Abstract

   Dampness-heat diarrhea (DHD), a common syndrome in Chinese dairy farms,
   is mainly resulted from digestive system disorders, and accompanied
   with metabolic disorders in some cases. However, the underlying
   mechanisms in the intestinal microbiome and plasma metabolome in calves
   with DHD remain unclear. In order to investigate the pathogenesis of
   DHD in calves, multi-omics techniques including the 16S rDNA gene
   sequencing and metabolomics were used to analyze gut microbial
   compositions and plasma metabolic changes in calves. The results
   indicated that DHD had a significant effect on the intestinal microbial
   compositions in calves, which was confirmed by changes in microbial
   population and distribution. A total of 14 genera were changed,
   including Escherichia-Shigella, Bacteroides, and Fournierella, in
   calves with DHD (P < 0.05). Functional analysis based on the Kyoto
   Encyclopedia of Genes and Genomes (KEGG) annotations indicated that 11
   metabolic functions (level 2) were significantly enriched in DHD cases.
   The untargeted metabolomics analysis showed that 440 metabolites
   including bilineurin, phosphatidylcholine, and glutamate were
   significantly different between two groups (VIP > 1 and P < 0.05), and
   they were related to 67 signal pathways. Eight signal pathways
   including alpha-linolenic acid, linoleic acid, and glycerophospholipid
   metabolism were significantly enriched (P < 0.05), which may be
   potential biomarkers of plasma in calves with DHD. Further, 107 pairs
   of intestinal microbiota-plasma metabolite correlations were
   determined, e.g., Escherichia-Shigella was significantly associated
   with changes of sulfamethazine, butyrylcarnitine, and 14 other
   metabolites, which reflected that metabolic activity was influenced by
   the microbiome. These microbiota-metabolite pairs might have a
   relationship with DHD in calves. In conclusion, the findings revealed
   that DHD had effect on intestinal microbial compositions and plasma
   metabolome in calves, and the altered metabolic pathways and
   microorganisms might serve as diagnostic markers and potential
   therapeutic targets for DHD in calves.

   Keywords: dampness-heat diarrhea, gut microbiome, metabolomics, 16S
   rDNA, pathogenesis

Introduction

   Diarrhea, the most common disease in calves, is one of the major causes
   of economic loss in cattle herds worldwide ([41]1, [42]2). Neonatal
   calves are susceptible to bovine coronavirus, Escherichia coli, bovine
   rotavirus, and Cryptosporidium parvum, which can cause diarrhea in calf
   ([43]3–[44]5). In addition, some non-infectious factors including
   colostrum management, calf housing, and hygiene are inducer of diarrhea
   in calf ([45]6, [46]7). The rational use of traditional Chinese
   medicine (TCM) to treat this disease has certain advantages ([47]8).
   Different types of diarrhea require different prescriptions based on
   the theory of TCM. According to the clinical symptoms, diarrhea can be
   divided into dampness-heat diarrhea (DHD), spleen deficiency diarrhea,
   and kidney deficiency diarrhea ([48]9–[49]11). DHD is one of the most
   common syndromes in Chinese cattle herds. According to traditional
   Chinese veterinary medicine (TCVM), DHD is usually caused by the
   exogenous heat and dampness toxin pathogens invading the intestine
   ([50]12). The main clinical symptoms of DHD in calves are hyperthermia,
   sticky and loose stools with blood and mucus, red tongue, and thick
   greasy tongue-coating ([51]9, [52]13). The mechanism of DHD has not
   been fully elucidated. This study provides new insights on
   understanding the pathogenesis and rational treatment of DHD in calves.

   The alteration of gut microbial composition is associated with many
   diseases ([53]14). Diarrheal diseases often alter the richness and
   diversity of intestinal flora ([54]15, [55]16). Therefore, the
   microbiota is an important factor in the maintenance of health and the
   development of disease ([56]17). The 16S rDNA gene amplicon sequencing,
   a powerful tool for routine microbial identification, can help
   researchers to identify, categorize, and understand the complex
   interactions between the host, pathogen, and microbiome ([57]18,
   [58]19). Studies have confirmed that the increase of Firmicutes and
   Bacteroidetes proportion is an important manifestation of diarrhea, and
   diarrhea could be alleviated when this abnormal proportion was adjusted
   to be normal ([59]20, [60]21). Gegenqinlian decoction, a classical TCM
   prescription, regulated the balance of intestinal mucosa flora in mice
   with diarrhea induced by high temperature and humidity ([61]22).
   Therefore, it might be a helpful strategy for elucidation of the
   pathogenic mechanism of DHD to study of gut microbial composition in
   calves.

