Liu et al. Microb Cell Fact (2020) 19:123 https://doi.org/10.1186/s12934-020-01383-4 Microbial Cell Factories RESEARCH Open Access Comparative analysis of microbial community structure between healthy and Aeromonas veronii-infected Yangtze fnless porpoise Zhigang Liu1,2,3* , Aoyun Li3*, Yaping Wang3, Mudassar Iqbal3,4, Aifang Zheng1,2, Mengmeng Zhao1, Zhongkai Li1, Nuo Wang1, Chao Wu1 and Daoping Yu1,2 Abstract Background: The gut microbiota is a complex ecosystem, which is essential for the metabolism, health and immu- nity of host. Many diseases have been shown to be closely related to the alteration of intestinal fora. Aeromonas vero- nii as a conditioned pathogen can cause disease in Yangtze fnless porpoise through intestinal infections. However, it is not clear whether the disease caused by Aeromonas veronii is related to changes of intestinal fora. In the current study, the diversity and composition of gut microbiota in the healthy and Aeromonas veronii-infected Yangtze fnless porpoise were evaluated by high-throughput sequencing to further investigate the potential association between intestinal fora alteration and pathogen invasion. Results: A total of 127,3276 high-quality sequences were achieved and 2465 operational taxonomic units (OTUs) were in common among all samples. The results of alpha diversity showed that there was no obvious diference in richness and diversity between healthy and Aeromonas veronii-infected Yangtze fnless porpoise. Firmicutes, Bac- teroidetes and Proteobacteria were the most dominant phyla in all samples. In addition, the healthy Yangtze fnless porpoise exhibited higher abundance of Firmicutes and Fusobacteria than Aeromonas veronii-infected Yangtze fnless porpoise, while, the level of Proteobacteria was decreased. At the genus level, Paeniclostridium and Paraclostridium were the predominant bacteria genera in the CK (healthy Yangtze fnless porpoise) group. In the DIS (Aeromonas veronii-infected Yangtze fnless porpoise) group, Lactobacillus and unidentifed_Enterobacteriaceae were the dominant bacteria genera and the proportion of Paeniclostridium, Paraclostridium, Terrisporobacter, Cetobacterium, Candidatus Arthromitus, Terrabacter and Dechloromonas were reduced. Conclusions: In conclusion, our results showed that Aeromonas veronii infection can alter the gut microbiota of the Yangtze fnless porpoise by afecting the number of harmful bacteria and benefcial bacteria. Keywords: Yangtze fnless porpoise, Aeromonas veronii, Gut microbiota, High-throughput sequencing Introduction Te Yangtze fnless porpoise (Neophocaena phocaeno- *Correspondence: [email protected]; [email protected] ides asiaeorientalis) is a rare species that mainly lives in 1 College of Life Science, Anqing Normal University, Anqing 246011, the Yangtze River basin, Dongting lake and Poyang lake China in China. Furthermore, the Yangtze fnless porpoise is 3 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China the only freshwater population of porpoises in the world. Full list of author information is available at the end of the article However, the survival of Yangtze fnless porpoise has © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Microb Cell Fact (2020) 19:123 Page 2 of 12 sufered serious threats due to decline in water quality porpoise. Terefore, the objective of the current study and overfshing over the last several decades. Accord- was to analyze the microbial diversity of healthy and ing to statistics, the number of Yangtze fnless porpoise Aeromonas veronii-infected Yangtze fnless porpoise by is gradually declining and less than 2000 are remaining high-throughput sequencing. [1, 2]. Te Yangtze fnless porpoise has been listed as an endangered species by the International Union for Con- Materials and methods servation of Nature (IUCN) since 2013. Multiple protec- Animals and sample collection tion measures including captive breeding, in situ and ex Yangtze fnless porpoises used in the present study are situ conservation have been applied to prevent the con- raised in Anqing Nature Reserve (Anqing, China). Te tinuous reduction of this unique porpoise since the end experimental animals (approximately 3 years old, and of the last century. At present, two semi-natural and four males