Original Article
Studying the efficacy of some commercial inactivated
avian influenza H5 vaccines against the currently circulating highly pathogenic
H5N8 virus in broiler chickens in Egypt
Estudio de la eficacia en pollos de engorde, de
vacunas comerciales inactivadas contra el virus de la influenza aviar de alta
patogenicidad H5N8 que circula actualmente en Egipto
Hussein AM1*
ORCID: https://orcid.org/0009-0001-8290-1317
El-Bagoury GF2 ORCID: https://orcid.org/0000-0002-2204-064X
El-Habbaa AS2 ORCID: https://orcid.org/0000-0003-3514-3573
Samir A Nassif3 ORCID: https://orcid.org/0000-0002-7907-0102
1 Armed Forces Laboratories for Medical Research and Blood Bank. Cairo, Egypt.
2 Department
of Virology, Faculty of Veterinary Medicine, Benha
university. Egypt.
3 Central
Laboratory for Evaluation of Veterinary Biologics (CLEVB), Agricultural
Research Center (ARC). Cairo, Egypt.
Corresponding author: dr_ah_hussien@yahoo.com
ABSTRACT
The avian influenza H5N8
virus is a highly pathogenic virus that can cause severe economic losses in
poultry species worldwide. In Egypt, highly pathogenic avian influenza virus of
subtype H5N8 clade 2.3.4.4b was reported in 2016, and despite vaccination
efforts, the virus has become endemic. The current study aims to evaluate the
efficacy of some commercial inactivated H5 vaccines from a different lineage
against the challenge with HPAI H5N8 virus in broiler chickens, in Egypt.
Groups of broilers vaccinated with different inactivated H5 vaccines and
unvaccinated controls were challenged with HPAI H5N8 virus. Antibody titer was calculated at the first 3 weeks after vaccination and the viral shedding titer was calculated at 3-, 5-, 7- and 10-days
post challenge.
Mortality rates
were monitored
daily during the first 10 days after the challenge to provide an estimate of
the protection level. According to our findings, following a challenge with the
HPAI H5N8 clade 2.3.4.4b virus, the inactivated vaccines offered varying
degrees of efficacy, HI titer and reduction of virus shedding titer, which may
be related to variations in the nucleotide sequence
identity percentages of the HA genes between the challenge virus and the
vaccine seeds. These findings emphasize the need for continual updating
of the H5 vaccines applied in Egypt to keep up with the continual mutations
that discovered in H5Nx viruses.
Keywords: avian
influenza; immune response;
quantitative Real Time PCR; vaccines.
RESUMEN
El virus de la gripe aviar
H5N8 es un virus altamente patógeno que puede causar graves pérdidas económicas
en especies avícolas de todo el mundo. En Egipto, el virus de la gripe aviar
altamente patógena del subtipo H5N8 clado 2.3.4.4b se
notificó en 2016 y, a pesar de los esfuerzos de vacunación, el virus se ha
vuelto endémico. El presente estudio tiene como objetivo evaluar la eficacia de
algunas vacunas comerciales inactivadas H5 de linaje diferente, contra el virus
de la influenza aviar de alta patogenicidad H5N8 en pollos de engorde, en
Egipto. Grupos de pollos de engorde vacunados con diferentes vacunas H5
inactivadas y controles no vacunados fueron retados con el virus de la
influenza aviar de alta patogenicidad H5N8. Se calculó el título de anticuerpos
a las 3 semanas de la vacunación y el título de excreción viral a los 3, 5, 7 y
10 días de la prueba. Las tasas de mortalidad se controlaron diariamente
durante los 10 días siguientes al reto para obtener una estimación del nivel de
protección. Según nuestros hallazgos, tras el reto con el virus de la influenza
aviar de alta patogenicidad H5N8 del clado 2.3.4.4b,
las vacunas inactivadas ofrecieron diferentes grados de eficacia, título de
inhibición de la hemaglutinación y reducción del título de excreción del virus,
lo que puede estar relacionado con las variaciones en los porcentajes de
identidad de la secuencia de nucleótidos de los genes HA entre el virus del
reto y las semillas de la vacuna. Estos hallazgos enfatizan la necesidad de una
actualización continua de las vacunas H5 aplicadas en Egipto para mantenerse al
día con las continuas mutaciones que se descubren en los virus H5Nx.
