Effects of three host-associated Bacillus species on mucosal immunity and gut health of Nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection

Fish and Shellfish Immunology 97 (2020) 83–95

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Effects of three host-associated Bacillus species on mucosal immunity and gut health of Nile tilapia, Oreochromis niloticus and its resistance against Aeromonas hydrophila infection

T

Felix K.A. Kuebutornyea,b,c,d,e,f, Zhiwen Wanga,b,c,d,e,f, Yishan Lua,b,c,d,e,f,∗, Emmanuel Delwin Abarikeg, Michael Essien Sakyia,c, Yuan Lia,b,c,d,e,f, Cai Xia Xiea,b,c,d,e,f, Vivian Hlordzih a

College of Fisheries, Guangdong Ocean University, Huguang Yan East, Zhanjiang, 524088, Guangdong, China Shenzhen Institute of Guangdong Ocean University, Shenzhen, 518120, Guangdong, China Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Animals, Zhanjiang, 524088, China d Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment, Shenzhen, 518120, China e Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen, 518120, China f Guangdong Key Laboratory of Control for Diseases of Aquatic Economic Animals, Zhanjiang, 524088, China g Department of Fisheries and Aquatic Resources Management, University for Development Studies, Tamale, Ghana h Laboratory of Aquatic Animal Nutrition and Feed, College of Fisheries, Guangdong Ocean University, Zhanjiang, Guangdong, 524088, China b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Probiotics Bacillus Tilapia Mucosal immunity Microbiota Intestinal morphology

Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Only few studies have investigated the influence of host-associated Bacillus on mucosal immunity. In this study, the effects of three host-associated Bacillus species on mucosal immunity, intestinal morphology, intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against Aeromonas hydrophila infection was evaluated. The fish were divided into five treatment groups and fed with diets containing no bacteria denoted as Control, Bacillus velezensis TPS3N denoted as group V, Bacillus subtilis TPS4 denoted as group S, Bacillus amyloliquefaciens TPS17 denoted as group A and a 5th group containing the three Bacillus species at a ratio 1:1:1 denoted as group CB. At the end of the feeding trial, significant enhancement of both skin mucus and intestinal immune titres were recorded in terms of nitric oxide (NO) (except in the mucus of V and S groups), immunoglobulin M (IgM) (except in the intestine of group V), lysozyme (LZM), and alkaline phosphatase (AKP) in all fish fed the Bacillus supplemented groups relative to the untreated group. Intestinal antioxidant enzymes (catalase (CAT) (except in the intestine of group S) and superoxide dismutase (SOD)) capacity of Nile tilapia were higher in the Bacillus groups. Intestinal lipase activity was elevated in the Bacillus supplemented groups. The intestinal morphological parameters (villus height, villus width, goblet cells count (except in group S and A), and intestinal muscle thickness) were significantly enhanced in the Bacillus supplemented groups relative to the Control group. Dietary probiotic supplementation also influenced the intestinal microflora composition of Nile tilapia. Proteobacteria recorded the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes at the phylum level in this study. At the genus level, the abundance of pathogenic bacteria viz Staphylococcus and Aeromonas were reduced in the Bacillus supplemented groups in comparison to the Control group. A challenge test with A. hydrophila resulted in lower mortalities (%) in the Bacillus treated groups thus 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. In conclusion, the inclusion of B. velezensis TPS3N, B. subtilis TPS4, and B. amyloliquefaciens TPS17 in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal health, and resistance of Nile tilapia against A. hydrophila infection.

∗

Corresponding author. College of Fisheries, Guangdong Ocean University, Huguang Yan East, Zhanjiang, 524088, Guangdong Province, China. E-mail address: fi[email protected] (Y. Lu).

https://doi.org/10.1016/j.fsi.2019.12.046 Received 25 October 2019; Received in revised form 4 December 2019; Accepted 13 December 2019 Available online 14 December 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.

