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American Journal of Life Science Researches

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426 October, 2014

© 2014, World of Researches Publication

Am. J. Life. Sci. Res. Vol. 2, Issue 4, 426-439, 2014

Serological Evaluation and Challenge Tests of the

Recombinant and Inactivated AI Vaccines to Egyptian HPAIV

H5N1 under Field Condition and Experimental Trails

Mahmoud E. Sediek*, Ashraf M. Awad

Department of Poultry and Fish Diseases, Faculty of Veterinary Medicine, Alexandria University,

Egypt

*Corresponding Author: [email protected]

INTRODUCTION

The Avian influenza viruses (AIVs) subtype H5N1, belonged to the family

Orthomyxoviridae, genus Influenza A, are negative single stranded, enveloped

RNA viruses. A few members of H5 and H7 subtypes cause major and frequently

Abstract: Four different commercial vaccines of AI were evaluated in these study (VolVac®

inactivated vaccine H5N2, VolVac® recombinant vaccine, Merial® inactivated vaccine H5N and

Merial® recombinant vaccine) by serological and challenge tests under field condition and

experimental trails. Egyptian HPAIV H5N1 (A_chicken_Egypt_11VIR4453-266_2010) used in

challenge tests .Five broiler farms in Alexandria governorates used in field trail evaluation and

the results indicated that the GM titers of the HI against AIV vaccine in the flocks (1, 2 & 3)

vaccinated with recombinant and oil inactivated vaccine of (Merial®) were 7, 6 and 7.8 log 2,

respectively, while in Flocks (4 & 5) vaccinated with recombinant and oil inactivated vaccine

(VolVac®) were 5 & 5.1 log 2, respectively. Flocks (1, 2, & 3) revealed 70, 20, and 40 % protection

respectively and flock (4 and 5) have 30 and 90 %, respectively.270 one day old commercial

broiler chicks (obtained from local hatchery were reared on a floor pens and divided into 9

groups) used in experimental trail evaluation were vaccinated S/C route and infected by HPAIV

H5N1 (A_chicken_Egypt_11VIR4453-266_2010) and the results showed that the highest HI titers

either at 28 days or 35 days of age was recorded in group (1) vaccinated with recombinant

Merial® 7.7 log 2 at 28 days and 7.8 log 2 at 35 days followed by group (7) vaccinated with

inactivated vaccine Merail® 7.4 log 2 at 28 days and 7.5 log 2 at 35 days then group (2)

vaccinated with recombinant and inactivated Merial® 6.8 log 2 at 28 days and 7.1 log 2 at 35

days. While, other groups vaccinated with recombinant and inactivated vaccine Volvac® or

inactivated vaccine of Volvac® gave the lowest titers ranged from 4.2 - 2.7 log 2. The highest

percent of protection after challenge was recorded in group (3) vaccinated with recombinant

vaccine Volvac® (90%) followed by group (1) vaccinate with recombinant vaccine Merial® (70%),

while, group (2) vaccinated with recombinant vaccine and inactivated Merial® and group (5)

vaccinated with inactivated Volvac® gave the same protection percent (50%) and the lowest

protection percent was reported in the groups (4, 6 & 7) it was 40, 30 and 14.2%, respectively.

Keyword: Avian Influenza, Serological, Vaccine, GM, Recombinant, Inactivated

ORIGINAL ARTICLE Received12 Aug. 2014 Accepted 18 Sep. 2014

Serological Evaluation and Challenge Tests …

fatal disease in experimental birds 1. At the beginning of 2004, the outbreaks of

avian influenza virus subtype H5N1 were reported in several Asian countries

including Vietnam, South Korea, Japan, Taiwan, Laos, Cambodia, China, Pakistan,

Indonesia and Thailand caused severe losses to the poultry industry 2.

Additionally, the H5N1 virus also poses a significant threat to human health 3.

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 which originated in

south east Asia in 1996,1997 has spread across Eurasia since 2003 and entered

Africa in 2005 4. In Egypt, H5N1 HPAI outbreaks were reported in February 2006 .

In March 2006, Egyptian official reported more than 900 H5N1 outbreaks

among poultry in governorates spanning the length of the Nile land in an effort to

control the outbreaks, authorities culled more than 34 million birds and

implemented a poultry vaccination programme, their efforts largely controlled

outbreaks in the second half of 20075.

When poultry are infected, they may have no disease, mild disease or very

sever disease. Chickens, quail and turkeys are especially susceptible, while ducks

more commonly show no disease but act as a reservoir for the virus. Other

poultry species including guinea fowl and pheasants, and also ostriches, can be

came affected. While wild birds are generally not affected by the AI viruses that

they carry, and can occasionally suffer disease. This has been observed as a

result of infection with the H5NI virus in Asia and parts of Europe and may be a

result of the virus’s first becoming highly virulent in domestic birds 6.

Vaccination efficacy in individual birds can be quantified using the following

three parameters: (1) degree of protection from infection when exposed to a

given amount of infectious virus, (2) the degree of reduction of morbidity and

mortality given infection occurred, and (3) the level of reduction of virus excretion

by infected poultry 7 .The efficacy of most commercially available vaccines has

been determined in studies with chickens or turkeys, since globally they

represent the economically most important poultry species. However, in many

developing countries, particularly in Southeast Asia, other species such as ducks,

Muscovy ducks, and quails also represent significant parts of the poultry sector.

Thus, vaccines use in poultry populations with significant shares of species other

than chickens and turkeys require efficacy testing in these species to contain viral

transmission. Although, almost all commercially available vaccines provide some

level of protection from infection with virulent virus and significantly reduce

mortality in infected chickens no HPAI vaccine has so far proved to satisfactory

perform on all three of the above parameters 8,9 .

The time lag between vaccination and protective immunity and the

respective duration of protection depends on the vaccine used, timing of

vaccination, number of doses given, species, immunologic condition of the birds,

and the challenge virus. The literature on vaccine efficacy is dominated by studies

with specific pathogen free (SPF) birds reared under laboratory conditions. The

results of these studies cannot safely be extrapolated to field conditions, and in

these protection is often assessed by serology, the assumption being that birds

with a specified antibody level (e.g., a hemagglutination inhibition [HI] titer ≥ 16)

Mahmoud et al., 2014

428 October, 2014

are protected. However, under conditions of antigenic variability and diversity of

the HPAI viruses circulating in the field, a given titer found against a laboratory

strain cannot unequivocally be interpreted as protective against field virus

challenge. Published literature on vaccine efficacy and the onset of protection

under field and laboratory conditions indicated that protection in chickens is in

no circumstances achieved prior to 11–13 days post vaccination 10, 11, 12. About

20.5 weeks after successful vaccination of layer chickens with two doses of killed

LPAI H6N2 vaccine, all birds tested sero-negative again 13.

Avian influenza viruses vary antigenically and evolve rapidly, which poses a

major challenge for the sustained use of vaccines as HPAI control measure.

Although, several studies demonstrated cross-protection for HPAI viruses, a

correlation between virus shedding and antigenic differences of vaccine and field

strains was shown 14 for the Mexican lineage H5N2 virus and 15 for a range of H5

HPAI viruses. During and after the extensive use of about two billion doses of

H5N2 vaccine in commercial poultry farms in Mexico, molecular drifts with a

yearly trend have been shown 16. Antigenic drift of avian influenza viruses was

also observed in the USA after vaccination programmes for LPAI in commercial

poultry 17. Variant field strains that escaped the protection by the commonly used

vaccines emerged in Shanxi China during 2006, in Egypt in late 2006, and in

Indonesia early 2007 18. Using a deterministic patch-structured model, 19 found

that an avian influenza vaccination campaign can lead to the prevalence of a

vaccine-resistant strain, which could result in the replacement of viral strains in

areas without vaccination via migration of asymptomatic birds.

The aim of this study is to evaluate different commercial vaccines

(recombinant and inactivated) by serological and challenge tests under field

condition and experimental trails.

MATERIAL AND METHODS

1. AI virus: Egyptian HPAIV H5N1 was isolated in embryonated chicken eggs

at the Faculty of Veterinary Medicine Alexandria Univ. Egypt, obtained during

routine surveillance of poultry holdings from a commercial broiler farm in

Alexandria governorate during late 2010 and 2011. These flocks suffering from

high mortality and respiratory distress. After RT-PCR test and gene sequencing

was done at to OIE, FAO National Reference Laboratory for Newcastle Disease

and AI, Istituto Zooprofilattico Sperimentale delle Venezie, Viale delle´ universita

(Padova)-Italy, was used for the challenge test.