and four females in each group) were selected seven natural reserves have been built. in the same water area (Table 1). Te clinical symptoms Aeromonas spp., as one of the main pathogens of of the diseased Yangtze fnless porpoise were depres- aquatic animals poses a huge threat to the health of sion, conjunctival hemorrhage and skin necrosis. Clini- aquatic animals [3, 4]. Liu et al. reported that Aeromonas cal observation, histopathological examination, bacterial veronii can cause the skin necrosis, visceral hemorrhage isolation and identifcation, PCR amplifcation and gene and even death of Yangtze fnless porpoise [5]. Not only sequence alignment were used to evaluate etiological that, Aeromonas veronii can cause a variety of diseases in agent and ultimately determined that the disease in Yang- terrestrial animals and humans, such as dysentery, sepsis tze fnless porpoises was due to Aeromonas veronii infec- and necrotizing fasciitis [6]. Most of the studies suggest tion. Te relevant research about disease assessment has that Aeromonas veronii is an opportunistic pathogen that been published in the Diseases of Aquatic Organisms [5]. regulates the expression of virulence factors according to Moreover, the Yangtze fnless porpoises in the control the surrounding environment [7, 8]. Te latest research group were declared healthy after being examined by the shows that the infection of Aeromonas veronii may inter- professional veterinarian. act with other bacteria and its pathogenicity may be Te medical infusion tube (cut the both ends of the related to the intestinal fora [9]. medical infusion tube by scissor) dipped in a small Te intestinal bacterial community consists of a vast amount of petroleum jelly was slowly and rotationally number of diferent microorganisms including com- inserted into the anus 10–15 cm of the Yangtze fnless mensals, pathogens and some conditioned pathogens porpoise to obtain fecal samples. In this study, a total of [10, 11]. Te intestinal fora plays a vital role in growth, metabolism and immunity of the host [12–14]. In addi- tion, intestinal microorganisms can inhibit the prolif- Table 1 The information of the Yangtze fnless porpoise eration of pathogenic bacteria in the host by competing Sample Gender Body length (cm) Body with the pathogens for nutrients and adhesion sites [15, weight 16]. Meanwhile, some gut microbes can also produce (kg) several metabolites with bacteriostatic efects to pre- CK1.1 Male 152 54.6 vent the reproduction of pathogenic bacteria [17]. Te CK1.2 Male 138 49.5 stability of intestinal fora is a prerequisite for the intes- CK1.3 Male 151 58.6 tine to play a mechanical and immune barrier against CK1.4 Male 132 46.7 the invasion of pathogenic microorganisms. However, CK1.5 Female 124 42.3 the stability of intestinal fora can be afected by many CK1.6 Female 149 52.8 intrinsic and extrinsic factors, including temperature, CK1.7 Female 128 45.5 environment, antibiotic, and host phenotypes [18]. Pre- CK1.8 Female 145 48.6 vious studies have shown that intestinal fora alteration DIS1.1 Male 137 44.2 is closely related to many diseases including diarrhea, DIS1.2 Male 133 43.5 rheumatoid arthritis, diabetes and obesity [19–21]. DIS1.3 Male 141 45.2 More importantly, intestinal fora imbalance can lead DIS1.4 Male 142 46.3 to some conditional pathogens which may show strong DIS1.5 Female 131 42.2 pathogenicity [22, 23]. Previous studies have reported DIS1.6 Female 137 42.5 diferences in intestinal microbes between healthy and DIS1.7 Female 150 50.4 Aeromonas veronii-infected grass carp [24]. However, DIS1.8 Female 128 44.5 still less is known about the intestinal fora structure in CK and DIS indicate the healthy and Aeromonas veronii-infected Yangtze fnless healthy and Aeromonas veronii-infected Yangtze fnless porpoise, respectively Liu et al. Microb Cell Fact (2020) 19:123 Page 3 of 12 16 fresh fecal samples were collected from sixteen Yang- Prior to the sequencing, the sequencing libraries were tze fnless porpoise (healthy Yangtze fnless porpoise: required to be inspected on Agilent Bioanalyzer via using CK1, CK2, CK3, CK4, CK5,CK6, CK7, CK8 and Aero- Agilent High Sensitivity DNA Kit. Te qualifed library monas veronii-infected Yangtze fnless porpoise: