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Composition of bacterial and archaeal communities in the rumen of

dromedary camel using cDNA-amplicon sequencing

Article  in  International Microbiology · August 2019

DOI: 10.1007/s10123-019-00093-1

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International Microbiology
https://doi.org/10.1007/s10123-019-00093-1

REVIEW

Composition of bacterial and archaeal communities in the rumen

of dromedary camel using cDNA-amplicon sequencing
Alaa E. Rabee 1 & Robert J. Forster 2 & Chijioke O. Elekwachi 2 & Khaled Z. Kewan 1 & Ebrahim Sabra 3 &
Hoda A. Mahrous 3 & Omaima A. Khamiss 3 & Safinaze M. Shawket 1

Received: 26 December 2018 / Revised: 30 June 2019 / Accepted: 21 July 2019

# Springer Nature Switzerland AG 2019

Abstract
The camel is known to survive in harsh environmental conditions, due to its higher digestive efficiency of high-fiber diets
compared with other ruminants. However, limited data are available on the microbial community in the rumen of a camel. In
this study, the Illumina sequencing of V4 region of 16S rRNA genes based on RNA isolation was employed to get insight into the
bacterial and archaeal communities associated with liquid and solid rumen fractions in eight camels under different feeding
systems. Camels in group C1 were fed Egyptian clover hay plus concentrates mixture and camels of group C2 were fed fresh
Egyptian clover. The results showed that liquid fraction has higher operational taxonomic units (OTUs) than solid fraction, and
camel group C1 showed a higher microbial diversity than C2. The UniFrac analysis indicated that the microbial communities in
camel groups are distinct. Moreover, phylum Firmicutes and Bacteroidetes dominated the bacterial community and Candidatus
Methanomethylophilus dominated the archaeal community with a significant difference in the relative abundance between camel
groups. Dominant bacterial genera were Prevotella, Fibrobacteres, Ruminococcus, and Butyrivibrio. There were many negative
and positive correlations between and within bacterial and archaeal genera. The composition of microbial community in the
rumen of a camel is similar to other ruminants with differences in the abundance.

Keywords Camel rumen . Microbial diversity . Bacteria . Archaea . cDNA-amplicon sequencing

Introduction The productivity of the camel is mainly affected by the quality

The dromedary camel (Camelus dromedarius) is found most-
ly in arid and semi-arid countries of Africa and Asia; it is
strongly adapted to live under severe environmental condi-
tions (Guerouali and Wardeh 1998). A camel in developing
countries has several purposes for production, including milk,
meat, and wool; meanwhile, it can be used in transport, tour-
ism, agricultural work, and sports contests (Rabee et al. 2019).
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10123-019-00093-1) contains supplementary
material, which is available to authorized users.
* Alaa E. Rabee
rabee_a_m@yahoo.com
1
Animal and Poultry Nutrition Department, Desert Research Center,
Cairo, Egypt
2
Lethbridge Research center, Lethbridge, AB, Canada
3
Genetic Engineering and Biotechnology Research Institute, Sadat
City University, Sadat City, Egypt
of the diet and the type of forage that constitute the diet (Faye
2013). Camels can utilize the low-quality and lignocellulolytic
feeds, which are avoided by other domestic ruminants (Iqbal
and Khan 2001). The degradation of ingested plant material in
the rumen depend mainly on microbial fermentations
(Henderson et al. 2015). Therefore, the high digestion effi-
ciency of the camel could be attributed to the composition of
microbial community in the rumen (Samsudin et al. 2011).
Furthermore, the camel has the ability to retain ingested ma-
terial in the rumen for longer time than other ruminant and the
pH in camel rumen is closer to neutral, which support the
lignocellulose degradation (Lechner-Doll and Engelhardt
1989; Russell and Wilson 1996).
Bacteria dominate the microbial community in the rumen
and make the greatest contribution in rumen fermentation. In
addition, it plays an important role in shaping animal produc-
tive traits like milk fat yield (Kim et al. 2011; Jami et al. 2014).
Also, archaea in the rumen achieve a vital role by preventing
the hydrogen (H2) accumulation in the rumen by using it to
reduce carbon dioxide (CO2) to methane (CH4) through
methanogenesis. The production of methane increases
 Int Microbiol

