Original Article
Molecular characterization and pathogenicity
evaluation of recent infectious bursal disease virus strains: implications for
Newcastle disease vaccine efficacy
Caracterización molecular y evaluación de la patogenicidad de cepas
recientes del virus de la bursitis infecciosa: implicaciones para la eficacia
de la vacuna contra la enfermedad de Newcastle
Ola Y. Abido1* ORCID: https://orcid.org/0000-0001-9768-045X
Karim M. Selim2 ORCID: https://orcid.org/0000-0002-5069-3947
Sara Abdel-Mawgod2
ORCID: https://orcid.org/0000-0002-0619-6142
Mohamed S. Sobh3 ORCID: https://orcid.org/0000-0002-6547-1255
Arwa
El Naggar1 ORCID: https://orcid.org/0000-0001-7184-3177
>Mohamed M. Shawki4 ORCID: https://orcid.org/0009-0003-6818-6358
Mohamed A. Elhady5 ORCID:
https://orcid.org/0000-0003-1308-4939
1 Central
Laboratory for Evaluation of Veterinary Biologics, Agriculture Research Center
(ARC), Cairo, Egypt.
2 Reference
Laboratory for Veterinary Quality Control on Poultry Production, Animal Health
Research Institute, Agriculture Research Center, Giza, Egypt.
3 Pathology
Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
4 College of
Avian and Rabbit diseases, Veterinary Hospital, Faculty of Veterinary Medicine,
Zagazig University, Zagazig, Egypt.
5 Department
of Toxicology and Forensic Medicine, Faculty of Veterinary Medicine, Cairo
University, Giza, Egypt.
Autor para correspondencia: ola.abido@yahoo.com
ABSTRACT
Infectious bursal disease
continues to cause significant economic losses in the Egyptian poultry industry
despite intensive vaccination programs. This study was aimed to molecularly
characterize the circulating infectious bursal disease virus strains in Egypt
and to compare the pathogenic and immunosuppressive effects of very virulent
and variant strains. Ten pooled bursal samples from suspected broiler flocks
were subjected to reverse transcription polymerase chain reaction for detection
and identification. Nine samples tested positive. Sequencing and phylogenetic
analysis identified seven samples (D1, D3, D4, D5, D6, D7, and D8) as very
virulent-like strains, showing 98.8-99.2% amino acid sequence identity among
them, but only 87.8-91% identity with vaccine strains used in Egypt. Two
samples (D9 and D10) were identified as variants with 96-96.4% identity with
other Egyptian variants. Two isolates (D8 and D10) were selected to study their
pathogenicity and immunosuppressive effects in specific pathogen free chickens,
which were orally infected with 105 egg infective dose 50% of each
isolate and vaccinated against Newcastle disease, 5 days before infection. The
variant strain caused earlier and more severe bursal damage without clinical
signs or mortality, while the very virulent strain led to typical disease
symptoms and 60% mortality. The mean hemagglutination inhibition titers were
lower in variant-infected chickens, while protection against Newcastle disease
virus was 60% and 40% in very virulent and variant-infected chickens,
respectively, compared to 90% in uninfected chickens. These findings indicate
that variant strains are more pathogenic and immunosuppressive than very
virulent strains, highlighting the need for effective control measures.
Keywords: infectious
bursal disease virus; RT-PCR; pathogenicity; Newcastle disease virus;
hemagglutination inhibition test.
RESUMEN
La bursitis infecciosa continúa causando importantes pérdidas económicas
en la industria avícola egipcia a pesar de los intensos programas de
vacunación. El objetivo de este estudio fue caracterizar molecularmente cepas
del virus de la bursitis infecciosa circulantes en Egipto y comparar los
efectos patógenos e inmunosupresores de cepas muy virulentas y variantes. Diez
muestras procedentes de pollos de engorde sospechosos de bursitis infecciosa fueron
sometidas a la reacción en cadena de la polimerasa de transcripción inversa
para su detección e identificación. Nueve muestras resultaron positivas.
Mediante la secuenciación y el análisis filogenético se identificaron siete
muestras (D1, D3, D4, D5, D6, D7 y D8) como cepas muy virulentas, que mostraban
una identidad de secuencia de aminoácidos del 98,8-99,2% entre ellas, pero sólo
del 87,8-91% con las cepas vacunales utilizadas en Egipto. Dos muestras (D9 y
D10) se identificaron como cepas variantes con un 96-96,4% de identidad con
otras variantes egipcias. Se seleccionaron dos aislados (D8 y D10) para
estudiar su patogenicidad y efectos inmunosupresores en pollos libres de patógenos
específicos, que fueron infectados por vía oral con 105 dosis
infectivas en huevos 50% de cada aislado y vacunados contra la enfermedad de
Newcastle, 5 días antes de la infección. La cepa variante causó lesiones bursales más tempranas y graves sin signos clínicos ni
mortalidad, mientras que la cepa muy virulenta provocó síntomas típicos de la
enfermedad y un 60% de mortalidad. El título medio de inhibición de la
hemaglutinación fue inferior en los pollos infectados por la cepa variante,
mientras que la protección contra el virus de la enfermedad de Newcastle fue
del 60% y el 40% en los pollos infectados con la muy virulenta y la variante,
respectivamente, en comparación con el 90% en los pollos no infectados. Estos
resultados indican que las cepas variantes son más patógenas e inmunosupresoras
que las cepas muy virulentas, lo que pone de relieve la necesidad de adoptar
medidas de control eficaces.
Palabras clave: virus de la enfermedad infecciosa
de la bolsa; reacción en cadena de la polimerasa de transcriptasa inversa;
patogenicidad; virus de la enfermedad de Newcastle; pruebas de inhibición de
hemaglutinación.
Recibido: 5 de agosto de 2024
Aceptado: 4 de noviembre de 2024
Introduction
Newcastle disease (ND) and infectious bursal disease (IBD) represent a
significant threat to the Egyptian poultry industry, leading to considerable
economic losses due to reduced productivity, immunosuppression, and increased
mortality. Despite ongoing control efforts, sporadic outbreaks continue to
affect poultry flocks. The infectious bursal disease virus (IBDV), responsible
for IBD, is classified into two serotypes, I and II, within the family Birnaviridae and genus Avibirnavirus.
Serotype I strains are pathogenic, targeting immunoglobulin M+ (IgM+) B cells
in the bursa of Fabricius, and this leads to immunosuppression, increased
susceptibility to secondary infections, and vaccine failures.(1)
In contrast, serotype II strains are non-pathogenic.
The viral genome consists of two segments of double-stranded RNA
(segments A and B), which are prone to frequent genetic mutations,
reassortments, and recombinations. These genetic
changes can affect virulence and antigenicity, thereby influencing vaccine efficacy.(2)
Segment A encodes structural proteins (VP2 and VP3), a viral protease (VP4),
and a nonstructural protein (VP5), while segment B encodes VP1, essential for
viral replication.(3) The VP2 protein, organized into base (B),
shell (S), and projection (P) domains, features a hyper-variable region (HVR)
spanning amino acid residues 206-350. This region contains hydrophilic sites at
residues 210-225 (peak A), 247-254 (minor peak 1), 281-292 (minor peak 2), and
312-324 (peak B), which are critical for pathogenicity and vary among strains.(4)
Serotype I strains are classified into four pathotypes: classical
virulent (cvIBDV), antigenic variant (VarIBDV), very virulent (vvIBDV),
and attenuated (atIBDV), with recent identification
of novel variants.(5) Recent
reports from Egypt highlight an increase in the virulence and severity of both vvIBDV and VarIBDV strains. These
strains pose significant challenges to conventional vaccines, penetrate
maternal immunity, and contribute to high mortality rates in young chickens.(6)
As IBDV targets the bursa of Fabricius and causes immunosuppression, it
makes chickens more susceptible to other diseases and reduces the effectiveness
of vaccines, including those for Newcastle disease virus (NDV). NDV is a highly
contagious virus that cause severe respiratory and neurological disease in
chickens. Despite vaccination efforts, NDV outbreaks persist, particularly in
farms where IBDV is prevalent.(7) The
emergence of novel VarIBDV strains exacerbates this
issue, as these new variants may penetrate maternal immunity, complicate the
immune response, and thereby potentially reduce the effectiveness of NDV
vaccines.(8)
Understanding how these novel IBDV variants impact NDV vaccine efficacy
is crucial for improving poultry health management. This research aimed to
molecularly characterize the currently circulating IBDV strains, assess the
pathogenicity of vvIBDV and VarIBDV
field isolates, investigate the effect of mutations on virulence, and explore
their implications for immune responses to ND vaccination. By providing these
insights, the study seeks to optimize vaccination strategies and improve
control measures against both IBD and ND in poultry.
Materials and
Methods
Specific pathogen free embryonated chicken eggs (SPF-ECE)
SPF-ECE were
obtained from the specific pathogen free (SPF) production farm, Koum Oshiem, El-Fayoum, Egypt and
were used for isolation and titration of IBDV field strains according to World
Organization for Animal Health (WOAH).(9)
Experimental SPF free
chickens
One hundred and
fifty 18-day-old SPF chickens, obtained from a farm in Koum
Oshiem, El Fayoum, Egypt, were raised on the floor
under strict hygienic conditions and fed a balanced commercial diet. They were
required for pathogenicity and immunosuppression assessment.
Reference virus
The Bursa-Vac®
IBD vaccine, supplied by Receiving sample office of Central Laboratory for
Evaluation of Veterinary Biologics (CLEVB), was used as a positive control for
virus detection by conventional RT-PCR.
Challenge ND virus
The virulent ND
virus, with an infectivity titer of 106 egg infective dose 50% (EID50)/mL,
was kindly supplied by the Newcastle Disease Research Department at the
Veterinary Serum and Vaccine Research Institute in Abbasia,
Cairo.
Sample collection and
preparation
Between 2022
and 2023, a total of 10 pooled hemorrhagic bursal tissues (seven bursas from
each farm) were collected from freshly dead chickens in El-Mansoura, El-Menia, and El-Sharkia
governorates. All farms had a vaccination history against IBDV, using
commercial live vaccines administered one or two times via drinking water. The
samples were prepared according to WOAH(9) and coded as D1 to D10.
Molecular identification of
IBDV by conventional one-step RT-PCR assay
Viral
RNAs were extracted from bursal homogenates using the QIAamp
Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's
guidelines. The RT-PCR was carried out using the forward primer AUS GU (5'-TCA
CCG TCC TCA GCT TAC CCA CAT C-3') and reverse primer AUS GL (5'-GGA TTT GGG ATC
AGC TCG AAG TTG C-3'), amplifying a 620 bp fragment within the HVR of the VP2
gene. The thermal profile employed was: 20 min at 50°C (RT reaction), followed
by 95°C for 15 min (initial PCR activation); then 40 three-step PCR cycles of
94°C for 30 s (denaturation), 59°C for 1 min (annealing), and 72°C for 1 min
(extension), with a final extension cycle at 72°C for 10 min on thermocycler (Biometra, Germany) according to Metwalley
et al., 2009.(10) The PCR products were analyzed by
electrophoresis in a 1.5% agarose gel containing ethidium bromide dye (final
concentration 0.5 μg/mL) to show amplification
at 620 bp.
Sequencing and phylogenetic
analysis
Positive
RT–PCR products were purified using the QIAquick PCR
Purification Kit (QIAGEN, Hilden, Germany) following the manufacturer’s
instructions. Sequencing of the purified PCR products was carried out using the
Bigdye TM Terminator V3.1 cycle sequencing kit
(Perkin-Elmer, Foster City, CA, USA) according to the manufacturer’s
instructions in the ABI PRISM 3130 genetic analyzer (Applied Biosystems) with
80 cm capillaries.
BLAST®
analysis was initially conducted to establish the identity of the obtained sequences
with other sequences in GenBank. The VP2 gene sequences were then submitted to
GenBank using the BankIt tool, the accession numbers
are provided in Table 1. The nucleotide and amino acid sequences of the
samples were aligned with sequences of IBDV strains, corresponding to different
groups (G1-G7), obtained from the National Center for Biotechnology
Information. The alignment was performed using the
CLUSTAL-W program and the Meg Align module of DNASTAR software (Laser gene
version 7.2; DNASTAR, Madison, WI, USA), then exported to MEGA 6
software for construction of a phylogenetic tree using the maximum likelihood
methodology with 1,000 bootstrap replicates to assess the robustness of the
tree topology. The pairwise nucleotide and amino acid
identity percent was calculated using DNA star
software (DNA Star, Madison, WI).
Table 1. Accession number for IBDV strains in the current study.
|
Samples Name |
Sample code |
Governorate |
Age at
sampling |
Phenotype |
GenBank
accession number |
|
IBDV-1/Mansoura/Egypt |
D1 |
El-Mansoura |
21d |
Very virulent |
PP508369 |
|
IBDV-2/Mansoura/Egypt |
D3 |
El-Mansoura |
30d |
Very virulent |
PP508370 |
|
IBDV-3/Mansoura/Egypt |
D4 |
El-Mansoura |
25d |
Very virulent |
PP508371 |
|
IBDV-4/Mansoura/Egypt |
D5 |
El-Mansoura |
21d |
Very virulent |
PP508372 |
|
IBDV-5/Mansoura/Egypt |
D6 |
El-Mansoura |
26d |
Very virulent |
PP508373 |
|
IBDV/Menia/Egypt (A) |
D7 |
El-Menia |
28d |
Very virulent |
PP508374 |
|
IBDV/Menia /Egypt (B) |
D8 |
El-Menia |
24d |
Very virulent |
PP508375 |
|
IBDV/sharkia/Egypt/ 2023 variant (C) |
D9 |
El-Sharkia |
18d |
Variant |
OR687651 |
|
IBD/Sharkia/Egypt/2023 variant (D) |
D10 |
El-Sharkia |
19d |
Variant |
OR687652 |
Virus isolation and
titration
After genetic
analysis, two PCR-positive samples were inoculated in SPF-ECE via chorioallantoic membranes (CAMs) route for isolation and
titration following standard protocols outlined by WOAH.(9) Subsequently, the isolated IBDV strains were diluted to 105 EID50/mL
for subsequent experimental infections.
Experimental design for
pathogenicity and immunosuppression assessment of IBDV isolates
A total of 150 SPF
chickens, 18-day old, were divided into three equal groups (50 chickens/each)
for experimental work as follow: the first group (G1) was infected orally with
105 EID50 of a vvIBDV strain,
the second group (G2) was infected orally with VarIBDV strain with the same dose, and the
third group (G3) was the uninfected control group. The three groups were housed
separately in strictly isolated and disinfected rooms and kept under daily
observation for clinical symptoms and mortality. All chickens were vaccinated
with inactivated ND vaccine 5 days before experimental infection via
subcutaneous route. Three chickens from each group were euthanized at 1st,
2nd, 3rd, 4th and 5th day
post-infection (dpi) for postmortem (PM) examination. The
bursas were collected and stored in 10% neutral formalin following the methods
described by Bancroft and Gamble(11) for pathological examination.
Bursa weight ratios (bursa-to-body weight ratio, BBR) were calculated using the
formula: bursa weight (g)/live body weight of individual bird (g ×1,000.
Additionally, the bursa: body weight index (BBIX) for each day was calculated,
including the standard deviation, using the formula [BBIX=
(Bursa: body weight ratios)/(Bursa:
body weight ratios in the negative group)]. A BBIX value below 0.7 was regarded
as an indicator of atrophy following the criteria established by Lucio and Hitchner.(12)
Individual blood samples
were collected from 10 chickens of each group at 7, 14, and 21 days
post-vaccination. The antibodies against NDV were measured in each collected
serum sample by hemagglutination inhibition (HI) test according to WOAH.(13) On 3rd week
post-vaccination, 10 chickens from each group were challenged intramuscularly
with NDV at a dose of 106 EID50/bird. The chickens were
observed daily for clinical symptoms and number of deaths in each group during
14 days post-challenge.
Protection (%) = Number of
survivals/Total number of challenged bird X 100.
Ethical approval
All animal experiments were
conducted in accordance with ethical standards and protocols approved by the
Institutional Animal Care and Use Committee of Zagazig University (ZU-IACUC
committee) under approval number ZU-IACUC/2/F/147/2023.
Statistical analysis
The statistical analysis
used ANOVA and t-test procedures via the InStat
GraphPad program. Differences between mean values were considered statistically
significant at P < 0.05.
Results
Molecular detection of IBDV
Out of the
total 10 of pooled bursal samples, only nine were found positive for IBDV by
VP2 gene-based RT-PCR.
Sequencing and phylogenetic
analysis
Phylogenetic
analysis of VP2 gene revealed that seven samples D1, D3, D4, D5, D6, D7, and D8
were clustered with vvIBDV strains. Conversely, the
remaining two isolates (D9 and D10) were grouped with VarIBDV
strains. The vvIBDV strains showed 96.8% to 98% amino
acid identity with other Egyptian vvIBDV strains, but
only 87.8-91% identity with vaccine strains used in Egypt. The VarIBDV strains (D9 and D10) showed 96% to 96.4% identity
with other Egyptian variants as shown in Figure 1.
None of the
examined samples were of attenuated or vaccinal origin due to absence of
253-Histidine and 284-Threonine substitutions typically found in attenuated
vaccine strains.
Fig. 1. Phylogenetic analysis of IBDV based on partial nucleotide sequences of
the VP2 gene. The isolates from this study are indicated by red circle. G2a and
G2b are the American variants; G2d are Chinese variants.
Virus isolation
and titration
Two PCR-positive samples (D8 and D10) were selected for isolation and
titration in SPF-ECE. The inoculated SPF-ECEs showed characteristic embryo
lesions during the second passage, including stunted growth, liver necrosis
with a mottled appearance, greenish coloration of the liver, and hemorrhage.
Additionally, the harvested CAMs exhibited thickening and petechial
hemorrhages.
The infectivity titration after the third passage revealed virus titers
of 106.5 and 105.6 log10 EID50/mL
for (D8 and D10) isolates, respectively.
Pathogenicity evaluation of
the two selected IBDV field isolates
Clinical signs and
mortality
Throughout the observation
period, the control group (G3) exhibited no mortality or clinical symptoms. In
group G1, birds displayed severe clinical manifestations, including depression,
anorexia, ruffled feathers, huddling, tremors, prostration and whitish watery
diarrhea, resulting in a 60% mortality rate. In contrast, the variant-infected
group G2 exhibited mild diarrhea and depression, without mortality. Notably,
clinical signs in group G1 were more severe, manifesting as early as 48-hour
post-infection.
PM examination
No gross lesions were
observed in the control group. In contrast, both infected groups exhibited the
characteristic PM lesions of natural IBDV infection.
Additionally, hemorrhagic thymic lobes were observed in group G1. As
quantified by the BBR, the severity of bursal atrophy was more pronounced in
group G2. The VarIBDV strain induced bursal atrophy
as early as 72-hour post-infection, while the vvIBDV
strain showed bursal atrophy from 96- hour post-infection.
Changes in body weight and
relative bursal weight
The infected birds exhibited a significant (P≤0.05) decrease in
both bursal weight and body weight compared to the control group. Additionally,
chickens in group G2 demonstrated significantly lower bursal and body weights
than those in group G1 throughout the experiment. The BBR decreased from 1.75±0.1 to 0.71±0.12 in group G1 and from
1.93±0.2 to 0.34±0.09 in group G2, in contrast to non-infected control birds in
group G3, where the BBR slightly increased from 2.2±0.1 to 2.52±0.1 throughout
the experiment. Furthermore, the BBIX decreased significantly (p < 0.05) in
infected birds compared to negative control group non-infected. The BBIX in group G2 was below 0.7 at 3 dpi and continued
to decrease to 0.1 until 5 dpi while in group G1, the BBIX was below 0.7 at 4th
dpi and then continued to decrease to 0.28 until 5 dpi. These results suggest
that the VarIBDV strain causes earlier and more
severe bursal atrophy than the vvIBDV strain, while
the control group did not exhibit bursal atrophy, as demonstrated in Table 2.
Table 2. Mean BBR and BBIX in
different groups.
|
Dpi |
Groups |
Body weight
(g) |
Bursa weight
(g) |
BBR |
BBIX |
|
1st |
G1 |
723± 0.00 |
1.3±0.1 |
1.75±0.1 |
0.795 |
|
|
G2 |
723±0.00 |
1.4 ±0.17 |
1.93±0.2 |
0.877 |
|
|
G3 |
739.3±0.57 |
1.7±0.08 |
2.2±0.1 |
- |
|
2nd |
G1 |
748.5±0.00 |
1.26±0.07 |
1.66±0.05 |
0.790 |
|
|
G2 |
748.6±1.15 |
1.3±0.1 |
1.7±0.1 |
0.809 |
|
|
G3 |
854.6±4.5 |
1.8±0.1 |
2.1±0.1 |
- |
|
3rd |
G1 |
984.6±4.2 |
1.8±0.1 |
1.8±0.1 |
0.782 |
|
|
G2 |
985±4.3 |
1.3±0.1 |
1.3±0.1 |
0.565 |
|
|
G3 |
1203±5.1 |
2.8±0.1 |
2.3±0.1 |
- |
|
4th |
G1 |
1206.3±5.5 |
1.56±0.3 |
1.29±0.25 |
0.586 |
|
|
G2 |
1200.6±0.57 |
0.73±0.2 |
0.58±0.16 |
0.263 |
|
|
G3 |
1605±4.58 |
3.6±0.26 |
2.2±0.15 |
- |
|
5th |
G1 |
1204.6±4.1 |
0.89±0.15 |
0.71±0.12 |
0.28 |
|
|
G2 |
1202±2.6 |
0.43±0.1 |
0.34±0.09 |
0.134 |
|
|
G3 |
1803.3±4.9 |
4.56±0.2 |
2.52±0.1 |
- |
Dpi: day post infection.
BBR: bursa-to-body weight ratio. BBIX: bursa body weight index. G1: group 1
infected with vvIBDV strain. G2: group 2 infected with VarIBDV
strain. G3: uninfected control group. -: BBIX equal to zero.
Immunosuppression effect
The absence of
clinical signs, coupled with marked bursal atrophy in chicks infected with the VarIBDV strain, prompted a focused evaluation of the
immunosuppressive properties of this virus compared to a vvIBDV
strain.
HI test
The mean HI
antibody titers were significantly reduced in the IBDV-infected groups. Group
G2 exhibited significantly lower mean HI antibody titers than group G1 at 1st,
2nd, and 3rd weeks post-vaccination. In contrast, control
group demonstrated a substantial increase in antibody titers, surpassing those
of both groups G1 and G2, as shown in Table 3.
Table 3. Mean HI antibody titer against NDV vaccine. Data are expressed as mean
±SD.
|
|
|
Mean HI
antibody titer (log2) |
|
|
Chicken group |
|
WPV |
|
|
|
1st |
2nd |
3rd |
|
G1 |
2.6
± 1.07d |
4.7
± 1.16d |
5.9
± 0.94a |
|
G2 |
1.8
± 0.63d |
3.8
± 1.03d |
5.1
± 0.94a |
|
G3 |
4.0
± 0.94d |
6.1
± 1.10d |
7.1
± 0.99c |
Different subscript letters
within the same column indicate significant difference at P<0.05, a =
non-significant (P>0.05), b = significant (P<0.05), c = highly significant (P<0.01) and d
= very highly significant (P<0.001). HI: hemagglutination inhibition. WPV:
weeks post-vaccination. G1: group 1 infected with vvIBDV
strain. G2: group 2 infected with VarIBDV strain. G3:
uninfected control group. Different superscript letters within the same column
indicate significant difference at P<0.05.
Protection against NDV challenge
Cumulative mortalities in groups G1 and G2 were statistically
significantly different from control group (P < 0.05). Protection was 60%,
40%, and 90% in groups G1, G2, and G3, respectively.
Histopathological findings
In
the assessment of bursal tissues, the control group displayed normal
histological architectures, while both infected groups demonstrated mild
interfollicular edema with lymphocyte depletion, congestion, and inflammatory
cell infiltration.
Significant
differences emerged by the 2nd dpi, with group G2 displaying cystic
follicles and necrosis, while group G1 exhibited compressed follicles and
moderate lymphocyte depletion. By the 3rd dpi, group G2 showed
severe hyperplasia of follicular epithelium alongside cystic follicles, severe
lymphocyte depletion, and interstitial edema with inflammatory cell
infiltration.
Notably,
at the 4th and 5th dpi, severe bursal atrophy was more
pronounced in group G2 compared to group G1. By the 5th dpi, group
G2 exhibited a complete absence of intact lymphoid follicles, substituted by fibrous
tissue, with a highly corrugated lining epithelium.
