Microbiome analysis of the skin of sheep that are resistant or susceptible to breech flystrike
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CSIRO PUBLISHING Special Issue: AAABG 2021
Animal Production Science, 2021, 61, 1774–1780
https://doi.org/10.1071/AN21063
Microbiome analysis of the skin of sheep that are resistant
or susceptible to breech flystrike
J. C. Greeff A,B,E, E. A. Paz B, K. Munyard C, A. C. Schlink A, J. Smith D,
L. J. E. Karlsson A, G. B. Martin B and D. Groth C
A
Department of Primary Industries and Regional Development, Perth, 3 Baron Hay Court, South Perth,
WA 6151, Australia.
B
Institute of Agriculture, University of Western Australia, Crawley, 35 Stirling Highway, Crawley,
WA 6009, Australia.
C
Curtin Medical School, Curtin University, Perth, WA 6102, Australia.
D
CSIRO, Livestock Industries, New England Highway, Armidale, NSW 2350, Australia.
E
Corresponding author. Email: johan.greeff@dpird.wa.gov.au
Abstract
Context. Breech strike is a serious disease for wool sheep. Skin wrinkle and dags are known predisposing factors for
breech strike; however, a large part of the variation among sheep is unknown.
Aims. We studied the natural diversity and difference in microbial populations in the skin around the breech area in
Merino sheep genetically resistant and susceptible to breech strike, by using 16S rRNA gene sequence analysis.
Methods. The sheep were from the breech strike flocks at the Mount Barker research station in Western Australia and
from the CSIRO research station near Armidale in New South Wales. Skin samples were collected from the breech of all
2013-born progeny in both flocks before they were struck. Yearling ewes and rams were then naturally exposed to
challenge by Lucilia cuprina blowflies. Breeding values for breech strike were estimated and used with phenotypic data
to identify breech strike-resistant and -susceptible sheep. Skin samples of 78 unstruck and 73 struck sheep were
selected, their microbiomes were analysed using 16S rRNA meta-barcoding, and operational taxonomic unit counts
were analysed.
Results. The diversity analyses showed that the two flocks in the different environments had different microbiome
profiles, but no difference was found between sexes or between breech strike-resistant and -susceptible sheep in either
flock.
Conclusions. The results indicated that microbial differences on the skin of sheep are not associated with differences
in susceptibility to breech strike.
Implications. Microbial differences do not offer opportunities to manage breech strike in Merino sheep.
Keywords: Merino sheep, breech strike, 16S rRNA gene, microbiome.
Received 8 February 2021, accepted 14 April 2021, published online 7 July 2021
Introduction area, making it more favourable for blowfly larvae to develop
Breech strike is a serious disease caused by the Australian (Seddon 1931). Greeff et al. (2018b) showed that sheep that
sheep blowfly, Lucilia cuprina. Greeff et al. (2014, 2018a, were struck before hogget shearing at 16 months of age, are
2018b, 2019) and Smith et al. (2009) showed that breech strike also more likely to be struck as adults under a management
is a heritable trait, and that large differences exist among sires regime where the sheep were annually crutched to prevent the
in the proportion of fly-struck sheep in both ewe and ram accumulation of dags. They showed that large differences exist
progeny groups. Faecal soiling (dags) and skin wrinkles of the among sire progeny groups for breech strike resistance that
breech explained most of the variation between struck and varied from only 3% of the progeny of the most resistant sire
unstruck animals in a Mediterranean environment (Greeff being struck, compared with 103% (some sheep were struck
et al. 2018a), whereas Smith et al. (2009) found that skin more than once) of the progeny of the most susceptible sire
wrinkles were the most important predisposing factor for being struck under the same environmental conditions. These
flystrike in a summer rainfall region. The presence of large differences among sire progeny groups contrast with the
moisture is the critical factor in breech strike, skin-wrinkles modest differences in visually detectable indicator traits such
and dags may change the micro-environment in the breech as dags, skin wrinkles and breech cover. They concluded that
Journal compilation CSIRO 2021 Open Access CC BY www.publish.csiro.au/journals/an
Microbiome of the skin of sheep Animal Production Science 1775
other, as yet unknown factors appear to contribute to Animals
differences among sheep in susceptibility to breech strike. Two Merino flocks were used in the present study. One flock
Susceptibility to breech strike is also a repeatable trait in both was located in a summer-rainfall region (SR) at the CSIRO
winter- and summer-rainfall regions. In a winter-rainfall research station near Armidale, New South Wales (Smith et al.
