Infectious bursal disease virus infection leads to changes in the gut associated-lymphoid tissue and the microbiota composition
Infectious bursal disease virus infection leads to changes in the gut associated-lymphoid tissue and the microbiota composition
Editor: Pierre Roques
Li Li 1
Tereza KubasovaÂ 0
Ivan Rychlik 0
Frederic J. Hoerr
Silke Rautenschlein 1
0 Veterinary Research Institute , Hudcova, Brno , Czech Republic, 3 Veterinary Diagnostic Pathology, Fort Valley, Virginia , United States of America
1 University of Veterinary Medicine Hannover, Clinic for Poultry, BuÈ nteweg , Hannover , Germany
Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease. IBD virus (IBDV) is the causative agent, which may lead to high morbidity and mortality rates in susceptible birds. IBDV-pathogenesis studies have focused mainly on primary lymphoid organs. It is not known if IBDV infection may modify the development of the gut associated lymphoid tissues (GALT) as well as the microbiota composition. The aim of the present study was to investigate the effects of IBDV-infection on the bursa of Fabricius (BF), caecal tonsils (CT) and caecum, and to determine the effects on the gut microbiota composition in the caecum. Commercial broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 (Experiment 2) or 15 (Experiment 1) days post hatch (dph). Virus replication, lesion development, immune parameters including numbers of T and B lymphocytes, macrophages, as well as the gut microbiota composition were compared between groups. Rapid IBDV-replication was detected in the BF, CT and caecum. It was accompanied by histological lesions including an infiltration of heterophils. In addition a significant reduction in the total mucosal thickness of the caecum was observed in vvIBDV-infected birds compared to virus-free controls (P < 0.05). vvIBDV infection also led to an increase in T lymphocyte numbers and macrophages, as well as a decrease in the number of B lymphocytes in the lamina propria of the caecum, and in the caecal tonsils. Illumina sequencing analysis indicated that vvIBDV infection also induced changes in the abundance of Clostridium XIVa and Faecalibacterium over time. Overall, our results suggested that vvIBDV infection had a significant impact on the GALT and led to a modulation of gut microbiota composition, which may lead to a higher susceptibility of affected birds for pathogens invading through the gut.
Data Availability Statement: All relevant data are
within the paper and its Supporting information
Funding: Li Li was supported by the Chinese
Scholar Council and Ivan Rychlik was partially
supported by AdmireVet project CZ.1.05/2.1.00/
01.0006 ± ED0006/01/01 from the Czech Ministry
of Education. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Veterinary Diagnostic Pathology, LLC provided
support in the form of salaries for authors [FH], but
Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease (IBD)
]. To date, this disease is prevalent in most of the poultry-producing regions of the world [2,
did not have any additional role in the study design,
data collection and analysis, decision to publish, or
preparation of the manuscript. The specific roles of
these authors are articulated in the `author
3]. Very virulent (vv) IBDV infection generally results in immunosuppression, which may lead
to high mortality rates in susceptible chickens. vvIBDV-induced immunosuppression in the
early phase of the chicken's growing period may result in subsequent problems with secondary
infections including also gut-associated diseases, which contribute to the economic losses in
the poultry industry [
IBDV targets IgM+ B cells leading to a severe damage of the bursa of Fabricius (BF). In
recent years, vvIBDV-pathogenesis studies have mainly focused on the primary lymphoid
tissues such as the BF and the thymus. However, only little is known about the effects of vvIBDV
on the gut-associated lymphoid tissues (GALT) besides the BF. These comprise organized
lymphoid tissues such as caecal tonsils (CT), peyer's patches (PP), Meckel's diverticulum and
other lymphoid aggregates located within the lamina propria (LP) along the gastrointestinal
]. These establish a first line of defense against invading pathogens and also contribute
to systemic immune responses [
]. Previous studies demonstrated that lymphocytes and
macrophages in the intestine play a role in vvIBDV transmission to the BF and other sites [
vvIBDV may impair the intestinal mucosal immunity. One recent study demonstrated that
vvIBDV infection led to a decrease in villus height, and a reduction in the number of intestinal
intraepithelial lymphocytes (IEL) and mast cells in the intestine of specific-pathogen-free
(SPF) chickens . These effects on gut associated immunity were observed during the first
three days after vvIBDV infection.
Little is known about the interaction between vvIBDV and the gut microbiota. The
interaction between viruses and the microbiota is presently an area of intensive research in
human and other animal models [
]. It has been shown that the immune system is also
likely to be an important contributor to host control over microbiota composition [
Several cell types such as goblet cells, IgA secreting B cells as well as intraepithelial lymphocytes
(IEL) function together to stratify luminal microbes and to minimize bacterial-epithelial
contact [12±14]. Likewise it has been shown that microbiota shapes immunity. Studies
comparing germ-free and microbiota colonized mice revealed an effect of microbial
colonization on the formation of lymphoid tissues and subsequent immune system development
]. We hypothesize that vvIBDV may lead to a modification of the GALT and
subsequently the gut microbiota composition, which enhances the risk of pathogen invasion of
the host through the gut [
]. Previously another important immunosuppressive virus
of chickens, Marek's disease virus (MDV), was shown to modify the core gut microbiota
Our objective was to investigate the effect of vvIBDV on the GALT of commercial
broiler chickens and the gut microbiota composition. In two experiments, broiler chickens
were experimentally inoculated with vvIBDV at 14 or 15 day post hatch, when the
maternally derived IBDV antibodies (MDA) were confirmed to be below the break through level
of the virus. Lesion development, viral antigen load, and local immune cell populations
were investigated in selected GALT such as the BF, CT and caecum. In addition, caecum
harbors a more diverse microbial community compared to other intestinal sections, and
it is physically associated with the CT, therefore caecal content was selected to determine
the gut microbiota by 16S rRNA sequencing. Our study clearly demonstrates that vvIBDV
not only modified immune cell populations in the BF but also in CT and caecum, and
subsequently led to changes in the microbiota composition. This indicates that not only
humoral immunity and innate immune parameters are affected in IBDV-infected birds,
but also the intestinal barrier is significantly altered, which could allow secondary
pathogens to colonize.
