Comparative Effectiveness of Two Oil AdjuvantInactivated Avian Influenza

H9N2 Vaccines
Author(s): Walid H. Kilany, Abdel-Hamid I. Bazid, Ahmed Ali, Ayman H. El-Deeb, Mohamed A. Zain
El-Abideen, Magdy El Sayed and Magdy F. El-Kady
Source: Avian Diseases, 60(1s):226-231.
Published By: American Association of Avian Pathologists
DOI: http://dx.doi.org/10.1637/11145-050815-Reg
URL: http://www.bioone.org/doi/full/10.1637/11145-050815-Reg

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AVIAN DISEASES 60:226231, 2016

Comparative Effectiveness of Two Oil AdjuvantInactivated Avian Influenza

H9N2 Vaccines
Walid H. Kilany,AF Abdel-Hamid I. Bazid,B Ahmed Ali,C Ayman H. El-Deeb,D Mohamed A. Zain El-Abideen,AE
Magdy El Sayed,E and Magdy F. El-KadyC
A
Reference Laboratory for Veterinary Quality Control on Poultry Production (RLQP), Animal Health Research Institute, P.O. Box, 264, Dokki,
Giza 12618, Egypt
B
Virology Department, Faculty of Veterinary Medicine, Sadat City University, Menoufia 32897, Egypt
C
Poultry Diseases Department, Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 65211, Egypt
D
Virology Department, Faculty of Veterinary Medicine, Cairo University, Giza 12511, Egypt
E
Middle East for Veterinary Vaccine Company, Second Industrial Area, El-Salhya El-Gededa, El-Sharqia 44671, Egypt
Received 13 May 2015; Accepted 5 February 2016; Published ahead of print 8 February 2016

SUMMARY. Low pathogenic avian influenza H9N2 virus infection has been an important risk to the Egyptian poultry industry
since 2011. Economic losses have occurred from early infection and co-infection with other pathogens. Therefore, H9N2
vaccination of broiler chicks as young as 7 days old was recommended. The current inactivated H9N2 vaccines (0.5 ml/bird)
administered at a reduced dose (0.25 ml/bird) do not guarantee the delivery of an effective dose for broilers. In this study, the
efficacy of the reduced-dose volume (0.3 ml/bird), compared with the regular vaccine dose (0.5 ml/bird) of inactivated H9N2
vaccines using two different commercially available adjuvants, was investigated. The vaccines were prepared from the local H9N2
virus (Ck/EG/114940v/NLQP/11) using the same antigen content: 300 hemagglutinating units. Postvaccination (PV) immune
response was monitored using the hemagglutination inhibition test. At 4 wk PV, both vaccinated groups were challenged using
the homologous H9N2 strain at a 50% egg infective dose (EID50) of 106 EID50/bird via the intranasal route. Clinical signs,
mortality, and virus shedding in oropharyngeal swabs were monitored at 2, 4, 6, and 10 days postchallenge (DPC). The reduced-
dose volume of vaccine induced a significantly faster and higher immune response than the regular volume of vaccine at 2 and 3
wk PV. No significant difference in virus shedding between the two vaccine formulas was found (P $ 0.05), and both vaccines
were able to stop virus shedding by 6 DPC. The reduced-dose volume of vaccine using a suitable oil adjuvant and proper antigen
content can be used effectively for early immunization of broiler chicks.

