STUDIES ON EGG DROP SYNDROME
343
STUDIES ON EGG DROP SYNDROME ... ANDREW CHANDRAMOHAN 1.0. No. 246 1334 DEPARTMENT OF MICROBIOLOGY , MADRAS VETERINARY COLLEGE MADRAS - 600 007 1994
Transcript of STUDIES ON EGG DROP SYNDROME
STUDIES ON EGG DROP SYNDROME
... ANDREW CHANDRAMOHAN 1.0. No. 246
1334
DEPARTMENT OF MICROBIOLOGY , MADRAS VETERINARY COLLEGE
MADRAS - 600 007
1994
STUDIES ON EGG DROP ·SYNDROME
A~DREW CHANDRA¥QBAN. 1.0. No. 246 '
Thesis sulnnitted in partial fulfilment of the
requirements for the degree of
POCTOR OF PHILOSOPHY
%TERINARY MICROBIOLOGY
to the
Tamil Nadu Veterinary and Animal Sciences University
Madras
. DEPARTMENT OF MICROBIOLOGY MADRAS VETERINARY COLLEGE
MADRAS - 600 007
1994
CERTIFICATE
This is to certify that the thesis entitled "STUDIES ON EGG DROP
SYNDROME" submitted in part fulfilment of the requirements for the degree of
DOcrOR OF PHILOSOPHY IN VETERINARY MICROBIOLOGY to the Tamil
Nadu Veterinary and Animal Sciences University, Madras is a record of bonafide
research carried out by Thiru. ANDREW CHANDRAMOHAN under my
supervision and guidance and that no part of this thesis has been submitted for the
award of any other degree, diploma, fellowship or other similar titles or prizes and
that the work has not been published in part or full in any scientific or popular journal
or magazines. _r
Date: 2..t..lf. '7 4- ~·V0b. 8-84-'
Place: Madras (DR. R.A. VENKATESAN) CHAIRMAN -./' Jf
APPROVED :1 ! ~ l q': CHAIRMAN (DR.R.AVENKATESAN)~' V j ..
MEMBER: 1. ~\~~ 0\1:( OR.V.D.PADMANABf'\N
MEMBER: 2. OR.AALBERT
MEMBER: 3. DR.P.RAMADASS
EXTERNAL EXAMINER ____.l_",-,ILJ'-':" ~~, 1tr1~op-
CURRICULUM VITAE
Name of the candidpte
Date of birth
Place of birth
Moior field of sQecfP'~siJf.fon
Educational status
Professional Experience I
Marital status .
Permanent addresl
Publications made
Memberships of Professional SocielY
\ ANDREW CHANDRAMOHAN \
22.11.1948
Madras
Veterinary Microbiology
B.v.Sc in 1974
M.V.Sc in 1984
11.9.74 to 30.10.76: Extension Veterinary Assistant Surgeon, Tamil Nadu Animal Husbandry Service.
31.10.76 to 25.6.87: Research Assistant , Institute of Veterinary Preventive Medicine, Ranipet.
26.6.87 to 25.2.90: Assistant Research Officer, Institute of Veterinary Preventive Medicine, Ranipet.
26.2.90 to the present: Asst. Professor, Dept. of Animal Biotechnology, Madras Veterinary College, Madras 600 007.
Married
M1, Rams Rats, West Mambalam, Madras 600 033.
Research papers - 6 Popular articles - 2
.~ 1. Tamil Nadu Veterinary Council. 2. Indian Society for Veterinary
Immunology & Biotechnology.
ACKNOWLEDGEMENT
I deem it my privilege and duty to express my heartfuI thanks to my respectful
guide, Dr.R.A.Venkatesan, MVSc, PhD, Professor & Head, Department of
Microbiology, Madras Veterinary College, Madras for his valuable guidance. He was
endlessly resourceful. His suggestions were always practical and constructive. But for
his encouragement this research work would not have been what it is now.
I acknowledge a special debt of gratitude to Dr.V.D.Padmanaban, MVSc,
PhD, Registrar, Tamil Nadu Veterinary & Animal Sciences University for his valuable
advice and encouragement as a member of the advisory committee.
I sincerely thank Dr.A.Albert, MVSc, PhD, Professor, Central University
('"\
Laboratory, Madhavaram and member of my advisory commiVee for his immense
support and prompt help.
I specially thank Dr.P.Ramadass, MVSc, DVPH, PhD., Professor, Dept. of
Animal Biotechnology and member of the advisory committee for his intellectual
company. The numerous discussions I had with him helped me to develop and
modify my ideas and give them a proper shape.
I place on record my deep sense of gratitude to Dr.K.Nachimuthu, MVSc,
PhD, Professor & Head, Dept. of Animal Biotechnology and all the staff of the
department of Animal Biotechnology for their ready assistance and kind help
throughout the period of this study.
extend my sincere thanks to Dr.M.Chandran, MVSc, Ph.D.,
Dr.V.Purushothaman, MVSc, PhD. Professors and Dr.T.G.Prabakar, MVSc,
Dr.R.Kalaimathi, MVSc, Assistant Professors, Dept. of Microbiology for their kind
help.
lowe my special thanks to Dr.Daniel Joy Chandran, MVSc, PhD, Professor
& Head, Dept. of Microbiology, Veterinary College and Research Institute, Namakkal
and Dr.K.Kumanan, MVSc, PhD, Associate Professor, Dept. of Animal
Biotechnology for their unflinching support.
I thank my friends and co-researchers, Dr.R.Govindarajan, Dr. A.M. Shaw,
Dr.Y.K.M.Reddy, Dr.Narasimha Reddy and Dr. Joseph Andrew Jesudas for
their co-operation and help.
Words will not suffice to express my gratitude to my mother, who planted the
seed of hope in me.
Above all, I thank God Almighty for blessing me with success in aU my
endeavours.
Title
Name of the student
Degree for which submitted
Name of the Chairman
Year and University
ABSTRACT
SllJDIES ON EGG DROP SYNDROME
ANDREW CHANDRAMOHAN
Ph.D in Veterinary Microbiology
Dr. RA.VENKATESAN, MVSc., Ph.D Professor & Head, Dept. of Microbiology, Madras Veterinary College, Madras 600 007.
1994, Tamil Nadu Veterinary and Animal Sciences, Madras 600 007
A study has been undertaken to assess the seroprevalence of Egg drop
syndrome-76 (EDS-76) in eight districts of Tamil Nadu, employing haem agglutination
inhibition (HI) and dot immunobinding assay (DIA). The overall incidence was 15.7
per cent. Madurai district recorded the highest percentage of incidence. The incidence
was more in birds above 40 weeks of age. HI test was found to be the test of choice
for serological screening.
Out of the 158 samples screened for virus isolation, five yielded virus isolates
from different areas and their identity was confirmed by HI test and immunoelectron
microscopy employing standard antiserum.
The replication behaviour of the isolates was studied in CEl, DEF and Duck
Pekin cell cultures. The infectivity titres were higher in DEF and Duck Pekin cell line.
The isolates were characterized and were found to be resistant to chloroform, ether
and acidic pH indicating the absence of envelop. The isolates were stable at 56°C for
30 min and resistant to 0.3 per cent trypsin. Although the isolates lost the infectivity
titre, the HA activity was retained when treated with 0.3 per cent formalin. Treatment
with nucleic acid inhibitor (IUDR) confirmed the DNA nature of the isolates.
Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed
the isolates to contain identical numbers of polypeptides. Restriction enzyme analysis
of the DNA with enzyme Pst I, revealed identical banding pattern thereby confirming
the genetic similarity between the isolates.
Experimentally infected birds excreted the virus in the faeces upto 13 days PI,
and for the detection of antigen, OIA was found to be the sensitive test. In
experimentally infected birds, EDS-76 antibody was first detected on 8th day PI and
rose gradually to a peak between 3rd and 4th week. HI test is the test of choice for
the routine detection of EDS-76 infection in poultry flocks.
CONTENTS
Page No.
UST OF TABLES
UST OF RGURES
UST OF ABBREVIA nONS
1. INTRODUCTION
2. REVIEW OF UTERA TURE
2.1 SEROPREVAlENCE OF AVIADENOVIRUS· /
2.1.1 Viral etiology
2.1.2 Avian adenoviruses :3
2.1.3 Properties of the avian adenovirus ~
2.1.4 General properties of EOS-76 virus '1
7
2.2 SEROPREVAlENCE OF EOS-76 "7
2.2.1 Seroprevalence in countries other than India
2.2.2 Seroprevalence in India l\
2.2.3 Haemagglutination-Inhibition (HI) Test I')
2.2.4 Dot Immunobinding Assay (DIA) 1 b
2.2.5 Comparison of serological tests for detection of antibodies to EOS-76 virus 17
2.3 ISOLATION OF 11iE VIRUS 18
2.4 CHARACTERIZATION OF THE VIRUS ~o
2.4.1 Physico-chemical characterization of ;1.0
EDS-76 virus
2.4.2 Electron microscopic studies of EDS-virus ~I
2.4.3 Growth characteristics of EDS-76 virus in :Z~ cell culture
2.4.3.1 Growth characters in priman; cell culture .~f..
2.4.3.2 Growth of EDS-76 in cell lines ;1.3
2.4.3.3 Cytopathic effect of EDS-76 virus in .2.~
cell culture
2.4.4 Cross haemaggiutination inhibition of ~>~
EDS-76 virus
2.5 PROTEIN FRACTIONATION STUDIES ~~
2.5.1 Protein fractions of avi-adenoviruses ?b
2.5.2 Protein fractions of EDS-76 virus ').t
?-7 2.5.3 Concentration and purification of the virus
2.5.4 Polyacrylamide gel electrophoresis (PAGE) ~g-
~g
2.5.5 Staining of gels ;2,,,
2.6 DNA ANALYSIS OF ADENOVIRUSES
~q
2.6.1 Restriction endonuclease fingerprinting
2.6.2 Restriction DNA analysis of aviadenoviruses :2.'1
2.6.3 Restriction DNA analysis of EDS-76 virus 30
30 2.7 EXPERIMENTAL INFECTION OF EDS-76 VIRUS
2.7.1 Histopathology of EDS-76 3f'.,
3 MATERIALS AND METI10DS 3-4
3.1 MATERIAL FOR SEROPREVALENCE 3~
3.1.1 Serum samples from chicken "3//
"3"'1 3.1.2 Referral virus
'3.>
3.1.3 Chicken erythrocytes '3>""
3.1.4 Phosphate buffer saline-Tween 20 ':35>
3.1.5 Bovine serum albumin
3.1.6 Enzyme substrate '35-
3.1.7 Horse radish peroxidase "3s-
"36
3.2 METHODS
3.2.1 Haemagglutination test "3~
3.2.2 HaemaggJutination inhibition 36
3.2.3 Dot immunobinding assay 3':1
3.3 iSOLATION AND iDENTIFICA110N OF VIRUS 3S>
3P 3.3.1 Collection of materials
3e 3.3.2 Duck embryos
3.3.3 Cell culture 3€
3.3.3.1 Hanks basal salt solution 3~
3.3.3.2 Trypsin 0.25 per cent $~
3.3.3.3 Trypsin versene glucose solution 3<::t
3.3.3.4 Serum supplement 34
3.3.3.5 T ryptose phosphate broth ),-0
3.3.3.6 Antibiotic solution ..It'()
3.3.3.7 Sodium bicarbonate solution /.to
3.3.3.8 Medium 199 40
3.3.3.9 Minimum essential medium itl
3.3.3.10 Fixative fluids r -41
3.3.3.10.1 Carnoy's fluid 41
3.3.3.10.2 Bouin's fluid .-4,
3.3.3.11 Staining solution 41
3.3.3.11.1 Rappaport stain 41
3.3.3.11.2 Haematoxylin stain 42-
3.3.3.11.3 Eosin stain J.r~
3.4 METI-IODS OF VIRUS IS OrA TION 4~
AND IDENTIFICATION
3.4.1 Virus isolation Lt£
3.4.1.1 Processing of faecal swabs 4:2-
3.4.1.2 Processing of shell glands "',3
3.4.1.3 Inoculation in duck embryo 4~
3.4.2 Preparation of EDS-76 virus hyperimmune serum 43
3.4.3 Cross haem agglutination inhibition test 4L,
3.5 CHARACTERIZATION OF TI-lE VIRAL ISOLATES 4~
3.5.1 Preparation of primary cell culture J.., .I,
3.5.2 Subculturing of duck Pekin cell line LtL,
3.5.3 Adaptation of EDS-76 viral isolates in CEL, 4-;;-DEF cell cultures and Duck Pekin cell line
3.5.4 Study on CPE 4>-
3.5.5 Titration of viral infectivity in ~'>'
primary cell culture and cell line
"'6 3.5.6 Physico-chemical properties
3.5.6.1 Sensitivity of the isolates to chloroform ~G
3.5.6.2 Sensitivity of the isolates to ethyl ether 46
3.5.6.3 Effect of pH on viral isolates Lj'1
3.5.6.4 Sensitivity of the isolates to trypsin ;"'1
3.5.6.5 Thermostability of the viral isolates ,J..,t
3.5.6.6 Effect of formalin on viral isolates l-j-;.
3.5.6.7 Determination of the type of viral 4f'>
nucleic acid
3.5.7 Electron microscopic studies of .Ij~
viral isolates
3.5.7.1 Direct negative contrast electron ' ),~
microscopy
3.5.7.2 Immuno electron microscopy 4'1
3.6 PROTEIN FRACTIONATION STUDIES 4C1
3.6.1 Solutions for SOS-Polyacrylamide ~0
gel electrophoresis
3.6.1.1 Acrylamide-bisacrylamide 4"
3.6.1.2 Separating gel .,If q
3.6.1.3 Stacking gel 5'0
3.6.1.4 Running buffer 50-0
>'0 3.6.1.5 Sample buffer
3.6.1.6 Staining solutions ~I
3.6.1.7 Destaining solution SI
3.6,2 Methods for protein fractionation S-I
3.6.2.1 Purification of EOS-76 viral isolates ~I
3.6.2.2 SDS-PAGE £'_z.
3.6.2.3 Estimation of molecular weight $"?,
3.7 RESTRICTION ENlYME ANALYSIS OF DNA OF ;,~:3
EDS-76 VIRAL ISOLA 1ES
3.7.1 Materials used for DNA analysis _;-s
3.7.1.1 Proteinase K S"'?>
3.7.1.2 Phenol chloroform isoamyl alcohol mixture s-~
3.7.1.3 Tris borate buffer 0-3
3.7.1.4 Agarose gel ~"4
3.7.1.5 Restriction enzymes ~-4
3.7.1.6 Gel loading buffer C:z,
3.7.1.7 DNA marker ,~~
3.7.2 Methods for DNA analysis :_,-s--
3.7.2.1 Purification of viral isolates ~'-S
3.7.2.2 Extraction, of viral DNA tlt;"
3.7.2.3 Enzyme digestion of viral DNA b ...... S'
3.7.2.4 Electrophoresis of DNA fragments ':>-b
3.8 EXPERIMENTAL INFECTION OF EDS-76 S'b
VIRAL ISOLA 1ES
~b 3.8.2 Chicken
~b 3.8.3 Methods in experimental infection
3.8.3.1 Inoculation of birds ~b
3.8.3.2 Collection of samples ~;-~
3.8.3.3 Antigen detection S'":i
3.8.3.4 Virus recovery ~':;
3.8.3.5 Monitoring of serum antibody titres $'f!
4 RESULTS ~ .... ~
4.1 SEROPREVAlENCE ~-q
4.2 ISOLATION bLI
4.2.1 Virus isolation from cloacal swabs 6'-1
4.2.2 Virus isolation from shell gland 6",
4.2.3 HA titre of isolates bL,
4.2.4 HI titre of reference serum with EDS-76 .. b?-isolates
4.2.5 Gross HI test of isolates with reference (,-;
strain 127
4.3 ADAPTATION OF EOS-76 ISOLATES IN 6'1 CEll CUl TURE
4.3.1 CEl cell culture b?-
4.3.2 DEF cell culture 6"'1-
4.3.3 Duck Pekin cell line '10
4.4 CYrOPATI-IOGENICIlY OF EDS-76 ISOLATES "H:1
4.4.1 CEl ceil culture "=10
4.4.2 DEF cell culture ":to
4.4.3 Duck Pekin cell line 10
4.5 INFECTIVIlY ASSAY OF EOS-76 ISOLATES 1--1 IN CELL CUl TURE SYSTEM
4.6 PHYSICO-CHEMICAL PROPERTIES ':l.1
4.6.1 Electron microscopic study '=1("
4.6.1.1 Direct negative contrast electron microscopy ~b
4.6.1.2 Immuno electron microscopy '='~
4.7 PROTEIN PRORLE OF EDS-76 -,.-6 VIRAL ISOLATES
4.8 RESTRICTION ENDONUCLEASE ANALYSIS "'=Jf'
OF EDS-76 VIRUS ISOLA.TES
4.9 EXPERIMENTAL INFECTION "':It
4.9.1 Clinical findings ,.~
4.9.2 Virus excretion ~o
4.9.3 Tests employed for virus detection 80
4.9.4 Virus resolution ~~
4.9.5 Detection of antibody in experimental birds ~!2
4.9.6 Tests employed for detection of EDS-76 antibody ~~
5 DISCUSSION '<--4
5.1 SEROPREVAlENCE {h,
5.2 ISOLATION t~
5.3 ADAPTATION OF EDS-76 ISOLATES IN ~ CELl.. CULTURE
~I 5.4 CHARACTERIZATION OF 111E EDS-76 ISOLATES
5.5 PROTEIN FRACTIONATION STUDIES 93
5.6 DNA RESTRICTION ENDONUCLEASE ANALYSIS OF Cf3
EDS-76 VIRUS ISOLATES
5.7 EXPERIMENTAL INFECTION OF EDS-76 OJt,
VIRAL ISOLATES
6 SUMMARY Ci";t
REFERENCES I 0 I
LIST OF TABLES
Table No. Title Page No.
SeropreValence of EDS-76 virus in Tamil Nadu 60
II Age-wise incidence of HI antibodies 6" to EDS-76 virus
III Comparison of results of HI test and DIA 6.2
IV Comparison of mean antibody titres of EDS-76 . 63 virus by HI test and DIA
V Comparative efficacy of DIA against HI in 63 detecting EDS-76 viral antibodies in serum
VI Details of area-wise sample collection ~$"
VII Details of source for EDS-76 viral isolation 6'
VIII HA titre of EDS-76 viral isolates 61
IX HI titre of referral EDS-76 antiserum with 60Q EDS-76 viral isolates
X Cross HI test of EDS-76 viral isolates with bq
referral strain 127
XI Infectivity titre (TCID50) of EDS-76 viral isolates in cell culture systems after fifth and 1-Jit
tenth passages.
XII PhYSico-chemical characters of the EDS-76 viral ~;:2.
isolate (Bl)
XIII Physico-chemical characters of the EDS-76 viral "3 isolate (Ml)
XIV Physico-chemical characters of the EDS-76 viral ~lt
isolate (01)
XV PhYSico-chemical characters of the EDS-76 viral 7'_ isolate (B2)
XVI Physico-chemical characters of the EDS-76 viral "').J:--
isolate (02)
XVII Viral protein bands of djfferent isolates and their ~~ molecular weights
XVIII EDS-76 virus DNA restriction fragments generated by ?q Restriction endonuclease Pst I
XIX Details of virus excretion in experimental birds ~{
XX Details of HA titre range of virus excreted gl
by experimental birds
XXI Comparison of results of HA, AGIO and DIA employed S3 for virus detection in cloacal swabs.
XXII Details of HI antibody titre range in experimentally '!s infected birds.
XXIII Comparison of results of HI, AGIO and DIA for EDS-76 tl3
antibody detection in serum samples
Fig.l
Ag.2
Ag.3
FigA
Fig.S
Ag.6
Fig.7
Fig.8
Ag.9
Fig.I0
Fig.II
LIST OF FIGURES
Quantitative dot immunobinding assay on nitrocellulose membrane to screen ED5-76 viral antibodies.
Chicken embryo liver monolayer infected with ED5-76 viral isolate, depicting derangements initially as rounding and grouping of cells. H & E staining.
Chicken embryo liver monolayer infected with ED5-76 viral isolate showing large vacuolations with cytoplasmic anastomosis of cells. H & E straining.
Chicken embryo liver monolayer- Normal. H & E staining.
Duck embryo fibroblast monolayer infected with ED5-76 viral isolate. Note the appearance of refractile cells and syncytia formation. H & E staining;
Duck embryo fibroblast monolayer infected with ED5-76 viral isolate, showing formation of giant cells with eosinophilic intranuclear inclusion bodies. H & E staining.
Duck embryo fibroblast monolayer. Normal. H & E staining.
Duck Pekin cell line monolayer infected with ED5-76 viral isolate, showing rounding and grouping of cells and syncytia formation. H & E staining.
Duck Pekin cell line monolayer infected with ED5-76 viral isolate showing fusion of nuclei. H & E staining.
Duck Pekin cell line monolayer- Normal. H & E staining.
Electron micrograph of ED5-76 viral isolate; DNCEM-icosahedral adenovirus particles scattered singly or in twos. X 1,00,000.
Page Nos.
.>"q - 60
':JD - ':/1
~o- ~)
Fig.12 Electron micrograph of EDS-76 viral isolate IEM- '?t 6 ..... clumping of viral particles. X 1,00,000.
Fig.13 SOS-PAGE of EDS-76 viral proteins stained with ....,6~
Coomassie Brilliant Blue stain.
Fig.14 Restriction endonuclease DNA profiles of EDS-76 ~ t-viral isolates and referral strain digested with Pst 1.
Fig. IS Restriction endonuclease DNA profile of EDS-76 -:J/f
viral isolates and referral strain digested with different enzymes.
Fig.16 Restriction endonuclease DNA profile of EDS-76 viral isolates digested with enzyme Pst I.