These findings collectively indicate a progressive histopathological
deterioration, for the VarIBDV strain compared to the
vvIBDV strain, as elucidated in Figures 2 and 3.
Fig. 2. Histopathology of bursa of
Fabricius in group G1. (A) Bursa at 1st dpi showing mild
interfollicular edema with mild lymphocyte depletion. The right (A) picture
shows congestion and edema in tunica muscularis of bursa. (B) Bursa at 2nd
dpi showing lymphocyte depletion, compressed follicles (indicated by arrow),
and interstitial edema with infiltration of inflammatory cells (star) H&E
X50. (C) Bursa at 3rd dpi showing lymphocyte
depletion, follicular cysts (arrow), and interstitial edema with
inflammatory cells infiltration (star) H&E X100. (D) Bursa at 4th
dpi showing hyperplasia of the epithelium (indicated by arrow) with cysts
(circled), lymphocyte depletion, compressed follicles, and interstitial edema
with inflammatory cells infiltration H&E X100. (E) Bursa at 5th
dpi showing depleted follicles (arrow) and proliferation of interstitial
connective tissue (star).
Fig. 3. Histopathology of bursa of
Fabricius in group G2. (1) Interfollicular
edema with mild lymphocyte depletion (arrow) H&E X100. (2) Bursa at 2nd
dpi showing cystic follicles (arrow) with interfollicular connective tissue
proliferation (circle) H&E X100, lymphocyte necrosis in medulla (arrow),
(3) severe hyperplasia (arrow) and cystic formation of
lining epithelium (star), with lymphocyte depletion H&E X100. (4)
Atrophy of the plicae with lymphocyte necrosis (circle) in medulla and
interfollicular connective tissue proliferation (arrow) H&E X100. (5)
Atrophy with severe follicular epithelization & interstitial lymphocytes aggregation.
Discussion
IBD is a highly contagious disease affecting chickens, leading to
significant economic losses worldwide through high mortality,
immunosuppression, and secondary infections. In Egypt, numerous IBDV outbreaks
have been reported within a short period following the emergence of novel VarIBDV strains in 2023.(6,14)
Then, updating the current situation is crucial to address this issue
effectively and to develop a new strategy for disease control. In this study,
IBDV was diagnosed from 10 infected farms in Egypt during 2022-2023. We
isolated and molecularly characterized IBDV strains from infected samples and
compared their pathogenic features by assessing clinical signs, mortality
rates, BBR, and histopathological changes in the bursa. Additionally, we evaluated
the impact of these strains on the effectiveness of the inactivated ND vaccine.
Initially, bursal samples were examined using VP2
based RT-PCR, nine out of 10 samples tested were positive. Phylogenetic
analysis of the VP2 protein’s hypervariable region classified the current
isolates into two main pathogenic subgroups. Seven samples (IBDV-1 through
IBDV-5), along with IBDV/Menia/Egypt (A) and (B),
were classified as vvIBDV, whereas two (IBDV/sharkia/Egypt/2023 variant (C) and (D) were identified as VarIBDV strains distinct from vaccine strains. The vvIBDV isolates exhibited 98.8% to 99.2% identity between
them while they showed a variability of 2-3.2 % with
other Egyptian vvIBDV strains at both nucleotide and
amino acid sequence. Comparative analysis with classical vaccine strains
showed identities ranging from 87.8% to 91%, with the closest similarity
observed with Bursa-Vac®. In contrast, VarIBDV
strains exhibited higher identities (96% to 96.4%) with other Egyptian variants
and approximately 93% with Chinese variants, but lower identities with American
strains (89%) and vaccine strains (83.5% to 85.1%). These findings indicate the
endemic circulation and rapid evolution of the virus in Egypt over years.(15)
Significant mutations were noticed in the VP2 region's hydrophilic peak
A (210-225), crucial for antibody binding, which likely influence virus
antigenicity and virulence.(16) VarIBDV strains displayed specific mutations like F220Y,
differing from F220S in vvIBDV, potentially impacting
immune evasion in vaccinated flocks.(17)
Moreover, specific residues
(222A, 256I, 279D, 294I, 299S) were consistently found in all vvIBDV isolates except IBDV-3, which exhibited a different
substitution (N instead of D at position 279) compared to other vvIBDV strains.(17) Mutation at position 222
(P222A) may facilitate immune escape, potentially explaining virus persistence
in vaccinated flocks.(2) The presence of amino acids 252I and 299S