2 / 25
Material and methods
One-day-old commercial broiler chicks (Ross 308, mixed sex) were purchased from the
hatchery BruÈterei Weser-Ems, GmbH & Co. KG, Visbek, Rechterfeld, Germany. All chicks were
kept in the same room at the Clinic for Poultry under isolation conditions on wood shavings
until the day of inoculation. On this day, the chicks were randomly distributed to different
isolation units and subsequently inoculated with virus or phosphate-buffered saline (PBS). All
groups received the same feed and water from the same commercial source ad libitum. The
chicks did not receive any vaccination. Experiments were conducted following the regulations
for animal welfare of Lower Saxony and were approved by the Lower Saxony State Office for
Customer Protection and Food Safety (LAVES: 33.12-42505-04-13/1215). Each bird was
individually marked with a wing tag.
All groups were evaluated daily for clinical signs. The following parameters were
determined to evaluate the health status of the animals and clinical scores/animal in case of illness.
If clinical disease would have been observed in one group, individual birds with clinical signs
such as huddling, ruffled feathers, separation from the group, loss of feathers and skin
integrity, dirty feathers, bleeding, nasal or conjunctival discharge would have been identified.
Individual birds were more closely investigated then to determine the clinical score. The clinical
score is based on the following criteria: breathing and excrement quality, injuries, conjunctiva
condition, modification at the blood sampling region, feed and water intake, and locomotion.
Each parameter was evaluated and scored from 0 to three ranging from no signs/normal
(score 0) to severe signs (score 3) (S1 Table). The maximum total clinical score for all six
parameters is 18. The following criteria led to the definition of the humane endpoint: one bird
shows at three subsequent controlling time points (at least two observation time points per
day) a total clinical score of at least 5 to 7 or for one or more than one parameter a score of 3. If
this would have been confirmed, birds would have been immediately taken to the necropsy
hall and sacrificed. A therapeutic approach was not followed due to the fact that therapeutic
intervention would modify the outcome and possible interpretation of the experiment.
In the case of minor injuries affected birds would have been separated within the isolation
room from the group and treated with a silver spray to protect the injured area and support
the healing process. The birds were placed back in the group, if a clear recovery was visible.
If more birds would have been injured due to pecking, the light intensity would have been
reduced to stop pecking.
If the group would have shown at least one of the following symptoms: depression, ruffled
feathers, closed eyes, reluctant to move, huddling, birds would have been clinically observed at
least two to three times/day, and the room temperature be raised.
Virus and inoculum preparation
The vvIBDV strain 89163/7.3, used in this study, was kindly provided by N. Eterradossi,
AFSSA, Ploufragan, France [
]. The preparation and the challenge dosage of vvIBDV with
103 egg infectious dose (EID)50 /bird via eye drop were described previously [
]. The virus
had been stored at -80ÊC.
Samples of the BF, CT and the middle region of the caecum were collected, fixed in 4% (w/v)
phosphate-buffered formalin for 48 hours, embedded in paraffin, sectioned (2 μm) and further
processed for histological examination following standard procedures as previously described
3 / 25
]. Bursal follicular lesion scores were determined according to previously described scores
(lesion scores: score 1 = 1±25%, score 2 = 26±50%, score 3 = 51±75% and score 4 = 76±100% of
bursal follicles showing more than 50% lymphoid cell depletion [
]. CT lesions included
edema, infiltration of plasma cells and heterophilic granulocytes.
Caecum lesions were characterized by the loss of epithelial integrity, edema, infiltration of
plasma cells as well as heterophils [
]. Total mucosal thickness, including the mucosal
epithelium and lamina propria of the cecum were determined by morphometric analysis. The
caecum mucosa was measured at 5 representative points in each caecum using ImageJ software
(National Institutes of Health, USA), with the line tool calibrated from pixels to micrometers
using the reference bar standard (calibration: 0.457 pixels/micrometers). The mean of mucosal
thickness was calculated for six birds per group.
Immunohistochemical staining of vvIBDV antigen
Sections of the BF, CT and the middle region of the caecum were prepared as previously
]. vvIBDV antigen was detected using a polyclonal rabbit anti-IBDV serum at a
dilution of 1:5000 [
]. The secondary anti-rabbit IgG biotinylated antibodies and ABC
reagent (Universal Vectastain 1Elite1ABC Kit, Vector Laboratories Inc.,
Wertheim-Bettingen, Germany) were used according to the manufacturer's instructions [
]. DAB (DAB
peroxidase substrate Kit, Vector Laboratories Inc.) was used to visualize the enzyme-linked
complex. Sections were investigated by light microscopy. The antigen score of each group is
based on the number of vvIBDV-antigen positive cells per field at a magnification of 200 x:
1 = 1±10, score 2 = 11±50, score 3 = 51±100 and score 4 = over 100 vvIBDV antigen-positive
cells in 10 randomly selected microscopic fields per bird (n = 6/group).