2. Specific pathogen free eggs (SPF) : Fertile, Specific pathogen free egg for

H5N1 propagation and titration were obtained from Specific pathogen free

governmental farm.

3. Washed chicken RBCs: Blood was collected from wing vein with sterile

syringe containing anticoagulant (3.8% sodium citrate) at the rate of 4:1.

Following collection, blood sample was mixed with equal amount of PBS (PH 7.2)

and centrifuged at 3000 rpm for 15 min. The supernatant was discarded and the

cell sediment was resuspended with PBS and centrifuged at 3000 rpm for 15 min


for three times and finally the supernatant was discarded. Finally, 1% and 10%

washed RBCs suspensions in PBS were prepared and used for HI and slide HA

test, respectively 20.

4. Determination of median embryo infective dose (EID50) of Egyptian

HPAIV: H5N1 (A_chicken_Egypt_11VIR4453-266_2010) virus for challenge tests:

This was carried out according to 21 .Allantoic fluid of the Egyptian HPAIV

H5N1 was tenfold diluted in PBS and inoculated in 10-day-old SPF embryonated

eggs via allantoic sac (5 eggs/dilution and 0.1ml /egg). The eggs were incubated at

37C and candled daily for mortality up to 3 days post inoculation (PI). The dead

embryo within 1st 24 hrs were discarded as nonspecific mortality. Embryos which

die later and survivors were opened 3 days PI and harvest allantoic fluid from

each egg and test for hemagglutination to determine the presence or absence of

Avian Influenza virus.

The EID50 was calculated after 22. The end point of the titration is used to

calculate the infectivity titer of the original suspension of virus. The Reed and

Muench equation is used to calculate this end point from the results of the

hemagglutination (HA) test on each of the inoculated eggs. A formula is used to

calculate an index (proportionate distance) that is then applied to the appropriate

dilution. The infectivity titer is expressed as EID50 per ml.

5. Slide hemagglutination test23: This test was done by placing one drop of

10% washed chicken RBCs suspension in sterile saline (0.8% sodium chloride)

onto a clean microscopic slide and thoroughly mixed with one drop of the

harvested allantoic fluid. The result was recorded within one minute. If the

haemagglutinating agent is present the RBCs will clump.

6. Mono specific antiserum :Mono specific antiserum for AI H5N1 kindly

supplied by Boehringer Ingelheim ® Company.

Mono specific antiserum for AI H5N2 kindly supplied by Merial® company.

7. Hemagglutination inhibition (HI) test:HI test was carried out according

to [23] to detect the H5-specific antibodies titer for each vaccine using mono

specific antisera of each vaccine individually.

8. Origin of vaccines: Table1. Four different commercial vaccines

Vaccine Strain

VolVac® inactivated vaccine H5N2 A/chicken/ Mexico/323/94

VolVac® recombinant vaccine A/chicken/Scotland/1959

Merial® inactivated vaccine H5N1 A/duck/Anhui/1/2006

Merial® recombinant vaccine A/turkey/Ireland/1378/83

9. Field experiments:

Five broiler farms in Alexandria governorates were vaccinated S/C route and

evaluate as follows: Table3. Five broiler farms in Alexandria governorates were vaccinated S/C route

Flock

No.

Vaccination regime

1 One day old recombinant vaccine

(Merial®)

Ten days old inactivated reassorrtant vaccine

(Merial®)


430 October, 2014


(Merial®)


(Merial®)


(Merial®)


(Merial®)


(VolVac®)


(VolVac®)


(VolVac®)


(VolVac®)

After 21 -22 days post last vaccination 10 blood samples were collected from

each flock and antibody titer was measured using HI test then 10 birds from each

flock were transferred to Department of Poultry and Fish Diseases, Fac. Vet. Med.,

Alexandria Univ., for the challenge test. Each bird was inoculated intranasal with

0.5 ml of fluid contain 106/EID50 viral particles of Egyptian HPAIV H5N1

(A_chicken_Egypt_11VIR4453-266_2010). The birds kept under daily observation

for 10 days. The signs, morbidity and mortality were recorded during the

observation period. Also, tracheal and lung samples from dead birds were

collected for reisolation of AI virus.

