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EQUINE
Effect of Manuka honey gel on the transforming growth factor β1
and β3 concentrations, bacterial counts and histomorphology of
contaminated full-thickness skin wounds in equine distal limbs
AS Bischofberger,a CM Dart,a N Horadagoda,a NR Perkins,b LB Jeffcott,a CB Littlec and AJ Darta*
Objective To investigate the effect of 66% Manuka honey gel
on the concentrations of transforming growth factor (TGF)-β1 and
TGF-β3, bacterial counts and histomorphology during healing of
contaminated equine distal limb wounds.
Methods In this experimental study of 10 Standardbred horses,
five full-thickness skin wounds (2 × 1.5 cm) were created on one
metacarpus and six similar wounds were created on the contralat-
eral metacarpus. Wounds were assigned to three groups: non-
contaminated control wounds; contaminated control wounds;
contaminated wounds treated daily with 1 mL Manuka honey
gel topically for 10 days. For the contaminated wounds, faeces
were applied for 24 h after wound creation. In five horses wounds
were bandaged and in the other five horses wounds were left
without a bandage. Biopsies were taken on days 1, 2, 7 and
10 after wounding to evaluate the effects of Manuka honey gel,
wound contamination and bandaging on TGF-β1 and TGF-β3
concentrations, aerobic and anaerobic bacterial counts, and
histomorphology.
Results Manuka honey gel had no significant effect on TGF-β1
and TGF-β3 concentrations or wound bacterial counts. Manuka
honey gel decreased wound inflammation (days 7, 10), increased
angiogenesis (days 2, 7, 10), increased fibrosis and collagen orga-
nisation (day 7) and increased epithelial hyperplasia (days 7, 10).
Conclusions Treatment with Manuka honey gel resulted in a
more organised granulation tissue bed early in wound repair,
which may contribute to enhanced healing of equine distal limb
wounds.
Keywords horses; Manuka honey gel; transforming growth
factor beta; skin wounds
Abbreviations cfu, colony-forming units; CI, confidence interval;
TGF-β, transforming growth factor beta; SEM, standard error of
the mean; UMF, unique Manuka factor
Aust Vet J 2016;94;27–34 doi: 10.1111/avj.12405
D
istal limb wounds in horses are often associated with severe
tissue avulsion and excessive contamination, so are left to
heal by second intention. These wounds are predisposed to
*Corresponding author.
a
Research and Clinical Trials Unit, University Veterinary Teaching Hospital Camden,
University of Sydney, Camden, New South Wales, Australia;
andrew.dart@sydney.edu.au
b
Ausvet Animal Health Services, Toowoomba, QLD, Australia
c
Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical
Research, Institute of Bone and Joint Research, University of Sydney at Royal North
Shore Hospital, St Leonards, NSW, Australia
© 2016 Australian Veterinary Association
EQUINE
the development of excessive granulation tissue and prolonged
wound healing because of greater wound retraction, slower rates and
earlier cessation of wound contraction, and slower rates of
epithelialisation.1–3 The different isoforms of transforming growth
factor beta (TGF-β) have been suggested to play a central role in
orchestrating equine wound healing.2–4 TGF-β1 is a pro-fibrotic and
pro-inflammatory cytokine that enhances the migration and growth
of fibroblasts and the accumulation of extracellular matrix; in con-
trast, TGF-β3 acts to downregulate the inflammatory process and
minimise scarring.1 The process of second-intention wound healing
in the horse is complex and not completely understood; however,
the concentrations and temporal expression patterns of these two
cytokines within the wound appear to be essential for the normal
progression of wound healing, particularly wounds on the distal
limb.2–4
The aim of applying topical medications to wounds left to heal by
second intention is to manipulate the wound environment. Some
medications work by modifying the production of cytokines and
growth factors from inflammatory cells in the wound bed.5,6 As a
topical preparation, Manuka honey has been shown to modulate the
early period of second-intention wound healing in the horse, but
its precise mechanism of action is unclear.7–14 Recently, a unique
5.8-kDa component found in Manuka honey was shown to engage
toll-like receptor 4 on monocytes to upregulate production of
tumour necrosis factor alpha, interleukins 1β and 6, and prostaglan-
din E-2, which in turn, enhances the inflammatory phase of
healing.7–9 Manuka honey also has potent antibacterial activity
against gram-negative and gram-positive wound pathogens that may
affect the progression of wound healing, particularly contaminated
wounds involving the distal limb of horses.10–12
Recent studies have shown that application of Manuka honey to
equine distal limb wounds enhances aspects of early wound heal-
ing.13,14 Those studies used a wound healing model aimed at produ-
cing an environment similar to naturally occurring wounds.13,14
Wounds were contaminated for 24 h with faeces to replicate the bac-
terial contamination seen in many naturally occurring wounds and a
bandage was applied to some wounds for up to 12 days to promote
the early stages of dysregulated healing.13–15 Using this model of
wound healing, distal limb wounds created surgically were treated
daily for 12 days with unique Manuka factor (UMF) 20 Manuka
honey or a 66% Manuka honey gel.13,14 The gel, made from 66%
Manuka honey and 34% water-based, pH neutral gel, was developed
to enhance wound contact. Manuka honey and 66% Manuka honey
gel had the similar beneficial effects on wounds. Treated wounds
retracted less and remained smaller than untreated wounds out to
Australian Veterinary Journal Volume 94, No 1-2, January/February 2016 27
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day 35 after wound creation.13,14 Furthermore, wounds treated daily
with 66% Manuka honey gel throughout healing healed faster than
untreated wounds.14 Although these studies did not establish the
precise mode of action of Manuka honey on equine wound healing,
the authors speculated that Manuka honey led to the early develop-
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ment of a mature granulation tissue bed, thereby optimising the
activity and organisation of wound fibroblasts and myofibroblasts
compared with untreated wounds.13,14 It is likely that more than one
property of Manuka honey contributes to the beneficial effect it has
on wound healing in horses, although, to date, the precise mechan-
ism of action has not been determined.