Mast and goblet cell staining
The preparation of the BF, CT and the middle of the caecum for mast and goblet cell staining
was done as previously described [
]. Briefly, for the mast cell staining, sections of 2 μm were
stained with 0.8% toluidine blue (Sigma Co., UK) for 2 min after the rehydration. Slides were
washed with distilled water for 1 min, immediately dehydrated using 95% alcohol, and 100%
alcohol for 2 min, cleaned with xylene and then mounted with neutral gums. For the goblet
cells staining, sections were stained with 1% Alcian blue 8GS in PBS (Sigma Co., UK) for 15
min, rinsed for 5 min with distilled water, dehydrated with 95% alcohol and 100% alcohol for
2 min, respectively, cleaned with xylene and then mounted with neutral gums. The number of
mast cells in the caecum was counted in five randomly selected fields per bird (n = 6/group)
under the microscope (200 x magnification) and the goblet cells were counted per villus, from
tip to the crypt and calculated as the mean number per villus.
Detection of immune cells by immunohistochemistry
Samples of BF, CT and the middle region of the caecum were snap frozen in liquid nitrogen.
Frozen sections of tissues of 4 μm were prepared. Immunohistochemical staining of immune
cells was conducted according to the manufacturer's instructions [
]. The following primary
antibodies were used at the following work concentration of 0.05 μg/ml: anti-CD4, anti-CD8β,
anti-Bu1, anti-KuL01 and anti-IgA (Southern Biotech, provided by Biozol, Eching, Germany).
Secondary anti-mouse IgG biotinylated antibodies, ABC reagent (Vectastain 1Elite1ABC
Kit, Vector Laboratories Inc., Wertheim-Bettingen, Germany), as well as DAB (DAB
peroxidase substrate Kit, Vector Laboratories Inc.) were used according to the manufacturer's
]. Sections were examined via light microscopy. The lymphocyte populations
and macrophages in the BF as well as the IgA-positive cell populations in the caecum were
4 / 25
evaluated by counting the number of stained cells at a magnification of 200x in five randomly
selected microscopic fields per bird (n = 6/group). The caecal Bu1+ and T LPL as well as
KuL01+ cells were evaluated by counting the number of positive cells per three crypt regions
at a magnification of 200x of five randomly selected fields per bird (n = 6/group) [
For the epithelial lymphocytes (IEL), the numbers of CD4+ and CD8û+ cells were evaluated
by counting the positive cells in the epithelial layer at a magnification of 200 × of five randomly
Gut microbiota composition
Microbiota composition was determined by sequencing of the V3/V4 variable region of 16S
rRNA genes exactly as described previously [
]. The resulting sequences were classified by
RDP Seqmatch with an OTU (operational taxonomic units) discrimination level set to 97%
using Qiime software.
vvIBDV-antibody detection by ELISA
Circulating anti-IBDV-specific IgG antibodies were detected by the commercially available
enzyme-linked immunosorbent assay (ELISA) ProFLOK1 IBD PLUS ELISA antibody test kit
(Synbiotics Co., Kansas City, Mo.). Anti-IBDV-antibody titers were calculated based on the
OD values and are presented as mean titer ± standard deviation (SD) per group.
Two experiments were conducted in the present study.
All people who participated in the animal experiments were either veterinarians with over
10 years of experience in conducting animal experiments with birds or specifically trained by
attending a FELASA C course, or were animal care takers, which were specialized in managing
poultry under experimental conditions. All participants were specifically approved by the
Lower Saxony State Office for Customer Protection and Food Safety (LAVES) to contribute to
these animal studies.
Experiment 1. Forty-eight one-day-old commercial broiler chickens were raised at the
Clinic for Poultry and were randomly divided into two groups (vvIBDV-inoculated group and
virus-free control). At seven and 14 days post hatch (dph), sera were collected for maternally
derived antibody (MDA) detection. Twenty-four chickens were inoculated with vvIBDV at the
age of 15 dph with a dosage of 103 egg-infectious dose (EID)50/bird via eye drop. Twenty-four
chickens were kept as virus-free controls which received PBS. Clinical signs were monitored
throughout the whole experiment. Six birds of each group were randomly selected and
sacrificed at three, seven, 14 and 21 days post inoculation (dpi), when the experiment was
terminated. Serum samples were collected for the detection of vvIBDV specific IgG antibodies by
ELISA. BF was weighted to calculate the organ to body weight ratio. Pathological lesions were
determined. Samples of BF, CT, as well as the middle of the caecum were formalin-fixed and
sectioned for the detection of histopathological lesions. Samples of BF, CT and the middle
region of the caecum were collected for immunohistochemical detection of vvIBDV-antigen,
immune cells, mast and goblet cells. In addition, caecum content was collected at necropsy for
gut microbiota composition analysis.
