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Vibrio coralliilyticus as an agent of red spotting disease in the sea urchin Strongylocentrotus intermedius
Aquaculture Reports 16 (2020) 100244
Contents lists available at ScienceDirect
Aquaculture Reports journal homepage: www.elsevier.com/locate/aqrep
Vibrio coralliilyticus as an agent of red spotting disease in the sea urchin Strongylocentrotus intermedius
T
Ruijun Li*, Huifeng Dang, Yuxi Huang, Zijiao Quan, Huijie Jiang, Weijie Zhang, Jun Ding* Agriculture Department Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, Dalian Ocean University, Dalian, 116023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sea urchin (Strongylocentrotus intermedius) Red spotting disease Vibrio coralliilyticus Isolation Identification
The bacterium, Vibrio coralliilyticus, is well known as one of the main pathogens involved in coral bleaching; however, there have been no previous reports of this bacterium in sea urchins. In the current study, a dominant bacterium, Rb102, was isolated from the coelomic fluid of a sea urchin (Strongylocentrotus intermedius) with red spotting disease, at a farm in Dalian, Liaoning Province. The isolates were identified based on Gram staining, morphological observations, 16S rDNA phylogenetic tree analysis, identification of physiological and biochemical characteristics, drug sensitivity tests, and artificial infection experiments. The identification and isolation results showed Rb102 to be V. coralliilyticus. Artificial infection experiments showed that Rb102 could infect S. intermedius by injection, causing the typical red spotting symptoms of this disease; the strains isolated from the diseased sea urchins were of the same strain type. The results of the bacterial susceptibility tests showed that Rb102 was highly susceptible to gentamicin, neomycin, tetracycline, cefradine, and ofloxacin, but showed strong resistance to vancomycin, ceftriaxone, kanamycin, rifampicin, midecamycin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin. The results of this study provide a reference for further study of the pathogenesis of V. coralliilyticus-induced erythema in sea urchins (S. intermedius) and its prevention and control.
1. Introduction The sea urchin Strongylocentrotus intermedius is an economical important marine echinoderm that is widely distributed and cultivated in North China, Korea, and Japan (Li et al., 2018; Wang et al., 2013). In recent years, with the rapid development of sea urchin farming, diseases of S. intermedius result in significant economic losses. Among the frequently reported diseases of S. intermedius, red spotting is one of the most serious and, thus, has received significant research attention. Some causative bacterial pathogens have been already reported. For example, in 1997, pathogenic bacteria isolated from diseased sea urchins in Japan were identified as Flexibacter sp. (Tajima et al., 1997). A new species of Vibrio causing red spotting disease was reported in 2005 (Wang et al., 2005). More recently, in 2011, an uncultured Tenacibaculum sp. was reported to induce red spotting disease in sea urchins (Masuda et al., 2004). Vibrio coralliilyticus is a temperature-regulated aquatic pathogen (Kimes et al., 2012; Ben-Haim et al., 2003) and has been identified as the main pathogen of coral bleaching and the cause of acute death and striping off disease in post larvae small abalone Haliotis diversicolor (Ushijima et al., 2014; Liu, 2006; Sussman et al., 2008); it has also been
⁎
reported to cause infection in other fish and shellfish, such as rainbow trout (Oncorhynchus mykiss), larval brine shrimp (Artemia spp.) (Austin et al., 2005), Pacific oyster larvae (Genard et al., 2013; KesarcodiWatson et al., 2012), great scallops (Pecten maximus), and the European flat oyster (Ostreaedulis). V. coralliilyticus forms a biofilm on the surface of aquatic animal tissues and organs that has high resistance to stress, antibiotics and the host immune system, resulting in mass mortality of infected animals. However, V. coralliilyticus as a cause of red spotting disease in sea urchins had not been previously reported. In the current study, we isolated and identified a bacterium from S. intermedius with red spotting disease from a sea urchin farm in Dalian, Liaoning Province and identified it as V. coralliilyticus. Thus, our results provide a reference for further study of the pathogenesis, transmission, and prevention of red spotting disease in sea urchins. 2. Materials and methods 2.1. Experimental animals S. intermedius with red spotting disease were collected from a farm in Dalian (121.5530 E, 38.8671 N). Healthy sea urchins were also
Corresponding authors at: 52# Heishijiao Street, Shahekou District, Dalian Ocean University, Dalian, 116023, China. E-mail addresses: [email protected] (R. Li), [email protected] (J. Ding).
https://doi.org/10.1016/j.aqrep.2019.100244 Received 22 June 2019; Received in revised form 23 October 2019; Accepted 25 October 2019 2352-5134/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Aquaculture Reports 16 (2020) 100244
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Fig. 1. Clinical symptoms of red spotting disease in sea urchins. A–D Different stages of disease progression during natural infection. Red arrows show typical symptom of red spotting disease.
