Inducing experimental bacterial chondronecrosis with osteomyelitis lameness
in broiler chickens using aerosol transmission model
Andi Asnayanti ,*,y,z Anh D. T. Do,*,y Khawla Alharbi,*,y and Adnan Alrubaye *,y,1
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA; yCenter of Excellence for
*
Poultry Science, University of Arkansas, Fayetteville, AR, USA; and zNational Agency of Drug and Food Control,
Jakarta, Indonesia
ABSTRACT Lameness disease attributed to bacterial
chondronecrosis with osteomyelitis in broilers affects
production, animal welfare, and food safety in the poul-
try industry. The disease is characterized by necrotic
degeneration of the rapidly growing femora and tibiae
due to bacterial translocation from the respiratory or
gastrointestinal tracts into the blood circulation, eventu-
ally colonizing the growth plate of the long bones. To
investigate the etiology, pathogenesis, and intervention
measures for BCO, developing an experimental model
that reliably induces BCO lameness is of the utmost
importance. In the past, we have employed a wire-floor-
ing model and a litter-flooring model administered with
a bacterial challenge to investigate strategies for miti-
gating BCO. However, multiple issues on labor-intensive
barn setup and cleanout efforts for the wire-flooring sys-
tem and concern of direct pathogenic exposure to the
broilers for the litter-flooring models rendered these
research models less effective. Thus, we investigated a
new approach to induce experimental BCO lameness
Key words: aerosol, broiler, lameness, chondronecrosis
INTRODUCTION
Bacterial chondronecrosis with osteomyelitis (BCO)
is a primary cause of lameness in the broiler industry,
leading to significant economic losses and tremendous
impacts on animal welfare (McNamee and Smyth, 2000;
Bradshaw and Kirkden, 2002; Wideman and Prisby,
2013; Wideman, 2016; Siegel et al., 2019). The latest
study reported that 19% of the birds in commercial
broiler farms exhibited moderate-to-severe lameness
symptoms (Granquist et al. 2019). The exceptional body
Ó 2024 The Authors. Published by Elsevier Inc. on behalf of Poultry
Science Association Inc. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
4.0/).
Received September 4, 2023.
Accepted January 9, 2024.
1
Corresponding author: aakhalaf@uark.edu
1
using an aerosol transmission model employing a group
of birds reared on wire-flooring pens as a BCO infection
source, and the disease is further disseminated through
the airborne transmission to other birds reared on litter
flooring in the same housing environment. The effective-
ness of the aerosol transmission model in inducing BCO
lameness was concluded from 4 independent experi-
ments. The cumulative lameness generated from the
BCO source group on the wire floors versus negative
control treatments on the litter floors from Experiments
1, 2, 3, and 4 were 84% vs. 69.33%, P = 0.09; 54.55% vs.
60%, P = 0.56; 78% vs. 73.50%, P = 0.64; 81% vs.
74.50%, P = 0.11. Overall, the cumulative lameness gen-
erated from the wire floors was successfully transmitted
to the birds on litter floors without significant statistical
differences (P > 0.05). The effectiveness of the aerosol
transmission model for experimentally triggering BCO
lameness provides a reliable system for evaluating prac-
tical intervention strategies for BCO lameness in
broilers.
2024 Poultry Science 103:103460
https://doi.org/10.1016/j.psj.2024.103460
weight gain of the modern broilers, which far outpaces
the maturity rate of cartilaginous growth plates in the
proximal leg bones, is considered the main determinant
of BCO susceptibility in the broiler industry (McNamee
and Smyth 2000; Bradshaw and Kirkden, 2002; Wide-
man and Prisby, 2013). The disease is characterized by
necrotic degeneration due to pathogenic infection on the
proximal growth plates of the femora and tibiae (McNa-
mee and Smyth, 2000; Li et al., 2012; Wideman et al.,
2012a; Wideman and Prisby, 2013; Prisby et al., 2014).