greenhouse gases emission (Moss et al. 2000) and represents a of V4 region of the 16S rRNA gene using Illumina MiSeq
loss in dietary gross energy intake (Van Nevel and Demeyer platform based on RNA isolation. Furthermore, the microbial
1996). The camel loses about 7–9% of dietary energy in the communities associated with solid and liquid rumen fractions
form of methane (Hook et al. 2010). The composition of mi- in the camels were examined.
crobial community in the rumen is mainly influenced by type
of animal diet (Henderson et al. 2015). For instance, cellulo-
lytic and hemicellulytic diets favor the fibrolytic microbes, Material and methods
while starch and sugars are the major fermentation compo-
nents of concentrate-based diets, thus, favoring the amylolytic Animals and sampling
microbes (Carberry et al. 2012). According to Carberry et al.
(2014) and Hook et al. (2010), the impact of diet on the diver- Eight adult dromedary camels (4–5 years) were used in this study.
sity of rumen archaea was reported to be due to its effects on Group C1 was represented by three fistulated camels housed in
fermentation patterns and rumen pH. Maryout Research Station, Alexandria, Egypt, and were fed
The study of rumen microbial communities offers the pos- Egyptian clover hay (Trifolium alexandrinum) plus concentrates
sibility of maximizing animal production as well as increasing mixture (50:50%). Animals of group C2 were represented by five
biofuel production (Zhou et al. 2009). Next-generation se- camels offered free fresh Egyptian clover, then slaughtered in the
quencing techniques expanded our knowledge regarding the Komhammada slaughtering house, Elbehera, Egypt. The proxi-
composition and the dynamics of microbial community in the mate chemical analyses of animal feeds are illustrated in Table 1.
rumen (Petri et al. 2013). The cDNA-amplicon (RNA- Diets fed to animals contained the same forage plant and were
amplicon) sequencing technique is more efficient than characterized based on the forage proportion in the diet; low
DNA-amplicon sequencing in characterizing the metabolical- forage (50%) in C1 group and high forage (100%) in C2 group.
ly active microbes in the rumen as it base on the isolation of Thereafter, the rumen contents were strained immediately by two
RNA, which has a short turnover (Nagalakshmi et al. 2010; Li layers of cheesecloth to separate the liquid (LF) and solid (SF)
et al. 2016). Additionally, DNA-based techniques cannot dis- fractions to evaluate differences in the microbial community as-
tinguish between active cells, inactive but alive, dead cells, sociated with either fraction. A total of sixteen samples from
and lysed cells (Gaidos et al. 2011). Wang et al. (2017) used liquid and solid fractions were frozen using liquid nitrogen and
cDNA-amplicon sequencing to explore metabolically active stored at − 80 °C for further processing. The project was ap-
bacteria in four rumen fractions in black goats. They have proved by the Institutional Animal Care and Use Committee,
reported that the active bacterial community were dominated Faculty of Veterinary Medicine, University of Sadat City, Egypt
by phyla Proteobacteria, Firmicutes, and Bacteroidetes. (Approval reference #VUSC00003).
Most of studies have focused on the composition and the
factors that alter the microbial communities in the rumen of RNA isolation, PCR amplification, and amplicon
bovine and other domesticated animals. On the other side, few sequencing
studies are interested in exploring the composition of micro-
bial community in the rumen of the dromedary camel. RNA was isolated and reverse-transcribed into first-strand
Additionally, all molecular-based studies that investigated cDNA according to the protocol of Wang et al. (2017).
the rumen microbial communities in the rumen of the camel Polymerase chain reaction (PCR) amplification was carried
were based on DNA isolation (Samsudin et al. 2011;
Gharechahi et al. 2015). In one study, Samsudin et al. (2011) Table 1 The chemical composition (%) of diets fed to camels under
used 16S rRNA clone library from DNA extracted from the investigation
rumen of Australian fair camels to identify rumen bacteria; the
Feeds DM Chemical analysis (%) on DM basis
detected major phyla were Firmicutes (67%) and
Bacteroidetes (25%). In another study, Gharechahi et al. Ash CP CF EE NFE
(2015), used the 16S rDNA-amplicon pyrosequencing to ex-
plore the diversity of bacterial community based on the DNA Concentrates mixture1 92.41 9.96 13.81 14.48 2.92 58.83
extracted from solid and liquid rumen fractions of grazing Fresh Egyptian clover2 18.00 12.94 14.23 31.38 1.34 40.11
camels in Iran. The results showed that the majority of bacteria Egyptian clover hay2 89.29 12.13 12.44 25.64 1.73 48.06
belonged to Bacteroidetes (51%), Firmicutes(31%), 1
Concentrates mixture composed of 30% wheat bran, 22% cotton seed
Proteobacteria (4.8%), Spirochaetes (3.5%), Fibrobacteres meal, 33% yellow corn, 10% sunflower meal, 3% molasses, 1.5% lime-
(3.1%), Verrucomicrobia (2.7%), and Tenericutes (0.95%). stone, and 0.5% salt
This study is aimed at determining the diversity and the struc- 2
Trifolium alexandrinum
ture of bacterial and archaeal communities in the rumen of DM, dry matter; CP, crude protein; CF, crude fiber; EE, ether extract;
camels under two different feeding systems by the sequencing NFE, nitrogen-free extract