RESUMEN. Eficacia comparativa de dos vacunas contra la influenza aviar H9N2 inactivadas y emulsionadas en aceite.
La infeccin por el virus de la influenza aviar de baja patogenicidad H9N2 ha sido un riesgo importante para la industria avcola
egipcia desde el ao 2011. Las prdidas econmicas se han producido por esta infeccin y por la coinfeccin con otros patgenos.
Por lo tanto, se recomienda la vacunacin contra este virus H9N2 en pollos de engorde tan temprano como a los siete das de edad.
Las vacunas inactivadas actuales H9N2 (0.5 ml/ave) administradas con una dosis reducida (0.25 ml/ave) no garantizan la aplicacin
de una dosis eficaz para pollos de engorde. En este estudio se investig la eficacia del volumen reducido de la dosis (0.3 ml/ave), en
comparacin con la dosis de vacuna regular (0.5 ml/ave) de las vacunas inactivadas H9N2 utilizando dos adyuvantes diferentes
disponibles en el mercado. Las vacunas se prepararon a partir del virus H9N2 local (Pollo/EG/114940v/NLQP/11), utilizando el
mismo contenido de antgeno: 300 unidades hemaglutinantes. Despus de la vacunacin, la respuesta inmune se determin
mediante la prueba de inhibicin de la hemaglutinacin. A las cuatro semanas despus de la vacunacin, ambos grupos vacunados
fueron desafiados con la cepa H9N2 homloga con una dosis de 106 dosis infectantes para embrin de pollo (EID50) por ave,
a travs de la ruta intranasal. Los signos clnicos, la mortalidad y la diseminacin del virus mediante hisopos orofarngeos se
determinaron a los dos, cuatro, seis y diez das despus del desafo. La vacuna con volumen reducido de dosis indujo una respuesta
inmune significativamente ms rpida y ms alta que el volumen regular de la vacuna a las dos y tres semanas despus de la
vacunacin. No se encontraron diferencias significativas en la diseminacin del virus entre las dos frmulas de vacunas (P $ 0.05) y
ambas vacunas fueron capaces de detener la propagacin del virus por el da seis despus del desafo. Se puede utilizar eficazmente el
volumen de la dosis reducida de la vacuna para la inmunizacin temprana de pollos de engorde usando el adyuvante oleoso y el
contenido de antgeno adecuados.
Key words: AIV, LPAI H9N2, SPF chickens, inactivated vaccine
Abbreviations: AI 5 avian influenza; AIV 5 AI virus; DPC 5 days post challenge; EID50 5 50% egg infective dose;
G1 5 genotype 1; HA 5 hemagglutinin; HI 5 hemagglutination inhibition; HPAI 5 highly pathogenic avian influenza; IL 5 inter-
leukin; ISA70 5 Montanide ISA 70 VG adjuvant; ISA71 5 Montanide ISA 71 VG adjuvant; LPAI 5 low pathogenic avian influ-
enza; NA 5 neuraminidase; PV 5 postvaccination; RT-PCR 5 reverse transcriptase; PCR; SPF 5 specific pathogen free

Avian influenza virus (AIV) is one of the most devastating viral dis-
eases in the poultry industry and has a worldwide distribution. AIV
infects many species of domesticated and wild birds (23). AIV is an
enveloped virus that belongs to the Orthomyxoviridae family and has
an eight-segmented single-stranded negative sense RNA genome.
AIVs are categorized into three distinct types, A, B, and C, based on
F
Corresponding author. E-mail: walidhamdy78@yahoo.com
the internal proteinsprincipally nucleoproteins and the M1 matrix
protein. All AIVs are type A. There are two surface glycoproteins:
hemagglutinin (HA) and neuraminidase (NA). AIVs are classified
into subtypes according to the combination of 18 HA and 11 NA
molecules (27). AIVs are classified into two distinct pathogenic
groups: highly pathogenic avian influenza (HPAI) and low pathogenic
avian influenza (LPAI) (24). LPAI H9N2 viruses isolated from poultry
in Middle Eastern countries give rise, in these countries, to four