region, the correlation between breech strike from birth to 2009), and the second flock was located at the Mount Barker
weaning and breech strike from weaning to hogget shearing research station of the Department of Agriculture and Food
was 0.29 (Greeff et al. 2014), and between breech strike from Western Australia in the winter-rainfall region (WR) of
birth to hogget age and breech strike in mature sheep, the Western Australia (Greeff et al. 2014). The SR flock
correlation was 0.14 (Greeff et al. 2021). In a summer-rainfall consisted of 439 sheep whereas the WR flock consisted of
region, Bird-Gardiner (2015) showed that the correlation 937 sheep. The animals in both flocks were born in 2013.
between breech strike over 4 years was 0.34. These
estimates are higher than the associated heritability, which Recording of breech strike
indicates that other permanent environmental factors contribute
None of the sheep in this experiment was mulesed. The WR
to making sheep more resistant or susceptible to breech strike.
sheep were crutched in May, at ~10 months of age, 4 months
Greeff et al. (2013) showed that trained sniffer dogs could
before sampling in August, whereas the SR sheep were
differentiate with a high degree of accuracy (>80%) between
crutched at weaning in January, and skin-sampled in
wool from resistant and wool from susceptible sheep that had
August. The sheep were then naturally exposed to
not been struck by blowflies. This indicates that odour may
blowflies. Daily inspections were undertaken to identify
play a role in attracting blowflies to sheep. Various studies
struck sheep. All blowfly strikes were recorded. Any struck
(Emmens and Murray 1983; Eisemann 1985, 1995; Morris
sheep was clipped clean around the strike site, was treated with
et al. 1997; Urech et al. 2004) have been conducted using
a short-acting insecticide (Extinosad®) at the manufacturers
mixtures of volatile compounds to determine blowfly
recommended rate, and was released back into the flock. A
attractants. Yan et al. (2019) used L. cuprina to compare
small proportion of sheep was struck more than once.
the attractiveness of wool from unstruck breech strike-
resistant and breech strike-susceptible sheep. They found
Estimating breeding values for breech strike
that blowflies do show a preference for wool from specific
sheep. Blowflies were more attracted to sheep whose wool The breech strike records were analysed with best linear
contained more of the semiochemicals octanal and nonanal. unbiased prediction mixed model methodology (Henderson
These compounds are well known attractants for a variety of 1984), using the ASRemL software package (Gilmour et al.
insects (https://www.pherobase.com/). However, the origin 2015). All previous years’ breech strike records (from 2006
of these compounds is unknown and may originate from onward) were included in the analysis. Separate analyses were
microbial populations on sheep, as Mulcock and Fraser performed for each site. Full pedigrees were available on all
(1958) indicated that the fleece is a suitable habitat for sheep, and an animal model was fitted with year of birth, sex of
microorganisms and that all the requirements for microbial the lamb, birth status (single or multiple) and age of the dam as
growth are present in the fleece. fixed effects. Animal was fitted as a random effect. The sheep
Jackson et al. (2002) used culture-based studies to were ranked within sex and within site on their breeding value
determine the bacterial composition in sheep fleece. for breech strike.