Int Microbiol
out using the V4 region of 16S rRNA gene primer set 338F
and 806R (Liu et al. 2016). For that, a mixture of 4 μl template
cDNA, 12.5 μl KAPA2G Robust Hot Start ready mix PCR kit
(KAPA BIO), 1.25 μl of 10 pmol/μl forward primer, and
1.25 μl of 10 pmol/μl reverse primer was added to 6 μL mo-
lecular biology grade water. The samples were then incubated
in a thermal cycler (PTC-220 DNA Engine Dyad Peltier
Thermal Cycler, Roche Molecular system) for 1 cycle at
95 °C for 3 min and 30 cycles at 94 °C for 20 s, 65 °C for
20 s and 72 °C for 50 s followed by 72 °C for 3 min. Two PCR
reactions were performed for each sample and PCR products
were then pooled together to form a final volume of 50 μL.
The PCR product was purified using QIAquick Gel
Purification Kit (Qiagen) and quantified by Quant-iTPico
Green dsDNA Assay Kit (Invitrogen) with a BioTek
Instruments microplate reader (BioTek). The final concentra-
tion of the library was determined by Applied Biosystem’s
7900HT Fast Real-Time PCR System (Life Technologies
Corporation). The libraries were then diluted to 4 nM and a
mixture of the amplicons was then sequenced in the Illumina
MiSeq system.
Data analysis
The pre-processing stage included quality checks, adaptor and
barcode removal, and trimming and assembling/merging of
forward and reverse reads of the paired-end sequence reads.
Fast QC version 0.11.4 (Andrews 2010) was used to evaluate
sequence quality. For data trimming and adaptor removal, the
Trimmomatic program version 0.35 (Bolger et al. 2014) was
applied. Pear version 0.9.6 (Zhang et al. 2014) was used to
merge corresponding read pairs (read1 and read2). The
merged sequence files were then subsampled as needed using
the fasta-subsample tool of MEME 4.10.2 (Bailey et al. 2009).
The amplicons analysis was performed using QIIME Version
1.9.0 (Caporaso et al. 2010). The analysis included the de
novo OTU picking, picking and assigning of a representative
sequence for each OTU, and filtering the alignment and build-
ing a phylogenetic tree. For each sample, 15,000 reads were
run through the pipeline. SILVA release 119 reference taxon-
omy and SILVA rep-set 97 genes databases were used for
reference. All sequences have been deposited in SRA under
study code SRP105269 with the accession numbers
SRX2765887–SRX2765902.
Diversity and statistical analysis
Alpha diversity indices Chao1, phylogenetic diversity,
Shannon, inverse Simpson’s, and the number of observed
OTUs were evaluated using QIIME. Additionally, beta diver-
sity was evaluated as the principal coordinate analysis (PCoA)
based on unweighted UniFrac analysis to compare the cluster-
ing of microbial communities in rumen samples. Data of
relative abundance of bacteria and archaea at phylum and
genus level was tested for normality and homogeneity using
the Shapiro-Wilk test and variables that were deemed non-
normal were then arcsine transformed. The significance of
differences in the relative abundances of bacterial phyla and
dominant genera were examined using unpaired T test with
SPSS 20 (SPSS 1999) at P < 0.05. Spearman’s correlation was
performed using SPSS. Correlation Heatmaps were drawn
online (http://www.heatmapper.ca.) (Babicki et al. 2016).
Results
Sequencing information and diversity indices
The sequencing of the variable region 4 (V4) of 16S rRNA in
sixteen solid and liquid rumen samples generated 237040
high-quality sequences in this study. The total number of se-
quence reads was 117680 reads in the solid fraction (SF) and
119360 in the liquid fraction (LF). Alpha diversity metrics
were used to assess the similarity in the community structure,
Chao1, Shannon, Inverse Simpson, and phylogenetic diversi-
ty (PD) whole tree. The average number of OTUs was rela-
tively higher in the LF compared with SF and the values of
alpha diversity metrics followed the same trend (Table 2).
Camel group C1 exhibited a higher sequence number, OTUs
number, Chao1 values, and phylogenetic diversity compared
with C2 without significant differences (Table 2). The beta
diversity of microbial communities within camel groups for
liquid and solid fractions were calculated using principal co-
ordinate analysis (Fig. 1) based on unweighted UniFrac. The
results showed that the microbial communities in the camel
group C1 were separated distinctly from those of C2 camels.
Analysis of bacterial community
The sequences in the current study were finally assigned as
bacteria (94.5%), archaea (1.4%), and 4.1% was unassigned
microorganisms. Unassigned reads and unclassified bacteria
were higher in LF; group C1 had the highest proportion of
unassigned reads with a significant difference in SF and LF
fractions (Table 3 and Fig. 2a). Overall, a total of 22 bacterial
phyla and a phylum belonging to archaea were detected. The
results revealed that the bacterial community was dominated
by six phyla; Firmicutes, Bacteroidetes, Fibrobacteres,
Spirochaetes, Proteobacteria, and Actinobacteria. Moreover,
phyla that was found to be between 0.5–1% were
Synergistetes and Lentisphaerae. While, phyla less than
0.5% were Cyanobacteria, Tenericutes, Verrucomicrobia,
Planctomycetes, Chloroflexi, Elusimicrobia, Candidate divi-
sion SR1, Candidate division TM7, and SHA-109 (Table 3).
Phylum Firmicutes were found dominant in the bacterial
community of the camel rumen; it was higher in SF-C2 (Solid
 Int Microbiol

