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Efficiency of a bacteriophage in controlling vibrio infection in the juvenile sea cucumber Apostichopus japonicus
Aquaculture 451 (2016) 345–352
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Efficiency of a bacteriophage in controlling vibrio infection in the juvenile sea cucumber Apostichopus japonicus Zhen Li a, Jiancheng Zhang a, Xiaoyu Li a, Xitao Wang a, Zhenhui Cao a,c, Lili Wang a, Yongping Xu a,b,⁎ a b c
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China Ministry of Education Center for Food Safety of Animal Origin, Dalian 116600, People's Republic of China Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, People's Republic of China
a r t i c l e
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Article history: Received 14 August 2015 Received in revised form 22 September 2015 Accepted 24 September 2015 Available online 28 September 2015 Keywords: Apostichopus japonicus Vibrio cyclitrophicus Bacteriophage Genome sequencing Skin ulceration syndrome Phage lyophilization
a b s t r a c t Vibro cyclitrophicus is thought to be responsible for the cause of severe infection in juvenile sea cucumber (Apostichopus japonicus). Increases in the prevalence of antibiotic resistant pathogens have promoted us to develop effective agents to antibiotics for combating microbial infection among animals as well as human. In the present study, we isolated a bacteriophage with the ability to cleave V. cyclitrophicus present in the sewage of sea cucumber farms. This bacteriophage was designated as phage vB_VcyS_Vc1 (Vibrio phage Vc1). A one-step growth curve analysis of the phage revealed eclipse and latent periods of 25 and 45 min, respectively, with a burst size of 215 PFU/infected cell. Morphological analysis revealed the phage belongs to family of Siphoviridae. Furthermore, genomic sequencing results revealed a double-stranded DNA containing 44,541 bp with a G + C content of 44.16%. Forty four coding sequences were annotated in the genome, and nineteen of these were associated with a known function. Genes related to virulence and toxins were not detected in the genome. In addition, a prevention experiment conducted in a marine environment demonstrated that the phage increased the survival rate of juvenile sea cucumbers (18 ± 2 g) from 18% to 81% when the sea cucumber were fed with feedstuff containing the freeze-dried phage powder, 58% when the sea cucumber was injected with purified phages (MOI = 10), and 63% when the sea cucumbers were immersed in a suspension containing purified phages. Notably, when the phage provided nearly the same protection to the sea cucumbers as antibiotic when it was fed to the sea cucumbers in the form of freeze-dried powder mixed with feedstuff. Taken together, the results demonstrated that the use of phage to control the infection of V. cyclitrophicus in sea cucumber may be a feasible alternative to antibiotics. Statement of relevance: The A. japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China, which is of high nutritional value and economic value. In the past decade, as the growth of the market demand, A. japonicus's artificial breeding scale expands rapidly and the sea cucumber industry has become a vigorous sector in China aquaculture. However, with the high density and intensive development model of A. japonicus breeding, the various diseases have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers. At present, sanitizer and antibiotics are still commonly used in farms or hatcheries to control the bacterial disease in juvenile sea cucumbers, which resulted in the environment pollution and antibiotic residues. Nowadays, increased appearance of antibiotic resistant phenomenon promoted us to develop effective agents. Hence, alternative strategies to antibiotics should be further developed. This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Apostichopus japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China. Due to its nutritional properties and high economical values, the farming ⁎ Corresponding author at: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.aquaculture.2015.09.024 0044-8486/© 2015 Elsevier B.V. All rights reserved.
of this cucumber has become a vigorous sector in the aquaculture of China (Zhang et al., 2003; Liu et al., 2010). In China, as much as 137,754 t of A. japonicus were produced in the provinces of Liaoning and Shandong alone in 2011, with a production value of nearly 4 billion dollars (He and Huang, 2014). However, the rapid expansion and intensification of sea cucumber farming has led to various problems, including the occurrence of various diseases that have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers (Deng et al.,
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2009), and it is mainly caused by the bacteria Vibrio splendidus, Vibrio alginolyticus, Vibrio cyclitrophicus and Shewanella marisflavi (Deng et al., 2009; Liu et al., 2010; Li et al., 2010, 2013). At present, antibiotics and sanitizer are still commonly used to control diseases in juvenile sea cucumbers. The problems of environment pollution and antibiotic residues in aquaculture have become a concern due to the frequent and widespread use of antibiotics (Nakai and Park, 2002; Defoirdt et al., 2011). Therefore, it is important to develop other strategies that could be used in place of antibiotics in aquaculture to protect the animals against diseases. Bacteriophages or phages are bacterial parasites with life cycles as short as 20–60 min (O'Flaherty et al., 2009). Phage therapy has many advantages over antibiotics, such as high abundance, host specificity, and rapid exponential replication (Matsuzaki et al., 2005). Phage therapy has already been tested in a variety of species and has achieved positive results in the control of bacterial infections in aquaculture, such as in the control of lactococcosis (caused Lactococcus garvieae) in yellowtail (Nakai et al., 1999), systemic bacterial coldwater disease (caused by Pseudomonas plecoglossicida) in ayu fish (Kim et al., 2010) and luminous vibriosis (caused by Vibrio harveyi) in shrimp (Shivu et al., 2007). Our previous work has focused on using phage as an alternative to antibiotics for controlling V. alginolyticus infection in A. japonicus (Zhang et al., 2015). The aim of this study was to evaluate the potential use of a lytic phage for the treatment of V. cyclitrophicus-inflicted skin ulceration syndrome in A. japonicus. 2. Materials and methods 2.1. Bacterial strain and animals The bacterial strain used for phage isolation was first isolated from diseased sea cucumbers obtained from a hatchery in Dalian Bay (Dalian, China) in 2012. The bacterium was isolated via the use of thiosulfate citrate bile salts–sucrose agar (TCBS agar, Difco) medium. It was subsequently grown in 2216E medium and stored as a bacterial suspension stock at − 80 °C. Genetic characterization of the strain was achieved by 16S ribosomal DNA (rDNA) sequence analysis, and the sequence was then submitted to the Genebank database. Homologous analysis was performed using NCBI BLAST. V. cyclitrophicus VC1 was recovered from a frozen stock by growth in 2216E liquid medium at 26 °C for 24 h. The bacterial suspension was subsequently centrifuged at 2500 × g for 10 min and the cell pellet was re-suspended in sterile physiological buffered saline (PBS) to a density. The bacterial cell counts were calculated according to the OD-cell counts coherence curve. Healthy sea cucumbers (15–20 g), which showed no symptoms of peristome dedma, skin ulceration, head shaking and visceral ejection (Deng et al., 2009), were purchased in the Dalianwan hatchery and kept in a temperature controlled mariculture room (14–16 °C) to provide the animals with normal growth condition. 2.2. Pathogenicity assay in sea cucumber One hundred and twenty healthy sea cucumbers (18 ± 2 g) were randomly divided into 12 groups, with 10 animals per group. Six groups were used for coelom injection while the other six groups were used for immersion challenge. For coelom injection, one group was designated as the control group, and was injected with 0.3 mL PBS per animal, whereas the remaining five groups were each injected with the above bacterial suspensions, which ranged from 8 × 105 to 8 × 109 CFU/mL through 10-fold dilution method. For immersion challenge, each group of sea cucumbers was placed in 4 L of sterilized seawater. For the control group, 10 mL of PBS was added to the water, whereas for the test groups, 10 mL of bacterial suspensions was added to the water, yielding a concentration ranging from 2 × 104 to 2 × 108 CFU/mL. According to the criteria described by Santos et al. (1988), a bacterial strain is considered highly virulent if the LD50 value is 1.7 × 104 to 1 × 106 CFU/g body weight,
