Reducing exposure to pathogens in the horse; A preliminary study into the survival of bacteria on a range of equine bedding types

Abstract

Aims:
Compare the rate of growth of four microbial strains that cause disease in the equine, on four commonly used types of bedding. The moisture holding capacity of each bedding type was also tested.

Methods and results:
Microbial strains included, Streptococcus equi, Streptococcus zooepidemicus, Fusobacterium necrophorum, Dichelobacter nodosus and Dermatophilus congolensis. The bedding types tested were Pinus syvestrus (Scots pine shavings), Pinus nigra (Corsican pine shavings), Picea sitchensis (Sitka spruce shavings), Cannabis sativa (hemp) and chopped wheat straw. A suspension of each microbial strain was spread in triplicate on agar media and incubated in its optimal growth conditions. The viable count (colony forming unit per ml) was determined for each bacterial strain for the five different bedding types. Pinus syvestrus bedding resulted in significantly less (p =0.001) bacterial growth of all strains tested.

Conclusions:
Factors resulting in the inhibition of bacterial growth include the anti-bacterial effects reported in the Pinacea family and the physical properties of the bedding substrate. Research is currently focussed on the diagnosis and management of disease. Prevention of disease is also important for matters of biosecurity. Strategies should include the provision of a hygienic environment and the use of specific types of bedding.

Significance and impact of the study:
Bedding choice has implications for global equine health and disease prevention as well as potential benefits in other animal species. This article is protected by copyright. All rights reserved.

Full text

ORIGINAL ARTICLE
Reducing exposure to pathogens in the horse: a
preliminary study into the survival of bacteria on a range
of equine bedding types
K. Yarnell
1
, M. Le Bon
1
, N. Turton
2
, M. Savova
2
, A. McGlennon
1
and S. Forsythe
2
1 School of Animal, Rural and Environmental Sciences, Nottingham Trent University, Southwell, Nottingham, UK
2 School of Science and Technology, Nottingham Trent University, Nottingham, UK
Keywords
antimicrobials, diseases, microbial
contamination, streptococci, veterinary.
Correspondence
Kelly Yarnell, School of Animal, Rural and
Environmental Sciences, Nottingham Trent
University, Brackenhurst Campus, Southwell,
Nottingham NG25 0QF, UK.
E-mail: kelly.yarnell@ntu.ac.uk
2016/1683: received 1 August 2016, revised
13 September 2016 and accepted 17
September 2016
doi:10.1111/jam.13298
Abstract
Aims: To compare the rate of growth of four microbial strains that cause
disease in the horse, on four commonly used types of bedding. The moisture-
holding capacity of each bedding type was also tested.
Methods and Results: Microbial strains included Streptococcus equi,
Streptococcus zooepidemicus,Fusobacterium necrophorum, Dichelobacter nodosus
and Dermatophilus congolensis. The bedding types tested were Pinus sylvestris
(Scots pine shavings), Pinus nigra (Corsican pine shavings), Picea sitchensis
(Sitka spruce shavings), Cannabis sativa (hemp) and chopped wheat straw. A
suspension of each microbial strain was spread in triplicate on agar media and
incubated in its optimal growth conditions. The viable count (colony-forming
unit per ml) was determined for each bacterial strain for the five different
bedding types. Pinus sylvestris bedding resulted in significantly less (P=0001)
bacterial growth of all strains tested.
Conclusions: Factors resulting in the inhibition of bacterial growth include the
antibacterial effects reported in the Pinacea family and the physical properties
of the bedding substrate. Research is currently focussed on the diagnosis and
management of disease. Prevention of disease is also important for matters of
biosecurity. Strategies should include the provision of a hygienic environment
and the use of specific types of bedding.
Significance and Impact of the Study: Bedding choice has implications for
global equine health and disease prevention as well as potential benefits in
other animal species.
Introduction
Management and control of disease is an ongoing con-
cern in the equine industry due to its potential economic
and welfare implications. One of the key aspects of dis-
ease control is decreasing exposure to pathogens which at
the most basic level includes the provision of a clean liv-
ing environment for the horse (Equus caballus). Many
disease-causing pathogens can be transmitted indirectly
through sharing of contaminated fomites (Timoney and
Kumar 2008) which could include horse equipment,
clothing or bedding.
Development of improved diagnostic tests and treat-
ment protocols are contributing to the improved
management of disease outbreaks in horses (Waller
2013). However, it is also important that preventative
measures be explored. Domestic horses may spend up to
23 h per day standing in their stables (Webster et al.
1987) and the bedding materials within those stables have
previously been implicated as being a primary source of
pathogenic bacteria (Hogan et al. 1990). Therefore, fac-
tors related to equine management need to be evaluated
including the provision of a suitable bedding material
that offers the most hygienic environment for the horse.
Bedding material has been evaluated for properties
such as dust production (Elfman et al. 2009), moisture
absorbency (Borhan et al. 2014), impact upon equine
behaviour (Werhahn et al. 2010) and airborne
Journal of Applied Microbiology 122, 23--29 ©2016 The Society for Applied Microbiology 23
Journal of Applied Microbiology ISSN 1364-5072

