pro— tective effect. Commercial poultry breeders (Arbor Acres, 1968) reported that chicks from dams hyperimmunized with one or more injections of infectious material were more resistant to challenge with virulent MDV than chicks from untreated dams. Eidson, et_al. (1968) reported that after inoculation with MDV, progeny from dams surviving an acute outbreak of MD were more resistant to challenge with MDV than progeny from a genetically comparable 17 control flock. Although selection for genetic resistance in surviving dams was not ruled out there is evidence to suggest an influence of antibody. Ball,et_§1, (1970, 1971) reported that chicks from hyperimmunized breeders were less susceptible to intra— peritoneal or natural contact challenge than chicks from non-immunized breeders, but in flocks undergoing early natural exposure, hyperimmunization did not seem to be of value in decreasing the incidence of MD in the progeny. Eidson, gt_g1.(1971) found that progeny of birds vaccinated with HVT were less susceptible to MD than progeny of a non—vaccinated flock or of a flock vaccinated with an attenuated strain of MD. Marek's disease virus may produce lesions in the bursa and thymus (Purchase and Biggs, 1967), organs important in the development of immunological competence in chickens (Cooper §t_al., 1965). In severly affected birds, lesions in these organs could be the reason for reduced antibody production and delayed graft rejection (Purchase gt_al., 1968). Although MD antibody production is reduced, levels of gamma globulin are elevated in birds with Marek's disease (Howard et_al., 1967; Samedieh §t_al., 1969). Losses from Marek's disease can be greatly reduced by the use of recently developed live virus vaccines. Three types of vaccines are available: 18' (a) an attenuated Marek's disease vaccine (Churchill 35:31., 1969), (b) a naturally occurring non—pathogenic strain of MDV (Rispens et_al., 1969, 1970), and (c) a herpes related virus isolated from turkeys (Witter gt_al., 1970b; Okazaki 32:31., 1970). Vaccination of day-old chicks with these vaccines does not prevent infection with the virulent viruses (Okazaki gt_al., 1970) but mortality and lesions are prevented. Although the mechanism of the protection is not known it seems to be independent of the humoral immune response. MATERIALS AND METHODS Experimental Chickens Single comb White Leghorn chickens from four genetic lines, 72, 151, 100 and a commercial line (designated as line B) were used. In order to obtain chicks with and without antibody, fertile eggs of each line were obtained from a MDV infected flock and from a MDV free flock. The latter were reared in isolators. All hens were inseminated twice weekly except non—isolated hens of line B which were pen—mated, using two males for fifteen females. Eggs were collected daily and stored at 55 C until set. Eggs from lines 72, 151, and 100 were stored under gaseous nitrogen in vinyl bags for four weeks (Proudfoot, 1966). Eggs from line B were stored for two weeks without nitrogen. Line 72 chickens are highly inbred (inbreeding coefficient > 0.95) and exhibit a high degree of suscepti- bility to MDV. This line, described by Waters and Prickett (1944), was developed and maintained at this laboratory (U.S.D.A. Regional Poultry Laboratory, East Lansing, Michigan). Marek's disease infection free and infected birds used in these trials were full sibs but hatched at different times. The infection free birds 19 20 consisted of 48 females and 12 males. The infected birds were hatched in isolators and later moved to conventional pens. These birds experienced a natural outbreak of MD in. which mortality reached 48%. Fifty-six of the survivors, 45 females and 12 males, were saved for this study. Line 151 chickens are highly inbred (inbreeding coefficient > 0.95) and exhibit a moderate degree of susceptibility to MDV. The line, described by Waters and Prickett (1944) was developed and maintained at this laboratory. Marek's disease infected and infection free birds used in these trails were full sibs but hatched at different times. The MD infection free group consisted of 64 females and 16 males. The infected birds were hatched in isolators and later moved to conventional pens, where 28% died of MD. One hundred-thirty—nine of the survivors, 114 females and 25 males, were saved for this study. Line 100 is an inbred line of chicken, with a high degree of susceptibility to MD, developed from crossing line 7 males with line 6 females and then backcrossing to line 7 for five generations (Stone, 1972). At the end of the fifth backcross the line was considered to be 98% line 7 and was subsequently maintained by brother and sister matings. The isolated and non—isolated flocks used in these studies originated as half sibs but have been separated for two generations. The MD infection free flock consisted of 21 64 females and 16 males. The MD infected flock consisted of 84 females and 12 males raised outside of plastic isolators. V ‘ Line B commercial chickens, 90 females and 15 males, were obtained from a commercial breeder just prior to sexual maturity and housed at this laboratory. Forty- five of the females had been vaccinated at one day of age with the FC126 strain of HVT and 45 were unvaccinated but all birds were reared in a common environment and received presumably equal exposure to MDV. Full sibs were hatched at the laboratory at a different time and reared in isola- tors. Six females were hept to sexual maturity and were found to be free of antibodies to MDV. Housing of the infected flocks of lines 7 15I 2, and B was in separate pens with fifteen to fifty birds per pen. Line 72 and 151 males were kept in individual cages in a separate room. The line 100 infected flock was housed in individual cages in a separate building. Infection free dams were housed in plastic isolators (Figure 1) supplied with filtered air under positive pres- sure. The isolators consist of a rectangular stainless steel base and a heavy vinyl plastic canopy, with glove ports and a pass through sleeve, sealed to the base. Air was passed through Astrocel filters (American Air Filter Co., Louisville, Ky.) for which the efficiency was rated at 99.97% for 0.3u particles. Figure 1.-—Plastic rearing isolators. Isolators supplied with filtered air under positive pressure used for maintaining dams free of MDV infection. e from these various bréfldrfia it 6 in clean incubators (Jamesway Incubators, “zon, Wis.) and after treatment, placed in ;t Horsfall-Bauer isolators. These isolators (Figure 2) were stainless units equipped with an automatic waterer and gravity flow feeder which could be filled through a port without opening the door. The units were under negative pressure with room air drawn into the isolator through three layers of 50-FG filter down (American Air Filter Co., Louisville, Ky.) for which the efficiency was rated at 93% for lOu particles and 72% for 0.3u particles. Cell Cultures Cultures of duck embryo fibroblast (DEF) or chick kidney cells (CK) were used in attempts to isolate MDV from experimental chickens. Cell cultures were prepared as described by Solomon, et a1. (1971). Fourteen day old 1 duck embryos were removed aseptically, decapitated and , placed in a trypsinization flask. Pools of three to five embryos were washed in phosphate buffered saline (PBS) three times to remove excess blood, macerated and washed again three times with PBS. After the final wash, 100 ml. of warm (37 C) 0.125% trypsin was added to the flask. The flask was then placed in a 37 C water bath and the contents stirred slowly for 30 to 60 minutes. The supernatant fluid, containing the released cells, was removed and Figure 2.--Stain1ess steel isolators. Isolators .used for housing of experimental birds. 26 1 in DEF growth medium (Appendix 5 b a hemocytometer. Cells were plated at 7 2 x 10 in 25 ml. of DEF growth medium in a 150 mm. diameter tissue culture dishes (Falcon Plastics, Oxnard, Calif.). Kidneys were removed aseptically from three to five line 72 chicks at one or two weeks of age, pooled in a flask, washed three times with PBS and minced with scissors. After mincing, the tissue was again washed three times with PBS. After the final wash the tissue was trypsinized with 100 ml. of warm 0.05% trypsin for three to five minutes, the supernatant fluid collected and stored on ice. This cycle was repeated six to eight times using separate tubes for each collection. The collections were centrifuged at 800 x g for five minutes and all except those containing excess blood were resus— pended in CK growth medium (Appendix I). Cells were 6 counted in a hemocytometer and plated at 8.0 x 10 cells per 60 mm diameter tissue culture dish. Virus Isolation Primary isolations of MDV from chickens were attempted by inoculating either DEF or CK cells with skin homogenate, kidney cells, spleen or white blood cells (WBC) (Calnek et al., 1970b; Witter et al., 1969b). 28 t Skin homogenate was used for assay of cell free ‘ MDV. Skin, including feather shafts, was obtained from individual birds form dorsal tracts and minced with scissors. Phosphate buffered saline or a buffer containing sucrose, phosphate, glutamine and bovine albumin (SPGA buffer) (Calnek §t_§1., 1970b) was added to make a 1:10 w/v suspension and sonicated for two minutes with a Bronwill Biosonik cell disrupter (Bronwill Scientific, Rochester, N.Y.) at a setting of 70 with the small probe. The fluid 1 portion was removed and inoculated onto drained CK or DEF cultures. Kidney cells were prepared by obtaining a piece of kidney from each bird and processing individually or by pooling tissue from birds of the same lot. The material was washed three times with PBS, passed through a syringe and again washed three times with PBS and then trypsinized in a tube at 37 C with occasional shaking for three cycles of ten minutes. After each cycle, cells were removed and stored on ice until after the final collection, then the collections were pooled and centrifuged at 800 x g for five minutes. Spleen cells were prepared from individual birds. Spleens were removed aseptically and minced with scissors until a fine homogenous suspension was obtained. Medium was then added and the suspension mixed thoroughly and allowed to stand until the large particles had settled. 