Palabras clave: influenza aviar; respuesta inmune; Reacción en Cadena en Tiempo Real de la Polimerasa; vacunas.
Recibido: 15 de julio de 2024
Aceptado: 14 de octubre de 2024
Introduction
Highly
pathogenic avian influenza (HPAI) H5N8 can infect wild and domestic birds. It
has a hemagglutinin (HA) segment of H5 clade 2.3.4.4, which emerged in 2010 in
China and other seven segments from multiple avian influenza viruses (AIV).
Outbreaks of the HPAI subtype H5N8 virus of clade 2.3.4.4 were firstly reported
in poultry farms and wetlands in January 2014 in South Korea.(1)
Then, the virus was discovered in Siberia, Beringia, and Japan by the
summer of 2014. By the end of 2014, the migration of waterfowl played a major
factor in the spread of highly pathogenic avian influenza virus (HPAIV) H5N8
throughout East Asia, North America, Africa and Europe.(2) This
virus has undergone several genetic changes and reassortments, resulting in
different subtypes and lineages.(3) The most recent wave of
H5N8 AIV outbreaks was caused by a novel reassortant
virus that originated in Russia and Kazakhstan and disseminated to Europe and
Asia.(4) In Egypt, The H5N8 AIV strain was first detected in late
2016 in a migratory bird (common coot).(4) Phylogenetically, the
original virus was closely linked to other H5N8 AIV of
clade 2.3.4.4b that were found in Russia in 2016.(5) Since then,
the H5N8 virus has spread rapidly among different poultry sectors in Egypt,
with multiple introduction events and genetic diversity observed among the
circulating viruses.(6) Six HPAI H5N8 genotypes were identified in
Egypt based on the genetic diversity of the internal gene segments. Among them,
the most common genotypes found in Egyptian poultry farms in 2019-2021 are G5
and G6.(7) According
to epidemiologic data, the Egyptian H5N1 virus (clade 2.2.1.2) has been
replaced by the HPAI H5N8 virus (clade 2.3.4.4b), which is now the most often
identified H5 subtype in Egyptian poultry sectors.(8)
Vaccines are one of the strategies to prevent and control the spread of
this virus among poultry and potentially humans.(9) But the
evaluation of H5 vaccines against H5N8 worldwide is a challenging task, as the
H5N8 virus is constantly evolving and diversifying into different subtypes and
lineages.(9) Egypt uses commercial AIV vaccines to combat H5
infections, but genetic differences between the recently mutated strains and
previously identified Egyptian HPAIV (H5N8) isolated in 2017 have led to
ongoing deaths in flocks that received vaccinations from H5N8 infection in
various governorates.(5,10) In the same context, a study(9)
mentioned that only
two commercial vaccines (Re-5 and Re-6) and an experimental vaccine based on
the Egyptian H5N8 strain showed complete protection and reduced virus shedding
in chickens; other six commercial vaccines failed to prevent virus shedding and
some of them did not protect chickens from mortality. Moreover, another study(11) evaluated a new bivalent
vaccine (Valley Vac H5Plus NDVg7) that contained the Egyptian H5N8 strain and a
Newcastle disease virus (NDV) strain and they found that this vaccine was more
effective than the commercial H5+ND7 vaccine in protecting chickens from both
H5N8 and NDV infection.
Based
on these findings, the most widely used commercial AIV vaccines need to be
regularly assessed against newly emerging H5N8 AIV with variable pathogenicity
and AIV clinical presentations in order to design the most effective
vaccination approach. Consequently, the present study was performed to study
the efficacy of some commercial inactivated H5 vaccines from a different
lineage against the challenge with HPAI H5N8 virus in broiler chickens, in
Egypt.
Materials and
Methods
Ethical
approval
The study was conducted
following the guidelines of the Animal Welfare Committee and protocols were
approved by the Research Ethics Committee, Faculty of Veterinary Medicine at Benha University, (Approval number BUFVTM 06-01-23).
Challenge
virus and antigen
The clade 2.3.4.4b HPAI
H5N8 challenge virus strain (A/chicken/Egypt/ v1526/2020) with accession no.
MW600499 was obtained from the National Laboratory for Veterinary Quality
Control on Poultry Production, Animal Health Research Institute, Giza, Egypt. This
virus was propagated in specified pathogen free (SPF) embryonated chicken eggs
and the allantoic fluid was harvested. The virus was
titrated in SPF embryonated chicken eggs after its purity was verified. To
create the viral challenge inoculum, it was diluted in PBS until reached the
desired final concentration of 107 median egg infectious dose (EID50)/mL.
The virus used in this study became inactive to be used as hemagglutination
inhibition (HI) antigen after treating it with 0.05% b-propiolactone for 2 h at
37.
Reassortant AIV vaccine (Re 13 & Re 14 strains): oil adjuvant commercial
inactivated reassortant avian influenza vaccine
prepared from H5N6 subtype, Re-13 strain (A/duck/Fujian/S1424/2020 clade
2.3.4.4h) and H5N8 subtype, Re-14 strain (A/whooper swan/Shanxi/4-1/2020), with
manufacture date: 1/2022 and expiry date: 1/2024.
Reassortment AIV vaccine (Re-5 strain): oil adjuvant inactivated reassortant avian influenza vaccine prepared from H5N1
subtype, Re-5 strain (A/duck/Anhui/1/2006 clade 2.3.4), with manufacture date:
1/2022 and expiry date: 1/2024.
Reassortment AIV vaccine
(Re-6 &Re-8 strains): oil adjuvant inactivated reassortant
avian influenza vaccine prepared from H5N1 subtype, Re-6 strain
(A/duck/Guangdong/s1322/10 Clade 2.3.2.1) and H5N1 subtype, Re-8 strain
(A/Chicken/Guizhou/4/13 clade 2.3.4.4g), with manufacture date: 3/2022 and
expiry date: 3/2024.
Nobilis influenza H5N2 vaccine: oil adjuvant inactivated reassortant avian influenza vaccine prepared from H5N2 subtype, LP strain (A/duck/Potsdam/1402-6/1986), with manufacture date:
3/2022 and expiry date: 3/2024.
MEFLUVAC vaccine: oil adjuvant reassortant avian influenza vaccine prepared from three strains H5N1 clade 2.2.1.1 (2016), H5N1 clade
2.2.1.2 (2017) and H5N8 clade 2.3.4.4b (2018), with manufacture date:
3/2022 and expiry date: 3/2024.
Genetic relatedness between the challenge
virus and vaccine seed strains
Avian
influenza HA gene nucleotide sequences of the challenge virus and the strains
used in each vaccine were obtained from the National Center for Biotechnology
Information database (http://www.ncbi.nlm.nih.gov). Next, these sequences were
aligned using (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
Broiler
chickens
A
total of 210 day-old (DO) commercial broiler chicks of the Ross breed were
kindly provided by El Wadi Company for poultry production. Chicks were housed
inside BSL3 chicken isolators during the whole experiment period and were
provided drinking water and feed ad libitum.
Vaccination
protocol
Two hundred and ten commercial broiler day old (DO) chicks were
allocated into seven groups (from Gp1 to Gp7) of 30 chickens each. At 10th
day of age, the chickens in Gp1, Gp2, Gp3, Gp4 and Gp5 were vaccinated with
inactivated reassortant avian influenza (Re-13 &
Re-14 strains) vaccine, inactivated reassortant avian
influenza (Re-5 strain) vaccine, inactivated reassortment avian influenza (Re-6
& Re-8 strains) vaccine, Nobilis avian influenza (H5N2) vaccine and
MEFLUVAC avian influenza vaccine, respectively. All vaccinations were performed
by subcutaneous injection route at the base of the neck (0.5 mL/ chicken). The
chickens in Gp6 served as challenged non-vaccinated control group (control
positive group) and the chickens in Gp7 were considered as non-vaccinated,
non-challenged group (control negative group).