contamination with straw bedding having higher levels of
microbial contamination compared to paper and wood
shavings (Tanner et al. 1997). Water holding capacity is
of particular importance, as excess moisture in the form
of urine in the horse’s bed can contribute to foot prob-
lems (Hinchcliff et al. 2013) and gaseous ammonia for-
mation which is known to have detrimental consequences
upon the equine respiratory tract (Clarke 1987).
Common plant-based bedding materials used in horse
stables include straw, hemp and wood shavings (Borhan
et al. 2014). Horse owners will select their bedding type
based on availability, cost and ease of disposal (Wheeler
and Zajaczkowski 2002). If the bedding used in horse sta-
bles were able to resist equine-specific pathogens then
this has implications for the prevention and control of
disease; yet to date, limited investigation of direct bacte-
rial resistance of the bedding types available to horse
owners has been carried out.
Tanner et al. (1997) found a significant increase in
airborne microbial contamination in stalls with
straw bedding when compared to stalls with wood
shavings and paper-based bedding. The authors sug-
gested the latter may be the most acceptable bedding
types when considering microbial contamination within
a stable.
Wood shavings used in equine bedding are sourced
from the Pinacea family and many species within this
family have been shown to have antibacterial effects
including Pinus nigra (Sarac et al. 2014), Pinus sylvestris
(Rauha et al. 2000) and Picea sitchensis (Liu et al. 2011).
Evidence shows the most consistent antibacterial and
antifungal properties are associated with extracts of
the Pinus species and these effects may be associated with
polyphenols in the wood (V€
alimaa et al. 2007). However,
research is primarily based on food hygiene and thera-
peutics (V€
alimaa et al. 2007) involving bacteria such as
Escherichia coli and Staphylococcus aureus (Rauha et al.
2000).
This study investigated the survival of four bacteria
that can cause disease in horses on four commonly used
equine bedding types.
Equine strangles is caused by the bacterium Streptococ-
cus equi and results in significant welfare and economic
cost throughout the world (Waller 2013) with approx.
1000 outbreaks of strangles in the UK alone each year
(Ivens et al. 2011). Strangles is currently the most
frequently diagnosed infectious disease of horses world-
wide, responsible for high morbidity and occasional mor-
tality of infected animals (Waller 2013). The bacterium
gains access to the horse through the nose or mouth and
transmission of the disease occurs by direct contact with
an infected horse or contaminated equipment (Lindahl
et al. 2011).
Streptococcus equi is believed to have evolved from an
ancestral strain of Strep. equi subspecies zooepidemicus
(Webb et al. 2008). Streptococcus zooepidemicus strains are
highly diverse and can cause disease in a variety of other
species including humans (Waller 2013). Streptococcus
zooepidemicus is part of the normal commensal popula-
tion of the upper respiratory tract and reproductive tract
in horses, however, Strep. zooepidemicus is also known as
an opportunistic pathogen during period of immune vul-
nerably or stress (Rasmussen et al. 2013; Velineni et al.
2014). In the equine species, Strep. zooepidemicus is asso-
ciated with inflammatory airway disease (Wood et al.
2005), and uterine infections in mares (Smith et al.
2003).
Thrush of the equine hoof is recognized as a bacterial
infection which in severe cases may result in permanent
lameness (Petrov and Dicks 2013). The condition is asso-
ciated with Fusobacterium necrophorum even in the
absence of Dichelobacter nodosus. This is different from
footrot in cattle and sheep, which is caused by the syner-
gistic action of both types of bacteria (Petrov and Dicks
2013). The condition is degenerative and is commonly
associated with poor foot hygiene and dirty bedding
(Hinchcliff et al. 2013).
Distal limb dermatitis (mudfever) has been associated
with the bacteria Dermatophilus congolensis (Colles et al.
2010) and although it is not found free living in the envi-
ronment, its zoospores can survive for long periods of
time (Pilsworth and Knottenbelt 2007). The condition
results in infection of the epidermis and is believed to be
spread by direct contact between animals and through
contaminated environments. Mudfever commonly affects
the lower limb of horses (Pilsworth and Knottenbelt
2007) and as such reduction of bacterial spores in bed-
ding that is in contact with the lower limb for prolonged
periods would be desirable.
Due to the potential risks associated with the contami-
nation of the horses’ environment and pathogen trans-
mission, it is important that equine bedding be
objectively assessed for its ability to resist bacterial sur-
vival and growth.
The aim of this study was to (i) compare the rate of
growth of the four bacteria described, on four commonly
used types of equine bedding under controlled laboratory
conditions and (ii) compare the moisture-holding capac-
ity of each bedding type.
Materials and methods
Bedding types, bacterial strains and reagents
The bedding types tested were P. sylvestris (Scots pine
shavings), P. nigra (Corsican pine shavings), Pi. sitchensis
Journal of Applied Microbiology 122, 23--29 ©2016 The Society for Applied Microbiology24
Bacteria and equine bedding K. Yarnell et al.