226
 Inactivated H9N2 vaccine dose reduction
distinct genetic groups: A, B, C, and D. All four groups belong to the
lineage (14).
The LPAI H9N2 virus was first reported in Egypt at a commercial
quail farm (10). The current endemic situation of both LPAI H9N2
and HPAI H5N1 viruses in commercial chicken farms in Egypt have
raised concern of the potential appearance of a reassortment virus and
increased human zoonotic risk (2,16). Further economic losses in the
poultry industry ensued when coinfection of LPAI H9N2 occurred
with other respiratory infections (9), causing increased severity and
high mortality rates in Egyptian broiler chickens.
In addition to biosecurity measures, the practice of vaccination is
an important tool in the prevention and control of avian influenza
infection in chickens. Several experimental studies have demonstrated
that inactivated avian influenza (AI) vaccines are capable of inducing
antibody response, which aids in the protection of the infected birds
(5,6). Because of the widespread nature of the LPAI H9N2 viruses
and their zoonotic potential, vaccination of susceptible birds with
inactivated oil emulsion vaccine was employed to control the disease
(19). Despite improper practices during the administration of the
H9N2 vaccine in the small broiler poultry sector (1), and despite pos-
sible coinfection with other poultry viruses (e.g., infectious bronchitis
virus) (9), only minor genetic changes were observed as detected by
HA gene analysis of recently isolated viruses (9,17).
Limited literature is available on the efficacy of H9N2 inactivated
oil emulsion commercial vaccines being used in Egypt. Full-dose AI
H9N2 vaccination in broiler chicks was recommended to be adminis-
tered as early as 7 days of agewith a booster doseto obtain high
and homogeneous antibody titers (3). Yet, the use of the full dose of
0.5 ml/bird is not applicable to young broiler chicks; on the other
hand, the reduction of dose volume without considering the antigen
load may hinder obtaining the required protection. The suggested
alternative is to reduce the vaccine dose volume while maintaining
antigen dose and using efficacious immune stimulant vaccine adju-
vants. The MontanideTM (SEPPIC, Paris, France) adjuvants have
been widely used in veterinary applications. Montanide ISA 70 and
ISA 71 VG are mineral oilbased adjuvants that have been proven
to improve cellular response, with only minimal side effects associated
with other mineral oil emulsions (8). Studies on immunization with
low doses of Eimeria profilin plus the ISA 70 VG or ISA 71 VG sub-
unit vaccine showed that Eimeria profilin plus ISA 71 VG reduced
fecal oocyst shedding and enhanced serum antibody levels and gene
transcripts encoding interleukin (IL)-2, IL-10, IL-17A, and interfer-
on- in intestinal intraepithelial lymphocytes. Such studies also
showed increased infiltration of lymphocytes, especially CD8+ lym-
phocytes, at the site of immunization in chickens (12).
The present work was conducted to study the efficacy of dose vol-
ume reduction (from 0.5 to 0.3 ml/dose) of inactivated H9N2
vaccines prepared from the A/CK/Eg/114940v/NLQP/2011(H9N2)
virus, utilizing the Montanide ISA 71 adjuvants, and compare this
with the alternate utilization of Montanide ISA 70 adjuvants. Com-
parisons were made in terms of immunity and reduction of viral
replication and shedding in broiler chickens challenged with
LPAI H9N2.
MATERIALS AND METHODS
Virus. The virus LPAI H9N2 virus [A/CK/Eg/114940v/NLQP/2011
(H9N2)] (GenBank Q440373) was isolated from a broiler flock, in
Menofia governorate, Egypt, in 2011 at the Reference Laboratory for Vet-
erinary Quality Control on Poultry Production, Animal Health Research
Institute, Dokki, Giza, Egypt. This virus was selected to be representative
of circulating LPAI virus (H9N2) in Egypt. The selected virus was propa-
gated using specific-pathogen-free (SPF) embryonated chicken eggs (Nile
227
SPF eggs; Koom Oshiem, Fayoum, Egypt) and was confirmed to be free
of other biological contaminants, including avian influenza H5N1, avian
leukosis subgroup J, infectious bursal disease virus, avian reovirus, infec-
tious bronchitis virus, Newcastle disease virus, chicken infectious anemia,
avian encephalomyelitis virus, adenoviruses, egg drop syndrome (EDS76)