The aim of the current study was to investigate the mechanism of
action of daily application of 66% Manuka honey gel on distal limb
wound healing in horses. Specifically, we were interested in the
effects of Manuka honey on wound bacterial counts, the expression
of TGF-β1 and TGF-β3 within the wounds, and the histomorphol-
ogy of wound healing of bandaged and unbandaged contaminated
distal limb wounds. We hypothesised that treatment with Manuka
honey gel would reduce bacterial numbers in contaminated wounds
and that it would alter the expression of TGF-β1 and TGF-β3 to
improve the early histological features of healing in treated wounds.
Materials and methods
Horses
Ten Standardbred horses (5–10 years of age), without evidence of
scars on their limbs, were used in the study. The experimental proto-
col was approved by the Institutions Animal Care and Ethics Com-
mittee. Seven days prior to surgery the horses were weighed, given
tetanus prophylaxis, an oral anthelmintic (ivermectin 200 μg/kg)
and housed in grass yards for the duration of the study. Lucerne hay
was fed at 2.5% body weight in divided feeds daily and all horses had
free access to water.
Surgical procedure
The horses were administered phenylbutasone (4.4 mg/kg IV),
sedated with xylazine (1.1 mg/kg IV) and butorphanol (0.01 mg/kg
IV) and anaesthetised with ketamine (2.2 mg/kg IV) and diazepam
(0.05 mg/kg IV). Horses were intubated and maintained under
anaesthesia using isoflurane in oxygen. Both metacarpi were clipped
and prepared for surgery. Five full-thickness skin wounds were cre-
ated on one foreleg and six full-thickness skin wounds were created
on the contralateral forelimb. The wound model was validated in
previous studies.2,14 Briefly, the wounds (2 × 1.5 cm) were evenly
spaced on the metacarpus, medial to the common digital extensor
tendon so that each wound was separated from the adjacent wound
by a minimum space of 1.5 cm. The wounds were created by incising
around a standardised template using a scalpel. A sample of the
excised skin from one wound on each leg of each horse was frozen
at −80 C to determine baseline concentrations of TGF-β1 and TGF-
β3 in the skin at a later date. The 11 wounds in each horse were ran-
domly assigned to three different groups: non-contaminated,
untreated control wounds (non-contaminated control wounds); con-
taminated, untreated control wounds (contaminated control
wounds); and contaminated wounds treated with Manuka honey gel
daily (treated, contaminated wounds). Following creation of the
28 Australian Veterinary Journal Volume 94, No 1-2, January/February 2016
wounds, contaminated wounds had 2 g of fresh faeces, which had
been retrieved from a single horse that was not in the study, applied
to the wounds for 24 h under a bandage. The remaining wounds
were bandaged and left uncontaminated. The bandage consisted of a
low-adherent, absorbent wound dressing (Melolin, Smith & Nephew
Medical Ltd, Hull, UK) cut to size to cover the individual wound
and treatment completely in order to reduce the risk of cross-
contamination, then all wounds were covered with gauze-coated
cotton wool (Veterinary Gamgee Roll, Robinson Animal Health
Care, Carlton-In-Lindrick, UK) and compressed under an elastic
adhesive tape (Tensoplast Vet, BSN Medical, VIC, Aust).
The bandages were removed from all wounds after 24 h and a moist
gauze swab was used to gently remove the faeces from the contami-
nated wounds before treatment commenced. Manuka honey gel
(1 mL), consisting of 66% Manuka honey (UMF 20) and 34% water-
based, pH neutral gel, was applied to the wounds designated as
treated wounds. No treatment was applied to the control wounds.
Following treatment, both forelimbs were bandaged throughout the
study in five horses and the bandages were changed daily to apply
Manuka honey gel to the treated wounds. In the remaining five
horses the wounds on both forelimbs were left open to heal and
Manuka honey gel was applied daily to the treated wounds. Wounds
were not cleaned between treatments.
Biopsies
Each wound was designated for a single biopsy time point. Full-
thickness punch biopsies (6 mm) of the wounds were obtained on
days 1, 2, 7 and 10 after wound creation. Because treatment com-
menced on day 1 after the first biopsy procedure, only two wounds
(a non-contaminated control wound and a contaminated control
wound) were needed for biopsy. On days 2, 7 and 10, three wounds
were biopsied on each day (a non-contaminated control wound, a
contaminated control wound and a treated, contaminated wound).
