Diagnosis of pregnancy toxemia in goats
Goats with suspected pregnancy toxemia exhibited clinical symptoms such as depressed spirits, loss of appetite, and an inability to lie on the ground (Fig. 1a), as well as tachypnea. Some diseased goats developed severe mental symptoms such as a head and neck tilt to the ventral costal region (Fig. 1b), and most of the sick goats died within 1–3 days. Autopsies showed that the livers of the sick goats were enlarged and earthy yellow, with rounded edges, a soft texture, reddish and yellow texture on the surface, and an enlarged gallbladder (Fig. 1c). Liver tissue sections showed numerous vacuoles in the cytoplasm of the cells, with variably sized vacuoles in the cytoplasm pushing the nuclei to one side (Fig. 1d).
The blood biochemical results (Table 1) showed that the level of AST in goats with suspected pregnancy toxemia was significantly higher than that in the control group (P < 0.05), the level of Ca ion was significantly lower than that of the control group (P < 0.01), the level of B-HB was significantly higher than that in the control group (P < 0.01), and the levels of the remaining 16 biochemical indexes were not significantly different from the control group (P > 0.05).
Table 1
Test results of blood biochemical indicators
Item
|
Groups
|
P-value
|
PT(n = 8)
|
NC(n = 8)
|
ALT (U/L)
|
28.08 ± 3.96
|
23.96 ± 1.67
|
0.247
|
AST (U/L)
|
183.46 ± 31.97
|
112.36 ± 4.51
|
0.017
|
ALP (U/L)
|
400.02 ± 225.37
|
158.38 ± 22.34
|
0.196
|
r-GGT (U/L)
|
45.58 ± 7.99
|
44.94 ± 2.73
|
0.929
|
TP (g/L)
|
60.08 ± 1.54
|
66.14 ± 2.40
|
0.095
|
ALB (g/L)
|
21.14 ± 0.41
|
23.09 ± 0.74
|
0.078
|
GLB (g/L)
|
38.94 ± 1.15
|
43.05 ± 1.90
|
0.142
|
A/G(%)
|
0.52 ± 0.020
|
0.52 ± 0.025
|
0.891
|
TBIL-V(µmol/L)
|
6.15 ± 0.79
|
5.07 ± 0.335
|
0.177
|
UREA(mmol/L)
|
6.12 ± 0.32
|
6.47 ± 0.37
|
0.533
|
GLU(mmol/L)
|
0.99 ± 0.48
|
1.38 ± 0.32
|
0.498
|
Ca(mmol/L)
|
1.78 ± 0.03
|
2.08 ± 0.02
|
0.000
|
P(mmol/L)
|
2.45 ± 0.19
|
2.24 ± 0.15
|
0.400
|
TC(mmol/L)
|
2.29 ± 0.40
|
3.01 ± 0.35
|
0.211
|
TG(mmol/L)
|
0.22 ± 0.04
|
0.37 ± 0.55
|
0.094
|
CK (U/L)
|
379.24 ± 156.77
|
189.93 ± 11.24
|
0.147
|
LDH (U/L)
|
281.18 ± 36.82
|
278.89 ± 15.40
|
0.948
|
FUN(µmol/L)
|
339.98 ± 139.76
|
198.30 ± 3.94
|
0.215
|
B-HB(mmol/L)
|
4.90 ± 0.59
|
0.39 ± 0.07
|
0.000
|
Overall structure of fecal bacterial communities
In the PT groups, 1402 OTUs were obtained,while the NC group 1586 OTUs.Combined,there were 1342 OTUs in both groups. Figure 2a shows that the NC group had more unique OTUs than the PT group. Beta diversity analysis revealed significant differences in microbial structural composition between the two groups (Fig. 2b). Meanwhile, Alpha diversity analysis showed that the ACE, Shannon, and Chao1 indices of the PT group were significantly lower than those of the NC group (P < 0.05), while the Simpson index was significantly higher than that of the NC group (P < 0.05) (Fig. 2c). These results indicate significant differences in the microbial composition, diversity, and abundance of goat feces between the PT and NC groups. Specifically, the PT group had a reduced microbial diversity and abundance.
