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[转载]肠道菌群紊乱：Enteritis in hybrid sturgeon caused by intestinal
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https://www.sciencedirect.com/science/article/pii/S2352513420305469
Xiaoli Huang, Liang Zhong, Wei Fan, Yang Feng, Guanqing Xiong, Sha Liu, Kaiyu Wang, Yi Geng, Ping Ouyang, Defang Chen, Shiyong Yang, Lizi Yin, Lili Ji,
Enteritis in hybrid sturgeon (Acipenser schrenckii♂ × Acipenser baeri♀) caused by intestinal microbiota disorder,
Aquaculture Reports, Volume 18, 2020, 100456, ISSN 2352-5134,
https://doi.org/10.1016/j.aqrep.2020.100456
(https://www.sciencedirect.com/science/article/pii/S2352513420305469)
Xiaoli Huang Liang Zhong Wei Fan Yang Feng Guanqing Xiong ShaLiu Kaiyu Wang Yi Geng Ping Ouyang Defang Chen Shiyong Yang Lizi Yin Lili Ji
aDepartment of Aquaculture, College of Animal Science & Technology, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China
bCollege of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China
cNeijiang City Academy of Agricultural Sciences, Neijiang 641000, Sichuan, China
dPharmacy and Bioengineering of Chengdu University, Chengdu 610106, Sichuan, China
Received 5 March 2020, Revised 1 July 2020, Accepted 13 August 2020, Available online 27 August 2020.
Abstract: Intestinal microbiota is crucial for the regulation of the immune system and feeding habits in aquatic animals, as well as preventing the invasion of pathogenic microorganisms. However, disorders of the intestinal microbiota can lead to numerous, possibly life-threatening diseases. In the present study, we reported a disease occurring in cultured hybrid sturgeons (Acipenser schrenckii♂×Acipenser baeri♀) in the Sichuan province, Southwest China. The diseased fish did not exhibit obvious skin lesions, with observed symptoms including intestine dilatation with large amounts of gas and a transparent intestinal wall. Pathological examinations, microbiological tests, and the high-throughput sequencing of intestinal microbiota were performed to investigate the cause of death of diseased sturgeons. Results demonstrated that no bacteria were isolated from the liver, spleen and kidney, and no obvious changes were observed in tissues, with the exception of the intestine. The intestines demonstrated marked necrotic enteritis with lumen dilatation and a large number of bacteria attached to the base of the intestinal villus. Furthermore, observations from an electron microscope revealed the invasion by bacteria of the striated border. Results from the 16 s rRNA high-throughput sequencing of intestinal microbiota indicate variations in the relative abundance and distribution uniformity of the intestinal microbiota in the diseased group. More specifically, the intestinal microbiota abundance in the diseased group was higher in Clostridium, Cetobacterium, and Lactococcus, and lower in Mycoplasma at the genus level compared to the healthy group. Our observations suggest that the death of the hybrid sturgeons was caused by intestinal enteritis, which itself was a result of the intestinal microbiota disorder. To our knowledge, this is the first study to reveal hybrid sturgeon death via enteritis caused by an intestinal microbiota disorder.
Keywords: Enteritis; Hybrid sturgeon; Intestinal microbiota disorder; High-throughput sequencing
1. Introduction
As one of the largest immune organs in the body (Kaldas and Farmer, 2008; Filipp et al., 2018), intestines prevent the invasion of pathogenic microorganisms by forming an intestinal immune barrier, thereby protecting the health of the body. Microbiota that resides in the intestines is an important part of the intestinal immune barrier, working together with the host's defense and immune system to defend against colonization and invasion by pathogens (Carding et al., 2015). Furthermore, intestinal microbiota is essential for the development and proper function of the immune system (Ma et al., 2018). Therefore, a balanced intestinal microbiota is crucial for maintaining the host’s health. However, under certain adverse conditions, when the balance is disturbed, the intestinal microbiota changes, consequently damaging the host’s intestines (Antonissen et al., 2016). Surprisingly, in a recent study, Zhang et al. identified the ability of intestinal microbiota to stimulate the host to feel hunger by damaging the host’s intestines, thus enticing the host to eat in order to acquire a carbon source for bacterial reproduction (Zhang, 2015; Zhang and Gong, 2017; Zhang et al., 2018). Based on the discovery that “hunger is derived from the intestinal microbiota”, a new theory of evolution, denoted as the gut flora-centric theory of evolution (GFCTE), was proposed by Zhang (2015). This theory states that microorganisms co-existing in the gastrointestinal tract transmit hunger signals to animals by decomposing the gastrointestinal mucosa (Zhang and Gong, 2017). In particular, if animals do not forage in time, the intestinal microbiota will continue to break down the gastrointestinal mucosa, causing gastrointestinal damage, which in turn may affect the animal's fitness and survival. Desai et al. (2016) demonstrated that the absence of dietary fiber, which was the primary energy source of the intestinal bacteria, could lead the intestinal microbiota to disrupt the intestinal mucosal barrier and increase the host’s vulnerability to pathogens. It is therefore assumed that insufficient feeding may stimulate intestinal microbiota to break down the mucosa in the host’s intestinal tract, causing intestinal damage.