29 The fluid and suspended cells were removed, counted and 25 x 106 cells inoculated onto DEF of CK monolayers. White blood cells (WBC) were obtained from blood of individual birds and processed either as individual samples or pooled by lots. Blood was layered on an equal quantity of 29.4% bovine albumin (Parker, 1961) and centri— fuged at 1000 x g for 14 minutes. The WBC's were collected at the interphase, washed with 10% bovine albumin, resuspended in medium and frozen at —l96 C until tested on DEF monolayers. Assay of MDV in CK Cell Cultures Primary chick kidney cells were prepared and plated at a concentration of 8.0 x 106 cells per 60 mm plastic tissue culture dish. Cells were allowed to grow, approxi- mately 48 hours, until a monolayer was established. For cell associated viral inocula, the medium was removed and replaced with fresh medium and the inoculum placed in the culture medium. After 18-24 hours the culture was drained, washed and fresh medium added. For cell-free viral inocula, the medium was removed and the inoculum placed directly on the cell layer. The inocula were adsorbed for 30 minutes at 37 C and then fresh medium added. After adsorption, cultures were allowed to grow for 8-10 days with medium replacement every second day. Cultures were 30 considered positive for MDV isolation if one or more typical plaques were present. Assay of MDV in DEF Cell Cultures ‘ Cells from primary DEF cultures were removed with 0.25% trypsin, centrifuged and suspended in growth medium and plated at a concentration of 1.0 x 106 cells per 60 mm plastic tissue culture dish. Cultures were inoculated when a monolayer was present, at approximately 24 hours. Inoculations were the same as for the CK cell assay. The medium was changed every other day and plaques were scored 10-14 days after inoculation. Cultures were considered positive for MDV isolation if one or more typical plaques were present. In experiments where virus isolations were attempted from kidneys, all inocula were assayed on CK cultures. When virus isolations were not attempted from kidneys inocula were assayed on DEF cultures because of the fewer difficulties encountered with the growth of DEF cells. Viruses The JM strain of MDV was obtained from Dr. M. Sevoian, University of Massachusetts, Amherst, Mass., and since 1966 has been serially passed in this laboratory in 72 chickens. Virus stocks consisted of either (1) pooled blood from infected chickens frozen and stored at —196 C with 15% dimethyl sulfoxide (DMSO) or (2) extract of MLrt from infected birds that was '- «C of 70 with the small probe of a Bronwill _c J cell disrupter for two minutes and then stored at -70 C. The FC126 strain of HVT was isolated by Witter et al. (1970b) from turkeys and was grown and passed in DEF cultures for 11 passages and stored as a cell suspension at -196 C. Hyperimmunization Hens of lines 7 and 151 were hyperimmunized with 2 a frozen pool of JM strain of MDV blood. Each hen was inoculated six times intra—abdominally (IA) with 0.5 ml. of a 1:10 dilution of blood in PBS. The first three inoculations were administered at weekly intervals followed by three inoculations at two month intervals. Each dose was calculated to contain 5 x 104 chick infec- tious doses. Hens for line B were hyperimmunized w'tu [he JM strain of MDV or with the FC126 strain of HVT Each dam was inoculated three times 1A at weekly intervals. Each dose was calculated to contain 1 x 104 plaque forming units (PFU's). Two tests were used to detect antibody, the agar gel precipitin (Chubb and Churchill, 1968a) and the l l l Antibody Determinations fluorescent antibody test (Purchase, 1969) 32 For the agar gel precipitin test (AGP), glass microscope slides 1" x 3" were lightly coated with 1% agar in PBS with 8% NaCl, and allowed to dry in air. When dry, 3.0 ml of the molten agar was dispensed onto each slide. After the agar had solidified the slides were stored in a humidified atmosphere and used 24 hours later. Patterns were cut into the agar with a template (National Appliance Co., Portland, Ore.) and the agar plugs removed with a pasteur pipette attached to a vacuum flask. For detection of antibody, serums were placed in peripheral wells and antigen in the central well. When detecting antigen, a known positive serum was placed in the central well and antigen smaples in the peripheral wells. Positive samples were identified by a precipitin line formed between the antigen and serum wells within three days (Figure 3). Stock antigens were obtained from CK cell cultures or from supernatant fluids from DEF cultures. Stock CK antigen was prepared from CK cell cultures infected with the JM strain of MDV. Cultures were serially passed onto feeder layers of CK cells until CPE involved approximately 75% of the monolayer. Cells were harvested with trypsin, centrifuged at 800 x g to pack the cells and resuspended in PBS at a concentration of 40 x 106 cells per ml. The suspension was frozen and thawed three times, dispensed into aliquots and stored at —20 C until used. Figure 3.--AGP test for MDV antibody. Test serums placed in wells 1-6. Well 7 contains MDV antigen. Wells 1 and 4 illustrate a positive test for MDV antibody. ’fluids were pooled and (NH 35 Stock DEF antigen was prepared from supernatant fluids collected from DEF cultures infected with the JM strain of MDV. The fluids were collected at 48 hour (intervals after plaques appeared in the cultures. The 4)2SO4 added slowly to the cold solution until 80% saturation was reached. The precipate was sedimented at a speed of 3000 x g for 20 minutes and dissolved in distilled water. The (NH4)ZSO was again 4 added until 60% saturation was reached. The precipate was again sedimented and resuspended in distilled water. The solution was then dialyzed against PBS until sulfate was no longer detectable by the addition of BaC12. After dialyzing, the volume was reduced to 1/100 of the original volume by perevaporation. The resultant antigen was stored at —20 C until used. The fluorescent antibody (FA) test was an indirect one performed by attaching coverslips, cell side up, to rubber stoppers and placing in a humidified atmosphere. A test serum was added to the coverslips and allowed to react for 30 minutes. The coverslips were then washed for 15 minutes in a buffer and a florescein isothiocynate labeled antichicken globulin reacted for 30 minutes. The coverslips were washed again for 15 minutes, rinsed in distilled water to remove the buffer and mounted on glass slides in elvanol (E. I. DuPont de Nemour and Co., Wilmington, Del.) (Rodriguez and Dienhardt, 1960). 36 The coverslips were examined with a fluorescence microscope using a HB200 mercury vapor lamp with a BG12 excitor filter and a 510 mu barrier filter. Positive samples were detected by bright yellow green staining of the plaques (Figure 4). The AGP test was used in an attempt to determine when virus antigens appear in the feather follicles of infected birds. Skin was obtained and an extract prepared and tested against a known positive MDV serum. Skin (including the proximal end of the feather shaft) was collected from individual birds, minced with scissors and a 1:10 w/v suspension made in SPGA buffer. The suspension was sonicated for two minutes with a Bronwill Biosonik cell disrupter at a setting of 70 using the small probe. The material was frozen at —70 C until tested. Gamma Globulin Preparations Gamma globulin for use in chicken inoculation trails was prepared by precipitation with NaZSO4 according to Orlans et a1. (1961). To 100 ml. of serum an equal volume of 34% NaZSO was added slowly and allowed to stir 4 for 30 minutes. The precipate was washed three times with 17% Na2S04 solution, dissolved in pH 8.0 Tris buffer and reprecipitated by the addition of 17g of NaZSO4 to each 100 m1. of solution. After stirring for 30 minutes the precipitate was washed three times with 17% NaZSO4 and -. .. ...-a; rest" -- '4': Figure 4.--Flourescent antibody test for MDV antibody. Serum testing positive for MDV antibody. Serum testing negative for MDV antibody. a. b. 38 39 and redissolved in Tris buffer and reprecipitated by the addition of 15g of NaZSO4 to each 100 ml. of solution. The precipitate was washed three times with 15% NaZSO4 and redissolved in PBS, pH 7.2-7.4, and dialyzed against PBS until no sulfate could be detected by the addition of BaClz. The gamma globulin was then titered and stored at —20 C until used. The MDV gamma globulin (two pools) was obtained (from the serum of 151 birds inoculated with MDV and had a precipitin titer of 1:64 and a FA titer of 1:160. The HVT gamma globulin was obtained from 72 birds inoculated with HVT and raised in isolators. Virus re-isolation attempts at the time of serum collection detected only HVT. This material did not react in the precipitin test but had a FA titer of 1:640. Normal gamma globulin, which failed to react with either MDV or HVT antigen in the AGP or FA test, was obtained from 72 birds raised in plastic isolators and were free of MDV or HVT infection. Diagnosis of Marek's Disease All birds were necropsied and examined for gross lesions of Marek's disease in the peripheral nerves or viscera. Figures 5 and 6 show characteristic gross lesions of Marek's disease. The gonads, vagus nerve and brachial and sciatic plexuses were examined histologically when gross lesions Figure 5.--Gross nerve lesions associated with MD. Normal vagus nerve of six week old chicken. Vagus nerve of six week old chicken with gross lesion. . ....r 41 Figure 6.