However, this technique identifies less than 1% of all
microbial species present in an environmental sample. The Collection of skin samples
use of 16S rRNA analysis (Hugenholtz 2002) facilitates the Skin samples were collected on all the sheep in both flocks
characterisation of mixed microbial communities without before their first shearing and before the onset of the fly season.
culturing. Dixon et al. (2007) used 16S rRNA gene analysis Both flocks were sampled in August 2014. No skin cleaning or
to investigate the natural bacterial diversity on the skin of clipping of the site was performed before collecting the skin
fleece rot-resistant and -susceptible sheep. They found that sample. The site was anaesthetised with 0.5 mL lignocaine
four operational taxonomic units (OTUs; groups of >97% intradermally (2% Lignocaine, Troy Animal Health,
sequence similarity) were present on susceptible, but absent Australia). A small (0.5–1.0 cm2) biopsy of skin was
from the resistant sheep. Hence, the current study was surgically removed with curved scissors from the
undertaken to determine whether the skin microbiome on designated breech area, ~2 cm to the right from the tip of
the breech contributes to differences in susceptibility to the docked tail, at 11 months after birth on the lambs in the SR
blowflies in unstruck resistant and susceptible Merino sheep flock, and at 14–15 months of age in September 2014 on all the
before the onset of the flystrike season, from both a summer- lambs in the WR flock. When an animal had excessive dags at
and winter-rainfall regions. sampling, care was taken to sample from a non-daggy area as
close as possible to the designated sampling site, so as to
ensure that the sample did not contain any dags. Some sheep in
Materials and methods the SR flock were already struck by blowflies at sampling;
The study was approved by the Animal Ethics Committee of however, their samples were not used in the study. None of the
the Department of Agriculture and Food Western Australia WR flock sheep was struck at, or before, sampling in spring. A
(Approval number AEC 17-4-09) and by the CSIRO Armidale separate sterilised pair of scissors was used on each lamb to
animal Ethics Committee (Approval number 13/35). prevent any cross-contamination. The wool on each skin
1776 Animal Production Science J. C. Greeff et al.
sample was trimmed back to a length of ~3 mm. The sample Merging of paired-end sequences and clustering de novo into
was stored in RNAlater, and held at –20C until processing. OTUs with a 97% similarity was performed using micca
v.1.7.0 (Albanese et al. 2015). Taxonomic assignment was
Selection of sheep for 16S rRNA analysis executed using the Bayesian Lowest Common Ancestor-based
All the lambs were skin sampled in each flock. After the fly taxonomic classification method (Gao et al. 2017) on the basis
season was completed, the phenotypic breech strike data were of the NCBI 16S microbial database. A phylogenetic tree was
analysed, as described above, to obtain breeding values on all built with FastTree, applying generalised time-reversible and
the animals in both flocks. The breeding values from both sites CAT approximation (Price et al. 2010). To normalise the
were standardised by subtracting the flock mean breeding number of reads in each sample, we rarefied to a depth of
value from the breeding value of each animal in the flock, 2732. Alpha and b diversities were estimated using QIIME
and by dividing the result with the standard deviation of the v.1.9.1 (Caporaso et al. 2010). To determine the significance
flock. This standardised value allowed for the identification of of the alpha metrics, a non-parametric two-sample Student’s
the most resistant and susceptible sheep in both locations. Any t-test with Bonferroni correction was performed. A principal
sheep with a high breeding value and that was not struck, and coordinates analysis was performed using unweighted UniFrac
any sheep with a low breeding value and that was struck was distance. The statistical significance of the distance matrix was
excluded from genetic analysis. The rams and ewes with the tested using analysis of similarity with 1000 permutations. The
highest (susceptible) and lowest (resistant) breeding values linear discriminant analysis effect size algorithm was used to
were then identified. Sixty-six sheep were selected from the identify significant taxonomic differences.