(Sitka spruce shavings), Cannabis sativa (hemp) and
chopped wheat straw. Prior to microbiological testing, all
bedding (2 g pooled sample) were placed in a glass bea-
ker covered with aluminium foil and autoclaved at 121°C
for 15 min. Isolates of E. coli and De. congolensis from
Nottingham Trent University’s culture collection were
retrieved from frozen storage (80°C). Streptococcus equi,
Strep. zooepidemicus and Di. nodosus were commercially
sourced (LGC Standards and Public Health, Middlesex,
England). All nutrient agar and broth were purchased
from Oxoid Thermo Fisher Scientific (Basingtoke, UK)
and Defibrinated Horse Blood was purchased from TCS
Biosciences (Buckingham, UK).
Bacterial count methodology
Each bacterial strain was streaked from the frozen stock
onto appropriate agar media. Microbial strains, media
and incubation conditions are listed in Table 1. After
incubation, purity of the cultures was assessed through
morphological assessment on the plate and Gram stain,
and a single fresh colony was used to inoculate the broth
culture. The culture was diluted at 1 in 10 in fresh broth
overnight and 07 ml of the dilution was used to inocu-
late 2 g of each bedding type. The inoculation dose was
determined for each strain; Strep. zooepidemicus 779
10
5
CFU, Strep. equi 55910
5
CFU, E. coli 529
10
6
CFU, Di. nodosus:15910
4
CFU and De. congolen-
sis:161 910
4
CFU. Bedding with bacterial inoculant
was incubated overnight at room temperature (Di. no-
dosus was incubated anaerobically). After incubation, the
bedding was mixed with 18 ml of 085% saline and
homogenized in a Stomacher 400 (Seward, Worthing,
UK) for 1 min at 265 rev min
1
. The suspension was
decimally diluted in saline and spread in triplicate on
their respective agar media and incubated in their
optimal growth conditions. The viable count (colony-
forming unit per ml) was determined for each bacterial
strain for the five different bedding types.
Bacterial count data analysis
All data were logarithmically (log10) transformed prior to
statistical analysis and are expressed as mean value and
standard error of three replicate per bedding. All log-
transformed data were normally distributed and one-way
ANOVA was used to compare bacterial count between the
bedding types. The Tukey post hoc test was used to deter-
mine pairwise significance between groups. Data analysis
was performed using IBM SPSS ver. 23 software (IBM Cor-
poration, Armonk, NY, USA) and Differences were con-
sidered significant at P≤005.
Moisture-holding capacity methodology
Each bedding type was investigated for basic moisture-
holding capacity under laboratory conditions. Bedding
was supplied in its original dried form by the manufac-
turer. One hundred grams of dried mass of each bedding
type was weighed (Salter, Kent, UK) and placed into a
mesh basket measuring 25 cm by 25 cm and weighing
350 g with a collecting tray placed underneath. Equine
urine collected noninvasively from one horse with a
specific gravity of 11 was then syringed into the bedding.
The container size was calculated by taking the approxi-
mate size of a stable of 35by35 m and scaling this
down to a workable size in the lab of 25 cm by 25 cm.
The same was done with the amount of urine, with an
average of 7 l of urine produced per day scaled down to
466 ml. Room temperature throughout the trial was
14 21°C and humidity was 47 56% rh.
Moisture-holding capacity was calculated on a weight
basis by periodically reweighing the basket (wet
weight container weight (350 g) weight of bedding
Table 1 Microbial strains used during the study
Microbial species
Collection
number Media Incubation requirements
Streptococcus equi NCTC 9682 Nutrient agar/broth 24 h at 37°C
Streptococcus
zooepidemicus
NCTC 7023 Nutrient agar/broth 24 h at 37°C
Fusobacterium
necrophorum
ATCC
25286
Blood agar base no. 2 +5% defribrinated horse blood/nutrient
broth
Anaerobically for 48 h at 37°C
Dermatophilus congolensis NCTC 5175 Blood agar base no. 2 +5% defribrinated horse blood/nutrient
broth
48 h at 37°C
Dicelobacter nodosus ATCC
25549
Tryptone soya agar +5% sheep blood/cooked meat broth +1%
glucose
5 days
This table provides details of the microbial strains used during the study, their collection number, growth media used and incubation
requirements.
Journal of Applied Microbiology 122, 23--29 ©2016 The Society for Applied Microbiology 25
K. Yarnell et al. Bacteria and equine bedding