--Gross visceral lesions associated with MD. a. Normal ovary of six week old chicken. b. Six week old chicken with ovarian tumor. . I) . 2. ”43’9"! ' . v." _ _ .1: ~. . L a"... . ’ -.{-.- I. .‘Vq _a1.sa ple size (Ruble an gan.Staté University computer EXPERIMENTAL DESIGN The objectives of this research, as previously. stated, were to: a. Determine if maternal antibody imparts 3 protection against MD. b. Determine what aspects of MDV infection and clinical disease differ in birds with and without maternal antibody. c. Determine if hyperimmunization increases the resistance of progeny to MD. d. Determine if infection of dams with a serologically related, non-pathogenic herpes virus from turkeys provides protection to progeny. e. Gain some insight into the possible mechanisms for the observed differences in susceptibility. Four types of experiments were used in an effort to realize these objectives. These experiments relate to the objectives as presented in Table 1. In order to carry out the experiments, groups of experimental dams with the appropriate antibody status were derived. 45 46 TABLE 1.--Relation of experiments to thesis objectives. Relates to Experiment Purpose Objectives 1 Compare mortality and lesions in a,b,c progeny of natural MDV exposed birds, MDV hyperimmunized birds and MDV free birds. 11 Compare mortality and lesions in a,b,c,d progeny of HVT vaccinated birds, natural MDV exposed birds, birds of both preceeding categories hyper- immunized with HVT or MDV and birds free of both HVT and MDV infections. III Administer immune gamma globulin to a,b,c,d progeny in an effort to simulate natural maternal antibody. IV Study the pathogenesis of the a,b,e disease in progeny for MDV exposed and MDV free birds. Experiment 1 Two lines of birds, 72 and 151, were utilized. Table 2 shows the parental treatment groups, designations and the number of dams in each group. The derivation and temporal relationships of instituted procedures for the groups are shown in Figures 7 and 8. The MDV exposed groups were chicks hatched in separate isolators and placed in conventional pens. At 223 days of age, the survivors were bled and separated .0 msoum Mom mfianOHumawm .n .mQOADmasoosw w>wooou “on cflb xz msoum umooxo m cam xz mmDOHm How mmflnmsoflmeom .m .Nh mafia mo wasp msflms mGOHuooHHoo mom can msoaumNHsssfiw .ms0fluowafioo Epsom soosuon mmflanOADMHoM oEHBII.> musmfim 48 A”: anocw ou...<._om_ an and *0 goo O¢m O¢N ON. _ _ , _ «I nr._.m: NLL r _J. 305 3:80: 13:00:00 mam “>05. 2 2:396 ozv 5:23. i]. wmaomo 2.: omN.z:22_mmm>I 024 szv omwomxw 453.22 mm oo< Co 2:5 06 o¢~ ow. o I 1 I 1 :1 _ x. w n e mm: doc. .02.: 09... c. .02: 256 v: PC. PC. NI :1 320 32m vagus: co:oo__oo uom .Illl norms co:o~_c:EE:0a>I >02 2 Samoaxm .0562 so weird venom 5:209 .U msoum How mflnmsowumaom .9 .mGOMDMHDUOGA o>flooou #0: cap xz msoum umooxo m can xz mmsoum How mmfismsofiumaom .m .HmH mafia mo mast @sflms mGOHDUoHHoo was was msoHsmNflnssfiw .ms0flbooaaoo Esnom soo3uon mmwsmCOHumaoH oEHB|I.w ousmflm 50 18 .525 8.5.69 3 ou< no «son. own ovm ON. 0 . ._. (— . . , 3.. m... Tit—v... 32m 3:22.. 6.32.00 93 A>oz 2 23.2.3 02. 3:28. i manomo 2.: owning—Emma»: oz< .xz. oumomxw ._05. 2 9.32.3 .9582 Co noted I voted c0298. 51 TABLE 2.--Designation of 72 and 151 parental groups of Experiment I. Exposure of . Number Dams Parental. Designation AEElEOdY ——————————— Groups to MDV a us L72 L151 Natural Nx + 24 55 Hyperimmunized H + 21 53 Not Exposed (MDV free) C — 48 64 into two groups. The groups designated as H were then hyperimmunized by inoculating with MDV three times, at weekly intervals and bled one week after the third inocu- lation (274 days). Boosters were given every 60 days thereafter until the end of the experimental period. All birds were bled at 580 days. The groups designated as NX were left untreated but were bled at the same dates as the treated groups. Serology indicated that these groups were infected with MDV prior to the 223rd day and both the Nx and H groups were maintained in an environment presumed to be contaminated with MDV. The MDV free birds, group C, were hatched and raised in isolators. This group was bled at 150 days and revealed no evidence of MDV infection. The MDV free dams were not bled at the end of the experimental period (580 days). Progeny from these dams were needed for repopula— tion of the MDV free flock and the risk of a loss of egg 52 production as a result of the bleeding procedure was deemed too great. Progeny from each group were challenged with MDV at one day of age by either 1A inoculation (three trials) or air exposure (four trials). Table 3 shows the basic design of the experiment. TABLE 3.—-Basic design of Experiment I. Number of Chicks Parental Antibody . Group . Status Line Challenge Trial No. Des1gnation l 2 3 4 Nx + 72 IA 7 15 8 Nx + 72 Air 8 9 10 10 H + 72 IA 8 14 10 H + 72 Air 8 10 10 8 C - 72 1A 8 l4 9 C — 72 Air 8 9 10 10 Nx + 151 IA 10 10 10 Nx + 151 Air 9 7 12 11 H + 151 IA 10 8 12 H + 151 Air 10 8 12 11 C - 15I IA 10 9 10 53 The 1A challenge consisted of inoculation of a dilution of a frozen pool of MD blood, containing 200 chick infectious doses per 0.2 m1. Air exposure was accomplished by connecting the exhaust outlet of the donor isolator, containing 25 three—week-old MD-infected chicks, to the air intake of the recipient isolator. Chicks from all three treatment groups (Nx, H, and C) were exposed to MDV simultaneously in the recipient isolator for four days. After exposure each lot was placed in a separate clean isolator and held for 12 weeks. All birds dying were necropsied and survivors were killed at 12 weeks and examined for gross lesions, antibody and virus levels. Antibody was determined by the AGP test on individual birds. Virus was isolated from pools of each group on CK cells using cells from trypsinized kidneys as inocula. Experiment 11 The design of Experiment 11 was similar to that of Experiment 1 except line B dams were utilized. The parental treatment groups and their designations appear in Table 4. The derivation and temporal relationships of instituted procedure for the groups are shown in Figure 9. The MDV—exposed birds were hatched at a commercial breeding Figure 9.--Time relationships between serum collections, immunizations and egg collections using dams of line B. a. Relationships for all antibody positive groups (V, VH-MDV, VH-HVT, Nx, H-MDV and H-HVT) except groups V and Nx did not receive immunizations. b. Relationship for group C. 55 (~— Period of Natural Exposure to MDV ——— Farm Laboratory Hyperimmuniz. Period Eco Coll Hatched Clmz Vaccinated Bleed Bleed Bleed Inoc. 1 . III . O 60 l20 240 Days of Age 93 VACCINATED AND NONVACCINATED Isolators (No exposure to MDV) EGO CoII HI H Hatched Bleed Bleed I I 0 60 |20 240 Days of Age 9b ISOLATED GROUP 56 TABLE 4.--Designation of Line B parental groups of Experiment 11. Virus Exposure of Parental Treatment Designation Agtiiody Ngmb:r Group a us am HVT Vaccinated V + 15 HVT Vaccinated and HVT Hyperimmunized VH—HVT + 15 HVT Vaccinated and MDV Hyperimmunized VH-MDV + 15 MDV Natural Exposed Nx + 15 HVT Hyperimmunized H—HVT + 15 MDV Hyperimmunized H-MDV + 15 No Exposure C - 6 farm. Half of the flock was vaccinated at one day of age with HVT and the other half left unvaccinated. All birds were then raised in a common commercial environment pre- sumed to be MDV contaminated. At 148 days 45 females from each of the vaccinated and nonvaccinated groups were brought to the laboratory, bled, divided equally into three groups and placed in conventional pens. Groups of the vaccinated and nonvaccinated chickens were either hyperimmunized with HVT, MDV or left untreated. All groups were bled one week after the final hyperimmuniza- tions (199 days) and again after egg collection at 269 days. 57 The MDV free dams of line B were brought to the laboratory as embryos and hatched and raised in isolators. Birds were bled prior to egg collection at 179 days of age and after egg collection at 249 days of age. Two trials with progeny from each of the seven groups were challenged by IA inoculation with a frozen pool of MD blood diluted to contain 200 chick infectious doses per 0.2 ml. Table 5 shows the groups and experi- mental number of chicks. TABLE 5.--Basic design of Experiment 11. Treatment Antibody Number of Progeny of Dams Status . . of Dams Tr1al 1 Trial 2 V + 42 44 VH-HVT + 47 42 VH-MDV + 4 5 45 Nx + 37 47 H—HVT + 45 40 H-MDV + 46 39 C — 26 24 After inoculation chicks were held for 12 weeks in conventional pens and specific mortality was recorded. All survivors were killed and examined for gross lesions of MD. 58 Experiment 111 ”Although in yiyg quantitative assays for virus activity have been deVeloped at other laboratories (Biggs and Payne, 1963; Anderson and Sevoian, 1969) it seemed desirable to work out the parameters of such a test with our own chickens and virus strains. The variables of most importance were the strain of host chickens, the response criteria and the time of response measurement. An ideal assay should have maximum sensitivity and should exclude all responses due to horizontal spread of MDV within test lots. Two trials were used in developing the assay system. In the first trial two groups of chickens, one from line 72 and one from a cross of lines 15 x 7 were used. Half of each group was inoculated with a frozen pool of MDV infected blood containing 2000 chick infectious doses per 0.2 ml. and half was left uninoculated to be exposed by contact. Twelve chicks, six of the inoculated and six of the contact exposed, were sacrificed from each group at 12, 15, 19, 22, 26, and 29 days and examined for gross and microscopic lesions of MD. In the second trial the optimum sacrifice times determined in the first trial were used to compare the relative sensitivity of gross examination and microscopic examination as assay procedures. Groups of 35 chicks each, one from line 72 and two from line 15 x 7, were used. 59 Serial lO—fold dilutions of a frozen pool of MDV-infected blood were inoculated IA into five chickens from each group. Ten chicks were held as contact controls. Each group of 35 chicks was placed in a single isolator. Two groups, one line 72 and one line 15 x 7, were killed and examined for microscopic lesions. The remaining one was held and sacrificed at the optimum time for gross examination. In Experiment III the quantitative assay procedure, developed as described above, was employed to measure the protection provided by antibody. Progeny from the MDV free dams were divided into six groups of 35 chicks, each of which was injected with 1.0 ml. of gamma globulin con- taining MDV or HVT antibodies or control gamma globulin. All birds were challenged after 24 hours (two days of age) with serial lO-fold dilutions of cell associated MDV or Icell free MDV, using five chicks per dilution (Table 6). An additional five uninoculated were sacrificed at five days to ascertain passive antibody levels and five were held as contact controls for the assay. All birds were killed at 19 days post inoculation and examined for histological lesions, antibody, and virus. Antibody was determined by the AGP or FA methods. Virus isolation was done on DEF using WBC's. Two trials were conducted with the above design. 