Experiment 2. Experiment 2 was partially a repeat of Experiment 1 with a total of
thirtysix broiler chickens. Eighteen chickens were inoculated with vvIBDV at a dose of 103 EID50/
bird at 14 dph. The remaining eighteen chickens were kept as virus-free controls. Six broilers
of each group were randomly selected and different to Experiment 1 necropsied at 10, 14 and
21 dpi, when the experiment was terminated. As in Experiment 1, serum samples were
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collected for MDA detection and at necropsy for the detection of vvIBDV specific IgG
antibodies by ELISA. Parameters, which were investigated at necropsy in Experiment 2 as a repeat
of Experiment 1 include: bursa/body weight ratio, histological lesions, viral-antigen detection,
immune cell populations including T and B lymphocytes, mast cell, and goblet cells in the BF,
CT, and caecum. Different to Experiment 1, gut microbiota composition of caecum content
was only determined at 14 dpi.
In all experiments birds were randomly selected for necropsy (n = 6/group and time point
in each experiment). They were stunned using blunt trauma, which was placed on the
frontoparietal region, and subsequently immediately killed by exsanguination, which was approved
by the animal welfare committee of the Lower Saxony State Office for Customer Protection
and Food Safety.
Statistical analysis was performed using Statistix version 9.0 (Analytical software, Thallahassee,
USA). Two Sample T test (two-tailed) was used to analyze the difference in the total mucosal
thickness, and LPL immune cells between groups because data were normally distributed as
tested by the Shapiro Wilk-Test. The Wilcoxon Rank Sum T test was used to analyze the
differences in IEL, mast cells, and antibodies titer between groups at the indicated time points
because data for these parameters were not-normally distributed as tested by the Sharpiro
Wilk-Test. P < 0.05 was considered as statistically significant. Graphs were prepared with
GraphPad v6 (Prism, LaJolla, USA).
Clinical signs and tissue lesion development
vvIBDV was inoculated when MDA were below the breakthrough titer of the virus in both
experiments. In Experiment 1 and 2, vvIBDV was inoculated at 15 and 14 days post hatch
based on the break-through levels as calculated by the Deventer formula and published
previously (data not shown) [
No chicken died due to vvIBDV-infection in either experiment, and none showed clinical
signs including ruffled feathers, huddling, respiratory distress or diarrhea after
virus-inoculation (S1 Table), which confirms previous experimental vvIBDV-infection studies in
commercial broilers [
Consistent with previous studies [
], vvIBDV inoculation induced a significant
increase in the bursa to body weight ratios (B/BW) at three dpi in comparison to the virus-free
controls (S2 Table, P < 0.05). Afterwards starting at seven dpi, bursal atrophy was observed in
vvIBDV-infected birds compared to virus-free controls. Comparable results were observed in
Experiment 2, with a significant decrease in B/BW at 21 dpi in comparison to the virus-free
control (S2 Table, P < 0.05). vvIBDV infection induced an increase in anti-IBDV IgG-specific
antibodies in both experiments. A significant upregulation was observed in both experiments
with ELISA-titers of log10 3.5 ± 0.2 at seven dpi in Experiment 1 and 3.2 ± 0.2 in Experiment 2
at 10 days post hatch compared to virus-free controls in Experiment 1 (2.4 ± 0.8) and 2
(1.6 ± 0.8), respectively (P < 0.05).
A depletion of lymphoid cells in bursal follicles was observed microscopically throughout
both experiments, and a trend of bursal recovery with beginning repopulation of follicles was
observed starting at 21 dpi (S1 Fig). Bursa lesion scores were 4.0 ± 0.0 starting at three dpi in
all vvIBDV-infected groups in both experiments (S3 Table).
An infiltration of heterophils was observed in the BF of vvIBDV-infected birds at three dpi
in Experiment 1 (Fig 1A and 1B). In the CT, vvIBDV-infected birds exhibited cellular
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Fig 1. vvIBDV infection led to histological lesions in the BF (A, B), CT (C, D) and caecum (E, F) (Experiment 1 as a representative experiment).
Control = virus-free control, vvIBDV = vvIBDV-infected group. A, C, E are representative sections from virus-free controls at three dpi, and B, D, F are
representative sections from vvIBDV-infected birds at three dpi.
destruction of germinal centers (Fig 1D), and a slight infiltration of heterophils in the
submucosal area at three and seven dpi (Experiment 1), these lesions were not observed at later time
points in either experiment. An infiltration of heterophils was also detected in the caecum of
7 / 25
vvIBDV±inoculated birds at three and seven dpi (Experiment 1) in comparison to virus-free
controls (Fig 1E and 1F). A significant decrease in the total mucosal thickness was observed at
seven, 14 and 21dpi in the caecum of vvIBDV±inoculated birds compared to virus-free
controls (Table 1, P < 0.05).