Table 2 Summary of OTU

numbers, and alpha-diversity es- Diversity indices C1 C2 Overall mean
timators for rumen archaea and
bacteria in ruminal solid (SF) and Sequences/sample/SF Av. 15,130 ± 1680 14,458 ± 1959.2 14,710 ± 1295
liquid (LF) fractions of camels Sequences/sample/LF Av 15,690 ± 1120 14,458 ± 1464.59 14,920 ± 975
under different feeding regimes OTUs/SF 4238.6 ± 302.9 3774.92 ± 396.6 3948.8 ± 270
(Mean ± SE)
OTUs/LF 4484.4 ± 313.8 4086.92 ± 339.27 4236 ± 238
PD (whole tree)/SF 224.2 ± 7.6 205.5 ± 12.9 212.5 ± 8.7
PD (whole tree)/LF 229.27 ± 9.25 215.3 ± 13.57 220 ± 9
Chao1/SF 18,120.9 ± 1518 14,735.21 ± 1702.84 16,004.8 ± 1290
Chao1/LF 18,294.9 ± 1396 16,061.63 ± 1322.6 16,899.1 ± 1000
Shannon/SF 9.7 ± 0.1 9.3 ± 0.1 9.5 ± 0.09
Shannon/LF 9.7 ± 0.1 9.7 ± 0.1 9.7 ± 0.08
Simpson/SF 0.99 ± 0.002 0.99 ± 0.005 0.99 ± 0.0009
Simpson/LF 0.99 ± 0.001 0.99 ± 0.0008 0.99 ± 0.0005

Av., average; PD, phylogenetic diversity

fraction of group C2) and the difference between groups was
significant in LF (P < 0.05) (Table 3). The classification of
Firmicutes to the family level revealed that the Firmicutes
members were assigned to 26 families dominated by
Ruminococcaceae and Lachnospiraceae. Moreover, phylum
Firmicutes was classified into 70 genera dominated by
Ruminococcus, Butyrivibrio, and uncultured
Ruminococcaceae which were higher in SF-C2 (Fig. 2a).
Phylum Bacteroidetes represented the second largest phy-
lum in the bacterial community. The results showed that
Bacteroidetes was highly abundant in LF-C1 and the differ-
ence was significant in SF (P < 0.01) (Table 3). The family
level classification of phylum Bacteroidetes revealed 27 fam-
ilies that were dominated by Prevotellaceae, S24-7,
Rikenellaceae, and Bs11 gut group. Additionally, the
Bacteroidetes at the genus level revealed 28 genera dominated
by Prevotella, which showed the highest relative abundance in
LF-C1 with a significant difference in SF (P < 0.05). RC 9 gut
group showed its highest percentage in SF-C1 with a signifi-
cant difference in LF (P < 0.01). Moreover, uncultured
Bacteriodetes were higher in SF-C1 (Fig. 2a).
Fibrobacteres made up 8.88% in SF-C1, 14.26% in SF-C2,
5.8% in LF-C1, and 7.27% in LF-C2. The sequence reads that
belong to the phylum Spirochaetes were higher in SF-C1. The

Fig. 1 Principal coordinates

analysis of camel rumen
microbial community based on
unweighted UniFrac distances.
The analysis was conducted
within two camel groups: blue
circles for samples of solid
fraction in group C1 (SF-C1), and
green circles for samples of liquid
fraction in C1 (LF-C1).
Moreover, yellow circles for
samples of solid fraction of C2
(SF-C2), and red circles for
samples of liquid fraction in C2
(LF-C2)
Int Microbiol