SR flock, and 85 from the WR flock (Table 1). The skin
samples from these 151 selected sheep were retrieved from the
stored library of skin samples and sent to the Australian Results
Genomic Research Facility (AGRF) for processing and In total, 15935704 raw reads were obtained from all samples
sequencing. combined. After trimming, filtering and merging of paired-end
reads, 4 654 408 tags were included in the workflow. In total,
DNA extraction and primary sequencing analysis 3212 OTUs were clustered at 97% of similarity. The Shannon
The extraction and isolation of DNA was undertaken by AGRF rarefaction curves reached a plateau and both groups had an
using the DNeasy PowerLyzer Powersoil Kit (QIAGEN 2017). acceptable sequence depth (Fig. 1). The rarefaction analysis
Conserved primers were used for the 16S rRNA amplification shows that the observed OTUs reached a plateau at a
(27F: AGAGTTTGATCMTGGCTCAG; and 519R: GWATT sequencing depth of 2000 bases at both flocks. This
ACCGCGGCKGCTG). The amplicon length was 300 bp. resulted in 232 993 (16.5%) features in 151 (86.3%)
The samples were sequenced using a paired-end protocol, subsamples of the total number of samples at this specified
and image analysis was performed in real time by the MiSeq depth.
Control Software v.2.6.1.1 and real-time analysis v.1.18.54. The Shannon diversity index plots are shown in Fig. 1 for
The Illumina bcl2fastq 2.17.4 pipeline was used to generate the the different samples, resistant or susceptible, location and sex,
de-multiplexed sequence files. The sequence data have been while the a diversity metrics, Chao1, observed species and
submitted to the NCBI Sequence Archive under the Bioproject Shannon analyses between the winter- and summer-rainfall
number PRJNA657495. regions are shown in Fig. 2. Higher microbial diversity was
measured in the WR flock than in the SR flock. However, no
Statistical analyses significant differences were found between rams and ewes, or
16S rRNA analysis between resistant or susceptible animals within flocks. There
was no correlation between microbial diversity and the
Trimming of raw reads was undertaken using Sickle, setting
individual standardised breeding value for breech strike for
the length-threshold at 200 bp and quality cut-off at 20 (https://
either flock (Fig. 3). The spearman test correlation between the
github.com/najoshi/sickle). The first 10 bases of the forward
standardised breeding value for breech strike and the 16S
and reverse reads were trimmed in the 16S rRNA analysis, and
rRNA sequences was very low, that is 0.0044 (P-value =
to ensure a minimum quality score of 30, the forward reads
0.9569).
were truncated at 190 bases, and the reverse reads at 145 bases.
The principal coordinate analysis showed a distinct
clustering of the samples by region (Fig. 4). Subsequent
Table 1. Number of sheep sampled per region and treatment analysis of similarity found highly significant differences
between the WR and SR regions (P = 0.00009; R2 = 0.724).
Flock Resistant Susceptible Total The microbial composition was evaluated at both phylum
and genus levels. Proteobacteria were the dominant
WR: winter rainfall (Mount Barker, WA)
identifiable phylum in the SR flock (5.3%), followed by
Rams 18 23 41
Ewes 21 23 44 Actinobacteria (1.4%) and Firmicutes (0.8%). Whereas in
the WR flock, Actinobacteria (15.6%), Proteobacteria
SR: summer rainfall (Armidale, NSW)
(11.2%) and Firmicutes (3%) were the most abundant
Rams 7 33 40
Ewes 10 16 26
identifiable phyla (Fig. 5). However, most of OTUs in both
Total 56 95 151 flocks were unable to be classified at the phylum level (92.4%
and 69.5%).
Microbiome of the skin of sheep Animal Production Science 1777
The top five most abundant genera in the study
Fig. 1. Shannon diversity index plot. (a) Shannon rarefaction curve based on sample ID. (b) Shannon rarefaction curve based on selection line (resistant or susceptible). (c) Shannon rarefaction curve
3000
were Dietzia (3.4%), followed by Corynebacterium
Ram
Ewe
(1.8%), Sphingomonas (1.1%), Paracoccus (1%) and
2500
Brevundimonas (1%). In the SR flock, Rhizobium (1.3%)
and Sphingomonas (1.3%) were the dominant taxa followed
2000
by Corynebacterium (0.5%). The genera most abundant in the
WR flock were Dietzia (6.6%), Corynebacterium (3.1%) and
1500
Sex
Brevundimonas (1.8%; Fig. 6). In both flocks, a large
proportion of the OTUs could not be classified to genus
1000
level (93.3% and 72.6%).
