As a commonly reported disease, cattle diarrhea causes considerable economic losses for cattle producers world-widely [33]. In Norway, about 10 million US dollars loss was noted due to calves death effected by diarrhea in 2006 [34]. As an agricultural country, the development of animal husbandry is important especially in Hongyuan (China) like plateau areas. In our study, the prevalence of diarrhea in yaks was estimated about 15–25% and 5–10% in yak calves and adults yaks respectively (Table 1). Diarrhea in yaks was significantly higher in yak calves (Fig. 2), which was in line with the widely accepted knowledge that morbidity and mortality of diarrhea in calves is more serious [35]. Therefore, discovering the potential causes of this emerging diarrhea is urgent and meaningful, especially on the remote plateau.
The intestinal microbiota is also considered as an additional organ, which comprises of billions of microorganisms. Intestinal microbiota is not only important in the synthesis and metabolism of nutrients, hormones, vitamins, but also plays role in drugs utilization, pathogen’s fortification, and immune system maturation [13, 36, 37]. Therefore, the imbalance of intestinal microbiota may lead to serious disease. Previously, we performed high-throughput sequencing of intestinal microflora from diarrheal yaks, our study found 41 genera of bacteria in perinatal healthy yaks, while 145 genera of bacteria were only tested in healthy perinatal yaks [5]. Moreover, 212 genera of fungus were found in healthy yaks, 373 and 208 genera of fungus were found in calves and diarrheal yaks respectively [38]. However, 16 s RNA sequencing was limited to genus level. In the current study, metagenomics sequencing was employed to explore the potential pathogens of diarrhea in yaks. Obvious difference was found in GC content (44.69%~46.08% vs 46.12%~46.38%) of two yak groups (p < 0.05) (Fig. 3). Violin box plot also showed the higher gene abundance in diarrhea yaks in concern with the degree of dispersion than normal yaks (Fig. 4). Such results may predict the different composition of microbiota in diarrhea and normal animals. In different levels, we found more significant lower species composition in diarrhea yaks (Fig. 12). Overall, 366163 obvious significant differential abundance genes with 141305 up-regulated and 224858 down-regulated genes was found in diarrheal yaks as compared with normal yaks via DESeq analysis (Fig. 15a). Metagenomics binning analysis with bin 33 (Bacteroidales) (p < 0.05) was significantly higher in diarrheal animals, while bin 10 (p < 0.0001), bin 30 (Clostridiales) (p < 0.05), bin 51 (Lactobacillales) (p < 0.05), bin 8 (Lachnospiraceae) (p < 0.05) and bin 47 (Bacteria) (p < 0.05) was obviously higher in normal animals (Fig. 16a & b).
Staphylococcus aureus is commonly known bacterium related to human and animal foodborne diseases [39]. This pathogen also causes orthopedic implant-associated infection, especially methicillin-resistant bacteria [40]. As infected animals are commonly treated with antimicrobial agents, thus the serious antimicrobial resistance is becoming a public concern world-widely [39]. Diseases such as gastroenteritis, nausea, vomiting, abdominal cramps and etc. are usually seen in infected individuals [41]. The increasing of Staphylococcus aureus in diarrheal yaks may indicate a potential threaten for local herdsmen. Bacteroides coprophilus was previously reported as pro-inflammatory in ankylosing spondylitis [42], which may infer with inflammatory status of diarrhea yaks. Bacteroides plebeius was previously found significantly higher in type 2 diabetes mellitus patients [43], which was also regarded as a biomarker of this disease. Thus the increase of Bacteroides plebeius in diarrhea animals means the abnormal glucose metabolism in yaks.
The butyrate-producing bacteria Butyricicoccus pullicaecorum is commonly linked with inflammatory conditions of intestinal ecosystem [44], which may cause inferred inflammatory response during diarrhea in yaks. Though Babesia ovata is a low pathogenic species, but its infection may lead to severe damages in cattle when co-infected with Theileria orientalis [45]. Previous study reported that the prevalence of T. orientalis in yaks was 9.7% on the plateau [46]. The infection of T. orientalis may be the main reason for bloody diarrhea in yaks (Fig. 1). Fusobacterium mortiferum was usually isolated from Crohn’s and Behcet’s disease patients [47], also Ruminococcus gnavus is a Crohn’s disease-associated pathobiont [48], which was in line with the diarrhea symptom in yaks. Anaplasma phagocytophilum is a commonly reported emerging tick-borne zoonotic pathogen causing anaplasmosis [49]. This bacterium primarily infects host neutrophils, which break the first-line immune defensive barrier in mammalians [50]. The infected animals show typically anemia [51], which reveal that A. phagocytophilum may contribute to diarrhea in yaks. Bacteroides fluxus is a pathogenic species of Bacteroides that displays numerous and high rate of antibiotic resistance. Higher abundance of Bacteroides fluxus means, this bacterium acting as potential role in diarrhea. Firmicutes bacterium was reported to be associated with lipogenesis metabolism in animals with nonalcoholic fatty liver disease [52]. The increased Firmicutes bacterium (CAG:424) in diarrhea yaks may cause dyslipidemia. Bovine viral diarrhea and Rotavirus were also reported in yaks [53, 54], which could be inferred that the increased abundance of these virus may cause diarrhea in yaks.