Challenge
protocol
At
31st day of age, challenge test was conducted on 20 chickens from
each vaccinated group (Gp1, Gp2, Gp3, Gp4 and Gp5), and in the Gp6 group using
the HPAI H5N8 virus (A/chicken/Egypt/v1526/2020). Each challenged chicken was
inoculated intranasally (IN) with 100 µL containing 106 egg
infective dose at 50% (EID50)/chicken, equivalent to 100 chicken
lethal dose at 50% (CLD50). All chickens were subjected to daily
observation and monitoring for 10 days post challenge (PC) in order to report
clinical signs and also to record mortality and detection of virus shedding
titer.
Clinical
data
Daily
observation of all experimental groups was carried out to report any clinical
signs or record any mortality throughout the experimental period (41 days).
Evaluation
of potency of avian influenza vaccines
The potency of
AIV vaccines was evaluated by testing their ability to induce seroconversion in
experimentally inoculated chicks.
Ten individual serum samples corresponding to 10 blood samples during the immunization
phase (at 1st, 10th,
17th, 24st and 31st DO) were collected from
each group (Gps1-5), as well as the control negative group (Gp7). The waning up
of the maternally derived antibodies was examined in serum samples from Gp7. Hemagglutination inhibition (HI) tests were conducted
using a heterologous inactivated HPAIV A/chicken/Egypt/v1526/2020 (H5N8)
antigen to detect H5 clade 2.3.4.4b specific antibodies. These were expected to
be induced by post-vaccination responses against inactivated vaccines. The
antigen was adjusted to 4 hemagglutinating units according to international standards.(12) Arithmetic means of HI
titers were expressed as reciprocal log2 and HI at a dilution
≥ 24 was considered for AIV-positive specific antibodies
The
seroconversion (seropositivity) rate was estimated as the proportion of
chickens with positive HI titers (≤24)
and was calculated using the following formula:
Evaluation
of viral load by quantitative real-time reverse transcriptase PCR
The shedding of
challenge virus from chickens from vaccinated challenged and non-vaccinated
challenged groups was monitored by quantitative
real-time reverse transcriptase PCR (qRT-PCR) to assess the effect of
vaccination on respiratory shedding of the virus.
The specimens
comprised 10 individual oropharyngeal swabs from each challenged group
collected onto dry swabs. Sampling was done at 3th, 5th,
7th and 10th days PC. The swabs were eluted by vortexing in 1 mL of PBS + 0.1% of an antibiotics stock
solution (penicillin, 100,000 units; streptomycin, 100 mg/mL) and kept frozen
at -70 until use. RNA
was extracted and detected by qRT-PCR using a primer
set and probe specific for the influenza matrix gene.(13) The
estimated viral shedding concentration in the specimens was extrapolated from
the values using a standard curve which was
constructed from 10-fold serial dilutions of the challenge material, similarly
to what has been done by others.(14) Results were expressed as log10
number of copies/PCR reaction.
The reduction
in virus shedding titers from respiratory tract should be at a minimum of 2 log10
(100-fold) less in vaccinated challenged chicken group compared to
non-vaccinated challenged group;(15) which is considered as a
minimum requirement for vaccine efficacy.
Mean shedding
titer = sum of shedding titer/number of shedders birds (10 from each
group).
Data
management and analysis
The
collected data were revised, coded, tabulated and introduced to a personal
computer using Statistical package for Social Science (SPSS 27). Data were
presented and suitable analysis was done according to the type of data obtained
for each parameter. The descriptive statistics included mean, standard
deviation (± SD) and range for parametric numerical data, while median and
interquartile range (IQR) for non-parametric numerical data. Analytical
statistics included ANOVA test (used to assess the statistical significance of
the difference between more than two study group means), the Kruskal-Wallis
test (used to assess the statistical significance of the difference between
more than two study group ordinal variables), repeated measure ANOVA test
(assess the statistical significance of the difference between means measured
more than two times for the same study group) and Post Hoc Test (used for comparisons of all possible pairs
of group means). P- value means level of significance: P>0.05,
non-significant (NS) and P< 0.05, significant (S).