according to Bergey's Manual (Sneath et al., 1986). Total bacterial DNA was extracted using a bacterial genome extraction kit (Tiangen, China). Two universal bacteria primers (F: 5′-AGAGTTTGATCCTGGCTCAG-5′; R: 5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991) for 16S rDNA gene were synthesized by Biotechnology Bioengineering (Shanghai) Ltd. The resulting products underwent PCR according to the following steps: predenaturation at 94 °C for 5 min; denaturation at 94 °C for 45 s, anneal at 55 °C for 45 s, extension at 72 °C for 90 s, 33 cycles; re-extension at 72 °C for 10 min.
sampled (shell diameter 4.15–5.22 cm, mean weight 6.70 ± 0.12 g in, aged 11 months). Experimental healthy S. intermedius were cultured for at least 7 days before any tests. All sea urchins were maintained in a recirculating aquaculture system, at a water temperature of 17 ℃ and room temperature of 20℃. The breeding and treatment of the experimental animals were based on animal experimental welfare and ethical management guidelines. 2.2. Isolation and purification of pathogenic bacteria The peristomal membrane of diseased S. intermedius was cut open using sterile scissors and 100 μL body coelomic fluid was removed using a pipette and spread evenly on Nutrient Agar (NA) plates (with 2 % NaCl). The plates were incubated in a constant temperature incubator at 28℃ for 24 h. The dominant bacterial colonies were observed and purified, and then stored in a −80 °C ultra-low temperature refrigerator in a 20 % glycerol protective solution until use.
2.4. Drug sensitivity test The antibiotic susceptibility of Rb102 strain was measured by using the disc diffusion method. A 0.2-mL bacterial culture was evenly coated on a NA plate. Drug-sensitive test papers were affixed onto the plate at equal distances apart. The plate was then placed in an incubator at 28 °C for 24 h. The diameter of the inhibitory zone was measured by using vernier calipers: 0 mm diameter was judged as insensitivity, < 10 mm was low sensitivity, 10–15 mm was medium sensitivity, and > 15 mm was high sensitivity. The drug sensitivity was determined according to the CLSI standard (Jorgensen and Turnidge, 2015). The experiment was repeated three times.
2.3. Identification of pathogenic bacteria Pathogenic bacteria were cultured by plate streaking. Each bacterial colony was then collected and stained using a Gram staining kit (Solarbio, China). Bacterial morphology was observed and recorded under an optical microscope. Bacterial cultures cultivated for 24 h were detected and analyzed by physiology and biochemistry experiments 2
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2.5. Artificial infection
Table 1 Comparison of the physiological and biochemical characteristics of strain Rb102 and Vibrio coralliilyticus.
The isolated strains were inoculated on 2 % NaCl NA plates and cultured at 28℃ for 24 h. Single colonies were then selected and inoculated into 2 % NaCl nutrient broth liquid. The bacteria were cultured for 12 h to logarithmic phase and centrifuged at 4000 r/min for 10 min. After centrifugation, the supernatant was discarded. And then precipitate was resuspended in sterilized saline. The number of bacteria were counted by hemocytometer. The bacterial solution concentration for challenging was respectively 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 CFU/mL. Fifteen sea urchins were assigned to each bacterial concentration and injected with 0.1 mL the relevant solution concentration. A control group was injected with 0.9 % sterile saline. The disease symptoms were observed and recorded daily for 7 days. Different group sea urchins were maintained in separate aquaculture system, at a water temperature of 17 ℃ and room temperature of 20 ℃. 3. Results 3.1. Symptoms of naturally infected sea urchins
Characteristic
Vibrio coralliilyticus
Rb102
0％ NaCl 3％ NaCl 6％ NaCl 8％ NaCl 10％ NaCl Glucose Galactose Citrate Gelatin L-Arginine Ornithine L-Arabinose Sucrose Trehalose VP Indole Inositol Erythritol Mannose
− ＋ ＋ － － ＋ ＋ ＋ － ＋ ＋ － － － － － － － ＋
－ ＋ ＋ － － ＋ ＋ ＋ － ＋ ＋ － － － － － － － ＋
There were two main symptoms in naturally diseased sea urchins. The first was the loss of spines, with purple-red viscous spots appearing at the points where the spines had dropped off, gradually enlarging until the surrounding areas also became covered with larger purple viscous plaques (Fig. 1A, C). The second symptom was that purple viscous spots appeared initially and, with the progress of the disease, larger purple viscous plaques appeared, with the concomitant loss of spines (Fig. 1B, D). 3.2. Isolation and identification of pathogenic bacteria A dominant bacterium, Rb102, was isolated from the coelomic fluid of diseased S. intermedius. On a 2 % NaCl NA plate, the strain colony was milky yellow, round and translucent, with a diameter of ∼2–3 mm. Gram staining showed the bacteria to be short, rod-shaped, red, and Gram-negative (Fig. 2). Combined with the results of physiological and biochemical identification (Table 1) and 16S rDNA sequence alignment, the strain Rb102 was identified as V. coralliilyticus. The 16S rDNA sequence of strain Rb102 was 1403 bp in size. The obtained gene sequence information was submitted to GenBank and the gene number of the strain was MK530200. Based on the Blast results of the sequences in GenBank, the similarity between the isolate and V. coralliilyticus (GenBank: KY229798.1) was as high as 99 %. Several strains of V. coralliilyticus 16S rDNA gene sequences and other Vibrio
Fig. 3. Phylogenetic relationship of related Vibrio sp. based on 16S r DNA gene sequences.