virus, avian mycoplasma, and Salmonella spp.
Reverse transcriptase PCR and HA gene sequencing. The viral RNA
was extracted from harvested allantoic fluids, by the BiofluxH viral RNA Mini
Spin column kit (BioFlux, Hangzhou China), following the manufacturers
instructions. Reverse transcriptase (RT)-PCR was used for HA gene amplifi-
cation using specific oligonucleotide primers (22). The final reaction volume
was 25 ml; including a 7-ml RNA template; 5 ml of 56 RT-PCR enhancer;
2.5 ml of 106 RT-PCR buffer; 1.5 ml of each forward and reverse primers;
1.25 ml of hot master Taq polymerase; 1 ml of Super pure dNTPs; 0.25 ml
of RNasin; 0.25 ml of Quant RTase; and 4.75 ml of RNase-free water. Ther-
mocycling RT-PCR conditions were 50 for 15 min, then 95 for 15 min,
followed by 35 cycles at 95 for 15 sec, then another 30 sec at 47 and 1
min at 65 . Final extension was performed at 65 for 10 min. Amplified
RT-PCR products were purified using the PCR purification KitH (Thermo
Scientific, Waltham, MA), following the manufacturers instructions and
were then sent for sequencing by Macrogen Inc. (the Republic of Korea).
A BLAST search was conducted (http://www.ncbi.nlm.nih.gov/BLAST).
Sequence comparisons and phylogenetic relationships through a bootstrap
of 1000 trials were determined with the MEGA version 6 program using
the Clustal W alignment algorithm (25). Additionally, nucleotide and amino
acid identities were determined using GeneiousH 7.1.3 (Biomatters Ltd.,
New Zealand).
Virus inactivation and vaccine formulation. The LPAI H9N2 virus
harvest inactivation was conducted using 0.2% formalin at 37 C for 24 hr
(Sigma, St. Louis, MO). Two different oil emulsioninactivated vaccines
were used in this experiment. The first was designated Vaccine A, contain-
ing the local (H9N2) isolate at a concentration of 300 hemagglutinating
units per dose and blended with Montanide ISA 70 VG adjuvant
(ISA70) at a dose of 0.5 ml/bird. The second vaccine was designated Vac-
cine B, containing the same antigen load but blended with Montanide
ISA 71 VG adjuvant (ISA71) at a dose of 0.3 ml/bird. The antigen aque-
ous phase was pre-emulsified with either ISA70 or ISA71 at a 30:70 ratio
(w/w) at 1000 rpm using the Silverson L5M high shear laboratory mixer
(Silverson Machines Inc., Buckinghamshire, UK). The speed of the mixer
was then increased to 3000 rpm and maintained for 30 min for the emul-
sification of 10,000 doses of the vaccine. During the emulsion formula-
tion, the temperature of the mixture was maintained between 18 and 22
C. The three different vaccine batches were inactivated H9N2 ISA70 vac-
cine batches (A1, A2, and A3) and inactivated H9N2 ISA71 vaccine
batches (B1, B2, and B3).
Birds and experimental design. Seventy SPF chickens 2 wk of age
were randomly divided into seven groups, including six experimental
and one control group (10 chicks/group; Table 1). Each group was reared
separately in chicken isolators at the Laboratory Animal Research Unit of
ME VACH Company (El-Sharqia, Egypt). Feed and water were available
ad libitum. At 2 wk of age, birds in Groups A1A3 were vaccinated with
the H9N2 AI vaccine A (0.5 ml/bird) via the intramuscular route in the
thigh muscle. The birds in Groups B1B3 were vaccinated with H9N2
vaccine B by the same route, but at a dose of 0.3 ml/bird. Finally, the
unvaccinated birds (control Group 7) were inoculated with an equal vol-
ume of placebo vaccine (normal saline). Sera were collected on a weekly
basis, for 4 wk postvaccination (PV) and at 14 days postchallenge (DPC).
Hemagglutination inhibition test. The hemagglutination inhibition
(HI) test was carried out according to the World Organization for Animal
Health Manual (20) to monitor the postvaccination humoral immune
response for each vaccine batch using the homologous HA antigen. Brief-
ly, twofold serial dilutions of sera were mixed with four hemagglutination
(HA) units of H9N2 AI antigen. The HI titer was determined using a
1% red blood cell suspension, which was collected from at least three
SPF chickens (8 wk of age) reared in our lab animal facility.
Challenge experiment. The challenge experiment was conducted in
Biosafety Level 3 chicken isolators, at ME VAC and according to the
ME VAC guidelines on animal research ethics. Because of the limited
228 W. H. Kilany et al.