Results
Genetic
relatedness between the challenge virus and vaccine seed strains
Nucleotide sequence
identity percentage of the HA genes between the challenge virus and the vaccine
seeds revealed
that Re-14 strain and EGY2018/H5N8 strain showed the highest identity
percentage with 97.83% and 96.84%, respectively; while, the other vaccines
strains (Re-5 strain, Re-8 strain, Re-6 strain, Re-13 strain, EGY2016/H5N1
strain, EGY2017/H5N1 strain, and Potsdam/H5N2) showed identity percentages
92.13%, 91.01%, 90.51%, 90.49%, 87.87%, 87.65%, and 86.60% with the challenge
virus, respectively, as shown in Table 1.
Table 1. Nucleotide
sequence identity percentage of the HA antigens of the challenge virus compared
to some commercially available H5 vaccines used in Egypt.
|
Group No. |
Vaccine tradename |
Vaccine seed virus |
Subtype |
Abbreviation |
Clade/Lineage |
Identity percentage compared to challenge virus H5N8 |
|
Gp1 |
Re-13 & Re-14 vaccine |
A/duck/Fujian/S1424/2020 |
H5N6 |
Re-13 |
clade 2.3.4.4h |
90.49% |
|
|
|
A/whooper
swan/Shanxi/4-1/2020 |
H5N8 |
Re-14 |
clade 2.3.4.4b |
97.83% |
|
Gp2 |
Re-5 vaccine |
A/duck/Anhui/1/2006 |
H5N1 |
Re-5 |
clade 2.3.4 |
92.13% |
|
Gp3 |
Re-6 & Re-8 vaccine |
A/duck/Guangdong/s1322/10 |
H5N1 |
Re-6 |
Clade 2.3.2.1 |
90.51% |
|
|
|
A/Chicken/Guizhou/4/13 |
H5N1 |
Re-8 |
clade 2.3.4.4g |
91.01% |
|
Gp4 |
Nobilis vaccine |
A/duck/Potsdam/1402-6/1986 |
H5N2 |
Potsdam/H5N2 |
Eurasian |
86.60% |
|
Gp5 |
MEFLUVAC vaccine |
RGA/chicken/ME-2018 |
H5N8 |
EGY18/H5N8 |
clade 2.3.4.4b |
96.84% |
|
|
|
RGA/CHICKEN/Egypt/ME1010/2016 |
H5N1 |
EGY16/H5N1 |
clade 2.2.1.1 |
87.87% |
|
|
|
A/Chicken/Egypt/
RG-173CAL/2017 |
H5N1 |
EGY17/H5N1 |
clade 2.2.1.2 |
87.65% |
Clinical signs
Pre-challenge
period
Chickens in all
groups in their first 31 days of life (which corresponds to the pre-challenge
or immunization period) were characterized as healthy by clinical inspection.
Challenge period (from day
31 to day 41)
In
the non-vaccinated challenged group (Gp6), all chickens developed clinical
illness following the challenge, exhibiting symptoms of HPAI including
hemorrhage on the legs and shanks, depression, ruffled feathers, cyanosis of
the combs and beaks, nervous signs (tremors, convulsions, incoordination),
respiratory signs (nasal discharges and breathing difficulties) and diarrhea
beginning 24 hours after challenge. Mortality begun on the third day of
challenge and by the fourth day all chickens were found dead.
While in the
vaccinated groups (Gp1 to Gp5), the majority of chickens remained healthy, with
the exception of a small number of chickens that developed clinical illness,
and began exhibiting slight depression, ruffled feathers and mild respiratory
signs (nasal discharges and rales), several days later than the non-vaccinated
challenged chickens. Following that, these sicker chickens developed severe
respiratory signs, cyanosis of beak and comb and severe nervous signs, and
finally were found dead. Mortality begun the fifth day of the challenge and by
the ninth day, there were no more dead or diseased chickens.
Seroconversion
Monitoring of maternally-derived antibodies
During the
first day, each tested chicken in the five vaccinated groups and the control
group were 100% seropositive.
At
one DO, the mean HI titers of the maternally-derived antibodies (MDAs) ranged
from 4.6 to 4.7 log in all groups, using a heterologous inactivated HPAIV H5N8
antigen. Then, the MDA titers decreased gradually until the second week in the
five vaccinated groups. But in the control group, the MDA
titers waned up nearly at the fourth week of age, as shown in Table 2.