To obtain the biopsies the horses were sedated with xylazine
(1.1 mg/kg IV) and anaesthetised with ketamine (2.2 mg/kg IV) and
diazepam (0.05 mg/kg IV). Wounds were not cleaned prior to
biopsy. A total of four biopsies were taken from each wound. One
biopsy was taken from the centre of the granulation bed using a ster-
ile biopsy punch and forceps and immediately submitted for quanti-
tative bacteriological analysis. Subsequently, three biopsies were
taken from the wound margin to include approximately 3 mm of
skin and 3 mm of granulation tissue. One marginal biopsy was
placed in 10% formalin for histology and two biopsies were frozen at
−80 C for analyses of TGF-β1 and -β3 at a later date.
Tissue bacterial counts
The tissue samples were collected using a sterile biopsy punch and
processed aseptically. Biopsies were weighed immediately after exci-
sion using a balance accurate to 0.01 g and then finely ground in
sterile mortars and pestles with 1 mL of sterile saline. The tissue sus-
pensions were further diluted from 101 to 108 in sterile saline and
100 μL of each suspension was plated aerobically and anaerobically
on horse blood agar (Oxoid Australia Pty Ltd, SA, Aust) and incu-
bated overnight at 37 C.16 Colony counts were completed within
24 h of culture and the colony-forming units (cfu) per gram of tissue
© 2016 Australian Veterinary Association

were determined after correcting for variations in sample size and
dilutions.
Histology
Tissue fixed in 10% neutral buffered formalin was processed into 5-
μm paraffin sections and stained with haematoxylin and eosin
(H&E) and van Giessen’s stain. All slides were examined by a pathol-
ogist (NH) blinded to the study using two established scoring sys-
tems.2,17 Briefly, the Theoret scoring system evaluates wound healing
variables semiquantitatively using a scale of 0–3 (none, mild, moder-
ate and marked).2 Variables evaluated included acute inflammation,
mononuclear cell infiltration, angiogenesis, fibrosis and collagen
organisation, epithelial coverage and epithelial hyperplasia.2 Van
Giessen’s stain was used to assess fibrosis and collagen organisation.
The Greenhalgh scoring system applied an overall histology score
ranging from 1 to 12, (with 1 corresponding to no healing and
12 corresponding to completely re-epithelialised), based on the
degree of cellular invasion, granulation tissue formation, vascularity
and re-epithelialisation.14
Growth factor analyses
Tissues for growth factor extraction were weighed and processed as
previously described.2 Briefly, the tissue was finely diced and homo-
genised in 1 mL of phosphate-buffered saline using a homogeniser.
The dispersed samples were centrifuged for 5 min at 1000g and the
supernatants were then centrifuged for 20 min at 12,000g. The final
supernatants were filtered (0.22-μm filters) and frozen at −80 C.
Analyses were performed using commercially available human cyto-
kine ELISA kits for TGF-β1 (DB100B) and TGF-β3 (DY243) ana-
lyses (R&D Systems, Inc., MN, USA). To activate latent TGF-β1 to
immunoreactive TGF-β1 detectable by the Quantikine TGF-β1
immunoassay, the samples were acid-activated. The TGF-β1 immu-
noassay was sensitive to 4.61 pg/mL and specific for natural and
recombinant TGF-β1. The paired ELISA TGF-β3 capture antibody
(MAB#643) and biotinylated detection antibody (BAF#243) and
standard (243-B3) provided by the supplier were used in the DuoSet
ELISA Development Kit. The antibodies do not cross-react with
TGF-β1 or TGF-β2.
Statistical analysis
Outcomes recorded as scores were analysed using non-parametric
methods (Mann-Whitney and Wilcoxon rank-sum tests). Results are
presented as descriptive summaries (median and 95% confidence
interval (CI) for median) and with P values derived from non-
parametric comparisons. Continuous outcomes were analysed using
multivariable, general linear models (xtmixed procedure within
STATA) with separate models run for each outcome.
Growth factor outcomes (TGF-β1 and TGF-β3) were analysed in
separate general linear models where each model included fixed
effects coding for treatment groups, bandage (yes or no), baseline
concentrations of TGF-β1 and TGF-β3 and interaction terms invol-
ving treatment group, bandage and day. Preliminary analyses indi-
cated evidence of heteroscedasticity in inspection of residuals, which
was corrected by natural logarithm transformation of the outcomes.
All analyses were conducted using natural log-transformed data for
© 2016 Australian Veterinary Association
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TGF-β1 and TGF-β3 (log (raw count + 1)). A random effect coding
for horse identity was added to each model to adjust correlations
between repeated measurements within each horse. Models were
developed by starting with all terms and then interaction terms were
removed from the model if they were not significant, starting first
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with the three-way interaction term (treatment group × bandage ×
day) and then considering the three different two-way interactions
(treatment group × bandage, treatment group × day, bandage ×
day). Interaction terms were retained in the model if they were asso-
ciated with a significant P value. Residuals were inspected for model
fit. Follow-up tests on marginal means were then performed to
investigate pairwise comparisons between group means. Pairwise
comparisons used standard error terms derived from the general lin-
ear model and involved t-tests with Benjamini Hochberg adjustment
to correct for the risk of elevated type I error because of multiple
comparisons.18 Marginal means were then back-transformed to gen-
erate estimates in the original scale for reporting.
