1.3.1 Vaginal pH
The vaginal pH of all 23 female Bactrian camels was measured. The results showed that the vaginal PH range of the healthy group of bactrian camels ranged from 7.47 to 8.23 with an average of 7.85 ± 0.13. The vaginal PH of the diseased group was in the range of 7.18- 7.61. Also, the average was between 7.41 ± 0.11. Such that the vaginal PH of bactrian camels was significantly different between the normal group and the group that was ill (p = 0.006). Also, the vaginal PH of the group that was ill was lower than that of the normal group.
1.3.2 Sequencing Information
After optimization of quality control and chimera removal, a total of 1644139 reads were obtained for all 23 samples. That had with an average of 71484 reads per sample (Table 1). Samples from the group that was ill were taken and received a total of 744455 reads, with an average of 77446±11214 reads per sample. The normal group samples received a total of 899684 reads with an average of 69206 ± 11047 reads per sample. The results showed that the statistically significant differences in the number of optimized sequences, obtained between the two groups were not significant (P>0.05).
1.3.3 Alpha- and Beta-Diversity
The sequence obtained above was subjected to merging, and revealed the OTU division by 97% sequence similarity. Also, the OTU having abundance value lower than 0.001% of the total. Also, the sample sequencing amount was removed (Bokulich et al., 2013). A total of 1845 OUTs were detected with an average of 1689. Also, 1267 OUTs were detected in the group that was ill. In addition, the normal environment vagina for each was maintained with 1111 OTUs shared between various vagina environments (Fig. 1).
Alpha-diversity was measured and observed using OTU Chao1, ACE, simpson and Shannon Diversity Index. The conclusive analysis presented in Table1. No significant differences existed in alpha-diversity between the normal samples of female Bactrian camels those that were ill and vaginal bacterial observed OTU Chao1 ACE simpson index and Shannon’s Diversity Index (p>0.05). But the illness bactrian camels vagina had a significantly greater number of OTU than did the normal bactrian camels vagina increased richness as measured by Chao1 and ACE and greater diversity as measured by Shannon’s Diversity Index and the Simpson Index all of which are presented in Table 1.
Table 1
Sequence and alpha-diversity statistics of the 16S rRNA gene sequences for bacterial populations in the vaginal of Illness and Normal environments.
Group
|
Samples No.
|
Average of Sequence No.
|
simpson
|
chao1
|
ACE
|
shannon
|
Illness
|
10
|
77446±11214
|
0.94±0.04
|
495.04±230.85
|
497.33±228.58
|
5.55±1.05
|
Normal
|
13
|
69206±11047
|
0.94±0.03
|
361.65±147.45
|
364.26±148.16
|
5.07±0.59
|
P
|
——
|
0.28
|
0.98
|
0.11
|
0.11
|
0.17
|
Beta-diversity was also analyzed to examine differences in microbial communities between samples. Using an OTU-centric approach PCoA matrices were employed using weighted and unweighted UniFrac distance matrices to compare the phylogenetic divergence among the OTU between samples from ill camels and healthy camel vaginal samples (Fig. 2). The results showed that the clustering of subsets of healthy camel vaginal samples was more closely clustered in the weighted and unweighted UniFrac distance matrix. In addition, ANOSIM analysis showed that there was a significant difference between the vaginal samples of ill female camels and normal camels (P=0.033). R statistics showed that the difference between the groups was significantly greater than within groups (R=0.1483) and the grouping effect was evidently well done.
1.3.4 Taxonomic composition analysis
According to the results of OTU classification and classification status identification, the dominant vaginal flora and average relative abundance of Bactrian camels in the normal group and the illness group were respectively identified at the phylum level: Firmicutes (39.33%±16.228.34%±19.39);Proteobacteria (28.48%±15.0635.23%±14.18);Fusobacteria (16.12%±11.3216.86%±13.55);Bacteroidetes(6.04%±3.58.95%±3.6);Actinobacteria (7.5%±3.07%±6.17%±4.21);The average relative abundance of unallocated taxa is:(1.45%±2.81%±1.72%±2.81). The relative abundance of the dominant vaginal flora of the bactrian camels was not significantly different between the healthy group and the diseased group (P>0.05).
At the level of genera the dominant Bacteria and their average relative abundance identified from the vaginas of bactrian camels in the normal group and the group of ill female camels were: Campylobacter(9.58%±7.03%±9.9%±10.05),Ochrobactrum (5.76%±6.037.56%±5.99), Fusobacterium (6.42%±5.596.6%±5.84), GW-34 (5.96%±7.535.58%±12.15), Porphyromonas (4.08%±3.574.96%±3.98), Facklamia (6.08%±4.582.33%±1.15), Sediminibacterium (1.45%±1.65%±2.48%±2.56), Helcococcus (1.25%±2.32.38%±4.05), Peptoniphilus (1.04%±1.052.11%±1.85), 1-68 (1.59%±2.220.93%±1.56), Clostridium (1.22%±1.071.23%±1.3), Acinetobacter (1.18%±1.281%±0.78, Fusibacter (1.29%±1.680.44%±0.62), Sphingomonas (0.68%±0.631.09%±0.86), ph2 (0.9%±0.830.53%±0.98). It can be seen that the relative abundance of the dominant vaginal flora of the bactrian camels was not significantly different between the normal group and the group that was ill (P>0.05).
The genus of vaginal specimens that cannot be identified or placed in undefined categories in either the normal or group that was ill of Bactrian camels and their average relative abundance are as follows family Leptotrichiaceae 9.58%±7.03%±9.9%±10.05, family Aerococcaceae (7.78%±5.182.71%±4.2, family Carnobacteriaceae 6.81%±7.080.3%±0.62), family Xanthomonadaceae (1.13%±1.092.97%±2.96) , family Pseudomonadaceae (1.9%±2.520.9%±0.75), family [Tissierellaceae] (1.39%±1.491.12%±1.75), family Ruminococcaceae (0.35%±0.391.69%±3.14, family Enterobacteriaceae (0.47%±0.781.39%±2.04, family Comamonadaceae (0.35%±0.391.02%±0.78).
Using the visualization tool GraPhlAn (Asnicar et al., 2015) to build a hierarchical tree of the composition of the sample population at each classification level (Fig. 3). More information is evident. While each classification unit was distinguished by different colors and their distribution in abundance was also reflected by the node size.
Using the Mothur software, called the statistical algorithm of Metastats (http://metastats.cbcb.umd.edu/) (White et al., 2009). We were able to determine the overall classification level of all classification units in the sample population. The difference of sequence quantity (i.e. absolute abundance) between each taxon at phylum and genus level was analyzed and compared (pin-wise). We found that there were 4 classification units with significant differences in gate levels (Fig. 4) namely: SR1 (p=0.030 q=0.120); Planctomycetes (p=0.030 q=0.120); Gemmatimonadetes (p= 0.041 q = 0.120); Elusimicrobia (p = 0.048 q = 0.120). There are 51 taxonomies with significant differences in levels (Fig. 5) mainly Anaerostipes (p=0.001 q=0.005); Caldilinea (p=0.001 q=0.005); Edwardsiella (p=0.001 q= 0.005); Lactobacillus (p=0.027 q=0.064) et al.