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 Page

1 The coefficient variation of MDA in chicks vaccinated via

different routes…..…………………………………………………33

List of Figures

Figure Page

1 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 non-

vaccinated 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 األطروحة

هذه الدراسة في آتاآيت الحم لتقييم نمط اضمحالل المناعة األمية ضد فيروس مرض أجريت

تم .عبر اعطاءه بطرق مختلفة(D78) لمعدي وتقييم االستجابة المناعية للقاحجراب فابريشس ا

وقسمت الي مجموعات ملقحه وغير ملقحه بوسطة االغراض لهذهتوآتكإستخدام مائه و خمسون

في المناعة االمية لمرض جراب فابريشس المعدي تبقتاظهرت هذة الدراسة ان. لقاح القمبورو

تى اإلسبوع السادس من عمر الكتاآيت بعد أن تم تحديدها بواسطة إختبار الطيور غير الملقحة ح

. هذا المرض قد خلصت باألسبوع الرابع مع أن آمية األجسام المضادة الكافية للحماية من. االليسا

قد عبر الطرق المختلفة D78)( في اليوم الثامن عشر بواسطة الكتاآيتعمليه تلقيح فشلعموما

ولكن الجرعه الثانية للتلقيح عند اليوم الخامس والعشرين اعطت نتائج . ه في دراستناتمت مالحظت

لذلك نقترح ان من اجل الحصول علي اليوم المثالى للتلقيح يجب ان تفحص المناعة االمية . جيدة

. اثناء نمو الكتاآيت مثال من اليوم العاشر الي الرابع عشر من عمر الكتاآيت

فى الطيور غير الملقحة تمت مقارنته مع تلك التى اعطيت محالل المناعة االميةمعيار نمط اض

الرش و ، التقطير باالنف، عبر طرق مختلفة هى ماء الشرب(D78)لقاح مرض جراب فابريشس

اظهرت النتائج ان االستجابة المناعية لكل طرق اعطاء اللقاح آانت جيدة وان . الحقن تحت الجلد

اظهرت النتائج ايضا بالنظر الى معامل ). (P > 0.05اييرهم لم يكن ذا مغذى االختالف بين مع

> P)لطرق اعطاء اللقاح المختلفه آان هنالك اختالف ذا مغذى بينهم) أخذ اللقاح(االختالف

الرش والتقطير باالنف وبين وآان هذا االختالف ظاهر بين ماء الشرب والتقطير باالنف (0.05

أخذ اللقاح عبرالتقطير باالنف للكتاآيت آان بالرغم من ان درجه. حقن تحت الجلد الرش والوبين

يتم فحص السيرم من اجل التلقيح في بان نوصى.االحسن بين الطرق المحتلفة عند آل االعمار

. من عمر الكتاآيت وبعد اسبوع الحقًا لمعرفة درجة أخذ اللقاح14 -10اليوم

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 (p-

Nitro 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 Antilog = Titre

S/P value Titre range 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).

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:

1 7 18 25 32 39 45 0.00

1.00

2.00

3.00

4.00

5.00

6.00

S

/P r

atio

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).

Age post-hatch (days)

Figure 2: Seroconversion of progeny chicks after vaccination with D78 via drinking

water

NB:

1 7 18 25 32 39 45 0.00

1.00

2.00

3.00

4.00

5.00

6.00

S

/P r

atio

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).

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

1 7 18 25 32 39 45 0.00

1.00

2.00

3.00

4.00

5.00

6.00

S/P

ratio

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).

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.

1 7 18 25 32 39 450.00

1.00

2.00

3.00

4.00

5.00

6.00

S/P

ratio

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).

.

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

1 7 18 25 32 39 45 0.00

1.00

2.00

3.00

4.00

5.00

6.00

S/P

ratio

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

chicks

(days)

CV% of

MDA

CV% of

drinking

water

CV% of

nasal

drops

CV% of

spray

CV% of

subcutaneous

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 30 to 50% Homogenous 50 to 80% poorly homogenous

80 % Heterogeneous 50 to 80% poorly homogenous > 150 % Very heterogeneous

CHAPTER FOUR

DISCUSSION

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. day10-

14. 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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