60 TABLE 6.--Basic design of Experiment III. Dams. GIZEEIin Challenge Virus MDV exposed None Cell associated MDV None Cell free MDV MDV free MD Cell associated MDV MD Cell free MDV HVT Cell associated MDV HVT Cell free MDV Normal Cell associated MDV Normal Cell free MDV Experiment 1V Two trials were conducted with the same design, which differed only in the line of birds used and in some of the treatment groups. These will be discussed separately in IVa and IVb. In trial IVa, chicks from the 72 parental treatment groups in Experiment 1 were used. Twenty chicks from each of the three treatment groups (Nx, H and C) were challenged with MDV by air exposure as in Experiment 1, and 40 chicks from group C were used as unchallenged controls. Starting at the 4th day, 2 or 3 chicks from each group were sacrificed and examined for the following: (1) weight 61 of bursa; (2) antibody; (3) virus isolation from kidney, spleen or skin; (4) microscopic lesions in nerves, gonad, liver, thymus, or bursa. Antibody was determined by the AGP method and virus was isolated from various tissues on CK cells. In trial IVb, chicks of line 100 were obtained from both the MDV exposed flock and the MDV free flock. Some chicks from the MDV free line 100 dams were treated with immune gamma globulin. The treatment groups are presented in Table 7. TABLE 7.--Design of Experiment IVb. Maternal Group Antibody Gamma Globulin Number of Birds 1 + — 50 2 — + 50 3 — — 50 4 + — 15 5 - — 15 Groups 1 to 3 were challenged with MDV by air exposure. Groups 4 and 5 were held as unchallenged isolated controls. Starting at day 6, predetermined random lots of five chicks were removed from the MDV exposed birds at two day intervals through the 25th day. . 15) precipitatifig a body was determined by the AGP me .iSOlated on DEFs. .7 ,‘ R- ~b thod and RESULTS Status of Experimental Dams and Chicks in Experiments 1 and II All of the trials of Experiments I and 11 required progeny chicks with identical genetic backgrounds but with varied parental antibody status. In order to supply such chicks, it was first necessary to obtain the appropriate groups of dams. The antibody status of the dams and their progeny are presented in Table 8. No major differences in antibody titers were detected between the virus exposed groups in either line 7 or 151. Antibody titers in line 7 2 increased slightly 2 during the experimental period but stayed relatively constant in line 151. Antibody titers determined by the AGP method were higher in both dams and progeny of line 151 than line 72 but antibody titers detected by the FA test were slightly lower in the 151 dams. No antibody was detected in the MDV—free birds of both lines by either test. Antibody titers of progeny, hatched when line 72 and line 151 dams were 550 days and 472 days of age respectively, revealed no difference between the MDV— exposed groups within the lines. Precipitin titers for 63 .msmc «senthH «when ommutus "mean no amen .mUHmHMm mo Hands: up cmww>flu meowusawp uswomvso mo mamooumwomu no 55w n .>oz on ousmomxmm o HH n o I o m.m m H t o I a sac men h.v Ha UH o n xz HmH numzmmoum o as omH o o o m.HH we omm s + ~.HH mm wen m + n.oa mm mum o + m v.oa om own 0 + 0.0a mm «em o + m.m mm mam o + xz HmH mama 0 0H H o t . u m. t ..M m n 1 - .. o Ithsowonm o on own o t u we m.m NH omm n + H.v Hm sen m + o.v HN mum o + m we H.m «a own 0 + m.m «a saw 0 + m m~.m an mma o + :2 5 mean #60 A s crummy 0 sense u nesz :ofiuoosaou Esuom um meowumqussaH .mxm usosumoua mad when :w mmd Mo Honssz mamnsumz Amundsen osflq mo mspmum uponwusflii.m mamma 65 line 151 chicks were slightly higher than line 72 but FA ! titers were equivalent. Progeny from the MDV free dams did not have detectable antibody by either method. The serological data from the parental treatment groups of line B and their progeny are presented in Table 9. The dams of group C had no detectable antibody to MD. The dams from the groups V, VH—HVT, and VH—MDV had similar antibody titers and revealed no effect of hyperimmunization and, in fact, appeared to exhibit a drop in antibody titers with time. The Nx groups showed a slight drop at 199 days, but 269 day titers and 148 day titers were similar. The H-HVT and H-MDV groups gave similar results and suggested a slight effect of hyperimmunization. Antibody titers of progeny obtained when the virus- exposed dams were 230 days of age revealed the H-HVT and the H-MDV groups to have slightly higher antibody titers than the other groups again suggesting a slight influence of hyperimmunization. The progeny from group C had no detectable antibodies. .msmw on... "were no amau .monfinm mo Hon—.5: ha pacific muoHusHHu unHomuso no mHmuoumHoou no flaws (was on chamomxmu o o mH H I o o GIN n.N mH H I 0 >911.— vuu . m.N mH H I c 92.11 N o.N mH H I. 0 £2 a o.N 3 H I 0 >921; N o.N mH H I 0 9211.5 N O.” 3 0H I o > Famous c o m mew I o o o w mnH I o o wHIH m.N mH mow + m uIH w.~ mH «.2 + m 0H m.H 3 m: + m >97: wIH m.N mH mom + m wIH o.~ mH mmH + m NIH m.H mH mvH + m e>mum wIH H.N vH mmm + o «IH m.H vH mmH + o mIH md 3 MW: + o xz NIH m.H mH mow + w oIH o.H 3 mg + v mIH v.m mH mvH + v >nzum> HIH o.H mH mom + w vIH m.H mH mmH + v NMIH H.m mH 2H + w Elm; NIH m.H mH mom + H «La m.H mH mmH + H NMIH m.m mH 9: + H > mama madam ado: n ”swam 538:8 Hun—HE DE: Esuom um .mxm meoHucNHcsEEH usmfiuuoua made 5” mg MHuusumz mo Hon—.52 Hmucohmm nouns—e a: and: flames mus .m on: no hammoum was neat chquHHomxm mo msumum \HHUOAHUEHII.m Human. 67 Experiments 1 and II: Susceptibility of Chicks from Naturally Exposed, Hyper— immunized and Control Dams to Mortality and Lesions of MD Influence of Parental MDV Exposure on Mortality and Lesions in Chicks after IA or Air Challenge Progeny from dams with MD antibody from natural infection (Nx), hyperimmunization (H), and with no antibody (C), were challenged with MDV by 1A inoculation or exposure to air from MDV infected birds. Table 10 presents the mortality and lesion data for the four trials. Responses of progeny chicks from the antibody positive groups (Nx and H) were similar within each line whether exposed with MDV by IA inoculation or air challenge. However, the antibody negative (C) groups were clearly more sensitive than groups Nx or H to MD challenge. Within line 72 mortality for the antibody positive groups ranged from 60% to 75%. In comparison antibody negative chicks responded with mortalities of 94% to 100%. A difference between chicks with antibody and without antibody is clearly shown by the mortality responses but no difference is apparent between the two antibody positive groups. Examinations of survivors for gross and microscopic lesions revealed the same pattern between the groups. 68 TABLE 10.-—Mortality, gross and microscopic lesions in progeny of 72 and 151 dams which received different MDV exposures. Mortality and Lesions Response Through 12 Weeks Line §::::;::t Trial IAa AIRb c d e Mort Gross Total Mort Gross Total 72 Nx l 2/7f 2/7 4/7 6/8 7/8 7/8 2 12/15 14/15 14/15 5/9 5/9 5/9 3 6/8 7/8 7/8 6/10 6/10 10/10 4 5/10 6/10 10/10 Percent 67 77 83 60 65 81 H 1 5/8 6/8 8/8 8/8 8/8 8/8 2 11/14 12/14 12/14 6/10 6/10 6/10 3 8/10 9/10 9/10 4/10 6/10 7/10 4 4/8 6/8 7/8 Percent 75 84 91 67 72 78 C 1 8/8 8/8 8/8 8/8 8/8 8/8 2 12/14 14/14 14/14 9/9 9/9 9/9 3 9/9 9/9 9/9 10/10 10/10 10/10 4 10/10 10/10 10/10 Percent 94 100 100 100 100 100 151 Nx 1 2/10 6/10 9/10 0/9 2/9 9/9 2 2/10 3/10 9/10 0/7 1/7 7/7 3 0/10 2/10 6/10 0/12 3/12 6/12 4 0/11 1/11 2/11 Percent 13 37 80 O 18 62 H 1 0/10 2/10 7/10 0/10 1/10 5/10 2 0/8 0/8 5/8 3/8 4/8 5/8 3 0/12 1/12 7/12 0/12 2/12 3/12 4 0/11 0/11 0/1 Percent O 10 63 7 17 32 C 1 3/10 8/10 10/10 3/10 8/10 10/10 2 5/9 8/9 9/9 1/10 8/10 10/10 3 0/10 6/10 10/10 2/11 10/11 11/11 4 13/19 19/19 19/19 Percent 28 76 100 38 90 100 aIntra-abdominal challenge. bChallenged by contact with air from MDV infected chickens. cMortality includes all birds that died of MD d . . . . . Gross p051tlve includes all birds dead and suervors at termination positive by gross examination. eTotal positive: includes all grossly positive birds and all others including survivors found microscopically positive. f . . . Number pOSltlve/number at rlsk. 