Staphylococcus aureus, Escherichia coli, Enterococcus
cecorum, and Staphylococcus hyicus were the most prev-
alent cultures from commercial broiler farms affected by
BCO, while Staphylococcus agnetis was the most pre-
dominant species discovered in our research facility at
the University of Arkansas Poultry Research Farm (Al-
Rubaye et al., 2015; Shwani et al., 2020; Ekesi et al.,
2021). Typically, these pathogens translocate from the
2 ASNAYANTI ET AL.
respiratory and gastrointestinal tracts into the blood-
stream, spreading hematogenously to invade the growth
plates of broilers (Wideman and Prisby, 2013; Alrubaye
et al. 2020a).
A reliable experimental model for triggering BCO in
broilers is essential for investigating the etiology, patho-
genesis, and intervention measures for BCO lameness.
Our group has developed 2 approaches to induce experi-
mental BCO lameness. The first model is a wire-floor
challenge model where wire floors impose mechanical
stress on susceptible joints of the long bone of the
broiler’s legs while the birds traverse to access feed and
water in opposite directions. Massive body weight accre-
tion exacerbates the skeletal fragility on wire floors,
weakening the immune system, and promoting patho-
genic translocation from the respiratory and intestinal
tracts to the bloodstream by compromising the tight
junction barrier (Al-Rubaye, 2013; Wideman et al.
2013; Wideman et al., 2014; Wideman, 2016; Al-Rubaye
et al. 2017; Alrubaye et al. 2020a; Alrubaye et al.
2020b). The second model is bacterial challenge adminis-
tered to the broilers reared on wood-shaving litter floor-
ing. We demonstrated that the application of
approximately 105 CFU/mL of S. agnetis in the drinking
water at 10, 20, or 30 d of age for 24 h induced almost
equal incidences of lameness in broilers (Al-Rubaye, et
al., 2017, Alrubaye et al. 2020b).
Rearing broilers on wire floors has been proven to be a
golden approach in inducing naturally occurring BCO
lameness in broilers through mechanical torque and
shear stress on susceptible leg joints (Wideman and
Prisby 2013; Wideman, 2016). Nevertheless, the use of
multiple wire-flooring chambers or pens in large-scale
trials requires highly laborious work for assembly, disas-
sembly, and thorough cleanup between experiments. On
the other hand, even though we have successfully repro-
duced BCO by challenging our S. agnetis 908 isolates to
the drinking water of broilers on litter-flooring (Al-
Rubaye et al. 2015; Al-Rubaye et al. 2017; Alrubaye
et al. 2020b), there is a significant drawback of exposing
pathogen to the surrounding environment including
potentially cross-contaminating of chicken flocks. To
address the issues observed in the previous models, we
propose a new strategy for inducing infectious BCO in
broilers located in the same housing environment. A
group of birds reared on wire floors is used to trigger
BCO in the research house, followed by the aerosol
spread of the pathogens to the hatch-mate birds reared
on litter floors. This report presents our findings on the
power of the aerosol transmission model to effectively
and efficiently transmit BCO lameness in broilers.
MATERIALS AND METHODS
Animal Use and Welfare
All animals used were approved by the University of
Arkansas Institutional Animal Care and Use Committee
(21,007, 21,066, 21,085, 21,144). One-day-old chicks rep-
resenting surplus male over female purchased from
Cobb-Vantress (Siloam Springs, AR, USA) were placed
in designated pens at approximately 750 cm2/chick (60
birds per pen) on d 1. The number of birds was culled to
50 per pen on d 14 to maintain optimal stocking density
(approximately 900 cm2/chick). The total number of
birds used depended on the experimental designs of each
study presented in this report.
Pen Design and Barn Layout
The wire-flooring pen is a rectangular pen with a
dimension of 3 m £ 1.5 m made of a size of 5 cm £ 5 cm
net. The panels were elevated on 30-cm high masonry
blocks to permit manure to pass through and accumu-
late underneath the wire surface. The litter-flooring pen
is a rectangular pen with dimensions of 3 m £ 1.5 m con-
sisting of wood-shaving litter bedding. The schematic
presentation for the aerosol transmission experiments is
shown in Figure 1. Two wire-flooring pens were set up on
the left and right sides at the upstream end of the ventila-
tory air flow, followed by empty spaces as buffers. Two
rows of litter flooring pens were assembled downstream
(downwind) on the left and right sides of the house. Four
exhaust fans at the end of the barn controlled the air circu-
lation assisted by automatic inlet ventilations on the sides
of the house. The overall design allows for a BCO source
upstream/upwind of the remaining unchallenged litter-
flooring pens, allowing the pathogens to spread downwind
via aerosol transmission. This infectious spread is distrib-
uted by air circulation, which is controlled by ventilation
airflow and exhaust fans.