Table 1. Postvaccination HI mean antibody titers and seroconversion rates in SPF chickens immunized with different formulas of inactivated A/
H9N2 vaccines against the homologous A/CK/Eg/114940v/NLQP/11(H9N2) antigen.
PostvaccinationB Postchallenge
14 days
Week 1 Week 2 Week 3 Week 4 postchallenge
Vaccine typeA Batch no. Mean % Sero. Mean % Sero. Mean % Sero. Mean % Sero. Mean % Sero.
A 1 1.7 0.9 a 20 4.3 0.9 a 100 8 0.7 a 100 9.5 0.5 a 80 10 0.4 100
2 1.7 0.9 a 20 4.4 0.7 a 100 7.7 0.5 a 100 9.6 0.5 a 100
3 1.6 1.1 a 20 3.9 0.6 a 100 7.6 0.7 a 100 9.5 0.7 a 100
B 1 1.6 1.1 a 20 5.2 0.8 b 100 9.1 0.7 b 100 9.8 0.4 a 100 10 0 100
2 1.5 1.4 a 30 4.9 1.1 b 100 8.4 0.5 b 100 9.8 0.4 a 100
3 1.5 1.2 a 20 4.8 0.8 b 100 8.3 0.7 b 100 9.5 0.5 a 100
A
Vaccine A: inactivated H9N2 ISA70 vaccine batches (A1, A2, and A3) 0.5 ml/dose; vaccine B: inactivated H9N2 ISA71 vaccine batches (B1, B2,
and B3) 0.3 ml/dose.
B
Mean HI antibody titers are log2 SD; % Sero. 5 seroconversion rate 5 (no. of birds in a group with HI titers $ 23/no. of birds in a group) 6
100. Titers in the same column followed by different lowercase letters are statistically different (P , 0.05).