Analyses of aerobic and anaerobic cfu counts were complicated by
the missing data on day 1 for the treated, contaminated wounds,
because wounds were not treated with Manuka honey gel until day
1, and on day 10 in bandaged wounds because the data were lost.
Consequently, analyses were conducted using separate general linear
models performed on each observation day and for each outcome
(aerobic cfu and anaerobic cfu), with fixed effects dependent on
availability of data for that day. Both aerobic and anaerobic cfu
count data were right skewed and required natural logarithm trans-
formation to normalise the data. All analyses were conducted using
log-transformed data. Day 1 models only included a fixed effect cod-
ing for treatment because all wounds were bandaged between days
0 and 1 and there was no opportunity to assess the effect of bandag-
ing. Day 10 models only included a fixed effect coding for treatment
because there were no cfu data available for bandaged wounds on
that day. Models for days 2 and 7 included fixed effects coding
for bandage (yes, no) and treatment (control, untreated contami-
nated and treated contaminated). The interaction term between
bandage and each of the treatment groups (control, untreated con-
taminated and treated contaminated wounds) for each recording day
was not significant, so bandage was not retained in any of the models
evaluating bacterial counts.
Marginal means were estimated in the log-transformed scale for aer-
obic and anaerobic cfu counts and pairwise comparisons performed
using t-tests. Residuals from each model were inspected for
model fit.
Analyses were performed in STATA (Stata Corp, TX, USA).
P ≤ 0.05 was considered significant.
Results
Horses remained healthy throughout the study. Following creation
of the wounds there was neither lameness nor swelling of the limbs.
None of the dressings or bandages slipped and there was no evidence
of cross-contamination of any of the treatments applied to the
wounds.
The effect of faecal contamination is shown in Table 1. Applying fae-
ces under a bandage for 24 h resulted in an increase in the mean log
Australian Veterinary Journal Volume 94, No 1-2, January/February 2016 29
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 SEM of aerobic and anaerobic cfu on day 1 compared with non- wounds were lost after processing so no comparisons could be made
contaminated control wounds. On day 2 the mean log of aerobic cfu for that day.
remained higher in contaminated control wounds compared with
non-contaminated control wounds, but the mean log of anaerobic
cfu was not significantly different. After day 2, bacterial counts Histological findings
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increased in non-contaminated control wounds up to day 7 and the The histological features of the biopsies obtained from unbandaged
mean log of aerobic and anaerobic cfu were higher compared with control, contaminated and Manuka honey gel-treated wounds are
contaminated control wounds. By day 10 there was no significant shown (Figure 1A–C). Results are presented as median, 95% CI for
difference between contaminated control wounds and non- median and P values.
contaminated control wounds in bacterial counts.
Effect of faecal application. Using the Greenhalgh scoring system,
The overall effect of Manuka honey gel treatment is shown in
there were no differences in the histology scores of the contaminated
Table 2. There was not a significant effect on the mean log  SEM
control wounds and non-contaminated control wounds at any of the
of aerobic cfu on day 2. By day 7, the treated, contaminated wounds
biopsy time points in bandaged or unbandaged wounds.17 Using the
had significantly higher aerobic bacterial counts than contaminated
Theoret scoring system, contaminated control wounds (3, 2.34–3.66)
control wounds. There was no difference in the mean log of anaero-
had higher acute inflammation scores on day 7 compared with non-
bic cfu between treated, contaminated wounds and contaminated
contaminated control wounds (2, 1.18–2.82, P = 0.046) when the
control wounds on day 7. On day 10 there were no differences in
data from bandaged and unbandaged wounds were combined.2
mean log cfu for aerobic or anaerobic cfu between treated and
There was no difference between any of the wound groups in acute
untreated contaminated wounds.
inflammation scores on day 7 when bandaged and unbandaged
The overall effect of bandage application is shown in Table 3. Band- wounds were analysed separately. There were no other differences in
aging wounds had no effect on the mean log  SEM of aerobic or acute inflammation scores. There was no significant difference in the
anaerobic cfu in biopsies taken from wounds on day 2. By day 7 the mononuclear infiltration score, the angiogenesis score, the fibrosis
mean log of the aerobic and anaerobic cfu in unbandaged wounds and collagen organisation score, the score for epithelial cover or the
was higher than in bandaged wounds. The results of the bacterial
counts from the tissue biopsies taken on day 10 from bandaged Table 3. Values (mean log  SEM) for aerobic or anaerobic colony-
forming units (cfu) in wounds contaminated with faeces for 24 h
Table 1. Effect of applying faeces to open wounds for 24 h on subse-
Day Bandage (cfu mean No bandage (cfu mean
quent bacterial contamination
log  SEM) log  SEM)
Day Non-contaminated control Contaminated by faeces Aerobic Anaerobic Aerobic Anaerobic
(cfu mean log  SEM) (cfu mean log  SEM)
2 12.37  1.63 8.28  2.6 11.83  1.63 7.23  2.6
Aerobic Anaerobic Aerobic Anaerobic
7 8.83  0.64a 9.34  0.61b 13.65  0.64a 13.8  0.61b
1 5.99  1.08a 1.48  1.53b 13.72  1.08a 13.72  1.53b
Bandages were applied for 24 h to maintain contact between faeces
2 7.43  2c 3.02  3.19 14.98  2c 10.08  3.19
and the wounds. After the faeces were removed, wounds were either
7 12.07  0.79d 12.38  0.75e 9.22  0.79d 10.24  0.75e left open or bandaged for the 10 days of the study. There are no
10 10.3  1.77 10.33  1.78 12.4  1.77 12.39  1.78 values for day 1 because all wounds were bandaged for the first 24 h
after wounds were created and there were no data for day 10 because
Values (mean log  SEM) for aerobic or anaerobic colony-forming the biopsy samples’ cfu counts from the bandaged wounds were lost.
units (cfu) with the same superscript are significantly different from Values with the same superscript are significantly different from each
each other: aP < 0.001, bP < 0.01, cP < 0.01, dP < 0.02, eP = 0.0. other: a,bP < 0.01.

Table 2. Values (mean log  SEM) for aerobic or anaerobic colony-forming units (cfu) in wounds contaminated with faeces for 24 h

Day Contaminated wounds treated with Manuka honey daily (cfu mean Contaminated by faeces but received no treatment
log  SEM) (cfu mean log  SEM)

Aerobic Anaerobic Aerobic Anaerobic

2 13.92  2 10.18  3.19 14.98  2 10.08  3.19

7 12.43  0.79a 12.10  0.75 9.22  0.79a 10.24  0.75
10 11.04  1.77 11.21  1.78 12.4  1.77 12.39  1.78

Faeces were removed at 24 h and wounds were either left untreated or treated daily with UMF 20 Manuka honey gel topically. Manuka honey
treatment commenced at 24 h so there are no values for day 1.
Values with the same superscript are significantly different from each other: aP < 0.01.

30 Australian Veterinary Journal Volume 94, No 1-2, January/February 2016 © 2016 Australian Veterinary Association

epithelial hyperplasia between contaminated control wounds com-
pared with non-contaminated control wounds at any time point.2
Effect of Manuka honey gel treatment. Using the Greenhalgh
scoring system there were higher histological scores (evidence of bet-
ter healing) in treated, contaminated wounds (day 7: 10, 9.7–10.3;
day 10: 10: 9.22–10.78) compared with contaminated control
(day 7: 7, 6.6–7.4, P = 0.005; day 10: 8, 7.54–8.46, P = 0.022) and
non-contaminated control (day 7: 8, 7.18–8.82, P = 0.008; day 10:
8, 6.93–9.06, P = 0.03) wounds on day 7 and 10, respectively,
when data from bandaged and unbandaged wounds were com-
bined.17 Similarly, when bandaged and unbandaged wounds were
analysed separately, higher scores were also found in the treated,
contaminated wounds (bandaged: 10, 9.18–10.82; unbandaged:
9, 7.43–10.57) compared with contaminated control wounds (ban-
daged: 7, 6.06–7.94, P = 0.041; unbandaged: 7, 6.46–7.54, P = 0.041)
on day 7, while bandaged and unbandaged treated, contaminated
wounds (bandaged: 10, 9.18–10.82; unbandaged: 9, 7.43–10.57) also
showed a trend towards higher scores compared with non-
contaminated control wounds (bandaged: 8.5, 7.3–9.7, P = 0.059;
unbandaged: 8, 7.04–8.96, P = 0.056).
© 2016 Australian Veterinary Association
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Figure 1. (A) Photomicrograph of a biopsy from an unbandaged, non-
contaminated untreated control wound at day 7 showing moderate
infiltration of the dermis with polymorphonuclear inflammatory cells
(arrowheads) and haemorrhage at the surface, mild epithelial hyper-
plasia ( ) and mild degree of angiogenesis in the dermis (arrows).
(B) Photomicrograph of a biopsy from an unbandaged, contaminated
untreated wound at day 7 showing marked infiltration of the dermis
with polymorphonuclear inflammatory cells and haemorrhage at the
wound surface (arrow heads). (C) Photomicrograph of a biopsy from
an unbandaged, contaminated wound treated with Manuka honey gel
at day 7 showing moderate epithelial hyperplasia ( ), mild infiltration
with polymorphonuclear inflammatory cells (arrowheads) and moder-
ate degree of angiogenesis in the dermis (arrows). (A–C: bar = 500μm;
H&E.)
Using the Theoret scoring system, the treated, contaminated wounds
(1.5, 0.65–2.35) had lower acute inflammation scores than the con-
taminated control wounds (3, 2.34–3.66, P = 0.007) and non-
contaminated control wounds (2, 1.18–2.82, P = 0.008) on day
7 when the data from bandaged and unbandaged wounds were com-
bined for analysis.2 When bandaged and unbandaged wounds were
analysed separately, only treated, contaminated wounds
(2, 1.11–2.89) that were bandaged had lower acute inflammation
scores on day 7 compared with non-contaminated control wounds
(2.5, 1.63–3.37, P = 0.046). There was a trend for treated, contami-
nated wounds that were bandaged (2, 1.11–2.89) or unbandaged
(1, 0.06–1.94) to have lower acute inflammation scores than con-
taminated control wounds (bandaged: 3, 2.11–3.89, P = 0.052;
unbandaged: 3, 2.13–3.87, P = 0.055) on day 7. By day 10 a similar
difference was only found between treated, contaminated wounds
(1, 0.37–1.63) and contaminated control wounds (2, 1.62–2.38,
P = 0.047) when bandaged and unbandaged wounds were combined
for analysis.