bacteria 16S rDNA sequences were selected from GenBank, and phylogenetic trees were constructed using Mega 5.0 software. The 16S rDNA phylogenetic tree results showed that Rb102 and V. coralliilyticus were clustered into one branch (Fig. 3). 3.3. Drug sensitivity test The antibiotic susceptibility of Rb102 strain was measured after 24 h by using the disc diffusion method. The results showed that the strain was insensitive to vancomycin, ceftriaxone, kanamycin, rifampicin, madithromycin, azlocillin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin, low sensitive to polymyxin B, moderately sensitive to gentamicin, neomycin, tetracycline and cefradine, and highly sensitive to ofloxacin (Table 2). 3.4. Artificial infection The control group S. intermedius injected with 0.9 % sterile saline remained alive and disease free throughout the experimental period. All mortalities after bacterial challenge occurred within 1 week after inoculation. Artificially infected S. intermedius (Fig. 4A, B) showed symptoms similar to the naturally diseased S. intermedius. The results showed that the isolated strain at concentrations of 1 × 107 CFU/mL and 1 × 106 CFU/mL caused obvious red spotting disease symptoms in healthy S. intermedius, and the cumulative rate of red spotting
Fig. 2. Gram staining of strain Rb102. (scale bar: 10 μm). 3
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Table 2 Drug sensitivity test results of Rb102 strain.a Drug Name
Inhibition zone diameter (mm)
Sensitivity
Vancomycin Cefatriaxone Kanamycin Rifampicin Medemycin Azlocillin Cefoperazone Ceftazidime Cotrimoxazole Penicillin Polymyxin B Gentamicin Cefradine Neomycin Tetracycline Ofloxacin
0 0 0 0 0 0 0 0 0 0 7 10 10 12.5 13 18
R R R R R R R R R R LS MS MS MS MS S
a Inhibition zone diameter 0 mm, insensitive (R); < 10 mm, low sensitivity (LS); 10–15 mm, medium sensitivity (MS); > 15 mm, high sensitivity (S).
symptoms reached 100 % and 33.3 %, respectively (Fig. 5). In addition, 1 × 108 and 1 × 109 CFU/mL V. coralliilyticus caused acute S. intermedius death, with the cumulative mortality rate reaching 100 % within 3 days of inoculation, without any symptoms of red spotting disease. And 1 × 105 CFU/mL V. coralliilyticus did not caused S. intermedius death and any symptoms of red spotting disease throughout the test period.
Fig. 5. Relationship between time and S. intermedius morbidity after artificial infection. A, 1 × 107 CFU/mL; B, 1 × 106 CFU/mL; C, control.
of red spotting disease in farmed S. intermedius. Further studies aim to analyze the transcriptome and metabolomics of S. intermedius with red spotting disease to reveal the pathogenic mechanisms involved. Based on previous studies, it was thought that red spotting disease in S. intermedius was not caused by a single specific pathogen. For example, Tajima et al. isolated pathogenic bacteria from coelomic fluid of S. intermedius with red spotting disease and identified the strain as Flexibacter maritimus, successfully using the bacteria to cause red spotting disease in artificially inoculated S. intermedius (Tajima et al., 1997). In 2006, Wang et al. isolated a causative pathogen of red spotting disease and identified it as a new Vibrio strain, 0205, which exhibited the highest 16S rRNA gene similarity to the strain Marine Bacterium AJ002566 (Genbank) (Wang et al., 2006). In 2011, Masuda et al. reported that the causative bacterium of red spotting disease in S. intermedius was Tenacibaculum sp. (Masuda et al., 2004). In the current study, based on the results of biological characteristics, 16S rDNA sequence alignment, phylogenetic analysis, and artificial infection experiments, a new causative bacterium of red spotting disease was confirmed as V. coralliilyticus. These multiple causative agents of disease are not unusual in echinoderms. For example, skin ulcerative syndrome in sea cucumbers is caused by a variety of pathogenic microorganisms
4. Discussion V. coralliilyticus has received research attention because of its role as temperature-controlled pathogen in corals, and its presence in marine environments globally. In 2003, Ben-Haim et al. reported V. coralliilyticus to be the main pathogenic bacteria isolated from diseased Pocillopora damicornis in the Indian Ocean, and confirmed the taxonomic position of the isolate; thus, it was thought that V. coralliilyticus only spread and caused coral disease above water temperatures of 25℃ (Ben-Haim et al., 2003). Kimes et al. identified potential virulence mechanisms using whole genome sequencing of V. coralliilyticus, to further define this mechanism of temperature-dependent pathogenicity (Kimes et al., 2012). The genus Vibrio is notorious for its pathogenic effects in numerous aquatic organisms and, thus, is an economically important genus in the marine aquaculture industry. Although previous studies have reported disease caused by V. coralliilyticus (e.g. Liu, 2006), the current study is the first to report V. coralliilyticus as a cause