(100 g)). This was carried out at 2, 4, 6, 8, 10 and 24 h
post introduction of urine. As it is recommended that
urine and faeces be removed from stables at least once a
day, no further measures were taken beyond 24 h. Each
trial was repeated three times for each bedding type and
a mean weight for each time point calculated. A one-way
repeated measures ANOVA was used to compare wet
weights between each bedding type.
Results
Microbial results
Results from the microbial count after overnight incuba-
tion with each bedding type are shown in Table 2. Three
of five strains tested, E. coli, Di. nodosus and De. con-
golensis were not detectable (limit of detection
10
2
CFU ml
1
) when incubated with Corsican pine, Scots
Pine and Sitka Spruce.
There was a significant difference in Strep. zooepidemi-
cus counts between bedding type as determined by the
one-way ANOVA (F(4, 10) =9291, P<0001). According
to the Tukey post hoc test, all bedding were statistically
different from each other except Corsican pine and Scots
pine, with straw showing the highest count, and Corsican
pine and Scots pine the lowest count for Strep. zooepi-
demicus.
There was also significant differences in Strep. equi
counts between bedding type (F(4, 10) =262,
P<0001). Counts in hemp and straw were significantly
higher than in Scots pine and Sitka spruce while Corsican
pine showed the lowest count for Strep. equi.
Escherichia coli counts were significantly higher in
hemp compared to straw (F(1, 4) =12506, P<0001).
However, there were no significant differences in the
Di. nodosus and D. congolesis counts between hemp and
straw (P>005).
Overall, hemp and straw showed the highest colony
counts for all bacterial strain tested while Corsican and
Scots pine showed the lowest colony counts.
Moisture-holding capacity results
There was a significant difference in the moisture-holding
capacity between all bedding types, F(3, 56) =1062,
P=0001 with the pine-based bedding absorbing signifi-
cantly more urine when compared to all other bedding
types (Fig. 1). The amount of urine absorbed increased
over time up to a period of 24 h for the pine and hemp
bedding at which point no further measurable absorption
took place. The amount of urine absorbed increased over
time up to a period of 8 h for the spruce and straw bed-
ding after which no further measurable absorption took
place (Fig. 2).
Discussion
Pine bedding presented with significantly less bacterial
growth for each of the bacteria types tested when
Table 2 Mean SE of Log viable count (CFU ml
1
;n=3) for each bacterial strain incubated overnight with different bedding type
Bedding type Streptococcus zooepidemicus Streptococcus equi Escherichia coli Dichelobacter nodosus Dermatophilus congolensis
Hemp 645 0020
a
526 0015
a
440 0026
a
2434 0219 483 0018
Straw 713 0008
b
489 0012
a
540 0010
b
3021 0126 473 0052
Corsican pine 285 0087
c
333 0039
b
<LOD <LOD <LOD
Scots pine 304 0111
c
411 0013
c
<LOD <LOD <LOD
Sitka spruce 436 0011
d
425 0322
c
<LOD <LOD <LOD
Pvalue <0001 <0001 <0001 0081 0130
LOD, limit of detection.
Mean with different letters differ significantly within column (P≤005).
Pine
100
150
200
250
300
95% CI weight (g)
350
Spruce Hemp Straw
Bedding type
Figure 1 Mean moisture-holding capacity by weight for each of four
bedding types tested. This figure shows the mean moisture-holding
capacity by weight (g) (SD) of each bedding type of pine, spruce,
hemp and straw. Pine bedding held significantly (P<0001) more
moisture when compared to all other bedding types.
Journal of Applied Microbiology 122, 23--29 ©2016 The Society for Applied Microbiology26
Bacteria and equine bedding K. Yarnell et al.