Table 3 The relative abundance

of bacterial and archaeal phyla in Phylum C1 C2 Overall mean P value
ruminal solid (SF) and liquid (LF)
fractions of camels under investi- Euryarchaeota 0.67 ± 0.02 2.34 ± 0.47 1.771 ± 0.4 P < 0.05
gation (Mean ± SE) Euryarchaeota 0.63 ± 0.09 1.4 ± 0.33 1.1 ± 0.25 –
Actinobacteria/SF 1.4 ± 0.15 2.1 ± 0.23 1.9 ± 0.2 –
Actinobacteria/LF 0.75 ± 0.05 2.27 ± 0.4 1.7 ± 0.4 P < 0.05
BD1–5/SF 0 0.008* –
Bacteroidetes/SF 33.4 ± 2.5 23.02 ± 1.2 26.9 ± 2.2 P < 0.01
Bacteroidetes/LF 45.7 ± 2.2 40.26 ± 1.1 42.3 ± 1.46 –
Candidate-SR1/SF 0.07 ± 0.01 0.24 ± 0.07 0.18 ± 0.05 –
Candidate-SR1/LF 0.12 ± 0.04 0.45 ± 0.09 0.33 ± 0.08 P < 0.05
Candidate-TM7/SF 0.06 ± 0.007 0.04 ± 0.01 0.05 ± 0.009 –
Candidate-TM7/LF 0.06 ± 0. 01 0. 045 ± 0. 01 0.05 ± 0.008 –
Chlorobi/SF 0.004 ± 0.00006 0.004 ± 0.002 0.004 ± 0.001 –
Chloroflexi/SF 0.068 ± 0.009 0.067 ± 0.01 0.067 ± 0.009 –
Chloroflexi/LF 0.03 ± 0.001 0.04 ± 0.01 0.04 ± 0.008 –
Cyanobacteria/SF 0.76 ± 0.1 0.13 ± 0.05 0.37 ± 0.13 P < 0.01
Cyanobacteria/LF 0.65 ± 0.08 0.47 ± 0.14 0.54 ± 0.1 –
Deinococcus-Thermus/SF 0.007* 0 –
Deinococcus-Thermus/LF 0.006* 0 –
Elusimicrobia/SF 0.17 ± 0.04 0.06 ± 0.03 0.1 ± 0.03 –
Elusimicrobia/LF 0.53 ± 0.13 0.31 ± 0.1 0.4 ± 0.09 –
Fibrobacteres/SF 8.88 ± 2.1 14.26 ± 2.7 12.25 ± 2.1 –
Fibrobacteres/LF 5.8 ± 1.2 7.27 ± 1.4 6.7 ± 1.08 –
Firmicutes/SF 39.97 ± 2 45 ± 2.4 43.30 ± 1.9 –
Firmicutes/LF 29.67 ± 2.9 37.62 ± 1.1 34.6 ± 1.9 P < 0.05
Fusobacteria/SF 0 0.01* –
Fusobacteria/LF 0.006* 0.014* –
Lentisphaerae/SF 0.27 ± 0.01 0.28 ± 0.07 0.28 ± 0.04 –
Lentisphaerae/LF 1.6 ± 0.24 1.2 ± 0.23 1.36 ± 0.18 –
Planctomycetes/SF 0.27 ± 0.01 0.28 ± 0.07 0.28 ± 0.044 –
Planctomycetes/LF 0.27 ± 0.02 0.28 ± 0.07 0.28 ± 0.05 –
Proteobacteria/SF 1.7 ± 0.15 1.8 ± 0.15 1.8 ± 0.1 –
Proteobacteria/LF 3 ± 0.4 2.8 ± 0.79 2.9 ± 0.5 –
SHA-109/SF 0.08 ± 0.03 0.44 ± 0.22 0.30 ± 0.15 –
SHA-109/LF 0.09 ± 0.03 0.36 ± 0.09 0.26 ± 0.076 –
Spirochaetae/SF 4.9 ± 0.9 7.7 ± 1.8 6.67 ± 1.23 –
Spirochaetae/LF 4.4 ± 0.9 3.2 ± 0.79 3.7 ± 0.6 –
Synergistetes/SF 1.46 ± 0.2 1.12 ± 0.25 1.24 ± 0.17 –
Synergistetes/LF 0.93 ± 0.1 0.52 ± 0.1 0.68 ± 0.1 –
Tenericutes/SF 0.41 ± 0.04 0.12 ± 0.03 0.23 ± 0.06 P < 0.01
Tenericutes/LF 1.04 ± 0.1 0.34 ± 0.1 0.6 ± 0.14 P < 0.01
Thermotogae 0.0035* 0 –
Verrucomicrobia/SF 0.13 ± 0.01 0.12 ± 0.04 0.12 ± 0.02 –
Verrucomicrobia/LF 0.37 ± 0.04 0.27 ± 0.08 0.31 ± 0.06 –
Other/SF 0.03 ± 0.009 0.02 ± 0.006 0.02 ± 0.005 –
Other/LF 0.02 ± 0.01 0.02 ± 0.003 0.02 ± 0.004 –
Unassigned/SF 6.8 ± 0.99 2.4 ± 0.3 4.08 ± 0.89 P < 0.01
Unassigned/LF 6.2 ± 0.78 2.6 ± 0.39 4.01 ± 0.7 P < 0.01

*Means that archaeal genera found in one animal

 Int Microbiol

Fig. 2 Relative abundances of the abundant active bacterial (a) and archaeal (b) genera in ruminal solid (SF) and liquid (LF) fractions of two camel
groups (C1 and C2) under investigation
Int Microbiol