Fig. 4. Symptoms of red spotting disease in artificially infected S. intermedius. Red arrows show typical symptom of red spotting disease. 4
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(Deng et al., 2009; Liu et al., 2010). Currently, the most efficient way to prevent and treat various aquatic animal diseases is generally considered to be the rational use of antimicrobial drugs. In this study, the result of drug sensitive tests showed that V. coralliilyticus was not sensitive to many antibiotics (showing low or no sensitivity to vancomycin, ceftriaxone, kanamycin, rifampicin, midecamycin, azlocillin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin). This might be because antibiotic overuse can lead to bacterial drug resistance or that V. coralliilyticus has evolved mechanisms to overcome the effects of antibacterials. Previous research has indicated that V. coralliilyticus can produce antimicrobial compounds and biofilms (Wietz et al., 2011; Vizcaino et al., 2010; Lu et al., 2014). Nevertheless, several high-sensitivity drugs are in use for treating red spotting disease in sea urchin framing. Therefore, the correct application of these drugs should be ensured to avoid their overuse. Meanwhile, the development of replacements for use in aquaculture requires research attention. To conclude, the causative bacterium isolated from the coelomic fluid of S. intermedius with red spotting disease in a farm in Dalian, Liaoning Province, was confirmed to be V. coralliilyticus. This study is the first to report V. coralliilyticus as a causative agent of red spotting disease and provides a reference for the further study of the pathogenesis, transmission, and prevention of this disease in S. intermedius.
vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii. Environ. Microbiol. 7 (9), 1488–1495. Ben-Haim, Y., Thompson, F., Thompson, C., Cnockaert, M., Hoste, B., Swings, J., Rosenberg, E., 2003. Vibrio coralliilyticus sp. nov., a temperature-dependent pathogen of the coral Pocillopora damicornis. Int. J. Syst. Evol. Microbiol. 53 (1), 309–315. 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 (1), 18–27. Genard, B., Miner, P., Nicolas, J.L., Moraga, D., Boudry, P., Pernet, F., Tremblay, R., 2013. Integrative study of physiological changes associated with bacterial infection in Pacific oyster larvae. PLoS One 8 (5), e64534. Jorgensen, J.H., Turnidge, J.D., 2015. Susceptibility test methods: dilution and disk diffusion methods. Manual of Clinical Microbiology, Eleventh edition. pp. 1253–1273 Am. Soc. Microbiol. Kesarcodi-Watson, A., Miner, P., Nicolas, J.L., Robert, R., 2012. Protective effect of four potential probiotics against pathogen-challenge of the larvae of three bivalves: Pacific oyster (Crassostrea gigas), flat oyster (Ostrea edulis) and scallop (Pecten maximus). Aquaculture 344 (1), 29–34. Kimes, N.E., Grim, C.J., Johnson, W.R., Hasan, N.A., Tall, B.D., Kothary, M.H., Kiss, H., Munk, A.C., Tapia, R., Green, L., 2012. Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus. ISME J. 6 (4), 835. Li, M., Wu, F., Lu, D., Zhuo, R., Chang, Y., 2018. Effects of Cu2+ on growth, immunity and gonad development of Strongylocentrotus intermedius. J. Fish. China 42 (2), 854–862. Liu, G., 2006. Studies on the pathogenic bacteria of acute death and striping off disease in cultured postlarvae small abalone Haliotis diversicolor Reeve. J. Fish. Sci. China 13 (4), 12–17. Liu, H., Zheng, 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 (3), 236–242. Lu, W., Liu, G., Guo, Z., Zhou, Y., Guan, S., 2014. Antimicrobial activities of Lonicera japonica Thunb. and Forsythia suspensa Thunb. Vahl on Vibrio coralliilyticus and its biofilm. J. Guangdong Pharm. Univ 3 (1), 691–700. Masuda, Y., Tajima, K., Ezura, Y., 2004. Resuscitation of Tenacibaculum sp., the causative bacterium of spotting disease of sea urchin Strongylocentroutus intermedius, from the viable but non-culturable state. Fish. Sci. 70 (2), 277–284. Sneath, P.H., Mair, N.S., Sharpe, M.E., Holt, J.G., 1986. Bergey’s Manual of Systematic Bacteriology Volume 2 Williams & Wilkins. Sussman, M., Willis, B.L., Victor, S., Bourne, D.G., 2008. Coral pathogens identified for white syndrome (WS) epizootics in the Indo-Pacific. PLoS One 3 (6), e2393. Tajima, K., Hirano, T., Shimizu, M., Ezura, Y., 1997. Isolation and pathogenicity of the causative bacterium of spotting disease of sea urchin Strongylocentrotus intermedius. Fish. Sci. 63 (2), 249–252. Ushijima, B., Videau, P., Burger, A.H., Shore-Maggio, A., Runyon, C.M., Sudek, M., Aeby, G.S., Callahan, S.M., 2014. Vibrio coralliilyticus strain OCN008 is an etiological agent of acute Montipora white syndrome. Appl. Environ. Microbiol. 80 (7), 2102–2109. Vizcaino, M.I., Johnson, W.R., Kimes, N.E., Williams, K., Torralba, M., Nelson, K.E., Smith, G.W., Weil, E., Moeller, P.D., Morris, P.J., 2010. Antimicrobial resistance of the coral pathogen Vibrio coralliilyticus and Caribbean sister phylotypes isolated from a diseased octocoral. Microb. Ecol. 59 (4), 646–657. Wang, B., Li, Y., Li, X., Qu, J., Zhao, X., 2005. Pathogenic mechanism of the causative vibrio found in ‘“red spotting”’ diseased sea urchin Strongylocentroyus intermedius. J. Dalian Fish. Univ. 20 (1), 11–15. Wang, B., Yan, L., Xia, L., Chen, H.X., Liu, M.Q., Kong, Y.T., 2006. Biological characteristic and pathogenicity of the pathogenic vibrio on the “redspot disease” of Strongylocentrotos intermedius. J. Dalian Fish. Univ. 30 (1), 371–376. Wang, Y., Feng, N., Qiang, L., Ding, J., Zhan, Y., Chang, Y., 2013. Isolation and characterization of bacteria associated with a syndrome disease of sea urchin Strongylocentrotus intermedius in North China. Aquacult. Res. 44 (5), 691–700. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697–703. Wietz, M., Mansson, M., Gram, L., 2011. Chitin stimulates production of the antibiotic andrimid in a Vibrio coralliilyticus strain. Environ. Microbiol. Rep. 3 (5), 559–564.
Ethics statement The breeding and treatment of the experimental animals were based on animal experimental welfare and ethical management guidelines. Author contributions Ruijun Li, drafted paper and analysed data; Huifeng Dang, conducted experiment and analysed data; Yuxi huang, conducted experiment; Zijiao Quan, conducted experiment; Huijie Jiang, conducted experiment; Weijie Zhang, analysed data；Jun Ding, designed and supported study. Declaration of Competing Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Acknowledgments This work was funded in part by the State Key Research Project "Marine environment safety" (2017YFC1404503), and Department of Science & Technology of Liaoning Province Nature Foundation Guidance Program (2019-ZD-0733). References Austin, B., Austin, D., Sutherland, R., Thompson, F., Swings, J., 2005. Pathogenicity of
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Contents lists available at ScienceDirect
Aquaculture Reports journal homepage: www.elsevier.com/locate/aqrep
Vibrio coralliilyticus as an agent of red spotting disease in the sea urchin Strongylocentrotus intermedius
T
Ruijun Li*, Huifeng Dang, Yuxi Huang, Zijiao Quan, Huijie Jiang, Weijie Zhang, Jun Ding* Agriculture Department Key Laboratory of Mariculture & Stock Enhancement in North China’s Sea, Dalian Key Laboratory of Marine Animal Disease Control and Prevention, Dalian Ocean University, Dalian, 116023, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sea urchin (Strongylocentrotus intermedius) Red spotting disease Vibrio coralliilyticus Isolation Identification
The bacterium, Vibrio coralliilyticus, is well known as one of the main pathogens involved in coral bleaching; however, there have been no previous reports of this bacterium in sea urchins. In the current study, a dominant bacterium, Rb102, was isolated from the coelomic fluid of a sea urchin (Strongylocentrotus intermedius) with red spotting disease, at a farm in Dalian, Liaoning Province. The isolates were identified based on Gram staining, morphological observations, 16S rDNA phylogenetic tree analysis, identification of physiological and biochemical characteristics, drug sensitivity tests, and artificial infection experiments. The identification and isolation results showed Rb102 to be V. coralliilyticus. Artificial infection experiments showed that Rb102 could infect S. intermedius by injection, causing the typical red spotting symptoms of this disease; the strains isolated from the diseased sea urchins were of the same strain type. The results of the bacterial susceptibility tests showed that Rb102 was highly susceptible to gentamicin, neomycin, tetracycline, cefradine, and ofloxacin, but showed strong resistance to vancomycin, ceftriaxone, kanamycin, rifampicin, midecamycin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin. The results of this study provide a reference for further study of the pathogenesis of V. coralliilyticus-induced erythema in sea urchins (S. intermedius) and its prevention and control.
1. Introduction The sea urchin Strongylocentrotus intermedius is an economical important marine echinoderm that is widely distributed and cultivated in North China, Korea, and Japan (Li et al., 2018; Wang et al., 2013). In recent years, with the rapid development of sea urchin farming, diseases of S. intermedius result in significant economic losses. Among the frequently reported diseases of S. intermedius, red spotting is one of the most serious and, thus, has received significant research attention. Some causative bacterial pathogens have been already reported. For example, in 1997, pathogenic bacteria isolated from diseased sea urchins in Japan were identified as Flexibacter sp. (Tajima et al., 1997). A new species of Vibrio causing red spotting disease was reported in 2005 (Wang et al., 2005). More recently, in 2011, an uncultured Tenacibaculum sp. was reported to induce red spotting disease in sea urchins (Masuda et al., 2004). Vibrio coralliilyticus is a temperature-regulated aquatic pathogen (Kimes et al., 2012; Ben-Haim et al., 2003) and has been identified as the main pathogen of coral bleaching and the cause of acute death and striping off disease in post larvae small abalone Haliotis diversicolor (Ushijima et al., 2014; Liu, 2006; Sussman et al., 2008); it has also been
⁎
reported to cause infection in other fish and shellfish, such as rainbow trout (Oncorhynchus mykiss), larval brine shrimp (Artemia spp.) (Austin et al., 2005), Pacific oyster larvae (Genard et al., 2013; KesarcodiWatson et al., 2012), great scallops (Pecten maximus), and the European flat oyster (Ostreaedulis). V. coralliilyticus forms a biofilm on the surface of aquatic animal tissues and organs that has high resistance to stress, antibiotics and the host immune system, resulting in mass mortality of infected animals. However, V. coralliilyticus as a cause of red spotting disease in sea urchins had not been previously reported. In the current study, we isolated and identified a bacterium from S. intermedius with red spotting disease from a sea urchin farm in Dalian, Liaoning Province and identified it as V. coralliilyticus. Thus, our results provide a reference for further study of the pathogenesis, transmission, and prevention of red spotting disease in sea urchins. 2. Materials and methods 2.1. Experimental animals S. intermedius with red spotting disease were collected from a farm in Dalian (121.5530 E, 38.8671 N). Healthy sea urchins were also
Corresponding authors at: 52# Heishijiao Street, Shahekou District, Dalian Ocean University, Dalian, 116023, China. E-mail addresses: [email protected] (R. Li), [email protected] (J. Ding).
https://doi.org/10.1016/j.aqrep.2019.100244 Received 22 June 2019; Received in revised form 23 October 2019; Accepted 25 October 2019 2352-5134/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Aquaculture Reports 16 (2020) 100244
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Fig. 1. Clinical symptoms of red spotting disease in sea urchins. A–D Different stages of disease progression during natural infection. Red arrows show typical symptom of red spotting disease.
according to Bergey's Manual (Sneath et al., 1986). Total bacterial DNA was extracted using a bacterial genome extraction kit (Tiangen, China). Two universal bacteria primers (F: 5′-AGAGTTTGATCCTGGCTCAG-5′; R: 5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991) for 16S rDNA gene were synthesized by Biotechnology Bioengineering (Shanghai) Ltd. The resulting products underwent PCR according to the following steps: predenaturation at 94 °C for 5 min; denaturation at 94 °C for 45 s, anneal at 55 °C for 45 s, extension at 72 °C for 90 s, 33 cycles; re-extension at 72 °C for 10 min.
sampled (shell diameter 4.15–5.22 cm, mean weight 6.70 ± 0.12 g in, aged 11 months). Experimental healthy S. intermedius were cultured for at least 7 days before any tests. All sea urchins were maintained in a recirculating aquaculture system, at a water temperature of 17 ℃ and room temperature of 20℃. The breeding and treatment of the experimental animals were based on animal experimental welfare and ethical management guidelines. 2.2. Isolation and purification of pathogenic bacteria The peristomal membrane of diseased S. intermedius was cut open using sterile scissors and 100 μL body coelomic fluid was removed using a pipette and spread evenly on Nutrient Agar (NA) plates (with 2 % NaCl). The plates were incubated in a constant temperature incubator at 28℃ for 24 h. The dominant bacterial colonies were observed and purified, and then stored in a −80 °C ultra-low temperature refrigerator in a 20 % glycerol protective solution until use.
2.4. Drug sensitivity test The antibiotic susceptibility of Rb102 strain was measured by using the disc diffusion method. A 0.2-mL bacterial culture was evenly coated on a NA plate. Drug-sensitive test papers were affixed onto the plate at equal distances apart. The plate was then placed in an incubator at 28 °C for 24 h. The diameter of the inhibitory zone was measured by using vernier calipers: 0 mm diameter was judged as insensitivity, < 10 mm was low sensitivity, 10–15 mm was medium sensitivity, and > 15 mm was high sensitivity. The drug sensitivity was determined according to the CLSI standard (Jorgensen and Turnidge, 2015). The experiment was repeated three times.
2.3. Identification of pathogenic bacteria Pathogenic bacteria were cultured by plate streaking. Each bacterial colony was then collected and stained using a Gram staining kit (Solarbio, China). Bacterial morphology was observed and recorded under an optical microscope. Bacterial cultures cultivated for 24 h were detected and analyzed by physiology and biochemistry experiments 2
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2.5. Artificial infection
Table 1 Comparison of the physiological and biochemical characteristics of strain Rb102 and Vibrio coralliilyticus.