number of Level 3 isolators, the challenge experiment was conducted for
only two vaccinated groups (A1, B1) and one nonvaccinated group (C)
using the homologous LPAI H9N2 virus, at 4 wk PV. The challenged
birds received a 10-ml dose at 106 50% egg infective dose (EID50) via
the intranasal route. At 2, 4, 6, and 10 DPC, oropharyngeal swabs
were collected from each challenged bird separately for virus detection
and titration.
Viral isolation and detection. Oropharyngeal swabs were collected in
a 1-ml phosphate-buffered saline, pH 7.2, containing gentamycin (50 g/
ml) and nystatin (1000 U/ml) (20). Swab samples were vortexed and then
centrifuged at 2000 rpm for 10 min at 4 C to pellet the debris. The
supernatants of individual samples were tested via virus titration to moni-
tor virus shedding in 10-day SPF embryonated chicken eggs. Infective
dose (EID50/ml) was then calculated (21).
Statistical analysis. Statistical analysis was performed using SPSS ver-
sion 13. The nonparametric Kruskal-Wallis test was used for the compar-
ison of vaccinated and unvaccinated groups. Differences were considered
significant at P , 0.01. The Mann-Whitney U-test was also used for the
comparison between groups. A P , 0.05 was considered statistically
significant.
RESULTS
Phylogenetic and sequence analysis of the AIV strain. The phyloge-
netic tree and sequence analysis of the partial sequence of the HA
gene showed that the strain Ck/EG/114940v/NLQP/11(H9N2)
has genetic traits similar to those of the currently circulating viruses
belonging to the G1-like lineages circulating in Egypt (Fig. 1). Minor
evolution was observed, with no significant differences in the HA
gene. The nucleotide identity showed that the virus was 99.2% simi-
lar to the recent Egyptian H9N2 strains.
Postvaccination humoral immune response. In general, there were
no significant differences (P $ 0.05) between the postvaccination
immune responses in the three different batches of each vaccine.
The vaccinated birds showed detectable HI antibody titers by week
2 PV in all groups of both vaccine batches ($4 log2). Vaccine B
showed faster and higher immune response than Vaccine A batches
by weeks 2 and 3 PV (P # 0.05). However, this significant difference
disappeared by week 4 PV (Table 1).
Challenge experiment in SPF chickens. To evaluate the protective
efficiency of both vaccine groups, A1 and B1 vaccinated SPF chickens
were challenged with the same LPAI H9N2 at week 4 PV. The mean
log2 HI titers of the A1 and B1 groups at age of challenge were 9.5
0.4 and 9.8 0.5 log2, respectively (Table 1). Neither vaccinated
challenged groups showed clear clinical signs, whereas the
nonvaccinated group showed mild respiratory signs and depression.
The virus shedding titers in both vaccinated groups at 2 and 4
DPC were significantly lower than the nonvaccinated challenge group
by at least 23 log EID50. Both vaccines were able to stop virus shed-
ding by 6 DPC. The challenge virus was detected in 30% and 10% of
challenged birds in Group A (vaccinated with ISA70) at 2 and 4
DPC, respectively; whereas 20% and 10% of challenged birds in
Group B (vaccinated with ISA71) showed detectable virus shedding
at 2 and 4 DPC, respectively (Table 2).
DISCUSSION
AI is one of the most devastating viral diseases in the poultry indus-
try and has a worldwide distribution (23). Since the first H9N2 LPAI
outbreak in Egypt (10), several outbreaks have occurred. This has
caused significant health problems and great economic losses. In
Egypt, the main adopted AI control measure is vaccination against
both A/H5N1 and A/H9N2. The endemic situation of both viruses,
as well as other poultry viruses, has complicated the current vaccina-
tion programs in all poultry sectors. Several experimental studies have
demonstrated that inactivated water in oil emulsion vaccines are capa-
ble of inducing antibody response to protect poultry (5,19). Howev-
er, the need for potent vaccines with full antigen dose that could be
used during an early stage of life has increased (3). To reduce dose
volume, a potent adjuvant to enhance immunogenicity of the antigen
was deemed important (4). A perfect oil adjuvant should enhance the
immune response and reduce the number of immunizations required.
The Montanide ISA71 adjuvants have been shown to stimulate both
humoral and cell-mediated immune response in different animal spe-
cies, including chickens (12,13). In this study, the efficacy of a
reduced dose volume of vaccine (from 0.5 to 0.3 ml) containing inac-
tivated H9N2 vaccines and formulated with Montanide ISA71 was
compared with a regular dose of vaccine (i.e., 0.5 ml/bird) formulated
with Montanide ISA70 adjuvant.
The phylogenetic analysis of the HA gene sequence showed that
the selected LPAI H9N2 virus strain had genetic and antigenic traits
similar to those of the field viruses currently circulating in Egypt (Fig.
1), with a 99.2% nucleotide identity. This high genetic similarity
with the recent Egyptian H9N2 LPAI and further vaccine efficacy
tests conducted on SPF chickens indicated that the virus can be
used as an effective vaccine, with no need for modification.
The two formulated H9N2 vaccines using Montanide ISA70 and
ISA71 developed a protective immune response in vaccinated SPF
birds by week 2 PV using a single-dose regime. Previous studies
 Inactivated H9N2 vaccine dose reduction 229