AGIO
BSA
CElO CEl CKC
CEF CPE
CBB
DIA
DNCEM
DEF DKC
DEL
EUSA
EDS-76
GALV
HA
HI
HEV
HBSS
h
IEM
KDa
Min
nm
NCM
PBST
PAGE
QBV
Sec
TCID
VNT
liST OF ABBREVIATIONS
Agar gel immunodiffusion
Bovine serum albumin
Chicken embryo lethal orphan
Chicken embryo liver
Chicken kidney cell
Chicken embryo fibroblast
Cytopathic effect
Coomassie Brilliant Blue
Dot immunobinding assay
Direct negative contrast electron microscopy
Duck embryo fibroblast
Duck kidney cell
Duck embryo liver
Enzyme linked immunosorbent assay
Egg drop syndrome-1976
Gal adeno-Iike virus
Haemagglutination
Haemagglutination inhibition
Haemarrhagic enteritis virus
Hanks balanced salt solution
Hour
Immunoelectron microscopy
Kilo dalton
Minute
Nanometer
Nitrocellulose membrane
Phosphate buffered saline-Tween
Polyacrylamide gel electrophoresis
Quail bronchitis virus
Seconds
Tissue culture infective dose
Virus neutralization test
or-
CHAPTER I
INTRODUCTION
Poultry industry has become popular among small and marginal farmers.
Progressive improvements in avian health and the use of new genetic standards
together with better feed and improved managerial practices have had an enormous
influence on the development of poultry industry.
The poultry industry requires scientific support and better understanding of
microbial diseases particularly of viral origin based on the health policies of the
country. There are protective vaccines in India, against certain devastating diseases
like Ranikhet disease, Infectious bursal disease, Fowl pox, Marek's disease and others.
But even after effective vaccination, there is a significant drop in egg production
which has become a burning problem to the poultry industry thereby causing greater
economic loss to the farmer. Drop in egg production in poultry is due to varied
etiological factors such as climatic conditions, stress, aflatoxicosis as well as various
infectious agents, viz., Ranikhet disease virus, Infectious bronchitis virus, Infectious
laryngotracheitis virus and aviadenovirus infections. Of these, Egg Drop Syndrome
1976 caused by aviadenovirus has become a major cause for loss of egg production
throughout the world. Once the infection is established in a poultry farm this
condition is more often the cause of failure to achieve the production targets.
There were very many outbreaks of syndrome of drop in egg production in
poultry farms in Tamil Nadu and also in the farms of the neighbouring states like
Andhra Pradesh and Kamataka, which warranted a detailed study. Henceforth, the
present work has been undertaken with the following objectives.
1. To undertake serological survey of avian adenovirus Egg Drop
syndrome (EDS~ 76) both in chicken flocks which fail to reach peak
production, as well as healthy flocks.
2. To isolate the cousative agent EDS-76 from indigenous source posing
the problem of drop in egg production.
3. To compare the physico-chemical and biochemical characteristics of the
isolates in conjunction with a referral strain 127.
4. To propagate and adapt the causative agent in tissue culture system.
5. To analyse and compare the protein fractions of the isolates with the
referral strain 127.
6. To analyse the DNA mapping pattern of the isolates and to check for
any strain variation.
7 . To assess the immune status in birds experimentally infected with the
indigenous isolate of EDS-76 virus.
CHAPTER II
REVIEW OF LITERATURE
~
Egg Drop Syndrome is currently one of the important economic proble~faced
by the poultry industry. The syndrome is caused by multiple agents, viz., bacteria ,
mycoplasma, fungi and viruses. However, the major cause of this syndrome is a .'
haemagglutinating aviadenovirus.
2. SEROPREVALENCE OF A V1ADENOVIRUS
2.1 Viral Etiology
In birds affected with acute respiratory form of Newcastle disease, a condition "
of drop in egg production was observed by Platt (1948).
Complete cessation of egg production was recorded for a period of three
weeks in birds affected with New Castle disease (Biswall and Morrill, 1954).
,,' Taylor et 01. (1955) recorded a drop in egg production in poultry fann affected
with avian encephalomyelitis infection.
2.1.1 Avian Adenoviruses
The avian adenoviruses comprises of Quail Bronchitis virus (QBV), Chick
Embryo Lethal Orphan (CELO) virus, Ganus Adeno-like virus (GAL) and Eg~ Dron
Syndrome-76 (EDS-76) virus.
Adenoviruses have been isolated from nonnal birds and birds suffering from
respiratory diseases, egg production problems, inclusion body hepatitis and other
conditions (Kawamura et 0/., 1964). There were a number of reports on the isolation
of avian adenoviruses in chicken from USA (Burke et 0/., 1968), Hungary (Khanna,
1966), India (Adlakha, 1966; Venna&Ma.Li.R 1970; Yadav et 01., 1975), Japan
/ (Kawamura et 01., 1966), Canada (Trewick and Lang, 1971) and Northern Ireland
,(McFerran et 01.,1978).
/ Yadav et 0/. (1975) in India reported isolation of CELO virus from faeces,
rectal mucous membrane and intestinal contents of both diseased and healthy
chickens.
In 1976, a new syndrome, characterized by a drop in egg production and egg
shell defects was described in the Netherlands (Van Eck et 0/., 1976).
/ McFerran et 01. (1977) reported an adenotype virus which affected the poultry
flocks resulting in a failure of flocks to reach peak egg production, accompanied by
loss of shell colour and laying of soft shelled eggs.
Egg drop syndrome has been reported in Europe (Baxendale, 1978) and North
America (Calnek, 1978) and EDS-76 also then posed a potential threat to Nigerian
poultry industry (Nawathe, 1980) .
. ' Higashihara et 0/. (1983) and Yamagushi et 0/. (198~) observed very many
outbreaks of EDS-76 over a period of one decade from 1970 to 1980.
In India too, tVenkata Reddy (1984), Satyanarayana Chetty (1985:{~ Ram
Kumar et 01. (1992), and Swain et 01. (1992) investigated the magnitude of egg drop
syndrome associated with ED5-76 virus.
2.1.2 Properties of the avian adenovirus
The property of avian adenovirus stability to organic solvents viz., ethyl ether,
chloroform and sodium desoxycholate had been recorded by many workers (Burke
et 01.,1968; Adlakha, 1966 andWinterfield et 01.,1973).
The relative heat resistance of aviadenovirus was first r{ported by Yates and
Fry (1957) for CELO virus tested at 56°C. The stability to heat for different CELO
virus strains had been reporf~d 30 min for "Conn" strain (Petek et 01., 1963) and 18 ; ,
h for "C 1903" strain and 22 h for "EV 89" strain IBurke et al., 1959, 1965).
'" Yates (1960) reported that the titre of CELO virus was not affected over a
wide range of pH from 2.0 to 9.0 but the titres were badly affected when pH was
lower or higher than this range. This was further confirmed by Burke et 01. (1965) and
, Adlakha (1966) .
. The structure of avian adenoviruses employing electron microscope was '-'
studied by Home et 01. (1959). This study revealed the typical icosahedral symmetry
of adenoviruses. In fine structure of GAL virus, the negatively stained particles
measured 95-100 nm with a polygonal profile revealing a core (Macpherson et 01.,
1961).
The CELO virus was purified and the ultrastructural studies were made by
Dutta and Pomeroy (1963). They found that the virus particles were naked measuring
73 nm and exhibited the characters which were similar to GAL adenovirus.
/Dutta and Pomeroy (1963) also reported the electron microscopic studies on
QBV. Their study revealed two types of particles one being the larger (70-75 nm)
similar to CELO virus and the other smaller (20-24 nm). The existence of the small
particles in QBV material but not in CELO virus material was made possible to
differentiate these two viruses as these are indistinguishable by serological tests.
Many workers have reported the lack of agglutination of chicJGn erythrocytes
by QBV, CELO, GAL or other serotypes of avian adenoviruses (Burke et al., 1959a;
.. Kawamura et al., 1964; Clemmer, 1964) .
• Krauss (1965) found absence of hemagglutination activity by CELO virus
isolate at 4°C employing erythrocytes of man, baboon, pigeon, mouse, rabbit, guinea
pig, rat, pig and horse.
The relationship of hemagglUtination and infectivity was observed by-Clemmer
(1964). The HA was first observed at 48 h and a ten fold rise in titre occurred
between 48-72 h, whereas the virus infective titre was stabilized after 48 h.
The HA property of CELO virus was tested by Elmishad et al. (1971)
employing erythrocytes from ten different species. Only rat erythrocytes were
agglutinated at 37°C. Specificity was determined by inhibition with antiserum. The
antigen was inactivated at 56°C for 15-30 min although the infectivity was retained
at least for one h at this temperature.
~ustaffer and Spradbrow (1975) characterized three strains of avian
adenoviruses using biochemical, physical and electron microscopic studies. The
isolates were relatively stable to the effect of chloroform, both sides of extreme pH,
trypsin, heat, lyophilization and ultrasonication.
2.1.3 General properties of EDS-76 virus
EDS-76 virus belongs to the genus aviadenovirus and the size of the virus as
determined by negatively stained preparations was found to be in the range of 67 to
80 nm (McFerran et al., 1978).
/ Todd and McNulty (1978) found that infectious EDS-76 virus particles banded
at densities of 1.32 to 1.30 glml in cesium chloride (CsCI). On the other hand Zask
and Kisary (1981a) reported that EDS-76 virus particles banded at densities of 1.36
to 1.31 glml.
The genome of EDS-76 virus contains DNA. The molecular weight of the DNA
is estimated at 22.6 x 1()6 daltons. EDS-76 virus agglutinates erythrocytes of chicken,
ducks, turkeys, geese, pigeons and peacocks, but does not agglutinate those of rat,
rabbit, horse, sheep, cattle, goat or pig (Todd and McNulty, 1978).
2.2 SEROPREVALENCE OF EDS-76
2.2.1 Seroprevalence in countries other than India
Serological SUNey by various workers from different parts of the world
revealed the presence of specific antibodies against EDS- 76 virus in chicken, ducks,
including a few wild birds.
The presence of antibodies to aviadenovirus 127 in poultry flocks with
decreased egg production was reported b(McFerran et 01. (1977).
/ Karlj et 01. (1977) detected the loss of productioH in laying flocks due to
adenovirus infections and the virus was isolated from the eggs.
In Denmark, the presence of antibodies to adenovirus in two layer flocks and
in a flock with a history of drop in egg production was reported by Beadstue and
Smidt (1978).
Antibodies to adenovirus BC-14 serologically identical to 127 virus were
observed by'Baxendale (1978) in England in poultry flocks that failed to reach a peak
egg production accompanied by the loss of shell colour and the laying of soft shelled
eggs.
Calnek (1978) conducted an examination of sera from commercial duck and
egg laying chicken flocks in New York State and confirmed that the EDS-76 agent is
present in duck flocks and the agent is indigenous in populations of ducks and may
well be a duck virus rather than a chicken virus.
Both laying and breeding flocks had antibodies to BC-14 strain in Italy and
Rampin et 01. (1978) reported the presence of HI antibodies to BC-14 virus in flocks
with and without the history of drop in egg production.
While screening 17 layer flocks in Belgium with a drop in egg production,
eJeven had antibodies against A 127 virus and Muelemans et 01. (1978) contended
that there was no correlation existed between the prevalence of antibodies and
specific disease.
In a serological survey conducted by/Durham and Cathcast (1979) in North
and South Islands of New Zealand, only one South Island flock had an adenovirus
antibody titre of 1: 1 O.
Of the 73 flocks screened at regular intervals for the presence of specific
antibodies to IS virus, EDS virus and BC-14 virus, Van Eck et al. (1980) reported that
eight flocks with drop in egg production problems were associated with antibodies to
BC-14 virus and aviadenovirus.
In Singapore, Ng et af. (1980) reported that flocks exhibited typical signs of
EDS associated with a low ebb in egg production lasted for 10 weeks and eighty three
per cent of flocks had HI antibodies to aviadenovirus.
In France, the birds with a fall in egg production coupled with the laying of soft
shelled eggs, when serologically examined were positive for the presence of antibodies
to aviadenovirus BC-14 and 127 (Bennejean et al., 1980).
A serological survey for EDS-76 viral antibodies using the HI test was
conducted on commercial poultry flocks in Nigeria and Nawathe (1980) reported that
antibody was detected in layers on most of the farms. EDS-76 poses a potential threat
to the Nigerian poultry industry.
Sera from layer and breeder flocks experiencing a fall in egg production in
New South Wales were tested for antibody against DC 61 strain. All were found to
give negative serological reactions. Virus DC 61 has been shown to be serologically
indistinguishable from recognized strains of EDS-76, 127 and BC-14 (Fry-Smith and
Gilchrist, 1981).
In Yugoslavia, out of 22 poultry flocks with a drop in egg production, Bidin
et 0/. (1981) reported that 12 flocks were found positive for antibodies against BC-14
strain of avian adenovirus.
Antibody to virus 127 was detected in 102 of 106 fowl sera tested, in an
Australian poultry flock. Rrth et 0/. (1981) reportsd that the affected flock consisted
of broiler breeder fowl be longing to a commercial producer and almost every flock
was affect ed by EDS-76 virus.
In Japan, an outbreak of EDS-76 occurred in 14 boiler breeding flock in the
age group of 30-55 weeks where the egg production has fallen suddenly ranging
between 6 and 25 per cent'{Yamaguchi et 01., 1981b}.
In Australia Wilcox et 01. (1983) conducted a serological survey of wild birds
which revealed the prevalence of antibodies to mixed infections of EDS-76 and IBD
viruses.
Akay et 0/. (1988) demonstrated HI antibodies to EDS-76 virus in a poultry
flock with a decrease in egg production in Turkey.
In Ukraine about 1000 selUll)amples collected from different age groups of both
layer and broiler flocks which were subjected to passive HA test for detection of
aviadenovirus, about 80 per cent were found to harbour the virus (Volosyanko and
German, 1990).
Six commercia! layer flocks not vaccinated against EDS-76 were tested by HI
test, since there was a severe decrease in egg production. All the birds in 4 flocks
reacted positively with titres of 1:40. This is the first report of aviadenovirus infection
in Egypt (Khafagi and Hamouda, 1991).
2.2.2 Seroprevalence in India
In 1980, Mohanty et 01. (1980) reported the egg drop syndrome in chickens
associated with EDS-76 virus in India.
A study was carried out during the period 1980-83 in different poultry farms
in India, which indicated the prevalence of infection of EDS-76 virus in flocks
experiencing a drop in egg production ranging between 22 and 64 per cent. The drop
in egg production lasted for 3-10 weeks (Mohanty et 01., 1984).
A serological survey in exotic and indigenous breeds of ducks, quails, turkeys,
pheasants and guinea fowls was conducted to determine the prevalence of EDS-76 ..._.,
virus infection. Most of the indigenous ducks and one quail have shown serum HI
antibody to EDS-76 virus. Sera from turkeys, pheasants and guinea fowls were
negative (Mohanty et 0/., 1985).
Sukumar and Suribabu (1986) made a study to assess the sero prevalence of
egg drop syndrome and infectious bronchitis in Andhra Pradesh. Venkata Reddy and
Raghavan (1987) conducted a sero- epidemiological investigation into the incidence
of EOS-76 in 16 flocks of white leghorn birds experiencing drop in egg production.
Out of 770 poultry sera an overall incidence of 27.3 per cent was recorded and birds
aged 44 to 66 weeks had the highest incidence.
A detailed survey on seroprevalence of EOS-76 virus infection in chickens with
a drop in egg production as well as apparently healthy chickens revealed the presence
of specific haemagglutination-inhibition and precipitating antibodies against EDS-76
virus' (Satyanarayana Chetty et 01., 1988).
Birds of 8-26 weeks of age, from 3 farms near Ludhiana with a history of drop
in egg production were tested for antibodies to EDS-76 and CELO strains of
aviadenovirus by HI and agar gel immunurecipitation test. Out of the samples
tested, 29.7 per cent of the sel1Jlf6amples were positive for EDS-76 and 20.3 per cent
were positive for CELO virus (Oberoi et 01., 1990).
Commercial layer and broiler chickens aged between 5-56 weeks in and
around Jabalpur area were screened for the presence of EDS- 76 antibodies and
/ Shakya and Ohawedkar (1991a) reported that the highest incidence of antibodies to
EDS-76 was recorded in the chicks between five and ten weeks of age group.
/ Oas and Pradhan (1992) detected HI antibodies to EDS-76 virus in two
different outbreaks both in quail flocks and chicken flocks with decrease in egg
production. The egg drop ranged between 10.6 and 50.6 per cent.
Serological screening of 323 serum samples received from different states
revealed seroprevalence of EDS-76 infection in several poultry flocks. The titre of HI
antibody level ranged between 1:4 to 1:32 (Ramkumar et 01., 1992).
2.2.3 Haemagglutination-Inhibition ( HI) test
Serological tests like haemagglutination inhibition, EUSA, serum neutralization,
fluorescent antibody and agar gel immunodiffusion are commonly employed for
detection of egg drop syndrome virus antibody. Among these tests, HI . is the
simplest and widely employed test in serological diagnosis of egg drop syndrome virus
infection (Parsons et 01., 1980).
Antibodies to EOS-76 virus were usually detected by HI test. SeltU'llsamples
from poultry flocks were tested for HI antibodies against strain 127 and detected
antibodies in 10 out of 219 chicken flocks (Siegmann et 0/., 1979). Meulemans et 0/.
(1979) detected HI antibodies in seven week old broilers .
. Kaleta et 0/. (1980) screened fowl serumsamples by HI test against EOS-76 virus
during 1973-79, procured from West Germany. For the first five years, antibody could
not be detected till 1978 and thereafter, 29 out of 1853 serum;amples and 167 out of
3095 sera samples during 1979 were found to be.positive by HI test.
In a serological survey undertaken amongst 30 adult poultry flocks in five
districts of Singapore, 25 flocks were found to be positive by HI test (Ng et o/., 1980).
Haemagglutination-inhibition (HI) testing was carried out on 106 samples using
a microtitre system and employing 4 hemagglutinating units of antigen per test and
test volumes of 0.025 ml of serum, 0.025 ml of antigen and 0.025 ml of 0.8% fowl
erythrocytes (Firth et 0/., 1981).
· / Piela and Yates (1983) compared HI, immunodifusion and EUSA tests for the
detection of antibody to haemagglutinating duck adenovirus and found that the
antibody response curves were same with both HI and EUSA, peak titres occurring
approximately 4 weeks PI.
HI antibodies to EDS-76 virus could be demonstrated from domesticated and
wild birds. The HI test was performed in a micro- system. Four HA units of
formalin-inactivated EDS antigen in 0.025 ml saline were used and reacted against
2-fold serial dilutions of test sera. A 0.5 per cent suspension of chicken red cells was '
used for the test. Titres of 1:4 or less were regarded as negative (Bartha et al., 1982).
Antibody titres of duck sera were determined in the HI test by using 4 units of
haemagglutinating antigen prepared from adenovirus 127. HI titres 1:8 or higher were
considered positive (Bartha, 1984).
" Adair et at. (1986) reported that among the serological tests employed, only
HI and SNT are specific for EDS and HI has become the accepted serological test for
EDS-76 virus.
Out of 770 pooled sera screened by HI test, an overall incidence of 27.3 per
cent was recorded. The titres were expressed as log1(Y'0.2 ml of the reciprocals of the
dilutions. A titre of 10g1.0 or more was considered as positive (Venkata Reddy and
Raghavan, 1987).
Two serological tests, viz., micro-HI and immunodiffusion tests were employed
to screen the serum samples. The micro-HI was performed employing two fold
dilutions of the pooled serum samples and 4 HA units of the reference virus. The
reciprocal of the high est dilution of serum where there was complete inhibition of HA
was taken as antibody titr{(s:tyanarayana Chetty et a1., 1988).
/ Akay et af. (1988) described HI antibody titre against EDS-76 virus in
vaccinated hens and yolk material of hens eggs. EDS-76 antibodies were not detected
in the egg yolk and sera of unvaccinated hens. A good correlation existed between
the sera and egg yolk HI titres.
Asi et af. (1990) detected HI antibody titre in birds vaccinated at the age of 16
weeks. Birds were challenged orally with the BC-14 strain at 20, 27, 34, 41, 48, 55
and 62 weeks of age. Vaccinated birds with a mean log2 titre upto 5 produced thin
shelled and shell-less eggs. Egg quality was restored 18-20 days after challenge
whereas birds with log2 titres of 6 and above resisted challenge.
, Oberoi et al. (1990) detected antibodies against two aviadenoviruses, EDS-76
and CELO virus in serL).JrSamples from layers suffering from drop in egg production.
Among the EDS-76 positive serUJ(lsamples, 16.7 per cent had antibody titre of 1:64
and above indicating a definite infection of this virus in birds,
// Shakya and Dhawedkar (1991a) considered the HI antibody titre of 8 (3 1092)
or above as positive when scr'eening 1024 sera samples for EDS-76 antibodies.
Serological screening of 323 serum samples received from different states
revealed prevalence of EDS-76 infection in several poultry flocks. The titre of HI
antibody was 1:4 in 7 flocks and 1:32 in 6 flocks (Ramkumar et al., 1989 and 1992).
2.2.4 Dot Immunobinding Assay (DIA)
Dot and Dipstick assays were developed from classical EUSA to reduce the
cost. The Dot EUSA proved to be economical with respect to the use of reagents and
improved perlormance. Interpretation could easily to made by visual inspection of
reaction end points on nitrocellulose discs. Dot-EUSA was highly specific compared
to that of EUSA and found to be an excellent test to be executed in the field during
sero-epidemiological surveys (Guimaraes et al., 1986).
/1-Ieberling et al. (1987) made a serodiagnosis of rabies by dot immunobinding
assay using inactivated antigen for the detection of rabies viral antibodies. DIA was
found to be a simple, specific and sensitive test for the determination of status of
rabies vaccines, including humans and canines, as well as for surveying animal
population for the occurrence of rabies infections.
/ Muneer et al. (1988) desc:ribed a dot immunobinding assay for detection of
serum antibodies to infectious bronchitis virus antigens absorbed on to filter paper
discs or NCM sheets. It was conduded that the technique was economical, easy to
perlorm, simple and more sensitive than HI and SNT.
The Dot-EUSA is a highly versatile solid-phase immunoassay for antigen or
antibody detection, which when used involves minute amount of reagents to be
dotted onto solid surlace such as nitriSellulose paper. The dot-EUSA has been used
extensively in the detection of human and veterinary parasitic diseases (Pappas,
1988).
Gupta et 0/. (1990) described a dot immunobinding assay for detection of
antibodies to blue tongue virus in sheep and for the control and surveillance in cattle,
exposed to blue tongue virus. The blocking (8)- Dot-EUSA could be considered.
which is rapid, simple and inexpensive (Afshar et 0/., 1992).
