Immune response of chickens to live and inactivated avian infectious bronchitis virus

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Iowa State University Capstones, Teses and
Retrospective Teses and Dissertations
Dissertations
1970
Immune response of chickens to live and
inactivated avian infectious bronchitis virus
Manuel Farnil Coria
Iowa State University
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Recommended Citation
Coria, Manuel Farnil, "Immune response of chickens to live and inactivated avian infectious bronchitis virus" (1970). Retrospective
Teses and Dissertations. 4827.
htps://lib.dr.iastate.edu/rtd/4827
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71-14,215
CORIA, Manuel Farnil, 1925-
IMMUNE RESPONSE OF CHICKENS TO LIVE AND
INACTIVATED AVIAN INFECTIOUS BRONCHITIS VIRUS.
Iowa State University, Ph.D., 1970
Veterinary Science
University Microfilms, A XEROX Company, Ann Arbor, Michigan
IMMUNE RESPONSE OF CHICKENS TO LIVE
AND INACTIVATED AVIAN INFECTIOUS
BRONCHITIS VIRUS
by
Manuel Farnil Coria
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Major Subject: Veterinary Microbiology
Approved:
Signature was redacted for privacy.
in Charge o± Major Work
Signature was redacted for privacy.
Head of Major Department
Signature was redacted for privacy.
Iowa State University
Of Science and Technology
Ames, Iowa
1970
ii
TABLE OF CONTENTS
Page
INTRODUCTION 1
REVIEW OF LITERATURE , 3
Etiology of Avian Infectious Bronchitis Virus 3
Isolation and Pathology of IBV in Chicken Embryos 4
Serologic Relationships Among Strains of IBV 5
Adaptation and Propagation of IBV in Cell Cultures 7
Infectivity of Cell Culture Propagated IBV for
Chicken Embryos 10
Detection of Intracellular IBV by
Immunofluorescence 11
Electron Microscopy of IBV 13
Immune Response of Chickens to Inactivated IBV 15
Immune Response of Chickens to Live IBV 19
MATERIALS AND METHODS 27
IBV Strains Used 27
Iowa 33 27
Beaudette 27
Massachusetts 82828 28
Iowa 97 28
Connecticut AS968 28
Cell Culture and Overlay Media 29
Initial culture medium 29
Maintenance medium 30
iii
Phosphate buffered saline 30
Trypsin solution 31
Agar-overlay medium 31
Neutral red solution 32
Chicken Embryo Kidney Cell Cultures 32
Adaptation of IBV to CEK Cells 34
IBV Propagation in Chicken Embryos 34
Antiserum Preparation 35
Antiserum to IBV-33 35
Antiserum to normal chicken globulins 36
Preparation of Conjugated Antiserums 37
BPL Inactivated IBV-33 41
Immune Response of Chickens 42
Chickens used 42
Challenge procedures 43
IBV isolation 43
CEX exposed 44
Challenged 44
Tissue smears 44
Conjugate staining and examination 45
Immune response to BPL inactivated IBV-33 46
Immune response to CEK adapted IBV 47
Determination of immunity 55
Negative Staining for Electron Microscopy 56
iv
RESULTS 58
Adaptation and Propagation of IBV-33 in CEK Cells 58
Infectivity of CEK Propagated IBV-33 for Chicken
Embryos 60
Immune Response of Chickens to BPL Inactivated
IBV 63
Serologic response 63
Neutralizing indices of pooled sera to 5
strains of IBV 67
Response to challenge 69
Immune Response of Chickens to CEK Adapted IBV 75
Isolation of virus in CEK cells 75
Intracellular IBV antigens of CEK adapted
IBV 78
Serologic response 78
Response to challenge 78
Electron Microscope Examination of Negatively
Stained IBV 86
DISCUSSION 90
SUMMARY 99
LITERATURE CITED 101
ACKNOWLEDGEMENTS 111
1
INTRODUCTION
Avian infectious bronchitis virus (IBV) has been deter-
mined to be the etiologic agent of an acute, highly conta-
gious, panzootic, respiratory disease occurring in chickens
of all ages. IBV has been implicated in ovarian lesions
with a resultant reduction in egg production and a nephritis-
nephrosis syndrome.