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1. Introduction

2. Materials and methods

Probiotics have long been used in aquaculture to enhance the growth and immunity of fish [1]. Earlier researches indicated that dietary supplementation of probiotics can be beneficial to the host's digestive enzyme activities, immune system, intestinal morphology, and intestinal microbiota [2,3]. However, these probiotics are mostly from terrestrial sources. Recently, attention has been drawn to the beneficial effects of host-associated probiotics (“bacteria originally isolated from the rearing water or the GI tract of the host to improve growth and health of the host”) relative to terrestrial probiotics use in aquaculture due to their better efficacies in the host [4,5]. It is well established that host-associated probiotics are more effective in the colonization process with a greater chance of competing with resident microbes thus dominating the gut within a short period [5,6]. Mucosal immunity in fish is made up of mucosa-associated lymphoid tissues which are divided into gut-associated lymphoid tissues, gill-associated lymphoid tissues, and skin-associated lymphoid tissues [7]. Skin and intestinal mucosa lymphoid tissues are known to be the fish's first line of defence since they serve as the first point of contact for pathogens. Mucosal surfaces contain immune titre such as proteolytic enzymes, piscidins, defensins calmodulin, antimicrobial peptides, lysozyme, complement, lectin, agglutinin C-reactive protein, interferon, vitellogenin, immunoglobulin, and glycoproteins; typically, intestinal mucosa also comprises macrophages, plasma cells, granulocytes, and lymphocytes [8–11] and play important roles in the defence against pathogens. Being the first line of defence, enhancement of its functions and understanding its mechanism is paramount for the protection of the fish against invasive pathogens. Furthermore, gut mucosal surfaces are the main sites for interaction between the host, antigens, and microorganisms [11]. However, relative to other immune components such as serum, the understanding of the mucosal immunity of fish and the influence of probiotics on mucosal immunity is still at the infant stage especially in Nile tilapia [7,12,13]. The intestinal microbiomes have been shown to influence fish's nutrition, metabolism, health, mucosal differentiation and development, and disease resistance [14]. The beneficial roles of a healthy gut microbiota include the secretion of digestive enzymes, vitamins production, synthesis of nutrients and metabolites required by the fish, and the enhancement of the gut epithelium architecture at the developmental and post-developmental stages [15,16]. Additionally, the intestinal mucosal immune system has been proposed to shape the intestinal microbial populations [4,11] whereas host-associated probiotics assist in returning a disturbed microbiota to its normal beneficial composition [17]. Thus probiotics can be used as a novel approach to improve gut health and the wellbeing of aquatic animals through dietary supplementation [6,18]. For instance, Bacillus subtilis was reported to improve the absorptive surface area, intestinal microvilli structure, and the gut health of rainbow trout [19] and likewise Bacillus licheniformis [18] and B. subtilis [14] in tilapia. Therefore understanding the influence of probiotics on the gut health and mucosal immunity of fish is necessary for the growth and enhancement of the immune status of the fish. Bacillus species have proven over the years to be beneficial to the aquaculture industry [20] and many studies have elucidated the effects of Bacillus species in Nile tilapia culture [18,21,22]. However, little information has been published on the effects of host-associated Bacillus in Nile tilapia, an important farmed fish [23], especially their individual or combined effects on gut health and mucosal immunity. Therefore, this study is aimed at evaluating the influence of the three Bacillus species (B. velezensis, B. subtilis, and B. amyloliquefaciens) on gut microbiota, immunity, digestive enzymes, and morphology as well as their influence on skin mucus immunity of Nile tilapia and its resistance against Aeromonas hydrophila infection.