For routine detection of antibodies to EDS-76 virus, the OIA was simple and
positive results were easily interpreted visually as coloured dots on the white et a.L.,
nitrocellulose membrane (Ranipremeela, 1992). ~
2.2.5 Comparison of serological tests for detection of antibody to EDS-76
virus
vtonreal and Dom (1981) made comparative studies between virus
~
neutralization test (VNT), EUSA and HI for demonstration of antiyodies to avian
adenovirus and EDS-76 virus. It was concluded that EUSA was more sensitive and
recommended as a routine method for the detection of avian adenovirus and EOS-76
antibodies.
'Kaleta et of. (1982) studied and compared kinetics of antibody formation
against EOS virus in pigeon, turkey, fowls and reported that the VNT was more
sensitive than HI test in wild birds .
.,. Piela and Yates (1983) compared EUSA, HI and immunodiffusion for
detection of antibodies to a duck adenovirus in experimentally infected chicken. The
EUSA was found to be a sensitive and reliable method for detecting antibody,
although positive titres did not agree with HI and 10 results at one week after
inoculation. On the other hand, Mesanjaz et al. (1982) reported that EUSA a~d HI
tests were comparable for detecting antibodies to EDS-76 virus.
/ Adair et al. (1986) 'compared the sensitivity of five serological tests, viz., HI,
EUSA, SNT, FAT and AGID for the detection of EDS-76 virus antibody. These
workers are of the opinion that HI or SNT was best used for the detection of infection
in commercial birds.
2.3 ISOLATION OF THE VIRUS
d Adenovirus"have been isolated from normal birds and birds with respiratory
disease, production problems, inclusion body hepatitis and other
conditions (McFerran et al., 1972).
Several workers have reported the isolation of avian adenoviruses in Australia.
Boyle (1976) reported the isolation in Queens land of avian adenoviruses from
poultry flocks with egg production losses.
McFerran and Connor (1978) described 3 new fowl adenovirus serotypes, two
A-2 and 62-B were isolated from the USA and one, 380 in Northern Ireland.
Baxendale (1978) isolated Be 14 adenovirus strain from a poultry flock that
failed to reach peak egg production. The virus was isolated from blood leukocytes of
the affected birds.
, Meulemans et al. (1979) recovered a strain of avian adenovirus from a Belgian
flock that had the problem of fall in egg production and laying of abnormal eggs. It
was serologically and morphologically identical with a virus isolated in Ireland. Villegas
et 01. (1979) isolated an adenovirus strain 127 in the chicken embryo liver cells from
the cloacal swabs of clinically nonnal ducks. The isolate haemagglutinated chicken
erythrocytes and this activity was inhibited by the antiserum to adenovirus 127.
In Italy, a viral strain E77 was isolated from cloacal swabs of hens affected with
decreased egg production. It showed virological features similar to adenovirus strains
127, BC 14 and 3877 (Zanella et 01., 1980).
Yamaguchi et 01. (1981a) reported a condition similar to egg drop syndrome
1976 and isolated haem agglutinating adenovirus from cloacal swabs and a uterus of
hen in one farm. Of the eleven isolates, one isolate, had the same antigenicity in
serologic tests and the same biological and physico-chemical properties as the BC 14
strain of EDS-76 virus.
Firth et 01. (1981) isolated two haem agglutinating viruses from a flock with
delayed onset of laying and a drop in egg production. Samples were taken one week
after the onset of an egg drop syndrome.
Higashihara et 01. (1983) isolated a virus strain (H-162) from the faeces of a
hen and the strain was shown to be identical to the BC-14, and JAP-1 strains of EDS
virus.
In Kamataka, Venkata Reddy (1984) isolated haemagglutinating adenoviruses
from serologically positive poultry and duck flocks. In Andhra Pradesh seven
haem agglutinating virus isolates obtained were specifically inhibited by reference
serum to EDS-76 virus (Satyanarayana Chetty, 1985).
In India/Ramkumar et al. (1991) isolated haemagglutinating adenovirus from
faecal samples of a few of the birds which had production failure associated with
laying of soft thin shelled eggs.
2.4 CHARACTERIZATION OF EDS-76 VIRUSES
2.4.1 Physico-chemical characterization of EDS-76 virus
Todd and McNulty in 1978 and Adair et al. in 1979 studied the biological and
physico-chemical properties of avian adenovirus strain 127 assodated with EDS-76.
The infectivity of the virus was found to be stable with ether treatment, extremes of
pH whereas IUDR inhibited the replication of the virus.
An avian adenovirus strain E-77 which was isolated by Zanella et af. (1980)
had similar physico-chemical properties as described by Adair et al. (1979).
An isolate of avian adenovirus strain JPA-1 was found to be stable against
organic solvents, pH 3, thermostable at 50 to 56°C, but the infectivity was destroyed
at 60°C (Yamaguchi et al., 1981a). The replication of a haemagglutinating virus strain
H-162 isolated by Higashihara etal. (1983) was inhibited with 5-iodo, 2'-deoxyuridine
and its infectivity was resistant to treatment by ethyl ether or chloroform. The virus
was stable at pH 3.7 .
. [ Roxhdestvenskii (1984) inactivated avian adenovirus strain 88/78 by exposure
to 60 to 700C for 40 min and 1 per cent formaldehyde solution for 24 to 48 h.
Venkata Reddy (1984) in Andhra Pradesh studied the physico- chemical
properties with local EDS virus isolates and found similar results as described by
Yamaguchi et al. (1981a).
Ramkumar et al. (1991) has reported that the avian adenoviral isolates
EDSV-IVRI/AD-86, EDSV-01/AD-86 and EDSV-02/AD-86, when treated with 10%
chloroform were found to be resistant to chloroform with retention of activity.
Characterization of a field isolate of EDS-76 virus revealed that the virus was
resistant to heat (56°C), ether, chloroform, acid reactions (pH 3) and trypsin, but
sensitive to formalin (0.3 per cent) (Swain et al., 1992).
2.4.2 Electron microscopic studies of EDS-76 virus
Direct negative contrast electron microscopy (DNCEM) of purified viral samples
consisted of rod-like elements which radiated from a central area (Todd and McNulty,
1978). fowl adenovirus strains. A-2 and C2-B isolated from USA and 380 from
Northern Ireland, all had typical adenovirus icosahedral morphology when negatively
stained and examined under electron microscope (McFerren and Connor, 1978).
Purified and negatively stained JPA-1 strain, when mounted on
micromesh-coated grids, consisted of both complete particles as well as disrupted and
penetrated viral particles (Yamaguchi et al., 1981a).
Negative contrast examination of duck embryo propagated IC 8055 virus
L revealed the presence of adenovirus like particles in large numbers (Rrth et 01.,1981).
Electron microscopy revealed typical adenovirus particies often aggregated in
paracrystalline array forming a cubic lattice in the nuclei of thin-sectioned cells
(Higashibara et al., 1983).
The electron microscopic observation of the EDS-76 virus isolate of Swain
et al. (1992) present in the allantoic fluid of embryonated duck eggs revealed the
presence of large and small hexagonal viral particles.
2.4.3 Growth characters of EDS-76 virus in cell culture
EDS-76 virus grows to higher titres in Duck Embryo Fibroblast (DEF), Duck
Kidney (OK) and Duck Embryo Liver (DEL) cells. It also grows well in Chicken
Embryo Liver (eEL) cells, but less well in Chicken Kidney Cells (CKC) and rather
poorly in Chicken Embryo Fibroblast (CEF) cultures. The virus d..u{ not replicate in
very many numbers of mammalian cell culture systems (Adair et al., 1979).
2.4.3.1 Growth characters in primary cell culture
Todd et 0/. (1978) propagated EDS-76 virus (127 strain) in CEL cells and then
purified for further studies on soluble haemagglutinins.
Yamaguchi et al. (1981a) isolated JPA-1 strain in CK cell culture and clone
purified in CEl cell culture. The virus clone was passaged 3 times in CEl cell culture
for comparative studies with BC 14 strain.
B8/78 adenovirus was grown well in CEl cell cultures. The virus replicated well
in tissue culture producing intranuclear inclusion bodies in the infected cells (Zsak and
Kisary, 1981).
Duck adenovirus (Cornell strain) was propagated in duck and chicken embryo
cells. In duck cells high viral titres were observed. Evidence of viral propagation was
not detected in CEl and CK cells (Gulka et al., 1982.).
Firth et al. (1981) successfully attempted to isolate a haemagglutinating
adenovirus from flocks showing an EDS infection from DEF cells.
,. Nikolaeva and Roxhdestvenskii (1982) and Ramkumar et al. (1993) propagated
EDS-76 virus in primary DEF cell cultures and confirmed that after 72 h there was a
massive release of virus from the cells.
A haemagglutinating virus (H-162 strain) was isolated from the faeces of a hen
in a broiler breeding flock. This virus multiplied with a characteristic CPE in cultures
""' of CEl and CK cells (Higashi hara et aI., 1983). '-""
+ Venkata Reddy (1984) and Sathyanarayana Chetty (1985) cultivated the local
EDS viral isolates in OEF cultures. The CPE was apparent at 24th and its peak
changes were observed at 72 h PI.
. Bragg et al. (1991) isolated the causative agent of EDS in CEl cell cultures
from a farm in the Western Cape followed by identification and confirmation as EOS
virus by HI and SN tests.
2.4.3.2 Growth of EDS-76 virus in cell lines
/-Martone et al. (1983) studied the replication and behaviour of aviadenovirus
EDS-76 strain in continuous cell culture and reported that the virus failed to multiply
in the cell lines of HeLa and Hep-2.
f" Higashihara et 0/. (1983) propagated the EDS-76 (H-162 strain) virus in
mammalian cell lines such as HeLa, Vero and HEl R-66 and concluded that no
replication of the H-162 strain could be observed in these cell lines.
2.4.3.3 Cytopathic effect of EDS-76-virusin cell cultures
. Burke et 01. (1968) studied the CPE of aviadenovirus in CK cell culture. The
early changes were of cells with nuclei much enlarged and containing nucleoli placed
eccentrically against the margin of the nuclear membrane.
The JP A-I strain of EOS virus clone purified in CEl cell culture produced
essentially the same CPE as that of the BC 14 strain (Yamaguchi et 0/., 1981a).
A CPE consisting of rounded, clumped aggregates of refractile cells were
detected in the DEF cultures infected with PO 1917 strain while Gouch et al. (1982)
reported that no CPE could be observed by the same strain in CK cultures.
The H-162 strain multiplied manifesting CPE in CEl cultures. The CPE was
characterized by the appearance of refractile round cells followed by their enlargement
and eventually detaching from the glass surface. Eosinophilic inclusion bodies were
observed in the nuclei of the swollen cells of the infected cultures (Higashi hara et al.,
1983).
Venkata Reddy (1984) observed changes in OEF cell cultures infected with
local EOS isolates of Andhra Pradesh. The changes observed after 24 h PI were
swelling of the cells and granulation of cytoplasm. The nucleus was denser and often
nucleolus was enlarged. Intranuclear inclusion bodies were observed 72 h PI.
Sathyanarayana Chetty (1985) reported that the CPE of the EDS isolates in
OEF cultures was milder during early passages but was prominent during subsequent
passages. The CPE includes rounding and swelling of cells, focal areas of enlarged
refractile cells and the appearance of intranuclear inclusion bodies by the tenth day
PI.
An indigenous strain EOS-76 isolate in CEl cell culture produced characteristic
CPE and eosinophilic intranuclear inclusion bodies (Swain et al., 1992).
2.4.4 Cross Haemagglutination Inhibition of EDS-76 virus
The JPA-1 strain possessed the same antigenicity as the BC 14 strain and
antiserum of each strain inhibited the other (Yamaguchi et a1., 1981a).
Gough et a1. (1982) carried out cross haem agglutination inhibition tests using
4 HA units of antigen against monospecific antisera of EOS-76 isolate PO 1917 strain
and adenovirus 127. The adenovirus 127 antiserum inhibited the isolate (PO 1917)
to a titre of 29 , which was the same as the homologous titre.
Higashihara et a1. (1983) studied the properties of a local isolate H-162 strain
and found that the H-162 strain showed no difference in antigenicity from the BC-14
strain of EDS-76 virus and cross HI test indicated that the H-162 strain was
serologically identical to the EOS-76 virus.
The results of the cross HI indicated that all the three strains 127, JBP and
SPC of EOS-76 virus were serologically indistinguishable from one another. The
AGPT and CIE with homologous and heterologous antigen and antibody system
further demonstrated the antigenic relationship (Shakya and Dawedkar, 1991b).
2.5 PROTEIN FRACTIONATION STUDIES
2.5.1 Protein fractions of Avi-adenovimses
The structural proteins of fowl adenovirus type-1 were analyzed (U et a/.,
1984) and this CELO virus contains at least 14 structural proteins with polypeptide
molecular weights ranging from lOOK to about 6K.
Zhang et al. (1991) studied the polypeptides of serologically related type-ll
avian adenoviruses, haemorrhagic enteritis virus (HEV), marble spleen disease virus
and the virus causing splenom{a1y in chickens. The polypeptides were separated by
50S-PAGE and analyzed by protein immunoblotting with polyclonal antibodies which
revealed antigenic differences between the three viruses.
The structural proteins of (HEV) a turkey adenovirus were analyzed by PAGE. -_:'~-~
In purified HEV preparation, Van-den-Hark (1992) reported eleven polypeptides with
apparent molecular weights ranging from 96,000 to 9,500.
2.5.2 Protein fractions of EDS-76 virus
The polypeptides present in purified virus preparation were analysed (Todd
and McNulty, 1978) by electrophoresis on gels containing 12.5% acrylamide and
0.375% bisacrylamide. The molecular weight of the virus polypeptides were
determined by comparing their mobilities relative to the tracker dye with those of the
reference proteins.
Swain et 0/. (1992) purified a field isolate EDS-76 virus and analysed for
proteins and found to contain 12 polypeptides of 10- 126 KD.
2.5.3 Concentration and purification of the virus
The five proteins V, VI, VII, VIII and X} of adenovirus type 2 were purified by
selective extraction in urea at high ionic strength and at low pH followed by
preparative polyacrylamide electrophoresis towards the cathode at pH 3-4 (Everitt
et 01., 1973).
Todd and McNulty (1978) pUrified the EDS virus 127 after growth in CEL cells.
Centrifugation of the clarified, sonicated cell lysate at 80,000 g for 2 h at 4°C allowed
a crude pellet. The crude pellet was resuspended in 0.1 M phosphate buffered saline
and further purified using CsCI equilibrium density gradient centrifugation.
Infected tissue culture harvest was subjected to salting out with 50% saturated
ammonium sulphate at 4°C overnight. After centrifugation the precipitate was
resuspended in about 1/5Oth of the original volume of phosphate buffered saline. The
suspension was ultracentrifuged at 25,000 rpm for 1 h at 4°. The crude virus pellet
was resuspended in 1/10Oth of the original volume of phosphate buffered saline. The
suspension was treated with an equal volume of fluorocarbon. One millilitre of the
fluorocarbon-treated viral material was overlaid on CsCI with density of 1.31 glml and
centrifuged at 40,000 rpm for 22 h at 4°C (Yamaguchi et 0/., 1981a).
2.5.4 Polyacrylamide gel electrophoresis (PAGE)
Electrophoresis in gels containing SOS was used for separating the polypeptide
chains from complex biological samples (Laemmli, 1970).
Everitt et al. (1973) analysed the polypeptides of adenovirus type 2 by gels
containing 75% (w/v) acrylamide, 0.3 per cent (w/v) N,N' Methylene bisacrylamide
in 5M urea and 0.1M atrate buffer of pH 3.4.
The polypeptides present in purified virus preparations were analyzed by
electrophoresis on gels containing 12.5 per cent acrylamide and 0.375 per cent .
bisacrylamide and the gels stained with Coomassie Brilliant blue (CBB).
Swain et aT. (1992) subjected an indigenous EOS-76 virus isolate after
purification to SOS-PAGE analysis . Ten percent separating gel and 4 per cent stacking
gel were prepared. The virus along with molecular markers were run initially at 40
volts for 1 h and then at 60 volts for 8 h. The gel was fixed and stained in CBB and (12. )
molecular weights of £OS-76 virus polypeptides were of JO_JJ.~ k.D .A
2.5.5 Staining of gels
Amido Black lOB, 0.3 per cent in 7 per cent acetic acid was used to stain the
gels to study the multiple proteins of adenovirus type 2, 7A and 12 (Maizel et al.,
1968).
Everitt et al. (1973) analysed the polypeptides of adenovirus type 2 by gels
and the gels were stained for 45 min in a solution of 0.2 per cent Coomassie Brilliant
Blue R 250 in 7 per cent acetic acid and 10 per cent methanol and destained in the
same solvent.
Silver staining procedure for polyacrylamide gels was used and was found that
this can detect concentrations as low as 0.01 ng of proteins per square millimetre
r (Merill et 0/., 1981).
Silver staining procedures are of the order of 100 times more sensitive than
Coomassie Blue staining and can be applied satisfactorily to gels that have already
been stained with Coomassie Blue stain (Andrews, 1986) .
. 2.6 DNA ANALYSIS OF ADENOVIRUSES
2.6.1 Restriction endonuclease fingerprinting
The primary tools used by the present day molecular biologists in manipulating
DNA are restriction enzymes and other DNA modifying enzymes. Restriction enzymes
bind specifically to and cleave double stranded DNA at specific sites within or
adjacent to a particular sequence known as the recognition sequence.
2.6.2 Restriction DNA analysis of avi-adenoviruses
The effect of specific endonucleases on CELO virus DNA was studied by
#' Denisova et 0/. (1979). it was shown that Hpa I, Eco HI and Hind III cleaved viral
DNA into 5,7 and 7 specific fragments,respectively.
Seventeen fowl adenovirus strains representing 11 serotypes were placed into
5 groups based upon the restriction patterns of DNAs generated by restriction
endonucelases Bam HI and Hind III (Zsak and Kisary, 1984).
Zhang and Nagaraj (1989) analysed three serologically indistinguishable viruses
of avian adenoviruses by restriction endonuclease fingerprinting. Markedly, different
DNA cleavage patterns were found in these viral isolates. Restriction endonuclease
analysis were found US e F u. t for distinglJishing genetically different and
yet serologically similar strains of avian adenovirus type II.
Homologous sequences between EDS-76 adenovirus strain 127 DNA and
bovine adenoVirus DNA were reported by Zakharchuk et 01. (1993\ usLng: Southern
blotting technique and no homology with CELO virus DNA was detected. These data
suggest a genetic similarity between EDS-76 virus and bovine adenovirus. The
fragments generated from EDS-76 adenovirus DNA by eight restriction endonucleases
were physically mapped.
2.6.3 Restriction DNA analysis of EDS-76 virus
The restriction endonuclease Eco HI cleaved at two sites of the EDS
adenovirus (strain 88/78) DNA generating 3 fragments and Kisary and Zsak (1980)
estimated the molecular weight of the whole undigested DNA to be about 22.9 x
10(6) daltons.
2.7 EXPERIMENTAL INFECTION OF EDS-76 VIRUS
- ~~,
, Monreal et al. (1979) inoculated three month~ old SPF chicks by intranasal and
intraocular routes with avian adenovirus FAV-l. The virus was detected in peripheral
lymphocytes, plasma and in faeces after two days PI. The virus was recovered from
various organs upto 12 days PI.
The spread of EDS-76 infection both under experimental and field condition'
in broiler and commercial flocks was investigated by Cook and Darbyshire (1980).
Under experimental conditions, infection was demonstrated serologically in broiler
farm and virus was isolated from faecal swabs in commercial farm.
Yamaguchi et of. (1981b) inoculated Rhode Island Red laying hens, orally with
JPA-1 strain of EDS-76 virus. Inoculated hens laid abnormal eggs and the virus was
recovered from various organs from 3 to 7 days PI.
Day:Old chicks were inoculated orally with EDS-76 virus, cloacal swabs were
regularly collected for 14 days PI and the swabs examined for the presence of EOS-76
virus (Cook and Darbyshire, 1981). Virus was recovered from cloacal swabs during
the first 14 days after inoculation.
Transovarial transmission of EDS-76 was evident on 7th day PI. Faecal swabs
were collected on 0, 5, 10, 15, 20 and 25 days PI which revealed the presence of
avian adenovirus from infected hens by 5th day PI (Dawson et 01., 1981).
The persistence of virus on different age groups of chicken was done by Kaleta
et of. (1982) following oral infection. Virus was first recovered from faeces from first
day to fourteenth day and could also be isolated from various organs.
Persistence of EDS virus in various internal organs and the rate of its excretion
in experimentally infected chicken declined rapidly with increasing age. EOS-76 virus
strain 127 was detectable in faeces upto 2 weeks and in organs of young chicken
upto 5 weeks post infection (Heffels et 0/., 1982).
Following oral infection of 60 laying hens with strain 127 of avian adenovirus.
Loupal et al. (1983) reported on the clinical and serological findings. The birds laid
smaller and lighter eggs 10 to 11 days PI. Antibody to EOS virus was first detected
on the 7th day and all the infected hens developed antibody.
Brugh et al. (1984) and Van Eck (1983) studied experimental infection of
commercially reared white leghorn hens with adenovirus 127 and found that the
production decrease was first evident as early as on the 6th or 7th day and reached
maximum between 20 and 24 days PI.
Higashihara et al. (1986) infected Brown and White layer hens orally with the
H-162 strain of the EOS-76. Egg production decrease in white layer hens was
significant as compared with the decrease in production observed in brown layer
hens. The spread of the virus between the two category of birds and to the contact
hens was very much apparent.
2.7.1 Histopathology of EDS-76
Van Eck et al. (1978) studied histopathological changes in the oviduct of hens
affected with EOS. Histopathological changes were confined to the uterus. No
macroscopic changes were noticed. Microscopically, extensive atrophy of the
glandular tissue, oedema and infiltration of the plasma cells and lymphocytes in the
uterine villi were observed.
Feherrari et al. (1979) recorded the inflammatory reaction in the propria of the
uterus with mononuclear infiltration and formation of lymphoid follicles.
