Download THE DECAYING PATTERN OF MATERNALLY DERIVED

THE DECAYING PATTERN OF MATERNALLY DERIVED ANTIBODIES
AGAINST INFECTIOUS BURSAL DISEASE VIRUS IN BROILER CHICKS
By
MURTADA ABUBAKR AHMED ABDALLA
(B.V.Sc, 1999)
University of Khartoum
Supervisor
Dr. ABDELWAHID SAEED ALI BABIKER
A Thesis submitted to the University of Khartoum in Partial Fulfilment of the
Requirements of the Degree of Master of Tropical Animal Health, (M.T.A.H)
Department of Preventive Medicine and Public Health
Faculty of Veterinary Medicine
University of Khartoum
October 2005
DEDICATION
To my mother, father, sisters and brothers
ACKNOWLEDGEMENTS
First of all my great thanks to Almighty Allah who made this work possible and for
giving me the health and strength to complete it.
I wish to express my sincere gratitude and appreciation to Dr. Abdelwahid Saeed Ali for
his keen supervision, valuable guidance, considerable assistance and constructive
criticism during this work.
My thanks is also extended to my colleagues, technical and assisting staff at the
Department of Preventive Medicine and Public Health, Faculty of Veterinary Medicine,
University of Khartoum for their assistance and patience with special thanks to Dr. Isam
M. A. Elgali the coordinator of Masters of Tropical Animal Health for his valuable
encouragement and care during the course of the study. And I am indebted to Dr. Atif
Alamin for his kind help.
My thanks to technical and assisting staff at the department of poultry production,
Faculty of Animal Production, University of Khartoum for their precious aid and
appreciable assistance.
To my friends and colleagues who assisted or advised me, I wish to express my sincere
thanks and appreciation.
Special thanks also to my Father and Mother for their extreme support from the
beginning of my life and without them I wouldn’t have been what I am.
List of Contents
Page
Dedication………………………………………………………………………………….Ι
Acknowledgements……………………………………………………………………... ΙΙ
List of
contents…………………………………………………………………………...ΙΙΙ
List of tables ………………………………………………………………………….. ..VΙ
List of figures ………………………………………………………………………… VΙΙ
Abstract ……………………………………………………………………………… VΙΙΙ
Arabic Abstract …...…………………………………………………………………... ...X
Introduction …………………………………………………………………………….1
Chapter one: - Literature review ……... ………………………………………………...3
Infectious bursal disease ………………………………………………………………....3
1.1. Definition …….. …………………………………………………………………. 3
1.2. History …….. …………………………………………………………………… 3
1.3. Economic importance ……...………………………………………………………. 3
1.4. IBD in Sudan ……………………………………………………………..………... 4
1.5. Etiology …….……………………………………………………………………… ..5
1.5.1. Classification and structure……… ……………………………………………… ..5
1.5.2. Physiochemical properties ……….………………………………………………...6
1.5.3. Serotypes and variants ………………………………………………… ………….6
1.6. Susceptible age ……...…………………………………………………………….….8
1.7. Transmission of IBD ………… …………………………………………………….8
1.8. Incubation period….………..……………………………………………………….8
1.9. Morbidity and mortality ………….………………………………………………...9
1.10. Pathogenesis ……………………………………………………………………….9
1.11. Gross lesions……. ………………………………………………………………...10
1.12. Immunosuppression…… ………………………………………………………… 10
1.13. Diagnosis of IBD
…………………………………………………………………13
1.13.1. Detection of viral antigens
...…………………………………..……………… .13
1.13.2. Detection of antibodies
………………………………….……………………..15
1.13.3. Isolation of IBDV ...………………………………………………………….....16
1.13.3.1. Cell culture …...…………………………………………………………… ...16
1.13.3.2. Egg embryos ………………………………………………………………....17
1.13.3.3. In vitro and in vivo methods ......…………………………………………..….18
1.14. Control of IBDV infection ……………………………………………………….18
1.14. Control by vaccination and types of vaccines used …………………… ………...18
1.14.1. Live vaccines …………………………………………………………………..19
1.14.1.1. Mild vaccines ………………………………………………………………...19
1.14.1.2. Intermediate vaccines ……………………………………………………….20
1.14.1.3. Hot vaccine Strains ………………………………………………………….20
1.14.2. Inactivated vaccines ……………………………………………………………20
1.15. Vaccination of broilers ………………………………………………………….21
1.16. Vaccination failure ……………………………………………………………….21
Chapter two: - Materials and Methods …………………………………………………23
2.1.1. Experimental chicks ……………………………………………………………..23
2.1.2. Housing of chicks……………………………………………………………….. 23
2.1.3. Preparation of D78 vaccine ………………………………………………………23
2.1.4. Vaccination program ……………………………………………………………23
2.1.5 Blood collection and serum preparation ……………………………….………... 24
2.2.1 Enzyme-Linked Immunosorbent Assay (ELISA)………………………………….
24
2.3. Experimental design………………………………………………………… …….25
2.3.1 Experiment 1 ……………………………………………………………………..25
2.3.2 Experiment 2 ……………………………………………………………………..25
2.4. Data analysis ………………………………………………………………………26
Chapter three: - Results ………………………………………………………………...28
Chapter four: -Discussion and conclusion………………………………………………34
Recommendations …………………………………………………………...………...37
References……………………………………………………………………….. …….38
List of tables
Table
1
Page
The coefficient variation of MDA in chicks vaccinated via
different routes…..…………………………………………………33
List of Figures
Figure
1
Page
The levels of maternal anti-Infectious Bursal Disease
antibodies in progeny chicks at various ages post hatch ……………………28
2
Seroconversion of progeny chicks after vaccination with D78
via drinking water…. ……………………………………………………….. 29
3
Seroconversion of progeny chicks after vaccination with D78
via nasal drops…..………………………..……………………………...……30
4
Seroconversion of progeny chicks after vaccination with D78
via spray…….…..…………………………………………………………...31
5
Seroconversion of progeny chicks after vaccination with D78
via subcutaneous Injection …………………………………………………32
ABSTRACT
This study was conducted to assess the decaying pattern of maternally derived antibodies
(MDA) in broiler chicks against infectious bursal disease virus (IBDV) and the immune
response for D78 vaccine via different routes of administration. For this purposes
hundred and fifty chicks were used, the chicks were divided into vaccinated and nonvaccinated groups with IBD vaccine. The study revealed that the maternally derived
antibodies (MDA) against Infectious bursal disease virus in unvaccinated chicks persisted
up to the 6th week as determined by ELISA. However the protective level of these
antibodies expired by the 4th week. Generally failure of vaccination by D78 at the 18th
day via different route of administration was observed in this study but the second
vaccination dose at 25 day of age gave good results. So we suggest that for appropriate
vaccination day, the level of MDAS must be evaluated while the chicks are growing at 10
–14 days of age. The declining pattern of maternally derived antibodies titres in
unvaccinated group was compared with those given IBD intermediate vaccine (D78) via
different routes namely drinking water, nasal drops, spray and subcutaneous. The results
obtained indicated that immune responses against D78 vaccine given via different routes
of administration were good and the difference between their S/P ratios was not
significant (P> 0.05). The result also revealed that with regard to coefficient variation
(vaccine take) there were significant (P<0.05) differences between the four routes of the
vaccine and this significances were clear between drinking water and nasal drops and
between nasal drops and spray and between spray and subcutaneous injection. However
the vaccine take via nasal drops at various ages was the best among others routes with
regard to coefficient variation. It is recommended to check serum for vaccination at day
10-14 day of age and one week later for vaccine take.