reads of this phylum were dominated by the Spirochaetaceae
family while at the genus level the members were dominated
by Treponema (Table 3 and Fig. 2a). Proteobacteria made up
1.7% in SF-C1, 1.8% in SF-C2, 3% in LF-C1, and 2.8% in
LF-C2. The sequence reads of Proteobacteria phylum were
distributed in 42 families which were dominated by
Campylobacteraceae, Succinivibrionaceae, uncultured family
0319-6G20, Rhodospirillaceae, and Rhodobacteraceae. On
the genus level, 69 genera were observed and the most prev-
alent were Ruminobacter, Desulfovibrio, and Succinivibrio
(Table 3 and Fig. 2a).
Actinobacteria made up 1.4% in SF-C1, 2.1% in SF-C2,
0.75% in LF-C1, and 2.27% in LF-C2. At the family level, most
of the sequence reads in this phylum were assigned to
Coribacteriaceae, Micrococcaceae, Corynebacteriaceae, and
Actinomycetaceae. On the other hand, members of this phylum
were distributed into 37 genera and the most prevalent were
Atopobium and Collinsella (Table 3 and Fig. 2a). Lentisphaerae
phylum was about 4-fold higher in LF relative to in SF and was
higher in C1 compared with in C2. Furthermore, the relative
abundance of Lentisphaerae was dominated by the genus
Victivallis. Synergistetes phylum was higher in SF-C1 with the
dominance of Pyramidobacter genus (Table 3 and Fig. 2a).
In this study, a small number of bacterial phyla had small
relative abundance such as Cyanobacteria and Elusimicrobia,
which were higher in LF and phylum Tenericutes and
Verrucomicrobia, which were higher in SF. Inspection of the
bacterial community to genus level revealed 411 genera, 172
of which were shared among all animals under investigation;
all the dominant genera in each phylum were shared among
the animals. All unshared genera were observed in traces (<
0.001%) and most of them were found in phylum Firmicutes
and Bacteriodetes. For example, genus Alistipes (phylum
Bacteriodetes) and Salinicoccus and Solibacillus (phylum
Firmicutes) were found only in C1. Moreover, genus
Actinomyces (phylum Actinobacteria) was found only in C2
(Supplementary file 1).
Analysis of archaeal community
The results revealed that all the archaeal sequence reads belonged
to phylum Euryarchaeota. The highest percentage of archaea in
the present study was observed in SF-C2 samples and the differ-
ence was significant in SF (P < 0.05) (Table 3). The archaeal
community in the rumen of camels under investigation was rep-
resented in seven genera, four of which were shared between all
animals, including Candidatus Methanomethylophilus,
Methanobrevibacter, M ethanosphaera, and
Methanimicrococcus. Candidatus Methanomethylophilus was
the dominant genus and made up 0.8% in SF-C2, 0.69% in LF-
C2, 0.2% in SF-C1, and 0.26% in LF-C1. Methanobrevibacter
made up 0.35% in SF-C2, 0.34% in LF-C2, 0.16% in SF-C1, and
0.18% in LF-C1. Methanosphaera, the second dominant genus
made up 1.14% in SF-C2, 0.33% in LF-C2, 0.27% in SF-C1, and
0.14 in LF-C1. Methanimicrococcus was higher in SF than LF
and was found to be higher in C2 than the C1. Methanosaeta,
Methanosarcina, and Methanomicrobium were observed only in
C1 (Table 4 and Fig. 2b).
Correlation between rumen microbes of dromedary
camel
Spearman’s correlation analysis was used to investigate the rela-
tionship between and within archaeal and bacterial genera. The
correlation was visualized as a heatmap (Fig. 3a, b). The corre-
lation analysis showed that the relative abundance of Prevotella
correlated positively with Elusimicrobium and Anaeroplasma
and correlated negatively with Ruminococcus and Blautia. The
relative abundance of Fibrobacteres correlated positively with
Blautia and Atopobium and correlated negatively with

Table 4 The relative abundance

of archaeal genera in ruminal Genera C1 C2 Overall mean P value
solid (SF) and liquid (LF) frac-
tions in the rumen of camels un- Methanobrevibacter/SF 0.16 ± 0.01 0.35 ± 0.1 0.28 ± 0.08 –
der investigation (Mean ± SE) Methanobrevibacter/LF 0.18 ± 0.04 0.34 ± 0.09 0.28 ± 0.06 –
Methanosphaera/SF 0.27 ± 0.04 1.14 ± 0.2 0.82 ± 0.2 P < 0.05
Methanosphaera/LF 0.14 ± 0.05 0.33 ± 0.09 0.26 ± 0.07 –
Methanomicrobium/SF 0.007* 0 –
Methanomicrobium/LF 0.015* 0.01* –
Methanosaeta/SF 0.015* 0 –
Methanimicrococcus/SF 0.019 ± 0.004 0.02 ± 0.01 0.025 ± 0.008 –
Methanimicrococcus/LF 0.026 ± 0.008 0.04 ± 0.02 0.02 ± 0.004 –
Methanosarcina/SF 0.007* 0 –
Methanosarcina/LF 0.006* 0 –
Methanomethylophilus/SF 0.2a ± 0.04 0.8 ± 0.2 0.58 ± 0.17 –
Methanomethylophilus/LF 0.26 ± 0.07 0.69 ± 0.19 0.53 ± 0.14 –