Sturgeons belong to the Osteichthyes, Actinopterygii, Ganoidomorpha, and Acipenseriformes, and are considered as some of the oldest living creatures. They are associated with high economic and nutritional values, and their production has increased gradually in recent decades (Bronzi et al., 2019). However, the rapid expansion of breeding scales has resulted in an increase in the number of farming enterprises and the density of animals, and consequently the more frequent occurrence of sturgeon diseases. As one of the most common diseases in sturgeon culture, enteritis affects the entire breeding cycle, incurring huge economic losses (Ma et al., 2009; Chen et al., 2012; Zheng et al., 2018; Di et al., 2018). In addition, it restricts both the cultivation process of sturgeons and the healthy development of related aquaculture industries. Despite increasing efforts, knowledge on enteritis in sturgeons remains relatively limited.
In the current study, we evaluated the occurrence of disease in a group of cultured hybrid sturgeons (Acipenser schrenckii♂×Acipenser baeri♀) in the Sichuan province, Southwest China during March 2019. Diseased fish exhibited clinical symptoms, including gathering close to the pond peripheries, mid-water swimming or floating at the surface with their belly facing up, and a bloated abdomen. Pathological examinations, microbiological studies, and high-throughput sequencing of intestinal microbiota were performed in order to determine the cause of the disease. To our knowledge, this was the first report on hybrid sturgeon mortality due to enteritis caused by intestinal microbiota disorder.
2. Materials and methods
2.1. Experimental fish
A total of 24 hybrid sturgeons (3−5 cm) were collected in the same breeding pond, including 9 and 15 healthy hybrid and diseased sturgeons, respectively. The healthy hybrid sturgeons were selected via the following criteria: (1) Fish swam at the pool bottom, with a typical black and gray body color, and a strong vitality and feeding ability; and (2) no obvious intestinal damage was detected through histopathological examination.
2.2. Bacteria isolation
The diseased hybrid sturgeons were initially sanitized with 75 % alcohol and subsequently dissected in the laboratory. Pathogens isolated from the kidneys, livers, and spleens were planted on a brain heart infusion (BHI) medium and incubated at 28 °C for 24−48 h.
2.3. Histopathological examination
Samples were fixed in 10 % neutral buffered formalin and the tissues were then trimmed into cassettes, dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin wax. Sections of 4 μm were prepared and mounted on slides for hematoxylin and eosin (H&E) staining, followed by the examination with an optical microscope.
2.4. Electron microscopy
Fresh intestine tissue was placed in fixative (2.5 % glutaraldehyde in pH 7.4 cacydolate buffer) washed three times in phosphate buffer saline (PBS), and post fixed with osmium tetroxide (1 %). Samples were then dehydrated with ascending concentrations of alcohol and post embedded in Araldite. Crossly oriented ultra-thin sections were cut and stained with uranyl acetate and lead citrate. Images were acquired using a HT7700 transmission electron microscope (HITACHI, Japan).