‫ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ‬
‫أﺟﺮﻳﺖ هﺬﻩ اﻟﺪراﺳﺔ ﻓﻲ آﺘﺎآﻴﺖ ﻻﺣﻢ ﻟﺘﻘﻴﻴﻢ ﻧﻤﻂ اﺿﻤﺤﻼل اﻟﻤﻨﺎﻋﺔ اﻷﻣﻴﺔ ﺿﺪ ﻓﻴﺮوس ﻣﺮض‬
‫ﺟﺮاب ﻓﺎﺑﺮﻳﺸﺲ اﻟﻤﻌﺪي وﺗﻘﻴﻴﻢ اﻻﺳﺘﺠﺎﺑﺔ اﻟﻤﻨﺎﻋﻴﺔ ﻟﻠﻘﺎح)‪ (D78‬ﻋﺒﺮ اﻋﻄﺎءﻩ ﺑﻄﺮق ﻣﺨﺘﻠﻔﺔ‪ .‬ﺗﻢ‬
‫إﺳﺘﺨﺪام ﻣﺎﺋﻪ و ﺧﻤﺴﻮن آﺘﻜﻮت ﻟﻬﺬﻩ اﻻﻏﺮاض وﻗﺴﻤﺖ اﻟﻲ ﻣﺠﻤﻮﻋﺎت ﻣﻠﻘﺤﻪ وﻏﻴﺮ ﻣﻠﻘﺤﻪ ﺑﻮﺳﻄﺔ‬
‫ﻟﻘﺎح اﻟﻘﻤﺒﻮرو‪ .‬اﻇﻬﺮت هﺬة اﻟﺪراﺳﺔ ان اﻟﻤﻨﺎﻋﺔ اﻻﻣﻴﺔ ﻟﻤﺮض ﺟﺮاب ﻓﺎﺑﺮﻳﺸﺲ اﻟﻤﻌﺪي ﺗﺒﻘﺖ ﻓﻲ‬
‫اﻟﻄﻴﻮر ﻏﻴﺮ اﻟﻤﻠﻘﺤﺔ ﺣﺘﻰ اﻹﺳﺒﻮع اﻟﺴﺎدس ﻣﻦ ﻋﻤﺮ اﻟﻜﺘﺎآﻴﺖ ﺑﻌﺪ أن ﺗﻢ ﺗﺤﺪﻳﺪهﺎ ﺑﻮاﺳﻄﺔ إﺧﺘﺒﺎر‬
‫اﻻﻟﻴﺴﺎ‪ .‬ﻣﻊ أن آﻤﻴﺔ اﻷﺟﺴﺎم اﻟﻤﻀﺎدة اﻟﻜﺎﻓﻴﺔ ﻟﻠﺤﻤﺎﻳﺔ ﻣﻦ هﺬا اﻟﻤﺮض ﻗﺪ ﺧﻠﺼﺖ ﺑﺎﻷﺳﺒﻮع اﻟﺮاﺑﻊ‪.‬‬
‫ﻋﻤﻮﻣﺎ ﻓﺸﻞ ﻋﻤﻠﻴﻪ ﺗﻠﻘﻴﺢ اﻟﻜﺘﺎآﻴﺖ ﻓﻲ اﻟﻴﻮم اﻟﺜﺎﻣﻦ ﻋﺸﺮ ﺑﻮاﺳﻄﺔ )‪ (D78‬ﻋﺒﺮ اﻟﻄﺮق اﻟﻤﺨﺘﻠﻔﺔ ﻗﺪ‬
‫ﺗﻤﺖ ﻣﻼﺣﻈﺘﻪ ﻓﻲ دراﺳﺘﻨﺎ‪ .‬وﻟﻜﻦ اﻟﺠﺮﻋﻪ اﻟﺜﺎﻧﻴﺔ ﻟﻠﺘﻠﻘﻴﺢ ﻋﻨﺪ اﻟﻴﻮم اﻟﺨﺎﻣﺲ واﻟﻌﺸﺮﻳﻦ اﻋﻄﺖ ﻧﺘﺎﺋﺞ‬
‫ﺟﻴﺪة‪ .‬ﻟﺬﻟﻚ ﻧﻘﺘﺮح ان ﻣﻦ اﺟﻞ اﻟﺤﺼﻮل ﻋﻠﻲ اﻟﻴﻮم اﻟﻤﺜﺎﻟﻰ ﻟﻠﺘﻠﻘﻴﺢ ﻳﺠﺐ ان ﺗﻔﺤﺺ اﻟﻤﻨﺎﻋﺔ اﻻﻣﻴﺔ‬
‫اﺛﻨﺎء ﻧﻤﻮ اﻟﻜﺘﺎآﻴﺖ ﻣﺜﻼ ﻣﻦ اﻟﻴﻮم اﻟﻌﺎﺷﺮ اﻟﻲ اﻟﺮاﺑﻊ ﻋﺸﺮ ﻣﻦ ﻋﻤﺮ اﻟﻜﺘﺎآﻴﺖ ‪.‬‬
‫ﻣﻌﻴﺎر ﻧﻤﻂ اﺿﻤﺤﻼل اﻟﻤﻨﺎﻋﺔ اﻻﻣﻴﺔ ﻓﻰ اﻟﻄﻴﻮر ﻏﻴﺮ اﻟﻤﻠﻘﺤﺔ ﺗﻤﺖ ﻣﻘﺎرﻧﺘﻪ ﻣﻊ ﺗﻠﻚ اﻟﺘﻰ اﻋﻄﻴﺖ‬
‫ﻟﻘﺎح ﻣﺮض ﺟﺮاب ﻓﺎﺑﺮﻳﺸﺲ)‪ (D78‬ﻋﺒﺮ ﻃﺮق ﻣﺨﺘﻠﻔﺔ هﻰ ﻣﺎء اﻟﺸﺮب‪ ،‬اﻟﺘﻘﻄﻴﺮ ﺑﺎﻻﻧﻒ‪ ،‬اﻟﺮش و‬
‫اﻟﺤﻘﻦ ﺗﺤﺖ اﻟﺠﻠﺪ‪ .‬اﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ ان اﻻﺳﺘﺠﺎﺑﺔ اﻟﻤﻨﺎﻋﻴﺔ ﻟﻜﻞ ﻃﺮق اﻋﻄﺎء اﻟﻠﻘﺎح آﺎﻧﺖ ﺟﻴﺪة وان‬
‫اﻻﺧﺘﻼف ﺑﻴﻦ ﻣﻌﺎﻳﻴﺮهﻢ ﻟﻢ ﻳﻜﻦ ذا ﻣﻐﺬى )‪ .(P > 0.05‬اﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ اﻳﻀﺎ ﺑﺎﻟﻨﻈﺮ اﻟﻰ ﻣﻌﺎﻣﻞ‬
‫اﻻﺧﺘﻼف )أﺧﺬ اﻟﻠﻘﺎح( ﻟﻄﺮق اﻋﻄﺎء اﻟﻠﻘﺎح اﻟﻤﺨﺘﻠﻔﻪ آﺎن هﻨﺎﻟﻚ اﺧﺘﻼف ذا ﻣﻐﺬى ﺑﻴﻨﻬﻢ < ‪(P‬‬
‫)‪ 0.05‬وآﺎن هﺬا اﻻﺧﺘﻼف ﻇﺎهﺮ ﺑﻴﻦ ﻣﺎء اﻟﺸﺮب واﻟﺘﻘﻄﻴﺮ ﺑﺎﻻﻧﻒ وﺑﻴﻦ اﻟﺮش واﻟﺘﻘﻄﻴﺮ ﺑﺎﻻﻧﻒ‬
‫وﺑﻴﻦ اﻟﺮش واﻟﺤﻘﻦ ﺗﺤﺖ اﻟﺠﻠﺪ‪ .‬ﺑﺎﻟﺮﻏﻢ ﻣﻦ ان درﺟﻪ أﺧﺬ اﻟﻠﻘﺎح ﻋﺒﺮاﻟﺘﻘﻄﻴﺮ ﺑﺎﻻﻧﻒ ﻟﻠﻜﺘﺎآﻴﺖ آﺎن‬
‫اﻻﺣﺴﻦ ﺑﻴﻦ اﻟﻄﺮق اﻟﻤﺤﺘﻠﻔﺔ ﻋﻨﺪ آﻞ اﻻﻋﻤﺎر‪ .‬ﻧﻮﺻﻰ ﺑﺎن ﻳﺘﻢ ﻓﺤﺺ اﻟﺴﻴﺮم ﻣﻦ اﺟﻞ اﻟﺘﻠﻘﻴﺢ ﻓﻲ‬
‫اﻟﻴﻮم ‪ 14 -10‬ﻣﻦ ﻋﻤﺮ اﻟﻜﺘﺎآﻴﺖ وﺑﻌﺪ اﺳﺒﻮع ﻻﺣﻘًﺎ ﻟﻤﻌﺮﻓﺔ درﺟﺔ أﺧﺬ اﻟﻠﻘﺎح‪.‬‬
Introduction
Infectious bursal disease (IBD), also known, as Gumboro disease, is an acute, highly
contagious viral infection of 3 to 6 weeks susceptible chickens caused by an avibirnavirus
genus. The causative virus has two serotypes 1 and 2. Both serotypes are present in
commercial chickens although serotype 2 is most prevalent in turkey. The disease is
characterized by pathological effects on the lymphoid organs of the chickens, primarily
the bursa of Fabricious, thus immunosuppression is a common consequence of the
infection. The disease gets its importance in poultry industry due to its significant
economic losses resulting from high mortality and immunosupression. The disease was
firstly reported in Sudan when an outbreak occurred at El-Obied in Northern Kordufan
state in 1980.
Many previous studies stated the role of maternally derived antibodies (MDA) in the
chicks in protection against infectious bursal disease virus (IBDV). The MDA is acquired
by chicks through the passage of IgG from hen serum to the embryo and remained
protective for certain period of time and then start to decay. The amount and duration of
these maternally derived antibodies (MDA) were variable in progeny chicks. In practice,
different vaccination schedules have been recommended and used but still outbreaks are
reported. The time of vaccination, maternal derived antibodies in chicks, type of vaccine,
routes of administration and pathogenicity of the IBDV challenge are important factors
determining the efficacy of Infectious bursal disease vaccination.
The objective of the present work:
The present study was conducted to investigate the decaying pattern of maternally
derived antibodies against IBDV and the immune responses of four administration routes
of D78 vaccine namely drinking water, nasal drops, spray and subcutaneous injection in
commercial broiler chickens. D78 vaccine was chosen because most poultry producers in
Sudan use it for routine vaccination against Gumboro disease and mostly via drinking
water .
CHAPTER ONE
LITERATURE REVIEW
Infectious bursal disease (IBD)
1.1. Definition
Infectious bursal disease is an acute, highly contagious viral disease of young
chickens. The signs of the disease as described by Cosgrove (1962) include soiled vent
feathers, ruffled feathers, whitish watery diarrhea, anorexia, depression, dehydration and
death. The causative virus primary target is the lymphoid tissue with special predilection
for the bursa of Fabricius (Lukert and Saif, 1991).
1.2. History
The disease was known in 1957, and observed firstly by Cosgrove in 1962 in
Gumboro, Delware, USA. This is why the disease is called Gumboro. Also the disease
used to be called (avian nephrosis) due to the kidney damage detected in infected birds.
The disease was also named infectious bursal disease when Wilter and Hitchner (1970)
isolated the causative virus and differentiated it from certain infectious bronchitis variant
virus, which gives similar kidney lesions.
1.3. Economic Importance
Infectious bursal disease became a big problem in poultry industry in many parts of
the world since when it was first reported in 1962 by Cosgrove. The disease has a
potential hazard to poultry industry due to the change of clinical picture to the sub
clinical form of the disease manifestation (Survache, 1987). The disease economic impact
is affected by strains of the virus; flocks breed susceptibility, intercurrent, primary and
secondary environmental factors. The infection influence seen at the level of flocks
livability, weight gain, feed conversion and reproductive efficiency (Shane et al., 1994).
Beside mortality IBD is an immunosuppressive disease (Rosenberger and Cloud, 1986).
Both broilers and pullet flocks infected with IBD frequently have reduced antibody
response to live attenuated vaccine against respiratory diseases such as infectious
bronchitis (IB) and Newcastle disease (ND) (Snyder et al., 1986), increased susceptibility
to respiratory viruses including Newcastle disease (Faragher et al., 1974), and infectious
bronchitis (Pejkoveski et al., 1979). A chronic form of the disease was associated with
gangrenous dermatitis, coccidiosis, chronic respiratory disease and hepatitis with varying
mortalities and adversely affected broilers flock performance (Mcllory, 1994). In addition
to death and immunosuppersion, losses from IBD including depressed growth rate, feed
conversion inefficiency were recorded in affected broilers flocks. Variability in flocks
uniformity and maturity occurred in affected pullet replacement flocks. Costs incurred as
a result of more thorough disinfections, poor quality of eggs and carcasses are further
reducing revenue and profit (Shane et al., 1994). Mcllory (1994) had also confirmed that
subclinical IBD has economic effects on broilers production.
1.4. IBD in Sudan
The disease was first reported in late December 1980 and early 1981 at El -Obied
government poultry farm in the Kordofan Region, Western Sudan. The mortality rate was
36% in 6- weeks old chicks and 22 % in one day old, a new batch introduced in the same
premises. In October of the same year, mortality was 17.8% in the first generation of
chicks from parent flocks that had the disease earlier and which were kept in the same
area (Shauib et al., 1982). The outbreak-involved chicks of mixed layers breeds (local,
crosses, Fayoumi, New Hampshire and White Leghorn)(Salman et al., 1983). Since then
the disease has been reported regularly from most part of the country and became a
serious problem to the poultry industry. In 1988, an outbreak of the disease was reported
in Kassala state (Gaffer et al., 1988). Later studies have shown that Kassala virus strain
and that of EL- Obied outbreak were similar, which were both isolated from chicks
imported from abroad. The authors suggested that the disease has been introduced to the
country through these imported chicks. Serological studies concluded that IBDV
antibodies could be detected in local and exotic breeds. The figures were 14.5% in
Kordofan Region and 44.21% in Khartoum State, Obeid and Kassala towns (El-Hassan et
al., 1989; Mahasin, 1998). Mahasin (1998) confirmed the existence of subclinical IBD in
Sudan. A serotyping study of representative local field IBDV strains was conducted in
order to identify the most suitable strain for vaccine production and it was proved that
Sudan IBDV isolates belongs to serotype 1 standard strain (Mahasin, 1998).
1.5. Etiology
1.5.1. Classification and structure
Infectious bursal disease virus (IBDV) is a member of Birnaviridae family,
avibirnavirus genus. The other genus in that family is aquabirnavirus, which include the
pancreatic necrosis virus of fish (Murphy et al., 1999). Virions are non-enveloped,
hexagonal in outline with icosahedral symmetry, 60 nm in diameter. The genome consists
of two molecules of linear double stranded RNA (Murphy et al., 1999). The smaller
genomic segment encodes viral protein (vp1) 98,000 daltons and the viral polymerase.
The larger segment encodes three proteins namely vp2, vp3 and vp4; of which vp2 and
vp3 are structural proteins while vp4 is viral protease (Fahey et al., 1991). Neutralizing
monoclonal antibodies (Mab) have been shown to bind to vp2 whereas vp3 doesn’t carry
neutralizing epitopes (Fahey et al., 1991; Synder et al., 1992).
1.5.2. Physiochemical Properties
The virus is stable, resistant to ether, chloroform, heating at 70 Cº for 30 minutes
and was unaffected by exposure to 0.5% phenol and 0.125% thimorsal for one hour at 30
Cº, but there was a marked reduction in virus infectivity when exposed to 0.5 formalin for
6 hours (Benton et al., 1967). Definitely the strong nature of the virus explains its long
survival in poultry houses even when thorough cleaning and disinfection procedures are
followed (Lukert and Saif, 1991).
1.5.3. Serotypes and Variants
Two serotypes of IBDV, 1 and 2, have been reported (McFerran et al., 1980;
Jackwood et al., 1982). The two serotypes can be differentiated by virus neutralization
test (VN) (Lukert and Saif, 1991). The two serotypes can be found in chickens but only
serotype 1 is pathogenic while serotype 2 is detected in turkeys (Ismail et al., 1988).
McFerran et al. (1980) reported antigenic variation among IBDV isolates and showed
only about 30% correlation between several strains of serotype 1 and designated them as
prototypes of that serotypes. The two serotypes share common antigens demonstrated in
agar gel precipitation test (Synder et al., 1992).
In the United State of America, variant viruses were isolated from specific pathogen free
(SPF) chickens, which were vaccinated with classical vaccines and mixed with naturally
infected broiler chickens. These viruses were found to differ from the standard strains
both antigenically and in terms of their pathogenicity for broilers and SPF leghorns
(Rosenberger and Cloud, 1986). These strains were breaking through maternal immunity
against "standard" strains, and they also differ from standard strains in their biological
properties (Rosenberger et al., 1985). Jackwood and Saif, (1987) found that of 13
serotype 1 strains tested by VN test six different IBDV subtypes were identified. Also
Saif and Jackwood (1987) isolated a virus from broiler chickens, which had bursal lesions
in spite of the presence of high maternal antibodies. Later, strain proved to be
antigenically different form those isolated before hence it was designated as variants,
whereas viruses isolated before 1984 were considered as classical viruses. McFerran et al.