Fournierella massiliensis is described as a new human-associated member of the family Ruminococcaceae [55], which may have little relationship with diarrhea. Bacteroides vulgatus was found a main cause of polycystic ovary syndrome through disrupted ovarian functions and aggravated insulin resistance [56]. This means increment of Bacteroides vulgatus in yaks may cause diarrhea via affect glycol metabolism. Klebsiella pneumonia causes many infections i.e. pneumonia, urinary tract infection, meningitis and bacteremia [57], which indicates the infection status of diarrheal yaks. While Firmicutes bacterium CAG:110 (p < 0.01), Clostridiales bacterium (p < 0.001), Ruminococcaceae bacterium (p < 0.001), Clostridium sp. CAG:413 (p < 0.001), Clostridia bacterium (p < 0.001), Firmicutes bacterium CAG:137 (p < 0.001), Methanobrevibacter olleyae (p < 0.001), Bacteroidales bacterium WCE2008 (p < 0.001), Ruminococcus flavefaciens (p < 0.001), Methanobrevibacter ruminantium (p < 0.001), Bacteroidete bacterium (p < 0.001), Anaerotruncus sp. Cag:390 (p < 0.001), Clostridium sp. CAG:448 (p < 0.001), Firmicutes bacterium (p < 0.01), Firmicutes bacterium CAG:124 (p < 0.001) and Firmicutes bacterium CAG:170 (p < 0.001) were significantly lower (Fig. 11c). Firmicutes bacterium CAG:110 was found to be potentially associated with swine feed efficiency variation in cecum microbiota via the utilization of dietary polysaccharides and dietary protein [58]. Its mean the dropped abundance of Firmicutes bacterium CAG:110 decrease the efficiency of feeds. Firmicutes bacterium CAG:137, Firmicutes bacterium (p < 0.01), Firmicutes bacterium CAG:124 (p < 0.001) and Firmicutes bacterium CAG:170 (p < 0.001) belongs to host energy uptake or storage limiting related Firmicutes, which are one of the most abundant bacteria in animals and human beings [59]. The lower of Firmicutes bacteria may linked to glycol metabolism, which may further cause diarrhea.
Butyrate producing Clostridiales bacterium is associated in protection of host from colorectal cancer, immune, and metabolic disorders [60]. It means dropped Clostridiales bacterium in yaks made contribute to diarrhea. Ruminococcus flavefaciens works with noncellulolytic Treponema or Butyrivibrio species that can accelerates the digestion of cellulose [61]. The lower Ruminococcus flavefaciens in diarrheal yaks may decrease the cellulose efficiency. Previously lower Ruminococcaceae bacterium was found in hospitalized patients, Cirrhosis [52], and diarrhea foals [62]. The deceased of this bacterium may insight that Ruminococcaceae bacterium has relationship with diarrhea in yaks. CAG:413, CAG:448 and Clostridia bacterium are belongs to Clostridium genus, which were recognized as beneficial bacteria to the host [63]. The lower of these three Clostridium spp. may promote diarrhea in yaks. Methanobrevibacter olleyae and Methanobrevibacter ruminantium composed the M. ruminantium clade, which belongs to ruminant Methanobrevibacter genus [64]. These two bacteria with other Methanobrevibacter spp. compose the rumen methanogenic community [65], which indicates that diarrheal yaks also have decreased production of methane. Bacteroidales bacterium WCE2008 is a Bacteroidales specie, which was accepted as“beneficial” microbes [66]. Previously dropped abundance of Bacteroidales was found in pediatric patients with CD [66], which may infer that the imbalance of this bacterium is related to diarrhea in yaks. Higher abundance of Bacteroidete bacterium is related to healthy lean of host, as it can generate three main SCFAs, butyrate, acetate and propionate [67]. The decreased Bacteroidete bacterium in animals contribute to diarrhea. Anaerotruncus can utilize cheese whey to produce acetic and butyric acids [68]. The decreased Anaerotruncus sp. Cag:390 in diarrhea may effect fatty acid metabolism in ruminants.