Effect of vvIBDV on goblet and mast cells
Consistent with previous studies [
], an increase in the number of goblet cells in the out layer
of the bursa was observed in the vvIBDV-infected chickens in comparison to virus-free
controls in both experiments (data not shown). In the caecum, vvIBDV-infected birds showed a
significant increase in the number of goblet cells at three, seven (Experiment 1, Fig 2) and 10
dpi (data not shown, Experiment 2) compared to virus-free controls (P < 0.05). While a
significant lower number of goblet cells was observed at 21 dpi in the caecum of vvIBDV-infected
birds compared to virus-free control (Fig 2 and S2 Fig, P < 0.05)
Mast cells were frequently detected in the mucosal LP of the caecum, while they were rarely
detected in the BF and CT of virus-free birds. The effect of vvIBDV on the mast cell numbers
varied between different tissues. A transient increase of mast cell numbers was observed at
three dpi in the BF and CT of vvIBDV-infected birds in comparison to virus-free controls in
Experiment 1 (Fig 3A±3D). Compared to virus-free controls, a significant decrease in the
number of mast cells in the caecum was observed at three, seven and 14dpi in Experiment 1
and at 10 and 14 dpi in Experiment 2 in the vvIBDV-infected birds (Fig 3E±3H, P < 0.05).
vvIBDV antigen detection
All virus-free birds were negative for vvIBDV. Consistent with previous studies, vvIBDV
replication was most vigorous in the BF [
]. vvIBDV antigen was detected at high levels in the
BF at three dpi (Experiment 1, Fig 4), and then the number of antigen-positive cells decreased
over time. At 21 dpi nearly no IBDV-positive cells were detected in the BF in either
experiment. vvIBDV-antigen positive cells were also observed at three and seven dpi in the CT and
caecum and at 14 dpi in the CT of vvIBDV-infected birds in comparison to virus-free controls
in Experiment 1 (Fig 4). No IBDV-positive cells were detected in the CT and caecum at 10, 14
and 21 dpi in Experiment 2. The score of vvIBDV antigen-positive cells was lower in these
tissues compared to the BF.
Effects of vvIBDV-infection on immune cells in the LP of the GALT
Consistent with previous studies, vvIBDV infection led to a depletion of B lymphocytes, a
significant increase in T lymphocytes, as well as transient infiltration of macrophages at three and
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Fig 2. Goblet cell staining in the caecum (A, B) of birds after three days post vvIBDV inoculation (Experiment 1) and average number of goblet cells at varies
time points post virus-inoculation (C). A, is the representative picture from virus-free controls, and B is the representative picture from a vvIBDV-infected bird. C is
the summary of the number of goblet cells in the caecum in Experiment 1. significantly different between groups at the indicated time points (Wilcoxon Rank Sum
Test, P < 0.05). control = virus-free control, vvIBDV = vvIBDV-infected group.
seven dpi in the BF (P < 0.05) [
]. In the caecum, vvIBDV-infected birds showed a significant
decrease in the number of LP B lymphocytes at seven and 14 dpi compared to virus-free
controls in Experiment 1 (Fig 5A and S3 Fig, P < 0.05), and this significant difference was also
observed at 10 and 14 dpi in Experiment 2 (P < 0.05). Compared to virus-free controls,
9 / 25
Fig 3. Mast cell detection in the BF (A, B), CT (C, D) and caecum (E, F) of control (A, C, E) and vvIBDV-inoculated (B, D, F)
birds after three days post vvIBDV inoculation (Experiment 1) and a summary of mast cell detection over time in the caecum
of animals in Experiment 1 (G) and Experiment 2 (H). Arrows indicate stained mast cells. Enlargements of indicated regions
with mast cells are presented in figures B, D, E, F. indicates significant differences between groups at the indicated time points
(Two-sample T test, P < 0.05). Control = virus-free control, vvIBDV = vvIBDV-infected group. n = 6 per group.
vvIBDV-infected birds had a reduced number of B cells and smaller sizes of germinal centers
in the CT throughout both experiments (Fig 6B). The decrease in the number of LP B
lymphocytes in the caecum coincided with a significant decrease in the number of IgA secreting cells
in the caecum in the area of the lamina propria in Experiment 1 of vvIBDV-infected birds in
comparison to virus-free controls (Table 2, S4 Fig). No IgA-positive cells were detectable in
Fig 4. Staining of IBDV-antigen in the BF (A, B, C), CT (D, E, F), and caecum (G, I, H) of chickens at three days post vvIBDV-inoculation (Experiment 1).
Control = virus-free control, vvIBDV = vvIBDV-infected group. A, D, G are sections from virus-free controls, and B, E, I are from vvIBDV-infected birds. C, F, H are the
summary of antigen positive cells in the BF, CT and caecum, respectively.
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Fig 5. Immunohistochemical detection of Bu1+ (A), CD4+ (B), CD8β+ (C), and KuL01+ (D) cells in the caecum of chickens at different days after
vvIBDVinfection (Experiment 1). indicates significant difference between groups at the indicated time points (Two-sample T test (A) or Wilcoxon Rank Sum Test (B, C, D),
P < 0.05). Control = virus-free control, vvIBDV = vvIBDV-infected group.
An increase in the number of CD4+ and CD8û+ LPL in the caecum was detected in both
experiments. This increase was significant at three, seven and 14 dpi when comparing
vvIBDV-infected birds and virus-free controls in Experiment 1 (Fig 5B and 5C, P < 0.05).
Comparable results with a significant increase in the number of CD4+ LPL at 10 dpi were
observed in the caecum of vvIBDV-infected birds compared to virus-free controls in
Experiment 2 (P < 0.05). Similar results were observed in the CT with an infiltration of T cells in the
submucosal area at three dpi (Fig 6D and 6F).
A significant increase in the number of macrophages was observed at three and seven dpi
in the caecal LP of vvIBDV-infected birds compared to virus-free controls (Fig 5D, P < 0.05).
An increase in the amount of macrophages was also induced by vvIBDV in the CT at three
and seven dpi, but not at later time points (Fig 6H).