(1980); Saif et al. (1987) reported failed vaccination program and failure of maternal
immunity to protect chicks due to antigenic differences between subtypes of serotype 1
IBDV. Using neutralizing monoclonal antibodies (Mabs) directed at vp2 of IBDV in
antigen capture enzyme linked immunoassay (Ac-ELISA), it was evident that variant
IBDVs had altered or deleted neutralization sites previously found on the standard or
classic IBDV strains and that neutralizing Mabs prepared against the classic IBDVs
would no longer bind to or neutralize the variant strains (Snyder et al., 1988). The very
virulent strains of IBDV were isolated from broilers and replacement layers in 1986 in
Europe (Chettle et al., 1989). These strains caused up to 70% mortality in laying pullets
and 30% in broilers and caused typical lesions of IBDV. These strains break through
maternal antibodies, which were protective against classical strains (Chettle et al., 1989;
VoB and Vielitz, 1994). Within few years, this highly pathogenic IBDV strains spread
over Europe, Middle East, South Africa and south East Asia (V o B and Vielitz, 1994). In
USA, highly virulent strains isolated were defined as variants by the fact they has been
isolated from vaccinated flocks and by specific reaction pattern with Mab (Synder et al.,
1988). In Sudan, the highly virulent strains were detected within the virus isolated since
1994 onwards (Mahasin, 2001).
1.6. Susceptible age
The clinical picture is most commonly recognized in 3-6 weeks old susceptible
chicks, birds younger than 3 weeks have subclinical infection (Lukert and Saif, 1991).
The disease outbreaks were also reported in 14 and 15-week white leghorns chickens
(Ley et al., 1997) and in 16 and 20 weeks old broilers (Durojaiye et al., 1984).
1.7. Transmission of IBD
The feces are the main source of the infection, the virus can be found there for up
to 2 weeks post infection. Aerial spread is not important nor is there evidence for egg
borne spread or virus carriers (Wiebe van der Sluis, 1994). Benton et al. (1967) found
that the infection persists in contaminated buildings for as long as 122 days after removal
of infected birds. Naturally infected wild birds, beetles (grass insects), sparrows, other
free-living birds and rodents can transmit mechanically the infection (Weibe van der
Sluis, 1994). The Aedes vexans mosquitoes and meal worms (Alphitobius diaperinus)
have been incriminated as even more important vectors (Howie and thorsen, 1981;
Snedeker et al., 1967). Parede et al. (1996) isolated infectious bursal disease virus from
darkling beetles (Carcinopes pumilio) and their larvae, which have been suspected to act
as vectors.
1.8. Incubation period
The disease incubation period is very short about 2-3 days (Lukert and Saif,
1991). Detection of histopathological lesion in the cloacal bursa could be during 24 hours
post infection (Helmboldt and Garner, 1964). Muller et al. (1979) detected gut cell
associated virus within 4-5 hours using immunofluorescence technique post-oral
infection. Viruses infected cells present in the cloacal bursa by 11 hours after direct
application of the virus to the bursa.
1.9. Morbidity and Mortality
The type and virulence of the infecting virus determine the morbidity and
mortality rates. Morbidity may reach 100% in highly susceptible birds (Lukert and Saif,
1991). Mortality starts after 3 days from the infection and rises to peak and reduce within
5-7 days. Mortality ranges from zero to 30 %( Lukert and Saif, 1991). However with very
virulent IBDV strains mortality can reach 70-80 % (Chettle et al., 1989; Murphy et al.,
1999).
1.10.Pathogenesis
Oral route is usually the natural way for infection of IBDV but the upper
respiratory tract and conjunctiva probably play also role in the transmission. The virus
travels to the bursa by blood, although direct spread from the gut cannot be excluded.
Once
arriving
the
bursa
there
is
massive
virus
replication,
with
strong
immunofluorescene reaction from 11 hours post infection onwards detected (Wiebe van
der Sluis, 1994). Histologic lesions in the cloacal bursa resemble an arthus reaction
(necrosis, hemorrhage, and large number of polymorph nuclear cells). This reaction is
type of localized immunologic injury induced by antigen –antibody complexes and
complement (Skeels et al., 1979; Ivany and Morris, 1976). The hemorrhagic lesions,
which were observed with the disease, were suggested to be due to an increased clotting
time in chicks infected with IBDV (Skeels et al., 1979; Skeele et al., 1980).
1.11. Gross lesions
The gross lesions of IBD includes dehydration, dark discoloration, petechial
hemorrhages of the pectoral, thigh muscles and in the junction between proventriculus
and gizzard, increased mucus in the intestine and renal changes (Cosgrove, 1962). The
primary target organ for IBDV is the bursa, which was found on the third day post
infection increased weight and size due to edema and hyperemia and the normal white
color changed to cream. By the fourth day it was double its normal weight then starts to
decrease in size .By the fifth day it returned to normal size and weight and became gray
but continued to decrease, and on the eight day onwards it was approximately one –third
its normal size (Cheville, 1967).
1.12. Immunosuppression
Is a state of temporary or permanent dysfunction of the immune response resulting
from insult to the immune system and leading to increased susceptibility to diseases
(Dohms and Saif, 1983).
Immunosuppression due to IBD is due to direct lyses of B-cells or their precursors
(Rosenberger, 1994). It was found that the degree of virulence of IBDV is an important
factor
in
exertion
of
immunosuppression
(Higashihar
et
al.,
1991).
The
immunosuppressive effect was found to be lower when chicks were infected with IBDV
at 21 days than at one day of age (Souza et al., 1987). In another study, it was shown that
immunosuppression could be produced even when exposure to IBDV was at one day old
but depending on the virus strain (Minta et al., 1986).
Faragher et al. (1974) in their first report on immunosuppression by IBDV stated that
suppression to Newcastle (ND) disease immunity was greatest in chicks infected at day
old, moderate at 7 days and neglible when infection was at two or three weeks.
Furthermore, Meulemans et al. (1977) mentioned that the immunosuppression due to
IBDV infection depends on the age of the birds, the time of ND vaccination and strains of
IBDV used.
To study the immunosuppressive effect of IBDV on vaccination against ND the response was compared in 2,3 and 4 weeks old
chickens inoculated with the highly virulent IBDV field isolate 90-11 and the reference serotype 1 strain GBF-1. And then vaccinated
with ND Lasota vaccine. In all age groups, isolate 90-11 severely lowered antibody response to ND vaccination on challenge. In
contrast, birds inoculated with reference strain GBF-1 and vaccinated with ND vaccine were protected from ND challenge (Nakamura
et al., 1992).
The influence of IBD infection on vaccination to ND in broiler chicks was greatly
dependent on the age of birds and time of vaccination (Rosendale et al., 1987).
IBD vaccine was reported to have adverse effect on the ND vaccine whereas the reverse
was not true (Ali et al., 2004).
The immunosuppressive potential and pathogenicity of field isolates in commercial
broilers were also investigated by Rosales et al. (1989) who showed that field isolates of
IBDV reduced the serological response to NDV vaccine.
It was reported that IBDV affects the response to many antigens. In a study by Dohms
and Jaeger (1988) broiler chickens infected at 3 weeks of age, were given Brucella
abortus (BA) or sheep red blood cells (SRBC) antigens intranasally and intraocularly
during and after the acute phase of the infection. Glands of Harder (GH) extract and
serum samples were used to measure systemic and local antibody titre to each antigen
seven days after antigen administration. Antibody titre to both BA and SRBC antigen
were less in GH extracts and serum on IBDV –infected chickens than uninfected controls.
Subclinical IBD was proved to have adverse effect also on ND vaccination of broilers
chickens. Okoye and Shoyinka (1983) inoculated 4500 broiler chicks with NDV vaccine
introculary (strain B1) at ten days of age. The chicks showed clinical signs of the disease
19 days later with 71% mortality rate and characteristic NDV lesions on post mortem
examination. Newcastle disease virus was isolated and identified by haemagglutination
(HA) test .In addition histopathological examination showed that follicles of the bursa
were adapted for lymphocytes with many large activities. Both the acute and
convalescent sera samples were positive for IBD antibodies in agar gel precipitation test
(AGPT). It was concluded that the vaccination failure might be due to the subclinical
IBD or the highly pathogenic character of the wild virus.