moderately virulent if it is 1.4 × 106 to 1.8 × 107 CFU/g body weight, and non-virulent if it is more than 108 CFU/g body weight. To protect animals' welfare, all sea cucumbers were cultured in a temperature controlled mariculture room (14–16 °C), and the pH, dissolved oxygen and salinity of the water were maintained at 7.5–8.0, 5–6 mg/mL, and 30–33 g/L, respectively. 2.3. Phage isolation, propagation and purification The phage was isolated from the raw sewage obtained from the drain-pipes from the same local hatchery (Dalian, China). Prior to the isolation, CaCl2 and MgCl2 were added to the water to a final concentration of 10 mM. After three rounds of plaque purification using the double-layered agar method described by Adams (1959), a single plaque was enriched using the method described by Maniatis et al. (1978) in 2216E broth and grown at 26 °C for overnight. The suspension was clarified by centrifugation (10,000 ×g, 10 min) and filtered through a 0.22-μm filter (EMD Millipore Co., Billerica, MA, USA). After that it was assayed for phage titers (plaque forming units, PFU) using the spot assay technique. Purification of the phage was carried out using the methods described in molecular cloning (Sambrook et al., 1989) with modification to obtain phage particles with high quality and quantity. The phage lysate was mixed with 1 M NaCl and 10% (w/v) polyethylene glycol (PEG) 8000 and dissolved by shaking. The lysate was allowed to stand at 4 °C for 10 h or overnight and then centrifuged at 10,000 × g for 20 min. The precipitate was then re-suspended in SM buffer (NaCl, MgSO4·7H2O, Tris·Cl, gelatin and double distilled water), and CsCl was added to the suspension to a concentration of 1.2 g/mL. The suspension was centrifuged at 150,000 ×g for 24 h along a CsCl density gradient using with a Beckman Optima L-100XP Ultracentrifuge (Fullerton, CA) (Sambrook et al., 1989). 2.4. Morphological observation of the phage CsCl-purified phage particles (≥1010 PFU/mL) were spotted onto a carbon-coated copper acid grid for 10 min, stained with 0.5% (w/v) uranyl acetate, and then examined by JEOL-1200EX transmission electron microscopy (JEOL USA, Inc., Peabody, MA, USA) at an accelerating voltage of 80 kV. 2.5. One-step growth curve of the phage One-step growth curve of the phage was obtained as described by Phumkhachorn and Rattanachaikunsopon (2010) with some modifications designed to calculate the latent period, burst time and burst size. Briefly, 200 mL of early log phase culture was centrifuged at 10,000 ×g for 10 min and then re-suspended in 10 mL of fresh 2216E broth to obtain a final concentration of 108 CFU/mL. In order to obtain a MOI (multiplicity of infection) of 0.1, of 1010 PFU/mL phage particles were added to the bacterial culture and allowed to absorb for 10 min at 26 °C. The suspension above was then centrifuged at 10,000 × g for 5 min, resuspended in 10 mL of fresh 2216E broth, and then incubated at 26 °C with shaking at 120 rpm. Samples were taken every 10 min and over a period of 2 h. The supernatant of each sample was subjected to phage titration using the spot assay technique. 2.6. In vitro inhibition of bacterial growth by phage To calculate the antimicrobial capacity of the phage, 96-well-micro plates were filled with 100 μL of inoculated double strengthen 2216E per well, and 100 μL of the prepared phage dilution was added to the well to provide three final doses of MOI to 0.1, 1 and 10. Controls for plate sterility, phage suspension sterility and bacterial culture without phage addition were also included, and each phage–host culture combination at specific MOI was performed in triplicate wells. Subsequently,
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these plates were incubated at 26 °C for 12 h and the optical density at 600 nm (OD600) was measured using a Micro-titer Plate Reader (Mutiskan Go, Thermo Scientific Co. Ltd., USA) at 30 min intervals. Each independent trial was repeated three times. 2.7. Phage genomic DNA extraction and sequencing analysis To find out whether the phage genome contains the genes related to toxin and virulence, the entire phage genome was extracted and sequenced. This safety issue would be particularly relevant for phage therapy. CsCl-purified phage particles (≥ 1010 PFU/mL) were used to extract the whole genomic DNA using a phenol–chloroform–isoamyl–alcohol method (Sambrook et al., 1989). The phage genomic DNA was subsequently analyzed by a garose gel electrophoresis (AGE) using 0.8% (w/v) agarose gel in Tris-acetate buffer at 100 V. The DNA extracted above was sequenced using Illumina High-Throughput Sequencing Platform (Illumina Hiseq™ 2000, Shenzhen, China). For genomic analysis, coding sequences (CDS) of the genome were determined using the Heuristic GeneMarkS software (Besemer and Borodovsky, 2005). Comparison of amino acid sequences against the NCBI non-redundant database and CDS functions were predicted by BLASTX (Altschul et al., 1990) and InterproScan (Fischetti, 2008), respectively. In addition, the conserved domains of CDSs were confirmed by the Conserved Domain Database (Marchler et al., 2005). Transfer RNA (tRNA) encoding genes were screened using tRNASscan at http:// greengene.uml.edu/programs/FindtRNA.html (Lowe and Eddy, 1997). Meanwhile, the annotation of the phage genome was identified using Artemis software (Rutherford et al., 2000). 2.8. Phage therapeutic study 2.8.1. Diet preparation The experimental diet contained 50% Laminaria japonica feedstuff, 20% sea mud, 15% soybeen meal, 10% fish meal, 3% shell powder 2% vitamin and mineral premixes (Wang et al., 2015), which were all provided by the SEM Feed Company (Dalian, China). The purified phage suspension was freeze-dried in the presence of skim milk using a laboratory freeze dryer (Beijing Biocool Company, Beijing, China). The drying process yielded a powder form of the phage. Two set of diets were made: the first set contained feedstuff mixed with the phage powder was named diet-1, whereas the second set consisted of only feedstuff, and was named diet-2 (Table 1). The standard of nutrition was based on early report described by Zhu et al. (2005). 2.8.2. Phage therapeutic experiment The therapeutic potential of the phage was evaluated by assessing its ability to protect sea cucumbers from developing skin ulceration syndrome. A total of 300 sea cucumbers (15 ± 2 g) were randomly divided into five groups (three test groups and two control groups). Each group consisted of three PVC tanks (38 cm × 54 cm × 80 cm) with 20 animals per tank as three parallel experiments. Each tank was filled with 40 L of aerated sand-filtered seawater and the temperature of the water was kept at 16–18 °C. Among the five groups, two groups were kept as control and three groups were treated with the phage. For the two control groups, one group was treated with antibiotic (positive control) whereas the other group was kept in sterilized seawater (negative control). As
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for the three test groups, one group was fed with diet-1 (group A), another group was immersed in water containing purified phages (group B), while the remaining group was injected with purified phages (group C) (Table 2). Sea cucumbers were fed at a rate of approximately 3% of the body weight and the feedstuff above contained 5% lyophilization powder per gram. After 60 days of feeding, After 60 days of feeding, all sea cucumbers were challenged with V. cyclitrophicus VC1. The animals were exposed to an initial bacterial concentration of 5 × 107 CFU/mL of 400 mL (bacterial suspensions re-suspended with PBS were diluted to 40 L seawater). Twenty-four hours later, positive control group was injected with 1 mL antibiotic (5 mg/mL doxycycline), negative control group, A and B groups were injected with the same volume of sterilized seawater, group C was injected with phage particles at 107 PFU/mL (MOI = 10), and group B was added the same concentration of phage suspensions to the tanks for phage immersion treatment (Table 2). Survival was then recorded for 10 days and the animals were not fed during this period. In addition, phage titers in the tank as well as the tracks of sea cucumbers were detected respectively in every ten days. At each time point, three sea cucumbers at random were sterilized with 70% ethanol for about 3 min and then dissected by aseptic surgery technique as described by Xing et al. (1998). The digestive tract of each animal (Fig. 1 F, G) was collected and homogenized (IKA R104, Germany) in cold 50 mM Tris–HCl (pH 7.8) buffer, followed by centrifugation at 10,000 ×g for 10 min at 4 °C (Wang et al., 2015).The final concentration of the phage in the tank was calculated as follows: Final phage titer (T2) = T1 (N W R r)/V, where T1 is the phage titer after lyophilization treatment, N is the number of animals in each tank, W is the body weight of each sea cucumber, R and r are, respectively, feed rate (3%) and phage lyophilization powder additive rate (5% in this study), and V is the seawater volume in the tank. 2.9. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) using Prism 5.0. The significance of differences between the treatments and controls were determined using Tukey's Multiple Comparison Test and Chi-square test. Statistical significance was considered at the P b 0.05 level. 3. Results 3.1. Pathogenicity test of the isolate V. cyclitrophicus VC1 The bacterial strain designated as V. cyclitrophicus VC1 was characterized by 16S-rDNA sequencing. The 16SrDNA sequence of 1442 bp was submitted to GenBank Database (GenBank: JX856180), showing 99% identity to V. cyclitrophicus strain LMG21359 (DQ481610) and 99% identity to V. cyclitrophicus strain VC2 (KC185413). The results of the pathogenicity test revealed mass mortalities on day 5 of postinfection by coelom injection and on day 4 by immersion challenge (Fig. 2). The LD50 of the V. cyclitrophicus VC1 was 3.37 × 104 CFU/g body weight as determined by coelom injection and 1.68 × 105 CFU/mL as determined by immersion challenge. Since the LD50 values obtained by different methods were within the range of 1.7 × 104 to 1 × 106 CFU/g, V. cyclitrophicus VC1 was considered highly virulent to sea cucumber.