The linear discriminant analysis effect size analysis (Fig. 7)
500
indicated that Rhizobium and Sphingomonas were
significantly more abundant in the SR flock, whereas
(d)
Dietzia, Brevundimonas and Corynebacterium were
0
3000
significantly more abundant in the WR flock.
Summer
Winter
2500
Discussion
The present study was conducted to determine whether the
2000
microbial populations on the skin in the breech of sheep
contribute to sheep being resistant or susceptible to breech
1500
Site
strike. On the basis of the work of Greeff et al. (2014), it was
speculated that unique odours that are more attractive or
repulsive to blowflies, and/or create a more favourable
1000
environment for blowfly larvae in the breech, may be
correlated with a sheep’s susceptibility or resistance to
500
Sequences per sample
breech strike. The present study is the first study of this
(c)
nature that has used proven genetically resistant and
3000 0
susceptible sheep. The only other related study was
conducted by Dixon et al. (2007) who used sheep resistant
Susceptible
and susceptible to fleece rot.
Resistant
2500
A large proportion of unknown OTUs was identified in the
study and could not be classified even at the phylum level. In
the summer-rainfall region, only 7.6% of the OTUs were
2000
associated with known microbial species, and 30.5% in the
Treatment
winter-rainfall region. Thus, sheep from a summer- and from a
1500
winter-rainfall region will host different microbial
populations, related to that environment. In the summer-
1000
rainfall site, the genera Rhizobium and Sphingomonas were
significantly more abundant, while at the winter-rainfall site,
500
Brevundimonas and Dietzia were the most abundant. These
based on flock. (d) Shannon rarefaction curve based on sex.
four genera are mostly associated with plants and soils. Dixon
(b)
et al. (2007) also found that 75% of the sequences in their
0
study had not previously been identified in fleece-rot studies.
3000
However, contrary to our results, they did find that four OTUs
were present on body strike-susceptible sheep, but absent from
2500
the body strike-resistant sheep. However, although they did
find differences, they sequenced only two resistant and two
2000
susceptible sheep in their study.
Sample ID
Moisture is a key requirement for blowfly eggs and larvae
1500
to develop. Rams and ewes are anatomically very different and
therefore ewes with urine stain may have more moist breeches
1000
that could be more conducive for blowfly larvae to develop.
Greeff et al. (2018b) also showed that sheep with urine stain
are more susceptible to breech strike. Therefore, these factors
500
may indicate that rams and ewes could harbour different
(a)
microbial species on their breeches. However, no significant
0
(P > 0.05) difference was found between males and females
6
5
4
3
2
1
0
Rarefaction measure: shannon
that were managed separately in different paddocks in both the
1778 Animal Production Science J. C. Greeff et al.
Chao1 Observed species Shannon
1000 600 6
900 P = 0.001
500 5
800
700 P = 0.001 P = 0.001
400 4
600
300 3
500
400 200 2
300
100 1
200
100 0 0
WR SR WR SR WR SR
Fig. 2. Alpha diversity metrics between winter-rainfall (WR) and summer-rainfall (SR) regions.
0.90
0.85
0.80
Pielou’s evenness
0.75
0.70
0.65
0.60
0.55
0.50
−2.0 −1.5 −1.0 −0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0
Standardised breeding value
Fig. 3. Relationship between species evenness and the standardised breeding value for breech strike.
SR
WR The present study has clearly shown that in a total of 3212
OTUs, no significant difference in microbial populations was
able to be detected between sheep resistant and susceptible
0.20
for breech strike. Thus, we conclude that the composition of
microbial populations in the breech area of unstruck sheep
PC3 - Percent variation
0.15
before the blowfly season does not contribute to making
explained 2.78%
0.10
sheep more or less susceptible to breech strike. Because
0.05
large genetic differences exist between sire progeny
0.00
groups for breech strike, the present study indicated that
−0.05 any difference between sheep is largely due to the animal
−0.10 itself, of which dags and breech wrinkle are the two most
−0.15 important predisposing factors. These two traits explain only
0.3 up to 40% of the variation in breech strike (Greeff et al.