2.5. Collection of intestinal contents
For the 16 s rRNA high-throughput sequencing of intestinal microbiota, 9 healthy and 9 diseased hybrid sturgeons were divided into 3 groups, respectively. All fish were euthanized using MS-222. The ventral belly surface of the fish was opened under sterile conditions to expose the peritoneal cavity. Following this, the entire intestine was excised and rinsed several times with 0.65 % sterile saline. Intestinal contents were then collected into a sterile Eppendorf tube. Samples were kept on ice for less than 2 h and stored at −80 °C prior to analysis.
2.6. DNA extraction and purification
Genomic DNA was extracted from the intestinal contents using a bacterial DNA isolation kit (Foregene Co., Ltd., China) according to the manufacturer's instructions. Following extraction, the genomic DNA was detected by 1% agarose gel electrophoresis. Samples were used for PCR amplification with a forward primer (338F: 5′- ACTCCTACGGGAGGCAGCAG-3′) and a reverse primer (806R: 5′- GGACTACHVGGGTWTCTAAT-3′). The PCR reactions were held at 95 °C for 3 min to denature the DNA, followed by 27 amplification cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s, and a final extension of 10 min at 72 °C. The PCR product was then detected by 2% agarose gel electrophoresis, purified using a AxyPrep DNA Gel Extraction Kit (Axygen, USA), quantified via the QuantiFluor™-ST Blue Fluorescence System (Promega, China), and subjected to next-generation sequencing.
2.7. Sequencing and processing
A library was constructed for the V3-V4 amplicons, and paired-end (PE) sequencing was performed on the MiSeq system (Illumina, San Diego, CA, USA). Based on the overlap relationship between PE reads, pairs of reads were merged into a sequence using Flash. Prior to analysis, the following was excluded from the dataset: (i) shorter than 50 bp; (ii) <10 bp in the libraries; and (iii) ambiguous nucleotides that constituted over 20 % of the sequence. The remaining sequences were clustered into operational taxonomic units (OTUs) with a similarity cutoff of 97 % using Uparse.
2.8. Bioinformatic analysis
OTU taxonomic analysis was performed using the Usearch (version 7.0 http://drive5.com/uparse/). Each OTU was compared with the 16 s rRNA database (Silva) using BLAST analysis to obtain species classification information. Species composition analysis was conducted via Circos. Species with a relative abundance rate of less than 0.01 in all samples were classified as “others”. The difference between the sample groups was determined by t-tests (P < 0.05). Statistical analyses were performed using SPSS v.22.0.
3. Results
3.1. Role of the intestine as the target organ in diseased hybrid sturgeons
The cultured hybrid sturgeons began to exhibit a high morbidity rate in March 2019, with water temperatures within 10−13 °C. Obvious lesions were not observed on the surface of the skin of the diseased fish, and the only visible sign was abdominal enlargement. The remaining organs did not exhibit any obvious changes. The intestines of the diseased fishes were dilatated due to large amounts of gas, however they were devoid of food (Fig. 1A), with transparent walls (Fig. 1B). Furthermore, following incubation at 28 °C for 48 h on BHI, no bacterial growth was isolated from the kidneys, livers, or spleens.
Fig. 1. Gross observation of diseased hybrid sturgeons. A Intestines contained large amounts of gas (arrowhead). B Intestines of diseased fishes were markedly extended when compared to healthy fish.
3.2. Severe intestinal mucosal necrosis and detachment revealed from pathological observations
The histopathological examination of the tissues revealed no obvious changes in the muscle, brain, gill, liver, kidney, pancreas, and stomach (Fig. 2A, C, D, E, F, G and H). However, changes in the intestine were marked (Fig. 2J), indicating marked necrotic enteritis with lumen dilatation (Fig. 3A). Not all areas of the diseased hybrid sturgeon intestines exhibited obvious changes (Fig. 3C). However, in several regions, the striated border was mildly ruptured or even completely disappeared (Fig. 3B), and a large number of bacteria were observed at the base of the intestinal villi (Fig. 3B). In addition, many mucosal epithelial cells across the majority of the intestine demonstrated marked necrosis, detachment, and shedding (Fig. 3D and 3E). The detached cells were observed in the enteric lumen yet minimal amounts of food were identified. The entire mucosal layer had totally disappeared in the most severely affected areas, and normal structures were lost (Fig. 3F). The wall of the intestine was obviously thinner due to intestinal distension and the disappearance of intestinal villi (Fig. 3G).