*Means that archaeal genera found in one animal

 Int Microbiol

Fig. 3 Heatmap shows the correlation between bacterial and archaeal genera in solid rumen fraction (a) and liquid rumen fraction (b) based on
Spearman’s correlation analysis
Int Microbiol
Desulfovibrio. Positive correlations were observed between the
relative abundance of Methanobrevibacter, Methanosphaera,
and Methanomethylophilus. Methanomethylophilus and
Methanobrevibacter had a positive correlation with
Fibrobacteres, Blautia, and Atopobium. Methanosphaera had a
positive correlation with Butyrivibrio, Blautia, and Atopobium.
Methanomethylophilus had a negative correlation with
Prevotella and Anaeroplasma.
Discussion
The interactions between rumen microbes are the main driver
of feed degradation and methane formation in the rumen,
which impact animal production (Carberry et al. 2012;
Kittelmann et al. 2014; Henderson et al. 2015). Therefore,
understanding the rumen microbial community leads to un-
derstanding rumen fermentation. This study demonstrate and
compare the composition of bacteria and archaea in the rumen
of camels under different feeding systems based on RNA
isolation.
Feeding system, including diet composition and feeding
plan, is the main determiner of the diversity of rumen micro-
bial communities (Henderson et al. 2015). The diets in our
study varied depending on forage proportions and forage type:
C1 diet contained 50% clover hay and 50% concentrates mix-
ture, and C2 diet contained 100% fresh clover. It is well
known that Egyptian clover is considered a balanced fodder,
which has a high nutritive value regarding crude protein, min-
eral content, soluble carbohydrate, and high-fiber contents
(Bakheit 2013). Concentrates mixture has low-fiber content
and has high content of protein, energy, and soluble carbohy-
drates (Carberry et al. 2012).
The rumen samples of C1 showed a higher OTUs number
and higher diversity indices compared with C2 animals
(Table 2). These results are supported by the variation in the
relative abundance of dominant bacterial groups and the result
of UniFrac analysis and was in agreement with findings
obtained by Petri et al. (2012) on the cattle. Most of the bac-
terial sequence reads were assigned to the Firmicutes and
Bacteroidetes phyla (Table 3), which was also indicated in
the previous studies on the camel (Samsudin et al. 2011),
cattle (Petri et al. 2013), and Surti buffalo (Pandya et al.
2010). Firmicutes was dominated by cellulolytic bacteria,
Butyrivibrio and Ruminococcus, which could illustrate its
higher population in SF-C2 and suggests that the high-
forage diet (C2) increased the population of cellulolytic bac-
teria compared with the low-forage diet (C1) (Fig. 2a) (Liu
et al. 2017; Gharechahi et al. 2015). Bacteroidetes varied be-
tween the camel groups and was higher in C1; members of this
phylum have the capability of degrading the protein and poly-
saccharides such as cellulose, pectin, and xylan (Pitta et al.
2014b), and the uncultured members of Bacteroidetes are
specialized in lignocellulose degradation (Naas et al. 2014),
which could explain their higher proportion in solid fraction.
B acte r oid et e s w as d om i n at ed by Prevotella an d
RC9_gut_group, these results were similar to previous find-
ings on bovine and camels (Fouts et al. 2012; Gharechahi et al.
2015). Prevotella was overrepresented in C1 diet that contains
concentrates mixture, which highlights its ability to degrade
hemicelluloses, pectin, starch, and protein and produce propi-
onate in the rumen (Russell and Rychlik 2001), which is used
as an energy source by the host animal and has a negative
impact on methanogenesis in the rumen (Nathani et al.
2015; Koike et al. 2003).
Most of the members in phylum Proteobacteria have proteo-
lytic activities (Liu et al. 2017). Therefore, Proteobacteria was
found to be abundant in the rumen of a camel fed low-forage diet
(C1). Furthermore, some members of Proteobacteria utilize
methane as a carbon source (Ishaq and Wright 2012). The mem-
bers of phylum Fibrobacteres are recognized as major cellulolytic
bacteria in the gut of herbivores (Ransom-Jones et al. 2012),
which might illustrate its lower relative abundance in the rumen
of camels fed C1 diet; a similar finding was observed by Petri
et al. (2012). The percentage of this phylum in the current study
was 6.7% in LF and 12.25% in SF, while it was 4.5–29% in
Mehshana buffalo (Pitta et al. 2014a) and 4.2–14.1% in wild
ruminant (Gruninger et al. 2014).
Genus Treponema that dominated Spirochaetes has the ability
of cellulose degradation (Leahy et al. 2013), which could explain
the higher percentage of Spirochaetes in the SF-C2 samples.
Elusimicrobium genus dominated the phylum Elusimicrobia, this
genus could have a role in fiber digestion (Herlemann et al.
2009). Verrucomicrobia was found also in the rumen of
Mehshana buffalo (Pitta et al. 2014a) and has the ability to de-
grade plant polysaccharides (Lee et al. 2009; Hou et al. 2008).
The sequence reads that belong to Chloroflexi, SR1, TM7,
and SHA-10 represented less than 1% in our study. TM7 was
found in the rumen of the Brazilian goat (Cunha et al. 2011).
Among the less prevalent bacterial phyla observed in this study,
Planctomycetes, Deinococcus-Thermus, Fusobacteria,
Cyanobacteria, and SR1 were also observed in the rumen con-
tents of Mehshana buffalo (Pitta et al. 2014a). Planctomycetes
was previously observed in the dromedary camel (Samsudin
et al. 2011). The presence of a high number of uncharacterized
bacteria in gut environments is commonly observed (Gruninger
et al. 2016) and suggests the existence of new bacteria that could
be involved in fiber digestion; unclassified bacteria in the current
study were lower, compared with the previous observations of
Muskoxen (Salgado-Flores et al. 2016).
The archaeal community in the camel rumen differed be-
tween the camel groups (Table 3), which could be attributed to
diet type. These findings were in agreement with Hook et al.
(2010), who indicated that the archaeal community composi-
tion could be changed by altering dietary composition or by
feed additives. Fiber breakdown increases the acetate in