Immunosuppression resulting from IBD infection weakens response to vaccination
and makes chickens more susceptible to a number of other infections including
coccidiosis, Marek’s disease, gangrenous dermatitis, infectious laryngotracheitis,
infectious bronchitis, chicken anaemia agent, salmonellosis and colibacillosis (Anon,
2000). IBDV has been shown to compromise the ability of chickens to react
immunologically to ND, Mycoplasma synovaie and Haemophilus gallinarum (Wiebe van
der Sluis, 1994).
The earlier in life a chick is infected the greater the immunosuppression. It has been
suggested that the longer period a bird retain an intact bursa, the smaller the chance of
immunosuppression (Wiebe van der Sluis, 1994).
1.13. Diagnosis of IBD
Diagnosis of IBD in chickens is historically based on clinical signs and gross lesions
seen at necropsy and by histopathology; confirmation of the diagnosis is based on
detection of viral antigens, detection of antibodies and isolation of the virus (Liiticken et
al., 1994).
1.13.1. Detection of viral antigens
Viral antigens can be detected in the bursa of Fabricius on 3-4 days post infection using
an agar gel immunodiffusion (AGID) test in which homogenized bursal tissue antigens
are precipitated by known specific positive antisera (Mekkes and Wit, 1994). A direct
immunoflourescence smears of bursa of Fabricius and direct electron microscopic
examination of bursa specimens had been compared with virus isolation in chick embryo
fibroblast and embryonated eggs for diagnosis of IBD (Allan et al., 1994). It has been
indicated that immunoflourescence was, over all, more sensitive method than virus
isolation and direct electron microscopy, and gave a good correlation with
histopathological diagnosis of IBD.
Antigen capture (Ac)-ELISA had been used for detection of IBDV antigens directly from
infected tissues (Snyder et al., 1988). The same method also used to differentiate IBDV
stains. Virus neutralization (VN) assay has been considered as only reliable technique for
differentiation of IBDV strains into antigenic serotypes and subtypes (Jackwood and Saif,
1987). However the test is time consuming and expensive to be conducted. Kataria et al.
(1998) applied a more recent method; a reverse transcriptase (RT) polymerase chain
reaction (RT/PCR) to differentiate IBDV antigenic serotypes and subtypes. Furthermore,
Jackwood and Nielsen (1997) tested bursa samples for the presence of IBDV using the
reverse transcriptase polymerase chain reaction –restriction endonuclease (RT-PCR-RE)
assay. The results of their studies demonstrated that the (RT/PCR-RE) assay can be used
to diagnose IBD in chickens and that IBDV strains exist in commercially reared chickens
that have restriction endonuclease (RE) patterns different from known IBDV strains.
Counter immuno electrophoresis (CIEP) technique was used for detecting viral antigen 4
hours after infection. This method was found more sensitive than agar gel precipitation
test (AGPT) (Dzhavador et al., 1988). For qualitative and quantitative estimation of
IBDV specific antigen, Raj et al. (2000) used the rocket electrophoresis by which IBDV
– specific antigen was detected in bursa of experimentally infected chickens up to 7 days
post infection, and in a significantly great number of samples collected from field
outbreaks, than the conventional AGPT. Immunoperoxidase and immunofloourescence
technique had been used for detection of IBDV in formalin fixed paraffin embedded
sections of the bursa of Fabricius of infected chickens (Cruz-coy et al., 1993) they
assumed that the immunoperoxidase test was more useful than the immunoflourescence
test, since the same section could be examined by immunoperoxidase and examined for
microscopic pathology. Pereira et al., 1998 applied a western blot for diagnosis of IBDV
infection, they found that by the use of blocking western blot test, two distinct viral
proteins (VP2) and (VP3) were detected. The use of this methodology for detection of
infection was then suggested in animals suspected of having IBD reinfection and a
chronic subclinical form of the disease. Immunorheophoresis technique was applied for
detecting IBDV antigen in infected bursa, the precipitin lines appear 3-5 hours in the
immunorheophoresis technique, whereas that of AGID appear 14-24 hours (Raj et al.,
1998). Rao and kumar, (1994) evaluated double sandwich ELISA, immunoperoxidase,
fluorescent antibody and radial immuodiffusion tests for detection of IBDV in the bursa
of Fabricius, they found that double sandwich ELISA, is specific, sensitive and the results
are reproducible with each sample and easy to perform. The reverse passive
haemagglutination test (RPHA) was used to detect IBDV antigen in various organs of
experimentally infected chickens and field cases (Nachimuthu et al., 1995). They
detected IBDV antigen in 86.4%, 80.4% and 80.5% of different organs by RPHA, latex
agglutination and AGID test respectively.
1.13.2. Detection of antibodies
The agar gel precipitation test (AGPT) and viral neutralization test (VN) tests are the
most immunological methods used to detect chickens exposure to IBDV (Weisman and
Hitchner, 1978). The AGPT is both economical to use and simple to perform and the
results could be obtained within 28 hours. Yet V N is laborious and time consuming than
the AGPT, but is more sensitive and can detect antigenic variations between IBDV
isolates (Lukert and Saif, 1991; Mekkes and De wit, 1994; Weisman and Hitchner, 1978).
An indirect ELISA was used for measuring IBDV antibodies in chickens (Mekkes and
Wit, 1994). Antibodies could be detected as early as 4 days post infection and the test is
safe, highly reproducible, specific and sensitive. Counter immuno electrophoresis (CIEP)
was also used to detect IBDV antibodies in chickens’ sera. The method was found to be
more sensitive, simple, reproductive and rapid, detecting precipitin in 15 minutes (Ninna,
1982). In addition, results when compared well with that obtained with AGPT could be
read visually without staining. Nakamura et al. (1993) used monoclonal antibody
bounded to polysterene latex microspheres for quantitative measuring of IBDV
antibodies. The results were obtained within 5 minutes. The titre of this rapid assay
showed a close relationship with that of VN test. Perera et al., (1996) developed an ultra
–micro ELISA technique for the detection of antibodies to IBD. This technique had high
sensitivity (99%) and specificity (95.6%) when compared to ELISA. It is concluded that
the technique is suitable for serological diagnosis of IBD.
In comparing serological methods used for detection of IBD antibodies ELISA was found
to be useful in detecting the maternal derived antibodies (MDA). This was because in
infectious bursal disease (IBD) the MDAS are mainly of immunoglobulin G (IgG) class
(Rose and Orlans, 1981) and ELISA system mainly detects the IgG class (Solano et al.,
1986).
1.13.3. Isolation of IBDV
1.13.3.1 Cell Culture
Virulent IBDV strains are very difficult to adapt to tissue culture and when adapted they
become less virulent (Synder et al., 1994). It was reported that both classic virulent and
very virulent IBDV strains cannot be propagated in vitro, hence should be propagated by
in vivo method (Brown et al., 1994). Isolation of IBDV from field specimens of the
disease was reported to be very difficult (Synder et al., 1994). Even in chicken embryo
growth was not satisfactory. In addition, it was very difficult to isolate and serially
passage the virus in cell culture of chicken embryo origin (McFerran et al., 1980).
However, Hirai and Calnek (1979) propagated virulent IBDV in normal chicken's
lymphocyte and in lymphoblastoid Bcell line derived from an avian leucosis virus
induced tumor cells. Many strains of IBDV could be adapted to cell cultures of chicken
embryo origin and cytopathic effect had been seen. Cell culture adapted virus is easy to
assay by plaque production method (Lukert and Saif, 1991). Many scientist were able to
culture egg adapted strains of IBDV in chick embryo fibroblast culture which was
considered as the method of choice for virus propagation (Rinaldi et al., 1992). Wild type
virus could also be adapted to grow successfully in culture prepared in chicken embryo
bursa after four serial passages. Then this virus was propagated in chicken kidney cells
and produced plaques under agar (Lukert and Davis, 1974). Mammalian continuous cell
lines known to be susceptible to IBDV include RK-13 derived from rabbit kidneys (Petek
et al., 1973), Vero cells derived from African green monkeys (jackwood et al., 1987), and
MA -104 cells from fetal rhesus monkey kidneys (Jackwood et al., 1987).