2.1. Diet and probiotics spore preparation The Bacillus species used in the present study were isolated from the gastrointestinal tract of Nile tilapia, O. niloticus [24]. The Bacillus spores were prepared in accordance with methods described by Ran et al. [25] with slight modifications. The spore preparation agar (peptone 3.3 g/l, beef extract powder 1.0 g/l, NaCl 5.0 g/L, K2HPO4 2.0 g/L, KCl 1.0 g/L, MgSO4. 7H2O 0.25 g/L, MnSO4 0.01 g/L, lactose 5 g/L, agar 15 g/L) was prepared. The cell suspension was activated in LB broth (37 °C for 6 h) and spread on the agar plate and later incubated at 28 °C for 6 days. In order to collect the spore suspensions, 5 ml of sterile distilled water was added to each agar plate and the spores were suspended with the help of an inoculation loop and transferred into a 15 ml tube. To kill the vegetative cells, the spores were incubated at 85 °C for 15 min. This was followed by 10-fold serial dilution in 1 x phosphate-buffered saline (PBS) to determine the concentration of the spore suspensions. The final concentration of the spores was then adjusted to 1.0 × 108 CFU/ml. Spore amended diets were prepared by adding 80 ml of spore suspensions to 1000 g of commercial feed (Heng Xing company, Gaozhou, China) using a bleach and ethanol sterilized pump sprayer to achieve approximately 8% (v/w) spore suspensions. The probiotic viability in feed was determined according to methods described by Abarike et al. [26]. The experimental feeds were prepared by adding bacteria suspensions (1 × 108 CFU/ml) of each of the Bacillus isolates thus B. velezensis TPS3N designated as group V, B. subtilis TPS4 designated as group S, B. amyloliquefaciens TPS17 designated as group A and a 5th group which was made up of the combination of the three Bacillus species at a ratio of 1:1:1 (with same concentration) denoted as group CB. The Control group, however, contains no bacteria suspension but rather equal volume of PBS. This experiment was designed with reference to Van Doan et al. and Xia et al. [22,27]. All diets were dried under 16 °C using air conditioning and later stored at room temperature for use [26]. 2.2. Experimental set-up and fish management A total of 900 O. niloticus of average weight 46.24 ± 0.48 g with no signs of hemorrhage, lethargic, ascites, and detachment of scales (healthy and devoid of any signs of disease) were used in this study. The fish were obtained from Langye fish farm Gaozhou, Guangdong province, China. Concrete tanks of 1000 L water capacity provided by Langye fish farm were used for the experiment. The fish were randomly distributed into concrete tanks containing 800 L of water (static) and allowed to acclimate for two weeks. The fish were fed a control diet twice daily at 2% of their average body weight during the acclimatization period. Later, the tanks were divided into five groups namely, Control, V, S, A, and CB in triplicates (60 fish per tank). The fish were fed with the experimental diets twice a day at 2% of their average body weight for 4 weeks. The amount of feed given was adjusted according to Abarike et al. [26]. Water quality was maintained by renewing about 30% of the rearing water 3 times a week. A temperature of 28 ± 2.0 °C, pH of 6.8 ± 0.30, and dissolved oxygen concentration of 6.05 ± 0.55 mg/L were maintained as the condition of water during the acclimatization and experimental period. 2.3. Sampling 2.3.1. Collection of mucus samples Skin mucus samples were collected after four weeks of feeding trial according to Ref. [28]. In brief, 12 fish from each treatment (4 fish from each triplicate) were placed into a sterile polyethene bag each containing 10 ml of 50 mM NaCl (prepared in our laboratory) and rubbed gently in a downward motion for about 1 min. Mucus samples were collected by centrifuging at 1500 g for 10 min at 4 °C. The supernatant 84

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was collected and stored in 1.5 ml tubes at −20 °C until use.

extension at 72 °C for 5 min) reactions were performed in triplicate 25 μL mixture containing 2.5 μL of TransStart Buffer, 2 μL of dNTPs, 1 μL of each primer, and 20 ng of template DNA. DNA libraries concentration was validated by Qubit3.0 Fluorometer. PCR products were detected by 1.5% agarose gel electrophoresis. The library was quantified to 10 nM by Illumina MiSeq (Illumina, San Diego, CA, USA). Sequencing was performed using PE250/PE300 double-end sequencing, MiSeq Control Software by MiSeq (MCS). The QIIME data analysis package was used for 16S/8S rRNA data analysis. The forward and reverse reads were joined and assigned to samples based on barcode and truncated by cutting off the barcode and primer sequence. Quality filtering on joined sequences was performed. Then the sequences were compared with the reference database (RDP Gold database) using UCHIME algorithm to detect chimeric sequence, and then the chimeric sequences were removed. The effective sequences were used in the final analysis. Sequences were grouped into operational taxonomic units (OTUs) using the clustering program VSEARCH(1.9.6) against the Silva 132 database pre-clustered at 97% sequence identity. The Ribosomal Database Program (RDP) classifier was used to assign taxonomic category to all OTUs at a confidence threshold of 0.8. The RDP classifier uses the Silva 128 database which has taxonomic categories predicted to the species level. The unweighted pair group method with arithmetic mean (UPGMA) was performed using QIIME for beta diversity analysis.

2.3.2. Collection of intestinal samples The intestinal samples of 12 fish from each treatment (4 fish from each triplicate) were collected, weighed, and homogenized in 0.9% sterilized saline at a ratio of 1 : 9 (tissue: saline) using a bead homogenizer in ice for 10 min. The homogenate was later centrifuged at 2500 rpm for 10 min at 4 °C. This was followed by the collection of the supernatant in 1.5 mL tubes and stored at −20 °C for the determination of enzyme activities. Furthermore, whole intestines were collected from 9 fishes from each treatment, divided into fragments and prepared for analysis by light microscopy (LM) and scanning electron microscopy (SEM). The remains of the intestinal samples were stored at – 80 °C for the analysis of intestinal microbiota. 2.4. Determination of enzymes activities Mucus and/or intestinal immune titres (alkaline phosphatase (AKP), immunoglobulin M (IgM), lysozyme (LZM), nitric oxide (NO)), antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)), digestive enzymes (amylase, lipase) and total protein (TP) activities were determined using commercial kits from Nanjing Jiancheng, Bioengineering Institute, China following strictly, the manufacturer's instructions. 2.5. Histological analysis