Table 2. HI mean titers
(log2±SD) against heterologous inactivated HPAI H5N8 challenge
antigen to some commercially available H5 vaccines used in Egypt.
Humoral response to vaccination
At
24 and 31 DO, sera collected from all vaccinated groups (from Gp1 to Gp5) and
tested against heterologous inactivated HPAI H5N8 challenge antigen revealed
detectable HI antibody response to vaccination at those ages. At time of
challenge (31 DO), the vaccinated group exhibiting the highest mean level of HI
titer was Gp1 (with a titer of 5.2 log2), followed by Gp3 (5.1 log2), then Gp2
(5 log2), following that Gp5 (4 log2) and, finally,
Gp4 exhibited the lowest HI titer (3.5 log2), as shown in Table 2.
Protective
efficacy following HPAI H5N8 challenge
The
efficacy of the vaccines was evaluated based on protection against mortality
after challenge and reduction of viral shedding. All non-vaccinated challenged
chickens showed no protection against the challenge (protection level 0%);
their mortality started on day 3 and ended on day 4 PC. On the other hand, in
the vaccinated challenged groups, the highest protection level was observed in
chickens that received the Re-13 & Re-14 vaccine (95%), followed by those
that received Re-5 (90%) and Re-6 & RE-8 (90%) vaccines, then the MEFLUVAC
vaccine recipients showed 80% protection and, finally, chickens that received
Nobilis vaccine showed the lowest level of protection (70%), as shown in Table
3 and Figure 1.
AIV shedding in
oropharyngeal swabs
Among
all the challenged groups, the highest viral shedding titer was obtained for
the positive control group (Gp6) at 3 days PC (5.4
log10). At the same time, compared to the positive
control group,
the five vaccinated challenged groups shed significantly less challenge virus
with a reduction of 3.8, 3.55, 3.7, 2.85 and 3.3 log10,
respectively. Moreover, there was a statistically significant difference
between vaccinated groups. Chickens in Gp1, Gp2, Gp3 and Gp5 displayed
significant reductions in H5N8 viral shedding titers compared to Gp4 on each
day PC. In addition, chickens in Gp1 and Gp3 comparing to that of Gp5 displayed
significant reductions in viral shedding. In contrast, no significant
difference in shedding levels were found between Gp1, Gp2 and Gp3. At 3rd
day, PC results revealed a higher rate of virus shed for Gp4 (3.9 log10)
and lower titers were recorded for Gp1 (2.8 log10); at 5th
day PC, there was a higher virus shedding titer for Gp4 (3 log10) and
lower shedding titers were detected for Gp1 (1.8 log10); at day 7
PC, results were significantly different with a high titer for Gp4 (1.8 log10)
and the low titer was presented in Gp1 (1.1 log10), as shown in
Table 3.
Table 3. Mean viral load of
challenge virus plus standard deviation (log10 EID50/mL±SD) in vaccinated and
non-vaccinated groups on days 3, 5, 7 and 10 PC with the HPAI H5N8 virus clade
2.3.4.4b (A/chicken/Egypt/ v1526/2020) and their protection percentages.
Discussion
The main approaches of controlling HPAI in poultry usually include rapid
eradication, animal movement restriction rules and early warning passive
surveillance technologies. These strategies might not be enough to prevent
introduction of the virus (mainly from infected wild birds) and dissemination
within the commercial poultry sector, as recent and recurrent epizootics caused
by different A/goose/Guang dong/1/1996-lineage HPAI
A(H5Nx) viruses still happened. So, it is necessary to regularly develop and
test vaccines that offer effective protection against HPAI (preventing
mortality and virus shedding); this should be done regularly to match the
emerging circulating virus strains in order to implement complementary
preventive vaccination against HPAI in poultry.(16)
In the present study, the protective efficacy
of five different types of commercially available inactivated avian influenza
vaccines was assessed in broiler chickens carrying MDAs against HPAI H5N8 virus
belonging to major HA clade 2.3.4.4b that circulates in Egypt, and is known to
have previously caused severe clinical signs and 100% mortality in
non-vaccinated chickens.