in VarIBDV strains, also found in Chinese variants,
underscores their evolutionary relationships.(8)
Amino acids at positions 253 and 284 (Q253H; A284T) are critical for
cell tropism and adaptation in cell culture.(18) All IBDV isolates in this
study had glutamine at position 253 and alanine at position 284, potentially
affecting their growth in cell culture and pathogenicity. Strains with glutamine at
253 are linked to higher pathogenicity compared to those with histidine. This
variation is significant given the extensive use of live attenuated viruses in
vaccination programs, indicating a potential for these viruses to evolve and
change their pathogenicity over time.(19)
Chickens infected with vvIBDV exhibited typical signs and high mortality (60%),
consistent with previous reports.(20)
In contrast, infection with VarIBDV strains led to
severe bursal atrophy without clinical signs, causing immunosuppression and
susceptibility to secondary infections.(8,21)
PM examinations revealed that chickens infected with vvIBDV
strains exhibited severe bursal atrophy and hemorrhages, including in the
thymus which aligns with other findings,(21) suggesting direct viral
injury or virus-induced
inflammatory responses. VarIBDV infections led to rapid bursal
depletion. Both groups had lower body weights compared to controls, with
continuous bursal atrophy until the end of the
experiment, as reported by others.(8) The BBIX indicated
significant atrophy in both groups, with group 2 showing earlier and more
severe decline. These findings suggest VarIBDV
strains cause more severe bursal atrophy and immunosuppression compared to vvIBDV strains.(8,21)
Histopathological analysis showed that chickens infected with the VarIBDV strain of IBDV experienced more severe and rapid
bursal damage compared to those infected with the vvIBDV
strain. This aligns with prior studies highlighting the aggressive nature of VarIBDV strains.(8)
By the 4th and 5th dpi, the VarIBDV
strain group exhibited significantly more pronounced bursal atrophy and
complete absence of intact lymphoid follicles by the 5th dpi, with
tissue replaced by fibrous tissue and highly corrugated epithelium, indicating
severe damage.
Furthermore, the immunosuppressive effect was evident in the VarIBDV strain group, as indicated by significantly lower
HI antibody titers against NDV compared to the vvIBDV
strain group. This compromised protection against virulent NDV, suggesting a
greater impact on NDV vaccine efficacy. This finding is consistent with
previous reports that highlight the association between high systemic antibody
levels and protection against NDV.(8,9) In contrast, group G3, comprising vaccinated chickens without infection,
showed a substantial increase in antibody titers, surpassing those of both
infected groups, indicating effective vaccine-induced immunity against NDV
despite exposure to VarIBDV. The results of the HI and challenge tests against the NDV vaccine suggest that both IBD strains induce immunosuppressive effects, with the
VarIBDV strain exhibiting a more pronounced effect
than the vvIBDV strain.
Our study highlights the
coexistence of both vvIBDV and VarIBDV
strains in Egypt, underscoring their progressive evolution and persistence. VarIBDV strains exhibit greater pathogenicity and induce
more pronounced immunosuppressive effects compared to vvIBDV
strains. These findings emphasize the critical need for continuous monitoring
and the development of effective vaccines to mitigate the evolving threats to
poultry health.
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Conflict of interest
The authors declare that
there is no conflict of interest.
Author’s
contributions
Ola Y. Abido:
study conception, molecular characterization of collected field samples, design
of the work, writing-review and editing.
Karim M. Selim: isolation of collected field samples.
Sara Abdel-Mawgod: molecular characterization of collected field
samples.
Mohamed S. Sobh: histopathology for bursa.
Arwa El Naggar: performed the
analytic calculation and statistics.
Mohamed M. Shawki: sample collection and study the pathogenicity of
variant and very virulent IBDV strains by experimental infection.
Mohamed A. Elhady: supervision, project administration, sample
collection.
All authors read and approved the final
manuscript.
*Researcher at Central
Laboratory for Evaluation of Veterinary Biologics, Agriculture Research Center
(ARC), Cairo, Egypt.