Treated, contaminated wounds had higher angiogenesis scores on
day 2 (2, 1.68–2.32), day 7 (3, 3–3) and day 10 (3, 3–3) compared
with contaminated control wounds (day 2: 1, 0.53–1.47, P = 0.046;
Australian Veterinary Journal Volume 94, No 1-2, January/February 2016 31
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day 7: 2, 2–2, P = 0.0056, day 10: 2, 2–2, P = 0.003) when data from
bandaged and unbandaged wounds were combined.2 When ban-
daged and unbandaged wounds were analysed separately, higher
angiogenesis scores were found in treated contaminated wounds
(3, 2.4–3.6) compared with contaminated control wounds
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(2, 1.72–2.28, P = 0.46) on day 10 in bandaged wounds, while in
unbandaged wounds, treated contaminated wounds (3, 2.72–3.28)
had higher angiogenesis scores than contaminated control wounds
(2, 2–2, P = 0.46) on days 7 and 10 (treated contaminated wounds:
3, 3–3; contaminated control wounds: 2, 2–2, P = 0.025). Angiogene-
sis scores were only higher in treated, contaminated wounds on day
7 (3, 3–3) and day 10 (3, 3–3) when compared with non-
contaminated control wounds (day 7: 2, 2–2, P = 0.005, day 10:
2, 2–2, P = 0.014) when data from bandaged and unbandaged
wounds were combined. When bandaged and unbandaged wounds
were analysed separately, the treated, contaminated wounds that
were bandaged (3, 2.46–3. 54) and those that were unbandaged
(3, 2.72–3.28) had higher angiogenesis scores than non-
contaminated control wounds (bandaged: 2, 1.43–2.57, P = 0.046,
unbandaged: 2, 2–2, P = 0.046) on day 7.
The fibrosis and collagen organisation scores were higher in treated,
contaminated wounds 2, 1.46–2.53) compared with contaminated
control wounds (1, 0.64–1.36, P = 0.015) on day 7 when data from
bandaged and unbandaged wounds were combined.2 There were no
differences when the data from bandaged and unbandaged wounds
were analysed separately. Epithelium hyperplasia scores were higher
in treated, contaminated wounds (day 7: 3, 3–3; day 10: 3, 3—3)
compared with contaminated control (day 7: 2, 1.24–2.76, P = 0.03;
day 10: 2, 1.69–2.31, P = 0.007) and non-contaminated control (day
7: 2, 2–2, P = 0.005; day 10: 2, 2–2, P = 0.005) wounds on days
7 and 10, respectively, when data from bandaged and unbandaged
wounds were combined.2 When bandaged and unbandaged wounds
were analysed separately, there were differences on days 7 and 10. In
bandaged wounds on day 10, treated, contaminated wounds (3, 3–3)
had higher epithelium hyperplasia scores than non-contaminated
control wounds (2, 1.72–2.28, P = 0.046). Treated, contaminated
wounds that were unbandaged had higher epithelium hyperplasia
scores on day 7 (3, 3–3) and day 10 (3, 3–3) compared with non-
contaminated control wounds (day 7: 2, 2–2, P = 0.025; day 10:
2, 2–2, P = 0.046).2 There were no other significant effects of treat-
ment with Manuka honey gel on any of the measured histological
variables at any time point.2
Effect of bandage application. Using the Greenhalgh scoring sys-
tem, bandaging of wounds had minimal effect on the histological
score for any of the wounds at any time point.16 The exception was
that on day 7 higher histological scores were found in treated, con-
taminated wounds that were bandaged (10, 9.18–10. 82) compared
with treated, contaminated wounds that were not bandaged
(9, 7.43–10.57, P = 0.033). Using the Theoret scoring system, band-
aging resulted in an increase in fibrosis and collagen organisation
scores on day 10 in bandaged wounds (bandaged: 2, 1.39–2.61;
unbandaged: 1, 0.69–1.31, P = 0.022 ) when scores from non-
contaminated control, contaminated control and treated, contami-
nated wounds were combined.2 When wounds were analysed sepa-
rately contaminated control wounds that were bandaged (2, 2–2)
had a higher fibrosis and collagen organisation scores on day
32 Australian Veterinary Journal Volume 94, No 1-2, January/February 2016
10 compared with contaminated control wounds (1, 1–1, P = 0.014)
that were left unbandaged. The epithelium cover score on day 7 was
higher in bandaged wounds (1, 1–1) compared with unbandaged
wounds (0, 0–0, P = 0.03) when scores from non-contaminated con-
trol, contaminated control and treated, contaminated wounds were
combined.2 There were no other significant effects of applying on
bandage on any of the measured histological variables at any of the
time points.2
TGF-β1 and TGF-β3 concentrations
The mean baseline levels (day 0) of TGF-β1 (994 pg/g  89) and
TGF-β3 (64  21 pg/g) were not significantly different in any of the
wounds. The interaction between treatment group, day and bandag-
ing was not significant for either TGF-β1 or TGF-β3.