House Installation and Environmental
Control
Each pen was equipped with nipple water lines on 1
side of the pen and 2 feeders were stationed on the oppo-
site side. Automatic controllers were used to regulate
temperature, photoperiod, and ventilation of the house.
The photoperiod was set for 23-h light: 1-h darkness for
the entire experiment. Thermoneutral/optimal brooding
and growing temperatures as follows: 32°C for d 1 to d 3;
31°C for d 4 to d 6; 29°C for d 7 to d 10; 26°C for d 11 to
d 14; and 24°C thereafter.
Feed
From d 0 to d 35, birds received a standard starter
diet (crumbles), after which a finisher diet (pellets) con-
tinued from d 35 to d 56. Basal diet used in the 4 experi-
ments met the broiler feed requirements. The same basal
diet was applied to the BCO source group in the wire-
flooring pens and the negative control group reared in
the litter-flooring pens in each experiment. Birds
accessed the feed and water ad libitum.
 EXPERIMENTAL LAMENESS BY AEROSOL TRANSMISSION
Experimental Designs
Preliminary Study (Spring, March 2020) This prelim-
inary study, designed to investigate the effect of specific
mycotoxins in BCO induction, was the first experiment
to use both the wire and litter-flooring models in the
same house. Six pens were allocated for each of the wire
flooring and litter flooring models, which were randomly
distributed within the house.
Aerosol Transmission Model Four consecutive inde-
pendent experiments presented below were employed to
investigate the effectiveness of the aerosol transmission
model in triggering BCO lameness. All studies shared a
similar experimental setup using the same research facil-
ities in Arkansas Agricultural Research and Extension
Center at building A365W.
Experiment 1 (Fall, October 2020) This study was
conducted to evaluate the efficacy of probiotic products
in reducing incidence of BCO lameness employing 5
treatment groups in a randomized block design. A BCO
infection source group were grown in 2 wire-flooring
pens, while a negative control diet group and 3 groups of
Biomin probiotics were reared in litter-flooring pens
with 3 replicate pens of each treatment.
Experiment 2 (Spring, February 2021) This study
investigated the effects of microalgae and organic trace
mineral supplemented diets on the incidence of BCO
lameness. There were 5 treatment groups in a random-
ized block design, with 2 pens of wire-flooring serving as
3
the source of BCO infection. Dietary treatments
included one group on a negative control diet and 3
groups on diets supplemented with Zinpro Availa ZMC
organic trace mineral at 1,500 mg/kg, microalgae (5%
spirulina algae), and a combination of organic trace min-
eral and microalgae (5% spirulina algae and Availa ZMC
at 1,500 mg/kg) in 5 replicates of litter-flooring pens in
each treatment.
Experiment 3 (Fall, December 2021) This study
investigated the potency of 3 herbal-supplemented diets
in reducing the incidence of BCO lameness. The experi-
ment was divided into 7 treatment groups with 2 wire-
flooring pens serving as the source of infection, and 6
treatment groups − including a negative control,
Nuproxa Panbonis in different concentrations, Indian
Herbs Natufeat, and thyme oil diet supplementations −
reared in the litter-flooring model with 4 pen replicates
of each treatment.
Experiment 4 (Fall, September 2022) This study was
conducted to evaluate the efficacy of the selected
herbal-supplemented diets with various concentra-
tions and treatment strategies for reducing the inci-
dence of BCO lameness. Two wire-flooring pens were
used as the BCO etiological source, and 6 treatment
groups − including a negative control group and 5
groups of Nuproxa Panbonis diet supplementations in
different concentrations and feeding strategies −
reared on litter-flooring pens with 4 pen replicates of
each treatment.