Fig. 2. Histopathological examination of lesions in diseased hybrid sturgeon. A Skin and muscles. B, I Sagittal section of a hybrid sturgeon. C-H Amplification of B and I: Brain, gill, liver, kidney, pancreas, and stomach, respectively. J Intestinal dilatation of a diseased hybrid sturgeon.
Fig. 3. Histopathological examination of lesions in diseased hybrid sturgeon intestines. ASagittal section of a hybrid sturgeon. B Large number of bacteria detected in the intestinal lumen (arrowhead); section of striate border was ruptured and disappeared (star). CPathological changes not obvious in these areas. D Intestinal microbiota was identified (arrowhead), with necrotic and shed mucosal epithelial cells (star). E Large number of intestinal mucosal epithelial cells appeared necrotic with shedding (star). F Large amount of necrotic mucosal epithelial cells (star). G Thinning of the intestinal muscle layer (arrowhead).
In order to further examine the pathological changes of the intestine, ultrastructural sections were performed. Results demonstrated an enlarged gap between the intestinal epithelial cells and numerous blank areas (Fig. 4A). Apoptotic cells were also identified in the intestines of diseased fish (Fig. 4B). Remarkably, many vacuoles were detected in the intestinal goblet cells, with the presence of unknown substances (Fig. 4C). The nucleus of the apoptotic cells disintegrated, and the mitochondrial crista were disturbed and even disappeared (Fig. 4D). Observations from the electron microscope revealed a large number of bacteria in the intestines of the diseased hybrid sturgeon (Fig. 4E) that invaded the striated border of the intestinal mucosa, resulting in its eventual disappearance (Fig. 4F).
Fig. 4. Ultrastructural examination of lesions in diseased hybrid sturgeon intestines.
A Widened gap between intestinal epithelial cells (arrowhead). B Apoptotic cells (star). CVacuoles in goblet cells containing unknown substances (arrowhead). D Disintegrated nucleus (star) and ruptured mitochondria (arrowhead) in apoptotic cells. E Bacteria in intestine cavity (arrowhead). F Invasion by intestinal microbiota of the striated border (arrowhead).
3.3. Disrupted intestinal microbiota
The results in Sections 3.1 and 3.2 revealed that no pathogens were isolated from the principle organs of the diseased fish, and that the intestine was the only target organ through necropsy and histopathological observations. Moreover, no obvious inflammatory cell infiltration was observed in the necrotic intestinal mucosa. Earle et al. (2015) demonstrated the reduction in the distance between microbes and epithelial cells, and increased the expression of the REG3 inflammatory marker due to diluted distal colon mucus from the lack of microbiota available carbohydrates (MACs) in the diet. Furthermore, the pathogenesis of inflammatory bowel disease (IBD) is associated with intestinal microbiota disorders (Marteau et al., 2004). These affected gut microbiota can pass through the intestinal mucosal barrier and contact the intestinal epithelial cells, thereby causing intestinal inflammation (Johansson et al., 2013). Thus, an intestinal bacterial disorder may have caused the enteric cell digestion, resulting in necrotic enteritis in the diseased sturgeons. In order to verify this, 16 s rRNA high-throughput sequencing was performed.
Results indicate an obvious variation in intestinal microbiota between the healthy and diseased groups. OTUs assigned at 97 % sequence similarity yielded a total of 1344 OTUs in the healthy group sample, and only 984 in the diseased group. The number of shared OTUs was observed as 705. The Rank-Abundance curve revealed the higher relative abundance of intestinal microbiota in the healthy group than that of the diseased group, with a smoother curve for the former. This indicates the more evenly distributed intestinal microbiota of the healthy group (Fig. 5). Thus, the diversity and relative abundance of intestinal microbiota in the diseased group was markedly reduced.
Fig. 5. Rank-Abundance curve for the diseased and healthy groups.
The relative abundance of colonies in the two groups varied greatly at the genus level. For example, the relative abundances of Clostridium, Mycoplasma, Lactococcus, and Cetobacterium were 23.28 %, 34.56 %, 4.72 %, and 0.03 % in the healthy group, and 40.81 %, 4.05 %, 7.83 %, and 15.76 % in the diseased group, respectively (Fig. 6).