TGF-β1 concentrations increased after wounding to a peak at 24 h
and thereafter gradually decreased. There was no significant change
in the mean TGF-β1 concentration from day 1 to day 10 for any of
the treatment groups. There was no significant difference in TGF-β1
concentrations between treatment groups or between bandaged and
unbandaged wounds.
TGF-β3 concentrations decreased in all treatment groups on the first
day after wounding, although this decrease was not significant. Con-
centrations remained unchanged from day 1 to day 2. By day 7 after
wound creation the TGF-β3 concentrations had increased and were
significantly different from day 1 (P < 0.001) and 2 (P < 0.001).
From day 7 to day 10, the TGF-β3 concentrations continued to
increase but were not significantly different. However, concentra-
tions on day 10 were significantly higher than on day 2 (P < 0.001)
and day 1 (P < 0.001). There was no significant difference in TGF-
β1 concentrations between treatment groups or between bandaged
and unbandaged wounds.
Discussion
The precise mechanism by which 66% Manuka honey gel enhances
second-intention healing of distal limb wounds in horses has not
been established; however, direct modulation of the inflammatory
phase of wound healing and an antibacterial effect may both play a
role.7–11,13,14,19,20 In the present study, daily application of 66% Man-
uka honey gel to wounds had no apparent effect on bacterial counts
during the first 10 days after wound creation, which was surprising
given the well-established antibacterial activity of Manuka
honey.10–12 In fact, aerobic cfu in contaminated wounds treated with
Manuka honey were higher than in untreated contaminated wounds
on day 7. Furthermore, bacterial counts in non-contaminated con-
trol wounds were higher than in contaminated control wounds on
day 7. Although it is possible that the techniques used to quantify
the bacterial counts in this study were inaccurate, the methods used
were based on established protocols and the samples were processed
by qualified laboratory technicians.16 It is more likely that the experi-
mental design complicated interpretation of the statistical analysis.
The study included several levels of treatment (bandage, contamina-
tion, honey). Wounds in all 10 horses were bandaged for 24 h, after
which the wounds remained bandaged in five horses but were
removed in the other five horses and the wounds were left open for
© 2016 Australian Veterinary Association

the remainder of the study. Bandages are known to provide protec-
tion from environmental contamination.21 On day 2, 24 h after the
bandages were removed from five of the horses, there was no differ-
ence in bacterial counts between bandaged and unbandaged wounds.
However, as expected, bacterial counts in the unbandaged wounds
increased between day 2 and day 7 in each of the treatment groups,
irrespective of treatment. During this time the interaction term
between bandage and treatment group was not significant, confirm-
ing that the change in bacterial counts over time was the same for all
treatment groups. More specifically, the results indicate that the
change in bacterial counts in the unbandaged, treated, contaminated
wounds was not different to the change in the bacterial counts in the
unbandaged wounds in all treatment groups. The results support a
finding that there was no effect of Manuka honey on bacterial counts
using this model.
In this study the Manuka honey was applied once daily. On each col-
lection day the honey was applied after the biopsies had been col-
lected for culture. It is possible that once-daily application of
Manuka honey was not sufficient to maintain the antimicrobial
effects for a full 24 h, particularly in the unbandaged wounds. More
frequent application of Manuka honey may be required to have a
sustained effect on bacterial numbers, particularly in open wounds
where exposure to contamination might be high and contact time is
likely to be limited.
Traditionally, in equine wound healing models, wounds have
been created surgically under aseptic conditions.13,22 In contrast, nat-
urally occurring wounds are commonly traumatic and associated
with extensive tissue avulsion and bacterial contamination.13,22
Recent studies investigating the effects of Manuka honey on second-
intention healing of distal limb wounds in horses have used a model
in which wounds are contaminated with faeces under a bandage for
24 h.13,14 In this model, wounds exposed to faeces for 24 h under-
went greater wound retraction but a shorter overall time to healing
compared with wounds that were not contaminated, irrespective of
whether or not wounds were treated with Manuka honey.14 Bacterial
contamination in these studies was not quantified, but the studies
established that exposing experimental wounds to faecal contamina-
tion for 24 h was sufficient to influence the pattern of healing.14
Applying faeces to wounds in the current study increased aerobic
bacterial counts for 48 h compared with non-contaminated control
wounds, suggesting this model is effective in increasing cfu counts,
at least transiently. Histologically, contaminated control wounds had
higher inflammation scores than non-contaminated control wounds
by day 7. It is well documented that a suboptimal, early inflamma-
tory response contributes to the dysfunctional healing characteristic
of some wounds in horses, particularly wounds to the distal limb.1,23
It would appear, in horses, that some contamination of the wound
with faeces for 24 h enhances the initial inflammatory response in
distal limb wounds and may play a role in improving wound healing.
However, the model is unlikely to replicate the level of contamina-
tion in many naturally occurring wounds. It is unlikely this model
achieved sufficient contamination to adequately assess the antibacte-
rial effects of Manuka honey on wound healing.