'I I 69 [Ii I. The mortality response for line 151 progeny \ revealed a similar pattern. Mortality for the Nx groups a was 13% by IA inoculation and 0% for air challenge and 1 the H group responded with 0% by IA inoculation and 7% by air challenge. In comparison, antibody negative progeny responded with 28% by IA challenge and 38% by air challenge. Histological examination revealed similar differences between the positive groups and the antibody negative groups. A least squares analysis of variance (Table 11) revealed significant variation in response due to line, trial, and parental treatment groups. The difference between the lines was expected. The variation between trials may be partially due to variation in inoculum but is not completely explainable. Further analysis of the variation within parental treatment groups showed no significant difference between groups Nx and H but there was a highly significant difference (p g .01) between the antibody positive and the antibody negative groups. Virus levels and antibody titers are presented in Table 12 for the survivors of the MD—exposed line 151 chickens. All chicks from the groups Nx and H were positive for precipitin antibodies at termination and mean titers for the groups were similar. However, mean titers of group C were substantially lower than for groups Nx and H and 30—40% of the birds tested had no detectable antibody at 12 weeks. Virus levels for group C were 4-6 7O .Ho>oH Ho.o om unmofleflcaam %% mme H Hensonmm mGOHmoH msonm moon m H o no UHmoomonon . H 0 D.H D 2 mo condom moMMDWm coo: .wonsmomxo mSMH> psoquMHc mo mEmc HmH one we mo woomoum EH msonoH OHQOQmOHoHE cam mmonm .hDHHouHOE mo oOQMHno> mo mHthmso monoswm DmooHII.HH mqmHc wQOHDsHHp psflompso mo HoooumHoou we 85mm oomHImmw meH NMIo m.v mm Hm Dumpsou ommIHNN use NMIo N.m no Hm «H O MOHIBH mm NMIN m.oH 00H mm pomusoo omImH om NmIN v.0H 00H om «H m oneINv NNN NMIN N.oH OOH mm poopsoo BHNIem omH NMIN m.HH OOH mm Q¢ kuHB w omsoHHono Dcofipmone mssfl> mUOQHpc< sHuHmHoonm >Q2 hsomomm Hopsoumm oQHH wHO>H>H5m Q0 mEOHHMNVwaQO .moHSmomxo msuH> usoanMHo wo mEoo HmH Eoum mxoos NH @cH>H>Hsm wsomonm sH mnouHu mcoofluso Use QOHDMHowH man> mo QOmHHmmEooII.mH mqmme 72 times greater than the Nx group and 17—18 times greater than the H group. Although antibody titers for group Nx were similar to group H, virus levels were 3-4 times greater. The small number of survivors in MD-exposed line 72,precluded51meaningful analysis of this line. In Experiment 11, progeny from variously treated line B dams were challenged with MDV by IA inoculation (Table 13). Group C was highly susceptible to challenge with MDV, with 84% of the chicks dying and 94% responding with gross lesions at termination. The susceptibility of birds in the other groups was less than for group C, as indicated by mortality responses of 41 to 63% and gross lesion responses of 53 to 76%. A least square analysis (Table 14) revealed significant differences between both parental treatment groups and trials. Further analysis between treatment group pairs in all combinations revealed differences between the groups as shown in Table 15. All progeny with maternal antibody differed from the antibody negative group C. Group VH—HVT differed significantly from groups Nx, H—HVT and H—MDV only at the gross lesion response. Group VH—MDV differed from group H—MDV only in the gross lesions response and from groups Nx and H-HVT in mortality and gross lesions, all other groups were not statistically different. 73 .meh we HoQEs:\o>Hunom Hwnfiszo .:0Hum:HEhou um mGOHmoH mmoum suHs mno>H>n5m cam a: mo cmoo moHHb HHm moosHosH n .o: no mafiso moans HHam em om\nv em om\mv mm ¢N\HN me vm\wH OOH wm\om mm om\vm U He mm\ow mm mm\wv Hm mM\vN He mm\mH we mv\mm Ho mv\mm >Qzlm Hr mm\ow mm mm\om mp ov\om om ov\mm no mv\om ow me\>m B>mIm or am\eo mo em\mm mo se\mm mm sa\mm om sm\mm on sm\mm xz mm om\ww He om\hm Nv mv\mH mm mv\mH we mV\mm me mv\mm >QEIE> mm mm\hv ow mm\v¢ mm Nv\wH Hm mv\MH mm PV\vm we hV\Hm B>mlm> No om\mm me om\mv mm ev\mm em ea\mH He NV\0m so ome\sm > w .02 w .OZ mm .02 w .02 m. .02 ea .02 muonw huHHmuHOE mmOHw huHHmuHoz mmono mmuHHmuuoz usofiumoue Hence m Hmflue H HmHns Hmoewumm mxomz NH Smsouna omsommwm EOHmoH one HDHHmuuoz .moMSmomxo mSHH> usonomme msH>HoooH mace m osHH mo mswmosd CH msonoH mmosm one thHouuozII.mH MHmHH .Ho>oH Ho.o pm HCMOHMHcmHm .3. mam Haeoe OHom.o msmm.o mmm aouum Hmmm.o msom.o e ropes m x a mxm HmuH> rrssmm.m Irmmao.m H .HBHHEV Booms *rvmmm.H reoth.H o ousmomxm mDHH> Hopsonom 4. 7 msonoH mmouw muHHmuHoz mo oousom monmswm goo: .moHSmomxo msuH> DconowwHo mo mEoo m osHH Eonm hsomonm mo mQOHmoH mmonm one WHHHMHHOE mo mmeHmso monoswm umooHII.vH mqmma 75 .nemammmHo HHHmoHomHomnm Hoz n mz Ho. Ho. 0 w> >ozIm Ho. Ho. 0 m> B>mIm m2 m2 >Qzlm m> B>mlm mo. mo. 0 m> xz m2 m2 >Q2Im m> xz m2 m2 B>mIm m> xz Ho. Ho. 0 m> >ozIm> mo. mz >QZIm m> >Qzlm> mo. mo. E>mlm m> >02Im> Ho. Ho. xz m> >02Im> Ho. Ho. 0 m> B>mlm> mo. mz >ozIm m> B>mim> mo. mz B>mIm m> B>mIm> Ho. mz xz m> B>mIm> m2 m2 >QZIm> m> B>mIm> Ho. Ho. 0 m> > m2 m2 >QEIm m> > m2 m2 e>mIm m> > m2 m2 xZ m> > mz mz >QEIE> m> > mz rmz B>mim> m> > mconoH mmouo muHHmuuoz pcosumoue Houseman cosmonHsmHm mo Ho>oH .wsomonm m mcHH mo masonm usoEumoHu cowsuon moosoHoMMHp mo soHumsHo>w HMUHumHDMDmII.mH mqmme 76 Data of Experiments 1 and 11 revealed that chicks from antibody positive dams were less susceptible to lesion induction, had lower virus levels and had more precipitating antibody at 12 weeks than chicks from dams without MD antibody. Experiment III: Simulation of the Protective Effect of Maternal Antibody by the Administration of Immune Gamma Globulin In an effort to further study the mechanism by which chicks from dams with antibody are resistant to MD, experiments were undertaken to develop methods to simulate, by the administration of immune gamma globulin to antibody free chicks, the levels and duration of maternal antibody which were comparable to those of chicks from immune dams. Gamma globulin was administered by various routes and at various doses. At selected tine intervals chicks were sacrificed and antibody levels determined. Table 16 presents the data from the five trials. In trial 1, l6 chicks with natural maternal anti- body, sacrificed at one day of age, were all positive for antibody and the mean AGP and FA titers were 3.26 and 38.3 respectively. Antibodies were detected through 12 days but were absent at 15, 20, and 24 days. Subsequent trials were designed to simulate this naturally occurring pattern of antibody. 77 .monfimw mo Hogan: >9 pooH>Ho mcoHDSHHo chomoco mo mHmooanown mo 85mg .omm mo hop wco um UoQHEnmpoc muouHu uponHusdm oH v.0H A om oz . o\o om o.H oso HIs>m I o mH v 0H A mm o.m o\m om o.o so m mH v OH A om m.m m\o am m.o so N oH v 0H A ms o.s m\m om o.H so mIoz I m oz o.m s\s >H o.o so m oz m.m s\s >H o.H so m os o.m s\s am o.o so N ms o.s w\o om o.H so mIoz I s o o m\o sH o.m o.s H o o NH\o om o.H o.s H o o NH\o oH wooansm Esnow mo soHDoscsHII.mH MHmHB 78 In the first attempts (trials 2 and 3) to induce a passive immune state in chicks from antibody free parents, one day old chicks were injected with 1.0-2.0 ml. of gamma globulin preparation 1. Detectable antibody was not found in chicks in either trial 2 or 3. In trial 4, a gamma globulin pool of high titer (preparation 2) was used. Subcutaneous administration of 1.0 m1. produced detectable serum antibody in 8/8 chicks with a mean titer of 4.0. In trial 5, gamma globulin pool 2 was administered subcutaneously and antibody levels determined at 1, 10 and 15 days. Figure 10 shows the decay curves for_ precipitating antibody in chicks inoculated with 1.0 m1. of gamma globulin and in chicks with naturally occurring maternal antibody. Titers obtained with administration of gamma globulin appear to be slightly higher but parallel closely the decay pattern of natural maternal antibody. Detectable antibody was absent at 15 days in chicks with natural maternal antibody and chicks with administered gamma globulin. Detection of antibody due to administration of immune HVT gamma globulin required the use of the FA technique. High titer immune HVT gamma globulin (pool 3) which had an FA titer of 1/640 against HVT antigen, and 1/160 against MD antigen, was administered subcutaneously sHHsnon oEEmm E>m conmpmHsHfico n o hconHasm Hmsuoumfi Handpos u < .sHHSQOHm mesmm oQSEEH B>m mo sOHHoHumHnHfioH .a GHHDHon ofifiom oz possumHsHficm n o moonHusm Hosuoums Handpms n < .sHHDQon magma osssfiH a: mo slomnumHsHEcm .m .hponHHGM Esnmm UoHHSUUo MHo>Hmmmm mo moooQII.OH onsmHm [0- I O N 40-. 30- (l6 I I l I InvION— J9I!.I. dev Io I°°°Jdi°°a 8 IO l2 l4 6 Days Duration 10a 81 in amounts of 1.0 ml. per bird in trial 6. Chicks were sacrificed at l, 6 and 15 days after administration and antibody levels for MD antigen determined. Figure 10 shows the decay curve for antibody as measured by the FA test in chicks receiving HVT gamma globulin and chicks with natural MDV maternal antibody. Both curves are similar and detectable antibody is absent at 15 days. Development of a Short Term in vivo Assay for MDV Short term in yiyg assay procedures suitable for quantitative measurement of MDV were required. Two pre— liminary trials dealt with developing the assay system. In the first tiral, lesions appeared in 4/5 inoculated 72 Chicks at 15 days and in 1/6 contact birds at 22 days post inoculation (Table 17). Microscopic lesions were not present in contact birds of line 15 x 7 at 22 days. Gross lesions were not detected in contact birds of either line throughout the experimental period. The optimum sacrifice times were selected by choosing the last times in which contact birds of either line were negative for lesions. Sacrifice times of 19 days for microscopic examina— tion and 29 days for gross examination were selected and their relative sensitivity and specificity compared in trial 2 (Table 18). The titer of a standard inoculum was .msop Hoz.m DZ 92 w\o o\m mm oz oz oxo oxo oN mxo o\o o\o o\s NN m\o oxo o\o o\o mH oxo o\o o\o m\o oH oz oz oxo o\o NH N x mH 92 oz m\o o\m mm oz oz o\o o\o oN o\H oxo o\o o\s NN m\o o\m o\o o\H mH o\o mxs o\o o\o mH N oz moz o\o o\o NH N uomunoo onMHsoocH uooucoo UopoHsoocH QOHHMHDUOGH osHH sOHuosHmem HMOHmOHoumHm coHuosHsoxm mmouw umom moo .soMoHco mo wooden osm omcommon .meu ooHMHHoom mo GOHumsHm>o ">92 Hem mowmm o>Humqusmsw o>H> sH so no ucoEQOHo>ooII.hH mqmfle m.o oIOH m\o oIOH .H.H mmo sIOH o s OH mxs MUCH .smxo mmouoINmoImN s x oH m\o osoz o\o oIoH s\o mIOH x om.H sms sIoH m o oH o\s MHoH HmonoHoumHmIHooImH s x mH N\O wqoz s\o oIoH sxo oIOH .x om.H mmm sIOH m m IOH s\s MIOH HmmHuoHoumHzIHmoImH NA murmflnvflmom aOz NMOD EHDOOCH wwgommmflm NEH-H t I .