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Fig 6. Immunohistochemical detection of Bu1+ (A, B) CD4+ (C, D), CD8β+ (E, F), and KuL01+ (G, H) in the CT of chickens after
three days post vvIBDV inoculation (Experiment 1). A, C, E, G are from virus-free controls, and B, D, F, H are from vvIBDV-infected
Effects of vvIBDV-infection on IEL in caecum
IEL B cells were rarely detectable in non-as well as infected birds. A significant decrease in the
number of CD4+ IEL was observed at three and seven dpi in the caecum of vvIBDV-infected
birds compared to virus-free controls in Experiment 1 (Fig 7A±7C; P < 0.05).
vvIBDVinfected birds also had a significant decrease in CD8û+ IEL numbers at seven dpi in the
caecum compared to virus-free controls (Fig 7D±7F; P < 0.05).
Effect of vvIBDV on caecum microbiota composition
To obtain a deeper insight into the changes occurring to the caecal microbiota during vvIBDV
infection, we investigated the caecal content at three, seven, 14 and 21 dpi in Experiment 1 and
14 dpi in Experiment 2. The sample with the lowest coverage was characterized by 3677
sequences, and 10283 sequences were available for the sample with the highest coverage.
Representatives of nine phyla were detected at all the investigated time points. Independent of
vvIBDV infection, the majority (over 95%) of microbiota was formed by representatives of
Firmicutes, Proteobacteria, Acitinobacteria and Bacteroidetes (Figs 8 and 9). The relative
representation of individual phyla in the caecal samples remained stable with Firmicutes forming more
than 90% of the microbiota between 18 dph to 36 dph [
A more detailed analysis was performed on the family and genus level. At the family level,
the majority of bacteria were Lachnospiraceae and Ruminococcaceae in both vvIBDV-infected
and virus-free control groups. Independent of vvIBDV inoculation, the abundance of
Lachnospiraceae decreased over time. It ranged from 54.4% at 18 dph to 42.2% at 36 dph.
Ruminococcaceae showed a reverse trend with an abundance ranging from 25.6% at 18 dph to 42.2% at 36
dph (Fig 8). At the genus level, the abundance of Faecalibacterium increased ranging from
0.5% at 18 dph to 13.2% at 29 dph, and decreased afterwards to 9.3% at 36 dph (Fig 9).
vvIBDV inoculation modified the gut microbiota. Independent of age, vvIBDV inoculation
led to a lower abundance of Clostridium XlVa at three dpi, which was followed by a higher
abundance at seven and 21 dpi compared to virus-free controls (Fig 9). A higher abundance of
Faecalibacterium at seven dpi, but a lower abundance at 14 and 21 dpi was observed in
vvIBDV-infected birds in comparison to virus-free controls (Fig 9). There was also a decrease
in abundance of Escherichia/Shigella detected at three, 14 dpi and 21 dpi in vvIBDV-infected
birds compared to virus-free controls (Fig 9), confirming the decrease in the abundance of
Dpi = days post inoculation; Control = virus-free control; vvIBDV = vvIBDV-infected group.
indicates significant differences between groups at the indicated time point (P < 0.05, n = 6/group).
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Fig 7. Average number of CD4 (A, B, C) and CD8β-positive (D, E, F) cells located within the epithelia (IEL) of the caecum of chickens at seven (A, B, D, E) or
different days post vvIBDV inoculation (C, F) (Experiment 1). indicates significant difference between groups at the indicated time points (Wilcoxon Rank Sum T
test, P < 0.05). Control = virus-free control, vvIBDV = vvIBDV-infected group. Arrows indicate IEL cells, n = 6 per group.
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Fig 8. Bacterial communities of caecal samples from chickens at the family level (Experiment 1). Data were analyzed
using QIIME. The x-axis represents the groups at different days post inoculation (dpi) and the y-axis represents the relative
abundance of sequences. Control = virus-free control, vvIBDV = vvIBDV-inoculated group. NA = not analysed.
Enterobacteriaceae in the vvIBDV-infected birds compared to virus-free controls (Fig 8).
Similar observations were made in Experiment 2 at 14 days pi.
Gut health is a very important aspect in poultry production and significantly contributes to the
overall health and performance of a flock [
]. If the gut immunity as well as the mucosal
intestinal barrier is disturbed, this may have a severe impact on the bird's development and may
enhance the risk for not only gut but also systemic infections [
diseases may influence the development of the gut immunity and possible the microbiota
composition and subsequently modify the intestinal barrier [
]. Neither the effect of vvIBDV
infection on the GALT nor the possible correlation to the gut microbiota composition has
been investigated so far. In the present study, commercial broiler chickens were infected with
vvIBDV at 14 (Experiment 2) or 15 (Experiment 1) days post hatch, when MDA had reached
the break-through levels of the virus. vvIBDV-infection was confirmed by viral antigen
detection in the BF, and bursal atrophy with a depletion of B lymphocytes. Seroconversion was
detected in vvIBDV-infected birds starting at seven dpi. Birds showed beginning recovery of
microscopical lesions in the BF at 21 dpi.