Taniguchi et 01. (1981) observed oedema of the uterus consequent to
experimental inoculation of EDS virus and microscopically, the visibility of
intranuclear inclusion bodies were apparent in the epithelial cells of the uterus and
isthmus after 14 days of infection. Lymphoid follides were formed in the mucosal
folds of some parts of the oviduct 21 days PI.
Yamaguchi et 01. (1981b) studied the pathogenicity and distribution of EDS-76
virus (JPA-1) in inoculated hens. Histological changes such as atrophy of the tubular
glands, degeneration and desquamation of the epithelial cells were predominant in
the uterus. It was concluded that the virus exerted a direct ill-effect on the uterus so
as to prevent the uterus from forming the outer shell of the egg.
Gylstorff and Rolf (1982) infected 60 laying hens orally with strain 127 of avian
adenovirus and attempted to study the histological changes. Histopathological studies
showed inflammatory infiltration with lymphocytes, small amounts of heterophil
granulocytes, plasma cells and macrophages. The inflammation was most evident in
the uterus, the seat of predilection for the virus. Atrophy of surface and glandular
epithelium in the uterus was adduced. In all parts of the oviduct oedema and follicles
of lymphocytes were present in variable frequency .
.._ Sathyanarayana Chetty et 01. (1985) studied the histopathology of various
organs viz., heart, lungs, kidneys, Bursa of Fabricius, liver, proventriculus, intestines
wherein they observed severe congestion, haemorrhagia, oedema together with
infiltration of mononuclear cells and degenerative changes in almost all organs.
CHAPTER III
MATERIALS AND METHODS,
3.1 MATERIALS FOR SEROPREVALENCE
3.1.1 Serum samples from chicken
Serum samples were collected from various poultry farms in different districts
of Tamil Nadu with a history of sudden drop in egg production in apparently healthy
chicken. Serum samples were also collected from normal healthy flocks. In a few of
the farms, where the birds had not reached the expected peak level of egg production
pooled serum samples, each pool comprising of five samples were collected during the
study.
The birds involved in the present study were of different strains such as white
Leghorns, Babeok, Ranishavers, etc. All birds of the flock had history of having
vaccinated against common viral diseases like Newcastle disease, Marek's disease,
Fowl Pox and infectious bursal disease ..
A total of 809 serum samples were collected during the study.
3.1.2 Referral virus
EDS-76 (H-127) virus obtained from Dr. J.B.McFerran, Veterinary Research
Laboratory, Stormont, belfast, Northern Ireland, was maintained in duck embryos and
used whenever its presence was warranted.
3.1.3 Chicken erythrocytes
Blood was collected in Alsever's solution from birds maintained in the r"\
Department of Microbiology, Madras Veterinary CoUege, for the preparation of 0.8
per cent erythrocytes.
3.1.4 Phosphate buffer saline - Tween 20 (PBST)
PBS containing 0.05 per cent Tween 20 (Sigma, USA) was used in Dot
Immunobinding Assay (DIA).
3.1.5 Bovine serum albumin (BSA)
il.3 per cent BSA (Sigma, USA) prepared in PBST was used in DIA.
3.1.6 Enzyme substrate
A freshly prepared substrate solution containing 1 mglml of diaminobenzidine
tetrahydrochloride (DAB, Sigma, USA) in distilled water and one uVml of 3 per cent
hydrogen peroxide was used in DIA (Chauhan and Singh, 1992).
3.1. 7 Horse radish peroxidase
Chicken IgG (whole molecule) peroxidase conjugate (Sigma, USA) was used
in DIA.
3.2 METHODS
Haemagglutination (HA) and Haemagglutination inhibition tests (HI) were
carried out as per the procedures of Shakya and Dhawedkar (1991a) with little
modification.
3.2.1 Haemagglutination test (HA)
The HA test was performed by the microtitre method in Laxbro microplates.
Two fold dilutions of the virus were made in 50 ul volumes, starting from 1: 2 dilution
using phosphate buffered saline (pH 7.2). To each dilution of the virus, an equal
volume of 0.8 per cent washed chicken erythrocytes were added and incubated at
room temperature for 30 min. The highest (lOg2) dilution of virus showing complete
HA pattern was taken as virus titre.
3.2.2 Haemagglutination inhibition test (HI)
HI test was also performed in Laxbro microtitre plates using 4 HA units of
antigen in 50 ul volumes. Two fold dilutions of the test sera samples in 50 ul volumes
were made serially with an initial dilution of 1 :2. Equal quantity of washed chicken
erythrocytes was added and incubated at room temperature for 30 min.
Titres were recorded based on the highest (lOg2) dilution of sera giving cent
percent inhibition of HA activity. ff
3.2.3 Dot immunobinding assay (DIA) -
OlA was performed in accordance with the method described by tPappas
(1988) for the detection of the antibodies to EDS-76 virus as well as for their
quantitation. Nitrocellulose (NC) membrane (Sigma, USA) with a pore size of 0.22
microns was used as the solid support in the present study. Thirty six per cent sucrose
cushion purified EDS-76 virus antigen at the rate of 2 ul (1:8000 dilution as
determined by checkerboard titration) was dotted at the centre of the pencil marked
squares on the NC membrane which was subsequently allowed to dry for 15 min at
37°C. The unsaturated binding sites of the NC membrane was blocked with the
blocking solution containing 3 per cent bovine serum albumin in PBST and incubated
at 37°C for 30 min. Serial 10-fold dilutionsof the test serum samples were prepared
in PBS and two microlitres of these suspected serumsamp\es were dotted in the
respective squares and incubated at 37°C for 1 h. The NC membrane was washed
thrice in PBST and was immersed in 1:200 (predetermined titre) of anti-chicken IgG
peroxidase conjugate to react. After 1 h incubation at 37°C the NC membrane was
again washed thrice in PBST. The NC membrane soaked in a freshly prepared
substrate solution containing 0.5 mglml of 3,3' diamino benzidine tetrahydrochloride
(DAB, Sigma, USA) and 1 uVml of 30 per cent hydrogen peroxide, for colour
development. Mer 10 min the NC membrane was washed in tap water and dried.
The test included controls for positive serum, negative serum, antigen and BSA.
3.3 ISOLATION AND IDENTIFICATION OF VIRUS
3.3.1 Collection of materials
Cloacal swabs and shell glands were collected in Hanks balanced salt solution
(HBSS) supposed to contain antibiotics from apparently healthy birds showing sudden
drop in egg production, from places in Tamil Nadu and bordering areas of Andhra
Pradesh and Kamataka. A total of 118 cloacal swabs and 40 shell glands were
collected.
3.3.2 Duck embryos
Fresh duck eggs were purchased at random, cleaned with anti septic lotion and
incubated at 37°C. Fertile eggs were selected on the 10th day and the biological
samples were inoculated in ten day old embryos through allantoic sac route.
3.3.3 Cell culture
3.3.3.1 Hanks basal salt solution (HBSS)
HBSS (Hi-media) was prepared by dissolving 9.9 g of the powder contained
in a bottle in one litre of sterile triple glass distilled water, sterilized by filtration and
tested for sterility. They were distributed in aliquots of 50 ml and stored at -20°C until
required for further use.
3.3.3.2 Trypsin (0.25 per cent)
One gram of trypsin (Difeo, USA) was dissolved in 400 ml of calcium and
magnesium free phosphate buffered saline (1 :250) at room temperature for 30 min
and filtered through membrane filter, subjected to sterility check, dispensed in 10 ml
aliquots and stored at -20ct::.
3.3.3.3 Trypsin-Versene-Glucose solution ('IVG)
Sodium chloride (AR, BDH)
Potassium chloride (AR, BDH)
Sodium phosphate dibasic (AR, BDH)
Potassium phosphate monobasic (AR, BDH)
Tnjpsin, 1:250 (Difco, USA)
Ethylene diamine tetracetate, disodium salt (AR, BDH)
Glucose (AR, BOH)
Sodium bicarbonate (AR, BDH)
Phenol red, 1 % (Difco, USA)
Sterile triple glass distilled water
0.8 g
0.02 g
0.19 g
0.02 9
0.10 9
0.20 9
0.05 9
0.05 g
1.00 ml
100 ml
Sterilized by filtration, subjected to sterility check, dispensed in 10 ml aliquots and
stored at 4°C until required for further use.
3.3.3.4 Serum supplement
Blood was collected from healthy goats maintained in the Department of
Microbiology, Madras Veterinary College and the serum was separated, inactivated
at 56°C for 30 min, sterilized through Seitz filter, followed by sterility check and
dispensed in 10 ml aliquots and stored at -20°C.
3.3.3.5 Tryptose phosphate broth (TPB)
Tryptose phosphate broth (Difco, USA) was prepared by dissolving 29.5 gin
1000 ml of triple glass distilled water, sterilized by autoclaving at 10 pounds pressure
for 15 min, cooled, dispensed in 50 ml aliquots and stored at 4°C until required for
further use.
3.3.3.6 Antibiotic solution
Twenty vials of crystalline sodium salt of penicillin (5 lakhs IU) and five vials
of streptomycin (1 g vials) were dissolved in 200 ml of sterile HBSS and distributed
in 10 ml aliquots and stored at -2ooC. One milli litre of this stock solution was added
to every 100 ml of medium so that one millilitre of the final medium contained SOD
IU of penicillin and 250 micro:)ram of streptomycin.
3.3.3.7 Sodium bicarbonate solution (8.8 per cent)
8.8 g of sodium bicarbonate (AR, SDH) was dissolved in enough triple glass
distilled water and made up to 100 ml. It was then sterilized by filtering through Seitz
filter (EKS Pad) tested for sterility, distributed in aliquots of 5 ml and stored at 4°C.
3.3.3.8 Medium 199 (MI99, Hi-media)
Medium 199 (Hi-media) was prepared by dissolving 15.6 g of Medium 199
powder in 1000 ml of triple glass distilled water and sterilized by filtration and used
for preparing both growth and maintenance medium pertaining to eEL and DEF
cultures.
3.3.3.9 Minimum Essential Medium (MEM Eagle's modified, Hi Media)
9.6 g of dehydrated medium was dissolved in 1000 ml of triple glass distilled
water to which 10 ml of antibiotics stock solution was added. The medium was
sterilized by filtration, checked for sterility and stored at 4°C. This medium was used
for growth and maintenance of Duck Pekin cell line. f
3.3.3.10 Fixative Fluids (Tissue culture)
3.3.3.10.1 Camoy's fluid (Merchant et al., 1964) .
Absolute alcohol
Glacial acetic acid
Chloroform
600 ml
100 ml
300 ml
This fluid was stored in tightly stoppered amber coloured bottles.
3.3.3.10.2 Bouin's fluid (Anant Rai, 1985)
1.2 per cent (saturated) picriC acid
Formalin
Glacial acetic acid
3.3.3.11 Staining solutions
75 parts
25 parts
5 parts
3.3.3.11.1 Rappaport stain (Rovozzo and Burke, 1973)
Crystal violet
Citric acid
0.10 g
1.92 g
Triple glass distilled water to make up to 100 ml.
Citric acid was dissolved in 75 ml of triple glass distilled water followed by the
addition of crystal violet and the final volume made upto 100 ml.
3.3.3.11.2 Haematoxylin stain
Haematoxylin stain was prepared as per John Paul (1973).
3.3.3.11.3.3 Eosin stain
A one per cent solution of eosin was prepared in 70. per cent ethyl alcohol.
3.4 METHODS OF VIRUS ISOLATION AND IDENTIFICATION
3.4.1 Virus isolation
Virus isolation was followed as per the method of Ramkumar et al. (1992).
3.4.1.1 Processing of faecal swabs
The faecal swabs were collected in HBSS directly from the live birds
manifesting the sign of drop in egg production, for the isolation of viral agents. The
collected specimens were homogenized and centrifuged at 2500 rpm for 15 min. The
supernatant was collected, filtered through millipore membrane filter, treated with
antibiotics and stored at -200C.
3.4.1.2 Processing of shell glands
Samples of shell gland collected in HBSS were processed by triturating with
sterile sand in a ~."or.ttr wi.th ft.!!. Le. by adding sufficient HBSS as diluent. Further
processing was identical as in the case of faecal swabs.
3.4.1.3 Inoculation in duck embryo
Each processed sample was inoculated into 10 day old duck embryonated eggs
through allantoic sac route using 0.2 ml. Inoculated eggs were incubated at 37°C for
4-5 days. After incubation the eggs were chilled and the allantoic fluid was harvested
and clarified at 5000 g for 15 min. Harvested fluids of each passage level were
screened for HA activity. A minimum of 3 passages for each sample were made
before declaring it as negative for the presence of viral agent. Viral isolates were
initially identified with HA and HI activities, employing fowl erythrocytes against
referral EDS antiserum.
3.4.2 Preparation of EDS-76 virus hyperimmune serum
Antiserum against EDS-76 virus strain 127 and one of the EDS isolates (Ml)
were prepared in rabbits and apparently healthy cockerals free from EDS-76 viral
antibodies, as ascertained by the method of Satyanarayana Cherty (1985) with slight
modification. Two rabbits and three cockerels were used for each strain of EDS-76
virus. The fifth egg passage virus grown in embryonated duck eggs was used as a
source of antigen. One milli litre of the virus suspension was mixed thoroughly, with
one millilitre of Freund's complete adjuvant and injected intramuscularly at the rate
of 1 ml per rabbit and 0.6 ml per cockerel. The second and third doses of antigen was
given without adjuvant at weekly intelVals. Ten days after the third injection, the
rabbits and cockerels were test bled and checked for HI titre. When satisfactory HI
titres were inferred the final halVest was done and the serum samples collected were
stored in one milli litre quantity at -2OOC
3.4.3 Cross haemagglutination inhibition test (Cross HI)
Cross HI with microtitre plate method (Shakya and Dhawedkar, 1991b) was
carried out with all the isolates and the referral strain 127, o-f EDS-76 virus. Each virus
isolate was titrated against the referral hyperimmune serum (strain 127) and the hyper
immune serum raised against the Ml isolate.
3.5 CHARACTERIZATION OF THE VIRAL ISOLATES
3.5.1 Preparation of primary cell culture
CEl cell cultures were prepared as per the method described by Higashihara
et of. (1983) by using 14=-day:"old chicken embryos while DEF cell cultures were
prepared as per the method followed by Gulka et 0/. (1982).
3.5.2 Sub_culturing of Duck Pekin cell line
The Duck Pekin cell line (obtained from National Institute of Virology, Pune)
was periodically sub-cultured and maintained using MEM (Eagle's modified) medium
and foetal calf serum. The cell seeding rate was 1()6 cells/ml. The cell line was
subcultured once in 8 days and the subculture ratio of 1:2 was maintained during the
study.
3.5.3 Adaptation of EDS-76 viral isolates in CEL. DEF cell cultures and
Duck Pekin cell line
The viral isolates at fifth passage in duck embryos were propagated in CEL,
DEF and Duck Pekin cell line. The fully formed monolayers in milk dilution bottles
were inoculated with one ml of the isolates and incubated at 37°e for 1 h for viru:s
absorption. After absorption, the inoculum was discarded and fresh maintenance
medium was added and incubated at 37°e and the bottles were examined
periodically for cytopathological changes. At the height of ePE the tissue culture fluid
was harvested by freezing and thawing for three times. All the viral isolates were
passaged 10 times in the above cell cultures. The HA activity of the virus isolates at ? .. _._--_.-
each passage level was assayed in accordance with Shakya and Dhawedkar (1991a).
3.5.4 Study on CPE
The primary cell culture and cell line adapted virus isolates at their fifth
passage level were used to infect the flying cover slip cultures and examined daily.
The covers lips showing ePE at different intervals were fixed in Carnoy's or Bouin's
fixatives. H & E staining of both the infected and control coverslips were done as per
the procedure described by Rovozzo and Burke (1973) . .Ii
3.5.5 Titration of viral infectivity in primary cell culture and cell line
Titration of all the isolates at their fifth and tenth passage level was carried out
using microtitre technique as per the method described by Kumanan (1989). Ten-fold
dilutions of the virus were made from 10-1 to 10-9 in separate sterile tubes with
maintenance medium. For each dilUtion, six wells were allotted alld 25 ul of virus
suspension was added. The last two rows of wells were kept as control. The plates
were sealed with adhesive tape and incubated at 37°C for 1 h for adsorption. After
the incubation period 25 ul and 50 ul of maintenance medium was added to each of
the infected and control wells, respectively. The plates were sealed again, incubated
at 37CC and observed periodically for the development of CPE. The highest dilution
showing ePE was taken as the end point. The TCIDso was calculated in accordance
with Reed and Muench method (1938).
3.5.6 Physico-chemical properties
3.5.6.1 Sensitivity of the isolates to chlorofonn
For the treatment with chloroform, 1.8 ml of viral suspension was mixed with
0.2 ml of chloroform (Higashihara et 01., 1983) and the mixture was allowed to stand
at room temperature for 15 min with intermittent vigorous shaking, followed by
centrifugation. Simultaneously, untreated virus control was also kept along with
experimental10ts. After centrifugation, the supernatantsfrom'both the tubes were used
for assessing the infectivity titre in DEF cell culture and also for inoculating duck of v~ru.s
embryos for assessment of HA titre 1\ :'1Lthe allantoic fluid.
3.5.6.2 Sensitivity of the isolates to ethyl ether
The method described by Higashihara et 01. (1983) was followed. A mixture
of 3 ml of the virus suspension and one milli litre of anaesthetic ether was kept at 4°C
for 18 h along with untreated control. The mixture was then centrifuged and the
supernatant was used for assay of infectivity and HA titres.
3.5.6.3 Effect of pH 0" viral isolates
The stability of the viral isolates at pH 3.0 was studied as described by
Higashihara et 0/. (1983). After incubation at room temperature for 1 h at acidic pH
3 in HBSS were subseqtJently titrated for the concentration of the virus in DEF cell
cultures and also with allantoic fluid followed by inoculation into embryonated duck
eggs for HA titre.
3.5.6.4 Sensitivity of the isolates to trypsin
The sensitivity of the viral isolates to trypsin was tested by the method
advocated by Swain et 0/. (1992). The viral material after treatment with 0.3 per cent
trypsin solution at 37°C for 30 min was checked for probable effect of infectivity and
HA activity.
3.5.6.5 Thermostability of the viral isolates
The stability of the viral isolates to temperature was done in accordance with
earlier researchers (Todd and McNulty, 1978; Ramku=mar et 0/., 1991 and Swain
et a/., 1992) by exposing the viral dilutions to 56°C for 30 min along with controls at
room temperature followed by assessment of infectivity and HA activity titres.
3.5.6.6 Effect of formalin on viral isolates
The viral isolates were exposed to 0.3 per cent formalin for 1 h and the
infectivity and HA activitY were assessed in accordance with the method followed by
Swain et 0/. (1992).
3.5.6.7 Detennination of the type of viral nucleic acid
The nucleic acid determination of the viral isolates was carried out following
the method of Higashihara et 0/. (1983). DEF cultures prepared in microplates were
inoculated with varying dilutions of the viral isolates with a minimum allocation of
three wells per dilution. After virus adsorption, each well was fed with 50 ul of
maintenance medium containing 100 uglml of 5-iodo-2' deoxyuridine (lUDR).
lnoculated wells fed with plain maintenance medium served as control. After
incubation at 37°C for 5 days the culture fluid was tested for HA and the TCID50
titres.
3.S.7 Electron microscopic studies of viral isolates
The method o(Swain et al. (1993) was followed with slight modification.
3. S. 7.1 Direct negative contrast electron microscopy (DNCEM)
The EDS-76 virus isolates propagated in duck embryos were used for this
study. Infected allantoic fluid of each isolate showing high HA titre (10 log2) was
clarified at 5000 9 for 15 min at 4°C. The clear supernatant was used for DNCEM.
Briefly, a drop of the clear supernatant was charged on to the collodion carbon
coated 200 mesh copper grids and kept at room temperature for virus adsorption in
a dust free chamber. After 20 min the excess fluid was removed by touching to filter
paper and the grid washed in distilled water. Then the grids were negatively stained
with 2 per cent phosphotungstic acid (pH 7.2) for 30 sec. Excess stain was removed
and the grids were air dried and examined in a Jeol JEM-100C Transmission electron
microscope (TEM) at 80 KV.
3.5.7.2 Immuno electron microscopy
Clarified infected allantoic fluid of each isolate was allowed to react with the
EDS-76 virus standard hyperimmune serum (1 in 20 dilution) for 1 h at 37°C before
being processed for TEM scanning.
3.6 PROTEIN FRACTIONATION STUDIES
3.6.1 Solutions for SDS-Polyacrylamide gel electrophoresis (SOS-PAGE) J_a.emmLi.., Jq~O
3.6.1.1 Acrylamide-bisacrylamide (30 per cent) stock
Acrylamide
Bisacrylamide
Distilled water to make
30 g
0.8 g
100 ml
Filtered through Whatman No.1 filter paper and stored at 4°C.
3.6.1.2 Separating gel (12 per cent)
30% Acrylamide stock 12.0 ml
Tris.HCI, l.5M, pH 8.8 7.5 ml
Sodium dodecyl sulphate, 10% 0.3 ml
Ammonium per sulphate, 10% 0.3 ml
TEMED 0.03 ml
Distilled water 9.9 ml
3.6.1.3 Stacking gel (5 per cent)
30% Acrylamide stock
1M Tris.HCI, pH 6.8
Sodium dodecyl sulphate, 10%
Ammonium per sulphate, 10%
TEMED
Distilled water
3.6.1.4 Running buffer
Glycine
Tris base
Sodium dodecyl sulphate, 10%
Distilled water added to make
3.6.1.5 2X Sample buffer
Tris base
Glycerol
1.7 ml
1.25 mt
0.1 ml
0.1 ml
0.01 ml
6.8 ml
.14.4 9
3.0 9
10m)
1 L
1.51 9
20ml
dissolved in 35 ml of distilled water, pH was adjusted to 6.75 with cone. He}.
Sodium dodecyl sulphate
2-Mercaptoethanol
Bromophenol Blue
Distilled water added to make
4.0 9
10 ml
0.002 9
100 ml
3.7.2. Methods for DNA analysis
3.7.2.1. Purification of the viral isolates
EDS-76 viral isolates grown in duck embryos were concentrated and purified
(Tood and McNulty, 1978).