>oz mo cowumuHusosu Iy;sfl o£# How moosgoe oumcHosmo mo >DH>HuHmcom o>HHMHomfiooII.mH mHmse 84 determined to be 1.58 x 105 lesion producing doses in both line 72 and 15 x 7 when measured by microscopic detection of lesions at 19 days and 1.19 x 104 lesion producing doses in line 15 x 7 by gross examination for lesions at 29 days. The sensitivity of an assay based on microscopic examination for the detection of lesions induced by MDV was 10~fold greater than an assay based on gross examination. Therefore the assay based on microscopic examination was used to compare the ability of MDV to induce lesions in antibody positive and antibody free chicks in Experiment III as a means of measuring the effect of antibody on MDV. Experiment III: Protection of Chicks by Maternal Antibody and Administered Immune Gamma Globulin Two trials comparing the susceptibility of chicks with maternal antibody and administered gamma globulin to graded doses of cell—associated MDV and cell—free MDV were conducted. Table 19 presents the data obtained using cell- associated MDV. Calculated LPD50 titers were 13.4 x 103 for the chicks with natural maternal antibody, 4.2 x 103 for the chicks receiving MD gamma globulin and 20.0 x 103 for the chicks receiving HVT gamma globulin. The titer in antibody negative chicks was 200 x 103, ten—fold greater. In vivo neutralization indicies were calculated by dividing the titers in antibody positive groups by 85 .ESHDUOCH HMCHmHuo mo .HE m.o mom .omcommmu COHmoH Eoum coumHsoHoo .momoc .e>= no >oz 0» mmHoooHocm oHomuuoumo 0: buss :HHsoon assoc .poHMHOmH omosH «0 Homes: mosmem >Dz mo Hogans ommuo>¢ .umou 3 pocHEkump HouHB .xmbcH COHDMNHHmuuso: o>H> :H Dix: .umou mwd >2 pocHEuouop HouHB (U l‘i-I "HauHu OmooH p o .meH um HonESC\m:OHme :qu Honfiszn .EsHsoosH HmchHno mo msoHusHHQo oz II s.HO oz oz O m\O O o\H Ns m\m mm o\o O sxo onsuoz I oz oo.o ON oz oz O m\O O m\O NN m\m mN o\s mom m\m e>z I oz mm.O o.mH oz oz 0 m\o O s\O mH o\N Hm o\m oo.N m\o o: I oz sO.H sm.m oz oz O m\O O m\O OH s\H oN m\o oN.m m\m ocoz + N O s\O II OON O s\o O s\o O s\o H N\H N N\N OH s\s O sxo onsuoz I O s\o O.H 0.0N o s\O O m\O O m\o O m\O o s\N s m\H mom s\s s>z I O s\O Os.H N.s O s\O O m\O O s\O O s\o mz N\H mz s\N oom.m s\s oz I O m\o AH.H s.mH O s\o O m\O O s\O O s\H N s\N N m\m ooN.m s\s ocoz + H > H sz > H > H > H > H > o o> oH uwuHe .moo omoooHcH museum OOH x ocoz OH OH OH OH O com: .oz :H sooHo HUHHO was Houucou UHouHB OI ml ml VI MMI H . s x .H hponHucm H . H poHMHomH owned 00 H pmom m on m w Hmcuouoz >02 suH3 :oHucHSOOCH mcHsoHHom omcommom o no NoooHucs .cHHsnon museum ossEEH mo coHumuuchHfiom mCHsoHHow >oz onMHOOmwm HHoo mo momoc poncho ou monco ooH osHH mo prHHoHonomsmII.OH MHmHB 86 that of the antibody negative group. The N15 ranged from 1.0 to 1.7. Antibody titers done at five days show that gamma globulin administration was successful in simulating natural antibody titers. In trial 2, titers for the three antibody positive groups ranged from 8.34 x 103 to 20 x 103 whereas the titers in antibody negative chicks was 91.4 x 103. In yiyg neutralization indices ranged from 0.66 to 1.04. In both trials virus was isolated only from those lots showing lesions and antibody could not be detected at 19 days. The susceptibility to graded doses of cell—free MDV is presented in Table 20. In trial 1, calculated LPD50 titers and in yiyg NIs, were 9.1 x 100 and 2.05 respectively for both chicks with maternal antibody and chicks administered MD gamma globulin. The titer in chicks administered HVT gamma globulin was 10.2 and the NI was 2.0. Chicks administered normal gamma globulin responded with a titer of 1026. Marek's disease virus was isolated only from lots in which lesions were present. Antibody was not detected at 19 days in any of the lots. In trial 2 similar results were obtained. 12 yiyg neutralizing indices were 2.17 for chicks with maternal antibody, 2.0 for chicks administered MD gamma globulin and 2.34 for chicks administered HVT gamma globulin. Marek's disease virus was isolated only from lots in which lesions .ss: no so: 0» mmHoooHusm oHoeuuouoo 0: oqu :HHnoon masses .umou so an OoaHsuouoo umust .onEom ozm .umwu moo so ooannuuoo uwuHs w .xop:H :OHuMNHHMHusoo 0>H> mmw we .HE m.o Hum .omsommoh oOHmoH Eonw moUMHsono .womoo OmomH mo Hones: "HouHu OmomH o .coumHOmH mosmem >oz mo Hones: ommuo>¢o .men no Hoofiss\m:onoH sum: Hoosszo .ESHsuooH HmonHuo mo mGOHusHHoM oz oz 0 s\o HH s\N Hm s\s Om s\v o v\o HHmENoz I oz oz 0 s\o o m\o O s\o NH s\H com m\m s>m I oz oz 0 s\o o s\o o. s\H HH s\N mm.N m\m .o: I oz oz o m\o O s\o o m\o NH m\N mN.m m\m 0:02 + N N\o o m\o O N\o H m\H m N\N OH m\m o s\o HHmshoz I ¢\o o m\o. o N\o o N\o O N\o m m\H :mm s\v B>= I «\O O sxo O N\O O s\O O s\o mz st uoN.m s\s oz I N\O O N\o O s\O O sxo O s\O Omz st me.m sxs ocoz + H .H > .H > _H > _H > .H o> AH “WWW“ . mwm monouw pwuomnoH HUHSU oodz OH OH OH OH OH IIIIIIIIIIIIIIII . . m s m N MHI .oosH umom mzmo :stoon zoooHuoH HoHHB >oz ouHB GOHUMHaoooH msHsoHHom wwoomwom m um zcooHusm Esme Hccuouoz as... I we .oHHSAOHO MEEmm ossEEH mo :oHHMHHmHoHEcm warm. >nz wouulHHoo mo momoc popcum 0» monzo OOH 95H m0 NUHHHnHumoomsmIION mam—<9 88 were present and antibody was not detected at 19 days in any of the lots. Data obtained in the foregoing experiments show that the administration of MDV or HVT immune gamma globulin at one day of age simulates the level of protection present in chicks with naturally occurring maternal antibody and appears to act against both cell-associated and cell—free MDV. However, antibody is more effective against cell— free MDV. Experiments IVa and IVb were designed in an effort to further elucidate the role of maternal antibody in MD. Experiment IV: Influence of Antibody on Various Host Responses to Infection Experiment IVa: Comparison of Chicks with and without Maternal Antibody Experiment IVa was designed to compare in a sequential manner the response of chicks with and without maternal antibody. Table 21 presents the data obtained on the appear— ance of microscope lesions in various tissues. Microscopic lesions were first observed at 10 and 13 days in the bursa of l of 2 progeny of group C and were present in all of the bursa of subsequent groups examined. Bursa lesions did not appear until 15 days in progeny of group Nx and 17 days in the progeny of group H. .m mH HmHu um mouHo Ho “onenzo mmmH mmooum HOHuooo omuMHOmH on» sH meH vs mpHHo mo Honsozo mmoHos N mH 0 com z .xz museum oH MmHH um mouHo mo “snooze O O O O O O uo>HH O O O O O O mango O O O O O O omcoo mHouueoo O O O O O HO o>umz omumHomH N O O O O O um>HH N H H O O O omuso H O O O O O omnoo N O O O O NO m>uoz O O O O O O O uo>HH O O O O O O «mono O O O O O O ooeoo O O O O O «O m>uoz o O O O O O O um>HH H O O O O O madam O O O O O O ooqoo O O O O O ...O o>umz xz oH MH OH O o s oommHB poofiuooue . HMHGQHmm 90 Bursa lesions in group C progeny were first observed as minor intrafollicular lesions as described by Fletcher (1971) and proceded to severe intrafollicular degeneration and cyst formation (Purchase and Biggs, 1967; Jakowski gt_al., 1968). Whereas only minor intrafollical lesions were observed in the bursa of progeny from the antibody positive groups (Nx and H). Figures 11 and 12 show examples of normal bursal follicles, follicles showing minor lesions and follicles showing severe lesions. Lesion severity in the bursa was associated with reduced size of the bursa. Mean bursa weights for groups Nx and H and the isolated controls were similar through- out the experimental period whereas the mean bursa weights of group C was markedly reduced after day 13 (Figure 13). At 22 days mean bursa weights from group C were approxi- mately 40% of the other three groups. Microscopic lesions were observed in the nerves, liver and gonad of progeny of group C at 15 days of age, whereas they were delayed four days in group Nx and seven days in group H. Lesions were not present in isolated controls. Nerve and gonad lesions appeared as minor to massive infiltration of lymphocytes as illustrated in Figures 14 and 15. Liver lesions were observed as numerous small infiltrations of lymphocytes in the portal area shown in Figure 16. Figure ll.—-Norma1 bursa of fabricius and bursa with minor MDV lesions. a. Normal bursa from 10 day old chick x 100. b. Bursa from 10 day old chick with minor intra follicular lesion x 100. 92 II' , i .a 3“ -_~ ", ff. .3 vhf. - up, ‘3 ' ' 5N.“ 93 Figure 12.—-Normal bursa of fabricius and bursa with severe degenerative MDV lesions. a. Normal bursa from 22 day old chick x 100. b. Bursa from 22 day old chick with severe degenerative lesion and cyst formation x 100. 94 .>oz ouHB QOHuoomoH 0o .msonm Houpooo coHMHOmH .>oz ouHs oouomoeH Ho ozonmv mroHoo mono NoooHuem .>oz ouHs pouoomoH Amooum zv mEmo ooNHooEEHHomzo Eoum mHOHoo .>oz oHHB OopoomoH Auschm sz moooHuow Hmouopmfi Houspmo suHs monoo < .NoooHuco psoouHs woo ouHs memo Eoum zoomoum mo mHHOHos omhoo some mo QOmHHooEOUII.MH chomHm one. He 23o ..@_@_¢_N_0_.00¢N0 .H. ..0 N.0 n0 .v.0 0.0 0.0 N0 0.0 0.0 0.. smms u! IqbgaM Figure l4.—-Norma1 nerve and nerve with MDV a. b. lesion. Normal nerve from 18 day old chicken x 100. Nerve from 18 day old chicken with MD lesion x 100. 98 Figure 15.--Norma1 gonad and gonad with MDV a. b. lesion. Normal gonad from 18 day old chicken x 100. - Gonad from 18 day old chicken with MD lesion x 100. ....sixy. .l..~vl.’vl.h I. I . ...}... . m... ,o. I!) r... .. .. Figure 16.--Normal liver and liver with MDV lesion. a. Normal liver from 14 day old chicken x 100. ' b. Liver from 14 day old chicken with MD lesion x 100. 102 . «I \— .I; A ‘.;;y “«R3”:5* ."3$$,?‘V%I~ i.