2.7. Challenge test

Light microscopy was carried out in accordance with methods described by Ref. [29]. Images were captured using Olympus, model BX51 microscope, serial number: 9K18395, Tokyo, Japan. Intestinal measurements such as villus width (VW), villus height (VH), crypt depth (CD), intestinal epithelial muscle thickness (MT), and goblet cells count (GC) were determined using Image J 1.52a software (National Institute of Health, USA) [14]. The VW was measured at the midpoint of each villus. Measurement from the inner edge of the muscularis mucosae to the outer edge of the serosa indicated intestinal MT (Fig. 6A). Measurements of VH (villus tip to crypt mouth) and CD (from crypt mouth to base) were taken from areas where the plane section ran vertically from the tip of a villus to the base of the adjacent crypt [30]. As many villi as possible were measured, up to 10 villi per slide and not less than five. If more than 10 were able to be measured, the villi were chosen to be as evenly spaced around the intestine sample as possible. Slides with fewer than six suitable villi per slide were excluded. For scanning electron microscopy (SEM), intestinal samples, divided into foregut (FG), midgut (MG), and hindgut (HG) were washed in 1% S-carboxymethyl-L-cysteine for about 30 s in order to remove mucus followed by fixing in 2.5% glutaraldehyde in sodium cacodylate buffer (0.1 M pH 7.2). The samples were then prepared according to Ref. [31] and screened with SU8100 SEM (Hitachi High-Technologies Corporation Corporate Manufacturing Strategy Group, Japan).

Pathogenic A. hydrophila previously used in our work [24] was used for the challenge test in this study. The lethal dose 50 (LD50) (1 × 108) was calculated by pre-challenge trial as previously described in our work [26]. The bacteria were enumerated by counting the colonyforming units (CFU) on LB agar plates. Intraperitoneal injection was carried out according to modified methods described by Ref. [32]. Briefly, thirty fish from each treatment and control group (10 fish from each replicate) were selected randomly and 0.2 ml of suspended A. hydrophila (1 × 108 CFU/ml) was injected intraperitoneally after the 4 weeks feeding trial. The same number of fish with similar size were injected 0.2 ml of PBS for the negative control group (NC). Mortality was recorded for 7 days after infection. Cumulative mortality (%) was calculated according to Ref. [33]. Also, relative percentage of survival (RPS) was calculated according to the equation provided by Amend [34]. A. hydrophila was re-isolated from the dead fish and confirmed using light microscope and 16S rDNA gene sequence analysis as described previously [26].

2.8. Statistical analysis All data were analyzed using the SPSS version 22 software for Windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) and Tukey's HSD test was used to determine significant variation (P < 0.05) among the indexes studied between the treatments and control groups. Means were calculated from the triplicates of each group and data were expressed as a mean ± standard error (SE) of the combined means.

2.6. Intestinal microbiota analysis Total genome DNA from samples were extracted using Soil DNA Kit according to the manufacturer's protocols. DNA concentration was monitored by Qubit3.0 Fluorometer. 20–30 ng DNA was used to generate amplicons from the V3–V4 (bacteria and Archaea) and V7–V8 (fungi) using 16S rDNA/18S rDNA primers using PCR. The 16S forward and reverse primers were “CCTACGGRRBGCASCAGKVRVGAAT” and “GGACTACNVGGGTWTCTAATCC” respectively and the 18S forward and reverse primers were “CGWTAACGAACGAG" and “AICCATTCAATCGG" respectively. Besides the 16S/8S target-specific sequences, the primers also contained adaptor sequences allowing uniform amplification of the library with high complexity ready for downstream NGS sequencing on Illumina Miseq platform. PCR (94 °C for 3 min, followed by 24 cycles at 94 °C for 5 s, 57 °C for 90 s, and 72 °C for 10 s and a final

3. Results 3.1. Fish response to experimental diets It was observed that fish fed with the Bacillus supplemented diets (groups V, S, A, and CB) were more receptive to the experimental diets thus consumed more feed in comparison to the Control group as determined by the 2% average body weight daily ration, resulting in higher weight gain. 85

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Fig. 2. Mucous antioxidant enzymes activity of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). CAT = catalase; SOD = superoxide dismutase.