Table 1 Proximate composition of the two experimental diets. Diet
Diet-1 Diet-2
Nutrient content Protein (%)
Lipid (%)
Ash (%)
Fiber (%)
Dry matter (%)
Additives (5% per gram feedstuff)
15.2 14.8
4.5 4.2
37.3 37.8
7.2 6.8
97.2 96.4
Phage freeze-dried powder with skim milk Only skim milk freeze-dried powder without phage
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Table 2 Experimental design for phage-based therapy in sea cucumber. Group
Negative control
Positive control
Diet Diet-2 Diet-2 After 60 days of feeding, challenged with V. Administration Coelom Coelom injection injection Seawater + − Antibiotics − + Phage − −
A group
B group
C group
Diet-1 Diet-2 Diet-2 cyclitrophicus VC1, 24 h later Coelom Immersion Coelom injection injection + − − − − − − + +
Survival was recorded for 10 days and animals were not fed in this period. “+”: related procedure was applied; “−”: related procedure was not applied.
For the group treated with the highest concentration of the culture, clinical symptoms of skin ulceration syndrome such as mouth tumidity, head shaking, enlarged ulcers and visceral ejection were detected after 20 h of infection, and sea cucumbers began to die two days after they were challenged with the bacteria. No mortality was observed in the control from the pathogenicity test conducted by coelom injection or immersion. 3.2. Isolation and identification of phage Using V. cyclitrophicus VC1 as a host organism, a bacteriophage was isolated from the water sample through the double layer method (Adams, 1959). The phage produced clear plaques on the lawn of V. cyclitrophicus VC1 cells, with a diameter of the plaques about 1.6 ± 0.2 mm (Fig. 3 A), indicating that the phage was a virulent phage toward V. cyclitrophicus VC1. Morphological analysis showed that the phage possessed a symmetry head (diameter 50 ± 3 nm) together with a long flexible non-contractile tail (length 150 ± 6 nm) (Fig. 3 B), suggesting that the phage belonged to the Siphoviridae of Caudovirales. The isolated phage was then designated as phage vB_VcyS_Vc1 (vibrio phage Vc1, CGMCC 9101). The one-step growth curve of the phage propagated on the host strain showed that the latent and burst periods were approximately 25 min and 45 min, respectively (Fig. 4). In addition, the average burst size of the phage was calculated as 215 PFU per infected cell. The effect of the phage on V. cyclitrophicus VC1 in vitro is shown in Fig. 5. Compared to the control group, the optical density of V. cyclitrophicus VC1
Fig. 2. Cumulative mortality of sea cucumber infected with Vibrio cyclitrophicus VC1 by coelom injection (A) or immersion (B) at different concentrations.
treated with different MOI phage stocks started to show significant decreases (P b 0.05) 3 h after infection. Four hours after infection, the OD600 decreased further and was still significant (P b 0.05) compared to the control group. Subsequently, the OD600 values of all three test groups with different MOI increased slightly after 10 h after infection.
3.3. Genome sequencing and annotation of the phage The complete sequencing data of the phage genome revealed a DNA sequence of 44,541 bp in length, with 44.61% GC content. Forty-four coding sequences (CDSs) comprising 88.6% of the genome were
Fig. 1. Photographs of diseased sea cucumbers suffering from skin ulceration syndrome. A and B: skin ulceration syndrome in dead sea cucumbers obtained from one hatchery in Dalian (China), C: rotting skin after day 2 post-infection (dashed arrow) and normal body wall (solid arrow), D: typical symptoms of skin ulceration syndrome (dashed arrow), E: healthy (solid arrows) and diseased (dashed arrows) juvenile sea cucumbers, F: diseased juvenile sea cucumbers with empty intestines and ulceration body wall, G: intestinal structure diagram of healthy juvenile sea cucumbers.
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Fig. 3. Phage plaques (A) in 2216E media and electron micrographs (B) negatively stained with uranylacetate. Scale bar represents 50 nm.
subsequently predicted (Table S1) and nearly all of them start with an ATG codon, except for CDS2, which starts with GTG and CDS19 and CDS22, which start with TTG. In addition, a tandem repeat sequence (5′-AACATCCGCTATCAAGCAGAG-3′) was observed and no tRNA genes were found in the genome. Moreover, the functions of 19 CDSs were clarified using Blastx against NR Database and two of them were hypothetically conserved (e-value b 0.01). Blastn analysis showed a low degree of homology between vibrio phage Vc1 and other phages. Furthermore, the genome also comprised four basic segments, including genes coding for packaging proteins and structural proteins, and DNA replication-related genes as well as other functional genes (Fig. 6). The structural and packaging proteins related genes of this phage were clustered in the first half of the genome. Putative scaffolding protein, coded by CDS6, has been proven to play an important role in virion particles formation by promoting the interaction between protein subunits and the stability of assembly intermediates (Dokland, 1999). However, the genes encoding lysozyme were not detected in the genome of vibrio phage Vc1, while a cell wall hydrolase-encoding gene was identified (CDS21), which was detected in the middle of the phage genome (Table S1). Besides, there were no genes related to the toxin and virulence of the phage, suggesting that the phage could have considerable potential for phage therapy.
Fig. 4. One-step growth curve of vibrio phage Vc1. Data represent are the means ± SD from three independent experiments. n = 3.
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Fig. 5. Inactivation of V. cyclitrophicus by vibrio phage Vc1 at different multiplication of infection (MOI) in vitro. n = 3.
3.4. Nucleotide sequence accession number and culture preservation The 16S rDNA sequence of V. cyclitrophicus VC1 and the complete genome sequence of phage vB_VcyS_Vc1 (vibrio phage Vc1) were available in GenBank under accession number JX856180 and KJ502657 respectively. The phage and the host strain were simultaneously preserved in China General Microbiological Culture Collection Center (Beijing, China) with CGMCC No. 9101.
3.5. Phage therapeutic study Skim milk was assayed as phage lyophilization media and the phage titer was significantly decreased (P b 0.05) as showed in Fig. 7, which was consistent with the results described by Dini and de Urraza (2013) before. The titer of initially purified phage suspension was 8 × 1011 PFU/mL. After freeze-drying treatment, the titer decreased to 6 × 109 PFU/mL, and the standard FT in each tank was about 9 × 105 PFU/mL (Fig. 9 Curve C). After 60 days of feeding with diet-1 and diet-2, all five groups of sea cucumbers were challenged with V. cyclitrophicus VC1, and the cumulative survival rate was determined. All three phage treatment groups showed significant (P b 0.001) increases in survival rate compared to negative control group (Fig. 8). Notably, group A displayed nearly the same survival rate with the antibiotic treatment group (P N 0.05). Group A appeared to have a better protection (81% survival rate) than group B (63% survival rate) and group C (58% survival rate), the difference between A group and B or C group was significant (P b 0.05) while no significant difference between B and C groups (P N 0.05). In the control group, sea cucumbers suffered skin ulceration syndrome and began with the appearance of a white lesion (Fig. 1 C, D and E), the animals began to die after day 2 post-infection. The phage titers in the water that housed group A sea cucumbers determined as well as the phage titer in the intestinal tracts of these sea cucumbers was also determined (Fig. 9). As shown by the light gray area, phage titer in the seawater (Fig. 9 Curve A) was always lower compared to the standard FT (Curve C), and in each sampling point, phage titer within the intestinal tract was more or less steady (P N 0.05) (Fig. 9 Curve B). These results indicated that the phage could exist in the seawater and intestinal tracts of the sea cucumbers when these animals were fed with diet-1. Ten days after challenged with V. cyclitrophicus VC1, the phage titer was significantly increased (P b 0.05) as shown by the gray area in Fig. 9, which suggested that the phage could protect the sea cucumbers against infection of V. cyclitrophicus VC1. Moreover, consumption of diet-1, which
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Fig. 6. Schematic representation of the complete genome of vibrio phage Vc1. Forty-four putative proteins are represented by arrows. Blue: mainly structural proteins; red: packaging proteins; pink: additional functional proteins; green: proteins related to DNA transcription, replication and repair; gray: hypothetical proteins (an e-value b 0.01 is considered significant).
contained the lyophilized phage could lead to a higher cumulative survival rate. 4. Discussion Nowadays, pathogens and bacterial infections have been recognized as an important limitation to Asian aquaculture production (BondadReantaso et al., 2005). The widespread and frequent uses of antibiotics in aquaculture have resulted in the emergence of antibiotic resistance and food safety hidden danger (Defoirdt et al., 2011). Early studies have reviewed the application of phage therapy in aquaculture and highlighted the use of phages as an alternative strategy to antibiotics (Nakai and Park, 2002; Oliveira et al., 2012), but the application of phage mainly focused on fishes and shrimps. In the past decade, semiintensive culturing method has made great contribution to the development of sea cucumber farming in China, and demands for sea cucumber in the world market have increased rapidly (Yuan et al., 2006; Xu and Li, 2013). However, the outbreaks of microbial diseases have resulted in important economic losses, with most of the pathogens that cause skin ulceration syndrome being Vibrio species (Deng et al., 2009; Zhang et al., 2015; Li et al., 2013).