\x:¥§‘i:\\»_fl I :3”?! A?! -)’(’wuwmom mwuwn mo Hmnfisz m .mmunu ma men um muufln mo HanESZw .uuuo: mwoans “dam ma macho Houu:00 MwUMHOmw wgu aw xmfin um muufin mo Munsnzv .mwsumam mo H0255: wmmnm>¢o .mshfi> manmuo>ouwu spas woman mo Honfisz A .vuuo: mmmana 03» mw U cum I .xz msoum :w Mmfiu um muufln mo umnfiszm o o o o o o mHouunou onMHOmH o o o o o o U o Ao.aym Ao.H.N .m.Hv~ Am.mvm Ao.NvN m o ho.HvN Ao.HvN Am.HVN Am.HVN mno.Nvmm xz >coa«»:¢ o o o o o o afixm o o o o o o mmnvfim maouusoo o o o o o myo comamm kumHOmH Anya o o o o o cwxm Aonga Reflflcm AmHVH o o o o soaunx 8.3. a 93 N 32 a 8?; N o o o 53% o o o o o o o o afixm o o ANVH o o o o hwawwm o o o Anya o o o :moamm = c o o o o o o nwxm Rm. n a: w o o o o 0 >983 ammdyn ANHVH o onfiunH o o no nomamm xz wfiHH> .. hH. . nu ma 0H m w v ucmsummue p. mammfla coaum>ummno .. dam uwom mfimo omnommwm hconwuafi can mflhw> Haucwnmm .mfiumum muonaunm mo meflAO wwmomxo >nz aw hwonfincm can m5HH> mo maoflum>nmmno Hafiuconwwmul.wm mamas 105 had bursal lesions and l of 5 had liver lesions (Table 23). Bursal and liver lesions did not occur in either of the groups with antibody until the 12th day, a delay of four days compared with the antibody negative group. Nerve and gonad lesions exhibited a similar relationship between antibody positive and negative groups but occurred slightly later than bursa or liver lesions. Degenerative lesions were first observed in the feather follicle epithelium at 12 days in antibody negative birds and at 16 days in both groups of birds with antibody. The follicular lesions were similar to those described by Purchase (1970) and Nazerian and Witter (1970) and in some cases were accompanied by lymphocytic infiltrations. Inclusion bodies could be observed in some of the degenerating cells in the epithelium (Figure 17). Follicular lesions were first noted in only a small number of the follicles present however the number of positive follicles seemed to increase rapidly. At 20 days the majority of follicles were positive. Virus was isolated from the white blood cells of all three infected groups at 14 days (Table 24). However, the levels of virus were always approximately four times greater in the group without antibody. Virus was first isolated from the skin of antibody negative birds at 16 days whereas isolation of virus from the skin from the 106 .B>m Ho >Qz ou .moucu ma xmflu um woman wo Hwnfidzu mmflponflucm wanmuoouwp o: :ufls SHHSAOHm mEEmw w .uDOM mH Mmfln um mpufln mo gmnEDZ 03» ma MmHH um mcufin mo HonESZ© .pmuo: mmmfics w>ww we xmfln um mamas mo HmnEdzm :flxm amusm uw>flq pmcow w>~mz I mHmEMOZ v m U U U U U cflxm mmusm Hw>wq vmcoo ®>H®Z I | + V U N N N m N M N V m V m m m m m 0C O O O O O O O O O U C D O O O O O O O O O O O O M m 0 O O O O O O O O O O :flxm mwusm Hw>Hq cmcow m>uwz + wamfiuoz I m £ Q 0 O O O O O O O O O O O O O O O O O O O O O O O O O V N N V V V V M V V V V V V V U VOMWM MVNLDN NNNNN D O O O M O O O O m 0 V V V V V O O O O O O O O O O :flxm mmusm um>flq pmcoo m>umz + a: I N D 9 £ 0 O H m N H H M V M m m V m m cflxm mwhsm Hm>flq vmcou ®>H®Z + I + H m m M m m V M N V V V V M V V O O O O O O O O O O O O O O O O O O O O O O H M O O O O m O O O 0 v 0 v 0 m m m L £ m m N Q mm 0N ma ma va NH OH m 6 pwcflfimxm .mxm :flHDQOHw hponwucc Hmcuwumz uoq mdmma w _Ea COwUMHDUOCH Umom mhmo mCOMmmq mo wucmuusooo .B SHH> m mo >02 .mponfipcm unonues paw hponflpcm Umumumfidflfipm .mponflucw HMHSpmc zpfiB mxoflno vwmomxm >92 GM mcofimma osmoomouofla mo coaum>uwmno Hafiucmsvmmlx.mm mqmda .uuuwu 50:0 .0950» muuwn Ho Hanan: Hu>o cinnamon 3.55 No hue—52m. . ..5: no >9. cu $333.3 933033 on 51. 53.3.3 «Esau .mmnuqnm no nuns—E Mann—main .vcumawuum Hons—E uo>o may?“ «JAE—3.000” nun; acuwn mo umnfinzm n\o m\o Qo I H3502 I m nxo 8. 5 «\n :4. n\m I I + v . mxo vxo mxo mxo mxo nxo + 35oz I n «\o mxo aém} 8.5m} $.cm\m aimxm + a: I N mxo 8.2m} €.3m\~ 8.8m}. 8.5m} 08.333. gm 523:: + I + H m\o «.3 Co I 3502 I m «.3 . n\o . mxo . I I + e m}. v\~ mxo mxo m\o 30 + 2502 I m «\o mxo m3 m\o m\o mxo + a: I N mxo m\o m\o mxo m\o «\o ism 5322 + I - + a m\o mxo Qo 8.86 Q0 Q0 m\o on: I $5qu I m m\o mxo «\o :33 m\o m\o mxo on: I I + q 5 m} «\o 30 m3 m\o mxo aim GB mxm $8.1m mxo m\o mxo 20 on: + 03502 I m m\o mxo mxo m\o m\o m}. .56 51>” Em}. mxo mxo mxo m\o was + a: I N m\o m\o mxo m\o m\o «\o 5% Ga. Pa 3 mxv Aa mxun mxo mxo mxo «\0 on: man: + I + H a." ma 3 3 ca w o 5% roofing . , Inns—Hana msofl cwmomxm 5.333on Hmnnwumz uoa nhln E owing $58.55 no coma»: .95.; wnmmfla nut—omno mad, mfinmo >nz . .huoaflunm ”39.3w: can hwonwunm wmuopmwswficn .huonwunm Han—sum: .. ufiubonomaw >3— 5” mammwunm Hands can macawunm .uowumdumun 93.? mo sojourn—mm”? Hmwucmsvwmluém mamas Figure l7.--Feather follicle of MDV infected chicken with lymphocytic infiltra- tion and inclusion bodies. a. Feather follicle with lymphocytic infiltration around the follicle x 100. b. Feather follicle epithelium with inclusion bodies x 440. a” 110 antibody positive groups was delayed four days and levels were 1/3 of those of the antibody negative group. Precipitating antigens appeared in the skin in antibody negative birds at 14 days and at 18 days in both groups of antibody positive birds. Table 24 shows the disappearance of antibody in the group of birds administered gamma globulin and the birds with maternal antibody. The decline of antibody in infected and non—infected birds (lots 1 and 4) as well as in gamma globulin—inoculated birds seems to be identical and detectable antibody is absent from all groups by 16 days. The mean bursa weights of each of the groups is presented in Figure 18. The effect of MDV infection on development of the bursa in antibody negative birds can be seen by 13 days and bursa weights at 25 days were only 50-60% of the bursa weights of birds from the antibody positive groups or from the isolated controls. .>QS ##HS nofl#ommcfl on .mHouuqoo wmgmHOmH u o .Umuowmcfi >Q2 can muonfivnm mo wmum mxoflno u o .vmuommcw >o2 6cm QHHDQon mfifimm mo cofiumuumflcflfiwm map scum moonfipcm spas mxoflno n < .Umuommnfl >QS can mwonflpcm qunmpma amusumc cufl3 meHso n D .mponflncm psonufls paw :fiHnnoam mfifimm mo noaumnumflcflfipm Scum mponflnsm .hconflpqm quuwumfi nufl3 mawmoum mo mvsmwms amusn same we QOmHHmmfiooln.mH musmflm Nd n6 ¢.o m.o 0.0 #6 w.o m6 0.. swme u! IqbgaM DISCUSSION Influence of Maternal MDV Antibody on MDV Susceptibility of Progeny The serological data (Tables 8 and 9) show that the required classes of antibody positive and antibody free experimental chickens were obtained. The mortality and lesion response of antibody positive and antibody free progeny of these lines were compared (Tables 10 and 13). The results show that anti- body free progeny of each of the three lines were signifi- cantly more susceptible than the antibody positive progeny. This is in agreement with the observations of Chubb and Churchill (1968b). The virological and serological examinations of line 151 survivors after exposure to MDV (Table 12) revealed that there were higher levels of virus but lower titers and percent occurrence of antibody among progeny free of antibody at hatching than among progeny with maternal antibody at hatching. These results suggest that maternal antibody acts by limiting viral multiplication thus reducing damage to the immune system. This in turn provides the basis for a greater immune response among birds with maternal antibody than among those without 113 114 maternal antibody. Due to the possibility that genetic selection for resistance in the MDV exposed dams might also be playing a role in the observed effect, administration of immune gamma globulin was used to establish the specificity of the action of antibody. The administration of immune globulin to antibody free progeny at one day of age produced levels of protection similar to those seen in progeny with maternal antibody (Tables 19 and 20). The difference between the groups in the LPD50 titer of the inoculum measures the susceptibility of the experimental groups. The greater titer denoting the more susceptible group. The difference in susceptibility, by design, is due to the presence of antibody and its ability to neutralize the inoculated virus. This is expressed as the in zivg neutralizing index (NI). Activity was present against both cell associated and cell—free MDV but higher in vivg neutralization indicies (NIs) Were obtained against cell-free MDV. Calnek and Adldinger (1971) using an in vitrg serum neutralization test, have shown similar NIs for serums from infected birds. The greater activity towards cell-free MDV indicates that antibodies directed against the virus were present and active. In pathogenicity studies, other beneficial effects of maternal antibody were observed. In antibody free chicks microscopic lesions were first detected in the 115 bursa of Fabricus (Tables 21—23) at 8-10 days of age and in other tissues at 12—15 days of age. Bursa lesions ‘progressed from minor degenerative lesions to severe atrophy of follicles and cyst formation. In antibody positive chicks the appearance of bursa lesions were delayed 4—7 days from the time of appearance in antibody free chicks and severe degenerative lesions were not observed. This sparing effect on the bursa is also well illustrated by the comparison of bursa weights (Figures 13 and 18). Mean bursa weights for the antibody positive groups and the isolated control groups were similar where as bursa weights of the antibody free groups were markedly lower after day 13. Microscopic lesions in other tissues showed the same delay in appearance as was found with bursal lesions. The first isolations of virus were from the white cells and occurred at the same time for both antibody positive and antibody free chicks. However, the mean levels of Virus in antibody free chicks were always greater than the mean levels of virus in antibody positive chicks. Good correlations were found between microscopic lesions in the skin, precipitating antigen