Analysis on composition and difference of microbiota
Figure 3a-3c shows the species composition of fecal microorganisms in the PT group and NC group. At the phylum level (Fig. 3a), the phylums with relative abundances greater than 10% in the PT group were Firmicutes (64.55%), Bacteroidetes (16.10%) and Proteobacteria (13.37%), and the phylum with relative abundances greater than 10% in the NC group were Firmicutes (68.34%) and Bacteroidetes (20.43%). At the family level (Fig. 3b), the families with relative abundances greater than 10% in the PT group were Ruminococcaceae (26.23%) and Lachnospiraceae (16.62%), and the families with relative divisions greater than 10% in the NC group were Ruminococcaceae (31.37%). At the genus level (Fig. 3c), the genera with relative abundances greater than 5% in the PT group were Clostridium_XlVa, (8.69%) and Bacteroides (7.03%), while genera with relative abundances greater than 5% in the NC group were Bacteroides (7.81%) and Clostridium_XlVa (5.03%).
The analysis of species difference in fecal microorganisms between the PT group and NC group is presented in Fig. 3d-f. At the phylum level (Fig. 3d), the relative abundance of Tenericutes was significantly lower in the PT group than in the NC group (P < 0.05), while the relative abundance of Proteobacteria was significantly higher in the PT group than in the NC group (P < 0.05). At the genus level (Fig. 3e), the relative abundance of Escherichia, Roseburia, Desulfovibrio, and Paenibacillus was significantly higher in the PT group than in the NC group (P < 0.05), While the relative abundance of Clostridium_Ⅳ, Intestinimonas, and Holdemania was significantly lower in the PT group than in the NC group(P < 0.05). At the species level (Fig. 3f), the relative abundance of Holdemania_filiformis, Desulfovibrio_simplex and Escherichia in the PT group was significantly higher than t in the NC group (P < 0.05). While the relative abundance of Odoribacter_laneus, Clostridium_scindens and Intestinimonas_butyriciproducens was significantly lower than in the NC group(P < 0.05).
To further investigate the key species differences between fecal microorganisms in the PT group and the NC group, the top 10 species with average relative abundance were selected for analysis. At the phylum level (Fig. 3g), the relative abundance of Proteobacteria was significantly higher in the PT group than in the NC group (P < 0.05), while the relative abundance of Tenericutes was significantly lower in the PT group than in the NC group (P < 0.05). At the family level (Fig. 3h), the relative abundance of Lachnospiraceae was significantly higher in the PT group than in the NC group (P < 0.05), while the relative abundance of Enterobacteriaceae was significantly higher in the PT group than in the NC group (P < 0.01). At the species level (Fig. 3i), the relative abundance of Escherichia was significantly higher in the PT group than in the NC group (P < 0.01).
Composition change and functional difference analysis of microbiota
As shown in Fig. 4a-4b, the relative abundance of Blautia, Faecalicoccus, Enterobacteriaceae, Enterobacteriales, and Escherichia in the PT group were significantly higher than in the NC group (P < 0.05). Conversely, Acidaminococcaceae, Phascolarctobacterium, Holdemania, Intestinimonas, Clostridium_IV, Clostridium_III, Odoribacter, and Acholeplasma had significantly lower than relative abundance in the PT group compared to the NC group (P < 0.05).
As shown in Fig. 4c, pregnancy toxemia goat fecal microorganisms are mainly involved in carbonic acid metabolism, cofactor and vitamin metabolism, terpene and polyketone metabolism, amino acid metabolism and other functions. The functions of fecal microbial amino acid metabolism, immune system, replication and repair, cell growth and death, and translation in the PT group were significantly lower than those in the NC group (P < 0.05), and the functions of membrane transport, transcription, infectious bacteria and signaling were significantly higher than those in the NC group (P < 0.05).
Classification of metabolites and functional notes
Quality control analysis indicates that the high-quality ion number meets the requirements and the model predictions are accurate (Additional file 1). In positive and negative ion modes, metabolites are mainly divided into compounds with biological effects, lipids, plant compounds, and others (Fig. 5a and b). Compounds with biological effects mainly included benzene and its derivatives, amino acids and peptide derivatives and organic acids, while lipids comprised polyketone compounds, fatty acyls, sterol lipids and pregnenol lipids. Plant compounds mainly include flavonoids and terpenoids. These metabolites were associated with functions such as environmental information processing, metabolism, and organic systems (Fig. 5c and d). Metabolites were primarily involved in environmental information processing, such as membrane transport and signaling molecules and their interactions. They also participated in metabolic processes such as amino acid and vitamin metabolism, and played a role in organic systems like the digestive, nervous and immune.