10. Experimental Trail:

* A total number of 270 one day old commercial broiler chicks obtained from

local hatchery were reared on a floor pens with straw letter and divided into 9

groups. Each group contain 30 chicks. The chicks were vaccinated against

Newcastle disease at 8 and 20 days of age using a clone 30 by eye drop vaccine

and against IBD using IBD vaccine at 12 and 20 days of age in drinking water.

* Groups (1-7) were vaccinated against AI using recombinant and/or

inactivated vaccines according to the following regime: Table4. Groups (1-7) were vaccinated against AI using recombinant and/or inactivated

vaccines

Group No. Treatment

1 Rec. vacc. Merial® ( 1st

day )*


day ) *+ inactivated Merial® ( 10th

day)

3 Rec. vacc. Volvac® ( 1st

day )*


day ) *+ inactivated Volvac® ( 10th

day)

5 Inactivated vacc. Volvac® ( 10th

day)

6 Inactivated 0.3ml Volvac®( 1st

day )* + 0.5 ml Volvac®( 10th

day)

7 Inactivated vacc. Merial® ( 10th

day)

8 Control positive challenged with AI virus

9 Control negative non Challenged with AI virus

* 0.3 ml was inoculated at 1 day of age

* The chicks were reared for 6 weeks the temperature adjusted according to

the age of birds. Water and feed were available addlibtum. Anticoccidial drugs

were added two times at 18 and 28 days of age for 3 successive days each.

* Blood samples were collected at one day old, 8th, 21st, 28th, 35th days of

age for determining the antibodies against AI virus using HI test with homologous

antigen for each vaccine.


* At 29 days of age 10 birds from each group transferred to the lab for

challenge test using HPAIV H5N1 (A_chicken_Egypt_11VIR4453-266_2010) virus.

Each bird inoculated through naso-ocular route with a dose 106/ EID 50 and kept

under daily observation for 10 days. The signs, morbidity and mortality were

recorded during the observation period. Also, tracheal and lung samples from

dead birds were collected for reisolation of AI virus.

RESULTS

Results of field trail evaluation:

1. Serological evaluation

Data of the GM titers against AI virus vaccines in ten serum samples were

collected at 30 days of age for each flock were presented in (Table 1).

The GM titers of the HI against AIV vaccine in the flocks (1, 2 and 3)

vaccinated with recombinant and oil inactivated vaccine of (Merial®) were 7, 6

and 7.8 log 2, respectively. While in Flocks (4 and 5) vaccinated with recombinant

and oil inactivated vaccine (VolVac®) the GM titers were 5 and 5.1 log 2,

respectively.

2. Challenge test: Data of mortality and protection percentages are

presented in (Table 5). It was clear that flocks 1, 2, and 3 revealed 70, 20, and 40

% protection respectively and flock 4 and 5 have 30 and 90 %, respectively.

Table 5. Results of mortality and protection percent against AI virus for field trail

No= numbers Gm = Geometric titers (log 2) at 30 days of age.

Results of experimental trail evaluation:

1. Serological evaluation

Data of the GM titers against AI virus vaccines in serum samples collected

weekly during the experimental period (35 days of age) were presented in (Table

2). The highest HI titers either at 28 days or 35 days of age was recorded in group

1 vaccinated with recombinant (Merial®) 7.7 log 2 at 28 days and 7.8 log 2 at 35

days followed by group 7 vaccinated with inactivated vaccine (Merail®) 7.4 log 2

at 28 days and 7.5 log 2 at 35 days then group 2 vaccinated with recombinant and

inactivated (Merial®) 6.8 log 2 at 28 days and 7.1 log 2 at 35 days. While, other

groups vaccinated with recombinant and inactivated vaccine (Volvac®) or

inactivated vaccine of (Volvac®) gave the lowest titers ranged from 4.2 - 2.7 log 2.

Farm

No.