The TGF-β isoforms have been reported to play a central role in
orchestrating the complex network of cytokines and growth factors
© 2016 Australian Veterinary Association
EQUINE
responsible for second-intention wound healing in the horse.1
Wound healing in the horse is often problematic and can lead to the
production of excessive granulation tissue.23 The difference in the
pattern of healing of limb and body wounds has been associated with
differences in the temporal expression of TGF-β1 and TGF-β3.1 In
EQUINE
the current study, wounds treated daily with 66% Manuka honey gel
were found to have lower inflammation scores and evidence of more
advanced healing on days 7 and 10 compared with untreated
wounds. Specifically, there was a generalised increase in angiogenesis
as early as day 2 following wound creation, followed by enhanced
fibrosis and collagen organisation and improved epithelialisation in
wounds treated with Manuka honey gel. However, these differences
were not associated with a difference in the concentrations or
expression of TGF-β1 and TGF-β3 in wounds treated with Manuka
honey gel compared with untreated wounds, and the expression of
TGF-β1 and TGF-β3 in all wounds was similar to that in a previous
equine study.2 This would suggest that the beneficial effects of Man-
uka honey on the early phase of wound healing are being modulated
by other growth factors or cytokines.
Recent studies have shown distal limb wounds in horses have lower
oxygen saturation values than body wounds early in the inflamma-
tory phase of wound repair.24 It is thought that occlusion of micro-
vessels within the granulation tissue of limb wounds may contribute
to a local state of hypoxia.25 Hypoxia stimulates angiogenesis, stimu-
lates synthesis of TGF-β1 and upregulates the expression of TGF-β1
receptors.26,27 Although we did not evaluate oxygen saturation or
microvessel occlusion, there was no difference in the concentrations
of TGF-β1 between any of the wounds during the study, further sup-
porting that the mechanism of action of Manuka honey is not
through modulation of the TGF isoforms. Wounds treated with
Manuka honey showed improved angiogenesis as early as 2 days
after wounding, lower inflammation scores and evidence of more
advanced healing on days 7 and 10 compared with untreated
wounds. It is reasonable to speculate that the beneficial effects of
Manuka honey in promoting the formation of a healthy granulation
tissue bed in the early phase of second-intention healing in horses
may, at least in part, be associated with a direct effect on angiogene-
sis. The effect of Manuka honey on angiogenic factors such as vascu-
lar endothelial growth factor and basic fibroblast growth factor,
among other cytokines that exhibit angiogenic activity, has not been
investigated and may be warranted.
Prolonged bandaging of wounds has been shown to progressively
induce dysregulated healing and ultimately the production of the
excessive granulation tissue that is commonly associated with natu-
rally occurring distal limb wounds.4,13,14,23,25 Semi-occlusive bandages
such as those used in the current experiment can reduce gas
exchange between the granulation bed and the surrounding environ-
ment, exacerbating local hypoxia and promoting excess granulation
tissue production in equine lower limb wounds.4,24,25 Recent studies
investigating the effects of Manuka honey on distal limb wounds
have applied a semi-occlusive bandage for approximately 12 days to
try and create a wound environment that better replicates the envi-
ronment in naturally occurring wounds.13,14 If the bandage is
removed approximately 12 days after wound creation, wounds gen-
erally do not progress to developing excessive granulation tissue;
however, the granulation bed does not appear as organised as that of
Australian Veterinary Journal Volume 94, No 1-2, January/February 2016 33
 EQUINE
comparable unbandaged wounds.13,14 Although this model has been
used, the histological effects of applying a bandage to the wound for
12 days have not been evaluated.13,14 Histologically, in the current
study there were few differences between bandaged and unbandaged
wounds in the first 10 days following wound creation, but where dif-
EQUINE
ferences were identified they consistently reflected higher fibrosis
and collagen organisation and higher epithelial hyperplasia scores in
bandaged wounds. These changes are similar to those described in a
previous study where limb wounds in horses were bandaged to
induce excessive fibroplasia to evaluate the role of micro-occlusion
of vessels and apoptosis in the process of wound healing.25 It is likely
that differences in the wound environment between bandaged and
unbandaged wounds in the first 10 days after wound creation do
exist, but that the histological assessment criteria used in the current
study were not sensitive enough to provide statistical differences so
early in the healing process.23,25
Treating equine distal limb wounds with Manuka honey gel resulted
in the earlier development of a more mature granulation tissue bed
characterised by lower inflammation, improved angiogenesis,
enhanced fibrosis and collagen organisation and improved epithelia-
lisation compared with untreated wounds. These effects of Manuka
honey gel did not appear to be mediated through modulation of the
TGF-β1 or TGF-β3 concentration or their temporal expression
within the wounds. It is possible other cytokines and growth factors,
particularly those with direct effects on angiogenesis, may play a role
and warrant further investigation. Despite the known antibacterial
effects of Manuka honey, in this model there was no difference in
aerobic and anaerobic bacterial counts between wounds treated with
Manuka honey and untreated wounds.10,11,20 Nonetheless the anti-
bacterial activity of Manuka honey is well documented and in heav-
ily contaminated, naturally occurring wounds Manuka honey
may provide additional beneficial effects on would healing. It is pos-
sible that more frequent application of the honey to the wound is
required to maintain antibacterial activity. Although the precise
mode of action of Manuka honey gel on second-intention healing
of distal limb wound in horses remains unclear, histologically there
was evidence that daily application Manuka honey gel enhanced the
development of a healthier bed of granulation tissue during the early
phase of wound healing compared with untreated wounds.
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(Accepted for publication 16 October 2014)
© 2016 Australian Veterinary Association