In addition to the changes in the BF, detectable histopathological lesions were also observed
in the CT and caecum of vvIBDV-infected birds. These findings coincide with the presence of
virus antigen. CT and caecum showed structural recovery starting at 14 dpi. Viral clearance
and recovery occurred faster in the CT and caecum compared to the BF. IBDV initially
replicates in lymphocytes and macrophages in the gut intestine [
]. It reaches the liver and
enters the bloodstream leading to a primary viremia within 11 hours post infection. The virus
starts replicating in proliferating B lymphocytes of the BF [
]. Afterwards it migrates into
the different tissues via blood circulation, causing secondary viremia [
]. In the present study,
we observed the viral antigen positive cells in the germinal centers together with lesions from
three to seven dpi. We speculate that the lesion in the CT and caecum are due to the secondary
Gut associated mucosal immunity is important as a first barrier of host defense against
pathogen invasion. An increase in the number of mast cells was observed in the BF and CT
during the acute phase of the disease what confirms previous studies showing IBDV infection
may affect the number and morphology of mast cells [
]. Our method does not allow the
differentiation if this was an increase in absolute numbers or just a change in relative cell
numbers due to the IBDV-mediated B cell depletion. However Wang et al.  also demonstrated
by using a comparable staining method a mast cell number increase in the thymus, a tissue,
which is not as severely affected by B cell depletion as the bursa, providing circumstantial
evidence that changes in mast cell numbers may not only be due to B cell depletion. vvIBDV
infection also led to a significant decrease in number of mast cells in the caecum. We propose
that this decrease contributes to a compromised gut mucosal immunity caused by vvIBDV
]. A significant increase in the number of goblet cells was observed in the caecum
of vvIBDV-infected birds compared to virus-free controls (P < 0.05). Goblet cells, together
with epithelial cells and macrophages are regarded as the major cellular constituents of the
innate defense system [
]. It was demonstrated that various enteric infections including
bacteria and viruses are associated with an alteration of the goblet cell response [41±43]. Goblet
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Fig 9. Bacterial communities of caecal samples from chickens at genus level (Experiment 1). Data were analyzed
using QIIME. The x-axis represents the groups at different days post inoculation (dpi) and the y-axis represents the
relative abundance of sequences. Control = virus-free control, vvIBDV = vvIBDV-inoculated group. NA = not
cell hyperplasia was suggested to be controlled by immunological mechanism during infection
]. vvIBDV-infection may also interfere with the function of goblet cells .
A significant increase in the number of T cells was observed in the BF and CT, as well as in
the LP of the caecum of vvIBDV-infected birds compared to virus-free controls (P < 0.05). It
has been shown that T lymphocytes infiltrate into the LP of the gut after various enteric
infections, such as with rotavirus [
] or Salmonella Enteritidis [
]. Previous studies had already
demonstrated that an infiltration of T cells into the BF starts at the early stage of
]. Cytotoxic T lymphocytes were suggested to play a role in the clearance of IBDV, but
also to contribute to lesion development in the bursa [
]. Interestingly also the number of
T LPL increased in the caecum of vvIBDV-infected birds, which suggests that the T cells are
not only activated in the BF but also in the gut. However, vvIBDV infection led to a significant
decrease in the number of CD4+ IEL at three and seven and CD8û+ IEL at seven dpi
(P < 0.05). The increase in T LPL might be due to the movement of CD4+ and CD8û+
lymphocytes from the intraepithelial to the submucosal area. LPL and IEL are two distinct
components of the GALT. Based on our investigation there is not clear correlation between the
increase in T LPL and IBDV-antigen, as there was no detectable IBDV-antigen in the LP.
A decrease in the number of IgA+ secreting cells was observed in the caecum of
vvIBDVinfected birds compared to virus-free controls. IgA is the most common immunoglobulin in
the mucosal tissue, being an important line of the immunological defense against invading
enteric pathogens. In addition, IgA regulates the ecological balance of the microbiota and has
a fundamental role in mucosal homeostasis [
]. This detected decrease in IgA+ cells may be
due to direct infection of these cells by IBDV. But older studies suggested that IBDV may
target receptors mainly presented on the surface of IgM-bearing cells. vvIBDV-exposure did not
reduce the levels of total serum IgA, IgG, and IgM, nor did it affect IgG and IgA B-cells in the
spleen, while the caecum was not investigated [
]. Therefore, we speculate that the decrease
in IgM+ B cells in the BF and CT possibly led to the subsequent reduction of IgA+ secreting
cells in the caecum. Further studies should be conducted to determine if local and systemic
IgA-levels may be reduced under comparable experimental conditions. Overall, this reduction
in IgA+ cells together with the decrease in the number of mast cells as well as T IEL may result
in insufficient protection against other pathogens, such as Salmonella [
], or Escherichia coli
]. Early findings indicated that IBDV-induced humoral immunity suppression led to failure
of seroconversion to other pathogens including infectious bronchitis virus (IBV) [
infectious anemia virus (CIAV) [
The gastrointestinal tract represents one of the primary sites of exposure to pathogens [
There is limited literature on dysbiosis caused by viruses. Recent studies targeted at the
influence of the human immunodeficiency virus (HIV) as well as the simian immunodeficiency
virus (SIV) on the gut microbiota and showed a selective enrichment of few phenotypes in the
gut microbiota after viral infection [
]. Interestingly, we observed a higher abundance of
Ruminococcaceae and Desulfovibrionaceae in vvIBDV-infected birds compared to virus-free
control. Similar finding were observed in HIV chronically infected patients [
]. It was also
shown that HIV infection leads to a lower level of local IgA. This possibly contributes to HIV
infection-associated enhanced microbial translocation, which may lead, in turn, to a chronic
state of immune activation as noted in many HIV patients [
]. HIV targets the CD4+ cells,
while IBDV targets B cells. However, both virus infections lead to a decrease in the number of
19 / 25
IgA+ cells in the intestine. We may speculate the decrease in IgA might attribute to the
dysbiosis of gut microbiota.