GM

titers

HI

Mortality Total Mortality

%

Protection

% 1st

day

2nd

day

3rd

day

4th

day

5th

day

6th

day

7th

day

8th

day

9th

day

10th

day

1 27

1 1 1 3 30 70 %

2 26

2 2 4 8 80 20 %

3 27.8

2 2 2 6 60 40 %

4 25

3 4 7 70 30 %

5 25.1

1 1 10 90 %


432 October, 2014

Table 6. Geometric means titers of H5 AI virus vaccines post vaccination

(Experimental trail 30 chicks/ group)

Group

No.

Vaccination program Geometric means titer

1 day

old

8

days

21

days

28

days

35

days


day )* 4.4 9.2 8.75 7.7 7.8


day ) *+ inactivated

Merial® ( 10th

day)

8 7.75 6.8 7.1


day )* 4.5 3.7 3 3.2


day ) *+ inactivated

Volvac® ( 10th

day)

4.5 3.5 3.9 4.2

5 Inactivated vacc. Volvac® ( 10th

day) 6 1.5 2.6 2.7

6 Inactivated 0.3ml Volvac®( 1st

day )* + 0.5 ml

Volvac®( 10th

day)

5 3.3 2.6 2.8

7 Inactivated vacc. Merial® ( 10th

day) 5.2 6.3 7.4 7.5

8 Control positive challenged with AI virus 5.2 0.5 0.5 0.4

9 Control negative non Challenged with AI virus 5.0 0.3 0.4 0.4

*3.0 ml was inoculated at 1 day of age

2. Challenge test:

Data of mortality and protection percentages are presented in (Table 3). It

was clear that the highest percent of protection after challenge was recorded in

group 3 vaccinated with recombinant vaccine Volvac® (90%) followed by group 1

vaccinate with recombinant vaccine Merial® (70%).While, group 2 vaccinated with

recombinant vaccine and inactivated Merial® and group 5 vaccinated with

inactivated Volvac® gave the same protection percent (50%).On the other hand,

the lowest protection percent was reported in the groups 4, 6 and 7 it was 40, 30

and 14.2%, respectively.

Table 7. Results of mortality and protection percent against H5N1 AI virus of experimental trail

Group

no.

Mortality Total** MDT/

Day*

Mortality

%

Protection

% 1st

day

2nd

day

3rd

day

4th

day

5th

day

6th

day

7th

day

1 1 1 1 3 4.3 30 70 %

2 1 1 1 2 5 4.8 50 50 %

3 1 1 4 10 90 %

4 1 1 1 6 4.3 60 40 %

5 1 2 1 1 5 5 50 50 %

6 1 2 4 7 3.4 70 30 %

7 1 3 1 1 6 4.3 85.8% 14.2 %

8 4 4 2 10 2.8 100% 0 %

9 - - - - - - - - - - -

•Challenge with HPAI H5N1 at 28 days of age.

• Birds kept under daily observation for 10 days.

*MDT : Mean death time.

** Total: Total mortality after 10 days post-challenge.

DISCUSSION


Avian influenza (AI) is a widely highly contagious disease of poultry. In Egypt,

in February 2006, sever outbreaks of HPAI, H5 NI; have emerged in several

governorates and were associated with drastic mortality up to 100 % infected

chickens 24 .

Egypt has been most severely affected by continuous outbreaks, resulting in

sever losses in the poultry industry. Efforts to control highly pathogenic H5NI

avian influenza virus in poultry have failed despite increased biosecurity,

quarantine and doses of oil emulsion H5N1 and H5N2 vaccines and the disease

became endemic in Egypt 23. The ongoing circulation of HP H5NI AI in Egypt has

caused >168 human infections and remains an unresolved threat to veterinary

and public health 25 .

Broiler production is the most important branch in the Egyptian poultry

industry. Annually, more than 600-700 million broilers are fattened within

approximately 5–6 weeks when more than 80% of them are traded as live bird

markets (LBMs) throughout the country 26. During trade to LBMs the risk of

exposure to endemically circulating HPAIV H5N1 is grossly increased. Marketing

and processing of HPAIV-infected broilers in LBMs would pose a high risk

interface of virus transmission to humans 27. Therefore, protection of broilers

when ready to trade is a highly desirable goal in the combat against HPAI in

Egypt. However, due to the short time life span of only 35–42 days of these birds

it is difficult to induce resilient specific immunity by (repeated) vaccination.