Fig. 7. Effect of lyophilization on the titer of vibrio phage Vc1. A: prior to lyophilization; B: after lyophilization. Means with different superscripts differ significantly from each other (P b 0.05). n = 3.
In the present study, a bacteriophage that targets V. cyclitrophicus VC1 was isolated and characterized. This study was also the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against Vibrio infection. V. cyclitrophicus is considered as one of the pathogens responsible for skin ulceration syndrome in sea cucumbers (Deng et al., 2009). The LD50 of V. cyclitrophicus VC1 was 3.37 × 104 CFU/g body weight by coelom injection, which showed that the pathogen exhibited greater virulence to juvenile sea cucumbers as previously noted by other investigators (Santos et al., 1988). Several phages against Vibrios have been described (Oliveira et al., 2012; Zhang et al., 2015), and the one that we isolated in this study, vibrio phage Vc1, belongs to Siphoviridae of Caudovirales as shown by morphological analysis. The phage was effective against V. cyclitrophicus VC1 in vitro, yielding a latent period of 25 min and a burst period of 45 min (Fig. 4), which were consistent with the description of phage lifecycle reported by O'Flaherty et al. (2009). Meanwhile, phage stocks with a higher MOI displayed more obvious antibacterial effect. Additionally, the OD600 values of the test groups increased slightly in the later incubation stage, which could be caused by the generation of phage-resistant strains, a phenomenon that was noted in previous studies (Labrie et al., 2010; Laanto et al., 2012; Mateus et al., 2014). Thus, further study is needed to confirm the existence of phage-resistant strains of V. cyclitrophicus VC1.
Fig. 8. Cumulative survival rates of sea cucumbers treated with phage and antibiotic. A group: diet-1 treatment group; B group: phage immersion group (MOI = 10); C group: phage injection group. Letters above the columns indicate significant differences at the P b 0.05 levels. Note: ***P b 0.001 (vs negative control group); #P b 0.05 (vs positive control group).
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5. Conclusions
Fig. 9. Variation trends of phage titers over a 71-days period for group C (60 days of feeding as shown in light gray area, one day of challenging period and 10 days of post-infection period as showed in gray area). A: standard/initial phage titers after lyophilization; B: phage titers in tank water; C: phage titers in the track of sea cucumbers. n = 3.
Genomic analysis of vibrio phage Vc1 revealed no significant similarity to any other phages from the NCBI database, with the highest sequence similarity being less than 1%. To our knowledge, vibrio phage Vc1 could be considered as the second phage specific to V. cyclitrophicus, and its genome, both in terms of DNA length and GC content, was quite different from phage φJM-2012, the first V. cyclitrophicus-specific phage that was isolated (Jang et al., 2013). Vc1 encodes its own RNA-polymerase (Table S1), suggesting that it is capable of self-duplication. It lacks the genes for generalized transduction and virulence factors (Table S1). This and the fact that it formed clear plaques (Fig. 3) indicated that it might be a lytic phage. However, further analysis is needed to provide more information on the evolutionary history of vibrio phage Vc1. A number of safety issues have been raised with respect to the therapeutic use of phages (Brüssow, 2005; Oliveira et al., 2012). Since no genes encoding toxin and virulence factors were detected in the phage genome, the issue of gene recombinants between phages and their host is not particularly relevant for phage therapy. Moreover, like mammalian, phage particles could be recognized by the immune system of them, whereas invertebrates such as sea cucumbers do not pose the problem that occurrence of an immune response because of their nonspecific immunity (Efrony et al., 2007). In general, phage treatment trials at the laboratory or pilot scale usually involve coelom injection or immersion treatment (Nakai and Park, 2002; Zhang et al., 2015). In addition to these two methods, we also fed the sea cucumbers with feedstuff containing the phage (freeze-dried powder). In this phage-based protection experiment, all five groups were challenged with V. cyclitrophicus VC1 after 60 days of feeding. MOI of 10 phage suspensions and a concentration of 5 mg/mL Doxycycline were respectively used for phage injection group (C group) and positive control group, and their doses were based on the experience of our previous work (Zhang et al., 2015). After 10 days of observation, all three experiment groups showed encouraging result (Fig. 8). Especially the group fed with diet-1 (group A), which achieved similar result as that obtained from antibiotic treatment group. The presence of phages in the tanks and the gut of sea cucumbers were measured as well (Fig. 9). During the 60 days of feeding, the phage titers in the tanks and gut of the sea cucumbers remained stable and the sea cucumbers also remained in a relatively healthy state, which proved the applicability of the administration of adding phage lyophilization powder to the feedstuff and adaptation of the sea cucumber. In addition, unlike chemicals and other substances, the dose of phage administered to the sea cucumbers via the feedstuff may not be too important because of the self perpetuating nature of the phages, which could increase the phage titer in the pathogen-contaminated water (Nakai, 2010), and this phenomenon was also illustrated in our study (Fig. 9, pink area).
This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against Vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture. However, using single phage to control the diverse bacterial strains is critical because of the need to find the right specificity and the issue posed by bacterial resistance to the phages. Given these two constrains, a cocktail of several phages might be necessary to combat a wide range of bacteria and reduce the development of resistance (Brüssow, 2005; Nakai, 2010). Furthermore, the impact of the phage on nonspecific immunerelated enzymes should be further studied. In short, the application of lytic phages as a way of combating diseases in the sea cucumber industry might represent a viable alternative to the use of antibiotics. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2015.09.024. Acknowledgments This work was supported by the National Public Science and Technology Research Funds Projects of Ocean (Grant No. 201405003) and the National High-Tech Research and Development Program of China (863 Program) (2013AA102805-03). References Adams, M.H., 1959. Bacteriophages. Interscience Publishers, New York. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 404–410. Besemer, J., Borodovsky, M., 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 33, W451–W454. Bondad-Reantaso, M.G., Subasinghe, R.P., Arthur, J.R., Ogawa, K., Chinabut, S., Adlard, R., Tan, Z., Shariff, M., 2005. Disease and health management in Asian aquaculture. Vet. Parasitol. 132, 249–272. Brüssow, H., 2005. Phage therapy: the Escherichia coli experience. Microbiology 151, 2133–2140. Defoirdt, T., Sorgeloos, P., Bossier, P., 2011. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251–258. Deng, H., He, C., Zhou, Z., Liu, C., Tan, K., Wang, N., Jiang, B., Gao, X., Liu, W., 2009. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. Aquaculture 287, 18–27. Dini, C., de Urraza, P.J., 2013. Effect of buffer systems and disaccharides concentration on Podoviridae coliphage stability during freeze drying and storage. Cryobiology 66, 339–342. Dokland, T., 1999. Scaffolding proteins and their role in viral assembly. Cell. Mol. Life Sci. 56, 580–603. Efrony, R., Loya, Y., Bacharach, E., Rosenberg, E., 2007. Phage therapy of coral disease. Coral Reefs 26, 7–13. Fischetti, V.A., 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11, 393–400. He, C., Huang, G., 2014. Analysis of sea cucumbers (Apostichopus japonicas) culture in China. Modern Fish. Inform. China 29, 24–30 (In Chinese, with English abstract). Jang, H.B., Fagutao, F.F., Nho, S.W., Park, S.B., Cha, I.S., Yu, J.E., Lee, J.S., Im, S.P., Aoki, T., Jung, T.S., 2013. Phylogenomic network and comparative genomics reveal a diverged member of the φKZ-related group, marine vibrio phage φJM-2012. J. Virol. 87, 12866–12878. Kim, J.H., Gomez, D.K., Nakai, T., Park, S.C., 2010. Isolation and identification of bacteriophages infecting ayu Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum. Vet. Microbiol. 140, 109–115. Laanto, E., Bamford, J.K.H., Laakso, J., Sundberg, L.R., 2012. Phage-driven loss of virulence in a fish pathogenic bacterium. PLoS One 7, e53157. Labrie, S.J., Samson, J.E., Moineau, S., 2010. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327. Li, H., Qiao, G., Li, Q., Zhou, W., Won, K.M., Xu, D., Park, S., 2010. Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus. J. Fish Dis. 33, 865–877. Li, Q., Sun, K., Zhang, X., 2013. Research progress on “Skin Ulceration Syndrome” of Apostichopus japonicas. J. Agr. Sci. Technol. China 15, 40–45 (In Chinese, with English abstract). Liu, H., Zhen, F., Sun, X., Hong, X., Dong, S., Wang, B., Tang, X., Wang, Y., 2010. Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka). J. Invertebr. Pathol. 105, 236–242. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15, 687–701.
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Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Efficiency of a bacteriophage in controlling vibrio infection in the juvenile sea cucumber Apostichopus japonicus Zhen Li a, Jiancheng Zhang a, Xiaoyu Li a, Xitao Wang a, Zhenhui Cao a,c, Lili Wang a, Yongping Xu a,b,⁎ a b c
School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China Ministry of Education Center for Food Safety of Animal Origin, Dalian 116600, People's Republic of China Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201, People's Republic of China
a r t i c l e
i n f o
Article history: Received 14 August 2015 Received in revised form 22 September 2015 Accepted 24 September 2015 Available online 28 September 2015 Keywords: Apostichopus japonicus Vibrio cyclitrophicus Bacteriophage Genome sequencing Skin ulceration syndrome Phage lyophilization
a b s t r a c t Vibro cyclitrophicus is thought to be responsible for the cause of severe infection in juvenile sea cucumber (Apostichopus japonicus). Increases in the prevalence of antibiotic resistant pathogens have promoted us to develop effective agents to antibiotics for combating microbial infection among animals as well as human. In the present study, we isolated a bacteriophage with the ability to cleave V. cyclitrophicus present in the sewage of sea cucumber farms. This bacteriophage was designated as phage vB_VcyS_Vc1 (Vibrio phage Vc1). A one-step growth curve analysis of the phage revealed eclipse and latent periods of 25 and 45 min, respectively, with a burst size of 215 PFU/infected cell. Morphological analysis revealed the phage belongs to family of Siphoviridae. Furthermore, genomic sequencing results revealed a double-stranded DNA containing 44,541 bp with a G + C content of 44.16%. Forty four coding sequences were annotated in the genome, and nineteen of these were associated with a known function. Genes related to virulence and toxins were not detected in the genome. In addition, a prevention experiment conducted in a marine environment demonstrated that the phage increased the survival rate of juvenile sea cucumbers (18 ± 2 g) from 18% to 81% when the sea cucumber were fed with feedstuff containing the freeze-dried phage powder, 58% when the sea cucumber was injected with purified phages (MOI = 10), and 63% when the sea cucumbers were immersed in a suspension containing purified phages. Notably, when the phage provided nearly the same protection to the sea cucumbers as antibiotic when it was fed to the sea cucumbers in the form of freeze-dried powder mixed with feedstuff. Taken together, the results demonstrated that the use of phage to control the infection of V. cyclitrophicus in sea cucumber may be a feasible alternative to antibiotics. Statement of relevance: The A. japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China, which is of high nutritional value and economic value. In the past decade, as the growth of the market demand, A. japonicus's artificial breeding scale expands rapidly and the sea cucumber industry has become a vigorous sector in China aquaculture. However, with the high density and intensive development model of A. japonicus breeding, the various diseases have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers. At present, sanitizer and antibiotics are still commonly used in farms or hatcheries to control the bacterial disease in juvenile sea cucumbers, which resulted in the environment pollution and antibiotic residues. Nowadays, increased appearance of antibiotic resistant phenomenon promoted us to develop effective agents. Hence, alternative strategies to antibiotics should be further developed. This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Apostichopus japonicus (sea cucumber) is the most profitable aquaculture animal with the highest output as single variety in China. Due to its nutritional properties and high economical values, the farming ⁎ Corresponding author at: School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, People's Republic of China. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.aquaculture.2015.09.024 0044-8486/© 2015 Elsevier B.V. All rights reserved.
of this cucumber has become a vigorous sector in the aquaculture of China (Zhang et al., 2003; Liu et al., 2010). In China, as much as 137,754 t of A. japonicus were produced in the provinces of Liaoning and Shandong alone in 2011, with a production value of nearly 4 billion dollars (He and Huang, 2014). However, the rapid expansion and intensification of sea cucumber farming has led to various problems, including the occurrence of various diseases that have resulted in serious economic losses. Skin ulceration syndrome (SUS) is one of the most epidemic and serious diseases that affect sea cucumbers (Deng et al.,
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2009), and it is mainly caused by the bacteria Vibrio splendidus, Vibrio alginolyticus, Vibrio cyclitrophicus and Shewanella marisflavi (Deng et al., 2009; Liu et al., 2010; Li et al., 2010, 2013). At present, antibiotics and sanitizer are still commonly used to control diseases in juvenile sea cucumbers. The problems of environment pollution and antibiotic residues in aquaculture have become a concern due to the frequent and widespread use of antibiotics (Nakai and Park, 2002; Defoirdt et al., 2011). Therefore, it is important to develop other strategies that could be used in place of antibiotics in aquaculture to protect the animals against diseases. Bacteriophages or phages are bacterial parasites with life cycles as short as 20–60 min (O'Flaherty et al., 2009). Phage therapy has many advantages over antibiotics, such as high abundance, host specificity, and rapid exponential replication (Matsuzaki et al., 2005). Phage therapy has already been tested in a variety of species and has achieved positive results in the control of bacterial infections in aquaculture, such as in the control of lactococcosis (caused Lactococcus garvieae) in yellowtail (Nakai et al., 1999), systemic bacterial coldwater disease (caused by Pseudomonas plecoglossicida) in ayu fish (Kim et al., 2010) and luminous vibriosis (caused by Vibrio harveyi) in shrimp (Shivu et al., 2007). Our previous work has focused on using phage as an alternative to antibiotics for controlling V. alginolyticus infection in A. japonicus (Zhang et al., 2015). The aim of this study was to evaluate the potential use of a lytic phage for the treatment of V. cyclitrophicus-inflicted skin ulceration syndrome in A. japonicus. 2. Materials and methods 2.1. Bacterial strain and animals The bacterial strain used for phage isolation was first isolated from diseased sea cucumbers obtained from a hatchery in Dalian Bay (Dalian, China) in 2012. The bacterium was isolated via the use of thiosulfate citrate bile salts–sucrose agar (TCBS agar, Difco) medium. It was subsequently grown in 2216E medium and stored as a bacterial suspension stock at − 80 °C. Genetic characterization of the strain was achieved by 16S ribosomal DNA (rDNA) sequence analysis, and the sequence was then submitted to the Genebank database. Homologous analysis was performed using NCBI BLAST. V. cyclitrophicus VC1 was recovered from a frozen stock by growth in 2216E liquid medium at 26 °C for 24 h. The bacterial suspension was subsequently centrifuged at 2500 × g for 10 min and the cell pellet was re-suspended in sterile physiological buffered saline (PBS) to a density. The bacterial cell counts were calculated according to the OD-cell counts coherence curve. Healthy sea cucumbers (15–20 g), which showed no symptoms of peristome dedma, skin ulceration, head shaking and visceral ejection (Deng et al., 2009), were purchased in the Dalianwan hatchery and kept in a temperature controlled mariculture room (14–16 °C) to provide the animals with normal growth condition. 2.2. Pathogenicity assay in sea cucumber One hundred and twenty healthy sea cucumbers (18 ± 2 g) were randomly divided into 12 groups, with 10 animals per group. Six groups were used for coelom injection while the other six groups were used for immersion challenge. For coelom injection, one group was designated as the control group, and was injected with 0.3 mL PBS per animal, whereas the remaining five groups were each injected with the above bacterial suspensions, which ranged from 8 × 105 to 8 × 109 CFU/mL through 10-fold dilution method. For immersion challenge, each group of sea cucumbers was placed in 4 L of sterilized seawater. For the control group, 10 mL of PBS was added to the water, whereas for the test groups, 10 mL of bacterial suspensions was added to the water, yielding a concentration ranging from 2 × 104 to 2 × 108 CFU/mL. According to the criteria described by Santos et al. (1988), a bacterial strain is considered highly virulent if the LD50 value is 1.7 × 104 to 1 × 106 CFU/g body weight,
moderately virulent if it is 1.4 × 106 to 1.8 × 107 CFU/g body weight, and non-virulent if it is more than 108 CFU/g body weight. To protect animals' welfare, all sea cucumbers were cultured in a temperature controlled mariculture room (14–16 °C), and the pH, dissolved oxygen and salinity of the water were maintained at 7.5–8.0, 5–6 mg/mL, and 30–33 g/L, respectively. 2.3. Phage isolation, propagation and purification The phage was isolated from the raw sewage obtained from the drain-pipes from the same local hatchery (Dalian, China). Prior to the isolation, CaCl2 and MgCl2 were added to the water to a final concentration of 10 mM. After three rounds of plaque purification using the double-layered agar method described by Adams (1959), a single plaque was enriched using the method described by Maniatis et al. (1978) in 2216E broth and grown at 26 °C for overnight. The suspension was clarified by centrifugation (10,000 ×g, 10 min) and filtered through a 0.22-μm filter (EMD Millipore Co., Billerica, MA, USA). After that it was assayed for phage titers (plaque forming units, PFU) using the spot assay technique. Purification of the phage was carried out using the methods described in molecular cloning (Sambrook et al., 1989) with modification to obtain phage particles with high quality and quantity. The phage lysate was mixed with 1 M NaCl and 10% (w/v) polyethylene glycol (PEG) 8000 and dissolved by shaking. The lysate was allowed to stand at 4 °C for 10 h or overnight and then centrifuged at 10,000 × g for 20 min. The precipitate was then re-suspended in SM buffer (NaCl, MgSO4·7H2O, Tris·Cl, gelatin and double distilled water), and CsCl was added to the suspension to a concentration of 1.2 g/mL. The suspension was centrifuged at 150,000 ×g for 24 h along a CsCl density gradient using with a Beckman Optima L-100XP Ultracentrifuge (Fullerton, CA) (Sambrook et al., 1989). 2.4. Morphological observation of the phage CsCl-purified phage particles (≥1010 PFU/mL) were spotted onto a carbon-coated copper acid grid for 10 min, stained with 0.5% (w/v) uranyl acetate, and then examined by JEOL-1200EX transmission electron microscopy (JEOL USA, Inc., Peabody, MA, USA) at an accelerating voltage of 80 kV. 2.5. One-step growth curve of the phage One-step growth curve of the phage was obtained as described by Phumkhachorn and Rattanachaikunsopon (2010) with some modifications designed to calculate the latent period, burst time and burst size. Briefly, 200 mL of early log phase culture was centrifuged at 10,000 ×g for 10 min and then re-suspended in 10 mL of fresh 2216E broth to obtain a final concentration of 108 CFU/mL. In order to obtain a MOI (multiplicity of infection) of 0.1, of 1010 PFU/mL phage particles were added to the bacterial culture and allowed to absorb for 10 min at 26 °C. The suspension above was then centrifuged at 10,000 × g for 5 min, resuspended in 10 mL of fresh 2216E broth, and then incubated at 26 °C with shaking at 120 rpm. Samples were taken every 10 min and over a period of 2 h. The supernatant of each sample was subjected to phage titration using the spot assay technique. 2.6. In vitro inhibition of bacterial growth by phage To calculate the antimicrobial capacity of the phage, 96-well-micro plates were filled with 100 μL of inoculated double strengthen 2216E per well, and 100 μL of the prepared phage dilution was added to the well to provide three final doses of MOI to 0.1, 1 and 10. Controls for plate sterility, phage suspension sterility and bacterial culture without phage addition were also included, and each phage–host culture combination at specific MOI was performed in triplicate wells. Subsequently,
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these plates were incubated at 26 °C for 12 h and the optical density at 600 nm (OD600) was measured using a Micro-titer Plate Reader (Mutiskan Go, Thermo Scientific Co. Ltd., USA) at 30 min intervals. Each independent trial was repeated three times. 2.7. Phage genomic DNA extraction and sequencing analysis To find out whether the phage genome contains the genes related to toxin and virulence, the entire phage genome was extracted and sequenced. This safety issue would be particularly relevant for phage therapy. CsCl-purified phage particles (≥ 1010 PFU/mL) were used to extract the whole genomic DNA using a phenol–chloroform–isoamyl–alcohol method (Sambrook et al., 1989). The phage genomic DNA was subsequently analyzed by a garose gel electrophoresis (AGE) using 0.8% (w/v) agarose gel in Tris-acetate buffer at 100 V. The DNA extracted above was sequenced using Illumina High-Throughput Sequencing Platform (Illumina Hiseq™ 2000, Shenzhen, China). For genomic analysis, coding sequences (CDS) of the genome were determined using the Heuristic GeneMarkS software (Besemer and Borodovsky, 2005). Comparison of amino acid sequences against the NCBI non-redundant database and CDS functions were predicted by BLASTX (Altschul et al., 1990) and InterproScan (Fischetti, 2008), respectively. In addition, the conserved domains of CDSs were confirmed by the Conserved Domain Database (Marchler et al., 2005). Transfer RNA (tRNA) encoding genes were screened using tRNASscan at http:// greengene.uml.edu/programs/FindtRNA.html (Lowe and Eddy, 1997). Meanwhile, the annotation of the phage genome was identified using Artemis software (Rutherford et al., 2000). 2.8. Phage therapeutic study 2.8.1. Diet preparation The experimental diet contained 50% Laminaria japonica feedstuff, 20% sea mud, 15% soybeen meal, 10% fish meal, 3% shell powder 2% vitamin and mineral premixes (Wang et al., 2015), which were all provided by the SEM Feed Company (Dalian, China). The purified phage suspension was freeze-dried in the presence of skim milk using a laboratory freeze dryer (Beijing Biocool Company, Beijing, China). The drying process yielded a powder form of the phage. Two set of diets were made: the first set contained feedstuff mixed with the phage powder was named diet-1, whereas the second set consisted of only feedstuff, and was named diet-2 (Table 1). The standard of nutrition was based on early report described by Zhu et al. (2005). 2.8.2. Phage therapeutic experiment The therapeutic potential of the phage was evaluated by assessing its ability to protect sea cucumbers from developing skin ulceration syndrome. A total of 300 sea cucumbers (15 ± 2 g) were randomly divided into five groups (three test groups and two control groups). Each group consisted of three PVC tanks (38 cm × 54 cm × 80 cm) with 20 animals per tank as three parallel experiments. Each tank was filled with 40 L of aerated sand-filtered seawater and the temperature of the water was kept at 16–18 °C. Among the five groups, two groups were kept as control and three groups were treated with the phage. For the two control groups, one group was treated with antibiotic (positive control) whereas the other group was kept in sterilized seawater (negative control). As
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for the three test groups, one group was fed with diet-1 (group A), another group was immersed in water containing purified phages (group B), while the remaining group was injected with purified phages (group C) (Table 2). Sea cucumbers were fed at a rate of approximately 3% of the body weight and the feedstuff above contained 5% lyophilization powder per gram. After 60 days of feeding, After 60 days of feeding, all sea cucumbers were challenged with V. cyclitrophicus VC1. The animals were exposed to an initial bacterial concentration of 5 × 107 CFU/mL of 400 mL (bacterial suspensions re-suspended with PBS were diluted to 40 L seawater). Twenty-four hours later, positive control group was injected with 1 mL antibiotic (5 mg/mL doxycycline), negative control group, A and B groups were injected with the same volume of sterilized seawater, group C was injected with phage particles at 107 PFU/mL (MOI = 10), and group B was added the same concentration of phage suspensions to the tanks for phage immersion treatment (Table 2). Survival was then recorded for 10 days and the animals were not fed during this period. In addition, phage titers in the tank as well as the tracks of sea cucumbers were detected respectively in every ten days. At each time point, three sea cucumbers at random were sterilized with 70% ethanol for about 3 min and then dissected by aseptic surgery technique as described by Xing et al. (1998). The digestive tract of each animal (Fig. 1 F, G) was collected and homogenized (IKA R104, Germany) in cold 50 mM Tris–HCl (pH 7.8) buffer, followed by centrifugation at 10,000 ×g for 10 min at 4 °C (Wang et al., 2015).The final concentration of the phage in the tank was calculated as follows: Final phage titer (T2) = T1 (N W R r)/V, where T1 is the phage titer after lyophilization treatment, N is the number of animals in each tank, W is the body weight of each sea cucumber, R and r are, respectively, feed rate (3%) and phage lyophilization powder additive rate (5% in this study), and V is the seawater volume in the tank. 2.9. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) using Prism 5.0. The significance of differences between the treatments and controls were determined using Tukey's Multiple Comparison Test and Chi-square test. Statistical significance was considered at the P b 0.05 level. 3. Results 3.1. Pathogenicity test of the isolate V. cyclitrophicus VC1 The bacterial strain designated as V. cyclitrophicus VC1 was characterized by 16S-rDNA sequencing. The 16SrDNA sequence of 1442 bp was submitted to GenBank Database (GenBank: JX856180), showing 99% identity to V. cyclitrophicus strain LMG21359 (DQ481610) and 99% identity to V. cyclitrophicus strain VC2 (KC185413). The results of the pathogenicity test revealed mass mortalities on day 5 of postinfection by coelom injection and on day 4 by immersion challenge (Fig. 2). The LD50 of the V. cyclitrophicus VC1 was 3.37 × 104 CFU/g body weight as determined by coelom injection and 1.68 × 105 CFU/mL as determined by immersion challenge. Since the LD50 values obtained by different methods were within the range of 1.7 × 104 to 1 × 106 CFU/g, V. cyclitrophicus VC1 was considered highly virulent to sea cucumber.
Table 1 Proximate composition of the two experimental diets. Diet
Diet-1 Diet-2
Nutrient content Protein (%)
Lipid (%)
Ash (%)
Fiber (%)
Dry matter (%)
Additives (5% per gram feedstuff)
15.2 14.8
4.5 4.2
37.3 37.8
7.2 6.8
97.2 96.4
Phage freeze-dried powder with skim milk Only skim milk freeze-dried powder without phage
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Table 2 Experimental design for phage-based therapy in sea cucumber. Group
Negative control
Positive control
Diet Diet-2 Diet-2 After 60 days of feeding, challenged with V. Administration Coelom Coelom injection injection Seawater + − Antibiotics − + Phage − −
A group
B group
C group
Diet-1 Diet-2 Diet-2 cyclitrophicus VC1, 24 h later Coelom Immersion Coelom injection injection + − − − − − − + +
Survival was recorded for 10 days and animals were not fed in this period. “+”: related procedure was applied; “−”: related procedure was not applied.
For the group treated with the highest concentration of the culture, clinical symptoms of skin ulceration syndrome such as mouth tumidity, head shaking, enlarged ulcers and visceral ejection were detected after 20 h of infection, and sea cucumbers began to die two days after they were challenged with the bacteria. No mortality was observed in the control from the pathogenicity test conducted by coelom injection or immersion. 3.2. Isolation and identification of phage Using V. cyclitrophicus VC1 as a host organism, a bacteriophage was isolated from the water sample through the double layer method (Adams, 1959). The phage produced clear plaques on the lawn of V. cyclitrophicus VC1 cells, with a diameter of the plaques about 1.6 ± 0.2 mm (Fig. 3 A), indicating that the phage was a virulent phage toward V. cyclitrophicus VC1. Morphological analysis showed that the phage possessed a symmetry head (diameter 50 ± 3 nm) together with a long flexible non-contractile tail (length 150 ± 6 nm) (Fig. 3 B), suggesting that the phage belonged to the Siphoviridae of Caudovirales. The isolated phage was then designated as phage vB_VcyS_Vc1 (vibrio phage Vc1, CGMCC 9101). The one-step growth curve of the phage propagated on the host strain showed that the latent and burst periods were approximately 25 min and 45 min, respectively (Fig. 4). In addition, the average burst size of the phage was calculated as 215 PFU per infected cell. The effect of the phage on V. cyclitrophicus VC1 in vitro is shown in Fig. 5. Compared to the control group, the optical density of V. cyclitrophicus VC1
Fig. 2. Cumulative mortality of sea cucumber infected with Vibrio cyclitrophicus VC1 by coelom injection (A) or immersion (B) at different concentrations.
treated with different MOI phage stocks started to show significant decreases (P b 0.05) 3 h after infection. Four hours after infection, the OD600 decreased further and was still significant (P b 0.05) compared to the control group. Subsequently, the OD600 values of all three test groups with different MOI increased slightly after 10 h after infection.
3.3. Genome sequencing and annotation of the phage The complete sequencing data of the phage genome revealed a DNA sequence of 44,541 bp in length, with 44.61% GC content. Forty-four coding sequences (CDSs) comprising 88.6% of the genome were
Fig. 1. Photographs of diseased sea cucumbers suffering from skin ulceration syndrome. A and B: skin ulceration syndrome in dead sea cucumbers obtained from one hatchery in Dalian (China), C: rotting skin after day 2 post-infection (dashed arrow) and normal body wall (solid arrow), D: typical symptoms of skin ulceration syndrome (dashed arrow), E: healthy (solid arrows) and diseased (dashed arrows) juvenile sea cucumbers, F: diseased juvenile sea cucumbers with empty intestines and ulceration body wall, G: intestinal structure diagram of healthy juvenile sea cucumbers.
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Fig. 3. Phage plaques (A) in 2216E media and electron micrographs (B) negatively stained with uranylacetate. Scale bar represents 50 nm.
subsequently predicted (Table S1) and nearly all of them start with an ATG codon, except for CDS2, which starts with GTG and CDS19 and CDS22, which start with TTG. In addition, a tandem repeat sequence (5′-AACATCCGCTATCAAGCAGAG-3′) was observed and no tRNA genes were found in the genome. Moreover, the functions of 19 CDSs were clarified using Blastx against NR Database and two of them were hypothetically conserved (e-value b 0.01). Blastn analysis showed a low degree of homology between vibrio phage Vc1 and other phages. Furthermore, the genome also comprised four basic segments, including genes coding for packaging proteins and structural proteins, and DNA replication-related genes as well as other functional genes (Fig. 6). The structural and packaging proteins related genes of this phage were clustered in the first half of the genome. Putative scaffolding protein, coded by CDS6, has been proven to play an important role in virion particles formation by promoting the interaction between protein subunits and the stability of assembly intermediates (Dokland, 1999). However, the genes encoding lysozyme were not detected in the genome of vibrio phage Vc1, while a cell wall hydrolase-encoding gene was identified (CDS21), which was detected in the middle of the phage genome (Table S1). Besides, there were no genes related to the toxin and virulence of the phage, suggesting that the phage could have considerable potential for phage therapy.
Fig. 4. One-step growth curve of vibrio phage Vc1. Data represent are the means ± SD from three independent experiments. n = 3.
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Fig. 5. Inactivation of V. cyclitrophicus by vibrio phage Vc1 at different multiplication of infection (MOI) in vitro. n = 3.
3.4. Nucleotide sequence accession number and culture preservation The 16S rDNA sequence of V. cyclitrophicus VC1 and the complete genome sequence of phage vB_VcyS_Vc1 (vibrio phage Vc1) were available in GenBank under accession number JX856180 and KJ502657 respectively. The phage and the host strain were simultaneously preserved in China General Microbiological Culture Collection Center (Beijing, China) with CGMCC No. 9101.
3.5. Phage therapeutic study Skim milk was assayed as phage lyophilization media and the phage titer was significantly decreased (P b 0.05) as showed in Fig. 7, which was consistent with the results described by Dini and de Urraza (2013) before. The titer of initially purified phage suspension was 8 × 1011 PFU/mL. After freeze-drying treatment, the titer decreased to 6 × 109 PFU/mL, and the standard FT in each tank was about 9 × 105 PFU/mL (Fig. 9 Curve C). After 60 days of feeding with diet-1 and diet-2, all five groups of sea cucumbers were challenged with V. cyclitrophicus VC1, and the cumulative survival rate was determined. All three phage treatment groups showed significant (P b 0.001) increases in survival rate compared to negative control group (Fig. 8). Notably, group A displayed nearly the same survival rate with the antibiotic treatment group (P N 0.05). Group A appeared to have a better protection (81% survival rate) than group B (63% survival rate) and group C (58% survival rate), the difference between A group and B or C group was significant (P b 0.05) while no significant difference between B and C groups (P N 0.05). In the control group, sea cucumbers suffered skin ulceration syndrome and began with the appearance of a white lesion (Fig. 1 C, D and E), the animals began to die after day 2 post-infection. The phage titers in the water that housed group A sea cucumbers determined as well as the phage titer in the intestinal tracts of these sea cucumbers was also determined (Fig. 9). As shown by the light gray area, phage titer in the seawater (Fig. 9 Curve A) was always lower compared to the standard FT (Curve C), and in each sampling point, phage titer within the intestinal tract was more or less steady (P N 0.05) (Fig. 9 Curve B). These results indicated that the phage could exist in the seawater and intestinal tracts of the sea cucumbers when these animals were fed with diet-1. Ten days after challenged with V. cyclitrophicus VC1, the phage titer was significantly increased (P b 0.05) as shown by the gray area in Fig. 9, which suggested that the phage could protect the sea cucumbers against infection of V. cyclitrophicus VC1. Moreover, consumption of diet-1, which
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Fig. 6. Schematic representation of the complete genome of vibrio phage Vc1. Forty-four putative proteins are represented by arrows. Blue: mainly structural proteins; red: packaging proteins; pink: additional functional proteins; green: proteins related to DNA transcription, replication and repair; gray: hypothetical proteins (an e-value b 0.01 is considered significant).
contained the lyophilized phage could lead to a higher cumulative survival rate. 4. Discussion Nowadays, pathogens and bacterial infections have been recognized as an important limitation to Asian aquaculture production (BondadReantaso et al., 2005). The widespread and frequent uses of antibiotics in aquaculture have resulted in the emergence of antibiotic resistance and food safety hidden danger (Defoirdt et al., 2011). Early studies have reviewed the application of phage therapy in aquaculture and highlighted the use of phages as an alternative strategy to antibiotics (Nakai and Park, 2002; Oliveira et al., 2012), but the application of phage mainly focused on fishes and shrimps. In the past decade, semiintensive culturing method has made great contribution to the development of sea cucumber farming in China, and demands for sea cucumber in the world market have increased rapidly (Yuan et al., 2006; Xu and Li, 2013). However, the outbreaks of microbial diseases have resulted in important economic losses, with most of the pathogens that cause skin ulceration syndrome being Vibrio species (Deng et al., 2009; Zhang et al., 2015; Li et al., 2013).
Fig. 7. Effect of lyophilization on the titer of vibrio phage Vc1. A: prior to lyophilization; B: after lyophilization. Means with different superscripts differ significantly from each other (P b 0.05). n = 3.