0.2 0.3
PC 0.1 0.1
0.2 2018a), which indicates that more research is needed to
2- on
P
exp ercen
0.0 0.0 riati determine why certain sheep are repeatedly struck over
−0.1 t va
lain
ed
t va
r
−0.1 −0.2 e r cen 6.19% their lifetime, even in the absence of dags. Research
3.2 iation −0.2 −0.3 -P d1
7% PC1 plaine
ex should focus on the micro-environment in the breech area,
to determine whether diurnal patterns of temperature and
Fig. 4. Principle-coordinates analysis of the number of strikes between moisture differ between breech strike-resistant and breech
the winter-rainfall (WR) and summer-rainfall (SR) regions. strike-susceptible sheep.
WR and SR flocks. These results indicate that the microbial
species on ewes and rams were very similar across paddocks in Conflicts of interest
both environments. The authors declare no conflicts of interest.
Microbiome of the skin of sheep Animal Production Science 1779
Summer rainfall Winter rainfall
1.4%
5.3% 3%
0.8% 11.2%
15.6%
Unclassified
Proteobacteria
Actinobacteria
Firmicutes
92.4% 69.5%
Fig. 5. Classification of the most abundant phyla present at the sampling location.
Summer rainfall Winter rainfall
0.1%
0.1% 0.7%
0.7%
0.2% 0.7%
0.2% 0.7%
0.3% 0.8%
0.9%
0.3% 1.7%
0.4% 1.8%
1.3% 0.5% 3.1%
1.3%
6.6%
Unclassified Unclassified
Rhizobium Dietzia
Sphingomonas Corynebacterium
Corynebacterium Brevundimonas
Paracoccus Paracoccus
Devosia Sphingomonas
Romboutsia Rhodococcus
Dietzia Devosia
Brevundimonas Romboutsia
Kocuria Ornithinimicrobium
Methylobacterium Kocuria
93.3% 72.6%
Fig. 6. Relative abundance of the principal genera in the breech of sheep in the summer-rainfall and winter-rainfall
regions.
SR WR
Dietzia
Brevundimonas
Corynebacterium
Sphingomonas
Rhizobium
−6.0 −4.8 −3.6 −2.4 −1.2 0.0 1.2 2.4 3.6 4.8 6.0
LDA score (log 10)
Fig. 7. Linear discriminant analysis (LDA) effect size (LEfSe) between summer-rainfall (SR) and winter-
rainfall (WR) flocks for the 16S rRNA analysis.1780 Animal Production Science J. C. Greeff et al.
Acknowledgements Greeff JC, Karlsson LJE, Schlink AC, Gilmour AR (2018a) Factors
explaining the incidence of breech strike in a Mediterranean
We thank the Department of Agriculture and Food Western Australia for
environment in unmulesed and uncrutched Merino sheep. Animal
their support in conducting this experiment on the Department’s and
CSIRO’s research stations, as well as wool producers and the Production Science 58, 1279–1288. doi:10.1071/AN16528
Commonwealth government, through Australian Wool Innovation Ltd, Greeff JC, Schlink AC, Karlsson LJE (2018b) The impact of sire on the
for their generous financial and positive constructive contributions lifetime susceptibility of their progeny to breech strike in a
throughout the lifetime of this project. We also express our Mediterranean environment. Animal Production Science 58,
appreciation to Nicola Stanwyck, the technical officer involved with 1522–1530. doi:10.1071/AN17559
this experiment, for her diligence and dedication in recording the data Greeff JC, Karlsson LJE, Schlink AC (2019) Are breech strike, dags and
on the animals in this flock. breech wrinkle genetically the same trait in crutched, uncrutched and
mulesed Merino sheep? Animal Production Science 59, 1777–1782.
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