1.13.3.2. Egg Embryos
Initial isolation of the virus in egg embryo was difficult to maintain in serial passage
(Lukert and Saif, 1991). When allantoic sac used as route of inoculation all inoculated
embryos died after the first passage, 30% in second passage and no mortality was
reported in third passage (Landgraf et al., 1967). Highest virus titre was obtained 72
hours after inoculation (Hitchner, 1970). The difficulty of virus isolation in egg embryos
refer to two factors (Hitchner, 1970), eggs from hens with IBDV antibodies were highly
resistant to infection, and in early passage all fluid had a very low virus titre chorio
allantoic membrane (CAM) had higher titre of the virus.
The best route of virus inoculation in egg embryo was found to be CAM of 9_11 day old
(Hitchner, 1970). Mortality of inoculated embryo 10 day old started from 3rd to 5th day
post inoculation.
1.13.3.3In vitro and in vivo methods
The amount of infective IBDV is determined by scoring mortality resulting in the chicken
lethal dose 50 (CLD50) or determination of the chicken infective dose scoring clinical
signs and/or bursal lesions (CID50)(Van Loon et al., 1994). In vitro methods are
determining of the embryo lethal dose (ELD50), embryo infective dose (EID50) or tissue
culture infective dose (TCID50) (Van Loon et al., 1994). Also Van Loon et al., (1994)
described amore recent diagnosis assay to determine the amount of IBDV based on
embryonated eggs as the most effective substrate for the virus and on the application of
Mabs directed against IBDV for titration. The authors method: ten- fold serial dilution of
IBDV challenge virus are inoculated on the CAM of ten to eleven day old fertilized
specific pathogen free (SPF) eggs. Five days after the inoculation the CAMS were
harvested and homogenized. The presence of virus in CAMS was then determined with
indirect ELISA assay making use of Mab-8 which can recognized all serotype I viruses.
Different viruses including challenge strains, classic and variants could be assayed by this
method.
1.14. Control of IBDV infection
1.14. Control by vaccination and types of vaccines used
Application of various management strategies was used to control the virulent IBDV.
They all proved to be of limited success (Kouwenhoven and Van Den Bos, 1994). These
include use of disinfections, use of mild and intermediate vaccine strain and different
vaccination programs such as day old application, multiple vaccination and vaccination
based on ELISA determination of maternally derived antibody (MDA) status (Gardin,
1994). Immunity against IBDV is mainly antibody mediated and maternal antibodies are
transmitted via the egg- yolk to the chick (Kouwenhoven, 1994). IBD is mainly
controlled by vaccination of breeder flocks so as to confer parental
immunity to their
progeny. Such maternal immunity will protect the chicks from early immunosuppressive
infections and naturally protect chicks for 1-3 weeks, but, by boosting the breeder flocks
with oil adjuvant vaccines, passive immunity may be extended to 4-5 weeks. However
such maternal antibodies can neutralize the vaccine virus (V o B and Vielitz, 1994).
According to Lukert and Saif, (1991) a universal vaccination program against IBD
cannot be offered and the major problem with active immunization of young maternally
immune chicks is the determination of the proper time of vaccination, and this varies with
level of maternal antibodies, route of vaccination and virulence with vaccine virus. Also
monitoring of antibody level in the parent flock or its progeny can aid in determining the
proper time of vaccination.
Types of Vaccines
There are two types of vaccines: live and inactivated vaccines.
1.14.1. Live Vaccines
The live vaccine strains could be classified into three groups by the determination of the
bursa body weight index in vaccinated and unvaccinated control birds (Mazariegos et al.,
1990). These are mild, intermediate and hot vaccine strains
1.14.1.1 Mild Vaccines
They do not induce significant difference in bursa /body weight index in vaccinated and
control birds (Mazariegos et al., 1990). They are mild field or attenuated strains. Mild
highly attenuated strains are non immunosuppressive to the bursa and can easily be
neutralized by maternal antibodies. They are suitable to use for prevention of early
exposure in one-day-old susceptible chicks and in chicks with low MDA initially or after
waning of MDA (Mazariegos et al., 1990).
1.14.1.2. Intermediate Vaccines
These induce temporary significant difference in vaccinated and control birds in bursa
/body weight index. Here the bursa weight in vaccinated birds is less than half that in
control unvaccinated ones (Mazariegos et al., 1990). They cause no lesion even in SPF
chickens. They include: CU, D78, IM, Lukert CK37, LZ228TC and Bursine 2. They are
used for vaccination of replacement pullets and for primary vaccination before
inactivated revaccination (Lohren, 1994).
1.14.1.3. Hot Vaccine Strains
These showed significant difference throughout the observation period, the bursa size in
vaccinated birds was more than or three times less than in the control (Mazariegos et al.,
1991) they include: LZ228E (intervet), Bursa plus (Solavy, Duphar, Sterwin) and TAD
LC75 (Gumboro vac forte). Its use should be restricted to very severely affected areas
where no other means of control exist. In Europe, its use needs permission from the
authorities (Kouwenhoven et al., 1994).
1.14.2. Inactivated Vaccines
Killed vaccines in oil emulsion give a depot effect and provide along uniform protection
and high antibody levels (Edison et al., 1980; Wyeth et al., 1992). However, it requires
priming with live virus exposure for effectiveness and should be given late in the rearing
period before lay (Abram, 1990).
1.15. Vaccination of broilers
Many European countries do not vaccinate broilers. They thought that by giving the
breeder
flock a booster dose of Oil adjuvant killed virus vaccines; the immunity
transferred to the off spring was enough to protect them during their short life span
(Lohren, 1994). Life long protection of broilers by maternal antibodies was reported to be
impossible (Gmeiner, 1983). The author’s study revealed that variation in maternal
antibodies was higher than expected in commercial broilers.
Killed adjuvant virus vaccine used to control IBD in Britain until 1988 by vaccination
primed parent chicks at 18-20 weeks of age (Chettle and Wyeth, 1994). The investigators
reported that the immunity transferred to the progeny was enough for protecting them for
the whole duration of their lives. But, since the emergence of the very virulent IBDV in
England in 1988, it became necessary to vaccinate broiler and parent chicks (Chettle et
al., 1989).
1.16. Vaccination failure
Many reasons were involved in IBD vaccination failure .Of these:
1) Improper timing of vaccination
The level of MDA at the time of vaccination is very important. When application is too
early the vaccine virus was neutralized by MDA (Solano et al., 1986). When applied too
late the virulent field virus had the chance to be the first to infect the chicks (Kreager,
1989). Serological evaluation of MDA determines the optimum time of the vaccine
(Gullen and Wyeth, 1975; Kouwenhoven and Van Den Bos, 1992). Considering that
MDA is related to the flock not individual birds and the vaccine application date must be
forecasted at least one week earlier and ELISA should be the serological tool to be used
(Kouwenhoven et al., 1994). The optimum age at which broilers coming from adjuvant
killed virus vaccine parents could be vaccinated with intermediate vaccines was 14-21
days (Kouwenhoven et al., 1994). However, according to the investigator these vaccines
were effective to some extent but they failed in situation of high infection pressure. More
recent reports stated that serological examination of day old chicks obtained from
similarly vaccinated flocks revealed that 8-10 days old was the most optimum time for
early vaccination (Lohren, 1994). Other reports stated that live vaccine were able to
reduce the number of susceptible chicks but there was a time during which chicks were
susceptible which corresponded to day 25 of age (Chettle et al., 1994). In fact the hardly
challenge virus could break through MDA at least one week earlier than live vaccines
could (Chettle et al., 1994).
2) The ability of the virulent field virus to break through high MDA levels i.e. to infect
broiler chicks at a much younger age than the intermediate vaccine virus (Chettle et al.,
1994)
3) Variation of maternal antibodies titre and deficient proper vaccination timing
(Kouwenhoven et al., 1994). However the problems of the less effective intermediate
vaccines was over come by the use of hotter vaccines or by multiple repeated
vaccinations by these intermediate vaccines, which in high infection pressure also failed
(Kouweenhoven et al., 1994).
To avoid the variation of maternal antibody hatcheries were advised to avoid as much as
possible combination of hatches from different breeder flocks and if possible to combine
off spring from flocks of similar titres (Kouwenhoven and van den Bos, 1994).