Figure 1. Schematic presentation of the aerosol transmission model to induce experimental BCO lameness. The BCO seeder birds are reared on
the wire floors placed upwind of the other treatment groups grown in the litter floors. Lameness causative agents follow the air flow from the wire
floors to the litter floors. Buffer (empty) zones in between the wire floors and the litter floors are created to prevent direct contact of the BCO seeder
birds and the treatment birds to mimic BCO outbreak in commercial broiler farms.
4 ASNAYANTI ET AL.

Lameness Assessment The treatment effects were analyzed with logistic

Assessment of the cumulative lameness was performed
by daily diagnosis of lame birds from d 22 to d 56. Birds
showing symptoms of goblet gait, kinky back, wing tip
dipping to support the body, and complete paralysis
were diagnosed as clinically lame birds. All lame birds
were humanely euthanized for necropsy and further
gross evaluation of tibial and femoral lesions based on
the classification presented by Wideman (2016) as fol-
lows: N = Femur head and proximal tibia appear
entirely normal; FHS = Proximal Femoral Head Separa-
tion (epiphyseolysis); FHT = Proximal Femoral Head
Transitional degeneration; FHN = Proximal Femoral
Head Necrosis; THN = Proximal Tibial Head Necrosis;
THNC = Proximal Tibial Head Necrosis Caseous;
THNS = Proximal Tibial Head Necrosis Severe. Other
symptoms are TD = Tibial Dyschondroplasia;
KB = Kinky Back (spondylolisthesis). Total lame
included all birds with FHS, FHT, FHN, THN, THNC,
THNS, TD, and KB lesions. Picture representation of
BCO lesion categories is shown in Figure 2.
regression (binomial distribution) using the generalized
linear model (GLM) procedure implemented in R
v4.2.2, (R Foundation for Statistical Computing,
Vienna, Austria). Cumulative lameness is mean value §
standard error of the mean (SEM), where significant sta-
tistical difference is determined at P ≤ 0.05.
RESULTS
Preliminary Data
The incidence of lameness was compared between
broilers reared in the wire-flooring pens and in the wood
shaving litter-flooring pens. Figure 3 presents data that
the wire flooring model can generate BCO lameness by
45% higher than the litter flooring model (P ≤ 0.05). In
this study, there were no specific arrangements for the
sites of the wire-flooring and litter-flooring pens. Lame-
ness incidence on both wire and litter-floorings were
noticeable after d 34 of age.

Airborne Transmission Model

Data Analyses
The percentage cumulative lameness from the lame-
ness trials was calculated using Microsoft Excel 2018
(Microsoft, Redmond, WA) with the following formula:
% cumulative lameness per treatment ¼
Number of lame bids for treatment
 100
Total number of birds in treatment on d14
The effectiveness of the aerosol transmission model of
BCO lameness was evaluated from 4 independent stud-
ies generating cumulative BCO lameness data by com-
paring the lameness incidence of the wire-flooring group
and the negative control group on litter floors in each
experiment. Data in Table 1 demonstrates the detail of
cumulative lameness of BCO source on wire floor versus
negative control on litter floors in Experiments 1, 2, 3,
and 4 were 84 vs. 69.33%, P = 0.09; 54.55 vs. 60%,

Figure 2. Classification of BCO lesion progressions in the femur and tibia. Femur (top row): 1. Normal femur (the head of the proximal femur is
fully intact), 2. FHS (femoral head separation), 3. FHT (femoral head transitional degeneration), and 4. FHN (femoral head necrosis, femoral head
is completely damage); Tibia (bottom row): 5. Normal tibia (no macroscopic abnormalities of the proximal tibia), 6. THN (tibial head necrosis), 7.
THNS (tibial head necrosis severe), 8. THNC (tibial head necrosis caseous, a visible caseous excretion), and 9. TD (tibial dyschondroplasia).
 EXPERIMENTAL LAMENESS BY AEROSOL TRANSMISSION 5

Figure 3. Preliminary data for cumulative lameness of birds grown on wire floor and litter floors. Six replicates of 25 birds were used for each
wire and litter floor. a-bValues denote significant statistical differences at P ≤ 0.05.