Microflora is a key regulator of digestion, extraction, synthesis, and absorption of many nutrients and metabolites i.e. bile acids, lipids, amino acids, vitamins, and short-chain fatty acids (SCFAs) [37]. SCFAs are not only principle nutrient substrates of intestinal epithelial cells, but also can regulate the epithelial barrier [69]. Previously, concentrations of SCFAs was related with diarrhea-predominant irritable bowel syndrome patients [69]. SCFAs could mitigate adenine-induced chronic kidney disease [70], Preoperative fecal levels of SCFAs had an important impact on the occurrence of postoperative infectious complications in patients with esophageal cancer [71]. SCFAs in fecal samples was commonly used as an approximation of gut levels, which can infer the relationship between intestinal SCFAs production and fecal levels [72]. In the current study, 6 out of 7 SCFAs were uncovered significantly lower in diarrhea yaks from 100 mixed fecal samples by employing GC-MS/MS (p < 0.05) (Fig. 5). It reveals that the imbalance of gut microbiota dropped the levels of SCFAs in diarrhea due to the extensive immunological and regulatory functions of SCFAs in the host-microbe interactions [73], activating anti-inflammatory signaling via acting as ligands of G-protein coupled receptors e.g. GPR109A, GPR41, and GPR43 [16]. The current results are in line with diarrhea-dominant IBS with lower levels of SCFAs [74]. Among the common SCFAs, acetate (C2), propionate (C3) and butyrate (C4) are the most in number, produced by anaerobic fermentation of dietary fibers in intestine [16]. Those three SCFAs are accounted for 90% of SCFAs produced by gut microbiota, which depicts the beneficial effects on intestinal epithelial cells and immune cells in the intestinal mucosa [75, 76].
Acetate was reported to mediate joint inflammation in a murine gout model via inflammasome assembly and IL-1β [77]. Propionic Acid was found to be increased in gut-associated Treg cells (relates to systemic immune reaction and disease amelioration) [78]. Butyrate is not only a primary energy source for colonocytes, but also can maintain intestinal homeostasis through anti-inflammatory actions via inhibiting nuclear factor kappa β, and histone deacetylation by promoting epithelial barrier function [16, 79]. Previously, lower abundance of butyrate-producing bacteria and fecal butyrate were found in stroke patients as higher risk factors [80]. Bacteroidetes from Firmicutes mainly produce acetate and propionate, while Butyrate is mainly produced by phylum Firmicutes i.e. Faecalibacterium prausnitzii, Clostridium leptum, Eubacterium rectale and Roseburia spp. [81]. In a previous study, Firmicutes phylum was found clearly lower in diarrheal yaks (p < 0.05) [5]. Also genus of Clostridium_IV (p < 0.01) and Clostridium_XI (p < 0.05) were found obviously lower in diarrheal yaks except Clostridium XVIII (p < 0.01). Genera of Bacteroides (p < 0.05) and Faecalibacterium (p < 0.05) were found significantly higher in diarrhea yaks, while no significant difference was found in genera of Eubacterium, Eubacterium and Roseburia [5]. However, among all those genera, Clostridium_IV and Clostridium_XI were the dominant [5], which may uncover that the decreasing of clostridium may cause the drop of SCFAs (C2-C4). In current study, Isobutyric acid, Isovaleric acid and Caproic acid were found significantly lower in diarrheal animals (p < 0.05), which was in line with patients suffering from cirrhosis and neuromyelitis optica spectrum disorders [82, 83]. As SCFAs plays a critical role in mucosal integrity and immune response [84]. So, the dropping of SCFAs (C4-C6) may mean the damage of mucosal and inflammation response. In a sentence we can say, SCFAs generation bacteria of Anaerotruncus sp. Cag:390, Clostridiales bacterium and Butyricicoccus pullicaecorum are lower in number in diarrheal yaks. Although Fusobacterium mortiferum producing butyric and acetic acids increase obviously [85]. But, it could not affect the dropping trend of SCFAs in diarrheal animals. Statistical analysis of SCFAs; relevant to dominant KEGG signal pathways related to SCFA Acetic acid (53.85%) (Fig. 19); which was the primary level of acetate (50–70%) in the intestine [86].
In conclusion, we estimated the prevalence of emerging diarrhea disease in yak calves (15–25%) and adults (5–10%). Besides the high prevalence of Staphylococcus aureus, Babesia ovata, Anaplasma phagocytophilum, Bacteroides fluxus, viruses, Klebsiella pneumonia, and inflammation-related bacteria, the decreased of SCFAs may potentially lead to emerging diarrhea in yaks. Our results will make insights to the prevention and treatment of emerging diarrhea disease in yaks on the cold plateau.