Furthermore, interference of vaccine efficacy in chicks hatching from H5-

vaccinated breeders by maternal derived antibodies has been claimed to further

jeopardize efforts to achieve protection of broiler chicks 28, 29, 30. Moreover,

another possible cause for the increased percent of infection in broiler chickens

is the presence of 21 commercial vaccines in Egypt during 2010 and 2011 leads to

subsequent selection of mutants escaping vaccine pressure. Interestingly, a

previous study estimated the emergence of antigenically distinct viruses in Egypt

as also occurring 18 months after the beginning of vaccination in poultry 31. In

China the emergence of vaccine escape variant viruses was reported one year

after the implantation of vaccination in poultry 32.

Regarding the vaccine evaluation, using homologous H5N2 antigens half-life

of maternally derived H5-specific antibodies in broiler chicks was approximately

4.4 log 2 at 1st day based on an initial mean HI titer of H5. This value was in

agreement with results obtained by 33 4.2 log 2 at 1st day and lower than that (5.2

log 2) reported by28.

Evaluation of the efficacy of the AI vaccines and vaccination program applied

in 5 broiler flocks under field condition was carried out. Serological monitoring of

immune response after 21 days post vaccination ( 30 days of age) indicated that

the GM titers (log 2) ranged from 5- 7.8 34,35. Mentioned that the protected titers

ranged from 4-6 log 2, this means that all the examined broiler flocks have a

protected titer. Simultaneously at 30 days of age 10 birds from each flock was

challenged using HPAI H5N1 virus. After 10 days of observation period the

protection percent ranged from 20 - 90% (Table 5).


434 October, 2014

Amazingly, the high protection percent (90%) was in a flock (No. 5) which had

a GM titers of 25. On the other hand flock No. 3 gave 40% protection while GM

titers was high 27.8 (Table5).

Under experimental condition evaluation of different AI vaccines included

recombinant and oil emulsion vaccine (H5N1 and H5N2) was studied. GM titers of

AI virus vaccines were determined weekly from 3rd to 5th weeks of age (2-3

weeks post vaccination). The data clearly indicated that after 3 weeks post

vaccination the recombinant vaccine of Merial® or recombinant vaccine plus oil

emulsion inactivated vaccine of Merial® gave the highest titers (27.8 - 27.1),

respectively (Table 6).

While, recombinant vaccine Volvac® or recombinant vaccine plus oil

emulsion inactivated vaccine gave low titers (23.2 - 24.2), respectively. On the

other hand, oil emulsion inactivated vaccine of Merial® administered alone gave

high titer 27.2 at the end of the 3rd week post vaccination. But, oil emulsion

inactivated vaccine in both groups gave a low titer 23.2, 22.8 (Table 6).

At 29 days of age (3 weeks post vaccination) the different experimental

groups were challenged with HPAIV H5N1 and kept under daily observation for

10 days. The high protection percent was recorded in the groups vaccinated with

recombinant vaccine of Volvac® and Merial® (90% and 70%), respectively. While,

the lowest protection percent was recorded in the group vaccinated with oil

emulsion inactivated vaccine of Merial® (14.3%) (Table 7).

In fact, the previous mentioned data clarify that the chicken groups ( 1,2 and

7) had the highest GM titers for AIV vaccines 27.2 , 27.1 and 27.5 respectively gave

only a protection of 70%, 50% and 14.3% respectively. While group 3 which had

only GM titer of 23.2 gave a protection of 90%. These data lead us to state that

there was no clear correlation between the GM titers and the protection rate.

This could be attributed to, under field conditions antigenic variability and

diversity of HPAI viruses circulating in the field, a given titer found against a

laboratory strain cannot unequivocally be interpreted as a protective against field

virus challenge. Also, serological monitoring of H5 vaccinated flocks by the HI test

using the homologous vaccine antigen is a routine laboratory procedure to

evaluate vaccination efficacy of poultry in Egypt and elsewhere [36].A correlation

between the level of HI antibody titer and protection against disease and virus

excretion was predicted by 34,35 to be at least 4.0 _ HI _ 6.0. However, results

obtained in this study showed that such relations are only valid if there is a close

antigenic match between the vaccine and the challenge viruses 37. Relying on the

homologous HI titer measured against the commercial vaccine H5N2 antigen may

result in an erroneous prediction of protection against currently circulating H5N1

viruses in Egypt. In countries endemically infected with divergent HPAI H5N1

viruses, antigens from the circulating field viruses must be used for HI assay-

based predictions. Using different H5N1 viruses circulating in China and

Vietnam38 found that the levels of cross reaction of HI antibody titer differed 8–32

fold between different strains of H5N1 even within the same clade.