Analysis of differential metabolites
The results of the volcano plot and cluster analysis showed that the fecal metabolites of the PT group and the NC group were quite different. In positive ion mode, the total number of differential metabolites was 125, of which 89 were upregulated and 36 were down-regulated. In negative ion mode, the total number of differential metabolites was 100, of which 51 were upregulated and 49 were downregulated (Fig. 6a-d, Additional file 2). Additional file 3 lists the top 10 up-regulated and down-regulated differential metabolites in positive and negative ion modes. The bubble plot shows the degree of enrichment of fecal differential metabolites in different metabolic pathways, as well as the number of differential metabolites enriched in each pathway. In positive ion mode, 12 metabolic pathways were enriched and 13 differential metabolites were significantly enriched. In negative ion mode, 7 metabolic pathways were enriched and 12 differential metabolites were significantly enriched (Fig. 6e-f, Additional file 4). Additional file 5 lists the metabolic pathways in which differential metabolites are significantly enriched in positive and negative ion modes.
In positive ion mode, the PT group showed upregulated levels of metabolites such as cortisol, dehydroepiandrosterone, β-estradiol, and testosterone in steroid hormone biosynthesis (Additional file 6). Additionally, in bile secretion (Additional file 7), the levels of metabolites such as thromboxane b2, cortisol and deoxycholate were upregulated in the PT group. In negative ion mode, the levels of metabolites such as chenodeoxycholate, deoxycholic acid and lithocholic acid were upregulated in bile secretion, while the content of glycocholate was down-regulated (Additional file 8). Moreover, in the metabolic pathways of phenylalanine, tyrosine and tryptophan biosynthesis, the level of chenodeoxycholate in fecal metabolites of the PT group was up-regulates, while the level of glycocholate was down-regulated (Additional file 9).
Comprehensive analysis of microbiome and metabolomics
The correlation and chord diagram between differential metabolites and microflora showed that, at the phylum level, the metabolic pathways negatively correlated with Proteus phylum abundance included pentose phosphate metabolism, glycerol ester metabolism, ubiquinone biosynthesis, and so on, while terpenoid metabolic pathways positively correlated with Proteus phylum abundance. Anti-folic acid and aldosterone synthesis were negatively correlated with the abundance of interoxophiles, while sphingolipid signaling pathway was positively correlated with them. The tyrosine and vitamin B6 metabolism were the metabolic pathways negatively correlated with Actinomyces phylum abundance. Bile metabolism was negatively correlated with the abundance of Firmicutes, while glycerol ester metabolism was positively correlated. The primary bile acid biosynthesis pathway was negatively correlated with spirochete abundance (Fig. 7a). At the genus level, metabolic pathways negatively correlated with Escherichia abundance included renin secretion, vascular smooth muscle contraction, cGMP-PKG signaling pathway, and regulation of lipolysis. Carbon metabolism and glyoxylate and dicarboxylic acid metabolism were negatively correlated with spirochete abundance. Phenylalanine, tyrosine, and tryptophan anabolism were positively correlated with odor bacillus abundance and Haldeman’s abundance, while bile secretion metabolic pathways were negatively correlated with odor bacillus abundance. Thermogenesis and unsaturated fatty acid biosynthesis in metabolic pathways negatively correlated with clostridium abundance. Amino acid metabolic pathways such as valine, leucine, and isoleucine were negatively correlated with the abundance of anaerobic protozoa (Fig. 7b).
The heatmap of correlation between differential metabolites and microflora showed that at the phylum level, N-benzyltrioxane ketone and dihydrokabasine were metabolites positively correlated with Tenericutes, while formamide, phenethyltheophylline, leucine, tryptophan, heptadecenedione, trienediol, and thyronine were negatively correlated with Tenericutes. Amyl cinnamyl alcohol and carboxy furosine alcohol were metabolites positively correlated with cyanophyta. Phenylsulfone azole and jatrorrhizine were metabolites negatively correlated with mycophenolate mofetil. Hexylresorcinol was negatively correlated with spirochetes (Fig. 7c). At the genus level, n-amylbenzene, diethyl hexyl phthalate, delphinine, and testosterone isoate were metabolites negatively correlated with Haldemanella, while thyronine was positively correlated with them. Metabolites of ammonium butyrate, diisononyl phthalate, and tenivastatin were positively correlated with Escherichia. Metabolites positively associated with the genus Odorbacillus were diosmin and hydrogenated dicarboxylic acids, with diphenyl octadecone negatively associated. Metabolites such as etomidate and Metominol were negatively correlated with coprococcus (Fig. 7d).