CHAPTER TWO
MATERIALS AND METHODS
2.1.1. Experimental chicks
One hundred and fifty, one day old, Hy-line broilers chicks were obtained from El Gharia
Company, Khartoum, Sudan. The broiler breeder parent flocks of these chicks were
vaccinated against IBD using killed IBD virus vaccine.
2.1.2. Housing of chicks:
The chicks were reared in isolated rooms with open system at the poultry houses of
Faculty of Animal Production, University of Khartoum. All chicks were fed, watered and
kept under the same environmental conditions throughout the experiments. Before and
after the experiment, the rooms were thoroughly cleaned, washed, disinfected and left for
4 weeks before being used for the experiment.
2.1.3. Preparation of D78 Vaccine:
This is an intermediate live vaccine strain (intervet). Every dose contains at least 4.0
log10 TCID50. It was obtained from Detasi Company, Khartoum. The manufacture
instructions for vaccine application with the standard procedure were strictly followed.
2.1.4. Vaccination program:
The vaccine D78 was given twice times, primary dose on the 18th day and booster dose at
day 25 of age. The vaccine was administered via drinking water, nasal drops, spray and
subcutaneous injection according to the experiment design.
2.1.5 Blood collection and serum preparation:
The blood was collected from the heart of chicks and the wing vein of older birds. Sterile
disposable syringe of 29 gauge and 1-millimeter length was used for blood collection. An
amount of 0.5 ml was collected from each bird. The blood was left for 2 hours at room
temperature, and then the clot was loosened from the surface of the syringe, kept
overnight at 4 degree centigrade. The serum was separated and clarified by centrifugation
at 2000 revolutions per minute (rpm) for 10 minutes. The serum was stored in test tube at
-20°C till use.
2.2. Enzyme-Linked Immunosorbent Assay (ELISA):
Infectious bursal disease antibody test kit was obtained from BioCheck B.V. Crabethstaat
38-C 2801 AN Gouda Holland.
The antigen coated plates (inactivated viral antigen on microtitre plates) and the ELISA
kit reagents were adjusted at room temperature prior to the test. The serum was diluted,
five hundred folds (1:500) prior to the assay with sample diluents provided. 100 µl of
diluted serum was then put into each well of the plate. This was followed by addition of
100 µl of undiluted negative control (Specific pathogen free serum in phosphate buffer
with protein stabilizers and sodium azide preservative (0.1% w/v)). 100µl of positive
control was also added. (Antibodies specific to IBD in phosphate buffer with protein
stabilizers and sodium azide preservative (0.1% w/v)). The plate was incubated for 30
minutes at room temperature. Each well was then washed 4 times with wash buffer, this
contains 0.05% Tween 20 in powdered phosphate buffered saline (300 µl per well). A
100 µl of conjugate reagent (Sheep anti-chicken: alkaline phosphatase in Tris buffer with
protein stabilizers, inert red dye and sodium azide preservative (0.15 w/v))) was added
into each well. The plate was incubated in room temperature for 30 minutes. Each well
was washed again 4 times with buffer (3oo ml per well). 100 µl of substrate reagent (pNitro phenyl phosphate was dissolved in Diethanolamine buffer with enzyme co-factors)
was dispensed into each well. The plate was then incubated at room temperature for 15
minutes. Finally 100 µl of stock solution (Sodium Hydroxide in Diethanolamine buffer)
was dispensed into each well to stop the reaction. The absorbance values were measured
and recorded at 405 nm wavelength using ELISA microtitre Plate reader.
2.3. Experimental design:
2.3.1 Experiment 1:
The objective of this experiment was to monitor the declining pattern of maternally
derived antibody (MDA) against infectious bursal disease virus (IBDV) in commercial
broiler chicks.
For this purpose, 25 broiler chicks of one day old were reared in separation. The chicks
were given infectious bronchitis (IB) Vaccine and Newcastle (ND) vaccine (colon 30) as
spray at day 3 of age and another dose of Newcastle vaccine (colon 30) spray at day 14 of
age for protection from IB and ND. But were not vaccinated against infectious bursal
disease (IBD).
Blood was collected from five chicks randomly selected at days 1, 18, 25, 32,39 and 45
day old. The serum prepared as mentioned in section 2.1.5
2.3.2 Experiment 2:
Four administration routes of D78 live vaccine were adopted in this experiment to study
the effect of IBD vaccine route on the depletion rate of IBDV maternally derived
antibody (MDA) titres in the chicks, along with the immune response in the IBDV
vaccinated birds with each route was examined.
The vaccine was administrated by four routes namely drinking water (A), nasal drops
(B), spray (C) and subcutaneous (D) and the test group was left without vaccination (F).
125 birds were divided into five groups (25 chicks of each). All groups were vaccinated
by infectious bronchitis (IB) vaccine and Newcastle (ND) vaccine (colon 30) as spray at
day 3 of age and another dose of Newcastle vaccine (colon 30) spray at 14 day of age for
protection against IB and ND.
The four groups were vaccinated separately by D78 on day 18 and 25 by either route (A,
B, C and D). The remaining 25 acted as control (F). From each group 5 birds were
selected randomly for blood collection every week starting from day 18 of age. The sera
were separated and saved at –20 ºC till used for antibodies detection by ELISA.
2.4. Data analysis:
IBD antibody titre was calculated automatically, using soft ware.
The ELISA data are presented as:
1- S/P ratio of the samples where (S) represented the absorbance value of the test serum
divided by the absorbance Value of the positive control (P) serum.
S/P =
Mean of Test Sample – Mean of negative Control
Mean of positive control –Mean of Negative Control
The following equation relates the S/P of a sample at a 1:500 dilution to an end point
titre
Log 10 Titre = 1.1 × Log (SP) + 3.361
S/P value
Titre range
Antilog = Titre
Antibody status
0.145 or less
284 or less
negative
0.150 – 0.199
285 – 390
suspect
0.200 or greater
391 or greater
positive
2- Coefficient of Variation (CV): is an indicator of individual value dispersal with
regards to titre mean.
Coefficient of Variation =
Standard deviation
Arithmetic mean titre
CV values is currently made according to the following threshold
< to 30% =Very homogenous
30 to 50% =Homogenous
50 to 80%= poorly homogenous
> 80 %= Heterogeneous
> to 150 % =Very heterogeneous
For statistical analysis stat graphics plus version 3.0 (1997) was employed for analysis.
One-way ANOVA was used in order to find out the relationship between different
vaccine administrations. With regard to S/P and CV%.
CHAPTER THREE
RESULTS
The declining pattern of maternally derived antibodies (MDA):
The decaying pattern of maternally derived antibody (MDA) against infectious bursal
disease virus was observed as the age of chick’s progress. High S/P ratio (5.4) of MDA
was obtained for day one, this level was almost stable up to the 7th day with S/P ratio of
5.06, and then the MDA started to decline rapidly. By the 18th day (S/P ratio = 0.62) and
with very low level at day 45 of age (S/P ratio 0.04) (Figure 1).
6.00
5.00
S/P ratio
4.00
3.00
2.00
1.00
0.00
1
7
18
25
32
39
45
Age post-hatch (days)
Figure 1: The levels of maternal anti-Infectious Bursal Disease antibodies in
progeny chicks at various ages post hatch
The effect of vaccine administration route on MDA:
Generally, the correlation between the four groups of vaccine administration routes with
regard to S/P ratio was found (F = 0.94 and P = 0.4519) which is statistically not
significant (P>0.05).
Following, the administration of the vaccine via drinking water, the result obtained
showed that at day 25 of age the level of immunity was very low (S/P ratio = 0.05).
Detection of serum at day 32 (one week after the second vaccination) indicated that the
S/P ratio was increased to 1.67. Then the S/P ratio was continuously increased up to day
45 (3.26) (Figure 2).
6.00
5.00
S/P ratio
4.00
3.00
2.00
1.00
0.00
1
7
18
25
32
39
45
Age post-hatch (days)
Figure 2: Seroconversion of progeny chicks after vaccination with D78 via drinking
water
NB:
Day 18 the first vaccination dose of D78
Day 25 the second vaccination dose of D78
S/P ratio indicates the amount of antibodies as determined by ELISA.
Following the administration of the vaccine via nasal drops, the S/P ratio was 0.1 when
the serum tested at day 25 of age, then the S/P ratio was increased at day 32 of age (S/P
ratio = 1.19) and it remained high (S/P ratio = 1.86) until the last day of the experiment
(Figure 3).