in extracts of skin and isolation of virus from the skin. It appears that either of the three methods could be used as an 116 indicator of virusv shedding.' A two day interval was >found between the appearance of lesions and precipitating antigens, and between precipitating antigens and virus isolation. This delay in detection is probably related to the concentration of virus in the tissue needed for detection by each of the methods; successful cell—free Virus isolation requiring a high concentration of virus, while at the time of appearance of lesions virus concen— tration is low. Several differences in the course of events following MDV infection in antibody free and antibody positive chicks have been observed. Briefly these are: (a) in antibody negative birds virus spreads more rapidly and reaches higher levels in all tissues than in antibody positive birds and (b) the development of the bursa in antibody negative birds is severely imparied presumably reducing the ability to elicit an acquired immune response while in antibody positive birds the severity of bursa lesions is reduced presumably allowing a more normal development of ability to elicit an acquired immune response. One explanation for the observed differences may be found in a possible differential rate of spread of infection within the chicken by cell—free and cell associated virus. Early infected tissues may liberate 117 cell—free and cell associated virus. Even small amounts of cell—free virus would be more efficient and result in a more rapid spread of infection than the cell associated virus. Rapid spread would lead to early involvement of the bursa resulting in severe lesions and an impaired ability to elicit an acquired immune response. However, when maternal antibody is present the cell—free virus is neutralized. This would leave only the cell associated virus and its inefficient method of cell to cell spread of infection. This method of spread would require a longer period for the spread of infection to pathologically sensitive tissues. During this interval there would be more time for the development of an immune response to protect the bird after maternal antibody had disappeared. The major drawback of this hypothesis is that to date cell—free virus has not been detected within the birds, however, this does not mean that cell-free virus is not present. Although other hypotheses for the mechanism of action of antibody are possible, this one seems best able to explain the observed results. Hyperimmunization of Dams as a Means of Decreasing the Susceptibility of Progeny Hyperimmunization of dams as a means of decreasing the susceptibility of their progeny was found to be of little value. Hyperimmunization, i.e., repeated 118 inoculation with MDV of dams, as shown in Tables 8 and 9, had no apparent effect on the serological status of these dams thus no differences in mean antibody titers were detected between the natural exposed and the hyperimmunized dams by either the AGP or FA tests. Challenge of progeny from hyperimmunized and natural exposed dams revealed no significant difference in susceptibility to MD (Tables 10 and 13), time of appearance of lesions (Table 21), mean bursa weights (Figure 13), or time of first recoverable virus (Table 22). Only in the case of virus levels in survivors at 12 weeks (Table 12) were differences noted in favor of progeny from hyperimmunized dams. The results of these studies indicate that hyper— immunization of dams as a means of decreasing the sus- ceptibility of progeny is of doubtful value. Although some reports indicate a decrease in susceptibility of progeny from hyperimmunized dams (Eidson, 1968; Arbor Acres, 1968; Ball et_31., 1970). Ball et_al. (1971) reported that in flocks experiencing early natural exposure, hyperimmunization of dams did not decrease the incidence of MD in their progeny. Witter (1971) has shown that under natural conditions antibody persists for long periods of time and that precipitin titers remain high when persistent viremias are present. The lack of effect of hyperimmuniza— tion could be due to MDV exposure from natural sources 119 providing full stimulation and in a sense naturally hyperimmunizing the birds. In such a situation further hyperimmunization, inoculation with additional MDV, would not be expected to have any effect. It is also possible that in hyperimmunizing with a cell associated virus, the virus is not presented in a form capable of stimulating the immune system to produce high titers of antibody. Influence of HVT Vaccination of Dams on the Susceptibility of Progeny Progeny from HVT vaccinated dams were found to be slightly less susceptible to MD than progeny from non- vaccinated dams. Mean MD antibody titers of HVT vaccinated dams did not differ from those of non—vaccinated dams (Table 9). Mean titers of maternal antibody in progeny from each of the groups were also similar, however, antibody levels against HVT were not determined. Challenge of progeny of vaccinated and non- vaccinated dams (Table 13) suggest a slight decrease in the susceptibility of progeny from HVT vaccinated dams. Statistical analysis (Table 15) of differences between groups shows significant difference between two of the HVT vaccinated groups, VH—HVT and VH-MDV, and the non— vaccinated groups. The remaining HVT vaccinated group, group V, is not significantly different from either of the HVT vaccinated groups or from the non-vaccinated .IV‘ 'ti_$ ' '2' ’3 OW- the virus particle. -_:%..IT6?713” Q ;:{11; .7.§ Although the vaccination of dams with HVTquQ§-.;r f5_ ‘.;;7 not seem to increase parental antibody titers to MDV. Sn V V ,f advantage to the progeny does seem to be present. This I advantage could be due to an increase in antibodies directed toward HVT since antibody to HVT does provide protection from MD. , $1.111. .. S UMMARY A. Maternal antibody markedly influences the course of MDV infection in young chickens. It was found that the presence of antibody: (1) reduces the incidence of mortality and lesions, (2) delays the onset of lesions in various organs, (3) reduces the recoverable levels of virus from survivors, (4) raises the incidence and mean titers of antibody in survivors, and (5) is effective against both cell-free and cell associated virus. The foregoing effects of antibody are the same whether it is naturally acquired via the egg or administered as immune gamma globulin at one day of age. B. Hyperimmunization of dams in attempts to increase antibody titers in these dams and thereby increase resistance to MD in progeny was found not to be effective. Higher antibody titers were not produced in the hyperimmunized dams and progeny from hyperimmunized dams did not differ from progeny of natural exposed dams in the following observations: (1) MD antibody titers at day of age, (2) incidence of mortality and lesions, (3) appearance of lesions, (4) antibody titers at 12 weeks, and (5) appearance of recoverable virus. 121 -Progeny from HVT vaccinated dams were observéa.%g . 7*. a slight increase in resistance over progeny frOm noné vaccinated dams with antibody to MDV. 'This resistance could not be related to the antibody detected. LITERATURE C ITED 123 LITERATURE CITED Ahmed, M., and G. Schidlovsky. 1968. Electron microscopic localization of herpes virus-type particles in Marek's disease. J. Virol._ 2:1443—1457. Anderson, K., and M. Sevoian. 1969. Bioassay of cell cultures containing a Type II leukosis virus (JM Strain). Avian Diseases. 13:486-499. Arbor Acres Review. 1968. Progress report No. 2. Leukosis (Marek's disease). Armed Forces Institute of Pathology. 1960. Manual of Histologic and Special Staining Techniques. 2nd ed. Ed. Helen K. Steward. McGraw-Hill Book Co., New York. Ball, R. F., J. F. Hill, J. Lyman and A. Wyatt. 1970. The resistence to Marek's disease of chicks from immunized breeders. Poultry Sci. 50:1084-1085. Ball, R. F., J. F. Hill, J. Lyman and A. Wyatt. 1971. The effect of early natural exposure to Marek's disease on immunization of breeders by vaccination. Poultry Sci. 50:648—649. Bankowski, R. A., J. E. Moulton, and T. Mikami. 1969. Characterization of the Cal—l strain of acute Marek's disease agent. Am. J. Vet. Res. 30: 1667—1676. Baxendale, W. 1969. Preliminary observations on Marek's disease in ducks and other avian species. Vet. Record. 85:341—342. Beasley, J. N., L. T. Patterson and D. H. McWade. 1970. Transmission of Marek's disease by poultry house dust and chicken dander. Am. J. Vet. Res. 31: 339-344. Biggs, P. M. 1966. Avian leukosis and Marek's disease. 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Avian Diseases. 15:346-365. APPENDIX 135 kw --~Tass @f 100 units of penicillin, 100 micrograms of strepto- mycin and 50 units of mycostatin per 1.0 ml. of media were added to control contamination. CK Growth Medium BME (Earls Salts) (10x)ac -- 40. Tryptose Phosphate Broth —- 49. Bovine Fetal Seraa —— 22. 2.8% Bicarb -- 22. H20 -- 360. Antibiotics added for DEF growth media. Phosphate Buffered Saline NaCl -- KC1 -- KH2PO4 ‘- NazHPO4 _- HzO -- pH adjusted to SPGA Buffer Sucrose KH2PO4 K2HPO4 8.0g 0.29 1.29 0.29 to 1000 ml 7.2-7.4. "" 7o -- 0. -- 0. 0 ml 6 5 0 0 49 0529 1639 Mono Sodium Glutamate Disodium Ethylenediamine— tetracetete Bovine Albumin Powderc H20 pH adjusted to 6.3 136 0.0839 0.29 1.09 to 1000 ml. "€929. ' Heat to boiling to dissolve: of phenol. Filter through tiSsue j.p--- Fluorescent Antibody Buffer FTA Hemagglutination Bufferg -- 9.239 H20 -- to 1000 m1. aGrand Island Biological Company P.O. Box 68 Grand Island, New York 14072 bDifco 920 Henry Street Detroit, Michigan 48201 CColorado Serum Company 4950 York Street Denver, Colorado 80261 eMiles Laboratory, Inc. P.O. Box 272 Kankokee, Illinois 60901 fConsolidated Laboratories, Inc. P.O. Box 234 Chicago Heights, Illinois 60412 gBaltimore Biological Laboratories P.O. Box 175 Cockeysville, Maryland 21030 f“"III'I‘II‘IIIIIIIII[IIIIIIIII’IIIIIIIII