3.7.2.2. Extraction of viral DNA and Quantification
Viral DNA was extracted from the purified virus particles by the phenol:
Chloroform extraction method as described by Sambrook et 01., 1989 with slight
modification. Briefly 100 ul of proteinase K (10 mg/ml) was added to 200ul of virus
suspension and mixed thoroughly. The mixture was incubated at 560 C for 2h. To this
double the volume of tris buffer saturated phenol: chloroform - isoamyl alcohol
mixture in the ratio 25:24: 1 was added and centrifuged at 15,000 rpm in Beckman
microfuge for 10 min. The upper aqueous phase was separated and treated twice
with pure chloroform (AR,BDH) and the mixture was subsequently centrifuged as
described earlier. The DNA molecule in the aqueous phase was then precipitated with
cold ethanol and kept overnight at-200C and centrifuged at 15,000 rpm for 20 min.
The pelleted DNA was washed with 70 per cent ethanol and dried in a vacuum oven
for 30 min.The DNA was then suspended in lNE buffer and stored.
The purity of the DNA was checked by measuring the OD at 260 and 280 nm
in a spectrophotometer and the ratio of 260/280 was calculated. DNA preparations
with the ratio of 1.80 and above was used for restriction enzymes analySis. The
concentration of the DNA was determined by using the relationships 1 OD at 260 nm
= SOug/mt.
3.7.2.3 Enzyme digestion of viral DNA
Approximately 5ug of purified viral DNA was mixed with 20 units of resbiction
endonuclease and 20u1 of enzyme buffer supplied by the manufacturer for enzyme
3.6.1.6 Staining Solution
Coomassie Brilliant Blue, R250
Methanol:water (1.1, v/v)
Glacial acetic acid
O.25g
90 ml
10 ml
Stain was filtered through Whatman No.1 filter paper.
3.6.1.7 Destaining Solution
Glacial acetic acid
Methanol
Distilled water
10 ml
250 ml
240ml
3.6.2 Methods for protein fractionation
3.6.2.1. Purification of EDS-76 viral isolates
EDS-76 isolates grown in ~uck embryos were used for concentration and
purification (Todd and McNulty, 1978 and Swain et 01., 1982}.lnfected allantoic fluid
was clarified at 5000g for 15 min. The supernatant was subjected to
ultracentrifugation in a Bechman ultracentrifuge (Model L7-80) 7011 rotor at 80,000g
for 2h at 4°C. The crude virus pellet obtained was resuspended in minimum quantity
of Tris Sodium EDTA(TNE) buffer. This was overlaid on 36 per cent sucrose cushion
centrifuged at 1,OO,000g for 4h at 4·C in a 6011 swingout rotor. The purity of the
virus was checked by running on SDS-PAGE. The protein concentration of the
Durified virus was measured in a spectrophotometer at 280nm using a standard curve
prepared with bovine serum albumin. The purified virus pellet was resuspended in
TNE and used for protein fractionation and restriction enzyme analyses of DNA
3.6.2.2 50S-PAGE
Thirty millilitre of 12 per cent separating gel was prepared for a plate of 14 x
15 cm dimension. The solution was degassed and then ammonium per sulphate and
TEMEO were added and poured benveen the glass plates and allowed to polymerize.
Distilled water was layered on top of the gel to make the surface smooth. When
polymerization was completed, the distilled water layer was removed and 10 ml of 5
per cent stacking gel was prepared and poured in between the glass plates with the
well former in position, and kept for polymerization. Then the plate was fitted in the
buffer reservoir and the samples were applied into each slot.
Fifty ul of each sample was mixed with 20 ul of sample buffer, kept in the
boiling water bath for 3 min and then loaded onto the gel. SOS (6H, Sigma)
molecular weight marker was included in the gel.
The electrophoresis was run for 4 h at 100 volts until the marker dye reaches
the lower level of gel. Once electrophoresis was completed, the gel was removed from
the glass plates, the stacking gel was discarded and the separating gel stained in
Coomassie Blue staining solution overnight. The gel was destained with destaining
solution containing methanol-acetic acid-water until the gel back ground becomes
clear.
3.6.2.3 Estimation of molecular weight
Molecular weight of different fractions of the virus isolates were estimated as
per the method of Shapiro et al. (1967). SDS- molecular weight marker (SDS-6H,
Sigma) was used. This marker contained six proteins, viz., carbonic anhydrase (MW
29,000), albumin, egg (MW 45,000), bovine serum albumin (MW 66,000),
phosphorylase B (MW 97,400), beta-galatosidase (MW 1,16,000) and myosin (MW
205,000). Relative migration of standard protein and different viral fractions were on Semi. LOB gra.ph fu-:pey
plotted "and their molecular weight estimated.
3.7 RESTRICfION ENZYME ANALYSIS OF DNA OF EDS-76 VIRAL
l.S0LATES .,.
3.7.1 Materials used for DNA analysis
3.7.1.1 Proteinase K (Sigma) 10 mglml
3.7.1.2 Phenol-chlorofonn .. ,isoamyl alcohol mixture(SllmbYDOX et aJ ., }qgG
Phenol 25 parts
Chloroform 24 parts
Isoamyl alcohol 1 part
3.7.1.3 Tris_borate buffer, lOX
Tris 108.0 9
EDTA, Sodium salt 9.3 9
Boric acid 55.0 g
Distilled water 1000 ml
pH adjusted to 8.2. Diluted 10 times before use and ethidium bromide is added to
buffer to a final concentration of 0.5 uglml.
L1.1.4 Agarose gel (0.1 per cent)
\.garose (Sigma) 0.35 gm
~ris borate buffer SOml
~oiled and poured belore electrophoresis.
1.1.1.5 Restriction ~nzymes (Boehringer Mannheim, Germany)
:co RI
-find III
>st I
~gl II
-Iae III
~am HI
l. 7.1. 6 Gel loading buffer
~romophenol Blue 0.25 per cent
(ylene cyanol, FF 0.25 per cent
31ycerol in water 30 per cent
l.1.1.1 DNA marker
t.coRr Lambda DNA (Sigma) digested with restriction enzyme was used.
"
digestion. The enzymes EcoRI, Hind III, Bgl I, Bam HI, Pst 1 and Hae III were
individually examined with viral DNA sample. The DNA preparation was digested
overnight at 37°C. After digestion, the DNA was mixed with gel loading buffer and
then layered on to the horizontal slab gel.
3.7.2.4 Electrophoresis of DNA fragments
The DNA restriction fragments were electrophoresed at 50V for 16h on a 0.7
per cent agarose gel (6 x 6.5 em size) using Tris-borate buffer containing ethidium
bromide. at a concentration of 0.5uglml. After electrophoresis, the DNA fragments
were visualized with a transilluminator and photographs were taken with highspeed
films. The relative molecular sizes of restriction fragments were estimated by
comparing with the corresponding relative electrophoretic mobilities of Eco RI cleaved
lambda DNA fragments of known molecular weights.
3.8 EXPERIMENTAL INFECTION OF EDS-76 VITAL ISOLATES
3.8.1 EDS-76 virus isolate
Indigenous virus isolate M1 was used for experimental infection.
3.8.2 Chicken
Twentyfive white leghorn birds in the age group of 24 to 28 weeks purchased
from Poultry Research Centre, T eynampet were used for experimental infection.
3.8.3 Methods in experimental infection
3.8.8.1 Inoculation of birds
The experimental birds were divided into two groups of 20 experimentally
infected birds and 5 control birds. The indigenous virus suspension containing 512
HA units was used for inoculation (Satyanarayana Chetty, 1985). The first group
of 20 birds were inoculated with the virus suspension both orally (0.25 ml) and
intramuscularly (0.25 ml) per bird and the birds were observed for a continuous
period of ten weeks PI.
3.8.3.2 Collection of samples
Faecal swabs were collected at twentietn dllU [w(:!lIlY luunrrtraurly initially
followed by examination at every 24 hourly intervals upto 15 days then at 20th, 25th
and 30th day PI.
From 5th day PI blood samples were collected by heart puncture daily till 28th
day and then at weekly intervals till the 10th week. Serum separated from the above
samples were pooled and stored at -20°C. Pooled serum samples were used for
detection of EOS-76 antibody.
Infected birds were sacrificed at weekly intervals and tissues for viral isolation
and histopathology were collected in HBSS and 10 per cent buffered formalin,
respectively.
3.8.3.3 Antigen detection
HA, AGIO and OIA were employed for the detection of antigen in the faecal
swabs.
3.8.3.4 Virus recovery
The virus was reisolated from samples collected from experimentally infected
birds as per the method employed by Ramkumar et 01. (1992).
3.8.3.5 Monitoring.of serum antibody titres
1""\
H1, OIA and AGIO were employed to monitor the specific antt90dy to EOS-76
in infected birds as per the procedure adopted by Shakya and Ohawedkar (1991a),
, Pappas (1988) and Sathyanarayana Chetty et al. (1985), respectiveJy.
CHAPTER IV
RESULTS
4.1. SEROPREVALENCE
The titres in the serological tests were expressed as log to the base 2 per 50
ul (log:/50 ul). The HI antibody titre of 8 (3 log2) or above was considered positive
in the present study. In DIA, the reaction was read and scored depending upon the
intensity of the colour reaction as 3+ (intense brown dot), 2+ (brown dot), 1 + (light
brown dot), trace (hardly any dot) and negative (no dot). The highest serum dilution
showing a 1 + reaction was taken as positive DIA titre (Fig.l).
A total of 809 set'l.'l16amples were randomly collected from various poultry farms
in 8 districts of Tamil Nadu both with or without the syndrome of drop in egg
production. All the samples were subjected to HI and orA to detect the presence of
EDS-76 viral antibodies. District wise distribution of EDS-76 viral antibodies are
presented in Table I.
The highest incidence of EDS-76 viral antibodies was recorded in Madurai with
a positive percentage of 53.21. Madras recorded the next higher incidence with a
positive percentage of 23.18 followed by North Arcot (16.52), Dharmapuri (10.47),
Kamarajar (8.3), Kanyakumari (5.31), Salem (5.26) and Periyar (3.4). Such similar
trend of prevalence of EDS-76 was apparent as tested by DIA.
Fig.1 Quantitative Dot ImmuTlobinding A'iSay on Nitrocellulose membrane to screen (rt)
EDS-76 viral antibodies. Bottom most horizontal"row- first three squares
denote positive controls, while the rest five are negative controls. Top most (I) (rfl- '1)
horizontal"row and six other horizontalArows below it show positive colour
reaction of varying intensities indicating positive DIA at varying dilution levels
(1 in 50 to 1 in 8000).
C/)
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Agewise incidence of HI antibodies in the serum;amples of chicken is presented
in Table II. The highest percentage of incidence of antibodies to the tune of 24.5 to
EOS-76 virus was apparent among the birds above 40 weeks, while 16.14 per cent
was observed among the birds aged between 30 and 40 weeks and 14.62 per cent
among the birds aged between 20 and 30 weeks. The least percentage of incidence
5.64 was adduced among the birds aged below 20 weeks. It is evident that as the age
increases the incidence of infection also increases as assessed by both HI and OrA
Of the 809 selWl'6amples screened both by HI and DIA, 127 were positive by
HI with a percentage of 15.69, whereas, 137 samples were positive by OIA with a
positive percentage of incidence of 16.93. The comparative results of both the
serological tests are presented in Table III. In this study, the percentage of positivity
of HI is slightly lower when compared to that of DIA. Inspite of this slight variation it
has been clearly shown by student 't' test (t = 0.722 NS, P < 0.05) that there is no
significant correlation between the two tests employed for the detection of EDS-76
viral antibody.
The HI titres of the semrnsamples from different districts in Tamilnadu ranges
from 8 to 1024, while the range of DIA titre is 100 to 8000. A comparison of mean
antibody titres of both HI and DIA are presented in Table IV. It is apparent from this
table that flocks showing higher range of HI titres between 120 and 1024 certainly
harbour EOS-76 virus infection or indicate that such birds have suffered from such
a syndrome of drop in egg production as ascertained by higher mean HI and DlA
titres.
The comparative efficacy of DIA over HI test is represented in Table V.
TABLE II: AGE-WISE INCIDENCE OF HI ANTIBODIES TO EDS-76 VIRUS
Number Number Percent
S.No. Age group
of positive by positive by (weeks) samples DIA HI HI DIA
1. Below 20 weeks 124 7 8 5.64 6.45
2. 20-30 weeks 212 31 33 14.62 5.16
3. 30-40 weeks 322 52 55 16.14 17.08
4. Above 40 weeks 151 37 41 24.50 27.15
TABLE III : COMPARISON OF RESULTS OF HI TEST AND DIA
Observation HI DIA
Total samples screened 809 809
Total positive 127 137
Percentage positive 15.69 16.93
t = 0.72 NS (P < 0.05)
NS = Not Significant.
TABLE IV: COMPARISON OF MEAN ANTIBODY TITHES OF EDS-76 VIRUS BY HI TEST AND DIA
Range of HI titres Mean HI titres Mean DIA titres
8 - 16 11.01 120.400
32 - 64 42.90 396.998
128 - 256 194.01 1414.213
512 - 1024 772.07 5388.175
A TABLE V: COM~RATIVE EFFICACY OF DIA AGAINST HI IN
DETECffNG EDS-76, VIRAL ANTIBODIES IN SERUM
Test Number of HI+ HI - HI+ HI - Correlation samples Test+ Test+ Test- Test- Co-efficient
OIA 809 127 10 0 672 0.99
Variations in HI is 98% in association with variation in OIA. Mean antibody titre by
OIA for samples that are positive by OIA and negative by HI is 190.95. Both OIA and
HI are positively correlated.
4.2 ISOLATION
Duck embryos were inoculated with both the processed cloacal swabs and shell
gland homogenates. In all, 118 cloacal swabs and 40 shell gland homogenates
collected from different areas in Tamilnadu and neighbouring Andhra Pradesh and
Kamataka were used for virus isolation. The details of samples collected and
the percentage of egg drop syndrome observed in different areas are tabulated in
Table VI.
4.2.1 Virus isolation from cloacal swabs
Out of the 118 cloacal swabs, attempted for virus isolation, allantoic fluid from
three samples at second passage level showed HA activity by spot test and the three
viral isolates procured from different areas were designated 'as 81, M1 and D1
representing Bangalore (81), Madurai (M1) and Oharmapuri (01), the details of which
are presented in Table VII.
4.2.2 Virus isolation from shell gland
Out of the 40 shell gland homogenates used for viral isolation, two isolates
could be procured at third passage level in duck embryos. These isolates are
designated as B2 and 02. The details of virus isolation are presented in Table VII.
4.2.3 HA titre of isolates
The HA titre of the five isolates were assessed, first on isolation and after 5
passages in duck embryos. The HA titre of the isolates on isolation ranged between
8 and 32 and after 5 subsequent passages, the HA titre ranged between 256 and
w -61: o a) ... u "'0 ... S,W LO LO 0 0 0 LO LO 1Il-= r-- \0 00 '<:j' r-- r-- LO
I I I I I I I
c."'O LO 0 0 l1J 0 0 0 0 W \0 '<:j' \0 N l1J \0 C") ... t: "'0
W O)fIl 0)..0
t.Ll 0
"'0 I: ~ -0)
w - LO C"') '<:j' N '<:j' ['.. LO - - ~ 'tl.. ~
6 ..c:: Ie CJ) fIl -0 w ..0 u ... Ie :: :3 0 III
CJ) - C"') \0 N \0 r-- \0 00 ~ u rl rl N rl ,...... ,...... rl
Ie 0
0
fIl W -c. 6 tC 00 0\ \0 00 ~ C"') C"') fIl rl N N ,...... N N N -0 0
Z
III
6 ... Ie - 0 "<::t 00 N 00 "<::t N -0
rl ~ ,...... ,...... ...... ...... 0 Z
E .t: (I)
ctl ::l ro :» ... ctl 0-'ra ..::.:: 0 ro ctl Ie
(I) ... ..::.:: ro (I) E ... ::l ctl ... 0-W 0 OJ 0 ... "'0 E c ctl ctl <t: (I) ctl ctl ctl (I) 'co ..c
Z :z z co > 0:::: 0
rl N (Y) .q: l1J \0 r--
TABLE VII: DETAILS OF SOURCE FOR EDS-76 VIRAL ISOLATION
Source of Number No. positive Percentage Viral sample tested for antigen positive designates
Cloacal swab 118 3 2.54 Bl, Ml, 01
Shell gland AO 2 5 82,D2
1024 (Table VIII). Significant increase in titre has been observed on embryonated
duck egg passage.
4.2.4 HI titre of reference serum with EDS-76 isolates
The HI titre of the reference serum with EDS-76 isolates were assessed. The
HI titre of the reference serum to all the isolates ranged from 32 to 128 (Table IX).
This has significantly established the specificity of the known antibody to the local
indigenous isolates.
4.2.5 Cross HI test of isolates with reference strain 127
The results of cross HI indicated that all the five isolates were serologically
identical to strain 127 (Table X).
4.3 ADAPTATION OF EDS-76 ISOLATES IN CELL CULTURE
4.3.1 CEL cell culture
All the EDS-76 isolates showed appreciable viral replication in eEL cultures.
First, a blind passage was made and at the second passage eEL culture exhibited
ePE from the 4th day PI. The culture fluid agglutinated chicken erythrocytes.
4.3.2 DEF cell culture
The isolates in DEF culture multiplied progressively from the first passage
onwards. Though ePE started from 3rd day PI during the first passage, the ePE was
adduced as early as 48 h PI in the subsequent passages involving 50 per cent of the
cells. The HA titre increased appreciably after every passage.
TABLE VIII: HA TITRE OF EDS-76 VIRAL ISOLATES
Viral isolates HA titre (lOg2) on HA titre (log2) after 5
isolation passages in duck embryos
B1 5 10
M1 6 10
01 5 9
B2 4 8
02 3 8
TABLE IX : HI TITRE OF REFERRAL EDS-76 ANTISERUM WITH EDS-76
VIRAL ISOLATES
--
Viral isolates HI titres (log2 )
81 5
M1 7
D1 5
82 5
02 6
TABLE X: CROSS HI TEST OF EOS-76 VIRAL ISOLATES WITH REFERRAL STRAIN 127
Viral isolates HI titres (log2) of antiserum to
Reference strain M1 isolate
81 6 5
M1 6 6
01 5 5
82 5 5
02 6 5
Strain 127 6 6
4.3.3 Duck Pekin cell line
All the isolates showed progressive viral replication and characteristic ePE from
the first passage as monitored by HA.
4.4 CYfOPATHOGENICIlY OF EDS-76 ISOLATES
4.4.1 CEl cell cultures
The ePE started in eEL cultures from the 4th day PI and reached a peak by
8th day PI. The ePE manifested itself in the form of rounding and grouping of
infected cells (Fig.2). Large vacuolations with cytoplasmic "nastomosis of cells together
with formation of multinucleated giant cell (Fig.3) were observed as compared to the
normal cell monolayer (Fig.4). H & E staining revealed eosinophilic inclusion bodies
in the nuclei of the infected cells on further observation.
4.4.2 DEF cell culture
The isolates produced ePE in DEF Cells from the first passage. The ePE
started with the appearance of refractile round cells and the formation of syncytia
(Fig.S) followed by formation of giant cell with eosinophilic intranuclear inclusion
bodies in the infect ed cells (Fig.6) as compared to normal cells (Fig.7). On continued
observation detachment of infected cells was adduced.
4.4.3 Duck Pekin cell line
The isolates produced identifical ePE in duck Perkin cell line to those
produced in DEF cells. Rounding and grouping of cells started by 24 h and from 4th
Fig.2 Chicken Embryo Liver monolayer infected with EOS-76 viral isolate, depicting
derangement initially as rounding and grouping of c~lIs. H&E staining. X100
Fig.3 Chicken Embryo Liver monolayer infected with EDS-76 viral isolate showing
large vacuolations with cytoplasmic anastomosis of cells. H & E staining. XIOO
FigA Chicken Embryo Liver monolayer - Normal. H & E staining. X 100
•
Fig.S Duck Embryo Fibroblast monolayer infected with EDS-76 viral isolate. Note
the appearance of refractile cells and syncytia formation. H & E staining. X
400
Fig.6 Duck Embryo Fibroblast monolayer infected with EOS-76 viral isolate, showing
formation of giant cells with eosinophilic intranuclear inclusion bodies. H&E
staining. X 400
Fig.7 Duck Embryo Fibroblast monolayer - Normal. H & E staining. X 100
day PI there was syncytia formation (Fig.8) and finally by 7th day PI there was fusion
of nuclei with giant cell formation (Fig.9) as compared to normal cells (Fig.l0).
4.5 INFECfIVllY ASSAY OF EDS-16 ISOLATES IN CELL CULTURE
SYSTEM
The 50 per cent tissue culture infectivity dose (TCIDso) of all the EDS-76 viral
isolates after 5th and 10th passages in the course of propagation in both primacy ceU
cultures and cell line are presented in Table XI. At the end of the 5th passage the titre
of the isolates ranged between 3.9 and 4.3 in CEl, 4.3 and 4.8 in DEF and 4.2 and
4.5 in. duck Pekin cell line. At the end of 10th passage the titre of the isolates ranged
between 4.1 and 4.4 in CEl, 4.3 and 4.6 in DEF and 4.3 and 4.6 in duck Pekin cell
line. The infectivity titres of the referral EDS strain 127 correlate well with limits of
TCIDso evinced by the indigenous isolates.
4.6 PHYSICO-CHEMICAL PROPERTIES
The results of the physico-chemical properties of five different EDS-76
: indigenous isolates are furnished in Tables XII to XVI.
Treatment of the isolates with chloroform, ether and acid pH did not influence
both the infectivity titres and HA activity significantly. Concentration of Trypsin at 0.3
per cent level also did not influence both the infectivity titre and the HA activity. All
the isolate were found to be stable on heat treatment at 56°C for 30 min.
The infectivity of the isolates was completely nullified when treated with
formalin at 0.3 per cent concentration.
Fig.8 Duck Pekin cell line monolayer infected with EDS-76 viral isolate, showing
rounding and grouping of cells and syncytia formation . H & E staining. X 100
Fig.9 Duck Pekin cell line monolayer infected with EDS-76 viral isolate showing
fusion of nuclei. H & E staining. X 100
Fig.I0 Duck Pekin cell line monolayer - Normal. H & E staining. X 100
•.
. -.. ~ _--- - ~ . .