Methods of controlling this disease by immunization
have produced inconclusive results. The use of live IBV
has been confined to egg passage material, that will pro-
duce immunity for periods up to 18 months. This method
of immunization amounts to controlled exposure and gener-
ally cannot be used to immunize chickens close to laying
age, due to losses in egg production.
In England and some western European countries the use
of live IBV vaccines is prohibited. The majority of IBV
strains isolated in these countries belong to the Massachu-
setts serotype. These strains have been used in production
of inactivated IBV vaccines. Immunizations with inactivated
vaccines have failed to induce neutralizing antibody against
standard Massachusetts type strains. One vaccine has been
reported to produce antibodies to its homologous strain used
in preparation of the vaccine.
Two aspects of the immune response of chickens to IBV
were selected for this study: 1. The adaptation and
2
propagation of IBV-33 in chicken embryo kidney cells and its
ability to induce immunity in susceptible chickens, to sub-
sequent challenge. Also determination of the effect of suc-
cessive passage in cell cultures on virulence for chicken
embryos. 2. Inactivation of IBV-33 by Beta-propiolactone
and its use as an immunizing agent.
3
REVIEW OF LITERATURE
Etiology of Avian Infectious
Bronchitis Virus
Schalk and Hawn (1) in 1931 authored the first descrip-
tion of a highly infectious respiratory disease that caused
heavy mortality in chicks and called it infectious bronchitis
of baby chicks. The virus etiology of this disease was sug-
gested by Bushnell and Brandly (2), but was first estab-
lished conclusively by Beach and Schalm (3), and Beaudette
and Hudson (4) by passing supernatant fluid of ground infec-
ted tissue through all grades of Berkefeld filters.
Infectious bronchitis virus (IBV) was tentatively class-
ified by Berry et al. (5) and Berry and Cruickshank (6) as a
Myxovirus on basis of ether sensitivity, size of 80 to 120
millimicrons, pear shaped surface projections, no hemaggluti-
nin or neuraminidase and a medium sized ribonucleoprotein
core. Corbo and Cunningham (7) have demonstrated presence
of a hemagglutinin in trypsin-modified IBV.
Almeida et al. (8) proposed the name of CORONAVIRUSES
for IBV and a group of IBV-like viruses of human and murine
origin. This suggestion has been accepted by members of the
Myxovirus Study Group under the International Committee for
Nomenclature of viruses.
4
Isolation and Pathology of
IBV in Chicken Embryos
Beaudette and Hudson (4) were the first to cultivate IBV
in chicken embryos. They observed that the first 6 passages
were relatively non-lethal to embryos. An increase in viru-
lence on further passage was noted, with death resulting in
a majority of embryos. Gross lesions were dwarfing of the
embryo, thinning of the chorioallantoic membrane and thick-
ening of yolk material. Delaplane and Stuart (9] confirmed
these findings and in addition found white, opaque, circular
lesions on chorioallantoic membranes at point of inoculation.
With successive passage in eggs, virus increased in lethal-
ity for embryos, with a progressive decrease in time required
to produce death of embryos. An increase in lethality for
embryos was accompanied by a decrease in virulence for
chicks.
Later, Delaplane (10] reported that an allantoic cavity
route of inoculation was superior to chorioallantoic membrane
route, for initial isolation of virus, as evidenced by
dwarfing in first passage.
Fabricant (11) confirmed findings of previous authors
as to gross pathologic changes of chicken embryos inoculated
with IBV. In addition he described "curling" of embryos into
a ball-like shape. This curling precedes dwarfing, and
occurs in infected embryos of normal size. Loomis et al.