The isolated strains were inoculated on 2 % NaCl NA plates and cultured at 28℃ for 24 h. Single colonies were then selected and inoculated into 2 % NaCl nutrient broth liquid. The bacteria were cultured for 12 h to logarithmic phase and centrifuged at 4000 r/min for 10 min. After centrifugation, the supernatant was discarded. And then precipitate was resuspended in sterilized saline. The number of bacteria were counted by hemocytometer. The bacterial solution concentration for challenging was respectively 1 × 105, 1 × 106, 1 × 107, 1 × 108, and 1 × 109 CFU/mL. Fifteen sea urchins were assigned to each bacterial concentration and injected with 0.1 mL the relevant solution concentration. A control group was injected with 0.9 % sterile saline. The disease symptoms were observed and recorded daily for 7 days. Different group sea urchins were maintained in separate aquaculture system, at a water temperature of 17 ℃ and room temperature of 20 ℃. 3. Results 3.1. Symptoms of naturally infected sea urchins
Characteristic
Vibrio coralliilyticus
Rb102
0％ NaCl 3％ NaCl 6％ NaCl 8％ NaCl 10％ NaCl Glucose Galactose Citrate Gelatin L-Arginine Ornithine L-Arabinose Sucrose Trehalose VP Indole Inositol Erythritol Mannose
− ＋ ＋ － － ＋ ＋ ＋ － ＋ ＋ － － － － － － － ＋
－ ＋ ＋ － － ＋ ＋ ＋ － ＋ ＋ － － － － － － － ＋
There were two main symptoms in naturally diseased sea urchins. The first was the loss of spines, with purple-red viscous spots appearing at the points where the spines had dropped off, gradually enlarging until the surrounding areas also became covered with larger purple viscous plaques (Fig. 1A, C). The second symptom was that purple viscous spots appeared initially and, with the progress of the disease, larger purple viscous plaques appeared, with the concomitant loss of spines (Fig. 1B, D). 3.2. Isolation and identification of pathogenic bacteria A dominant bacterium, Rb102, was isolated from the coelomic fluid of diseased S. intermedius. On a 2 % NaCl NA plate, the strain colony was milky yellow, round and translucent, with a diameter of ∼2–3 mm. Gram staining showed the bacteria to be short, rod-shaped, red, and Gram-negative (Fig. 2). Combined with the results of physiological and biochemical identification (Table 1) and 16S rDNA sequence alignment, the strain Rb102 was identified as V. coralliilyticus. The 16S rDNA sequence of strain Rb102 was 1403 bp in size. The obtained gene sequence information was submitted to GenBank and the gene number of the strain was MK530200. Based on the Blast results of the sequences in GenBank, the similarity between the isolate and V. coralliilyticus (GenBank: KY229798.1) was as high as 99 %. Several strains of V. coralliilyticus 16S rDNA gene sequences and other Vibrio
Fig. 3. Phylogenetic relationship of related Vibrio sp. based on 16S r DNA gene sequences.
bacteria 16S rDNA sequences were selected from GenBank, and phylogenetic trees were constructed using Mega 5.0 software. The 16S rDNA phylogenetic tree results showed that Rb102 and V. coralliilyticus were clustered into one branch (Fig. 3). 3.3. Drug sensitivity test The antibiotic susceptibility of Rb102 strain was measured after 24 h by using the disc diffusion method. The results showed that the strain was insensitive to vancomycin, ceftriaxone, kanamycin, rifampicin, madithromycin, azlocillin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin, low sensitive to polymyxin B, moderately sensitive to gentamicin, neomycin, tetracycline and cefradine, and highly sensitive to ofloxacin (Table 2). 3.4. Artificial infection The control group S. intermedius injected with 0.9 % sterile saline remained alive and disease free throughout the experimental period. All mortalities after bacterial challenge occurred within 1 week after inoculation. Artificially infected S. intermedius (Fig. 4A, B) showed symptoms similar to the naturally diseased S. intermedius. The results showed that the isolated strain at concentrations of 1 × 107 CFU/mL and 1 × 106 CFU/mL caused obvious red spotting disease symptoms in healthy S. intermedius, and the cumulative rate of red spotting
Fig. 2. Gram staining of strain Rb102. (scale bar: 10 μm). 3
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Table 2 Drug sensitivity test results of Rb102 strain.a Drug Name
Inhibition zone diameter (mm)
Sensitivity
Vancomycin Cefatriaxone Kanamycin Rifampicin Medemycin Azlocillin Cefoperazone Ceftazidime Cotrimoxazole Penicillin Polymyxin B Gentamicin Cefradine Neomycin Tetracycline Ofloxacin
0 0 0 0 0 0 0 0 0 0 7 10 10 12.5 13 18
R R R R R R R R R R LS MS MS MS MS S
a Inhibition zone diameter 0 mm, insensitive (R); < 10 mm, low sensitivity (LS); 10–15 mm, medium sensitivity (MS); > 15 mm, high sensitivity (S).
symptoms reached 100 % and 33.3 %, respectively (Fig. 5). In addition, 1 × 108 and 1 × 109 CFU/mL V. coralliilyticus caused acute S. intermedius death, with the cumulative mortality rate reaching 100 % within 3 days of inoculation, without any symptoms of red spotting disease. And 1 × 105 CFU/mL V. coralliilyticus did not caused S. intermedius death and any symptoms of red spotting disease throughout the test period.
Fig. 5. Relationship between time and S. intermedius morbidity after artificial infection. A, 1 × 107 CFU/mL; B, 1 × 106 CFU/mL; C, control.
of red spotting disease in farmed S. intermedius. Further studies aim to analyze the transcriptome and metabolomics of S. intermedius with red spotting disease to reveal the pathogenic mechanisms involved. Based on previous studies, it was thought that red spotting disease in S. intermedius was not caused by a single specific pathogen. For example, Tajima et al. isolated pathogenic bacteria from coelomic fluid of S. intermedius with red spotting disease and identified the strain as Flexibacter maritimus, successfully using the bacteria to cause red spotting disease in artificially inoculated S. intermedius (Tajima et al., 1997). In 2006, Wang et al. isolated a causative pathogen of red spotting disease and identified it as a new Vibrio strain, 0205, which exhibited the highest 16S rRNA gene similarity to the strain Marine Bacterium AJ002566 (Genbank) (Wang et al., 2006). In 2011, Masuda et al. reported that the causative bacterium of red spotting disease in S. intermedius was Tenacibaculum sp. (Masuda et al., 2004). In the current study, based on the results of biological characteristics, 16S rDNA sequence alignment, phylogenetic analysis, and artificial infection experiments, a new causative bacterium of red spotting disease was confirmed as V. coralliilyticus. These multiple causative agents of disease are not unusual in echinoderms. For example, skin ulcerative syndrome in sea cucumbers is caused by a variety of pathogenic microorganisms
4. Discussion V. coralliilyticus has received research attention because of its role as temperature-controlled pathogen in corals, and its presence in marine environments globally. In 2003, Ben-Haim et al. reported V. coralliilyticus to be the main pathogenic bacteria isolated from diseased Pocillopora damicornis in the Indian Ocean, and confirmed the taxonomic position of the isolate; thus, it was thought that V. coralliilyticus only spread and caused coral disease above water temperatures of 25℃ (Ben-Haim et al., 2003). Kimes et al. identified potential virulence mechanisms using whole genome sequencing of V. coralliilyticus, to further define this mechanism of temperature-dependent pathogenicity (Kimes et al., 2012). The genus Vibrio is notorious for its pathogenic effects in numerous aquatic organisms and, thus, is an economically important genus in the marine aquaculture industry. Although previous studies have reported disease caused by V. coralliilyticus (e.g. Liu, 2006), the current study is the first to report V. coralliilyticus as a cause
Fig. 4. Symptoms of red spotting disease in artificially infected S. intermedius. Red arrows show typical symptom of red spotting disease. 4
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(Deng et al., 2009; Liu et al., 2010). Currently, the most efficient way to prevent and treat various aquatic animal diseases is generally considered to be the rational use of antimicrobial drugs. In this study, the result of drug sensitive tests showed that V. coralliilyticus was not sensitive to many antibiotics (showing low or no sensitivity to vancomycin, ceftriaxone, kanamycin, rifampicin, midecamycin, azlocillin, cefoperazone, ceftazidime, cotrimoxazole, and penicillin). This might be because antibiotic overuse can lead to bacterial drug resistance or that V. coralliilyticus has evolved mechanisms to overcome the effects of antibacterials. Previous research has indicated that V. coralliilyticus can produce antimicrobial compounds and biofilms (Wietz et al., 2011; Vizcaino et al., 2010; Lu et al., 2014). Nevertheless, several high-sensitivity drugs are in use for treating red spotting disease in sea urchin framing. Therefore, the correct application of these drugs should be ensured to avoid their overuse. Meanwhile, the development of replacements for use in aquaculture requires research attention. To conclude, the causative bacterium isolated from the coelomic fluid of S. intermedius with red spotting disease in a farm in Dalian, Liaoning Province, was confirmed to be V. coralliilyticus. This study is the first to report V. coralliilyticus as a causative agent of red spotting disease and provides a reference for the further study of the pathogenesis, transmission, and prevention of this disease in S. intermedius.
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Ethics statement The breeding and treatment of the experimental animals were based on animal experimental welfare and ethical management guidelines. Author contributions Ruijun Li, drafted paper and analysed data; Huifeng Dang, conducted experiment and analysed data; Yuxi huang, conducted experiment; Zijiao Quan, conducted experiment; Huijie Jiang, conducted experiment; Weijie Zhang, analysed data；Jun Ding, designed and supported study. Declaration of Competing Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. Acknowledgments This work was funded in part by the State Key Research Project "Marine environment safety" (2017YFC1404503), and Department of Science & Technology of Liaoning Province Nature Foundation Guidance Program (2019-ZD-0733). References Austin, B., Austin, D., Sutherland, R., Thompson, F., Swings, J., 2005. Pathogenicity of
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