TABLE XI : INFECTIVITY TITRE (TCIDso) OF EDS-76 VIRAL ISOLATES IN CELL CULTURE SYSTEMS AFTER FIFTH AND TENTH PASSAGES
Viral isolates
Bl
Ml
Dl'
B2
D2
Stra· m'~' In" ...
TABLE XII:
Treatments
Chlorofrom
Ether
pH 3
Trypsin 0.3%
Infectivity titre (log10 i TCIDso /0" »'lL)
CEL ceJ) DEF cell Duck Pekin Culture culture cell line
5th 10th 5th 10th 5th lOth
4.1 4.3 4.4 4.5 4.4 4.4
4.2 4.2 4.5 4.5 4.5 4.5
3.9 4.1 4.4 4.6 4.2 4.4
4.3/ 4.4 4.3 4.4 4.2 4.3
4.1 4.3 4.3 4.3 4.3 4.4
4.4 4.4 4.8 4.9 4.5 4.6
PHYSICO-CHEMICAL CHARACTERS OF THE EOS-76 VIRAL ISOLATE (81)
Infectivity titre HA titre 10gl(/fCIDso (log2)
Untreated Treated Untreated Treated
4.4 4.4 9 9
4.4 4.4 9 9
4.4 4.2 9 9
4.4 4.3 9 9
Temp. 56oC, 30 min 4.4 4.4 9 9
Formalin 0.3% 4.4 - 9 8
IUDR 4.4 - 9 3
,
Treatments ' - !
Choroform \
Ether
pH 3
Trypsin 0.3%
PHYSICO-CHEMICAL CHARACTERS OF mE EOS-76 VIRAL ISOLATE (MI)
Infectivity titre HA titre I091tlfCIDso (1092)
Untreated Treated Untreated Treated
4.5 4.4 11 11
4.5 4.5 11 11
4.5 4.5 11 11
4.5 4.4 11 11
Temp. 56oC, 30 min 4.5 4.5 11 11
Formalin 0.3% 4.5 - 11 11
IUDR 4.5 2.1 11 3
TABLE XIV:
Treatments
Chlorofoml
Ether
pH3
Trypsin 0.3%
PHYSICO-CHEMICAL CHARACTERS OF THE EDS-76 VIRAL ISOLATE (Dt)
Infectivity titre HA titre IOgllY'fCI Dso (Jog2)
Untreated Treated Untreated Treated
4.4 4.4 7 7
4.4 4.3 7 7
4.4 4.4 7 7
4.4 4.4 7 6
Temp. 56oC, 30 min 4.4 4.3 7 7
Formalin 0.3%
IUDR
TABLE XV:
Treatments
Chloroform
Ether
pH 3
Trypsin 0.3%
4.4 - 7 6
4.4 2.2 7 4
PHYSICO-CHEMICAL CHARACTERS OF THE EOS-76 VIRAL ISOLATES (B2)
Infectivity titre HA titre IOgllY'fCIDso (log2)
Untreated Treated Untreated Treated
4.3 4.3 7 7
4.3 4.2 7 7
4.3 4.3 7 7
4.3 4.2 7 7
Temp. 56oC, 30 min 4.3 4.3 7 7
Formalin 0.3% 4.3 - 7 6
IUDR 4.3 2.1 7 3
TABLE XVI :
Treatments
Chloroform
Ether
pH 3
Trypsin 0.3%
PHYSICO-CHEMICAL CHARACTERS OF THE EDS-76 VIRAL ISOLATES (02)
Infectivity titre HA titre logltlfCIOso (log2 )
Untreated Treated Untreated Treated
4.3 4.3 6 6
4.3 4.3 6 6
4.3 4.3 6 6
4.3 4.2 6 5
Temp. 56oC, 30 min 4.3 4.3 6 6
Formalin 0.3% 4.3 - 6 5
IUDR 4.3 2.1 6 3
All the isolates suffered a significant fall in their infectivity titres as well as with
their HA activity when treated with IUOR, thus confirming the presence of
deOxyribonucleic acid in their genome.
4.6.1 Electron microscopic study
4.6.1.1 Direct negative contrast electron microscopy (DNCEM)
The electron microscopic observation of the different viral isolates present in
the allantoic fluid of embryonated duck eggs by DNCEM revealed the presence of
typical adenovirus particles scattered singly or in twos. The icosahedral particles
measured approximately 80 nm in diameter Fig.ll.
4.6.1.2 Immuno electron microscopy (IEM)
The presence of adenovirus particles in the allantoic fluid and freedom from
contamination with other viruses was confirmed by IEM. IEM revealed the presence
of typical adenovirus particles clumped together when treated with standard
hyperimmune serum (Fig.12).
4.7 PROTEIN PRORLE OF EDS-76 VIRAL ISOLATES
For fractionation of proteins of the five isolates and to compare with the
referral strain SOS-PAGE was used by employing conventional Coomassie Blue
staining. By this technique a total of 13 polypeptides were observed (Fig.13). The
molecular weight of different polypeptides of the isolates ranged between 11 KDa and
126 KDa (Table XVII). SOS6H (Sigma) protein markers were used for comparing the
Fig.II Electron micrograph of EDS-76 viral isolate, DNCEM- icosahedral adenoviral
particles scattered singly or in twos. X 1,00,000.
., .
, I
Fig.12 Electron micrograph of E05-76 viral isolate IEM- Clumping of viral particles.
X 1,00,000.
t + '1
Fig.13 SDS-PAGE analysis of EOS-76 viral proteins stained with Coomassie Brilliant
Blue stain.
Lane] - Protein profile of Bl isolate Lane 2 - Protein profile of B2 isolate Lane 3 - Protein profile of referral strain 127 Lane 4 - Molecular weight markers - SOS-6H Lane 5 - Protein profile of Ml isolate Lane 6 - Protein profile of 01 isolate Lane 7 - Protein profile of D2 isolate
i TABLE XVII:
Viral Protein
band
1
2 < ,
3\
4
5 ,
6 «
7 ,
8
9
10
11
12
13
VIRAL PROTEIN BANDS OF DIFFERENT ISOLATES AND THEIR MOLECULAR WEIGHTS
Molecular weight (KDa)
Bl B2 127 Ml 01 02
125.8 125.8 125.8 125.8 125.8 125.8
93.3 93.3 93.3 93.3 93.3 93.3
85.1 85.1 85.1 85.1 85.1 85.1
70.7 70.7 70.7 70.7 70.7 70.7 ,
51.2 51.2 51.2 51.2 51.2 51.2
40.7 40.7 40.7 40.7 40.7 40.7
33.1 33.1 33.1 33.1 33.1 33.1
27.5 27.5 27.5 27.5 27.5 27.5
22.3 22.3 22.3 22.3 22.3 22.3
20.0 20.0 20.0 20.0 20.0 20.0
14.1 14.1 14.1 14.1 14.1 14.1
12.0 12.0 12.0 12.0 12.0 12.0
11.0 11.0 11.0 11.0 11.0 11.0
relative migration of the polypeptides of the isolates and the molecular weight of
different fractions were estimated as per the method of Shapiro et af. (1967).
4.8 RESTRICTION ENDONUCLEASE ANALYSIS OF EDS-76 VIRAL DNA
The DNA extracted from different field isolates were checked for their whole
DNA purity and then subjected to digestion with. six restriction endonulceases, viz.,
Eco RI, Hind III, Pst I, BgI II, Hae III and Bam I-ll. Of these six enzymes, Pst I
generated more number of DNA fragments (Fig.14). The digestion products were
analyzed on 0.7 per cent w/v agarose (Sigma) gel using ethidium bromide stain in the
buffer. The DNA restriction fragment patterns generated by Pst I is presented in Fig.IS
& 16. The relative molecular sizes of restriction fragments were estimated by
comparing with the corresponding relative electrophoretic mobilities of Eco RI cleaved
lambda DNA fragments of known molecular weights. The number of restriction
fragments and their corresponding molecular sizes (kbp) generated by these restriction
enzymes are presented in Table XVIII.
4.9 EXPERIMENTAL INFEC110N
4.9.1 Clinical findings
The indigenous EDS-76 virus isolate M1 did not induce clinical signs of disease
in any chicken of the age group 24-28 weeks under this study. The chickens remained
healthy throughout and no mortality was recorded.
8
,.....---4) -So f? I-i b 5 .6 kb
(Mi ) Fig.14 Restriction endonuclease DNA profile of EDS-76 viral isolate digested with
f...
different enzymes.
Lane 1 - Eco HI Lane 2 - Hind III Lane 3 - Pst I Lane 4 - Blank Lane 5 - 8g1 I Lane 6 - Hae III Lane 7 - Bam HI Lane 8 - Lambda DNA cut with Eco RI
6 '7
___ --7~J.~ kb
tel '-t.4 kb I)
5'. g k b
5.6 hh
4. Q 111:>
--~-~3.q Kb
Fig.15 Restriction endonuclease DNA profile of EDS-76 viral isolates and referral
strain digested with enzyme Pst l.
Lane 1 - B1 did not cut due to unavoidable reasons Lane 2 - B2 did not cut due to unavoidable ,reasons Lane 3 - Referral strain 127 Lane 4 - Ml Lane 5 - 01 ~ne6-D2 ~ Lane 7 - Lambda DNA cut with Eco RI ,\6
2. 3
---+5.81'\0 ---+'0.6 Kb
-----? it·q lih
Fig.16 Restriction endonuclease DNA profile of EDS-76 viral isolates digested with
enzyme Pst I.
Lane 1 - EDS-76 viral isolate B1 Lane 2 - EDS-76 viral isolate B2 Lane 3 - Lambda DNA marker.
T~LE· XVIII : EDS-76 VIRUS DNA RESTRICTION FRAGMENTS GENERATED BY RESTRICTION ENDONUCLEASE Pst I.
Fragment Molecualr Size (Kbp) No.
Bl B2 127 Ml D1 . D2
1. > 21.2 > 21.2 > 21.2 > 21.2 > 21.2 > 21.2
: 2. > 21.2 > 21.2 > 21.2 > 21.2 > 21.2 > 21.2
3. > 21.2 > 21.2 > 21.2 > 21.2 > 21.2 > 21.2
4. 18.8 18.8 . 18.8 18.8 18.8 18.8
5. 16.2 16.2 16.2 16.2 16.2 16.2
6. 13.8 13.8 13.8 13.8 13.8 13.8 ~
7. 12.6 12.6 12.6 12.6 12.6 12.6
8. 9.5 9.5 9.5 9.5 9.5 9.5
9. 8.7 8.7 8.7 8.7 8.7 8.7
10. 7.7 7.7 7.7 7.7 7.7 7.7
11. 7.2 7.2 7.2 7.2 7.2 7.2
12. 6.4 6.4 6.4 6.4 6.4 6.4
13 5.0 5.0 5.0 5.0 5.0 ,-,/>
5.0
14. 4.7 4.7 4.7 4.7 4.7 .4.7
4.9.2 Virus ~
The experimental birds excreted the virus in the faeces from 24 h PI and up
to 13 days PI. The virus was detected from the cloacal swabs as assessed by the HA
activity and subsequent treatment of these samples with E05-76 antiserum inhibited
the HA activity.
Nearly, 45 per cent of the birds excreted the virus upto 5 days PI, 85 per cent
of the birds excreted the virus between 6 and 10 days PI. The percentage of virus
excretion was 80 between 11 and 13 days PI and after 14 days PI, there was no
excretion of virus. The details of virus excretion are presented in Table XIX.
The HA titre !"Clnge of virus excreted by the experimental birds are furnished
in Table XX. The HA titre (log 2) ranged between 2 and 4 upto 5 days PI, 4 and 8
between 6 and 10 days PI, 8-16 between 11 and 13 days PI and there was no HA
titre after 14 days PI. Large percentage of the bird population has been excreting
virus in higher concentration between day 6 and day 13 PI (Table XIX and XX).
4.9.3 Tests employed for virus detection
HA, AGIO and OIA were employed for detection of the E05-76 virus in the
faecal swabs. Of the 384 samples tested for virus detection, 208 were positive by HA,
211 by AGIO and 216 were positive by DIA. The details are furnished in Table XXI.
The samples positive by HA and AGID were detected by OIA. The positive results of
HA, AGIO and DIA are comparable. Eight samples which were negative by HA test
were found to be positive by OIA. DIA was found to be the most sensitive test for the
detection of virus in cloacal swabs.
TABLE XIX: DETAILS OF VIRUS EXCRETION IN EXPERIMENTAL BIRDS EXCRETORY PATIERN OF VIRUS·
Average number of Period of Percentage of birds birds excreting the observation excreting the virus
virus (Days PI)
9 0-5 45
17 6-10 8S
16 11-13 80
0 14 and above Ni!
* Total number of experimental birds examined per day was 20.
1,'ABLE XX: DETAILS OF HA TITRE RANGE OF VIRUS EXRETED BY EXPERIMENTAL BIRDS ,_
Period of observation (days PI) Range of HA titre (1092)
0-5 2 to 4
6-10 4 to 8
11-13 8 to 16
Above 14 Nil
4.9.4 Virus reisolation
Virus was reisolated in duck embryos from the pooled cloacal swab samples
collected on the 7th and 8th day PI. Virus could not be recovered from the ovary
homogenates of these experimental birds, upto 10 weeks of observation under this
study.
4.9.5 Detection of antibody in experimental birds
In the experimental birds EDS-76 antibody could be detected in serum samples
from 8 day PI. The HI antibody titre rose gradually and reached a peak between 3
and 4 weeks PI and maintained at that level upto 10 weeks. The HI antibody titre
range recorded at different week intervals after experimental inoculation are furnished
in Table XXII. There is an increased antibody titre against EDS-76 for the period
between 3rd and 8th week PI.
4.9.6 Tests employed for detection of EDS-76 antibody
HI, AGIO and DIA were employed for the detection of antibody to EDS-76
virus, in serum samples collected from experimentally infected birds. Out of the 176
pooled serum samples tested, 141 samples were positive by HI, 118 samples were
positive by AGIO and 138 samples were positive by DIA. The details of inference are
furnished in Table XXIII. It is apparent from Table XXIII that HI test may be employed 1\
as the test of choice for detection of anti bodies to EDS-76 virus apart from its u
advantage in quantifying the same.
TABLE XXI: COMPARISON OF RESULTS OF HA, AGIO AND DIA EMPLOYED FOR VIRUS DETECION IN CLOACAL SWABS:
Observation HA AGIO DIA
Total positive 208 211 216
Percentage positive 54.16 54.94 56.25
* Total cloacal swabs screened were 384.
TABLE XXII : DETAILS OF HI ANTIBODY TITRE RANGE IN EXPERIMENTALLY INFECTED BIRDS
Duration of observation following Range of HI titre experimental infection (log2 )
(Week)
0- 1 Nil 1 - 2 4-9 2-3 6 - 12 3-4 9 - 12 4-8 9 - 12
8 - 10 9 - 10
TABLE XXIII : COMPARISON OF RESULTS OF HI, AGIO AND DIA FOR EDS. -76 ANTIBODY DETECTION IN SERUM SAMPLES
Observation HI AGIO DIA
Total positive 141 118 138
Percentage positive 80.11 67.04 78.40
* Total serum samples screened 176.
CHAPTER V
DISCUSSION
The success of Poultry Industry in the modem era for the small and marginal
farmers is to achieve the production targets. However, with the present introduction
of progressive improvements in avian health, major threat to the poultry industry is ("\
the eco nomic loss due to significant drop in egg production. Various infectious agents I.)
such as Newcastle disease virus, Infectious laryngeotracheitis virus, Infectious bursal
disease virus and fowl pox virus are attributed as etiological agents of egg drop
syndrome in poultry. Better understanding of these diseases, absence of specific
symptoms associated with these diseases as well as protective vaccination of the flocks
with improved vaccines and vaccination schedules ruled out their possible
involvement in this episode. Many workers have reported the involvement of an
aviadenovirus in apparently healthy flocks which fail to reach expected level of peak
production. Of late, Tamil Nadu, Kamataka and Andhra Pradesh faced egg drop and
hence a detailed study was undertaken to establish the existence of this agent causing
egg drop syndrome.
5.1 SEROPREVALENCE
Various sero-diagnostic tests like EUSA, SN, HI, AGIO and DIA were
employed by different workers to detect specific antibodies to EDS-76 virus infected . ~ /
flocks(Piela and Yates, 1983; Higashihara et 01.,1983; Bartha, 1984; Sathyanarayana
1""\
Chetty et 01., 1988; Ramku mar et 01., 1989 ana Raniprameela, 1992}. Of these tests, v
specificity, sensitivity and rapidity together with a test being employed as an onsite
test are factors considered most important for quick diagnosis of this infection. HI and
OIA were found to be simple, convenient for on-site performance, rapid as well as
economical and equally sensitive for screening a large number of birds. Hence, HI
and OIA were used in the present seroprevalence study of egg drop syndrome '1t.')'\-oS.
The present investigation revealed the presence of haemagglutination inhibiting
and immuno-binding antibodies against EOS-76 referral virus in 8 districts (Table I)
of Tamil Nadu. Madurai recorded the highest positive percentage (53.21) followed by
Madras (23.18 per cent), North Arcot (16.52 per cent), Dharmapuri (10.47 per cent),
Kamarajar (4.3 per cent), Kanyakummi (5.31 per cent), Salem (5.26 per cent) and
Periyar (3.4 per cent). In Madurai, the samples were collected mostly in birds above
40 weeks of age when there was a significant drop in egg production at the time of
collection, consequently a higher percentage of incidence of sero positivity. Though, Of
Madurai recorded the maximum incidence EOS-76, the mean HI titre was found to "-
be low during the phase of reduced egg production. This finding is in accordance with
that of earlier workers (Mohanty et al., 1984) where they observed a low HI titre
during the phase of drop in egg production.
The overall incidence of seropositivity in Tamil Nadu (Table I) was 15.70 per
cent. In the neighbouring states of Kamataka and Andhra Pradesh, earlier workers
have reported an overall incidence of 27.3 per cent and 28.15 per cent, respectively
-rVenkata Reddy, 1984 :, Satyanarayana Chetty et al., 1988). This may be
suggestive of the fact that the virus could have gained entry into poultry populations
of Tamil Nadu as inapparent infection from the neighbouring states, through duck
population.
Apart from Madurai and Madras, six other districts recorded a low percentage
(3.4 per cent to 16.52 per cent) of seropositivity, without manifestation of classical
forms which is suggestive of the widespread prevalence of inapparent infection with
EOS-76 virus in the poultry flocks of Tamil Nadu. The occurrence of inapparent
infection with EOS-76 virus has also been reported by many re}earchers ~(Rampin
et al., 1978 and Shakya and Dhawedkar, 1991a).
Age wise incidence of IiI antibodies to EDS-76 virus (Table II) in the present
study clearly shows that birds of all ages are susceptible to the infection. Similar
findings were reported earlier by McFerran et al. (1978). However, in birds above 40
weeks of age, the prevalence of HI antibodies was the highest and the prevalence
decreased in younger birds with the least reading recorded in birds below 20 weeks.
These results concur with the fil)dings of Venkata Reddy and Raghavan (1987) where
they have reported the highest prevalence of HI antibodies in the age group of 44-60
weeks while the least in the ag~ group of 20 weeks. According to them the reason for
higher incidence of infection in older age group of birds could be attributed to the
stress and strain factors experienced during the phase of peak egg production.
Of the two tests, HI and OrA (Table III) employed for the detection of EOS-76
antibodies, with 809 sem.'>'nsarnples, 127 were positive by HI (15.69 per cent) as
compared to 137 positive samples detected by DIA (16.93 per cent). In the present
study, it is a fact that OIA has detected more number of positive cases than HI. In this
investigation, a HI titre of 3 (lOg2) and above has been taken as positive in
accordance with earlier worken; (Bartha et al., 1982; Shakya and Ohawedkar, 1991a; /' Sekar et aI., 1992) while Mohanty et al. (1984) have taken HI titre of 1 (lOg2) as
positive HI titre. Therefore, higher HI titre considered in this study could be the
reason attributed to the lower incidence of detection of EDS-76 by HI in comparison
with DIA. There is no significant correlation between the two tests employed for the
detection of EDS-76 virus al1tibody. Though DIA is a simple test, cost effective and
the results can easily be interpreted visually, HI test for all practical purposes can
exclusively be employed as the serological test of choice for the detection of EDS-76
virus antibody since it is simple to perform, sensitive, specific and economical as well
apart from the fact that the antibody level can be assayed quantitatively. This is in
concurrence with the finding of the earlier report by Adair et al. (1986).
5.2 ISOLATION
The second objective of this study is to isolate the etiological agent, EDS-16
virus from apparently healthy poultry population with the history of drop in egg
production. Different workers have isolated the virus in field outbreaks from different
samples like blood leucocytes (Baxendale, 1978; Yamaguchi et al. 1981a; Higashihara
et al., 1983; Ramkumar et al., 1992) and oviduct (Yamaguchi et al., 1981b). The fact
that the virus is excreted in the faeces for a considerable period, cloacal swabs
constituted the main source in the present study for virus recovery from the affected
population. EDS-76 virus replicates in the epithelial cells of the infundibulum, tubular
shell gland, pouch shell gland and isthmus (McFerran, 1978). Hence, pouch shell
gland homogenates were also used for virus isolation.
The most sensitive indicator system for virus isolation is the embryonated duck
eggs or duck cell culture (Gough et ai., 1982; "McFerran et ai., 1978). They also
observed that the EDS-76 virus multiplied rapidly and reached higher virus titres in
the duck embryos rather than in duck tissue culture monolayers. Obviously, the choice
of the indicator system in the present study for virus isolation was duck embryos. Out
of the 118 cloacal swabs and 40 shell gland homogenates tested, a total of five
isolates were obtained. The time of collection of samples is extremely important for
successful virus isolation. Many workers have isolated the virus within 15 days of the
flocks showing signs of drop in egg production. due to E05-76 (Baxendale, 1978; //
Yamaguchi et al., 1981a; Higashihara et al., 1983). Perhaps, the virus excretion in the
faeces is maximum during the first 15 days of infection. It is quite interesting to note
(Table VI) that the virus could be isolated from the poultry population where the egg
drop percentage is less than 65 per cent. In other words, successful isolation of virus
could be made during the early phase of manifestation of the egg drop syndrome.