Fig. 1. Mucous immune parameters of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). NO = nitric oxide; LZM = lysozyme; IgM = immunoglobulin M; AKP = alkaline phosphatase.

3.2. Mucus immune parameters After 4 weeks of feeding, the influence of the treatment on mucus immune titres namely NO, LZM, IgM and AKP were assayed. The results revealed a significant increase (P < 0.05) in NO activities only in the groups A and CB with A showing the highest NO activity (P > 0.05). There was no significant difference between the NO activities between the Control, V and S group. LZM activity was high (P < 0.05) in all the Bacillus treatment groups relative to the Control group with the CB group displaying the highest LZM activity (P < 0.05). Again, an increasing trend was observed in the IgM activity as shown in Fig. 1. All the Bacillus treatment groups showed significantly high IgM activities in comparison to the Control. Significantly low AKP activity was recorded in the Control group relative to the V, S, A, and CB. The highest (P < 0.05) AKP activity was recorded in the CB group. 3.3. Mucus antioxidant enzymes activity Fig. 3. Intestinal immune parameters of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). LZM = lysozyme; IgM = immunoglobulin M; AKP = alkaline phosphatase.

Fig. 2 represents the mucous antioxidant activities of Nile tilapia fed the experimental diets. No detectable difference (P > 0.05) was observed in the CAT activity of Nile tilapia fed Bacillus supplemented diets. Meanwhile, all treated groups exhibited significantly higher CAT relative to the untreated Control group. The CB group showed the highest (P < 0.05) SOD activity. There was insignificant (P > 0.05) difference between the SOD activities of group V and A, and group V and S, however, all the Bacillus supplemented diets resulted in higher (P < 0.05) SOD activities in comparison to the untreated diet.

with the CB group displaying the highest (P < 0.05).

3.5. Intestinal antioxidant enzymes activity Lower (P < 0.05) CAT and SOD activities were observed in the Control group when compared to V, S, A, and CB groups. Again, the CB group displayed the highest CAT and SOD activities as shown in Fig. 4. There was no detectable difference between the CAT activities of groups V and A, and groups V and S. Similarly, there was no significant difference between the SOD activities of groups V and A, and groups S and A.

3.4. Intestinal immune parameters The intestinal immunological parameters of O. niloticus fed the experimental diets are displayed in Fig. 3. Compared with the Control group, AKP and LZM activities were significantly higher (P < 0.05) in O. niloticus fed Bacillus supplemented diets. No difference (P > 0.05) was observed in the IgM activity between the Control and V group, however, groups S, A, and CB showed significantly high IgM activity 86

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compared to the untreated group (Control) (Fig. 6A). Dietary Bacillus treatment resulted in taller villus tissues as well as higher goblet cells count in comparison to the untreated Control group (Fig. 6A). Significantly higher (P < 0.05) VL, VW, and MT measurements were recorded in the V, S, A, and CB groups as compared to the untreated Control group (Fig. 7). Crypt depth (CD) measurements were, however, significantly higher (P < 0.05) in the untreated Control group (but not higher than group V (P > 0.05)) than the treated groups. Goblet cells count did not differ significantly between Control, S, and A groups as shown in Fig. 7, however, CB recorded the highest GC (P < 0.05) followed by V. The SEM results revealed that unlike the treated groups, the untreated Control group had fewer microvilli density (arrowheads) in the HG and weaker (*) microvilli in the FG (Fig. 6B). Fewer microvilli density was also observed in the HG of groups V and S in comparison to A and CB. 3.8. Intestinal microbiota A total of 52 OTUs were shared among all the treatments; the CB, S, and A group had 11, 3, and 1 unique OTUs respectively whereas the Control and V groups had no unique OTU (Fig. 8). The unweighted pair group method with arithmetic mean (UPGMA) tree from the beta diversity distance matrix showed a clear separation with the Control group indicating that dietary Bacillus supplementation modulated the overall structure of intestinal microbiota in Nile tilapia (Fig. 9). Cyanobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes, Fusobacteria, Tenericutes, Firmicutes, and Proteobacteria were the predominant bacteria observed at the phylum level in the current study with Proteobacteria recording the highest abundance followed by Firmicutes, Fusobacteria, and Bacteroidetes (Fig. 10). The composition of the gut microbiome of the untreated Control group was dominated by members of Proteobacteria and Firmicutes (Fig. 10). At the family level, Enterobacteriaceae, Fusobacteriaceae, and Clostridiaceae were the most abundant bacteria observed. At the genus level, the inclusion of Bacillus species in the diet of Nile tilapia resulted in the reduction of Plesiomonas in the V, S, and CB groups relative to the Control group. Increased Cetobacterium was detected in the treated groups as compared to the untreated group except in the CB group. Relatively higher Clostridium, and Cellulosilyticum (except group A) populations were induced in all Bacillus supplemented groups when compared to the untreated Control group (Fig. 11). No Hathewaya was detected in the Control group except in the Bacillus treated groups. Relatively lower Staphylococcus, Bacillus (dominated by Bacillus thuringiensis at the species level), Bacteroides (except S and A), Macrococcus, and Kurthia (except V) was detected in the treated groups when compared to the untreated Control group. Unlike the other treatment groups, CB group did not record any Aeromonas.

Fig. 4. Intestinal antioxidant enzymes activity of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). CAT = catalase; SOD = superoxide dismutase.

3.6. Intestinal digestive enzymes activity The results of the intestinal amylase and lipase activities are displayed in Fig. 5. Insignificantly high amylase (P > 0.05) activity was recorded in the treated groups in comparison to the untreated group. However, higher lipase activity (P < 0.05) was detected in the treated groups compared to the untreated group. The CB group showed the highest lipase activity (P < 0.05). 3.7. Histology of the intestine Fig. 6(A and B) shows the photomicrographs and Fig. 7 shows the histological measurements of the intestinal tract of O. niloticus after four weeks of dietary Bacillus treatment. The intestinal epithelial cells were observed to be closely arranged in the treated groups with clear gaps as

3.9. Challenge test After 7 days of A. hydrophila infection, the cumulative mortality rates of Nile tilapia are shown in Fig. 12. The cumulative mortality of the treatment groups was significantly (P < 0.05) lower than in the untreated Control group, that is, 86.67%, 50.00%, 43.33%, 63.33%, and 30.00% for Nile tilapia fed Control, V, S, A, and CB diets respectively. The relative percent survival (RPS %) was highest in the CB group (65.28%), followed by group S (49.54%), group V (42.13%) and group A (26.85%). 4. Discussion Host-associated probiotics, formerly part of the host's microbiome has been shown to be helpful not only in improving the growth and immunity of the host but also aid in shaping a disturbed host's microbiome as well as shaping the intestinal morphology for better absorption. The use of host-associated probiotics in aquaculture has been

Fig. 5. Intestinal digestive enzymes activity of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). 87

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Fig. 6A. Photomicrographs showing the general cross section of the intestine of O. niloticus treated with the various Bacillus species after 4 weeks. Arrows heads represent goblet cells; VH = villus height; VW = villus width; MT = muscle thickness; CD = crypt depth.

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Fig. 6B. SEM showing the general cross section of the intestine of O. niloticus treated with the various Bacillus species after 4 weeks. Lane FG = foregut; lane MG = midgut; lane HG = hind gut. * indicate weak microvilli; arrow heads indicate fewer microvilli density.

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Fig. 7. Intestinal morphological parameters of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. Values are presented as mean ± SE. Significant differences are indicated by different letters (P < 0.05). VL = villus length; VW = villus width; CD = crypt depth; GC = goblet cells count; MT = intestinal epithelial muscle thickness.

mucosal immune titres by probiotics is scarce, however, increased LZM activity by B. subtilis AB1 in rainbow trout has been documented [42]. Likewise, enhanced intestinal AKP, IgM and LZM activities have been reported in Cyprinus carpio after probiotic Lactobacillus delbrueckii diet inclusion [43]. The higher mucosal immune parameters recorded in the CB group is perhaps due to the synergistic effects of the three Bacillus species and affirms that Bacillus stimulates mucosal immune parameters in fish. CAT and SOD are the common antioxidant enzymes in fish and are notable for the protection of fish against oxidative stress [44]. Bacillus acts as an antigen, thus stimulating the secretion of antioxidant enzymes by the fish's body and produces antioxidant enzymes as well [45–47]. O. mossambicus fed diets containing B. licheniformis Dahb1 exhibited increased skin mucous SOD activity [10] and feeding Nile tilapia with mixed Bacillus species resulted in enhanced mucous SOD and CAT activities [26]. Similarly, CAT and SOD activities were increased in both skin mucus and intestine in the current study suggesting that dietary addition of indigenous Bacillus species especially CB (Figs. 2 and 4.) can boost the antioxidant capacity of Nile tilapia. The genus Bacillus secretes a wide range of enzymes which enhance the nutrition of the host [48]. In agreement with this, significantly higher lipase (known to catalyse the hydrolysis of fats (lipids)) activities were recorded in the intestine of the treatment groups relative to the untreated group in our study. Similarly, increased intestinal digestive enzymes of Nile tilapia have been reported after host-associated B. subtilis HAINUP40 [33] and Bacillus paralicheniformis SO‐1 treatment [49]. Contrarily, no observable difference was recorded in the amylase

recently reviewed by Van Doan et al. [5] and it was made known that host-associated probiotics are the future of sustainable aquaculture. Research in this area is much advancing, however, less attention is paid to the effects of host-associated probiotics on fish's mucosal immunity and the gut microflora, especially in Nile tilapia. This study was conducted to assess the singular or the combined effects of three host-associated Bacillus species on the mucosal immunity, intestinal morphology, intestinal digestive enzymes, and the gut microflora of Nile tilapia as well as its resistance against pathogenic A. hydrophila infection. Increase in AKP activity indicates increased enzyme production by macrophage cells [10,35]; LZM possess antibacterial activity [36,37]; NO is an effector molecule involved in the activation of granulocytes and immune response [38]; IgM plays an important role in bacterial opsonization, toxin and virus neutralization [39,40]. Dietary supplementation of Bacillus species for 4 weeks resulted in enhanced skin mucus and intestinal immune parameters namely NO, LZM, IgM and AKP in Nile tilapia. Interactions between probiotic Bacillus and immune cells of fish leading to enhanced immune response have been documented by other scientists. For instance, increased AKP, NO, and LZM activities were recorded in Oreochromis mossambicus after host-associated Bacillus licheniformis Dahb1 dietary treatment [10]. Similarly, Catla catla fed host-associated B. subtilis and B. amyloliquefaciens showed enhanced mucous AKP [38] and LZM and NO [41] activities respectively. Van Doan and colleagues [22] also recorded increased skin mucous LZM activities in Nile tilapia after host-associated B. velezensis H3.1 dietary treatment. Literature on the enhancement of gut 90

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Fig. 8. Venn diagram demonstrating the distribution of OTUs shared by O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. CT = Control.

A healthy intestine is determined by biological markers such as muscular layer thickness, goblet cells count, and intestinal villi height [51]. An increase in VL and VW leads to increase in the intestinal absorption surface. Goblet cells, for instance, secrete mucus which possesses bactericidal properties and expedite transport through the intestinal epithelium thus preventing entry of pathogens [52]. B. amyloliquefaciens was reported to enhance the intestinal morphology in

activity (P > 0.05) of the treatment groups when compared to the control group (Fig. 5). B. subtilis Ch9 (1.0 × 109 CFUKg−1 and 3.0 × 109 CFUKg−1) isolated from the gut of grass carp did not significantly enhance the amylase activity of grass carp in the foregut after 28 days treatment except in the fish fed the highest dose (5.0 × 109 CFUKg−1) [50]. They [50] however, recorded significantly high lipase activities in the gut which agrees with the current study.

Fig. 9. UPGMA-clustering tree of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. CT = Control. 91

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Fig. 10. Heatmap of the abundance of the intestinal bacteria of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks at the phylum level. Colour intensity indicates the relative enrichment of OTUs. CT = Control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

decrease when Nile tilapia were fed Bacillus diets (Fig. 11). Plesiomonas is part of the pathogenic genera of the phylum Proteobacteria and is part of the dominant indigenous microbiome of freshwater fish [57,58]. Similarly, reduction in the Plesiomonas in the gut of Nile tilapia has been documented after probiotics treatment [59]. Some members of the genus Clostridium have been used as probiotics in fish [60,61] and Cellulosilyticum genus is able dissolve cellulose [62]. Dietary Bacillus supplementation resulted in the abundance of the genus Clostridium and Cellulosilyticum in the gut of Nile tilapia suggesting enhanced digestive capacity of the fish. The combined administration of Bacillus species (CB group), inhibited the proliferation of the genus Aeromonas, which could be a possible reason for the higher survival recorded in the CB group after A. hydrophila infection (Fig. 12). In summary, the inclusion of Bacillus species in the diet of Nile tilapia could positively shape intestinal microbiota thus enhancing the health of the fish. A. hydrophila is a common pathogen of freshwater fish including tilapia. Immunostimulation of fish, as well as bactericidal effects of Bacillus species against A. hydrophila, have been recorded [10,24,63,64]. Feeding fish with dietary supplements and subsequently challenging them with pathogenic bacteria has been a useful method of assessing the efficacy of the supplements in terms of resistance to disease infections. In the current study, all fish fed probiotic Bacillus supplemented diets recorded significantly lower mortalities after A. hydrophila infection in Nile tilapia. It is no doubt that the CB group recorded the lowest mortality since this group induced the best immune titres among all the treatment groups. Additionally, intestinal microbiota analysis revealed that the combined group (CB) inhibited the growth of the genus Aeromonas which could be the possible reason for the high survival of Nile tilapia post-infection. Similar to our findings, Gobi et al. [10] also recorded high survival in O. mossambicus infected with A. hydrophila after Bacillus treatment. However, the high lethal dose 50 (LD50) (1 × 108) used in this experiment (which was determined in a pre-challenge trial) may be due to pre-exposure of the fish to Aeromonas species as seen in the microbiota results in Fig. 11. In conclusion, this study showed that the inclusion of potential probiotic Bacillus (B. velezensis TPS3N, B. subtilis TPS4 and B. amyloliquefaciens TPS17) in the diet of Nile tilapia singularly or in combination, could enhance the mucosal immunity, intestinal morphology,

terms of VL and GC of Nile tilapia which was attributed to the utilization of large amounts of carbohydrates and the production of shortchain fatty acids by the Bacillus resulting in the stimulation of gastrointestinal peptides [53]. Feeding Nile tilapia with Bacillus supplemented diets resulted in enhanced intestine morphological parameters (VW, VL, MT, GC) in the current study, especially in the CB group. This result suggests that combination of Bacillus species could better enhance intestinal absorption surface and mucous secretion (GC) in Nile tilapia indicating better nutrient utilization (Fig. 7). Similar to our findings, a mix of Bacillus species (B. subtilis, B. licheniformis, and B. pumilus) enhanced the intestinal morphology of Nile tilapia after dietary treatment [15]. Furthermore, the increase in microvilli density indicates an increased absorptive surface area and reduces the exposure of enterocyte junctions thereby reducing the rate of colonization by pathogenic microbes thus increasing the fish's resistance [27,54]. Four weeks dietary Bacillus supplementation resulted in stronger and higher intestinal microvilli density of Nile tilapia relative to the untreated group as seen in Fig. 6B. In agreement with this result, Xia et al. [27] recorded higher intestinal microvilli density in Nile tilapia fingerlings when treated singularly or in combination with host-associated Bacillus cereus NY5 and B. subtilis. The gut of fish is made up of complex microbiota and the interactions of these microbes with one another and the epithelial cells influence various host functions including nutrition, digestion development, immunity, and disease resistance [55]. Notably, probiotics have been documented to modulate the intestinal microbiota of fish. Indeed the current study demonstrates that probiotic Bacillus can improve and maintain a balanced intestinal microflora to enhance the health and wellbeing of Nile tilapia. The findings of this current research did not differ entirely from Baldo et al. [56] who documented that Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Planctomycetes, Verrucomicrobia, and Proteobacteria were the common bacteria phyla in the gut microbiome of cichlids. Proteobacteria was the dominant bacteria phyla recorded in this study, however, relatively lower populations were recorded in the treated groups. This agrees with Yang et al. [17] who also recorded reduced Proteobacteria in grouper after hostassociated B. pumilus SE5 treatment. At the genus level, the gut microbiome was dominated by Plesiomonas, which was observed to 92

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Fig. 11. Taxonomy classification of reads of intestinal bacteria at genus level of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks. CT = Control.

immunity and intestinal microbiome of Nile tilapia.

intestinal digestive enzymes activity, intestinal microbiome and resistance of Nile tilapia against A. hydrophila infection. To the best of our knowledge, this study is the first to assess the combined probiotic effects of these three host-associated Bacillus species on mucosal

Fig. 12. Cumulative mortality (%) of O. niloticus fed diets supplemented with different Bacillus species for 4 weeks after 7 days post challenge with A. hydrophila. Values are presented as mean ± SE. Negative control = NC; CT = Control. 93

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Funding Shenzhen strategic emerging and future industrial development funds (20170426231005389) supported this work.

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Animal rights

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All fish were handled following the U.K animal act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments.

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Declaration of competing interest

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The authors declare that they have no conflicts of interest. References

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