In the present study, a bacteriophage that targets V. cyclitrophicus VC1 was isolated and characterized. This study was also the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against Vibrio infection. V. cyclitrophicus is considered as one of the pathogens responsible for skin ulceration syndrome in sea cucumbers (Deng et al., 2009). The LD50 of V. cyclitrophicus VC1 was 3.37 × 104 CFU/g body weight by coelom injection, which showed that the pathogen exhibited greater virulence to juvenile sea cucumbers as previously noted by other investigators (Santos et al., 1988). Several phages against Vibrios have been described (Oliveira et al., 2012; Zhang et al., 2015), and the one that we isolated in this study, vibrio phage Vc1, belongs to Siphoviridae of Caudovirales as shown by morphological analysis. The phage was effective against V. cyclitrophicus VC1 in vitro, yielding a latent period of 25 min and a burst period of 45 min (Fig. 4), which were consistent with the description of phage lifecycle reported by O'Flaherty et al. (2009). Meanwhile, phage stocks with a higher MOI displayed more obvious antibacterial effect. Additionally, the OD600 values of the test groups increased slightly in the later incubation stage, which could be caused by the generation of phage-resistant strains, a phenomenon that was noted in previous studies (Labrie et al., 2010; Laanto et al., 2012; Mateus et al., 2014). Thus, further study is needed to confirm the existence of phage-resistant strains of V. cyclitrophicus VC1.
Fig. 8. Cumulative survival rates of sea cucumbers treated with phage and antibiotic. A group: diet-1 treatment group; B group: phage immersion group (MOI = 10); C group: phage injection group. Letters above the columns indicate significant differences at the P b 0.05 levels. Note: ***P b 0.001 (vs negative control group); #P b 0.05 (vs positive control group).
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5. Conclusions
Fig. 9. Variation trends of phage titers over a 71-days period for group C (60 days of feeding as shown in light gray area, one day of challenging period and 10 days of post-infection period as showed in gray area). A: standard/initial phage titers after lyophilization; B: phage titers in tank water; C: phage titers in the track of sea cucumbers. n = 3.
Genomic analysis of vibrio phage Vc1 revealed no significant similarity to any other phages from the NCBI database, with the highest sequence similarity being less than 1%. To our knowledge, vibrio phage Vc1 could be considered as the second phage specific to V. cyclitrophicus, and its genome, both in terms of DNA length and GC content, was quite different from phage φJM-2012, the first V. cyclitrophicus-specific phage that was isolated (Jang et al., 2013). Vc1 encodes its own RNA-polymerase (Table S1), suggesting that it is capable of self-duplication. It lacks the genes for generalized transduction and virulence factors (Table S1). This and the fact that it formed clear plaques (Fig. 3) indicated that it might be a lytic phage. However, further analysis is needed to provide more information on the evolutionary history of vibrio phage Vc1. A number of safety issues have been raised with respect to the therapeutic use of phages (Brüssow, 2005; Oliveira et al., 2012). Since no genes encoding toxin and virulence factors were detected in the phage genome, the issue of gene recombinants between phages and their host is not particularly relevant for phage therapy. Moreover, like mammalian, phage particles could be recognized by the immune system of them, whereas invertebrates such as sea cucumbers do not pose the problem that occurrence of an immune response because of their nonspecific immunity (Efrony et al., 2007). In general, phage treatment trials at the laboratory or pilot scale usually involve coelom injection or immersion treatment (Nakai and Park, 2002; Zhang et al., 2015). In addition to these two methods, we also fed the sea cucumbers with feedstuff containing the phage (freeze-dried powder). In this phage-based protection experiment, all five groups were challenged with V. cyclitrophicus VC1 after 60 days of feeding. MOI of 10 phage suspensions and a concentration of 5 mg/mL Doxycycline were respectively used for phage injection group (C group) and positive control group, and their doses were based on the experience of our previous work (Zhang et al., 2015). After 10 days of observation, all three experiment groups showed encouraging result (Fig. 8). Especially the group fed with diet-1 (group A), which achieved similar result as that obtained from antibiotic treatment group. The presence of phages in the tanks and the gut of sea cucumbers were measured as well (Fig. 9). During the 60 days of feeding, the phage titers in the tanks and gut of the sea cucumbers remained stable and the sea cucumbers also remained in a relatively healthy state, which proved the applicability of the administration of adding phage lyophilization powder to the feedstuff and adaptation of the sea cucumber. In addition, unlike chemicals and other substances, the dose of phage administered to the sea cucumbers via the feedstuff may not be too important because of the self perpetuating nature of the phages, which could increase the phage titer in the pathogen-contaminated water (Nakai, 2010), and this phenomenon was also illustrated in our study (Fig. 9, pink area).
This study was the first to monitor the effectiveness of using feedstuff mixed with freeze-dried phage powder as a form of protection for sea cucumber against Vibrio infection, and demonstrated the effectiveness of phage in the control of pathogen in aquaculture. However, using single phage to control the diverse bacterial strains is critical because of the need to find the right specificity and the issue posed by bacterial resistance to the phages. Given these two constrains, a cocktail of several phages might be necessary to combat a wide range of bacteria and reduce the development of resistance (Brüssow, 2005; Nakai, 2010). Furthermore, the impact of the phage on nonspecific immunerelated enzymes should be further studied. In short, the application of lytic phages as a way of combating diseases in the sea cucumber industry might represent a viable alternative to the use of antibiotics. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2015.09.024. Acknowledgments This work was supported by the National Public Science and Technology Research Funds Projects of Ocean (Grant No. 201405003) and the National High-Tech Research and Development Program of China (863 Program) (2013AA102805-03). References Adams, M.H., 1959. Bacteriophages. Interscience Publishers, New York. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 404–410. Besemer, J., Borodovsky, M., 2005. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 33, W451–W454. Bondad-Reantaso, M.G., Subasinghe, R.P., Arthur, J.R., Ogawa, K., Chinabut, S., Adlard, R., Tan, Z., Shariff, M., 2005. Disease and health management in Asian aquaculture. Vet. Parasitol. 132, 249–272. Brüssow, H., 2005. Phage therapy: the Escherichia coli experience. Microbiology 151, 2133–2140. Defoirdt, T., Sorgeloos, P., Bossier, P., 2011. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251–258. Deng, H., He, C., Zhou, Z., Liu, C., Tan, K., Wang, N., Jiang, B., Gao, X., Liu, W., 2009. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. Aquaculture 287, 18–27. Dini, C., de Urraza, P.J., 2013. Effect of buffer systems and disaccharides concentration on Podoviridae coliphage stability during freeze drying and storage. Cryobiology 66, 339–342. Dokland, T., 1999. Scaffolding proteins and their role in viral assembly. Cell. Mol. Life Sci. 56, 580–603. Efrony, R., Loya, Y., Bacharach, E., Rosenberg, E., 2007. Phage therapy of coral disease. Coral Reefs 26, 7–13. Fischetti, V.A., 2008. Bacteriophage lysins as effective antibacterials. Curr. Opin. Microbiol. 11, 393–400. He, C., Huang, G., 2014. Analysis of sea cucumbers (Apostichopus japonicas) culture in China. Modern Fish. Inform. China 29, 24–30 (In Chinese, with English abstract). Jang, H.B., Fagutao, F.F., Nho, S.W., Park, S.B., Cha, I.S., Yu, J.E., Lee, J.S., Im, S.P., Aoki, T., Jung, T.S., 2013. Phylogenomic network and comparative genomics reveal a diverged member of the φKZ-related group, marine vibrio phage φJM-2012. J. Virol. 87, 12866–12878. Kim, J.H., Gomez, D.K., Nakai, T., Park, S.C., 2010. Isolation and identification of bacteriophages infecting ayu Plecoglossus altivelis altivelis specific Flavobacterium psychrophilum. Vet. Microbiol. 140, 109–115. Laanto, E., Bamford, J.K.H., Laakso, J., Sundberg, L.R., 2012. Phage-driven loss of virulence in a fish pathogenic bacterium. PLoS One 7, e53157. Labrie, S.J., Samson, J.E., Moineau, S., 2010. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 8, 317–327. Li, H., Qiao, G., Li, Q., Zhou, W., Won, K.M., Xu, D., Park, S., 2010. Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus. J. Fish Dis. 33, 865–877. Li, Q., Sun, K., Zhang, X., 2013. Research progress on “Skin Ulceration Syndrome” of Apostichopus japonicas. J. Agr. Sci. Technol. China 15, 40–45 (In Chinese, with English abstract). Liu, H., Zhen, F., Sun, X., Hong, X., Dong, S., Wang, B., Tang, X., Wang, Y., 2010. Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka). J. Invertebr. Pathol. 105, 236–242. Lowe, T.M., Eddy, S.R., 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 25, 955–964. Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15, 687–701.
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