The initial HA titre of the isolate ranged between 8 and 32. The virus titre
increased considerably after passage in duck embryos and after 5 passage in duck
embryos the titre of the isolates ranged between 256 and 1024. This finding concur
with that of earlier researchers (Gough et al., 1982; Ramkumar et al., 1992), wherein
they have reported that the HA titre of each isolate increased during every successive '-"
passage in duck embryos. The HA activity of the indigenous E05-76 viral isolates,
was specifically inhibited by reference antiserum to 127 strain but not with hyper
immune serum to Newcastle disease virus, thus confirming the virus isolates as
E05-76 virus.
Cross HI test was performed to assess the sharing of antigenic components
between local isolates and reference E05-127 virus. All the five isolates were titrated
against reference serum to strain 127 and hyperimmune serum raised against the
isolate M1. The results of cross HI indicated that all the five isolates were serologically
identical to strain 127 of EDS-76 virus. Higashihara (1983), Venkata Reddy (1984)
and'""'Shakya and Dhawedkar (1991b) also observed sharing of antigens among the
local isolates with that of EDS-127 virus.
5.3 ADAPTATION OF EDS-76 ISOLATES IN CELL CULTURE
To study the replication behaviour of the EDS-76 isolates in cell culture system,
two primary cell culture and one cell line of duck origin were chosen. In eEL cell
cultures, mild ePE started at the second passage level only. eEL cultures began to
exhibit ePE from 4th day PI and extended upto 8th day PI, indicating its mild
replication behaviour in eEL culture. Higashihara et al. (1983) and Swain et al.
(1992) also successfully adapted field isolates in eEL cultures.
Both in DEF cell cultures and Duck Pekin cell line, the isolates propagated
itself progressively from the very first passage. Characteristic CPE involving more than
50 per cent of the cells, induced by the field isolates were well observed in both DEF
and Duck Pekin cell line. This observation coroborrated well with Calnek (1978), /
Firth et al. (1981) and Swain et al. (1993), who have stated that the EDS-76 virus
(strain 127) had an in vitro preference for duck cells because EDS-76 virus is in fact
a duck adenovirus.
All the five EDS-76 indigenous viral isolates produced appreciable CPE in CEl
cell cultures. On comparison of the extent of propagation and the onset of CPE it was
observed that the CPE started from the 4th day PI and reached a peak by 8th day
PIon eEL cell cultures in contrast to the onset of CPE in DEF and Duck Pekin cell
line. Although all the isolates produced progressive ePE it is interesting to mention
that both in DEF and Duck Pekin cell line the ePE started much earlier than in eEL
cell cultures, indicating the rapid multiplication of the virus and its affinity to tissue of /'
duck origin. Similar findings were observed by Nokolaeva and Roxhdestvenskii
(1982). However, these findings are conflicting to the observations made by
Ramkumar et aT. (1993) wherein they have reported that the multiplication of EDS-76
virus occurred in DEF cell culture and the multiplication was confined to the nuclei
of infected cells and no appredable ePE could be observed during the first five serial
passages made in DEF.
The ePE in all the three cell cultures manifested itself in the form of rounding
and grouping of cells. Large vacuolations with cytoplasmic anastomosis of cells were
observed as the CPE progressed. The final stage of ePE include formation of syncytia
and multinucleated giant cells finally leading to the detachment of cells from the glass
surface. These observations are in conjunction with the findings of many researchers
(Zanlella et aT., 1980; Yamaguchi et aT., 1981a; Higashihara et aT., 1983;
'Satyanarayana Chetty, 1985 and Swain et aT., 1993).
Infected coverslips of all these cultures on H & E staining revealed eosinophilic
intranuclear inclusion bodies as adduced earlier by Adair et aT. (1979), Zsak and
Kisary (1981), Higashihara et aT. (1983) and Swain et aT. (1993).
The infectivity titres of the isolates at the end of 5th and 10th passage levels
are on the higher range particularly in DEF and Duck Pekin cell line systems (Table
XI) than eEL cell cultures. The higher infectivity titres of the isolate is due to the fact
that both DEF and Duck Pekin cell line are of tissue from duck origin and are
homologous systems to the virus. This is in agreement with the concept originally put
,..:: fOIward by MeFerran (1978) and later confirmed by Baxendale (1978) that the cause
of EDS-76 is in fact a duck adenovirus.
5.4 CHARACTERIZATION OF THE EDS-76 ISOLATES
The indigenous EDS-76 isolates were characterized using certain
c.. physio-chemical parameters as suggested by Yamaguchi et 01. (1981a).
~
The treatment of the isolates to chloroform and ethyl ether did not result in
any significant fall in either infectivity titre or HA titre. This suggests the absence of
envelope a well known character of adenoviruses (Burke et 0/., 1968). Similar
observations were made by Villegas et 01. (1979) and Yamaguchi et 01. (1981a).
There was no fall in the infectivity titre of all the five isolates when exposed
to pH 3. Stability to acid pH is also one of the well known properties of
aviadenoviruses (Adair et 01., 1979; Yamaguchi et 0/., 1981a). There is a direct
correlation between resistance to lipid solvent and stability to pH 3.0 among all naked
viruses.
The isolates in the present study were found to be resistant to the action of 0.3
per cent trypsin concurring with the findings of Swain et 0/. (1992). However, Todd
an~ McNulty (1978) have contradicted this finding and have reported the virus to be
sensitive. This is due to the fact that in their study, they have used a purified soluble
haemagglutinin sample as against the whole virus.
All the five isolates were found to be stable at 56°C for 30 min. Both the HA
activity and infectivity were not affected and this observation concurs with the earlier
/ reports (Todd and McNulty, 1978; Ramkumar et a1., 1991 and Swain et a1., 1992).
All the isolates showed a loss in infectivity titre when treated with 0.3 per cent
formalin. The HA activity;> however, was not affected at this concentration. This
property of the virus could be best made use of, while evolving an inactivated
vaccine.
Halogenated uridines have a major role in the suppression of viral DNA
synthesis. Accordingly, in the present study, IUDR (a nucleic acid inhibitor) was
employed to determine the type of genome in the indigenous viral isolates. The
treatment with IUDR resulted in more than 3 log loss in viral infectivity titre confirming
the DNA nature of the nucleic acid and this is in agreement with the findings of
Adair et af. (1979) and Yamaguchi (1981a).
Examination of the allantoic fluid of embryonated duch eggs infected with
different indigenous isolates, under transmission electron microscope by DNCEM
reveBled virus particles scattered singly or in twos. The virions were identified as
adenoviruses based upon the icosahedral morphology and average particle diameters
of 70 nm. Similar findings were reported by Swain et a1. (1992).
The same samples when subjected to IEM at a serum dilution of 1 :20 revealed
the adenoviral particles in clumps thus confirming the isolates as EDS-76 virus.
5.5 PROTEIN FRACTIONATION STUDIES
The sodium dodecyl sulphate-polyacrylamide gel electrophoresis (50S-PAGE)
is being used for studying various isolates based on the viral protein bandings.
Purified virus on 50S-PAGE contains at least 13 structural proteins with polypeptide
molecular weights ranging from 11 KDa to 126 KDa. The major structural protein of
the virus was found to be protein 1 with a molecular weight of 125.8 KDa. The
protein bands of the isolates are identical to the protein bands of referral strain. Todd
and McNulty (1978) observed 13 bands on 50S-PAGE with adenovirus-127 strain of
EOS-76 virus. They also reported that the molecular weight of the polypeptides range
from 11 to 126 KDa. However, Swain et of. (1992) in contrast observed only 12
polypeptides with an indigenous EOS-76 viral isolate.
5.6 DNA RESTRICfION ENDONUCLEASE ANALYSIS OFEDS-76 VIRAL
ISOLATES
DNA finger printing is a modem technique for the present day molecular
biologists to distinguish genetically different yet serologically similar strains of viruses.
In general, adenoviruses contain more number of inverted repeat sequences of the
viral DNA. Considerable fingerprint variations occur since sequences may have been
added to or deleted from the existing fragments, resulting in mobility differences in
the corresponding fragments from different isolates. This type of interstrain fingerprint
variations occur more frequently in restriction fragments containing inverted repeat
sequences of viral DNA (Zhang and Nagaraja, 1989).
Restriction endonuclease fingerprinting will also help in quickly characterizing
a viral strain in a disease epizootic. The DNA from three serologically indistinguishable
viruses of aviad()1Ovirus Type II were analysed by six restriction endonucleases
(Zhang and Nagaraj a, 1989). ~
In the present study, the DNA of one indigenous isolate as well as the referral
strain were digested with 6 restriction endonucleases. Of the 6 enzymes, Pst I cleaved
the DNA into more number of fragments compared with five other endonucleases
(Fig. 14) and hence Pst I was selected for digestion of the DNA of different isolates.
Comparison of the DNA fingerprint of all the five isolates revealed identical banding h(l.ve.
patterns. Hence, in the present study all the five isolates were found to ; genetic<' ;..
5.8 EXPERIMENTAL INFECTION OF EDS-76 VIRAL ISOLATE
In the present study, chicken of the age group 24-28 weeks were infected with
the indigenous EDS-76 viral isolate (M1) and after infection, the birds remained
healthy throughout the study period. This finding concurs with Heffels et af. (1982).
The experimental birds excreted the virus in the faeces from 24 h upto 13th
day PI .1::ook and Darbyshire (1981) also recovered the virus from the swabs of the
cloaca during the first 14 days PI following oral infection with EDS-76 virus strain
061.
In the present study, nearly 45 per cent of the birds excreted the virus upto 5th
day PI and more than 80 per cent of the birds excreted the virus between 6th day
and 13th day PI in contrast to observation made by Heffels et al. (1982) where in
they have stated that the excretion of the virus in faeces lastsfor only five days PI.
1""'. This may be attributed to the fact that virus excretion by adult hens is erratic (Cook
and Darbyshire, 1980). This often results in a low incidence of infection in such flocks.
The viral concentration as observed by HA titre was maximum between 10th
and 13th day PI and this gives an indication that the causative agent could be
successfully isolated if samples are collected between 10th and 13th day after the
onset of infection in a flock.
Of the three tests employed for the detection of virus, DIA detected the
presence of antigen in more number of samples. This explains the sharpness in
sensitivity of this test over HA and AGIO in detection of EOS-76 antigen in the faecal
samples.
In the present study, the infected virus was reisolated from the faecal samples
on the 7th day. Yamaguchi et af. (1981b) have also reported that the inoculated virus
was recovered from 3 out of 4 faecal samples of the inoculated group of birds on the
10th day PI. The shedding of virus in the first week itself indicated the active viraemic
state of virus more likely in view of quick absorption of virus inoculated through
combined routes of oral and intra muscular. Further, resolution of the virus in faeces
upto 15th day PI suggested the possibility of lateral spread of infection (Par sons et
al., 1980 and Cook and Darbyshire, 1981). However, in the present study, it is
unfortunate that the virus could not be reisolated from the ovary homogenate. This
failure to reisolate the virus may indicate inefficient sampling rather than true failure
to reisolate the virus.
In experimental birds, EOS-76 antibody could be detected in serum samples
from 8th day PI. Similar observations were made by Heffels et al. (1982), where
specific HI antibodies were first detected in sera of birds killed on 7th day PI, in all
age groups. The I-ll antibody titre rose gradually and reached a peak between 3rd
and 4th week and maintained itself at this level upto 70th day PI. These findings
concurred well with the findings of the earlier workers (Brugh et 0/., 1984 and Van
Eak, 1983). They also observed consistently high I-ll titres associated with adenovirus
experimental infection.
On comparison on the sensitiveness of three serological tests viz., I-ll, AGIO
and OIA for the detection of EOS-76 viral antibody the present study suggest that
field infections may be easily and reliably confirmed by HI serology. It is concluded
that only I-ll should be used for detection of EOS-76 infection in poultry flocks.
CHAPTER VI
SUMMARY
In the present study, seroprevalence of EDS-76 in 8 districts of Tamil Nadu
was taken up both in chicken flocks which failed to reach peak production as we)) as
healthy flocks. Inspite of the fact that birds screened in this study were apparently
normal, birds in many flocks experienced a drop in egg production.
The seroconversion study was conducted by employing both Hemagglutination
inhibition (HI) test and Dot immunobinding assay (OIA) as quantitative serological
tests using referral EOS-76 viral strain 127.
Out of the 809 serum samples screened, an overall incidence of 15.7 per cent
was recorded. Madurai district recorded the highest percentage of inCidence, where
there was a significant drop in egg production. The lower percentage of incidence in
many other districts is due to the prevalence of inapparent infection.
Though birds of all ages are susceptible to the infecti01) the highest incidence
of HI antibodies was observed in birds above 40 weeks of age.
The present study revealed no significant difference in the efficacy between the
two tests employed for the detection of EOS-76 viral antibody. Hence, HI test was
found to be the serological test of choice for the detection of EOS-76 viral antibodies.
Virus isolation was attempted from sample collected from poultry flocks which
suffered a drop in egg production. The egg drop percentage in such farms ranged
between 25 and 80 per cent. Out of the 118 cloacal swabs and 40 shell gland
homogenates of chicken tested, 5 samptes from 5 different farms yielded virus isolates.
The present study revealed that the samples should be collected during the early
phase of the syndrome for successful virus isolation.
r">
Specific inhibition of the HA activity of the different iso lates by referral '-../
antiserum to strain 127 confirmed the virus isolates as EDS-76 virus, and cross HI test
indicated that all the five indigenous isolates were serologically identical to referral
strain 127.
The replication behaviour of the indigenous isolates was studied in three ceU
culture systems: CEl, DEF and Duck Pekin cell line.
The isolates produced CPE in all the three cell culture systems. The changes
inc)udEdrounding and grouping of cells with large vacuolation and fusion of nuclei
leading to the formation of giant cell. In H & E stained preparations, eosinophilic
intranuclear inclusion bodies were demonstrable.
In the present study of the three cell culture systems employed DEF and Duck
Pekin cell line systems gave higher infectivity titre than CEL. It was observed that all
the indigenous isolates had an in vitro preference for duck cells rather than chicken
cells.
The physico-chemical properties of the isolates were studied by standard
procedures. The isolates withstood the action of chloroform, ether and acidic pH at
3.0, thus suggesting the absence of an envelo~ They were resistant to 0.3 per cent
trypsin and stable at 56°C for 30 min.
When treated with 0.3 per cent formalin, the isolates showed a loss in
" infectivity titre; however, the HA activity was not affect ed and this property can be u
best made use of while evolving an inactivated vaccine for EDS.
The isolates recorded a significant fall in infectivity titre when treated with
IUDR thereby indirectly confirming the DNA gen.ome of the isolates.
The isolates were first identified as adenovirus by direct negative contrast
electron microscopy (DNCEM) and later confirmed as EDS-76 virus by
immuno-electron microscopy.
All the isolates, when purified and subjected to SDS-PAGE, exhibited 13
structural proteins with molecular weight ranging from 11 KDa to 126 KDa. The
protein bands of the isolates were found to be identical to the protein bands of the
referral strain 127.
Restriction DNA fingerprinting of all the isolates was carried out in conjunction
with the referral strain to check for any genetic variation between the strains
Comparison of the DNA fingerprint of all the five isolates digested with restriction
endonuclease Pst I, revealed identical banding pattern thereby confirming the genetic
similarity of the isolates.
In the present study, experimentally infected birds excreted the virus in the
faeces upto 13 days PI.
The serological tests (HA, AGIO and DIA) were employed for the detection of .
antigen and OIA was found to be the most sensitive test in the detection of antigen.
Reisolation of the virus from the faecal samples was successful in the present
study. In experimental birds, EOS-76 antibody was first detected on 8th day PI. The
HI antibody titre rose gradually and reached a peak between 3rd and 4th week PI
and maintained at this level upto 10 weeks.
Among the three serological tests, viz., HI, AGIO and OIA employed for the
detection of EOS-76 viral antibody, HI test was found to be sensitive, simple and
reliable. It is concluded that HI test should be used for the detection of EDS-76
infection in poultry flocks.
REFERENCES
"Adair, B.M., J.B. McFerran, T.J. Connor, M.S. McNulty, and E.R. McKillop. 1979.
Biological and physical properties of a virus (strain 127) associated with the
egg drop syndrome-76. Avian Path., 8: 249-264.
,-/ Adair, B.M., D. Todd, J.B. McFerran, and E.R. Mckillop. 1986. Comparative
serological studies with Egg drop syndrome virus. Avian Path., 15: 677-686.
L Adlaka, S.c. 1966. A serological investigation to determine respiratory infections of
poultry in India. Avian Dis., 10: 401-404.
/' Afshar, A., C. Dubuc, G.c. Dulac, C. Thomas, K. Nielson, and D. Henning. 1991. Dot
immunoperoxidase assay using monoclonal antibody for detection of
bluetongue virus antigens. J. virol. Method., 31: 105-112 .
.e:: Akay, 0., M. IzGur, H. Ayhan. 1988. Detection of antibodies to the egg drop
syndrome in the egg yolk and serum of vaccinated and non- vaccinated
chickens by the hemagglutination inhibition test. Etlik Veteriner
Mickrobiyologi Desgiri., 6: 57-64.
~ Anant Rai, 1985. Methods in cell culture and virology. Allied Publishers Private Ltd,
New Delhi (India).
• Andrews,A.T. 1986. Electrophoresis. 2nd Edn. Theory techniques of biochemical and
clinical applications- Monographs on physical biochemistry. Clarendon Press.
Oxford.
(Asi, Y and S. Iyisan. 1990. Standardization of the HI test for the egg drop syndrome
and determination of protective antibody levels in hens with an EDS-76
vaccine. Doga Turki Veterinerlik Ve Hay Vancilik Desgiri., 14: 96-111.
Bartha, A. 1984. Dropped egg production in ducks associated with adenovirus
infection. Avian Path., 13: 119-126.
Bartha, A., J. Meszaros, and T. Tanyi. 1982. Antibodies against EDS-76 avian
adenovirus in birds before 1975. Avian Path., 11: 511- 512.
~xendale, w. 1978. Egg drop syndrome-76. Vet. Rec., 102: 285-286.
,/ Beadstue, P.B. and B. Smidt. 1978. Egg drop syndrome in Danish poultry. Nord.
Vet. Med., 30: 498-505.
Bennejean, G., M. Guillet and J.P. Picault. 1980. Fall in egg production associated
with the laying of soft shelled eggs, a new infectious disease of the hen.
Science Veterinaires et de medicine Compaya delycn 81: 159-165.
(Vet.Bull. 50: Abst. 648)
-13idin, Z., H. Mazija and M. Kralj. 1981. Investigations of the EDS in Yugoslavia with
reference to pure adenovirus infection and superimposed virus infections.
Praxis Veterinaria, 29: 87-88. (Vet. Bull. 51: Abst. 7420).
.::' Biswal, G. and C.C: Morrill. 1954. The pathology of the reproductive tract of laying
pullets affected with Newcastle disease. Poultry Sci., 33: 880-897.
/Boyle, D.B. and J.B. McFerran. 1976. Avian adenovirus isolated from poultry in
Queensland. Aust. Vet. J., 52: 587-589 .
.....-sragg, R.R, D.M. Allwright and L. Coetzee. 1991. Isolation and identification of
adenovirus 127, the causative agent of EDS from commercial laying hens in
South Africa. J. Vet. Res., 58: 309-310.
, Brugh, M., C.W. Beard and p.villegas. 1984. Experimental infection of laying
chickens with adenovirus 127 and with a related virus isolated from ducks.
Avian Dis., 28: 168-178.
CBurke, eN., R.E. Luginbuhl and C.F. Helmboldt. 1965. Isolation of latent adeno-like
virus from chicken kidney cell cultures. Avian Dis., 9: 31-43.
/'Burke, C.N., RE. Luginbuhl and E.L. Jungherr. 1959a. Avian enteric cytopathogenic
viruses (ii) characteristics of prototype. Avian Dis., 3: 419-427.
'Burke, C.N., RE. Luginbuhl and L.F.Williams. 1968. Avian adeno- like viruses.
Characterization and comparison of seven isolates. Avian Dis., 12: 483-505.
Calnek, B. W. 1978. Hemagglutination inhibition antibodies against an adenovirus
(virus-127) in white Pekin ducks in the United States. Avian Dis., 22:
798-801.
(' Chauhan, RS and N.P. Singh. 1992. Rapid diagnosis of rotavirus infection in calves
by dot immunobinding assay. Vet. Rec., 130: 381.
<' Clemmer, OJ. 1964. Characterization of agents isolated from market chickens on a
quest for enteric viruses. J. Infect. Dis., 114: 386-400.
'Cook, J.K.A and J.H. Darbyshire. 1980. Epidemiological studies with egg drop
syndrome-1976 (EDS-76). Avian Path., 9: 437-443.
-Cook, J.K.A. and J.H. Darbyshire, 1981. Longitudinal studies on egg drop syndrome
1976 in the fowl following experimental infection at day old. Avian Path., 10:
449-459.
f Dawson, J., V.J.Yates., P.W. Chang and W.Wattanavijarus. 1981. Egg transmission
of Avian Adenovirus-Associated virus and CELO virus during experimental
infections. Am. J. Vet. Res., 42: 1833-1837.
L Das, B.B. and H.K. Pradhan. 1992. Outbreaks of egg drop syndrome due to EDS-76
virus in Quail (C;otumix cotumix janponica). Vet. Rec., 131: 264-265 .
.. Denisova, T.S., B.S.Sitnickov and RA. Gibadulin. 1979. DNA fragmentation of
chicken adenovirus CELO by specific endonucleases Hpa I, EcoRI, Hind III.
Mol. BioI. Mosk., 13: 1021-1034.
-Durham, P.J.K. and Cathcast. 1979. A serological survey for the presence of
antibodies to "Egg drop syndrome 1976" adenovirus. New Zealand Vet. J.,
27: 196-198.
/'Dutta, S.K and B.S. Pomeroy. 1963. Electron microscopic structure of chicken
embryo lethal orphan virus. Proc. Soc. Exp. BioI. Med., 114: 541-549.
Elmishad, AM., K.J.McCormic., J.P.Anderson, N.A Stenbach and J.J. Trentin. 1971.
Studies on hemagglutination by CELO and oncogenic avian adenovirus. Bact.
Proc., 189.