Also, Variation in the protection percent of challenged birds either with low

or high GM titer could be explained as follow, vaccination of MDA chicks with the

same vaccine that has been used for breeder immunization was useless in terms

of inducing protection, a vaccine that was based on the antigenically highly

distinct HPAIV EGYvar/H5N1 strain (clade 2.2.1) did not suffer from interference

with MDA. HI antibodies of similar titers were induced in chicks independent of

their MDA status and their age. These chicks were clinically protected when

challenged with EGYvar/ H5N137.

Our findings support studies by 29, 38, 39, who mentioned that the antigenic

match between the vaccine strain and the circulating field virus is one of the most

decisive factors in determining the H5 vaccine efficacy to prevent replication and

transmission of H5N1 virus. However, previous studies stated that homology

between H5 vaccine strains and challenge viruses did not influence virus

excretion and/or protection 1, 40, 41. This means that the strain selection should be

based not only on genotype but also protectotype.

In fact, in the field and experimental trails the broiler chicks with MDA were

delivered from broiler breeder flocks vaccinated several times with inactivated oil

emulsion vaccine of AI virus, . Vaccination of MDA-positive chickens at a later age

(10 days) seems to be a valuable, although MDAs may still interfere with

vaccination to a lesser extent because they are present up to 3 weeks post

hatching. Therefore, in areas with high infection pressure, when possible, two

vaccinations are recommended for optimal protection 28 .The induction of

antibody titers by vaccination was severely inhibited by maternal antibodies, with

only a few chickens showing responses similar to the control chickens. It is

concluded that high maternal antibody titers are required for clinical protection

and reduction of virus titers after infection of chickens, whereas low antibody

titers already interfere with vaccine efficacy 30 Also, this idea in accordance with

field observations from Egypt in late 2006 and early 2007 where 295 out of 355

(83%) examined broiler flocks possessing MDA did not seroconvert upon

vaccination with vaccines similar to those received by the breeders [41]. This may

lead us to a state that it might be advisable to take into account day-old AI MDA

titers when one is determining the optimal age of vaccination, as the MDA make

clearance of the viral antigen within the vaccine thus the vaccine efficacy reduced.

Generally, recombinant vaccines of Merial® and Volvac® gave a protection

of 70% and 90% in group 1 and 3 respectively either with low or high GM titers

27.8, 23.2.

The chickens with high levels of maternal antibodies against NDV and AIV

may be well and fully protected when vaccinated with 106.8 and 107.8 CEID50 of

a live recombinant LaSota Newcastle disease virus (NDV) - Avian Influenza H5

vaccine (rNDV-LS/AI-H5) respectively. Lower doses (105.8 or less) of the same

vaccine conferred poor protection. There was no correlation of protection

induced by rNDV-LS/AI-H5 with NDV and AIV HI antibody levels 21 days after

vaccination. Even those groups that were fully protected by 107.8 CEID50 had HI

antibody titers as low as 20.7 against NDV and 22.4 against AIV. This may indicate


436 October, 2014

that the induced protection is from enhanced innate immunity or cell mediated

immunity 32.

Therefore, annual or biannual evaluation of the vaccine efficacy in the face

of antigenic drift due to the immune pressure exerted by the vaccine has been

suggested 9,27. For the foreseeable future routine update of vaccines and

diagnostics will remain the major challenge of all attempts to restrict circulation

of HPAIV H5N1 in poultry populations of endemically infected regions and to

decrease risks of human exposure.

In addition to, high tittered vaccines, more aggressive vaccine strains,

multiple immunization especially if high MDA in chicks, or a combination of all

three approaches all these are required for successive vaccination as supported

by idea of33.

Finally, we can concluded that continuous active and passive surveillance is

very necessary for monitoring virus evolution, manager of infected flock

appropriately, improve vaccination protocols and identify or exclude the

presence of other strain. Also, AI vaccines should be evaluated regularly to

ensure that they are still protective against the circulating virus strain.

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