In the present study, vvIBDV-infected birds had a lower abundance of Clostridium XIVa at
three dpi compared to virus-free birds. This trend reversed between seven and 21dpi. An
increase in the abundance of Faecalibacterium was observed at three and seven dpi in the
vvIBDV-infected birds compared to virus-free controls, while this trend also reversed starting
at 14 dpi. The acute phase of IBDV infection is between three to five dpi, which includes the
peak of IBDV replication, and a strong inflammatory response with a `cytokine storm'. After
the acute phase, all these reactions decrease over time, possibly coinciding with modification
in the gut microbiota composition. Previous studies showed that Clostridium spp. are strong
inducers of colonic T regulatory (Treg) cells [
]. Treg cells are primary mediators in
maintaining the immune homeostasis and play a critical role in the suppression of extensive
intestinal inflammation. In inflammatory bowel disease also a decrease in the abundance of
Clostridium XIVa and Faecalibacterium had been observed [62±64]. Therefore, the decrease in
the abundance of Clostridium XIVa at three days post vvIBDV infection might suggest that
vvIBDV interferes with the delicate balance of gut mucosal immunity and may support
harmful intestinal inflammation. The role of Faecalibacterium is unknown in chickens. In human
studies, it was demonstrated that Faecalibacterium prausnitzii is a sensor and a marker of
human health [
]. Intestinal disorders, such as inflammatory bowel disease [
colorectal cancer [
] are associated with a diminished abundance of Faecalibacterium prausnitzii. If
this observation can be transferred to chicken, our data provides circumstantial evidence that
changes in the abundance of Faecalibacterium for example through vvIBDV infections are an
indicator for intestinal disorders.
In conclusion, this study shows for the first time the influence of vvIBDV on the gut
associated immune system and the microbiota composition. These results may help to understand
the far-reaching consequences of immunosuppressive diseases in poultry. The change of the
microbial populations correlated well with changes in immune cell populations such as mast
cells, B cells especially IgA+ cells in the LP of the caecum and CT of vvIBDV-infected birds.
Due to the complexity of the viral pathogenesis and the GALT system of the host, it is difficult
to pinpoint the exact mode of action of the virus on the microbiota. It is not clear if there were
direct or indirect effects of the virus. This has to be evaluated further to be able to improve
chicken's health in the field in the future.
S1 Fig. Histological bursa lesions of virus-free control (A, C, E, G) and vvIBDV-inoculated
(B, D, F, H) chickens at three, seven, 14 and 21 dpi. Arrows indicate beginning recovery in
some bursa follicles.
S2 Fig. Goblet cell staining in the caecum of virus-free control (A) and vvIBDV-inoculated (B) birds after 21 days post virus-inoculation (Experiment 1).
S3 Fig. Immunohistochemical detection of Bu1+ (A, B), CD4+ (C, D), CD8β+ (E, F), and
KuL01+ (G, H) cells in the caecum of virus-free control (A, C, E, G) and vvIBDV-inocu
lated chickens after three days post virus-inoculation (Experiment 1). Arrows indicate the
positive immune cells in the lamina propria of the caecum.
20 / 25
S4 Fig. Immunohistochemical detection of IgA in the caecum of virus-free control (A) and vvIBDV-inoculated (B) chicken after three days post virus-inoculation (Experiment 1).
Arrows indicate IgA positive cells in the lamina propria of the caecum.
S1 Table. Clinical scoring of virus-free control and vvIBDV-inoculated birds during Experiments 1 and 2.
S2 Table. Bursa to body weight ratio of chicken after vvIBDV inoculation. Dpi = days post
inoculation; Control = PBS-inoculated control; vvIBDV = vvIBDV-infected group. letter
indicates significant differences between groups at the indicated time point (P < 0.05, n = 6).
S3 Table. Bursa lesion score after vvIBDV-inoculation (Experiment 1 as a representative
experiment). dpi = days post inoculation; control = PBS-inoculated control;
vvIBDV = vvIBDV-infected group. indicates significant differences between groups at the
indicated time point (P < 0.05, n = 6/group).
We would like to thank Sonja Bernhardt, Christine Haase and Katja Stolpe for their excellent
technical assistance and support. In addition we would like to thank Marina and Thomas for
help with the necropsy work. We also would like to thank August C. Hoerr for morphometric
assessment of the cecum mucosal.
Data curation: Li Li.
Formal analysis: Tereza KubasovaÂ.
Funding acquisition: Ivan Rychlik, Silke Rautenschlein.
Investigation: Li Li, Ivan Rychlik, Silke Rautenschlein.
Methodology: Li Li, Tereza KubasovaÂ, Ivan Rychlik, Frederic J. Hoerr.
Project administration: Silke Rautenschlein.
Software: Tereza KubasovaÂ, Frederic J. Hoerr.
Supervision: Ivan Rychlik, Silke Rautenschlein.
Validation: Frederic J. Hoerr.
Writing ± original draft: Li Li, Silke Rautenschlein.
Writing ± review & editing: Silke Rautenschlein.
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