relation to propionate; the acetate provides a methyl group for
methanogenesis and subsequently increases the methane pro-
duction (Johnson and Johnson 1995). Therefore, the high-
forage diet (C2) might increase archaea population (Ishaq
et al. 2015). Digested starch and rapidly fermentable carbohy-
drates could decrease the methanogens by increasing the rate
of passage from the rumen and decreasing the ruminal pH as a
result of more VFA production (Hindrichsen and Wettstein
2006; Plaizier et al. 2008; Tapio et al. 2017). Moreover,
starch-utilizing bacteria tend to produce less H2 and acetate
than other bacteria.
The dominant archaeal genera found in this study were
Methanomethylophilus, Methanobrevibacter,
Methanosphaera, and Methanomicrobium (Table 4 and Fig.
2b). This finding was similar to the previous study on camels
(Gharechahi et al. 2015). The genus Methanobrevibacter was
dominant in a wide range of animals such as Alpaca and
Muskoxen (St-Pierre and Wright 2012; Salgado-Flores et al.
2016). The high percent of Methanobrevibacter in C2 could
be illustrated by the availability of H2 and acetate as a result of
fiber breakdown (Tapio et al. 2017). Candidatus
Methanomethylophilus was observed also in bovine rumen,
this culture uses trimethylamine and methanol to produce
methane (Noel et al. 2016). Methanosarcina was also ob-
served in the rumen of bovine, alpaca, and sheep (Whitford
et al. 2001; Cai-Xia et al. 2010). Moreover, Methanosaeta and
Methanimicrococcus were found in small proportions in the
cow (Kong et al. 2013) and sheep rumen (Wright et al. 2004).
The composition of bacterial and archaeal communities in
the rumen of dromedary camels was studied using16S rDNA-
clone library in Samsudin et al. (2011) and 16S rDNA-
amplicon pyrosequencing in Gharechahi et al. (2015); the re-
sults showed some similarities and discrepancies between our
findings and the findings of those studies. For instance, the
composition of dominant bacterial and archaeal groups was
similar between the studies with some differences in the rela-
tive abundances. For example, Fibrobacteres was higher in
our study than other studies, which might refer to the activity
of this cellulolytic bacteria. More details regarding the simi-
larities and differences between our study and previous studies
could be found in Supplementary Table S1.
Some bacteria and archaea in the rumen of the camel ex-
hibited strong positive and negative correlation (Fig. 3a, b).
These microbes may cooperate to facilitate the fermentation of
ingested feed in the rumen or share similar growth require-
ments; additionally, end products of one group may form the
substrates of another (Henderson et al. 2015). For instance, the
fibrolytic bacteria produce the substrates that methanogens
use for growth, mainly hydrogen and methyl groups
(Johnson and Johnson 1995), which might demonstrate the
positive correlation between, Fibrobacteres and the
methanogens and the high archaeal population in high-
forage diet (C2). A similar finding was observed by
Int Microbiol
Kittelmann et al. (2013) in different ruminants. Some
methanogens had a negative correlation with Prevotella; this
relationship was also observed in the buffalo (Iqbal et al.
2018). This trend is expected as Prevotella is a hydrogen
utilizer and produces propionate, which has a negative impact
on methanogenesis in the rumen (Nathani et al. 2015; Stewart
et al. 1997; Koike et al. 2003). Consequently, the high
Prevotella population in group C1 might impact the archaeal
population negatively. Some of the dominant bacteria differed
between the rumen fractions such as Butyrivibrio,
Fibrobacteres, and Ruminococcus that were observed with a
higher proportion in the solid fraction of camels fed high-
forage diet (C2) indicate their major role in fiber digestion
(Nathani et al. 2015). Furthermore, different bacteria prefer
particular metabolic substrates and rumen environment and
this might be distributed differently in disparate phases (Ji
et al. 2017). Therefore, investigating the microbial community
in both solid and liquid fractions could indicate the function
and growth requirements of a specific group of bacteria in the
rumen.
This study applied the cDNA-amplicon sequencing to get
insight on the bacterial and archaeal communities in the rumen
of a dromedary camel. However, using the DNA-amplicon
with cDNA-amplicon sequencing is recommended in future
studies to compare the composition of active microbial groups
(from cDNA-amplicon sequencing) with the composition of
the whole microbial community. On the other hand, using the
metatranscriptomics technique that was applied in the protocol
of Elekwachi et al. (2017) is recommended to get insight into
all the active microbial groups.
Funding information This study was supported by the Desert Research
Center and Lethbridge Research Center, Canada.
Compliance with ethical standards
The project was approved by the Institutional Animal Care and Use
Committee, Faculty of Veterinary Medicine, University of Sadat City,
Egypt (Approval reference #VUSC00003).
Conflict of interest The authors declare that they have no conflict of
interest.
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