Evernt E., B.O. Sunquist., U.L.E Petterson and P. Lennart. 1973. Structural proteins
of adenovirus. Isolation and topography of low molecular weight antigens from
the virion of adenovirus type 2. Virology, 52: 130-147.
,. Feherrari, T., R.Glavits and F.Rats. 1979. Egg shell formation disorder (egg drop
syndrome) in a large laying flock in Hungary. Magyow Allatorvosok Lapja
34: 291-296. (Vet. Bull. 49: Abst. 7288)
C Firth, G.A, M.J. Hall and J.B. McFerran. 1981. Isolation of a hemagglutinating
adeno-Iike virus related to virus 127 from an Australian poultry flock with an
egg drop syndrome. Aust. Vet. J., 51: 239-242.
<t~ Fry Smith, DJ. and P.T. Gilchrist. 1981. Avian hemagglutinating adenovirus
antibodies in Australian poultry. Aust. Vet. J., 51: 395.
Gulka, C.M., V.J. Yates., F.W. Chang and E.C. Sadasiv. 1982. The effect of
incubation temperature on the propagation of duck adenovirus {virus-127 like)
in duck and chicken cells. Avian Dis., 26: 354-359.
~ough, R.E., M.S.Coliins and D. Spackman. 1982. Isolation of a hemagglutinating
adenovirus from commerdal ducks. Vet. Rec., 110: 275-276.
,.-c:fui~araes, 5., M. Casolina, J. Beatriz and E.L. Franco. 1986. Evaluation of dot
enzyme linked immunosorbent assay for Mucocutaneous Leishmaniasis and
comparison with microplate enzyme immunoassays. J. Clin. Microbiol., 24:
364-367.
t. Gupta, Y., P.Chand., A Singh and N.C. Jain. 1990. Dot immunobinding assay in
comparison with EUSA for the detection of BlV anti bodies in sheep. Vet.
Microbiol., 22: 365-371.
£:_ Gylstorff, I and J. Rolf. 1982. Egg drop syndrome of hens. II. Pathology and
histopathology. Deutesche Tierarzttiche Wochens chriff, 89: 14-16. (Vet.
Bull., 52: Abst. 5477)
/Heberling, R.L., 5.5. Kalter., J.S. Smith and D.G. Hilderbrand. 1987. Serodiagnosis
of rabies by dot immunoblnding assay. J. Clin. Microbiol., 25: 1262-1264 .
.t!" Heffels, V., S.ED.Khalaf and E.F. Khaleta. 1982. Studies on the persistance and
excretion of EOS-76 virus in chickens. Avian Path., 11: 441-452 .
.r Higashihara, M., M. Hiruma., T. Houdatsu., S. Takai and M. Matumo to. 1986.
Experimental infection of laying chickens with egg drop syndrome 1976 virus.
Avian Dis., 31: 193-196.
/"Higashihara, M., S.Takai., AHidaka., T. Houdatsu., M. Hisuma., M. Watanabi and
M. Matumoto. 1983. Isolation of the virus of egg drop syndrome 1976 in a
Japanese outbreak. Jpn. J. Vet. Sci., 45: 603- 612.
/ Home, R.W., S. Brenner., AP. Waterson and P. Wildy. 1959. The icosahedral form
of an adenovirus. J. Mol. BioI., 1: 84-86 .
..........-Kaleta, E.F., S.E.D. Khalaj and Y. Heffels. 1982. Egg drop syndrome 76. Persistence
and excretion of virus 127 in susceptible and immunized chicken of differen1
age groups. Dev. BioI. Stand., 51: 151-160.
,1<aleta, E.F., S.E.D. Khalaj., O. Siegmann and H.J. Busche. 1980. Demonstration 01
antibodies against EDS-76 adenovirus in domestic and wild birds. Praktische
Tierarzt., 61: 948-952. (Vet. BulL, 51: Abst. 3223)
Kralj, M., H. Maziji., M. Hercej., J. Nemanic., P. Borzickovic., 1. Dijansic and 1. £'
Jeskovic. 1977. Adenovirus infection in poultry. 1. Etiology in laying hens and
their effect on egg production. Veteri narski Arhin., 47: 1-24. (Vet. Bull., 48:
Abst. 2973)
fe' Kawamura, H.T., T. Horiuchi., S. Shoya. and F. Shimizu. 1966. Mixed infection with
avian adenovirus and fowl pox virus. Natl. Inst. Anim. Hlth. Quarterly,
Tokyo., 6: 216-222.
/Kawamura, H.T, F. Shimizu and H. Tsubahara. 1964. Avian adenovirus. Its
properties and serologic.al classification. Natl. Inst. Anim. Hlth. Quarterly,
Tokyo., 4: 183-193.
/ Khanna, P.N. 1966. Studies on cytopathogenic avian enteroviruses. II. Influence of
age on virus excretion and incidence of certain serotypes in a colony of chicks.
Avian Dis., 10: 27-32.
Khafagi, AK. and M.S.M. Hamouda. 1991. Studies on egg drop syndrome-76. l.
Existence of antibodies in Egyptian commercial chicken. Vet. Med. J. GIZA., ..._;
39: 287-289. (Vet. Bull., 62: Abst. 3261)
Kisary, J. and L. Zsak. 1980. The molecular weight of the egg drop syndrome (EDS)
avian adenovirus (strain 88/78) DNA estimated by digestion with restriction
endonuclease enzyme EcoR 1. Brief re port. Arch. Virol., 65: 63-65.
~uss, H. 1965. Untersuchungen Zur characterisierurg Von aus Hugnem isolierten
orphan virus. Arch. Exp. Vet. Med., 19: 1077- 1090 (Vet. Bull., 44: 531) .
. Kumanan, K. 1989. Studies on immunogneic proteins of Newcastle disease virus.
Ph.D thesis, submitted to Tamil Nadu Veterinary and Animal Sciences
University.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature, (London), 127: 680-685.
Li, P., AJ. Bellett and c.R. Parish. 1984. The structural proteins of chick embryo
lethal orphan virus (fowl adenovirus type 1). J. Gen. Virol., 65: 1803-1815.
Loupal, G., W. Spadint and L. Vasicek. 1982. Egg drop syndrome of hens. I. Clinical
and serological findings. Dent. Tier. Wechen., 89: 11-14 (Vet. Bul!., 53:
Abst.5476).
"" Macpherson, I., P. Wildy., M. Strokes and R.W. Home. 1961. The fine structure of
GAL- an avian orphan virus. Virology, 13: 146-149.
( Maizel, Jr, V. Jacob., D.o. White and MD. Scharff. 1968. The polypeptides of
adenovirus. Virology, 36: 115-125.
\ . 1
~ C'Maniatis, T., J. Sambrook and E.F.Fritsch. 1989. Molecular cloning. A laboratory
manual. Second edition. Cold Spring Harbour Laboratory Press, USA.
" Martone, E, P. Pagnine, G. Ivone.; E. Biondi and G. Ricci. 1983. Replication and
behaviour of aviadenovirus strain EDS-76 in primary and continuous cell
culture. Acta Med. Veterinaria, 29: 3-15 (Vet. Bull., 54: Abst. 3315).
McFerran. J.B., and T.J. Connor. 1978. Further studies on the classification of fowl
adenovirus. Avian Dis., 21: 585-595.
/McFerran, J.B., J.K. Clark and T.J. Connor. 1972. Serological classification of avian
adenoviruses. Arch. fur die garanite virus forschung., 39: 132-139 (Vet.
BulL, 44: Abst. 551).
, McFerran, J.B., RM. McCracken., E.R McKillop., M.S. McNulty and d.S. Collins.
1978. Studies on a depressed egg production syndrome in Northern Ireland.
Avian Path., 7: 35-45.
, McFerran, J.B., HM. Rowley., M.S. McNulty and L.T. Montgomery. 1977. Serological
studies on flocks showing depressed egg production. Avian Path., 6: 405-413.
-t' Merchant, D.J., RM. Khan and Jr.W.H.Murphy. 1964. Handbook of cell and organ
culture. lInd edition. BurQes PublishinQ Co, Minneapolis. Minnesota.
( Merril, c.R., O. Goldman., S.A Sedman and M.H. Ebent. 1981. Ultra sensitive stain
for proteins in polyacrylamide gels showing region al variation in cerebrospinal
fluid proteins. Science, 211: 1437- 1438.
/"Mesanjaz, T.R., J.M. Sanchez-Vizeaino and J. Barrera. 1982. Adaptation of EUSA to.
detect antibodies in fowls to aviadenovirus strain 127. Anales de Institute
National de Investigaciones Agravias, Canadera, Spain. 77: 111-127
(Vet.BuIL, 53: Abst. 6420).
Meulemans, G., D. Oekegel., J. Peters., E. Meirhaeghe and P. Van Helen. 1979.
Isolation of an adenovirus- egg drop syndrome-76. V1aams die I'
geneeskundig, 48: 151-157 (Vet. Bull. 49: Abst. 6606).
C:Meulemans, G., R. Forymans and P. Helen. 1978. Hemagglutination inhibition
antibodies against EOS-76 virus in broilers. Avian Path., 8: 483-495 .
..... Mohanty, G.c., H.K Pradhan., KC. Verma and AS. Panisup. 1980. Adenovirus and
egg drop syndrome. Ind. Poult. Rev., 21: 29-31.
.r Mohanty, G.c., KC. Verma., H.K. Pradhan and Ramkumar. 1984. Egg drop
syndrome (EOS-76) in India. Seroprevalence of EOS-76 virus infection in
poultry flocks. Ind. J. Poult. Sci., 19: 15-18.
<"/Monreal, G. and R. Dom. 1981. Comparative studies on the demonstration of
antibodies in the course of avian adenovirus and egg drop syndrome 1976
virus infection. (Vet. Bull. 52: 3820).
/Monreal, G., R. Dorn and M.H.A. Kassim. 1979. Distribution of adenovirus in the
internal organs of chickens (Preliminary report). Bertiner und munchemer
Tierarzthiche. Wochenschrift, 92: 264-266 (Vet. Bull. 49: Abst. 7290).
rMuneer, M.A., J.A. Newman., V. Sivanandam and D.A. Halvorson. 1988. A dot
immunobinding assay for infectious bronchitis virus. Avian Dis., 32: 137-139.
~" Mustaffer, B.A. and e.B. Spradbrow. 1975. Characterization of three strains of avian
adenovirus isolated in Queensland. (ii) Biochemical, biophysical and electron
microscopic studies. Avian Dis., 19: 175-184.
/' Nawathe, D.R. and A. Abegunde, 1980. Egg drop syndrome'76 in Nigeria.
Serological evidence on commercial farms. Vet. Rec., 107: 466- 467.
tN~, F.K., K.V. Sing., S.e. Yeo and S.H. Ng. 1980. Studies on egg drop syndrome
1976 in Singapore. Sing. Vet. J., 4: 36-51 (Vet. Bull., 54: 4189).
Nikolaeva, J.P. and I.K. Roxhdest Venskii. 1982. Propagation of the aviadenovirus
egg drop syndrome-76 in cell cultures. Veterinariya, Moscow, 4: 28-30
(Vet.Bull., 52: Abst. 6202).
Oberoi, M.S., P.N. Dwivedi., J. Misra., S.N. Sharma and B.Singh. 1990.
Sero-epidemiology of avian adenoviruses in layers. Ind. J. Virol., 6: 104-107.
,., Pappas G. Michael. 1988. Recent application of Dot-EUSA in immuno parasitology.
Vet. Parasitol., 29: 105-129.
'Parsons, D.G., C.D. Bracewell and G. Parsons. 1980. Experimental infection of
turkeys with egg drop syndrome-76 virus and studies on the application of the
haemagglutination inhibition test. Res. Vet. Sci., 29: 89-92.
/ Paul, J. 1973. Cell and tissue culture. 5th Edn. Churchill Uving stone, Edinburgh,
London and New York.
_,/ Petek, M., B. Felluga and R. Zoletto. 1963. Biological properties of CELO virus:
Stability to various agents and electron microscopic study. Avian Dis., 7:
38-49.
,r Piela, T.H. and V.J. Yates. 1983. Comparison of EUSA with HI and Immunodiffusion
tests for detection of antibodies to a hemagglutinating duck adenovirus in
chickens. Avian Dis., 27: 724-730.
Platt, C.S. 1948. Some observations on the effect of Newcastle disease upon laying
fowl. Poult. Sci., 27: 201-206.
,A(amkumar, Kataria, K.c. Verma., KL. Sah and G.c. Mohanty. 1989. Comparative
antibody response of indigenous and standard virus strains of E05-76 in
poultry. Ind. J. Vet. Path., 13: 28-32.
" Ramkumar, J.M. Kataria and G.c. Mohanty. 1991. Isolation and characterization of
haemagglutinating viral agents resembling EDS- 76 virus from poultry flocks.
Ind. J. Virol., 7: 53-58.
r'Ramkumar, G.c. Mohanty and KC. Verma. 1992. Epizootiological studies on egg
drop syndrome in poultry. Ind. J. Ani. Sci., 62: 497-501.
Ramkumar, G.C. Mohanty and KC.verma. 1993. Response of duck embryo
fibrobalsts to EOS-76 virus infection. Ind. J. Vet. Path., 17: 18-21.
/Hampin, T., F. Enice and G. Mandelli. 1978. Antibodies against the BC-14 virus
antigen in laying and breeding flocks in Italy. Clinica Veterinaria. 101:
265-272 (Vet. Bull. 49: Abst. 1318).
Raniprameela, D., K Varalakshmi and M.v.Subbarao. 1992. Dot immunobinding
assay for EDS-76 virus antibody. Paper presented at First Symposium of
Indian Society for Veterinary Immunology and biotechnology (lSVIB).
( Rovozzo, e.G. and N.C. Burke, 1973. A manual of basic virological techniques.
Prentice-Hall, Inc., Englewood Cliff, N.J., USA
Roxhdestvenskii, I.K 1984. Inactivating the aviadenoviruses of the egg drop
syndrome (EOS-76). Veterinaria Moscow. 4: 61-62 (Vet. Bull., 54:
Abst.6361).
Satyanarayana Cherty, M. 1985. Studies on egg drop syndrome virus (EDS-76) in
chickens in AP. Ph.D Thesis submitted to AP. Agricultural University,
Hyderabad.
i Satyanarayana Chetty, M., A Seshagiri Rao and T. Venkata Reddy. 1988.
Seroprevalence of egg drop syndrome-76 (EOS-76) virus infection in chicken
in Andhra Pradesh. Cheiron, 17: 34-38.
/' Sekar, M., J. Ramakrishna and N. Raghavan. 1992. Seroprevalence of egg drop
syndrome'76 in and around Madras. Cheiron, 21: 189-190.
...... Shakya, S. and KG. Dhawedkar. 1991a. Seroepidemiology of egg drop syndrome
1976 in and around Japalpur. Ind. Vet. J., 68: 406-408.
r Shakya, S. and KG. Dhawedkar. 1991b. Antigenic relationship among egg drop
syndrome 1976 virus strains. Ind. Vet. J., 68: 510-513.
Shapiro, A.L., E. Vinnela and JV. Maizel. 1967. Molecular weight estimation of
polypeptide chains by electrophoresis in SDS- polyacrylamide gels. Biochem.
Biophys. Res. Commun. 28: 815-820.
Siegmann, 0., E.F. Kalatar and S.E.D. Khalaf. 1979. Detection of antibodies against
adeno-like virus in commercial poultry flocks. Berliner and Munchener
Tierarztliche Wochenschrift. 92: 262-264 (Vet. Bull., 50: Abst. 647).
Sukumar, M.N.B. and 1. Suri Babu. 1986. Sero-prevalence of "Egg drop syndrome"
in poultry in Andhra Pradesh. Ind. Vet. J., 63: 353-355.
, Swain, P., J.M. Kataria and K.c. Venna. 1993. Biological chracterization of an Indian
isolate of egg drop syndrome-76 virus. Res. Vet. Sci., 55: 396-397.
Swain, P., J.M. Kataria., K.c. Verma and Satish Kumar. 1992. Characterization of a
field isolate of egg drop syndrome-76 (EDS-76) virus. Ind. J. Virol., 8: 8-14.
~niguchi, 1., S. Yamaguchi., H. Maeda, M. Kawamusa and 1. Harivchi. 1981.
Pathological changes in laying hens inoculated with the JPA-1 strain of egg
drop syndrome-76 virus. Natl. Inst. Anim. H)th. Quarterly, Japan, 21:
83-93 (Vet. Bull. 52: Abst. 1661).
;' Taylor, L.W., D.C. Lowry and L.G. Raggi. 1955. Effects of an out break of avian
encephalomyelitis (epidemic tremor) in a breeding flock. Poult. Sci., 34:
1036-1045.
/' Todd, D and M.S. McNulty. 1978. Biochemical studies on a virus associated with egg
drop syndrome 1976. J. Gen. Virol., 40: 63-75.
,.
Trewick, E.G. and G. Lang. 1971. The passive hemagglutination test in avian
virology. Proc. 74th Ann. Meet. US Anim. Hlth. Assn., 1970: 631.
Van Eck, J.H.H. 1983. Effect of experimental infection of fowl with EDS-76 virus,
Infectious bronchitis virus and/or fowl adenovirus on laying performance. Vet.
Quarterly, 5: 11-25.
/Van Eck, J.H.H., F.G. Davellaar., T.A.M. Meurelplerman., Koluene den., N. Yan., B.
Kouwenhoven and F.H.M. Guldie. 1976. Dropped egg production, soft shelled
and shell-less eggs associated with appearance of precipitins to adenovirus in
flocks of laying fowl. Avian Path., 5: 261-272.
/'\Ian Eck, J.H.H., L. Elenbaas, T.P. Wensvoor and B. Kouwenhoven. 1978.
Histopathological changes in the oviduct of hens producing shell-less eggs
associated with precipitins to adenovirus. Avian Path., 7: 279-289.
, Van Eck, J.H.H., N. Vankol and B. Kouwenhoven. 1980. Egg production in relation
to the results of long term serological survey of 73 flocks of fowl. Vet.
Quarterly, 2: 15-24.
Van-den-Hurk, JV. 1992. Characterization of the structural proteins of haemorrhagia
enteritis virus. Arch. ViroL, 126: 195-213.
; V'enkata Reddy, T. 1984. Isolation, characterization and serology of adenovirus from
egg drop syndrome (EOS) in poultry. Thesis submit ted to University of
Agricultural Sciences, Bangalore.
Venkata Reddy, T and R. Raghavan. 1987. Seroepidemiological studies on EDS-76
in poultry and ducks. Ind. Vet. J .• 64: 1-6.
Verina, KC. and B.S. Malik. 1970. Incidence of infectious bronchitis of poultry by gel
precipitation test. Ind. J. Poult. Sci., 37:
Volosyanko, EV. and VV. German. 1990. Distribution of aviadenovirus type 1
infection in Ukranian poultry farm. (Vet. Bull. 62: Abst.2).
_,""'Villegas, P., S.H. Kleven., C.S. Eidson and O. Tampel. 1979. Isolation of
hemagglutinating adenovirus serologically related to adenovirus 127. Avian
Dis., 23: 507-514 .
...,/ Wilcox, G.E., RL. Rower, W. Baxendale and J.S. Mackenzie. 1983. Serological
survey of wild birds in Australia for the prevalence of antibodies to EDS-76
and infectious bursal disease virus. Avian Path., 12: 135-139.
~interfield, RW., A.M. Fadly and A.M. Gallina. 1973. Adenovirus infection and
disease. I. Some characteristics of an isolate from chickens in Indiana. Avian
Dis., 17: 334-342.
Yadav, M.P., N.S. Parihar and S. Kumar. 1975. Pathogenicity of avian adenoviruses
in chicken. Ind. J. Anim. Sci., 45: 28-35.
Yadav, M.P., N.S. Parihar and S. Kumar. 1977. Studies on the adoption and
pathogenicity of adenoviruses in chicken embryos. Ind. J. Anim. Sci. 45:
474-478.
Yamaguchi, S., T. Imada., H. Kawamura., T. Taniguchi and K. Kawakan. 1981b.
Pathogenicity and distribution of egg drop syndrome- 1976 virus (JPA-1) in
inoculated laying hens. Avian Dis., 25: 642-649.
Yamaguchi, S., T. Imada., H. Kawamura., S. Taniguchi., H. Saio and K. Shimamatsu.
1981a. 06tbreak of egg drop syndrome- 1976 in Japan and its etiological
agent. Avian Dis., 25: 628-641.
Yates, V.J. 1960. Characterization of the chicken embryo lethal orphan (CELO) virus.
Avian Dis., 20: 146-152.
Yates, V.J. and D.E. Fry. 1957. Observations on a chicken embryo lethal orphan
(CELO) virus. Am. J. Vet. Res., 18: 657-660 .
...... Zakharchuk, AN., V.A Kruglyak., T.A Akopian., B.s. Naroditsky and TJ.
Tikchonenko. 1993. Physical mapping and homology studies of egg drop
syndrome (EDS-76) adenovirus DNA Arch. Virol., 128: 171-176.
/" Zanella, A, A Donato., A Nigrelli and G. Pali. 1980. Egg drop syndrome (EDS-76):
Etiopathogenesis, epidemiology, Immunology and control of the disease.
Clinica Vetrinaria 103: 459-460 (Vet. Bull., 50: Abst. 3219).
, Zsak, L., A. Bartha., J. Mesgaros and I. Szusop. 1981. Immunization trials against the
egg drop syndrome (EDS) in laying hens. (Vet. Bull., 51: Ahst. 7423) .
.r Zsak, L. and J. Kisary. 1981. Some biological and physico-chemical properties of egg
drop syndrome (EDS-76) avian adenovirus strain B8/78. Arch. Virol., 68:
211-219.
Zsak, L. and J. Kisary. 1984. Grouping of fowl adenoviruses based upon the
resniction patterns of DNA generated by Bam HI and Hind III. IntelVirology,
22: 110-1~hang, c.L. and K.V. Nagaraja. 1989. Differentiation of avian
~ adenoviruse type II strains by restriction endocuclease fingerprinting. Am. J.
Vet. Res. 50: 1466- 1470.
Zhang, c.L., KV. Nagaraja and V. Swanandan and J.A. Newman. 1991.
Identification and characterization of viral polypeptides from type II avian
adenoviruses. Am. J. Vet. Res., 52: 1137-1141.