P = 0.56; 78 vs. 73.50%, P = 0.64; 81 vs. 74.50%, experimental flocks (Wideman and Pevzner, 2012;
P = 0.11. Generally, the 4 experiments successfully gen- Wideman and Prisby, 2013; Wideman et al. 2013; Wide-
erated lameness in the wire-flooring pens with cumula- man et al., 2014; Wideman, 2016). In this report, broilers
tive incidences of BCO lameness ranging from 50 to reared in the wire-flooring pens showed cumulative
84%. The litter-flooring pens developed cumulative lameness of up to 84%, which reinforces previous reports
BCO lameness incidences ranging from 60% to 73%. of BCO exceeding 50% using the same model (Wideman
From Experiments 1 to 4, lameness incidences were also and Pevzner, 2012; Wideman et al., 2014; Alrubaye
recorded starting in litter floors from d34 and spread to et al. 2020a; Alrubaye et al. 2020b). A significant impact
other groups until the end of the experiment. of the wire-flooring model design lies in its ability to
impose mechanical stress and damage to the bones, ren-
dering the broiler susceptible to pathogens infection,
DISCUSSION weakening immune response, and promoting leaky gut.
Discussions related to the model’s mode of action agree
The wire-flooring model has consistently proven an on the physical and physiological stress imposed as the
effective method to generate BCO lameness in animals traverse the length of the wire flooring to access
feed and water. This continuous locomotion on unstable
Table 1. Percentage cumulative lameness in 4 independent flooring is postulated to exacerbate conditions such as
experiments using the airborne transmission model. epiphyseolysis, osteochondrotic cleft formation in the
Cumulative growth plates, and microfractures on the long bones
Experiments Treatment groups lameness (%)1 N2 of the animal’s legs − the onset of which seems to be
1 BCO source in wire floors 84.0 § 2.5a
2 imposed by a combination of rapidly increasing body
Negative control diet 69.3 § 2.2a 3 weights, growing trends, and inherent nature of
Probiotic 1 37.3 § 1.2b 3 broiler skeletal development (Wideman et al., 2012b;
Probiotic 2 37.3 § 3.2b 3
Probiotic 3 33.3 § 1.9b 3 Wideman, 2016). Potential pathogenic translocation
2 BCO source in wire floors 54.5 + 3.0a 2 of bacteria from other sites in the body leads to even-
Negative control diet 60.0 + 2.5a 5 tual colonization, lesion formation, and tissue necrosis
Availa ZMC 29.3 § 1.3b 5
Microalgae 63.8 § 1.5a 5 at these damaged points (McNamee and Smyth 2000;
Microalgae + Availa-ZMC 50.0 § 1.6c 5 Wideman et al. 2012b;Wideman, 2016; Weimer et al.,
3 BCO source in wire floors 78.0 + 3.0a 2 2021). On the other hand, the wood-shaving litter
Negative control diet 73.5 + 1.3a 4
Panbonis high 1,000 g/ton 41.5 § 1.9b 4 floor exaggerates the broiler’s vulnerability to BCO
Panbonis low 100 g/ton 32.5 § 1.9c 4 infection due to direct contact to the fecal aggrega-
Panbonis low + NatuFeat 38.5 § 1.3b,c 4 tion on which they stand.
Panbonis low + Animunin 35.0 § 2.2c 4
Panbonis low + Thyme oil 29.0 § 1.3c 4 Considering the advantage of the wire-flooring for
4 BCO source in wire floors 81.0 + 3.0a 2 inducing BCO lameness in broilers, we expanded our
Negative control diet 74.5 + 2.1a 4 strategy to combine the wire-flooring and litter-flooring
Panbonis 50 g/ton 60.5 § 1.5b 4
Panbonis 100 g/ton 34.5 § 3.6c 4 models in order to reduce the number of wire flooring
Panbonis 200 g/ton 36.0 § 3.9c 4 pens required in experiments. Our hypothesis for the
Panbonis 100g/ton (d29-56) 68.0 § 3.5a 4 aerosol transmission model focused on the potential for
Panbonis 100g/ton (d1-28) 35.0 § 4.0c 4
a-c
excreta from affected wire-flooring broilers to carry the
Superscript letters represent significant statistical differences at
P ≤ 0.05 against negative control diets.