Fig. 1. Phylogenetic evolutionary analyses of A/H9N2 viruses (conducted through a bootstrap trial of 1000 using MEGA version 6).

have shown that AI vaccine emulsified with Montanide ISA70 oil
adjuvant provided good protection to immunized chickens against
H9N2 infection (7).
In this study, the antibody levels elicited by reduced ISA71 (0.3 ml)
vaccine were faster and higher than with a regular dose (0.5 ml) of
ISA70 vaccine at as early as weeks 2 and 3 PV (P # 0.05). These results
indicate that the dose volume reduction adds to the benefits of early
immunization against infection. This might be attributable to the
reduction of oil volume used, having the effect of accelerating the anti-
gen absorption rate. On the other hand, the stimulatory effect of the
oil adjuvant used cannot be ignored. Montanide ISA71 has been
shown to enhance both serum antibody levels and cell-mediated
immunity in chickens vaccinated with low doses of Eimeria profilin
subunit vaccines (12).
Although the pathogenicity of H9N2 was previously reported (18),
the role of secondary agents in the pathogenesis of AIV H9N2,
including environmental and other pathogens, cannot be ignored
(11,19). Because of the low pathogenic nature of H9N2 in the
absence of complicating agents, quantitation of viral replication and
shedding in infected birds were used for the evaluation of both vac-
cines in this study. It has been documented that an effective vaccine
not only prevents clinical disease, but it also reduces viral shedding
compared with unvaccinated control birds, thereby decreasing the
likelihood of transmission of the virus to new susceptible hosts (24).
230 W. H. Kilany et al.

Table 2. Mean titer of virus shedding and number of sheddersB of different vaccine formulas and nonvaccinated SPF chickens challenged with A/
A

CK/Eg/114940v/NLQP/11(H9N2).
Vaccine AC Vaccine BD Unvaccinated challengedE
DPC Titer (mean SD) No. shedders (pos/total) Titer (mean SD) No. shedders (pos/total) Titer (mean SD) No. shedders (pos/total)
2 2.5 0.7 3/10 2.4 0.5 2/10 4.4 0.5 10/10
4 1.5 1/10 1.9 1/10 4.4 1.1 7/10
6 0 0/10 0 0/10 3.6 0.5 3/10
10 0 0/10 0 0/10 0 0/10
A
Titers expressed as EID50/ml SD.
B
No. of positive (pos) samples/total positive tested samples; shedding rate 5 number of shedder birds/total tested birds in the group.
C
Vaccine A: inactivated H9N2 ISA70 vaccine (0.5 ml/dose) challenged with A/CK/Eg/114940v/NLQP/11(H9N2) virus at 6 log EID50/bird.
D
Vaccine B: inactivated H9N2 ISA71 vaccine (0.3 ml/dose) challenged with A/CK/Eg/114940v/NLQP/11(H9N2) virus at 6 log EID50/bird.
E
Unvaccinated challenged group challenged with A/CK/Eg/114940v/NLQP/11(H9N2) virus at 6 log EID50/bird.

The results of virus isolation and viral shedding in both vaccines indi-
cated that the vaccine-challenged chickens were protected against viral
shedding. There was no significant difference in the virus shedding
titers. The percentage of shedder birds in the ISA71 vaccinated group
however was lower than that in the ISA70 group, although not statisti-
cally significant. The ability of inactivated oil emulsion vaccines to pro-
tect chickens against AI clinical disease, both in experimental (7) and
field conditions (26), has previously been documented. The endemic
situation of H9N2 virus in Egypt could be due to the shortage of vacci-
nation coverage in different poultry sectors (15). Not only is an effective
mass vaccination strategy required for proper AI control in endemic
countries, but focus also should be put on using potent vaccines that
are capable of inducing an early postvaccination immune response.
To conclude, although both vaccine formulas (ISA70 and ISA71)
were potent and lower virus shedding was observed, the dose-reduced
inactivated vaccine with a potent adjuvant showed good efficacy, as
observed by earlier and higher immune responsesas early as 2 wk
PV. Thus it may be stated that the dose-reduced vaccine could be
used effectively to achieve earlier protection, especially in broiler
chicks in endemic countries.
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