Fig. 6. Intestinal microbiota composition at the genus level in different groups of the hybrid sturgeons.
Three species were identified at the species level, namely, Streptococcus parauberis, Lactococcus raffinolactis, and Plesiomonas shigelloides. The relative abundances of these bacteria were observed as 0.03 %, 3.22 %, and 0.01 % in the healthy group and 1.22 %, 0.94 %, and 5.87 % in the diseased group, respectively (Fig. 7). Results indicate a clear increase of P. shigelloides in the diseased group.
Fig. 7. Intestinal microbiota composition at the species level in different groups of the hybrid sturgeons.
The relative abundance values observed for each genus in the healthy group were as follows: Leucobacter 1.38 %, Streptococcus 0.24 %, Bosea 0.06 %, Phyllobacterium 1.16 %, Gemmobacter 0.79 %, Ruminococcaceae 4.32 %, Acinetobacter 4.39 %, Lactobacillus 2.95 %, Plesiomonas 0.01 %, Lactococcus 4.72 %, Cetobacterium 0.03 %, Mycoplasma 34.56 %, Clostridium sensu stricto 23.28 %, and others 22.11 %. The corresponding values for the diseased group were: Leucobacter 0.23 %, Streptococcus 1.27 %, Bosea 1.61 %, Phyllobacterium 1.24 %, Gemmobacter 1.68 %, Ruminococcaceae 0.09 %, Acinetobacter0.54 %, Lactobacillus 1.85 %, Macellibacteroides 6.12 %, Plesiomonas 6.56 %, Lactococcus7.83 %, Cetobacterium 15.76 %, Mycoplasma 4.05 %, Clostridium sensu stricto 40.81 %, and others 10.37 %.
Relative abundance values within each species in the healthy group were as follows: Streptococcus parauberis 0.03 %, Leucobacter 1.38 %, Bosea 0.06 %, Phyllobacterium 1.16 %, Gemmobacter 0.79 %, Acinetobacter 3.66 %, Lactococcus raffinolactis 3.22 %, Ruminococcaceae 4.32 %, Plesiomonas shigelloides 0.01 %, Lactococcus 1.47 %, Cetobacterium 0.03 %, Mycoplasma 34.56 %, Clostridium sensu stricto 23.13 %, and others 26.19 %. The corresponding values for the diseased group were: Streptococcus parauberis 1.22 %, Leucobacter 0.23 %, Bosea 1.61 %, Phyllobacterium 1.24 %, Gemmobacter 1.68 %, Acinetobacter 0.40 %, Lactococcus raffinolactis 0.94 %, Ruminococcaceae 0.09 %, Plesiomonas shigelloides 5.87 %, Macellibacteroides 6.12 %, Lactococcus 6.87 %, Cetobacterium 15.76 %, Mycoplasma 4.05 %, Clostridium sensu stricto40.79 %, and others 13.15 %.
4. Discussion
The relationship between the intestinal microbiota and host has been considered as symbiotic for many years. However, the recent discovery of the inseparable relationship between the host and the intestinal microbiota has resulted in widespread concern among scientists. Intestinal microbiota plays an important role in animals, affecting many functions of the host organism, including the immune function (Yap and Mariño, 2018), the Microbiota-Gut-Brain Axis adjustment (Serra et al., 2019), and the feeding regulation function (Zhang and Gong, 2017). Intestinal microbiota disorder can seriously affect the health of the host, with possible life-threatening effects. For example, intestinal microbiota disorder in humans is closely related to type 2 diabetes (Horie et al., 2017), inflammatory bowel disease (IBD) (Babickova and Gardlik, 2015), and obesity (Gao et al., 2018). Similarly, intestinal microbiota imbalance has been reported to cause diarrhea in pigs (Yu et al., 2000) and necrotic enteritis in chickens (Stanley et al., 2012). Furthermore, intestinal microbiota imbalances in aquatic animals have been observed to lead to enteritis in grass carp (Ctenopharyngodon idellus) (Tran et al., 2018), intestinal diseases in seahorses (Hippocampus trimaculatus, H. erectus, and H. spinosissimus) (Li et al., 2015), and “Red-Operculum” disease in Crucian Carp (Carassius auratus) (Li et al., 2017). In the present study, gross examination revealed the flatulence and expansion in the intestines of diseased hybrid sturgeon. Histopathological observations identified the intestine as the sole organ with marked necrotic enteritis. This indicated that the intestine was the target organ of the diseased hybrid sturgeons. In addition, no pathogen was isolated from the kidneys, livers, or spleens. This suggests that the occurrence of diseased fish enteritis may be attributed to the intestinal microbiota disorder. The 16S high-throughput sequencing results confirmed the obvious intestinal microbiota disorder in diseased fish, demonstrated that the death of the hybrid sturgeon was a result of enteritis caused by intestinal microbiota disorder.