According to the results, the Gp1 group that received the Re-13 &
Re-14 vaccine showed the highest protection level (95%), the highest HI titer
(5.2 log2) and a significant reduction in viral shedding titer and this may be
explained by considering that the vaccine is made up of two seed strains: the
Re-14 strain, which is H5N8 subtype clade 2.3.4.4b and closely related to the
challenge H5N8 clade 2.3.4.4b with identity percentage (97.83%) and the Re-13
strain, which is H5N6 subtype clade 2.3.4.4h, which raises the antigenic mass
of H5. These
results were in agreement with those of other authors(17,18) who
reported that the vaccine produced with the seed viruses H5-Re13 and H5-Re14
carrying the HA and NA genes of a newly detected H5N6 virus and H5N8 virus,
respectively, provided complete protection against challenge with H5N8 virus
bearing the clade 2.3.4.4b in chickens.
Subsequently, we found that Gp2 and Gp3 groups immunized with Re-5 and
Re-6 & Re-8 vaccines, respectively, attained 90% protection levels for both
of the groups, along with demonstrating significantly high
HI titers (5 and 5.1 log2, respectively) and significantly reduced
viral shedding titers. These could be related to the fact that the Re-5 vaccine is made up of a seed strain (Re-5 strain, H5N1
subtype clade 2.3.4) closely related to the challenge H5N8 clade 2.3.4.4b with
identity percentage of 92.13% and the Re-6 & Re-8 vaccine is made up of two
seed strains (the Re-6 and Re-8 strains) which are H5N1 subtype bearing clade
2.3.2.1 and clade 2.3.4.4g, respectively and these strains were found to be
closely related to the challenge H5N8 clade 2.3.4.4b with identity percentage
90.51% and 91.01%, respectively and also believed to increase the H5 antigenic
mass. These findings
were supported by other studies(18,19)
and disagreed with the findings of Kilany, et al.(20)
Furthermore,
the Gp5 group which received MEFLUVAC vaccine achieved a protection level of
80%, a significant high HI titer (4 log2) and a significant
reduction in viral shedding titer; these findings are possibly explained by the
fact that the vaccine consists of three seed strains: one of them is EGY18/H5N8 clade 2.3.4.4b, which is closely related to the
challenge virus and demonstrated 96.84% identity percentage and the other two
strains are Egy2016/H5N1 clade 2.2.1.1 and Egy2017/H5N1 clade 2.2.1.2, which expected to increase the antigenic mass of H5. These results are
consistent with those obtained in another study.(10)
Finally, the
Gp4 group, which demonstrated the least protection level of 70%, received the
Nobilis vaccine; it also had low levels of HI titers (3.5 log2) and
reduction of viral shedding titers.
This could be attributed to the genetic differences between the vaccinal strain
and the challenge virus, as it demonstrated 86.6% similarity in genetic
relatedness between the vaccinal strain and the challenge virus; these results
were in agreement with findings of other authors.(19)
Conclusion
The present study, aimed to assess the efficacy of five
commercially inactivated H5 vaccines belonging to a different lineage against
challenge with HPAI H5N8 virus in broiler chickens in Egypt, revealed that the Re-13 & Re-14 vaccine gives a
superior humoral immune response and lower virus shedding resulting in a higher
protection level, whereas the Nobilis vaccine achieves the lowest humoral
immune response and protection percentage. This could show the importance of
continuously evaluating and updating avian influenza poultry vaccines in Egypt,
as vaccination is used as an effective method to prevent and control of AIV
spread.
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Conflict of interest
The authors declare that
there is no conflict of interest.
Author’s
contributions
Hussein AM: conducted the experiment, drafted
the manuscript, designed the study, followed up the experiment and critically
reviewed the manuscript.
El-Bagoury GF:
designed the study, followed up the experiment, critically reviewed the
manuscript and followed up the practical work.
El-Habbaa AS:
designed the study, followed up the experiment and critically reviewed the
manuscript and followed up the practical work.
Samir A Nassif: conducted the experiment,
drafted the manuscript, designed the study, followed up the experiment,
critically reviewed the manuscript and followed up the practical work.
All authors read and approved the final
manuscript.
* Veterinarian.
Armed Forces Laboratories for Medical Research and Blood Bank. Cairo, Egypt.