compared to other commonly used equine bedding sub-
strates including hemp and straw. Furthermore, wood-
based bedding of pine and spruce absorbed and retained
significantly more urine and continued to do so for up to
24 h when compared to the other bedding types tested
which absorbed urine for up to 8 h.
With regard to the moisture-holding capacity, it must
be stressed that this trial took place in a laboratory set-
ting. In a horse’s stable, moisture will be introduced spo-
radically to the bedding over a longer period each time
the horse passes urine. Therefore, the bedding will have a
chance to absorb this moisture and dry out before the
introduction of further urine. This part of the trial was
conducted to gain a basic idea of the moisture-holding
capacity. Results may be different if this trial were to be
repeated in field conditions and is an area of future
investigation.
Hemp and straw bedding continued to absorb mois-
ture for a total of 8 h. This is still satisfactory in terms of
equine hygiene as most horse owners will remove dirty
bedding twice per day. Further work must now be carried
out in field conditions to ascertain the rate at which
moisture is removed by each bedding type and how
much moisture each bedding type is capable of absorb-
ing. This is important as the amount of free urine in the
horses bed could contribute to bacterial growth by pro-
viding a source of nitrogen.
The bacteria types tested pose varying levels of disease
risk for the equine species ranging from the discomfort
of distal limb dermatitis associated with De. congolensis
to the significant welfare and economic implications of
strangles caused by the bacterium Strep. equi. Recent
work on pathogen control strategies in sheep suggest that
the environment may assist persistence of footrot caused
by Di. nodosus, one of the bacteria tested during this
study and one which costs the UK farming industry £24
million per year (Nieuwhof and Bishop 2005). Footrot
occurs in winter in housed sheep, therefore, Di. nodosus
must also survive on bedding long enough to be trans-
mitted between animals and Green and George (2008)
suggest that damp bedding is an ideal medium for sur-
vival. As pine bedding produced the least bacterial growth
and the largest moisture-holding capacity during this
study, this suggests pine-based bedding to be a suitable
choice for housed sheep as well as for horses to prevent
spread of disease and poor foot health. Transmission of
the bacteria from one animal to another will always occur
via the surface on which they are kept Green and George
(2008). It is therefore important that further work be
carried out to improve knowledge on bacterial survival
and transmission in a range of bedding substrates and
surfaces.
It is not clear at this stage for this particular study why
pine-based bedding produced the least bacterial growth
but there are a number of factors to consider. Evidence
suggests that equine health improves (foot and respira-
tory health in particular) when horses are stabled on
pine-based product and this could be due to the reported
pathogen-resisting properties of pine (Kim and Shin,
2005; Zeng et al., 2012).
V€
alimaa et al. (2007) tested 30 species of hard and soft
wood trees for their antimicrobial activity and reported
that by far the most consistent antibacterial and antifun-
gal properties were associated with extracts of Pinus spe-
cies. The authors suggest that this antimicrobial effect is
associated with stilbenes, a polyphenol found in the
wood. This compound may be lost in the processing of
the bedding or with age of the wood and so other factors
that could contribute to the bacterial resistance must be
considered.
One factor to consider would be the physical proper-
ties of the bedding itself, including moisture holding
capacity. Animal urine provides a source of nitrogen
which assists bacterial growth. If the bedding substrate
0
50
100
150
200
250
300
350
400
246824
Weight (g)
Time (h)
Figure 2 Mean (SD) wet weight over time
for each of four bedding substrates tested.
This figure shows the mean (SD) wet
weight of each bedding type of pine shavings
(black), spruce shavings (white), hemp (grey)
and straw (hatched) over a 24-h period.
Bedding was weighed at 2 h post
introduction of urine (466 ml) and then at 4,
6, 8 and 24 h post introduction of urine.
Each bedding was tested three times and a
mean calculated.
Journal of Applied Microbiology 122, 23--29 ©2016 The Society for Applied Microbiology 27
K. Yarnell et al. Bacteria and equine bedding