According to the gut flora-centric theory of evolution (GFCTE), the intestinal microbiota first colonize in the human body following birth. After feeding, the intestinal microbiota decompose and digest the food to acquire nutrients and energy for reproduction, avoiding attacks and destroying the gastrointestinal mucosa (Zhang and Gong, 2017). However, if the animal is not able to forage in time, the intestinal microbiota continue to attack the intestinal mucosa of the host, causing severe damage and enhancing the sensitivity of the pathogens (Desai et al., 2016). In the present study, the hybrid sturgeon juvenile density was markedly high, possibly preventing the hybrid sturgeons to obtain sufficient amounts of food.
Numerous recent studies have identified changes in intestinal microbiota for humans and animals with enteritis. The bacterial species exhibiting variations were, however, distinct. For example, in the necrotizing enterocolitis (NEC) of premature infants, the relative abundances of Clostridium sensu stricto, Escherichia, and Shigellawere significantly higher (Zhou et al., 2015). In the necrotic enteritis of livestock and poultry, Clostridium perfringens was generally identified at significantly higher levels (Allaart et al., 2013; Shojadoost et al., 2012). In zebrafish, intestinal inflammatory responses were associated with an increase in Cetobacterium and Lactobacillus (Zheng et al., 2019). In rainbow trout (Oncorhynchus mykiss, Walbaum), Lactococcus garvieaewas identified as a possible cause for enteritis outbreaks (Avci et al., 2010). In the present study, the relative abundances of Clostridium, Cetobacterium, and Lactococcusin the diseased group were observed to be higher than those in the healthy group, while the relative abundance of Mycoplasma was lower in the diseased group. Furthermore, clostridium, the gas producing bacteria (Wiegel et al., 2006), may be the cause of intestinal flatulence. The relative abundances of P. shigelloides, an important pathogen of aquatic animals (Hu et al., 2014; Behera et al., 2018; Zhang et al., 2019), clearly increased in the diseased group, indicated its potential role in the death of hybrid sturgeon.
Data availability
All data generated or analyzed are included in the article. All materials are available from the corresponding author, on reasonable request.
Authors contributions
Xiaoli Huang, Liang Zhong, Fan Wei contributed the work equally. All authors read and approved the final manuscript.
Funding
This research was supported by grants from the double support plan of Sichuan Agricultural University (NO. 1921993230); and Sichuan Provincial Key Laboratory of Meat Processing Fund (No. 19-R-03).
CRediT authorship contribution statement
Xiaoli Huang: Conceptualization, Methodology, Writing - original draft. Liang Zhong: Conceptualization, Software, Investigation, Writing - review & editing. Wei Fan: Software, Formal analysis, Resources. Yang Feng: Data curation, Supervision, Investigation. Guanqing Xiong: Software, Validation, Data curation. Sha Liu: Formal analysis, Supervision, Visualization. Kaiyu Wang: Methodology, Formal analysis. Yi Geng: Conceptualization, Formal analysis. Ping Ouyang: Methodology, Project administration. Defang Chen: Writing - original draft, Visualization. Shiyong Yang:Conceptualization, Methodology, Project administration. Lizi Yin: Software, Supervision. Lili Ji: Software, Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank all authors for stimulating discussions and support.
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These authors contributed to this work equally.
http://wap.sciencenet.cn/blog-40692-1272668.html
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