   Qualitative and quantitative analysis of low molecular weight
   metabolites in biological samples can reflect the influence of diseases
   on metabolic in body ([62]23, [63]24). In recent years, a great deal of
   research has been conducted on the metabolomics in a lot of diseases
   ([64]25–[65]28). The 16S rRNA gene sequencing with untargeted
   metabolomics can present much better perspectives on physiological and
   metabolic mechanisms involved in the pathogenesis of DHD. Some
   metabolites in plasma such as chenodeoxycholic acid and creatinine were
   found to the biomarkers for colorectal cancer diagnosis and prognosis
   ([66]29). Metabolomics was utilized to study the molecular mechanisms
   of Atractylodis Rhizoma in the treatment of spleen deficiency ([67]30).
   Therefore, we hypothesized that the changes of metabolic and intestinal
   microbiological might be related to DHD in calves. In this study, the
   difference in fecal microbiome and plasma metabolic profile between DHD
   calves and healthy calves was detected by 16S ribosomal DNA gene
   sequencing and metabolomics, and the results revealed that DHD had
   effect on intestinal microbial compositions and plasma metabolome, and
   the altered metabolic pathways and microorganisms might serve as
   diagnostic markers and potential therapeutic targets for DHD in calves.

Materials and Methods

Experimental Animals

   The experiment was carried out in a dairy farm in north-western China.
   The calves in this study were similar in genetic background and age,
   and all of enrolled animals were housed and fed under same conditions.
   A total of 6 DHD calves and 6 healthy calves were enrolled in this
   study. The standards of dampness-heat diarrhea in the literature are
   mostly determined by primary and secondary symptoms ([68]9, [69]10,
   [70]13). The inclusion criteria for DHD cases were required two main
   symptoms and secondary symptoms, or three main symptoms ([71]Table 1).
   The specific clinical syndrome differentiation was carried out by the
   author and a Chinese veterinary expert. According to the diagnostic
   criteria, we screened 6 eligible calves from 129 diarrhea cases as DHD
   group and six healthy calves as the control group. The enrolled calves
   were not given any probiotics or antibiotics prior to sample
   collection. All animal procedures were carried out in accordance with
   the Guidelines on Laboratory Animal Ethics Commission of the Lanzhou
   Institute of Husbandry and Pharmaceutical Sciences of CAAS (SYXK [Gan]
   2019-0002).

Table 1.

   Diagnostic criteria for DHD calves.
   Main symptoms Diarrhea, mucus or bloody purulent stool, red tongue,
   thick greasy tongue-coating
   Secondary symptoms Hyperthermia, shortness of urination, abdominal
   pain, anal burning loose stools like water, tenesmus, dry nose, thirst
   and small amount
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Fecal Sample Collection and DNA Extraction

   Fresh fecal samples were collected by inserting an anal swab into the
   anus. The anal swabs were deposited into a sterile sampling tube and
   sent to the laboratory within 2 h. The fecal samples were immediately
   frozen in liquid nitrogen until the microbial DNA was extracted.

   Microbial DNA was extracted from each fecal sample using the HiPure
   Fecal DNA Kit (Magen, Guangzhou, China) according to kit instructions.
   The DNA purity was determined using a Nanodrop microspectrophotometer
   (NanoDrop 2000, Thermo Fisher Scientific, America) and DNA integrity
   was investigated using the agarose gel electrophoresis.

Plasma Sample Collection and Metabolite Extraction

   Blood was collected from the jugular vein of the calves and stored in
   an anticoagulant tube containing ethylenediaminetetraacetic acid
   (EDTA), and was temporarily stored in an incubator at 4°C and sent to
   the laboratory within 2 h. The plasma was separated after
   centrifugation at 3,000 rpm, and 4°C for 15 min, and then frozen at
   −80°C until metabolite extraction.

   Metabolites were extracted separately from plasma. After addition of
   300 μL of methanol and 20 μL internal references in 100 μL plasma, the