used can remove and hold urine then this will inhibit the
growth of the bacteria. During this study pine-based bed-
ding produced the least bacterial growth and was also the
most absorbent substrate which supports this theory.
During this study pine and spruce bedding reduced the
growth rate of bacteria in a controlled laboratory setting.
It is therefore important that these lab findings be tested
in field conditions. If the bedding substrate used as part
of a horse’s management can reduce survival and spread
of disease-causing pathogens, then this has an application
in biosecurity measures for the equine industry. Bedding
is used not only in the horse’s own stable but in horse
transporters, temporary stables at competitions and
equine stabling at veterinary centres, all of which are uti-
lized by many different horses and are key areas where
disease prevention is paramount. Bedding choice may be
one way of reducing disease risk in these scenarios.
As well as identifying and managing disease, prevention
of pathogen transmission needs further research. Horse
husbandry, in particular choice of bedding substrate, can
potentially make an important contribution to prevention
or reduction of disease outbreak.
Acknowledgements
The authors thank BEDMAXTM for providing the bed-
ding materials tested during the study and for part fund-
ing of the project in collaboration with Nottingham
Trent University. We also thank Samantha York and her
stallion Oakring Chadun for on demand provision of the
urine used in the trial.
Conflict of Interest
This project was part funded by BEDMAXTM who also sup-
plied three of the bedding substrates tested. Nottingham
Trent University and the authors of this study have no
commercial or personal relationships with this company.
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