6.00
S/P ratio
5.00
4.00
3.00
2.00
1.00
0.00
1
7
18
25
32
39
45
Age post-hatch (days)
Figure 3: Seroconversion of progeny chicks after vaccination with D78 via nasal
drops
NB:
Day 18 the first vaccination dose of D78
Day 25 the second vaccination dose of D78
S/P ratio indicates the amount of antibodies as determined by ELISA.
The application of the vaccine through spray route revealed that the S/P ratio was low
(0.19) at day 25 of age, and then the S/P ratio was raised up to day 45 of age (1.54)
(Figure 4).
6.00
5.00
S/P ratio
4.00
3.00
2.00
1.00
0.00
1
7
18
25
32
39
45
Age post-hatch (days)
Figure 4: Seroconversion of progeny chicks after vaccination with D78 via spray .
NB:
Day 18 the first vaccination dose of D78
Day 25 the second vaccination dose of D78
S/P ratio indicates the amount of antibodies as determined by ELISA.
Following the administration of vaccine via subcutaneous injection, the level of
antibodies was very low at day 25 of age (S/P ratio = .06) . Then the S/P ratio began to
increase up to the last day detection of serum at day 45 (S/P ratio = 3.29) (Figure 5).
.
6.00
S/P ratio
5.00
4.00
3.00
2.00
1.00
0.00
1
7
18
25
32
39
45
Age post-hatch (days)
Figure 5: Seroconversion of progeny chicks after vaccination with D78 via
subcutaneous injection
NB:
Day 18 the first vaccination dose of D78
Day 25 the second vaccination dose of D78
S/P ratio indicates the amount of antibodies as determined by ELISA.
The homogeneity of the results was measured with regard to coefficient variation (CV
%), which is an indication for vaccine take. The difference between the four different
types of vaccine administration was statistically significant (F=6.95
P< 0.05). This
significance was clear between the group of nasal drops and drinking water, and nasal
drops and spray route, subcutaneous injection and spray route.
The result of CV% is
summarized in Table 1.
Table 1: The coefficient variation of MDA in chicks vaccinated via different routes
Age of
CV% of
CV% of
CV% of
CV% of
CV% of
chicks
MDA
drinking
nasal
spray
subcutaneous
water
drops
(days)
injection
0
27
27
27
27
27
7
29
29
29
29
29
18
68
68
68
68
68
25
51
134
47
177
62
32
15
66
29
130
48
39
10
71
28
70
46
45
10
78
15
90
33
< to 30% Very homogenous
80 % Heterogeneous
30 to 50% Homogenous
50 to 80% poorly homogenous
CHAPTER FOUR
DISCUSSION
50 to 80% poorly homogenous
> 150 % Very heterogeneous
Infectious bursal disease (IBD) or Gumboro is an old disease of chicks caused by a
Birnavirus; which targets the bursa of Fabricius, most of the economic losses associated
with IBD are due to its immunosuppressive effects. The disease is mainly controlled by
vaccination. To achieve a successful vaccination program; an effective vaccine
administered by an appropriate route is necessary. This study was conducted to assess the
immune response of D78 vaccine administrated by drinking water, nasal drops, spray and
subcutaneous injection; the declining pattern of maternally derived antibodies (MDA)
was also investigated.
From the results obtained in the present study, it was clear that, the level of maternally
derived antibodies (MDA) or passively transferred antibodies against infectious bursal
disease virus (IBDV) were very high at day one of age as they hatched from vaccinated
dams. Which indicates that, the parent's flocks of these chicks were hyper immunized.
The dams transmit this high level of antibodies in the form of maternal derived antibodies
to the progeny chicks. This was previously published by Sharma et al. (1989). These high
levels of maternal derived antibodies protect the chicks from infection by IBDV in early
ages, but will hinder vaccination against IBD as the vaccine will be neutralized by the
circulating MDA and rendered infective. This fact was previously confirmed by (Solano
et al., 1986).
In this study, the maternally derived antibodies was observed to decline rapidly after the
first week and was noted to remain detectable in chicks up to 45 days of age with
appreciable magnitude log low (S/P of 0.04). However the protective limit of these
antibodies level expired by the 4th week. The finding of this study looks in agreement
with Kenzevic et al. (1987) who reported that the progeny antibodies persisted up to 6
weeks of age and diminished after the first week. While this result is somehow in
disagreement with Zaheer and Saeed, (2003) who reported that the progeny antibodies
persisted up to 4 weeks and the protective limit of them expired by the second week. The
difference in the results of our study and above mentioned study could be due to the
difference in the initial titer of IBD in chicks, which is a direct reflection of the immune
status against IBD in the parent flock. This explains the individual variation among
chicks to take the vaccine
The maternally derived antibodies declined rapidly after the first week of age, however
were still detectable by the end of the study (45 days). This is not surprising since these
chicks were not raised on specific pathogen free environment and were perhaps still
exposed to low levels of antigenic challenge from external sources. Similar levels of
maternally derived antibodies decay were observed in another study (Ahmed and Akhter,
2003). The reason for such rapid declining at this period of age attributed to the
proteolytic degradation of antibodies or neutralization because of naturally occurring
infectious bursal disease virus challenge. It is clear evident that an appropriate time for
maternal derived antibodies level testing in chicks is while they are growing i.e. day1014. Since the rate of decrease in the level of MDA is affected by existence of the
pathogen in the environment, metabolism and growth rate.
The second part of the study showed that the S/P ratios for all different routes of
administration of the vaccine D78 was decreased post the first vaccination day and the
increase to the protective level after the second vaccination was observed and remained
high up to the 45th day of age. This seroconversion indicated that the immune response
for all different routes of administration of the vaccine is good, and the decrease was a
result of neutralization of the vaccine by the high MDA at the first vaccination day hence
the intermediate vaccine D78 can not break through high MDA like the hot vaccines.
This phenomenon was reported in previous studies (Zaheer and Saeed, 2003) which
suggested that a customized vaccination regime with regard to the type of vaccine and
level of MDA is crucial for complete protection rather than the route of the vaccine
administration which does not have great influence on the protection level of antibodies.
And the increase in the immunity due to the second vaccination dose is observed and in
fact because the level of MDA was not high enough to hinder the work of the vaccine at
this time.
The difference between the S/P ratios of the birds against different routes of
administration was not significant (P> 0.05) and this agreed with Nakamura et al. (1995)
who confirmed the insignificance between vaccination routes with regard to immune
response when he studied intratrachea, intranasal, per os, by crop gavage and
intramuscular routes for vaccine administration. Giambrone and Hathcock (1991)
reported similar findings when they studied coarse spray and dinking water routes for
vaccination against IBD. This indicates that D78 vaccine can be administrated by all
routes used in the study without detectable variation in their immune response elicited.
On the other hand the S/P ratio was seen useful as it allows to estimate whether the birds
were immunized or not, but it doesn't allow us to compare the vaccine take. So the most
significant criterion is to find out whether the batch as a whole is immunized or not, and
this is established with homogeneity or coefficient variation and the differences between
the four different routes of administration of the vaccine was statistically significant in
our study (P<0.05). This significance was clear between drinking water and nasal drops,
and nasal drops and spray, spray and subcutaneous injection. And it was clear from Table
1; the nasal drops route of administration at various ages had the best interpretation with
regard to the coefficient variation and this because this route gives the birds equal
opportunities for vaccine antigens exposure
In conclusion, this study revealed that the decay of maternal derived antibodies in chicks
started rapidly after the first week and remained detectable to the end of the study under
our experimental condition whereas the naturally occurring IBDV challenge played role
in this decay. While the protective limit of antibodies expired by the end of the 4th week.
It was also clear that improper vaccination time due to vaccination at early age (high
MDA) leads to depression period of immunity between time of vaccine neutralization and
protective antibodies resulting from the second vaccine dose. Concerning routes of
vaccination our study revealed that there was no significant difference between drinking
water, spray, subcutaneous injection and nasal drops as routes of administrations of the
vaccine D78 with regard to the immune response. Although the vaccination take by nasal
drops was the best.
So it is recommended to devise vaccination schedule in the light of prevailing maternal
antibody titre while chicks are growing i.e. 10-14 day of age. And nasal drops is the best
administration route of D78.
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