etiological agent for BCO lameness − mainly reported
1
Cumulative lameness is mean § SEM. Staphyloccocus species in the current literature − and
2
N is number of pens with n = 50 birds per pen. disperse them to litter-flooring birds throughout the
6 ASNAYANTI ET AL.
research barn when placed at a strategic location that
maximizes the downstream ventilatory distribution of
air flow. This model involves placement of 2 wire-floor-
ing pens serving as the infection source at the north end
of the house where ventilation curtains are located, with
exhaust fans located at the south end, and all other
experimental pens situated between these 2 points,
allowing for constant unidirectional airflow (Figure 1).
The arrays of the wire floors, litter floors, and exhaust
fans are pivotal in the aerosol transmission model. In the
preliminary study, the wire floors and litter floors were
randomly assigned resulting in a significant difference of
cumulative lameness. While the results of 4 experiments
having a consistent setup displayed in Figure 1 demon-
strated cumulative lameness incidence values ranging
from 50 to 84% on wire flooring pens. Furthermore, the
negative control groups reared on wood-shaving litter
also demonstrated high cumulative incidences of BCO
lameness, ranging from 60 to 73%, without significant
differences when compared to their respective wire floor-
ing pens (P > 0.05). As a point of clarification, we
acknowledge that these experiments did not solely focus
on the airborne transmission model as a research objec-
tive, but rather on the investigation of multiple feed sup-
plements and their effects in reduction of BCO lameness.
However, as all 4 experiments involved the consistent
use and placement strategy of similarly designed wire
flooring and negative control (litter flooring) pens, the
observed cumulative lameness incidences affirm the
validity of this model.
This aerosol transmission model for inducing experi-
mental BCO lameness resembles the pathogenic aerosol-
ized transmission occurred within the livestock industry,
including poultry. Extensive research effort has been
carried out in the wake of widespread epizootic and zoo-
notic pathogenic transmission (CDC), which focuses on
the understanding of contagious viral and pathogenic
aerosol transmission in order to mitigate its detrimental
effects (Zhong et al., 2009; Liu et al., 2012; Alonso et al.,
2017; Bertran et al., 2017). Within the poultry industry,
aerosolized transmission is mostly facilitated by ventila-
tion equipment and open air. Pathogenic debris and par-
ticles, mainly in the form of dust, are agitated from a
source and spread through air currents to animals living
within such an enclosed environment, as well as those
living in other barns and complexes in close vicinity
(Harbaugh et al., 2006; Zhong et al., 2009; Liu et al.,
2012).
Furthermore, we observed that massive lameness
symptoms in the birds grown in the wire flooring pens
occurred after d 35 and the progression was prompt.
From early clinical symptoms, such as goblet gait, reluc-
tance to move, and slight wing-tip dipping, to full immo-
bilization − or even death − can happen in only 24 to 48
h, which is similar to the observation reported in previ-
ous studies (Wideman and Pevzner, 2012; Wideman,
2016). Further research into factors that could poten-
tially mitigate this acute progression may be of high
industrial importance in reducing mortality and eco-
nomic losses.
In conclusion, the aerosol transmission model − air-
borne transmission as an extension to the already effec-
tive wire-flooring model − is a remarkably simple and
powerful tool to induce BCO lameness in an experimen-
tal broiler population. The effectiveness of the aerosol
transmission model potentially provides a reliable sys-
tem for evaluating practical intervention strategies for
reducing BCO lameness, particularly with the applica-
tion of feed supplements, as well as prophylactic and
therapeutic measures.
ACKNOWLEDGMENTS
This research was funded by a grant to Alrubaye from
Biomin DSM and a grant 2019-69012-29905 to Walter
Bottje from USDA Sustainable Agricultural Systems.
DISCLOSURES
The authors declare that they have no competing
interests. The funders played no role in the design of the
experiments, the interpretation of the data, the writing
of this manuscript, or the decision to publish this manu-
script.
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